1
Hematopathology
CHRONIC LYMPHOCYTIC LEUKEMIA
Prof. Pier Paolo Piccaluga
Department of Experimental, Diagnostic and Specialty Medicine, Bologna University
Department of Pathology JKUAT, Nairobi
2
CLL - Definition
• CLL is a clonal B cell malignancy characterizedby proliferation and accumulation (blood,marrow and lymphoid organs) ofmorphologically mature but immunologicallydysfunctional lymphocytes
Cancer statastics 2000; CA J Clin 2000; 50:7-33
CLL - Epidemiology
• Most common leukemia in 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 indifferent races.
CLL - Etiology
• The cause of CLL is unknown
• There is increased incidence in farmers, rubbermanufacturing workers, asbestos workers, and tirerepair workers
• Genetic factors have been postulated to play a role inhigh incidence of CLL in some families
• Antigen stimulation (BCR)
CLL – Initial symptoms
• Approximately 40% are asymptomatic atdiagnosis – discovered by a CBC
• In symptomatic cases the most commoncomplaint is fatigue
• Well’s syndrome – increase sensitivity to insectsbites
• B symptoms – fever, sweats, weight loss
• Less often the initial complaint are enlargednodes or the development of an infection(bacterial)
CLL - Clinical findings
• The lymph nodes are usually discrete, freely movable,
and nontender
• Splenomegaly is not rare
• Hepatomegaly may occurr
• Less common manifestation are infiltration of tonsils,
mesenteric or retroperitoneal lymphadenopathy, and
skin infiltration
CLL – Laboratory findings
Blood test lymphocytosis ≥ 5,000/MMC(4 weeks)
Morphology monoconal population of smallmature lymphocyte
B-cell CLL phenotype clonal CD5+/CD19+ populationof lymphocyte
Markers of clonality κ/λ light chain restriction; cytogenetic abnormalities
Bone marrow infiltrate > 30% of nuceated cells on aspirate
Lymph node diffuse infiltrate of small lymphocye
CLL - Peripheral Blood
• Absolute lymphocytosis
• Lymphocytes = B cells– Thin cytoplasm
– Dense nucleus
– Partially aggregated
chromatin
– No recognizable
nucleoli
• Smudge cells (Gumprecht's shadowcells)
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
– CD27
– Light chains (κ, λ) 9
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
10
CLL - Differential diagnosis
• Infectious causes of lymphocytosis
– bacterial (tuberculosis)
– viral (mononucleosis)
• Malignant causes
– B-cell
• leukemic phase of non-Hodgkin lymphomas
• Hairy-cell leukemia
• Waldenstrom macroglobulinemia
• Large granular lymphocytic leukemia
Rai and Binet staging systems for CLL
Rai and Binet staging systems for CLL
MORPHOLOGY
CLL – Bone Marrow aspirate
• Cytological examination:
– extensive replacement of
marrow element by mature
lymphocytes (more than
30%)
– Causes neutropenia,
anemia, thrombocytopenia
Small lymphocytes • Dense chromatin, • high nuclear/cytoplasmic ratio• Dark areas at light microscopy
Small/Medium size prolymphocytes• Dense chormatin, • conspicuous nuclolus
Large paraimmunoblasts• Open chromatin, • conspicuous nucleolus
Cytology
Bone marrow involvement
Interstitial
Nodular: more often paratrabecular
Diffuse (worse prognosis)Proliferation centers
In all tissues: pseudofollicular growth in 85% of cases
Three cell types: small Ly (Ly), prolymphocytes (PL) and paraimmunoblasts (PI)
Ly
Ly
Ly
PL
PI
Para-immunoblastic growth pattern
Spleen
• Red pulp effacement • White pulp expansion
Liver
PHENOTYPE
CD79a+ /CD19+
CD20+-: weak or negative in small Ly; stronger expression in PI and PL
sIgM or sIgM/IgD, rare IgG: low intensity (> differ. Pc)
CD79a CD20
PHENOTYPE
CD5+ (neg in 7-20%)
CD23+ more intense in prolympho/paraIb
LEF-1 expression in CLL
290 cases of B-cell lymphomas
100% positive BCLL/SLL(92 of 92, including two
CD5- cases)strongest staining in
Richter's sdNegative in all
MCL (53), low-grade FL (31), MZL (31)
12 FL grade 3: 5-15% poscells
DLBCL positive in 38%.
