Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.

BACKGROUND & AIMS: The antiviral function of peripheral hepatitis B virus (HBV)-specific T cells can be increased in patients with chronic hepatitis B by blocking the interaction of programmed death (PD)-1 with its ligand PD-L1. However, no information is available about the effects of this blockade on intrahepatic lymphocytes. We studied T-cell exhaustion and the effects of PD-1/PD-L1 blockade on intrahepatic and circulating HBV-specific T cells in patients with chronic hepatitis B. METHODS: A total of 42 patients with chronic HBV infection who underwent liver biopsy were studied. The ex vivo phenotype of peripheral and intrahepatic HBV-specific CD8(+) T cells was assessed by flow cytometry with class I tetramers and antibodies to T-cell differentiation molecules. Functional recovery was evaluated by analyzing expansion and production of interferon (IFN)-gamma and interleukin (IL)-2 after short-term incubation of T cells with HBV peptides in the presence of anti-PD-L1 or control antibodies. RESULTS: Intrahepatic HBV-specific CD8(+) cells expressed higher levels of PD-1 and lower levels of CD127 than their peripheral counterparts. Blockade of PD-1/PD-L1 interaction increased CD8(+) cell proliferation and IFN-gamma and IL-2 production by circulating intrahepatic lymphocytes, even though anti-PD-L1 had a stronger effect on intrahepatic compared with peripheral T cells. CONCLUSIONS: T-cell exhaustion by high antigen concentrations promotes HBV-specific T-cell dysfunction by affecting phenotype and function of peripheral and intrahepatic T cells. By restoring antiviral T-cell functions, not only in peripheral but also in intrahepatic lymphocytes, anti-PD-L1 might be a good therapeutic candidate for chronic HBV infection.

Extragenital Müllerian adenosarcoma with pouch of Douglas location.

BACKGROUND: Of all female genital tract tumors, 1-3% are stromal malignancies. In 8-10% of cases, these are represented by Müllerian adenosarcoma an extremely rare tumor characterized by a stromal component of usually low-grade malignancy and by a benign glandular epithelial component. Variant that arises in the pouch of Douglas is scarcely mentioned in the medical literature. CASE PRESENTATION: A 49-year-old para-0 woman, was seen at our OB/GYN-UNIT because she complained vaguely of pelvic pain. She had a mass of undefined nature in the pouch of Douglas. A simple excision of the mass showed low-grade Müllerian adenosarcoma with areas of stromal overgrowth. One and a half year after surgery, at another hospital, a mass was detected in the patient's posterior vaginal fornix and removed surgically. Six months later she came back to our observation with vaginal bleeding and mass in the vaginal fornix. We performed radical surgery. The pathological examination showed recurrent adenosarcoma. Surgical treatment was supplemented by radiation therapy. CONCLUSIONS: The case of Müllerian adenosarcoma reported here is the third known so far in the literature that was located in the pouch of Douglas. To date, only two other such cases have been reported, including one resulting from neoplastic degeneration of an endometriotic cyst.

Histopathological grading affects survival in patients with IDH-mutant grade II and grade III diffuse gliomas.

