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Clinical Genotyping of Lung Cancer in the Era of Personalized Medicine
Laura J. Tafe, MDAssistant Professor of Pathology
Assistant Director, Molecular Pathology
CTOP Retreat May 23, 2014
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Overview
• Overview of molecular workflow• NGS 50 gene panel experience• Mass spec ALK project
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Histology mattersLung Cancer Histology
AdenocarcinomaSquamous cell carcinomaLarge cell car-cinomaSmall cell car-cinoma
Any primary lung cancer with adenocarcinoma histologyMay be mixed (ADC-SQC, ADC-SCLC)No pure SQC, SCLC or neuroendocrinePoorly differentiated tumors should be tested
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MG Pathologist review of H&E for adequacy and % tumor
DNA extracted from USS in molecular laboratory for PCR
Molecular testing ordered by surgical pathologist 2 H&E and 10 USS
1 H&E and 2 USS to FISH lab to hold for additional testing as needed (rearrangements by FISH)
Pre-analytical Workflow
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NGS (Analytical) Workflow
Total time: ~9hHands on time: ~3h
Sample Preparation
Library Preparation
Emulsification and Enrichment
Sequencing and Data Analysis
DNA Extraction -minimum tumor cellularity: 10%
-8 unstained slides
PCRAmpliSeq
HotSpot Cancer Panel
• 201 amplicons• 50 genes• Require 10ng
DNADNA
Quantification PicoGreen Method
FuPa Treatment
Barcode Adaptor Ligation
Library Quantification and
Pooling(qPCR)
Emulsification PCRClonal amplification of DNA on Ion Spheres
(ISP’s)
ISP’s quantification
Enrichment of ISP’s with DNA
318 IonChip• Majority of amplicon
coverage >500X
Variant Calling• Ion Torrent Variant
Caller Plugin• Reference genome:
hg19
ReportingGolden Helix SVS
Software• Variant Call Summary• Variant Prediction
Data Annotation, Review and Sign-out
Total time: ~8hHands on time: ~4h
Total time: ~7hHands on time: ~1h
Total time: ~14hHands on time: ~5h
Courtesy of F. de AbreuDay 1-2 Day 3-4 Day 5 Day 6-7
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Ion Torrent Technology • Simple, robust, scalable and cost effective.
Low cost+, convenient, single use device.
Easy, automatic fluid connections.Match the size of the Ion chip to your application.
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AmpliSeq Cancer Hotspot Panel v2 Single pool of primers
• 207 Primer Pairs• 50 Genes• 10 ng input DNA
Targets genomic "hot spots“
1 year: ~ 500 clinical samples + ~ 100 research samples
Weekly run: ~ 20 samples
TAT: 7 days (samples in the lab)
ABL1 EGFR GNAS KRAS PTPN11
AKT1 ERBB2 GNAQ MET RB1
ALK ERBB4 HNF1A MLH1 RET
APC EZH2 HRAS MPL SMAD4
ATM FBXW7 IDH1 NOTCH1 SMARCB1
BRAF FGFR1 IDH2 NPM1 SMO
CDH1 FGFR2 JAK2 NRAS SRC
CDKN2A FGFR3 JAK3 PDGFRA STK11
CSF1R FLT3 KDR PIK3CA TP53
CTNNB1 GNA11 KIT PTEN VHL
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Post-analytical WorkflowAnalysis Pipeline:
Variant-Calling and Annotation
Run Variant Caller Version
4.0
Upload VCF File to Golden
Helix SVS (Version 7.7.8) and Annotate
Variants
FILTERNon-Coding
Variants
FILTERSynonymous
Variants
FILTER<5% SNVs
<20%INDELS
Review Remaining Variants in
IGV, FILTER Homopolymeric Variants and
Sequencing Artifacts
Report Remaining Variants To Clinicians
Variant calls and annotation:• Initially filtered to remove non-coding and synonymous mutations. • Golden Helix then used to annotate and help predict pathogenicity. • All reported variants received sufficient coverage and were of high
enough frequency to be annotated as true variants.
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EGFR Exon 21 p.L858R (c.2573T>G)
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EGFR Exon 19 18bp deletion
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Example reportINDICATION FOR STUDY: Lung, right (CT-guided needle core biopsy): Adenocarcinoma
SPECIMEN ANALYZED: Cytology or surgical #, Block #
Analysis: Examination of DNA extracted from formalin-fixed paraffin-embedded tumor tissue for somatic mutation analysis.Results: The following gene variants were identified in the submitted tissue:
CLINICALLY ACTIONABLE: BRAF: NORMALEGFR: MUTATION c.2573T>G p.L858R Exon 21KRAS: NORMALPIK3CA: NORMAL
NOT CLINICALLY INDICATED:TP53 c.421C>T p.R141C Exon 4
Interpretation: After review of the pathology report and slides, the specimen (N-14-00257, Block A2) was selected for mutation analysis from a panel of 50 genes. The results of this test indicate that tumor cells comprising 25.0% of the tissue specimen analyzed were normal for BRAF, KRAS and hotspots in 46 other genes. A p.L858R activating mutation was detected in exon 21 of the EGFR gene suggesting that this patient may benefit from anti-EGFR therapy. In addition, a mutation of unknown clinical significance was detected in the TP53 gene. Therapeutic options related to the presence or absence of mutations should be carefully assessed. Availability of other therapeutic indications and clinical trials may be possible. For additional information on reported variants please visit:http://www.mycancergenome.org/content/disease/lung-cancer
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203 non-squamous NSCLC cases on Ion Torrent AmpliSeq Hotspot
Panel v2 (May 2013 – May 2014)
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Specimen types tested
Cell Block: 33%
Needle Core: 30%
Consult: 13%
Resection: 24%
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Types of Mutations
VUS: 31%
Actionable: 48%
Wild Type: 13%QNS: 8%
EGFRKRASBRAFERBB2 insPIK3CA
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TP53: 32%
KRAS: 30%
EGFR: 12%
STK11: 10%
Other = Mutations in 32 additional genes were seen in 1-7 cases each
Most Frequent Mutations
Other: 16%
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Uncommon mutations
• EGFR– 2 – Exon 20 insertion (1%)– 3 – Exon 18 (1.5%)– 3 – T790M (1.5%)
• BRAF– 7 mutations (only 3 - V600E) (3%)
• ERBB2– 2 – exon 20 insertion (1%)
• PIK3CA– 9 mutations (4%)
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Limitations of AmpliSeq
• CNVs• Structural variants (rearrangements/translocations)
• mRNA
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Quantification of ALK from Formalin-Fixed Paraffin-Embedded Non-small Cell Lung Cancer (NSCLC) Tissue by Mass Spectrometry
Christopher P. Hartley 1, Wei-Li Liao2, Jon Burrows2, Todd Hembrough2, and Laura J. Tafe1
1Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH and 2OncoPlex Diagnostics, Rockville, MD
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Selected Reaction Monitoring (SRM) - MS
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ALK exons
Wang R et al. Clin Cancer Res 2012;18:4725-4732
5’ NH2 3’ COOH
SRM peptide (outside KD)
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11 samples from 10 patients (6 with ALK rearrangement)
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Heterozygous Single Nucleotide Point Mutation in ALK for DH9(ALK kinase domain: 1116-1392, peptide 1417D P E G V P P L L V S Q Q AK1431 is C-terminal to the KD)
C0481-T2LR-C (DH1)
Homozygous G results in DPEGVPPLLVQQAK (WT) from both DNA alleles.
Homozygous (G in both alleles)
C0483-T2LR-C (DH9)Heterozygous (T in one allele and G in the other)
Heterozygous G/T results in DPEGVPPLLVQQAK (WT) from one allele and DPEGVPPLLVSQ*AK (Q to stop codon*) in the second allele introducing a stop codon (p.Q1429X) within the MS targeted peptide (missing aa 1429-1620).
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Shaw. JCO. 2013. 31(8):1105-1111
Crizotinib resistance in ALK-positive lung cancer
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Hypothesis: Missing 192aa might alter the function of the ALK fusion protein and response to ALK inhibitors
DH# M/F Age at dx Smoking hx StageProgression free
survival Status
DH1 F 68Non-smoker (3 pyr;
40 yrs prior) pT2a N2 7 mos + (?) NED - lost to f/u
DH2 M 54 Never smoker pT2a pN2 14 mos NED
DH3 F 49 Never smoker pT1a N2 19 mos NED
DH4 M 76Former (stopped -
40 pyr) stage 4 12 mosAWD -Stable brain
met
DH5/6 M 62
Never smoker (second hand
smoke exposure 22 yrs) pT2b pN1 no crizotinib tx NED
DH9 M 65Smoker
(quit 1 yr ago) pT2 pN1 4 mos AWD
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Conclusions
• The Ion Torrent Ampliseq technology: Successfully performed on small biopsy / cytology
specimens Requires very little input DNA (10ng)
• Mass Spectrometry proteomic techniques are complementary to molecular analysis and have potential to identify clinically meaningful biomarkers