non-UC Patients Research Articles

P103 Natural compounds in Ulcerative Colitis and Irritable Bowel Syndrome: Insights from immune-metabolic profiles

Abstract Background Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) represent chronic and occasionally disabling conditions, necessitating innovative treatment strategies. Covering 14% of Ireland's landscape, bogland ecosystems house unique plant biodiversity, yielding biologically active secondary metabolites. Informed by traditional medicinal knowledge and in vitro screening for immunomodulatory potential, three plant extracts (NTP0226A, NTP0127B, NTP0206EA) were selected for exploration of their potential to treat IBD and IBS within the "Unlocking Nature’s Pharmacy from Bogland Species" project. Methods Patients undergoing colonoscopy were prospectively recruited in three cohorts: healthy controls, IBS, and ulcerative colitis (UC). Recto-sigmoid biopsies were collected and cultured for 24 h with treatments (Infliximab, NTP0226A, NTP0127B, NTP0206EA). Colonic tissue explants underwent real-time energy metabolism profiling and quantification of 10 inflammatory mediators using Seahorse Xfe24 analyser and multiplex ELISA, respectively. P values <0.05 were considered significant in analyses. Results Twenty-seven patients were recruited (6 healthy controls, 6 IBS, and 15 UC). UC patient explants exhibited elevated glycolytic metabolism and increased IL-4, IL-6, and IL-12p70 secretion compared to non-UC patients. No differences were observed between healthy controls and IBS patients. Linear regression did not show correlation between age and mitochondrial bioenergetics. There was no change in metabolism profiles of any cohort following treatment with Infliximab or the 3 novel extracts. In the UC tissue, treatment with Infliximab reduced IL-12p70 secretion (p=0.0098), this reduction was not seen in the other 2 cohorts. NTP0206EA reduced IL-10 secretion in the UC cohort (p=0.0255). In the IBS cohort; NTP0127B reduced IL-10 secretion (p=0.0048) and NTP0226A reduced IL-4 (p=0.004) and IL-6 (p=0.0219) secretion. Principal component analysis demonstrated reduced separation between the immune-metabolic profiles of UC patients and healthy controls following treatment with NTP0226A (Figure 1). Conclusion Using an explant model, we have shown that UC patients have a distinct immune-metabolic profile modifiable by a standard IBD therapy and novel plant extracts. Blockade of IL-6 trans-signalling has recently shown promise as an effective treatment target in IBD. Our natural extract NTP0226A reduced IL-6 secretion in IBS patients and is a promising candidate for therapeutic exploration. These findings contribute to our understanding of the complex interplay in the colonic microenvironment and highlight potential avenues for novel therapeutic interventions.

Quantitative analysis of the three gut microbiota in UC and non-UC patients using real-time PCR

Backgroundand study aims: Gastrointestinal microbiota are closely related to the pathogenesis of ulcerative colitis (UC). This study aimed at quantification of F. prausnitzii, Provetella, and Peptostreptococcus in UC and non-UC patients using Real-Time PCR and a new set of primers were also validated for this purpose. Materials and methodsIn this study, the relative abundance of microbial populations between the UC and non-UC subjects were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). DNA extraction from biopsies and polymerase chain reaction (PCR) amplification of bacterial 16S rRNA gene-targeted species-specific primers was performed to detect the anaerobic bacterial species. The qRT-PCR was used to show the relative change in the bacterial populations of F. prausnitzii, Provetella, and Peptostreptococcus in the UC and non-UC subjects. ResultsOur data for detection of the anaerobic intestinal flora showed Faecalibacterium prausnitzii, Provetella and Peptostreptococcus were the predominant microflora in the controls and showed significant differences (p = 0.002, 0.025 and 0.039, respectively). The qRT-PCR analyses of F. prausnitzii, Provetella and Peptostreptococcus were 8.69-, 9.38- and 5.77-higher, respectively, in the control group than in the UC group. ConclusionThe results of this study showed decreased abundance of F. prausnitzii, Provetella and Peptostreptococcus in the intestine of UC patients in comparison to non-UC patients. Quantitative RT-PCR, as a progressive and sensitive method, could be useful for evaluation of bacterial populations in patients with inflammatory bowel diseases to attain appropriate therapeutic strategies.

