simposio sies patogenesi molecolare dei linfomi non … · lymphoma malt-1 (infectious agents)...

PATOGENESI MOLECOLARE DEI

LINFOMI NON-HODGKIN

Divisione di Ematologia

Dipartimento di Medicina Clinica e Sperimentale

Università del Piemonte Orientale Amedeo Avogadro

ASO Maggiore della Carità

Novara

Gianluca Gaidano

SIMPOSIO SIES

MOLECULAR PATHWAYS in B-CELL LYMPHOMAGENESIS

GERMINALCENTER

MANTLE

MARGINALZONE

PLASMA CELL

MEMORYB-CELS

VIRGINB-CELLS

BCL-2

DLBCL

?

p53p15, p16ASHM

Mantle

cell

lymphoma

CYCLIN D1

Primary effusion

lymphoma

HHV-8

Lymphoplasmacytoid

lymphoma

PAX-5

MALT

lymphoma

MALT-1(Infectious Agents)

c-MYCp53

BCL-6Burkitt

lymphoma

Follicular

lymphoma

DIFFUSE LARGE B CELL LYMPHOMA

Division of Hematology

DIMECS & IRCAD

Novara

Heterogeneity due to:

• clinical presentation

• response to therapy & survival

• genotype

• phenotype

• GEP

• host’s immunity (HIV, PTLD)

• de novo vs transformed

Plasmacells

Virgin B-cells

Germinal centre B-cellsMemory B-cells

BCL6

CD138MUM1

SHM

Burkitt/Burkitt-like lymphoma

DLBCL-centroblastic

BCL6+

/MUM1–

/CD138–

BCL6–

/MUM1+

/CD138–

Polymorphic PTLD

DLBCL-immunoblastic DLBCL-immunoblastic

BCL6–

/MUM1+

/CD138+

HISTOGENESIS and PATHOGENESIS of PTLD

Capello, Blood 2003 Rinaldi, BJH 2005 Lucioni, Transplantation 2006

AIDS- BL

AIDS- LCL BL-lines LCL PEL

AIDS- IBL MM

BCL6CD27CD10CD23CD30CD39IRF-4BLIMP-1CD138

lymphoblastoid-associated

plasma cell-associated

germinal center immuno-blastic

plasma cell

4-4 0-2 2

MOLECULAR PATHOLOGY of HIV-NHL and of PEL

Naïve B Centroblast Centrocyte

Plasma Cell

Post-GCGerminal Center (GC)Pre-GCMemory B

HL

PAX-5

LCL: EBV-immortalized lymphoblastoid cell lines; MM: multiple myeloma cell lines

Blood 2004

Blood 2005

Adv Cancer Res 2005

A: Expression of BCL6 and/or

CD10 but NOT MUM1/CD138

B: Expression of BCL6 and/or

CD10 and MUM1and/or CD138

C: Expression MUM1and/orCD138

but NOT BCL6 and/or CD10

HISTOGENETIC MARKERS and PROGNOSIS in HIV-NHL

(Hoffman et al., Blood 2005)

DIFFUSE LARGE B CELL LYMPHOMA

Heterogeneity due to:

• clinical presentation

• response to therapy & survival

• genotype

• phenotype

• GEP

• host’s immunity (HIV, PTLD)

• de novo vs transformed:

from follicular lymphoma

from CLL (Richter’s syndrome)

FL

DLBCL

CLL

SOMATIC HYPERMUTATION:BEYOND IgV GENES

physiologicalphysiological aberrant (DLCL)aberrant (DLCL)

BCL-6BCL-6Fas/CD95Fas/CD95IgVIgV PIM-1PIM-1c-MYCc-MYCPAX-5PAX-5

RhoHRhoH/TTF/TTF

ASHM in TRANSFORMATION of FOLLICULAR LYMPHOMA to DLBCL

Case PAX-5 RhoH/TTF PIM-1

1A

1B

2A

2B

3A

3B

4A

4B

5A

5B

6A

6B

7A

7B

8A

8B

9A

9B

10A

10B

-

-

-

-

-

-

-

-

-

-

-

-

-

-

G847A

G847A, C938T

-

G1025A

G656T

G656T, C1207T,

G1325T, G1333A

-

T319C, T495G, A518T, T562A, G694A, T732C,

C788G, A848C, A857C, A880G, G884T, C890G, T895C, A985C

-

-

-

-

G938A

G938A, G963A

-

-

-

-

-

-

-

-

-

-

-

T732G

-

-

-

-

-

-

-

C1458T

-

-

-

-

-

-

-

C1540G

-

-

-

-

c-MYC

-

G4652T, C4653G

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Rossi et al., Haematologica 2006

