Multicenter Harmonization Study of Pan-Trk Immunohistochemistry for the Detection of NTRK3 Fusions.

Pan-Trk immunohistochemistry has been described as a screening test for the detection of NTRK fusions in a broad spectrum of tumor types. However, pan-Trk testing in the clinical setting may be limited by many factors, including analytical parameters such as clones, platforms, and protocols used. This study aimed to harmonize pan-Trk testing using various clones and immunohistochemical (IHC) platforms and to evaluate the level of analytical variability across pathology laboratories. We developed several IHC pan-Trk assays using clones EPR17341 (Abcam) and A7H6R (Cell Signaling Technology) on Ventana/Roche, Agilent, and Leica platforms. To compare them, we sent unstained sections of a tissue microarray containing 9 cases with NTRK3 fusions to participating laboratories, to perform staining on Ventana/Roche (10 centers), Agilent (4 centers), and Leica (3 centers) platforms. A ready-to-use pan-Trk IVD assay (Ventana/Roche) was also performed in 3 centers. All slides were centrally and blindly reviewed for the percentage of stained tumor cells. Laboratory-developed tests with clone EPR17341 were able to detect pan-Trk protein expression in all cases, whereas lower rates of positivity were observed with clone A7H6R. Moderate to strong variability of the positive cases rate was observed with both antibodies in each IHC platforms type and each of the positivity cut points evaluated (≥1%, ≥10%, and ≥50% of stained tumor cells). 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 laboratory-developed tests were able to detect NTRK3-fusion proteins; however, a significant analytical variability was observed between antibodies, platforms, and centers.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Idylla MSI test combined with immunohistochemistry is a valuable and cost effective strategy to search for microsatellite instable tumors of noncolorectal origin.

Recent diagnostic and therapeutic progresses have increased the need of searching for microsatellite instability (MSI) in cancer samples beyond colorectal cancer (CRC) ones. The availability of the fully-automated Idylla MSI test (Biocartis), implementable easily in pathology laboratories, offers the opportunity to reconsider MSI diagnostic strategies towards rapid and in-house diagnosis. In this study, we evaluate the performances and cost-effectiveness of an in-house Idylla MSI testing in comparison with an externalized testing of about 54 non-CRC tumor samples. The Idylla MSI test concluded in valid analyses in 53/54 (98.1%) tumor samples with MSI statuses concordant with external molecular and immunohistochemical testing in 50/53 (94.3%) samples. Wrong Idylla MSI test results were obtained in 3/53 (5.7%) samples. Manual checking of microsatellite analyses results and confrontation between the results of Idylla and immunohistochemical analyses have permitted detection and correction of the discrepancies. The implementation of an in-house Idylla MSI testing for non-CRC tumors, necessarily combined with immunohistochemistry searching for MSI tumors, appeared not only valuable in terms of performances, but also in terms of cost-effectiveness without increasing the analyses-related costs but decreasing dramatically their turnaround times to one single working day.

PD-L1 expression in recurrent head and neck squamous cell carcinoma.

PURPOSE: To evaluate the Programmed Cell Death Ligand (PD-L1) expression at diagnosis and relapse in patients with head and neck carcinoma (HNSCC) treated with radio(chemo)therapy. METHODS: PD-L1 immunohistochemistry was performed in tumor cells (TC) and immune cells (IC) in 44 patients and scored as 0 = 0%, 1 = < 5%, 2 = 6-49% or 3 = ≥ 50% cells. RESULTS: PD-L1 expression on TC before RT was scored as 0, 1, 2 and 3 in 28, 4, 8 and 4 patients, respectively. In 10 patients, IC did not show any PD-L1 expression; while in 8, 16, and 10 patients, PD-L1 expression was scored 1, 2 and 3, respectively. At relapse, 7/36 patients had a PD-L1 expression positivation in TC, while the opposite was observed in 6 patients. Overall, survival at 2 years was higher in patients with PD-L1 expression (90% versus 62.5%, p = 0.032). CONCLUSION: PD-L1 expression may vary throughout the course of the disease. A re-evaluation of PD-L1 expression on biopsies at the time of recurrence should be recommended.

[Rapid Idylla™ mutational testing: Current and future theranostic applications]. / Diagnostic mutationnel rapide Idylla™ : applications théranostiques actuelles et futures.

