Sporadic Colorectal Cancer Cases Research Articles

Establishment of a large-scale patient-derived high-risk colorectal adenoma organoid biobank for high-throughput and high-content drug screening

BackgroundColorectal adenoma (CA), especially high-risk CA (HRCA), is a precancerous lesion with high prevalence and recurrence rate and accounts for about 90% incidence of sporadic colorectal cancer cases worldwide. Currently, recurrent CA can only be treated with repeated invasive polypectomies, while safe and promising pharmaceutical invention strategies are still missing due to the lack of reliable in vitro model for CA-related drug screening.MethodsWe have established a large-scale patient-derived high-risk colorectal adenoma organoid (HRCA-PDO) biobank containing 37 PDO lines derived from 33 patients and then conducted a series of high-throughput and high-content HRCA drug screening.ResultsWe established the primary culture system with the non-WNT3a medium which highly improved the purity while maintained the viability of HRCA-PDOs. We also proved that the HRCA-PDOs replicated the histological features, cellular diversity, genetic mutations, and molecular characteristics of the primary adenomas. Especially, we identified the dysregulated stem genes including LGR5, c-Myc, and OLFM4 as the markers of adenoma, which are well preserved in HRCA-PDOs. Based on the HRCA-PDO biobank, a customized 139 compound library was applied for drug screening. Four drugs including metformin, BMS754807, panobinostat and AT9283 were screened out as potential hits with generally consistent inhibitory efficacy on HRCA-PDOs. As a representative, metformin was discovered to hinder HRCA-PDO growth in vitro and in vivo by restricting the stemness maintenance.ConclusionsThis study established a promising HRCA-PDO biobank and conducted the first high-throughput and high-content HRCA drug screening in order to shed light on the prevention of colorectal cancer.

Colitis-Associated Colorectal Cancer Survival is Comparable to Sporadic Cases after Surgery: a Matched-Pair Analysis

BackgroundInflammatory bowel disease (IBD) confers an increased lifetime risk of colorectal cancer (CRC). The pathogenesis of colitis-associated CRC is considered distinct from sporadic CRC, but existing is mixed on long-term oncologic outcomes. This study aims to compare clinicopathological characteristics and survival between colitis-associated and sporadic CRC. MethodsData was retrospectively extracted and analyzed from a single institutional database of patients with surgically resected CRC between 2004 and 2015. Patients with IBD were identified as having colitis-associated CRC. The remainder were classified as sporadic CRC. Propensity score matching was performed. Univariate and survival analyses were carried out to estimate the differences between the two groups. ResultsOf 2275 patients included in this analysis, 65 carried a diagnosis of IBD (2.9%, 33 Crohn’s disease, 29 ulcerative colitis, 3 indeterminate colitis). Average age at CRC diagnosis was 62 years for colitis-associated CRC and 65 for sporadic CRC. The final propensity score matched cohort consisted of 65 colitis-associated and 130 sporadic CRC cases. Patients with colitis-associated CRC were more likely to undergo total proctocolectomy (p < 0.01) and had higher incidence of locoregional recurrence (p = 0.026) compared to sporadic CRC patients. There were no significant differences in time to recurrence, tumor grade, extramural vascular invasion, perineural invasion, or rate of R0 resections. Overall survival and disease-free survival did not differ between groups. On multiple Cox regression, IBD diagnosis was not a significant predictor of survival. ConclusionsPatients with colitis-associated CRC who undergo surgical resection have comparable overall and disease-free survival to patients with sporadic CRC.

