Implementing Clinical Whole Exome Sequencing For The Care .
Implementing clinical whole exomesequencing for the care of childrenwith Mendelian disorders and cancerSharon E. Plon, MD, PhD, FACMGDepartments of Pediatrics/Hematology-Oncologyand Molecular and Human GeneticsHuman Genome Sequencing CenterBaylor College of MedicineNational Institute of General Medical SciencesHUMAN GENOME SEQUENCING CENTER
Disclosures – Sharon E. Plon, MD, PhD I have the following financial relationships todisclose:– I am a member of the Baylor Genetics Scientific AdvisoryBoard I will not discuss specific off label use and/orinvestigational use in my presentation.
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Clinical Exome Sequencing G IFguidelinesNEJM250CasesJAMA 2000and 800cases; GIM500 cases2015ACMG/AMPVariantGuidelinesYaping Yang201220152015NHGRICSERU01Exomerelated CPTcodesCritical andprenatalexomeChristine Eng
Analysis of Next Gen Data for Clinical ReportingAnalysis focuses on genes with rare, protein-altering changes with appropriatemechanism of inheritance, in genes associated with disease.Exome capture (VCRome 2.1)Sequencing (Illumina HiSeq PE)Genotyping (Atlas2 SNP/Indel)Annotation (Type of Variant;Known Disease Allele)Potential DiseaseVariant selection Rare: given the severity of thephenotypes, the allele should not bepresent at polymorphism frequency(1%) in control populations Protein-altering: most likely to havebiological consequence (especiallyloss of function mutations) Disease genes: is this variant in agene known to be associated withMendelian disease (OMIM, Pubmed) What is known about this particularvariant (HGMD, ClinVar) ACMG/AMP Guideline for VariantInterpretation (Richards GIM, 2015)
Yang et al., JAMA, 2014 – Description of 2000WES clinical cases 1780 predominantly pediatricpatients (89%) 1440 (72%) have intellectualdisability, seizure disorder orautism Diagnostic rate 25% forpatients referred forproband only WES. Now completed over 12,000clinical cases
Mutations in Positive WES Cases1.1% 0.8%0.7% .9%48.9%in-framelarge deletionsstart codonstoploss21.0%promoter regionmitochondrial708 Mutant Alleles in the 504 Positives, 409 (58%) novel attime of reporting
Most Mutant Alleles Arose de Novo(AD: 74%; XL: 62%)X-LINKED, 13%Mito, 0.2%de novo,74%AD, 53%AR, 36%unknown, 14%inherited, 11%
Multiple Mendelian Diagnoses in WES Cases7374 sequential cases submittedfor proband WES Diagnosis in 28.2% (2076/7374) Two or more diagnoses relatedto phenotype in 4.9%(101/2076) of diagnosed casesPercent of all diagnoses5974321313402Posey et al, NEJM, 2017Number of diagnoses
Gene AMoleculardiagnosis:Gene BGene AGene BXerodermaKBG syndrome Ichthyosis vulgaris kinSkeletalDistinct phenotypes0.8OverlapDistinct0.60.40.20Distinct(n 42)SkinCancerOverlapping(n 29)CancerOverlapping phenotypesDisease pair phenotype similarityIncreasing phenotype similarityConcept:Skin0.8p 5.1e-09 (Wilcoxon)0.60.40.20Distinct Overlapping(n 42)(n 29)
Diagnostic rate heavily dependent on newlydiscovered disease 1126241713342419 18151383Year in which a gene was first reported as disease er of molecular diagnoses90
WES re-analysis increases diagnostic rateover timePengfei Liu
Discovery of new disease genes is the greatestcontributor to improved diagnostic rate
