Engineered bacteria detect tumor DNA.

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Correlation of Morphological Features of Chromosomal Instability and Flow Cytometric DNA Ploidy Analysis in Aspirates of Breast Carcinoma.

OBJECTIVE: Morphological indicators of chromosomal instability (CI), including multipolar mitoses, chromatin bridges (CB), strings, nuclear buds (NB), micronuclei (MN), and deoxyribonucleic acid (DNA) ploidy analysis help in prognostication of breast carcinoma. The present study was done to evaluate CI in breast carcinoma and correlate with DNA ploidy and tumor grade. STUDY DESIGN: Fifty cases of carcinoma breast diagnosed by fine-needle aspiration cytology were included. Robinson's grading method was used on smears to grade breast carcinoma. To assess the morphological features of CI, the best May-Grünwald Giemsa stained smear was chosen. At least 1,000 epithelial cells on oil immersion magnification (×100 objective) were counted. DNA ploidy on the aspirates was done by flow cytometry. RESULTS: All the patients were female, diagnosed as infiltrating ductal carcinoma on cytology. Eight tumors were grade I, 32 were grade II, and 10 were grade III. MN was seen in 48 cases, NB in 45, and CB in 12 cases. Mean MN, NB, and CB scores in aneuploid (24) cases were 9.96 ± 8.42, 5.29 ± 4.71, and 1.08 ± 1.84 while 6.19 ± 6.67, 1.92 ± 1.79, and 0.11 ± 0.33 were seen in diploid (26) cases. Statistically significant positive correlation was observed between CI and DNA ploidy. CONCLUSIONS: Morphological evaluation of CI by light microscopy on routinely stained breast aspirates is feasible, although a meticulous search is required. Cytomorphological features of CI and ploidy have a positive correlation with increasing tumor grade.

DNA image cytometry parameters to identify high-grade cervical lesions.

OBJECTIVE: Evaluate the performance of different DNA image cytometry (DNA-ICM) ploidy parameters in the categorisation of DNA-ICM results and identification of high-grade cervical intraepithelial neoplasia or worse (≥ CIN2). METHODS: Cervical samples from 232 women were collected for DNA-ICM analysis and biopsy confirmation. Five DNA parameters were used to define DNA aneuploidy: number of cells with exceeding events (EE) over 2.5cEE, 4cEE, 5cEE and 9cEE, and aneuploid stemlines. DNA-ICM results were categorised as normal, suspicious, and abnormal. RESULTS: For individual DNA ploidy parameters, sensitivity values for 50 cells with 2.5cEE, 45 cells with 4cEE, 1 cell with 9cEE and aneuploid stemline were 72.95%. 54.1%, 69.67% and 54.1%, while specificity values were 80.0%, 90.0%, 89.09% and 95.45%, respectively. For the 5cEE parameter, the sensitivity values for 1, 2, 3, 4 and 5 cells were 93.44%, 85.25%, 81.97%, 77.87% and 75.41%, while specificity values were 46.36%, 63.64%, 74.55%, 76.36% and 80.91%, respectively. For categorised DNA-ICM results, a suspicious result showed superior sensitivity than an abnormal result (87.70% vs 82.79%, P = 0.031), but lower specificity (54.55% vs 75.45%, P < 0.001). Both types of DNA-ICM result were statistically significantly different from a normal result (P < 0.05). CONCLUSION: For prognostic purposes, 1 cell with 9cEE, 45 cells with 4cEE and aneuploid stemline are the best parameters with which to categorise an abnormal DNA-ICM result, followed by 50 cells with 2.5cEE and 4 cells with 5cEE. For screening purposes, 10 cells with 2.5cEE, 10 cells with 4cEE, and 2 cells with 5cEE are suitable parameters with which to categorise a suspicious DNA-ICM result.

Health Care Provider Characteristics Associated With Colorectal Cancer Screening Preferences and Use.

