NILM Pap Slides From Women 30 Years of Age and Older With Positive High-Risk HPV DNA

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1 NILM Pap Slides From Women 30 Years of Age and Older With Positive High-Risk HPV DNA Focused Rescreening Prior to Report Issuance, An Enhanced Quality Control Measure Karen Cormier, CT(ASCP), 1 Michael Schaaf, CT(ASCP), 1 Stephanie Hamilton, EdD, SCT(ASCP)MB, 1 Ronald J. Tickman, MD, 1 Nuria Perez-Reyes, MD, 1 and Charles D. Sturgis, MD 1,2 From 1 CellNetix Pathology and Laboratories, Seattle, WA, and 2 Department of Pathology, Providence Regional Medical Center, Everett, WA. Key Words: CLIA 88; Negative for intraepithelial lesion (NILM); Pap smears; High-risk HPV DNA; Quality control ABSTRACT Objectives: Clinical Laboratory Improvement Amendments of 1988 (CLIA 88) regulations specify that at least 10% of negative Papanicolaou (Pap) slides be rescreened as a quality control (QC) measure. With incorporation of human papillomavirus (HPV) DNA testing into screening guidelines for women aged 30 years or older, a population of patients exists who are HPV positive as well as negative for intraepithelial lesion or malignancy (NILM). Methods: In this 9-month retrospective review with follow-up, 26,501 women 30 years of age and older underwent liquid-based Pap screening with concomitant high-risk HPV DNA testing at CellNetix Pathology and Laboratories, Seattle, WA. Of these women, 1,096 (4.1%) were originally interpreted by cytotechnologists as NILM with HPV DNA positivity. Results: On rescreening, 13.9% (152/1,096) of patients were upgraded to atypical squamous cells and higher, with 2.8% being upgraded to low-grade squamous intraepithelial lesion (LSIL) and higher. Historical routine QC measures from the same period showed that 0.3% of cases were upgraded to LSIL and higher, representing a statistically significant increase in the detection of cases with LSIL and higher (c 2 two-tailed P <.0001). Conclusions: Focused rescreening of this enriched subpopulation of patients who are NILM and high-risk HPV DNA positive enhances QC. An inherent potential bias in study design is recognized because results of DNA testing were, by definition, known at the time of rescreening result interpretations. In the mid-1980s, multiple media reports focused public and US legislative attention on deficiencies in the quality of services provided by some clinical laboratories. The Clinical Laboratory Improvement Amendments of 1988, or CLIA 88, resulted from congressional examination. The CLIA 88 standards were designed to improve quality in all laboratory testing and included specifications for quality control (QC)/ quality assurance, patient test management, and proficiency testing. The CLIA regulations were implemented to ensure accuracy, reliability, and timeliness of patient test results regardless of where the test was performed. 1 One of the mandates set for Papanicolaou (Pap) smear testing by CLIA 88 was that a minimum of 10% of negative cervicovaginal cytology screening samples be rescreened by a pathologist or qualified senior cytotechnologist, with a portion of these cases being randomly selected. This mandate was made with the hope that if a primary Pap test screener was identified as making repeated screening or interpretive errors, then the individual could be counseled and re-educated to improve performance and prevent false-negative results. Since the implementation of the CLIA 88 guidelines, many additional advances have been made in Pap test quality assurance, and many peer-reviewed articles have been published on the topic. Various authors and investigators have probed the number of slides that must be re-evaluated to make statistically valid assessments of screening performance, and others have noted the practical problem with calculating the false-negative rate of Pap smears by rescreening negative cases alone. 2,3 In recent years, several articles have focused on measures other than traditional 10% rescreening of negative slides to increase quality. Some of these have included 100% rapid rescreening of negative slides and rapid prescreening of slides. 4-6 Many 494 Am J Clin Pathol 2014;141: Downloaded 494 from

