NTRK Gene Fusion Detection in a Pan-Cancer Setting Using the Idylla GeneFusion Assay.

Recently, approval of tyrosine receptor kinase (TRK) inhibitors by Food and Drug Administration and European Medicines Agency in NTRK fusion-positive cancer types has led to a variety of proposed testing algorithms. In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small-cell lung cancer, microsatellite instability-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK immunohistochemical staining. Compared with the RNA-based Oncomine Focus Assay, the Idylla GeneFusion Assay demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 92.7%, 81.8%, and 93.8%, respectively; and the pan-TRK immunohistochemistry demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 82.1%, 45.5%, and 85.7%, respectively. These findings highlighted the importance of tailoring NTRK testing algorithms per cancer type. In a small subset, data from the RNA-based Archer FusionPlex Assay were also available. NTRK fusion detection efficiency was compared between the four NTRK testing modalities, with a high concordance between the PCR-based methods. Last, RNA degradation was observed when using the Idylla GeneFusion Assay on snap frozen tissue samples as these are nonfixated. This might be countered by increasing the amount of sample input. To conclude, the Idylla GeneFusion Assay has shown a clear potential in identifying NTRK fusions.

Excellent Response to MEK Inhibition in an AGK-BRAF Gene Fusion Driven Carcinoma: Case Report and Literature Review.

BACKGROUND: Soft tissue myoepithelial carcinomas (STMC) are a rare, malignant subgroup of myoepithelial tumors that arise typically in glandular or ductal tissues, but also in the bone and soft and cutaneous tissues. Due to its rarity, there is no consensus regarding the treatment of STMC, including chemotherapy or other systemic agents for metastatic STMC. CASE REPORT: A chemotherapy- and regorafenib-refractory STMC, harboring an AGK-BRAF fusion, was successfully treated using MEK-inhibition with cobimetinib in monotherapy. MEK-inhibition with cobimetinib effectively silenced paradoxical MAP kinase/ERK-signaling pathway activation after regorafenib monotherapy, and resulted in a significant and durable clinical response. CONCLUSION: This effect of MEK-inhibition in STMC harboring an AGK-BRAF fusion has not been previously reported and contributes to the existing, yet limited, knowledge on the treatment of BRAF fusion-driven tumors. Also, our case highlights the importance of next generation sequencing in driving further rational therapeutic choices to provide disease control and palliation.

The ß2-adrenergic receptor in the apical membrane of intestinal enterocytes senses sugars to stimulate glucose uptake from the gut.

The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the ß2-adrenergic receptor (ß 2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the ß 2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a ß 2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a ß 2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the ß 2-AR. Oral administration of glucose with a non-bioavailable ß 2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical ß 2-ARs in stimulating gut glucose uptake, and suggesting enterocyte ß 2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether ß 2-ARs mediate glucose sensing also in other tissues.

Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial.

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.

Clinical Performance of the Idylla MSI Test for a Rapid Assessment of the DNA Microsatellite Status in Human Colorectal Cancer.

In this study, the clinical performance of the Idylla MSI test (investigational use only) was evaluated in 330 colorectal carcinoma samples (all stages). This test is fully automated, from formalin-fixed, paraffin-embedded slide to result, and gives a result in <2.5 hours. Compared with the Promega MSI Analysis System version 1.2, an overall agreement, sensitivity, and specificity of 99.7%, 98.7%, and 100%, respectively, was reached. Whereas seven samples were invalid with the Promega MSI Analysis System, only two were invalid with the Idylla MSI test. Compared with the historical immunohistochemistry (IHC) data, overall agreement, sensitivity, and specificity of 98.7%, 94.4%, and 100%, respectively, were observed. Tumor mutation burden analysis of the discordant IHC cases was in favor of the Idylla MSI test result in three of the four samples. Furthermore, for those cases where the IHC data were invalid or hard to interpret because sole loss of one DNA mismatch repair deficiency marker was observed, Idylla MSI test results were always valid and accurate. Herein, the Idylla MSI test has been shown to be an accurate, fast screening assay for the detection of microsatellite status in colorectal cancer patients, with a low number of invalid results.

Complete tumor response of a locally advanced lung large-cell neuroendocrine carcinoma after palliative thoracic radiotherapy and immunotherapy with nivolumab.