Abstract P014: A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer

Abstract Colorectal cancer (CRC) is the 3rd most common type of cancer in the U.S., and it is estimated it will account for an alarming 151,030 (8%) of new total cancer cases and 52,580 (9%) of total cancer deaths in 2022. Remarkably, dysregulation of the Wnt signaling pathway has been linked to ~90% of all colorectal cancer (CRC) cases. The vast majority (&amp;gt;80%) of sporadic CRC cases display mutations in Adenomatous Polyposis Coli (APC), a central Wnt signaling regulator. Loss of APC function causes aberrant stabilization of β-catenin (βcat), a crucial step in CRC initiation. Notably, attempts towards “drugging” this pathway still poses multiple hurdles due to poor tumor cell targeting, negative side effects associated with required long-term treatments and poorly understood mechanisms of action. Consequently, there is an urgent need to gather additional mechanistic insights associated with the Wnt pathway to develop novel therapeutic approaches. With respect to Wnt biology, two key plasma membrane receptors, LRP5/6 and Frizzled (Fzd) require lipid raft localization and nanoclustering for efficient signaling. Interestingly, various effectors that disrupt raft dynamics, alter LRP5/6-Fzd clustering, leading to reduced βcat stabilization. Thus, we examined the effect of mutant APC (mAPC) on plasma membrane lipid/protein-mediated interactions of Wnt-associated effectors in a CRC mouse model, human mAPC-expressing organoids and cell lines, and Drosophila. We show for the first time that mAPC significantly increased the levels of plasma membrane free cholesterol, a major component of lipid rafts known to selectively activate canonical Wnt signaling, in the intestinal epithelium including Lgr5+ colonic stem cells. Moreover, this change in plasma membrane cholesterol was associated with alterations in colonocyte membrane rigidity and raft organization. We subsequently examined the effect of mAPC on the interactions between Wnt signaling receptors/effectors in mAPC-expressing cell lines. mAPC significantly increased LRP6 and Fzd7 homo-clustering and LRP6-Fzd7 hetero-clustering as well as their interactions with key lipids, e.g., PIP2, resulting in the enhancement of Wnt signaling activation. Interestingly, the effects of mAPC were recapitulated by the addition of exogenous cholesterol to wild type colonocytes. Finally, we demonstrate that n-3 polyunsaturated fatty acids (n3-PUFA), i.e., DHA and EPA, reduces mAPC-driven colonic polyp formation in mice in part by reducing plasma membrane cholesterol, rigidity and LRP6-Fzd7 clustering in colonocytes. Collectively, these findings indicate that mAPC can directly perturb lipid homeostasis in the colon, thereby altering raft stability, LRP6-Fzd7 membrane nanoclusters and downstream Wnt signaling and supports the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane lipid composition, to reduce Wnt signaling and cancer risk. Citation Format: Alfredo Erazo-Oliveras, Mohamed Mlih, Mónica Muñoz-Vega, Eunjoo Kim, Venkat Thiriveedi, Michael L. Salinas, Xiaoli Wang, Jatin Roper, Jason Karpac, Robert S. Chapkin. A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P014.

KRAS Promoter Methylation Status and miR-18a-3p and miR-143 Expression in Patients With Wild-type KRAS Gene in Colorectal Cancer.

Although some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC. A total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated. In the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed. No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.

Cancer stem cell markers CD44v9+/CD133- are associated with low apoptosis in both sporadic and ulcerative colitis-associated colorectal cancers.

To elucidate tumor cell behavior associated with cancer stem cell (CSC) marker expression, the expression of CD133, CD44v9, and ALDH1A1, which are considered markers of CSCs, was examined in sporadic and ulcerative colitis (UC)-associated colorectal tumors. A total of 23 cases of sporadic colorectal cancer and 44 cases of adenoma were collected. Additionally, 22 cancer lesions and 38 dysplasia lesions were selected from 28 colectomy cases of UC with neoplastic lesions. Lesions were examined by immunohistochemistry using primary antibodies against CD133, CD44v9, ALDH1A1, Ki-67, cleaved-Caspase 3, and p53. CD133, CD44v9, and ALDH1A1 showed higher expression in both sporadic and UC-associated tumors than in the normal mucosa. ALDH1A1 expression in sporadic cancer was higher in the right colon than in the left colon (p=0.0089). ALDH1A1 expression in UC-associated cancer was higher in those with longer disease duration than in those with shorter disease duration (p=0.019). The CD44v9+/CD133- region had fewer cleaved-Caspase 3 positive cells in both sporadic and UC-associated cancers. In sporadic cancer, CD133+/ALDH1A1+ regions had fewer apoptotic cells than CD133+/ALDH1A1- regions, while CD133+/ALDH1A1- regions were less proliferative than CD133+/ALDH1A1+ regions in UC-associated cancer. CD44+/CD133- regions were commonly associated with low apoptosis in sporadic and UC-associated cancers; thus, these were considered target areas for CSCs. Additionally, the combination of markers comprising CSCs may differ between sporadic and UC-associated cancers.

