Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer.

Background: Expression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been associated with improved efficacy to anti-PD-1/PD-L1 inhibitors in patients with advanced-stage non-small-cell lung cancer (NSCLC) and emerged as a potential biomarker for the selection of patients to cancer immunotherapies. We investigated the utility of circulating tumor cells (CTCs) and circulating white blood cells (WBCs) as a noninvasive method to evaluate PD-L1 status in advanced NSCLC patients. Patients and methods: CTCs and circulating WBCs were enriched from peripheral blood samples (ISET® platform; Rarecells) from 106 NSCLC patients. PD-L1 expression on ISET filters and matched-tumor tissue was evaluated by automated immunostaining (SP142 antibody; Ventana), and quantified in tumor cells and WBCs. Results: CTCs were detected in 80 (75%) patients, with levels ranging from 2 to 256 CTCs/4 ml, and median of 60 CTCs/4 ml. Among 71 evaluable samples with matched-tissue and CTCs, 6 patients (8%) showed ≥1 PD-L1-positive CTCs and 11 patients (15%) showed ≥1% PD-L1-positive tumor cells in tumor tissue with 93% concordance between tissue and CTCs (sensitivity = 55%; specificity = 100%). From 74 samples with matched-tissue and circulating WBCs, 40 patients (54%) showed ≥1% PD-L1-positive immune infiltrates in tumor tissue and 39 patients (53%) showed ≥1% PD-L1 positive in circulating WBCs, with 80% concordance between blood and tissue (sensitivity = 82%; specificity = 79%). We found a trend for worse survival in patients receiving first-line cisplatin-based chemotherapy treatments, whose tumors express PD-L1 in CTCs or immune cells (progression-free and overall survival), similar to the effects of PD-L1 expression in matched-patient tumors. Conclusions: These results demonstrated that PD-L1 status in CTCs and circulating WBCs correlate with PD-L1 status in tumor tissue, revealing the potential of CTCs assessment as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with advanced-stage NSCLC.

Comparative study of the PD-L1 status between surgically resected specimens and matched biopsies of NSCLC patients reveal major discordances: a potential issue for anti-PD-L1 therapeutic strategies.

BACKGROUND: High expression of programmed death ligand-1 (PD-L1) on tumor cells (TC) and/or on tumor-infiltrating immune cells (IC) is associated with a high response rate in patients with advanced nonsmall-cell lung cancer (NSCLC) treated with PD-L1 inhibitors. The use of a PD-L1 immunohistochemical (IHC) test in determining the responsiveness to immunotherapy has raised the question of the reliability and reproducibility of its evaluation in lung biopsies compared with corresponding resected surgical specimens. PATIENTS AND METHODS: PD-L1 expression in TC and IC was assessed in 160 patients with operable NSCLC on both whole surgical tissue sections and matched lung biopsies, by using a highly sensitive SP142 IHC assay. The specimens were scored as TC 0-3 and IC 0-3 based on increasing PD-L1 expression. RESULTS: PD-L1 expression was frequently discordant between surgical resected and matched biopsy specimens (the overall discordance rate = 48%; 95% confidence interval 4.64-13.24) and κ value was equal to 0.218 (poor agreement). In all cases, the biopsy specimens underestimated the PD-L1 status observed on the whole tissue sample. PD-L1-positive IC tumors were more common than PD-L1-positive TC tumors on resected specimens. The discrepancies were mainly related to the lack of a PD-L1-positive IC component in matched biopsies. CONCLUSIONS: Our results indicate relatively poor association of the PD-L1 expression in TC and IC between lung biopsies and corresponding resected tumors. Although these results need to be further validated in larger cohorts, they indicate that the daily routine evaluation of the PD-L1 expression in diagnostic biopsies can be misleading in defining the sensitivity to treatment with PD-L1 targeted therapy.

Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK 'borderline'-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies.

