Hereditary Colorectal Cancer Research Articles

Whole-Exome Sequencing Identifies Pathogenic Germline Variants in Patients with Lynch-Like Syndrome.

Simple SummaryA significant proportion of families with a clinical suggestion of Lynch syndrome and screened for the known MMR genes remain without a molecular diagnosis. These patients, who generally show a suggestive family pedigree or early-onset tumors with MMR deficiency and no detectable germline variants, are referred to as having Lynch-like syndrome. To investigate underlying and potentially predisposing variants related to Lynch-like syndrome, we performed whole-exome sequencing in patients with clinical criteria for Lynch syndrome, MMR deficiency and without germline variants. This approach allowed for the identification of new variants potentially associated with Lynch-like syndrome, providing new clues to explain the familial predisposition to Lynch syndrome-related tumors in these patients, which could lead to new screening strategies for the identification of families at risk of developing cancer.Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, characterized by germline pathogenic variants in mismatch repair (MMR)-related genes that lead to microsatellite instability. Patients who meet the clinical criteria for LS and MMR deficiency and without any identified germline pathogenic variants are frequently considered to have Lynch-like syndrome (LLS). These patients have a higher risk of CRC and extracolonic tumors, and little is known about their underlying genetic causes. We investigated the germline spectrum of LLS patients through whole-exome sequencing (WES). A total of 20 unrelated patients with MMR deficiency who met the clinical criteria for LS and had no germline variant were subjected to germline WES. Variant classification was performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Pathogenic/likely pathogenic variants were identified in 35% of patients in known cancer genes such as MUTYH and ATM. Besides this, rare and potentially pathogenic variants were identified in the DNA repair gene POLN and other cancer-related genes such as PPARG, CTC1, DCC and ALPK1. Our study demonstrates the germline mutational status of LLS patients, a population at high risk of colorectal cancer.

A case of early onset adenocarcinoma associated with colorectal polyposis with an unknown germline mutation

BackgroundTypically, in cases of adenomatous polyposis, colorectal cancer develops in the third or fourth decade of life. We report the case of a female patient with colorectal polyposis who developed adenocarcinoma at 8 years of age.Case presentationAn 8-year-old girl was admitted with a 4-year history of occasional bloody stools. Colonoscopy revealed colon polyposis and histopathological assessment confirmed a well-differentiated adenocarcinoma in the adenomatous polyps, so laparoscopy-assisted proctocolectomy was performed in the lithotomy position by a simultaneous abdominal and anal approach. To completely resect the rectal mucosa, excision was commenced just distal to the dentate line. After the mucosal resection up to the peritoneal reflection level, an inverted muscular cuff was cut circumferentially, and the terminal ileum was pulled through the muscular cuff and anastomosed to the anal canal. Histopathology revealed multiple adenomatous polyps and scattered well-differentiated tubular adenocarcinomas (tub1) in the adenomatous polyps and the non-polypoid mucosal lesions. Because complete resection was achieved, additional adjuvant chemotherapy was not administered. Polymerase chain reaction (PCR)-direct sequencing of the entire coding region and the exon–intron junctions, and real-time PCR of DNA extracted from blood cells, revealed no mutations of either APC or MUTYH. No deletions, duplications, translocations or inversions of APC, MUTYH and GREM1 genes were found using multiplex ligation-dependent probe amplification (MLPA) and G-banding analysis. Multi-gene panels sequencing for polyposis syndromes or hereditary colorectal cancers, and trio-whole exome sequencing was conducted. However, no candidate pathogenic variants of genes were detected in de novo dominant or autosomal recessive model. Somatic mutation of APC was not detected in 4 polyps by loss of heterozygosity analysis at a single nucleotide polymorphism in intron 14. The patient has remained disease-free for 5 years. Currently, the patient is on loperamide and passes stool 5 times/day without any soiling.ConclusionsThe genetic analysis suggests that she may have a germline mutation at unscreened region of these genes or in unidentified FAP gene. The patient will be carefully followed up for residual rectal carcinoma and for the development of other cancers.

Identifying Patients Who Meet Criteria for Genetic Testing of Hereditary Cancers Based on Structured and Unstructured Family Health History Data in the Electronic Health Record: Natural Language Processing Approach.

BackgroundFamily health history has been recognized as an essential factor for cancer risk assessment and is an integral part of many cancer screening guidelines, including genetic testing for personalized clinical management strategies. However, manually identifying eligible candidates for genetic testing is labor intensive.ObjectiveThe aim of this study was to develop a natural language processing (NLP) pipeline and assess its contribution to identifying patients who meet genetic testing criteria for hereditary cancers based on family health history data in the electronic health record (EHR). We compared an algorithm that uses structured data alone with structured data augmented using NLP.MethodsAlgorithms were developed based on the National Comprehensive Cancer Network (NCCN) guidelines for genetic testing for hereditary breast or ovarian and colorectal cancers. The NLP-augmented algorithm uses both structured family health history data and the associated unstructured free-text comments. The algorithms were compared with a reference standard of 100 patients with a family health history in the EHR.ResultsRegarding identifying the reference standard patients meeting the NCCN criteria, the NLP-augmented algorithm compared with the structured data algorithm yielded a significantly higher recall of 0.95 (95% CI 0.9-0.99) versus 0.29 (95% CI 0.19-0.40) and a precision of 0.99 (95% CI 0.96-1.00) versus 0.81 (95% CI 0.65-0.95). On the whole data set, the NLP-augmented algorithm extracted 33.6% more entities, resulting in 53.8% more patients meeting the NCCN criteria.ConclusionsCompared with the structured data algorithm, the NLP-augmented algorithm based on both structured and unstructured family health history data in the EHR increased the number of patients identified as meeting the NCCN criteria for genetic testing for hereditary breast or ovarian and colorectal cancers.

