Phospho-RNA-seq: a modified small RNA-seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma.

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non-coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5' phosphate or presence of 3' phosphate. These fragments were revealed using a modified protocol ("phospho-RNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5' and 3' ends, as well as human plasma exRNA Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-RNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

Performance of multitarget stool DNA testing in African American patients.

BACKGROUND: Multitarget stool DNA (mt-sDNA) is an approved method for colon cancer screening that is especially relevant for patients who cannot undergo colonoscopy. Although the test performance has been evaluated in a large clinical trial, it was limited to a predominantly white population. Given differences in the epidemiology and biology of colon cancer in African American individuals, the authors sought to compare the performance of mt-sDNA between racial groups. METHODS: The authors prospectively identified patients aged ≥40 years who were referred for colonoscopy at an academic medical center and 2 satellite facilities. Prior to the colonoscopy, the authors collected stool for mt-sDNA and fecal immunochemical testing (FIT). They compared the sensitivity, specificity, and receiver operating characteristic curve between African American and white patients for the detection of advanced lesions or any adenoma. RESULTS: A total of 760 patients were included, 34.9% of whom were African American. The prevalence of any adenoma (38.9% for African American patients and 33.9% for white patients) and that for advanced lesions (6.8% and 6.7%, respectively) were similar between groups. The overall sensitivities of mt-sDNA for the detection of advanced lesions and any adenoma were 43% and 19%, respectively, and the specificities were 91% and 93%, respectively. In general, mt-sDNA was more sensitive and less specific than FIT. When stratified by race, the sensitivity, specificity, and receiver operating characteristic curve area were similar between African American and white patients for both mt-sDNA and FIT. CONCLUSIONS: Test performance characteristics of mt-sDNA were comparable in African American and white patients. Given the lower uptake of colonoscopy in African American individuals, mt-sDNA may offer a promising screening alternative in this patient population.

Evaluation of Patients with an Apparent False Positive Stool DNA Test: The Role of Repeat Stool DNA Testing.

BACKGROUND: There is uncertainty as to the appropriate follow-up of patients who test positive on multimarker stool DNA (sDNA) testing and have a colonoscopy without neoplasia. AIMS: To determine the prevalence of missed colonic or occult upper gastrointestinal neoplasia in patients with an apparent false positive sDNA. METHODS: We prospectively identified 30 patients who tested positive with a commercially available sDNA followed by colonoscopy without neoplastic lesions. Patients were invited to undergo repeat sDNA at 11-29 months after the initial test followed by repeat colonoscopy and upper endoscopy. We determined the presence of neoplastic lesions on repeat evaluation stratified by results of repeat sDNA. RESULTS: Twelve patients were restudied. Seven patients had a negative second sDNA test and a normal second colonoscopy and upper endoscopy. In contrast, 5 of 12 subjects had a persistently positive second sDNA test, and 3 had positive findings, including a 3-cm sessile transverse colon adenoma with high-grade dysplasia, a 2-cm right colon sessile serrated adenoma with dysplasia, and a nonadvanced colon adenoma (p = 0.045). These corresponded to a positive predictive value of 0.60 (95% CI 0.17-1.00) and a negative predictive value of 1.00 (95% CI 1.00-1.00) for the second sDNA test. In addition, the medical records of all 30 subjects with apparent false positive testing were reviewed and no documented cases of malignant tumors were recorded. CONCLUSIONS: Repeat positive sDNA testing may identify a subset of patients with missed or occult colorectal neoplasia after negative colonoscopy for an initially positive sDNA. High-quality colonoscopy with careful attention to the right colon in patients with positive sDNA is critically important and may avoid false negative colonoscopy.

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.

Missed adenomas during colonoscopic surveillance in individuals with Lynch Syndrome (hereditary nonpolyposis colorectal cancer).

