Background As knowledge of mechanisms that drive the development of cancer grows, there has been corresponding growth in therapies specific to a mechanism. While these therapies show improvements in patient outcomes, they can be expensive and are effective only for a subset of patients. These treatments drive interest in research focused on the assignment of cancer therapies based on aberrations in individual genes or biomarkers that assess the broader mutational landscape, including microsatellite instability (MSI) and tumor mutational burden (TMB).Methods Here we describe the TruSight™ Oncology 500 (TSO500; Research Use Only) bioinformatics workflow. This tumor-only approach leverages the next-generation sequencing-based assay TSO500 to enable high fidelity determination of DNA variants across 523 cancer-relevant genes, as well as MSI status and TMB in formalin-fixed paraffin-embedded (FFPE) samples.Results The TSO500 bioinformatic workflow integrates unique molecular identifier (UMI)-based error correction and a dual approach variant filtering strategy that combines statistical modeling of error rates and database annotations to achieve detection of variants with allele frequency approaching 5% with 99.9998% per base specificity and 99% sensitivity in FFPE samples representing a variety of tumor types. TMB determined using the tumor-only workflow of TSO500 correlated well with tumor-normal (N =170, adjusted R 2=0.9945) and whole-exome sequencing (N=108, adjusted R 2=0.933). Similarly, MSI status determined by TSO500 showed agreement (N=106, 98% agreement) with a MSI-PCR assay.Conclusion TSO500 is an accurate tumor-only workflow that enables researchers to systematically characterize tumors and identify the next generation of clinical biomarkers.### Competing Interest StatementAll authors are employees and shareholders of Illumina Inc.* MSI : microsatellite instability TMB : tumor mutational burden TSO500 : TruSight™ Oncology 500 FFPE : formalin-fixed paraffin-embedded UMI : unique molecular identifier NGS : next-generation sequencing IHC : immunohistochemistry PCR : polymerase chain reaction WES : whole exome sequencing NRUMIs : non-random unique molecular identifiers AQ : variant quality score LQ : likelihood ratio score COSMIC : Catalogue of Somatic Mutations in Cancer CNV : copy number variant VAFs : Variant allele frequencies

Because of its non-invasive nature urine testing may enable increased screening for HPV in women who avoid cervical sampling. Comparisons have shown fewer HPV positives in urine. The objectives were to compare first-void urine (FVU) treated with proteinase K (PK) to untreated FVU and cervical samples collected from women attending a colposcopy clinic using an Aptima HPV mRNA assay, and comparing the HPV rates to cytology and pathology results. Female FVU (n = 433) was treated with Aptima Transfer Solution (ATS) containing PK within 24 h or after months of storage. Untreated female FVU samples were HPV-positive in 20.8-27.6% compared to 34.4-45.6% of ATS-treated FVU and 44.9-48.4% of PreservCyt samples. Good overall agreement for HR-HPV detection between ATS-FVU and PreservCyt was observed (81.1%; k 0.63). Validation of ATS treatment was performed on 356 male FVU, detecting 6.7% HPV positive compared to 3.4% of untreated samples (p = 0.059). Although HPV presence in ATS FVU and PreservCyt samples were similar, significantly more women with abnormal cervical cytology and histopathology were HPV-positive in cervical specimens than in ATS-treated FVU.

Objective The aim of the study was to evaluate the performance of the HPV-HR test to detect high-risk human papillomavirus (HPV) in urine samples in comparison with a commercial molecular HPV test. Materials and Methods This is a prospective study, in which 350 patients diagnosed previously with cervical intraepithelial neoplasia (CIN) grade 2 or higher were enrolled. Urine and cervical specimens were collected. Urine was tested with the HPV-HR test and cervical specimens were tested with the Cobas. Results Of the 336 evaluable patients, there were 271 cases of CIN 2+, of which 202 were CIN 3+ and the remaining 65 patients were less than CIN 2. Positivity was 77.1% (95% confidence interval [CI] = 72.5–81.5) for the urine samples and 83.6% (95% CI = 79.6–87.6) for the cervical samples. Agreement between cervical and urine samples for HPV detection was 79.8% (κ = 0.363; 95% CI = 0.243–0.484). Sensitivity for CIN 2+ was 83.4% (95% CI = 78.4–87.6) for urine and 90.8% (95% CI = 86.7–92.9) for cervical samples. The sensitivity for CIN 3+ was 85.6% (95% CI = 80.0–90.2) for urine and 92.6% (95% CI = 88.0–95.8) for cervical samples. Specificity for worse than CIN 2 was 50.8% (95% CI = 33.7–59.0) and 46.2% (95% CI = 33.7–59.0) for urine and cervical samples, respectively. Conclusions Although these results demonstrated slightly higher detection rates for HR-HPV and clinical sensitivity in cervical samples than in urine, when compared with histological diagnoses, urine sampling is a viable alternative to access women who do not participate in routine screening programs.