Results of WES testing for 278 critically illinfants 100 days Overall 36.7% received a genetic diagnosis. Critical trio (14 day TAT) had a higher yield with 32of 63 infants achieving diagnosis (50.8%). Diagnostic rate lower in children withcardiovascular disorders. Medical management was affected for 52.0% withdiagnoses. These included:– Changing care or adding needed diagnostic testing.– Withdrawal of care in children with lethal diagnoses
Critical Trio Example Clinical presentation:– 4-day-old male– IUGR, admitted to NICU due to respiratory distress, paleskin, petechiae and bruising on chest and back Initial lab work revealed pancytopenia Critical trio WES (TAT 10d):– FANCA, c.154C T (p.R52X), c.2852G A, p.R951Q, bothpathogenic, compound heterozygous Fanconi anemia, complementation group A [MIM:227650]
Newborn diagnosis of Fanconi Anemia Represents an extraordinarily early presentation ofFA– Average age of bone marrow failure – 6 years– Only a few other case reports of newborn presentation Clinical management after WES:– Postpone bone marrow biopsy– Early plan for bone marrow transplantation– Monitoring for other systems: renal ultrasound,echocardiogram– Early discharge and close follow up in clinic
Baylor College of Medicine BASIC3 Key oyDoloresLopez-Terrada
3BASICBaylor College of Medicine Advancing Sequencing Into Childhood Cancer CarePATIENTS280 children newly diagnosedCNS and non-CNSsolid le exomesequencingRETURN OF ilyNo relapseMUTATIONREPORTSStudy objectives: To integrate information from CLIA-certified germlineand tumor exome sequencing into the care of newlydiagnosed solid and brain tumor patients at TexasWill ParsonsChildren’s Cancer CenterPediatric Oncology To perform parallel evaluation of the impact of tumorand germline exomes on families and physicians
Race/Ethnicity of BASIC3 Subjects areRepresentative of Houston Population6% Other38% Latino11% Black45% AngloTexasCharacteristics of patients enrolled and not enrolled on study - updatedEnrolledDeclinedCharacteristic - no. (%)P Value(n 239)(n 103)Ethnicity0.54Hispanic111 (46%)41 (40%)Non-Hispanic119 (50%)52 (50%)Not reported10 (4%)10 (10%)Race0.11White141 (59%)74 (72%)Black or African American25 (10%)12 (12%)Asian7 (3%)4 (4%)American Indian or Alaska Native10 (4%)2 (2%)Multiple14 (6%)--Not reported42 (18%)Updated from Scollon et al., Genome Medicine 2014
BASIC3 DIVERSE PEDIATRIC TUMOR DIAGNOSESNON-CNSCNS81/94 (86%)40/56 (71%)Tumor available for WES
Tumor WES Results (n 230)HIGHEST category of mutation PER PATIENTCat. 4(50%)Cat. 1(2%)Cat. 2(23%)Cat. 3(25%)Now converting to new AMP variantcuration
Germline and/or somatic mutations with potentialclinical relevance found in 40% of casesParsons et al., JAMA Oncology, 2016
Diversity of germline results returnedTUMORREPORTAllGERMLINEREPORTGeneCancer or OtherPatient nsenseRare WT1missenseSCN5AmutCYP2AmutCFTRDF508Opt-InScollon et al., Genome Medicine, 2014
Two Exome Reporting Teams Work in ParallelSomaticMutationsMolecularPathologyReview &AnnotationTumorExomeSignout ularDiagnosticianreviewGermlineExomeSignout hPipelineAnnotation
Variants of Uncertain “Clinical” Significance (VUS) Predominantly missense mutations in proteinregions with or without known function. A variety of approaches including conservation,computational predictions, segregation with cancerand population studies are utilized to try anddetermine the significance. Different laboratories may report out same variantas a VUS or likely pathogenic or likely benign basedon their laboratory’s criteria.– Data sharing through ClinVar and other databases helpsto decrease discordance across laboratories.
