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Aim: Programmed cell death-ligand 1 (PD-L1) predicts response to immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) patients. Most NSCLCs are diagnosed at an advanced stage and using minimally invasive diagnostic procedures that yield small biopsies or cytological samples. Methods: Cytological smears and paired histological samples from 52 advanced NSCLC patients were tested for PD-L1 expression by immunocyto/histochemistry (ICC/IHC) and for PD-L1 gene status by FISH. Results: PD-L1 was overexpressed in 9/52 (17%) cytological samples and in seven (13.5%) matched biopsies. The concordance between immunocytochemistry and IHC was 92.3% (48/52; p

Background: With age, aortic valve cusps undergo varying degrees of sclerosis which, sometimes, can progress to calcific aortic valve stenosis (AVS). To perform a retrospective clinico-pathologic investigation in patients with calcific AVS.Methods: We characterized and graded the structural remodeling in 236 aortic valves (200 tricuspid and 36 bicuspid) from patients with calcific AVS (148 males; average 72 years); possible relationships between general/clinical/echocardiographic characteristics and the histopathologic changes were explored. Twenty autopsy aortic valves served as controls. In 40 cases, we also tested the immunohistochemical expression of metalloproteinases and cytokines, and characterized the inflammatory infiltrate. In 5 cases, we cultured cusp stem cells and explored their potential to differentiate into osteoblasts/adipocytes.Results: AVS cusps showed structural remodeling as severe fibrosis (100%), calcific nodules (100%), neoangiogenesis (81%), inflammation (71%), bone metaplasia with or without hematopoiesis (6% and 53%, respectively), adipose metaplasia (16%), and cartilaginous metaplasia (7%). At multivariate analysis, AVS degree and interventricular septum thickness were the only predictors of remodeling (barring inflammation). All the tested metalloproteinases (except MMP-13) and cytokines were expressed in AVS cusps. Inflammation mainly consisted of B and T lymphocytes (CD4+/CD8+ cell ratio 3:1) and plasma cells. AVS changes were mostly different from typical atherosclerosis. Cultured mesenchymal cusp stem cells could differentiate into osteoblasts/adipocytes.Conclusions: Structural remodeling in AVS is peculiar and considerable, and is related to the severity of the disease. However, the different newly formed tissues-where "valvular interstitial cells" play a key role-and their wellknown slow turnover suggest a reverse structural remodeling improbable. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Objective Chronic periaortitis (CP) is a rare disease characterized by periaortic and periiliac fibroinflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. This study was undertaken to explore the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. Methods We studied 44 patients with newly diagnosed CP and 30 healthy controls. Circulating fibrocytes were identified as Col1+CD45+ cells using flow cytometry. Retroperitoneal tissue biopsy samples from 9 CP patients were stained with anti-type I procollagen, anti-CXCR4, and anti-CD45 antibodies and analyzed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We also characterized T helper polarization in biopsy samples from CP patients and measured serum levels of a panel of cytokines that are hallmarks of T helper responses and capable of influencing fibrocyte differentiation. Results The frequency of circulating Col1+CD45+ fibrocytes was higher in patients than in controls (P = 0.0371). CD45+proCol1+ and CXCR4+proCol1+ cells were detected in all examined biopsy samples from CP patients. Serum levels of CXCL12 were also higher in CP patients than controls (P = 0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2 cytokines (e.g., interleukin-13 [IL-13] and IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2 polarization; Th2-skewed responses are known to promote fibrocyte differentiation. Conclusion Our findings indicate that fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions. The accumulation of fibrocytes in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.

OBJECTIVES: Lymphangiogenesis plays a critical role in the immune response, tumour progression and therapy effectiveness. The aim of this study was to determine whether the interplay between the lymphatic and the blood microvasculature, tumour-infiltrating lymphocytes and the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint constitutes an immune microenvironment affecting the clinical outcome of patients with non-small-cell lung cancer. METHODS: Samples from 50 squamous cell carcinomas and 42 adenocarcinomas were subjected to immunofluorescence to detect blood and lymphatic vessels. CD3pos, CD8pos and PD-1pos tumour-infiltrating lymphocytes and tumour PD-L1 expression were assessed by immunohistochemical analysis. RESULTS: Quantification of vascular structures documented a peak of lymphatics at the invasive margin together with a decreasing gradient of blood and lymphatic vessels from the peritumour area throughout the neoplastic core. Nodal involvement and pathological stage were strongly associated with vascularization, and an increased density of vessels was detected in samples with a higher incidence of tumour-infiltrating lymphocytes and a lower expression of PD-L1. Patients with a high PD-L1 to PD-1 ratio and vascular rarefaction had a gain of 10 months in overall survival compared to those with a low ratio and prominent vascularity. CONCLUSIONS: Microvessels are an essential component of the cancer immune microenvironment. The clinical impact of the PD-1/PD-L1-based immune contexture may be implemented by the assessment of microvascular density to potentially identify patients with non-small-cell lung cancer who could benefit from immunotherapy and antiangiogenic treatment.

Immunologically based clinical trials performed thus far have failed to cure type 1 diabetes (T1D), in part because these approaches were nonspecific. Because the disease is driven by autoreactive CD4 T cells, which destroy β cells, transplantation of hematopoietic stem and progenitor cells (HSPCs) has been recently offered as a therapy for T1D. Our transcriptomic profiling of HSPCs revealed that these cells are deficient in programmed death ligand 1 (PD-L1), an important immune checkpoint, in the T1D nonobese diabetic (NOD) mouse model. Notably, the immunoregulatory molecule PD-L1 plays a determinant role in controlling/inhibiting activated T cells and thus maintains immune tolerance. Furthermore, our genome-wide and bioinformatic analysis revealed the existence of a network of microRNAs (miRNAs) controlling PD-L1 expression, and silencing one of key altered miRNAs restored PD-L1 expression in HSPCs. We therefore sought to determine whether restoration of this defect would cure T1D as an alternative to immunosuppression. Genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited the autoimmune response in vitro, reverted diabetes in newly hyperglycemic NOD mice in vivo, and homed to the pancreas of hyperglycemic NOD mice. The PD-L1 expression defect was confirmed in human HSPCs in T1D patients as well, and pharmacologically modulated human HSPCs also inhibited the autoimmune response in vitro. Targeting a specific immune checkpoint defect in HSPCs thus may contribute to establishing a cure for T1D.

DNA content was determined by flow cytometry in a series of 51 paired fresh tissue samples of primary colorectal carcinomas and the respective non-neoplastic adjacent mucosa in order to assess the relationship between DNA ploidy and the most commonly used prognostic factors. Aneuploidy was observed in 70.6% of the tumors and more than one aneuploid peak was present in 3.9%. Aneuploid tumor frequency was higher in left (93.3%) and right colon (64.7%) cancers than in rectal carcinomas (60.0%), and multiple aneuploid clones were detected more frequently in men than in women and in patients with advanced disease (Dukes stage D). Non-neoplastic mucosa adjacent to aneuploid tumors showed aneuploidy in 4 out of 51 samples (7.8%). The mucosa adjacent to diploid cancers had only diploid characteristics. Polidy did not correlate with histological abnormalities. These findings suggest that DNA content as determined by flow cytometry needs further study with adequate follow-up to evaluate possible correlations with relapse-free and overall survival. Furthermore the aneuploidy of non-neoplastic mucosa provides evidence for a field defect in mucosa adjacent to colorectal cancer and supports the concept that this alteration may be of influence on carcinogenesis.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. All the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for all the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 espression and nodal status and ERICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.

Two hundred and fifty bone marrow and 140 lymph nodal biopsies were analyzed immunocytochemically, using a mouse monoclonal antibody b-12 (MAb b-12), which reacts with MCA (mucinous-like carcinoma-associated antigen). The presence of MCA in bone marrow specimens was demonstrated in 102 out of 105 (97.1%) breast cancer metastases, 5 out of 8 (62.5%) gastric cancers, 5 out of 6 (83.3%) colon cancers, 3 out of 5 (60%) prostate cancers, 11 out of 26 (42.3%) lung cancers and 25 out of 30 (83.3%) unknown primary cancers, while no positivity to anti-MCA antibody was found in 30 cases of normal bone marrow biopsies, 5 cases of non epithelial malignancies and 30 cases of hemolymphoproliferative disease. Normal lymph nodes and non-epithelial lymph node metastases did not show any reaction to MAb b-12; on the contrary MCA positive staining was observed in 75 out of 75 (100%) lymph nodal metastases in breast cancer. These results suggest that application of MAb b-12 in immunohistochemistry is valid for the detection of bone marrow and lymph nodal micrometastases of epithelial origin.

CEA and TPA were studied in sera and in histologic specimens of 200 patients with benign (77) or malignant (123) breast pathology. The frequency and expression of the two markers was different in benign and in cancer tissues. Histologic positivity and high levels of circulating markers were observed more frequently in cancer patients than in patients with benign disease. Tissue positivity for the two tumor markers did not always correlate with elevated levels of circulating markers. Positive CEA and TPA incidence was higher in tissue samples than in serum samples. In the breast cancer group, among 33 patients with histologic positivity for CEA, only 5 cases had circulating CEA levels higher than 5 ng/ml; among 91 patients with histologic positivity for TPA, only 45 cases showed circulating levels for TPA higher than 95 U/l. These findings confirm that tumor size, secretory characteristics and vascular supply are factors affecting the achievement of high circulating marker levels. Combined marker measurement in serum and tissues can provide more information about the presence of a given tumor marker. A limited evaluation of the prognostic meaning of the study of combined CEA and TPA in sera and in tissues was carried out during the follow-up of 60 evaluable patients. Only 5 patients had cancer relapses in the first 12 months from surgery; in 2 of 5 patients TPA was positive initially and at the time of recurrence, in serum as well as in tissues. Circulating CEA gave negative findings in all relapsed patients; 2 of them showed weak positivity only in the histologic staining at the time of presentation.