Tumor mutational burden and its transition towards the clinic · 2021. 2. 10. · Tumor mutational burden and its transition towards the clinic Wolfram Jochum Institute of Pathology

Tumor mutational burden and its transition towards the clinic

Wolfram JochumInstitute of PathologyKantonsspital St.GallenCH-9007 [email protected]

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30th European Congress of Pathology10 September 2018 │ Bilbao, Spain

First-line systemic treatment for stage IV NSCLCASCO recommendations (2017)

Immune checkpoint inhibitors

Pembrolizumab PD-L1 tumor proportion score [TPS] ≥ 50%

Tyrosinkinase inhibitors (TKI)

EGFR TKI (afatinib, erlotinib, gefitinib)

EGFR mutation

ALK TKI (crizotinib)

ALK gene rearrangement

Crizotinib ROS1 gene rearrangement

Chemotherapy (CT)

Combination cytotoxic CT (platinum-based)

In the absence of EGFR mutation and ALK/ROS1 gene rearrangement, and TPS <50%

Hanna N et al. Systemic Therapy for Stage IV Non–Small-Cell Lung Cancer: ASCO Clinical Practice Guideline Update. J Clin Oncol 35:3484 (2017)

PD-L1 expression scoring of tumor cells: Staining pattern Quantification: Tumor proportion score (TPS)

PD-L1 (SP142)

Tissue-based biomarkers for immune checkpoint inhibition

Gibney GT et al. Lancet Oncol 16:e542 (2016)Jenkins RW et al. Annu Rev Med 69:25.1 (2018)

Tumor cells Tumor microenvironment

PD-L1 expression CD8+ tumor infiltrating lymphocytes (TILs)

Microsatellite instability T-cell receptor clonality

Tumor mutational load (TML)

Immune gene signatures

Neoantigen burden

Resistance mutations

Rizvi NA et al. Science 348:124 (2015)

Tumor mutational burden (TMB) predicts response to PD-1 inhibitor pembrolizumab in NSCLC

Whole exome sequencing (WES)

Cohort Treatment TMB assessment Association between TMB and response

Ref.

CheckMate 026 Nivolumab vs. chemotherapy WES ✔ 1

CheckMate 012 Nivolumab plus ipilimumab vs. chemotherapy

WES ✔ 2

CheckMate 227 Nivolumab plus ipilimumab vs. chemotherapy

FoundationOne®Assay

✔ 3

Tumor mutational burden (TMB) assessmentin prospective NSCLC immuno-oncology trials

1: Carbone DP et al. N Engl J Med 376:2415 (2017) 2: Hellmann MD et al. Lancet Oncol 18:31 (2017); Hellmann MD et al. Cancer Cell 33:1 (2018)3: Hellmann MD et al. N Engl J Med 378:2093 (2018)

Tumor mutational burden (TMB) testing project at Institute of Pathology, Kantonsspital St.Gallen Aim: To introduce an assay for TMB testing intoroutine use in a clinical molecular pathology laboratory

Ion GeneStudio™ S5 System(Thermo Fisher Scientific)

OncomineTM Tumor Mutation Load Assay Can be run locally on the available NGS platform

(Ion GeneStudio™S5 System) and bioinformatics tools (Ion Reporter™ Software)

Complements the test portfolio of the institute

Oncomine™ Tumor Mutation Load Assay

Sequencing

Primary data analysis

Sample selection

DNA extraction

Library/template preparation

Tumor cell dissection

TML quantification

Reporting

Assay characteristics:

Targeted NSG assay 1.7 Mb genomic footprint 409 genes 15’513 amplicons 2 pool assay Low DNA input requirement (20 ng) Manual or automated library preparation Up to 8 samples per Ion 540™ Chip Ion GeneStudio™ S5 Systems

Oncomine™ Tumor Mutation Load AssayIon Reporter™ Software 5.6 analysis

All single nucleotide variants (SNVs)

Remove polymorphismsGermline variant filtering using 1000 Genomes, 5000 Exomes, ExAC databases

Variant calling (substitutions, allelic frequency ≥5%)

Display somatic SNVs (including nonsynonymous)

Sequencing results

1.22 Mb of coding regions

BAM files from Ion Reporter™ Server System

Calculate Mutation Load (all somatic SNVs)

~1.7 Megabases sequenced

Updated Mutations/Mb:Missense and Nonsense only

Workflow w1.0

Workflow w1.2

TMB Estimates on Control Samples

Robustness and Reproducibility of theOncomine™ Tumor Mutation Load Assay

NA12878 DNA sample obtained from the NIGMS Human Genetic Cell

Repository at the Coriell Institute for Medical Research

Reproducibility of the Oncomine™ Tumor Mutation

Load Assay

Replicates of 10 FFPE samples(Lung, CRC, Melanoma) and 2 cell lines

(HCC1143 and NA12878)

r2 = 0.98

Comparison of Oncomine™ Tumor Mutation LoadAssay To Whole Exome Sequencing

Correlation (r2) 0.925

WES was performed on nine tumors and their matched normal to compute WES TMB. The Oncomine™ TML assay TMB was derived from tumor samples only.

