Int. J. Med. Sci. 2014, Vol. 11

Ivyspring International Publisher

80

International Journal of Medical Sciences

Research Paper

2014; 11(1):80-86. doi: 10.7150/ijms.5585

Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology Sung-Jong Lee1, Ah-Won Lee2, Chang-Suk Kang2, Jong-Sup Park3, Dong-Choon Park1, Eun-Young Ki3, Keun-Ho Lee3, Joo-Hee Yoon1, Soo-Young Hur3, Tae-Jung Kim2 1. 2. 3.

Department of Obstetrics and Gynecology, St. Vincent’s Hospital, College of Medicine, The Catholic University of Korea, Suwon, Republic of Korea Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Department of Obstetrics and Gynecology, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea

 Corresponding author: Tae-Jung Kim, Department of Hospital Pathology, Yeouido St. Mary’s Hospital, 62 Yeouido-dong Yongdeungpo-gu, Seoul, Republic of Korea (Postal code: 150-010). Phone: +82-2-3779-2157; Fax: +82-2-783-6648; Email: [email protected] © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

Received: 2012.11.22; Accepted: 2013.12.11; Published: 2013.12.20

Abstract Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women. Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens. Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004) Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is strongly associated with high-grade histopathology diagnoses. The multiplicity of HPV infections may have an important role in high-grade histopathology diagnoses (≥CIN3) in HPV L1-positive cases. Key words: cervical cancer, cervical intraepithelial neoplasia, human papillomavirus, immunocytochemistry, cervical cytology

http://www.medsci.org

Int. J. Med. Sci. 2014, Vol. 11

Introduction Cervical cancer is the second most common type of cancer in women worldwide [1] and the fourth most common female malignancy in Korea [2, 3]. Human papillomavirus (HPV) infection is a well-known prerequisite for the development of cervical cancer. Moreover, the integration of high-risk HPV is a key step in cervical neoplastic progression [4]. HPV type 16 (HPV16) is regarded as the most oncogenic in cervical carcinogenesis and is detectable in more than 50% of cervical cancers [5, 6]. HPV testing in addition to a Papanicolaou smear has helped reduce the development of grade 2 and 3 cervical intraepithelial neoplasia (CIN) and cancer [7]. Furthermore, the epidemiological and biological effects of multiple-type HPV infections in the presence of high-risk HPV infections have been documented with the increasing administration of HPV vaccines [8]. HPV L1 capsid protein comprises 90% of HPV viral surface proteins and is typically expressed during the late phase of viral replication [9]. Thus far, most immunocytochemical studies on the HPV L1 capsid protein have investigated the predictive role of the protein in low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of unknown significance (ASCUS) [10-12]. Unfortunately, few comprehensive studies have investigated HPV L1 protein immunoreactivity in abnormal cytology, including high-grade squamous intraepithelial lesions (HSIL) and cancer according to the multiplicity of HPV infections in the presence of HPV16. This study investigated the expression of HPV L1 in abnormal cervical cytology with HPV16 infection and analyzed its association with HPV multiplicity and cervical histopathology diagnoses in Korean women.

Materials and methods Patient selection We analyzed 475 abnormal cervical cytology samples from patients with HPV16 infection confirmed by HPV DNA genotyping at Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Korea, from January 2007 to December 2012. The cervical cytology samples were gently scraped from the exocervix and endocervix with a cervical brush. Cytological diagnosis was carried out according to the 2001 Bethesda reporting system [13], and HPV type was determined by a DNA genotyping polymerase chain reaction-based DNA microarray system (Mygene, Seoul, Korea) that detected 16 types of high-risk HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68) and 8 types of low-risk HPV (6,

81 11, 34, 40, 42, 43, 44, and 70) [14]. The results of tissue diagnosis were obtained from records of patients who underwent colposcopic biopsy, loop electrosurgical excision procedures, or total hysterectomy. The following parameters were collected from medical records: age, cervical cytology, HPV infection type, and results of cervical histopathology of surgical specimens. The study design, data collection, and analysis followed the principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board of The Catholic University of Korea.

