Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

European e-Delphi process to define expert consensus on electrochemotherapy treatment indications, procedural aspects, and quality indicators in melanoma.

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.

Electrochemotherapy is an effective locoregional therapy for skin metastases from melanoma, a problem faced by almost half of patients with metastatic disease. The lack of comparative studies and the heterogeneity of its clinical application among centres make it challenging to support consistent, evidence-based recommendations. To address this unmet need, a three-round online survey was conducted to establish a consensus on treatment indications, standard operating procedures, and quality indicators. In the survey, a panel of 100 European melanoma experts agreed on 56 statements that can be used to improve patient selection, homogenize treatment application, and monitor outcomes.

The importance of mitosis ≥2 in selecting patients with T1 cutaneous melanomas for sentinel lymph node biopsy.

The 8th Edition of the American Joint Committee on Cancer (AJCC) Staging Manual removed the mitotic rate (MR) as a staging criterion for T1 melanomas, thus leading to a debate on sentinel lymph node biopsy (SLNB) in thin melanomas. This study investigates whether MR plays a role in selecting patients with T1 melanoma for SLNB. We analyzed clinical and histological data from the Florence Melanoma & Skin Cancer Unit database for 313 patients with a single thin melanoma who had undergone SLNB. We determined sentinel lymph node (SLN) positivity percentages in T1 melanomas according to the AJCC 8th Edition focusing on MR. Of the 313 T1 patients, 108 had MR = 0, 127 had MR = 1 and 78 had MR ≥2. The overall SLN positivity rate was 8.6%, (5.6% with MR = 0, 6.3% with MR = 1 and 16.7% with MR ≥2). The SLNB positivity rate in T1b melanomas was 12.1%, (8.5% with MR = 0, 5.7% with MR = 1 and 24.4% with MR ≥2), whereas in T1a melanomas it was 5.8%, (3.3% with MR = 0, 6.8% with MR = 1 and 8.1% with MR ≥2). In a logistic regression analysis, MR ≥2 had an odds ratio of almost three in comparison with MR = 0/1 also adjusting for thickness. Thus, MR ≥2 significantly predicted SLN metastases in T1 melanomas. Of those patients with positive SLN, 37% were classified as T1a according to the AJCC 8th edition. These findings underline the importance of MR ≥2 in selecting patients with T1 cutaneous melanomas for SLNB.

The role of sentinel node tumor burden in modeling the prognosis of melanoma patients with positive sentinel node biopsy: an Italian melanoma intergroup study (N = 2,086).

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.

Tolerogenic IDO1+CD83- Langerhans Cells in Sentinel Lymph Nodes of Patients with Melanoma.

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin+ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1−CD83+ LCs; real immature IDO1−CD83− LCs; tolerogenic mature IDO1+CD83+ LCs; tolerogenic immature IDO1+CD83− LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.

Psychological impact of BRAF mutational status in advanced melanoma patients.

BACKGROUND: The aim of the study was to highlight the psychological aspects involved in patients with advanced melanoma and to describe the differences between subjects who are positive and negative for the BRAFv600e genetic mutation, a variable that leads to a different medical approach to cancer therapy. The hypothesis is that following knowledge of the genetic mutation and the therapeutic possibilities inherent to it, mutation positive patients (BRAF+) exhibit fewer negative psychological reactions than negative patients (BRAF-) at the time of diagnosis. METHODS: The tests used (SF-12, MHQ) were administered at the time of diagnosis and after three months. RESULTS: The main findings suggest a greater impairment of quality of life at T1 than at T0, regardless of the mutation; BRAF mutated patients show more favorable scores at diagnosis and a reversal of the trend at three months after diagnosis. CONCLUSIONS: The results obtained, in line with the literature under review, show a significant general psychological distress in the present oncological sample, suggesting the importance of a psychological, as well as medical, care of the patient and the family.

Melanoma diagnosis: traumatic impact of the event on the patient.

BACKGROUND: The aim of our study was to consider the distressing impact of the diagnosis in a group of patients with metastatic melanoma, and the effects it could have on the quality of life of the patients. METHODS: We proposed an Impact Event Scale (IES-R) to a group of 31 patients. The patients were positive to the distress thermometer (DS) and accepted the psychological support. After six months from the start of the treatment we made a semi-structured interview of 10 multiple choice questions. RESULTS: Sixty-five per cent of women and 50% of men report that all the event related to the disease, cause emotions that recall the disease. Eighty-two per cent of women compared to 50% of men, report that the thought of their medical condition tends to affect their quality of sleep; the patients report feelings of anger and irritation (41% of the women and 78% of the men). CONCLUSIONS: The traumatic aspects following the diagnosis of melanoma burst powerfully into the life of these patients, who show different reactions, also according to gender.

