ECP 2023 Abstracts
S104 Virchows Archiv (2023) 483 (Suppl 1):S1–S391 13 Background & objectives: Fusobacterium nucleatum is an oral pathogen implicated in colorectal cancer (CRC) progression. Colorectal polyps are thought to initiate colorectal carcinogenesis. This study aimed to investi- gate novel targeted RNA sequencing technology TempO-Seq for detecting bacteria in formalin-fixed paraffin-embedded (FFPE) polyp tissue. Methods: TempO-Seq depends on hybridizing detector oligos (DOs) to proximal sequences. Excess DOs are enzymatically removed, the juxtapositional DOs ligated, and amplified by PCR before sequencing. DOs were designed to target F. nucleatum ’s (ATCC 10953) 16S region. DOs were quality checked (QC) on purified human and Fusobacterium RNA, before TempO-Seq analysis of a pilot study of 120 polyp samples. Results: Eight probes were designed, and five passed in silico QC. Three probes passed in assay QC. TempO-Seq analysis using Fusobac- terium DOs was performed on FFPE tissue lysates from 120 screened patients. Human DOs were added as assay controls. 8% were F. nuclea- tum + for Probe 1, 23% for Probe 2 and 9% for Probe 3. Samples were stratified for metachronous polyps (MPs) development as an outcome, and 3%, 24%, and 6% of those who developed MPs were F. nucleatum + for Probe 1, Probe 2, and Probe 3 respectively. Alternatively, 12% of patients that didn’t develop MPs were F. nucleatum + for Probe 1, 21% for Probe 2, and 12% for Probe 3. Conclusion: These results suggest that TempO-Seq is a robust technol- ogy that detects bacterial RNA in archival FFPE tissue. Three probes displayed different trends based on their specificity. Since F. nucleatum has been associated with poor prognostic factors in CRC, this technol- ogy can be investigated as a prognostic technique. Up-scaled future work will use TempO-Seq to detect F. nucleatum in FFPE polyp lysates and will use that information to predict the development of MPs and explore any associations with other clinical characteristics. Funders of this work include Medical Research Scotland BioClavis Ltd. Innovate UK PS-17-005 Immunohistochemical assessment of Ki67 and pathway analysis in pre-malignant colonic polyps suggest Ki67 as a potential prognostic maker for metachronous disease A. Ammar* *University of Glasgow, United Kingdom Background & objectives: Bowel screening program risk stratification tool uses polyps’ number and size to assess the need for surveillance colonoscopy following polypectomy. The prognostic significance of index polyps Ki67 for metachronous disease and molecular differences between normal and dysplastic epithelia were explored. Methods: Whole tissue index polyps (n=153) were stained for Ki67 using immunohistochemistry. 48.4% developed metachronous polyps (median time 36.5 months (2006-2019)) and 63% had BSG20 high risk score. QuPath software was used to quantify Ki67 in annotations of luminal/basal epithelium. Temp-O-Seq RNA sequencing for nor- mal and dysplastic epithelium (n=40) and ssGSEA/GSEA methods for pathways and leading- edge gene analyses were applied. Results: Ki67 percentages were higher in luminal (46.4%) vs basal (24.8%) epithelium (p<0.0001). High luminal Ki67 significantly associated with metachronous disease (p=0.013) and polyp histology (p=0.051) and was an independent prognostic factor for metachro- nous disease (HR=2.6, CI(1.238-5.46), p=0.012; compared to num- ber of polyps and BSG20 risk score using Cox-regression analysis). Myc targets-V1, G2M checkpoint and E2F-targets were enriched in dysplastic epithelium (NES=2.98, 2.88 and 2.7; respectively) whereas epithelial-mesenchymal transition was mostly enriched in normal epithelium (NES=2.33) and that was confirmed on single sample level (ssGSEA p<0.0001). Ki67 gene