| 摘要: | 累積的研究證據顯示，在子宮頸癌病患的血液內可發現腫瘤去氧核醣核酸，而血液內的腫瘤去氧核醣核酸具有診斷和臨床預後的價值。人類乳突病毒去氧核醣核酸，在子宮頸癌病患的血清和血液中，被偵測到的比率是百分之七至四十五， 而這差異可能是由於偵測樣品的不同，例如血清或血漿、萃取去氧核醣核酸的方法不同、或是使用分析的工具不同，例如傳統的聚合酶反應 (PCR)，即時定量PCR、或是利用酵素免疫的PCR和不同的引子 (primers)，例如L1、E6、E7，因此在過去的文獻報告中，只有少數是有關在血液中偵測HPV DNA的比較。
因為即時定量PCR可用來偵測在血液中HPV DNA的低病毒量，所以本論文的第一部採用即時定量PCR來改善病毒的偵測率，提供一前瞻性的人類乳突病毒偵測方法，使病人在被診斷出疾病的同一時間偵測其HPV DNA，並討論其臨床意義。研究的結果顯示：以即時定量PCR的方法可以在超過四分之一(27%)的子宮頸癌病患血液中偵測到HPV DNA。且在帶有HPV 16, 18 或是52型的子宮頸癌病患中，幾乎有百分之五十的病患，可在血液裡偵測到HPV DNA。本研究提供了子宮頸癌病患在治療後，其血液中HPV DNA病毒量連續追蹤的數據並強調其臨床意義的重要性。在本研究中，有6位子宮頸癌病患於治療完成後，其血液中偵測不到HPV DNA，且在後續的追蹤中都沒有復發。而在連續追蹤中，有10位子宮頸癌病患在治療完後三個月，仍可在血液裡偵測到HPV DNA，其中有8位發生轉移，且有7位發生的是遠端轉移。雖然本研究局限於不多的病人數，及追蹤時間不夠長，但經統計分析結果顯示：在血液裡被偵測出定量的HPV DNA的確反應了腫瘤轉移與否，且具有其預後價值。換言之、子宮頸癌病患在治療完後仍可在其血液中偵測到HPV DNA，是可用來預測該病患未來復發與否的一個有用指標，並可用以決定哪一些子宮頸癌病患需要更積極的治療。
HPV DNA嵌入宿主細胞基因體，被認為是子宮頸癌癌化轉變的重要事件，而且發生在早期子宮頸癌的階段，然而大部分的研究報告都著重在16型HPV病毒，少數著重在18型上。在美國、歐洲、非洲、及東南亞洲，52型和58型發生在子宮頸癌病患相對是較低的，而在台灣和亞洲地區，52型和58型是較常見的致癌型別。為瞭解HPV 52型及58型，是否和16型及18型一樣其嵌入宿主細胞基因體，或是高病毒量是癌化過程中必要的，本論文第二部份的研究著重於HPV 52型和58型嵌入宿主細胞基因體的致癌過程。分析子宮頸分泌物採自178位連續病患、包含81位子宮頸癌和97位子宮頸中度至重度細胞病變之患者，利用基因晶片和定量即時PCR技術，檢查並判定HPV16、18、52、58型的盛行率，和其嵌入狀態和病毒量。
研究結果發現，於台灣婦女子宮頸癌病患中並不常見到HPV 52型和58型病毒DNA嵌入到人類基因體中，可見病毒52型和58型其DNA嵌入宿主基因體並非是子宮頸癌發展必須的因素。相反的，16型及18型病毒DNA嵌入宿主基因體是導致子宮頸癌非常重要的步驟。而16、18、52型的高E6病毒量，則具有預測高度細胞病變轉變至子宮頸癌的能力。可見HPV DNA是否嵌入宿主基因體和其病毒量，在子宮頸癌癌化過程中，可能因病毒型別的差異而扮演不同的角色。在此研究中我們成功地利用HPV 16、18、52型的病毒量(the median log of viral loads) 來預測子宮頸癌，選擇的分界點可以達到預測子宮頸癌62.5% ~83.3% 的敏感度，及0-25%的偽陽性機率。ROC曲線分析顯示所建立的模型能準確地預測、鑑別及診斷出高度細胞病變，或是子宮頸癌發生在病人感染HPV 16型、18型或52 型分別達73.8%，92.9%，88.57%的正確率。
最後本論文的研究著重在低度細胞病變與病毒的相關性。幾乎50%的非典型鱗狀上皮細胞和80%的低度細胞病變，會被致癌型的HPV感染；而HPV DNA的測試對非典型鱗狀上皮細胞患者，可提供訊息轉介這些病患做陰道鏡檢查並查出潛藏的高度細胞病變和子宮頸癌。相反的，致癌型的HPV DNA檢測並無法提供低度細胞病變更一步的訊息供臨床做進一步的選擇性處理；因為在低度細胞病變患者身上有相當高的比率可偵測到HPV DNA。而臨床上，對於低度細胞病變的處理是在3至6個月後重複做抹片檢查，或是直接陰道鏡切片檢查，所以發展出一個可供低度細胞病變患者臨床處理選擇的模式，以區分哪些患者可能會進展到高度細胞病變，哪些會自動痊癒，就非常有價值。
本研究測試在低度及高度細胞病變中，於亞洲最常見的癌前病變致癌病毒型別HPV 16、18、52、58四型之病毒量，並評估2年累積進展到高度細胞病變的危險性，且探討病毒DNA嵌入宿主基因體是否為導致低度細胞病變進展到高度細胞病變的主要原因。此外，並探討在6個月後重複做抹片檢查時，E6病毒量的改變是否會導致進展成高度細胞病變。研究結果發現低度細胞病變患者在6個月後，病毒量增加者比病毒量沒有增加的患者有45%的危險性會進展成高度細胞病變。利用定量即時PCR偵測病毒量，發現病毒量增加者比沒有增加的患者有大於7倍的危險性會進展成高度細胞病變；若用HC2技術偵測病毒量發現病毒量增加者比病毒量沒有增加的患者有大於6倍的危險性會進展成高度細胞病變。這兩種技術偵測到的病毒量是一致的 (Person’s coefficient, r=0.687, p<0.001)。結果亦指出，6個月後重複檢測的HPV DNA病毒量，病毒量的改變可用來預測感染HPV DNA 16、18、52、58型的低度細胞病變患者，是否進展成高度細胞病變且與臨床結果相符。