Immunocytochemistry The immunocytochemical reaction for HPV L1 capsid protein in abnormal cervical cytology was performed using the Cytoactiv® HPV L1 screening set (Cytoimmun Diagnostics GmbH, Pirmasens, Germany) according to the manufacturer’s protocols. In brief, the coverslip and mounting medium of Papanicolaou-stained cervical smear slides in xylene were separated, and the slides were washed using absolute alcohol to eliminate the remaining xylene. To retrieve the antigen, 0.01 mol/L citrate buffer (pH 6.0) was used, and the sample was heated in a microwave vacuum histoprocessor (RHS-1; Milestone, Bergamo, Italy) at a controlled final temperature of 121°C for 15 minutes. The slides were kept at room temperature for 10 minutes to cool down. Afterwards, one droplet of HPV L1 capsid antibody was applied to the cervical smear slides, and the slides were incubated for 1 hour at room temperature. A drop of detection reagent (ready to use) was applied and incubated for 10 minutes. Next, a drop of chromogen solution (3-amino-9-ethylcarbazole, ready to use) was applied to the slide for 5 minutes. Finally, Mayer’s hematoxylin counterstaining was applied to the samples. We included at least 1 positive control slide supplied by the manufacturer in each batch. The slides were examined by 2 pathologists (TJ Kim and AW Lee) and scored using a 3-step algorithm [15]: (1) the adequacy of the specimen was determined according to the cervical cytology criteria of the 2001 Bethesda System; (2) whether there was red nuclear staining in the squamous cells was determined; and (3) whether the stained cells showed abnormalities that conformed to the diagnostic criteria was determined.

Statistical analysis We investigated the expression of HPV L1 in cervical cytology and compared it with that of cervical histopathology. Analysis of variance, Pearson’s χ2, and Fisher’s exact tests were used to determine the associations between the immunocytochemical expression of HPV L1 capsid and other variables. All http://www.medsci.org

Int. J. Med. Sci. 2014, Vol. 11

82

calculations were performed using SAS version 8.0 (SAS institute Inc., Cary, NC, USA). The level of significance was set at p < 0.05.

Results The immunocytochemical expression of the HPV L1 capsid protein was observed mainly in the nuclei of cervical cells (Fig.1). Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. Three hundred sixty (75.8%) patients were infected with only HPV16, whereas 115 (24.2%) patients exhibited multiple-type HPV infections, including HPV16. Further, the patients comprising the cancer group were older (mean age: 56.6 years) than those of any other groups, with a

statistically significant difference (p < 0.0001) (Table 1). The immunocytochemical expression rates of HPV L1 in ASCUS, LSIL, HSIL, and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity among the 4 groups (p < 0.0001). Although the expression of L1 tended to be negatively associated with the cytology grades, a high percentage (78.2%) of samples without L1 were observed in cases of ASCUS (Table 1). HSIL showed the highest rate (38.3%) of HPV16 multiple infections, and cancer cytology expressed the lowest rate (7.3%) of multiple-type HPV infections (p < 0.0001) (Table 1).

Fig. 1. The immunocytochemical expression of HPV L1 capsid protein in the nuclei of cervical cells.

Table 1. Clinicopathological parameters and HPV L1 protein expression of HPV 16-positive abnormal cervical cytology (N =475). Total (N = 475) number (%)

ASCUS† (n = 87) number

(%)

LSIL‡ (n = 253) number (%)

HSIL§ (n = 94) number

HPV* L1 capsid immunocytochemistry Negative Positive

287 188

(60.4) (39.6)

HPV 16 infection Single Multiple

360 115

Age (years) Mean age 20 - 29 30 - 39 40 - 49 ≥50

38.7 129 163 123 60

(%)

Cancer (n = 41) number

(%)



68 19

(78.2) (21.8)

102 151

(40.3) (59.7)

76 18

(80.9) (19.1)

41 0

(100.0) (0)

Clinicopathological implications of human papilloma virus (HPV) L1 capsid protein immunoreactivity in HPV16-positive cervical cytology.

The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV1...
619KB Sizes 0 Downloads 0 Views