Assessment of fusion genes: pros and cons of

the different testing strategies

Frédérique Penault-Llorca, MD, PhD

Centre Jean PERRIN – 58 Rue Montalembert - 63011 CLERMONT-FERRAND CEDEX

Links

• Abbvie, AstraZeneca, Bayer, BMS, Lilly, Merck, Merck Lifa, Novartis, Pfizer, Puma, Roche, Sanofi, Takeda, honoraria and/or research grants in the field of biomarkers

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Known fusion

Known kinase-novel partner

Known kinase-novel indication

New fusion

Gene fusions represent an important class of oncogenes

• Receptor tyrosine kinases altered by gene fusions are associated with a diverse range of hematologicalmalignancies and solid tumours,

•Stransky N et al. Nat Commun 2014;5:846

Samples n= 498 157 513 374 461 286 411 103 1072

ALK 3 5

BRAF 9 4

EGFR 5 1

FGFR1 1

FGFR2 2

FGFR3 2 3 2

FGR 1

MET 1 1 1

NTRK1 5 1 1

NTRK2 1 2 1

NTRK3 7 1 2 1 1

PDGFRA 1 1

PIK3CA 2

PKN1

PRKCA

PRKCB 1 1

RAF1 7 4

RET 31 2 1 1

ROS1 1 8

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Landscape of recurrent kinase fusions in solid tumours

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• The prevalence of gene fusions varies widely between cancer types • From 90% of all lymphomas (often driver),

• To >1/2 leukemias

• 1/3 soft tissue tumors.

• In prostate cancer, one specific fusion (TMPRSS2-ERG) is the most common genetic alteration, being found in over 50% of patients

• However, many recurrent gene fusions occur at low frequencies, such as the KIF5B-RET fusion, which is present in 1–2% of lung adenocarcinomas

Nature Clin Review 2018 https://doi.org/10.1038/s41571-018-0113-0

~0,05% of all cancers?

Quantitative aspect of the presence of fusions among all tumor types

Schram et al doi:10.1038/nrclinonc.2017.127

Stransky N et al. Nat Commun 2014;5:846

Different aspects of fusions• Diagnostic utility of fusions e.g. for salivary

glands CRTC1–MAML2 in mucoepidermoidcarcinoma, MYB–NF-IB in adenoid cysticcarcinoma, EWSR1–ATF1 in hyalinizing clear-cell carcinoma

• Prognostic implications of fusions e.g. RCC worse prognosis if TFE3-containing fusions

• Targeted therapy and resistance e.g. ALK fusions in lung adenocarcinoma, NTRKfusions in different tumor types

Stransky N et al. Nat Commun 2014;5:846

Clinically relevant fusions

Fusions gene testing: methods & challenges accross different

tumor types

Types of Molecular Tumor Testing

• Next-generation sequencing (NGS)• Detects known and novel fusions with arbitrary

breakpoints in DNA or RNA

• Exact capabilities depend on enrichment strategy

• Reverse transcription polymerase chain reaction (RT-PCR)• Detects known and unknow fusion transcripts in RNA

• Detects 5′/3′ imbalance as a fusion signature, but cannot determine novel partner

• Fluorescence in situ hybridization (FISH)• Detects known gene rearrangements in DNA that may generate a fusion

transcript

• Immunohistochemistry (IHC)• Detects protein expression, which may be attributable to a fusion event

1. Pierce KE, et al. Proc Natl Acad Sci USA. 2005;102:8609–8614; 2. Rimm DL. Nat Methods. 2014;11:381–383; 3. Vaishnavi A, et al. Nat Med. 2013;19:1469–1472; 4. Wu LR, et al. Nat Methods. 2015;12:1191–1196.

