Non-dysplastic Mucosa Research Articles

Deep learning-based pathology image analysis predicts cancer progression risk in patients with oral leukoplakia.

Oral leukoplakia (OL) is associated with an increased risk for oral cancer (OC) development. Prediction of OL cancer progression may contribute to decreased OC morbidity and mortality by favoring early intervention. Current OL progression risk assessment approaches face large interobserver variability and is weakly prognostic. We hypothesized that convolutional neural networks (CNN)-basedhistology image analyses could accelerate the discovery of better OC progression risk models. Our CNN-based oral mucosa risk stratification model (OMRS) was trained to classify a set of nondysplastic oral mucosa (OM) and a set of OC H&E slides. As a result, the OMRS model couldidentify abnormal morphological features of the oral epithelium. By applying this model to OL slides, we hypothesized that the extent of OC-like features identified in the OL epithelium would correlate with its progression risk. The OMRS model scored and categorized the OL cohort (n=62) into high- and low-risk groups. OL patients classified as high-risk (n=31) were 3.98 (95% CI 1.36-11.7) times more likely to develop OC than low-risk ones (n=31). Time-to-progression significantly differedbetween high- and low-risk groups (p=0.003). The 5-year OC development probability was 21.3% for low-risk and 52.5% for high-risk patients. The predictivepower of the OMRS model was sustained even after adjustment for age, OL site, and OL dysplasia grading (HR=4.52, 1.5-13.7). The ORMS model successfully identified OL patients with a high risk of OC development and can potentially benefit OC early diagnosis and prevention policies.

Further Study on Field Cancerization in the Human Colon.

Nearly 70 years ago, Slaughter launched the hypothesis of field cancerization for oral carcinomas; that hypothesis was subsequently also claimed for carcinomas in other organs. We previously found in the colon mucosa adjacent to nonpolypoid adenomas, branching crypts lined by normal epithelium (BCNE). Here, we explored whether BCNE could also be found in the colon mucosa adjacent to sporadic polypoid tubular adenomas (TA), the most prevalent of all colon adenomas. Nondysplastic mucosa adjacent to TA was found in 103 out of 131 TA. All BCNE adjacent to TA were recorded. In 98 (95.1%) out of 103 TA having nondysplastic adjacent mucosa, 645 BCNE were registered: 82.6% were in asymmetric branching and 17.4% in symmetric branching. Thus, BCNE in asymmetric branching predominated. The frequency of BCNE adjacent to TA was influenced by the adenoma size and degree of dysplasia severity. Contrarywise, the frequency of BCNE adjacent to TA was neither influenced by the age or sex of the patients, nor by the colon localization of TA. BCNE often occur in the normal mucosa adjacent to TA. BCNE emerge as integral components of TA. The majority of the BCNE were in asymmetric branching, considered as aberrations of cryptogenesis. We propose that the accretion of asymmetric BCNE adjacent to TA supports Slaughter's hypothesis of field cancerization.

Genetic Profiling of Colorectal Carcinomas of Patients with Primary Sclerosing Cholangitis and Inflammatory Bowel Disease.

BackgroundPatients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) run a 10-fold increased risk of developing colorectal cancer (CRC) compared to patients with IBD only. The aim of this study was to perform an extensive screen of known carcinogenic genomic alterations in patients with PSC-IBD, and to investigate whether such changes occur already in nondysplastic mucosa.MethodsArchival cancer tissue and nondysplastic mucosa from resection specimens of 19 patients with PSC-IBD-CRC were characterized, determining DNA copy-number variations, microsatellite instability (MSI), mutations on 48 cancer genes, and CpG island methylator phenotype (CIMP). Genetic profiles were compared with 2 published cohorts of IBD-associated CRC (IBD-CRC; n = 11) and sporadic CRC (s-CRC; n = 100).ResultsPatterns of chromosomal aberrations in PSC-IBD-CRC were similar to those observed in IBD-CRC and s-CRC, MSI occurred only once. Mutation frequencies were comparable between the groups, except for mutations in KRAS, which were less frequent in PSC-IBD-CRC (5%) versus IBD-CRC (38%) and s-CRC (31%; P = .034), and in APC, which were less frequent in PSC-IBD-CRC (5%) and IBD-CRC (0%) versus s-CRC (50%; P < .001). Cases of PSC-IBD-CRC were frequently CIMP positive (44%), at similar levels to cases of s-CRC (34%; P = .574) but less frequent than in cases with IBD-CRC (90%; P = .037). Similar copy number aberrations and mutations were present in matched cancers and adjacent mucosa in 5/15 and 7/11 patients, respectively.ConclusionsThe excess risk of CRC in patients with PSC-IBD was not explained by copy number aberrations, mutations, MSI, nor CIMP status, in cancer tissue, nor in adjacent mucosa. These findings set the stage for further exome-wide and epigenetic studies.

