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Objec ves
Gene cs of Select • Define tumor suppressor gene and oncogene Lymphoprolifera ve Neoplasms: • Correlate genotypes Pathophysiology, Prognos ca on, and Therapy with prognosis in lymphoprolifera ve neoplasms
Kris n Landis-Piwowar PhD, MLS(ASCP)CM • Recognize the targets of lymphoprolifera ve Clinical Laboratory Hematology, 3/E, McKenzie • Williams neoplasm therapies
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Leukemia Lymphoma WHO 2008: Mature B-Cell Neoplasms
• Chronic lymphocy c leukemia/small lymphocy c • Mantle cell lymphoma lymphoma • Diffuse large B-cell lymphoma (DLBCL), NOS • Primarily BM and • Primarily lymph • B-cell prolymphocy c leukemia • T-cell/his ocyte rich large B-cell lymphoma • Primary DLBCL of the CNS • Splenic marginal zone lymphoma • Primary cutaneous DLBCL, leg type blood involvement node / solid ssue • Hairy cell leukemia • EBV-posi ve DLBCL of the elderly • Splenic lymphoma/leukemia, unclassifiable • DLBCL associated with chronic inflamma on • Splenic diffuse red pulp small B-cell lymphoma • Lymphomatoid granulomatosis • Hairy cell leukemia variant involvement • Primary medias nal (thymic) large B-cell lymphoma • Myeloid or • Lymphoplasmacy c lymphoma • Intravascular large B-cell lymphoma • Waldenström macroglobulinemia • ALK-posi ve large B-cell lymphoma lymphoid origin • Lymphoid origin • Heavy chain diseases • α Heavy chain disease • Plasmablas c lymphoma • γ Heavy chain disease • Large B-cell lymphoma arising in HHV8-associated • May secondarily • May secondarily • μ Heavy chain disease mul centric Castleman disease • Plasma cell myeloma • Primary effusion lymphoma • Solitary plasmacytoma 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 • Follicular lymphoma Hodgkin lymphoma • 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 prolymphocy c leukemia • • Hodgkin lymphoma • B lymphoblas c leukemia/lymphoma Sézary syndrome • Nodular lymphocyte predominant Hodgkin lymphoma • B lymphoblas c leukemia/lymphoma, NOS • T-cell large granular lymphocy c • Primary cutaneous CD30+ T-cell • Classical Hodgkin lymphoma • B lymphoblas c leukemia/lymphoma with recurrent leukemia • Nodular sclerosis classical Hodgkin lymphoma gene c abnormali es lymphoprolifera ve disorders • Lymphocyte-rich classical Hodgkin lymphoma • B lymphoblas c leukemia/lymphoma with t(9;22) (q34;q11.2);BCR-ABL 1 • • Chronic lymphoprolifera ve disorder of • Lymphomatoid papulosis Mixed cellularity classical Hodgkin lymphoma • B lymphoblas c leukemia/lymphoma with t(v; NK cells • Lymphocyte-depleted classical Hodgkin lymphoma 11q23);MLL rearranged • Primary cutaneous anaplas c large cell • B lymphoblas c leukemia/lymphoma with t(12;21) • Aggressive NK-cell leukemia lymphoma (p13;q22) TEL-AML1 (ETV6-RUNX1) • His ocy c and dendri c cell neoplasms • B lymphoblas c leukemia/lymphoma with • Systemic EBV-posi ve T-cell • Primary cutaneous γδ T-cell lymphoma • His ocy c sarcoma hyperdiploidy lymphoprolifera ve disease of • Langerhans cell his ocytosis • B lymphoblas c leukemia/lymphoma with hypodiploidy • Langerhans cell sarcoma • B lymphoblas c leukemia/lymphoma with t(5;14) • Primary cutaneous CD8+ aggressive (q31;q32) IL3-IGH childhood • Interdigita ng dendri c cell sarcoma epidermotropic cytotoxic T-cell • B lymphoblas c leukemia/lymphoma with t(1;19) • Hydroa vacciniforme-like lymphoma • Follicular dendri c cell sarcoma (q23;p13.3);TCF3-PBX1 lymphoma • Fibroblas c re cular cell tumor • Adult T-cell leukemia/lymphoma • Intermediate dendri c cell tumor • T lymphoblas c leukemia/lymphoma • Primary cutaneous CD4+ small/medium • • Extranodal NK/T-cell lymphoma, nasal Disseminated juvenile xanthogranuloma T-cell lymphoma • Acute leukemias