Multisite Performance Evaluation of the cobas 5800 System and Comparison to the cobas 6800/8800 Systems for Quantitative Measurement of HBV, HCV, and HIV-1 Viral Load.

The cobas 5800 System ("cobas 5800") is a new low- to mid-throughput PCR-based nucleic acid testing system which performs both qualitative and quantitative testing, including viral load (VL) determination. cobas 5800 shares numerous design elements and technical characteristics with the existing cobas 6800/8800 Systems. We compared HBV, HCV, and HIV-1 VL results from cobas 5800 in three different laboratories to those from the same specimens tested on a cobas 6800 system. We also assessed cobas 5800 assay reproducibility by repetitive testing of specimens with VL close to values used as thresholds for patient management or classification. The correlation between VL measurements generated using cobas 5800 versus 6800 was extremely high, with r2 correlation coefficients between 0.990 and 0.999 for the three targets at the different sites. The slope of the Deming regression line ranged from 0.994 (HBV, site 3) to 1.025 (HIV-1, site 1). The standard deviation values ranged from 0.04 to 0.19 log10 IU/mL for HBV, 0.06 to 0.33 log10 IU/mL for HCV, and 0.05 to 0.34 log10 copies/mL for HIV-1. In general, variability was higher at lower VL. Between 98.6% and 100% of results fell within the allowable total difference zone that defines expected variability on the existing 6800/8800 system. This multisite comparison study demonstrates equivalent performance of the new cobas 5800 system compared with cobas 6800. This establishes cobas 5800 as a new option for low- to mid-throughout laboratories seeking to optimize efficiency of their viral molecular testing. IMPORTANCE These are the first published data that demonstrate equivalent performance of the new cobas 5800 system compared with cobas 6800. This fulfills an unmet need for low- to mid-throughout laboratories seeking to optimize efficiency of their viral molecular testing.

The IMproving Primary Screening And Colposcopy Triage trial: human papillomavirus, cervical cytology, and histopathologic results from the baseline and 1-year follow-up phase.

BACKGROUND: An increase in human papillomavirus test volumes is expected in the near future because human papillomavirus-based screening protocols are expected to become more widely adopted. OBJECTIVE: The IMproving Primary Screening And Colposcopy Triage trial, a prospective, multicenter, US-based cervical cancer screening trial, was conducted to obtain US Food and Drug Administration approvals for the new, high-throughput cobas human papillomavirus (cobas HPV) test for use on the cobas 6800/8800 Systems (cobas HPV) for detecting cervical precancerous and cancerous cells (cervical intraepithelial neoplasia of grade 2 or worse and grade 3 or worse). Here, the baseline demographics, human papillomavirus test results, cervical cytology, and histopathologic results are presented. In addition, the baseline and 1-year risks of cervical intraepithelial neoplasia grade 2 or worse and grade 3 or worse associated with the human papillomavirus results are reported. STUDY DESIGN: In total, 35,263 women aged between 25 and 65 years undergoing routine screening were enrolled; liquid-based cytology and 2 polymerase chain reaction-based tests for high-risk human papillomavirus were performed. Women with abnormal Papanicolaou cytology, women positive for high-risk human papillomavirus by either of the 2 human papillomavirus tests, and a random subset of women negative according to the Papanicolaou cytology and the 2 human papillomavirus tests were referred for a colposcopy and cervical biopsy. Women who did not meet the study endpoint were eligible for the 1-year follow-up study phase. Verification bias-adjusted cervical disease prevalence and risks and 95% confidence intervals were computed. RESULTS: The prevalence of atypical squamous cells of undetermined significance and worse than atypical squamous cells of undetermined significance cytology were 6.5% and 3.2%, respectively. Prevalence of high-risk human papillomavirus, human papillomavirus 16, and human papillomavirus 18 based on the new cobas HPV test were 15.1%, 3.1%, and 1.4%, respectively. Both cytologic abnormalities and human papillomavirus positivity declined with increasing age. Among women who had a colposcopy and biopsy, the prevalence of cervical intraepithelial neoplasia grade 2 or worse and grade 3 or worse were 8.8% and 3.6%, respectively. The baseline and 1-year cumulative risks for cervical intraepithelial neoplasia grade 3 or worse were 13.6% and 16.9%, respectively, among women who tested positive for human papillomavirus 16. Women who tested negative for human papillomavirus had the lowest 1-year cumulative risk for cervical intraepithelial neoplasia grade 3 or worse (0.06%). CONCLUSION: The contemporary, age-specific prevalence of human papillomaviruses (including human papillomavirus 16 and 18), cytologic abnormalities, and cervical intraepithelial neoplasia in a large, US-based cervical cancer screening population provides benchmarks for healthcare policies, screening programs, and for laboratories and clinicians.

