BACKGROUND:Mucinous rectal cancer is associated with a higher incidence of microsatellite instability and a poorer response to neoadjuvant chemoradiotherapy compared to other subtypes of rectal adenocarcinoma. Immune checkpoint inhibitors are an emerging family of anticancer therapeutics associated with highly variable outcomes in colorectal cancer. Although the immune landscape of mucinous rectal cancer has not been fully explored, the presence of mucin is thought to act as a barrier preventing immune-cell infiltration. OBJECTIVE:The aim of this study was to determine the immune properties of mucinous rectal cancer and investigate the degree of lymphocyte infiltration in this cohort.DESIGN: This is a retrospective cohort study that involved multiplexed immunofluorescence staining of tumor microarrays. SETTINGS:Samples originated from a single university teaching hospital. PATIENTS:Our cohort included 15 cases of mucinous and 43 cases of nonmucinous rectal cancer. MAIN OUTCOME MEASURES:Immune cells were classified and quantified. Immune-cell counts were compared between mucinous and nonmucinous cohorts. Immune marker expression within tumor epithelial tissue was evaluated to determine the degree of lymphocyte infiltration.

The importance of next generation sequencing (NGS) rises in cancer research as accessing this key technology becomes easier for researchers. The sequence data created by NGS technologies must be processed by various bioinformatics algorithms within a pipeline in order to convert raw data to meaningful information. Mapping and variant calling are the two main steps of these analysis pipelines, and many algorithms are available for these steps. Therefore, detailed benchmarking of these algorithms in different scenarios is crucial for the efficient utilization of sequencing technologies. In this study, we compared the performance of twelve pipelines (three mapping and four variant discovery algorithms) with recommended settings to capture single nucleotide variants. We observed significant discrepancy in variant calls among tested pipelines for different heterogeneity levels in real and simulated samples with overall high specificity and low sensitivity. Additional to the individual evaluation of pipelines, we also constructed and tested the performance of pipeline combinations. In these analyses, we observed that certain pipelines complement each other much better than others and display superior performance than individual pipelines. This suggests that adhering to a single pipeline is not optimal for cancer sequencing analysis and sample heterogeneity should be considered in algorithm optimization.

There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the impact of Fusobacterium prevalence on immune cell expression and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumor microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with increased CD8 + lymphocyte (p = 0.018), regulatory T-cell (p = 0.001) and M2 macrophage (p = 0.001) expression. Similarly in the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. Elevated Fusobacterium expression was associated with increased CD4+ (p = 0.031) and M1 macrophage (p = 0.006) expression, whilst M2 macrophages (p = 0.043) were under-expressed in the TCGA cohort. Increased Fusobacterium relative abundance in mucinous CRC was associated with improved clinical outcomes in our TCGA cohort despite having no association with MSI status (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance is associated with improved outcomes in mucinous CRC, possibly due its modulatory effect on the host immune response.

