Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer

Introduction

Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.

Materials and Methods

Total RNA was extracted from feces using Qiagen''s miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.

Results

All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.

Conclusion

Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.     

Altered microRNA expression in bovine subcutaneous and visceral adipose tissues from cattle under different diet

Background

MicroRNAs (miRNAs) are a class of molecular regulators found to participate in numerous biological processes, including adipogenesis in mammals. This study aimed to evaluate the differences of miRNA expression between bovine subcutaneous (backfat) and visceral fat depots (perirenal fat) and the dietary effect on miRNA expression in these fat tissues.

Methodology/Principal Findings

Fat tissues were collected from 16 Hereford×Aberdeen Angus cross bred steers (15.5 month old) fed a high-fat diet (5.85% fat, n = 8) or control diet (1.95% fat, n = 8). Total RNA from each animal was subjected to miRNA microarray analysis using a customized Agilent miRNA microarray containing 672 bovine miRNA probes. Expression of miRNAs was not equal between fat depots as well as diets: 207 miRNAs were detected in both fat depots, while 37 of these were found to be tissue specific; and 169 miRNAs were commonly expressed under two diets while 75 were diet specific. The number of miRNAs detected per animal fed the high fat diet was higher than those fed control diet (p = 0.037 in subcutaneous fat and p = 0.002 visceral fat). Further qRT-PCR analysis confirmed that the expression of some miRNAs was highly influenced by diet (miR-19a, -92a, -92b, -101, -103, -106, -142–5p, and 296) or fat depot (miR-196a and -2454).

Conclusions/Significance

Our results revealed that the miRNA may differ among adipose depots and level of fat in the diet, suggesting that miRNAs may play a role in the regulation of bovine adipogenesis.     

Biotinidase is a Novel Marker for Papillary Thyroid Cancer Aggressiveness

Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer (PTC, 34 benign thyroid tissues and 43 FNA samples and correlated with patients’ prognosis. Overall biotinidase expression was decreased in PTC compared to benign nodules (p = 0.001). Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival (p = 0.019, Hazards ratio (HR) = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival (p = 0.014, HR = 5.4). Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors (p = 0.002, Odds ratio (OR) = 0.29) which was evident by its significant association with advanced T stage (p = 0.003, OR = 0.28), nodal metastasis (p<0.001, OR = 0.16), advanced TNM stage (p<0.001, OR = 0.21) and extrathyroidal extension (p = 0.001, OR = 0.23). However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas (p = 0.015, HR = 12.8). In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness.     

miR-16 and miR-21 expression in the placenta is associated with fetal growth

Background

Novel research has suggested that altered miRNA expression in the placenta is associated with adverse pregnancy outcomes and with potentially harmful xenobiotic exposures. We hypothesized that aberrant expression of miRNA in the placenta is associated with fetal growth, a measurable phenotype resulting from a number of intrauterine factors, and one which is significantly predictive of later life outcomes.

Methodology/Principal Findings

We analyzed 107 primary, term, human placentas for expression of 6 miRNA reported to be expressed in the placenta and to regulate cell growth and development pathways: miR-16, miR-21, miR-93, miR-135b, miR-146a, and miR-182. The expression of miR-16 and miR-21 was markedly reduced in infants with the lowest birthweights (p<0.05). Logistic regression models suggested that low expression of miR-16 in the placenta predicts an over 4-fold increased odds of small for gestational age (SGA) status (p = 0.009, 95% CI = 1.42, 12.05). Moreover, having both low miR-16 and low miR-21 expression in the placenta predicts a greater increase in odds for SGA than having just low miR-16 or miR-21 expression (p<0.02), suggesting an additive effect of both of these miRNA.

Conclusions/Significance

Our study is one of the first to investigate placental miRNA expression profiles associated with birthweight and SGA status. Future research on miRNA whose expression is associated with in utero exposures and markers of fetal growth is essential for better understanding the epigenetic mechanisms underlying the developmental origins of health and disease.     

Identification of mouse serum miRNA endogenous references by global gene expression profiles

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments.     

The tissue microlocalisation and cellular expression of CD163, VEGF, HLA-DR, iNOS, and MRP 8/14 is correlated to clinical outcome in NSCLC

Background

We have previously investigated the microlocalisation of M1 and M2 macrophages in NSCLC. This study investigated the non-macrophage (NM) expression of proteins associated with M1 and M2 macrophages in NSCLC.

Methods

Using immunohistochemistry, CD68⁺ macrophages and proteins associated with either a cytotoxic M1 phenotype (HLA-DR, iNOS, and MRP 8/14), or a non-cytotoxic M2 phenotype (CD163 and VEGF) were identified. NM expression of the markers was analysed in the islets and stroma of surgically resected tumours from 20 patients with extended survival (ES) (median 92.7 months) and 20 patients with poor survival (PS) (median 7.7 months).

