Targeted Sequencing of Cytokine-Induced PI3K-Related Genes in Ulcerative Colitis, Colorectal Cancer and Colitis-Associated Cancer.

Chronic relapsing inflammatory bowel disease is strongly linked to an increased risk of colitis-associated cancer (CAC). One of the well-known inflammatory carcinogenesis pathways, phosphatidylinositol 3-kinase (PI3K), was identified to be a crucial mechanism in long-standing ulcerative colitis (UC). The goal of this study was to identify somatic variants in the cytokine-induced PI3K-related genes in UC, colorectal cancer (CRC) and CAC. Thirty biopsies (n = 8 long-standing UC, n = 11 CRC, n = 8 paired normal colorectal mucosa and n = 3 CAC) were subjected to targeted sequencing on 13 PI3K-related genes using Illumina sequencing and the SureSelectXT Target Enrichment System. The Genome Analysis Toolkit was used to analyze variants, while ANNOVAR was employed to detect annotations. There were 5116 intronic, 355 exonic, 172 untranslated region (UTR) and 59 noncoding intronic variations detected across all samples. Apart from a very small number of frameshifts, the distribution of missense and synonymous variants was almost equal. We discovered changed levels of IL23R, IL12Rß1, IL12Rß2, TYK2, JAK2 and OSMR in more than 50% of the samples. The IL23R variant in the UTR region, rs10889677, was identified to be a possible variant that might potentially connect CAC with UC and CRC. Additional secondary structure prediction using RNAfold revealed that mutant structures were more unstable than wildtype structures. Further functional research on the potential variants is, therefore, highly recommended since it may provide insight on the relationship between inflammation and cancer risk in the cytokine-induced PI3K pathway.

Incidence and clinicopathological features of colorectal cancer among multi-ethnic patients in Kuala Lumpur, Malaysia: a hospital-based retrospective analysis over two decades.

BACKGROUND: The incidence rate of colorectal cancer (CRC) in Asian countries is increasing. Furthermore, recent studies have shown a concerning rise in the incidence of CRC among younger patients aged less than 50 years. This study aimed to analyze the incidence trends and clinicopathological features in patients with early-onset CRC (EOCRC) and later-onset CRC (at age ≥ 50 years). METHODS: A retrospective analysis was performed on 946 patients with CRC diagnosed from 1997 to 2017 at Universiti Kebangsaan Malaysia Medical Centre. The time trend was assessed by dividing the two decades into four 5-year periods. The mean age-standardized and age-specific incidence rates were calculated by using the 5-year cumulative population of Kuala Lumpur and World Health Organization standard population. The mean incidence was expressed per 100,000 person-years. RESULTS: After a stable (all age groups) CRC incidence rate during the first decade (3.00 per 100,000 and 3.85 per 100,000), it sharply increased to 6.12 per 100,000 in the 2008-2012 period before decreasing to 4.54 per 100,000 in the 2013-2017 period. The CRC incidence trend in later-onset CRC showed a decrease in the 2013-2017 period. Contrariwise, for age groups of 40-44 and 45-49 years, the trends showed an increase in the latter 15 years of the study period (40-44 years: 1.44 to 1.92 to 2.3 per 100,000; 45-49 years: 2.87 to 2.94 to 4.01 per 100,000). Malays' EOCRC incidence rate increased from 2008-2012 to 2013-2017 for both the age groups 40-44 years (1.46 to 2.89 per 100,000) and 45-49 years (2.73 to 6.51 per 100,000). Nearly one-fifth of EOCRC cases were diagnosed at an advanced stage (Dukes D: 19.9%), and the majority of them had rectal cancer (72.8%). CONCLUSION: The incidence of EOCRC increased over the period 1997-2017; the patients were predominantly Malays, diagnosed at a later stage, and with cancer commonly localized in the rectal region. All the relevant stakeholders need to work on the management and prevention of CRC in Malaysia.

The Effect of Probiotics (MCP® BCMC® Strains) on Hepatic Steatosis, Small Intestinal Mucosal Immune Function, and Intestinal Barrier in Patients with Non-Alcoholic Fatty Liver Disease.

