Follow up of Atypical Squamous Cell Pap Smears in Iraqi Women

Atypical squamous cell (ASC) is a name given, in a Pap smear or cervicovaginal cytology, to squamous cells that do not have a normal appearance but are not clearly precancerous. Being equivocal, the term ASC is categorized by The Bethesda System (TBS) 2001 into atypical squamous cells of undetermined significance (ASC-US) where the cytologic changes are suggestive of but, quantitatively or qualitatively, are insufficient for a definitive interpretation of squamous intraepithelial lesion (SIL) and into atypical squamous cells-cannot exclude high squamous intraepithelial lesion (ASC-H) where the atypical squamous cells exhibit some equivocal features suggestive of but not sufficient to call high squamous intraepithelial lesions (HSIL) (NCI Bethesda System, 2001; Selvaggi, 2003). In addition to the possible progression of ASC-US and ASC-H into HSIL or invasive malignancy, the link between these lesions and high risk-human papillomavirus (HR-HPV) creates


Introduction
Atypical squamous cell (ASC) is a name given, in a Pap smear or cervicovaginal cytology, to squamous cells that do not have a normal appearance but are not clearly precancerous.Being equivocal, the term ASC is categorized by The Bethesda System (TBS) 2001 into atypical squamous cells of undetermined significance (ASC-US) where the cytologic changes are suggestive of but, quantitatively or qualitatively, are insufficient for a definitive interpretation of squamous intraepithelial lesion (SIL) and into atypical squamous cells-cannot exclude high squamous intraepithelial lesion (ASC-H) where the atypical squamous cells exhibit some equivocal features suggestive of but not sufficient to call high squamous intraepithelial lesions (HSIL) (NCI Bethesda System, 2001;Selvaggi, 2003).In addition to the possible progression of ASC-US and ASC-H into HSIL or invasive malignancy, the link between these lesions and high risk-human papillomavirus (HR-HPV) creates

Follow up of Atypical Squamous Cell Pap Smears in Iraqi Women
Intisar S Pity*, Maida Y Shamdeen, Shawnim A Wais a great incentive to investigate the clinically significant association with these two cytologic entities (Solomon et al., 2001;Chivukula and Shidham, 2006;Bandyopadhyay et al., 2008).To clarify this paradox we propose initiated this study to monitor the cytological and histological results of women with ASC-US and ASC-H Pap smears for a 7 year period to determine the possible association of these abnormal Pap smears with HR-HPV and HSIL in women from Duhok Governorate, Iraq.The prevalence of ASC-US and ASC-H was also evaluated.To the best of our knowledge, this is the first prospective clinical study in Iraq.

