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.

Modulation of vulvovaginal atrophy (VVA) by Gelam honey in bilateral oophorectomized rats.

Introduction: Vulvovaginal atrophy (VVA) is a common condition in post-menopausal women. Symptoms of VVA include dyspareunia, vaginal dryness, vaginal and/or vulvar itching, burning and soreness, dysuria and vaginal bleeding accompanying sexual activity. These symptoms are physiological responses to hypoestrogenicity, inducing atrophy of the vagina epithelia and sudden reduction in mucous production. Prevailing therapy for VVA is hormone replacement therapy (HRT), notably estrogen, progesterone or a combination of the two. However, using HRT is associated with an increased incidence of breast and endometrial cancer, venous thromboembolism in the lungs and legs, stroke and cardiovascular complications. Methods: This study evaluated Malaysian Gelam honey as a nutraceutical alternative to estrogen HRT (ERT) in alleviating VVA. A total of 24 female 8-weekold Sprague Dawley rats underwent bilateral oophorectomy. A minimum of 14 days elapsed from the time of surgery and administration of the first dose of Gelam honey to allow the female hormones to subside to a stable baseline and complete recovery from surgery. Vaginal tissues were harvested following a 2-week administration of Gelam honey, the harvested vagina tissue underwent immunohistochemistry (IHC) analysis for protein localization and qPCR for mRNA expression analysis. Results: Results indicated that Gelam honey administration had increased the localization of Aqp1, Aqp5, CFTR and Muc1 proteins in vaginal tissue compared to the menopause group. The effect of Gelam honey on the protein expressions is summarized as Aqp1>CFTR>Aqp5>Muc1. Discussion: Gene expression analysis reveals Gelam honey had no effect on Aqp1 and CFTR genes. Gelam honey had up-regulated Aqp5 gene expression. However, its expression was lower than in the ERT+Ovx group. Additionally, Gelam honey up-regulated Muc1 in the vagina, with an expression level higher than those observed either in the ERT+Ovx or SC groups. Gelam honey exhibits a weak estrogenic effect on the genes and proteins responsible for regulating water in the vaginal tissue (Aqp1, Aqp5 and CFTR). In contrast, Gelam honey exhibits a strong estrogenic ability in influencing gene and protein expression for the sialic acid Muc1. Muc1 is associated with mucous production at the vaginal epithelial layer. In conclusion, the protein and gene expression changes in the vagina by Gelam honey had reduced the occurrence of vaginal atrophy in surgically-induced menopause models.

Roles of phosphatidylinositol-3-kinases signaling pathway in inflammation-related cancer: Impact of rs10889677 variant and buparlisib in colitis-associated cancer.

BACKGROUND: Phosphatidylinositol-3-kinases (PI3K) is a well-known route in inflammation-related cancer. Recent discovery on PI3K-related genes revealed a potential variant that links ulcerative colitis (UC) and colorectal cancer (CRC) with colitis-associated cancer (CAC). PI3K/AKT pathway has been recommended as a potential additional therapeutic option for CRC due to its substantial role in modifying cellular processes. Buparlisib is a pan-class I PI3K inhibitor previously shown to reduce tumor growth. AIM: To investigate the regulation of rs10889677 and the role of buparlisib in the PI3K signaling pathway in CAC pathogenesis. METHODS: Genomic DNA from 32 colonic samples, including CAC (n = 7), UC (n = 10) and CRC (n = 15), was sequenced for the rs10889677 mutation. The mutant and wildtype fragments were amplified and cloned in the pmirGLO vector. The luciferase activity of cloned vectors was assessed after transfection into the HT29 cell line. CAC mice were induced by a mixture of a single azoxymethane injection and three cycles of dextran sulphate sodium, then buparlisib was administered after 14 d. The excised colon was subjected to immunohistochemistry for Ki67 and Cleaved-caspase-3 markers and quantitative real-time polymerase chain reaction analysis for Pdk1 and Sgk2. RESULTS: Luciferase activity decreased by 2.07-fold in the rs10889677 mutant, confirming the hypothesis that the variant disrupted miRNA binding sites, which led to an increase in IL23R expression and the activation of the PI3K signaling pathway. Furthermore, CAC-induced mice had a significantly higher disease activity index (P < 0.05). Buparlisib treatment significantly decreased mean weight loss in CAC-induced mice (P < 0.05), reduced the percentage of proliferating cells by 5%, and increased the number of apoptotic cells. The treatment also caused a downward trend of Pdk1 expression and significantly decreased Sgk2 expression. CONCLUSION: Our findings suggested that the rs10889677 variant as a critical initiator of the PI3K signaling pathway, and buparlisib had the ability to prevent PI3K-non-AKT activation in the pathophysiology of CAC.

