The 2001 Bethesda System for gynecologic cervical cytology reporting classifies squamous intraepithelial lesions into low-grade (LSIL) and high-grade (HSIL) lesions. An intermediate term, “low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H),” has been used in a small percentage of LSIL cases. To the authors' knowledge, little is known regarding the human papillomavirus (HPV) status in patients with LSIL-H.
A total of 808 SurePath specimens obtained between December 2009 and April 2011 were tested for 40 HPV genotypes using DNA microarray, followed by a confirmatory DNA sequencing assay.
The infection rate for high-risk HPV in women with LSIL-H (92%) was strikingly close to that for women with HSIL (91%), which was higher than that for those with LSIL (74%); atypical squamous cells, cannot rule out high-grade lesion (ASC-H) (78%); or LSIL and ASC-H combined (74%). HPV type 16, the most common carcinogenic HPV genotype, was detected in 36% of women with LSIL-H, which was significantly higher than that in women with LSIL and ASC-H combined (13.8%), but less than that in women with HSIL (44.6%). Patients with LSIL-H and HSIL had similar infection rates for low-risk/intermediate-risk HPV genotypes, which were lower than those in LSIL or LSIL and ASC-H combined.
The Bethesda System (TBS 2001) for gynecologic cervical cytology reporting classifies squamous intraepithelial lesions (SILs) into low-grade (LSIL) and high-grade (HSIL) categories.1 The classifications reflect the biological differences between these lesions and have a significant impact on patient management. According to the 2006 American Society for Colposcopy and Cervical Pathology (ASCCP) consensus guidelines, patients with histologically confirmed HSIL are treated with a loop electrocautery excision procedure or cervical conization, whereas those with biopsy-confirmed LSIL are closely monitored with an additional Papanicolaou (Pap) test and/or biopsy.2
Although an unequivocal diagnosis of LSIL can be made for the majority of such cases, there is a small percentage of outliers with rare atypical cells that are insufficient qualitatively or quantitatively to diagnose as HSIL. An intermediate term, “low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion (LSIL-H),” has been used in clinical practice for these cases. Currently, LSIL-H is not a diagnostic category in TBS 2001.1 Furthermore, to our knowledge, no management guideline has been defined for patients with a cytologic interpretation of LSIL-H.2
It has recently been debated whether LSIL-H represents an independent subgroup of lesions with distinct biologic and clinical behavior that deserve a unique treatment strategy. More than a decade ago, Walsh et al reported 50 women with an LSIL-H interpretation and found that 38% of these patients had high-grade cervical intraepithelial lesions (CIN2+) on follow-up biopsy compared with 3.5% of patients with unequivocal LSIL.3 Many subsequent studies reported similar findings and indicated that patients with LSIL-H appeared to have an intermediate risk of harboring or developing high-grade squamous lesions.4-24 Although LSIL-H has been commonly used in practice to indicate an increased risk of high-grade squamous lesions, the diagnostic criteria of LSIL-H are not well defined and little is known about the human papillomavirus (HPV) status of these patients. Therefore, we analyzed the frequency and distribution of 40 HPV genotypes among the cytology categories including LSIL-H.
