Stefano Pepe

A phase II study of biweekly oxaliplatin plus infusional 5-fluorouracil and folinic acid (FOLFOX-4) as first-line treatment of advanced gastric cancer patients

The aim of the study was to assess the toxicity and the clinical activity of biweekly oxaliplatin in combination with infusional 5-fluorouracil (5-FU) and folinic acid (FA) administered every 2 weeks (FOLFOX-4 regimen) in patients with advanced gastric cancer (AGC). A total of 61 previously untreated AGC patients were treated with oxaliplatin 85 mg m−2 on day 1, FA 200 mg m−2 as a 2 h infusion followed by bolus 5-FU 400 mg m−2 and a 22 h infusion of 5-FU 600 mg m−2, repeated for 2 consecutive days every 2 weeks. All patients were assessable for toxicity and response to treatment. Four (7%) complete responses and 19 partial responses were observed (overall response rate, 38%). Stable disease was observed in 22 (36%) patients, with progressive disease in the other six (10%) patients. Median time to progression (TTP) and median overall survival (OS) were 7.1 and 11.2 months, respectively. National Cancer Institute Common Toxicity Criteria grade 3 and 4 haematologic toxicities were neutropenia, anaemia and thrombocytopenia in 36, 10 and 5% of the patients, respectively. Grade 3 peripheral neuropathy was recorded in three (5%) patients. FOLFOX-4 is an active and well-tolerated chemotherapy. Response rate (RR), TTP and OS were comparable with those of other oxaliplatin-based regimens, suggesting a role for this combination in gastric cancer.

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor, in head and neck squamous carcinoma cells.

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous‐cell carcinomas of head and neck (SCCHN). ZD1839 (‘Iressa’) is an orally active, selective EGFR‐TKI (EGFR‐tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host‐dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR‐mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27KIP1 and p21CIP1/WAF1 cyclin‐dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose‐dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27KIP1 and p21CIP1/WAF1 proteins associated with CDK2‐cyclin‐E and CDK2‐cyclin‐A complexes. In addition, ZD1839‐induced growth inhibition was significantly reduced in cell transfectants expressing p27KIP1 or p21CIP1/WAF1 antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27KIP1 and p21CIP1/WAF1 upregulation, suggest a mechanistic connection between these events.

Epidermal Growth Factor Receptor (EGFr) Expression in Non-small Cell Lung Carcinomas Correlates with Metastatic Involvement of Hilar and Mediastinal Lymph Nodes in the Squamous Subtype

Epidermal growth factor receptor (EGFr) levels were evaluated in paraffin-embedded tumour specimens of non-small cell lung cancer (NSCLC) from 176 patients who underwent surgical resection. The EGFr expression was evaluated by immunocytochemical assay using a monoclonal antibody which recognises the external domain of the receptor. EGFr immunoreactivity was significantly higher in squamous than in non-squamous cell carcinomas (P = 0.0009). Hilar and/or mediastinal nodal involvement was found in 29 of 105 (27.4%) squamous cancers, and in this group of patients, the mean of EGFr positive cells was significantly higher than that of patients without nodal involvement (P = 0.01). No significant correlations were found between the expression of EGFr and other clinicopathological or biological parameters such as T-status, grading, proliferative activity. EGFR is suggested to represent a useful indicator of nodal metastasis in NSCLC.

Desferioxamine increases iron depletion and apoptosis induced by ara‐C of human myeloid leukaemic cells

We investigated whether changes in iron metabolism and the transferrin receptor (TRF‐R) expression were involved in the antileukaemic effects of arabinoside cytosine (ara‐C). Treatment with 100 n M ara‐C for 48 h reduced thymidine uptake and increased the surface expression of the TRF‐R on leukaemic blasts derived from 13/16 (81%) patients and on the HL‐60 and U‐937 cell lines. Whereas intracellular non‐haem iron was strongly depleted 24 h after ara‐C addition, TRF‐R up‐regulation and recovery of intracellular non‐haem iron concentration occurred together after a longer exposure of the cultured cells to the drug. Since iron is an essential regulator of cell proliferation we have evaluated the effects of the combination between ara‐C and the iron chelator desferioxamine (DSF) on the growth of HL‐60 and U‐937 cells. We found that desferioxamine strongly potentiated the effects of ara‐C on leukaemic cell growth inhibition and apoptosis. This is the first report of a positive interaction between ara‐C and an iron chelator in terms of antileukaemic effects.

The expression of proliferating cell nuclear antigen in paraffin sections of peripheral, node-negative non-small cell lung cancer.