Activated protein kinase C-alpha as a simple test for urothelial cancer screening.

e16552 Background: The development of a non-invasive, highly predictive diagnostic test for urothelial cancer (UC) would greatly benefit patients. Although numerous urine-based tests have been developed, it is not widely used, except for urine cytology, due to insufficient sensitivity, negative predictive value and high cost. Previously we reported that protein kinase C-alpha (PKCα) is overexpressed or activated in several cancer tissues but not in normal tissue, and we developed an activated PKCα-specific peptide substrate (FKKQGSFAKKK; Patent No. 5476559). The purpose of this study was to investigate the potential for UC screening of simple test detecting activated PKCα. Methods: This institutional review board-approved prospective study included patients with bladder cancer or upper urinary tract UC who underwent surgery at Kyushu University Hospital between September 2018 and December 2020. Patients with benign urologic disorders or healthy volunteers were included as a control group. Patients with prostate cancer (PC) or renal cell carcinoma (RCC) who underwent curative surgery were included as other genitourinary (GU) cancer group. Patients with other coexisting malignant tumors were excluded. Urine samples were collected prior to surgery, 20 mL of urine was centrifuged, and the pellet was lysed for analysis. We evaluated the phosphorylation status of the activated PKCα-specific peptide substrate by MALDI-TOF mass spectrometry. The utility of activated PKCα as a urinary biomarker was evaluated by its sensitivity and negative predictive value when compared to control group or other GU cancers group. Results: Urine samples were obtained from 226 individuals, which included 98 patients with UC, 51 with other GU caners, and 77 with control group. Of the 98 patients with UC, activated PKCα was detected in 75 patients, with sensitivity of 76.5%. The sensitivity in patients with high-grade and low-grade UC were 84% and 62%, respectively. The sensitivity in those with high-grade UC increased to 93% when combined with urine cytology. On the other hand, activated PKCα were rarely detected in non-UC patients which includes other GU cancers group and control group. The specificity among the non-UC patients was 74.2%. In the comparison between patients with UC and non-UC, the negative predictive value of activated PKCα was 80.5%. The area under the receiver operating characteristic curve (AUC) for detecting UC of activated PKCα was 0.80. Conclusions: This study demonstrates the utility of activated PKCα as a novel urine screening test for UC. (Patent Application No. PCT/JP2020/045257) Since this test is based on a single target, inexpensive detection devices can be developed.

Solute carrier family 12 member 2 as a proteomic and histological biomarker of dysplasia and neoplasia in ulcerative colitis.

Ulcerative colitis (UC) patients have a greater risk of developing colorectal cancer through inflammation-dysplasia-carcinoma sequence of transformation. The histopathological diagnosis of dysplasia is therefore of critical clinical relevance, but dysplasia may be difficult to distinguish from inflammatory changes. A proteomic pilot study on 5 UC colorectal dysplastic patients highlighted proteins differentially distributed between paired dysplastic, inflammatory and normal tissues. The best candidate marker was selected and immunohistochemistry confirmation was performed on AOM/DSS mouse model lesions, 37 UC dysplasia, 14 UC cancers, 23 longstanding UC, 35 sporadic conventional adenomas, 57 sporadic serrated lesions and 82 sporadic colorectal cancers. Differential proteomics found 11 proteins significantly more abundant in dysplasia compared to inflammation, including Solute carrier family 12 member 2 (SLC12A2) which was confidently identified with 8 specific peptides and was below the limit of quantitation in both inflammatory and normal colon. SLC12A2 immunohistochemical analysis confirmed the discrimination of preneoplastic and neoplastic lesions from inflammatory lesions in mice, UC and in sporadic contexts. A specific SLC12A2 staining pattern termed "loss of gradient" reached 89% sensitivity, 95% specificity and 92% accuracy for UC-dysplasia diagnosis together with an inter-observer agreement of 95.24% (multirater κfree of 0.90; IC95%: 0.78 - 1.00). Such discrimination could not be obtained by Ki67 staining. This specific pattern was also associated with sporadic colorectal adenomas and cancers. We found a specific SLC12A2 immunohistochemical staining pattern in precancerous and cancerous colonic UC-lesions which could be helpful for diagnosing dysplasia and cancer in UC and non-UC patients.