MATERIALS AND METHODS
Study Population and Cytology Classification
The study was conducted at The Methodist Hospital in Houston, Texas and was approved by the Institutional Review Board of The Methodist Hospital Research Institute (IRB1210-0221). The study included 808 women who were referred to the study institution from December 2009 through April 2011 for further evaluation of abnormal Pap tests. The average age of the patients was 36.5 years (range, 19 years-85 years). All patients underwent liquid-based Pap tests (SurePath; BD, Franklin Lakes, NJ) that were interpreted according to the criteria set by TBS 2001 for cervicovaginal cytology reporting,1 with minimal modification to include LSIL-H. The cytologic interpretations of the 808 cases included negative for intraepithelial lesion and malignancy (497 cases); atypical squamous cells of undetermined significance (ASCUS) (48 cases); atypical squamous cells, cannot rule out high-grade lesion (ASC-H) (9 cases); atypical glandular cells (AGC) (2 cases); LSIL (167 cases); LSIL-H (25 cases); HSIL (56 cases); adenocarcinoma (1 case); and unsatisfactory (3 cases). The diagnosis of LSIL-H was defined as the presence of ASC-H cells in a background of unequivocal LSIL (Fig. 1).4-8, 25, 26
HPV Genotyping by DNA Microarray
HPV DNA was extracted from the residual SurePath specimens and amplified with polymerase chain reaction in the L1 region of the HPV genome with human beta actin as the reference gene. The amplified HPV L1 gene and human beta actin gene were labeled with cyanine 5 (Cy5) and hybridized with a HPV40 DNA chip (GG HPV DNA Genotyping Chip Kit; GoodGene Inc, Seoul, Korea), and the signal was observed using a GenePix 4000B Microarray Scanner (Molecular Devices, LLC, Sunnyvale, Calif). The detection limit of the HPV40 DNA microarray chip is between 10 to 100 copies of HPV DNA per sample. DNA microarray provided high sensitivity for HPV subtypes in the specimens and identified cases infected with multiple HPV subtypes (Fig. 2).
HPV Genotyping by Confirmatory DNA Sequencing
The HPV DNA microarray data were confirmed further by conventional direct DNA sequencing methods in all samples. The sequence data obtained by automated DNA sequencing were analyzed by the Basic Local Alignment Search Tool (BLAST) search (http://www.ncbi.nlm.nih. gov/BLAST/) for HPV genotypes. DNA sequencing identified the most dominant genotype in a given specimen, and served as a confirmatory assay.
Categorization of HPV Genotypes and Data Analysis
The 2009 recommendations from the expert working group at the International Agency for Research on Cancer (IARC) categorized HPV into 4 groups: carcinogenic (group 1), probably carcinogenic (group 2A), possibly carcinogenic (group 2B), and not classifiable (group 3).27 The HPV genotypes in IARC groups 1 and 2A are commonly referred to as high-risk HPV (HR-HPV) and are included in the Digene Hybrid Capture 2 test (Qiagen, Valencia, Calif). These genotypes include HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68A/68B. In the current study, HPV genotypes in IARC groups 2B and 3 will be referred to as intermediate-risk HPV (IR-HPV) and low-risk HPV (LR-HPV), respectively. The chi-square test was used in data analysis to determine the significance of differences in HPV infection rates among the diagnostic categories.
Among the 808 cases, there were 308 (38.1%) with abnormal cytologic diagnoses including ASCUS (48 cases; 5.9%), ASC-H (9 cases; 1.1%), AGC (2 cases; 0.25%), LSIL (167 cases; 20.7%), LSIL-H (25 cases; 3.1%), HSIL (56 cases; 6.9%), and adenocarcinoma (1 case; 0.12%). The LSIL-H cases corresponded to 3.1% of women in the cohort and 13% of all LSIL cases (LSIL and LSIL-H; 192 cases).
HPV genotyping and sequencing data were obtained from all 808 samples. The frequency of HPV infection was extremely high in this cohort of women, with 93% being infected and 62.2% of women having HR-HPV genotypes. Infection with multiple HPV genotypes was detected in 38% of the women, with up to 16 different HPV genotypes noted within a single specimen.
Patients with LSIL-H and HSIL were found to have similar infection rates for LR- and IR-HPV genotypes (8% and 7%, respectively), which were lower than those in patients with LSIL or LSIL and ASC-H combined (25% and 24%, respectively) (Fig. 3A). However, patients diagnosed as LSIL-H had a much higher frequency of HR-HPV infection (92%) than those with ASC-H (78%), LSIL (74%), or LSIL and ASC-H combined (74%). The infection rate of HR-HPV in patients with LSIL-H was strikingly close to that of HSIL (91%) (Fig. 3B).
