Background and objectives: Cancer stem cells (CSCs) have been implicated in tumor initiation, recurrence, metastatic spread and poor survival in multiple tumor types, breast cancers included. CSCs selectively overexpress key mitochondrial-related proteins and inhibition of mitochondrial function may represent a new potential approach for the eradication of CSCs. Because mitochondria evolved from bacteria, many classes of FDA-approved antibiotics, including doxycycline, actually target mitochondria. Our clinical pilot study aimed to determine whether short-term pre-operative treatment with oral doxycycline results in reduction of CSCs in early breast cancer patients.Methods: Doxycycline was administered orally for 14 days before surgery for a daily dose of 200 mg. Immuno-histochemical analysis of formalin-fixed paraffin-embedded (FFPE) samples from 15 patients, of which 9 were treated with doxycycline and 6 were controls (no treatment), was performed with known biomarkers of “stemness” (CD44, ALDH1), mitochondria (TOMM20), cell proliferation (Ki67, p27), apoptosis (cleaved caspase-3), and neo-angiogenesis (CD31). For each patient, the analysis was performed both on pre-operative specimens (core-biopsies) and surgical specimens. Changes from baseline to post-treatment were assessed with MedCalc 12 (unpaired t-test) and ANOVA.Results: Post-doxycycline tumor samples demonstrated a statistically significant decrease in the stemness marker CD44 (p-value < 0.005), when compared to pre-doxycycline tumor samples. More specifically, CD44 levels were reduced between 17.65 and 66.67%, in 8 out of 9 patients treated with doxycycline. In contrast, only one patient showed a rise in CD44, by 15%. Overall, this represents a positive response rate of nearly 90%. Similar results were also obtained with ALDH1, another marker of stemness. In contrast, markers of mitochondria, proliferation, apoptosis, and neo-angiogenesis, were all similar between the two groups.Conclusions: Quantitative decreases in CD44 and ALDH1 expression are consistent with pre-clinical experiments and suggest that doxycycline can selectively eradicate CSCs in breast cancer patients in vivo. Future studies (with larger numbers of patients) will be conducted to validate these promising pilot studies.

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC.

Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma.

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Uveal melanoma is one of the most deadly diseases in ophthalmology for which markers able to predict the appearance of metastasis are needed. The study investigates the role of circulating tumor cells (CTC) as a prognostic factor in this disease. We report the detection of circulating tumor cells by Isolation by Size of Epithelial Tumor cells (ISET) in a cohort of 31 uveal melanoma patients: we identified single CTCs or clusters of cells in 17 patients, while the control population, subjects with choroidal nevi, showed no CTC in peripheral blood. The presence of CTCs did not correlate with any clinical and pathological parameter, such as tumor larger basal diameter (LBD), tumor height and TNM. By stratifying patients in groups on the basis of the number of CTC (lower or higher than 10 CTC per 10 mL blood) and the presence of CTC clusters we found a significant difference in LBD (p = 0.019), Tumor height (p = 0.048), disease-free and overall survival (p < 0.05). In conclusion, we confirm the role of CTC as a negative prognostic marker in uveal melanoma patients after a long follow-up period. Further characterization of CTC will help understanding uveal melanoma metastasization and improve patient management.

Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.

Adrenocortical cancer (ACC) is a rare aggressive malignancy. Recent ACC integrated genomics analysis contributed to redefine the risk groups on molecular basis, including tumor microRNAs (miRs), detectable also in the bloodstream. We developed a quantitative real-time (RT) assay for the measurement of miR483 and miR483-5p absolute levels in plasma samples. miR483/miR483-5p levels were evaluated in plasma samples of 27 patients with ACC before surgery and at follow-up.Statistically significant differences in miR483-5p and miR483 levels were found between stage 1/2 and stage 3/4 ACCs in pre-surgery and post-surgery samples. ROC curve analysis of miR483–5p levels gave a prediction of the clinical stage (accuracy 0.917±0.084), with the best cut-off value of 0.221 ng/ml, prognosticating overall and recurrence-free survival. In a multivariate Cox analysis (HR 16.2, 95%CI[1.39-188.6, P<0.026]), miR483-5p was the only variable that significantly predicted recurrence, but not overall survival. In addition, miR483 and miR483-5p levels correlated with the number of circulating tumor cells (CTCs) detected in the same blood samples, independently of the timing of sampling. In conclusion, we demonstrated that miR483-5p absolute plasma levels in ACC patients are powerful molecular markers that may help in the follow-up of patients after surgery and chemotherapy, and contribute to more accurately classify and predict tumor progression.

Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite the combination of novel therapeutical approaches, it remains a deadly malignancy with an abysmal prognosis. GBM is a polymorphic tumour from both molecular and histological points of view. It consists of different malignant cells and various stromal cells, contributing to tumour initiation, progression, and treatment response. GBM’s microenvironment is multifaceted and is made up of soluble factors, extracellular matrix components, tissue-resident cell types (e.g., neurons, astrocytes, endothelial cells, pericytes, and fibroblasts) together with resident (e.g., microglia) or recruited (e.g., bone marrow-derived macrophages) immune cells. These latter constitute the so-called immune microenvironment, accounting for a substantial GBM’s tumour volume. Despite the abundance of immune cells, an intense state of tumour immunosuppression is promoted and developed; this represents the significant challenge for cancer cells’ immune-mediated destruction. Though literature data suggest that distinct GBM’s subtypes harbour differences in their microenvironment, its role in treatment response remains obscure. However, an in-depth investigation of GBM’s microenvironment may lead to novel therapeutic opportunities to improve patients’ outcomes. This review will elucidate the GBM’s microenvironment composition, highlighting the current state of the art in immunotherapy approaches. We will focus on novel strategies of active and passive immunotherapies, including vaccination, gene therapy, checkpoint blockade, and adoptive T-cell therapies.