How To Write A Cancer Sequencing

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1 Evalua&ng tumor exome sequencing in the oncology clinic: Lessons from the BASIC3 study GBM Cancer Risk? A Clinical Sequencing Exploratory Research (CSER) project Supported by NHGRI/NCI 1U01HG ISMB2014 Post- genomic medical decision making in cancer Will Parsons, MD PhD July 14, 2014

2 Objec:ve precision oncology To bring genomic sequencing technologies from the laboratory into the pediatric oncology clinic, providing real- :me gene:c informa:on that will guide the care of each pa:ent and family.

3 Challenges (par:al list) 1. Limited understanding of the biologically and clinically- relevant gene:c altera:ons 2. Limited number of preclinical models 3. Limited number of available drugs 4. Inexperience with clinical applica:on of genomic technologies 5. Challenges of clinical trial design Small numbers of pa:ents with each tumor subtype Biopsies of refractory tumors oken not performed

4 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Study objec:ves: To integrate informa:on from CLIA- cer:fied germline and tumor exome sequencing into the care of newly diagnosed solid and brain tumor pa:ents at Texas Children s Cancer Center To perform parallel evalua:on of the impact of tumor and germline exomes on families and physicians Sharon E. Plon, MD PhD

5 BASIC 3 inclusion criteria Children less than 18 years old having surgery for CNS or non- CNS solid tumors at Texas Children s Hospital Study enrollment within 60 days of final pathology report Parents of these children Primary oncologists of these children

6 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Provide germline exome results to clinicians and families Measure impact on cancer surveillance and gene:c tes:ng of family members Iden&fy and consent pa&ents PROJECT 1 (CLINICAL) Provide tumor exome results to clinicians and families Measure frequency of incidental findings Measure impact on treatment decisions at relapse (2 yrs)

7 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Complete whole exome sequencing of tumor/blood Provide annotated tumor exome results Provide annotated germline exome results Novel repor:ng pla^orm (ipad disclosure app) PROJECT 2 (SEQUENCING/REPORTING)

8 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Study physician- parent communica&on of exome results (audio- recording of disclosure visits) PROJECT 3 (ELSI) Describe oncologist preferences and values for sharing and use of exome data (through longitudinal interviews) Describe family preferences and values for receipt and use of exome data (through surveys & longitudinal interviews) Development of ethical framework to guide shared decision- making for parents and pediatric specialists

9 Enrollment and sample status 150 pa:ents, 223 parents, and 16 oncologists No (17%) Yes (83%) Overwhelmed (10%) Privacy risks (3%) Gene:c anxiety (2.5%) Blood draw (2%)

10 Tumor Diagnoses of 100 subjects Non- CNS (n=68) CNS (n=32) DYSGERMINOMA ALVEOLAR SOFT PART SARCOMA ADRENAL CORTICAL CARCINOMA YOLK SAC TUMOR HEPATO BLASTOMA HCC PHEOCHROMO CYTOMA NEURO BLASTOMA HIGH GRADE GLIOMA CHOROID PLEXUS TUMORS MENINGIOMA PINEO BLASTOMA LOW GRADE GLIOMA OSTEOSARCOMA MIXED MALIGNANT GERM CELL TUMOR EWING SARCOMA RHABDOMYO SARCOMA OTHER NON- CNS SOLID TUMORS WILMS TUMOR OTHER CNS TUMORS EPENDYMOMA Tumor available for WES MEDULLO BLASTOMA 59/68 (87%) 22/32 (69%)

11 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Provide germline exome results to clinicians and families Measure impact on cancer surveillance and gene:c tes:ng of family members Iden&fy and consent pa&ents PROJECT 1 (CLINICAL) Provide tumor exome results to clinicians and families Measure frequency of incidental findings Measure impact on treatment decisions at relapse (2 yrs)

12 Clinical whole exome sequencing Using 1 ug of DNA isolated from frozen :ssue Illumina Sequencing Pla^orm Capture exome on Roche- Nimblegen VCRome 2.1 from BCM HGSC Paired tumor/blood samples on one HiSeq lane Analysis using pla^orm developed by BCM- HGSC informa:cs team WES sample Total Gb Unique per sample aligned Gb Mean coverage Bases 20X coverage Bases 40X coverage Germline % 95.7% Tumor % 95.1%

13 BCM Mercury pipeline Reads pile- up over exons Variant call format (.vcf)

14 Iden:fica:on of soma:c muta:ons tumor.vcf normal.vcf Soma&c caller (T- N) COSMIC: Var_ID Var_Gene Prim_Site Source PMID Soma:c Muta:on Repor:ng Annota&on Rare Variants OMIM, 1000G, dbsnp, HGMD Germline Muta:on Repor:ng

