Jane V. Carter

Blood-based microRNAs as biomarkers for the diagnosis of colorectal cancer: a systematic review and meta-analysis.

Background:Colorectal cancer (CRC) is common and associated with significant mortality. Current screening methods for CRC lack patient compliance. microRNAs (miRNAs), identified in body fluids, are negative regulators of gene expression and are dysregulated in many cancers, including CRC. This paper summarises studies identifying blood-based miRNAs dysregulated in CRC compared with healthy controls in an attempt to evaluate their use as a screening tool for the diagnosis of CRC.Methods:A search of electronic databases (PubMed and EMBASE) and grey literature was performed between January 2002 and April 2016. Studies reporting plasma or serum miRNAs in the diagnosis of CRC compared with healthy controls were selected. Patient demographics, type of patient sample (serum or plasma), method of miRNA detection, type of normalisation, and the number of significantly dysregulated miRNAs identified were recorded. Statistical evaluation of dysregulated miRNAs using sensitivity, specificity, and area under the curve (AUC) was performed.Results:Thirty-four studies investigating plasma or serum miRNAs in the diagnosis of CRC were included. A total of 31 miRNAs were found to be either upregulated (n=17) or downregulated (n=14) in CRC cases as compared with controls. Fourteen studies identified panels of ⩾2 dysregulated miRNAs. The highest AUC, 0.943, was identified using a panel of 4 miRNAs with 83.3% sensitivity and 93.1% specificity. Meta-analysis of studies identifying a single dysregulated miRNA in CRC cases compared with controls was performed. Overall sensitivity and specificity of 28 individual miRNAs in the diagnosis of CRC were 76% (95% CI 72%–80%) and 76% (95% CI 72%–80%), respectively, indicating good discriminative ability of miRNAs as biomarkers for CRC. These data did not change with sensitivity analyses.Conclusions:Blood-based miRNAs distinguish patients with CRC from healthy controls with high sensitivity and specificity comparable to other common and invasive currently used screening methods for CRC. In future, miRNAs may be used as a relatively non-invasive blood-based marker for detection of CRC.

A Highly Predictive Model for Diagnosis of Colorectal Neoplasms Using Plasma MicroRNA: Improving Specificity and Sensitivity.

Objective: To develop a plasma-based microRNA (miRNA) diagnostic assay specific for colorectal neoplasms, building upon our prior work. Background: Colorectal neoplasms [colorectal cancer (CRC) and colorectal advanced adenoma (CAA)] frequently develop in individuals at ages when other common cancers also occur. Current screening methods lack sensitivity, specificity, and have poor patient compliance. Methods: Plasma was screened for 380 miRNAs using microfluidic array technology from a “Training” cohort of 60 patients, (10 each) control, CRC, CAA, breast cancer, pancreatic cancer, and lung cancer. We identified uniquely dysregulated miRNAs specific for colorectal neoplasia (P < 0.05, false discovery rate: 5%, adjusted &agr; = 0.0038). These miRNAs were evaluated using single assays in a “Test” cohort of 120 patients. A mathematical model was developed to predict blinded sample identity in a 150 patient “Validation” cohort using repeat-sub-sampling validation of the testing dataset with 1000 iterations each to assess model detection accuracy. Results: Seven miRNAs (miR-21, miR-29c, miR-122, miR-192, miR-346, miR-372, and miR-374a) were selected based upon P value, area under the curve (AUC), fold change, and biological plausibility. Area under the curve (±95% confidence interval) for “Test” cohort comparisons were 0.91 (0.85–0.96) between all neoplasia and controls, 0.79 (0.70–0.88) between colorectal neoplasia and other cancers, and 0.98 (0.96–1.0) between CRC and colorectal adenomas. In our “Validation” cohort, our mathematical model predicted blinded sample identity with 69% to 77% accuracy, 67% to 76% accuracy, and 86% to 90% accuracy for each comparison, respectively. Conclusions: Our plasma miRNA assay and prediction model differentiate colorectal neoplasia from patients with other neoplasms and from controls with higher sensitivity and specificity compared with current clinical standards.

