ACY446271.indd E-Mail karger@karger.com Review Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 Review of Cytology Practice at Thomas Jefferson University Hospital before and after High-Risk Human Papillomavirus Testing Marluce Bibbo a Zi-Xuan Wang b Krister Jones a Charalambos Solomides a Rossitza Draganova-Tacheva a Robert Stapp c a Cytopathology Laboratory, b Molecular Biology Laboratory and c Pathology Informatics, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pa. , USA testing was due to the lengthening of the test interval when cotesting results were negative. Practitioners adhering to guidelines accounts for increased molecular testing volume. A trend towards higher-grade cervical intraepithelial neo- plasia in the follow-up of detected HPV 16/18 was noted. So far there has been no demand for HPV as a stand-alone test. © 2016 S. Karger AG, Basel Introduction Over time, cervical cancer screening has evolved from a single glass slide smear to a test involving liquid-based processing and molecular HPV testing in the residual ma- terial. Since 1988 the Bethesda System for reporting cervical/ vaginal cytology introduced the atypical squamous cells of undetermined significance (ASCUS) category under epithelial squamous cell abnormality [1] . It was noted, however, by the editors of the 1994 monograph [2] that cervical/vaginal cytopathology includes an element of subjective interpretation and consequently the applica- tion of criteria should be viewed within this context. Nu- Key Words ThinPrep test · Papanicolaou test · Human papillomavirus · Reflex test · Cotesting · Cervical intraepithelial neoplasia Abstract Objective: We performed a retrospective review of Papani- colaou (Pap) testing to assess whether the cytology practice in our institution was affected by the introduction of high- risk (HR) human papillomavirus (HPV) assays over time. Study Design: Cytology, HPV and histopathology records were retrieved from our laboratory information system from 2003 to 2015. Records for Digene Hybrid Capture 2 ® , Hologic Cervista ® and Roche Cobas ® HPV assays were ob- tained. A 3-month follow-up for HPV detected cases was performed, and results were correlated with cytology and biopsies. A 1-year follow-up of HPV 16/18 and other HR HPV detected cases was also performed. Results: From 2008 to 2015, a noticeable decrease in Pap testing volume occurred, from 11,792 to 4,664, while the percentage of HPV testing increased from 19 to 59%. Similar HPV detection rates and follow-up results for both reflex and cotesting were ob- served in the 3 HPV assays. Conclusions: The decrease in Pap Received: April 13, 2016 Accepted after revision: April 19, 2016 Published online: June 2, 2016 Correspondence to: Dr. Marluce Bibbo Department of Pathology, Anatomy and Cell Biology Thomas Jefferson University Hospital , Main Building, Suite 260, 132 South 10th Street Philadelphia, PA 19107 (USA) E-Mail Marluce.Bibbo   @   Jefferson.edu © 2016 S. Karger AG, Basel 0001–5547/16/0606–0534$39.50/0 www.karger.com/acy http://dx.doi.org/10.1159%2F000446271 Cytology Practice before and after HR HPV Testing Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 535 merous clinicians were unhappy with the growing num- ber of ASCUS interpretations issued by cytology labora- tories. In 2001 the America Society for Colposcopy and Cervical Pathology (ASCCP) issued guidelines for human papillomavirus (HPV) testing [3] , and reflex HPV testing was recommended for cases interpreted as ASCUS in pa- tients >20 years of age. When high-risk (HR) HPV was positive, the patient was referred to colposcopy and, when negative, returned to routine screening. The use of HPV cotesting was approved as a cervical cancer screening test in the USA in 2003 and endorsed by the American Cancer Society (ACS) [4] . It was approved in combination with Papanicolaou (Pap) testing, for women 30 years or older every 3–5 years, but it was not approved as a stand-alone test. Updated consensus guidelines from the ACS, ASCCP and American Society for Clinical Pathology were issued in 2012 [5] . In 2014 Roche Cobas ® became the first HPV test approved by the FDA to replace the Pap test for pri- mary screening of cervical cancer [6] . Assays for HPV DNA testing in our practice have in- cluded Digene