TEST:

VENTANA pan-TRK (EPR17341) Assay

Pan-TRK IHC shows some utility as a diagnostic and surrogate marker for NTRK screening however, physiologic or non-specific expression may lead to false-positive results.

No NTRK or ALK translocations or increased copy number/amplification were identified in all eight cases which had fluorescence in situ hybridisation and/or next generation sequencing for NTRK1-3 and ALK available for assessment. None of the cases expressed BRAF-V600E.Although our study is limited, our report is the first to document PanTRK expression in AFH in the absence of identifiable NTRK1-3 gene alterations.

Even though pan-TRK immunohistochemical expression is not entirely sensitive or specific for NTRK-rearranged sarcomas, these neoplasms tend to exhibit diffuse staining of moderate/strong intensity, unlike its mimics. Pan-TRK should be performed in monomorphic uterine (corpus and cervix) spindle cell neoplasms that are negative for smooth muscle markers and hormone receptors and positive for CD34 and/ or S100. Ultimately, the diagnosis requires molecular confirmation.

CANTRK illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytic sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Conclusion This study supports the known conclusion that the diagnosis of a NTRK-related fusion-driven neoplasm cannot be made based solely on the immunohistochemical expression of panTRK. The significance of panTRK immunohistochemical expression in multiple different uterine mesenchymal neoplasms without NTRK-related fusions is, as of yet, not entirely clear but continues to raise questions regarding a) the exact mechanisms causing this, and b) the possible therapeutic utility of selective TRK inhibitors in the treatment of these neoplasms.

After reviewing the literature, possible NTRK fusions justified by their spindle-shaped morphology have not been studied. We carried out this study not for diagnostic purposes but to analyze possible new molecular associations and treatment targets.

Pan-TRK should be performed in uterine monomorphic spindle neoplasms negative for smooth muscle markers and hormone receptors and variably positive for CD34 and S100. The diagnosis of an NTRK-rearranged sarcoma requires molecular confirmation but pan-TRK immunohistochemistry is a useful screening tool to direct which cases require molecular testing

With this approach, the detection rate of NTRK fusions was 20 times higher (5.57%) by only screening 323 patients than the largest cohort in the literature (0.30%) comprising several hundred thousand patients. Based on our findings, we propose a multiparametric strategy (ie, "supervised tumor-agnostic approach") when pathologists start searching for NTRK fusions.

The rate of false negative cases was lower when pan-Trk staining was assessed with the lowest positivity threshold (≥1%). In conclusion, most evaluated pan-Trk IHC LDTs were able to detect NTRK3-fusion proteins, however a significant analytical variability was observed between antibodies, platforms and centers.

P=N/A; Recruiting --> Active, not recruiting

Given the therapeutic importance, testing for NTRK rearrangements in daily practice has become necessary and despite IHC being a fast and affordable tool, using it in routine diagnostics is complicated and requires a high level of expertise.

These patients may potentially benefit from immune checkpoint inhibitor therapy. Frequent HER2-low may offer novel anti-HER2 treatment options.

While low expression with Pan-TRK is common in TNBC, intermediate to high expression is rare (<5%). Carcinomas with no special type morphology and intermediate to high Pan-TRK expression should be tested for fusion transcript for potential therapeutic value.

Pan-Trk IHC has outstanding sensitivity for NTRK fusions detection, regardless of staining pattern, and can be used as an inexpensive screening test for NTRK fusions. Pan-Trk IHC has superior sensitivity compared to Idylla GeneFusion assay, especially for NTRK2 fusion detection. Interestingly, IHC+/NGS fusion- cases may have other kinase alterations, including fusions and copy number gains.

Our results indicate that positive nuclear staining for pan-Trk is highly specific for secretory carcinoma. In low to intermediate grade invasive breast carcinomas that share histologic features with secretory carcinomas, the addition of pan-Trk to a routing panel of ER/PR/HER2 is highly diagnostic. Our results also support that pan-Trk can differentiate secretory carcinoma from its triple-negative histological mimics such as adenoid cystic carcinoma, matrix-producing carcinoma and apocrine carcinoma, although this finding needs to be validated in larger studies.

NTRK fusions were not uncommon and detected in all NSCLC histologies as well as with concurrent mutations.

