The relevance of the BRAF V600E mutation in papillary thyroid carcinoma (PTC) as a negative prognostic factor is a subject of intense debate. This mutation has been associated with several clinicopathological features, but the lack of consistency among data does not support its usefulness as marker of tumor aggressiveness and poorer outcome. Due to the genetic heterogeneity of the tumor, both the occurrence and the allele percentage of the BRAF mutation should be considered to unravel this controversy. We aimed to evaluate the impact of the BRAF V600E mutation occurrence and the allele percentage on the metastatic process in PTCs. The presence and allele percentage of the BRAF mutation were determined by pyrosequencing in 132 cases of well-differentiated PTCs with (n = 37) or without lymph node metastases (LNMs) (n = 95) and in 40 LNMs matched with 35 PTCs. No significant differences were observed in either the occurrence or the allele percentage of V600E mutation between the 2 groups of PTCs with or without LNMs. The LNMs were heterogeneous for the V600E mutation as the primary lesions. In this study, the occurrence and percentage of the BRAF V600E mutated allele was not preferentially associated with the development of metastases and the average mutated allele percentage decreased as the tumor progresses from the primary site to the lymph node metastatic sites. These observations support the need to reevaluate the role of the BRAF V600E mutation as a negative prognostic marker in PTCs.

Aberrant reactivation of embryonic pathways is a common feature of cancer. RUNX2 is a transcription factor crucial during embryogenesis that is aberrantly reactivated in many tumors, including thyroid and breast cancer, where it promotes aggressiveness and metastatic spreading. Currently, the mechanisms driving RUNX2 expression in cancer are still largely unknown. Here we showed that RUNX2 transcription in thyroid and breast cancer requires the cooperation of three distantly located enhancers (ENHs) brought together by chromatin three-dimensional looping. We showed that BRD4 controls RUNX2 by binding to the newly identified ENHs and we demonstrated that the anti-proliferative effects of bromodomain inhibitors (BETi) is associated with RUNX2 transcriptional repression. We demonstrated that each RUNX2 ENH is potentially controlled by a distinct set of TFs and we identified c-JUN as the principal pivot of this regulatory platform. We also observed that accumulation of genetic mutations within these elements correlates with metastatic behavior in human thyroid tumors. Finally, we identified RAINs, a novel family of ENH-associated long non-coding RNAs, transcribed from the identified RUNX2 regulatory unit. Our data provide a new model to explain how RUNX2 expression is reactivated in thyroid and breast cancer and how cancer-driving signaling pathways converge on the regulation of this gene.

Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis. Thirty patients with histologically proven PCa, 33 with benign prostate hyperplasia (BPH) and 21 healthy controls were enrolled. cfDNA was extracted from seminal fluid samples. cfDNA quantification and analysis were performed using Qubit ssDNA Kit and Agilent 2100 Bioanalyzer. Statistical analysis included: Levene's test, Shapiro-Wilk, Kolmogorov-Smirnov and Kruskal Wallis tests. Median cfDNA was significantly higher in PCa patients 428.45 ng/mL (173.93-1159.62) compared to BPH patients 77.4 ng/mL (18.23-501) and healthy controls 25.4 ng/mL (15.37-76.62). scfDNA fragments longer than 1000 base-pairs were more common in patients with PCa compared to those with BPH and controls. scfDNA concentration and fragment size differed significantly in the three groups of PCa, BPH and healthy controls. Both parameters are potential clinical biomarkers for PCa and can be used in both early diagnosis and follow-up. Using automated systems for high-throughput cfDNA quantification could improve the reproducibility of the method and facilitate the implementation of liquid biopsies in the clinical setting.

Purpose: Pulmonary Sarcomatoid Carcinoma (PSC) is an aggressive form of NSCLC. Rarity and poor characterization have limited development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment. Experimental Design: A training set of 17 biphasic PSCs was tested for the expression of a large panel of 770 genes related to cancer progression using Nanostring technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype. Results: We demonstrated that transcriptional activation of an Epithelial Mesenchymal Transition (EMT) program drives PSC phylogeny in vivo. We showed, that loss of the epithelial-associated transcription factor (TF) OVOL2 characterizes the transition to sarcomatoid phenotype triggering the expression of EMT promoting TFs, including TWIST,ZEB and the expression of the membrane kinase DDR2. Finally, using a drug repurposing approach, we identified dasatinib as potential inhibitor of the PSC-gene expression signature and we confirmed in vitro that this drug efficiently restrains proliferation and reverts the sarcomatoid-associated phenotype. Conclusions: Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.