Lung large-cell neuroendocrine carcinoma (L-LCNEC) is a rare subset of lung carcinoma associated with poor overall survival. Due to its rarity, little has been established about its optimal treatment in the advanced stage. We report the case of a 41-year-old woman diagnosed with an unresectable locally advanced L-LCNEC who presented an impressive tumor response to immunotherapy with nivolumab after non-curative thoracic radiotherapy. Salvage surgery was then performed, and pathologic analysis of the resected piece revealed the absence of residual viable tumor cells. Based on this case report, we discuss the literature regarding the efficacy of inhibitors of programmed death-1 protein (PD-1) in L-LCNEC and their use in association with radiotherapy and in the neoadjuvant setting.

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples.

BACKGROUND: Testing for KRAS mutations in metastatic colorectal cancer (mCRC) on formalin-fixed, paraffin embedded (FFPE) tumor tissue has become standard of care. Different molecular methods exist to determine hotspot KRAS mutations in exon 2, 3 and 4, but testing is often limited by the sensitivity and the speed of analysis. The aim of this retrospective study was to establish the clinical performance of the Idylla™ KRAS Mutation Test on FFPE tumor samples of patients with mCRC. METHODS: KRAS mutation analysis was performed using the therascreen KRAS on the RotorGene Q platform (CE-IVD; Qiagen) and results were subsequently compared to the Idylla™ KRAS Mutation Test. Discordant result testing was performed with massive parallel sequencing or alternative routine approaches. RESULTS: Data from 182 samples were used to show that the overall agreement between the two methods for mutation characterization was 96.7% [95%CI: 93.0%-98.5%]. Six out of 182 samples (3.3%) showed true discordant results. CONCLUSION: The Idylla™ KRAS Mutation Test allows for a fast and reliable analysis of FFPE samples with a turnaround-time of two hours without the need of molecular infrastructure or expertise in order to guide the personalized treatment of colorectal cancer patients.

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

Detection of ALK expression in non-small-cell lung cancer with ALK gene rearrangements--comparison of multiple immunohistochemical methods.

AIM: Testing for ALK rearrangements in advanced, non-squamous non-small-cell lung cancers that are wild-type for activating EGFR mutation has become standard care. Fluorescence in-situ hybridization is considered the gold standard for this evaluation. Pre-screening with immunohistochemistry has been suggested, to reduce testing costs and to make testing more widely available. By analysing the sensitivity and specificity of different ALK immunohistochemical assays, we aimed to identify the most reliable assay to detect ALK rearrangement. METHODS AND RESULTS: ALK screening performed by FISH analysis was compared with three different immunohistochemical assays, in which two ALK antibody clones (5A4 and D5F3) were used on two detection platforms (Dako AutostainerLink 48 and Ventana Benchmark GX). Data from 30 ALK FISH-positive cases show that the sensitivity of the immunohistochemical assays varies from 93.3% to 96.6%. Head-to-head comparison of the 5A4 and D5F3 ALK antibody clones demonstrates similar staining potency. In general, homogeneous, intermediate to strong staining of the ALK-positive samples was obtained. CONCLUSIONS: ALK immunohistochemistry can be considered as a pre-screen method if one accepts a sensitivity of 93.3-96.6%. Because ALK immunohistochemical staining needs to be performed close to the detection limit of the assay, vigilant quality control monitoring is required to guarantee trustworthy results.

Diet-induced obesity in gravid rats engenders early hyperadiposity in the offspring.

Exposure to a dysmetabolic in utero environment may be one of the mechanisms to explain why individuals with high birth weight are more likely to remain overweight. We explored this hypothesis in an animal model of diet-induced obesity (DIO). We studied adipose tissue development and glucose tolerance in the offspring of rat dams fed a diet rich in milk and sugar from early adulthood until day (d) 2 postpartum. This diet promoted body weight (BW) gain and was previously shown to produce insulin resistance and gestational glucose intolerance. The DIO offspring showed a higher BW in early life (between d7 and d35), with a maximum of 1 SD above the mean BW of controls; however, BW in DIO offspring after d35 was comparable with that of controls. Neonatal DIO offspring also showed larger fat depots, adipocyte hypertrophy (P

Adipose tissue in offspring of Lepr(db/+) mice: early-life environment vs. genotype.

