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Hematopathology

CHRONIC LYMPHOCYTIC LEUKEMIA

Prof. Pier Paolo Piccaluga

Department of Experimental, Diagnostic and Specialty Medicine, Bologna University

Department of Pathology JKUAT, Nairobi

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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

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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

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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

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