Malignant pleural mesothelioma immune microenvironment and checkpoint expression: correlation with clinical-pathological features and intratumor heterogeneity over time.

Background: Tumor immune microenvironment (TME) plays a key role in malignant pleural mesothelioma (MPM) pathogenesis and treatment outcome, supporting a role of immune checkpoint inhibitors as anticancer approach. This study retrospectively investigated TME and programmed death ligand 1 (PD-L1) expression in naïve MPM cases and their change under chemotherapy. Patients and methods: Diagnostic biopsies of MPM patients were collected from four Italian and one Slovenian cancer centers. Pathological assessment of necrosis, inflammation, grading, and mitosis was carried out. Ki-67, PD-L1 expression, and tumor infiltrating lymphocytes were detected by immunohistochemistry. When available, the same paired sample after chemotherapy was analyzed. Pathological features and clinical characteristics were correlated to overall survival. Results: TME and PD-L1 expression were assessed in 93 and 65 chemonaive MPM samples, respectively. Twenty-eight samples have not sufficient tumor tissue for PD-L1 expression. Sarcomatoid/biphasic samples were characterized by higher CD8+ T lymphocytes and PD-L1 expression on tumor cells, while epithelioid showed higher peritumoral CD4+ T and CD20+ B lymphocytes. Higher CD8+ T lymphocytes, CD68+ macrophages, and PD-L1 expression were associated with pathological features of aggressiveness (necrosis, grading, Ki-67). MPM cases characterized by higher CD8+ T-infiltrate showed lower response to chemotherapy and worse survival at univariate analysis. Patients stratification according to a combined score including CD8+ T lymphocytes, necrosis, mitosis, and proliferation index showed median overall survival of 11.3 months compared with 16.4 months in cases with high versus low combined score (P < 0.003). Subgroup exploratory analysis of 15 paired samples before and after chemotherapy showed a significant increase in cytotoxic T lymphocytes in MPM samples and PD-L1 expression in immune cells. Conclusions: TME enriched with cytotoxic T lymphocytes is associated with higher levels of macrophages and PD-L1 expression on tumor cells and with aggressive histopathological features, lower response to chemotherapy and shorter survival. The role of chemotherapy as a tumor immunogenicity inducer should be confirmed in a larger validation set.

Endocrine Pancreas in Cats With Diabetes Mellitus.

Pancreatic amyloidosis and loss of α and ß cells have been shown to occur in cats with diabetes mellitus, although the number of studies currently available is very limited. Furthermore, it is not known whether pancreatic islet inflammation is a common feature. The aims of the present study were to characterize islet lesions and to investigate whether diabetic cats have inflammation of the pancreatic islets. Samples of pancreas were collected postmortem from 37 diabetic and 20 control cats matched for age, sex, breed, and body weight. Histologic sections were stained with hematoxylin and eosin and Congo red; double labeled for insulin/CD3, insulin/CD20, insulin/myeloperoxidase, insulin/proliferating cell nuclear antigen, and glucagon/Ki67; and single labeled for amylin and Iba1. Mean insulin-positive cross-sectional area was approximately 65% lower in diabetic than control cats (P = .009), while that of amylin and glucagon was similar. Surprisingly, amyloid deposition was similar between groups (P = .408). Proliferation of insulin- and glucagon-positive cells and the number of neutrophils, macrophages, and T (CD3) and B (CD20) lymphocytes in the islets did not differ. The presence of T and B lymphocytes combined tended to be more frequent in diabetic cats (n = 8 of 37; 21.6%) than control cats (n = 1 of 20; 5.0%). The results confirm previous observations that loss of ß cells but not α cells occurs in diabetic cats. Islet amyloidosis was present in diabetic cats but was not greater than in controls. A subset of diabetic cats had lymphocytic infiltration of the islets, which might be associated with ß-cell loss.

Exocrine Pancreas in Cats With Diabetes Mellitus.