Gravidas with obesity and diabetes ("diabesity") may transmit this syndrome to their children through genetic and nongenetic mechanisms. Here, we used the Lepr(db/+) diabese mouse to examine the magnitude of these transmission modes, focusing on adipose tissue (AT). We compared the following six groups: wild-type (+/+) offspring from +/+ or db/+ dams (different early life environment) and db/+ offspring from db/+ dams, fed a standard or high-fat diet. Weight gain (0-8 wk) was higher in +/+ offspring from db/+ vs. +/+ mothers, and even higher in db/+ vs. +/+ offspring from db/+ mothers. In addition, we observed a stepwise increase in AT and adipocyte size in +/+ from +/+ mice, +/+ from db/+ mice, and db/+ mice at 8 wk. Differences in weight and adiposity between +/+ offspring from db/+ vs. +/+ dams were more pronounced in males than in females. Leptin and apelin mRNA levels in white and brown AT were higher in +/+ offspring from db/+ vs. +/+ dams; however, leptin, apelin, and tumor necrosis factor-alpha expression were boosted more robustly in db/+ offspring. The high-fat diet amplified AT differences between db/+ vs. +/+ offspring from db/+ dams, but not between +/+ offspring from db/+ vs. +/+ dams. Moreover, db/+ but not +/+ offspring from db/+ mothers were insulin-resistant and hyperinsulinemic after a glucose challenge. In conclusion, the genetic transmission of the diabesity phenotype clearly prevailed, but the early-life diabesity environment had discernible effects on postnatal weight gain as well as on adipocyte size and adipokine expression at a postpubertal age.

Chronic tumor necrosis factor-alpha infusion in gravid C57BL6/J mice accelerates adipose tissue development in female offspring.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is thought to mediate, in part, the link between obesity and insulin resistance, and women with gestational diabetes mellitus (GDM) have raised serum TNF-alpha concentrations. Our objective was to investigate whether systemic TNF-alpha administration into gravid C57BL6/J mice causes a GDM-like syndrome and affects growth and adipose tissue (AT) development in the offspring. METHODS: We assessed glucose tolerance and reproductive outcome in mice infused with saline, or 2 mug or 4 mug recombinant mouse (rm)TNF-alpha by subcutaneous mini-osmotic pumps between days (d)11.5 and 18.5 of gestation. Subsequently, we studied the effects of the 2-mug dose on maternal AT metabolism. Finally, the growth of offspring exposed to 2 mug rmTNF-alpha in utero was followed until 8 weeks postnatal age. At 8 weeks, we assessed AT accumulation, as well as adipocyte area in white AT and insulin sensitivity in males, and adipokine mRNA levels in various AT depots in females. RESULTS: The peak glucose response to an intraperitoneal glucose stimulus in late-gravid mice and fetal weight were higher with 2 mug but not 4 mug rmTNF-alpha compared with saline; however, 2 mug TNF-alpha did not affect AT parameters. The female but not male offspring of these mice showed accelerated growth, hyperadiposity, robustly increased leptin expression in all AT depots, and raised fasting blood glucose. CONCLUSIONS: TNF-alpha infusion (2 mug for 7 days) in gravid mice resulted in a mild GDM syndrome and accelerated AT development in the offspring in a sex-specific manner. The data suggest that TNF-alpha mediates in part the effects of GDM on fetal growth and postnatal adiposity, and constitutes a potential mediator of intrauterine programming.

Adipokine profile and C-reactive protein in pregnancy: effects of glucose challenge response versus body mass index.

OBJECTIVE: To test the hypothesis that gravidas who have an abnormal response to glucose loading have dysfunctional adipose tissue cells that produce more insulin resistance-inducing and proinflammatory adipokines but less insulin-sensitizing adipokines. METHODS: We performed a nested case-control study within a larger sample of gravidas who had a glucose challenge test (GCT) at 24-29 weeks; we compared 73 cases with an abnormal GCT (>8.3 mM) and 146 controls with a strictly normal GCT (<7.2 mM) matched for body mass index (BMI) and height (mean difference between cases and controls: 0.1 kg/m(2) and 1 cm, respectively). We measured plasma insulin, adipokines (leptin, adiponectin, resistin, tumor necrosis factor [TNF]-alpha, interleukin [IL]-6), soluble leptin receptor (sOb-R), the main leptin-binding protein, and C-reactive protein (CRP). RESULTS: The cases showed a 48% increase in insulin concentrations and a 27% increase in TNF-alpha concentrations compared to the controls (both P < .0001), but leptin, sOb-R, IL-6, and adiponectin, as well as CRP, concentrations were comparable between cases and controls. In the whole group (n = 219), BMI was correlated with insulin, leptin, IL-6, and CRP, and inversely with sOb-R and adiponectin concentrations (all P < .0003). CONCLUSIONS: Plasma leptin, sOb-R, IL-6, and adiponectin, as well as CRP, are strongly related to BMI in gravidas at 24-29 weeks gestational age but not to the glucose loading response. However, TNF-alpha is higher in women with an abnormal GCT. Further studies should disclose the source of increased TNF-alpha in these women, and to assess whether TNF-alpha is causally related to glucose intolerance during pregnancy.