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.

[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.

MSI-High RAS-BRAF wild-type colorectal adenocarcinomas with MLH1 loss have a high frequency of targetable oncogenic gene fusions whose diagnoses are feasible using methods easy-to-implement in pathology laboratories.

Targetable kinase fusions are extremely rare (<1%) in colorectal cancers (CRCs), making their diagnosis challenging and often underinvestigated. They have been shown particularly frequently among MSI-High, BRAF/KRAS/NRAS wild-type CRCs with MLH1 loss (MLH1loss MSI-High wild-type). We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation. Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1loss MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1loss MSI-High wild-type CRCs contained targetable kinase fusions. Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1loss MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.

Fluorescent In Situ Hybridization Must be Preferred to pan-TRK Immunohistochemistry to Diagnose NTRK3-rearranged Gastrointestinal Stromal Tumors (GIST).

Tyrosine kinase inhibitors have revolutionized the treatment of patients with gastrointestinal stromal tumors (GISTs). Nevertheless, some GISTs do not contain any targetable KIT or PDGFRA mutations classically encountered in this field. Novel approved therapies targeting TRK chimeric proteins products of NTRK genes fusions consist in a promising approach to treat some patients with GISTs lacking any identified driver oncogenic mutation in KIT, PDGFRA or BRAF genes. Thus, an adequate testing strategy permitting to diagnose the rare NTRK-rearranged GISTs is required. In this work, we studied about the performances of pan-TRK immunohistochemistry (IHC) and NTRK1/2/3 fluorescent in situ hybridization in a series of 39 GISTs samples. Among 22 patients with GISTs lacking KIT or PDGFRA mutations, BRAFV600E IHC permitted to diagnose 2/22 (9%) BRAFV600E-mutated GISTs and, among the 20 KIT, PDGFRA, and BRAF wild type tumors, 1/20 (5%), NTRK3-rearranged tumor was diagnosed using NTRK3 fluorescent in situ hybridization. Pan-TRK IHC using EPR17341 and A7H6R clones was negative in this NTRK3-rearranged sample. Pan-TRK IHC was frequently positive in NTRK not rearranged tumors without (24 samples analyzed) or with (15 samples analyzed) KIT or PDGFRA mutations with major discrepancies between the 2 IHC clones (intraclass correlation coefficient of 0.3042). 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.

A 60-Year-Old Woman With Recurrent Hemoptysis.

CASE PRESENTATION: A 60-year-old non-smoker white woman presented with a new episode of hemoptysis. She reported recurrent hemoptoic sputum in the past month. She had no relevant medical history, except presumed resolved bacterial pneumonia 1 year ago. She denied taking immunosuppressive treatment and was not exposed to lung irritants. She had not traveled recently. General health status was good. She denied fever, dyspnea, chest pain, and extra-pulmonary symptoms.

Evaluation of KRAS, NRAS and BRAF mutational status and microsatellite instability in early colorectal carcinomas invading the submucosa (pT1): towards an in-house molecular prognostication for pathologists?

AIM: We aimed to study the prognostic value of KRAS, NRAS, BRAF mutations and microsatellite stable (MSS)/instable (MSI) in the field of colorectal cancer invading the submucosa (ie, pT1 colorectal cancer (CRC)). METHODS: We led a case-control study in tumour samples from 60 patients with pT1 CRC with (20 cases) and without (40 cases) metastatic evolution (5 years of follow-up) which were analysed for KRAS, NRAS, BRAF mutations (Idylla testing and next generation sequencing, NGS) and MSS/MSI status (Idylla testing and expression of mismatch repair (MMR) proteins using immunohistochemistry). RESULTS: KRAS mutations were encountered in 11/20 (55%) cases and 21/40 (52.5%) controls (OR=1.11 (0.38 to 3.25), p=0.8548), NRAS mutations in 1/20 (5%) cases and 3/40 (7.5%) controls (OR=3.08 (0.62 to 15.39), p=0.1698) and BRAF mutations in 3/20 (15%) cases and 6/40 (15%) controls (OR=1.00 (0.22 to 4.5), p=1.00). A MSI status was diagnosed in 3/20 (15%) cases and 5/40 (12.5%) controls (OR=1.2353 (0.26 to 5.79), p=0.7885). Beyond the absence of significant association between the metastatic evolution and any of the studied molecular parameters, we observed a very good agreement between methods analysing KRAS, NRAS and BRAF mutations (Kappa value of 0.849 (0.748 to 0.95) between Idylla and NGS) and MSS/MSI (Idylla)-proficient MMR/deficient MMR (immunohistochemistry) status (Kappa value of 1.00). CONCLUSION: Although being feasible using the fully automated Idylla method as well as NGS, the molecular testing of KRAS, NRAS, BRAF and MSS/MSI status does not seem useful for prognostic purpose in the field of pT1 CRC.

Microsatellite instability diagnosis using the fully automated Idylla platform: feasibility study of an in-house rapid molecular testing ancillary to immunohistochemistry in pathology laboratories.

AIM: To study the performances of the Idylla MSI Assay in the diagnosis of microsatellite instability (MSI) or microsatellite stability (MSS). METHODS: We selected 12 tumour samples previously tested for MSI focusing on cases with discrepant results between MLH1, PMS2, MSH2 and MSH6 immunohistochemistry and microsatellite molecular analyses (five cases) or doubtful immunohistochemistry (two cases). Idylla MSI Assay was compared with retrospective immunohistochemistry and molecular results. RESULTS: Idylla MSI Assay showed an almost perfect concordance with microsatellite analysis results previously obtained (only one case with not fully conclusive analysis due to sample exhaustion). The full molecular analysis took less than 150 min per sample and revealed no mutation in any of the seven microsatellite sequences in five MSS samples and four to six mutated ones in seven MSI-High samples. CONCLUSION: At the era when the determination of MSI/MSS status is becoming important for rapid treatment choices, the Idylla MSI Assay consists of a valuable easy-to-perform diagnostic tool that allows, complementary to MLH1, PMS2, MSH2 and MSH6 immunohistochemistry, the diagnosis of MSI/MSS status in a single day.

Histopathological factors help to predict lymph node metastases more efficiently than extra-nodal recurrences in submucosa invading pT1 colorectal cancer.

The therapeutic management of patients with endoscopic resection of colorectal cancer invading the submucosa (i.e. pT1 CRC) depends on the balance between the risk of cancer relapse and the risk of surgery-related morbidity and mortality. The aim of our study was to report on the histopathological risk factors predicting lymph node metastases and recurrences in an exhaustive case series comprising every pT1 CRC (of adenocarcinoma subtype only) diagnosed in Finistère (France) during 5-years. For 312 patients with at least 46 months follow-up included in the digestive cancers registry database, histopathological factors required for risk stratification in pT1 CRC were reviewed. Patients were treated by endoscopic resection only (51 cases), surgery only (138 cases), endoscopic resection followed by surgery (102 cases) or transanal resection (21 cases). Lymph node metastases were diagnosed in 19 patients whereas 15 patients had an extra-nodal recurrence (7 local recurrences only, 4 distant metastases only and 4 combining local and distant recurrences). Four patients with distant metastases died of their cancer. Poor tumor differentiation, vascular invasion and high grade tumor budding on HES slides were notably identified as strong risk-factors of lymph node metastases but the prediction of extra-nodal recurrences (local, distant and sometimes fatal) was less obvious, albeit it was more frequent in patients treated by transanal resection than with other treatment strategies. Beyond good performances in predicting lymph node metastases and guiding therapeutic decision in patients with pT1 CRC, our study points that extra-nodal recurrence of cancer is more difficult to predict and requires further investigations.

High reproducibility is attainable in assessing histoprognostic parameters of pT1 colorectal cancer using routine histopathology slides and immunohistochemistry analyses.

Assessment of the risk of lymph node invasion and tumour recurrence is critical to determine whether additional surgery is required in patients with endoscopically-removed pT1 colorectal cancer (CRC). A reproducible assessment of this risk of recurrence based on histopathological parameters is crucial for relevant therapeutic decisions. The inter-observer reproducibility of these parameters was the subject of our study. Two pathologists independently analysed 163 endoscopically-removed pT1 CRC recorded in a local digestive cancer registry database (Finistère, France). Using haematoxylin-eosin-saffron (HES) and immunohistochemistry slides, they evaluated several parameters related to the risk of tumour recurrence according to the international pT1 CRC-dedicated guidelines. Based on Kappa and intra-class correlation coefficients, good to very good inter-observer agreement was obtained by analysing vertical and lateral margins, submucosal invasion, tumour differentiation and lymphovascular invasion. The reproducibility of tumour budding quantification was only fair on the basis of HES slides but reached a very good agreement using cytokeratin immunohistochemistry. Dual colour cytokeratin and podoplanin immunohistochemistry also improved inter-observer agreement for the detection of lymphovascular invasion. All patients with loco-regional nodal metastases (7 of 101 who underwent complementary surgery) or distant metastases (3 patients) were diagnosed as having a high risk of recurrence and requiring an additional surgery by the two observers. Our study showed that good to very good inter-observer agreement is achievable in evaluating the pathological parameters of recurrence risk in endoscopically-removed pT1 CRC. In addition to HES slides, the detection of lymphovascular invasion and tumour budding can benefit with more reproducible immunohistochemical analyses.

Sequential analysis of 18 genes in polycythemia vera and essential thrombocythemia reveals an association between mutational status and clinical outcome.

Philadelphia-negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular-based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing-based 18-gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3-year follow-up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression. © 2017 Wiley Periodicals, Inc.

NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

BACKGROUND: The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas. METHODS: 142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry. RESULTS: 23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity. CONCLUSIONS: In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.

Severe hydrocephalus caused by diffuse leptomeningeal and neurocutaneous melanocytosis of antenatal onset: a clinical, pathologic, and molecular study of 2 cases.

Diffuse leptomeningeal melanocytosis (DLM) is a rare nevomelanocytic proliferation arising in the meninges. Despite their lack of morphological features of malignancy, these clonal nevomelanocytic cells are capable of extensive invasion and of malignant behavior. When associated with congenital melanocytic nevi, the disorder is named neurocutaneous melanocytosis (NCM). When symptomatic, DLM is usually revealed during childhood, but some cases remain clinically silent. The aim of this study was to analyze melanocytic proliferation in 2 rare and severe cases of isolated DLM and NCM of prenatal onset by neuropathologic and molecular analysis. We performed neuropathologic examination, comparative genomic hybridization arrays, fluorescence in situ hybridization, BRAF and NRAS pyrosequencing in the 2 cases, and next-generation sequencing in the case of isolated DLM. The neuropathologic examination showed diffuse meningeal melanocytic proliferation involving the whole central nervous system with multiple areas of intraneural invasion, associated with large nevi in 1 case. We did not find any chromosomal imbalances. A NRAS(Q61K) mutation was found in the cutaneous and meningeal lesions from the NCM. No mutation was found within a panel of oncogenes including BRAF, NRAS, HRAS, KIT, GNAQ, and GNA11 concerning the isolated DLM. We report 2 exceptional cases of hydrocephalus of prenatal onset related to DLM and NCM. The molecular mechanisms underlying our case of DLM remain unsolved despite the panel of analysis applied.

Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease.