As of 26 Feb 2020

ORAL HANDOUTS BOOK

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TABLE OF CONTENTS

Handout List

Session B: CLINICOPATHOLOGICAL CORRELATION B-03 PLAQUE STAGE FOLLICULOTROPIC MYCOSIS FUNGOIDES: HISTOPATHOLOGIC FEATURES AND PROGNOSTIC FACTORS IN A SERIES OF 40 PATIENTS B-04 ADNEXOTROPISM: AN UNDER-RECOGNIZED HISTOPATHOLOGICAL FINDING IN AGGRESSIVE CYTOTOXIC CUTANEOUS LYMPHOMAS. B-05 SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA (SPTCL): A LARGE US CASE SERIES REVEALS A LYMPHOMA WITH LIMITED GROWTH POTENTIAL IN NEED OF DIAGNOSTIC AND THERAPEUTIC GUIDELINES B-06 GRANULOMATOUS MYCOSIS FUNGOIDES: A CLINICOPATHOLOGICAL STUDY OF 41 CASES FROM A TERTIARY CANCER CENTER

Session C: PROGNOTIC FACTORS IN CTCL C-01 PROGNOSTIC FACTORS FOR STAGE PROGRESSION AND SURVIVAL IN MYCOSIS FUNGOIDES AND SEZARY SYNDROME: THE PROCLIPI STUDY C-02 REVIEW OF THE MEASUREMENTS OF ABNORMAL LYMPH NODES ON IMAGING AND THE CORRELATION WITH N CLASS FROM THE PROSPECTIVE CUTANEOUS LYMPHOMA INTERNATIONAL PROGNOSTIC INDEX STUDY C-03 T-CELL MONOCLONALITY IN BLOOD AND SKIN CORRELATES WITH POOR RESPONSE TO TREATMENT IN MYCOSIS FUNGOIDES C-04 ERYTHRODERMA: ANALYSIS OF SÉZARY SYNDROME CRITERIA IN 292 ERYTHRODERMIC PATIENTS

Session D: EPIDEMIOLOGY D-01 GLOBAL EPIDEMIOLOGY OF PRIMARY CUTANEOUS LYMPHOMAS: A SYSTEMATIC REVIEW OF RELATIVE FREQUENCIES AND META-ANALYSIS D-02 EPIDEMIOLOGY OF PRIMARY CUTANEOUS CD8+ T CELL LYMPHOMA: A UNITED STATES POPULATION-BASED COHORT ANALYSIS USING THE SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS DATABASE D-03 RACIAL AND AGE DISPARITIES IN CARDIOVASCULAR EVENTS MAY EXIST IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMAS. D-04 MYCOSIS FUNGOIDES IN THE CHILEAN PEDIATRIC POPULATION: A RETROSPECTIVE EPIDEMIOLOGICAL AND CLINICAL-PATHOLOGICAL STUDY IN A CHILEAN REFERRAL CENTER D-05 MYCOSIS FUNGOIDES IN CHILDREN D-06 THE EPIDEMIOLOGY OF PRIMARY CUTANEOUS GAMMA/DELTA T-CELL LYMPHOMA: A SEER-18 ANALYSIS

Session E: IMMUNOLOGY I E-01 PROINFLAMMATORY AND IMMUNOSUPPRESSIVE CYTOKINES POLARIZE THE MICROENVIRONMENT IN CUTANEOUS T CELL LYMPHOMA AND REGULATE PD-L1 EXPRESSION E-02 NORMAL FIBROBLASTS PROMOTE TH1 CYTOKINE EXPRESSION IN MYLA CELLS BY SUPPRESSING TWIST1 EXPRESSION E-03 ANTI-CD47 IMMUNOTHERAPY IS MEDIATED BY CYTOTOXIC CD107A+IFN-Γ- NK CELLS AND CAN BE POTENTIATED BY INTERFERON-Α IN CUTANEOUS LYMPHOMA

Session F: IMMUNOLOGY II F-01 MAC-1/MAC-2A CELLS REPRESENT A POTENTIAL MODEL TO STUDY IL-13 SIGNALING IN SATB1 POSITIVE CUTANEOUS ALCL F-03 PROTEIN AND MRNA EXPRESSION LEVELS OF IL-17A, IL-17F AND IL-22 IN PATIENTS WITH MYCOSIS FUNGOIDES. F-04 INDUCTION OF ANTI-TUMOR EFFECT BY CD8+ T-CELLS VIA CADM1/CRTAM INTERACTION IN PATIENTS WITH ADULT T-CELL LEUKEMIA LYMPHOMA

Session G: PATHOGENESIS OF CUTANEOUS LYMPHOMAS G-01 ROLE OF TOX1 AND STAT3 PATHWAYS IN THE PATHOGENESIS OF CUTANEOUS T-CELL LYMPHOMA G-03 STANDARDIZED FLOW CYTOMETRY (EUROFLOW) DEMONSTRATES HETEROGENEOUS T-CELL ORIGIN OF SÉZARY LYMPHOMA CELLS G-04 MUTATIONAL SIGNATURE ANALYSIS REVEALS A KEY ROLE FOR UV RADIATION IN THE ACCUMULATION OF MUTATIONS IN CUTANEOUS T-CELL LYMPHOMA. G-05 EPIDERMAL FATTY ACID-BINDING PROTEIN IS NOT EXPRESSED BY TUMOR CELLS IN ADVANCED MYCOSIS FUNGOIDES G-06 PATCH LESIONS OF MYCOSIS FUNGOIDES PATIENTS HAVE A SIMILAR SKIN MICROBIOME PROFILE COMPARED TO NON-LESIONAL AND HEALTHY SKIN

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Session H: BIOMARKERS I H-01 EVALUATION OF BLOOD SÉZARY MARKERS BY MEANS OF CORRELATION WITH APOPTOSIS RESISTANCE AND CLONALITY H-02 THE A ALLELE OF RS7096317 IS PERMISSIVE FOR CD39 EXPRESSION IN CTCL AND IS A NEGATIVE PROGNOSTIC FACTOR IN OLDER PATIENTS.

Session I: BIOMARKERS II I-01 IDENTIFICATION OF CD39 AS A POTENTIAL THERAPEUTIC TARGET IN SEZARY SYNDROME I-02 THE CORRELATION OF FLOW CYTOMETRY ANALYSIS ON SKIN TISSUE WITH IMMUNOHISTOCHEMISTRY IN CUTANEOUS T-CELL LYMPHOMA I-03 OVEREXPRESSION OF STAT4 AT EARLY STAGES OF MYCOSIS FUNGOIDES: COINCIDENCE OR NOT?

Session J: GENETIC PATHOGENESIS J-01 COMPARISON OF EARLY WITH ADVANCED MYCOSIS FUNGOIDES LESIONS ON A SINGLE CELL LEVEL TO ASSESS POTENTIAL MEDIATORS OF DISEASE PROGRESSION J-03 GENIC SIGNATURE OF CUTANEOUS T CELL LYMPHOMA-ASSOCIATED FIBROBLASTS FROM MYCOSIS FUNGOIDES AND SEZARY SYNDROME. J-04 THE EFFECT OF EXTRACELLULAR MATRIX AND FIBROBLASTS ON PROLIFERATION AND SURVIVAL OF MALIGNANT CELLS IN MYCOSIS FUNGOIDES

Session K: EPIGENETIC ABNORMALITIES K-01 HYPOMETHYLATION-MEDIATED ACTIVATION OF TMEM244 GENE IN SÉZARY CELLS K-03 MYCOSIS FUNGOIDES CELL LINE – DERIVED EXOSOMES SHOW A DISTINCT SIGNATURE OF ELEVATED EXPRESSION OF miR-155, miR-1246, AND OX40 TO PROMOTE TUMOR PROGRESSION K-02 EPIGENETIC INVOLVEMENT IN CUTANEOUS T-CELL LYMPHOMA LYMPHOMAGENESIS K-04 TRANSFORMED MYCOSIS FUNGOIDES SHOWS DISTINCT MIRNA AND MRNA EXPRESSION PROFILES COMPARED TO CLASSIC MYCOSIS FUNGOIDES.

Session L: GENOMIC INSIGHTS L-03 FUNCTIONAL BIOLOGICAL CLASSIFICATION OF DIFFERENTIAL GENE EXPRESSION IN SÉZARY SYNDROME L-04 HIGH-THROUGHPUT SEQUENCING REVEALS T-CELL REPERTOIRE RESTRICTION IN SÉZARY SYNDROME AND MYCOSIS FUNGOIDES

Session M: MOLECULAR CHARACTERIZATION OF RARE CUTANEOUS LYMPHOMAS I M-01 MOLECULAR STUDY OF SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA, LUPUS PANNICULITIS AND OVERLAP CASES M-02 GERMLINE TIM-3 MUTATIONS CHARACTERIZE SUB-CUTANEOUS PANNICULITIS-LIKE T2 CELL LYMPHOMAS WITH HEMOPHAGOCYTIC LYMPHOHISTIOCYTIC SYNDROME M-03 COMPARATIVE GENOMIC ANALYSIS OF CD30+ LYMPHOPROLIFERATIVE DISORDERS M-05 CLONAL RELATIONSHIP BETWEEN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM AND MYELOID NEOPLASMS

Session N: MOLECULAR CHARACTERIZATION OF RARE CUTANEOUS LYMPHOMAS I N-01 CELLULAR ORIGINS AND GENETIC LANDSCAPE OF CUTANEOUS GAMMA DELTA T CELL LYMPHOMAS N-03 WHOLE-GENOME ANALYSIS UNCOVERS RECURRENT IKZF1 INACTIVATION AND ABERRANT CELL ADHESION IN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM.

Session O: CUTANEOUS B-CELL LYMPHOMAS O-01 PRIMARY CUTANEOUS FOLLICULAR LYMPHOMA IS GENETICALLY DISTINCT FROM SECONDARY CUTANEOUS FOLLICULAR LYMPHOMA O-02 CHALLENGES IN THE DIAGNOSIS OF PRIMARY CUTANEOUS LARGE B-CELL-LYMPHOMAS O-03 MUTATIONS OF THE B-CELL RECEPTOR PATHWAY CONFER CHEMORESISTANCE IN PRIMARY CUTANEOUS DIFFUSE LARGE B-CELL LYMPHOMA LEG-TYPE O-05 PRIMARY CUTANEOUS MARGINAL ZONE B-CELL LYMPHOMA IN CHILDREN AND YOUNG ADULTS O-06 FIRST - LINE RADIOTHERAPY DOES NOT IMPROVE THE RISK OF DISEASE PROGRESSION IN PRIMARY CUTANEOUS DIFFUSE LARGE B CELL LYMPHOMA, LEG TYPE

Session P: QUALITY OF LIFE P-01 THE EVALUATION OF THE QOL AND DAILY FUNCTIONING OF POLISH PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA COGNOSCO LLC COMMISSIONED BY THE FIRM TAKEDA PHARMA LLC P-02 SKIN-SPECIFIC QUALITY OF LIFE IN CUTANEOUS T-CELL LYMPHOMA COMPARED WITH OTHER DERMATOLOGIC DISEASES P-03 QUALITATIVE ASSESSMENT OF THE QUALITY OF LIFE IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA (CTCL) P-04 THE IMPACT OF GENDER, AGE, RACE/ETHNICITY, AND STAGE ON QUALITY OF LIFE IN A SPECTRUM OF CUTANEOUS LYMPHOMAS

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P-05 PSYCHIATRIC COMORBIDITY IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA (CTCL)

Session R: PRECLINICAL DEVELOPMENT OF PERSONALIZED THERAPIES I R-02 SYNERGISTIC THERAPEUTIC DRUG SCREENING USING JAK INHIBITION FOR CTCL. R-04 PHOSPHOLIPASE C GAMMA 1 (PLCG1) MUTATIONS IN SÉZARY CELLS DRIVE NFKB, AP-1 AND NFAT SIGNALLING AND MEDIATE RESISTANCE TO THE CALCINEURIN INHIBITOR TACROLIMUS. R-05 SCREENING FOR NOVEL COMBINATION TREATMENTS FOR CUTANEOUS T CELL LYMPHOMA FOR EXPEDITED DEVELOPMENT R-06 THE SYNERGISTIC PRO-APOPTOTIC EFFECT OF HDAC AND PARP-1 INHIBITION IN CUTANEOUS T- CELL LYMPHOMA IS MEDIATED VIA BLIMP-1

Session S: PRECLINICAL DEVELOPMENT OF PERSONALIZED THERAPIES II S-01 A SMALL MOLECULE CCR2 ANTAGONIST DEPLETES TUMOR MACROPHAGES AND SYNERGIZES WITH ANTI-PD1 IN A MURINE MODEL OF CUTANEOUS T CELL LYMPHOMA S-02 INVESTIGATING THE ROLE FOR POT1 GENE DYSFUNCTION IN PRIMARY CUTANEOUS T-CELL LYMPHOMA S-04 INTEGRIN ΑVΒ3 INHIBITION IMPROVES REXINOIDS ANTITUMORAL ACTIONS ON CUTANEOUS T CELL LYMPHOMA (CTCL) S-05 HDAC INHIBITOR RESMINOSTAT COUNTERACTS DISEASE-RELATED GENE EXPRESSION AND CYTOKINE SECRETION IN CTCL CELLS. S-06 SINGLE CELL RNA SEQUENCING ANALYSIS TO DEFINE THERAPEUTIC TARGETS IN CUTANEOUS T- CELL LYMPHOMA

Session T: NOVEL THERAPEUTICS T-01 CUSATUZUMAB FOR TREATMENT OF CD70-POSITIVE RELAPSED/REFRACTORY CUTANEOUS T-CELL LYMPHOMA IN A PHASE 1/2 CLINICAL TRIAL T-02 A PHASE 1B STUDY EVALUATING THE SAFETY AND EFFICACY OF TOPICAL ADMINISTRATION OF WP1220, A STAT3 INHIBITOR, FOR MYCOSIS FUNGOIDES (MF) T-03 HIGH DOSE RATE BRACHYTHERAPY FOR THE TREATMENT OF PRIMARY CUTANEOUS LYMPHOMA AT COMPLEX SITES AND COMPLEX CURVES SKIN SURFACES. T-04 INTRALESIONAL ONCOLYTIC VIROTHERAPY RESULTS IN TUMOR REGRESSION ASSOCIATED WITH THE INFLUX OF CYTOTOXIC T CELLS IN CUTANEOUS B-CELL LYMPHOMA

Session U: STEM CELL TRANSPLANT U-01 A PHASE II PROSPECTIVE STUDY USING NON-MYELOABLATIVE ALLOGENEIC TRANSPLANTATION IN PATIENTS WITH ADVANCED STAGE MYCOSIS FUNGOIDES AND SEZARY SYNDROME U-02 ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION AS A CURATIVE TREATMENT STRATEGY FOR PATIENTS WITH ADVANCED MYCOSIS FUNGOIDES AND SÉZARY SYNDROME: 2019 UPDATE OF THE MILAN EXPERIENCE U-03 NON-MYELOABLATIVE ALLOGENEIC STEM CELL TRANSPLANTATION USING TSEB TLI AND ATG FOR MYCOSIS FUNGOIDES(MF) AND SEZARY SYNDROME(SS). MEDIUM TERM RESULTS FROM A LARGE SINGLE CENTRE COHORT.

Session V: THERAPEUTICS I V-03 REAL-LIFE EXPERIENCE ON THE USE OF BRENTUXIMAB VEDOTIN ON NINE MYCOSIS FUNGOIDES PATIENTS: TIME TO RE-DISCUSS TREATMENT PROTOCOLS? V-04 BRENTUXIMAB VEDOTIN (BV) AND LENALIDOMIDE (LEN) IN RELAPSED/REFRACTORY (R/R) CUTANEOUS (CTCL) AND PERIPHERAL (PTCL) T-CELL LYMPHOMAS; INTERIM RESULTS OF A PHASE II TRIAL V-05 FINAL ALCANZA RESULTS: BRENTUXIMAB VEDOTIN VERSUS PHYSICIAN'S CHOICE IN PREVIOUSLY TREATED CD30-POSITIVE CUTANEOUS T-CELL LYMPHOMA (MYCOSIS FUNGOIDES OR PRIMARY CUTANEOUS ANAPLASTIC LARGE CELL LYMPHOMA) V-06 BRENTUXIMAB VEDOTIN FOR RELAPSED/REFRACTORY SÉZARY SYNDROME: A SINGLE -CENTER EXPERIENCE

Session W: THERAPEUTICS & CLINICAL TRIALS II W-01 RESOLUTION OF SÉZARY SYNDROME AFTER COMBINATION OF MOGAMULIZUMAB WITH PEGYLATED INTERFERON Α2-A IS MEDIATED BY CD56DIM NKP30+ IFN-Γ+ NK CELLS W-03 CLINICAL ACTIVITY AND SAFETY OF LOW-DOSE TOTAL SKIN ELECTRON BEAM THERAPY COMBINED WITH MOGAMULIZUMAB IN REFRACTORY SÉZARY SYNDROME: SUPPORT FOR A CLINICAL TRIAL IN CTCL

Session X: OBSERVATIONS AND PROSPECTIVES X-03 IMPROVED SURVIVAL FOR SKIN-PRIMARY PRESENTATION OF ADULT T-CELL LEUKEMIA/LYMPHOMA (ATLL) X-04 TREATMENT AND PROGNOSIS OF RARE PATIENTS WITH A PRIMARY CUTANEOUS CD30-POSITIVE LYMPHOPROLIFERATIVE DISORDER WHO DEVELOP EXTRACUTANEOUS LOCALISATIONS X-05 WINKELMANN REGIMEN: ORAL CHLORAMBUCIL FOR MYCOSIS FUNGOIDES AND SEZARY SYNDROME

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X-06 ORAL ABSTRACT X-06: CLINICAL CHARACTERIZATION OF MOGAMULIZUMAB-ASSOCIATED RASH X-07 CHARACTERIZATION OF THE HISTOPATHOLOGIC AND MOLECULAR FEATURES OF RASH ASSOCIATED WITH MOGAMULIZUMAB

Session Y: TOPICAL CHEMOTHERAPY Y-01 REAL-LIFE EXPERIENCE WITH CHLORMETHINE GEL: MOVING BEYOND CLINICAL TRIAL DATA Y-02 MECHLORETHAMINE TREATMENT DURATION AS A FUNCTION OF CLINICIAN-LEVEL PATIENT VOLUME FOR MYCOSIS FUNGOIDES CUTANEOUS T-CELL LYMPHOMA (MF-CTCL) Y-03 THE PROVE STUDY: REAL-WORLD EXPERIENCE WITH CHLORMETHINE GEL AND OTHER THERAPIES IN THE TREATMENT OF MYCOSIS-FUNGOIDES CUTANEOUS T-CELL LYMPHOMA PATIENTS Y-04 INCIDENCE AND TYPES OF CONTACT DERMATITIS AFTER CHLORMETHINE GEL TREATMENT IN PATIENTS WITH MYCOSIS FUNGOIDES-TYPE CUTANEOUS T-CELL LYMPHOMA: THE MIDAS STUDY

Session Z1: CLINICAL OBSERVATIONS I

Z-01 PARADOXICAL RESPONSE OF CUTANEOUS T-CELL LYMPHOMA IN A PATIENT WITH CONCOMITANT ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA Z-02 PROGRESSION OF CUTANEOUS T-CELL LYMPHOMA AFTER DUPILUMAB: CASE REVIEW OF 6 PATIENTS Z-04 PRIMARY CUTANEOUS B-CELL LYMPHOMA – CASE SERIES OF TWO UNIQUE CUTANEOUS PRESENTATIONS TREATED WITH RITUXIMAB. Z-05 TONSIL INVOLVEMENT AS A MARKER OF ADVANCED DISEASE IN MYCOSIS FUNGOIDES Z-06 EPSTEIN-BARR VIRUS-POSITIVE MUCOCUTANEOUS ULCER IN A PATIENT WITH DYSKERATOSIS CONGENITA

Session Z2: CLINICAL OBSERVATIONS II Z-08 CLINICAL, HISTOLOGICAL AND MOLECULAR CHARACTERISTICS OF ANAPLASTIC LYMPHOMA KINASE-POSITIVE PRIMARY CUTANEOUS ANAPLASTIC LARGE CELL LYMPHOMA Z-09 LUPUS ERYTHEMATOSUS TUMIDUS MIMICKING PRIMARY CUTANEOUS MARGINAL ZONE B CELL LYMPHOMA Z-10 MYCOSIS FUNGOIDES – GRANULOMATOUS SLACK SKIN IN ASSOCIATION WITH HODGKIN LYMPHOMA Z-11 AGGRESSIVE CUTANEOUS T-CELL LYMPHOMAS: A SERIES OF 19 CASES. Z-12 ANETODERMA: SECUNDARY TO SYPHILIS OR MYCOSIS FUNGOIDES? Z-13 CD8+ MYCOSIS FUNGOIDES PALMARIS ET PLANTARIS WITH PERIPHERAL BLOOD INVOLVEMENT. Z-14 LYMPHOMATOID DRUG ERUPTION AFTER TREATMENT OF HEPATITIS C VIRUS INFECTION WITH SOFOSBUVIR: A NEW DESCRIBED ADVERSE REACTION Z-15 MYCOSIS FUNGOIDES, LYMPHOMATOID PAPULOSIS AND HODGKIN’S LYMPHOMA IN THE SAME PATIENT: APROPOS OF A POSSIBLE MONOCLONAL ORIGIN. Z-16 PAGETOID RETICULOSIS: A RARE VARIANT OF MYCOSIS FUNGOIDES

Session Z3: CLINICAL OBSERVATIONS III Z-17 MAINTENANCE THERAPY – A NEGLECTED TOPIC Z-18 ROBUST GRAFT-VERSUS-LYMPHOMA (GVL) RESPONSE MIMICKING CUTANEOUS RELAPSE OF T- CELL LYMPHOMA Z-19 SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA IN THE MESENTERY WITH ASSOCIATED HEMOPHAGOCYTIC SYNDROME TREATED BY CHEMOTHERAPY CHOEP, MODIFIED HLH 2014 PROTOCOL AND HDT AND AUTO SCT. Z-20 AGGRESSIVE EPIDERMOTROPIC CD8+ T CELL LYMPHOMA PRESENTING WITH TARGETOID LESIONS Z-21 CD4/CD8 DOUBLE-NEGATIVE FOLLICULOTROPIC MYCOSIS FUNGOIDES/SEZARY SYNDROME PRESENTING AS SEVERE LEONINE FACIES

Session Z4: CLINICAL OBSERVATIONS IV Z-22 AIN-SHAMS CUTANEOUS LYMPHOMA CLINIC – SUGGESTED GUIDELINES FOR THE TREATMENT OF MYCOSIS FUNGOIDES IN COUNTRIES WITH LIMITED RESOURCES Z-23 PATIENTS WITH MYCOSIS FUNGOIDES ARE AT INCREASED RISK OF LUNG CANCER: A SYSTEMATIC REVIEW AND POPULATION-BASED ANALYSIS Z-24 EPIDEMIOLOGICAL ANALYSIS AND REVIEW OF HISTOPATHOLOGICAL PARAMETERS OF PATIENTS DIAGNOSED WITH MYCOSIS FUNGOIDES AND ITS VARIANTS IN FOLLOW-UP AT STATE UNIVERSITY OF CAMPINAS Z-25 AN OVERVIEW: OUR EXPERIENCE OF MICROBIOLOGICAL CAUSES OF INPATIENT ADMISSIONS FOR PATIENTS WITH MYCOSIS FUNGOIDES AND SÉZARY’S SYNDROME AT PETER MACCALLUM CANCER CENTRE. Z-26 THE USE OF GATA3 IN CHALLENGING CASES OF CD30+ MYCOSIS FUNGOIDES (MF) VERSUS ANAPLASTIC LARGE CELL LYMPHOMA (ALCL) Z-27 CD30 EXPRESSION IS DETECTABLE IN MOST RARE CUTANEOUS LYMPHOMA SUBTYPES Z-28 CD8+ MYCOSIS FUNGOIDES WITH DIVERSE ATYPICAL CLINICAL AND PATHOLOGICAL PRESENTATIONS Z-29 THE THERAPEUTIC POTENTIAL OF CANNABINOIDS FROM CANNABIS SATIVA EXTRACTS FOR MYCOSIS FUNGOIDES / SÉZARY SYNDROME - AN IN VITRO AND EX VIVO STUDY

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HANDOUTS LIST

B-03 PLAQUE STAGE FOLLICULOTROPIC MYCOSIS FUNGOIDES: HISTOPATHOLOGIC FEATURES AND PROGNOSTIC FACTORS IN A SERIES OF 40 PATIENTS Van Santen, S.; Jansen, P.; Quint, K.; Vermeer, M.; Willemze, R. Leiden University Medical Center, Leiden, Netherlands.

B-04 ADNEXOTROPISM: AN UNDER-RECOGNIZED HISTOPATHOLOGICAL FINDING IN AGGRESSIVE CYTOTOXIC CUTANEOUS LYMPHOMAS Walker, C.J.; Martinez-Escala, E.; Espinosa, M.L.; Tan, T.; Guitart, J. Feinberg School of Medicine, Northwestern University, Chicago, United States.

B-05 SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA (SPTCL): A LARGE US CASE SERIES REVEALS A LYMPHOMA WITH LIMITED GROWTH POTENTIAL IN NEED OF DIAGNOSTIC AND THERAPEUTIC GUIDELINES Guitart, J.1; Walker, C.J.1; Martinez-Escala, E.1; Comfere, N.I.2; Pulitzer, M.3; Rieger, K.4; Torres Cabala, C.A.5; Pincus, L.6; Duvic, M.5; Park, K.E.5; Espinosa, M.L.1; Kumar, E.S.7; Horwitz, S.3; Kim, Y.H.8; Mangold, A.R.9 1Feinberg School of Medicine, Northwestern University, Chicago, United States; 2Mayo Clinic, Rochester, United States; 3Memorial Sloan Kettering Cancer Center, New York, United States; 4Stanford University, Palo Alto, United States; 5MD Anderson Cancer Center, Houston, United States; 6University of California San Francisco, San Francisco, United States; 7Pathology Laboratory Associates/Regional Medical Laboratory, Tulsa, United States; 8Stanford University, Stanford, United States; 9Mayo Clinic, Phoenix, United States.

B-06 GRANULOMATOUS MYCOSIS FUNGOIDES: A CLINICOPATHOLOGICAL STUDY OF 41 CASES FROM A TERTIARY CANCER CENTER Alani, A.1; Navarrete-Viveros, J.2; Aung, P.2; Nagarajan, P.2; Tetzlaff, M.2; Curry, J.2; Prieto, V.2; Huen, A.2; Miranda, R.2; Duvic, M.2; Torres-Cabala, C.2 1Baylor College of Medicine, Houston, United States; 2The University of Texas MD Anderson Cancer Center, Houston, United States.

C-01 PROGNOSTIC FACTORS FOR STAGE PROGRESSION AND SURVIVAL IN MYCOSIS FUNGOIDES AND SEZARY SYNDROME: THE PROCLIPI STUDY Scarisbrick, J.1; Quaglino, P.2; Prince, M.3; Papadavid, E.4; Hodak, E.5; Whittaker, S.6; Bagot, M.7; Querfeld, C.8; Akilov, O.9; Servitje, O.10; Berti, E.11; Ortiz, P.12; Stadler, R.13; Knobler, R.14; Mitteldorf, C.15; Estrach, T.10; Marschalko, M.16; Guenova, E.17; Pimpinelli, N.18; Beylot-Barrie, M.19; Wobser, M.20; Wehkemp, U.21; Cowan, R.22; Vakeva, L.23; Busschots, A.M.24; Matin, R.25; Evison, F.1; Hong, E.26; Cerroni, L.27; Kempf, W.17; Gru, A.28; Battistella, M.7; Vermeer, M.29; Willemze, R.29; Kim, Y.26 1University Hospital Birmingham, Birmingham, United Kingdom; 2Turin, Italy; 3PeterMac, Melbourne, Australia; 4Athens, Greece; 5Rabin, Tel Aviv, Israel; 6GSTT, London, United Kingdom; 7St Louis, Paris, France; 8City of Hope, Los Angeles, United States; 9Pittsburgh, United States; 10Barcelona, Spain; 11Milan, Italy; 12Madrid, United Kingdom; 13Minden, Minden, Germany; 14Vienna, Austria; 15Hildesheim, Germany; 16Semmelweis, Hungary; 17Zurich, Switzerland; 18Florence, Italy; 19Bordeaux, France; 20Wuerzburg, Germany; 21Kiel, Germany; 22Manchester, United Kingdom; 23Helsinki, Finland; 24Leuven, Belgium; 25Oxford, United Kingdom; 26Stanford, United States; 27Graz, Austria; 28Virginia, United States; 29Leiden, Netherlands.

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C-02 REVIEW OF THE MEASUREMENTS OF ABNORMAL LYMPH NODES ON IMAGING AND THE CORRELATION WITH N CLASS FROM THE PROSPECTIVE CUTANEOUS LYMPHOMA INTERNATIONAL PROGNOSTIC INDEX STUDY Yoo, J.1; Bagot, M.2; Hodak, E.2; Servitige, O.2; Pujol, R.2; Marschalko, M.2; Papadavid, E.2; Cowan, R.2; Mitteldorf, C.2; Jonak, C.2; Querfeld, C.3; Busschots, A.2; Martin, R.2; Wehkamp, U.2; Hong, E.3; Battistella, M.2; Evison, F.1; Kim, Y.3; Scarisbrick, J.2 1University Hospitals Birmingham, Birmingham, United Kingdom; 2Cutaneous Lymphoma Task Force EORTC, Belgium; 3Cutaneous Lymphoma International Consortium, United States.

C-03 T-CELL MONOCLONALITY IN BLOOD AND SKIN CORRELATES WITH POOR RESPONSE TO TREATMENT IN MYCOSIS FUNGOIDES Geller, S.; Fajnerman Tel-Dan, S.; Solar, I.; Sprecher, E.; Goldberg, I. Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

C-04 ERYTHRODERMA: ANALYSIS OF SÉZARY SYNDROME CRITERIA IN 292 ERYTHRODERMIC PATIENTS Miyashiro, D.; Cury-Martins, J.; Abdo, A.; Pereira, J.; Sanches, J.A. University of São Paulo Medical School, São Paulo, Brazil.

D-01 GLOBAL EPIDEMIOLOGY OF PRIMARY CUTANEOUS LYMPHOMAS: A SYSTEMATIC REVIEW OF RELATIVE FREQUENCIES AND META-ANALYSIS Dobos, G.; Ram-Wolff, C.; Bagot, M.; De Masson, A. Hopital Saint-Louis, APHP, Paris, France.

D-02 EPIDEMIOLOGY OF PRIMARY CUTANEOUS CD8+ T CELL LYMPHOMA: A UNITED STATES POPULATION-BASED COHORT ANALYSIS USING THE SURVEILLANCE, EPIDEMIOLOGY, AND END RESULTS DATABASE Mirza, F.N.; Yumeen, S.; Girardi, M. Yale School of Medicine, New Haven, United States.

D-03 RACIAL AND AGE DISPARITIES IN CARDIOVASCULAR EVENTS MAY EXIST IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMAS Johnson, C.; Rozati, S. Johns Hopkins School of Medicine, Baltimore, United States.

D-04 MYCOSIS FUNGOIDES IN THE CHILEAN PEDIATRIC POPULATION: A RETROSPECTIVE EPIDEMIOLOGICAL AND CLINICAL-PATHOLOGICAL STUDY IN A CHILEAN REFERRAL CENTER Molgó, M.; Reyes-Baraona, F.; Downey, C.; Giordano, M.C.; Acle, R.; González, S. Pontificia Universidad Católica de Chile, Santiago, Chile.

D-06 THE EPIDEMIOLOGY OF PRIMARY CUTANEOUS GAMMA/DELTA T-CELL LYMPHOMA: A SEER-18 ANALYSIS Goyal, A.; Pearson, D.; Bohjanen, K. University of Minnesota, Minneapolis, United States.

E-01 PROINFLAMMATORY AND IMMUNOSUPPRESSIVE CYTOKINES POLARIZE THE MICROENVIRONMENT IN CUTANEOUS T CELL LYMPHOMA AND REGULATE PD-L1 EXPRESSION Gunes, E.G.; Rosen, S.T.; Querfeld, C. City of Hope National Medical Center, Duarte, United States.

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E-02 NORMAL FIBROBLASTS PROMOTE TH1 CYTOKINE EXPRESSION IN MYLA CELLS BY SUPPRESSING TWIST1 EXPRESSION Mehdi, S.J.; Moerman-Herzog, A.; Wong, H.K. University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States.

E-03 ANTI-CD47 IMMUNOTHERAPY IS MEDIATED BY CYTOTOXIC CD107A+IFN- Γ- NK CELLS AND CAN BE POTENTIATED BY INTERFERON-Α IN CUTANEOUS LYMPHOMA Kruglov, O.1; Johnson, L.2; Uger, R.2; Wong, M.2; Wu, X.3; Hwang, S.3; Akilov, O.1 1University of Pittsburgh, Pittsburgh, United States; 2Trillium Therapeutics, Mississauga, Canada; 3University of California Davis, Davis, United States.

F-01 MAC-1/MAC-2A CELLS REPRESENT A POTENTIAL MODEL TO STUDY IL-13 SIGNALING IN SATB1 POSITIVE CUTANEOUS ALCL Kadin, M.E.1; Kouttab, N.1; Morgan, J.1; Xu, H.1; Wang, Y.2 1Boston University, Boston. Roger Williams Medical Center, Providence, United States; 2Peking University First Hospital, Beijing, China.

F-03 PROTEIN AND MRNA EXPRESSION LEVELS OF IL-17A, IL-17F AND IL-22 IN PATIENTS WITH MYCOSIS FUNGOIDES Papathemeli, D.; Patsatsi, A.; Koletsa, T.; Pikou, O.; Lazaridou, E.; Georgiou, E. Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece.

F-04 INDUCTION OF ANTI-TUMOR EFFECT BY CD8+ T-CELLS VIA CADM1/CRTAM INTERACTION IN PATIENTS WITH ADULT T-CELL LEUKEMIA LYMPHOMA Tatsuno, K.; Shimauchi, T.; Tokura, Y. Hamamatsu University School of Medicine, Hamamatsu, Japan.

G-01 ROLE OF TOX1 AND STAT3 PATHWAYS IN THE PATHOGENESIS OF CUTANEOUS T-CELL LYMPHOMA Seffens, A.1; Koralov, S.2; Geskin, L.3 1Columbia University Vagelos College of Physicians and Surgeons, New York, United States; 2New York University School of Medicine, New York, United States; 3Columbia University Irving Medical Center, New York, United States.

G-03 STANDARDIZED FLOW CYTOMETRY (EUROFLOW) DEMONSTRATES HETEROGENEOUS T-CELL ORIGIN OF SÉZARY LYMPHOMA CELLS Najidh, S.1; Van Der Sluijs-Gelling, A.1; Zoutman, W.H.1; Tensen, C.P.1; Van Hall, T.1; Almeida, J.2; Van Dongen, J.1; Vermeer, M.H.1 1Leiden University Medical Center, Leiden, Netherlands; 2Centro de Investigación del Cáncer, Salamanca, Spain.

G-04 MUTATIONAL SIGNATURE ANALYSIS REVEALS A KEY ROLE FOR UV RADIATION IN THE ACCUMULATION OF MUTATIONS IN CUTANEOUS T-CELL LYMPHOMA Jones, C.1; Degasperi, A.2; Grandi, V.1; Mitchell, T.1; Nik-Zainal, S.2; Whittaker, S.1 1King's College London, London, United Kingdom; 2University of Cambridge, Cambridge, United Kingdom.

G-05 EPIDERMAL FATTY ACID-BINDING PROTEIN IS NOT EXPRESSED BY TUMOR CELLS IN ADVANCED MYCOSIS FUNGOIDES Takahashi-Shishido, N.1; Morimura, S.1; Suga, H.2; Oka, T.2; Kamijo, H.2; Miyagaki, T.2; Sato, S.2; Sugaya, M.1

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1International University of Health and Welfare, Chiba, Japan; 2The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

G-06 PATCH LESIONS OF MYCOSIS FUNGOIDES PATIENTS HAVE A SIMILAR SKIN MICROBIOME PROFILE COMPARED TO NON-LESIONAL AND HEALTHY SKIN Ulrike, W.1; Britt M, H.2; Marion, J.1; John F, B.1; Jürgen, H.1 1University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 2Max Planck Institute for Evolutionary Biology, Plön, Germany.

H-01 EVALUATION OF BLOOD SÉZARY MARKERS BY MEANS OF CORRELATION WITH APOPTOSIS RESISTANCE AND CLONALITY Sven, S.1; Chalid, A.2; Max, S.3; Rudolf, S.4; Rene, S.4; Ulrike, W.5; Marion, W.6; Jana, B.1; Sergij, G.1; Jan, N.1 1University Medical Center Mannheim, Mannheim, Germany; 2HELIOS Klinikum Krefeld, Krefeld, Germany; 3University Hospital Munich, Munich, Germany; 4University Hospital Minden, Minden, Germany; 5University Hospital Kiel, Kiel, Germany; 6University Hospital Würzburg, Würzburg, Germany.

H-02 THE A ALLELE OF RS7096317 IS PERMISSIVE FOR CD39 EXPRESSION IN CTCL AND IS A NEGATIVE PROGNOSTIC FACTOR IN OLDER PATIENTS Jones, C.; Devaney, A.; Grandi, V.; Samuel, S.; Whittaker, S.; Mitchell, T. King's College London, London, United Kingdom.

I-01 IDENTIFICATION OF CD39 AS A POTENTIAL THERAPEUTIC TARGET IN SEZARY SYNDROME De Masson, A.1; Battistella, M.1; Sonigo, G.1; Janela, B.2; Thonnart, N.1; Musette, P.1; Ginhoux, F.2; Bensussan, A.1; Bagot, M.1; Marie-Cardine, A.1 1Saint-Louis Hospital, Paris, France; 2Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

I-02 THE CORRELATION OF FLOW CYTOMETRY ANALYSIS ON SKIN TISSUE WITH IMMUNOHISTOCHEMISTRY IN CUTANEOUS T-CELL LYMPHOMA Geller, S.1; Ho, C.2; Noor, S.2; Roshal, M.2; Myskowski, P.2; Moskowitz, A.2; Horwitz, S.2; Pulitzer, M.2 1Sourasky Medical Center and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 2Memorial Sloan Kettering Cancer Center, New York, United States.

I-03 OVEREXPRESSION OF STAT4 AT EARLY STAGES OF MYCOSIS FUNGOIDES: COINCIDENCE OR NOT? Olisova, O.; Grekova, E.; Zaletaev, D.; Alekseeva, E. I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation.

J-01 COMPARISON OF EARLY WITH ADVANCED MYCOSIS FUNGOIDES LESIONS ON A SINGLE CELL LEVEL TO ASSESS POTENTIAL MEDIATORS OF DISEASE PROGRESSION Farlik, M.1; Bauer, W.1; Shaw, L.1; Porkert, S.1; Halbritter, F.2; Jonak, C.1; Brunner, P.M.1 1Medical University of Vienna, Vienna, Austria; 2Children’s Cancer Research Institute, Vienna, Austria.

J-03 GENIC SIGNATURE OF CUTANEOUS T CELL LYMPHOMA-ASSOCIATED FIBROBLASTS FROM MYCOSIS FUNGOIDES AND SEZARY SYNDROME Habib, Z.1; Gabor, D.2; Adèle, D.M.3; Sophie, L.K.S.2; Christophe, B.4; Anne, B.5; Martine, B.3; Armand, B.2; Jean-François, D.5; Laurence, M.2

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1CEA, PARIS, France; 2INSERM U976, PARIS, France; 3AP-HP, PARIS, France; 4CEA, Grenoble, France; 5CEA, Evry, France.

J-04 THE EFFECT OF EXTRACELLULAR MATRIX AND FIBROBLASTS ON PROLIFERATION AND SURVIVAL OF MALIGNANT CELLS IN MYCOSIS FUNGOIDES Beksac, B.1; Baik, S.2; O'donnell, M.2; Porcu, P.2; Nikbakht, N.2 1University of Health Sciences Gulhane Research and Training Hospital, Ankara, Turkey; 2Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, United States.

K-01 HYPOMETHYLATION-MEDIATED ACTIVATION OF TMEM244 GENE IN SÉZARY CELLS Iżykowska, K.1; Rassek, K.1; Żurawek, M.1; Nowica, K.1; Paczkowska, J.1; Olek-Hrab, K.2; Giefing, M.1; Przybylski, G.1 1Institute of Human Genetics PAS, Poznań, Poland; 2University of Medical Sciences, Poznań, Poland.

K-02 EPIGENETIC INVOLVEMENT IN CUTANEOUS T-CELL LYMPHOMA LYMPHOMAGENESIS Chebly, A.1; Ropio, J.1; Baldasseroni, L.1; Prochazkova-Carlotti, M.1; Idrissi, Y.1; Ferrer, J.1; Poglio, S.1; Pham-Ledard, A.1; Beylot-Barry, M.1; Merlio, J.P.1; Tomb, R.2; Soares, P.3; Chevret, E.1 1University of Bordeaux, Bordeaux, France; 2Saint Joseph University, Lebanon; 3University of Porto, Porto (i3s/ipatimup), Portugal.

K-03 MYCOSIS FUNGOIDES CELL LINE – DERIVED EXOSOMES SHOW A DISTINCT SIGNATURE OF ELEVATED EXPRESSION OF miR-155, miR-1246, AND OX40 TO PROMOTE TUMOR PROGRESSION Lilach Moyal,1,2 Coral Arkin,1,2 Batia Gorovitz,1,2 Jamal Knaneh1,2 Hadas Prag1,2 Iris Amitay-Laish 1,2 Emmilia Hodak1,2 1Division of Dermatology, Rabin Medical Center – Beilinson Hospital, Petach Tikva, and 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

K-04 TRANSFORMED MYCOSIS FUNGOIDES SHOWS DISTINCT MIRNA AND MRNA EXPRESSION PROFILES COMPARED TO CLASSIC MYCOSIS FUNGOIDES Di Raimondo, C.1; Wu, X.2; Zain, J.2; Abdulla, F.2; Rosen, S.T.2; Querfeld, C.2 1City of Hope Medical Center/University of Rome Tor Vergata, Duarte, United States; 2City of Hope Medical Center, Duarte, CA, United States.

L-03 FUNCTIONAL BIOLOGICAL CLASSIFICATION OF DIFFERENTIAL GENE EXPRESSION IN SÉZARY SYNDROME Moerman-Herzog, A.; Acheampong, D.; Rahmatallah, Y.; Glazko, G.; Medhi, S.J.; Wong, H.K. University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States.

L-04 HIGH-THROUGHPUT SEQUENCING REVEALS T-CELL REPERTOIRE RESTRICTION IN SÉZARY SYNDROME AND MYCOSIS FUNGOIDES Blanco, G.1; López-Aventín, D.1; Pujol, R.M.1; Gómez-Llonín, A.1; Puiggros, A.1; López- Sánchez, M.2; Estrach, T.3; Garcia-Muret, M.P.4; Lopez-Lerma, I.5; Servitje, O.6; Muro, M.2; Espinet, B.1; Rabionet, R.7; Gallardo, F.1 1Hospital del Mar, Barcelona, Spain; 2Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; 3Hospital Clínic de Barcelona, Barcelona, Spain; 4Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Hospital Universitari Vall d´Hebrón, Barcelona, Spain; 6Hospital Universitari de Bellvitge, L´Hospitalet de Llobregat, Spain; 7Universitat de Barcelona, Barcelona, Spain.

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M-01 MOLECULAR STUDY OF SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA, LUPUS PANNICULITIS AND OVERLAP CASES Machan, S.1; Rodríguez Moreno, M.2; Manso Aonso, R.2; Rodríguez-Peralto, J.L.3; Cerroni, L.4; García, C.5; González Núñez, M.Á.6; García Toro, E.7; Estrach, T.8; Ferrer, B.9; Torres, Á.10; Lobo, C.11; Sigues, N.11; Alfonso Martin, J.L.12; Borrego, L.13; Montenegro, T.13; Monteagudo, C.14; Limeres Gonzalez, M.Á.15; Córdoba, R.1; Piris, M.Á.16; Requena, L.1; Rodríguez-Pinilla, S.M.17 1Fundación Jiménez Díaz, Madrid, Spain; 2Fundación Jiménez Díaz; CIBERONC., Madrid, Spain; 3Hospital Universitario 12 de Octubre, Madrid, Spain; 4Medical University of Graz, Graz, Austria; 5Hospital Universitario de Canarias, Tenerife, Spain; 6Hospital Ciudad de Coria; Hospital San Pedro de Alcántara, Cáceres, Spain; 7Hospital Universitario de Burgos, Burgos, Spain; 8Hospital Clínic de Barcelona, Barcelona, Spain; 9Hospital Vall d’Hebron, Barcelona, Spain; 10Hospital Universitario Río Hortega, Valladolid, Spain; 11Hospital Universitario Donostia, San Sebastián, Spain; 12Hospital Materno-Infantil, Las Palmas de Gran Canaria, Spain; 13Hospital del Henares, Madrid, Spain; 14Hospital de la Malva-Rosa, Valencia, Spain; 15Hospital Universitario de Gran Canaria Doctor Negrín, Las Palmas de Gran Canaria, Spain; 16Fundación Jiménez Díaz, CIBERONC, Madrid, Spain; 17Fundación Jiménez Díaz. CIBERONC, Madrid, Spain.

M-02 GERMLINE TIM-3 MUTATIONS CHARACTERIZE SUB-CUTANEOUS PANNICULITIS-LIKE T2 CELL LYMPHOMAS WITH HEMOPHAGOCYTIC LYMPHOHISTIOCYTIC SYNDROME Mccormack, C.1; Khuong-Quang, D.A.2 1Peter Macallum Cancer Center, Melbourne, Australia; 2Royal Children's Hospital, Melbourne, Australia.

M-03 COMPARATIVE GENOMIC ANALYSIS OF CD30+ LYMPHOPROLIFERATIVE DISORDERS Abdulla, F.1; Parekh, V.1; Song, J.1; Querfeld, C.1; Zain, J.1; Rosen, S.1; Honda, K.2 1City of Hope, Duarte, United States; 2University Hospitals Cleveland, Cleveland, United States.

M-05 CLONAL RELATIONSHIP BETWEEN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM AND MYELOID NEOPLASMS Colomo, L.; Fernandez, C.; Papaleo, N.; Vazquez, I.; Gallardo, F.; Calvo, X.; Bellosillo, B.; Pujol, R.; Arenillas, L. Hospital del Mar, Barcelona, Spain.

N-01 CELLULAR ORIGINS AND GENETIC LANDSCAPE OF CUTANEOUS GAMMA DELTA T CELL LYMPHOMAS Daniels, J.1; Doukas, P.1; Martinez Escala, M.1; Ringbloom, K.1; Shih, D.J.2; Yang, J.1; Tegtmeyer, K.1; Park, J.1; Thomas, J.J.1; Selli, M.E.1; Altunbulakli, C.1; Gowthaman, R.3; Mo, S.H.1; Jothishankar, B.4; Pease, D.R.1; Pro, B.1; Abdulla, F.R.5; Shea, C.4; Sahni, N.2; Gru, A.A.6; Pierce, B.G.3; Louissaint Jr., A.7; Guitart, J.1; Choi, J.1 1Northwestern University, Chicago, United States; 2University of Texas MD Anderson Cancer Center, Houston, United States; 3University of Maryland, College Park, United States; 4University of Chicago, Chicago, United States; 5City of Hope, Duarte, United States; 6University of Virginia, Charlottesville, United States; 7Massachussets General Hospital, Boston, United States.

N-03 WHOLE-GENOME ANALYSIS UNCOVERS RECURRENT IKZF1 INACTIVATION AND ABERRANT CELL ADHESION IN BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM Bastidas Torres, A.1; Cats, D.1; Mei, H.1; Fanoni, D.2; Gliozzo, J.3; Corti, L.3; Paulli, M.4; Vermeer, M.1; Willemze, R.1; Berti, E.3; Tensen, C.1

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1Leiden University Medical Center, Leiden, Netherlands; 2University of Milan, Milan, Italy; 3Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; 4University of Pavia, Pavia, Italy.

O-01 PRIMARY CUTANEOUS FOLLICULAR LYMPHOMA IS GENETICALLY DISTINCT FROM SECONDARY CUTANEOUS FOLLICULAR LYMPHOMA Zhou, X.(.1; Yang, J.1; Ringbloom, K.G.1; Martinez-Escala, M.E.1; Wenzel, A.1; Moy, A.P.2; Morgan, E.A.3; Harkins, S.4; Paxton, C.N.5; Hong, B.6; Andersen, E.F.6; Guitart, J.1; Weinstock, D.M.7; Cerroni, L.8; Choi, J.1; Louissaint, Jr, A.9 1Northwestern University Feinberg School of Medicine, Chicago, United States; 2Northwestern University Feinberg School of Medicine, Boston, United States; 3Brigham and Women's Hospital, Boston, United States; 4Massachusetts General Hospital, Boston, United States; 5ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, United States; 6University of Utah School of Medicine, Salt Lake City, United States; 7Dana-Farber Cancer Institute, Broad Institute of Harvard and MIT, Boston, United States; 8Medical University of Graz, Graz, Austria; 9Massachusetts General Hospital, Dana-Farber Cancer Institute, Boston, United States.

O-02 CHALLENGES IN THE DIAGNOSIS OF PRIMARY CUTANEOUS LARGE B- CELL-LYMPHOMAS Sarah, M.1; Audrey, G.2; Philippe, R.3; Marie, P.2; Eric, F.2; Anne, P.L.2; Marie, B.B.2; Fabrice, J.4; Béatrice, V.2; Jean-Philippe, M.2 1Université Bordeaux, Bordeaux, France; 2CHU et Université Bordeaux, Bordeaux, France; 3Centre Henri Becquerel, Rouen, France; 4Centre Henri Becquerel, Bordeaux, France.

O-03 MUTATIONS OF THE B-CELL RECEPTOR PATHWAY CONFER CHEMORESISTANCE IN PRIMARY CUTANEOUS DIFFUSE LARGE B-CELL LYMPHOMA LEG-TYPE Océane, D.1; Marie, B.B.1; Anne, P.L.1; Elodie, B.2; Pierre-Julien, V.2; Thomas, B.3; Nicolas, F.3; Eric, F.1; Béatrice, V.1; Fabrice, J.2; Jean-Philippe, M.1; Audrey, G.1 1CHU et Université Bordeaux, Bordeaux, France; 2Centre Henri Becquerel, Rouen, France; 3CHU Bordeaux, Bordeaux, France.

O-05 PRIMARY CUTANEOUS MARGINAL ZONE B-CELL LYMPHOMA IN CHILDREN AND YOUNG ADULTS Jo-Velasco, M.1; Matteo, E.2; Machan, S.1; Vivanco Allende, B.3; De Dios Velazquez, A.3; Garcia Solano, M.4; Sánchez Frías, M.E.5; Requena Caballero, L.1; Rodriguez-Pinilla, S.M.1; Piris Pinilla, M.A.1 1Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 2Hospital de Niños Dr. Ricardo Gutiérrez, Buenos Aires, Argentina; 3Hospital Universitario Central de Asturias, Oviedo, Spain; 4HCU Arrixaca, Murcia, Spain; 5Hospital Universitario de Córdoba, Córdoba, Spain.

O-06 FIRST - LINE RADIOTHERAPY DOES NOT IMPROVE THE RISK OF DISEASE PROGRESSION IN PRIMARY CUTANEOUS DIFFUSE LARGE B CELL LYMPHOMA, LEG TYPE Dimitriou, F.1; Zehnder, M.2; Amarov, B.3; Saulite, I.4; Dummer, R.1; Cozzio, A.4; Anzengruber, F.1; Mühleisen, B.1; Navarini, A.2; Guenova, E.1 1University Hospital of Zurich, Zurich, Switzerland; 2University Hospital of Basel, Basel, Switzerland; 3Sofia University "St. Kliment Ohridski", Sofia, Bulgaria; 4Kantonsspital St Gallen, St. Gallen, Switzerland.

P-01 THE EVALUATION OF THE QOL AND DAILY FUNCTIONING OF POLISH PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA COGNOSCO LLC COMMISSIONED BY THE FIRM TAKEDA PHARMA LLC Giza, A.1; Sokołowska-Wojdyło, M.2; Ciepłuch, H.3

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1Collegium Medicum of the Jagiellonian Univeristy, Krakow, Poland; 2Medical University of Gdansk, Gdansk, Poland; 3Regional Oncology Centre, Gdansk, Poland.

P-02 SKIN-SPECIFIC QUALITY OF LIFE IN CUTANEOUS T-CELL LYMPHOMA COMPARED WITH OTHER DERMATOLOGIC DISEASES Bhat, T.1; Herbosa, C.1; Rosenberg, A.1; Mehta-Shah, N.1; Semenov, Y.2; Musiek, A.1 1Washington University School of Medicine in St. Louis, St. Louis, MO, United States; 2Massachusetts General Hospital, Boston, MA, United States.

P-03 QUALITATIVE ASSESSMENT OF THE QUALITY OF LIFE IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA (CTCL) Bhat, T.1; Herbosa, C.1; Rosenberg, A.1; Jeffe, D.1; Mehta-Shah, N.1; Semenov, Y.2; Musiek, A.1 1Washington University School of Medicine in St. Louis, St. Louis, MO, United States; 2Massachusetts General Hospital, Boston, MA, United States.

P-04 THE IMPACT OF GENDER, AGE, RACE/ETHNICITY, AND STAGE ON QUALITY OF LIFE IN A SPECTRUM OF CUTANEOUS LYMPHOMAS Martinez, X.; Stiller, T.; Palmer, J.; Loscalzo, M.; Barrios, E.; Abdulla, F.; Zain, J.; Rosen, S.; Querfeld, C. City of Hope, Duarte, United States.

P-05 PSYCHIATRIC COMORBIDITY IN PATIENTS WITH CUTANEOUS T-CELL LYMPHOMA (CTCL) Bhat, T.1; Herbosa, C.1; Rosenberg, A.1; Mehta-Shah, N.1; Musiek, A.1; Semenov, Y.2 1Washington University School of Medicine in St. Louis, St. Louis, MO, United States; 2Washington University School of Medicine in St. Louis, Boston, MA, United States.

R-02 SYNERGISTIC THERAPEUTIC DRUG SCREENING USING JAK INHIBITION FOR CTCL Yumeen, S.; Mirza, F.N.; Lewis, J.M.; King, A.L.O.; Kim, S.R.; Carlson, K.; Foss, F.; Girardi, M. Yale School of Medicine, New Haven, United States.

R-04 PHOSPHOLIPASE C GAMMA 1 (PLCG1) MUTATIONS IN SÉZARY CELLS DRIVE NFKB, AP-1 AND NFAT SIGNALLING AND MEDIATE RESISTANCE TO THE CALCINEURIN INHIBITOR TACROLIMUS Flanagan, C.E.; Patel, V.M.; Jones, C.L.; Whittaker, S.J.; Mitchell, T.J. King’s College London, London, United Kingdom.

R-05 SCREENING FOR NOVEL COMBINATION TREATMENTS FOR CUTANEOUS T CELL LYMPHOMA FOR EXPEDITED DEVELOPMENT Mirza, F.N.; Yumeen, S.; Lewis, J.M.; King, A.L.O.; Kim, S.R.; Carlson, K.R.; Foss, F.M.; Girardi, M. Yale School of Medicine, New Haven, United States.

R-06 THE SYNERGISTIC PRO-APOPTOTIC EFFECT OF HDAC AND PARP-1 INHIBITION IN CUTANEOUS T-CELL LYMPHOMA IS MEDIATED VIA BLIMP-1 Kruglov, O.1; Wu, X.2; Hwang, S.2; Akilov, O.1 1University of Pittsburgh, Pittsburgh, United States; 2University of California Davis, Davis, United States.

S-01 A SMALL MOLECULE CCR2 ANTAGONIST DEPLETES TUMOR MACROPHAGES AND SYNERGIZES WITH ANTI-PD1 IN A MURINE MODEL OF CUTANEOUS T CELL LYMPHOMA

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Wu, X.1; Singh, R.2; Hsu, D.1; Zhou, Y.1; Yu, S.1; Han, D.1; Shi, Z.1; Huynh, M.1; Campbell, J.2; Hwang, S.1 1University of California Davis, Sacramento, United States; 2ChemoCentryx, Inc., Mountain View, United States.

S-02 INVESTIGATING THE ROLE FOR POT1 GENE DYSFUNCTION IN PRIMARY CUTANEOUS T-CELL LYMPHOMA Bakr, F.; Holdich, A.; Jones, C.; Whittaker, S.; Mitchell, T. Kings College London, London, United Kingdom.

S-04 INTEGRIN ΑVΒ3 INHIBITION IMPROVES REXINOIDS ANTITUMORAL ACTIONS ON CUTANEOUS T CELL LYMPHOMA (CTCL) Cayrol, M.F.1; Revuelta, M.V.2; Debernardi, M.M.1; Phillip, J.M.2; Zamponi, N.2; Diaz Flaque, M.C.1; Magro, C.2; Ruan, J.2; Cremaschi, G.1; Cerchietti, L.2 1Instituto de Investigaciones Biomédicas UCA CONICET, Buenos Aires, Argentina; 2Weill Cornell Medicine, New York, United States.

S-05 HDAC INHIBITOR RESMINOSTAT COUNTERACTS DISEASE-RELATED GENE EXPRESSION AND CYTOKINE SECRETION IN CTCL CELLS Streubel, G.1; Bretz, A.C.1; Wulff, T.1; Parnitzke, U.1; Hannah, K.2; Kronthaler, K.2; Borgmann, M.2; Hamm, S.1 14SC AG, Planegg-Martinsried, Germany; 24SC AG, Planegg-Martinsried, Germany.

S-06 SINGLE CELL RNA SEQUENCING ANALYSIS TO DEFINE THERAPEUTIC TARGETS IN CUTANEOUS T-CELL LYMPHOMA Querfeld, C.; Wu, X.; Gunes, E.G.; Jonsson, V.; Rosen, S.T. City of Hope, Duarte, United States.

T-01 CUSATUZUMAB FOR TREATMENT OF CD70-POSITIVE RELAPSED/REFRACTORY CUTANEOUS T-CELL LYMPHOMA IN A PHASE 1/2 CLINICAL TRIAL Bagot, M.1; Maerevoet, M.2; Zinzani, P.L.3; Offner, F.4; Morschhauser, F.5; Michot, J.M.6; Ribrag, V.7; Battistella, M.8; Moins-Teisserenc, H.1; Alleri, A.C.9; Dalle, S.10; Beylot-Barry, M.11; Zwanenpoel, K.12; De Winne, K.13; Marie-Cardine, A.1; Cayuela, J.M.1; Tabanelli, V.9; Motta, G.9; Melle, F.9; Hultberg, A.14; Gandini, D.14; Moshir, M.14; Delahaye, T.14; Zabrocki, P.14; Silence, K.14; Van Rompaey, L.14; Bensussan, A.1; De Haard, H.14; Pauwels, P.13; Leupin, N.14; Pileri, S.9 1Saint Louis Hospital, Paris, France; 2Jules Bordet, Brussels, Belgium; 3University of Bologna, Bologna, Italy; 4University Hospital Gent, Gent, Belgium; 5University Lille, Lille, France; 6Gustave Roussy, Villejuif Cedex, France; 7Gustave Roussy, Villjuif Cedex, France; 8Saint Louis Hospital, Paris, France; 9European Institute of Oncology (IEO), IRCCS, Milan, Italy; 10Centre Hospitalier Lyon Sud, Pierre Bénite, France; 11Hôpital Saint-André, Centre Hospitalier, Bordeaux, France; 12University Hospital Antwerp, Antwerp, Belgium; 13University Hospital Antwerp, Antwerp, Belgium; 14Argenx, Zwijnaarde, Belgium.

T-02 A PHASE 1B STUDY EVALUATING THE SAFETY AND EFFICACY OF TOPICAL ADMINISTRATION OF WP1220, A STAT3 INHIBITOR, FOR MYCOSIS FUNGOIDES (MF) Sokołowska-Wojdyło, M.1; Błażewicz, I.2; Olszewska, B.1; Zak, E.3; Silberman, S.4; Priebe, W.5 1Medical University of Gdańsk (GUMed), Gdansk, Poland; 2Medical University in Gdansk, Gdansk, Poland; 3Dermin Spz.oo, Warsaw, Poland; 4Moleculin Biotech, Houston, Texas, United States; 5MD Anderson Cancer Center, Houston, TX, United States.

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T-03 HIGH DOSE RATE BRACHYTHERAPY FOR THE TREATMENT OF PRIMARY CUTANEOUS LYMPHOMA AT COMPLEX SITES AND COMPLEX CURVES SKIN SURFACES Sim, V.R.1; Defrancesco, I.1; Child, F.2; Wain, M.2; Whittaker, S.2; Freeman, K.3; Jones, E.3; Aldridge, S.1; Morris, S.1 1Guy's Cancer Centre, London, United Kingdom; 2St John's Institute of Dermatology, Guy's Hospital, London, United Kingdom; 3Guy's Cancer Centre, Guy's Hospital, London, United Kingdom.

T-04 INTRALESIONAL ONCOLYTIC VIROTHERAPY RESULTS IN TUMOR REGRESSION ASSOCIATED WITH THE INFLUX OF CYTOTOXIC T CELLS IN CUTANEOUS B-CELL LYMPHOMA Ramelyte, E.1; Tastanova, A.1; Balazs, Z.1; Menzel, U.2; Turko, P.1; Beisel, C.2; Krauthammer, M.1; Levesque, M.P.1; Dummer, R.1 1University Hospital Zurich, Zurich, Switzerland; 2Department of Biosystems Science and Engineering, Basel, Switzerland.

U-01 A PHASE II PROSPECTIVE STUDY USING NON-MYELOABLATIVE ALLOGENEIC TRANSPLANTATION IN PATIENTS WITH ADVANCED STAGE MYCOSIS FUNGOIDES AND SEZARY SYNDROME Weng, W.K.; Arai, S.; Khodadoust, M.; Hoppe, R.T.; Kim, Y.H. Stanford University, Stanford, United States.

U-02 ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION AS A CURATIVE TREATMENT STRATEGY FOR PATIENTS WITH ADVANCED MYCOSIS FUNGOIDES AND SÉZARY SYNDROME: 2019 UPDATE OF THE MILAN EXPERIENCE Onida, F.1; Valli, V.1; Saporiti, G.1; Alberti-Violetti, S.1; Grifoni, F.1; Goldaniga, M.1; Casarin, F.1; Schiavone, C.2; Fanoni, D.1; Baldini, L.1; Grillo Ruggieri, F.2; Berti, E.1 1Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico - University of Milan, Milano, Italy; 2Ospedali Galliera, Genova, Italy.

U-03 NON-MYELOABLATIVE ALLOGENEIC STEM CELL TRANSPLANTATION USING TSEB TLI AND ATG FOR MYCOSIS FUNGOIDES(MF) AND SEZARY SYNDROME(SS). MEDIUM TERM RESULTS FROM A LARGE SINGLE CENTRE COHORT Morris, S.1; Palanicawandar, R.2; Grandi, V.1; Defrancesco, I.1; Child, F.1; Mary, W.1; Whittaker, S.1; Mangar, S.2; Kanfer, E.2 1Guys Hospital, London, United Kingdom; 2Hammersmith Hospital, London, United Kingdom.

V-03 REAL-LIFE EXPERIENCE ON THE USE OF BRENTUXIMAB VEDOTIN ON NINE MYCOSIS FUNGOIDES PATIENTS: TIME TO RE-DISCUSS TREATMENT PROTOCOLS? Cury-Martins, J.1; Miyashiro, D.1; Neder Ramires Abdo, A.1; Pereira, J.1; Sanches, J.A.2 1University of Sao Paulo, Sao Paulo, Brazil; 2University of Sao Paulo, Madrid, Brazil.

V-04 BRENTUXIMAB VEDOTIN (BV) AND LENALIDOMIDE (LEN) IN RELAPSED/REFRACTORY (R/R) CUTANEOUS (CTCL) AND PERIPHERAL (PTCL) T-CELL LYMPHOMAS; INTERIM RESULTS OF A PHASE II TRIAL William, B.; Johnson, A.; Huang, Y.; Reneau, J.; Brammer, J.; Chung, C. The Ohio State University, Columbus, United States.

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V-05 FINAL ALCANZA RESULTS: BRENTUXIMAB VEDOTIN VERSUS PHYSICIAN'S CHOICE IN PREVIOUSLY TREATED CD30-POSITIVE CUTANEOUS T-CELL LYMPHOMA (MYCOSIS FUNGOIDES OR PRIMARY CUTANEOUS ANAPLASTIC LARGE CELL LYMPHOMA) Scarisbrick, J.1; Horwitz, S.2; Dummer, R.3; Whittaker, S.4; Duvic, M.5; Kim, Y.6; Quaglino, P.7; Zinzani, P.L.8; Bechter, O.9; Eradat, H.10; Pinter-Brown, L.11; Akilov, O.12; Geskin, L.J.13; Sanches, J.14; Ortiz Romero, P.L.15; Weichenthal, M.16; Fisher, D.17; Walewski, J.18; Trotman, J.19; Taylor, K.20; Dalle, S.21; Stadler, R.22; Lisano, J.23; Brown, L.23; Palanca-Wessels, M.C.23; Bunn, V.24; Little, M.24; Prince, H.M.25 1University Hospital Birmingham, Birmingham, United Kingdom; 2Memorial Sloan Kettering Cancer Center, New York, United States; 3University Hospital Zürich and University Zürich, Zürich, Switzerland; 4St John’s Institute of Dermatology, Guys and St Thomas NHS Foundation Trust, London, United Kingdom; 5The University of Texas MD Anderson Cancer Center, Houston, United States; 6Stanford University School of Medicine and Stanford Cancer Institute, Stanford, United States; 7University of Turin, Turin, Italy; 8Institute of Haematology, University of Bologna, Bologna, Italy; 9University Hospitals Leuven, KU Leuven, Belgium; 10David Geffen School of Medicine at UCLA, Los Angeles, United States; 11Chao Family Comprehensive Cancer Center, University of California, Irvine, United States; 12University of Pittsburgh, Pittsburgh, United States; 13Columbia University, New York, United States; 14University of São Paulo Medical School, São Paulo, Brazil; 15University Hospital 12 de Octubre, Institute i+12 Medical School, University Complutense, Madrid, Spain; 16University Hospital of Schleswig-Holstein, Kiel, Germany; 17Dana-Farber Cancer Institute, Boston, United States; 18Maria Sklodowska-Curie Institute and Oncology Centre, Warsaw, Poland; 19Concord Repatriation General Hospital, University of Sydney, Concord, Australia; 20ICON Cancer Care, South Brisbane, Australia; 21Hospices Civils de Lyon, Claude Bernard Lyon 1 University, Lyon, France; 22University Clinic for Dermatology, Johannes Wesling Medical Centre, Minden, Germany; 23Seattle Genetics, Inc., Bothell, United States; 24Millennium Pharmaceuticals, Inc., Cambridge, United States; 25Peter MacCallum Cancer Centre and Sir Peter MacCallum Department of Oncology, The University of Melbourne, Victoria, Australia.

