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Background: Melanoma guidelines recommend surgical excision with 2-cm margins for melanomas >2 mm in thickness. However, this procedure may be problematic at critical anatomic sites. We aimed to compare the outcomes of wide (2 cm) versus narrow (1 cm) excision margins in patients with melanoma >2 mm in thickness near critical structures., Patients and Methods: We retrospectively examined 736 patients undergoing excision with wide versus narrow margins at the National Cancer Institute in Milan, Italy, between 2001 and 2015., Results: A total of 265 (36.0%) patients received a wide local excision-82 (30.9%) with linear repair and 183 (69.1%) with flap or graft reconstruction. A total of 471 (64.0%) patients received a narrow excision-320 (67.9%) with linear repair and 151 (32.1%) with flap or graft reconstruction (P<.001). The 10-year overall survival rate was 69.5% (95% CI, 63.3%-76.2%) in the wide group and 68.7% (95% CI, 63.8%-74.0%) in the narrow group (P=.462); 10-year crude cumulative incidence (CCI) of local recurrence was 5.4% (95% CI, 3.2%-9.2%) in the wide and 8.8% (95% CI, 6.4%-12.1%) in the narrow group (P=.150). Multivariable Fine-Gray modeling of the CCI of local recurrence showed that Breslow thickness (P=.010) was the only statistically significant parameter. Multivariable Cox models for overall survival showed that age (P<.001), Breslow thickness (P<.001), and sentinel lymph node status (P=.019) were statistically significant covariates. Excision margin was not a significant parameter affecting patients' outcome., Conclusions: Wide local excision with 1-cm margins for melanoma >2 mm in thickness was not associated with an increased risk of local recurrence and did not affect overall survival.

Background: This study aimed to improve the understanding of the prognostic value of tumor mitotic rate (TMR) in cutaneous melanoma and assessed its significance as a predictor for overall, melanoma-specific, and recurrence-free survival., Patients and Methods: This is a retrospective multicenter Italian cohort study of 13,016 patients diagnosed with and treated for invasive primary melanoma between 2005 and 2020 with median follow-up of 5.5 years. The survival probability was assessed by Kaplan-Meier method, hazard ratios (HRs), and corresponding 95% confidence interval (CI) of all-cause mortality and recurrence/death by multivariable Cox proportional hazards models., Results: Higher dermal mitoses number was associated with decreased overall survival. Among patients with TMR 0/mm 2 , 1/mm 2 , 2/mm 2 -3/mm 2 , 4/mm 2 -10/mm 2 , and >10/mm 2 , 5-year overall survival (OS) was 97.3%, 93.6%, 88.3%, 73.0%, and 60.9%, respectively. In multivariate analyses, compared to TMR of 0/mm 2 , HRs for all-cause mortality were 1.35 (95% CI, 1.08-1.68), 1.70 (95% CI, 1.40-2.07), 2.04 (95% CI, 1.67-2.49), and 2.39 (95% CI, 1.90-3.00) for 1 mitoses/mm 2 , 2 mitoses/mm 2 -3 mitoses/mm 2 , 4 mitoses/mm 2 -10 mitoses/mm 2 , and >10 mitoses/mm 2 , respectively. A similar increase in risks was observed in melanoma-specific survival (MSS) and recurrence-free survival (RFS). The HRs for MSS and RFS for the highest compared to the lowest TMR category were 3.01 (95% CI, 2.20-4.11) and 2.26 (95% CI, 1.88-2.73), respectively. Sentinel lymph-node biopsy positivity was significantly associated with TMR increase even with adjustment for several potential confounders., Conclusions: A clear association was demonstrated between an increasing TMR and decreased OS, MSS, and RFS, suggesting a reconsideration of TMR prognostic role for future inclusion in the melanoma staging system., Plain Language Summary: The 8th American Joint Committee on Cancer for melanoma staging removed tumor mitotic rate (TMR) from the staging criteria for T1 melanomas, giving way to ulceration and tumor thickness as stronger prognostic predictors. However, it is still recommended that TMR should be assessed and recorded in all primary invasive melanomas. In a large retrospective multicenter study on primary invasive melanomas, we investigated the prognostic value of TMR to assess its significance as survival predictor. Our results showed a clear association between increasing TMR and decreased patients' survival, suggesting that TMR should be considered for inclusion in the melanoma staging system., (© 2023 American Cancer Society.)

Melanoma patients have a high risk of developing subsequent primary melanomas, a condition known as multiple primary melanoma (MPM). We aimed to compare risk factors of patients with MPM and single primary melanoma (SPM). Primary MPM and SPM consecutively treated at the National Cancer Institute in Milan, Italy, from 1978 to 2021 were retrospectively investigated. Demographic and clinicopathological characteristics were analyzed. Multivariate hazard ratios and mortality were estimated using Cox proportional hazards regression models. Overall, 9122 patients with SPM and 944 with MPM were included. A total of 1437 and 85 deaths occurred in SPM and MPM group, respectively. Of these, 1315 (14.4%) within SPM patients and 60 (6.4%) in MPM group were melanoma-specific deaths (MSDs). Males had a higher risk for MPM (hazard ratio = 1.29), while age was not associated with MPM (hazard ratio = 0.98). The risk of MPM decreased by about 50% for Breslow thickness >1 mm, and by about 45 and 75% in presence of mitoses and ulceration, respectively. The multivariate hazard ratio of death for MPM compared to SPM patients was 0.85 (95% confidence interval, CI: 0.67-1.06), while considering MSD the corresponding hazard ratio was 0.93 (95% CI: 0.71-1.22). Melanoma patients should receive regular follow-up with complete skin examination to detect early subsequent primary melanoma. Patients with more advanced primary have decreased risk of MPM, while males have higher risk. Our study reported no significant difference in mortality between SPM and MPM, but the issue is still open for discussion and further studies., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Melanoma guidelines recommend surgical excision with 10 mm margins for T1 melanomas (invasive melanomas with Breslow thickness ≤1 mm), including those in radial growth phase, which are without metastatic potential; however, such margins may be problematic on head-and-neck., Objective: We compared outcomes of wide (10 mm margins) versus narrow (5 mm margins) excisions in patients with radial growth phase T1 melanoma on head-and-neck including face., Methods: We retrospectively examined 610 consecutive patients excised with wide versus narrow margins, from 2001 to 2018, at six European centres. In all cases, radial growth phase, and clear margins with 5 or 10 mm of clearance, were ascertained histologically. Multivariable models investigated associations of margins and other factors with overall survival and local recurrence., Results: Three hundred and sixteen (51.8%) patients received wide excision, 219 (69.3%) with primary wound closure, 97 (30.7%) with reconstruction; 294 (48.2%) patients received narrow excision, 264 (89.8%) with primary wound closure, 30 (10.2%) with reconstruction (p < 0.001). Median follow-ups were 88 months (wide) and 187 months (narrow) (inter-quartile ranges 43-133 and 79-206, respectively). Ten-year overall survival (95% confidence interval) was 96.7% (94.2%-99.3%) in wide and 98.2% (96.4%-100%) in narrow patients. Ten-year local recurrence incidence was 6.4% (4.1%-10.1%) in wide and 7.8% (5.3%-11.6%) in narrow groups. Lentigo maligna melanoma subtype appeared associated with increased risk of local recurrence in narrow versus wide patients (15.0% vs. 7.5%; p = 0.190)., Conclusions: Narrower excision margins for T1 radial growth phase melanoma are not associated with worse overall survival (hazard ratio 0.97, p = 0.996) or increased local recurrence (subdistribution hazard ratio: 0.87; p = 0.751) compared to wider margins, and may be safely applied to such lesions, although caution may be required in the presence of lentigo maligna melanoma., (© 2023 European Academy of Dermatology and Venereology.)

