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BACKGROUND Survival of patients with metastatic colorectal cancer (mCRC) varies. We aim to develop a prognostic score for mCRC after emergency surgery to guide treatment decisions. METHODS Newly diagnosed mCRC patients who presented with primary tumor-related complications and underwent emergency surgery between January 1999 and December 2013 were included. Univariate and multivariate Cox regression analyses were performed to identify covariates significantly associated with the time to death following surgery. A survival score was derived using the Cox regression equation. RESULTS The study cohort comprised 248 patients. Median patient age was 66 ± 13 years. Primary tumor was located in the left colon and rectum in 211 patients (85.1%) while 37 patients (14.9%) had primaries in the right colon. Liver, lung, and peritoneal metastases occurred in 161 patients (64.9%), 59 patients (23.8%), and 96 patients (38.7%), respectively. Majority of patients presented with either obstruction (174 patients, 70.1%) or perforation (52 patients, 21%). On multivariate analysis, age of 60 years or older (p = 0.007), carcinoembryonic antigen levels greater than 45 ng/ml (p = 0.022), presence of liver metastases (p = 0.024), and peritoneal carcinomatosis (p

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-β, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

Worldwide, colorectal cancer (CRC) is the third highest incidence cancer and a leading cause of cancer mortality. Metastasis to distal organ is the major cause of cancer mortality. However, chemotherapeutic treatment of metastatic CRC has a dismayed success rate of less than 30%. Patients who are resistant to chemotherapy and who are KRAS wildtype are sometimes offered anti-EGFR as a second line therapy. Nevertheless, only about ½ of these patients respond and those who do inevitably develop resistance within months due to selection for downstream KRAS mutations. Moreover, most (80%) sporadic CRCs are microsatellite-stable and are refractory to immune checkpoint blockade therapy. Hence, there is an unmet need to identify novel therapeutics for CRC. KRAS is a gatekeeper gene in colorectal tumorigenesis but is ‘undruggable’ due to its structure not being easily amenable to inhibitor docking. Focus has been diverted to develop small molecule inhibitors for its downstream effectors such as ERK/MAPK and AKT. Despite intense research efforts for the past few decades, however, no small molecule inhibitor has been in clinical use for CRC. Recently developed Sotorasib inhibitor specific for KRAS G12C mutations (rare in CRC) is not effective for CRC. Antibodies have high affinity to their target proteins without having to bind to specific pocket and are fully human in nature and hence less toxic. Antibody targeting KRAS itself is thus an attractive alternative. We developed a transient ex vivo patient-derived matched mucosa-tumor primary culture to assess whether anti-KRAS antibody can be internalized to bind and inactivate KRAS. We showed that anti-KRAS antibody can enter live matched mucosa-tumor cells and specifically aggregate KRAS in the cytoplasm, thus hindering its trafficking to the inner plasma membrane. The mis-localization of KRAS reduces KRAS dwelling time at the site where it tethers to activate downstream effectors. We previously showed that expression of SOX9 was KRAS-mutation-dependent and possibly a better effector than ERK in CRC. In the present study, we showed that anti-KRAS antibody treated tumor cells have less intense SOX9 cytoplasmic and nuclear staining compared to untreated cells suggesting down-regulation of KRAS signaling. Our results demonstrated that anti-KRAS antibody can be internalized and specifically inhibits KRAS function in live tumor cells. With an efficient intracellular antibody delivery system, this can be further developed as combinatorial therapeutics for CRC and other KRAS-driven cancers. Citation Format: Peh Yean Cheah, Kuen Kuen Lam, Yee Syuen Low, Michelle Lo, Michelle Wong, Choong Leong Tang, Emile Tan, Aik Yong Chok, Issac Seow En, Siew Heng Wong. Anti-KRAS antibody aggregates KRAS in the cytoplasm of live ex vivo cultured human colorectal adenocarcinoma cells and prevents its trafficking to the plasma membrane. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4119.

INTRODUCTION Successful stenting of an obstructing colorectal tumor can avert upfront emergency surgery in malnourished obstructed patients with metastatic disease and poor physiological condition. This study aims to evaluate the outcomes of stenting followed by primary colorectal tumor resection among patients with obstructed stage IV colorectal cancer at presentation, over a 10-year period. METHODS From 2007 to 2016, a cohort comprising 25 consecutive patients were retrospectively reviewed from a prospectively collected database. The durability of palliation of bowel obstruction, oncological outcomes and factors influencing overall survival were analyzed. RESULTS No re-interventions were required for bowel obstruction during the study period. The overall perioperative morbidity rate was 16%, with no postoperative 90-day mortality. Laparoscopic resection rate was 52% and stoma formation rate was 8%. The median overall survival was 24 months for the entire cohort, and the 1-, 3- and 5-year survival rates were 80%, 35% and 23.33% respectively. More than one site of distant metastases, peritoneal involvement, and elevated carcinoembryonic antigen levels were significantly associated with poorer survival outcomes. Patients with peritoneal-only metastasis had worse outcomes, with a median survival of 7 months and no patients surviving beyond 18 months. CONCLUSION Stenting followed by resection of the primary obstructing colorectal cancer provides durable palliation among patients with stage IV disease, with low perioperative morbidity and stoma formation rates. Superior survival was observed among patients with single-site, non-peritoneal distant metastases.