2 laboratories now also rely on automation to help with increasing Pap test accuracy, timeliness, and efficiency. 7 Recently, 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors were issued. 8 These guidelines were published in follow-up to the American Cancer Society, American Society for Colposcopy and Cervical Pathology (ASCCP), and American Society for Clinical Pathology (ASCP) screening guidelines for the prevention and early detection of cervical cancer. 9 These consensus publications recommend cytology combined with high-risk human papillomavirus (HPV) DNA cotesting for women of 30 to 65 years of age. Based on the guidelines, women aged 30 to 65 years who undergo cervicovaginal screening and receive dual-negative cytomorphology and high-risk HPV DNA test results may not need to have their Pap test repeated for an interval of up to 5 years. This extended collection interval has the potential to result in financial savings for the individual and the health care system without compromising care. It is imperative that interpretive accuracy and testing sensitivity be optimized if screening intervals are to be increased. One outcome of the broad-scale application of these guidelines to clinical practice has been the creation of a relatively new (albeit small) interpretive subset of women who are 30 years of age or older and are morphologically negative for intraepithelial lesion or malignancy (NILM) but are simultaneously positive for high-risk HPV by DNA testing. Immunocompetent women who are NILM and HPV DNA test negative may not need to return for another Pap test for 5 years, whereas those who are NILM and HPV DNA test positive are recommended by the guidelines to either undergo repeat cotesting in 1 year or to have HPV DNA genotyping to ascertain whether their high-risk HPV infection is positive for viral types 16/18. If the patient is HPV16/18 positive, she should be referred to colposcopy. If HPV16/18 negative, 12-month follow-up with cotesting is recommended. 8 In the past, we have studied a small cohort of patients in the morphologically negative category who were found to be HPV DNA positive on molecular testing. We discovered that rescreening this targeted population may be an effective QC measure. 10 In our previous investigation, we found that in some instances rare lesional cells can be missed in screening and that focusing rescreening efforts on HPV DNA positive, NILM-cotested patients can improve screening accuracy. 10 Women who are older than 30 years and have cytologic interpretations of atypical squamous cells (ASC) and above with positive HPV DNA testing results should be referred to colposcopy. Even minor one-step upgrades (such as from NILM to ASC) in this population will trigger different management paradigms. Herein, we undertake a similar investigation on a larger number of patient samples. Our data and conclusions further support the findings of our original pilot study. Materials and Methods This retrospective review of the 9-month period from July 2011 through March 2012 revealed a total of 26,501 women who were 30 years of age or older and who underwent dual liquid-based morphologic Pap screening (mixture of SurePath [Becton Dickinson, Franklin Lakes, NJ] and ThinPrep [Hologic, Marlborough, MA] cytology samples) and simultaneous high-risk HPV DNA screening by Hybrid Capture II (HCII; Qiagen, Valencia, CA) at CellNetix Pathology and Laboratories, Seattle, WA. HCII tests for 13 HPV types including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. This assay is based on a sandwich capture method followed by molecular hybridization and subsequent nonradioactive chemiluminescence measurements from microplates. HCII testing was performed following commercially available manufacturer s guidelines using Digene standard transport medium as approved by the Food and Drug Administration for the ThinPrep samples. Triage of SurePath samples to HCII was performed relying on internally validated procedures and using SurePath proprietary fluid. (Internal validation of DNA testing of SurePath vials was conducted to meet criteria/ recommendations from the College of American Pathologists and guidelines as per the Clinical and Laboratory Standards Institute MM17-A for the verification and validation of nucleic acid assays.) For the SurePath samples, the cellular pellets were