The role of the miR-200 family in epithelial-mesenchymal transition in colorectal cancer: a systematic review: Role of miR-200 family in EMT in colorectal cancer

Colorectal cancer (CRC) is associated with significant morbidity and mortality as many patients are diagnosed with advanced stage disease. MicroRNAs are small, noncoding RNA molecules that have a major role in gene expression regulation and are dysregulated in CRC. The miR‐200 family is involved in epithelial–mesenchymal transition (EMT). This systematic review describes the roles of the miR‐200 family in EMT in CRC. A search of electronic databases (PubMed and Embase) was conducted between January 2000 and July 2017. Both in vitro and human studies reporting on the miR‐200 family and CRC were included. Studies describing molecular pathways and the role of the miR‐200 family in the diagnostic and therapeutic management of CRC were analyzed. Thirty‐four studies (22 in vitro and 18 human studies) were included. miR‐200 family expression is regulated epigenetically and via transcriptional factor regulation. In vitro studies show that transfection of miR‐200 family members into chemo‐resistant colon cancer cell lines results in improved chemo‐sensitivity and epithelial phenotype restoration. There is intra‐tumoral variability in the tissue expression of miR‐200 family members with decreased expression at the invasive front. Clinical studies in CRC patients have shown decreased primary tumor tissue expression of miR‐429, miR‐200a and miR‐200c may be associated with worse survival. Conversely, increased blood levels of miR‐141, miR‐200a and miR‐200c may be associated with worse outcomes. The miR‐200 family has a central role in EMT. The miR200 family has potential for both prognostic and therapeutic management of CRC.

IκK-16 decreases miRNA-155 expression and attenuates the human monocyte inflammatory response

Excessive inflammatory responses in the surgical patient may result in cellular hypo-responsiveness, which is associated with an increased risk of secondary infection and death. microRNAs (miRNAs), such as miR-155, are powerful regulators of inflammatory signalling pathways including nuclear factor κB (NFκB). Our objective was to determine the effect of IκK-16, a selective blocker of inhibitor of kappa-B kinase (IκK), on miRNA expression and the monocyte inflammatory response. In a model of endotoxin tolerance using primary human monocytes, impaired monocytes had decreased p65 expression with suppressed TNF-α and IL-10 production (P < 0.05). miR-155 and miR-138 levels were significantly upregulated at 17 h in the impaired monocyte (P < 0.05). Notably, IκK-16 decreased miR-155 expression with a corresponding dose-dependent decrease in TNF-α and IL-10 production (P < 0.05), and impaired monocyte function was associated with increased miR-155 and miR-138 expression. In the context of IκK-16 inhibition, miR-155 mimics increased TNF-α production, while miR-155 antagomirs decreased both TNF-α and IL-10 production. These data demonstrate that IκK-16 treatment attenuates the monocyte inflammatory response, which may occur through a miR-155-mediated mechanism, and that IκK-16 is a promising approach to limit the magnitude of an excessive innate inflammatory response to LPS.

Long-term outcomes following ileal pouch-anal anastomosis in patients with indeterminate colitis

Background. The advisability of performing ileal pouch–anal anastomosis for patients with indeterminate colitis is debated. Indeterminate colitis is found in up to 15% of inflammatory bowel disease colectomy specimens. We determined long‐term outcomes in patients diagnosed with indeterminate colitis undergoing ileal pouch–anal anastomosis. Methods. Fifty‐six patients were included with a mean follow‐up of 14 ± 7 years. Long‐term behavior was defined based on surgeon assessment as “Crohn disease–like” in patients who subsequently developed clear signs of Crohn disease and as “non‐Crohn disease–like.” Long‐term function was assessed using the Cleveland Global Quality of Life and Pouch Functional Score. Results. Thirty‐nine percent of patients developed Crohn disease–like behavior, and 61% developed non‐Crohn disease–like behavior. Both groups experienced a high rate of pouchitis (57%). Crohn disease–like patients required more anti‐inflammatory/immunomodulatory medications (95% vs 18%, P < .001), dilatations for afferent‐limb strictures (41% vs 0%, P < .001), and pouch reoperations (32% vs 6%, P = .02). Eight patients required pouch excision or diversion (7 with Crohn disease–like behavior). The Pouch Functional Score was equivalent between groups. Conclusion. Long‐term function after ileal pouch–anal anastomosis for the majority of indeterminate colitis patients was good. Approximately 40% eventually exhibited Crohn disease–like behavior, but the majority had acceptable function and quality of life. Ileal pouch–anal anastomosis is an appropriate surgical option for indeterminate colitis patients after informed consent.