BACKGROUND With advanced age and chronic illness, the life expectancy of a patient with colorectal cancer (CRC) becomes less dependent on the malignant disease and more on their pre-morbid condition. Justifying major surgery for these elderly patients can be challenging. An accurate tool demonstrating post-operative survival probability would be useful for surgeons and their patients. AIM To integrate clinically significant prognostic factors relevant to elective colorectal surgery in the elderly into a validated pre-operative scoring system. METHODS In this retrospective cohort study, patients aged 70 and above who underwent surgery for CRC at Singapore General Hospital between 1 January 2005 and 31 December 2012 were identified from a prospectively maintained database. Patients with evidence of metastatic disease, and those who underwent emergency surgery or had surgery for benign colorectal conditions were excluded from the analysis. The primary outcome was overall 3-year overall survival (OS) following surgery. A multivariate model predicting survival was derived and validated against an equivalent external surgical cohort from Kyungpook National University Chilgok Hospital, South Korea. Statistical analyses were performed using Stata/MP Version 15.1. RESULTS A total of 1267 patients were identified for analysis. The median post-operative length of stay was 8 [interquartile range (IQR) 6-12] d and median follow-up duration was 47 (IQR 19-75) mo. Median OS was 78 (IQR 65-85) mo. Following multivariate analysis, the factors significant for predicting overall mortality were serum albumin < 35 g/dL, serum carcinoembryonic antigen ≥ 20 µg/L, T stage 3 or 4, moderate tumor cell differentiation or worse, mucinous histology, rectal tumors, and pre-existing chronic obstructive lung disease. Advanced age alone was not found to be significant. The Korean cohort consisted of 910 patients. The Singapore cohort exhibited a poorer OS, likely due to a higher proportion of advanced cancers. Despite the clinicopathologic differences, there was successful validation of the model following recalibration. An interactive online calculator was designed to facilitate post-operative survival prediction, available at http://bit.ly/sgh_crc. The main limitation of the study was selection bias, as patients who had undergone surgery would have tended to be physiologically fitter. CONCLUSION This novel scoring system generates an individualized survival probability following colorectal resection and can assist in the decision-making process. Validation with an external population strengthens the generalizability of this model.

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-β, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

Colorectal cancer (CRC) is the third highest incident cancer worldwide and a major cause of cancer death. KRAS protein gain-of-function point mutations play major roles in colorectal tumorigenesis. However, direct inhibition of KRAS is challenging as its protein structure is inadmissible to inhibitor docking. Efforts have been diverted to inhibit downstream RAF/MEK/ERK and PI3K/Akt signaling. Nevertheless, none of these inhibitors has progressed to clinical use in CRC despite extensive trials over the past few decades. We examined levels of phospho-ERK1/2(T202/Y204) and phospho-Akt1/2/3(S473) in human colorectal tumor and matched mucosa at least 10 cm away with quantitative near infrared western blot and immunofluorescence imaging. Surprisingly, 75.5% (25/33) of tumors has lower or equivalent phospho-ERK1/2 and 96.9% (31/32) of tumors has lower phospho-AKT1/2/3 compared to matched mucosa, irrespective of KRAS mutation status and tumor stage. In contrast, we discovered KRAS-dependent SOX9 upregulation in 28 of the 31 (90.3%) tumors. CPTAC phosphoproteomics data similarly showed significantly lower phospho-ERK1/2 levels in tumor compared to matched mucosa. Analysis of transcriptome data from TCGA and GEO series GSE95132 also showed no activation of RAF/MEK/ERK signaling in CRC tumors. These data indicate that the pursue of targeted therapy inhibiting RAF/MEK/ERK and PI3K/Akt signaling is unlikely to be effective in sporadic CRC and possibly harmful as phospho-ERK1/2 are highly expressed in the matched mucosa suggesting a role in normal colonic homeostasis. The widespread use of RAF/MEK/ERK or PI3K/Akt signaling downregulation as a biomarker in drug testing on CRC cell lines needs to be re-evaluated. The data suggest further that SOX9 could be an alternative effector and biomarker of KRAS signaling. Citation Format: Peh Yean Cheah, Kuen Kuen Lam, Choong Leong Tang, Emile Tan, Siew Heng Wong. ERK/AKT pathways may be the wrong effectors for KRAS signaling inhibition in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6192.

Background Laparoscopic low anterior resection for rectal cancer has superior short-term benefits compared to open surgery. When operative conditions do not favour a totally-laparoscopic (TL) approach, a hybrid operation can be performed. In this laparoscopic-assisted (LA) approach, mobilization and vessel ligation are performed laparoscopically, with total mesorectal excision and distal transection performed either partially or totally in an extra-corporeal fashion. We compared short-term post-operative and oncological outcomes of both approaches. Methods A prospectively collected database of patients who underwent laparoscopic low anterior resection for rectal cancer between January 2009 and December 2014 was retrospectively analysed. Demographics, post-operative and oncological outcomes were compared. Results Of 174 patients, 97 were completed by TL, 62 by LA and the remaining 15 were converted to open. Baseline demographics were similar. LA group compared to TL group had bulkier rectal cancers (6.75 cm3 versus 4.50 cm3 , P = 0.04) which were lower (6 cm versus 7 cm from anal verge, P = 0.02). They were of a more advanced tumour grade and had greater incidence of lymphovascular invasion. Yet, post-operative outcomes such as time to diet, pain scores, hospitalization duration, wound-related and anastomotic complications, 30-day morbidity and mortality were similar. There was no difference in oncological adequacy, including circumferential resection margins, distal margins, lymph node harvest and 2-year local recurrence rates. Conclusion Laparoscopic-assisted low anterior resection enables minimally invasive rectal surgery to be performed despite unfavourable tumour factors and technical challenges; and compares favourably with TL approach in terms of short-term outcomes and oncological safety.

The role of stem cells in the development of solid tumors remains controversial. In colorectal cancers (CRC), this is complicated by the conflicting “top-down” or “bottom-up” hypotheses of cancer initiation. We profiled the expressions of genes from the top (T) and bottom (B) crypt fractions of normal-appearing human colonic mucosa (M) at least 20 cm away from the tumor as a baseline and compared this to the genes of matched mucosa adjacent to tumors (MT) in twenty-three sporadic CRC patients. In thirteen patients, the genetic distance (M-MT) between the B fractions is smaller than the distance between the T fractions, indicating that the expressions diverge further in the top fractions (B T). Assuming that a greater genetic divergence in the top or bottom fractions indicates that position as the initiation site, it is thus equally likely that human CRC initiates from ‘top-down’ via de-differentiated colonocytes or ‘bottom-up’ via dysregulated intestinal stem cells. Dysregulated genes that persist until tumor stage are not limited to tumor suppressors or oncogenes but include metabolic and transporter genes such as CA7, PHLPP2, and AQP8.

