Polyketide synthase positive Escherichia coli one-time measurement in stool is not informative of colorectal cancer risk in a screening setting.

Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 µg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Overexpression of the miR-17-92 cluster in colorectal adenoma organoids causes a carcinoma-like gene expression signature.

Gain of chromosome arm 13q is one of the most prevalent DNA copy number alterations associated with colorectal adenoma-to-carcinoma progression. The oncogenic miR-17-92 cluster, located at 13q, was found to be overexpressed in colorectal cancer and in adenomas harboring 13q gain. However, to what extent overexpression of this group of microRNAs actually drives progression to cancer remains to be resolved. Therefore, we aimed to clarify the role of miR-17-92 cluster in the progression from colorectal adenoma to carcinoma. The miR-17-92 cluster was overexpressed in human colorectal adenoma organoids without 13q gain and downstream effects on mRNA expression were investigated, along with functional consequences in vitro and in vivo. Comparison of mRNA sequencing results of organoids overexpressing miR-17-92 and cultures transduced with control vector revealed a miR-17-92 expression signature. This signature appeared to be enriched in an independent series of colorectal cancers and adenomas with 13q gain, confirming that miR-17-92 expression is associated with malignant progression. However, tumor-associated characteristics, such as increased proliferation rate, were not observed in miR-17-92 overexpressing adenoma organoids in vitro. In addition, subcutaneous injection of these organoids in immunodeficient mice was insufficient to cause tumor outgrowth. In conclusion, this study showed that miR-17-92 expression contributes to 13q gain-associated adenoma-to-carcinoma progression, however, this is insufficient to cause malignancy.

Genetic Profiling of Colorectal Carcinomas of Patients with Primary Sclerosing Cholangitis and Inflammatory Bowel Disease.

BACKGROUND: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) run a 10-fold increased risk of developing colorectal cancer (CRC) compared to patients with IBD only. The aim of this study was to perform an extensive screen of known carcinogenic genomic alterations in patients with PSC-IBD, and to investigate whether such changes occur already in nondysplastic mucosa. METHODS: Archival cancer tissue and nondysplastic mucosa from resection specimens of 19 patients with PSC-IBD-CRC were characterized, determining DNA copy-number variations, microsatellite instability (MSI), mutations on 48 cancer genes, and CpG island methylator phenotype (CIMP). Genetic profiles were compared with 2 published cohorts of IBD-associated CRC (IBD-CRC; n = 11) and sporadic CRC (s-CRC; n = 100). RESULTS: Patterns of chromosomal aberrations in PSC-IBD-CRC were similar to those observed in IBD-CRC and s-CRC, MSI occurred only once. Mutation frequencies were comparable between the groups, except for mutations in KRAS, which were less frequent in PSC-IBD-CRC (5%) versus IBD-CRC (38%) and s-CRC (31%; P = .034), and in APC, which were less frequent in PSC-IBD-CRC (5%) and IBD-CRC (0%) versus s-CRC (50%; P < .001). Cases of PSC-IBD-CRC were frequently CIMP positive (44%), at similar levels to cases of s-CRC (34%; P = .574) but less frequent than in cases with IBD-CRC (90%; P = .037). Similar copy number aberrations and mutations were present in matched cancers and adjacent mucosa in 5/15 and 7/11 patients, respectively. CONCLUSIONS: The excess risk of CRC in patients with PSC-IBD was not explained by copy number aberrations, mutations, MSI, nor CIMP status, in cancer tissue, nor in adjacent mucosa. These findings set the stage for further exome-wide and epigenetic studies.

The excessive risk of colorectal carcinoma (CRC) in patients with both primary sclerosing cholangitis and inflammatory bowel disease (IBD) was not explained by an extensive screen of copy number aberrations, mutations, microsatellite instability, and CpG island methylator phenotype status when compared with patients with IBD-CRC and sporadic CRC.

Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

Molecular characterization of colorectal adenomas reveals POFUT1 as a candidate driver of tumor progression.

Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. The present study aimed to characterize adenomas by in-depth molecular profiling, to obtain insights into altered biology associated with the colorectal adenoma-to-carcinoma progression. We obtained low-coverage whole genome sequencing, RNA sequencing and tandem mass spectrometry data for 30 CRCs, 30 adenomas and 18 normal adjacent colon samples. These data were used for DNA copy number aberrations profiling, differential expression, gene set enrichment and gene-dosage effect analysis. Protein expression was independently validated by immunohistochemistry on tissue microarrays and in patient-derived colorectal adenoma organoids. Stroma percentage was determined by digital image analysis of tissue sections. Twenty-four out of 30 adenomas could be unambiguously classified as high risk (n = 9) or low risk (n = 15) of progressing to cancer, based on DNA copy number profiles. Biological processes more prevalent in high-risk than low-risk adenomas were related to proliferation, tumor microenvironment and Notch, Wnt, PI3K/AKT/mTOR and Hedgehog signaling, while metabolic processes and protein secretion were enriched in low-risk adenomas. DNA copy number driven gene-dosage effect in high-risk adenomas and cancers was observed for POFUT1, RPRD1B and EIF6. Increased POFUT1 expression in high-risk adenomas was validated in tissue samples and organoids. High POFUT1 expression was also associated with Notch signaling enrichment and with decreased goblet cells differentiation. In-depth molecular characterization of colorectal adenomas revealed POFUT1 and Notch signaling as potential drivers of tumor progression.

Consensus molecular subtype classification of colorectal adenomas.

Consensus molecular subtyping is an RNA expression-based classification system for colorectal cancer (CRC). Genomic alterations accumulate during CRC pathogenesis, including the premalignant adenoma stage, leading to changes in RNA expression. Only a minority of adenomas progress to malignancies, a transition that is associated with specific DNA copy number aberrations or microsatellite instability (MSI). We aimed to investigate whether colorectal adenomas can already be stratified into consensus molecular subtype (CMS) classes, and whether specific CMS classes are related to the presence of specific DNA copy number aberrations associated with progression to malignancy. RNA sequencing was performed on 62 adenomas and 59 CRCs. MSI status was determined with polymerase chain reaction-based methodology. DNA copy number was assessed by low-coverage DNA sequencing (n = 30) or array-comparative genomic hybridisation (n = 32). Adenomas were classified into CMS classes together with CRCs from the study cohort and from The Cancer Genome Atlas (n = 556), by use of the established CMS classifier. As a result, 54 of 62 (87%) adenomas were classified according to the CMS. The CMS3 'metabolic subtype', which was least common among CRCs, was most prevalent among adenomas (n = 45; 73%). One of the two adenomas showing MSI was classified as CMS1 (2%), the 'MSI immune' subtype. Eight adenomas (13%) were classified as the 'canonical' CMS2. No adenomas were classified as the 'mesenchymal' CMS4, consistent with the fact that adenomas lack invasion-associated stroma. The distribution of the CMS classes among adenomas was confirmed in an independent series. CMS3 was enriched with adenomas at low risk of progressing to CRC, whereas relatively more high-risk adenomas were observed in CMS2. We conclude that adenomas can be stratified into the CMS classes. Considering that CMS1 and CMS2 expression signatures may mark adenomas at increased risk of progression, the distribution of the CMS classes among adenomas is consistent with the proportion of adenomas expected to progress to CRC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

MACROD2 expression predicts response to 5-FU-based chemotherapy in stage III colon cancer.