expression was higher in dysplastic than in normal epithelium (p<0.001). Myc, MAD2L1, KPNA2, CDK4 and MCM5/MCM6 genes were highly enriched in dysplastic regions. Conclusion: Ki67 is an independent prognostic marker for metachro- nous polyps and may help improve the current BSG20 criteria. Ki67 scores can be combined with BSG20 risk score and number of polyps excised at index colonoscopy to better stratify patients who are at a higher risk of developing metachronous polyps. Dysplasia-enriched pathway and leading-edge genes suggest an active biological process relating to cell cycle and replication. Genes enriched in dysplastic pol- yps require further validation on the protein level using INCISE polyp cohorts. Funding: Title: Integrated Technologies for Improved Polyp Surveil- lance Project number: 42497 Funder: Innovate UK 2022 – 2023 Title: Risk Stratification Tool for Colorectal Polyp Surveillance Project num- ber: 10054829 Funder: Innovate UK 2023 – 2025 PS-17-006 Dysplasia in sessile serrated lesions: prevalence, interobserver vari- ability and value of immunohistochemistry V. Angerilli*, M.E. Vink-Börger, G. van Lijnschoten, N.C. van Grieken, M. Fassan, R.S. van der Post, I.D. Nagtegaal *Department of Medicine, Surgical Pathology Unit, University of Padua, Italy Background & objectives: Sessile serrated lesions with dysplasia (SSLd) are the precursors of 15% of colorectal carcinomas (CRCs). We aim to estimate the prevalence of dysplasia in sessile serrated lesions (SSLs), (ii) evaluate interobserver variability, and (iii) assess additional value of immunohistochemistry (IHC). Methods: (i) We retrieved histology reports from colonoscopy patients between 2014 and 2022 from the Dutch Nationwide Pathol- ogy Database. (ii) Histological slides of SSLd from all laboratories were reviewed by the pathology panel of the Dutch CRC screening program. (iii) A cohort of 212 SSLs/SSLd of >=1cm was reviewed to assess dysplasia and expression of MLH1, beta-catenin, Myc, p16, and p53. Results: (i) Out of the 186,427 SSLs 17,456 showed dysplasia (9.4%). (ii) Twelve laboratories submitted their SSLd cases. No IHC stain was originally performed in any of the cases. The diagnosis of SSLd was confirmed by the pathology panel in 44 cases (75.9%). The remaining 14 cases were reclassified as SSL (n=8), traditional serrated adenoma (n=5), and serrated adenocarcinoma (n=1). (iii) The hematoxylin and eosin (HE) slides of SSL/SSLd cohort were subject to an IHC-blinded and IHC-unblinded revision. The IHC-blinded and IHC-unblinded revi- sions revealed the presence of dysplasia in 20/212 (9.4%) and 26/212 (12.3%) cases, respectively. The six additional SSLd had minimal deviation dysplasia and were identified by loss of MLH1. Conclusion: This study shows that considerable variability is present in the histopathological assessment of SSLd. The accurate diagnosis of subtle dysplasia can be challenging and MLH1 expression should be used when suspected on routine stains. PS-17-007 Early-onset small bowel adenocarcinomas (EO-SBAs) are more frequently associated with a predisposing condition compared to late-onset SBAs: an international multicentre study G. Arpa*, C. Guerini, M.V. Lenti, E. Quaquarini, F. Grillo, F. Antoci, E. Travaglino, M. Fassan, R.P. Graham, A. Casadei Gardini, M. Fer- rante, A. Moens, G. De Hertog, A. Di Sabatino, A. Vanoli *Department of Molecular Medicine, Unit of Anatomic Pathology, University of Pavia, Unit of Anatomic Pathology, ICS Maugeri-IRCCS SpA SB, Pavia, Italy Background & objectives: The incidence of early-onset gastrointes- tinal cancers (i.e., age at diagnosis <50 years) is increasing and most of them are supposedly sporadic. However, data on early-onset small
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