Role of Cyclooxygenase-2 in colorectal cancer patients.

Cyclooxygenase-2 (COX-2) is an inducible enzyme which triggers the biosynthesis of prostaglandins. COX-2 is activated in response to inflammatory stimuli and is one of the major molecules that is involved in the development and progression of colorectal cancer (CRC). Consistent with such a conceptual framework, it has been shown that COX-2 inhibitors prevent the carcinogenesis of CRC and aid in the treatment of sporadic or familial cases of CRC as shown by an overall increase in the survival rate. However, prolonged use of these inhibitors is associated with increase risk of development of cardiovascular complications, implying that further research is required to identify COX-2 inhibitors that are associated with lower risks of such complications.

Colorectal Cancer, Age, and Obesity-Related Comorbidities: A Large Database Study.

The association between obesity and colorectal cancer (CRC) is well established in older individuals, but evidence is limited in the younger population. The study aims to analyze the relationship of obesity and its related comorbidities in early-onset CRC (E-CRC) and compare it to late-onset CRC (L-CRC). A retrospective, cross-sectional study was performed on average-risk individuals ≥ 20years who were active patients in the commercial database, IBM Watson Health Explorys in the last 5years. Individuals with CRC were compared to those without CRC across different age groups (20-39, 40-49, and 50-74years). Individuals with CRC diagnosed < 50years (E-CRC) were compared to those with CRC between 50 and 74years (L-CRC). Variables included sex, smoking, obese BMI, diabetes mellitus type 2 (DM2), hypertension (HTN), and hyperlipidemia (HLD). Since Explorys aggregates population-level, de-identified data, approval from institutional review board was not required. Among 37,483,140 individuals, 162,150 cases of sporadic CRC were identified. Compared to the general population, obesity and HLD were independent risk factors for CRC across all age groups; DM2, HTN, and smoking were independent risk factors for CRC in men of all age groups and women with L-CRC. Compared to L-CRC, individuals with E-CRC had lower percentages of obesity-related comorbidities. In E-CRC, obesity, DM2, HTN, HLD, and smoking are independent risk factors for CRC among men; obesity and HLD are independent risk factors for CRC in women. These subgroups may benefit from a personalized screening approach to detect early-onset CRC.

COVID-19: An Opportunity to Reimagine Colorectal Cancer Diagnostic Testing—A New Paradigm Shift

ABSTRACT Colorectal cancer is the third leading cause of cancer related deaths and to date, the COVID-19 pandemic has led to major disruptions of the diagnosis and treatment pathways of this cancer, which may adversely affect survival outcomes for many years. Colorectal cancer pathways must be maintained at a near normal throughput with urgent attention to the backlog of patients to avoid a potential crisis of avoidable cancer deaths. A radical review and revamping of endoscopy services to improve efficiency during COVID-19 is urgently required. To date most endoscopy units have severely restricted their activity, except for time-critical diagnostic and therapeutic procedures. In the post- COVID-19 recovery phase, access to endoscopy services will increase but capacity will be reduced compared to before the pandemic, due to infection control measures. A pragmatic and safe strategy is urgently needed to clear the backlog of endoscopic procedures and for managing the new symptomatic referrals. We propose the use of non-invasive tests, such as FIT and mSEPT9 combined with “safety netting” to “rule out” colorectal cancer and to reduce the utilization of colonoscopy by up to 80% and limit the procedure to those patients at high risk of cancer.

Comparison of Tissue-Based Molecular Markers in Younger versus Older Patients with Colorectal Neoplasia.

