Bethesda Classification of Cervicovaginal Smears: Reproducibility and Viral Correlates NANCY E. JOSTE, MD, LYNDA RUSHING, MD, ROSARIO GRANADOS, MD, JULIA C. ZITZ, BS, DAVID R. GENEST, MD, CHRISTOPHER P. CRUM, AND EDMUND S, CIBAS, MD MD, Fifty-fivecervieovaginai smears from women with squamons in- traepithelial lesions (SILs) were independently evaluated on two sepa- rate occasions by four cytopathologists using a binary classification system (the Bethesda system). Smears were categorized as low-grade (LSIL) or high-grade (HSIL) using previously published criteria. All women had subsequent cervical biopsies containing human papillo- mavlrns (HPV) DNA amplified with the polymerase chain reaction and typed by restriction fragment polymorphism analysis. Three or more observers agreed on classification in 49 of 55 cases (87%); unanimous diagnoses were rendered in 31 cases (56%). Interobserver and intraobserver reproducibility ranged from fair to near-excellent (Kvalues 0.40 to 0.63; 0.63 to 0.74, respectively). HPV types included HPV 16 (27%), 18 (7%), 31 (9%), 35 (4%), 39 (4%), 6 (10%), 11 (2%), novel types (30%), and multiple types (4%). High-risk HPV types (16, 18, 31, 35, and 39) were significantlyassociated (P = .03) with consensus HSIL diagnoses (agreement of three or more observ- ers). This was primarily because of the strong association of HPV 16 with HSIL (P = .001). After excluding HPV 16, the other high-risk HPV types (18, 31, 35, and 39) were no longer significantly associated with consensus HSIL diagnoses (P > .5). Conversely, LSIL diagnoses were significandy associated with non-hlgh-risk HPV types (all HPV types except 16, 18, 31, 35, and 39; P = .006). Binary cytological classification of cervicovaginal SILs is reproducible among cytopa- thologists. Such classification correlates well with most low-risk HPV types and with the prototypic high-rlsk HPV 16 but not with other high-risk HPV types. HUM PATHOL 27:581--585. Copyright © 1996 by W.B. Saunders Company Key words: cervlcovaglnal smear, cervical dysplasia, human papil- lomavirus, reproducibility, K statistics, cervix, squamous intraepithe- lial lesion. Abbrev/at/ons: SIL, squamous intraepithefial lesion; LSIL, low grade intraepithefial lesion; HSIL, high-grade intraepithelial lesion; HPV, human papillomavlrus; PCR, polymerase chain reaction. Squamous intraepithelial lesions are a morphologi- cally and biologically diverse group of abnormalities, some of which, if untreated, progress to invasive can- cer. a Almost all of these lesions contain human papillo- mavirus (HPV), and the HPV subtypes are classified as low or high risk, depending on the frequency with which they are present in invasive cancers. Although HPV types 6 and 11 are low-risk types and identified mainly in genital condylomata, types 16, 18, 31, 35, and 39 are high-risk types often present in invasive tumor. 2'3 The histological and cytological separation of lesions into lower and higher grades is based on the increasing probability of association with high-risk HPV types and subsequent progression to invasive squamous carci- n o m a . 3-5 Despite these associations, the correlation between lesion grade and HPV type has not been exact. In partic- ular, it is well known that some morphologically low- grade cervical lesions harbor high-risk HPV types and vice versa. 4'6'7 The authors, therefore, set out to deter- mine the degree to which cytology predicted different HPV subtypes. To address the preceding issue, it is important to consider the reproducibility of the cytological diagnosis of a low-grade or highgrade lesion. Several investigators have studied interobserver and intraobserver concor- dance in the evaluation of cervicovaginal smears, s-a~The agreement among cytopathologists is generally related to the complexity of the grading scheme: More compli- cated schemes with many diagnostic categories produce lower concordance than simpler systems. 14 The Bethesda classification, with its two categories for squa- mous intraepithelial lesions, is the simplest system. TM A previous study by our group addressed the rela- tionship between HPV types and morphology in cervical biopsies. 4 The authors found good reproducibility in grading lesions using the Bethesda system, and the cor- relation between HPV type and grade varied, being greater for some HPV types than for others. The purpose of this study is twofold: to evaluate the reproducibility among cytopathologists in using the Bethesda system to grade precursor lesions identified on smears, and to evaluate the association between cyto- logical grade and specific HPV type. From the Department of Pathology, Divisions of Cytology and Women's and Perinatal Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA. Accepted for publication December 5, 1995. Presented at the 83rd annual meeting of the United States-Cana- dian Academy of Pathology, San Francisco, CA, March 12-18, 1994. Address correspondence and reprint requests to NancyJoste, MD, Department of Pathology, Universityof New Mexico, School of Medicine, Albuquerque, NM 87131. Copyright © 1996 by W.B. Saunders Company 0046-8177/96/2706-001255.00/0 MATERIALS AND METHODS The study set was composed of cases of squamous intraep- ithelial lesions previously analyzed for HPV nucleic acids. 4 In the cytology study, the files of the Brigham and Women's Hospital were searched for cervical smears that corresponded to these cervical lesions. In all cases the cervical biopsy con- tained human papillomavirus nucleic acids determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analyses. 4 581