Significant discordance of missensepredications across algorithms in current use
Assessment of algorithm performance acrossdifferent disease mechanisms
VUS REPORTED in CANCER SUSCEPTIBILTY GENES(n 215 germline exome reports)FREQUENCY5040median of 3 VUS(range from 0 to 10)3020100012345VUS #678910
Evaluation of VUS reports in cancer susceptibility genes based on: Ethnicity (Hispanic vs non-Hispanic) – median 3 Race - increased VUS reported in African-Americans – median 5p. 0.0003VUSVUSp. 0.65HispanicNon-HispanicAfrican- WhiteAmericanOther
Cancer susceptibility molecular diagnosis in 9.8%(27/278) pediatric cancer patientsAutosomal dominant 26(P/LP)19 different genesGenes associated w/specific childhoodcancer15Examples include DICER1,VHLx3, MSH2, WT1x2,TP53x3Genes not previouslyassociated w/specific childhoodcancer11Examples include BRCA1x2,BRCA2, PALB2, CHEK2x2,FLCN, SMARCA4Autosomal recessive 1(biallelic)TJP2No one gene was reported in more than 3 BASIC3 patients:3 each for VHL and TP53.
Germline results can have an impact onmultiple family members 14 yo girl with glioblastoma– Mother aware of cancer family historybut not in electronic medical record– Sequencing revealed c.1697delAframeshift mutation in MSH2transmitted from her mother. MSH2 mutation associated withLynch syndrome and glioma.GBM– Cancer screening recommendationsmade for siblings, mother and otherMSH2 positive family members– Now important for treatment decisions
Example of unexpected finding of mosaic WT1mutation in patient with Wilms tumor Subject 223202 – 9 mo malewith Stage III Wilms tumor. No FH of cancer, no congenitalanomalies and no genetictesting recommended.– WES revealed mosaicism forframeshift in WT1.– Complete loss ofheterozygosity in tumor.– Finding of WT1 mutationresulted in long-term renalfunction assessment and morefrequent contralateral kidneysurveillance.TumorNormalAngshumoy Roy
Newly described TSG with unexpected tumor:SMARCA4 LOF w/ neuroblastoma tumor
Can we predict which patients have findings?Column1AgeCancer Diagnostic Finding 22-12 12n 278Yes (n 27) 9.8%No (n 251)43159766 (14%)14 (8.8%)7 (9.2%)37 (86%)145 (91.2%)69 (90.8%)Genderp*0.6324for trend (p 0.40.6898FemaleMale13514312 (8.9%)15 (10.5%)123 (91.1%)128 (89.5%)Ethnicity0.8372Hispanic or LatinoNon-HispanicNA133136912 (9%)14 (10.3%)1121 (91%)122 (89.7%)8Race0.6453WhiteBlackother (American Indian,Asian, 1 race)NA15927375513 (8.2%)2 (7.4%)1 (2.7%)146 (91.8%)25 (92.6%)36 (97.3%)1144Tumor type1CNSNon-CNS97181* p-values were calculated by Fisher's exact test9 (9.3%)18 (9.9%)88 (90.7%)163 (90.1%)
Also little correlation with histologicdiagnosis except rare tumors, e.g. PHEO, PPBHistologyColumn1Column2Column3Cancer Diagnostic FindingColumn4Column5n 278Yes (n 27)No (n 251)p*ATRTCARCINOMA OTHERCNS OTHEREPENDYMOMAEWING SARCOMAGERM CELL TUMORHIGH GRADE GLIOMALIVER TUMORLOW GRADE GLIOMAMEDULLOBLASTOMANEUROBLASTOMANON-CNS OTHEROSTEOSARCOMARHABDOMYOSARCOMASARCOMA OTHERWILMS TUMOR414201113247931183023141519261 (25%)3 (21.4%)1 (5%)01 (7.7%)01 (14.3%)2 (22.2%)3 (9.7%)1 (5.6%)3 (10%)7 (30.4%)01 (6.7%)03 (11.5%)3 (75%)11 (78.6%)19 (95%)11 (100%)12 (92.3%)24 (100%)6 (85.7%)7 (77.8%)28 (90.3%)17 (94.4%)27 (90%)16 (69.6%)14 (100%)14 (93.3%)19 (100%)23 0.00310.374610.23260.7271any SARCOMASARCOMA w/out EWING61482 (3.3%)1 (2.1%)59 (96.7%)47 (97.9%)0.0830.0587HISTOLOGY