Emerging colorectal cancer trends demonstrate increased incidence and mortality in younger populations, prompting consideration of average-risk colorectal cancer screening initiation at age 45 versus 50 years. However, screening test performance characteristics in adults 45-49 years have been minimally described. To inform the biologic rationale for multi-target stool DNA (mt-sDNA) screening in younger patients, we analyzed and compared tissue levels of methylation (BMP3, NDRG4) and mutation (KRAS) markers included in the FDA-approved, mt-sDNA assay (Cologuard; Exact Sciences Corporation). Within 40-44, 45-49, and 50-64 year age groups, archived colorectal tissue specimens were identified for 211 sporadic colorectal cancer cases, 123 advanced precancerous lesions (APLs; adenomas >1 cm, high-grade dysplasia, ≥25% villous morphology, or sessile serrated polyp; 45-49 and 50-64 age groups only), and 204 histologically normal controls. Following DNA extraction, KRAS, BMP3, and NDRG4 were quantified using QuARTS assays, relative to ACTB (reference gene). None of the molecular marker concentrations were significantly associated with age (P > 0.05 for all comparisons), with the exception of NDRG4 concentration in APL samples (higher in older vs. younger cases; P = 0.008). However, NDRG4 levels were also statistically higher in APL case versus normal control samples in both the 45-49 (P < 0.0001) and 50-64 (P < 0.0001) year age groups. Overall, these findings support the potential for earlier onset of average-risk colorectal cancer screening with the mt-sDNA assay. These novel data address an identified knowledge gap and strengthen the biologic basis for earlier-onset, average-risk screening with the mt-sDNA assay.

P-242 Germline mutations identified in Colombian patients with Lynch syndrome

Colorectal cancer (CRC) is the third cause of death from cancer in the world; in Colombia, it is the fourth neoplasm in men and the third in women. Approximately 70% can be classified as sporadic CRC cases and about 30% of CRC cases show a family history of the disease and about 6% have familial aggregation. In our previous studies, we identified 69 family cases among 1,278 patients, 14 were carriers of 48 mutations in high-risk genes such as APC, MLH1, MSH2, PMS2, PTEN, SMAD4, STK11, POLD1, and POLE, while for the remaining 55 family cases, the genetic basis was not established.

Candida albicans culture from a rectal swab can be associated with newly diagnosed colorectal cancer.

Plenty of metagenomic studies have suggested possible associations between microbiome composition and colorectal cancer (CRC). However, these techniques are not economic enough for routine use so far. Therefore, we explored the possibility to detect species associated with colorectal cancer by conventional culture from rectal swab. Fifty-two patients newly diagnosed for adenoma/CRC and 52 age-matched controls were recruited and sampled. Rectal swabs were inoculated on several types of plates and incubated appropriately under both aerobic and anaerobic conditions. All colonial morphotypes were subcultured and identified using MALDI-ToF MS. Although no bacterial species was significantly associated with CRC in our study, we surprisingly observed a strong and significant overrepresentation of the yeast Candida albicans in cases (P = 0.0066, odds ratio 5.444 [95% CI 1.449-20.462]). Potential confounding factors were associated neither with CRC (history of CRC in first-degree relatives, a personal history of appendectomy and cholecystectomy, increased BMI (body mass index), and the percentage of males) nor with C. albicans presence (preexisting diabetes and PPI medication) in our cohort. A growing body of evidence supports the view that C. albicans does cause cancer in humans. We hypothesize that presence of C. albicans in the gut may induce or facilitate some part of the sporadic CRC cases. Our observation should be a strong incentive to verify the potential usefulness of the easily culturable C. albicans yeast as a screening marker for patients at risk of CRC or those suffering an early asymptomatic stage of CRC.