A qualitative analysis of patient- and provider-perceived barriers and facilitators to cascade genetic testing for hereditary breast, ovarian, endometrial and colorectal cancer (423)

A qualitative analysis of patient- and provider-perceived barriers and facilitators to cascade genetic testing for hereditary breast, ovarian, endometrial and colorectal cancer (423)

Routine Immunohistochemical Analysis of Mismatch Repair Proteins in Colorectal Cancer-A Prospective Analysis.

Simple SummaryRecognition of a hereditary colorectal cancer (CRC) syndrome is crucial. Our aim was to assess the value of routine immunohistochemistry screening for mismatch repair proteins deficiency in CRC patients under 70 years-old. In our cohort, this inclusive strategy allowed the identification of Lynch Syndrome patients that could otherwise be missed using a restrictive approach that relies only on Amsterdam and Bethesda criteria. This study strengthens current recommendations and highlights the role of universal CRC screening for MMR protein status.Recognition of a hereditary colorectal cancer (CRC) syndrome is crucial and Lynch Syndrome (LS) is the most frequent immunohistochemistry (IHC)—screening for mismatch repair proteins (MMR) deficiency in CRC is therefore advocated. An unicentric cohort study was conducted in a central Oncological Hospital to assess its results. All patients under 70 years-old admitted between July 2017–June 2019 and submitted to surgery for CRC were included. Of 275 patients, 56.0% were male, median age 61.0 (IQR:54.5–65.0), with synchronous tumors in six. Histology revealed high grade adenocarcinoma in 8.4%; mucinous and/or signet ring differentiation in 11.3%; and lymphocytic infiltration in 29.8%. Amsterdam (AC) and Bethesda (BC) Criteria were fulfilled in 11 and 74 patients, respectively. IHC revealed loss of expression of MMR proteins in 24 (8.7%), mostly MLH1 and PMS2 (n = 15) and PMS2 (n = 4). Among these, no patients fulfilled AC and 13 fulfilled BC. BRAF mutation or MLH1 promoter hypermethylation was found in four patients with MLH1 loss of expression. Genetic diagnosis was performed in 51 patients, 11 of them with altered IHC. LS was diagnosed in four, and BC was present in three. One patient would not have been diagnosed without routine IHC screening. These results strengthen the important role of IHC screening for MMR proteins loss of expression in CRC.

Extracellular Nucleic Acids in the Diagnosis and Progression of Colorectal Cancer.

Simple SummaryColorectal cancer (CRC) is a disease that usually shows no evident clinical symptoms in the early stages, often leading to late diagnosis. Over the past few years, a new approach based on liquid biopsy has gained far-reaching applications in less-invasive CRC diagnosis and management, allowing for the use of extracellular nucleic acids as promising biomarkers to detect CRC at an early stage and monitor disease recurrence. That is why an up-to-date review and discussion of in-depth liquid biopsy-derived DNA and RNA biomarkers is essential. We hereby offer an overview of known predisposing genetic factors for developing sporadic and hereditary CRC, and an extensive repertoire of available extracellular DNA/RNA molecules with their potential clinical applications and shortcomings. Our review may be of value to experts dealing with CRC at the molecular level as well as to clinical professionals aiming for a better understanding of state-of-the-art techniques in CRC diagnosis and management.Colorectal cancer (CRC) is the 3rd most common malignant neoplasm worldwide, with more than two million new cases diagnosed yearly. Despite increasing efforts in screening, many cases are still diagnosed at a late stage, when mortality is high. This paper briefly reviews known genetic causes of CRC (distinguishing between sporadic and familial forms) and discusses potential and confirmed nucleic acid biomarkers obtainable from liquid biopsies, classified by their molecular features, focusing on clinical relevance. We comment on advantageous aspects such as better patient compliance due to blood sampling being minimally invasive, the possibility to monitor mutation characteristics of sporadic and hereditary CRC in a disease showing genetic heterogeneity, and using up- or down-regulated circulating RNA markers to reveal metastasis or disease recurrence. Current difficulties and thoughts on some possible future directions are also discussed. We explore current evidence in the field pointing towards the introduction of personalized CRC management.

Fusobacterium nucleatum load in MSI colorectal cancer subtypes.

Fusobacterium nucleatum (F. nucleatum) infection may lead to colorectal cancer (CRC) development in the context of microsatellite instability (MSI). To date, however, the relationship between F. nucleatum load and MSI CRC subtypes has not been clarified. One hundred seventy-nine consecutive patients with CRC were enrolled in the present study. In 94 patients with MSI CRC, 32 had hereditary MSI CRC from Lynch syndrome, 62 had sporadic MSI CRC, while the remaining 85 had microsatellite stable (MSS) CRC. The association of the F. nucleatum load with each CRC subtype and the patients' clinicopathological characteristics was examined. Of the 179 patients with CRC, 158 (88.3%) were F. nucleatum-positive. A high F. nucleatum load was found in 84.4% (27/32), 96.8% (60/62), and 83.5% (71/85) of the patients with hereditary MSI CRC, sporadic MSI CRC, and MSS CRC, respectively (P = 0.024). In terms of clinicopathological features, a high F. nucleatum load was significantly associated with female, right-sided CRC, BRAF V600E, CpG island methylator phenotype-positive CRC, and MSI CRC (P = 0.008, P = 0.015, P = 0.007, P = 0.006, and P < 0.001, respectively). However, the clinicopathological characteristics did not differ significantly by F. nucleatum load between hereditary and sporadic MSI CRCs without tumor depth. The F. nucleatum load was higher in hereditary MSI CRC than in MSS CRC as well as sporadic MSI CRC. These findings may contribute to preventing CRC in hereditary MSI CRC through appropriate intervention.

