Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.

Improved Anti-Tumoral Therapeutic Efficacy of 4-Hydroxynonenal Incorporated in Novel Lipid Nanocapsules in 2D and 3D Models.

4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.

Melanoma of the lower extremities: foot site is an independent risk factor for clinical outcome.

BACKGROUND: Despite the better prognosis of melanomas localized on lower extremities, some studies have suggested that melanomas on the foot are related to a poorer survival and should be considered separately. OBJECTIVE: To review our case series of cutaneous melanomas on the lower extremities and to analyze the clinicopathological association, time course, types of progression, and survival differences. METHODS: We included 1671 patients (stage 0-II) with a cutaneous melanoma on the lower extremities (subungual melanomas were excluded). Of these, 327 were localized on the foot. Multivariate analyses were performed to evaluate disease-specific survival and disease-free interval. RESULTS: Distribution of known prognostic factors and patterns of progression of foot and leg melanoma differ across genders. The foot site was confirmed as a negative independent prognostic factor on disease-specific survival and disease-free interval. CONCLUSION: Foot melanoma could represent a particular subgroup, which could require specific management in the future.

Melanoma density and relationship with the distribution of melanocytic naevi in an Italian population: a GIPMe study--the Italian multidisciplinary group on melanoma.

The most frequent site for melanoma is the back in men and the lower limbs in women, where intermittent sun exposure has been reported to be an environmental agent, although studies on age-specific incidence have suggested that melanoma in chronically sun-exposed areas, such as the face, increases with age. To identify the preferential development of melanoma in chronically or intermittently sun-exposed areas and the relationship between body site distribution and parameters such as sex, age, distribution of melanocytic naevi, atypical naevi and actinic keratoses, a prospective epidemiological multicentre study was carried out on all the consecutive melanoma cases diagnosed in a 2-year period from 27 Italian GIPMe centres (GIPMe: the Italian Multidisciplinary Group on Melanoma). Both the relative density of melanoma (RDM), defined as the ratio between observed and expected melanoma for a specific body site, and the average nevi density were identified. The most common melanoma site was the back, a factor that was not affected by either age or sex, even if men had higher density values. Statistically significant higher RDM values were observed in women aged more than 50 years for leg lesions and in the anterior thighs for young women (<50 years), whereas the lowest values were observed in the posterior thighs in women of any age. Facial RDM was statistically significantly higher than expected in both male and female patients more than 50 years of age. Melanoma was associated with a significantly higher atypical naevi density only for the back, chest and thighs. Indeed, facial melanoma was related to the presence of more than four actinic keratoses and not naevi density. To the best of our knowledge, the RDM method was applied for the first time together with naevus density calculation to obtain these data, which strongly substantiate the 'divergent pathway' hypothesis for the development of melanoma, but not find a direct correlation between melanoma and nevi for each anatomical site.

Clinical experience with ipilimumab 3 mg/kg: real-world efficacy and safety data from an expanded access programme cohort.

BACKGROUND: Ipilimumab improves survival in patients with advanced melanoma. The activity and safety of ipilimumab outside of a clinical trial was assessed in an expanded access programme (EAP). METHODS: Ipilimumab was available upon physician request for patients aged 16 or over with pretreated stage III (unresectable)/IV melanoma, for whom no other therapeutic option was available. Patients received ipilimumab 3 mg/kg every 3 weeks for four doses. Patients with stable disease or an objective response to ipilimumab were eligible for retreatment upon disease progression. Tumour assessments were conducted at baseline and week 12. Patients were monitored for adverse events (AEs) within 3 to 4 days of each scheduled visit. RESULTS: Of 855 patients participating in the EAP in Italy, 833 were evaluable for response. Of these, 13% had an objective immune response, and the immune-related disease control rate was 34%. Median progression-free survival and overall survival were 3.7 and 7.2 months, respectively. Efficacy was independent of BRAF and NRAS mutational status. Overall, 33% of patients reported an immune-related AE (irAE). The frequency of irAEs was not associated with response to ipilimumab. CONCLUSIONS: Outside of a clinical trial setting, ipilimumab is a feasible treatment option in patients with pretreated metastatic melanoma, regardless of BRAF and NRAS mutational status. Data from this large cohort of patients support clinical trial evidence that ipilimumab can induce durable disease control and long-term survival in patients who have failed to respond to prior treatment.