V-06 BRENTUXIMAB VEDOTIN FOR RELAPSED/REFRACTORY SÉZARY SYNDROME: A SINGLE-CENTER EXPERIENCE Lewis, D.; Haun, P.; Samimi, S.; Landsburg, D.; Svoboda, J.; Barta, S.; Berg, S.; Del Guzzo, C.; Jariwala, N.; Vittorio, C.; Villasenor-Park, J.; Nasta, S.; Schuster, S.; Rook, A.; Kim, E. Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States.

W-01 RESOLUTION OF SÉZARY SYNDROME AFTER COMBINATION OF MOGAMULIZUMAB WITH PEGYLATED INTERFERON Α2-A IS MEDIATED BY CD56DIM NKP30+ IFN-Γ+ NK CELLS Patino, P.; Kruglov, O.; Akilov, O. University of Pittsburgh, Pittsburgh, United States.

W-03 CLINICAL ACTIVITY AND SAFETY OF LOW-DOSE TOTAL SKIN ELECTRON BEAM THERAPY COMBINED WITH MOGAMULIZUMAB IN REFRACTORY SÉZARY SYNDROME: SUPPORT FOR A CLINICAL TRIAL IN CTCL Fong, S.; Hong, E.; Khodadoust, M.S.; Hiniker, S.; Li, S.; Wang, E.B.; Hoppe, R.T.; Kim, Y.H. Stanford University, Stanford, United States.

X-03 IMPROVED SURVIVAL FOR SKIN-PRIMARY PRESENTATION OF ADULT T- CELL LEUKEMIA/LYMPHOMA (ATLL) Goyal, A.; O'leary, D.; Bohjanen, K. University of Minnesota, Minneapolis, United States.

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X-04 TREATMENT AND PROGNOSIS OF RARE PATIENTS WITH A PRIMARY CUTANEOUS CD30-POSITIVE LYMPHOPROLIFERATIVE DISORDER WHO DEVELOP EXTRACUTANEOUS LOCALISATIONS Melchers, R.1; Willemze, R.1; Vermaat, J.1; Jansen, P.1; Daniëls, L.1; Putter, H.1; Bekkenk, M.2; De Haas, E.3; Horvath, B.4; Van Rossum, M.5; Sanders, C.6; Veraart, J.7; Vermeer, M.1; Quint, K.1 1Leiden University Medical Center, Leiden, Netherlands; 2Academic Medical Centre and Vrije University Medical Centre, Amsterdam, Amsterdam, Netherlands; 3Erasmus Medical Center, Rotterdam, Netherlands; 4University Medical Center of Groningen, Groningen, Netherlands; 5Radboud University Medical Center, Nijmegen, Netherlands; 6University Medical Center Utrecht, Utrecht, Netherlands; 7Maastricht University Medical Center, Maastricht, Netherlands.

X-05 WINKELMANN REGIMEN: ORAL CHLORAMBUCIL FOR MYCOSIS FUNGOIDES AND SEZARY SYNDROME Doss, G.; De Francesco, I.; Grandi, V.; Child, F.; Wain, M.; Whittaker, S.; Morris, S. Guys & St Thomas NHS trust, London, United Kingdom.

X-07 CHARACTERIZATION OF THE HISTOPATHOLOGIC AND MOLECULAR FEATURES OF RASH ASSOCIATED WITH MOGAMULIZUMAB Wang, J.Y.; Hirotsu, K.E.; Neal, T.M.; Kwong, B.Y.; Kim, Y.H.; Rieger, K.E. Stanford University School of Medicine, Stanford, United States.

Y-01 REAL-LIFE EXPERIENCE WITH CHLORMETHINE GEL: MOVING BEYOND CLINICAL TRIAL DATA Prag Naveh H1, Amitay-Laish I1, 2, Zidan O1, Hodak E1,2 Division of Dermatology, Rabin Medical Center, Petach Tikva1,and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv2

Y-02 MECHLORETHAMINE TREATMENT DURATION AS A FUNCTION OF CLINICIAN-LEVEL PATIENT VOLUME FOR MYCOSIS FUNGOIDES CUTANEOUS T-CELL LYMPHOMA (MF-CTCL) Querfeld, C.1; Pacheco, T.2; Haverkos, B.2; Binder, G.3; Angello, J.3; Poligone, B.4 1City of Hope National Medical Center, Duarte, United States; 2University of Denver, Denver, United States; 3Helsinn Therapeutics US, Inc., Iselin, United States; 4Rochester Skin Lymphoma Medical Group, Fairport, United States.

Y-03 THE PROVE STUDY: REAL-WORLD EXPERIENCE WITH CHLORMETHINE GEL AND OTHER THERAPIES IN THE TREATMENT OF MYCOSIS-FUNGOIDES CUTANEOUS T-CELL LYMPHOMA PATIENTS Kim, E.1; Geskin, L.2; Querfeld, C.3; Girardi, M.4; Guitart, J.5; Musiek, A.6; Mink, D.7; Williams, M.7; Angello, J.8; Bailey, W.8 1Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States; 2Columbia University, New York, United States; 3City of Hope, Duarte, United States; 4Yale School of Medicine, New Haven, United States; 5Northwestern University, Feinberg School of Medicine, Chicago, United States; 6Washington University School of Medicine, St. Louis, United States; 7ICON Clinical Research, Dublin, Ireland; 8Helsinn Therapeutics (U.S.) Inc., Iselin, United States.

Y-04 INCIDENCE AND TYPES OF CONTACT DERMATITIS AFTER CHLORMETHINE GEL TREATMENT IN PATIENTS WITH MYCOSIS FUNGOIDES- TYPE CUTANEOUS T-CELL LYMPHOMA: THE MIDAS STUDY Gilmore, E.S.1; Alexander-Savino, C.V.1; Chung, C.G.2; Poligone, B.1 1Rochester Skin Lymphoma Medical Group, Fairport, United States; 2The Ohio State University, Columbus, United States.

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Z-01 PARADOXICAL RESPONSE OF CUTANEOUS T-CELL LYMPHOMA IN A PATIENT WITH CONCOMITANT ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA Johnson, A.; Ren, R.; William, B.; Chung, C. The Ohio State University, Columbus, United States.

Z-02 PROGRESSION OF CUTANEOUS T-CELL LYMPHOMA AFTER DUPILUMAB: CASE REVIEW OF 6 PATIENTS Espinosa, M.L.1; Nguyen, M.T.1; Agirre, A.S.1; Martinez-Escala, M.E.1; Walker, C.J.1; Pontes, D.1; Silverberg, J.I.2; Choi, J.1; Pro, B.3; Pincus, L.4; Guitart, J.1; Zhou, X..1 1Northwestern University Feinberg School of Medicine, Chicago, United States; 2The George Washington University School of Medicine and Health Sciences, Washington DC, United States; 3Northwestern University Feinberg School of Medicine, Chicago, United States; 4University of California, San Francisco, San Francisco, United States.

Z-04 PRIMARY CUTANEOUS B-CELL LYMPHOMA – CASE SERIES OF TWO UNIQUE CUTANEOUS PRESENTATIONS TREATED WITH RITUXIMAB Stoll, J.; Pulitzer, M.; Moskowitz, A.; Myskowski, P.; Noor, S. Memorial Sloan Kettering Cancer Center, New York/Memorial Sloan Kettering Cancer Center, United States.

Z-05 TONSIL INVOLVEMENT AS A MARKER OF ADVANCED DISEASE IN MYCOSIS FUNGOIDES Csányi, I.1; Ócsai, H.1; Varga, E.1; Hideghéty, K.1; Marschalkó, M.2; Krenács, L.3; Borbényi, Z.1; Gurbity Pálfi, T.1; Oláh, J.1; Kemény, L.1; Baltás, E.1 1Albert Szent-Györgyi Health Centre - University of Szeged - Faculty of Medicine, Szeged, Hungary; 2Semmelweis University - Faculty of Medicine, Budapest, Hungary; 3Laboratory of Tumor Pathology and Molecular Diagnostics, Szeged, Hungary.

Z-06 EPSTEIN-BARR VIRUS-POSITIVE MUCOCUTANEOUS ULCER IN A PATIENT WITH DYSKERATOSIS CONGENITA Fornons, R.1; Climent, F.1; González-Barca, E.M.2; Muniesa, C.1; Bauer, A.1; Llobera, C.1; Servitje, O.1 1Hospital Universitari de Bellvitge, L´Hospitalet de Llobregat, Spain; 2Institut Catalá d´Oncología - Hospital Duran i Reynals, L´Hospitalet de Llobregat, Spain.

Z-08 CLINICAL, HISTOLOGICAL AND MOLECULAR CHARACTERISTICS OF ANAPLASTIC LYMPHOMA KINASE-POSITIVE PRIMARY CUTANEOUS ANAPLASTIC LARGE CELL LYMPHOMA Melchers, R.1; Willemze, R.1; Van De Loo, M.1; Jansen, P.1; Cleven, A.1; Solleveld, N.1; Bekkenk, M.2; Van Kester, M.3; Diercks, G.3; Vermeer, M.1; Quint, K.1 1Leiden University Medical Center, Leiden, Netherlands; 2Academic Medical Center and Vrije University Medical Center Amsterdam, Amsterdam, Netherlands; 3University Medical Center of Groningen, Groningen, Netherlands.

Z-09 LUPUS ERYTHEMATOSUS TUMIDUS MIMICKING PRIMARY CUTANEOUS MARGINAL ZONE B CELL LYMPHOMA Trager, M.1; Ram-Wolff, C.2; Bouaziz, J.D.2; Battistella, M.2; Vignon-Pennamen, M.D.2; Rivet, J.2; Brice, P.2; Michel, L.3; De Masson, A.2; Bagot, M.2; Dobos, G.2 1Columbia University, New York City, United States; 2Saint-Louis Hospital, Paris, France; 3Université de Paris, Paris, France.

Z-10 MYCOSIS FUNGOIDES – GRANULOMATOUS SLACK SKIN IN ASSOCIATION WITH HODGKIN LYMPHOMA Della Ripa Rodrigues Assis, G.; De Melo Miranda, V.J.; Visentainer, L.; Machado De Moraes, A.; Fantelli Stelini, R.; Nunes Secamilli, E.; Massuda, J.Y.

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State University of Campinas, Campinas, Brazil.

Z-11 AGGRESSIVE CUTANEOUS T-CELL LYMPHOMAS: A SERIES OF 19 CASES Molgó, M.1; Reyes-Baraona, F.1; Ogueta, I.1; Acle, R.1; González, S.2 1Pontificia Universidad Católica de Chile, Santiago, Chile; 2Private Practice, Santiago, Chile.

Z-12 ANETODERMA: SECUNDARY TO SYPHILIS OR MYCOSIS FUNGOIDES? Lopes Iori, N.; Della Ripa Rodrigues Assis, G.; Cintra, M.L.; Massuda, J.Y.; Fantelli Stelini, R.; Nunes Secamilli, E. State University of Campinas, Campinas, Brazil.

Z-13 CD8+ MYCOSIS FUNGOIDES PALMARIS ET PLANTARIS WITH PERIPHERAL BLOOD INVOLVEMENT Yumeen, S.; Mirza, F.N.; Lewis, J.M.; Carlson, K.R.; King, B.; Cowper, S.; Bunick, C.G.; Mcniff, J.; Girardi, M. Yale School of Medicine, New Haven, United States.

Z-14 LYMPHOMATOID DRUG ERUPTION AFTER TREATMENT OF HEPATITIS C VIRUS INFECTION WITH SOFOSBUVIR: A NEW DESCRIBED ADVERSE REACTION Michel, M.; Farouk, M.; Ibrahim, M. Ain Shams University, Cairo, Egypt, Cairo, Egypt.

Z-15 MYCOSIS FUNGOIDES, LYMPHOMATOID PAPULOSIS AND HODGKIN’S LYMPHOMA IN THE SAME PATIENT: APROPOS OF A POSSIBLE MONOCLONAL ORIGIN Molgó, M.; Espinoza-Benavides, L.; Rojas, P.; González, S. Pontificia Universidad Católica de Chile, Santiago, Chile.

Z-16 PAGETOID RETICULOSIS: A RARE VARIANT OF MYCOSIS FUNGOIDES Secamilli, E.N.; Massuda, J.Y.; Stelini, R.F.; Adad, M.A.H.; Magalhães, R.F.; Cintra, M.L.; Souza, E.M. State University of Campinas, Campinas, Brazil.

Z-17 MAINTENANCE THERAPY – A NEGLECTED TOPIC Stadler, R. University Clinic for Dermatology, Minden, Germany.

Z-18 ROBUST GRAFT-VERSUS-LYMPHOMA (GVL) RESPONSE MIMICKING CUTANEOUS RELAPSE OF T-CELL LYMPHOMA Johnson, A.; Ren, R.R.; William, B.; Chung, C. The Ohio State University, Columbus, United States.

Z-19 SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA IN THE MESENTERY WITH ASSOCIATED HEMOPHAGOCYTIC SYNDROME TREATED BY CHEMOTHERAPY CHOEP, MODIFIED HLH 2014 PROTOCOL AND HDT AND AUTO SCT Giza, A.1; Jońca, M.2; Raźny, M.2; Zimowska-Curyło, D.1; Wilk, M.1; Goldman- Mazur, S.1; Piątkowska- Jakubas, B.1; Sacha, T.1 1Collegium Medicum of the Jagiellonian University, Krakow, Poland; 2Rydygier Hospital, Krakow, Poland.

Z-20 AGGRESSIVE EPIDERMOTROPIC CD8+ T CELL LYMPHOMA PRESENTING WITH TARGETOID LESIONS El-Zawahry, K.; Daruish, M.; Ibrahim, M.A.H.

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Ain shams university, Cairo, Egypt.

Z-21 CD4/CD8 DOUBLE-NEGATIVE FOLLICULOTROPIC MYCOSIS FUNGOIDES/SEZARY SYNDROME PRESENTING AS SEVERE LEONINE FACIES Merkel, E.; Chovatiya, R.; Guggina, L.; Pro, B.; Guitart, J.; Zhou, X. Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States.

Z-22 AIN-SHAMS CUTANEOUS LYMPHOMA CLINIC – SUGGESTED GUIDELINES FOR THE TREATMENT OF MYCOSIS FUNGOIDES IN COUNTRIES WITH LIMITED RESOURCES Ibrahim, M.A.H.; El-Tayeb, N.; Michel, M.; Nassar, A.; Mohamed, A.; Daruish, M.; El-Zimaity, M.; El-Afifi, A.; Abdelbary, H.; El-Lithy, M.; Mostafa, A.; El-Sayed, M.H. Ain Shams University hospitals, Cairo, Egypt.

Z-23 PATIENTS WITH MYCOSIS FUNGOIDES ARE AT INCREASED RISK OF LUNG CANCER: A SYSTEMATIC REVIEW AND POPULATION-BASED ANALYSIS Goyal, A. University of Minnesota, Minneapolis, United States.

Z-24 EPIDEMIOLOGICAL ANALYSIS AND REVIEW OF HISTOPATHOLOGICAL PARAMETERS OF PATIENTS DIAGNOSED WITH MYCOSIS FUNGOIDES AND ITS VARIANTS IN FOLLOW-UP AT STATE UNIVERSITY OF CAMPINAS Suzuki, N.1; Machado Toribio, J.2; Abdala, S.M.2; Ferreira Magalhães, R.2; Nunes Secamilli, E.2; Fantelli Stelini, R.2; Massuda, J.Y.2 1State University of Campinas, Campinas, Italy; 2State University of Campinas, Campinas, Brazil.

Z-26 THE USE OF GATA3 IN CHALLENGING CASES OF CD30+ MYCOSIS FUNGOIDES (MF) VERSUS ANAPLASTIC LARGE CELL LYMPHOMA (ALCL) Parente Almeida, I.; Cury Martins, J.; Miyashiro, D.; Neder Ramires Abdo, A.; Abrantes Giannotti, M.; Barros Domingues, R.; Pereira, J.; Sanches, J.A. Hospital das Clinicas, University of São Paulo Medical School, São Paulo, Brazil.

Z-27 CD30 EXPRESSION IS DETECTABLE IN MOST RARE CUTANEOUS LYMPHOMA SUBTYPES Ulrike, W.1; Christina, M.2; Marion, W.3; Roland, S.4; Uwe, H.5; Sarja, S.1; Mehmet, B.6; Bernd, H.6; René, S.7; Jan, N.8; Claus-Detlev, K.9; Werner, K.10; Chalid, A.6 1University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 2University Medical Center Goettingen, Göttingen, Germany; 3University Hospital Würzburg, Würzburg, Germany; 4Ludwigshafen, Ludwigshafen, Germany; 5Vivantes Klinikum Neukölln, Berlin, Germany; 6Helios-Klinikum Krefeld, Krefeld, Germany; 7Johannes Wesling Klinikum, UKRUB, University Bochum, Bochum, Germany; 8University Hospital Mannheim, Mannheim, Germany; 9Städtisches Klinikum Karlsruhe, Akademisches Lehrkrankenhaus der Universität Freiburg, Karlsruhe, Germany; 10University Hospital Zurich, Zurich, Switzerland.

Z-28 CD8+ MYCOSIS FUNGOIDES WITH DIVERSE ATYPICAL CLINICAL AND PATHOLOGICAL PRESENTATIONS Ibrahim, M.; Nassar, A.; Abdelbary, H. Ain Shams University hospitals, Cairo, Egypt.

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Z-29 THE THERAPEUTIC POTENTIAL OF CANNABINOIDS FROM CANNABIS SATIVA EXTRACTS FOR MYCOSIS FUNGOIDES / SÉZARY SYNDROME - AN IN VITRO AND EX VIVO STUDY Amitay-Laish, I.1; Moyal, L.2; Tiroler, A.3; Mazuz, M.3; Stalin, R.N.3; Ajjampura, V.C.3; Gorovitz-Haris, B.4; Lubin, I.4; Drori, A.5; Drori, G.5; Van Cauwenberghe, O.6; Namdar, D.3; Koltai, H.3; Hodak, E.7 1Rabin Medical Center - Beilinson Hospital, and Sackler Faculty of Medicine, Tel Aviv University, Kiryat-Ono, Israel; 2Felsenstein Medical Research Center, Rabin Medical Center - Beilinson Hospital, Sackler Faculty of Medicine, Tel Aviv University, Petach Tikva, Israel; 3Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel; 4Felsenstein Medical Research Center, Rabin Medical Center - Beilinson Hospital, Petach Tikva, Israel; 5MedC Biopharma Corporation, MedC Biopharma Corporation, Canada; 6AgMedica Bioscience Inc, Chatham, Ontario, Canada; 7Felsenstein Medical Research Center, Rabin Medical Center - Beilinson Hospital, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Petach Tikva, Israel.

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ORAL HANDOUTS

B-03

Handout Presentation B-03 Plaque stage folliculotropic mycosis fungoides: histopathologic features and prognostic factors in a series of 40 patients . Introduction: Folliculotropic mycosis fungoides (FMF) is a distinct variant of mycosis fungoides. Recent studies recognized indolent and aggressive subgroups of FMF, but there is controversy how patients presenting with plaques should be classified. The present study describes the histopathologic features of 40 FMF plaques. The aim of the study was to identify risk factors for disease progression and poor outcome in this group.

Methods: Clinical, histopathological, and immunophenotypical data from 40 patients with plaque stage FMF were reviewed and analysed for risk factors for disease progression and survival.

Results: After a median follow-up of 80 months, disease progression occurred in 20 of 40 patients. Percentage of atypical cells, cell size, percentage of Ki-67+ cells, and co-existent interfollicular epidermotropism, but not the extent of perifollicular infiltrates, were associated with disease progression and reduced survival, while extensive follicular mucinosis was associated with increased survival (see Table 1).

Conclusions: This study underlines that FMF patients presenting with plaques represent a heterogeneous group and that a subgroup of these patients may have an indolent clinical course. It further shows that histological examination is a valuable tool to differentiate between indolent and aggressive disease.

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Table 1 Univariate and multivariate analysis of relevant histopathologic features in plaque stage folliculotropic mycosis fungoides

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B-04

Adnexotropism: an under-recognized histopathological finding in aggressive cytotoxic cutaneous lymphomas

Christina J. Walker, MD, Estela Martinez-Escala, MD, PhD, Maria L. Espinosa, BS, Timothy Tan, DO, Joan Guitart, MD

Department of Dermatology, Northwestern University, Feinberg Medical School, Chicago, IL, USA

1. Introduction and Objectives: Primary cutaneous gamma-delta T-cell lymphoma (PCGDTCL)[1] and primary cutaneous aggressive epidermotropic T-cell lymphomas (PCAETCL)[2] are rare aggressive cytotoxic cutaneous lymphomas (CyCL) often difficult to diagnose. Adnexotropism, including folliculotropism and syringotropism, are usually associated with mycosis fungoides and its occurrence in CyCL has been rarely described. Our aim is to evaluate histopathological adnexotropism in CyCL with clinical correlation. 2. Material and Methods: Retrospective single-center review of medical records, clinical photographs and histopathological material from CyCL patients with pathological annotation of adnexotropism. 3. Results: We reviewed 110 skin biopsies of 40 patients (median age 72.5 years; 33M:7F) diagnosed with PCAETCL (17), PCGDTCL (15) and cytotoxic CTCL, unspecified (8) with notable adnexotropism (Table 1). Clinically, most patients had extensive patches/plaques (92.5%) and tumors (42.5%) occupying a mean body surface area of 20%. Patches of alopecia (15.0%) were noted in 6 cases, mostly correlating with extensive adnexotropism. Palmoplantar involvement was associated with syringotropism in 8 cases. The pathological pattern was primarily epidermotropic (82.8%) without predominantly panniculitic lymphomas. Folliculotropism (74.2%) was observed more frequently than syringotropism (57.0%) and combined adnexotropism was noted in 44.1% of cases. Follicular mucinosis (3), hyperplasia of the eccrine unit (2) and disruption of the follicular epithelium with granulomatous reaction (1) were rarely observed. Eosinophils were abundant in 27.8% of biopsies. Direct comparison between PCGDTCL and PCAETCL showed slightly higher presence of folliculotropism in PCAETCL (Figure 1). 4. Conclusions: We estimate that the majority of epidermotropic CyCL demonstrate a variety of adnexotropism, which in some cases may be the salient location of the tumor infiltrate. This histological finding is rarely accompanied by clinical signs of adnexotropism. Raising awareness of this phenomenon is important for a timely diagnosis of these aggressive lymphomas.

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5. References: 1. Guitart, J., et al., Cutaneous gammadelta T-cell lymphomas: a spectrum of presentations with overlap with other cytotoxic lymphomas. Am J Surg Pathol, 2012. 36(11): p. 1656- 65. 2. Guitart, J., et al., Primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphomas: reappraisal of a provisional entity in the 2016 WHO classification of cutaneous lymphomas. Mod Pathol, 2017. 30(5): p. 761-772.

Table 1: Patient population characteristics. CyCL with 100,0% adnexotropism (n=40)

80,6% Age (median) 72.5 80,0% Age range M:F ratio 4.7 64,7% 65,0% 62,1% Cytotoxic Subtypes 60,0% PCGDTCL 15 37.5% PCAETCL 17 42.5% CTCL, NOS 8 20% 40,0% Clinical presentation Patches/plaques 37 92.5% Tumor 17 42.5% 20,0% Ulceration 31 77.5% Alopecia 6 15.0%

Palmoplantar 0,0% involvement 8 20% Syringotropism Folliculotropism Pathology Folliculotropism 49/66 74.2% PCGDTCL PCAETCL

Syringotropism 49/86 57.0% Figure 1: Syringotropism, folliculotropism and alopecia in Combined PCGDTCL and PCAETCL. folliculotropism and syringotropism 26/59 44.1%

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B-05

Subcutaneous panniculitis-like T-cell Lymphoma (SPTCL): a large US case series reveals a lymphoma with limited growth potential in need of diagnostic and therapeutic guidelines

Joan Guitart (1), Christina J. Walker (1), Estela Martinez-Escala (1), Nneka I. Comfere (2), Melissa Pulitzer (3), Kerri Rieger (4), Carlos A. Torres-Cabala (5), Laura Pincus, (6) Katherine E. Park (5), Maria L. Espinosa (1), Madeleine Duvic (5), Steven Horwitz (3), Youn H. Kim (4), Aaron R. Mangold (2)

From: (1) Northwestern University, Chicago IL (2) Mayo Clinic Rochester Mn & Scottsdale AZ, (3) Memorial Sloan-Kettering Hospital, New York NY, (4) Stanford University, Palo Alto Ca, (5) MD Anderson Cancer Center, Houston TX, (6) University of California San Francisco, CA

1. Introduction & objectives: The knowledge of SPTCL is limited by the lack of multicenter large cohort data. We aimed to review the collaborative experience of 6 US academic centers.

2. Materials & Methods: Retrospective clinic-pathological review of 93 cases during 2 workshops.

3. Results: We identified a spectrum of cases including atypical lymphocytic lobular panniculitis (ALLP) characterized by mixed lymphohistiocytic infiltrate with T-cell clonality, subtle atypia and indolent chronic course (19 cases) preceding SPTCL in 2 cases. We also identified 74 SPTCL cases (mean age 38; F:M ratio: 2.7) fulfilling rigorous clinic-pathological criteria. Most patients had multiple deep painful nodules (85%) involving legs (67.7%)> arms (52.7%) with 11.8% unilesional cases. Lesional and/or non-lesional lipoatrophy was observed in 29.1% of the cases and a personal (26%) or family history (13%) of autoimmunity was recognized. While fever and other B-symptoms were common (75%) only 13.5% were reported to have HLH (mean 3.6 criteria). An abnormal bone marrow biopsy was reported in 30%, including HLH 11, SPTCL 4. Most abnormal PET/CT involved the subcutaneous tissue with 2 PET-avid nodal cases with negative biopsy. With a mean follow up of 54 months 66.6% achieved CR with 3 median cumulative therapies. Relapse was common (61%) with a gradual decrease in recurrence after each subsequent treatment. None of our patients died of disease progression or HLH.

4. Conclusions: This is the largest reported series of SPTCL to date demonstrating no evidence of systemic tumoral progression beyond the subcutaneous fat. Rare involvement of marrow adipose tissue or perinodal fat does not seem to imply systemic progression. Clonal ALLP poses a diagnostic challenge and may precede SPTCL. Our data shows favorable responses with a variety of treatments from immune-suppression to polychemotherapy with morbidity mostly related to HLH. Our observations highlight the need for consensus guidelines for the diagnosis and treatment of SPTCL.

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Table 1: Patient population characteristics.

SPTCL and ALLP SPTCL ALLP (n=93) (n=74) (n=19) Age (median) 38 38.5 32 Age range 2-81 2-80 3-81 F:M ratio 2.7 2.5 3.8 Duration until 26.4 18.7 56.6 diagnosis (months) Disease Extend (T1-T3) T1 11 11.8% 11 14.9% 0 0% T2 36 38.7% 28 37.8% 8 42.1% T3 46 49.5% 35 47.3% 11 57.9% Lipoatrophy 23/79 29.1% 19/63 30.2% 4/16 25.0% Anatomical involvement Head and 29.0% 28.4% 31.6% Neck Trunk 54.8% 58.1% 42.1% Upper 52.7% 52.7% 52.6% Extremities Lower 67.7% 63.5% 84.2% Extremities

Table 2: Pathology SPTCL vs. ALLP

SPTCL ALLP (n=74) (n=19) CD4:CD8 ratio 1:5.2 (p<0.05) 1:2.9 Ki67% mean 45% (p<0.01) 17% Clonal TCR 37/47 78.7% 11/17 64.7% Interface 19/50 38.0% 9/17 52.9% dermatitis Mucin deposits 35/50 70.0% 9/17 52.9% Density Low 18/68 26.5% 11/19 57.9% Intermediate 35/68 51.5% 5/19 26.3% High 15/68 22.1% 3/19 15.8% Atypia 14 Low 20/50 40.0% 10/14 71.4% Intermediate 22/50 44.0% 1/14 7.1% High 8/50 16.0% 1/14 7.1% HPS in skin biopsy None 26/61 42.6% 8/17 47.1% Low 23/61 37.7% 9/17 52.9% Intermediate 8/61 13.1% 1/17 5.9 High 4/61 6.6% 0/17 0.0%

27

B-06

Granulomatous Mycosis Fungoides: a Clinicopathological Study of 41 Cases from a Tertiary Cancer Center Ali M Alani MD1, Jesus Navarrete-Viveros MD2, Phyu P Aung MD PhD2, Priyadharsini Nagarajan MD PhD2, Michael T Tetzlaff MD PhD2, Jonathan L Curry MD2, Victor G Prieto MD PhD2, Auris Huen MD2, Roberto N Miranda MD2, Madeleine Duvic MD2 , Carlos Torres-Cabala MD2 1 Baylor College of Medicine, 2 The University of Texas MD Anderson Cancer Center, Houston, TX, USA Introduction & Objectives: Our current knowledge of the clinicopathological features of granulomatous mycosis fungoides (GMF) is very limited. We aimed to evaluate a large series of GMF in order to elucidate histopathological features and their impact on prognosis. Material & Methods: 47 skin biopsies from 41 GMF patients seen at our institution between 2010 and 2019 were evaluated. Medical records and skin biopsies were examined. Clinical and pathological findings were recorded. Results: The median age of our 41 patients (28 Caucasian, 11 African American, 2 Hispanic) was 63 years. M:F was 1.4:1. Biopsies were taken from upper extremities (34%), lower extremities including hip (22%), head & neck (20%), back (15%), and chest and abdomen (9%). Most patients presented with stage I (68%), followed by stage II (25%) and stage IV (7.5%); no patients were classified as stage III. Most of the lesions were plaques (80%) and tumors (18%) with moderate to high cell density (93%) and diffuse (pure or in combination, 50%) or nodular (pure or in combination, 47.5%) pattern of infiltration. Most lesions were composed of small/medium sized cells (88%). Epidermotropism was common (95%) but predominantly mild (80%). Folliculotropism was frequent (70%); syringotropism was detected in 33% of the cases. Four cases (10%) showed large cell transformation. The granulomatous component of the lesions showed: poorly formed granulomas (73%), well-formed granulomas (28%), multinucleated giant cells (67%), elastophagocytosis (36%), and emperipolesis (23%). The infiltrate was CD3-positive in all the cases, with the following phenotypes: CD4+CD8- (89%), CD4-CD8+ (2%), CD4+CD8+ (at least a subset, 7%), and CD4-CD8- (at least a subset 2%). All the tested cases showed alpha/beta phenotype. Special stains (GMS, Gram, Fite) were negative in all tested cases. Other epidermal and dermal changes were also recorded (Table 1). 41.5% of our GMF patients were dead on a median follow up of 67.2 months. All the large cell transformation patients were alive at last follow up. Histopathological features that appeared to be associated with survival were: presence of well-formed granulomas (80% patients alive), elastophagocytosis (79%), and emperipolesis (100%). Conclusions: GMF frequently occurs on upper and lower extremities. Folliculotropism is frequently seen and syringotropism is not uncommon. Cases showing well-formed granulomas, elastophagocytosis, and emperipolesis on biopsy examination may be associated with a better survival.

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Table 1. Pathological characteristics of cases of granulomatous mycosis fungoides Characteristic N % Type of lesion Patch 1/40 (2.5%) Plaque 32/40 (80.0%) Tumor 7/40 (17.5%) Density Sparse 3/40 (7.5%) Moderate 23/40 (57.5%) Dense 14/40 (35.0%) Nodular 11/40 (27.5%) Nodular+ diffuse 2/40 (5.0%) Nodular+ interstitial 1/40 (2.5%) Diffuse 19/40 (47.5%) Interstitial 1/40 (2.5%) Cell size Small 21/40 (52.5%) Small + medium 2/40 (5.0%) Medium 12/40 (30.0%) Medium + large 3/40 (7.5%) Large 2/40 (5.0%) Epidermotropism Absent 2/39 (5.1%) Mild 31/39 (79.5%) Moderate 3/39 (7.7%) Severe 3/39 (7.7%) Pautrier microabcesses Absent 31/38 (81.6%) Present 7/38 (18.4%) Large cell transformation Absent 35/39 (89.7%) Present 4/39 (10.3%) Presence of other inflammatory cells Neutrophils 10/40 (25.0%) Plasma cells 10/40 (25.0%) Eosinophils 20/40 (50.0%) Granulomas Not well formed 29/40 (72.5%) Well formed 11/40 (27.5%) Multinucleated giant cells Absent 13/39 (33.3%) Present 26/39 (66.7%) Elastophagocytosis Absent 25/39 (64.1%) Present 14/39 (35.9%) Emperipolesis Absent 31/40 (77.5%) Present 9/40 (22.5%) Inmunoprofiling n (%) CD3+ 47/47 (100.0%) CD4+ 46/47 (97.9%) CD8+ 4/34 (11.8%) Loss of expression of CD7 (at least partial) 36/39 (92.3%) CD30+ 0 to 5% 17/39 (43.5%) 5 to 50% 19/39 (48.7%) > 50% 3/39 (7.7%) Parakeratosis Absent 22/39 (56.4%) Present 17/39 (43.6%) Presence of other inflammatory cells Neutrophils 10/40 (25.0%) Plasma cells 10/40 (25.0%) Eosinophils 20/40 (50.0%) Spongiosis Absent 26/39 (66.7%) Present 13/39 (33.3%) Interface changes Absent 28/39 (71.8%) Present 11/39 (28.2%) Fibrosis in dermis Absent 10/39 (25.6%) Present 29/39 (74.4%) Solar elastosis Absent 23/40 (57.5%) Present 17/40 (42.5%)

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C-01

Prognostic Factors in Early Stage Mycosis Fungoides: The PROCLIPI Study Julia Scarisbrick, Pietro Quaglino, Miles Prince, Evangelina Papadavid, Emilia Hodak, Sean Whittaker, Martine Bagot, Christina Querfeld, Oleg Akilov, Octavio Servitje, Emilio Berti, Pablo Ortiz-Romero, Rudolf Stadler, Robert Knobler, Christina Mitteldorf, Teresa Estrach, Marta Marschalko, Emmanuella Guenova, Nicola Pimpinelli, Marie Beylot-Barry, Marion Wobser, Ulrike Wehkamp, Richard Cowan, Liisa Vakeva, Anne-Marie Buschots, Rubeta Matin, Felicity Evison, Eric Hong, Lorenzo Cerroni, Werner Kempf, Ale Gru, Maxime Battistella, Rein Willemze, Youn Kim

Introduction: Mycosis fungoides (MF) is the most prevalent form of primary cutaneous T-cell lymphoma. Patients frequently present with early-stage disease typically associated with a favourable prognosis and survival of 10–35 years, but over 25% may progress to advanced disease with a median survival < 4 years and just 13 months in those with nodal involvement and 30% present with the advanced stages of MF. A recent international study in advanced MF found age >60 years, large cell transformation in skin, stage IV and raised LDH all to be associated with a worse prognosis but the significance of these findings in early stage MF is not known1. Identifying prognostic factors in early stage disease may allow better management and improve survival and the development of a prognostic index could help identify patients at risk of progression. In order to investigate prognostic factors in mycosis fungoides and Sezary syndrome the PROCLIPI Study; a prospective international study was launched in 2015.

Methods: This study collects pre-defined clinical, haematological, radiological, immunohistochemical, genotypic, treatment data including treatment responses and health related quality of life.

Results: 1485 patients have been recruited at 50 sites, from 19 countries across 6 continents. 1105 early-stage patients (male:female ratio=1.7:1) and 380 advanced (male;female ratio 1.8:1). Early-stage includes IA;n=557, IB;n=471, IIA;n=77 with median age=57years. Blood involvement (B1) was present in 8% and 15% had an identical clone in blood to skin. 387 patients (37%) had imaging and 59 patients (15.2%) had lymph nodes ≥1.5cm. Only16/59 had a LN biopsy which was N1;n=13 and N2;n=3. Factors associated with progression to advanced disease included higher mSWAT;p<0.001, raised LDH;p=0.027, LCT skin;p<0.001 and age>60yrs;p=0.0126 but not B1;p=0.154 or identical blood clone;p=0.221. Stage IIA patients had a higher mSWAT;p=0.0012 but were not more likely to be B1 than IB patients;p=0.5852. 380 advanced stage patients are recruited stage IIB;n=128,IIIA;n=45,IIIB;n=44,IVA1;n=114, IVA2;n=36,IVB;n=113. The median age=65.5yrs (significantly higher than early-stage cohort at 57yrs p<0.0001). In advanced stages disease-specific deaths were more frequent with LCT skin;p=0.011, age>60yrs;p=0.0197 and N3;p=0.048 but not raised LDH;p=0.317.

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Table showing Recruitment

Conclusions This is the first prospective study recording pre-defined parameters for skin, lymph nodes, viscera and blood in MF aiming to identify factors associated with disease progression and survival. Treatments and responses are being recorded to identify best regimes for survival alongside quality of life.. In early stage progression to advanced disease is associated with higher mSWAT, raised LDH, LCT in skin and age>60yrs. In advanced disease poor survival was also associated with raised LDH and LCT in skin but not age or mSWAT. N3 nodes were also a poor prognostic marker for survival. Identifying prognostic markers may help select patients for more aggressive treatment options and better management and survival.

References 1 Scarisbrick JJ, Prince HM, Vermeer MH et al Cutaneous Lymphoma International Consortium (CLIC) Study of Outcome in Advanced Stages of Mycosis Fungoides & Sézary Syndrome: Effect of specific prognostic markers on survival and development of a prognostic model. J Clin Oncology. 2015;33(32):3766-73 2. Scarisbrick JJ, Quaglino P, Prince HM, et al. The PROCLIPI international registry of early stage mycosis fungoides identifies substantial diagnostic delay in most patients. Br J Dermatol. 2019 Aug;181(2):350-357

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C-02

Review of the measurements of abnormal lymph nodes on imaging and the correlation with N class from the PROCLIPI study

J Yoo, M Bagot, E Hodak, O Servitige, R Pujol, M Marschalko, E Papadavid, R Cowan, C Mitteldorf, C Jonak, C Querfeld, A Busschots, R Matin, U Wehkemp, Y Ma, E Wong, M Battistella, A Gru, F Evison, A Gru, Y Kim, J Scarisbrick

Introduction: Current skin lymphoma guideline suggests that long axis ≥15mm on imaging is considered abnormal which requires further histological investigation.1 However, this is based on systemic lymphoma guidelines which does not reflect the presence of dermatopathic lymph nodes (LNs). Furthermore, excision LN biopsies are associated with morbidities such as infection and it may not be deemed necessary in patients with recurrent infection, who had previous LN biopsies or in patients whose biopsy would not alter the management. Therefore, we need a better predictor to detect abnormal LNs to reduce the complications which can be associated with LN excision.

Methods: We reviewed PROCLIPI database to review information on imaging and LN biopsy and to identify better predictor for N stage. Out of 82 patients who had LN biopsy, we reviewed their demographic details, mSWAT, imaging including the type and measurements of the biopsies LN on scan (long axis, short axis, product of dimension (PD), sum of product of dimension (SPD), total nodal score). The correlation between the N stage and the measurements on the imaging was also reviewed.

Results: In PROCLIPI, 82 patients had LN biopsy following imaging. Five patients had two biopsies. Gender was 56 males:26 females (2:1). Median age at diagnosis was 58 yrs (IQR=50- 70yrs). Stage at diagnosis was IB=5, IIA=20, IIB=8, IIIA=4, IIIB=8, IVA1=9, IVA2=25 & IVB=3. In 74(85%) this was a biopsy at diagnosis and in 13 (15%) it was at a further visit. Median time from diagnosis to biopsy was 1 month (IQR=0-4months). The N-class of the biopsied node was N1=37 (43%), N2=17 (20%), N3=33 (38%). Imaging was ultrasound scan; n=6(7%), CT; n=63(72%) and PET/CT; n=15(17%). 68/82 (83%) had the biopsied node with long axis ≥15mm on imaging. The measurements of biopsied LN on imaging were reviewed and compared with histological N-class (N1/2 vs N3), which is summarised in the table 1. Median number of LN sites with enlarged LN (total-nodal-score) was 3 in both N1/2 and 4 in N3 (p=0.1190). mSWAT was significantly higher in N3 at 93 (IQR=68-110) compared with 68 (IQR=31-90) in N1/2(p=0.0051). Blood-class was more likely to be B2 in N3;15/31(48%) compared to in N1/2;7/45 (15.6%) (p=0.009).

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Table 1. Comparison between measurements on imaging and N-class

N1/2 N3 p value

Median long axis 23 (IQR=19-27) 30 (24-40) 0.0011

Median short axis 12 (11-15) 18.5 (14.5-20) 0.0006

Median PD 291 (228-378) 522 (400-805) 0.0001

Median SPD 751 (479-1129) 1300 (800-1900) 0.0062

Conclusion: Our study showed that the product of dimensions in mm2, referred to as PD (long axis mm x short axis mm), is the better predictor for N3 node than any single axis measurement. The use of PET/CT has been increasing and this study showed that 17% of imaging were PET/CT. In 2014, Lugano Classification incorporated PET/CT in the staging and treatment response assessment in systemic lymphomas.2 However, this classification is not suitable to be applied to cutaneous lymphomas due to the presence of dermatopathic node. Developing specific imaging guideline for cutaneous lymphomas will better identify LN suspicious for N3 and thereby improve LN selection for biopsy and our diagnosis and avoid unnecessary LN biopsies. Currently, our PROCLIPI database does not include data on metabolic activity (SUV) on PET/CT. Large-scale central review of SUV measurement on PET/CT will aid us to incorporate PET/CT criteria to the imaging guideline specific for cutaneous lymphomas.

Reference: 1. Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler R, et al. Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORC). Blood. 2007 Sept 15;110(6):1713-22. 2. Cheson BD, Fisher RI, Barrington SF, Cavalli F, Schwartz LH, Zucca E, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014 Sep 20;32(27):3059-68.

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C-03

T-cell monoclonality in blood and skin correlates with poor response to treatment in mycosis fungoides

Shamir Geller1,3,+, Shira Fajnerman Tel-Dan1,3, Irit Solar2,3, Eli Sprecher1,3, Ilan Goldberg1,3

Department of Dermatology1 and Pathology2, Sourasky Medical Center, Tel Aviv; 3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv. [email protected]

Introduction & Objectives: The prognostic value of T-cell receptor (TCR) clonality in the blood and skin in mycosis fungoides (MF) is a matter of debate. We aimed at ascertaining the relation between the presence of a monoclonal T-cell population in the skin and circulation and the response to treatment in a large cohort of MF patients. Materials & Methods: We identified 94 adult patients with MF who had polymerase chain reaction (PCR) results for TCR-gamma gene rearrangement in both the skin and the blood and were followed by the cutaneous lymphoma clinic at Tel Aviv Sourasky Medical Center for at least three years. Data including age, sex, stage at diagnosis and response to treatment were retrospectively collected. Results: TCR clonality was assessed in 84 patients with a diagnosis of early-stage MF and in 10 patients with advanced-stage disease. T-cell monoclonality was detected in the skin, blood or both tissues in 32%, 13% and 6% of all cases, respectively. 64 patients achieved complete clinical response (CR, 68%), 13 - partial response (PR, 14%), 10 had stable disease (SD, 11%) and 7 had progression of disease (PD, 7%). T-cell polyclonality in the circulation was associated with higher rates of CR (P=0.006). The presence of a monoclonal T-cell population in skin (P=0.009), blood (P=0.002) and in both tissues (P<0.001) were found to be statistically associated with lack of response to treatment (SD and PD) and this correlation was found to be statistically significant also after adjusting for age, sex and disease stage in a multivariate analysis. Conclusions: T-cell monoclonality in the circulation and the skin correlates with poorer response to therapy in MF. Assessment of skin and blood T-cell clonality should be considered as part of the routine initial workup in patients with early-stage disease.

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C-04

Erythroderma: analysis of Sézary syndrome criteria usefulness in 309 erythrodermic patients Authors: Miyashiro D¹; Cury-Martins J¹; Abdo ANR²; Pereira J²; Sanches JA¹ ¹ Department of Dermatology, University of São Paulo Medical School, São Paulo, Brazil. ² Department of Hematology, University of São Paulo Medical School, São Paulo, Brazil.

Introduction: Distinction between Sézary syndrome (SS) and benign inflammatory erythroderma (BIE) is difficult, and many cases remain idiopathic. Our objective was to analyze SS criteria and compare with BIE, erythrodermic mycosis fungoides (MF), and idiopathic erythroderma. Methods: Data of 309 patients with acquired erythroderma was collected in a single center from 2007 to 2018. The ISCL/EORTC criteria for SS were collected at first evaluation.(1) Immunophenotypic abnormalities were calculated based on the whole lymphocyte population.(2) Results: There were 39 patients with SS (13%), 17 with MF (6%), 200 with BIE (65%), and 53 with idiopathic erythroderma (16%) (Figure 1). SS and idiopathic erythroderma patients were older (median 64 years and 68 years, respectively) than BIE (53 years) (p=0.008 and p<0.001, respectively). SS had higher levels of leukocytes (median 13575/mm3 vs 7320/mm3 MF, p<0.001; 9570/mm3 BIE, p=0.0017; 9555/mm3 idiopathic erythroderma, p=0.0231), lymphocytes (median 4400/mm3 vs 1700/mm3 MF, p=0.0001; 1800/mm3 BIE, and 1585/mm3 idiopathic erythroderma, p<0.0001), absolute manual Sézary cell count (median 248/mm3 vs 0/mm3 MF, p=0.0281; 0/mm3 BIE, p=0.0004; and 0/mm3 idiopathic erythroderma, p=0.0113). Increased LDH was more frequent in SS (76.3%) and idiopathic erythroderma (76.5%) than BIE (58.7%) and MF (58.8%) (p=0.043). On immunophenotyping of lymphocytes by flow cytometry, SS had higher percentages of CD3+ cells (92% vs 80% MF, p=0.0104; 78% BIE, p=0.0005; 78% idiopathic erythroderma, p=0.0097), percentage of CD4+ cells (87% vs 49% MF, p=0.0037; 49.5% BIE, p<0.0001; 54% idiopathic erythroderma, p=0.0002), CD4+ absolute count (2697/mm3 vs 936.7/mm3, p=0.0045; 770/mm3 BIE, p<0.0001; 803/mm3 idiopathic erythroderma, p=0.0002), CD4/CD8 ratio (18.25 vs 1.85 MF, p=0.0014; 1.97 BIE, p<0.0001; 2.9 idiopathic erythroderma, p=0.0001), percentage of CD4+CD7- cells (45% vs 12% MF, p=0.0181; 8% BIE, p=0.0173; 11.5% idiopathic erythroderma, p=0.0008), absolute CD4+CD7- count (1344/mm3 vs 136/mm3 MF, p=0.0023; 154/mm3 BIE, p=0.0059; 179/mm3 idiopathic erythroderma, p=0.0059), percentage of CD4+CD26- cells (76.5% vs 13% MF, p=0.0061; 16% BIE, p<0.0001; 17% idiopathic erythroderma, p=0.0008), absolute CD4+CD26- count (2776/mm3 vs 210/mm3, p=0.0082; 225/mm3 BIE, p<0.0001; 271/mm3 idiopathic erythroderma, p=0.0017); but had lower percentage of CD8+ cells (4.7% vs 24% MF, p=0.0013; 25% BIE, p<0.0001; 20% idiopathic erythroderma, p=0.0001), and CD8+ absolute count (240/mm3 vs 351/mm3 BIE, p=0.0451; no statistically significant difference compared to MF, 283/mm3, and idiopathic erythroderma, 319/mm3). T-cell receptor (TCR) monoclonality was more frequent in SS in blood, skin, and lymph nodes (90.9%, 75.9%, 88.2%, respectively), than BIE (7.5%, 8.4%, 0%) and idiopathic erythroderma (12.2%, 6.8%, 0%) (p<0.001). TCR clonality on skin (50%) and lymph nodes (50%) of MF had no statistically significant difference compared to SS, but TCR monoclonality on blood of MF (38.5%) was less frequent than SS (90.9%, p<0.001). Criteria for SS observed on our patients are summarized in Table 1. Sensitivity/specificity were (Table 2): 65.8%/99.0% for CD4/CD8 ratio ≥ 10; 57.1%/77.5% for CD4+CD7- ≥ 40%; 92.9%/86.9% for CD4+CD26- ≥ 30%; 32%/100% for Sézary cells ≥ 1000/mm3; 90.9%/92.4% for TCR monoclonality on the blood; 75.9%/91.5% for TCR monoclonality on the skin. Other immunophenotypic alterations included: loss of CD2, observed on 4 patients with SS (18.2%); 1 with MF (7.1%); 1 with BIE (1.7%, psoriasis). Loss of CD5 was observed only in 1 patient with BIE (1.9%, eczema).

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Sézary syndrome Idiopathic erythroderma 13% 16% MF 6%

BIE 65% Figure 1. Diagnosis of erythrodermic patients

Table 1. Criteria for SS according to the etiology of erythroderma Idiopathic SS MF BIE erythroderma Sézary cells ≥ 8/25 (32%)* 0/16 (0%) 0/86 (0%) 1/43 (2,3%) 1.000/mm3 CD4/CD8 ≥ 10 25/38 (65,8%)* 1/16 (6,25%) 1/104 (0,9%) 2/49 (4,1%) CD4+CD7- ≥ 40% 12/21 (57,1%)** 0/7 (0%) 9/40 (22,5%) 4/20 (20%) CD4+CD26- ≥ 26/28 (92,9%)* 0/11 (0%) 8/61 (13,1%) 6/42 (14,3%) 30% Skin 22/29 (75,9%)* 6/12 (50%)* 6/71 (8,4%) 3/44 (6,8%) Blood 30/33 (90,9%)* 5/13 (38,5%) 4/53 (7,5%) 5/41 (12,2%) Lymph node 15/17 (88,2%)* 2/4 (50%)* 0/6 (0%) 0/8 (0%) *p<0.001; p=0.005

Table 2. Sensitivity and specificity according to SS criteria Sensitivity Specificity Sézary cells ≥ 1.000/mm3 32% 100% CD4/CD8 ≥ 10 65,8% 99,0% CD4+CD7- ≥ 40% 57,1% 77,5% CD4+CD26- ≥ 30% 92,9% 86,9% Clonality - Blood 90,9% 92,4% Clonality - Skin 75,9% 91,5%

Conclusions: Erythroderma is a challenging syndrome. We described laboratory, immunophenotypic, and molecular differences between SS, MF, BIE, and idiopathic erythroderma. Flow cytometry studies use different protocols that impede interpretation and comparison of results. We highlight the importance in standardizing laboratory techniques to facilitate diagnosis and management of erythroderma.

References: 1. Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler R, et al. Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (EORTC). Blood. 2007;110(6):1713-22. 2. Roelens M, de Masson A, Ram-Wolff C, Maki G, Cayuela JM, Marie-Cardine A, et al. Revisiting the initial diagnosis and blood staging of mycosis fungoides and Sezary syndrome with the KIR3DL2 marker. Br J Dermatol. 2019.

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D-01

Global epidemiology of primary cutaneous lymphomas: a systematic review of relative frequencies and meta-analysis

G. Dobos, C. Ram-Wolff, M. Bagot, A. de Masson

1 Department of Dermatology, APHP, Saint-Louis Hospital, Paris, France 2 Université de Paris, INSERM U976, Paris, France

Introduction: Cutaneous lymphomas (CL) are a heterogenous group of lymphomas primarily affecting the skin. There is strong evidence supporting geographic inequalities in the epidemiology of other frequent skin cancers. However, there was no systematic comparison in the epidemiology of cutaneous lymphomas among different geographic regions.

Methods: A systematic literature review and meta-analysis were conducted to compare the relative frequencies of CLs in different countries. Based on a previously registered protocol, standardized searches were performed in Medline and the Cochrane Library. Peer-reviewed articles reporting at least 100 patients were included if detailing at least 3 CLs according to the WHO, EORTC or WHO-EORTC classifications. Publications before 1999 were excluded. We extracted study characteristics, reporting quality and relative frequencies of CLs. References of included articles were screened for additional records. Articles were grouped by geographical regions. For each cutaneous lymphoma the heterogeneity between the studies was calculated, this calculation was repeated within each group. Statitistical analysis was conducted using R.

Figure 1. Study selection

Results: Altogether 3,752 records were identified, 501 remained after removal of duplicates, of which 92 were screened in full text for eligibility. Twenty-five publications were included in the meta-analysis (fig. 1). The articles reported on 120-4,551 patients. The majority of the studies reported on European populations. More than half of them presented survival data. The reporting quality of the studies improved over time, most authors used the WHO-EORTC 2005 classification. Only one study compared the different classification systems.

Cutaneous T-cell lymphomas (CTCL) frequency among CLs was stable and varied Figure 2. Relative frequency of MF between 63-98%. The group of mycosis compared to CTCL fungoides (MF) and Sézary syndrome together

37

made up 52-93% of CTCLs. The average relative frequency of MF among CTCL was 62%, although the heterogeneity between studies was important (fig. 2). CD30-positive CTCLs represented 3.5-40% of CTCL. The relative frequencies of subcutaneous panniculitis-like lymphomas and NK/T-cell lymphomas varied the most, being more frequent in Asians. When focusing on NK/T cell lymphomas, a grouping of studies by geographic regions could decrease heterogeneity between studies by 19%, underlining this observation (fig. 3).

The proportion of marginal zone B-cell lymphomas (MZL) among CBCLs was 8-54%. This lymphoma appeared to be less frequent when using the WHO classification. The follicle center lymphomas (FCL) varied between 5-73% of CBCL, on average 41% when using the WHO-EORTC 2005 classification.

Conclusions: CTCLs made up approximately two thirds of cutaneous lymphomas worldwide. MF and SS accounted for almost two thirds of CTCLs. There was an important heterogeneity between the investigated registry data. Rare CTCLs seemed to be more frequent in Asia, which was supported by the statistical analysis. Figure 2. Relative frequency of NK/T lymphoma, nasal type compared to CTCL

The proportion of CBCL compared to all cutaneous lymphomas was stable. Different classification systems impacted the observed relative frequencies of CBCL.

Figure 2. Relative frequency of marginal zone B-cell lymphoma, compared to CBCL

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D-02

Epidemiology of Primary Cutaneous CD8+ T Cell Lymphoma: A United States Population- Based Cohort Analysis using the Surveillance, Epidemiology, and End Results Database

Authors: F. N. Mirza1, S. Yumeen1, M. Girardi1.

Affiliations: 1Department of Dermatology, Yale School of Medicine, New Haven, CT, USA

Introduction: While the most common immunophenotype of primary cutaneous T cell lymphoma (CTCL) is CD4+, CD8+ CTCL variants have also been described. Recent epidemiologic studies have identified African-American race, advancing age, and male gender as risk factors for CTCL, but little is known about the epidemiology of CD8+ CTCLs; thus, we sought to characterize demographic and baseline characteristics of patients diagnosed with CD8+ variants (e.g. aggressive primary cutaneous epidermotropic cytotoxic, acral CD8+ T-cell lymphoma, indolent cutaneous CD8+ lymphoid proliferation), independent of specific subtype.

Methods: A population-based cohort analysis was conducted in the Surveillance, Epidemiology, and End Results (SEER) database, 2010-2016, for all patients diagnosed with CD8+ CTCL, ICD- 9 9709, removing historic cases, death certificate only, and pathology-only cases. Cases coded with a primary skin site (C440-449) were included; age <18 years were excluded.

Results: CD8+ CTCLs were diagnosed and reported in 1271 patients during the 6-year period studied, revealing a slight predominance in males (55.9%), and with lesion location on the face (21.5%), trunk (20.5%), and extremities (24%). The incidence rate increased with age, with 63 as the median age of diagnosis. Majority of cases presented at stage I (75.4%). Highest incidence was in individuals of Caucasian race (75.7%) followed by African-Americans (15.4%). Patients received surgical management for their diagnosis in 28.8% of cases, radiation in 26.0% of cases, and chemotherapy in 77.4% of cases. Since 2010, 264 (20.7%) patients died of their disease, with mean time to death in these cases as 65.5 months.

(A) Overall survival (B) Disease-specific survival

(C) Overall survival (D) Disease-specific survival

Conclusions: Higher incidences of primary CD8+ CTCLs are found with Caucasian race, male gender, and increasing age, and lesions are found generally distributed across skin sites. While our study presents population-level data on CD8+ CTCL collectively, the SEER database does not allow for such analysis across each CD8+ variant.

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D-03

Introduction Cutaneous T-cell lymphoma (CTCL) is a heterogenous group of neoplasms of skin-homing T cells associated with a chronic low-grade systemic inflammatory state and has a median age of diagnosis of 55-60 years of age.1 Survival for CTCL patients depends on various factors including stage of disease with those diagnosed at an early-stage having a median survival of 13 years, while late-stage diagnosis has a much lower survival.1-2 Racial disparities exist in the CTCL population with higher incidence rates and more advanced disease found among African American than white individuals in the United States. In general, African Americans and Hispanics have an earlier onset of mycosis fungoides (MF), a subset of CTCL, with African American women in particular demonstrating the earliest onset with a poorer prognosis. Moreover, after accounting for disease characteristics, socioeconomic factors, and types of treatments, the overall survival of African Americans with MF was poorer than white patients. As non-white patients are likely to have more cardiovascular risk factors and to have higher rates of mortality from cardiovascular disease (CVD), it is of utmost importance to determine if and how the inflammatory nature of CTCL may contribute to coronary artery disease.

Methods We performed a retrospective analysis of all patients in the Johns Hopkins Health System medical database from January 2011 to June 2019 to create a CTCL population. This population includes all patients above the age of 18 with the following ICD-9 and ICD -10 diagnosis codes for Mycosis Fungoides, and Sézary disease. A matched control population of patients without the prior diagnoses were compiled for comparison studies. Cardiovascular events for both populations included any diagnosis of the following: coronary artery disease, ischemic heart disease, myocardial infarction, cardiovascular disease, cerebrovascular accident, transient ischemic angina, peripheral arterial occlusive disease, aortic aneurysm, coronary artery finding, myocardial ischemia, peripheral vascular disease, and unstable angina. Cardiac risk factors as defined by the ACC/AHA guidelines included any diagnosis of chronic kidney disease, essential hypertension, hyperlipidemia, tobacco use, type 2 diabetes mellitus and obesity were also recorded. Appropriate statistical analysis and propensity matching was used to assess the prevalence and odds ratio of cardiovascular events in non-white CTCL patients as compared to a matched control population with respect to cardiovascular risk factors.

Results Within the CTCL cohort, the average age was 62 years old with 40.1% consisting of non-white individuals and 48.6% females. 47.7% of this population had cardiovascular risk factors and consisted of a non-white population of 42.8% with 46.3% females. Of the CTCL cohort without risk factors, the non-white population was 39.3% and 51.2% female. In the CTCL cohort with cardiovascular events, the average age was 66 years old with 41.5% consisting of non-white individuals and 44.7% female. Of this group, 77.3% had cardiovascular risk factors and consist of 43.6% non-white individuals and 43.1% females. Of the group without cardiovascular risk factors, 31.3% were non-white and 50% were female. The average age of the control population was 49 years of age with 45.1% non-white individuals and 66.4% females. We found a slight increased prevalence of CVD in the CTCL population as compared to the control population (p=0.036) with a similar result among all racial groups. CTCL patients had a 16% increased association of CVD (95% CI 1.012-1.330, p=0.034) with a similar result in all racial groups. However, by age stratification, the younger population of non-white CTCL patients (age 18-64) had a significantly increased association (OR 1.32, 95% CI 1.008-1.733, p=0.043) of CV events. Furthermore, known cardiovascular risk factors increased the association of CVD in the total population and the younger population across all racial groups with young non-white patients having the highest association of 70% (95% CI 1.213-2.384, p=0.0022).

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Conclusions: The increased association of CVD in young non-white CTCL patients warrants further analysis as this may present an opportunity for population-specific targeted preventative therapies. Furthermore, the difference in CV events by race and age may reflect a unique immunosuppressive role of the malignant T-cell clones requiring further studies to elucidate relevant markers and pathways.

References: 1. Wilson LD, Hinds GA, Yu JB. Age, race, sex, stage, and incidence of cutaneous lymphoma. Clinical Lymphoma, Myeloma & Leukemia. 2012;12(5):291-296. https://www.clinicalkey.es/playcontent/1-s2.0-S2152265012001437. doi: 10.1016/j.clml.2012.06.010. 2. Kim YH, Liu HL, Mraz-Gernhard S, Varghese A, Hoppe RT. Long-term outcome of 525 patients with mycosis fungoides and sezary syndrome: Clinical prognostic factors and risk for disease progression. Arch Dermatol. 2003;139(7):857-866.

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D-04

Title: Mycosis fungoides in the Chilean pediatric population: a retrospective epidemiological and clinical-pathological study at the dermatology department of Pontificia Universidad Católica de Chile. Authors: Montserrat Molgó, Francisco Reyes-Baraona, Camila Downey, María Consuelo Giordano, Renata Acle, Sergio González

Introduction: Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma in adults and children. Clinical presentation of MF, its treatment and prognosis are poorly described in the pediatric population, with no treatment guidelines made especially for this group. Hypopigmented MF is the most prevalent variant of MF in children and adolescents. Its diagnosis is usually delayed due to the similarity with other common childhood hypopigmented diseases. Despite having a good prognosis, its relapse rate is high, which requires long-term follow-up. There are scarce reports of MF in childhood in Latin America, with no evidence of its prevalence in the Chilean population. In this study we describe the epidemiological, clinical-pathological characteristics, therapeutic response and prognosis of MF in pediatric population at a Chilean referral center for cutaneous lymphomas.

Methods: A retrospective review of epidemiological, clinical-pathological characteristics, therapeutic response and evolution of patients under 18 years-old diagnosed with MF between 1986 and 2019 was performed at the dermatology department of Pontificia Universidad Católica de Chile.

Results: 23 pediatric patients diagnosed with MF were included. The most frequent clinical variant was hypopigmented MF (91.3%), with only 1 case of classical MF and 1 case of folliculotropic MF (Figure 1). Demographic characteristics, latency in diagnosis, treatment and its response, and evolution are detailed in Table 1.

Figure 1. Variants of MF in the pediatric population.

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Table 1. Characteristics of pediatric patients diagnosed with MF. Distribution by sex 1:2 (Female:Male) Mean age at diagnosis 10 years-old (range: 4 to 18 years-old inclusive) Time from evolution to diagnosis (average) 27 months Treatment of choice Nb-UVB phototherapy (100%) Favorable response to phototherapy 14 of 23 (60%) Extracutaneous involvement 0 of 23 (0%) Recurrence of skin lesions 9 of 23 (39%) Patients who continue treatment and follow- 5 of 23 (21%) up

Conclusions: Hypopigmented MF was the most common variant of MF in children and adolescents, as it is described in the literature, showing good prognosis in all cases with no extracutaneous involvement nor progression to advanced stages. Recurrence after treatment was frequent. This study reports the largest number of cases of MF in the Chilean pediatric population, which gives an approximation to the absent national statistics in our country.

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D-05

Mycosis Fungoides in children Paula Enz MD, Silvina Bruey MD, Valeria Angles MD, Andrea Andrade MD, Victoria Volonteri MD. Dermatology Department. Hospital Italiano de Buenos Aires. Argentina.

Introduction: Mycosis Fungoides is the most frequent t-cell skin lymphoma. It rarely occurs in pediatrics. It is for this reason that we present a series of five cases of children with mycosis fungoides along with its treatment for a specific length of time. Method: all the patients came to the dermatology department at Hospital Italiano de Buenos Aires along last year. Patient 1: Eleven year old boy, presents generalized hypochromic macules all over the skin. The lesions had evolved for a year with slight flaking and pruritus. Patient 2: Nine year old girl presented hypopigmented lesions in her abdomen, thighs and legs for two months. Patient 3: Fifteen year old girl presented lychenoid lesions and was diagnosed with lichenoid pityriasis, those lesions later evolved to hypopigmented macules which evolved for 7 years. Patient 4: Eighteen year old girl who was diagnosed with atopic dermatitis starts showing hypopigmented as well as some lichenoid lesions which evolved for 2 months. Patient 5: Six year old boy who starts showing some hypopigmented lesions in his torso and later all over the rest of the tegument, evolved for two years. All the patients had a skin biopsy done and the results indicated that they all had epidermotropism of the tumoral infiltrate, atypical T lymphocytes which were positive for CD 3 and CD8+, and negative for CD4. The treatment of choice was narrow band UVB with good response, showing an improvement after session 15 approximately, depending on the case. Most of them completed 30 treatment sessions and then had maintenance follow up. A patient received PUVA sessions since her lesions were not only hypopigmented but some of them were lichenoid. Since she was 18 years old she was able to receive PUVA. She completed 50 sessions and responded well to the treatment. The last patient is still in treatment, with UVB, showing good response so far. Conclusion: Mycosis fungoides is a rare entity in pediatrics, it is important to suspect it and have a biopsy done to be able to diagnose it. In general it is a late diagnosis since it is similar to other hypopigmented entities in childhood. It has a good prognosis but it carries a high relapse rate which is why it requires long term follow ups. Bibliography. 1) Cervini AB, Torres-Huamani AN, Sanchez-La-Rosa C, et al. Mycosis Fungoides: Experience in a Pediatric Hospital. Actas Dermosifiliogr. 2017; 108:564-570. 2) Virmani P, Levin L, Myskowski PL, et al. Clinical Outcome and Prognosis of Young Patients with Mycosis Fungoides. Pediatr Dermatol. 2017; 34:547-553. 3) Abeldaño A, Enz P, Maskin M, et al. Primary cutaneous lymphoma in Argentina: a report of a nationwide study of 416 patients. Int J Dermatol. 2019; 58:449-455.

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D-06

Epidemiology of primary cutaneous gamma/delta T-cell lymphoma and subcutaneous panniculitis-like T-cell lymphoma in the United States from 2006-2015: A Surveillance, Epidemiology, and End Results-18 analysis Amrita Goyal MD, Kavita Goyal, MD, Kimberly Bohjanen MD, and David Pearson MD Department of Dermatology, University of Minnesota, Minneapolis, MN Contact: [email protected] Background: Primary cutaneous gamma/delta T-cell lymphoma (pcGDTCL) is a rare, aggressive T-cell lymphoma that presents with ulcerated nodules, dissemination to extranodal sites, constitutional symptoms, and often, hemophagocytic lymphohistiocytosis (HLH). pcGDTCL is so named because the T-cell receptor (TCR) has a gamma/delta phenotype. pcGDTCL is a histological mimicker of subcutaneous panniculitis-like T-cell lymphoma (SPTCL), a lymphoma with an alpha/beta TCR phenotype. Prior to 2005, cases of pcGDTCL and SPTCL were grouped as SPTCL. Here we seek to more clearly delineate the demographic characteristics of patients with pcGDTCL and SPTCL in the SEER-18 database in patients diagnosed from 2006-2015.

Methods: We identified all patients with SPTCL and pcGDTCL diagnosed between 2006-2015 in the 18 SEER cancer registries. Cumulative incidences were calculated using the SEER*Stat software package, v. 8.3.4 (Surveillance Research Program, National Cancer Institute, Bethesda, MD).

Results: Search of the SEER-18 registries identified 37 cases of pcGDTCL and 132 cases of SPTCL. Cumulative incidences pcGDTCL and SPTCL were 0.40 and 1.51 per 10 million (95% confidence interval (CI) 0.28-0.56 and 1.26-1.80), respectively. Patients with pcGDTCL were significantly older than those with SPTCL (median 63 years, interquartile range [IQR] 49.5-72 years vs. median 45 years, IQR 23-62; p<0.001). Patients with pcGDTCL were more likely to be male than those with SPTCL (p<0.013). There was no significant difference between the groups in race/ethnicity, stage at diagnosis, cause of death, location of tumors, or presence of B symptoms. The majority of patients with either pcGDTCL and SPTCL who died did so of their lymphoma (83% and 86% of cases, respectively). There was no significant difference in median time to death between the two groups (pcGDTCL 6 months, IQR 1.5-10.25; SPTCL 10.5 months, IQR 2-40; p>0.05).

