Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

GAPPS - Gastric Adenocarcinoma and Proximal Polyposis of the Stomach Syndrome in 8 Families Tested at Masaryk Memorial Cancer Institute - Prevention and Prophylactic Gastrectomies.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare variant of familial adenomatous polyposis. It is an autosomal-dominant cancer-predisposition syndrome with massive polyposis of the stomach and a significant risk of gastric adenocarcinoma. Li et al., 2016, described point mutations in the Ying Yang 1 binding site of the APC gene 1B promoter associated with GAPPS syndrome. The first GAPPS syndrome in a Czech family was described in 2016. At Masaryk Memorial Cancer Institute, GAPPS syndrome was diagnosed in eight families using Sanger sequencing. In all families, one mutation in promoter 1B of APC gene NM_001127511: c.-191T>C was detected. This mutation was not found in any patient with multiple colon polyposis without a detected classic mutation in the APC gene. In total, 24 carriers of this mutation in promoter 1B of the APC gene were detected. Out of those 24 carriers, 20 had massive gastric polyposis with more than 100 fundic glandular polyps diagnosed between the age of 22 and 65, 5 had already died of adenocarcinoma of the stomach (at the ages of 29, 40, 59, 60 and 64, respectively) and another woman was treated at the age of 29. Two female carriers do not yet have polyposis of the stomach at the ages of 31 and 65, respectively; one female carrier has incipient polyposis at the age of 58. A male carrier does not have any clinical symptoms, gastroscopy was not indicated because of his age. Prophylactic total gastrectomy with D2 lymphadenectomy has already been performed 6 times at Masaryk Memorial Cancer Institute, in 5 cases without adenocarcinoma at the ages of 27, 34, 44, 51 and 66, respectively; in one female carrier adenocarcinoma of the stomach was detected in a histology specimen. Two prophylactic gastrectomies with D1 lymphadenectomy were performed at University Hospital Brno at the ages of 42 and 50, respectively. In the Czech Republic point mutation c.-191T>C (rs879253783) in the 1B promoter of the APC gene is a frequent cause of gastric polyposis with a high risk of gastric adenocarcinoma, even at a young age. Positively tested individuals are recommended to high-risk oncology clinic. A necessary part of the discussion with the patient is information about a preventive gastrectomy.

Circulating PIWI-Interacting RNAs piR-5937 and piR-28876 Are Promising Diagnostic Biomarkers of Colon Cancer.

Background: The early detection of colon cancer is one of the main prerequisites for successful treatment and mortality reduction. Circulating PIWI-interacting RNAs (piRNA) were recently identified as novel promising biomarkers. The purpose of the study was to assess the profiles of piRNAs in blood serum of colon cancer patients with the aim to identify those with high diagnostic potential.Methods: Blood serum samples from 403 colon cancer patients and 276 healthy donors were included in this 3-phase biomarker study. Large-scale piRNA expression profiling was performed using Illumina small RNA sequencing. The diagnostic potential of selected piRNAs was further validated on independent training and validation sets of samples using RT-qPCR.Results: In total, 31 piRNAs were found to be significantly deregulated in serum of cancer patients compared with healthy donors. Based on the levels of piR-5937 and piR-28876, it was possible to differentiate between cancer patients and healthy donors with high sensitivity and specificity. Moreover, both piRNAs exhibited satisfactory diagnostic performance also in patients with stage I disease and enabled detection of colon cancer with higher sensitivity than currently used biomarkers CEA and CA19-9. Finally, the expression of analyzed piRNAs in blood restored significantly 1 month after the surgical resection.Conclusions: Based on our findings, piRNAs are abundant in human blood serum. Furthermore, their levels in colon cancer have been observed to be significantly deregulated. However, their involvement in carcinogenesis must be further established.Impact: piRNAs could serve as promising noninvasive biomarkers for early detection of colon cancer. Cancer Epidemiol Biomarkers Prev; 27(9); 1019-28. ©2018 AACR.

Tumor Expression of miR-10b, miR-21, miR-143 and miR-145 Is Related to Clinicopathological Features of Gastric Cancer in a Central European Population.