P0768NOVEL URINARY PROTEIN BIOMARKERS FOR UROTHELIAL CANCER IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Introduction: Chronic kidney disease (CKD) patients are known to have greater risk of urothelial cancer (UC) than general population. Urine is the most non-invasive examination and is directly linked to UC. Urinalysis and urine cytology are the major screening tools; however, these tests have low sensitivity or specificity in early disease. In this study, we used both normal and Non-UC CKD patients as controls to discover novel and more specific protein biomarkers in urine for early UC diagnosis. Aims To discover novel and more specific urinary protein biomarkers for screening of urothelial cancer (UC) in CKD patients. Method This is a multicenter prospective cohort study, carried out at 10 nationwide hospitals covering different regions of Taiwan from 2013-2018. Subjects were Taiwanese healthy adults (healthy controls, n=210), patients with CKD but without UC (disease controls, n=273) and patients with ongoing UC (n=211). For non-UC patients, urine and blood specimens were collected at the time of the structured interview. For UC patients, urine and blood samples were collected before operation. iTRAQ-LC-MS/MS approach was applied to compare protein expression among normal, CKD and UC cohorts. Results A total of 3838 proteins in urine were identified and 2497 proteins of them can be quantified. By filtering with fold changes, published papers and Human ATLAS, 27 candidates were selected as potential biomarkers and were validated with ELISA assay in a large sample size of urine specimens. Among the 27 candidates, we found BRDT, CYBP, GARS and HDGF were highly-expressed in the UC cohort when compared to normal and CKD cohorts (Figure 1). To increase the diagnostic value of UC, we combined ROC of our newly discovered 4 protein biomarkers. The AUC values of our newly discovered 4-biomarker panel were higher than the AUC values of the published biomarker panel, including the FDA-approved protein biomarkers (Figure 2). Conclusion We discover and validate a highly specific urinary protein biomarker panel (BRDT, GARS, HDGF and CYBP) to distinguish UC from CKD and healthy controls. It may be used for high throughput screening of subjects, such as CKD patients, who are at high risk to develop UC.

Prognostic significance of non-urothelial carcinoma of bladder: analysis of nationwide hospital-based cancer registry data in Japan.

To identify the prognosis of pure non-urothelial carcinoma (non-UC) of bladder and to compare them with those of pure urothelial carcinoma (UC). We used Japan's nationwide hospital-based cancer registry data to extract histologically confirmed pure non-UC and UC cases of bladder diagnosed in 2008-2009. We estimated the 5-year overall survival (OS) by a Kaplan-Meier analysis. A total of 8094 patients with confirmed histological subtypes of bladder cancer were identified. The most common pure non-UC was squamous cell carcinoma (SQ, n=192, 2.4%) followed by adenocarcinoma (AC, n=138, 1.7%) and small cell neuroendocrine carcinoma (SmC, n=54, 0.7%). The proportion of female patients (48%) was significantly higher in the SQ group compared with the pure UC group (P<0.001). The 5-year OS rate of the non-UC patients was significantly worse than that of the UC patients (40 vs. 61%, P<0.001). According to stages, the 5-year OS rates of the stage I and III non-UC patients were significantly worse than those of the UC patients (P=0.001). Considering histologic subtypes and stages, the 5-year OS rates of the stage I SQ patients were worse than those of the AC and SmC patients (46, 68 and 64%, respectively). The prognosis of pure non-UC was worse than that of pure UC, especially in the stage I and III non-UC patients. To improve these patients' oncologic outcomes, a more aggressive surgical approach may be necessary in stage I patients with non-UC, especially in pure SQ.

