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Objecves

Genecs of Select • Define tumor suppressor gene and oncogene Lymphoproliferave Neoplasms: • Correlate genotypes Pathophysiology, Prognoscaon, and Therapy with prognosis in lymphoproliferave neoplasms

Krisn Landis-Piwowar PhD, MLS(ASCP)CM • Recognize the targets of lymphoproliferave Clinical Laboratory Hematology, 3/E, McKenzie • Williams neoplasm therapies

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Leukemia WHO 2008: Mature B-Cell Neoplasms

• Chronic lymphocyc /small lymphocyc • lymphoma • Diffuse large B-cell lymphoma (DLBCL), NOS • Primarily BM and • Primarily lymph • B-cell prolymphocyc leukemia • T-cell/hisocyte rich large B-cell lymphoma • Primary DLBCL of the CNS • Splenic marginal zone lymphoma • Primary cutaneous DLBCL, leg type blood involvement node / solid ssue • • EBV-posive DLBCL of the elderly • Splenic lymphoma/leukemia, unclassifiable • DLBCL associated with chronic inflammaon • Splenic diffuse red pulp small B-cell lymphoma • Lymphomatoid granulomatosis • Hairy cell leukemia variant involvement • Primary mediasnal (thymic) large B-cell lymphoma • Myeloid or • Lymphoplasmacyc lymphoma • Intravascular large B-cell lymphoma • Waldenström macroglobulinemia • ALK-posive large B-cell lymphoma lymphoid origin • Lymphoid origin • Heavy chain diseases • α • Plasmablasc lymphoma • γ Heavy chain disease • Large B-cell lymphoma arising in HHV8-associated • May secondarily • May secondarily • μ Heavy chain disease mulcentric Castleman disease • Plasma cell myeloma • Primary effusion lymphoma • Solitary of bone • Burki lymphoma involve lymph involve BM & • Extraosseous plasmacytoma • B-cell lymphoma, unclassifiable, with features intermediate • Extranodal marginal zone lymphoma of mucosa-associated between diffuse large B-cell lymphoma and Burki nodes and solid blood; “leukemic lymphoid ssue (MALT lymphoma) lymphoma • Nodal marginal zone lymphoma • B-cell lymphoma, unclassifiable, with features intermediate ssues phase” • Pediatric nodal marginal zone lymphoma between diffuse large B-cell lymphoma and classical • • Pediatric follicular lymphoma • Primary cutaneous follicle centre lymphoma Adapted from: Clinical Laboratory Hematology, 3/E, McKenzie • Williams 4 3

