CHRONIC LYMPHOCYTIC

LEUKEMIA (CLL)
Curs an IV – limba engleza
2012-2013
 CLL - Definition
• CLL is a neoplastic disease characterized by
proliferation and accumulation (blood, marrow
and lymphoid organs) of morphologically
mature but immunologically dysfunctional
lymphocytes

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 CLL - Definition
• Clonal B cell malignancy.
• Progressive accumulation of long lived
mature lymphocytes.
• Increase in anti-apoptotic protein bcl-2.
• In most cases, the cells are monoclonal B
lymphocytes that are CD5+
• Intermediate stage between pre-B and mature
B-cell.
• T cell CLL can occur rarely
 CLL - Epidemiology
• Most common leukemia of Western world.
• Less frequent in Asia and Latin America.
• Male to female ratio is 2:1.
• Median age at diagnosis is 65-70 years.
• Uncommon (10%) in patients under 50 years
• In US population incidence is similar in
different races.

Cancer statastics 2000; CA J Clin 2000; 50:7-33

 CLL - Etiology (1)

• The cause of CLL is unknown

• There is increased incidence in farmers, rubber

manufacturing workers, asbestos workers, and
tire repair workers

• Genetic factors have been postulated to play a

role in high incidence of CLL in some families
 CLL - Etiology (2)
• Cytogenetics
– clonal chromosomal abnormalities are detected in
approximately 50% of CLL patients
– the most common clonal abnormalities are:
• trisomy 12
• structural abnormalities of chromosomes 13, 14 and 11
– patients with abnormal karyotypes have a worse
prognosis
• Oncogenes
– in most cases of CLL is overexpressed the proto-
oncogene c-fgr 9a member of the src gene family of
tyrosine kinases
 CLL – Initial symptoms
• Approximately 40% are asymptomatic at
diagnosis – discovered by a CBC
• In symptomatic cases the most common
complaint is fatigue
• Well’s syndrome – increase sensitivity to insects
bites
• B symptoms – fever, sweats, weight loss
• Less often the initial complaint are enlarged
nodes or the development of an infection
(bacterial)
 CLL - Clinical findings
• Most symptomatic patients have enlarged lymph nodes
(more commonly cervical and supraclavicular) and
splenomegaly
• The lymph nodes are usually discrete, freely movable,
and nontender
• Hepatomegaly may occure
• Less common manifestation are infiltration of tonsils,
mesenteric or retroperitoneal lymphadenopathy, and
skin infiltration
• Patients rarely present with features of anemia, and
bruising or bleeding
 CLL – Lab findings
a) Blood test lymphocytosis ≥ 5G/l (4 weeks)

b) Morphology monoconal population of small mature

lymphocyte

c) B-cell CLL phenotype clonal CD5+/CD19+ population

of lymphocyte

d) Markers of clonality κ/λ light chain restriction; cytogenetical

abnormalities

e) Bone marrow infiltrate > 30% of nuceated cells on aspirate

f) Lymph node diffuse infiltrate of small lymphocye

 CLL - Laboratory findings (1)
• The blood lymphocyte count above 5.000/mmc

• Leukemic cells have the morphologic appearance of

normal small lymphocytes
• In the blood smears are commonly seen ruptured
lymphocytes (“basket” or “smudge” cells)
• Careful examination of the blood smear can usually
differentiate CLL, and the diagnosis can be confirmed by
immunophenotyping
 CLL - Peripheral Blood
• Absolute lymphocytosis
• Lymphs = B cells
– Thin cytoplasm
– Dense nucleus
– Partially aggregated
chromatin
– No recognizable
nucleoli
• Smudge cells

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 CLL - Laboratory findings (2)

• Clonal expansion of B (99%) or T(1%)

lymphocyte
– In B-cell CLL clonality is confirmed by
• the expression of either  or  light chains on the cell surface
membrane
• the presence of unique idiotypic specificities on the
immunoglobulins produced by CLL cells
• by immunoglobulin gene rearrangements
• typical B-cell CLL are unique in being CD19+ and CD5+
 CLL - Immunophenotype
• Detect antigens on surface of cells
– Specific antibodies
– Use flow cytometry or immunohistochemistry
• CLL = mature B cells
– CD5
– CD19
– CD20 - low
– CD22 - low
– CD23
– Light chains (κ, λ)
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 CLL - Immunophenotype scoring
system
Scoring system for B-CLL

Points

Membrane 1 0
marker
Smlg Weak Moderate/strong

CD5 Positive Negative

CD23 Positive Negative

FMC7 Negative Positive

CD79b (SN8) Negative Positive

1. Matutes E, et al. Leukemia. 1994;8:1640-1645.

2. Moreau EJ, et al. Am J Clin Pathol. 1997;108:378-382.
 CLL – Other lab tests
• Hypogammaglobulinemia or agamma-
globulinemia are often observed
• 10 - 25% of patients with CLL develop
autoimmune hemolytic anemia, with a positive
direct Coombs’ test
• The marrow aspirates shows greater than 30%
of the nucleated cells as being lymphoid