Efficacy and safety of ipilimumab in elderly patients with pretreated advanced melanoma treated at Italian centres through the expanded access programme.

BACKGROUND: Elderly patients with metastatic melanoma have different disease characteristics and a poorer prognosis than younger patients. Data from clinical trials and expanded access programmes (EAPs) suggest ipilimumab confers a consistent survival benefit and has a similar safety profile across different age groups of patients with metastatic melanoma. Here we report the efficacy and safety of ipilimumab 3 mg/kg in elderly patients enrolled in an EAP in Italy. METHODS: Patients aged > 70 years with pretreated melanoma received ipilimumab 3 mg/kg every 3 weeks for four doses through an EAP. Tumour response was evaluated at baseline and after completion of induction therapy using immune-related response criteria and patients were monitored throughout the treatment period for adverse events (AEs), including immune-related AEs. RESULTS: The immune-related disease control rate among 188 evaluable patients was 38%, including four patients with an immune-related complete response, 24 with an immune-related partial response and 44 with immune-related stable disease. Median progression-free survival (PFS) was 4.0 months and the 1- and 2-year PFS rates were 21% and 12%, respectively. Median overall survival (OS) was 8.9 months; 1- and 2-year OS rates were 38% and 22%, respectively. The safety profile of ipilimumab was consistent with that observed in the general population of the Italian EAP and treatment-related AEs generally resolved within a median of 2 weeks with treatment as per protocol-specific guidelines. CONCLUSIONS: These results suggest ipilimumab is a feasible treatment option in elderly patients with metastatic melanoma. Ipilimumab treatment was generally well tolerated and resulted in clinical benefit and extended survival in elderly patients treated at centres in Italy.

Sequential treatment with ipilimumab and BRAF inhibitors in patients with metastatic melanoma: data from the Italian cohort of the ipilimumab expanded access program.

Of 93 patients with pretreated, BRAF(V600) mutation-positive advanced melanoma who received vemurafenib or dabrafenib before (n = 45) or after (n = 48) treatment with ipilimumab 3 mg/kg, median overall survival (mOS) from first treatment was 9.9 and 14.5 months, respectively. Among patients treated with a BRAF inhibitor first, mOS from the end of BRAF inhibition was 1.2 months for those who did not complete ipilimumab treatment as per protocol, compared with 12.7 months for those who did (p < .001). Prospective, randomized studies are required to determine the optimal sequencing of ipilimumab and BRAF inhibitors in patients with BRAF-mutated metastatic melanoma.

A retrospective analysis of the costs and management of genital warts in Italy.

