Detection Of Human Papillomavirus DNA And Expression Of P16, P53 And Ki67 Proteins In Cervical Intraepithelial Neoplasia

Cervical cancer is the second leading cause of cancer-related deaths in women worldwide,and the principle cancer among women in most developing countries. With the development of cervical exfoliative cytology,the incidence of cervical carcinoma decreases,however,the incidence of Cervical Intraepithelial Neoplasia, (CIN) steps up in recent years.Currently,management of preinvasive cervical cancer largely relies on histologic examination to confirm cervical intraepithelial neoplasia(CIN) and ultimately lead to efficient treatment of lesions that are classified as higher grades (CINâ…¡-â…¢).Although the histologic features of CIN are well described in World Health Organization(WHO),inconsistent use and misinterpretation of such features may lead to significant intra- and inter-observer variability.On the basis of morphology alone,distinction of squamous metplasia coupled with hyperplasia from CINâ… and distinction of CINâ…¡from CINâ…¡-â…¢can be very difficult in some cases. Therefore,objective diagnostic methods in addition to histology are needed in the assessment of biopsies for cervical intraepithelial neoplasia.Human papillomavirus(HPV) plays an etiologic role in cervical carcinogenesis and high-risk subtypes are detectable in 90%invasive cervical epithelial neoplasms. The most common high-risk HPV subtypes include types 16 and 18,which account for approximately 70%of HPV species detected in cervical cancers.Low-risk types 6 and 11 account for approximately 90%of HPV species present in genital warts,and infections with low-risk types has been known to cause genital warts and low-grade squamous intraepithelial lesions.Uncontrolled cell proliferation and malignant transformation are the basic elements in the development of cancer including cervical cancer and its precursors. Better understanding of control of cell cycle and cell proliferation activity allows a rational therapeutic approach,p 16 is a cyclin-dependent kinase inhibitor that regulates the transition from G1 to S phase of the cell cycle and normally functions as a tumor suppressor.Although p 16 levels are reduced in a variety of malignances,this gene product has been shown to be up-regulated and over-expressed in most high-grade cervical dysplasias and carcinomas induced by high-risk HPV subtypes.P53,as a tumor suppressor,is one of the major factors controlling cell proliferation.As the "guardian of the genome",it can arrest the cell cycle in response to DNA damage or direct the damaged cell to an apoptotic pathway.Ki67 is a cell proliferation marker that is expressed in all phases of the cell cycle except GO.The aim of our study was to investigate the significance of HPV infection and the potential role of P16,P53 and Ki67 immunohistochemical expression in diagnosis and grading of cervical intraepithelial neoplasia(CIN).Immunohistochemical staining was used for detection of expression of these proteins in 191 various cervical lesions.In 104 of 191 cases,the presence of HPV-6/11 and 16/18 DNA were determined by Polymerase Chain Reaction(PCR). Nuclear and/or nuclear and cytoplasmic staining was considered as positive for P16 when present in＞10%of squamous cells.Two patterns of positive P16 staining were observed:(1)"spotty" in which positive cells were scattered throughout the lesion and (2) "band" in which＞90%of contiguous cells in the lesion stained positive.P53 staining was located in nuclear and was considered as positive when present＞5%of squamous cells.Ki67 staining was also located in nuclear and Ki67 proliferaton index and the location of Ki67 positive cells in cervical epithelium(low third,two thirds,or full epithelium) were recorded.Results were as follows:in 191 various cervical lesions,the expression rate of P16 was 73.3%(140/191),and 75.0%of these 140 cases were band-like and 25.0% were spotty-like.In CINâ… ,P16 expression rate(78.8%,26/33) was significantly higher than squamous metplasia coupled with hyperplasia(33.4%,13/39)(p＜0.0001); In CINâ…¡-â…¢,P16 expression rate(96.1%,74/77) was significantly higher than CINâ… (p=0.004);However,no differences were found between cervical cancer group(94.4%,17/18) and CINâ…¡-â…¢group.For the expression pattern of P 16 staining, squamous metplasia coupled with hyperplasia group displayed predominately spotty or negative expressions(92.3%,36/39);Cerical condyloma group also displayed predominately spotty or negative expressions(91.7%,22/24);CINâ… group displayed similarly distributed patterns of the scattered spotty and contiguous band-like expressions(40.5%,15/37);In contrast,both CINâ…¡-â…¢group and cervical cancer group showed mainly contiguous band-like patterns(92.2%,94/102).The expression patterns of P16 changed from the scattered spotty to contiguous band-like with increasing grade of cervical lesions,and a significant difference achieved in the P16 staining pattern(band versus spotty or negative ) among cervical tissues with progression of lesions(p＜0.0001).The expression rate of P53 protein is 45.5%(87/191) in 191 cases,and the highest group is cervical condyloma group(75.0%,18/24).Significant differences were observed between cervical condyloma group and other groups except cervical cancer group(p＜0.05).The Ki67 proliferation index was 0.1874±0.1879 in 191 cervical cases and increased gradually with progression of lesions.As well as P16 protein,Ki-67 proliferation index(0.1345±0.1034) in CINâ… group was significantly higher than in squamous metplasia coupled with hyperplasia group(0.0594±0.0415)(p＜0.0001);In CINâ…¡-â…¢group,Ki-67 proliferation index(0.2375±0.2020) was significantly higher than in CINâ… group(p=0.0036);No differences were found between cervical cancer group(0.3361±0.2306) and CINâ…¡-â…¢group in Ki-67 proliferation index(p=0.0853). For the distribution of Ki-67 staining,Ki-67 positive cells were distributed mainly in the lower third of the epithelial layer in CINâ… group cases.In CINâ…¡-â…¢group,Ki-67 positive cells were distributed in up half of the epithelium.Distribution of the Ki-67 positive cells in cervical epithelium were significantly different in different groups of cervical lesions(X²=87.8387,p＜0.0001)。HPV16/18 DNA were present in 50 of 104 specimens(48.1%,50/104).The frequency of HPV16/18 in C1Nâ… group was significantly higher than squamous metplasia coupled with hyperplasia group(p=0.018) and no difference was observed between CINâ… group and CINâ…¡-â…¢group(p=0.986).HPV6/11 DNA were present in 9 of 104 specimens(8.7%,9/104),and the frequency of HPV6/11 in cervical condyloma group was the highest,was significantly higher than in other groups (p＜0.05).For the relationship of expression of P16,P53 and Ki67 proteins and infection of HPV,the presence of P16 band-like reactivity correlated with HPV16/18 infection (r=1.0000,p＜0.0001).However,there were no differences in the relationship of spotty or negative expression of p 16^INK4A to the HPV 16/18 infection(r=0.7906, p=0.1114).No difference were observed between HPV16/18 positive cases and HPV negative cases(X²=0.2189,p=0.640) in the expression of P53 protein,however, significant difference were observed between HPV6/11 positive cases and HPV negative cases(X²=4.1640,p=0.041).Expression of Ki67 was significantly correlated with HPV16/18 infection(r=1.0000,p＜0.0001 ).In our present study,P16 and Ki67 can be used as surrogate markers to distinguish CINâ… from squamous metplasia coupled with hyperplasia,as well as CINâ… from CINâ…¡-â…¢,which improves the results of histologic classification.A band-like pattern of P16 staining was strongly associated with HPV16/18 infection. Expression of P53 protein was not related to progression of CIN or infection of HPV16/18,however,may be correlated with infection of HPV6/11.