Current management status of hereditary colorectal cancer

Hereditary colorectal cancer accounts for approximately 5% of all colorectal cancer cases, mainly including familial adenomatous polyposis and Lynch syndrome. Total proctocolectomy plus ileal pouch-anal anastomosis and total colectomy plus ileorectal anastomosis are two major procedures for familial adenomatous polyposis, however, the exact impact of these two procedures on surgical efficacy, oncologic efficacy as well as functional results still remains uncertain. Segmental colectomy and total colectomy are two major procedures for Lynch syndrome, each of them both has advantages and disadvantages, and there still lacks a consensus about the optimal strategy because of the nature of retrospective study with a relatively insufficient evidence support. As a result, we would make a review about the current surgical treatment status and future perspectives of hereditary colorectal cancer.

Shared environment and colorectal cancer: A Nordic pedigree registry-based cohort study.

Risk of colorectal cancer (CRC) increases in relatives of patients with CRC. The extent to which this is attributable to genetic predisposition or shared environment is unclear. We explored this question using nationwide cohorts from Denmark, Finland and Sweden. From 1977 to 2013, we identified 359 879 individuals with a CRC diagnosis and 2 258 870 of their relatives who we followed for CRC incidence. We calculated standardized incidence ratios (SIR) and 95% confidence intervals (CI) for CRC in individuals with an affected relative. We used nationwide household and pedigree data along with national SIR estimates to calculate risk ratios (RR) for the contribution of shared household environment, childhood environment and genetic relationship to CRC risk in those with an affected relative. SIR of CRC was increased for individuals with an affected relative, across all countries and ages. For those with an affected parent, the SIR was 1.65 (95% CI: 1.61‐1.69), 1.98 (95% CI: 1.87‐2.09), for those with an affected sibling and 2.14 (95% CI: 1.84‐2.49) for those with an affected halfsibling. In those <65 years old, shared childhood (RR: 1.41, 95% CI: 1.26‐1.57) and household (RR: 2.08, 95% CI: 1.25‐3.46) environments were significantly greater contributors to familial risk of CRC than genetics (RR: 0.88, 95% CI: 0.53‐1.46). This large‐scale Nordic population‐based study of excess risk of CRC among relatives of those with CRC addresses the difficult disentangling of shared environment from genetic predisposition in the heritability of CRC. We found shared environment to be the most important contributor to CRC risk.

Abstract 5773: A one-stop approach in diagnosing hereditary colorectal cancer: A prospective, proof-of-principle, single-center study

Abstract Background: Colorectal cancer (CRC) remains one of the most commonly diagnosed cancers and a leading cause of cancer-related deaths worldwide. Between 3 and 6% of all CRC cases are attributed to hereditary colorectal cancer (HCRC). It is important to distinguish CRCs that arise hereditarily from those that arise sporadically, because patients with HCRC and their relatives can benefit from intensive clinical management and surveillance. The detection of germline variants using tumor tissue and paired non-cancerous tissue/whole blood cells (WBC) (conventional method) is considered as the gold standard for germline confirmation, but which could introduce high cost and long turnaround time. In this work, we aimed to investigate the feasibility of diagnosing HCRC using only tumor next-generation sequencing data without normal tissue/WBC data. Methods: One-hundred patients with suspected HCRC were prospectively enrolled according to the clinical diagnosis. Both tumor tissue and paired WBC were collected from each patient. Capture-based targeted sequencing using a panel consisting of 41 cancer-related genes was performed on both tumor tissue and WBC sample. The germline variants were identified using conventional and ColonCore method, respectively. The sensitivity and positive predictive value (PPV) of ColonCore method in identifying germline alterations were calculated, when conventional method was used as a reference. Results: A total of 636 alterations were identified using ColonCore method, including 606 somatic alterations and 30 germline alterations. Among the somatic alterations detected from this panel, 599 single-nucleotide variants and 7 copy number variants were observed. The most frequently mutated genes were APC, TP53, and KRAS, occurring in 76%, 66%, and 46% of patients, respectively. For ColonCore analysis, 17, 9, 2, and 2 patients harbored germline pathogenic variants in DNA mismatch repair (MMR) genes, APC, MUTYH, and ATM, respectively. Compared to the conventional method, the sensitivity of ColonCore method in the diagnosis of HCRC achieved 88.5% with a PPV of 82.1%. Next, the diagnostic performance of ColonCore method in Lynch syndrome (LS) and familial adenomatous polyposis (FAP) was subsequently investigated. We found its sensitivity in the diagnosis of LS achieved 100% (with a PPV of 98.8%), which was obviously higher than that in the diagnosis of FAP (70.0% with a PPV of 63.6%). Twenty-three patients identified as having a microsatellite instability (MSI)-high tumor using ColonCore method were also identified as having an MMR-deficient (dMMR) tumor using immunohistochemistry staining analysis. Conclusions: Our study suggests that ColorCore method is a one-stop approach for detecting germline/somatic alterations and MSI/dMMR status for suspected HCRC. Furthermore, ColorCore method is a feasible and valid tool to diagnose HCRC, especially LS. Citation Format: Cong Li, Yun Xu, Fangqi Liu, Ye Xu. A one-stop approach in diagnosing hereditary colorectal cancer: A prospective, proof-of-principle, single-center study [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 5773.

