esmo advanced course on ntrk gene fusion · modifiedfrom cocco e et al, cancerres 2019. ihc =>...

ESMO ADVANCED COURSEON NTRK GENE FUSION:IDENTIFICATION/TESTING METHODOLOGIES AND

CHALLENGES

Caterina Marchiò - University of Turin, Pathology Unit at FPO-IRCCS Candiolo Cancer Institute

Barcelona, October 21-22 2019

[email protected]

DISCLOSURE OF INTEREST

Consultancy fees from Daiichi Sankyo, MSD, Bayer, Roche, Cor2ED

IDENTIFICATION/TESTING METHODOLOGIES AND

CHALLENGES

Outline

• Brief summary of what we need to know before getting into NTRK gene fusion testing

• Available techniques: rationale, outputs, caveats

• Strategy for testing: possible algorithms

• Open questions, new challenges

NEUROTROPHIC TROPOMYOSIN RECEPTOR KINASE (NTRK)

• NTRK1- 1q21-q22 – TRKA

• NTRK2- 9q22.1 – TRKB

• NTRK3- 15q25 – TRKC

• Tyrosine kinases that play roles in- Neuronal differentiation and development,

including the growth and function of neuronal synapses and memory development

Cocco E, Scaltriti M & Drilon A, Nature Reviews Clinical Oncol 2018

Transactivation of the

intracellular tyrosine

kinase domains

Receptor homodimerization

Recruitment of various cytoplasmic adaptors

Activation downstream

signalling pathways,

including the MAPK, PI3K

and PKC pathways

Differentiation and survival in neuronal cells

Binding of neurotrophins to the

extracellular region of TRK proteins





• Tyrosine kinases that play roles in- Neuronal differentiation and development,

including the growth and function of neuronal synapses and memory development

- Expression restricted to specific tissues (in adult tissues: smooth muscle, testes and neuronal components)





Congenital fibrosarcoma

Cellular mesoblasticnephroma

Secretorycarcinoma

t(12;15)(p13;q25)

NTRK FUSIONS ACROSS TUMOR TYPES

High frequency in special histologic types

Low frequency in common forms of different types of cancers

• Secretory breast carcinoma• Mammary analogue secretory carcinoma of the

salivary glands (MASC)• Congenital infantile fibrosarcoma

• Thyroid PTC• GIST (lacking canonical KIT/PDGFRA/RAS alterations) • Lung cancer• Carcinomas of the GI tract• Melanoma• Sarcomas• Gliomas• Acute myeloid and acute lymphoblastic leukemias

ETV6-NTRK3 rearrangement

NTRK1, NTRK2, NTRK3 rearrangements

DETECTION OF GENE REARRANGEMENTS ACROSS DIFFERENT TUMOR TYPES

Zehir A et al, Nature Medicine 2018

Spectrum of kinase fusionsidentified by MSK-IMPACT

NTRK3

NTRK1

10 cases

8 cases

0.2% of the assayed

population


This may stem from intra-chromosomal or inter-chromosomal rearrangements

NTRK rearrangements create chimaeric genes

NTRKs are promiscuous: multitude of 5’ partners

Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204

Many 5’ gene partners (at least 25) described All rearrangements share an in-frame, intact kinase domain

In-frame fusion

Transcription and translation


Amatu A et al, ESMO open 2016

PROLIFERATION

DIFFERENTATION

SURVIVAL

NTRK fusions have oncogenic properties:

- induction of cancer cell proliferation

- activation of critical cancer-related downstream signalingpathways (e.g. MAPK and PI3K/AKT)

NTRK RECEPTOR SIGNALING=> The fusions typically occur in a mutually exclusive fashion with other strong mitogenicdrivers, i.e. genetic alterations affecting the most common driver genes belonging to the MAPK signalling pathway (KRAS, NRAS and BRAF)

NTRK ROS1 ALK

Drilon A et al, Cancer Discovery 2017 Drilon A et al, NEJM 2018

Change in tumor diameter

EFFICACY OF NTRK INHIBITORS

FDA drug approvals in 2018

Nature Reviews Clinical Oncology 2019

FDA drug approvals in 2018

Nature Reviews Clinical Oncology 2019

2019 update :FDA approval- Larotrectinib- Entrectinib

EMA approval- Larotrectinib

HOW CAN WE IDENTIFY THE PATIENTS?

