Clinical Cancer Genotyping Using Next-Generation Sequencing

Long Phi Le, MD, PhD

Department of Pathology Center for Integrated Diagnostics Massachusetts General Hospital

Boston, MA lple@partners.org

Disclosures

Co-Founder of ArcherDx Enzymatics: Equity holder, Consultant Broad Clinical Research Sequencing Platform: Medical Director

The case for broad clinical cancer genotyping Our current approach at MGH Challenges

The New Paradigm in Cancer Treatment

EGFR: Erlotinib/ Gefitinib 20% Lung adenocarcinomas

ALK: Crizotinib 3-5% Lung adenocarcinoma

BRAF V600E: PLX4032 60% Melanoma

BRAF

1799 T>A

V600E

KRAS: Cetuximab resistance 36-50% Colon adenocarcinoma

Molecular Markers and Targeted Therapy

Dr. Ignatius Ou, UC Irvine

Rapid Response to Crizotinib

Identification of PF2341066

PF2341066 Inhibits ALK activity

PF2341066 demonstrates cytocidal activity in cells exhibiting ALK fusion (Pfizer in house)

PF2341066 activity in cells exhibiting ALK fusion in broad screen (MGH-McDermott)

Discovery of EML4-ALK fusions in NSCLC (CREST) Japan Science & Technology Agency)

2007

PF2341066 FIP

May

2005 2006 2008 2009

Objective responses demonstrated in ALK fusion positive NSCLC and IMT

Phase III study of PF02341066 in ALK positive NSCLC starts

Slide Courtesy of Ross Camidge

Timeline for PF2341066 and ALK in NSCLC

ROS1-translocated NSCLC Crizotinib Response

Considerations in Clinical Genotyping Platform

Logistics • Clinical patient coordinator • Accessioning • Tracking • Automation • Test all tumors

Clinical Test • Archived FFPE tissue • Analytical sensitivity (5%) • 2 week turnaround time • Report in patient’s record • Performed in a CLIA lab

Actionable Targets • Predicts response/resistance • Clinches diagnosis • Yields prognosis • Stratify patients for trials • Adaptability for new targets

Other • Economics • Insurance and billing • Translational research • Bioinformatics • Patient Consent

COSMIC, Wellcome Trust Sanger Institute, 2010

Distribution of KRAS Mutations

ddNTP

ddNTP

ddNTP

loci of interest

Multiplex PCR Single Base Extension Reaction Capillary Electrophoresis

Electrophoretic Output

Increasing

molecular weight

Rela

tive

flu

ore

sce

nce

A B D C F E

SNaPshot Genotyping

KRAS 23%

No Mutation 42%

EGFR 15%

TP53 5%

IDH1 <1%

NRAS 1% BRAF 2%

HER2 2%

PIK3CA 4%

ALK 3%

CTNNB1 2%

AKT 1%

N=650

Lung Cancers: SNaPshot

More Than Just Point Mutations

copy number indels

SNVs

epigenetics

non-coding RNAs

proteomics

gene expression

rearrangements

Not So Common Recurrent Mutations

Real Life in a Molecular Diagnostic Lab

More With Less

MGH Center for Integrated Diagnostics

• HER2, EGFR, MET, PDGFRA, FGFR1, PIK3CA (many tumors)

• Oligodendroglioma 1p/19q FISH analysis (glioma)

• Sarcoma translocation FISH: Synovial Sarcoma, Ewing’s Sarcoma, Alveolar Rhabdomyosarcoma, Myxoid Liposarcoma • ALK, RET, ROS1 FISH (lung) • MYC FISH (lymphoma) (all FISH: 60-100)

• Hereditary Hemochromatosis/HFE (heme, 5-10)

• MGMT promoter methylation (glioma, 1-5) • Microsatellite instability (colon, 1-3) • SNAPSHOT-NGS (many tumors, 40-60)

• Chimerism (BMT transplant, 8-15)

• Array CGH (ASD, MCA, ID, 4-8)

• NGS AMP-Translocation V1 (10-15)

• Cancer genetics has rapidly expanded with high complexity • Demands for clinical molecular profiling face some challenges: many targets, limited sample quantity/quality, fast turnaround time, uncertain reimbursement • Continued need for higher-throughput cancer genotyping

Summary

Sanger Sequencing

Started in 1990, initially led by James Watson and then Francis Collins took over in 1993

International effort (US, China,

France, Germany, Japan, UK)

Public: $2.7 billion effort (FY 1991) funded by the NIH, Wellcome Trust, and other groups (13 years)

Private: $300 million effort led by

Craig Venter and Celera Genomics

(3 years)

