Beatriz Carvalho

Early detection of colorectal cancer (CRC) through screening is the most effective method to reduce morbidity as well as mortality from this disease. Fecal immunochemical test (FIT)-based screening has shown to be effective, especially in multi-round population-based screening. However, its sensitivity and specificity are suboptimal, leaving room for improvement. In this perspective, the development journey of a new screening test, the multitargetFIT (mtFIT), which outperforms FIT, is described from discovery down to large-scale clinical utility testing and health technology assessment.

Supplementary Figure S1. Evaluation of screening model DELFI-Pro scores and comorbidities in individuals without cancer. Supplementary Figure S2. DELFI-Pro score evaluation in available clinical characteristics of patients with cancer. Supplementary Figure S3. Stability analysis across fold repeats and collection source. Supplementary Figure S4. Detection of ovarian cancer subtypes using DELFI-Pro screening model. Supplementary Figure S5. ROC analyses of asymptomatic individuals in the using screening or diagnostic models in the Discovery Cohort. Supplementary Figure S6. Performance of ichorCNA and median cfDNA fragment lengths in the Discovery Cohorts. Supplementary Figure S7. Detection of ovarian cancer subtypes using DELFI-Pro at high specificity. Supplementary Figure S8. Genome-wide fragmentation profiles are altered in patients in the Validation Cohort with ovarian cancer. Supplementary Figure S9. Analyses of chromosomal changes in Discovery and Validation cohorts. Supplementary Figure S10. Performance of DELFI-Pro for detection of ovarian cancer in the Validation Cohort. Supplementary Figure S11. Correlation of rank ordered DELFI-Pro scores for the Screening and Diagnostic models. Supplementary Figure S12. Assessment of DELFI-Pro in women with benign lesions. Supplementary Figure S13. Performance of DELFI-Pro for distinguishing ovarian cancer from benign masses. Supplementary Figure S14. Performance of DELFI-Pro for distinguishing ovarian cancer subtypes from benign masses. Supplementary Figure S15. Performance of DELFI-Pro for distinguishing ovarian cancer subtypes from benign masses in Validation Cohort. Supplementary Figure S16. Evaluation of overall tumor burden using the sum of reported lesion diameters. Supplementary Figure S17. Evaluation of CA1-25 and HE4 blood concentrations measured at different centers.

Supplementary Table S1. Summary of clinical information. Supplementary Table S2. Summary of sample characteristics. Supplementary Table S3. Summary of whole-genome sequencing and fragment analyses. Supplementary Table S4. Summary of protein analyses. Supplementary Table S5. Summary of machine learning models and scores. Supplementary Table S6. Summary of performance for ovarian cancer detection.

Background: Non‐invasive biomarkers may reduce post‐colonoscopy colorectal cancer (CRC) rates and colonoscopy overuse in Lynch syndrome. Unlike faecal immunochemical test (FIT), faecal volatile organic compounds (VOCs) may accurately detect both advanced and non‐advanced colorectal neoplasia.Aim: The aim of this study was to evaluate the potential of faecal VOCs—separately and with FIT—to guide optimal colonoscopy intervals in Lynch syndrome.Methods: Prospective longitudinal multicentre study in which individuals with Lynch syndrome collected faeces before and after high‐quality surveillance colonoscopy. VOC‐patterns were analysed using field asymmetric ion mobility spectrometry (FAIMS) and gas chromatography‐ion mobility spectrometry (GC‐IMS) followed by machine learning pipelines, and combined with FIT at 2.55 μg Hb/g faeces. Gas chromatography time‐of‐flight mass spectrometry analysed individual VOC abundance.Results: Among 200 included individuals (57% female, median 51 years), 62 had relevant neoplasia at colonoscopy: 3 CRC, 6 advanced adenoma (AA), 3 advanced serrated lesion (ASL), and 50 non‐advanced adenoma (NAA). Respective sensitivity and negative predictive value for CRC and AA (and also ASL in case of FAIMS) were 100% and 100% using FAIMS (54% specificity), and 89% and 99% using GC‐IMS (58% specificity). Respective sensitivity and specificity for any relevant neoplasia were 88% and 44% (FAIMS) and 84% and 28% (GC‐IMS); accuracy did not significantly improve upon VOC‐FIT. VOC‐patterns differed before and after polypectomy (AUC 0.70). NAA showed decreased faecal abundance of butanal, 2‐oxohexane, dimethyldisulphide and dimethyltrisulphide.Conclusions: In Lynch syndrome, faecal VOCs may be a promising strategy for postponing colonoscopy and for follow‐up after polypectomy. Our results serve as a stepping stone for large validation studies.Trial Registration: NL8749

