临床医学进展  >> Vol. 8 No. 8 (October 2018)

P16和Ki-67与宫颈癌患者HPV持续性感染的相关性研究
Relationship between Expression of P16 and Ki-67 and Persistent Infection of HPV in Cervical Carcinoma Patients

DOI: 10.12677/ACM.2018.88130, PDF, HTML, XML, 下载: 505  浏览: 1,520  科研立项经费支持

作者: 黄群欢, 金绍燕, 陆 欢:上海健康医学院附属第六人民医院南院,上海;洪 岭:同济大学附属第一妇婴保健院,上海

关键词: 宫颈上皮内瘤变免疫组织化学P16Ki-67人乳头瘤病毒Cervical Intraepithelial Neoplasia (CIN) Immunohistochemistry P16 Ki-67 Human Papillomavirus (HPV)

摘要: 目的:探讨P16和Ki-67表达与宫颈病变之间的关系,评估P16和Ki-67作为高危型人乳头瘤病毒(hr-HPV)持续感染预后标记物的意义。方法:纳入宫颈活检标本1369例,无癌前病变(NEG) 392例,宫颈上皮内瘤变I (CIN I) 519例,CIN II 257例,CIN III 154例,鳞状细胞癌(SCC) 47例。此外,306名CIN I患者被纳入为期12个月的随访中,使用HPV特异性引物通过聚合酶链反应方法检测宫颈分泌物中hr-HPV,随访期内每6个月检测一次。133名患者持续感染hr-HPV,94名一过性感染,79名未感染hr-HPV。免疫组织化学法检测P16和Ki-67的表达。P16和Ki-67的染色结果分为四类:阴性,1+,2+和3+。结果:与NEG相比CINs和SCC中P16和Ki-67的表达均显著增加(P < 0.01)。CIN III中P16和Ki-67的表达与CIN II相比均无显著性差异(P > 0.05)。P16阳性率与hr-HPV持续感染正相关(P < 0.05)。hr-HPV感染患者中P16以及Ki-67阳性率显著高于未感染hr-HPV的患者(P < 0.05)。结论:P16和Ki-67可提高宫颈病变的诊断准确性,P16表达与CIN I期患者hr-HPV持续感染存在相关性。
Abstract: Objective: To determine the association between the expression of P16 and Ki-67 and cervical le-sions, and to evaluate the role of P16 and Ki-67 as prognostic markers for persistent high risk human papillomavirus (hr-HPV) infection. Methods: Totally 1369 cases of cervical biopsies were enrolled, 392 cases with negative for dysplasia (NEG), 519 with cervical intraepithelial neoplasia I (CIN I), 257 with CIN II, 154 with CIN III and 47 with cervical squamous cell carcinoma (SCC). Fur-thermore, 306 women with CIN I were recruited into 12-month follow-up, and HPV specific gene detection by polymerase chain reaction was used to detect hr-HPV of cervical secretions at 6-month-interval for 12-month follow-up period. 133 women were infected with persistent hr-HPV, 94 with transient infection and 79 not infected with hr-HPV. The expression of P16 and Ki-67 was evaluated by immunohistochemical method. The immunostaining results of P16 and Ki-67 were classified into four categories: negative, 1+, 2+ and 3+. Results: There was significant increase in the expression of P16 and Ki-67 (P < 0.01) from NEG to SCC. There was no significant difference in the expression of P16 and Ki-67 between CIN II and CIN III (P > 0.05). Ratio of P16 positivity was positive correlated with persistent hr-HPV infection (P < 0.05). Ratios of P16 and Ki-67 in hr-HPV infection group were significantly higher than that of non-infection group (P < 0.05). Conclusion: P16 and Ki-67 can improve the diagnostic accuracy of cervical lesions. P16 expression is correlated with persistent hr-HPV infection in CIN I patients.

