人類乳突病毒變異株與子宮頸癌變風險之分子流行病學研究

Abstract

目前在全世界，人類乳突病毒第十六型與第十八型是最具致癌性的。其他也具致癌性的第五十二型與第五十八型在亞洲特別流行。這個研究主要是在探討在台灣的人類乳突病毒第十六型、第十八型、第五十二型與第五十八型四型的型內變異株的情形，與他們和發生持續性感染與子宮頸癌變之間的相關性。在1991至1992年間，從台灣七鄉鎮，我們研究團隊共收案11,923婦女。當時抽取了這些婦女的子宮頸細胞內的DNA，並且以EasyChip HPV blot作型別鑑定。其中具有人類乳突病毒第十六型、第十八型、第五十二型與第五十八型四型任一型陽性的婦女共有807人，這807人的檢體便進一步進行變異株的檢測，針對人類乳突病毒基因上E6、E7與LCR三個區域的PCR反應與DNA定序，有653個檢體可決定出變異株的型別。 在人類乳突病毒第十六型與第十八型分別有170與116樣本可決定出變異株，其中占最多的型分別為Asian型(139, 81.8%))與Asian American型(95, 81.9%)。在人類乳突病毒第五十二型與第五十八型占最多的型分別為B型與A3型。在280個可決定出變異株的人類乳突病毒第五十二型陽性樣本中，B型與C型是屬於非似原型變異株，而且可被偵測於278個(99.3%)樣本中。在134個可決定出變異株的人類乳突病毒第五十八型陽性樣本中，A型包括了原型與似原型變異株，可被偵測於132個(98.5%)樣本中。我們發現在台灣人類乳突病毒第十六型、第十八型、第五十二型與第五十八型這四型的變異株的分布，是很不同於歐洲與美洲族群。 先前好幾個研究已指出人類乳突病毒變異株和發生子宮頸癌變危險性之間的相關，但是大多主要在探討人類乳突病毒第十六型。經這個研究發現，與European變異株比較，人類乳突病毒第十六型Asian變異株會分別與細胞診斷的高度子宮頸鱗狀上皮癌變和組織診斷的高度子宮頸內上皮癌變有相關，呈現年齡調整後的危險對比值分別為4.44 (1.26-15.64)與9.99 (1.30-77.10)。屬於Asian變異株分支內不同的單點變異包括T178A/G、A647G、A7730C/G、T7781C、G7842A與C24T/G，也是與細胞診斷的高度子宮頸鱗狀上皮癌變和組織診斷的高度子宮頸上皮內癌變有相關。因此，本研究發現，人類乳突病毒第十六型非European變異株，尤其是Asian變異株，是有比European變異株具有較高的子宮頸癌變發生的危險性。 在被人類乳突病毒第五十二型單型別感染的婦女與B型變異株比較，C型變異株會分別與細胞診斷的高度子宮頸鱗狀上皮癌變和組織診斷的高度子宮頸上皮內癌變有相關，呈現年齡調整後的危險對比值分別為5.2 (1.0-27.6).與7.6 (1.3-43.8)。但是人類乳突病毒第五十二型C型變異株與子宮頸癌變發生的危險性之相關，仍需要更進一步的確定。 在那些進入研究當時細胞學診斷為正常的婦女，有被人類乳突病毒第十六型、第十八型、第五十二型與第五十八型四型感染者，她們的型內變異株與後來發生高度子宮頸鱗狀上皮癌變與子宮頸癌之間的相關性也被研究。研究發現，與European變異株比較，感染人類乳突病毒第十六型Asian變異株的婦女後來會有3.15倍(0.73-13.57)發生高度子宮頸鱗狀上皮癌變與子宮頸癌的危險性，但是可能因為新發個案數目太少，導致統計上無顯著意義 先前有些研究已指出人類乳突病毒變異株和病毒發生持續性感染的相關，但是研究很少，且尚未有一致結論。在這個研究，後來在1993至1995年間對收案婦女做追蹤，共有6,383人完成追蹤檢測。當時也抽取了這些婦女的子宮頸細胞內的DNA，並且以EasyChip HPV blot作型別鑑定。在基線可決定變異株的653樣本中，有281個樣本是基線為細胞學正常且有參與基線與追蹤檢測的婦女的。在人類乳突病毒第十八型Asian American變異株(55.6%)比European變異株(0%)有較高的病毒持續性感染，而且以Fisher’s exact test檢定是具有統計上顯著意義的相關(p=0.00022)。人類乳突病毒第五十八型A1型變異株(5.9%)比A3型變異株(64.7%)有較低的病毒持續性感染，經年齡與病毒量調整後的危險對比值為0.08 (0.01-0.71)。因此，人類乳突病毒第十八型Asian-American變異株與第五十八型A3型變異株是有比其他變異株有較高的傾向於持續性感染。 人類乳突病毒第十六型、第十八型、第五十二型與第五十八型四型的型內變異株的盛行情形，與第五十二型與第五十八型的演化樹分析，在台灣是第一篇的報告。至今，也是第一篇在台灣探討人類乳突病毒變株與病毒發生持續性感染、與細胞診斷的高度子宮頸鱗狀上皮癌變和組織診斷的高度子宮頸上皮內癌變，與後來發生高度子宮頸鱗狀上皮癌變與子宮頸癌之間的相關性。的確，不同的人類乳突病毒變株，與病毒發生持續性感染、與子宮頸癌變，與後來發生高度子宮頸鱗狀上皮癌變與子宮頸癌之間，是具有不同程度的危險性。