Downregulation of Lipid Phosphate Phosphatase 3 Correlates With Tumor-Infiltrating Immune Cells in Oral Cancer.

BackgroundSphingosine-1-phosphate (S1P) is a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P-metabolizing enzymes. S1P synthesis is catalyzed by two sphingosine kinases, i.e., sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Five lipid phosphatases (two S1P phosphatases and lipid phosphate phosphatases (LPPs) 1, 2, and 3) reversibly convert S1P back to sphingosine. Previously, we have determined the mRNA expression profile of eight S1P-metabolizing enzymes in tumor tissues and adjacent normal tissues from oral squamous cell carcinoma (OSCC) patients. Except for SphK1, the role of S1P-metabolizing enzymes in OSCC has been poorly studied.MethodsWe have determined the protein expression of four S1P-metabolizing enzymes (SphK1, SphK2, sphingosine-1-phosphate phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3)) by immunohistochemistry (IHC) in tumor tissues of 46 OSCC patients. Six subjects with non-dysplastic oral mucosa were also included in the study. The immunoreactivity score (IRS) was calculated for each protein in every subject. Further, we determined the associations of expression of S1P-metabolizing enzymes with clinicopathological features of OSCC patients.ResultsWe demonstrate the low IRS for SphK2 and LPP3 in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant epithelial cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with tumor‑node‑metastasis (TNM) staging of patients (r = −0.307, p = 0.043). Importantly, expression of LPP3 in tumors was found to be an independent predictor of perinodal extension (b = −0.440, p = 0.009), lymphovascular invasion (b = −0.614, p < 0.001), lymph node ratio (b = 0.336, p = 0.039), and TNM staging (b = −0.364, p = 0.030).ConclusionTaken together, our data show that expression of SphK2 and LPP3 is decreased compared to normal mucosa. Thus, the S1P signaling pathway could represent a potential therapeutic target.

Expression of the EGFR-RAS Inhibitory Proteins DOK1 and MTMR7 and its Significance in Colorectal Adenoma and Adenoma Recurrence.

Colorectal adenomas are precursor lesions for colorectal cancer (CRC), a major cause of cancer-related death. Despite all molecular insights, there are still unknown variables in the development of CRC as well as uncertainties regarding adenoma recurrence after resection. We aimed to characterize the expression of docking protein 1 (DOK1) and myotubularin-related protein 7 (MTMR7), which share inhibiting functions on EGFR-RAS-signalling, a major oncogenic driver in CRC, and their association with clinical variables and adenoma recurrence. This observational study is based on clinical data obtained from patients who underwent routine endoscopy and consecutive follow-up examinations. Immunohistochemistry was conducted both in dysplastic tissue and adjacent non-dysplastic mucosa followed by microscopical assessment. Recurrence was differentiated between local, segmental and distant relapse. A total of 56 patients (23 females) gathering 96 adenomas/polyps were included. 36 patients experienced a metachronous lesion, 23 patients had simultaneous lesions in their index endoscopy. Female patients showed lower levels of MTMR7 in adenomas (p=0.0318). Adenomas of young patients showed lower DOK1 than those of older patients (p=0.0469). Big adenomas showed a higher expression of DOK1 than small lesions (p=0.0044). In serrated lesions, DOK1 was reduced (p=0.0026) and correlated with the quantity of lesions (p < 0.001). MTMR7 was significantly reduced in distant (p=0.05) and local segmental recurrence (p=0.0362), while DOK1 showed higher expression in recurrence (p=0.0291). We found ambivalent results regarding the role of the markers as potential tumor suppressors, implying a context-dependent function of these molecules which might change in the course of time. DOK1 may play an inhibiting role in the serrated pathway. Remarkably, molecular markers have the potential to predict recurrence, since a combined expression analysis of high DOK1 and low MTMR7 correlated with the likelihood of segmental adenoma recurrence.

Toward improved endoscopic surveillance with multidiameter single fiber reflectance spectroscopy in patients with Barrett's esophagus.

Patients with Barrett's esophagus are at an increased risk to develop esophageal cancer and, therefore, undergo regular endoscopic surveillance. Early detection of neoplasia enables endoscopic treatment, which improves outcomes. However, early Barrett's neoplasia is easily missed during endoscopic surveillance. This study investigates multidiameter single fiber reflectance spectroscopy (MDSFR) to improve Barrett's surveillance. Based on the concept of field cancerization, it may be possible to identify the presence of a neoplastic lesion from measurements elsewhere in the esophagus or even the oral cavity. In this study, MDSFR measurements are performed on non-dysplastic Barrett's mucosa, squamous mucosa, oral mucosa, and the neoplastic lesion (if present). Based on logistic regression analysis on the scattering parameters measured by MDSFR, a classifier is developed that can predict the presence of neoplasia elsewhere in the Barrett's segment from measurements on the non-dysplastic Barrett's mucosa (sensitivity 91%, specificity 71%, AUC = 0.77). Classifiers obtained from logistic regression analysis for the squamous and oral mucosa do not result in an AUC significantly different from 0.5.