文章引用: 黄群欢, 金绍燕, 陆欢, 洪岭. P16和Ki-67与宫颈癌患者HPV持续性感染的相关性研究[J]. 临床医学进展, 2018, 8(8): 776-783. https://doi.org/10.12677/ACM.2018.88130

1. 引言

宫颈癌是全球女性中第二大常见恶性肿瘤 [1] ,其起源于宫颈上皮内瘤变(CIN),目前可通过对CIN的治疗有效预防宫颈癌进展。对于CIN I病例,通常建议严格随访,无需治疗 [2] 。而CIN II和CIN III病例则需要积极治疗,包括宫颈锥切或宫颈环电外科切除术等。宫颈活检的组织学检查通常被认为是诊断“金标准”,但该方法可能受到观察者和观察者间差异的影响 [3] [4] 。此外,非肿瘤病变与CINs之间的差异和CIN I与CIN II/III之间的差异在形态学上很难找出明确界限以有效区分,从而容易导致过度治疗或不治疗 [5] [6] 。因此,宫颈病变的准确诊断对于临床医生的决策和患者的治疗均十分重要。

大多数高危型人乳头瘤病毒(hr-HPV)感染自发退化,只有一小部分持续存在。持续的hr-HPV感染是CIN和宫颈癌发生、发展的必需条件之一 [7] 。因此,使用合适的标记预测持续性hr-HPV感染将具有相当大的临床价值。在当前研究中,hr-HPV的持续时间定义为2~14个月,中位数为6个月 [8] [9] [10] 。Syrjänen, K.等报道人乳头状瘤病毒持续存在6个月以上对疾病进行有预测意义 [11] 。P16INK4a (P16)是一种细胞周期蛋白依赖性激酶抑制剂,可调节细胞周期从G1期向S期的转变 [12] [13] 。Ki-67是最近发现的一种检测细胞增殖的可靠指标 [14] 。已经显示P16和Ki-67在诊断CIN中的价值。然而,关于P16和Ki-67与hr-HPV持续感染的关系鲜见研究报道。本研究选择2015年底至2018年初在我院行阴道镜活检切除手术的患者,对其P16和Ki-67表达进行检测,并分析了hr-HPV感染与P16和Ki-67表达的相关性。

2. 材料与方法

2.1. 病例样本

本研究纳入2015年10月至2018年2月在上海健康医学院附属第六人民医院南院就诊的1369例细胞学检查异常的(非典型鳞状上皮细胞) ASCUS,(上皮内低度病变) LSIL,(上皮内高度病变) HSIL或宫颈鳞癌(SCC)患者。年龄17~69岁(平均30.39岁)。纳入标准:KPS ≥ 70分;未接受过宫颈癌根治性手术治疗;未接受过放化疗;无其他系统严重合并症;无其他系统恶性肿瘤。排除标准:不符合纳入标准者;患有严重精神疾病者;临床资料不全影响判断者。每个受试者接受阴道镜检查活检切除手术,通过聚合酶链式反应(PCR)检测hr-HPV基因。上海健康医学院附属第六人民医院南院伦理委员会批准了所有实验程序,并根据获得患者书面知情同意书。

将福尔马林固定的石蜡包埋组织块切成3 μm厚度,切片用苏木精和伊红(H&E)染色进行常规检查。在所有活检样品中进行P16和Ki-67免疫组织化学(IHC)染色。所有HE染色切片首先由2名独立病理学家审查,将共识诊断作为黄金标准。若诊断意见不同,则由病理专家小组确认共识诊断。共有306名CIN I女性被纳入12个月的随访。在12个月的随访期间,每6个月进行一次宫颈分泌物的HPV基因检测。

2.2. 免疫组织化学染色

所有标本通过间接法IHC染色。切片用二甲苯去石蜡,在分级醇中水合,用PBS缓冲液洗涤,在EDTA溶液(pH8.0,100℃,2.5分钟)中煮沸以进行抗原修复并冷却。鼠抗人P16抗体(1:200稀释,武汉博士德,BM0266),兔抗人Ki-67抗体(1:50稀释,武汉博士德,BM4381)。IHC染色结果的判断:P16在细胞质或细胞核中表达,Ki-67观察在细胞核中表达情况。P16表达情况分级描述如下:阴性(低于5%),弱阳性(1+,5%~25%),中等阳性(2+,26%~50%),强阳性(3+,大于50%)。为了确定Ki-67表达的等级,在高倍视野(×400)中检查位于整个上皮层的200个上皮细胞的细胞核。Ki-67阳性细胞的百分比低于5%、5%~25%、26%~50%和大于50%分别描述为阴性、1+、2+和3+。