WHO 2008: Mature T-Cell Neoplasms WHO 2008: More Neoplasms

• T-cell prolymphocyc leukemia • • Hodgkin lymphoma • B lymphoblasc leukemia/lymphoma Sézary syndrome • Nodular lymphocyte predominant Hodgkin lymphoma • B lymphoblasc leukemia/lymphoma, NOS • T-cell large granular lymphocyc • Primary cutaneous CD30+ T-cell • Classical Hodgkin lymphoma • B lymphoblasc leukemia/lymphoma with recurrent leukemia • classical Hodgkin lymphoma genec abnormalies lymphoproliferave disorders • Lymphocyte-rich classical Hodgkin lymphoma • B lymphoblasc leukemia/lymphoma with t(9;22) (q34;q11.2);BCR-ABL 1 • • Chronic lymphoproliferave disorder of • Lymphomatoid papulosis Mixed cellularity classical Hodgkin lymphoma • B lymphoblasc leukemia/lymphoma with t(v; NK cells • Lymphocyte-depleted classical Hodgkin lymphoma 11q23);MLL rearranged • Primary cutaneous anaplasc large cell • B lymphoblasc leukemia/lymphoma with t(12;21) • Aggressive NK-cell leukemia lymphoma (p13;q22) TEL-AML1 (ETV6-RUNX1) • Hisocyc and dendric cell neoplasms • B lymphoblasc leukemia/lymphoma with • Systemic EBV-posive T-cell • Primary cutaneous γδ T-cell lymphoma • Hisocyc sarcoma hyperdiploidy lymphoproliferave disease of • Langerhans cell hisocytosis • B lymphoblasc leukemia/lymphoma with hypodiploidy • Langerhans cell sarcoma • B lymphoblasc leukemia/lymphoma with t(5;14) • Primary cutaneous CD8+ aggressive (q31;q32) IL3-IGH childhood • Interdigitang dendric cell sarcoma epidermotropic cytotoxic T-cell • B lymphoblasc leukemia/lymphoma with t(1;19) • Hydroa vacciniforme-like lymphoma • Follicular dendric cell sarcoma (q23;p13.3);TCF3-PBX1 lymphoma • Fibroblasc recular cell tumor • Adult T-cell leukemia/lymphoma • Intermediate dendric cell tumor • T lymphoblasc leukemia/lymphoma • Primary cutaneous CD4+ small/medium • • Extranodal NK/T-cell lymphoma, nasal Disseminated juvenile xanthogranuloma T-cell lymphoma • Acute of ambiguous lineage type • Posransplantaon lymphoproliferave disorders • Peripheral T-cell lymphoma, NOS (PTLDs) • Acute undifferenated leukemia • Enteropathy-associated T-cell • Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); • Early lesions BCR-ABL1 lymphoma • Angioimmunoblasc T-cell lymphoma • Plasmacyc hyperplasia • Mixed phenotype acute leukemia with t(v;11q23); MLL • Hepatosplenic T-cell lymphoma • Infecous mononucleosis–like PTLD rearranged • Anaplasc large cell lymphoma, ALK- • Polymorphic PTLD • Mixed phenotype acute leukemia, B-myeloid, NOS • • Subcutaneous panniculis-like T-cell posive Monomorphic PTLD (B- and T/NK-cell types) • Mixed phenotype acute leukemia, T-myeloid, NOS • lymphoma Classical Hodgkin lymphoma type PTLD • Provisional enty: natural killer (NK) cell lymphoblasc • Anaplasc large cell lymphoma, ALK- leukemia/lymphoma • negave 5 6

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Posion effect leads to new Pathophysiology Gene A promoter Gene B coding region expression rate

• Chromosome Alteraons • Unbalanced: Change in total genec informaon Gene B coding region (addions / deleons) Gene A coding region • Balanced: Genec informaon Del 17p (8%) rearranged Expression of trisomy 12 fusion genes with (12–16%) • DNA alteraons new funcon Del 11q • Single nucleode (18%) • Inserons Del 13q Breakpoint effect • Deleons (55%) CLL deletes coding • Duplicaons region and leads 7 to loss of funcon 8

Chromosomal Endogenous translocaons Growth Growth factor Exogenous factors receptors • Free radicals Gene • DNA replicaon errors • Ionizing radiaon amplificaon Signal Transcripon • V(D)J recombinaon • Chemicals transducers factors • Class switching Proto- • Drugs • Somac hypermutaon Retroviral oncogenes transducon Double Strand Breaks Inseronal Gain of mutagenesis Funcon M G Point Oncogenes 1 mutaons Promote Cell Death G2 S DNA Repair proliferaon DNA Incorrect Neoplasc cells in damaging peripheral blood event Genomic Instability Carcinogenesis

Carcinogenesis Normal Cells in peripheral blood

Bone marrow HSC pool 10 Adapted from: Clinical Laboratory Hematology, 3/E, McKenzie • Williams

Growth Growth factor Oncogenes Promote factors receptors Lymphoproliferaon Signal Transcripon transducers factors Signal Transcripon Dimerizaon / Receptors Transducers Factors Transmembrane Acvaon Receptors (RTKs) BRAF NOTCH STAT3 BCL2 1. Receptor-growth factor BCR-ABL1 ETV6-RUNX1 BCR binding MYD88 BIRC3 TCF3-PBX1 2. Signal transducon JAK3 CD79A/B 3. Gene expression NOTCH MYC-IGH2

DNA RNA Protein 12

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M G1 Growth Growth factor factors receptors Both alleles Cell Death G2 S DNA Repair must be affected Signal Transcripon Two-hit Incorrect transducers factors Inherited / hypothesis Sporadic Dimerizaon / Loss of Funcon Transmembrane Acvaon Tumor Receptors (RTKs) Suppressor Genes Inhibit proliferaon 1. Receptor-growth factor binding Carcinogenesis 2. Signal transducon 3. Gene expression

DNA RNA Protein 13

Tumor Suppressors Inhibit Lymphoproliferaon Epigenecs • “On top of genecs” Signal Transcripon Epigenec • Informaon carried by the Transducers Factors Regulators genome that is not encoded Brooker by DNA ATM SF3B1 • DNA provides the genec plan P53 • Other factors affect how that plan will be expressed

SF3B1 miR15/16 • DNA methylaon Brooker • Histone modificaons miRNA • Non-coding RNA (miRNA) 15 16

RNA interference Cancer Evolves by Natural Selecon Depletes mRNA via (miRNA) sequence-specific pairing • Cancer is mulple different (sub-clonal) cancers occupying the same or disnct ssue environments microRNA: Non-protein coding RNA Establishes and • Clonal expansion results due to compeve maintains ssue- advantage (proliferave or survival) over other (18 – 24 nucleodes) binds mRNA specific expression sub-clones profile • Number of mutaons can vary from few (10–20) to hundreds of thousands • Most are ‘passengers’ (no effect on growth advantage) mRNA Translaonal • Others are funconally relevant ‘drivers’ (confers degradaon inhibion selecve growth advantage) 18 17

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Diffuse Walden- Chronic Hairy cell T-cell large Plasma cell T-cell acute Clonal Evoluon is an Interplay of large B-cell ström lympho- leukemia granular myeloma lympho- lymphoma macro- cyc lympho- blasc Genec Changes: (DLBCL), globulin- leukemia cyc leukemia NOS emia leukemia CD79A 20% ------Selecvely • Survival/an-apoptosis mutaon advantageous ‘driver’ CD79B 20% ------• Oncogenes mutaon lesions MYD88 30-75% 68-100% 2-10% - - - - mutaon Selecvely neutral • Mono-alleleic loss NOTCH1 8% - 5-20% - - - >60% mutaon ‘passenger’ lesions • Tumor suppressors NOTCH2 8% ------mutaon BRAF 4% - - 99-100% - ??? - • Increase the rate of other genec changes mutaon ‘Mutator’ lesions - - - - • DNA repair genes SF3B1 - - 5-23% STAT3 3-5% - - - 28-40% 50% -

P53 - - 10% - - 10% - Changes to the micro-environment miR 15a/ ??? - 55-66% - - Up to 50% - 19 16-1 20

MYD88 MYD88

• Toll-like receptors (TLR) are central to B-cell • MYD88 L265P missense clonal ‘driver’ receptor-independent angen response mutaon • Sense pathogen-associated molecular paerns • Idenfied in 2011 in DLBCL (2012; WM) (bacteria, viruses, and fungi) • Gain of funcon to drive NF-kB • Iniates intracellular signaling (MYD88) Toll-interleukin-1 receptor domain DD TIR NF-kB IRAK interacon L265P Promotes cell survival • TIR mutaon: homo-dimerizaon; Pro- inflammatory uncontrolled formaon of MYD88–IRAK cytokines Inhibits apoptosis signaling complex 21 22 Sakata-Yanagimoto

MYD88 L265P Prognoscs and Therapeucs BRAF • MYD88 L265P (and other mutaons) • Serine/threonine kinase that regulates • Poor prognosc indicator mitogen-acvated protein kinase (MAPK) • DLBCL, WM signaling pathway • Cell proliferaon • Small molecule inhibitors target MYD88 • Differenaon dimerizaon in pre-clinical trials • Survival • Hydrocinnamoyl-l-valyl pyrrolidine (compound 4a) • BRAF V600E missense ‘driver’ mutaon • Pephinh-MYD88 • • Idera Pharmaceucals: Clinical trial of Idenfied in 2011 in HCL IMO-8400 • 90% of BRAF mutaons • Relapsed or refractory DLBCL paents • Directly acvates MEK with MYD88 L265P mutaon CR1 CR2 Kinase domain • ClinicalTrials.gov idenfier NCT02252146

Krieg 23 V600E 24

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BRAF V600E Prognoscs and Therapeucs SF3B1

