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.

Prospective evaluation of NGS-based liquid biopsy in untreated late stage non-squamous lung carcinoma in a single institution.

BACKGROUND: NGS from plasma samples in non-squamous cell lung carcinoma (NSCC) can aid in the detection of actionable genomic alterations. However, the absolute clinical value of NGS in liquid biopsy (LB) made at baseline is currently uncertain. We assessed the impact of plasma-based NGS using an in-house test and an outsourced test in comparison to a routine molecular pathology workflow. METHODS: Twenty-four advanced/metastatic treatment-naïve NSCC patients were prospectively included. NGS analyses were conducted both in-house using the Oncomine cfTNA Panel and in an external testing center using the Foundation Liquid assay. NGS analysis and/or specific molecular based assays were conducted in parallel on tissue or cytological samples. RESULTS: Both LB tests were well correlated. Tissue NGS results were obtained in 67% of patients and demonstrated good correlation with LB assays. Activating EGFR mutations were detected using LB tests in three patients. PD-L1 expression assessed in tissue sections enabled the initiation of pembrolizumab treatment in five patients. CONCLUSION: NGS from LB is feasible in routine clinical practice using an in-house or an outsourced test at baseline. However, the impact on therapy selection was limited in this small series of patients and LB was not able to replace tissue-based testing in our hands.

Immunohistochemistry as a potential tool for routine detection of the NRAS Q61R mutation in patients with metastatic melanoma.

BACKGROUND: It can be useful to assess the NRAS mutation status in patients with metastatic melanoma because NRAS-activating mutations confer resistance to RAF inhibitors, and NRAS-mutated patients appear to be sensitive to mitogen-activated protein kinase (MEK) inhibitors. OBJECTIVE: We aimed to assess the diagnostic accuracy of an immunohistochemistry (IHC) approach using a novel anti-NRAS (Q61R) monoclonal antibody on formalin-fixed paraffin-embedded tissue samples from patients with metastatic melanoma. METHODS: We conducted a retrospective multicenter cohort study on 170 patients with metastatic melanoma. The automated IHC assay was performed using the SP174 clone, and compared with results of the molecular testing. RESULTS: Evaluation of a test cohort with knowledge of the mutation status established a specific IHC pattern for the mutation. In the independent blinded analysis of the remaining cases, the anti-NRAS (Q61R) antibody accurately identified all NRAS Q61R-mutated tumors, and demonstrated 100% sensitivity and specificity. LIMITATIONS: Limitations include retrospective design and lack of multicenter interobserver reproducibility. CONCLUSION: The NRAS (Q61R) IHC assay is reliable and specific for the evaluation of the Q61R mutation status in metastatic melanoma and may be an alternative to molecular biology in evaluation of metastatic melanoma in routine practice.

Shifting from Immunohistochemistry to Screen for ALK Rearrangements: Real-World Experience in a Large Single-Center Cohort of Patients with Non-Small-Cell Lung Cancer.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3-7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

[Accreditation of the activity of molecular pathology according to ISO 15189: key steps to follow and the main potential pitfalls]. / Accréditation de l'activité de pathologie moléculaire selon la norme ISO 15189. Principales étapes à respecter et principaux écueils possibles.

The quick emerging of the several targeted therapies and the concept of personnalized medicine underlie the necessity to develop and to well organize a molecular biology (or molecular pathology) unit of high quality, dedicated to clinical care, in order to look for tissular and cellular theragnosis biomarkers. This new and sudden area of activity for a clinical pathologist is strongly linked to the knowledge of a new medical speciality in health care institutions. Thus, the molecular pathology (or molecular biology made from cellular or tissular samples) can nicely be implemented in a clinical pathology laboratory. This new mission for a pathologist has to be done in respect with a great quality assurance which should allow obtaining in a short-term an ISO 15189 accreditation to keep going to perform this activity. The present work aims to describe the main steps to be set up in the order to get an ISO 15189 accreditation in molecular pathology. The different chapters of this norm will not be described in their exhaustivity, but in their large lines. Finally, we will describe the potential difficulties and pitfalls to be avoided before getting this accreditation.

[The accreditation of a surgical pathology and somatic genetic laboratory (LPCE, CHU of Nice) according to the ISO 15189 norm: Sharing of experience]. / L'accréditation selon la norme ISO 15189 d'un laboratoire de pathologie et de génétique somatique (LPCE, CHU de Nice) : retour d'expérience.

Accreditation is going to be vital and unavoidable in the medium term for medical biology laboratories in France. This accreditation will certainly condition the authorization to conduct biological testing in the health care system. All the biological specialities are now affected by this procedure, including the somatic genetics. The anatomo-pathology, which is a medical speciality in France, may be also concerned by the accreditation. However, the nature and the practices of this specialty increase the complexity of this approach to be implemented according to the standard requested by the authorities, i.e. the ISO 15189 normative standard (standard on "specific requirements for quality and competence for medical biology analysis laboratories"). The present article recounts the experience of a hospital laboratory (LPCE, Nice University Hospital) composed of a surgical pathology and a somatic genetics unit: (1) in the accreditation process according to the ISO 15189 standard, (2) at the time of the audit made by the team of "COFRAC" evaluators, and, (3) in evaluating the strategy implemented following the audit.

[Setting up a department of molecular pathology in oncology in a pathology laboratory (LPCE, CHU de Nice)]. / Mise en place d'un secteur de pathologie moléculaire en oncologie au sein d'un laboratoire d'anatomie pathologique (LPCE, CHU de Nice).

The advent of targeted therapies and personalized medicine in oncology has led in France to the settlement and organisation of a network of hospital molecular genetic platforms under the impetus of the National Cancer Institute (INCa). These platforms are, according to the concerned sites, integrated or not in pathology laboratories. The development of molecular biology methods, the choice of the procedures, the establishment of sample workflow, the quality control and the selection of the genomic alterations to be detected on each platform, have been left to the discretion of the different laboratories. Based on calls for project made by the INCa, hospital molecular genetic platforms were able to adapt their activity according to the assigned budgets. While the presence of some genomic alterations (i.e. KRAS gene mutations in metastatic colon adenocarcinoma or EGFR gene mutations in lung adenocarcinomas), may lead to administration of targeted therapies under the Marketing Authorization Application (MAA), others are associated with therapeutic clinical trials. However, increasing number of MAA for new molecules targeting genomic alterations is likely in the near future. In this context, it is necessary to quickly adapt the organisation of work of the hospital pathology laboratories performing molecular biology tests in order to meet the growing demand of oncologists in the field of targeted therapies. The purpose of this article is to describe the different steps of the settlement of a molecular genetic platform in an academic pathology laboratory (LPCE, CHU de Nice) and to show the experience of this laboratory specifically oriented on the support of the morphological and molecular diagnosis of lung cancer, thyroid cancer and malignant melanoma.

Need for a Dedicated Ophthalmic Malignancy Clinico-Biological Biobank: The Nice Ocular MAlignancy (NOMA) Biobank.

Ophthalmic malignancies include various rare neoplasms involving the conjunctiva, the uvea, or the periocular area. These tumors are characterized by their scarcity as well as their histological, and sometimes genetic, diversity. Uveal melanoma (UM) is the most common primary intraocular malignancy. UM raises three main challenges highlighting the specificity of ophthalmic malignancies. First, UM is a very rare malignancy with an estimated incidence of 6 cases per million inhabitants. Second, tissue biopsy is not routinely recommended due to the risk of extraocular dissemination. Third, UM is an aggressive cancer because it is estimated that about 50% of patients will experience metastatic spread without any curative treatment available at this stage. These challenges better explain the two main objectives in the creation of a dedicated UM biobank. First, collecting UM samples is essential due to tissue scarcity. Second, large-scale translational research programs based on stored human samples will help to better determine UM pathogenesis with the aim of identifying new biomarkers, allowing for early diagnosis and new targeted treatment modalities. Other periocular malignancies, such as conjunctival melanomas or orbital malignancies, also raise specific concerns. In this context, the number of biobanks worldwide dedicated to ocular malignancies is very limited. The aims of this article were (i) to describe the specific challenges raised by a dedicated ocular malignancy biobank, (ii) to report our experience in setting up such a biobank, and (iii) to discuss future perspectives in this field.

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.

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.

Characterisation of the protein expression of the emerging immunotherapy targets VISTA, LAG-3 and PRAME in primary uveal melanoma: insights from a southern French patient cohort.

Uveal melanoma (UM) is the most common intraocular tumour in adults, with dismal prognosis once metastases develop, since therapeutic options for the metastatic disease are ineffective. Over the past decade, novel cancer therapies based on immunotherapy have changed the landscape of treatment of different forms of cancer leading to many hopes of improvement in patient overall survival (OS). VISTA, LAG-3 and PRAME are novel promising targets of immunotherapy that have recently gained attention in different solid tumours, but whose relevance in UM remained to be comprehensively evaluated until now. Here, we studied the protein expression of VISTA, LAG-3 and PRAME using immunohistochemistry in representative whole tissue sections from primary UM cases in a cohort of 30 patients from a single centre (Nice University Hospital, Nice, France). The expression of each of these markers was correlated with different clinical and pathological parameters, including onset of metastases and OS. We demonstrated the protein expression of VISTA and LAG-3 in small lymphocytes infiltrating the tumour, while no expression of the proteins was detected in UM cells. For PRAME, nuclear expression was observed in UM cells, but no expression in tumour infiltrating immune cells was identified. Increased levels of VISTA expression in tumour infiltrating lymphocytes (TILs) were associated with nuclear BAP1 expression and better prognosis. Higher levels of LAG-3 in TILs were associated with higher levels of CD8-positive TILs. PRAME nuclear positivity in melanoma cells was associated with epithelioid cell dominant (>90%) UM histological subtype, higher mitotic numbers and a higher percentage of chromosome 8q gain. This study proposes VISTA as a novel relevant immune checkpoint molecule in primary UM and contributes to confirm LAG-3 and PRAME as potentially important immunotherapy targets in the treatment of UM patients, helping to expand the number of immunotherapy candidate molecules that are relevant to modulate in this aggressive 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.