A Pooled Analysis to Compare the Clinical Characteristics of Human Papillomavirus-positive and -Negative Cervical Precancers.

Given that high-risk human papillomavirus (HPV) is the necessary cause of virtually all cervical cancer, the clinical meaning of HPV-negative cervical precancer is unknown. We, therefore, conducted a literature search in Ovid MEDLINE, PubMed Central, and Google Scholar to identify English-language studies in which (i) HPV-negative and -positive, histologically confirmed cervical intraepithelial neoplasia grade 2 or more severe diagnoses (CIN2+) were detected and (ii) summarized statistics or deidentified individual data were available to summarize proportions of biomarkers indicating risk of cancer. Nineteen studies including 3,089 (91.0%) HPV-positive and 307 (9.0%) HPV-negative CIN2+ were analyzed. HPV-positive CIN2+ (vs. HPV-negative CIN2+) was more likely to test positive for biomarkers linked to cancer risk: a study diagnosis of CIN3+ (vs. CIN2; 18 studies; 0.56 vs. 0.24; P < 0.001) preceding high-grade squamous intraepithelial lesion cytology (15 studies; 0.54 vs. 0.10; P < 0.001); and high-grade colposcopic impression (13 studies; 0.30 vs. 0.18; P = 0.03). HPV-negative CIN2+ was more likely to test positive for low-risk HPV genotypes than HPV-positive CIN2+ (P < 0.001). HPV-negative CIN2+ appears to have lower cancer risk than HPV-positive CIN2+. Clinical studies of human high-risk HPV testing for screening to prevent cervical cancer may refer samples of HPV test-negative women for disease ascertainment to correct verification bias in the estimates of clinical performance. However, verification bias adjustment of the clinical performance of HPV testing may overcorrect/underestimate its clinical performance to detect truly precancerous abnormalities.

Approaches to triage optimization in HPV primary screening: Extended genotyping and p16/Ki-67 dual-stained cytology-Retrospective insights from ATHENA.

The objective of our study was to assess the performance of different triage strategies for high-risk human papillomavirus (hrHPV)-positive results utilizing either extended genotyping or a p16/Ki-67 dual-stained cytology (DS) approach, with or without partial genotyping. A subset of women with hrHPV infections participating in the Addressing the Need for Advanced HPV Diagnostics (ATHENA) study were analyzed to determine the number of cervical intraepithelial neoplasia grade 3 or worse (≥CIN3) cases detected, and the absolute risk for ≥CIN3 of each genotype. A clinical utility table was constructed to compare the impact of different triage strategies. In all, 2,339 women with single-genotype hrHPV infections were identified. Among these were 171 ≥CIN3 cases. The U.S. Food and Drug Administration (FDA)-approved algorithm (HPV16/18 positive, or 12-other hrHPV positive and Pap positive, i.e., ≥ atypical squamous cells of undetermined significance) for primary HPV screening detected 132/171 (77.2%) ≥CIN3 cases and required 964 colposcopies (colposcopies per ≥CIN3 ratio: 7.3). An approach that uses DS instead of cytology in the FDA-approved algorithm detected 147/171 (86.0%) ≥CIN3 cases, requiring 1,012 colposcopies (ratio: 6.9). Utilizing DS for triage of all hrHPV-positive women identified 126/171 (73.7%) ≥CIN3 cases, requiring 640 colposcopies (ratio: 5.1). A strategy that detected HPV16/18/31/33/35+ captured 130/171 (76.0%) ≥CIN3 cases, requiring 1,025 colposcopies (ratio: 7.9). Inclusion of additional genotypes resulted in greater disease detection at the expense of higher colposcopy ratios. Substituting cytology with a DS triage approach improved disease detection and the colposcopy detection rate. Further reduction of colposcopy rates can be achieved by using DS without partial genotyping. Extended genotyping strategies can identify a comparable number of cases but requires an increased number of colposcopies.

Performance of the cobas HPV Test for the Triage of Atypical Squamous Cells of Undetermined Significance Cytology in Cervical Specimens Collected in SurePath.

OBJECTIVES: Determine performance of the cobas human papillomavirus (HPV) test for triage of atypical squamous cells of undetermined significance (ASC-US) in SurePath. METHODS: Women presenting for routine screening had cervical specimens collected in SurePath and specimen transport medium (STM); those with ASC-US cytology underwent colposcopy. Performance of cobas HPV in SurePath specimens that had undergone a preanalytic procedure to reverse possible cross-linking of HPV DNA was compared with Hybrid Capture 2 (hc2) specimens in STM. RESULTS: Among 856 women, HPV prevalence was 45.8%; HPV 16 and HPV 18 prevalences were lower than expected in the 21- to 29-year-old group in this highly vaccinated population. cobas HPV performance in SurePath was comparable to hc2 in STM. Sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 3 or worse were 87.5% (95% confidence interval [CI], 71.9%-95.2%) and 55.5% (95% CI, 52.1%-58.9%) for cobas and 85.3% (95% CI, 69.9%-93.6%) and 54.7% (95% CI, 51.4%-57.9%) for hc2. Sensitivity was negatively affected by random biopsies performed at colposcopy; comparable sensitivities were achieved in the nonvaccinated and vaccinated populations with disease determined by directed biopsy only. CONCLUSIONS: Performance of cobas HPV for ASC-US triage in pretreated SurePath specimens meets criteria for validation. Preliminary data indicate reliable performance of HPV testing in a highly vaccinated population.

Development and Validation of a Preanalytic Procedure for Performing the cobas HPV Test in SurePath Preservative Fluid.

The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (CT) values for cobas HPV detection were evaluated over time and various temperatures, and mean CT differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) CT differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: -2.58 (-3.16 to -2.01), -2.63 (-3.62 to -1.64), and -3.39 (-4.95 to -1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) CT differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: -0.48 (-0.98 to 0.02) and -0.23 (-0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [-0.35 (-0.57 to -0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath.

Cervical Precancer and Cancer Risk by Human Papillomavirus Status and Cytologic Interpretation: Implications for Risk-Based Management.

BACKGROUND: Cervical cancer risks, estimated by using cervical intraepithelial neoplasia grade 3 (CIN3) or more severe diagnoses (≥CIN3) endpoints, have not been quantified for different combinations of results from currently approved screening methods. Understanding these risks will guide optimal patient management. METHODS: Women aged ≥25 years (n = 7,823) underwent high-risk human papillomavirus (hrHPV) and liquid-based cytology (LBC) testing. Women with hrHPV-positive results and/or abnormal LBC, plus a random subset of hrHPV and LBC negatives, underwent colposcopy; those without ≥CIN2 at baseline were screened annually by LBC and referred to colposcopy for an abnormal LBC (n = 7,392). One- and 3-year ≥CIN3 risks with 95% confidence intervals (95% CI) were calculated for paired hrHPV and LBC (hrHPV/LBC) results. RESULTS: One-year ≥CIN3 risks ranged from 81.27% (95% CI, 66.02%-90.65%) for HPV16 positive/high-grade to 0.33% (95% CI, 0.18%-0.62%) for hrHPV negative/negative for intraepithelial lesion or malignancy (NILM). One-year ≥CIN3 risk for HPV16/NILM (13.95%; 95% CI, 10.98%-17.58%) was greater than low-grade squamous intraepithelial lesion (LSIL; 7.90%; 95% CI, 5.99%-10.37%; P = 0.002) and similar to hrHPV-positive/LSIL (11.45%; 95% CI, 8.61%-15.07%; P = 0.3). Three-year ≥CIN3 risks for HPV16 positive/LSIL and HPV16/atypical squamous cells of undetermined significance was 24.79% (95% CI, 16.44%-35.58%) and 24.36% (95% CI, 15.86%-35.50%), respectively, and 0.72% (95% CI, 0.45%-1.14%) for hrHPV negative/NILM. CONCLUSIONS: hrHPV and LBC results stratify cervical cancer risk by more than two orders of magnitude. HPV16-positive women, regardless of the LBC result, warrant immediate colposcopy. Women with concurrent HPV16 and high-grade LBC might consider treatment without a confirmatory biopsy with informed decision-making with their provider. IMPACT: These results provide relevant benchmarks for risk-based cervical cancer screening and management. Cancer Epidemiol Biomarkers Prev; 25(12); 1595-9. ©2016 AACR.