Assessment of clinical activity and severity using serum ANCA and ASCA antibodies in patients with ulcerative colitis

BackgroundUlcerative colitis (UC) is a chronic, non-specific inflammatory bowel disease (IBD) with unknown etiology. The lack of specific clinical manifestations, standard diagnostic criteria, objective and accurate indicators to the severity of the disease and the efficacy of the treatment, often results in difficulties in diagnosis and timely treatment of UC. Therefore, there is a need to develop a clinically suitable serum biomarker assay with high specificity and sensitivity.Objective and methodsTo explore the significance of anti-neutrophil cytoplasmic antibodies (ANCA) and anti-saccharomyces cerevisiae antibodies (ASCA) in the diagnosis, differential diagnosis and treatment assessment in patients with ulcerative colitis (UC). Serum levels of ANCA-IgG, ASCA-IgA and ASCA-IgG were measured by an enzyme-linked immunosorbent assay (ELISA) in 105 UC patients, 52 non-UC patients and 100 healthy controls.Results(1) Both the ANCA-IgG level and its positive rate in UC patients were significantly higher than those in non-UC controls and healthy controls (p < 0.01). However, the levels of ASCA-IgA, ASCA-IgG and the positive rates in UC patients had no statistical differences when compared with those in non-UC controls or healthy controls (p > 0.05). (2) The sensitivity of ANCA+ and ANCA+/ASCA− in detecting UC patients was 61.90% and 55.24%, respectively, whereas the specificity was 91.45% and 94.08%, respectively. The sensitivity of ASCA+ and ASCA+/ANCA− in non-UC disease controls was 5.33% and 3.85%, respectively, and specificity was 83.9% and 88.78%, respectively. (3) When UC patients were grouped into mild, moderate or severe subtypes, the ANCA-IgG levels were correlated with the severity of UC, and the differences of the ANCA-IgG levels were statistically different among the three subtypes (p < 0.05). There was no correlation between the levels of ANCA-IgG and the disease locations of UC.Conclusions(1) Serum levels of ANCA may be useful in the diagnosis of UC. (2) Dynamic quantitation of ANCA-IgG levels may be helpful in determining the severity of UC and therefore, may guide treatment of UC.

Prevalence of Ulcerative Colitis in Patients Who Underwent Colonoscopy in Benha University Hospitals

Abstract Background: Colonoscopy is the most accurate diagnostic tool in IBD. It is useful in detection of the severity and extent of UC. the incidence of IBD especially UC increased significantly among Egyptian patients as evidenced in previous studies in the past ten years. Aim of Study: To study the prevalence of UC among patients referred to the internal medicine endoscopy unit to perform colonoscopy and evaluate the most common presenting symptoms of UC. Patients and Methods: Cross sectional study encompassed 190 patients who underwent colonoscopy during the period from October 2018 to October 2019, the main presenting complaint was recorded and the result of colonoscopy. Results: Prevalence of UC was about 11.05% on the other side, internal hemorrhoids was the commonest diagnosis in group I (Non-UC patients) 26.6% followed by nonspecific colitis in 23.6%. Bleeding per rectum was the main symptom and indication for colonoscopy (50.5%) but was not signifi-cantly different among studied groups. Conclusion: Prevalence of UC raised in Egypt in the last years. In this study, it was the 3rd diagnosis after internal hemorrhoids and non-specific colitis constituting about 11.05%.

Clinicopathological features and their impacts on the prognoses of patients with nonurothelial carcinoma: A hospital-based cancer registry in Japan.

555 Background: Urothelial carcinoma (UC) is the most common histology of genitourinary (GU) tract cancer. Non-UC tumors in the GU tract appear to be more aggressive than UC at that location, but the clinicopathological features and their impacts on the prognoses of the non-UC patients are not known due to the low numbers of these patients. Methods: We used Hospital-Based Cancer Registry (HBCR) data in Japan to extract non-UC cases, i.e., patients with adenocarcinoma (AC), squamous cell carcinoma (SCC), or small cell carcinoma (SmCC) of the GU tract who were diagnosed in 2008–2009 with histological confirmation and had received first course of treatment. We retrospectively analyzed the clinicopathological features of these patients, stratified by the bladder and the upper urinary tract (UUT) as tumor locations. We used a Cox proportional hazards regression to identify prognostic factors associated with the overall survival (OS). Results: Of the 8,095 cases at the bladder and 2,580 cases at the UUT, 384 (4.7%) and 131 (5.1%) non-UC cases were identified, respectively. The proportions of histologic subtypes in the bladder were 1.7% AC, 2.4% SCC, and 0.7% SmCC. In the UUT group, these proportions were 1.3%, 3.4%, and 0.4%, respectively. At both tumor locations, the distribution of ages was similar across all subtypes, with the age peak in the 70s. More patients with non-UC were diagnosed at an advanced clinical stage compared to the patients with UC at either location. The 5-yr OS rates of the non-UC patients with a tumor in the bladder and at the UUT were 40% and 26%, whereas the corresponding 5-yr OS rates among the UC patients were 61% and 52%, respectively. A multivariate analysis revealed that the presence of non-UC was significantly associated with increased mortality (hazard ratio 1.66, 95% confidence interval 1.48–1.87) regardless of the tumor location. Conclusions: The clinicopathological features of the non-UC patients were similar between both tumor locations. The presence of non-UC was associated with poor prognosis regardless of the tumor location. A standard of care must be established for non-UC patients, since the prognoses of these patients are not satisfactory.

