Roundabout 4在原發性急性骨髓性白血病的研究

Roundabout 4 (Robo4) 是一種僅被發現存在於造血幹細胞及血管內皮細胞的膜蛋白。最近的研究發現造血幹細胞Robo4的表現與其在骨髓微環境中維持造血穩態有密切關係。骨髓微環境(bone marrow microenvironment, niche)提供了造血幹細胞(hematopoietic stem cell, HSC) 進行自我更新 (self-renewal)、冬眠 (quiescence)、歸巢 (homing) 與植入(engraftment)、分化 (differentiation) 及增生的特殊環境。愈來愈多的研究顯示骨髓微環境可能與急性骨髓性白血病 (acute myeloid leukemia, AML) 的致病機轉 (leukemogenesis) 有關。至今尚無針對Robo4是否表現於急性骨髓性白血病細胞的研究。因此為了解急性骨髓性白血病細胞Robo4表現的高低與急性骨髓性白血病患者臨床表現及預後、分子生物學特徵如細胞表面標記、染色體及非染色體基因異常等的關聯性，我們利用即時定量聚合酶連鎖反應合成法 (real-time quantitative polymerase chain reaction, RQ-PCR) 分析急性骨髓性白血病患者初診斷時骨髓 (bone marrow , BM) 檢體中Robo4 mRNA的表現。骨髓檢體來自1995年至2006年間於台大醫院血液科診斷為急性骨髓性白血病且接受標準化學治療的病患共計148位，藉由即時定量聚合酶連鎖反應合成(RQ-PCR) 的方法分析初診斷時骨髓單核細胞Robo4 mRNA的表現。臨床表現分析包含性別、年齡、FAB分類、初診斷時的周邊白血球及芽細胞 (blast) 數目、血色素、血小板數目、LDH數值、染色體變化及預後影響的評估。分析Robo4 mRNA表現的高低與總體存活期 (overall survival)、無病存活期 (disease-free survival)、緩解率 (remission rate) 的相關性來評估其對預後的影響。 148位病患中有78位是男性、70位是女性，中位數年齡為46歲。研究結果發現急性骨髓性白血病患者初診斷時骨髓單核細胞Robo4 mRNA的表現都顯著高於正常對照組 (P=0.0016)。以0.010 (Robo4/RPLP0) 作為閥值 (cut-off value)，患者分成Robo4高表現 (n=61) 與低表現 (n=87) 兩組。臨床表現方面，兩組的性別、年齡、初診斷時周邊白血球及芽細胞數目、血色素、血小板數目、LDH數值無顯著差。表面標記特徵方面，Robo4高表現病患的急性骨髓性白血病呈HLA-DR陽性(85.0% vs. 62.4%, P=0.003) 及CD56 陽性(30.0% vs. 8.4%, P=0.001) 的比例顯著較高。 142位 (96 %) 病患具初診段時有效的傳統染色體分析結果，其中67位 (47.2 %) 檢出同源性染色體異常。Robo4高表現的病患發生t(8;21) 的頻率(20% vs. 2.3%, P=0.0009) 顯著較高，t(15;17) 的頻率則顯著較低(1.6% vs. 14.9%, P=0.0081)。在染色體檢查無法驗出異常的基因變異方面，Robo4高表現的病患發生DNMT3A 突變的頻率 (P=0.0543) 顯著較高，CEBPAdouble mutation的頻率(P=0.024) 則顯著較低。 148位病患中有107位 (72.3%) 在接受標準治療後達到完全緩解 (complete remission, CR)。在病患的預後方面，Robo4高表現的病患有治療效果較差的傾向(CR rate, 63.9% vs.78.2%, P=0.0643)。在中位數為31個月的追蹤期內 (範圍介於1.0至160個月)，Robo4高表現病患的總體存活期 (中位數, 17.0個月vs. 95.0個月, P =0.023) 及無病存活期 (中位數, 5.0個月vs. 15.0個月, P=0.024) 均顯著較差；分析其中99位中度風險染色體核型 (intermediate-risk cytogenetics) 的病患，Robo4高表現病患的總體存活期 (中位數, 13.5個月vs. 95.0個月, P =0.007) 及無病存活期 (中位數, 4.0個月vs. 10.0個月, P=0.025) 也顯著較差。 Cox proportional hazards 多變項分析可以發現，年齡大於50歲、初診斷時周邊白血球數目大於50,000/μL、預後不佳之染色體核型 (unfavorable cytogenetics) 及Robo4的高表現 (Hazard ratio 1.779, 95% CI 1.005-3.149, P=0.048)為總體存活期較差的獨立預後預測因子，CEBPAdouble mutation與NPM1mutation+/FLT3-ITDmutation- 則為總體存活期較佳的獨立預後預測因子；年齡大於50歲、初診斷周邊白血球數目大於50,000/μL、預後不佳之染色體核型(unfavorable cytogenetics) 及Robo4的高表現(Hazard ratio 1.779, 95% CI 1.005-3.149, P=0.048) 也是無病存活期較差的獨立預後預測因子，CEBPAdouble mutation與NPM1mutation+/FLT3-ITDmutation- 則也是無病存活期較佳的獨立預後預測因子。本次研究的結果顯示急性骨髓性白血病患者初診斷時骨髓單核細胞Robo4 mRNA的高表現為預後不佳的象徵；總體存活期的預後預測在中度風險染色體核型的病患顯得更有意義。在白血病致病機轉 (leukemogenesis) 中，Robo4本身的作用機轉及其與t(8;21)、DNMT3A突變的交互作用值得進一步研究。