總結，本論文的研究對HPV DNA病毒量和嵌入宿主基因體的狀態，在子宮頸癌癌化過程中所扮演的角色，提出其指標性的臨床運用，以預測癌前病變及子宮頸癌患者的疾病進展。
Accumulating evidence shows that tumor DNA can be found in the circulation of patients with cervical cancer. The presence of such tumor DNA in the blood may be of diagnostic and prognostic value. HPV DNA has been found in serum or plasma samples from cervical cancer patients with detection rates varying from 7% to 45%. The discrepancy may be due to different target materials (serum or plasma), method of extracting DNA, tools of analysis (conventional PCR, real-time PCR, or PCR-enzyme immunoassay), and differing primers used (L1, E6, E7). Therefore, information regarding the comparison of detection rates of HPV DNA in circulating blood is limited.
The first part of this study provides a prospective study of HPV DNA detection at a single diagnostic time point. Real-time PCR is used to detect the low viral loads of HPV DNA in blood. The results show that more than one-fourth (27%) of patients with invasive cervical cancer had HPV DNA detected in their blood samples. Approximately 50% of patients with confirmed HPV 16, 18 or 52 positive cervical cancers had HPV DNA detected in their blood. This study also used serial follow-up data on HPV DNA viral load among cervical cancer patients after treatment to understand its clinical significance. Six cervical cancer patients with HPV DNA viral loads undetectable in their blood after treatment showed no recurrence during follow-up. In longitudinal follow-up, eight out of ten cervical cancer patients with viral loads of HPV DNA detectable in the blood at 3 months after treatment were associated with recurrence. Among these, seven of eight patients had distant metastases. Although the study was limited to a small number of patients and a short period of follow-up, it is worth pointing out that detection of circulating HPV DNA after treatment could predict recurrence. It is postulated that blood HPV DNA might be a useful marker to select subsets of patients who need more aggressive treatment. The presence and quantity of HPV DNA in blood are likely to be a reflection of metastasis and may be of prognostic value.