Cancer testis antigen PASD1 expression and immunogenicity in human colorectal cancer and polyps.

Colorectal cancer (CRC) is a malignant tumor arising from a human inner colon lining that may spread to other organs such as the liver and lungs. Per ARNT Sim domain containing 1 (PASD1) is a cancer-testis antigen expressed in cancers including CRC but not in normal tissues except for normal testes. This study aims to study PASD1 protein as a potential target for CRC immunotherapy. A total of 90 CRC and polyps tissue samples were investigated for PASD1 RNA and protein expression using a real-time polymerase chain reaction and immunohistochemical staining, respectively. Matched patients' peripheral blood mononuclear cells were pulsed with PASD1 peptides and measured for immunogenicity, cell cytotoxicity, and cytokine assays. The clinical data were collected and analyzed accordingly. Our results show that PASD1_v2 mRNA expression was highly expressed in CRC (46.0%) and polyps samples (33.3%). Both PASD1-1 and PASD1-2 proteins were expressed in 31.7% of CRC and 29.4% of polyps samples. Protein expression was weak to moderate positive in the cytoplasm and/or nucleus of the tissues. Immune responses towards CD4-specific PASD1 peptides were detected in 21.7% of CRC and 23.5% of polyps patients. The most immunogenic peptide was PASD1 (1) in CRC while PASD1 (3) in polyps. Cytotoxicity effects were detected up to 57.20% observed in CRC samples while IL-17A and IL-6 cytokines were highly expressed. The demographic data suggest that Chinese female patients more than 60 years old, diagnosed with late-stage rectosigmoid tumors may benefit from the PASD1 peptide immunotherapy approach. This is the first report describing CD4-positive T-helper response to the PASD1 positive CRC patients and its cytotoxicity.

Placental Cyclophilin A Expression in Pregnancies Complicated with Hypertension.

Introduction: Cyclophilin A was reported to be increased in the serum of mothers with preeclampsia, and is implicated in its pathogenesis. This study aimed to determine the expression of cyclophilin A in the placenta of mothers with and without hypertension, and to correlate its expression with maternal complications and adverse perinatal outcomes. Materials and Methods: This study consisted of a total of 70 cases (35 cases of mothers with hypertension, and 35 normotensive mothers as a control). Cyclophilin A immunohistochemistry was performed on a paraffin-embedded tissue section of placenta submitted at full thickness in order to evaluate the expression in fetal endothelial cells, cytotrophoblasts, syncytiotrophoblasts, maternal endothelial cells and decidual cells. The cyclophilin A expression was scored as weak, moderate or strong intensity. Results: The hypertensive group was more likely to have preterm deliveries (p < 0.0001), caesarean sections (p < 0.0001), and infants admitted to the intensive care unit (p < 0.001). Fifty-one percent of the fetal endothelial cells and cytotrophoblasts expressed cyclophilin A in the hypertensive group, compared to only 28.6% in the normotensive group. However, the difference was not statistically significant (p = 0.086). Conclusion: We found no significant difference in placental cyclophilin A expression between hypertensive and normotensive mothers. There was also no difference in expression in mothers with and without maternal complications and adverse perinatal outcomes.