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.

[Setting up indicators in biobanking: why and how?]. / Mise en place d'indicateurs de suivi au sein d'une tumorothèque et/ou d'un centre de ressources biologiques : pourquoi et comment ?

The biobanking area is highly complex, and its complexity is increasing along with its growth and demand. Due to the advancements in genetic research, stem cell research and regenerative medicine, biobanking has become ever more important and plays a key role in biomedical research. The robustness and the reproducibility of research results depend greatly on the quality and on the number of the samples used, and thus on the expertise of biobanks having supplied these samples. Undoubtedly, the recognition of a research biobank depends on the impact of the research projects conducted with samples obtained from tumour bank(s), but also on many other criteria. It thus seems important to determine a number of indicators within a biobank to estimate objective criteria for the performance of these structures. These indicators can allow to make some strategic decisions knowing that biobanks are expensive structures to maintain in the present hospital context. The use of these indicators could also contribute to the elaboration of an "biobank impact factor of" or so called "bioresource research impact factor" (BRIF). We describe here four major categories of indicators (quality, activity, scientific production, visibility), which seem to be useful for the evaluation of a biobank by making a proposition of allocation of coefficients for the various considered items.

Setting Up an Ultra-Fast Next-Generation Sequencing Approach as Reflex Testing at Diagnosis of Non-Squamous Non-Small Cell Lung Cancer; Experience of a Single Center (LPCE, Nice, France).

The number of genomic alterations required for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has increased and become more complex these last few years. These molecular abnormalities lead to treatment that provides improvement in overall survival for certain patients. However, these treated tumors inexorably develop mechanisms of resistance, some of which can be targeted with new therapies. The characterization of the genomic alterations needs to be performed in a short turnaround time (TAT), as indicated by the international guidelines. The origin of the tissue biopsies used for the analyses is diverse, but their size is progressively decreasing due to the development of less invasive methods. In this respect, the pathologists are facing a number of different challenges requiring them to set up efficient molecular technologies while maintaining a strategy that allows rapid diagnosis. We report here our experience concerning the development of an optimal workflow for genomic alteration assessment as reflex testing in routine clinical practice at diagnosis for NS-NSCLC patients by using an ultra-fast-next generation sequencing approach (Ion Torrent Genexus Sequencer, Thermo Fisher Scientific). We show that the molecular targets currently available to personalized medicine in thoracic oncology can be identified using this system in an appropriate TAT, notably when only a small amount of nucleic acids is available. We discuss the new challenges and the perspectives of using such an ultra-fast NGS in daily practice.

Evaluation of Sample Pooling for SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples with the Idylla SARS-CoV-2 Test.

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.

Salivary detection of COVID-19: clinical performance of oral sponge sampling for SARS-CoV-2 testing.

BACKGROUND: The current diagnostic standard for coronavirus disease 2019 (COVID-19) is reverse transcriptase-polymerase chain reaction (RT-PCR) testing with nasopharyngeal (NP) swabs. The invasiveness and need for trained personnel make the NP technique unsuited for repeated community-based mass screening. We developed a technique to collect saliva in a simple and easy way with the sponges that are usually used for tamponade of epistaxis. This study was carried out to validate the clinical performance of oral sponge (OS) sampling for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. METHODS: Over a period of 22 weeks, we collected prospectively 409 paired NP and OS samples from consecutive subjects presenting to a public community-based free screening centre. Subjects were referred by their attending physician because of recent COVID-19 symptoms (n = 147) or by the contact tracing staff of the French public health insurance because they were considered as close contacts of a laboratory-confirmed COVID-19 case (n = 262). RESULTS: In symptomatic subjects, RT-PCR SARS-CoV-2 testing with OS showed a 96.5% (95% CI: 89.6-94.8) concordance with NP testing, and a 93.2% (95% CI: 89.1-97.3) sensitivity when using the IdyllaTM platform and a sensitivity of 76.3% (95% CI: 69.4-83.2) on the Synlab Barla laboratory platform. In close contacts the NP-OS concordance (93.8%, 95% CI: 90.9-96.7) and OS sensitivity (71.9%, 95% CI: 66.5-77.3) were slightly lower. CONCLUSION: These results strongly suggest that OS testing is a straightforward, low-cost and high-throughput sampling method that can be used for frequent RT-PCR testing of COVID-19 patients and mass screening of populations.