Background: Human papillomavirus (HPV) testing from clinician-collected cervical and self-collected cervico-vaginal samples is more sensitive for detecting CIN2+/CIN3+ than cytology-based screening, stimulating interest in HPV testing from urine. The objective was to determine the performance of the Trovagene HPV test for the detection of CIN2+ from urine and PreservCyt cervical samples. Methods: Women referred for colposcopy at St Mary's Hospital (London, United Kingdom), following abnormal cytology, were recruited to this diagnostic accuracy study by convenience sampling (September 2011 to April 2013). A total of 501 paired urine and cervical samples were collected. Primary outcomes were sensitivity for CIN2+/CIN3+ and specificity for <CIN2; secondary outcomes were comparisons with other HPV tests and agreement/kappa values between urine and cervical samples. Results: Trovagene HPV test sensitivity and specificity from PreservCyt were similar to well-established tests [sensitivity for CIN3+ (n = 145) 96.3% (95% confidence interval (CI), 89.6–99.2); CIN2+ (n = 81) 94.5% (95% CI, 89.4–97.6); specificity for <CIN2 25.3% (95% CI, 20.8–30.1)]. Sensitivity from urine was slightly, but not significantly, lower [CIN3+ 91.4% (95% CI, 83.0–96.5), P = 0.3; CIN2+ 88.3% (95% CI, 81.9–93.0), P = 0.06]. Specificity for <CIN2 was similar: 24.7% (95% CI, 20.3–29.5), P = 0.9. A total of 403 Trovagene cervical and 396 urine HPV tests were positive. Overall agreement between paired samples was 82.6% (95% CI, 79.3–86.0). Conclusions: Trovagene HPV test's performance on PreservCyt cervical samples was comparable with established HPV tests. Sensitivity in urine, although slightly lower, may nevertheless be adequate for self-sampling. This referral population's higher HPV positivity rate affects specificity, warranting further studies in a screening population. Impact: This may prove useful for women not attending for cervical screening. Cancer Epidemiol Biomarkers Prev; 26(7); 1053–9. ©2017 AACR.

SurePath specimens from women referred to colposcopy were treated with Aptima Transfer Solution (ATS) before testing in Aptima HPV (AHPV) and Aptima HPV 16, 18/45 (AHPV-GT) assays. Untreated SurePath specimens were tested with the cobas HPV test. PreservCyt specimens were assessed for cytology and tested with AHPV. High-grade cervical intraepithelial neoplasia lesions served as the reference standard. Excellent agreement (95.5%; k=0.91) was observed for ATS-treated SurePath specimens between Tigris and Panther systems and between the PreservCyt and ATS-treated SurePath specimens (91.1%, k=0.81) with the AHPV assay on Tigris. Agreement between the AHPV and cobas assays with SurePath specimens was substantial (89.9%, k=0.80). AHPV sensitivity for CIN2+(n=147) was 91.2% for SurePath and PreservCyt. Cobas HPV sensitivity was 93.9% for SurePath specimens. AHPV testing of SurePath specimens was more specific (59.4%) than cobas (54.7%) (p<0.001). Detection and genotyping showed similar absolute and relative risks. ATS-treated SurePath specimens tested with AHPV and AHPV-GT assays showed similar performance with greater specificity than cobas HPV on SurePath specimens. Similar overall results were seen using a CIN3 disease endpoint.