HOW CAN WE RELIABLY DETECT NTRKFUSION GENES?

ESMO PRECISION MEDICINE WORKING GROUP ACTIVITY

• Identification of experts in the field• Experts in contact via first Tele-Conference (TC)• Creation of a detailed overview of available techniques and assays:

• i) reported in the literature and applied to different cohorts• ii) commercialized by different companies

• Second TC for discussion of key points

• Drafting of the manuscript and proposed recommendations

April 2018

NTRK FUSION DETECTION: POSSIBLE TOOLS


IMMUNOHISTOCHEMISTRY (IHC)

Advantages:

� it is a rapid method that can be easily employed in different laboratory environments => quick turnaround time

� it is able theoretically to detect only transcribed and translated fusion proteins

� It is (relatively) inexpensive: LDT versus Kit preparation


IHC

Colorectal adenocarcinoma

� anti-TRKA Ab� anti-TRKB Ab� panTRK Ab� Cocktail of Abs

- KM12 (TPM3-NTRK1), MO-91 (ETV6-NTRK3) and CUTO-3.29 (MPRIP-

NTRK1) cells

- Peripheral nerves

Pos controls

Non-neoplastic tissues

Neg controls


SOME CONSIDERATIONS ON IHC

� TRKA, TRKB and TRKC expression is restricted in adult tissues to smooth

muscle, testes and neuronal components

=> IHC is highly sensitive (from 95% to 100%) and specific (from 93% to 100%) for the

detection of NTRK rearrangements

� IHC is not a good assay when investigating CNS tumours; specificity is high only in

lesions/organs without smooth muscle/neuronal differentiation

� Sensitivity has been demonstrated to depend on the type of antibody used (false

negatives reported mainly for NTRK3 fusions)

IHC

The pattern of TRK expression detected by IHC can be variable in intensity and subcellular localisation

⇒ Nuclear pan-TRK IHC can be considered a diagnostic surrogate of NTRK3 fusions

⇒ Moderate to strong diffuse cytoplasmicpan-TRK IHC staining can be considered diagnostic of NTRK1/NTRK2 fusions

ETV6-NTRK3 fusion positive case

from Am J Surg Pathol 2017;41:1547–1551


IHC

The pattern of TRK expression detected by IHC can be variable in intensity and subcellular localisation

⇒ Nuclear pan-TRK IHC can be considered a diagnostic surrogate of NTRK3 fusions

⇒ Moderate to strong diffuse cytoplasmicpan-TRK IHC staining can be considered diagnostic of NTRK1/NTRK2 fusions

ETV6-NTRK3 fusion positive case


Intrahepatic cholangiocarcinoma

with a PLEKHA6-NTRK1 fusion

Secretory carcinoma of the salivary

gland with the canonical ETV6-

NTRK3 fusion

Colonic adenocarcinoma with an

LMNA-NTRK1 fusion

Modified from Cocco E et al, Cancer Res 2019

IHC

=> For tumours with only weak cytoplasmic expression of pan-TRK, an NTRK fusion should be confirmed by other molecular/cytogenetic methods to ensure that a fusion is present in patients being considered for targeted therapeutic agents

“Two-step approach”

IHC as a screening method

NGS to confirm the presence of rearrangement

Sensitivity is

crucial


Modern Pathology 2019; doi.org/10.1038/s41379-019-0324-7

Immunohistochemistryshowed overallsensitivity of 87.9% and specificity of 81.1%, with high sensitivity for NTRK1 (96%) and NTRK2 (100%) fusionsand lower sensitivity for NTRK3 fusions (79%).

