TP53 mutational profile in CLL : A retrospective study of the FILO

group.

Fanny Baran-Marszak Hopital Avicenne Bobigny France

2nd ERIC workshop on TP53 analysis in CLL, Stresa 2017

Döhner et al, NEJM, 2000 Hallek M, Lancet, 2010

Adverse prognosis Fludarabine resistance

TP53 abnormalities : Clinical impact

Prognostic marker Predictive marker

Del17p (+ mutTP53?)

Del17p (+ mutTP53?)

Similar unfavorable prognostic influence.

TP53 mutations and (del17p) : Clinical impact

Do all TP53 mutations have the same clinical impact?

Subclones of TP53 : Clinical impact

Rossi et al, Blood 2014

Similar unfavorable prognostic influence.

Does the clinical impact of single or multiple clones is the same?

Spectrum of TP53 mutations : Clinical impact

Patients carrying missense mutations in DBMs seem to have shorter time to first treatment and OS compared with other missense mutations and non-missense alterations but larger studies are needed to be significant

Does the clinical impact of missense and other mutations is the same?

Collado R et al Cancer Letter 2017

Aims

• Harmonization of the analysis: Sanger / NGS • Guidelines for genetic testing : When to search for the mutations of

TP53? Alone or associated with other genes? • Create a database of TP53 mutations associated with clinical data • Identify CLL mutational TP53 profile • Identify the functional effect and the clinical impact of the various

alterations

French laboratories of the GBMHM

1st Quality control p53

14

13 NGS

Sanger

Working on technical harmonization

and

Before treatment FISH 17p, 11q, karyotype 1 st line TP53 mutation (NGS) IGHV status ? Other mutations ? SF3B1, NOTCH1, FBXW7, BIRC3, ATM…? At relapse FISH 17p, 11q, karyotype? Subsequent lines TP53 mutation (NGS) After 2-3 years of ibrutinib BTK, PLCG2, TP53 mutations (NGS)

In a routine setting

Clinician prescription

Prognostic and theranostic marker

Materiel

PBMC

frozen

Purified B cells

DNA extraction

Results depend on % of clonal B lymphocytes in the sample

Before treatment: >50% of B lymphocytes in PBMC of CLL patients is usual At relapse: if lymphocytosis <10G/L use purified B cells

Cytogenetics fixed pellets can be analysed as well

Pellets

EDTA

564

1

3

1 1

10

100

1000

blood skin lymph nodes pleura

Chip 316

10 samples

2M reads

PGM Methods Sequencing

PGM targeted Panel ampliseqTP53:

24 amplicons (exons 2-11)

2 pools

1,28kb 100% coverage

PGM targeted Panel ampliseq

32 amplicons (TP53 exons 2-11)(BTK

exon15)(PLCG2 exons 19, 20, 24, 27, 30)

2 pools

4,86kb 100% coverage

Variant caller: TMAP software

Methods Alignment

SNP indel

List of variants Polymorphism or mutation?

# locus type ref genotype pvalue coverage allele_coverage maf transcript location function codon exon protein coding 5000Exomes clinvar cosmic dbsnp

chr17:7577539 SNV G G/A 0.0 1992 1253,739 NM_000546.5 exonic missense TGG 7 p.Arg248Trp c.742C>T pathogenic 6546:6545:44920:1640831:120006:120005:1189381:120007:11564:10656:1189383:1189382rs121912651

chr17:7578115 SNV T C/C 0.0 1988 0,1988 0.137 NM_000546.5 intronic rs1625895

chr17:7578210 SNV T T/C 0.0 1999 1351,648 0.007 NM_000546.5 exonic synonymous CGG 6 p.(=) c.639A>G AMAF=0.0027:EMAF=0.0193:GMAF=0.0137 45513:249885:45753rs1800372

chr17:7578645 SNV C T/T 0.0 1308 0,1308 0.132 NM_000546.5 intronic rs2909430

chr17:7579472 SNV G G/C 0.0 1974 1347,622 0.398 NM_000546.5 exonic missense CGC 4 p.Pro72Arg c.215C>G AMAF=0.4051:EMAF=0.2548:GMAF=0.37 non-pathogenic 45985:250061 rs1042522