Median follow-up for pcGDTCL was significantly shorter than that for SPTCL, at 13 months vs 23 months (p<0.0007). All deaths in the pcGDTCL population occurred within the first 24 months of diagnosis, yielding a 2-year and 5-year disease specific survival (DSS) of 73% and a 2-year and 5-year overall survival (OS) of 67% (Fig. 1A). This is compared to a 2-year DSS of 95%, 5- year DSS of 91%, 2-year OS of 90%, and 5-year OS of 86% for SPTCL.

Cox proportional hazards modeling revealed that pcGDTCL patients were at significantly higher risk of death than SPTCL patients (HR 5.00, 95% confidence interval [CI] 1.8-14.3, p=0.005) (Fig 1B). Increasing age (HR 1.31 per 10 years, 95% CI 1.0-1.7, p=0.04) and stage (HR 1.52, 95% CI 1.1-2.1, p=0.023) were also significant factors. Multivariate Cox proportional hazard models for pcGDTCL revealed decreased survival with stage IV disease as compared to stages I- III (HR 1.65, 95% CI 1.07-2.54, p=0.022) when controlling for age at diagnosis, stage, sex, and race/ethnicity. Modeling for SPTCL revealed increasing age to be associated with increased risk of death (HR 1.36 per 10 years, 95% CI 1.1-1.7, p=0.005) when controlling for stage, location of tumor, sex, and race.

Discussion: pcGDTCL and SPTCL are exceedingly rare cutaneous lymphomas. They require accurate diagnosis as pcGDTCL needs rapid recognition and treatment. These two lymphomas demonstrate significant differences in demographics and survival. Notably, the 5-year DSS for pcGDTCL here is markedly better than that found by Willemze et al. (2008). This deviation may

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reflect the small number of cases available for in our analysis, miscoding of patients, or insufficient follow-up to capture late deaths.

A likely primary complicating factor in this report is accurate diagnosis and classification, as correct diagnosis of pcGDTCL can be extremely challenging. Gamma and delta TCR subunits can be expressed on many other lymphomas including mycosis fungoides, although there is controversy around the significance of low levels of gamma/delta TCR expression in other lymphomas. When gamma/delta T-cells are present in inflammatory skin conditions, they tend to comprise a minority of the overall infiltrate with the exception of some rare T-cell dyscrasias. The overlap of these panniculitis-like lymphomas with lupus panniculitis, mycosis fungoides, and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) may create additional diagnostic ambiguity.

There is marked difference in age and gender distribution between the two diseases: pcGDTCL patients are more likely to be older and male than SPTCL patients. While these conclusions are limited by small sample size, they may offer some guidance in constructing a differential diagnosis and providing prognostic guidance.

Differentiating these two diseases is crucial because they have different treatments. There is no standard of care for SPTCL, and treatments for localized disease include radiation therapy or immunosuppressives, including systemic corticosteroids, methotrexate, bexarotene, and cyclosporine. Treatments for refractory disease or hemophagocytic lymphohistiocytosis include combination chemotherapy, denileukin difitox, and autologous stem cell transplant. Treatment for pcGDTCL is almost exclusively based on combination chemotherapy regimens, although allogeneic stem cell transplantation may be a promising therapeutic option.

Conclusion: pcGDTCL and SPTCL affect different patient populations and have distinct clinical courses. Patients with pcGDTCL were five times more likely to die than those with SPTCL. Improved understanding of the epidemiology of these diseases will lead to more accurate diagnosis and treatment.

A. Kaplan-Meier plot. Kaplan-Meier plot describing the survival distribution function for and 37 cases of primary cutaneous gamma/delta T-cell lymphoma (pcGDTCL) and 132 cases of subcutaneous panniculitis-like T-cell lymphoma (SPTCL). Log-Rank p=0.0045.

B. Multivariate survival analysis. Survival curves of 37 cases of pcGDTCL and 132 of SPTCL using a Cox proportional hazards model with diagnosis age, stage, site, sex, and race/ethnicity as covariates. The hazard ratio for pcGDTCL was 5.00 (95% confidence interval 1.8-14.3, p=0.005).

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E-01

Proinflammatory and immunosuppressive cytokines polarize the microenvironment in cutaneous T cell lymphoma and regulate PD-L1 expression E. Gulsen Gunes1, Steven T. Rosen1,2, Christiane Querfeld1,3 1Beckman Research Institute, 2Dept. of Hematology, and 3Division of Dermatology, City of Hope National Medical Center, Duarte, CA, USA

Introduction: Cutaneous T-cell lymphoma (CTCL) develops from clonally expanded CD4+ T cells in a background of chronic inflammation [1]. Macrophages are important regulators in the tumor microenvironment by presenting antigens to T cells and having key roles in tumor immune surveillance and tolerance hereby showing flexibility/plasticity in producing distinct cytokine and chemokine that can modify tumor cell migration and proliferation in the tumor microenvironment (TME). Tumor‐ associated macrophages (TAMs) exhibit a CD163+ M2 macrophage phenotype and represent the most abundant phenotype in skin lesions of MF. In addition, neoplastic T cells escape immune surveillance via immune checkpoint signaling such as PD1/PD-L1 axis [2]. It remains unknown how cytokines promote tumor-growth and polarize the TME. Methods and Results: To determine whether PD-L1 plays a role on TAMs in CTCL, we first examined PD-L1 expression on CD163+ M2 macrophage subsets in mycosis fungoides (MF) skin lesions and in blood of Sézary syndrome (SS) patients by multicolor immunohistochemistry staining (IHC) and flow cytometry (FACS), respectively. IHC demonstrated co-localization of PD-L1 with CD163+ cells, but not with CD3+ cells in MF lesions. Similarly, we observed elevated PD-L1 expression on CD16++/CD14+/HLA-DR+/ CD163+ M2 macrophages; CD16neg/CD14+/HLA-DR+/ CD163+ M2 macrophages and increased number of CD163+ M2 cells in blood specimens Figure 1. CD163+ M2 macrophages and PD-L1 expression is upregulated on CD163+ M2 in PBMCs of SS patients compared to HDs. of SS patients compared to healthy controls (Fig 1). These results indicate that M2 macrophages have a role in CTCL tumorigenesis and CD163+ M2 macrophages express high levels of PD-L1 in the presence of the tumor cells. To gain further mechanistic insight into regulation of PD-L1 we treated healthy donor (HD) peripheral blood mononuclear cells (PBMCs) with supernatant (SN) from two CTCL cell lines, MyLa and Hut78. Expression levels of PD-L1 were examined at mRNA level by qRT-PCR after 24h and cell surface levels on CD163+ M2 macrophages after 48h by FACS.

47

As shown in Fig 2, Hut78 and MyLa SNs induced expression levels of PD-L1 on CD163+ M2. Our initial experiments demonstrated that PD‐ L1 expression was regulated in a cytokine/STAT‐ 1 and 3‐ dependent manner on PBMCs from SS patients. Of note, CD14+ cells from HD PBMCs display differentiation to CD163+ M2 macrophages in conditioned culture PDL1 Figure 2. Representative flow histogram and graphs show a medium from Hut78 and MyLa. Analysis of significant increase of PD-L1 on CD163+ M2 macrophages in the cell from HDs treated with Hut78 and MyLa SNs. monocytes revealed a reduced number of CD14+ cells, while significant increase in CD163+ M2 macrophages was found. Of note, PD-L1 expression (measured by median fluorescence intensity [MFI]) was much higher on these cells

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0.0 0 T 6 0 g a N IL 1 F F T 6 0 g a IL N N L 1 F recombinant IL-6, IL-10, IFN-γ and I N I N T IL F N I T Figure 3. A) Dot graph shows a significant increase in PD- TNF-α for 48h. PD-L1 protein and L1 expression on CD163+ M2 with exogenously IFNγ and TNFα stimulation B) and an induction at mRNA expression mRNA expression levels were levels of PD-L1 with IFNγ and TNFα stimulation. significantly elevated on CD163+ M2 macrophages, particularly with IFN-γ and TNF-α (Fig. 3A-B). We observed that TNF-α significantly induced/increased pSTAT3 expression in CD163+ M2 compared to the other cytokines. HD-PBMCs were then cultured with conditioned SN from Hut78 or MyLa cells with/ without a pan-STAT inhibitor or anti-human IFN-γ, IL-10 or TNF-α antibodies. We found that blocking of TNF-α in conditioned culture media from Hut78 and MyLa cell lines has let to reduced PD-L1 expression on CD163+ M2 macrophages. Furthermore, blocking STAT activation with a pan-STAT inhibitor also reduced PD-L1 expression on CD163+ M2 macrophages. Conclusions: Our results highlight that the CTCL cytokine profile bias PD-L1 expression and polarizes towards CD163+ M2 macrophages to potentiate T cell exhaustion in the TME. TNF-α appears to be the most potent inducer of pSTAT3. Our results identify potential targets for combination with immune checkpoint therapy. References: 1. Gonzalez, B.R., et al., Tumor microenvironment in mycosis fungoides and Sezary syndrome. Curr Opin Oncol, 2016. 28(1): p. 88-96. 2. Querfeld, C., et al., Primary T Cells from Cutaneous T-cell Lymphoma Skin Explants Display an Exhausted Immune Checkpoint Profile. Cancer Immunol Res, 2018. 6(8): p. 900-909.

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E-02 Normal Fibroblasts promote Th1 cytokine expression in Myla cells by suppressing TWIST1 expression Mehdi, S.J.; Moerman-Herzog, A.; Wong, H.K. Dermatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States. INTRODUCTION & OBJECTIVES: The interaction of tumor cells with fibroblasts serves an important role intumor cell biology, and normal fibroblasts can exert suppressive functions against cancer initiating andmetastatic cells. The role of fibroblasts in mycosis fungoides (MF) tumorigenesis is largely unknown. The aimof this study is to decipher the effect of fibroblasts on MF cells, and identify pathways that mediate theseeffects. MATERIALS & METHODS: Fibroblasts were isolated from normal skin using collagenase. Myla cells werecultured either alone or with normal fibroblasts for 5 days, trypsinized and replated, followed by serial washingto collect floating Myla cells. Myla were subjected to RT- PCR for MF biomarkers (TWIST1 and TOX), Th1subtype (IFNɣ), and Th2 subtype (GATA3, IL-5). Western blot was performed to detect TWIST1 expression inMyla cells. RESULTS: Myla cells have high TWIST1 expression which is significantly suppressed after co- culturing withnormal fibroblasts (p < 0.0001), suggesting that fibroblasts promote Th1 cell transcriptional network in Mylacells. Since TWIST1 limits IFNɣ expression in Th1 cells, we found that IFNɣ expression was increased in Mylacells after co-culture (p < 0.0001), suggesting that IFNɣ increased expression is related to TWIST1 suppression.GATA3 deletion allows the appearance of IFNɣ producing cells, we found that normal fibroblasts modulatesTh2 cells gene expression in Myla cells by suppressing GATA3 expression (p < 0.0001). TOX expression wassuppressed in Myla cells after co-culture (p< 0.0001), suggesting that fibroblasts might regulate T cellsexhaustion during the disease progression. CONCLUSIONS: We conclude that normal fibroblasts promote Th1 cell transcriptional network via TWIST1-GATA3 axis. Fibroblasts are crucial components of the tumor microenvironment, and normal fibroblasts canpromote Th1 cytokine expression by attenuating Th2 processes. Increased expression of IFNɣ producingTh1 cells might enhance immune responses against the tumor cells.

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E-03

Anti-CD47 immunotherapy is mediated by cytotoxic CD107a+IFN-γ- NK cells and can be potentiated by interferon-α in cutaneous lymphoma

Oleg Kruglov,1 Lisa D.S. Johnson,2 Robert A. Uger,2 Mark Wong,2 Xuesong Wu,3 Sam T. Hwang,3 Oleg E. Akilov,1

1Cutaneous Lymphoma Program, Department of Dermatology, University of Pittsburgh, Pittsburgh, PA, USA; 2Trillium Therapeutics Inc., Mississauga, ON, Canada; 3Department of Dermatology, University of California Davis, USA

Introduction/Aim: CD47 has been identified as a “do-not-eat” signal which is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. Material and Methods: We utilized depletion of NK cells and mice genetically deficient in IFN- γ to elucidate the mechanism of anti-CD47 therapy in the murine MBL2 model of cutaneous T- cell lymphoma. Results: After intraepidermal inoculation of MBL2 cells, treatment with anti-CD47 led to significant reduction of the thickness of the tumors as early as four days after the first treatment, which was accompanied by an increase in the number of cytotoxic NK cells at the tumor site (Fig. 1). While depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti- CD47, IFN-γ was not required. An improved therapeutic effect was observed when anti-CD47 therapy was combined with IFN-α. An increased number of cytotoxic CD107a+IFN-γ- NK1.1 cells and intermediate CD62L+ NKG2a- NK1.1 cells was observed during anti-CD47+IFN-α therapy in comparison with either treatment as a monotherapy. Conclusions: CD47 blockade is an effective therapy for cutaneous lymphoma via engagement of cytotoxic NK cells rather than IFN-γ-producing NK cells, and this effect can be potentiated by IFN-α.

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Figure 1. CD47 blockade efficient in control of malignant lymphoma growth in a murine model of mycosis fungoides and its effect accompanied by accumulation of CD26L+ NKG2A- NK cells in tumor microenvironment. (a) CD47 expression on CD3+TOX+ MBL2 cells. (b) Cytospin preparation of representative samples 2 hours after phagocytosis in the presence of MIAP301. Red arrows identify MBL2 cells that are external to macrophages in the absence of MIAP301 but phagocytosed in the presence of MIAP301. (c) Phagocytosis of MBL2 cells by BMDM in the presence of anti-CD47 antibody (MIAP301) or IgG control (2A3). *, p<0.05. (d) Design of the anti-CD47 experiments. I.P., intraperitoneal. (e) Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n=5 mice for each group). ***, p<0.001. (f) Percentage of TOX+ tumor cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation. The percentage of caspase-3+ cells outlined in black columns (n=5 mice for each group). Tx, treatment, *, p<0.05, ***, p<0.001. (g) Percentage of F4/80+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). ns, non-significant. (h) Percentage of MHC class II+ cells among F4/80+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). **, p<0.01. (i) Percentage of TNF-α+ cells among F4/80+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). *, p<0.05; **, p<0.01. (j) Percentage of NK1.1+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). **, p<0.01. (k) Percentage of IFN-γ+ cells among NK1.1+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). ns, non-significant. (l) Percentage of CD62L+ NKG2A- cells among NK1.1.+ cells in the inflammatory infiltrate of the auricular skin. Day 24 after implantation (n=5 mice for each group). *, p<0.05; **, p<0.01.

51

F-01

Presentation code: F-01

Abstract title: Mac-1/Mac-2A cells represent a potential model to study IL-13 signaling in SATB1 positive cutaneous ALCL

Marshall Kadin MD 1, 2, John Morgan PhD1, Nick Kouttab PhD1, Haiying Xu1,Yang Wang MD 3

1- Department of Dermatology, Boston University and Roger Williams Medical Center, Providence RI, USA 2- Department of Pathology and Laboratory Medicine, Rhode Island Hospital, USA 3- Department of Dermatology and Venereology, First Hospital Peking University, Beijing China

Introduction: Our publication (Sun et al, JID, 2018,138, 1795-1804) demonstrated that pcALCL could be divided into 2 main groups; a SATB1(-) group and a SATB1(+) group that had Th17 polarization, prominent epidermal and granulocytic hyperplasia, but also unexplained high expression of genes comprising the Th2-IL13 signaling pathway (IL13, IL13R1, IL4R, JAK1, TYK2, pSTAT6). Mac2A and Mac1 cells appear to be derived from this SATB1 positive group.

Methods: Clonally related cell lines Mac1 and Mac2A derived from progression of lymphomatoid papulosis (LyP) to ALCL were studied for expression of IL13, IL4 and their receptors by flow cytometry. Cytokines secreted by Mac1 and Mac2A were measured by ELISA. Clinical samples of pcALCL and LyP were studied by immunohistochemistry for expression of SATB1, IL13, IL4 and pSTAT6. qPCR was performed for IL13, IL13R1, IL13R2, IL4R, and GATA3. Effects of silencing SATB1 on expression of GATA3 and IL13 in Mac1 cells were investigated. The concentration dependent effects of a pSTAT6 inhibitor (AS1517499) on viability of Mac1, Mac2A and control cell lines were evaluated.

Results: The primary skin tumor from which Mac2A was derived had epidermal hyperplasia, was infiltrated by neutrophils and eosinophils, and contained anaplastic tumor cells with a Th2 phenotype including GATA3, IL4 and IL13 but also a Th17 phenotype with RORt, IL17F and IL22 (see top figure). The primary tumor cells also expressed SATB1, pSTAT3 and pSTAT6. Mac2A cells secreted IL13, IL17A, IL17F and IL22. Mac1 cells derived from leukemic Sezary- like cells secreted IL17A and IL17F but low level of IL13. Unexpectedly then, over-expression of SATB1 up-regulated IL13 in Mac1 cells without altering GATA3 expression, suggesting that IL13 is regulated by SATB1 via an alternative mechanism.

Both Mac1 and Mac2A had high expression of IL4R and lower expression of IL13R1 and IL13R2. Mac2A cells expressed higher levels of pSTAT6 than Mac1. A specific inhibitor of pSTAT6 reduced cell viability in a concentration dependent manner, similar to Sezary cells as described by Geskin L et al (Blood, 2015, 125: 2798-2805).

Tumor cells in clinical samples of pcALCL and LyP often expressed pSTAT3, pSTAT6 and IL13 with low or absent expression of GATA3, thus resembling Mac1 cells. It is presently unclear how IL13 is up-regulated in SATB1+ pcALCL. A possibility under investigation is Thymic Stromal Lymphopoietin (TSLP) which acts through Th2 cytokine production to stimulate CTCL lines and primary CTCL cells (Takahashi N et al, Cancer Res; 2016, 76: 6241–52. TSLP is derived from keratinocytes which are hyperplastic in SATB1+ CD30+ cutaneous lymphoproliferative disorders.

Conclusions:

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1. These results suggest that Mac 2A cells and some CD30+ CLPD are derived from a novel subset of CD4 Th2 memory/effector cells that produce inflammatory Th17 cytokines and promote the exacerbation of chronic allergic asthma (Wang, YH et al, J Exp Med 2010, 207:2479). 2. Mac1 cells demonstrate a predominant Th17 phenotype which can be polarized towards Th2 by over-expression of SATB1 as described for a subset of pcALCL. 3. Suppression of SATB1+ cell proliferation by a specific pSTAT6 inhibitor suggests possible future applications for therapy of pcALCL

Original skin tumor cells co-produce Th2 and Th17 cytokines

H&E GATA3 IL-13 IL-4

RORgt IL-17F IL-22

Concentration dependent effect of pSTAT6 inhibitor (AS1517499) on viability of Mac-1, Mac-2A and myeloid cell line K562

53

F-02

Expression heterogeneity and clinical significance of SATB1 in Mycosis fungoides

Introduction: SATB1 is an important T-cell specific nuclear matrix protein. Recent studies from our group and others have demonstrated involvement of SATB1 in the pathogenesis of cutaneous T-cell lymphoma. However, the correlation between SATB1 expression and the clinicopathological features and its utility in clinical settings in MF have not been determined. The mechanism underlying the discrepancy in SATB1 expression and its stage-dependent decrease remains largely unknown.

Methods: The mRNA and protein expression levels of SATB1 and their clinicopathological correlation were evaluated in a cohort of 170 MF patients. Lentivirus mediated gene silencing was performed in MJ cells to explore the key downstream genes regulated by SATB1.

Results: Heterogeneity of SATB1 expression existed in all stages of mycosis fungoides（Figure a）, and decrease of SATB1 in tumor stage was observed compared to patch/plaque stage MF (Figure b). SATB1 high expression cases showed higher eosinophil infiltrates（Figure c）and favorable prognosis（Figure d）. SATB1 and CD30 double positivity favored a diagnosis of cutaneous CD30+ lymphoproliferative disorder, which is a major differential diagnosis for MF large-cell transformation（Figure e）. SATB1 silencing in MJ cells showed that SATB1 upregulated genes enriched in JAK-STAT and cytokine-cytokine receptor interaction pathways （Figure f）, including the eosinophil recruiting cytokine IL13（Figure g-h）. IL13 expression level was correlated with eosinophil infiltration in MF tumor lesions (Figure i). SATB1 downregulated genes enriched in cell cycle pathway, including E2F1 and MCM3 (Figure f, j-k), which may account for the favorable prognosis for SATB1 high expression cases. Moreover, SATB1 positivity was inversely correlated with PD-1 expression (Figure l-m), and was positively correlated with TLRs expression (Figure n), indicating innate immunity activation may play pivotal role in the pathogenesis of SATB1 high expressing cases.

54

Conclusions: Collectively, the divergence of SATB1 expression reflected the intrinsic heterogeneity in MF. MF cases with high SATB1 expression may have distinct etiology, clinic- pathological phenotype, and prognosis. SATB1 may serve as a reliable marker for prognosis and differential diagnosis, as well as a potential therapeutic target for intervention.

55

F-03

Protein and mRNA expression levels of IL-17A, IL-17F and IL-22 in patients with mycosis fungoides.

Despoina Papathemeli, Aikaterini Patsatsi, Triantafyllia Koletsa, Olga Pikou, Elizabeth Lazaridou, Elisavet Georgiou

Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki

Introduction & Objectives: It is well known that IL-17A, IL-17F and IL-22 play a crucial role in psoriasis and other inflammatory diseases. The aim of this study is to elucidate the possible role of IL-17A, IL-17F and IL-22 in mycosis fungoides.

Patients & Methods: The peripheral blood protein and mRNA levels of IL-17A, IL-17F and IL- 22 were examined in 50 MF patients and 50 healthy controls. The ♂:♀ ratio in our sample was 2,1. Among patients, 39 (78%) had early-stage disease (IA-IIA) and 11 (22%) had advanced stage disease (IIB-IV). Also 18 patients (36%) were newly diagnosed and had received no previous treatment for MF and 32 (64%) had a previous history of MF and were under treatment, having active disease. The protein level was evaluated in the serum using ELISA and the mRNA levels were evaluated in peripheral blood by quantitative real-time PCR. Mean expression levels in patients were compared to controls by unpaired Student’s t-test.

Patient characteristics Value Sample n 50 Clinical features Age (median, range) 60,62 (27-86) Male 34 (68%) Female 16 (32%) Newly diagnosed 18 (36%) Patients with previous history of MF 32 (64%) Died 2 (4%) Erythrodermic 5 (10%) Patch stage 16 (32%) Plaque stage 23 (46%) Tumor stage 6 (12%) Pathological characteristics Large Cell Transformation 5 (10%) CD8+ 1 (2%) Folliculotropic 3 (6%) Poikilodermic 3 (6%) Stage IA 26 (52%) IB 11 (22%) IIA 2 (4%) IIB 5 (10%) IIIA 5 (10%) IV 1 (2%) Therapy None 22 (44%) Skin-directed therapy 3 (6%) Systemic therapy 17 (34%) Combination of skin directed & systemic therapy 8 (16%)

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Results: Protein levels did not differ significantly among patients and controls for IL-17A, IL- 17F and IL-22 (p:0,212, 0,131 and 0,944, respectively). Likewise, mRNA relative quantities were not found significantly different between patients and controls (p: 0,146, 0,910 and 0,201 respectively). Also protein expression levels did not differ significantly among early and advanced stage patients. Interestingly, significantly higher IL-22 mRNA expression levels were detected in early stage patients (p=0,026), a finding which must be further confirmed. In general, protein and mRNA expression levels of the examined interleukins in peripheral blood were extremely low, with few exceptions.

IL-17A IL-17F IL-22 Protein mRNA Protein mRNA Protein mRNA Patients vs Control 0,212 0,146 0,131 0,910 0,944 0,201 Individuals Early vs Advanced 0,534 0,772 0,529 0,307 0,798 0,026 Stage Patients

p=0,026

Conclusions: No significant differences in circulating IL-17A, IL-17F and IL-22 were found. Increased IL-22 mRNA expression levels in early stage MF remains to be interpreted. The interplay among cytokines in mycosis fungoides and the similarities or differences in immunological pathways among early MF and psoriasis or eczema may be important for therapeutic or prognostic purposes.

57

F-04

Induction of anti-tumor effect by CD8+ T-cells via CADM1/CRTAM interaction in patients with adult T-cell leukemia lymphoma

Kazuki Tatsuno, Takatoshi Shimauchi*, Yoshiki Tokura Department of Dermatology, Hamamatsu University School of Medicine

Introduction : Adult T cell leukemia/lymphoma (ATL) cells express CADM1 (Cell Adhesion Molecule 1), and its expression is increased upon disease progression1. CRTAM (Class I Restricted T-cell Associated Molecule), a CADM1 ligand, is expressed on activated CD8+ T cells, and CRTAM-CADM1 interaction leads to increased IFN- production and cell lytic function against CADM1 expressing target cells. However, CADM1-expressing ATL cells seem to cunningly evade the host immunity against itself. The aim of study is to explore the association of CADM1 and CRTAM interaction in ATL patients. Methods: Activated PBMCs from ATL patients (n=6) were evaluated for CRTAM expression on CD8+ T cells with or without autologous CD4+ ATL cells by flow cytometry. Isolated CD8+ T cells were co-cultured with autologous CD4+ ATL cells in transmembrane co-culture system and analyzed again (n=4). Isolated and pre-activated CD8+ CRTAM+ T-cells were co-cultured with autologous CD4+ ATL cells with or without anti-CADM1 blocking antibody, and then, casepase- 3 activity on tumor cells were analyzed by flow cytometry (n=5). Results: We found that CRTAM expression on CD8+ T cells in ATL patients were significantly suppressed compared to normal subjects. Interestingly, depletion of ATL cells lead to the recovery of CRTAM expression on CD8+ T cells. Co-culture of ATL cells and CD8+ T cells using transmembrane culture device did not suppress the CRTAM expression. Furthermore, casepase- 3 expression on CD4+ ATL cells was induced by co-culture with autologous CRTAM+ CD8+ T- cells, which was efficiently hampered by pretreatment with anti-CADM1 blocking antibody. Conclusions: These observations underscore the importance of CRTAM in host defense against ATL cells, and it’s down-modulation by direct cell-cell contact with tumor cells. Thus, the control of CRTAM expression on CD8+ T cells suggests high therapeutic potential for the treatment of ATL (Figure 1).

References: 1) Kobayashi S, Watanabe E, Ishigaki T, et al. Advanced human T-cell leukemia virus type 1 carriers and early-stage indolent adult T-cell leukemia lymphoma are indistinguishable based on CADM1 positivity in flow cytometry. Cancer Sci. 106:598-603, 2015.

58

1. Down regulate CRTAM ATL tumor cells

X CADM-1 CRTAM

CD8+ cytotoxic T-cells 2. Can not kill tumor efficiently

Figure 1. CRTAM expression on CD8+ T cells is suppressed directly by HTLV-1 infected tumor cells in ATL patients, and at least by part, contributes to the tumor’s escape from host immunity.

59

G-01

Role of TOX1 and STAT3 in the pathogenesis of cutaneous T-cell lymphoma Angelina M. Seffens1,2, Sergei B. Koralov2, and Larisa J. Geskin3 1Columbia University Vagelos College of Physicians and Surgeons, New York, NY 2Department of Pathology, New York University School of Medicine, New York, NY 3Department of Dermatology, Columbia University Irving Medical Center, New York, NY

Introduction Sézary Syndrome (SS) and Mycosis Fungoides (MF) are the most common clinical variants of cutaneous T-cell lymphoma (CTCL), a group of lymphomas characterized by the accumulation of malignant T cells in the skin. Thymocyte selection associated high mobility group box 1 (TOX1), a transcription factor that is required to establish the CD4+ lineage, is overexpressed in malignant cells found in the skin and blood of patients with CTCL. Knockdown of TOX1 results in decreased malignant cell viability, while treatment with HDAC inhibitors results in normalization of TOX1 expression in patient-derived cell lines1. Another gene which is consistently overexpressed in CTCL is signal transducers and activators of transcription 3 (STAT3), a transcription factor critical for the differentiation of Th17 and follicular helper T cells. Treatment of CTCL cell lines with a STAT3 inhibitor leads to increased apoptosis, demonstrating that this pathway is also important for malignant cell survival. The Koralov lab has developed a mouse model which constitutively expresses a hyperactive STAT3 allele, STAT3C, selectively in T lymphocytes that recapitulates several key features of MF2. We hypothesize that both TOX1 and STAT3 play an important role in CTCL pathogenesis, and that their upregulation contributes to the ability of malignant cells to survive, proliferate, migrate, and invade tissues. We expect that overexpression of both of these genes, which we believe contribute to T-cell lymphomagenesis, will result in a phenotype that more closely mimics aggressive forms of CTCL.

Methods We have taken advantage of a conditional gene targeting approach by introducing Tox1 cDNA downstream of a floxed stop cassette into the ubiquitously expressed Rosa26 locus of C57Bl/6J embryonic stem (ES) cells. We have used PCR and Southern blotting to select positive clones. Furthermore, we have treated targeted ES clones with a transducible Cre protein (TAT-Cre) to demonstrate appropriate deletion of the floxed stop cassette and subsequent expression of Tox1 cDNA. The TAT domain of the fusion protein promotes translocation of the Cre to the nucleus, enabling the excision of the loxP-flanked stop cassette. We have generated R26Tox1stopfl mice using tetraploid complementation to generate 100% ES cell derived animals and genotyped these animals to ensure that they express TOX1 cDNA. To validate that the floxed stop allele within the targeted R26TOX1stopfl locus is functional, we exposed CD4+ T cells isolated from the blood of two mice carrying the R26TOX1stopfl allele to the TAT-Cre fusion protein. R26TOX1stopfl mice have been crossed to CD4Cre and STAT3Cstopfl CD4Cre strains, thus enabling us to examine synergy between TOX1 overexpression and hyperactive JAK/STAT signaling in CTCL. We have taken peripheral blood from R26Tox1stopfl CD4 Cre mice and R26STAT3Cstopfl Tox1stopfl CD4Cre mice and checked for Tox1 cDNA expression.

60

Results

300 TOX1 Negative H O Control 2 200

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A B . . Tox1 Gene Targeting Strategy and PCR Screen of clones A. Schematic map of the wild-type Rosa26 locus and he targeting vector used to generate the conditional R26TOX1stopfl allele. Filled triangles, loxP sites; SA, splice acceptor; CAG, CAG promoter; Neo, neomycin resistance gene; IRES, internal ribosome entry site that will drive concomitant expression of the truncated human CD2 (hCD2) to be used as a marker of cells expressing transgenic TOX1 cDNA. B. Representative PCR for Tox1 cDNA on DNA from ES clone

Tox1 Expression

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Conclusions We have developed a relevant small animal model to study CTCL pathogenesis, including the role of TOX1 in T-cell lymphomagenesis, early hematopoiesis, and other biological processes. We have validated the function of the conditional allele in vitro and in vivo. We will continue detailed FACS and immunohistochemical analyses of these mice as they age to study the function of TOX1 in T cell lymphomagenesis and the synergy between TOX1 and STAT3 overexpression.

References 1. Dulmage BO, A. O., Vu JR, Falo LD, and Geskin LJ. Dysregulation of the Tox-Runx3 pathway in cutaneous t-cell lymphoma. Oncotarget (2015). 2. Fanok, M. H., Sun, A., Fogli, L. K., Narendran, V., Eckstein, M., Kannan, K., Dolgalev, I., Lazaris, C., Heguy, A., Laird, M. E., Sundrud, M. S., Liu, C., Kutok, J., Lacruz, R. S., Latkowski, J. A., Aifantis, I., Odum, N., Hymes, K. B., Goel, S. & Koralov, S. B. Role of dysregulated cytokine signaling and bacterial triggers in the pathogenesis of cutaneous t-cell lymphoma. J Invest Dermatol 138, 1116-1125 (2018).

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G-03

Standardized flow cytometry (EuroFlow) demonstrates heterogeneous T-cell origin of Sézary lymphoma cells

Safa Najidh, A.J. van der Sluijs-Gelling, Willem H. Zoutman, Cornelis P. Tensen, Thorbald van Hall, J. Almeida, J.J.M. van Dongen, Maarten H. Vermeer

Introduction Sézary syndrome (SS) is generally considered as a T-helper central memory (TCM) cell of T- helper (Th)2-subset. However, Sézary cells (SCs) remain incompletely characterized and sensitive monitoring tools are lacking for their identification. Therefore, our goal was to identify the immunophenotypic profiles of SS and to investigate expression of characteristic T-helper subset markers by SCs to increase our general understanding of SS.

Methods We applied fully standardized flow cytometric protocols as developed by EuroFlow Consortium on freshly isolated SCs from 20 SS patients and 7 follow-up samples using immunophenotypic markers included in the Lymphoid Screening Tube (LST) and T-cell Lymphoproliferative Disease (T-CLPD) antibody panels. Moreover, T-cell origin of SCs was investigated using a recently developed EuroFlow CD4+ T-cell characterization Tube including a variety of chemokine receptor, activation and maturation markers. Results Dim expression of CD3 and lack of CD26 surface marker were the most commonly observed immunophenotypic aberrancies, followed by CD7-/dim and CD2-/dim. Atypical cells were then re-defined in the CD4+ T-cell Tube based on their patient-specific phenotypic profile in the LST and T-CLPD panels. Markers CD45RA, CD27, and CD62L then allowed for different maturation stages to be defined. Subsequently, functionally distinct T-helper subsets were discriminated using chemokine receptors CXCR3, CCR4, CCR6, and CCR10. SCs were mostly of TCM origin and shared overexpression of chemokine receptor CCR4 which is preferentially expressed on Th2 differentiated CD4+ T cells. Nevertheless, some samples showed surface phenotypes resembling Th17 (CCR4+CCR6+), Th22 (CCR4+CCR6+CCR10+) or other T-helper functional subsets. Interestingly, we observed overlapping but sometimes diverse phenotypic profiles within one patient, showing intra-patient dynamics in SC differentiation. Furthermore, SCs from follow-up samples showed a phenotypic shift over time.

Conclusions Here, we demonstrated distinct phenotypic profiles in SS reflecting SC heterogeneity which is suggestive of a more inter- and intratumor heterogeneous nature of the disease than previously appreciated.

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Two 2D-plots showing the expression of 4 Sézary-specific markers: CD28, CD26, CD2, and CD7. The green lines represent the 1.0 and 2.0 SD contour lines of normal CD4+ T cells from 10 healthy donors and their corresponding population medians (green). CD4+ T cells from 20 SS patients are shown in red illustrating the loss of CD26, CD2, and CD7 in most cases.

TE TE TE

PM PM PM

TM TM N TM N N

CM CM CM 2nd principal component principal 2nd 1st principal component Three automatic population separator (APS) plots comparing the maturation stages of normally distributed CD4+ T cell populations (depicted as 2.0 SD contour lines) from 10 healthy donors with their corresponding medians for each maturation stage. The maturation pathway for normal CD4+ T cells starting from naïve and maturating into terminally effector cells. Right: all aberrant CD4+ T cells from 20 Sézary syndrome (SS) patients (red dots) with their representative population medians (red) for each patient. Abbreviations: N, naïve; CM, central memory; TM, transitional memory; PM, peripheral memory, TE; terminally effector cells.

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G-04

Mutational signature analysis reveals a key role for UV radiation in the accumulation of mutations in cutaneous T-cell lymphoma.

Christine L Jones, Andrea Degasperi, Vieri Grandi, Tracey J Mitchell, Serena Nik-Zainal,

Sean J Whittaker

Introduction & Objectives: T-cell lymphomas, including Mycosis Fungoides and Sezary

Syndrome, develop following transformation of tissue resident T-cells. Although a diverse set of driver gene mutations contribute to constitutive TCR signalling and evasion of apoptosis, little is understood about the underlying mechanisms driving these mutations.

Materials & Methods: We performed a meta-analysis of mutational catalogues derived from whole exome sequencing data from 403 patients with eight subtypes of T-cell lymphoma (Table

1) to identify mutational signatures and recurrent gene mutations associated with specific causal peaks within these signatures.

AITL ATLL EATL HSTL MF NKTCL PTCL SS Choi 2015 ------33 33 da Silva Almeida 2015 - - - - 8 - - 26 34 Jiang 2015 - - - - - 25 - - 25 Kataoka 2015 - 81 ------81 McKinney 2017 - - - 64 - - - - 64 Moffitt 2017 - - 58 - - - - - 58 Palomero 2014 3 - 1 - - 2 6 - 12 Prasad 2016 ------12 12 Roberti 2016 - - 15 - - - - - 15 Sakata-Yanagimoto 2014 3 - - - - - 3 - 6 Ungewickell 2015 - - - - 6 - - 5 11 Wang 2015 ------37 37 Woollard 2016 ------10 10 Yoo 2014 5 ------5 11 81 74 64 14 27 9 123

Table 1: Details of the studies included and the number of samples with each disease subtype.

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Results: Signature 1, indicative of age-related deamination, was prevalent across all T-cell lymphoma subtypes, reflecting derivation of these malignancies from memory T- cell subsets.

Signature 7, implicating UV exposure as a potential initiating factor was uniquely identified in cutaneous T-cell lymphoma, contributing 52% of the mutational burden in Mycosis Fungoides and 23% in Sezary Syndrome. Importantly whilst the Mycosis Fungoides samples were obtained from skin biopsies, in Sezary Syndrome the UV signature was observed in tumour enriched T- cells isolated from blood.

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1 and Signature 7 with the combined mutational catalogues observed in Sezary Syndrome and

Mycosis Fungoides.

Conclusions: Our analysis identifies UV exposure as a key contributor to the mutational burden in cutaneous T-cell lymphoma. The identification of this signature in tumour enriched T-cells isolated from blood indicates prolonged exposure of these T-cells to UV during re-circulation through or residence in the skin.

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G-05

TITLE Epidermal fatty acid-binding protein is not expressed by tumor cells in advanced mycosis fungoides * Naomi Takahashi-Shishido,1 Soshi Morimura,1 Hiraku Suga,2 Tomonori Oka,2 Hiroaki Kamijo,2 Tomomitsu Miyagaki,2 Shinichi Sato,2 Makoto Sugaya1 1Department of Dermatology, International University of Health and Welfare, Chiba, Japan. 2Department of Dermatology, the University of Tokyo Graduate School of Medicine, Tokyo, Japan

Introduction: Epidermal fatty acid-binding protein (E-FABP) is predominantly expressed by keratinocytes, contributing to their differentiation. It is also expressed by immune cells like macrophages. The importance of E-FABP in the maintenance of skin-resident memory CD8+ T cells was recently reported. Mycosis fungoides (MF) is regarded as malignancy of skin-resident T cells and it is important to know whether or not tumor cells in MF express E-FABP. In this study, we investigated E-FABP expression in psoriasis vulgaris (PV), atopic dermatitis (AD), MF, and Sézary syndrome (SS).

Methods: Messenger RNA (mRNA) levels of E-FABP were examined by quantitative RT-PCR. Skin samples were collected from 23 patients with MF/SS, five patients with PVF, 12 patients with AD and eight healthy control subjects. We also performed immunohistochemical staining using skin samples from 28 patients with MF/SS, 10 patients with PV, five patients with AD and 11 healthy subjects.

Results: E-FABP mRNA levels were increased in lesional skin of PV, AD, and MF/SS, compared to those of normal skin (Fig.1-2). While E-FABP mRNA levels in patch, plaque, and erythrodermic MF/SS were significantly higher than normal skin, those in MF tumor lesions were not elevated (Fig.3). Immunohistochemical staining showed that E-FABP was strongly positive in the suprabasal epidermal layers of PV skin. Epidermis of AD and MF/SS skin was slightly positive. Dermal cells such as macrophages and endothelial cells also expressed E-FABP in these skin diseases. In MF/SS, some lymphocytes infiltrating into the epidermis were positive, suggesting that a part of tumor cells expressed this protein. In tumor MF lesions, however, most dermal atypical lymphocytes were negative for E-FABP (Fig.4-5).

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Fig.1 Fig.2 Fig.3

Fig.4 Fig.5

Conclusions: E-FABP is expressed by some tumor cells and surrounding inflammatory cells in early MF and SS. In advanced MF, tumor cells do not express E-FABP, suggesting that this protein cannot be a therapeutic target.

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Patch lesions of mycosis fungoides patients have a similar skin microbiome profile compared to non-lesional and healthy skin Authors: U. Wehkamp1, B.M. Hermes2,3,4, M. Jost1, J.F. Baines2,3, J. Harder1

1Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 2Evolutionary Genomics, Max Planck Institute for Evolutionary Biology, Plön, Germany 3Institute for Experimental Medicine, Kiel University, Kiel, Germany 4 Lübeck Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany

Staphylococci are known to play a role in cutaneous T-cell lymphoma. Previously, it has been shown that staphylococcal enterotoxin-producing bacteria may promote activation of the oncogenic pathway via STAT3 in the malignant T-cell population. In addition, many patients in advanced disease stages decease due to septic complications often mediated by S. aureus. The here presented study aimed to characterize the microbiome profile in patch lesions of mycosis fungoides (MF) patients. The skin microbiome was investigated compared to non-lesional and healthy skin by 16S amplicon sequencing. Skin washing solutions were retrieved from MF patch lesions and non-lesional skin of the same patient at 5 cm distance to the lesion in the same area. Age-, localization- and sex-matched healthy individuals were used as controls. 8 MF patients were analyzed, with 7 of 8 in early disease stage (IA, IB). The relatively most abundant genera across the samples were Staphylococcus, Bacteroidales spec., and Erysipeloptrichaceae spec. No significant differences between the patch lesions, non- lesional MF and healthy control skin were detected. Interestingly, lesional and non-lesional skin of MF patients displayed a consistent intraindividual microbiome profile. Further analysis including plaque and tumor lesions of patients are necessary to gain insight whether a shift in the microbiome might be present in more infiltrated skin lesions or more advanced disease stage, hereby potentially promoting or triggering disease progression.

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H-01

Evaluation of blood Sézary markers by means of correlation with apoptosis resistance and clonality

Sven Schneider1, Chalid Assaf2, Christina Fassnacht3, Emmanuella Guenova3, Max Schlaak4, Rudolf Stadler5, René Stranzenbach5, Ulrike Wehkamp6, Marion Wobser7, Jana Braun8, Sergij Goerdt8, Jan P. Nicolay8,9# 1Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Mannheim, Ruprecht-Karls-University of Heidelberg, Mannheim, Germany, 2Department of Dermatology, HELIOS Klinikum Krefeld, Germany, 3Department of Dermatology, University Hospital Zurich, Switzerland, 4Department of Dermatology, University Hospital Munich, Germany, 5Deparment of Dermatology, Johannes-Wesling-Klinikum Minden and University of Bochum, Germany, 6Department of Dermatology, University Hospital Kiel, Germany, 7Department of Dermatology, University Hospital Würzburg, Germany, 8Department of Dermatology, Venereology and Allergy, University Medical Center Mannheim, Ruprecht-Karls-University of Heidelberg, Mannheim, Germany, 9Department of Immunogenetics, German Cancer Research Center, Heidelberg, Germany,

Introduction and objectives: To date, the diagnosis of Sézary syndrome (SS) is often impaired by the distinct lack of unique diagnostic blood markers that clearly allow the identification of the malignant T cell population in the blood. Several markers are known and characterized, such as CD7 and CD26 loss as well as gain of PD-1 and CD158k. Nevertheless, it is yet to be clarified which of these markers characterize the malignant population best and how these markers correlate with T cell receptor (TCR) clonality, the defining feature of the malignant Sézary cells. In addition, the correlation of these markers with the distinct apoptosis resistance described for Sézary cells is still not sufficiently clarified.

Methods: We isolated CD4+ T cells from the peripheral blood of Sézary patients. As controls, we also isolated CD4+ T cells from patients with mycosis fungoides without blood involvement and with psoriasis as a non-malignant inflammatory disease. These T cells were separated into different populations according to their positivity for CD7, CD26, PD-1 and CD158k by flow-cytometry. Cell death was measured in these populations following different conditions as well as apoptotic and therapeutic stimuli. In addition, we determined TCR clonality by flow cytometry or TCR sequencing to further correlate marker expression within the populations with TCR clonality.

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Results: We found significant intra-individual differences in cell death sensitivity and correlation with clonality between the different marker-positive T cell populations. For example, our data confirm the preferred clonal expansion of specific TCR chains in different patients. In addition, apoptosis sensitivity significantly correlates with CD7 and CD26 loss. The exact and final results will be presented at the WCCL 2020.

35

30 CD7-

25 CD7+ 20 15

10 Cell death [%] Cell death 5 [%] Cell death 0 0 h 48 h

30 25 CD26- CD26+ 20 15 10 5 0 0 h 48 h

Figure 1.: Spontaneous cell death in CD4+ T cells of patients with Sézary syndrome, after 0h and 48h without stimulation. Populations are differentiated by their CD7 expression (left panel) or CD26 expression (right panel) showing that the negative population for both markers shows relative cell death resistance after 0h, an effect that disappears after 48h in ex vivo culture.

Conclusions: The established Sézary cell markers vary in specificity and sensitivity and can only be used in combination. Correlation of surface markers with clonality and apoptosis increases the sensitivity of flow cytometry in SS. This underlines the need to establish novel unique markers and highly standardized panels. In addition, we can show that the putative Sézary populations show the postulated, yet not sufficiently proven cell death resistance.

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H-02

The A allele of rs7096317 is permissive for CD39 expression in CTCL and is a negative prognostic factor in older patients.

Christine L Jones, Alice Devaney, Vieri Grandi, Samira Samuel, Sean J Whittaker, Tracey

J Mitchell

Introduction & Objectives: CD39 overexpression has been observed in a proportion of patients with cutaneous T-cell lympyhoma. GWAS analysis of immune traits has shown that expression of CD39 is strongly associated with the genotype of the rs7096317 SNP in healthy T-cells. As the rate-limiting enzyme in the generation of immunosuppressive adenosine, overexpression of CD39 could be influential in in the tumour microenvironment in CTCL.

Materials & Methods: We performed flow cytometry and genotyping on CD4+ T-cells from 46

CTCL patients to examine the relationship between cell surface expression of CD39 and genotype at the rs7096317 SNP in CTCL. Subsequently we performed genotyping on a cohort of 543 CTCL patients to investigate whether rs7096317 genotype had any influence on survival.

Results: A substantially increased proportion of CD39+ cells was observed within the CD4+ T- cell population of CTCL patients with the A/A or A/G genotype at rs7096317 but not in those with the G/G genotype (Figure 1). Within the 543 patient cohort the allele frequency for A was

0.53, which is in agreement with the allele frequency reported in dbSNP. Overall survival was reduced in patients harbouring an A allele and analyses of clinically relevant subsets revealed that an A allele confers significantly worse survival in patients who present aged 60 or above. This difference was independent of gender, disease, or staging at diagnosis.

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% 25

0 A/A A/G G/G rs7096317 Genotype

Figure 1: Percentage CD39+ cells within the CD4+ T-cell population in patients with the A/A,

A/G and G/G genotype at the rs7096317 SNP.

Conclusions: We confirm that CD39 overexpression is frequent in CTCL patients and is genetically regulated. The genotype permissive for CD39 expression is a negative prognostic factor in patients who present over the age of 60 suggesting that CD39 expression is playing a pathogenic role. In addition to the prognostic potential, this data could be of value in the targeting of anti-CD39 antibody therapy to the subgroup of patients likely to see the greatest benefit.

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I-01

Identification of CD39 as a potential therapeutic target in Sezary syndrome

Adèle de Masson1,2,3, Maxime Battistella1,2,4, Gabrielle Sonigo1,2,3, Baptiste Janela5, Nicolas Thonnart1,2, Philippe Musette1,2, Florent Ginhoux5, Armand Bensussan1,2, Martine Bagot1,2,3, and Anne Marie-Cardine1,2 1INSERM U976, Team 1, Onco-Dermatology and Therapies, Paris, France 2Université de Paris, Paris, France 3Saint Louis Hospital, Department of Dermatology, Paris, France 4Saint Louis Hospital, Department of Anatomo-Pathology, Paris, France 5Agency for Science, Technology and Research (A*STAR) and Skin Research Institute of Singapore (SRIS), Singapore 1. Introduction and objectives. Sezary syndrome (SS) is a cutaneous T-cell lymphoma characterized by the presence of malignant CD4+ T-cells that accumulate mainly in the skin and peripheral blood. Flow cytometry is now the method of choice for evaluating blood tumor burden in SS. Some years ago, we identified KIR3DL2 as a unique and specific positive marker for Sezary cells, expressed in 90% of SS patients. CD39 is an ectonucleotidase involved in ATP/ADP hydrolysis, leading to the generation of adenosine. Its expression by tumor cells in cancer patients was recently correlated to their ability to inhibit T-cell proliferation and generation of cytotoxic effector CD8+ T-cells in an adenosine-dependent manner. We aimed to investigate CD39 expression in SS. 2. Material and methods. CyTOF phenotyping was conducted on peripheral blood mononuclear cells from SS patients (n=2) and healthy donors (HDs) (n=5). Flow cytometry was performed on blood from HDs (n=30) and SS patients (n=30). CD39 expression was studied by immunohistochemistry in lesional skin of 36 SS patients. 3. Results. Most CD4+ T cells of SS patients segregated in a single cluster by CyTOF, corresponding to the malignant T-cell clone (as confirmed by its high expression levels of KIR3DL2 and CCR4) that co-expressed CD39. CD39 expression was detected by immunohistochemistry in lesional skin of 34 out of 36 studied SS patients (mean % of positive cells:45%;range:5-90%). Classical flow cytometry analysis confirmed CD39 over-expression on SS patients malignant CD4+ T-cells. Moreover, an unusual percentage of CD39-positive cells was also detected in normal CD4+ and CD8+ T cell and NK cell populations when compared to HDs. 4. Conclusions. These findings support the possibility of using CD39 as a diagnostic marker of SS but also as a promising therapeutic target with the current development of CD39- blocking antibodies that may restore efficient antitumor responses.

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The correlation of flow cytometry analysis on skin tissue with immunohistochemistry in cutaneous T-cell lymphoma (CTCL)

Authors: Shamir Geller MD1,2, Caleb Ho MD2, Sarah Noor MD2, Mikhail Roshal MD2, Patricia Myskowski MD2, Alison Moskowitz MD2, Steven Horwitz MD2, and Melissa Pulitzer, MD2 Sourasky Medical Center, Tel Aviv and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv.1

Memorial Sloan Kettering Cancer Center, New York, NY, USA.2 [email protected]

Introduction & Objectives: Flow cytometric analysis (FC) of fresh skin specimens is infrequently used to diagnose and manage cutaneous T-cell lymphoma (CTCL). Our goal was to compare objective and interpretive characteristics of contemporaneous formalin-fixed paraffin- embedded (FFPE) and FC skin biopsies in CTCL. Materials & Method: Flow plots from 31 consecutive skin samples from 25 WHO/EORTC- classified CTCL patients with 27/31 concomitant H&E and immunohistochemically stained tissue sections were retrospectively identified and reviewed. Histology, clonality, flow sample quality, cell yield, skin lesion and procedure type were analyzed for association with identification of an abnormal population. Antibodies employed and immunophenotypic interpretation of abnormal lymphocytes on FC and FFPE were compared. Results: FC specimens included 23 punch biopsies, 6 shave biopsies and two excisions. Cell counts varied (70-180,000, mean 14,215) and correlated with procedure type (p=0.01). FC identified an abnormal T-cell population in 71% of cases. Identification of an abnormal population by FC was associated with lesion type (p<.001) and not with CTCL classification (p=.09), histological localization (p=0.4) or monoclonality (p=0.1). FC did not detect an abnormal population in 9 cases; three with no or histologically equivocal lymphoma, three with sparse diagnostic infiltrates, and one with necrosis. Comparison of aberrant cells between FC and FFPE skin samples showed differences in CD4 or CD8 labeling/interpretation (5/17 cases), and in pan- T-cell markers (CD3/CD5/CD7, 10/18 cases) but not in CD2. In 2 patients FC identified immunophenotypically identical populations in temporo-spacially distinct skin. In 8 patients FC of extracutaneous tissue showed the same abnormal phenotype as in skin. Conclusions: FC detects abnormal T-cell populations in CTCL tissues with small numbers of analyzed cells and can identify immunophenotypically similar populations in temporo-spacially distinct biopsies, and extracutaneous sites, with diagnostically insignificant differences in individual marker labeling/interpretation. The implementation of skin FC with FFPE should be considered routinely for patient management.

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I-03

Title: Overexpression of STAT4 at early stages of mycosis fungoides: coincidence or not? Authors: Olga Olisova,1 Ekaterina Grekova1, Dmitry Zaletaev2,3, Ekaterina Alekseeva2,3. Affilation 1Department of Dermatology and Venereology, Sechenov University, 4 Bolshaya Pirogovskaya Street, Building 1, 119991, Moscow, Russia; 2Laboratory of Medical Genetics, Institute of Molecular Medicine, Sechenov University, 8 Trubetskaya Street, Building 2, 119048, Moscow, Russia; 3Research Centre for Medical Genetics, Russian Academy of Sciences, 1 Moskvorechye Street, 115522, Moscow, Russia.

Introduction Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphomas (CTCL) accounting for approximately 65-85% of all CTCL. MF is characterized by proliferation and accumulation of malignant monoclonal T-lymphocytes or natural killer cells in the skin, which may also subsequently involve lymph nodes, internal organs and bone marrow.1 In the early stages, the prognosis is favorable, while the progression to the point of nodal or visceral involvement is indicative of a poor prognosis.1 The early diagnosis of MF is challenging due to the existence of different clinical forms and absence of definitive diagnostic criteria. The molecular pathogenesis of CTCL is only partially understood. Activation of the signal transducers and activators of transcription (STAT) protein family has been involved in the pathogenesis for leukemias, Hodgkin lymphomas and CTCL.2 In particular, STAT4 is essential for T-helper (Th) 1 differentiation which is induced by IL-12 cytokine. Then IL-12 activates TYK2 and JAK2 tyrosine kinase receptors.3 Previous studies showed that expression of STAT genes has potential diagnostic and prognostic value in CTCL patients. 1,2 The objective of our pilot study was to investigate the expression levels of STAT4 at different stages of MF and inflammatory skin disorders (ISDs).

Material and Methods The study included patients with early MF (stages I-IIA; n=19), advanced MF (stages IIB-IV; n=10), ISDs that may mimic CTCL (atopic dermatitis, chronic eczema, psoriasis) (n=13) and healthy controls (n=10). MF was diagnosed using clinical examination and skin biopsies for histological, immunohistochemical and molecular tests. Molecular tests entailed detection of TCRγ gene rearrangement by PCR. RNA expression of STAT4 was evaluated by quantitative real-time PCR (qPCR) using FAM-labeled hydrolysis probes (Bio-Rad, USA). To normalize the level of STAT4 gene expression in each individual sample, B2M and ACTB reference genes with stable expression during cell activity were used as endogenous control. To assess the reproducibility of the results, three replicates of each sample were examined. A p value of 0.05 was chosen as a threshold for statistical significance.

Results As demonstrated in Fig.1, STAT4 was significantly elevated in early MF lesional skin compared to healthy skin (p=0,001), as well as ISDs skin samples (p=0.01). Our study showed that STAT4 expression, which is essential for Th1 differentiation, is decreased at advanced stages (p=0,05), where the disease shifts to the Th2 phenotype and produces malignant T-cells.

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Litvinov et al. demonstrated that STAT4 is one of the targets of miR-155.1 Hence, STAT5-driven upregulation of miR-155 leads to a subsequent loss of STAT4 expression.2 Netchiporouk E et al. mentions that STAT5 is involved in the transcription of anti-apoptotic genes (bcl-x, bcl-2) and cell cycle genes (c-myc, Cyclin D) which facilitate cancerogenesis.2 Furthermore, one of the essential functions of STAT5 in CTCL is to upregulate oncogenic miR-155 microRNA, which activates immune cells and increases genomic instability.1,2

Conclusions Thus, our pilot study showed that overexpression of STAT4 was associated with early stages of MF, and it may become a potentially useful diagnostic marker. Further studies may fully assess the usefulness of STAT4 in early diagnosis of MF and help develop effective treatment options. . References 1. Litvinov I, Cordeiro B, Fredholm S, Ødum N, Zargham H, Huang Y. Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines. J Am Acad Dermatol. 2015;72(5). 2. Johnson VE, Vonderheid EC, Hess AD, Eischen CM, McGirt LY. Genetic markers associated with progression in early mycosis fungoides. J Eur Acad Dermatol Venerol. 2014; 28(11): 1431-5.

Figure 1. Quantitative real-time PCR of mRNA in healthy volunteers, patients with MF and ISDs. STAT4 is overexpressed in early stages of MF. *p=0,05; **p=0,01; *** p=0,001.

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J-01

Comparison of early with advanced Mycosis fungoides lesions on a single cell level to assess potential mediators of disease progression

Matthias Farlik, PhD,1 Wolfgang M. Bauer, MD,1 Lisa Shaw, MSc1, Stefanie Porkert, MD,1 Florian Halbritter, PhD,2 Constanze Jonak, MD,1 Patrick M. Brunner, MD, MSc1

1 Department of Dermatology, Medical University of Vienna, Austria 2 Children’s Cancer Research Institute - CCRI, Vienna, Austria

Introduction: Mycosis fungoides (MF) has been identified as a malignancy of tissue resident memory T-cells, but mechanisms associated with stable vs. progressive disease are still only incompletely understood. We sought to characterize MF-cells and their skin microenvironment, comparing longstanding stable patches with rapidly growing tumors. Methods: We investigated skin cells from patch and tumor lesions of early and advanced MF, respectively, by using single cell RNA sequencing. Results: MF-cells were CD3+, CD4+ and TOX+ in all stages, while benign bystander T-cells were mostly TOX- and displayed distinct clustering in UMAP plots. MF-cells expressed high levels of the activation marker NKG7 and were hyperproliferative as indicated by broad MKI67 upregulation. In line with published literature, we detected high levels of IL13 and IL26 in MF- cells (but not in bystander T-cells), paralleled by upregulated IL13RA1 in nearly all other identified cell populations. Higher numbers of malignant T-cells in tumors were paralleled by relative increases in macrophage, dendritic cell and endothelial cell counts, but a decrease in benign T-cells. Differential gene expression analysis of MF-cells identified increases in GZMB and GNLY in advanced disease, with concomitant decreases in CCL5 and CD7. In parallel, myeloid cells acquired fatty-acid binding protein 5 (FABP5) and S100A9 in tumor vs. patch stage. FABP5 is a negative regulator of IL-12 production in dendritic cells, and S100A9, despite its general role as a pro-inflammatory antimicrobial, has been shown to attenuate inflammatory capabilities of dendritic cells. Thus, these two markers might reflect dendritic cell exhaustion. We also found fibroblasts and endothelial cells to upregulate the tissue inhibitor of metalloproteinases TIMP1 in tumors vs. patches, likely impacting on extracellular matrix remodeling with progressive disease. Conclusions: Single-cell transcriptomics shed a new light on interactions between MF-cells and their skin microenvironment, revealing novel potential regulatory mechanisms facilitating disease progression.

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J-03

Genic signature of Cutaneous T cell lymphoma-associated fibroblasts from Mycosis Fungoides and Sezary Syndrome.

H. Zouali1, G. Dobos2,3, A. de Masson 2,3,4, S. Ly Ka So2,4, C. Battail5, A. Boland6, M. Bagot2,3,4 , A. Bensussan2,4, J.-F. Deleuze 1,6, L. Michel 2,3,4

1 Fondation Jean Dausset-CEPH, Paris, France, ² INSERM UMR-S U976, Centre de Recherche sur la Peau, Hôpital Saint-Louis, Paris, France, 3 Service de Dermatologie, APHP, Hôpital Saint- Louis, Paris, France, 4 Université de Paris, Paris, France, 5 UMR_S 1036, CEA, Univ. Grenoble Alpes, Grenoble, France, 6 Centre National de Recherche en Génomique Humaine, CEA-Institut François Jacob, Evry, France.

Corresponding author: [email protected]

Background

Mycosis fungoides (MF) is the most prevalent form of primary cutaneous T-cell lymphoma with cutaneous localization and is usually indolent whereas Sezary syndrome (SS), is rare and aggressive. Both SS and MF are derived from the clonal expansion of CD4+ resident cutaneous T cells in response to chronic antigen stimulation although etiology remains unknown. Cancer-associated fibroblasts (CAFs), a component of tumor microenvironment play a key role in oncogenesis, and promote growth, invasion, metastasis and resistance to treatment of tumor cells. Here, we aim to determine the contribution of skin microenvironment in the development and maintenance of tumor lesions of CTCL by characterizing the gene expression profile of the dermal fibroblasts obtained from skin lesions of patients with MF and SS and compared with fibroblasts from healthy donor (NF) skin.

Materials and methods Patients: A well-defined cohort of 14 patients with CTCL (6 SZ, 8 MF) including (10 males, 4 females) were recruited in the Department of Dermatology of Saint-Louis hospital and 15 skin pieces from healthy mammary reduction performed in the Plastic Surgery Department. All individuals had signed informed consent. Ethics approval was obtained from the Human Research Ethics Committee of the Saint- Louis Hospital (Paris, France). The median and range of ages of CTCL patients and control donors were 63 (min 28 – max 88) and 58 (min 20 – max 82) years old, respectively. The study was conducted according to the principles of the Declaration of Helsinki. The revised diagnostic criteria for staging of MF and SS by the ISCL/ EORTC were used, as detailed in the table 1.

Table 1. Patient’s characteristics

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Methods: Stranded RNA-sequencing (RNA-seq) was performed on non-activated fibroblast primary cultures from skin biopsies of MF and SS patients versus 15 cultures of normal fibroblasts (NF). RNA-seq was carried out using Illumina TruSeq V3 reagents with TruSeq library construction and selection for polyadenylated mRNA generating an average of ~50 million paired-end 100-bp reads per sample. The resulting sequencing reads were mapped to human reference genome (GRCh38_r79) using STAR (version 2.5.3a) generating raw gene-count table. Quantification of expression level and analysis of differential expression of RNA-Seq data were done using DESeq2 packages (version 1.24.0) implemented in R (version R 3.4). DESeq2 enable the identification of differentially expressed genes (DEG) whose expression abundance is significantly increased (up-regulated) or decreased (down-regulated) in a pathological condition compared to a control condition. The results of the differential expression obtained by DESeq2 were expressed in fold change (FC), p-value (estimated by Wald test) and adjusted p-value (padj) computed by Benjamini-Hochberg calculation to correct for multiple testing. The selection of differentially expressed genes (DEGs) is based on an arbitrary criteria. Only the genes with an adjusted p-value (padj) < 5% were selected. DEGs with log2FoldChange ≤ -1 were considered down-regulated and those with log2FoldChange ≥ 1 were up-regulated. The functional analyzes of the DEGs making it possible to define their biological significance in the group of CTCLs were carried out respectively by the "Ingenuity Pathway Analysis" (IPA®, Ingenuity Systems, Inc., QIAGEN Redwood City, CA, www.qiagen.com/ingenuity) and "Gene Set Enrichment Analysis" (GSEA) (http://www.broad.mit.edu/gsea/index.jsp). Results The results showed that 1044 differential gene expressions (DEGs) were displayed between normal fibroblasts (NF) and CTCL-associated fibroblasts (CTCL-AF or CLAF), with 542 genes (52%) under- expressed and 502 (48%) overexpressed. The Differential Expression Analysis by DESEq2 of the different sample groups enables to distinct gene clusters with a number of 1345 genes in MF-associated fibroblasts versus NF, 457 genes in SS ones versus NF and only 45 genes in SS- versus MF- associated fibroblasts, as depicted in Venn diagram Figure 1. Venn diagram: DEG (Figure 1). Gene Set Enrichment analysis (GSEA) of in fibroblasts from MF (left) or CAF and NF data against the "hallmark gene sets" SS (right) patients vs NF (h.all.v6.2 containing 50 gene sets) identified 3 enriched signaling pathways (NES ≥ 1.5): "HALLMARK INTERFERON  RESPONSE", "HALLMARK INTERFERON γ RESPONSE", and "HALLMARK MYC TARGETS". In addition, the transcription factor analysis carried out by the Ingenuity Pathway Analysis (IPA) software makes it possible to highlight sets of genes associated with several "pathways" such as “Granulocyte Adhesion and Diapedesis” or “Inhibition of Matrix Metalloproteases”. Using IPA, a total of 25 networks (Networks) with between 17 and 33 molecules among the DEGs were identified. Validation of main genes of interest is under current investigation.

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Conclusion The present data demonstrate the alteration of gene expression in CTCL-associated dermal fibroblasts, suggesting the major involvement of these mesenchymal cells on the behavior of CTCL and offering a new potent tool for establishing diagnosis.

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The Effect of Extracellular Matrix and Fibroblasts on Proliferation and Survival of Malignant Cells in Mycosis Fungoides Burcu Beksaç, Sarah Baik, Megan O'Donnell, Pierluigi Porcu, Neda Nikbakht Introduction & Objectives: Early stage Mycosis Fungoides (MF) is restricted to the skin, an organ abundant in fibroblasts and extracellular matrix (ECM). While cutaneous lymphomas may be the only type of lymphoproliferative disorder in such abundant contact with fibroblasts and ECM, little is known about the effect of stromal microenvironment on the growth and survival of malignant lymphocytes. In many solid tumor types, as well as some types of Hodgkin lymphomas, fibroblasts promote tumor growth, angiogenesis, drug resistance and immune escape through several mechanisms (1). Fibronectin is a major component of ECM, aberrantly expressed in the many tumor microenvironments. Through binding to its integrin receptors, fibronectin mediates cell adhesion-mediated drug resistance for many different cancer cell lines, including leukemic Jurkat cells (2). Our aim was to investigate the effects of the fibroblasts and fibronectin on the proliferation and survival of lymphocytes from MF lesions and MF cell lines. Materials and Methods: Cancer associated fibroblasts (CAFs) and malignant skin-derived lymphocytes (MF-SDL) were obtained through explant method from MF skin biopsies. Healthy fibroblasts (HF) and healthy skin-derived lymphocytes (H-SDL) were also obtained through explant method from normal skin leftovers from plastic surgery operations. For proliferation assays, lymphocytes were activated with anti-CD3/28 microbeads and co-cultured with fibroblasts or grown on fibronectin-coated culture plates. Their proliferation rates were assessed at the end of 96 hours’ culture through flow cytometry for CFSE dye dilution. Integrin expression on MF-SDL and MF cell lines was assessed through flow cytometry. To assess cell adhesion-mediated drug resistance, HUT-78 and MyLa cell lines were cultured with or without fibronectin and treated with doxorubicin. Rescue from chemotherapy and overall apoptosis were assayed by Annexin V staining. The effect of fibroblasts on chemoresistance and survival of malignant lymphocytes was also investigated. Malignant lymphocytes were co-cultured with fibroblasts and treated with doxorubicin for 24-hours before measuring apoptosis with Annexin V assay. Results: Fibroblasts, both healthy and cancer-associated, increased proliferation of malignant lymphocytes (MF-SDL and HUT-78 cells) while dramatically suppressing the growth of healthy lymphocytes (peripheral blood CD4+ T cells and H-SDL, activated with anti-CD3/28 microbeads). Fibroblasts, however, did not protect malignant lymphocytes from doxorubicin-induced apoptosis. Fibronectin-binding integrins were present on all cell types we analyzed (HUT-78, MyLa, MF- SDL, and peripheral blood mononuclear cells). However, only HUT-78 adhered to fibronectin- coated plates with any degree of specificity measured via adhesions assays. Fibronectin did not increase proliferation of HUT-78 or MF-SDL, but did increase proliferation of MyLa cells. Fibronectin also did not mediate chemoresistance to doxorubicin, and it did not decrease apoptosis in HUT-78 or MyLa cell lines. Table 1 summarizes all the fibroblast and fibronectin data.

