Plasma miRNA-based signatures in CRC screening programs.

Colorectal cancer (CRC) screening programs help diagnose cancer precursors and early cancers and help reduce CRC mortality. However, currently recommended tests, the fecal immunochemical test (FIT) and colonoscopy, have low uptake. There is therefore a pressing need for screening strategies that are minimally invasive and consequently more acceptable to patients, most likely blood based, to increase early CRC identification. MicroRNAs (miRNAs) released from cancer cells are detectable in plasma in a remarkably stable form, making them ideal cancer biomarkers. Using plasma samples from FIT-positive (FIT+) subjects in an Italian CRC screening program, we aimed to identify plasma circulating miRNAs that detect early CRC. miRNAs were initially investigated by quantitative real-time PCR in plasma from 60 FIT+ subjects undergoing colonoscopy at Fondazione IRCCS Istituto Nazionale dei Tumori, then tested on an internal validation cohort (IVC, 201 cases) and finally in a large multicenter prospective series (external validation cohort [EVC], 1121 cases). For each endoscopic lesion (low-grade adenoma [LgA], high-grade adenoma [HgA], cancer lesion [CL]), specific signatures were identified in the IVC and confirmed on the EVC. A two-miRNA-based signature for CL and six-miRNA signatures for LgA and HgA were selected. In a multivariate analysis including sex and age at blood collection, the areas under the receiver operating characteristic curve (95% confidence interval) of the signatures were 0.644 (0.607-0.682), 0.670 (0.626-0.714) and 0.682 (0.580-0.785) for LgA, HgA and CL, respectively. A miRNA-based test could be introduced into the FIT+ workflow of CRC screening programs so as to schedule colonoscopies only for subjects likely to benefit most.

Detection of (pre)cancerous colorectal lesions in Lynch syndrome patients by microsatellite instability liquid biopsy.

Lynch syndrome (LS) is an inherited condition characterized by an increased risk of developing cancer, in particular colorectal cancer (CRC). Microsatellite instability (MSI) is the main feature of (pre)cancerous lesions occurring in LS patients. Close endoscopic surveillance is the only option available to reduce CRC morbidity and mortality. However, it may fail to intercept interval cancers and patients' compliance to such an invasive procedure may decrease over the years. The development of a minimally invasive test able to detect (pre)cancerous colorectal lesions, could thus help tailor surveillance programs in LS patients. Taking advantage of an endoscopic surveillance program, we retrospectively assessed the instability of five microsatellites (BAT26, BAT25, NR24, NR21, and Mono27) in liquid biopsies collected at baseline and possibly at two further endoscopic rounds. For this purpose, we tested a new multiplex drop-off digital polymerase chain reaction (dPCR) assay, reaching mutant allele frequencies (MAFs) as low as 0.01%. Overall, 78 plasma samples at the three time-points from 18 patients with baseline (pre)cancerous lesions and 18 controls were available for molecular analysis. At baseline, the MAFs of BAT26, BAT25 and NR24 were significantly higher in samples of patients with lesions but did not differ with respect to the grade of dysplasia or any other clinico-pathological characteristics. When all markers were combined to determine MSI in blood, this test was able to discriminate lesion-bearing patients with an AUC of 0.80 (95%CI: 0.66; 0.94).

Colorectal carcinoma peritoneal metastases-derived organoids: results and perspective of a model for tailoring hyperthermic intraperitoneal chemotherapy from bench-to-bedside.

BACKGROUND: Peritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been reported to improve survival, but peritoneal recurrence rates are still high and there is no consensus on the drug of choice for HIPEC. The aim of this study was to use patient derived organoids (PDO) to build a relevant CRCPM model to improve HIPEC efficacy in a comprehensive bench-to-bedside strategy. METHODS: Oxaliplatin (L-OHP), cisplatin (CDDP), mitomycin-c (MMC) and doxorubicin (DOX) were used to mimic HIPEC on twelve PDO lines derived from twelve CRCPM patients, using clinically relevant concentrations. After chemotherapeutic interventions, cell viability was assessed with a luminescent assay, and the obtained dose-response curves were used to determine the half-maximal inhibitory concentrations. Also, induction of apoptosis by different HIPEC interventions on PDOs was studied by evaluating CASPASE3 cleavage. RESULTS: Response to drug treatments varied considerably among PDOs. The two schemes with better response at clinically relevant concentrations included MMC alone or combined with CDDP. L-OHP showed relative efficacy only when administered at low concentrations over a long perfusion period. PDOs showed that the short course/high dose L-OHP scheme did not appear to be an effective choice for HIPEC in CRCPM. HIPEC administered under hyperthermia conditions enhanced the effect of chemotherapy drugs against cancer cells, affecting PDO viability and apoptosis. Finally, PDO co-cultured with cancer-associated fibroblast impacted HIPEC treatments by increasing PDO viability and reducing CASPASES activity. CONCLUSIONS: Our study suggests that PDOs could be a reliable in vitro model to evaluate HIPEC schemes at individual-patient level and to develop more effective treatment strategies for CRCPM.

Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

Exploring the association with disease recurrence of miRNAs predictive of colorectal cancer.

INTRODUCTION: Disease recurrence after surgery is a crucial predictor of poor prognosis in colorectal cancer, where disseminated disease at the time of intervention can also be observed in localized early-stage cases. We evaluated the ability to predict disease recurrence of miRNAs from two signatures that we have found linked to the presence of colorectal cancer (CL signature) or adenoma (HgA signature) in higher-risk subjects. METHODS: miRNAs from the signatures were studied longitudinally by quantitative real-time polymerase chain reaction in plasma from 24 patients with resectable colorectal cancer collected at the time of surgery and during scheduled follow-up across 36 months. Patients either showed relapse within 36 months (alive with disease (AWD)), or remained disease-free (no evidence of disease (NED)) for the same period. RESULTS: Although the signatures did not predict recurrence, expression of the miRNAs from the CL signature decreased 1 year after surgery, and one miRNA of the signature, miR-378a-3p, almost reached significance in the NED subgroup (Wilcoxon signed-rank test: p-value = 0.078). Also, miR-335-5p from the HgA signature was higher in AWD patients before surgery (Kruskal-Wallis test: p-value = 0.019). CONCLUSIONS: These data, although from a small cohort of patients, support the possible use of miRNAs as non-invasive biomarkers in liquid biopsy-based tests to identify patients at risk of relapse and to monitor them during follow-up.

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.

Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production.

Metformin is a widely used and well-tolerated anti-diabetic drug that can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear. We studied the anti-cancer activity of metformin on colorectal cancer (CRC) by using the drug to treat HT29, HCT116 and HCT116 p53-/- CRC cells. Metformin reduced cell proliferation and migration by inducing cell cycle arrest in the G0/G1 phase. This was accompanied by a sharp decrease in the expression of c-Myc and down-regulation of IGF1R. The anti-proliferative action of metformin was mediated by two different mechanisms: AMPK activation and increase in the production of reactive oxygen species, which suppressed the mTOR pathway and its downstream targets S6 and 4EBP1. A reduction in CD44 and LGR5 expression suggested that the drug had an effect on tumour cells with stem characteristics. However, a colony formation assay showed that metformin slowed the cells' ability to form colonies without arresting cell growth, as confirmed by absence of apoptosis, autophagy or senescence. Our finding that metformin only transiently arrests CRC cell growth suggests that efforts should be made to identify compounds that combined with the biguanide can act synergistically to induce cell death.

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.

miRNA profiling in colorectal cancer highlights miR-1 involvement in MET-dependent proliferation.

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression.

Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction.

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2 and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19 promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19 during human development in 60 normal and 66 idiopathic IUGR (Intrauterine Growth Restriction) pregnancies, studying embryonic (cord blood) and extraembryonic (placenta and umbilical cord) tissues. We found ICR1 normal methylation levels ( approximately 50%) and H19 promoter/DMR2 hypomethylation in extra-embryonic tissues. In contrast, in embryonic samples the three loci displayed normal methylation values comparable to those in postnatal blood. This feature is stably maintained throughout gestation and does not vary in IUGR cases. We reported asymmetric allelic expression of H19 and IGF2 as a common feature in pre- and post-natal tissues, independent of H19 promoter and DMR2 methylation levels. In addition, we excluded in IUGR post-transcriptional IGF2 interference possibly related to miRNA 483-3p (IGF2, intron 2) expression defects. Through LINE1 methylation analysis, we observed a methylation gradient with increasing methylation from pre- to post-natal life. The involvement of UPD (Uniparental Disomy) in IUGR aetiology was excluded. Our data indicate that: (1) ICR1 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2 and H19 present in embryonic as well as in extra-embryonic tissues; (2) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2 and H19; (3) there is a gradient of global methylation, increasing from extra-embryonic to embryonic and adult tissues. Finally, because of placental hypomethylation, cautions should be exercised in diagnosis of imprinting diseases using chorionic villi.

Angiotensin-converting enzyme and adducin-1 polymorphisms in women with preeclampsia and gestational hypertension.

The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism and the Adducin-1 (ADD1) G460W nonsense single nucleotide polymorphism (SNP) have previously been associated to hypertension, whereas their association with preeclampsia (PE) and gestational hypertension (GH) is still controversial. We genotyped ACE I/D, ADD1 G460W, and ADD1 S586C polymorphisms in 672 unrelated pregnant women: 204 PE (81/204 mild PE), 56 GH, and 412 controls, evaluating both their single and combined effects on these pathologies. The genotype combination of the 3 polymorphisms was not statistically different in cases versus controls, nor were ACE and ADD1 polymorphisms in GH. Nevertheless, the distribution of ACE genotypes was different in PE. This was confirmed in mild PE, whereas no significance was found in severe PE. This could suggest that different factors may lead to mild and severe PE, with ACE polymorphism playing a more important role in the mild form.