Systemic Inflammatory Protein Profiles Distinguish Irritable Bowel Syndrome (IBS) and Ulcerative Colitis, Irrespective of Inflammation or IBS-Like Symptoms.

Inflammatory mechanisms of ulcerative colitis (UC) and irritable bowel syndrome (IBS) may overlap or are part of different spectrums. However, potential links between inflammation and IBS-like symptoms in these patient groups are still unclear. The aim of this study was to determine if the systemic inflammatory protein (SIP) profiles differ between UC patients, with presence of inflammation or in remission with or without IBS-like symptoms, and IBS patients. Serum from patients with active UC (UCA), UC patients in remission with or without IBS-like symptoms (UCR + IBS, UCR-IBS), IBS patients (IBS), and healthy subjects (HS) was analyzed using the ProSeek Multiplex Inflammation kit, which detects 92 proteins. The exploratory cohort consisted of 166 subjects (UCA, n = 40; UCR-IBS, n = 45; UCR + IBS, n = 20; IBS, n = 40; HS, n = 21). Systemic inflammatory protein profiles separated UC from non-UC (HS and IBS) patients in multivariate analysis, revealing caspase 8, axin 1, sulfotransferase 1A1, and tumor necrosis factor superfamily member 14 as the variables most important to clustering. Although minor differences were detected between UCR + IBS and UCR-IBS, SIP profiles discriminated UCA from UCR, and interleukin (IL) 17C, IL17A, chemokine ligand 9, and transforming growth factor-α characterized active inflammation. SIP profiles weakly discriminated HS from IBS, although fibroblast growth factor 21 and IL6 serum levels were higher in IBS. Results were confirmed in a validation cohort (UCA, n = 15; UCR + IBS, n = 9; IBS, n = 14). SIP profiles distinguish UC patients from IBS patients, irrespective of inflammation or IBS-like symptoms, suggesting that inflammatory mechanisms of the diseases are part of different spectrums.

Indoleamine-2,3-dioxygenase-1 expression predicts poorer survival and up-regulates ZEB2 expression in human early stage bladder cancer

PurposesIndoleamine-2,3-dioxygenase-1 (IDO1) is a key enzyme of tryptophan metabolism which regulates T cell function in immune cells and little is known about the role of IDO1 expression in bladder cancer cells. The study is aimed to evaluate the clinical relevance of IDO1 expression in human bladder urothelial carcinoma (UC). Materials and MethodsOne hundred and sixty paraffin-embedded UC tissues (130 bladder, 30 upper urinary tract) and 47 adjacent normal tissues were retrieved for IDO1 immunostaining. Urine samples from UC and non-UC patients were collected before surgery for measuring the concentration of tryptophan and its metabolites. Clinicopathological correlates of IDO1 expression and the prognostic values in human bladder cancer were explored. External validation was performed with 4 published bladder cancer datasets, as well as in vitro studies. ResultsAs compared with normal adjacent tissues, UC exhibited a higher frequency of IDO1 expression (chi-square, P = 0.0005). IDO1 expression is an independent poor prognostic factor for disease progression [hazard ratio and 95% confidence interval, 3.80 (1.46–9.86), P = 0.006], which is associated with decreased number of intratumoral infiltrating CD8+ lymphocyte (unpaired t test, P = 0.026). External validation showed that patients with higher IDO1 expression exhibit decreased disease-specific survival than those with lower IDO1 expression. Furthermore, IDO1 expression correlated positively with the expression of several EMT markers, including ZEB2, fibronectin and vimentin. The in vitro T24 cell subline demonstrated that IDO1 expression can up-regulate ZEB2 expression probably through miR-200c signaling. ConclusionIDO1 expression predicts poorer survival and up-regulates ZEB2 expression in human bladder cancer.

