Abstracts S47

abnormal cases were adjudicated and biopsied. Patients that had negativeresults for HPV, standard LBC and SPP were not biopsied and wereconsidered disease negative; patients lost to follow-up were excluded fromthe analysis.Results: Preliminary data were collected from 3613 women. SPP identified97 additional HSIL cases. With a CIN2+ endpoint, SPP improved diseasedetection by increasing the sensitivity relative to standard LBC by 13.9% -21.7% while decreasing specificity only 3.8% - 1.2%. In the <35 years ofage patients SPP improved sensitivity 13.6% - 23.3% with a loss ofspecificity of only 3.7% - 1.1%. In the high risk subgroup, SPP improvedsensitivity 12.5% - 26.8% while decreasing specificity only 5.1% - 1.6%.Conclusions: Within our cohort, SurePath Plus identified more cases withhigh grade cytologic abnormalities and biopsy confirmed CIN2+ diseasecompared to standard LBC. This novel combination of biomarker detectionwithin a standard LBC Pap may improve clinical management of youngwomen and those at increased risk for developing invasive cervical cancer.

74

Accuracy of FocalPoint GS Imaging: Can We Trust the System?

Hedieh Honarpisheh, MD, Kevin Schofield, CT(ASCP),Malini Harigopal, MD, David Chhieng, MD, Angelique Levi, MD.Pathology, Yale University, New Haven, Connecticut

Introduction: Recently, the Food and Drug Administration approved theuse of the location guided imaging system FocalPoint GS (FPGS) to assistin the primary screening of SurePath Papanicolaou (pap) tests. A fewstudies have demonstrated a significant decrease in screening time anda substantial increase in the detection of squamous abnormalities. Theobjective of the current prospective study is to analyze the accuracy ofFPGS in detecting squamous intraepithelial lesions in the clinical setting.Materials and Methods: All SurePath pap tests being evaluated with theassistance of FPGS during 2011 were included in the current study. Falsenegative cases that were discovered during quality control (QC) reviewwere retrieved. The majority of the cases that were selected for QC reviewwere high-risk i.e. cases with previous cervical abnormalities and/orpositive HPV co-testing. Only false negative cases with the final diagnosisof low-grade squamous intraepithelial lesion (LSIL) or above wereincluded. The original 10 fields of view (FOV) were then reviewed bya senior cytotechnologist to determine if any abnormal cells were present inany of the original 10 FOVs.Results: A total of 66,863 SurePath pap tests were evaluated with theassistance of the FPGS imaging system during the study period. A total of9,762 (14.6%) underwent full manual QC review. Among these cases, 105(1.1%) cases were reclassified as LSIL or above. No abnormal cells werepresent in the original FOVs in 15 (14.3%) out of the 105 false negativecases. All 15 cases were reclassified as LSIL; 7 were subsequentlyconfirmed histologically; 2 additional cases tested positive for high-riskHPV. No HSIL cases were missed by FPGS imaging system.Conclusions: On prospective analysis, based on the results of QC review of9,762 cases, only a small number (0.15%) of abnormal squamous cells/cellclusters were not presented in the 10 FOVs by the FPGS imaging system. Itis reasonable to conclude that the FPGS imaging system is relativelysensitive in detecting clinically relevant squamous cell abnormalities.

75

p16 and Ki-67 Dual Immunohistochemical Staining in CervicalCytology Specimens Diagnosed as Squamous Lesions Indeterminatefor High Grade Dysplasia

Kirsten Woolf, MD, Sharlin Varghese, MD, Kunchang Song, MD,Loralee McMahon, BA, HTL, Donna Russell, MS, CT(ASCP),Michael Facik, MPA, CT(ASCP), Thomas Bonfiglio, MD. Pathologyand Laboratory Medicine, University of Rochester Medical Center,Rochester, New York

Introduction: Patients with high grade squamous intraepithelial lesions(HSIL) of the cervix have a significant risk of progression to invasive

cancer. Papanicolaou smear cervical cytology screening is a well estab-lished method of detecting precancerous lesions and aids in selection ofpatients to undergo further testing. Correct diagnosis of HSIL is extremelyimportant, but can be difficult due to low numbers of atypical cells,extensive reactive changes, or difficulty distinguishing glandular cells fromdysplastic squamous cells. Hence some cases are diagnosed as atypicalsquamous cells or low grade squamous intraepithelial lesion, cannotexclude high grade (ASC-H, LSIL-H). Many of these patients havea high grade lesion at follow up colposcopic biopsy. It would be helpfulfor clinical management to have a test to supplement routine cytologyevaluation and more accurately determine those patients who harbor a highgrade lesion.Materials and Methods: After institutional review board approval, allliquid-based gynecologic cytology cases received from July 1, 2010 witha diagnosis of ASC-H or LSIL-H with unused sample remaining, wereprospectively collected for study inclusion. Residual material was pro-cessed and the unstained slide was stained with the CINtec� PLUS Kit(Roche mtm laboratories AG, Heidelberg, Germany). Cases with one ormore cells with clear dual staining for p16 and Ki-67 were considered posi-tive. Stained slides were independently scored by three pathologists (KW,SV, KS) and two cytotechnologists (DR, MF) and were reviewed forconsensus when necessary. Additional remaining sample was sent forhuman papilloma virus (HPV) testing by Digene Hybrid Capture (Qiagen,Germantown, MD), performed in the virology laboratory at our institution.The surgical pathology files were searched for follow up material and thediagnoses were reviewed. Statistical analysis was performed to determinethe sensitivity and specificity of adjunctive CINtec� PLUS staining for pre-dicting HSIL.Results: Of 293 cases, 226 (77.1%) cases were positive for dual stainingand 248 (84.6%) cases were positive for HPV. However, only 228 (77.8%)cases had available biopsy follow up to date. 82 (35.9%) of these caseswere diagnosed as HSIL. The sensitivity of p16 and Ki-67 dual staining forHSIL was 90.2%, but specificity for HSIL was only 28.8%. Similarly, thesensitivity of HPV positivity for HSIL was 96.3%, while specificity was21.2%.Conclusions: p16 and Ki-67 dual staining showed a high sensitivity forpredicting HSIL. However, the specificity was low as most of the caseswere positive for the dual stain. p16/Ki-67 dual staining may not be usefulin this cohort of patients since the majority of patients in this study showedpositive staining that did not necessarily correlate with a HSIL diagnosis onbiopsy. However, the study to date is limited by the relatively short durationof follow up (1-2 years).

76

Cervical Cytology of the Diagnosis Atypical Squamous Cells, CannotExclude High Grade Squamous Intraepithelial Lesion: A Follow-upStudy with Corresponding Histology and Significance of PredictingDysplasia by Human Papillomavirus DNA Testing

Syed Gilani, Lamia Fathallah. Pathology, St.John Hospital &Medical Center, Detroit, Michigan

Introduction: Atypical squamous cells, cannot exclude high gradesquamous intraepithelial lesion (ASC-H) is a less frequent diagnosis onPapanicolaou (pap) smears, with varying results for high risk humanpapillomavirus (HPV) testing. In this study, we evaluated the clinicalsignificance of ASC-H by performing cytohistologic correlation and its’association with high risk HPV.Materials and Methods: A Total of 23,5518 ThinPrep Pap tests were donefrom 2008-2010 in our institution and only 727 (0.3%) cases werediagnosed as ASC-H. Of these 727 cases we had histologic follow-up of309 cases (32.53 + 11.52 years old, mean + SD) with mean follow-up of4.09 + 5.32 months (mean + SD). Cases with previous history of any typeof dysplasia were excluded from the study.Results: Of these 309 cases, 120 (38.83%) cases were proved to be cervicalintraepithelial neoplasia (CIN) 2/3 on the corresponding histology follow-up. 59 (19%) cases were CIN1, 16 cases showed koilocytic changes on thesubsequent histology. On follow-up histology inflammation or reactive

S48 Abstracts

changes were found in 55 cases. Interestingly, no transformation zone wasfound in 20 cases. We have observed that the incidence of dysplasia inpatients below 40 is 64.3% vs. 53.8% (pZ0.147) in patients above 40years. If we combine the dysplastic cases, the difference is more significant,of these 309 cases, 179 (57.9%) cases showed dysplastic changes includingCIN-1, CIN-2 or CIN-3.

There were 67 cases with high risk HPV DNA Testing. HPV DNA waspositive in 39 of dysplastic cases [(14 CIN-1), (24 CIN-2 or CIN-3)] andnegative in 5 (1 CIN-1 & 4 CIN2/3) cases; for non-dysplastic cases (benign,inflammation, koilocytic), 14 cases were HPV positive and 9 cases negative(Sensitivity 73.6%, Specificity 64.3%, PPV 88.6%, NPV 39.1%, pZ0.012).Conclusions: We concluded that cases diagnosed as ASC-H should befollowed-up with caution as they are strongly associated with dysplasia(57.9%) especially CIN2/3 (38.83%,high grade dysplasia). Reflex HPVtesting is an important predictor of dysplasia with 88.63% positivity indysplastic cases. Reactive changes and koilocytic atypia can result in thereactive atypia. Sampling errors, such as lack of transformation zone andsampling of the non-representative areas are the most common factors ofdiscrepancy between the pap smear and subsequent biopsy. Close follow-upof ASC-H cases is recommended.

77

The Effect of Age, Race, Pregnancy and Treatment Facility on theFollow-up Biopsy Rate for Atypical Squamous Cells, Cannot ExcludeHigh-grade Squamous Intraepithelial Lesion (ASC-H) and High-gradeSquamous Intraepithelial Lesion (HSIL) Diagnoses at Two NewOrleans Teaching Hospitals

Farrukh Gill, MD1, Sharon Hirsh, MD1, Hillary Kimbrell, MD1,Michelle Lacey, PhD2, Monica Goswami, MD1, Krzysztof Moroz, MD1.1Pathology and Laboratory Medicine, Tulane University School ofMedicine, New Orleans; 2Mathematics, Tulane University, New Orleans,Louisiana

Introduction: The study objective was to determine whether the biopsy ratefor inner city women of New Orleans with ASC-H and HSIL diagnosesdiffers as a function of race, age, pregnancy status, and/or treatment facility.Two teaching hospitals were considered, namely the state-operated InterimLSU Public Hospital (ILH) and the for-profit Tulane Medical Center(TMC).Materials and Methods: The laboratory information systems of bothhospitals were searched from July 1, 2008 to June 30, 2010 for cervicalpap test cases with ASC-H and HSIL diagnoses. The age, race, pregnancystatus and the follow up information with the biopsy diagnosis, whenavailable, were recorded for each case. Analysis of race was simplified bycreating a binary “AA” variable to indicate whether or not each patient wasAfrican American (AA). The data was analyzed for each set of ASC-H andHSIL patients by a logistic regression analysis with “biopsy” as a responseand the other variables as predictors. Odds ratios (OR) were calculated foreach of the model parameters.Results: Review of 12703 pap tests at ILH and 9238 cases at TMCgenerated 210 cases included in the study. The mean age of women was33 years (range 18-74), 155 were AA and 27 patients were pregnant.Follow-up biopsies were performed in 177 individuals, 125 of which hadCIN 2+ (Table 1). The likelihood of having a follow-up biopsy variedaccording to age and diagnosis. Younger patients with an ASC-Hdiagnosis were less likely to have a biopsy than older patients with thesame diagnosis. For example, the predicted biopsy rate for an 18-year-oldnon-pregnant, non-AA woman with an ASC-H diagnosis was 0.697, butfor a 65-year-old non-AA woman, the predicted rate of biopsy was 0.979(Figure 1). The opposite trend was found for HSIL diagnoses, with thebiopsy rate for a 65-year-old woman slightly less than that for an 18-year-old woman. Pregnant patients were less likely to have follow-upbiopsies (ORZ0.174), as were AA women (ORZ0.378). The treatmentfacility was not a significant predictor of biopsy rate in any of the modelsthat were considered.