Abstract 721: Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer

Abstract Introduction: Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC) affecting &amp;gt;1 million Americans. LS is caused by germline mutations in one of four DNA mismatch repair (MMR) genes, generating several hundreds of mutations in microsatellite coding sequences. Although defects in the MMR machinery are the principal mechanism driving LS carcinogenesis, the role of the epigenome through histone and DNA methylation remains unexplored. Hypermethylation of MLH1 is detected in 15% of sporadic MMR-deficient CRC and also in LSCRC, indicating a pivotal role of DNA methylation in LSCRC. In this study, we investigated the landscape of DNA methylation and EZH2-mediated histone H3 lysine 27 methylation in LS tumor samples, and tested the preventive effects of GSK-503, on tumor formation using a novel LS mouse model. Methods: We profiled the somatic mutational, global DNA methylation landscape, and performed ChIP-seq in LS precancers and tumors. Patient-derived organoids (PDO) from colonic tissues treated with EZH2 and 5-AZA were used for gene expression analysis. LS mice (Villin-Cre;Msh2fl/fl;TgfβRII-KI) were treated for 9 weeks with EZH2 inhibitor GSK503 to assess the effect on polyp formation in vivo monitored through murine colonoscopy. Results: Our mutational profiling via whole-exome-seq and gene expression survey of 10 LSCRC and 33 pre-cancers showed accumulation of somatic mutations and aberrant expression patterns in key epigenetic regulators including EZH2 and DNMT3A, which suggests dysregulation of epigenetic control in LS. Global DNA methylation using RRBS in tumors of 3 LS patients showed that gene promoters were more hypermethylated when compared to matched normal samples. Immunohistochemistry data showed higher expression of EZH2 in tumors when compared to precancerous and normal colonic mucosa. ChIPseq analysis demonstrated high levels of H3K27me3 present in gene promoters of stem cell marker genes (WNT9A, LGR5, and ASCL2), and immune-related genes (CD4, CD8a, and HLA-E). Interestingly, 577 out of 2756 DNA hypermethylated genes from RRBS analysis displayed high levels of H3K27me3, thus indicating a central role of EZH2 in epigenetic programming of LS neoplasms. RNAseq data from PDOs treated with 5-AZA (DNMT inhibitor) and GSK503 (EZH2 inhibitor) showed that both epigenetic inhibitors led to the reactivation of silenced genes in colorectal carcinogenesis such as CDKN2A, RUNX3, IGFBP3, and immune genes, including CD40, CD4, SEMA3B, CXRC6/6, and ICAM1. Finally, a 9-week chemo-preventive treatment of a cohort of LS mice with GSK503 significantly decreased polyp formation when compared to control, and polyp reduction was associated with increased infiltration of cytotoxic CD8a+ Tcells and NK in the large intestine mucosa. Conclusions: These results demonstrate the chemopreventive potential of EZH2 inhibitors in prevention of LS-associated tumors through an immune modulation approach. Citation Format: Laura Reyes Uribe, Charles M Bowen, Kyera Evans, Hong Wu, Fahriye Duzagac, Krishna Sinha, Eduardo Vilar. Epigenetic intervention in the prevention of Lynch Syndrome Colorectal Cancer [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 721.

Abstract 3386: Detection of TGFbR2 frameshift mutation

Abstract TGFβR2 a10→a9 frameshift mutation is an early occurring cancer driver mutation and mutant TGFβR2 cell free DNA (cfDNA) is therefore a potential biomarker for early detection of cancer (E.G. Lynch Syndrome) as well as for monitoring response to cancer treatment. The frameshift mutation is associated with micro satellite instability (MSI) and is found in 80% of MSI-colorectal cancers (MSI-CRC) and in over 90% of hereditary nonpolyposis colorectal cancer. Here we show that cell free mutant (a9) TGFβR2 DNA is present in liquid biopsies from MSI-CRC patients and that the a9 micro satellite of mutant TGFβR2 can be selectively detected by using specially designed PCR primers modelled after the a9 microsatellite. PCR and primers defining a fragment of TGFβR2 comprising the a10/a9 microsatellite was used to amplify cfDNA extracted from MSI-CRC and microsatellite stable-CRC (MSS-CRC) patients. The resulting PCR products were analysed and confirmed by DNA sequencing after gel electrophoresis. In addition, primers modelled after the shorter a9 microsatellite was designed and tested using synthetic DNA of both TGFβR2 variants. Despite the naturally established mismatch between the a10 sequence and primers modelled after the a9 sequence, conventionally designed primers could not differentiate enough for specific annealing to the a9 sequence. A further mismatch with the a10 sequence was introduced at the 3’ end of the primer, which, in combination with the already established mismatch, denied the primer tail-end annellation with the a10 sequence that is necessary for polymerase amplification of this sequence. Experiments to optimise PCR conditions and buffer solution were conducted prior to analysing the cfDNA extracted from the patient samples. It was found that TGFβR2 DNA was present in cfDNA from all (10/10) CRC patients tested and that TGFβR2 a10→a9 frameshift was present in all (5/5) MSI-CRC patients and none (0/5) of MSS-CRC patients tested, shown by sequencing. PCR using the selected a9 modelled primer yielded a PCR product that was clearly detectable on the electrophoresis gel only for cfDNA from the MSI-CRC patients (5/5). The results show that TGFβR2 a10→a9 frameshift DNA is present in cfDNA of liquid biopsies from MSI-CRC patients which establishes cell free TFGβR2 frameshift DNA as a potential biomarker for early detection of hereditary CRC (Lynch Syndrome) as well as for monitoring cancer progression and remission of sporadic MSI-CRC. Citation Format: Henrik K. Eriksen, Jon A. Eriksen. Detection of TGFbR2 frameshift mutation [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 3386.

Prevalence and risk factors of barrett's esophagus in lynch syndrome.