MDR1A deficiency restrains tumor growth in murine colitis-associated carcinogenesis.

Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.

Abstract 5754: The genetic aberrations in carcinogenic sequence of colitis-associated cancer

Abstract Colitis-associated cancer (CAC) is a common complication among patients with a long history of ulcerative colitis (UC). Previous studies revealed that dysplasia precedes CAC development, and more than 20% of CACs are multicentric, suggesting the presence of a field effect on colorectal carcinogenesis in UC patients. However, the genetic basis of the field effect in UC patients is poorly understood.To address this issue, we isolated single crypts from non-tumorous colon mucosa from 6 UC patients, as well as those from apparently normal mucosa from non-UC patients, and “single crypt-derived DNA” was analyzed for somatic mutations using whole-exome sequencing (WES). In total, 19 crypts from histologically non-dysplastic, but inflamed mucosa, and 2 from apparently normal mucosa were isolated from 6 UC patients, including 4 with pancolitis, 1 with left-sided colitis and 1 with proctitis, while 32 crypts were obtained from normal mucosa from 20 non-UC patients, including 10 with colorectal cancer, 6 with adenoma and 4 healthy volunteers. Regardless of the disease status, mutations were detected in all crypts analyzed with the median of 43/crypt (3 − 121), showing high variant allele frequencies (VAFs) (~0.5), suggesting that these mutations were shared by all cells within single crypts and therefore by crypt-maintaining stem cells. The number of mutations increased with age, in crypts from both UC and non-UC patients (P &amp;lt; 10-9), showing clear age-related C to T transition in both normal and UC-inflamed crypts. Strikingly, however, the age-adjusted number of mutations was significantly higher in UC-derived crypts than in those from non-UC individuals (P &amp;lt; 10-3). KRAS and TP53 mutations were reported to be the most prevalent alterations in UC-associated CAC, but only rarely seen in UC-derived normal crypts. Next, to evaluate clonal expansion of mutated clones, we performed WES for the bulk specimens composed of 20 - 30 mutually adjacent crypts. However, this time, mutations and copy number alterations (CNAs) with high VAFs were detected only in UC-inflamed mucosa, suggesting that clonal crypt expansion occurred only in UC-derived mucosa. WES was also performed for 14 CACs. The number of non-synonymous mutations (101/sample) in CAC were significantly higher than that in UC-derived normal crypts (38/sample) and recurrently involved known driver genes, such as TP53 (71%) and KRAS (36%). CNAs were also more frequent in CAC than UC-derived, normal crypts. In conclusion, our study revealed that somatic mutations accumulated in normal mucosa of both UC and non-UC cases, but the mutational burden increased in UC-inflamed mucosa. Chronic inflammation caused clonal expansion of crypts even in histologically non-dysplastic mucosa in UC case, accompanied by acquisition of driver mutations and CNAs, which could trigger the development of CAC. Our findings provide novel insights into the carcinogenic sequence in UC-derived CAC. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Akira Yokoyama, Keisuke Kataoka, Yoshikage Inoue, Yasuhide Takeuchi, Yasunori Kogure, Ayana Kon, Masahiro Nakagawa, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Kenji Kawada, Hideaki Okajima, Yoshiharu Sakai, Takaki Sakurai, Hironori Haga, Hiroshi Nakase, Motoi Uchino, Hiroki Ikeuchi, Takako Kihara, Seiichi Hirota, Takahiro Horimatsu, Minoru Matsuura, Hiroyuki Marusawa, Hiroshi Seno, Seishi Ogawa. The genetic aberrations in carcinogenic sequence of colitis-associated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5754. doi:10.1158/1538-7445.AM2017-5754

The potential of volatile organic compounds for the detection of active disease in patients with ulcerative colitis.