Figure 1 Biopsy rates as a function of age and diagnosis. PG Z pregnant.

Conclusions: Our data shows that the rate of follow-up biopsies did notdiffer between a state-run hospital and a for-profit hospital in New Orleans.Biopsy rates varied significantly according to the age of patients and theinitial Pap test diagnosis. AA women, particularly when pregnant, were lesslikely to have a follow-up biopsy compared to non-AA women. The causesbehind the racial differences in our study should be further investigated.Age and race may have important implications for the development of moreeffective approaches to improve quality of healthcare and reduce healthinequities.

Table 1 ASC-H and HSIL data distribution with the biopsy follow-up

Pap Dx_ Facility No of Race AA(%) Mean age Pregnant Yes(%) Bx Outcome NFU Bx(%)

cases non-AA (Range) Neg CIN1 CIN2+ Neg CIN1 CIN2+(%)

ASC-H_ILH

54 39 (72) 15 35 (18-74) 6 (11) 1 0 1 0 15 39 (70) 8 13 18 (46) ASC-H_TMC 18 10 (56) 8 33 (18-58) 0 0 0 0 0 3 15 (75) 3 3 9 (60) HSIL_ILH 109 89 (82) 20 34 (18-58) 15 (13) 8 0 1 7 11 98 (84) 5 11 82 (84) HSIL_TMC 29 17 (59) 12 30 (22-64) 6 (19) 5 1 0 4 4 25 (81) 8 1 16 (64)

NFU - no follow-up information

78

Utility of P16 Expression and Ki-67 Proliferation in Addition toStandard HPV Analysis in Pap Smears of ASCUS and ASC-H

Adam Toll, MD, Deidra Kelly Technician, Zahra Maleki, MD.Cytopathology, Johns Hopkins Hospital, Baltimore, Maryland

Introduction: Current management of cervical screening uses a combina-tion of cytology and analysis of high-risk human papillomavirus (HR-HPV)in cases of atypical squamous cells of undetermined significance (ASCUS)and atypical squamous cells cannot rule out high-grade intraepithelial lesion(ASC-H). Cytologic diagnoses of ASCUS, and ASC-H are subject tointerobserver variability and require correlation with HR-HPV status.Although highly sensitive, HR-HPV analysis can be limited by samplinginadequacy. This study was designed to correlate the immunocytochemicalexpression of HR-HPV surrogates P16INK4A (P16) and Ki-67 prolifera-tion index from cytospin prepared with residual thin-layer cervicovaginalmaterial against cytologic diagnoses and HR-HPV status.Materials and Methods: Eighteen cervicovaginal cytology specimens wereselected based on diagnosis (average age 35, range 22-60). Eight (8) casesof ASCUS and 4 cases of ASC-H were collected along with 6 positivecontrols (2 cases of LSIL and 4 HSIL). Digene Hybrid Capture II in situhybridization was used to detect 13 strains of HR-HPV. Pap smears wereprepared in a liquid-based BP Surepath preparation. Residual 1-2 ml ofcervicovaginal material was vortexed, and the residual pellet was dilutedwith buffer and placed in 10% formalin for 30 minutes, air dried, andstained for P16 and Ki-67. Nuclear staining of lesional cells by P16 wasconsidered positive expression. The Ki-67 index of lesional cells wasscored as follows: rare (1-3 cells), mild (4-7 cells), moderate (8-10 cells)and high (11 or more cells).