of ambiguous lineage type • Pos ransplanta on lymphoprolifera ve disorders • Peripheral T-cell lymphoma, NOS (PTLDs) • Acute undifferen ated leukemia • Enteropathy-associated T-cell • Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); • Early lesions BCR-ABL1 lymphoma • Angioimmunoblas c T-cell lymphoma • Plasmacy c hyperplasia • Mixed phenotype acute leukemia with t(v;11q23); MLL • Hepatosplenic T-cell lymphoma • Infec ous mononucleosis–like PTLD rearranged • Anaplas c large cell lymphoma, ALK- • Polymorphic PTLD • Mixed phenotype acute leukemia, B-myeloid, NOS • • Subcutaneous panniculi s-like T-cell posi ve Monomorphic PTLD (B- and T/NK-cell types) • Mixed phenotype acute leukemia, T-myeloid, NOS • lymphoma Classical Hodgkin lymphoma type PTLD • Provisional en ty: natural killer (NK) cell lymphoblas c • Anaplas c large cell lymphoma, ALK- leukemia/lymphoma • Mycosis fungoides nega ve 5 6
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Posi on effect leads to new Pathophysiology Gene A promoter Gene B coding region expression rate
• Chromosome Altera ons • Unbalanced: Change in total gene c informa on Gene B coding region (addi ons / dele ons) Gene A coding region • Balanced: Gene c informa on Del 17p (8%) rearranged Expression of trisomy 12 fusion genes with (12–16%) • DNA altera ons new func on Del 11q • Single nucleo de (18%) • Inser ons Del 13q Breakpoint effect • Dele ons (55%) CLL deletes coding • Duplica ons region and leads 7 to loss of func on 8
Chromosomal Endogenous transloca ons Growth Growth factor Exogenous factors receptors • Free radicals Gene • DNA replica on errors • Ionizing radia on amplifica on Signal Transcrip on • V(D)J recombina on • Chemicals transducers factors • Class switching Proto- • Drugs • Soma c hypermuta on Retroviral oncogenes transduc on Double Strand Breaks Inser onal Gain of mutagenesis Func on M G Point Oncogenes 1 muta ons Promote Cell Death G2 S DNA Repair prolifera on DNA Incorrect Neoplas c 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 Lymphoprolifera on Signal Transcrip on transducers factors Signal Transcrip on Dimeriza on / Receptors Transducers Factors Transmembrane Ac va on Receptors (RTKs) BRAF NOTCH STAT3 BCL2 1. Receptor-growth factor BCR-ABL1 ETV6-RUNX1 BCR binding MYD88 BIRC3 TCF3-PBX1 2. Signal transduc on 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 Transcrip on Two-hit Incorrect transducers factors Inherited / hypothesis Sporadic Dimeriza on / Loss of Func on Transmembrane Ac va on Tumor Receptors (RTKs) Suppressor Genes Inhibit prolifera on 1. Receptor-growth factor binding Carcinogenesis 2. Signal transduc on 3. Gene expression
DNA RNA Protein 13
Tumor Suppressors Inhibit Lymphoprolifera on Epigene cs • “On top of gene cs” Signal Transcrip on Epigene c • Informa on carried by the Transducers Factors Regulators genome that is not encoded Brooker by DNA ATM SF3B1 • DNA provides the gene c plan P53 • Other factors affect how that plan will be expressed
SF3B1 miR15/16 • DNA methyla on Brooker • Histone modifica ons miRNA • Non-coding RNA (miRNA) 15 16
RNA interference Cancer Evolves by Natural Selec on Depletes mRNA via (miRNA) sequence-specific pairing • Cancer is mul ple different (sub-clonal) cancers occupying the same or dis nct ssue environments microRNA: Non-protein coding RNA Establishes and • Clonal expansion results due to compe ve maintains ssue- advantage (prolifera ve or survival) over other (18 – 24 nucleo des) binds mRNA specific expression sub-clones profile • Number of muta ons can vary from few (10–20) to hundreds of thousands • Most are ‘passengers’ (no effect on growth advantage) mRNA Transla onal • Others are func onally relevant ‘drivers’ (confers degrada on inhibi on selec ve growth advantage) 18 17
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Diffuse Walden- Chronic Hairy cell T-cell large Plasma cell T-cell acute Clonal Evolu on is an Interplay of large B-cell ström lympho- leukemia granular myeloma lympho- lymphoma macro- cy c lympho- blas c Gene c Changes: (DLBCL), globulin- leukemia cy c leukemia NOS emia leukemia CD79A 20% ------Selec vely • Survival/an -apoptosis muta on advantageous ‘driver’ CD79B 20% ------• Oncogenes muta on lesions MYD88 30-75% 68-100% 2-10% - - - - muta on Selec vely neutral • Mono-alleleic loss NOTCH1 8% - 5-20% - - - >60% muta on ‘passenger’ lesions • Tumor suppressors NOTCH2 8% ------muta on BRAF 4% - - 99-100% - ??? - • Increase the rate of other gene c changes muta on ‘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 an gen response muta on • Sense pathogen-associated molecular pa erns • Iden fied in 2011 in DLBCL (2012; WM) (bacteria, viruses, and fungi) • Gain of func on to drive NF-kB • Ini ates intracellular signaling (MYD88) Toll-interleukin-1 receptor domain DD TIR NF-kB IRAK interac on L265P Promotes cell survival • TIR muta on: homo-dimeriza on; Pro- inflammatory uncontrolled forma on of MYD88–IRAK cytokines Inhibits apoptosis signaling complex 21 22 Sakata-Yanagimoto
MYD88 L265P Prognos cs and Therapeu cs BRAF • MYD88 L265P (and other muta ons) • Serine/threonine kinase that regulates • Poor prognos c indicator mitogen-ac vated protein kinase (MAPK) • DLBCL, WM signaling pathway • Cell prolifera on • Small molecule inhibitors target MYD88 • Differen a on dimeriza on in pre-clinical trials • Survival • Hydrocinnamoyl-l-valyl pyrrolidine (compound 4a) • BRAF V600E missense ‘driver’ muta on • Pephinh-MYD88 • • Idera Pharmaceu cals: Clinical trial of Iden fied in 2011 in HCL IMO-8400 • 90% of BRAF muta ons • Relapsed or refractory DLBCL pa ents • Directly ac vates MEK with MYD88 L265P muta on CR1 CR2 Kinase domain • ClinicalTrials.gov iden fier NCT02252146
Krieg 23 V600E 24
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BRAF V600E Prognos cs and Therapeu cs 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 • Poten al targets: FOXP1, P53, ATM, Epigene c modifiers • Vemurafenib (Zelboraf®; BRAF inhibitor) • Subclonal ‘driver’ muta on in CLL; • FDA approved for melanoma (2011) promotes disease progression • A Phase II Study of the BRAF Inhibitor, Vemurafenib, in Pa ents With • K700E most frequent muta on (50% of Relapsed or Refractory Hairy Cell Leukemia (h p://www.clinicaltrials.gov/ct2/show/ reported cases) NCT01711632?term=hairy+cell+leukemia&recr=Open&rank=4) • DNA hypomethyla on • Dabrafenib (Tafinlar®; BRAF inhibitor), Trame nib (Mekinist®; MEK inhibitor) • A Phase II, Open-label, Study in Subjects With BRAF V600E-Mutated Rare Cancers With Several Histologies to Inves gate the Clinical Efficacy and Safety of the Combina on Therapy of Dabrafenib and Trame nib (h p:// www.clinicaltrials.gov/ct2/show/NCT02034110?term=hairy+cell+leukemia+BRAF +V600&rank=2)
25 26 Wan Brooker
SF3B1 miR 15a/16-1 Prognos cs • Non-protein coding micro RNA involved in ssue SF3B1 muta ons associated specific gene regula on (B-cells) with poorer clinical outcome • Highly expressed in normal CD5+ lymphocytes in CLL pa ents independent of other prognos c 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 Transla on of mRNA into protein Ini a on Disease survival miR Progression
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miR 15a/16-1 miR 15a/16-1
• Inhibits the an - apopto c BCL-2 gene • Located on chromosome 13q14 (B cell lymphoma 2) expression • Commonly deleted in MM and CLL • Clonal ‘driver’ muta on in CLL; contribute to disease • BCL2 gene is progression overexpressed in 65– 70% of B cells in CLL • Iden fied in 2002 in CLL • t(14;18)(q32;q21) • Germline muta ons (reduce expression) places BCL2 gene under control of BCL-2 mRNA immunoglobulin Transla on of BCL-2 mRNA into protein heavy-chain enhancers miR15a/16-1
• Rare in CLL Co er 29 30
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miR 15a/16-1 NOTCH1 Prognos cs and Therapeu cs • Prognosis: Accelerates clinical course • Transmembrane protein: Ligand-ac vated transcrip on 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 ac vators to drive • ABT-199 combina on treatment transcrip on 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) • Prolifera on • Bortezomib for MM pa ents (NCT01794507) • Survival 31 Anderson 32
NOTCH1 Prognos cs and Therapeu cs Chronic Lymphocy c Leukemia • Puta ve ‘driver’ muta on Very • 17p13- (P53 mut) • Iden fied in the early 1990s, defined in 2004 in T-ALL Poor High MYD88 SF3B1 • 11q22- (ATM mut) • Muta ons in 5–10% newly diagnosed CLL, 15–20% in progressive and prognos c Risk relapsed cases markers • Muta on in > 60% of T-ALL cases (most commonly mutated gene) miR 15a/ • BCR ac vity 16-1 NOTCH1 • IGHV UM High/ • NOTCH1 mut Inter CD38 and • SF3B1 mut P53 and ATM unmutated 17p13.1 muta ons IGHV gene dele on • None of above Low
Stransky • Prognosis: Neutral to poor The heterogeneous clinical course of lymphoprolifera ve neoplasms is • Therapeu cs are pre-clinical: likely explained by underlying molecular prognos c factors • Specific an bodies directed against NOTCH • Chemically modified pep des block NOTCH transcrip onal complexes Molecular analysis at diagnosis provides be er prognos ca on
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Case 1 Case 1: Addi onal Workup
• Pa ent: 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 matura on, cells with a ‘fried • Family Hx: Father diagnosed with chronic lymphocy c 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 re culin fibers • Splenomegaly HGB 10.9 g/dL Neutro 20 % 1.0 x 109/L • Cells posi ve 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 • Cytogene cs: normal male karyotype or inguinal regions MCHC 34.2 g/dL Baso 0 • Differene al Diagnosis: Disorders associated with • No hepatomegaly, RDW 14.2 % splenomegaly petechiae, purpura, PLT 95 x 109/L or bruises MPV 6.1 fL • CLL, prolymphocy c leukemia, splenic marginal zone lymphoma, HCL, variant HCL and mantle cell lymphoma
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Case 1: Addi onal Workup Case 2
• Real me PCR: BRAF V600E • Pa ent: 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 Pa ent 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: Addi onal Workup Case 2: Addi onal Workup
• Serum protein electrophoresis with • Pyrosequencing immunofixa on showed an M spike of • MYD88 L265P IgM κ paraprotein Pa ent • Bone marrow: • hypercellular, small lymphocytes, plasmacytoid lymphocytes and plasma cells • Cells posi ve for: CD19, CD20, CD22, and surface IgM • Cytogene cs: 6q- Pa ent was • Differene al Diagnosis: Disorders assoc with hyperviscosity treated with cladribine • Monoclonal gammopathy of undertermined significance, lymphoplamacy c lymphoma/Waldenström macroglobuliniemia, marginal zone lymphoma 39 40
References
• Mckenzie, Williams. Clinical Laboratory Hematology. Perason. 3rd ed. 2014. • Brooker, Concepts of Gene cs. McGraw Hill. 1st ed. 2011. • Sakata-Yanagimoto, Disease-specific muta ons in mature lymphoid neoplasms: recent advances. Cancer Sci. 2014. • Krieg, AIMing 2 defend against intracellular pathogens. Nat Immuno. 2010. • Wan, Wu. SF3B1 muta ons in chronic lymphocy c leukemia. Blood. 2013. • Co er. 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 Muta onal 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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