BACKGROUND/AIM: In Western countries, most patients with gastric cancer (GC) present in advanced stages. Therefore, there is imminent clinical need for novel diagnostic and prognostic biomarkers. Deregulation of microRNAs has been reported as a frequent event in GC development in a number of studies. Our study validated the potential of microRNAs to serve as diagnostic and prognostic biomarkers in patients with GC from the Central European population. MATERIALS AND METHODS: Using quantitative real-time polymerase chain reaction, expression levels of six microRNAs (miR-10b, -21, -93, -107, - 143, and -145) were examined in 67 tumor tissues and 67 paired adjacent gastric tissues, and correlated with clinicopathological features of GC patients. RESULTS: Expression levels of miR-10b, miR-21, miR-93, and miR-107 were significantly higher in GC samples compared to non-tumor tissue. Furthermore, the expression levels of miR-10b, miR-143, and miR-145 positively correlated with advanced stages, and increased expression of miR-10b, miR-21 and miR-145 was significantly associated with worse prognosis of gastric cancer patients. CONCLUSION: Our results indicate that selected tissue microRNAs have the potential to serve as relevant diagnostic and prognostic biomarkers of GC in a central European population.

Serum-based microRNA signatures in early diagnosis and prognosis prediction of colon cancer.

Early detection of colorectal cancer is the main prerequisite for successful treatment and reduction of mortality. Circulating microRNAs were previously identified as promising diagnostic, prognostic and predictive biomarkers. The purpose of this study was to identify serum microRNAs enabling early diagnosis and prognosis prediction of colon cancer. In total, serum samples from 427 colon cancer patients and 276 healthy donors were included in three-phase biomarker study. Large-scale microRNA expression profiling was performed using Illumina small RNA sequencing. Diagnostic and prognostic potential of identified microRNAs was validated on independent training and validation sets of samples using RT-qPCR. Fifty-four microRNAs were found to be significantly deregulated in serum of colon cancer patients compared to healthy donors (P < 0.01). A diagnostic four-microRNA signature consisting of miR-23a-3p, miR-27a-3p, miR-142-5p and miR-376c-3p was established (AUC = 0.917), distinguishing colon cancer patients from healthy donors with sensitivity of 89% and specificity of 81% (AUC = 0.922). This panel of microRNAs exhibited high diagnostic performance also when analyzed separately in colon cancer patients in early stages of the disease (T1-4N0M0; AUC = 0.877). Further, a prognostic panel based on the expression of miR-23a-3p and miR-376c-3p independent of TNM stage was established (HR 2.30; 95% CI 1.44-3.66; P < 0.0004). In summary, highly sensitive signatures of circulating microRNAs enabling non-invasive early detection and prognosis prediction of colon cancer were identified.

MiR-205 functions as a tumor suppressor in adenocarcinoma and an oncogene in squamous cell carcinoma of esophagus.

Esophageal cancer is a malignant disease with poor prognosis, increasing incidence, and ineffective treatment options. MicroRNAs are post-transcriptional regulators of gene expression involved in many biological processes including carcinogenesis. We determined miR-205 expression levels in tumor/non-tumor tissues of 45 esophageal cancer patients using qPCR and found that decreased level of miR-205 in tumor tissue correlates with poor overall survival in esophageal adenocarcinoma patients. Further, we observed significantly higher levels of miR-205 in tumor tissue of esophageal squamous cell carcinoma. Ectopic overexpression of miR-205 in adenocarcinoma cell line SK-GT-4 led to decreased cell proliferation, cell cycle arrest in G1, and decreased migration ability. Conversely, in squamous cell line KYSE-150, same effects like inhibition of proliferation, migration, and colony-forming potential and cell cycle arrest in G2 were observed after silencing of miR-205. We performed global gene expression profiling and revealed that suppressive functioning of miR-205 in adenocarcinoma could be realized through regulation of epithelial-mesenchymal transition (EMT), whereas oncogenic in squamous cell carcinoma by regulation of metalloproteinase 10. Our results suggest that miR-205 could serve as biomarker in esophageal cancer and acts as a tumor suppressor in esophageal adenocarcinoma and oncogene in esophageal squamous cell carcinoma.

Diagnostic and prognostic potential of miR-21, miR-29c, miR-148 and miR-203 in adenocarcinoma and squamous cell carcinoma of esophagus.

BACKGROUND: Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential. FINDINGS: Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients. CONCLUSIONS: Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4646922201567057.

Prognostic value of scores based on malnutrition or systemic inflammatory response in patients with metastatic or recurrent gastric cancer.

Cancer patients are frequently affected by malnutrition and weight loss, which affects their prognosis, length of hospital stay, health care costs, quality of life and survival. Our aim was to assess the prognostic value of different scores based on malnutrition or systemic inflammatory response in 91 metastatic or recurrent gastric cancer patients considered for palliative chemotherapy at the Masaryk Memorial Cancer Institute. We investigated their overall survival according to the following measures: Onodera's Prognostic Nutritional Index (OPNI), Glasgow Prognostic Score (GPS), nutritional risk indicator (NRI), Cancer Cachexia Study Group (CCSG), as previously defined, and a simple preadmission weight loss. The OPNI, GPS, and CCSG provided very significant prognostic values for survival (log-rank test P value < 0.001). For example, the median survival for patients with GPS 0 was 12.3 mo [95% confidence interval (CI): 7.7-16.7], whereas the median survival for patients with GPS 2 was only 2.9 mo (95% CI: 1.9-4.8). A significantly worse survival of malnourished patients was also suggested by a multivariate model. The values of GPS, OPNI, and CCSG represent useful tools for the evaluation of patients' prognosis and should be part of a routine evaluation of patients to provide a timely nutrition support.

Identification of microRNAs regulated by isothiocyanates and association of polymorphisms inside their target sites with risk of sporadic colorectal cancer.

Sporadic colorectal cancer (CRC) is a typical multifactorial disease. Isothiocyanates (ITC) have been recently shown to inhibit development of CRC in many experimental models. MicroRNAs (miRNAs) are short noncoding RNAs that posttranscriptionally regulate gene expression through binding to 3' untranslated regions (3'UTR) of target mRNAs. MiRNAs are regulated by natural agents, ITCs included. In our study, using global expression profiling based on TaqMan Low-Density Arrays, we identified 3 common miRNAs (miR-155, miR-23b, miR-27b) regulated by ITCs (sulforaphane, iberin) in colonic epithelial cell lines NCM460 and NCM356. In silico predictions allowed us to find 9 relevant single nucleotide polymorphisms (SNPs) localized within the 3'UTRs of genes (AGTR1, TNFAIP2, PRKCB, HSPA9, RABGAP1, DICER1, ADAM19, VWA5A, and SIRT5) targeted by these ITC-related miRNAs. Finally, we observed that homozygous CC genotype of DICER1, rs1057035, was significantly associated with decreased risk of CRC (odds ratio = 0.49; 95% confidence interval: 0.25-0.95, P = 0.036) when compared to TT homozygote genotype; also, the C allele tended to have a protective effect (P = 0.072). This study showed that miRNAs could be involved in chemoprotective effects of natural agents; their function alteration through SNPs in their binding sites and flanking regions presents a new class of CRC risk factors.

Evaluation of SNPs in miR-196-a2, miR-27a and miR-146a as risk factors of colorectal cancer.

AIM: To investigate whether selected single nucleotide polymorphisms (SNPs) in miR-196a2, miR-27a and miR-146a genes are associated with sporadic colorectal cancer (CRC). METHODS: In order to investigate the effect of these SNPs in CRC, we performed a case-control study of 197 cases of sporadic CRC and 212 cancer-free controls originating from the Central-European Caucasian population using TaqMan Real-Time polymerase chain reaction and allelic discrimination analysis. RESULTS: The genotype and allele frequencies of SNPs were compared between the cases and the controls. None of the performed analysis showed any statistically significant results. CONCLUSION: Our data suggest a lack of association between rs11614913, rs895819 and rs2910164 and colorectal cancer risk in the Central-European Caucasian population, a population with an extremely high incidence of sporadic colorectal cancer.

Common polymorphisms in GSTM1, GSTT1, GSTP1, GSTA1 and susceptibility to colorectal cancer in the Central European population.