OBJECTIVESThe Aptima human papillomavirus (HPV) 16 18/45 Genotype (GT) assay (AHPV-GT) is a qualitative E6/ E7 oncogene messenger RNA test that detects HPV 16 and a pool of HPV 18 and 45. The CLEAR (Clinical Evaluation of APTIMA mRNA) study was the pivotal, prospective, multicenter US clinical study to validate the Aptima HPV (AHPV) assays.METHODSIn this analysis, we evaluated the clinical performance of AHPV and AHPV-GT assays for detection of cervical intraepithelial neoplasia grade 2 or more severe (CIN2 +) and grade 3 (CIN3) or adenocarcinoma in situ in 912 women with atypical squamous cells of undetermined significance (ASC-US) Papanicolaou result. The AHPV-GT assay was performed on high-risk HPV (hrHPV) positives as determined by the AHPV assay.RESULTSOverall, the percent positive for hrHPV was 38.8% (354/912), of which 34.2% (121/354) were GT positive. Among hrHPV-positive women, the risks of CIN2 + were 37.0% for HPV 16 positives, 15.9% for HPV 18/45 positives, 14.3% for other hrHPV positives, and 2.2% for AHPV negatives. The risks of CIN3 + were 20.5% for HPV 16 positives, 9.1% for HPV 18/45 positives, 4.3% for other hrHPV positives, and 0.7% for HPV negatives.CONCLUSIONSWe demonstrated that AHPV-GT is a reliable and effective test for cervical cancer risk stratification in women with an ASC-US cytology diagnosis.

Background: The Aptima HPV assay (AHPV) for high-risk human papillomavirus (hrHPV), and the Aptima HPV 16 18/45 Genotype assay (AHPV GT) for HPV16 and for HPV18 and/or HPV45 (HPV18/45) genotypes are approved for cervical cancer screening by the U.S. Food and Drug Administration. There are limited data on the reliability of these tests for detection of hrHPV, HPV16, and HPV18/45.Objectives: To assess the reliability of AHPV and AHPV GT on paired specimens from women with atypical squamous cells of undetermined significance (ASC-US) cytology. Study design: In a population of women with ASC-US cytology (n = 988), cervical specimens were collected at a routine screening baseline visit and at the colposcopy visit that occurred a median of 29 days later. Specimens were tested by AHPV and if positive, by AHPV GT.Results: There was no significant difference in the percent AHPV positive between the colposcopy and baseline specimens (41.9% vs. 43.0%, respectively, p = 0.3). The percent agreement, percent positive agreement, and the kappa value were 88.6%, 76.3%, and 0.766, respectively. There were no significant differences between AHPV testing of the colposcopy and baseline specimen in the sensitivity (95.2% vs. 92.9%, respectively, p = 1) and specificity (60.5% vs. 59.2%, respectively, p = 0.3) for CIN3+. Comparing the hierarchical AHPV and AHPV GT results on the two specimens, the percent exact agreement was 86.2%, the percent positive agreement was 68.4%, and the kappa value was 0.746.Conclusions: AHPV and AHPV GT demonstrated good reliability for hrHPV detection and risk stratification. (C) 2015 The Authors. Published by Elsevier B.V.

OBJECTIVES:This study determined the longitudinal clinical performance of a high-risk human papillomavirus (HR-HPV) E6/E7 RNA assay (Aptima HPV [AHPV]; Hologic, San Diego, CA) compared with an HR-HPV DNA assay (Hybrid Capture 2 [HC2]; Qiagen, Gaithersburg, MD) as an adjunctive method for cervical cancer screening.METHODS:Women 30 years or older with a negative result for intraepithelial lesions or malignancy cytology (n = 10,860) positive by AHPV and/or HC2 assays and randomly selected women negative by both assays were referred to colposcopy at baseline. Women without baseline cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+) continued into the 3-year follow-up.RESULTS:The specificity of AHPV for CIN2 or lower was significantly greater at 96.3% compared with HC2 specificity of 94.8% (P < .001). Estimated sensitivities and risks for detection of CIN2+ were similar between the two assays. After 3 years of follow-up, women negative by either human papillomavirus test had a very low risk of CIN2+ (<0.3%) compared with CIN2+ risk in women with positive AHPV results (6.3%) or positive HC2 results (5.1%).CONCLUSIONS:These results support the use of AHPV as a safe and effective adjunctive cervical cancer screening method.