Human papillomavirus (HPV) 16 and 18 are the most oncogenic HPV types worldwide. Other oncogenic HPV 52 and 58 are prevalent in Asia. This study aims to elucidate intratypic variants of HPV 16/18/52/58 and their associations with risk of viral persistence / cervical neoplasia in Taiwan. A total of 11,923 women were enrolled from seven townships in 1991-1992. The HPV DNA in cervical cells was detected and typed by EasyChip HPV blot. Among 807 participants infected with one or more types of HPV 16, 18, 52 and 58, nucleotide variations were determined in cervical cell samples of 653 participants by the polymerase chain reaction sequencing of the long control region (LCR), E6 and E7 genes. Among 170 and 116 samples determinable for variant analysis of HPV 16 and 18, HPV16 Asian variant and HPV18 Asian-American variant were the most prevalent variants in Taiwan, about 139 (81.8%) and 95 (81.9%) samples, respectively. The large majority of HPV52 and HPV58 samples belonged to HPV52 B variant and HPV58 A3 variant, respectively. Among 280 samples determinable for variant analysis of HPV 52, non-prototype-like variants including lineage B and C were detected in 278 (99.3%) samples. Among 134 samples determinable for variant analysis of HPV 58, the prototype and prototype-like group (lineage A) of HPV58 was found in 132 (98.5%) samples, with sub-lineage A1, A2, and A3 variant in 14.2%, 27.6%, and 56.7%, respectively. It was concluded that frequency distributions of HPV 16, 18, 52 and 58 variants in Taiwan were different from those in European and American populations. Several previous studies have documented the association between intratypic variants of HPV and risk of cervical neoplasia, but mainly focused on those for HPV type 16. Compared with HPV 16 European variant, the Asian variant was associated with an increased prevalence of cytologically identified high-grade cervical squamous intraepithelial lesion or worse lesions (HSIL+) and histologically confirmed cervical intraepithelial neoplasia or worse lesions (CIN3+) showing an age-adjusted odds ratio (95% confidence interval) of 4.44 (1.26-15.64) and 9.99 (1.30-77.10), respectively. Distinct nucleotide substitutions within Asian lineage including T178A/G, A647G, A7730C/G, T7781C, G7842A, and C24T/G were significantly associated with an increased prevalence of HSIL+ and/or CIN3+. It was concluded that non-European variants of HPV16, predominantly Asia variants, had an increased risk of cervical neoplasia relative to European variant. Among women infected with single HPV 52 type, the C variant (vs. B variant) was associated with an increased prevalence of cytologically diagnosed high-grade squamous intraepithelial lesion or worse lesions showing an age-adjusted odds ratio (95% confidence interval) of 5.2 (1.0-27.6) and an increased prevalence of histologically confirmed high-grade cervical intraepithelial neoplasia or more severe lesions with an age-adjusted odds ratio (95% confidence interval) of 7.6 (1.3-43.8). The association between C variant of HPV 52 and prevalence of cervical neoplasia needs further validation. Among normal cytological women at study entry, the association between intratypic variants of HPV 16/18/52/58 and risk of incident HSIL and cancer was examined. HPV16 Asian variant at baseline had a 3.15–fold risk to develop HSIL and cervical cancer (95% confidence interval, 0.73-13.57) after adjustment of age, but not statistically significant due to the small number of newly developed cases. Some studies have documented significant associations of HPV intratypic variants with viral persistent infection, but still limited data concerned. In this study, there were 6,383 women completed follow-up visit from 1993 to 1995. The HPV DNA in their cervical cells was also detected and typed by EasyChip HPV blot. Among these 653 participants determinable for variant analysis, the persistent infection of HPV 16/18/52/58 was determined in 281 subjects who were cytologically normal at baseline examination and attended follow-up examination. HPV 18 Asian-American variant had a similar proportion of persistent infection (55.6%) with the European variant (0%) showing a statistically significant association (p=0.00022) by Fisher exact test. The HPV 58 A1 variant (5.9%) had a lower proportion of persistent infection than the HPV 58 A3 variant (64.7%) showing an odds ratio of 0.08 (95% confidence interval, 0.01-0.71) after adjustment of age and viral load. HPV 18 Asian-American variant and HPV 58 A3 variant have a general tendency to persist more frequently than other variants. The prevalence of HPV 16/18/52/58 variants and the phylogeny of HPV 52 and HPV 58 in Taiwan were first documented in this report. This study is, to date, the first report of HPV 16/18/52/58 variants associated with viral persistence, or high-grade cytological or histological diagnoses of cervical neoplasia, or incident HSIL and cancer in Taiwan. The variant groups of HPV 16/18/52/58 were associated with different risks of viral persistence or cervical neoplasia.