Pan-Trk immunohistochemical reactions with antibody clone EPR17341 (Abcam, Cambridge, MA)

IHC - PITFALLS

• Most of the available antibodies are RUO• Ventana pan-TRK is CE-IVD: kit preparation

KIT PREPARATION:

ANTIBODY

+

OPTIVIEW DAB IHC DETECTION KIT

+

RABBIT MONOCLONAL NEGATIVE CONTROL Ig

+

SPECIFIC SOLUTIONS/WASHINGS

IMMUNOHISTOCHEMISTRY (IHC)

Advantages:

� it is a rapid method that can be easily employed in different laboratory environments => quick turnaround time

� it is able theoretically to detect only transcribed and translated fusion proteins

� It is (relatively) inexpensive: LDT versus Kit preparation


FISH

� Genes on a glass slide

Hicks and Tubbs, Human Pathology 2005

Amp

Del

Translocations/fus

FISH

• It is a commonly used method for detecting chromosomal rearrangement fusions in solid tumours (see ALK, ROS1 and RET…)

• Split-apart rearrangement probes are invariably easier in FFPE samples

FISH

Secretory carcinoma of the breastETV6/NTRK3 split apart probe

Normal Aberrant

=> FISH cannot ascertain the 5’ partner or whether the rearrangement results in a productive in-frame chimaeric transcript

=> Three separate FISH assays would have to be run in parallel => expensive and time consuming


FISHSecretory carcinoma of the breast

ETV6/NTRK3 split apart probe

The utility of FISH for screening cancers with NTRK1/2/3 fusions is limited, given the multitude of partners involved, the expertise required and its labour-intensive nature

=> Ideal technique when we have to confirm the presence of NTRK rearrangements in lesions where it is predicted to be found at high frequency => ETV6-NTRK3


NTRK FUSION DETECTION: POSSIBLE TOOLS

IN VITRO NUCLEIC ACID-BASED ASSAYS


IN VITRO NUCLEIC ACID-BASED ASSAYS OTHER THAN NGS

RT-PCR

• Typically used as an orthogonal

validation method in studies

exploring the genetic landscape

of subgroups of neoplasms by

high-throughput techniques

• The partner has to be known

• Specific primers to be designed

• Used in the context of

confirmation of ETV6-NTRK3 in

several studies


Nanostring

• nCounter Lung Fusion Panel

(only selected fusions)

• Custom-made panels

• Technology used also for

other types of diagnostic

testings

• No specific studies so far

Real Time PCR

• Just developed and soon

available

• Short TAT

• Low costs


RT-PCR










several studies


Nanostring






testings


Real Time PCR


available

• Short TAT

• Low costs


RT-PCR










several studies

Confirmatory technique Can be used for screening Can be used for screening


Real Time PCR


available

• Short TAT

• Low costs


RT-PCR










several studies

Confirmatory technique Can be used for screening Can be used for screening

Nanostring






testings


NGS

RNA next generation targeted sequencing assays

It enables de novo detection of fusion genes that are transcribed

Anchored Multiplex PCR (AMP) has become a widely adopted methodology for fusion gene detection

=> commercial ready to use kits as well customisable assays are available=> high technical sensitivity and specificity even in FFPE-derived RNA samples

The sequencing library targets fusion exons in multiple oncogenes including NTRK1 and/or NTRK3, or all of the three members of the NTRK family


AMPA target enriched chemistry that creates target enriched libraries for NGS

Works on both Illumina and Ion Torrent platforms

It uses a combination of gene specific primers and

molecular barcodes for multiplex targeted NGS using

low input clinical samples (FFPE tissues)

Images from https://archerdx.com

Able to detect and identify gene fusions without prior knowledge of fusion

partners

NGS

� the Oncomine assays (ThermoFisher Scientific), which cover fusion variants including NTRK1, NTRK2 and NTRK3.

� GeneTrails Solid Tumor Fusion Gene Panel (Knight Diagnostic Laboratories), designed to detect fusions involving 20 target genes including NTRK1, NTRK2, NTRK3

� the Universal Fusion/Expression Profile (Neogenomics), an assay capable of detecting different classes of genomic abnormalities such as fusion transcripts and transcriptomic gene expression levels in 1,385 genes (NTRK1, NTRK2, NTRK3 included)

RNA-BASED ASSAYS

Quality of RNA extracted

from FFPE samples?

Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204; Church AJ et al, Mod Pathol 2018; 31(3): 463–473.