BACKGROUND AND AIMS: Lynch syndrome (also known as hereditary nonpolyposis colon cancer) is associated with an increased risk for colorectal cancer, which can arise despite frequent colonoscopic exams. We evaluated the adenoma miss rate of conventional colonoscopy in patients with Lynch syndrome, and compared the sensitivity of chromoendoscopy versus intensive inspection for detecting polyps missed by conventional colonoscopy. METHODS: Fifty-four subjects with Lynch syndrome underwent tandem colonoscopies at four centers of the Great Lakes-New England Clinical Epidemiology and Validation Center of the Early Detection Research Network. All participants first had a conventional colonoscopy with removal of all visualized polyps. The second endoscopy was randomly assigned as either pancolonic indigo carmine chromoendoscopy or standard colonoscopy with intensive inspection lasting >20 minutes. Size, histology, and number of polyps detected on each exam were recorded. RESULTS: After undergoing standard colonoscopy, 28 individuals were randomized to a second exam with chromoendoscopy and 26 underwent intensive inspection. The mean interval since last colonoscopy was 17.5 months. Seventeen polyps (10 adenomas and 7 hyperplastic polyps) were identified on the first standard colonoscopies. Twenty-three additional polyps (12 adenomas and 11 hyperplastic polyps) were found on the second exams, yielding an adenoma miss rate of 55%. Fifteen polyps (5 adenomas and 10 hyperplastic polyps) were found in subjects who had chromoendoscopy and 8 polyps (7 adenomas and 1 hyperplastic polyp) in those who had intensive inspection. Chromoendoscopy was associated with more normal tissue biopsies (11 versus 5) and longer procedure times compared with intensive inspection (29.8 +/- 9.5 versus 25.3 +/- 5.8 minutes; P = 0.04). Controlling for age, number of previous colonoscopies, procedure time, and prior colonic resection, chromoendoscopy detected more polyps (P = 0.04), but adenoma detection was not significantly different compared with intensive inspection (P = 0.27). CONCLUSIONS: Small adenomas are frequently missed in patients with Lynch syndrome. Although chromoendoscopy did not detect more missed adenomas than intensive inspection in this pilot study, larger trials are needed to determine optimal surveillance techniques in this high-risk population.

Chromoendoscopy detects more adenomas than colonoscopy using intensive inspection without dye spraying.

Conventional colonoscopy misses some neoplastic lesions. We compared the sensitivity of chromoendoscopy and colonoscopy with intensive inspection for detecting adenomatous polyps missed by conventional colonoscopy. Fifty subjects with a history of colorectal cancer or adenomas underwent tandem colonoscopies at one of five centers of the Great Lakes New England Clinical Epidemiology and Validation Center of the Early Detection Research Network. The first exam was a conventional colonoscopy with removal of all visualized polyps. The second exam was randomly assigned as either pan-colonic indigocarmine chromoendoscopy or standard colonoscopy with intensive inspection lasting >20 minutes. Size, histology, and numbers of polyps detected on each exam were recorded. Twenty-seven subjects were randomized to a second exam with chromoendoscopy and 23 underwent intensive inspection. Forty adenomas were identified on the first standard colonoscopies. The second colonoscopies detected 24 additional adenomas: 19 were found using chromoendoscopy and 5 were found using intensive inspection. Chromoendoscopy found additional adenomas in more subjects than did intensive inspection (44% versus 17%) and identified significantly more missed adenomas per subject (0.7 versus 0.2, P < 0.01). Adenomas detected with chromoendoscopy were significantly smaller (mean size 2.66 +/- 0.97 mm) and were more often right-sided. Chromoendoscopy was associated with more normal tissue biopsies and longer procedure times than intensive inspection. After controlling for procedure time, chromoendoscopy detected more adenomas and hyperplastic polyps compared with colonoscopy using intensive inspection alone. Chromoendoscopy detected more polyps missed by standard colonoscopy than did intensive inspection. The clinical significance of these small missed lesions warrants further study.

Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot.

Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.