• Hairy cell leukemia; good prognosis • Splicing factor 3b1 (component of U2 • 81% experience complete hematologic remission snRNP) • Median period of disease-free survival is 16 years • Potenal targets: FOXP1, P53, ATM, Epigenec modifiers • Vemurafenib (Zelboraf®; BRAF inhibitor) • Subclonal ‘driver’ mutaon in CLL; • FDA approved for melanoma (2011) promotes disease progression • A Phase II Study of the BRAF Inhibitor, Vemurafenib, in Paents With • K700E most frequent mutaon (50% of Relapsed or Refractory Hairy Cell Leukemia (hp://www.clinicaltrials.gov/ct2/show/ reported cases) NCT01711632?term=hairy+cell+leukemia&recr=Open&rank=4) • DNA hypomethylaon • Dabrafenib (Tafinlar®; BRAF inhibitor), Tramenib (Mekinist®; MEK inhibitor) • A Phase II, Open-label, Study in Subjects With BRAF V600E-Mutated Rare Cancers With Several Histologies to Invesgate the Clinical Efficacy and Safety of the Combinaon Therapy of Dabrafenib and Tramenib (hp:// www.clinicaltrials.gov/ct2/show/NCT02034110?term=hairy+cell+leukemia+BRAF +V600&rank=2)

25 26 Wan Brooker

SF3B1 miR 15a/16-1 Prognoscs • Non-protein coding micro RNA involved in ssue SF3B1 mutaons associated specific gene regulaon (B-cells) with poorer clinical outcome • Highly expressed in normal CD5+ lymphocytes in CLL paents independent of other prognosc markers • Involved in normal CD5+ B-cell homeostasis • Target oncogenes: BCL2, MCL1, CCND1, WNT3A, NFkB, VEGF Earlier me- Shorter Target mRNA to-treatment Faster overall Translaon of mRNA into protein Iniaon Disease survival miR Progression

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miR 15a/16-1 miR 15a/16-1

• Inhibits the an- apoptoc BCL-2 gene • Located on chromosome 13q14 (B cell lymphoma 2) expression • Commonly deleted in MM and CLL • Clonal ‘driver’ mutaon in CLL; contribute to disease • BCL2 gene is progression overexpressed in 65– 70% of B cells in CLL • Idenfied in 2002 in CLL • t(14;18)(q32;q21) • Germline mutaons (reduce expression) places BCL2 gene under control of BCL-2 mRNA immunoglobulin Translaon of BCL-2 mRNA into protein heavy-chain enhancers miR15a/16-1

• Rare in CLL Coer 29 30

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miR 15a/16-1 NOTCH1 Prognoscs and Therapeucs • Prognosis: Accelerates clinical course • Transmembrane protein: Ligand-acvated transcripon factor • BCL-2 inhibitors • Intracellular domain • ABT-263 (Navitoclax) inhibits translocates to nucleus BCL-2, clinical trials (2006) for • NOTCH recruits co- relapsed and refractory CLL acvators to drive • ABT-199 combinaon treatment transcripon of target genes (MYC, NF-kB) • An-CD20 • – CLL (NCT01682616, NCT01671904, Commitment of HPCs to T- NCT01889186, NCT01685892, cells, marginal zone B cells NCT02005471) • Cell growth – Lymphoma (NCT01594229) • Proliferaon • Bortezomib for MM paents (NCT01794507) • Survival 31 Anderson 32

NOTCH1 Prognoscs and Therapeucs Chronic Lymphocyc Leukemia • Putave ‘driver’ mutaon Very • 17p13- (P53 mut) • Idenfied in the early 1990s, defined in 2004 in T-ALL Poor High MYD88 SF3B1 • 11q22- (ATM mut) • Mutaons in 5–10% newly diagnosed CLL, 15–20% in progressive and prognosc Risk relapsed cases markers • Mutaon in > 60% of T-ALL cases (most commonly mutated gene) miR 15a/ • BCR acvity 16-1 NOTCH1 • IGHV UM High/ • NOTCH1 mut Inter CD38 and • SF3B1 mut P53 and ATM unmutated 17p13.1 mutaons IGHV gene deleon • None of above Low