ABSTRACT Few studies have compared the cobas HPV test to the Aptima HPV assay (AHPV) and the Aptima HPV 16 18/45 genotype assay (AHPV GT) for high-risk human papillomavirus (hrHPV) detection, clinical performance in detecting cervical intraepithelial neoplasia grade 2 (CIN2) or more severe (CIN2+) diagnoses, and risk stratification by partial HPV genotyping. The cobas HPV test is a DNA test that separately and concurrently detects HPV16, HPV18, and a pool of 12 other hrHPV types. AHPV is an RNA test for a pool of 14 hrHPV genotypes, and AHPV GT is an RNA test run on AHPV-positive results to detect HPV16 separately from HPV18 and HPV45, which are detected together. In a population of patients ( n = 988) referred for colposcopy because of a cervical Pap cytology result of atypical squamous cells of undetermined significance (ASC-US), a cervical scrape specimen was taken, placed into a ThinPrep Pap test vial containing PreservCyt liquid cytology medium, and tested in a blinded fashion with cobas and AHPV and with AHPV GT for AHPV-positive results. The final diagnoses were based on a consensus panel review of the biopsy specimen histology. AHPV and cobas were equally sensitive for CIN2+ diagnoses (89.4% each; P = 1.000), and AHPV was more specific than cobas (63.1% versus 59.3%; P ≤ 0.001). The percent total agreement, percent positive agreement, and kappa value were 90.9%, 81.1%, and 0.815, respectively. Risk stratification using partial HPV genotyping was similar for the two assays. AHPV and AHPV GT had similar sensitivity and risk stratification to cobas HPV, but they were more specific than cobas HPV.

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BACKGROUND:An APTIMA specimen collection and transportation (SCT) kit was developed by Hologic/Gen-Probe. OBJECTIVES:To compare cervical SCT samples to PreservCyt and SurePath samples and self-collected vaginal samples to physician-collected vaginal and cervical SCT samples. To determine ease and comfort of self-collection with the kit. STUDY DESIGN:Each woman (n = 580) self-collected a vaginal SCT, then filled out a questionnaire (n = 563) to determine ease and comfort of self-collection. Colposcopy physicians collected a vaginal SCT and cervical PreservCyt, SCT, and SurePath samples. Samples were tested by APTIMA HPV (AHPV) assay. RESULTS:Agreement between testing of cervical SCT and PreservCyt was 91.1% (κ = 0.82), and that of SurePath samples was 86.7% (κ = 0.72). Agreement of self-collected vaginal SCT to physician-collected SCT was 84.7% (κ = 0.68), and that of self-collected vaginal to cervical SCT was 82.0% (κ = 0.63). For 30 patients with CIN2+, AHPV testing of cervical SCT was 100% sensitive and 59.8% specific compared with PreservCyt (96.6% and 66.2%) and SurePath (93.3% and 70.9%). Vaginal SCT sensitivity was 86.7% for self-collection and 80.0% for physician collection. Most patients found that vaginal self-collection was easy, 5.3% reported some difficulty, and 87.6% expressed no discomfort. CONCLUSIONS:Cervical samples collected with the new SCT kit compared well to traditional liquid-based samples tested by AHPV. Although there was good agreement between self-collected and physician-collected samples with the SCT, in a limited number of 30 women, vaginal sampling identified fewer with CIN2+ precancerous cervical lesions than cervical SCT sampling. Comfort, ease of use, and detection of high-risk HPV demonstrated that the kit could be used for cervical and vaginal sampling.

OBJECTIVE: The objective of the study was to determine the sensitivity and specificity of the APTIMA human papillomavirus (AHPV) assay for high-grade cervical intraepithelial neoplasia (CIN) in women 21 years old and older with atypical squamous cells of undetermined significance (ASC-US) cytology. STUDY DESIGN: Women 21 years old and older with ASC-US cytology had colposcopy/biopsy and molecular human papillomavirus testing. Performance of the AHPV and Hybrid Capture 2 assays was compared with a clinical diagnosis of CIN grade 2, CIN grade 3, or adenocarcinoma in situ (CIN2 or greater). RESULTS: Of 939 evaluable subjects, CIN2 or greater and CIN3 or greater prevalence was 9.7% and 4.4%, respectively. AHPV sensitivity and specificity was 86.8% and 62.9% for CIN2 or greater detection and 90.2% and 60.2% for CIN3 or greater, respectively. AHPV had a similar sensitivity to Hybrid Capture 2 but a significantly higher specificity (62.9% vs 55.8%, P < .001) for CIN2 or greater detection. CONCLUSION: Among women with ASC-US cytology, detection of high-risk human papillomavirus E6/E7 oncogenic messenger ribonucleic acid is an effective triage method for colposcopy referral.

