Modulation of faecal miRNAs highlights the preventive effects of a Mediterranean low-inflammatory dietary intervention.

BACKGROUND: Dietary interventions have been proposed as therapeutic approaches for several diseases, including cancer. A low-inflammatory Mediterranean dietary intervention, conducted as a pilot study in subjects with Familial Adenomatous Polyposis (FAP), reduced markers of local and systemic inflammation. We aim to determine whether this diet may modulate faecal microRNA (miRNA) and gene expression in the gut. METHODS: Changes in the faecal miRNome were evaluated by small RNA sequencing at baseline (T0), after the three-month intervention (T1), and after an additional three months (T2). Changes in the transcriptome of healthy rectal mucosa and adenomas were evaluated by RNA sequencing at T0 and T2. The identification of validated miRNA-gene interactions and functional analysis of miRNA targets were performed using in silico approaches. RESULTS: Twenty-seven subjects were included in this study. It was observed that the diet modulated 29 faecal miRNAs (p < 0.01; |log2 Fold Change|>1), and this modulation persisted for three months after the intervention. Levels of miR-3612-3p and miR-941 correlated with the adherence to the diet, miR-3670 and miR-4252-5p with faecal calprotectin, and miR-3670 and miR-6867 with serum calprotectin. Seventy genes were differentially expressed between adenoma and normal tissue, and most were different before the dietary intervention but reached similar levels after the diet. Functional enrichment analysis identified the proinflammatory ERK1/2, cell cycle regulation, and nutrient response pathways as commonly regulated by the modulated miRNAs and genes. CONCLUSIONS: Faecal miRNAs modulated by the dietary intervention target genes that participate in inflammation. Changes in levels of miRNAs and genes with oncogenic and tumour suppressor functions further support the potential cancer-preventive effect of the low-inflammatory Mediterranean diet. CLINICAL TRIAL NUMBER REGISTRATION: NCT04552405, Registered in ClinicalTrials.gov.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance.

Background: The secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation. Methods: KiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays. Results: We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium. Conclusions: Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

End-of-neoadjuvant treatment circulating microRNAs and HER2-positive breast cancer patient prognosis: An exploratory analysis from NeoALTTO.

Background: The absence of breast cancer cells in surgical specimens, i.e., pathological complete response (pCR), is widely recognized as a favorable prognostic factor after neoadjuvant therapy. In contrast, the presence of disease at surgery characterizes a prognostically heterogeneous group of patients. Here, we challenged circulating microRNAs (miRNAs) at the end of neoadjuvant therapy as potential prognostic biomarkers in the NeoALTTO study. Methods: Patients treated within the trastuzumab arm (i.e., pre-operative weekly trastuzumab for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks; post-operative FEC for 3 cycles followed by trastuzumab up to complete 1 year of treatment) were randomized into a training (n= 54) and testing (n= 72) set. RT-PCR-based high-throughput miRNA profile was performed on plasma samples collected at the end of neoadjuvant treatment of both sets. After normalization, circulating miRNAs associated with event free survival (EFS) were identified by univariate and multivariate Cox regression model. Results: Starting from 23 circulating miRNAs associated with EFS in the training set, we generated a 3-circulating miRNA prognostic signature consisting of miR-185-5p, miR-146a-5p, miR-22-3p, which was confirmed in the testing set. The 3-circulating miRNA signature showed a C-statistic of 0.62 (95% confidence interval [95%CI] 0.53-0.71) in the entire study cohort. By resorting to a multivariate Cox regression model we found a statistical significant interaction between the expression values of miR-194-5p and pCR status (p.interaction =0.005) with an estimate Hazard Ratio (HR) of 1.83 (95%CI 1.14- 2.95) in patients with pCR, and 0.87 (95%CI 0.69-1.10) in those without pCR. Notably, the model including this interaction along with the abovementioned 3-circulating miRNA signature provided the highest discriminatory capability with a C-statistic of 0.67 (95%CI 0.58-0.76). Conclusions: Circulating miRNAs are informative to identify patients with different prognosis among those with heterogeneous response after trastuzumab-based neoadjuvant treatment, and may be an exploitable tool to select candidates for salvage adjuvant therapy.

What if the future of HER2-positive breast cancer patients was written in miRNAs? An exploratory analysis from NeoALTTO study.

