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IMPLICATIONS OF NGS AND MOLECULAR TESTING BEYOND RAS, RAF AND MSINicola Normanno
ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI-IRCCS-FONDAZIONE G. Pascale – NAPOLISC Biologia Cellulare e Bioterapie
DISCLOSURE SLIDE
Personal financial interests (speaker’s fee and/or advisory boards): MSD, Qiagen, Bayer, Biocartis, Incyte, Roche, BMS, MERCK, Thermofisher, Boehringer Ingelheim, Astrazeneca, Sanofi, Eli Lilly
Institutional financial interests (financial support to research projects):MERCK, Sysmex, Thermofisher, QIAGEN, Roche, Astrazeneca, Biocartis
Non-financial interests: President, International Quality Network for Pathology (IQN Path); President Elect, Italian Cancer Society (SIC)
Outline of the talk
• Different applications of NGS• Genomic, trascriptomic, liquid biopsy
• Genomic biomarkers• HER2 (CNV, variants), Fusions, POLE, TMB, etc.
• Transcriptomic subtypes of CRC• CMS classification, therapeutic implications
Current and future applications of next generation sequencing (or massive parallel sequencing) in cancer
Frese Biology 2013
Targeted sequencing of DNA and RNA is the main NGS application currently used in the clinic
NGS panels may vary from fewgenes to hundred genes
An increasing number of centers are using panelscovering large number of genes
CNB=core needle biopsy; CNV=copy number variation; FFPE=formalin-fixed paraffin-embedded; FNA=fine needle aspirate; indel=insertion and deletion; Mb=megabase; MNV=multi-nucleotide variants; mut=mutations; no=number; SNV=single-nucleotide variant; TMB=tumour mutational burden
NGS panels for comprehensive genomic profiling
5
Assay name FoundationOneCDx™ MSK-IMPACT™ Molecular
Intelligence® Tempus xT ACE ImmunoID™16-18
TruSightOncology 500(assay under development)
OncomineTM
TumourMutational Load
assay
NeoTYPE® Discovery Profile CANCERPLEX
Sequencingplatform
Illumina HiSeq4000
Illumina HiSeq2500 Illumina NextSeq Illumina HiSeq
4000Illumina
NovaSeq 600Illumina
NextSeq 550DxIon GeneStudio
S5 series Not reported Illumina NextSeq
No. of genes 324 468 592 595 >20,000 523 409 326 435
Types of alterations
Substitutions,indels, CNVs,
rearrangements
Somatic SNVs, indels, CNVs,
rearrangements
Somatic non-synonymous
missense mutations
SNVs, indels, CNVs,
rearrangements
SNVs, indels, fusions
SNVs, MNVs, indels
Nonsynonymous and synonymous
SNVs
SNPs, indels, rearrangements
SNVs, indels, CNVs,
rearrangements
Tumour slide specifications
10 FFPE slides (20% tumour
nuclei)
FFPE tissue(50–250 ng) 15 FFPE slides
FFPE slides (20% tumour
nuclei)
FFPE tissue (50 ng DNA) Not reported FFPE tissue
(20 ng DNA) FFPE tissue
FFPE slides/blocks,
CNB, FNA (>20% tumour nuclei)(>50 ng DNA)
Patient-matched normal sample No Yes No Yes No No No No No
Reported TMB value (determination for TMB high)
mut/Mb(≥20)
mut/Mb(≥13.8)
mut/Mb(≥17)
mut/Mb(not reported)
Non-synonymous mut/Mb
(not reported)
mut/Mb(≥15)
mut/Mb(not reported)
mut/Mb(not reported) Not reported
Turnaround time <2 weeks <3 weeks 10–14 days 2–3 weeks 2–4 weeks Not reported 2–3 days 14–22 days 7–10 days
Analytical Sensitivity of the Different Approaches Used for cfDNA Analysis
Normanno CTR 2018
Methods based on quantitative PCR have a limit of detection (LoD) up to 0.005%
The Emulsion PCR-based technologies [Droplet Digital PCR (ddPCR) and Beads, Emulsion, Amplification, and Magnetics (BEAMing)] have a LoD ranging from 0.01 to 0.001%