OBJECTIVE: To assess health care provider (HCP) preferences related to colorectal cancer (CRC) screening overall, and by HCP and patient characteristics. PARTICIPANTS AND METHODS: We developed a survey based on the Theoretical Domains Framework to assess factors associated with CRC screening preferences in clinical practice. The survey was administered online November 6 through December 6, 2019, to a validated panel of HCPs drawn from US national databases and professional organizations. The final analysis sample included 779 primary care clinicians (PCCs) and 159 gastroenterologists (GIs). RESULTS: HCPs chose colonoscopy as their preferred screening method for average-risk patients (96.9% (154/159) for GIs, 75.7% (590/779) for PCCs). Among PCCs, 12.2% (95/779) preferred multi-target stool DNA (mt-sDNA), followed by fecal immunochemical test (FIT), (7.3%; 57/779) and guaiac-based fecal occult blood test (gFOBT) (4.8%; 37/779). Preference among PCCs and GIs generally shifted toward noninvasive screening options for patients who were unable to undergo invasive procedures; concerned about taking time from work; unconvinced about need for screening; and refusing other screening recommendations. Among PCCs, preference for mt-sDNA over FIT and gFOBT was less frequent in larger compared with smaller clinical practices. Additionally, preference for mt-sDNA over FIT was more likely among PCCs with more years of clinical experience, higher patient volumes (> 25/day), and practice locations in suburban and rural settings (compared to urban). CONCLUSION: Both PCCs and GIs preferred colonoscopy for CRC screening of average-risk patients, although PCCs did so less frequently and with approximately a quarter preferring stool-based tests (particularly mt-sDNA). PCCs' preference varied by provider and patient characteristics. Our findings underscore the importance of informed choice and shared decision-making about CRC screening options.

Plant material selection, collection, preservation, and storage for nuclear DNA content estimation.

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed.

Epitachophoresis is a novel versatile total nucleic acid extraction method.

Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

Multiple Biomarker-Combined Screening for Colorectal Cancer Based on Bisulfate Conversion-Free Detection of Fecal DNA Methylation.

To evaluate the applicability of bisulfate conversion-free methylation assay based on enzyme digestion in fecal screening for colorectal cancer (CRC). Stool samples were collected from a total of 1142 participants with intestinal abnormalities, including 180 positive cases, 60 advanced adenomas, and 902 negative cases. DNA from reference cell lines and clinical samples was extracted and digested with an enzyme to detect the methylation of CRC markers SEPT9, SDC2, NDRG4, SFRP2, and BMP3 genes. Statistical analysis was then used to determine the ability of the markers, both individually and in combination, to detect CRC and adenoma. Our results showed that the enzyme digestion method could suitably detect DNA marker methylation in as low as 1% of the cell lines. BMP3 had a considerably low detection rate in all clinical samples, with only 6 positive cases detected out of 180 cancer samples. Our findings showed that the combination of SEPT9, SDC2, and SFRP2 had an area under the receiver operation curve of 0.937, sensitivity of 94.11%, and specificity of 89.21% for detecting CRC. Moreover, the detection sensitivity of adenoma can also reach 38.33%. After innovatively utilizing bisulfate conversion-free methylation assay for CRC screening, this study verified the potential clinical applicability of combining multiple biomarkers for CRC screening in a large number of samples.

Multi-region sequencing reveals genetic correlation between esophageal squamous cell carcinoma and matched cell-free DNA.

PURPOSE: This study aimed to determine if both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) in patients with esophageal squamous cell carcinoma (ESCC). METHODS: Paired multi-regional tumor tissues, cfDNA, and white blood cells (WBCs) were collected from five ESCC patients before treatment, as part of an ongoing prospective study (NCT02395705). Samples from Cohort 1 were sequenced by whole-exome sequencing and samples from Cohort 2 were sequenced by targeted capture sequencing. Somatic single-nucleotide variations (SNVs) were detected by comparing solid tumor or cfDNA with matched WBCs, with a minimum variant allele frequency (VAF) of 0.1% and P value <0.05. RESULTS: Genomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were sequenced successfully. In Cohort 1, a significant linear relationship between the tumor and cfDNA VAFs (R2= 0.78, P < 0.0001) was found. In Cohort 2, cfDNA could recover an average of 60.7% (31/51; range, 35.7-76.2%) of somatic mutations present in matched solid tumors. There was a significant positive correlation in VAFs between cfDNA and matched solid tumor tissues (R2= 0.92, P < 0.0001). CONCLUSIONS: Both sequencing approaches revealed high intratumoral heterogeneity in ESCC, and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA.