RNA-BASED ASSAYS


from FFPE samples?

Pathology

Lab

Surgical

Theater

Grossing

Paraffin

embedding

Sectioning Staining

Fixation

Processing

RNA-BASED ASSAYS


from FFPE samples?

PlosOne 2007

Red indicates no amplification,

yellow–weak amplification,

green–good amplification

Influence of fixation time =>

RNA-BASED ASSAYS


from FFPE samples?

PlosOne 2007

Influence of storage =>

NGS

Targeted next generation DNA sequencing assays

Memorial Sloan Kettering Integrated Mutation Profil ing of Actionable Cancer Targets (MSK-IMPACT™) assay, a deep-coverage assay encompassing the entire coding regions and selected intronic and regulatory regions of >400 key cancer genes

Zehir A et al, Nature medicine 2018

This tumour-profiling multiplex panel has been recently

cleared by the U.S. Food and Drug Administration (FDA)

as an in vitro diagnostic test that can identify somatic

genetic alterations

NGS

� MSK-IMPACT™ can detect missense mutations, indels, copy number alterations, and selected gene fusions=> Probes for introns 3, and 7 to 12 of NTRK1, and intron 15 of NTRK2=> Probes for ETV6 introns 4 and 5 included to detect ETV6-NTRK3rearrangements

=> Other introns affected by NTRK rearrangements not included because too large for a DNA-based capture approach (approximate upper limit: 25 kb)

Targeted next generation DNA sequencing assays

NGS

Other DNA targeted sequencing assays that can be em ployed in the detecting of NTRK rearrangements

• FoundationOneCDx test (Foundation Medicine)• UW Oncoplex and the UCSF500 Cancer Gene Panel• SmartGenomics Complete –(PathGroup) Expanded Solid Tumor• Solid Tumor Focus Oncomine NGS Panel (Cancer Genetics)

NGS

1) DNA-based NGS has proven to be effective to detect gene rearrangementsand predicted fusions

2) Detected rearrangements by DNA-based assays may not result in fusions

=> correlation with surgical pathology and predicted transcript (for sequencing) is needed

3) NOT ALL of the NTRK rearrangements can be practically detected usingtargeted assays, especially those fusions involving NTRK2 and NTRK3where large intronic regions can render DNA-based detection challenging

Targeted next generation DNA sequencing assays – key concepts

METHODS FOR THE DETECTION OF NTRK FUSION GENES: BALANCING THE PROS & CONS

Method Sensitivity Specificity Detection of all fusion genes

Detection of partner

Detection of

expression

Screening

IHC High Moderate/ High

Yes No Yes Yes

FISH High High One per probe No No No

RNA seq NGS High High Yes Yes Yes Yes

DNA seq Moderate High Yes Yes No Yes


RECOMMENDATIONS FOR THESCREENING OF NTRK FUSIONS

Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019

Is the histologic tumor type known to harbor highly recurrent NTRK

fusions?

YES NO

As a confirmatory technique use FISH, RT-PCR, or RNA-

NGS assays with specific probes for the fusion

involving theknown NTRK gene

Is there a sequencing

platform available?

YESNO

Use front line NGS reliably detecting NTRK

fusions, preferably including RNA testing

when possible

Use IHC as a screening tool

Detection of TRK expression

NO TRK expression

IHC to confirm

expression


Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019

FOR PEDIATRIC TUMORS

Albert et al. J Clin Oncol 2018

OPEN QUESTIONS

OPEN QUESTIONS1. Which patients should be tested?

ARE THERE ANY CLINICO-PATHOLOGICAL

CORRELATIONS WE MAY USE?

=> The fusions typically occur in a mutually exclusive fashion with other strong mitogenicdrivers, i.e. genetic alterations affecting the most common driver genes belonging to the MAPK signalling pathway (KRAS, NRAS and BRAF)

=> They have also been reported as significantly more frequently encountered in microsatellite instability (MSI)-high tumours in the context of colorectal carcinoma patients

According to a recent study the association between NTRK fusions with MSI-high colorectal carcinomas seems to be strictly connected with MLH1 deficiency associated with MLH1 promoter hypermethylation in the context of a non-Lynch syndrome scenario

Church AJ et al, Mod Pathol 2018; 31(3): 463–473

Lezcano C et al, Am J Surg Pathol 2018; 42(8): 1052–1058.