obtained when the SurePath slides were made, and then 3.0 ml of SurePath fluid was added to the remaining 1.0 ml. This specimen was then used for HCII HPV DNA testing. Of these 26,501 women 30 years of age or older who were dual morphology and DNA screened, 1,096 (4.1%) were found to have NILM liquid-based Pap interpretations coupled with positive high-risk HPV HCII results. These 1,096 slides were then rescreened by laboratory cytotechnologists with more than 3 years of experience. All rescreened slides judged as ASC or higher were then reviewed by three additional senior cytotechnologists and by one or more cytopathologists (C.D.S./R.J.T.). One cytopathologist (C.D.S.) assigned a final rescreening interpretation. CellNetix Pathology and Laboratories is a pathologistowned and -operated company based in the Puget Sound region of Washington state. The annual Pap test volume at CellNetix during calendar year 2011 was 109,162, with 23.8% of tests being ThinPrep samples, 74.7% being BD SurePath samples, and 1.5% being conventional non liquid-based smears. During the study period, the laboratory employed 14 ASCP-certified cytotechnologists (11.6 fulltime equivalent positions), and the overall ASC/squamous intraepithelial lesion (SIL) ratio was 2.0:1 for the laboratory as a whole. During the study period, 40% of atypical squamous cells of undetermined significance (ASC-US) cases reflexed to high-risk HPV DNA testing within the laboratory were found to be positive. Downloaded from Am J Clin Pathol 2014;141:

3 Cormier et al / NILM Pap Slides With Positive High-Risk HPV DNA The average number of whole slide equivalents (with liquid-based Pap test slides read with automated assistance counting as half of a slide) per cytotechnologist per 8-hour screening interval was 64. The overall QC rate during the study period was 19.1%, with 20.6% of QC slides being SurePath samples and 15.4% of slides being ThinPrep samples. The overall narrow definition of false-negative fraction (FNF) before implementing focused rescreening of NILM HPV-positive Pap smears was 6.5%. On implementing the focused rescreening in our laboratory, the overall FNF of the laboratory has increased. In July 2012, at the end of the study period, the FNF was 9.4%. After study closure, the FNF has trended downward and is now approaching previous rates. All ThinPrep slides were originally screened using the Hologic ThinPrep Imaging System. All BD SurePath slides were originally screened using the BD FocalPoint Slide Profiler. CellNetix policy indicates that all Pap smears (even those in the FocalPoint no further review needed group) are fully screened by a cytotechnologist before being classified into a definitive interpretive category. Results The results of rescreening are presented in Table 1. Of the 1,096 women who were 30 years of age or older and were originally interpreted as morphologically negative with positive high-risk HPV DNA testing with HCII, 152 (13.9%) were judged as ASC or higher on rescreening. Of these, 113 (10.3% of total) were upgraded to ASC-US and 39 (3.6% of total) were upgraded to atypical squamous cells, cannot rule out high-grade SIL (ASC-H) and above. ASC-H represented six upgraded cases (0.5% of total), low-grade SIL (LSIL) represented 23 upgraded cases (2.1% of total), high-grade SIL (HSIL) represented eight cases (0.7% of total), and atypical glandular cells (AGC) represented two cases (0.2% of total). The 39 cases that were upgraded to ASC-H and above had a mean follow-up period of 17 months, with the shortest followup period for an individual case being 11 months. All patients except one had either histologic study on biopsy or additional Pap testing during the follow-up interval. Follow-up data for patients judged to have ASC-H and above is presented in Table 2. Of the six ASC-H cases, four had biopsy on follow-up with three confirming dysplasia. Neither of the AGC cases showed lesional biopsy findings on follow-up. Of the 23 cases upgraded to LSIL, 22 had follow-up biopsies with six showing dysplasia, and five had follow up cytology with three showing either SIL or a positive high-risk HPV DNA result. Of the eight HSIL cases, seven showed dysplasia on followup biopsy with six showing high grade. Fifteen (33.3%) of the liquid-based