Normal Mucosa Samples Research Articles

STLA101 assay for the detection of cancerous progression in Barrett’s esophagus: A multi-institutional study.

249 Background: Since 1975, Esophageal adenocarcinoma (EAC) incidence has increased more than any other cancer (̃700%). Pre-malignant metaplastic tissue, or Barrett's Esophagus (BE), is the only known precursor for EAC. Periodic histological assessment of endoscopic biopsy along dysplasia-carcinoma sequence is used to assess progression risk. Current surveillance protocols have clinical limitations as 80% of patients still present with stage II-IV EAC. The aim of this study is to compare novel proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval patients with BE/GERD were identified. Two cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (̃10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 24 normal esophageal mucosa samples with no h/o of BE were also retrieved for baseline expression levels. Tissue sections were microdissected to isolate pure BE cells from surrounding stromal cells. BE cells underwent a multi-step process where tissue was heated and digested to produce a liquid biopsy for mass spec. Isotopically labeled control peptides representing the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Triple-quad mass spectrometer. Results: A total of 95 tissues were analyzed. 29 BE biopsies progressed to EAC within a mean of 380 days while the remaining 28 BE and 14 GERD specimens did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to BE/GERD tissue that remained stable over 10 years. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusions: The differential overexpression of the STLA101 panel on progressive and non-progressive BE tissues demonstrates the clinical utility of STLA101 as a predictive biomarker panel for early detection of esophageal carcinogenesis. Non-progressive BE tissues were similar to normal mucosa, therefore, patients whose BE tissue samples resemble this profile may receive a reduced screening cadence. On the other hand, patients with overexpression of the STLA101 panel may be eligible to receive more frequent screening or therapeutic intervention.

Quantitative proteomic profiling of esophageal adenocarcinoma tumors to assess prevalence of approved targets and elucidate novel biomarkers.

343 Background: Esophageal adenocarcinoma (EAC) continues to have extremely poor prognosis despite advances in surgical and oncological regimens. Targeted and immune therapy hold promise to improve outcomes in solid tumors including EAC. At present HER-2 and anti-PD-L1 therapies are approved by the FDA for EAC. Additionally, off-label use of EGFR directed therapy has been reported. The aim of this study is to measure expression of 80+ oncoproteins in EAC using multiplexed mass spectrometry to elucidate expression of currently approved-drug targets and explore promising new targets for drug development. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC), EAC patients whose tumors were resected via esophagectomy before chemotherapy and/or radiation were identified. Normal esophageal mucosa samples with no history of EAC were also retrieved for analysis. Tissue sections were microdissected to exclude stroma from adenocarcinoma cells. Isolated EAC cells were solubilized for mass spectrometry-based quantitation of more than 80 clinically relevant tumor markers. Results: 55 EAC tissues were analyzed. HER2, PD-L1 and EGFR were overexpressed only in 16.3%, 0%, and 0% of patients, respectively. New oncoproteins #2 and #6 were noted to have high (7,634.0 and 27,222.6 attomoles/ug) and consistent (98.1% and 94.5%) overexpression in EAC samples. Expression of HER2, oncoproteins #2 and #6 were significantly higher in EAC compared to normal esophageal mucosa. There was no statistical difference in the expression of PD-L1 and EGFR between EAC and normal samples. Conclusions: Mass spectrometry revealed a very low prevalence of target oncoproteins in EAC for currently used drugs. Our study potentially reveals alternate markers which are near universally present at high levels and should be further explored for targeted treatment.[Table: see text]

IMMUNOHISTOCHEMICAL EXPRESSION OF P53 IN ORAL SQUAMOUS CELL CARCINOMA, ORAL EPITHELIAL PRECURSOR LESIONS, AND NORMAL ORAL MUCOSA

Abstract
 Objective: To assess the immunohistochemical expression of p53 in tissue samples of oral squamous cell carcinoma (OSCC), oral epithelial precursor lesions and normal oral mucosa.
 Methods: A comparative cross-sectional study was jointly conducted at the Departments of Pathology and Oral and Maxillofacial Surgery of various medical and dental institutes of the country from April 2016 to March 2017. A total of 180 subjects were included in the study. Oral tissue specimens were collected for lab investigations after obtaining written consent from all subjects. TP53 was assessed by means of immunohistochemistry in tissue samples of 60 cases of OSCC, 60 cases of epithelial precursor lesions and normal oral mucosal samples of 60 healthy individuals. Data was recorded, evaluated and analysed by SPSS version 20.
 Results: p53 protein expression was noted in 85% OSCC and 73% oral epithelial precursor lesions. Among healthy individuals ,one subject showed p53 immunoreactivity in normal oral mucosa.
 Conclusion: Raised TP53 overexpression in OSCC and oral precursor lesions, compared to normal oral mucosa make it a probable candidate for potential predictive biomarker in oral premalignancy and malignancy.

Expression of Ki67 as potential biomarker in oral submucous fibrosis: An immunohistochemical study.

Oral submucous fibrosis (OSMF) is a potentially malignant disorder (PMD) known to transform into oral cancer. One of the important hallmarks of malignant transformation is the uncontrolled growth rate, commonly reflected as increased cell proliferation which can be significantly detected by proliferative markers such as a high Ki-67 index. The aim of this study is to evaluate the degree and pattern of expression of Ki67 in OSMF, oral squamous cell carcinoma (OSCC) and in normal mucosal (NOM) patients and to correlate the Ki67 expression with clinical and histological grading of OSMF and OSCC patient. A prospective cross-sectional study was conducted over a duration of two years. An immunohistochemical study was performed for Ki76 expression on 35 cases of OSMF, 10 cases of OSCC and 10 normal mucosal patients. Data were analysed using SPSS version 21. Chi-squared test was used to analyse the differences between the intensity levels in OSMF, OSCC and NOM. Expression of Ki67 was significantly higher in OSMF than that of NOM samples but less than that of OSCC samples. Expression of Ki67 increased with increasing grade of clinical and histological stages. The study demonstrated a high incidence of Ki67 overexpression in OSMF and OSCC and showed a correlation between clinical and histological grading of OSMF and OSCC. Identification of high-risk oral PMDs and intervention at premalignant stages could constitute one of the key steps in reducing the mortality, morbidity and cost of treatment associated with malignant transformation of these diseases.

ANALYSIS OF EXPRESSION OF MIRNA AND MRNA IN THE CELLULAR MATERIAL OF THE STOMACH LINING OBTAINED BY ESOPHAGOGASTRODUODENOSCOPY IN ORDER TO DETECT DYSPLASIA AND STOMACH CANCER

miRNA and mRNA are highly specific molecular markers, with their own unique expression profile for each type of tissue. They can become a valuable tool in clinical practice in addition to routine esophagogastroduodenoscopy.Purpose. To study the prospects of using a classifier based on miRNA and mRNA profiling in cytological samples for detecting dysplasia and stomach cancer, as well as to compare the obtained data with the results of profiling of 7 miRNAs used for the analysis of both histological and cytological material.Materials and methods. The study included 221 cytological preparations: 108 samples of adenocarcinoma, 27 samples of dysplasia, 86 samples of normal mucosa. The expression level of miRNA-145-5p, -150-5p, -21,-20a, -31-5p, -34a-5p, -375, -125b, -196b, -106b and mRNA of the following genes: TERT, CDKN2A, CKS2, FN1, was determined using real-time reverse transcription polymerase chain reaction. Quantitative comparison of two independent samples for was performed using the Mann-Whitney test. The samples were stratified into different groups using the C5.0 algorithm.Results: when examining cytological samples in cases of miRNA-125b, 145, -196b, -21, -375 and TERT, СKS2, FN1 mRNA, there was achieved a high level of significance of differences in the cancer/norm group; miRNA-375 and FN1 mRNA in the cancer/dysplasia group and miRNA-145, -196b, -20a, and СKS2, TERT mRNA in the norm/dysplasia groups. When comparing the results for the 7 miRNAs that were used to analyze both histological and cytological material, several differences was noted.Conclusion. The practical use of miRNA and mRNA expression (in cytological material data mining technology does not allow differentiating dysplasia and cancer but can help in identifying "high-risk patients" who should repeat esophagogastroduodenoscopy with multifocal forceps biopsy, with mandatory molecular genetic research.