Inheritance pattern of diagnostic mutationsDiagnostic findingN 27Parental SamplesAvailable20 80% of alleles inherited!Inherited from aparent16 Equivalent maternal andpaternal inheritanceDe novo (3) ormosaic (1)4Proportioninherited from aparent80% Parents have been veryinterested in having atrisk siblings tested for themutations identified
Early Data on Clinical Utility: Cancer SurveillanceRecommendations for Germline FindingsExamples of relevanceKindreds Impacted NumberPatient and sibling5Parent only7Both11None3 Both parents & siblings:TP53, VHL Parents only: BRCA1, CHEK2 No recommendations: KRAS,PTPN11, TJP2 Cancer screening in siblings hasbeen initiated through dedicatedpediatric cancer screening clinic. Major focus of our CSER2 project.
Single pathogenic variants in genes forautosomal recessive cancer syndromes Total of 18/278 BASIC3 (6.5%) pediatric cancerpatients had P/LP variants in a variety of recessivecancer syndrome gene. We subsequently reviewed medical findings at entryinto study.– 0 of 18 subjects had clinical features of the recessivedisorder except one patient with PFO and FANCL variant. Several of these reported variants were withinFanconi anemia genes (FANCC, FANCL, FANCM).
What is the expected frequency of Fanconianemia pathway variants in pediatric patientsundergoing WES? Evaluated the frequency of pathogenic or likelypathogenic (P/LP) variants in genes in the Fanconipathway from Baylor clinical whole exomesequencing patients referred for non-cancer findings. We evaluated this frequency in each of 15 FA genes:FANCA, B, C, D1/BRCA2, D2, E, F, G, I, J/BRIP1, L, N/PALB2,O/RAD51C, P/SLX4 and BRCA1 (FA-like condition,FANCS)
Clinical BCM non-cancer WES Cohort (n 9986) As previously reported (Yang et al., JAMA, 2014)patients referred for clinical WES are predominantly inpediatric age range: 88% 18 years Referred for WES from a wide variety of medicalcenters. Most common indications are neurologic, intellectualdisability and/or congenital anomalies. Data provided here is variants detected in proband:
Frequency of 3 autosomal dominant cancersusceptibility genes: BRCA1, BRCA2, 20%BRCA2310.31%PALB2100.10%Gene
FA Carrier Status per Gene – summing across allFA gene 2.92%
Nature of the pathogenic FA alleles found innon- cancer WES cohort 10% of BRCA1 and 5% of BRCA2 reported P/LPvariants were missense alleles, whereas all othervariants in FA genes were predicted to betruncating. Similarly, 90% of BRCA1 and 92% of BRCA2 mutationswere previously reported in the literature whereonly 47% of the pathogenic variants in the other FAgenes were previously reported.
Conclusions of Fanconi/BRCA Analysis Clinical WES of a large primarily pediatric cohort:– Approximately 2.9% are carriers of a Fanconi allele– This includes 0.5% with either BRCA1 or BRCA2 Now doing a comparison with Geisinger 10Kpediatric exomes to generalize the findings. This data provides framework for comparingfindings in these genes in pediatric cancer cohorts,BASIC3, PCGP, TARGET, etc.
