Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia David W. Bahler MD PhD Martha J. Glenn MD March 16, 2012 Talk Outline • David Bahler – Background on CLL – Pathogenesis...
Author: Wesley Knight
0 downloads 2 Views 3MB Size
Chronic Lymphocytic Leukemia David W. Bahler MD PhD Martha J. Glenn MD March 16, 2012

Talk Outline • David Bahler – Background on CLL – Pathogenesis • Antigen receptor stimulation

– IgVH analysis and ZAP-70 tests

• Martha Glenn – Clinical work up of CLL patients – Treatment

Chronic Lymphocytic Leukemia (CLL) • Neoplasm of small mature B-cells • Most common leukemia affecting adults in North America and Europe – 30% of all leukemias

• Incidence : 4 cases / 100,000 people / year (10,000 new cases / year in US) • Median age at diagnosis: 65 years – Only 15% of patients under 50 years

• Male predominance (M:F = 2)

CLL Morphology (small mature appearing lymphocytes)

Diagnosis of CLL • Requires 5000 or more CLL-phenotype cells /µl of peripheral blood (2008 IWCLL guidelines) – CD5+, CD19+, weak CD20+, CD23+, weak monotypic light chain expression

• Fewer than 5000 CLL-phenotype cells / µl in asymptomatic patients is termed CLL-like monoclonal B-cell lymphocyotsis (MBL) – Typically benign expansions that occasionally progress to CLL

Clinical Course of CLL • Most patients are asymptomatic at diagnosis – Picked up by routine screening tests

• Highly variable clinical behavior – Interest in prognostic markers

• Patients are not treated unless symptomatic – Early treatment doesn’t improve survival and can generate drug resistance

B-cell Antigen Receptor (BCR) • Key molecule for normal B-cell differentiation, survival, and proliferation • Also important for the development of B-cell neoplasms, especially the more indolent less transformed types

Immunoglobulin (antigen binding BCR)

Immunoglobulin Heavy Chain Gene Rearrangement

Immunoglobulin Variable Gene Somatic Hypermutation • Mechanism in normal B-cells to generate antibodies with high binding affinities • Mostly occurs in lymph node germinal centers – Point mutations are generated in both heavy and light chain variable genes – B-cells that express higher affinity Ig variants are then selected for clonal expansion through interactions with antigen presenting cells and Tcells (affinity maturation)

Mutational status of CLL IgVH predicts survival • Median survival – Mutated: 25 years – Unmutated: 8 years

• References – Damle et al, Blood 1999, 94:1840-47 – Hamblin et al, Blood 1999, 94: 1848-54

IgVH Mutation Analysis of CLL • Point mutations in heavy chain variable gene segments used by CLL clone are quantified • Separates CLL into two similar size groups with different clinical behaviors – Mutated IgVH: good prognosis, Unmutated IgVH: poor prognosis – Typical cut off is 98% or more homology to germline IgVH segments = Unmutated – No mixing between mutated and non-mutated types – Suggestive of different cells of origin

Why is CLL prognosis related to IgVH mutation status ? • Antigen receptor stimulation is important in the development and progression CLL – Biased or non-random use of IGHV segments • Different among mutated and unmutated groups

– More than 20% of cases express remarkably similar IGHV genes (V-D-J) • Virtually identical in 1-2% of cases • More evident among IGHV unmutated cases • Recognize the same antigens

IgVH Gene Complimentarity Determining Regions • Three areas that encode the traditional antigen binding site – Also called hypervariable regions

• Two from VH gene segment (CDR1 & CDR2) • CDR3 from N nucleotides, D and part of the J segment – Most variable part of VH gene (clonal marker) – Key determinant of antibody specificity CDR1

CDR3

CDR2

V

NNNNNN

D

NNNN

J

Biased CLL VH Segment Use Related to Mutation Status

Mauerer et al, BJH 2005, 129:499-510

Identical and Similar VH CDR3s Used by Unmutated VH1-69 Cases

Mauerer et al, BJH 2005, 129:499-510

Identical and Similar VH and VL CDR3s Used by VH3-21 expressing CLL Cases

Blood 2006, 107:2889-94

CLL cases using VH3-21 have poor prognosis cases regardless of mutation status

Blood 2008; 111:5101-08

ARUP IgVH Analysis Test • RNA is reversed transcribed and amplified with VH family specific leader and JH primers – Also use a VH3-21 gene specific leader primer

• Products are sequenced and clones compared to a database of germline VH, DH, and JH gene segment sequences • Results include the VH gene segment used and % homology to the germline counterpart J. Mol. Diagn. 2010, 12:244-49

Electrophoresis of IgVH PCR products from CLL cases using VH gene segments from different families

Gene Expression Array Analysis of Mutated and Unmutated CLL (J Exp Med 2001;194:1625 &1639)

• Mutated and unmutated CLL share a common signature – Distinct from other mature B-cell neoplasms – Most similar to normal memory B-cells

• A limited number of genes can distinguish mutated from unmutated CLL – ZAP-70 best discriminator

ZAP-70 (zeta associated protein of 70,000 KD) • Expression was initially thought to be restricted to Tcells prior to CLL expression array studies – Critical for T-cell antigen receptor signaling – Member of the Syk tyrosine kinase family

• Typically expressed in CLL with unmutated IGHV – Enhances antigen receptor signaling

• Proposed as a surrogate of IGHV mutation status – Protein detection usually easier than sequencing – Some degree of discordance is typical

Clinical Tests for ZAP-70 • Typically flow cytometry based – Separately analyze T-cells and CLL cells • Built in positive (and negative) controls

– Not standardized and typical weak staining can be difficult to interpret • Choice of negative control is critical

– Results are usually not validated with VH mutational status or clinical outcome • Continuous distributions of ZAP-70 complicate separation into good and poor prognostic groups

ZAP-70 flow cytometry test at ARUP • Lymphocytes are stained for CD5, CD19, and ZAP-70 • An optimized isotypic control is used to set the negative threshold – Normal B-cells appear ZAP-70 negative as expected

Normal B-cell and optimized isotypic controls can yield different ZAP-70 results Normal B-cells ZAP-70 stained

27% ZAP 70+

CLL cells Isotypic Control

77% ZAP 70+

ZAP-70 test at ARUP using an optimized isotope control yields a bimodal distribution 100

% of ZAP-70 positive CLL cells

90 80

Isotype cutoff Normal B-cell cutoff

70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

Samples

Clin. Cytom. 2012,82b:78-84

ZAP-70 test at ARUP also correlates well with IgVH mutational status

B-cell Cutoff (% of ZAP-70 positive cells)

120

100

Mutated Unmutated

80

60

40

20

0 0

20

40

60

80

100

Isotype Antibody Cutoff (% of ZAP-70 positive cells)

120

IgVH vs ZAP-70 testing • VH more objective and less subject to laboratory variations/methodologies • Surrogates for VH mutation status are not required- sequencing is not difficult/expensive • VH testing identifies the VH segment used which may have clinical significance • ZAP-70 results may provide independent prognostic information

Cytogenetics of CLL • FISH – 80% of cases abnormal • • • •

13q14 deletion (55%) miR-15a/mir16-1 11q22 deletion (18%) ATM 12 trisomy (16%) 17p13 deletion (7%) P53

• Conventional – 20-40% of cases abnormal – 14q32 translocations uncommon (< 5%)

17p or 11q Deletions Can Trump VH mutational Status

Krober et al, Blood 2002;100:1410

Unmutated CLL is More Likely to Have Deletions of 17p or 11q

Krober et al, Blood 2002;100:1410

Clonal Evolution Occurs Primarily in Unmutated CLL – Median observation time • 42.5 months

– Clonal evolution • Only in unmutated cases • 11 of 64 patients (11%) – – – –

del 17p del 6q21 del 11q23 +8q 24

(4 ) (3) (2) (1)

Haematologica 2007; 92:1242-24

Monoclonal B-cell Lymphocyotsis (2008 IWCLL guidelines)

• Less than 5000 monotypic B-cells /µl – Most cases have CLL phenotypes

• No history or symptoms of a B-cell neoplasm • Common and increases with age – < 40 years 0.3%, > 40 years 3.5%

• Precede all cases of CLL (NEJM 2009 360:659-67) • Only occasionally evolves into CLL – 1.1% of cases/yr. with > 4000 lymph /µl , median follow up 6.7 yrs. (NEJM 2008;359:575)

Normal (red) and CLL phenotype MBL cells (blue)

Most CLL-type MBL cases have mutated IgVH genes and good prognosis cytogenetics

N Engl J Med 2008;359:575

The IgVH repertoire in low count MBL appears to differ from CLL

Blood 2009;114:26-32

Model for CLL Development

Klein and Della-Favera, SemCanBio 2010, 20:377-83

Overview of clinical topics in CLL • • • • •

Clinical aspects of CLL Clinical spectrum of disease Factors affecting prognosis Treatment New approaches

Clinical Presentation • Incidentally noted elevated lymphocyte count • Asymptomatic lymphadenopathy • Median age about 72 – 80% > 60 yrs old

• Males > females – 60:40

• European > African Amer > Asian/Pacific Islander

Diagnosis • Requires: – >5000 monoclonal B lymph/ul – CD5/19/23 positive – CD20/79a/sIg—dim – Cyclin D1 negative • SLL – LAD or splenomegaly –

Suggest Documents