Knowledge of Patients' Human Papillomavirus Status at the Time of Cytologic Review Significantly Affects the Performance of Cervical Cytology in the ATHENA Study.

OBJECTIVES: With human papillomavirus (HPV) testing, patients' HPV status may be known when reviewing cytology specimens. METHODS: 41,955 women 25 years or older had cytology and HPV screening. Originally, cytology was reviewed blinded to HPV status. We re-reviewed unblinded to HPV status a subset of 428 cytology slides from women with cervical intraepithelial neoplasia grade 2 + (CIN2+) and 1,287 from women without CIN2+. RESULTS: Of the original interpretations of atypical squamous cells of undetermined significance (ASC-US), 33.7% were downgraded to negative after unblinded review, and 8.7% were upgraded to atypical squamous cells, cannot rule out a high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion. Of the original interpretations of ASC-US, 66.7% were downgraded on unblinded review in HPV-negative women and 30.2% were upgraded in HPV 16+/HPV 18+ women. Unblinding increases the sensitivity for cervical intraepithelial neoplasia grade 3+ of cotesting from 54.1% to 62.4% (P = .0015) and the sensitivity of HPV primary screening from 72.2% to 77.1% (P = .0029). With cotesting, specificity with unblinding is improved, whereas with HPV primary screening, there would be a small decrease in specificity. CONCLUSIONS: Unblinded cytology increases overall sensitivity with either cotesting or HPV primary screening; specificity is either slightly improved or is not affected by unblinding.

Novel Approach for Clinical Validation of the cobas KRAS Mutation Test in Advanced Colorectal Cancer.

AIM: Our objective was to assess the performance of the cobas test versus comparators for KRAS mutation status and predicting clinical response to anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). METHODS: mCRC samples from 398 patients from Roche study NO16968 (XELOXA) and 82 supplemental samples were tested with the cobas(®) KRAS mutation test (cobas test), the therascreen(®) KRAS RGQ PCR kit test (therascreen test), and Sanger sequencing as the reference method for detecting mutations in codons 12/13. RESULTS: For 461 eligible samples, the cobas test, therascreen test, and sequencing had invalid results for 5.2, 10.8, and 2.6 % of specimens, respectively. Valid cobas and therascreen test results had similar KRAS mutation-positive rates (37.3 vs. 36.3 %, respectively); sequencing was 28.5 %. Positive and negative percent agreement (PPA/NPA) between the cobas test and sequencing was 96.9 % (95 % confidence interval [CI] 92.2-98.8), and 88.7 % (95 % CI 84.7-91.8), respectively. PPA/NPA between the cobas and therascreen tests was 93.3 % (95 % CI 88.1-96.3) and 96.5 % (95 % CI 93.5-98.1), respectively. Bridging analysis from NCIC-CO.17 and NCT00113763 using the cobas test yielded modeled hazard ratios for overall survival and progression-free survival (PFS) of 0.558 (95 % CI 0.422-0.752) and 0.413 (95 % CI 0.304-0.550), respectively, for cetuximab and 0.989 (95 % CI 0.778-1.299) and 0.471 (95 % CI 0.360-0.626), respectively, for panitumumab, demonstrating significant efficacy in the KRAS-negative population for PFS. CONCLUSION: The cobas test showed similar accuracy to the therascreen test for detecting KRAS mutations and could appropriately identify mCRC patients ineligible for anti-EGFR therapy as demonstrated by bridging analysis results.

Evaluation of the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HCV Test, v2.0 and comparison to assays used in routine clinical practice in an international multicenter clinical trial: The ExPECT study.