An optimized protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in oral squamous cell carcinomas

Approximately 93% of the human genome is translated into RNAs, of which only 2% code for proteins and the rest 98% are noncoding RNAs. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs of > 200 nucleotides length that are emerging as novel players in the field of cancer diagnostics or prognostics. Recently, lncRNAs are known to be associated with oral squamous cell carcinomas (OSCC). The demonstration of stable lncRNA has been a challenge in formalin-fixed paraffin-embedded tissues (FFPE). The survivability and expression level of lncRNA in FFPE tissues compared with fresh tissues is not well documented in the literature. Hence, we designed the current pilot study with the main aim to optimize modified TRI (Total RNA isolation) reagent RNA isolation protocol to identify the lncRNA expression in archived FFPE tissues of OSCC in comparison to the standard RNA isolation kit method. The findings of our study demonstrated that the RNA quantity and quality were comparatively better with the optimized TRI reagent modified protocol than the standard RNA isolation kit method. Furthermore, ct (cycle threshold) values after reverse-transcription and qRT-PCR (Quantitative Real time PCR) were comparable and almost equal in both the methods for normal mucosa (control) and OSCC samples.

The upregulation of proteins light chain 3 and autophagy-related 5 and the occurence of intestinal-type gastric cancer.

This study aims to investigate the expression levels and values of autophagy genes light chain 3 (LC3) and autophagy-related 5 (ATG5) in intestinal-type gastric cancer. Ninety samples of normal gastric mucosa, intraepithelial neoplasia, and gastric cancer tissue were used in this study. The messenger ribonucleic acid (mRNA) and protein expression levels of autophagy genes LC3 and ATG5 were detected using quantitative reverse transcription polymerase chain reaction, Western blot, and the immunohistochemistry method. The correlations of the autophagy genes and certain clinical pathological parameters were analyzed. The results showed that LC3 mRNA expression was 43.76 ± 20.31 in the normal group, 111.29 ± 18.65 in the intraepithelial neoplasia group, and 131.78 ± 26.29 in the gastric cancer group, while ATG5 mRNA expression was 4.52 ± 2.37 in the normal group, 7.09 ± 1.88 in the intraepithelial neoplasia group, and 10.25 ± 2.81 in the gastric cancer group. The differences between the groups were statistically significant (P < 0.05). The protein expression of LC3 in the normal group was 1.05 ± 0.41, 1.53 ± 0.36 in the intraepithelial neoplasia group, and 1.99 ± 0.14 in the gastric cancer group. The protein expression of ATG5 was 0.78 ± 0.24 in the normal group, 1.37 ± 0.39 in the intraepithelial neoplasia group, and 2.04 ± 0.63 in the gastric cancer group. The differences between the groups were statistically significant (P < 0.05). The positive rate of LC3 protein expression was 33.3% in the normal group and 60% in the intraepithelial neoplasia group, and the difference was statistically significant (χ2 = 4.89; P = 0.04). In the gastric cancer group, the positive rate of LC3 protein expression was 83.3%, making it significantly higher than the intraepithelial neoplasia group, with a statistically significant difference (χ2 = 4.02, P = 0.045). The positive rate of ATG5 protein expression was 23.3% in the normal group, 50.0% in the intraepithelial neoplasia group, and 76.7% in the gastric cancer group. The expression in the intraepithelial neoplasia group was much higher than in the normal group, with a statistically significant difference (χ2 = 4.59, P = 0.03), and that of the gastric cancer group was much higher than that of the intraepithelial neoplasia group, with a statistically significant difference (χ2 = 4.59, P = 0.03). LC3 protein expression was significantly correlated with depth of infiltration, and lymph node status. ATG5 protein expression was significantly correlated with age, depth of infiltration, and lymph node status. There was also a correlation between the LC3 and ATG5 proteins (correlation coefficient r = 0.72, P = 0.001). The enhanced autophagy activity of LC3 and ATG5 may participate in the occurrence and development of intestinal gastric cancer, and they may play a synergistic role in promoting the occurrence and development of intestinal gastric cancer. These findings provide clinical value for the diagnosis of intestinal gastric cancer.