BACKGROUND: Patients with advanced lung adenocarcinomas expressing ALK rearrangements are highly responsive to crizotinib, a dual ALK/c-MET inhibitor. Immunohistochemistry (IHC) is an easy clinically and routinely applicable cost-effective assay for ALK, c-MET and ROS1 protein expression for potential treatment with crizotinib. The purpose of this study was to evaluate the percentage and the pattern of ALK-rearranged cells, the variation in the native ALK copy number, as well as ALK, c-MET and ROS1 protein expression, and their significance on outcome of crizotinib-treated lung adenocarcinoma patients. PATIENTS AND METHODS: Consecutive lung adenocarcinoma specimens (n = 176) 'double-negative' (wild-type EGFR and KRAS) were tested for ALK rearrangements/copy number alterations and for ALK, c-MET and ROS1 protein expression using automated standardized protocols. Preliminary data on the outcome of crizotinib-treated patients were recorded. RESULTS: FISH analysis identified 26/176 (15%) cases with ALK rearrangements. Seven cases had discordant results between the ALK FISH and IHC. Five cases with discordant FISH-positive/IHC-negative revealed FISH 'borderline' positivity (15%-20%). Three cases overexpressed c-MET and responded to crizotinib, and two cases with ALK-'borderline' rearranged cells only, not associated with c-MET expression, progressed under crizotinib. Two cases with discordant FISH-negative/IHC-positive revealed ALK gene amplification without associated c-MET or ROS1 protein expression. CONCLUSIONS: The discrepancies observed between the IHC and FISH data revealed unexpected biological events, rather than technical issues, which potentially can have a strong impact on the therapeutic strategy with crizotinib.

Early infectious acute respiratory distress syndrome is characterized by activation and proliferation of alveolar T-cells.

Acute respiratory distress syndrome (ARDS) in humans is characterized by the infiltration of polymorphonuclears in the alveolar spaces. However, the role of T-cells in ARDS is unknown. Our aim was to characterize the T-cell phenotype in bronchoalveolar lavage (BAL) during the early phase of acute lung infection(ALI)/ARDS-infected patients in comparison to a control group (CG). BAL lymphocyte phenotypes of two ALI, 16 ARDS, and eight CG were examined by flow cytometry. ALI/ARDS showed a significant increase in CD4 and CD8 T-cell activation as compared to CG. Moreover, a significant level of proliferation was observed using the Ki67 marker in ARDS patients as compared to controls (median): 37 versus 6 % for CD4 T-cells (p = 0.022) and 34 versus 2 % for CD8 T-cells (p = 0.009). In contrast, the percentage of T-regulatory cells and apoptotic T-cells were similar in both groups. Among costimulatory molecules, we observed an overexpression of CTLA-4/CD152 on CD4 T-cells in ALI/ARDS as compared to CG: 30 versus 7 %, respectively (p = 0.063). In further characterizing T-cell subsets expressing high levels of CD152, we found the presence of IL-17 secreting CD4 T-cells in ALI/ARDS. In humans, ALI/ARDS due to infection is associated with a high level of T-cell activation and proliferation, along with the presence of Th17 cells, which are known to attract polymorphonuclears.

Clinical value of circulating endothelial cells and of soluble CD146 levels in patients undergoing surgery for non-small cell lung cancer.

BACKGROUND: Previous studies indicate that endothelial injury, as demonstrated by the presence of circulating endothelial cells (CECs), may predict clinical outcome in cancer patients. In addition, soluble CD146 (sCD146) may reflect activation of angiogenesis. However, no study has investigated their combined clinical value in patients undergoing resection for non-small cell lung cancer (NSCLC). METHODS: Data were collected from preoperative blood samples from 74 patients who underwent resection for NSCLC. Circulating endothelial cells were defined, using the CellSearch Assay, as CD146+CD105+CD45-DAPI+. In parallel, sCD146 was quantified using an ELISA immunoassay. These experiments were also performed on a group of 20 patients with small-cell lung cancer, 60 healthy individuals and 23 patients with chronic obstructive pulmonary disease. RESULTS: The CEC count and the plasma level of sCD146 were significantly higher in NSCLC patients than in the sub-groups of controls (P<0.001). Moreover, an increased CEC count was associated with higher levels of sCD146 (P=0.010). Both high CEC count and high sCD146 plasma level at baseline significantly correlated with shorter progression-free survival (P<0.001, respectively) and overall survival (P=0.005; P=0.009) of NSCLC patients. CONCLUSIONS: The present study provides supportive evidence to show that both a high CEC count and a high sCD146 level at baseline correlate with poor prognosis and may be useful for the prediction of clinical outcome in patients undergoing surgery for NSCLC.

