Multicenter evaluation of an automated, multiplex, RNA-based molecular assay for detection of ALK, ROS1, RET fusions and MET exon 14 skipping in NSCLC.

The current study assessed the performance of the fully automated RT-PCR-based Idylla™ GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK, ROS1, RET, and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The Idylla™ GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK, 33 ROS1, 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the Idylla™ GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK, 96.7%/99.0% for ROS1, 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The Idylla™ GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK, ROS1, RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

De novo thrombotic microangiopathy after kidney transplantation in adults: Interplay between complement genetics and multiple endothelial injury.

De novo thrombotic microangiopathy (dnTMA), after renal transplantation may significantly alter graft outcomes. However, its pathogenesis and the role of complement alternative pathway dysregulation remain elusive. We studied all consecutive adult patients with a kidney allograft biopsy performed between January 2004 and March 2016 displaying dnTMA. Ninety-two patients were included. The median time of occurrence was 166 (IQR 25-811) days. The majority (82.6 %) had TMA localized only in the graft. Calcineurin inhibitor toxicity and antibody-mediated rejection (ABMR) were the 2 most frequent causes (54.3% and 37.0%, respectively). However, etiological factors were multiple in 37% patients. Interestingly, pathogenic variants in the genes of complement alternative pathway were significantly more frequent in the 42 tested patients than in healthy controls (16.7% vs 3.7% respectively, P < .008). The overall graft survival after biopsy was 66.0% at 5 years and 23.4% at 10 years, significantly worse than a matched cohort without TMA. Moreover, graft survival of patients with TMA and ABMR was worse than a matched cohort with ABMR without TMA. The 2 main prognostic factors were a positive C4d staining and a lower estimated glomerular filtration rate at diagnosis. DnTMA is a severe and multifactorial disease, induced by 1 or several endothelium-insulting conditions, mostly calcineurin inhibitor toxicity and ABMR.

c-Met immunohistochemistry as reflex test at diagnosis for non-small cell lung cancer: a real-world experience from a monocentric case series.

AIMS: Recent clinical trials have shown promising results with drugs targeting the hepatocyte growth factor receptor (c-Met) for advanced non-small cell lung cancers overexpressing c-Met. We assessed reflex testing of c-Met immunohistochemistry (IHC) at diagnosis for NSCLC in the real-world. METHODS: We retrospectively collected clinical, pathological and molecular data of cases diagnosed with NSCLC in our institution from January 2021 to June 2023. We performed c-Met IHC (SP44 clone) and scored the expression using a H-score and a three-tier classification. RESULTS: 391 cases with interpretable c-Met IHC staining were included. The median age at diagnosis was 70 years (range 25-89 years) including 234 males (male/female ratio 1:5). 58% of the samples came from surgical resections, 35% from biopsies and 8% from cytological procedures. 52% of cases were classified as c-Met-positive (H-score≥150) and 19% were classified as c-Methigh (≥50%, 3+). 43% of the c-Metneg presented with lymph node and/or visceral metastases at diagnosis vs 55% for c-Methigh (p=0.042). 23% of the adenocarcinomas showed c-Methigh expression vs 3% for squamous cell carcinomas (p=0.004). 27% of the c-Metneg cases had a high PD-L1 expression vs 58% of c-Methigh cases (p<0.001). MET ex14 skipping was present in 8% of the c-Methigh cases. CONCLUSIONS: Systematic c-Met testing in daily routine for NSCLC patients is feasible, highlighting a potential correlation with clinicopathological and molecular features.

Prognostic Role of KRAS G12C Mutation in Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis.