Background: Human papillomavirus (HPV) DNA testing is widely used in conjunction with Papanicolaou (Pap) testing in cervical cancer screening programs to improve the detection of high-grade lesions. While HPV DNA test sensitivity is good, an improvement in specificity is desired. Detection of HPV mRNA may improve specificity. The APTIMA(R) HPV Assay detects the mRNA of 14 high-risk HPV types in liquid-based cytology specimens.Objective: To evaluate APTIMA HPV Assay performance for detection of high-risk HPV and high-grade cervical intraepithelial neoplasia (CIN) compared to Qiagen's Hybrid Capture 2 HPV DNA (HC2) test.Study design: Liquid Pap specimens were collected from 800 women referred to colposcopy and tested with the APTIMA HPV Assay and the HC2 test. Complete results were available for 753 subjects. A subset of samples (n = 393) were typed using Roche's Linear Array HPV Genotyping Test.Results: Sensitivity and specificity for detection of high-risk HPV were >92% and 99% for the APTIMA HPV Assay and 93% and 82% for the HC2 test. Clinical sensitivity and specificity were 91% and >55% for detection of CIN 2+, and 98% and 53% for detection of CIN 3+ for the APTIMA HPV Assay; values for the HC2 test were 95% and 47% for CIN 2+, and 99% and 44% for CIN 3+.Conclusions: The APTIMA HPV Assay is sensitive and very specific for detection of high-risk HPV. The APTIMA HPV Assay had similar clinical sensitivity for disease detection but higher clinical specificity than the HC2 test, which may improve patient management and reduce the cost of care. (C) 2009 Elsevier B.V. All rights reserved.

Background: The APTIMA(R) HPV Assay (AHPV) is designed to detect HPV E6/E7 mRNA from 14 high-risk types in Cytyc PreservCyt liquid-based cytology specimens.Objectives: To compare AHPV analytical sensitivity for RNA and DNA; to compare the sensitivity of AHPV and Hybrid Capture(R) 2 (HC2) assays for HPV DNA detection; to compare assay performance with and without sample denaturation; to compare assay results with cytology.Study design: Analytical sensitivity of AHPV for detecting E6/E7 RNA was assessed by spiking samples with various quantities of HPV 16 E6/E7 in vitro RNA transcript or HPV 16-positive SiHa cells. AHPV and HC2 analytical sensitivity for HPV 16 DNA was evaluated by spiking samples with various quantities of a plasmid vector containing cloned HPV 16 DNA, or with purified SiHa cell genomic DNA containing integrated HPV 16 genome. Samples were tested using standard AHPV and HC2 protocols. Endocervical samples from 568 women were collected in Digene Specimen Transport Medium. Non-denatured and denatured samples were tested in AHPV and denatured samples with HC2. Assay results were compared to each other, and to cytology.Results: AHPV had substantially higher (2-4 log(10)) analytical sensitivity for HPV 16 RNA than for HPV 16 DNA. AHPV also had substantially lower (3 log(10)) analytical sensitivity for HPV 16 DNA compared to HC2. The overall agreement between assay results in clinical specimens was 94.2%, but AHPV had fewer positives than HC2 (48.4% positive agreement). In denatured samples, the number of samples testing positive in AHPV increased two-fold, yielding a positive agreement rate of 88.7%. When assay results were compared with cytology, AHPV had fewer positives in samples with normal or ASC-US diagnoses than did HC2.Conclusions: AHPV is much more efficient at detecting HPV 16 RNA than DNA. Selective capture, amplification, and detection of HPV RNA by AHPV may improve the specificity of molecular HPV testing. (C) 2009 Elsevier B.V. All rights reserved.

Background: Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA(R) HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.Objective: To determine the analytical performance characteristics of the APTIMA HPV Assay.Study Design: Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems.Results: The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or tow-risk HPV types. Spermicides, antifungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-tot, and inter-run signal variability were <10% for >99% of the data. Intra-run variability was <15%, except for those samples with concentrations at or below the 95% detection limit of the assay. Conclusions: Based upon the analytical sensitivity, analytical specificity, and low variability, the APTIMA HPV Assay showed excellent performance and robustness. (C) 2009 Elsevier B.V. All rights reserved.