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Table 1. A summary of our results.

Experiment Type Cell Fibroblast Fibroblas Fibroblast Fibronecti Fibronecti Fibronectin Type Proliferati t Rescue n n Rescue on Apoptosis (with Proliferati Apoptosis (with (No Doxorubic on (No Doxorubici treatment in) treatment n) ) ) MyLa No change No change ↑Apoptosis ↑Proliferati No change No change on

HUT-78 ↑Proliferat No change ↑Apoptosis ↓Proliferati No change No change ion on MF-SDL ↑Proliferat ↑Apoptosi ↑Apoptosis ↓Proliferati ion s on Periphera ↓Proliferat l CD4+ ion H-SDL ↓Proliferat ion

Conclusions: Fibroblasts appear to provide a proliferative advantage to MF cells. This is an interesting finding, considering that in many solid malignancies, fibroblasts gain an activated phenotype that promotes tumor growth. While we could not demonstrate a significant functional difference between healthy and cancer associated fibroblasts in these in vitro experiments, the proliferative advantage of fibroblasts was unique to MF cells and not healthy lymphocytes. However, the presence of fibroblasts did not decrease MF cell apoptosis, nor did it rescue MF cells from chemotherapy. While cellular competition might have contributed to this observation in fibroblast co-cultures, further characterization of fibroblasts is necessary to explain these findings. Although all MF cells expressed fibronectin binding integrins, fibronectin did not appear to produce consistent survival or proliferative advantage for MF cells. Unlike the previous results reported for leukemic Jurkat cells (2), fibronectin did not rescue MF cells from doxorubicin- induced apoptosis. This lack of rescue was apparent in both fibronectin adherent and non-adherent MF cells. Overall, results of our in vitro experiments demonstrate a role for fibroblasts, but not fibronectin, in MF propagation.

References 1. Menter T, Tzankov A. Lymphomas and Their Microenvironment: A Multifaceted Relationship. Pathobiology. 2019;86(5-6):225-36. 2. Liu CC, Leclair P, Yap SQ, Lim CJ. The membrane-proximal KXGFFKR motif of alpha- integrin mediates chemoresistance. Mol Cell Biol. 2013;33(21):4334-45.

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Hypomethylation-mediated activation of TMEM244 gene in Sézary cells Katarzyna Iżykowska1, Karolina Rassek1, Magdalena Żurawek1, Karina Nowicka1, Julia Paczkowska1, Karolina Olek-Hrab2, Maciej Giefing1, Grzegorz K. Przybylski1 1. Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland 2. Department of Dermatology, University of Medical Sciences, Poznań, Poland INTRODUCTION Sézary syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T-cells (Sézary cells) with many complex changes in genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. Our previous study revealed ectopic expression of TMEM244 gene, with unknown biological function, in SS patients but not in healthy individuals. In this study we used CRISPR-dCas9 epigenome editing system to prove that methylation of the promoter can be a mechanism responsible for regulation of TMEM244 expression. MATERIALS AND METHODS  DNA and RNA were isolated from 5 SS patients, 3 mycosis fungoides samples, 13 blood and 3 bone marrow samples from different hematological malignancies, 5 healthy individuals and 5 established T-cell lines (SeAx, Hut78, HH, Jurkat, HDLM2)  TMEM244 expression was analyzed by qRT-PCR (TaqMan gene expression assay)  DNA methylation level was analyzed at three CG dinucleotides (genomic position: chr6:130,182,479-130,182,514; GRCh37/hg19) by bisulfite pyrosequencing  Demethylation of TMEM244 promoter in vitro was conducted using CRISPR-dCas9 epigenome editing approach. Two CRISPR-dCas9 vectors, with active and inactive domain of Tet1 demethylase were used, 4 single guided RNAs were designed to target chosen CG sites, 2 non-targeting guided RNAs were used as controls  Expression of dCas9 –TET fusion protein was confirmed by Western Blot

Figure 1. TMEM244 promoter, localization of three CpG dinucleotides and sgRNAs RESULTS 1. TMEM244 is expressed in SS patients and CTCL cell lines with hypomethylation of the promoter  TMEM244 expression was detected in all SS and MF patients, SS derived cell lines: SeAx, Hut78, HH and T-cell derived Hodgkin lymphoma HDLM2 cell line  No TMEM244 expression was detected in mononuclear cells from healthy donors and T- ALL Jurkat cell line  Bisulfite sequencing revealed 85-89% (MV=86%) methylation of promoter in all samples without TMEM244 expression and hypomethylation, 2-68% (MV=44%), in samples expressing TMEM244  TMEM244 expression was inversely correlated with promoter DNA methylation (r=-.7592; p<.00001)

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Figure 2. Correlation between promoter methylation and TMEM244 expression in hematological malignancies and in T-cell lines 2. In vitro demethylation of TMEM244 promoter activates its expression  Transduction of Jurkat T-cell line with catalytically inactive Cas9 protein (dCas9) fused to Tet1 active domain resulted in marked demethylation of TMEM244 promoter with 2 single guide RNAs (sgRNAs) (60.2%) and less prominent in with 2 other sgRNAs and 2 NT sgRNAs (84%) or 3 different combinations of those sgRNAs (79.5), as compared to cells transduced with Tet1 inactive domain (84.5%, 90% and 90.3% respectively; p<.00001)  Activation of TMEM244 expression in modified cells was negatively correlated with the level of promoter demethylation (R=-0.4766, p= .000178)

Figure 3. Correlation between methylation and TMEM244 expression CONCLUSIONS  Our study shows that methylation is a key regulatory mechanism of TMEM244 expression  Negative correlation between TMEM244 expression and methylation was confirmed in hematological malignancies and in T-cell lines  CRISPR-dCas9 induced demethylation of TMEM244 promoter further supports the hypothesis of epigenetic regulation of TMEM244 expression ACKNOWLEDGEMENTS National Science Centre, Poland, Grant no. 2017/27/B/01540

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Epigenetic involvement in cutaneous T-cell lymphoma lymphomagenesis

Introduction:

Telomeres and telomerase (hTERT), considered as biomarkers of cancer cells, were found implicated in the lymphomagenesis of cutaneous T-cell lymphoma (CTCL). Nevertheless the mechanism responsible for telomerase expression in this pathogenesis is enigmatic. Epigenetic changes were reported to play a role in the increased expression of biomarkers in CTCL, especially in epidermotropic form (Mycosis Fungoides (MF) and Sézary Syndrome (SS)). This epigenetic involvement is supported by the efficacy of evolving therapeutic strategies, such as histone deacetylase inhibitors (HDACi) and methylation inhibitors in the treatment of CTCL patients.

Methods:

In this study, we investigated the implication of epigenetic events in telomeres biology, focusing on hTERT gene promoter methylation status and the transcription of subtelomeric regions towards chromosome ends into long non-coding RNAs (TERRA). Bisulfite Sanger sequencing was performed to evaluate hTERT promoter methylation in five CTCL cell lines, four cultured SS patients' cells and fresh cells of six SS patients. This CTCL cells were compared to healthy lymphocytes and normal stem progenitor cells. RT-qPCR was used to evaluate hTERT expression and to assess TERRA expression.

Results:

Comparing with normal cells, we observed the implication of hTERT Hypermethylated Oncologic Region (THOR) in CTCL. This hypermethylation is responsible for hTERT re-expression, and offers important insights that may be used for diagnosis, prognosis, and treatment monitoring. We also found that besides enriched in heterochromatic marks, telomeres are transcribed from multiple chromosome ends (1q, 9p, 10q, 11q, 15q, 16p and XpYp). This TERRA expression correlates with telomere length and allows distinguishing different CTCL subtypes. Moreover, one TERRA exhibited interesting results, showing that it could be involved in CTCL lymphomagenesis. However, the molecular details of TERRA functions remain to be elucidated.

Conclusions:

In this work we report for the first time the methylation pattern of hTERT promoter in CTCL and we unveil the epigenetic mechanism behind telomerase re-activation in CTCL. We identify as well a potential new biomarker valuable in CTCL lymphomagenesis.

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Mycosis fungoides cell line – derived exosomes show a distinct signature of elevated expression of miR-155, miR-1246, and OX40 to promote tumor progression Lilach Moyal,1,2 Coral Arkin,1,2 Batia Gorovitz,1,2 Jamal Knaneh1,2 Hadas Prag1,2 Iris Amitay- Laish 1,2 Emmilia Hodak1,2 1Division of Dermatology, Rabin Medical Center – Beilinson Hospital, Petach Tikva, and 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Introduction- Exosomes are endosome-derived nano-sized extra-cellular vesicles that mediate intercellular communication by transferring biological information into the recipient cells. Cancer exosomes create a favorable tumor microenvironment (TME) by mediating immune escape, and promote tumor progression by enhancing tumor growth, drug-resistance, and metastasis. Clinically, exosome miRNAs and proteins might serve as minimally invasive diagnostic, prognostic, and treatment response biomarkers of cancer, and even as therapeutic targets. We have previously shown that cutaneous T-cell lymphoma (CTCL) cell lines secrete exosomes with oncogenic cargo known to be involved in CTCL. Herein, we extended our in-vitro study to explore the functional oncogenic role of exosomes in mycosis fungoides (MF) mediated by their exosomal miRs and proteins, and their in-vivo potential as biomarkers for MF. Methods- Exosomes were isolated from CTCL cell lines (Hut78, HH, MJ and MyLa) by differential ultracentrifugation, with further confirmation by electron microscopy, nanoparticle tracking analysis and FACS analysis for CD81 (exosome surface marker). The exosomes were profiled for miR content by Affymetrix microarray, followed by qPCR for specific miRs. The protein content of the exosomes was profiled by proteomic mass spectrometry, followed by FACS analysis of specific proteins. Normal PBMCs (NPBMCs) were isolated from blood samples of healthy individuals. Exosome internalization was monitored by: fluorescence microscopy and FACS analysis of PKH26 and CD81 staining; exosomal miR uptake- by qPCR; and protein uptake- by immunofluorescence. Survival assay was done by MTT and cell migration by transwell migration assay. MF cell line with manipulated expression of a specific miR was used for survival and migration assay in order to demonstrate exosomal-miR dependency. AMD3100 is CXCR4 antagonist was used to block CXCR4. MF-exosomal miRs were analyzed by qPCR as cell-free RNA in plasma samples of early-stage MF patients (n=15) and healthy individuals (n=12). Results- Microvesicles isolated from all CTCL cell lines had more than 95% CD81 positive vesicles, indicating for exosome enriched samples. MF cell lines (MJ and MyLa) were characterized by abundant exosomal miRs and proteins, with an overlapping signature. miR-155 and miR-1246 had the highest expression, both known as exosomal oncomiRs in hematologic and solid neoplasms, respectively. In contrast, Sezary (Hut78) and aggressive CTCL cell line (HH) had low content exosomes. MJ cell line is a unique cell line derived from the peripheral lymphoma cells of a patient with MF, and represents invasive high-grade MF cells, unlike MyLa cells, which are skin MF cells representing localized low-grade MF. Therefore, we used exosomes from MJ cell line to study their effect on MyLa cell line and the effect of MyLa exosomes on NPBMCs. MJ-derived exosomes were internalized into MyLa cells, which in turn increased the cellular expression of miR-155 and miR-1246, protected them from doxorubicin- induced cell death, and enhanced their cell motility. Similar results were obtained with MyLa-exosomes that were internalized into NPBMCs. In contrast, exosomes isolated from MJ cells expressing miR-155 inhibitor had low content of miR-155, and reduced the migration of MyLa cells. Cell-free miR- 155 and miR-1246 were significantly higher in plasma samples of early MF patients than healthy individuals. In addition, proteomic analysis showed that exosomes derived from MF cell lines carry several immune related and lymphoma related proteins among them are: 1. OX40- member of the TNF superfamily, known as an activator of Tregs in the tumor microenvironment. 2. GITR- glucocorticoid-induced TNFR-related gene, known as an immune checkpoint co-activator. 3. CXCR4- Chemokine receptor of CXC chemokine stromal cell-derived factor 1 (SDF-1), involved in trafficking and homeostasis of immune cells. 4. CD44- cell-surface glycoprotein involved in

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cell-cell interactions, cell adhesion and migration. Cytoplasmic foci of OX40, GITR, CXCR4 and CD44 were observed by immunofluorescence staining of MyLa cells that internalized MJ- exosomes or NPBMCs that internalized MyLa exosomes. FACS analysis of MJ and MyLa exosomes that were pre-conjugated to beads, demonstrated positive membrane staining of GITR, while, the other three proteins apparently are intra-exosome proteins, with negative FACS staining. The resistance of MyLa and NPBMCs to Doxorubicin mediated by MF-exosomes was prevented by blocking of CXCR4. Conclusions- miR-155 and miR-1246 are the most abundant miRs in exosomes of MF cell lines, and their higher cell-free expression in the plasma of early-stage MF patients compared with healthy individuals, might be used as a biomarker for the disease. Further ongoing studies are being done to investigate the potential of cell free miR-155 and miR-1246 as biomarker for MF progression and response to treatment. Exosomes of MF cell lines enhance the migration of recipient T cells in dependency on exosomal miR-155, and protect the cells from dox-induced death in dependency on CXCR4. It is conceivable that this may serve as novel machinery for overcoming tumor heterogeneity, by delivering exosome capsulated miR-155 or CXCR4 from high-grade lymphoma cells to low-grade cells and normal cells. Two immune regulators were found to be highly expressed in MF exosomes- OX40 and GITR; the former might enhance the suppression activity of Tregs in the TME of MF lesions, and the latter might compete with cytotoxic T cells on GITR ligand expressed by dendritic cells leading to interference in the activation of T cytotoxic cells against MF. Further experiments are currently ongoing to examine our hypothesis for the role of exosomal OX40 and GITR in regulating the cancer supportive TME in MF.

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Transformed mycosis fungoides shows distinct miRNA and mRNA expression profiles compared to classic mycosis fungoides Cosimo Di Raimondo1,5, Xiwei Wu2,4, Jasmine Zain3, Farah Abdulla1, Steven T. Rosen3,4, Christiane Querfeld1,4 1Division of Dermatology, 2Department of Molecular Medicine, 3Department of Hematology/ Hematopoietic Cell Transplantation, and 4Beckman Research Institute, City of Hope, Duarte, CA, USA and 5Dept. of Dermatology, University of Roma Tor Vergata, IT

Introduction and Objectives: Mycosis Fungoides (MF) is the most common primary cutaneous T cell lymphoma (CTCL) and clinically characterized by an evolution of patches, plaques to tumors and/or erythroderma. Patients with early stage disease have a 10-year survival of 97-98% but patients with advanced stage disease have a poor prognosis with a 10-year survival of 42%. Large cell transformation of MF (MF-LCT), defined as > 25% overall or microscopic nodules of atypical lymphocytes being 4x greater than normal size, occurs in 20–50% of advanced MF and is generally associated with poor response to treatment regimens and dismal prognosis [1]. Genome profiling analyses in MF/SS identified genomic alterations in T cell signaling and differentiation genes, several tumor suppressor genes, including CCR4, TP53, NF-κB and Janus Kinase signaling members. In addition, microRNAs (miRNAs), small noncoding regulatory RNAs, have been implicated in the pathogenesis and progression of classic (non-transformed) MF with or without folliculotropism. However, the molecular background and mechanisms involved in large cell transformation remains largely unknown. The objective of our study was to identify differences in microRNA (miR) and gene expression profiles of MF-LCT when compared to classic MF using high-throughput sequencing. In addition, we analyzed clinical data and 5-year overall survival of MF-LCT vs classic MF. Materials and Methods: Total RNA from 50 FFPEs taken from skin biopsies of lesional skin of 46 patients with MF, stages IA-IV was extracted with miRNAeasy FFPE kit (Qiagen) that included 15 samples with large cell transformation (5 plaques, 10 tumors) and 35 non-transformed lesions (12 patches/ 16 plaques, 7 tumors). Total RNA, which includes miRNA, was extracted from CTCL (MF and MF-LCT) samples. Library preparation and high-throughput next generation sequen cing was performed on an Illumina Hiseq

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2500. Differences in miR and RNA expression between MF and MF-LCT cases were analyzed. Gene ontology (GO) term enrichment analysis and Hallmark and Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis, based on R software, were applied for the identification of pathways in which DEGs significantly enriched. Results: Our analysis revealed a distinct miR expression profile for MF-LCT when compared to classic MF. Unsupervised heatmap of miRNAs revealed specific clustering for transformed cases (top half; plaques and tumors) compared to non-transformed cases (bottom half) (Fig 1A). Twenty-seven upregulated miRs correlated with MF-LCT; the highest ranked miRs (controlled for age, gender and stage) included miR-146a-3p, miR-18b-5p, miR-21-3p, miR- 136-5p, miR-323b-3p, miR- 889 and miR-539-3p (Fig 1B). The top downregulated miRs included miR-708-5p, miR- 5701, miR-3065-3p and miR- Figure 2. Unsupervised heatmap of mRNA reveals distinct cluster for 3653 that are widely viewed as LCT and non-LCT. Hallmark pathway analysis reveals activation of signaling pathways such as for TCR, TNFα/NfΚB & inflammatory tumor suppressor miRs and responses. downregulated in many cancers and during tumor progression. Comparing mRNA of transformed vs non-transformed cases identified 3 distinct clusters shown in the unsupervised mRNA heatmap; top cluster reveals transformed cases (plaques and tumors), 2nd cluster reveals non-transformed patches/plaques and bottom cluster highlights non-transformed tumors (Fig 2A). GSEA analysis of Hallmark pathways identified differentially expressed genes (DEGs) in T cell activation, T cell receptor and TNF-alpha/NF-kappa B signaling pathways and inflammatory response highlighting the importance of the microenvironment. When compared with clinical course and outcome the 5- year OS for classic MF was 95% compared to 50% in patients with MF-LCT. Conclusions: We identified key miRs and gene signaling pathways in MF-LCT that provide insight into pathogenesis and may show promising therapeutic targets.

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Functional biological classification of differential gene expression in Sézary syndrome Andrea Moerman-Herzog, Daniel Acheampong, Yasir Rahmatallah, Galina Glazko, Syed Jafar Medhi, Henry K. Wong University of Arkansas for Medical Sciences, Little Rock, Arkansas

INTRODUCTION T cell lymphomas are a heterogenous subset of lymphoid malignancies with highly variable clinical course. Thus, diagnosis of erythrodermic skin diseases can be a challenge. We previously identified gene expression abnormalities unique to Sézary syndrome (SS) CD4+CD45RO+ T cells by excluding gene expression shared by a novel disease control, lymphocytic-variant hypereosinophilic syndrome (L-HES), a benign T-lymphoproliferation with clinical findings similar to SS [1]. Here, we sought to validate this gene expression signature in an independent cohort of SS cases, and gain insight from the genes assigned to biological function classifications. METHODS Transcriptome-wide RNA sequencing was conducted on purified T cells from 6 SS cases (CD4+ or CD4+CD45RO+) and 6 normal donors (CD4+CD45RO+). The microarray derived, SS-unique gene expression signature was defined as genes with significant differential expression in SS, but not significant or oppositely regulated in L-HES (Figure 1) [1]. This microarray-derived gene expression signature was compared to differential gene expression detected by RNA sequencing, and concordant gene expression abnormalities were identified (Figure 2). The Molecular Signatures Database [2] was queried to identify enriched Gene Ontology (GO) processes in the concordant gene set. Figure 1. Microarray-derived SS-unique gene Figure 2. Concordant gene expression in expression signature (gold overlap). microarray and RNAseq gene sets.

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RESULTS A validated SS-unique gene expression Figure 3. Expression of established SS signature of 79 upregulated and 67 biomarker genes in the SS-unique gene downregulated genes was identified (Figure expression signature. 2), including increased expression of FCRL3, HDAC9, NEDD4L, TIGIT and TOX, and decreased expression of DPP4 and STAT4 (Figure 3). GO results for all 146 genes included “Regulation of Cell Population Proliferation” and “Negative Regulation of Cytokine Production.” When analyzed separately, upregulated genes were enriched in 12 categories for morphogenesis- and development-related processes. Downregulated genes were enriched in 2 proliferation and 16 immune-related categories (Figure 4). CONCLUSIONS These results suggest that Figure 4. Selected functional associations identified within the abnormal gene expression SS-unique gene expression signature contributes to proliferative processes in SS. Up- and down-regulated genes were associated with different GO processes. Elevated ectopic gene expression may contribute to or reflect altered cell fate specification in SS, while reduced expression of genes normally expressed in memory T cells may contribute to dysregulated immune phenotypes in SS. REFERENCES 1. Moerman-Herzog A, Acheampong D, Brooks A, Hsu P-C, Blair S, Wong HK (2019) Transcriptome analysis of Sézary syndrome and lymphocytic-variant hypereosinophilic syndrome T cells reveals common and divergent genes. Oncotarget, 10(49):5052-5069 PMC6707948 2. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P, Mesirov JP (2011) Molecular signatures database (MSigDB) 3.0. Bioinformatics. 27: 1739-40 PMC3106198

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High-throughput sequencing reveals T-cell repertoire restriction in Sézary syndrome and mycosis fungoides Gonzalo Blanco1,2, Daniel López-Aventín3, Ramon M. Pujol3,4, Andrea Gómez-Llonín1,2, Anna Puiggros1,2, Manuela López-Sánchez5, Teresa Estrach6, Mª Pilar Garcia-Muret7, Ingrid Lopez-Lerma8, Octavio Servitje9, Manuel Muro5, Blanca Espinet1,2, Raquel Rabionet10,11,12,13 and Fernando Gallardo1,3 1Grup de Recerca Translacional en Neoplàsies Hematològiques, Cancer Research Programme, IMIM-Hospital del Mar, Barcelona, Spain; 2Laboratori de Citogenètica Molecular, Laboratori de Citologia Hematològica, Servei de Patologia, Hospital del Mar, Barcelona, Spain; 3Servei de Dermatologia, Hospital del Mar, Barcelona, Spain; 4Grup de Recerca en Malalties Inflamatòries i Neoplàsiques Dermatològiques, Inflamation and Cardiovascular Disorders Research Programme, IMIM-Hospital del Mar, Barcelona, Spain; 5Servicio de Inmunología, Hospital Clínico Universitario Virgen de la Arrixaca-IMIB, Murcia, Spain; 6Servei de Dermatologia, Hospital Clínic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain; 7Servei de Dermatologia, Hospital de Sant Pau, Barcelona, Spain; 8Servei de Dermatologia, Hospital Vall d’Hebron, Barcelona, Spain; 9Servei de Dermatologia, Hospital de Bellvitge, Barcelona, Spain; 10Departament de Genètica, Microbiologia i Estadística, Universitat de Barcelona, Barcelona, Spain; 11Institut de Recerca Sant Joan de Déu, Hospital Sant Joan de Déu, Barcelona, Spain; 12Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain; 13CIBER en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain.

Introduction. Prior studies suggested a role for antigen stimulation in Sézary syndrome (SS) and mycosis fungoides (MF) pathogenesis. We aimed to characterize the T-cell Receptor Alfa (TRA) and Beta (TRB) CDR3 repertoire of CD4+ T-cells and the HLA haplotypes in SS/MF. Methods. Peripheral blood CD4+ cells were isolated in 11 SS and one MF patients. mRNA was sequenced on a HiSeq2500 (Illumina). Sequencing data was QC’d with FastQC and aligned to reference genome with STAR. High-resolution HLA typing was performed employing PCR-SSO on microbeads array (One Lambda). CDR3 amino acid regions were obtained using IMGT/V-QUEST, and those almost identical to the predominant (CDR3-AIP) were defined as having identical length and one different amino acid. Antigen matches (AgM) were identified employing VDJdb. Results. Overall, 82216 CDR3 sequences were obtained. An important restriction of the CD4+ T- cell repertoire was observed in SS, with a median frequency of the predominant CDR3 (CDR3-P) of 94.4% for TRA and 93.7% for TRB (Table 1). Both parameters strongly correlated (r=0.832, P=0.002).

TRA TRB Patient CDR3 CDR3 ID V gene J gene V gene J gene Sequence % Sequence % SS01 TRAV6 TRAJ31 ALDNNARLM 94.6 TRBV7-2 TRBJ1-2 ASSPGQPNYGYT 94.9 SS02 TRAV1-2 TRAJ4 AVDGGYNKLI 94.4 TRBV19 TRBJ1-2 ASSPDRGRNYGYT 87.4 SS03 TRAV29/DV5 TRAJ40 AASASSGTYKYI 21.8 TRBV21-1 TRBJ2-7 ASRQGADEQY 26.2 SS04 TRAV9-2 TRAJ36 AHQTGANNLF 55.0 TRBV11-3 TRBJ2-7 ASSGDRGREQY 11.7 SS05 TRAV19 TRAJ27 ALMGNAGKST 95.9 TRBV7-9 TRBJ2-6 ANSFGRSGANVLT 94.2 SS07 TRAV26-1 TRAJ27 IVRVNTNAGKST 96.5 TRBV20-1 TRBJ2-7 SARGLAKIDEQY 93.7 SS13 TRAV13-1 TRAJ29 AARNSGNTPLV 65.5 TRBV7-9 TRBJ1-1 ASSLGQNTEAF 80.0 SS14 TRAV23/DV6 TRAJ50 AASMKTSYDKVI 95.3 TRBV28 TRBJ1-1 ASSLWRRRGTEAF 95.4 SS15 TRAV12-3 TRAJ29 AMSSGNTPLV 96.3 TRBV19 TRBJ2-1 ASSRTGGSYNEQF 96.8 SS16 TRAV17 TRAJ29 ATSRRSGNTPLV 69.8 TRBV29-1 TRBJ2-3 SVGPSGSHTQY 95.0 SS17 TRAV6 TRAJ23 ALAPIYNQGGKLI 87.7 TRBV10-3 TRBJ2-3 AISEPREGPDTQY 90.6 Table 1. V and J genes and CDR3 regions corresponding to the predominant clonotypes found in each SS patient.

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Frequencies of CDR3-P and CDR3-AIP in the same SS patient were exponentially correlated (Figure 1).

Data 1 Data 1

A 44 B 9090 AIP AIP

P<0.001 AIP P<0.001

- - R2=0.654 7575 R2=0.918 33 CDR3 6600 of of TRA 22 4545 TRB 3300 11

1515 Frequency (%) (%) Frequency 00 (%) of Frequency CDR3 00 0 2020 4400 6060 8800 100100 0 2020 4400 6060 8800 100100 Frequency (%) of CDR3-P Frequency (%) of CDR3-P

Figure 1. Relationship between the frequencies of CDR3-P and CDR3-AIP in the total of sequences (A) or in non-predominant sequences (B).

Identical CDR3 were frequently detected among different SS/MF patients. VDJdb analysis considering CDR3 with ≥3 reads revealed 21 AgM, including CMV, EBV and self-antigens. CDR3 regions shared by SS/MF patients showed an increased frequency of AgM compared to unique CDR3 (TRA: 15.7% vs. 1.4%, P<0.001; TRB: 11.8% vs. 0.6%, P=0.005; respectively). HLA similarities were frequently detected, including a high frequency (25%) of the Western-European A*29:02-B*44:03-C*16:01-DRB1*07:01-DQA1*02:01-DQB1*02:02 haplotype (Table 2).

ID HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQA1 HLA-DQB1 SS02 02:01,11:01 52:01,55:01 03:03,12:02 01:01,15:02 01:01,01:03 05:01,06:01 SS03 03:01,11:01 35:01,35:01 04:01,04:01 01:01,07:01 01:01,02:01 02:02,05:01 SS04 03:01,03:01 18:01,35:01 02:02,04:01 04:03,14:01 01:01,03:01 03:02,05:03 SS15 02:01,02:01 27:05,51:01 01:01,02:02 01:01,13:01 01:01,01:03 05:01,06:03 SS16 02:01,03:01 07:02,40:02 03:04,15:05 01:01,15:01 01:01,01:02 05:01,06:02 SS07 02:02,11:01 18:01,49:01 07:01,07:01 09:01,11:04 03:02,05:05 02:02,03:01 SS13 23:01,34:02 14:01,44:03 04:01,08:02 11:03,11:04 05:05,05:05 03:01,03:01 SS14 01:01,02:01 44:02,57:01 05:01,06:02 07:01,11:01 02:01,05:05 03:01,03:03 SS17 02:01,02:01 38:01,51:01 02:02,12:03 11:01,13:02 01:06,05:05 03:01,06:04 SS01 29:02,31:01 44:03,53:01 04:01,16:01 07:01,13:02 01:02,02:01 02:02,06:04 SS05 02:01,29:02 13:02,44:03 06:02,16:01 07:01,15:01 01:02,02:01 02:02,05:02 SS06 11:01,29:02 18:01,44:03 05:01,16:01 03:01,07:01 02:01,05:01 02:01,02:02

Table 2. HLA haplotypes in SS and one MF patient (SS06). Similarities are highlighted in bold.

Conclusions. CD4+ T-cells of SS/MF express restricted antigen receptors (highly expanded and correlated TRA and TRB CDR3-P, CDR3-AIP in the same SS patient, and shared CDR3 regions enriched in AgM between distinct SS/MF patients) and HLA similarities. These findings allude to increased stimulation by common antigens, potentially inducing malignant T-cell proliferation. Acknowledgements. PI18/00021, FEHH.

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Title: MOLECULAR STUDY OF SUBCUTANEOUS PANNICULITIS-LIKE T- CELL LYMPHOMA, LUPUS PANNICULITIS AND OVERLAP CASES

Introduction: Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare cytotoxic T-cell lymphoma, with mostly an indolent behavior and peculiar clinicopathological features. Up to 20% of patients can associate an autoimmune disease, mainly systemic lupus erythematosus. Differential diagnosis with lupus panniculitis, also known as lupus erythematosus profundus (LEP), use to be straightforward, however overlapping cases which are difficult to chategorize have been described.1 In this study, we have analyzed 22 cases with diagnosis of SPTCL, LEP and overlapping cases using a customized NanoString platform that includes 208 genes related to T-cell differentiation, stromal signatures, oncogenes and tumor suppressor genes.

Results: By means of the unsupervised analysis of the gene expression of the samples, 2 clusters of samples were found recognizing LEP cases versus a bimodal group conformed of neoplastic samples; and an heterogenous group including the overlep case. A comparative analysis identified 46 up-regulated genes and 34 down-regulated genes. The Gene Set Enrichment Analysis (GSEA) recognized gene sets defining cytotoxic T-cells; and mast cells and B-cells defining both group of cases (LEP versus SPTCL). Specific diagnostic markers are suggested by the analysis.

GSEA, SPTCL: GSEA, Lupus Panniculitis:

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Conclusions: Our study demonstrates up-regulation of T-cell cytotoxicity genes compared to lupus profundus (i.e. PRF1, KLRD1, GZMA, NKG7, GZMB, KLRK1, GNLY, GZMH, CTSW). In addition, LAG3, PTGER4, TBX21 and EOMES wich are related to exhausted CD8+ cells were overexpressed as well. The overexpression of genes implicated on mast cell pathways in lupus profundus (i.e. MS4A2, CPA3, HDC, TPSAB1/B2, KIT, ENPP3) highlight a presumptive role of these cells in the pathogenesis of LEP that has been poorly studied before. This study adds to the scarce knowledge2 about molecular characterization of SPTCL.

Bibliography: 1. Pincus LB, LeBoit PE, McCalmont TH, et al. Subcutaneous Panniculitis-like T-Cell Lymphoma with Overlapping Clinicopathologic Features of Lupus Erythematosus: Coexistence of 2 Entities? Am J Dermatopathol 2009; 31: 520-26. 2. Maliniemi P, Hahtola S, Ovaska K, et al. Molecular characterization of subcutaneous panniculitis-like T-cell lymphoma reveals upregulation of immunosuppression- and autoimmunity-associated genes. Orphanet J Rare Dis. 2014;9:160.

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Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome. Subcutaneous panniculitis-like T cell lymphoma (SPTCL) is an uncommon cytotoxic T cell non- Hodgkin lymphoma3 in which CD8+ T cells that express α /β T cell antigen receptors (TCRs) infiltrate subcutaneous adipose tissue, rimming adipocytes in a lace-like pattern. Both children and adults can be affected, with a median age at diagnosis of 36 years and a female gender bias. Affected individuals typically present with multiple subcutaneous nodules, systemic B-cell symptoms, and, in ~20% of cases, associated autoimmune disorders, most commonly systemic lupus erythematosus. SPTCL can follow a fulminant course when associated with hemophagocytic lymphohistiocytosis (HLH), which accompanies ~20% of cases and decreases 5 year survival from 90% to less than 50%. There is no standardized therapy, and although multidrug chemotherapy and radiotherapy are commonly used, immunosuppressive regimens, particularly cyclosporine A, seem more effective, possibly owing to the immunologic features of this lymphoma. The underlying genetic cause of SPTCL remains unknown, but a familial predisposition has been suggested. We studied a series of 27 SPTCLs to identify genetic variants underlying this disease. All samples were collected with informed consent following approval of the institutional review boards of the respective institutions. We performed whole-exome sequencing (WES) on 17 cases and further validated results by targeted sequencing on all cases included in this series. We identified missense variants in HAVCR2, which encodes T cell immunoglobulin mucin 3 (TIM-3), with c.245A> G (p.Tyr82Cys; NP_116171) and c.291A> G (p.Ile97Met; NP_116171) observed in 16 of 27 patients. The 12 SPTCL patients of Polynesian and East Asian origin harbored the homozygous p.Tyr82Cys TIM-3 missense variant in tumors and in the corresponding germline in 10 of 16 patients for whom this material was available. In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two and heterozygous in one. There were no other relevant or recurrent mutations on analyses of the WES dataset, including for the 11 SPTCLs wild-type for TIM-3. TIM-3 acts as a negative immune checkpoint regulating peripheral tolerance, antitumoral immunity, and innate immune responses. p.Tyr82Cys and p.Ile97Met TIM-3 are predicted to be deleterious and these variants are highly conserved across species as well as among other TIM family members Fig. 1b). They map to the membrane distal immunoglobulin variable (IgV) domain of the protein, which is critical for engaging galectin-9 and TIM-3 function in terminating immune responses. Compared with TIM-3 wild-type patients (P17–P27), patients with mutations showed a much younger median age at onset (15 versus 41 years), with onset below the age of 22 in 10 cases, and a more severe disease course. Most TIM-3-mutant SPTCL patients had HLH (14 of 16 versus 3 of 11; P < 0.01) and adipocyte rimming on bone marrow biopsies (11 of 13 versus 0 of 11; P < 0.0001) All samples with p.Tyr82Cys and p.Ile97Met mutant TIM-3 showed an aggregate staining pattern in the peri-Golgi apparatus with limited plasma membrane expression in panniculitis biopsies compared with the expected plasma membrane expression seen in all TIM-3 wild-type samples. The intracellular peri-Golgi aggregates and lack of TIM-3 detection using the N-terminal TIM-3 antibody suggested protein misfolding, as also predicted by FoldX force-field calculations. This was further confirmed by in vitro protein-folding experiments followed by size-exclusion chromatography; by native polyacrylamide gel electrophoresis (PAGE), which showed that only wildtype IgV TIM-3 retained a normal secondary structure migrating at 62 kDa; and by the disruption of the known amount of IgV domain N-glycosylation14. Taken together, our results show that the Tyr82 and Ile97 are critical residues needed for TIM-3 localization at the cell surface, and that the two identified substitutions impair proper folding and posttranslational modifications of the TIM-3 protein. HLH appears as a defining feature of TIM-3-mutant SPTCL. The TIM-3 negative checkpoint is also a critical regulator of innate immunity and inflammatory responses. It suppresses effector T cell (the TH1 subset of helper T cells) responses by decreasing interferon-γ (IFN-γ )-driven inflammation, and defects in its function may thus account for the HLH manifestations seen in TIM-3-mutant LPS stimulation alone induced significant release of

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mature IL-1β by TIM-3-mutant P4 macrophages. The results indicate that misfolding of TIM-3 promotes secretion of inflammatory cytokines and activation of the inflammasome. It is likely that defective TIM-3 function on macrophages lowers the threshold for inflammasome activation. An intriguing and central clinical feature of SPTCL is the selective infiltration of pathogenic T cells within the subcutis with no indications of a local infectious trigger. There is a high frequency of regulatory T lymphocytes (Treg cells) within adipose tissues in normal skin. Interestingly, Tim- 3+ Treg cells exert a more potent suppressor function than Tim-3− Treg cells, and their depletion in mice results in severe cutaneous inflammation. In keeping with these observations, we identified a drastic decrease in FOXP3+CD4+ T cells in TIM-3 mutants compared with TIM-3 wild-type SPTCL. This further supports the notion that TIM-3 potentially plays a gatekeeper role for inflammation in these tissues. This is especially true in cancer, where upregulation of TIM-3 following manipulation of the programmed cell death 1 checkpoint limits therapeutic responses and is actively being targeted. Our observation of clinical consequences of TIM-3 deficiency calls for cautious use of this strategy. In summary, we report a new inherited autosomal recessive condition characterized by HLH–SPTCL that results from TIM-3 deficiency and leads to uncontrolled immune activation. p.Tyr82Cys and p.Ile97Met TIM-3 pathogenic variants induce protein misfolding, abrogate expression of the receptor, and lead to the loss of the TIM-3 immune checkpoint and its multiple regulatory functions, promoting disease. TIM-3-mutant SPTCL should therefore be viewed as an inflammatory condition. Initial and possibly longterm control of the disease could be attained with immunosuppression and more novel agents targeting IL-1 and possibly IFN-γ that are both safe and effective in treating HLH–SPTCL.

Reference: Tenzin Gayden , Fernando E. Sepulveda , Dong-Anh Khuong-Quang , Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome. Nature Genetics 50(12), 1650-1657, 2018

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Principal Investigator: Dr. Farah Abdulla, MD

Co PI: Kord Honda, MD; Vishwas Parekh, MD; Christiane Querfeld, MD, PhD; Steve Rosen, MD; Joo Song, MD, Xiwei Wu, PhD; Jasmine Zain, MD

1.Introduction & Objectives: A sentence describing the background and a sentence describing the purpose of the study. Primary cutaneous CD30+ T-cell lymphoproliferative disorders (CD30+ LPD) are the second most common cutaneous lymphomas after mycosis fungoides (MF) and Sezary Syndrome (SS), representing around 30% of all cutaneous lymphomas. According to the World Health Organization (WHO), CD30+ LPD include primary cutaneous anaplastic large cell lymphoma (pcALCL) and lymphomatoid papulosis (LyP) as well as borderline lesions. pcALCL and LyP represent two ends of a spectrum of diseases that have different clinical presentations, clinical courses, and prognosis in their classic forms. However, the two entities, in particular LyP types A and C, do have overlapping histopathologic and immunophenotypic features with pcALCL, which can make diagnosis of borderline lesions difficult. Histologically, both share medium to large CD30+ atypical lymphoid cell infiltrates. In addition, this histologic feature is not unique since similar histology is also seen in MF with large cell transformation (MF-LCT), correlating with a more aggressive clinical course, as well as secondary cutaneous ALCL (scALCL), Hodgkin lymphoma, and various reactive conditions. The purpose of our study is to use whole exome sequencing to gain a better understanding of the genomics of LyP and pcALCL, in which no consistent somatic mutations have been identified.

2.Materials & Methods: Briefly, describe your methodology. Histopathologic features and immunophenotyping identified the number of large CD30+ T cells and the level of background inflammation including eosinophils, neutrophils and histiocytes. We performed whole-exome sequencing on of LyP and pcALCL (5 cases each) tumors along with paired normal samples. Mean exome sequencing depth was 30×. Findings were correlated with clinicopathologic features and TCR rearrangement status. Only cases of LyP Type A and C were included because of the significant overlapping histologic and immunophenotypic features with pcALCL. Gene sequencing profiles of LyP and pALCL were compared to each other and against matched normal skin specimens. GATK-MuTect2 (v4.0.11.0) was used to identify novel somatic mutations. After potential mutations were identified, they were compared to the COSMIC database, the Catalogue of Somatic Mutations in Cancer, which is most comprehensive resource of somatic mutations in human cancer.

3.Results: Describe your results in a logical sequence. 240 somatic mutations were identified. The number of mutations was higher in pcALCL compared to LyP. 32 of those mutations were found in at least two specimens, 22 of which were limited only to either LyP or pcALCL. SETD2 was the only mutation identified affecting the lymphogenesis pathway. A frameshift, loss of function, mutation in SETD2 was found in two samples of LyP, type C. This mutation was not found in pcALCL.

4.Conclusions: Emphasize new and important aspects of the study and conclusions that are drawn from them. In looking to identify any somatic mutations that may be novel drivers in CD30+ LYPD, we set out to see if the mutations were the same or different in LyP for pcALCL. With regards to known drivers of lymphogenesis, only frameshift mutations in SETD2 were identified. SETD2 protein is a histone methyltransferase that is specific for lysine-36 of histone H3, and methylation of this residue is associated with active chromatin.

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The trimethylation of lysine-36 of histone H3 (H3K36me3) is required in human cells for homologous recombinational repair and genome stability. This mutation leads to silencing of the gene and a loss of function. SETD2 is also mutated in other malignancies of lymphoid cell lineage such as acute lymphoblastic leukemia, enteropathy associated T-cell lymphoma, and hepatosplenic T- cell lymphoma. At this time, no definitive conclusions can be drawn. Additional samples of both LyP and pcALCL need to be analyzed with WES to determine if in fact this mutation is limited to LyP or exists in pcALCL.

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Clonal relationship between Blastic Plasmacytoid Dendritic Cell Neoplasm and Myeloid Neoplasms Introduction & Objectives: Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive tumor derived from the precursors of plasmacytoid dendritic cells. Rare cases of BPDCN associate with another myeloid neoplasm (MN), mainly myelodysplastic syndromes (MDS), acute myeloid leukemia, and chronic myelomonocytic leukemia (CMML). To evaluate the relationship between both diseases, we studied a series cases with concurrent BPDCN and MN. Materials & Methods: We included 5 patients with concurrent BPDCN and MN (2 with refractory cytopenia with multilineage dysplasia -RCMD-, 2 CMML and 1 MDS with isolated del5q). The tumors were characterized by their morphology, immunophenotype by flow cytometry and/or immunohistochemistry, cytogenetics and targeted sequencing of the whole codifying sequence of 21 genes recurrently mutated in MN. Results: The patients were 3 males and 2 females with a median age of 74 years (range 61-82 years). BPDCN and MDS were synchronic in 2 cases (both RCMD), whereas BPDCN followed the MN in 3 cases (2 CMML, 1 MDS 5q-). All cases had involvement of the skin by BPDCN except the case of MDS 5q- (bone marrow involved only). In this case, del5q was identified in the tumor cells of BPDCN. In one case, trisomy 8 was identified in the tumor cells of BPDCN and CMML. All cases had TET2 mutations. Mutations in the splicing factors ZRSR2 or U2AF1 were observed in 2 CMML and 1 RCMD. The majority of mutations seen in the MDS/CMML phase were also observed when BPDCN appeared, with the exception of TP53, IKZF1 and NRAS mutations, only detected in BPDCN. Conclusions: A clonal relationship between BPDCN and myeloid neoplasms is established in this series of patients. Notably, mutations involving DNA demethylation and spliceosome machinery were the mutations in these cases.

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Abstract Title: Cellular Origins and Genetic Landscape of Cutaneous Gamma Delta T Cell Lymphomas Presenting Author: Jay Daniels Authors: Jay Daniels†, Peter G. Doukas†, Maria E. Martinez Escala, Kimberly G. Ringbloom, David J. H. Shih, Jingyi Yang, Kyle Tegtmeyer, Joonhee Park, Jane J. Thomas, Mehmet E. Selli, Can Altunbulakli, Ragul Gowthaman, Samuel H. Mo, Balaji Jothishankar, David R. Pease, Barbara Pro, Farah R. Abdulla, Christopher Shea, Nidhi Sahni, Alejandro A. Gru, Brian G. Pierce, Abner Louissaint Jr*, Joan Guitart*, Jaehyuk Choi* †These authors contributed equally to this work. *Corresponding authors.

Introduction Cutaneous  T cell lymphomas are a group of heterogenous and rare skin lymphomas with a poor prognosis. Patients diagnosed with primary cutaneous  T cell lymphoma have a median survival of 31 months and a five-year survival of 19%1. Apart from hematopoietic stem cell transplant, there are no effective or targeted therapies for this disease. Histologically and clinically, cutaneous  T cell lymphomas are diverse (Figure 1). Some patients present with disease mainly involving the epidermis and/or dermis, while other patients have disease focused in the subcutaneous adipose tissue (termed panniculitic disease)1,2. Furthermore, patients can present with thin patches or thick tumors, with or without ulcerations. Figure 1. Clinical and histological heterogeneity of cutaneous  Currently, the molecular basis lymphomas. Schematic showing clinical photographs, H&E staining, underlying the clinical and and  T cell receptor immunostaining for representative patients. histological heterogeneity of cutaneous gamma delta T cell lymphomas is unknown. There have been few prior efforts to understand the molecular basis for this disease. The cell of origin has been long presumed to be a V2 cell, based on Southern blot analysis of four cases3. The genetics of this disease have been largely obscure, except for STAT3 and STAT5B mutations uncovered in several of a small cohort of patients4. Because of these gaps in the understanding of this disease, we sought to uncover the molecular underpinnings of this disease by genomic analysis of patient samples.

Methods A clinically annotated cohort of 42 cases was collected. All samples were confirmed to express the  T cell receptor by immunohistochemistry and/or next generation sequencing analysis. Our cohort included patients diagnosed as primary cutaneous  T cell lymphoma (PCGDTL) (n=25),  MF (n=16), and one case of intravascular  T cell lymphoma presenting in the skin. For samples with sufficient tissue available, genomic analysis was performed. We analyzed 29 cutaneous  T cell lymphomas by DNA-sequencing, RNA-sequencing, and/or T cell receptor sequencing. DNA-sequencing was performed by whole-genome, whole-exome, and targeted sequencing for 20 samples. 11 samples were analyzed by RNA-sequencing, and 8 samples analyzed by high throughput T cell receptor (TCR) sequencing (Adaptive

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Biotechnologies). T cell receptor sequences were inferred from whole genome sequencing and RNA-sequencing data using MiXCR software.

Results Epidermal/dermal and panniculitic  T cell lymphomas have distinct cells of origin We originally sought to determine  TCR usage by next generation sequencing to confirm the diagnosis and the immunohistochemistry results of  TCR expression in these samples. The  chain usage was determined for 15 samples. Based on the literature3, we expected all cases to be derived from V2 cells. Strikingly, we found that the histological phenotype determined  chain usage. 8/8 samples that predominantly involved the epidermis and/or dermis expressed V1, and 7/7 samples predominantly Figure 2: Distinct  chain usage involving the subcutaneous adipose tissue were V2 in origin by depth of infiltrate. (Figure 2). This difference was statistically significant (P = 0.0002; Fisher’s exact test). Based on this data, we refer to epidermal/dermal lymphomas as V1 lymphomas and panniculitic as V2 lymphomas. V1 and V2 lymphomas are transcriptionally distinct Next, we asked whether transcriptomic differences of V1 and V2 accounted for differences in the phenotypes of the tumors. Principal component analysis showed separate clustering of V1 and V2 samples (Figure 3a). Importantly, analysis of differentially expressed genes and Figure 3: Transcriptional analysis of V1 and V2 lymphomas. a) Distinct clustering of subtypes by principal component analysis. pathway analysis suggested that V2 b) Enriched gene signatures in V2 lymphomas. lymphomas harbor a more inflammatory phenotype. V2 lymphomas expressed significantly higher levels of genes associated with cytotoxic and inflammatory function, including granzymes K and H, IFN-G, and STAT4. Pathway analysis showed enrichment of interferon alpha and interferon gamma signaling signatures in the V2 lymphomas (Figure 3b). In accordance with the more inflammatory gene signatures observed in V2 lymphomas, these lymphomas had higher incidence of inflammatory cytokine driven symptoms and syndromes. B symptoms (fevers, night sweats, and and/or weight loss) was observed in a significantly higher proportion of patients with V2 lymphomas (58% vs 15%, P = 0.02, Fisher’s exact test). Hemophagocytic lymphohistiocytosis (HLH), a potentially fatal condition characterized by overproduction of inflammatory cytokines, was observed exclusively in V2 lymphomas (36% vs 0%, P = 0.005, Fisher’s exact test).

Genomic landscape of cutaneous  T cell lymphomas Genomic analysis of somatic single nucleotide variants and somatic copy number variants confirmed the previously reported JAK/STAT pathway mutations and implicated new pathways including MAPK, MYC, and Figure 4: Landscape of genomic alterations in cutaneous  T cell chromatin modification genes as lymphomas identify commonly mutated pathways. 102

being frequently mutated in cutaneous  T cell lymphomas (Figure 4). Interestingly, V1 and V2 lymphomas shared mutations in each of these pathways, suggesting common genetic features. Furthermore, these findings have clinical relevance. We identified activating mutations in the MAPK pathway as a negative prognostic marker in cutaneous  T cell lymphomas (P = 0.001, log rank test). Additionally, by comparing mutations we found to those in precision medicine databases, we identified mutations previously identified as targetable in nearly a quarter of samples analyzed by WES, including mutations targetable by MEK, combined MEK/AKT, or JAK inhibition.

Conclusions Our study provides molecular insights into a rare subset of cutaneous lymphomas. Importantly, we find that different histological subtypes are derived from distinct cells of origin that results in transcriptional and clinical differences. Genetically, both V1 and V2 lymphomas share mutations in the MAPK, MYC, JAK/STAT, and chromatin remodeling pathways. This work paves the way for the development of molecularly targeted therapeutics for the treatment of this aggressive disease.

References 1. Guitart, J., et al. Cutaneous γδ T-cell Lymphomas. The American Journal of Surgical Pathology 36, 1656-1665 (2012). 2. Merrill, E.D., et al. Primary Cutaneous T-Cell Lymphomas Showing Gamma-Delta (γδ) Phenotype and Predominantly Epidermotropic Pattern are Clinicopathologically Distinct From Classic Primary Cutaneous γδ T-Cell Lymphomas. The American Journal of Surgical Pathology 41, 204-215 (2017). 3. Przybylski, G.K., et al. Hepatosplenic and Subcutaneous Panniculitis-Like γ/δ T Cell Lymphomas Are Derived from Different Vδ Subsets of γ/δ T Lymphocytes. The Journal of Molecular Diagnostics 2, 11-19 (2000). 4. Küçük, C., et al. Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells. Nature Communications 6(2015).

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Whole-genome analysis uncovers recurrent IKZF1 inactivation and aberrant cell adhesion in blastic plasmacytoid dendritic cell neoplasm.

A. N. Bastidas Torres1, D. Cats2, H. Mei2, D. Fanoni3, J. Gliozzo4, L. Corti4, M. Paulli5, M. H. Vermeer1, R. Willemze1, E. Berti4 and C. P. Tensen1

1Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands 2Sequencing analysis support core, Leiden University Medical Center, Leiden, The Netherlands 3Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy 4Department of Dermatology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy 5Unit of Anatomic Pathology, Department of Molecular Medicine, University of Pavia and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Presenting author: A. N. Bastidas Torres (email: [email protected]) Corresponding author: C. P. Tensen (email: [email protected])

Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematological malignancy with a poorly understood pathobiology and no effective therapeutic options. Despite a few recurrent point mutations and indels have been identified in BPDCN, none are disease-specific, and more importantly, none explain its genesis or clinical behavior. This study aimed at characterizing the landscape of structural genomic alterations in BPDCN by employing Whole Genome Sequencing (WGS) and RNA-sequencing (RNA-seq).

Materials and Methods BPDCN tumors were subjected to WGS (10 patients) and RNA-seq (4 patients) on the Illumina HiSeq X-Ten and HiSeq-4000 platforms, respectively. Raw data were processed using in-house customized pipelines. Differential expression (DE) analysis was performed by comparing the transcriptomes of BPDCN tumors with publicly available transcriptomes of normal plasmacytoid dendritic cells. Select genomic alterations were validated by sanger sequencing and digital droplet PCR (ddPCR).

Results We have characterized for the first time the landscape of genomic rearrangements and copy number alterations of BPDCN using high resolution next-generation sequencing. Our analysis identified 54 genes disrupted by rearrangements across 10 BPDCN genomes, 13 of which are involved in malignancies at present (i.e. AHI1, CD36, FAT1, IKZF1, IQGAP2, MLLT4, MYB, NRG1, PIK3C2G, PMS1, PPFIBP1, PTPRD, TFG). We also identified 6 recurrently deleted genomic regions enclosing recognized tumor suppressors (i.e. CDKN1B, ETV6, HNRNPK, IKZF1, RB1, SFRP4). Notably, IKZF1, a critical gene for the development of plasmacytoid dendritic cell precursors, was found to be recurrently inactivated by structural alterations in 7 of 10 sequenced patients, and recurrent structural alterations involving this gene were confirmed in an independent cohort by ddPCR. Transcriptome analysis revealed a loss-of-IKZF1 expression pattern as well as up-regulation of the PI-3-K/Akt pathway and cellular processes responsible for cell-cell and cell-ECM interactions, which is a hallmark of IKZF1 deficiency in immune cells.

Conclusions Our findings suggest that IKZF1 inactivation plays a central role in the pathobiology of the disease, and consequently, therapeutic approaches directed at reestablishing the function of this gene might be beneficial for patients.

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Figure 1. (A) Diagram representing the hemizygous loss of IKZF1 (217 Kb-3.8 Mb) in BPDCN tumors. Blue bars depict deleted areas at 7p12.2 in tumors from patients with BPDCN. (B) Diagram showing inactivating structural changes at the IKZF1 locus in BPDCN tumors. iDel, interstitial deletion; ITX, intrachromosomal rearrangement; CTX, interchromosomal rearrangement.

Figure 2. (A) Conserved IKZF1 target genes follow a loss-of-IKZF1 expression pattern in BPDCN; that is, genes repressed by IKZF1 are up-regulated while genes activated by IKZF1 are down-regulated. (B) Pathway analysis using DAVID uncovered up-regulation of cellular processes responsible for cell-cell and cell-ECM interactions, the PI-3-K/Akt pathway, the Rap1 pathway and the cell cycle. (C) Down-regulated profiles included ribosome, oxidative phosphorylation and protein processing in the endoplasmic reticulum (ER)

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Primary Cutaneous Follicular Lymphoma is Genetically Distinct from Typical Nodal Follicular Lymphoma Xiaolong Alan Zhou, MD1, Jingyi Yang, BS4,5, Kimberly G. Ringbloom BS4, Maria Estela Martinez-Escala MD, PhD1, Alexander Wenzel, BS4, Haley Martin2, BS, Andrea P. Moy, MD2,3, Elizabeth A. Morgan, MD6, Shannon Harkins BS2, Christian N. Paxton, PhD7, Bo Hong, MD8, Erica F. Andersen, PhD8, Joan Guitart, MD1, David M. Weinstock, MD3,11, Lorenzo Cerroni, MD10, Abner Louissaint, Jr., MD, PhD,^2,3 Jaehyuk Choi, MD, PhD^1,4,5

1Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. 2Department of Pathology, Massachusetts General Hospital, Boston, MA 3Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. 4Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. 5Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA. 6Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 7ARUP Institute for Clinical and Experimental Pathology®, Salt Lake City, UT, USA. 8Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA. 9Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. 10Department of Dermatology, Medical University of Graz, Graz, Austria 11Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA.

Introduction & Objectives Cutaneous follicle center lymphomas comprise primary cutaneous follicle center lymphomas (PCFCLs) that originate and stay in the skin, PCFCLs that subsequently spread to other sites (~10% of all PCFCLs*), and typical nodal follicular lymphomas (TNFLs) that occasionally spread to the skin secondarily. The purpose of this study is to identify clinicopathologic and genetic features that differentiate PCFCLs that remain skin-limited from those that subsequently spread systemically and from TNFLs that secondarily spread to the skin. Materials & Methods We collected clinicopathologic data from 41 cases of cutaneous follicular lymphomas from 3 academic institutions, including 27 skin-limited PCFCL, 4 PCFCLs that subsequently spread systemically, and 10 secondary cutaneous follicular lymphomas (SCFL). Whole exome sequencing was performed on 19 PCFCLs and 6 SCFLs and copy number analysis on 5 PCFCLs. Results Skin-limited PCFCL have very little genetic overlap with SCFL, which resemble TNFL. PCFCLs that later spread systemically resemble SCFL and TNFL more than skin-limited PCFCL. Histologically, skin-limited PCFCLs are associated with reduced CD21+ follicular dendritic cell meshworks and increased proliferation indices. Genetically, they possessed frequent damaging mutations or deletions in TNFRSF14 and lacked BCL2 translocations or recurrent epigenome mutations in CREBBP, KMT2D, ARID1A, EP300 and EZH2 seen in TNFL. In contrast, SCFL and PCFCL that spread systemically closely mirror TNFL with frequent CREBBP, KMT2D and EZH2 mutations and BCL2 translocation in nearly every sample (Figure). Among follicular lymphoma subtypes, skin-limited PCFCL most closely resembled pediatric type follicular lymphoma (PTFL), which is similarly anatomically localized and has less aggressive course. Conclusions A unique combination of clinicopathologic and genetic features differentiates follicle center lymphomas that stay localized to the skin from those that spread systemically or those that started nodally and spread to the skin. These may enhance diagnosis and treatment strategies for these cohorts. References

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*Swerdlow SH CE, Harris NL, et al. ed World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008.

10 a b

M

/ 5

t

u CFCL Subtype M 0 SCFL b 100% PCFCL-SSD C>A C>G PCFCL-PCR C>T T>A 50% T>C T>G Mutation Type 0% Gain of function SNV c ONC NU MG MG MG MG NU MG MG MG MG AU AU AU AU AU MG AU MG MG MG AU MG AU MG MG GENE Inactivating SNV /TSG 212 721 816 503 126 029 885 486 475 275 16 01 13 23 05 276 03 274 886 487 19 383 08 272 485 Damaging / Loss of function n. Not functionally validated BCL2 t(14;18) + + a. + + + + + + + ------CR EB B P TSG Chromatin remodeling KM T2D TSG Chromatin remodeling EZ H2 ONC Chromatin remodeling STAT6 ONC JAK-STAT signaling IL4R ONC JAK-STAT signaling KR AS ONC Ras-MAPK / Ras-PI3K pathways M YC ONC Transcription factor CAR D11 ONC NF-kB pathway F O XO 1 ONC Transcription factor R HO A ONC Cell cycle / cytoskeleton JAK3 ONC JAK-STAT signaling F AS TSG Apoptosis IKZF 1 TSG Transcription factor EP300 TSG Chromatin remodeling SO CS1 TSG JAK-STAT signaling TN F R SF 14 TSG Immune modulation HIST1H1E TSG Chromatin remodeling TET2 TSG Chromatin remodeling IR F 8 TSG B cell development EB F 1 TSG B cell development M SH2 TSG DNA damage / cell cycle control M LH1 TSG DNA damage / cell cycle control PALB 2 TSG DNA damage / cell cycle control R B 1 TSG DNA damage / cell cycle control SETD2 TSG Chromatin remodeling KM T2C TSG Chromatin remodeling G N A13 TSG B cell lymphoma related B TG 1 TSG B cell lymphoma related F B XO 11 TSG B cell lymphoma related B 2M TSG Immune modulation CD58 TSG Immune modulation EPHA 7 TSG DNA damage / cell cycle control PTCH1 TSG Hedgehog pathway

d SCFL Similarity Index 1.0

0.5

0.0 SCFL Typical FL Diffuse FL PCFCL-PCR PTNFL

Figure. Landscape of somatic alterations in primary cutaneous follicle center lymphoma with persistent cutaneous restriction (PCFCL-PCR), primary cutaneous follicle center lymphoma with subsequent systemic dissemination (PCFCL-SSD), and secondary cutaneous follicle center lymphoma (SCFL). (A) Number of somatic mutations in each sample in mutations per megabase (green = PCFCL-PCR, yellow = PCFCL-SSD, orange = SCFL) (B) The relative ratios of single somatic nucleotide variant (SSNV) types in each sample. C) Oncoplot - red denotes recurrent mutations in putative oncogenes; black denotes damaging mutations in putative tumor suppressor genes; grey denotes missense mutations that have not been functionally validated. (D) Similarity index comparison with combined SCFL and PCFCL-SSD.

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O-02

Challenges in the diagnosis of primary cutaneous large B-cell-lymphomas Sarah Menguy, Audrey Gros, Philippe Ruminy, Marie Parrens, Eric Frison, Anne Pham-Ledard, Marie Beylot-Barry, Fabrice Jardin, Béatrice Vergier, and Jean-Philippe Merlio for the French Group of Cutaneous Lymphoma. Departments of Dermatology, Pathology, Methodology and Tumor Biology. INSERM U1053. CHU and University of Bordeaux, Bordeaux, France. INSERM U1245, Departments of Biological and Clinical Hematology, Centre Henri Becquerel, UNIROUEN, University of Normandie, Rouen, France.

Introduction and objectives: The 2017 WHO and 2018 updated WHO/EORTC criteria distinguishes primary cutaneous follicle center lymphoma (PCFCL) with a diffuse growth pattern from primary cutaneous diffuse large B-cell lymphoma-leg type (PCDLBCL-LT) which is crucial for therapy as both entities display different prognosis especially after relapse/progression. Such event is a very bad prognostic factor in PCDLBCL-LT while recurrence in PCFCL may be controlled by local therapy at local or regional stages. These criteria primarily based on morphology, architecture and phenotype may have limitations to differentiate germinal-center derived PCFCL, large cell (LC) from non- germinal center derived PCDLBCL, LT which was found to display an activated B-cell phenotype. Indeed pathologists have to face some specific limitations on skin sections such as the lack of reproducibility in CD10 immunostaining, which is a pivotal biomarker for Hans’ algorithm classification of diffuse large B-cell lymphomas (DLBCL). Alternative criteria have been proposed such as the presence of confluent sheets of centroblasts/immunoblasts seen in PCDLBCL-LT while their predominance but not in confluent sheets should favor PCFCL, LC. Cases with an ambiguous morphology or incomplete phenotype have been classified as an “other” category that should no longer be used but such situation may be encountered. Materials & Methods: To assess the relevance of the 2017 WHO classification criteria, we retrieved 64 unambiguous primary cutaneous diffuse large B-cell lymphomas (PCLBCL) with a diffuse growth pattern containing ≥80% of large cells and a proliferative rate of ≥40%. We also employed a new cost- effective RT-MLPA technique allowing both MYD88 status determination and genomic profiling of large B-cell lymphoma as germinal center (GC) or non-GC in parallel with the modified Hans algorithm. Another assessment of MYD88 mutation was performed by a more sensitive real-time PCR technique. Results: Morphology and phenotype identified 32 PCLBCL-LT and 25 PCFCL-LC. Seven cases (11%) remained borderline or undetermined. Phenotype analysis was found more reproducible than morphological classification between observers. Relevant markers for the differential diagnosis were CD10, MUM1 and FOXP1. Dual expression of MYC and BCL2 also favored PCDLBCL- LT diagnosis. BCL6 determination was neither reproducible nor discriminant between the two categories. The use of the modified Hans’ algorithm classified 26 cases as germinal center (GC) and a 38 as non-GC. The use of RT-MPLA profiling on 21 PCFCL, LC cases confirmed their categorization as GC and of 23 PCDLBCL-LT as non-GC. The MYD88 mutation was specifically present in the PCDLBCL-LT group at high prevalence (around 70%). Overall survival was poorer for patients with PCLBCL-LT than with PCFCL-LC (p = 0.0002). In parallel, non-GC cases had poorer overall survival than GC cases (p = 0.0007). Evaluating MYC, BCL2 and BCL6 status by interphase fluorescence in situ hybridization on a subset of patients, we also observed that MYC rearrangement was uncommon. Alternatively, the coexpression of MYC and BCL2 appeared to define patients with impaired prognosis even in the PCDLBCL-LT subgroup. Conclusions:

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For borderline cases, the WHO/EORTC update recommends to use morphological criteria such as confluent sheets of centroblast for PCDLBCL-LT whatever CD10 expression or to use the term PCLBCL, not otherwise specified (NOS) for rare unclassified cases. Our data strongly support the use of the simplified Hans algorithm as a first line histopathological tool. Dual expression of MYC and BCL2 appears a diagnostic criteria for PCDLBCL-LT diagnosis and defines patients with an impaired prognosis. However, the exclusive use of histopathological criteria may be limited for several reasons: Reproducibility of morphological classification is challenging. Consensus multi-head review is useful but does not evaluate interobserver reproducibility in routine evaluation. More challenging is the fact that PCDLBCL-LT usually contain a high number of large cell corresponding to M2 macrophages that may limit the use of cut-off thresholds for establishing positivity of a phenotypic biomarker unless double immunostaining with image analysis is employed. Our study indicates that genomic profiling with a simple and cost-effective RT-MLPA technique was able to differentiate PCFCL, large cell from PCDLBCL-LT according to their GC or non- GC profile. Mutational analysis and especially MYD88 status determination may also challenge or complement other diagnostic criteria especially for samples with less abundant tumor cells when using a sensitive molecular techniques. Besides this diagnostic challenge, establishing the mutational profile of cutaneous large B-cell lymphoma may also contribute to a better understanding of therapeutic response in such patients. References: 1. Menguy S, Frison E, Prochazkova-Carlotti M, Dalle S, Dereure O, Boulinguez S, et al. Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas. Mod Pathol. 2018;31(8):1332‑ 42.

2. Menguy S, Beylot‐ Barry M, Parrens M, Ledard A, Frison E, Comoz F, et al. Primary cutaneous large B‐ cell lymphomas: relevance of the 2017 World Health Organization classification: clinicopathological and molecular analyses of 64 cases. Histopathology. 2019;74(7):1067‑ 80.