Results

The NM expression of NM-HLA-DR (p<0.001), NM-iNOS (p = 0.02) and NM-MRP 8/14 (p = 0.02) was increased in ES compared to PS patients in the tumour islets. The tumour islet expression of NM-VEGF, was decreased in ES compared to PS patients (p<0.001). There was more NM-CD163 expression (p = 0.04) but less NM-iNOS (p = 0.002) and MRP 8/14 (p = 0.01) expression in the stroma of ES patients compared with PS patients. The 5-year survival for patients with above and below median NM expression of the markers in the islets was 74.9% versus 4.7% (NM-HLA-DR p<0.001), 65.0% versus 14.6% (NM-iNOS p = 0.003), and 54.3% versus 22.2% (NM-MRP 8/14 p = 0.04), as opposed to 34.1% versus 44.4% (NM-CD163 p = 0.41) and 19.4% versus 59.0% (NM-VEGF p = 0.001).

Conclusions

Cell proteins associated with M1 and M2 macrophages are also expressed by other cell types in the tumour islets and stroma of patients with NSCLC. Their tissue and cellular microlocalisation is associated with important differences in clinical outcome.     

miRNA expression in colon polyps provides evidence for a multihit model of colon cancer

Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n = 53) and inherited colon cancer (n = 11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (≥4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (≥2-fold change).     

Association of the myostatin gene with obesity, abdominal obesity and low lean body mass and in non-diabetic asian indians in north India

Background

To determine the association of the A55T and K153R polymorphisms of the Myostatin gene with obesity, abdominal obesity and lean body mass (LBM) in Asian Indians in north India.

Materials and Methods

A total of 335 subjects (238 men and 97 women) were assessed for anthropometry, % body fat (BF), LBM and biochemical parameters. Associations of Myostatin gene polymorphisms were evaluated with anthropometric, body composition and biochemical parameters. In A55T polymorphism, BMI (p = 0.04), suprailiac skinfold (p = 0.05), total skinfold (p = 0.008), %BF (p = 0.002) and total fat mass (p = 0.003) were highest and % LBM (p = 0.03) and total LBM (Kg) were lowest (p = 0.04) in subjects with Thr/Thr genotype as compared to other genotypes. Association analysis of K153R polymorphism showed that subjects with R/R genotype had significantly higher BMI (p = 0.05), waist circumference (p = 0.04), %BF (p = 0.04) and total fat mass (p = 0.03), and lower %LBM (p = 0.02) and total LBM [(Kg), (p = 0.04)] as compared to other genotypes. Using a multivariate logistic regression model after adjusting for age and sex, subjects with Thr/Thr genotype of A55T showed high risk for high %BF (OR, 3.92, 95% Cl: 2.61–12.41), truncal subcutaneous adiposity (OR, 2.9, 95% Cl: 1.57–6.60)] and low LBM (OR, 0.64, 95% CI: 0.33–0.89) whereas R/R genotype of K153R showed high risk of obesity (BMI; OR, 3.2, 95% CI: 1.2–12.9; %BF, OR, 3.6, 95% CI: 1.04–12.4), abdominal obesity (OR, 2.12, 95% CI: 2.71–14.23) and low LBM (OR, 0.61, 95% CI: 0.29–0.79).

Conclusions/Significance

We report that variants of Myostatin gene predispose to obesity, abdominal obesity and low lean body mass in Asian Indians in north India.     

Elevated expression of squamous cell carcinoma antigen (SCCA) is associated with human breast carcinoma

Squamous cell carcinoma antigen (SCCA) belongs to the serine protease inhibitor (Serpin) family of proteins. Elevated expression of SCCA has been used as a biomarker for aggressive squamous cell carcinoma (SCC) in cancers of the cervix, lung, head and neck, and liver. However, SCCA expression in breast cancer has not been investigated. Immunohistochemical analysis of SCCA expression was performed on tissue microarrays containing breast tumor tissues (n = 1,360) and normal breast epithelium (n = 124). SCCA expression was scored on a tiered scale (0-3) independently by two evaluators blind to the patient''s clinical status. SCCA expression was observed in Grade I (0.3%), Grade II (2.5%), and Grade III (9.4%) breast cancers (p<0.0001). Comparing tissues categorized into the three non-metastatic TNM stages, I-III, SCCA positivity was seen in 2.4% of Stage I cancers, 3.1% of Stage II cancers, and 8.6% of Stage III breast cancers (p = 0.0005). No positive staining was observed in normal/non-neoplastic breast tissue (0 out of 124). SCCA expression also correlated to estrogen receptor/progesterone receptor (ER/PR) double-negative tumors (p = 0.0009). Compared to SCCA-negative patients, SCCA-positive patients had both a worse overall survival and recurrence-free survival (p<0.0001 and p<0.0001, respectively). This study shows that SCCA is associated with both advanced stage and high grade human breast carcinoma, and suggests the necessity to further explore the role of SCCA in breast cancer development and treatment.     