2.3. hr-HPV基因检测

采用凯普HPV分型检测试剂盒(广东凯普生物有限公司),通过聚合酶链反应(PCR)体外扩增和DNA芯片技术进行DNA 提取、扩增、杂交、洗膜、显色及结果读取。15种高危型别包括:HPV16、18、31、33、35、39、45、51、52、53、56、58、59、66、68。在12个月的随访期间,被诊断为CINⅠ的306名参与者未接受治疗。一过性感染为首次检测阳性,而在下一次检测时针对相同hr-HPV基因型检测阴性。持续性hr-HPV感染为12个月的随访期间连续检测到相同的hr-HPV类型,未感染表示随访期间hr-HPV基因检测始终为阴性结果。

2.4. 统计学分析

使用SPSS 16.0软件进行统计学分析。使用卡方检验分析频率表,利用Spearman相关法分析有序变量之间的二元相关性,采用分层法分析hr-HPV感染与P16和Ki-67表达的相关性。双侧P < 0.05认为差异具有统计学意义。

3. 结果

3.1. P16和Ki-67在宫颈病变组织中的表达

表1所示,P16和Ki-67的表达与宫颈病变的等级有关。NEG,CIN I,CIN II,CIN III和SCC中P16的阳性率分别为20.66%,82.47%,98.83%,98.70%和100%;Ki-67的阳性率分别为31.38%,80.15%,97.28%,98.05%和100%。随病变分级的上升,P16 (P < 0.01,图1)和Ki-67 (P < 0.01,图2)表达显著增加。但CINⅡ和CINⅢ中P16 (P > 0.05)和Ki-67 (P > 0.05)阳性率无显著差异。

Figure 1. Immunohistochemical staining results of P16

图1. P16免疫组织化学染色结果。(A) 无癌前病变(NEG)阴性对照;(B) 宫颈上皮内瘤变I (CIN I);(C) CINI I;(D) CIN III;(E) 鳞状细胞癌(SCC),标尺60 µm

Figure 2. Immunohistochemical staining results of Ki-67

图2. Ki-67免疫组织化学染色结果。(A) 无癌前病变(NEG)阴性对照;(B) 宫颈上皮内瘤变I (CIN I);(C) CIN II;(D) CIN III;(E) 鳞状细胞癌(SCC),标尺60 µm

Table 1. Expression of P16 and Ki-67 in different cervical lesions

表1. P16和Ki-67在不同宫颈病变中的表达情况

注:NEG:无癌前病变;CIN:宫颈上皮内瘤变;SCC:宫颈鳞癌。

3.2. P16的表达与Ki-67表达正相关

P16的表达水平与Ki-67的表达水平呈正相关(r = 0.641,P = 0.008)。359例P16和Ki-67双阴性病理诊断样本均为NEG或CIN I (表2)。在P16和Ki-67表达均呈强阳性(3+)的163例样本中有151例为高分级CIN (CIN II和CIN III)或SCC。2例P16表达阴性的高分级CIN中Ki-67表达呈阳性。1例Ki-67表达阴性的高分级CIN中P16表达呈阳性。

3.3. P16和Ki-67表达与hr-HPV感染状态的相关性

306名CIN I患者中,hr-HPV持续性感染133例,一过性感染94例。P16阳性率与HPV持续感染状态之间存在显著相关性(P16,r = 0.364,P = 0.011)。Ki-67阳性率与HPV持续感染状态(Ki-67,r = −0.009,P = 0.974)无显著相关性。hr-HPV感染患者中P16以及Ki-67阳性率显著高于未感染hr-HPV的患者(P16,P = 0.003;Ki-67,P = 0.019) (表3)。