Background: Skin metastases are an important co-morbidity in melanoma. Despite broad adoption, electrochemotherapy implementation is hindered by a lack of treatment indications, uncertainty regarding procedural aspects, and the absence of quality indicators. An expert consensus may harmonize the approach among centres and facilitate comparison with other therapies., Methods: An interdisciplinary panel was recruited for a three-round e-Delphi survey. A literature-based 113-item questionnaire was proposed to 160 professionals from 53 European centres. Participants rated each item for relevance and degree of agreement on a five-point Likert scale, and received anonymous controlled feedback to allow revision. The items that reached concordant agreement in two successive iterations were included in the final consensus list. In the third round, quality indicator benchmarks were defined using a real-time Delphi method., Results: The initial working group included 122 respondents, of whom 100 (82 per cent) completed the first round, thus qualifying for inclusion in the expert panel (49 surgeons, 29 dermatologists, 15 medical oncologists, three radiotherapists, two nurse specialists, two clinician scientists). The completion rate was 97 per cent (97 of 100) and 93 per cent (90 of 97) in the second and third rounds respectively. The final consensus list included 54 statements with benchmarks (treatment indications, (37); procedural aspects, (1); quality indicators, (16))., Conclusion: An expert panel achieved consensus on the use of electrochemotherapy in melanoma, with a core set of statements providing general direction to electrochemotherapy users to refine indications, align clinical practices, and promote quality assurance programmes and local audits. The residual controversial topics set future research priorities to improve patient care., (© The Author(s) 2023. Published by Oxford University Press on behalf of BJS Society Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Importance: Melanoma guidelines recommend surgical excision with 10-mm margins for T1 melanoma. However, this procedure may be problematic at sites close to critical structures such as the scalp, face, external genitalia, acral, periumbilical, and perineal areas., Objective: To compare outcomes of wide (10-mm margins) vs narrow (5-mm margins) excision in patients with T1a melanoma near critical structures., Design, Setting, and Participants: This cohort study was a retrospective comparison of 1341 consecutive patients aged 18 years or older from the National Cancer Institute of Milan, Italy, diagnosed between 2001 and 2020 with T1a cutaneous melanoma close to critical structures who accepted wide excision vs narrow excision., Exposures: Local recurrence and melanoma-specific mortality (MSM) rates with 5-mm vs 10-mm excision margins., Main Outcomes and Measures: The primary aim of the study was to ascertain whether a narrower (5-mm) vs wider (10-mm) excision margin was associated with local recurrence and MSM. The secondary aim was to compare the need for reconstructive surgery in the groups defined by excision margin width. Between April 28 and August 7, 2022, associations were assessed by weighted Cox and Fine-Gray univariable and multivariable models., Results: A total of 1179 patients met the inclusion criteria (median [IQR] age, 50.0 [39.5-63.0] years; female, 610 [51.7%]; male, 569 [49.3%]). Six hundred twenty-six patients (53.1%) received a wide excision (434 [69.3%] with linear repair and 192 [30.7%] with flap or graft reconstruction) and 553 (46.9%) received a narrow excision (491 [88.8%] with linear repair and 62 [11.2%] with flap or graft reconstruction). The weighted 10-year MSM was 1.8% (95% CI, 0.8%-4.2%) in the wide group and 4.2% (95% CI, 2.2%-7.9%) in the narrow group; the weighted 10-year local recurrence rate was 5.7% (95% CI, 3.9%-8.3%) in the wide group and 6.7% (95% CI, 4.7%-9.5%) in the narrow group. Breslow thickness greater than 0.4 mm (subdistribution hazard ratio [sHR] for 0.6 vs 0.4 mm, 2.42; 95% CI, 1.59-3.68; P < .001) and mitotic rate greater than 1/mm2 (sHR for a single increment, 3.35; 95% CI, 2.59-4.32; P < .001) were associated with worse MSM. Multivariable analysis showed that acral lentiginous melanoma, lentigo maligna melanoma, and increasing Breslow thickness were associated with a higher incidence of local recurrence., Conclusions and Relevance: The study's findings suggest that local excision with 5-mm margins for T1a melanoma may not be associated with an increased risk of local recurrence. Breslow thickness greater than 0.4 mm, mitotic rate greater than 1/mm2, and acral lentiginous melanoma and lentigo maligna melanoma subtypes were associated with a higher risk of recurrence. These findings may be useful for future melanoma treatment guidelines.

Background: The management of melanoma patients with metastatic melanoma in the sentinel nodes (SN) is evolving based on the results of trials questioning the impact of completion lymph node dissection (CLND) and demonstrating the efficacy of new adjuvant treatments. In this landscape, new prognostic tools for fine risk stratification are eagerly sought to optimize the therapeutic path of these patients., Methods: A retrospective cohort of 2,086 patients treated with CLND after a positive SN biopsy in thirteen Italian Melanoma Centers was reviewed. Overall survival (OS) was the outcome of interest; included independent variables were the following: age, gender, primary melanoma site, Breslow thickness, ulceration, sentinel node tumor burden (SNTB), number of positive SN, non-sentinel lymph nodes (NSN) status. Univariate and multivariate survival analyses were performed using the Cox proportional hazard regression model., Results: The 3-year, 5-year and 10-year OS rates were 79%, 70% and 54%, respectively. At univariate analysis, all variables, except for primary melanoma body site, were found to be statistically significant prognostic factors. Multivariate Cox regression analysis indicated that older age (P < 0.0001), male gender (P = 0.04), increasing Breslow thickness (P < 0.0001), presence of ulceration (P = 0.004), SNTB size (P < 0.0001) and metastatic NSN (P < 0.0001) were independent negative predictors of OS., Conclusion: The above results were utilized to build a nomogram in order to ease the practical implementation of our prognostic model, which might improve treatment personalization., (© 2022. The Author(s).)