Interruption and defaulting of multidrug therapy against leprosy: population-based study in Brazil's Savannah Region

Background

Low adherence to multidrug therapy against leprosy (MDT) is still an important obstacle of disease control, and may lead to remaining sources of infection, incomplete cure, irreversible complications, and multidrug resistance.

Methodology/Principal Finding

We performed a population-based study in 78 municipalities in Tocantins State, central Brazil, and applied structured questionnaires on leprosy-affected individuals. We used two outcomes for assessment of risk factors: defaulting (not presenting to health care center for supervised treatment for >12 months); and interruption of MDT. In total, 28/936 (3.0%) patients defaulted, and 147/806 (18.2%) interrupted MDT. Defaulting was significantly associated with: low number of rooms per household (OR = 3.43; 0.98–9.69; p = 0.03); moving to another residence after diagnosis (OR = 2.90; 0.95–5.28; p = 0.04); and low family income (OR = 2.42; 1.02–5.63: p = 0.04). Interruption of treatment was associated with: low number of rooms per household (OR = 1.95; 0.98–3.70; p = 0.04); difficulty in swallowing MDT drugs (OR = 1.66; 1.03–2.63; p = 0.02); temporal non-availability of MDT at the health center (OR = 1.67; 1.11–2.46; p = 0.01); and moving to another residence (OR = 1.58; 95% confidence interval: 1.03–2.40; p = 0.03). Logistic regression identified temporal non-availability of MDT as an independent risk factor for treatment interruption (adjusted OR = 1.56; 1.05–2.33; p = 0.03), and residence size as a protective factor (adjusted OR = 0.89 per additional number of rooms; 0.80–0.99; p = 0.03). Residence size was also independently associated with defaulting (adjusted OR = 0.67; 0.52–0.88; p = 0.003).

Conclusions

Defaulting and interruption of MDT are associated with some poverty-related variables such as family income, household size, and migration. Intermittent problems of drug supply need to be resolved, mainly on the municipality level. MDT producers should consider oral drug formulations that may be more easily accepted by patients. Thus, an integrated approach is needed for further improving control, focusing on vulnerable population groups and the local health system.     

The proteomics of colorectal cancer: identification of a protein signature associated with prognosis

Colorectal cancer is one of the commonest types of cancer and there is requirement for the identification of prognostic biomarkers. In this study protein expression profiles have been established for colorectal cancer and normal colonic mucosa by proteomics using a combination of two dimensional gel electrophoresis with fresh frozen sections of paired Dukes B colorectal cancer and normal colorectal mucosa (n = 28), gel image analysis and high performance liquid chromatography–tandem mass spectrometry. Hierarchical cluster analysis and principal components analysis showed that the protein expression profiles of colorectal cancer and normal colonic mucosa clustered into distinct patterns of protein expression. Forty-five proteins were identified as showing at least 1.5 times increased expression in colorectal cancer and the identity of these proteins was confirmed by liquid chromatography–tandem mass spectrometry. Fifteen proteins that showed increased expression were validated by immunohistochemistry using a well characterised colorectal cancer tissue microarray containing 515 primary colorectal cancer, 224 lymph node metastasis and 50 normal colonic mucosal samples. The proteins that showed the greatest degree of overexpression in primary colorectal cancer compared with normal colonic mucosa were heat shock protein 60 (p<0.001), S100A9 (p<0.001) and translationally controlled tumour protein (p<0.001). Analysis of proteins individually identified 14-3-3β as a prognostic biomarker (χ² = 6.218, p = 0.013, HR = 0.639, 95%CI 0.448–0.913). Hierarchical cluster analysis identified distinct phenotypes associated with survival and a two-protein signature consisting of 14-3-3β and aldehyde dehydrogenase 1 was identified as showing prognostic significance (χ² = 7.306, p = 0.007, HR = 0.504, 95%CI 0.303–0.838) and that remained independently prognostic (p = 0.01, HR = 0.416, 95%CI 0.208–0.829) in a multivariate model.     

PTEN as a prognostic and predictive marker in postoperative radiotherapy for squamous cell cancer of the head and neck

Background

Tumor suppressor PTEN is known to control a variety of processes related to cell survival, proliferation, and growth. PTEN expression is considered as a prognostic factor in some human neoplasms like breast, prostate, and thyroid cancer.

