Gynecologic Cytopathology Received: September 29, 2010 Accepted: November 4, 2010 Published online: April 27, 2011
Acta Cytologica 2011;55:251–254 DOI: 10.1159/000323320
Infectious Organisms on Papanicolaou Smears Should Not Influence the Diagnosis of Atypical Squamous Cells of Undetermined Significance Haitham Nasser a Sylvia Hayek a Mamtha Balasubramaniam b Tomi J. Kuntzman a
Departments of a Cytology and b Biostatistics, William Beaumont Hospital, Royal Oak, Mich., USA
Key Words Atypical squamous cells of undetermined significance ⴢ Infection ⴢ Human papilloma virus ⴢ Cervical dysplasia
Abstract Background: Atypical squamous cells of undetermined significance (ASCUS) remain the center of diagnostic controversy and patients’ stress despite recent advances in cervical cancer screening and the introduction of human papilloma virus (HPV) testing. The role of infectious agents in the induction of such changes is not well understood. Aim: We aim at reviewing the effect of the different infectious organisms in Papanicolaou (PAP) smears on the ASCUS diagnosis. Material: 133 ASCUS cases associated with variable infectious organisms (ASCUS-infection group) with secondary HPV testing and appropriate follow-up studies were reviewed. A control group of 310 ASCUS cases without any organisms (ASCUS-only group) was selected for comparison. Results: The ASCUS-infection group had a significantly higher proportion of HPV-positive tests than the ASCUS-only group (p = 0.0027). There was no significant difference on followup PAPs and biopsies between the two groups (p = 0.4272).
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They showed an overall mean of 75% negative, 20% lowgrade lesions, and 5% high-grade lesions/carcinoma in situ on follow-up. Conclusion: Our study demonstrates no significant effect of infections on the cytological changes diagnostic of ASCUS. The cytologist should make this diagnosis neglecting any background infections even when predominant. Copyright © 2011 S. Karger AG, Basel
Papanicolaou (PAP) smears revolutionized the screening for cervical cancer, especially with the introduction of liquid-based preparations, automation, and imaging with the ability and simplicity of running molecular testing for high-risk human papilloma virus (HR-HPV) as a follow-up in inconclusive cases [1–3]. According to the Bethesda classification of cervical cytology [4], the diagnostic spectrum varies; on one end it is negative for intraepithelial lesion or malignancy and squamous cell carcinoma on the other end. The category of atypical squamous cells of undetermined significance (ASCUS) remains the least reproducible, yet a financial burden especially when estimating their numbers from the pool
Correspondence to: Dr. Haitham Nasser 515 Tobin Drive, Apt 312 Inkster, MI 48141 (USA) Tel. +1 248 898 0117, Fax +1 248 898 1257 E-Mail haitham.nasser @ beaumont.edu
of around 4.8 million PAPs evaluated yearly [5–7]. The cytological criteria for ASCUS, whether on conventional smears or liquid-based preparations, consist of 2.5–3 times enlarged nuclei, minimal hyperchromasia, slight increase in the nuclear-cytoplasmic ratio, and nuclear abnormalities associated with dense orangeophilic cytoplasm. These features fall short of squamous intraepithelial lesions, especially low-grade squamous intraepithelial lesion and HPV cytopathic effect. The rate of ASCUS should be limited to less than 5% in any accredited laboratory [5, 8]. There is unanimous agreement on the malignant predisposition of the HR-HPV in cervical cancer, especially strains HPV16 and 18 [9, 10]. However, there is controversy in the literature about the influence of other infectious agents on the development of cervical dysplasia and malignancy [11–15]. Few studies addressed the effect and potential pitfall attributed to infectious agents and reparative changes on the diagnosis of ASCUS [16, 17]. Although features attributed to infectious agents such as candida, trichomonas, bacterial vaginosis, and herpes virus are well established, they do not include the changes previously described with ASCUS. However, they may include reactive changes of squamous cells with nuclear enlargement and sometimes perinuclear clearing that may be confused with ASCUS or HPV effect. We aim in this study to evaluate the influence of concomitant infections on the ASCUS diagnosis and to observe their effect on the cytological changes in squamous cells. Subjects and Methods The cytological reports at the Pathology Department at William Beaumont are reviewed for all ASCUS cases between March 2007 and June 2009. We selected all cases with concomitant infectious agents and designated this group as ASCUS infection. The distribution of the cases is shown in table 1. A reference group of ASCUS cases without any infectious organisms which we designated ASCUS-only was also selected from the same time interval. All cases had follow-up studies including HPV testing, with an additional PAP, colposcopic cervical biopsy, or rarely cervical conization (independent of HPV result). PAP smears were prepared according to the ThinPrep automated method (ThinPrep 3000, Cytyc Corp., Hologic Inc., Marlborough, Mass., USA). HPV testing was performed using the polymerase chain reaction technique (Applied Biosystems, Foster City, Calif., USA). Statistical Analysis Categorical variables were summarized using frequencies and percentages and compared using Pearson’s 2 test, Fisher’s exact test or Cochran-Mantel-Haenszel statistics, wherever appropriate. p values !alpha of 0.05 (probability of type I error) were considered statistically significant. Statistical analysis was performed using the SAS System for Windows version 9.2.