Cutaneous squamous cell carcinomas (CSCC) account for about 20% of all keratinocyte carcinomas, which are the most common form of cancer. Heterogeneity of treatments and low mortality are a challenge in obtaining accurate incidence data and consistent registration in cancer registries. Indeed, CSCC mostly presents as an indolent, low-risk lesion, with five-year cure rates greater than 90% after surgical excision, and only few tumors are associated with a high-risk of local or distant relapse; therefore, it is particularly relevant to identify high-risk lesions among all other low-risk CSCCs for the proper diagnostic and therapeutic management. Chemotherapy achieves mostly short-lived responses that do not lead to a curative effect and are associated with severe toxicities. Due to an etiopathogenesis largely relying on chronic UV radiation exposure, CSCC is among the tumors with the highest rate of somatic mutations, which are associated with increased response rates to immunotherapy. Thanks to such strong pre-clinical rationale, clinical trials led to the approval of anti-PD-1 cemiplimab by the FDA (Food and Drug Administration) and EMA (European Medicines Agency), and anti-PD-1 pembrolizumab by the FDA only. Here, we provide a literature review and clinical recommendations by a panel of experts regarding the diagnosis, treatment, and follow-up of CSCC.

Background: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity after sustained response are unknown., Materials and Methods: This retrospective case series analysis conducted at a single Cancer Center in Italy included patients with BRAF mutated metastatic melanoma treated with a BRAF inhibitor as a single agent or in combination with a MEK inhibitor between June 1, 2011 and January 1, 2020 and interrupted treatment due to cumulative toxicity after achieving complete response (CR) or long-lasting partial response (PR; i.e. >12 months)., Results: We included 24 patients with a median treatment duration of 59.4 months (95% confidence interval [CI], 55.4-63.4; range, 12-88). CR and PR were achieved in 71% and 29% of patients, respectively. At a median follow-up after treatment discontinuation of 37.8 months (95% CI, 33.7-41.9), the 12-month progression-free survival after discontinuation (dPFS) rate was 70.8% (95% CI 54.8-91.6) and 24-month dPFS rate was 58.3% (95% CI, 41.6-81.8). Baseline patient and tumor characteristics as well as treatment duration and best response did not significantly impact on dPFS. Patients with CR and negative circulating tumor DNA (ctDNA) at time of discontinuation had a significantly improved dPFS compared with patients with either radiological residual disease or ctDNA positivity (p = .007). No patient in CR with undetectable ctDNA experienced progression., Conclusion: The risk of progression is high even in patients with sustained sensitivity to BRAF/MEK inhibitors. Integration of liquid biopsy in clinical trials investigating the optimal management of patients with sustained sensitivity to BRAF/MEK inhibitors is warranted., Implications for Practice: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity are unknown. This study analyzed patients with sustained responses (median treatment duration 59.4 months). Twelve- and 24-month progression-free survival following discontinuation were 70.8% and 58.3%, respectively. Complete response and negative circulating tumor DNA at time of discontinuation are promising prognostic biomarkers in this setting., (© 2021 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background: Prognostic parameters in sentinel node (SN)-positive melanoma are important indicators to identify patients at high risk of recurrence who should be candidates for adjuvant therapy. We aimed to evaluate the presence of melanoma cells beyond the SN capsule-extranodal extension (ENE)-as a prognostic factor in patients with positive SNs., Methods: Data from 1,047 patients with melanoma and positive SNs treated from 2001 to 2020 at the Istituto Nazionale dei Tumori in Milano, Italy, were retrospectively investigated. Kaplan-Meier survival and crude cumulative incidence of recurrence curves were estimated. A multivariable logistic model was used to investigate the association between ENE and selected predictive factors. Cox models estimated the effect of the selected predictors on survival endpoints., Results: Median follow-up was 69 months. The 5-year overall survival rate was 62.5% and 71.7% for patients with positive SNs with and without ENE, respectively. The 5-year disease-free survival rate was 54.0% and 64.0% for patients with positive SNs with and without ENE, respectively. The multivariable logistic model showed that age, size of the main metastatic focus in the SN, and numbers of metastatic non-SNs were associated with ENE (all P<.0001). The multivariable Cox regression models showed the estimated prognostic effects of ENE associated with age, ulceration, size of the main metastatic focus in the SN, and number of metastatic non-SNs (all P<.0001) on disease-free survival and overall survival., Conclusions: ENE was a significant prognostic factor in patients with positive-SN melanoma. This parameter may be useful in clinical practice as a selection criterion for adjuvant treatment in patients with stage IIIA disease with a tumor burden <1 mm in the SN. We recommend its inclusion as an independent prognostic determinant in future updates of melanoma guidelines.

Background: Sentinel Node Biopsy (SNB) is routinely performed for primary melanoma, but its role in the treatment of Local Recurrence (LR) and In-Transit metastasis (IT) is controversial. This study aims to assess the role of SNB in melanoma patients who developed first loco-regional recurrence., Methods: A series of consecutive melanoma patients who received SNB for a first IT or LR at the National Cancer Institute of Milan, Italy, from 2000 to 2015 were selected from a prospective database. Clinicopathological characteristics were analyzed., Results: Seventy-two patients met selection criteria. Forty-three patients (59.7%) received SNB for LR and 29 (40.3%) for IT. The average interval between treatment of primitive melanoma and first recurrence diagnosis was 19 months (interquartile range: 6.9-49.0). SN identification rate was 97.2%. SN positivity was detected in 26 (37.1%) patients. The SN-positive ratein melanoma patients who had LR or IT was significantly higher than reported for primary tumours. Of patients with nodal involvement 17 had LR and 9 IT lesions. Disease Free Survival (DFS) was slightly higher in SN negative patients, in the absence of statistically significant differences. Overall Survival (OS) analysis showed similar values in the two groups., Conclusion: Since DFS and OS do not show significant differences between SN negative and positive patients, our data do not give clear indications about performing SNB in case of first LR or IT. However, we suggest submitting patients with LR to this procedure to obtain a more accurate staging and eventually candidate these patients to adjuvant treatment., Competing Interests: Declaration of competing interest All authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Introduction: Treatment of metastatic melanoma has rapidly changed during the last years, and patients often require a multidisciplinary approach to achieve effective results. We aimed to assess the survival benefit achieved through surgical approach to patients with small bowel (SB) metastases from cutaneous melanoma, to emphasize the potential role of surgery in association with novel therapies., Methods: Ninety consecutive patients with cutaneous melanoma diagnosed as having resectable SB metastases from 1995 to 2015 were retrospectively investigated., Results: Median age at surgery of melanoma metastases was 53.4 years. Among 30 patients who had a curative-intent resection, the 5- and 10-year survival rates were 61% and 54%, respectively, while among 60 patients treated with a palliative surgery the corresponding rates were both 4%. Among 29 patients, for whom the interval time between the occurrence of SB metastases and the previous surgical event on GI tract was ≥36 months, the 5-year overall survival rate was 42%; for 56 patients who had an interval time <36 months the corresponding survival rate was 14%. Within the whole series, an absence of any residual disease after surgery (R0) was a factor affecting better survival, regardless of the evidence of metastases in other organs., Conclusion: Our observational data showed that surgical treatment for patients with SB metastases from melanoma might increase survival, but further studies are needed to confirm this finding. In the age of novel available therapies, the increase in survival time given by surgery may offer important chances for patients to benefit from systemic therapies., Competing Interests: Declaration of competing interest All authors disclose that there are no financial and personal relationships with other people or organizations that could inappropriately influence their work., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Background: Atypical melanocytic tumors (AMTs) include a wide spectrum of melanocytic neoplasms that represent a challenge for clinicians due to the lack of a definitive diagnosis and the related uncertainty about their management. This study analyzed clinicopathologic features and sentinel node status as potential prognostic factors in patients with AMTs., Patients and Methods: Clinicopathologic and follow-up data of 238 children, adolescents, and adults with histologically proved AMTs consecutively treated at 12 European centers from 2000 through 2010 were retrieved from prospectively maintained databases. The binary association between all investigated covariates was studied by evaluating the Spearman correlation coefficients, and the association between progression-free survival and all investigated covariates was evaluated using univariable Cox models. The overall survival and progression-free survival curves were established using the Kaplan-Meier method., Results: Median follow-up was 126 months (interquartile range, 104-157 months). All patients received an initial diagnostic biopsy followed by wide (1 cm) excision. Sentinel node biopsy was performed in 139 patients (58.4%), 37 (26.6%) of whom had sentinel node positivity. There were 4 local recurrences, 43 regional relapses, and 8 distant metastases as first events. Six patients (2.5%) died of disease progression. Five patients who were sentinel node-negative and 3 patients who were sentinel node-positive developed distant metastases. Ten-year overall and progression-free survival rates were 97% (95% CI, 94.9%-99.2%) and 82.2% (95% CI, 77.3%-87.3%), respectively. Age, mitotic rate/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss were factors affecting prognosis in the whole series and the sentinel node biopsy subgroup., Conclusions: Age >20 years, mitotic rate >4/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss proved to be worse prognostic factors in patients with ATMs. Sentinel node status was not a clear prognostic predictor.