To optimise treatment of ulcerative colitis (UC), patients need repeated assessment of mucosal inflammation. Current non-invasive biomarkers and clinical activity indices do not accurately reflect disease activity in all patients and cannot discriminate UC from non-UC colitis. Volatile organic compounds (VOCs) in exhaled air could be predictive of active disease or remission in Crohn's disease. To investigate whether VOCs are able to differentiate between active UC, UC in remission and non-UC colitis. UC patients participated in a 1-year study. Clinical activity index, blood, faecal and breath samples were collected at each out-patient visit. Patients with clear defined active faecal calprotectin >250 μg/g and inactive disease (Simple Clinical Colitis Activity Index <3, C-reactive protein <5 mg/L and faecal calprotectin <100 μg/g) were included for cross-sectional analysis. Non-UC colitis was confirmed by stool culture or radiological evaluation. Breath samples were analysed by gas chromatography time-of-flight mass spectrometry and kernel-based method to identify discriminating VOCs. In total, 72 UC (132 breath samples; 62 active; 70 remission) and 22 non-UC-colitis patients (22 samples) were included. Eleven VOCs predicted active vs. inactive UC in an independent internal validation set with 92% sensitivity and 77% specificity (AUC 0.94). Non-UC colitis patients could be clearly separated from active and inactive UC patients with principal component analysis. Volatile organic compounds can accurately distinguish active disease from remission in UC and profiles in UC are clearly different from profiles in non-UC colitis patients. VOCs have demonstrated potential as new non-invasive biomarker to monitor inflammation in UC.

MiRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis.

Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

The value of serum CYFRA21-1 examination in the detection of urothelial carcinoma

Objective To evaluate the value of CYFRA21-1 examination in the detection of urothelial carcinoma (UC).Methods Serum concentrations of CYFRA21-1 were examined by electrochemiluminescence technique in 65 cases with UC,61 cases of non-UC patients (including 21 cases of renal carcinoma,20 cases of benign prostatic hyperplasia,10 cases of prostatic carcinoma,10 cases of urolithiasis) and 43 hea]thy individuals.Sixty-five UC cases including 9 renal pelvis UC cases,4 ureter UC cases,52 bladder UC cases,61 non-UC cases including 21 renal carcinoma cases,20 benign prostatic hyperplasia cases,10 prostatic carcinoma cases,10 urolithasis cases.Results The serum CYFRA21-1 concentration in UC patients (7.57± 12.76) μg/L was significantly higher than the healthy controls (2.58±0.97) μg/L and also the non-UC patients (2.75± 1.34) μg/L (P＜0.01).The difference between the healthy individuals and the nonUC patients was not significant (P＞0.05).According to the manual in the reagent kit,the positive value of serum CYFRA21-1 was defined as greater than 3.3 μg/L.The false positive rate in healthy controls and the non-UC patients was 10％-30％.The positive percentage of serum CYFRA21-1 of the 65 UC patients was 67.7％ (44/65).In low,middle and high grade UC cases,the positive rates were 50.0％ (10/20),62.5％ (5/8) and 78.3％ (29/37) respectively.The difference of the positive rates between the high grade group and middle,low grade groups was significant (x2 =4.55,P＜0.05).The positive rates in non-muscle invasive (Ta-Ti) UC and in healthy controls were 40.9％ (9/22) and 23.2％ (10/43) respectively,and the difference between the two groups was not significant (x2=2.19,P＞0.05).The positive rate in muscle invasive (T2-T3) UC was 78.6％ (22/28) and the difference compared with healthy controls was significant (x2=20.96,P＜0.01).The positive rate in local advanced and metastatic UC (＞T3) was 86.7％ (13/15).The positive rates were not significantly deferent between the T2-T3 group and the ＞ T3 group (x2 =1.97,P＞0.05).Conclusions The serum CYFRA21-1 positive rate is 67.7％ in UC patients,which was higher than non-UC patients and healthy controls,and associated positively with the pathologic grade and the clinical stage of UC.Monitoring the serum CYFRA21-1 is effective in assessing the outcomes and prognosis of UC,if the serum CYFRA21-1 was positive.The serum CYFRA21-1 test has some significance in distinguishing UC from renal carcinoma and prostatic carcinoma. Key words: Urothelium; Carcinoma; Tumor markers; Keratins

Clinical significance of expression of tissue factor and tissue factor pathway inhibitor in ulcerative colitis.

To investigate the clinical significance of expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in ulcerative colitis (UC). Thirty UC specimens taken by colonoscopy from patients with active UC treated at the Department of Pathology, Central Hospital Affiliated to Shenyang Medical College from February 2010 to January 2012 were included in an experimental group, and 30 normal colon tissue samples taken by colonoscopy from non-UC patients were included in a control group. Expression of TF and TFPI in UC and normal colon tissue samples was detected by immunohistochemistry. The positive rate of TF in UC was significantly higher than that in normal colon tissue (63% vs 33%, χ(2) = 5.41, P < 0.05). The positive rate of TFPI in UC was also significantly higher than that in normal colon tissue (43% vs 17%, χ(2) = 5.08, P < 0.05). Positive rates of TF and TFPI expression in UC are significantly higher than those in normal colon tissue. TF and TFPI may play an important role in the pathogenesis of UC.

Epithelial Expression of Vasoactive Intestinal Peptide in Ulcerative Colitis: Down-Regulation in Markedly Inflamed Colon

Vasoactive intestinal peptide (VIP) has a number of important effects in intestinal physiology and pathology, including in ulcerative colitis (UC). The expression patterns of the predominant VIP receptor in the mucosa (the VPAC1 receptor) are unknown for the mucosa in UC. It is assumed that the sources of VIP in the intestine are the innervation and the inflammatory cells. The VIP and VPAC1 receptor expression patterns in the epithelial layer of UC and non-UC patients were examined in the present study. The influence of marked inflammation of the mucosa was evaluated. Specimens of the human colon, including the colon of UC patients, were examined concerning expressions of VIP and VPAC1 receptor, focusing on the epithelial layer. Immunohistochemistry and in situ hybridization were utilized. There were VIP mRNA reactions and also marked VPAC1 receptor immunoreactions in the normal and slightly/moderately affected epithelium. VIP mRNA reactions were not detected and VPAC1 immunoreactions were minimal in response to marked mucosal derangement. The findings suggest that there is a local production of VIP in the epithelial cells in normal and slightly/moderately inflamed mucosa but not in severely inflamed mucosa. Furthermore, a marked downregulation in VPAC1 receptor expressions occurs in the epithelium in severe UC. Based on the knowledge that VIP can have trophic, healing and anti-inflammatory effects, it is likely that the decrease in VIP mRNA and VPAC1 receptor reactions seen in severely affected mucosa in UC may be associated with adverse effects on intestinal function.

Perceived and Actual Quality of Life With Ulcerative Colitis: A Comparison of Medically and Surgically Treated Patients

Patients with chronic ulcerative colitis (UC) often refuse colectomy, despite data indicating that it might improve quality of life. We hypothesized that perceived utility values are different for patients living with UC compared with UC patients after total proctocolectomy. Our aims were to compare the perceived utility assigned by UC patients with and without a colectomy to standardized chronic UC and post-colectomy scenarios, and to compare the utility of actual health states among groups. We surveyed patients in a tertiary referral center from three groups, including non-UC, UC patients without colectomy, and UC patients who were post-colectomy. We measured the Time-Trade-Off (TTO) utilities of subjects for standardized scenarios, describing moderate UC and a post-colectomy state. Among all UC patients (with and without colectomy), we measured TTO utility for their own health state. Responses were obtained from 150 patients per group (n=450). The non-UC patients considered UC and colectomy scenarios equally (0.92), which was similar to UC patients without colectomy (0.90 and 0.91). Post-colectomy patients strongly preferred the colectomy scenario to the UC scenario (0.86 vs. 0.92, P<0.001). The median utility of UC patients without colectomy for their actual health state was higher than that of post-colectomy patients (0.96 and 0.92, P<0.05). Patients with more social support were more likely to have undergone colectomy compared with patients with little social support (odds ratio=1.20 per dependent/supporter). Patients living with UC prefer their actual health state to a perceived UC scenario or a post-colectomy scenario. Patients who have undergone colectomy equate the quality of life in their actual state with that in a post-colectomy scenario, and prefer each to a perceived chronic UC state. Given the variety of preferences and the importance of social support, opportunities to interact with UC patients who have previously undergone colectomy could help patients living with UC and their physicians to navigate these complex choices.

MiR‐96 and miR‐183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology

A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.