BACKGROUND: In Italy the prevalence of genital warts in women (15-64 years) is approximately 0.6% with an incidence of 0.4% per year. Treatments for GW are usually long, with moderate success and high costs. The aim of the study was to evaluate the diagnostic-therapeutic pathway, duration and setting of treatment, costs of episodes of condyloma in a population attending a regional STI clinic in Piedmont. METHODS: This was a retrospective observational study conducted using medical records of outpatients who first visited the STI Clinic of San Lazzaro Dermatological Hospital in 2008. The patients' medical histories were analysed for episodes that occurred and were cleared in 18 months following the initial visit. Data on screening methods for STIs, type of diagnosis for condyloma, treatment type, treatment setting, and anatomic lesion site were obtained from medical records. The costs were calculated for each episode. RESULTS: A total of 450 episodes were analysed (297 men,153 women). The most frequently affected anatomic site was the genital area (74%) in both genders. With regard to treatment setting, 78.44% of patients received outpatient treatment at the STI clinic, 4% were treated at home, and 0.22% were hospitalised; 11.11% were treated in multiple settings. The mean number of treatments per episode was 2.03; although many patients received only 1 treatment (n = 207, 46%), exspecially cryotherapy or diathermy coagulation (64.73% versus 28.02% of episodes, respectively). The mean episode duration was 80.74 days. The mean cost (in 2011 euros) for an episode was €158.46 ± 257.77; the mean costs were €79.13 ± 57.40 for diagnosis and €79.33 ± 233.60 for treatment. The mean cost for treatment in a STI-Clinic setting was €111.39 ± 76.72, that for home treatment was €160.88 ± 95.69, and that for hospital care was €2825.94. CONCLUSIONS: The treatment of and associated costs for genital warts are significant. Several factors affect the cost, and internal STI clinic protocols, such as the 6 month window used to consider a recurrence or new diagnosis, create bias. Nonetheless, our findings how costs similar to those reported in the international literature and should be considered when deciding on which HPV vaccination programs should be provided by the public health system.

Adjuvant ganglioside GM2-KLH/QS-21 vaccination versus observation after resection of primary tumor > 1.5 mm in patients with stage II melanoma: results of the EORTC 18961 randomized phase III trial.

PURPOSE: The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation. PATIENTS AND METHODS: A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat. RESULTS: After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity. CONCLUSION: GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

Diagnostic and therapeutic approaches in Italian hospitals: adjuvant and metastatic therapy in melanoma.

Melanoma incidence and mortality rates are rising in Italy, indicating that more effective treatments are required both in the adjuvant and metastatic settings. We analyzed clinical practices in the adjuvant and metastatic settings by conducting a nationwide survey of clinicians responsible for managing melanoma treatment and follow-up in a representative sample of Italian hospitals. 95% of participating hospitals completed the panel of questions on adjuvant and metastatic treatment, making it likely that these results give a realistic picture of treatment and follow-up of melanoma patients in Italy. In low-volume hospitals (<25 new melanoma diagnoses yearly) adjuvant therapy was significantly more used than in large-volume hospitals for patients in stage III and IV (82 versus 66% and 56 versus 30%, respectively), and only 11% of patients were enrolled in clinical trials. In the metastatic setting dacarbazine was the preferred first-line treatment (32%) followed by polychemotherapy (23%); 12% of patients were enrolled in clinical trials and less than 10% received interleukin-2, usually subcutaneously. The information provided by this study was used by the Italian Melanoma Intergroup to improve the quality of care and to redirect financial resources.

Intralesional cidofovir for the treatment of multiple and recalcitrant cutaneous viral warts.

Cidofovir is a nucleoside analog of deoxycytidine with a strong activity against a broad spectrum of DNA viruses, including human papillomavirus. The first objective was to evaluate efficacy of cidofovir for the treatment of cutaneous viral warts, recalcitrant after conventional therapies or where the surgery approach is difficult for their location or extension. Second, the present authors propose to point out possible local and systemic side effects consequent to treatment. Two-hundred eighty patients affected by recalcitrant cutaneous viral warts, were treated with intralesional cidofovir 15 mg/mL once a month. The present authors stated that candidates were those who had made before at least two other treatments reported in the guideline for management of cutaneous viral warts. In 276 cases, warts completely cleared: 158 of those have a follow-up period longer than 12 months and 118 have a follow-up of 6 months. On the average, 3,2 injections were enough to solve the problem. Local side effects consisted of pain and burning sensation during the injections; itching, erythema, and post-inflammatory hyperpigmentation were observed. No cases of systemic side effects were noted. The treatment was well tolerated, and the warts were completely cleared without relapses. Intralesional cidofovir is emerging as an effective therapeutic alternative for warts that are unresponsive to conventional treatments.

Time course, clinical pathways, and long-term hazards risk trends of disease progression in patients with classic mycosis fungoides: a multicenter, retrospective follow-up study from the Italian Group of Cutaneous Lymphomas.