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 CLL - Bone Marrow
• Infiltrates of small lymphocytes
– Causes neutropenia, anemia,
thrombocytopenia
• Extramedullary expansion
– Splenomegaly
– Hepatomegaly
– Lymphadenopathy

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 CLL – Lymph node
• Loss of nodal architecture
• Diffuse infiltration of small
lymphocytes
• Lymphadenopathy

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 CLL - diagnostic criteria
1) A peripheral blood lymphocyte count of greater
than 5 G/L, with less than 55% of the cells being
atypical

2) The cell should have the presence of Bcell-specific

differentiation antigens (CD19, CD20, and CD24)
and be CD5(+)

3) A bone marrow aspirates showing greater than

30% lymphocytes
 CLL - Differential diagnosis
• Infectious causes
– bacterial (tuberculosis)
– viral (mononucleosis)
• Malignant causes
– B-cell
– T-cell
• leukemic phase of non-Hodgkin lymphomas
• Hairy-cell leukemia
• Waldenstrom macroglobulinemia
• large granular lymphocytic leukemia
 Investigations
• Pretreatment studies of patients with CLL should
include examination of:
– complete blood count
– peripheral blood smear
– reticulocyte count
– Coomb’s test
– renal and liver function tests
– serum protein electrophoresis
– immunoglobulin levels
– plasma 2 microglobulin level
• If available immunophenotyping should be carried
out to confirm the diagnosis
• Bone marrow biopsy and cytogenetic analysis is not
routinely performed in CLL
 CLL – Rai staging system

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CLL – Rai stages
 CLL – Binet staging system

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CLL – Binet stages
 Staging: Rai and Binet staging systems for CLL

Clinical staging systems for CLL

Stage
Value Rai Binet Median survival
Lymphocytosis 150 months
0 -
(>15,000/mm3) (12.5 years)
Lymphocytosis plus <3 101-108 months
I A
nodal involvement node groups (8.5-9 years)
Lymphocytosis plus >3 60-71 months
II B
organomegaly node groups (5-6 years)
Hgb <10 g/dL
III 19-24 months
Anemia (RBCs)
Hgb <11 g/dL (1.5-2 years)
C
Lymphocytosis plus
IV
thrombocytopenia PLT <100,000/mm3
PLT <100,000/mm3
(platelets)
CLL - Markers of poor prognosis
• Advanced Rai or Binet stage
• Peripheral lymphocyte doubling time <12
months
• Diffuse marrow histology
• Increased number of prolymphocytes or cleaved
cells
• Poor response to chemotherapy
• High 2- microglobulin level
• Abnormal karyotyping
 Prognosis: histologic bone
marrow patterns
Nodular Interstitial Diffuse
(low risk) (low risk) (high risk)

• The different bone marrow patterns probably reflect

variations in amount of lymphoid accumulation during
the natural course of the disease

Randy Gascoyne, MD., Montserrat E, et al. Cancer. 1984;54:447-451.

 Prognosis: lymphocyte doubling
time
Survival time according to LDT (all stages)
1.0
0.9
0.8
Doubling time ≤12 months
Probability of survival

0.7
Doubling time >12 months
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 20 40 60 80 100 120 140 160

Months

1. Montserrat E, et al. Br J Haematol. 1986;62:567-575.

 CLL - Genetic abnormalities
Median
Genetic Incidence survival
abnormality (%) (months) Clinical correlation
13q14 55-62 133-292 Typical morphology
Mutated VH genes
Stable disease
+ 12 16-30 114-122 Atypical morphology
Progressive disease
del 11q23 18 79-117 Bulky lymphadenopathy
Unmutated VH genes
Progressive disease
Early relapse
post autograft
p53 7 32-47 Atypical morphology
loss/mutation Unmutated VH genes
Advanced disease
Drug resistance

1. DÖhner H, et al. N Engl J Med. 2000;343:1910-1916.

2. Oscier DG, et al. Blood. 2002;100:1177-1184.
 Effect of genetic abnormalities
on survival

1. Döhner H, et al. N Engl J Med. 2000;343:1910-1916.

 CLL - VH Status
• “Variable” region of immunoglobulin gene
• H=heavy chain

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 CLL - VH Mutation
• Naïve B cells have unmutated V regions
– Make initial IgM and IgG that bind Ag poorly
• Somatic hypermutation of V region creates memory
B cells
– Make Ab with higher affinity for Ag

CLL progression is dependent on mutation status of

B cells
– Unmutated VH = rapid progression, short survival
– Mutated VH = slow progression, long survival

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 CLL - VH Mutation

Naïve B cells Memory B cells

Unmutated VH Mutated VH

CLL CLL
Fast progression Slow Progression
Short survival Long Survival
 Prognosis: effect of VH gene
mutations on survival
100 Unmutated VH gene
90 Median = 117 months
Mutated VH gene
Percent surviving (%)

