Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2

Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2

The clinical significance of MiR-148a as a predictive biomarker in patients with advanced colorectal cancer

Aim: Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies. Methods: We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival. Results: Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93). Discussion: MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy

An evaluation of the SENTiFIT 270 analyser for quantitation of faecal haemoglobin in the investigation of patients with suspected colorectal cancer.

BACKGROUND: An evaluation of SENTiFIT® 270 (Sentinel Diagnostics, Italy; Sysmex, Spain) analyser for the quantitation of faecal haemoglobin (f-Hb) was performed. METHODS: The analytical imprecision, linearity, carry over and f-Hb stability were determined. Evaluation of the diagnostic accuracy was performed on 487 patients. RESULTS: Within-run and between-run imprecision ranged 1.7%-5.1% and 3.8%-6.2%, respectively. Linearity studies revealed a mean recovery of 101.1% (standard deviation, 6.7%) for all dilutions. No carry over was detected below 7650 μg Hb/g faeces. Decay of f-Hb in refrigerated samples ranged 0.2%-0.5% per day. f-Hb in patients with advanced colorectal neoplasia (ACRN) (colorectal cancer [CRC] plus advanced adenoma [AA]) were significantly higher than from those with a normal colonoscopy. Sensitivity for ACRN at f-Hb cutoffs from 10 to 60 μg Hb/g faeces ranged from 28.9% (95% confidence interval [CI], 21.7%-37.2%) to 46.5% (95% CI, 38.1%-55%), the specificity ranged from 85% (95% CI, 82.3%-87.3%) to 93.2% (95% CI, 91.2%-94.8%), positive predictive values for detecting CRC and AA ranged from 11.6% (95% CI, 7.6%-17.2%) to 20.6% (95% CI, 13.3%-30.3%) and from 34.7% (95% CI, 28.1%-42%) to 42.3% (95% CI, 32.4%-52.7%), respectively, and the negative predictive value for ACRN ranged from 90.2% (95% CI, 87.9%-92.2%) to 88.4% (95% CI, 86%-90.4%). Using two samples per patient sensitivity increased with a slight decrease in specificity. CONCLUSIONS: The analytical and clinical performances of SENTiFIT assay demonstrate a specific and accurate test for detecting ACRN in symptomatic patients and those undergoing surveillance. KEYWORDS: adenoma; analyser evaluation; colorectal cancer; faecal haemoglobin; faecal immunochemical tes

Glyceraldehyde-3-phosphate dehydrogenase is overexpressed in colorectal cancer onset

Background Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential regulator of glycolysis used as a housekeeping marker for gene/protein normalisation. Given the pivotal role of GAPDH in tumour metabolism, our aim was to correlate its protein expression with tumour staging and prognosis of colorectal cancer. Methods GAPDH expression was immunohistochemically analysed in tumour tissues from 62 colorectal cancer (CRC) patients, and validated at mRNA level in an independent dataset comprising 98 paired stage II CRC and normal samples. Staining quantification was performed by computational image analysis, and correlations between GAPDH expression and tumour progression stage were assessed. Gene expression profiling was performed using Affymetrix microarrays. Probability of patient survival and disease-free survival were analysed by the univariate product-limit method of Kaplan-Meier. Groups were compared using Kruskal-Wallis tests. Results Overexpression of GAPDH is positively associated with early stage tumours without regional lymph node and distant metastases involved. These results were reinforced by those obtained at mRNA level. Conclusion Studying the role of GAPDH in malignant transformation can shed new light on the understanding of tumour onset and lead to the design of more efficient personalised therapies

Novel Circulating miRNA Signatures for Early Detection of Pancreatic Neoplasia

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) presents the lowest survival rate of all cancers because only 6% of patients reach five-year survival. Alterations in the expression of several microRNAs (miRNAs) occur in the tumor of PDAC and in preneoplastic lesions as the called intraductal papillary mucinous neoplasm (IPMN). Here, we aimed at identifying which miRNAs are significantly altered in liquid biopsies from patients with PDAC and IPMN to find new noninvasive biomarkers for early detection of PDAC. METHODS: We analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) the expression of 17 circulating miRNAs, previously found to be significantly overexpressed in tissue pancreatic neoplasms, in a set of 182 plasma samples (94 PDAC, 19 IPMN, 18 chronic pancreatitis, and 51 disease-free controls). Then, we analyzed CA19.9 levels in the same plasma set, and we assessed the diagnostic values of differentially expressed miRNAs, CA19.9, and all possible combinations. RESULTS: Of note, 16, 14, and 9 miRNAs were significantly increased in PDAC, IPMN, and chronic pancreatitis, respectively, compared with control plasmas. miR-21-5p, miR-33a-3p, miR-320a, and miR-93-5p showed the highest discriminating capacity for pancreatic neoplasia (PDAC or IPMN) with an area under the receiver operating characteristic curve (AUC) of 0.86, 0.85, 0.85, and 0.80, respectively. 2-miRNA combinations improved these performances reaching AUC = 0.90 for "miR-33a-3p+miR-320a." Addition of CA19.9 increased the diagnostic potential of miRNA signatures even further achieving an AUC of 0.95 (93% sensitivity and 85% specificity) for the combination of "miR-33a-3p+miR-320a+CA19.9." CONCLUSIONS: Novel signatures combining miRNAs and CA19.9 could be used as noninvasive biomarkers for early detection of PDAC

Evaluation of alpha 1-antitrypsin and the levels of mRNA expression of matrix metalloproteinase 7, urokinase type plasminogen activator receptor and COX-2 for the diagnosis of colorectal cancer

Background Colorectal cancer (CRC) is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. Aim Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. Methods In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7), urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF), cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2), and CD44) and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA) and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. Results Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79±0.25 in the CRC group vs 1.27±0.25 in the control group, P<0.0005). The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96). The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81) and tetranectin (0.80), COX-2 (0.78), uPAR (0.78) and carbonic anhydrase (0.77). The markers which identified early stage CRC (Stages I and II) were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. Conclusions Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages

Serum methylation of GALNT9, UPF3A, WARS, and LDB2 as noninvasive biomarkers for the early detection of colorectal cancer and advanced adenomas

Background Early detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicenter cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC.Results First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9, UPF3A, WARS, and LDB2 as new noninvasive biomarkers for the early detection of AN. The combination of GALNT9/UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test.Conclusions Overall, this study highlights the utility of cfDNA methylation for CRC screening. Our results suggest that the combination methylated GALNT9/UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC

Budget Impact Analysis of Molecular Lymph Node Staging Versus Conventional Histopathology Staging in Colorectal Carcinoma

Background: The presence of lymph node (LN) metastasis is a critical prognostic factor in colorectal cancer (CRC) patients and is also an indicator for adjuvant chemotherapy. The gold standard (GS) technique for LN diagnosis and staging is based on the analysis of haematoxylin and eosin (H&E)-stained slides, but its sensitivity is low. As a result, patients may not be properly diagnosed and some may have local recurrence or distant metastases after curative-intent surgery. Many of these diagnostic and treatment problems could be avoided if the one-step nucleic acid amplification assay (OSNA) was used rather than the GS technique. OSNA is a fast, automated, standardised, highly sensitive, quantitative technique for detecting LN metastases. Objectives: The aim of this study was to assess the budget impact of introducing OSNA LN analysis in early-stage CRC patients in the Spanish National Health System (NHS). Methods: A budget impact analysis comparing two scenarios (GS vs. OSNA) was developed within the Spanish NHS framework over a 3-year time frame (2017-2019). The patient population consisted of newly diagnosed CRC patients undergoing surgical treatment, and the following costs were included: initial surgery, pathological diagnosis, staging, follow-up expenses, systemic treatment and surgery after recurrence. One- and two-way sensitivity analyses were performed. Results: Using OSNA instead of the GS would have saved 1,509,182, 6,854,501 and 10,814,082 during the first, second and third years of the analysis, respectively, because patients incur additional costs in later years, leading to savings of more than 19 million for the NHS over the 3-year time horizon. Conclusions: Introducing OSNA in CRC LN analysis may represent not only an economic benefit for the NHS but also a clinical benefit for CRC patients since a more accurate staging could be performed, thus avoiding unnecessary treatments

CNApp, a tool for the quantification of copy number alterations and integrative analysis revealing clinical implications.

Somatic copy number alterations (CNAs) are a hallmark of cancer, but their role in tumorigenesis and clinical relevance remain largely unclear. Here, we developed CNApp, a web-based tool that allows a comprehensive exploration of CNAs by using purity-corrected segmented data from multiple genomic platforms. CNApp generates genome-wide profiles, computes CNA scores for broad, focal and global CNA burdens, and uses machine learning-based predictions to classify samples. We applied CNApp to the TCGA pan-cancer dataset of 10,635 genomes showing that CNAs classify cancer types according to their tissue-of-origin, and that each cancer type shows specific ranges of broad and focal CNA scores. Moreover, CNApp reproduces recurrent CNAs in hepatocellular carcinoma and predicts colon cancer molecular subtypes and microsatellite instability based on broad CNA scores and discrete genomic imbalances. In summary, CNApp facilitates CNA-driven research by providing a unique framework to identify relevant clinical implications. CNApp is hosted at https://tools.idibaps.org/CNApp/

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS-and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT