CHRONIC LYMPHOCYTIC LEUKEMIA - unibo.it · •Hairy-cell leukemia •Waldenstrom macroglobulinemia...

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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

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

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

MORPHOLOGY

CLL – Bone Marrow aspirate

• Cytological examination:

– extensive replacement of

marrow element by mature

lymphocytes (more than

– 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)

Para-immunoblastic growth pattern

Spleen

• Red pulp effacement • White pulp expansion

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)

Prognosis

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

1600 20 40 60 80 100 120 140

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

225 3000 50 100 150 200 25025 75 125 175 275

Unmutated VH geneMedian = 117 months

Mutated VH geneMedian = 293 months

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)

N=286(44.8%)

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%)

NOTCH1 M

SF3B1 M

BIRC3 M

del13q14

TP53 M

del11q22-q23MYD88 M

BIRC3 deldel17p13 *

Mutational complementation groups in newly diagnosed CLL

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

Years from diagnosis

lative

bility o

del13q14

Normal/+12

NOTCH1 M/SF3B1 M/del11q22-q23

TP53 DIS/BIRC3 DIS

Matched general population

0 2 4 6 8 10 12 14

Years from diagnosis

lative

bility o

del13q14

Normal

del17p13

Matched general population

del11q22-q23

NObserved

eventsExpected events*

5-year OS (%)10-year OS

(%)10-year relative

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

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

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

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