Background:A compromised base excision repair (BER) promotes carcinogenesis by accumulating oxidative DNA-damaged products as observed in MUTYH-associated polyposis, a hereditary colorectal cancer syndrome marked by adenomas and cancers with an accumulation of 8-oxoguanine. Remarkably, DNA global demethylation has been shown to be mediated by BER, suggesting a relevant interplay with early colorectal tumourigenesis. To check this hypothesis, we investigated a cohort of 49 adenomas and 10 carcinomas, derived from 17 MUTYH-associated polyposis patients; as adenoma controls, we used a set of 36 familial adenomatous polyposis and 24 sporadic polyps.Methods:Samples were analysed for their mutational and epigenetic status, measured as global LINE-1 (long interspersed nuclear element) and gene-specific LINE-1 MET methylation by mass spectrometry and pyrosequencing.Results:MUTYH-associated polyposis adenomas were strikingly more hypomethylated than familial adenomatous and sporadic polyps for both DNA demethylation markers (P=0.032 and P=0.007 for LINE-1; P=0.004 and P<0.0001 for LINE-1 MET, respectively) with levels comparable to those of the carcinomas derived from the same patients. They also had mutations due mainly to KRAS/NRAS p.G12C, which was absent in the controls (P<0.0001 for both sets).Conclusions:Our results show that DNA demethylation, together with specific KRAS/NRAS mutations, drives the early steps of oxidative damage colorectal tumourigenesis.

Background Aberrant DNA hypomethylation of the long interspersed nuclear elements (LINE-1 or L1) has been recognized as an early event of colorectal transformation. Simultaneous genetic and epigenetic analysis of colorectal adenomas may be an effective and rapid strategy to identify key biological features leading to accelerated colorectal tumorigenesis. In particular, global and/or intragenic LINE-1 hypomethylation of adenomas may represent a helpful tool for improving colorectal cancer (CRC) risk stratification of patients after surgical removal of polyps. To verify this hypothesis, we analyzed a cohort of 102 adenomas derived from 40 high-risk patients (who developed CRC in a post-polypectomy of at least one year) and 43 low-risk patients (who did not develop CRC in a post-polypectomy of at least 5 years) for their main pathological features, the presence of hotspot variants in driver oncogenes (KRAS, NRAS, BRAF and PIK3CA), global (LINE-1) and intragenic (L1-MET) methylation status. Results In addition to a significantly higher adenoma size and an older patients’ age, adenomas from high-risk patients were more hypomethylated than those from low-risk patients for both global and intragenic LINE-1 assays. DNA hypomethylation, measured by pyrosequencing, was independent from other parameters, including the presence of oncogenic hotspot variants detected by mass spectrometry. Combining LINE-1 and L1-MET analyses and profiling the samples according to the presence of at least one hypomethylated assay improved the discrimination between high and low risk lesions (p = 0.005). Remarkably, adenomas with at least one hypomethylated assay identified the patients with a significantly (p < 0.001) higher risk of developing CRC. Multivariable analysis and logistic regression evaluated by the ROC curves proved that methylation status was an independent variable improving cancer risk prediction (p = 0.02). Conclusions LINE-1 and L1-MET hypomethylation in colorectal adenomas are associated with a higher risk of developing CRC. DNA global and intragenic hypomethylation are independent markers that could be used in combination to successfully improve the stratification of patients who enter a colonoscopy surveillance program. Graphic abstract

Background: BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes.Patients and methods: The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients' clinical characteristics. KaplaneMeier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression. Results: A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P ¼ 0.017), to melanoma thickness according to Clark levels, (level V versus III, P ¼ 0.004; level V versus IV, P ¼ 0.04), to lymph node metastases rather than cutaneous (P ¼ 0.04) or visceral (P ¼ 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P ¼ 0.04; PFS P ¼ 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P ¼ 0.014 and P ¼ 0.003, respectively). Conclusion: These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.

