“Next Generation Sequencing: What Will it Mean for the ... · PDF fileTopics •Brief...
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“Next Generation Sequencing: What Will
it Mean for the Pathologist?”
Christopher Corless, MD, PhD
Professor of Pathology, Oregon Health & Science University
Chief Medical Officer, Knight Diagnostic Laboratories
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Disclosures
Company Activity
Ion Torrent/ThermoFisher Travel support; reagents
Ventana/Roche Travel support
Genentech/Roche Honoraria & consulting fees
Novartis Consulting fees
I will mention off-label uses of cancer therapeutics
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Topics
• Brief introduction to next-gen sequencing (NGS)
• Critical pre-analytical factors
• Usefulness of quantitative output from NGS
– Detecting mutations – correlation with specimen
– Fusion gene detection
– Copy number assessment
• Data overload and implications for pathologists
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42 year old male with ‘glioblastoma’ treated with surgery, temozolomide and radiation
Bone and lymph node mets appeared at 20 months (what is this tumor?)
Admitted to OHSU to manage pain, monitor pending cord compression
Another round of chemo failed
BRAF V600E mutation identified
Patient started on dabrafenib
Excellent clinical response
Individualized Cancer Medicine - An Example -
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Garraway, JCO epub April 15, 2013
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SHC GRB2
SOS
BRAF
MEK
ERK
P
P
P
P P P
P
P
P
P
PI3K PDK
AKT
P
mTOR
S6K
P
P
P
PTEN
>500 Targeted Therapeutics in Development
Receptor tyrosine kinases
ALK or ROS1
Erlotinib
Lapatinib
Sorafenib
Imatinib
Sunitinib
Afatinib
Dovitinib
Vemurafenib
Dabrafenib
LGX818
Trametinib
Selumetinib
Benitinib
Everolimus
Temsirolimus
Buparlisib
BGT226
BYL719
Crizotinib
Ceritinib
MK2206
SR13668
Trastuzumab
Cetuximab
Panitumumab
METMab
CDK4/6 P1446A-05
LEE001
NRAS
KRAS
HRAS
BEZ235
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DNA Sequencing Traditional Sanger Method
Capillary electrophoresis Final Sequence
BRAF V600E
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Next-Generation DNA Sequencing • Massively parallel sequencing (many
sequencing reactions performed simultaneously)
ACTGGTCCTGCTGGTTAG
ACTGGTCCTGCTGGTTAG
ACTGGTCCTGCTGGTTAG
ACTGGTCCTGCTGGTTAG
ACTGGTCCTGCTGGTTAG
ACTGGTCCAGCTGGTTAG
ACTGGTCCAGCTGGTTAG
ACTGGTCCAGCTGGTTAG
ACTGGTCCAGCTGGTTAG
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G T C
V
842
Frameshift
G A C A T C A T G C ……
G T C A T A T G C …… [Stop]
V A L
PDGFRA D842V and M844fs*16
Gastrointestinal Stromal Tumor (GIST)
Known GIST driver mutation Truncates kinase domain
A S P
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Applications of Next-Gen Sequencing
• Broad-based approaches
– Whole genome: all 3.2 billion base pairs
– Whole transcriptome: all mRNAs in a sample
– Whole exome: ~1.6% of genome that encodes proteins
• Targeted approaches
– Panels of genes (10-400)
– Limited to targets regarded as actionable or prognostic
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Ion Torrent Proton Illumina NextSeq 500
Ion Torrent PGM Illumina MiSeq (FDA-approved version available)
NGS Sequencers in Clinical Labs
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Next-Gen Sequencing Read Out
1. Base-calling: sequencer generates millions of ‘reads’ (50 to 200 base pair stretches of DNA)
2. Alignment: software matches the reads to the known genome
3. Variant calling: software determines where the sequence differs from a standard reference genome
4. Variant annotation: software programs + manual review to determine whether a variant is a:
– Benign polymorphism
– Variant of unknown significance
– Mutation (single nucleotide change, deletion, etc)
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NGS Sequencing Methodology
Hybrid-Capture Amplicon
Applications Whole exome; larger panels
Smaller panels
Input DNA 100 ng 10-20 ng
Overnight hybridization Yes No
Gene copy number Yes Yes
Fusion gene detection Yes Yes (RNA)
• 5 x 4 μm unstained sections of 1 cm core biopsy
is sufficient for amplicon-based method • New extraction method may work with just 4 mm2 from a single section
How Much Tissue Is Needed?
