Next-Generation Multitarget Stool DNA Test for Colorectal Cancer Screening.

BACKGROUND: A next-generation multitarget stool DNA test, including assessments of DNA molecular markers and hemoglobin level, was developed to improve the performance of colorectal cancer screening, primarily with regard to specificity. METHODS: In a prospective study, we evaluated a next-generation multitarget stool DNA test in asymptomatic adults 40 years of age or older who were undergoing screening colonoscopy. The primary outcomes were sensitivity of the test for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions). Advanced precancerous lesions included one or more adenomas or sessile serrated lesions measuring at least 1 cm in the longest dimension, lesions with villous histologic features, and high-grade dysplasia. Secondary objectives included the quantification of sensitivity for advanced precancerous lesions and specificity for nonneoplastic findings or negative colonoscopy and comparison of sensitivities for colorectal cancer and advanced precancerous lesions between the multitarget stool DNA test and a commercially available fecal immunochemical test (FIT). RESULTS: Of 20,176 participants, 98 had colorectal cancer, 2144 had advanced precancerous lesions, 6973 had nonadvanced adenomas, and 10,961 had nonneoplastic findings or negative colonoscopy. With the next-generation test, sensitivity for colorectal cancer was 93.9% (95% confidence interval [CI], 87.1 to 97.7), and specificity for advanced neoplasia was 90.6% (95% CI, 90.1 to 91.0). Sensitivity for advanced precancerous lesions was 43.4% (95% CI, 41.3 to 45.6), and specificity for nonneoplastic findings or negative colonoscopy was 92.7% (95% CI, 92.2 to 93.1). With the FIT, sensitivity was 67.3% (95% CI, 57.1 to 76.5) for colorectal cancer and 23.3% (95% CI, 21.5 to 25.2) for advanced precancerous lesions; specificity was 94.8% (95% CI, 94.4 to 95.1) for advanced neoplasia and 95.7% (95% CI, 95.3 to 96.1) for nonneoplastic findings or negative colonoscopy. As compared with FIT, the next-generation test had superior sensitivity for colorectal cancer (P<0.001) and for advanced precancerous lesions (P<0.001) but had lower specificity for advanced neoplasia (P<0.001). No adverse events occurred. CONCLUSIONS: The next-generation multitarget stool DNA test showed higher sensitivity for colorectal cancer and advanced precancerous lesions than FIT but also showed lower specificity. (Funded by Exact Sciences; BLUE-C ClinicalTrials.gov number, NCT04144738.).

Algorithm Training and Testing for a Nonendoscopic Barrett's Esophagus Detection Test in Prospective Multicenter Cohorts.

BACKGROUND & AIMS: Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. METHODS: Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. RESULTS: Training (N = 352) and test (N = 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. CONCLUSIONS: A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

Assessment of Stool DNA Markers to Detect Colorectal Neoplasia in Patients with Inflammatory Bowel Disease: A Multi-site Case-control Study.

BACKGROUND AND AIMS: The FDA-approved multitarget stool-DNA [mt-sDNA] test is a successful colorectal cancer [CRC] screening tool in average-risk individuals but is not indicated for patients with inflammatory bowel disease [IBD]. We determined the performance of the mt-sDNA assay without the haemoglobin component [mt-sDNAHgb-] in patients with IBD, while measuring sensitivity for colorectal cancer and advanced colorectal neoplasia [ACRN]. METHODS: This was a multi-centre, proof-of-concept investigation in persons aged 18-84 years with a diagnosis of IBD, or primary sclerosing cholangitis [PSC] with IBD. Enrolment occurred between March 2013 and May 2016. Stool was tested with the mt-sDNA molecular markers only, minus the immunochemical haemoglobin component. RESULTS: The analysis set contained 355 samples. The median age was 52 [range 39-62] years, 45.6% were female and 93% were White. Two-thirds [63%] had ulcerative colitis [UC] and 10.1% had PSC/IBD. Colonoscopy revealed cancer in 8.5% [N = 30], advanced precancerous lesions [APLs] in 9.3% [N = 33] and non-advanced precancerous lesions in 7.6% [N = 27], and three-quarters [74.7%, N = 265] had negative findings. mt-sDNAHgb- sensitivity was 73.3% for any stage cancers, and 76.2% for ACRN. Sensitivity was highest for IBD-associated high-grade dysplasia at 100% and 84.6% for IBD-associated low-grade dysplasia ≥1 cm. The test showed higher sensitivity and lower specificity in UC than in Crohn's disease. Increasing inflammation score was associated with a significant decrease in mt-sDNAHgb- test score [ = 0.028] amongst neoplasia-negative individuals, but not in patients with ACRN. CONCLUSIONS: These data highlight the potential of multitarget stool-DNA marker testing as an important addition to colorectal cancer surveillance by complementing colonoscopic evaluations in IBD patients.

Validation of a Novel Multitarget Blood Test Shows High Sensitivity to Detect Early Stage Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although biannual ultrasound surveillance with or without α-fetoprotein (AFP) testing is recommended for at-risk patients, sensitivity for early stage HCC, for which potentially curative treatments exist, is suboptimal. We conducted studies to establish the multitarget HCC blood test (mt-HBT) algorithm and cut-off values and to validate test performance in patients with chronic liver disease. METHODS: Algorithm development and clinical validation studies were conducted with participants in an international, multicenter, case-control study. Study subjects had underlying cirrhosis or chronic hepatitis B virus; HCC cases were diagnosed per the American Association for the Study of Liver Diseases criteria and controls were matched for age and liver disease etiology. Whole blood and serum were shipped to a central laboratory and processed while blinded to case/control status. An algorithm was developed for the mt-HBT, which incorporates methylation biomarkers (HOXA1, TSPYL5, and B3GALT6), AFP, and sex. RESULTS: In algorithm development, with 136 HCC cases (60% early stage) and 404 controls, the mt-HBT showed 72% sensitivity for early stage HCC at 88% specificity. Test performance was validated in an independent cohort of 156 HCC cases (50% early stage) and 245 controls, showing 88% overall sensitivity, 82% early stage sensitivity, and 87% specificity. Early stage sensitivity in clinical validation was significantly higher than AFP at 20 ng/mL or greater (40%; P < .0001) and GALAD (gender, age, Lens culinaris agglutinin-reactive AFP, AFP, and des-γ-carboxy-prothrombin score) of -0.63 or greater (71%; P = .03), although AFP and GALAD at these cut-off values had higher specificities (100% and 93%, respectively). CONCLUSIONS: The mt-HBT may significantly improve early stage HCC detection for patients undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality. ClinicalTrials.gov number NCT03628651.

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).

Improved efficacy with targeted pharmacogenetic-guided treatment of patients with depression and anxiety: A randomized clinical trial demonstrating clinical utility.

The objective of this study was to evaluate the effect of pharmacogenetics-guided treatment on patients diagnosed with depression and/or anxiety, in a diverse set of clinical settings, as compared to the standard of care. The trial design followed a prospective, randomized, subject- and rater-blinded approach enrolling 685 patients from clinical providers specializing in Psychiatry, Internal Medicine, Obstetrics & Gynecology, and Family Medicine. The NeuroIDgenetix® test uses a genetic variant panel of ten genes, along with concomitant medications, to make medication management recommendations based on gene-drug and drug-drug interactions for over 40 medications used in the treatment of depression and anxiety. Pharmacogenetic testing was performed at the initial screening visit and baseline patient assessments were determined using the 17-item Hamilton Rating Scale for Depression (HAM-D17) and the Hamilton Rating Scale for Anxiety (HAM-A). Following enrollment and randomization, pharmacogenetic results for subjects assigned to the experimental group were provided to physicians to guide treatment selection, while control subjects were treated according to the usual standard of care. HAM-D17 and HAM-A assessments were collected at 4 weeks, 8 weeks, and 12 weeks after baseline to assess the efficacy of therapeutic selection. In patients diagnosed with depression, response rates (p = 0.001; OR: 4.72 [1.93-11.52]) and remission rates (p = 0.02; OR: 3.54 [1.27-9.88]) were significantly higher in the pharmacogenetics-guided group as compared to the control group at 12 weeks. In addition, patients in the experimental group diagnosed with anxiety showed a meaningful improvement in HAM-A scores at both 8 and 12 weeks (p = 0.02 and 0.02, respectively), along with higher response rates (p = 0.04; OR: 1.76 [1.03-2.99]). From these results, we conclude that pharmacogenetic-guided medication selection significantly improves outcomes of patients diagnosed with depression or anxiety, in a variety of healthcare settings.

