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.

Patterns of IgE sensitization in house dust mite-allergic patients: implications for allergen immunotherapy.

BACKGROUND: Understanding patterns of IgE sensitization in Dermatophagoides-allergic patients living in various geographical areas is necessary to design a product suitable for worldwide allergen immunotherapy (AIT). METHODS: Using a HIFI Allergy customized microarray assay, IgEs specific for 12 purified allergens from Dermatophagoides pteronyssinus or D. farinae were assessed in sera from 1302 house dust mite (HDM)-allergic patients living in various areas. Comprehensive mass spectrometric (MS) analyses were conducted to characterize HDM extracts, as well as purified bodies and feces. RESULTS: Patterns of IgE reactivity to HDM allergens are comparable in all cohorts of patients analyzed, encompassing adults and 5- to 17-year-old children, as well as American, Canadian, European, and Japanese patients. Overall, >70% and >80% of HDM-allergic patients are sensitized to group 1 and group 2 allergens, respectively, from D. pteronyssinus and/or D. farinae species. Furthermore, 20-47% of patients also have IgEs to allergens from groups 4, 5, 7, 13, 15, 21, and 23. All patients have IgEs to allergens present in mite bodies and feces. MS-based analyses confirmed the presence of mite allergens recorded by IUIS in D. pteronyssinus and D. farinae extracts, with groups 2, 8, 10, 11, 14, and 20 prominent in bodies and groups 1, 6, 18, and 23 well represented in feces. CONCLUSIONS: Mite-specific AIT should rely upon a mixture of D. pteronyssinus and D. farinae extracts, manufactured from both feces and bodies. Such a combination is appropriate to treat children and adult Dermatophagoides-allergic patients from Asia, Europe, and North America.

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.

DNA biosensor/biochip for multiplex blood group genotyping.

At present, 33 blood groups representing over 300 antigens are listed by the International Society of Blood Transfusion (ISBT). Most of them result from a single nucleotide polymorphism (SNP) in the corresponding DNA sequence, i.e. approx. 200 SNPs. In immunohematology laboratories, blood group determination is classically carried out by serological tests, but these have some limitations, mostly in term of multiplexing and throughput. Yet, there is a growing need of extended blood group typing to prevent alloimmunization in transfused patients and transfusion accidents. The knowledge of the molecular bases of blood groups allows the use of molecular biology methods within immunohematology laboratories. Numerous assays focused on blood group genotyping were developed and described during the last 10 years. Some of them were real biochips or biosensors while others were more characterized by the particular molecular biology techniques they used, but all were intending to produce multiplex analysis. PCR techniques are most of the time used followed by an analytical step involving a DNA biosensor, biochip or analysis system (capillary electrophoresis, mass spectrometry). According to the method used, the test can then be classified as low-, medium- or high-throughput. There are several companies which developed platforms dedicated to blood group genotyping able to analyze simultaneously various SNPs or variants associated with blood group systems. This review summarizes the characteristics of each molecular biology method and medium-/high-throughput platforms dedicated to the blood group genotyping.

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.

Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies.

TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors).

Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair.

The recent advances in 3D-printed silicone (PDMS: polydimethylsiloxane) implants present prospects for personalized implants with highly accurate anatomical conformity. However, a potential adverse effect, such as granuloma formation due to immune reactions, still exists. One potential way to overcome this problem is to control the implant/host interface using immunomodulatory coatings. In this study, a new cytokine cocktail composed of interleukin-10 and prostaglandin-E2 was designed to decrease adverse immune reactions and promote tissue integration by fixing macrophages into M2 pro-healing phenotype for an extended period of time. In vitro, the cytokine cocktail maintained low levels of pro-inflammatory cytokine (TNF-α and IL-6) secretions and induced the secretion of IL-10 and the upregulation of multifunctional scavenging and sorting receptor stabilin-1, expressed by M2 macrophages. This cocktail was then loaded in a gelatine-based hydrogel to develop an immunomodulatory material that could be used as a coating for medical devices. The efficacy of this coating was demonstrated in an in vivo rat model during the reconstruction of a tracheal defect by 3D-printed silicone implants. The coating was stable on the silicone implants for over 2 weeks, and the controlled release of the cocktail components was achieved for at least 14 days. In vivo, only 33% of the animals with bare silicone implants survived, whereas 100% of the animals survived with the implant equipped with the immunomodulatory hydrogel. The presence of the hydrogel and the cytokine cocktail diminished the thickness of the inflammatory tissue, the intensity of both acute and chronic inflammation, the overall fibroblastic reaction, the presence of oedema and the formation of fibrinoid (assessed by histology) and led to a 100% survival rate. At the systemic level, the presence of immunomodulatory hydrogels significantly decreased pro-inflammatory cytokines such as TNF-α, IFN-γ, CXCL1 and MCP-1 levels at day 7 and significantly decreased IL-1α, IL-1ß, CXCL1 and MCP-1 levels at day 21. The ability of this new immunomodulatory hydrogel to control the level of inflammation once applied to a 3D-printed silicone implant has been demonstrated. Such thin coatings can be applied to any implants or scaffolds used in tissue engineering to diminish the initial immune response, improve the integration and functionality of these materials and decrease potential complications related to their presence.

