Developing a rapid clinical sequencing system to classify meningiomaWebinars @ QIAGEN 19/02/2016Hiroshi Nishihara, MD, PhD

Department of Translational Pathology,Hokkaido University, Graduate School of MedicineTranslational Research Laboratory,Hokkaido University HospitalSapporo, Japan

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1Hokkaido University

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Hokkaido Univ. TokyoKyotoLocated in Sapporo cityInitially, Sapporo Agricultural Collage was built by Dr. ClarkPopulation Sapporo city; 2,000,000 Hokkaido state; 5,000,000

Hokkaido UniversitySapporo

Beautiful Campus Conveniently Located

Our location is very convenient and we have wide and beautiful campus including 200ha of primeval foreestOver ten thousands of people visit our campus every year just for sightseeing. 5

Self Introduction

Individualized medicine based on gene profile

Hiroshi Nishihara MD, PhD.Professor, Dept of Translational Pathology, Sch. of MedDirector, Translation Research Laboratory, Hokkaido Univ. Hosp.

Development of Clinical Science

Clinical ResearchARO)

Development of genomic medicine promotes Individualized Medicine.Integrate multiple patients genomic data

High qualityClinical InformationBiostatistics

SNVsCNVs

Clinical SequencingReal-time genomic analysisAnalysis of individual samplesClinically relevant outputsIndividualized Medicine

Clinical BioBank (Translational Research Laboratory)8

Biobank JapanNational Center.

Clinical Biobank in Hospital

Sampling

Rapid and adequate sample processingClinical sequencing in TR Laboratory

Rapid feedback to physicians

High quality sample linked with clinical information

Promote specific clinical studiesUniversitiesInstitutes

High quality biospecimenMass AnalysisSpecific institutes or universities

Research paper

Drug discovery

Government oriented clinical studiesPharmaceutical companies

9For use, not for storage of biospecimenOn-Demand type banking and advanced analysis of biospecimen

Valued biospecimen, especially for clinical studiesAvailability for genomic and molecular analyses

Physical support for clinical studies Core facility for clinical study

Sampling and storage based on specific protocolsProject-based repository systemA novel system of biospecimen repository which vigorously promotes variable clinical studies and clinical sequence

Clinical BioBankClinical BioBank (Translational Research Laboratory)

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Tissue sampling

PFPE tissue blocksPMBC for controlFFPE blocks from pathological materialsFrozen materials

Pathological evaluationTumor content rate, viabilityExtraction of DNA, RNA

dsDNAQubit

CTRT-PCR

RINBioanalyzer

QIA symphony

Targeted Amplicon Sequence with GeneReadMiSeqIllumina

Original pipeline for SNV, CNV analysis

Team conference for final diagnosis with physician, bioinformatican

Individualized MedicineClinical Sequence System in Hokkaido University Hospital

Clinical Sequence System for Brain Tumor (Meningioma)11Meningioma, arising from meningothelial cells, is the most common primary brain tumor, and accounts for about 25% of all intracranial tumors.Loss of neurofibromin 2 (NF2) has been found in about half of sporadic meningiomas.Mutations in TRAF7, KLF4, AKT1 and SMO were recently reported in non-NF2 meningiomas by NGS analysis.We established a clinical sequence system for meningioma to determine the genotype as a routine laboratory examination in addition to concurrent pathological diagnosis.

PFPE Tissue Specimen (Brain Tumor biobank)

Extraction of DNA

Targeted Amplicon Sequence; GeneRead(Whole exon for NF2, TRAF7, KLF4, AKT, SMO)

NF2; Mutation and LOHTRAF7, KLF4, AKT, SMO: Mutation

GeneRead DNAseq Targeted Panel V2Multiplex PCR-enabled enrichment of any region, gene, or set of genes in the human genome average amplicon size 150 bpNeed just 10 ng of DNA/poolTakes only 3 hours for target enrichment Integrated data analysis and biological interpretation

Vikram Devgan

Sample to InsightClinically Relevant Panels

13TypePanel nameSolid tumorClinically Relevant TumorTumor Actionable MutationsHematologic malignanciesMyeloid NeoplasmsTissue-specificBreast CancerColorectal CancerLiver CancerLung CancerOvarian CancerProstate CancerGastric CancerCardiomyopathyComprehensiveCancer PredispositionComprehensive CancerCarrier TestingGene specificBRCA1/2 Panel

Reference databases:

The Cancer Genome AtlasNational Comprehensive Cancer NetworkCOSMIC Cancer Genome CensusOMIMClinVar (NCBI)

