Baseline neutrophils and derived neutrophil-to-lymphocyte ratio: prognostic relevance in metastatic melanoma patients receiving ipilimumab.

BACKGROUND: Clinical responses to ipilimumab are variable in terms of onset, magnitude and duration. Upfront identification of patients who are more likely or unlikely to benefit from treatment is a major need. PATIENTS AND METHODS: Prospectively collected data from 720 advanced melanoma patients treated with ipilimumab 3 mg/kg within the Italian expanded access program were analyzed. The derived neutrophil-to-lymphocyte ratio (dNLR) was calculated from baseline peripheral blood cell counts, and receiver operating characteristic curve was used to evaluate the best cutoff for this marker. Patients were stratified according to dichotomized baseline absolute neutrophil counts (ANC), dNLR and their combination. The prognostic values of ANC and dNLR for survival were assessed using multivariate Cox proportional hazard models. A subgroup analysis including LDH in the models was also carried out. RESULTS: The median follow-up was 16.5 months. The optimal cutoff for dNLR was 3. Baseline ANC and dNLR were significantly associated with the outcome of ipilimumab-treated melanoma patients, in terms of disease progression and death (P < 0.0001 for all). Furthermore, for each elevated variable, prognosis worsened. Patients with both ANC ≥ 7500 and dNLR ≥ 3 had a significantly and independently increased risk of death [hazard ratio(HR) = 5.76; 95% confidence interval (CI) 4.29-7.75] and of progression (HR = 4.10; 95% CI 3.08-5.46) compared with patients with both lower ANC and dNLR. Patients with one of the two factors elevated displayed an intermediate risk of progression and death. The 1- and 2-year survival rates were 2% and 0%, respectively, for patients with ANC ≥ 7500 and dNLR ≥ 3, and 43% and 24%, respectively, for patients with both lower ANC and dNLR. CONCLUSIONS: Although these findings need to be confirmed and validated, we suggest that a neutrophil-based index may help risk-group stratification and assist disease-management strategies. Furthermore, the potential predictive value of this index for response to ipilimumab should be investigated in randomized clinical trials.

Three-year follow-up of advanced melanoma patients who received ipilimumab plus fotemustine in the Italian Network for Tumor Biotherapy (NIBIT)-M1 phase II study.

BACKGROUND: In the NIBIT-M1 study, we reported a promising activity of ipilimumab combined with fotemustine in metastatic melanoma (MM) patients with or without brain metastases. To corroborate these initial findings, we now investigated the long-term efficacy of this combination. PATIENTS AND METHODS: This analysis captured the 3-year outcome of MM patients who received ipilimumab combined with fotemustine as first- or second-line treatment. Median overall survival (OS), 3-year survival rates, immune-related (ir) progression-free survival (irPFS), brain PFS, and ir duration of response (irDOR) for the entire population and for patients with brain metastases were assessed. Clinical results were correlated with circulating CD3(+)CD4(+)ICOS(+)CD45RO(+) or CD45RA(+) T cells, neutrophil/lymphocyte (N/L) ratios, and tumorBRAF-V600 mutational status. RESULTS: Eighty-six MM patients, including 20 with asymptomatic brain metastases that had been pre-treated with radiotherapy in 7 subjects, were enrolled in the study. With a median follow-up of 39.9 months, median OS and 3-year survival rates were 12.9 months [95% confidence interval (CI) 7.1-18.7 months] and 28.5% for the whole study population, and 12.7 months (95% CI 2.7-22.7 months) and 27.8% for patients with brain metastases, respectively. Long-term ir adverse events consisting of G1 rush and pruritus occurred in 21% of patients. The absolute increase from baseline to week 12 in 'memory' but not in 'naïve' T cells identified patients with a better survival (P = 0.002). The N/L ratio correlated with a significantly better survival at early time points. BRAF status did not correlate with clinical outcome. CONCLUSIONS: Long-term analysis of the NIBIT-M1 trial continues to demonstrate efficacy of ipilimumab combined with fotemustine in MM patients. Fotemustine does not seem to impair the immunologic activity of ipilimumab. EUDRACT NUMBER: 2010-019356-50. CINICALTRIALSGOV: NCT01654692.

Combined and sequential expression of p53, Rb, Ras and Bcl-2 in bronchial preneoplastic lesions.