Treatment for non-alcoholic fatty liver disease (NAFLD) currently consists of lifestyle modifications such as a low-fat diet, weight loss, and exercise. The gut microbiota forms part of the gut-liver axis and serves as a potential target for NAFLD treatment. We investigated the effect of probiotics on hepatic steatosis, fibrosis, and biochemical blood tests in patients with NAFLD. At the small intestinal mucosal level, we examined the effect of probiotics on the expression of CD4+ and CD8+ T lymphocytes, as well as the tight junction protein zona occluden-1 (ZO-1). This was a randomized, double-blind, placebo-controlled trial involving ultrasound-diagnosed NAFLD patients (n = 39) who were supplemented with either a probiotics sachet (MCP® BCMC® strains) or a placebo for a total of 6 months. Multi-strain probiotics (MCP® BCMC® strains) containing six different Lactobacillus and Bifidobacterium species at a concentration of 30 billion CFU were used. There were no significant changes at the end of the study in terms of hepatic steatosis (probiotics: -21.70 ± 42.6 dB/m, p = 0.052 vs. placebo: -10.72 ± 46.6 dB/m, p = 0.29) and fibrosis levels (probiotics: -0.25 ± 1.77 kPa, p = 0.55 vs. placebo: -0.62 ± 2.37 kPa, p = 0.23) as measured by transient elastography. Likewise, no significant changes were found for both groups for the following parameters: LiverFAST analysis (steatosis, fibrosis and inflammation scores), alanine aminotransferase, total cholesterol, triglycerides, and fasting glucose. In the immunohistochemistry (IHC) analysis, no significant expression changes were seen for CD4+ T lymphocytes in either group (probiotics: -0.33 ± 1.67, p = 0.35 vs. placebo: 0.35 ± 3.25, p = 0.63). However, significant reductions in the expression of CD8+ T lymphocytes (-7.0 ± 13.73, p = 0.04) and ZO-1 (Z-score = -2.86, p = 0.04) were found in the placebo group, but no significant changes in the probiotics group. In this pilot study, the use of probiotics did not result in any significant clinical improvement in NAFLD patients. However, at the microenvironment level (i.e., the small intestinal mucosa), probiotics seemed to be able to stabilize the mucosal immune function and to protect NAFLD patients against increased intestinal permeability. Therefore, probiotics might have a complementary role in treating NAFLD. Further studies with larger sample sizes, a longer duration, and different probiotic strains are needed to evaluate the real benefit of probiotics in NAFLD.

Serum pepsinogen and gastrin-17 as potential biomarkers for pre-malignant lesions in the gastric corpus.

There is a lack of non-invasive screening modalities to diagnose chronic atrophic gastritis (CAG) and intestinal metaplasia (IM). Thus, the aim of the present study was to determine the sensitivity and specificity of serum pepsinogen I (PGI), PGI:II, the PGI:II ratio and gastrin-17 (G-17) in diagnosing CAG and IM, and the correlations between these serum biomarkers and pre-malignant gastric lesions. A cross-sectional study of 72 patients (82% of the calculated sample size) who underwent oesophageal-gastro-duodenoscopy for dyspepsia was performed in the present study. The mean age of the participants was 56.2±16.2 years. Serum PGI:I, PGI:II, G-17 and Helicobacter pylori antibody levels were measured by enzyme-linked immunosorbent assay. Median levels of PGI:I, PGI:II, the PGI:II ratio and G-17 for were 129.9 µg/l, 10.3 µg/l, 14.7 and 4.4 pmol/l, respectively. Subjects with corpus CAG/IM exhibited a significantly lower PGI:II ratio (7.2) compared with the control group (15.7; P<0.001). Histological CAG and IM correlated well with the serum PGI:II ratio (r=-0.417; P<0.001). The cut-off value of the PGI:II ratio of ≤10.0 demonstrated high sensitivity (83.3%), specificity (77.9%) and area under the receiver operating characteristic curve of 0.902 in detecting the two conditions. However, the sensitivity was particularly low at a ratio of ≤3.0. The serum PGI:II ratio is a sensitive and specific marker to diagnose corpus CAG/IM, but at a high cut-off value. This ratio may potentially be used as an outpatient, non-invasive biomarker for detecting corpus CAG/IM.

Molecular Characterization of Somatic Alterations in Dukes' B and C Colorectal Cancers by Targeted Sequencing.

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes' B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes' B (n = 10) and Dukes' C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes' B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes' B and C). In addition, TP53 was more frequently altered in Dukes' C (n = 7) compared to Dukes' B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-ß, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape.

A 19-Gene expression signature as a predictor of survival in colorectal cancer.

BACKGROUND: Histopathological assessment has a low potential to predict clinical outcome in patients with the same stage of colorectal cancer. More specific and sensitive biomarkers to determine patients' survival are needed. We aimed to determine gene expression signatures as reliable prognostic marker that could predict survival of colorectal cancer patients with Dukes' B and C. METHODS: We examined microarray gene expression profiles of 78 archived tissues of patients with Dukes' B and C using the Illumina DASL assay. The gene expression data were analyzed using the GeneSpring software and R programming. RESULTS: The outliers were detected and replaced with randomly chosen genes from the 90 % confidence interval of the robust mean for each group. We performed three statistical methods (SAM, LIMMA and t-test) to identify significant genes. There were 19 significant common genes identified from microarray data that have been permutated 100 times namely NOTCH2, ITPRIP, FRMD6, GFRA4, OSBPL9, CPXCR1, SORCS2, PDC, C12orf66, SLC38A9, OR10H5, TRIP13, MRPL52, DUSP21, BRCA1, ELTD1, SPG7, LASS6 and DUOX2. This 19-gene signature was able to significantly predict the survival of patients with colorectal cancer compared to the conventional Dukes' classification in both training and test sets (p < 0.05). The performance of this signature was further validated as a significant independent predictor of survival using patient cohorts from Australia (n = 185), USA (n = 114), Denmark (n = 37) and Norway (n = 95) (p < 0.05). Validation using quantitative PCR confirmed similar expression pattern for the six selected genes. CONCLUSION: Profiling of these 19 genes may provide a more accurate method to predict survival of patients with colorectal cancer and assist in identifying patients who require more intensive treatment.

Molecular characterization of serous ovarian carcinoma using a multigene next generation sequencing cancer panel approach.

BACKGROUND: High grade serous ovarian cancer is one of the poorly characterized malignancies. This study aimed to elucidate the mutational events in Malaysian patients with high grade serous ovarian cancer by performing targeted sequencing on 50 cancer hotspot genes. RESULTS: Nine high grade serous ovarian carcinoma samples and ten normal ovarian tissues were obtained from Universiti Kebangsaan Malaysia Medical Center (UKMMC) and the Kajang Hospital. The Ion AmpliSeq™ Cancer Hotspot Panel v2 targeting "mutation-hotspot region" in 50 most common cancer-associated genes was utilized. A total of 20 variants were identified in 12 genes. Eleven (55%) were silent alterations and nine (45%) were missense mutations. Six of the nine missense mutations were predicted to be deleterious while the other three have low or neutral protein impact. Eight genes were altered in both the tumor and normal groups (APC, EGFR, FGFR3, KDR, MET, PDGFRA, RET and SMO) while four genes (TP53, PIK3CA, STK11 and KIT) were exclusively altered in the tumor group. TP53 alterations were present in all the tumors but not in the normal group. Six deleterious mutations in TP53 (p.R175H, p.H193R, p.Y220C, p.Y163C, p.R282G and p.Y234H) were identified in eight serous ovarian carcinoma samples and none in the normal group. CONCLUSION: TP53 remains as the most frequently altered gene in high grade serous ovarian cancer and Ion Torrent Personal Genome Machine (PGM) in combination with Ion Ampliseq™ Cancer Hotspot Panel v2 were proven to be instrumental in identifying a wide range of genetic alterations simultaneously from a minute amount of DNA. However, larger series of validation targeting more genes are necessary in order to shed a light on the molecular events underlying pathogenesis of this cancer.

The effect of eurycoma longifolia Jack on spermatogenesis in estrogen-treated rats.

INTRODUCTION: There is little data concerning the ability of Eurycoma longifolia Jack (EL) to reverse the inhibitory effects of estrogen on testosterone production and spermatogenesis. The aim of the present study was to determine the effect of EL on testicular histology and sperm count in estrogen-treated male rats. METHODS: Adult male Sprague-Dawley rats weighing 200-250 g were divided into four groups of six rats each. Group A (control) was given solvent in the same manner as the treated groups were given EL. Group B was treated with EL (8 mg/kg body weight) orally. Group C was treated with estradiol (E(2)) (intramuscular dose of 500 microg/kg body weight), and group D received a combined treatment of oral EL and intramuscular E(2). After fourteen consecutive days of treatment, rats from all groups were sacrificed and subjected to spermatogenic and epididymal sperm cell counts. RESULTS: The spermatogenic cell count in the E(2)-treated group was significantly decreased as compared to the control (p < 0.05) and EL+E(2)-treated groups (p < 0.05). A similar finding was found for the epididymal sperm count; the E(2)-treated group had a significant decrease in the count compared to the control (p < 0.05) and EL+E(2)-treated groups (p < 0.05). Rats that were treated with EL alone exhibited significantly higher sperm counts and sperm motility when compared to the control group (p < 0.05). CONCLUSIONS: EL extract acts as a potential agent for reversing the effects of estrogen by increasing spermatogenesis and sperm counts in rats after fourteen consecutive days of treatment.