15 Molecular Pathology variant soma.vcf : >20 quality metrics >50 genotype + annota:on features review Apply Filters Total Cov T > 50X Q20 Var Cov T > 6X Exon non- synon SNV + Splicing + Remove misaligned ar:facts Tumor type Total variants Reported variants Neuroblastoma 94 4 Medulloblastoma Neuroblastoma Confirma&on: Sanger seq Wilms tumor Anaplas:c ependymoma 75 2 Interpret and Rank Report

16 Clinical u:lity of tumor WES Category I muta:ons Established clinical u&lity for the tumor type tested Category II muta:ons MET - neuroblastoma Poten&al clinical u&lity Genes that are members of cancer pathways, gene families, or func:onal groups; targets of approved or inves:ga:onal therapeu:c agents Category III muta:ons PHF6 - neuroblastoma In consensus cancer genes (not included in I and II) Category IV muta:ons All other muta&ons ALK - neuroblastoma XIAP - neuroblastoma A. Roy & F. Monzon

17 Tumor exome results Recurrently- mutated genes CTNNB1 (8/80; 10%) TP53 (4/80, 5%) BRAF (3/80, 4%) DOES NOT INCLUDE COPY NUMBER DATA KIT, KRAS, ARID1A, TSC2 (each 2/80, 2%) Genes mutated in only one tumor: ALK, NRAS, MET, JAK2, JAK3, FGFR3, NTRK2, FBXW7, TSC2, SMARCA4, DDX3X, NF2, DICER1, NOTCH3, BRCA1, BRCA2, WT1 n = 81 exomes

18 Tumor exome results Median of 8 soma&c muta&ons PER PATIENT *Note: Cat 4 muta:ons have not been validated 1st 67 exomes

19 Tumor exome results HIGHEST category of muta:on PER PATIENT Cat. 1 (2%) Muta:ons of known clinical u:lity in that tumor type Cat. 4 (49%) Only muta:ons in non- cancer genes n = 81 exomes Cat. 2 (26%) Muta:ons of poten:al clinical u:lity Cat. 3 (23%) Muta:ons in other cancer genes

20 14 y/o girl with metasta:c OS Tumor exome sequencing result: frameshik muta&on in TSC2 (and loss of other allele) N T TSC2 c.2764_2765del, p.l922fs Diagnosis: metasta:c osteosarcoma Prognosis: poor Tumor exome result: TSC2 muta:on Courtesy of A. Roy Clinical considera:on: use of mtor inhibitor?

21 7 y/o boy with malignant liver tumor * NRAS c.181c>a, p.q61k Diagnosis: malignant epithelial hepa:c neoplasm Prognosis: poor Tumor exome result: NRAS muta:on Clinical considera:on: targe:ng RAS pathway?

22 14 y/o boy with medullobastoma Courtesy of A. Roy Tumor exome sequencing results: β- catenin: subset of tumor cells with nuclear reac:vity N N T T CTNNB1 p.d32y DDX3X p.v345l TP53 p.r282w p.e286x ARID1B c g>a FOXO3 p.w233x

23 16 year old girl with GBM Tumor exome sequencing results: TP53 N N T T H3F3A p.g34r ATRX p.q2192x TP53 p.r273h BCOR p.s1261fs MED12 p.r1994w

24 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer PARENTS Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Addi:onal pa:ent blood sample Addi:onal parental blood sample Tumor sample from first surgery Tumor samples from addi:onal surgeries COMPLEMENTARY GENOMIC ANALYSES CGH/SNP arrays Whole genome sequencing RNA sequencing Tumor heterogeneity Treatment response/resistance Other research studies

25 RNAseq integra:on with WES TVarRa:o gdna TVarRa:o cdna /44 (32%) Category 4 muta:ons expressed Linghua Wang & David Wheeler, Baylor College of Medicine HGSC

26 Preclinical model development Integrated genomic characteriza:on ü Primary tumor, blood normal, xenograk, cell line(s) ü WES, RNA- seq, CGH/SNP array, WGS Xiao- Nan Li, MD PhD

27 BASIC 3 Baylor Advancing Sequencing Into Childhood Cancer Care PATIENTS SEQUENCING RETURN OF RESULTS FOLLOW- UP 280 children - newly diagnosed CNS and non- CNS solid tumors Blood Tumor CLIA- cer:fied whole exome sequencing Germline Soma:c EMR MDs GCs Family Relapse No relapse SAMPLES MUTATION REPORTS Provide germline exome results to clinicians and families Measure impact on cancer surveillance and gene&c tes&ng of family members Iden&fy and consent pa&ents PROJECT 1 (CLINICAL) Provide tumor exome results to clinicians and families Measure frequency of incidental findings Measure impact on treatment decisions at relapse (2 yrs)

28 Diversity of germline exome results TUMOR REPORT GERMLINE REPORT All Gene Cancer or Other Pa:ent Phenotype Other Medically Ac:onable PCG Genes Recessive Carrier Genes All Soma:c Muta&on FDA Pathogenic VUS Pathogenic Pathogenic Indica:on BRAF V600E Example DICER1 nonsense Rare WT1 missense SCN5A mut CYP2A mut CFTR DF508 Opt- In

