Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme.

BACKGROUND: Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme. METHODS: Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4(+) and CD8(+) T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10. RESULTS: Median overall survival among 27 patients was 9.6 months (95 % CI 3.2-16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived >4 years. Patients with an increase in the number of circulating ICOS(+) T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median. CONCLUSIONS: Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS(+) T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.

Expression and regulation of B7-H3 immunoregulatory receptor, in human mesothelial and mesothelioma cells: immunotherapeutic implications.

No treatment prolongs the survival of malignant mesothelioma (MM) patients. Since MM elicits anti-tumor host's immune responses, immunotherapy represents a promising strategy for its control. Immunomodulatory antibodies against components of the B7 family of immunomodulatory molecules that regulate T cell activation are being investigated in human malignancies including MM. The expression of B7-H3, a new component of the B7 family was investigated in primary cultures of human mesothelial cells (HMC) and in MM cell lines by flow cytometry and molecular analyses, and in MM tissues by immunohistochemistry. The role of DNA hypomethylating agents in modulating levels of B7-H3 expression in MM cells was also studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that B7-H3 mRNA was consistently detectable in mesothelial and MM cells investigated; however, real-time quantitative RT-PCR analyses showed highly heterogeneous levels of B7-H3 mRNA among investigated MM cells. The analysis of B7-H3 protein expression indicated that comparable levels of B7-H3 were expressed on both cell types. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine did not significantly affect the expression of B7-H3 mRNA in MM cells. In vivo, while B7-H3 was expressed in all 13 tumor biopsies of the epithelial variant, with high levels in 54% of cases, it was rarely detectable in spindle type MM in which 1/5 biopsies weakly expressed B7-H3. These findings suggest that B7-H3 is a promising target for new immunotherapeutic strategies in MM, with particular emphasis in the epithelial variant.

Ipilimumab experience in heavily pretreated patients with melanoma in an expanded access program at the University Hospital of Siena (Italy).

AIM OF STUDY: To evaluate the feasibility of ipilimumab treatment for metastatic melanoma outside the boundaries of clinical trials, in a setting similar to that of daily practice. METHODS: Ipilimumab was available upon physician request in the Expanded Access Programme for patients with life-threatening, unresectable stage III/IV melanoma who failed or did not tolerate previous treatments and for whom no therapeutic option was available. Induction treatment with ipilimumab 10 mg/kg was administered intravenously every 3 weeks, for a total of 4 doses, with maintenance doses every 12 weeks based on physicians' discretion and clinical judgment. Tumors were assessed at baseline, Week 12, and every 12 weeks thereafter per mWHO response criteria, and clinical response was scored as complete response (CR), partial response (PR), stable disease (SD), or progressive disease. Durable disease control (DC) was defined as SD at least 24 weeks from the first dose, CR, or PR. RESULTS: Disease control rate at 24 and 60 weeks was 29.6% and 15%, respectively. Median overall survival at a median follow-up of 8.5 months was 9 months. The 1- and 2-year survival rates were 34.8% and 23.5%, respectively. Changes in lymphocyte count slope and absolute number during ipilimumab treatment appear to correlate with clinical response and survival, respectively. Adverse events were predominantly immune related, manageable, and generally reversible. One patient died from pancytopenia, considered possibly treatment related. CONCLUSION: Ipilimumab was a feasible treatment for malignant melanoma in heavily pretreated, progressing patients. A sizeable proportion of patients experienced durable DC, including benefits to long-term survival.

CD40 expression by human melanocytic lesions and melanoma cell lines and direct CD40 targeting with the therapeutic anti-CD40 antibody CP-870,893.

Anti-CD40 antibodies are in clinical development in patients with metastatic melanoma, a cancer that has been reported earlier to express CD40. The antitumor activity of anti-CD40 antibodies may be mediated by direct cytotoxic effects on CD40-positive melanoma cells or indirectly through modulation of host cells. In these studies, biopsies of patients with primary and metastatic melanoma, short-term cultures, and established melanoma cell lines were analyzed for CD40 expression using a combination of methods including immunohistochemistry, flow cytometry, and gene expression profiling, and the cytotoxic effects of the anti-CD40 antibody CP-870,893 on melanoma cell lines were tested using cell viability assays. CD40 was expressed at a higher frequency in metastatic melanoma lesions compared with primary melanomas. There was a variable expression of CD40 in synchronous and metachronous melanoma metastases. Expression of CD40 was present in slightly over half of a large panel of short-term primary melanoma cultures, with a wide range of expression levels by flow cytometry. Similar results were obtained in established melanoma cell lines when analyzed at the mRNA level or by surface protein staining. In approximately one-third of cell lines, the expression of CD40 could be up-regulated with a histone deacetylase inhibitor. Treatment with increasing concentrations of CP-870,893 had no antitumor activity against either CD40-positive or negative melanoma cell lines. In conclusion, approximately one-third to one-half of melanomas expresses CD40 at variable levels. Direct exposure to a CD40-activating antibody does not lead to antitumor activity in melanoma cell lines, suggesting that the antitumor effects of these antibodies in the clinic may be indirectly mediated.

Relationship between Hpt polymorphisms and serum protein electropherogram.

Serum protein electrophoresis is routinely used to identify pathologies involving dysproteinemia. The electropherogram mainly represents the most abundant serum proteins, one of which is the polymorphic haptoglobin (Hpt), characterized by a molecular heterogeneity with three major phenotypes (Hpt 1-1, 2-1, and 2-2). To improve the interpretation of electropherogram and possibly to extend its applicability, we aimed to explore the relationship between Hpt phenotypes (determined by immunoblotting) and protein profiles. Serum samples were separated by CZE with PROTEIN 6 and high-resolution methods. The PROTEIN 6 analysis showed significant associations between alpha2 zone profiles and Hpt phenotypes (chi-square=154.06, p<0.0001). The high-resolution method indicated significant differences between Hpt 2-2 and Hpt 1-1 peak mobilities, evidenced by receiver operating characteristic analysis, (area under the receiver operating characteristic curve=0.98, p<0.0001, standard error=0.01346, likelihood ratios=21.39), with 98.7% sensitivity, and 95.4% specificity. However, the structural heterogeneity of Hpt 2-1 made it difficult to relate with a particular profile. Thus, we developed an alternative approach that excluded the Hpt 1-1 or Hpt 2-2 phenotypes. This may prove to be a useful technique in clinical applications considering the involvement of Hpt 2-2 or Hpt 1-1 in various pathologies.

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract.

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.

Simultaneous determination of serum concentrations of levodopa, dopamine, 3-O-methyldopa and alpha-methyldopa by HPLC.

Levodopa is the medication of choice for Parkinson's disease. The biological complexity of levodopa and of its main derivatives makes their determination important in the clinical field. The aim of this study was to develop an HPLC method for the simultaneous determination of serum concentrations of levodopa, dopamine, 3-O-methyldopa and alpha-methyldopa. We compared UV and fluorimetric detection of native and derivatised compounds. Though less sensitive than other methods, UV detection is important to exclude naturally fluorescent, interfering substances. Fluorimetric detection of derivatised compounds is more sensitive than UV detection. Since 3-O-methyldopa does not react with the derivatising agent 1,2-diphenylethylenediamine, it cannot be detected. For simultaneous determination of the four compounds after pharmacological treatment of patients we therefore advise fluorimetric detection of the native compound.