esmo advanced course on ntrk gene fusion...marchiò c et al, on behalf of the esmo tr and pm working...
TRANSCRIPT
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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
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DISCLOSURE OF INTEREST
Consultancy fees from Daichii Sankyo, MSD, Bayer, Roche
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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
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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
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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
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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)
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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)
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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
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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
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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
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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
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In-frame fusion
Transcription and translation
Cocco E, Scaltriti M & Drilon A, Nature Reviews Clinical Oncol 2018
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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)
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NTRK ROS1 ALK
Drilon A et al, Cancer Discovery 2017 Drilon A et al, NEJM 2018
Change in tumor diameter
EFFICACY OF NTRK INHIBITORS
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FDA drug approvals in 2018
Nature Reviews Clinical Oncology 2019
HOW CAN WE IDENTIFY THE PATIENTS?
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HOW CAN WE RELIABLY DETECT NTRKFUSION GENES?
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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
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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
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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
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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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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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)
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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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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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)
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Modern Pathology 2019; doi.org/10.1038/s41379-019-0324-7
ETV6-NTRK3
Pan-Trk immunohistochemical reactions with antibody clone EPR17341 (Abcam, Cambridge, MA)
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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
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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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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FISH
Genes on a glass slide
Hicks and Tubbs, Human Pathology 2005
Amp
Del
Translocations/fus
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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
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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
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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
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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
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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
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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 diagnostictestings
• No specific studies so far
Real Time PCR
• Just developed and soonavailable
• Short TAT
• Low costs
• No specific studies so far
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
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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 diagnostictestings
• No specific studies so far
Real Time PCR
• Just developed and soonavailable
• Short TAT
• Low costs
• No specific studies so far
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
Confirmatory technique Can be used for screening Can be used for screening
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IN VITRO NUCLEIC ACID-BASED ASSAYS OTHER THAN NGS
Real Time PCR
• Just developed and soonavailable
• Short TAT
• Low costs
• No specific studies so far
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
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 diagnostictestings
• No specific studies so far
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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
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Ann Oncol. 2019 Jul 3. pii: mdz204
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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)
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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.
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RNA-BASED ASSAYS
Quality of RNA extractedfrom FFPE samples?
Pathology Lab
SurgicalTheater
Grossing
Paraffinembedding
Sectioning Staining
Fixation
Processing
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RNA-BASED ASSAYS
Quality of RNA extractedfrom FFPE samples?
PlosOne 2007
Red indicates no amplification, yellow–weak amplification, green–good amplification
Influence of fixation time =>
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RNA-BASED ASSAYS
Quality of RNA extractedfrom FFPE samples?
PlosOne 2007
Influence of storage =>
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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
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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
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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)
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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
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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
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RECOMMENDATIONS FOR THESCREENING OF NTRK FUSIONS
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Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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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
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Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019
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Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019
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Penault-Llorca F, Rudzinski ER, Sepulveda AR, J Clin Pathol 2019
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FOR PEDIATRIC TUMORS
Albert et al. J Clin Oncol 2018
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OPEN QUESTIONS
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OPEN QUESTIONS1. Which patients should be tested?
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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.
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MOST EXHAUSTIVE APPROACH WHEN SCREENING
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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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’.
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
SCREENING
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THE EXAMPLE OF LUNG CANCERNCCN guidelines 2019 for NSCLC
https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf
Latest version: August 2019
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OPEN QUESTIONS2. Any other relevant issues for NTRK testing?
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2. ANY OTHER RELEVANT ISSUES FOR NTRK TESTING?
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
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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)
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RESISTANCE TO THERAPY
Marchiò C et al, on behalf of the ESMO TR and PM Working Group, Annals of Oncology 2019
“On target” resistance
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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
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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
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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
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OPEN QUESTIONS3. Is there a gold standard technique?
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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
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WE STILL NEED MORE METHODOLOGICAL STUDIES ON REPRODUCIBILITY AND HEAD TO HEAD COMPARISONS!
Thank you for for attention – Questions?
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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!