Clinical Expectations/Utility in BASIC3 We prospectively evaluated whether standard clinicalpractice for genetic testing could predict the WESfindings (or did the exome provide more information):– At entry, the BASIC3 clinical genetics team reviewed tumorpathology, family and medical history in the EMR and anystudy related surveys:– We determined if genetic testing would be considered forthe patient based on clinical features?– If so, what genes or tests would be ordered?Any testingconsidered?YesNo#pts113176Gene testconsideredTP53microarray#pts3519Katie Bergstrom, CGC Sarah Scollon, CGC and Sharon Plon, FACMG
We found poor ability to predict which BASIC3subjects would have molecular diagnosis Only 11 of 27 (41%) patients with diagnostic cancersusceptibility findings were predicted at entry. Variety of reasons subjects were missed:– Didn’t recommend testing for genes like BRCA1– Diagnoses that we might think are obvious(PTPN11/Noonan) were not considered byoncologists prior to the WES results.– Clinically, relevant molecular findings like de novo ormosaic WT1 mutations in unilateral Wilms patients.
Need to anticipate ongoing evolution ofvariant interpretation (first reports in 2012) Child with pleomorphic xanthoastrocytoma and delayedspeech History of tumors in maternal & paternal lineage Germline WES – pathogenic variant in DKC1 - geneassociated with dyskeratosis congenita– C.-142c G in DKC1 shared by mother; reported in article in HumanGenetics 2001 in patient with DKC and functional study showedthat it disrupted sp1 binding site Referred to Alison Bertuch, who tested patient forperipheral blood telomere length, which was normal Now in gnomad database of 100K individuals– There are 16 hemizygotes (from 50K males)– Unlikely this variant would be called pathogenic today
BASIC3 Conclusions and Recommendation Multiple studies demonstrate that 10% of diversepediatric cancer populations carry P/LP variants inwide range of dominant cancer susceptibility genes.– Mixture of genes with with and without prior associationwith the child’s tumor diagnosis– Another 6% carry single recessive alleles (no clear clinicalsignificance or evidence of enrichment over controls). Current clinical practice for genetic evaluation maymiss 50% of these children including clinicallyrelevant germline findings for patient families. Time to develop clinical guidelines with germlinepanel/WES for all childhood cancer patients.
Contrasting WES results in pediatric cancerand neurodevelopmental cohortsPediatric Cancer Diagnostic rate of 10% Autosomal dominantdisorders predominate Small numbers but 80%inherited from parent Results frequently impactscreening & surveillancerecommendations Tumor data can be used toaid interpretation ofgermline genomeNeurodevelopmental Diagnostic rate of 25% More equal mixture of AD,AR and XLR De novo mutations ( 70%)predominate (multiple DNM) Results used for diagnosisand refining recurrence riskfor parents Relatively rapididentification of newgermline disease genes
KidsCanSeq – Next phase of CSER projectFigure 1CookCHOSA,UTHSC-SATCH/BCMVCCCStudy sites TCH/BCM (Houston)VCCC: TCH Vannie Cook Cancer Clinic (McAllen)Cook: Cook Children’s (Fort Worth)CHOSA: Children’s Hospital of San AntonioUTHSC-SA: UT Health Science Center – San Antonio
Sequencing plan – direct comparisons ofclinical utility with targeted panelsFigure 2Newly diagnosed patients (n 250/yr)Non-high riskn 200/yrGermline (blood)sequencing:all patientsHigh riskn 50/yr(n 300/yr)Relapsedn 50/yrRelapsed patients (n 50/yr)Tumor (FFPE)sequencing:High risk & relapsedpatients only(n 100/yr)
A Clinical Sequencing Exploratory Research (CSER) projectSupported by NHGRI/NCI 1U01HG006485BASIC3 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)BASIC3 Clinical Project Team TXCCC pediatric oncologists Robin Kerstein, MT, CCRA Sarah Scollon, MS, CGC Katie Bergstrom, MS, CGC Stephanie Gutierrez (Data manager) Ryan Zabriskie (Laboratory manager)TCH/BCM Pathology Angshumoy Roy, MD, PhD Dolores López-Terrada, MD, PhD Adekunle Adesina, MD, PhDTCH Surgery and NeurosurgeryBCM/TCH leadership David Poplack, MD Susan Blaney, MD Arthur Beaudet, MD James Versalovic, MD, PhD Jed Nuchtern, MDBASIC3 Project 2 (sequencing and reporting) Richard Gibbs, PhD (co-PI) Christine Eng, MD (co-PI) Yaping Yang, PhD (co-I) Angshumoy Roy, MD, PhD (co-I) David Wheeler, PhD (Co-I) Donna Muzny, MSBASIC3 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)
