Chronic Lymphocytic Leukemia - University of Utah Mutation Analysis of CLL • Point mutations in heavy chain variable gene segments used by CLL clone are quantified • Separates

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

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

% o

f ZAP

-70

posi

tive

CLL

cel

ls

Samples

Isotype cutoff

Normal B-cell cutoff

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

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

0

20

40

60

80

100

120

0 20 40 60 80 100 120

B-c

ell C

utof

f (%

of Z

AP-7

0 po

sitiv

e ce

lls)

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

Mutated

Unmutated

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

0

100

200

300

400

500

600

700

2/1/

1998

7/1/

1998

12/1

/199

8

5/1/

1999

10/1

/199

9

3/1/

2000

8/1/

2000

1/1/

2001

6/1/

2001

11/1

/200

1

4/1/

2002

9/1/

2002

2/1/

2003

7/1/

2003

12/1

/200

3

5/1/

2004

10/1

/200

4

3/1/

2005

8/1/

2005

1/1/

2006

6/1/

2006

11/1

/200

6

4/1/

2007

9/1/

2007

2/1/

2008

7/1/

2008

12/1

/200

8

5/1/

2009

10/1

/200

9

3/1/

2010

8/1/

2010

1/1/

2011

6/1/

2011

11/1

/201

1

WBC

Hgb

Plts

10 yr 5 yr 1 yr 14 yr

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


0

20

40

60

80

100

120

140

160

180

2009/

1/20

0810

/1/2

008

11/1

/200

812

/1/2

008

1/1/

2009

2/1/

2009

3/1/

2009

4/1/

2009

5/1/

2009

6/1/

2009

7/1/

2009

8/1/

2009

9/1/

2009

10/1

/200

911

/1/2

009

12/1

/200

91/

1/20

102/

1/20

103/

1/20

104/

1/20

105/

1/20

106/

1/20

107/

1/20

108/

1/20

109/

1/20

1010

/1/2

010

11/1

/201

012

/1/2

010

1/1/

2011

2/1/

2011

3/1/

2011

4/1/

2011

5/1/

2011

6/1/

2011

7/1/

2011

8/1/

2011

9/1/

2011

10/1

/201

111

/1/2

011

WBC

HGB

PLTS

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.)


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%


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









Case #1

101 months

Case #2



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



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.

Documents

Chronic Lymphocytic Leukemia - University of Utah Mutation Analysis of CLL • Point mutations in heavy chain variable gene segments used by CLL clone are quantified • Separates