HER2 testing by immunohistochemistry in breast cancer: A multicenter proficiency ring study.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) over-expression in breast cancer is associated with aggressive tumor behavior and predicts response to targeted therapy. Accurate HER2 result is paramount for optimal patient management. However, routine HER2 immunohistochemistry (IHC) testing are subjected to intra- and inter-laboratory variability. OBJECTIVE: This study aims to determine inter-laboratory variation in HER2 IHC testing through a slide-exchange program between five main reference laboratories. METHOD: A total of 20 breast carcinoma cases with different known HER2 expression and gene status were selected by the central laboratory in five testing rounds. Three unstained tissue sections from each case were sent to participating laboratories, which immunostained and interpreted the HER2 immunohistochemistry result. One of the stained slides was sent to one designated participating laboratory for evaluation. Results were analyzed by the central laboratory. RESULTS: A complete concordance was achieved in six IHC-positive and six IHC-negative cases, its gene status of which was confirmed by in-situ-hybridization (ISH) study. The discordant results were observed in six equivocal cases, one negative case and one positive case with a concordance rate of 50-88.3%. Interestingly, the negative discordant case actually displays tumor heterogeneity. Good inter-observer agreement was achieved for all participating laboratories (k = 0.713-1.0). CONCLUSION: Standardization of HER2 testing method is important to achieve optimum inter-laboratory concordance. Discordant results were seen mainly in equivocal cases. Intra-tumoral heterogeneity may impact the final HER2 IHC scoring. The continuous quality evaluation is therefore paramount to achieve reliable HER2 results.

Prospective evaluation of using multiparametric magnetic resonance imaging in cognitive fusion prostate biopsy compared to the standard systematic 12-core biopsy in the detection of prostate cancer.

PURPOSE: There is mounting evidence to suggest that multiparametric magnetic resonance imaging (mpMRI)-guided biopsy is better than systematic biopsy for the diagnosis of prostate cancer (PCa). Cognitive fusion biopsy (CFB) involves targeted biopsies of areas of suspicious lesions noted on the mpMRI by transrectal ultrasound (TRUS) operator. This study was undertaken to determine the accuracy of mpMRI of the prostate with Prostate Imaging-Reporting and Data System (PI-RADS) version 2 in detecting PCa. We also compare the cancer detection rates between systematic 12-core TRUS biopsy and CFB. MATERIALS AND METHODS: Sixty-nine men underwent mpMRI of the prostate followed by TRUS biopsy. In addition to 12-core biopsy, CFB was performed on abnormal lesions detected on MRI. RESULTS: Abnormal lesions were identified in 98.6% of the patients, and 59.4% had the highest PI-RADS score of 3 or more. With the use of PI-RADS 3 as cutoff, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MRI for the detection of PCa were 91.7%, 57.8%, 53.7%, and 92.8%, respectively. With the use of PI-RADS 4 as cutoff, the sensitivity, specificity, PPV, and NPV of mpMRI were 66.7%, 91.1%, 80%, and 83.7%, respectively. Systematic biopsy detected more PCa compared to CFB (29% vs. 26.1%), but CFB detected more significant (Gleason grade ≥7) PCa (17.4% vs. 14.5%) (P < 0.01). CFB cores have a higher PCa detection rate as compared to systematic cores (P < 0.01). CONCLUSIONS: mpMRI has a good predictive ability for PCa. CFB is superior to systematic biopsy in the detection of the significant PCa.

Delayed Presentation of Metastatic Renal Cell Carcinoma as an Arteriovenous Malformation Mimicking Vascular Tumour of the Forearm.

INTRODUCTION: The development of metastatic renal cell carcinoma (RCC) many years after a nephrectomy is not common but has been reported. A metastasis appearing as a hypervascular tumour, mimicking an arteriovenous malformation (AVM), is a highly unusual phenomenon, with a biopsy required for diagnostic confirmation. Surgery is an option for a solitary metastatic lesion amenable to complete excision, with proven survival benefits. However, widespread metastatic disease carries a very poor prognosis, and is best treated with systemic agents such as anti-angiogenic drugs or tyrosine kinase inhibitors. REPORT: A 58 year old man developed an AVM mimicking a vascular tumour within his left brachioradialis muscle 10 years after a nephrectomy for RCC. Ultrasound and magnetic resonance imaging did not reveal any suspicious features of the vascular lesion.The lesion was successfully removed surgically, and was later proven histopathologically to be metastatic RCC. Further imaging showed widespread metastatic disease, and the patient survived only 15 months after receiving tyrosine kinase inhibitor therapy. DISCUSSION: This case report aims to highlight a few important points: RCC metastases may be hypervascular, mimicking an AVM. A long disease free interval does not necessarily exclude recurrence or metastasis, as in this case, therefore long term surveillance is recommended. A high index of suspicion must be maintained to avoid delay in treatment, and biopsy of any suspicious lesion for histological examination is mandatory, albeit after many years of cancer remission. Whole body imaging with computed tomography or positron emission tomography computed tomography may detect clinically occult recurrence or metastases, and is important to guide further treatment.