PEC-MATCH studyPrecision oncology trialObjective: to open a COG-wide single stage phase II trial of genomicallydirected therapies for children with refractory solid tumors and lymphomas
Primary objectives To determine the objective response rate in patientswith a priori specified genomic alterations treated withpathway-targeting agents To determine the proportion of patients whosetumors have pathway alterations that can be targetedby existing drugs To demonstrate the feasibility of analyzing geneticpathway alterations in refractory/recurrent pediatrictumors in a timeframe that permits use of the resultsto guide therapy choices Germline
Implementing clinical whole exome sequencing for the care of children with Mendelian disorders and cancer HUMAN GENOME SEQUENCING CENTER National Institute of General Medical Sciences Sharon E. Plon, MD, PhD, FACMG Departments of Pediatrics/Hematology-Oncology and Molecular and Human Genetics Human Genome Sequencing Center Baylor College of .
Ambry ARUP Baylor Emory GeneDx UCLA Name of test Clinical Diagnostic ExomeTM Exome Sequencing With Symptom-Guided Analysis Whole Exome Sequencing EmExome: Clinical Whole Exome Sequencing XomeDx Clinical Exome Sequencing Began offering 09/2011 04/2012 10/2011 06/2012 01/2012 01/2012 Turn around time (weeks) 8–16 12–16 15 15 12–16 11–12 .
Purpose: Reports of the use of whole-exome sequencing in clini-cal practice are limited. We report our experience with whole-exome sequencing in 115 patients in a single center and evaluate its feasibil-ity and clinical usefulness in clinical care. Methods: Whole-exome sequencing was utilized based on the judg-ment of three clinical geneticists.
different clinical laboratories offering exome sequencing as a clinical service is unknown, and detailed guidance from regulatory and professional organizations is still under development. Finally, exome sequencing has some similar limitations as Sanger sequencing; e.g., it will not
different platforms and procedures used by different clinical laboratories offering exome sequencing as a clinical service is unknown. Clinical utility The clinical utility of exome sequencing lies in the influence of the results on medical decision making and patient outcomes. There are several ways in which clinical utility can be demonstrated.
Next Generation Sequencing - Whole Exome Sequencing (WES) The exome accounts for only 1.5% of the human genome, and yet includes 85% of all disease-causing mutations (10). Whole exome sequencing (WES) examines all protein-coding regions in the human genome. In this method, DNA fragments are hybridized in solution to sequence-specific capture
Genetic Testing – Whole Genome-Exome Sequencing Policy Number: PA-204 Last Review Date: 11/14/2019 Effective Date: 01/01/2020 Policy Evolent Health considers Whole Genome-Exome Sequencing (WGS/WES) Genetic Testing medically necessary for the following indications provided that the results could have a direct influence on clinical management:
Baylor College of Medicine offers Whole exome sequencing: Whole Genome Sequencing (WGS) & Whole Exome Sequencing (WES) is in clinical practice . WGS/WES is now in clinical practice Partners HealthCare System, which is affiliated with Harvard Medical School and includes Massachusetts General
Accounting Standard (IAS) terminology and requiring pre sentation in International Standard format. Approach – These qualifications were designed using Pearson’s Efficacy Framework. They were developed in line with World-Class Design principles giving students who successfully complete the qualifications the opportunity to acquire a good knowledge and understanding of the principles .
