MicroRNA profiling in women with migraine: effects of CGRP-targeting treatment.

BACKGROUND: Migraine lacks biomarkers that can trace the biological pathways of the disease and predict the effectiveness of treatments. Monoclonal antibodies targeting calcitonin gene-related peptide pathway - including erenumab - offer the opportunity of investigating potential migraine biomarkers due to their specific mechanism of action in preventing both episodic (EM) and chronic (CM) migraine. Our study aims at evaluating the expression levels of circulating microRNAs (miRNAs) according to migraine type, before and after treatment with erenumab and based on treatment response, in order to identify miRNAs with potential role as epigenetic biomarkers. METHODS: The study included women aged 25-50 years with EM or CM treated with erenumab according to clinical indications. MiRNAs expression levels were assessed before (baseline) and after a 16-week treatment with erenumab, 140 mg every four weeks (post-treatment). An extensive miRNAs profiling was performed by qRT-PCR in small, pooled groups of ≤ 8 women each, classified according to migraine frequency (EM and CM) and the degree of response to erenumab. The expression levels of selected miRNAs were also validated using single miRNA assays in each woman with EM and CM. RESULTS: During the study, 36 women with migraine (19 with EM and 17 with CM) out of 40 who were initially screened, performed the assessment of miRNA expression at baseline and post-treatment, Erenumab treatment significantly improved migraine burden in both EM and CM. MiRNA profiling revealed differential expression levels of a wide set of miRNAs (hsa-let-7d-3p, hsa-miR-106b-3p, hsa-miR-122-5p, hsa-miR-143-3p, hsa-miR-144-3p, hsa-miR-16-5p, hsa-miR-181a-5p, hsa-miR-221-3p, hsa-miR-25-3p, hsa-miR-29b-2-5p, hsa-miR-326, miR-363-3p, hsa-miR-424-5p, hsa-miR-485-3p, hsa-miR-532-5p, hsa-miR-543, hsa-miR-629-5p, hsa-miR-660-5p, hsa-miR-92a-3p) depending on treatment response. Among them, single miRNA assays confirmed the progressive decrease of hsa-miR-143-3p expression levels in relation to increasing response to erenumab in women with EM (7 with low, 6 with medium, and 6 with high response; p = 0.02). Additionally, single assays showed higher hsa-miR-34a-5p and hsa-miR-382-5p expression levels at baseline in women with CM compared with those with EM (p = 0.0002 and p = 0.0007, respectively), as well as their expression level decrease in women with CM from baseline to follow-up (p = 0.04 and p = 0.02, respectively). CONCLUSIONS: Our study suggests that targeting the CGRP pathway in migraine changes the expression levels of certain miRNAs. These miRNA levels are linked to the levels of response to CGRP receptor blockage. Future research challenges include assigning specific functions to the modulated miRNAs to unravel pathways modulated by the disease and the treatment. TRIAL REGISTRATION: The study was registered in clinicaltrials.gov with code NCT04659226 and in the Novartis database with code CAMG334AIT05T.

Classification of tumor types using XGBoost machine learning model: a vector space transformation of genomic alterations.

BACKGROUND: Machine learning (ML) represents a powerful tool to capture relationships between molecular alterations and cancer types and to extract biological information. Here, we developed a plain ML model aimed at distinguishing cancer types based on genetic lesions, providing an additional tool to improve cancer diagnosis, particularly for tumors of unknown origin. METHODS: TCGA data from 9,927 samples spanning 32 different cancer types were downloaded from cBioportal. A vector space model type data transformation technique was designed to build consistently homogeneous new datasets containing, as predictive features, calls for somatic point mutations and copy number variations at chromosome arm-level, thus allowing the use of the XGBoost classifier models. Considering the imbalance in the dataset, due to large difference in the number of cases for each tumor, two preprocessing strategies were considered: i) setting a percentage cut-off threshold to remove less represented cancer types, ii) dividing cancer types into different groups based on biological criteria and training a specific XGBoost model for each of them. The performance of all trained models was mainly assessed by the out-of-sample balanced accuracy (BACC) and the AUC scores. RESULTS: The XGBoost classifier achieved the best performance (BACC 77%; AUC 97%) on a dataset containing the 10 most represented tumor types. Moreover, dividing the 18 most represented cancers into three different groups (endocrine-related carcinomas, other carcinomas and other cancers),such analysis models achieved 78%, 71% and 86% BACC, respectively, with AUC scores greater than 96%. In addition, the model capable of linking each group to a specific cancer type reached 81% BACC and 94% AUC. Overall, the diagnostic potential of our model was comparable/higher with respect to others already described in literature and based on similar molecular data and ML approaches. CONCLUSIONS: A boosted ML approach able to accurately discriminate different cancer types was developed. The methodology builds datasets simpler and more interpretable than the original data, while keeping enough information to accurately train standard ML models without resorting to sophisticated Deep Learning architectures. In combination with histopathological examinations, this approach could improve cancer diagnosis by using specific DNA alterations, processed by a replicable and easy-to-use automated technology. The study encourages new investigations which could further increase the classifier's performance, for example by considering more features and dividing tumors into their main molecular subtypes.