Hybrid Capture 2 (HC 2) ® approved by the FDA in 1999, Hologic Cervista ® approved in 2009 for detection of HR HPV, and Roche Cobas ® approved in 2011 for detection of HR HPV and genotyping HPV types 16 and 18. The purpose of this retrospective review on Pap testing was to assess whether the cytology practice in our institu- tion was affected by the introduction of HR HPV assays over time. Materials and Methods With institutional review board approval, we retrospectively reviewed cytology, HPV and histopathology records from our lab- oratory information system from 2003 to 2015. These records were obtained in collaboration with the pathology informatics director (R.S.). No records for 2003, 2004, and 2005 HR HPV assays were found in our practice. Records for HC 2 ® HPV testing were ob- tained from 2006 to 2008, for Cervista ® from 2009 to 2011 and for the Cobas ® from 2012 to 2015. The HPV assays were performed in the residual material of ThinPrep specimens by the molecular pathology laboratory. A 3-month (January to March) follow-up of cases with detect- ed HR HPV was performed for 2008 HC 2 ® , for 2011 Cervista ® , and for 2013 Cobas ® . The results of the 3 HR HPV assays were correlated with Pap cytology results. In addition, HR HPV positiv- ity of various cytologic interpretations in Pap specimens and fol- low-up biopsies were obtained. A 1-year follow-up of ASCUS cases with HPV 16/18 and other HR HPV detected by Roche Cobas ® was also obtained for 2014. Results Table 1 displays the volume of Pap tests (conventional and ThinPrep) and HR HPV assays performed from 2003 to 2015. No HPV tests are listed for 2003, 2004, and 2005, and a small number of tests are listed for 2006 and 2007. This table also shows the number and percent of reflex HPV and cotesting from 2006 to 2015. From 2008 to 2015, a noticeable decrease in Pap testing occurred from 11,792 to 4,664, while the percentage of HPV testing in- creased from 19 to 59%. The percentage of reflex HPV tests remained stable from 2008 to 2011 and showed a decrease from 2012 to 2015. The percentage of cotesting during the same time period remained relatively stable. Testing volumes of all Pap tests, ThinPrep, and HR HPV from 2003 to 2015 appear in figure 1 a. A progressive decrease in Pap test volume and an increase in HPV test- ing are seen over time. Figure 1 b shows the volume of HPV reflex and cotesting for the years 2006–2015. A higher volume of cotesting compared to reflex testing is evident. Table 2 shows a 3-month window of HR HPV testing at Thomas Jefferson University Hospital by 3 different as- says: HC 2 ® in 2008, Cervista ® in 2011 and Cobas ® in 2013. The percentage of molecular tests increased from 18% in 2008 to 33% in 2011 and to 47% in 2013. Similar HPV detection rates in both reflex (32, 34, 27%) and co- testing (12, 10, 10%) in the 3 HR HPV assays were ob- served. Table 1. Testing volume and percentage of HR HPV tests Year Pap, n Thin- Prep, n HPV Reflex Cotest n % n % n % 2003 18,271 17,180 0 0 – – – – 2004 14,858 14,389 0 0 – – – – 2005 13,893 13,667 0 0 – – – – 2006 13,518 13,294 12 0 12 100 0 0 2007 11,687 11,388 218 0 123 56 95 44 2008 12,158 11,792 2,239 19 441 20 1,798 80 2009 10,557 10,166 2,534 25 425 17 2,109 83 2010 9,583 9,235 2,920 31 650 22 2,270 78 2011 8,611 8,352 2,676 32 607 22 2,069 77 2012 6,870 6,708 2,841 42 448 15 2,393 84 2013 5,991 5,814 2,788 48 334 12 2,454 88 2014 5,234 4,999 2,862 57 344 12 2,518 88 2015 4,923 4,664 2,746 59 267 10 2,479 90 Total Pap tests, ThinPreps and HR HPV testing including reflex and co- testing for the years 2003 – 2015. http://dx.doi.org/10.1159%2F000446271 Bibbo/Wang/Jones/Solomides/ Draganova-Tacheva/Stapp Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 536 0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 PAP totals ThinPrep totals HPV totals 20,000 Te st in g v o lu m e (n ) 2003 2004 2005 2006 2007 2008 2009 Year 2010 2011 2012 2013 2014 2015 a 0 Te st in g v o lu m e (n ) 2003 2004 2005 2006 2007 2008 2009 Yearb 2010 2011 2012 2013 2014 2015 500 1,000 1,500 2,000 2,500 Cotest HPV Reflex HPV 3,000 3,500 Fig. 1. a Testing volume of all Pap tests, from ThinPrep and HR HPV testing for the years 2003–2015; see table  1 for details. b HPV reflex and cotesting for the years 