The second part of this study focuses on the role of integration of HPV type 52 and 58 in cervical cancer patients. The integration of HPV DNA into the host genome is thought to occur early in cancer development and to be an important event in malignant transformation of cervical cancer. However, most studies on the integration of HPV DNA focus on type 16 and a few on type 18. While HPV type 52 and 58 are oncogenic types with relatively low prevalence in cervical cancer in the Americas, Europe, Africa and Southeast Asia, they are as prevalent as the known high-risk (for cervical cancer) HPV types 16 and 18 in Taiwan and other Asian countries. To analyze whether integration or high viral loads of human papillomavirus (HPV) are essential for malignant transformation of HPV type 52 and 58 as well as type 16 and 18, cervical swabs from 178 consecutive patients, including 81 with invasive cervical cancers and 97 with cervical intraepithelial neoplasias (CIN) II-III, were collected and examined to determine the prevalence, physical status and viral load of HPV type 16, 18, 52 and 58 DNA using genechip and real-time PCR (polymerase chain reaction) analysis.
The infrequent integration of HPV 52 and 58 DNA in cervical cancer suggests that it is not a prerequisite for progression to cervical cancer. By contrast, integration appears to be a critical step for carcinogenesis of HPV 16 and 18 DNA. High viral loads (E6) of HPV 16, 18 and 52 DNA may be predictive of the transition of CIN II-III to cervical cancer. The results indicate that both viral DNA physical status and viral loads of HPV are important factors in the carcinogenesis of different HPV types. This study successfully used the median log of viral loads of HPV 16, 18 and 52 DNA to predict the presence of cervical cancer. The selected cut-off values of the median log of viral loads in HPV 16, 18 and 52 DNA achieved 62.5-83.3% sensitivity and a 0-25% false positive rate in predicting the presence of cervical cancer. The ROC curve analyses indicated that the model could accurately predict the diagnostic group of CIN II-III or cervical cancer in 73.8%, 92.9%, and 88.5% of patients with positive HPV 16, 18 and 52, respectively.
The third part of this study focuses on low-grade squamous intraepithelial lesions (LSILs). Approximately 50% of atypical squamous cells of undetermined significance (ASCUS) and 80% of LSILs are infected by oncogenic types of HPV. HPV DNA testing for patients with ASCUS provides useful information and allows referral of patients for immediate colposcopy to detect high-grade squamous intraepithelial lesions (HSILs) and cancer. By contrast, oncogenic HPV DNA testing is not informative for triage of patients with LSILs because a high percentage of LSIL patients are HPV positive. A repeat Pap smear in 3 to 6 months or direct biopsy under colposcopy is generally used in clinical practice. Development of alternative triage strategies for women with LSILs would be valuable in distinguishing women with LSILs that have high probabilities of progression to HSILs from women with LSILs that have spontaneously regressed.
The 2-year cumulative risks were evaluated for HSIL attributable to HPV 16, 18, 52, and 58, the most common oncogenic types in pre-invasive cervical lesions including LSILs and HSILs in Asia, and questioned as to whether the integration of HPV oncogenes into the host genome contributed to the risk of LSILs progressing to HSILs. In addition, it was determined whether or not E6 viral load and its change contributed to the risk of LSILs progressing to HSILs during the interval between baseline diagnosis of LSIL by Pap smear and a 6-month follow-up visit by repeat Pap smear. It was found that women with LSILs whose viral loads increased between baseline and 6 month follow-up had a 45% risk of developing HSIL, which was seven-fold greater than those without increased viral loads (OR = 7.6, 95% CI = 1.9 to 29.4, p < 0.01), as evaluated by real-time PCR. The risk was calculated at 44%, a six-fold greater risk than those without increased viral loads (OR = 6.1, 95% CI = 1.6 to 22.7, p < 0.01), as evaluated by HC2. The two viral load measures correlated well (Person’s coefficient, r = 0.687, p < 0.001). The results indicate that evaluation of viral load changes (increased or not increased) through repeat HPV DNA testing could predict progression of disease in LSIL cases of HPV types 16, 18, 52, and 58, which correlates to clinical implications.
In summary, this research strives to understand the role of HPV DNA viral loads and integration in the carcinogenesis of cervical cancer by searching for a useful marker applicable in clinical practice to predict disease progression in pre-invasive and invasive cervical cancer. |