Tandon et al. Mod pathol 2011
Gutierrez A et al. Blood 2010LEF-1 is a prosurvival factor in chronic lymphocytic leukemia and is
also expressed in the preleukemic state of monoclonal B-cell lymphocytosis.
CD200 expression in CLL
HCL (%) HCL-v SMZL SDRPL SBCL B-CLL LPL/WM MCL
CD200 32/32 (100) 3/4 (75) 6/8 (75) 1/2 (50) 3/3 (100) 11/11 (100) 3/3 (100) 0/10 (0)
From Toth-Liptak J et al. Pathol Oncol Res 2014
Ki-67: PC Ki-67: Reactive GC
CD23 more intense
Proliferation center: variation of phenotype
CD20 more intense
Ki67 >
Survivin (anti-apoptotico)
IRF4
Prognosis
30
Prognostication
• Clinical heterogeneity:
– Rapidly progressive to severe/refractory CLL of poor prognosis
– Essentially indolent disease (life expectancy similar norm)
1) Binet Rai classifiation scheme: first staging system, based on clinical features, still widely maintained nowadays
2) Biomarkers: serum levels of β2-microglobulin and thymidine kinase, or expression of CD38 or CD49d and ZAP70
3) Genetic approach: Mutational status of IgVH genes
Prognosis: lymphocyte doubling time
Survival time according to LDT (all stages)
Months
Pro
bab
ility
of
surv
ival
1600 20 40 60 80 100 120 140
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Doubling time ≤12 months
Doubling time >12 months
Montserrat E, et al. Br J Haematol. 1986;62:567-575.
Proliferation center and prognosis
Ciccone et al, Leukemia 2012
Gaidano JCI 2012
suggests that M-CLL and U-CLL derive from
progenitors that are reminiscent of antigen-experienced B cells
Cell of origin and IGHV mutation status
1. Hamblin TJ, et al. Blood. 1999;94:1848-1854.
Prognosis: effect of VH gene mutations on survival
90
225 3000 50 100 150 200 25025 75 125 175 275
100
80
60
40
0
20
70
50
30
10
Unmutated VH geneMedian = 117 months
Mutated VH geneMedian = 293 months
325
Perc
ent
surv
ivin
g (%
)
Months
Recurrent genetic imbalances in CLL
Found in 8% cases
• Delete tumor suppressor TP53
• Many cases of CLL with del17p13
display inactivation of the second
TP53 allele by point mutation (in
70% deleted cases).
• A fraction (10%) display TP53 mutations
in the absence of Del17p13
• Such cases are not recognized by
FISH analysis, and their
identification requires mutation
analysis of the TP53 geneUnfavourable prognosis
Del17p13
Genetic imbalances and prognosis
• del 17p
• >3 chrom. abnormalities
• del 11q
High Risk
• Translocations 14q32
• Trisomy 12
• del 6q
• del 13q (isolated)