2003–2015. http://dx.doi.org/10.1159%2F000446271 Cytology Practice before and after HR HPV Testing Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 537 Table 3 shows the follow-up of HR HPV detected cas- es in both reflex and cotesting categories for the same 3-month window of HR HPV testing using HC 2 ® , Cer- vista ® and Cobas ® . For ASCUS cases where biopsy re- sults were available, cervical intraepithelial neoplasia (CIN) 1 was frequently diagnosed, followed by CIN 2 and negative for CIN. Forty-three percent of ASCUS HPV cases detected by HC 2 ® (13/30 = 43%) and Cobas ® (13/30 = 43%) showed CIN 1 or 2 on a follow-up biopsy Table 2. Three-month analysis of HPV testing from 3 assays used at Thomas Jefferson University Hospital Digene HC 2® Hologic Cervista® Roche Cobas® Date range January to March 2008 2,757 January to March 2011 2,236 January to March 2013 1,547ThinPrep tests, n Total molecular tests 514/2,757 (18%) 735/2,236 (33%) 732/1,547 (47%) Reflex HPV 94/514 (18%) 207/735 (28%) 110/732 (15%) HPV detected 30/94 (32%) 71/207 (34%) 30/110 (27%) Cotesting 420/514 (82%) 528/735 (72%) 622/732 (85%) HPV detected 50/420 (12%) 51/528 (9.6%) 64/622 (10%) Table 3. Follow-up of HPV detected cases HPV Digene HC 2® Hologic Cervista® Roche Cobas® Reflex from ASCUS Reflex total cases 94 207 110 HPV positive cases 30 71 30 Follow-up CIN 1 9 (30%) 25 (35%) 9 (30%) CIN 2 4 (13%) 3 (4%) 4 (13%) Negative CIN 5 (17%) 10 (14%) 5 (17%) Negative Pap 7 (23%) 25 (35%) 7 (23%) No follow-up 5 (17%) 8 (12%) 5 (17%) Cotest Cotest total cases 420 528 622 HPV positive cases 50 51 64 Initial diagnosis Negative 35 33 47 Follow-up CIN 1 4 (11%) 1 (3%) 5 (11%) Negative CIN 3 (9%) 4 (12%) 10 (21%) Negative Pap 15 (43%) 12 (36%) 16 (34%) No follow-up 13 (37%) 16 (49%) 16 (34%) LSIL 13 11 13 Follow-up CIN1 7 (54%) 7 (64%) 7 (54%) Negative Pap 6 (46%) 4 (36%) 6 (46%) HSIL 2 7 4 Follow-up CIN 1 2 (100%) CIN 2 7 (100%) 4 (100%) CIN 1/2 = Cervical intraepithelial neoplasia; LSIL = low-grade squamous intraepithelial lesion; HSIL = high-grade squamous in- traepithelial lesion. Table 4. ASCUS reflex HPV test by Roche Cobas®, 1-year follow-up Cases, n Percent ASCUS reflex HPV by Roche Cobas® Total ASCUS cases 344 100 Total detected 138 40 Total not detected 206 60 HR 16/18 positive 29 8 HR other positive 109 32 Follow-up biopsy for positive HPV 16/18 Total biopsies 20/29 69 CIN 1 5 25 CIN 2 2 10 CIN 3 4 20 Negative CIN 9 45 Follow-up biopsy for positive HR HPV Total biopsies 55/109 50 CIN 1 21 38 CIN 2 2 4 CIN 3 3 5 Negative CIN 29 53 LSIL = Low-grade squamous intraepithelial lesion; HSIL = high-grade squamous intraepithelial lesion. http://dx.doi.org/10.1159%2F000446271 Bibbo/Wang/Jones/Solomides/ Draganova-Tacheva/Stapp Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 538 and 39% (28/71 = 39%) by Cervista ® . Some cases were followed by repeat Pap tests, and some cases had no fol- low-up, as shown in table 3 . Fewer cases of CIN 1 (4/35 = 11%, 1/33 = 3%, 5/47 = 11%) were found in the follow-up of negative cases with HPV detected by the 3 cotesting as- says. Most cases diagnosed as low- and high-grade squa- mous intraepithelial lesions were confirmed by CIN 1 and CIN 2/3, respectively, in the histologic follow-up. No cas- es of invasive carcinoma were found. Table 4 demonstrates the total number of HPV reflex tests of ASCUS by Roche Cobas ® in 2014. One hundred thirty-eight cases out of 344 (40%) had HPV detected: 29 HPV 16/18 and 109 other HR HPV. The follow-up of these cases is also shown in table 4 . A higher percentage of CIN 2 and CIN 3 was found in the follow-up biopsies of HPV 16/18 detected cases. No statistical analysis was performed, as the total case number is small. To date, the molecular laboratory has no record of Cobas ® HR HPV testing alone. Discussion In the last decade it has become clear that infection with HR HPV is required for the development of most cervical cancers and high-grade precursor lesions. Recent emphasis on molecular testing seeks to identify infection with HPV strains considered a high risk for carcinogen- esis. The clinical use of HPV testing started as additional screening for patients with ASCUS cervical cytology re- sults in order to determine the need for colposcopy [3] . In our hospital practice, HR HPV reflex tests, automati- cally