NGS-BASED GENE FUSION DETECTIONHOUSTON METHODIST HOSPITAL EXPERIENCE

USCAP - Pulmonary Pathology SocietyBryce Portier MD, PhDDirector Solid Tumor Molecular Diagnostics

DISCLOSURES

Financial Disclosures:• No financial association with any of the companies or products in this presentation

ALK TRANSLOCATIONS AND RESPONSE TO TARGETED THERAPY

Kwak et al. N Engl J Med. 2010;363:1693‐1703.. 

MOLECULAR DIVERSITY CAUSES DIAGNOSTIC CHALLENGES

Modified from: Horn and Pao , J Clin Oncol. 2009 Sep 10;27(26):4232‐5. 

MULTIPLE METHODS FOR GENE FUSION DETECTION

FISHFluorescent in situ hybridization

RT‐PCRReverse Transcriptase PCR

IHCImmunohistochemistry

DNA ProteinRNA

Limitations of Current Methods:• Throughput limitations due to limited multiplexing capability (FISH and IHC)• Subjective results, cannot determine breakpoint or fusion partner (FISH and IHC)• Requires knowledge of fusion partners and breakpoints for assay design (RT‐PCR)• Decreased sensitivity due to poor quality of RNA from FFPE (RT‐PCR)

NGSNext Generation Sequencing

cDNA

Murakami Y, Mitsudomi T, Yatabe Y., Front Oncol 2012

MULTIPLE METHODS FOR GENE FUSION DETECTION

FISHFluorescent in situ hybridization

RT‐PCRReverse Transcriptase PCR

IHCImmunohistochemistry

DNA ProteinRNA

Advantages of Current Methods:• Screening method used for clinical trials (FISH and IHC)• Established techniques and workflows in many labs (FISH and IHC)• Applicable to archival specimens (FISH and IHC)• Rapid turn around time and inexpensive (RT‐PCR)

NGSNext Generation Sequencing

cDNA

Murakami Y, Mitsudomi T, Yatabe Y., Front Oncol 2012

MOLECULAR DIVERSITY CAUSES DIAGNOSTIC CHALLENGES

MLL (KMT2A) Gene Rearrangements66 Partner genes

Catalogue of somatic mutations in cancer v71, 2015 http://cancer.sanger.ac.uk/cosmic/gene/overview?ln=ALK

MOLECULAR DIVERSITY CAUSES DIAGNOSTIC CHALLENGES

ALK Gene Rearrangements19 Partner genes

Catalogue of somatic mutations in cancer v71, 2015 http://cancer.sanger.ac.uk/cosmic/gene/overview?ln=ALKSasaki et al, Eur J Cancer 2010

MOLECULAR DIVERSITY CAUSES DIAGNOSTIC CHALLENGES

ALK, RET, ROS1 Gene Rearrangements~36 Partner gene fusions>100 with multiple fusion break points

www.archerdx.com

NGS-BASED FUSION DETECTION WORKFLOW

Frampton, et. al Nature Biotechnology Nov 2013 (modified)

RNAExtraction Sequencing

Analysis &Interpretation

Library Generation1‐ Target (hybridization) Capture

“Foundation Medicine”2‐ Opposing Primers

“Ion Life Technologies”3‐ Anchored Multiplex PCR

“Archer”

LimitationsHigher input requirements (not ideal for biopsies/limited FFPE samples)

Fusion partner and breakpoint must be knownOpposing primer pair needed for every fusion partner and breakpointFixed start site for every amplicon (problematic with fragmented FFPE)

Frampton, et. al Nature Biotechnology Nov 2013 (modified)

RNAExtraction Sequencing

Analysis &Interpretation

NGS-BASED FUSION DETECTION WORKFLOW

ADVANTAGES OF NGS GENE FUSION DETECTION BY ANCHORED MULTIPLEX PCR

Features of  Anchored Multiplex PCR (AMP™)

Low input requirements (nanograms) ‐ FFPE, fresh frozen tissue or blood

Bar codes available for MiSeq or PGM sequencing

Molecular indexing measures number of unique fusion events

Up to 600 amplicons can be multiplexed in a single tube‐ DNA or RNA targets

Thermal cycling maintain linearity during amplification ‐ Enabling quantitation

Random start sites improve coverage

www.archerdx.com

• Library generated with as little as 1 ng of RNA

• Library complexity increases with input levels

• Uniform read coverage and excellent library diversity across sample input quantities

55ng RNA input

27.5ng RNA input

5ng RNA input

Library Input Library Uniformity

ADVANTAGES OF NGS GENE FUSION DETECTION BY ANCHORED MULTIPLEX PCR

CLINICAL ASSAY VALIDATION PLANAccuracyCorrelation to FISH results

Analytical Sensitivity (LOD)A dilution series utilizing a translocation positive Geneblock mixed with Ambion normal lung. 

Analytical Specificity/Interfering SubstancesAnalytical specificity‐ correlation with FISH and known Geneblock constructsInterfering substances‐ to minimize the possibility of interference, validation and testing will be restricted to RNA isolated from formalin fixed paraffin embedded tissue from samples with >20% tumor burden quantified by H&E examination.

PrecisionIntra‐ and inter‐run reproducibilityInter‐technologist reproducibility

Reference values and reportable rangesReference range:  Negative for translocation Reportable range:  Positive or negative (qualitative) for translocation

CAP Molecular Pathology ChecklistsCLSI guidelinesArch Pathol Lab Med, Test verification and validation for molecular diagnostic assays, 2012. 

CLINICAL ASSAY VALIDATION SAMPLES

Anonymized Samples25 samples negative for translocation• 1 Cell line• 7 Geneblocks• 17 FISH negative patient samples

25 samples positive for translocation• 1 Cell line • 7 Geneblocks• 17 FISH positive patient samples 

Total number of samples: 50

GENERATION OF CONTROLS FOR CLINICAL ASSAY VALIDATION

Cell lines:• HCC‐78 (SLC34A2‐ROS1 S4:R32 & S4:R34)• H2228 (EML4‐ALK E6:A20)• Normal Lung RNA (Ambion) 

Engineered Controls:• ALK‐ EML4‐ALK E13:A20

EML4‐ALK E20:A20• RET‐ CCDC6‐RET C1:R12

KIF5B‐RET K15:R12NCOA4‐RET N6:R12

• ROS1‐ CD74‐ROS1 C6:R32CD74‐ROS1 C6:R34

Homo sapiens mRNA for fusion protein EML4‐ALK variant 1, complete cds (Variant 1 = EML4 ex 13 and ALK ex20)  GenBank: AB274722.1

1621 gggaaatatg aaaagccaaa atttgtgcag tgtttagcat tcttggggaa tggagatgtt

1681 cttactggag actcaggtgg agtcatgctt atatggagca aaactactgt agagcccaca

1741 cctgggaaag gacctaaagt gtaccgccgg aagcaccagg agctgcaagc catgcagatg

1801 gagctgcaga gccctgagta caagctgagc aagctccgca cctcgaccat catgaccgac

1861 tacaacccca actactgctt tgctggcaag acctcctcca tcagtgacct gaaggaggtg

Step 1: ID Variant of Interest Break Point 

Step 2: Download cDNA sequence (Genebank)

Step 3: Locate Break Point

Step 4: Order Oligonucleotide “Engineered Control”

Controls:

PREBUILT BIOINFORMATIC PIPELINEArcher Analysis Pipeline Version 3.0

Virtual machine based prebuilt pipeline• Download with all dependences included• Locked down version for clinical use• Future updates can be loaded as independent

virtual machine images for testing• Password protected data and login• All data and analysis done locally

Analysis &Interpretation

AUTOMATED BIOINFORMATICNGS ANALYSIS

1 2

3

AUTOMATED BIOINFORMATICNGS ANALYSIS

4

5

VALIDATION DATA SUMMARY

Anonymized Samples25 samples negative for translocation• 1 Cell line No Fusion Detected 1 of 1• 7 Geneblocks No Fusion Detected 7 of 7• 17 FISH negative patient samples No Fusion Detected 17 of 17

25 samples positive for translocation• 1 Cell line Detected 1 of 1• 7 Geneblocks Detected 7 of 7• 17 FISH positive patient samples Low Detection Rate

0

20

40

60

80

100

2013‐2014 2011‐2012 2005‐2010

% of C

ases 

Passing QC

Cases by Surg‐Path Accession Year

0

20

40

60

80

100

2013‐2014 2011‐2012 2005‐2010

% of C

ases 

Passing QC

Cases by Surg‐Path Accession YearNGS RT‐PCR

SUMMARYNGS BASED FUSION DETECTION

Methodology selection for ALK Fusion detection will depend on multiple factors• Sample volume• Recent vs. archive cases• In‐house expertise• Cost/reimbursement

Selection of sequencing platform

RNA degradation in FFPE• Best results: Extract RNA from fresh cut block

THANK YOUNGS-BASED GENE FUSION DETECTIONHOUSTON METHODIST HOSPITAL EXPERIENCE

AcknowledgmentsPhilip Cagle MDRandall Olsen MD, PhDNeal Lindeman MDLynette Sholl MDJohn Iafrate MD, PhDKirtee Raparia MBBSJan Nowak MD, PhD