Belang van cytogenetica bij CLL

Barbara Cauwelier

Voorjaarssymposium Hematologie 23.4.2016

FISH

Conventional karyotyping

Recurrent aberrations in CLL

Gahrton, Blood 1980

• Low mitotic index : only 40% aberrations >> 80% after B cell stimulation CpG-IL2

• sub-optimal quality of oncological metaphases

• Low sensitivity: 3/20 metaphases

• Low resolution (10 Mb) : del 13q > del 17p often missed

• Laborintensive (2-3 W)

• Expertise

• Whole genome

• Balanced / unbalanced translocations

• Cheap

Fluorescence In Situ Hybridization

FISH

Cytogenetic prognostic markers of CLL

Interphase FISH ; 325 CLL

>80% aberrations 5 cytogenetic risk groups:

17p deletion (TP53) 7% 4%11q deletion (ATM) 18% 9%Trisomy 12 16% 12%Normal 18% 33%13q deletion (RB1) 55% 46%

clinical characteristics : •TP53 deletion: fludarabine resistence•Trisomy 12 : atypical morphology•ATM deletion: lymphadenopathy

Döhner et al N Engl J Med 2000Hernandez et al Haematologica, 2009

Interphase FISH ; 350 CLL

• No need for dividing cells (smears, imprints, FFPE)

• Higher sensitivity : 5/100

• Higher resolution : 100-200 kb vb del 13q/del 17p

• Fast (2d)

• Semi-quantitative

• Targeted :TP53,ATM,RB1,CEP12

• New translocations

• Expensive : € 20 per probe (€ 100 per patient)

• Not suited for MRD ; only at diagnosis/transformation

• Expertise interpretation/labourintensive

Fluorescence In Situ Hybridization

Comparative Genomic Hybridization on array

Array : slide imprinted with BAC (FISH) clones or oligonucleotides selected throughoutthe whole genome (or region of interest)

Hybridisation of tumor DNA vs normal DNA (Comparative)

High resolution *BAC clones: cfr FISH (100-200kb)

*oligonucleotides: 60 bp

* SNPs (single nucleotide) : aUPD

Array CGH

BAC aCGH

Oligo aCGH

• Whole genome

• High resolution

• Deletions/amplifications (incl cryptic or new)

• Unbalanced translocations

• Low quality DNA (FFPE)

• ‘Simple’ interpretation

• Balanced translocations

• Normal copy number variants (CNV)

• Tumorsize at least 20%

• No quantitative result

Molecular karyotyping

Detection rate of prognostic markers

by different cytogenetic techniques

Karyotype aCGH FISH

del (13)(q14) 3(17 missed :

0.6 Mb-16 Mb)

20 20

afwijkend mannelijk:

del(13)(q14.11q21.1)

del(13)(q14) aanwezig in

45% van de cellen 46,XY,del (13)(Q14Q21) (8)

Karyotype aCGH FISH

+12 6

(1 missed : 8%)

5

(2 missed: 8-12%)

7

Detection rate of prognostic markers

by different cytogenetic techniques

Karyotype aCGH FISH

del (11)(q22) 3

(1 missed : 10%)

4 4

Detection rate of prognostic markers

by different cytogenetic techniques

Karyotype aCGH FISH

del (17)(p13) 3

(2 missed : 10-

25%)

3

(2 missed: 10-

25%)

5

Detection rate of prognostic markers

by different cytogenetic techniques

13q14 deletions :

55% of CLL, similar frequency in CLL-MBL (early event)

Isolated : Good prognosis

Array CGH Heterogeneous !

MDR: miR-15a and miR16-1 : tumor suppressor genes ; downregulation leads to increased BCL2 expression

Cytogenetic prognostic markers of CLL : What’s new ?

RB1

MDR: miR-15a and miR16-1

Y Pekarsky1and C M Croce1

Cell Death and Differentiation (2015)

2 types 13q14 deletions with different prognostic value

type I deletions (80%) : small deletion 13q involving only miR-15aand miR16-1

type II deletions (20%) : 13q loss involving RB1 gene : shorter TTFT and OS

> 2 Mb

strong association with elevated genomic complexity ( copy number aberrations)

may contribute to CLL disease evolution through genomic destabilization

P.Ouilette et al. Clin Cancer Res. 2011

Cytogenetic prognostic markers of CLL : What’s new ?

normal type II type I

Biallelic loss of 13q14 in 30% of 13q deleted CLLs (2013)

small type I deletions evolution of monoallelic 13q14 deletion : more aggressive ? no significant differences in clinical outcome by different studies

Higher percentage of 13q14 deleted nuclei : > 80 % deleted nuclei

overexpression of genes involved in proliferation ≈ % deleted nuclei Higher lymphocyte count, diffuse bone marrow infiltration significant shorter TTFT and OS than normal karyotype gene expression pattern similar to 11q- cases

Although heterogeneity of 13q deletion, studies that integrate molecular

and cytogenetic data keep 13q deletion as very low-risk group

Cytogenetic prognostic markers of CLL : What’s new ?