BACKGROUND: Mycosis fungoides (MF) is an indolent primary cutaneous T-cell lymphoma. To the authors' knowledge, no data currently are available regarding the evolution over time of the risk of developing specific pathways of disease progression. METHODS: This retrospective study analyzed 1422 patients with MF who were diagnosed and followed from 1975 through 2010 in 27 Italian Study Group for Cutaneous Lymphoma centers. The primary objectives were to ascertain the time course, pathways, and hazards risk trends of cutaneous/extracutaneous disease progression; to evaluate whether different tumor-lymph node-metastasis-blood (TNMB) stages have different pathways of disease progression; and to analyze differences between tumor-stage and erythrodermic MF with regard to clinical onset, disease evolution, and prognosis. The secondary objective was to provide a further validation for the revised International Society for Cutaneous Lymphomas and the Cutaneous Lymphoma Task Force of the European Organization of Research and Treatment of Cancer (ISCL/EORTC) classification. RESULTS: The median follow-up was 14.5 years; stage progression occurred in 29.7% of patients and blood involvement was the most frequent extracutaneous site of disease progression. Patients with stage IA to stage IB disease demonstrated a steady low annual incidence of disease progression to tumor-stage (1%-2%); patients with stage IIA disease had a higher risk within the first years (up to 9.4%). Erythroderma evolved with a significantly higher frequency from patches/plaques (13.9%/28.2%) than tumors (P = .028 and P = .013, respectively). Hazards rates of extracutaneous involvement were low (< 1%). The T-score was found to be associated with extracutaneous involvement site, tumor-stage disease with lymph node/visceral lesions, and erythroderma with blood involvement. TNMB classification and stage progression resulted as independent prognostic variables being detected on multivariate analysis; the type of extracutaneous involvement was found to affect survival . CONCLUSIONS: The data from the current study support the need for a stage-tailored follow-up, suggest that the classification of tumor-stage disease at a stage below erythroderma could be modified, and offer a further validation for the revised TNMB classification.

Functional and phenotypical impairment of polymorphonuclear cells in atopic dermatitis: an additional cause for the known susceptibility to infections?.

BACKGROUND: Atopic dermatitis (AD) patients present an high susceptibility to infections. The phagocytic activity of polymorphonuclear granulocytes (PMNs) is mediated by the interactions between Toll-like receptors (TLRs) and pathogen-associated molecular patterns. OBJECTIVE: To investigate functional activity and phenotype of PMNs in AD patients. METHODS: In vitro PMN phagocytosis and intracellular killing towards Klebsiella pneumoniae were evaluated in 24 AD patients; flow cytometry was applied to analyze PMN phenotype. RESULTS: PMNs from AD patients displayed both reduced phagocytic activity and intracellular killing against K. pneumoniae than healthy subjects (HS). CD11b, CD66b, TLR2, TLR4 and TLR5 median fluorescence intensity (MFI) on PMN membrane were significantly higher in AD patients than in HS. CONCLUSION: PMN functional impairment in AD patients could represent an additional cause of skin infections, coupled with other known defects in the innate immune system. The increased MFI of adhesion molecules and TLRs is rather a consequence of the increased skin barrier permeability to bacterial molecules capable of stimulating immunological reactions.

Thymopentin down-regulates both activity and expression of iNOS in blood cells of Sézary syndrome patients.

While thymopentin has been used for many years in the experimental treatment of Sézary syndrome (SS), a rare and very aggressive lymphoma, its mechanism of action is still not known. Herein we show that this peptide acts as an inhibitor of isolated iNOS and nNOS isoforms, and reduces iNOS protein/mRNA levels and iNOS activity in blood cells obtained from both healthy donors and SS patients. Similar results were obtained with TPN-2, the N(ω)-nitro analogue of the Arg-Lys motif present in thymopentin. Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of iNOS down-regulation in the therapeutic action of these peptides against SS.