80
Median = 293 months
70
60
50
40
30
20
10
0
0 25 50 75 100 125 150 175 200 225 250 275 300 325

Months

1. Hamblin TJ, et al. Blood. 1999;94:1848-1854.

 Clinical Course of CLL
• Disease has a variable course; however, it often progresses
from an indolent lymphocytosis without other evident
disease to one of generalized lymphatic enlargement with
concomitant pancytopenia

• Progression: bone marrow impairment,  susceptibility to

infection

• Complications of pancytopenia, including hemorrhage and

infection, represent a major cause of death in these patients
CLL - Immunologic Complications
• Autoimmune hemolytic anemia
– Coombs’ positive
– Clinical hemolysis
• Pure red cell aplasia
• Immune-mediated thrombocytopenia
• Depressed immunoglobin levels
• Granulocytopenia
• Hypogammaglobulinemia  with advanced disease
• Long-term complications: autoimmune phenomena,
Richter’s transformation
 CLL - Complications
• Severe systemic infections
• Bleeding
• Richter’s transformation
• Prolymphocytoid transformation
• Secondary malignancies
• Acute myeloid leukemia
Transforming the way we approach
CLL therapy
• Traditional treatment goal: Palliation
– Age is a factor
– Treat to relieve symptoms
– Continuous/intermittent treatment
– No survival advantage for any tested treatment to date
• New advances may allow treatment for remission
and survival
– Treat to complete remission (CR)
– Eliminate minimal residual disease
– Response predicts for survival: More CRs
 CLL – treatment (1)
• Watch and wait
• Monotherapy
– glucocorticoids
– alkylating agents (Chlorambucil, Cyclophosphamide)
– purine analogues (Fludarabine, Cladribine, Pentostatin)
• Combination chemotherapy
– Chlorambucil/ Cyclophosphamide + Prednisone
– Fludarabine + Cyclophosphamide +/- Mitoxantrone
– CVP, CHOP
• Monoclonal antibodies (monotherapy and in combination)
– Alemtuzumab (anti-CD52)
– Rituximab (anti-CD20)
• Splenectomy
• Radiotherapy
 CLL – treatment (2)
• Hematopoietic stem cell transplantation
– allogeneic with reduced intesity conditioning
– autologous
• New and novel agents
– Oblimersen – bcl2-directed antisense oligonucleotide
– Lenalidomide
– Flavopiridol
– Anti-CD23
– Anti-CD40
• Vaccine strategies
• Supportive therapy (allopurinol, G-CSF, blood and platelet transfusion,
immunoglobulins, antibiotics)
 NCI-WG: Indications to Initiate
Treatment for CLL
• Constitutional symptoms referable to CLL (B-
symptoms)
• Progressive marrow failure
• Autoimmune anemia and/or thrombocytopenia
poorly
responsive to corticosteroids
• Massive or progressive splenomegaly
• Massive or progressive lymphadenopathy
• Rapid lymphocyte doubling time
 MoAbs citotoxic mechanisms

Effector cells/ Apoptosis Radionuclide Toxin/Antibiotic

Complement
 MoAbs for CLL
Antibody Antigen

Alemtuzumab (Campath-1H) CD52

Rituximab (Rituxan, Mabthera) CD20
Epratuzumab (LymphoCide) CD22
Hu-1D10 (Apolizumab) HLA-DR
IDEC-152 (Lumiliximab) CD23
IDEC-114 CD80
Bevacizumab (Avastin) VEGF
BL-22 CD22( conjugate
with Pseudomonas)
MabThera®: a chimeric murine/human
MoAb

Variable murine regions bounding

CD20 on B cells

Human kappa costant regions

Human domain IgG1 Fc, synergistic

with human effector mechanisms

Chimeric IgG 1
 Rituximab as part of first-line
therapy for CLL: Rationale
• Rituximab monotherapy is moderately active in CLL1,2
– Activity is dose dependent (between 500–2250 mg/m2)1

• Rituximab acts synergistically with other cytotoxic

agents in vitro
– Increases fludarabine activity in NHL cell lines3
– Increases activity of bendamustine, mitoxantrone and other
chemotherapeutic agents in CLL cells4

• Rituximab combination therapies (e.g. R-F, R-FC, R-PC, R-FCM,

R-Bendamustine, R-Chlorambucil) are now being assessed
Alemtuzumab (anti-CD52) antibody
IgG1 humanised antibody:
Low immunogenicity

 CD52 antigen:
 Highly expressed on
 all lymphocytes
 monocytes and macrophages
 spermatozoa
 eosinophils
 Not expressed on haemopoietic stem
cells
 Does not modulate/shed
 Also expressed on the majority of
malignant lymphocytes
Treatment of CLL
 CLL – Treatment strategy

Gribben J G Blood 2010;115:187-197