Clinical Impact of Pharmacogenetic-Guided Treatment for Patients Exhibiting Neuropsychiatric Disorders: A Randomized Controlled Trial.

Objective: Pharmacogenetic testing holds promise as a personalized medicine tool by permitting individualization of pharmacotherapy in accordance with genes influencing therapeutic response, side effects, and adverse events. The authors evaluated the effect on outcomes for patients diagnosed with neuropsychiatric disorders of pharmacogenetics (PGx)-guided treatment compared to usual standard of care. Methods: This was a prospective, randomized study of 237 patients at an outpatient community-based psychiatric practice conducted between April 2015 and October 2015. Baseline patient assessments and a buccal swab were collected for pharmacogenetic testing at study initiation. For the experimental group, PGx results were provided to the clinicians as guides to treatment. Control subjects were treated according to the usual standard of care with no clinician reference to their PGx results. Neuropsychiatric Questionnaire (NPQ) and Symbol Digit Coding Test (SDC) scores and adverse drug events, hospitalizations, and medication information were collected at 30, 60, and 90 days. Results: More than half (53%) of patients in the control group reported at least 1 adverse drug event compared to 28% of patients with PGx-guided medication management (P = .001). NPQ and SDC scores improved for both groups, but no statistical difference in efficacy as measured by these assessments was observed within the 90-day observation period. Conclusions: Pharmacogenetic testing may facilitate psychiatric drug therapy with greater tolerability and similar efficacy compared to standard of care. Trial Registration: ClinicalTrials.gov Identifier: NCT02411123​​.

Rate of detection of high-risk HPV with two assays in women ≥ 30 years of age.

BACKGROUND: High-risk (HR) human papillomavirus (HPV) prevalence rates, as determined by the Cervista(®) HPV HR test, in women aged ≥30 years in a routine screening population have not been studied. OBJECTIVES: The primary objective of this study was to estimate HR HPV prevalence in women negative for intraepithelial lesion or malignancy (NILM) cytology using the CERVISTA HPV HR test. The study also compared HR HPV prevalence rates in women aged ≥30 years and NILM cytology using the CERVISTA HPV HR and Hybrid Capture(®) 2 (hc2) tests. STUDY DESIGN: A multi-center study was conducted to analyze HR HPV prevalence rates using the CERVISTA HPV HR test from residual ThinPrep(®) specimens. HR HPV positive rates were determined for hc2; percent agreement between the CERVISTA HPV HR and the hc2 tests were reported. RESULTS: HR HPV prevalence rates among women with NILM cytology were not statistically different between the CERVISTA HPV HR and hc2 tests (6.92% [98/1417] versus 5.93% [84/1417], respectively; P>0.05). The overall percent agreement between the tests was 95.3% (1351/1417; 95% confidence interval [CI]: 94.1-96.3; κ=0.61, 95% CI: 0.53-0.70). There were no statistically significant differences between tests across age groups or investigational sites. For both tests, there was a statistically significant decrease in HR HPV positive results as age increases (CERVISTA HPV HR, P=0.0009; hc2, P<0.0001). DISCUSSION: There is no statistically significant difference between HR HPV prevalence rates obtained with the CERVISTA HPV HR and hc2 tests in women aged ≥30 years with NILM cytology.

Analytical performance of Cervista HPV 16/18 genotyping test for cervical cytology samples.

BACKGROUND: Human papillomavirus (HPV) types 16 and 18 are the 2 most frequent types associated with cervical cancer. Identifying their presence or absence in cervical samples may assist in triaging women for subsequent management. The Cervista HPV 16/18 genotyping test specifically detects the presence of HPV 16 and 18 in ThinPrep cervical specimens. OBJECTIVES: The objective was to establish the analytical performance of the CERVISTA HPV 16/18 genotyping test. STUDY DESIGN: These studies were performed in support of a regulatory submission to the US Food and Drug Administration. Here we report the analytical sensitivity (limit of detection), accuracy compared to consensus L1 gene PCR/bi-directional sequencing, precision, reproducibility, and cross-reactivity (specificity) of the genotyping test. RESULTS: Analytical sensitivity for detection of HPV 16 and 18 ranged between 625 and 1250 copies/reaction for both types. When compared to PCR/sequencing for women with atypical squamous cells of undetermined significance cytology, the positive percent agreement was 94.1% (95% confidence interval [CI], 89.8-96.7) and the negative percent agreement was 85.7% (95% CI, 82.4-88.4). The test demonstrated high within-laboratory and inter-operator precision. Reproducibility within sites and between 3 testing sites resulted in 100% agreement with expected results (150 positive, 90 negative results). The genotyping test did not exhibit cross-reactivity to DNA from common low-risk HPV types and other microorganisms found in the human female reproductive tract. CONCLUSIONS: These analytical performance data support the use of CERVISTA HPV 16/18 genotyping test for the detection and differentiation of HPV 16 and 18 in ThinPrep cervical cytology specimens.

