tp53 mutational profile in cll : a retrospective … · tp53 mutational profile in cll : a...
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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.