Background: Sentinel lymph node biopsy (SNB) still remains a key procedure to appropriately stage melanoma patients and to select those who are candidate to novel treatments with immunotherapy and targeted therapy in the adjuvant setting. The impact of timing of SNB on disease-free survival (DFS) and overall survival (OS) is still unclear., Material and Methods: The study was conducted at 6 Italian Melanoma Intergroup (IMI) centres and included 8953 consecutive clinical stage I-II melanoma patients who were diagnosed, treated, and followed up between November 1997 and March 2018. All patients were prospectively included in dedicated IMI database. Multivariable Cox regression analyses were performed to investigate how baseline characteristics and time interval until SNB are related to DFS and OS., Results: Considering the whole population, at multivariable analysis, after adjusting for age, gender, Breslow thickness, site, ulceration, and the SNB status, a delay in the timing of SNB was associated with a better DFS (adjusted hazard ratio [aHR, delayed versus early SNB] 0.98, 95% confidence interval [CI] 0.97-0.99, p < 0.001) and OS (aHR 0.98, 95% CI 0.97-0.99, p = 0.001). Specifically, in patients with a negative SNB status, a beneficial impact of delayed SNB (i.e. at least 32 days after primary excision) was confirmed for DFS (aHR 0.70, 95%CI 0.63-0.79, p < 0.001) and OS (aHR 0.69, 95%CI 0.61-0.78, p < 0.001), whereas in those with a positive SNB status, DFS (aHR 0.96, 95%CI 0.84-1.09, p = 0.534) and OS (aHR 0.94 95%CI 0.81-1.08, p = 0.374) were not significantly different in patients with early or delayed SNB., Conclusions: Our study does not support a strict time interval for SNB. These results may be useful for national guidelines, for counselling patients and reducing the number of high urgency referrals., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Thin melanomas (T1; ≤ 1 mm) constitute 70% of newly diagnosed cutaneous melanomas. Regional node metastasis determined by sentinel node biopsy (SNB) is an important prognostic factor for T1 melanoma. However, current melanoma guidelines do not provide clear indications on when to perform SNB in T1 disease and stress an individualized approach to SNB that considers all clinicopathologic risk factors. We aimed to identify determinants of sentinel node (SN) status for incorporation into an externally validated nomogram to better select patients with T1 disease for SNB., Patients and Methods: The development cohort comprised 3,666 patients with T1 disease consecutively treated at the Istituto Nazionale Tumori (Milan, Italy) between 2001 and 2018; 4,227 patients with T1 disease treated at 13 other European centers over the same period formed the validation cohort. A random forest procedure was applied to the development data set to select characteristics associated with SN status for inclusion in a multiple binary logistic model from which a nomogram was elaborated. Decision curve analyses assessed the clinical utility of the nomogram., Results: Of patients in the development cohort, 1,635 underwent SNB; 108 patients (6.6%) were SN positive. By univariable analysis, age, growth phase, Breslow thickness, ulceration, mitotic rate, regression, and lymphovascular invasion were significantly associated with SN status. The random forest procedure selected 6 variables (not growth phase) for inclusion in the logistic model and nomogram. The nomogram proved well calibrated and had good discriminative ability in both cohorts. Decision curve analyses revealed the superior net benefit of the nomogram compared with each individual variable included in it as well as with variables suggested by current guidelines., Conclusion: We propose the nomogram as a decision aid in all patients with T1 melanoma being considered for SNB.

Background: Recently the 8th version of the American Joint Committee on Cancer (AJCC) classification has been introduced, and has attempted to define a more accurate and precise definition of prognosis in line with the major progresses in understanding the biology and pathogenesis of melanoma. This new staging system introduces major changes in the stage III staging system. Indeed, surgical practice is changing in stage III patients, since, according to recent evidence, there is no survival benefit in radical lymph node dissection following a positive sentinel lymph node dissection. Therefore, some patients currently staged IIIB-C after dissection could be downgraded to IIIA (as in the case of patients with metastatic non-sentinel lymph nodes) since many completion lymph node dissections will no longer be performed. Moreover, new and effective targeted and immune strategies are being introduced in the pharmacological armamentarium in the adjuvant setting, showing major efficacy., Conclusions: This article provides the authors' personal view on the above-mentioned topics.

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14+ monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460., (© 2016 AlphaMed Press.)

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass.

Advances in the understanding of tumor immunology and molecular biology of melanoma cells have favored a larger application of immunotherapy and targeted therapies in the clinic. Several selective mutant gene inhibitors and immunomodulating antibodies have been reported to improve overall survival or progression-free survival in metastatic melanoma patients. However, despite impressive initial responses, patients treated with selective inhibitors relapse quickly, and toxicities associated to the use of immunomodulating antibodies are not easily manageable. In this sense, the concept of using antibodies as delivery vehicles for the preferential in vivo localization of the drug at the site of disease with reduction of side effects has raised particular interest. Antibody-cytokine fusion proteins (termed immunocytokines) represent a new simple and effective way to deliver the immunomodulatory payload at the tumor site, with the aim of inducing both local and systemic antitumoral immune responses and limiting systemic toxicities. Several clinical trials have been conducted and are actually ongoing with different immunocytokines, in several tumor histotypes. In metastatic melanoma patients, different drug delivery modalities such as systemic, loco-regional and intratumoral are under investigation. In this review, the rationale for the use of L19-IL2 and L19-TNF, two clinical stage immunocytokines produced by the Philogen group, as well as opportunities for their future development will be discussed.