ordered by the cytology laboratory for ASCUS re- sults, were performed from 2006 (when covered by insur- ance) and continue through the present. In women 30 years and older, the HR HPV assay can be used in combination with cervical cytology to adjunc- tively screen for the presence or absence of HR HPV types. This information, together with the physician’s as- sessment of the cytology history, other risk factors, and professional guidelines [5] , may be used to guide patient management. Table 1 shows that HPV cotesting at Thom- as Jefferson University Hospital started in 2007 and pro- gressively increased from 44 to 90% by 2015. At the same time, the decrease in Pap testing is striking from 11,792 in 2008 when the percentage of HPV testing was 19%, to 4,664 in 2015 with 59% utilizing HPV testing. It is known that a negative HR HPV test represents a low risk of de- veloping disease over 5 years and safely allows lengthen- ing of the test interval. This rationale is reasonable to ex- plain our observed decrease in Pap testing. Also notice- able is the percentage decrease in HPV reflex testing and increase in HPV cotesting. Most likely this was the result of increased adherence by practitioners to cotesting guidelines [5] . Of interest is the similar HR HPV detection rate in both reflex (32, 34, 27%) and cotests (12, 10, 10%) for the 3 HPV assays, as shown in table 2 . The follow-up biopsies for ASCUS cases, when avail- able, revealed CIN 1 or 2 in 43% of cases detected by HC 2 ® and Cobas ® and 39% by Cervista ® . Several biopsies showed absence of CIN, and quite a few cases were fol- lowed only by negative Pap tests. For cotesting, 3–11% of negative Pap tests showed CIN 1 in the follow-up bi- opsy. The majority of cases had only negative Pap tests for follow-up. Most low-grade squamous intraepithelial lesion cases were confirmed by cervical biopsy and all high-grade squamous intraepithelial lesion cases were confirmed by histology as shown in table 3 . It is not sur- prising that no cases of cervical carcinoma were found, since our female population is at low risk for cervical cancer. Table 4 illustrates the number of HPV reflex tests with the Cobas ® platform in 2014, separating the HR HPV 16/18 (29 = 8%) from other HR HPV types (109 = 32%). Follow-up shows 69% of the HPV 16/18 cases had subse- quent biopsies, in comparison to 50% for other HR HPV cases. A trend towards higher CIN 2/3 for HPV 16/18 was noted. The lack of demand for HR HPV stand-alone testing in our institution is of interest. Major changes to screen- ing guidelines in the last decade include initiation of screening at the age of 21 years, conservative manage- ment of young women with abnormal cytology, ex- tended screening intervals for women aged ≥ 30 years and cessation of screening in low-risk women over the age of 65 years [5] . HR HPV is a prerequisite for the development of almost all types of cervical cancer, therefore HR HPV has become an integral part of new screening strategies. With the FDA approval of the first HPV test for primary cervical cancer screening of wom- en ≥ 25 years [6] , clinicians in the USA now have 3 dif- ferent first-line screening options that they may offer to patients: the Pap test, cotesting with Pap and HPV, and HPV testing as a stand-alone test [7] . The choice of the cervical screening method may vary for a variety of reasons including patient and provider preference along with geographic, socioeconomic, and practice settings. http://dx.doi.org/10.1159%2F000446271 Cytology Practice before and after HR HPV Testing Acta Cytologica 2016;60:534–539 DOI: 10.1159/000446271 539 Conclusions It is evident in our practice that ThinPrep Pap testing decreased from 11,792 to 4,664 (60%) over 8 years, while the percentage of HPV molecular testing (reflex plus co- testing) increased from 19 to 59%. The decrease in Pap testing is most likely due to an increased interval between tests when cotesting results were negative. The HPV de- tection rates assessed during a 3-month interval showed that the 3 HR HPV assays used, namely HC 2 ® , Cervis- ta ® , and Cobas ® , detected similar rates of HR HPV. A trend was noted towards higher-grade CIN in the follow- up of HPV 16/18 detected cases. 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