Plasma microRNA Profile Differentiates Crohn’s Colitis From Ulcerative Colitis

Abstract Background Inflammatory bowel disease (IBD) is commonly divided into 2 entities: Crohn’s disease (CD) and ulcerative colitis (UC). Differentiating between these entities when dealing with IBD confined to the colon is important, especially when planning surgical treatment. Due to ambiguous histological or endoscopic findings, accurate diagnosis is not possible in up to 15% of cases. The aim of this study was to determine whether plasma microRNAs (miRNAs) can help differentiate Crohn’s colitis (CC) from ulcerative colitis. Methods Patients with isolated CC and with UC were enrolled in our study from January 2010 to May 2016. Peripheral blood was collected, and total RNA was isolated from plasma. Screening was performed for 380 common miRNAs. miRNAs that were differentially expressed between these 2 groups were chosen, and their differential expression was confirmed using single miRNA assays in a larger sample size. A predictive model was generated using these data. Significantly differentially expressed miRNAs were then validated utilizing the predictive model to assess blinded data from the single assays. Results Screening was performed on 8 patients from each group. Seven differentially expressed miRNAs were chosen for single assay confirmation. Two miRNAs (miR-598, miR-642) were consistently different between the patient groups (P = 0.013, P = 0.005). Using blinded data, these 2 miRNAs were validated using the predictive model, achieving an overall accuracy of 75% (95% confidence interval, 40.7–92.9). Conclusions We identified 2 plasma miRNAs that differentiated CC from UC. Our data indicate the promise and feasibility of a plasma miRNA–based assay to distinguish between these 2 conditions.

Genetic polymorphisms predict response to anti-tumor necrosis factor treatment in Crohn’s disease

AIM To investigate genetic factors that might help define which Crohn’s disease (CD) patients are likely to benefit from anti-tumor necrosis factor (TNF) therapy. METHODS This was a prospective cohort study. Patients were recruited from a university digestive disease practice database. We included CD patients who received anti-TNF therapy, had available medical records (with information on treatment duration and efficacy) and who consented to participation. Patients with allergic reactions were excluded. Patients were grouped as ever-responders or non-responders. Genomic DNA was extracted from peripheral blood, and 7 single nucleotide polymorphisms (SNPs) were assessed. The main outcome measure (following exposure to the drug) was response to therapy. The patient genotypes were assessed as the predictors of outcome. Possible confounders and effect modifiers included age, gender, race, and socioeconomic status disease, as well as disease characteristics (such as Montreal criteria). RESULTS 121 patients were included. Twenty-one were non-responders, and 100 were ever-responders. Fas ligand SNP (rs763110) genotype frequencies, TNF gene -308 SNP (rs1800629) genotype frequencies, and their combination, were significantly different between groups on multivariable analysis controlling for Montreal disease behavior and perianal disease. The odds of a patient with a Fas ligand CC genotype being a non-responder were four-fold higher as compared to a TC or TT genotype (P = 0.009, OR = 4.30, 95%CI: 1.45-12.80). The presence of the A (minor) TNF gene -308 allele correlated with three-fold higher odds of being a non-responder (P = 0.049, OR = 2.88, 95%CI: 1.01-8.22). Patients with the combination of the Fas ligand CC genotype and the TNF -308 A allele had nearly five-fold higher odds of being a non-responder (P = 0.015, OR = 4.76, 95%CI: 1.35-16.77). No difference was seen for the remaining SNPs. CONCLUSION The Fas-ligand SNP and TNF gene -308 SNP are associated with anti-TNF treatment response in CD and may help select patients likely to benefit from therapy.