BACKGROUND: The COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. OBJECTIVES: The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS(®) TaqMan(®) HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. STUDY DESIGN: Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS(®) AmpliPrep(®)/COBAS(®) TaqMan(®) HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. RESULTS: All quantifiable WHO dilutions were within ±0.3log10IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5log10IU/mL. When low viral load results (25-3500IU/mL) were compared, values obtained by the ART assay were significantly lower (p<0.0001) than those obtained with the CAP/CTM2. CONCLUSIONS: The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens.

HCV RNA detection in HCV antibody-positive patients with the COBAS AmpliPrep/COBAS TaqMan HCV test, v2.0 in comparison with FDA-approved nucleic acid tests.

BACKGROUND: Analysis of hepatitis C virus (HCV) RNA levels is critical for assessing the efficacy of antiviral therapy and the achievement of a sustained virologic response. OBJECTIVE AND STUDY DESIGN: This study evaluated the clinical performance of the COBAS AmpliPrep/COBAS TaqMan HCV quantitative test, version 2.0 (TAQMAN v2.0) with the COBAS AmpliPrep/COBAS TaqMan HCV quantitative test, version 1.0 (TAQMAN v1.0), the VERSANT HCV qualitative assay (VERSANT), and the COBAS AMPLICOR HCV test, v2.0 (AMPLICOR) qualitative test for the detection of HCV RNA in serum or EDTA plasma from patients who are or have been infected with HCV and carry HCV antibodies. RESULTS: A total of 277 participants were evaluable for the percent agreement analysis of the TAQMAN v2.0 with the VERSANT and with the AMPLICOR. The overall percent agreement between the TAQMAN v2.0 and the VERSANT or the AMPLICOR was 99.3% (95% CI: 97.4%, 99.8%) or 98.9% (95% CI: 96.9%, 99.6%), respectively. The overall percent agreement between the TAQMAN v2.0 and the TAQMAN v1.0 when 267 of the original samples were assessed was 98.9% (95% CI=96.7%, 99.6%). CONCLUSION: The TAQMAN v2.0 demonstrated high correlation with the previously approved HCV RNA quantitative and qualitative tests.

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients.

BACKGROUND: Sensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice. OBJECTIVES/STUDY DESIGN: Using 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS(®) AMPLICOR MONITOR CMV tests. RESULTS: The median time between transplantation and testing samples was 57 days (range, 0-207 days). The median CMV load (log(10)) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (-0.15 [95% CI, -0.18 to -0.13] copies/mL), but slope differences indicated only limited co-linearity. CONCLUSIONS: The CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.

An international multicenter performance analysis of cytomegalovirus load tests.

BACKGROUND: Quantification of cytomegalovirus (CMV) load is central to the management of CMV infections in immunocompromised patients, but quantitative results currently differ significantly across methods and laboratories. METHODS: The COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV test), developed using the first World Health Organization CMV standard in the calibration process, was compared to local assays used by 5 laboratories at transplant centers in the United States and Europe. Blinded plasma panels (n = 90) spiked with 2.18-6.7 log(10) copies/mL and clinical plasma samples from immunocompromised patients (n = 660) were tested. RESULTS: Observed mean panel member concentrations by site and 95% confidence intervals (CIs) of the data combined across sites were narrower for CAP/CTM CMV test compared with local assays. The 95% CI in log(10) copies/mL of the combined data per panel member for CAP/CTM CMV test vs comparator assays was .17 vs 1.5 at 2.18 log(10) copies/mL; .14 vs .52 at 2.74 log(10) copies/mL; .16 vs .6 at 3.3 log(10) copies/mL; .2 vs 1.11 at 4.3 log(10) copies/mL; .21 vs 1.13 at 4.7 log(10) copies/mL; and .18 vs 1.4 at 6.7 log(10) copies/mL. In clinical specimens, constant and variable quantification differences between the CAP/CTM CMV test and comparator assays were observed. CONCLUSIONS: High interlaboratory agreement and precision of CAP/CTM CMV test results across 5 different laboratories over 4 orders of magnitude suggest that this assay could be valuable in prospective studies identifying clinical viral load thresholds for CMV treatment.