Epithelial and fibroblast SPARC expression patterns in oral leukoplakia and oral squamous cell carcinoma

This study evaluated and compared the expression of secreted protein acidic and rich in cysteine (SPARC) in epithelial cells and fibroblasts of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) using normal oral mucosa as a control. The expression of SPARC was determined in samples of normal oral mucosa (n=12), OL without dysplasia (n=31), OL with dysplasia (n=54), and OSCC (n=69) using immunohistochemistry. The percentage of positive cells in epithelial cells and fibroblasts was independently evaluated. Epithelial SPARC was found in 33.3%, 35.5%, 25.9%, and 66.7% of normal oral mucosa, OL without dysplasia, OL with dysplasia, and OSCC, respectively. Fibroblast SPARC was found in 50.0%, 29.0%, 46.3%, and 84.1% of normal oral mucosa, OL without dysplasia, OL with dysplasia, and OSCC, respectively. OSCC had higher epithelial and fibroblast SPARC expression than normal oral mucosa, OL without dysplasia, and OL with dysplasia (P < .05). No significant differences were observed in epithelial and fibroblast SPARC among normal oral mucosa or OL with and without dysplasia. Overexpression of epithelial and fibroblast SPARC was observed in OSCC but not in OL, suggesting that SPARC is involved in the late stage of oral carcinogenesis.

Identification and verification of key cancer genes associated with prognosis of colorectal cancer based on bioinformatics analysis.

The biomarkers targeting colorectal cancer (CRC) prognosis are short of high accuracy and sensitivity in clinic. Through bioinformatics analysis, we aim to identify and confirm a series of key genes referred to the diagnosis and prognosis of CRC. GSE31905, GSE35279, and GSE41657 were selected as complete RNA sequencing data sets of CRC and colorectal mucosa (CRM) tissues from the NCBI-GEO database, and the differentially expressed genes (DEGs) were analyzed. The common DEGs in these 3 data sets were obtained by Venn map, and enriched by STRING network system and Cytoscape software. The Kaplan-Meier plotter website was used to verify the correlation between the enriched genes and the prognosis of CRC. For the whole RNA sequencing data sets of CRC and normal intestinal mucosa samples, the DEGs of CRC and CRM in the 3 data sets (|log2FC|>2 and P<0.05) were screened by GEO2R tool in NCBI-GEO database. By using Venn graph analysis software, the intersection of up-regulated/down-regulated genes in 3 GSE datasets was obtained, and a total 105 up-regulated genes and 140 down-regulated genes were found in the 3 samples. The up-regulated/down-regulated genes were introduced into the STRING network system to obtain the interacting genes. The interacting gene sets were introduced into Cytoscape software, and 61 up-regulated genes were found by Molecular Complex Detection (MCODE) plug-in. Through the Kaplan-Meier plotter website, we found that EPHB2, KLK8, DIAPH3, STC2, OXTR, MMP7, MET, KRT85, KRT6B, KRT23, and KLK10 genes were highly expressed in CRC, and were related to the prognosis. The above 11 genes verified by bioinformatics retrieval and analysis can predict the poor prognosis of CRC to a certain extent, and they provide a possible target for the diagnosis and treatment of CRC.

Characterization of immune cell infiltrate in tumor stroma and epithelial compartments in oral squamous cell carcinomas of Sudanese patients.

BackgroundTumor immune infiltrate has been explored in oral squamous cell carcinoma (OSCC), but studies on simultaneous characterization of multiple immune cell subtypes separately in stromal and intraepithelial tumor compartments are limited.ObjectivesWe aimed to investigate the immune cell infiltrate in OSCC by using immunohistochemistry (IHC) for a panel of inflammatory cells in stromal and epithelial tumor compartments for a better characterization of the tumors.MethodsThirty‐six OSCC lesions and nine normal oral mucosa (NOM) samples from patients attending Khartoum Dental Teaching Hospital, Sudan were investigated for presence of tumor infiltrating lymphocytes, tumor‐associated macrophages, tumor‐associated neutrophils, and PD‐L1 positive cells in the inflammatory infiltrate by single and double IHC. Digital quantitative analysis (Aperio Technologies Inc.) was performed separately for stromal and epithelial compartments.ResultsOSCC cases displayed a higher inflammatory infiltrate in the associated stroma, but not in the epithelial compartment when compared to NOM. The immunosuppressive type of inflammatory infiltrate, that is, T regulatory cells (FoxP3+ cells) was identified to be significantly higher in the epithelial compartment of tumors with advanced clinical state. An immunoscore developed by combining intraepithelial FoxP3+ and CD4+ cells was found significantly higher in lesions from elderly patients, localized at toombak dipping‐related sites, poorly differentiated OSCCs, or with loco‐regional lymph node spreading.ConclusionsDespite heavy immune cell infiltration in tumor‐associated stroma, the majority of OSCCs in this cohort displayed a low intraepithelial immune infiltration. An immunoscore based on combined CD4 and FoxP3 intraepithelial expression may serve as an indicator of advanced tumor progression and should be further investigated for its use as potential prognostic biomarker in OSCC.

