shashikant kulkarni, m.s (medicine)., ph.d., facmg associate professor of pediatrics, genetics,...
TRANSCRIPT
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Cancer Next Generation Sequencing Clinical Implementation in CLIA/CAP facility
Shashikant Kulkarni, M.S (Medicine)., Ph.D., FACMGAssociate Professor of Pediatrics, Genetics, Pathology and Immunology
Medical Director of Genomics and Pathology Services
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Why do we need NGS for clinical cancer diagnostics?
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Advantages of detecting mutations with next-generation sequencing
High throughput Test many genes at once
Systematic, unbiased mutation detection All mutation types
▪ Single nucleotide variants (SNV), copy number alteration (CNA)-insertions, deletions and translocations
Digital readout of mutation frequency Easier to detect and quantify mutations in a
heterogeneous sample Cost effective precision medicine
“Right drug at right dose to the right patient at the right time”
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Unique challenges for implementing NGS for clinical cancer diagnostics
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Complexity of Cancer genomes
Cancer genomes are extremely complex and diverse Mutation frequency
▪ Degree of variation in cancer cells compared to reference genome
Copy number/ploidy▪ Most tumors are aneuploid▪ Bioinformatic software assume diploid status
Genome structure
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Cancer-specific challenges Genomic alterations in cancer found at low-
frequency Samples vary in quantity, quality and purity from
constitutional samples Quantity
▪ Limiting for biopsy specimens▪ Whole genome amplification not ideal
Quality▪ Most biopsies are formalin fixed, require special protocols ▪ Often include necrotic, apoptotic cells
Purity (tumor heterogeneity)▪ Admixture with normal cells (need pathologists to ensure test
is performed on DNA from tumor cell)▪ Within cancer heterogeneity (different clones)
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Sample procurement and pre-analytical issues
FFPE (formalin-fixed, paraffin-embedded) samples Age, temperature, processing
Fresh tissues Not ideal without accompanying pathology
investigation and marking of tumor cell population to guard against dilution effect on total DNA extracted
Fine needle biopsies Very few cells available NGS methods will need to work by decreasing
minimum inputs of DNA
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Implementation of NGS for clinical cancer diagnostics
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Clinical Next Generation Sequencing in Cancer Goals
High throughput, cost effective multiplexed sequencing assay with deep coverage
Target clinically actionable regions in clinically relevant time
Challenges Huge infrastructure costs Bioinformatic barriers
Solution Leverage expertise and resources across
Pathology, Bioinformatics and Genetics
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Example process of targeted sequencing panel in cancer
From “soup to nuts”
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Test overview
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Cancer Gene Panel
Genes DiseaseALK Lymphoma, LungBRAF Brain, Colon, Lung, Melanoma, Thyroid
CEBPA AMLCTNNB1 Colon, Desmoid Tumor, Liver, Lung, Prostate, Renal, ThyroidCHIC2 Myeloid Neoplasms w/EosinophiliaCSF1R AML, GISTDNMT3A AMLEGFR Colon, LungFLT3 AMLIDH1 AML, BrainIDH2 AML, BrainJAK2 Myeloproliferative NeoplasmsKIT AML, GIST, Systemic MastocytosisKRAS Colon, Endometrium, Lung, Melanoma, Pancreatic, ThyroidMAPK1(ERK) Lung, MelanomaMAPK2(MEK) Lung, MelanomaMET Lung, MelanomaMLL AMLNPM1 AMLNRAS Colon, Lung, Melanoma, Pancreatic, ThyroidPDGFRA GIST, SarcomaPIK3CA Colon, Lung, Melanoma, PancreaticPTEN Brain, Endometrium, Melanoma, Ovarian, Prostate, TestisPTPN11 JMML, MDSRET MEN2A/2B (adrenal), ThyroidRUNX1 AMLTP53 Colon, Lung, PancreaticWT1 AML, Renal, Wilms Tumor
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Target definitions
Exons +/- 200 bp, plus 1000 bp +/- each gene
AUG STOPTSS poly(A)
promoter
splice signals
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Getting started
Capture efficiency and coverage Overall and by gene
Specimen type differences Fresh-frozen vs. FFPE specimens
Detection of single nucleotide variants (SNVs) Methods Filters
Detection of indels and other mutation types Methods
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First steps
HapMap samplesKnown genotypes
lung adenocarcinomasKnown genotypes
frozen DNA sample+
FFPE DNA sample
Library prep, target enrichment
Multiplex sequencing
Analysis (coverage and comparison with genotypes)
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Significant variation in coverage by geneC
over
age
Capture baits
Target region
1000x
500 bp
Cov
erag
e
1000x
Capture baits
Target region
500 bp
Good coverage of EGFR Poor coverage of CEBPA
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Significant variation in coverage by gene
NA19129 coverage distribution by gene (black bar = median; box = 25-75%ile)
* *
Capture for genes with poor coverage have been redesigned
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Fresh vs. FFPE: Coverage by gene
Tumor 1 normalized coverage, by gene(solid = frozen, hatched = FFPE)
Only minor differences are apparent between fresh-frozen and FFPE data
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Re-designing of capture set
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Defining clinical NGS guidelines
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http://www.cdc.gov/genomics/gtesting/ACCE/
ACCE
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Defining clinical validation
AccuracyDegree of agreement between the nucleic acid sequences derived from the assay and a reference sequence
Precision
Repeatability—degree to which the same sequence is derived in sequencing multiple reference samples, many times. Reproducibility—degree to which the same sequence is derived when sequencing is performed by multiple operators and by more than one instrument.