Pietrantonio F et al, J Natl Cancer Inst 2017; 109(12): djx089.

Cocco E et al, Cancer Res 2019; 79(6): 1047–1053.

Cocco E et al, Cancer Res 2019; 79(6): 1047–1053.

MOST EXHAUSTIVE APPROACH WHEN SCREENING


1. WHICH PATIENTS SHOULD BE TESTED?

This population would be

likely represented by

‘any malignancy at an

advanced stage, in

particular if it has been

proven wild type for

other known genetic

alterations tested in

routine practice, and

especially if diagnosed in

young patients’.


SCREENING

THE EXAMPLE OF LUNG CANCER

NCCN guidelines 2019 for NSCLC

https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf

Latest version: August 2019

OPEN QUESTIONS2. Any other relevant issues for NTRK testing?

2. ANY OTHER RELEVANT ISSUES FOR NTRK TESTING?

Despite durable responses to TRK kinase-directed therapy in patients with NTRK-rearranged

tumours, it is expected that acquired resistance to therapy will ultimately emerge in most patients

…

=> Description of acquisition of secondary mutations in the TRK kinase domain after treatment

with entrectinib in two patients:

- NTRK1 G595R and G667C substitutions in a patient with LMNA– NTRK1 fusion-positive

colorectal cancer

- NTRK3 G623R substitution (homologous to TRKA G595R) in a patient with ETV6–NTRK3

fusion-positive secretory carcinoma of the salivary glands

Drilon A et al, Cancer Discov 2017; 7(9): 963–972.

Other TRK TKI active!

1. BAY2731954 (LOXO-195)

2. TPX-0005 (repotrectinib)

RESISTANCE TO THERAPY


“On target” resistance

Amatu A et al, ESMO open 2016

PROLIFERATION

DIFFERENTATION

SURVIVAL

NTRK fusions have oncogenic properties:

- induction of cancer cell proliferation

- activation of critical cancer-related downstream signalingpathways (e.g. MAPK and PI3K/AKT)

NTRK RECEPTOR SIGNALING

“Off target” resistance

RESISTANCE TO

THERAPY

Cocco E et al (Scaltriti’s lab at MSKCC), Nature Medicine Aug 25 2019

=> Convergent MAPK pathway

activation:

KRAS/BRAF mut

MET amplification

“Off target” resistance

Modified from Supplementary material by Marchiò C et

al, on behalf of the ESMO TR and PM Working Group,

Annals of Oncology 2019

DNA/RNA panelsTruSight Oncology 500

DNA + RNA* assay targeting523 genes for assessment of small variants, TMB, MSI, splice variants, and fusions

WE STILL NEED MORE METHODOLOGICAL STUDIES ON REPRODUCIBILITY AND HEAD TO HEAD COMPARISONS!

Thank you for your attention

ESMO PROJECT GROUP MEMBERS

•Caterina Marchiò , FPO-IRCCS Candiolo, University of Turin, Italy•Maurizio Scaltriti , MSKCC, USA

•Marc Ladanyi , MSKCC, USA•A. John Iafrate , Massachusetts General Hospital, Harvard Medical School, USA

•Frederique Bibeau , Caen University Hospital , France•Manfred Dietel , Charité, University Medicine Berlin, Germany•Teresa Troiani , Medical Oncology, Department of Precision Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy •Jaclyn Hechtman , MSKCC, USA

•Fernando López-Rios , Hospital Universitario HM Sanchinarro, Madrid, Spain•Jean-Yves Douillard , European Society for Medical Oncology, Lugano, Switzerland•Fabrice Andrè , Institut Gustave Roussy, Villejuif, France

•Jorge S. Reis-Filho , MSKCC, USAThank you to Svetlana Jezdic for coordination of activities!

esmo advanced course on ntrk gene fusion · modifiedfrom cocco e et al, cancerres 2019. ihc =>...

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