S431 Proteomic Assay for Barrett’s Esophagus Progression: A Multi-Institutional Retrospective Study

Introduction: Incidence of Esophageal adenocarcinoma (EAC) in the US has increased at a greater rate than any other cancer. EAC arises from pre-malignant metaplastic tissue - Barrett’s Esophagus (BE). Periodic histological assessment of an endoscopic biopsy is used to assess progression along dysplasia-carcinoma sequence. Current surveillance protocols have clinical limitations as 80% of EAC patients present in advanced cancer stages. The aim of this study is to compare 8 proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC) patients with BE were identified. 2 cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (who had 10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 11 normal esophageal mucosa samples were also retrieved for analysis. Serial tissue sections were microdissected to reduce proteomic noise from surrounding stromal cells. Isolated cells underwent a multi-step process where cross-links were reversed, and proteins were digested and reduced into a mass spec-compatible lysate. Isotopically labeled control peptides of the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Q-Exactive HFX mass spectrometer in a CAP/CLIA setting. Results: A total of 42 tissues were analyzed. Cohort tissue descriptions can be found in Table 1. 13 BEs progressed to EAC within a mean of 334 days while the remaining 18 BEs did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level when comparing dysplastic BE tissue that progressed to cancer to BE tissue that remained stable over 10 yrs and normal esophageal mucosa. There was not a significant difference in age, sex and length of BE between the groups. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusion: The overexpression of the STLA101 panel in dysplastic BE tissue that progressed to cancer demonstrates the potential predictability of the assay when assessing molecular hallmarks of carcinogenesis. The expression quantities of the non-progressive BE tissues were closer to normal mucosa and may warrant patients with this profile type to be screened less. Patients with high expression quantities of the STLA101 panel may be candidates for a more frequent screening cadence or therapeutic intervention.Figure 1.: Mass spectrometry-based expression patterns of 2 markers in the STLA101 panel demonstrating clear overexpression of proteins involved in the malignant transformation of Barrett’s precancerous tissue into esophageal adenocarcinoma. The expression mean of non-diseased esophageal squamous mucosa, labeled as “normal”, are represented by blue bars. The green bars represent non-progressive BE tissue that remained stable for over 10-years according to collaborating institutional databases. The red bars represent progressive BE tissue retrieved at a mean of 334 days before adenocarcinoma diagnosis.Table 1.: Specimen types analyzed in this study

Expression of Interleukin-1ß and Interleukin-8 in Oral Potentially Malignant Disorders and Carcinomas.

Objective: To evaluate interleukin-1ß (IL-1ß) and interleukin-8 (IL-8) epithelial expressions in potentially malignant disorders of the oral mucosa as malignant predictive markers.Study design: About 55 tissues embedded in paraffin, comprising 15 oral lichen planus (OLP) lesions, 15 leukoplakias, 15 oral squamous cell carcinomas (OSCC), and 10 samples of normal oral mucosa were included in the study. IL-1ß and 8 expressions were assessed by immunohistochemistry using antibodies antihuman IL-1ß human (sc-7884, Santa Cruz® H-153) and antihuman IL-8 (ab7747, abcam®). The number of positive cells was compared using Student's t-test. Any p-value < 0.05 was considered statistically significant.Results: Nuclear and cytoplasmatic keratinocyte staining were positive for both cytokines in all study groups. However, a statistically significant decrease was observed within all cases compared to normal mucosa, both staining for IL-1β and 8. Moreover, IL-8 showed significant differences between OLP and leukoplakia, and when compared to OSCC.Conclusions: Oral epithelial expression of IL-1β and 8 seems to decrease when the malignant transformation of the oral mucosa increases.

