Dr. Edward P. Fox February 26, 2016

Chronic Lymphoproliferative disorder Progressive accumulation functionally incompetent lymphocytes Monoclonal Overview

Epidemiology Most common leukemia – Western countries 30% in US 16,000 total 9500 male / 6500 females Male:female ratio = 1.7: / 3.65 per 100,000 – US 5.87 / 4.01 per 100,000 - Europe

Epidemiology Disease of Elderly Median age of diagnosis – 70 Can be diagnosed in year-olds Incidence increases rapidly with age

Epidemiology Race Higher in Caucasians Lower in –African Americans - Asian and Pacific Islanders 90% lower in China and Japan

Epidemiology Geography Point to genetic rather than environmental factors Japanese in Hawaii – incidence = Japan Japan – cytogenetics and molecular genetics the same

Epidemiology Environmental Risks None known No increase in atomic bomb survivors Possibly Rubber manufacturers Farmers Benzene / solvents Multiple pneumonias

Epidemiology Family Studies Family members of CLL patients have greater risks: Lymphoid malignancies Hematologic malignancies Solid malignancies 17% of first degree relatives have MBL

Epidemiology Multi-Hit Hypothesis Median age parent (72), child (51) DNA analysis of monozygotic twins: Genetically distinct cells Transforming event occurs later in life

Clinical Presentation Wide variety of presentations Commonly asymptomatic lymphadenopathy 25% abnormal CBC w/o symptoms

Clinical Presentation 10% have “B” symptoms Fevers (>38 o, > 2 weeks, no infection) Sweats (drenching, no infection) Weight loss (≥ 10% in 6 months) Extreme fatigue (ECOG ≥ 2)

Clinical Presentation ITP AIHA PRCA Exaggerated reaction to insect bites

Clinical Presentation Signs: Lymphadenopathy = 50-90% Splenomegaly = 25-55% Hepatomegaly = 10-25% Cutaneous = <5% Renal = MPGN Rarely GI or CNS

Clinical Presentation Laboratory abnormalities Lymphocytosis > 5,000/microL – requisite > 100,000 – 30% > 200,000 – rare, usually normal viscosity

Clinical Presentation Labs: Cytopenias – usually mild ITP = 2-3% AIHA = 11% PRCA = 0.5% Agranulocytosis = 0.5%

Clinical Presentation Labs: Hypogammaglobulinemia = 8% at diagnosis Usually all 3 classes IgG, A, M Monoclonal paraprotein = 5% Polyclonal increase up to 15%

Pathology Initial evaluation should include: CBC Peripheral smear exam Peripheral blood flow cytometry

Pathology Not necessary* Bone marrow biopsy / aspirate Lymph node biopsy * but occasionally useful

Morphology Lymphocytosis Small mature appearing Dense nucleus No nucleoli, aggregated chromatin Narrow rim of cytoplasm

Morphology “Smudge cells” – common Prolymphocytes – small number Larger cells More open, notched nuclei Lacy chromatin, nucleoli

Immunophenotyping Key component to diagnosing CLL Cells appear mature Immature functionally and developmentally

Immunophenotyping B cell antigens: CD19, CD20, CD23 – present CD21, CD24 – sometimes Co-expression of CD5 – T cell antigen

Immunophenotyping Sn Ig : weakly expressed IgM or IgM and IgD Single Ig light chain expressed “light chain restriction” – monoclonal Negative cyclin D1, CD10

Immunophenotyping (+)(-) IgMCD10 IgD, IgMCyclin D1 CD19 CD20 CD21 CD5

Evaluation and Diagnosis 2008 NCI guidelines: 1. Absolute B lymphocytes >5,000/microL Morphologically mature appearing 2. Flow cytometry demonstrates monoclonality CD19, CD20, CD23 CD5 Kappa or lambda light chain restriction

Monoclonal B cell lymphocytosis (MBCL) + flow cytometry Lymphocytosis < 5,000 No adenopathy, hepatomegaly, splenomegaly

Small Lymphocytic Lymphoma (SLL) Lymphadenopathy ALC <5,000 No hepatosplenomegaly

CLL vs SLL vs MBCL Sometimes fuzzy Controversy ongoing Usually irrelevant to treating patients

B-CELL LINEAGE AND DIFFERENTIATION Gaidano et al. Leukemia. 2000;14: LPL MCL HHV-8 SLL/B-CLL PEL Germinal Center p53 C-MYC BCL-2 FL p53, p16 DLCL Plasma Cells Follicular Mantle Memory B Cells Memory B Cells CD5+ B Cells CD5+ B Cells Marginal Zone BL BCL-6 ? Virgin B Cells Virgin B Cells PAX-5 ? BCL-1 ?