HPV-16, the most common carcinogenic HPV genotype worldwide, was detected in 36% of patients with LSIL-H, which was significantly higher than in those with LSIL and ASC-H combined (14%; P = .007), but was slightly less than in those with HSIL (44.6%; P = .6) (Fig. 3C). The HPV distribution patterns did not differ significantly between younger (aged < 30 years) and older (aged ≥ 30 years) age groups (data not shown).
The carcinogenic role of HPV, especially with regard to the high-risk genotypes, in the development of cervical dysplasia and malignancy has been well established, and the cervical cancer screening program has been the most successful model of cancer prevention in modern history.28 In the past decade, TBS 2001 for gynecologic cervical cytology reporting and the 2006 ASCCP consensus guidelines have had a significant impact on patient care by providing cytologic classification of cervical lesions and corresponding management guidelines.1, 2 Despite the success of TBS 2001 and improved communication between gynecologists and pathologists, diagnostic dilemmas and confusion still remain because some lesions do not fit into the current TBS. In practice, LSIL-H is a term used by many pathologists to report lesions that have rare ASC-H cells within a background of unequivocal LSIL, a pattern not included in the current TBS.
When TBS 2001 was published, there were insufficient data to clarify whether LSIL-H was a distinct cytologic category or simply a combination of LSIL and ASC-H. Over the past decade, accumulating data have suggested that the category of LSIL-H corresponds to a small group of women with intermediate risk, between that of LSIL and HSIL, of harboring or developing high-grade cervical lesions. Therefore, a distinct diagnostic category of LSIL-H was recommended.4-24 A few studies, with small sample sizes, suggested that women with LSIL-H had a high prevalence of HR-HPV infection as determined by the Digene Hybrid Capture 2 test.6, 17 However, a large-scale study on the distribution of HPV genotypes among cytology categories, including LSIL-H, has not been performed to date.
To the best of our knowledge, the current study is the first comprehensive study of the frequency and distribution of HPV genotypes in a large cohort of women with simultaneous cytologic evaluation. The data enable us to analyze the entire spectrum of HPV infection across all cytology categories, including LSIL-H. The results of the current study demonstrate that patients with an interpretation of LSIL-H on cytology have an HPV infection pattern similar to that of women with HSIL with regard to the frequency and composition of HPV genotypes. Specifically, the patients with LSIL-H and HSIL were found to have a much higher infection rate for HR-HPV genotypes than those with LSIL, ASC-H, or LSIL and ASC-H combined. In addition, the patients with LSIL-H and HSIL also were found to share similar low infection rates for LR/IR-risk HPV genotypes (8% and 7%, respectively), which were much lower than those for women with LSIL (25%) or LSIL and ASC-H combined (24%). When we narrowed the focus of the current study to individual HPV genotypes, the most common and potent carcinogenic genotype, HPV-16, was detected in 36% of patients with LSIL-H, which was lower than the rate of 45% in patients with HSIL but was significantly higher than that in those with LSIL and ASC-H combined (14%; P = .007). The HPV distribution pattern clearly separates the patients with LSIL-H from those with LSIL or ASC-H. The data suggest that LSIL-H does not appear to be simply a combination of LSIL and ASC-H, but rather a small group of patients with an HPV infection pattern that is clearly deviated from LSIL or ASC-H and likely to carry a higher risk of developing high-grade cervical lesions compared with LSIL or ASC-H alone.
Since LSIL-H was adopted by many as a practical category of diagnosis over the past decade, data have accumulated regarding the follow-up of patients with LSIL-H.4-24 The detection rate for cervical intraepithelial neoplasia 2 or worse (CIN2+) lesions on follow-up biopsy among cytology categories is summarized from published large-scale studies in Table 1.6, 7, 15, 17, 21, 24 The combined data indicate that CIN2+ lesions were detected in 36% of patients with LSIL-H on follow-up biopsy, which was significantly higher than for LSIL (15%; P = .001) and significantly lower than noted in HSIL (71%; P = .001). The follow-up histology data, along with the results of the current study on HPV genotype distribution, support the notion that the LSIL-H category may represent a subset of patients who carry a relatively high risk of high-grade cervical lesions, which necessitates a vigilant surveillance and treatment strategy.