Highly sensitive fecal DNA testing of NDRG4 12b methylation is a promising marker for detection of colorectal precancerosis.

PURPOSE: The purpose of this study was to research and validate techniques for extracting DNA from human genomes, explore the sensitivity and specificity of known nucleic acid markers of intestinal malignancy in Chinese patients with early colorectal cancer. We also tried to find adenoma-specific biomarkers in human DNA in feces. METHODS: We compared the ability of fecal DNA testing, Fecal Occult Blood Testing (FOBT) and serum tumor markers to diagnose different types of polyps, and DNA testing was significantly superior to the other two methods. We also found a dominant expression of NDRG12b methylation in multi-target DNA testing, which may be a promising marker for detection of colorectal precancerosis. RESULTS: The sensitivity of NDRG4 12b methylation was 85.7% for advanced adenomatous polyp (AP), and 62.6% for non-advanced AP, respectively, with specificity of 70.8%. The diagnostic efficacy of NDRG4 12b methylation for detecting advanced AP was significantly higher than FOBT (sesitivity: 85.7% vs. 42.9%, p<0.05). The receiver operating characteristics (ROC) curve for NDRG4 12b methylation in detecting AP showed a relatively high area under the curve (AUC = 0.807). CONCLUSIONS: Our results indicate that highly sensitive fecal DNA testing of NDRG4 12b methylation is a promising marker for detection of colorectal precancerosis, especially in detecting adenomatous polyp.

Association Between LIN28A Gene Polymorphisms and Glioma Susceptibility in Chinese Children.

Gliomas are the most prevalent brain tumors among children and adolescents. The occurrence and development of various malignant tumors is closely related with LIN28A gene, but its relationship with glioma susceptibility has not been widely discovered. In this case-control study, we conducted four single nucleotide polymorphisms (SNPs) (rs3811464 G>A, rs3811463 T>C, rs34787247 G>A, and rs11247957 G>A) of LIN28A gene to investigate whether they increase the risk of glioma. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate their relationship. There was no significant correlation between four SNPs and glioma risk in single polymorphism and conjoint analysis. However, in stratification analysis, we found that rs3811463 TC/CC may add to the risk of glioma with clinical stage III (adjusted OR = 3.16, 95% CI = 1.15-8.70, P = .026) or stage III+IV patients (adjusted OR = 2.05, 95% CI = 1.02-4.13, P = .044). Our research suggested that four SNPs of LIN28A gene have a weak relationship with the risk of glioma in Chinese children. LIN28A rs3811463 TC/CC may increase the possibility of glioma in clinical stage III or stage III+IV patients which need larger samples and further confirmation.

Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study.

BACKGROUND: The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS AND FINDINGS: We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4-38.9; p = 0.02) compared to utDNA MRD-negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point. CONCLUSIONS: utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.

Comparative study of BRAF gene mutations in ovarian serous tumors by immunohistochemistry and DNA sequencing.