Lynch syndrome (LS), the most common hereditary cause of colorectal cancer, predisposes to upper gastrointestinal neoplasia. The prevalence of Barrett's esophagus (BE) is elevated in some hereditary gastrointestinal cancer syndromes but has not been systematically evaluated in LS. We assessed the prevalence of BE, BE-related dysplasia, esophageal adenocarcinoma (EAC), and factors associated with BE in LS. Asymptomatic patients with a germline pathogenic variant (PV) in the DNA mismatch repair (MMR) genes undergoing EGD for LS surveillance were identified from a hereditary colorectal cancer registry. We assessed the prevalence of BE and compared demographic, clinical, and endoscopic factors in LS patients with and without BE by logistic regression analysis. 323 patients were included. 21 patients (6.5%) were diagnosed with BE including 38% of females and 33% without gastroesophageal reflux disease (GERD). Dysplasia was diagnosed in two patients (9.5%) and EAC in one (4.8%) patient. Factors associated with BE included male gender (OR 3.00, 1.21-7.46), age at last LS EGD (OR 1.04, 1.01-1.08), presence of hiatal hernia (OR 20.09, 4.57-88.23), hiatal hernia > 3cm (OR 11.25, 2.41-51.94), and GERD (OR 3.39, 1.32-8.67). No MMR PV was associated with BE. BE was diagnosed in 1 of 15 patients undergoing EGD surveillance for LS and nearly 10% had dysplasia including one EAC. Risk factors associated with BE in LS are similar to those established for BE in the general population. More studies are needed to evaluate if an association between BE and LS exists.

Variants of unknown significance (VUS) in patients with hereditary CRC without a known pathogenic variant.

10599 Background: Nearly 5-10% of all newly diagnosed colorectal cancer (CRC) cases develop due to the presence of a highly penetrant pathogenic variant in one of the hereditary colorectal cancer (hCRC) associated genes. NGS technology raised the opportunity for fast and efficient detection of these variants, but still in ̃20-30% of patients with hCRC the genetic defect remains unknown. Recent data shows that 6-8% of these cases harbor pathogenic/VUS variant in low/moderate penetrance genes not directly associated with hCRC. Methods: A total of 109 patients with hCRC (43 with polyposis and 66 with non-polyposis syndromes) were analyzed by NGS covering coding and exon/intron sequences of 109 genes, of which 25 associated with known hCRC syndromes and 83 other cancer predisposition genes. Results: Pathogenic variants were detected in 63/109 (57.7%) of analyzed patients; 54/63 (85.7%) of these had a pathogenic variant in one of the genes associated with hCRC (APC, MMR genes, MUTYH, NTHL1, BMPR1A) and 9/63 (14.3%) had a pathogenic variant detected in genes not directly related to hCRC (CHEK2, FANCL, FANCM, ERCC2, BRIP1, FLCN, BLM). In 26/109 (23.8%) patients a rare VUS variant was detected, of which 14/26 (53.8%) in double strand repair (DRG) genes (BLM, CHEK2, PALB2, ATM, MRE11A, BRIP1, FANCM, FANCL and ERCC2), 8/26 (30.8%) in one of the known hCRC genes (APC, MSH6, PMS2 and POLE) and 4/26 (15.4%) in other cancer predisposition genes (KIT, NSD1, CDH1, EZH1 and FH). VUS variants in DRG genes were more common in patients with MSI- HNPCC (9/15, 60%), compared to patients with polyposis syndromes (6/15, 40%). The VUS variants in other cancer predisposition genes were dominantly present in patients with oligopolyposis. Most (21/26, 80.8%) VUS variants were detected as single variants, while only five patients (5/26, 19.2%) had two different variants in two different genes. In 20/109 (18.3%) patients who presented primarily with MSI- HNPCC or oligopolyposis phenotypes, no pathogenic and/or VUS variants were detected in the 109 analyzed genes. Conclusions: Genetic basis of hereditary CRC was not clearly defined in a large proportion (42.3%) of patients in our cohort. Although a VUS variant was detected in a significant portion (23.8%), a major fraction (18.3%) of patients had no known genetic variant detected. The presence of a high frequency of VUS variants in DRG genes indicates that this pathway plays an important role in CRC carcinogenesis. Although we cannot exclude the presence of deep intronic and/or regulatory region pathogenic variants in the analyzed genes, it appears that the current list of identified cancer predisposition genes responsible for the hereditary CRC is far from being complete. The influence of environmental factors in conjunction with polygenic inheritance might also play a key role in a fraction of hCRC in our population.

Evaluation of Lynch syndrome risk models in a multicenter diverse population.

10597 Background: Lynch syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC) with an increased CRC lifetime risk of 70-80%. LS affects 1:250 individuals and is caused by pathogenic variants in the mismatch repair (MMR) genes. Statistical prediction models such as MMRpro and PREMM5 are widely used to identify LS carriers. However, these models are trained and validated in mostly white populations, and there remains a gap in understanding their performance in Hispanic populations. The purpose of this study was to evaluate the performance of MMRpro and PREMM5 on a large Hispanic cohort from the Clinical Cancer Genomics Community Research Network (CCGCRN). Methods: We validated MMRpro and PREMM5 on 3,490 CCGCRN families, of which 1,122 are Hispanic and 2,062 Non-Hispanic. The two models were evaluated for discrimination using the C-statistic, calibration using the observed to expected ratio (O/E), and overall performance using the root Brier score and negative and positive predictive value (NPV/PPV) at the 5% carrier probability threshold. Evaluations were stratified by ethnicity, and 95% confidence intervals were obtained via bootstrapping for all measures. Results: The C-statistic is 0.90 for both MMRpro (95% CI: 0.88, 0.92) and PREMM5 (95% CI: 0.87, 0.92). When stratified by ethnicity, the C-statistics are 0.96 (95% CI: 0.94, 0.97) and 0.86 (95% CI: 0.83, 0.89) for Hispanics and Non-Hispanics, respectively, in MMRpro, and 0.96 (95% CI: 0.94, 0.97) and 0.84 (95% CI: 0.79, 0.88) in PREMM5. Both models underpredict mutation probabilities, with O/E ratios ranging from 1.79 to 1.96. At a 5% threshold, variations in PPV between Hispanics and Non-Hispanics are observed in both models: 0.72 (95% CI: 0.63, 0.80) and 0.43 (95% CI: 0.37, 0.50) in Hispanic and Non-Hispanic groups in MMRpro; 0.50 (95% CI: 0.43, 0.57) and 0.25 (95% CI: 0.20, 0.30) in PREMM5. We observe less variation and higher values in NPVs in both models. Conclusions: Overall, MMRpro and PREMM5 perform well in this cohort in predicting the probability of having a pathogenic variant in an MMR gene, with modest underprediction. While these results offer reassurance for the clinical use of MMRpro and PREMM5 in Hispanic populations, further validation studies in underrepresented racial and ethnic populations are crucial.