There has been much recent interest in the use of procalcitonin (PCT) as a marker of intra-abdominal infection (IAI) following colorectal surgery. However, the literature remains divided on the value of PCT in this setting. This meta-analysis aims to evaluate the value of PCT in predicting IAI after colorectal surgery.Systemic literature search was performed using MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) and the Cochrane Database of Systematic Reviews to identify studies evaluating the diagnostic accuracy of PCT as a predictor for detecting IAI on postoperative days (POD) 3 to 5 following colorectal surgery. A meta-analysis was performed using random effect model and pooled predictive parameters as well as cut-off values for POD 3 to 5 were derived.Eight studies consisting 1629 patients were included. The pooled prevalence of IAI was 5.7% on POD 3, 9.7% on POD 4, and 6.3% on POD 5. The pooled AUC for POD 3 to 5 were 0.83 (95% CI 0.78-0.88), 0.79 (95% CI 0.64-0.93), and 0.94 (95% CI 0.91-0.97), respectively. The derived PCT cut-off values were 1.45 ng/ml on POD 3, 1.28 ng/ml on POD 4, and 1.26 ng/ml on POD 5. PCT had the highest diagnostic capability on POD 5 with diagnostic odds ratio of 32.9 (95% CI 15.01-69.88), sensitivity of 0.78 (95% CI 0.65-0.89), and specificity of 0.88 (95% CI 0.85-0.90).PCT is a useful diagnostic predictor of IAI after colorectal surgery. It has the greatest diagnostic accuracy on POD 5 and can help guide safe discharge of patients after colorectal surgery.

BackgroundMultiple single nucleotide polymorphisms (SNPs) have been associated with colorectal cancer (CRC) risk. The role of structural or copy number variants (CNV) in CRC, however, remained unclear. We investigated the role of CNVs in patients with sporadic CRC.MethodsA genome-wide association study (GWAS) was performed on 1000 Singapore Chinese patients aged 50 years or more with no family history of CRC and 1000 ethnicity-matched, age-matched and gender-matched healthy controls using the Affymetrix SNP 6 platform. After 16 principal component corrections, univariate and multivariate segmentations followed by association testing were performed on 1830 samples that passed quality assurance tests.ResultsA rare CNV region (CNVR) at chromosome 14q11 (OR=1.92 (95% CI 1.59 to 2.32), p=2.7e-12) encompassing CHD8, and common CNVR at chromosomes 3q13.12 (OR=1.54 (95% CI 1.33 to 1.77), p=2.9e-9) and 12p12.3 (OR=1.69 (95% CI 1.41 to 2.01), p=2.8e-9) encompassing CD47 and RERG/ARHGDIB, respectively, were significantly associated with CRC risk. CNV loci were validated in an independent replication panel using an optimised copy number assay. Whole-genome expression data in matched tumours of a subset of cases demonstrated that copy number loss at CHD8 was significantly associated with dysregulation of several genes that perturb the Wnt, TP53 and inflammatory pathways.ConclusionsA rare CNVR at 14q11 encompassing the chromatin modifier CHD8 was significantly associated with sporadic CRC risk. Copy number loss at CHD8 altered expressions of genes implicated in colorectal tumourigenesis.

Approximately 20% early-stage (I/II) colorectal cancer (CRC) patients develop metastases despite curative surgery. We aim to develop a formalin-fixed and paraffin-embedded (FFPE)-based predictor of metastases in early-stage, clinically-defined low risk, microsatellite-stable (MSS) CRC patients. We considered genome-wide mRNA and miRNA expression and mutation status of 20 genes assayed in 150 fresh-frozen tumours with known metastasis status. We selected 193 genes for further analysis using NanoString nCounter arrays on corresponding FFPE tumours. Neither mutation status nor miRNA expression improved the estimated prediction. The final predictor, ColoMet19, based on the top 19 genes' mRNA levels trained by Random Forest machine-learning strategy, had an estimated positive-predictive-value (PPV) of 0.66. We tested ColoMet19 on an independent test-set of 131 tumours and obtained a population-adjusted PPV of 0.67 indicating that early-stage CRC patients who tested positive have a 67% risk of developing metastases, substantially higher than the metastasis risk of 40% for node-positive (Stage III) patients who are generally treated with chemotherapy. Predicted-positive patients also had poorer metastasis-free survival (hazard ratios [HR] = 1.92, design-set; HR = 2.05, test-set). Thus, early-stage CRC patients who test positive may be considered for adjuvant therapy after surgery.

The intensity and duration of surveillance for rectal cancer after surgical resection remain contentious. We evaluated the pattern of recurrences in a rectal cancer cohort followed up beyond 10 years. An analysis was performed on a retrospective database of 326 patients with rectal cancer who underwent curative surgical resection from 1999 to 2007. The above study duration was chosen to ensure at least 10 years of follow-up. Data on patient demographics, peri-operative details, and follow-up outcomes were extracted from the database. The pattern of recurrences and investigative modality that detected recurrences was identified. Patients were followed up until either year 2016 or the day of their demise. Two hundred seventeen patients (66.6%) were male and 109 patients (33.3%) female. Median age was 64 years old. Close to a third of the patients received adjuvant therapy (34%). Among the 326 patients studied, 29.8% of (97/326) patients developed recurrence. 7.7% (25/326) had loco-regional recurrence while 22.1% (72/326) had distant metastasis. Median time to recurrence was 16 months (4–83) and 18 months (3–81), respectively. Computed tomography scan was the best modality to detect both loco-regional and distant recurrences (48% in loco-regional and 41.7% in distant metastasis). The most common site of distant metastasis is the lung (34.7%). The salvage rate for loco-regional and distant recurrences was 52 and 12.5%, respectively. The predominant pattern of recurrence in rectal cancer is distant disease. Surveillance regimes may need to be altered to increase early detection of distant metastases.