KRAS G12C mutation (mKRAS G12C) is the most frequent KRAS point mutation in non-small cell lung cancer (NSCLC) and has been proven to be a predictive biomarker for direct KRAS G12C inhibitors in advanced solid cancers. We sought to determine the prognostic significance of mKRAS G12C in patients with NSCLC using the meta-analytic approach. A protocol is registered at the International Prospective Register for systematic reviews (CRD42022345868). PubMed, EMBASE, The Cochrane Library, and Clinicaltrials.gov.in were searched for prospective or retrospective studies reporting survival data for tumors with mKRAS G12C compared with either other KRAS mutations or wild-type KRAS (KRAS-WT). The hazard ratios (HRs) for overall survival (OS) or Disease-free survival (DFS) of tumors were pooled according to fixed or random-effects models. Sixteen studies enrolling 10,153 participants were included in the final analysis. mKRAS G12C tumors had poor OS [HR, 1.42; 95% CI, 1.10-1.84, p = 0.007] but similar DFS [HR 2.36, 95% CI 0.64-8.16] compared to KRAS-WT tumors. Compared to other KRAS mutations, mKRAS G12C tumors had poor DFS [HR, 1.49; 95% CI, 1.07-2.09, p < 0.0001] but similar OS [HR, 1.03; 95% CI, 0.84-1.26]. Compared to other KRAS mutations, high PD-L1 expression (>50%) [OR 1.37 95% CI 1.11-1.70, p = 0.004] was associated with mKRAS G12C tumors. mKRAS G12C is a promising prognostic factor for patients with NSCLC, negatively impacting survival. Prevailing significant heterogeneity and selection bias might reduce the validity of these findings. Concomitant high PD-L1 expression in these tumors opens doors for exciting therapeutic potential.

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer.

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

The Need to Set up a Biobank Dedicated to Lymphoid Malignancies: Experience of a Single Center (Laboratory of Clinical and Experimental Pathology, University Côte d'Azur, Nice, France).

Several therapies to improve the management of lymphoma are currently being investigated, necessitating the development of new biomarkers. However, this requires high-quality and clinically annotated biological material. Therefore, we established a lymphoma biobank including all available biological material (tissue specimens and matched biological resources) along with associated clinical data for lymphoma patients diagnosed, according to the WHO classification, between 2005 and 2022 in the Laboratory of Clinical and Experimental Pathology, Nice, France. We retrospectively included selected cases in a new collection at the Côte d'Azur Biobank, which contains 2150 samples from 363 cases (351 patients). The male/female ratio was 1.3, and the median age at diagnosis was 58 years. The most common lymphoma types were classical Hodgkin lymphoma, diffuse large B-cell lymphoma, and extra-nodal marginal zone lymphoma of MALT tissue. The main sites of lymphoma were the mediastinum, lymph node, Waldeyer's ring, and lung. The Côte d'Azur Biobank is ISO 9001 and ISO 20387 certified and aims to provide high quality and diverse biological material to support translational research projects into lymphoma. The clinico-pathological data generated by this collection should aid the development of new biomarkers to enhance the survival of patients with lymphoid malignancies.

Detection of Nine Oncogenes Amplification in Lung and Colorectal Cancer Formalin-Fixed Paraffin-Embedded Tissue Samples using Combined Next-Generation Sequencing-Based Script and Digital Droplet Polymerase Chain Reaction.

INTRODUCTION: Gene copy number variations have theranostic impact and require reliable methods for their identification. We aimed to evaluate the reliability of combined next-generation sequencing (NGS) and digital droplet PCR (ddPCR) method for gene amplification evaluation. METHODS: We conducted a retrospective multicentric observational study. MET/ERBB2 amplifications were assessed in patients with lung or colorectal carcinoma (cohort A), from 2016 to 2020, by fluorescence in situ hybridization (FISH)/immunohistochemistry (IHC), NGS and ddPCR. NGS-based script and ddPCR were then used to detect amplifications of 7 additional oncogenes (EGFR, KRAS, BRAF, FGFR1, FGFR2, FGFR3, PIK3CA) in a cohort of patients (cohort B). RESULTS: 55 patients (9 control, 25 ERBB2-amplified and 21 MET-amplified) out of 3779 patients tested were included in cohort A. Correlation coefficient between NGS-based script and FISH/IHC results were .88 for MET (P < .001) and .89 (P < .001) for ERBB2. Using a threshold ratio of 1.56 with the NGS-based script, the sensitivity was 100% for both genes and the specificity 69% for MET and 90% for ERBB2, respectively. With an alternative 1.76 threshold, sensitivity was 94% for MET and 96% for ERBB2, while specificity was 85% for MET and 90% for ERBB2. Correlation coefficient between FISH and ddPCR ratio was .90 for MET and .88 for ERBB2. In both cohorts, NGS-based script and ddPCR results were significantly correlated regarding all genes (P < .001). CONCLUSION: Combined NGS-based script and ddPCR method is reliable and easily feasible for the detection of gene amplifications, providing useful data for guided therapy in cancer.