Mo1651 GENOMIC LANDSCAPES OF EARLY-STAGE UC-ASSOCIATED CANCER IN THE JAPANESE POPULATION

Patients with ulcerative colitis (UC) have an increased risk of developing colorectal cancer, known as UC-associated cancer (UCAC). Previous studies have shown that UCACs differ from colorectal tumors that develop in patients without UC, known as sporadic colorectal cancer (CRC), by mutation analysis of a limited number of genes. Although several studies on genomic alterations of advanced-stage UCACs have been reported, there are no reports on UCACs at early-stage. To fill in this knowledge gap, we investigated somatic alterations in UCACs atearly-stage. We extracted DNA from 36 early-stage colorectal tumor tissues (T1 cancer 9, Dysplasia 27) from 32 UC patients using microdissection and performed targeted sequencing of 43 genes that were reported to be strongly associated with Japanese UCACs in a previous report. Mutations from each cancer tissue were compared with counterpart dysplasia tissue and normal colitic mucosa tissue. We found 16 genes that were mutated in more than 3 patients and had a mutation rate of more than 10 mutations/Mb. The most frequently mutated gene in cancers of UC patients was APC (mutated in 47.2% of our cohort), followed by TP53 (44.4%), KRAS (27.8%), PRKDC (27.8%), CSMD3 (22.2%), LTBP4 (13.9%), KIF26B (13.9%), CREBBP (13.9%), TCF7L2 (11.1%), EP300 (11.1%), and ATM (11.1%). Next, we divided 36 early-stage colorectal tumors into 23 UC-associated colorectal neoplasms (UCANs) and 13 colorectal sporadic neoplasms with UC (UCSNs) and compared the mutated frequency of each gene between the UCAN and UCSN groups. We found a significantly higher frequency of somatic mutations of TP53 in the UCAN group than in the UCSN group. Moreover, only 1 out of 6 low-grade dysplasia surrounding UCANs with TP53 mutations had TP53 mutations themselves. In addition, among 11 UCACs tested for p53 immunostaining, all 7 lesions with TP53 somatic mutation were positive for p53 immunostaining and all 4 lesions without the TP53 somatic mutation were negative for p53 immunostaining. Finally, we compared the mutated frequency of each gene in the UCAN and UCSN groups with that of advanced UCAC and sporadic CRCs cases from the literature and The Cancer Genome Atlas. As a result of consideration, there was no difference in the frequency of TP53 somatic mutations between UCAN group, advanced UCAC cases, and TCGA's sporadic CRCs; however, the frequency of TP53 somatic mutations in UCSN groups was significantly lower than that in other 3 groups. TP53 mutations could be biomarkers to distinguish between UCAN and UCSN in early stages. Furthermore, the results of this research suggest that TP53 mutations could occur during the transition from low-grade dysplasia to high-grade dysplasia in UCANs.

Hypothesis: Retinoblastoma protein inactivation mediates effects of histone deacetylase inhibitor-induced Wnt hyperactivation in colorectal cancer cells.

Butyrate, a product of dietary fiber and a histone deacetylase inhibitor, induces apoptosis of colorectal cancer cells; this effect of butyrate is in part mediated by its ability to hyperactivate Wnt signaling, and may in part explain the preventive action of dietary fiber against colorectal cancer. However, the mechanisms by which Wnt hyperactivation promotes apoptosis are unknown. Inactivation of the retinoblastoma tumor suppressor occurs in some cancers and can lead to context-dependent cell proliferation or cell death/apoptosis. The function of retinoblastoma protein (Rb) in normal cells is modulation of cell cycle; inactivation of Rb allows for cell cycle progression and, hence, cell proliferation. Wnt signaling is upregulated in a variety of cancers, and deregulated Wnt signaling is a key initiating event in most cases of sporadic colorectal cancer. It has been shown that Wnt signaling activated by APC inactivation can synergize with the inactivation of Rb to induce apoptosis in a manner mediated by increased TORC1 activity, leading to induced metabolic and energy stress. Rb is typically not inactivated in colorectal cancer; however, Rb is phosphorylated and deactivated during cell cycle G1/S transition. This manuscript posits that it is during this time that butyrate/histone deacetylase inhibitor-induced Wnt hyperactivation induces apoptosis in colorectal cancer cells. Thus, the inactivation of Rb in cell cycle progression may synergize with Wnt hyperactivation to induce apoptosis in response to histone deacetylase inhibitors. The hypothesis is that hyperactivation of Wnt signaling enhances colorectal cancer cell apoptosis via the interaction between upregulated Wnt signaling and inactivated Rb during cell cycle progression. This paper discusses this hypothesis and offers initial experimental approaches for testing the hypothesis. A better understanding of how histone deacetylase inhibitors induce colorectal cancer cell apoptosis through hyperactivation of Wnt signaling, and of cross-talk between repression of cell cycle and induction of apoptosis that occurs with treatment with histone deacetylase inhibitors, can assist in the development of novel therapies for colorectal cancer.