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O-03

Mutations of the B-cell receptor pathway confer chemoresistance in primary cutaneous diffuse large B-cell lymphoma leg-type Océane Ducharme1,2, Marie Beylot-Barry1,2, Anne Pham-Ledard1,2, Elodie Bohers6, Pierre-Julien Viailly6, Thomas Bandres3, Nicolas Faur3, Eric Frison4, Béatrice Vergier2,5, Fabrice Jardin6, Jean- Philippe Merlio2,3, Audrey Gros2,3 Authors’ affiliations : 1Service de Dermatologie, CHU de Bordeaux, Bordeaux, France 2INSERM U1053, Equipe Oncogenèse des lymphomes cutanés, Université de Bordeaux 3Service de Biologie des tumeurs, CHU de Bordeaux, Pessac, France 4 Service d'information médicale, CHU Bordeaux, Bordeaux, France. 5Service d’Anatomie pathologique, CHU de Bordeaux, Pessac, France 6INSERM U1245 and Centre Henri Becquerel 76038 Rouen, France

ABSTRACT Introduction Primary cutaneous diffuse large B-cell lymphoma leg-type (PCLBCL-LT) is the most aggressive cutaneous B-cell lymphoma requiring a combination of poly-chemotherapy with Rituximab as first line therapy. About 50% of patients will experience progression or relapse without so far any predictive biologic marker. Our group first showed the original prevalence around 70% of MYD88L265P mutation in PLCBCL-LT that could serve as a diagnostic feature versus follicular large B-cell lymphoma. We also first characterized the specific mutational profile of PLCBCL- LT with mutations leading to constitutive activation of the NF-κB and B-cell receptor (BCR) signaling pathways. Such profile resemble to that of central nervous system or testicular lymphoma and the recent cytogenomic classification of large B-cell lymphomas pooled them into a common C5 or MCD subgroup derived from activated B-cells displaying MYD88 and CD79B mutations. Within such specific cutaneous lymphoma subtype, we tried here to determine if the genomic profile may predict therapeutic response and help to design personalized second line therapy.

Material and Methods Using lymphopanel next generation sequencing, we analyzed 14 PCLBCL-LT cases with complete response and 18 with relapsing/refractory disease. Among the latter, 14 tumor pairs at diagnosis and relapse/progression were analyzed to assess whether genetic changes could be associated with or selected by disease progression.

Results PCLBCL-LT patients harboring one mutation that targets one of the following BCR signaling genes (CD79A/B or CARD11) displayed a reduced progression-free survival and specific survival

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(median 18 months, P=0.002 and 51 months, P=0.03 respectively, whereas median duration in the wild type group was not reached) and were associated with therapeutic resistance (P=0.0006). Longitudinal analyses showed that both MYD88 and CD79B were the most conserved mutated genes at high allelic frequency between primary samples and those obtained at relapse or progression.

Conclusion Our study supports that evaluating the genomic profile of cutaneous large B-cell lymphoma has not only a descriptive/classification interest but also predicts in patients with BCR mutations therapeutic resistance to conventional first line immunochemotherapy. These patients may therefore benefit from adjuvant or second-line selected therapy.

References Ducharme, O., Beylot-Barry, M., Pham-Ledard, A., Bohers, E., Viailly, P.-J., Bandres, T., Faur, N., Frison, E., Vergier, B., Jardin, F., et al. (2019). Mutations of the B-cell receptor pathway confer chemoresistance in primary cutaneous diffuse large B-cell lymphoma leg-type. J. Invest. Dermatol. https://doi.org/10.1016/j.jid.2019.05.008.

Mareschal, S., Pham-Ledard, A., Viailly, P.J., Dubois, S., Bertrand, P., Maingonnat, C., Fontanilles, M., Bohers, E., Ruminy, P., Tournier, I., et al. (2017). Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg-type by massive parallel sequencing. J. Invest. Dermatol. 137, 1984–1994.

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O-05

Primary Cutaneous Marginal Zone B-Cell Lymphoma in Children and Young Adults Introduction Primary cutaneous marginal zone B-cell lymphoma is rare in children and adolescents (PCPMZL) with only 23 previously reported cases in patients below 20 years-old. We present 5 cases of PCPMZL and review the previously published cases. Methods We collected 5 cases of PCMZL in pediatric and young adult patients at our institution from 2016 to 2019. Ten biopsies were available for histopathological and molecular analysis. In all cases, routine light microscopy, immunohistochemical studies, and molecular biologic studies (IgH rearrangements) were performed. Clinical data of all cases was reviewed. Results Patients (3 females and 2 males ) age ranged from 9 to 21 years, with median age at diagnosis of 12 years. Two patients had clinical history of chronic diarrhea; one suffered from atopic dermatitis, one of hereditary increase of lipoprotein A and the other one a previous history of lymphoblastic B-cell leukemia. Four patients presented with multiple lesions involving different anatomic sites: trunk, limbs and arms. Whereas in one patient, 3 small papules on the left elbow were seen. Two patients, suffered recurrence of the disease two years after diagnosis. Histopathologically, the characteristic appearance of PCMZL was found in 8 of 10 specimens, with nodular infiltrates composed of small lymphocytes in the interfollicular compartment, surrounding reactive germinal centers, with presence of clusters of monotypic plasma cells situated mainly at the interface with the normal dermis or epidermis. Expression of MNDA, PD1, CD123, CD30 and SIgG is still being evaluated. IgH and TCR PCR studies were performed. Conclusions PCMZL in pediatric or young patients are rare. They differed from cases published in adults in their initial multicentric location and frequent rate of recurrence. Biological features that could be related to this peculiar biological behavior are still being investigated. References 1: Amitay-Laish I, Tavallaee M, Kim J, Hoppe RT, Million L, Feinmesser M, Fenig E, Wolfe MEL, Hodak E, Kim YH. Paediatric primary cutaneous marginal zone B-cell lymphoma: does it differ from its adult counterpart? Br J Dermatol. 2017 Apr;176(4):1010-1020. doi: 10.1111/bjd.14932. Epub 2017 Feb 20. PubMed PMID: 27501236.

2. Kempf W, Kazakov DV, Buechner SA, Graf M, Zettl A, Zimmermann DR, Tinguely M. Primary cutaneous marginal zone lymphoma in children: a report of 3 cases and review of the literature. Am J Dermatopathol. 2014 Aug;36(8):661-6. doi: 10.1097/DAD.0000000000000062. Review. PubMed PMID: 24698939.

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O-06

Introduction

Primary cutaneous diffuse large B cell lymphoma, leg type (PCDLBCL, LT) represents an aggressive lymphoma variant with unfavorable prognosis, frequent relapses and often extracutaneous spreading. Due to its aggressive course and its common clinical behavior to the systemic diffuse, large B-cell lymphoma (DLBCL), an anthracycline – based chemotherapy based on rituximab is recommended as first – line treatment for PCLBCL, LT. Although radiotherapy

(RT) is less effective than in other PCLBCL subtypes, it has been considered as a therapeutic option for local disease control. Herein, we report the results of a single institution retrospective analysis of PCDLBC, LT patients treated either with RT alone or with physician`s decision as first – line treatment, aiming to assess disease progression and/or recurrence as well as overall survival (OS) on these treatment groups.

Results

We retrospectively analyzed 20 patients diagnosed with PCDLBCL, LT, treated either with RT alone as first – line treatment (N = 8) or with investigator`s choice (N = 12), including chemotherapy alone or combined with RT and local excision. Complete response was achieved in 8 patients from the first group and 9 patients from the second group, with one treatment failure.

Six patients treated with RT – alone progressed with median time to progression (TTP) 12.5 months. In the second group, 5 patients progressed with median TTP 5.2 months. RT showed high rates of local disease control in both groups without skin relapses.

Conclusion

Although our data did not reveal any systematic differences in the disease progression distribution between the RT – alone and investigator`s decision, systemic treatment with chemotherapy was eventually applied in the majority of the patients (60%), either as first – line treatment or upon

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progression to RT – alone. RT as a first – line monotherapy did not improve the risk of disease progression, but significantly improved the local disease in both groups.

Table 1. Disease characteristics. RT: radiotherapy, ID: investigator`s decision

RT group ID group Sex Male 2 7 Female 6 5 Age at diagnosis (median) 83 (67 – 95) 69 (47 – 94) Clinical localization Lower leg 8 7 Upper extremities - 1 Trunk - 1 Scalp - 3 Extent of involved skin Solitary 2 3 Multiple 6 8 Generalized - 1 Lesion types Plaques - 2 Tumors 6 6 Both 1 4 Ulceration Yes 4 - No 4 12 Figure 1. Progression probability for both treatment groups. RT: radiotherapy, ID: investigator`s decision

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P-01

The evaluation of the quality of life of Polish patients with Cutaneous T-Cell Lymphoma and of the influence of the disease on their daily functioning. P-01 The study was conducted by Cognosco LLC commissioned by Takeda Pharma Poland. Data available courtesy of Takeda Pharma Poland. All opinions and conclusions are my own.

Agnieszka Giza1, Małgorzata Sokołowska-Wojdyło2, Hanna Ciepłuch3

1 Department of Hematology, Collegium Medicum of the Jagiellonian University, Krakow, Poland 2 Department of Dermatology, Venerology and Allergology, Medical University of Gdansk, Poland 3 Copernicus. Regional Oncology Centre, Gdańsk, Poland Introduction and Objectives: Cutaneous T cell lymphomas (CTCLs) which constitute about 75- 80% of primary cutaneous lymphoma is a chronic, incurable disease with involvement of a visible organ, and thereby having a profound impact on patients’ quality of life (QoL) well-being. The goal of our study is the evaluation of QoL of the Polish CTCLs pts and of the influence of the disease on their daily functioning. Materials and methods: The study was divided into two parts. Part I (explorative) conducted from November to December 2018 as an interview with pts and physicians about various aspects of the disease resulted in the design of questionnaire. Part II (quantitative) was carried out from January to March 2019 in which 67 pts with CTCL and 20 physicians taking care of them completed this and SKINDEX-29 questionnaire. The study involved patients with CTCL after at least one systemic treatment line treated under NHS program with Bexarotene or those who stopped program or who were not eligible to this program with mycosis fungoides or Sezarys’ syndrome or patients with primary cutaneous anaplastic large cell lymphoma (pcALCL) or with other CTCL subtypes. Results: Of the studied population: 63% were men, and 37% women. The mean age was 60 years, 42% pts were between 40-59 years. 64% pts live with family and have support from them and 22% were alone. The mean time of the disease duration is 7 years. The mean time from the occurrence of symptoms to diagnosis was 2,8 years and in 29% pts even longer. Statistically, until pts received the proper diagnosis, they had visited 3 physicians, 25% patients even 5. 74% pts manifested an anxiety associated with the diagnosis and were afraid of the worst. At the time of diagnosis 82% of pts trusted the skills of the physician who took care of them. 35% pts declared they had to take a day off work. 46% pts felt that their work is at risk and less effective. 19% of pts spoke about negative attitude of their boss and colleagues from the work due to disease. The daily functioning is impaired by CTCL in the opinion of 87% pts and 95% physicians, physical activity respectively, of 52% pts and 62% physicians, and insomnia was reported as a problem by 54% a pts and by 16 % physicians. 34% pts reported difficulties with dressing and 55% needed the help of another person. 18% of pts had problems with moving independently. The impairment of the emotional functioning is declared by 79% pts and by an even larger (95%) number of physicians. 50% pts feel overwhelmed by the disease,64% pts spoke about longing for an earlier lifestyle, 27% pts relied on others,31% pts hid their disease. More physicians (71%) than pts (57%) point at the need of psychological/psychiatric support for CTCL pts. The pts wanted to be informed at the time of diagnosis about treatment options available 40% of pts, side effects of treatment 39% of pts, specialists treated CTCL 33% of pts and would like to know stories and experience of other patients 39% of pts. These interests did not change in the course of treatment of the disease. The level of the general public awareness of CTCL was evaluated as very low according to 84% of pts and 95% physicians. 46% of pts met with wrong judgment of their disease by other people. The ignorance of society had a large impact on well-being of pts according to 58% pts and 90% physicians.

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Conclusions: Our analysis has demonstrated profound impact of CTCL on each aspect of QoL (physical, emotional and professional).Patients considered diagnostic process too long and anxiety evoking, but after diagnosis they trusted physicians taking care of them. The symptoms of the disease worsen in time, even when treated. Symptoms decrease pts’ physical functioning and make them dependent on others. CTCL affects pts’ emotions – there is a strong need for psychological support. CTCL changes pts’ professional lives - wages, working hours and work conditions are reduced. CTCL awareness in society is low .

References: 1. Willemze R, Hodak E, Zinzani PL, Specht, Ladetto. (2018) Primary cutaneous lymphomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 29(suppl_4):iv30-iv40. 2. Sampogena F, Frontani M, Baliva G , Lombardo G.A, Alvetreti G, Di Pietro C, Tabolli S, Russo G, Abeni G. Quality of life and psychological distress in patients with cutaneous lymphoma. Br J of Derm 2009, 160: 815-822.

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P-02

Skin-specific quality of life in cutaneous T-cell lymphoma compared to other dermatologic diseases Bhat TS1, Herbosa CM1, Rosenberg AR1, Mehta-Shah N2, Semenov YR3,4, Musiek AC1

1Washington University School of Medicine, Division of Dermatology; 2Washington University School of Medicine, Division of Oncology; 3Massachusetts General Hospital, Department of Dermatology; 4Harvard Medical School, Department of Biomedical Informatics

Introduction Health-related quality of life is exceedingly poor among patients with cutaneous T-cell lymphoma (CTCL). There are multiple instruments currently in use to measure health-related quality of life (HRQoL). This includes generic instruments such as the Health Utilities Index Mark 3 (HUI3) and the RAND 36-Item Short-Form Health Survey (SF-36), which are designed to be applicable across a wide range of populations and interventions, as well as dermatology-specific instruments such as the Skindex-29. The Skindex-29 is a validated dermatology-specific 30-item instrument with three domains: symptoms, emotions, and functioning. Higher domain scores, expressed on a 100-point scale, indicate lower levels of quality of life. Published cutoff scores of ≥39, ≥42, and ≥52 on the Symptoms subscale; ≥24, ≥35, and ≥39 on the Emotions subscale; and ≥21, ≥32, and ≥37 on the Functioning subscale indicate mildly, moderately, and severely impaired quality of life, respectively.(1) The objective of this study was to assess the skin-specific HRQoL in patients with CTCL compared to other dermatologic diseases using the Skindex-29.

Methods Patients with mycosis fungoides (MF) or Sézary syndrome (SS) were recruited from a multidisciplinary CTCL clinic or general dermatology clinic at Washington University School of Medicine between 5/2017 and 9/2019. Patients completed the paper-based version of the Skindex- 29. Scores were compared to published data from Klein et al for various dermatologic diseases using individual t-tests.(2)

Results 130 patients completed more than 75% of the Skindex-29. The median age was 66 (range 24-91), 81% of the patients were white, and 19% of the patients were advanced-stage (stages IIB-IV). Advanced-stage patients had worse QoL than early-stage patients in all domains. Study Symptoms p value Emotions p value Functioning p value Group , mean , mean , mean (SD) (SD) (SD)

Early- 31.7 (23.3) 29.6 16.6 (22.7) stage (24.5) (n=105) <.0001 <.0001 <.0001 Late- 59.4 (20.4) 57.6 47.9 (30.9) stage (26.4) (n=25) *Scores are on a scale from 0 to 100. Higher scores indicate worse QoL.

Early-stage patients met criteria for mildly impaired quality of life based on the Skindex threshold for the Emotions domain. Advanced-stage patients met criteria for severely impaired quality of life for the Emotions domain. For the Symptoms and Functioning domains, early-stage patients did not meet criteria for any overall impairment of quality of life. However, advanced-stage patients still met criteria for severely impaired quality of life for both the Symptoms and Functioning domains.

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Simple linear regression revealed that among patients with CTCL, age was a significant predictor for the Emotions subscore (p=0.0069). The Emotions subscore decreased (improved) with increased age. Age remained a significant predictor in multivariable regression models controlling for demographics and stage. Though increased age also corresponded to improved Symptoms and Functioning subscores (better QoL), these associations did not reach significance (p=0.6536 and p=0.0899, respectively).

The Symptoms subscore was worse in patients with all-stage CTCL than vitiligo and non- melanoma skin cancer (NMSC). Emotions and Functioning subscores were also worse in all-stage CTCL than NMSC. Sample Symptoms, Emotions, Functioning, Disease p value p value p value size Mean(SD) Mean(SD) Mean(SD) All-stage 130 37.1(25.2) − 35.0(27.1) − 22.7(27.3) − CTCL Vitiligo 245 13.9(14.6) <.0001* 35.9(23.6) 0.7336 16.7(19.5) 0.0286 NMSC/AK 136 29(20) 0.0043* 20(19) <.0001* 9(14) <.0001* Without 107 14(12) <.0001* 9(13) <.0001* 4(8) <.0001* skin disease

Patients with advanced-stage CTCL had worse QoL than patients with acne and psoriasis (all domains); pemphigus (Symptoms, Emotions); cutaneous lupus erythematosus (Symptoms, Functioning); eczema and epidermolysis bullosa (Emotions, Functioning); and dermatomyositis (Functioning). Functionin Sample Symptoms, Emotions, Disease p value p value g, p value size Mean(SD) Mean(SD) Mean(SD) Advanced-stage 25 59.4(20.4) − 57.6(26.4) − 47.9(30.9) − CTCL Acne vulgaris 63 30(19) <.0001* 41(25) 0.0070* 16(16) <.0001* Psoriasis 44 42(21) 0.0013* 39(27) 0.0072* 23(27) 0.0008* Pemphigus 126 37(22) <.0001* 37(22) <.0001* 33(23) 0.0294 CLE 178 41.3(23.8) 0.0004* 49.1(27.8) 0.1515 28.4(25.6) 0.0006* Eczema 102 48(23) 0.0247 41(27) 0.0065* 26(26) 0.0004* Epidermolysis 75 49(25) 0.0623 35(26) 0.0003* 31(24) 0.0056* Bullosa Dermatomyositis 41 44.9(24.3) 0.015 50.4(26.1) 0.2833 28.2(26.6) 0.0078*

Conclusions

Compared to other dermatologic diseases, CTCL, especially in advanced stages, is associated with poorer HRQoL. The improvement in Emotions subscore with age mirrors findings in psoriasis and CLE and suggests that the psychosocial impact of cutaneous disease may be greater in younger patients. The Emotions subscore must therefore be interpreted cautiously as a comparator measure, as age may be a confounding factor when comparing QoL in skin diseases with different age distributions. The marked findings in the CTCL population underscore the importance of including HRQoL as an essential outcome measure when evaluating clinical progress and designing clinical trials in this patient population.

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1. Prinsen CAC, Lindeboom R, Sprangers MAG, Legierse CM, de Korte J. Health-Related Quality of Life Assessment in Dermatology: Interpretation of Skindex-29 Scores Using Patient- Based Anchors. Journal of Investigative Dermatology. 2010;130(5):1318-22. 2. Klein R, Moghadam-Kia S, Taylor L, Coley C, Okawa J, LoMonico J, et al. Quality of life in cutaneous lupus erythematosus. Journal of the American Academy of Dermatology. 2011;64(5):849-58.

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P-03

Qualitative assessment of quality of life in patients with cutaneous T-cell lymphoma Bhat TS1, Herbosa CM1, Rosenberg AR1, Jeffe D2, Mehta-Shah N3, Semenov YR4,5, Musiek AC1

1Washington University School of Medicine, Division of Dermatology; 2Washington University School of Medicine, Division of Oncology; 3Massachusetts General Hospital, Department of Dermatology; 4Harvard Medical School, Department of Biomedical Informatics

Introduction While the quality of life (QoL) burden associated with CTCL has been reported previously, our group and others have largely relied on QoL instruments that were not developed specifically for patients with CTCL. Our objective was to examine the lived experience of CTCL and capture the aspects of QoL affected by CTCL that are not currently represented in existing QoL instruments.

Methods Participants were recruited using purposive sampling from a multidisciplinary CTCL clinic. Patients were recruited by stage and symptom severity. Eighteen one-on-one semi-structured interviews were conducted with patients with mycosis fungoides (MF) or Sézary syndrome (SS) between May 2019 and June 2019 by a medical researcher trained in qualitative methods using a topic guide developed empirically and from literature review. We used a grounded theory approach as described by Glaser and Strauss in order to analyze our qualitative interview data, with inductive thematic analysis used to identify themes that emerged from the data.(1) Interviews were coded using a three-step process involving open coding (bits of meaning that generated codes), axial coding (defining themes), and selective coding (developing an overarching model of the themes).

Results

Psychological and social functioning Lack of awareness Even after receiving a diagnosis, the rarity of the disease and the lack of understanding surrounding it were frustrating to many patients. Patients struggled to explain CTCL to friends and family members, and there were few online or written resources available. Many assumed that patients’ disease was contagious, with patients mentioning that others assumed they had chicken pox or even leprosy. Patients often had to adjust and correct for this assumption, including obtaining documentation for contact sports and for travel on public transport.

Self-image and concern about appearance Sixteen patients reported significant concern about their appearance, with 10 patients noting specifically that they felt self-conscious and embarrassed. The visibility of CTCL set it apart from other diseases and opened patients to scrutiny, curiosity and ridicule, which largely prevented patients from participating in their normal daily activities, including going out in public. Patients described feelings of physical and social isolation and felt that the disease affected not only the way that others viewed them, but also the way that they viewed themselves. Seventeen patients noted a feeling of “otherness,” the perception that they were different than others and not ‘normal’, in irrevocable ways.

Depression, hopelessness, and uncertainty These changes resulted in significant mood symptoms, with 11 patients reporting sadness or depression related to their disease and 4 patients reporting suicidal ideation. Additionally, half of the patients interviewed noted that they had experienced hopelessness related to their disease, especially in relation to the pervasiveness of their clinical symptoms. Patients also reported significant distress associated with the uncertainty in prognosis.

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Clinical symptoms and associated concerns (among 18 patients)

Itch 16 Heat regulation 6 Pain 12 Skin dryness 6 Fatigue 12 Constitutional symptoms 3 Sleep 11 Hair loss 2 Skin breaks (skin fissuring, 11 Weight loss 2 lacerations, and sores) Eyes tearing up 1 Skin flaking 10 Wrinkled skin 1 Skin redness 8

16/18 patients reported pruritus. Patients reported significant psychological distress associated with their itch, including frustration, depression, and frank suicidality. Many patients also reported pain associated with their disease, which affected the way that patients interacted with the world in addition to their mobility and ability to perform activities of daily living. Some patients attributed their pain specifically to skin breaks, including skin fissuring, lacerations, and sores. Skin flaking was also associated with significant impairments in social functioning, particularly as it contributed to patients’ feelings of humiliation and the feeling of being examined by others. Managing this symptom was particularly time-consuming.

A majority of patients (11/18) also reported that their symptoms interfered with sleep, which significantly impacted their quality of life and functioning in other realms. Patients reported difficulty falling asleep due to symptoms, particularly itch, physical discomfort, and pain, as well as worry about their disease. These symptoms also woke patients during the night and prevented them from going back to sleep.

Treatment burden Treatment for CTCL was a significant burden for patients in terms of logistics, finances, and side effects. Many patients described the distress associated with fighting with insurance companies for coverage of needed therapy. Side effects of therapy included, among others, skin burning and redness, additional skin rashes, skin blistering, burning eyes, fatigue, nausea and vomiting, and generally ‘feeling sick’. Patients also gained new medical conditions as a result of therapy that required lifelong management. Patients noted that they would value their quality of life over trying to cure (or continuing to treat) their CTCL if side effects of therapy grew too severe.

Conclusions Our findings confirm the utility of many of the questions in the recently published MF/SS QoL survey, with many patients reporting burden of treatment (15/18), trouble coping with daily demands (13/18), uncertainty (12/18), depression (11/18) and suicidality (4/18), and hopelessness (9/18), but demonstrate that it is incomplete. Identifying the concerns and priorities that separate CTCL patients from other patient populations, including problems with sleep and concern about appearance, is paramount in developing a comprehensive instrument that adequately captures their illness experience.

References 1. Glaser BG SA. The Discovery of Grounded Theory: Strategies for Qualitative Research New York: Aldine de Gruyter; 1967 [Available from: http://www.worldcat.org/title/discovery-of- grounded-theory-strategies-for-qualitative-research/oclc/253912.]

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P-04

The impact of gender, age, race/ethnicity, and stage on quality of life in a spectrum of cutaneous lymphomas Xochiquetzal U. Martinez1, Tracey Stiller2, Joycelynne Palmer2, Matthew Loscalzo3, Estella Barrios3, Farah R. Abdulla1, Jasmine Zain4, Steven T. Rosen4,5, Christiane Querfeld1,5 1Division of Dermatology, 2Division of Biostatistics, 3Patient & Family Resource Center, 4Department of Hematology/Hematopoietic Cell Transplantation, & 5Beckman Research Institute, City of Hope, Duarte, CA, USA

Introduction: Cutaneous lymphomas (CLs) are a group of rare, potentially disfiguring and disabling cancers that can have a significant impact on quality of life (QoL). While previous studies have examined QoL in patients with mycosis fungoides (MF) and Sézary syndrome (SS), QoL in patients with other types of CL has not been evaluated. The major aims of this study were to determine what factors impact QoL in all CL patients and in demographic and clinical sub- populations of patients; and secondarily, to understand the efficacy of our use of social work in the clinic. Methods: The Cutaneous Lymphoma Distress Questionnaire (CL-DQ) was used to assess QoL in all CL patients that were seen in a multidisciplinary CL clinic setting. The CL-DQ scores were analyzed to identify which factors most negatively influenced QoL in CL patients. In addition, the scores were compared to patient demographics, i.e. gender, age, and race/ethnicity, and clinical data, i.e. type of CL, clinical stage for patients with MF/SS, type of treatment, and involvement of social work. Results: The study population consisted of 151 patients presenting with distinct types of cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL). Notable across our study population was the presence of pruritus (32%), worry about disease progression (43%), frustration with disease (44%), and feelings of shame or embarrassment regarding the appearance of the skin (28%). Significantly, when examining sub-populations of patients, QoL was found to be most negatively affected in females, younger patients, Blacks, and patients with advanced MF and SS (Figures 1 and 2). Furthermore, CL was discovered to have the most widespread effect on QoL in SS patients and, to a lesser degree, Black patients. Examination of non-MF/SS CL patients revealed findings that reflected those noted in the entire study population. CD30+ LPD patients and those with other sub-types of CTCL reported frustration, most frequently, at 33% and 50%, respectively. Whereas, 25% of patients with CBCL expressed worry regarding disease progression. No significant difference in responses was noted when comparing those with and without SW involvement. Figure 1. Skin disease interferes with social life and interactions most significantly in SS patients.

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Figure 2. Skin disease interferes with social life and interactions most significantly in Black patients.

Conclusions: QoL is most significantly impaired in patients with advanced MF/SS, and correlates with other characteristics such as gender, age, and race/ethnicity. However, other distinct types of CL also affect QoL and are associated with psychosocial distress. Our findings highlight the need for QoL assessment in all CL patients and further examination of disparities noted across demographic groups. Additionally, our results provide a rationale for expanding the role of SW to target specific patient needs. Social work intervention, education, and counseling may improve symptoms.

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Psychiatric comorbidity in patients with cutaneous T-cell lymphoma Bhat TS1, Herbosa CM1, Rosenberg AR1, Mehta-Shah N2, Musiek AC1, Semenov YR3,4

1Washington University School of Medicine, Division of Dermatology; 2Washington University School of Medicine, Division of Oncology; 3Massachusetts General Hospital, Department of Dermatology; 4Harvard Medical School, Department of Biomedical Informatics

Introduction Hodak et al found that MF/SS is significantly associated with depression and anxiety in the Israeli population, but the extent of psychiatric comorbidity among CTCL patients across the United States has not been explored.(1)

Methods Patients with MF/SS were recruited from a multidisciplinary CTCL clinic or general dermatology clinic of a CTCL specialist between May 2017 and October 2019. Controls without a diagnosis of cutaneous lymphoma were recruited from the same provider’s general dermatology clinic. Demographics, comorbidities, and medication use were collected at the time of questionnaire completion. Patients completed paper-based versions of the Health Utilities Index Mark 3 (HUI3), RAND 36-Item Short-Form Survey (SF-36), and Skindex-29. Medication use was excluded if specified for indications outside of symptoms of depression or anxiety, including pruritus, pain, and insomnia. Individual items included in the emotional and mental health subscores for each HRQoL instrument were included in analysis.

Results 132 MF/SS patients and 132 general dermatology control patients were included in the study.

Psychiatric Comorbidity The prevalence of documented depression or anxiety disorders was similar between CTCL patients and controls (p>0.05). Items of the SF-36 assessing whether patients’ feelings of depression or anxiety had interfered with work or other daily activities (SF17-19) were highly predictive of provider-assigned depression or anxiety diagnosis (OR 2.68-4.094). However, more severe scores on most individual Skindex items and other generic HRQoL items were not significantly associated with provider-assigned depression or anxiety diagnosis.

Psychiatric medication The prevalence of use of psychiatric medication at the time of questionnaire completion was similar between CTCL patients and controls (p>0.05). Among medication users, significantly fewer CTCL patients had previously received a corresponding psychiatric diagnosis compared to controls (56.10% vs. 83.72%) (p<0.05). Of patients who had received a psychiatric diagnosis, a greater proportion of MF/SS patients were not taking psychiatric medication compared to controls (30.00% vs. 15.22%) (p=0.0993). As a result, the correlation between depression or anxiety diagnosis and current use of psychiatric medication was high in controls but moderate in patients with CTCL (rs = 0.81 vs. 0.55, respectively).

Individual Item Analysis In both CTCL and control populations, patients with documented depression or anxiety reported more severe symptoms than patients without a psychiatric diagnosis. On all questions and subscales, CTCL patients with documented depression or anxiety had the most severe QoL impairments. Notably, CTCL patients without a psychiatric diagnosis still reported worse HRQoL and more severe symptoms of depression or anxiety than the general control population. Independent of baseline depression or anxiety diagnosis, CTCL patients were more likely to report higher severity of depressive or anxiety symptoms compared to controls. CTCL

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significantly increased the odds of a higher response by a factor of 2.1-5.7 on generic HRQoL items and a factor of 2.7-9.5 on skin-specific items.

HRQoL Summary Scores Neither control patients with a psychiatric diagnosis nor CTCL patients with a psychiatric diagnosis met criteria for psychiatric morbidity based on the published threshold for mean SF-36 Mental Health score (≤52). CTCL patients both with and without a psychiatric diagnosis met criteria for overall QoL impairment based on the Skindex Emotions score (≥24), with CTCL patients with a psychiatric diagnosis meeting criteria for severe QoL impairment and surpassing the recommended threshold to detect psychiatric morbidity (≥39). In contrast, control patients with a diagnosis of depression or anxiety did not meet criteria for psychiatric comorbidity and in fact did not meet criteria for any QoL impairment based on Skindex Emotions cutoffs.

Conclusions Psychiatric comorbidity is underdiagnosed and suboptimally managed in patients with cutaneous T-cell lymphoma. MF/SS has a profound and pervasive impact on patients’ quality of life, and patients report considerable symptoms of depression and anxiety compared to general dermatologic controls. Even after adjusting for the baseline prevalence of diagnosed psychiatric comorbidity, CTCL significantly increased the odds of worse psychiatric symptoms on both generic and skin-specific HRQoL items. Patient-reported severity of psychiatric symptoms was not predictive of provider-assigned psychiatric diagnosis, indicating that the diagnosis of psychiatric comorbidity in this population does not accurately reflect the health status of these patients. Additionally, though the use of psychiatric medication was similar between CTCL patients and controls, medication use was not highly correlated with previous psychiatric diagnosis in the CTCL population. Among patients taking psychiatric medication, significantly fewer CTCL patients had previously received a corresponding psychiatric diagnosis compared to controls. This may indicate that providers recognize some symptoms of depression or anxiety in these patients, but the appropriate diagnosis and management of underlying depression or anxiety can be improved. Among patients who had received a psychiatric diagnosis, a greater proportion of MF/SS patients were not taking psychiatric medication compared to controls, suggesting that even when these patients are appropriately diagnosed, they may suffer from mismanagement and undertreatment. Existing QoL instruments are not sufficient to accurately detect psychiatric morbidity in a dermatologic patient population. Neither CTCL patients nor controls with pre-existing psychiatric diagnoses met criteria for psychiatric morbidity using the SF-36 Mental Health score threshold despite the severity of their psychiatric symptoms, indicating that published SF-36 cutoffs may not be sufficiently sensitive to detect psychiatric morbidity in a dermatologic patient population. The published psychiatric cutoff for the Skindex-29 was sufficient to accurately detect psychiatric morbidity in CTCL patients with psychiatric diagnoses, the patients who reported the most severe symptoms of depression and anxiety. However, it was not sufficient to accurately detect psychiatric morbidity in control patients with pre-existing psychiatric diagnoses. The current psychiatric screening threshold for the Skindex is therefore adequate to identify patients with the most severe psychological impairments but is not suitable for screening for early or less severe signs of mental illness. Our findings demonstrate that it is vital to routinely assess psychological well-being in patients with MF/SS, even in patients who have not previously received psychiatric diagnoses, with a low threshold for referral for further psychiatric evaluation. A directed, disease-specific QoL instrument that incorporates somatic symptoms is necessary to address the unique and considerable QoL burden in the CTCL population.

References 1. Hodak E, Lessin S, et al. New insights into associated co-morbidities in patients with cutaneous T-cell lymphoma (mycosis fungoides). Acta dermato-venereologica. 2013;93(4):451-5.

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Synergistic Therapeutic Drug Screening Using JAK Inhibition for CTCL Authors: S. Yumeen1, F. N. Mirza1, J. M. Lewis1, Amber L. O. King1, S. Kim1, K. Carlson1, F. Foss2, M. Girardi1. Affiliations: 1Department of Dermatology, Yale School of Medicine, New Haven, CT, USA; 2Department of Internal Medicine, Hematology and Oncology, Yale School of Medicine, New Haven, CT, USA; Introduction Cutaneous T cell lymphoma (CTCL) is a rare malignancy of skin-homing T lymphocytes that is more likely to involve the peripheral blood in advanced stages. For such patients, there are few available systemic treatment options and prognosis remains poor. High-throughput screening has emerged as a method for identification of novel therapeutic compounds. Cutaneous T cell lymphoma (CTCL) is driven by single nucleotide and copy number variants in pathways of T cell activation, cell cycle regulation, and DNA structural regulation, and dysregulation of JAK/STAT signaling has also been described. Ruxolitinib is a small molecule inhibitor of JAK1/2 and has been reported to inhibit in vitro proliferation of three CTCL cell lines. We sought to examine its effects on CTCL cells in vitro and evaluate synergy with known and putative therapies for CTCL. Methods Malignant CTCL cells were antibody magnetic-bead isolated from patients’ peripheral blood and exposed to high-throughput single and combination drug screening. Cytotoxic effects were evaluated as cell viability via ATP quantitation at 72h. Synergy was evaluated by the Chou- Talalay method. Changes in gene expression were evaluated by qRT-PCR at 24h. Results CTCL patient-derived cells showed differential responses to ruxolitinib. Of seventeen patient samples tested, seven appeared to have low-response to ruxolitinib, with a mean IC50 of 0.15µM, while ten were sensitive with a mean IC50 of 205.6 µM. There was no statistically significant difference in response to ruxolitinib between patients classified as B1 and B2. Ruxolitinib- sensitive samples showed synergy when combined with either a BCL2 (venetoclax), proteasome (bortezomib), BET (mivebresib) or HDAC inhibitor (vorinostat) with combination index (CI) <1. Ruxolitinib-resistant samples also showed strong kill potentiation with these same inhibitors. BCL2 gene expression was repressed in sensitive samples treated with ruxolitinib alone or in combination with venetoclax, suggesting a mechanism for cooperative activity.

Table 1. Patient derived malignant cells had variable response to JAK inhibition.

Code IC50 Ruxolitinib (µM) 1 1.85E-05 2 0.00017 3 0.0021 4 0.0048 5 0.1919 6 0.26 7 0.6384 8 1.7 9 1.788 10 4.882 11 9.4 12 21

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13 83.72 14 36.87 15 319 16 16 17 934.2 Mean 84.10 Standard Deviation 232.30

Conclusions JAK monotherapeutic targeting has differential response on the viability of CTCL cells. However, when used in combination with a BCL2, proteasome, BET or HDAC inhibitor, JAK inhibition demonstrates consistent synergistic efficacy, regardless of monotherapy sensitivity. Thus, JAK inhibition, in combination with other agents, may represent a novel therapeutic strategy in treatment of CTCL. References 1. Chou T-C. Drug combination studies and their synergy quantification using the Chou- Talalay method. Cancer Res. 2010;70(2):440–446. 2. Pérez C, González-Rincón J, Onaindia A, et al. Mutated JAK kinases and deregulated STAT activity are potential therapeutic targets in cutaneous T-cell lymphoma. Haematologica. 2015;100(11):e450–3.

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Phospholipase C Gamma 1 (PLCG1) mutations in Sézary cells drive NFkB, AP-1 and NFAT signalling and mediate resistance to the calcineurin inhibitor Tacrolimus.

Charlotte E Flanagan, Varsha M Patel, Christine L Jones, Sean J Whittaker, Tracey J Mitchell.

Introduction & Objectives: Somatic mutations in PLCG1 frequently occur in Mycosis Fungoides and Sézary Syndrome (SS). R48W and S345F are the most common PLCG1 mutations across all T-cell malignancies. The aims of this in vitro study were to determine the effect of PLCG1 mutations on i) PLCγ1 downstream signalling pathways and ii) the activity of the calcineurin inhibitor Tacrolimus.

Materials & Methods: Reporter constructs for 9 PLCG1 mutations (p.R48W1, p.S312L1, p.D342N1, p.S345F1, p.S520F1, p.R1158H2, p.E1163K1, p.D1165H1 and the in-frame indel p.VYEEDM1161V2) identified in SS, were generated by site directed mutagenesis of wild-type PLCG1. PLCγ1 expression was confirmed by western blotting. NFAT, AP-1 and NF-kB transcriptional activity was determined by luciferase reporter assays in HEK293 cells and J.gamma1 cells (PLCγ1 null cell line derived from the Jurkat T-cell leukaemia cell line). Cell staining for caspase activation and flow cytometry were used to determine cellular apoptosis in response to Tacrolimus treatment (30 μM;20 hrs).

Results: Our data demonstrates in basal conditions, the majority of the mutations confer PLCγ1 gain-of- function activity through significant (p≤0.03) downstream activation of NFκB and NFAT transcriptional activity in HEK293 cells. Results for R48W and S345F, the two commonest PLCG1 mutations reported in CTCL, were confirmed in J.gamma1 cells. Interestingly, the gain of function activity associated with R48W was significantly higher in the T-cell derived cell line than in HEK293 cells. Furthermore, in contrast to wild-type PLCγ1, activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 activity in HEK293 cells, and these results were also confirmed in J.gamma1 cells using p.S345F mutation as a proof of principle. J.gamma1 cells transduced R48W and S345F, followed by treatment with the calcineurin inhibitor Tacrolimus were shown to be significantly (p<0.01) less apoptotic than cells transduced with wild-type PLCγ1.

Conclusions: These data show that PLCG1 mutations commonly reported in CTCL lead to constitutive activation of PLCγ1 (Figure 1) and enhance cell survival in response to Tacrolimus treatment. This study provides compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.

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Figure 5 Summary of gain-of function activity of PLCγ1 mutant p.S345F vs wildtype PLCγ1. In the presence of p.S345F mutation, AP-1, NFAT and NFkB activity increased and this activity is independent of phosphorylation at p.Y783.

Funding: CEF was supported by the King’s Bioscience Institute and the Guy’s and St Thomas' Charity Prize PhD Programme in Biomedical and Translational Science. The authors would like to thank the National Institute for Health Research (NIHR) Biomedical Research Centre at the Guy’s and St Thomas’ NHS Foundation Trust and King’s College London for funding this independent research.

References: 1. Patel V.M.* and Flanagan C.F.* et al. Frequent and Persistent PLCG1 Mutations in Sézary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling. J Invest Dermatol. pii: S0022-202X(19)32679-X [Epub ahead of print] (2020). *These authors contributed equally to this work. 2. Kiel, M. J. et al. Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK–STAT pathway in Sézary syndrome. Nat. Commun. 6, 8470 (2015).

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R-05

Screening for Novel Combination Treatments for Cutaneous T Cell Lymphoma for Expedited Development

Authors: F. N. Mirza1, S. Yumeen1, J. M. Lewis1, A. O. King1, S. Kim1, K. Carlson1, F. Foss2, M. Girardi1.

Affiliations: 1Department of Dermatology, Yale School of Medicine, New Haven, CT, USA; 2Department of Internal Medicine, Hematology and Oncology, Yale School of Medicine, New Haven, CT, USA;

Introduction:

Cutaneous T cell lymphoma (CTCL) is incurable and often fatal in advanced stages, with an overall response rate (~30-50%) to current FDA-approved therapies that warrants a search for new and more effective treatments. High-throughput screening has emerged a method for identification of novel therapeutic compounds. A 1000+ agent panel rich in phosphatase or kinase inhibitors and natural compounds (Selleckchem Kinase Inhibitors, Enzo Phosphatase Inhibitors, MicroSource Gen-Plus) was screened on patient-derived CTCL cells. Several promising agents that demonstrated over 80% effective kill were further evaluated for their synergistic/potentiating effects in combination in vitro.

Methods:

Malignant cells were isolated from the peripheral blood from 13 CTCL patients using antibody- magnetic bead sorting for CD3+CD4+ and CD7- and/or CD26- based on the known aberrant phenotype of the patient; peripheral blood from 8 healthy controls was sorted for CD3+CD4+ cells, and 5 CTCL cell lines – HH, Hut78, Sez4, SeAx, and MyLa – were cultured. Cells were exposed individually and in combination to: (1) seven agents with prior FDA-approval or available over the counter as natural compounds  sanguinarine  pyrvinium  quinacrine  ciclopirox  salinomycin  gentian violet, and  certinib; (2) and two additional agents in phase II clinical trials  BIIB021  NVP-BGT226.

Cell viability was assessed using an ATP luminescence assay at 72h, and synergy or potentiation was evaluated by the Chou-Talalay method.1

Results:

Single agent titrations identified pyrvinium as demonstrating the following the highest average potency against patient-derived malignant cells. Patient-derived CTCL cells were found to be statistically significantly more susceptible to salinomycin and gentian violet than normal cells. Interestingly, patient-derived CTCL cells and CTCL cell lines behaved significantly differently when incubated with sanguinarine, BIIB021, quinacrine, and certinib, demonstrating that cell lines do not faithfully recapitulate CTCL pathophysiology.

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Patient Control Cell Lines

IC50 of Single Drugs Patient Control Cell Lines

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In combination, the average Chou-Talalay synergy indices revealed synergy in patient-derived CTCL cells for the following combinations:

 salinomycin + ciclopirox, gentian violet, or sanguinarine, and  gentian violet + sanguinarine

While several patient samples demonstrated resistance to NVP-BGT226 and BIIB021 as single agents, combinations revealed statistically significant potentiation for salinomycin and NVP- BGT226 in comparison to normal cells. Furthermore, both NVP-BGT226 and BIIB021 demonstrated at least moderate average potentiation for nearly all drug combinations in patient- derived CTCL cells.

Conclusions:

Taken together, these results suggest that salinomycin and pyrvinium, alone and in combination, may represent novel therapies for the treatment of CTCL, while NVP-BGT226 and BIIB021 may represent important potentiators in other drug combination strategies. The compounds validated in this screen may allow for expedited development for use in CTCL.

References: 3. Chou T-C. Drug combination studies and their synergy quantification using the Chou- Talalay method. Cancer Res. 2010;70(2):440–446.

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R-06

The synergistic pro-apoptotic effect of HDAC and PARP-1 inhibition in cutaneous T-cell lymphoma is mediated via Blimp-1

Oleg Kruglov, MD,1 Xuesong Wu,2 Sam T. Hwang, MD, PhD, 2 Oleg E. Akilov, MD, PhD1

1Cutaneous Lymphoma Program, Department of Dermatology, University of Pittsburgh, Pittsburgh, PA, USA 2Department of Dermatology, University of California Davis, USA

Introduction and Objectives: The therapy of advanced mycosis fungoides (MF) still presents a therapeutic challenge, and search for new therapeutic targets is ongoing. Poly(ADP-ribose) polymerase 1 (PARP-1) was shown to be upregulated in patients with advanced MF and could be druggable by a new class of chemotherapeutic agents, PARP1 inhibitors, which are already in clinical trials for other malignancies. However, the role of PARP1 inhibitors in MF has never been established. Materials and Methods: We used MBL2 murine model of cutaneous lymphoma to investigate the clinical benefit of PARP1 inhibitor, talazoparib alone and in combination with other anti- lymphoma medications. Mechanism of action was investigated with RNA sequencing assay and confirmed by flow cytometry and Western blotting. Results: The cytotoxic effect of talazoparib on MBL2 cells was due to G2/M cell cycle arrest via the upregulation of MDM4 and CDKN1A (Fig.1). The in vivo experiments confirmed the clinical effect of talazoparib on cutaneous lymphoma tumors. When talazoparib was combined with HDAC inhibitor, romidepsin, the cytotoxic effect was synergized via downregulation of DNA- repair genes, FANCA, FANCD2, and TOPBP1 and stimulation of apoptosis via Blimp-1/Bax axis. Romidepsin increased the expression of IRF8 and Bcl-6, leading to upregulation of Blimp1 and Bax; while talazoparib upregulated Blimp-1 and Bax via upregulation IRF4 leading to cleavage of caspases 6 and 7. Conclusions: Thus, a combination of talazoparib with romidepsin demonstrated the synergistic anti-lymphoma effect and warranted further investigation in a clinical trial.

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Figure 1. Talazoparib arrests the cell cycle of lymphoma cells at G2/M. (A) Dose-response cell-viability curves at 24 hrs for MBL2 and MDA-MB-436. (B) Changes in transcriptome associated with the effect of talazoparib at IC25 on the survival properties of MBL2 cells identified by RNA sequencing. Volcano plot (C) The transcriptional changes in apoptosis pathway in MBL2 cells treated with IC25 of talazoparib. (D) The transcriptional changes in the cell cycle pathway in MBL2 cells treated with IC25 of talazoparib. (E) Cell cycle assessment after treatment of MBL2 cells treated with IC25 of talazoparib. Click-iT EdU with FxCycle Violet Ready Assay. Representative flow cytometry. (F) The differences between the phases of cell cycle of cells treated with IC25 of talazoparib and controls. ****, p<0.0001. (G) Hoechst 33258 fluorescent staining to show DNA breakage in MBL2 after exposure to Talazoparib. Representative images, Fluorescent macroscopy (x100). (I) An outline of the murine experiment. (J) The measurement of the thickness of the orthotopic tumors after treatment with talazoparib. n = 5 mice per group. 21 days after treatment. ***, p<0.001 (K) The measurement of the diameter of the regional lymph nodes after treatment with talazoparib. n = 5 mice per group. 21 days after treatment. *, p<0.05 (L) Representative images of regional lymph nodes treated with PBS or talazoparib. (M) Flow cytometry-based analysis of early (Annexin V+ PI-) and late (Annexin V+ PI+) apoptosis in MBL2 cells treated with talazoparib alone, romidepsin alone, or talazoparib with romidepsin. (N) Analysis of early and later apoptosis in flow-cytometry based assay (Annexin V and PI) in MBL2 cells treated with talazoparib alone, romidepsin alone, or talazoparib with romidepsin. ***, p<0.001, ****, p<0.0001.

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S-01

A small molecule CCR2 antagonist depletes tumor macrophages and synergizes with anti- PD1 in a murine model of cutaneous T cell lymphoma (CTCL)

Xuesong Wu1, Rajinder Singh2, Daniel K. Hsu1, Yan Zhou1, Sebastian Yu1, Dan Han1, Zhenrui Shi1, Mindy Huynh1, James J. Campbell2, and Sam Hwang1 1 Department of Dermatology, School of Medicine, University of California Davis, Sacramento, California, United States; 2 ChemoCentryx, Inc. Mountain View, California, United States Address: Suite 1300, 3301 C St., Sacramento, CA 95816 Phone: 916-734-6377; Email: [email protected]

Extended abstract

Tumor-associated macrophages (TAMs) recruited from blood monocytes are key in establishing an immunosuppressive tumor microenvironment (TME). Besides secreting immunosuppressive cytokines and producing angiogenetic factors to support tumor growth, TAMs bear PD-L1 on cell surface which confers them direct suppressive function via antigen-specific tolerance. Accumulating evidence shows high macrophage density in TME is associated with poor patient prognosis. Of note, the presence of TAMs in human non-Hodgkin’s lymphoma has been shown to not only correlate with patient’s survival but also with response to treatment (Steidl et al., 2010). In skin lesions of mycosis fungoides or Sezary syndrome, macrophages exist abundantly and produce chemokine ligand 18 (CCL18) which can induce chemotaxis for Th2 cells. A tumor microenvironment such as this is thought to allow or foster tumor progression in CTCL. The CCL2-CCR2 axis mediates monocyte trafficking into tumors, which has become an attractive therapeutic target in cancer therapy. Preclinical studies demonstrated a remarkable anti-tumor activity axis in pancreatic cancer by applying CCR2 inhibitors to block monocyte recruitment. As a result, CCR2 inhibitors are now in clinical trials for patients with pancreatic cancer. Herein, we examine a small molecule compound of CCR2 inhibitor, called “CCR2i”, with CTCL mouse models to assess the possibility of controlling tumor growth, investigate its influence for TME on immune cell composition, and optimize the treatment effect by combinatory application of CCR2i and anti-PD1.

We started with the examination of CCR2i in our previously established syngeneic mouse T cell lymphoma in ear skin (Wu et al., 2014). Following oral CCR2i administration, depletion of macrophages was achieved as early as two days after tumor implantation in the ear TME. The CCR2i-mediated macrophage depletion by blocking monocyte recruitment; by contrast, neutrophil recruitment was not affected in the TME. Quantitative RT-PCR detected increases in the ear tumors of immune stimulatory inflammatory cytokines, e.g. IFN-γ and IL-12, in CCR2i-

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vs. vehicle-treated mice. Within two weeks the tumors from control groups attained the maximum size, while CCR2i-treated mice exhibited much smaller tumor sizes. Immunohistochemistry also revealed CCR2i-treated tumors possessed significantly more CD8+ T cells, which demonstrated their essential role in CCR2i-induced tumor inhibition with a following study of using CD8 depleting antibody. In addition to this syngeneic mouse tumor model, we also administrated CCR2i in a xenograft model by implanting HH cells, a human-derived CTCL line, in immunodeficient NSG mice. Two weeks of daily dosing for CCR2i resulted in significant tumor growth inhibition accompanied with reduced macrophage infiltration and decreased CD31 expression. Thus, CCR2i blocked tumor growth in both an inflammation-dependent syngeneic murine T cell lymphoma model and in a xenograft human CTCL tumor model.

The role of checkpoint inhibitor anti-PD1 is well established in so far as tumor cells as well as antigen-presenting cells, such as macrophages, expressing PD-L1 and their interaction with PD- 1 positive CD8-T cells to render them anergic with respect to antitumor activity. We found that tumors formed in our syngeneic model exhibited a significant increase of PD-L1 compared to cultured cells in vitro, implying presence of cancer immune evasion. Antibody blockade of PD- 1/PD-L1 axis in this mouse model could retard the tumor growth but was unable to eliminate the tumors, which prompted us to combination therapy over a single agent in order to achieve enhanced anti-tumor effects. In the combination therapy for syngeneic tumor mice, anti-PD1 began on the same day of first CCR2i treatment and tumor initiation. When mice were euthanized after completing the two weeks treatment, the number of IFN-γ-producing CD8-T cells in the spleen was significantly increased in the group treated with the combination of CCR2i and anti- PD1, suggesting these mice exhibited more robust anti-tumor immunity. While the anti-PD1 or CCR2i single treatment resulted in no to mild reduction of tumor size, the combination therapy showed significantly higher efficacy in inhibiting tumor growth, with ~80% rate of tumor-free survival.

Overall, our findings provide strong evidence that the CCR2i, particularly in combination with an immune checkpoint inhibitor, reduces tumor growth and is a potential future treatment of cutaneous T cell lymphomas.

References

Steidl C, Lee T, Shah SP, Farinha P, Han G, Nayar T, et al. Tumor-associated macrophages and survival in classic Hodgkin's lymphoma. N Engl J Med 2010;362(10):875-85. Wu X, Schulte BC, Zhou Y, Haribhai D, Mackinnon AC, Plaza JA, et al. Depletion of M2-like tumor-associated macrophages delays cutaneous T-cell lymphoma development in vivo. J Invest Dermatol 2014;134(11):2814-22.

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S-02

Title: Investigating the role for POT1 gene dysfunction in Primary Cutaneous T-cell Lymphoma

Introduction & Objectives: Somatic single nucleotide variants (SSNVs) in the POT1 (protection of telomeres 1) gene are frequently occurring in CTCL. This study investigates the hypothesis that POT1 mutations lead to telomere length (TL) dysregulation and inappropriate activation of DNA damage repair responses (DDRR), which contribute to genomic instability in CTCL.

Materials & Methods: Tetracycline inducible stably transfected Flp-In cell lines were generated with the integration of a single copy of either wild-type (WT) or mutant POT1. Q-PCR was used to measure TL in both WT and mutant cell lines and telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay. The effects of POT1 mutation on the DDRR was determined by FLOW cytometry.

Results: TL Q-PCR revealed up to a 3-fold increase in telomere length in 2 out of 5 mutants compared to WT and there was also increased telomerase activity in these cell lines. Mutant cells also displayed significantly increased expression of DNA damage markers including yH2AX, p53, 53BP1 and RPA.

Conclusions: This study demonstrates telomere length dysregulation as well as providing evidence for exaggerated DNA damage responses in the context of POT1 mutation, that may contribute to genomic instability, a key feature of CTCL. Crucially, increased expression of the ATR DNA damage pathway marker (RPA) provide a POT1-specific mechanism. Future work will focus on determining whether the ability of the mutated POT1 protein to bind telomeric DNA are compromised and whether the effects of POT1 mutation can be attenuated by pharmacological means.

Funding: I would like to thank my funding body the Wellcome trust who have supporting this work in its entirety.

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S-04

INHIBITION OF INTEGRIN αVβ3 IMPROVES REXINOID ANTITUMORAL ACTION ON CUTANEOUS T CELL LYMPHOMA (CTCL) F. Cayrol1, MV. Revuelta 2, MM. Debernardi1, A. Paulazo1, JM. Phillip 2, N. Zamponi2, HA. Sterle1, MC. Díaz Flaqué1, C. Magro3, J. Ruan 2, GA. Cremaschi1* L. Cerchietti2* 1. Instituto de Investigaciones Biomédicas (BIOMED-UCA-CONICET)), BsAs, Argentina. 2. Hematology and Oncology Division, Weill Cornell Medicine, NY. 3. Department of Pathology Weill Cornell Medicine, NY. Introduction: Bexarotene (Bex) is an oral RXR agonist used for the treatment of early and advanced-stage CTCL. Bex is associated with hypothyroidism in about 95% of patients, which are prophylactically managed with the administration of high doses of the thyroid hormone (TH) levothyroxine (T4). Paradoxically, we have previously found that physiological levels of TH increase the proliferation of CTCL cells mostly by activating the membrane integrin αVβ3 receptor. Here, we determined the influence of TH replacement therapy on the anti-lymphoma activity of Bex, an unknown topic with clinical implications. Methods: We performed in vitro and in vivo experiments with HuT78 and MJ human CTCL cells and EL4 murine cells, using RNA sequencing, RT-qPCR techniques and functional assays to determine cell viability, apoptosis and motility. Results: We first analyzed changes in cell viability and transcriptomics in HuT78 and MJ cells. We found that Bex decreases cell viability and induces apoptosis in both CTCL cell lines (Fig.1A). Regarding the effects of Bex in transcriptomics we found, as expected, an enrichment of genes involved in cell proliferation, differentiation and apoptosis. We also found regulation of genes involved in cell chemotaxis, motility and the immune response like TBX21 and IFNG (Fig.1B). These results suggest that Bex treatment could also regulates anti-lymphoma immunity by increasing IFN-gamma release from CTCL cells.

We then evaluated the effect of physiological levels of TH on Bex activity in vitro and found that TH decreased the anti-lymphoma effect of Bex measure as increased viability or decreased apoptosis in both CTCL cells (Fig.2). These results support the notion that Bex should not be administered with TH replacement. However, this is not possible because hypothyroidism is associated with marked immunosuppression that could favor CTCL progression. We thus determined the impact of TH replacement on the anti- lymphoma effect of Bex using a murine orthotopic T-cell lymphoma model. We implanted murine EL4 TCL cells in the hypodermis of immunocompetent mice. Once tumors developed, mice were randomized into treatment with vehicle (Veh) and Bex alone (Bex) or with TH replacement (BexT4+). Compared to Veh, the administration of Bex significantly decreased lymphoma growth in

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both conditions although slightly better in mice without T4 replacement (Fig.3A). However, mice treated with Bex alone showed a significant decrease of total and activated CD8 T cells in the tumor draining lymph nodes (Fig.3B) and increase of myeloid-derived suppressor cells in the spleen. We then investigated whether integrin αVβ3 inhibition would be sufficient to blunt the TH- induced decrease on the antineoplastic efficacy of Bexarotene. The lack or low expression of integrin aVb3 in normal T-cells offers a rationale for a selective effect on CTCL cells whithout affecting the immune microenviroment. We found that integrin V3 mRNA silencing increases Bex-induced apoptosis and decrease proliferation of CTCL cells. The same effect was found using cilengitide, a selective pharmacological inhibitor of integrin V3 (data not shown). Finally, we tested if this mechanism could be therapeutically capitalized to improve Bex treatment using our murine TCL model. This time mice with tumors were randomized into Veh, BexT4+, cilengitide alone (Cil) or in combination with BexT4+ (BexT4+Cil). After 12 days of treatment, mice treated with BexT4+Cil significantly smaller tumors despite TH replacement therapy compare to all of the treatments (Fig.4).

Taken together, our data indicates that inhibition of the integrin αvβ3 is an effective strategy to improve bexarotene-based treatments in CTCL and sufficient to avoid the up-regulation of tumor growth that would result from TH replacement therapy while maintaining the lymphoma immunity.

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S-05

Presentation code: S-05 HDAC inhibitor resminostat counteracts disease-related gene expression and cytokine secretion in CTCL cells

G. Streubel, A.C. Bretz, T. Wulff, U. Parnitzke, H. Kallus, K. Kronthaler., M. Borgmann, S. Hamm. 4SC AG, Martinsried, Germany.

Introduction: Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma characterized by skin- infiltrating malignant T cells with an increasing bias towards the T helper cell type 2 (Th2) during disease progression. The majority of CTCL patients suffer from severe itching (pruritus), which has a significant negative impact on the patient’s quality of life. Importantly, systemic and chemotherapeutic therapies for CTCL last only about four months until disease progression (TTNT = 4.5 months). Therefore, a key challenge in the treatment of CTCL is to maintain and to stabilize the initial therapeutic response. Addressing this urgent clinical need, the class I, IIb, IV HDAC inhibitor resminostat is currently under clinical evaluation for disease control after systemic therapy (RESMAIN, NCT02953301). Here, we explore the mode-of-action of resminostat and evaluate its potential for the treatment of CTCL.

Material & Methods CTCL cell lines representing mycosis fungoides (My-La CD4+, HH) and Sézary syndrome (HuT- 78) were used in cell proliferation assays and cell cycle analysis. Genome-wide gene expression alterations upon resminostat treatment were explored by RNA-Seq. Then, RT-qPCR and antibody-based techniques (ELISA, Western blot) were used to further investigate the changes on mRNA and protein level.

Results Resminostat mediated anti-proliferative and pro-apoptotic effects on both MF and SzS cells. In addition to cytotoxic and cytostatic effects, HDAC inhibitors have been reported to change the chromatin landscape and to modulate gene transcription. Indicating an interference with epigenetic processes, resminostat induced the hyperacetylation of specific histone residues linked with transcriptional regulation. In line with this finding, global gene expression analysis upon resminostat treatment uncovered extensive gene expression changes in CTCL cells upon resminostat treatment. Gene ontology analysis indicated an enrichment of oncogenic driver signaling pathways in the down-regulated gene set. Moreover, resminostat modulated the mRNA level of genes associated with the pathogenesis of CTCL (Figure 1). The expression of skin- homing receptors, which mediate the infiltration of malignant T cells into the skin, was reduced by resminostat. Furthermore, resminostat up-regulated a gene expression signature representative of the Th1 cell type, and down-regulated genes of the Th2 cell type, thus favoring the beneficial Th1 cell phenotype. In agreement with this observation, resminostat decreased the mRNA level and protein secretion of the Th2 and itch-mediating cytokine IL-31, suggesting that resminostat might improve pruritus (Figure 2).

Conclusion Taken together, these findings corroborate that resminostat has the potential to counteract the malignant T cell population and to improve itching in CTCL patients. In conclusion, our preclinical data support the hypothesis that resminostat treatment will improve or stabilize CTCL and its symptoms. In the ongoing RESMAIN trial (NCT02953301), resminostat-mediated gene expression changes from patient samples as well as cytokine level from the blood, e.g. IL-31, will be determined in the translational biomarker program.

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Figure 1. Resminostat modulates the expression of disease-associated genes in CTCL cells A. Gene expression analysis in My-La CD4+, HH and HuT-78 treated with vehicle control (0.1% DMSO) or 4 µM Resminostat. Heatmaps depict transcript levels in TPM (Transcripts Per Kilobase Million) of skin homing T cells receptors, genes linked with advanced disease progression, and genes representing the Th2 or Th1 cell phenotype. (n = 3) B. RT-qPCRs of the Th1 cell transcription factor STAT4 and the Th2 cell transcription factor STAT6. C. RNA expression of the Th1 T cell fate transcription factor TBX21/T-bet. TBX21/T-bet acts in concert with STAT4 to coordinate Th1 T cell differentiation. D. Upregulation of the TBX21/T-bet protein upon resminostat treatment.

Figure 2. Resminostat down- regulates the Th2 cytokine IL- 31 A. RNA-Seq reveals a decrease of IL-31 mRNA after treatment of My-La CD4+ MF cells with resminostat for 24 h compared to vehicle control (0.1% DMSO), (n = 3). B. Changes of relative IL-31 mRNA level following treatment of My-La CD4+ cells with resminostat or vehicle control. C. ELISA for secreted IL-31 protein [pg/mL] from supernatants of My-La CD4+ cells treated with 0.3, 1.0 and 3.0 µM resminostat for 24 h. The right-hand graph depicts the cell viability.

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S-06

Single cell RNA sequencing analysis to define therapeutic targets in cutaneous T-cell lymphoma Christiane Querfeld, MD, PhD1,3, Xiwei Wu, PhD2, Emine Guelsen Gunes, PhD2, Steven T. Rosen, MD3 1Division of Dermatology, 2Department of Molecular Medicine, 3Hematologic Malignancies and Stem Cell Transplantation Institute, City of Hope, Duarte, CA, USA

Introduction: T cell exhaustion is a hallmark of CTCL and alterations in mRNA profiles correlate with immune checkpoint expression, with potential clinical relevance (Querfeld et al. 2018). There is no marker that can distinguish malignant CD4+ T cells from benign CD4+ T cells in the infiltrate and intratumoral heterogeneity poses a major challenge to treatments and long term remissions. The microenvironment in CTCL harbors multiple immune cells that may contribute to the development of resistance to drug treatments; however, the genomic and molecular determinants of response to therapeutic agents remain incompletely understood. The aim of our study was to distinguish malignant from non-malignant T cells based on TCR α/β repertoires and to understand the transcriptional landscapes of malignant and non-malignant cells in the TME while on anti-PD-L1 therapy. Methods: Migrated cells from skin explants were harvested and subsequently analyzed by single cell sequencing method. We performed paired single-cell RNA and T cell receptor (TCR; alpha/beta) sequencing on ~ 3000-4000 cells (for each treatment naïve and anti-PDL1 treated) from skin lesions of 6 MF patients at baseline and during treatment (cycle 1 day 15) with anti-PD-L1 + lenalidomide. Results: Results identified 14 clusters. Differential expression (DE) of genes in each of the unique clusters were identified by comparing gene expression from cells in each cluster to that of all other cells in the dataset, using a cut-off of P < 0.05 and further requiring expression of the gene from >25% of cells in the cluster. Thus, DE- identified genes are expressed either uniquely or by a large proportion of cells within each cluster compared to all other clusters. TCR clones in these cells were also characterized. Through this combined analysis, we revealed differences in the diversity, clonal expansion and T cell phenotype that differentiated expanded malignant T cell populations (cluster 0-3) from non- malignant cells including tumor infiltrating lymphocytes (TILs), regulatory T cells (Tregs), NK/T cells, B cells, antigen presenting cells (dendritic cells, macrophages) and other cells (stromal, epithelial cells) (cluster 4-13). (Fig 1B). Notably, of the single cells with productive TCRs in the treatment-naïve skin lesion, 70% consisted of a single highly expanded T cell clonotype containing TCR variable regions TRAV12-2/TRBV9 exhibited gene enrichment related to high proliferation (GIMAP), and high T cell activation (CD69, ICOS). We have characterized the

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expression and significance of PD1, LAG3, CTLA4, TIM3 and ICOS in malignant and non- malignant T cell clusters. Our results demonstrated differential expression of these targets in

malignant T cells (clusters 0-4). However, non-malignant T cell phenotyping revealed an enriched tumor-infiltrating CD8+ T cell population at baseline with upregulation of LAG3 gene expression, and FOXP3+ CD4+ regulatory T cell population with high expression of CTLA4 and ICOS in all, but one baseline sample. Conclusions: Single cell analysis revealed differences in the diversity, clonal expansion and T cell phenotype that differentiated expanded malignant T cell populations from non-malignant tumor-infiltrating lymphocytes and other immune cells. A total of 14 stable clusters emerged, including 4 clusters for malignant CD4+ T cells, 1 cluster for tumor- infiltrating CD8+ T cells and 1 cluster for FOXP3+ Tregs each with its unique signature genes. Our preliminary results suggest that potential targeting of ICOS, CTLA4 and/or LAG3 will reverse T cell dysfunction in TILS and Tregs, respectively and increase clinical benefit.

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T-01

T-01: Cusatuzumab for Treatment of CD70-positive Relapsed/Refractory Cutaneous T-cell Lymphoma in a Phase 1/2 Clinical Trial Introduction Cutaneous T-cell lymphoma (CTCL), a heterogeneous group of rare non-Hodgkin’s lymphomas, is characterized by infiltration of malignant clonally expanded T lymphocytes in the skin and is very challenging to treat at advanced stages. Fig. 1 Cusatuzumab Modes of Action We observed that CD70 is overexpressed on the circulating malignant cells of a responding CTCL patient included in the phase 1 trial of cusatuzumab1. Cusatuzumab (ARGX-110) is a monoclonal antibody (mAb) targeting CD70 with multiple Modes-of-Action: 1) blocking of proliferation and survival signals; 2) direct cell killing via its effector functions including enhanced antibody dependent cell-mediated cytotoxicity (ADCC) and 3) inducing anti-tumor immunity2 (Fig. 1). With only limited expression in normal tissues and strong expression on tumor cells, CD70 is a very attractive target for antibody-based therapy in cancer. The role of CD70 and treatment with cusatuzumab was further investigated as part of a Phase 1/2 trial in relapsed/refractory (R/R) CTCL patients.

Methods As part of the trial (NCT01813539), adult patients with CD70+ R/R CTCL of different subtypes and stages were included. The primary objectives were to perform a dose-finding study in this patient population further expanding on the recommended cusatuzumab dose based on the Phase 1 experience (1 mg/kg intravenously Q3W)2, and to assess safety and exploratory efficacy. Secondary objectives were investigations of pharmacokinetics (PK), immunogenicity and biomarkers of drug activity. Adverse events were graded according to NCI-CTCAE v. 4.03.