235 Background: Molecular subtyping in colorectal cancer provided novel insights into tumour heterogeneity and delivered new prognostic signatures. Existing methods (such as CMS-based subtyping) were generated without much consideration of differences in expression profiles between colon and rectal cancer tissues. Moreover, stage 2 and 3 rectal cancer patients often receive neo-adjuvant radio-chemotherapy which may impact on gene expression when determined from resection samples. Methods: We investigated possible differences between treatment-naive colon and rectal cancers at the molecular level. We collected mRNA expression profiles for rectal and colon cancer samples available from Gene Expression Omnibus (GEO) database (n=2139) and applied batch-effect correction using ComBat. Results: To explore whether CMS subtypes changed after neoadjuvant chemoradiation in rectal cancers, we analysed rectal tumour samples from 40 patients where both pre-operative biopsies and resections after chemoradiotherapy were available. More than half of samples shifted from CMS1-3 to CMS4 after patients received chemoradiotherapy. We therefore focused our further analysis on treatment-naive rectal cancers. We observed that CMS-based subtyping showed different prognostic potential in treatment-naïve colon vs. rectal cancers. Rectal cancers had significantly better disease-free survival (DFS) when assigned to CMS 2 or 4 compared to colon cancers. This suggested differences in disease biology that were not captured by CMS subtyping. We next aimed to identify molecular subtypes in rectal cancers, and identified three prevalent rectal-specific subtypes (RSS) by applying hierarchical clustering in 182 untreated rectal cancer samples. We used gene co-expression network analysis to define functional gene modules to identify these subtypes. Based on the gene modules’ signatures, we built a classifier to predict our new subtypes in different transcriptomics datasets (n=692). We observed that RSS1 had better disease-free survival (DFS), whereas RSS2 exhibited worse short-term DFS (less than 3 years) and RSS3 showed worse long-term DFS (3+ years). We discovered the deactivation of EGFR, MAPK and WNT pathways on RSS3 as well as high CD8+ infiltration and low CD4+ counts in these tumours. RSS2 exhibits low plasma cell and regulatory T-cell abundance and high activations of TGF-β, NF-κB, and TNF-α. We also observed high EMT, angiogenesis and inflammatory response in this subtype. Lastly, RSS1 showed high expression on MYC target and low activity on angiogenesis genes. Moreover, the immune and inflammatory responses were also lower than in the other subtypes. Conclusions: Rectal-specific subtypes are of prognostic importance and provide new insight into molecular disease pathways and potential targets.

e22508 Background: Abuja, Nigeria’s annual World Cancer Day Walk (WCDW) is a tool for promoting public awareness of cancer risk factors, preventative lifestyle strategies, and the importance of early screening as critical elements of prevention and control. The day includes physical activities (walk, race, ride, skate, cycle, marathon), as well as health education and free breast cancer, cervical, and prostate screenings. The effectiveness of the event to attract the most vulnerable Nigerian populations has not been studied. Aim: To determine the social-demographic characteristics of participants and evaluate the impact of outreach campaigns. Methods: Approximately 2,000 Nigerians attended Abuja WCDW on February 1, 2020. A similar number attended Abuja WCDW on February 5, 2022. Trained research assistants recruited participants to complete a one-sheet questionnaire that assessed basic demographic, social, and lifestyle information. Participants were given informed consent. In 2020, 237 (11%) participants, aged 18-68 years voluntarily completed survey (ClinicalTrials #NCT04248881). In 2022, 111 (6%), aged 17-74 years voluntarily completed survey (ClinicalTrials #NCT05239325). Note: In 2021, Abuja WCDW was canceled due to COVID. Results: In 2020, the mean age for participants was 28; SD 7.71. Sixty-eight percent were women. Eighty-seven percent had at least an undergraduate education. Of the 237 participants, 65% reported that they attended to obtain free cancer screening. More than 50% reported they had no health insurance. Of those insured, more had National Health Insurance Scheme (NHIS) than private insurance. The average body mass index (BMI) was between 24-28; BMI was highest among the mid-20 age group. Lifestyle data revealed more men than women were concerned with their health; those with health worries were more likely to have had cancer screening at the event. The 2022 WCDW data were being analyzed at the time of abstract submission; findings will be presented at the meeting. Analysis: Data from 2020 revealed participants are predominately associated with a lower risk of developing cancer. They have higher levels of knowledge about cancer and lifestyle/health-related behaviors beneficial for early detection and prevention. WCDW is a great avenue for cancer awareness/lifestyle prevention interventions yet there is an urgent need to evaluate efficacy of current outreach to target underserved members of Nigerian population: those with lower levels of education, unemployed, lower income, and without insurance. Conclusions: Attendees in 2020 are younger people of higher socioeconomic status with lifestyle practices that could reduce cancer risk. If our 2022 data are similar, we must elaborate better strategies to reach populations at greater risk and encourage them to attend future events to have more impactful lifestyle/prevention outcomes. Clinical trial information: NCT05239325.