BACKGROUND: Diffuse grade II and grade III gliomas are actually classified in accordance with the presence of isocitrate dehydrogenase mutation (IDH-mut) and the deletion of both 1p and 19q chromosome arms (1p/19q codel). The role of tumour grading as independent prognostic factor in these group of tumours remains matter of debate. The aim of this study was to determine if grade is an independent prognostic factor and not somehow associated to IDH mutation and 1p/19q status of the tumour. METHODS: We analysed 399 consecutive patients with newly diagnosed, histologically proven World Health Organisation (WHO) 2016 grade II or grade III IDH-mut gliomas, assessed by polymerase chain reaction, immunohistochemistry or next-generation sequencing (NGS). RESULTS: The analysis included 399 patients with grade II (n = 250, 62.7%) or grade III (n = 149, 37.3%) diffuse gliomas. Median follow-up time was 105.3 months. Median survival was 148.1 months. In multivariate analysis, grade II (hazard ratio [HR] = 0.342, 95% confidence interval [CI]: 0.221-0.531; P < 0.001) and 1p/19q codeletion (HR = 0.440, 95% CI: 0.290-0.668; P < 0.001) were independently associated with a lower risk for death. The difference in survival remained significant (p = 0.006 in astrocytomas, p = 0.014 in oligodendrogliomas) when adjusted for histological subtype. Residual disease after surgery (or biopsy) negatively affected survival (HR: 2.151, 95% CI: 1.375-3.367, P = 0.001). Post-surgical treatment with radiotherapy + adjuvant chemotherapy improved survival compared with follow-up and other treatments (HR: 0.316, 95% CI: 0.156-0.641, P = 0.001). CONCLUSIONS: In our study, histopathological grade still affects survival in IDH-mutant WHO grade II and III diffuse gliomas. This effect appears to be independent from molecular features, extension of surgical resection and post-surgical treatments. Therefore, physicians should continue to take into account tumour grade, along their molecular characteristics, for a better clinical and therapeutic management of the patients.

Metformin prevents liver tumourigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma.

Metformin is a hypoglycaemic agent used to treat type 2 diabetes mellitus (DM2) patients, with a broad safety profile. Since previous epidemiological studies had shown that the incidence of hepatocellular carcinoma (HCC) decreased significantly in metformin treated DM2 patients, we hypothesised that intervention with metformin could reduce the risk of neoplastic transformation of hepatocytes. HCC is the most common primary liver malignancy and it generally originates in a background of liver fibrosis and cirrhosis. In the present study, we took advantage of a transgenic mouse (TG221) characterized by microRNA-221 overexpression, with cirrhotic liver background induced by chronic administration of carbon tetrachloride (CCl4). This mouse model develops fibrosis, cirrhosis and liver tumours that become visible in 100% of mice at 5-6 months of age. Our results demonstrated that metformin intervention improves liver function, inhibits hepatic stellate cell (HSC) activation, reduces liver fibrosis, depletes lipid accumulation in hepatocytes, halts progression to decompensated cirrhosis and abrogates development HCC in CCl4 challenged transgenic mouse model. The study establishes the rationale for investigating metformin in cirrhotic patients regardless of concomitant DM2 status.

Immune context characterization and heterogeneity in primary tumors and pulmonary metastases from renal cell carcinoma.

AIM: The knowledge of the immune context of renal cell carcinoma (RCC) is useful to predict benefit from immunotherapy. We retrospectively characterized the immune context of RCC patients underwent primary nephrectomy and pulmonary metastasectomy. MATERIALS & METHODS: Intratumoral infiltrating lymphocytes and peritumoral renal infiltrating lymphocytes, lymphocyte subpopulations (CD4+, CD8+), PD-1, PD-L1 were explored in paired samples of primary RCC (T) and respective pulmonary metastases (M). RESULTS: The immune variables demonstrated intralesional and intratumoral heterogeneity. Intralesional lymphocyte heterogeneity reached 76% of cases in T, 28% in M. The heterogeneity rate for PD-L1 expression was from 44% (T) to 56% (M); it correlated with better survival. CONCLUSION: The immune context of RCC is highly variable both within a given tumor and among primary and metastases.

Prognostic modeling of oral cancer by gene profiles and clinicopathological co-variables.

Accurate staging and outcome prediction is a major problem in clinical management of oral cancer patients, hampering high precision treatment and adjuvant therapy planning. Here, we have built and validated multivariable models that integrate gene signatures with clinical and pathological variables to improve staging and survival prediction of patients with oral squamous cell carcinoma (OSCC). Gene expression profiles from 249 human papillomavirus (HPV)-negative OSCCs were explored to identify a 22-gene lymph node metastasis signature (LNMsig) and a 40-gene overall survival signature (OSsig). To facilitate future clinical implementation and increase performance, these signatures were transferred to quantitative polymerase chain reaction (qPCR) assays and validated in an independent cohort of 125 HPV-negative tumors. When applied in the clinically relevant subgroup of early-stage (cT1-2N0) OSCC, the LNMsig could prevent overtreatment in two-third of the patients. Additionally, the integration of RT-qPCR gene signatures with clinical and pathological variables provided accurate prognostic models for oral cancer, strongly outperforming TNM. Finally, the OSsig gene signature identified a subpopulation of patients, currently considered at low-risk for disease-related survival, who showed an unexpected poor prognosis. These well-validated models will assist in personalizing primary treatment with respect to neck dissection and adjuvant therapies.