BACKGROUND: Colorectal cancer (CRC) is caused by genetic aberrations. MACROD2 is commonly involved in somatic focal DNA copy number losses, in more than one-third of CRCs. In this study, we aimed to investigate the association of MACROD2 protein expression with clinical outcome in stage II and stage III colon cancer. METHODS: Tissue microarrays (TMA) containing formalin-fixed paraffin-embedded tissue cores from 386 clinically well-annotated primary stage II and III colon cancers were stained by immunohistochemistry and evaluated for MACROD2 protein expression. Disease-free survival (DFS) analysis was performed to estimate association with clinical outcome. RESULTS: Loss of nuclear MACROD2 protein expression in epithelial neoplastic cells of stage III microsatellite stable (MSS) colon cancers was associated with poor DFS within the subgroup of 59 patients who received 5-fluorouracil (5-FU)-based adjuvant chemotherapy (p=0.005; HR=3.8, 95% CI 1.4-10.0). CONCLUSION: These data indicate that low nuclear expression of MACROD2 is associated with poor prognosis of patients with stage III MSS primary colon cancer who were treated with 5-FU-based adjuvant chemotherapy.

Evaluation of Cancer-Associated DNA Copy Number Events in Colorectal (Advanced) Adenomas.

About 5% of colorectal adenomas are estimated to progress to colorectal cancer. However, it is important to identify which adenomas actually carry a high risk of progression, because these serve as intermediate endpoints, for example, in screening programs. In clinical practice, adenomas with a size of ≥10 mm, villous component and/or high-grade dysplasia, called advanced adenomas, are considered high risk, although solid evidence for this classification is lacking. Specific DNA copy number changes are associated with adenoma-to-carcinoma progression. We set out to determine the prevalence of cancer-associated events (CAE) in advanced and nonadvanced adenomas. DNA copy number analysis was performed on archival tissues from three independent series of, in total, 297 adenomas (120 nonadvanced and 177 advanced) using multiplex ligation-dependent probe amplification or low-coverage whole-genome DNA sequencing. Alterations in two or more CAEs were considered to mark adenomas as high risk. Two or more CAEs were overall present in 25% (95% CI, 19.0-31.8) of advanced adenomas; 23% (11/48), 36% (12/33), and 23% (22/96) of the advanced adenomas in series 1, 2, and 3, respectively, and 1.7% (1/58) and 4.8% (3/62) of the nonadvanced adenomas, in series 1 and 2, respectively. The majority of advanced adenomas do not show CAEs, indicating that only a subset of these lesions is to be considered high risk. Nonadvanced adenomas have very low prevalence of CAEs, although those with CAEs should be considered high risk as well. Specific DNA copy number alterations may better reflect the true progression risk than the advanced adenoma phenotype. Cancer Prev Res; 11(7); 403-12. ©2018 AACR.

Identification of Differentially Expressed Splice Variants by the Proteogenomic Pipeline Splicify.

Proteogenomics, i.e. comprehensive integration of genomics and proteomics data, is a powerful approach identifying novel protein biomarkers. This is especially the case for proteins that differ structurally between disease and control conditions. As tumor development is associated with aberrant splicing, we focus on this rich source of cancer specific biomarkers. To this end, we developed a proteogenomic pipeline, Splicify, which can detect differentially expressed protein isoforms. Splicify is based on integrating RNA massive parallel sequencing data and tandem mass spectrometry proteomics data to identify protein isoforms resulting from differential splicing between two conditions. Proof of concept was obtained by applying Splicify to RNA sequencing and mass spectrometry data obtained from colorectal cancer cell line SW480, before and after siRNA-mediated downmodulation of the splicing factors SF3B1 and SRSF1. These analyses revealed 2172 and 149 differentially expressed isoforms, respectively, with peptide confirmation upon knock-down of SF3B1 and SRSF1 compared with their controls. Splice variants identified included RAC1, OSBPL3, MKI67, and SYK. One additional sample was analyzed by PacBio Iso-Seq full-length transcript sequencing after SF3B1 downmodulation. This analysis verified the alternative splicing identified by Splicify and in addition identified novel splicing events that were not represented in the human reference genome annotation. Therefore, Splicify offers a validated proteogenomic data analysis pipeline for identification of disease specific protein biomarkers resulting from mRNA alternative splicing. Splicify is publicly available on GitHub (https://github.com/NKI-TGO/SPLICIFY) and suitable to address basic research questions using pre-clinical model systems as well as translational research questions using patient-derived samples, e.g. allowing to identify clinically relevant biomarkers.