Hernandez et al Haematologica, 2009

Trisomy 12

Clonal driver mutation (early in CLL )

10-20% of CLL, 8-22% of MBL (both HC and LC-MBL)

Often unique cytogenetic aberration (40-60%)

Atypical morphology and immunophenotype

Atypical morphology : CLL with > 10% larger lymphocytes with cleft and folded nuclei, CLL-PL

Atypical immunophenotype : FMC7+, bright sIg expression, Catovsky score < 3/5

Diagnostic for (atypical) CLL : DD Mantle cell lymphoma

CLL with trisomy 12 : mostly catovsky score < 3/5 and CD200 – (cfr MCL), CCND1/BCL1 negative

Cytogenetic prognostic markers of CLL : What’s new ?

Trisomy 12

- Prognosis : First studies : agressive clinical coarse low - intermediate

• depending on the presence of concurrent mutations or deletions

• Patients with trisomy 12 in the absence of TP53, NOTCH1 or other mutations have similarsurvival than those with normal FISH findings

• Patients with trisomy 12 and NOTCH 1/ SF3B1 mutations have similar survival as del 11q22

Del 17p (TP53)

3-8% of CLL diagnosis; up to 30% in refractory CLL patiënts Highest risk category No response to standard treatment for CLL

(FC: fludarabine-cyclofosfamide) or FCR (FC+rituximab)

Percentage of 17p deleted nuclei prognostic important

cut-off : 20-25%

clone size has a negative impact on OS and response to treatment as a continious variable

Rare cryptic deletions 17p can be detected by aCGH

smaller than FISH probes

Additional mutations in TP53 non-deleted allele :

75% of del 17p, 5% in non del 17p Sanger sequencing/NGS Monoallelic inactivation of TP53 is enough for resistance to treatment and clonal selection

Delgado et al. BJH 2012

Del 11q (ATM)

10-15% of CLL diagnosis; 30% in refractory CLL

Large deletions (>20 Mb) including entire ATM gene

Clinically progressive disease , lymphadenopathy, shorter TTFT, lower treatment response following standard chemotherapy (FC)

Percentage of 11q deleted nuclei prognostic important

<25% 11q deleted nuclei experienced longer TTFT compared with patients with ≥25% 11q deleted nuclei and also showed better response to treatments

(Marasca et al, Hematolgical Oncology 2013)

Genetic mutations associated with higher percentage (>40%) of 11q deleted cells :

SF3B1, NOTCH1, TP53, BIRC3 (not for ATM)

(Hernandez et al, PLOS 2015)

Del 11q (ATM)

ATM deletions mostly include the BIRC3 gene

11q deleted CLL : 83% including BIRC3 gene ATM or BIRC3 responsible for worse prognosis ?

ATM mutation rather than BIRC3 deletion or mutation predicts reduced survival in 11q deleted CLL (Rose-Zerilli, Haematologica 2014)

Additional mutations in ATM in non-deleted allele :

31% ATM mutations in del 11q (ex 3-59, no hotspots)

5% BIRC3 mutations , mostly associated with BIRC3 deletion Monoallelic inactivation of ATM by eiter deletion or mutation is not enough for

impaired therapy response; biallelic ATM lesions as bad as TP53 alterations

Which cytogenetic test at CLL diagnosis/ evolution ?

Karyotyping aCGH FISH

Known

prognostic

factors

+12, del11q +12, del11q, del 13q, del17p

except for small clones ( < 20%)

+12, del11q, del 13q, del17p

(cut-off 5%)

Additional

findings

Chromosomal translocations :

27%

IgH, 13q14, other B-NHL

other genome loss or gain

detection of (novel)

cryptic aberrations (del17p)

Clinical

importance

Complex karyotype (> 3) or

unbalanced translocations :

13,6% poor prognosis (often

associated with del11q or del17p)

6q deletions (9 %): ?

2p gain (MYCN): up to 28% , risk

of transformation Richter, shorter

OS

8p+, 8q+ : 2-5% : shorter OS?

MYC : < 1% ; transformation;

poor prognosis

diagnosis

evolution