PURPOSE: Somatic mutations in the BRAF gene are common in several types of cancer, especially in ovarian serous cancer (OSC). Normally, the BRAF protein is switched on and off in response to signals that control cell growth and development. METHODS: To investigate the correlation between the mutation of BRAF gene and the expression of BRAF protein in OSC, pyrosequencing was performed to detect the mutation of the 600th codon in BRAF gene (written as Val600Glu or V600E) in 23 cases of high-grade serous ovarian cancer (HGSC), 28 cases of low-grade serous ovarian cancer (LGSC) and 72 cases of serous borderline ovarian tumors (SBT). Meanwhile, immunohistochemistry which stained with the specific antibody VE1 were used to clarified the expression level of BRAF V600E mutant protein. RESULTS: Finally, we found that V600E mutation in LGSC and SBT of occurred in 2 of 23 (7.1%) and 21of 72 (29.2%), respectively. The V600E mutation was not detected in 23 cases of HGSC. One case of HGSC (1, 4.3%), 3 cases of LGSC (3 of 28, 10.7%) and 25 cases of SBT (25 of 72, 34.7%) were positive expression detected by immunohistochemistry. Compared with BRAF gene mutation, the sensitivity, specificity and consistency of BRAF V600E protein were 91.3%, 92% and 91.9%, respectively. CONCLUSION: Our findings indicate that BRAF mutations in LGSC and SBT, which are closely related to tumor staging. The specific antibody VE1 could be used as a preliminary screening for the mutation of BRAF gene.

Brief Research Report Regional Difference in TRAF2 and TRAF3 Gene Mutations in Colon Cancers.

TRAF2 and TRAF3 genes of tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family are involved in diverse cell signaling, and function as both tumor suppressor gene and oncogene. Alterations of TRAF2 and TRAF3 in colon cancer (CC) along with their regional difference and microsatellite instability (MSI) are largely unknown. In the present study, we analyzed TRAF2 and TRAF3 frameshift mutations in 168 sporadic CCs (100 high MSI (MSI-H) and 68 microsatellite-stable (MSS) CCs). We identified TRAF2 and TRAF3 frameshift mutations in 4 (4%) and 3 CCs (3%) with MSI-H, respectively, but none in 68 cases of MSS CCs. Of the 168 CCs, we analyzed the mutations in multi-regions for 39 CCs (16 MSI-H and 23 MSS CCs), and discovered that 12.5% (2/16) and 6.3% (1/16) of MSI-H CCs exhibited regional difference in TRAF2 and TRAF3 mutations, respectively. In the multi-region samples of 23 MSS CCs, neither TRAF2 nor TRAF3 frameshift mutation was found. In 40% of CCs, both TRAF2 and TRAF3 expressions were increased compared to normal colon cells. Our data indicate that TRAF2 and TRAF3 frameshift mutations and their regional difference as well as altered expressions are present in MSI-H CCs, which could contribute to MSI-H cancer development.

Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

Detection of Postcolonoscopy Colorectal Neoplasia by Multi-target Stool DNA.

INTRODUCTION: Significant variability between colonoscopy operators contributes to postcolonoscopy colorectal cancers (CRCs). We aimed to estimate postcolonoscopy colorectal neoplasia (CRN) detection by multi-target stool DNA (mt-sDNA), which has not previously been studied for this purpose. METHODS: In a retrospective cohort of patients with +mt-sDNA and completed follow-up colonoscopy, positive predictive value (PPV) for endpoints of any CRN, advanced adenoma, right-sided neoplasia, sessile serrated polyps (SSP), and CRC were stratified by the time since previous colonoscopy (0-9, 10, and ≥11 years). mt-sDNA PPV at ≤9 years from previous average-risk screening colonoscopy was used to estimate CRN missed at previous screening colonoscopy. RESULTS: Among the 850 studied patients with +mt-sDNA after a previous negative screening colonoscopy, any CRN was found in 535 (PPV 63%). Among 107 average-risk patients having +mt-sDNA ≤9 years after last negative colonoscopy, any CRN was found in 67 (PPV 63%), advanced neoplasia in 16 (PPV 15%), right-sided CRN in 48 (PPV 46%), and SSP in 20 (PPV 19%). These rates were similar to those in 47 additional average risk persons with previous incomplete colonoscopy and in an additional 68 persons at increased CRC risk. One CRC (stage I) was found in an average risk patient who was mt-sDNA positive 6 years after negative screening colonoscopy. DISCUSSION: The high PPV of mt-sDNA 0-9 years after a negative screening colonoscopy suggests that lesions were likely missed on previous examination or may have arisen de novo. mt-sDNA as an interval test after negative screening colonoscopy warrants further study.

