B型肝炎病毒相關B細胞淋巴癌之研究

Abstract

背景 許多研究已證實B型肝炎病毒(HBV)表面抗原(HBsAg)陽性的慢性HBV感染和B細胞非何杰金氏淋巴癌(B-cell NHLs)的發生有正向關係，尤其是B-cell NHL中的瀰漫性大B細胞淋巴癌(DLBCL)和濾泡型淋巴癌(FL)。然而，DLBCL或FL的患者同時有慢性HBV感染時的臨床特色和預後，目前尚不清楚。此外，因慢性HBV感染和B-cell NHL的發生有正向關係，發掘和探討影響HBV相關B細胞淋巴癌(HBV-associated B-cell NHL)預後和致癌機轉的因子值得我們深入研究。 研究目標 本研究分析了在現今標靶合併化學藥物治療的時代，HBsAg於DLBCL和FL患者所代表的預後意義。亦探討其中一個可能影響HBV-associated B-cell NHL的致癌機轉因子，HBV DNA能否嵌入(HBV integration)淋巴癌細胞的基因體？其嵌入的模式為何？及HBV integration是否影響預後和淋巴癌發生。 病人和實驗方法 本研究建構了兩組回溯性世代研究群體(cohort studies)。一是DLBCL cohort study, 收錄從2002至2016年於台大醫院新診斷DLBCL，並接受全身標準標靶合併化學治療(R-CHOP)的患者。另一是FL cohort study，收錄了從2006至2016年於台大醫院新診斷FL，並接受包含標靶藥物rituximab合併其它全身化學藥物治療的患者。本研究分析了HBsAg的有無於此兩群cohorts中的臨床特色和預後相關性。 此外，本研究利用捕獲型次世代基因定序平台(capture-NGS platform)來發掘45個B-cell NHL的腫瘤檢體中，是否存有HBV integration，及其嵌入的模式為何。 結果 首先在包括416位病患的DLCBL cohort中，98位(23.6%)為HBsAg陽性。HBsAg陽性的DLBCL患者，比起HBsAg陰性的患者而言，罹病年齡較年輕，腫瘤侵犯期別較廣泛，治療反應和預後皆較差。而多變項存活分析更發現HBsAg陽性對於DLBCL患者是一個獨立的預後不佳因子。 接著於擁有149位患者的FL cohort中，32位(21.5%)為HBsAg陽性。FL同時帶有HBsAg的慢性HBV感染患者，經常出現脾腫大，胸水或腹水，和治療期間肝指數異常等等臨床徵兆。另外，比起HBsAg陰性的患者，HBsAg陽性的FL病人對於標靶合併化學藥物的治療有較低的完全緩解率，較差的整體存活期和較短的無病存活期。在多變項存活分析中，HBsAg仍舊是一個獨立的預後不佳因子。有趣的是，比起HBsAg陰性的患者，HBsAg陽性的FL病人有較高機率發生疾病早期惡化(progression of disease within 24 months)。 最後藉由capture-NGS platform，在45個B-cell NHL的腫瘤檢體中，有28.9％(13/45)發現有HBV integration的現象，共偵測到354個HBV-lymphoma基因的嵌入接合片段。不過淋巴瘤的基因組並沒有發現有嵌入熱點(hotspot integration)，而這些嵌入接合片段僅有極少數產生單株性(clonality)現象。另外，HBV integration並非穩定的存在於成對的初診斷和復發的腫瘤檢體中，且嵌合現象的有無並不影響DLBCL患者的預後。 結論 本研究發現同時有慢性HBV感染的DLBCL或FL患者，擁有特殊的臨床表徵，較低的治療反應和較差的存活率。本研究亦指出，HBsAg對於DLBCL或FL的患者而言，可當成是一個新的預測預後和存活的因子。 雖然HBV integration的現象確實存在於HBV-associated B-cell NHL，但其在腫瘤檢體中發生的頻率遠低於HBV-associated肝細胞癌。這些病毒-宿主基因嵌入接合片段於淋巴瘤的基因組中並無形成嵌入熱區，也僅有極少數發展成有意義的clonality現象。此外，HBV integration的現象於B-cell NHL的病程中並非穩定的存在，且其存在與否並不影響DLBCL患者預後。此皆暗示了HBV integration於HBV-associated B-cell NHL的生物學功能影響或許有限。 HBV-associated B-cell NHL的患者於現今的治療模式下，確實具有較差的預後。雖然嵌入式致突變進而導致淋巴瘤生成的機轉或許不能應用於HBV相關B-cell NHL，但是後續仍有許多值得研究的方向，如慢性HBV感染是否導致腫瘤免疫微環境(Tumor immune microenvironment)改變，進而影響患者的治療預後等。我們將進行更多，更深入的研究來確立HBV感染於B-cell NHL所扮演的真實生物學角色，並用以發展新的治療策略，來改善此類患者的預後。

