Atypical Glandular Cells of Undetermined Significance Cytologic Criteria to Separate Clinically Significant From Benign Lesions

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1 ANATOMIC PATHOLOGY Original Article Atypical Glandular Cells of Undetermined Significance Cytologic Criteria to Separate Clinically Significant From Benign Lesions STEPHEN S. RAAB, MD, CHRISTINA ISACSON, MD, LESTER J. LAYFIELD, MD, JULIA C. LENEL, PHD, DANIEL D. SLAGEL, MD, AND PATRICIA A. THOMAS, MD Histologic followup of the cervicalvaginal smear diagnosis of atypical glandular cells of undetermined significance (AGUS) shows a broad spectrum of clinically significant (preneoplastic or neoplastic) and benign lesions. There are few statistical studies that have attempted to separate these AGUS categories based on select cytologic criteria. The authors retrospectively reviewed AGUS without concurrent squamous dysplasia smears ( clinically significant and 5 benign lesions), and used logistic regression analysis to identify the cytologic criteria of irregular nuclear membranes, atypical single cells, and decreased cyto Atypical glandular cells of undetermined significance (AGUS) is a Bethesda system diagnosis that categorizes glandular cells exhibiting changes beyond reactive/reparative changes, but lacking unequivocal features of invasive adenocarcinoma. " Because AGUS is a relatively new term and there is a lack of conformity among laboratories as to the meaning of "glandular atypia," the exact incidence of AGUS is difficult to assess. Goffand colleagues reported the incidence of "endocervical atypia" to be.%, whereas Nasu and associates 5 reported the incidence of "reactive glandular atypia" combined with "lowgrade atypia" to be.5%. Based on histologic followup, the reported range of patients with AGUS diagnoses who have clinically significant lesions is % to 8%. "'' We use the term "clinically significant" to denote any lesion that is malignant or premalignant, and include low grade squamous intraepithelial lesions (LGSILs), high grade squamous intraepithelial lesions (HGSILs), endocervical adenocarcinomas, and en Frotn the' University of Iowa. Iowa City. Iowa: and Duke University Medical Center. Durham. North Carolina. plasm as useful in separating clinically significant from benign lesions. Using contingency tables, these criteria in combination had a sensitivity 9% and a specificity of 9% in the diagnosis of clinically significant lesions. If any single criterion was present, the sensitivity and specificity were % and 8%, respectively. In conclusion, by using key cytologic criteria, a percentage of benign AGUS lesions can be separated from clinically significant AGUS lesions. (Key words: Atypia; Cytology; Bethesda system; Endocervix; Dysplasia; Cancer; Cervix; Glandular; Regression) Am J Clin Pathol 995; :5758. dometrial adenocarcinomas and hyperplasias. Of these clinically significant lesions, % to % are of glandular origin, indicating that in some studies AGUS primarily describes lesions of squamous origin. "" Of clinically significant AGUS lesions, % to 8% are HGSILs. " ' Histologic diagnoses in patients without clinically significant lesions have included repair, tubal, metaplasia, polyp, microglandular hyperplasia, AriasStella change, and no diagnostic abnormality. " Because a wide spectrum of lesions are included under the AGUS umbrella, several studies have attempted to identify cytologic criteria that may be used to separate these lesions. " ' Some of the reported features indicative of a clinically significant lesion include crowded, multilayered groups, coarse chromatin, irregular nuclear borders, micronucleoli, and elongated nuclear shape. "" However, these criteria also may be seen in benign lesions and have not been examined in arigorouslystatistical manner. In this study, we performed a stepwise logistic regression analysis to identify key cytologic criteria for the separation of benign from clinically significant AGUS lesions. Downloaded from by guest on March, Manuscript received March : revision accepted June Address reprint requests to Dr. Raab: University of Iowa Hospitals and Clinics, Department of Pathology, Hawkins Drive 5 RCP, Iowa City, IA 59. MATERIALS AND METHODS The cytopathology files from the University of Iowa Hospitals and Clinics were retrospectively reviewed for the 57