Molecular characterization of ulcerative colitis-associated colorectal carcinomas.

Patients with ulcerative colitis (UC) are at increased risk for developing colorectal cancer (CRC). In contrast to sporadic colorectal tumorigenesis, TP53 mutations occur early in the progression from inflamed colonic epithelium to dysplasia to CRC, and are sometimes readily detectable in inflamed, (yet) non-dysplastic mucosa. Here, we analyzed formalin-fixed paraffin-embedded tissue samples from 19 patients with long-standing UC (median 18 years, range 3 to 34) who had developed CRC as a consequence of chronic inflammation of the large bowel. We performed microsatellite instability testing, copy number analysis by array-based comparative genomic hybridization, mutation analysis by targeted next generation sequencing (48-gene panel) and TP53 immunostaining. The results were compared to The Cancer Genome Atlas (TCGA) data on sporadic CRC. All UC-CRC lesions in our cohort were microsatellite stable. Overall, genomic imbalances of UC-CRCs showed patterns of chromosomal aneuploidies characteristic for sporadic CRC with the exception of gains of chromosome arm 5p (12 of 23 UC-CRC, 52%), which are rare in sporadic CRCs from TCGA (21 of 144, 15%; FDR adjusted P = 0.006). UC-CRCs showed a predilection for TP53 alterations, which was the most frequently mutated gene in our cohort (20 of 23, 87%). Interestingly, spatially separated tumor lesions from individual patients tended to harbor distinct TP53 mutations. Similar to CRCs arising in a background of Crohn’s colitis, the genetic landscape of UC-CRCs was characterized by TP53 mutations and chromosomal aneuploidies including gains of chromosome arm 5p. Both alterations harbor the potential for early detection in precursor lesions, thus complementing morphologic diagnosis.

Diagnosis, risk stratification, and management of ampullary dysplasia by DNA flow cytometric analysis of paraffin-embedded tissue

The limited accuracy of endoscopic biopsy in detecting high-grade dysplasia or adenocarcinoma within ampullary adenoma or dysplasia has been reported. The natural history of ampullary dysplasia is also unclear, and there are no established guidelines to determine which patients with ampullary dysplasia require resection versus surveillance endoscopy. DNA flow cytometry was performed on 47 ampullary biopsies with low-grade dysplasia, 18 high-grade dysplasia, and 23 negative for dysplasia, as well as 11 cases of ampullary adenocarcinoma. Abnormal DNA content (aneuploidy or elevated 4N fraction > 6%) was identified in 9 (82%) of adenocarcinoma, 13 (72%) of high-grade dysplasia, 7 (15%) of low-grade dysplasia, and none (0%) of non-dysplastic mucosa. One-, 2-, and 7-year detection rates of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with abnormal DNA content were 57%, 86%, and 88%, respectively, whereas low-grade dysplasia patients in the setting of normal DNA content had 1-, 2-, and 7-year detection rates of 10%, 10%, and 10%, respectively. The univariate and multivariate hazard ratios (HRs) for subsequent detection of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 16.8 (p = <0.01) and 9.8 (p = <0.01), respectively. Among the 13 high-grade dysplasia patients with DNA content abnormality, 5 patients (38%) were subsequently found to have adenocarcinoma within a mean follow-up time of 3 months, whereas only 1 (20%) of the remaining 5 patients in the setting of normal DNA content developed adenocarcinoma in a month (HR = 2.6, p = 0.39). The overall 1- and 2-year detection rates of adenocarcinoma in all high-grade dysplasia patients (regardless of flow cytometric results) were 34% (95% confidence interval = 16–63%) and 47% (95% confidence interval = 23–79%), respectively. In conclusion, the majority of low-grade dysplasia patients (86%) in the setting of abnormal DNA content developed high-grade dysplasia or adenocarcinoma within 2 years and thus may benefit from resection, whereas those with normal DNA content may be followed with surveillance endoscopy. The presence of DNA content abnormality can also confirm a morphologic suspicion of high-grade dysplasia, which should be managed with resection, as nearly 50% of the high-grade dysplasia patients were found to have adenocarcinoma within 2 years.

Weak Cytotoxic T Cells Activation Predicts Low-Grade Dysplasia Persistence in Ulcerative Colitis.