273 Multi-Target DNA Aberrations in Sporadic Colorectal Cancer Tissues Do Not Differ Between Younger and Older Patients

INTRODUCTION: Colorectal cancer (CRC) screening is broadly endorsed for individuals 50–75 years of age. Given recent increases in CRC incidence and mortality for patients &lt;50 years old, the American Cancer Society now recommends initiation of average-risk CRC screening at age 45 (Wolf AMD CA Cancer J Clin 2018). The performance of recommended tests, including the multi-target stool DNA (mt-sDNA; marketed as Cologuard™) test, has been minimally described in populations under 50 years old. To address this knowledge gap, we conducted a retrospective study of DNA markers (KRAS, BMP3, NDRG4) included in the mt-sDNA assay to quantify and compare tissue marker levels in sporadic CRC cases and normal colon controls from patients in 45–49 and 50–64 year-old age groups. Our primary hypothesis was that mt-sDNA marker levels would be statistically similar between cases in both age groups. METHODS: Patient samples were identified from a pathology tissue archive at Mayo Clinic, excluding those with inflammatory bowel disease or familial cancer syndromes. Formalin-fixed, paraffin-embedded tissues were reviewed by a GI pathologist for 45–49 year-old (n = 90) and 50–64 year-old (n = 96) CRC cases; normal colon samples (45–49, n = 76; 50–64, n = 92) were also included for comparison. DNA extracted from core punches was blinded and processed in a randomized order. Quantitative Allele specific Realtime Target and Signal amplification (QuARTS) assays were used to quantify KRAS mutations, and BMP3 and NDRG4 methylation. Marker level in each sample was reported as the QuARTS product of mutant KRAS, methylated BMP3 or NDRG4 relative to the ACTB product in each sample, reported as a percentage. At least 60 patients per group provided sufficient power to demonstrate differences in marker levels of ±25% between case groups and between control groups using the Wilcoxon Rank Sum test. RESULTS: CRC cases in the 45–49 vs 50–64 year-old groups did not differ significantly with respect to sex (P = 0.77) or race/ethnicity (P = 0.06); left-sided CRC was more common in the 45–49 vs 50–64 year-old group (87% vs. 72%; P = 0.02). None of the marker levels were statistically different between cases of the younger vs older groups or between controls of these groups (Figure 1). CONCLUSION: These data support the expectation of no age-related differences in CRC detection by mt-sDNA, as the relevant molecular markers, when they occur, are present at similar levels across age groups 45–49 and 50–64 years.

Abstract 3473: Comprehensive genetic screening in hereditary colorectal cancer

Abstract High-penetrant pathogenic variants in established genes associated with hereditary colorectal cancer (CRC) explain the disease in approximately 5-6% of cases. In as much as 20%-30% of all, including sporadic CRC cases, genetic factors are thought to play a significant role. Dependent on technical as well as limitations due to interpretation challenges, the high-penetrant pathogenic variants are gathered in the exonic regions of the genes. Contributions of pathogenic variants in genes, not traditionally associated with hereditary CRC have also recently been recognized. It is crucial for the patients to get a correct molecular diagnosis that allows for adequate follow-up since the majority of the syndromes include predisposition also for tumors in other organs. A comprehensive panel including high-penetrant susceptibility genes as well as genes not generally associated with hereditary CRC was used for the analyses. Sequencing of 50 kb upstream and downstream of all genes including intronic regions were performed. All SNVs and CNVs in exons were thoroughly evaluated concerning pathogenicity and also intronic and intragenic sequences were screened for the presence of possibly pathogenic variants. In addition, cDNA were analysed to detect variants in splice-sites as well as aberrations causing other effects on mRNA level. The study included 206 patients referred to the Cancer Genetics Counselling Clinic in Gothenburg, Sweden. The spectra of pathogenic variants in well-known high-penetrance predisposition genes as well as the contribution of pathogenic variants in low-penetrant genes were determined. The contribution of pathogenic variants also in “non-CRC” genes was examined. The findings include identification of previously unreported variants and putative causative variants in unexplained patients with hereditary CRC. An attempt to identify putative genetic variants that could explain the variability in cancer risk among carriers of specific variants in high-penetrance genes was also made. Citation Format: Anna Rohlin, Frida Eiengård, Emma Mårtensson, Theofanis Zagoras, Samuel Gebre-Medhin, Margareta Nordling. Comprehensive genetic screening in hereditary colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3473.

Shortcuts to intestinal carcinogenesis by genetic engineering in organoids.