Microsatellite instability in colorectal cancer (CRC) and its long-term outcomes remains poorly studied in Asians. We investigate the prognostic significance of microsatellite instability in an Asian population and assess its clinical impact in patients who undergo adjuvant chemotherapy. Six hundred fifty-four consecutive CRC patients who underwent surgical resection between January 2010 and December 2012 were recruited. Survival was estimated using the Kaplan-Meier approach. Univariate Cox proportional hazard models were used to estimate the hazard ratios for variables associated with survival. A subgroup analyses was performed for stage III patients who underwent chemotherapy to evaluate the prognostic significance of microsatellite instability in this group. Five hundred ninety-one (90.4%) patients were microsatellite stable (MSS) while 63 (9.6%) were microsatellite instable (MSI). Three years recurrence-free survival (RFS) and disease-specific survival (DSS) were 83.7 versus 73.7% (p = 0.295) and 87.1 versus 91.2% (p = 0.307) in MSS and MSI tumors, respectively. Among stage III patients who received adjuvant therapy, MSI status was found to be an adverse prognostic factor for RFS (HR 2.74 (95% CI 1.43–5.26), p = 0.002). This remained significant on multivariate analysis (HR 2.38 (95% CI 1.15–4.93), p = 0.018). Adjuvant chemotherapy was associated with survival benefit for patients with MSS tumors (HR 0.35, 95% CI 0.17–0.69, p = 0.002) but not MSI tumors (HR 0.67, 95% CI 0.08–8.15, p = 0.750). MSI status is not a prognostic indicator in the general CRC population but appears to be an adverse prognostic indicator for RFS in stage III CRC patients who received adjuvant chemotherapy.

Colorectal carcinoma (CRC) is the third highest incidence cancer and leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. This trend is expected to be exacerbated by an aging population in the developed world. Genetic predisposition plays a greater role in disease progression than disease etiology in sporadic CRC. However, the underlying genetic factors for metastasis are currently unclear. We identified germline single nucleotide polymorphism (SNP) variants contributing to metastasis risk from a case-case (metastasis-positive vs metastasis-negative) genome-wide association study (GWAS) comprising 3,000 sporadic Chinese CRC cases with definitive metastasis status. Metastasis-positive case is defined as one with distal-organ involvement attributable to primary CRC; metastasis-negative case is defined as metastasis-free with 5 years or more follow-up. By interrogating summarized SNP statistics from the case-case GWAS using the Functional Mapping and Annotation (FUMA) software, we identified 5 genomic risk loci and 74 disease-relevant genes based on gene set enrichment analysis and the Molecular Signatures Database. These genes are in various pathways implicated in different metastasis steps, from epithelial-mesenchymal transition (EMT) to implantation at distal organs. They are different from the risk variants identified by case-control GWAS for disease occurrence. We will expression-profile 250 metastasis-positive vs metastasis negative cases from the GWAS panel to validate the bioinformatics analysis by quantitative real-time PCR. We will focus first on the five genes implicated in glycolysis as this is a biochemical fingerprint of malignant cells that represents one of the ‘hallmarks of cancer'. Expression profile of these genes will be validated in a second independent panel. Kaplan Meier survival analysis as function of the median expression value will be performed to assess prognostic value of these genes. The ultimate goal is to improve metastasis risk assessment for secondary prevention to reduce cancer mortality and morbidity for individual patient as well as reducing public health expenditure. Citation Format: PehYean Cheah, Lai Fun Thean, Michelle Wong, Michelle Lo, Emile Tan, Choong Leong Tang. Identifying germline variants for metastatic risk assessment in sporadic colorectal carcinomas [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 2829.

Colorectal cancer (CRC) is the third most frequent cancer and the second leading cause of cancer mortality in the developed world including Singapore. Despite an aging populace which is now more compliant to having a baseline colonoscopy at age 50 or earlier, interval cancer (cancer that develops in between colonoscopies) is an unmet critical problem. The identification of early driver genes that can serve as biomarkers for CRC risk will aid in clinical management decision to counter cancer-associated mortality and morbidity. We profiled the genome-wide expression of matched mucosa (M), mucosa next to tumor (MT) and tumor (T) samples of a series of 23 patients using Affymetrix Gene Array. M samples are at least 20 cm away from the tumor and MT samples are adjoining the T samples but maintained the normal colonic crypt architecture of M. We discovered that thirty-nine dysregulated genes in MT samples compared to M samples persists until tumor formation. These early driver genes comprise not only tumor suppressors or oncogenes but included metabolic and transporter genes indicating that transformation towards malignancy requires the disruption of many pathways. Majority of these genes were down-regulated; the top three most drastically down-regulated genes include AQP8 (a water transporter), CA7 (a metabolic gene) and PHLPP2 (a tumor suppressor). Further analysis indicates that the three genes were already down-regulated in the normal appearing mucosa of a ‘healthy' individual compared to age-, gender-, ethnicity-matched (AGE-) controls seven years before recto-sigmoid cancer was diagnosed. This suggests that the dysregulation can occur several years before tumor formation and hence could possibly be used as early or pre-cancer biomarker. We have validated the down-regulation of the 3 genes in an independent series of 135 CRC samples from the same center using real-time RT-PCR assays. Correlation of the expression with clinicopathological features of the patients (gender, tumor site, age-group) and molecular subtypes (CMS 1-4) of the tumors will be ascertained to assess whether the signature is applicable to all CRC subtypes. Furthermore, the expression in matched blood samples will also be determined to explore feasibility of using these samples for assessing interval colorectal carcinoma risk. Citation Format: Peh Yean Cheah, Lai Fun Thean, Kuen Kuen Lam, Soo Chin Liew, Michelle Wong, Nicholas Koh, Michelle Lo, Emile Tan, Choong Leong Tang. Developing an early expression-based signature to assess interval colorectal carcinoma risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1134.

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco-secretome). The HCT-116 and E1 glyco-secretomes were compared using the label-free quantitative SWATH-MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin β-1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.