DNA hypermethylation analysis in sputum of asymptomatic subjects at risk for lung cancer participating in the NELSON trial: argument for maximum screening interval of 2†years.

AIMS: Lung cancer is the major contributor to cancer mortality due to metastasised disease at time of presentation. The current study investigated DNA hypermethylation of biomarkers RASSF1A, APC, cytoglobin, 3OST2, FAM19A4, PHACTR3 and PRDM14 in sputum of asymptomatic high-risk individuals from the NELSON lung cancer low-dose spiral CT screening trial to detect lung cancer at preclinical stage. METHODS: Subjects were selected with (i) lung cancer in follow-up (cases; n=65), (ii) minor cytological aberrations (controls; n=120) and (iii) a random selection of subjects without cytological aberrations (controls; n=99). Median follow-up time for controls was 80 months. Cut-off values were based on high specificity to assess diagnostic value of the biomarkers. RESULTS: RASSF1A may denote presence of invasive cancer because of its high specificity (93% (95% CI 89% to 96%); sensitivity 17% (95% CI 4% to 31%), with best performance in a screening interval of 2 years. The panel of RASSF1A, 3OST2 and PRDM14 detected 28% (95% CI 11% to 44%) of lung cancer cases within 2 years, with specificity of 90% (95% CI 86% to 94%). Sputum cytology did not detect any lung cancers. CONCLUSIONS: In a lung cancer screening setting with maximum screening interval of 2 years, DNA hypermethylation analysis in sputum may play a role in the detection of preclinical disease, but complementary diagnostic markers are needed to improve sensitivity.

Methylation analysis in spontaneous sputum for lung cancer diagnosis.

OBJECTIVES: Lung cancer is the most fatal cancer in the developed world due to presence of metastases at time of diagnosis. The aim of this study is to examine DNA hypermethylation in sputum compared to sputum cytology for the diagnosis of lung cancer. A novel risk analysis is introduced, using the distinction between diagnostic and risk markers. METHODS: Two independent sets were randomly composed from a prospectively collected sputum bank (Set 1: n = 98 lung cancer patients, n = 90 controls; Set 2: n = 60 lung cancer patients, n = 445 controls). Sputum cytology was performed for all samples. The following DNA hypermethylation markers were tested in both sets: RASSF1A, APC and cytoglobin (CYGB). Two statistical analyses were conducted: multivariate logistic regression and a risk classification model based on post-test probabilities. RESULTS: In multivariate analysis, RASSF1A was the best of the three markers in discriminating lung cancer cases from controls in both sets (sensitivity 41-52%, specificity 94-96%). The risk model showed that 36% of lung cancer patients were defined as "high risk" (≥ 60% chance on lung cancer) based on RASSF1A hypermethylation in Set 1. The model was reproducible in Set 2. Risk markers (APC, CYGB) have less diagnostic value. Sensitivity of cytology for lung cancer diagnosis was 22%. RASSF1A hypermethylation yielded a sensitivity of 45%. The combined sensitivity for RASSF1A with cytological diagnosis increased to 52% with similar specificity (94%). CONCLUSION: In a diagnostic setting, hypermethylation analysis in sputum is possible when a diagnostic marker is used. However, risk markers are insufficient for this purpose.

Alternative cleavage and polyadenylation during colorectal cancer development.