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TMB (Mutations/Mb), Whole Exome Sequencing

N=37 TML #

Mean 10.2

Range 1.6 - 29.5

25th percentile 6.2

Median 9.0

75th percentile 11.9

90th percentile 18.0

# Non-synonymous somatic SNV per megabasecoding regions (Oncomine™ Tumor Mutation Load –w1.2 – DNA – Single Sample Workflow)

All NIVO

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Retrospective TMB analysis of nivolumab-treated NSCLC using Oncomine™ Tumor Mutation Load Assay

37 NSCLC samples of patients with nivolumab therapy and clinical follow-up information 31 FFPE/6 cytological samples

DCB: Durable clinical benefit (complete response, partial response, or stable disease for 6 months and more)

Non-D

CB

DCB

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Mu

tati

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p = 0.004

Retrospective TMB analysis of nivolumab-treatedNSCLC using Oncomine™ Tumor Mutation Load Assay

Median TML

Non-DCB (n=22) 8.2

DCB (n=15) 11.5

Retrospective TMB analysis of an extended NSCLC cohort using Oncomine™ Tumor Mutation Load Assay

N=74 TML #

Mean 8.7

Range 1.6 – 29.5

25th percentile 4.9

Median 7.8

75th percentile 11.5

90th percentile 15.2

# Non-synonymous somatic SNV per megabase coding regions(Oncomine™ Tumor Mutation Load – w1.2 – DNA – Single Sample Workflow)

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# genes Sequencedregion (Mb)

TML definition

Oncomine™ Tumor Mutation Load Assay

409 1.7 Number of somatic mutations per Mb coding region

FoundationOne®Assay

324 0.8 Total number of somatic mutations (non-synonymous and synonymous) and small insertions/deletions (indels) per Mb

MSK-IMPACT 468 (341, 410)

1.22(0.98, 1.06)

Total number of non-synonymous single nucleotide or insertion/deletion mutations per Mb coding region

Whole-exome sequencing (WES)

22‘000 30 Total number of non-synonymous somatic mutations

Comparison of tumor mutational burden (TMB) assays

Chalmers ZR et al. Genome Medicine 9:34 (2017)Rizvi H et al. J Clin Oncol 36:633 (2018)Alexandrov LB et al. Nature 500:415 (2013)

Assay Median (mutations/Mb)

Our results Oncomine™ Tumor Mutation Load Assay

NSCLC: 7.8

Chalmers,2017

FoundationOne®Assay

Lung squamous cell carcinoma: Lung adenocarcinoma:NSCLC, NOS:

9.06.38.1

Rizvi, 2018 MSK-IMPACT NSCLC: 6.1/7.5/7.8(341/410/486-gene panel)

Alexandrov, 2013

Whole-exome sequencing (WES)

Lung squamous cell carcinoma: Lung adenocarcinoma:

10.0≈ 9.0

Comparison of Oncomine™ TML Assay results with published NSCLC TMB data

Chalmers ZR et al. Genome Medicine 9:34 (2017)Rizvi H et al. J Clin Oncol 36:633 (2018)Alexandrov LB et al. Nature 500:415 (2013)

Conclusions and outlook

The Oncomine™ TML assay can be used to analyze tumor DNA obtained from various types of clinical NSCLC samples (surgical resection/biopsy/cytological samples).

The results of our pilot study indicate that the Oncomine™ TML assay is able to retrospectively identify NSCLC patients who may benefit from nivolumab.

Future developments should address the following issues: Consensus on TMB definition Identification of TMB cut-off values for sample stratification Detection of mutations associated with resistance to immune checkpoint

inhibitors (e.g. KRAS/STK11 mutations) Combined workflows for TML testing and mutational profiling ( selection for

targeted therapies)

Skoulidis F et al. Cancer Discov 8:822 (2018)

Updated Ion Reporter™Software 5.10 analysis forthe Oncomine™ Tumor Mutation Load Assay

New Features: Updated Mutational Burden calculation based

on nonsynonymous SNVs and Indels View variants to support mutational profiling Compatibility with the Oncomine™

Knowledgebase Reporter Software Support for the Ion 550 Chip

Acknowledgments

Institute of PathologyMolecular Pathology Division

Izadora Demmer BuchsDiana Förbs

Claudia ZimmermannJasmin GermannKarin Baruschke

Department of Medical Oncology/Hematology

Simone Schmid Martin FrühStefan DiemLukas Flatz

Financial support by Thermo Fisher Scientific

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