IHC

IHC

• Advantages• Less expensive, reimbursed in many countries

• Fast turn around time

• Provides clues about specific fusions by protein localization

• Disadvantages• Rare low expression of fusion may yield false negative result

• Constitutive expression or low expression not associated with fusion (low specificity)

• Does not detect involved genes and exons

• Does not detect concomitant alterations

Immunohistochemistry (IHC)For the fusions, many antibodies are commercially available.Most of them are RUO tests Thus, clinical performance of available antibodies

https://rockland-inc.com/ihc

Testing methodologies: Immunohistochemistry

RET protein

SECRETORY BCAbcam EPR17341 Optiview+ amplification

ETV6-NTRK3 fusion➔ nuclear staining

Invasive BC nosAbcam EPR17341 Optiview + amplification

Absence of rearrangement

panTRK IHC breast cancer Cocktail TRK1, 2 3, ROS1, ALK IHC CRC

Cocktail of Pan-Trk (C17F1, active against TrkA, TrkB, and TrkC), ROS1(D4D6), and ALK (D5F3).

TPM3/NTRK1 fusion

Alk IHC if 3+➔ no FISH

Results from the three larger FISH / IHC concordance studies*

• 1,117 total samples analysed

• 100% concordance between FISH– and IHC 0

• 100% concordance between FISH+ and IHC 3+

0

1+

2+

3+

Novocastra 5A4 Antibody 1:50;photographs from Park H, et al. WCLC 2011

*Paik JH, et al. J Thorac Oncol. 2011;6:466–72; Yi ES, et al. J Thorac Oncol. 2011;6(3):459–65; update at EMCTO by Yang, et al.; Park H, et al. Presented at WCLC 2011; Abstract 05.07.

FISH

Fluorescence in situ hybridization

• Advantages• Commonly used method for detecting gene fusions

• Fast turnaround time

• Disadvantages• Tests have to be run for each gene

• While break-apart FISH can identify gene disruptions in DNA, it cannot confirm in-frame, functional fusions

• No indication about the partner gene

• Subjective interpretation

• Requires specific equipment

• May show up to 30% false negative results

/// Bayer Oncology /// 201918

Fluorescence In Situ Hybridization (FISH)

DNA FISH can be used to detect gene fusions however, in order to detect fusions from different genes multiple FISH tests would need to be run, but there is utility in using DNA FISH in diseases such as infantile fibrosarcoma is ETV6-NTRK3, or in Ewing sarcoma EWSR1-ETS fusion

Fluorescence In Situ Hybridization

Bourgeois JM, et al. Am J Surg Pathol. 2000;24(7):937–946.

Fluorescence in situ hybridization (FISH): NTRK1–3

• ZytoLight users’ manual.

Ideogram of chromosome 1 indicating hybridization locations

NTRK1

Ideogram of chromosome 9 indicating hybridization locations

NTRK2NTRK3

Ideogram of chromosome 15 indicating hybridization locations

Lung cancer tissue section

SPEC NTRKNormal interphase cells

SPEC NTRKNormal interphase cells

Secretory breast carcinoma

SPEC NTRKNormal interphase cells

NTRK1 NTRK2 NTRK3

ALK FISH break apart assay

Wild Type

Inversion

+ =A yellow signal

indicates no ALK fusion

Splitting probes of the red and green signals

indicates ALK fusion*

*Assay is positive if fusion can be detected in ≥15% of cells Vysis probe

RT-PCR or RNA targeted fusions panels(non NGS)

RT-PCR

BENEFITS • Detects known and unknown fusion

transcripts• Proven• Inexpensive

LIMITATIONS• Too many TRK fusion partners• Cannot detect novel fusion partners

Reverse transcriptase polymerase chain reaction (RT-PCR)

• Unique primers are needed to identify each respective fusion partner; therefore, the fusion partner must be known prior to testing1

• Development of a comprehensive multiplex RT-PCR assay would likely be technically challenging4

• RT-PCR may be practical in certain rare tumor types where a particular NTRK gene fusion is essentially pathognomonic (e.g. ETV6-NTRK3 in SBC, MASC, IFS), 4

• Parameters that may influence assay:• Type of gene• Fusion partner• Location of fusion break point in the genes,