Diagnostic value of immunohistochemistry for the detection of the BRAFV600E mutation in primary lung adenocarcinoma Caucasian patients.

BACKGROUND: Non-small-cell lung carcinoma (NSCLC) patients with a BRAF(V600E) mutation benefit from targeted therapy. The usefulness of immunohistochemistry (IHC) as an alternative approach for the detection of BRAF(V600E) in NSCLC patients has not been evaluated until now. This study compared the specificity and sensitivity of IHC with other methods for the detection of BRAF(V600E) in primary lung adenocarcinoma. PATIENTS AND METHODS: BRAF mutations were analysed by DNA sequencing of a Caucasian subpopulation of selected 450 of 1509 (30%) EGFR, KRAS, PI3KA, Her2 and EML4-ALK wild-type (wt) primary lung adenocarcinomas. Detection of the BRAF(V600E) mutation was carried out by IHC using the VE1 clone antibody and compared with the results of other molecular methodologies. RESULTS: Of 450 (9%) of tumours, 40 harboured a BRAF mutation, which corresponded to either a BRAF(V600E) or a non-BRAF(V600E) mutation in 21 of 450 (5%) and 19 of 450 (4%) cases, respectively. The IHC VE1 assay was positive in 19 of 21 (90%) BRAF(V600E)-mutated tumours and negative in all BRAF(nonV600E)-mutated tumours. CONCLUSION: IHC using the VE1 clone is a specific and sensitive method for the detection of BRAF(V600E) and may be an alternative to molecular biology for the detection of mutations in NSCLC.

ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma.

BACKGROUND: A subgroup of anaplastic lymphoma kinase (ALK)-rearranged lung tumours can respond to ALK inhibitors. Until now, the ALK status in circulating tumour cells (CTCs) isolated from patients with lung cancer has not been characterised. We assessed the ALK status in CTCs detected in patients with lung cancer and correlated the results to the ALK status defined in the corresponding tumour tissue. PATIENTS AND METHODS: A total of 87 patients with lung adenocarcinoma showing CTCs isolated using the isolation by size of epithelial tumour cell method were screened for their ALK status both in tumour samples and in CTCs. ALK break-apart fluorescence in situ hybridisation (FISH) and immunoreactivity analyses using an anti-ALK antibody (5A4 clone) were carried out on CTCs and compared with the results obtained in the corresponding tissue specimens. RESULTS: A total of five patients showed ALK-gene rearrangement and strong ALK protein expression in CTCs and in the corresponding tumour samples. Both ALK-FISH and ALK immunoreactivity analyses show negative results in CTCs and corresponding tumour samples for 82 patients. Conclusions We demonstrated that the ALK status can be determined in CTCs isolated from patients with lung cancer by immunocytochemistry and FISH analyses. These results favour non-invasive, ALK-gene status pre-screening on a routine basis on CTCs isolated from patients with lung cancer and open new avenues for real-time monitoring for adapted targeted therapy.

Morphological analysis of circulating tumour cells in patients undergoing surgery for non-small cell lung carcinoma using the isolation by size of epithelial tumour cell (ISET) method.

