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
TP53 abnormalities : Clinical impact Prognostic marker Predictive marker Del17p (+ muttp53?) Del17p (+ muttp53?) Döhner et al, NEJM, 2000 Hallek M, Lancet, 2010 Adverse prognosis Fludarabine resistance
TP53 mutations and (del17p) : Clinical impact Similar unfavorable prognostic influence. Do all TP53 mutations have the same clinical impact?
Subclones of TP53 : Clinical impact Similar unfavorable prognostic influence. Does the clinical impact of single or multiple clones is the same? Rossi et al, Blood 2014
Spectrum of TP53 mutations : Clinical impact Does the clinical impact of missense and other mutations is the same? Collado R et al Cancer Letter 2017 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
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
1st Quality control p53 French laboratories of the GBMHM 13 14 NGS Sanger and Working on technical harmonization
Clinician prescription In a routine setting Prognostic and theranostic marker Before treatment FISH 17p, 11q, karyotype 1 st line TP53 mutation (NGS) IGHV status? Other mutations? SF3B1, NOTCH1, FBXW7, BIRC3, ATM? At relapse Subsequent lines After 2-3 years of ibrutinib FISH 17p, 11q, karyotype? TP53 mutation (NGS) BTK, PLCG2, TP53 mutations (NGS)
Materiel 1000 564 100 10 3 Results depend on % of clonal B lymphocytes in the sample 1 EDTA PBMC 1 blood skin lymph nodes pleura Purified B cells 1 Before treatment: >50% of B lymphocytes in PBMC of CLL patients is usual At relapse: if lymphocytosis <10G/L use purified B cells Pellets frozen DNA extraction Cytogenetics fixed pellets can be analysed as well
Sequencing PGM Methods Chip 316 10 samples 2M reads 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
Alignment Variant caller: TMAP software Methods SNP indel List of variants Polymorphism or mutation?
Analysis Methods Ion reporter # 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 6546:6545:449rs121912651 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:rs1800372 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 P value <0.05 Allele variant > 10 reads Exon, 5 UTR Clinician report VAF>1%
Analysis Thierry Soussi Data Base and Variant Caller
Confirmation Methods
Data base 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. 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 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%. Sanger NGS 59% 41% variants 51% 49% patients NGS sanger multiclonal 14% single clone 86% P<0,0001 patients multiclonal 32% single clone 68%
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)) 6% Results of the FILO study: P53 functional impact 11% 9% 74% missense nonsense indel splice patients 160 140 120 100 80 60 40 20 0 80 72 single mutation misense P= 0,03 21 Dominant negative effect 11 37 35 multiclonal mutations single mutation null Loss of p53 Expression or truncated p53 TP53 mutation without del(17p) TP53 mutation +del(17p)
TP53 variants distribution 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 FILO base (511) 175 234 273
CLL mutational profile 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 160 patients 140 120 49% 51% 23% 77% sanger NGS NGS VAF<12% NGS VAF>12% Misense mutations 100 80 60 40 20 83% 68% unmutated IGHV Mutated IGHV 0 hotspot 175 248 273 280 282 179 others
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 We would like to thank all the network collaborators for sharing their experience and data. Tours Caen Rouen Rennes Lille Avicenne St Louis Henri Mondor Pitié Salpétrière Nancy Reims Bordeaux Clermont Ferrand Lyon Service d hématologie biologique Hopital Avicenne Bobigny Toulouse Montpellier Nice