Periklis G. Foukas

High risk HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa.

Studies on the involvement of the human papillomavirus (HPV) in initiation and progression of oral neoplasia have generated conflicting results. The observed discrepancy is attributable mainly to the varying sensitivity of the applied methodologies and to epidemiologic factors of the examined patient groups. To evaluate the role of HPV in oral carcinogenesis, we analyzed 53 potentially neoplastic and neoplastic oral lesions consisting of 29 cases of hyperplasia, 5 cases of dysplasia, and 19 cases of squamous cell carcinomas, as well as 16 oral specimens derived from healthy individuals. A highly sensitive nested polymerase chain reaction (PCR) assay was used, along with type-specific PCR, restriction fragment length polymorphism analysis, dot blotting, and nonisotopic in situ hybridization. Nested PCR revealed the presence of HPV DNA in 48 of the 53 (91%) pathologic samples analyzed, whereas none (0%) of the normal specimens was found to be infected. Positivity for HPV was independent of histology and the smoking habits of the analyzed group of patients. At least one “high risk” type, such as HPV 16, 18, and 33, was detected by type-specific PCR in 47 (98%) infected specimens, whereas only 1 (2%) squamous cell carcinoma was solely infected by a “low risk” type (HPV 6). HPV 16 was the prevailing viral type, being present in 71% of infected cases. Single HPV 16 and HPV 18 infections were confirmed by restriction fragment length polymorphism. HPV 58 was detected by dot blotting in three hyperplastic lesions. HPV positivity and genotyping were further confirmed, and the physical status of this virus was evaluated by nonisotopic in situ hybridization. Diffuse and punctate signals, indicative of the episomal and integrative pattern of HPV infection, were observed for low- and high-risk types, respectively. Our findings are suggestive of an early involvement of high-risk HPV types in oral carcinogenesis.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Significance mAbs directed to inhibitory immune receptors represent a very promising class of immunotherapeutics. This study suggests a potential mechanism of action of ipilimumab (a fully human anti–cytotoxic T lymphocyte–associated antigen 4), by which FcγRIIIA (CD16)-expressing nonclassical monocytes kill regulatory T cells ex vivo via antibody-dependent cell-mediated cytotoxicity. Notably, at baseline, responder patients display significantly higher peripheral frequencies of nonclassical monocytes and a selective enrichment in tumor-infiltrating CD68+CD16+ macrophages compared with nonresponder patients. If further confirmed, these findings may contribute to the generation of predictive biomarker panels, antibody design, and the development of rational combination therapies to promote antitumor immunity. Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4–specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14++CD16− monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68+/CD163+ macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti–CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.

The Regulation of Liver Cell Survival by Complement

Complement effectors are known to contribute to host cell injury in several inflammatory diseases. Contrary to this paradigm, in this study utilizing surgical liver resection (partial hepatectomy) in various complement-deficient mice as a model, we have demonstrated that complement anaphylatoxins C3a and C5a are required for the survival of liver cells during regeneration. The mechanisms of these cytoprotective functions of complement were related to the regulation of IL-6 and TNF production or release after liver resection. Disturbances in the cytokine milieu, induced by a loss of complement activity, were found to alter prosurvival signaling, including the IL-6/STAT3 and PI3K/Akt/mammalian target of rapamycin pathways. In conclusion, this study documents functions of complement proteins as prosurvival factors that, through their interactions with cytokines, inhibit apoptotic signaling in proliferating cells of epithelial origin.

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

BackgroundRecent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors.Material and MethodsA total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods.ResultsAberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this “full abnormal” phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the “normal” phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the “normal” and “full abnormal” phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055).ConclusionsOur findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.

Expression of HLA-DR, costimulatory molecules B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1) and Fas ligand (FasL) on gastric epithelial cells in Helicobacter pylori gastritis; influence of H. pylori eradication

There is evidence that Helicobacter pylori infection up‐regulates the expression of HLA class II molecules by gastric epithelial cells (GEC). In this study we evaluated whether GEC are capable of expression of costimulatory molecules in H. pylori gastritis. The expression of FasL by GEC, before and after eradication of H. pylori, was also studied. Thirty patients (23 men) aged 27–81 years (53·67 ± 13·99 years (mean ± s.d.)) with dyspepsia were studied. Upper gastrointestinal endoscopy was performed and six biopsies were obtained (antrum, n= 3; corpus, n= 3) for Campylobacter‐Like Organisms (CLO) test and histology; 23 (16 men) were H. pylori+ and seven (all men) were H. pylori− by both methods and served as controls. Helicobacter pylori eradication therapy was given to H. pylori+ patients and all patients were re‐endoscoped after 116 ± 9 days. Formalin‐fixed paraffin‐embedded tissue sections were stained by the ABC immunoalkaline phosphatase method. In H. pylori gastritis HLA‐DR was expressed and correlated with disease activity (P < 0·01). No HLA‐DR was observed in controls. In H. pylori‐eradicated patients significant decrease of HLA‐DR was found (antrum, P < 0·001). ICAM‐1 was expressed by GEC in 80% of H. pylori+ patients; ICAM‐1 expression did not correlate with gastritis parameters and decreased significantly after eradication (antrum, P < 0·01). B7‐1 and B7‐2 were expressed on H. pylori+ samples and their expression decreased after eradication, albeit not significantly. Weak epithelial expression of both B7 molecules was observed in all the controls. FasL was steadily expressed by GEC in both H. pylori+ and H. pylori− patients and remained almost unchanged after eradication. These findings suggest that GEC may acquire antigen‐presenting cell properties in H. pylori infection through de novo expression of HLA‐DR and costimulatory molecules. This seems to be attenuated after eradication and resolution of mucosal inflammation. The same cells exhibit the capacity to control the inflammatory process, probably by inducing apoptotic cell death to Fas‐bearing infiltrating lymphocytes.

Human Papillomavirus (HPV) Detection Using In Situ Hybridization in Histologic Samples Correlations With Cytologic Changes and Polymerase Chain Reaction HPV Detection

Although in situ hybridization (ISH) and polymerase chain reaction (PCR) have extensively been used on cytology specimens, there have been limited reports of the usefulness of these techniques in relation to confirmed histologic findings. In this study, we used PCR and ISH to detect human papillomavirus (HPV) in cytologic and histologic specimens, respectively. By using positive and negative likelihood ratios, we attempted to identify any predictive role of ISH testing alone or in combination with PCR for the development of high-grade histologic lesions (cervical intraepithelial neoplasia [CIN] 2+). In our study, ISH was a useful method for detection of HPV, even in a large fraction of samples with normal cytologic or biopsy findings. We suggest that when used together and evaluated in conjunction with histologic sections, ISH is a useful tool for ancillary molecular testing of HPV infection in cervical lesions, especially in CIN 2+ histological lesions where its analytic sensitivities and specificities were as good as those of PCR testing.

γ-H2AX expression detected by immunohistochemistry correlates with prognosis in early operable non-small cell lung cancer

Background Phosphorylation of the H2AX histone is an early indicator of DNA double-strand breaks and of the resulting DNA damage response. In the present study, we assessed the expression and prognostic significance of γ-H2AX in a cohort of 96 patients with operable non-small cell lung carcinoma. Methods Ninety-six paraffin-embedded specimens of non-small cell lung cancer patients were examined. All patients underwent radical thoracic surgery of primary tumor (lobectomy or pneumonectomy) and regional lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Follow-up was available for all patients; mean duration of follow-up was 27.50 ± 14.07 months (range 0.2–57 months, median 24 months). Results Sixty-three patients (65.2%) died during the follow-up period. The mean survival time was 32.2 ± 1.9 months (95% confidence interval [CI]: 28.5–35.8 months; median 30.0 months); 1-, 2- and 3-year survival rates were 86.5% ± 3.5%, 57.3% ± 5.1%, and 37.1% ± 5.4%, respectively. Low γ-H2AX expression was associated with a significantly better survival as compared with those having high γ-H2AX expression (35.3 months for low γ-H2AX expression versus 23.2 months for high γ-H2AX expression, P = 0.009; hazard ratio [HR] 1.95, 95% CI: 1.15–3.30). Further investigation with multivariate Cox proportional hazards regression analysis revealed that high expression of γ-H2AX remained an independent prognostic factor of shorter overall survival (HR 2.15, 95% CI: 1.22–3.79, P = 0.026). A combined p53/γ-H2AX analysis was performed, and we found that the p53 low/γ-H2AX low phenotype was associated with significantly better survival compared with all other phenotypes. Conclusion Our study is the first to demonstrate that expression of γ-H2AX detected by immunohistochemistry may represent an independent prognostic indicator of overall survival in patients with non-small cell lung cancer. Further studies are needed to confirm our results.

Alterations in the Proliferating Compartment of Gastric Mucosa during Helicobacter Pylori Infection: The Putative Role of Epithelial Cells Expressing p27kip1

The proliferating zone contains stem cells that give rise to all epithelial cells of the gastric mucosa. In the present study, we investigated the turnover of gastric epithelial cells in the proliferating zone of Helicobacter pylori–infected mucosa, with or without intestinal metaplasia, before and after eradication of the microorganism. In addition, we studied the topographical distribution of the cyclin dependent kinase inhibitor p27Kip1, which plays a critical role in cell cycle progression and differentiation programs. Twenty-eight patients (22 male), aged 32–78 years and with dyspeptic symptoms, were endoscoped, and gastric biopsies were obtained from antrum and corpus for histopathological examination and the Campylobacter-like organisms test; eradication therapy was given to infected patients, and all patients were re-endoscoped after 105 ± 33 days (mean ± SD). The kinetics of gastric epithelial cells and p27Kip1 status was assessed by means of immunohistochemistry and TUNEL (Tdt-mediated dUTP-biotin nick end labeling) assay. Twenty-one (21) of 28 patients were H. pylori positive, and 7 were found H. pylori negative and served as controls. In antrum, intestinal metaplasia was detected in 7/21 (33.3%). In H. pylori gastritis, Ki67 expression was found increased in the proliferating zone, compared with normal (P = .03); analogous results were obtained with the other proliferation markers, namely retinoblastoma protein and topoisomerase IIα. An inverse relationship between proliferation index and atrophy was disclosed (P = .02). A reduction in the proliferation index was observed after eradication, albeit not significant. Apoptotic epithelial cells were found significantly increased (P < .01) in H. pylori gastritis, and a significant reduction was observed after eradication (P < .01). In addition, apoptotic index was found to correlate with H. pylori density. The topographical study of p27Kip1 revealed a p27kip1-positive epithelial cell population that resided deep in the proliferating zone; these cells were considered to be stem cells and were found significantly increased in areas with intestinal metaplasia (P < .05); in H. pylori gastritis, there was also an increase that did not reach statistical significance. H. pylori infection induces apoptosis and increases proliferation in the proliferating zone. The increased cellular turnover, together with the increased number of putative p27Kip1-positive stem cells in the context of intestinal metaplasia, provides further evidence for the role of H. pylori infection in gastric carcinogenesis.

Multiplexed tissue biomarker imaging

Multiplexed Tissue Biomarker Imaging The detection of structural and functional proteins in cells within the tumor microenvironment in tissue samples is achieved by immunolabeling with specific antibodies. These target proteins are visualized with the subsequent application of either an enzymatic

Complement Deficiency Promotes Cutaneous Wound Healing in Mice

Wound healing is a complex homeostatic response to injury that engages numerous cellular activities, processes, and cell-to-cell interactions. The complement system, an intricate network of proteins with important roles in immune surveillance and homeostasis, has been implicated in many physiological processes; however, its role in wound healing remains largely unexplored. In this study, we employ a murine model of excisional cutaneous wound healing and show that C3−/− mice exhibit accelerated early stages of wound healing. Reconstitution of C3−/− mice with serum from C3+/+ mice or purified human C3 abrogated the accelerated wound-healing phenotype. Wound histology of C3−/− mice revealed a reduction in inflammatory infiltrate compared with C3+/+ mice. C3 deficiency also resulted in increased accumulation of mast cells and advanced angiogenesis. We further show that mice deficient in the downstream complement effector C5 exhibit a similar wound-healing phenotype, which is recapitulated in C5aR1−/− mice, but not C3aR−/− or C5aR2−/− mice. Taken together, these data suggest that C5a signaling through C5aR may in part play a pivotal role in recruitment and activation of inflammatory cells to the wound environment, which in turn could delay the early stages of cutaneous wound healing. These findings also suggest a previously underappreciated role for complement in wound healing, and may have therapeutic implications for conditions of delayed wound healing.