Table 1. Literature Review: Positive Predictive Value of the Cytological Diagnostic Categories for Detecting CIN2 or Worse (CIN2+) Lesions in Follow-Up Biopsy
Although LSIL-H has been used more frequently by pathologists in recent years, the terminology is not included in TBS 2001 and the corresponding guidelines for the management of these patients have not been established. Lack of recognition of LSIL-H as an independent category has resulted in tremendous confusion for gynecologists regarding clinical management. Patients are treated variably for LSIL or ASC-H according to current guidelines. Therefore, the follow-up for patients with LSIL-H is based mainly on the clinical judgment of the treating gynecologist.2, 29, 30 Recent studies have shown a wide range of rates of follow-up biopsy (30%-70%) in patients with LSIL-H,6, 7, 15, 17, 21, 24 although no data currently are available with which to evaluate the adequacy of those management strategies.
Based on the striking similarities with regard to the HPV infection pattern and high infection rates of HR-HPV between LSIL-H and HSIL, the results of the current study strongly suggest that women with LSIL-H are a group of high-risk patients at an increased risk of harboring or developing CIN2+ lesions, comparable to those with HSIL. Therefore, the current guidelines for managing patients with LSIL or ASC-H may not be sufficient for patients with LSIL-H, and a management strategy similar to that for patients with HSIL may be warranted.
The data from the current study should be interpreted with caution for several reasons. There was a significantly higher rate of LSIL-H cases in this study cohort (3.1%) than the rates of 0.15% to 0.35% reported in the literature.4, 6, 7, 9, 21, 24 This was most likely because of the cohort not being a general screening population but rather a cohort of referred patients with a history of abnormal Pap tests. For the same reason, our patients had a very high rate of HPV infection (93% for any HPV genotype and 62% for HR-HPV) compared with that observed in general screening populations. Even with the highly concentrated high-risk population, the absolute number of LSIL-H cases was still relatively small compared with other cytology categories. Because of the relatively small group of LSIL-H cases, there was insufficient power to demonstrate the statistical significance of overall HR-HPV infection rates among the categories, although trends were observed. However, the study did demonstrate that the rate of HPV-16 infection was significantly higher in patients with LSIL-H than those with LSIL, ASC-H, or a combination of the 2 (P = .007). Further studies with larger patient cohorts may provide valuable information that could have a significant impact on the management of these patients. In addition, the current study was unable to address the correlation of HPV infection with the results of follow-up biopsy because the patients were inconsistently treated and followed with various strategies because of a lack of consensus guidelines concerning management. However, accumulated data have demonstrated that patients with LSIL-H have a higher rate of high-grade lesions on follow-up biopsy than patients with LSIL, but less than that in patients with HSIL (Table 1). The current study provides valuable information regarding the distribution pattern and infection rate of HPV genotypes in patients with LSIL-H that, in our view, is consistent with the histologic follow-up data in the literature.6, 7, 15, 17, 21, 24
In conclusion, the results of the current study, along with published histologic follow-up data, suggest that LSIL-H has an intermediate risk status between LSIL and/or ASC-H and HSIL, leaning more toward HSIL. Recognizing LSIL-H as a distinct diagnostic category in TBS would facilitate the establishment of diagnostic criteria, the standardization of management guidelines, and better coordination between pathologists and gynecologists for the appropriate management of these patients.
We thank Woo-Chul Moon, MD, PhD; Myung-Ryul Oh, PhD; and Jin-Kyung Lee, PhD for sponsoring the human papillomavirus genotyping tests and Mr. Christopher Nelsen, Ms. Mun Jung Han, and Ms. Hyejung Park for their excellent technical assistance. We also thank Steven Shen, MD, PhD, for assistance with the statistical analysis and Mr. Philip Randall for scientific editing of the article.
No specific funding was disclosed.
CONFLICT OF INTEREST DISCLOSURES
The authors have no conflict of interest to disclose.