半乳糖凝集素-3的表現在急性骨髓性白血病之研究

Abstract

背景 半乳糖凝集素-3(galectin-3)為一35kDa的蛋白質，擁有特殊的嵌合結構，廣泛存於許多細胞和組織中，主要藉由本身的醣類辨識結構域(carbohydrate-recognition domains)結合至β-半乳糖(β-galactosides)。Galectin-3對腫瘤生物學有許多功能，如癌症進展(progression)，附著(adhesion)，凋亡(apoptosis)，血管新生(angiogenesis)，腫瘤發生(transformation)和轉移(metastasis)。近來有許多研究指出galectin-3的表現於多種癌組織中扮演重要的角色。但至今為止，galectin-3的表現於急性骨髓性白血病(AML)的臨床意義仍不清楚。 研究目標 我們將探討galectin-3的基因，LGALS3，於骨髓內的表現量於成人非M3型急性骨髓性白血病(non-M3 AML)中所扮演的臨床意義及角色。 病人和實驗方法 我們回溯性收集280位年紀大於15歲且在台大醫院確診為成人非M3型急性骨髓性白血病的患者，利用即時定量聚合酶連鎖反應合成法(RQ-PCR)評估其骨髓內LGALS3基因的表現量，並將此結果和病人的臨床特色，染色體變化，分子基因突變及預後做相關性分析。我們另外完成30位病患骨髓切片檢體galectin-3的免疫組織化學染色(Immunohistochemical staining)及使用酵素連結免疫分析法(Enzyme-linked immunosorbent assay; ELISA)檢測骨髓血漿中galectin-3 蛋白質的表現量，並依此結果來做LGALS3基因和galectin-3蛋白質表現量兩者間的關連性比較。最後LGALS3表現量所代表的預後意義亦在另一個包含42位成人非M3型急性骨髓性白血病患的獨立cohort中接受驗證。 結果 於280位病患中，我們以LGALS3基因表現量的中間值當成分界點，將其區分成較高表現量(higher expression group)和較低表現量(lower expression group)兩群。我們發現，擁有較高表現量的病患比起較低表現量的患者年紀較大，白血病較常屬於French-American-British (FAB) subtypes M4和M5，和白血病芽細胞上常表現CD14，但較少為FAB M1 subtype和較少於白血病芽細胞上表現CD7。另外，擁有較高LGALS3基因表現量的病患常帶有PTPN11基因的突變，但較不常出現FLT3-ITD和CEBPA基因突變。然而LGALS3基因的表現量則和染色體的變化無相關性。 預後的分析我們只比較280位病患中有接受標準強力化學治療的211位患者。其中，擁有較高LGALS3基因表現量的病患比起較低表現量的患者有較差的完全緩解率(complete remission rate) (61.5% vs. 82.5%, P=0.001)和較高的原發性頑固比率(primary refractory rate) (23.1% vs. 10.8%, P=0.023)。在整體存活(overall survival)方面，擁有較高LGALS3基因表現量的病患有較差的整體存活期中位數(median overall survival) (16.3 months vs. 39.8 months, P=0.02)。除此之外，多變項分析指出，在整體存活上，骨髓內帶有較高LGALS3基因的表現量為預後不佳的獨立因子，而在染色體正常(normal karyotype)的族群中也是如此。更甚者，此一預後不佳因子亦在另一獨立的cohort中獲得確認和驗證。 我們將骨髓內有較高LGALS3基因的表現量，合併其它八個預後因子，包含年紀，初診斷時周邊白血球數目，染色體變化，以及有無NPM1/FLT3-ITD, MLL-PTD, CEBPAdouble-mut 和是否有AML1/RUNX1和WT1的基因突變等整合成一評分系統(scoring system)。我們發現此一評分系統非常有效地將急性骨髓性白血病患區分成四個擁有不同預後的族群(P<0.001)。 結論 我們的研究指出骨髓內LGALS3基因的表現量於急性骨髓性白血病中有其獨特的生物影響和臨床意義。骨髓內有較高LGALS3基因的表現量為急性骨髓性白血病整體存活上預後不佳的獨立因子。於是我們認為，骨髓內LGALS3基因表現的程度可當作預測急性骨髓性白血病患臨床預後的新生物標記(new biomarker)，而針對galectin-3蛋白質的抑制策略或許對急性骨髓性白血病有高度表現此一因子的患者可望當成一個新的治療契機。

Background Galectin-3, a β-galactoside-binding lectin, plays an important role in cancer cell progression, adhesion, apoptosis, transformation, angiogenesis and metastasis. A lot of researches also demonstrated an important role of galectin-3 expression in several types of malignancies. However, the studies concerning clinical implications of galectin-3 expression in patients with acute myeloid leukemia (AML) are scarce. Aims In this study we aimed to determine the clinical implication of the expression of LGALS3, the gene encoding galectin-3, in adult de novo non-M3 AML patients. Methods We investigated the expression of LGALS3 in the bone marrow (BM) by reverse-transcriptase real-time polymerase chain reaction in a test cohort consisting of 280 adults 15 years of age or older with newly diagnosed de novo non-M3 AML at the National Taiwan University Hospital and correlated the results with clinical features and outcomes of the patients. The prognostic impact of BM LGALS3 expression was also validated in an independent cohort comprised 42 de novo non-M3 AML patients. Results Among 280 patients, those with higher BM LGALS3 expression were older (P<0.001), more frequently had French-American-British (FAB) M4 and M5 subtypes (both P=0.003), and CD14 expression on leukemic cells (P=0.009), but less commonly had FAB M1 subtype (P<0.001). In addition, higher LGALS3 expression was closely associated with PTPN11 mutation but negatively associated with FLT3-ITD and CEBPA mutation. There was no correlation between the cytogenetic abnormalities and BM LGALS3 expression. Survival analysis was performed in 211 non-M3 AML patients who received standard intensive cure-intent chemotherapy. Patients with higher BM LGALS3 expression, compared to those with lower expression, had lower CR rates (61.5% vs. 82.5%, P=0.001) and higher primary refractory rates (23.1% vs. 10.8%, P=0.023). With a median follow-up time of 69.5 months, patients with higher BM LGALS3 expression had a shorter overall survival than those with lower expression (median 16.3 months vs. 39.8 months, P=0.02). Moreover, multivariate analysis demonstrated that higher BM LGALS3 expression was an independent poor prognostic factor for overall survival among total patients and patients with normal karyotype. The unfavorable prognostic impact of higher BM LGALS3 expression was also confirmed in the independent validation cohort. A scoring system incorporating higher BM LGALS3 expression and eight other prognostic factors, including age, white blood cell count, cytogenetics, NPM1/FLT3-ITD, MLL-PTD, CEBPAdouble-mut and mutations of AML1/RUNX1 and WT1, into survival analysis was proved to be very useful to stratify AML patients into different prognostic groups (P<0.001). Conclusions Our research provides evidences that BM LGALS3 expression is associated with distinct biologic and clinical characteristics in AML. Higher BM LGALS3 expression is significantly correlated with poor prognosis in AML patients. BM LGALS3 expression may serve as a new biomarker to predict clinical outcome. Galectin-3 may be a potential target for the treatment of AML patients with higher expression of this protein.