Italian Euromelanoma Day Screening Campaign (2005-2007) and the planning of melanoma screening strategies.

Although no study has definitively shown that unfocused screening of skin cancer is effective, many campaigns have been organized with the aim of increasing awareness on melanoma risk factors. The objective of this study was to analyse the results of the Skin Cancer Screening Day in Italy during the period 2005-2007, to determine the priorities for melanoma control plans in a Mediterranean country. A total of 5002 patients were screened by dermatologists in 31 cities. Individuals who considered themselves to have many naevi and those with a family history of melanoma showed a higher number of common and atypical naevi. Ten melanomas, 20 basal cell carcinomas and two squamous cell carcinomas were histopathologically confirmed. Our observations provide the following suggestions for melanoma prevention strategies: (a) an unfocused campaign is suitable to inform the public about the importance of self-examination of the skin, but is not useful to identify a larger number of melanomas; and (b) melanoma screening campaigns should focus on a selected population, which meets rigorous risk criteria to maintain higher cost-effectiveness. The financial support to effective melanoma screening programmes could be increased, especially in southern populations where lower levels of self-surveillance and socioeconomic conditions represent risk factors for late identification of melanoma.

Electrochemotherapy in the treatment of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial.

BACKGROUND: Electrochemotherapy (ECT) is an emerging treatment for cutaneous lesions of different tumor types. The combination of chemotherapy and electroporation enhances drug uptake into tumoral cells. However, its role in the treatment of Kaposi sarcoma (KS) has not yet been well defined, and to date, literature reports are scarce. We prospectively evaluated clinical activity and safety of ECT in KS patients. METHODS: Twenty-three patients with histologically confirmed unresectable KS, not treatable by radiotherapy or intralesional vincristine therapy, were enrolled onto the study according to the European Standard Operating Procedures of Electrochemotherapy (ESOPE) guidelines and treated with a pulse generator. RESULTS: A response to the first ECT session was obtained in all patients, with a complete response (CR) in 14 (60.9%) of 23 patients. A second ECT was performed in 5 (21.7%) and a third in 2, with a median interval between two sessions of 5.1 (range 2.5-25.5) months. Overall, a total of 15 patients (65%) experienced a CR. After a median follow-up of 1.5 years (range 2 months to 4.2 years), 16 patients maintained the response, 4 after repeated courses. Sustained local control of treated lesions was present in 20 of 23 patients. The overall survival rate was 74.4% at 2 years. CONCLUSIONS: ECT represents an additional therapeutic tool for the management of KS cutaneous lesions, characterized by a definite clinical activity and long-lasting remissions. The absence of systemic side effects and the low impact on the immune system also make this treatment suitable for elderly people, even with repeated courses.

Functional and phenotypical alterations of polymorphonuclear cells in Sézary syndrome patients.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma (CTCL), has a poor prognosis and infections represent the most frequent cause of death. Polymorphonucleate granulocytes (PMNs) constitute an essential part of the innate immune system: their phagocytic and killing activity against pathogens is mediated by the interactions between Toll-like receptors (TLRs) and the Pathogen-associated molecular patterns (PAMPs). The aim of this study was to investigate PMN functional activity and phenotype in SS patients and their correlation with the onset of infectious complications. This prospective study enrolled 18 consecutive SS patients; PMN functional activity was evaluated by phagocytosis and intracellular killing tests towards Klebsiella pneumoniae. Flow-cytometry was applied to analyze PMN phenotype. PMNs from SS patients displayed a reduced phagocytic activity and intracellular killing against K. pneumoniae at 30 min and 60 min, more pronounced in SS patients with recurrent infections. CD11b and CD66b median fluorescence intensity (MFI) was significantly higher in SS than in healthy subjects, whereas CD62L MFI was decreased. No significant differences in TLR2, 4, 8 and 9 percentage expression or MFI were found. An increased TLR5 percentage expression was documented. The impairment in PMN functional activities in SS could favour the immune-suppression and raise infection risk.