TP63 mutations are frequent in cutaneous melanoma, support UV etiology, but their role in melanomagenesis is unclear

In contrast to TP53, cancer development is rarely associated with mutations in the TP63 and TP73 genes. Recently, next generation sequencing analysis revealed that TP63 mutations are frequent, specifically in cutaneous melanomas. Cutaneous melanoma represents 4% of skin cancers but it is responsible for 80% of skin cancer related deaths. In the present study, we first determined whether all three members of the P53 family of transcription factors were found mutated in cutaneous melanomas by retrieving all TP53, TP63 and TP73 mutations from cBioPortal (http://www.cbioportal.org/). TP53 and TP63 were frequently mutated [15.0% (91/605) and 14.7% (89/605), respectively], while TP73 [1.5% (9/605)] was more rarely mutated (p<0.0001). A UV-mutation fingerprint was recognized for TP63 and TP73 genes. Then, we tried to evaluate the potential role of TP63 mutations as drivers or passengers in the tumorigenic process. In the former case, the amino acid substitutions should cause significant functional consequences on the main biochemical activity of the P63 protein, namely transactivation. The predicted effects of specific amino acid substitutions by two bioinformatics tools were rather different. Using a yeast-based functional assay, the observed hotspot mutant R379CP63 protein exhibited a substantial residual activity compared to the wild-type (>70%). This result does not support a major role of the mutant P63 protein in melanomagenesis while it is still consistent with the TP63 gene being a recorder of UV exposure. The TP63 mutation spectrum from cutaneous melanomas, when compared with that observed at the germinal level in patients affected by P63-associated diseases [ectodermal dysplasia syndromes, (EDs)], revealed significant differences. The TP63 mutations were more frequent at CpGs sites (p<0.0001) in EDs and at PyPy sites (p<0.0001) in cutaneous melanomas. The two spectra differed significantly (p<0.0001). We conclude that TP63 mutations are frequent in cutaneous melanoma, support UV etiology, but their role in melanomagenesis is unclear

Combining molecular and immunohistochemical analyses of key drivers in primary melanomas: Interplay between germline and somatic variations

Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants. BRAF mutations were present in 68% of cases, while CDKN2A germline mutations were found in 16 % and p16 loss in tissue was found in 63%. TERT promoter somatic mutations were detected in 38% of cases while the TERT -245T > C polymorphism was found in 51% of cases. NRAS mutations were found in 39% of BRAF negative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a positive, although not significant association between CDKN2A germline mutations, but not MC1R variants, and BRAF somatic mutation was found, we did not observe other associations between germline and somatic events. A yet undescribed inverse correlation between TERT -245T > C polymorphism and the presence of BRAF mutation was found. It is possible to hypothesize that -245T > C polymorphism could be included in those genotypes which may influence the occurrence of BRAF mutations. Further studies are needed to investigate the role of -245T > C polymorphism as a germline predictor of BRAF somatic mutation status

EGFR-TKI plus anti-angiogenic drugs in EGFR-mutated NSCLC: a meta-analysis of randomized clinical trials

Abstract Background Results of several RCTs testing the combination of an EGFR-TKI plus an anti-angiogenic drug in advanced EGFR-mutated NSCLC were reported. Methods We first report a systematic-review and meta-analysis of all RCTs, to estimate effectiveness and toxicity of such new therapeutic approach as compared with first-generation EGFR-TKIs monotherapy. Subsequently, we present a network meta-analysis (NMA) comparing the combination of an EGFR-TKI plus an anti-angiogenic drug with other two new treatment-options: combination of an EGFR-TKI plus chemotherapy or new EGFR-TKIs of second or third generation as monotherapy. Results Five RCTs were included in the first meta-analysis. The PFS was statistically significantly larger in patients treated with an EGFR-TKI plus an anti-angiogenic drug as compared with EGFR-TKI monotherapy: the pooled PFS-HR was 0.59 (95% CI = 0.51 to 0.69). The pooled median-PFS was 17.8 months (95% CI = 16.5 to 19.3) for the combination versus 11.7 months (95% CI = 11.1 to 12.7) for EGFR-TKI as monotherapy. No statistically significant differences between the two treatment-arms were observed in terms of both OS and ORR. The rate of grade equal or higher than 3 AEs was statistically significantly higher in patients treated with EGFR-TKI plus an anti-angiogenic drug: the pooled-Relative Risk was 1.72 (95% CI = 1.43 to 2.06). Ten RCTs were included in the NMA. All the three experimental treatments were associated with a statistically significant improvement of PFS as compared with first-generation EGFR-TKIs. When compared to each other, none of the three experimental treatments was statistically significantly associated with larger PFS or lower rate of grade ≥3AEs. Conclusion Patients with EGFR-mutated NSCLC derived clinically meaningful larger PFS-benefit from the addition of an anti-angiogenic drug to a first-generation EGFR-TKI, at the cost of an increase of toxicitie

