6/12/2018. Disclosures. Clinical Genomics The CLIA Lab Perspective. Outline. COH HopeSeq Heme Panels

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1 Clinical Genomics The CLIA Lab Perspective Disclosures Raju K. Pillai, M.D. Hematopathologist / Molecular Pathologist Director, Pathology Bioinformatics City of Hope National Medical Center, Duarte, CA I do not have anything to disclose. Outline CLIA genomics workflow and tools classification Challenges in clinical interpretation HopeSeq clinical workflow solution COH HopeSeq Heme Panels COH HopeSeq Solid Tumor Panels CLIA Genomic Testing ver Single nucleotide variants (SNV) Insertion deletions (Indels) Copy number variants (CNV) Structural s (Gene fusions) Microsatellite instability MGMT methylation TERT PDL1 IHC 1

2 Analysis Workflow Interpretation Calling Annotation Interpretation Actionability Calling Annotation Interpretation Actionability Map to reference Population Effect Type Diagnosis Databases Indel realignment Cancer Databases In silico Predictions Prognosis All variant interpretation in the CLIA setting should be performed by laboratory professionals taking into consideration the clinical context, pathologic diagnosis and other laboratory data available (eg. Cytogenetics) Base recalibration Transcript Pathogenicity Therapeutics calling Protein Structure Drugs Clincial Trials recalibration BRAF c.1799t>a, p.v600e Diagnostic Prognostic Therapeutic Colorectal Poor outcome Melanoma NSCLC Thyroid Diagnostic Poor outcome Hairy cell Diagnostic leukemia Erdheim Chester Diagnostic Glioma Classification ACMG AMP Guidelines 1. Pathogenic 2. Likely pathogenic 3. Benign 4. Likely benign 5. of Uncertain Significance ACMG AMP Guidelines Evidence of Pathogenicity PVS1, PS1 4, PM1 6, PP1 5 PVS1 Nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion in a gene where LOF is a known mechanism of disease PS1 Same amino acid change as a previously established pathogenic variant PS2 De novo (both maternity and paternity confirmed) in a patient with the disease and no family history PS3 Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product PS4 The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls PM1 Located in a mutational hot spot and/or critical and well-established functional domain (e.g., active site of an enzyme) without benign variation ACMG AMP Guidelines Evidence of Pathogenicity PVS1 Nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion in a gene Is LOF a known disease mechanism? Is it an LOF variants at the extreme 3 end of a gene? Is it a splice variants that is predicted to lead to exon skipping but leave the remainder of the protein intact? Are there multiple transcripts? Does the variant affect all? Alternative splicing in the tissue? 2

3 JAK2: c.1849g>t; V617F in Primary Myelofibrosis PS3 Well-established in vitro or in vivo functional studies supportive of a damaging effect (Strong) PM2 Absent from controls (or at extremely low frequency if recessive) in gnomad [<= 0.001%] (Moderate) PM1 Located in a mutational hot spot and in a critical and well-established functional domain (PKc_like) without benign variation (Moderate) PM5 Novel missense change at an amino acid residue where a different missense change (p.v617i) determined to be pathogenic has been seen before (Moderate) PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product [CADD = 33.0] (Supporting) PP5 Reputable source recently reports variant as pathogenic (Supporting) NM_ : ASXL1 - c.1135g>t; p.e379* PVS1 Null variant (nonsense, frameshift, canonical +/-1 or 2 splice sites, initiation codon) in a gene where loss of function is a known mechanism of disease (Very Strong) PS4 The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls [odds ratio = 6.86] (Strong) PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product [CADD = 35.0] (Supporting) Functional Effect Prediction of Missense s dbnsfp 83,422,341 nssnvs and sssnvs (splicing site SNVs) Compiles prediction scores from 20 prediction algorithms (SIFT, Polyphen2 HDIV, Polyphen2 HVAR, LRT, MutationTaster2, MutationAssessor, FATHMM, MetaSVM, MetaLR, CADD, VEST3, PROVEAN, FATHMM MKL coding, fitcons, DANN, GenoCanyon, Eigen coding, Eigen PC, M CAP, REVEL, MutPred) 6 conservation scores PhyloP x 2, phastcons x 2, GERP++ and SiPhy Therapeutic Significance AMP/ASCO/CAP Guidelines Therapeutic Significance of Biomarker 1A Predicts response or resistance to an FDA or EMA approved therapy, according to drug label or professional guidelines for this diagnosis 1B Predicts response or resistance to a therapy for this diagnosis based on well powered studies 2C Off Label use Associated with response or resistance to an FDA or EMA approved therapy, according to drug label or professional guidelines but only for different diagnosis. Biomarker is an inclusion criterion for an active clinical trial 2D Biomarker shows plausible response or resistance based on case or preclinical studies 3 Biomarker has uncertain clinical significance and not known to be likely benign or benign 4 Biomarker is likely benign or benign Therapeutic Biomarker Specificity of Evidence Exact variant (same alteration) Same amino acid position Same exon or domain Amplification or Loss (CNV) Gene fusion or overexpression (RNA or protein) Activating / Inactivating / Any mutation positive Expressed / Not expressed (RNA or protein) Targeted (drug known to target protein) Prognostic Significance AMP/ASCO/CAP Guidelines Prognostic Significance 1A Biomarker included in professional guidelines is prognostic for this diagnosis 1B Biomarker is prognostic for this diagnosis based on well powered studies 2C Biomarker is prognostic based on multiple small studies 2D Biomarker may assist in disease prognosis based on small studies 3 Biomarker has uncertain clinical significance and not known to be likely benign or benign 4 Biomarker is likely benign or benign 3

4 JAK2: c.1849g>t; V617F in Primary Myelofibrosis Evidence for prognostic significance Pietra D et al. (2015) Differential clinical effects of different mutation subtypes in CALR mutant myeloproliferative neoplasms. Leukemia 30(2): PMID: Male / female; years Mutant CALR gene median overall survival duration of 17.7 years Mutant human JAK2 protein (p.v617f) median overall survival duration of 12.8 years Interpretation in the CLIA lab Every variant in a patient needs an evidence based interpretation that is documented in the laboratory database HIGH IMPACT: Clinical grade evidence based cancer specific variant database Requirements for a CLIA Lab Solution Patient Database Annotation and Filtering Repository of all relevant variant annotations Pathogenicity evidence levels Clinical significance type? evidence level?? Reporting Tool Integration with EMR (HL7 or FHIR) Knowledge Base Hopeseq Patient Database Hopeseq Patient Database Hopeseq Data Chr. Position Gene Gene Dir. RNA Accession CDS Chr Reference Nucleotide Score A(#F,#R) C(#F,#R) G(#F,#R) T(#F,#R) Ins(#F,#R) Del(#F,#R) A Ratio% C Ratio% G Ratio% T Ratio% Ins Ratio% Del Ratio% Genotype Amino Acid Change Mutation Call: HGVS Mutant Allele Frequency Function Coverage 4

5 Hopeseq Population Data Hopeseq Database Hopeseq Final Report Future Directions Incorporation of RNA sequencing Integrated analysis for comprehensive genomic characterization Gene targeting vs Pathway targeting Epigenetics and microrna Clinical grade cancer variant databases with evidence and clinical actionability data (ClinOnc) Immunotherapy support Detection of neoantigens Cellular profiling of the tumor microenvironment Liquid Biopsy Minimal Residual Disease testing 5