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• Specimen decalcification
• Acid is often used to soften the bone for sectioning and this degrades nucleic acids
• EDTA-based solutions are better
Result
Mimics a C>T mutation
• DNA deamination is common in older paraffin blocks
Pre-Analytic Factors Influencing NGS
• Tumor fraction: % of specimen comprised of tumor
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Impact of Tumor Purity
Mutant Allele Ratio in Tumor Cells
Percent Tumor in Specimen
Mutant Allele Ratio in Sequenced DNA
50% 90% 45%
50% 70% 35%
50% 50% 25%
50% 30% 15%
50% 10% 5%
• Each cellular nucleus in a specimen contributes DNA • % Tumor fraction is based on tumor nuclei, not area • The lower limit of sensitivity for most NGS panels is ~2-3%
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Selecting Tumor-Rich Material
for DNA Extraction
Coring
a block Scraping
slides
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Metastatic
Melanoma in Lung
Tumor purity: ~90%
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Metastatic
Colorectal
Adenocarcinoma
in Mesentery
Tumor purity: ~10-20%
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Transbronchial Biopsy
Adenocarcinoma of the Lung
Please,
get another
sample!
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• NRAS p.Q61R (27% mutant allele)
• TP53 p.R282W (26% mutant allele)
• Tumor fraction: 52-54%
Metastatic Colorectal Adenocarcinoma
Estimated Tumor Fraction
• Microscope: 60%
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Quantitating
Tumor Nuclei
Using
CellProfiler
Semi-automated
using
machine learning
algorithm
• Tumor nuclei
• Normal nuclei
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KRAS
EGFR
ALK BRAF
PIK3CA ERBB2
MAPK1
NTRK1
NTRK3
MET ROS1
HRAS
RET
DDR2
PTEN
ARAF
FGFR3
FGFR2
FGFR1
Unknown
Molecular Subtypes of NSCLC Before 2000
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KRAS
EGFR
ALK BRAF
PIK3CA ERBB2
MAPK1
NTRK1
NTRK3
MET ROS1
HRAS
RET
DDR2
PTEN
ARAF
FGFR3
FGFR2
FGFR1
Unknown
Molecular Subtypes of NSCLC in 2003
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KRAS
EGFR
ALK BRAF
PIK3CA ERBB2
MAPK1
NTRK1
NTRK3
MET ROS1
HRAS
RET
DDR2
PTEN
ARAF
FGFR3
FGFR2
FGFR1
Unknown
Molecular Subtypes of NSCLC in 2015
FDA approved drugs
FDA approved drugs in other cancers
In clinical trials
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NSCLC Case Example
• 47 y/o woman with treatment refractory
bronchioalveolar carcinoma
• Genotyping: BRAF V600E mutation
• Phase I study combining BRAF + MEK inhibitors: 6 month response
• Disease controlled for 7 months; she died about 1 year later
Baseline 2 months
http://www.oregonlive.com/health/index.ssf/
2012/12/new_oregon_health_science_univ_1.html
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Interim results of phase II study BRF113928 of dabrafenib in BRAF V600E
mutation–positive non-small cell lung cancer (NSCLC) patients.
J Clin Oncol 31, 2013 (suppl; abstr 8009)
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KRAS
EGFR
ALK BRAF
PIK3CA ERBB2
MAPK1
NTRK1
NTRK3
MET ROS1
HRAS
RET
DDR2
PTEN
ARAF
FGFR3
FGFR2
FGFR1
Unknown
GENE FUSIONS
Molecular Subtypes of Lung Cancer 2015
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ROS1 fusions in lung adenocarcinoma
• Discovery published in Jan. 2012
• Trial published Nov. 2014
• FDA-approved tx in Apr. 2015
• 2nd Line drug published in Oct. 2014
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Kinase
Fusion FISH IHC RNAseq
ALK Yes Yes Yes
AXL Yes ?? Yes
BRAF Yes No Yes
FGFR1 Yes No Yes
FGFR2 Yes No Yes
FGFR3 Yes No Yes
MET Yes No Yes
NTRK1 Yes ?? Yes
NTRK3 Yes ?? Yes
PGDFRA Yes No Yes
RET Yes ?? Yes
ROS1 Yes Yes Yes
Detecting Actionable Gene Fusions In NSCLC
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Kohno et al. Trans Lung Cancer Res. Vol 4, No 2 (April 2015)
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Target Genes (169 potential fusions / 94 partners)
ALK BRAF FGFR1 FGFR2 FGFR3 MET
NTRK1 NTRK2 NTRK3 PDGFRA PDGFRB RAF1
RET ROS1 TMPRSS2 EGFR EGFRvIII ERBB4
NRG1 AKT3
Gene Expression