BACKGROUND AND OBJECTIVE: Recurrence rates after surgery for non-small cell lung cancer (NSCLC) range from 25 to 50% and 5-year survival is only 60-70%. Because no biomarkers are predictive of recurrence or the onset of metastasis, pathological TNM (pTNM) staging is currently the best prognostic factor. Consequently, the preoperative detection of circulating tumour cells (CTCs) might be useful in tailoring therapy. The aim of this study was to characterize morphologically any circulating non-haematological cells (CNHCs) in patients undergoing surgery for NSCLC using the isolation by size of epithelial tumour cell (ISET) method. METHODS: Of 299 blood samples tested, 250 were from patients with resectable NSCLC and 59 from healthy controls. The presence of CNHCs was assessed blindly and independently by 10 cytopathologists on May-Grünwald-Giemsa stained filters and the cells classified into three groups: (i) malignant cells, (ii) uncertain malignant cells, and (iii) benign cells. We assessed interobserver agreement using Kappa (κ) analysis as the measure of agreement. RESULTS: A total of 123 out of 250 (49%) patients showed CNHCs corresponding to malignant, uncertain malignant and benign cells, in 102/250 (41%), 15/250 (6%) and 6/250 (2%) cases, respectively. No CNHCs were detected in the blood of healthy subjects. Interobserver diagnostic variability was absent for CNHCs, low for malignant cells and limited for uncertain malignant and benign cells. CONCLUSION: Identification of CTCs in resectable NSCLC patients, using ISET technology and according to cytopathological criteria of malignancy, appears to be a new and promising field of cytopathology with potential relevance to lung oncology.

[French practical guidelines for the diagnosis and management of IPF - 2021 update, full version]. / Recommandations pratiques pour le diagnostic et la prise en charge de la fibrose pulmonaire idiopathique ­ Actualisation 2021. Version intégrale.

BACKGROUND: Since the previous French guidelines were published in 2017, substantial additional knowledge about idiopathic pulmonary fibrosis has accumulated. METHODS: Under the auspices of the French-speaking Learned Society of Pulmonology and at the initiative of the coordinating reference center, practical guidelines for treatment of rare pulmonary diseases have been established. They were elaborated by groups of writers, reviewers and coordinators with the help of the OrphaLung network, as well as pulmonologists with varying practice modalities, radiologists, pathologists, a general practitioner, a head nurse, and a patients' association. The method was developed according to rules entitled "Good clinical practice" in the overall framework of the "Guidelines for clinical practice" of the official French health authority (HAS), taking into account the results of an online vote using a Likert scale. RESULTS: After analysis of the literature, 54 recommendations were formulated, improved, and validated by the working groups. The recommendations covered a wide-ranging aspects of the disease and its treatment: epidemiology, diagnostic modalities, quality criteria and interpretation of chest CT, indication and modalities of lung biopsy, etiologic workup, approach to familial disease entailing indications and modalities of genetic testing, evaluation of possible functional impairments and prognosis, indications for and use of antifibrotic therapy, lung transplantation, symptom management, comorbidities and complications, treatment of chronic respiratory failure, diagnosis and management of acute exacerbations of fibrosis. CONCLUSION: These evidence-based guidelines are aimed at guiding the diagnosis and the management in clinical practice of idiopathic pulmonary fibrosis.

[French practical guidelines for the diagnosis and management of IPF - 2021 update, short version]. / Recommandations pratiques pour le diagnostic et la prise en charge de la fibrose pulmonaire idiopathique ­ Actualisation 2021. Version courte.

BACKGROUND: Since the previous French guidelines were published in 2017, substantial additional knowledge about idiopathic pulmonary fibrosis has accumulated. METHODS: Under the auspices of the French-speaking Learned Society of Pulmonology and at the initiative of the coordinating reference center, practical guidelines for treatment of rare pulmonary diseases have been established. They were elaborated by groups of writers, reviewers and coordinators with the help of the OrphaLung network, as well as pulmonologists with varying practice modalities, radiologists, pathologists, a general practitioner, a head nurse, and a patients' association. The method was developed according to rules entitled "Good clinical practice" in the overall framework of the "Guidelines for clinical practice" of the official French health authority (HAS), taking into account the results of an online vote using a Likert scale. RESULTS: After analysis of the literature, 54 recommendations were formulated, improved, and validated by the working groups. The recommendations covered a wide-ranging aspects of the disease and its treatment: epidemiology, diagnostic modalities, quality criteria and interpretation of chest CT, indication and modalities of lung biopsy, etiologic workup, approach to familial disease entailing indications and modalities of genetic testing, evaluation of possible functional impairments and prognosis, indications for and use of antifibrotic therapy, lung transplantation, symptom management, comorbidities and complications, treatment of chronic respiratory failure, diagnosis and management of acute exacerbations of fibrosis. CONCLUSION: These evidence-based guidelines are aimed at guiding the diagnosis and the management in clinical practice of idiopathic pulmonary fibrosis.

