Maurizio Biagioli

Therapeutic efficacy of ipilimumab, an anti-CTLA-4 monoclonal antibody, in patients with metastatic melanoma unresponsive to prior systemic treatments: clinical and immunological evidence from three patient cases

The management of unresectable metastatic melanoma is a major clinical challenge because of the lack of reliably effective systemic therapies. Blocking cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has recently been proposed as a strategy to enhance cell-mediated immune responses to cancer, and clinical trials have demonstrated that anti-CTLA-4 therapy can produce durable outcomes with different response patterns than cytotoxic chemotherapy. We enrolled eight out of 155 patients with advanced melanoma in a multicentre phase II trial that evaluated the activity and tolerability of ipilimumab, a fully human, anti-CTLA-4 monoclonal antibody (www.clinicaltrials.gov; NCT00289627; CA184-008). Here we report our experience with three of these patients, who experienced progressive disease after a variety of previous therapies, including prior immunotherapies, and who achieved good outcomes with ipilimumab. One patient had a partial response ongoing at 17+ months on ipilimumab despite failure with four prior therapies, and the other two patients showed durable stable disease, both still ongoing at 17+ and 20+ months, respectively. The patient achieving a partial response experienced no side effects while receiving ipilimumab. The other two patients developed immune-related adverse events (irAEs) including rash (one case; grade 2) and diarrhoea (both cases; grades 1 and 2, respectively); the histopathology of colon biopsy samples from both was suggestive of colitis, with an abundant CD8+ T-cell infiltrate. Nausea, vomiting and acute pancreatitis were also observed in one patient. In addition, immunohistochemical findings of a dense CD8+, TIA1+ and granzyme B+ lymphoid infiltrate within a biopsied lesion provide indirect evidence of functional T-cell activation induced by treatment. These case reports highlight the potential for anti-CTLA-4-based therapy in previously treated patients with advanced melanoma. Moreover, because the patterns of response to ipilimumab differ from chemotherapy, we need to understand how and when patients may respond to treatment so that appropriate clinical decisions can be made.

Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions.

Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.

A new model of epidermal culture for the surgical treatment of vitiligo.

Background Vitiligo can be successfully treated with grafts of autologous cultured epidermal cells.

S100A13 is a new angiogenic marker in human melanoma

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called ‘dysplastic’ nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clarks level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7±0.7) to vertical growth phase (3.6±3.1) to metastases (7.0±7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

Novel variants of muscle calpain 3 identified in human melanoma cells: cisplatin- induced changes in vitro and differential expression in melanocytic lesions

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Different patterns of cell proliferation and death and oncogene expression in cutaneous malignant melanoma

Ninety‐six cutaneous melanomas (CMs) were investigated aiming at finding differences, if any, among tbe main four chnicopatho‐logical types, for Bcl‐2. c‐myc and p53 protein expression, and for tumor cell proliferation and death indices. Proliferation was assessed by calculating die mitotic index (MI, number of mitoses) and the MIBl labelling index (M‐LI, number of MIBI+ nuclei), and tumor cell death by calculating tbe apoptotic index (AI, number of apoptoses) among 1000 tumor cells. CMs were subdivided into thin (<1 mm) and intermediate thickness (1‐4 mm) tumors. Bcl‐2 expression did not significantly change among different types, c‐myc Expression decreased expecially in thicker superficial spreading (SSM) and lentigo maligna melanoma (LMM) types. p53 Expression was higher in nodular melanoma (NM) and in acral lentigiuous melanoma(ALM), which also showed the highest degrees of proliferation. AI was significantly higher in thin rather than in intermediate thickness SSMs, LMMs and ALMs (8.4 vs. 2; 6.1 vs. 2.3. and 5.8 vs. 3.6, respectively). AI was low in thin (1.7) and intermediate thickness (1.9) NMs. Which also showed high MI (3.9 and 4.5, respectively), and M‐LI (16.7 and 2.9, respectively). Thin and intermediate thickness ALMs also showed high MI and M‐LI (4.1 vs. 5.2 and 11.3 vs. 14.6, respectively). Bcl‐2 is among genes which inhibit apopiotic death, whereas c‐myc and p‐53 genes promote this process. In CMs. no relation was found between BcI‐2 expression, MI, PI, and AI. All SSMs, LMMs, and ALMs with a high AI showed a high c‐myc expression and were negative for p53. c‐myc, Although highly expressed, did not promote a significant apoptotic death in NM type. Bc12, c‐myc, and p53 were not equally expressed nor equally related to tumor cell turnover in all CMs, suggesting their different influence on the various types and stages, and the role of other factors in CM growth control.

