Purpose The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. Patients and Methods In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. Results BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. Conclusion In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

There is increasing evidence that mast cells (MCs) and their mediators are involved in the remodeling of the tumor microenvironment and promote tumor growth, angiogenesis and metastasis. We have found that an increased density of MCs in thyroid cancer (TC) correlates with enhanced invasiveness. However, the MC-derived factors responsible for this activity and the mechanisms by which they enhance TC invasiveness remain unidentified. Here, we report that MCs, when activated by TC cells, produce soluble factors that induce epithelial-to-mesenchymal transition (EMT) and stemness features of TC cells. We identified CXCL8/interleukin (IL)-8 as the main mediator contained in activated MC conditioned media (CM) capable of inducing both EMT and stemness of TC cells. Mechanistically, MC CM or exogenous IL-8 stimulated Akt phosphorylation and Slug expression in TC cells. The inhibition of the Akt pathway or depletion of the Slug transcription factor by RNA interference, reverted EMT and stemness responses. TC cells stably transfected with exogenous IL-8 underwent EMT, displayed increased stemness and enhanced tumorigenicity with respect to control cells. The analysis of TC surgical specimens by immunohistochemical analysis demonstrated a positive correlation between MC density (Tryptase(+) cells) and stemness features (OCT4 staining). Taken together, our data identify an MC-dependent IL-8-Akt-Slug pathway that sustains EMT/stemness of TC cells. The blockade of this circuit might be exploited for the therapy of advanced TC.

Purpose: CXCR4 receptor and its unique ligand, the CXCL12 chemokine, have been recently implicated in cancer metastasis. Evidence about the role of CXCR4/CXCL12 axis has been reported in several cancers including melanoma. Our goal was to investigate if CXCR4 expression has a prognostic value in malignant melanoma.Experimental Design: Immunohistochemical expression of CXCR4 was evaluated on 71 specimens of primary cutaneous melanoma with a Breslow tumor thickness of >1 mm after radical resection. Associations between baseline patient features and tumors were analyzed by C 2 test. The prognostic value of CXCR4 expression was evaluated by univariate and multivariate analyses adjusted by age, sex, Breslow tumor thickness, presence of ulceration, and sentinel lymph node metastases.Results: CXCR4 expression was detected in 31 of 71 (43.6%) primary cutaneous melanomas. Membrane or cytoplasmic staining for CXCR4 protein was absent in 56% of the tumors. The positive cases were divided into three score classes according to their staining: low in 15 cases (21%), moderate in 10 (14%), and high in 6 (8%). After a median follow-up of 38 months, 26 patients progressed (16 of 26 expressed CXCR4) and 19 died (12 of 19 expressed CXCR4). The CXCR4 expression on tumor cells was correlated with an unfavorable prognosis with a median disease-free and overall survival of 22 and 35 months, respectively. The hazard ratios of relapse and death, compared with patients with CXCR4-negative tumors, were 2.5 (95% confidence interval, 1.2-6.1) and 3.1 (95% confidence interval, 1.1-7.2), respectively. Median time-to-event (progression and survival) was not reached in patients with CXCR4-negative tumors. In the multivariate analysis, CXCR4 expression, presence of ulceration, and sentinel lymph node status emerged as independent prognostic factors.Conclusions: This article provides the first evidence that CXCR4 expression could be an independent and powerful prognostic marker in primary cutaneous malignant melanomas.

Here, we report a retrospective series of 47 EBV-positive diffuse large B-cell lymphoma associated with advanced age. Histopathology allowed to the identification of different histological patterns: cases with polymorphic diffuse large B-cell lymphoma (29 cases), Hodgkin-like (8 cases) and polymorphic lymphoproliferative disorder-like (9 cases) patterns. One case was purely monomorphic diffuse large B-cell lymphoma. We show that this lymphoma type is a neoplasm with prominent classical and alternative nuclear factor-kB pathway activation in neoplastic cells (79% of the cases showed nuclear staining for p105/p50, 74% for p100/p52 and 63% for both proteins), with higher frequency than that observed in a control series of EBV-negative diffuse large B-cell lymphoma (v 2 o0.001). Most cases showed an activated phenotype (95% non-germinal center (Hans algorithm); 78% activated B cell (Choi algorithm)). Clonality testing demonstrated IgH and/or K/Kde/L monoclonal rearrangements in 64% of cases and clonal T-cell populations in 24% of cases. C-MYC (1 case), BCL6 (2 cases) or IgH (3 cases) translocations were detected by FISH in 18% cases. These tumors had a poor overall survival and progression-free survival (the estimated 2-year overall survival was 40±10% and the estimated 2-year progression-free survival was 36±9%). Thus, alternative therapies, based on the tumor biology, need to be tested in patients with EBV-positive diffuse large B-cell lymphoma of the elderly. Modern Pathology (2012) 25, 968-982;

We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.