MiR-182-5p Is Upregulated in Hepatic Tissues from a Diet-Induced NAFLD/NASH/HCC C57BL/6J Mouse Model and Modulates Cyld and Foxo1 Expression.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

MicroRNAs Expression in Response to rhNGF in Epithelial Corneal Cells: Focus on Neurotrophin Signaling Pathway.

PURPOSE: Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. METHODS: Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. RESULTS: Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. CONCLUSIONS: MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.

Next-generation sequencing: recent applications to the analysis of colorectal cancer.

Since the establishment of the Sanger sequencing method, scientists around the world focused their efforts to progress in the field to produce the utmost technology. The introduction of next-generation sequencing (NGS) represents a revolutionary step and promises to lead to massive improvements in our understanding on the role of nucleic acids functions. Cancer research began to use this innovative and highly performing method, and interesting results started to appear in colorectal cancer (CRC) analysis. Several studies produced high-quality data in terms of mutation discovery, especially about actionable or less frequently mutated genes, epigenetics, transcriptomics. Analysis of results is unveiling relevant perspectives aiding to evaluate the response to therapies. Novel evidences have been presented also in other directions such as gut microbiota or CRC circulating tumor cells. However, despite its unquestioned potential, NGS poses some issues calling for additional studies. This review intends to offer a view of the state of the art of NGS applications to CRC through examination of the most important technologies and discussion of recent published results.

Identification of New Natural CphA Metallo-ß-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy.

Two new natural CphA metallo-ß-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.

Mitotic phosphorylation of Tau/MAPT modulates cell cycle progression in prostate cancer cells.

PURPOSE: Tau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases. METHODS: In our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain. RESULTS: The analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity. CONCLUSIONS: We hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.

Concurrent RAS and RAS/BRAF V600E Variants in Colorectal Cancer: More Frequent Than Expected? A Case Report.

The assessment of RAS and BRAF mutational status is one of the main steps in the diagnostic and therapeutic algorithm of metastatic colorectal cancer (mCRC). Multiple mutations in the BRAF and RAS pathway are described as a rare event, with concurrent variants in KRAS and BRAF genes observed in approximately 0.05% of mCRC cases. Here, we report data from a case series affected by high-risk stage III and stage IV CRC and tested for RAS and BRAF mutation, treated at our Medical Oncology Unit. The analysis of KRAS, NRAS (codons 12, 13, 59, 61, 117, 146), and BRAF (codon 600) hotspot variants was performed in 161 CRC tumors from August 2018 to September 2021 and revealed three (1.8%) patients showing mutations in both KRAS and BRAF (V600E), including two cases with earlier CRC and one with metastatic disease. We also identified one patient (0.6%) with a mutation in both KRAS and NRAS genes and another one (0.6%) with a double KRAS mutation. Notably, the latter was characterized by aggressive behavior and poor clinical outcome. The mutational status, pathological features, and clinical history of these five CRC cases are described. Overall, this study case series adds evidence to the limited available literature concerning both the epidemiological and clinical aspects of CRC cases characterized by the presence of concurrent RAS/BRAF variants. Future multicentric studies will be required to increase the sample size and provide additional value to results observed so far in order to improve clinical management of this subgroup of CRC patients.