Cell proliferation of 40 peripheral, node‐negative non‐small cell lung cancers (NSCLC) treated with surgery alone was investigated by immunohistochemical analysis with the monoclonal antibody (MoAb) PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin‐fixed and paraffin‐embedded material. Results were correlated with DNA ploidy and S‐phase fraction (SPF) analyzed by DNA flow cytometric study. Mitotic count (MC) was analyzed by light microscopic study and histopathologic features. PCNA immunoreactivity was seen in all samples and confined to the nuclei of cancer, but not to the surrounding, tumor‐negative cells; its frequency ranged from 0–70% (median, 15%), and tumors expressed either a low (0–25%, n = 25) or intermediate (26–75%, n = 15) proliferative activity. There was no relationship between PCNA immunoreactivity and tumor stage or among size, histologic type, and mitotic count (MC). Tumors with intratumoral blood vessel invasion (BVI) showed a significantly higher (P < 0.005) PCNA immunoreactivity than BVI‐negative tumors. PCNA scores were significantly higher (P < 0.005) in DNA aneuploid (n = 22) than in DNA diploid (n = 18) tumors and correlated significantly with the SPF of DNA aneuploid tumors (r = 0.825, P < 0.0001), but not with diploid tumors (r = 0.002, P = < 0.9). Intermediate proliferating tumors had a significantly higher (P < 0.01) MC than their counterparts. In univariate analysis, significant predictors of survival were tumor classification (T1 versus T2), tumor size (less than or equal to 2.6 cm versus more than 2.6 cm), BVI (BVI‐negative versus BVI‐positive), MC (less than or equal to 8 versus more than 8), and PCNA immunoreactivity (low versus intermediate). DNA ploidy and SPF did not influence survival significantly. Only PCNA immunoreactivity retained its independent level of significance (P = 0.02) by multivariate analysis. It was concluded that PCNA immunostaining is a simple and clinically useful method for estimating cell proliferation in formalin‐fixed, paraffin‐embedded tissue of resected peripheral, node‐negative NSCLC.

Interferon-α induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor

We have demonstrated that interferon-α2-recombinant (IFNα) at growth inhibitory concentrations enhances the expression and signalling activity of the epidermal growth factor receptor (EGF-R) in human epidermoid carcinoma KB cells. Here we report that KB cells exposed to IFNα underwent apoptotic cell death and this effect was antagonized by EGF. We have also found that IFNα enhanced the expression of heat shock proteins (HSP) HSP-70, HSP-90 and HSP-27 and activated the NH2-terminal Jun kinase-1 (JNK-1) and p38 mitogen activated protein kinase, the target enzymes of a stress-dependent intracellular transduction pathway. Moreover, the overexpression of the wild-type JNK-1, obtained through plasmid transfection of KB cells, induced apoptosis which was potentiated by the exposure of wild-type JNK-1 (JNK-1wt)-transfected cells to IFNα. All these effects were neutralized by the addition of EGF to parental and JNK-1wt-transfected KB cells exposed to IFNα. In conclusion, EGF has a protective effect on KB cells from apoptosis while antagonizing a stress response elicited by IFNα and targeted on the stress pathway terminal kinases.

EGF activates an inducible survival response via the RAS-> Erk-1/2 pathway to counteract interferon- α -mediated apoptosis in epidermoid cancer cells

AbstractThe mechanisms of tumor cell resistance to interferon-α (IFNα) are at present mostly unsolved. We have previously demonstrated that IFNα induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNα-induced apoptosis depends upon activation of the NH2-terminal Jun kinase-1 (Jnk-1) and p38 mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNα increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNα occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNα via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNα-treated cells. Moreover, IFNα induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNα. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNα-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNα-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNα.

Synergistic Antitumor Activity of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Gefitinib and IFN-α in Head and Neck Cancer Cells In vitro and In vivo

Purpose: Epidermal growth factor receptor (EGFR) overexpression has been implicated in the development of head and neck squamous cell carcinomas (HNSCC) and represents a potential therapeutic target for this disease. We have reported previously that growth inhibitory concentrations of IFN-α enhance the expression and activity of EGFR and that this effect could represent an escape mechanism to the growth inhibition and apoptotic cell death induced by IFN-α. In this study, we investigate whether the combination of IFN-α and gefitinib (Iressa, AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom), a selective EGFR tyrosine kinase inhibitor, might have a cooperative antitumor effect on HNSCC-derived cell lines. Experimental Design: The interaction of IFN-α and gefitinib was evaluated in vitro on HNSCC-derived cell lines by median drug effect analysis calculating a combination index with CalcuSyn software and in vivo by using HNSCC xenografts in nude mice. The mechanism of gefitinib and IFN-α interactions was also studied by analysis of cell cycle kinetics, apoptosis assays, and Western blotting of EGFR signal transduction components. Results: Simultaneous exposure to gefitinib and IFN-α produced synergistic antiproliferative and proapoptotic effects compared with single drug treatment. Furthermore, daily treatment of gefitinib (50 mg/kg p.o.) in combination with an IFN-α regimen (50,000 units s.c. three times weekly) induced tumor growth delay and increased survival rate on established HNSCC xenografts in nude mice. Moreover, the concomitant treatment with gefitinib suppressed the stimulation of extracellular signal-regulated kinase phosphorylation/activity induced by IFN-α both in vitro and in vivo. Conclusion: The observed cooperative antitumor effects could be, at least in part, explained by the inhibition exerted by gefitinib of an IFN-α-induced EGF-dependent survival pathway, which involves extracellular signal-regulated kinase activation. These results provide a rationale for the clinical evaluation of gefitinib in combination with IFN-α in HNSCC.

ipilimumab in the treatment of metastatic melanoma: management of adverse events

Recently, “ipilimumab,” an anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, has been demonstrated to improve overall survival in metastatic melanoma. “CTLA-4” is an immune-checkpoint molecule that downregulates pathways of T-cell activation. Ipilimumab, by targeting CTLA-4, is able to remove the CTLA-4 inhibitory signal, allowing the immune system to react to cancer cells. Due to its immune-based mechanism of action, ipilimumab causes the inhibition of CTLA-4-mediated immunomodulatory effects, the enhancement of antitumor specific immune response mediated by the weakening of self-tolerance mechanisms while exacerbating the development of autoimmune diseases and immune-related adverse events, including dermatitis, hepatitis, enterocolitis, hypophysitis, and uveitis.

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.