Next-generation Multi-target Stool DNA Panel Accurately Detects Colorectal Cancer and Advanced Precancerous Lesions.

The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case-control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). PREVENTION RELEVANCE: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93.

The 17-Gene Genomic Prostate Score Test Is Prognostic for Outcomes After Primary External Beam Radiation Therapy in Men With Clinically Localized Prostate Cancer.

PURPOSE: The Oncotype DX Genomic Prostate Score (GPS) assay has been validated as a strong prognostic indicator of adverse pathology, biochemical recurrence, distant metastasis (DM), and prostate cancer (PCa)-related death (PCD) in men with localized PCa after radical prostatectomy. However, it has yet to be tested in men undergoing external beam radiation therapy (EBRT), for whom assessing PCa progression risk could inform decisions on treatment intensity. We analyzed whether GPS results are associated with time to biochemical failure (BCF), DM, and PCD after EBRT in men with localized PCa and whether the association is modified by race. METHODS AND MATERIALS: We conducted a retrospective study of men with localized PCa treated with EBRT at the VA Health Care System in Durham, NC from 2000 to 2016. Study endpoints were time to BCF per the Phoenix criteria, DM, and PCD. The association of GPS results, per 20-unit increase or dichotomous variable (0-40 vs 41-100), was evaluated with each endpoint using univariable and multivariable Cox proportional hazards models. Results were then stratified by race. RESULTS: A total of 238 patients (69% Black) met the eligibility criteria. Median follow-up for patients who did not experience BCF was 7.6 years. GPS results per 20-unit increase were significantly associated with BCF (hazard ratio [HR], 3.62; 95% confidence interval [CI], 2.59-5.02), DM (HR, 4.48; 95% CI, 2.75-7.38), and PCD (HR, 5.36; 95% CI, 3.06-9.76) in univariable analysis. GPS results remained significant in multivariable models adjusted for baseline clinical and pathological factors, with HRs being similar to the univariable analysis. There was no significant interaction between the GPS assay and race (P = .923). HRs for BCF were similar in Black men (HR, 3.88; 95% CI, 2.40-6.24) versus non-Black men (HR, 4.01; 95% CI, 2.42-6.45). CONCLUSIONS: Among men treated with EBRT, the GPS assay is a strong, independent prognostic indicator of time to BCF, DM, and PCD, and performs similarly in Black and non-Black men.

Usability of an At-Home Anterior Nares SARS-CoV-2 RT-PCR Sample Collection Kit: Human Factors Feasibility Study.

BACKGROUND: Readily available testing for SARS-CoV-2 is necessary to mitigate COVID-19 disease outbreaks. At-home collection kits, in which samples are self-collected without requiring a laboratory or clinic visit and sent to an external laboratory for testing, can provide convenient testing to those with barriers to access. They can prevent unnecessary exposure between patient and clinical staff, increase access for patients with disabilities or remote workers, and decrease burdens on health care resources, such as provider time and personal protective equipment. Exact Sciences developed an at-home collection kit for samples to be tested to detect SARS-CoV-2 that includes an Instructions for Use (IFU) document, which guides people without prior experience on collecting a nasal swab sample. Demonstrating successful sample collection and usability is critical to ensure that these samples meet the same high-quality sample collection standards as samples collected in clinics. OBJECTIVE: The aim of this study was to determine the usability of a SARS-CoV-2 at-home nasal swab sample collection kit. METHODS: A human factors usability study was conducted with 30 subjects without prior medical, laboratory, or health care training and without COVID-19 sample self-collection experience. Subjects were observed while they followed the IFU for the at-home sample collection portion of the SARS-CoV-2 test in a setting that simulated a home environment. IFU usability was further evaluated by requiring the subjects to complete a survey, answer comprehension questions, provide written feedback, and respond to questions from the observer about problems during use. RESULTS: All 30 subjects successfully completed the sample collection process, and all 30 samples were determined by reverse transcription-polymerase chain reaction (RT-PCR) testing to meet quality standards for SARS-CoV-2 testing. The subjects' written feedback and comments revealed several recommendations to improve the IFU. CONCLUSIONS: The study demonstrated the overall usability of an at-home SARS-CoV-2 collection kit. Various feedback mechanisms provided opportunities to improve the wording and graphics for some critical tasks, including placing the label correctly on the tube. A modified IFU was prepared based on study outcomes.