•Normal Karyotype
Intermediate risk
Low risk
Effect of genetic abnormalities on survival
1. Döhner H, et al. N Engl J Med. 2000;343:1910-1916.
Newly diagnosed CLL (N=637)
Fre
qu
ency
N=286(44.8%)
N=637
N=105(16.4%)
N=46(7.2%)
N=62(9.7%) N=54
(8.4%)
N=71(11.1%)
N=43(6.7%)
N=17(2.6%)
N=22(3.4%)
N=26(4.0%)
N=26(4.0%)
NOTCH1 M
SF3B1 M
BIRC3 M
del13q14
+12
TP53 M
del11q22-q23MYD88 M
BIRC3 deldel17p13 *
* **
Mutational complementation groups in newly diagnosed CLL
del13q14Normal/+12NOTCH1 M/SF3B1 M/del11q22-q23TP53 DIS/BIRC3 DIS
del13q14Normal/+12NOTCH1 M/SF3B1 M/del11q22-q23TP53 DIS/BIRC3 DIS
OS Treatment
Integrated mutational and cytogenetic model for CLL prognostication
N % 10-years OS155 26% 69%228 39% 57%99 17% 37%
101 17% 29%
p<0.0001 p<0.0001
N % Treated at 10 years155 26% 41%228 39% 50%99 17% 83%
101 17% 100%
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
Years from diagnosis
Cu
mu
lative
pro
ba
bility o
f O
S (
%)
del13q14
Normal/+12
NOTCH1 M/SF3B1 M/del11q22-q23
TP53 DIS/BIRC3 DIS
Matched general population
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
Years from diagnosis
Cu
mu
lative
pro
ba
bility o
f O
S (
%)
del13q14
Normal
+12
del17p13
Matched general population
del11q22-q23
NObserved
eventsExpected events*
5-year OS (%)10-year OS
(%)10-year relative
OS*p*
155 27 20.4 86.9 69.3 84.2% 0.1455228 53 30.9 77.6 57.3 70.7% <0.000199 41 10.4 65.9 37.1 48.5% <0.0001101 57 12.6 50.9 29.1 37.7% <0.0001
NObserved
eventsExpected events*
5-year OS (%)10-year OS
(%)10-year relative
OS*p*
194 44 24.9 83.9 53.3 68.1% <0.0001212 50 24.7 78.0 57.1 72.8% <0.000182 30 12.4 62.5 47.5 62.4% <0.000139 23 6.1 54.7 41.3 54.4% <0.000156 31 6.1 48.5 30.6 38.1% <0.0001
Inclusion of mutations in addition to FISH abnormalitiessignificantly improves the accuracy of CLL prognostication (20% of
normal Karyotype reclassified)
c-index=0.617 c-index=0.642
Therapeutic implications of genetic patterns
• Different sensitivity to CHT
– CHT may favor emergence of fludarabine
resistant clones
• Novel targets:
– NOTCH1 (gamma -secretase inhibitors)
– BIRC3 (NFkB inhibitors)
– BCR signaling (Bruton tyrosine kinase
inhibitor/ibrutinib; PI3K-δ inhibitors/GS-1101)
SINDROME DI RICHTER
LLCLGCBD
RS: CLL transformation into aggressive lymphoma (DLBCL)
Residual CLL
DLBCL
CD20
The definition of Richter syndrome (RS) encompasses two biologically
different conditions:
1) transformation of chronic lymphocytic leukemia (CLL) to clonally
related diffuse large B-cell lymphoma (DLBCL) – 50/75%
2) development of a DLBCL unrelated to the CLL clone
Clonal evolution Non-ralted DLBCL
Thorthon PD et al. Leuk Res 2005
RS (clonally related to BCLL) • Cumulative incidence of RS at 5 and 10 years after CLL diagnosis is
approximately 10% and 15%, respectively
• Additional genetic lesions acquired by a cell belonging to the initial CLL clone or (2) selective pressure of CLL treatment might have advantaged the growth of a preexisting drug resistant clone
• Most frequent recurrent genetic lesions in RS– TP53 disruption (50%–60%)
– NOTCH1 activation (30%) - mutually exclusive with MYC oncogenic activation (NOTCH1 directly stimulates MYC transcription)
– MYC abnormalities (20%)
Take home message
• CLL is a quite common disease
– Minority of cases to Pathologist
• Diagnosis is relatively simple but pay attention!
• Prognostic evaluation
– Immunophenotype
– Molecular genetics (FISH)
– Molecular biology (somatic mutations): NGS
THANKS YOU
Q/A
51