samples in this set were ThinPrep, while the other 26 (66.7%) were SurePath. Of the 13 ThinPrep cases upgraded to ASC-H or higher, three (23.1%) were upgraded to ASC-H, one (7.7%) was upgraded to AGC, six (46.2%) were upgraded to LSIL, and three (23.1%) were upgraded to HSIL. Of the 26 SurePath cases upgraded to ASC-H or higher, three (11.5%) were upgraded to ASC-H, one (3.8%) was upgraded to AGC, 17 (65.4%) were upgraded to LSIL, and five (19.2%) were upgraded to HSIL. As stated before, ThinPrep samples accounted for 23.8% of the Pap volume and SurePath samples accounted for 74.7% of the Pap volume at the laboratory at the time of the study. Table 3 shows the relative frequencies of upgraded categories for the two sample preparation types. Discussion This study focuses on the rescreening of Pap test cervicovaginal slides from a cohort of women who were 30 years of age or older at the time of sample collection and who were found to be morphologically NILM while being simultaneously positive for high-risk HPV DNA with the HCII method. This unique cohort of patients was created by application of ASCCP/American College of Gynecologists (ACOG) guidelines to a screening population of patients who obtained care in Washington state s Puget Sound region in a consecutive 9-month period that overlapped portions of calendar years 2011 and Of the 1,096 women (Table 1) who were 30 years of age or older and were originally interpreted as morphologically negative with a positive high-risk HPV DNA test by HCII, 152 (13.9%) were judged as ASC or higher on rescreening. Of these, 113 (10.3% of total) were upgraded to ASC-US and 39 (3.6% of total) were upgraded to ASC-H and Table 1 Rescreening Results (All Women Initially NILM and HCII Positive) Originally Upgraded to Upgraded Upgraded Upgraded Upgraded Upgraded NILM ASC and Higher to ASC to ASC-H to LSIL to HSIL to AGC No. 1, % AGC, atypical glandular cells; ASC, atypical squamous cells; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; HCII, Hybrid Capture II; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy. 496 Am J Clin Pathol 2014;141: Downloaded 496 from

4 above. ASC-H represented six upgraded cases (0.5% of total), LSIL represented 23 upgraded cases (2.1% of total), HSIL represented eight cases (0.7% of total), and AGC represented two cases (0.2% of total). As a means of comparison, routine combined QC (encompassing random, FocalPoint enriched, and historically high-risk cases) was performed on 19.1% of Pap tests during the sample period, with 0.3% of cases being upgraded to LSIL or higher. Focused rescreening of the NILM patients with positive HCII high-risk HPV DNA test results compared with routine combined QC revealed a statistically significant increase in the detection of cases that were LSIL and higher (c 2 two-tailed P <.0001). The current study was pursued in follow-up to a previous smaller pilot investigation of a similar population of women from a shorter 4-month period at the end of When the data from the pilot study are combined with the data from the current investigation, a total of 1,482 women in the category of 30 years of age or older with original NILM cytology and positive HPV DNA testing exists in the aggregated cohort (combined data shown in Table 4 ). The combined numbers from CellNetix during the aggregated periods show a 13.6% upgrade rate to ASC and higher, with a 2.2% increase in LSIL interpretations and a 0.6% increase in HSIL interpretations. Focused rescreening of the patients who were originally Table 2 Patients Upgraded to ASC-H and Higher Senior Cytotechnologist Pap Follow-Up Retrospective Cells No. of Case Age, Missed by Final Lesional Follow-Up HPV Cytopathologist No. y a Imager? b Interpretation Cells Present Method Biopsy Interpretation DNA No. 1 No. 2 No. 3 Retrospective 1 59 No ASC-H 4 TP None NILM Pos ASC-H ASC-H ASC-H ASC-H 2 35 No ASC-H >10 TP NILM NILM Pos AGC HSIL ASC-H HSIL 3 42 No ASC-H 6 TP None None None ASC-US HSIL ASC-US ASC-H 4 53 No AGC >10 TP NILM None None AGC AIS AGC AGC 5 39 No LSIL >10 TP NILM NILM Neg LSIL LSIL LSIL LSIL 6 49 Yes LSIL 5 TP NILM LSIL Pos LSIL LSIL LSIL LSIL 7 38 No LSIL >10 TP NILM ASC-US Pos ASC-H HSIL LSIL ASC-H 8 30 No LSIL >10 TP LSIL NILM Neg LSIL LSIL LSIL LSIL 9 37 No LSIL >10 TP NILM None None LSIL HSIL LSIL LSIL No LSIL 5 TP HSIL ASC-US Neg LSIL LSIL LSIL LSIL No HSIL >10 TP HSIL ASC-US Neg HSIL HSIL HSIL HSIL No HSIL >10 TP HSIL None None HSIL HSIL HSIL HSIL No HSIL >10 TP LSIL None None HSIL HSIL HSIL HSIL ASC-H >10 SP HSIL None None HSIL HSIL ASC-H HSIL ASC-H 7 SP LSIL None None ASC-H ASC-H ASC-US ASC-H ASC-H >10 SP HSIL None None HSIL HSIL ASC-H HSIL AGC 6 SP Unsat None None NILM HSIL ASC-US AGC LSIL >10 SP NILM None None LSIL LSIL LSIL LSIL LSIL >10 SP NILM NILM Neg LSIL LSIL LSIL LSIL LSIL 6 SP NILM None None LSIL LSIL LSIL LSIL LSIL >10 SP NILM NILM Pos ASC-H HSIL LSIL LSIL LSIL 5 SP LSIL NILM None LSIL LSIL LSIL LSIL LSIL 8 SP NILM NILM Neg LSIL LSIL LSIL LSIL LSIL >10 SP None ASC-H None LSIL LSIL LSIL LSIL LSIL 1 SP NILM None None LSIL ASC-US LSIL LSIL LSIL 5 SP NILM NILM Neg LSIL HSIL LSIL LSIL LSIL >10 SP LSIL None None LSIL LSIL LSIL LSIL LSIL 2 SP Unsat None None LSIL LSIL LSIL LSIL LSIL >10 SP LSIL None None LSIL HSIL LSIL LSIL LSIL >10 SP None ASC-US Neg LSIL LSIL LSIL LSIL LSIL 6 SP NILM None None LSIL LSIL LSIL LSIL LSIL 2 SP NILM None None LSIL LSIL LSIL LSIL LSIL 5 SP NILM NILM Neg LSIL LSIL LSIL LSIL LSIL >10 SP LSIL None None LSIL LSIL LSIL LSIL HSIL >10 SP HSIL NILM Neg HSIL HSIL HSIL HSIL HSIL >10 SP HSIL None None HSIL HSIL HSIL HSIL HSIL >10 SP None ASC-H Pos HSIL HSIL ASC-H HSIL HSIL >10 SP HSIL None None HSIL HSIL HSIL HSIL HSIL >10 SP HSIL NILM Pos HSIL HSIL HSIL HSIL AGC, atypical glandular cells; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; Neg, negative; NILM, negative for intraepithelial lesion or malignancy; Pap, Papanicoloau; Pos, positive; SP, SurePath; TP, ThinPrep; Unsat, unsatisfactory. a Mean age, 40 years. b Candida species were found in cases 24 and 37 and bacterial vaginosis in case 29. Downloaded from Am J Clin Pathol 2014;141:

5 Cormier et al / NILM Pap Slides With Positive High-Risk HPV DNA NILM with positive HCII high-risk HPV DNA test results showed a statistically significant increase in the detection of cases that were LSIL and higher compared with routine combined QC from the aggregate period (c 2 two-tailed P <.0001). Table 3 Comparative Upgrades Between ThinPrep and SurePath Sampling No. (%) No. (%) No. (%) No. (%) Upgraded Upgraded Upgraded Upgraded to ASC-H to LSIL to HSIL to AGC ThinPrep 3 (23.1) 6 (46.2) 3 (23.1) 1 (7.7) SurePath 3 (11.5) 17 (65.4) 5 (19.2) 1 (3.8) AGC, atypical glandular cells; ASC-H, atypical squamous cells, cannot exclude highgrade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion. To the best of our knowledge, this submission is the second report in the literature specifically detailing previously overlooked abnormalities that are detectable by focused QC rescreening of NILM Pap test slides in women aged 30 years or older with positive high-risk HPV DNA test results, the first report being the prior CellNetix pilot investigation. Other investigators who have focused attention on women aged 30 years or older (but not from a slide-rescreening QC perspective) have reported that the percentage of women in their data sets who are NILM but positive for high-risk HPV DNA ranges from 3.99% to 5.4%. 11,12 The percentage of patients in this group at CellNetix falls within this range. Additional authors have reported on this population of patients from the perspective of routine laboratory QC but have not described specific abnormalities detected with this measure of QC Table 4 Rescreening Results (Current Data Combined With Previously Published Data a ) Originally Upgraded to Upgraded Upgraded Upgraded Upgraded Upgraded NILM ASC and Above to ASC to ASC-H to LSIL to HSIL to AGC No. 1, % AGC, atypical glandular cells; ASC, atypical squamous cells; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy. a Data from the current study period are combined with similar data from our previously published results on a 2009 cohort. 