Clinical implications of germline genetic testing stratified by ethnicity in a large colorectal cancer cohort.

10504 Background: Germline genetic testing for patients (pts) with colorectal cancer (CRC) is currently recommended for those diagnosed prior to age 50, as well as other select cases based on personal and/or family history criteria. Whether universal germline multi-gene panel testing (MGPT) should be performed in all pts with CRC remains uncertain given the lack of large-scale studies examining MGPT in CRC across a diverse population. The present study aims to determine the yield, and potential clinical impact, of MGPT across a large CRC cohort. Methods: We conducted a de-identified retrospective cohort study of all pts with CRC who underwent MGPT (excluding patients who underwent testing limited to ≤10 genes) at a commercial laboratory between 03/2015-05/2021. We collected pts demographics from test requisition forms and results of germline MGPT. Clinically actionable PGVs were defined as variants in genes with reported CRC or polyposis risk, as well as other actionable genes associated with clinical management and/or therapeutic implications. Results: A total of 34,210 pts with a history of CRC underwent germline MGPT. These pts were primarily female (60.7%), White (70.6%), and 50 or older (68.8%), with 35.5% reporting an extra-colonic malignancy. Of this cohort, 4,577 (13.4%) were found to carry at least one PGV, with 2,925 (8.6%) having a PGV associated with increased risk of CRC or polyposis. Of all positive pts, 3,038 (66%) had PGVs with precision therapy or clinical trial implications while another 33% had PGVs with published management implications. Among pts under the age of 30 when tested, 23.3% had a clinically actionable PGV compared to 14.7% at age 30-39, 11.7% at 40-49, 12.9% at 50-59, 11.6% at 60-69, 8.9% 70-79, and 7.8% over the age of 80. When compared to pts identified as White, PGVs were more frequently identified in pts of Ashkenazi Jewish descent (p &lt; 0.001) and less frequently identified in those who identified as Hispanic (p &lt; 0.001). VUS was more frequently identified in pts identified as Black, Asian, or Hispanic (p &lt; 0.001). Conclusions: This is the largest study to date examining MGPT in CRC, where we demonstrate high rates of clinically actionable variants across all age groups, self-reported racial/ethnic groups, and panel sizes, with 13% of all CRC pts having a PGV with precision therapy, clinical trial and/or published management implications.This is likely an underestimate as patients with strong clinical suspicion of hereditary CRC (Lynch, FAP) often receive a targeted panel of &lt;10 genes and were excluded. The lower rate of PGVs in Hispanic pts and higher rate of VUS in Black, Asian and Hispanic pts underscores the historical underrepresentation of these pts and ongoing need to mitigate the associated healthcare disparities. Overall, this work supports consideration of broadening germline genetic testing criteria for patients with CRC.

Colorectal cancer screening participation among citizens not recommended to be screened: a cohort study

BackgroundGuidelines on colorectal cancer (CRC) screening recommend screening of average-risk adults only. In addition, screening of individuals with active inflammatory bowel disease (IBD) might result in too many false-positive cases. However, the organisers of CRC screening programmes are often uninformed of whom to exclude due to an elevated CRC risk or active IBD. It is therefore unknown how often high-risk individuals (i.e. individuals with a previous diagnosis of CRC or polyps associated with hereditary CRC syndromes and certain patient groups with a diagnosis of inflammatory bowel disease (IBD) or multiple polyps) and individuals with active IBD participate in CRC screening following invitation.Materials and methodsWe used data from the first two years of the Danish CRC screening programme (2014–2015). Information on invitations, participations and FIT test results were obtained from the national screening database, while information on previous CRC, hereditary CRC syndromes, IBD or multiple polyps diagnoses were obtained from the Danish Cancer Registry and the Danish Patient Register. Screening participation rates and FIT-positive rates were calculated and compared for high-risk invitees, invitees having IBD and an average risk group of remaining invitees not diagnosed with colorectal polyps in 10 years preceding the invitation.ResultsWhen invited to CRC screening, 28–48% of high-risk residents (N: 29; 316; 5584) and 55% of residents with IBD (N: 2217; 6927) chose to participate. The participation rate was significantly higher (67%) among residents without previous colorectal disease, i.e. the average risk group (N = 585,624). In this average group 6.7% of the participants had a positive FIT test. The proportion of positive FIT results was higher among all disease groups (7.7–14.8%), though not statistically significant for participants with prior CRC diagnosis and participants with high-risk IBD.ConclusionWhen high-risk residents and residents with IBD receive an invitation to CRC screening, many participate despite being recommended not to. The screening program was not intended for these groups and further research is needed as several of these groups have a higher rate of positive screening result than the average risk population.

Colon Tumors in Enterotoxigenic Bacteroides fragilis (ETBF)-Colonized Mice Do Not Display a Unique Mutational Signature but Instead Possess Host-Dependent Alterations in the APC Gene.