Results Characteristics: Skin biopsies of 35/48 R/R CTCL patients (73%) were CD70 positive (>10%) and 27 met the criteria and were enrolled in the trial. The primary diagnosis was Mycosis Fungoides (MF, N=14), Sézary Syndrome (SS, N=9), one patient with subcutaneous panniculitis- like T-cell lymphoma (SPTCL), two patients with follicular T-helper-cell lymphoma (FTH), and one patient with anaplastic large-cell lymphoma (ALCL). Ten (37%) patients had Stage IV disease and the median age was 67 years (range: 25-84 years). Eleven patients were dosed at 1 mg/kg and 16 at 5 mg/kg. Recommended dose: After evaluation of safety, PK and anti-drug antibodies (ADA) for the 11 patients receiving 1 mg/kg cusatuzumab Q3W, the dose was increased to 5 mg/kg for 16 patients. In about half of the patients dosed with 1 mg/kg, ADA affected the minimal concentration (Cmin) at the end of first treatment cycle resulting in cusatuzumab concentrations Below Level of Quantification (BLQ: 0.5 μg/ml). ADA levels increased with the number of administrations, leading to increased clearance of cusatuzumab that in a few patients also lowered maximal concentrations (Cmax) of cusatuzumab. In contrast, the majority of patients administered with 5 mg/kg cusatuzumab did not develop ADA with the exception of three patients, who developed a transient low titer ADA response that did not influence Cmax and Cmin levels.

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An example of how this impacted response was observed for a 79-year old patient (MF-1), with MF Stage IB and six lines of prior treatments, including mogamulizumab, who reached a PR (red circle in Fig. 2A and Fig. 2C) with plaque-to-patch regression after five doses of 1 mg/kg cusatuzumab (Fig. 2A-B). Cmin reached BLQ after cycle 2 (C2), whereas Cmax was not measurable after C8 until EOT and the levels of ADA reached a plateau after C6 (Fig. 2D). A rapid decrease in mSWAT score was observed until at C6, when high ADA titers were measured; the response was lost after C8 and progression occurred at C17.

Table 1. AE in ≥2 patients Safety: Cusatuzumab was safe and well Cohort A Cohort B Preferred term N G1-2 G3 G4-5 N G1-2 G3 G4-5 tolerated at both doses with a total of 106 Pyrexia 4 4 0 0 1 1 0 0 adverse events (AE) reported in 26/27 Pruritus 2 2 0 0 2 2 0 0 Asthenia 2 2 0 0 3 3 0 0 patients. Most common was pyrexia and Back pain 2 2 0 0 0 0 0 0 asthenia (5 patients each) (Table 1). Forty Chills 2 2 0 0 0 0 0 0 Diarrhoea 2 2 0 0 0 0 0 0 events in 16 patients were drug-related as Dyspnoea 2 2 0 0 1 1 0 0 Oedema Peripheral 2 2 0 0 0 0 0 0 considered by the investigator, of which Staphycoccal infection 2 2 0 0 0 0 0 0 infusion-related reactions (IRRs) were the GGT increased 0 0 0 0 2 1 1 0 Abdominal pain upper 1 1 0 0 1 1 0 0 most common (22 events in 8 patients). ECG QT prolonged 1 0 1 0 1 1 0 0 Eighteen SAEs were reported in 11 Fatigue 1 1 0 0 1 1 0 0 General physical health deterioration 1 1 0 0 1 0 0 1 patients, one was considered drug related. Headache 1 1 0 0 1 1 0 0 No differences between the doses. Vomiting 1 1 0 0 1 1 0 0 Cohort A: 11 at 1 mg/kg and 2 at 5 mg/kg; Cohort B: 14 at 5 mg/kg Responses: Best response was one complete response (CR) and five partial responses (PR, two at 1 mg/kg and three at 5 mg/kg) in 26/27 patients evaluable for response (overall response rate (ORR) of 23%). Mean duration on the study was four months with two patients still on the study at cut-off (June 2018): one patient in CR (SPTCL at 1 mg/kg for 10 cycles then 5 mg/kg Q6W, 24 months), one patient in PR (SS, 6.5 months) (Fig. 3A). Figure 2B (spider plot, N=24) shows that for several patients, the progression of the disease in the skin was halted after three to four doses of cusatuzumab with continued regression over time resulting in overall clinical activity in 15 of the 26 evaluable patients (58%) (Fig. 3B and 3A). SS patients dosed at 5 mg/kg resulted in three PR and two SD versus one PR and two SD for the patients dosed at 1 mg/kg. With an ORR of 50% in SS, cusatuzumab seems to be more efficient in this patient population than in MF.

Fig. 3. Clinical activity of cusatuzumab monotherapy (1 or 5 mg/kg) in 26 CTCL patients. A B

Conclusions Clinical anti-tumor activity in patients with R/R CTCL was observed after treatment with cusatuzumab at both 1 and 5 mg/kg, indicating a safe and promising treatment option for advanced CTCL. To achieve better exposure, 5 mg/kg (or higher) given Q3W (or more frequently) would

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be preferred. The drug gave better responses in Sézary Syndrome than in Mycosis Fungoides patients, but further investigation in different CTCL subtypes would be interesting.

References 1. Aftimos P, Rolfo C, Rottey S, et al. Phase I dose-escalation study of the anti-CD70 antibody ARGX-110 in advanced malignancies. Clinical cancer research 2017;23(21):6411-6420. 2. Silence K, Dreier T, Moshir M, et al. ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade. MAbs 2014;6(2):523-32.

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T-02

T-02: A Phase 1b Study Evaluating the Safety and Efficacy of Topical Administration of WP1220, a STAT3 Inhibitor, for Mycosis Fungoides (MF)

Malgorzata Sokołowska-Wojdyło1, Izabela Błażewicz 2, Berenika Olszewska 1, Ewa Zak 3, Sandra Silberman 4, Waldemar Priebe 5

1Medical University of Gdańsk (GUMed), Gdansk, 2Medical University in Gdansk, Gdansk, 3Dermin Spz.oo, Warsaw, 4Moleculin Biotech, Houston, Texas, 5MD Anderson Cancer Center, Houston, TX

Introduction & Objectives: MF, the most common variant of cutaneous T-cell lymphoma (CTCL) is a disease with symptomatic, disfiguring skin lesions. Standard and experimental treatments include topcialas and systemic therapies as well as allo-stem cell transplantation. STAT3 (Signal transducer and activator of transcription 3) has been identified as one of the regulator of MF, activation leading to tumor proliferation and suppression of immune responses. WP1220, a synthetic compound potentially inhibits p-STAT3 and the growth of CTCL cell lines. This Phase 1b study was designed to demonstrate safety and efficacy of WP1220 during and after treatment of MF. Materials and Methods: Topical treatment of 2-6 baseline index lesions in adults with stable disease ( at lest three month without treatment (1) or on the same treatment ( 3 on bexarotene and IFNa ) at stage IA (2 patients ), IIA (1), IIB (1) and 1 erythrodermic MF was with a 10% w/w strength ointment. Eligible subjects with progressive MF applied ointment twice a day. Primary endpoint was shift from baseline CAILS (Composite Assessment of Index Lesion Severity describing erythema, scaling, plaque elevation, hyper and hypopigmentation) scores to 84 day of treatment and day of follow-up (day 112). Secondary objectives included photographic validation by independent dermatologists. Biopsies were evaluated for STAT3 pathway activation. Adverse events (AE) were monitored. Results: Of 5 subjects enrolled, 9 lesions were assessed by the CAILS score from screening to day 84 and then to day 112 ( follow up after 1 month without treatment). The only AE was mild transient contact dermatitis in one subject. CAILS scores on index lesions were decreased in all subjects, but significantly decreased - in four of thems, with a median reduction of 56% (range 25%-94%) ( Table 1). Improvement was noted by 7 days of treatment and maintained in 4 of 5 cases in 1 month after discontinuation (Day 112).

Table 1A. CAILS of lesions ((*) (total sum of subtotals/ mean of subtotals )

1001 (2 1002 ( 2 1005( 2 1006 (2 CAILS lesions) (*) lesions) (*) 1003 ( 1 lesion) lesions) (*) lesions) (*)

screening 29/ 14,5 24/12 19 15/7,5 30/15

baseline 29/ 14,5 24/12 19 15/7,5 30/15

D7 21/ 10,5 17/8,5 19 15/7,5 30/16

D28 18/09 10/5 13 11/5,5 28/14

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D56 13/6,5 10/5 10 11/5,5 28/14

D84 11/5,5 8/4 4 11/5,5 21/10,5

D112 follow 12.cze 7/3,5 up 3 15/7,5 22/11

Independent dermatologic review based on photographic documentation is providing ( Example of result: Figure 1), as well as evaluations of the biopsy samples for the status of p-STAT3 in skin lesions (ongoing).

Figure 1. Patient No. 3 CAILS reduction from 19 to 3

Baseline Day 26 Day 56 Day 84 Day 112 CAILS 19 CAILS 13 CAILS 10 CAILS 4 CAILS 3

Conclusions: WP1220, an inhibitor of p-STAT3 pathway, shows safety and some efficacy in MF after 3 months of topical treatment in stable MF. This is the demonstration that inhibition of the STAT3 activation pathway with topical therapy has revealed efficacy in CTCL. A larger Phase 2 study is now being planned.

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T-03

High dose rate brachytherapy for the treatment of primary cutaneous lymphoma at complex sites and complex curves skin surfaces. Sim VR1, DeFrancesco I1, Child F2, Wain M2, Whittaker S2, Freeman K3, Jones E3, Aldridge S3, Morris S1 1 Clinical Oncology, Guys Hospital, London 2 Dermatology, Guys Hospital, London 3 Radiotherapy Department, Guy’s Cancer Centre, London Introduction Radiotherapy is an important treatment for cutaneous lymphomas with high response rates. Some sites of skin involvement are difficult to treat with standard orthovoltage or electron beam radiotherapy. We present a service evaluation of our experience using HDR brachytherapy. Methods Patients referred for radiotherapy to primary cutaneous lymphomas at difficult complex curves sites were selected for treatment with HDR brachytherapy. Two methods were used; Friesburg flap or 3D printed mould therapy. The patients were CT planned using Oncentra planning system and treated with I-192 on the Flexitron afterloader system. Patient’s response and toxicity outcomes were collected with photographs of the areas treated pre and post treatment.

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Results 19 patients (18 Mycosis Fungoides, 1 PCMZL treated) were treated using HDR brachytherapy from February 2014 – September 2019. 4 patients were planned using 3D printed mould technique and 15 patients were planned with a Friesburg flap applicator. The sites treated are Sites Hand Forearm Lower Foot Nose Eyebrow Limb No 5 5 3 3 2 1

Small fields were treated with 8Gy in 2 fractions over 2 days or 12Gy in 3 fractions over 3 days. Larger fields and re irradiation were treated with 24Gy in 12 fractions over 2.5 weeks or 20Gy in 10 fractions over 2 weeks. All patients had complete response to HDR Brachytherapy locally. Toxicities recorded CTCAE Grading No Radiation dermatitis Grade 2 1 Cellulitis Grade 2 1 Pain Grade 2 2

5 patients had a small local recurrence within the treated field that responded to superficial radiotherapy. Conclusion HDR brachytherapy is a useful technique for treating complex surfaces involved with cutaneous lymphoma, with a very high response rate and minimal toxicity.

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T-04

Intralesional oncolytic virotherapy results in tumor regression associated with the influx of cytotoxic T cells in cutaneous B-cell lymphoma

Ramelyte E1,2,*, Tastanova A1,2, Balazs Z3,4, Menzel U5, Turko P1,2, Beisel C5, Guenova E1,2, Krauthammer M3,4, Levesque MP1,2, Dummer R1,2

Introduction

Cutaneous B cell lymphomas (CBCL) are a group of B-cell derived lymphoproliferative diseases with cutaneous tropism. We initiated an investigator-initiated trial to investigate the impact of oncolytic virus on the immune compartment and clinical anti-tumor efficacy in CBCL.

Methods

Patients with CBCL limited to skin received intralesional oncolytic herpes virus Talimogene

108 PFU/ml 3 weeks later and every two weeks up to 9 injections. Biopsies were taken at baseline, after 2 and 5 injections. Samples were stained for H&E, CD79a and CD8. Clinical response was defined as reduction of erythema and infiltration. Histological response was defined as reduction of CD79a+ cells compared to baseline biopsy. In 3 patients sequential fine needle aspirations were collected for single cell RNA sequencing.

Results

To date, seven CBCL patients were treated in this study. The median age was 62y (range 46-79). 4 patients had cMZL, 2 had cDLBCL and 1 had cFL. All patients developed clinical and histological response in the injected lesions. 5 patients developed complete or near complete clinical response, 2 patients showed partial response. In histology, all patients showed reduction of CD79a+ and increase in CD8+ cells in after 5 injections compared to baseline, 2 patients developed histological complete response. All patients developed adverse events, 97% of those were grade 1-2. Single cell RNA sequencing of lesions demonstrated substantial alterations in the composition of the immune compartment and the activation status of malignant and reactive cell populations.

pcDLBCL, LT pcFCL pcMZL Overall (n=2) (n=1) (n=5) (n=8) Age (mean, SD) 63.5 (12.0) 69 (NA) 59 (10.0) 61.4 (9.56) Previous therapy Systemic therapy 1 (50%) 0 (0%) 2 (40%) 1 (35.5%) Surgery 0 (0%) 0 (0%) 2 (40%) 2 (25%) Radiotherapy 1 (50%) 0 (0%) 2 (40%) 3 (37.5%) Received injections Median [Min, Max] 8.00 [8.00, 8.00] 6.00 [6.00, 6.00] 8.00 [5.00, 8.00] 8.00 [5.00, 8.00] Therapy after EoT ChT, SCT, RT 1 (50%) 0 (0%) 1 (20%) 2 (25%) No therapy 1 (50%) 1 (100%) 4 (80%) 6 (75%) Response CR 1 (50%) 1 (100%) 3 (60%) 5 (62.5%) PR 1 (50%) 0 (0%) 2 (40%) 3 (37.5%)

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Conclusions

Oncolytic virotherapy with intralesional T-VEC shows efficacy in CBCL lesions. It causes clinical response and reduction of CD79a+ B-cell infiltration. The adverse events were mostly mild and self-limited, suggesting good safety profile.

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U-01

A phase II prospective study using non-myeloablative allogeneic transplantation in patients with advanced stage mycosis fungoides and Sezary syndrome

W-K Weng, S Arai, M Khodadoust, R Hoppe, YH Kim BMT/Oncology/Radiation Oncology/Dermatology, Stanford University School of Medicine, Stanford, CA, USA

Introduction: We report a phase II study of allogeneic transplant using a novel non- myeloablative conditioning in stage IIB-IV MF/SS patients.

Methods: Patients received TSEBT (20-36 Gy) as part of conditioning, immediately followed by total-lymphoid-irradiation (TLI, 8-12 Gy) and anti-thymocyte-globulin (ATG), and received allograft infusion as outpatients. This unique TSEBT-TLI-ATG conditioning may have reduced the disease burden in skin, lymph node and blood, respectively. The primary efficacy end point is day+180 PFS with a target rate of 75%.

Results: The study has completed the enrollment and transplanted 35 patients as planned (13 MF, 22 SS; median age 62 years (range 20-74); median prior systemic therapies of 5 (range 2-13); 7 stage IIB, 28 stage IV). All had active disease at the time of conditioning (skin: 100%, blood: 34%, lymph node: 63%, visceral: 14%). Patients tolerated the transplant well with 1- and 2-year NRM of 3% and 14%, respectively. The day+180 grade II-IV acute GVHD incidence was 16% and the 2-year moderate/severe chronic GVHD incidence was 32%. The 3-month post-transplant global ORR is 80% (20 CR, 8 PR). The day+180 PFS was estimated to be 73% (95% CI, 55- 85%). The 2-, 3- and 5-year OS were 68%, 62% and 56%, respectively. Using HTS of the T-cell receptor for MRD monitoring, we observed that 43% of the patients achieved molecular remission, which was associated with significantly lower incidence of disease progression/relapse (13% vs 94%, p<0.0001). Our study also included a much older population than other reported series with 8 patients 60-64 y/o and 12 patients > 65 y/o at the time of transplant. However, the 5-year OS is not different between patients who were > 65 y/o and < 65 y/o (50% vs 60%, p=0.791).

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Conclusions: We have developed an effective and potentially curative non-myeloablative allogeneic transplant for advanced stage MF/SS patients.

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U-02

Allogeneic hematopoietic stem cell transplantation as a curative treatment strategy for patients with advanced mycosis fungoides and Sézary syndrome: 2019 update of the Milan experience Onida F1, Valli V1, Saporiti G1, Alberti Violetti S2, Grifoni F1, Goldaniga M1, Casarin F1, Schiavone C3, Fanoni D2, Baldini L1, Grillo Ruggieri F3, Berti E2 1Hematology - BMT Center and 2Dermatology Unit, Fondazione Ca’ Granda IRCCS Ospedale Maggiore Policlinico, University of Milan, Italy. and 3 Radiotherapy Unit , Ospedali Galliera, Genova, Italy. Introduction: Although a few novel drugs have recently shown promising activity in mycosis fungoides (MF) and Sézary syndrome (SS), prognosis of patients with advanced stages or refractory disease remain poor, with median survival ranging from 1.4 to 3.4 years (Agar NS et al. JCO 2010). High-dose CT followed by autologous SCT allows achievement of clinical CR in a quite large proportion of patients but high rate of relapse leads to disappointing long-term results. Based on immunological rather than cytotoxic effect, allogeneic-SCT is the only potentially curative treatment option in carefully selected patients. However, morbidity and mortality related to myeloablative regimens counterbalances survival benefit of allotransplant. By decreasing non relapse mortality, reduced intensity (RIC) and nonmyeloablative conditioning (NMA) regimens lead to better outcomes in comparison to myeloablative ones. Here we update the long-term outcome of our RIC allo-HSCT experimental program. Methods: Since 09/2000, 46 patients (median age 54 years) with stage IIB/IV refractory MF (n=32) or SS (n=14) and a median time from diagnosis of 46 months underwent allo-HSCT. Donors were HLA-identical sibling in 18, unrelated in 23, and haploidentical related in 5 patients. The source of stem cells has been peripheral blood in 35 patients (87.5%), bone marrow in 4 (10%) and cord blood in 1 (2.5%). Median number of previous treatment lines was 6. Bridge to transplant strategy included TSEB in 14 patients and Brentuximab Vedotin in 2, of whome one showed pulmonary CD30+ disease infiltration. Conditioning regimens included FC/TBI200 or pentostatin/TBI200 in case of HLA-identical sibling donor, fludarabine/melphalan in MUD and Thiotepa/CTX/Flu/TBI in case of haploidentical donor. GvHD prophylaxis included CsA/MMF in all patients, with the addition of ATG in cases with UD and post-transplant CTX (50 mg/kg giorni +3 e +4) in haploidentical transplants. Results: Full donor chimerism was obtained in 38 out of 43 evaluable patients, in a median time of 2 months (range 1-12). A clinical CR was achieved in 29 patients (63%). As of January 2020, with a median follow-up of 82 months, 24 patients were alive with confirmed CR in 23 (96%). Out of the 17 patients who did not achieve CR, 13 died from progressive disease, 3 were treated with TSEB +/- donor lymphocyte infusion achieving a new durable CR status, while 1 is alive with disease 74 months after transplant. Transplant-related death occurred in 7 patients. Acute grade II-IV GvHD occurred in 15 patients (33%). Chronic GvHD occurred in 20 of the 42 evaluable patients (48%), being severe in 3 (7%). Outcomes at 5-years were: OS 49% (33%-65%),

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DFS 38% (23%-53%), NRM 14% (3%-24%) and relapse incidence 48% (33%-63%). However, when MF and SS were analysed separately, 5-yrs DFS were 27.5% (95% CI 11-44) and 56% (95% CI 32-81), respectively [Fig.1]. Apart from diagnosis, outcome appeared to be primarily associated with chemosensitivity and status of disease at transplantation. Of note, all the five patients who underwent haploidentical transplantation were alive and disease-free at the time of last f-up.

Figure 1.

Conclusions: After a median follow-up longer than 7 years, we confirm the efficacy of RIC allo- HSCT as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage CTCL, with results particularly encouraging in SS.

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Non-myeloablative allogeneic stem cell transplantation using TSEB TLI and ATG for Mycosis Fungoides(MF) and Sezary Syndrome(SS). Medium term results from a large single centre cohort.

Morris S L1, Palanicawandar R2, Grandi V1, De Francesco I1, Child F1, Wain M1, Whittaker S1, Mangar S2, Kanfer E2.

1. Guys Hospital, London, UK 2. Hammersmith Hospital, London, UK

Introduction Advanced Stage MF and SS has a poor prognosis. In 2012 we introduced a protocol involving TSEB, TLI and ATG conditioning. We report out medium term outcomes.

Methods Patients were selected with stage IIB to IVB MF and SS who had failed at least one prior systemic therapy. Patients were referred for transplant once a global partial or complete response was attained. The conditioning protocol involved TSEB (12Gy/8,24Gy/16 or 30Gy/20) followed by TLI 8Gy/10 and ATG 0.5mg/kg per day on Day-11 to Day -7, cyclosporine was started on day -3 and on day 0 oral MMF was started. Outcomes on response, toxicity and survival were collected.

Transplant protocol:

Results 30 patients were transplanted between August 2012 and May 2019. Median follow-up 3.38 years (range 0.5 – 7.0 years). 27 patients had MF and 3 patients had SS. Stage prior to transplant was IIB=6, IIIA=2, IVA2=16, IVB=6. Median number of prior systemic treatments was 4. The Global response prior to TSEB/TLI/ATG was CR=6, PR =22, SD=1, PD=1

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The 1-year OS was 73.2%, 2-year OS 65.7% and 3-year OS 59.7%. The median progression free survival was 1.29 years and 1-year PFS 51.6%, 2 year PFS 39.7%, 3-year PFS 37.3%.

PFS TSEB TLI ATG Allo for MF SS

100

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v

i

v

r

u 60

s

t

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e 40

c

r

e

P 20

0 0 2 4 6 8 Time

14 patients relapsed with a median time to relapse of 3.77 months (0.43 to 55.4 months).

9 patients received Donor lymphocyte infusions (7 for relapse and 2 for low chimerism). 2 patients went into CR following DLI.

At last follow up 18 patients are alive. 14 patients are alive in CR; 4patients are alive with disease

12 patients have died. 5 from MF/SS Disease progression 1 from Sepsis/GVHD and MF/SS 6 from Sepsis/GVHD in complete Remission

19 patients developed acute GVHD most commonly mild involving the skin. 5 patients (16%) had grade 2 or higher acute GVHD.

The 1- year non relapse mortality was 10%.

Conclusion The TSEB TLI ATG allogeneic stem cell transplant conditioning protocol is associated with durable remissions, high medium-term overall survival rates and low rates of NRM.

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V-03

Real-life experience on the use of brentuximab vedotin on nine mycosis fungoides patients: time to re-discuss treatment protocols? Authors: Cury-Martins J¹; Miyashiro D¹; Abdo ANR2; Pereira J²; Sanches JA¹ ¹ Department of Dermatology, Hospital das Clinicas, University of São Paulo Medical School, São Paulo, Brazil. ² Department of Hematology, Hospital das Clinicas, University of São Paulo Medical School, São Paulo, Brazil.

INTRODUCTION & OBJECTIVES: Brentuximab vedotin (BV) is an anti-CD30 antibody– drug conjugate that has received regulatory approval for CD30+ cutaneous T-cell lymphomas (CTCL) after the results of a phase 3 trial. Based on this study, recommended treatment regimen is 1.8 mg/kg, intravenous, once every 3 weeks, for up to 16 cycles. The aim of this study is to report a single center experience of BV use for mycosis fungoides (MF) in real-life setting, with particularities not included on a clinical trial, such as BV associated to conventional chemotherapy or to phototherapy, lower dose regimens or longer intervals between cycles and re- exposure to BV.

METHODS: retrospective analysis of MF patients treated with BV with special focus on treatment regimens (dosing, schedule, associated treatments).

RESULTS: From the nine patients treated with BV, 55% were male (5), 2 had folliculotropic MF and all presented large cell transformation. Median CD30+ on skin biopsies was 30% (<10-100), with 6 patients presenting at least 1 prior biopsy with no or scarce CD30+ cells. At BV start, median age was 40 years (29-70), median number of prior treatments was 5 (2-8); regarding TNMB stage: T – all were T3; N: N0=3, N1=1, N3=1, Nx=4; M – all were M0; B: B0=8, B1b=1. Patients were treated for a median of 14 cycles (4 still ongoing) with a 21d interval in 7 cases, one was treated every 30d with good response and one was treated every 6 weeks after tumor regression. Standard 1.8 dose was used in 6 cases, two were treated with 1.2 since start, and in one, dose was reduced to 1.2 after G2 neuropathy. Neuropathy was the most frequent adverse event in 8/9 patients (only 1 had grade 2). Main deviations of the recommended protocol were: interval – one patient was treated every 30d with good response and one was treated every 6 weeks after tumoral lesions regressed; dose: two were treated with 1.2mg/kg since start; combined therapies: PUVA was added in 1 patient, one had PD after cycle7 with association of local radiotherapy, followed by chemotherapy, one used BV in association to methotrexate (due to a rheumatological disease). Eight patients had partial response after 1st cycle; three progressed

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while on treatment (on cycles 2 – rapid progression, 7 and 14). Two patients were re-exposed to BV after a complete treatment and one is continuing BV after 16th cycle. Main characteristics of patients, biopsies and treatment are summarized on Tables 1 and 2, with main protocol deviations highlighted in red.

Table 1. Patients characteristics: main demographic and histological data

* at start of BV treatment ** Nx – PET suggestive of involvement, but not biopsied

Table 2. Main data on treatment regimens, prior treatments and associated therapies

CONCLUSION: Treatment protocols are fundamental for an initial evaluation of a new drug, but they do not always reflect real-life scenario, since they restrict the use of associated therapies, comorbidities and other variables. Most treatments available for MF rarely induce long-term remissions, and cure is not the main objective. Therefore, BV use in lower doses, larger intervals and/or in combination to other therapies (skin-directed or systemic) might help prolonging clinical benefits and time to next treatment, while reducing its main related adverse event, neuropathy. V-04

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Brentuximab vedotin (BV) and lenalidomide (Len) in relapsed and refractory (r/r) cutaneous (CTCL) and peripheral (PTCL) T-cell lymphomas; interim analysis of a phase II trial Basem M. William, MD, MRCP(UK), FACP, Amy Johnson, MD, Ying Huang, MA, MS, John Reneau, MD, PhD, Jonathan E Brammer, MD, and Catherine G. Chung, MD Multidisciplinary Cutaneous Lymphoma Program- The Ohio State University Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH, USA Introduction: Patients with r/r tumor stage CTCL and/or PTCL have a poor prognosis. BV is currently FDA approved for CD30 positive CTCL and anaplastic large cell lymphoma (ALCL) with single agent activity in additional PTCL subtypes. Len also has single agent activity in patients with r/r CTCL/PTCL. The safety of the combination was established in a phase I trial in patients with r/r diffuse large B-cell lymphoma. Methods: We conducted a single-institution phase II trial to determine the safety and efficacy of BV+Len combination in patients with r/r CTCL/PTCL. Simon’s 2-stage optimal design was followed to test the null hypothesis of overall response rate (ORR) ≤0.3 versus the alternative hypothesis of ORR≥0.5. Patients with ≥ 1 line of systemic therapy or 2 lines of skin directed therapy, at least stage IB (for CTCL), and no prior progression on BV were eligible regardless of CD30 staining. All patients were treated with BV 1.2 mg/kg IV and Len 20 mg PO daily q3 weeks for a maximum 16 cycles. After 7 patients were treated, we reduced Len to 10 mg given safety/tolerability concerns. Responses are assessed by the International Society for Cutaneous Lymphomas and the cutaneous lymphoma task force of the European Organization of Research and Treatment of Cancer (ISCL/EORTC) Global response criteria (for CTCL) and Cheson year criteria (for PTCL). The effect of treatment on quality of life is assessed by Skindex-16. Results: As of July 1, 2019, 17 subjects were treated; 10 (59%) with mycosis fungoides (MF), 2 (12%) with Sezary syndrome (SS), 2 (12%) with CD30+ lymphoproliferative disorder, and 3 (18%) with PTCL. Median age was 60 (49-90) years and 76% were males. CD30 was completely negative (<1%) in 3 (18%) of patients and median CD30 staining (by immunohistochemistry) was 7.5% (range 1-75%). Of 12 patients with MF/SS, 5 (42%) had evidence of large cell transformation at accrual. Of 14 patients with CTCL, median baseline mSWAT was 54.5 (range 4.4-190). Median number of prior therapies was 5 (range 1-9). Grade 3 adverse events (AEs) were reported in 11/17 patients; including neutropenia (4), thrombocytopenia (1), bronchitis (1), dyspnea (1), abdominal pain (2), vertigo (1), , DRESS (Drug Rash with Eosinophilia and Systemic Symptoms) syndrome (1), urinary tract infection (1), and tumor flare (2). Median number of cycles received was 4 (range 1-17). Best response in 14 evaluable patients were 2 (14%) complete response, 3 (21%) partial response, and 8 (57%) stable disease with ORR of 33% (95% confidence interval: 12-62%) (Figure 1). Of 17 patients, 5 (29%) remain on treatment, and 12 (71%) discontinued treatment because of disease progression (7; 58%), AEs (4; 33%), or patient preference (1; 2%). Median duration of response was 3.2 (range 2.5-13) months. Median progression-free survival (PFS) was 7 months (Figure-2). Of note, 7/14 patients (50%) patients with CTCL had >50% reduction in their Skindex-16 scores after a median of 2 cycles (range 1-3) (Figure-3).

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Figure-1: Response to Treatment

Figure-2: Progression-Free survival Figure-3: Skindex scores

Conclusions: BV + Len is combination is safe and efficacious in a heavily pre-treated patients with T-cell lymphomas. Len doses higher than 10 mg daily are poorly tolerated and associated with excess tumor flare. Recruitment of both CTCL and PTCL patients for this trial is ongoing.

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V-05

Title: Final ALCANZA results: brentuximab vedotin versus physician's choice in previously treated CD30-positive cutaneous T-cell lymphoma (mycosis fungoides or primary cutaneous anaplastic large cell lymphoma) Authors: Julia Scarisbrick, Steven M. Horwitz, Reinhard Dummer, Sean Whittaker, Madeleine Duvic, Youn H. Kim, Pietro Quaglino, Pier Luigi Zinzani, Oliver Bechter, Herbert Eradat, Lauren Pinter-Brown, Oleg Akilov, Larisa Geskin, Jose Sanches, Pablo Ortiz-Romero, Michael Weichenthal, David Fisher, Jan Walewski, Judith Trotman, Kerry Taylor, Stephane Dalle, Rudolph Stadler, Julie Lisano, Lisa Brown, Maria Corinna Palanca-Wessels, Veronica Bunn, Meredith Little, H. Miles Prince. Affiliations: For a full list of author affiliations please refer to abstract V-05

Introduction: ALCANZA (NCT01578499) is an international, open-label, randomised phase 3 trial comparing brentuximab vedotin vs physician’s choice (PC) of methotrexate or bexarotene in patients with previously treated mycosis fungoides (MF) or primary cutaneous anaplastic large cell lymphoma (pcALCL). The primary analysis,1 performed 10 months after the last patient's end of treatment visit (data cut-off: May 31, 2016) and a median follow-up of 22.9 months, demonstrated superiority of brentuximab vedotin over physician’s choice in terms of:  Significantly improved objective response rate lasting ≥4 months (ORR4) (56% vs 13%; p<0.0001)  Significantly higher CR rate (16% vs 2%; adjusted p=0.0046)  Significantly longer PFS (median 16.7 vs 3.5 months; HR=0.270, 95% CI: 0.169–0.430; adjusted p<0.0001)  Significant reduction in patient-reported symptoms per Skindex-29 symptom domain (– 27.96 vs –8.62; adjusted p<0.0001). We report the following final ALCANZA results; long-term efficacy (ORR4, progression-free survival [PFS], overall survival [OS], time to next treatment [TTNT), response by disease subtype (MF or pcALCL), and safety data including resolution and improvement of peripheral neuropathy (PN) (data cut-off: September 28, 2018). Methods: Adults (aged ≥18 years) with previously treated CD30+ MF (including transformed MF) or pcALCL were randomised 1:1 to brentuximab vedotin or PC of methotrexate or bexarotene. Study design is shown in Figure 1. Detailed methods have been published previously.1 Objective response, including ORR4, and disease progression were determined per independent review facility (IRF). TTNT was defined as the time from randomisation to the date of the first documented antineoplastic therapy or last contact date for patients who never received antineoplastic therapy. Treatment-emergent AEs, including PN (SMQ; included peripheral motor neuropathy and peripheral sensory neuropathy), were evaluated and graded according to NCI CTCAE version 4.03.

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Figure 1. ALCANZA study design

Results: Between August 2012 and July 2015, 131 patients were enrolled at 34 global sites, with 128 patientsanalysed in the ITT population (MF n=97, pcALCL n=31; 3 patients with MF were excluded for insufficient CD30 expression).The intent-to-treat population comprised 128 patients with either MF (n=97) or pcALCL (n=31). Final results demonstrated improved efficacy with brentuximab vedotin vs PC. ORR4 per IRF was 54.7% with brentuximab vedotin vs 12.5% with PC (p<0.001); complete response (CR) rate per IRF was 17.2% with brentuximab vedotin vs 1.6% with PC (p=0.002). ORR4, ORR, and CR rates were higher with brentuximab vedotin than with PC in patients with MF and pcALCL (Table 1).

Table 1: Patient response per IRF by disease subtype (ITT population)

Patients, n Brentuximab vedotin (n=64) Physician’s choice (n=64) (%) Total ORR4 ORR CR Total ORR4 ORR CR MF 48 (75) 24 (50) 31 (65) 5 (10) 49 (77) 5 (10) 8 (16) 0 pcALCL 16 (25) 11 (69) 11 (69) 6 (38) 15 (23) 3 (20) 5 (33) 1 (7)

With a median follow-up 36.8 months, median PFS per IRF was 16.7 months in the brentuximab vedotin arm vs 3.5 months with PC (p<0.001; Figure 2).

Figure 2: PFS per IRF (ITT population)

With a median overall follow-up for TTNT of 37.3 months, in the brentuximab vedotin and PC arms, 78% and 75% of patients had received subsequent antineoplastic therapy, respectively (Figure 3). Median TTNT was improved with brentuximab vedotin vs PC (14.2 [95% CI: 12.2– 16.4] vs 5.6 months [95% CI: 4.2–7.3]; HR=0.269; 95% CI: 0.171–0.424; p<0.001). In the brentuximab vedotin arm, median TTNT in the MF and pcALCL groups was 13.4 months (95% CI: 11.4–15.3) and 20.6 months (95% CI: 7.0–32.8), respectively.

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Figure 3: TTNT (ITT population)

With median follow-up of 45.9 months, 3-year OS estimates were 64.4% (95% CI: 50.7–75.2) with brentuximab vedotin and 61.9% (95% CI: 47.3–73.6) with PC.

In the brentuximab vedotin arm safety population (n=66), 44 patients treated with brentuximab vedotin experienced PN; most events were Grade 1 (n=18) or 2 (n=20); 6 patients had Grade 3 PN. At final data cut-off, 86% of brentuximab vedotin-treated patients who experienced PN had complete resolution (n=26) or improvement (by ≥1 grade; n=12) of all PN events and all ongoing cases of PN were Grade 1 or 2 (Table 2). Brentuximab vedotin Physician’s choice (n=44) (n=4) Data cut-off May 31, Sep 28, May 31, Sep 28, 2016 2018 2016 2018 Patients with resolution or 36 (82) 38 (86) 1 (25) 2 (50) improvement of PN events, n (%) Patients with resolution of all PN 22 (50) 26 (59) 1 (25) 2 (50) events, n (%) Median time to resolution, weeks 27.0 33.0 2.0 10.5 Patients with improvement in PN 14 (32) 12 (27) 0 0 events, n (%) Median time to improvement, 8.0 15.0 – – weeks Patients with ongoing PN events, 22 (50) 18 (41) 3 (75) 2 (50) n (%) Maximum severity grade 1, n (%) 17 (39) 15 (34) 1 (25) 1 (25) Maximum severity grade 2, n (%) 5 (11) 3 (7) 2 (50) 1 (25) Conclusions: Final analyses from ALCANZA demonstrate that with longer follow-up, treatment with brentuximab vedotin in patients with CD30+ MF and pcALCL provides durable, robust benefits with prolonged PFS vs physician's choice of methotrexate or bexarotene. Brentuximab vedotin extended TTNT vs physician’s choice, suggesting that durable brentuximab vedotin responses were clinically meaningful. In addition, with additional follow-up, 86% of PN cases in patients treated with brentuximab vedotin had either resolved or decreased in severity by ≥1 grade. References: 1. Prince HM, et al. Lancet 2017;390:555–66.

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Title: Brentuximab vedotin for relapsed/refractory Sézary syndrome: a single-center experience

Authors: Daniel J. Lewis MD,1 Paul L. Haun MD,1 Sara S. Samimi MD,1 Daniel J. Landsburg MD,2 Jakub Svoboda MD,2 Stefan K. Barta MD,2 Sara A. Berg MD,1 Christina A. Del Guzzo MD,1 Neha N. Jariwala MD,1 Carmela C. Vittorio MD,1 Jennifer Villasenor-Park MD PhD,1 Sunita D. Nasta MD,2 Stephen J. Schuster MD,2 Alain H. Rook MD,1 Ellen J. Kim MD1

1 Department of Dermatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA 2 Department of Oncology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

Introduction Brentuximab vedotin (BV) is a CD30-directed antibody-drug conjugate approved for CD30+ cutaneous T-cell lymphoma. BV has been studied primarily in plaque- and tumor-stage mycosis fungoides, whereas limited data exist on its efficacy in erythrodermic Sézary syndrome (SS), including in the pivotal phase III ALCANZA trial.1 Treatment options for SS remain limited and have been associated with low response rates.

Methods Between 2017-2019, nine patients with SS at the University of Pennsylvania received infusions of BV 1.8 mg/kg every 21 days. Only SS patients with B2 blood involvement at the time of BV treatment were included, as defined by the 2011 EORTC/ISCL criteria: an absolute Sézary cell count ≥1000 cells/μL or (2) a CD4/CD8 ratio ≥10 or (3) increased CD4+ cells with CD4+CD7− cells ≥40% or CD4+CD26− cells ≥30%, with a positive TCR gene rearrangement.2 Outcomes were assessed in the skin and lymph nodes per the EORTC/ISCL response criteria and in the blood per the PROCLIPI revised blood response criteria. CD30 expression level was assessed in skin biopsy tissue using standard immunohistochemistry.

Results Nine patients (6 males, 3 females, median age 67) received BV after a median of six prior systemic therapies (range 0–12) (Table 1). Skin biopsies showed CD30 positivity (>10%) in 5 of 9 patients. Two of 9 patients (22%) had a global response (1 CR with 20% skin CD30 expression; 1 PR with 1% skin CD30 expression) (Figure 1). Time to response was 6 weeks with a treatment duration of 13 cycles (CR) and 9 cycles (PR) with a duration of response of 105 weeks (ongoing) and 22 weeks, respectively. In the 5 patients in whom blood response could be assessed, 3 responded (1 CR, 2 PR). Two of 4 patients exhibited a response (1 PR, 1 CR) in lymph nodes (LN) on PET/CT. One patient with 95% skin CD30 expression received BV as first-line therapy and achieved SD (PR in blood; SD in skin and LN). Grade 1-2 neuropathy occurred in 4 of 9 patients, lasted a median of 62 weeks (range, 23-91 weeks), and resolved in 1 of 4 patients. No grade 3-4 adverse events occurred.

Conclusions This study suggests a therapeutic role of BV in SS even in the setting of low skin CD30 expression, although with a lower global response rate than that observed in the ALCANZA trial (22% vs 56.3%). Generally well-tolerated and administered infrequently, BV may represent an additional therapeutic option for SS and a potential alternative to agents such as alemtuzumab, pembrolizumab, or single-agent chemotherapy.

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Table 1. Patient characteristics and responses and adverse events to BV

Pt Age Sex Race Stage Cycles CD30% Skin Blood LN Global AEs 1 61 M W IVA1 2 5 SD SD SD None 2 56 M W IVA1 6 95 SD PR SD SD PN (G2) 3 71 M W IVA1 9 1 PR PR PR PR PN (G1) 4 87 M W IVA1 8 7 SD SD PN (G1) 5 69 F W IVA1 6 25 SD SD SD None Constipation (G1) 6 62 F W IVA1 3 15 SD SD Insomnia (G2) 7 78 F W IVA2 2 25 SD SD None Alopecia (G1) 8 59 M W IVA2 3 10 SD SD SD Diarrhea (G1) PN* (G2) 9 67 M B IVB 13 20 CR CR CR CR Diarrhea (G1)

Abbreviations: Pt, patient; M, male; F, female; W, Caucasian; B, African American; SD, stable disease; CR, complete response; PR, partial response; PN, peripheral neuropathy; G, grade *Neuropathy resolved after 68 weeks

Figure 1. Patient 9’s skin disease (a) at the start of BV – mSWAT of 41.2 (b) at initial follow-up after completing 13 cycles of BV – mSWAT of 0

References 1. Prince HM, Kim YH, Horwitz SM, et al. Brentuximab vedotin or physician's choice in CD30- positive cutaneous T-cell lymphoma (ALCANZA): an international, open-label, randomised, phase 3, multicentre trial. Lancet. 2017 Aug 5;390(10094):555-566. 2. Olsen EA, Whittaker S, Kim YH, et al. Clinical end points and response criteria in mycosis fungoides and Sézary syndrome: a consensus statement of the International Society for Cutaneous Lymphomas, the United States Cutaneous Lymphoma Consortium, and the Cutaneous Lymphoma Task Force of the European Organisation for Research and Treatment of Cancer. J Clin Oncol. 2011;29(18):2598-2607.

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W-01

Resolution of Sézary Syndrome after Combination of Mogamulizumab with Pegylated Interferon α2-a is Mediated by CD56dim NKp30+ IFN-γ+ NK Cells

Patricia Patino, MD1, Oleg Kruglov, MD1, Oleg E Akilov, MD, PhD1

Introduction and Objectives: The recent advances in the immunobiologic therapy of cancer made a significant impact on patients' life. Combination strategies demonstrated to be more advantageous in comparison with monotherapy leading to faster disease resolution and prolonged disease-free survival. Materials and Methods: Targeted single-cell RNA sequencing and flow cytometry were performed on samples from 3 patients before and after the combinational therapy of mogamulizumab with pegylated interferon α2-a. Results: We report an exceptional outcome of the combination of mogamolizumab and pegylated interferon-α2a resulting in 3 patients with a partial clinical response as soon as two weeks after initiation of the therapy and complete response after only two months of therapy (Fig. 1). Targeted single-cell RNA sequencing with subsequent flow cytometry demonstrated that the activation of CD56dim NKp30 NK cells by pegylated interferon-α2a was crucial in the induction of antibody-dependent cytotoxicity against CCR4+ Sezary cells (Fig. 2).

Figure 1. Resolution of Sezary syndrome after treatment with mogamulizumabFigure 2. Resolution and pegylated of Sezary interferonsyndrome -α2. after A.treatment Clinica withl Conclusions: Our presentationmogamulizumab before and andp egylated after clinical data and evidence treatment.interferon -B.α2 Targetedaccompanied single- cell by of efficacy justify future RNAexpansion sequencing of NK demonstrated cells in the a immunotherapy peripheral blood. A. A decrease of decrease in number of Sezary cells combinations that can be andVβ17 clonal expansion population of of NK malignant cell populationcells after treatment.. B. An expansion evaluated in the clinical of CD3-CD56dim NK cells after trial for patients with treatment. C. Expansion of CD3- Sezary syndrome. CD56dim CD62L (green) NKG2A (blue) NKp30 (red) IFNγ NK cells after treatment. D. Expansion of NKp30+IFNγ+ NK cells after treatment.

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W-03

Clinical activity and safety of low-dose total skin electron beam therapy combined with mogamulizumab in refractory Sézary syndrome: support for a clinical trial in CTCL Authors: Sophia Fonga, Eric Honga, Michael S Khodadoustb, a, Susan Hinikerc, Shufeng Lia, Erica B Wanga, Richard T Hoppec, Youn H Kima, b a Department of Dermatology, Stanford University, Stanford, California b Department of Medicine, Division of Medical Oncology, Stanford University, Stanford, California c Department of Radiation Oncology, Stanford University, Stanford, California

Introduction: Management of patients with refractory mycosis fungoides (MF) and Sézary syndrome (SS) is often challenging as available therapies lack durable response and/or consistent activity across disease compartments. Combining low-dose total skin electron beam therapy (LD-TSEBT) upfront with mogamulizumab could optimize the clinical outcome of these patients. LD-TSEBT is effective in clearing skin disease, and mogamulizumab is an anti-tumor immunotherapy with long-term tolerability suggesting its potential as a maintenance therapy after maximal response. To generate supportive experience for a clinical trial, we examined the combination regimen in previously treated SS patients. Methods: Two patients with SS were treated following the planned study design (Figure 2) of an investigator-initiated, phase 2 clinical trial of LD-TSEBT and mogamulizumab (TSE-Moga) in previously treated patients with MF/SS. Both patients received mogamulizumab 1mg/kg weekly x 4 and then bi-weekly; LD-TSEBT (12 Gy) was initiated within 2 days of starting mogamulizumab and given over 2-3 weeks. Safety and clinical response were evaluated. Results: TSE-Moga was well-tolerated without any unanticipated adverse events. Patient 1 (T4N2M0B2) is a 63 yo female with 3 prior systemic therapies; time to global response (TTR) with TSE-Moga was 9 weeks. Patient 2 (T4NxM0B2) is a 75 yo male with 5 prior systemic therapies; TTR was 4 weeks. Both patients lacked global response to their prior therapies but achieved global complete response (CR, blood and skin) with TSE-Moga. After a follow-up of 58 weeks and 28 weeks respectively, global CR continues. Conclusions: TSE-moga demonstrated excellent tolerability and promising clinical activity with ongoing global complete responses in two patients with refractory SS. This encouraging experience supports our upcoming clinical trial evaluating the efficacy and safety of TSE-Moga in MF/SS. Comprehensive translational studies are planned.

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Figure 1. Global CR; Patient 1 (A-B), Patient 2 (C-D)

Figure 2. TSE-Moga Phase 2 Trial Study Design

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X-03

Improved survival for skin-primary presentation of adult T-cell leukemia/lymphoma (ATLL) Meera Jain BS,1 Kavita Goyal MD,2 Daniel O’Leary MD,3 Nathan Rubin MS,4 Kimberly Bohjanen MD,2 Amrita Goyal MD2

1Drexel University College of Medicine, 2Department of Dermatology, University of Minnesota, Minneapolis MN, 3Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis MN, 4Biostatistics Core, Masonic Cancer Center, University of Minnesota

Background: Adult T-cell leukemia/lymphoma (ATLL) is an uncommon and aggressive systemic leukemia that can present in the liver, spleen, CNS, bone marrow, GI tract, and skin. ATLL is caused by viral integration of the human T-cell leukemia virus-1 (HTLV-1) into the host genome. ATLL is most commonly found in Japanese and Caribbean populations, with an age of onset of 60 years and 40 years, respectively. The Shimoyama classification divides ATLL into four subtypes, each with their own cutaneous manifestations: smoldering, chronic, lymphomatous, and acute, in order of worsening prognosis.

Methods: In this study, we examined 3140 cases of ATLL from the U.S. in the Surveillance, Epidemiology, and End Results-18 (SEER-18) database from 1975-2016 to assess differences in survival based on initial site of diagnosis (skin, lymph node, bone marrow, or other) as well as differences in race, sex, and age of diagnosis (Table 1).

Results: Of the 3140 cases, 3126 patients had complete data for further analysis. Shimoyama classification is not recorded in SEER. Data were subject to two-sided t-test and chi-square with p<0.05 being significant. We identified 24 patients whose disease presented primarily in the skin (0.76%), 804 in the lymph node (25.61%), 2245 in the bone marrow (71.50%), and 53 other presentations (1.69%) (Table 1). Of the 24 with skin-primary disease, 7 (29%) had localized disease, 2 (8%) had regional disease, 10 (41%) had distant disease. There was no gender predilection for skin-primary disease. Patients who presented with skin-primary disease had markedly better 2- and 5-year survival rates than patients with other primary sites of diagnosis (Figure 1). Cox regression model confirmed that skin as site of primary diagnosis was a potent protective factor (Table 1).

Discussion: It has been documented that approximately 50% of patients with ATLL develop cutaneous involvement during their disease course,4 however, only a small fraction of patients will present with primarily cutaneous disease. In this analysis, we report that patients with primary cutaneous disease have improved survival compared to patients with other primary sites of diagnosis. This knowledge will allow us to better prognosticate and offer patients guidance on their expected survival. This pattern may reflect differences in the underlying pathobiology of the disease or differences in environmental factors.

Of note, median age in this study was substantially lower than documented in the Japanese and Caribbean population. which may reflect differences in biology of patients with ATLL in United States; it has been previously hypothesized that there are differences in age of onset of disease in Japan and the Caribbean due to environmental factors.

This study is limited in that it is a retrospective analysis. We are unable to assess what Shimoyama subtype the skin-primary patients had, and it is possible that skin-primary presentation may be skewed to more indolent subtypes. We also lack information about disease progression or treatment course. In summary, we find that patients with skin-primary ATLL have improved survival as compared to patients with other primary sites.

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Table 1. Population characteristics and survival. All sites* Skin Lymph Bone Other p-value n=3126 n=24 node marrow n=53 n=804 n=2245 Age, median 23 (0-85) 47.5 (1- 25 (0-85) 22 (0-85) 47 (2-85) Skin vs. LN p=0.0022 (range), years 83) Skin vs. BM p=0.019 Skin vs. other p=0.81

Age, mean (st dev), 31.5 (24.9) 43.8 30.4 (21.7) 31.4 (25.8) 45.3 (25.3) Skin vs. LN p=0.006 years (23.4) Skin vs. BM p=0.017 Skin vs. other p=0.805 Gender Male 2104 12 (50%) 557 1500 35 (66.0%) Skin vs. LN p=0.0493 (67.0%) (68.9%) (66.5%) Skin vs. BM p=0.088 Female 1036 12 (50%) 251 755 (33.5%) 18 (34.0%) Skin vs. other p=0.181 (33.0%) (31.1%)

Survival Median follow-up 24 (0-201) 49 (0-95) 22 (0-198) 25 (0-201) 29 (0-188) Skin vs. LN p=0.044 (range), months Skin vs. BM p=0.91 Skin vs. other p=0.90 Median survival 76 (71-81) NA (NA- 77 (68-125) 76 (70-82) 50 (22- time (95% CI), NA) NA) months # Alive at end of 1906 22 548 1310 26 follow-up # Dead at end of 1234 2 260 945 27 follow-up

2-year OS (95% 0.75 (0.73- 1.0 (1.0- 0.78 (0.74- 0.74 (0.72- 0.62 (0.50- CI) (Non-age 0.77) 1.0) 0.81) 0.76) 0.78) adjusted) 5-year OS (95% 0.56 (0.54- 0.85 0.57 (0.52- 0.56 (0.53- 0.46 (0.34- CI) (Non-age 0.58) (0.67-1) 0.62) 0.58) 0.63) adjusted) St dev—standard deviation; LN—lymph node; BM—bone marrow; CI—confidence interval; NH—non-Hispanic *Patients were identified using SEER*Stat V. 8.3.6 (NCI/NIH, Bethesda, MD) in the “SEER-18 Regs Research Data + Hurricane Katrina Impacted Louisiana Cases, Nov 2018 Sub (1975-2016)” registry via Lymphoma subtype recode/WHO 2008 “2(b)2.3 Adult T-cell leukemia/lymphoma.” Survival analysis was performed in R Version 3.6.0 using survminer package V 0.4.4. Cases were limited to those microscopically confirmed; diagnoses made on autopsy were excluded. **Percentage is percent of all cases at that site

Figure 1. Kaplan-Meier survival curves demonstrating significantly improved survival for skin- primary ATLL as opposed to other primary sites of diagnosis.

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Treatment and prognosis of rare patients with a primary cutaneous CD30-positive lymphoproliferative disorder who develop extracutaneous localisations Introduction Development of extracutaneous disease in patients with a primary cutaneous CD30+ lymphoproliferative disease (pcCD30+LPD; lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma) is uncommon and patients’ outcomes are largely unknown. This study evaluated current treatment results and prognosis of rare patients with pcCD30+LPDs who developed extracutaneous disease. Methods In this multicentre study, 51/785 (6.5%) pcCD30+LPD patients, selected from the Dutch registry of cutaneous lymphomas between October 1985 and December 2017, developed histologically proven extracutaneous localizations. Ultimately, 43 patients were suitable for further analyses. Results (including tables and figures) Extracutaneous disease developed after a median of 35 months (range 5-264). Most patients (34/43; 79%) were treated with anthracycline-based chemotherapies (Table 1). A complete response (CR) was observed in 26/43 (61%) patients, and a 48% 5-year cumulative incidence of relapse was reported (of which 6 extracutaneous). Five-year progression free survival (PFS5) and overall survival (OS5) after extracutaneous development were 39% and 47%, respectively, and similar to ALK-negative systemic anaplastic lymphoma. Thirteen patients with localized lesions and a solitary regional involved lymph node reported superior responses and survival compared with patients with more extensive extracutaneous disease (85% versus 29% PFS5; log-rank p=0.03, 62% versus 41% OS5; log-rank p=0.16).

Table 1. Treatment responses in patients with C-ALCL or LyP developing extracutaneous disease (n=43) C-ALCL (n=30) LyP (n=13) Overall (n=43) Therapy N CR Relap Syste N CR Relap Syste N CR Relap PFS DS OS o (%) se mic o (%) se mic o (%) se 5 D5 5 skin relaps * skin relaps (%) (%) (%) (% limite e (%) limite e (%) ) d (%) d (%) Anthracycli 2 16/2 3/16 6/16 9 5/9 3/5 0/5 (0) 3 21/3 12/21 34 46 46 ne-based 5 5 (19) (38) (56) (60) 4 4 (57) chemothera (64) (62) py RT 3 3/3 2/3 0/3 (0) 1 1/1 1/1 0/1 (0) 4 4/4 3/4 100 0 10 (10 (67) (10 (100) (10 (75) 0 0) 0) 0) No therapy 2 0/2 - - 3 1/3 1/1 0/1 (0) 5 1/5 1/1 20 80 20 (0) (33) (100) (20) (100) Total 3 19/3 5/19 6/19 1 7/1 5/7 0/7 (0) 4 26/4 16/26 39 47 47 0 0 (26) (32) 3 3 (71) 3 3 (62) (63) (54) (61) C-ALCL, cutaneous anaplastic large T-cell lymphoma; LyP, lymphomatoid papulosis; RT, radiotherapy; PFS5, five-year progression free survival; DSD5, five-year cumulative incidence of disease specific death; OS5, five-year overall survival.

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*CR refers to complete remission of extracutaneous localisations.

Figure 1. PFS, DSD and OS of pcCD30+LPD patients after extracutaneous development, compared between patients with localised lesions and a solitary regional involved lymph node (T1-2;N1;M0), and patients with more extensive extracutaneous disease (non-T1-2;N1;M0). PFS, DSD and OS were analysed using the Kaplan-Meier method. (A) Progression free survival). (B) Disease specific death (one minus survival). (C) Overall survival.

Conclusions Development of extracutaneous manifestations in pcCD30+LPDs is rare and can develop after many years. In the current study we show that prognosis of patients with a pcCD30+LPD who develop extracutaneous localisations corresponds with ALK-negative systemic ALCL and provide a benchmark for novel targeted therapies. Given the therapeutic success in ALK-negative systemic ALCL, BV-based regimens should be considered in patients with a pcCD30+LPD who develop extracutaneous localisations. Radiotherapy may suffice in patients with localised lesions and a solitary regional involved lymph node (supplementary Figure 2).

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Winkelmann regimen – oral Chlorambucil for Mycosis Fungoides and Sezary syndrome Doss G, Francesco I, Grandi V, Child F, Wain M, Whittaker S, Morris S Clinical Oncology - GSTT, Guys Cancer Centre, London Dermatology - GSTT, St. John’s dermatology Institute, London Introduction: Advanced Stage Mycosis Fungoides and Sezary Syndrome have a poor prognosis and are resistant to multiple treatments. Treatment can cause significant morbidity. We report our experience of oral chlorambucil using the Winkelmann regimen. Methods: We retrospectively reviewed case treated at our institution with Winkelmann regimen of 2 mg daily oral chlorambucil on a 28 day cycle. Cases were identified from the skin tumour unit research data base. Outcomes and toxicity results were obtained from the staging and progression information recorded on the database and from the patient electronic notes. Results:

Patients & Treatment Characteristics 26 Median Age at Diagnosis Male: female 17:9 MF:SS 11:15 Median No. of Prior treatments 4 (0-10) Stage before Chlorambucil IIB=1, IIIA=2, IIIB=3, IVA1=6, IVA2=12, IVB=2. B2=15, B1=4, B0=7 Median time from diagnosis to start of 1144 (89-5313) Chlorambucil in days Response rate

After 3 cycles PR 38.5%, SD 42.3% and PD 19.2%. After 3 cycles with Blood Involvement ORR 55.5% (PR 33.3, SD 22.2) and PD (9/14) 44.5%

Toxicity (2/26) Febrile Neutropenia – Nil, GI – 1, Others - 1 Median OS in days 225 (40% at 1 year) Median PFS in days 469 (51% at 1 year) Median time to retreat in days 225 (at 180days 53% on different treatment)

Conclusions: The Winkelmann regimen of oral Chlorambucil is a useful low toxicity palliative treatment option for patients with advanced Mycosis Fungoides and Sezary Syndrome.

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Reference: 1. Hamminga L, Hartgrink-Groeneveld CA, van Vloten WA. Sezary’s syndrome: a clinical evaluation of eight patients. Br J Dermatol 1979; 100(3):291–6. 2. Winkelmann RK, Diaz-Perez JL, Buechner SA. The treatment of Sezary syndrome. J Am Acad Dermatol 1984; 10:1000–4.

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Oral Abstract X-06: Clinical Characterization of Mogamulizumab-associated Rash

Kelsey E. Hirotsu1, Tatiana M. Neal1, Jennifer Y. Wang1,2, Kerri E. Rieger1,2, Jennifer Strelo1, Michael Khodadoust3, Youn H. Kim1,3, and Bernice Y. Kwong1.

Departments of 1Dermatology, 2Pathology, and 3Oncology, Stanford University School of Medicine, California, USA

Introduction:  Mogamulizumab is a humanized defucosylated monoclonal antibody targeting CC chemokine receptor 4 recently approved for mycosis fungoides (MF) and Sézary syndrome (SS). It was associated with reported drug rash in 24% (44/186) of patients in the pivotal MAVORIC trial [1].  Given the difficulty of clinically differentiating mogamulizumab-associated rash (MAR) and CTCL disease, we aimed to characterize the clinical presentation of MAR, including real-world patient experiences.

Methods:  Utilizing our institution’s cutaneous lymphoma database, we searched for patients treated with mogamulizumab.  Included were patients with MF or SS who presented with a new rash after starting mogamulizumab, with a skin biopsy consistent with drug eruption including a polyclonal TCR high-throughput sequencing molecular profile.  We excluded patients with no biopsy, those lost-to-follow-up, and those with a diagnosis of ATL.  We retrospectively identified basic demographics, dates of mogamulizumab infusions and rash onset, skin biopsy results, morphology and distribution (with standardized patient photos), and management of MAR and response. New medications initiated up to 4 weeks prior to rash onset were reviewed.

Results:  Sixteen patients with Sézary Syndrome and MAR were identified (stage IV), with a median age of 65 years [38-78]. Seven patients were female and nine were male. The median time from first mogamulizumab dose to rash onset was 105 days [56-301]. Eight patients reported pruritus associated with MAR. Most patients had near-complete clearing of their Sézary Syndrome disease at the time of MAR onset.  Three common presentations of MAR included: head and neck dermatitis, alopecia, and a photo-distributed dermatitis. The overall distribution of MAR was most commonly on the head & neck, trunk, and/or extremities, and was often accentuated on photo-exposed or scalp areas. Many patients had MAR that clinically mimicked their disease.  Histopathology demonstrated psoriasiform and spongiosis, interface and lichenoid, and granulomatous reaction patterns. Immunohistochemistry frequently showed CD8 predominance among epidermotropic lymphocytes. All patients had molecular diagnostic evaluation with TCR high-throughput sequencing demonstrating a predominantly polyclonal process.  For management of MAR, six patients improved with topical steroids (2 to 11 months), ten improved with systemic steroids (2 weeks to 5 months). Five patients required prolonged steroids and improved with the addition of methotrexate.  Three patients with worsening SS were retreated with mogamulizumab and experienced recurrent rash. These patients were managed with prednisone +/- methotrexate with stable to resolved MAR.

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Conclusion:  Differentiating MAR from disease progression is difficult given their similar cutaneous presentation, however determining this is crucial for patient management.  MAR usually occurs a few months after initiating treatment (median onset time of 105 days) similar to drug eruptions observed with other immune therapies.  MAR was manageable with topical or systemic steroids in the majority of patients, but for some patients prolonged rash required a steroid-sparing agent.  Given the difficulty of clinically differentiating MAR from disease progression and the heterogeneous pathology, TCR high-throughput sequencing can aid in diagnosis. Additionally, over time prolonged MAR can blend with increasing cutaneous disease, and TCR HTS results can reflect this process.  Limitations of our study include small cohort size and combined review of on-trial and real-world patients.  Study is ongoing to understand the mechanism of MAR

References: 1. Kim et al. Mogamulizumab versus vorinostat in previously treated cutaneous T-cell lymphoma (MAVORIC): an international, open-label, randomised, controlled phase 3 trial. Lancet Oncology. 2018 August 9.

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Oral Abstract X-07: Characterization of the Histopathologic and Molecular Features of Mogamulizumab-Associated Rash (MAR)

Jennifer Y. Wang1,2, Kelsey E. Hirotsu1, Tatiana M. Neal1, Bernice Y. Kwong1, Youn H. Kim1, and Kerri E. Rieger1,2

Departments of 1Dermatology and 2Pathology, Stanford University School of Medicine, California, USA

Introduction  Drug rash is one of the most common adverse events of mogamulizumab in the treatment of adult T-cell leukemia/lymphoma (ATLL) and mycosis fungoides/Sezary Syndrome (MF/SS), affecting up to 25% of treated MF/SS patients [1].  There has been limited histopathologic characterization of mogamulizumab-associated rash (MAR). Limited case reports describe interface dermatitis with exocytosis of CD8- positive lymphocytes in the setting of ATLL, while others describe a granulomatous dermatitis in the setting of MF/SS [2].  We aim to characterize the histopathologic and molecular features of MAR in a larger cohort of patients.

Methods  Retrospective analysis of suspected MAR in MF/SS cases at Stanford University  Inclusion criteria: o Skin biopsy specimen(s) of suspected MAR available for histopathologic evaluation o Patient has an established malignant T-cell receptor (TCR) gene sequence by high-throughput sequencing, and this malignant sequence is not detected in a dominant profile on suspected MAR skin biopsy o Other primary causes of new rash ruled out or considered unlikely  Exclusion criteria: o Dominant malignant TCR sequence not established on diagnostic skin biopsy prior to mogamulizumab start o New emerging dominant TCR sequence discovered on suspected MAR biopsy  Histologic features were scored by a board-certified dermatopathologist (KER) and dermatopathology fellow (JYW)

Results  Total of 47 biopsies from 16 patients  Three main reaction patterns identified. Most cases with multiple concurrent reaction patterns: o Spongiotic/psoriasiform dermatitis: primary pattern in 29/47 biopsies (61%) o Interface/lichenoid dermatitis: primary pattern in 11/47 biopsies (23%) o Granulomatous dermatitis: primary pattern in 8/47 biopsies (17%)  Variable levels of dermal inflammation, often lymphohistiocytic with variable levels of admixed eosinophils, neutrophils, and plasma cells  Features mimicking MF present in several cases: o Lymphocyte tagging and exocytosis o Follicular destruction o Lamellar fibroplasia  Immunohistochemistry o Inverted CD4:CD8 ratio within the epidermis

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o Normal/mixed ratio of CD4 and CD8 within the dermis and follicles o CD7 sometimes lost, sometimes retained  Molecular studies o In few cases, a previously identified dominant/malignant clone from the patient’s MF/SS was detected at low levels in MAR skin biopsy. However, the sequence was not present in a dominant profile and is of unclear clinical significance.

Conclusions  MAR most commonly shows a psoriasiform/spongiotic reaction pattern, followed by interface/lichenoid and granulomatous patterns.  MAR may have features that mimic MF on histopathology  Immunohistochemistry tends to show CD8 predominance in the intra-epidermal lymphocytes. o However, utility may be limited if immunophenotype of patient’s disease prior to mogamulizumab is CD8-positive, CD4/CD8 double-negative, or unknown  High-throughput/next generation sequencing to detect presence or absence of dominant TCR clone can be helpful in distinguishing MAR from residual/recurrent disease  Limitations and future directions: o Small, single-center study with only MF/SS patients o Inclusion of additional MAR cases in study cohort anticipated with continued use of mogamulizumab o Additional immunohistochemical characterization to identify other histopathologic characteristics of MAR and elucidate mechanism of rash o Identification of molecular and genomic biomarkers of MAR

References 1. Kim, Y.H., et al., Mogamulizumab versus vorinostat in previously treated cutaneous T-cell lymphoma (MAVORIC): an international, open-label, randomised, controlled phase 3 trial. Lancet Oncol, 2018. 19(9): p. 1192-1204. 2. Chen, L., et al., Mogamulizumab-Associated Cutaneous Granulomatous Drug Eruption Mimicking Mycosis Fungoides but Possibly Indicating Durable Clinical Response. JAMA Dermatol. 2019 May 29. doi: 10.1001/jamadermatol.2019.0369.

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Real-life experience with chlormethine gel: moving beyond clinical trial data Background Compounded topical chlormethine (mechlorethamine) has been successfully used to treat mycosis fungoides (MF) since 1950s. Topical chlormethine as a gel formulation for the treatment of MF received FDA approval in 2013 and EMA approval in 2017, on the basis of the results of a phase 2, multicenter, randomized, observer-blinded, non-inferiority trial, comparing chlormethine gel with compounded ointment in MF patients, stage I–IIA. In Israel, the gel was approved in 2016 for patients with stage IA,IB MF as a third-line treatment (for those who received at least topical steroids and phototherapy) , and in 2018 as a second line therapy (for those who failed on topical steroids). Currently, data on real-life experience with this novel 0.016% chlormethine gel for topical treatment of early-stage MF is strikingly sparse. Objective To assess the efficacy and safety of chlormethine gel in early-stage MF patients in a real-life setting. Methods Data on early-stage MF patients treated with chlormethine gel during 2016-2019 was retrospectively collected from the records of the MF Clinic of Rabin Medical Center. All patients had at least one follow-up visit after initiation of treatment, and in each patient the best response was assessed. Results Overall, 66 early-stage MF patients received chlormethine gel: 51 male (77%), mean age 57 years (range 24-83), 37 had stage IA, 27-stage IB, and 2 stage IIA. Seven patients had early- stage folliculotropic MF. Regional application of chlormethine gel was given to 57 patients (86%), and total body application- to 9 (14%), as monotherapy (most of the time)- in 48 patients (73%), in combination with topical corticosteroids- in 13 (20%), and in combination with systemic treatment- in 5 (7%), for and an average duration of 342 days (7-1096). Overall response rate (≥50% clearance from baseline) of the entire group was 52% (34/66), and of patients with treated at least 6 months - 65%. Longer treatment periods (1 year or more) led to an increase in the overall response (75%). Only 2 patients achieved complete response (100% clearance). Cutaneous side effects (SE) Overall, 32 patients suffered from SE (48%) that were mild in the vast majority of the patients. Dermatitis, specifically irritation, and localized pruritus were the most frequently observed SE and were manageable by a reduction in application frequency and/ or application of topical steroids, or temporary treatment interruption. Eight patients had unmasking effect, and 7- hyperpigmentation at application site. Treatment withdrawals for SE occurred in 13 patients, and in 7 during the first month.

Summary and conclusions Our single-center experience showed that topical chlormethine gel improves the skin lesions of early- stage MF. Patients treated for more than 6 months achieved an overall response of 65% based on body surface area assessment. These results are comparable to the mSWAT -based overall response of 63% reported by Lessin et al. in early-stage MF patients treated for more than 6 months. Cutaneous SE were reported in 48% of our patients, compared to 62% by Lessin et al, a difference which may be attributed to the difference in protocol treatment between the 2 studies: a gradual increase in application frequency in our cohort in almost all of our patients, as well as the combination treatment with topical corticosteroids in 20% of our patients, as opposed to once daily application and avoidance of topical corticosteroids in Lessin’s study. SE resulted in treatment withdrawal in 20% of the patients (13/66), a rate that is similar to that reported by Lessin et al (26/128, 20.3%). It should be noted, however, that of the 32 patients suffering SE 40% stopped treatment, more than half of them within 30 days of treatment initiation. Almost a third of the patients treated for over a year had SE, mostly irritation and hyperpigmentation. SE were managed by topical corticosteroids, temporary interruption in therapy and reduction in application

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frequency. Intertriginous areas and skin folds were prone to irritation due to occlusion and friction. In conclusion, our single-center experience is largely in agreement with the results of the clinical study reported by Lessin et al, with similar overall response rates and treatment withdrawal rates due to SE, though the rate of cutaneous SE was lower in our cohort. According to our experience, chlormethine gel is well tolerated, especially if treatment is initiated gradually. Prompt treatment of SE allows patients to continue therapy, with 75% overall response of patients treated for over a year. It should be noted, however that only 2 patients in our cohort achieved CR, thus clinical benefit is moderate.

Reference 1. Lessin SR, Duvic M, Guitart J, Pandya AG, Strober BE, Olsen EA, Hull CM, Knobler EH, Rook AH, Kim EJ, Naylor MF, Adelson DM, Kimball AB, Wood GS, Sundram U, Wu H, Kim YH. Topical chemotherapy in cutaneous T-cell lymphoma: positive results of a randomized, controlled, multicenter trial testing the efficacy andsafety of a novel mechlorethamine, 0.02%, gel in mycosis fungoides. JAMA Dermatol. 2013 Jan;149(1):25-32.

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Mechlorethamine treatment duration as a function of clinician-level patient volume for mycosis fungoides cutaneous T-cell lymphoma (MF-CTCL)

Christiane Querfeld1, Theresa Pacheco2, Bradley Haverkos3, Gary Binder4, James Angello4, Brian Poligone5 1 City of Hope Comprehensive Cancer Center, Duarte, CA, USA; 2University of Colorado, Dept of Dermatology, Denver, CO, USA; 3University of Colorado, Division of Hematology, Denver, CO, USA; 4Helsinn Therapeutics US, Inc., Iselin, NJ, USA; 5Rochester Skin Lymphoma Medical Group, Fairport, NY, USA

Introduction & Objectives: NCCN guidelines recommend skin-directed therapies for early- stage MF-CTCL, including topical mechlorethamine. Mechlorethamine yields high response rates, particularly with treatment >3 months (Lessin, 2013, Kim 2003); early discontinuation is often attributed to dermatitis. We evaluated the association of patient volume with early discontinuation and overall treatment duration for United States clinicians prescribing standardized 0.016% gel formulation mechlorethamine. Methods: We evaluated dispensing records 10/2013-4/2019 (>99% a one-month supply) representing the majority of US utilization, and for each prescriber calculated average cumulative quantity of dispenses per patient. We excluded patients initiating treatment ≤100 days of data cutoff. Clinicians were grouped by number of patients treated with mechlorethamine gel. Kruskal-Wallis significance testing was performed on groups and logistic regression on early discontinuation. Results: We assigned 4922

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patients to 2004 clinicians. The monthly discontinuation rate was 33% in treatment months 1-3; thereafter rates averaged 16%. Patients receiving >1 dispense had 5 months median treatment duration (range 2-65). The 52 clinicians with >15 patients (mean 41.3) treated 44% of total patients, with 6.3 median dispenses/patient. 128 clinicians with 5-15 patients had 4.3 median dispenses and the interquartile range was 6.625-3.325. As volume further declined, variability and early discontinuation increased; 1348 clinicians with a single patient had 2 median dispenses, with 33% having only one dispense (p<0.0001). Early discontinuation was significantly associated with lower volume (OR 0.80; CI 0.754-0.842). Conclusions: Individual clinicians prescribing mechlorethamine gel for MF-CTCL varied considerably in patient volume and treatment duration. Clinicians with higher patient volume consistently sustained longer treatment duration and, importantly, avoided early discontinuation, perhaps attributable to experience managing the condition and dermatitis, and setting patient expectations. The early discontinuation noted may identify lack of patient education on how to adhere to treatment.

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Figure 1:

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The PROVe study: real-world experience with chlormethine gel and other therapies in the treatment of mycosis fungoides cutaneous T-cell lymphoma patients Authors: E.J. Kim1, L.J. Geskin2, C. Querfeld3, M. Girardi4, J. Guitart5, A. Musiek6, J.T. Angello7, W.L. Bailey7 Affiliations: 1Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 2Department of Dermatology, Columbia University, New York, NY, USA; 3Division of Dermatology, City of Hope, Duarte, CA, USA; 4Department of Dermatology, Yale School of Medicine, New Haven, CT, USA; 5Northwestern University Feinberg School of Medicine, Chicago, IL, USA; 6Division of Dermatology, Washington University School of Medicine, St. Louis, MO, USA; 7Helsinn Therapeutics (U.S.) Inc., Iselin, NJ, USA Introduction Topical chlormethine (mechlorethamine; nitrogen mustard) 0.016% w/w gel (equivalent to 0.02% chlormethine HCl) is a skin-directed therapy approved for treatment of mycosis fungoides cutaneous T-cell lymphoma (MF-CTCL) on the basis of results of the 201 registration study.1 A better understanding of real-world use of chlormethine gel may help improve the management of patients with MF-CTCL. Here we present data from the PROVe study, which assessed treatment patterns and efficacy, safety, and health-related quality of life (HRQoL) after chlormethine gel treatment in a real-world setting in the US. Methods This prospective, open-label, single-arm, multicenter, observational study (NCT02296164) enrolled adult patients with any stage MF-CTCL in 46 centers (US university-affiliated and community hospitals). The patients were either initiating or continuing chlormethine gel treatment and were followed for an observation period of 24 months during standard-of-care visits, regardless of whether the gel was discontinued. Clinical response, determined as a ≥50% reduction from baseline in body surface area (BSA), was evaluated at 12 months in patients with baseline and post-baseline assessments. A by-time analysis of the BSA response was performed to determine the proportion of responders over time (up to 24 months) and to assess when the maximum response occurred. HRQoL was assessed by the Skindex-29, a skin disease-specific questionnaire that includes 3 domains: emotions, symptoms, and functioning. Higher scores in the Skindex-29 indicate lower HRQoL or higher impact of disease. Results In total, 298 patients were included, with a mean age of 61.7 years and mean disease duration of 5.5 years at baseline. At 12 months, the proportion of stage IA–IB patients with a ≥50% reduction in the pre-enrollment baseline BSA percentage (%BSA; responders) was 45.1% (37/82). The by- time analysis showed there was variation in the response to treatment over time. Clinical responses were already seen after 1 month (36.7%) and the peak clinical response occurred at 18 months, when 66.7% of patients had a ≥50% reduction in %BSA (Figure 1). Over a 24-month period, clinical response was associated with an improved Skindex-29 score. The weighted mean scores for emotions, symptoms, and functioning were significantly lower in patients who responded to treatment (26.6, 25.3, and 13.3, respectively) compared with non-responders (36.2, 34.4, and 21.2; all p<0.001).

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Fig 1. Patients With ≥50% Reduction From Baseline in %BSA at 1, 3, 6, 9, 12, 15, 18, 21, and 24 Months (± 45 Days) – Patients With IA–IB Staging Only

The most commonly occurring adverse events (AEs) that were related to chlormethine gel were dermatitis, pruritus, skin irritation, erythema, and skin burning sensation. The frequency of most of these AEs appeared to be lower during the PROVe study than during the 201 registration study (Table 1). This could be due to concomitant use of topical corticosteroids and more flexibility in the dosing schedule in the PROVe study. No unexpected or serious chlormethine gel-related AEs were observed in either study. Table 1. Adverse Events: PROVe versus 201 Study PROVe Registration Study 2011 Chlormethine Gel Only AEs N=298 N=128 Overall, n (%) 125 (41.9) 108 (84.4)

CL gel-related AEs occurring in ≥3% of patients, n (%) Dermatitis 37 (12.4) 14 (10.9) Pruritus 22 (7.4) 20 (15.6) Skin irritation 21 (7.0) 31 (24.2) Erythema 12 (4.0) 17 (13.3) Skin burning sensation 10 (3.4) 2 (1.6) AE: adverse event; CL: chlormethine. Conclusions PROVe is the largest prospective, observational study of real-world chlormethine gel usage in the US. The peak clinical response by %BSA among stage IA–IB patients with available disease assessment data was higher (66.7%) than that observed in the post-hoc by-time analysis of the 201 study (55.7%) but took longer to occur. The by-time analysis showed a real-world fluctuation of MF-CTCL over time, with a trend showing steady improvement during treatment. This indicates that close monitoring of patients is important and continued use of chlormethine gel treatment is required to reach the maximum response. The by-time analysis results can also help patients and healthcare practitioners to set expectations regarding typical response timelines. Responders had improved HRQoL compared with non-responders, as determined by the Skindex- 29. Treatment with chlormethine gel was well tolerated, with a lower rate of skin-related AEs than observed in the 201 study. References 1. Lessin SR, et al. JAMA Dermatol. 2013;149:25–32.

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Presentation Summary

Incidence and types of contact dermatitis after chlormethine gel treatment in patients with mycosis fungoides-type cutaneous T-cell lymphoma: the MIDAS study

Authors: E.S. Gilmore1*, C.V. Alexander-Savino1, C.G. Chung2, B. Poligone1,3 1Rochester Skin Lymphoma Medical Group, Fairport, NY, USA; 2Departments of Dermatology and Pathology, The Ohio State University, Columbus, OH, USA; 3Rochester General Hospital Research Institute, Rochester, NY, USA

Introduction Chlormethine (mechlorethamine; nitrogen mustard) is an efficacious therapy for patients with stage IA and IB MF-CTCL, with higher response rates compared with other available treatments (Lessin et al, 2013; Hoppe et al, 1987). However, chlormethine use may be limited by cutaneous intolerance, such as contact dermatitis at the site of application. Chlormethine gel formulation has been developed to minimize these skin reactions, which, although they still occur, do so less frequently than with the aqueous formulation (Lessin et al, 2013; Hoppe et al, 1987). The Mechlorethamine Induced Contact Dermatitis Avoidance Study (MIDAS; NCT03380026) is evaluating the incidence and types of contact dermatitis following treatment with chlormethine gel monotherapy or chlormethine gel combined with triamcinolone ointment in patients with MF- CTCL.

Methods MIDAS is a non-randomized, open-label, split-face 2-arm study undertaken in patients aged ≥18 years with histologically confirmed stage IA or IB CTCL (which can include folliculotropic, granulomatous slack skin, or syringotropic MF, or large cell transformation). Patients received 2 different therapies concurrently to 2 discrete lesions that had similar characteristics. The interventions were 0.016% w/w topical chlormethine gel (once nightly) or 0.016% w/w topical chlormethine gel (once nightly) and triamcinolone 0.1% ointment once daily both applied over a minimum of 8 cm2, over a period of 4 months. Endpoints included percentage of dermatitis in lesions for both treatments, nature of contact dermatitis, efficacy, severity of dermatitis (assessed by a modified SCORAD), and etiology of dermatitis.

Results In total, 28 patients enrolled in the MIDAS study. Of these, 19 patients developed no or mild- moderate dermatitis, while 9 patients developed severe dermatitis (32.1%), requiring interruption of therapy. In total, 7/8 (87.5%) patients with severe dermatitis showed 2-3+ reactions at the 96- hour reading; 1/8 (12.5%) patients with severe dermatitis showed irritant-type reactions, and 5/8 (62.5%) patients with severe dermatitis also showed reactions to vehicle alone. Patch testing revealed that there were no reactions to 20% propylene glycol or 1% menthol in those tested.

One 72-year-old patient who presented with contact dermatitis with a delayed hypersensitivity response received 10 weeks of chlormethine treatment. Despite treatment suspensions in months 1, 2, 3, and discontinuation during month 4, the patient achieved a complete response by month 9. Another patient who was 81 years old had contact dermatitis without a delayed hypersensitivity response. They spent a total of 3.5 months on chlormethine (stopping 3 times for approximately one week over 4 months) and achieved a partial response at month 5; they then resumed treatment 3 times weekly and remain on treatment. Novel clones as well as expansion of pre-existing clones were identified in skin biopsies from the contact dermatitis.

Conclusions

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Contact dermatitis with and without hypersensitivity responses was reported in patients with MF- CTCL who had skin reactions to chlormethine treatment. Interestingly, the histology of the hypersensitivity reactions was often not classic for allergic contact dermatitis. The patients on the study who developed contact dermatitis exhibited differences in T-cell receptor repertoire with both new clones and previously identified clones that were expanded.

References Lessin SR, et al. JAMA Dermatol. 2013;149:25–32. Hoppe RT, et al. J Clin Oncol. 1987;5:1796–803.

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Paradoxical response of cutaneous T-cell lymphoma in a patient with concomitant angioimmunoblastic T-cell lymphoma Amy G. Johnson, MD, Rongqin Ren MD, PhD, Basem M. William, MD, MRCP(UK), FACP, and Catherine G. Chung, MD Multidisciplinary Cutaneous Lymphoma Program- The Ohio State University Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH, USA A 69-year-old Hispanic woman with a 2-year history of relapsed angioimmunoblastic T-cell lymphoma (AITL) presented for evaluation of violaceous discoloration of the right third toe [Figure 1]. She was diagnosed with stage III AITL 2 years prior and achieved remission following 6 cycles of CHOP. She experienced disease relapse 18 months later and was treated with rituximab + ICE with disease progression followed by a clinical trial where she received combination therapy with brentuximab vedotin and lenalidomide that resulted in complete remission. She noted swelling and violaceous discoloration of the right third toe that preceded any treatment for AITL that became more prominent immediately following each infusion with brentuximab vedotin before subsequent return to baseline. She denied associated symptoms including pruritus or tenderness. Skin biopsy demonstrated a diffuse proliferation of small to intermediate-sized pleomorphic CD3-positive T cells. A large number of lesional cells also expressed PD-1. T-cell gene rearrangement studies by PCR confirmed a monoclonal population of T-cells distinct from that observed in her prior lymph node biopsy for AITL [Figure 2]. This case highlights two unusual phenomena, the first being a co-occurring primary cutaneous T- cell lymphoma with a T-follicular helper phenotype similar to AITL, but with a distinctly separate clonal population of T-lymphocytes from her systemic disease. Her lack of clinical response despite complete remission of systemic disease further supports two discrete lymphomas. The second unusual finding is paradoxical response of her cutaneous disease to treatment. Despite a similar immunophenotype to her systemic lymphoma, she experienced a “tumor flare” with treatment rather than clinical response. These findings suggest that a deeper understanding of the molecular basis of some T-cell lymphomas may be necessary for more targeted therapy and response to treatment.

Figure 1. Violaceous edematous R 3rd toe.

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Figure 2. PCR showing separate monoclonal TCR-B peaks between marrow and toe biopsies.

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Progression of Cutaneous T-Cell Lymphoma after dupilumab: Case review of 6 patients

Maria L Espinosa BS,1 Morgan T Nguyen BA,1 Amaia Saenz Agirre MD,1 Maria Estela Martinez- Escala MD PhD,1 Christina J Walker MD,1 David S Pontes BS,1 Jonathan I Silverberg MD PhD MPH,2 Jaehyuk Choi MD PhD,1Barbara Pro MD,1 Laura B. Pincus MD,3 Joan Guitart MD,1 Xiaolong Alan Zhou MD1

1Northwestern University, Chicago, IL, USA. 2The Georgetown University School of Medicine, Washington D.C., USA. 3University of California San Francisco, San Francisco, California, USA.

Introduction: Dupilumab is a biologic approved for treating moderate-to-severe atopic dermatitis (AD) with efficacy in reducing pruritus. MF/SS and AD share similarities in clinical features including pruritus, skin impetiginization by Staphylococcus aureus, disruption of the skin barrier, and upregulation of T-helper 2 (Th2) cytokine pathways.

Methods: Retrospective review of 6 patients treated with dupilumab and either diagnosed with CTCL/mycosis fungoides (MF) following initial diagnosis of AD, or experienced rapid progression of previously diagnosed CTCL.

Results: Six patients (2 female; median age=66 [range 58-77] years) were identified. Dupilumab was initiated for clinically presumed AD in three patients and used off-label in CTCL (stages IB- IIIB) with pruritus in three patients (mean duration= 8 months; range 3-27 months). Some (n=5) experienced initial improvement, followed by worsening body surface area (n=6), pruritus (n=4), lymphadenopathy (n=3), systemic symptoms (n=3), and progression of blood involvement in all with previously diagnosed MF. The three patients with clinically presumed AD were diagnosed with CTCL/MF. After stopping dupilumab, all three stage IVA patients developed higher Sézary counts (two died of disease progression); the stage IIIA and IB patients experienced skin improvement with narrowband UVB and topical corticosteroids; the remaining stage IA patient continued dupilumab given atopic benefits but weighed discontinuation. See Table I on next page.

Conclusion: Dupilumab should be avoided in patients with MF/SS. Warning signs suggestive of CTCL for patients with presumed atopic dermatitis while on dupilumab therapy include new eczematous plaques in locations different than original sites, worsening pruritus, and lymphadenopathy.

References: Serrano L, Martinez-Escala ME, Zhou XA , Guitart J. Pruritus in Cutaneous T-Cell Lymphoma and Its Management. Dermatologic Clinics 2018;36:245-58.

Saulite I, Hoetzenecker W, Weidinger S, Cozzio A, Guenova E , Wehkamp U. Sezary Syndrome and Atopic Dermatitis: Comparison of Immunological Aspects and Targets. Biomed Res Int 2016;2016:9717530.

Table I. “Patient characteristics and response to dupilumab therapy

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Cas Sex/ Diagnosis Total Concomitant Length of Worsening on Sézary cell CTCL e Age prior to treatment therapies time dupilumab count staging, (y) dupilumab time on improved Outcome dupiluma on upon b dupilumab discontinuati (months) (months); on of description dupilumab of initial improveme nt 1 M/6 Presumed AD 8 Azathioprine 1; Decrease Palmoplantar N/A CTCL-NOS 4 (retrospective (PO), in BSA desquamation, Stage IIIA, ly diagnosed diphenhydrami (40% to severe skin Improvement as CTCL- ne (PO) 32%+) and burning/prurit with NBUVB NOS gabapentin pruritus us, and topical stage IB) (PO), development corticosteroids glucocorticoids of (T), prednisone erythroderma (PO) (BSA 95%), impetigization with S. aureus

2 M/7 Presumed AD 4 Methotrexate 1.5; Thickening of N/A MF Stage IB, 2 (PO) Decrease in plaques with Improvement BSA (80% superimposed with NBUVB to 60%) and papules and topical pruritus corticosteroids

3 F/59 Presumed AD 27 Gabapentin 8; Decrease Enlargement N/A MF Stage IA, (PO), in BSA neck of facial plaque Dupilumab glucocorticoids down (40% and onset of continued at (T), non- to 5%), fatigue and longer medication decreased weight loss intervals emollient(T), pruritus (600mg every tacrolimus(T) 3 weeks) given patient preference and atopic benefits.

4 M/6 MF Stage 3 Bexarotene 2; Decrease Palmoplantar 575/uL*, MF/SS Stage 7 IIIB (PO), in BSA desquamation, 1022/uL** IVA and hydroxyzine(P (80%+ to increase in death. O), interferon-γ 60%+), BSA (100%), (IM), decreased LAD, glucocorticoids pruritus worsening (T), Prednisone pruritus, (PO), fatigue, pregabalin (PO) impetiginizatio n with S. aureus

5 M/5 MF Stage IIA 3 Bexarotene 1.75; Increase in <100/uL*, MF/SS Stage 8 (PO), Improved BSA (60%+), 6,000/uL** IVA and chlormethine(T asthma and development , death. ), intravenous mild of LAD, 9,000/uL** immunoglobuli decrease in worsening * n (IV), BSA (15%+ pruritus, glucocorticoids to 13%+) fatigue. (T), tacrolimus(T) 6 F/77 MF Stage IB 3 Non-medication 0; N/A Development 1150/uL**, MF/SS Stage emollient (T), of 1296/uL** IVA, glucocorticoids erythroderma * endocarditis, (T) (BSA 80%) partial and LAD,

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worsening response with pruritus romidepsin. BSA, body surface area; IM, intramuscular; IV, intravenous; LAD, lymphadenopathy; MF, mycosis fungoides; NBUVB, narrow-band ultraviolet B phototherapy; PO, Oral; SS, Sézary syndrome; T, topical. * pre-dupilumab treatment, ** during dupilumab treatment, *** after dupilumab treatment. + Estimated body surface area from clinical chart.

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4th World Congress of Cutaneous Lymphomas- Oral Handout

Title: Primary cutaneous B-cell lymphoma – case series of two unique cutaneous presentations treated with rituximab.

Joseph R. Stoll BTL, Melissa Pulitzer M.D., Alison Moskowitz M.D., Steven Horwitz M.D., Patricia Myskowski M.D., Sarah J. Noor M.D.

Introduction: Primary cutaneous B-cell lymphomas (PCBCL) make up 25% of primary cutaneous lymphomas including primary cutaneous follicle center lymphoma (PCFCL) and primary cutaneous marginal zone lymphoma (PCMZL). Most cases of PCBCL present with solitary or grouped lesions that are typically treated with local treatment modalities (e.g. topical/intralesional steroids, excision, radiation), and less commonly, systemic treatment (rituximab), with possibility of recurrence but overall indolent course. We present two unique presentations of multifocal PCBCL (1 PCMZL and 1 PCFCL) that were ultimately treated with rituximab.

Case 1: A 43-year-old male with no significant past medical history presented with erythematous indurated plaque of 15-month duration involving bilateral ear helices and earlobes that was intermittently painful and pruritic. A skin biopsy showed primary cutaneous marginal zone lymphoma, and systemic workup including imaging was negative for extracutaneous involvement. He was treated with Rituximab 375 mg/m2 weekly for four weeks, with clinical improvement, with no recurrence at three-month follow-up. (Figure 1.)

Case 2: A 35-year-old female with no past medical history presented with a five-year history of asymptomatic erythematous papulonodules on the face, previously treated as cutaneous lupus (due to elevated ANA titers) with hydroxychloroquine, prednisone, and mycophenolate mofetil. Biopsy was consistent with primary cutaneous follicle center lymphoma, with negative systemic workup. She was similarly treated with rituximab 375 mg/m2 weekly for four weeks, with significant flattening of the papules and reduced erythema. She received a maintenance dose at one month, and skin was noted to be clear at six-month follow-up.

Discussion:

PCMZL usually presents as solitary or multiple erythematous to brown-colored papules, nodules, or plaques on the trunk and upper extremities. PCFCL similarly presents with solitary or grouped papules predominantly across the scalp, forehead, and posterior torso, with unique presentations reported including include rosacea-like, infiltrative lesions of the nose and/or rhinophyma and a scalp lesion of scarring alopecia within a cluster of tumid annular erythematous plaques.1

Management: Some European studies have found an association with Borrelia and PCBCL, and therefore the EORTC-CLG/ISCL recommends antibiotic therapy prior to initiation of invasive therapy in antibody positive individuals. However, in the United States, antibiotics are not recommended, with first line treatment of PCMZL and PCFCL often consisting of localized radiation, surgical excision, or topical/intralesional steroids. In patients with multifocal skin disease, systemic therapies including monoclonal chimeric anti-CD20 antibody have been used with varying therapeutic benefit. We present two unique clinical presentation of primary cutaneous B-cell lymphoma that were multifocal, symptomatic, and cosmetically bothersome, and not easily treatable with skin-directed modalities.

There have been a few case series of rituximab used in treatment of PCBCL - two case series of PCMZL and PCFCL patients who were treated with IV rituximab demonstrated excellent

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response rates (though patients with PCFCL had a better response).2, 3 In one study, all patients (10) with PCFCL had a response with 80% achieving complete response, while 3/5 patients with PCMZL had either partial or complete response, with clinical response observed in most patients at the end of induction therapy (median time to response 30 days).The second study (11 PCFCL, 5 PCMZL) similarly demonstrated an 87.5% complete remission rate for the cohort (with the PCFCL group having better response.) As with the two patients we are presenting, in these studies, rituximab was given IV 375 mg/m2 once weekly usually for 4-6 consecutive weeks, with variable maintenance regimens and duration of response (6-37 months) 2,3. It should however be noted that these series are too small to determine optimal duration of therapy for sustained response.

Conclusion: Multifocal or unusual presentations of PCBCL can complicate management. Although first-line treatment of PCBCL is typically localized/skin-directed treatment, the diffuse multifocal presentation in these patients made rituximab a more feasible choice with clinical improvement and no adverse effects. Duration of response and maintenance regimen remains to be determined. Further studies evaluating impact of PCBCL on quality of life may also help guide treatment decision, and determine which patients would benefit from systemic treatment.

Figure 1. Primary Cutaneous Marginal Zone Lymphoma presenting as indurated plaques on bilateral earlobes. Reduction in erythema and induration after treatment with weekly Rituximab 375 mg/m2 for four weeks.

1. Suárez AL, Pulitzer M, Horwitz S, Moskowitz A, Querfeld C , Myskowski PL. Primary cutaneous B-cell lymphomas: Part I. Clinical features, diagnosis, and classification. Journal of the American Academy of Dermatology 2013;69:329.e1-.e13. 2. Morales AV, Advani R, Horwitz SM, Riaz N, Reddy S, Hoppe RT et al. Indolent primary cutaneous B-cell lymphoma: Experience using systemic rituximab. Journal of the American Academy of Dermatology 2008;59:953-7. 3. Valencak J, Weihsengruber F, Rappersberger K, Trautinger F, Chott A, Streubel B et al. Rituximab monotherapy for primary cutaneous B-cell lymphoma: response and follow-up in 16 patients. Annals of Oncology 2009;20:326-30.

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Tonsil involvement as a marker of advanced disease in mycosis fungoides

I Csányi1, H Ócsai1, E Varga1, K Hidehéty2, M Marschalkó3, L Krenács4, Z Borbényi5, T Gurbity Pálfi5, J Oláh1,2, L Kemény1, E Baltás1

1 Department of Dermatology and Allergology, Albert Szent-Györgyi Health Centre, University of Szeged, Faculty of Medicine, Szeged, Hungary 2 Department of Oncotherapy, Albert Szent-Györgyi Health Centre, University of Szeged, Faculty of Medicine, Szeged, Hungary 3 Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Faculty of Medicine, Budapest, Hungary 4 Tumor Pathology and Molecular Diagnostic Laboratory, Szeged, Hungary 5 Second Department of Internal Medicine and Cardiology Centre - Haematology Unit, Albert Szent-Györgyi Health Centre, University of Szeged, Faculty of Medicine, Szeged, Hungary

Introduction: Oral involvement has been reported in less than 1% in mycosis fungoides (MF), but based on autopsy studies it is more frequent (7-18%) and is associated with poor prognosis.

Case 1: The 75-year-old male patient was first presented at our department in November, 2017 in poor general condition with a 3-month history of livid-erythematous plaques and ulcerated tumours. He had hypertension and insulin dependent diabetes mellitus as comorbidities. Histology of the skin biopsy revealed tumor stage of mycosis fungoides with large cell transformation. Flow cytometry showed elevated CD7-, CD26- and CD4+/CD8+ cell population. CT scans described axillar and inguinal lymhadenomegaly with no lymphoma involvement by histological examination. Regarding to the high ECOG status and age of the patient, PUVA therapy was initiated in combination with acitretin. Some of the lesions regrediated, nevertheless novel tumors occurred continuously. Thus low dose interferone (IFN) therapy was started with local irradiation of the larger tumors. Complete clinical remission was achieved with this combined therapy. Five months later severe dyspnea occurred necessitating tracheostomy for maintaining the patient’s breathing. The histology of the right tonsil revealed the infiltration of an aggressive peripheral T-cell lymphoma involving the Waldeyer lymphatic ring as extracutaneous manifestation of cutaneous T-cell lymphoma (CTCL). Local targeted irradiation was started immediately resulting significant tumor regression, cessation of the dyspnea and removal of the tracheostomy tube. Unfortunately, a few months later further tumor progression was formed in different locations on the skin. Nearly complete clinical remission was achieved again with palliative irradiation. However, we lost our patient due to the rapid deterioration of his general condition. Case 2: The 63-year-old male patient was first presented at our department in December, 2017 with a 10-year history of scaly, erythematous, non-itching plaques. He had no relevant comorbidities. The first histology showed lymphomatoid papulosis C type. Narrow-band UVB therapy were initiated with low effectiveness. Thus we repeated the skin biopsy and the second histology revealed plaque stage of mycosis fungoides. The skin directed phototherapy was combined with acitretin. Unfortunately 1 month later, tumorous lesions occurred on both legs and on the scalp. Our third skin biopsy confirmed the diagnosis of mycosis fungoides nevertheless in tumor stage with large cell transformation. Flow cytometry and bone marrow biopsy were negative. PET-CT described cutaneous manifestations. Interferone therapy was initiated. In October, 2018, the patient presented with progressive loss of hearing and constant sore throat. IFN therapy was stopped and the histological examination of the tonsil revealed extracutaneous manifestation of CTCL. Chemotherapy was started at the heamatology department, and later they switched to brentuximab-vedotin and bendamustine combined therapy to achieve complete remission. After the effective treatment combination, some novel skin lesions occurred, which proved to be cutaneous manifestations of CTCL. To maintain the remission, autologous stem cell

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transplantation was made. Unfortunately the patient died 2 months later in the complication of the transplant. Conclusions: Oropharyngeal manifestation of MF is extremely rare. It has been reported in less than 1% of the cases. However, based on autopsy studies it is more frequent, around 7-18% and is associated with poor prognosis. Clinicians must be aware of tonsil involvement not because based on the few reported cases and our experiences it can be a marker for advanced, aggressive and rapidly progressive cases in this otherwise indolent disease, but also because it can lead to severe complications requiring urgent interventions.

References: 1. Le BT, Setlur J, Sikora AG, Lee KC. Mycosis fungoides A Case of Tonsil Involvement. Arch Otolaryngol Head Neck Surg, 2006, 132(7):794-96. 2. TJ Phillips, IM Leigh, M Keir. Mycosis fungoides of the tonsil: a T-cell lymphoma involving the skin and tonsil. J R Soc Med, 1985, 78(Supll 11):25-27.

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EPSTEIN-BARR VIRUS-POSITIVE MUCOCUTANEOUS ULCER IN A PATIENT WITH DYSKERATOSIS CONGENITA Rosa Fornons1, Fina Climent1, Eva M González-Barca2, Cristina Muniesa1, Andrea Bauer1, Clàudia Llobera1, Octavio Servitje1. Hospital Universitari de Bellvitge1, Institut Català d’Oncologia2.

Introduction: Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBVMCU) has been included as a provisional entity in the 2016 World Health Organization Classification of Lymphoproliferative Disorders. EBVMCU was initially described as a solitary, sharply circumscribed ulcer in the oropharyngeal mucosa, skin or gastrointestinal tract in patients with age-related or iatrogenic immunosuppression. However, recent reports have shown that its clinicopathologic spectrum is wider than initially described and that other immunosuppressive conditions could be associated.

Case report: We report a case of a 53-year-old man with a history of repeated respiratory infections and bronchiectasis, who was referred to our clinic for an oral ulcer of 3 months duration (Figure 1). Additional skin examination revealed nail dystrophy and reticular skin pigmentation in the upper chest and neck, which are suggestive of dyskeratosis congenita (Figure 2). He has a twin with the same skin and nail changes and a 3-year-old niece diagnosed with dyskeratosis congenita in another hospital. The biopsy of the oral ulcer showed a polymorphous lymphoid infiltrate with abundant large lymphoid cells, some of them with Reed-Sternberg-like features, which stained positive for CD20, PAX-5, CD30 and EBERs (Figure 3). PET-CT revealed FDG activity only of oral lesions, without other remarkable findings. Serum EBV PCR was negative. HIV testing was negative. Routine blood tests were normal except for a reduced number of B and CD4+ T lymphocytes. Genetic studies revealed a mutation in TERC (48A>T) with relative telomeric length about 50-25%. The oral lesion underwent complete spontaneous remission after the biopsy was performed. Discusion: The EBVMCU was recognized as provisional entity by the WHO since 2016 but its real prevalence could be yet underestimated because of the rarity of this lesion and the often self- limited course. Clinically, EBVMCU may resemble nonlymphoid conditions, usually non hematological neoplasia. Therefore, biopsies of these lesions were often performed with another diagnostic suspicion (typically squamous cell carcinoma in cutaneous and oropharyngeal lesions). The lesions were characterized by the proliferation of EBV-positive, variably sized, atypical B-cells that may resemble Hodgkin and Reed-Sternberg-like cells. Once the biopsy findings are consistent with a lymphoproliferative disease associated with EBV, the clinical characteristics are fundamental to distinguish EBVMCU from other EBV-related lymphoproliferative disorders. The main differential diagnoses include lymphomatoid granulomatosis, EBV-positive diffuse large B- cell lymphoma, and EBV-positive classical Hodgkin Lymphoma. EBVMCU is usually an indolent process. Although radiotherapy or chemotherapy may be considered as therapeutic options, most patients have spontaneous regression or complete remission after withdrawal of underlying immunosuppressive drug. The ulcerated lesions rarely spread to distant sites, but locally spreads and relapses after regression have been reported. Only one disease-associated death has been reported. The EBVMCU seems to be developed in patients with a certain level of immunosuppression, which allows the development of an often self-limited local lymphoproliferative disease but not of a systemic infection. Therefore, in the presence of active viremia the EBVMCU diagnosis should be questioned and other lymphoproliferative disorders with a more profound level of immunosuppression should be suspected. EBV is a member of the herpes virus family (herpes virus 4) and approximately 95% of people become infected with this virus during childhood. In most people, EBV infections are transient, but some malignant tumors are associated with EBV infections.

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The first cases of EBVMCU were reported in patients who were undergoing iatrogenic immunosuppression or with age-associated immunosenescence. However, there were subsequent reports of EBVMCU in patients with other immunosuppressive conditions such as primary immunodeficiencies, HIV infection/acquired immune deficiency syndrome (AIDS) and recipients of solid organ or bone marrow transplant. Dyskeratosis congenita is a multisystem inherited syndrome caused by mutations in genes regulating telomere maintenance, exhibiting marked clinical and genetic heterogeneity. In its classic form, it is usually characterized by the mucocutaneous abnormalities (triad of abnormal skin pigmentation, nail dystrophy, and leucoplakia), bone marrow failure and a predisposition to cancer. Bone marrow failure is the principal cause of early mortality. Mutations in 8 different genes have been characterized in patients with dyskeratosis congenita (DKC1, TERC, TERT, NOP10, NHP2, TIN2, C16orf57, and TCAB1). Our patient has an autosomal dominant form, with a TERC mutation, which directly affects the telomerase (an essential protein to maintain telomere length). Without telomerase, the telomeres shorten with each successive round of replication, and when they reach a critical length the cells enter senescence. Conclusions: In conclusion, this case was consistent with an EBVMCU associated with immunosuppression in the context of dyskeratosis congenita. To the extent of our knowledge, this association has not been previously reported. Therefore, it is important to consider this entity in the differential diagnosis among different kinds of immunosuppressed patients. References 1.Dojcinov SD, Venkataraman G, Raffeld M, Pittaluga S, Jaffe ES. EBV positive mucocutaneous ulcer-a study of 26 cases associated with various sources of immunosuppression. Am J Surg Pathol. 2010;34(3):405-17. 2.Ikeda T, Gion Y, Yoshino T, Sato Y. A review of EBV-positive mucocutaneous ulcers focusing on clinical and pathological aspects. J Clin Exp Hematop. 2019;59(2):64-71.

Figure 1: oral ulcer after Figure 2: reticular skin pigmentation and nail dystrophy. biopsy.

A B

C D Figure 3: polymorphous lymphoid infiltrate with abundant large lymphoid cells, some of them with Reed- Sternberg-like features in H&E (A), which stained positive for CD20, CD30 (B), PAX-5 (C) and EBERs (D).

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Clinical, histological and molecular characteristics of anaplastic lymphoma kinase-positive primary cutaneous anaplastic large cell lymphoma Introduction: Unlike systemic anaplastic lymphoma (sALCL), the vast majority of primary cutaneous anaplastic large cell lymphomas (C-ALCL) do not carry translocations involving the ALK gene and do not express ALK. Expression of ALK protein therefore strongly suggests secondary cutaneous involvement of a sALCL. Recent studies described a small subgroup of ALK-positive C-ALCL, but information on frequency, prognosis and translocation partners is virtually lacking. Methods: A total of 6/309 (2%) C-ALCL patients included in the Dutch registry for cutaneous lymphomas between 1993 and 2019 showed immunohistochemical ALK expression. Clinical and histopathological characteristics, immunophenotype and disease course were evaluated. Underlying ALK translocations were analyzed with anchored multiplex PCR based targeted next- generation sequencing (FusionPlex, ArcherDX, Boulder, CO). Results: Median age at diagnosis was 39 years (range 16-53). All patients presented with a solitary lesion. Treatment with radiotherapy (n=5) or anthracycline-based chemotherapy (n=1) resulted in complete responses in all 6 patients. Three patients developed a relapse, of whom two extracutaneous. After a median follow-up of 41 months, 5 patients were alive without disease and one patient died of lymphoma (Table 1). Immunohistochemically, three cases (50%) showed combined nuclear and cytoplasmic ALK expression with underlying NPM1-ALK fusions, while three cases (50%) showed solely cytoplasmic ALK expression with variant ALK fusion partners (TRAF1, ATIC, TPM3; Table 2). Conclusion: ALK-positive C-ALCL is extremely uncommon, has a comparable favorable prognosis as ALK-negative C-ALCL, and should be treated in the same way with radiotherapy as first-line treatment.

Table 1. Clinical characteristics of 6 ALK positive C-ALCL patients Case 1 2 3 4 5 6 Age at onset 34 44 16 50 53 30 (y) Sex F F M F F M Clinical Solitary Solita Solitary Clustered Solitary Solita presentation tumor ry tumor left lesions tumor right ry abdomen tumor arm right lower elbow tumor left leg left breast arm Initial RT RT Anthracycli RT RT RT treatment ne-based chemothera py Response CR CR CR CR CR CR Relapses (time to relapse) + (4 - - - - - only skin months) - - + (11 + (4 - extracutane - months) months) ous

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Treatment Anthracycl - - Anthracycl Anthracycl after ine based ine based ine based - relapse chemother chemothera chemothera apy py py Response CR PD CR Follow-up Status last AWD AWD AWD DOD AWD AWD follow up 101 65 36 19 46 23 Duration (months) F; female, M; male, ALK; anaplastic lymphoma kinase, C-ALCL; primary cutaneous anaplastic large cell lymphoma, RT; local radiotherapy, CR; complete remission of 100% disappearance of lesions, PD; progressive disease, AWD; alive without disease, DOD; death due to disease.

Table 2. Immunophenotypic and genetic features of 6 ALK positive C-ALCL patients Case 1 2 3 4 5 6 Immuno- phenotype + + + + + + CD30 - - - + + + CD2 - + - - - - CD3 ------CD5 ND - ND - - + CD7 + - + + + - CD4 - + - - - - CD8 + + ND + + + Granzyme B + + ND - - + TIA-1 ND >80% ND 50% >60% >75% Ki-67 ALK1 + + + - - - Nuclear + + + + + + Cytoplasmic Genetics Translocation NPM1- NPM1- NPM1- TRAF1- ATIC-ALK TPM3-ALK Exon ALK ALK ALK ALK 7 :: 20 7 :: 20 4 :: 20 4 :: 20 4 :: 20 6 :: 20 ALK; anaplastic lymphoma kinase, C-ALCL; primary cutaneous anaplastic large cell lymphoma, ND; not done, NPM1; nucleophosmin 1, TRAF1; TNF Receptor Associated Factor 1, ATIC; 5'aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase, TPM3; tropomyosin 3

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Not Everything That Shines is Lymphoma: A Case Series of lupus erythematosus mimicking cutaneous marginal-zone B-cell lymphoma

M. H. Trager,1, C. Ram-Wolff2, J.D. Bouaziz2,3, M. Battistella3,4, M.D. Vignon-Pennamen4, J. Rivet4, P. Brice5, A. de Masson2, M. Bagot*2,3, G. Dobos*2,3

1Columbia University Vagelos College of Physicians and Surgeons 2Department of Dermatology, APHP, Saint-Louis Hospital, Paris, France 3Université de Paris, INSERM U976, Paris, France 4Pathology Department, APHP, Saint-Louis Hospital, Paris, France 5Hematological Oncology, APHP, Saint-Louis Hospital, Paris, France *these authors contributed equally

Introduction Marginal zone B-cell lymphoma (MZL) is a neoplastic proliferation of cells ranging from marginal zone B cells to plasma cells. A key component of the diagnosis are light chain restricted plasma cells seen on immunohistochemistry, and in cases with insufficient numbers of plasma cells clonal immunoglobulin heavy chain rearrangements will be detected. It typically affects adults aged 35-60 years and presents as multiple red to violaceous plaques or nodules preferentially located on the trunk and arms. On biopsy, patchy, nodular, or diffuse infiltrates are seen with sparing of the epidermis. Lupus erythematosus tumidus (LET) is a rare form of cutaneous lupus erythematosus that was first described in 1930. Clinically, it is characterized by nonscarring, erythematous, succulent, urticaria-like plaques without surface changes and is slightly more frequent in males. Association with systemic lupus erythematosus (SLE) is not frequent, and only 10% of patients have an elevated anti-nuclear antibody (ANA) level. Similar to MZL, biopsy classically shows perivascular and periadnexal superficial and deep lymphocytic infiltration. Here we present the cases of two young female patients with a challenging diagnoses of LET that were initially diagnosed as MZL. Clinicopathological correlation led to revision of the diagnosis of LET leading to a change in treatment and symptom improvement. These cases highlight the importance of considering LET in cases of difficult to treat MZL.

Methods In this retrospective case series two patients are presented from the Hopital Saint Louis with initial diagnoses of MZL. Here we review the clinical course and biopsies leading to their final diagnosis of LET.

Results Case Presentation 1

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A 19-year-old female was referred to the Hopital Saint Louis for evaluation and management of papulonodules on the arm and forearm, present for 4 years (Figure 1). Biopsies at outside hospitals were inconclusive but were read as MZL versus pseduolymphoma without B cell clonality or light chain restriction. At Hopital Saint Louis, a diagnosis of pseudolymphoma rather than MZL was made based on histology. However, the clinical presentation and chronicity of the lesions was more suggestive of MZL. A complete autoimmune work-up was negative, clinicopathologic correlation favored a diagnosis of LET. The patient was started on empiric treatment with hydroxychloroquine Figure 1. Patient 1 papulonodules and plaque in September 2010 which was self-discontinued after one month on the right cheek and B) arm. C) Patient 2 although no intolerability was experienced. Treatment with papulonodules and plaque on the right cheek chloroquine was initiated in December 2010 given concern for LET and D) left arm and back. but was discontinued after seven days due to development of urticarial lesions. Hydroxychloroquine (200 mg daily) was started in January 2011 and continued for four months without improvement. Another biopsy was performed in June 2011 showing normal epidermis, conserved papillary structures, and no vacuoles at the basal layer. In the dermis, there was moderately dense infiltration of small lymphocytes, numerous histiocytes, and plasmocytes at the dermal- hypodermal junction organized along the capillaries. Immunohistochemistry showed an infiltrate comprised of 60% CD3+ T lymphocytes (70% CD4+, 30% CD8+), 40% CD20+ B lymphocytes, and numerous CD68+ histiocytes. Biopsy was read as a non-specific lymphohistiocytic infiltrate with an important B-cell component unusual for lupus (Fig 2). Given the clinical picture, treatment with topical steroids and doxycycline was initiated in October 2011 leading to stable disease. Thalidomide (50 mg daily) was started in April 2013 with significant improvement in lesions without side effects and dosage was increased to 100 mg daily. At the two-year follow-up visit in 2015 improvement persisted.

Case Presentation 2 A 20-year-old female was referred to the Hopital Saint Louis in 2019 for papulonodules on the trunk, thighs, and face present for four years. Previously, three biopsies over nine months at outside hospitals were read as MZL versus pseudolymphoma. Despite the lack of clonality and light chain restriction, diagnosis of MZL was validated by the tumor board based clinicopathological correlation and the strong B-cellular lymphocytic infiltrate in October 2018. Treatment with rituximab was ineffective and she was referred to Hopital Saint Louis for further evaluation. A complete workup was performed including an autoimmune panel and a new skin biopsy (April 2019), which Figure 2. Pathology images from case presentation was read as LET. Morphology showed preserved thickness of 2 biopsy in June 2011. A) Immunohistochemistry the epidermis and perivascular infiltrate across the entire staining for CD3. Infiltrate was comprised of 60% CD3+ T lymphocytes (70% CD4+, 30% CD8+) B) dermis with small lymphocytes and some eosinophils. Immunohistochemistry staining for CD20. + Immunohistochemistry showed predominance of CD3 Infiltrate was comprised of 40% CD20+ lymphocytes with occasional B lymphocytes (CD79+ and lymphocytes C) H&E stain at 4x magnification D) + - H&E stain at 10x magnification. Biopsy was read PAX5 ). The B lymphocytes were CD20 , probably secondary as a non-specific lymphohistiocytic infiltrate with to treatment with rituximab. Some follicular helper T cells an important B-cell component. were observed (PD1+, ICOS+, BCL6+, focal CXCL13+). CD123 staining showed a large population of plasmocytoid dendritic cells often clustered over the entire dermis. There was no light chain restriction and clonality was negative, making MZL unlikely. Given the diagnosis of LET, the patient began treatment with prednisolone and chloroquine leading to improvement in symptoms. Thalidomide

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was subsequently started and lesions significantly improved after 6 weeks of treatment. The dosage of prednisone was decreased from 10 mg to 5 mg and she will continue follow-up.

Conclusions These cases highlight the difficulty in distinguishing marginal zone B-cell lymphoma from lupus erythematosus tumidus both clinically and histopathologically. It is important to consider lupus erythematosus tumidus as a differential diagnosis of marginal zone B-cell lymphoma in cases of discrete lymphoid infiltrates or if refractory to standard treatments and of atypical evolution. Positive B-cell clonality or light-chain restriction are essential for diagnosing marginal zone B-cell lymphoma.

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Mycosis Fungoides – Granulomatous Slack Skin in association with Hodgkin Lymphoma Assis GDRRA, Miranda VJM, Visentainer L, de Moraes AM, Stelini RF, Secamilli EN, Massuda JY. State University of Campinas, São Paulo, Brazil

INTRODUCTION Granulomatous Slack Skin (GSS) is a variant of mycosis fungoides (MF), a form of cutaneous T-cell lymphoma, with pendulous slack skin in flexural areas. The disease preferentially affects adult men. It is considered a chronic, progressive and atrophying dermatological condition. However, in half of the cases, it may occur with other lymphoproliferative diseases, such as Hodgkin Lymphoma (HL).

CASE REPORT A 59-year-old man had been diagnosed with HL in inguinal and cervical lymph nodes two months before dermatological evaluation. He reported erythema and itchy desquamation in the abdomen and inguinal region for years, worsening in the last two months. He presented erythematous atrophic and elastotic plaques in the axillary region, medial aspects of the arm, hypogastrium, inguinal and scrotal region. Histology of cutaneous fragments from the left arm, left hemithorax and hypogastrium showed marked atrophy of dermal collagen and elastic fibers, replaced by loose and well-vascularized connective tissue. It was also demonstrated moderate interstitial lymphocytic infiltrate in the dermis extending to the epidermis with small lymphocytes, lined up along the dermoepidermal junction, with high lymphocyte density and Pautrier microabscesses. Verhoeff staining showed a numerical reduction and fragmentation of elastic fibers. In immunohistochemistry, infiltrate was positive for CD2 and CD5 in most part of lymphocytes, with CD8 and CD20 positive in 5% and CD56 negative. The hypothesis of MF - GSS in association with HL was corroborated. Patient underwent 6 cycles of chemotherapy, with remission of HL. Treatment with topical corticosteroids and PUVA was instituted with improvement of lesions and good clinical control.

Fig 1: Pendulous slack skin in right armpit Fig 2: Slack skin in inguinal region

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Fig 3: Slide stained in hematoxylin eosin. On the left: lymphocytic infiltrate in the upper dermis, with lymphocytes permeating the epidermis and fibrosis of the superficial dermis; On the right: lymphocyte detail permeating the epidermis.

Fig 4: Verhoeff staining slide comparing the superficial dermis with very rare, almost imperceptible elastic fibers (on the left) and the deep dermis with elastic fibers present (on the right).

DISCUSSION GSS is classified as a MF variant. It is characterized by atrophic erythematous or violaceous plaques affecting flexural folds, such as the armpits and inguinal region. With evolution, folds of redundant and pendular skin form. It can occur at any age, with a predilection for male adults. Histology shows a lymphohistiocytic infiltrate, mainly in the upper dermis, with T lymphocytes with a cerebriform nucleus. There is loss of elastic fibers and elastophagocytosis, with multinucleated giant cells and elastic fibers phagocytosis. There may be epidermotropism and Pautrier microabscesses. In immunohistochemistry, CD4, CD45RO, CD8, CD30 cells predominate, with loss of T cell markers such as CD3, CD5, CD7. Among the differential diagnoses, anetoderma stands out among clinical diagnoses and Granulomatous MF among histological differentials. CLG can be an indolent disease, of slow evolution, however, in half of the cases it is associated with lymphoproliferative diseases, being Hodgkin's lymphoma the most common, exactly as the case demonstrated. Treatment consists of topical and systemic corticosteroids, PUVA, radiotherapy, immunosuppressants, immunomodulators (interferon) and surgical treatment. In this patient, topical corticosteroids and PUVA were performed, with good response. The purpose of this presentation was to demonstrate a case of GSS clinically exuberant, with didactic histology, and association with another lymphoproliferative disease.

REFERENCES Gangar P, Venkatarajan S. Granulomatous Lymphoproliferative Disorders: Granulomatous Slack Skin and Lymphomatoid Granulomatosis. Dermatol Clin. 2015;33(3):489–496. Motta LMD, Soares CT, Nakandakari S, Silva GVD, Nigro MHMF, Brandão LSG. Granulomatous slack skin: a rare subtype of mycosis fungoides. An Bras Dermatol. 2017;92(5):694–697.

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Title: Aggressive cutaneous T-cell lymphomas at the dermatology department of Pontificia Universidad Católica de Chile: a series of 20 cases. Authors: Montserrat Molgó, Francisco Reyes-Baraona, Isabel Ogueta, Renata Acle, Sergio González

Introduction: Cutaneous T-cell lymphomas (CTCL) are heterogeneous, with a prognosis determined in large part by clinical, histopathologic and immunophenotypic features. Certain subtypes have been proven to be associated with a poor response to therapy and/or short survival.1 These aggressive subtypes of CTCL include Sézary syndrome, extranodal NK/T-cell lymphoma nasal type and other EBV-associated neoplasms, adult T-cell leukemia/lymphoma, primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma, primary cutaneous gamma delta T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified. Also, some indolent subtypes of CTCL may be associated with an aggressive course, including large-cell transformed mycosis fungoides and subcutaneous panniculitis-like T cell lymphoma.1 We report the aggressive subtypes of primary CTCL evaluated in the Dermatology department of our center.

Methods: Clinical cases, demographic and histopathologic characteristics, treatment and evolution of patients with diagnosis of primary CTCL at the dermatology department of Pontificia Universidad Católica de Chile between 1986 and 2019 were analyzed.

Results: 195 cases of primary CTLC were found. Of this, 20 cases were aggressive primary CTCL: 7 Sézary syndrome (SS), 2 large-cell transformed mycosis fungoides (T-MF), 4 subcutaneous panniculitis-like T-cell lymphoma (SPL), 1 primary cutaneous gamma delta T-cell lymphoma (γ/δ), 4 extranodal NK/T-Cell lymphoma nasal type (NK/T), 1 hydroa vacciniforme- like lymphoproliferative disorder (HVL) and 1 peripheral T-cell lymphoma not otherwise specified (NOS). Demographic characteristics, inmunophenotype, treatment and evolution of this 20 cases area detailed in Table 1.

Conclusions: These cases show us the importance of an early clinical suspicion and of the knowledge of the ominous prognosis of these aggressive subtypes.

References: 1. Junkins-Hopkins JM. Aggressive cutaneous T-cell lymphomas. Semin Diagn Pathol. 2017;34:44-59.

Table 1.

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Ca Se Ag Diagnosis Immunophenotyp Treatment Evolution se x e e 1 F 75 Sézary CD4+CD7-CD8- Chlorambucil Death (3 syndrome years) 2 F 83 Sézary CD4+CD7-CD8- PUVA, Death (2 syndrome Chlorambucil years) 3 M 82 Sézary CD4+CD7-CD8- Chlorambucil Death (2 syndrome years) 4 M 70 Sézary CD4+CD7-CD8- Chlorambucil Death (2 syndrome years) 5 M 64 Sézary CD4+CD7-CD8- CHOP, ECP Death (3 syndrome years) 6 F 57 Sézary CD4+CD7+CD8- Electrom beam, Death (1 syndrome Gemcitabine, year) Bexarotene 7 F 56 Sézary CD4+CD7-CD8- ECP, Alive syndrome Methotrexate 8 F 65 Transformed CD4+CD-CD8- CMT Death Mycosis CD30+ Fungoides 9 M 29 Transformed CD4+CD-CD8- CMT (R-COP), Death (6 Mycosis CD30+ Bexarotene months) Fungoides 10 F 22 Subcutaneous CD3+CD4-CD8- CHOP Death (<1 panniculitis-like CD56- year) 11 F 42 Subcutaneous CD3+CD4-CD8- CHOP, Interferon Death (<1 panniculitis-like CD56- year) 12 M 59 Subcutaneous CD3+CD4-CD8- CHOP Death (<1 panniculitis-like CD56-EBER+ year) 13 M 27 Subcutaneous CD3+CD4-CD8- CHOP, Death (3 panniculitis-like CD56-EBER+ Radiotherapy years) 14 M 36 Gamma/delta CD4+CD8+CD30 CMT (E-CHOP, Death (1 + CD56+EBER- SMILE) month) 15 F 37 NK/T nasal type CD3+CD56+EBE CHOP, Death (9 R+ Methotrexate months) 16 F 62 NK/T nasal type CD3+CD56+EBE 1° E-CHOP, 2° Death (9 R+ RDT, 3° month) intrathecal CMT, 4° MINE 17 F 56 NK/T nasal type CD3+CD56+EBE CHOP Death (3 R+ months) 18 F 14 NK/T nasal type CD3+CD56+EBE CHOP Death (5 R+ months) 19 F 29 Hydroa CD3+CD56+EBE Thalidomide Alive vacciniforme- R+ like 20 M 53 Peripheral T- (-) CMT, Death (2 cell lymphoma Radiotherapy years) NOS

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Abbreviations: CHOP: Cyclophosphamide, Doxorubicin, Vincristine and Prednisone. ECP: Extracorporeal photopheresis. CMT: chemotherapy. R-COP: Rituximab, Cyclophosphamide, Vincristine and Prednisone. E-CHOP: Etoposide, Cyclophosphamide, Doxorubicin, Vincristine and Prednisone. RDT: Radiotherapy. MINE: Mesna, Ifosfamide, Mitoxantrone and Etoposide. SMILE: Dexamethasone, Methotrexate, Ifosfamide, L-asparaginase and Etoposide.

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Anetodermia: secondary to syphilis or mycosis fungoides? Nathalia Lopes Iori NL, Assis GDRR, Cintra ML, Massuda JY, Stelini RF, Secamilli EM. State University of Campinas, São Paulo, Brazil

Introduction Cutaneous lymphomas are a group of diseases defined by malignant skin lymphocytes that do not present extracutaneous manifestations. More than half are T cell lymphomas, most of which correspond to mycosis fungoides (MF). The classic form of the disease presents itself in variable forms, ranging from macules, plaques and tumors, mostly located in photoprotected skin areas, to erythroderma in late stages. MF is considered one of the “great imitators”, along with syphilis, because of their wide range of clinical presentations. Unusual cases such as the one reported here may poses diagnostic challenge. Since there is no cure for MF, treatment seeks control of the disease and symptoms, along with improvement in the patient’s quality of life.

Case Report 68 years old woman refers erythematous and itchy skin lesions in flanks and right thigh, which appeared 15 years ago, with symptoms worsening in the last 2 years. Physical exam showed erythematous papules converging in a horseshoe form and lesions with an anetodermic aspect in flanks, lumbar region and right thigh (Fig. 01). Sensibility tests were normal. The hypotheses of MF was suggested, and a skin biopsy of the right thigh lesion was made, along with blood exams, which showed positivity in treponemal test and negativity in nontreponemal test. All other tests were normal. Three doses of benzathine penicillin (2.4 million IU) were prescribed for latent syphilis treatment, since patient referred no previous treatments, with reduction in lesions size, along with itching improvement. Histopathological exam revealed band-like lymphocyte infiltrate in the subepidermal layer, permeating the epidermis in a few regions, with slight associated spongiosis, outlining an intraepidermal lymphocyte aggregate. The lymphocytes had predominantly small volume and the papillary dermis exhibited fibroplasia. Immunohistochemically, lymphocytes expressed CD2, CD3 and CD5; some of them expressed CD7 and granzyme B and TIA-1 in spare lymphomononuclear cells. Therefore, MF was diagnosed, associated with latent syphilis, and treatment with UVB-NB phototherapy was initiated, alongside with clinical revaluation.

Discussion Cutaneous lymphomas are a group of Non-Hodgkin T, B and NK lymphomas, that manifest themselves on the skin, without evidence of extracutaneous disease at diagnosis. They are defined by presence of malignant lymphocytes on the skin. T cell lymphomas are three times more frequent than B cell lymphomas. MF is responsible for most cases of T cell lymphomas, being more common in adults, men and caucasians. Its etiology is still unknown, but theories suggest that chronic stimulation of T lymphocytes by a persistent antigen would lead to transformation of benign lymphocytes into neoplastic ones. According to the most recent WHO-EORTC classification, MF can be described in its classic form and in folliculotropic, pagetoid reticulosis and granulomatous cutis laxa subtypes. The classic form is characterized by hyper or hypochromic erythematous macules, that can be scaly (patches) or not, and by infiltrated scaly erythematous plaques, mainly localized in photoprotected areas. In advanced stages, it can present as tumorous lesions and erythroderma, which have worse prognosis. In MF initial stages, histopathology reveals lymphocytic infiltrate in papillary and subpapillary dermis, with mild or absent epidermotropism. Pautrier’s microabscess, a characteristic finding of the disease, is observed in only 10% of the initial stages, making histological diagnosis difficult in this period. In second stage, with the appearance of plaques,

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the lymphocytic infiltrate becomes dense and presents in band-like in the papillary dermis and epidermotropism becomes more evident. In the tumorous stage, epidermotropism is no longer so evident and there is intense infiltration of atypical mononuclear cells throughout papillary and reticular dermis. Finally, in the erythroderma stage, the neoplastic cells reach the bloodstream. Immunophenotyping of most cases shows memory T cells pattern, positive for CD2, CD3, CD4, CD5, CD45Ro and TCRbeta; and usually negative for CD30, loss of CD7 and CD26 antigens expression are frequent. Anetodermia is a benign condition that causes flaccidity of the skin and herniary sensation, because it alters or reduces the elastic fibers in a specific region of the skin. The main histopathologic feature of anetoderma is this elastic tissue loss in the dermis. It is more frequent in women and usually affects the upper trunk and proximal extremities. It can be classified as primary or secondary. Secondary anetoderma is more frequent and appears during the course of other systemic diseases. There are variable etiologies, ranging from genetic, autoimmune and infectious causes, and, among all possible causes are MF and syphilis, and cases like this, rarely reported, with the association of these two conditions. The diagnosis of MF is often challenging, both in its early stages, when there are few clinical manifestations and a lack of more specific histological changes, and in its unusual presentations. Therefore, it is important that we maintain a high index of suspicion, even when other diseases are identified that could justify the atypical condition.

References:

1) Girardi M, Heald PW, Wilson LD. The pathogenesis of mycosis fungoides. N Engl J Med. 2004; 350(19):1978-88. 2) Andrés-Ramos I, Alegría-Landa V, Gimeno, Pérez-Plaza A, Rütten A, Kutzner H, Requena L. Cutaneous Elastic Tissue Anomalies. Am J Dermatopathol. 2019 Feb;41(2):85-117.

Figures:

Figure 01. Clinical features of the physical exam. A. Erythematous papules converging in a horseshoe form in the right thigh. B, C. Lesions with an anetodermic aspect, with flaccid, finely wrinkled and circumscribed areas of slack skin in flanks.

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CD8+ Mycosis Fungoides Palmaris et Plantaris with Peripheral Blood Involvement Authors: S. Yumeen1, F. N. Mirza1, J. M. Lewis1, K. R. Carlson1, B. King1, S. Cowper1,2, C. G. Bunick1, J. McNiff2, M. Girardi1. Affiliations: 1Department of Dermatology, Yale School of Medicine, New Haven, CT, USA; 2Department of Pathology, Yale School of Medicine, New Haven, CT, USA; Introduction The majority of CTCL variants typically show a T-helper CD4+ phenotype. MF palmaris et plantaris represents a subtype of MF limited to the palms and soles. CTCL variants with CD8+ immunophenotype are relatively rare and include primary cutaneous aggressive epidermotropic cytotoxic T cell lymphoma and CD8+ variants of mycosis fungoides, including a reported case of CD8+ MF palmaris et plantaris limited to the skin. Blood involvement with a CD8+ CD4- immunophenotype remains a rare entity. Case Report We describe a rare case of CD8+ MF palmaris et plantaris with peripheral blood involvement. The patient presented with a longstanding history of relapsing-remitting palmoplantar skin disease recalcitrant to numerous therapies for dyshidrotic eczema and psoriasis, including immunomodulatory therapies.

Fig 1. images of palms, and right sole (a, b) before treatment.

Skin biopsy histology, immunohistochemistry (IHC), and molecular analyses eventually indicated the presence of an epidermotropic clonal CD8+ (CD4-) T cell population of the palms and soles. High-throughput sequencing of the T cell receptor gene rearrangements of unsorted peripheral blood leukocytes and samples from the palms and soles revealed precise clonal matches in blood to both palm and sole skin sites, with sequence frequencies >10%. Positron emission tomography-computed tomography imaging did not reveal changes of lymph node or visceral involvement. Collectively, these findings define the rare diagnosis of CD8+ MF palmaris et plantaris with peripheral blood involvement.

Figure 2. (a) Haematoxylin and eosin x 200 original magnification photomicrograph: dense lymphocytic infiltrate in the dermis, single and nested atypical lymphocytes within the epidermis. (b) CD8 immunohistochemical stain x 200 original magnification photomicrograph: positive staining is seen in the majority of lymphocytes.

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Discussion This case presents a rare instance of CD8+ MF palmaris et plantaris with peripheral blood involvement. The majority of previously reported cases of primary cutaneous CD8+ cytotoxic T cell lymphomas show metastatic involvement of distal anatomic sites and poor prognosis, however blood involvement is uncommon. Our patient presented with palmoplantar skin disease recalcitrant to numerous therapies, with skin biopsy histology, immunohistochemistry (IHC), and molecular analyses eventually indicating the presence of an epidermotropic clonal CD8+CD4- T cell population in the palms and soles. Notably, our case arrived at a diagnosis of MF palmaris et plantaris after trials of multiple therapies, including TNF⍺ inhibition. Previous reported cases of CTCL have revealed unmasking and progression after anti-TNF therapy, and such may have contributed to the pathoetiology in our patient. Our case highlights the importance of high clinical suspicion for CTCL in patients with chronic unremitting inflammatory dermatoses recalcitrant to treatment, especially when systemic immunomodulatory treatments are considered or utilized, as such may exacerbate underlying malignancy. References 1. Resnik KS, Kantor GR, Lessin SR, et al. Mycosis fungoides palmaris et plantaris. Arch Dermatol. 1995;131(9):1052-1056. 2. Weed J, Gibson J, Lewis J, et al. FISH Panel for Leukemic CTCL. J Invest Dermatol. 2017;137(3):751-753. doi:10.1016/j.jid.2016.10.037.

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Lymphomatoid drug eruption after treatment of Hepatitis C virus infection with Sofosbuvir: A new described adverse reaction Mirna Michel, Mohamed Farouk, Mona Ibrahim Dermatology, Venereology and Andrology Department, Ain Shams University Hospital; Cairo, Egypt

I. Introduction: Lymphomatoid drug eruption is a type of T-cell pseudo-lymphoma where there is atypical lymphoid infiltrate that simulates cutaneous T-cell lymphoma. It is most commonly anticonvulsants induced. Sofosbuvir (Sovaldi®) is an anti-viral drug used successfully in treatment of hepatitis C virus (HCV) infection that was not reported to cause lymphomatoid drug eruption.

II. Case Report:  A 53-year-old HCV positive male patient presented with itchy scaly erythematous macules and papules over face and trunk one month after his last dose of Sofosbuvir.  There was no associated fever or hepato-splenomegaly.  Two weeks later he developed areas of exfoliations all over his body along with generalized lymphadenopathy.  Clinical differential diagnosis included maculopapular drug eruption and pityriasis rosea.  Histopathological examination of the skin biopsy showed moderate superficial and mid- dermal perivascular and perifollicular lymphocytic infiltrate, infiltrating the overlying epidermis with epidermotropism and many haloed lymphocytes were seen displaying mild atypia, the papillary dermis also showed fibroplasia however no eosinophils could be identified.  Immunohistochemistry revealed that the infiltrate was positive for CD3 and negative for CD7.  Investigations were done in the form of pan CT scans, CBC, LDH, β2 microglobulin, comprehensive metabolic profile; and all were found to be normal except for Pan CT scans with contrast that showed generalized benign reactionary lymphadenopathy.  The provisional diagnosis was primary cutaneous T-cell lymphoma (classic mycosis fungoides).  Interestingly, during follow up the patient showed complete resolution of all the lesions within two months.  The final diagnosis was lymphomatoid drug eruption reporting a new adverse reaction that may occur with anti-viral treatment of HCV infection.