Short-term safety of thoracoscopic talc pleurodesis for recurrent primary spontaneous pneumothorax: a prospective European multicentre study.

The safety of talc pleurodesis is under dispute following reports of talc-induced acute respiratory distress syndrome (ARDS) and death. We investigated the safety of large-particle talc for thoracoscopic pleurodesis to prevent recurrence of primary spontaneous pneumothorax (PSP). 418 patients with recurrent PSP were enrolled between 2002 and 2008 in nine centres in Europe and South Africa. The main exclusion criteria were infection, heart disease and coagulation disorders. Serious adverse events (ARDS, death or other) were recorded up to 30 days after the procedure. Oxygen saturation, supplemental oxygen use and temperature were recorded daily at baseline and after thoracoscopic pleurodesis (2 g graded talc). During the 30-day observation period following talc poudrage, no ARDS (95% CI 0.0-0.9%), intensive care unit admission or death were recorded. Seven patients presented with minor complications (1.7%, 95% CI 0.7-3.4%). After pleurodesis, mean body temperature increased by 0.41°C (95% CI 0.33-0.48°C; p<0.001) at day 1 and returned to baseline value at day 5. Pleural drains were removed after day 4 in 80% of patients. Serious adverse events, including ARDS or death, did not occur in this large, multicentre cohort. Thoracoscopic talc poudrage using larger particle talc to prevent recurrence of PSPS can be considered safe.

[The biological rationale for immunotherapy in cancer]. / Rationnel biologique de l'immunothérapie des cancers.

INTRODUCTION: Immunotherapy aims to promote the immune system's activity against malignant cells by stimulating the response to several tumor antigens. STATE OF THE ART: Immunosurveillance may adjust the immunogenicity of tumors. To be effective, immunity must induce the specific activation of CD4+ and CD8+ T lymphocytes, as well as activation of innate immunity. Activator and inhibitory costimulatory molecules regulate T lymphocyte activation at immunity checkpoints such as PD-1/PD-L1 and CTLA-4. Adaptive immune resistance confers tumour resistance to immunosurveillance through these immune checkpoints. PERSPECTIVES: Approaches involving the combination of several immunotherapies with each other or with chemotherapy and radiotherapy and antibodies against other molecules of costimulation are under development. The development of biomarkers, which can select a targeted population and predict therapeutic response, represents a major challenge. Tumour high-throughput sequencing could refine "immunoscore". Intratumoral T cell receptor seems to represent a promising biomarker. CONCLUSIONS: Numerous challenges still remain in developing research approaches for the development of immunotherapies.

[Emphysema, did you say emphysema?] / Emphysème, vous avez dit emphysème ?

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a common condition that may initially look simple but may conceal other diseases capable of accelerating its natural history or even simulating it. We describe four cases presenting as COPD with emphysema that were reclassified on the basis of certain clinical characteristics and the radiological pattern. CASE REPORTS: A 52 year old never smoking woman presenting with emphysema was eventually diagnosed as having lymphangioleiomyomatosis on the basis of an abdominal CT scan showing kidney angiomyolipomas. A 44 years old smoker presenting with rapidly evolving emphysema was eventually diagnosed as having Langerhans cell histiocytosis on the basis of a previous chest CT (four years earlier) showing cavitating nodules. An airport refueler, 73 years old, with severe emphysema despite never having smoked, was eventually diagnosed as suffering from alpha-1 antitrypsin deficiency. The last patient was a 54 year old man, a never smoker, who presented with severe airflow limitation and multilobar hyperlucency, with bronchiectasis in the same areas. He was eventually diagnosed as having a severe form of the Swyer-James-MacLeod syndrome. CONCLUSION: These four case reports underline the importance of questioning the diagnosis of COPD when certain particular phenotypic characteristics are identified.