Role of exosomal microRNAs in cancer therapy and drug resistance mechanisms: focus on hepatocellular carcinoma.

Extracellular vesicles (EVs), defined as intercellular messengers that carry their cargos between cells, are involved in several physiological and pathological processes. These small membranous vesicles are released by most cells and contain biological molecules, including nucleic acids, proteins and lipids, which can modulate signaling pathways of nearby or distant recipient cells. Exosomes, one the most characterized classes of EVs, include, among others, microRNAs (miRNAs), small non-coding RNAs able to regulate the expression of several genes at post-transcriptional level. In cancer, exosomal miRNAs have been shown to influence tumor behavior and reshape tumor microenvironment. Furthermore, their possible involvement in drug resistance mechanisms has become evident in recent years. Hepatocellular carcinoma (HCC) is the major type of liver cancer, accounting for 75-85% of all liver tumors. Although the improvement in HCC treatment approaches, low therapeutic efficacy in patients with intermediate-advanced HCC is mainly related to the development of tumor metastases, high risk of recurrence and drug resistance. Exosomes have been shown to be involved in pathogenesis and progression of HCC, as well as in drug resistance, by regulating processes such as cell proliferation, epithelial-mesenchymal transition and immune response. Herein, we summarize the current knowledge about the involvement of exosomal miRNAs in HCC therapy, highlighting their role as modulators of therapeutic response, particularly chemotherapy and immunotherapy, as well as possible therapeutic tools.

Anthracycline-Free Neoadjuvant Treatment in Patients with HER2-Positive Breast Cancer: Real-Life Use of Pertuzumab, Trastuzumab and Taxanes Association with an Exploratory Analysis of PIK3CA Mutational Status.

HER2 is considered one of the most traditional prognostic and predictive biomarkers in breast cancer. Literature data confirmed that the addition of pertuzumab to a standard neoadjuvant chemotherapy backbone (either with or without anthracyclines), in patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC), leads to a higher pathological complete response (pCR) rate, which is known to correlate with a better prognosis. In this retrospective analysis, 47 consecutive patients with HER2-positive EBC received sequential anthracyclines and taxanes plus trastuzumab (ATH) or pertuzumab, trastuzumab and docetaxel (THP). Despite the limited sample size, this monocentric experience highlights the efficacy (in terms of pCR) and safety of THP in the neoadjuvant setting of HER2-positive EBC as an anthracycline-free approach. Given the role of PIK3CA as a prognostic and therapeutic target in breast cancer, tumors were also analyzed to assess the PIK3CA mutational status. Thirty-eight out of forty-seven patients were evaluated, and PIK3CA variants were identified in 21% of tumor samples: overall, one mutation was detected in exon 4 (2.6%), two in exon 9 (5.3%) and four in exon 20 (10.5%). Of note, one sample showed concurrent mutations in exons 9 (codon 545) and 20 (codon 1047). Among patients reaching pCR (n = 13), 38.5% were PIK3CA mutants; on the other hand, among those lacking pCR (n = 25), just 12% showed PIK3CA variants. Regarding THP-treated mutant patients (n = 5), 80% reached pCR (three hormone-receptor-negative, one hormone-receptor-positive). Interestingly, the only patient not achieving pCR had a tumor with two co-occurring PIK3CA mutations. In conclusion, this study provides new evidence about the efficacy and good safety profile of THP, compared to the ATH regimen, as an anthracycline-free neoadjuvant treatment of HER2-positive EBC. Further studies on larger/multicentric cohorts are planned for more in-depth analysis to confirm our molecular and clinical results.

Investigational neuroprotective compounds in clinical trials for retinal disease.