Materials and Methods
This is a seven year prospective, observational and descriptive study conducted at Azadi Teaching Hospitalgynecologic clinic and Central laboratory, Duhok, Iraq, during a period extending from January 2005 to December 2011.Ethical approval to conduct the study was obtained from the Faculty of Medicine Research Ethics Committee, and patient consent was obtained before enrolling participants in the study.Cervical smears were taken from 10,338 women with wooden Ayre's spatula and directly wet fixed by 95% ethanol.Pap smear results were reported according to the 2001 Bethesda System (NCI Bethesda System., 2001;Solomon et al., 2001).A slide showing ASC-US or ASC-H was initially reevaluated and further scrutinized by a cytopathologist for final report.When the diagnosis of atypical squamous cells was confirmed upon review, an attempt was made to keep patients under follow up for at least 36 months.Follow up included a repeat Pap smear twice at 4 month interval.Cohorts with persistent abnormal Pap smears were referred for colposcopic examination using aqueous solution of 5% glacial acetic acid and lugols' solution.When changes were observed, patients subsequently underwent a direct punch biopsy using Gaylor-Medina forceps.Other histological samplings included cone biopsy, endocervical curettage and total hysterectomy.No loop electrosurgical excision procedure (LEEP) was available at that time of the study.The biopsy specimens were formalin fixed, paraffin embedded and stained with Hematoxylin and Eosin stains (H&E).Those, in whom histological material was not obtained, were followed up by Pap smear with or without colposcopy.
Cytologic and histologic results were classified and reported as follows: NILM (negative for intraepithelial lesion or malignancy) for negative, reactive or inflammatory lesions; ASC-US for equivocal cases where the cytologic changes are suggestive of but inadequate for a definitive interpretation of SIL; ASC-H where the atypical squamous cells exhibit some equivocal features suggestive of but not sufficient to call HSIL; low squamous intraepithelial lesion (LSIL) for koilocytotic atypia and cervical intraepithelial neoplasia type-1 (CIN I); HSIL for cervical intraepithelial neoplasia type-2 (CIN II) and type-3 (CIN III); and malignancy when definite invasion was confirmed.
Continuous follow up Pap smear at 6 month interval was then recommended for all participants with abnormal cervical cytology even those who underwent total hysterectomy.The mean number of subsequent cervical smearing was 6.1 (range, 4-21) and the average follow-up duration was 41.2 months (Range: 36 to 84).The least favorable histologic or cytologic result on follow up, after being confirmed, was considered as the final outcome.High-risk human papillomavirus DNA testing was performed on exfoliated cervical cells from 106 women after at least 36 months from the initial Pap test.Crude extracts of the cervical scrapes were used, as described previously (de Roda Husman et al, 1995).High-risk HPV DNA analysis protocol was performed by the conventional PCR-based assays using the HPV high-risk screen PCR-mix kit provided by Sacace Biotechnology (Sacace Company, Italy, and Catalog No. V-26-50R).The kit contained primers directed against 13 HR-HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68).PCR technique and interpretation were carried out according to recommendations supplied by the manufacturer's (Sacace Company, Italy, and Catalog No. V-26-50R) and as described previously (de Roda Husman et al., 1995;Jacobs et al., 1997;van den Brule, 2002).Negative control (DNA buffer) and positive HPV (type 31, 39 and 56) controls were run with the technique.Participants were scheduled for a return visit to obtain test results 2 weeks after testing.Because there was no current standard protocol in the clinic for positive HR-HPV DNA results, women were encouraged to continue cervical cytology screening or follow-up visits as recommended (Solomon et al., 2001).Interpretation of cytology, histology and HR-HPV testing was performed blinded to the other test results.The results were then analyzed by comparing cytological and histological criteria and HPV profile.
Statistical analysis: Significant associations between categorical variables were performed using chi-squared test and p< 0.05 was considered to be significant.Descriptive statistics were used to summarize demographic variables.
Two hundred and fourteen women were excluded from the study as there was no follow up (n=117), ASC-US/ASC-H diagnosis was not confirmed on further review (n=81) or when patients had clinically significant diagnoses defined as HSIL, LSIL, cervical adenocarcinoma in situ or invasive cervical carcinoma prior to the ASC Pap smear (n=16).The remaining 689 cases (596 ASC-US and 93 ASC-H) for whom follow up was possible, were recruited in the study.Patients' ages ranged from 18 to 79 years (median 48.5 years).
Table 1 demonstrates consensus cytology and histology    2 and Table 2. Significant percentages of both ASC-US (87.9%) and ASC-H (62.4%) groups showed no significant lesion on subsequent follow up, reported as NILM (p<0.001).On the other hand, 5.2% revealed LSIL and above.Significantly higher HSIL rate and higher HSIL: LSIL ratio were observed among ASC-H than ASC-US group (P<0.001).The single squamous cell carcinoma case was observed among ASC-H women.The remaining 9.6% of ASC-US cases showed persistent equivocal changes (ASC) versus 15.1% of ASC-H group.The difference was statistically not significance (p= 0.105).
High-risk human papillomavirus DNA testing was performed on 106 women, 65 (10.9%)ASC-US and 41 (44.1%)ASC-H.These cases comprised all patients showing SIL and more severe epithelial abnormalities (n=36) and randomly selected cases from NILM results (n=27) and from those showing persistent equivocal cytologic changes (n=43).Table 3 compares the HR-HPV DNA status with the follow up findings.Overall, 20 (18.9%) patients were positive for HR-HPV DNA.These included 10.8% of ASC-US and 31.7% of ASC-H subjects.No significant viral DNA difference was noted between the two groups (P > 0.05).Patients with follow-up proven HSIL of both ASC-US and ASC-H had positive HR-HPV in 60% while LSIL cases showed positive HR-HPV in 33.3% cases (P > 0.05).The single carcinoma case was positive for HR-HPV.On the other hand, persistent ASC revealed positive HR-HPV in 4.7% of cases.None of NILM cases showed evidence of HR-HPV.