The Human Genome Project

Cost of Next Generation Sequencing

Adapted from Nature 1 April 2010

Library Preparation Cost

Illumina Next-Generation Sequencing

NGS Assays Comparison

Assay Sample Input Cost Turnaround

Time Informatics Interpretation Discovery Potential

Hotspot + TSP + $ 1-2 weeks + + +

Rearrangement + $ 1-2 weeks + + +

100-200 Gene Panel ++ $$ 2-4 weeks +++ +++ ++

1000+ Gene Panel ++ $$$ 2-4 weeks ++++ ++++ ++++

Exome ++ $$$$$ 3-4 weeks +++++ ++++++ ++++++

Whole Genome +++ $$$$$$$ 4-8 weeks ++++++++ +++++++++++ ++++++++

Tiered Clinical Cancer Genotyping

Disease Progression

2nd Tier Result 1st Tier

Hotspot+

Rearrangement

$

Positive None

Comprehensive Panel

$$$

Negative

Comprehensive Panel

$$$

Comprehensive Panel

$$$

Target Capture Technologies

Solution-phase Capture

Microdroplet PCR

Reaction Array

TruSeq Amplicon

Haloplex

Ion Torrent AmpliSeq

Anchored Multiplex PCR (AMP)

Cancer: More Than Just Point Mutations

copy number indels

SNVs

epigenetics

non-coding RNAs

proteomics

gene expression

rearrangements

Detecting Rearrangements Status Quo

Immunohistochemistry

- Antigen expression

in tumor but not in

normal

- Ab availability

- Ab specificity

- Qualitative analysis

FISH

- Technically

challenging

- Expert analysis

- Poor scalability and

multiplexing ability

RT-PCR

- Requires knowledge

of both partners

- Limited scalablity

and multiplexing

ability

ROS1 Heterogeneous Partner Breakpoints

Unbiased Rearrangement Detection

bwa unmapped reads blat annotation/filter

Complex Rearrangement Detection

Indeterminate ROS1 FISH Confirmed Positive by AMP

Indeterminate ALK FISH Confirmed Negative by AMP

AMP Positive KIF5B-RET; RET FISH Negative

AMP Positive KIF5B-RET; RET FISH Negative

Interesting TRIM24-RET Clinical Case

Pileup of Interesting Clinical Case

Pileup of Interesting Clinical Case

Hotspot+ Panel

Clinical targeted sequencing of FFPE DNA

• ~370 amplicon targets w/ bi-directional coverage (hotspots + TP53 + PTEN + CDKN2A)

• 1000X median coverage

• 4-500 Mb data per tumor

• 2-5% analytical sensitivity

• 2-week turnaround time

Desired

• Sensitivity for hotspots

• SNV, indel, copy number

The Problem

Report

Results

Worksheets

Path Review

Requisition

Consent

Path Report

* 1 Patient with 1 surgical specimen may have 6+ different tests

Office/Lab

MGH MDX and TRL Informatics 2005-2012

PowerPath

CoPath TRL DB

FISH SpreadSheet

GeneInsight

NAE SpreadSheet

File Maker Post-it Notes

Molecular Lab Asset Graph

CIDSuite

Clinical NGS Workflow

Order Sample

Submission Pre-

Analytical Wet Lab Sequencing

Analysis Variant Store

Variant Filtering

Variant Vetting

Variant Curation

Reporting Decision Support

Action Outcome New

Knowledge

Clinical NGS Workflow

Order Sample

Submission Pre-

Analytical Wet Lab Sequencing

Analysis Variant Store

Variant Filtering

Variant Vetting

Variant Curation

Reporting Decision Support

Action Outcome New

Knowledge

CIDWikiLIMS

Pipeline VarVault VarVetter WikiVar

CIDPortal

NGS Variant Interpretation Complexity

4th Interpretation

Variant1 1st

Interpretation 2nd

Interpretation 3rd

Interpretation

NGS Variant Interpretation Complexity

Variant2

Variant3

(Versioned)

• Next-generation sequencing offers the throughput and scalability to achieve broad-based clinical cancer genotyping

• Challenges in broad deployment:

• Single gene tests vs. panel testing

• Complex assay validation for multi-gene/target tests

• Complex variant assessment/interpretation with dynamic evidence

• Lack of end-to-end informatics solution

• Complex reporting

• NGS reimbursement still not clear

• FDA regulation of laboratory developed tests (LDTs)

Summary

Acknowledgments

John Iafrate

Zongli Zheng

Marianne Boswell

Gaddy Getz

Martin Aryee

Spring Liu

Alex Ramos

Dora Dias-Santagata

Hayley Robinson

Darrell Borger

Julie Batten

Kerry Lynch

Yi Cao

David Louis

Elliott Moy

Sekhar Duraisamy