BACKGROUND: Central Europe presents with the highest incidence of sporadic colorectal cancer (CRC) worldwide. As sporadic CRC represents a typical multifactorial disease, it is characterized by intense interaction of the genetic background with the environment. Glutathione S-transferases could act as attractive susceptibility genes for CRC, as they are directly involved in conjugation between glutathione and chemotherapeutics, environmental pollutants and a wide spectrum of xenobiotics. METHODS: In this study, we investigated associations of polymorphisms in glutathione S-transferases (GSTs) genes, that is GSTA1, GSTT1, GSTM1 and GSTP1, with CRC in a total of 197 cases and 218 controls originating from the Czech Central European population. Polymorphisms were assessed by polymerase chain reaction/restriction fragment length polymorphism-based methods, allele-specific multiplex and allelic discrimination by real-time polymerase chain reaction. RESULTS: None of investigated polymorphisms showed any associations with CRC, with the exception of GSTP1; where the heterozygote genotype Ile105Val was associated with decreased risk of CRC (P = 0.043). CONCLUSIONS: The frequencies observed in our study are in accordance with those from other European Caucasian populations. Based on our studies, examined variability in GST genes is not a major determinant of CRC susceptibility in the Central European population.

Circulating miR-17-3p, miR-29a, miR-92a and miR-135b in serum: Evidence against their usage as biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Therefore, there is a high demand for cost-effective and non-invasive biomarkers that would enable an early detection of asymptomatic and curable disease with high sensitivity and specificity. OBJECTIVE: The main objective of this study was to investigate the potential of circulating miRNAs as biomarkers of CRC. METHODS: Total RNA enriched for small RNAs was isolated from 100~sera of patients with CRC and 30 sera of healthy donors. The expression levels of miR-17-3p, miR-29a, miR-92a and miR-135b were determined using quantitative real-time PCR. The average expression levels of particular miRNAs were normalized to miR-16 levels and statistically evaluated. RESULTS: Using Mann-Whitney U test, no significant differences were observed in miR-17-3p (P=0.18), miR-29a (P=0.14) and miR-92a (P=0.60) levels between sera of CRC patients and controls. The levels of miR-135b in serum were too low to be quantified accurately. Subsequently, we tried to correlate expression levels of analyzed miRNAs to clinical-pathological features of CRC patients. Only levels of mir-29a were correlated with the clinical stage (P=0.04). Expression levels of the other miRNAs were correlated neither with the clinical stage, nor with the grade. CONCLUSIONS: Interestingly, our results are contradictory to previous studies performed on the CRC patients from Chinese population, providing an evidence against usage of serum miR-17-3p, miR-29a, miR-92a and miR-135b as new biomarkers for early detection of CRC.

Gene expression of somatostatin receptor 4 predicts clinical outcome of patients with metastatic neuroendocrine tumors treated with somatostatin analogs.

Somatostatin analogs (SSA) are the standard diagnostic and treatment tools in the clinical management of patients with neuroendocrine tumors (NETs) expressing somatostatin receptors (SSTRs). Although symptomatic and biochemical control is obtained with SSA in the majority of functional NETs, antineoplastic effects of SSA are partial and of limited duration. The aim of this study was to quantify expression levels of five SSTR subtypes (SSTR1-SSTR5) and correlate them with the clinical outcomes of patients with NETs who underwent SSA therapy. The expression levels were analyzed using real-time polymerase chain reaction in a series of 22 metastatic NETs with a median time of 10 months on the SSA therapy (range 2-82 months). The median duration of disease stabilization in patients who developed progression (n = 14) was 9 months (range 3-92 months). The median survival period for all patients was 44 months (range 3-175 months). According to RECIST criteria, one (5%) partial objective tumor response was obtained, disease stabilization was achieved in 10 (45%) patients, and progressive disease was observed in 11 (50%). Analysis of mRNA expression of the SSTR subtypes showed that SSTR2 and SSTR5 were expressed in all of the studied NETs; SSTR1 and SSTR4 in all but 3 tumors (86%); and SSTR3 in only 10 NETs (49%). Interestingly, our preliminary data suggest that only the levels of SSTR4, though it has the lowest affinity for SSA of all SSTR subtypes, were significantly associated with the stabilization of disease during SSA therapy (p = 0.0357). These levels correlated with time to progression (p = 0.0015) and overall survival (p = 0.0017) in NET patients.