ALK ROS1 RET EGFR NTRK1 NTRK2
NTRK3 BRAF Housekeeping genes (5)
Also useful for: • Cholangioca • Bladder ca • HNSQCC
Beadling et al., submitted
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Detecting Copy Number Alterations by NGS
FGFR1
ERRB2/HER2
Normal
(2 Copies)
Breast Carcinoma With FGFR1 and HER2 Amplification
Validation
Samples
•Known
FISH status
•Microarray
results
Grasso et al. J Mol Diag 17:53-63; 2015
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FFPE Ductal Carcinoma of the Breast
EGFR
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EGFR Amplification in Breast Carcinoma
68 year old F
Tumor metastatic
to the liver
HER2-negative
[6% of breast
carcinomas have
EGFR amplif]
Bhargava et al. Mod Pathol. 2005 Aug;18(8):1027-33
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Example of Variant List For a 37-Gene Panel Head & Neck Squamous Cell Carcinoma
Chrom Position_Start Position_End Ref Variant Type Consequence Zygosity Var_Freq Gene p_AA_change
chr4 55968089 55968089 T G SNP nonsynonymous Het 61.29 KDR p.K747N
chr9 21971096 21971096 C A SNP nonsynonymous Het 83.33 CDKN2A p.E88*
chr17 7577548* 7577548 CGCCCA C DEL stop gain Het 30.52 TP53 p.G245G
chr17 7577557* 7577557 A C SNP stop gain Het 31.28 TP53 p.C242G
chr17 7577558* 7577558 G C SNP stop gain Het 32.33 TP53 p.S241S
chr7 55229255 55229255 G A SNP nonsynonymous Het 23.13 EGFR p.R521K
chr17 7579472 7579472 G C SNP nonsynonymous Het 31.25 TP53 p.P72R
chr17 37884037 37884037 C G SNP nonsynonymous Het 60 ERBB2 p.P1170A
chr17 37879588 37879588 A G SNP nonsynonymous Het 65.48 ERBB2 p.I655V
chr2 29416366 29416366 G C SNP nonsynonymous Het 92.68 ALK p.D1529E
chr2 29416572 29416572 T C SNP nonsynonymous Hom 99.34 ALK p.I1461V
chr9 139418260 139418260 A G SNP synonymous Het 7.37 NOTCH1 p.N104N
chr2 29455267 29455267 A G SNP synonymous Het 8.61 ALK p.G845G
chr7 55238874 55238874 T A SNP synonymous Het 23.68 EGFR p.T629T
chr17 29508775 29508775 G A SNP synonymous Het 24.79 NF1 p.L234L
chr4 55602765 55602765 G C SNP synonymous Het 34.95 KIT p.L862L
chr17 29553485 29553485 G A SNP synonymous Het 52.07 NF1 p.P678P
chr12 25362777 25362777 A G SNP synonymous Het 57.24 KRAS p.D173D
chr7 116436097 116436097 G A SNP synonymous Het 80 MET p.P1382P
chr7 116435768 116435768 C T SNP synonymous Het 82.81 MET p.D1304D
chr7 116436022 116436022 G A SNP synonymous Het 85.36 MET p.A1357A
chr2 29416615 29416615 G A SNP synonymous Het 85.88 ALK p.T1446T
chr2 29445458 29445458 G T SNP synonymous Het 86.96 ALK p.G1125G
chr2 30143499 30143499 G C SNP synonymous Hom 97.3 ALK p.L9L
chr7 55214348 55214348 C T SNP synonymous Hom 99.26 EGFR p.N158N
chr7 55266417 55266417 T C SNP synonymous Hom 99.5 EGFR p.T903T
chr12 25368462 25368462 C T SNP synonymous Hom 99.61 KRAS p.R161R
chr2 29940529 29940529 A T SNP synonymous Hom 99.62 ALK p.P234P
chr2 29543663 29543663 T C SNP synonymous Hom 99.65 ALK p.Q500Q
chr7 55249063 55249063 G A SNP synonymous Hom 99.87 EGFR p.Q787Q
chr4 1807894 1807894 G A SNP synonymous Hom 100 FGFR3 p.T651T
Gene
Amino acid
Change
KDR K747N
CDKN2A E88*(stop)
TP53 G245G
TP53 C242G
KDR encodes VEGFR2
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Head & Neck SQCC
Before Treatment Treatment Day 15
TKI With VEGFR2 Activity
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“We need to provide knowledge, not just data” Dr. Kojo Elenitoba-Johnson, Nov. 2013
• 40 year old male with thymic carcinoma and no good treatment options
• Tumor sequenced by a commercial laboratory (large gene panel)
–No actionable mutations reported
–Variants of unknown significance at very bottom of report included “KIT Y646D”
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KIT-Mutant Thymic Carcinoma
Patient Exon Mutation Treatment Response Reference
54 yr M KIT 11 Deletion V560D
Imatinib Mixed 6 mo
N Engl J Med. 2004 Jun 17;350(25):2625-6
46 yr M KIT 17 D820E Sorafenib PR 15+ mo
J Thorac Oncol. 2009 Jun;4(6):773-5
47 yr F KIT 11 Deletion 557-559
Sorafenib PR 6+ mo
Lung Cancer. 2011 Jan;71(1):109-12
48 yr M KIT 11 Y553N Imatinib PR 9+ mo
J Clin Oncol. 2011 Nov 20;29(33):e803-5