Efficacy of Electrochemotherapy in the Treatment of Cutaneous Melanoma Metastases and Rare Non-melanoma Skin Cancer.

BACKGROUND: We aimed to verify the clinical efficacy and safety of the electrochemotherapy in melanoma metastases and in cases of rare non-melanoma tumors that were difficult to treat for the specific anatomical site or for patient comorbidities. PATIENTS AND METHODS: We treated 68 patients (699 cutaneous nodules), 44 patients with metastatic melanomas and 24 patients with non-melanoma tumors, at the Melanoma & Skin Cancer Unit, Florence, Italy. RESULTS: We obtained an objective response of 89.7% (88.6% in melanomas and 91.7% in non-melanoma tumors), complete response 54.4% and partial response 35.3%. CONCLUSION: This study showed that electrochemotherapy is effective in the treatment of melanoma metastases and in rare types of non-melanoma tumors. In particular, we successfully treated rare tumors as angiosarcoma, pleomorphic sarcoma, myxofibrosarcoma, sarcoma di Kaposi, porocarcinoma, sebaceous carcinoma, Merkel cell carcinoma, malignant blue nevus, undifferentiated epitheliomorphic cell neoplasia and metastases from thyroid carcinoma. No serious adverse events were observed.

Factors Affecting Sentinel Node Metastasis in Thin (T1) Cutaneous Melanomas: Development and External Validation of a Predictive Nomogram.

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.

Correction to: Thick melanoma in Tuscany.

This corrects the article DOI: 10.23736/S0392-0488.17.05584-5.

Correction to: New paradigm for stage III melanoma: from surgery to adjuvant treatment.

Following publication of the original article [1], the authors reported that one of the authors, Corrado Caracò, has been accidentally omitted from the author list. In this Correction the author has been added to the author list.

New paradigm for stage III melanoma: from surgery to adjuvant treatment.

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.

Enhancing the prognostic role of melanoma sentinel lymph nodes through microscopic tumour burden characterization: clinical usefulness in patients who do not undergo complete lymph node dissection.

This study aimed to investigate the sentinel lymph node (SLN) tumour burden to predict the non-SLN positivity rate and the survival of melanoma patients to evaluate whether SLN microstaging could predict the prognosis, similar to what is currently performed by examining the lymph nodes excised by complete lymph node dissection. Of 1130 consecutive melanoma patients who underwent SLN biopsy, 226 were tumour-positive and 204 were included in this study. SLN metastases were classified on the basis of dimensional (Rotterdam) and topographic (Dewar) criteria either separately or combined. SLN metastases more than 1 mm in diameter had the highest non-SLN positivity rate (31%) compared with metastases 0.1-1 mm (10%) and less than 0.1 mm (4%). The non-SLN positivity rate was 45% for extensive metastases, 5% for subcapsular metastases and 23-29% for parenchymal, combined and multifocal classes, therefore suggesting a simplification of the parenchymal SLN metastases into only two classes: extensive and 'not extensive'. The dimension of the metastasis was correlated with a different non-SLN positivity rate only when the metastasis was in the parenchyma (20-36%) and not when it was in the subcapsular location (4-7%). Interestingly, the 5-year melanoma-specific survival (MSS) was 89% for patients with subcapsular less than 0.1 mm metastases and 45% for patients with nonsubcapsular more than 1 mm metastases (P=0.017). In the parenchyma, larger metastases (>1 mm) were related to a lower 5-year MSS (46%) than smaller (<1 mm) metastases (MSS 77%). SLN tumour burden characterization can be simplified and it can provide prognostic information on non-SLN positivity and survival, which is especially useful in patients who do not undergo complete lymph node dissection.

Thick melanoma in Tuscany.

BACKGROUND: The epidemiologic trends of cutaneous melanoma are similar in several countries with a Western-type lifestyle, where there is a progressively increasing incidence and a low but not decreasing mortality - even increasing in selected cases, especially in the older age groups. Also in Tuscany there is a steady rise in the incidence with prevalence of in situ and invasive thin melanomas, with also an increase of thick melanomas. It is necessary to reduce the frequency of thick melanomas to reduce specific mortality. The objective of the current survey has been to compare, in the Tuscany population, by a case-case study, thin and thick melanoma cases, trying to find out those personal and tumor characteristics which may help to customize preventive interventions. METHODS: The study included nine centers involved in the melanoma diagnosis. A consecutive series of incident invasive melanomas diagnosed in a period of about 18 months (July 2010 to December 2011) was collected and matched according in a ratio of one thick melanoma (cutoff thickness: 1 mm) every two thin melanomas. The investigators filled in a questionnaire on patients' self-reported sun exposure, way of melanoma detection, awareness and performance of self-skin examination, as well as propensity to prevention in general. RESULTS: The results of this survey confirm that older age and the lower education level are associated with a later detection. The habit of performing skin self-examination is crucial in the early diagnosis of thick melanoma. The results of this survey seem to suggest that population aged over 50 years, with few total and few atypical nevi, and limited sun exposure and burning are at higher risk of late diagnosis. It can be assumed that part of the population is not effectively reached by prevention campaigns because they do not recognize themselves as being at risk for skin cancers. CONCLUSIONS: In order to achieve a higher rate of early diagnosis of skin melanoma, a new strategy must be implemented. It could be useful to rethink educational campaigns - which seem to unintentionally leave out subjects more at risk for melanoma - and to renew the active involvement of the general practitioners.

