Next Generation Sequencing For Thyroid Cancer Diagnosis And Treatment

Next Generation Sequencingfor Thyroid Cancer Diagnosis andTreatmentMarina N. Nikiforova, MDUniversity of Pittsburgh Medical CenterMGP

Principles of Molecular Biology of Thyroid CellTHYROIDFollicular cellsDNA(Genes)C cellsmRNAProtein

Normal Thyroid CellsTHYROIDThyroglobulinFollicular cellsDNAmRNA(Genes)C cellsCalcitoninProtein

Thyroid TumorsTHYROIDThyroid TumorDNAmRNA(Genes) Mutations ChromosomalvariationsProtein Gene fusions Alterations ingene expressionMOLECULAR TECHNIQUESNext Generation SequencingGene Expression Profiling

Genomic Revolution:Next Generation SequencingSanger SequencingNext Generation SequencingACTG Interrogates multiple regions of genome at once Sequence DNA/RNA in massively parallelconfiguration

Next Generation Sequencing ApproachesDNAGenomeDNAExomeTranscriptome Discovery tool Expensive, time consuming, complex BI analysis and resultsinterpretationAdopted from Simon et al. Nature 2013

Next Generation Sequencing ApproachesTargeted Sequencing of multiplepreselected genes orgene regions Used in clinical practice

Next Generation Sequencing ApproachesHybrid CaptureTargetedAmplificationDNA/RNA isolationFragmentationHybridization to probesAmplificationNGS librarypreparationSequencingSequencing,BI AnalysisEmulsion PCRSequencing

NGS Sequence AnalysisSequenceReadsReferenceHumanGenomeDepth ofsequencing ordepth ofcoverage:Number of timesgenome positionis sequenced

Advantages of NGS

Technology that allows for detection of all types ofgenetic alterations in a single workflowNGSPoint mutationsIndelsCopy numberalterationGene fusionsGene expressionAdopted from Simon et al. Nature 2013

Allows quantitation(mutation frequency, gene expression)NGS allows to quantitate number of sequencing reads with mutationBRAF V600E 24% of alleles or 48% of cells with heterozygous mutationSequenceReadsReferenceHumanGenomeDepth ofsequencing ordepth ofcoverage:Number of timesgenome positionis sequenced

Requires small amount of DNA/RNA (5-50 ng) Works on any type of material: FNA (fresh orfixed slides), FFPE tissue, blood Highly sensitive detection of genetic alterationsSanger Sequencing15-20% of allelesor30-40% of cellsNGS3-5% of allelesor6-10% of cellsAllows to overcome limited sampling of the lesion, can be used for earlydetection of cancer

MorphologyMolecularAllows quantitation(mutation frequency, gene expression)RAS 3% AFBenignnoduleObservation,Mutation monitoringRAS 15% AFNIFTP/Fv, PTCLobectomyRAS 30% AFTERT 30% AFInvasive PTCTotalthyroidectomyMolecular monitoring of mutant clone expansion anddisease progression, personalized management

Detects both germ line and somatic variants Provides genetic mechanism of diseaseMedullary Thyroid CarcinomaRET p.M918TmutationGerm line or somaticPapillary Thyroid CarcinomaRET/CCDC6 (RET/PTC1)fusionSomatic

Clinical Applications of NGSin Thyroid

DiagnosticsDiagnosis of benign or malignant thyroid nodules in FNAsamples with indeterminate cytology Cancer hot spot panel (ThermoFisher Scientific) 50 genes Thyroid specific panel (UPMC) 56 genes

ThyroSeq v.2 14 genes for mutations, 1000 hotspots 42 fusion types, 16 genes for expression Ion Torrent/Proton based targeted NGSDNA LibraryGENE MUTATIONSBRAFRETNRASRNA Library5-10 ngDNA/RNAGENE FUSIONSGENE F1AXCALCAOTHER

ThyroSeq v2 Genomic ClassifierAnalytical PipelineDNARNAFNADNA/RNA retation PipelineReportingUPMC DatabaseThyroSeq v2 Genomic ClassifierSpecimen type: FNA, Left lower poleTEST RESULTS SUMMARYTest ResultProbability of CancerPositiveHigh ( 99%)Variants AnnotationPotential ManagementSurgical excision**See Interpretation below for detailsINTERPRETATIONBRAF V600E mutation found in thyroid FNA sample is associated with 99% risk of cancer and more specifically of papillarythyroid carcinoma (PTC) or related lesions. In addition, BRAF V600E mutation may correlate with more aggressive tumorbehavior, which is more likely when BRAF is found in combination with other mutations. This sample was NEGATIVE forTERT and other known to occur in combination with BRAF in tumors with high propensity for dedifferentiation andunfavorable outcome. According to the ATA risk stratification of thyroid cancer, BRAF V600E-positive intrathyroidal PTC 1cm in size are expected to be of intermediate risk and tumors 1cm in size of low risk for structural disease recurrence (1).Potential clinical management of patients with BRAF V600E mutations is discussed in the Background section and on Figures1 and 2 and should involve correlation of the results of ThyroSeq testing with cytology, imaging and clinical --------------------------------------DETAILED RESULTSSpecimen cellularity/adequacy for interpretation: ADEQUATEMarker TypeMarkerResultAmino on allelefrequency(mutations only)Gene mutationsBRAFp.V600Ec.1799T A23%Gene fusionsNegativeGene expression profilePositiveEstimated Riskof DiseaseRecurrenceThyroid Cancer RiskAssessmentIntermediate(if nodule 1 cm)Low(if nodule 1cm)CytologydiagnosisMutationsGene expressionSequencingBioinformatics

Clinical Applications of NGS in ThyroidPrognosticPrediction of clinical outcome based on genetic alterations(e.g. BRAF TERT or RAS TERT aggressive disease)

Multiple Mutations by ThyroSeq v20.6 cm noduleBRAF /TERT AKT1 /PIK3CA 35 (55%) BRAF Another HR mutation18 (29%) RAS Another HR mutation3 (5%) Other Multiple HR mutations55 (98%) Thyroid CancerAUS cytologymPTC withextrathyroid ext51 (93%) Cancers with Aggressive Features: Extrathyroidal extension (55%) Vascular invasion (53%) Lymph node macrometastasis (47%) Poorly differentiated/anaplastic carcinomaareas (14%) Distant metastasis (8%)

Clinical Applications of NGS in ThyroidTherapeuticIdentify potential markers (BRAF, RET, HRAS, PPARG, ALK,NTRK) for targeted chemotherapyFagin J and Wells S. N Engl J Medicine 2016; 375:1054-1067

Clinical Applications of NGS in ThyroidTherapeuticIdentify potential markers (BRAF, RET, HRAS, PPARG, ALK,NTRK) for targeted chemotherapyPan-cancer NGS Panels:341 genes315 genesThyroid NGS Panel:

Summary Next-gen sequencing allows simultaneous analysis formultiple genomic alterations with high accuracy and sensitivity It requires minimal amount of DNA and RNA and can beperformed on FNA and paraffin tissue samples Quantitate mutation frequency, gene fusion transcript, andgene expression, can be used for monitoring of mutant cloneand disease progression Used diagnostically in FNA samples with indeterminatecytology and in surgically removed samples forprognostication and treatment of patients with thyroid cancer

Next Generation Sequencing Approaches . DNA/RNA isolation NGS library preparation Sequencing, BI Analysis . Reference Human Genome Depth of sequencing or depth of coverage: Number of times genome position is sequenced Sequence Reads . NGS Sequence Analysis . Advantages of NGS .