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used. Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable. Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples. Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered

Efficacy of novel immunotherapy regimens in patients with metastatic melanoma with germline CDKN2A mutations

Background: Inherited CDKN2A mutation is a strong risk factor for cutaneous melanoma. Moreover, carriers have been found to have poor melanoma-specific survival. In this study, responses to novel immunotherapy agents in CDKN2A mutation carriers with metastatic melanoma were evaluated. Methods: CDKN2A mutation carriers that have developed metastatic melanoma and undergone immunotherapy treatments were identified among carriers enrolled in follow-up studies for familial melanoma. The carriers' responses were compared with responses reported in phase III clinical trials for CTLA-4 and PD-1 inhibitors. From publicly available data sets, melanomas with somatic CDKN2A mutation were analysed for association with tumour mutational load. Results: Eleven of 19 carriers (58%) responded to the therapy, a significantly higher frequency than observed in clinical trials (p=0.03, binomial test against an expected rate of 37%). Further, 6 of the 19 carriers (32%) had complete response, a significantly higher frequency than observed in clinical trials (p=0.01, binomial test against an expected rate of 7%). In 118 melanomas with somatic CDKN2A mutations, significantly higher total numbers of mutations were observed compared with 761 melanomas without CDKN2A mutation (Wilcoxon test, p<0.001). Conclusion: Patients with CDKN2A mutated melanoma may have improved immunotherapy responses due to increased tumour mutational load, resulting in more neoantigens and stronger antitumorous immune responses

Efficacy of novel immunotherapy regimens in patients with metastatic melanoma with germline CDKN2A mutations

Inherited CDKN2A mutation is a strong risk factor for cutaneous melanoma. Moreover, carriers have been found to have poor melanoma-specific survival. In this study, responses to novel immunotherapy agents in CDKN2A mutation carriers with metastatic melanoma were evaluated

Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

BACKGROUND: Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting. METHODOLOGY/PRINCIPAL FINDINGS: We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called "unlicensed" NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells. CONCLUSIONS/SIGNIFICANCE: We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma

Nationwide multidisciplinary consensus on the clinical management of Merkel cell carcinoma: a Delphi panel