2 RAAB ET AL. 575 Cytologic Criteria in AGUS TABLE. CLASSIFICATION OF ATYPICAL GLANDULAR CELLS OF UNDETERMINED SIGNIFICANCE (9999) Year TABLE. CLASSIFICATION OF CLINICALLY SIGNIFICANT HISTOLOGIC DIAGNOSES IN CASES WITH CERVICAL VAGINAL DIAGNOSIS OF ATYPICAL GLANDULAR CELLS OF UNDETERMINED SIGNIFICANCE Diagnosis (%) Year NOS Favor endocervical Favor endometrial Favor endocervical and endometrial NOS with ASCUS NOS with LGSIL NOS with HGSIL (.9) 9(.5) (5.8) (.) 8(8.) 5(7.) 8(5.) NOS = not otherwise specified: ASCUS = atypical squamous cells of undetermined significance: LGSIL = low grade squamous intraepithelial neoplasia: HGSIL = high grade squamous intraepithelial neoplasia. time period from January, 99 to June, 99. During this period, there were,59 cervicalvaginal smears and (.7%) AGUS diagnoses (from patients). The AGUS category has been used at the University of Iowa since 99. We classify AGUS into three subcategories: not otherwise specified (NOS), favor endocervical, and favor endometrial. _ We do not further subclassify AGUS as favor reactive or favor neoplastic. The AGUS diagnoses were equally distributed over the years. The four cytology faculty and six cytology fellows who participated on the service during this time interval made the AGUS diagnosis with relatively equal frequency. In this time period, there were,58 (5.5%) clinically significant lesions including,5 LGSILs, 8 HGSILs, 9 carcinomas in situ, and 8 carcinomas. Table shows the breakdown of the AGUS cases. Our study was specifically directed at AGUS, favor endocervical origin, and AGUS, NOS. We excluded AGUS cases with concurrent squamous abnormalities, because the presence of squamous abnormalities already indicated that a clinically significant lesion could be present and that probably there would be some sort of followup. Of the cases of AGUS, favor endometrial origin, histologic followup was obtained in cases; 7 were benign endometrial lesions, and were endometrial adenocarcinomas. Because this category did not contain endocervical glandular or squamous lesions, the cases of AGUS, favor endometrial origin category also were excluded from the study. The total number of cases classified as AGUS, NOS, AGUS favor endocervical origin, and AGUS favor endocervical and endometrial origin was 55. Histologic followup was obtained in 7 (9%) of these cases and the time interval between the AGUS diagnosis to the histologic followup ranged from to 9 days (mean Diagnosis LGSIL HGSIL LGSIL and endocervical adenocarcinoma HGSIL and endocervial adenocarcinoma Endometrial adenocarcinoma (%) (5.5) (.) (.) (.5) (.7) LGSIL = low grade squamous intraepithelial neoplasia: HGSIL = high grade squamous intraepithelial neoplasia. days). Histologic followup consisted of cone biopsies; 5 cervical biopsies and endocervical curettages; 8 cervical biopsies only; 5 endocervical curettages only; hysterectomies; endometrial curettages and cervical biopsies; endometrial and endocervical curettages; endometrial and endocervical curettages and cervical biopsies; and cytologic fluid. The histologic slides were not reviewed. Of the 7 cases with followup, (from patients) had cervicalvaginal smears available for review. The other cases primarily were sent to the University of Iowa for consultation and were not available for review. The patients who had slides available for review ranged in age from 9 to 7 (mean age 9.7) and included 8 postmenopausal patients. Thirtyseven patients had a previous history of a clinically significant lesion (5 LGSILs, 8 HGSILs, and adenocarcinomas). One patient had a previous history of AGUS. As shown in Tables and, histologic followup showed clinically significant lesions in (5.9%) cases and benign lesions in 5 (. %) cases. Fiftyfive (8%) of TABLE. CLASSIFICATION OF BENIGN HISTOLOGIC DIAGNOSES IN 5 CASES WITH CERVICALVAGINAL DIAGNOSIS OF ATYPICAL GLANDULAR CELLS OF UNDETERMINED SIGNIFICANCE Diagnosis Endocervical polyp Immature metaplasia Dysynchronous endometrium No diagnostic abnormality Endometritis 99 8 Year (%) 5(.) (.) (.) (8.) (.) 5 Downloaded from by guest on March, Vol..No. 5

