DNA and other genomic analysis- What a practicing oncologist needs to know

Jennifer Morrissette, PhD, FACMG Clinical Director, Center for Personalized Diagnostics Scientific Director, Clinical Cancer Cytogenetics Cancer: a disease of the genome

Every tumor is different: no way to predict the mutations based on tumor type or morphologic characteristics

Reasons to consider genomic testing

1. Identify mutations • Actionable, prognostic, therapeutic

2. Detection of evolution • Tumor heterogeneity is common

3. Drug repurposing: Application of known/approved drugs to new indications/cancer types

Next generation sequencing has advantages over traditional methods

• Simultaneous detection of many different mutation types in many different • Single nucleotide variants (SNV), copy number alteration (CNA), insertions, deletions, and translocations • Easy to detect and quantify mutations in a heterogeneous sample • Normal tissue • Tumor heterogeneity • Unbiased mutation detection • Different cancers have different mutational loads • Targetable mutations Next generation sequencing: Multiple DNA regions and multiple patients in one reaction

MiSeq HiSeq 25 million molecules 300 million molecules sequenced HiSeq 1.25x1010bases sequenced 1.5x1011 bases sequenced 2.4 billion molecules sequenced 1.2x1012 bases sequenced Current panels available at the CPD Solid tumor testing Heme malignancy testing ABL1, ASXL1, ATM, BCOR, BCORL1, BIRC3, BRAF, ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, BRCA1, BRCA2, ESR1 CALR, CBL, CDH2, CDKN2A, CEBPA, CSF1R, CSF3R, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, DNMT3A, DDX3X, ETV6, EZH2, FAM5C, FBXW7, FLT3, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, AKT1, ALK, BRAF, CSF1R, EGFR, GATA2, GNAS, HNRNPK, HRAS, IDH1, IDH2, IL7R, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, KLHL6, KIT, KRAS, MAPK1, MPL, MLL2, PHF6, ERBB2, HRAS, IDH1, IDH2, KIT, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, PRPF40B, PTPN11, MAP2K1, miR-142, MYC, MYCN, MPL, NOTCH1, NPM1, NRAS, PDGFRA, KRAS, MAP2K1, NRAS, NOTCH1, MYD88, NF1, NOTCH1, NOTCH2, NPM1, NRAS, POT1, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, MET, NRAS, PDGFRA, PIK3CA, PTEN, RAD21, RIT1, RUNX1, SRSF2, SETBP1, SMC1A, SMARCB1, SMO, SRC, STK11, TP53, VHL PTEN, RET, TP53 SF1, STAG2, SF3A1, SF3B1, TBL1XR1, TET2, TP53, TPMT, U2AF1, U2AF2, WT1, XPO1, ZRSR2, ZMYM3 ABL1,AKT1,AKT2,AKT3,ALK,APC,AR,ARAF,ARID1A,ARID2, ATM, ATRX,AURKA, BAP1, BRAF,BRCA1,BRCA2, BRIP, BTK, CBP, CCND1, CCND2, CCND3 , CCNE1, CDH1, CDK4, CDK6, CDKN2A, CHEK2,CIC, CRKL, CSF1R, CTNNB1, DAXX, DDR2, DNMT3A,EGFR, EIF1Ax, EPHA3,ERBB2, ERBB3, ERBB4, ERCC2, ERG, ESR1, ESR2, EZH2, FBXW7, FGF3, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FUBP1, GATA3, GNA11, GNAQ, GNAS, HRAS, H3F3A, IDH1, IDH2, IGF1R, JAK1, JAK2, JAK3, KCNG1, KDM5A, KDM5C, KDM6A, KDR, KIT, KMT2C, KRAS, Germline testing LRRK2, MAP2K1, MAP2K2,MAP2K4 ,MAPK1 ,MAPK3, MAX, MCL1, MDM2 ,MDM4,MED12, MEN1, MET, MITF, MLH1, MRE11A, MSH2, MSH6, MTOR, MYC, MYCN, NBN, NF1, NF2, NKRT1, NKRT2, NKRT3, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NRAS, EP300, PAK1, PALB2, PBRM1, PDGFRA, PIK3CA, PIK3CB, PIK3R1, PTCH1, AJ founder BRCA1, BRCA2 PTEN, PTPN11, RAB35, RAC1, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAF1, RB1, RET,RHOA, RNF43, SETD2, SF3B1, SLIT2, SMAD4, SMARCA4, SMO, SPOP, SRC, STAG2, STK11, SUFU, SUZ12, SYK, TERT, TET2, TGFBR2, TP53, TRAF7, TSC1, TSC2, TSHR, U2AF1, VHL, WT1, XRCC2 Next Generation Sequencing at Penn

Hematologic Malignancies Solid Tumors Germline testing

Blood/BM FFPE slides/rolls

NGS based assays

EGFRvIII assay BRCA1/2 AJ • Heme NGS panel: 68 genes Glioblastoma • Solid tumor: 47 genes ->154 genes, August 2016 founder mutations • PPP: 20 genes • BRCA1, BRCA2, ESR1 Characteristics of the cancer specimen can influence results

• Variable purity of tumor in the specimen • Normal tissue • Tumor heterogeneity • Low quantity • Poor quality Quantity of tumor can be reflected in the mutational allele frequency 50% Tumor 25% Tumor

25% variant allele frequency Few tumor cells or low allele burden can be more challenging 10% Tumor 5% Tumor

5% variant allele frequency Actionable mutations detected in lung tumors

38.35 40.00

35.00 Includes primary and secondary mutations 30.00

25.00 22.40 % 20.14 20.00

15.00

10.00 3.51 5.00 2.83 2.49 1.47 1.36 1.13 1.02 0.79 0.68 0.68 0.57 0.45 0.45 0.45 0.23 0.23 0.23 0.11 0.11 0.11 0.11 0.11 0.00

• Actionable mutations included: genes targetable by FDA approved therapies: EGFR, BRAF, ERBB2, KIT, JAK2 • Clinical trials: PIK3CA, CTNNB1, IDH1, AKT1 • ALK, ROS1, and RET rearrangements (~5%, not listed above) Mechanisms of resistance: EGFR

• Classical activating mutations, exon 19 deletions and exon 21 L858R, are associated with good responses to EGFR targeted therapies • Most patients develop resistance to treatment: ~1 year

• Primary resistance • EGFR mutations with differential sensitivities • Co-occurrence of T790M: can occur as a minor clone in treatment naïve patients • Acquired resistance • New mutations in EGFR • Mutations, amplifications, increased E= Expression expression of other genes A= Amplification Stewart et al Transl Lung Cancer Res 2015 R=Regulation The future: Liquid biopsies

• As tumors grow some cells die, releasing DNA and RNA into the circulation • Cell-free, circulating tumor DNA(ctDNA) in the patient’s plasma can act as a minimally-invasive cancer biomarker

mycancergenome.org Liquid biopsies

• Closely mirror mutations detected from traditional tumor biopsies • 15,000 patients and 50 tumor types (ASCO 2016)

• Preliminary data suggests that liquid biopsies offer an accurate alternative when invasive tumor biopsies cannot be performed

• Clinical correlations are underway between liquid vs traditional biopsies

ASCO 2016, # LBA 11501 Summary

• Next generation sequencing allows for an assessment of a wide range of mutations in a single test

• Mutations can be useful for diagnosis, prognosis or to guide therapeutic decisions

• Tumors can change over the course of treatment, gaining and losing mutations in response to therapy, so dynamic testing can be useful