Stransky • Prognosis: Neutral to poor The heterogeneous clinical course of lymphoproliferave neoplasms is • Therapeucs are pre-clinical: likely explained by underlying molecular prognosc factors • Specific anbodies directed against NOTCH • Chemically modified pepdes block NOTCH transcriponal complexes Molecular analysis at diagnosis provides beer prognoscaon

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Case 1 Case 1: Addional Workup

• Paent: A 46-year-old male visited his primary care • Bone marrow: physician for a two month history of backache and fever • hypercellular, all states of maturaon, cells with a ‘fried • Family Hx: Father diagnosed with chronic lymphocyc egg’ appearance leukemia 4 years ago • M:E = 2:1

• Physical Exam: RBC 3.66 x 1012/L WBC 4.2 x 109/L • Moderate increase in reculin fibers • Splenomegaly HGB 10.9 g/dL Neutro 20 % 1.0 x 109/L • Cells posive for: CD19, CD20, CD22, CD25, CD11c, FMC7 • No palpable HCT 31 % Lymph 80% 4.0 x 109/L and kappa light chain (mature B cells) lymphadenopathy MCV 88.1 fL Mono 0 in cervical, axillary MCH 31.3 pg Eos 0 • Cytogenecs: normal male karyotype or inguinal regions MCHC 34.2 g/dL Baso 0 • Differeneal Diagnosis: Disorders associated with • No hepatomegaly, RDW 14.2 % splenomegaly petechiae, purpura, PLT 95 x 109/L or bruises MPV 6.1 fL • CLL, prolymphocyc leukemia, splenic marginal zone lymphoma, HCL, variant HCL and mantle cell lymphoma

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Case 1: Addional Workup Case 2

• Real me PCR: BRAF V600E • Paent: A 59-year-old male visited his primary care physician for presents with a two-week history of headaches, blurred vision, and bruising • Family Hx: Unremarkable • Physical Exam: RBC 3.4 x 1012/L WBC 8.1 x 109/L 9 • No palpable HGB 10.2 g/dL Neutro 61.2 % 5.0 x 10 /L HCT 30 % Lymph 28.2% 2.3 x 109/L lymph- 9 adenopathy MCV 97 fL Mono 9.0% 0.7 x 10 /L MCH 32.7 pg Eos 1.2% 0.1 x 109/L Paent was • No spleno- MCHC 33.7 g/dL Baso 0.4% 0.0 x 109/L hepatomegaly, treated with RDW 14.8 % petechiae, PLT 193 x 109/L cladribine purpura, or MPV 7.2 fL Total 10.6 g/dL (6.4-8.3) bruises protein 37 38

Case 2: Addional Workup Case 2: Addional Workup

• Serum protein electrophoresis with • Pyrosequencing immunofixaon showed an M spike of • MYD88 L265P IgM κ paraprotein Paent • Bone marrow: • hypercellular, small lymphocytes, plasmacytoid lymphocytes and plasma cells • Cells posive for: CD19, CD20, CD22, and surface IgM • Cytogenecs: 6q- Paent was • Differeneal Diagnosis: Disorders assoc with hyperviscosity treated with cladribine • of undertermined significance, lymphoplamacyc lymphoma/Waldenström macroglobuliniemia, marginal zone lymphoma 39 40

References

• Mckenzie, Williams. Clinical Laboratory Hematology. Perason. 3rd ed. 2014. • Brooker, Concepts of Genecs. McGraw Hill. 1st ed. 2011. • Sakata-Yanagimoto, Disease-specific mutaons in mature lymphoid neoplasms: recent advances. Cancer Sci. 2014. • Krieg, AIMing 2 defend against intracellular pathogens. Nat Immuno. 2010. • Wan, Wu. SF3B1 mutaons in chronic lymphocyc leukemia. Blood. 2013. • Coer. Apoptosis and cancer: the genesis of a research field. Nat Rev Can. 2009. • Andersen, Uosaki, Shenje, Kwon. Non-canonical Notch signaling: emerging role and mechanism. Trends in Cell Biology. 2012. • Stransky et al. The Mutaonal Landscape of Head and Neck Squamous Cell Carcinoma. Science. 2011. • HCL and WM images provided by John Landis: [email protected] 41

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