Aberrantly methylated genes contribute to the landscape of epigenetic alterations in colorectal adenocarcinoma. The global CpG Island methylator phenotype (CIMP) and individually methylated genes are ...potential prognostic/predictive biomarkers. Research suggests an association between methylated DCR1 (mDCR1) and lack of benefit with irinotecan (IFL) treatment. We assessed the association between DCR1 methylation status and survival in patients receiving adjuvant fluorouracil/ leucovorin (5-FU/LV) or IFL. We analysed data from patients with stage III colon adenocarcinoma randomly assigned to adjuvant 5-FU/LV or IFL in CALGB 89803 (Alliance). The primary endpoint was overall survival (OS), and the secondary endpoint was disease-free survival (DFS). Using tumour sample DNA, we evaluated the association between survival, DCR1 methylation status, and molecular subgroups (BRAF, KRAS, mismatch repair status, CIMP status) using Kaplan-Meier estimator and Cox proportional hazard model. mDCR1 was observed in 221/400 (55%) colon cancers. Histopathologic features were similar between mDCR1 and unmethylated DCR1 (unDCR1) colon cancers. There was no difference in OS (p = 0.83) or DFS (p = 0.85) based on DCR1 methylation status. There was no association between methylation status and response to IFL . In patients with unDCR1 and KRAS-wildtype tumours, those who received IFL had a nearly two-fold worse DFS compared to patients who received 5-FU/LV (HR = 1.85, 95% CI (0.97-3.53, p = 0.06). This relationship was not notable among other subgroups. In stage III colon cancer patients, mDCR1 status did not associate with response to irinotecan. Larger studies may suggest an association between the iridocene response and molecular subgroups.
The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement.
To identify novel ...protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas.
Case-control study.
Colonoscopy-controlled referral population from several centers.
315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples).
Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples.
In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus control stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P = 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001).
Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples.
Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening.
Center for Translational Molecular Medicine, International Translational Cancer Research Dream Team, Stand Up to Cancer (American Association for Cancer Research and the Dutch Cancer Society), Dutch Digestive Foundation, and VU University Medical Center.
With more than 70 different histological sarcoma subtypes, accurate classification can be challenging. Although characteristic genetic events can largely facilitate pathological assessment, ...large-scale molecular profiling generally is not part of regular diagnostic workflows for sarcoma patients. We hypothesized that whole genome sequencing (WGS) optimizes clinical care of sarcoma patients by detection of diagnostic and actionable genomic characteristics, and of underlying hereditary conditions. WGS of tumor and germline DNA was incorporated in the diagnostic work-up of 83 patients with a (presumed) sarcomas in a tertiary referral center. Clinical follow-up data were collected prospectively to assess impact of WGS on clinical decision making. In 12/83 patients (14%), the genomic profile led to revision of cancer diagnosis, with change of treatment plan in eight. All twelve patients had undergone multiple tissue retrieval procedures and immunohistopathological assessments by regional and expert pathologists prior to WGS analysis. Actionable biomarkers with therapeutic potential were identified for 30/83 patients. Pathogenic germline variants were present in seven patients. In conclusion, unbiased genomic characterization with WGS identifies genomic biomarkers with direct clinical implications for sarcoma patients. Given the diagnostic complexity and high unmet need for new treatment opportunities in sarcoma patients, WGS can be an important extension of the diagnostic arsenal of pathologists.
Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which ...methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa 29.5% (18/61), adenomas 81.0% (47/58) and carcinomas 82.7% (62/75) (P = 8.6 × 10−9)} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2′-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.
Abstract
Purpose of the study: Molecular tests have the potential to improve current non-invasive faecal immunochemical test (FIT) screening for colorectal cancer (CRC) and advanced precancerous ...lesions. We examined the performance of a panel of faecal DNA (sDNA) markers and FIT in archival samples from an invitational CRC screening population.
Methods: Whole stool samples were prospectively collected from individuals participating an invitational primary colonoscopy-screening program (COCOS trial). Only participants that provided stool, performed FIT (OC-Sensor) and underwent colonoscopy were selected. The sDNA panel included quantitative molecular assays for KRAS mutations and for aberrant NDRG4 and BMP3 methylation. The performance of the sDNA plus FIT panel was compared to the FIT results alone, by Receiver Operator Characteristic (ROC) analyses.
Results: A total of 1047 individuals (51% male) with a median age of 60 years (range 50-75) were included, of which 7 (0.7%) had colorectal cancer and 104 (9.9%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps ≥ 1 cm).
The combination of sDNA and FIT was more sensitive than FIT alone for detecting advanced precancerous lesions (49% (50/102) and 25% (26/102), respectively). Specificities among individuals with non-advanced or negative findings (controls) were 89% and 96% for sDNA and FIT testing, respectively.
ROC analysis of CRC and advanced precancerous lesions compared to controls revealed an Area Under the Curve (AUC) of 0.75 for the sDNA plus FIT test, compared to 0.68 for FIT alone. At an equal specificity of 95%, advanced precancerous lesions were detected with a higher sensitivity by the sDNA plus FIT test compared to FIT alone (36% vs 28%, p = 0.08).
Conclusions: In an invitational colorectal cancer screening cohort, combining stool DNA markers with FIT detected more advanced neoplasia than FIT alone, primarily due to detecting more advanced adenomas.
Citation Format: Linda J.W. Bosch, Veerle Melotte, Sandra Mongera, Kathleen L.J. Daenen, Veerle H.M. Coupe, Sietze T. van Turenhout, Esther M. Stoop, Thomas R. de Wijkerslooth, Chris J.J. Mulder, Ernst J. Kuipers, Evelien Dekker, Michael Domanico, Graham P. Lidgard, Barry M. Berger, Beatriz Carvalho, Manon van Engeland, Gerrit A. Meijer. Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4338.
Abstract
Introduction and objectives: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. The fecal immunochemical test (FIT) ...is a non-invasive CRC screening test that detects small traces of the blood protein hemoglobin. Although beneficial in its current form, the FIT test characteristics leave room for improvement. The aim of the present study was to identify and validate novel protein biomarkers in stool that complement or outperform the current hemoglobin-based test, to improve its diagnostic accuracy.
Methods: Proteins isolated from stool from 10 subjects without any signs of colorectal neoplasia (controls) at colonoscopy and from 12 CRC patients were analyzed by GeLC-MS/MS (discovery set). Data were analyzed by comparing protein abundancies, measured as spectral counts. Analysis of differential proteins was performed using the beta-binomial test. Findings were validated by mass spectrometry (Q-Exactive) in an independent series of 292 stool samples obtained from control subjects (n = 109) and subjects with adenomas (n = 55), advanced adenomas (n = 53), or CRCs (n = 75).
Results and Discussion: In total 830 human proteins were identified in the discovery set, of which 134 were significantly enriched in CRC. These included 78 proteins that were significantly more enriched in FIT-negative CRC stool samples compared to controls. Preliminary analysis of the validation set indicates that more than half of these markers are significantly more abundant in CRC samples compared to controls.
Conclusion: Proteome profiling of stool samples revealed novel candidate biomarkers to improve current CRC screening tests. More data analysis is currently ongoing to select most promising protein biomarkers for clinical assay development.
Citation Format: Linda JW Bosch, Meike de Wit, Annemieke C. Hiemstra, Sander Piersma, Thang Pham, Gideon Oudgenoeg, George Scheffer, Sandra Mongera, Malgorzata Komor, Jochim Terhaar Sive Droste, Frank A. Oort, Sietze van Turenhout, Ilhame Ben Larbi, Chris JJ Mulder, Beatriz Carvalho, Remond JA Fijneman, Connie Jimenez, Gerrit A. Meijer. Stool proteomics reveals novel candidate biomarkers for colorectal cancer screening. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1563. doi:10.1158/1538-7445.AM2015-1563
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Background: Heterogeneity in the biology of colorectal cancer (CRC) is associated with variable responses to standard chemotherapy. We aimed to identify DNA hypermethylated genes ...as predictive biomarkers for irinotecan treatment of patients with metastatic CRC. Methods: The presence of DNA methylation for a selected panel of 22 genes was assessed by methylation specific PCR (MSP) on primary tumors of 185 patients with metastatic CRC treated with first-line capecitabine (CAP, n=90) or a combination of capecitabine and irinotecan (CAPIRI, n=95) in the phase 3 CAIRO trial. Methylation status of each gene was correlated to progression free survival (PFS) by treatment regimen. Genes for which methylation status associated with response to irinotecan, were validated in 166 patients treated with first-line CAP (n=78) or CAPIRI (n=88). Results: Decoy Receptor 1 (DCR1) was identified as a novel hypermethylated gene in CRC. In CAPIRI treated patients, DCR1 methylation was correlated to a shorter PFS compared to patients with unmethylated DCR1 (hazard ratio HR=0.4 (95%CI =0.3-0.7), p = 0.0009). In patients with methylated DCR1 PFS did not improve with CAPIRI treatment, compared to treatment with CAP (discovery set: HR=0.8 (95%CI=0.5-1.3, p=0.4); validation set: HR=1.1 (95%CI 0.7-1.7, p=0.6)), in contrast to patients with unmethylated DCR1 (discovery set: HR=2.5 (95%CI 1.7-3.3, p=0.00004); validation set: HR=1.7 (95%CI 1.1-2.0, p=0.004)). Conclusions: CRC patients with methylated DCR1 did not benefit from adding irinotecan to capecitabine therapy, indicating that DCR1 methylation status may guide selecting metastatic CRC patients for irinotecan-based therapy.
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