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

Overview

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

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

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

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

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 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

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.

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

EGFR Exon 19 18bp deletion

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

203 non-squamous NSCLC cases on Ion Torrent AmpliSeq Hotspot

Panel v2 (May 2013 – May 2014)

Specimen types tested

Cell Block: 33%

Needle Core: 30%

Consult: 13%

Resection: 24%

Types of Mutations

VUS: 31%

Actionable: 48%

Wild Type: 13%QNS: 8%

EGFRKRASBRAFERBB2 insPIK3CA

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%

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

• CNVs• Structural variants (rearrangements/translocations)

• mRNA

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

ALK exons

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

5’ NH2 3’ COOH

SRM peptide (outside KD)

11 samples from 10 patients (6 with ALK rearrangement)

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).

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

Crizotinib resistance in ALK-positive lung cancer

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

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