Background Studies have shown a positive association between hepatitis B surface antigen (HBsAg)-positive hepatitis B virus (HBV) infection and B-cell non-Hodgkin lymphomas (NHLs), especially for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Nevertheless, the clinical implications of HBV infection in patients with DLBCL and FL are unclear. Moreover, factors that contribute to lymphomagenesis of HBV-associated B-cell NHL remain to be explored. Aims We aimed to examine the prognostic value of HBsAg in DLBCL and in FL. We further explored the factor that might lead to the development of HBV-associated lymphoma, the integration of HBV DNA into the genome of lymphoma cells. Methods We investigated the clinical relevance of HBsAg in immunocompetent patients with DLBCL and in those with FL, respectively. For the DLBCL cohort, patients who had received homogeneous rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone as the frontline therapy at National Taiwan University Hospital (NTUH) between 2002 and 2016 were selected. For the FL cohort, patients treated with frontline rituximab-containing chemoimmunotherapy at NTUH between 2006 and 2016 were recruited. Additionally, we selected 45 B-cell NHL cases and used the Hybridization capture-based next generation sequencing (NGS) to characterize the pattern of HBV DNA integration into the genome of lymphoma cells in their tumor samples. Results Among 416 analyzed patients in the DLBCL cohort, 98 (23.6%) were HBsAg-positive. HBsAg positivity was associated with a younger age and more advanced stage at diagnosis, more frequent hepatic impairment during perichemotherapy, and a trend of higher National Comprehensive Cancer Network-International Prognostic Index score. Compared with the HBsAg-negative patients, the HBsAg-positive patients had a poorer 5-year overall survival (OS) rate and shorter 5-year progression-free survival (PFS) rate. Multivariate analyses showed HBsAg was an independent unfavorable prognostic indicator. Regarding the FL cohort including 149 patients, 32 (21.5%) were HBsAg-positive. HBsAg positivity was positively associated with symptomatic splenomegaly, significant serous effusions, and peritreatment hepatic dysfunction. HBsAg-positive patients had significantly poorer OS and shorter PFS than had HBsAg-negative patients. Multivariate analysis still revealed that HBsAg is an independent adverse prognostic factor for OS. Intriguingly, HBsAg-positive patients had a higher incidence of progression of disease within 24 months than had HBsAg-negative patients (cumulative incidence rate, 25.8% vs. 12.4%, P = 0.045). Finally, by conducting high-throughput viral integration detection, a total of 354 HBV integrations were identified from 28.9% (13/45) of HBV-associated NHLs. Nevertheless, there was no clustered genomic hotspot of viral integration. Additionally, only few HBV integrations had clonality in the genome of B-cell NHL. Moreover, the HBV integration did not show persistence and stability by analyzing the paired diagnosis-relapse samples. Furthermore, the presence of HBV integration was not associated with the survival of patients with DLBCL. Conclusions We demonstrated that HBV infection is uniquely relevant to DLBCL and FL. Our research also provides evidences that HBsAg was an independent unfavorable factor significantly associated with survival, highlighting its potential as a novel prognostic predictor of the survival of patients with DLBCL or with FL. Although HBV DNA could indeed integrate into the genome of B-cell NHL, the sample frequency of integration events was far less than that in hepatocellular carcinoma. No significantly enriched viral integration hotspot was discovered. Additionally, only few cases were identified to have clonally expanded lymphoma cells carrying the same integrated viral DNA. Moreover, HBV integration was unstable during disease evolution and not associated with prognosis. Hence, the mechanism of insertional mutagenesis to drive lymphomagenesis might not be applicable to HBV-associated lymphoma. The other factor such as the influence of chronic HBV infection on the tumor immune microenvironment merits further investigation. We look forward to exploring the biological role of HBV infection in B-cell NHL and developing novel therapeutic strategies for this group of patients.