Detection of telomerase activity and correlation with mitotic and apoptotic indices, Ki‐67 and expression of cyclins D1 and A in cutaneous melanoma

Telomerase plays a key role in carcinogenesis. It is activated in most immortal cell lines and human cancers, including cutaneous melanoma (CM). Increased cell proliferation and deregulation of the cell cycle occur in human cancers. Links between telomerase activity (TA), cell proliferation, cell death and expression of cell‐cycle regulators have not been extensively elucidated in CM. In this study, we investigated TA, mitotic index (MI), apoptotic index (AI), Ki‐67 and nuclear positivity of cyclins D1 and A (Ki‐67+N/1,000, cyclin D1+N/1,000, cyclin A+N/1,000) in 42 primary cutaneous melanomas (PCMs). TA was detected in all cases and directly correlated with MI, Ki‐67+N/1,000, cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001); it was not correlated with AI. When subdividing PCMs into radial and vertical growth phase melanomas (RGPMs, VGPMs), a correlation was maintained only with MI (p < 0.005) and cyclin D1+N/1,000 (p < 0.005). Although MI and Ki‐67+N/1,000 were highly correlated with cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001) when considering all cases together, a high correlation was found in the RGPM and VGPM groups between cyclin A+N/1,000 and Ki‐67+N/1,000 only (p < 0.001), thus suggesting that cyclin A is more closely correlated with cell proliferation than cyclin D1. Our results further support the association between TA, tumor cell proliferation and cyclin D1 and A expression in PCM, though it is possible that links between TA and proliferation, on the one hand, and TA and cyclin D1 expression, on the other, might occur following various pathways. Int. J. Cancer 88:411–416, 2000.

Composite cutaneous haemangioendothelioma: case report and review of the literature.

Composite cutaneous haemangioendothelioma is a recently characterized rare tumour of vascular origin. As there are only a few reported cases, the biological behaviour of the tumour and appropriate therapeutic approaches are not yet clear. We report a new case of composite cutaneous haemangioendothelioma and discuss prognostic and therapeutic aspects of this neoplasm.

Posttransplant primary cutaneous CD30(Ki-1)-positive anaplastic large T-cell lymphoma. A case report

agents in this setting is growing. Voriconazole is a new broad-spectrum antifungal agent active on Aspergillus spp., Candida spp. and other fungi. Voriconazole has previously been used as a salvage treatment for five patients with phaeohyphomycosis, all of whom were cured. The in vitro voriconazole mean MIC for three isolates of M. mycetomatis was 0Æ05 mg mL, compared with 0Æ08 and 0Æ03, respectively, for itraconazole and amphotericin B. The favourable outcome for our patient shows that voriconazole can be a valuable nonsurgical treatment for M. mycetomatis eumycetoma, although it requires high daily doses and lengthy administration. Dose and duration of antifungal treatment, as well as absence of bone involvement, seem predictive of the success of eumycetoma medical therapy. Without bone involvement initial medical treatment may facilitate limited surgical excision or even, as in our case, may be sufficient for a total cure. All authors stress the importance of lengthy follow-up before concluding that treatment has been successful. Our patient was disease-free 4 years after the end of treatment, confirming the efficacy of voriconazole treatment alone. To our knowledge, this is the first report of voriconazole treatment of eumycetoma. Voriconazole could represent a real progress in the antifungal treatment of mycetoma due to M. mycetomatis. Unfortunately, the prohibitive cost of this drug may limit its use, especially in endemic countries.

Nucleolin protein expression in cutaneous melanocytic lesions

Background:  Nucleolin is a major nucleolar argyrophilic protein involved in carcinogenesis. There are only few studies on its tissue expression in human cancer and none in melanoma. We aimed at exploring this protein and its prognostic impact in cutaneous melanocytic lesions.