Chronic Lymphocytic Leukemia - University of Utah Mutation Analysis of CLL • Point mutations in...
Transcript of Chronic Lymphocytic Leukemia - University of Utah Mutation Analysis of CLL • Point mutations in...
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 T-cells (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
NNNNNN NNNN CDR1 CDR2 CDR3
J D V
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 T-cells 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
CLL cells Isotypic Control
27% ZAP 70+ 77% ZAP 70+
ZAP-70 test at ARUP using an optimized isotope control yields a bimodal distribution
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Clin. Cytom. 2012,82b:78-84
ZAP-70 test at ARUP also correlates well with IgVH mutational status
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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
Haematologica 2007; 92:1242-24
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 (4 ) – del 6q21 (3) – del 11q23 (2) – +8q 24 (1)
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)
N Engl J Med 2008;359:575
Most CLL-type MBL cases have mutated IgVH genes and good prognosis cytogenetics
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 – <5K ly/ul
• MBL – <5K monoclonal B lymphs/ul, no LAD – 1-2%/yr progress to CLL
IWCLL. Blood. 2008;111:5446-5456)
CLL: Staging
• Rai – 0 = Lymphocytosis only – 1 = Lymphocytosis + LAD – 2 = Hepatosplenomegaly – 3 =plts < 100K/ul – 4 = Hgb< 11 gm/dl
• Modified Rai – Low Risk = Lymphocytosis only – Intermediate Risk = LAD and/or HSM – High Risk = Hgb < 11gm/dl and/or plts <100K/ul
• Binet – A = < 2 involved nodal areas and Hgb >10gm/dl and plts
>100K/ul – B = neither A nor C – C = Hgb < 10gm/dl or plts <100K/ul
Clinical Staging
Rai Stage
Rai et al, Blood 1975
Case #1
• Oct 1997: 61 yo woman inc WBC noted at health fair – WBC 27K – PE: Small cervical, axillary LNS <2cm, no HSM
• Flow cytometry (bone marrow): – monoclonal kappa restricted B cells,
CD5/CD19/CD20(dim)/CD23+ – Bone marrow diffusely infiltrated
• =Rai Stage 1 CLL/SLL
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Diagnosis Splenectomy Spleen tip
Case #2
• Sept 2008: 41 yo man with DM, HTN, hyperlipidemia went to PCP with rash. – WBC 27K – PE: No LAD, no HSM
• Flow cytometry (blood): – 53% cells monoclonal lambda restricted B cells,
CD5/CD19/CD20(dim)/CD23+; CD38 neg
• =Rai Stage 0 CLL/SLL
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Diagnosis FCR XRT Ofa AlloBMT
12 mo 24 mo 36 mo Died 42 mo
Cancer 1999;86:2684–92. OS 48.2 % at 5 yrs; 22.5 % at 10 yrs. Of those who die, 69% die of CLL
Overall Prognosis
CLL: Staging Prognosis factors
• 20th Century – Stage – Age – Pattern of bone marrow involvement – Lymphocyte doubling time
• 21st Century – Stage, age – “Biologic” prognostic markers
• Specific chromosomal abnormalities – Del13q, tri12, del11q, del17p
• IgVH somatic mutation • CD38, ZAP70
Cancer 1999;86:2684–92.
Survival relative to unaffected population in same age group. Most patients are dying of CLL.
Effect of Age
Cytogenetic abnormalities
Dohner NEJM 2000;343:1910-6.)
Cytogenetic abnormalities
Goorha. Genet Med 2004:6(1):48–53.
FISH Result Utah study Dohner et al Others
13 q Del 52% 55% 51-64%
Tri 12 23% 16% 10-25%
ATM del 7% 18% 11-25%
P53 del 2.3% 7% 3-8%
Normal 25% 18% 19-23%
Total abnormalities 75% 82% 77-81%
Cytogenetic abnormalities
Figure 1. Probability of Survival from the Date of Diagnosis among the Patients in the Five Genetic Categories. Median OS for 17p deletion = 32 mos, 11q deletion = 79 mos, 12q trisomy = 114 mos, normal karyotype = 111 mos, and 13q deletion = 133 mos.
Dohner NEJM 2000;343:1910-6.)