Validation of immunogenic PASD1 peptides against HLA-A*24:02 colorectal cancer.

Colorectal cancer is the third commonest malignancy in Asia including Malaysia. The immunogenic cancer-testis antigens, which are expressed in a variety of cancers but with limited expression in normal tissues except the testis, represent an attractive approach to improve treatment options for colorectal cancer. We aimed to validate four PASD1 peptides as the immunotherapeutic targets in colorectal cancer. First, PASD1 mRNA and protein expression were determined via real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. The PASD1 peptides specific to HLA-A*24:02 were investigated using IFN-y-ELISpot assay, followed by the cytolytic and granzyme-B-ELISpot assays to analyze the cytolytic effects of CD8+ T cells. Gene and protein expressions of PASD1 were detected in 20% and 17.3% of colorectal cancer samples, respectively. PASD1(4) peptide was shown to be immunogenic in colorectal cancer samples. CD8+ T cells raised against PASD1(4) peptide were able to lyze HLA-A*24:02+ PASD1+ cells. Our results reveal that PASD1(4) peptide represents a potential target for colorectal cancer.

Identification of differentially expressed circular RNAs in chemoresistant colorectal cancer.

Aim: Chemoresistance in colorectal cancer (CRC) has become a burden in treating the disease effectively. Circular RNAs (circRNAs) are a type of noncoding RNA that were found to be important in cellular homeostasis. The involvement of circRNAs in relation to chemoresistance in other types of cancers has also been reported. This study aims to identify the differentially expressed circRNAs between chemoresistant and chemosensitive CRC cells. Materials & methods: We developed a chemoresistant cell line model and profiled the circRNAs via microarray. We further validated the expression of two circRNAs in 25 formalin-fixed paraffin-embedded (FFPE) tissue specimens (13 nonresponders and 12 responders) via quantitative polymerase chain reaction (qPCR).  Results & conclusion: We found that there were 773 upregulated and 732 downregulated circRNAs between the chemoresistant and chemosensitive HCT-116 cells. We found that hsa_circ_32883 could be a promising biotarget.

A Rare Cause of Acute Kidney Injury: Primary Renal Lymphoma in a Patient with Human Immunodeficiency Virus.

We reported a case of primary renal lymphoma (PRL) presented with non-oliguric acute kidney injury and bilateral kidney infiltrates in an individual with human immunodeficiency virus (HIV) disease. Acute kidney injury secondary to lymphoma infiltrates is very rare (less than 1% of hematological malignancy). A 37-year-old gentleman with underlying human immunodeficiency virus (HIV) disease was on combined antiretroviral therapy since diagnosis. He presented to our center with uremic symptoms and gross hematuria. Clinically, bilateral kidneys massively enlarged and were ballotable. Blood investigations showed hemoglobin of 3.7 g/L, urea of 65.6 mmol/L, and serum creatinine of 1630 µmol/L with hyperkalemia and metabolic acidosis. An urgent hemodialysis was initiated, and he was dependent on regular hemodialysis subsequently. Computed tomography renal scan showed diffuse nonenhancing hypodense lesion in both renal parenchyma. Diagnosis of diffuse large B cell lymphoma with germinal center type, CD20 positive, and proliferative index 95% was confirmed via renal biopsy, and there was no bone marrow infiltrates. Unfortunately, the patient succumbs prior to initiation of chemotherapy.

Urethral Leiomyoma: A Rare Clinical Entity.

Extrauterine leiomyomas are encountered occasionally, which can pose a diagnostic dilemma and challenge to the gynaecologist. We report a rare case of urethral leiomyoma. A 31-year-old woman with history of primary subfertility presented with mass at her urethral meatus and lower urinary tract symptoms. She underwent examination under anaesthesia and excision of the urethral mass. Histopathological examination confirmed leiomyoma. Diagnosis and management of this common growth situated at a rare location were reviewed and discussed.