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Table 1. Distribution of ASCUS-infection group Infectious organism
Number HPV– HPV+ Nega- LGLs HGLs of cases tive
Herpes virus 3 Trichomonas 9 Bacterial vaginosis 17 Candida 104 Total 133
3 5 9 56 73
0 4 8 48 60
2 9 12 73 96
1 0 4 26 31
0 0 1 5 6
Results
Three hundred and ten cases of ASCUS without infections were selected and designated the ASCUS-only control group. One hundred and thirty-three cases of ASCUS with infectious organisms and proper follow-up were identified and designated the ASCUS-infection group. HR-HPV testing and follow-up were available for all cases. HPV testing was positive in 94 (30%) cases of ASCUS only. Follow-up on the ASCUS-only cases showed 241 (78%) negative results, low-grade lesions (LGLs) including low-grade squamous intraepithelial lesions and cervical intraepithelial neoplasia 1 in 52 (17%) cases, and highgrade lesions (HGLs) including high-grade squamous intraepithelial lesions, cervical intraepithelial neoplasia 2–3 and squamous cell carcinoma in 17 (5%) cases. In the ASCUS-infection group, 133 cases fulfilled all inclusion criteria. HPV was positive in 60 (45%) cases. Ninety-six (72%) cases were negative on follow-up. LGLs and HGLs were seen in 31 (23%) and 6 (5%) cases, respectively. Statistical analysis demonstrated a statistically higher percentage of HR-HPV in the ASCUS-infection group compared to the ASCUS-only group (p = 0.0027). In other words, ASCUS-only patients with HPV-negative results were 0.5292 times as likely to have infection as those without infection. No significant difference was noted between the two groups when it came to the comparison of follow-up findings (p = 0.4272). Results and findings are shown in table 2. Due to the low numbers in some infectious categories statistical analysis showed weak individual comparative strength except for the candida subgroup. Larger numbers are required for higher statistical power. HR-HPV was present in 73% of LGLs and 82% of HGLs in the ASCUS-only group on follow-up. It was present in 84% of LGLs and 67% of HGLs in the ASCUSinfection group on follow-up. In general, there was an average of 77% HR-HPV among all LGLs and 78% HRHPV among all HGLs. These results are shown in table 3. Nasser /Hayek /Balasubramaniam / Kuntzman
Table 2. Comparison between both groups with statistical p values
Group
ASCUS-infection (n = 133) ASCUS-only (n = 310) Statistical p value
HPV negative
positive
negative
LGLs
HGLs
73 (55%) 216 (70%) 0.0027
60 (45%) 94 (30%)
96 (72%) 241 (78%)
31 (23%) 52 (17%) 0.4272
6 (5%) 17 (5%)
Discussion
Since the introduction of PAP smears as a screening tool for cervical cancer, it gained success and had a tremendous effect on the reduction of the cervical cancer rate, making it a rare cancer in developed countries. More recently, and with the introduction of more sophisticated methods such as liquid-based preparations, and molecular testing for HR-HPV, there was a great shift in the management of cervical dysplasia with excellent surveillance results. However, the ASCUS diagnosis remains a challenge to the cytologist and the clinician and not all clinicians have a similar approach to its management despite the clear proposed guidelines by the American Society for Colposcopy and Cervical Pathology (ASCCP) [18, 19]. These guidelines included either HR-HPV reflex testing, repeat PAP at 6 and 12 months, or colposcopy, with reflex HPV being the preferred approach. In the case of a positive