3 57 ANATOMIC PATHOLOGY Original Article the clinically significant lesions were squamous dysplasia and only 5 endocervical adenocarcinomas, all of them invasive, were present. Glandular dysplasia and endocervical adenocarcinoma in situ were not observed on histologic followup. Additional cervicalvaginal cytologic followup was obtained in (8%) of the 5 benign cases. The mean followup time was. months. Three cases were determined to have clinically significant lesions ( HGSIL and LGSILs), all approximately year after the original AGUS diagnosis. However, in each case, intervening cervicalvaginal smears had been interpreted as negative. Thus, for the purposes of this study, these cases were considered to have benign followup. The cases were reviewed by two cytopathologists who had no knowledge of the original cytologic or corresponding histologic diagnosis. Each case was scored for the presence or absence of cytologic criteria. An additional scoring category reflected the presence or absence of separate endocervical and cervical components. These cervicalvaginal smears consisted of two slides, whereas the remainder of the cases consisted of one slide. All cases were examined together, and a consensus opinion was reached. The criteria are listed in Table. Although these cytologic features have been discussed in detail elsewhere, " several deserve further definition: high cellularity of atypical cells consisted of greater than seven groups of atypical cells or greater than single atypical cells; cellular dissociation consisted of single atypical cells adjacent to and "breaking off' of groups of atypical cells; increased nuclear/cytoplasmic (N/C) ratio consisted of a N/C ratio greater than : TABLE Smear Pattern. SIXTYONE CYTOLOGIC CRITERIA EXAMINED Architecture of Atypical Groups or :; increased cell size, increased nuclear size, or increased or decreased amount of cytoplasm consisted of atypical cells exhibiting these features in comparison to benign endocervical cells. Some cytologic features, such as increased N/C ratio, increased nuclear size, and decreased cytoplasm, were related and often observed together. Many cells that exhibited an increased N/C ratio also had a larger nucleus and less cytoplasm. However, these three criteria were not always found in conjunction. Not all cells with increased nuclear size had an increased N/C ratio, because these cells also had a corresponding increase in cytoplasmic area. A twoway logistic regression analysis was used to model the relationships between the cytologic variables and the probability of a clinically significant lesion. 7 The technique of logistic regression in cytopathology has been previously described. 8 To briefly summarize, variables to be entered into the logistic regression were identified using univariate analyses of association. These analyses were performed between the dependent variable (clinically significant lesion) and the presence or absence of each independent variable (cytologic criterion). Independent variables with P < A using the two tail Fisher's exact test were included in the actual model. In the logistic regression, it is assumed that the relationship between the dependent and independent variables can be explained by the logistic distribution, an Sshaped curve that ranges from to. The formula for the logistic distribution is: afv) e/soxl/9x /Skxk/i, e(so/xl/x /Skxk where g(x) is the expected value of the dependent variable, given X, the independent variable(s). IN LOGISTIC REGRESSION ANALYSIS Cytoplasmic Individual Cellular Features Nuclear Downloaded from by guest on March, Diathesis Benign endocervical groups High cellularity of atypical cells Neutrophils in atypical groups Atypical stripped nuclei Benign endometrial cells Parakeratotic cells Benign single endocervical cells Endocervical repair Crush artifact Lymphocytes in atypical groups Benign metaplastic cells Spindle cells Irregular group borders Cellular dissociation Strips (singlefileatypical cells) Palisading Monolayers Threedimensional groups (syncytia) Cellular crowding Acini Large groups (> cells/group) Cell balls Papillae Anisonucleosis Nuclear overlap Feathering (nuclear protrusion) Atypical single cells Small groups (< cells/group) Nuclear molding Increased cell size Irregular borders Increased cytoplasm Vacuolization Metaplastic Cilia Columnar shape Cuboidal shape Foamy Decreased cytoplasm Keratinized cytoplasm Increased N/C ratio Absent borders Distinct borders Distinct membranes Vacuolization Mitotic figures Round shape Oval shape Cigar shape Irregular membranes Thick membranes Micronucleoli Macronucleoli Coarse chromatin Fine chromatin Smudged chromatin Multinucleated Increased size Grooves Red nucleoli N/C = nuclear cytoplasmic. A.J.C.P. November 995