ABSTRACTEnterotoxigenic Bacteroides fragilis (ETBF) is consistently found at higher frequency in individuals with sporadic and hereditary colorectal cancer (CRC) and induces tumorigenesis in several mouse models of CRC. However, whether specific mutations induced by ETBF lead to colon tumor formation has not been investigated. To determine if ETBF-induced mutations impact the Apc gene, and other tumor suppressors or proto-oncogenes, we performed whole-exome sequencing and whole-genome sequencing on tumors isolated after ETBF and sham colonization of Apcmin/+ and Apcmin/+Msh2fl/flVC mice, as well as whole-genome sequencing of organoids cocultured with ETBF. Our results indicate that ETBF-induced tumor formation results from loss of heterozygosity (LOH) of Apc, unless the mismatch repair system is disrupted, in which case, tumor formation results from new acquisition of protein-truncating mutations in Apc. In contrast to polyketide synthase-positive Escherichia coli (pks+ E. coli), ETBF does not produce a unique mutational signature; instead, ETBF-induced tumors arise from errors in DNA mismatch repair and homologous recombination DNA damage repair, established pathways of tumor formation in the colon, and the same genetic mechanism accounting for sham tumors in these mouse models. Our analysis informs how this procarcinogenic bacterium may promote tumor formation in individuals with inherited predispositions to CRC, such as Lynch syndrome or familial adenomatous polyposis (FAP).IMPORTANCE Many studies have shown that microbiome composition in both the mucosa and the stool differs in individuals with sporadic and hereditary colorectal cancer (CRC). Both human and mouse models have established a strong association between particular microbes and colon tumor induction. However, the genetic mechanisms underlying putative microbe-induced colon tumor formation are not well established. In this paper, we applied whole-exome sequencing and whole-genome sequencing to investigate the impact of ETBF-induced genetic changes on tumor formation. Additionally, we performed whole-genome sequencing of human colon organoids exposed to ETBF to validate the mutational patterns seen in our mouse models and begin to understand their relevance in human colon epithelial cells. The results of this study highlight the importance of ETBF colonization in the development of sporadic CRC and in individuals with hereditary tumor conditions, such as Lynch syndrome and familial adenomatous polyposis (FAP).

Nonspecific, Acute Pouchitis in Patients With Familial Adenomatous Polyposis: Less Common Than We Think.

Nonspecific acute pouchitis is common in patients with ulcerative colitis who undergo IPAA, but there is disagreement about the rate at which this occurs in patients with familial adenomatous polyposis. This study aimed to define the rate at which nonspecific acute pouchitis occurs in patients with familial adenomatous polyposis. This study was conducted at a hereditary colorectal cancer center in a large academic medical center. This is a retrospective cohort study using prospectively gathered data. Patients with familial adenomatous polyposis who have had IPAA were included. Symptoms, pouchoscopy and pouch radiography, and pouch biopsy were evaluated. The primary outcomes measured were the presence of nonspecific acute pouchitis and the alternative diagnosis of pouch dysfunction. There were 250 patients with familial adenomatous polyposis who had undergone IPAA; 38 (15.2%) had been labeled as having "pouchitis": 23 men and 15 women. Median age was 55 years (range, 18-90 years). No patient had nonspecific, acute pouchitis as defined by our criteria. In 9 cases, the diagnosis was derived from benign ulcers common in ileal pouches. Stool frequency was the most common symptom associated with the "pouchitis" label, but actual causes for the frequency included poor pouch emptying, poor eating habits, poor bowel habits, and afferent limb syndrome. Median stool frequency was 9, ranging from 4 to 15 per day. Every patient had at least 1 pouch endoscopy, and the median number was 19 per patient (range, 1-21). Nine patients had pouch biopsies to rule out inflammation; all showed chronic active enteritis, a normal finding in ileal pouches. This study was limited by its retrospective nature. We could not confirm any case of nonspecific acute pouchitis in patients with familial adenomatous polyposis. Those caring for patients with familial adenomatous polyposis who have a pouch should seek causes for pouch-related symptoms other than "pouchitis." See Video Abstract at http://links.lww.com/DCR/B666.POUCHITIS AGUDA INESPECÍFICA EN PACIENTES CON POLIPOSIS ADENOMATOSA FAMILIAR: MENOS COMÚN DE LO QUE PENSAMOSANTECEDENTES:La pouchitis aguda e inespecífica es común en pacientes con colitis ulcerativa, sometidos a anastomosis de bolsa ileoanal, pero no hay acuerdo sobre la frecuencia en que se presenta en pacientes con poliposis adenomatosa familiar (PAF).OBJETIVO:Definir la frecuencia en la que se presenta pouchitis aguda inespecífica en pacientes con poliposis adenomatosa familiar.ENTORNO CLINICO:Centro de cáncer colorrectal hereditario en un gran centro médico académico.DISEÑO:Estudio de cohorte retrospectivo utilizando datos recopilados prospectivamente.PACIENTES:Pacientes con poliposis adenomatosa familiar sometidos a bolsa con anastomosis ileoanal.INTERVENCIONES:Síntomas, proctoscopia, radiografía y biopsia de bolsa ileoanal.PRINCIPALES MEDIDAS DE VALORACION:Presencia de pouchitis aguda inespecífica; diagnósticos alternos de disfunción de la bolsa ileal.RESULTADOS:De 250 pacientes con poliposis adenomatosa familiar sometidos a una anastomosis anal de la bolsa ileal; 38 (15,2%), habían sido etiquetados como "pouchitis", 23 hombres y 15 mujeres. La mediana de edad fue de 55 años (rango de 18 a 90 años). Ningún paciente tuvo pouchitis aguda inespecífica según lo definido por nuestros criterios. En 9 casos el diagnóstico se derivó de úlceras benignas frecuentes en bolsas ileales. La frecuencia de las evacuaciones fue el síntoma más común asociado con la etiqueta "pouchitis", pero las causas reales de la frecuencia, incluyeron un mal vaciado de la bolsa, malos hábitos alimenticios, malos hábitos intestinales y síndrome de asa aferente. La frecuencia media de las evacuaciones fue de 9, con un rango de 4 a 15 por día. A cada paciente se le realizó al menos una endoscopia de la bolsa: la mediana fue de 19 por paciente (rango de 1 a 21). A 9 pacientes se les realizó biopsia de bolsa para descartar inflamación: todos mostraron enteritis activa crónica, un hallazgo normal en las bolsas ileales.LIMITACIONES:Naturaleza retrospectiva del estudio.CONCLUSIONES:No pudimos confirmar ningún caso de pouchitis aguda e inespecífica en pacientes con PAF. Aquellos que atienden a pacientes con PAF con bolsa ileal, deben buscar otras causas para los síntomas relacionados con la bolsa que no sean "pouchitis". Consulte Video Resumen en http://links.lww.com/DCR/B666. (Traducción-Dr. Fidel Ruiz Healy).