關鍵字

急性骨髓性白血病；骨髓微環境； Robo 4

並列摘要

Background and Purpose Roundabout 4 (Robo4) is a transmembrane protein expressed specifically in endothelial cells and hematopoietic stem cells (HSCs). Recently, Robo4 expression was shown to be tightly associated with bone marrow (BM) microenvironment and involved in HSC homeostasis. BM microenvironment provides support for self-renewal, quiescence, homing, engraftment and proliferative potential for HSCs. Emerging evidence suggested that BM microenvironment may play a role in the leukemogenesis of acute myeloid leukemia (AML). Till now, there has been no study concerning the prognostic implication of Robo4 expression in de novo AML. Methods and Materials We investigated the RNA expression of genes encoding Robo4by real-time quantitative polymerase chain reaction in the BM from a cohort of 148 newly diagnosed de novo AML patients who received standard conventional chemotherapy and 20 healthy BM donors at the National Taiwan University Hospital. The expression of the target gene was normalized to that of the housekeeping gene RPLP0.The result was correlated with clinical features, cytogenetics, other genetic alterations and treatment outcomes. Results Among the 148 AML patients recruited, 78 were males and 70 were females with a median age of 46 years. Median levels of Robo4 expression were significantly higher in AML patients than in normal BM donors (P=0.0016). The patients were then divided into two groups, one with low expression of Robo4 (n=87) and the other with high expression (n=61), by using a cut-off point of 0.010 (Robo4/RPLP0). There was no difference in age, gender, hemogram and LDH levels between the patients with high and low Robo4 expression. Patients with high Robo4 expression had higher incidence of HLA-DR and CD56 expression on the leukemia cells (85.0% vs. 62.4%, P=0.003 and 30.0% vs. 8.4%, P=0.001, respectively). However, there was no difference in the expression of other antigens between the patients with high and low Robo4 expression. Chromosome data were available in 142(96%) patients at diagnosis and clonal chromosomal abnormalities were detected in 67 patients (47.2%). High Robo4 expression was closely association withchromosomal abnormalities t(8;21), but inversely correlated with t(15;17) (20% vs. 2.3%, P=0.0009 and 1.6% vs. 14.9%, P=0.0081, respectively). To investigate the interaction between Robo4expression and other genetic alterations in the pathogenesis of AML, a mutational screening of 16 other genes was also performed. We found that Robo4 expression was significantly higher in AML patients with DNMT3A mutation (P=0.0543), but lower in those with CEBPA mutation (P=0.0240). Among the 148 AML patients, 107 (72.3%) patients achieved a complete remission (CR) after standard intensive chemotherapy. High Robo4 expression was associated with a trend of inferior response (CR rate, 63.9% vs.78.2%, P=0.0643). With a median follow-up of 31 months (ranges, 1.0-160), patients with high Robo4 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) than those with low Robo4 expression (median, 17.0 months vs. 95.0 months, P =0.023, and medium, 5.0 months vs. 15.0 months, P=0.024, respectively). In the subgroup of 99 patients with intermediate-risk cytogenetics, the differences between patients with high and low Robo4 expressionin OS (median, 13.5 months vs. 95.0 months, P= 0.007) and DFS (median, 4.0 months vs. 10.0 months, P=0.025) were still significant. In multivariate analysis, the independent poor risk factors for OS were older age > 50 years, high WBC count >50,000/μL, unfavorable karyotype, and high Robo4 expression (Hazard ratio 1.779, 95% CI 1.005-3.149, P=0.048). On the other hand, CEBPAdouble mutation and NPM1mutation+/FLT3-ITDmutation- were independent favorable prognostic factors. The independent poor risk factors for DFS included older age > 50 years, high WBC count >50,000/μL, unfavorable karyotype, and high Robo4 expression (Hazard ratio 1.600, 95% CI 1.026-2.495, P=0.038). CEBPAdouble mutation and NPM1mutation+/FLT3-ITDmutation- were also independent favourable factors for DFS. Conclusion Our results demonstrated that high pre-treatment expression of Robo4 in the BM indicates an unfavorable prognosis in de novo AML patients and the prognostic significance of Robo4 expression for OS in the subgroup of patients with intermediate-risk cytogenetics was even more obvious. Further studies are needed to explore the mechanisms of this gene expression and its interaction with t(8;21) and DNMT3A mutation in the leukemogenesis of AML.

並列關鍵字

acute myeloid leukemia ； microenvironment ； Robo 4

參考文獻

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Roundabout 4在原發性急性骨髓性白血病的研究

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