大腸直腸癌預後學之研究－ 著重表觀遺傳學和腫瘤微環境之解析

Abstract

CpG島甲基化表現型(CpG island methylator phenotype) 大腸直腸癌是一種在DNA啟動子許多CpG島有廣泛性高度甲基化的一種亞型，和許多臨床病理特徵有高度相關，例如：老年、女性、右側大腸癌、BRAF V600E突變和偶發性錯配修復缺陷大腸直腸癌。CpG島甲基化表現型的預後角色仍存有爭議。為了探究CpG島甲基化表現型的預後角色，我們先前針對450位大腸直腸癌病人的檢體分析CpG島甲基化表現型等生物標記。結果顯示CpG島甲基化表現型在早期癌並不是預後因子，但是在第四期的轉移性癌症中，則病人顯著地有較短的存活期。我們也發現在「年輕型」大腸直腸癌(小於50歲診斷癌症)中，CpG島甲基化表現型大腸直腸癌的比率達14.3%，明顯較西方族群的比例(約5%)高。 為了進一步驗證CpG島甲基化表現型在台灣「年輕型」大腸直腸癌的高發生率，以及探究大腸直腸癌危險因子與台灣CpG島甲基化表現型大腸直腸癌的相關性，我們於2016-2019年間「前瞻性」收集大腸直腸癌病人的腫瘤檢體、臨床病理資料和大腸直腸癌危險因子。研究結果顯示，小於50歲和大於等於50歲的病人中各有15.7% (14/89)和15.2% (31/204)是CpG島甲基化表現型大腸直腸癌。另外，我們也發現CpG島甲基化表現型大腸直腸癌在小於50歲的病人中，與較多的第四期診斷、BRAF V600E突變、和高身體質量指數 (≥ 27.5 kg/m2)有相關。在多變相分析中，只有高身體質量指數 (≥ 27.5 kg/m2)這個危險因子和CpG島甲基化表現型在小於50歲的大腸直腸癌病人有顯著相關。這結果驗證了在台灣年輕型大腸直腸癌有高比例是CpG島甲基化表現型，而且這些腫瘤與高身體質量指數 (≥ 27.5 kg/m2)有顯著相關。 許多研究指出BRAF V600E突變型在轉移癌與較差的預後有關。我們團隊之前的研究也發現雖同屬腫瘤細胞基因型BRAF V600E突變型，在「晚期大腸直腸癌」與存活期較短相關，但在「早期大腸直腸癌」則與預後無關。有鑑於腫瘤微環境中的免疫組成也可以是大腸直腸癌的預後因子，我們假設BRAF V600E突變的大腸直腸癌腫瘤內免疫組成有預後影響。因為轉移性BRAF V600E突變大腸直腸癌的免疫組成很少被研究，我們納入54個第四期未接受過治療BRAF V600E突變的轉移性大腸直腸癌的病人，收集他們的檢體和臨床資料。這些檢體的RNA被萃取出來後利用PanCancer Immune Panel (nanoString)進行免疫基因表現分析。我們的研究結果顯示，許多腫瘤內補體基因的表現與病人預後有關。我們也發展出「補體分數」這個工具。「補體分數」高的腫瘤相較於「補體分數」低的腫瘤有較短的疾病無惡化存活期和總存活期。我們發現「補體分數」也與C4d (一個補體路徑活化的生物標記)染色的強度有顯著正相關，驗證了RNA分析的結果。最後，我們發現，「補體分數」與腫瘤微環境中的M2型巨噬細胞所代表的基因表現也是有顯著相關性。因為M2型巨噬細胞被認為有促進腫瘤生長的作用。這也許是「補體分數」高的腫瘤有比較差的預後的可能原因之一。 綜合以上，我們的研究首次點出東西方大腸直腸癌在CpG島甲基化高表現型大腸直腸癌有不同的發生率，也提醒國人適當身體質量指數(< 27.5 kg/m2)對大腸癌預防的重要性。我們的研究結果亦將有助於未來對於BRAF V600E突變大腸直腸癌發展新的治療策略。

The CpG island methylator phenotype-high (CIMP-high) subtype of colorectal cancer (CRC) is characterized by widespread hypermethylation in the CpG islands of DNA promoters. Our previous study demonstrated that CIMP-high was not associated with poor prognosis in early-stage CRC but an independent prognostic factor in Stage 4 CRC in a multivariate analysis model. Among patients with early-onset CRC (EOCRC) diagnosed before 50 years old (y/o), the frequency of CIMP-high was 14.3%, significantly higher than that in the Western population (5%). Thus, we collected specimens from patients with CRC and analyzed the clinicopathologic characteristics and CRC risk factors of patients during 2016-2019. We analyzed the tumor’s KRAS/NRAS mutations, BRAF V600E mutation, microsatellite instability (MSI), and CIMP. The results revealed that CIMP-high tumors represented 15.7% (14/89) in EOCRC and 15.2% (31/204) in late-onset CRC (LOCRC, diagnosed at 50 y/o or older), respectively. In addition, we observed that in a multivariate analysis, a high body mass index (BMI) of ≥ 27.5 kg/m2 was significantly correlated with CIMP-high CRC in those younger than 50 yrs. These findings validated that the frequency of CIMP-high CRC in EOCRC is high in Taiwan and demonstrated the significant association between these tumors and a high BMI. We previously demonstrated the significant prognostic role in late-stage BRAF V600E mutant CRC but not in early-stage tumors. We hypothesized that immune contextures in the tumor microenvironment of BRAF V600E mutant CRC have prognostic implications in CRC. We enrolled 54 patients with untreated, metastatic microsatellite-stable BRAF V600E-mutated CRC and analyzed the expression of immune-related genes in these tumors. The results showed that many complement genes were associated with patient’s survival outcomes. We developed a complement score and observed that BRAF V600E-mutated CRC with a high complement score was associated with shorter progression-free survival and overall survival compared to that with a low complement score. Finally, we identified that complement scores were significantly associated with M2 macrophage signatures. This may contribute to the phenomenon that tumors with a high complement score are associated with poor survival. Therefore, our study indicates different incidences of CIMP-high CRC in Eastern and Western populations, and reminds the importance of a proper BMI (<27.5 kg/m2) for Taiwanese population. The findings in our study could provide insight into developing novel treatments for BRAF V600E mutant CRC.