Background: The clinical significance of occult hepatitis B virus (HBV) infection, defined as the presence of HBV DNA in individuals with HBV core antibodies (anti-HBc) in the absence of HBV surface antigen (HBsAg), is unclear in HIV-infected patients. This information is needed to determine the importance of detecting and treating occult HBV in this population.Objective: To determine if HIV-infected patients with occult HBV infection have an increased incidence of transaminitis.Study design: We performed a cohort study among randomly selected HBsAg -/anti-HBc+HIV-infected patients in the Penn CFAR Database and Specimen Repository. HBV DNA was qualitatively detected using a transcription-mediated amplification assay. Hepatic transaminase levels, the main study outcome, were collected at 6-month intervals from the time of occult HBV determination.Results: Among 97 randomly selected subjects without baseline transaminitis, 13 (13%) had occult HBV. These subjects more frequently had detectable HIV RNA. The 2-year incidence of transaminitis among HIV-infected subjects with occult HBV (50 events/100 person-years) was not significantly different from those without occult HBV (38 events/100 person-years; adjusted incidence rate ratio = 1.36 [95% CI, 0.72-2.59]).Conclusions: Occult HBV did not increase the incidence of hepatic transaminitis over 2 years. Future studies should determine whether occult HBV is associated with other clinically important outcomes, particularly hepatocellular carcinoma. (C) 2008 Elsevier B.V. All rights reserved.

Background: The APTIMA HPV Assay (AHPV) is a qualitative nucleic acid test designed to detect the E6/E7 mRNA of 14 high-risk HPV types in liquid based cytology (LBC) specimens. In this study the performance of the assay was evaluated in specimens collected in Digene Specimen Transport Media (DG-STM).Objectives: A method for processing DG-STM specimens compatible with the AHPV assay was developed. Its analytical sensitivity, as compared to LBC, was evaluated using HPV in vitro transcripts and HPV infected SiHa cells.Materials and Methods: To evaluate clinical performance, DGSTM specimens (n=483), collected primarily from patients undergoing routine screening, were tested in both the AHPV assay and the hc2 HPV DNA Test or hc2 HR HPV DNA Test (hc2). AHPV assay performance was compared to hc2 as well as cytology for a subset of the specimens (n=98).Results: DG-STM samples diluted into GP-STM at very low volumes or diluted in PBS and then GP-STM yield comparable analytical sensitivity to LBC samples diluted in GP-STM. Overall agreement of the AHPV assay with hc2 from the testing of the clinical specimens was 93%, with 99% negative agreement and 47% positive agreement. Cytology results were available for 28 of the 31 samples that were discordant between the AHPV and hc2 tests. Twenty-three were within normal limits, 3 were ASCUS, 1 was LSIL and 1 was HSIL.Conclusions: These results demonstrate that specimens collected in Digene STM may be tested in the APTIMA HPV Assay. Additional evaluations are required to fully assess clinical performance of the APTIMA HPV Assay in this sample type.

Background: The APTIMA HPV Assay (AHPV, Gen-Probe Incorporated) is a qualitative nucleic acid test designed to detect the E6/E7 mRNA of 14 high-risk HPV types in liquid based cytology (LBC) specimens. In this study, the performance of the assay was evaluated in a set of Cytyc LBC specimens stored for up to 3 years at room temperature and the results compared to histology.Materials and Methods: More than 400 clinical specimens were collected from patients with abnormal cytology over the course of more than 2 years (Universitats-Frauenklinik, Freiburg). Cytology, both conventional and liquid based, as well as histology results were evaluated. Subsequently, samples were stored at room temperature for up to 3 years and a subset (n=47) recently tested for HPV mRNA in the AHPV assay. AHPV results were compared to histology results.Results: The AHPV assay yielded a positive result in all 10 CIN 3 and cervical carcinoma specimens. All 6 CIN 2-3 specimens, 6 of 8 CIN 2 specimens, and 3 of 4 CIN 1-2 specimens were positive in the AHPV assay. Of the 19 CIN1 and normal specimens, 15 were negative in the AHPV assay.Conclusions: The results from this subset of specimens tested to date indicates that the APTIMA HPV assay is able to detect HPV high-risk mRNA in retrospective LBC specimens stored at room temperature for up to three years with strong correlation to disease. Testing of additional specimens from this sample set will be completed as well as HPV DNA testing.