Molecular analyses have become mandatory for treatment choices in patients with various advanced cancers. Beside next generation sequencing (NGS) analyzing genes panels, non-NGS targeted analyses about the main biomarkers remain of interest. In this article, we review the data about the fast and fully automated real-time PCR platform Idylla™ (Biocartis, Mechelen, Belgium) permitting the mutational analyses of BRAF, KRAS, NRAS, EGFR and microsatellite instability notably in melanoma, non-small-cell lung cancer and colorectal cancer samples. Future applications as well as the implementation of Idylla™ in the workflow of pathology and/or molecular biology laboratories are also discussed.

[How to test for NTRK gene fusions: A practical approach for pathologists]. / Recherche de fusions des gènes NTRK : guide pratique pour les pathologistes.

The recent availability of targeted anti-TRK therapies represents a new opportunity to treat patients with advanced cancers harboring NTRK gene fusions. In this article, we present an update on the practical modalities of implementing a "NTRK testing" to search for these fusions in view of the performances and availability of the different testing methods and the epidemiological characteristics of the tumors liable to present the NTRK1, NTRK2 or NTRK3 gene fusions.

Decalcification can cause the failure of BRAF molecular analyses and anti-BRAFV600E VE1 immunohistochemistry.

BRAF mutation detection is worthwhile for the management of patients with some advanced cancers. The tumor samples are sometimes difficult to analyze using DNA-based molecular methods because of poor tumor DNA quality or quantity. Anti-BRAFV600E VE1 immunohistochemistry (IHC) has been proposed as a valuable ancillary tool to analyze some "molecularly challenging" tumor samples. In this technical study, we focused on its application in the field of decalcified tumor samples. We selected four patients with known BRAFV600E-mutated cancer (3 metastatic melanomas and 1 hairy cell leukemia) and paired non-decalcified/decalcified tumor samples. Molecular analyses failed in the four decalcified samples (3 bone metastases and 1 osteo-medullar biopsy) with non-contributive mutation status. Whereas non-decalcified tumor samples were all positive using anti-BRAFV600E VE1 IHC, the four decalcified samples were concluded negative. Because decalcified tumor samples are difficult to analyze from a molecular point of view, it is tempting to use IHC instead of DNA-based methods searching for BRAFV600E mutations in these samples. Nevertheless, the decalcification process may also cause false-negative results using VE1 IHC. Decalcified samples require specific and optimized IHC and molecular protocols and quality controls.

Non-secretory breast carcinomas lack NTRK rearrangements and TRK protein expression.

Anti-TRK targeted therapies offer opportunities to treat patients with advanced NTRK1/2/3-rearranged cancers. Beyond NTRK-rearranged secretory breast carcinomas, little is known about NTRK rearrangements and the expression of TRK proteins in non-secretory breast carcinomas. We search for TRK proteins expressions using pan-TRK immunohistochemistry and NTRK1, NTRK2 and NTRK3 rearrangements using fluorescent in situ hybridization (FISH) tests in a set of tissue microarray included breast carcinomas. Only 1/339 invasive breast carcinomas, the only example of secretory subtype, was positive using pan-TRK immunohistochemistry and harboured a NTRK-rearrangement (NTRK1 positive FISH test). According to our results, druggable NTRK rearrangements and related-TRK proteins expression are not encountered in non-secretory breast carcinomas.

[Meningeal melanoma arising from a preexisting meningeal melanocytoma: A clinical, pathological and cytogenetic study about one case]. / Transformation d'un mélanocytome méningé en mélanome : étude clinique, histopathologique et cytogénétique.

Meningeal melanocytic tumors are rare. We report an exceptional case of transformation of a meningeal melanocytoma in a malignant melanoma. The course of the disease extents from 61-years to 85-years and ends with the death of the patient. Besides histopathological and immunohistochemical data, we also report the array CGH study of the melanocytoma and melanoma components suggesting the malignant transformation from whole chromosome gains in the melanocytoma to additional segmental aberrations in the malignant melanoma. Beyond the rarity of this tumor subtype, this case report highlights the potential interest of molecular analyses for diagnostic and prognostic purposes in the field of meningeal melanocytic tumors.

Evaluation of a Rapid, Fully Automated Platform for Detection of BRAF and NRAS Mutations in Melanoma.

