Ultrafast Gene Fusion Assessment for Nonsquamous NSCLC.

Introduction: Gene fusion testing of ALK, ROS1, RET, NTRK, and MET exon 14 skipping mutations is guideline recommended in nonsquamous NSCLC (NS-NSCLC). Nevertheless, assessment is often hindered by the limited availability of tissue and prolonged next-generation sequencing (NGS) testing, which can protract the initiation of a targeted therapy. Therefore, the development of faster gene fusion assessment is critical for optimal clinical decision-making. Here, we compared two ultrafast gene fusion assays (UFGFAs) using NGS (Genexus, Oncomine Precision Assay, Thermo Fisher Scientific) and a multiplex reverse-transcriptase polymerase chain reaction (Idylla, GeneFusion Assay, Biocartis) approach at diagnosis in a retrospective series of 195 NS-NSCLC cases and five extrapulmonary tumors with a known NTRK fusion. Methods: A total of 195 NS-NSCLC cases (113 known gene fusions and 82 wild-type tumors) were included retrospectively. To validate the detection of a NTRK fusion, we added five NTRK-positive extrathoracic tumors. The diagnostic performance of the two UFGFAs and standard procedures was compared. Results: The accuracy was 92.3% and 93.1% for Idylla and Genexus, respectively. Both systems improved the sensitivity for detection by including a 5'-3' imbalance analysis. Although detection of ROS1, MET exon 14 skipping, and RET was excellent with both systems, ALK fusion detection was reduced with sensitivities of 87% and 88%, respectively. Idylla had a limited sensitivity of 67% for NTRK fusions, in which only an imbalance assessment was used. Conclusions: UFGFA using NGS and reverse-transcriptase polymerase chain reaction approaches had an equal level of detection of gene fusion but with some technique-specific limitations. Nevertheless, UFGFA detection in routine clinical care is feasible with both systems allowing faster initiation of therapy and a broad degree of screening.

Telomeric Repeat-Containing RNA (TERRA): A Review of the Literature and First Assessment in Cutaneous T-Cell Lymphomas.

Telomeric Repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from telomeric DNA sequences from multiple chromosome ends. Major research efforts have been made to understand TERRA roles and functions in several physiological and pathological processes. We summarize herein available data regarding TERRA's roles in human cells and we report the first investigation in cutaneous T-cells lymphomas (CTCL) using real-time PCR. Among the TERRA analysed, our data suggest a particular role for TERRA 16p downregulation and TERRA 11q upregulation in CTCL lymphomagenesis.

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.

Assessment of BRAFV600E mutation in pulmonary Langerhans cell histiocytosis in tissue biopsies and bronchoalveolar lavages by droplet digital polymerase chain reaction.

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF V600E and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAFV600E detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS). The presence of BRAFV600E mutation was assessed by at least two concordant techniques to further evaluate specificity and sensitivity of each method. The BRAF V600E mutation prevalence was detected in 18 out of 41 cases by ddPCR, 10 out of 36 cases by HRM PCR, and 16 out of 31 cases by NGS. BRAFV600E immunohistochemistry sensitivity was 94%, and specificity was 79%. HRM PCR sensitivity was only 59%, and specificity was 100%. NGS sensitivity and specificity were 100% for interpretable cases (n = 31), but in 11 cases, this technique was non-contributive. The analysis of BAL samples by ddPCR revealed a BRAFV600E mutation both in tissue and in BAL samples in one patient, a wild-type status both in tissue and in BAL samples in two patients, and a wild-type BRAF status in BAL and a BRAFV600E mutation in tissue samples in four patients. The study supports the usefulness of ddPCR for BRAF status assessment in either tissue or BAL samples to increase the accuracy of PLCH diagnosis.

TP53 alterations in primary and secondary Sézary syndrome: A diagnostic tool for the assessment of malignancy in patients with erythroderma.

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.

Performance and cost efficiency of KRAS mutation testing for metastatic colorectal cancer in routine diagnosis: the MOKAECM study, a nationwide experience.

PURPOSE: Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs. METHODS: 96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists. RESULTS: This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0. CONCLUSION: The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.

[Impact of pathological review by an expert on the diagnosis and management of patients with cancer in Aquitania]. / Impacts des avis diagnostiques de cancérologie en Aquitaine. Etude quantitative, qualitative et médicoéconomique rétrospective sur une année.

AIMS: The goal of this work was to evaluate the impact of expert pathological second opinion on the diagnosis and management of patients with cancer, in a French region (Aquitaine) and with an economic point of view. MATERIAL AND METHODS: The study was first quantitative, performed retrospectively on all cases of cancer, voluntary sent for a second opinion to an expert pathologist of two centers. Secondly, we restricted the study to lymphoid, melanocytic and soft tissue tumors sent for second opinion. We considered that the expert review had an important diagnostic impact either when the initial pathologist sent the specimen to identify or classify malignant tumor or hesitated between benign and malignant tumor or had no hypothesis, or if there were discordant diagnoses (malignant/benign) between the two pathologists. We considered that the expert review had a high therapeutic impact if the disagreement between initial and expert diagnoses induced a complete modification in therapy. We evaluated the cost of second opinion for the expert centers and the cost of care management. RESULTS: Over the year 2006, the expert centers received 5077 lesions for consultation: 3769 specimens were sent by a pathologist for a second review, 1324 by pathologists of Aquitania and of these, 751 samples were submitted for lymphoid (55%), soft tissues (30%) or melanocytic tumors (15%). There was an important diagnostic impact for 75% of the samples; the impact of the expert review on patient management was considered high for 46% of specimens and the expert pathological diagnosis modified the clinical prognosis for 40% of the specimens. We estimated that for 53 discordant diagnoses (malignant/benign), second opinion allowed an economy of 500,000 euro. CONCLUSION: Expert second opinion is very important not only for diagnosis and management for patient with cancer but also for economic reasons.

Automatized assessment of 1p36-19q13 status in gliomas by interphase FISH assay on touch imprints of frozen tumours.

Molecular genetic analyses have demonstrated that combined losses in 1p36 and 19q13 were associated with a good response to treatment and a higher survival rates in oligodendrogliomas (O). The presence of such deletions in a subset of mixed oligoastrocytomas (OA) also suggests that 1p-19q status may assist the histological classification of gliomas. Representative frozen fragments of 25 patients with a primary grade II or III glioma [2 O, 8 astrocytic tumours (A), 15 OA] were selected and a sensitive interphase fluorescent in situ hybridization (FISH) analysis was developed on serial touch preparations using two sets of probes. A positive detection threshold at 6% was reached by the use of both touch imprints and a new set of LSI 1p36/19q13 Abbott-Vysis probes. Strong and discrete hybridization signals of these probes facilitated the following FISH analysis; indeed, an automatic analysis of the hybridization patterns (Metafer 4, Metasystems, Althlussheim, Germany) was compared with visual counting. Both methods were highly correlated and combined 1p-19q losses found in five tumours with an oligodendroglial component (2 O, 3 OA). The automatic system allowed the capture and storage of hybridization patterns and the processing of several slides. A convenient checking of the nuclei gallery was done with direct recall and visual verification on the slides of nuclei with ambiguous hybridization patterns. The recently developed probes together with automatic counting may facilitate multicentric evaluation and standardization of 1p-19q assessment in gliomas.