Pancreatitis has been described in cats with diabetes mellitus, although the number of studies currently available is very limited. In addition, ketoacidosis has been hypothesized to be associated with pancreatitis in diabetic cats. The aims of the present study were to investigate whether diabetic cats have pancreatitis and to determine if pancreatitis is more frequent with ketoacidosis. Samples of pancreas were collected postmortem from 37 diabetic cats, including 15 with ketoacidosis, and 20 control cats matched for age, sex, breed, and body weight. Sections were stained with hematoxylin and eosin, double-labeled for insulin/CD3, insulin/CD20, insulin/myeloperoxidase, insulin/PCNA, and glucagon/Ki67, and single-labeled for Iba1. A previously proposed semiquantitative score was used to characterize pancreatitis, along with counts of inflammatory cells. Scores of pancreatitis and the number of neutrophils, macrophages, and lymphocytes in the exocrine pancreas did not differ between diabetic and control cats or between diabetic cats with and without ketoacidosis. Of note, PCNA-positive acinar cells were increased (P = .002) in diabetic cats, particularly near islets (P < .001). Ki67-positive acinar cells were increased only near islets (P = .038). Ketoacidosis was not linked to proliferation. The results suggest that histopathologic evidence of pancreatitis may not be more frequent in diabetic cats and that ketoacidosis may not be associated with it at the time of death. Augmented PCNA-positive acinar cells might indicate increased proliferation due to chronic pancreatitis. The reason behind the prevalent proliferation of acinar cells surrounding pancreatic islets deserves further investigation.

Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro.

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

Inhaled mucoactive drugs for treating non-cystic fibrosis bronchiectasis in children.

Non-cystic fibrosis bronchiectasis (nCFb) is an acquired condition of variable etiology. Medical treatment basically involves antibiotics and chest physiotherapy. An impaired mucociliary clearance seems to be one of the mechanisms behind nCFb, and inhaled therapy with mucoactive agents has frequently been used to try to correct it. The most often used mucoactive agents in this setting are N-acetylcysteine, hypertonic saline solution (HS), mannitol powder and recombinant human DNase (rhDNase). Reviewing the international medical literature on the use of these drugs for patients with nCFb from 1992 to the present day, we retrieved 88 articles, only 12 of which met our selection criteria for this analysis. We found only 2 papers and 2 reviews on the use of rhDNase in children, and in adults 3 trials on HS, 5 on mannitol powder and 2 on rhDNase. In conclusion, no observational or randomized controlled trials (RCT) have been published on the use of these drugs in children with nCFb, while the few conducted on adult patients report some evidence of their effects. Further studies are needed on inhaled mucoactive drugs for the treatment of children with nCFb.

Acute cellular rejection and Epstein-Barr virus-related post-transplant lymphoproliferative disorder in a pediatric lung transplant with low viral load.

We report the case of an 18-year-old male who underwent bilateral lung transplantation for end-stage cystic fibrosis. No Epstein-Barr virus (EBV) or cytomegalovirus serology mismatch was detected on pre-transplant evaluation (donor and recipient were both positive). Two months after lung transplantation a computed tomography scan showed multiple nodules throughout both lungs. At that time a low EBV DNA blood level was detected (<300 copies/100,000 lymphomonocytes). Scheduled follow-up transbronchial biopsy (TBB) revealed a prevalent finding characterized by perivascular lymphoid infiltrates with endothelitis. Extensive tissue coagulative necrosis with peripheral areas of dense aggregates of larger lymphoid cells were detected in the trans-thoracic fine needle core biopsy (FNCB) performed on the largest nodule. The immunophenotypic profile characterized the perivascular lymphoid cells in TBB as mainly composed of T lymphocytes (CD3 positive) while the larger number of lymphocytes in FNCB as B cells (CD20 positive). In situ hybridization for EBV (EBER mRNA) was negative in TBB while it was positive in many lymphocytes of the FNCB. Real-time polymerase chain reaction (PCR) for EBV was performed on paraffin-embedded FNCB and detected a high quantity of EBV genomes (1260 copies/cell). IgH gene rearrangement using a fragment size PCR technique revealed a monoclonal B-cell population in FNCB. Morphological and molecular findings suggest a final diagnosis of acute cellular rejection and a post-transplant lymphoproliferative disorder (PTLD) EBV-related in a lung transplant recipient with a low EBV DNA blood level. A possible coexistence of PTLD and acute rejection should be considered both for diagnosis and treatment. EBV PCR in the peripheral blood is a useful screening tool in transplant recipients; however, rare cases with PTLD may not have detectable levels of EBV DNA. This aspect should be taken into consideration to avoid false negatives.