Stage IV colorectal cancer patients with unresectable metastasis who undergo elective primary tumour resection experience heterogeneous post-operative survival. We aimed to develop a scoring model for predicting post-operative survival using pre-operative variables to identify patients who are least likely to experience extended survival following the procedure.Survival data were collected from stage IV colorectal cancer patients who had undergone elective primary tumour resection between January 1999 and December 2007. Coefficients of significant covariates from the multivariate Cox regression model were used to compute individual survival scores to classify patients into three prognostic groups. A survival function was derived for each group via Kaplan-Meier estimation. Internal validation was performed.Advanced age (hazard ratio, HR 1.43 (1.16-1.78)); poorly differentiated tumour (HR 2.72 (1.49-5.04)); metastasis to liver (HR 1.76 (1.33-2.33)), lung (HR 1.37 (1.10-1.71)) and bone (HR 2.08 ((1.16-3.71)); carcinomatosis (HR 1.68 (1.30-2.16)); hypoalbuminaemia (HR 1.30 (1.04-1.61) and elevated carcinoembryonic antigen levels (HR 1.89 (1.49-2.39)) significantly shorten post-operative survival. The scoring model separated patients into three prognostic groups with distinct median survival lengths of 4.8, 12.4 and 18.6 months (p 0.0001). Internal validation revealed a concordance probability estimate of 0.65 and a time-dependent area under receiver operating curve of 0.75 at 6 months. Temporal split-sample validation implied good local generalizability to future patient populations (p 0.0001).Predicting survival following elective primary tumour resection using pre-operative variables has been demonstrated with the scoring model developed. Model-based survival prognostication can support clinical decisions on elective primary tumour resection eligibility.

Introduction: The Singapore Polyposis Registry (SPR) was established in 1989 in Singapore General Hospital (SGH). The aims were to provide a central registry service to facilitate identification, surveillance and management of families and individuals at high risk of colorectal cancer. Materials and Methods: This is a review of published literature in the department. Results: The registry currently has 253 families with several genetic conditions—93 familial adenomatous polyposis (FAP) families, 138 Amsterdam-criteria positive presumed Lynch syndrome (LS) families, 12 families with Peutz Jeghers syndrome, 2 families with Cowden’s syndrome, and 8 families with hereditary mixed polyposis syndrome (HMPS). There are also 169 families with a strong family history of colorectal cancer but no abnormal genes yet identified. In FAP, a diagnostic tool developed has allowed a 94% local APC germline detection rate in FAP families. Knowledge obtained studying the phenotype of FAP patients has allowed better choice of surgery between ileal pouch anal anastomosis (IPAA) against an ileal-rectal anastomosis (IRA). In LS, our review has noted a highly heterogenous mutational spectrum and novel variants made up 46.7% (28/60) of all variants identified in this cohort. This may suggest that our Southeast Asian ethnic groups have distinct mutational variants from Western populations. Pathogenic mutations were only confined to MLH1 and MSH2, and identified in 28.8% of families. Conclusion: The impact of predictive gene testing for hereditary cancer risk in clinical practice has allowed evolution of care. Risk-reducing surgery and aggressive surveillance allows reduction in morbidity and mortality of patients. The SPR will continue to grow and improve outcomes in hereditary colorectal cancer patients and families. Key words: Familial adenomatous polyposis, Genetic testing, Lynch syndrome

Laparoscopic low anterior resection for rectal cancer has superior short-term benefits compared to open surgery. When operative conditions do not favour a totally-laparoscopic (TL) approach, a hybrid operation can be performed. In this laparoscopic-assisted (LA) approach, mobilization and vessel ligation are performed laparoscopically, with total mesorectal excision and distal transection performed either partially or totally in an extra-corporeal fashion. We compared short-term post-operative and oncological outcomes of both approaches.A prospectively collected database of patients who underwent laparoscopic low anterior resection for rectal cancer between January 2009 and December 2014 was retrospectively analysed. Demographics, post-operative and oncological outcomes were compared.Of 174 patients, 97 were completed by TL, 62 by LA and the remaining 15 were converted to open. Baseline demographics were similar. LA group compared to TL group had bulkier rectal cancers (6.75 cmLaparoscopic-assisted low anterior resection enables minimally invasive rectal surgery to be performed despite unfavourable tumour factors and technical challenges; and compares favourably with TL approach in terms of short-term outcomes and oncological safety.

OBJECTIVE The overall aim was to develop and validate a risk prediction score for laparoscopic colorectal surgery training cases. BACKGROUND Published risk prediction scores are not transferable between hospitals because they are derived from a single institution's data and are not designed for use in training situations. METHODS Cases from the prospectively collected database of the National Training Programme in Laparoscopic Colorectal Surgery, between July 2008 and July 2012, were analyzed. Independent risk factors for conversion were identified by the logistic regression. Converting the odds ratios into integers created a risk prediction score for conversion. The clinical impact of this score was investigated by comparing postoperative complications and the level of trainer input in high- and low-risk cases. To study whether adverse outcomes in predicted high-risk cases occur outside the National Training Programme in Laparoscopic Colorectal Surgery, 2 external data sets were examined. RESULTS A total of 2341 cases carried out in 42 hospitals were analyzed. Significant risk factors for conversion were body mass index, American Society of Anesthesiology classification, male sex, prior abdominal surgery, and resection type. At a risk score of more than 6, complication rates increased, including mortality (2.9% vs 0.5%, P < 0.001), anastomotic leak (4.3% vs 1.4%, P = 0.002), and a higher level of trainer input (32.2% vs 19.9% of cases, P < 0.001). Analysis of 786 external cases showed that high-risk cases had higher conversion (18.8% vs 7.1%, P < 0.001), overall complication (36.4% vs 15.0%, P < 0.001), and leak rates (4.0% vs 1.3%, P = 0.015). CONCLUSIONS A risk predication score to facilitate case selection in laparoscopic colorectal surgery training was developed and validated.