10 A B Image 1 A, Thick low-grade squamous intraepithelial lesion (LSIL) group. This cervicovaginal cytology ThinPrep sample was from a 49-year-old woman whose slide originally screened as negative for intraepithelial lesion or malignancy (NILM) by a cytotechnologist. This microscopic field was not selected by the ThinPrep imager. When a Hybrid Capture (HCII) DNA test result was positive, the case was rescreened before final reporting and upgraded by the second cytotechnologist and cytopathologist to LSIL. The slide showed a rare group of thick LSIL cells without singly dispersed koilocytes in the background or singly dispersed cells of mild squamous dysplasia. B, Thick LSIL group. This cervicovaginal cytology ThinPrep sample was from a 57-year-old woman whose SurePath slide originally screened as NILM. When an HCII DNA test result was positive, the case was rescreened before final reporting and upgraded by the second cytotechnologist and cytopathologist to LSIL. The slide showed a rare group of thick LSIL cells without singly dispersed koilocytes in the background or singly dispersed cells of mild squamous dysplasia. (A and B, Papanicolaou stain, 630.) 498 Am J Clin Pathol 2014;141: Downloaded 498 from

6 rescreening. 13,14 Our experience and that of others support the conclusions of a prior publication from the Netherlands (more than a decade earlier) which showed that high-risk HPV DNA types could be detected in archival materials classified as false-negative on cytology and that cytologic screening errors might be reduced if combined with molecular testing. 15 On review of the 39 cases upgraded to ASC-H and higher, we found that lesional cells were not identified by the automated screening instrument in one of 13 ThinPrep samples. Five (22%) of the total 23 cases (ThinPrep and SurePath combined) that were originally interpreted as NILM but were later upgraded to LSIL were discovered on review to show rare thick clusters of LSIL; this included the one ThinPrep sample in which the automated instrument did not detect the lesional cells. These thick groups were characterized by three-dimensional aggregates of overlapping cells with convincing LSIL cytomorphology, including perinuclear clearings, irregular nuclear membranes, nucleomegaly, and altered chromatin. These thick LSIL groups were originally overlooked by both experienced cytotechnologists and the ThinPrep imager in rare cases Image 1. Four of the 13 Thin- Prep samples had rare lesional cells with less than 10 lesional cells present, and 11 of the 26 SurePath samples had less than 10 lesional cells present. Excluding the one case not identified by the ThinPrep imager, all cases were felt to be screening errors. Although the numbers of upgraded cases in the current study set that were reclassified as ASC-H and higher were relatively small (Table 3), approximately twice as many ASC-H upgrades were noted for ThinPrep samples compared with SurePath samples (23.1% vs 11.5%). Alternatively, more LSIL upgrades were noted for SurePath samples than for ThinPrep samples (65.4% vs 46.2%). Monitoring interpretive rate categories for Pap tests, concordance of cytotechnologist and pathologist interpretations before sign out, and turnaround time are three categories of monitors that have been reported to be useful for quality assurance in gynecologic cytology. 16 Recent conclusions from the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference Working Group 2 suggest that the number of Pap tests from high-risk patients should be maximized in prospective and retrospective rescreening; a document from Working Group 3 points out that many quality monitors are based on tradition and empirical good intentions with no established standards. 17,18 Focused rescreening of negative slides from patients 30 years of age and older who are initially NILM and are simultaneously proven to be HPV positive on molecular testing seems intuitively commonsensical, and patients shown to be carriers of high-risk HPV DNA could certainly be considered a high-risk group. Recent follow-up and outcomes studies of women who are cytology negative and HPV DNA positive appear to support this conclusion. 