Overexpression of squamous cell carcinoma antigen in idiopathic pulmonary fibrosis: clinicopathological correlations.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disorder with a poor prognosis. Epithelial instability is a crucial step in the development and progression of the disease, including neoplastic transformation. Few tissue markers for epithelial instability have been reported in IPF. Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor typically expressed by dysplastic and neoplastic cells of epithelial origin, more often in squamous cell tumours. At present, no information is available on its expression in IPF. METHODS: SCCA and transforming growth factor beta (TGFbeta) expression in surgical lung biopsies from 22 patients with IPF and 20 control cases was examined. An in vitro study using A549 pneumocytes was also conducted to investigate the relationship between SCCA and TGFbeta expression. SCCA and TGFbeta epithelial expression was evaluated by immunohistochemistry and reverse transcription-PCR (RT-PCR). SCCA values were correlated with different pathological and clinical parameters. Time course analysis of TGFbeta expression in A549 pneumocytes incubated with different SCCA concentrations was assessed by real time RT-PCR. RESULTS: SCCA was expressed in many metaplastic alveolar epithelial cells in all IPF cases with a mean value of 24.9% while it was seen in only two control patients in up to 5% of metaplastic cells. In patients with IPF, SCCA correlated positively with extension of fibroblastic foci (r = 0.49, p = 0.02), expression of TGFbeta (r = 0.78, p<0.0001) and with carbon monoxide transfer factor decline after 9 months of follow-up (r = 0.59, p = 0.01). In vitro experiments showed that incubation of cultured cells with SCCA induced TGFbeta expression, with a peak at 24 h. CONCLUSION: Our findings provide for the first time a potential mechanism by which SCCA secreted from metaplastic epithelial cells may exert a profibrotic effect in IPF. SCCA could be an important biomarker in this incurable disease.

What's new in mesothelioma.

Malignant pleural mesothelioma is a neoplasm characterized by a very poor prognosis and medico-legal implications. Diagnosis, prognosis and therapy are often challenging and include several issues. Cytological diagnosis is frequently the first step of the diagnostic process, and although its sensitivity may be somewhat lower, diagnostic criteria should be taken into account. When effusion cytology is inconclusive for the diagnosis, tissue biopsies should be taken. Even if the morphologic criteria for deciding whether a mesothelial proliferation is a benign or a malignant process have been defined, the separation of benign from malignant mesothelial proliferation is often a difficult problem for the pathologist, particularly on small biopsies. Thirdly, when the diagnosis is made, despite many efforts have been made to identify possible new biomarkers for early diagnosis, prognostic stratification and also predictive tools should be defined. Nowadays, the main prognostic parameter is still represented by the histological subtype, having the epithelioid MPM a better outcome than the sarcomatoid or biphasic MPM. A nuclear grading system have been also proposed to stratify patient outcome. Reliable predictive biomarkers are still lacking in MPM and a personalized therapeutic concept is eagerly needed. Mesothelioma occurs mostly as sporadic cancer and the main risk factor is asbestos exposure, but it also occurs among blood relatives suggesting possible increased genetic susceptibility besides shared exposures. Recently the study of genetic predisposition syndrome raised new aspect in the occurrence of mesothelioma cases.This review summarize these most important issues.

Cryopreservation of domestic cat (Felis catus) ovarian tissue: Comparison of two vitrification methods.

We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3-4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue.

Overexpression of Hypoxia-Inducible Factor-1α in Primary Graft Dysfunction Developing in an Orthotopic Lung Transplantation Rat Model.

BACKGROUND: Primary graft dysfunction (PGD) is the major cause of early morbidity and mortality after transplantation. A high rate of PGD is a frequent complication in orthotopic lung transplantation (OLT) models, which are currently used to investigate acute and chronic rejection pathways. Hypoxia-inducible factor (HIF)-1α is a heterodimeric αß transcription factor that mediates tissue response to hypoxia. In other solid organ transplantations, a significant correlation between HIF-1α expression and PGD was detected. To our knowledge no data are available on HIF-1α expression in PGD developing in lung transplantation. The aims of this study were to investigate HIF-1α expression (using immunohistochemistry) and correlate it to the main histological parameters related to ischemia-reperfusion (IR) injury, including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) -positive apoptotic cells). METHODS: OLT was performed in 32 inbred rat strains and 11 of them died in the early postoperative period (from day 0-3) for IR injury. The histological and molecular evaluations were done in all lung tissues. Unimplanted donor rat lungs were used as controls. HIF-1α expression was correlated with all morphological parameters. RESULTS: Lung samples of animals with IR injury showed high scores of HIF-1α expression, edema, blood extravasation, granulocyte margination, apoptotic index, and necrosis in 91% of cases. Tissue overexpression of HIF-1α was detected in all lung samples with high scores of histological parameters and with high apoptotic indexes. CONCLUSION: Our data demonstrate that HIF-1α was overexpressed in more severe rat lung IR injury. The use of HIF-1α inhibitors could provide a translatable route into manipulating this complex system in vivo.