Lack of correlation between MET and PD-L1 expression in non-small cell lung cancer revealed by comparative study of matched biopsies and surgical resection samples.

INTRODUCTION: Both MET expression and the PD-L1 tumor proportion score (TPS) are companion diagnostics for treatment of advanced non-small cell lung carcinoma (aNSCLC) patients. We evaluated the rate of correlation between MET expression and the PD-L1 TPS in matched biopsies and surgically resected specimens from NSCLC patients. PATIENTS AND METHODS: This retrospective analysis assessed the prevalence and correlation between MET expression (SP44 clone) and the PD-L1 TPS (22C3 clone) by immunohistochemistry together with molecular alterations determined by targeted next-generation sequencing in matched lung biopsy and surgically lung resected specimens from 70 patients with NSCLC. RESULTS: The study found a significant correlation between the MET H-score in surgical samples and matched biopsies (P-value < 0.0001), as well as between the PD-L1 TPS in paired biopsies and surgical samples (P-value < 0.0001). However, there was no significant correlation between the MET H-score or expression subgroups and the PD-L1 TPS in both types of paired samples (P-value = 0.47, and P-value = 0.90). The MET H-score was significantly higher in adenocarcinoma compared to squamous cell carcinoma (P-value < 0.0001). A mutational analysis showed that the MET H-score was significantly higher in NSCLC cases with targetable molecular alterations (P-value = 0.0095), while no significant correlation was found for the PD-L1 TPS. CONCLUSIONS: Our study found no significant correlation between PD-L1 and MET expression in samples from NSCLC patients, highlighting the importance of personalized treatment strategies based on individual expression profiles. These findings provide valuable insight into the development of effective immunotherapy and targeted therapy for NSCLC patients.

The significance of co-mutations in EGFR-mutated non-small cell lung cancer: Optimizing the efficacy of targeted therapies?

Non-small cell lung cancer (NSCLC) is the most common cause of cancer death worldwide. In non-squamous NSCLC, the identification of oncogenic drivers and the development of target-specific molecules led to remarkable progress in therapeutic strategies and overall survival over the last decade. Nevertheless, responses are limited by systematically acquired mechanisms of resistance early on after starting a targeted therapy. Moreover, mounting evidence has demonstrated that each oncogenic-driven cluster is actually heterogeneous in terms of molecular features, clinical behaviour, and sensitivity to targeted therapy. In this review, we aimed to examine the prognostic and predictive significance of oncogene-driven co-mutations, focusing mainly on EGFR and TP53. A narrative review was performed by searching MEDLINE databases for English articles published over the last decade (from January 2012 until November 2022). The bibliographies of key references were manually reviewed to select those eligible for the topic. The genetic landscape of EGFR-mutated NSCLC is more complicated than what is known so far. In particular, the occurrence of TP53 co-mutations stratify patients carrying EGFR mutations in terms of treatment response. The study provides a deeper understanding of the mechanisms underlying the variability of the genetic landscape of EGFR-mutated NSCLC and summarizes notably the clinical importance of TP53 co-mutations for an open avenue to more properly addressing the clinical decision-making in the near future.

A Real-World Experience from a Single Center (LPCE, Nice, France) Highlights the Urgent Need to Abandon Immunohistochemistry for ROS1 Rearrangement Screening of Advanced Non-Squamous Non-Small Cell Lung Cancer.

The detection of ROS1 rearrangements in metastatic non-squamous non-small cell lung carcinoma (NS-NSCLC) permits administration of efficient targeted therapy. Detection is based on a testing algorithm associated with ROS1 immunohistochemistry (IHC) screening followed by ROS1 FISH and/or next generation sequencing (NGS) to confirm positivity. However, (i) ROS1 rearrangements are rare (1-2% of NS-NSCLC), (ii) the specificity of ROS1 IHC is not optimal, and (iii) ROS1 FISH is not widely available, making this algorithm challenging to interpret time-consuming. We evaluated RNA NGS, which was used as reflex testing for ROS1 rearrangements in NS-NSCLC with the aim of replacing ROS1 IHC as a screening method. ROS1 IHC and RNA NGS were prospectively performed in 810 NS-NSCLC. Positive results were analyzed by ROS1 FISH. ROS1 IHC was positive in 36/810 (4.4%) cases that showed variable staining intensity while NGS detected ROS1 rearrangements in 16/810 (1.9%) cases. ROS1 FISH was positive in 15/810 (1.8%) of ROS1 IHC positive cases and in all positive ROS1 NGS cases. Obtaining both ROS1 IHC and ROS1 FISH reports took an average of 6 days, while obtaining ROS1 IHC and RNA NGS reports took an average of 3 days. These results showed that systematic screening for the ROS1 status using IHC must be replaced by NGS reflex testing.

Multicenter automatic detection of invasive carcinoma on breast whole slide images.

Breast cancer is one of the most prevalent cancers worldwide and pathologists are closely involved in establishing a diagnosis. Tools to assist in making a diagnosis are required to manage the increasing workload. In this context, artificial intelligence (AI) and deep-learning based tools may be used in daily pathology practice. However, it is challenging to develop fast and reliable algorithms that can be trusted by practitioners, whatever the medical center. We describe a patch-based algorithm that incorporates a convolutional neural network to detect and locate invasive carcinoma on breast whole-slide images. The network was trained on a dataset extracted from a reference acquisition center. We then performed a calibration step based on transfer learning to maintain the performance when translating on a new target acquisition center by using a limited amount of additional training data. Performance was evaluated using classical binary measures (accuracy, recall, precision) for both centers (referred to as "test reference dataset" and "test target dataset") and at two levels: patch and slide level. At patch level, accuracy, recall, and precision of the model on the reference and target test sets were 92.1% and 96.3%, 95% and 87.8%, and 73.9% and 70.6%, respectively. At slide level, accuracy, recall, and precision were 97.6% and 92.0%, 90.9% and 100%, and 100% and 70.8% for test sets 1 and 2, respectively. The high performance of the algorithm at both centers shows that the calibration process is efficient. This is performed using limited training data from the new target acquisition center and requires that the model is trained beforehand on a large database from a reference center. This methodology allows the implementation of AI diagnostic tools to help in routine pathology practice.

Accurate Detection of SARS-CoV-2 by Next-Generation Sequencing in Low Viral Load Specimens.

As new SARS-CoV-2 variants emerge, there is an urgent need to increase the efficiency and availability of viral genome sequencing, notably to detect the lineage in samples with a low viral load. SARS-CoV-2 genome next-generation sequencing (NGS) was performed retrospectively in a single center on 175 positive samples from individuals. An automated workflow used the Ion AmpliSeq SARS-CoV-2 Insight Research Assay on the Genexus Sequencer. All samples were collected in the metropolitan area of the city of Nice (France) over a period of 32 weeks (from 19 July 2021 to 11 February 2022). In total, 76% of cases were identified with a low viral load (Ct ≥ 32, and ≤200 copies/µL). The NGS analysis was successful in 91% of cases, among which 57% of cases harbored the Delta variant, and 34% the Omicron BA.1.1 variant. Only 9% of cases had unreadable sequences. There was no significant difference in the viral load in patients infected with the Omicron variant compared to the Delta variant (Ct values, p = 0.0507; copy number, p = 0.252). We show that the NGS analysis of the SARS-CoV-2 genome provides reliable detection of the Delta and Omicron SARS-CoV-2 variants in low viral load samples.

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.

Deciphering the Impact of HER2 Alterations on Non-Small-Cell Lung Cancer: From Biological Mechanisms to Therapeutic Approaches.

Despite the recent increase in the number of types of treatments, non-small-cell lung cancer (NSCLC) remains the major cause of death from cancer worldwide. So, there is an urgent need to develop new therapeutic strategies. The HER2 gene codes for tyrosine kinase receptor whose alterations are known to drive carcinogenesis. HER2 alterations, including amplification, mutations, and overexpression, have been mainly described in breast and gastric cancers, but up to 4% of NSCLC harbor actionable HER2 mutations. HER2-targeted therapy for NSCLC with trastuzumab, pertuzumab, and trastuzumab emtansine has failed to demonstrate an improvement in survival. Nevertheless, recent data from phase II trials have shed light on promising specific therapies for HER2-mutant NSCLC such as trastuzumab deruxtecan. Herein, we aimed to provide an updated review on the biology, epidemiology, molecular testing, and therapeutic strategies for NSCLC with HER2 molecular alterations.

Molecular Profiling in Non-Squamous Non-Small Cell Lung Carcinoma: Towards a Switch to Next-Generation Sequencing Reflex Testing.

Molecular diagnosis of lung cancer is a constantly evolving field thanks to major advances in precision oncology. The wide range of actionable molecular alterations in non-squamous non-small cell lung carcinoma (NS-NSCLC) and the multiplicity of mechanisms of resistance to treatment resulted in the need for repeated testing to establish an accurate molecular diagnosis, as well as to track disease evolution over time. While assessing the increasing complexity of the molecular composition of tumors at baseline, as well as over time, has become increasingly challenging, the emergence and implementation of next-generation sequencing (NGS) testing has extensively facilitated molecular profiling in NS-NSCLC. In this review, we discuss recent developments in the molecular profiling of NS-NSCLC and how NGS addresses current needs, as well as how it can be implemented to address future challenges in the management of NS-NSCLC.

[Myeloid neoplasms associated with rearrangement of PDGFRB: A rare and tricky disease]. / La néoplasie myéloïde associée à un réarrangement de PDGFRB : une pathologie rare de diagnostic difficile.

In the latest World Health Organization classification (WHO), eosinophilic disorders represent a group of rare pathologic conditions with highly heterogeneous pathophysiology. In this report, we describe a case of myeloid neoplasm associated with eosinophilia and rearrangement of PDGFRB gene in a 67-year-old-male patient hospitalized with cerebellous ataxia. Initial investigations showed a bicytopenia with hypereosinophilia varying from 1.1 to 1.6×109/L. Bone marrow aspiration was rich and showed a heterogeneous distribution of myeloid cells with clusters of promyelocytes and proerythroblasts associated with numerous eosinophils and spindle-shaped mast cells but without excess of blasts, dysplasia nor maturation skewing. These aspects suggested an atypical myeloproliferative neoplasm. Bone marrow biopsy was performed showing also a very high cellularity with area of myeloid and erythroid precursors associated with numerous spindle-shaped mast cells. Diagnoses of unclassified myeloid neoplasm and/or systemic mastocytosis were then proposed. Further chromosome analysis showed a t(5;8) translocation with PDGFRB rearrangement revealed in fluorescent in situ hybridization. Patient was treated with imatinib and intravenous immunoglobulin therapy allowing a significant improvement in neurological symptoms and biological results. Patient condition is currently stable after six lines of treatment. This rare hematopoietic neoplasm displays unusual histological and cytological features and can mimic other myeloproliferative neoplasm. Specific cytogenetics analysis should be considered for such cases with hypereosinophilia to select patients that may benefit from targeted therapy.

The Third Joint Meeting on Lung Cancer of the FHU OncoAge (University Côte d'Azur, Nice, France) and the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Understanding New Therapeutic Options and Promising Predictive Biomarkers for Lung Cancer Patients.

We are proud and happy to present this Special Issue, a follow-up to the third joint meeting on lung cancer of the FHU OncoAge (University Côte d'Azur, Nice, France) and the University of Texas MD Anderson Cancer Center (Houston, TX, USA), which was held virtually on 4 October 2021 [...].