Methodology/Principal Findings

In this study we analyzed the influence of PTEN expression on the outcome of a randomized clinical trial of conventional versus 7-days-a-week postoperative radiotherapy for squamous cell cancer of the head and neck. The patients with cancer of the oral cavity, oropharynx, and larynx were randomized to receive 63 Gy in fractions of 1.8 Gy given 5 days a week (CF) or 7 days a week (p-CAIR). Out of 279 patients enrolled in the study, 147 paraffin blocks were available for an immunohistochemical assessment of PTEN. To evaluate the prognostic value of PTEN expression and the effect of fractionation relative to PTEN, the data on the outcome of a randomized clinical trial were analyzed. Tumors with a high intensity of PTEN staining had significant gain in the loco-regional control (LRC) from p-CAIR (5-year LRC 92.7% vs. 70.8%, for p-CAIR vs. CF, p = 0.016, RR = 0.26). By contrast, tumors with low intensity of PTEN did not gain from p-CAIR (5-year LRC 56.2% vs. 47.2%, p = 0.49, RR = 0.94). The intensity of PTEN highly affected the LRC in a whole group of 147 patients (5-year LRC 80.9% vs. 52.3% for high vs. low PTEN, p = 0.0007, RR = 0.32). In multivariate Cox analysis, including neck node involvement, EGFR, nm23, Ki-67, p53, cyclin D1, tumor site and margins, PTEN remained an independent predictor of LRC (RR = 2.8 p = 0.004).

Conclusions/Significance

These results suggest that PTEN may serve as a potent prognostic and predictive marker in postoperative radiotherapy for high-risk squamous cell cancer of the head and neck.     

MicroRNA expression aberration as potential peripheral blood biomarkers for schizophrenia

Since brain tissue is not readily accessible, a new focus in search of biomarkers for schizophrenia is blood-based expression profiling of non-protein coding genes such as microRNAs (miRNAs), which regulate gene expression by inhibiting the translation of messenger RNAs. This study aimed to identify potential miRNA signature for schizophrenia by comparing genome-wide miRNA expression profiles in patients with schizophrenia vs. healthy controls. A genome-wide miRNA expression profiling was performed using a Taqman array of 365 human miRNAs in the mononuclear leukocytes of a learning set of 30 cases and 30 controls. The discriminating performance of potential biomarkers was validated in an independent testing set of 60 cases and 30 controls. The expression levels of the miRNA signature were then evaluated for their correlation with the patients'' clinical symptoms, neurocognitive performances, and neurophysiological functions. A seven-miRNA signature (hsa-miR-34a, miR-449a, miR-564, miR-432, miR-548d, miR-572 and miR-652) was derived from a supervised classification with internal cross-validation, with an area under the curve (AUC) of receiver operating characteristics of 93%. The putative signature was then validated in the testing set, with an AUC of 85%. Among these miRNAs, miR-34a was differentially expressed between cases and controls in both the learning (P = 0.005) and the testing set (P = 0.002). These miRNAs were differentially correlated with patients'' negative symptoms, neurocognitive performance scores, and event-related potentials. The results indicated that the mononuclear leukocyte-based miRNA profiling is a feasible way to identify biomarkers for schizophrenia, and the seven-miRNA signature warrants further investigation.     

Microarray-based oncogenic pathway profiling in advanced serous papillary ovarian carcinoma

Introduction

The identification of specific targets for treatment of ovarian cancer patients remains a challenge. The objective of this study is the analysis of oncogenic pathways in ovarian cancer and their relation with clinical outcome.

Methodology

A meta-analysis of 6 gene expression datasets was done for oncogenic pathway activation scores: AKT, β-Catenin, BRCA, E2F1, EGFR, ER, HER2, INFα, INFγ, MYC, p53, p63, PI3K, PR, RAS, SRC, STAT3, TNFα, and TGFβ and VEGF-A. Advanced serous papillary tumours from uniformly treated patients were selected (N = 464) to find differences independent from stage-, histology- and treatment biases. Survival and correlations with documented prognostic signatures (wound healing response signature WHR/genomic grade index GGI/invasiveness gene signature IGS) were analysed.

Results

The GGI, WHR, IGS score were unexpectedly increased in chemosensitive versus chemoresistant patients. PR and RAS activation score were associated with survival outcome (p = 0.002;p = 0.004). Increased activations of β-Catenin (p = 0.0009), E2F1 (p = 0.005), PI3K (p = 0.003) and p63 (p = 0.05) were associated with more favourable clinical outcome and were consistently correlated with three prognostic gene signatures.

Conclusions

Oncogenic pathway profiling of advanced serous ovarian tumours revealed that increased β-Catenin, E2F1, p63, PI3K, PR and RAS –pathway activation scores were significantly associated with favourable clinical outcome. WHR, GGI and IGS scores were unexpectedly increased in chemosensitive tumours. Earlier studies have shown that WHR, GGI and IGS are strongly associated with proliferation and that high-proliferative ovarian tumours are more chemosensitive. These findings may indicate opposite confounding of prognostic versus predictive factors when studying biomarkers in epithelial ovarian cancer.