• IFS, infantile fibrosarcoma; MASC, mammary analog secretory carcinoma of the salivary gland; RT-PCR, reverse transcriptase polymerase chain reaction; SBC, secretory breast cancer

• Farrell RE. RT-PCR: A Science and an Art Form. In: A Laboratory Guide for Isolation and Characterization. 2010;385-448; Garibyan L, Avashia N. J Invest Dermatol. 2013;133:e6. Fehr A, et al. Am J Surg Pathol. 2011;35:1600-1602; Kummar S, Lassen UN. Targeted Oncol. 2018;13(5):545-556.

Detection of ETV6-NTRK3 fusion transcripts in MASC tumors by

RT-PCR analysis3

Commercially available RNA-based targeted assays

Vendor Kit Name Additional Information

NanoString nCounter® Gene Fusion panelshttps://www.nanostring.com/products/gene-expression-panels/gene-

expression-panels-overview

Biocartis Idylla™ assay https://www.biocartis.com/meet-idylla/idylla-assays

HTGEdgeSeq miRNA Whole Transcriptome

Assayhttps://www.htgmolecular.com/assays/mirna-wta

/// Bayer Oncology /// 2019 26

NGS

Next-generation sequencing (NGS)

• Advantages

• All in-one test

• Detects concomitant alterations

• Detects involved genes and exons

• Disadvantages

• Expensive

• Time-consuming

• Lower sensitivity due to lower coverage of introns for specific genes (only when DNA based)

• Limited sensitivity with low tumor purity specimens

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DNA-based NGS methods

• In most cases, DNA-based sequencing methods are fusion-partner agnostic and are able to capture the diversity of gene fusions

• Hybridization capture-based NGS is recommended to detect known and novel XX gene fusions due to its ability to capture and amplify surrounding targeted sequences

• DNA-based methods can be more challenging to design for NTRK gene fusions because of the large NTRK2&3 introns and low sequence complexity (i.e., low sequence specificity, longer sequence reads, more intensive bioinformatics)

Advantages Drawbacks

• DNA is more stable than RNA• Detection independent of

expression

• Detection of novel fusions might be limited with DNA-based methods, especially when large intronic regions are involved

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Examples of commercially available NGS tests

Platform Company Details Website

FoundationOne® Foundation Medicine

• 315 genes included; 28 genes for fusions• Includes NTRK1 and ETV6-NTRK3

www.foundationmedicine.com/genomic-testing/foundation-one

FoundationOne® HemeFoundation Medicine

• 406 genes included + 265 genes for fusions• Can be ordered for solid tumors• Includes NTRK1, NTRK2, and NTRK3

www.foundationmedicine.com/genomic-testing/foundation-one-heme

GeneTrails® Solid Tumor Fusion Gene Panel

Knight Diagnostic Laboratories

• 20 genes for fusions• Includes NTRK1, NTRK2, and NTRK3

https://www.ohsu.edu/custom/knight-diagnostic-labs

MI Profile™ Caris Life Sciences• 592 genes included (fusion analysis optional)• TRKA/B/C available

www.carismolecularintelligence.com

NGS ALK, NTRK, RET, ROS1 FusionProfile

Neogenomics• 5 gene fusions• Includes NTRK1 and NTRK3

https://neogenomics.com/test-menu/ngs-alk-ntrk-ret-ros1-fusion-profile

OmniSeq ComprehensiveSM OmniSeq• 144 genes, including fusions• Includes NTRK1, NTRK2, and NTRK3

www.omniseq.com/comprehensive

Oncomine™ Focus Assay Sirona Dx• 52 genes, including 23 genes for fusions• Includes NTRK1, NTRK2, and NTRK3

www.sironadx.com/assay-menu/oncomine-focus-assay

SmartGenomics™ NGS Solid Tumor

PathGroup • 160 genes, with 126 genes for fusionswww.pathgroup.com/oncology/smartgenomics

