Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Proposal of a classification of cannulation damage in vascular access grafts based on clinical, ultrasound, and microscopic observations.

INTRODUCTION: Arteriovenous grafts (AVGs) serve as an alternative to native arteriovenous fistulas (AVFs) in the context of hemodialysis patient life planning. AVGs are more susceptible to developing outflow stenosis (due to intimal hyperplasia), thrombosis, and infections. However, an often overlooked contributor to AVG failure is cannulation damage. The objective of this paper is to assess the impact of cannulations on AVGs. We aim to establish a classification of AVG damage by comparing clinical data and ultrasound images with microscopic morphological findings obtained from explanted grafts. MATERIALS AND METHODS: This study is conducted at a single center. We included all patients who underwent AVG creation between 2011 and 2019. Comprehensive data on clinical history, follow-up, and complications were collected and reviewed. Duplex ultrasound (DUS) characteristics were documented, and all grafts explanted during the analysis period underwent optical microscopy evaluation. Finally, clinical data, along with DUS and microscopic findings, were integrated to derive a damage classification. RESULTS: During the study period, 247 patients underwent 334 early cannulation AVGs. The median follow-up duration was 714 days (IQR 392, 1195). One hundred eleven (33%) grafts were explanted. Clinical data and DUS findings were utilized to formulate a four-grade classification system indicating increasing damage. CONCLUSION: Cannulation damage alone does not solely account for AVG failure. It results from a biological host-mediated process that promotes the growth of intimal hyperplasia at the cannulation sites. This process is not clinically significant within the initial 2 years after AVG creation.

Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade.

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.