Ion reporter

Methods Analysis

P value <0.05

Allele variant > 10 reads

Exon, 5’UTR

Clinician report VAF>1%

Thierry Soussi Data Base and Variant Caller

Analysis

Methods Confirmation

single clone 86%

multiclonal 14%

Sanger

single clone 68%

multiclonal 32%

NGS

511 TP53 variants were retrospectively collected from centers of the FILO group (French Innovative Leukemia Organization) and laboratories of the GBMHM (Groupe des biologistes moleculaires des hémopathies malignes) Variants were identified in 370 patients mostly with relapse/refractory disease. 212 variants were evidenced in 183 patients by Sanger sequencing (exons 4 to 9) with a sensitivity around 15% 299 variants were evidenced in 187 patients by Next Generation Sequencing (exons 2 to 11) with a VAF (variant allelic frequency) >1%.

Data base

41% 49%

59% 51%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

variants patients

NGS

sanger

patients

P<0,0001

• 74% of the variants were misense mutations

• 112/256 patients (44%) had no del(17p) by FISH and TP53 alteration would have been missed

before. - 93 patients (36%) had only a single TP53 mutation - among them 80 (86%) had a TP53 missense mutation The dominant negative effect alleviates the need of a second event such as loss of 17p. Multiple clones are often associated with presence of del(17p)(64%) • Only 11 patients (4%) (6 NGS, 5 Sanger) harboured only a partial loss of p53 expression

(null mutation with no del(17p))

pat

ien

ts

Loss of p53 Expression or truncated p53

Dominant negative effect

Results of the FILO study: P53 functional impact

74%

6%

11% 9%

missense

nonsense

indel

splice

72

37 35

80

21 11

0

20

40

60

80

100

120

140

160

singlemutationmisense

multiclonalmutations

singlemutation null

TP53 mutationwithout del(17p)

TP53 mutation+del(17p)

P= 0,03

All the variants were found in exons 4 to 9 except 2 in exon 10 One third of the missense variants occurred in known hot spot codons. 21 patients had a mutation at codon 234 (p.Y234C) (6%) significantly higher than in: • solid tumors (389/76738=0.5%, p<10-9) • myeloid (6/1300=0.4%, p<10-9) • lymphoid malignancies (17/1358= 1.2 %, p<10-6) • low grade lymphomas (5/361=1,3%, p=0,01) This mutation might represent a CLL specific hotspot

TP53 variants distribution

234 273

175 FILO base (511)

• 84 patients (22%) harbored multiclonal TP53 mutations 70% were detected by NGS 10 patients had between 4 and 8 clones. • 42 patients analyzed by NGS (23% ) harbored only small clones (VAF<12%), among

them 18/27 (67%) had no del(17p) detectable by FISH neither and would have been missed before NGS.

• IGHV status was mutated in 45/ 179 (25%) patients

Hotspot mutations were more often associated with unmutated IGHV Presence of several clones appeared independent of IGHV status

CLL mutational profile

patients M

isen

se m

uta

tio

ns

49%

23%

77% 51%

sanger

NGS

NGS VAF<12%

NGS VAF>12%

0

20

40

60

80

100

120

140

160

hotspot 175 248 273 280282 179

others

unmutated IGHV

Mutated IGHV

83%

68%

Conclusion

• Clinical data are currently being collected to analyse the impact of the various mutations

• TP53 variants collection is still ongoing for identification

of a CLL mutational profile • Guidelines for genetic testing are warranted

FILO study : patients-clinicians-biologists-scientists

Avicenne St Louis Henri Mondor Pitié Salpétrière

Nice

Bordeaux

Rennes

Montpellier

Tours

Lyon

Clermont Ferrand

Caen

Toulouse

Lille

Reims

Rouen

Nancy

Service d’hématologie biologique Hopital Avicenne Bobigny

We would like to thank all the network collaborators for sharing their experience and data.