Rossi et al., Leukemia 2006

RICHTER’s SYNDROME

BM PB

• Transformation to DLBCL occurs in 2-10% CLL, depending on length of follow-

up and re-biopsy policy (Tsimberidou et al, 2005)

• Evidence of clinical predictors of Richter syndrome is scattered

• The value of molecular and phenotypic risk factors of CLL progression as

predictors of transformation is unknown

Lymph node

Q1: Are transforming CLL biologicaly different from non-

transforming CLL?

Q2: Is it possible to identify predictors of CLL transformation to

DLBCL?

Q3: Which lesions are acquired at the time of transformation?

CLL DLBCL

Issues To Be Addressed


DIMECS & IRCAD

Novara

55.2%

25.5%

21.8%

10.9%

6.7%

0 10 20 30 40 50 60 70 80 90 100

del 13q

+12

Normal

del 17p

del 11q

FISH karyotype

IGHV mutated63.7%

IGHV unmutated

36.3%

IGHV mutation

BIOLOGICAL FEATURES OF THE CLL COHORT (n=185)

CD38 positive32.8%

CD38 negative67.2%

CD38 expression

ZAP70 positive31.0%

ZAP70 negative61.0%

ZAP70 expression

90%

10%

0 50 100

Mutated p53

Wt p53

16%

84%

0 50 100

p53 inactivation

No p53 inactivation

p53 status

BIOPSY POLICY

Patients were biopsied at CLL diagnosis or at CLL progession in the case of:

• Lymph node size >5 cm

• Doubling of lymph node size <3 months

• Extranodal lesions


DIMECS & IRCAD

Novara

CUMULATIVE INCIDENCE of

CLL TRANSFORMATION to RICHTER SYNDROME

months

n=185

Cu

mu

lati

ve

in

cid

en

ce

of

tra

ns

form

ati

on

(%

)

5-year: 13.6% (95% CI: 7.0-20.1% )

10-year: 16.2% (95% CI: 8.0-24.4% )


DIMECS & IRCAD

Novara

Median time to transformation:

23.0 months

(95%CI: 15.9-30.1 months)

Cu

mu

lati

ve

in

cid

en

ce

of

tra

ns

form

ati

on

(%

)months

CLINICAL FEATURES AT DLBCL DIAGNOSIS (n =17)

Age (years) 70

Male: female 11:6

ECOG PS >1 6/17 (35.2%)

Rai III-IV 3/17 (17.7%)

Binet B-C 14/17 (82.4%)

Ann Arbor III-IV 17/17 (100.0%)

B symptoms 9/17 (52.9%)

Bulky >10 cm 7/17 (43.7%)

Largest lymph node >5 cm 9/16 (56.2%)

Extranodal sites >1 9/16 (56.2%)

Splenomegaly 5/16 (31.2%)

Peripheral blood involvement by large

cells/immunoblasts3/15 (20.0%)

Peripheral blood lymphocytes ( x 109/l) 4.7

Hb (g/dl) 12.7

Platelets (x 109/l) 204

Beta-2-microglobulin (mg/l) 3

LDH (U/l) 500

Alkaline phosphatase (U/l) 173

Albumin (g/l) 39

IPI 0-2 4/15 (26.7%)

IPI 3-5 11/15 (73.3%)Division of Hematology

DIMECS & IRCAD

Novara

6% (1/16)

94% (15/16)

Clonally related Clonally unrelated

IGHV GENE ANALYSIS

35.3%

64.7%

32.9%

67.1%

TRANSFORMING CLL (n=17) NON-TRANSFORMING CLL (n=168)

p=0.009

IGHV homology >98%

IGHV homology <98%Division of Hematology

DIMECS & IRCAD

Novara

FISH KARYOTYPE

0% 10% 20% 30% 40% 50% 60% 70%

p=0.002

19.1% (30/157)