HR-HPV it is recommended to go to colposcopy. Despite efforts to limit the numbers of ASCUS cases, their follow-up still carries an economical burden, not to forget the emotional stress for the patients. Methods to reduce this diagnostic category would definitely gain popularity. Hall and Kendall [20] detected a higher incidence of high-risk HPV in cases with ASCUS and concomitant candida, suggesting that features of ASCUS should not be entirely attributed to reactivity to the fungus especially in younger women. An older but a smaller study by Miguel et al. [16] proposed an opposite approach, attributing the ASCUS changes to the candida infection. Tirone et al [17], in their large study, attribute some of the ASCUS changes and diagnoses to bacterial vaginosis. However some studies considered some infectious organisms – other than HPV – to be agents of cervical dysplasia [11, 17]. In this study we aimed to evaluate the effect of infectious agents in PAP smears on the diagnosis of ASCUS. We tried to answer the question of whether we should downgrade the ASCUS diagnosis in the presence of conInfections Should Not Influence the ASCUS Diagnosis
Follow-up
Table 3. Frequencies of HR-HPV among the different follow-up categories in the different groups
LGLs HPV– ASCUS-infection ASCUS-only Total
HGLs HPV+
5 (16%) 26 (84%) 14 (27%) 38 (73%) 19 (23%) 64 (77%)
HPV–
HPV+
2 (33%) 4 (67%) 3 (18%) 14 (82%) 5 (22%) 18 (78%)
comitant infection, and whether the squamous cytological changes should be attributed to these infectious organisms. We gathered our ASCUS cases and compared the findings on HPV testing and follow-up studies between cases with infections and those without. Our ASCUS-infection group did not include equivalent numbers of different infectious agents due to a lack of concomitant HR-HPV and/or cervical biopsy follow-up. Another reason is the low prevalence of these infections in the study population. The prevalence of HR-HPV is reported in the literature to be 40–51% in ASCUS cases, with HGLs being present in up to 7% of ASCUS cases on follow-up [21–23]. In our study HR-HPV was present in 35% of all ASCUS cases (with and without infections). We also demonstrate a higher prevalence of HR-HPV among cases showing LGLs and HGLs on follow-up, a finding that confirms the oncogenic nature of this virus in the development of cervical cancer. More importantly, our study demonstrates no significant difference between ASCUS with and without infection, a finding that should make us more confident about the ASCUS diagnosis in an infectious background on the PAP smears. The presence of a significantly higher percentage of HR-HPV in the ASCUS-infection group was expected. This can be explained by the high possibility of concomitant sexual transmission of HR-HPV and other sexually transmitted diseases, some of which can be easily detected on PAP Acta Cytologica 2011;55:251–254
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smear. The reactive changes present in concomitance with ASCUS had no influence on the ASCUS diagnosis in such cases. The criteria for ASCUS were respected and great caution was taken so that these criteria do not overlap.
Until there is a larger and more powerful study where larger numbers of infectious organisms are collected and studied better individually, the cytological findings mimicking ASCUS changes seen in squamous cells on PAP smears from patients with infections should not be attributed to the infectious organisms.