Actionable aberrations were most often located in PIK3CA (n = 18, 15%), ERBB2 (n = 15, 12%), HRAS and NOTCH1 (both n = 9, 7%). Actionable genetic aberrations were seen in 53.7% of all SGC cases on the RNA and DNA level, with varying percentages between subtypes.

P=N/A, N=3820, Completed, Institut Bergonié | Unknown status --> Completed

Interestingly, pan-TRK positivity was observed in one case with precursor lesions in both precancerous and cancerous regions, whereas MLH1 loss was restricted to the cancerous region. In summary, an optimized multi-step algorithm using pan-TRK IHC as a screening method was proposed to identify CRC patients harboring NTRK fusions.

We identified NTRK3 expression imbalance in one (1.1%) of 93 CCA tumours but this could not be confirmed with NGS. Our results show limited specificity of pan-TRK IHC to identify NTRK fusions. Pan-TRK IHC can thus be utilized as a screening technique for NTRK fusions in CCA, but positive samples require confirmatory testing with RNA-based methods.

This is the largest case series of PSCN/RN tested for NTRK fusions. We found that a subgroup of PSCN/RN is char-acterized by NTRK fusions, with a percentage of positive cases (13-14%) similar to what was previously shown by Kervarrec T. et al. Furthermore, we found that PSCN/RN pan-TRK(+) are pref-erentially localized on trunk/dorsum and extremities, and display moderate/high cytological atypia.

A screening based on IHC analysis showed limited sensitivity and specificity in the identification of NTRK-rearranged tumors. Since falsely negative results could preclude the administration of effective targeted drugs, alternative detection strategies should be considered for thyroid cancer.

The frequency of NTRK fusions among Hispanic patients with NSCLC is similar to that previously reported worldwide. Negative results on NGS may be related to pre-analytical factors such as DNA quality. To the best of our knowledge, this is the first study using a systematic population-based identification of NTRK fusion genes in Latin America.

"The detection of NTRK- fusions using NGS had high specificity over NTRK-fusion detection by using Pan-Trk IHC. Pan-Trk IHC is not a suitable tissue-efficient biomarker to screen for NTRK-fusions in CNS tumours, however RNA-based NGS sequencing should be used as an alternative method."

"Our results show that kinase fusions (20/30, 67%) and most importantly targetable NTRK1 fusions (7/30, 23%) are frequent in CRCs with dMLH1/BRAFV600Ewt/MLH1ph/RASwt. NGS was the most comprehensive method in finding the fusions, of which a subset can be screened by Idylla or IHC, provided that the result is confirmed by NGS."

TRK inhibitors such as larotrectinib and entrectinib have shown anti-tumor activity regardless of tumor types. NTRK gene fusion is extremely rare in primary liver tumors. Although equivocal/weak pan-TRK expression by immunohistochemistry can be seen in some HCC, no NTRK gene fusion was detected by RNA-seq confirmatory test, suggesting that equivocal/weak pan-TRK expression may not be used as a surrogate for NTRK gene fusion. Nevetheless, RNA-seq detected many other gene fusions in HCC, the function of which warrant further studies.

This case demonstrates the value of pan-TRK immunohistochemistry in identifying these rare but prognostically significant tumors, particularly among older patients, in whom the diagnosis may be less expected. Pan-TRK immunohistochemistry may be especially useful in low-grade–appearing but hormone receptor–weak or –negative tumors, as most triple-negative breast cancers are generally higher grade, whereas most low-grade invasive ductal carcinomas have strong estrogen receptor expression.

After the US Food and Drug Administration approval of Larotrectinib, the detection of NTRK fusion in many late-stage cancers have become a standard part of management... We expand the list of tumors that may be positive for NTRK fusion protein by IHC. The high diagnostic sensitivity, cost-effectiveness, and rapid turnaround time, the monoclonal antibody (EPR17341) may potentially be used as a screening marker to rule in NTRK positive tumors especially when DNA or RNA-based molecular testing is not available.

In contrast to other cancers, pan-TRK IHC appears of limited interest in this field because there is no 'on/off' IHC positivity criterion to distinguish between NTRK-rearranged and non-NTRK-rearranged gliomas. RNA sequencing analyses are necessary in FISH positive cases with less than 30% positive nuclei, to avoid false positivity when scoring is close to the detection threshold.

The first NTRK multicentre research have found 8.7% IHC panTRK-positive tumours. Positive staining with confirmed NTRK fu- sion was seen in thyroid carcinoma, breast and salivary gland secretory carcinoma and infantile fibrosarcoma and mesoblastic nephroma.