BACKGROUND: Neoadjuvant therapy with dual HER2 blockade improved pathological complete response (pCR) rate in HER2-positive breast cancer patients. Nevertheless, it would be desirable to identify patients exquisitely responsive to single agent trastuzumab to minimize or avoid overtreatment. Herein, we evaluated the predictive and prognostic value of basal primary tumor miRNA expression profile within the trastuzumab arm of NeoALTTO study (ClinicalTrials.gov Identifier: NCT00553358). METHODS: RNA samples from baseline biopsies were randomized into training (n = 45) and testing (n = 47) sets. After normalization, miRNAs associated with Event-free survival (EFS) and pCR were identified by univariate analysis. Multivariate models were implemented to generate specific signatures which were first confirmed, and then analyzed together with other clinical and pathological variables. RESULTS: We identified a prognostic signature including hsa-miR-153-3p (HR 1.831, 95% CI: 1.34-2.50) and hsa-miR-219a-5p (HR 0.629, 95% CI: 0.50-0.78). For two additional miRNAs (miR-215-5p and miR-30c-2-3p), we found a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Besides, a two-miRNA signature was predictive of pCR (hsa-miR-31-3p, OR 0.70, 95% CI: 0.53-0.92, and hsa-miR-382-3p, OR: 1.39, 95% CI: 1.01-1.91). Notably, the performance of this predictive miRNA signature resembled that of the genomic classifiers PAM50 and TRAR, and did not improve when the extended models were fitted. CONCLUSION: Analyses of primary tumor tissue miRNAs hold the potential of a parsimonious tool to identify patients with differential clinical outcomes after trastuzumab based neoadjuvant therapy.

Different pixel pitch and maximum luminance of medical grade displays may result in different evaluations of digital radiography images.

AIM: To evaluate the effects of display pixel pitch and maximum luminance on intra- and inter-observer reproducibility and observer performance when evaluating chest lesions and bone fractures. MATERIALS AND METHODS: This was a multi-institutional study for a retrospective interpretation of selected digital radiography images. Overall, 82 images were selected by senior radiologists, including 50 cases of chest lesions and 32 cases of bone fractures. These images were displayed at two pixel pitches (0.212 and 0.165 mm size pixels) and two maximum luminance values (250 and 500 cd/m2) and reviewed twice by senior and junior radiologists. All the observers had to indicate the likelihood of the presence of the lesions and to rate the relative confidence of their assessment. Cohen Kappa statistic was computed to estimate the reproducibility in correctly identifying lesions; for multi-reader-multi-case (MRMC) analysis, weighted Jackknife Alternative Free-response Receiver Operating Characteristic (wJAFROC) statistical tools was applied. RESULTS: The intra-radiologist and inter-observer reproducibility values were the highest for the 0.165 mm size pixel at 500 cd/m2 display, for both chest lesions and bone fractures evaluations. As regards chest lesions, observer performances were significantly greater with 0.165 mm size pixel display at 500 cd/m2 than with lower maximum luminance and/or larger pixel pitch displays. Concerning bone fractures, the performance obtained with 0.212 mm size pixel display at 250 cd/m2 was statistically lower than that obtained with 0.165 mm sixe pixel display at 500 cd/m2. CONCLUSION: Our results indicate that an increased maximum luminance level and a decreased pixel pitch of medical-grade display improve the accuracy of detecting both chest lesions and bone fractures.

In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating.

The aim of this study is to compare 2 in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, that is, the RNAscope 2.0 High Definition (HD) and the upgraded RNAscope 2.5 HD version. The RNAscope 2.5 HD has recently replaced the RNAscope 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma with the final goal of applying it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify oropharyngeal squamous cell carcinoma that are truly driven by high-risk HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real-time reverse-transcription polymerase chain reaction in a fraction of cases where material for HPV E6/E7 mRNA real-time reverse-transcription polymerase chain reaction was available. Furthermore, the algorithm that associates p16 immunohistochemistry with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 immunohistochemistry with the identification of HPV DNA by ISH.

Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

A methodological procedure for evaluating the impact of hemolysis on circulating microRNAs.

Circulating microRNAs (miRNAs) are promising non-invasive biomarkers whose expression may be affected by confounding factors, including hemolysis, that should be considered in studies of miRNA discovery. The present study proposes a methodology for evaluating the impact of hemolysis on the expression of miRNAs. An experiment of in vitro controlled hemolysis was designed for assessing if changes in the expression of eight miRNAs observed to be circulating in plasma may be associated with hemolysis, and also to estimate the level of red blood cell (RBC) contamination in plasma samples where the expression of these miRNAs will be measured. It was confirmed that four miRNAs, miR-16, miR-92a, miR-451 and miR-486, known to be present in blood cells, were influenced by contamination of RBCs. Furthermore, it was demonstrated that miR-378 and miR-30c are hemolysis-independent and that the expression of miR-320 and miR-324-3p was associated with the level of RBC contamination. This procedure is proposed as a tool for the evaluation of the influence of hemolysis on candidate circulating miRNA biomarkers prior to their analysis in plasma samples.

Moving from discovery to validation in circulating microRNA research.

BACKGROUND: MicroRNAs (miRNAs), small noncoding RNAs, are involved in tumorigenesis and in the development of various cancers. Quantitative real-time polymerase chain reaction (qPCR) is the most commonly used tool to investigate miRNA expression, and qPCR low-density arrays are increasingly being used as an experimental technique for both the identification of potentially relevant miRNAs and their subsequent validation. Due to the reduced number of microRNAs to be validated, this phase is generally performed on ad hoc customized cards for which a technical robustness is assumed similar to that of the high-throughput cards used during the identification phase. METHODS: With the aim of investigating the degree of reproducibility between the 2 types of cards, we analyzed plasma-circulating miRNAs evaluated in 60 subjects enrolled in a colorectal cancer screening program. RESULTS: Our results showed a reproducibility between the 2 methods that was not fully satisfactory, with a concordance correlation coefficient equal to 0.69 (95% confidence interval, 0.12-0.92). CONCLUSIONS: This report highlights the need to add a technical validation step to the high-throughput-based miRNA identification workflow, after their discovery and before the validation step in an independent series.

Histone deacetylase inhibitor-temozolomide co-treatment inhibits melanoma growth through suppression of Chemokine (C-C motif) ligand 2-driven signals.

Target-specific agents used in melanoma are not curative, and chemokines are being implicated in drug-resistance to target-specific agents. Thus, the use of conventional agents in rationale combinations may result in optimization of therapy. Because histone deacetylases participate in tumor development and progression, the combination of the pan-inhibitor SAHA and temozolomide might provide a therapeutic advantage. Here, we show synergism between the two drugs in mutant BRAF cell lines, in association with decreased phosphorylation of cell survival proteins (e.g., C-Jun-N-terminal-kinase, JNK). In the spontaneous ret transgenic mouse melanoma model, combination therapy produced a significant disease onset delay and down-regulation of Chemokine (C-C motif) ligand 2 (CCL2), JNK, and of Myeloid-derived suppressor cell recruitment. Co-incubation with a CCL2-blocking-antibody enhanced in vitro cell sensitivity to temozolomide. Conversely, recombinant CCL2 activated JNK in human tumor melanoma cells. In keeping with these results, the combination of a JNK-inhibitor with temozolomide was synergistic. By showing that down-regulation of CCL2-driven signals by SAHA and temozolomide via JNK contributes to reduce melanoma growth, we provide a rationale for the therapeutic advantage of the drug combination. This combination strategy may be effective because of interference both with tumor cell and tumor microenvironment.

Circulating free DNA in a screening program for early colorectal cancer detection.

AIMS AND BACKGROUND: The quantification and molecular characterization of circulating free DNA (cfDNA) have attracted much interest as new and promising, noninvasive means of detecting and monitoring the presence of surgical resectable colorectal cancer (CRC). Instead, the role of cfDNA in the early detection of malignant and premalignant colorectal lesions is still unclear. The aim of this study was to evaluate the predictive power of the quantification and KRAS status of cfDNA in detecting early colorectal lesions in plasma from healthy high-risk subjects. METHODS: The study population consisted of 170 consecutive healthy high-risk subjects aged >50 years who participated in the screening program promoted by the Local Health Service (ASL-Milano) for early CRC detection and who underwent endoscopic examination after being found positive at fecal occult blood test (FOBT). Thirty-four participants had malignant lesions consisting of 12 adenocarcinomas (at an early stage in half of the cases) and 22 instances of high-grade intraepithelial neoplasia (HGIN) in adenomas; 73 participants had premalignant lesions (adenomas and hyperplasia), and 63 participants had no lesions. Plasma cfDNA was quantified by quantitative real-time PCR and analyzed for KRAS mutations by a mutant-enriched PCR. KRAS status was assessed also in matched adenocarcinoma and HGIN tissues. The distribution of cfDNA concentrations among FOBT-positive subjects with diagnosed lesion (cases) was compared with that of FOBT-positive subjects without lesions (controls) and its predictive capability (AUC) was assessed. RESULTS: The predictive capability of cfDNA levels was satisfactory in predicting adenocarcinomas (AUC 0.709; 95% CI, 0.508-0.909) but not HGIN and premalignant lesions. The rate of KRAS mutations in plasma was low (5/170 = 3%) compared with the rate observed in the matched adenocarcinoma and HGIN tissues (45%). CONCLUSIONS: The use of cfDNA quantification to predict adenocarcinoma at an early stage in high-risk (aged >50 years and FOBT positive) subjects seems to be promising but needs more sensitive methods to improve cfDNA detection.

Biomarkers for Early Cancer Detection - Methodological Aspects.

The development of a new breast cancer biomarker for early detection is a process that begins with biomarker discovery, followed by a rigorous definition and evaluation of the whole process of biomarker determination (analytical validation). It terminates with the assessment of the impact of the biomarker on clinical practice (clinical validation). A 4-phase scheme for the analytical validation process of the biomarkers for early diagnosis has recently been proposed with the aim of covering the need for standardized operating procedures as well as the need for monitoring and maintaining their quality. As far as clinical validation of biomarkers for early diagnosis is concerned, however, a well established phased approach exists, and guidelines are available for both planning studies and reporting results. Although analytical and clinical validation should be logically linked, often this is not the case in real-word practice, especially in the early phases of biomarker development. This is also the case with breast cancer biomarkers for early detection.