29 14 year old girl with GBM GBM Sequencing revealed c.1697dela frameshik muta&on in MSH2 transmited from her mother. MSH2 muta:ons are associated with increased risk of mul:ple cancers, including glioma. Cancer screening recommenda&ons made for pa:ent, mother and other MSH2 posi:ve family members

30 Muta:ons related to phenotype Mechanism N Genes Autosomal dominant Suspected clinically and ordered as part of care Not considered by clinical team but FH+ Autosomal recessive; single muta:on Diagnos:c for other medical problems 8 4 DICER1, VHL, TP53 x 2* 3 MSH2, BRCA1, BRCA2 1 WT1 (mosaic) 4 FANCL x 2, FANCA, MUTYH; Oncologist discre:on to pursue work- up further 3 TJP2 - Liver disease/hcc; CLCN5- renal disease; MNX1- neurologic disorders *One TP53 described as likely deleterious in text of report

31 Muta:ons unrelated to phenotype 3 pa:ents with muta:ons on the ACMG list SCN5A (long QT syndrome) DSP (cardiomyopathy) LDLR (familial hypercholesterolemia) 1 pa:ent withtnfrsf13b immunodeficiency muta:on 2 pa:ents with mtdna muta:ons including MELAS Referrals to appropriate TCH clinics made and evalua:on ini:ated based on incidental finding Larger dataset of 2000 exomes at BCM- WGL found closer to 4.5% of ac:onable muta:ons reported

32 Other common germline findings Median of 3 VUS in genes related to phenotype 98% carry at least one VUS in cancer genes Generally no further follow- up of these variants at this :me Median of 2 recessive carrier muta:ons Majority are very rare syndromes Only 2-3% carry muta:on in a gene on the ACMG popula:on screening list for recessive disorders Can iden:fy X- linked carrier status as well Median of 2 FDA pharmacogene:c variants

33 Yield of germline/tumor WES (n=100) Germline Tumor Diagnos:c - cancer l l l l l l l l Diagnos:c - other l l l Incidental l l l l l l l Category I l l Category II l l l l l l l l l l l l l l l l l l Germline Diagnos:c cancer suscep:bility Diagnos:c non- cancer Medically ac:onable Tumor Category 1 Category %

34 Conclusions Pipeline for clinical sequencing of pa:ent tumor and blood samples successfully established Muta:ons of poten:al clinical u:lity iden:fied by tumor/ germline WES in ~40% of pediatric solid tumors Diversity of muta:ons iden:fied suggests that genome- scale (or more pediatric- specific targeted) diagnos:c approaches may be favored The most useful genomic test (or combina:on of tests, e.g WES and RNAseq) remains to be proven Further characteriza:on of both temporal and intra- tumoral heterogeneity will be necessary

35 Looking forward There is significant pa:ent/family interest in these clinical genomic personalized approaches Specialized exper:se/training is necessary for informed consent and result repor:ng for clinical genomics Major interpre:ve challenges remain (the defini:on of ac:onable is a fairly loose one) It will be cri:cal to understand the preferences of physicians and families for repor:ng of genomic data For any given pa:ent the tumor or germline report may be more informa:ve Clinical trials are needed to test if clinical genomics will be beneficial for pediatric oncology pa:ents

36 A Clinical Sequencing Exploratory Research (CSER) project Supported by NHGRI/NCI 1U01HG BASIC 3 Project 1 (clinical) Sharon Plon, MD, PhD (Project PI) Will Parsons, MD, PhD (Project PI) Murali Chintagumpala, MD (co- I) Stacey Berg, MD (co- I,) Susan Hilsenbeck, PhD (co- I) Tao Wang, PhD (co- I) BASIC 3 Clinical Project Team TXCCC pediatric oncologists Robin Kerstein, MT, CCRA Sarah Scollon, MS, CGC Ka:e Bergstrom, MS, CGC Stephanie Gu:errez (Data manager) Ryan Zabriskie (Laboratory manager) TCH/BCM Pathology Angshumoy Roy, MD, PhD Dolores López- Terrada, MD, PhD TCH Adekunle Surgery and Adesina, Neurosurgery MD, PhD BASIC 3 Project 2 (sequencing and repor&ng) Richard Gibbs, PhD (co- PI) Chris:ne Eng, MD (co- PI) Yaping Yang, PhD (co- I) Angshumoy Roy, MD, PhD (co- I) Federico Monzon, MD (co- I) David Wheeler, PhD (Co- I) Donna Muzny, MS BASIC 3 Project 3 (ELSI) Laurence McCullough, PhD (co- PI) Richard Street, Jr., PhD (co- PI) Amy McGuire, JD, PhD (co- I) Melody Slashinski, PhD (co- I) BCM/TCH leadership David Poplack, MD Susan Blaney, MD Arthur Beaudet, MD James Versalovic, MD, PhD Jed Nuchtern, MD