55 yr F KIT 11 Deletion V560D
Imatinib SD 12+ mo
J Thorac Oncol. 2014 Feb;9(2):e12-6
58 yr M KIT 13 K642E Sorafenib PR 6 mo
Onco Targets Ther. 2014, 7:697-702
• Y646D is a non-conservative substitution located near K642E
• Recommendation to oncologist: try a KIT inhibitor
• KIT Mutations occur in ~10% of thymic carcinomas
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Sorafenib x 12 days
Patient remains on treatment and is doing well at 15 months
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Illumina NextSeq500
Clinical Genomics Database
Clinical Reports
Public Databases COSMIC
dbSNP, Clinvar PCT (MD Anderson) My Cancer Genome
CIViC
Exacloud Computing Cluster
LIS
Ion Torrent PGM
Turning Data Into Knowledge
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Whole Exome Sequencing (Sequencing All Protein-Coding Regions Across 20,000 Genes)
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OHSU/Intel Collaboration
Clinical Sequence Data
Research Genomic Data
Exacloud Computing
Center Variant
Annotation
Sequence alignment Variant calling
Data Reporting
Clinical Genomics Database
7,000 Processors for data crunching 220 Terabytes of storage for clinical datasets
1200 Terabytes of storage for research samples
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‘Liquid’ Biopsies DNA in Plasma and Urine
Commercial Labs
• Small fragments of DNA are present in normal plasma and urine
• Increased levels occur in patients with advanced cancer
• New, high sensitivity tests based on next-gen sequencing can be used to detect mutations in such samples
• May be useful in monitoring treatment responses
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Summary
• Advances in DNA sequencing technology are supporting the new era of ‘precision’ cancer care
• NGS supports: o Determination of exact allele frequency o Detection of gene fusion events o Assessment of gene copy number
• The pathologist plays a critical role in selecting material appropriate for testing
• NGS panels are becoming very routine and can be integrated into practice as another tool alongside IHC, FISH, etc.
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Acknowledgements
Carol Beadling, PhD
Richard Press, MD, PhD
Tanaya Neff, MA
Fei Yang, MD
Rebecca O’Gara
Marina Pukay
Andrea Warrick
Cara Poage
Amy Schilling
Catie Grasso, PhD
With Assistance From:
• Knight Cancer Institute
• Ion Torrent/ThermoFisher
• Intel
• GIST Cancer Research Fund
• LifeRaft Group
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AKT1 1%
BRAF 44%
CTNNB1 2%
GNAQ 1% KIT
1% MAP2K1
1%
NRAS 16%
PIK3CA 1%
TP53 1%
No Mutation
34%
Cutaneous Melanoma Cases (n=140)
Updated from Beadling et al. J Molec Diag, 2011 Sep;13(5):504-13
BRAF V600E(K)
• Vemurafenib
• Dabrafenib
• Trametinib
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Best Response to Vemurafenib in
Metastatic BRAFV600E Melanoma
Flaherty , et al. N Engl J Med. 2010 Aug 26;363(9):809-19
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KRAS p.A146T
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Metastatic BRAFV600E Papillary Thyroid Carcinoma
Treated With Vemurafenib
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AKT1 1%
BRAF 44%
CTNNB1 2%
GNAQ 1% KIT
1% MAP2K1
1%
NRAS 16%
PIK3CA 1%
TP53 1%
No Mutation
34%
Cutaneous Melanoma Cases (n=140)
Updated from Beadling et al. J Molec Diag, 2011 Sep;13(5):504-13
Would imatinib
work in
melanoma?
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KIT-Mutant Mucosal Melanoma
Hodi et al. J Clin Onc 2008 Apr 20;26(12):2046-51
Response to Imatinib
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Targeting KIT-Mutant Melanoma
Phase II Clinical Trials
Drug
# Pts
Clinical Benefit Rate
Carvajal et al. JAMA 2011;305(22):2327-2334
Imatinib 25 72%
Guo et al. JCO 2011;29(21):2904-2909
Imatinib 43 53%
Minor et al. Clin Cancer Res 2012; 18(5):1457-63
Sunitinib 4 75%
Hodi et al. JCO (epub Jul 8, 2013)
Imatinib 13 77%
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Left Lower Lobe Left Upper Lobe
72 Year Old Female Smoker
KRAS G12C KRAS G12V