INTRODUCTION: Retinal neurodegeneration causes irreversible vision loss, impairing quality of life. By targeting neurotoxic conditions, such as oxidative stress and ischemia, neuroprotectants can slow or stop sight loss resulting from eye disease. Despite limimted clinical use of neuroprotectants, there are several promising compounds in early clinical trials (pre-phase III) which may fulfil new therapeutic roles. Search terms relating to neuroprotection and eye disease were used on ClinicalTrials.gov to identify neuroprotective candidates. AREAS COVERED: Research supporting neuroprotection in eye diseases is focused on, ranging from preclinical to phase II, according to the ClinicalTrials.gov database. The compounds discussed are explored in terms of future clinical applications. EXPERT OPINION: The major challenge in neuroprotection research is translation from basic research to the clinic. A number of potential neuroprotectants have progressed to ophthalmology clinical trials in recent years, with defined mechanisms of action - saffron and CoQ10 - targeting mitochondria, and both CNTF and NGF showing anti-apoptotic effects. Enhancements in trial design and patient cohorts in proof-of-concept trials with enriched patient populations and surrogate endpoints should accelerate drug development. A further important consideration is optimising drug delivery to improve individualised management and patient compliance. Progress in these areas means that neuroprotective strategies have a much improved chance of translational success.

Emerging Role of isomiRs in Cancer: State of the Art and Recent Advances.

The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.

Ultrasound-Based Method for the Identification of Novel MicroRNA Biomarkers in Prostate Cancer.

The detection of circulating microRNA (miRNA)-based biomarkers represents an innovative, non-invasive method for the early detection of cancer. However, the low concentration of miRNAs released in body fluids and the difficult identification of the tumor site have limited their clinical use as effective cancer biomarkers. To evaluate if ultrasound treatment could amplify the release of extracellular cancer biomarkers, we treated a panel of prostate cancer (PCa) cell lines with an ultrasound-based prototype and profiled the release of miRNAs in the extracellular space, with the aim of identifying novel miRNA-based biomarkers that could be used for PCa diagnosis and the monitoring of tumor evolution. We provide evidence that US-mediated sonoporation amplifies the release of miRNAs from both androgen-dependent (AD) and -independent (AI) PCa cells. We identified four PCa-related miRNAs, whose levels in LNCaP and DU145 supernatants were significantly increased following ultrasound treatment: mir-629-5p, mir-374-5p, mir-194-5p, and let-7d-5p. We further analyzed a publicly available dataset of PCa, showing that the serum expression of these novel miRNAs was upregulated in PCa patients compared to controls, thus confirming their clinical relevance. Our findings highlight the potential of using ultrasound to identify novel cell-free miRNAs released from cancer cells, with the aim of developing new biomarkers with diagnostic and predictive value.

Circulating MicroRNAs as Prognostic and Therapeutic Biomarkers in Breast Cancer Molecular Subtypes.

Breast cancer (BC) is a common and heterogeneous disease, of which six molecular subtypes, characterized by different biological features and clinical outcomes, were described. The identification of additional biomarkers able to further connote and distinguish the different BC subtypes is essential to improve the diagnostic, prognostic and therapeutic strategies in BC patients. MicroRNAs (miRNAs) are short non-coding RNA involved in several physiological and pathological processes, including cancer development and progression. In particular, circulating miRNAs, which can be found in an adequately stable structure in serum/plasma of cancer patients, are emerging as very promising non-invasive biomarkers. Several studies have analyzed the potential role of circulating miRNAs as prognostic and therapeutic markers in BC. In the present review we describe circulating miRNAs, identified as putative biomarker in BC, with special reference to different BC molecular subtypes.

Applications of Next Generation Sequencing to the Analysis of Familial Breast/Ovarian Cancer.

Next generation sequencing (NGS) provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). The generated high-throughput data are particularly suitable for enhancing the understanding of the genetic bases of complex, multi-gene diseases, such as cancer. Among the various types of tumors, those with a familial predisposition are of great interest for the isolation of novel genes or gene variants, detectable at the germline level and involved in cancer pathogenesis. The identification of novel genetic factors would have great translational value, helping clinicians in defining risk and prevention strategies. In this regard, it is known that the majority of breast/ovarian cases with familial predisposition, lacking variants in the highly penetrant BRCA1 and BRCA2 genes (non-BRCA), remains unexplained, although several less penetrant genes (e.g., ATM, PALB2) have been identified. In this scenario, NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. In this review, we summarize and discuss the state of the art applications of NGS gene panels, WES and WGS in the context of familial breast/ovarian cancer.