Application of Multigene Panel Testing in Patients With High Risk for Hereditary Colorectal Cancer: A Descriptive Report Focused on Genotype-Phenotype Correlation.

The genetic test solely based on the clinical features of hereditary colorectal cancer has limitations in clinical practice. This study aimed to analyze the results of comprehensive multigene panel tests based on clinical findings. This was a cross-sectional study based on a prospectively compiled database. The study was conducted at a tertiary hospital. A total of 381 patients with high risk for hereditary colorectal cancer syndromes were enrolled between March 2014 and December 2019. The primary outcome was to describe the mutational spectrum based on genotype-phenotype concordance and discordance. Germline mutations were identified in 89 patients for polyposis hereditary colorectal cancer genes (76 in APC; 4 in PTEN; 4 in STK11; 3 in BMPR1A; 1 in POLE; 1 in POLD1), 89 patients for nonpolyposis hereditary colorectal cancer genes (41 in MLH1; 40 in MSH2; 6 in MSH6; and 2 in PMS2), and 12 patients for other cancer predisposition genes (1 in ATM; 2 in BRCA1; 1 in BRCA2; 1 in BRIP1; 1 in MLH3; 1 in NBN; 1 in PMS1; 1 in PTCH1; 1 in TP53; and 2 in monoallelic MUTYH). If we had used direct sequencing tests of 1 or 2 major genes based on phenotype, 48 (25.3%) of 190 mutations would not have been detected due to technical differences (12.1%), less frequent genotype (4.2%), unclear phenotype (3.7%), and genotype-phenotype discordance (4.7%). The genotype-phenotype discordance is probably linked to compound heterozygote, less distinctive phenotype, and insufficient information for colorectal cancer risk. This study included a small number of patients with insufficient follow-up duration. A comprehensive multigene panel is expected to identify more genetic mutations than phenotype-based direct sequencing, with special utility for unclear phenotype or genotype-phenotype discordance. See Video Abstract at http://links.lww.com/DCR/B844. ANTECEDENTES:La prueba genética basada únicamente en la característica clínica del cáncer colorrectal hereditario tiene limitaciones en la práctica clínica.OBJETIVO:Este estudio tuvo como objetivo analizar el resultado de pruebas integrales de panel multigénico basadas en hallazgos clínicos.DISEÑO:Este fue un estudio transversal basado en una base de datos recopilada prospectivamente.AJUSTE:El estudio se realizó en un hospital terciario.PACIENTES:Se inscribió un total de 381 pacientes con alto riesgo de síndromes de cáncer colorrectal hereditario entre marzo del 2014 y diciembre del 2019.PRINCIPALES MEDIDAS DE RESULTADO:El resultado principal fue describir el espectro mutacional basado en la concordancia y discordancia genotipo-fenotipo.RESULTADOS:Se identificaron mutaciones de la línea germinal en 89 pacientes para genes de cáncer colorrectal hereditario con poliposis (76 en APC; 4 en PTEN; 4 en STK11; 3 en BMPR1A; 1 en POLE; 1 en POLD1), 89 pacientes para genes de CCR hereditario sin poliposis (41 en MLH1; 40 en MSH2; 6 en MSH6; y 2 ​​en PMS2) y 12 pacientes por otro gen de predisposición al cáncer (1 en ATM; 2 en BRCA1; 1 en BRCA2; 1 en BRIP1; 1 en MLH3; 1 en NBN; 1 en PMS1; 1 en PTCH1; 1 en TP53; y 2 ​​en MUTYH monoalélico). Si hubiéramos utilizado pruebas de secuenciación directa de uno o dos genes principales basados ​​en el fenotipo, 48 (25,3%) de 190 mutaciones no se habrían detectado debido a diferencias técnicas (12,1%), genotipo menos frecuente (4,2%), fenotipo poco claro (3,7%) y discordancia genotipo-fenotipo (4,7%). La discordancia genotipo-fenotipo probablemente esté relacionada con el heterocigoto compuesto, el fenotipo menos distintivo y la información insuficiente para el riesgo de cáncer colorrectal.LIMITACIONES:Este estudio incluyó una pequeña cantidad de pacientes con una duración de seguimiento insuficiente.CONCLUSIONES:Se espera que un panel multigénico completo identifique más mutaciones genéticas que la secuenciación directa basada en el fenotipo, con especial utilidad para la discordancia de fenotipo o genotipo-fenotipo poco clara. Consulte Video Resumen en http://links.lww.com/DCR/B844. Traducción- Dr. Francisco M. Abarca-Rendon).