Technologies based on quantitative PCR and emulsion PCR can analyze up to hundreds bases and interrogate a limited number of loci, usually up to 10
Massively parallel or next-generation sequencing (NGS) technologies allow sequencing from 200 Kb to 3.2 Gb with a sensitivity up to 0.01% for targeted panels
NGS assays for cfDNA testing
bTMB=blood TMB; cfDNA=cell-free DNA; CNA=copy number alteration; CNV=copy number variation; indel=insertion and deletion; NGS=next-generation sequencing; no=number; seq=sequencing; SNV=single nucleotide variant; TMB=tumour mutational burden
Test name* FoundationOne®
Liquid/bTMBGuardant360/
GuardantOMNI™ MutatorDETECT Unnamed PredicineATLASOncomine Pan-
Cancer Cell-Free Assay
AVENIO ctDNAKits
No. of genes measured 70/394 73/500+ 64 508 600 52 17/77/197
Sequencing platform
Illumina HiSeq 4000
Guardant Health Digital Seq
PlatformIllumina NGS‡ Illumina NGS Not reported Ion GeneStudio
S5 seriesIllumina NextSeq
Types of alterations
SNVs, indels, fusions, CNAs
SNVs, indels, fusions, CNAs
SNVs, indels, fusions, CNVs
SNVs, indels, CNVs
SNV, CNV, rearrangements
SNVs, indels, fusions, CNVs
SNVs, indels, fusions, CNVs
Sample requirement (20 ng cfDNA) 1–2 mL plasma
(5–30 ng cfDNA)
Two 10 mL tubes of peripheral
whole blood or 6–10 mL plasma‡
Plasma (single blood
draw)Plasma (5 mL)§ 20 ng cfNA 10-50 ng
cfDNA
Genomic alterations detected by NGS testing of cfDNA in CRC patients
Kato JCO Precis Oncol. 2019
Outcomes of mCRC patients receiving matched vs. unmatched treatment based on NGS testing of cfDNA
Kato JCO Precis Oncol. 2019
Outline of the talk
• Different applications of NGS• Genomic, trascriptomic, liquid biopsy
• Genomic biomarkers• HER2 (CNV, variants), Fusions, POLE, TMB, etc.
• Transcriptomic subtypes of CRC• CMS classification, therapeutic implications
BRAF V600EBRAF non-V600MSIDDR
POLE mutHER2 ampl
MET ampl
Gene fusion
RAS mut +/-PIK3CA/PTEN mut
PIK3CA/PTEN mut
Wild-type
anti-EGFR therapies
BRAF inh + anti-EGFR +/- MEK inh
anti-PD1/L1
double anti-HER2
Kinase inh
45% 8%
26%
8%2%2%
1%
1%
2%2%
2%MET inh
To be defined
To be defined
To be defined
Genomic markers in CRC
Slide courtesy of R. Dienstmann
Gene mutations assessed by NGS in mCRC patients enrolled in the CAPRI-GOIM trial
Genes with >10 mutated cases
Total mutated cases, n (N=182 analyzed)
Cases with multiple mutations, n Types of concomitant mutations (n)
KRAS 45 30* TP53 (18), PIK3CA ex9 (9), PIK3CA ex20 (5), FBXW7 (5), BRAF (4), MET (1), EGFR (1), SMAD4 (1), FGFR3 (1), ERBB2 (1), PTEN (1)
NRAS 13 5 TP53 (3), PIK3CA ex9 (1), MET (1)
BRAF 15 12† TP53 (9), KRAS (4), PIK3CA ex20 (3), FBXW7 (2), PIK3CA ex9 (1), SMAD4 (1), FGFR3 (1), FGFR2 (1)
PIK3CA ex9 16 14‡ KRAS (9), TP53 (8), PIK3CA ex 20 (2), NRAS (1), BRAF (1), MET (1), EGFR (1), ERBB2 (1)
PIK3CA ex20 10 7‡ KRAS (5), BRAF (3), TP53 (3), PIK3CA ex9 (2), FBXW7 (2), ERBB2 (1)
TP53 72 36KRAS (18), BRAF (9), PIK3CA ex9 (8), FBXW7 (5), NRAS (3), PIK3CA ex20 (3), MET (1), EGFR (1), SMAD4 (1), CTNNB1 (1), FGFR3 (1), ERBB2 (1)
*11 cases with KRAS mutated tumors had >2 concomitant mutations (maximum 5 mutations); †5 cases with BRAF mutated tumors had >2 concomitant mutations (maximum 4 mutations); ‡9 cases with PIK3CA mutated tumors had >2 concomitant mutations (maximum 4 mutations)
Ciardiello, Normanno et al Ann Oncol 2014