4 RAAB ET AL. 577 Cytologic Criteria in AGUS TABLE 5. COMPARISON OF CYTOLOGIC FEATURES OF CLINICALLY SIGNIFICANT AND BENIGN LESIONS No. of Clinically Significant No. of Benign Lesions Criterion Lesions (%) (n = ) (%) (n = 5) Diathesis Benign endocervical groups Irregular group borders Benign single endocervical cells Threedimensional groups (syncytia) Irregular cytoplasmic borders Cellular dissociation Strips (single file atypical cells) Palisading Monolayers Feathering Acini Large groups (> cells/group) Cell balls High cellularity of atypical cells Papillae Increased nuclear/cytoplasmic ratio Increased cytoplasm Distinct nuclear membranes Cytoplasmic vacuolization Nuclear vacuolization Mitotic figures Benign metaplastic cells Round atypical nuclei Anisonucleosis Irregular nuclear membranes Thick nuclear membranes Micronucleoli Macronucleoli Coarse nuclear chromatin Fine nuclear chromatin Smudged nuclear chromatin Neutrophils in atypical groups Increased nuclear size Nuclear overlap Keratinized cytoplasm Increased cell size Atypical stripped nuclei Benign endometrial cells Atypical single cells Atypical metaplastic cells Parakeratotic cells Atypical multinucleated cells Cigarshaped atypical nuclei Cilia Endocervical repair Absent cytoplasmic borders Small groups (< cells/group) Red nucleoli Spindle cells Atypical columnar cells Atypical cuboidal cells Foamy cytoplasm Distinct cytoplasmic borders Cell crowding Oval atypical nuclei Separate endocervical sample Decreased cytoplasm Nuclear grooves Crush artifact Lymphocytes in atypical groups Nuclear molding (.5) (97. 5(77. (9. (7. 5(75.8 (. (. (9.7 (8. 5(78.8 (.7 (5.5 (9.) 5(5. (.) (9.9 (. (9.9 8 (. (8. (. 5(8. 5(8.8 (9. (. (8.5 9(. (.7 (. 9(. 9(8.8 (9.9 5( (87.9 8(57. (.5) (5. 9(8.8 7(. 8(. (.) (5. (5.5 5(78.8 (.) 7(. 8(7. 7(5. (.8 5(98.5 (95.5 7(5.8 9(.9 (.8 (.) 5(7.9 (.) 9 (98.) (.) 9 (98.) (.) (.) 7(.) 8(.) (.) (.) 5 (7.) 8(.) (.) (.) 5 (5.) (.) (8.) 5 (5.) (9.) (.) (.) (.) (.) 8 (7.) (8.) (.) 7(.) (.) (.) (.) (.) (.) 9(8.) (8.) (.) (.) 9(78.) (.) (.) (8.) (.) (.) (.) (.) (.) (8.) 5(.) (8.) (.) (.) (.) 8(.) (.) (.) 8 (9.) (9.) (8.) 8(.) (.) 5(.) (8.) 5(.) Downloaded from by guest on March, Vol. No. 5

5 578 ANATOMIC PATHOLOGY Original Article TABLE. PROBABILITY OF CLINICALLY SIGNIFICANT LESION BASED i ON THREE CYTOLOGIC FEATURES Irregular Nuclear Membranes Criteria Atypical Single Cells Decreased Cytoplasm Probability of CS Lesion (%) Number of Patients Clinically Significant Lesion HGSIL' LGSILf Adeno Benign CS = clinically significant; HGSIL = high grade squamous intraepithelial lesion: LGSIL = low grade squamous intraepithelial lesion: Adeno = adenocarcinoma. Includes I case of adenocarcinoma and HGSIL: flncludcs cases of adenocarcinoma and LGSIL. In a stepwise logistic regression, variables are selected one at a time. In the first step, the cytologic criterion that is the most strongly associated with malignancy (lowest P value from univariate analysis) is entered into the model. The model is checked with a P value associated with the improvement chisquare statistic to determine if the addition of the variable improves the fit of the model (eg, P <.). In subsequent steps, the remaining independent variables are reanalyzed, taking into account the effect of the variable already in the model. Again, the criterion with the strongest association is entered into the model. This procedure is repeated until none of the remaining variables improve the goodness of fit. The regression also examines the variables in the model to see if any variables should be removed. A threeway logistic regression analysis was used to model the relationships among the cytologic variables and the probability of a squamous dysplasia, adenocarcinoma, or benign lesion. RESULTS Table 5 shows the number of cases in which each cytologic feature was observed in the clinically significant and benign diagnostic categories. Features observed in more than 9% of the clinically significant cases included increased N/C ratios, distinct nuclear membranes, irregular nuclear membranes, increased nuclear size, atypical oval nuclei, cell crowding, and benign single endocervical cells. Thirtyseven of the criteria were present in fewer than 5% of the clinically significant cases. Only four criteria (diathesis, papillae, mitotic figures, and cilia) were observed exclusively in clinically significant lesions, although these criteria were observed in few cases. Using univariate analyses of association, cytologic criteria were significant at P <. and these criteria were entered into the stepwise logistic regression model. These criteria were threedimensional groups (syncytia), dissociation, palisading, increased cytoplasm, nuclear vacuolization, anisonucleosis, irregular nuclear membranes, thick nuclear membranes, coarse chromatin, atypical single cells, absent cytoplasmic borders, decreased cytoplasm, nuclear grooves, and nuclear molding. The twoway logistic regression model identified three cytologic features as useful in discriminating between clinically significant and benign lesions. These features were irregular nuclear membranes, atypical single cells, and decreased cytoplasm in atypical cells. The formula for the logistic regression is: g(x) =..8(irregular nuclear membranes).95 (atypical single cells).7(decreased cytoplasm) The number of patients with clinically significant and benign disease in whom these criteria were observed are shown in Table. Nineteen (9%) patients with histologically proved clinically significant disease had all three cytologic criteria and 8 (7%) patients had at least two of the criteria. Thirteen of 5 (87%) patients with HGSIL had either all three criteria or the combined criteria of irregular nuclear membranes and atypical single cells. Eleven of (9%) patients with adenocarcinoma had at least two of the criteria. The contingency table sensitivity, specificity, positive predictive value, and negative predictive value for clinically significant lesions using the three combined cytologic criteria are 9%, 9%, 8%, and 5%, respectively (Table 7). Only patients exhibited all features and 9 (8%) had a clinically significant lesion. The contingency table sensitivity, specificity, positive predictive value, Downloaded from by guest on March, A.J.C.P. November 995

6 RAAB ET AL. 579 Cytologic in AGUS TABLE 7. NUMBER OF PATIENTS WITH CLINICALLY SIGNIFICANT LESIONS PREDICTED BY THREE CYTOLOGIC CRITERIA AND OBSERVED BY HISTOLOGY Histology Presence of Three Cytologic Features Clinically Significant Lesion Benign Lesion Present Absent and negative predictive value for clinically significant lesions using at least one cytologic criterion are %, 8%, 5%, and %, respectively (Table 8). Figures through show examples of these three key cytologic features. Threeway logistic regression did not identify any cytologic features as useful in discriminating among the cytologic variables and the probability of a squamous dysplasia, adenocarcinoma, or benign lesion. In other words, criteria were not identified that could differentiate among the lesions (squamous versus true glandular) in the clinically significant category. DISCUSSION The separation of clinically significant from benign AGUS lesions is important because currently the clinical treatment of patients with AGUS is problematic. If the AGUS category contains a high percentage of clinically significant lesions and patients with these lesions cannot be differentiated from patients with benign lesions, then some form of clinical followup for all AGUS patients is necessary. Exclusion of patients with benign AGUS lesions from followup is desirable. A dilemma for the cytologist is that the AGUS category is a category of exclusion. Lesions are classified as AGUS if they cannot be placed in definitively benign or premalignant/malignant categories.'" This ambiguity may indicate why there is interlaboratory discrepancy in FIG.. Atypical glandular cells of undetermined significance. There is a cluster of glandular cells with irregular nuclear membranes. Histologic followup showed a LGSIL (Papanicolaou. X5). AGUS incidence and demographics: either there truly is a difference in hospital AGUS rates or cytologists use this category differently. In our laboratory, the incidence of AGUS and the percentage of AGUS lesions that were clinically significant were.7% and 5.9%, respectively. These numbers fall within the range of previously reported results. "" Wilbur and colleagues' and Bose and associates, among others, have argued that new sampling devices provide new patterns of normal and neoplastic endocervical and squamous cells. 9 " The challenge lies in interpreting these patterns and determining which cases are AGUS and which cases are truly benign or clinically significant. Wilbur and colleagues reported that with additional experience, the diagnostic accuracy of the " *. * " ' Downloaded from by guest on March, TABLE 8. NUMBER OF PATIENTS WITH CLINICALLY SIGNIFICANT LESIONS PREDICTED BY AT LEAST ONE CYTOLOGIC CRITERION AND OBSERVED BY HISTOLOGY Histology mm * Presence of at Least One Cytologic Feature Clinically Significant Lesion Benign Lesion Present Absent 5 FIG.. Atypical glandular cells of undetermined significance. Scattered atypical single cells with vacuolated, glandular cytoplasm arc present. Stripped nuclei also are present. Histologic followup showed a HGSIL (Papanicolaou, X5). Vol. No. 5

7 58 ANATOMIC PATHOLOGY Article FIG. 5. Atypical glandular cells of undetermined significance. A cluster of glandular cells with nuclear overlapping and irregularity and decreased cytoplasm is observed. Histologic followup showed a LGSIL and endocervical adenocarcinoma (Papanicolaou, X5). AGUS category can be improved as these patterns become more familiar. To increase this familiarity, there have been a number of retrospective, descriptive studies that have attempted to characterize these patterns and determine the features that are useful in separating benign from clinically significant lesions."'' Logistic regression analysis provides the advantage of examining these features in a blinded and rigorously statistical fashion. In our study, as expected, individual criteria, although often observed more frequently in clinically significant lesions, were not definitive if used alone. However, combinations of criteria as identified by regression analysis were helpful. If used in combination, the criteria of irregular nuclear membranes, single atypical cells, and decreased cytoplasm separated clinically significant from benign lesions with a sensitivity and specificity of 9% and 9%, respectively. The relatively low sensitivity reflects the fact that clinically significant lesions may exhibit a wide range of features and only infrequently were all three features present. This is understandable if one considers that clinically significant AGUS lesions have different morphologies and consist of lesions ranging from LGSIL to adenocarcinoma."" In general, such a low sensitivity is not satisfactory for a screening test, and consequently a tradeoff of lower specificity for higher sensitivity must be made. If any one of the three criteria were present the sensitivity was %, al FIG.. Atypical glandular cells of undetermined significance. A tight cluster of glandular cells with decreased cytoplasm and irregular nuclear membranes is seen. Histologic followup showed a LGSIL (Papanicolaou. X5). FIG.. Atypical glandular cells of undetermined significance. A loose cluster of glandular cells with irregular nuclear membranes is present, some cells have large nuclei and a decreased amount of cytoplasm. Histologic followup snowed a LGSIL (Papanicolaou, X5). A.J.C.P.November 995 Downloaded from by guest on March, FIG.. Atypical glandular cells of undetermined significance. Clusters of atypical glandular cells with decreased cytoplasm. Occasional cells have nuclei with irregular nuclear membranes. Detached, single cells are seen in the lower right. Histologic followup showed a LGSIL (Papanicolaou. X5).