Merkel cell carcinoma (MCC) is a rare and highly aggressive cutaneous neuroendocrine carcinoma. The MCC incidence rate has rapidly grown over the last years, with Italy showing the highest increase among European countries. This malignancy has been the focus of active scientific research over the last years, focusing mainly on pathogenesis, new therapeutic trials and diagnosis. A national expert board developed 28 consensus statements that delineated the evolution of disease management and highlighted the paradigm shift towards the use of immunological strategies, which were then presented to a national MCC specialists panel for review. Sixty-five panelists answered both rounds of the questionnaire. The statements were divided into five areas: a high level of agreement was reached in the area of guidelines and multidisciplinary management, even if in real life the multidisciplinary team was not always represented by all the specialists. In the diagnostic pathway area, imaging played a crucial role in diagnosis and initial staging, planning for surgery or radiation therapy, assessment of treatment response and surveillance of recurrence and metastases. Concerning diagnosis, the usefulness of Merkel cell polyomavirus is recognized, but the agreement and consensus regarding the need for cytokeratin evaluation appears greater. Regarding the areas of clinical management and follow-up, patients with MCC require customized treatment. There was a wide dispersion of results and the suggestion to increase awareness about the adjuvant radiation therapy. The panelists unanimously agreed that the information concerning avelumab provided by the JAVELIN Merkel 200 study is adequate and reliable and that the expanded access program data could have concrete clinical implications. An immunocompromised patient with advanced MCC can be treated with immunotherapy after multidisciplinary risk/benefit assessment, as evidenced by real-world analysis and highlighted in the guidelines. A very high consensus regarding the addition of radiotherapy to treat the ongoing focal progression of immunotherapy was observed. This paper emphasizes the importance of collaboration and communication among the interprofessional team members and encourages managing patients with MCC within dedicated multidisciplinary teams. New insights in the treatment of this challenging cancer needs the contribution of many and different experts

The Multidisciplinary Management of Cutaneous Squamous Cell Carcinoma: A Comprehensive Review and Clinical Recommendations by a Panel of Experts

Simple Summary Cutaneous squamous cell carcinoma is one of the most common forms of cancer. Although most cases are cured with surgical excision, a few tumors are associated with a high risk of local or distant relapse; therefore, it is 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. Recently, PD-1 inhibitor cemiplimab was approved by the regulatory authorities for the treatment of advanced cutaneous squamous cell carcinoma; subsequently, the anti-PD-1 agent pembrolizumab received the approval by the FDA only in the same setting. Here, we provide a literature review and clinical recommendations by a panel of experts regarding the diagnosis, treatment, and follow-up of cutaneous squamous cell carcinoma. 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

Clinicopathological predictors of recurrence in nodular and superficial spreading cutaneous melanoma: A multivariate analysis of 214 cases

Abstract Background Nodular melanoma (NM) accounts for most thick melanomas and because of their frequent association with ulceration, fast growth rate and high mitotic rate, contribute substantially to melanoma-related mortality. In a multicentric series of 214 primary melanomas including 96 NM and 118 superficial spreading melanoma (SSM), histopathological features were examined with the aim to identify clinicopathological predictors of recurrence. Methods All consecutive cases of histopathologically diagnosed primary invasive SSM and NM during the period 2005–2010, were retrieved from the 12 participating Italian Melanoma Intergroup (IMI) centers. Each center provided clinico-pathological data such as gender, age at diagnosis, anatomical site, histopathological conventional parameters, date of excision and first melanoma recurrence. Results Results showed that NM subtype was significantly associated with Breslow thickness (BT) at multivariate analysis: [BT 1.01–2 mm (OR 7.22; 95% CI 2.73–19.05), BT 2.01–4 mm (OR 7.04; 95% CI 2.54–19.56), and BT > 4 mm (OR 51.78; 95% CI 5.65–474.86) (p  5 mitoses/mm2 (OR 4.87; 95% CI 1.77–13.40) (p = 0.002)]. The risk of recurrence was not significantly associated with NM histotype while BT [BT 1.01–2.00 mm (HR 1.55; 95% CI 0.51–4.71), BT 2.01–4.00 mm (HR 2.42; 95% CI 0.89–6.54), BT > 4.00 mm. (HR 3.13; 95% CI 0.95–10.28) (p = 0.05)], mitotic rate [MR > 2 mitoses/mm2 (HR 2.34; 95% CI, 1.11–4.97) (p = 0.03)] and the positivity of lymph node sentinel biopsy (SNLB) (HR 2.60; 95% CI 1.19–5.68) (p = 0.007) were significantly associated with an increased risk of recurrence at multivariate analysis. Conclusions We found that NM subtype was significantly associated with higher BT and MR but it was not a prognostic factor since it did not significantly correlate with melanoma recurrence rate. Conversely, increased BT and MR as well as SNLB positivity were significantly associated with a higher risk of melanoma recurrence