Purpose: Cutaneous melanoma incidence is increasing. Most new cases are thin (≤ 1 mm) with favorable prognoses, but survival is nonetheless variable. Our aim was to investigate new prognostic factors and construct a nomogram for predicting survival in individual patients., Patients and Methods: Data from 2,243 patients with thin melanoma were retrieved from prospectively maintained databases at six centers. Kaplan-Meier survival and crude cumulative incidences of recurrence were estimated, and competing risks were taken into account. Multivariable Cox regression was used to investigate survival predictors., Results: Median follow-up was 124 months (interquartile range, 106 to 157 months); 12-year overall survival was 85.3% (95% CI, 83.4% to 87.2%). Median times to local, regional, and distant recurrence were 79, 78, and 107 months, respectively. Relapse was significantly related to age, Breslow thickness, mitotic rate (MR), ulceration, lymphovascular invasion (LVI), and regression; incidence was lower and subgroup differences were less marked for distant metastasis than for regional relapse. The worst prognosis categories were age older than 60 years, Breslow thickness more than 0.75 mm, MR ≥ 1, presence of ulceration, presence of LVI, and regression ≥ 50%. Breslow thickness more than 0.75 mm, MR ≥ 1, presence of ulceration, and LVI (all P = .001) were significantly associated with sentinel node positivity. Age, MR, ulceration, LVI, regression, and sentinel node status were independent predictors of survival and were used to construct a nomogram to predict 12-year overall survival. The nomogram was well calibrated and had good discriminative ability (adjusted Harrell C statistic, 0.88)., Conclusion: Our findings suggest including LVI and regression as new prognostic factors in the melanoma staging system. The nomogram appears useful for risk stratification in clinical management and for recruiting patients to clinical trials., (© 2014 by American Society of Clinical Oncology.)

Importance: Although the number of excised lymph nodes (LNs) represents a quality assurance measure in lymphadenectomy for many solid tumors, the minimum number of LNs to be dissected has not been established for melanoma., Objective: To investigate the distribution of the number of excised LNs in a large patient series (N = 2526) to identify values that may serve as benchmarks for monitoring the quality of lymphadenectomy in patients with melanoma., Design, Setting, and Participants: A retrospective multicenter study was conducted (1992-2010) in tertiary referral centers for treatment of cutaneous melanoma. Medical records on 2526 patients who underwent lymphadenectomy for regional LN metastasis associated with cutaneous melanoma were examined., Exposure: Patients had undergone lymphadenectomy for regional LN metastasis., Main Outcomes and Measures: The mean, median, and 10th percentile of the number of excised LNs were calculated for the axilla (3 levels), neck (≤3 or ≥4 dissected levels), inguinal, and ilioinguinal LN fields., Results: After 3-level axillary (n = 1150), 3-level or less neck (n = 77), 4-level or more neck (n = 135), inguinal (n = 209), and ilioinguinal (n = 955) dissections, the median (interquartile range [IQR]) and mean (SD) number of excised LNs were as follows: 3-level axillary dissection, 20 (15-27) and 22 (8); 3-level or less neck, 21 (14-33) and 24 (15); 4-level or more neck, 29 (21-41) and 31 (14); inguinal, 11 ( 9-14) and 12 (5); and ilioinguinal, 21 (16-26) and 22 (4). A total of 90% of the patients had 12, 7, 14, 6, and 13 excised LNs (10th percentile of the distribution) after 3-level axillary, 3-level or less neck, 4-level or more neck, inguinal, and ilioinguinal dissections, respectively. More excised LNs were detected in younger (21 for those <54 years of age and 19 for ≥54 years, P < .001) and male (21 for male sex and 19 for female sex, P < .001) patients from high-volume institutions (21 for volume of ≥300 vs 18 for volume <300, P < .001) with a more recent year of diagnosis (21 for years 2002-2010 vs 18 for years 1992-2001, P < .001), LN micrometastasis vs macrometastasis (20 vs 19, P = .005), and more positive LNs (R² = 0.03, P < .001); however, the differences between median values were small., Conclusions and Relevance: These minimum numbers of excised LNs are reproducible across the institution, patient, and tumor factors evaluated. They can be taken into consideration when monitoring the quality of lymphadenectomy in melanoma and can represent entry criteria for randomized trials investigating adjuvant therapies.

Background: Debate remains around the accuracy and prognostic implications of sentinel lymph node biopsy (SLNB) for melanoma arising in the head and neck (HN) areas because several analyses have shown discordances between clinically predicted lymphatic drainage pathways and those identified by lymphoscintigraphy. This study assesses the accuracy and prognostic value of SLNB in this critical anatomic region., Methods: Retrospective review of a prospectively collected melanoma database identified 331 patients with HN melanomas from January 2000 to December 2012. Primary end points included SLNB result, time to recurrence, site of recurrence, and survival. Multivariate models were constructed for analyses., Results: A sentinel lymph node (SLN) was identified in all 331 patients. There were 59 patients with a positive SLN (17.8%) with a recurrence rate of 88.1% compared with 22.4% in SLN-negative patients (P < 0.0001). The 5-y overall survival was 91.2% for SLN-negative patients and 48.7% for SLN-positive patients (P < 0.0001). Patients with scalp melanoma had thicker lesions and an elevated risk of SLN positivity, recurrence, and death compared with those with other sites. Among the 272 SLN-negative patients, four patients developed regional nodal disease in the same basin and had undergone a previous SLNB procedure for a false-omission rate of 1.45%. Risks for false-negative SLN occurrences included thick and scalp melanomas. Multivariate analysis on prognostic factors affecting relapse-free survival showed positive SLNB status to be the most prognostic clinicopathologic predictor of recurrence (hazard ratio, 20.56; P < 0.0001)., Conclusions: SLNB for patients with HN melanomas is an accurate procedure and has prognostic value., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: We investigated whether the nonsentinel lymph node (NSLN) status in patients with melanoma improves the prognostic accuracy of common staging features; then we formulated a proposal for including the NSLN status in the current melanoma staging system., Patients and Methods: We retrospectively collected the clinicopathologic data of 1,538 patients with positive SLN status who underwent completion lymph node dissection (CLND) at nine Italian centers. Multivariable Cox regression survival analysis was used to identify independent prognostic factors. Literature meta-analysis was used to summarize the available evidence on the prognostic value of the NSLN status in patients with positive SLN., Results: NSLN metastasis was observed in 353 patients (23%). After a median follow-up of 45 months, NSLN status was an independent prognostic factor for melanoma-specific survival (hazard ratio [HR] = 1.34; 95% CI, 1.18 to 1.52; P < .001). NSLN status efficiently stratified the prognosis of patients with two to three positive lymph nodes (n = 387; HR = 1.39; 95% CI, 1.07 to 1.81; P = .013), independently of other staging features. Searching the literature, this patient subgroup was investigated in other two studies. Pooling the results (n = 620 patients; 284 NSLN negative and 336 NSLN positive), we found that NSLN status is a highly significant prognostic factor (summary HR = 1.59; 95% CI, 1.27 to 1.98; P < .001) in patients with two to three positive lymph nodes., Conclusion: These findings support the independent prognostic value of the NSLN status in patients with two to three positive lymph nodes, suggesting that this information should be considered for the routine staging in patients with melanoma.

