Importance of minor TP53 mutated clones in the clinic

Transcription

1 Importance of minor TP53 mutated clones in the clinic Davide Rossi, M.D., Ph.D. Hematology IOSI - Oncology Institute of Southern Switzerland IOR - Institute of Oncology Reserach Bellinzona - Switzerland

2 Disclosure The Diagnostic Lab of the IOSI analyzes TP53 mutations by Sanger sequencing: 1. The evidence that TP53 mutations associate with chemorefractoriness comes from Sanger sequencing-based studies 2. The current evidence support small TP53 mutated subclones as prognostic 3. Lack of harmonizantion for small TP53 mutated subclone identification 4. Patients/week number does not cost/effectively support the switch to NGS

3 TP53 lesions are mostly subclonal Landau et al, Cell 2013

4 Detection limit of Sanger sequencing Scenario 3 Scenario 2 Scenario 1 Nadeu F et al Blood 2016

5 Backtracking of TP53 mutations clonally acquired at later stages of the disease NOTCH 1 EX34: c.7544_7545delct p.p2515fs*4 (heterozygous) TP53 EX7: c.716a>c p.n239t (heterozygous) 5.6% 58% 0.39% 43% CLL 30 months CLL 62 months RS precursor CLL diagnosis CLL progression RS transformation Fabbri G, et al, J Exp Med 2011; 208: ; Malcikova et al. Leukemia. 2015;29:

6 corrected for tumor representation Small TP53 mutated subclones account for ~3 of all cases harboring TP53 defects Sanger sequencing positive n=35 TP53 mutations Ultra deep-ngs 85 TP53 mutations in 46/309 (15%) CLL Sanger sequencing negative n=50 Median allele frequency: 2.1% (range: %) 6% TP53 mutations N=309 N=309 TP53 lesions* (N=18) 85% 7% 84% (N=263) (N=22) (N=259) 3% (N=10) 6% (N=18) 4% (N=13) 5% (N=15) subclonal M clonal M * TP53 mutations and 17p13 deletion clonal M+subclonal M wt subclonal M clonal M+subclonal M clonal M+del17p del17p subclonal M+del17p clonal M+subclonal M+del17p clonal M wt Rossi, Blood 2014

7 Frequency Suclonal TP53 mutations have the same detrimental impact on TP53 as clonal lesions Frequency TP53 residual transactivation of CDKN1A Subclonal TP53 mutations are mainly missense substitutions Subclonal TP53 mutations are preferentially transitions % 78% p= % 4% p=.0408 Clonal M % 8% p=1 Subclonal M % 1 p=.0808 Missense Insdel Nonsense Splicing sites % 4 p= % p= % 13% p= % 8% p=1 Clonal M % 4% p=1 Subclonal M % 8% p=1 GC>AT AT>GC GC>CG GC>TA AT>CG AT>TA Transitions Transversions Subclonal TP53 mutations affect hot spot codons within the DBD Subclonal TP53 mutations negatively impact on TP53 function Subclonal M p=.6674 L 1 L 2 DNA binding L 3 3 Missense Nonsense Frameshift indel Splice site In frame indel Clonal M 2 1 Clonal M 14% 11% Subclonal M Clonal mutations from Zenz et al, Leukemia 2010 CDKN1A promoter transcriptional activity measured in yeast functional assays from the IARC database

8 Small TP53 mutated subclones have the same unfavorable prognostic impact as clonal TP53 defects TP53 unmutated Solely subclonal TP53 M Clonal TP53 M TP53 WT Solely subclonal TP53 M Solely clonal TP53 lesion Clonal lesion + Subclonal TP53 M p< p< No. at risk Events Total 5-year OS 95% CI % % % % % % No. at risk Events Total 5-year OS 95% CI % % % % % % p from pairwise comparisons p from pairwise comparisons < < < < Rossi, Blood 2014

9 Small TP53 mutated subclones account for ~3 of all cases harboring TP53 defects Minimal mutant allelic fractions: 0.3% for TP53 Clonal M Subclonal M Nadeu F et al Blood 2016