Collagen deposition correlates with loss of E-cadherin and increased p63 expression in dysplastic conditions of oral submucous fibrosis

This paper focuses on the status of epithelial markers, E-cadherin, and p63 in the backdrop of an abnormal amount of collagen in the sub-mucosa of dysplastic and non-dysplastic grades of OSF. Histologically confirmed OSF and normal oral mucosa samples were procured. Samples were stained by Van Gieson's stain (VG) and immunohistochemistry. The captured images were analyzed by ImageJ software to quantify their grayscale intensities. There was a gradual increase in the intensity of VG stain from normal to non-dysplastic and dysplastic OSF and the differences in their mean grayscale values were found to be significant (p < 0.00001). The intensity of E-cadherin was found to be the highest in non-dysplastic conditions and lowest in dysplastic conditions. The intensity difference of E-cadherin between normal and non-dysplastic OSF was found to be significant (p < 0.00001). The grayscale scale intensity values for p63 in whole epithelium depicted significant differences between normal and diseased conditions but for its intensity, in basal cells, significant differences were found between non-dysplastic and other classes of tissues. There was a positive correlation observed between VG and p63 staining intensity. The diseased oral epithelium demonstrated greater deposition of sub-epithelial collagen fibers along with subsequent loss of E-cadherin and an increased p63 expression.

A Diagnostic Method for Gastric Cancer Using Two-Photon Microscopy With Enzyme-Selective Fluorescent Probes: A Pilot Study.

BackgroundEndoscopy is the most important tool for gastric cancer diagnosis. However, it relies on naked-eye evaluation by endoscopists, and the histopathologic confirmation is time-consuming. We aimed to visualize and measure the activity of various enzymes through two-photon microscopy (TPM) using fluorescent probes and assess its diagnostic potential in gastric cancer.Methodsβ-Galactosidase (β-gal), carboxylesterase (CES), and human NAD(P)H: quinone oxidoreductase (hNQO1) enzyme activities in the normal mucosa, ulcer, adenoma, and gastric cancer biopsy samples were measured using two-photon enzyme probes. The fluorescence emission ratio at long and short wavelengths (Ch2/Ch1) for each probe was comparatively analyzed. Approximately 8,000 – 9,000 sectional images in each group were obtained by measuring the Ch2/Ch1 ratio according to the tissue depth. Each probe was cross-validated by measuring enzymatic activity from a solution containing lysed tissue.ResultsTotal of 76 subjects were enrolled in this pilot study (normal 21, ulcer 18, adenoma 17, and cancer 20 patients, respectively). There were significant differences in the mean ratio values of β-gal (0.656 ± 0.142 vs. 1.127 ± 0.109, P < 0.001) and CES (0.876 ± 0.049 vs. 0.579 ± 0.089, P < 0.001) between the normal and cancer, respectively. The mean ratio value of cancer tissues was different compared to ulcer and adenoma (P < 0.001). The hNQO1 activity showed no significant difference between cancer and other conditions. Normal mucosa and cancer were visually and quantitatively distinguished through β-gal and CES analyses using TPM images, and enzymatic activity according to depth, was determined using sectional TPM ratiometric images. The results obtained from lysis buffer-treated tissue were consistent with TPM results.ConclusionsTPM imaging using ratiometric fluorescent probes enabled the discrimination of gastric cancer from normal, ulcer, and adenoma. This novel method can help in a visual differentiation and provide quantitative depth profiling in gastric cancer diagnosis.

Lipidomic Profiling of Colorectal Lesions for Real-Time Tissue Recognition and Risk-Stratification Using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) is a metabolomic technique analyzing tissue metabolites, which can be applied intraoperatively in real-time. The objective of this study was to profile the lipid composition of colorectal tissues using REIMS, assessing its accuracy for real-time tissue recognition and risk-stratification. Metabolic dysregulation is a hallmark feature of carcinogenesis; however, it remains unknown if this can be leveraged for real-time clinical applications in colorectal disease. Patients undergoing colorectal resection were included, with carcinoma, adenoma and paired-normal mucosa sampled. Ex vivo analysis with REIMS was conducted using monopolar diathermy, with the aerosol aspirated into a Xevo G2S QToF mass spectrometer. Negatively charged ions over 600 to 1000 m/z were used for univariate and multivariate functions including linear discriminant analysis. A total of 161 patients were included, generating 1013 spectra. Unique lipidomic profiles exist for each tissue type, with REIMS differentiating samples of carcinoma, adenoma, and normal mucosa with 93.1% accuracy and 96.1% negative predictive value for carcinoma. Neoplasia (carcinoma or adenoma) could be predicted with 96.0% accuracy and 91.8% negative predictive value. Adenomas can be risk-stratified by grade of dysplasia with 93.5% accuracy, but not histological subtype. The structure of 61 lipid metabolites was identified, revealing that during colorectal carcinogenesis there is progressive increase in relative abundance of phosphatidylglycerols, sphingomyelins, and mono-unsaturated fatty acid-containing phospholipids. The colorectal lipidome can be sampled by REIMS and leveraged for accurate real-time tissue recognition, in addition to riskstratification of colorectal adenomas. Unique lipidomic features associated with carcinogenesis are described.