Tumour molecular annotation is mandatory for biomarker discovery and personalised approaches, particularly in triple-negative breast cancer (TNBC) lacking effective treatment options. In this study, the interleukin-3 receptor α (IL-3Rα) was investigated as a prognostic biomarker and therapeutic target in TNBC. IL-3Rα expression and patients’ clinical and pathological features were retrospectively analysed in 421 TNBC patients. IL-3Rα was expressed in 69% human TNBC samples, and its expression was associated with nodal metastases (p = 0.026) and poor overall survival (hazard ratio = 1.50; 95% CI = 1.01–2.2; p = 0.04). The bioinformatics analysis on the Breast Invasive Carcinoma dataset of The Cancer Genome Atlas (TCGA) proved that IL-3Rα was highly expressed in TNBC compared with luminal breast cancers (p = 0.017, padj = 0.026). Functional studies demonstrated that IL-3Rα activation induced epithelial-to-endothelial and epithelial-to-mesenchymal transition, promoted large blood lacunae and lung metastasis formation, and increased programmed-cell death ligand-1 (PD-L1) in primary tumours and metastases. Based on the TCGA data, IL-3Rα, PD-L1, and EMT coding genes were proposed to discriminate against TNBC aggressiveness (AUC = 0.86 95% CI = 0.82–0.89). Overall, this study identified IL-3Rα as an additional novel biomarker of TNBC aggressiveness and provided the rationale to further investigate its relevance as a therapeutic target.

The activation of the LINE-1 sequence located within the second intron of MET leads to the onset of L1-MET transcript. We recently characterized the full L1-MET structure in breast cancer and showed that high levels of this transcript recognize a subset of more aggressive breast carcinomas, mainly of triple negative phenotype. However, at present, the relationship between L1-MET and MET is still poorly understood. In order to elucidate this function, we silenced L1-MET transcription using cells expressing different levels of L1-MET/MET, including lung cancer (A549 and EBC1), gastric cancer (GTL16), and breast cancer (MDA-MB231), that were transiently transfected with Gapmers-LNA (Exiqon), specifically targeting L1-MET sequence. Cell viability and apoptosis were evaluated after 24 h by cell count, Cell Titer Glow (Promega) and propidium iodide/annexin based-cytofluorimeter assays. RNA was purified from sample and control cells to assess the L1-METsilencing by qRT-PCR and to evaluate the gene-expression of a subset of cancer-related genes using Nanostring Technology, whereas western blot analyses were carried out to measure the protein expressions. A significant decrease of cell viability was detected in A549, EBC1 and GTL16, but not in MDA-MB231 cells, characterized by the lowest level of L1-MET overall. In parallel, the highly expressing L1-MET cells showed an increased rate of early and late apoptosis together with a strong reduction of MET gene and its protein expression. On the contrary, in MDA-MB231 cells L1-MET silencing induced only a slight MET gene and protein impairment. Overall, L1-MET knock-down caused a decrease expression of a conserved gene cluster, including a marked reduction of EGFR protein expression. Moreover, L1-MET silenced cells showed lower MET and EGFR phosphorylation, with a downstream silencing effect on pERK and pAKT. Results of cell treatment with the inhibitors of lysosome and proteasome activity bafilomycin and MG-132 ruled out the interaction of L1-MET silencing with protein degradation pathway. This is the first study investigating the function of the L1-MET transcript in cancer models. Our results show that although L1-MET is unable to encode for a protein, its silencing exerts a strong phenotypic effect on different tumor cell types, suggesting potential regulations at the transcriptional level. Note: This abstract was not presented at the meeting. Citation Format: Enrico Berrino, Umberto Miglio, Valentina Miano, Letizia Lanzetti, Silvia Benvenuti, Carla Debernardi, MIchele De Bortoli, Caterina Marchio, Tiziana Venesio, Anna Sapino. L1-MET transcription silencing modulates MET and EGFR gene and their protein expression and induces apoptosis and cell-death in different types of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 261.

No abstract

Background: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. Methods: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. Results: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. Conclusions: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate.

No abstract