INTRODUCTION:In patients with ulcerative colitis (UC), dysplasia develops in 10%–20% of cases. The persistence of low-grade dysplasia (LGD) in UC in 2 consecutive observations is still an indication for restorative proctocolectomy. Our hypothesis is that in the case of weak cytotoxic activation, dysplasia persists. We aimed to identify possible immunological markers of LGD presence and persistence.METHODS:We prospectively enrolled 112 UC patients who underwent screening colonoscopy (T0) who had biopsies taken from their sigmoid colon. Ninety of them had at least a second colonoscopy (T1) with biopsies taken in the sigmoid colon and 8 patients had dysplasia in both examinations suggesting a persistence of LGD in their colon. Immunohistochemistry and real time polymerase chain reaction for CD4, CD69, CD107, and CD8β messenger RNA (mRNA) expression and flow cytometry for epithelial cells expressing CD80 or HLA avidin-biotin complex were performed. Non-parametric statistics, receiver operating characteristic curves analysis, and logistic multiple regression analysis were used.RESULTS:Thirteen patients had LGD diagnosed at T0. The mucosal mRNA expression of CD4, CD69, and CD8β was significantly lower than in patients without dysplasia (P = 0.033, P = 0.046 and P = 0.007, respectively). A second colonoscopy was performed in 90 patients after a median follow-up of 17 (12–25) months and 14 of the patients were diagnosed with LGD. In these patients, CD8β mRNA expression at T0 was significantly lower in patients without dysplasia (P = 0.004). A multivariate survival analysis in a model including CD8β mRNA levels and age >50 demonstrated that both items were independent predictors of dysplasia at follow-up (hazard ratio [HR] = 0.47 [95% confidence interval [CI]: 0.26–0.86], P = 0.014, and HR = 13.32 [95% CI: 1.72–102.92], P = 0.013).DISCUSSION:These data suggest a low cytotoxic T cell activation in the colonic mucosa of UC patients who do not manage to clear dysplasia. Thus, low level of CD8β mRNA expression in non-dysplastic colonic mucosa might be considered in future studies about the decision making of management of LGD in UC.

Whole Exome Sequencing of Ulcerative Colitis-associated Colorectal Cancer Based on Novel Somatic Mutations Identified in Chinese Patients.

Carcinogenesis is a severe consequence of chronic ulcerative colitis. We investigated the somatic mutations and pathway alterations in ulcerative colitis-associated colorectal cancer (CRC) in Chinese patients compared with sporadic CRCs to reveal potential therapeutic targets in ulcerative colitis-associated CRC. Whole exome sequencing was performed on archival tumor tissues and paired adjacent nondysplastic mucosa from 10 ulcerative colitis-associated CRC patients at a high risk of carcinogenesis. Genomic alteration profiles from 223 primary CRCs from The Cancer Genome Atlas served as sporadic CRC controls. A meta-analysis was performed to investigate differences in major genetic mutations between ulcerative colitis-associated and Crohn's disease-associated CRCs. We identified 44 nonsilent recurrent somatic mutations via whole exome sequencing, including 25 deleterious mutations involved in apoptosis and the PI3K-Akt pathway (COL6A3, FN1), autophagy (ULK1), cell adhesion (PODXL, PTPRT, ZFHX4), and epigenetic regulation (ARID1A, NCOR2, KMT2D, NCOA6, MECP2, SUPT6H). In total, 11 of the 25 mutated genes significantly differed between ulcerative colitis-associated CRC and sporadic CRC (APC, APOB, MECP2, NCOR2, NTRK2, PODXL, RABGAP1, SIK3, SUPT6H, ULK1, USP48). Somatic TP53 mutations occurred in 33% of ulcerative colitis-associated CRCs. Subsequent meta-analysis revealed distinct mutation profiles for Crohn's disease- and ulcerative colitis-associated CRCs. Mutations involving the NF-kB pathway and epigenetic regulation were more common in ulcerative colitis-associated CRCs than in sporadic CRCs. Distinct genomic alteration profiles of deleterious somatic mutations were found in ulcerative colitis-associated and sporadic CRCs. Mutations of epigenetic regulators, such as KMT2D and NCOA6, were common, suggesting an epigenetic pathomechanism for colitis-associated carcinoma in Chinese patients.

Tenascin-C Produced by Intestinal Myofibroblasts Promotes Colitis-associated Cancer Development Through Angiogenesis.

Colitis-associated cancer (CAC) is one of the prognostic factors in inflammatory bowel disease (IBD), and prevention of CAC is a critical concern for patients with IBD. Component cells of the microenvironment, especially myofibroblasts, are known to affect tumor development, but the role of intestinal myofibroblasts (IMFs) in CAC has not been clarified. Here, we explored the role of IMFs in CAC and sought to identify candidate genes as novel therapeutic targets for the prevention of CAC. We used the azoxymethane (AOM)/dextran sodium sulfate (DSS) model for dysplasia and CAC. Flow cytometry and RNA sequencing (RNA-seq) were performed to obtain an unbiased gene expression profile of IMFs. The transcriptome of significantly differentially expressed genes was analyzed by RNA-seq, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. Comparison of normal intestinal fibroblasts and IMFs revealed 1045 genes with significantly differential expression. Among them, we focused on tenascin-C (TNC; q = 0.00232, Log2(Fold Change) = 3.87). Tenascin-C gene expression was markedly increased in the dysplasia model compared with control and CAC model (P < 0.05). Tenascin-C protein was barely expressed in normal and nondysplastic mucosa but strongly expressed in the stroma around dysplastic lesions. Moreover, TNC surrounded and enclosed integrin αvβ3-positive microvessels. Administration of ATN-161, an antagonist of αvβ3-integrin, significantly suppressed tumorigenesis of CAC through inhibition of angiogenesis (P < 0.05). In the early stages of CAC, TNC produced by IMFs affects tumor development via integrin αvβ3-mediated angiogenesis. Intestinal myofibroblasts might be a novel therapeutic target for preventing CAC.

Dynamics of Genome Alterations in Crohn's Disease-Associated Colorectal Carcinogenesis.

Purpose: Patients with inflammatory bowel diseases, that is, ulcerative colitis and Crohn's disease (CD), face an increased risk of developing colorectal cancer (CRC). Evidence, mainly from ulcerative colitis, suggests that TP53 mutations represent an initial step in the progression from inflamed colonic epithelium to CRC. However, the pathways involved in the evolution of CRC in patients with CD are poorly characterized.Experimental Design: Here, we analyzed 73 tissue samples from 28 patients with CD-CRC, including precursor lesions, by targeted next-generation sequencing of 563 cancer-related genes and array-based comparative genomic hybridization. The results were compared with 24 sporadic CRCs with similar histomorphology (i.e., mucinous adenocarcinomas), and to The Cancer Genome Atlas data (TCGA).Results: CD-CRCs showed somatic copy-number alterations (SCNAs) similar to sporadic CRCs with one notable exception: the gain of 5p was significantly more prevalent in CD-CRCs. CD-CRCs had a distinct mutation signature: TP53 (76% in CD-CRCs vs. 33% in sporadic mucinous CRCs), KRAS (24% vs. 50%), APC (17% vs. 75%), and SMAD3 (3% vs. 29%). TP53 mutations and SCNAs were early and frequent events in CD progression, while APC, KRAS, and SMAD2/4 mutations occurred later. In four patients with CD-CRC, at least one mutation and/or SCNAs were already present in non-dysplastic colonic mucosa, indicating occult tumor evolution.Conclusions: Molecular profiling of CD-CRCs and precursor lesions revealed an inflammation-associated landscape of genome alterations: 5p gains and TP53 mutations occurred early in tumor development. Detection of these aberrations in precursor lesions may help predicting disease progression and distinguishes CD-associated from sporadic colorectal neoplasia. Clin Cancer Res; 24(20); 4997-5011. ©2018 AACR.

A Case of Incidentally Found CD3+ Lymphocytic Infiltration at Ileocecal Valve During Colonoscopy - Why Should This Be Concerning?

We present an unusual finding of CD 3+ granulomatous patch located within the Ileocecal valve (IC), in an asymptomatic patient. Intestinal intraepithelial lymphocytes (IELs) belong to a unique T-cell population interspersed between epithelial cells of both the small and large intestine. Increased IEL in bowel is associated with lymphocytic colitis, celiac disease, enteropathy associated T cell lymphoma (EATL) or Indolent T cell lymphoproliferative disease. Guidelines for endoscopic investigation of the small intestine are well defined for diagnosis of celiac disease, however there is no indication for colonic investigation, which could leave IEL's, EATL's or isolated celiac disease undetected within the colon. A 47 year old African male with history of hypertension, schizophrenia, was seen in clinic and scheduled a screening colonoscopy. He denied nausea, vomiting, abdominal pain, loss of appetite, unintentional weight loss, hematemesis, melena or hematochezia and reported regular bowel movements. He had no family history of GI malignancy. His physical exam was unremarkable. Colonoscopy revealed areas of erythema at the ileocecal valve. Pathology from biopsies showed polypoid fragments of non-dysplastic colonic mucosa with dilated ectatic lymphatics and mucosal edema. Additionally, mucosal lymphoid aggregates with a mild increase in intraepithelial CD3-positive T-cells and CD 3+ granulomatous patch within the Ileocecal valve (IC) were present. Further studies revealed; TTG 11 (0-5U/mL), Gliadin IGA 47 (>30 strong positive), Endomysial antibody IGA negative, B12 levels- 284 (low normal) and HIV negative. An upper endoscopy performed showed normal gastric and duodenal mucosa, with duodenal biopsies showing normal intraepithelial lymphocytes without villous atrophy. Oncology was consulted for increased GI mucosal CD-3 cells, likely non-specific but concerning for cryptic/ intermediate enteropathy-type intestinal T cell lymphoma(EITCL) with impression that the biopsy revealed solely incidentally reactive T cell infiltrates. In our patient, the CD3-positive T-cells and CD 3+ granulomatous patch at Ileocecal valve (IC) with positive Celiac serological studies established the diagnosis of subclinical celiac. This is of potential importance for four reasons: the danger of malignancy, the presence of unsuspected nutritional deficiencies, the association with low-birth weight infants in affected mothers, and the occurrence of autoimmune disorders.

Evolutionary history of human colitis-associated colorectal cancer

ObjectiveIBD confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of...

Abstract 5368: The evolutionary history of human colitis-associated colorectal cancer

Abstract Introduction: Inflammatory bowel disease (IBD) confers an increased lifetime risk of developing colorectal cancer (CRC). Our study aimed to compare the molecular and genetic features of colitis-associated CRC (CA-CRC) to the more common sporadic CRC (S-CRC), and to dissect the evolutionary history of CA-CRC using multi-region next generation sequencing. Methods: Fresh frozen colectomy specimens were collected from 12 patients with CA-CRC. For each case, whole exome sequencing was performed on multiple regions of tumor, adjacent normal mucosa and blood. Variants in key genes were validated by Sanger sequencing and BaseScope in situ hybridization, and copy numbers were validated by FISH. Copy number profiling (by SNP array or low-pass whole genome sequencing) was performed on low grade dysplasia (LGD; n=28), high grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=15). Phylogenetic trees were reconstructed for each case, and evolutionary analysis was used to reveal the temporal sequence of events leading to CA-CRC. Results: The majority of sequenced tumors (10/12) were microsatellite stable, and these had a median mutation rate of 3.0 single nucleotide alterations (SNAs) per Mb, ~20% higher than that of S-CRC. Mutational signatures identified ‘accelerated ageing' in the colitic bowel, likely a consequence of repeated inflammation and regeneration cycles. There was considerable mutational burden in non-dysplastic IBD mucosa (median 47 SNAs), with a median of 24% of these SNAs also clonal within the neighboring CA-CRC, indicating a mutational field. In CA-CRC the most commonly mutated gene was TP53, occurring more frequently than in S-CRC (80% vs. 58%), whereas APC mutations were significantly less common (40% vs. 75%, p=0.03). We analyzed the genetic heterogeneity of CA-CRCs and found that the number of clonal SNAs per tumor was not significantly different to S-CRCs, however CA-CRCs had significantly more SNAs that were unique to one region (p=0.04); this increase in diversity is likely due to an elevated mutation rate. CA- CRCs were often near-triploid (42%) or near-tetraploid (21%), with many other recurrent copy-number alterations that were distinct from those observed in S-CRC. Phylogenetic analysis revealed that copy number alterations (CNAs) accrue in non-dysplastic bowel, but the transition from LGD to HGD involves a punctuated ‘catastrophic' increase in CNA burden. Conclusions: Multi-region sequencing of CA-CRC has revealed a distinct pathway of colon carcinogenesis, with an increase in mutational burden and heterogeneity compared to S-CRC. Copy-number profiling indicated extensive genomic alterations, with dramatic accrual at the LGD to HGD transition. The significant mutational burden in surrounding normal mucosa indicates field cancerization, which is an encouraging prospect for screening programs; however the likelihood of punctuated evolution may offer a limited window for early detection. Citation Format: Ann-Marie Baker, William Cross, Kathleen Curtius, Chang-ho Ryan Choi, Ibrahim Al-Bakir, Daniel Temko, Pierre Martinez, Marc Williams, Hayley Davis, Sujata Biswas, Nicholas A. Wright, Morgan Moorghen, Stephen J. Hayes, Manuel Rodriguez-Justo, Andrew Silver, Lai Mun Wang, Marnix Jansen, Ailsa L. Hart, Simon J. Leedham, Trevor A. Graham. The evolutionary history of human colitis-associated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5368.

Abstract 3600: Early detection of esophageal adenocarcinoma using near-infrared lectin-based imaging

Abstract Background: Long procedure times and sampling error frequently limit the efficacy of endoscopic surveillance for Barrett's esophagus (BE). Near-infrared (NIR) fluorescence imaging minimizes optical scattering and tissue autofluorescence. We assessed, in an ex vivo setting, the feasibility of using a topically applied NIR dye-labeled lectin for the detection of early neoplasia in BE. Methods: We recruited consecutive patients undergoing endoscopic mucosal resection (EMR) for BE-related early neoplasia. EMR specimens were freshly collected and sprayed at the bedside with a fluorescent lectin and then imaged ex vivo. A maximum of two punch biopsies were collected from each EMR under NIR light guidance. Fluorescence intensity from dysplastic and nondysplastic areas within EMRs were compared and from punch biopsies with different histological grades. Results: We analyzed 29 EMR specimens from 17 patients. Dysplastic regions showed significantly lower fluorescence across whole EMR specimens (P &amp;lt; 0.001). A 41% reduction in fluorescence was found for dysplastic compared to nondysplastic punch biopsies (P &amp;lt; 0.001), with a sensitivity and specificity for dysplasia detection of 80% and 82.9%, respectively. Conclusion: NIR imaging using fluorescently-labeled lectins can distinguish dysplastic from nondysplastic Barrett's mucosa ex vivo. We are currently planning a first-in-human study to validate this imaging platform. Citation Format: Andre A. Neves, Massimiliano Di Pietro, Maria O'Donovan, Dale J. Waterhouse, Sarah E. Bohndiek, Kevin M. Brindle, Rebecca C. Fitzgerald. Early detection of esophageal adenocarcinoma using near-infrared lectin-based imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3600.

P058 Phosphodiesterase 8A domains at the site of leucocyte–endothelial cell adhesion in ulcerative colitis submucosa

Development of anti-adhesion therapies like Etrolizumab and Alicaforsen highlights the potential for inhibiting recruitment pathways in ulcerative colitis (UC). Recent in vitro mouse work from our group shows that disruption of Phosphodiesterase (PDE) 8A domains inhibits T cell adhesion to ICAM-1 and endothelial cells.1 PDE inhibitors are known immunosuppressive agents, yet no PDE inhibitors are approved in UC due to dose-limiting side effects. As PDE8A is the most efficient in vivo hydrolyser of cAMP, PDE8A inhibitors are good candidates for overcoming the challenges presented mainly by PDE4 inhibitors. PDE8A expression in colorectal cancer (CRC) is well documented and PDE8A is reported in UC intestinal crypts by gene and protein array. However, the in situ localisation of PDE8A in UC submucosa during recruitment of leukocytes is unknown. Therefore, we developed an image analysis method to highlight PDE8A domains and investigated whether PDE8A domains were present at the site of leucocyte-endothelial cell adhesion in the submucosa of inflamed UC resections. PDE8A protein expression in five involved and two uninvolved UC colon resections was determined by immunohistochemistry using anti-PDE8A (ab61815) or IgG isotype (ab125938). Six paired biopsies from CRC (involved and uninvolved) were used as controls. Antibodies were titrated to 1:250 to reveal subcellular domains masked by diffuse staining at 1:100. Images were analysed using the saturation feature (0-255) in Image J software to assign PDE8A staining intensity as a representation of subcellular domains (Figure A). This in situ study performed in human UC tissue reveals PDE8A domains in the plasma membrane of submucosal leucocytes and endothelial cells participating in adhesion and transmigration (see Figure B). The PDE8A domains and high intensity staining were seen in both involved and uninvolved UC. In contrast, the control CRC dysplasia showed increased PDE8A staining compared with adjacent non-dysplastic mucosa. A) PDE8A domain visualisation using Image J saturation analysis B) PDE8A domains are present at the site of leukocyte-endothelial cell adhesion (black arrows). Images from involved UC resections of one (A) and three (B) patients. The in situ localisation of PDE8A in UC is consistent with its role in regulating inflammation and adhesion. Together with a favourable therapeutic index, the presence of PDE8A domains at the plasma membrane in involved and uninvolved UC suggests PDE8A is a promising target for anti-adhesion therapy in UC and other immune mediated diseases, perhaps in early disease. Future studies are planned to determine the ability of a PDE8A disruptor to inhibit adhesion molecules in whole blood cultures and intestinal explants. 1. Basole CP, Nguyen RK, Lamothe K, et al. PDE8 controls CD4+ T cell motility through the PDE8A-Raf-1 kinase signaling complex. 2017. http://www.sciencedirect.com/science/article/pii/S0898656817302231

MiR-4728-3p Functions as a Tumor Suppressor in Ulcerative Colitis-associated Colorectal Neoplasia Through Regulation of Focal Adhesion Signaling.

