Anne S. Bolijn

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. Clin Cancer Res; 18(19); 5256–66. ©2012 AACR.

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

Can free DNA be detected in sputum of lung cancer patients

SUMMARY Free DNA is present in the serum of cancer patients in a higher concentration than that in non-cancer patients. Free DNA in sputum may originate from malignant or inflammatory diseases. The aim of the study was to examine the presence of free DNA in sputum and the relationship to lung cancer. The contribution of inflammatory cells was established as well. The amount of free and cellular DNA in sputum was determined using real-time beta-globin PCR in 28 lung cancer patients and 68 controls. Free DNA was present in sputum samples of the cancer patients and controls. We found no differences in DNA concentration in sputum of patients with and without lung cancer. For all patients combined the amount of free DNA was related to the amount of inflammation. Further, we found increased hypermethylation of RASSF1A in lung cancer patients compared to controls to show that tumour related DNA is present in sputum. In conclusion, free DNA can be detected in sputum of lung cancer patients. The amount of free DNA is related to the amount of inflammation, but not to the presence of lung cancer.

Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

BackgroundMicroRNAs (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.ResultsTo 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.ConclusionsWith the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.

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.

Mo1620 Chromosomal Aberrations Implicated in Colorectal Adenoma to Carcinoma Progression as Markers of High Risk Colorectal Adenomas

Background: Accumulated evidence has reveled implications of chemokines and their receptors in the pathophysiology of cancer. Anaphylatoxin C5a, a fragment of the complement 5th component (C5), is a potent chemokine for leukocytes that express the C5a receptor (C5aR). Recent reports indicate production of C5a by proteases of various origins, besides a by-product of activation of the complement system, and expression of C5aR in nonmyeloid cells, suggesting an involvement of C5a in these cell biology. However, little is known about roles of C5aR in human cancer. Aim of our study is to investigate the expression and the role of C5aR on cancer cells in invasion. Method: C5aR expression was investigated in surgically resected cancer tissues and cancer cell lines. The effects of C5a on cancer cell shape and invasion activity were examined. Results: By immunohistochemical staining, C5aR was found in a wide variety of human cancers and in several human cancer cell lines. C5a triggered cytoskeletal rearrangement of cancer cells in a C5aR-dependent manner. Timelapse analysis and Matrigel chamber assay revealed that C5a enhances cell motility 3-fold and invasiveness 13-fold of C5aR-overexpressing cancer cells, but such enhancement by C5a was not observed in control cells. Interestingly, cancer cell invasion was still enhanced after removal of C5a stimulation, suggesting that C5a-triggered cancer cell invasion is caused by random cell locomotion rather than chemotaxis. C5a increased the release of matrix metalloproteinases (MMPs) from cancer cells by 2 to 11-fold, and inhibition of MMP activity abolished the C5a enhancing effect on cancer cell invasion. When C5aR-expressing cells were treated with C5a and implanted together with control cells into nude mouse skin, these cells spread 1.8-fold more broadly than control cells, whereas no such difference in spreading was observed in the absence of C5a treatment. Conclusion: These results illustrate a novel activity of C5a-C5aR axis that promotes cancer cell invasion through cytoskeletal rearrangement and MMP release. Thus, targeting this signaling pathway may provide a useful therapeutic option for cancer treatment.

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.

Abstract 1559: Proteogenomic analysis of alternative splicing in colorectal adenoma-to-carcinoma progression

Background Early diagnosis of colorectal cancer (CRC) and identification of its precursor lesions (adenomas) is crucial in reducing CRC mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects human protein hemoglobin. Although FIT is beneficial in its current form with a sensitivity of ~65% for detection of CRC and ~27% for adenomas, its performance is still suboptimal and needs to be further improved. Adenoma-to-carcinoma progression is accompanied by alternative splicing, which results in expression of tumor-specific protein variants. These may yield novel biomarkers suitable for improving detection of progressive adenomas and CRCs. Aim We aim to identify novel biomarkers to improve early detection of CRC. Materials and methods RNA was isolated from 3D organoid cultures derived from 5 adenomas and 4 CRC tissues. RNA and proteins were isolated from 18 healthy human colon tissues, 30 adenomas and 30 CRCs. Samples were analyzed by RNA sequencing (Illumina) and in-depth tandem mass spectrometry proteomics (QExactive). For both organoid- and tissue-datasets differential splicing analysis was performed on RNA level to enrich the sequence database, against which mass spectra were searched, with predicted protein isoforms. Results Comparative splicing analysis between CRC and adenoma organoids revealed ~90 differentially spliced genes, yielding candidate biomarkers from epithelial origin. In the tissues, differential splicing analysis between CRCs and controls and between CRCs and adenomas identified over 1000 of splice variants. These include known alternatively spliced genes involved in cancer such as CD44 and VEGFA and a number of candidates overlapping with the isoforms derived from the organoids. Proteomics analysis revealed that approximately 150 of the splice variants were expressed on protein level. Conclusion and Discussion We have confirmed that adenoma-to-carcinoma progression is accompanied by aberrant splicing. Analysis of the organoid cultures allowed us to identify gene isoforms from (neoplastic) epithelial origin. Tissue analysis yielded tumor-specific splice variants that represent novel protein candidate biomarkers for early detection of CRC. The diagnostic performance of these splice variant proteins will be validated in series of stool and FIT samples. Citation Format: Malgorzata A. Komor, Thang V. Pham, Sander R. Piersma, Anne S. Bolijn, Tim Schelfhorst, Pien M. Delis-van Diemen, Marianne Tijssen, Annemieke C. Hiemstra, Meike de Wit, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond J. Fijneman. Proteogenomic analysis of alternative splicing in colorectal adenoma-to-carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1559. doi:10.1158/1538-7445.AM2017-1559