Sentinel lymph nodes set the stance of the immune system to a localized tumor and are often the first site to be colonized by neoplastic cells that metastasize. To investigate how the presence of neoplastic cells in sentinel lymph nodes may trigger pathways associated with metastatic progression, we analyzed the transcriptional profiles of archival sentinel node biopsy specimens obtained from melanoma patients. Biopsies from positive nodes were selected for comparable tumor infiltration, presence or absence of further regional node metastases, and relapse at 5-year follow-up. Unsupervised analysis of gene expression profiles revealed immune response to be a major gene ontogeny represented. Among genes upregulated in patients with progressing disease, the TNF receptor family member CD30/TNFRSF8 was confirmed in biopsy specimens from an independent group of patients. Immunohistochemical analysis revealed higher numbers of CD30(+) lymphocytes in nodes from progressing patients compared with nonprogressing patients. Phenotypic profiling demonstrated that CD30(+) lymphocytes comprised a broad population of suppressive or exhausted immune cells, such as CD4(+)Foxp3(+) or PD1(+) subpopulations and CD4(-)CD8(-) T cells. CD30(+) T lymphocytes were increased in peripheral blood lymphocytes of melanoma patients at advanced disease stages. Our findings reinforce the concept that sentinel nodes act as pivotal sites for determining progression patterns, revealing that the presence of CD30(+) lymphocytes at those sites associate positively with melanoma progression.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti-vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine-specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE-A3(+) melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE-A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV-TK). HSV-TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti-TK and anti-MAGE-A3 T-cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE-A3-specific immune responses showed a clinical benefit. Additionally, we report that responder and non-responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses., (Copyright © 2012 UICC.)

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8(+) T cell effectors when used in the inflammatory environment of vaccination.

Background: L19-TNF is a tumor-targeting immunocytokine composed of the human L19 antibody binding to extra domain B (ED-B) of fibronectin of newly formed blood vessels, and of human TNF. This exploratory trial evaluates safety and clinical activity of L19-TNF plus melphalan-containing isolated limb perfusion (ILP) in extremity melanoma patients., Methods: Seven and 10 patients received 325 µg and 650 µg of L19-TNF, respectively, during the ILP. Patients were studied for safety, tolerability, and clinical activity of this experimental L19-TNF ILP procedure., Results: Non-hematologic toxicity of L19-TNF ILP was very low, but severe myelosuppression was seen in four patients. Although L19-TNF was administered at a TNF-equivalent dose of only 3.13 and 6.25% of the approved TNF (Beromun®) dose of 4 mg, L19-TNF ILP induced objective responses in 86 and 89% of patients, respectively, including a complete response (CR) in 5/10 patients treated with L19-TNF ILP at 650 µg that was durable at 12 months in four patients. No CR was seen at 325 µg of L19-TNF., Conclusions: ILP with L19-TNF had a favorable safety and a promising activity profile at a dose of 650 µg of L19-TNF, supporting the exploration of higher L19-TNF doses and a Phase II trial comparing L19-TNF ILP with standard melphalan-containing ILP., (Copyright © 2012 Wiley Periodicals, Inc.)

Introduction: Melanoma with recurrent loco-regional metastases to limbs often makes difficult a second surgical approach because of the adhesions affecting the vascular access. Our aim was to evaluate whether the placement of a polytetrafluoroethylene (PTFE) membrane around vessels might facilitate a surgical re-approach., Presentation of Case: We reported a case of a 64-year-old male with a melanoma on the left foot who developed in transit metastases after LND. While performing the inguinopelvic LND we coated the iliac vessels with PTFE patch to facilitate the vascular access in case of re-intervention for a ILP. In the second surgical approach we made a cutaneous incision in the left iliac region and we proceeded through the subcutaneous tissue until detection of iliac vessels, more clearly visible because of the PTFE patch fixed around vascular walls to minimize adhesions. We removed the PTFE coating and easily performed arteriotomy and venotomy for the completion of the ILP., Discussion: This case report seems to demonstrate the efficacy of a PTFE membrane applied in a patient around iliac vessels during inguinopelvic dissection, to reduce adhesion density. In fact this membrane provided a barrier to adhesions of the iliac vessels, decreasing the risk of vascular injury thereby facilitating a subsequent vascular access. Re-coating of the iliac vessels with PTFE could be preparatory to a better identification of the vascular structures in cases of a surgical re-approach., Conclusion: The use of PTFE effectively simplifies the second approach to vessels in event of a melanoma metastasizing to limbs., (Copyright © 2012 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III)., Experimental Design: Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201-restricted modified peptides (Melan-A/MART-1([27L]), gp100([210M]), NY-ESO-1([165V]), and Survivin([97M])) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m(2)) and low-dose IL-2 (3 × 10(6) IU). The immune responses were monitored using ex vivo IFN-γ-ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays., Results: Vaccination induced a rapid and persistent increase in specific effector memory CD8(+) T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells., Conclusions: Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8(+) T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease., (©2012 AACR.)

Objective: The purpose of the study was to evaluate the excision margin necessary for desmoplastic melanoma (DM)., Background: DM consists of 2 histologic subtypes, pure DM (PDM) and mixed DM (MDM), differing in extent of fibrotic component. We investigated clinical and therapeutic determinants of prognosis in these DM entities., Methods: We reviewed 118 PDM and 124 MDM treated at our Institute over 25 years. Local relapse, distant metastasis, and survival were studied., Results: Most (91.7%) distant metastases in PDM developed after 1 or more local recurrences; whereas distant metastasis usually (79.6%) occurred as first event in MDM. Overall mortality trends in relation to lesion-thickness-plus-excision-width differed for PDM (P = 0.014) but not MDM (P = 0.185). For PDM, 5-year crude cumulative incidence (CCI) of mortality was higher (40.0%) for thin tumors (≤ 2 mm thick) excised with 1 cm margin than those excised with 2 cm (14.8%); CCI of mortality for PDM > 2 mm thick excised with 2 cm margins (13.4%) was similar to that for thin PDM lesions excised with 2 cm (14.8%). CCI of local recurrence was also greater in PDM excised with 1 cm margins. In MDM, mortality increased with stage but was independent of excision width (CCI: 29.4% for ≤ 2 mm/2 cm, 31.3% for ≤ 2 mm/1 cm, and 48.3% for > 2 mm/2 cm); a similar trend was found for MDM distant metastases., Conclusions: In PDM, limited excision width is associated with significantly greater local recurrence and mortality; treatment should be excision with 2 cm margins even for thin lesions. MDM behaves similarly to other melanomas; treatment should follow guidelines on melanoma management.