4. 讨论

目前只有少数可用于预测持续hr-HPV感染的生物标志物 [15] [16] ,已发现16,18,31,33,35,39,45,51,52,56,58,59和68型HPV持续感染引起绝大多数CIN和宫颈癌,故上述hr-HPV基因型被定义为“致癌”病毒类型 [17] [18] 。许多关于P16和Ki-67免疫染色在宫颈病变中价值的研究主要集中在高分期CIN的诊断 [19] [20] 。在本研究中,我们收集了大量的样本,并评估宫颈病变中P16和Ki-67免疫组织化学结果的价值。本研究还调查了P16和Ki-67在CIN I患者hr-HPV持续感染中的预后价值。

结果显示,CIN II,CIN III和SCC中P16和Ki-67的表达高于CIN I,且CIN I和NEG之间有显着性差异。随着疾病进展,P16和Ki-67表达显着增加。这表明P16和Ki-67免疫组织化学染色可用于评估宫颈病变。值得一提的是,2例P16阴性CIN II/III阳性表达Ki-67,1例Ki-67阴性CIN II/III阳性表达P16。它表明P16免疫染色结合Ki-67可以提高高级别CIN的诊断准确性,并且P16和Ki-67是互补的生物标志物。P16和Ki-67表达在CIN II和CIN III中无显著性差异,提示P16过表达是早期事件,且Ki-67在CIN发展过程中起作用,但需要进一步的研究来验证。

hr-HPV感染导致病毒基因整合到宿主基因组中,早期基因E7是hr-HPV的重要致癌基因 [21] 。E7与肿瘤抑制剂视网膜母细胞瘤蛋白(pRB)结合使pRB失活,导致细胞周期的G1期到S期转变 [22] 。随后细胞中游离E2F的增加 [23] ,导致异常增殖(以Ki-67表达水平增加为标志)。P16是一种pRB调节因子,它是受pRB控制的负反馈的基础,其失活导致P16的过表达 [24] 。对于hr-HPV与P16以及Ki-67表达

Table 2. Expression correlation of P16 and Ki-67

表2. P16和Ki-67表达相关性

Table 3. Correlation between P16 and Ki-67 expression and hr-HPV infection

表3. P16和Ki-67表达与hr-HPV 感染的相关性

之间的关系,多数研究者认为P16可作为hr-HPV诱导CIN的标志物之一 [25] ,并且P16表达阳性的hr-HPV阴性样本通常被认为是假阴性。我们的研究证实,P16的阳性率与HPV感染状态之间存在显著相关性(P16,r = 0.364,P = 0.011)。但是,Ki-67阳性率与HPV感染状态之间的相关性很低(Ki-67,r = −0.009,P = 0.974)。为了研究P16和Ki-67在持续性hr-HPV感染中的作用,我们对hr-HPV检测进行了12个月的随访。在我们的研究中,由于所有入选病例都被诊断为CIN I,保证了研究人群的同质性。同时,CIN I反映了HPV的复制活性,但临床往往通过随访观察而非立即治疗,以避免过度治疗。有研究报道HPV阴性的一些病例中,P16也为阳性 [26] 。与未感染hr-HPV的患者相比,在hr-HPV持续感染和一过性感染患者的P16和Ki-67阳性率显著增加(P16,P = 0.003;Ki-67,P = 0.019)。这提示P16和Ki-67蛋白标志物在预测HPV感染方面具有一定的价值。但是hr-HPV感染导致P16和Ki-67表达增加的机制尚未完全明了,值得进一步研究。此外,由于本研究仅对CIN I患者进行了随访和hr-HPV感染跟踪监测,因此对于高级别宫颈病变中P16和Ki-67表达与hr-HPV持续感染的相互关系还有待研究。