Somatic Mutation of IgVH
Hamblin,T.Blood(1999)94:1848-1854
Unmutated: 8.4 yrs
Mutated: >25 yrs
CLL Prognostic markers: CD38 and ZAP70
ZAP 70 Neg 24.4 yrs
ZAP 70 Pos 9.3 yrs
CD38 Neg 24 yrs CD38 Pos
13.6 yrs
Case #1
• Oct 1997: 61 yo woman inc WBC noted at health fair – WBC 27K – PE: Small cervical, axillary LNS <2cm, no HSM
• Flow cytometry (bone marrow): – monoclonal kappa restricted B cells,
CD5/CD19/CD20(dim)/CD23+ – Bone marrow diffusely infiltrated
• =Rai Stage 1 CLL/SLL
• Oct 1997: 61 yo woman inc WBC noted at health fair – WBC 27K – PE: Small cervical, axillary LNS <2cm, no HSM
• Flow cytometry (bone marrow): – monoclonal kappa restricted B cells,
CD5/CD19/CD20(dim)/CD23+ – Bone marrow diffusely infiltrated
• =Rai Stage 1 CLL/SLL
Case #1
101 months
Case #2
• Sept 2008: 41 yo man with DM, HTN, hyperlipidemia went to PCP with rash. – WBC 27K – PE: No LAD, no HSM
• Flow cytometry (blood): – 53% cells monoclonal lambda restricted B cells,
CD5/CD19/CD20(dim)/CD23+; CD38 neg • =Rai Stage 0 CLL/SLL
– Cytogenetics: 46 XY(20) – CLL FISH panel: 17p13.1 del (250/500); trisomy 12
(15/500) – Zap 70 + by flow – IgVH mutation: Not available
Case #2
• Sept 2008: 41 yo man with DM, HTN, hyperlipidemia went to PCP with rash. – WBC 27K – PE: No LAD, no HSM
• Flow cytometry (blood): – 53% cells monoclonal lambda restricted B cells,
CD5/CD19/CD20(dim)/CD23+; CD38 neg • =Rai Stage 0 CLL/SLL
– Cytogenetics: 46 XY(20) – CLL FISH panel: 17p13.1 del (250/500); trisomy 12
(15/500) – Zap 70 + by flow – IgVH mutation: Not available
>150 months
>150 months
>150 months
32 months
112 months
• Incurable with standard approaches, but highly treatable.
• No randomized trial showing overall survival benefit for early treatment
• Patients can remain asymptomatic for years • Goal of treatment is palliation of symptoms,
avoidance of complications. • Quality of response is correlated with
response duration but not OS
Treatment
Triggers for Treatment
• Symptoms clearly referable to CLL • Progressive disease
– Worsening cytopenias (not autoimmune) • Plts consistently <100K/ul • Hgb < 10 gm/dl
– Bulky adenopathy • >7cm or symptomatic • Symptomatic splenomegaly
• Elevated WBC alone is not indication for treatment.
• Poor risk biologic predictors alone are not indication for treatment.
Available Treatments • Chemotherapy
– Alklyator: Chlorambucil, cyclophosphamide – Purine analogs: Fludarabine (FAMP), Pentostatin – Bendamustine
• Immunotherapy – Rituximab (chimeric antiCD20 mAB) – Ofatumumab (2nd gen humanized antiCD20 Ab) – Alemtuzumab (humanized antiCD52 Ab)
• Chemoimmunotherapy • Corticosteroids—high dose MP • IMiDs--Lenalidomide • Allogeneic bone marrow transplant • Splenectomy • Radiotherapy
• Patient related factors: – Age; Co-morbidities; preferences
• Clinical disease factors – Bulky vs non-bulky; Degree of cytopenias – Duration/quality of response to prior regimen
• Biologic predictors – Response prediction? – Duration of response prediction?
Risk adapted initial therapy
• Other – ATM del
• Better response to alkylator – p53 del
• HD corticosteroids + Rituximab • Alemtuzumab overcomes resistance—limited by bulky disease
• Biologic predictors of poor duration of response – IgVH unmutated – p53 del
• AlloBMT (“reduced intensity”) with Graft vs Leukemia effect can overcome negative prognostic factors – EFS @ 4 yrs 42%; no impact of neg prog factors seen – Non-relapse related mortality @ 4 yrs = 23%
Risk adapted therapy
P53 Del ATM del 13qDel Tri12, nl HD Rituximab
Effective? NO YES YES NO
Biologic predictors of chemoresistance
CD38
• All CLL is not the same – Probably two main biologic subsets reflected by
IgVH mutation status. – Clinical behavior further determined by genetic
abnormalities impacting proliferation, apoptosis, and drug resistance.
• Better understanding of biology is giving clues to divergent clinical behavior and uncovering novel targets for treatment.
Day -4: PC
Day 0,1,2: 1.42E7 transduced T cells
Day -1: BM w/40% CLL cells, p53del Day 14: Fevers, diarrhea etc
Day 22: TLS Day 23: neg BM
Day 28: no LAD
Disappearance of CLL cells from blood
Expansion and Persistence of Chimeric Antigen Receptor T Cells In Vivo.