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Clinical Genotyping of Lung Cancer in the Era of Personalized Medicine

Clinical Genotyping of Lung Cancer in the Era of Personalized Medicine. Laura J. Tafe, MD Assistant Professor of Pathology Assistant Director, Molecular Pathology CTOP Retreat May 23, 2014. Overview. Overview of molecular workflow NGS 50 gene panel experience Mass spec ALK project.

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Clinical Genotyping of Lung Cancer in the Era of Personalized Medicine

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  1. Clinical Genotyping of Lung Cancer in the Era of Personalized Medicine Laura J. Tafe, MD Assistant Professor of Pathology Assistant Director, Molecular Pathology CTOP Retreat May 23, 2014

  2. Overview • Overview of molecular workflow • NGS 50 gene panel experience • Mass spec ALK project

  3. Histology matters • Any primary lung cancer with adenocarcinoma histology • May be mixed (ADC-SQC, ADC-SCLC) • No pure SQC, SCLC or neuroendocrine • Poorly differentiated tumors should be tested

  4. Pre-analytical Workflow Molecular testing ordered by surgical pathologist 2 H&E and 10 USS MG Pathologist review of H&E for adequacy and % tumor 1 H&E and 2 USS to FISH lab to hold for additional testing as needed (rearrangements by FISH) DNA extracted from USS in molecular laboratory for PCR

  5. NGS (Analytical) Workflow Sample Preparation Library Preparation Emulsification and Enrichment Sequencing and Data Analysis • 318 IonChip • Majority of amplicon coverage >500X • PCR • AmpliSeqHotSpot Cancer Panel • 201 amplicons • 50 genes • Require 10ng DNA DNA Extraction -minimum tumor cellularity: 10% -8 unstained slides Emulsification PCR Clonal amplification of DNA on Ion Spheres (ISP’s) • Variant Calling • Ion Torrent Variant Caller Plugin • Reference genome: hg19 DNA Quantification PicoGreen Method ISP’s quantification Enrichment of ISP’s with DNA FuPa Treatment • Reporting • Golden Helix SVS Software • Variant Call Summary • Variant Prediction Barcode Adaptor Ligation Data Annotation, Review and Sign-out Library Quantification and Pooling (qPCR) Total time: ~14h Hands on time: ~5h Total time: ~9h Hands on time: ~3h Total time: ~8h Hands on time: ~4h Total time: ~7h Hands on time: ~1h Day 3-4 Day 5 Day 1-2 Day 6-7 Courtesy of F. de Abreu

  6. 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.

  7. 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)

  8. Post-analytical WorkflowAnalysis Pipeline:Variant-Calling and Annotation • 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.

  9. EGFR Exon 21 p.L858R (c.2573T>G)

  10. EGFR Exon 19 18bp deletion

  11. Example report INDICATION 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: NORMAL EGFR: MUTATION c.2573T>G p.L858R Exon 21 KRAS: NORMAL PIK3CA: 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

  12. 203 non-squamous NSCLC cases on Ion Torrent AmpliSeq Hotspot Panel v2 (May 2013 – May 2014)

  13. Resection: 24% Specimen types tested Consult: 13% Needle Core: 30%

  14. EGFR KRAS BRAF ERBB2 ins PIK3CA QNS: 8% Types of Mutations Wild Type: 13% Actionable: 48% VUS: 31%

  15. Most Frequent Mutations Other: 16% KRAS: 30% STK11: 10% EGFR: 12% TP53: 32% Other = Mutations in 32 additional genes were seen in 1-7 cases each

  16. 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%)

  17. Limitations of AmpliSeq • CNVs • Structural variants (rearrangements/translocations) • mRNA

  18. 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

  19. Selected Reaction Monitoring (SRM) - MS

  20. ALK exons SRM peptide (outside KD) 5’ NH2 3’ COOH Wang R et al. Clin Cancer Res 2012;18:4725-4732

  21. 11 samples from 10 patients (6 with ALK rearrangement)

  22. 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) 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 aa1429-1620). Homozygous (G in both alleles) C0481-T2LR-C (DH1) Homozygous G results in DPEGVPPLLVQQAK (WT) from both DNA alleles.

  23. Crizotinibresistance in ALK-positive lung cancer Shaw. JCO. 2013. 31(8):1105-1111

  24. Hypothesis: Missing 192aa might alter the function of the ALK fusion protein and response to ALK inhibitors

  25. 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

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