CD8(+) T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8(+) T cells expressing FOXP3. CD8(+) FOXP3(+) CD25(+) T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8(+) FOXP3(+) T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8(+) T lymphocytes. Instead, this subset showed an antigen-experienced "EM1" phenotype (CCR7(-) CD45RA(-) CD28(+) CD27(+)) and exhibited a CD127(-), KLRG1(-), HLA-DR(+), CD38(+), T-bet(+), perforin(+) "early effector" profile predicted by current models. CD8(+) FOXP3(+) T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8(+) FOXP3(+) T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8(+) FOXP3(+) "early effector" subset may be an invaluable tool for monitoring immunity at tumor site., (©2010 AACR.)

Melanospheres, the melanoma cells that grow as nonadherent colonies and that show in vitro self-renewing capacity and multipotency, were selected from melanoma specimens or from melanoma cell lines. Melanospheres were highly tumorigenic, and intradermal injections in severe combined immunodeficient (SCID) mice of as few as 100 cells generated tumors that maintained tumorigenic potential into subsequent recipients. Primary and serially transplanted xenografts recapitulated the phenotypic features of the original melanoma of the patient. Melanoma cells cultured in the presence of fetal calf serum (FCS) were also tumorigenic in SCID mice, although with lower efficiency; these xenografts showed a homogeneous phenotype for the expression of melanoma-associated markers, Melan-A/Mart-1, HMB45, and MITF, and contained cells with features of fully differentiated cells. Melanospheres were heterogeneous for the expression of stem cell markers and showed a significantly enhanced expression of the Nanog and Oct3/4 transcription factors when compared with adherent melanoma cells. No direct and unique correlation between any of the examined stem cell markers and in vivo tumorigenicity was found. Taken together, our data provide further evidence on the heterogeneous nature of human melanomas and show that melanospheres and their corresponding tumors, which are generated in vivo in immunocompromised mice, represent a model to investigate melanoma biology.

Aim: To determine the long-term outcomes of early-stage Merkel cell carcinoma (MCC) patients managed with surgery alone., Methods: Ninety-five consecutive patients were reviewed. Patients were treated by wide local excision. Clinically negative regional nodes were either followed up (n=42) or staged with sentinel lymph node biopsy (n=21), and clinically positive nodes underwent lymph node dissection (n=32)., Results: Median follow-up was 65 months. A total of 45 (47%) patients relapsed, with 80% of the recurrences occurring within 2 years and 96% within 5 years. The 5-year crude cumulative incidence (CCI) of recurrence and disease-specific survival (DSS) were 52% and 67%, respectively. CCI of local 5-year recurrence was 5% for the study cohort. Patients with MCC in the head and neck region had a 5-year local-recurrence CCI of 19%, and patients with MCC in the extremity and trunk region had a 5-year local-recurrence CCI of 2% (P=0.007). Comparing patients with 2 metastatic lymph nodes, the 5-year regional-recurrence CCI was 0% versus 39% (P=0.004). The 5-year distant-recurrence CCI was higher in clinically node-positive patients compared with node-negative patients (37% versus 12%; P=0.005). Patients with MCC in the head and neck region experienced no distant recurrences, patients with MCC in the extremity and trunk region had a 5-year distant-recurrence CCI of 22%, and patients with occult primary had a 5-year distant-recurrence CCI of 49% (P=0.023). The 5-year DSS rate was 80% for pathologically node-negative patients., Conclusion: The prognosis for surgically managed early-stage MCC is variable. Thus multidisciplinary tumor-board consultation is needed to optimize individual patient management.

Since the identification of tumor associated antigens (TAA) in different tumor histotypes, many vaccination strategies have been investigated, including peptide-based vaccines. Results from the first decade of clinical experimentation, though demonstrating the feasibility and the good toxicity profile of this approach, provided evidence of clinical activity only in a minority of patients, despite inducing immunization in up to 50% of them. In this review, we discuss the different approaches recently developed in order to induce stronger peptide-induced immune-mediated tumor growth control, possibly translating into improved clinical response rates, with specific focus on multipeptide-based anti-cancer vaccines. This strategy offers many advantages, such as the possibility of bypassing tumor heterogeneity and selection of antigen (Ag)-negative clones escaping peptide-specific immune responses, or combining HLA class I- and class II-restricted epitopes, thus eliciting both CD4- and CD8-mediated immune recognition. Notably, advances in Ag discovery technologies permit further optimization of peptide selection, in terms of identification of tumor-specific and unique TAA as well as Ags derived from different tumor microenvironment cell components. With the ultimate goal of combining peptide selection with patient-specific immunogenic profile, peptide based anti-cancer vaccines remain a promising treatment for cancer patients, as attested by of pre-clinical and clinical studies.

The discovery of tumour antigens recognized by T cells and the features of immune responses directed against them has paved the way to a multitude of clinical studies aimed at boosting anti-tumour T cell immunity as a therapeutic tool for cancer patients. One of the different strategies explored to ameliorate the immunogenicity of tumour antigens in vaccine protocols is represented by the use of optimized peptides or altered peptide ligands, whose amino acid sequence has been modified for improving HLA binding or TCR interaction with respect to native epitopes. However, despite the promising results achieved with preclinical studies, the clinical efficacy of this approach has not yet met the expectations. Although multiple reasons could explain the relative failure of altered peptide ligands as more effective cancer vaccines, the possibility that T cells primed by modified tumour peptides might may be unable to effectively cross-recognize tumour cells has not been sufficiently addressed. Indeed, the introduction of conservative amino acid substitutions may still produce diverse and unpredictable changes in the HLA/peptide interface, with consequent modifications of the TCR repertoire that can interact with the complex. This could lead to the expansion of a broad array of T cells whose TCRs may not necessarily react with equivalent affinity with the original antigenic epitope. Considering the results presently achieved with this vaccine approach, and the emerging availability of alternative strategies for boosting anti-tumour immunity, the use of modified tumour peptides could be reconsidered.