BRAF and NRAS genetic analyses are time-consuming and can delay treatment choices in patients with metastatic melanomas presenting with acute deterioration. We compared the rapid, real-time, fully automated molecular diagnosis platform Idylla™ with next-generation sequencing (NGS) and immunohistochemistry for detection of BRAF and NRAS mutations in 36 patients with metastatic melanomas. The Idylla™ NRAS-BRAF-EGFRS492R mutation assay (110 min per sample) detected BRAF and NRAS mutations in 15 and 17 samples, respectively. One NRAS mutation was different between NGS and Idylla™ (NRASG13C vs. NRASG12A/D). Four samples were BRAF and NRAS wild-type. The global concordance between NGS and Idylla™ assays was 97.2% (35/36 cases). Immunohistochemistry was positive only in 9/9 BRAFV600E- and 6/6 NRASQ61R-mutated samples with VE1 and SP174 antibodies, respectively. The Idylla™ platform is a valuable rapid molecular diagnosis tool to reduce the delay in BRAF and NRAS analyses-related treatment choices for patients with metastatic melanoma presenting with acute deterioration.

A transcriptome-based protein network that identifies new therapeutic targets in colorectal cancer.

BACKGROUND: Colon cancer occurrence is increasing worldwide, making it the third most frequent cancer. Although many therapeutic options are available and quite efficient at the early stages, survival is strongly decreased when the disease has spread to other organs. The identification of molecular markers of colon cancer is likely to help understanding its course and, eventually, to uncover novel genes to be targeted by drugs. In this study, we compared gene expression in a set of 95 human colon cancer samples to that in 19 normal colon mucosae, focusing on 401 genes from 5 selected pathways (Apoptosis, Cancer, Cholesterol metabolism and lipoprotein signaling, Drug metabolism, Wnt/beta-catenin). Deregulation of mRNA levels largely matched that of proteins, leading us to build in silico protein networks, starting from mRNA levels, to identify key proteins central to network activity. RESULTS: Among the analyzed genes, 10.5% (42) had no reported link with colon cancer, including the SFRP1, IGF1 and ADH1B (down), and MYC and IL8 (up), whose encoded proteins were most interacting with other proteins from the same or even distinct networks. Analyzing all pathways globally led us to uncover novel functional links between a priori unrelated or rather remotely connected pathways, such as the Drug metabolism and the Cancer pathways or, even more strikingly, between the Cholesterol metabolism and lipoprotein signaling and the Cancer pathways. In addition, we analyzed the responsiveness of some of the deregulated genes essential to network activities, to chemotherapeutic agents used alone or in presence of Lovastatin, a lipid-lowering drug. Some of these treatments could oppose the deregulations occurring in cancer samples, including those of the CHECK2, CYP51A1, HMGCS1, ITGA2, NME1 or VEGFA genes. CONCLUSIONS: Our network-based approach allowed discovering genes not previously known to play regulatory roles in colon cancer. Our results also showed that selected drug treatments might revert the cancer-specific deregulation of genes playing prominent roles within the networks operating to maintain colon homeostasis. Among those genes, some could constitute novel testable targets to eliminate colon cancer cells, either directly or, potentially, through the use of lipid-lowering drugs such as statins, in association with selected anticancer drugs.

Cosmetic Outcome and Chronic Breast Toxicity After Intraoperative Radiation Therapy (IORT) as a Single Modality or as a Boost Using the Intrabeam® Device: A Prospective Study.

PURPOSE: We aim to report our results in terms of chronic toxicities and cosmetic outcomes after intraoperative radiotherapy (IORT) using kV X-rays in women treated for early breast cancer at our institution. METHODS: Patients with early breast carcinoma were recruited between April 2011 and November 2014. After breast-conserving surgery, patients were treated with IORT using the Intrabeam® device. IORT was completed by whole-breast radiotherapy (WBRT) at a dose of 46-50.4 Gy in 23-28 fractions in case of adverse pathologic criteria on the final specimen examination. Skin toxicity was graded using the Late Effects in Normal Tissues-Subjective, Objective, Management and Analytic (LENT-SOMA) scale every 6 months, and cosmetic outcomes were evaluated at 36 months by patient self-evaluation and by two radiation oncologists, on a 1-10 scale. RESULTS: Forty-one women received IORT only and 30 patients received IORT followed by WBRT (IORT + WBRT group). After a median follow-up of 38.9 months, no locoregional or distant recurrence occurred. After IORT only, 2.4% of grade 2 or higher breast fibrosis, and no other grade 2 or higher disease, was observed. In the IORT + WBRT group, grade 2 or higher fibrosis and grade 2 or higher breast retraction were observed in 43.3 and 23.3% of patients, respectively. Objective cosmetic outcomes were very good and significantly better in the IORT-only group compared with the IORT + WBRT group (8.87 vs. 6.96) (p < 0.001). CONCLUSION: IORT using the Intrabeam® is well-tolerated, with very little chronic toxicity and good cosmetic outcome. However, a high rate of grade 2 or higher chronic breast toxicity was observed when IORT had to be completed by WBRT.

Upfront immunohistochemistry improves specificity of Helicobacter pylori diagnosis. A French pathology laboratory point of view.

BACKGROUND: There is no consensus about the histopathologic methods to detect Helicobacter pylori in gastric biopsies to date. We aimed to question about the value of upfront anti-H. pylori immunohistochemistry in this field. MATERIAL AND METHODS: We led a retrospective study about the rate of H. pylori-positive gastric biopsies before and after the implementation of upfront immunohistochemistry, the inter-rater and intermethods agreements in H. pylori identification about Hematoxylin-Eosin Saffron (HES), Giemsa, and immunohistochemistry stains and the histopathologic features associated with low amounts of H. pylori. RESULTS: First, the rate of H. pylori-positive gastric biopsies significantly diminished after the implementation of upfront immunohistochemistry (from 21.15% to 12.56%, P<.0001), suggesting potential overdiagnosis of H. pylori infection before the use of immunohistochemistry. Secondly, immunohistochemistry was the most reproducible and performing stain (kappa values >0.80), but HES and Giemsa stains also presented good-to-very good agreements. Finally, less than 1% of gastric biopsies with inconspicuous H. pylori infection showed no mucosal injury pointing out that any HES-detected mucosal injury could help to preselect the gastric biopsies requiring ancillary stains for the detection of H. pylori. CONCLUSIONS: Albeit being considered as a gold standard in the detection of H. pylori, the interest of using immunohistochemistry as an upfront stain on gastric biopsies is still debated. In our opinion, its use in second line in case of ambiguous HE/HES-Giemsa result is more appropriate. Further effort is needed to optimize the inexpensive but feasible HE/HES-based detection of H. pylori.

Mutational status of synchronous and metachronous tumor samples in patients with metastatic non-small-cell lung cancer.

BACKGROUNDS: Despite reported discordance between the mutational status of primary lung cancers and their metastases, metastatic sites are rarely biopsied and targeted therapy is guided by genetic biomarkers detected in the primary tumor. This situation is mostly explained by the apparent stability of EGFR-activating mutations. Given the dramatic increase in the range of candidate drugs and high rates of drug resistance, rebiopsy or liquid biopsy may become widespread. The purpose of this study was to test genetic biomarkers used in clinical practice (EGFR, ALK) and candidate biomarkers identified by the French National Cancer Institute (KRAS, BRAF, PIK3CA, HER2) in patients with metastatic non-small-cell lung cancer for whom two tumor samples were available. METHODS: A retrospective study identified 88 tumor samples collected synchronously or metachronously, from the same or two different sites, in 44 patients. Mutation analysis used SNaPshot (EGFR, KRAS, BRAF missense mutations), pyrosequencing (EGFR and PIK3CA missense mutations), sizing assays (EGFR and HER2 indels) and IHC and/or FISH (ALK rearrangements). RESULTS: About half the patients (52%) harbored at least one mutation. Five patients had an activating mutation of EGFR in both the primary tumor and the metastasis. The T790M resistance mutation was detected in metastases in 3 patients with acquired resistance to EGFR tyrosine kinase inhibitors. FISH showed discordance in ALK status between a small biopsy sample and the surgical specimen. KRAS mutations were observed in 36% of samples, six patients (14%) having discordant genotypes; all discordances concerned sampling from different sites. Two patients (5%) showed PI3KCA mutations. One metastasis harbored both PI3KCA and KRAS mutations, while the synchronously sampled primary tumor was mutation free. No mutations were detected in BRAF and HER2. CONCLUSIONS: This study highlighted noteworthy intra-individual discordance in KRAS mutational status, whereas EGFR status was stable. Intratumoral heterogeneity for ALK rearrangement suggests a limitation of single-biopsy analysis for therapeutic strategy with crizotinib.

ROS1 fusions in cancer: a review.

The ROS1 gene belongs to the sevenless subfamily of tyrosine kinase insulin receptor genes. A literature review identified a ROS1 fusion in 2.54% of the patients with lung adenocarcinoma and even higher frequencies in spitzoid neoplasms and inflammatory myofibroblastic tumors. At present, 26 genes were found to fuse with ROS1, some of them already known to fuse with RET and ALK. All the fusion proteins retain the ROS1 kinase domain, but rarely its transmembrane domain. Most of the partners have dimerization domains that are retained in the fusion, presumably leading to constitutive ROS1 tyrosine kinase activation. Some partners have transmembrane domains that are retained or not in the chimeric proteins. Therefore, different ROS1 fusions have distinct subcellular localization, suggesting that they may activate different substrates in vivo.