ESMO ADVANCED COURSE ON NTRK GENE FUSION · Modifiedfrom Cocco E et al, CancerRes 2019. IHC => For...
Transcript of ESMO ADVANCED COURSE ON NTRK GENE FUSION · Modifiedfrom Cocco E et al, CancerRes 2019. IHC => For...
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
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
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
- Expression restricted to specific tissues (in adult tissues: smooth muscle, testes and neuronal components)
NEUROTROPHIC TROPOMYOSIN RECEPTOR KINASE (NTRK)
• NTRK1- 1q21-q22 – TRKA
• NTRK2- 9q22.1 – TRKB
• NTRK3- 15q25 – TRKC
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
Cocco E, Scaltriti M & Drilon A, Nature Reviews Clinical Oncol 2018
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
Cocco E, Scaltriti M & Drilon A, Nature Reviews Clinical Oncol 2018
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
NTRK FUSION DETECTION: POSSIBLE TOOLS
IN VITRO NUCLEIC ACID-BASED ASSAYS
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
IN VITRO NUCLEIC ACID-BASED ASSAYS OTHER THAN NGS
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
• No specific studies so far
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
IN VITRO NUCLEIC ACID-BASED ASSAYS OTHER THAN NGS
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
• No specific studies so far
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
Confirmatory technique Can be used for screening Can be used for screening
IN VITRO NUCLEIC ACID-BASED ASSAYS OTHER THAN NGS
Real Time PCR
• Just developed and soon
available
• Short TAT
• Low costs
• No specific studies so far
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
Confirmatory technique Can be used for screening Can be used for screening
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
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Quality of RNA extracted
from FFPE samples?
Pathology
Lab
Surgical
Theater
Grossing
Paraffin
embedding
Sectioning Staining
Fixation
Processing
RNA-BASED ASSAYS
Quality of RNA extracted
from FFPE samples?
PlosOne 2007
Red indicates no amplification,
yellow–weak amplification,
green–good amplification
Influence of fixation time =>
RNA-BASED ASSAYS
Quality of RNA extracted
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019
Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019
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.
Cocco E et al, Cancer Res 2019; 79(6): 1047–1053.
MOST EXHAUSTIVE APPROACH WHEN SCREENING
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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’.
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
“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!