Sentinel Node in Oral Cancer: The Nuclear Medicine Aspects. A Survey from the Sentinel European Node Trial.

PURPOSE: Nuclear imaging plays a crucial role in lymphatic mapping of oral cancer. This evaluation represents a subanalysis of the original multicenter SENT trial data set, involving 434 patients with T1-T2, N0, and M0 oral squamous cell carcinoma. The impact of acquisition techniques, tracer injection timing relative to surgery, and causes of false-negative rate were assessed. METHODS: Three to 24 hours before surgery, all patients received a dose of Tc-nanocolloid (10-175 MBq), followed by lymphoscintigraphy. According to institutional protocols, all patients underwent preoperative dynamic/static scan and/or SPECT/CT. RESULTS: Lymphoscintigraphy identified 723 lymphatic basins. 1398 sentinel lymph nodes (SNs) were biopsied (3.2 SN per patient; range, 1-10). Dynamic scan allowed the differentiation of sentinel nodes from second tier lymph nodes. SPECT/CT allowed more accurate anatomical localization and estimated SN depth more efficiently. After pathological examination, 9.9% of the SN excised (138 of 1398 SNs) showed metastases. The first neck level (NL) containing SN+ was NL I in 28.6%, NL IIa in 44.8%, NL IIb in 2.8%, NL III in 17.1%, and NL IV in 6.7% of positive patients. Approximately 96% of positive SNs were localized in the first and second lymphatic basin visualized using lymphoscintigraphy. After neck dissection, the SN+ was the only lymph node containing metastasis in approximately 80% of patients. CONCLUSIONS: Best results were observed using a dynamic scan in combination with SPECT/CT. A shorter interval between tracer injection, imaging, and surgery resulted in a lower false-negative rate. At least 2 NLs have to be harvested, as this may increase the detection of lymphatic metastases.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells.

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.

Sentinel European Node Trial (SENT): 3-year results of sentinel node biopsy in oral cancer.

PURPOSE: Optimum management of the N0 neck is unresolved in oral cancer. Sentinel node biopsy (SNB) can reliably detect microscopic lymph node metastasis. The object of this study was to establish whether the technique was both reliable in staging the N0 neck and a safe oncological procedure in patients with early-stage oral squamous cell carcinoma. METHODS: An European Organisation for Research and Treatment of Cancer-approved prospective, observational study commenced in 2005. Fourteen European centres recruited 415 patients with radiologically staged T1-T2N0 squamous cell carcinoma. SNB was undertaken with an average of 3.2 nodes removed per patient. Patients were excluded if the sentinel node (SN) could not be identified. A positive SN led to a neck dissection within 3 weeks. Analysis was performed at 3-year follow-up. RESULTS: An SN was found in 99.5% of cases. Positive SNs were found in 23% (94 in 415). A false-negative result occurred in 14% (15 in 109) of patients, of whom eight were subsequently rescued by salvage therapy. Recurrence after a positive SNB and subsequent neck dissection occurred in 22 patients, of which 16 (73%) were in the neck and just six patients were rescued. Only minor complications (3%) were reported following SNB. Disease-specific survival was 94%. The sensitivity of SNB was 86% and the negative predictive value 95%. CONCLUSION: These data show that SNB is a reliable and safe oncological technique for staging the clinically N0 neck in patients with T1 and T2 oral cancer. EORTC Protocol 24021: Sentinel Node Biopsy in the Management of Oral and Oropharyngeal Squamous Cell Carcinoma.

Codon 72 polymorphism of P53 gene does not affect the risk of cirrhosis and hepatocarcinoma in HCV-infected patients.