Disentangling tumorigenesis-associated DNA methylation changes in colorectal tissues from those associated with ageing

ABSTRACT Physiological ageing and tumorigenesis are both associated with epigenomic alterations in human tissue cells, the most extensively investigated of which entails de novo cytosine methylation (i.e., hypermethylation) within the CpG dinucleotides of CpG islands. Genomic regions that become hypermethylated during tumorigenesis are generally believed to overlap regions that acquire methylation in normal tissues as an effect of ageing. To define the extension of this overlap, we analysed the DNA methylomes of 48 large-bowel tissue samples taken from women of different ages during screening colonoscopy: 18 paired samples of normal and lesional tissues from donors harbouring a precancerous lesion and 12 samples of normal mucosa from tumour-free donors. Each sample was subjected to targeted, genome-wide bisulphite sequencing of ~2.5% of the genome, including all CpG islands. In terms of both its magnitude and extension along the chromatin, tumour-associated DNA hypermethylation in these regions was much more conspicuous than that observed in the normal mucosal samples from older (vs. younger) tumour-free donors. 83% of the ageing-associated hypermethylated regions (n = 2501) coincided with hypermethylated regions observed in tumour samples. However, 86% of the regions displaying hypermethylation in precancerous lesions (n = 16,772) showed no methylation changes in the ageing normal mucosa. The tumour-specificity of this latter hypermethylation was validated using published sets of data on DNA methylation in normal and neoplastic colon tissues. This extensive set of genomic regions displaying tumour-specific hypermethylation represents a rich vein of putative biomarkers for the early, non-invasive detection of colorectal tumours in women of all ages.

Genomic Analysis Reveals Heterogeneity Between Lesions in Synchronous Primary Right-Sided and Left-Sided Colon Cancer.

Background: The synchronous primary right-sided and left-sided colon cancer (sRL-CC) is a peculiar subtype of colorectal cancer. However, the genomic landscape of sRL-CC remains elusive.Methods: Twenty-eight paired tumor samples and their corresponding normal mucosa samples from 14 patients were collected from the Second Affiliated Hospital of Harbin Medical University from 2011 to 2018. The clinical–pathological data were obtained, and whole-exome sequencing was performed based on formalin-fixed and paraffin-embedded samples of these patients, and then, comprehensive bioinformatic analyses were conducted.Results: Both the lesions of sRL-CC presented dissimilar histological grade and differentiation. Based on sequencing data, few overlapping SNV signatures, onco-driver gene mutations, and SMGs were identified. Moreover, the paired lesions harbored a different distribution of copy number variants (CNVs) and loss of heterozygosity. The clonal architecture analysis demonstrated the polyclonal origin of sRL-CC and inter-cancerous heterogeneity between two lesions.Conclusion: Our work provides evidence that lesions of sRL-CC share few overlapping mutational signatures and CNVs, and may originate from different clones.

Expression of Orai1 and STIM1 in human oral squamous cell carcinogenesis

Background/purposeReturn of Ca2+ to endoplasmic reticulum is mediated by Orai/STIM-mediated store-operated Ca2+ entry (SOCE) channel. We aimed to investigate Orai1 and STIM1 expressions in human oral carcinogenesis. Materials and methodsSixty-six oral squamous cell carcinomas (OSCCs), 14 oral potentially malignant disorders (OPMD) with moderate-severe oral epithelial dysplasia (OED), 19 OPMD with mild OED, and 14 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot and real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated for proliferation, migration, and invasion assays. ResultsOrai1 and STIM1 protein and mRNA expressions in OSCC were significantly enhanced as compared with normal samples. Protein expressions of Orai1 and STIM1 in OCCLs were significantly enhanced as compared with HOK. Significant decreases in degrees of proliferation, migration and invasion were noted in OCCLs with Orai1 and STIM1 siRNA transfection as compared with those without transfection. Significantly increased Orai1 and STIM1 protein levels were noted in OPMD with moderate-severe OED as compared with those with mild OED. ConclusionOur results indicate that Orai1 and STIM1 overexpression is associated with human oral carcinogenesis.