References 1 Linder J, Zahniser D: The ThinPrep Pap test. A review of clinical studies. Acta Cytol 1997; 41:30–38. 2 Alves VA, Bibbo M, Schmitt FC, Milanezi F, Longatto Filho A: Comparison of manual and automated methods of liquid-based cytology. A morphologic study. Acta Cytol 2004;48:187–193. 3 Miller FS, Nagel LE, Kenny-Moynihan MB: Implementation of the ThinPrep imaging system in a high-volume metropolitan laboratory. Diagn Cytopathol 2007;35:213–217. 4 Solomon D, Davey D, Kurman R, Moriarty A, O’Conor D, Prey M, et al: The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287: 2114–2119. 5 Davey DD, Neal MH, Wilbur DC, Colgan TJ, Styer PE, Mody DR: Bethesda 2001 implementation and reporting rates: 2003 practices of participants in the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med 2004; 128:1224–1229. 6 Stoler MH, Schiffman M: Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASC-US-LSIL Triage Study. JAMA 2001;285:1500–1505. 7 Gatscha RM, Abadi M, Babore S, Chhieng D, Miller MJ, Saigo PE: Smears diagnosed as ASC-US: interobserver variation and followup. Diagn Cytopathol 2001;25:138–140. 8 Kurman J, Henson DE, Herbst AL, et al: The 1992 National Cancer Institute work-shop. Interim guidelines for management of abnormal cervical cytology. JAMA 1994; 271: 1866–1869.
254
9 Schiffman M, Herrero R, Desalle R, Hildesheim A, Wacholder S, Rodriguez AC, Bratti MC, Sherman ME, Morales J, Guillen D, Alfaro M, Hutchinson M, Wright TC, Solomon D, Chen Z, Schussler J, Castle PE, Burk RD: The carcinogenicity of human papillomavirus types reflects viral evolution. Virology 2005; 337:76–84. 10 Cogliano V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F: Carcinogenicity of human papillomaviruses. Lancet Oncol 2005;6:204. 11 Misra JS, Srivastava S, Singh U, Srivastava AN: Risk-factors and strategies for control of carcinoma cervix in India: hospital based cytological screening experience of 35 years. Indian J Cancer 2009;46:155–159. 12 Kwaśniewska A, Korobowicz E, Zdunek M, Skoczyński M, Kwaśniewski W, Daniłoś J, Goździcka-Józefiak A: Prevalence of Chlamydia trachomatis and herpes simplex virus 2 in cervical carcinoma associated with human papillomavirus detected in paraffinsectioned samples. Eur J Gynaecol Oncol 2009;30:65–70. 13 Evans MF, Adamson CS, Schned LM, St John TL, Leiman G, Ashikaga T, Cooper K: HPV is detectable in virtually all abnormal cervical cytology samples after reinvestigation of HPV negatives with multiple alternative PCR tests. Diagn Mol Pathol 2010; 19: 144– 150. 14 Moody CA, Laimins LA: Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer 2010;10:550–560. 15 Engberts MK, Verbruggen BS, Boon ME, van Haaften M, Heintz AP: Candida and dysbacteriosis: a cytologic, population-based study of 100,605 asymptomatic women concerning cervical carcinogenesis. Cancer 2007; 111: 269–274. 16 Miguel NL Jr, Lachowicz CM, Kline TS: Candida-related changes and ASCUS: a potential trap! Diagn Cytopathol 1997;16:83–86.
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17 Tirone NR, Souza CE, Michelin MA, Murta EF: Frequency of infectious agents for vaginitis in patients with a cytological diagnosis of atypical squamous cells of undetermined significance. Eur J Gynaecol Oncol 2008;29: 144–147. 18 Wright TC Jr, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D: 2006 Consensus Guidelines for the management of women with abnormal cervical screening tests. J Low Genit Tract Dis 2007;11:201–222. 19 Wright TC Jr, Cox JT, Massad LS, Twiggs LB, Wilkinson EJ: 2001 Consensus Guidelines for the management of women with cervical cytological abnormalities. JAMA 2002; 287: 2120–2129. 20 Hall J, Kendall B: High risk human papillomavirus DNA detection in pap tests with both atypical squamous cells of undetermined significance and Candida. Acta Cytol 2009;53:150–152. 21 ASC-US-LSIL Traige Study (ALTS) Group: Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003;188:1383– 1392. 22 Manos MM, Kinney WK, Hurley LB, Sherman ME, Shieh-Ngai J, Kurman RJ, et al: Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou results. JAMA 1999;281:1605–1610. 23 Lonky NM, Felix JC, Naidu YM, Wolde-Tsadik G: Triage of atypical squamous cells of undetermined significance with hybrid capture. II. Colposcopy and histologic human papillomavirus correlation. Obstet Gynecol 2003;101:481–489.
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