Analytical Sensitivity
The likelihood that the assay will detect a sequence variation, if present, in the targeted genomic region.
Analytical Specificity
The probability that the assay will not detect a sequence variation, if none are present, in the targeted genomic region.
Diagnostic Specificity
The probability that the assay will not detect a clinically relevant sequence variation, if none are present, in the targeted genomic region.
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Reproducibility
Test Type Definitions
Inter-Tech (Stringent)
The technicians performing the run were different, but the experiment and lanes were the same.
Inter-Tech (Relaxed) The technicians performing the run were different for each comparison. We did not control for the experiment or lane.
Intra-Tech The technician performing the run was the same. The experiment was different.
Inter-Lane (All) The lanes are different. These experiments, the techs were different in two, and the same in two.
Inter-Lane & Intra-Tech
The lanes are different. In these experiments, the techs were the same.
Intra-Lane & Inter-Tech
The lanes are the same. In these experiments, the techs were different.
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Reproducibility
Inter-Tech (Stringent)
Inter-Tech (Relaxed)
Intra-Tech Inter-Lane (All)
Inter-Lane & Intra-Tech
Intra-Lane & Inter-Tech
90.0%
92.0%
94.0%
96.0%
98.0%
100.0%
98.1% 97.9%97.1%
98.6% 98.7% 98.4%
Reproducibility
Variability Method
Perc
ent A
gree
men
t
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Major barriers for clinical implementation of NGS
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Data tsunami
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1. Need expertise in Biomedical Informatics
2. Need clinical grade “user-friendly-soup to nuts” software solution
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3. Hardware
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Informatics pipeline workflow
Patient Physician
Sample
OrderSequence
Tier 1:Base CallingAlignment
Variant Calling
Tier 2:Genome Annotation
Medical Knowledgebase
Tier 3: Clinical Report
EHR
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Order Intake
• Patient samples accessioned in Cerner CoPath• Gene panels ordered through CoPath• Orders received will initiate workflow
HL7
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Order Intake
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Tier 1 Informatics
• Optimized pipelines using several base callers, aligners, and variant calling algorithms to meet CAP/CLIA standards– Easily customizable and updateable
• Facilitates new panel introduction and the rapid delivery of novel analytical tools and pipelines
– Seamless to the clinical genomicist
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Inspection of coverage for each run
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QC metrics (sample level)
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QC metrics (exon level)
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Tier 1 Informatics
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Cancer specific analysis pipeline
Data Output
FASTQ Sequence
Output
HiSeqMiSeq
NovoalignTM
SNVCalls
IndelCalls
TranslocationValidation
GATK/Samtools
Pindel
Breakdancer SLOPE
Parse Data
SNVFiltering
MergedVCF file
TranslocationCalls
Read Alignment
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Tier 2 Informatics
• Deliver a clinical grade variant database that meets CAP/CLIA standards– Requires combined expertise of
informaticians and clinical genomocists/pathologists
• Future interoperability with (inter)national variant databases that meet CAP/CLIA standards
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Tier 2 Informatics
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Tier 3 Informatics
EGFR (L858R)
Response rates of >70% in patients with non-small cell lung cancer treated with either erlotinib or gefitinib
KRAS (G12C)
Poor response rate in patients with non-small cell lung cancer treated with either erlotinib or gefitinib
+
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Tier 3 Informatics: Variant classificaiton
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Clinical NGS process map
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Conclusions• Cancer NGS gene panel helps in
multiplexing key actionable genes for a cost effective, accurate and sensitive assay
• Targeted cancer panel are useful for “drug repurposing” of small molecule inhibitors
• Clinical validation of NGS assays in cancer is complex and labor intensive but basic principles remain
• Bioinformatic barriers are the most challenging
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Karen Seibert, John Pfiefer, Skip Virgin, Jeffrey Millbrandt, Rob Mitra, Rich HeadRakesh Nagarajan and his Bioinf. teamDavid Spencer, Eric Duncavage, Andy Bredm.Hussam Al-Kateb, Cathy CottrellDorie Sher, Jennifer StratmanTina Lockwood, Jackie PaytonMark Watson, Seth Crosby, Don ConradAndy Drury, Kris Rickoff, Karen NovakMike Isaacs and his IT TeamNorma Brown, Cherie Moore, Bob FeltmannHeather Day, Chad Storer, George BijoyDayna Oschwald, Magie O Guin, GTAC teamJane Bauer and Cytogenomics &Mol path team
MANY MORE!