Progestin regulates chemokine (C-X-C motif) ligand 14 transcript level in human endometrium.

Leukocyte populations change profoundly in the human endometrium during the menstrual cycle. However the predominant cell, the uterine natural killer (uNK) cell does not contain steroid receptors. From gene array analysis we identified a transcript encoding chemokine (C-X-C motif) ligand 14 (CXCL14) which is markedly up-regulated in the secretory phase of the cycle. We confirm this data by northern blotting and quantitative PCR. Using in situ hybridization we localized CXCL14 mRNA to the glandular epithelial cells where it was detected only in the secretory phase of the cycle. Candidate progesterone response elements were identified at positions -2028/-2007 and -722/-697 (PRE1 and PRE2, respectively) relative to the translation start site. These were functionally tested using luciferase reporter deletion constructs, electrophoretic mobility shift assays and site-directed mutagenesis. The deletion/mutation of these sites reduced progesterone induction by 40 and 20%, respectively. Finally, we demonstrated that recombinant CXCL14 stimulated uNK cell chemotaxis in vitro. We therefore conclude that CXCL14 is likely to be regulated by progesterone in human endometrium and that it may exert a chemoattractive effect on uNK cells and in part be responsible for their clustering around the epithelial glands.

The effect of eurycoma longifolia jack on spermatogenesis in estrogen-treated rats

Introduction: There is little data concerning the ability of Eurycoma longifolia Jack (EL) to reverse the inhibitory effects of estrogen on testosterone production and spermatogenesis. The aim of the present study was to determine the effect of EL on testicular histology and sperm count in estrogen-treated male rats. Methods: Adult male Sprague-Dawley rats weighing 200-250 g were divided into four groups of six rats each. Group A (control) was given solvent in the same manner as the treated groups were given EL. Group B was treated with EL (8 mg/kg body weight) orally. Group C was treated with estradiol (E2) (intramuscular dose of 500 ìg/kg body weight), and group D received a combined treatment of oral EL and intramuscular E2. After fourteen consecutive days of treatment, rats from all groups were sacrificed and subjected to spermatogenic and epididymal sperm cell counts. Results: The spermatogenic cell count in the E2-treated group was significantly decreased as compared to the control (p < 0.05) and EL+E2-treated groups (p < 0.05). A similar finding was found for the epididymal sperm count; the E2-treated group had a significant decrease in the count compared to the control (p < 0.05) and EL+E2-treated groups (p < 0.05). Rats that were treated with EL alone exhibited significantly higher sperm counts and sperm motility when compared to the control group (p < 0.05). Conclusions: EL extract acts as a potential agent for reversing the effects of estrogen by increasing spermatogenesis and sperm counts in rats after fourteen consecutive days of treatment.

Deleterious effects of nicotine on the ultrastructure of oocytes: role of gamma-tocotrienol.

BACKGROUND: Previous studies have shown that nicotine enhances oxidative DNA damage and leads to increased lipid peroxidation, which affects embryo development. The present study investigated the effect of daily supplementation of gamma-tocotrienol on oocytes of nicotine-treated mice. MATERIAL/METHODS: Immature female mice (18-25 g) were divided into three groups. For 30 days, group A (control group) received saline (0.2 ml/day s.c.), group B nicotine (5 mg/kg/day s.c. in saline), and group C nicotine with gamma-tocotrienol (60 mg/kg/day p.o.). The animals were superovulated following these schedules. RESULTS: Scanning electron microscopy (SEM) showed that the nicotine-treated oocytes appeared nonspherical with rough surface and the zona pellucida (zp) was torn and became irregular. Supplementation with gamma-tocotrienol in the nicotine-treated mice retained the spherical shape of the oocytes with intact zp; however, the surfaces of the oocytes remained irregular and rough. Transmission electron microscopy (TEM) following chronic nicotine treatment showed loosening of the boundary and tearing of the zp. The perivitelline space was also widened. The cytoplasm of the oocytes retained abundant rough endoplasmic reticulum (rER) with numerous vesicles. Mitochondria were highly dense, with no cristae. The administration of gamma-tocotrienol partially reduced the detrimental effects of nicotine by retaining the smooth boundary of the zp with the tight perivitelline space. There was less rER with no visible vesicle and a lower amount of dense mitochondrial matrix. CONCLUSIONS: This study documented that chronic nicotine treatment adversely affects the ultrastructure of oocytes, while gamma-tocotrienol treatment at least minimizes the nicotine-induced damage to oocytes.