Lung function in Birt-Hogg-Dubé syndrome: a retrospective analysis of 96 patients.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a rare autosomal dominant disorder caused by mutations in the FLCN gene coding for folliculin. Its clinical expression includes cutaneous fibrofolliculomas, renal tumors, multiple pulmonary cysts, and recurrent spontaneous pneumothoraces. Data on lung function in BHD are scarce and it is not known whether lung function declines over time. We retrospectively assessed lung function at baseline and during follow-up in 96 patients with BHD. RESULTS: Ninety-five percent of BHD patients had multiple pulmonary cysts on computed tomography and 59% had experienced at least one pneumothorax. Mean values of forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and total lung capacity were normal at baseline. Mean (standard deviation) residual volume (RV) was moderately increased to 116 (36) %pred at baseline, and RV was elevated > 120%pred in 41% of cases. Mean (standard deviation) carbon monoxide transfer factor (DLco) was moderately decreased to 85 (18) %pred at baseline, and DLco was decreased < 80%pred in 33% of cases. When adjusted for age, gender, smoking and history of pleurodesis, lung function parameters did not significantly decline over a follow-up period of 6 years. CONCLUSIONS: Cystic lung disease in BHD does not affect respiratory function at baseline except for slightly increased RV and reduced DLco. No significant deterioration of lung function occurs in BHD over a follow-up period of 6 years.

Disposable screen-printed chemiluminescent biochips for the simultaneous determination of four point-of-care relevant proteins.

A screen-printed (SP) microarray is presented as a platform for the achievement of multiparametric biochips. The SP platform is composed of eight (0.28-mm(2)) working electrodes modified with electroaddressed protein A-aryl diazonium adducts. The electrode surfaces are then used as an affinity immobilisation support for the orientated binding of capture monoclonal antibodies, having specificity against four different point-of-care related proteins (myoglobin, cardiac troponin I, C-reactive protein and brain natriuretic peptide). The immobilised capture antibodies are involved in sandwich assays of the four proteins together with biotinylated detection antibodies and peroxidase-labelled streptavidin in order to permit a chemiluminescent imaging of the SP platform and a sensitive detection of the assayed proteins. The performances of the system in pure buffered solutions, using a 25-min assay duration, were characterised by dynamic ranges of 0.5-50, 0.1-120, 0.2-20 and 0.67-67 microg/L for C-reactive protein, myoglobin, cardiac troponin I and brain natriuretic peptide, respectively. The four different assays were also validated in spiked 40-times-diluted human sera, using LowCross buffer, and were shown to work simultaneously in this complex medium.

3D Bioprinting:principles, fantasies and prospects.

Conventional three-dimensional (3D) printing techniques have been growing in importance in the field of reconstructive surgery. Three-dimensional bioprinting is the adaptation of 3D printing techniques to tissue engineering, through the use of a bio-ink containing living cells and biomaterials. We hereby describe the principles of bioprinting, its main current limitations, and the prospects of this technique. A PubMed/MEDLINE search was performed. A total of 40 publications were included. To date, most of the tissues have been printed with promising results in vitro (e.g., skin, cartilage, and muscle). The first animal studies are promising for small-scale defects. Vascularization issues are the main limitation to printing large constructs. Once the barrier of vascularization is overcome, printing organs and composite tissues of any size could be possible, opening the doors for personalized medicine based on medical imaging. Printing custom-made autologous grafts or flaps could minimize donor site morbidity and maximize the morphological results. Considering the potential future applications of bioprinting in the field of reconstructive surgery, one has to be aware of this tool, which could drastically change our practice.

[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.