Chronic hepatitis C virus (HCV) infection is the most frequent cause of progressive liver disease and liver cancer in the West. The p53 tumor suppressor gene is known to play an important role in carcinogenesis of different tissues being involved in gene transcription, DNA synthesis and repair and somatic mutations of p53 are common in primary liver cancer. The p53 gene displays a common genetic Arg/Pro polymorphism at codon 72 with functional significance, that has been investigated as risk factor in several cancer models. We analyzed p53 codon 72 polymorphism in a group of 340 HCV-infected subjects at different stages of disease, including 84 hepatocellular carcinoma patients. No association between codon 72 genotypes and disease severity or liver cancer was observed.

An heteroduplex mobility analysis assay based on capillary electrophoresis for the study of HCV quasispecies.

The quasispecies nature of the hepatitis C virus (HCV) genome is central to the transmission, persistence and pathogenesis of the infection. Heteroduplex mobility analysis (HMA) is a simple and an inexpensive technique for the qualitative and quantitative analysis of genetic variation of viral quasispecies. An original HMA for the HVR1 region of HCV was developed, based on a semi-automated, non-radioactive capillary electrophoresis system, which allows the processing of large numbers of samples in short times, the accurate measure of mobility shifts and the quantitation of heteroduplexes. A set of 120 HVR1 clones of known sequence was used to develop the assay, which was tested on HVR1 sequences amplified directly from sera of 17 HCV-infected patients. HVR1 sequence divergence directly correlated with the heteroduplex mobility ratio (HMR) of hybrid molecules between six and 40 mismatches. Heteroduplexes between one and six mismatches were resolved, although HMRs were not proportional to base changes, likely due to an effect of type and position of the substitutions. The assay sensitivity was 1% of the total sample size. This assay may allow the application of quasispecies analysis to a wider range of clinical and basic investigations.

EGFR mutation-specific antibodies in pulmonary adenocarcinoma: a comparison with DNA direct sequencing.

INTRODUCTION: Epidermal growth factor receptor (EGFR) gene mutations are usually detected by direct sequencing to identify patients with advanced pulmonary adenocarcinomas as candidates for tyrosine kinase inhibitor therapy. The aim of the study was to evaluate the efficacy of EGFR mutation-specific antibodies in identifying EGFR-mutated adenocarcinomas. MATERIALS AND METHODS: Thirty-three consecutive cases of pulmonary adenocarcinomas sequenced for EGFR mutations were retrieved from our files. Immunohistochemistry was performed with the rabbit monoclonal antibodies E746-A750del (6B6) and L858R (43B2). The results obtained using the 2 procedures were statistically compared by Coehn κ and by calculation of sensitivity and specificity. RESULTS: There were 21 women and 12 men, ranging in age from 48 to 78 years. All cases were lung adenocarcinomas, 23 primaries and 10 metastatic. The mutational spectrum was as follows: 12 cases mutated in exon 19 (9 with E746-A750del, 1 with homozygote L747-T751del, 1 with L747-P753del, 1 with E747-S752del), 6 in exon 21 (5 with L858R, 1 with L861Q+L862L), and 15 EGFR wild type. Immunohistochemistry detected 6/9 cases with an E746-A750del mutation (κ=0.744, sensitivity: 66.7%, specificity: 100%) and 5/5 cases with an L858R mutation (κ=1, sensitivity: 100%, specificity: 100%). Four cases showed faint and focal immunostaining and were interpreted as negative. All other cases were negative. Overall, the 2 antibodies had 61.1% sensitivity and 100% specificity for EGFR mutations. CONCLUSIONS: EGFR mutation-specific antibodies may represent a first-line screening tool to identify patients as candidates for tyrosine kinase inhibitor therapy.

Implications of a positive sentinel node in oral squamous cell carcinoma.