BACKGROUND A prognostic scoring model has been devised previously to predict survival following primary tumor resection in patients with metastatic colorectal cancer and unresectable metastases. This has yet to be validated. OBJECTIVE The main objectives of this study are to validate the proposed prognostic scoring model and create an interactive online calculator to estimate an individual's survival after primary tumor resection. DESIGN Clinical data and survival outcomes of patients were extracted from a prospectively maintained database. Patients were categorized into good, moderate, or poor survivor groups based on the previously proposed scoring algorithm. Discrimination was assessed and recalibration was performed, with the recalibrated model implemented as an interactive Web application to provide individualized survival probability. SETTINGS This study was conducted at a tertiary referral center. PATIENTS The study included 324 consecutive patients with metastatic colorectal carcinoma and unresectable metastases who underwent primary tumor resection between January 2008 and December 2013. MAIN OUTCOME MEASURES The primary outcome measured was overall survival. RESULTS Three hundred twenty-four patients were included in the study. Median survival in the good, moderate, and poor prognostic groups was 56.8, 25.7, and 19.9 months (log rank test, p = 0.003). The κ statistic was 0.638 and RD was 0.101. Significant differences in survival were found between the moderate and good prognostic groups (HR, 2.79; 95% CI, 1.51-5.15; p = 0.001) and between poor and good prognostic groups (HR, 4.12; 95% CI, 1.98-8.55; p < 0.001). The model was implemented as an interactive online calculator to provide individualized survival estimation after primary tumor resection (http://bit.ly/Stage4PrognosticScore). LIMITATIONS Selection bias and single-center data preclude the generalizability of the proposed model. Information regarding the severity or likelihood of developing symptoms from the primary tumor were also not accounted for in the prognostic scoring model proposed. CONCLUSIONS The prognostic scoring model provides good prognostic stratification of survival after primary tumor resection and may be a useful tool to predict survival after primary tumor resection. See Video Abstract at http://links.lww.com/DCR/A330.

The ideal incision for laparoscopic specimen extraction is not known. There has been no randomised study thus far evaluating extraction site in laparoscopic colorectal surgery. The aim of our study was to compare post-operative outcomes, pain scores and quality of life scores of vertical periumbilical (VW) versus transverse left iliac fossa (TW) incisions for specimen extraction in laparoscopic anterior resections. Using an assumption of pain score of 5 in the VW group versus pain score of 2 in the TW group, on day one post-operatively, and based on a 80 % statistical power of analysis to achieve a statistical difference with reduction in pain scores, the sample size per arm calculated was 16. Forty patients undergoing laparoscopic anterior resection were randomised to VW (n = 20) or TW (n = 20). Primary endpoint was post-operative pain. Secondary endpoints were post-operative outcomes, wound cosmesis using Hollander Cosmesis Score and quality of life assessment using EQ-5D at 2 weeks and 2 months post-operatively. Median pain score on the first post-operative day was 2 in both groups (p = 0.360). There was no significant difference in wound infection rates, operative time or post-operative recovery. Cosmesis scores and EQ-5D scores were also similar in both groups. At a median follow-up of 30 months, the incidence of extraction site incisional hernia was similar. Transverse and vertical incisions in laparoscopic colorectal surgery have similar post-operative outcomes, with similar pain scores, cosmesis scores, quality of life scores and incisional hernia rates.

BACKGROUND Serum carcinoembryonic antigen (CEA) is the only marker recommended for surveillance of colorectal cancer (CRC) recurrence; its sensitivity and specificity, however, are suboptimal. This study sought to evaluate the values of postoperative serum methylation levels of 7 genes for prognostication and especially for recurrence detection after curative resection. METHODS This prospective cohort study included 150 patients with stage I-III CRC from whom 3 consecutive blood sampling was taken 1 week before, and 6 months and 1 year after operation. Methylation levels of 7 genes were evaluated via quantitative methylation-specific polymerase chain reaction. Serum CEA was measured in parallel. Univariate and multivariate survival analyses were followed by construction of receiver operating characteristic curves for recurrence detection. RESULTS After a median follow-up of 59 months, 43 patients (28.7%) developed recurrent lesions. High serum methylation levels of TAC1 in serum at 6-month follow-up (6M-FU), and SEPT9 at 1-year follow-up (1Y-FU) were independent predictors for tumor recurrence and unfavorable cancer-specific survival (CSS) (P

The incidence of mismatch repair deficiency in colorectal cancer (CRC) in young people remains unknown in Asians. The present study assessed the clinicopathological features and efficacy of immunohistochemistry screening for Lynch syndrome in young Asian CRC patients. This was a retrospective review conducted in Singapore General Hospital between January 2006 and December 2010 of 240 unrelated patients under the age of 50. All patients had immunohistochemical (IHC) staining for mismatch repair proteins in resected CRC specimen data retrieved from a prospective computerized database. A total of 21 % (n = 51) of the patients had abnormal IHC staining. Loss of staining for MLH1, MSH2, MSH6, and PMS2 proteins was observed in 10, 4, 6, and 13 % of tumors, respectively. Of the 22 patients who had abnormal staining of MLH1, 13 had concomitant abnormal staining for PMS2. One tumor specimen had abnormal staining in all four proteins. If the Amsterdam criteria alone were to be used, 86 % (n = 44) of the cohort would have not been detected for mismatch repair gene defects. The overall burden of germline mismatch repair deficiency in the Singapore population may be as high as 21 %. The Amsterdam criteria alone are inadequate to detect Lynch syndrome patients. The use of IHC staining of at least four mismatch repair proteins is a useful screening strategy for Lynch syndrome diagnosis. Routine screening of mismatch repair deficiency may be recommended for all young Asian CRC patients.