PURPOSE: Alternative cleavage and polyadenylation (APA) of mRNAs is a phenomenon that alters 3'-untranslated region length leading to altered posttranscriptional regulation of gene expression. Changing APA patterns have been shown to result in misregulation of genes involved in carcinogenesis; therefore, we hypothesized that altered APA contributes to progression of colorectal cancer, and that measurement of APA may lead to discovery of novel biomarkers. EXPERIMENTAL DESIGN: We used next-generation sequencing to directly measure global patterns of APA changes during colorectal carcinoma progression in 15 human patient samples. Results were validated in a larger cohort of 50 patients, including 5 normal/carcinoma pairs from individuals. RESULTS: We discovered numerous genes presenting progressive changes in APA. Genes undergoing untranslated region (3'UTR) shortening were enriched for functional groups such as cell-cycle and nucleic acid-binding and processing factors, and those undergoing 3'UTR lengthening or alternative 3'UTR usage were enriched for categories such as cell-cell adhesion and extracellular matrix. We found indications that APA changes result from differential processing of transcripts because of increased expression of cleavage and polyadenylation factors. Quantitative PCR analysis in a larger series of human patient samples, including matched pairs, confirmed APA changes in DMKN, PDXK, and PPIE genes. CONCLUSIONS: Our results suggest that genes undergoing altered APA during human cancer progression may be useful novel biomarkers and potentially targeted for disease prevention and treatment. We propose that the strategy presented here may be broadly useful in discovery of novel biomarkers for other types of cancer and human disease.

Functional microRNA screening using a comprehensive lentiviral human microRNA expression library.

BACKGROUND: MicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs. RESULTS: To systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs. CONCLUSIONS: With the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

High-resolution array comparative genomic hybridization in sporadic and celiac disease-related small bowel adenocarcinomas.

PURPOSE: The molecular pathogenesis of small intestinal adenocarcinomas is not well understood. Understanding the molecular characteristics of small bowel adenocarcinoma may lead to more effective patient treatment. EXPERIMENTAL DESIGN: Forty-eight small bowel adenocarcinomas (33 non-celiac disease related and 15 celiac disease related) were characterized for chromosomal aberrations by high-resolution array comparative hybridization, microsatellite instability, and APC promoter methylation and mutation status. Findings were compared with clinicopathologic and survival data. Furthermore, molecular alterations were compared between celiac disease-related and non-celiac disease-related small bowel adenocarcinomas. RESULTS: DNA copy number changes were observed in 77% small bowel adenocarcinomas. The most frequent DNA copy number changes found were gains on 5p15.33-5p12, 7p22.3-7q11.21, 7q21.2-7q21.3, 7q22.1-7q34, 7q36.1, 7q36.3, 8q11.21-8q24.3, 9q34.11-9q34.3, 13q11-13q34, 16p13.3, 16p11.2, 19q13.2, and 20p13-20q13.33, and losses on 4p13-4q35.2, 5q15-5q21.1, and 21p11.2-21q22.11. Seven highly amplified regions were identified on 6p21.1, 7q21.1, 8p23.1, 11p13, 16p11.2, 17q12-q21.1, and 19q13.2. Celiac disease-related and non-celiac disease-related small bowel adenocarcinomas displayed similar chromosomal aberrations. Promoter hypermethylation of the APC gene was found in 48% non-celiac disease-related and 73% celiac disease-related small bowel adenocarcinomas. No nonsense mutations were found. Thirty-three percent of non-celiac disease-related small bowel adenocarcinomas showed microsatellite instability, whereas 67% of celiac disease-related small bowel adenocarcinomas were microsatellite unstable. CONCLUSIONS: Our study characterized chromosomal aberrations and amplifications involved in small bowel adenocarcinoma. At the chromosomal level, celiac disease-related and non-celiac disease-related small bowel adenocarcinomas did not differ. A defect in the mismatch repair pathways seems to be more common in celiac disease-related than in non-celiac disease-related small bowel adenocarcinomas. In contrast to colon and gastric cancers, no APC nonsense mutations were found in small bowel adenocarcinoma. However, APC promoter methylation seems to be a common event in celiac disease-related small bowel adenocarcinoma. Clin Cancer Res; 16(5); 1391-401.

Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer.

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost beta-catenin binding sites. APC-free beta-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3' untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis.