ultra-fast-amplicon-based-next-generation-sequencing-non-squamous-non

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 ...

Université Côte d'Azur, Hôpital Pasteur, CHU de Nice

Université Côte d'Azur

Immunotherapy aims to promote the immune system's activity against malignant cells by stimulating the response to several tumor antigens.

[The biological rationale for immunotherapy in cancer].

: With the integration of various targeted therapies into the clinical management of patients with advanced lung adenocarcinoma, next-generation sequencing (NGS) has become the technology of choice and has led to an increase in simultaneously interrogated genes. However, the broader adoption of NGS for routine clinical practice is still hampered by sophisticated workflows, complex bioinformatics analysis and medical interpretation. Therefore, the performance of the novel QIAGEN GeneReader NGS system was compared to an in-house ISO-15189 certified Ion PGM NGS platform. : Clinical samples from 90 patients (60 Retrospectively and 30 Prospectively) with lung adenocarcinoma were sequenced with both systems. Mutations were analyzed and , , , , ,  and  genes were compared and sampling time and suitability for clinical testing were assessed. : Both sequencing systems showed perfect concordance for the overlapping genes. Correlation of allele frequency was ² = 0.93 for the retrospective patients and ² = 0.81 for the prospective patients. Hands-on time and total run time were shorter using the PGM system, while the GeneReader platform provided good traceability and up-to-date interpretation of the results. : We demonstrated the suitability of the GeneReader NGS system in routine practice in a clinical pathology laboratory setting.

Use of the Ion PGM and the GeneReader NGS Systems in Daily Routine Practice for Advanced Lung Adenocarcinoma Patients: A Practical Point of View Reporting a Comparative Study and Assessment of 90 Patients.

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact.

Ensuring the Safety and Security of Frozen Lung Cancer Tissue Collections through the Encapsulation of Dried DNA.

Lung cancer is the major cause of death from cancer in the world and its incidence is increasing in women. Despite the progress made in developing immunotherapies and therapies targeting genomic alterations, improvement in the survival rate of advanced stages or metastatic patients remains low. Thus, urgent development of effective therapeutic molecules is needed. The discovery of novel therapeutic targets and their validation requires high quality biological material and associated clinical data. With this aim, we established a biobank dedicated to lung cancers. We describe here our strategy and the indicators used and, through an overall assessment, present the strengths, weaknesses, opportunities and associated risks of this biobank.

Establishing a Dedicated Lung Cancer Biobank at the University Center Hospital of Nice (France). Why and How?

With underrepresentation of elderly patients with lung adenocarcinoma (LADC) in anti-PD-1/PD-L1 clinical trials, better understanding of the interplay of PD-L1 and tumor-associated immune cells (TAICs) could assist clinicians in stratifying these patients for immunotherapy. One hundred and one patients with LADCs, stratified by age, were included for analysis of PD-L1 expression and density of TAICs expressing CD4, CD8, and CD33, by using multiplex chromogenic immunohistochemistry (IHC) assays and automated digital quantification. The CD4⁺/CD8⁺ ratio was significantly higher in elderly patients. In patients <75 years, the density of CD4⁺, CD8⁺, and PD-L1 in TAICs showed a positive significant correlation with PD-L1 expression in tumor cells (TCs), while a lower correlation was observed in the elderly population. In the latter, a high CD4⁺/CD8⁺ ratio, and combined PD-L1 expression ≥1% TCs with a low CD8⁺ density, low CD33⁺ density, and a high CD4⁺ density correlated to worse overall survival. We identified differences according to age in the CD4⁺/CD8⁺ ratio and in correlation between PD-L1 expression and the density of TAICs in LADC patients. Distinct groups of tumor microenvironments had an impact on the OS of elderly patients with LADC.

Chromogenic Multiplex Immunohistochemistry Reveals Modulation of the Immune Microenvironment Associated with Survival in Elderly Patients with Lung Adenocarcinoma.

