Abstract
目的
了解Runt相关的转录因子1（RUNX1）在急性髓系白血病（AML）中的突变率及分布情况；探索RUNX1基因突变与临床特征的相关性及其对患者预后的影响。
方法
收集158例初发AML患者的骨髓标本，提取骨髓基因组DNA，采用聚合酶链式反应技术（PCR）扩增目的基因片段，应用基因测序分析RUNX1基因突变情况。同时检测ASXL1、DNMT3A、TET2、FLT3、CEBPA、NPM1、IDH2、NRAS及c-KIT突变情况，分析其突变与RUNX1突变的关系。用Kaplan-Meier方法进行生存分析，并行Log-rank检验比较生存时间差异。
结果
158例初发急性髓系白血病患者中，共发现RUNX1突变19例，突变率为12.0%，其中A166G 9例，A142T 6例，A162L 4例。RUNX1基因突变组与野生型组的年龄差异有统计学意义（P < 0.01），突变组患者年龄偏大，在M4和M5型中的发生率较高，较野生型组更易表达CD36、CD7。RUNX1突变发生于正常核型或预后中等风险核型的患者中，但差异均无统计学意义（P > 0.05）。突变组的完全缓解率和生存率均低于野生型组（P < 0.05），而两组在性别、初诊时白细胞计数、血红蛋白、血小板计数、骨髓原始细胞比例以及乳酸脱氢酶水平的差异无统计学意义（P > 0.05）。
结论
RUNX1基因突变对AML患者的预后具有不良影响。
Keywords: RUNX1基因, 急性髓系白血病, 基因突变
Abstract
Objective
To explore the rate and distribution of Runt- related transcription factor 1 (RUNX1) gene mutations in patients with acute myeloid leukemia (AML) and the correlation of these mutations with the clinical characteristics and survival outcomes of the patients.
Methods
The genomic DNA extracted from the bone marrow of 158 patients with newly diagnosed AML for PCR amplification of RUNX1 gene and sequence analysis to identify the mutations. The mutations of ASXL1, DNMT3A, TET2, FLT3, CEBPA, NPM1, IDH2, NRAS and c-KIT genes were also examined to analyze their association with RUNX1 gene mutations.
Result
Among the 158 AML patients, 19 (12.0%) were found to have RUNX1 mutations in A166G (9 cases), A142T (6 cases) and A162L (4 cases). RUNX1 mutations were more frequent in elderly patients (P < 0.01) and in cases of AML subtypes M4 and M5, and were associated with more frequent CD36 and CD7 expression as compared with the wild type. RUNX1 mutations were more likely to occur in patients with normal karyotype or karyotypes associated with moderate prognostic risks, but the difference was not significant (P > 0.05). The patients with RUNX1 mutations had significantly lower complete remission (CR) rate and overall survival (OS) rate than those without the mutations (P < 0.05). RUNX1 mutations were not associated with gender, white blood cell count upon diagnosis, hemoglobin level, platelet count, bone marrow blast cell ratio or lactate dehydrogenase level (P > 0.05).
Conclusion
RUNX1 gene mutations are associated with an adverse prognosis of patients with AML.
