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
Laura J. Tafe, MD
Assistant Professor of Pathology
Assistant Director, Molecular Pathology
CTOP Retreat May 23, 2014
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
Emulsification and Enrichment
Sequencing and Data Analysis
-minimum tumor cellularity: 10%
-8 unstained slides
Clonal amplification of DNA on Ion Spheres (ISP’s)
DNA Quantification PicoGreen Method
Enrichment of ISP’s with DNA
Barcode Adaptor Ligation
Data Annotation, Review and Sign-out
Library Quantification and Pooling
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
Courtesy of F. de Abreu
Low cost+, convenient, single use device.
Easy, automatic fluid connections.
Match the size of the Ion chip to your application.
Single pool of primers
207 Primer Pairs
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)
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:
EGFR: MUTATION c.2573T>G p.L858R Exon 21
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:
203 non-squamous NSCLC cases on Ion Torrent AmpliSeq Hotspot Panel v2
(May 2013 – May 2014)
Specimen types tested
Needle Core: 30%
Wild Type: 13%
Most Frequent Mutations
Other = Mutations in 32 additional genes were seen in 1-7 cases each
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
SRM peptide (outside KD)
Wang R et al. Clin Cancer Res 2012;18:4725-4732
Heterozygous Single Nucleotide Point Mutation in ALK for 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)
Homozygous G results in DPEGVPPLLVQQAK (WT) from both DNA alleles.
Shaw. JCO. 2013. 31(8):1105-1111