19,20 Findings from the ATHENA HPV study confirm a greater absolute risk of moderate squamous dysplasia and higher in women with negative cytology and positive HPV genotyping for HPV types 16 and Although the ASC category is poorly reproducible, the ASCCP/ACOG management guidelines for NILM with positive high-risk HPV DNA test results differ from those of ASC with positive high-risk HPV DNA testing. As such, the increase in QC upgrading to ASC and higher shown in this investigation has the potential to be of clinical relevance for the involved patients. Certainly, those patients upgraded to SIL would experience different management. Focused rescreening of this patient population may enhance QC in laboratories using liquid-based cytology preparations. An inherent potential bias in study design is recognized because results of DNA testing were by definition known at the time of rescreening. (Turnaround times for HPV DNA testing at CellNetix are in general equivalent to or shorter than the lengths of time needed for cytopathology processing and interpretation. Hence, there is no delay in finalizing cases). All NILM slides from liquid-based Pap test samples obtained from women aged 30 years and older who are simultaneously found to be high-risk HPV positive by HCII are now QC rescreened at CellNetix before they are assigned to a final morphologic interpretive category. Address reprint requests to Dr Sturgis: CellNetix Pathology and Laboratories, 1321 Colby Ave, Ste A2-300, Everett, WA References 1. Clinical Laboratory Improvement Amendments of 1988: final rule. Federal Register. 1992;57: Krieger PA, Cohen T, Naryshkin S. 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Image-guided ThinPrep Papanicolaou tests and cotesting with high-risk human papillomavirus in women aged 30 years and older in a low-risk private practice population. Cancer. 2009;117: Massad LS, Einstine MH, Huh WK, et al updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. Obstet Gynecol. 2013;121: Downloaded from Am J Clin Pathol 2014;141:

7 Cormier et al / NILM Pap Slides With Positive High-Risk HPV DNA 9. Saslow D, Solomon D, Lawson HW, et al. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. Am J Clin Pathol. 2012;137: Sturgis CD, Schaaf MR, Tickman RJ. Focused rescreening of NILM Pap slides from women 30 years of age with positive high risk HPV DNA: an enhanced quality control measure. Diagn Cytopathol. 2013;41: Castle PE, Fetterman B, Poitras N, et al. Five-year experience of human papillomavirus DNA and Papanicolaou test cotesting. Obstet Gynecol. 2009;113: Thrall MJ, Russell DK, Facik MS, et al. High-risk HPV testing in women 30 years or older with negative Papanicolaou tests: initial clinical experience with 18-month follow-up. Am J Clin Pathol. 2010;133: Zhao C, Elishaev E, Yuan K, et al. Very low human papillomavirus DNA prevalence in mature women with negative computer-imaged liquid-based Pap tests. Cancer. 2007;111: Bansal M, Austin RM, Zhao C. High-risk HPV DNA detected in less than 2% of over 25,000 cytology negative imaged liquid-based Pap test sampled from women 30 and older. Gynecol Oncol. 2009;115: Walboomers JM, De Roda Husman AM, Snijders PJ, et al. Human papillomavirus in false negative archival cervical smears: implications for screening for cervical cancer. J Clin Pathol. 1995;48: Clary KM, Davey DD, Naryshkin S, et al. The role of monitoring interpretive rates, concordance between cytotechnologist and pathologist interpretations before sign-out, and turnaround time in gynecologic cytology quality assurance: findings from the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference Working Group 1. Arch Pathol Lab Med. 2013;137: Brainard JA, Birdsong GG, Elsheikh TM, et al. Prospective and retrospective review of gynecologic cytopathology: findings from the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference Working Group 2. Arch Pathol Lab Med. 2013;137: Tworek J, Nayar R, Savaloja L, et al. General quality practices in gynecologic cytopathology: findings from the College of American Pathologists Gynecologic Cytopathology Quality Consensus Conference Working Group 3. Arch Pathol Lab Med. 2013;137: Zhao C, Chen X, Onisko A, et al. Follow-up outcomes for a large cohort of US women with negative imaged liquidbased cytology findings and positive high risk human papillomavirus test results. Gynecol Oncol. 2011;122: Katki HA, Schiffman M, Castle PE, et al. Five-year risks of CIN3+ and cervical cancer among women who test Pap-negative but are HPV-positive. J Low Genit Tract Dis. 2013;17:S56-S Wright TC, Stoler MH, Sharma A, et al. Evaluation of HPV16 and HPV18 genotyping for triage of women with high-risk HPV+ cytology-negative results. Am J Clin Pathol. 2011;136: Am J Clin Pathol 2014;141: Downloaded 500 from