Solid Tumor FOCUS: Oncomine™ NGS Panel

Cancer Genetics • Fusion analysis optional www.cancergenetics.com

xO Onco-seq Tempus• ~1700 genes, including fusions• Includes NTRK1, NTRK2, and NTRK3

www.tempus.com/providers.html

Oncomine Focus Assay Thermo Fisher• 52 cancer genes, 23 genes with fusion drivers,

including 30 NTRK fusion pairs

https://www.thermofisher.com/us/en/home/clinical/preclinical-companion-diagnostic-development/oncomine-oncology/oncomine-focus-assay.html

TruSight Tumor 170 Illumina • 170 cancer genes, including all NTRK fusionshttps://www.illumina.com/products/by-type/clinical-research-products/trusight-tumor-170.html

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Advantages of RNA NGS in detecting gene fusions

31 /// Bayer Oncology /// 2019 • Benayed et al Clinical Cancer Research doi:10.1158/1078-0432.CCR-19-0225

Prospective DNA NGS MSK-IMPACT of 2522 lung adenocarcinomas ➔195 (7.7%) fusions ➔Among 254 driver-negative ➔RNAseq➔ 27 in-frame fusions

Advantages of NGS in detecting gene fusions

RNA-based NGSAble to distinguish in-frame, transcribed gene fusions versus out-of-frame fusions

Avoids difficulties of sequencing large intronic regions such as in the NTRK genes

• General NGS

• High sensitivity and specificity potential

• Multiplexing: simultaneously queries multiple potentially actionable targets (e.g. NTRK, ALK, ROS1, RET)

• Detects both known and novel fusions regardless of break point or fusion partner (depending on library preparation method)

• Kumar S, et al. Wiley Interdiscip Rev RNA. 2016;7:811–823.

Challenge : Detecting fusions in NTRK1–3 genes

With such large intronic spaces (and difficult regions within them), fusion detection by DNA NGS can be complicated • Lower capacity (fewer samples to multiplex to get appropriate coverage) • More complications with difficult introns

RNA-based NGS methods

• More suitable for specific genes fusions in FFPE samples

• Compared to other genes with fusion inclination (e.g., ALK, ROS1, and RET), NTRK genes have introns where fusion breakpoint events occur

• RNA-based methods that can capture the diversity of NTRK gene fusions might provide better sensitivity for their detection compared with DNA-based NGS assays

• Tognon C, et al. Cancer Cell. 2002;2(5):367–376.

Advantages Drawbacks

• Novel gene fusions may be detected• Sensitivity depends on expression level• RNA is less stable than DNA

Vendor Kit Name Sample# of Genes Included

Library Prep Method

DNA/RNA NTRK1, 2, and 3Unknown

NTRK FusionsIntegrated

BioinformaticsRecommended

Sequencer

Illumina

TruSight Oncology 500 DNA/RNA Kit1

FFPE

523

Hybrid Capture

DNA/RNA Y Y

BaseSpace

NextSeq

TruSight Tumor 1702170 DNA/RNA Y Y NextSeq/HiSeq

TruSight RNA Fusion Panel3 507 RNA Y Y MiniSeq/MiSeq/NextSeq

TruSight RNA Pan-Cancer Panel4

1385 RNAY Y MiniSeq/MiSeq/NextSeq

Thermo Fisher

OncomineTM Focus Assay5

FFPE

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Amplicon

DNA/RNA N N

Ion Reporter/Oncomine Report

Ion GeneStudio S5/Chef Automation

Oncomine™ Comprehensive Assay v3M6 161 DNA/RNA Y N

Oncomine™ Childhood Cancer Research Assay7

FFPE, blood and bone marrow

203 DNA/RNA Y N

Archer DX

FusionPlex®

Solid Tumor Kit8

FFPE

53

Anchored Multiplex PCR

RNA Y Y

Archer Analysis Illumina/Ion Torrent

FusionPlex® Oncology Research Kit9 74 RNA Y Y

FusionPlex®

Sarcoma Kit10 26 RNA N (NTRK3 only) Y

FusionPlex® CTL Kit11 36 RNA Y ?