Early-stage treatment improves prognosis of lung cancer and two large randomized controlled trials have shown that early detection with low-dose computed tomography (LDCT) reduces mortality. Despite this, lung cancer screening (LCS) remains challenging. In the context of a global shortage of radiologists, the high rate of false-positive LDCT results in overloading of existing lung cancer clinics and multidisciplinary teams. Thus, to provide patients with earlier access to life-saving surgical interventions, there is an urgent need to improve LDCT-based LCS and especially to reduce the false-positive rate that plagues the current detection technology. In this context, LCS can be improved in three ways: (1) by refining selection criteria (risk factor assessment), (2) by using Computer Aided Diagnosis (CAD) to make it easier to interpret chest CTs, and (3) by using biological blood signatures for early cancer detection, to both spot the optimal target population and help classify lung nodules. These three main ways of improving LCS are discussed in this review.

Lung Cancer Screening, Towards a Multidimensional Approach: Why and How?

It is generally accepted that carcinogenesis and aging are two biological processes, which are known to be associated. Notably, the frequency of certain cancers (including lung cancer), increases significantly with the age of patients and there is now a wealth of data showing that multiple mechanisms leading to malignant transformation and to aging are interconnected, defining the so-called common biology of aging and cancer. OncoAge, a consortium launched in 2015, brings together the multidisciplinary expertise of leading public hospital services and academic laboratories to foster the transfer of scientific knowledge rapidly acquired in the fields of cancer biology and aging into innovative medical practice and silver economy development. This is achieved through the development of shared technical platforms (for research on genome stability, (epi)genetics, biobanking, immunology, metabolism, and artificial intelligence), clinical research projects, clinical trials, and education. OncoAge focuses mainly on two pilot pathologies, which benefit from the expertise of several members, namely lung and head and neck cancers. This review outlines the broad strategic directions and key advances of OncoAge and summarizes some of the issues faced by this consortium, as well as the short- and long-term perspectives.

The OncoAge Consortium: Linking Aging and Oncology from Bench to Bedside and Back Again.

The understanding of the natural history and biology of lung cancer has been enhanced by studies into circulating tumor cells (CTCs). Fundamental and translational research, as well as clinical trials in the characterization and behavior of these cells, have constantly contributed to improving understanding within the domain of thoracic oncology. However, the use of these CTCs as prognostic and predictive biomarkers has not been adopted to the same extent as circulating free DNA (cf-DNA) in plasma, in the daily practice of thoracic oncologists. However, recent technological advances have firmly put the detection and characterization of CTCs in thoracic oncology back on the agenda, and have opened up perspectives for their routine clinical use. This review discusses the major advances of using CTCs in the domain of thoracic oncology, as well as the envisaged short- and long-term prospects.

Circulating Tumor Cell Detection in Lung Cancer: But to What End?

Tumor banks are asked to clinical and translationnal research project development in oncology. They strongly participate to the assessment, then to the validation of diagnostic, prognostic and predictive biomarkers. The progressive change of these structures leads to induce a professionalization of their functioning and to identify them as key actors in oncology by the stakeholders of the public and private worlds. The progresses made in biotechnologies and therapeutics are rapidly modifying the impact and the proper functioning of the biobanks. These latter are now facing different challenges, in particular for their sustainability. Among the major issues, the integration of the clinical and biological data becoming increasingly complex leads to urgently consider an optimization of the role of different biobanks in France. Their goal is to be an attractive counterpart face to the international competition. The purpose of this review is to briefly describe the current evolution of the biobanks, then their present and future challenges, and finally the role made by the pathologists in these new issues in oncology field.

[Tumor banks and complex data management: Current and future challenges].

Lung cancer is one of the most lethal malignancies worldwide, mainly due to its late diagnoses. The detection of molecular markers on samples provided from routine bronchoscopy including several liquid-based cytology tests (e.g., bronchoaspirate, bronchoalveolar lavage) and/or on easily obtained specimens such as sputum could represent a new approach to improve the sensitivity in lung cancer diagnoses. Recently growing interest has been reported for "noninvasive" liquid biopsy as a valuable source for molecular profiling. Unfortunately, a biomarker and/or composition of biomarkers capable of detecting early-stage lung cancer has yet to be discovered even if in the last few years there has been, through the use of revolutionary new technologies, an explosion of lung cancer biomarkers. Assay sensitivity and specificity need to be improved particularly when new approaches and/or tools are used. We have focused on the most important markers detected in tissue, and on several cytological specimens and liquid biopsies overall.

Are There New Biomarkers in Tissue and Liquid Biopsies for the Early Detection of Non-Small Cell Lung Cancer?

Exogenous lipoid pneumonia is a rare disease resulting from intra-alveolar accumulation of lipids of mineral, vegetal, or animal origin, that induce a foreign body type of inflammatory reaction in the lungs. Gastroesophageal reflux disease and other esophageal abnormalities have often been associated with this disease.

A case report of exogenous lipoid pneumonia associated with avocado/soybean unsaponifiables.

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.

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

Lung cancer screening with low-dose chest CT (LDCT) reduces the mortality of eligible individuals. Blood signatures might act as a standalone screening tool, refine the selection of patients at risk, or help to classify undetermined nodules detected on LDCT. We previously showed that circulating tumour cells (CTCs) could be detected, using the isolation by size of epithelial tumour cell technique (ISET), long before the cancer was diagnosed radiologically. We aimed to test whether CTCs could be used as a biomarker for lung cancer screening.

Circulating tumour cells as a potential biomarker for lung cancer screening: a prospective cohort study.

[Primary spontaneous pneumothorax: We have time to get some fresh air…].

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.

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).

Article	Year
[The biological rationale for immunotherapy in cancer]. Revue des maladies respiratoires\| PubMed ID: 29428191	2018
Use of the Ion PGM and the GeneReader NGS Systems in Daily Routine Practice for Advanced Lung Adenocarcinoma Patients: A Practical Point of View Reporting a Comparative Study and Assessment ... Cancers\| PubMed ID: 29561830	2018
Ensuring the Safety and Security of Frozen Lung Cancer Tissue Collections through the Encapsulation of Dried DNA. Cancers\| PubMed ID: 29891792	2018
Establishing a Dedicated Lung Cancer Biobank at the University Center Hospital of Nice (France). Why and How? Cancers\| PubMed ID: 29966305	2018
Chromogenic Multiplex Immunohistochemistry Reveals Modulation of the Immune Microenvironment Associated with Survival in Elderly Patients with Lung Adenocarcinoma. Cancers\| PubMed ID: 30216999	2018
Lung Cancer Screening, Towards a Multidimensional Approach: Why and How? Cancers\| PubMed ID: 30759893	2019
The OncoAge Consortium: Linking Aging and Oncology from Bench to Bedside and Back Again. Cancers\| PubMed ID: 30795607	2019
Circulating Tumor Cell Detection in Lung Cancer: But to What End? Cancers\| PubMed ID: 30813420	2019
[Tumor banks and complex data management: Current and future challenges]. Annales de pathologie\| PubMed ID: 30819623	2019
Are There New Biomarkers in Tissue and Liquid Biopsies for the Early Detection of Non-Small Cell Lung Cancer? Journal of clinical medicine\| PubMed ID: 30917582	2019
A case report of exogenous lipoid pneumonia associated with avocado/soybean unsaponifiables. BMC pulmonary medicine\| PubMed ID: 31795982	2019
Prospective evaluation of NGS-based liquid biopsy in untreated late stage non-squamous lung carcinoma in a single institution. Journal of translational medicine\| PubMed ID: 32066459	2020
Circulating tumour cells as a potential biomarker for lung cancer screening: a prospective cohort study. The Lancet. Respiratory medicine\| PubMed ID: 32649919	2020
[Primary spontaneous pneumothorax: We have time to get some fresh air…]. Revue des maladies respiratoires\| PubMed ID: 36710208	2023
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). Journal of personalized medicine\| PubMed ID: 37511690	2023

Charles-Hugo Marquette

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