A rapid near-patient RT-PCR test for suspected COVID-19: a study of the diagnostic accuracy.

BACKGROUND: Management of large numbers of reverse transcriptase-polymerase chain reactions (RT-PCR) for diagnosis of coronavirus 2019 disease (COVID-19) requires robust infrastructures, located in dedicated premises with a high standard of biosafety procedures, and well-trained personnel. The handling of a "run-of-river sample" to obtain rapid reporting of results is challenging. METHODS: We studied the clinical performance of the Idylla™ SARS-CoV-2 Test (index test) on a platform capable of fully automated nucleic acid testing including extraction, amplification, and detection in a single-use cartridge to establish the diagnosis of COVID-19. The study was conducted on a prospective cohort of 112 volunteers with recent symptoms and an unknown SARS-CoV-2 status who came to free screening centers of the Nice metropolitan area. All subjects underwent bilateral nasopharyngeal sampling. One sample was processed using the index test, the other using the standard of care RT-PCR. Samples were treated blind. RESULTS: Most of the participants (70%) were sampled within 4 days of symptom onset. Forty-five (40.2%) were positive for COVID-19. No clinical symptoms were distinguished between SARS-CoV-2 RT-PCR positive and negative subjects except anosmia and dysgeusia. Positive and negative agreement between the index and the standard of care test was 100%. CONCLUSIONS: The Idylla™ SARS-CoV-2 Test is very sensitive, specific, rapid and easy to use in a near-patient RT-PCR approach to distinguish between symptomatic SARS-CoV-2 positive and negative patients in selected settings.

Setting-Up a Rapid SARS-CoV-2 Genome Assessment by Next-Generation Sequencing in an Academic Hospital Center (LPCE, Louis Pasteur Hospital, Nice, France).

Introduction: Aside from the reverse transcription-PCR tests for the diagnosis of the COVID-19 in routine clinical care and population-scale screening, there is an urgent need to increase the number and the efficiency for full viral genome sequencing to detect the variants of SARS-CoV-2. SARS-CoV-2 variants assessment should be easily, rapidly, and routinely available in any academic hospital. Materials and Methods: SARS-CoV-2 full genome sequencing was performed retrospectively in a single laboratory (LPCE, Louis Pasteur Hospital, Nice, France) in 103 SARS-CoV-2 positive individuals. An automated workflow used the Ion Ampliseq SARS-CoV-2 panel on the Genexus Sequencer. The analyses were made from nasopharyngeal swab (NSP) (n = 64) and/or saliva (n = 39) samples. All samples were collected in the metropolitan area of the Nice city (France) from September 2020 to March 2021. Results: The mean turnaround time between RNA extraction and result reports was 30 h for each run of 15 samples. A strong correlation was noted for the results obtained between NSP and saliva paired samples, regardless of low viral load and high (>28) Ct values. After repeated sequencing runs, complete failure of obtaining a valid sequencing result was observed in 4% of samples. Besides the European strain (B.1.160), various variants were identified, including one variant of concern (B.1.1.7), and different variants under monitoring. Discussion: Our data highlight the current feasibility of developing the SARS-CoV-2 next-generation sequencing approach in a single hospital center. Moreover, these data showed that using the Ion Ampliseq SARS-CoV-2 Assay, the SARS-CoV-2 genome sequencing is rapid and efficient not only in NSP but also in saliva samples with a low viral load. The advantages and limitations of this setup are discussed.

[Role of the surgical pathology laboratory in the pre-analytical approach of molecular biology techniques]. / Rôle du laboratoire d'anatomie pathologique dans l'approche pré-analytique des examens de biologie moléculaire réalisés en pathologie tumorale.

The advent of the targeted cancer therapies administered to patients, according to the results of molecular biology techniques (in particular, in situ hybridization, "polymerase chain reaction" amplification and sequencing), has modified the practice of the surgical pathology laboratories. The necessity to answer to the needs of physicians for optimizing the medical care for patients who develop cancer has led to a policy of national debate, spurred by the National Institute of Cancer (INCa), in order to implement new procedures in the pathology laboratories. Thus, in addition to the structuring of molecular biology platforms and their labeling by INCa, the upstream control of the steps present between resection of tumor samples and molecular analysis has proved to be crucial. Indeed, the quality of this upstream time, called "pre-analytical" phase, determines the reliability of the molecular biology results and therefore the therapeutic strategy. We describe here the main steps to be checked in the pre-analytical phase. The optimization of this pre-analytical phase within the surgical pathology laboratory aims to reduce or render insignificant the risk of errors of molecular biology tests. These errors can indeed lead to false negative or false positive results whose therapeutic consequences can be particularly harmful to patients with cancer.