INTRODUCTION The use of faecal immunochemical occult blood test (FIT) has been reported to decrease mortality from colorectal cancer. The Singapore Cancer Society (SCS) gives out FIT kits to encourage opportunistic screening of colorectal cancer. Any Singapore citizen or permanent resident aged ≥ 50 years is eligible to receive two FIT kits. Participants with at least one positive FIT are referred for further evaluation. We aimed to analyse the results of SCS data from the year 2008. METHODS The factors evaluated included compliance, positive test rate (PR) and positive predictive value (PPV) of FIT. RESULTS 20,989 participants received 41,978 kits in 2008. Compliance was 38.9%, with 8,156 participants returning at least one kit. 8% of participants tested positive, and 75% of these test-positive participants agreed to undergo further investigations. 33 participants had colorectal cancers, 45 had advanced polyps (≥ 1 cm) and 90 had polyps < 1 cm. Histologically, 114 polyps were adenomatous, 20 were hyperplastic and 1 was serrated. PPV of colorectal neoplasia for those who underwent further colonoscopy was 34%. Over half of the participants who had only one positive test had colorectal neoplasia. CONCLUSION PR and PPV of FIT in our study were comparable to that in the literature. However, compliance was low and a quarter of all participants who tested positive refused further investigations. Extensive population education programmes are required to improve compliance and tackle inhibitions among the masses. It is also important to take steps to enhance the cost effectiveness of future screening programmes.

Familial adenomatous polyposis (FAP) is an autosomal-dominantly inherited form of colorectal cancer (CRC) caused by mutation in the adenomatous polyposis coli (APC) gene. Our ability to exhaustively screen for APC mutations identify microsatellite-stable and APC-mutation negative familial CRC patients, enabling us to search for novel genes. We performed genome-wide scan on two affected siblings of one family and 88 ethnicity- and gender-matched healthy controls to identify deletions shared by the siblings. Combined loss of heterozygosity, copy number and allelic-specific copy number analysis uncovered 5 shared deletions. Long-range polymerase chain reaction (PCR) confirmed chromosome 19q13 deletion, which was subsequently found in one other family. The 32 kb deleted region harbors the CYP2A7 gene and was enriched with enhancer, repressor and insulator sites. The wildtype allele was lost in the polyps of the proband. Further, real-time RT-PCR assays showed that expressions of MIA and MIA-RAB4B located 35 kb upstream of the deletion, were up-regulated in the polyps compared to the matched mucosa of the proband. MIA-RAB4B, the read-through long non-coding RNA (lncRNA), RAB4B, PIM2 and TAOK1 share common binding site of a microRNA, miR-24, in their 3'UTRs. PIM2 and TAOK1, two target oncogenes of miR-24, were co-ordinately up-regulated with MIA-RAB4B in the polyps, suggesting that MIA-RAB4B could function as competitive endogenous RNA to titrate miR-24 away from its other targets. The data suggest that the 19.13 deletion disrupted chromatin boundary, leading to altered expression of several genes and lncRNA, could contribute to colorectal cancer via novel genetic and epigenetic mechanisms.

Background: Endoscopic polypectomy, although routinely used for the treatment of colorectal polyps, may be limited by polyp size, location and histology. Laparoscopic resection for malignant polyps and polyps not amenable to endoscopic removal has the advantage of adequate disease clearance as well as the short-term benefits of laparoscopic surgery. This study evaluates the outcomes of such an approach. Methods: Patients who had laparoscopic resection for colorectal polyps between January 2005 and July 2008 were identified from a prospective database. Polyps that were malignant, large, difficult to snare or incompletely excised, were included. Demographics, perioperative details and histopathology were analysed. Results: Seventy-eight patients (44 male) with a median age of 62.5 years (range 24–86) were studied. The majority (79%) were laparoscopic anterior resections for sigmoid or rectal polyps. Median operating time was 125 min (range, 65–225). Eight cases (10.3%) were converted to open mainly due to adhesions. There was no post-operative mortality. Perioperative complications occurred in seven patients (8.9%). Median hospital stay was 6 days (range 4–78). Median polyp size was 20 mm (range, 5–75). There were 44 benign polyps (55.7%); majority were tubulovillous adenomas (n= 22), and tubular adenomas (n= 10). Thirty-five patients (44.3%) had invasive cancer, with T1 (n= 27) and T2 (n= 2) tumours. Three of these patients (8.6%) had lymph node metastases. Median number of lymph nodes sampled was six (range 0–23). Conclusion: Laparoscopic resection is safe and effective for colorectal polyps not amenable to colonoscopic removal, and is especially important for adequate clearance in the case of malignant polyps.

Background: This study aims to evaluate the results of all 7302 stapled haemorrhoidectomy operations performed in a single centre. Method: A retrospective review of all 7302 patients who underwent stapled haemorrhoidectomy at our department over seven years was conducted. The hospital database was reviewed for subsequent readmissions and re-operations. A questionnaire survey was also sent out to all the patients. Results: A total of 302 patients (4.1%) were admitted with post-operative bleeding and 281 stopped with conservative measures. Twenty one required surgical haemostasis. A total of 301 patients (4.1%) were admitted for an inability to void and 191 (2.6%) had true acute retention of urine, requiring catheterization. There were 124 patients (1.7%) admitted for pain, but all resolved with oral analgesia subsequently. Anal stricture requiring surgery occurred in only 86 patients (1.2%). Serious complications such as staple line dehiscence or anorectal sepsis occurred in seven patients. There were a total of 14 recurrences requiring readmission over this seven-year period, of which 12 were treated successfully with a second haemorrhoidectomy. A total of 1834 patients returned their questionnaire surveys and 95% of the patients reported complete resolution or improvement of their symptoms. Only 27 patients reported subsequent severe bleeding requiring medical attention. Conclusion: Stapled haemorrhoidectomy is safe, and most patients are satisfied with the long-term outcome.

Background: Somatic mutations are attractive therapeutic targets for “individualized neoantigen vaccines” because of lack of host central tolerance and reduced risk of autoimmunity. Here, we perform large-scale-omic analyses to assess the neoantigen landscape of colorectal cancer (CRC), a cancer largely refractory to immune-checkpoint inhibition. Methods: We performed whole genome sequencing (WGS) (60x tumor, 30x normal) and deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 158 colorectal cancers of which 32 are microsatellite instability high (MSI-H) tumours and 126 are microsatellite stable (MSS). Whole exome sequencing (200x tumor, 100x normal) was also performed on 120 tumours. HLA typing, somatic mutations, gene expression and neoepitope predictions were computationally evaluated. Inferred HLA-A alleles were orthogonally validated with Pacbio long-read sequencing. Tissue microarrays (TMAs) with tumour core, tumour edge and normal adjacent tissue of these 158 CRCs were constructed. Histopathological analyses using multiplex immunohistochemistry (mIHC) to simultaneously evaluate 7 markers, i.e. cytokeratin (CK), CD3, CD8, FOXP3, CD68, PD-L1, DAPI, have been performed. Results: The most common HLAs were, by allele count: A*11:01: 56; A*33:03: 38; B*58:01: 33; B*46:01: 29; B*40:01: 26; C*01:02: 41; C*07:02: 33. Inferred HLA-A alleles from WGS data was largely concordant (>90%) with Pacbio long-read sequencing. There were a median of 2,850 (1229-6909) [MSI] & 213 (27-13,835) [MSS] coding variants, from which 10,487 (4,307-27,365) [MSI] & 726.5 (50-59,096) [MSS] possible neoepitopes were derived, after accounting for epitope processing, the normal proteome and general population variome based on dbSNP, Of these, 5,707 (2,608-15,218) [MSI] & 320 (14-25,243) [MSS] neoepitopes are expressed (based on RNA-Seq). Epitope prediction algorithms revealed a median of 423 (17-1,056) [MSI] & 26 (0-1,102) [MSS] bound & expressed neoepitopes. 5 MSS tumors did not have any predicted bound nor expressed neoepitopes, 112 of 126 (89%) of MSS tumors had at least 5 predicted bound, expressed neoepitopes. Histopathological correlations between extent of immune infiltrates in fixed tissues, tumour PD-L1 expression and neoantigen burden is ongoing. Conclusions: There is substantial variability in the neoantigen landscape amongst MSI & MSS CRCs. MSI contains multiple-fold higher neo-antigens. Amongst MSS tumours, 89% of patients have at least 5 predicted bound and expressed neo-epitopes that could be targeted in neoantigen-based vaccines for personalized immunotherapy. Citation Format: Si-Lin Koo, Joe Poh Sheng Yeong, Andy Nguyen, Clarinda Wei Ling Chua, J Zachary Sanborn, Steve Benz, Wah Siew Tan, Choong Leong Tang, Su Yan, Min Hoe Chew, Brian Goh, Chung Yip Chan, Xiao Qing Koh, Alexander Lezhava, Tony Kiat Hon Lim, Shahrooz Rabizadeh, Anders Skanderup, Iain Beehuat Tan. Systematic identification of tumour-specific neoantigens(by whole-genome sequencing) and correlation between tumour neoantigen burden, PD-L1 expression and immune infiltrates in 158Asian colorectal cancers [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 5725.

Colorectal Cancer (CRC) is the third leading cause of cancer death worldwide. Majority of mortality is attributable to metastasis to distal organs. Current treatment targeting the ERK/MAPK pathway used monoclonal anti-EGFR antibody after the first line of chemotherapy fails. However these treatments are only effective for a small percentage of patients with wild-type KRAS, and these same patients eventually develop resistance. Small molecule drugs are toxic and have limited success. A combination of antibodies is an attractive alternative. This study aims to investigate whether anti-KRAS antibody can enter live cells in an individualized ex vivo culture system and whether it can bind and inactivate KRAS. The transient culture has advantages over cell line study as it better mimics the human colonic environment and tumors can be compared to their matched normals to partially address the problem of heterogeneity. Samples were obtained from patients' resected tumour and matched mucosa, cultured ex vivo, and immunostaining performed. We show that anti-KRAS antibody is internalized in both the cultured mucosa and tumor crypt cells prior to fixation. Subcellular localization of the internalized antibody in the mucosa cells is similar in the pre-fixed and post-fixed cells. In contrast, the internal cell membrane staining observed in post-fixed tumor cells appear more cytoplasmic and 'punctated' in the pre-fixed tumors, mimicking that of the mucosa cells suggesting that the internalized anti-KRAS may have altered the sub-cellular localization and hence cycling of KRAS. Furthermore, staining with anti-keratin antibody indicates that live tumor cells treated with anti-KRAS has partial reversal of the phenotype to become more like the regular striated pattern of the matched mucosa. Preliminary double labelling of cultured tumours with monoclonal anti-KRAS prior to fixation and polyclonal anti-KRAS post-fixation revealed punctate staining of KRAS in the cytoplasm that were mainly non-overlapping suggesting that the punctate structures are in different compartments. Citation Format: Peh Yean Cheah, Yee Syuen Low, Michelle Lo, See Aik Tang, Alphaeus Ng, Choong Leong Tang, Siew Heng Wong. Developing an individualized human colonic crypt cell culture as an ex vivo model for immunotherapy [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 1147.

Stapled haemorrhoidectomy has become popular for the treatment of symptomatic bleeding or prolapsing piles. There are concerns about the safety of another stapled low colorectal anastomosis after rectal resection if a patient who has had previous stapled haemorrhoidectomy subsequently develops colorectal neoplasia requiring an anterior resection. A retrospective review of patients who underwent stapled haemorrhoidectomy and subsequently had anterior resection from 1999 to 2008 was performed. Five patients (all male) were found to have distal sigmoid or rectal tumours on surveillance colonoscopy after stapled haemorrhoidectomy. Median age was 65 years (range 58–71). All underwent anterior resection with stapled end-to-end colorectal anastomosis at median of 29 months (range 18–60 months) after the initial stapled haemorrhoidectomy. Median anastomotic height was 12 cm (range 1–12 cm). A defunctioning ileostomy was created for three out of five patients. All the colorectal anastomoses healed uneventfully. Stapled colorectal anastomosis may be safely performed after previous stapled haemorrhoidectomy.