Early detection of pancreatic cancer using DNA-based molecular approaches.

Due to its poor prognosis and the late stage at which it is typically diagnosed, early detection of pancreatic cancer is a pressing clinical problem. Advances in genomic analysis of human pancreatic tissue and other biospecimens such as pancreatic cyst fluid, pancreatic juice and blood have opened the possibility of DNA-based molecular approaches for early detection of pancreatic cancer. In this Review, we discuss and focus on the pathological and molecular features of precancerous lesions of the pancreas, including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm and mucinous cystic neoplasm, which are target lesions of early detection approaches. We also discuss the most prevalent genetic alterations in these precancerous lesions, including somatic mutations in the oncogenes KRAS and GNAS as well as tumour suppressor genes CDKN2A, TP53 and SMAD4. We highlight the latest discoveries related to genetic heterogeneity and multifocal neoplasia in precancerous lesions. In addition, we review specific approaches, challenges and clinically available assays for early detection of pancreatic cancer using DNA-based molecular techniques. Although detection and risk stratification of precancerous pancreatic neoplasms are difficult problems, progress in this field highlights the promise of molecular approaches for improving survival of patients with this disease.

EGFR mutation analysis on circulating free DNA in NSCLC: a single-center experience.

PURPOSE: Monitoring mutation status in circulating free DNA (cfDNA) during target therapy could hold significant clinical importance in non-small cell lung cancer (NSCLC). Our aim is to establish if EGFR mutational status change on cfDNA has predictive value that can impact clinical management of NSCLC patients care. METHODS: This study included 30 patients with EGFR-mutated NSCLC. Blood samples were collected at diagnosis (T0) and in 19 patients during therapy (T1). RESULTS: Concordance between T0 and T1 EGFR mutation status for patients evaluable for both samples (n = 19) was 79%, with a sensitivity of 100% (95% CI: 55.5-100.0) and specificity of 60.0% (95% CI: 26.2-86.8). For the patients in oncological therapy with targeted drug and with T1 sample available (n = 18), survival outcomes were evaluated. For both mutation-negative T0 and T1 patients, 12-month progression-free survival (PFS) was 66.7% (95% CI: 27.2-100.0) and 12-month overall survival (OS) was 100% (95% CI: 1.00-1.00); for patients mutated both at T0 and T1, PFS was 22.2% (95% CI: 6.5-75.4%) and OS was 55.6% (95% CI: 20.4-96.1%). CONCLUSION: EGFR mutation status can be assessed using cfDNA for routine purposes and longitudinal assessment of plasma mutation is an easy approach to monitor the therapeutic response or resistance onset.

Off-Label Use of Multitarget Stool DNA Testing in Primary Care.

INTRODUCTION: We evaluated the off-label use of multitarget stool DNA (mt-sDNA) testing in the primary care setting. METHODS: We reviewed all mt-sDNA orders between July 1, 2018, and June 30, 2019, to determine the frequency of off-label mt-sDNA orders. RESULTS: Nine hundred two patients with mt-sDNA orders were evaluated, of which 160/902 patients (17.7%) met at least 1 criterion for off-label mt-sDNA order. Increasing age was associated with off-label order (Odds Ratio [OR] 2.32 [95% CI, 1.86-2.89] for every 10-year increase in age, P < 0.0001). On multivariate analysis, increased age (OR 1.04 [1.02-1.06], P = 0.001) and need for diagnostic colonoscopy (OR 2.9 [1.01-8.34], P = 0.048) were associated with a positive mt-sDNA result. DISCUSSION: Off-label mt-sDNA testing is common, and further efforts are needed to educate patients and providers on appropriate use of mt-sDNA for colorectal cancer screening.