As mechanisms of neoplasia in patients with ulcerative colitis (UC) remain poorly understood, we sought to identify pathways of carcinogenesis in this high-risk population. MicroRNA (miRNA) and mRNA expression was examined in nondysplastic rectosigmoid mucosa from UC patients with (n = 19) or without remote colon neoplasia (n = 23). We developed a method to identify miRNA-regulated pathways based on differentially expressed miRNAs and their putative mRNAs targets in the same samples. One key pathway identified in the analysis, miR-4728-3p regulation of focal adhesion signaling was further evaluated in vitro and through examination of expression in UC-cancers. There were 101 significantly up-regulated and 98 down-regulated miRNAs (adjusted P < 0.05) in the rectal mucosa of UC patients harboring proximal neoplasia. Bioinformatic analysis identified miR-4728-3p as a regulator of 3 proteins involved in focal adhesion signaling, CAV1, THBS2, and COL1A2. Real-time PCR validated down-regulation of miR-4728-3p in nondysplastic tissue remote from UC-neoplasia and in UC-associated colon cancers. miR-4728-3p transfection into colon cancer cells down-regulated expression levels and decreased luciferase activities in cells expressing a wild type 3' untranslated region compared with a mutant 3' untranslated region for all 3 genes. Exogenous transfected miR-4728-3p also delayed wound healing and decreased formation of focal adhesion complexes. Patients with long-standing UC who harbor neoplasia can be identified based on miRNA and mRNA profiles in nondysplastic tissue. Using a method to analyze miRNA and mRNA expression from the same tissues, we identified that miR-4728-3p is likely an important tumor suppressor in UC-associated colon carcinogenesis.

Abstract 1437: Differential role of mutations in clonal evolution in esophageal mucosa at risk for cancer

Abstract Background Esophageal squamous cell carcinoma (ESCC) develops in close association with a chronic exposure to heavy drinking and smoking, frequently in a metachronous manner, suggesting a strong field effect on ESCC carcinogenesis. However, it is poorly understood how the field effect promotes clonal evolution in esophageal mucosa to induce dysplasia, eventually leading to the development of ESCC. Patients &amp; Methods A total of 221 samples were obtained by multiple sampling from cancer tissues (n = 53) as well as dysplastic (n = 12) and non-dysplastic (n = 156) mucosae from 49 cancer-bearing patients and 2 high-risk and 10 healthy patients either by endoscopy or surgery with different sampling sizes (regular size: 4 - 8 mm2 and small size: 0.2 or 0.8 mm2). All the samples were subjected to whole exome (WES) (n = 99) and/or targeted-capture (TS) (n = 221) sequencing. Copy number alterations (CNAs) were also investigated using SNP array- or sequencing-based karyotyping. Results WES and TS in regular size samples revealed clonal proliferation in most of the non-dysplastic samples (98%), although the number of accompanying mutations and their clone size were significantly lower compared to those in samples from cancer and dysplasia (respectively, P = 0.021 and P &amp;lt; 0.001). Non-dysplastic samples were most frequently accompanied by NOTCH1 mutations (75%), followed by TP53 mutations (50%). By contrast, cancer and dysplasia samples showed much lower frequencies of NOTCH1 mutations (19% and 66%), but instead characterized by higher frequency of TP53 mutations (100% and 92%), and CNAs (83% and 17%), especially those focally involving CCND1, CDKN2A, PIK3CA, and EGFR, respectively. Mutation signatures also differed significantly between samples from non-dysplastic mucosa, and cancer/dysplasia tissues; cancer/dysplasia samples exhibited an enhanced APOBEC-related and decreased transcription-coupled repair-related signatures, compared to non-dysplastic samples. In non-dysplasia samples, both NOTCH1 and TP53 mutations were significantly enriched in heavy drinkers compared to non- or occasional drinkers (p &amp;lt; 0.001 and p = 0.023, respectively). To decipher the clonal architecture in more detail, we also sequenced 58 samples collected from non-dysplastic mucosae with a smaller sampling size. Clonal proliferation was confirmed in all the samples analyzed; NOTCH1 and TP53 mutations were found in 79% and 55% of the samples, where 78% and 27% of the mutated area showed biallelic alteration, respectively. Conclusions Clonal proliferation in non-cancer esophageal epithelia is quite common even in non- or occasional drinkers and could be extensive in heavy drinkers. NOTCH1 and TP53 mutations as well as CNAs play major roles in clonal evolution but may have differential impacts on esophageal carcinogenesis, where TP53 mutation and CNAs are likely to make major contributions possibly under the influence of a higher APOBEC activity. Citation Format: Akira Yokoyama, Hiromichi Suzuki, Tetsuichi Yoshizato, Yuusuke Shiozawa, Yusuke Sato, Kosuke Aoki, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Yoichi Fujii, Hideki Makishima, Kenichi Yoshida, Keisuke Kataoka, Yaichi Shiraishi, Masashi Sanada, Shigeru Tsunoda, Sachiko Minamiguchi, Satoru Miyano, Manabu Muto, Seishi Ogawa. Differential role of mutations in clonal evolution in esophageal mucosa at risk for 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 1437. doi:10.1158/1538-7445.AM2017-1437

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