10 Small TP53 mutated subclones have the same unfavorable prognostic impact as clonal TP53 defects Nadeu F et al Blood 2016

11 Small TP53 mutated subclones detected before treatment subsequently expand under the selective pressure of therapies del17p ID % p.r273c TP53 M (p.r273c) months ID9245 del17p p.g244d TP53 M (p.g244d) % months CLB CR FCR CR Diagnosis Relapse Refractoriness Diagnosis Relapse Refractoriness ID5564 del17p p.r248w TP53 M (p.r248w) % % months ID10642 del17p p.r248q TP53 M (p.r248q) % % 5.5% months FCR CR BR FCM PR RDHAP PR Diagnosis Relapse PD Refractoriness Diagnosis Relapse Richter Relapse Richter Refractoriness

12 TP53 subclone evolution in CLL treated within the CLL8 trial INCREASING CCF Landau, Nature 2015

13 Small TP53 mutated subclones do not evolve under watch and wait Rossi, Blood 2014 ID4326 p.y220c % 0.8% months Watch and wait Diagnosis Last follow-up ID9240 p.l252v % 0.3% months Watch and wait Diagnosis Last follow-up

14 Scenarios of TP53-mutated subclones evolution UNTREATED RELAPSE 1 RELAPSE 2-n NO TREATMENT Slow expansion Persistence Malcikova, Leukemia 2015

15 Challenges in the identification of small TP53 mutated subclones Variant frequency (%) Sensitivity threshold True mutations Background noise of NGS Variant position (NM_ ) Library preparation chemistry? Coverage? Bioinformatic pipeline for variant calling?

16 The mutational landscape of CLL The wordcloud shows the genes that are reported as mutated in CLL by the v77 of the Catalogue of Somatic Mutations in Cancer (COSMIC). The size of the font is proportional to the mutation frequency

17 Subclonal mutations impact on CLL outcome Landau DA, Nature 2015

18 Small NOTCH1, SF3B1 and BIRC3 subclones occur in a significant fraction of CLL 6 5 Sanger sequencing positive n=32 Ultra deep-ngs 46 NOTCH1 mutations in 43/304 (14%) CLL NOTCH1 mutations Sanger sequencing negative n=14 Median allele frequency: 3.9% (range: 1.4-9%) 1 (N=31) 4% (N=12) NOTCH1 mutations subclonal M clonal M wt Sanger sequencing positive n=18 Ultra deep-ngs 43 SF3B1 mutations in 35/304 (11%) CLL SF3B1 mutations Sanger sequencing negative n=25 Median allele frequency: 4.3% (range: %) 5% (N=16) 1% (N=2) 6% (N=17) subclonal M clonal M SF3B1 mutations clonal M+subclonal M wt Rasi, Haematologica 2016 Sanger sequencing positive n=7 Ultra deep-ngs 37 BIRC3 mutations in 26/304 (8%) CLL BIRC3 mutations Sanger sequencing negative n=30 Median allele frequency: 1.4% (range: 0.2-6%) 1% (N=4) 1% (N=3) 6% (N=19) subclonal M clonal M BIRC3 mutations clonal M+subclonal M wt

19 The molecular spectrum of NOTCH1, SF3B1 and BIRC3 subclonal mutations was consistent with that of clonal mutations BIR1 BIR2 UBA BIR3 CARD UBA RING CARD Rasi, Haematologica 2016 RING Missense Nonsense Frameshift indel Splice site In frame indel 1 EGF repeats (1-36) LNR HD RAM Ankyrin 2556 TAD PEST 2514 Subclonal M TAD PEST Clonal M NOTCH p14 binding domain HEAT repeats Subclonal M Subclonal M SF3B Clonal M BIRC3 547 Clonal M

20 Small mutated subclones of NOTCH1, SF3B1 and BIRC3 genes are clinically irrelevant NOTCH1 wild type Solely subclonal NOTCH1 M Clonal NOTCH1 mutation SF3B1 wild type Solely subclonal SF3B1 M Clonal SF3B1 mutation BIRC3 wild type Solely subclonal BIRC3 M Clonal BIRC3 lesions NOTCH1 SF3B1 BIRC3 p from pairwise comparisons p from pairwise comparisons p from pairwise comparisons < < No. at risk Events Total Median OS 95% CI No. at risk Events Total Median OS 95% CI No. at risk Events Total Median OS 95% CI Rasi, Haematologica 2016