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc‐mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long‐term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus‐based RNAi‐mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full‐blown cancer, recapitulating multi‐step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid‐based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

Tranlesionsynthesis and clinical characteristics of tumor aggression in sporadic colorectal cancer

Background: Colorectal cancer (CRC) is the third most common cause of death by cancer worldwide. About 85% of those cases are sporadic colorectal cancer (CRC).Translesion synthesis (TLS) is a pathway of the DNA damage tolerance system. TLS bypasses DNA lesions, through specialized DNA polymerases, allowing the completion of replication of damaged genomes. This process which is error prone can lead to genomic instability, and may be linked to CRC carcinogenesis. Having in mind the link between TLS and instability, we believe that TLS may influence CRC.This study aims to evaluate gene expression of translesion synthesis (TLS) polymerases and the protein expression status of mismatch repair (MMR) in sporadic CRC cases resected during surgery and infer its influence in the main prognostic clinical and pathological features.

O3-17-1 - Importance to clarify germline hMLH1 K618A missense mutation for clinical surveillance and genetic counseling

Abstract Colorectal cancer (CRC) is the third common cancer and the fourth leading cause of cancer death worldwide. Subproportion CRC with defect function in one of the mismatch repair gene (MMR) are classed as inherited non-polyposis colorectal cancer (HNPCC) later called Lynch syndrome (LS). Disease causing germline mutations have so far been identified in five MMR genes. Among other MMR genes, germline mutations in hMLH1 account up to 50% LS patients and lead to increased risk of cancer development. hMLH1 K618A variant was first identified in our lab and was considered as a disease causing pathogenic alteration, our further investigation also support our previous findings. Although this variant has been investigated intensively during past decade, however the pathogenicity still remain unclear due to controversial data published up to date. In present study, hMLH1 K618A variant were genotyped in 2,743 individuals (1,087 sporadic CRC patients, 239 CRC patients with at least one affected close relative, 69 CRC cases with familial history and 1,348 controls) by TaqMan genotyping assay. Our results demonstrate that 30 individuals were heterozygous for the K618A variant; thirteen normal control had this variants and measurement of allele frequency was 0.96 % which is remarkably higher than previous published data, while eight were in sporadic CRC cases and allele frequency was 0.73 %, six were in CRC patients with at least one affected relative and allele frequency was 2.51 %, three were in CRC patients with familial history and allele frequency was 4.34 %. We conclude that hMLH1 K618A variant may not have effect in sporadic CRC development, in contrast that K618A may significantly increase CRC risk in CRC patients with strong familial history in Swedish population.

CTLA4 tagging polymorphisms and risk of colorectal cancer: a case-control study involving 2,306 subjects.

BackgroundCTLA4 is a candidate gene which has been implicated in the development of colorectal cancer (CRC).Patients and MethodsTo determine the important role of CTLA-4 polymorphisms on risk of CRC, we genotyped four CTLA-4 tagging polymorphisms and calculated crude/adjusted ORs with their 95% CIs. We recruited 1,003 sporadic CRC cases and 1,303 controls.ResultsThe findings suggested that CTLA-4 rs231775 G>A polymorphism increased the risk of CRC (homozygote model: adjusted OR=1.40, 95% CI=1.05–1.87, P=0.022; dominant model: adjusted OR=1.19, 95% CI=1.00–1.41, P=0.047; and recessive model: adjusted OR=1.38, 95% CI=1.05–1.82, P=0.021). In a stratified analysis by site of tumor, this association was also found in colon cancer. We also found that CTLA-4 rs231775 GA/AA genotypes might be associated with an increased risk of CRC in Zhenjiang cohort. In addition, we found the CTLA-4 rs16840252 C>T polymorphism was associated with the risk of colon cancer. Haplotype comparison analysis showed that CTLA-4 Grs3087243Crs16840252Crs733618 Ars231775, Grs3087243Crs16840252Trs733618Ars231775, and other haplotypes increased the risk of CRC (P<0.001, <0.001, and 0.002, respectively).ConclusionThis study evidences an association of CTLA-4 tagging polymorphisms and haplotypes with CRC risk. Additional well-designed studies with large sample sizes are required to confirm our findings.

Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.