CAPP-seq. Davide Rossi, M.D., Ph.D.

Transcription

1 CAPP-seq Davide Rossi, M.D., Ph.D. Hematology IOSI - Oncology Institute of Southern Switzerland IOR - Institute of Oncology Research Bellinzona - Switzerland

2 CAPP-seq capture and sequencing approach Exon capture SeqCap EZ Choice Nimbelgen 7 PCR cycles Amplified target pool 14 PCR cycles Sequencing NextSeq5 Illumina Ultra Deep-Sequencing

3 Bioinformatic pipeline for variant calling FASTQ deduplication using FastUniq v1.1 software Reads alignment on the reference genome GRCh37/hg19 using BWA v..6.2 software Variant calling with VarScan2 software using somatic function Comparing cfdna variants allele frequency with paired germline gdna (Bonferroni-adjusted Fisher test) Annotation using Seattle to filter out polymorphisms, synonymous variants Comparing cfdna variants to variants of the other cfdna occurring in the same position in (Bonferroni-adjusted Z test)

4 Target region DRIVER GENES - ASXL1 - ATM - BIRC3 - EGR2 - FBXW7 - IKZF3 - IRF4 - KRAS - MAP2K1 - BRAF - MGA - MYD88 - NFKB2 - NFKBIE - NOTCH1 - PAX5 - POT1 - RPS15 - SAMHD1 - SF3B1 - TP53 - XPO1 - ZMYM3 TARGET REGION: bp 23 recurrently mutated CLL genes Coding exon plus splice site 3 UTR of NOTCH1 and promoter/enhancer of PAX5 Landau et al., Nature 215; Puente et al., Nature 215

5 Overview of mutational profile of 173 FCR-treated CLL patients Mutation type Mutation frequency (%) 5 missense nonsense frameshift splice site NOTCH1 SF3B1 TP53 ATM XPO1 MGA IKZF3 RSP15 NFKBIE BIRC3 ZMYM3 EGR2 BRAF FBXW7 IRF4 KRAS SAMHD1 MYD88 ASXL1 POT1 MAP2K1 NFKB2 PAX5 IGHV status Del11q Del17p Low-risk (IGHV mutated) Mutated sample IGHV unmutated IGHV mutated Intermediate-risk (IGHV unmutated and/or Del11q) Not assessed Del11q/Del17p/IGHV status Del11q/Del17p High-risk (Del17p) NA 16.18% 12.72% 8.67% 6.36% % 3.47% 3.47% 2.89% 2.89% 2.89% 2.31% 1.73% 1.73% 1.73% 1.16% 1.16%.58%.58%.58%.58%

6 Liquid biopsy is a source of tumor DNA for lymphoma genotyping Plasma cell free DNA: Easily accessible Real time monitoring Plasma cell free DNA COSMIC v71 Crowley E, et al. Nat Rev Clin Oncol 213

7 cfdna load in lymphomas Hohaus S et al. Ann Oncol. 29;2(8):

8 Applications of circulating tumor DNA Genotyping Clonal evolution MRD monitoring

9 Sensitivity and validation of the CAPP-seq Allele frequency depth of coverage depth of coverage age Allele frequency in ctdna from the validation experiment a 15K c 8 R² = K 6 5K 5 4 5Kb 1Kb 15Kb 2Kb target region (nucleotide resolution) 3 15K 2 b 1 1K 1 5K Mutations VAF>1% Mutations VAF<1% Allele frequency in ctdna from the discovery experiment 1% 5Kb 1Kb 15Kb 2Kb d target region (nucleotide resolution) 8K 23bp 158bp 6K Mutations identified in 8 newly diagnosed chl patients ARID1A WT c.376c>t ARID1A M ATM WT c.97a>g ATM M

10 Robustness in avoiding biological background Allele frequency Density Density Allele frequency Frequency distribution of variants in normal cfdna Frequency distribution of variants in tumor cfdna Allele frequency Allele frequency Allele frequency Variants Variants Rossi D, et al. Blood 217