TRANSFORMING CLL NON-TRANSFORMING CLL

18.7% (3/16)

21.7% (34/157)18.7% (3/16)

18.7% (3/16)

58.6%

(92/157)

Normal

del13q14

del 17p13

+12

del11q22-23

> 2 FISH lesions

18.7% (3/16)

18.7% (3/16)

50.0% (8/16)

5.6% (9/157)

23.6% (37/157)

10.2% (16/157)


DIMECS & IRCAD

Novara

CD38 AND ZAP70 EXPRESSION

0% 20% 40% 60% 80% 100%

p=0.001

p=0.004

TRANSFORMING CLL NON-TRANSFORMING CLL

ZAP70>20%

CD38>30%

35.8% (43/120)

87.5% (7/8)

29.2% (49/168)

73.3% (11/15)


DIMECS & IRCAD

Novara


transforming CLL?

Q2: Is it possible to identify predictors of DLBCL

transformation in CLL?

Q3: Which lesions are acquired at the time of transformation?

CLL DLBCL



DIMECS & IRCAD

Novara

IGHV homology >98%

IGHV homology <98%

p=0.006

months

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

BIOLOGICAL PREDICTORS of RS: IGHV HOMOLOGY

Transformation at 5 years

IGHV homology >98% 28.3%

IGHV homology <98% 7.0%Division of Hematology

DIMECS & IRCAD

Novara

IGHV4-39

No IGHV4-39

p<0.001

months

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)


IGHV4-39 56.2%

No IGHV4-39 11.1%

BIOLOGICAL PREDICTORS of RS: IGHV GENE USAGE


DIMECS & IRCAD

Novara

months

del13q14

p=0.004

No del13q14

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

Transformation at 5

years

No del13q14 25.4%

del13q14 3.8%

BIOLOGICAL PREDICTORS of RS: FISH KARYOTYPE


DIMECS & IRCAD

Novara

CD38 >30%

CD38 <30%

p<0.001

months


CD38 >30 35.4%

CD38<30 4.7%

BIOLOGICAL PREDICTORS OF RS: CD38 EXPRESSION

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)


DIMECS & IRCAD

Novara

ZAP70 >20%

ZAP70 <20%

p=0.007

months


ZAP70 >20 22.3%

ZAP70 <20 2.3%

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

BIOLOGICAL PREDICTORS OF RS: ZAP70 EXPRESSION


DIMECS & IRCAD

Novara


Lymph node >3 cm 49.9%

Lymph node <3 cm 6.1%

Lymph node >3 cm

Lymph node <3 cm

p<0.001

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

months

CLINICAL PREDICTORS OF RS: LIMPH NODE SIZE


DIMECS & IRCAD

Novara


LDH > ULN 42.3%

LDH <ULN 8.6%

LDH >ULN

LDH < ULN

p=0.001

months

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

CLINICAL PREDICTORS OF RS: LDH


DIMECS & IRCAD

Novara


Binet B/C 29.6%

Binet A 7.1%

Binet B/C

Binet A

p=0.004

months

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

CLINICAL PREDICTORS OF RS: BINET STAGE


DIMECS & IRCAD

Novara

• Absence of del13q14 is:

an independent predictor of RS by multivariate analysis

significantly more frequent in transforming CLL

may be acquired at transformation to DLBCL

• del13q14 has been shown to activate molecular pathways that are

distinct from those of CLL without del13q14 (Calin et al, N Engl J Med 2005;

Kienle et al, J Clin Oncol 2005)

• Transformation to DLBCL of another indolent B-cell disorder, i.e.

MALT lymphoma, has been documented to be influenced by

chromosomal aberrations and differences in disease pathogenesis(Starostik et al, Blood 2002)

del13q14 and CLL TRANSFORMATION to DLBCL

CD38:

is an independent predictor of RS

is significantly more frequent in transforming CLL

signalling activation induces transformation of CLL cells to

proliferating plasmablasts reminiscent of RS blasts

(Deaglio et al, Blood. 2003; 102:2146)

CD38 and TRANSFORMATION to RICHTER’s SYNDROME

Role of CD38 polymorphism

Deaglio et al., Blood 2006


transforming CLL?

Q2: Is it possible to identify predictors of DLBCL transformation

in CLL?

Q3: Which lesions are acquired at the time of

transformation?

CLL DLBCL


KARYOTYPE EVOLUTION at TRANSFORMATION to DLBCL

Case FISH

CLL DLBCL

GG normal normal

KB normal del 17p13

MF +12, +12 +12, +12

MP +12+12

del 17p13

PA del 11q22-23 del 11q22-23

PLdel 11q22-q23

del 13q14normal

PM normal normal

RO del 17p13 del 17p13

TSdel17p13

del 13q14

-12

del17p13

del 13q14

CLL DLBCL

KB / LSIp53 PL / LSID13S319 (O) + Rb1 (G)

CLL DLBCL

p53 MUTATIONS at CLL TRANSFORMATION to DLBCL

Case p53 status

CLL DLBCL

BA wt C13208T (Pro177Ser)

BF wt wt

BG wt G14099A (Glu258Lys)

CAM wt G14487A (Arg273His)

GG wt wt

GL wt wt

GM C14512G(Arg281Glu)C14512G(Arg281Glu)

C13397T (Arg213STOP)

KB wt C13397T (Arg213STOP)

MF wt wt

MP wt wt

NGL wt del13069-13071

PA wt wt

PL +C13134(Pro152frameshift) +C13134(Pro152frameshift)

PM wt wt

RO wt G14038A(Met237Ile)

TS wt A13215T(His179Leu)

VF wt wt

• Lymph node size >3 cm predicts RS independent of clues of

leukemic disease

• Extensive lymph node colonization by CLL, rather than

colonization of blood, marrow or spleen, appears to be a

prerequisite for RS transformation

• Lymph node colonization may expose CLL cells to the

genetic instability of the lymph node microenvironment

LIMPH NODE SIZE and

RICHTER’s SYNDROME TRANSFORMATION

ABERRANT SOMATIC HYPERMUTATION IS RARELY

INVOLVED in CLL TRANSFORMATION to DLBCL

0

4/9

(44%)

0

1/9

(11%)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

FL DLBCL from FL B-CLL DLBCL from B-CLL

mutations in >1 gene

Rossi et al, Haematologica 2006 Division of Hematology

DIMECS & IRCAD

Novara

PERSPECTIVES

• SNP array on CLL/DLBCL pairs

• Telomere length

• CD49d expression

• GEP of transforming CLL vs non-transforming CLL

• GEP of RS vs de novo DLBCL


DIMECS & IRCAD

Novara

BCL2-938C>A POLYMORPHISM DOES NOT PREDICT RS

p=0.990

AAAC

months

Cu

mu

lative

in

cid

en

ce o

f tr

an

sfo

rma

tio

n (

%)

CC

5-year transformation

CC 12.7%

AC 14.5%

AA 14.9% Rossi et al., Blood, in press

• High risk of transformation to DLBCL is indicated by:

no del13q14 + lymph node size >3 cm

CD38 positivity + IGHV4-39 usage

• In CLL at high risk of transformation, a close

monitoring and a careful biopsy policy may be helpful

for prompt recognition of DLBCL

• Prompt recognition of DLBCL may improve RS

prognosis (Tsimberidou et al, J Clin Oncol. 2006)

CLUES FOR CLINICAL MANAGEMENT

Division of HematologyDepartment of Clinical and Experimental Medicine & IRCAD

Università del Piemonte Orientale Amedeo Avogadro

& ASO Maggiore della Carità

Novara

Clinical teamDavide Rossi

Silvia Franceschetti

Monia Lunghi

Lorenzo De Paoli

Paola Riccomagno

Chiara Vendramin

Paola Zigrossi

Annarita Conconi

Lab teamDaniela Capello

Michaela Cerri

Clara Deambrogi

Silvia Rasi

Valeria Spina

Stefania Cresta

Division of Statistics

& Clinical Epidemiology

AAUEP - Novara

Division of Pathology

AAUEP - Novara

Corrado Magnani

Antonio Ramponi

Guido Monga

Hematology Unit

CRO - Aviano

Experimental Oncology

IOSI - Bellinzona

Francesca Rossi

Antonella Zucchetto

Valter Gattei

Andrea Rinaldi

Ivo Kwee

Francesco Bertoni


University of Torino

Marco Ladetto

simposio sies patogenesi molecolare dei linfomi non … · lymphoma malt-1 (infectious agents)...

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