Largest collection of wet-bench verified catalog gene panelsVikram Devgan

Sample to Insight

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GeneRead DNAseq Custom Panel V214Online custom panel builder:

Increase breadth of coverageAdjust amplicon length Allow denser tilingGene(s) or genomic region(s) of interest Fully customized panel

Turnaround time: 3 weeks

Vikram DevganWhole exon for NF2, TRAF7, KLF4, AKT, SMO

Sample to InsightTitle, Location, Date15Simple Protocol

GeneRead DNAseq Targeted Panel V2PCR primer mixAdd genomic DNA (10 ng/reaction) andGeneRead DNAseq Panel PCR Kit V2Pool reactions for each sample and purify (AMPure bead purification)

PCR amplification3 hours

NGS library preparation

Sample to InsightStandardized and rapid workflow16

AMPure bead purification

GeneRead amplification

GeneRead Library Prep

GeneRead DNAseq Panel

GeneRead Size Selection

QIAquick PCR Purification RUO Hybrid Workflow

GeneRead Library Quant Kit

GeneRead QuantiMIZE Kit

FFPE DNA isolation

Sequencing

CLC Cancer Work bench(hh:mm)

3:45

2:00

3:00

1:00

2:00

1:15

1:00

0:45

0:30

3:00

24:00

5:00

Day 1Day 2Day 3For 12 samples

Turnaround time:4 days

AMPure bead purificationDay 4

Sample to Insight

PAXgene-fixed formalin-embedded (PFPE) blocks

Confirmation of histology

GeneRead Mix-n-Match Panel - NF2, AKT1, SMO, ERBB2, KIT, METGeneRead Custom Panel - TRAF7, KLF4GeneRead DNAseq Targeted Panel V2GeneRead DNAseq Variant Calling Service (QIAGEN)BioReT System (Amelieff)

Frozen tumor samples(median size in diameter 7 mm, range 2.512)Workflow of NGS

Mix-n-Match Panel (559 genes)17

MRI and HE staining of representative cases

CNA analysis by NGS

The score Q was used to set a threshold (Q 50) to identify clusters with significant copy number changes and socre P correlated with the precisionof the copy number estimate.

Score QScore PCopy number min maxBMC Bioinformatics. 2015 Jan 28;16:17.GeneRead DNAseq Variant Calling Service(http://ngsdataanalysis.sabiosciences.com/NGS2/)

The score P was a useful indicator for the precision of the assigned copy number. Assigned copy numbers with P above 20 generally differ by less than 20 percent from the real copy number. Assigned copy numbers with P greater than 30 roughly show a maximal difference of 10 percent from the true copy number.19

Individualized medicine based on gene profile

StaffDoctor, Technical staffTR LaboratoryDivision of Cancer-Genetic DiagnosisProf. NishiharaSample preparationNGS sequencingData analysis

Data analysisAnnotationDept. of OncologyCancer Chemo. CenterMSSMitsubishi Space SoftwareRaw FastQ dataCancer genetic diagnosis for out patients without health insuranceOncoPrime, MSK-IMPACT (outsourcing)In house targeted sequencing Team conference for final diagnosis

OncoPrimeMSK-IMPACTDivision of Cancer-Genetic DiagnosisOut PatientOutsourcing

Genetic MedicineGene counseling for hereditary cancer (BRCA1, Rinch-syndrome)CollaboResult

Dept. of PathologyCompanion diagnosis for insurance-covered gene (Ras, EGFR, RAF for specific types of cancer)Collabo

PresidentHiroshi Nishihara MD. PhD (Hokkaido University)Vice president Shinichi Toyooka MD, PhD (Okayama University) DirectorManabu Mutoh MD, PhD (Kyoto University)Director Kazuyuki Matsushita MD, PhD (Chiba University)DirectorKazuhiro Okano PhD (QIAGEN)Clinical BioBank Study Group Handing of high quality biospecimenSOP for tissue samplingSOP for sample storage Linkage of sample and clinical information

Analysis of biospecimen for clinical sequenceQuality certification of biospecimenIn house analysis using desktop type NGSAdequate system for supporting clinical research

Networking of clinical biobank in JapanCollaboration with Mega-Bank in JapanSharing the banking system and data base

A couple of months ago, we just started Clinical biobank study group in Japan by collaboration of 4 national universities. We named Clinical Biobank on our banking system designed for supporting clinical research and clinical sequence. In this study group, we discuss about the SOP for handing of high quality biospecimen and establishment of clinical sequence in Japan. We hope that our concept strongly promotes clinical research and clinical sequence.Thank you for your attention. 26

Thank you for your attention!

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