[Association of tracheobronchopathia osteochondroplastica and ozène]. / Association trachéobronchopathie chondro-ostéoplastique et ozène.

INTRODUCTION: We report a case of tracheobronchopathia osteochondroplastica associated with Ozena (atrophic rhinitis). OBSERVATION: Fibreoptic bonchoscopy showed irregular tracheal stenosis and histopathological examination displayed zones of bone metaplasia in the tracheal submucosa. We isolated the bacteria Klebsiella pneumoniae sp ozaenae from bronchial aspirate. CONCLUSION: This organism is frequently isolated in both conditions suggesting some link between the two diseases.

Immunotherapy in Non-Small Cell Lung Cancer: Biological Principles and Future Opportunities.

Immunotherapy aims to amplify the anticancer immune response through reactivation of the lymphocytic response raised against several tumor neo-antigens. To obtain an effective immune response, this therapeutic approach requires that a number of immunological checkpoints be passed, such as the activation of excitatory costimulatory signals or the avoidance of coinhibitory molecules. Among the immune checkpoints, the interaction of the membrane-bound ligand PD-1 and its receptor PD-L1 has received much attention because of remarkable efficacy in numerous clinical trials for various cancer types, including non-small cell lung cancer (NSCLC). However, several limitations exist with these therapeutic agents when used as monotherapy, with objective responses observed in only 30-40% of patients, with the majority of patients demonstrating innate resistance, and approximately 25% of responders later demonstrating disease progression. Recent developments in the understanding of cancer immunology have the potential to identify mechanisms of innate and acquired resistance to immune checkpoint inhibitors through translational research in human samples. This review focuses on the biological basic principles for immunological checkpoint blockade, and highlights the current status and the perspectives of this therapeutic approach in NSCLC patients.

Invasive pulmonary aspergillosis in chronic obstructive pulmonary disease: an emerging fungal pathogen.

Acute invasive pulmonary aspergillosis occurs predominantly in immunocompromised hosts, with increasing numbers of cases of invasive aspergillosis among patients with chronic obstructive pulmonary disease (COPD) being reported. Among 13 cases of invasive aspergillosis diagnosed in COPD patients admitted to the intensive care unit with acute respiratory distress, the only risk factor for invasive fungal infection was corticosteroid treatment. Invasive aspergillosis should be suspected in COPD patients receiving steroid treatment who have extensive pulmonary infiltrates. Survival depends on rapid diagnosis and early appropriate treatment. A decrease or interruption of steroid treatment should be considered as part of the overall therapeutic strategy.

KRAS Mutations in Lung Adenocarcinoma: Molecular and Epidemiological Characteristics, Methods for Detection, and Therapeutic Strategy Perspectives.

KRAS mutations are detected in over one third of lung adenocarcinomas, most frequently in Caucasian and smoker patients. The impact of KRAS mutations on lung adenocarcinoma prognosis is currently subject to debate, as is their impact on the response to chemotherapy and EGFR tyrosine kinase inhibitors. The different methods for KRAS status assessment, based on histological and cytological samples or biological fluids, offer varying sensitivities. Since no treatments are available in clinical routine for KRAS-mutated lung cancer patients, one of the current major challenges in thoracic oncology is developing new dedicated strategic therapies. Different molecules can be developed that act on a post-transcriptional KRAS protein level, blocking its cytoplasmic membrane recruitment. The efficacy of these molecules' targeting of the different signaling pathways activated by the KRAS mutation (such as the MEK and BRAF pathways) is related to the particular KRAS mutation subtype. New therapeutic strategies are currently focused on certain genes linked with KRAS inducing a synthetic lethal interaction. The purpose of this work is to provide an overview of i) the recent epidemiological and molecular findings concerning KRASmutated lung adenocarcinoma, ii) the prognostic impact of KRAS mutations, in particular during response to treatment, iii) the available methods for detecting this mutation, and iv) the current molecules under development for new therapeutic strategies and the clinical trials targeting this genomic alteration.