esmo advanced course on ntrk gene fusion...marchiò c et al, on behalf of the esmo tr and pm working...

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

Caterina Marchiò - University of Turin, Pathology Unit at FPO-IRCCS Candiolo Cancer InstituteLyon, 13-14 September 2019

[email protected]

DISCLOSURE OF INTEREST

Consultancy fees from Daichii Sankyo, MSD, Bayer, Roche

IDENTIFICATION/TESTING METHODOLOGIES AND CHALLENGESOutline

• 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, PI3Kand PKC pathways

Differentiation and survival in neuronal cells

Binding of neurotrophins to the extracellular region of TRK proteins


- 1q21-q22 – TRKA



• 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)


- 1q21-q22 – TRKA



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• Congenital mesoblastic nephroma

• 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 assayedpopulation


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

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- Podocytes

Pos controls

Non-neoplastic tissues (skin, blood vessels, inflammatory cells)

Neg controls


SOME CONSIDERATIONS ON IHC

TRKA, TRKB and TRKC expression is restricted in adult tissues to smoothmuscle, testes and neuronal components

=> IHC is highly sensitive (from 95% to 100%) and specific (from 93% to 100%) for thedetection of NTRK rearrangements

IHC is not a good assay when investigating CNS tumours; specificity is high only inlesions/organs without smooth muscle/neuronal differentiation

Sensitivity has been demonstrated to depend on the type of antibody used (falsenegatives reported mainly for NTRK3 fusions)

IHCThe 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

=> 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 iscrucial


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)

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

ETV6-NTRK3

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:

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


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 orthogonalvalidation method in studiesexploring the genetic landscapeof 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 diagnostictestings

• No specific studies so far

Real Time PCR

• Just developed and soonavailable

• Short TAT

• Low costs


RT-PCR






Nanostring





Real Time PCR


• Short TAT

• Low costs


RT-PCR





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


Real Time PCR


• Short TAT

• Low costs


RT-PCR





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

Nanostring





NGSRNA 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 known fusion exons in multiple oncogenes including NTRK1 and/or NTRK3, or all of the three members of the NTRK family


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 extractedfrom 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


Pathology Lab

SurgicalTheater

Grossing

Paraffinembedding

Sectioning Staining

Fixation

Processing

RNA-BASED ASSAYS


PlosOne 2007

Red indicates no amplification, yellow–weak amplification, green–good amplification

Influence of fixation time =>

RNA-BASED ASSAYS


PlosOne 2007

Influence of storage =>

NGSTargeted next generation DNA sequencing assays

Memorial Sloan Kettering Integrated Mutation Profiling 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

NGSOther DNA targeted sequencing assays that can be employed in the

detecting of NTRK rearrangements

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

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): 463–473Lezcano 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.

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 beenproven wild type for other known geneticalterations tested in routine practice, and especially if diagnosed in young patients’.


SCREENING

THE EXAMPLE OF LUNG CANCERNCCN 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?


2. ANY OTHER RELEVANT ISSUES FOR NTRK TESTING?

Despite durable responses to TRK kinase-directed therapy in patients with NTRK-rearrangedtumours, 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 pathwayactivation:

KRAS/BRAF mutMET 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

OPEN QUESTIONS3. Is there a gold standard technique?

3. IS THERE A GOLD STANDARD TECHNIQUE?

There is NOT a single technique that outperforms the others• IHC and RNA panels may be preferred but be aware of the limitations

There are techniques strategically better in some scenarios than others(histology-driven versus histology-agnostic)

A path lab should choose/adopt the technique(s) that guarantee high sensitivityand specificity in the context of screening and/or confirmation scenarios

In addition => There are also other pragmatical factors to be taken into account:- workload of cases/week- types of techniques already present in the path lab

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

Thank you for for attention – Questions?

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, USA

Thank you to Svetlana Jezdic for coordination of activities!

esmo advanced course on ntrk gene fusion...marchiò c et al, on behalf of the esmo tr and pm working...

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