Purpose: To assess the extent of signal transducer and activator of transcription (STAT) activation in response to interleukin 2 (IL-2) in melanoma patients' T cells, along with clinical stage of tumor progression., Experimental Design: T lymphocytes from peripheral blood of healthy donors and of American Joint Committee on Cancer stage I to IV melanoma patients, as well as from metastatic lymph nodes of patients, were evaluated for responsiveness to IL-2. CFSE assays and single-cell phospho-STAT-specific flow cytometry screening were used. Results. T cells from advanced melanoma patients, in comparison with healthy donors, showed reduced proliferation to IL-2 and IL-15, but not to anti-CD3 monoclonal antibody. Impaired response occurred in CCR7(+) and CCR7(-) T-cell subsets, but not in CD3(-) CD8(+) natural killer (NK) cells, and was not explained by induction of apoptosis, increased cytokine consumption, or altered IL-2R subunit expression in patients' T lymphocytes. By phospho-specific flow cytometry, defective STAT1 and STAT5 activation in response to IL-2 was found mainly in T lymphocytes from peripheral blood and/or tumor site of American Joint Committee on Cancer stage III and IV patients, compared with stage I and II patients and to donors, and in melanoma antigen-specific T cells isolated from metastatic lymph nodes. At tumor site, impaired STAT activation in T cells did not correlate with frequency of CD4(+) CD25(+) Foxp3(+) T cells. Serum from advanced melanoma patients inhibited IL-2-dependent STAT activation in donors' T cells and a neutralizing monoclonal antibody to transforming growth factor beta1 counteracted such inhibition., Conclusions: These results provide evidence for development of impaired STAT signaling in response to IL-2, along with clinical evolution of the disease, in melanoma patients' T cells.

The aim of this retrospective study was to analyze the incidence of further nonsentinel node metastases at completion lymphadenectomy of the groin after a positive sentinel node biopsy to evaluate whether radical dissection remains the treatment of choice for these patients. Patients treated at the National Cancer Institute of Milan between January 1999 and December 2006 were reviewed retrospectively. The analysis included patients with a diagnosis of positive sentinel node biopsy of the groin (clinically negative) who underwent completion groin, iliac, and obturatory dissections. The primary melanoma was located on the lower extremities and trunk in 82.5 and 17.5%, respectively. The median follow-up was more than 30 months. The number of positive sentinel nodes was considered, as well as the size and location of the metastases (micro vs. macro). After radical dissection, the number and the location (groin, iliac, or groin+iliac nodes) of further nonsentinel node metastases were analyzed. The frequency of further nonsentinel node metastases at completion of groin dissection was correlated to Breslow's thickness and to the characteristics of the positive sentinel node biopsy. A total of 1581 patients with primary melanoma (>1 mm, or Clark's IV-V) underwent lymphatic mapping and sentinel node biopsy: 752 patients had sentinel node biopsy at the groin basin; among these, 150 (20%) patients presented positive sentinel node biopsy and underwent completion radical dissection (groin, obturatory, and external iliac+obturatory radical node dissections). We found further positive nonsentinel node metastases in 36 of 150 (24%) patients, 69% (25 of 36) of which were located in the iliac-obturator area and 31% in the groin area only: 16 patients (44.4%) had one additional metastatic node and seven patients (19.4%) had two, whereas 13 (36.1%) had three or more. In 22 cases (61.1%), the sentinel node showed a macrometastasis (>2 mm deposit in the node) and in 14 cases (38.9%) a micrometastasis (<2 mm deposit). In conclusion, there is clear evidence that patients with a positive sentinel node biopsy could have further positive nonsentinel node metastases (in our series, 24%). Although it is well known that the impact of sentinel node biopsy on survival of melanoma patients has yet to be defined, to obtain a clear nodal basin and regional control a groin+iliac-obturatory radical node dissection remains an appropriate procedure in the presence of a positive sentinel node biopsy at the groin level. This could be considered a standard treatment until new data, provided by ongoing studies, indicate new parameters for selecting patients for completion lymph node dissection.

BRAFV600E is the most represented somatic point mutation in cutaneous melanoma, thus providing a unique molecular marker for this disease. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools. With this aim, we evaluated whether BRAFV600E represents a detectable marker in the plasma/serum from melanoma patients in a pilot study. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either presurgery or after surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained presurgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I-II; half of the positives were obtained presurgery and half at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. In conclusion, this method can be utilized for monitoring the disease in stage IV melanoma patients but it appears unsatisfactory for the early detection of melanoma.

Purpose: To evaluate the prognostic significance of sentinel node biopsy in the management of stage IB and II melanoma patients, and to evaluate the status of nonsentinel nodes as a "second step key factor" to assess the prognosis of these patients., Patients and Methods: We conducted an analysis of data collected in a prospective database., Results: From February 1994 to June 2005, 1,108 consecutive patients with stage IB and II melanoma were submitted to sentinel node biopsy; 176 patients (15.9%) had occult node metastases. The frequency of positive nodes increased with increasing Breslow's thickness. The largest diameter of metastatic foci and their localization within the lymph node were associated with the risk of nonsentinel node metastases only. The 5-year survival of patients with positive sentinel nodes was 81.4% in patients with one positive node and 39.6% in patients with two positive nodes (P = .056). Multivariate analysis indicated that status of sentinel nodes is a key factor and that sex and Breslow's thickness maintain statistically significant relevance. Ulceration, which was associated with survival when considered as single factor (P < .001) had no impact on survival in the multivariate analysis (P = .10). To evaluate the relevance of metastases to nonsentinel nodes, we identified four groups of patients., Conclusion: Evaluation of the sentinel node is a useful procedure to identify patients to be submitted for complete lymph node dissection. The procedure makes it possible to assess the best prognosis of patients.

Based on the poor impact on overall survival obtained by systemic chemotherapy in metastatic melanoma and the identification of many melanoma antigens recognized by T cells, in the last decade many efforts have been devoted to the development of active specific immunotherapy as a promising systemic treatment for this neoplastic disease. A number of Phase I-II clinical trials have been performed with different vaccination approaches that included whole tumor cells, antigen peptides, antigen-pulsed dendritic cells, recombinant viruses, plasmids or naked DNA, and heat-shock proteins. Despite some promising immunological and clinical results obtained in these studies, melanoma-specific vaccines have altogether failed to prove their efficacy in the few large Phase III randomized clinical trials performed. Nonetheless, the possibility of activating the human immune system to recognize and destroy tumor cells remains a challenging investigative field, considering that the new knowledge of the intricate cellular and molecular mechanisms that regulate the immune function and tumor-host interactions may allow the development of new clinically relevant melanoma vaccination strategies.

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage given with GM-CSF and IFN-alpha in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days -1, 0 and +1, while IFN-alpha (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-alpha is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.

The use of IFN-alpha in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-alpha also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-alpha, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8(+) T cells recognizing modified and native MART-1 and gp100 peptides and MART-1(+)gp100(+) melanoma cells was observed. Moreover, vaccination induced an increase in CD8(+) T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA(+)CCR7(-) (terminally differentiated effectors) and CD45RA(-)CCR7(-) (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14(+) monocytes and particularly of the CD14(+)CD16(+) cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8(+) T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-alpha, the present data represent an important starting point for considering a new clinical use of IFN-alpha and new immunologic end points, potentially predictive of clinical response.

Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin beta receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin beta receptor expression on the stromal cells was significantly associated with a "brisk" T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT(+) melanoma-derived microvesicles or even with LIGHT(+) melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3(+)CD8(+) T-cell proliferation. However, lymphocyte coculture with LIGHT(+) microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3(+)CD8(+) T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.