"Our study shows that Pan-Trk IHC detects NTRK1 fusions with 100% specificity in CRC. Our results confirm that NTRK1 fusions are frequently detected in dMLH1/BRAFV600Ewt (11%) and especially in dMLH1/MLH1ph/BRAFV600Ewt (16%) CRCs justifying screening for NTRK fusions in these subsets of CRCs. Our findings of NTRK1 fusions with partners LMNA, PLEKHA6 and TPM3 in CRC are consis- tent with previous studies but noteworthy, we introduce a novel IRF2BP2-NTRK1 fusion in CRC."

Frequency of NTRK1/2/3 fusions was 5.8% in our dMMR/MSI mCRCs cohort. These fusions were not restricted to sporadic cases. The diagnostic accuracy of pan-TRK IHC was low.

NTRK1-3 fusions using an RNA-based sequencing approached could not be detected. This stresses the importance of confirmation of immunohistochemistry results by molecular methods.

Frequency of NTRK1/3 fusions is 6.3% in our dMMR/MSI mCRCs population . These fusions are not restricted to sporadic cases . The diagnostic accuracy of pan-TRK IHC is low .

Given the new therapeutic opportunity offered by anti-TRK targeted therapies to treat patients with advanced cancers including GISTs, it is worth to extend molecular analysis to NTRK fusions testing in KIT, PDGFRA, and BRAF wild type GISTs. Pan-TRK IHC appears not relevant in this field but performing a simple NTRK3 fluorescent in situ hybridization test consists in a valuable approach to identify the rare NTRK3-rearranged GISTs treatable using anti-TRK therapies.

Purpose: Despite the low prevalence of oncogenic neurotrophin receptor kinase (NTRK) fusion among most solid tumors, the encouraging results of tropomyosin receptor kinase (Trk) inhibitors such as larotrectinib and entrectinib have prompted the screening of eligible patients. NGS was effectively help to screen patients with novel or reported NTRK fusion, who may benefit from NTRK inhibitor therapy. And NGS showed good consistency with IHC. Further prospective study is needed to compare the impact of the NTRK status identified by NGS and IHC on NTRK inhibitors efficacy.

We described for the first time a follicular patterned tumor with histological features reminiscent of NIFTP, but harbouring NTRK1 rearrangement and we open the question whether NTRK fusion may represent a very early genetic event in molecular pathogenesis of thyroid neoplasia and may occur in NIFTP. Further research is needed to elucidate the clinicopathological landscape of NTRK-rearranged thyroid carcinoma (NTRC), the pathogenic role and clinical significance of NTRK fusions found in thyroid nodules and cancer.

Molecular alterations can be effectively identified routinely in formalin-fixed paraffin-embedded ( FFPE) tissue samples. Patients presenting thyroid tumors with targetable alterations could be eligible for specific therapy such as larotrectinib or entrectinib targeting NTRK fusion or selpercatinib and pralsetinib targeting RET fusion. Immunotherapy alone or in combination with other drugs may be a promising treatment for aggressive types of thyroid cancers.

This study provides a framework to assess the inter-reader agreement of the automated VENTANA pan-TRK (EPR17341) Assay in NGS-confirmed NTRK gene fusion-positive and fusion-negative cases. Overall agreement between readers was high, but varied based on fusion partner and staining pattern observed with this analytic assay.

The presence of NTRK fusion genes in non-small cell lung cancer is exceedingly rare. The use of the immunohistochemical NTRK assay will result in a small number of false positive cases. This should be considered when the assay is applied as a screening tool in clinical diagnostics.

Availability of NTRK+ samples for validation of NTRK tests is challenging. CANTRK provides a framework for biomarker assay validation projects involving rare cancers. A multi-centre collaboration is instrumental in this setting to overcome the challenges faced by individual laboratories to source sufficient positive samples for independent validation and clinical implementation.

Pan-TRK (clone EPR17341, Roche/Ventana) immunhistochemistry is a reliable, highly sensitive but less specific diagnostic marker that can be expressed in non-NTRK-rearranged mesenchymal neoplasms. Conclusion Pan-TRK (clone EPR17341, Roche/Ventana) can be used as a surrogate marker for an identification of NTRK fusion, nevertheless, an RNA-based NGS for detection of the specific fusion should be performed to confirm the rearrangement if patients are undergoing targeted therapy.