IMPRESS: Improved methylation profiling using restriction enzymes and smMIP sequencing, combined with a new biomarker panel, creating a multi-cancer detection assay.

BACKGROUND: Despite the worldwide progress in cancer diagnostics, more sensitive diagnostic biomarkers are needed. The methylome has been extensively investigated in the last decades, but a low-cost, bisulfite-free detection method for multiplex analysis is still lacking. METHODS: We developed a methylation detection technique called IMPRESS, which combines methylation-sensitive restriction enzymes and single-molecule Molecular Inversion Probes. We used this technique for the development of a multi-cancer detection assay for eight of the most lethal cancer types worldwide. We selected 1791 CpG sites that can distinguish tumor from normal tissue based on DNA methylation. These sites were analysed with IMPRESS in 35 blood, 111 tumor and 114 normal samples. Finally, a classifier model was built. RESULTS: We present the successful development of IMPRESS and validated it with ddPCR. The final classifier model discriminating tumor from normal samples was built with 358 CpG target sites and reached a sensitivity of 0.95 and a specificity of 0.91. Moreover, we provide data that highlight IMPRESS's potential for liquid biopsies. CONCLUSIONS: We successfully created an innovative DNA methylation detection technique. By combining this method with a new multi-cancer biomarker panel, we developed a sensitive and specific multi-cancer assay, with potential use in liquid biopsies.

Simultaneous detection of eight cancer types using a multiplex droplet digital PCR assay.

DNA methylation biomarkers have emerged as promising tools for cancer detection. Common methylation patterns across tumor types allow multi-cancer detection. Droplet digital PCR (ddPCR) has gained considerable attention for methylation detection. However, multi-cancer detection using multiple targets in ddPCR has never been performed before. Therefore, we developed a multiplex ddPCR assay for multi-cancer detection. Based on previous data analyses using The Cancer Genome Atlas (TCGA), we selected differentially methylated targets for eight frequent tumor types (lung, breast, colorectal, prostate, pancreatic, head and neck, liver, and esophageal cancer). Three targets were validated using ddPCR in 103 tumor and 109 normal adjacent fresh frozen samples. Two distinct ddPCR assays were successfully developed. Output data from both assays is combined to obtain a read-out from the three targets together. Our overall ddPCR assay has a cross-validated area under the curve (cvAUC) of 0.948. Performance between distinct cancer types varies, with sensitivities ranging from 53.8% to 100% and specificities ranging from 80% to 100%. Compared to previously published single-target parameters, we show that combining targets can drastically increase sensitivity and specificity, while lowering DNA input. In conclusion, we are the first to report a multi-cancer methylation ddPCR assay, which allows for highly accurate tumor predictions.

Clinicopathological and molecular differences between stage IV screen-detected and interval colorectal cancers in the Flemish screening program.

Introduction: Interval cancer (IC) is an important quality indicator in colorectal cancer (CRC) screening. Previously, we found that fecal immunochemical test (FIT) ICs are more common in women, older age, right-sided tumors, and advanced stage. Here, we extended our existing stage IV patient cohort with clinicopathological and molecular characteristics, to identify factors associated with FIT-IC. Methods: Logistic regression models were fit to identify variables associated with the odds of having a stage IV FIT-IC. Multivariate models were corrected for gender, age, and location. Results: A total of 292 screen-detected (SD) CRCs and 215 FIT-IC CRCs were included. FIT-IC CRC had 5 fold higher odds to be a neuroendocrine (NET) tumor and 2.5 fold higher odds to have lymphovascular invasion. Interestingly, some variables lost significance upon accounting for location. Thus, tumor location is a critical covariate that should always be included when evaluating factors related to FIT-IC. Conclusions: We identified NETs and lymphovascular invasion as factors associated with increased odds of having a stage IV FIT-IC. Moreover, we highlight the importance of tumor location as a covariate in evaluating FIT-IC related factors. More research across all stages is needed to clarify how these insights might help to optimize the Flemish CRC screening program.

Epigenome-wide methylation analysis of colorectal carcinoma, adenoma and normal tissue reveals novel biomarkers addressing unmet clinical needs.

BACKGROUND: Biomarker discovery in colorectal cancer has mostly focused on methylation patterns in normal and colorectal tumor tissue, but adenomas remain understudied. Therefore, we performed the first epigenome-wide study to profile methylation of all three tissue types combined and to identify discriminatory biomarkers. RESULTS: Public methylation array data (Illumina EPIC and 450K) were collected from a total of 1 892 colorectal samples. Pairwise differential methylation analyses between tissue types were performed for both array types to "double evidence" differentially methylated probes (DE DMPs). Subsequently, the identified DMPs were filtered on methylation level and used to build a binary logistic regression prediction model. Focusing on the clinically most interesting group (adenoma vs carcinoma), we identified 13 DE DMPs that could effectively discriminate between them (AUC = 0.996). We validated this model in an in-house experimental methylation dataset of 13 adenomas and 9 carcinomas. It reached a sensitivity and specificity of 96% and 95%, respectively, with an overall accuracy of 96%. Our findings raise the possibility that the 13 DE DMPs identified in this study can be used as molecular biomarkers in the clinic. CONCLUSIONS: Our analyses show that methylation biomarkers have the potential to discriminate between normal, precursor and carcinoma tissues of the colorectum. More importantly, we highlight the power of the methylome as a source of markers for discriminating between colorectal adenomas and carcinomas, which currently remains an unmet clinical need.

Towards Novel Non-Invasive Colorectal Cancer Screening Methods: A Comprehensive Review.

Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Since the 70s, many countries have adopted different CRC screening programs, which has resulted in a decrease in mortality. However, current screening test options still present downsides. The commercialized stool-based tests present high false-positive rates and low sensitivity, which negatively affects the detection of early stage carcinogenesis. The gold standard colonoscopy has low uptake due to its invasiveness and the perception of discomfort and embarrassment that the procedure may bring. In this review, we collected and described the latest data about alternative CRC screening techniques that can overcome these disadvantages. Web of Science and PubMed were employed as search engines for studies reporting on CRC screening tests and future perspectives. The searches generated 555 articles, of which 93 titles were selected. Finally, a total of 50 studies, describing 14 different CRC alternative tests, were included. Among the investigated techniques, the main feature that could have an impact on CRC screening perception and uptake was the ease of sample collection. Urine, exhaled breath, and blood-based tests promise to achieve good diagnostic performance (sensitivity of 63-100%, 90-95%, and 47-97%, respectively) while minimizing stress and discomfort for the patient.