FusionPlex® Lung Kit12 14 RNA Y Y

QiagenGeneRead QIAact Lung RNA

Fusion UMI Panel13FFPE/

liquid biopsy19 UMI/PCR RNA N (NTRK1 only) N

Qiagen Clinical Insight

GeneReader

Commercially available NGS kits

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Strategy for testing-algorithmsTumor agnostic biomarkers

Strategy is linked to several factors

• Rarity of the fusion

• Relation genotype/phenotype

• Previous target testing strategy in place or/not withNGS in a specific tumor setting

• Reflex testing by the pathologist/biologist is important as oncologist’s testing exhaustion represents a major risk

Rare cancers with high fusion frequency

Rare cancer types that have a high frequency of EFTV6-NTRK3 gene fusions include:

• MASC

• Secretory carcinoma

• Infantile fibrosarcoma

• Congenital nephroma

Salivary gland cancer with specific fusions

• CRTC1–MAML2 in mucoepidermoidcarcinoma

• MYB–NF-IB in adenoid cystic carcinoma,

• EWSR1–ATF1 in hyalinizing clear-cellcarcinoma

Coco et al Nature Clin Review 2018 https://doi.org/10.1038s41571-018-0113-0

fusion gene is «pathognomonic»

FISH or IHC followed by confirmatory FISH

Screening

Routine pan-cancer screening: Tumor agnostic biomarker i.e. NTRK, RET, NRG1 fusions

• Screening for rare molecular events that occur across a variety of cancers can provide treatment options for patients with advanced cancers

• NGS is broad and can detect many mutations, copy number variations, certain rearrangements, and microsatellite status

• Mutation- and fusion-specific IHC have advantages in small samples or low tumor content cases

• RNA testing (Archer vs RNAseq) can identify a variety of fusions in ‘driver-negative’ cancers

• IHC with pan TRK or RET clones represent an interesting screening tools for frequent tumors

Strategy for testing

• NTRK, neurotrophic tyrosine receptor kinase; 1. Vaishnavi A et al. Cancer Discov 2015;5:25-34; 2. Tognon C et al. Cancer Cell 2002;2:367-76; 3. Pishvaian MJ et al. J Clin Oncol2018;36(Suppl.):521; 4. Brenca M et al. J Pathol 2016;238:543-9; 5. Stransky N et al. Nat Commun 2014;5:4846; 6. Wiesner T et al. Nat Commun 2014;5:3116; 7. Ricarte Filho JC et al. J Clin Invest 2013;123:4935-44; 8. Prasad ML et al. Cancer 2016;122(7):1097-107; 9. Bourgeois JM et al. Am J Surg Pathol 2000;24:937-46

Adult Brain cancers (0.4–3.1%)1

Salivary (MASC; 90–100%)1

Secretory breast cancer (92%)2

Pancreatic cancer3

Cholangiocarcinoma (3.6%)1

Thyroid cancer (1.5–14.5%)1

Lung cancer (0.2–3.3%)1

GIST (3.2%)4

Colon cancer (1.5%)1

Melanoma (0.3%)1,5

Sarcoma (1%)5

Pediatric

Spitzoid neoplasms (16.4%)6

Gliomas (7.1%)1

Thyroid (9.4–25.9%)7,8

Infantile fibrosarcoma (91–100%)9

Congenital nephroma (83%)1

Secretory breast cancer (92%)2

Sarcoma (1%)5

Finding the responders

If molecular screening is not routinely performed:

Enrichment with IHC + detection with NGS

In NSCLC, adding IHC for NTRK to ALK, ROS1 and RET is not cost effective

Finding the responders If molecular screening is routinely performed: DNA + RNA NGS (fusion panels)

IHC as a screening tool in TRK fusion cancer

• Albert CM, et al. J Clin Oncol. 2019;37(6):513–524./// Bayer Oncology /// 201943

Conclusion

Diagnostic testing overview

• Testing for gene fusions is essential to identify patients who harbor these genomic alterations for

• DIAGNOSIS

• PERSONALIZED TREATMENT

NGSFISHRT-PCR IHC*

Diagnostic testing methods summary: Benefits and drawbacks for fusions

• NTRK, neurotrophic tyrosine receptor kinase; TRK, tropomyosin receptor kinase• 1. Bubendorf L et al. Virchows Arch 2016;469:489-503; 2. Doshi S et al. Diagnostics 2016;6:4; 3. Bourgeois JM et al. Am J Surg Pathol 2000;24:937-46; 4. https://www.cms.gov/apps/physician-fee-schedule/search/search-

results.aspx?Y=0&T=0&HT=0&CT=0&H1=88342&M=1; 5. Data from Diaceutics Laboratory Database; 6. Kerr KM, Lopez-Rios F. Ann Oncol 2016;27:iii16-24; 7. Cui C et al. Front Cell Dev Biol 2016;4:1-11; 8. Argani P et al. Mod Pathol2000;13:29-36; 9. Church AJ et al. Mod Pathol 2017:modpathol2017127; 10. Kwang Chae Y et al. Oncotarget 2017;8:100863-98; 11. Drilon A et al. N Engl J Med 2018;378:731-9; 12. https://www.aacc.org/publications/cln/articles/2015/may/next-generation-sequencing-in-the-clinical-laboratory; 13. Paasinen-Sohns A et al. Neoplasia 2017;19:196-206; 14. Data from Diaceutics PayerLandscape Database; 15. https://neogenomics.com/test-menu/ngs-alk-NTRK-ret-ros1-fusion-profile; 16. Tannenbaum-Dvir S et al. Cold Spring Harb Mol Case Stud 2015;1:a000471

Immunohistochemistry

(IHC)

Fluorescence in situ

hybridisation (FISH)

Polymerase chain reaction

(PCR)

Next-generation sequencing (NGS)

Benefits • Cost effective (inexpensive)1,2

• Widely available3

• Established reimbursement

codes4

• Turnaround time = days5

• High sensitivity

• Low tissue input

• Can detect novel fusions if break-

apart probes used

• Low cost

• Established reimbursement codes

• Widely available

• Turnaround time = days

• High sensitivity and specificity7

• Can detect novel fusions if break-

apart probes used

• Low cost1

• Established reimbursement

codes

• Widely available

• Turnaround time = days

• High sensitivity and specificity3,8

• Low tissue input

• Current targets screening conducted via

NGS11

• Relevance of NGS increases as number of

actionable targets grows1

• High sensitivity and specificity potential1

Drawbacks • Low specificity3

• Scoring algorithms are not

standardised1

• Detects wild-type proteins as well

as fusions

• Target sequence must be known9

• High tissue input (at least 3

assays)

• Multiple tests needed to detect

fusions in multiple locations

• Break-apart probes can’t detect

whether the fusions are in-frame

and functional

• Cannot detect novel fusions as

target sequences must be

known9

• Expensive

• Turnaround time = weeks5

• Highly centralised testing model

(academia and reference labs)9

• Reimbursement currently restricted

(although developing)14

• The sensitivity and specificity of NGS

assays vary widely

• Some assays require high tissue input

NTRK fusion detection: The ESMO proposal

• MPS, massively parallel sequencing.• Marchiò C on behalf of the ESMO 2018 Working Group, in preparation.

Consider FISH or RT-PCR assays with specific probes for the rearrangement involving

the known NTRK gene

Is the histologic tumor type known

to harbor highly recurrent NTRK

rearrangements?

Is there a sequencing

platform available?

YES NO

Use IHC as a screening tool If cost effectiveUse frontline MPS-targeted gene

panel including NTRK genes(and possibility to investigate

rearrangements)Detection of TRK expression

No TRK expression

NO YES

/// Bayer Oncology /// 201947