11 CAPP-seq Lymphoma mutations as tumor fingerprint November 213 October 216 cfdna from plasma PB 3 ml gdna from granulocytes CAPP-seq DLBCL with Tumor gdna from LNF (n=36) Tumor gdna from LNF cfdna from plasma gdna from granulocytes DLBCL without Tumor gdna from LNF (n=14) PB 3 ml cfdna from plasma collected at every RCHOP treament cycle Ultra-deep NGS coverage >2x Diagnosis RCHOP Cycle 1 RCHOP Cycle 2 RCHOP Cycle 3 RCHOP Cycle 4 RCHOP Cycle 5 RCHOP Cycle 6 End of treatment Rossi D, et al. Blood 217

12 Gene panel 25 kb 133 genes recurrently mutated in CLL and lymphomas Non-coding region targeted by aberrant somatic hypermutation

13 Mutation Frequency (%) DLBCL mutations from the liquid biopsy N. of mutations N. of mutations Mutation Frequency (%) N. of mutations N=9 23% N=7 2 N=6 16% N=5 13% N=4 1 N=3 Training 6% N=2 N=3 GC Non-GC 3% N= N=6 2 N=4 15% N=3 Validation 1 N=2 5% N=1 N=2 GC Non-GC KMT2D TP53 CREBBP TBL1XR1 EZH2 BCL2 TNFAIP3 STAT6 PCLO CCND3 TNFRSF14 SPEN PIM1 NOTCH2 MYC KLHL6 CD79B B2M ARID1A ATM CARD11 CD58 EP3 FBXW7 GNA13 HIST1H1E ID3 IKBKB ITPKB KRAS MAP2K1 MEF2B MYD88 NOTCH1 POT1 XPO1 ZMYM3 BRAF ID7 Training N=2 ID2 ID23 ID9 ID4 ID1 ID12 ID15 ID8 ID28 ID16 ID6 ID13 ID14 ID5 ID11 ID1 ID3 ID21 ID17 25% 5 Frequency KMT2D CREBBP STAT6 TNFRSF14 GNA13 EZH2 BCL2 TP53 TNFAIP3 MYD88 MYC ITPKB HIST1H1E CD79B TBL1XR1 NOTCH1 EP3 SPEN ZMYM3 PIM1 NOTCH2 KRAS MEF2B CD58 CCND3 CARD11 BRAF B2M ARID1A 25% 5 Frequency missense nonsense indel in frame frameshift splice site GC ID36 ID35 ID47 ID31 ID51 ID48 ID39 ID52 ID42 ID43 ID34 ID38 ID32 ID49 ID37 ID41 non-gc Validation N=16 Mutated sample Rossi D, et al. Blood 217

14 Biopsy confirmed mutations identified In plasma Biopsy confirmed mutations identified In plasma N=21 N=18 N=13 N=12 Plasma cfdna genotyping vs tumor gdna genotyping Training Validation % % 6 6 N= N= Sensitivity Sensitivity Mutation identified both in gdna and in cfdna Mutation identified in gdna only Mutation identified in cfdna only 89% % 75% 75% 8 183% 75% 66% 66% Rossi D, et al. Blood 217

15 Longitudinal cfdna genotyping allows real time recovery of treatment-associated clonal evolution Mutated molecules per ml of plasma Mutated molecules per ml of plasma Mutated molecules per ml of plasma Mutated molecules per ml of plasma ID13 ID PIM1 PIM BIRC3 1 PIM1 Pre-treatment PIM1 Refractoriness 1 BIRC3 Pre-treatment Refarctoriness RCHOP Months 3 RCHOP Months 3 ID1 ID PIM1 Pre-treatment Remission (cycle 5) Remission (post-therapy) Relapse PIM FBXW7 Pre-treatment RCHOP Remission FBXW7 RCHOP FU Months 5 Months Rossi D, et al. Blood 217

16 Allele frequency in plasma cfdna Allele frequency in plasma cfdna Mutations are cleared from plasma cfdna in respondng patients but not in refractory patients A 8 Responding patients Pre-treatment R-CHOP cycle 2 R-CHOP cycle 3 R-CHOP cycle 4 R-CHOP cycle 5 R-CHOP cycle 6 End of treatment B Timepoints Resistant patients Pre-treatment R-CHOP cycle 2 Timepoints R-CHOP cycle 3 End of treatment Rossi D, et al. Blood 217