5. 结论

综上所述,P16蛋白的表达对CIN I患者hr-HPV持续感染的预后具有重要意义,可考虑作为预测宫颈病变发展的辅助指标。

基金项目

上海健康医学院2016年度校种子基金重点项目(HMSF-16-21-021)。

NOTES

*通讯作者。

参考文献

[1] Chih, H.J., Lee, A.H., Colville, L., et al. (2013) A Review of Dietary Prevention of Human Papillomavirus-Related in-fection of the Cervix and Cervical Intraepithelial Neoplasia. Nutrition and Cancer, 65, 317-328.
https://doi.org/10.1080/01635581.2013.757630
[2] Rouzier, R. (2008) Management of CIN1. Journal de Gyné-cologie Obstétrique et Biologie de la Reproduction, 37, S114-S120.
https://doi.org/10.1016/j.jgyn.2007.11.017
[3] Martin, C.M. and O’Leary, J.J. (2011) Histology of Cervical In-traepithelial Neoplasia and the Role of Biomarkers. Best Practice & Research Clinical Obstetrics & Gynaecology, 25, 605-615.
https://doi.org/10.1016/j.bpobgyn.2011.04.005
[4] Munro, A., Leung, Y., Spilsbury, K., et al. (2015) Comparison of Cold Knife Cone Biopsy and Loop Electrosurgical Excision Procedure in the Management of Cervical Adenocarcinoma in Situ: What Is the Gold Standard? Gynecologic Oncology, 137, 258-263.
https://doi.org/10.1016/j.ygyno.2015.02.024
[5] Chen, E.Y., Tran, A., Raho, C.J., et al. (2010) Histological ‘Progression’ from Low (LSIL) to High (HSIL) Squamous Intraepithelial Lesion Is an Uncommon Event and an Indi-cation for Quality Assurance Review. Modern Pathology, 23, 1045-1051.
https://doi.org/10.1038/modpathol.2010.85
[6] Creagh, T., Bridger, J.E., Kupek, E., et al. (1995) Pathologist Variation in Reporting Cervical Borderline Epithelial Abnormalities and Cervical Intraepithelial Neoplasia. Journal of Clinical Pathology, 48, 59-60.
https://doi.org/10.1136/jcp.48.1.59
[7] Cheol Park, G., Roh, J.L., Cho, K.J., et al. (2017) 18F-FDG PET/CT vs. Human Papillomavirus, p16 and Epstein-Barr Virus Detection in Cervical Metastatic Lymph Nodes for Identifying Primary Tumors. International Journal of Cancer, 140, 1405-1412.
https://doi.org/10.1002/ijc.30550
[8] Woodman, C.B., Collins, S.I. and Young, L.S. (2007) The Natural History of Cervical HPV Infection: Unresolved Issues. Nature Reviews Cancer, 7, 11-22.
https://doi.org/10.1038/nrc2050
[9] Kim, Y.H., Chang, B., Choi, J.H., et al. (2016) Biochemical Fingerprints of Human Papillomavirus Infection and Cervical Dysplasia Using Cervical Fluids: Spectral Pattern Investigation. Mi-croscopy Research and Technique, 79, 966-972.
https://doi.org/10.1002/jemt.22728
[10] Wang, L., Wang, P., Ren, Y., et al. (2016) Prevalence of High-Risk Human Papillomavirus (HR-HPV) Genotypes and Multiple Infections in Cervical Abnormalities from Northern Xinjiang, China. PLoS One, 11, e0160698.
https://doi.org/10.1371/journal.pone.0160698
[11] Syrjanen, K. (2011) Persistent High-Risk Human Papilloma-virus (HPV) Infections as Surrogate Endpoints of Progressive Cervical Disease. Potential New Endpoint for Efficacy Studies with New-Generation (Non-HPV 16/18) Prophylactic HPV Vaccines. European Journal of Gynaecological Oncology, 32, 17-33.
[12] Kim, T.E., Kim, H.W. and Lee, K.E. (2014) Distribution of Human Papillomavirus 52 and 58 Genotypes, and Their Expression of p16 and p53 in Cervical Neoplasia. Korean Journal of Pathology, 48, 24-29.
https://doi.org/10.4132/KoreanJPathol.2014.48.1.24
[13] Wei, Q., Fu, B., Liu, J., et al. (2013) Combined Detection of p16 (INK4a) and IMP3 Increase the Concordance Rate between Cervical Cytologic and Histologic Diagnosis. International Journal of Clinical and Experimental Pathology, 6, 1549-1557.
[14] Xing, Y., Wang, C. and Wu, J. (2017) Expression of Geminin, p16, and Ki67 in Cervical Intraepithelial Neoplasm and Normal Tissues. Medicine (Baltimore), 96, e7302.
https://doi.org/10.1097/MD.0000000000007302
[15] Serour, Y., Bendahmane, M., Abbou Baker, F., et al. (2017) HPV Test by Hybrid Capture II for the Diagnosis of HR-HPV Persistent Infection. Médecine et Maladies Infectieuses, 47, 484-489.
https://doi.org/10.1016/j.medmal.2017.05.013
[16] Seraceni, S., De Seta, F., Colli, C., et al. (2014) High Preva-lence of HPV Multiple Genotypes in Women with Persistent Chlamydia Trachomatis Infection. Infect Agent Cancer, 9, 30.
https://doi.org/10.1186/1750-9378-9-30
[17] Halec, G., Alemany, L., Lloveras, B., et al. (2014) Pathogenic Role of the Eight Probably/Possibly Carcinogenic HPV Types 26, 53, 66, 67, 68, 70, 73 and 82 in Cervical Cancer. The Journal of Pathology, 234, 441-451.
https://doi.org/10.1002/path.4405
[18] Das, C.R., Tiwari, D., Dongre, A., et al. (2018) Deregulated TNF-Alpha Levels along with HPV Genotype 16 Infection Are Associated with Pathogenesis of Cervical Neoplasia in Northeast Indian Patients. Viral Immunology, 31, 282-291.
https://doi.org/10.1089/vim.2017.0151
[19] Sari Aslani, F., Safaei, A., Pourjabali, M., et al. (2013) Evaluation of Ki67, p16 and CK17 Markers in Differentiating Cervical Intraepithelial Neoplasia and Benign Lesions. Iranian Journal of Medical Sciences, 38, 15-21.
[20] You, K., Guo, Y.L., Geng, L., et al. (2013) Correlation of Cervical Intraepithelial Neoplasia with Expressions of p16 and Ki67 in Exfoliated Cervical Cells in Fluid-Based Thin-Layer Samples. European Journal of Gynaecological Oncology, 34, 535-539.
[21] Volpi, C.C., Ciniselli, C.M., Gualeni, A.V., et al. (2018) In Situ Hybridization Detection Methods for HPV16 E6/E7 mRNA in Identifying Transcriptionally Active HPV Infection of Oropharyngeal Carcinoma: An Updating. Human Pathology, 74, 32-42.
https://doi.org/10.1016/j.humpath.2017.09.011
[22] Rabachini, T., Boccardo, E., Andrade, R., et al. (2018) HPV-16 E7 Expression Up-Regulates Phospholipase D Activity and Promotes Rapamycin Resistance in a pRB-Dependent Manner. BMC Cancer, 18, 485.
https://doi.org/10.1186/s12885-018-4392-8
[23] Guo, C.P., Liu, K.W., Luo, H.B., et al. (2011) Potent Anti-Tumor Effect Generated by a Novel Human Papillomavirus (HPV) Antagonist Peptide Reactivating the pRb/E2F Pathway. PLoS ONE, 6, e17734.
https://doi.org/10.1371/journal.pone.0017734
[24] Dreyer, J.H., Hauck, F., Barros, M.H.M., et al. (2017) pRb and CyclinD1 Complement p16 as Immunohistochemical Surrogate Markers of HPV Infection in Head and Neck Cancer. Applied Immunohistochemistry & Molecular Morphology, 25, 366-373.
https://doi.org/10.1097/PAI.0000000000000309
[25] Liu, Y., Alqatari, M., Sultan, K., et al. (2017) Using p16 Immunohistochemistry to Classify Morphologic Cervical Intraepithelial Neoplasia 2: Correlation of Ambiguous Staining Patterns with HPV Subtypes and Clinical Outcome. Human Pathology, 66, 144-151.
https://doi.org/10.1016/j.humpath.2017.06.014
[26] Golusinski, P., Pazdrowski, J., Szewczyk, M., et al. (2017) Is Immunohistochemical Evaluation of p16 in Oropharyngeal Cancer Enough to Predict the HPV Positivity? Reports of Practical Oncology and Radiotherapy, 22, 237-242.
https://doi.org/10.1016/j.rpor.2017.01.003