III. Discussion:

 Lymphomatoid drug eruption is a type of drug induced T-cell pseudo-lymphomas that is characterized by atypical lymphoid infiltrates which simulates cutaneous T- cell lymphomas.  It may occur with many medications as antidepressants, phenothiazines, benzodiazepines, lithium, antihistamines, calcium channel blockers, ACE inhibitors, beta blockers, and statins but occurs most commonly with anticonvulsants.

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 It can present with many clinical variants as; macules and patches, localized papules, plaques, localized nodules, single or generalized papulonodules or even erythroderma simulating sézary syndrome.  Eruption mostly occurs within 1–11 months following drug intake and its regression takes longer than other drug reactions as it can persist for 6 months or more with an average of 7 weeks from drug cessation. It can rarely turn into malignant lymphomas.  Histopathological findings are superficial and deep peri-adnexal and interstitial infiltrate that is T-cell-dominated with nuclear atypia. CD7 can be lost as in lymphoma while molecular analysis of TCR genes usually shows polyclonal pattern.  Differentiation between lymphomatoid drug reactions and cutaneous T-cell lymphomas needs clinicopathological, immunohistochemical and clonal correlation. Sometimes post withdrawal clinical improvement is the only definitive measure to identify the benign nature of the atypical lymphoid infiltrates.  So, we report a new adverse reaction that may occur with anti-viral treatment of HCV infection (Sofosbuvir).

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4WCCL 2020 - World Congress of Cutaneous Lymphomas Oral Presentation Hand Out

Mycosis Fungoides, Lymphomatoid Papulosis and Hodgkin’s Lymphoma in the Same Patient: Apropos of a Possible Monoclonal Origin.

M. Molgó,1 L. Espinoza-Benavides,1 P. Rojas,2 S. González.3 Departments of Dermatology,1 Hematology,2 and Pathology,3 Pontificia Universidad Católica de Chile.

Introduction Mycosis fungoides (MF), lymphomatoid papulosis (LyP) and Hodgkin’s lymphoma (HL) have been defined independently, yet case reports in which two of these diseases coexist suggest the possibility that there might be a common pathogenesis underlying these entities. Presence of all the three conditions in the same patient had not yet been reported.

Case report A 59-year-old man was referred by a hematologist for cutaneous lesions. His hematological history began 3 months prior to his dermatological visit and consisted of diarrhea, diffuse eczema, and a weight loss of 12 kg, without fever or night sweat. An etiological study was performed to assess the diarrheic episode to rule out an infectious disease. An axillary adenopathy was discovered, and the biopsy was compatible with HL. Enzyme-linked immunosorbent assay (ELISA) test for HIV was negative. Positron emission tomography–computed tomography (PET- CT) showed a supra and infra-diaphragmatic nodal compromise with an associated pleural effusion. A bone marrow biopsy was performed which reported normal hematopoiesis. A diagnosis of nodular sclerosis–type Hodgkin lymphoma stage IIIB was made, and chemotherapy was started with adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) regimen. At the time of his dermatological consultation, the patient was still under this treatment, with no other relevant medical background. He had a history of 1.5 years of generalized xeroderma and skin desquamation, initially asymptomatic and subsequently pruritic. The physical examination revealed a generalized ichthyosiform desquamation and the presence of erythematous, scaly plaques on the left groin area, abdomen, and upper extremities. A provisional diagnosis of MF was made, and 5 punch biopsies were taken. The serology for human T-lymphotropic virus (HTLV) type 1 and type 2 and Epstein Barr virus were negative; LDH level was normal.

The patient came for his dermatology follow-up having finished his hematology treatment. The result of the skin biopsy showed orthokeratosis, mild spongiosis, and multiple foci of epidermotropism with some roughly shaped Pautrier’s microabscesses. The dermis had a superficial infiltrate of histiocytes and atypical lymphocytes with large, hyperchromatic nuclei with an irregular contour. Immunohistochemistry was positive for CD4 in 80 percent of the lymphocytes and positive for CD8 and CD7 in 15 and 5 percent of the lymphocytes, respectively. These findings were compatible with the diagnosis of MF. During his medical visit, the patient complained of appearance of some new painful nodules. Physical examination revealed nodules with an erythematous halo and superficial crust on his abdomen and lower extremities. An infectious component was suspected, a cutaneous culture was obtained, and our empirical treatment began with cefadroxil and cold cream, having the intention of starting psoralen and ultraviolet A (PUVA) therapy once the infection was resolved. Vitamins D and B12 levels were also requested. Due to a nonresolving clinical course of the painful nodules after 10 days of treatment, a biopsy was performed. Histological studies showed orthokeratosis, irregular

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acanthosis, moderate spongiosis, and exocytosis of small and medium lymphocytes, with an ulcerated area. The dermis had a nodular, perivascular and interstitial, superficial and deep infiltrate, mainly composed of neutrophils and atypical lymphocytes containing irregular, vesicular nuclei and visible nucleoli. Periodic acid–Schiff (PAS) staining was negative for fungi. Immunohistochemistry was positive for CD3 in 90 percent of the lymphocytes and positive for CD25 and CD30 in 50 percent of the lymphocytes. These findings were compatible with the diagnosis of LyP. The skin culture showed normal skin flora. Vitamin D level was 7.5 ng/ml and that of B12 was 262 pg/ml.

Molecular clonality testing was performed using polymerase chain reaction (PCR) to assess for T cell receptor (TCR) gene rearrangement. The result was positive for monoclonal TCR-gamma rearrangement, a possible evidence of a same clonal origin for both the MF and the LyP. Due to technical limitations, we could not asses the clonality of the Reed–Sternberg cells in this case. During next medical visit, the physical examination of the patient showed clinical improvement, but with a diffuse desquamation on his extremities, abdomen and torso, and an erythematous scaly plaque on his left leg. PUVA-therapy was started with vitamin D supplementation and cold cream application. A good clinical response to phototherapy could be observed. After a year of remission, the patient presented with a relapse of his HL. Second line chemotherapy was started with anifosfamide, carboplatin, and etoposide (ICE) regimen. After completing two cycles, an autologous bone marrow transplant was performed. One month later, new scaly plaques and nodules appeared on his arms. Biopsy was compatible with mycosis fungoides with large cell transformation. The patient was subsequently planned to begin treatment with systemic bexarotene.

Lymphocyte T clonality test for VJ-A and VJ-B: (A) Lymphomatoid papulosis specimen. (B) Mycosis fungoides specimen. (C) Polyclonal control. (D) Monoclonal control.

Discussion: There is a biological plausibility for a unique neoplastic clone of T lymphocyte to manifest a clinical concomitance of more than one type of lymphomas. Likewise, epidemiology supports these associations. Notwithstanding such findings, when it comes to clinical corroboration, there is scarcity of publications, for which reason the monoclonal origin hypothesis is still controversial, requiring yet greater amount of evidence to establish a possible pathological spectrum. Our case represents, to our knowledge, the first report of MF, LyP, and HL diagnosis in the same patient.

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Pagetoid reticulosis: a rare variant of mycosis fungoides Secamilli EN, Massuda JY, Stelini RF, Adad MAH, Magalhães RF, Cintra ML, Souza EM

Pagetoid reticulosis, also known as Woringer-Kolopp disease, is a variant of mycosis fungoides. It is characterized by persistent psoriasiform or verrucous plaque localized on the limbs and its name derives from its histological features showing intraepidermal pagetoid distribution of the neoplastic lymphocytes. We report a case of a 49-year-old man that presented in 2012 to our dermatologic clinic complaining of itching and burning in a thickened plaque on the back of his left hand continuously growing for 18 months. At dermatologic examination, there was an erythematous and squamous plaque of 10 centimeters localized on the dorsal aspect of the left hand (figure 1A).

1A 1B

Figure 1A - Erythematous and squamous plaque of 10 centimeters on the dorsal aspect of the left hand. 1B – Cicatricial plaque after radiotherapy, 7 years of follow up.

The complementary exams, including CBC, serologies for HIV and HTLV, and thorax, abdomen and pelvis computed tomography scanning were normal. Skin biopsy was performed, and its histopathological examination showed epidermal hyperplasia and a prominent intraepidermal lymphocyte infiltration, with cerebriform nuclei. Immunohistochemistry staining was CD3-, CD8+ and CD30+.

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2 2 A B

Figure 2A- H&E: Epidermal hyperplasia and an extensive intraepidermal lymphocyte infiltration in a pagetoid distribution, with cerebriform nuclei and a perinuclear clearing (halo). 2B- CD8 antibody staining: atypical intraepidermal lymphocytes are CD8+.

The diagnosis of pagetoid reticulosis was made, and the patient was treated with localized radiotherapy. He has been followed for seven years and he is free of disease since then (Figure 1B). Pagetoid reticulosis was firstly described by Woringer and Kolopp in 1939. Since 1984, about 50 cases have been reported. The diagnosis is made by clinical-histological correlation, when solitary or localized psoriasiform or verrucous plaques in the acral region presents with the histologic features of epidermal hyperplasia and parakeratosis with epidermotropic and pagetoid atypical lymphocytes. In most of the cases, the lymphocytic infiltrate is CD3+CD8+. The clinical differential diagnoses include psoriasis and verrucous infections like tuberculosis and paracoccidiodomycosis. Histologically, it should be distinguished from primary CD8+ cytotoxic T-cell lymphoma. Treatment should be skin directed, and localized radiotherapy, phototherapy, surgical excision and topical steroids are the main options. The prognosis is excellent since neither extracutaneous dissemination nor disease related deaths have been reported.

References: 1. Corbeddu M, Ferreli C, Pilloni L, Faa G, Cerroni L, Rongioletti F. Pagetoid reticulosis (Woringer-Kolopp disease) in a 2-year-old girl—Case report and review of the literature. JAAD Case Rep. 2019 Jan; 5(1): 104–107. 2. Larson K, Wick MR. Pagetoid Reticulosis: Report of Two Cases and Review of the Literature. Dermatopathology (Basel). 2016 Mar 4;3(1):8-12.

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WCCL 2020 Abstract Title: „Maintenance Therapy – a neglected topic“, Presentation Summary Session Z3 – Clinical Observatons III, 2020.02.13, 11:20 am, Room 1 Presenting Author: Rudolf Stadler Patients with advanced stage (IIB – IVB) mycosis fungoides (MF) or Sezary’s Syndrome (SS) have a high symptom burden (e.g. pruritus) and a poor prognosis (median overall survival < 5 years [1]. The majority of patients with advanced disease is responding to systemic treatment approaches, but responses are generally not durable (median time to next treatment, TTNT: 5.4 months) [2]. Charlotte F. M. Hughes et al. have shown that most of the current therapeutics lack a durable disease control, especially with the use of chemotherapy for MF and SS. Even with the use of total skin electron beam therapy as skin directed therapy for MF, the median PFS in stage IIB and stage III lasts between 10.2 to 11.3 months. Only two new compounds, the antibody drug conjugate brentuximab vedotin has shown a PFS of 16.8 months, and the CCR4 antibody mogamulizumab has demonstrated a median PFS of 7.7 months. After inducing a clinically relevant remission the key questions, how to prolong the remission and avoid disease relapse or progression, remain. Currently there are no standardized strategies for maintenance therapy in patients with cutaneous T-cell lymphoma (CTCL) who achieve disease control. Moreover, evidence-based treatment options or drugs that are approved for maintenance treatment in advanced stages after systemic remission are still lacking. Thus, for patients requiring an effective palliation, new maintenance approaches are needed that provide a good safety profile, are convenient to apply, and do not affect quality of life. The majority of available systemic treatment options rarely induce long-term remissions; shorter duration of remission in most patients with advanced stage disease / median duration of response declines as the severity of the disease increases. In patients with advanced CTCL treatment decisions are individualized; however, treatment heterogeneity in advanced CTCL does not seem to influence survival. There is no guiding evidence on the indication and selection of maintenance therapies in MF and SS patients. Maintenance therapy can be defined as a continuous exposure to a skin directed or systemic therapy once remission has been achieved with the aim to maintain response and prevent relapse and progression. Qualifying criteria for the use as maintenance modality should be effectiveness of the compound, availability, safety, only small interference with quality of life as much as possible, and convenience. Following this approach Martine Bagot et al. started with the EORTC group a phase III trial of lenalidomide maintenance after debulking therapy in patients with advanced CTCL. In this patient cohort, the study showed 32 % progression within 4 weeks after registration. The high number of patients with disease progression after having immediately achieved a response after debulking, has emphasised the need for immediate maintenance therapy in advanced CTCL and further trials are urgently needed. The Italian lymphoma group showed in a retrospective pilot study that bexarotene as maintenance treatment after therapies other than skin-directed therapy in advanced stage MF can increase time to next treatment. However, their data are not based on a prospective trial and do have a limitation in the selection of patients featuring a better prognosis after the first line of therapy; thus, leading to a high TTNT value. As it is well recognized that deregulated expression of epigenetic regulatory proteins play the crucial role for cancer cell growth in many human tumour types (including malignant T-cells of CTCL), the use of small molecule inhibitors of histone deacetylases (HDACs) could provide a rational approach for halting disease progression. Resminostat - a broad spectrum HDACi - might be regarded as a possible candidate for maintenance therapy in CTCL. The compound influences gene regulation of the Th1/Th2 phenotype as well as homing receptors and induces the reduction of Il-31. Currently the largest maintenance study ever undertaken is a multicentre, double blind, randomized, placebo controlled, phase II trial to evaluate resminostat for maintenance treatment of patients with advanced stage (stage IIB - IVB) mycosis fungoides (MF) or Sézary Syndrome (SS) that have achieved disease control with systemic therapy - the RESMAIN study.

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It is currently in the recruitment phase and will answer important clinical and scientific questions about maintenance therapy. The concept of maintenance therapy is shown in figure 1, the study design is shown in figure 2 Literature references: 1.Dummer R et al. Eur J Cancer. 2007;43: 2321-29, 2.Hughes CFM et al. Blood 2015; 125: 71- 81

Figure 1 Concept Maintenance Therapy

Zytoreductive Maintenance Zytoreductive Maintenance therapy therapy therapy therapy PUVA + Bexarotene Brentuximab Vedotin PUVA Bexarotene Interferon Mogamulizumab Bexarotene Resminostat Local Radiation, chlormethine Gemcitabine TSEB Resminostat? Doxorubicine

Figure 2

Modified European Journal of Cancer 2007 Patch Plaque Tumour

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Robust graft-versus-lymphoma (GVL) response mimicking cutaneous relapse of T-cell lymphoma Amy G. Johnson, MD, Rongqin Ren MD, PhD, Basem M. William, MD, MRCP(UK), FACP, and Catherine G. Chung, MD Multidisciplinary Cutaneous Lymphoma Program- The Ohio State University Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH, USA A 65-year-old woman with a history of peripheral T-cell lymphoma, unspecified, characterized by intermittent nodules on the trunk and extremities was well-controlled on oral methotrexate without evidence of systemic disease Initial skin biopsy demonstrated an atypical CD4-positive lymphocytic infiltrate with aberrant loss of CD7 and diffuse PD-1 expression. After three years, she experienced rapid progression of nodules on the trunk and extremities with similar histopathologic findings to prior biopsies, now with marrow and peripheral blood involvement. She achieved remission after multiple lines of systemic therapy and underwent allogeneic stem cell transplant from a fully HLA-matched unrelated male donor. She engrafted with no evidence of graft versus host disease and was discharged home at day 21 post-transplant. At D+48 after transplant, she developed edematous pink plaques on the hands and axillae [Figure 1]. Biopsy demonstrated a dermal infiltrate of pleomorphic lymphocytes with diffuse CD3, CD4, and PD-1 expression, concerning for disease relapse [Figures 2, 3]. FISH demonstrated XY pattern in 99% of lymphocytes with XX pattern in squamous cells and fibroblasts, consistent with a donor-driven process [Figure 4]. She had no evidence of peripheral blood or visceral disease on imaging and flow cytometry. She was treated with immune suppression withdrawal and 12 Gy total skin electron beam radiation with resolution of skin lesions and is currently without evidence of disease 9 months post-transplant. This case highlights a novel presentation of an immunologic GVL response that has not been previously reported, to the best of our knowledge, where cutaneous infiltrate by donor lymphocytes resembles cutaneous relapse of T-cell lymphoma, with similar histologic and immunophenotypic features.

Figure 1. Post-transplant skin lesions suspicious for disease relapse

Figure 2. H&E stained pre-transplant (left) and post-transplant (right) biopsies with dermal atypical lymphocytic infiltrate

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Figure 3. Pre and post-transplant skin Figure 4. FISH of post-transplant skin biopsy showing similar immunophenotype biopsy showing majority of lymphocytes of T-cells of donor origin (Y chromosome=Red; X chromosome=Green)

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Subcutaneous panniculitis-like T-cell lymphoma in the mesentery with associated hemophagocytic syndrome treated by chemotherapy CHOEP, modified HLH 2014 protocol and consolidated by HDT and auto SCT. Agnieszka Giza1, Marcin Jońca2, Małgorzata Raźny2, Dagmara Zimowska-Curyło1, Krystyna Gałązka3, Mateusz Wilk1, Sarah Goldman- Mazur 1,Beata Piątkowska- Jakubas1, Tomasz Sacha1. 1Department of Hematology, Collegium Medicum of the Jagiellonian University, Krakow, Poland 2Department of Hematology Rydygier Hospital, Krakow, Poland 3 Department of Clinical Patomorphology, Collegium Medicum of the Jagiellonian University, Krakow, Poland

Background: Subcutaneous panniculitis –like T cell lymphoma (SPTCL) is a rare CD8+cytotoxic lymphoma primary localized in subcutaneous adipose tissue. SPTCL accounts for fewer than 1% of all NHL and fewer than 1% of CTCL. The neoplastic cells express cytotoxic T –cell phenotype , CD8, TIA1 (T - cell intracellular antigen1) , Granzyme B , perforin, but not CD56 and CD4. Disease has indolent clinical course with 5 year overall survival (OS) of 82% .SPTCL previously was divided into two subtypes: alfa/beta T cell phenotype and gamma/delta phenotype -now refers only alfa/beta phenotype. Nowadays term SPTCL is restricted to alfa/beta phenotype. Median age of patients is 35 years and 20% of pts are aged <20 years. SPTL more common occurs in young women. In about 20% of patients with SPTCL is also diagnosed autoimmune disease, mainly systemic lupus erythematosus. Typical clinical presentations are subcutaneous nodular lesions mainly on the extremities and trunk. Currently no standard treatment is available and reported therapies included: corticosteroids, immunosuppressive therapy, chemotherapy CHOP, or CHOP –like, radiotherapy, autologous stem cell transplantation ( auto- SCT) or allogeneic hematopoietic stem cell transplantation (allo-SCT) In 15-20% of cases of SPTCL is seen hemophagocytic syndrome (HPS). HPS is the severe hyperinflammatory disease, with prolonged fever, cytopenias, hepatosplenomegaly, hemophagocytosis, hyperferritinemia, hypertriglyceridemia, hyperfibrinogenemia, high-soluble interleukin -2 receptor levels and low or absent NK cells activity. In HLH dysfunctional CD8+lymphocytes and NK cells are unable to initiate appropriate response to malignant cells. The immune system is unable to control the hyperinflammatory response and histiocytes produce storm of cytokines invading organs. In SPTCL with HPS prognosis is poor with 5 –year OS 46%. There have been only few published cases of successfully managed SPTL with HPS and standard treatment has to be established.

Case presentation: A 35 -year old non- Caucasian man previously fit presented with a one-month history of fever, cough, fatigue and weight loss with the suspicion of Still’s disease was admitted to hospital on January 2019. At presentation blood tests showed leucopenia 2,66 x 10^3/L, (ref:4,0-10,0),Hb 12,3 g/dl, ( ref: 12,0-17,0), increased lactate dehydrogenase 1340 U/L (ref:<248), ferritin 27 219 ng/ml(ref:21,81-274,66), sIl-R2 4899U/ml (ref:<100 U/ml), and triglycerides 4,32 mmol/l (ref <1,7). On physical palpation there were found hepatosplenomegaly and right supraclavicular tumor about 15mm. The ultrasound of the neck showed right supraclavicular lymph node to 11x5 mm and intensive hypoechoic adipose tissue around lymph nodes. The whole-body CT (computed tomography) scan revealed small lymph nodes mediastinum to 15mm, effusion in right pleura to 15mm, hepatomegaly 185mm splenomegaly 125mm and ascites. The abdomen MRI presented generalized edema of adipose tissue of mesentery and mucosa of colon, hepatomegaly 180mm and splenomegaly 153 mm. Trephine biopsy revealed reactive bone marrow. Histopathology examination adipose tissue adjoined lymph nodes showed infiltration of lymphocytes surrounding adipocytes and high probability of secondary HPS. Immunohistochemically lymphocytes were: CD20-/CD3+( in some cells weak), CD5+/-, CD2+, CD56-, CD4-, CD8+, granzyme B+, CD25-, CD30-, CD15-, ALK1-. The proliferation index Ki67 was 60-70%. Finally the diagnosis of SPTCL with HPS was established. The patient was subjected to CHOEP and modified HLH 2014 protocols ( CyA and dexamethasone). Due to hepatotoxicity during chemotherapy doses of etoposide were delayed.

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Maximum level of ferritin on treatment was 156422ng/ml. The first cycle of CHOEP was complicated by pancytopenia and the abdominal pain, the second by pneumonia and left lung abscess. The MRI scan of abdomen after second cycle of CHOEP chemotherapy showed partial regression of infiltration of mesentery by adipose tissue and hepatosplenomegaly. After prolong antibiotics administration and resolution of pneumonia chemotherapy was continued. The ferritin level dropped to 1459ng/ml . The PET CT performed after third cycle of CHOEP revealed uptake 18 F-FDG in left lung 68X65mm. In this time Patient was not qualified to allo SCT due to high risk of procedure. The chemotherapy was continued. The 5th cycle of chemotherapy CHOEP was complicated by pancytopenia and neutropenic fever and skin infection of the parietal- occipital part of head. The PET CT after 5th cycle of CHOEP showed significant regression of active mass in lung and no active signs of disease. After the 6th cycle of CHOEP and 2 doses of plerixafor we successfully collected patient’s hematopoietic stem cells (HSC) 2,96x10^6/kg CD34+ cells. Finally, In the October 2019 the patient was subjected to high dose chemotherapy HDT (BEAM- busulfan, etoposide, cytarabine and melphalan) supported by auto SCT. The outcome of the procedure was safe, the only complication was mucositis grade 2 according to CTCAE . We did not observe any hepatic, GI and renal toxicity and any fever and infectious complication. Hematopoietic recovery was supported by filgrastim (G CSF) administered from +4 to +12 day after transplantation, and neutrophils recovery > 0,5 x 109/L was observed in + 12 day , platelet count > 20 x109/L in +12 day and patient was discharged from hospital in +13 day after auto SCT. Nowadays the patient is in a good clinical condition 3 months after completion of treatment in complete remission confirmed in PET CT scan. Conclusion: We presented good outcome of SPTCL with HPS treated by intensive protocol but longer follow-up is required. References: 1. Hrudka J, Eis V, Herman J, Prouzowa Z, Rosenwald A Duska F. Panniculitis like-T-cell lymphoma in the mesentery associated with hemophagocytic syndrome : autopsy case report. Diagnostic Pathology 2019, 14:80 . 2. Tring- An Lin, Ching-feu Yang, Yao Chung Liu, Jin-Hwang Lin et al. Hematopoietic Stem Cell Transplantation for Subcutaneous Panniculitis-Like T-cell Lymphoma: Single Center Experience in an Asian Population. Int J Hematol, 2019 109 (2), 187-196

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Aggressive Epidermotropic CD8+ T Cell Lymphoma presenting with targetoid lesions Khaled El-Zawahry, Maged Daruish, Mona Abdel-Halim Ibrahim Department of Dermatology, Venereology and Andrology, Ain Shams University, Cairo, Egypt Introduction Primary Cutaneous Aggressive Epidermotropic CD8+ T Cell Lymphoma (PCAETCL) is a rare subtype of cutaneous T-cell lymphomas that presents with rapidly progressive widespread plaques and ulcerated and hemorrhagic tumors. Herein, we report a case of PCAETCL with a rare clinical presentation in the form of targetoid lesions and necrotizing eschar formation. Case report A 49 years old male from Libya presented with progressive generalized ulcerations 2 years duration. The patient was previously diagnosed histologically as vacuolar interface dermatitis and received corticosteroids with no improvement. On examination, there were multiple eschar formation, massive pustulation over the scalp and targetoid lesions over the upper and lower extremities. The patient's general condition was poor. Our differential diagnosis was vasculitis or febrile ulceronecrotic Mucha-Habermann disease.

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On Histopathological examination, there were many atypical lymphocytes invading the epidermis massively. Blood vessels in the upper dermis showed vasculitic changes with angioinvasion and angiodestruction. Immunomarkers revealed the infiltrating cells were CD3, CD7 and CD8 positive, while CD4, CD20, CD30 and CD56 were negative. Unfortunately, cytotoxic markers were not done because they are not available in Egypt. Internal organs involvement was excluded by PET- CT scan.

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The patient was diagnosed as PCAETCL based on the presence of widespread progressive hemorrhagic ulcerations with bad general condition clinically, prominent epidermotropism with angioinvasion histopathologically, together with positive CD8 and negative CD30. The patient received CHOP but died one month after the diagnosis. Discussion PCAETCL is classified as a provisional entity in the 2016 lymphoid neoplasm classification. It is characterized by abrupt onset of rapidly progressing multiple, generalized, ulcerating and hemorrhagic plaques, or tumors with possible extra-cutaneous involvement. 1 The presence of targetoid lesions is a rare finding that poses a diagnostic pitfall for this rare disease.2 The tumor cells release cytotoxic granules destroying the epidermis when activated. The disease progresses rapidly with poor prognosis and high mortality rate due to extensive epidermal necrosis. 1 Owing to the aggressive behaviour of PCAETCL, many therapeutic options failed to induce remission and the patients usually die shortly after diagnosis. Hematopoietic stem cell transplantation may be the only option that can lead to durable partial or complete remission. In conclusion, we report a case with PCAETCL with targetoid lesions. To the best of our knowledge, this is the first case reported from African countries. References: 1- Guitart J, Martinez-Escala ME, Subtil A, et al. Primary cutaneous aggressive epidermotropic cytotoxic T-cell lymphomas: reappraisal of a provisional entity in the 2016 WHO classification of cutaneous lymphomas. Mod Pathol. 2017;30(5):761–772. doi:10.1038/modpathol.2016.240 2- Tomasini C, Novelli M, Fanoni D, Berti EF. Erythema multiforme-like lesions in primary cutaneous aggressive cytotoxic epidermotropic CD8+ T-cell lymphoma: A diagnostic and therapeutic challenge. J Cutan Pathol. 2017;44(10):867–873. doi:10.1111/cup.12995

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CD4/CD8 double-negative folliculotropic mycosis fungoides/Sézary syndrome presenting as severe leonine facies

Emily A. Merkel, MD1, Raj Chovatiya, MD, PhD1, Lauren Guggina, MD1, Barbara Pro, MD2, Joan Guitart, MD1, Xiaolong A. Zhou, MD1

1- Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago Illinois, Chicago, IL 60611, USA 2- Division of Hematology and Oncology, Northwestern University Feinberg School of Medicine, Chicago Illinois, Chicago, IL 60611, USA

A 64-year-old man presented with severe facial swelling and generalized pruritic rash. The rash had been previously treated with multiple biologics, including TNF and IL-17A inhibitors, systemic corticosteroids and cyclosporine. Physical examination revealed thick, scaly, indurated nodules and plaques over the face and ears that formed deep furrows over the forehead and cheeks (Figure 1). Erythematous, scaly plaques were seen throughout the trunk, arms and proximal thighs, covering >50% body surface area. Laboratory examination revealed significant leukocytosis (52,600/uL) with lymphocytosis (15,300/uL) and eosinophilia (23,100/uL). Lactic dehydrogenase (LDH) was elevated (1189 U/L) and flow cytometry revealed a large abnormal CD4-, CD8-, CD3+, CD7+, CD26-, alpha-beta+ T cell population (~10,200/uL). T-cell gene rearrangement testing demonstrated monoclonality from peripheral blood and skin samples with identical clonal bands. Skin biopsies of the face and trunk showed numerous atypical lymphocytes infiltrating the follicular units and adjacent perivascular lymphohistiocytic infiltrate with scattered eosinophils. Immunohistochemical profile was similar to the peripheral blood and demonstrated atypical CD4- /CD8- T-cells. The patient was diagnosed with CD4/CD8 double-negative folliculotropic cutaneous T- cell lymphoma (CTCL) with leukemic blood involvement. He progressed on gemcitabine and developed symptoms of hemophagocytic lymphohistiocytosis (HLH), including high ferritin (8000 ng/ml), severe thrombocytopenia and anemia, fevers, splenomegaly, low fibrinogen, rapidly rising D-dimer and periportal edema on imaging (H-score 178, 54-70% probability of HLH). He received etoposide-based chemotherapy (COEP) but continued to develop worsening facial tumor burden that on biopsy revealed large cell transformation. Despite palliative radiation and one cycle of brentuximab vedotin, his disease continued to worsen, and he ultimately passed away in home hospice. Leonine facies is a rare manifestation of CTCL, but has been described in patients with mycosis fungoides, folliculotropic mycosis fungoides, and Sézary syndrome. Among such patients, advanced disease and folliculotropism on facial biopsy is common. Unfortunately, prognosis is generally poor, with an overall survival of 26% at 5 years.1 Our patient was diagnosed with CTCL after failing biologics and immunosuppressants for presumed psoriasis. According to a recent series of patients who were similarly diagnosed with CTCL after exposure to anti-TNFα therapy,2 most patients experienced rapid worsening of their disease, resulting in a median time to diagnosis of CTCL of 6 months. Advanced disease was common.2 It is possible that chronic lymphocye-driven inflammatory states, such as psoriasis and atopic dermatitis, along with the use of immunosuppressive therapies may help in selecting a malignant T-cell clone. Alternatively, immunosuppression may unmask a previously quiescent low-grade primary cutaneous lymphoma. In clinically ambiguous or atypical cases, close monitoring and repeat biopsies with the use of immunohistochemistry and T-cell receptor (TCR) gene rearrangement should be considered. In patients with extensive skin disease or erythroderma, flow cytometry and TCR gene rearrangement studies of the peripheral blood can further aid in diagnosis.

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Figure 1: Thick, scaly, indurated nodules and plaques that formed deep furrows over the forehead and cheeks were seen.

References 1. Brown DN, Wieser I, Wang C, Dabaja BS, Duvic M. Leonine facies (LF) and mycosis fungoides (MF): A single-center study and systematic review of the literature. J Am Acad Dermatol. 2015;73(6):976-986. 2. Martinez-Escala ME, Posligua AL, Wickless H, et al. Progression of undiagnosed cutaneous lymphoma after anti-tumor necrosis factor-alpha therapy. J Am Acad Dermatol. 2018;78(6):1068-1076.

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Ain-Shams Cutaneous Lymphoma Clinic – suggested guidelines for the treatment of mycosis fungoides in countries with limited resources Mona Abdel-Halim Ibrahim1, Nada El-Tayeb1, Mirna Michel1, Ahmed Nassar1, Abdelrahman Mohamed1, Maged Daruish1, Maha El-Zimaity2, Amal El-Afifi2, Haitham Abdelbary2, Mahmoud El-lithy3, Ahmed Mostafa3, Mahira Hamdy El-Sayed1 1 Dermatology Department, Aim-Shams University, Cairo, Egypt 2 Hematology Department, Ain-Shams University, Cairo, Egypt 3 Oncology Department, Ain-Shams University, Cairo, Egypt Introduction: Though many treatment options for mycosis fungoides (MF) are emerging, many of the currently used treatment modalities (e.g. bexarotene, extracorporeal photophoresis, histone deacetylase inhibitors, and biologics) are unavailable in Egypt and other African/Arab countries. In addition, there is a lack of consensus for the treatment of hypopigmented MF that is frequently encountered in our population. Our aim is to figure out a practical treatment algorithm based on our institutional experience, treatment availability and consistent with international guidelines. Methodology: Treatment approach is based on literature review for the international guidelines for the treatment of MF as European Organization for Research and Treatment of Cancer (EORTC), 2017;1 European Society for Medical Oncology (ESMO), 2019; National Comprehensive Cancer Network (NCCN), 2019 and; British Association of Dermatologists and U.K. Cutaneous Lymphoma Group guidelines (BAD-UKCLG), 2018.2 Special attention is also given to studies conducted on patients with skin phototype (III-IV). Literature review was done to January 2020 on PubMed, Embase databases, the Cochrane Library for meta-analysis, systemic reviews, randomized and non-randomized clinical studies, cohort, case-control studies, case series, and case reports of mycosis fungoides and Sezary syndrome (MF/SS). Patients with specific variants are discussed separately owing to the variation in their prognosis from classic MF. These include patients with hypopigmented MF -that has more favorable prognosis- and; transformed, folliculotropic MF that showed poor prognosis. Only English language studies were included. This consensus was established among dermatologists, hematologists and oncologists at Ain-Shams cutaneous lymphoma clinic through series of consecutive discussion. Recommended treatment options are presented as an algorithm according to the disease stage. Dose and treatment duration are provided; and maintenance/follow-up protocol is designed. Level of evidence is specified for each treatment option using Oxford Centre for Evidence-Based Medicine 2011 (OCEBM). Results: Treatment options are presented in a stepwise pattern as the main objective of the treatment is to control the patients' disease with minimal intervention that yields the least side effects. Therefore, skin directed therapy (SDT) is given as a frontline in patients with early classic MF and hypopigmented MF, while systemic and combined therapy are reserved for the late cases and transformed MF. For early classic MF, phototherapy is given (PUVA is superior to UVB in inducing remission in darker skin type and for patients unresponsive to initial treatment by NB-UVB) (level 2). Potent steroids (level 3) and moisturizers (level 2) are additional basic treatment. Bath PUVA can be used for patients that are not amenable for oral PUVA (level 4). Topical mechlorethamine and topical bexarotene are unavailable; therefore, gentian violet is sometimes used owing to its chemotherapeutic effect in an in-vitro study (level 5). Addition of either methotrexate or acitretin -an alternative to the unavailable bexarotene- is recommended as a second line (level 5, level 4), total skin electron beam (TSEB) is considered a third line option (level 2). Expectant therapy is not appropriate in our community due to the uncertainty of patients' adherence to regular follow- up. Topical tazarotene can be added to resistant patches (level 3); and localized radiotherapy can be added to resistant plaques (level 4). Patients with stages (IB-IIA) who show complete response should enter a consolidation phase followed by a maintenance phase using phototherapy (level 2).

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For stage IIB with up to 3 nodules, localized radiotherapy can be added to SDT (level 4). For stage IIB with multiple nodules, stage III and SS, PUVA plus acitretin (level 4) or methotrexate (level 5) is recommended as first line, TSEB is considered a second line option (level 2). In addition, monochemotherapy with gemcitabine or liposomal doxorubicin can be given instead (level 4). Polychemotherapy and allogenic stem cell transplantation are regarded as a final option (level 3). Phototesting and slow dose escalation is mandatory in cases of erythroderma in stage III and IV. For stage IV, monochemotherapy is the first line (level 4), followed by polychemotherapy and allogenic stem cell transplantation (level 3). Regarding hypopigmented MF, phototherapy is recommended as a first line treatment, while low dose methotrexate is considered a second line (level 5). Moisturizers and mid potent steroid are basic treatment for all patients (level 5). For folliculotropic MF, distinction between indolent and aggressive variants is crucial. Early follicular MF can be treated as early stage classic MF (topical steroids and PUVA), while PUVA plus retinoids, localized radiotherapy, TSEB are reserved for advanced stages (level 3). No enough studies are available for transformed MF; however, aggressive therapy with monochemotherapy can be considered (level 4). Adjuvant treatment for pruritus includes gabapentin and mitrazapine (level 5). Decolonization with intranasal mupirocin 1% and oral antibiotics may be given to late recalcitrant cases to improve the response to therapy (level 3). Conclusion: We suggest a practical algorithm for the treatment of MF in patients with darker phenotype, in adherence with the international guidelines and in the context of limited medical resources. References:

1- Trautinger F, Eder J, Assaf C, et al. European Organisation for Research and Treatment of Cancer consensus recommendations for the treatment of mycosis fungoides/Sézary syndrome: Update 2017. Eur J Cancer. 2017;77:57-74 2- Gilson D, Whittaker SJ, Child FJ, et al. British Association of Dermatologists and U.K. Cutaneous Lymphoma Group guidelines for the management of primary cutaneous lymphomas 2018. Br J Dermatol. 2019; 180:496–526

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Patients with mycosis fungoides are at increased risk of lymphoma, melanoma, lung cancer, and bladder cancer: a systematic review and meta-analysis Amrita Goyal MD,1 Kavita Goyal MD,1 Daniel O’Leary MD,2 Krishnan Patel MD,2 Maria Hordinsky MD,1 Kimberly Bohjanen MD,1 David Pearson MD,1 Murali Janakiram MD3 1Department of Dermatology, University of Minnesota, 2Division of Hematology, Oncology, and Transplantation, University of Minnesota, 3Department of Radiation Oncology, University of Minnesota

Background: Patient with mycosis fungoides (MF) are at higher risk of developing secondary primary malignancies based on prior studies; however, rates have secondary malignancies have varied significantly across various studies. In order to determine an estimate of the standardized incidence and the type of secondary malignancies we performed a systematic review and meta- analysis of the literature.

Methods: A systematic review of manuscripts published between 1950-2019 was performed to assess the risk of second malignancies in patients with MF.

Results: Based on our inclusion and exclusion criteria, we identified 12 studies of interest, yielding 14 patient cohorts. Six were university-based registries and five were population-based cohorts (US SEER and national registries). Three of the population-based analyses were based on SEER data and their composition had minimal temporal overlap. Sample size ranged from 63 to 1798 patients, with 5.9-16.8% developing second malignancies. The studies were of poor to moderate quality. All studies showed a male predominance for patients developing second malignancies, with male-to-female ratios ranging from 1.2:1 to 2.9:1. Mean age across the studies ranged from 44.6 to 68.0, with a weighted mean age of 58.4 years [95% CI 51.7-65.1 years]. Latency (the time between diagnosis of MF and diagnosis of a second malignancy) ranged from 2.1-5.4 years (mean 3.29 years, [95% CI 2.69-5.15]). The meta-analysis revealed a combined SIR of 2.18 [95% CI 1.43-2.93] for all malignancies. For specific cancers, the SIR was 15.25 [7.70- 22.79] for Hodgkin lymphoma, 4.96 [3.58-6.33] for non-Hodgkin lymphoma, 1.89 [1.19-2.59] for lung cancer, 1.75 [1.10-2.40] for bladder cancer, and 3.24 [1.65-4.83] for melanoma.

Conclusions: We find that patients with MF are at increased risk of secondary malignancies, especially Hodgkin and non-Hodgkin lymphoma, lung cancer, bladder cancer, and melanoma. These findings provide moderate quality evidence of a population at increased risk of malignancy wherein early detection may improve survivorship by decreasing the morbidity burden of secondary malignancies via early detection. This study provides a strong rationale for prospective screening study of secondary malignancies in CTCL.

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Figure 1. Meta-analysis of institutional and registry studies examining the incidence of second malignancies in patients with mycosis fungoides.

Figure 2. A. Meta-analysis of studies including data on the incidence of Hodgkin and non- Hodgkin lymphoma in patients with mycosis fungoides. Overall SIR is considered a measurement of all patients with lymphoma, but does not include patients with leukemia. B. Meta-analysis of studies including data on the incidence of bladder cancer, lung cancer, and melanoma in patients with mycosis fungoides. A B

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“Epidemiological analysis and review of histopathological parameters of patients diagnosed with mycosis fungoides and its variants in follow-up at State University of Campinas”

Introduction Mycosis fungoides and its variants represent a challenge in diagnostics and therapeutics. The clinical presentations are broad and the findings of anatomopathological examinations are often nonspecific, which makes it difficult to diagnose and delays adequate therapy, compromising patient's prognosis. This retrospective study evaluated patient's histological criteria and allowed us to establish diagnosis of mycosis fungoides and its variants. In addition, the epidemiology of patients with this diagnosis in our institution was detailed.

Methods Retrospective study conducted at the hospital of the Faculty of Medical Sciences at the State University of Campinas (São Paulo, Brazil). Cases of mycosis fungoides (classical form and variants) followed up in the dermatology department were selected. Exclusion criteria: cases without histopathological evaluation by our team, of patients under 18 years of age or who started follow-up from 2017. Information from medical records was collected, including anatomopathological reports, focusing on the epidemiological profile and histological findings of each case. Histopathological slides were reviewed when necessary. Analyzed data: gender, age, race/phototype, education level, other dermatological conditions, evolution from another previous disease, type of epidermotropism, cerebriform atypia, lymphocyte cell volume (usual versus medium/large increase; mature T-helper lymphocyte as reference), blast cell type atypia, epidermal spongiosis, papillary dermal edema, papillary dermal fibrosis and hypoimmunoexpression of pan-T markers by lymphocytes. The type of epidermotropism was divided into three groups: linear (I), Pautrier's microabscesse (II) and sparse small aggregates plus isolated cells (so called pagetoid) (III). The work was approved by the ethics committee of our institution.

Results We found 19 cases of classical mycosis fungoides, 1 of pagetoid reticulosis, 1 of granulomatous slack skin and 1 of folliculotropic mycosis fungoides. The sample of patients was balanced with respect to gender (10 female and 12 male; p=0.6749). Almost 70% of patients had a low Fitzpatrick scale phototype. The mean age found at the time of diagnosis was 56.5 years (median: 57.5, minimum: 23 and maximum: 86). Three patients exhibited a previous condition interpreted with subsequent evolution to mycosis fungoides. Those conditions were pityriasis lichenoides chronica, parapsoriasis and a case in which the diagnosis of the previous condition has not been clearly established. Two patients had a previous history of lymphoma at the time of diagnosis of mycosis fungoides (about 9% of patients). The previously identified lymphomas

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were Hodgkin lymphoma (nodular sclerosis) and follicular lymphoma. Other skin diseases were found in 18.18% of cases (folliculitis decalvans, dysplastic nevi, tumid lupus erythematosus and onychomycosis). Epidermotropism was present in 16 cases (72.72%). Linear epidermotropism was present in 36.36% of the cases and in 18.18% Pautrier's microabscesses. Pagetoid epidermotropism (sparse small clusters with isolated lymphocites) was found in 18,18%, significantly different in genders, being observed only in female patients (p=0.0287; 40% of cases of female patients). In 90.91% of the cases, cerebriform lymphocytes were detected in the epidermis infiltrate. Atypical lymphocytes were present in all cases. Cerebriform lymphocytes of medium to large volume in the epidermis were detected in only 13.64% of the cases. Clusters of medium to large cerebriform lymphocytes in the dermis was detected in 36,36% of cases. Blast cell type atypia was not found. Papillary dermal fibrosis was observed in 72.73%, while edema in only one case. All cases of female patients exhibited fibrosis of the papillary dermis, whereas in males, only 50% (p=0.0152). In 86.36% of the cases, epidermal spongiosis was not observed. Only one case exhibited lympocytes with hypoimmunoexpression of pan-T markers (CD 2, CD3 and/or CD5) within less than 50% of the infiltrate.

Conclusions As previously established worldwide, in the population of this study, mycosis fungoides was more prevalent in patients over 40 years of age and slightly more prevalent in males. More confidente and objectives histological findings, such as Pautrier's microabscesses or cerebriform lymphocytes of medium to large volume in the epidermis was observed only in a minority of cases. Papillary dermal fibrosis, although non-specific, remains as an important and more sensitive finding to achieve the diagnosis of mycosis fungoides. Interestingly we found fibrosis of the papillary dermis in all cases of female patients, but in only 50% of male patients. The differentiation between early lesions of mycosis fungoides and inflammatory skin diseases remains a challenge for pathologists. Histological criteria, when analyzed in isolation, are of limited importance due to low specificity. Therefore, the correlation between clinical and histological findings is fundamental to establish the early diagnosis of mycosis fungoides.

References 1) Pulitzer M. Cutaneous T-cell Lymphoma. Clin Lab Med. 2017 Sep;37(3):527-546. doi: 10.1016/j.cll.2017.06.006. 2) Larocca C, Kupper T. Mycosis Fungoides and Sézary Syndrome: An Update. Hematol Oncol Clin North Am. 2019 Feb;33(1):103-120. doi: 10.1016/j.hoc.2018.09.001.

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Topic: Patient Care in Cutaneous Lymphoma An overview: Our experience of microbiological causes of inpatient admissions for patients with Mycosis Fungoides and Sézary’s Syndrome at Peter MacCallum Cancer Centre. Authors: Odette Buelens, Associate Professor Chris McCormack, Dr Belinda A Campbell, Dr Carrie Van Der Weyden, Professor Karin Thursky, Dr Olivia Bhupa-Intra, Dr Ben Teh, Robert Twigger, Professor Monica Slavin, Jake Valentine, Professor Miles Prince. Introduction and Objectives: Cutaneous Lymphomas have a profound impact on many facets of patients’ lives and the lives of those caring for them. Patients with disease-related pruritus and wound care issues may also experience complications from bacterial, fungal and viral infections. Infection may also aggravate patients’ cutaneous lymphoma disease (Lindahl et al, 2019). These problems affect patient care, comfort, and longevity of stay in the hospital, future treatment plans and long-term survival. In our team’s experience, this group of patients may experience complex, prolonged inpatient stays and multiple hospital re-admissions. In this retrospective study we review the recent hospitalisations for patients with a diagnosis of Mycosis Fungoides and/or Sezary’s Syndrome (MF/SS), admitted to Peter MacCallum Cancer Centre from 1/1/2018- 31/1/2019, for the management of sepsis. The objective of this study was to review the length of stay, number of re-admissions and primary causes for admission. Materials and methods: This was a retrospective audit of patients with MF/SS at Peter Mac admitted as an inpatient with sepsis identified as a reason for admission. Patients over the age of 18 were included. Ethics authorisation was received for this project by the local ethics reviewing committee and a waiver of consent was obtained. The Australian National Cutaneous Lymphoma database was utilised to identify eligible patients in addition to reviewing discharge summaries and electronic patient records of inpatients. Investigations including bloods and microbiological results were reviewed. Results: A total of 21 patients were eligible for audit review. The total number of occupied bed days for this group of 21 patients was 618 days. The highest number of admissions for the 21 patients was 8. The longest length of stay on the ward was 114 days (range 1-114 days). Causes for hospital admission were frequently multifactorial. Fever accounted for 16% of admissions, sepsis 13% and skin breakdown (including cellulitis) was 13%. Skin flares accounted for 9% of admissions. The fevers were categorised into three groups: 1.Microbiologically defined (MDI) in which bacteremia was identified, 2. Clinically defined (CDI)- when a site of infection was diagnosed but no microbiologic pathogenesis can be defined, and 3. Fever of unknown focus (FUF), in which no clinically or microbiological evidence of infection can be found (The Immunocompromised Host Society, 1990). See (Figure 1). Types of infections and incidences as outlined microbiologically and in discharge summaries were identified , (Figure 2). Staph aureus was the most common infection. Conclusions: Sepsis can result in prolonged inpatient stays for patients with MF/SS. Ongoing collaborative research projects are underway between the multi-disciplinary Cutaneous Lymphoma Service and the Infectious Diseases Service at Peter MacCallum Cancer Centre, with the driving goal to provide optimal care for this unique group of patients. References: Lindahl LM, Willerslev-Olsen A, Gjerdrum LMR, Nielsen PR, Blümel E, Rittig AH, Celis P, Herpers B, Becker JC, Stausbøl-Grøn B, Wasik MA, Gluud M, Fredholm S, Buus TB,

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Johansen C, Nastasi C, Peiffer L, Kubat L, Bzorek M, Eriksen JO, Krejsgaard T, Bonefeld CM, Geisler C, Mustelin T, Langhoff E, Givskov M, Woetmann A, Kilian M, Litman T, Iversen L, Odum, N.(2019). Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma, Blood, .Sep 26;134(13):1072-1083. The Immunocompromised Host Society. (1990) The design, analysis, and reporting of clinical trials on the empirical antibiotic management of the neutropenic patient. Report of a consensus panel. The Journal of infectious diseases. 161(3):397-401. PubMed PMID

Types of fevers in admitted patients U S Q O M K

Patient I G E C A 0 1 2 3 4 5 6 7 8 9 Number of admissions per patient Fever of unknown focus Clinically Defined Infection Microbiologically defined Infection

Figure 1. Types of fevers identified in admitted patients. Types of Infections in 21 patients admitted

10 9 8 7 6 5 4 Incidence 3 2 1 0

Figure 2. Types of infections in admitted patients. 236

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THE USE OF GATA3 IN CHALLENGING CASES OF CD30+ MYCOSIS FUNGOIDES (MF) versus ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

Authors: Almeida IP¹; Cury-Martins J¹; Miyashiro D¹; Abdo ANR²; Giannotti MA³; Domingues RB³; Pereira J²; Sanches JA¹

¹ Department of Dermatology, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil. ² Department of Hematology, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil. ³ Department of Pathology, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, Brazil

INTRODUCTION CD30 is a cell surface receptor that is classically expressed in lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma (cALCL). Some cases of mycosis fungoides (MF), transformed MF (tMF), and systemic lymphomas with cutaneous involvement like systemic anaplastic large cell lymphoma (sALCL) can also express CD30. Some of these disorders can be associated and have overlapping features, making the diagnosis a challenge. GATA 3, a TH2 differentiation transcriptional regulator, may be useful in the differentiation between tumoral MF and cALCL. This marker is frequently positive in cases of advanced MF. This study proposed the use of GATA3 as an additional tool for the distinction of four doubtful cases of CD30-positive lymphomas involving the skin.

METHODS Retrospective analysis of clinical charts, histological and immunohistochemical data, including GATA3 staining of four challenging cases, where the differentiation between MF, ALCL, or overlap of both entities could not be well defined with regular markers. Formalin-fixed, paraffin-embedded tissue sections were incubated with pretreatment buffer and were reacted with CD30 and GATA3 antibodies. GATA3 expression on the dermis was calculated in percentage of the infiltrate and was compared to the biopsies of typical cases of MF, cALCL, tMF and sALCL. Results were also compared to the data available in the literature. A positive GATA3 stain was defined as nuclear labeling at least 10% of infiltrating cells. Positive results were graded on scale of 10% to 100% of positive cells.

RESULTS Four questionable cases were reported below, with the respective positivity for the GATA3 staining. Case 1. 76-years-old woman with a plaque stage MF on phototherapy developing an extensive unilateral inguinal adenopathy with histology suggestive of an ALK-negative ALCL, with no other involved sites (Figure 1). Patient was treated with polychemotherapy, radiotherapy with complete nodal response and was maintained on phototherapy for MF. One year later, skin tumors composed of large anaplastic CD30+ cells developed with no signs of extracutaneous involvement. Expression of GATA3 was observed in 80% of infiltrating and Figure 1. A) Plaque stage MF. B) PET with large cells. localized inguinal nodal involvement. 237

Case 2. A 49-year-old woman with the previous diagnostic of lymphomatoid papulosis in treatment with phototherapy and topical steroids developed an erythematous, infiltrated and arciform skin plaque in lumbar region. GATA 3 was positive in 70 to 80% of infiltrating cells and negative in large cells.

Case 3. 59-years-old man presented with patches and a large tumor on the buttock (Figure 2). Histology revealed on tumor sample large anaplastic CD30+ cells and patch histology was compatible with CD30- MF. A diagnosis of transformed MF on the buttock was made, but on follow up, crops of papular lesions suggestive of LyP developed. None or minimal GATA3 expression was observed.

Case 4. 49-years-old woman with MF for for 20 years with Figure 2. A) Patches and tumor. B) multiple prior therapies (interferon, PUVA, Epidermotropism (patch). C) Infiltrate with monochemotherapy and radiotherapy) with partial responses pleomorphic atypical lymphocytes (tumor) (Figure 3). These lesions exhibited diffuse (90%) GATA3 expression in large cells. Recently presented controlled cutaneous disease with localized patches and plaques, when developed unilateral inguinal adenopathy with histology showing anaplastic large CD30+ cells, with no other involved sites. Patient expired due to sepsis during 2nd chemotherapy

Figure 3. A) Plaque stage. B) cycle. It was not possible to affirm that it represented nodal Histopathological examination of lymph node involvement of transformed MF or a systemic ALCL. No (H&E). C) Immunohistochemical findings GATA3 expression was observed in lymph node. showing CD30 positivity (lymph node).

Table 1. GATA3 expression in doubtful cases. Biopsy Area GATA % Positive infiltrating cells Case 1 Skin tumor + 80% Case 2 Lumbar region + 70-80% Case 3 Tumor on buttock 0 Case 4 Patches and Plaques + 90% Lymph node 0

Among four doubtful cases, a high expression of GATA3 was observed in tumoral lesions of three patients and no expression in one patient. Involved lymph node was also negative for GATA3 in one patient (Table

1).

CONCLUSION GATA3 is a marker of Th2 cell differentiation. Literature shows that while early MF presents a Th1 profile, the late-stage exhibits a Th2 profile. Some studies showed that tumor lesions of MF highly expressed GATA3, as opposed to lesions of cALCL. Immunohistochemical analyzes of confirmed cases of typical M F, tMF, cALCL and sALCL are being concluded to work as controls and help in the comparison with the doubtful cases. This might allow a better conclusion about the usefulness of GATA3 in such cases.

REFERENCES

1. Hsi AC, Lee SJ, Rosman IS. Expression of helper T cell master regulators in inflammatory

dermatoses and primary cutaneous T-cell lymphoma: diagnostic implications. J Am Acad Dermatol. 2015; 72: 159-167. 2. Elbendary A, Parikh K, Elattar I, Truong J, Elston DM. Expression of T-bet and GATA238-3 in early mycosis fungoides and spongiotic dermatitis. J Am Acad Dermatol. 2015; 74: 1012-1014.

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CD30 expression is detectable in most rare cutaneous lymphoma subtypes

Autoren: Ulrike Wehkamp1, Christina Mitteldorf2, Marion Wobser3, Roland Schneiderbauer4, Uwe Hillen5, Sarja Stendel1, Mehmet Baltaci6, Bernd Hemmerlein7, René Stranzenbach8, Jan Nicolay9, Claus- Detlev Klemke10, Werner Kempf11,12, Chalid Assaf6

1Department of dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 2Department of dermatology, University Medical Center Goettingen, Germany 3Department of dermatology, University Hospital Würzburg, Germany 4Department of dermatology, Ludwigshafen, Germany 5Department of dermatology, Vivantes Klinikum Neukölln, Berlin, Germany 6Department of dermatology, Helios-Klinikum Krefeld, Germany 7Department of pathology, Helios-Klinikum Krefeld, Germany 8Department of dermatology, Johannes Wesling Klinikum, UKRUB, University Bochum, Minden, Germany 9Department of dermatology, University Hospital Mannheim, Germany 10Hautklinik, Städtisches Klinikum Karlsruhe, Akademisches Lehrkrankenhaus der Universität Freiburg, Karlsruhe, Germany 11Department of dermatology, University Hospital Zurich, Switzerland 12Kempf und Pfaltz Histologische Diagnostik, Zurich, Switzerland

Introduction: CD30-targeted treatment has been explored and approved for CD30+ cutaneous T- cell lymphomas (TCL). Treatment for the rarer cutaneous lymphoma (CL) subtypes is a challenge, and clinical trials in this indication are scarce. The present study adds 20% more evaluable cases to our previously presented data and details the CD30 expression in rare CL.

Material/methods: 105 biopsies of 101 patients with rare CL from 10 centers were collected. 47 TCLs: 18 CD4+ small/medium sized lymphoproliferations, 9 subcutaneous panniculitis-like TCL, 6 peripheral TCL-NOS, 6 CD8+ aggressive epidermotropic cytotoxic TCL, 4 γ/δ, 4 NK/TCL; and 39 B-cell lymphomas (BCL): 38 diffuse large BCL (DLBCL)–leg-type, 1 EBV+DLBCL; and 14 blastoid plasmacytoid dendritic cell neoplasms. CD30 stains were performed in one center evaluated by six dermatologist/dermatopathologists.

Results: 100 rare CL cases were appropriate for CD30 evaluation. In more than two third of cases, an expression of CD30 was detectable (71/100; 71%). Most cases showed a low degree of CD30 expression (<1-5% (59/71; 83.1%). Staining intensity was regarded as weak or moderate (61/71; 85.9%). Expression was detected mostly in the cytoplasm with or without combined membrane and/or Golgi-reactivity (64/71; 90.1%).

Conclusion: CD30 expression can be detected in the majority of rare lymphoma cases. However, the expression is limited in most cases to a low percentage of the infiltrate and shows weak to moderate staining intensity, predominantly in the cytoplasm. For NK-TCL and other primary cutaneous TCL cases with low expression of CD30 have previously been treated with good results with targeted therapy; thus, these results might represent the basis for the initiation of a clinical study for rare CL.

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CD8+ mycosis fungoides with diverse atypical clinical and pathological presentations Mona Abdel-Halim Ibrahim1, Ahmed Nassar1, Haitham Abdelbary2 1 Dermatology Department, Aim-Shams University, Cairo, Egypt 2 Hematology Department, Ain-Shams University, Cairo, Egypt Introduction Mycosis fungoides (MF) is a great masquerader that can mimic many dermatoses either clinically or histopathologically. Herein, we present a rare case of MF that has multiple atypical presentations and poses a diagnostic dilemma. Case report 25 year-old female patient complained of recurrent abscesses on the groin, axillae and the trunk of 6 years' duration. The patient was previously diagnosed clinically as hidradenitis suppurativa, for which she received isotretinion and systemic antibiotics with no improvement. Skin biopsy was taken and revealed the presence of eosinophilic grauloma suggestive of parasitic etiology. She performed a blood film for detection of filaria and was found to be negative. She then received empirical ivermectin and antihelminthic drugs; however, she didn't show any improvement. On examination, there were painful purulent nodules on the axillae; along with ichthyosis and comedones allover her back and abdomen. Additionally, there were painful indurated dusky red plaques/ nodules on the groin and mons pubis associated with scaly hyperpigmented patches over the thighs. Multiple biopsies were taken; the first from the ichthyotic skin with comedones and the second from a nodule on the axilla. Both biopsies revealed non-epidermotropic superficial dermal perivascular infiltrate of hyperchromatic mildly atypical lymphocytes associated with deep dermal nodular lymphocytic infiltrate and multiple granulomas. The third biopsy was taken from an indurated plaque over the groin which showed band-like dermal infiltrate of hyerchromatic mildly atypical lymphocytes. The fourth biopsy was taken from a patch on the thigh which revealed perifollicular and periappendageal infiltrate, where atypical lymphocytes were seen infiltrating the follicular epithelium and the eccrine glands forming folliculotropism and syringotropism, respectively. Immunohistochemistry was done which revealed positive staining for CD3 and CD8 with negative staining for CD4, CD7 and CD30. Multiple reactive CD20 follicles were detected surrounded by positive CD3 cells. In addition, T cell receptor gene rearrangement was monoclonal for all types of lesions. Based on the clinical, pathological, immunohistochemical and molecular findings, we reached the diagnosis of CD8+ MF with atypical clinical presentations (hidradenitis suppurativa and ichthyosis) and atypical pathological presentations (granulomatous, follicular and syringotropic) associated with reactive B-cell proliferation. Laboratory and radiological investigations were done for staging which revealed no abnormality. Therefore, the patient was regarded as stage IIB.

Discussion Mycosis fungoides should be kept in mind for any dermatosis not responding to the usual treatment modalities because MF can imitate different inflammatory conditions; therefore, skin biopsy should be considered in such cases. In our case, the patient presented with hidradenitis suppurativa and acneiform lesions (comedones). These presentations may actually reflect the presence of folliculotropic MF (with or without syringotropism) histopathologically. The presence of tissue eosinophilia is also more common in the folliculotropic variant. In addition, the presence of granuloma may represent a foreign body tissue reaction to a ruptured hair follicle and points to the histopathological diagnosis of granulomatous MF. Granulomatous MF is a rare histopathological variant representing 6.3% of all MF. It presents as the classic variant (patches/plaques and nodules). Rarely, it presents with ichthyosis-like picture as in our case. It represents a major diagnostic challenge because epidermotropism is absent in about 47% of the cases and granulomas may obscure the lymphomatous component. This leads to a delay in diagnosis with an average of 8.4 years.1

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Reactive B cell proliferation had been previously reported in granulomatous MF and adds more to the difficulty in diagnosis; however, its significance is still unclear. It is important to point out that the simultaneous co-existence of different clinical and histopathological variants of MF is rarely encountered. Similar case had been reported with granulomatous, follicular and syringotropic MF that showed reactive B-cell proliferation.2 Most of granulomatous MF is CD4+, CD8-. CD8+ granulomatous MF is very rare; fortunately, CD8‐ positive phenotype does not influence the prognosis. Granulomatous, folliculotropic and syringotropic MF carry less favorable prognosis; accordingly, this warrants early systemic treatment. Many systemic treatments were found to induce successful remission as PUVA, skin electron irradiation, oral etretinate, systemic interferon‐ γ, gemcitabine, bexarotene and bath PUVA. Our patient received pegylated interferon alpha and PUVA for 6 months with good response. After 3 months, the lesions had relapsed; accordingly, she received gemcitabine and she had good response. PUVA was administered for 3 months after gemcitabine as a maintenance therapy. The patient relapsed 2 months after stopping PUVA; therefore, methotrexate was then given in a dose of 25mg/ week for 2 months with mild improvement. The dose was increased to 50mg/ week for 6 months and the patient achieved excellent response. Remission was maintained for one year follow-up. In conclusion, we report a case of MF with multiple rare atypical presentations that showed significant response and relative durable remission with methotrexate.

References 1. Li JY, Pulitzer MP, Myskowski PL, et al. A case-control study of clinicopathologic features, prognosis, and therapeutic responses in patients with granulomatous mycosis fungoides. J Am Acad Dermatol.2013;69:366–374. 2. Wang L, Wang G, Gao T. Granulomatous syringotropic mycosis fungoides with two lesions having reactive B-cell proliferation. J Cutan Pathol. 2014;41:400–6.

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The therapeutic potential of Cannabinoids from Cannabis sativa extracts for mycosis fungoides / Sézary syndrome - an in vitro and ex vivo study

Amitay-Laish Iris1, Moyal Lilach1,2, Tiroler Amir3,4, Mazuz Moran3, Stalin N. Rajan3, Ajjampura C Vinayaka3, Gorovitz-Haris Batia2, Lubin Ido2, Drori Avi5, Drori Guy5, Van Cauwenberghe Owen6, Namdar Dvora3, Koltai Hinanit3, and Hodak Emmilia1

1Department of Dermatology, and 2Laboratory for Molecular Dermatology, Felsenstein Medical Research Center, Rabin Medical Center - Beilinson Hospital, Petach Tikva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel, 4The Mina and Everard Goodman Faculty of Life Sciences, Bar- Ilan University, Ramat Gan, Israel, 5MedC Biopharma Corporation, Canada, 6AgMedica Bioscience Inc, Chatham, Ontario, Canada

Introduction: Cannabis sativa (C.sativa), produce more than 600 different metabolites. Studies have provided evidence that different cannabinoids exhibit antitumor effects in a wide array of animal cancer models, including leukemia cell-lines, when used alone or combined. There is no data on the effect of cannabinoids on mycosis fungoides (MF)/Sézary syndrome (SS). Our objective was to study the effect of different fractions of C.sativa and their combinations on MF/SS, and to identify a mixture with the optimal cytotoxic effect. Methods: Ethanol extracts of C.sativa were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry, and their cytotoxic activity was determined, using Alamar Blue-based assay (Resazurin) and tetrazolium dye-based assay (XTT), on My-La and HUT-78 cell-lines and on peripheral blood lymphocytes from Sézary patients (SPBL). Apoptotic effect was determined by FACS analysis of Annexin V-staining and PI. RNA sequencing was used to determine gene expression of My-La and HUT-78 cells following treatments with individual compounds and synergistic combinations. Results: Active fractions from cannabis that have cytotoxic activity against My-La and HUT-78 were identified, (fractions with the highest cytotoxic activity were named S4, and S5) (A). The optimal concentration of each fraction, attaining a synergistic-cytotoxic effect of S4+S5- combined against the cell-lines, was specified. This mixture (S4+S5) led to apoptosis of My-La (B), HUT-78 (C), and to a higher apoptosis of CD4+CD26- SPBLs (malignant sézary enriched cell population) than non-CD4+CD26- SPBLs (D), (higher apoptosis was attained with S4+S5, compared with each fraction alone). Gene expression profile was determined in My-La and HUT- 78 cells following treatments with S4, S5, or S4+S5 synergistic combination, showing 947 common genes in both My-La and HuT-78 cell-lines differentially expressed following treatment with the synergistic combination (compared to the control and to cells treated with separate fractions, S4, or S5). Accordingly, treatment with the synergistic combination led to the induction

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of several different biological pathways, including, among others, PI3K-AKT pathway, and pathways in cancer.

A A 140 Viability of My-La cells treated with A 120 different fractions of C. sativa SCBD AB extracts. Cells were seeded and treated 100

B with C. sativa extracts of SCBD, and viability 80 different fractions named: S2, S4, S5, S6

60 * and S7, at an arbitrary constant relatively

La La Cell C - high concentration for 48 h. Doxorubicin 40 * *

My CD (DOXO, 300 nM) served as a positive 20 * * D % D D control. Methanol (control) served as 0 solvent control. Error bars indicate ± SE (n = 3). Levels with different letters are significantly different from all combinations of pairs by Tukey-Kramer honest significant difference (HSD; P ≤ B C 0.05). * indicates significantly different mean from the control based on Student T-

test (P ≤ 0.05).

Proportion of viable, apoptotic or necrotic cells following treatment with S4, S5, or S4+S5 on My-La (B) or HuT-78 (C) cell-lines. Cells were treated with S4, S5, S4+ S5 and methanol (control) for 48 h. Shown are the percentages of viable, necrotic, and apoptotic cells. Error bars indicate ± SE (2 biological replicates, in each n = 3). Levels with different letters are significantly different from all combinations of pair, (Tukey-Kramer honest significant difference; P ≤ 0.05).

D S4+S5 D treatment treatment The percent of apoptotic-induced cells following the combined treatment of S4+S5 was compared between CD4+CD26- cells and non-CD4+CD26- cells of SPBL; (n=6); **denote significant difference between means (paired student T test; 0.001

Conclusions: Fractions of C.sativa and their specific combinations induce cytotoxic activity against MF/SS cells. This provides the first indication that C.sativa specific compound may hold promise as a possible new therapy for MF/SS.

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References: 1. Scott KA, et al. Anticancer effects of phytocannabinoids used with chemotherapy in leukaemia cells can be improved by altering the sequence of their administration. Int J Oncol. 2017;51:369- 77. 2. Lombard C, et al. Targeting cannabinoid receptors to treat leukemia: role of cross-talk between extrinsic and intrinsic pathways in Delta9-tetrahydrocannabinol (THC)-induced apoptosis of Jurkat cells. Leuk Res. 2005;29:915-22.

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Technical Secretariat Grupo Pacifico C/ Maria Cubi, 4. 08006 Barcelona Spain [email protected]