8 RAAB ET AL. 58 Cytologic Criteria in AGUS though the specificity was 8%, indicating that many of the benign lesions were captured by a less stringent application of the criteria. Even with this low specificity, of 5 cases of benign AGUS could have been excluded from additional followup. The acceptable sensitivity and specificity of the AGUS category has not been directly addressed and probably will change as the AGUS category is better understood. The regression results show that both architectural and cellular morphologic features are important. The three key cytologic features already are well known as excellent descriptors of squamous dysplasias. 8 This is not surprising, because there is uniformity among observers that the majority of AGUS lesions are squamous dysplasias. "" In fact, Currie and coworkers'' reported that cytologic followup of 7 clinically significant AGUS cases showed 98.% squamous dysplasias. In our study, 87% of the HGSILs exhibited all three criteria or the two criteria of irregular nuclear membranes and single atypical cells. An interesting finding is that these criteria applied equally to adenocarcinomas. A bias that we had before the study in examining AGUS lesions was that effort was concentrated on groups of cells that appeared "glandular." The regression results stress that there should be a diligent search for single atypical cells as well. The criteria of irregular nuclear membranes and decreased cytoplasm may be observed in both cell clusters and single cells. Nasu and associates, 5 Goff and colleagues, and Bose and colleagues previously emphasized the importance of nuclear membrane regularity in reactive lesions. A number of cytologic features described by others as important were not identified by regression analysis. For example, such features have included atypical metaplastic cells as reported by Bose and colleagues, and high N/C ratio and loss of polarity as reported by Goff and colleagues in clinically significant lesions. The failure to be identified by logistic regression does not indicate that these features are not important, only that in our study, these criteria were not definitive for the separation. One explanation is that these criteria appear in combination with other criteria that are actually the key criteria. The results of our study were influenced by our case mixture that was predominated by squamous lesions. Consequently, cytologic criteria characteristic of clinically significant glandular lesions, such as endocervical adenocarcinoma and adenocarcinoma in situ, were not identified in the twoway regression analysis. Previously reported cytologic criteria for endocervical adenocarcinoma in situ included cigarshaped nuclei, feathered edges on cell clusters, and nuclear crowding.'" 5 No cytologic criteria were identified in the threeway regression analysis to separate adenocarcinoma, squamous dysplasia, and benign lesion. The major limiting factor of the threeway logistic regression was that there were too few cases in the adenocarcinoma group. Thus, with the addition of cases of clinically significant glandular lesions, previously described "glandular" features could be identified as important in the separation of AGUS subcategories. Based on our results, we are confident that some cases in the AGUS category can be further separated into clinically significant and benign subcategories. In a sense, the Bethesda Committee has attempted to facilitate this separation by using the categories favor reactive and probably neoplastic. It is uncertain the extent to which the Bethesda criteria useful in this separation have concentrated on squamous or on glandular neoplasia. If the Bethesda criteria are only useful to separate reactive from neoplastic true glandular lesions, these criteria have a limited value for the identification of the majority of clinically significant AGUS lesions, which are of squamous origin. It is important to recognize reactive and neoplastic criteria for both squamous and glandular lesions. Ours was an attempt to identify some of these criteria, particularly for squamous lesions; additional studies are needed to identify key criteria for clinically significant glandular lesions, a smaller subset of AGUS lesions. Evaluation of criteria naturally was dependent on subjective viewer identification. A number of factors, such as viewer experience, skill and bias, played a role in the recognition of these criteria and consequently were incorporated into the regression model. We currently are investigating the practicality of the model by measuring diagnostic accuracy of multiple observers. The identified criteria are being applied to characterize newly diagnosed cases of AGUS. In summary, the cervicalvaginal diagnostic category of AGUS is composed of a spectrum of benign and clinically significant lesions. For clinical treatment, one important distinction is in the separation of these two categories. The key criteria identified by logistic regression provide a means for this separation. REFERENCES. The Bethesda Committee. The Bethesda system for reporting cervical/vaginal cytologic diagnoses. Ada Cylol 99; 7:5.. The Bethesda Committee. The Bethesda System for Reporting Cervical/Vaginal Diagnoses. New York: SpringerVerlag, 99.. The Bethesda Committee. Current issues: the 99 Bethesda system for reporting cervical/vaginal diagnoses. Diagn Cvtopathol 99;9:59.. Goff BA, Atanasoff P, Brown E, et al. Endocervical glandular atypia in Papanicolaou smears. 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