Multiple structural and epigenetic defects in the human leukocyte antigen class I antigen presentation pathway in a recurrent metastatic melanoma following immunotherapy.

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP(684delA)) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.

Enoxaparin prevents portal vein thrombosis and liver decompensation in patients with advanced cirrhosis.

BACKGROUND & AIMS: We performed a randomized controlled trial to evaluate the safety and efficacy of enoxaparin, a low-molecular-weight heparin, in preventing portal vein thrombosis (PVT) in patients with advanced cirrhosis. METHODS: In a nonblinded, single-center study, 70 outpatients with cirrhosis (Child-Pugh classes B7-C10) with demonstrated patent portal veins and without hepatocellular carcinoma were assigned randomly to groups that were given enoxaparin (4000 IU/day, subcutaneously for 48 weeks; n = 34) or no treatment (controls, n = 36). Ultrasonography (every 3 months) and computed tomography (every 6 months) were performed to check the portal vein axis. The primary outcome was prevention of PVT. Radiologists and hepatologists that assessed outcomes were blinded to group assignments. Analysis was by intention to treat. RESULTS: At 48 weeks, none of the patients in the enoxaparin group had developed PVT, compared with 6 of 36 (16.6%) controls (P = .025). At 96 weeks, no patient developed PVT in the enoxaparin group, compared with 10 of 36 (27.7%) controls (P = .001). At the end of the follow-up period, 8.8% of patients in the enoxaparin group and 27.7% of controls developed PVT (P = .048). The actuarial probability of PVT was lower in the enoxaparin group (P = .006). Liver decompensation was less frequent among patients given enoxaparin (11.7%) than controls (59.4%) (P < .0001); overall values were 38.2% vs 83.0%, respectively (P < .0001). The actuarial probability of liver decompensation was lower in the enoxaparin group (P < .0001). Eight patients in the enoxaparin group and 13 controls died. The actuarial probability of survival was higher in the enoxaparin group (P = .020). No relevant side effects or hemorrhagic events were reported. CONCLUSIONS: In a small randomized controlled trial, a 12-month course of enoxaparin was safe and effective in preventing PVT in patients with cirrhosis and a Child-Pugh score of 7-10. Enoxaparin appeared to delay the occurrence of hepatic decompensation and to improve survival.

Early menopause is associated with lack of response to antiviral therapy in women with chronic hepatitis C.

BACKGROUND & AIMS: Chronic hepatitis C (CHC) and liver fibrosis progress more rapidly in men and menopausal women than in women of reproductive age. We investigated the associations among menopause, sustained virologic response (SVR), and liver damage in patients with CHC. METHODS: We performed a prospective study of 1000 consecutive, treatment-naïve patients 18 years of age and older with compensated liver disease from CHC. Liver biopsy samples were analyzed (for fibrosis, inflammation, and steatosis) before patients received standard antiviral therapy. From women (n = 442), we collected data on the presence, type, and timing of menopause; associated hormone and metabolic features; serum levels of interleukin-6; and hepatic tumor necrosis factor (TNF)-α. RESULTS: Postmenopausal women achieved SVRs less frequently than women of reproductive age (46.0% vs 67.5%; P < .0001) but as frequently as men (51.1%; P = .283). By multivariate regression analysis, independent significant predictors for women to not achieve an SVR were early menopause (odds ratio [OR], 8.055; 95% confidence interval [CI], 1.834-25.350), levels of γ-glutamyl transpeptidase (OR, 2.165; 95% CI, 1.364-3.436), infection with hepatitis C virus genotype 1 or 4 (OR, 3.861; 95% CI, 2.433-6.134), and cholesterol levels (OR, 0.985; 95% CI, 0.971-0.998). Early menopause was the only independent factor that predicted lack of an SVR among women with genotype 1 hepatitis C virus infection (OR, 3.933; 95% CI, 1.274-12.142). Baseline levels of liver inflammation, fibrosis, steatosis, serum interleukin-6 (P = .04), and hepatic TNF-α (P = .007) were significantly higher among postmenopausal women than women of reproductive age. CONCLUSIONS: Among women with CHC, early menopause was associated with a low likelihood of SVR, probably because of inflammatory factors that change at menopause.

Human melanoma metastases express functional CXCR4.

PURPOSE: The chemokine receptor CXCR4 was identified as an independent predictor of poor prognosis in primary melanoma. The aim of the study was to investigate the role of CXCR4 in human melanoma metastases. EXPERIMENTAL DESIGN: CXCR4 expression was evaluated in melanoma metastases and in metastatic cell lines through immunohistochemistry, immunoblotting, immunofluorescence, and reverse transcription-PCR. The function of CXCR4 was tested in the presence of the ligand, CXCL12, through induction of extracellular signal-regulated kinase-1 and -2 (Erk-1 and -2) phosphorylation, proliferation, apoptosis, and migration capabilities. RESULTS: CXCR4 expression was detected in 33 out of 63 (52.4%) metastases from cutaneous melanomas. Metastatic melanoma cell lines expressed cell surface CXCR4; PES 43, Alo 40, and COPA cell lines showed the highest levels of CXCR4 (>90% of positive cells); PES 41, Alo 39, PES 47, POAG, and CIMA cell lines showed low to moderate degrees of expression (5-65% of positive cells). Other chemokine receptors, CCR7 and CCR10, were detected on the melanoma cell lines; CXCL12 activated Erk-1 and Erk-2, the whose induction was specifically inhibited by AMD3100 treatment. CXCL12 increased the growth in PES 41, PES 43, and PES 47 cells under suboptimal (1% serum) and serum-free culture conditions; AMD3100 (1 mumol/L) inhibited the spontaneous and CXCL12-induced proliferation. No rescue from apoptosis was shown but PES 41, PES 43, and PES 47 cells migrate toward CXCL12. CONCLUSIONS: These findings indicate that CXCR4 is expressed and active in human melanoma metastases, suggesting that active inhibitors such as AMD3100 may be experienced in human melanoma.

Acute hepatitis B caused by a vaccine-escape HBV strain in vaccinated subject: sequence analysis and therapeutic strategy.

HBV vaccine contains the 'a' determinant region, the major immune-target of antibodies (anti-HBs). Failure of immunization may be caused by vaccine-induced or spontaneous 'a' determinant surface gene mutants. Here, we evaluate the possible lack of protection by HBV vaccine, describing the case of an acute hepatitis B diagnosed in a 55-year-old Caucasian male unpaid blood donor, vaccinated against HBV. Sequencing data for preS-S region revealed multiple point mutations. Of all the substitutions found, Q129H, located in the "a" determinant region of HBsAg, can alter antigenicity, leading to mutants. This mutant may cause vaccine failure especially when associated with high viremia of infecting source.

Interleukin 28B genotype determination using DNA from different sources: A simple and reliable tool for the epidemiological and clinical characterization of hepatitis C.

Recent studies reported a close correlation between polymorphisms in the Interleukin (IL)28B gene and rates of resolution of hepatitis C virus infection occurring spontaneously or induced by treatment. The diagnostic utility of IL28B genotype, however, is not understood completely. For rapid data collection on the natural history of HCV infection in patients with different IL28B genotype, simple, sensitive and rapid methods suitable for non-invasive and archival clinical samples are needed urgently. A real-time polymerase chain reaction (PCR) method for IL28B typing (rs12979860) was developed using very small DNA quantities extracted from different biological specimens. Consistent IL28B genotyping of at least two DNA samples obtained from different sources such as whole blood, buccal swab, serum, and formalin fixed paraffin-embedded liver tissue was obtained from 58 patients with liver disease of mixed etiology. IL28B genotype prevalence in 170 patients with liver disease in this region of Italy was consistent with data reported in Caucasian populations. Differential distribution of genotypes was observed according to response to treatment in 68 patients infected with HCV, with higher prevalence of CC genotype in responders (50%) compared to non-responders (17.85%; p=0.015). These results indicate that the possibility of reliable IL28B genotyping using different DNA sources may represent a useful tool for both clinical research and characterization of patients with hepatitis C.

Pretreatment with pegylated interferon prevents emergence of lamivudine mutants in lamivudine-naive patients: a pilot study.

BACKGROUND: In patients with advanced fibrosis, primary end points of long-term or possibly indefinite antiviral therapy are sustained inhibition of viral replication and avoidance of emergence of resistance. In lamivudine-treated patients, the strongest predictor of emergence of YMDD mutations is baseline hepatitis B virus (HBV) DNA viral load. We aimed to verify whether abatement of viraemia by a short course of pegylated interferon (PEG-IFN-alpha2a) treatment before lamivudine treatment could prevent the emergence of lamivudine-associated mutations during long-term therapy. METHODS: A total of 14 patients with hepatitis B e antigen (HBeAg)-negative infection (3 lamivudine-experienced and 11 lamivudine-naive), with moderate/high viraemia (>10(6) copies/ml) and with Ishak stage 4-6 at liver biopsy were sequentially treated with 180 microg PEG-IFN-alpha2a for a period long enough to reach HBV DNA levels < or =10(3) copies/ml or have a decrease of 3 log(10) copies/ml from baseline. Lamivudine was then added to PEG-IFN-alpha2a treatment for 1 month and finally continued as monotherapy for 2 years or until viral breakthrough. RESULTS: Baseline HBV DNA (mean +/-se 2.3 x 10(7) +/-7.2 x 10(7) copies/ml) decreased with PEG-IFN-alpha2a treatment to target value in mean +/-se 3.7 +/-1.3 months. None of the 11 lamivudine-naive patients developed genotypic resistance and were still HBV-DNA-negative after a mean +/-se observation period of 23 +/-2 months, whereas the three lamivudine-experienced patients developed YMDD mutations after 6, 9 and 12 months of lamivudine monotherapy (P=0.003, Fisher's exact test). CONCLUSIONS: In lamivudine-naive patients, abatement of HBV DNA<10(3) copies/ml by pretreatment with PEG-IFN-alpha2a completely prevents the emergence of YMDD mutants after 24 months of lamivudine monotherapy. This sequential schedule can optimize the use of a well tolerated, effective and inexpensive drug, such as lamivudine, in highly viraemic HBV patients.