The effect of IκK-16 on lipopolysaccharide-induced impaired monocytes

This study focuses on impaired monocyte function, which occurs in some patients after trauma, major elective surgery, or sepsis. This monocyte impairment increases the risk of secondary infection and death. We aimed to determine the influence IκK-16 had on monocytes using an ex-vivo model of human monocyte impairment. We included the effects of the well-studied comparators interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on impaired monocytes. Primary human monocytes were stimulated with 10ng/mL of lipopolysaccharide (LPS) for 16h and then challenged with 100ng/mL LPS to assess the monocyte inflammatory response. Treatment regimens, consisting of either IκK-16, IFN-γ, or GM-CSF, were administered to impaired monocytes near the time of initial LPS stimulation. Stimulation with 10ng/mL LPS initially promoted a pro-inflammatory response but subsequently impaired production of both tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) and decreased HLA-DR expression. IκK-16 treatment attenuated TNF-α production and programmed death-ligand 1 (PD-L1) expression and increased IL-10 and CD14 expression. IFN-γ treatment increased TNF-α production as well as PD-L1 and HLA-DR expression. In conclusion, limiting early inflammation with IκK-16 suppresses TNF-α production and PD-L1 expression but enhances IL-10 production and preserves CD14 expression for potential future exposure to infective stimuli.

Abstract B32: Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia

Introduction: The adenoma-carcinoma sequence is well-recognized as a stepwise pattern of mutational activation of oncogenes and inactivation of tumor suppressor genes resulting in sporadic colorectal cancer (CRC) development from colorectal adenomas (CRA) within the chromosomal instability pathway. MicroRNAs (miRNA) play an important role in oncogenesis by regulating gene expression and are known to be actively released from cells. They are found in body fluids such as plasma, saliva, feces and urine. We have previously demonstrated differential expression of plasma miRNAs in patients with CRC as compared to that of controls. Methods: Plasma was isolated from 10 patients: 5 patients with advanced colorectal adenoma (>0.6cm diameter) and 5 patients with stage II or III colorectal cancer prior to treatment and 4-6 weeks following endoscopic removal or surgical resection, respectively. RNA was extracted from plasma (Qiagen® miRNeasy), RT-PCR performed and 768 miRNAs were screened using microfluidic array technology (Applied BioSystems®) in pre- and post-treatment samples for each patient. Data were analyzed using paired t-tests after normalizing raw cycle threshold data to endogenous RNU6 for pre- and post-treatment samples. In addition, miRNA expression data from both pre- and post-treatment CRA and CRC samples were compared to that of plasma from 10 individuals without neoplasia (controls) (ANOVA). Results: Significant differential expression of plasma miRNAs was observed between pre- and post-treatment samples in both CRA and CRC groups. Interestingly, there was generalized miRNA upregulation in CRA when comparing pre-treatment samples to post-treatment samples. Conversely there was miRNA down-regulation in pre-treatment CRC plasma as compared to post-resection samples. Utilizing significantly dysregulated miRNAs, as well as p-values, AUC, fold-change and biological significance, miRNA panels were selected to facilitate detection of CRA recurrence (following complex polypectomy for large polyps) or for detection of recurrent CRC following resection. The resulting panel was able to differentiate between pre- and post-treatment samples for CRA (miR-186 and miR-623) with AUC 0.94 (95% CI 0.76 – 1.00) and for CRC (miR-324-5p, miR-30d and miR-766) with AUC 0.88 (95% CI 0.63 – 1.00). In addition, while miRNA expression of pre-treatment CRA and CRC samples differed significantly from controls, post-treatment CRA and CRC samples did not. Conclusion: Comparison of pre- and post-treatment plasma samples reveals differing patterns of changes in miRNA expression in benign colorectal neoplasms (CRA) as opposed to invasive neoplasia (CRC). The observed miRNA down-regulation prior to surgical resection of CRC might indicate decreased expression of miRNAs inhibiting oncogenes prior to cancer treatment, while the miRNA upregulation seen in pre-treatment CRA plasma could indicate inhibition of tumor suppressors. Such observations will perhaps elucidate the role of miRNA in carcinogenesis and may provide for a relatively non-invasive method of detecting such lesions. Further validation is needed to develop a miRNA panel that will allow monitoring for evidence of recurrent disease. Citation Format: Jane Carter, Vanessa States, Uri Netz, Jianmin Pan, Shesh Rai, Susan Galandiuk. Longitudinal changes in plasma miRNA in patients with benign and malignant colorectal neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B32.