Heterogeneity Score (HS) in mCRC patients enrolled in the CAPRI trial
Normanno et al Ann Oncol 2015
The heterogeneity score (HS) was obtained by normalizing the frequency of mutant alleles for the fraction of neoplastic cells
The HS virtually corresponds to the fraction of neoplastic cells that carry a specific mutation
Trastuzumab plus lapatinib inHER2-positive mCRC (HERACLES) 914 patients with KRAS exon 2 (codons 12 and 13) wild-type mCRC were screened 48 (5%) had HER2-positive tumors
Sartore-Bianchi Lancet Oncol 2016
RR: 30%, SD 44% Median PFS 21 weeks
Plasma copy number of HER2 correlates with activity of trastuzumab + lapatinib in mCRC pts (Heracles A)
Siravegna CCR 2019
Pertuzumab plus trastuzumab for HER2-amplified mCRC (MyPathway)
Meric-Bernstam Lancet Oncol 2019
Raghav JCO Precis Oncol 2019
HER2 amplification in mCRC
Found in about 2% of CRC More frequent in patients with KRAS/NRAS/BRAF wild type tumors (5%) Associated with resistance to anti-EGFR moAbs in retrospective analyses (2nd/3rd line
treatment)
HER2 amplification as predictive biomarker of resistance to anti-EGFR MoAbs in mCRC
Sartore Bianchi Oncologist 2019
HER2 mutations predictive value in mCRC
Kavuri et al, Cancer Discov 2015
Neratinib in HER2/HER3 mutant cancer
Hyman Nature 2018
Gene fusions predictive value in metastatic CRC
Alteration Prevalence Targetabilityevidence Enrichment
NTRK1-3 fusion < 1% Case reports (> if right colon, RAS/BRAF wt, MSI)1
ALK fusion < 1% Case reports (> if right colon, RAS/BRAF wt, MSIcolitis-associated)2
ROS1 fusion < 1% Other tumors (> if right colon, RAS/BRAF wt)3
RET fusion < 1% Other tumors (> if right colon, RAS/BRAF wt)3
FGFR2-3 fusion <1% Other tumors (> if RAS mut)4
1Russo et al, Cancer Discov 2016; 2Yaeger et al, Gastroenterology 2016; 3Kloosterman et al, Cancer Res 2017; 4Clifton et al, ASCO 2018
Slide courtesy of R. Dienstmann
Best change in tumor size in adult patients with NTRK fusion cancer treated with larotrectinib
Hong ASCO 2019
Fusions
Presented By Scott Kopetz at 2019 Gastrointestinal Cancer Symposium
Presented By Katherine Clifton at 2018 ASCO Annual Meeting
Presented By Katherine Clifton at 2018 ASCO Annual Meeting
Tumor Mutation Burden (TMB) is a prognostic factor in mCRC
Innocenti J Clin Oncol 2019
Distribution of TMB in advanced colorectal cancer
Fabrizio J Gastrointest Oncol 2018
MSI and TMB in mCRC patients treated with ICIs
Schrock Ann Oncol 2019
MSI CRC heterogeneity
Marisa JNCI 2018
Mutation frequencies in human CRC
TCGA Nature 2012
The somatic mutation rates varied considerably among the samples
84% of the cases with a mutation rate of 8.24 per 106 (median number of non-silent mutations, 58) and 16% with mutation rates of >12 per 106 (median number of total mutations, 728), which we designated as hypermutated
POLE mutations in CRC
Domingo Lancet Gastro Hepat 2016
Negative Hyperselection of RAS and BRAF Wild-Type mCRC Patients Who Received Panitumumab-BasedMaintenance Therapy
The PRESSING panel: HER2 amplification;MET amplification;ALK/ROS1/NTRKs/RET fusions;SNVs in HER2/PIK3CAex.20/PTEN/AKT1;SNVs RAS (low mutant allele fraction);MSI
Morano JCO 2019
Genomic profile of quadruple-wt mCRC tumors
Rachiglio Cancers 2019
Genomic Profiles of Cases with Prolonged CetuximabBenefit and Primary Progressors
Woolston Cancer Cell 2019
What are the actionable alterationsobserved in a cancer type ?
(breast, lung, CRC, pancreas, prostate, stomach, liver)
ESCAT ranking of each alteration
% of patients presenting a Level I-II alteration
(minus the most frequent hotspot)
Value (nb to test to get a benefit)
ASSESSMENT OF THE VALUE OF MULTIGENE SEQUENCING: METHOD
Which alterations must betested in each disease ?
Recommendation on the use of multigene sequencing
Open questions
• Which patients should receive a comprehensive genomic profiling (CGP)?• Advanced or metastatic• Failure of previous lines of standard therapy• Progression after target therapy• Young patients, rare diseases, unusual clinical presentation, CUP…..• Fit for enrollment in a clinical trial or to receive additional treatments
• How should the result of a CGP test be interpreted?• Tumor heterogeneity: multiple actionable mutations in the same tumor• Clonal vs sub-clonal driver alterations• Tissue and cfDNA testing to assess clonal architecture of the disease• Monitoring clonal evolution of the disease• Results of CGP should be discussed in the Molecular Tumor Board
Outline of the talk
• Different applications of NGS• Genomic, trascriptomic, liquid biopsy
• Genomic biomarkers• HER2 (CNV, variants), Fusions, POLE, TMB, etc.
• Transcriptomic subtypes of CRC• CMS classification, therapeutic implications
Gene expression–based subtyping of CRC
CIMP, CpG island methylator phenotype; MSI, microsatellite instability; SCNA, somatic copy number alterations
Guinney Nat Med 2015
Loree CCR 2018
Relative prevalence of CMS at specific primary tumor locations
Intratumor heterogeneity of CMS
Alderdice J Pathol 2018
Central tumor Invasive front
Prognostic value of the CMS classification
Guinney Nat Med 2015
Overall Survival Relapse-free Survival Survival after relapse
CMS1; CMS2; CMS3; CMS4
Predictive value of the CMS classification
Aderka Lancet Oncol 2019
Transcriptomic Subtypes of Cases with Prolonged Cetuximab Benefit and Primary Progressors
Woolston Cancer Cell 2019
Immune phenotypes associated with CMS of CRC
Roelands Int J Mol Sci 2017
Schematic representation of CRC subtypes
Dienstmann Nat Rev Cancer 2017
Conclusions• Comprehensive genomic profiling may identify relevant prognostic and predictive
biomarkers in patients with mCRC• Comprehensive genomic profiling should be offered to patients who failed standard
lines of treatment for enrollment in clinical trials• In the near future, the availability of more targeted therapies for CRC patients will
lead to a greater use of comprehensive genomic profiling in the initial phases of the disease for a better molecular classification
• Integration of trascriptomics with genetic and clinical data might further improveprecision/personalized treatment of mCRC patients
• Results of molecular profiling of tumors sould be discussed within the MolecularTumor Board