BACKGROUND: The role of sentinel node biopsy in head and neck cancer is currently being explored. Patients with positive sentinel nodes were investigated to establish if additional metastases were present in the neck, their distribution, and their impact on outcome. METHODS: In all, 109 patients (n = 109) from 15 European centers, with cT1/2,N0 tumors, and a positive sentinel lymph node were identified. Kaplan-Meier and univariate and multivariate logistic regression analysis were used to identify variables that predicted for additional positive nodes and their position within the neck. RESULTS: A total of 122 neck dissections were performed in 109 patients. Additional positive nodes were found in 34.4% of cases (42/122: 18 same, 21 adjacent, and 3 nonadjacent neck level). Additional nodes, especially if outside the sentinel node basin, had an impact on outcome. CONCLUSIONS: The results are preliminary but suggest that both the number and the position of positive sentinel nodes may identify different prognostic groups that may allow further tailoring of management plans.

Glutamine synthetase immunostaining correlates with pathologic features of hepatocellular carcinoma and better survival after radiofrequency thermal ablation.

PURPOSE: Activation of the wnt pathway identifies a subgroup of hepatocellular carcinomas (HCC) with specific epidemiologic and genetic profiles. Wnt activation is predicted by mutation and/or nuclear translocation of beta-catenin and by glutamine synthetase (GS) immunoreactivity. We investigated whether GS staining associates with specific pathologic features of HCC and with survival after radiofrequency thermal ablation. EXPERIMENTAL DESIGN: Monoistitutional retrospective-prospective study in a tertiary hospital setting. Two hundred and seven cirrhotics (mean age, 70 years; 63% males, 82.1% hepatitis C virus positive) with early HCC were consecutively treated with radiofrequency thermal ablation (RFTA). Mean tumor size was 2.7 cm; 20.3% of patients had multiple nodules; and median follow-up was 36 months with 54.6% overall mortality. Tumor samples were mainly obtained by biopsy (92,5%) and examined by H&E and immunostaining for beta-catenin and GS. Main outcome measures were overall and tumor-specific mortality by Kaplan-Meier analysis and Cox proportional hazard models corrected for competing risks. RESULTS: Ninety-one patients (43.9%) had GS-positive HCCs by immunostaining. These tumors had larger size (P = 0.012) and characteristic histology (low grade, pseudoacini, hydropic changes, bile staining, lack of steatosis, and fibrosis). Other clinical or treatment variables were similar between groups. Variables correlating with tumor-specific and overall mortality by univariate analysis were tumor recurrence, advanced disease, posttreatment alpha-fetoprotein levels, and GS staining. Yearly, overall mortality rate was lower in GS-positive patients (12.4 versus 20% yearly; P = 0.006). By multivariate analysis, GS immunostaining correlated with reduced specific (hazard ratio, 0.58; 95% confidence interval, 0.34-0.97) and overall mortality (hazard ratio, 0.62; 95% confidence interval, 0.40-0.96). CONCLUSIONS: Standard histology and GS status identify a HCC subset with distinct clinical and pathologic features.

CYP enzyme polymorphisms and susceptibility to HCV-related chronic liver disease and liver cancer.

Cancer risk can be influenced by the exposure to endogenous or environmental toxins. Polymorphic enzymes involved in the metabolic activation/detoxification of carcinogens may account for individual variations of risk. We studied the polymorphisms of five enzymes of the P450 superfamily, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CY3A4, as risk factors for liver disease progression and cancer in hepatitis C virus-infected patients. CYP genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism or allele-specific PCR. Different stages of disease were considered, as follows: 90 asymptomatic carriers and 87 chronic hepatitis, 92 cirrhosis and 91 hepatocellular carcinoma (HCC) cases. Reference allele frequencies were obtained from 99 blood donors. Allele distributions among categories were compared using the chi(2) test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to express relative risks. Independent associations were modeled by correspondence analysis and logistic regression. Frequencies of the CYP1A1 highly inducible alleles, MspI m2 and Val, were increased in liver disease patients compared with carriers; no specific association with HCC was found. The high-activity CYP2E1 c2 allele was underrepresented among HCC patients with respect to other HCV categories, including cirrhosis. CYP2D6 poor metabolizer (PM) genotypes were significantly more frequent in healthy subjects (7.1%) and carriers (11.1%) than in hepatitis/cirrhosis (4.6%) and HCC (1.2%) patients. This was confirmed by multivariable analysis. PM genotypes protected against progressive disease as ORs reduced proportionally to stage. The age at diagnosis for HCC was anticipated in non-PM individuals. No differences were seen for CYP1A2 and CYP3A4 genes. Polymorphic variants of CYP genes may contribute to the progression of liver disease and HCC risk in HCV-infected subjects.