17 MRD monitoring on the liquid biopsy informs on treatment boutcome Courtesy of Ash Alizadeh

18 Diffuse large B-cell lymphoma vs classical Hodgkin lymphoma Pasqualucci L, et al. Semin Hematol 215 Reichel J, et al. Blood 215 DLBCL Tumor cells are enriched in the mass chl Tumor cells are rare in the mass Exome sequencing data from >1 cases Exome sequencing data from only 1 cases

19 Workflow of the project Time fixed study cohort N=8 N=32 N=24 N=12 Longitudinal study cohort N=8 N=5 Refractory Pre-treatment Interim PET Pre-treatment Interim PET EOT PET Baseline Relapse Baseline Immunotherapy timpepoints N=15 Diagnosis CAPP-seq PB granulocytes Plasma Macrodissected RS from FFPE biopsy Newman et al. Nat Med 214 Rossi et al. Blood 217

20 Biopsy confirmed mutations identified in ctdna 91% 75% 66.6% 66.6% 5 4 N. of mutations Biopsy confirmed mutations N. of mutations STAT6 ITPKB TNFAIP3 B2M GNA13 HIST1H1E CIITA IRF8 ARID1A BTG1 IRF4 PCBP1 PIM1 STAT3 ATM BCL6 BTK CCND3 CD58 CXCR4 ID3 KMT2D MYC NFKBIE NOTCH1 PRDM1 SPEN TET2 TNFRSF14 TP53 TRAF3 XPO1 % of mutated cases The liquid biopsy mirrors the genetics of chl a 8 (12/15) 8 53% 6 (8/15) 27% 4 (4/15) 2 (3/15) 2 13% (2/15) 7% (1/15) b ITPKB TNFAIP3 1 1 CATALYTIC domain 946 OTU ZF ZF ZF ZF ZF ZF ZF 79 c missense nonsense frameshift splicing start loss 3 -UTR d STAT6 STAT STAT_int STAT_bind SH2 STAT6_C alpha % missense nonsense splicing frameshift e *chemorefractory sample Patient Mutation identified both in gdna and in ctdna Mutation identified in ctdna Mutation identified in gdna

21 Mutational landscape of newly diagnosed chl N=8

22 Mutated pathways in newly diagnosed chl NF-κB Epigenetic genes PI3K-AKT 46.2% (37/8) 35% (28/8) immune surveillance genes 46.2% (37/8) 27.5% (22/8) Cytokine signaling NOTCH pathway 37.5% (3/8) 2 (16/8)

23 Branch of clonal evolution in refractory chl cases and immunotherapy effects on chl Germline clone Common ancestral clone Baseline clone Relapse clone Clonal divergence

24 Changes in tumor cfdna complement ipet Log fold change in tumor ctdna Log fold change in tumor ctdna chl 66 UPN6 UPN25 chl 85 chl 67 chl 52 chl 19 UPN4 chl 23 chl 8 UPN3 chl 63 chl 78 chl 64 chl 65 chl 13 UPN11 chl 74 chl 75 chl 76 chl 79 chl 8 chl 83 chl 86 Standardized log-rank statistic Standardized log-rank statistic a b Deauville PD PD PD PD PD PD CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR Outcome d p=.1 c ND -5 1 Score e Log Log > -2 log fold reduction ND Days from start of therapy < -2 log fold reduction p<.1 ipet positive Progressive disease ipet positive Cured ipet negative Progressive disease ipet negative Cured

25 Experimental Hematology Alessio Bruscaggin Adalgisa Condoluci Gabriela Forestieri Valeria Spina Lymphoma & Genomics Francesco Bertoni Franco Cavalli Lymphoma Unit Bernhard Gerber Alden Moccia Anastasios Stathis Georg Stüssi Emanuele Zucca Nuclear Medicine Luca Ceriani Michele Ghielmini Hematology Annarosa Cuccaro Stefan Hohaus Pathology Maurizio Martini Luigi Larocca Martina Di Trani Silvia Locatelli Carmelo Carlo-Stella Martina Manzoni Antonino Neri Clara Deambrogi Lorenzo De Paoli Fary Diop Luca Nassi Gianluca Gaidano Unrestricted research grant from Gilead and Abbvie