A Novel Blood-Based Panel of Methylated DNA and Protein Markers for Detection of Early-Stage Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) can be treated effectively if detected at an early stage. Recommended surveillance strategies for at-risk patients include ultrasound with or without α-fetoprotein (AFP), but their sensitivity is suboptimal. We sought to develop a novel, blood-based biomarker panel with improved sensitivity for early-stage HCC detection. METHODS: In a multicenter, case-control study, we collected blood specimens from patients with HCC and age-matched controls with underlying liver disease but without HCC. Ten previously reported methylated DNA markers (MDMs) associated with HCC, methylated B3GALT6 (reference DNA marker), and 3 candidate proteins, including AFP, were assayed and analyzed by a logistic regression algorithm to predict HCC cases. The accuracy of the multi-target HCC panel was compared with that of other blood-based biomarkers for HCC detection. RESULTS: The study included 135 HCC cases and 302 controls. We identified a multi-target HCC panel of 3 MDMs (HOXA1, EMX1, and TSPYL5), B3GALT6 and 2 protein markers (AFP and AFP-L3) with a higher sensitivity (71%, 95% CI: 60-81%) at 90% specificity for early-stage HCC than the GALAD score (41%, 95% CI: 30-53%) or AFP ≥7.32 ng/mL (45%, 95% CI: 33-57%). The AUC for the multi-target HCC panel for detecting any stage HCC was 0.92 compared with 0.87 for the GALAD score and 0.81 for AFP alone. The panel performed equally well in important subgroups based on liver disease etiology, presence of cirrhosis, or sex. CONCLUSIONS: We developed a novel, blood-based biomarker panel that demonstrates high sensitivity for early-stage HCC. These data support the potential for liquid biopsy detection of early-stage HCC to clinically benefit at-risk patients. This study was registered on ClinicalTrials.gov (NCT03628651).

Colorectal cancer outcomes after screening with the multi-target stool DNA assay: protocol for a large-scale, prospective cohort study (the Voyage study).

Introduction: Population-level screening has been shown to reduce the incidence and mortality of colorectal cancer (CRC). Unfortunately, adherence to screening recommendations among eligible US adults remains below national goals. A relatively new non-invasive screening modality, the Food and Drug Administration-approved multi-target stool DNA (mt-sDNA) assay (commercialised as Cologuard), which combines the detection of haemoglobin and DNA abnormalities, has been completed by more than 3 million individuals. Given mt-sDNA's recent availability, the effectiveness of mt-sDNA screening with respect to CRC incidence and mortality reduction has not yet been established. Methods and analysis: Through an academic-industry collaboration, a prospective cohort study (Voyage) was designed with an initial enrolment target of 150 000 individuals with mt-sDNA ordered by their healthcare provider for CRC screening. Consented participants will be asked to complete a baseline questionnaire to collect sociodemographic and health information. Additional questionnaires will be provided after 1 year, and every 3 years thereafter, to collect data regarding CRC screening follow-up in order to estimate rates of CRC incidence and other health outcomes. Linkage to the National Death Index will be used to estimate mortality rates. Ethics and dissemination: The Voyage study will be conducted in accordance with international guidelines and local regulatory requirements and laws. Data will be stored and retained at Mayo Clinic. Only limited data elements required for research purposes will be transmitted between Mayo Clinic and Exact Sciences Laboratories. Results of the Voyage study will be disseminated through scientific presentations and publications. Trial registration number: NCT04124406.

Targeting of colony-stimulating factor 1 receptor (CSF1R) in the CLL microenvironment yields antineoplastic activity in primary patient samples.

In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Hedgehog signaling pathway molecules and ALDH1A1 expression in early-stage non-small cell lung cancer.

INTRODUCTION: The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. METHODS: A tissue microarray representing 248 clinically annotated stage I-II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). RESULTS: In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO (p=0.017) and transcription factor GLI1 (p=0.001), while SMO correlated with GLI1 (p=0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression (p=0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. CONCLUSIONS: Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.