AIMS AND BACKGROUND: Several simple molecular abnormalities have been detected in bronchial preneoplastic lesions, but the simultaneous presence of these alterations has been scarcely investigated. METHODS: We studied, by an immunohistochemical method, the expression of p53, Rb, Ras and Bcl-2 in 65 samples from surgical specimens and diagnostic biopsies selected for the presence of preneoplastic changes in the bronchial epithelium. To perform an analysis of the combined expression of all markers in the same areas, we accurately mapped every consecutive section on which immunohistochemical reactions were performed, subdividing each specimen into 25x microscopic fields, which allowed good topographical mapping. RESULTS: It was found that the frequency of p53-positive and Rb-negative microscopic fields was directly related to the morphological grading of lesions. On the other hand, Ras expression characterized high-grade lesions not showing squamous differentiation (non-squamous Cis). Regarding Bcl-2 expression, only slight differences in positivity distribution were found between the different lesions. More interesting was the parallel evaluation of all markers in the same areas: one of the main patterns, found to be correlated with the severity of histopathological features, was characterized by combined p53 hyperexpression/Rb hypoexpression; furthermore, when Ras and Bcl-2 hyperexpression were superimposed to the above pattern, the former mainly characterized non-squamous Cis, while the latter was present only in high-grade squamous lesions. However, the most frequently encountered pattern did not show any alteration of the studied markers, suggesting that other mechanisms could be involved in bronchial carcinogenesis. CONCLUSIONS: The detection of combined molecular abnormalities in bronchial preneoplasia could clarify the steps involved in lung carcinogenesis; furthermore, a simple and inexpensive method, such as immunohistochemistry, could be routinely applied also to cytologic specimens in order to detect those lesions, or patients, that are prone to progression towards lung cancer.

Liver metastases from melanoma: hepatic intra-arterial chemotherapy. A retrospective study.

The liver is the primary site of metastases in most uveal melanoma patients. We retrospectively investigated intraarterial chemotherapy (IAC) as treatment for patients with hepatic melanoma metastases.Twenty-three patients (18 with uveal melanoma) received fotemustine (14 patients, 61.9%) or carboplatin (9 patients, 31.1%) via hepatic IAC delivery. The catheter was introduced through percutaneous access to the femoral artery with drugs delivered directly to the hepatic artery, and was removed at the end of each treatment cycle. A total of 3 cycles was planned, repeated every 21 days. However, patients with a clinical response could receive more than 3 cycles, provided that the toxic effects were acceptable.IAC was well tolerated and no catheter-related complications or grade 4 toxicities were reported. Considering only uveal melanoma patients, the overall response rate and disease control rate was 16.7% and 38.9%, respectively. Median time to progression was 6.2 months (95% CI 3.7-10.5) and median overall survival was 21 months (95% CI 8-39).IAC is well tolerated and is a valid choice for patients with a poor prognosis since median survival rates are among the longest reported.

Epigenetic remodelling of DNA in cancer.

DNA methylation regulates gene expression in normal cells. This epigenetic mechanism acts in at least two different ways: at global genomic level by targeting repetitive sequences distributed among the whole genome (LINEs, SINEs, satellite DNA, transposons) and at local level by targeting CpG islands in promoter regions. Both epigenetic mechanisms are involved in the carcinogenetic process; however, different evidences suggest that promoter hypermethylation occurring in genes involved in cell-cycle regulation, DNA repair, cell signalling, transcription and apoptosis likely plays a prominent role. Opposite to genetic defects DNA hypermethylation is a reversible process that can be handled through "epigenetic drugs" in a wide spectrum of tumors. Along this line, recent data have demonstrated the ability of DNA hypomethylating agents to up-regulate and/or induce the expression of genes silenced by promoter hypermethylation in cancer. Particularly relevant seems the ability of these drugs to modulate the expression of genes coding for molecules crucial for tumor immunogenicity and immune recognition of neoplastic cells by host's immune system, such as Cancer Testis Antigens, HLA class I molecules, costimulatory molecules. These evidences, coupled to the well-known cytotoxic, pro-apoptotic, and differentiating activities of epigenetic drugs, encourage to design and to develop new therapeutic strategies able to circumvent the immune escape of neoplastic cells and to potentiate the efficacy of immunotherapy in cancer patients. This review will provide an update on the most recent information about aberrant DNA methylation in cancer and on innovative therapeutic strategies of "epigenetic remodelling" of human malignancies, with particular attention to the immunologic and immunotherapeutic potential of this approach.

Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus.

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

Clinical implications of hepatitis C virus infection in MALT-type lymphoma of the ocular adnexa.

BACKGROUND: A pathogenic link between hepatitis C virus (HCV) and MALT-type lymphomas has been suggested. However, studies assessing the role of HCV infection separately in different forms of MALT lymphomas are not available. PATIENTS AND METHODS: The prevalence and clinical implications of HCV seropositivity were analyzed in 55 patients with ocular adnexa lymphoma (OAL) of MALT-type. RESULTS: HCV seropositivity was detected in seven (13%) patients. At presentation, HCV infection was significantly associated with concomitant extra-orbital disease, lymph node dissemination and involvement of additional extranodal organs. HCV seropositivity was associated also with a higher relapse rate and worse progression-free survival. In fact, 16 patients experienced relapse after first-line treatment: five (71%) were HCV-seropositive and 11 (23%) were HCV-seronegative, with a median TTP of 31 and 50+ months (P = 0.01), and a 5-year progression-free survival of 43 +/- 18% and 77 +/- 7% (P = 0.005), respectively. HCV-seropositive patients experienced frequent relapses despite further lines of therapy; relapses were systemic in all cases but one; multiple subcutaneous nodules were common at relapse. CONCLUSIONS: HCV seropositivity is present in 13% of OAL of MALT-type. Concomitant HCV infection is associated with more disseminated disease and aggressive behavior in OAL, with a consequent potential negative impact in patients managed with radiotherapy alone.

Opposite effects of alfa-actinin and of fructose 1,6-bisphosphate aldolase on the microfilament network. The role of orthophosphate revisited.

At pH 7.5, in the presence of 0.1 M KCl, 2 mM MgCl2 and 15 mM phosphate, the binding of 1 molecule of alfa-actinin for each strand of 1000 actin monomers doubles the apparent viscosity of an F-actin solution (12 microM as the monomer). Further binding of one molecule of aldolase for each strand of 280 actin monomers halves the apparent viscosity of the alfa-actinin-F-actin system without any desorption of alfa-actinin. The effect of aldolase is not hindered by the addition of 0.1 mM fructose 1,6-bisphosphate. It is shown that orthophosphate acts as a damper of the regulatory effect of fructose bisphosphate on the interaction between aldolase and microfilaments.

Binding of alpha-actinin to F-actin or to tropomyosin F-actin is a function of both alpha-actinin concentration and gel structure.

We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of alpha-actinin on the gel formed at 37 degrees C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature, alpha-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37 degrees C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2-2.4 microM). The binding of alpha-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound alpha-actinin increases when actin concentration increases from 0 to 1.2 microM but does not change significantly when actin concentration is further increased up to 48 microM. A similar result is obtained with tropomyosin-decorated F-actin. These observations can be explained by an hypothesis that binding is a function of the alpha-actinin - F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with alpha-actinin and, consequently, a larger alpha-actinin concentration is required to attain the same ratio of bound alpha-actinin to actin monomers in the filaments.

Microfilament gel rigidity cooperates negatively with the binding of actin gelling proteins.

At 37 degrees C, in the presence of 0.1 M KC1 and 2 mM MgCl2, the binding of alpha-actinin to F-actin increases with the concentration of alpha-actinin but not with the concentration of F-actin. This implies that binding is determined by additional factors, beside the alpha-actinin - F-actin association constant. We propose that one of these factors is the rigidity of the gel, which cooperates negatively to the binding by increasing the work needed to bring two actin filaments at the reaction distance with alpha-actinin.

Divergent effects of filamin and tropomyosin on actin filaments bundling.

Filamin increases and tropomyosin decreases the susceptibility of F-actin to form bundles of filaments in the presence of polyethylene glycol 6000. The two proteins, which are located in the leading edge and in the internal part of the cell, respectively, are thus likely to display divergent effects on the microfilaments into bundles transition in these two areas of the cell.

Substoichiometric concentrations of ATP-G-actin are required to anneal actin polymerized by calcium ions.