Ovarian cancer is a leading cause of death for women worldwide, in part due to ineffective screening methods. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome and protein biomarker [cancer antigen 125 (CA-125) and human epididymis protein 4 (HE4)] analyses to evaluate 591 women with ovarian cancer, with benign adnexal masses, or without ovarian lesions. Using a machine learning model with the combined features, we detected ovarian cancer with specificity >99% and sensitivities of 72%, 69%, 87%, and 100% for stages I to IV, respectively. At the same specificity, CA-125 alone detected 34%, 62%, 63%, and 100%, and HE4 alone detected 28%, 27%, 67%, and 100% of ovarian cancers for stages I to IV, respectively. Our approach differentiated benign masses from ovarian cancers with high accuracy (AUC = 0.88, 95% confidence interval, 0.83–0.92). These results were validated in an independent population. These findings show that integrated cfDNA fragmentome and protein analyses detect ovarian cancers with high performance, enabling a new accessible approach for noninvasive ovarian cancer screening and diagnostic evaluation. Significance: There is an unmet need for effective ovarian cancer screening and diagnostic approaches that enable earlier-stage cancer detection and increased overall survival. We have developed a high-performing accessible approach that evaluates cfDNA fragmentomes and protein biomarkers to detect ovarian cancer.

INTRODUCTION: Colonoscopy surveillance for Lynch syndrome is burdensome and postcolonoscopy colorectal cancers (CRCs) still occur. The noninvasive fecal immunochemical test (FIT) might guide optimal colonoscopy intervals. METHODS: Prospective, multicenter observational study in which individuals with Lynch syndrome performed a quantitative FIT before high-quality surveillance colonoscopy. Diagnostic performance of FIT at various thresholds ≤20 μg Hb/g feces was assessed for relevant neoplasia, including advanced neoplasia (CRC, advanced adenomas [AAs] and advanced serrated lesions [ASLs]) and non-advanced adenomas (NAAs). RESULTS: Of the 217 included individuals (59% female, median age 51 years), 4 had CRC, 5 AA, 4 ASL, and 57 NAA as most relevant neoplasia. The lowest FIT positivity threshold (2.5 μg Hb/g feces, 14% positivity rate) maximized detection: 4/4 CRCs, 4/5 AA, 1/4 ASL, and 9/57 NAA were detected, resulting in a sensitivity and negative predictive value of, respectively, 89% and 99% for CRC plus AA, 69% and 97% for advanced neoplasia, and 26% and 72% for all relevant neoplasia (91% specificity for all groups). At equal sensitivity and negative predictive value, specificity for advanced neoplasia optimized to 94% at threshold 4.1 μg/g. Per 100 FITs at threshold 4.1 μg/g, 11 individuals would test positive and thus proceed to colonoscopy, 2 individuals with advanced neoplasia would be missed and 3 individuals would need colonoscopy to detect 1 advanced neoplasia. DISCUSSION: FIT at thresholds ≤4.1 μg Hb/g feces may be a promising strategy to postpone colonoscopy in approximately 9 of 10 individuals with Lynch syndrome. Large validation studies that also provide gene variant-specific outcomes should be prioritized.

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.