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 –