At 3 degrees C and pH 7.0, the addition of 40 nM ATP-G-actin to F-actin (12 microM as the monomer), polymerized in the presence of 4 mM CaCl2, determines a substantial and rapid increase of the viscosity of the solution, which is accompanied by the incorporation of the ATP-G-actin added into the polymer. The hypothesis that the presence of ATP-actin at the filament end(s) promotes the annealing reaction is substantiated by the finding that, after the addition of ATP-G-actin, the average filament length is increased. This finding is relevant, not only because it provides evidences in favour of the existence of annealing but also because it shows that the concentration of ATP-G-actin influences the filaments length distribution through a mechanism different from the elongation reaction.

Activation of infiltrating cytotoxic T lymphocytes and lymphoma cell apoptotic rates in gastric MALT lymphomas. Differences between high-grade and low-grade cases.

In this study, we have characterized infiltrating T lymphocytes from 13 low-grade and 17 high-grade primary gastric MALT lymphomas by immunohistochemistry, with particular regard to the presence, activation, and topographic distribution of cytotoxic effectors. Although the prevalence of CD4+ and CD8+ cells was similar in low- and high-grade lymphomas, higher numbers of TIA-1+ cytotoxic effectors were found in this latter group of cases (11.6 versus 7. 8%; P = 0.004). Activation of CD8+ cytotoxic T lymphocytes (CTLs) was significantly more pronounced in high- than in low-grade lymphomas, as shown by immunostaining for perforin (8.7 versus 4.0%; P = 0.001) and granzyme-B (GrB) (8.7% versus 3.0%; P < 0.0001). Of note, CD20/GrB double labeling showed that high-grade lymphomas carried a markedly higher content (about ninefold) of activated CTLs relative to the number of CD20+ lymphoma B cells (0.081 +/- 0.076 versus 0.009 +/- 0.011; P < 0.0001). Moreover, high-grade lymphomas showed significantly increased apoptotic rates compared to low-grade cases (5.3 and 1.1% of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, respectively; P < 0.0001). In the whole series, the percentage of GrB+ cells and the GrB+/CD20+ ratio showed a strong linear correlation with the number of TUNEL-labeled cells. These findings, together with the frequent colocalization of CTLs and TUNEL+ neoplastic cells, suggested that apoptotic death of lymphoma cells may be due at least in part to the killing by cytotoxic effectors. Our results are consistent with the occurrence of host antitumor cell-mediated immune responses in gastric MALT lymphomas. Moreover, the finding of stronger cytotoxic responses in high- than in low-grade cases is of potential usefulness in the design of more effective therapeutic strategies for the management of these disorders.

High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability.

Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI- cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI- CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 +/- 6.2 versus 4.6 +/- 4.1, P < 0.001), CD8 (13 +/- 6.4 versus 3.7 +/- 3.8, P < 0.001), and TIA-1 (11.2 +/- 6.5 versus 1.9 +/- 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI- tumors, as revealed by the expression of granzyme B (5.3 +/- 4.5 versus 0.6 +/- 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 +/- 2.1 versus 2.6 +/- 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients.

Microsatellite instability and high content of activated cytotoxic lymphocytes identify colon cancer patients with a favorable prognosis.

Colorectal cancers with high-frequency microsatellite instability show peculiar clinicopathological features and a favorable clinical outcome. We investigated whether the improved prognosis for these cancers is related to the content of activated cytotoxic intraepithelial T lymphocytes. Microsatellite instability and the amount of activated cytotoxic lymphocytes were analyzed according to clinicopathological features, survival, and disease recurrence in 109 right-sided colon carcinomas from 245 consecutive patients with stage II/III colon cancer that underwent radical surgery. High-frequency microsatellite instability was found in 43% of stage II/III proximal colon cancers and was associated with significantly higher numbers of activated cytotoxic lymphocytes. High-frequency microsatellite instability, as well as the content of intratumoral-activated cytotoxic T lymphocytes correlated with improved overall and disease-free survival, particularly in patients with stage III tumors. Multivariate analysis revealed that patients with both features had a risk of death and relapse markedly lower than that associated with microsatellite status or intratumoral cytotoxic lymphocytes separately. The presence of local cytotoxic immune responses is probably the major determinant of the good clinical course of patients with microsatellite unstable colon cancer. Furthermore, high-frequency microsatellite instability coupled with a high content of intratumoral cytotoxic lymphocytes may identify a subset of colon cancer patients with a favorable clinical outcome, particularly in stage III disease.