Quality assurance in melanoma care: The EU-MELACARE study.

BACKGROUND: A significant disparity regarding survival outcome for melanoma among European regions is well recognized and access to high quality care for European melanoma patients needs to be improved. There is an unmet need for the implementation of minimal standard of care within defined clinical pathways and Quality Assurance (QA) indicators. OBJECTIVE: The EU-MELACARE study aims to identify shared variables for cutaneous melanoma cases recorded in melanoma registries across Europe. MATERIAL AND METHODS: Opinion leaders involved in melanoma data registration and care quality analysis in 34 European countries were invited to respond to an expert survey covering questions regarding the melanoma registration practice in their countries and the characteristics, coverage and variables collected by the relevant melanoma registries. RESULTS: Data regarding 13 melanoma registries from 11 European countries contributed to the study. The majority (61,5%) were population based registries and more than half (62%) had national coverage. The included registries collected a median of 38 variables (Interquartile Range, IRQ 21-76). We identified 24 shared variables available in >70% of registries. CONCLUSIONS: This study provides valuable specific information on information recorded for melanoma cases are registered within Europe. A core of shared variables has been identified, which will constitute the basis for a standardized set of QA indicators for assessing and monitoring melanoma care across European countries.

ERK5 is activated by oncogenic BRAF and promotes melanoma growth.

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

Prediction of Non-sentinel Node Status in Patients with Melanoma and Positive Sentinel Node Biopsy: An Italian Melanoma Intergroup (IMI) Study.

BACKGROUND AND PURPOSE: Approximately 20% of melanoma patients harbor metastases in non-sentinel nodes (NSNs) after a positive sentinel node biopsy (SNB), and recent evidence questions the therapeutic benefit of completion lymph node dissection (CLND). We built a nomogram for prediction of NSN status in melanoma patients with positive SNB. METHODS: Data on anthropometric and clinicopathological features of patients with cutaneous melanoma who underwent CLND after a positive SNB were collected from nine Italian centers. Multivariate logistic regression was utilized to identify predictors of NSN status in a training set, while model efficiency was validated in a validation set. RESULTS: Data were available for 1220 patients treated from 2000 through 2016. In the training set (n = 810), the risk of NSN involvement was higher when (1) the primary melanoma is thicker or (2) sited in the trunk/head and neck; (3) fewer nodes are excised and (4) more nodes are involved; and (5) the lymph node metastasis is larger or (6) is deeply located. The model showed high discrimination (area under the receiver operating characteristic curve 0.74, 95% confidence interval [CI] 0.70-0.79) and calibration (Brier score 0.16, 95% CI 0.15-0.17) performance in the validation set (n = 410). The nomogram including these six clinicopathological variables performed significantly better than five other previously published models in terms of both discrimination and calibration. CONCLUSIONS: Our nomogram could be useful for follow-up personalization in clinical practice, and for patient risk stratification while conducting clinical trials or analyzing their results.

High Antigen Processing Machinery component expression in Langerhans cells from melanoma patients' sentinel lymph nodes.

Langerhans cells (LCs) from melanoma patients sentinel lymph nodes (SLN) are poor T cell activators mostly due to an immature immunophenotype. However Antigen Presenting Machinery (APM) role is unknown. We investigated HLA-class I APM components (Delta, LMP-7/10, TAP-1, Calnexin, Tapasin, ß2-microglobulin and HLA-A,B,C) in LCs from healthy donors skin and melanoma patients SLN. APM component levels were low in immature epidermal LCs and significantly increased after maturation (p<0.05); their levels were significantly high in SLN LCs (p<0.01). APM component expression correlated with melanoma Breslow's thickness and SLN metastases: HLA-A,B,C level was significantly lower in SLN LCs from thick lesions patients compared with those from thin/intermediate lesions (p<0.05); ß2-microglobulin level was significantly higher in positive SLN LCs compared to negative ones (p<0.05). Functionally, SLN LCs did not phagocytose exogenous antigens. These findings extend LCs knowledge indicating that they are not fully impaired by melanoma, contributing to design new LCs-based therapeutic approaches.