Yes-associated protein expression is associated with poor prognosis in patients with colorectal cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. The aim of the present study was to investigate the expression of yes-associated protein (YAP) in CRC tissues, and to determine the relationship between the expression levels of YAP and the clinicopathological characteristics and prognosis of patients with CRC. Bioinformatics analysis was conducted to examine the expression of YAP and its correlation with clinicopathological characteristics and key genes, using functional enrichment analysis. Immunohistochemistry was used to detect YAP expression in 181 CRC tissue samples and 30 normal colorectal mucosa samples. Western blotting and reverse transcription-quantitative PCR were performed to detect the expression of YAP and β-catenin in CRC cells, and cellular proliferation was assessed using a Cell Counting Kit-8 assay. Finally, apoptosis was analyzed using flow cytometry. Immunohistochemical staining indicated that the positive expression rate of YAP in CRC tissues was 73.5%, which was significantly higher than that in normal colorectal mucosa samples. The expression of YAP in CRC was associated with histological differentiation, lymph node metastasis and Duke's stage. However, no significant associations were observed between YAP expression and age, sex and T stage. Downregulation of YAP promoted the proliferation and the inhibited apoptosis of CRC cells, and YAP expression was positively correlated with that of β-catenin in both CRC tissues and cells. Furthermore, YAP expression was upregulated in CRC tissues, which was correlated with tumor progression and prognosis. Therefore, YAP expression may be used as an independent predictor of poor prognosis in patients with CRC, and the underling molecular mechanism may be associated with the combined effect of Hippo and Wnt/β-catenin signaling.

Abstract 477: Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps

Abstract The purpose of this study is to evaluate the use of molecular markers for more accurately predicting the individual risk of metachronous polyps or cancer during colonoscopic surveillance. Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the United States. The risk of CRC varies among people; individuals who develop adenomas are at heightened risk of forming metachronous adenomas and interval cancer, compared to those without a personal history of adenoma or CRC. Yet, even in these high risk individuals, only 25-30% will develop subsequent adenomas. To date, few studies have evaluated the use of molecular markers for predicting the risk of metachronous lesions. We have demonstrated that aberrantly methylated mir342/EVL is present at a higher frequency in the normal colon of people with CRC compared to people with no colon cancer or polyps, suggesting it may indicate a field cancerization process. Further, we developed an ultra-sensitive quantitative Methylation-specific ddPCR (MS-ddPCR) assay to accurately determine the level of rare EVL methylation in the normal colon mucosa. To investigate the correlation of EVL methylation in normal mucosa and the risk of developing polyps during the follow-up colonoscopy, we conducted a retrospective cohort study (N=136 subjects) and EVL methylation status was determined in normal colon mucosa samples from these subjects. Among them, 71 subjects had polyps detected within 12 years of the index colonoscopy, and 11 subjects were polyp-free at all subsequent colonoscopic exams done within 8-16 years of the index colonoscopy (54 subjects did not meet the criteria for follow up, so were excluded from the following analysis). Applying the standard Inverse Probability Weighted Logistic Regression (IPWLR) model, we found that higher level of EVL methylation in the normal colon mucosa at the time of the index colonoscopy (which was when the tissue sample was collected) indicated an increased risk of having polyps detected within 12 years, compared to subjects with no polyps detected by colonoscopy done within 8-16 years (Odds Ratio (95% CI) = 4.35 (1.14, 16.60); p-value = 0.0317). Our results suggest that EVL methylation level in the normal colon mucosa can be used as a risk biomarker for metachronous adenomas. Citation Format: Ming Yu, Ting Wang, Kelly T. Carter, Kelsey Baker, Mary W. Redman, Kathy Vickers, Lynda Ann Dzubinski, Robert E. Schoen, William M. Grady. Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 477.