Genetic polymorphisms of steroid hormone metabolizing enzymes and risk of liver cancer in hepatitis C-infected patients.

BACKGROUND/AIMS: Genetic polymorphisms of enzymes involved in hormone metabolism can influence hormonal activities and risk of hormone-dependent cancers. As progression of chronic hepatitis C and risk of liver cancer is higher in males than in females, we evaluated whether the polymorphisms of three enzymes participating in the pathway of estrogen and androgen biosynthesis and inactivation, 5alpha-reductase type II (SRD5A2), cytochrome P450c17alpha (CYP17) and catechol-O-methyltransferase (COMT), might affect the expression of hepatitis C virus (HCV)-related liver disease. METHODS: The study included 78 healthy subjects and 387 HCV patients: 100 asymptomatic carriers, 105 hepatitis, 90 cirrhosis and 92 hepatocellular carcinomas (HCC). Variant positions SRD5A2 V89L and A49T, CYP17 (-34)T/C and COMT V108M were analysed by polymerase chain reaction and restriction fragment length polymorphism. A cross-sectional study of association was performed, considering carriers as reference category. RESULTS: The CYP17 (-34)C/C genotype was over-represented in HCC patients as compared to carriers (22.5 vs. 11.2%, odds ratio (OR): 2.29, P: 0.05). Females mostly contributed to this association (OR: 4.95, P: 0.01) and OR values increased in post-menopausal women (OR: 6.00, P: 0.03). No differences were observed for SRD5A2 and COMT gene polymorphisms. CONCLUSIONS: CYP17 high-activity alleles associated with increased circulating levels of estrogens and androgens may affect liver cancer risk in HCV-infected women.

Polymorphisms of microsomal epoxide hydrolase gene and severity of HCV-related liver disease.

Factors influencing the progression of liver disease and the development of hepatocellular carcinoma (HCC) in chronic hepatitis C virus (HCV) infection are poorly understood. Inherited variations of drug-metabolizing enzyme (DME) activities may affect liver damage and cancer risk by modifying individual susceptibility to endogenous or exogenous toxic compounds. We investigated the association of liver disease severity with common alleles of microsomal epoxide hydrolase (mEH), an enzyme involved in the metabolism of highly reactive epoxide intermediates. Three polymorphisms (Tyr113His, His139Arg, and -613C/T) were analyzed by polymerase chain reaction (PCR) restriction fragment length polymorphisms (RFLPs) in 394 patients at different stages of disease, including 92 asymptomatic carriers, 109 patients with chronic hepatitis, 100 patients with cirrhosis, and 93 patients with HCC. Reference allele frequencies were obtained from 99 healthy blood donors. Allele distributions between categories were compared using the chi(2) test; odds ratios (ORs) and 95% CI were calculated to express relative risks. Allele frequencies among 99 healthy controls were as follows: 15.1% for 113His/His, 4.0% for 139Arg/Arg, and 46.5% for -613C/T. mEH 113His/His homozygotes were overrepresented in advanced stages of disease, in particular among HCC patients (27.9%; P =.03; OR, 2.2; 95% CI, 1.0-4.6). Differences were more pronounced among men and between extreme patient categories. When mEH genotypes were combined to express a metabolic phenotype, very slow metabolizers were highly prevalent among cirrhotic and HCC patients (18% vs. 3.3% in carriers; P <.001). In conclusion, mEH gene polymorphisms were significantly associated with HCV-related liver disease severity and HCC risk. Men were at higher risk than women; this might be explained by hormonal regulation of gene expression or by differential exposure to environmental toxins.