Clinical validation of the Cervista HPV HR and 16/18 genotyping tests for use in women with ASC-US cytology.

OBJECTIVE: High-risk (HR) human papillomavirus (HPV) testing is important in cervical cancer screening for triage to colposcopy. This study evaluated the clinical performance of the Cervista HPV HR and 16/18 genotyping tests for detection of HPV in cervical cytology specimens. METHODS: The tests were prospectively evaluated in a multicenter clinical study. DNA was extracted from approximately 4000 residual liquid-based cytology specimens collected during routine liquid-based Papanicolaou tests at standard of care visits and was assessed for the presence of HR HPV and/or HPV types 16 and 18. All women with cytology results of atypical squamous cells of undetermined significance (ASC-US) or greater underwent colposcopic examination and biopsies were collected. Test results were compared with local colposcopy and histology results from a central pathology review panel. RESULTS: There were 1347 subjects with complete data sets of cytology, HR HPV, colposcopy, and histology included in the analysis of the HPV HR test. Sensitivity of the HPV HR test for detection of cervical intraepithelial neoplasia (CIN) 2+ among women with ASC-US cytology was 92.8% (95% confidence interval [CI]: 84.1-96.9) and the negative predictive value (NPV) was 99.1% (95% CI: 98.1-99.6). Sensitivity for detection of > or =CIN 3 in women with ASC-US was 100% (95% CI: 85.1-100) and the NPV was 100% (95% CI: 99.4-100). The specificity of the test for detection of > or =CIN 2 and > or =CIN 3 was 44.2% (95% CI: 41.5-46.9) and 43% (95% CI: 40.3-45.7), respectively. The HPV 16/18 genotyping test also performed as expected in women with ASC-US cytology who were positive for HR HPV. CONCLUSION: The Cervista HPV HR test can be clinically used for detecting HR HPV types in conjunction with cervical cytology for use in triage of women with ASC-US cytology during routine cervical cancer screening.

Invader assay for RNA quantitation.

The Invader assay is a homogeneous, isothermal, signal amplification system for the quantitative detection of nucleic acids. The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the hybridization of two oligonucleotides to the target sequence. The combination of sequence-specific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms directly from genomic DNA as well as highly homologous mRNAs in closely related gene families. Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence.

Invader technology for DNA and RNA analysis: principles and applications.

Concomitant advances made by the Human Genome Project and in the development of nucleic acid screening technologies are driving the expansion of pharmacogenomic research and molecular diagnostics. However, most current technologies are restrictive due to their complexity and/or cost, limiting the potential of personalized medicine. The invader assay, which can be used for genotyping as well as for gene expression monitoring without the need for intervening target amplification steps, presents an immediate solution that is accurate, simple to use, scaleable and cost-effective.

Multiplexed gene expression analysis using the invader RNA assay with MALDI-TOF mass spectrometry detection.

A mass spectrometric approach for measuring gene expression levels has been developed. This technique utilizes a signal amplification system and analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Signal amplification from the targeted RNA employs a recently developed invasive cleavage assay that does not require prior PCR amplification. The assay uses a set of target-specific probes (oligonucleotides), which hybridize to the RNA being measured to create an overlap structure with a single-stranded flap. This flap is enzymatically cleaved and accumulates linearly in a target-specific manner. The products of the reaction, short DNA oligomers, are well suited for quantitative detection by MALDI-TOF mass spectrometry. Multiplexing is achieved by designing the assays so that reaction products for different mRNA targets have discrete masses that can be resolved in a single mass spectrum. Simultaneous analysis of human cytokine in vitro transcripts IL-1beta, TNF-alpha, and IL-6, with GAPDH as a reference standard, was used as a model system to demonstrate this novel method of gene expression analysis.