Differential Diagnosis Sustained lymphocytosis, monoclonal Mononucleosis Whooping cough - pertussis Toxoplasmosis

Differential Diagnosis Monoclonal B cell lymphocytosis (MBCL) Clonal B lymphocytes <5,000 No B symptoms No organomegaly, lymphadenopathy

MBCL phenotype 5% of normal CBC’s > 60 y.o. 14% > 60 with lymphocytosis 1-2% per year progression to CLL Virtually 100% CLL patients

Prolymphocytic leukemia Larger cells - >90% CD5 negative SmIg normal

COMPARISON OF CLL AND PLL CLLPLL sIgfaintstrong CD19+++ CD20+++ CD5++-

Mantle Cell Lymphoma May have leukemic phase Cyclin D1, SmIg positive + (11, 14) CD23 negative

Others Lymphoplasmacytic lymphoma Hairy cell leukemia Follicular lymphoma Splenic marginal zone lymphoma

Genetic Features Apoptosis Used to be felt to be the major cause BCL2 overexpression Cell birth rates > 0.35%/day More likely progress, need treatment

Cytogenetics 5 categories of karyotypes (median survival) 17 p deletions (32 months) 11 q deletions (79 months) 12q trisomy (114 months) Normal (111 months) 13 q deletions (133 months)

Cytogenetics Abnormalities occur over time Karyotopic evolution – 38% over 5 years Associated with more aggressive disease

Cytogenetics 17 p deletions = 10-14% P53 present on 17 p Normally helps trigger apoptosis Poor prognosis More aggressive Less responsive

Micro RNA (miRNA) Mutations are common miR-21, miR nearly 100% CLL Upregulate BCL-2 P53 might regulate miRNA expression

Staging Systems RAI vs. BINET RAI l O – lymphocytosis l I – lymphadenopathy l II – hepatosplenomegaly l III – anemia l IV - thrombocytopenia BINET l A - lymphocytosis, < 3 LN groups, nl Hgb and platelets l B - > 3 LN groups l C - anemia, thrombocytopenia

Prognosis by Risk Groups RISKRAIBINETSURVIVAL LOWO, I A SAME MED. II B 84 mos HIGHIII, IV C 24 mos

Prognosis RAI Risk Group Median Survival 0 L 150 mos Low I L 101 mos Low II I 71 mos Int III H 19mos High IV H 19mos High

Prognosis Important distinction in the source of cytopenias Secondary to immune dysfunction = 7.2 years Secondary to marrow infiltration/failure = 2.4 years

Prognosis Genetic Abnormalities Risk % Abn High 27 IP53 BIRC3 Int 39 Notch 1 SF3B1 Low +12 or nl Very Low del 13q14 O.S. 5 years10 years * * same as age matched population

Prognosis Others Vitamin D Low Poorer Il-8 High Independent poor CD-20 High Independent poor Free Light Chains Abnormal Poor TPO High Independent MicRNA Low Poor

Prognostic Factors - Other Lymphocyte Doubling time # months to double the lymphocyte count < 12 months – aggressive > 12 months - indolent

B 2 microglobulin Regulated by exogenous cytokines - ?IL-6 Renal clearance Increased level – poor prognosis

IgVH Mutated – better – 50% Unmutated – worse – 50% May relate to developmental stage of B cell Not useful, difficult technically

CD38 Present in serum Correlates with IgVH mutation status Higher – poor prognosis

IG V GENE MUTATIONS AND CD38: NEWER PROGNOSTIC FACTORS IN CLL Damle et al. Blood. 1999;94: Ig V CD38 Years From Diagnosis Percent Surviving Unmutated Mutated  30% <30%

SURVIVAL CLL BY CD38

ZAP 70 Tyrosine kinase Not normally expressed in B cells Strong association with unmutated IgVH

Others Vitamin D levels VEGFR-2 receptors TNF Il-8 CD20 TPO miRNA

Treatment of CLL Decision to treat Evaluate prognostic factors Period of observation Symptoms

Treatment Chemotherapy Fludarabine Fludarabine + Cytoxan Fludarabine + Cytoxan + Rituxan

Antibodies 1. Alemtuzumab (Campath) Anti-CD52 Wipes out B cells for awhile Increased risk of infection 2. Rituximab Anti-CD20

On RxPost Rx

Others Bendamustine Chlorambucil Pentostatin 2-CDA Ibrutinib

NEW INITIATIVES IN CLL BCL2 Antisense Proteosome Inhibitor - Velcade Combination Rituxan + Campath Clofarabine (Adenosine Analog) Nelarabine (Guanosine Analog) 2 Methoxy-estradiol: (SOD Inhibitor)

Complications of CLL Infections – 50% of CLL deaths Fungi: Candida, Aspergillus, Crypto Bacteria: Listeria, Mycobacteria Viruses: CMV, Zoster, Simplex

Infection - Treatment Anti-microbials - ? Prophylaxis IVIG Growth factors – G-CSF

Cytopenias Anemia AIHA Marrow suppression/infiltration PRCA GI blood loss

Second Cancers Richter’s transformation AML MDS ? Solid tumors

Infection – Greater Risk Hypogammaglobulinemia Purine analogs Anti CD52 (Campath), Anti CD20 (Rituxan) Advanced stage diseases

Disease related complications B symptoms Symptomatic anemia / thrombocytopenia AIHA, ITP Infectious complications

Transformation Richters  DLCL Prolymphocytic Hodgkin’s Multiple Myeloma AML Frequency % – rare

THE END Thank you!