21 Small NOTCH1, SF3B1 ATM and BIRC3 subclones occur in a significant fraction of CLL Nadeu F et al Blood 2016

22 Small mutated subclones of NOTCH1, SF3B1 ATM and BIRC3 genes are clinically irrelevant Nadeu F et al Blood 2016

23 Treatment W&W Mixed shift in clonal representation of NOTCH1 and SF3B1 clones Diagnosis NOTCH1 Last follow-up Q2503* P2514fs P2514fs2 P2514fs3 Q2501* P2514fs4 P2514fs5 4 35% 3 25% 2 15% 1 5% Diagnosis SF3B1 Last follow-up K700E E622D K700E2 K666E K700E3 I704N K700E4 K700E5 K700E6 3 25% 2 15% 1 5% Diagnosis Last follow-up p=.124 p=.102 p=.345 R392fs SHIFTING CCF R555fs E546fs Diagnosis Last follow-up Diagnosis Last follow-up Diagnosis Last follow-up 1 8% 6% 4% 2% Pre-treatment Relapse P2514fs P2514fs2 P2514fs Pre-treatment Relapse E622D E622D2 I704F K666E K666E3 I704F2 E622D3 3 25% 2 15% 1 5% Pre-treatment Relapse p=.027 p=.001 p=.<.001 I427fs E429fs Q547fs IRE( )- Q547fs2 R432fs Q547fs3 R432fs2 E546fs Relapse Relapse Pretreatment Pretreatment Pretreatment Relapse Nadeu F et al Blood 2016 Rasi, Haematologica 2016 Landau, Nature 2015

24 Biomarkers that are inert under chemoimmunotherapy may become dengerous under new agents and vice versa KI resistant subclone FCR resistant subclone FCR Kinase inhibitor

25 Timepoints Clinical trial samples IOSI-EMA001 baseline week 2 week 24 week 48 week 72 week 96 yearly Patients Biobanked Pending

26 Longitudinal assessment of CLL clonal architecture under ibrutinib Cell sorting Ibrutinib Baseline Week 2 Week 24 Week 48 Week 72 Week 96 Yearly Progression End of treatment NGS cfdna CLL T Tumor gdna cfdna Tumor gdna Normal gdna T G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G A A T T T T A A T G G T C C G G G G A T C C Normal gdna T G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T T G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G C A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G A A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G A A T C C C A A G C T A G A A T T G G C A T T G C G C A T C G A A C A A G C T A G A A T T G G C A T T G C G C A T C G A A T T A G A A T T G G C A T T G C G C A T C G A A T T T T A A G G C A T T G C G C A T C G A A T T T T A A T G C A T T G C G C A T C G A A T T T T A A T G G C A T T G C G C A T C G A A T T T T A A T G T G C G C A T C G A A T T T T A A T G G T C G C G C A T C G A A T T T T A A T G G T C C G C G C A T C G A A T T T T A A T G G T C C C G C A T C G A A T T T T A A T G G T C C G C A T C G A A T T T T A A T G G T C C G G G G T C G A A T T T T A A T G G T C C G G G G A T C C

27 (%) TP53 clone dynamics under ibrutinib hge/ml of plasma Changes of TP53 clone abundance Changes of tumor burden VAF > 1.5% VAF <1.5% Time elapsed from ibrutinib start Time elapsed from ibrutinib start

28 Summary Conclusions Small TP53 mutated sunclones occur in 5% untreated CLL CLL patients harboring small TP53 mutated subclones have the same clinical outcome as patients with clonally represented TP53 lesions (prognostic) Chemoimmunotherapy invariably select small TP53 mutated subclones Small TP53 mutated subclones are inert under watch and wait Perspectives Meta-analysis of ultra-deep-ngs studies to identify a cut-off (if any) Comparative assessment of NGS protocols (and validation by PCR) Small TP53 mutated subclones upon treatment with novel agents