Clinical genotyping of lung cancer in the era of personalized medicine
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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. 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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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

  • Overview of molecular workflow

  • NGS 50 gene panel experience

  • Mass spec ALK project


Histology matters

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


Clinical genotyping of lung cancer in the era of personalized medicine

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


Ngs analytical workflow

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


Ion torrent technology

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.


Ampliseq cancer hotspot panel v2

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)


Post analytical workflow analysis pipeline variant calling and annotation

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.


Egfr exon 21 p l858r c 2573t g

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


Egfr exon 19 18bp deletion

EGFR Exon 19 18bp deletion


Example report

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


203 non squamous nsclc cases on ion torrent ampliseq hotspot panel v2 may 2013 may 2014

203 non-squamous NSCLC cases on Ion Torrent AmpliSeq Hotspot Panel v2

(May 2013 – May 2014)


Clinical genotyping of lung cancer in the era of personalized medicine

Resection: 24%

Specimen types tested

Consult: 13%

Needle Core: 30%


Types of mutations

EGFR

KRAS

BRAF

ERBB2 ins

PIK3CA

QNS: 8%

Types of Mutations

Wild Type: 13%

Actionable: 48%

VUS: 31%


Clinical genotyping of lung cancer in the era of personalized medicine

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


Uncommon mutations

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%)


Limitations of ampliseq

Limitations of AmpliSeq

  • CNVs

  • Structural variants (rearrangements/translocations)

  • mRNA


Clinical genotyping of lung cancer in the era of personalized medicine

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


Selected reaction monitoring srm ms

Selected Reaction Monitoring (SRM) - MS


Alk exons

ALK exons

SRM peptide (outside KD)

5’ NH2

3’ COOH

Wang R et al. Clin Cancer Res 2012;18:4725-4732


11 samples from 10 patients 6 with alk rearrangement

11 samples from 10 patients (6 with ALK rearrangement)


Clinical genotyping of lung cancer in the era of personalized medicine

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.


Crizotinib resistance in alk positive lung cancer

Crizotinibresistance in ALK-positive lung cancer

Shaw. JCO. 2013. 31(8):1105-1111


Clinical genotyping of lung cancer in the era of personalized medicine

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


Conclusions

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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