Discussion
During the seven year study-period, ASC-US represented 7.8% of total Pap tests done while ASC-H accounted 0.9% of cases.These findings fall within the rate ranges reported in different studies (Table 4).The wide ranges of ASC-US (1.2-39%) and, to a lesser extent, ASC-H (0.06-2%) may be contributed to the Bethesda system applied for categorization of ASC into ASC-US and ASC-H and further subcategorization of ASC-US into reactive and neoplastic counterparts (NCI Bethesda System, 2001;Solomon et al., 2001Selvaggi, 2003;Barcelos et al., 2011).In addition, the heterogeneous morphology and lack of well-defined criteria for both ASC-US and ASC-H reflected in their poor interobserver agreement between laboratories or even the variable experience of the microscopists may account for these differences (Saad et al., 2006;Chivukula and Shidham;CytrynI et al., 2009;Tewari and Chaudhary., 2010;Barcelos et al., 2011).The rate of Pap smears could be also influenced by the type of Pap test applied, the age group of the studied patients as well as the population sampled (Chivukula and Shidham, 2006;Saad et al., 2006;Sherman et al., 2006;Bandyopadhyay et al., 2008;Tewari and Chaudhary, 2010;Zhao et al., 2010).
On subsequent follow up, our clinic experienced low overall rates of HSIL among both ASC-US (0.8%) and ASC-H (16.1%) women when compared with other Pap reports (Table 5).It may be argued that our test comparisons were based, not on a single criterion but on many subjective criteria.As well, we did not instruct the physicians to use liquid based cervical brush in addition to the Ayres spatula every time they obtained the repeat Pap smear, liquid based cytology was not available within the Iraqi public health sector at time of the study.Furthermore, it is of some concern that 136 patients (18% of ASC-US women and 1.7% of ASC-H) had only cytologic sampling without histologic examination, a fact which may reflect a lack of knowledge about the appropriate follow-up of these equivocal Pap tests.However, modest decreases in rates of HSIL biopsy findings among ASC-H are also reported by other investigators (Alli and Ali, 2003;Bansal et al., 2012) suggesting that the diagnostic thresholds and disease  prevalence can be variable, or at least some portions of the rare atypical cells are not really premalignant cells.Saad et al who tracked patients with ASC-H for 18 months, reported higher HSIL rate among perimenopausal women (22%) but much lower rate among postmenopausals (6%) (Saad et al., 2006) a finding that has been further documented by others (Chivukula and Shidham, 2006;Sherman et al., 2006;Bandyopadhyay et al., 2008;Zhao et al., 2010).Nevertheless, in the present study we experienced a significantly higher LSIL and HSIL rates with higher HSIL: LSIL ratio among ASC-H group than ASC-US.This finding is comparable to other reports (Chivukula and Shidham, 2006;Duncan andJacob, 2005 Bandyopadhyay et al., 2008;Barcelos et al., 2011).
There is no consensus on the optimal management of women with an ASC-US and ASC-H.It has been anticipated that although the vast majority of these lesions often regress spontaneously and do not require immediate colposcopic examination or aggressive management, considerable percentages might harbor an underlying HSIL, a finding also observed in the current study.These HSIL cases require immediate management (Solomon et al., 2001;Chivukula and Shidham, 2006;Bandyopadhyay et al., 2008;Tewari and Chaudhary, 2010;Barcelos et al., 2011).It is also of clinical interest that in our series, one patients of ASC-H group had a histologic diagnosis of cervical cancer.This emphasizes the potential significance of ASC-H Pap smear owing to its association with an appreciable risk of clinically significant disease, and patients should undergo timely colposcopic and histologic assessment to rule out HSIL, and cervical cancer (Srodone et al., 2005;Barcelos et al., 2011).The ALTS study (ASCUS/LSIL Triage Study), a large randomized trial designed to evaluate different methods for managing patients with cytological findings of low grade squamous lesions, concluded that two repeat cytologic examinations performed at six-month intervals, testing for human papillomavirus (HPV) and a single colposcopic examination are all safe and effective approaches to managing women with ASC-US (ASCUS-LSIL Traige Study (ALTS) Group, 2003;Solomon et al, 2001).The ASCCP (American Society for Colposcopy and Cervical Pathology) recommends that any of the three steps could be taken (Solomon et al., 2001;Sherman et al, 2001).The interim guidelines published by a study done in Iran by Ghaemmaghami et al. and by the National Cancer Institute suggest that patients should be referred for colposcopy after persistent ASC-US diagnosis within 2 years (Ghaemmaghami et al., 2005;NCI Bethesda System, 2001).However, referring all women with minor cytologic lesions for colposcopic exploration and further evaluation means an increase in overdiagnosis and overtreatment, and this may result in substantial costs for the health care system (Solomon et al., 2001;Chivukula and Shidham, 2006;Bandyopadhyay et al., 2008;Tewari and Chaudhary, 2010;Barcelos et al., 2011).Saad et al. (2006), Srodon et al. (2005) and Sherman et al. (2006) suggested that HPV testing may be helpful for selecting patients with ASC-H who should undergo colposcopic examination.The presence of higher rates of CIN II/III among HPV-positive ASC-US in a study done by Barcelos et al. (2011) in Brazil and among HPV-positive ASC-H group in another study done in US by Srodon et al.' (2005) study suggest that HPV positivity may be associated with an increased probability of CIN among both groups.These studies along with the ASCCP recommend that HR-HPV DNA triage is pertinent to cervical Pap tests for atypical squamous cells (Solomon et al., 2001;NCI Bethesda System;Sherman et al., 2001;Srodone et al., 2005;Sherman et al., 2006;Barcelos et al., 2011).In the present study, we demonstrated positive HR-HPV DNA in 10.8% of ASC-US and 31.7% of ASC-H cases.Variations in HR-HPV DNA association with both ASC-US and ASC-H women have been reported (Table 6).These variations may reflect the significant differences in cytologic interpretation thresholds for ASC-US and ASC-H interpretations (Chivukula and Shidham, 2006;Saad et al., 2006;Bandyopadhyay et al., 2008).HR-HPV rate variation is further contributed to the impact of employing different techniques for HPV DNA identification (de Roda Husman et al., 1995;Jacobs et al., 1997;Sherman et al., 2001;van den Brule et al., 2002;Saad et al., 2006;Bandyopadhyay et al., 2008;Zhao et al., 2010).The use of different ranges of high and low risk HPV DNA tested (de Roda Husman et al., 1995;Jacobs et al., 1997;Srodone et al., 2005;Bandyopadhyay et al., 2008) as well as the Pap smearing applied (Louro et al., 2003;Bandyopadhyay et al., 2008).Another plausible explanation for reported variations might be that different HR-HPV DNA detection rates reflect different demographic population profiles with different HPV prevalence influenced in part by HR-HPV infection rates (Solomon et al., 2001;Sherman et al., 2006;Bandyopadhyay et al., 2008).The information obtained in this study provided an analysis of only 65 (10.9%) of ASC-US and 41 (44.1%) of ASC-H.When the cytologies of ASC-US and ASC-H were compared, the presence of high-risk HPV DNA seemed to be greater among ASC-H group (31.7%) versus 10.8% of ASC-US cases and ASC-H associated SIL lesions were observed to have higher prevalence HR-HPV DNA than their counterpart ASC-USassociated lesions.Although the differences didn't reach the level of significance (p > 0.05), subcategorization of ASC into ASC-US and ASC-H seems to be of a great value for detection of clinically significant diseases.
In conclusions, Since both ASC-US and ASC-H identify patients who are at an increased risk for the development of high squamous intraepithelial lesions and a considerable percentage harbor high-risk HPV, both should be retained as diagnostic categories and patients warrant a diligent follow up, and testing for High-risk HPV-DNA, colposcopic evaluation and biopsy when indicated are a must.

Figure 1 .Figure 2 .
Figure 1.Percentage of Women with Atypical Squamous Cells (ASC) Out of Total Pap Tests.NILM: Negative for intraepithelial lesion or malignancy, Neoplasia included premalignant and malignant lesions, ASC: Atypical squamous cells

Table 2 . Detailed Final Diagnoses after Follow up of ASC-US and ASC-H Cases
ASC-US, Atypical squamous cells of undetermined significance; ASC-H, Atypical squamous cells cannot exclude HSIL; NILM, Negative for intraepithelial lesion or malignancy; LSIL, Low-grade squamous intraepithelial lesion; HSIL, High-grade squamous intraepithelial lesion; *Chi-square used; **Persistent equivocal