Keywords: RUNX1 gene, acute myeloid leukemia, gene mutation
目前急性髓系白血病（AML）的诱导化疗可使75%的年轻患者（< 60岁）获得完全缓解，但是大多数患者注定要复发，而AML的复发性质被认为是由于克隆异质性造成的，其中包括白血病干细胞群体中存在的起始或驱动突变。因此，阐明每种基因突变在AML中的存在意义对于理解疾病进展和开发针对驱动突变的有效疗法至关重要[1]。Runt相关转录因子1（RUNX1）是造血功能的关键调节因子，参与造血干细胞的出现和调节，其通过染色体易位、体细胞突变等方式影响造血干细胞的自我更新以及细胞谱系的分化，从而参与AML的发生发展[2-4]。
RUNX1基因虽然在AML中处于不利预后地位，但国内关于RUNX1基因突变在AML中的报道甚少，与患者临床特征之间的关系亦不明确。因此，本研究旨在检测RUNX1基因在成人AML中的突变率，分析基因突变组患者的临床特征、免疫表型、染色体核型、以及与其他分子突变包括ASXL1、DNMT3A、CEBPA、TET2、FLT3、NPM1、IDH2、NRAS及c-KIT等的关系。同时分析RUNX1突变对化疗反应的完全关节率（CR）以及生存率的影响，以期为AML的诊断和治疗提供一定的参考。
1. 资料和方法
1.1. 研究对象
本研究收集2016年6月~2019年10月在我院血液内科被诊断为初发AML患者的骨髓标本158例，其中男89例，女69例，中位年龄55岁（13~79岁）。纳入标准：初发急性髓系白血病；年龄≥12岁；有完整的白血病MICM分型。排除标准：年龄 < 12岁、 > 80岁；不愿参加本研究的。患者临床分型：除去急性混合细胞白血病1例，其中M1 13例，M2 62例，M3 15例，M4 29例，M5 37例，M6 1例。所有患者均经细胞形态学、免疫分型、细胞遗传学、分子生物学检查，并按照FAB国际分型及WHO诊断标准明确诊断为急性髓系白血病。诊断标准符合文献标准[5]。本研究获得蚌埠医学院第一附属医院伦理委员会批准，同时标本采集得到患者及家属的知情同意，并签署知情同意书。
1.2. 基因组DNA的制备
使用DNA提取试剂盒（上海源奇生物医药科技有限公司）提取基因组DNA，应用紫外分光光度仪测定样本的吸光度A值，所有用于PCR的DNA样本的A260/280比值均 > 1.8，去离子水稀释基因组DNA样品至50~150 ng/μL。
1.3. 目的基因的PCR扩增
根据GenBank上目标基因序列，参照文献，由生工生物工程（上海）股份有限公司合成：RUNX1基因（第7外显子）：正义链5'-AATCCCACCCCACTTTACAT-3'，反义链：5'-CTCAGCTGCAAAGAATGTGT-3'，扩增产物长度300 bp。RUNX1基因的PCR扩增，共50 μL反应体系：10×PCR Buffer（Mg2+plus）5 μL+dNTP Mixture 4 μL+引物F 0.5 μL+引物R 0.5 μL+Tap酶0.25 μL+DNA标本2 μL，去离子水补足体积（PCR试剂盒来自宝日医生物技术有限公司）。PCR反应条件：95 ℃预变性5 min；然后95 ℃变性30 s，58 ℃退火1 min，72 ℃延伸1 min，共34个循环，最后72 ℃延伸10 min。扩增产物经2%琼脂糖凝胶电泳后，在紫外灯下观察并照相，确定成功扩增出目标条带。
1.4. 基因测序
将成功扩增的158例PCR产物送至上海生工生物工程股份有限公司进行测序。
1.5. 染色体核型分析
采用短期培养胰酶消化G显带技术，核型分析在杭州艾迪康医学检验中心有限公司完成，染色体核型按《人类细胞遗传学国际命名体制》判定。
1.6. 免疫表型分析
流式细胞术（FCM）检测骨髓标本，使用CD34，HLA-DR，CD117，cMPO，CD13，CD33，CD11b，CD36，CD38，CD56，CD64，CD5，CD7，CD8，CD10，CD19，CD20等相关抗原检测白血病细胞的免疫表型。
1.7. 统计学分析
采用SPSS 21.0软件进行数据处理。呈正态分布的计量资料，以均数±标准差表示，组间比较采用t检验；非正态分布的计量资料，以M（P25~P75）表示，组间比较采用秩和检验（Mann-Whitney）；计数资料以率表示，组间比较采用卡方检验或Fisher确切概率法。生存分析用Kaplan-Meier方法，并行Log-rank检验比较生存时间差异。P < 0.05为差异有统计学意义。
2. 结果
2.1. RUNX1基因突变率、临床特征及免疫表型
RUNX1基因PCR扩增产物琼脂糖凝胶电泳结果及基因测序图（图 1、2），158例AML患者中发现19例RUNX1基因突变，突变率为12.0%。RUNX1基因突变组与野生型组的年龄差异有统计学意义（P < 0.01），突变组患者年龄偏大。其在FAB分型中的M4、M5亚型中的发生率高于其他FAB亚型，但差异无统计学意义（P > 0.05）。突变组与野生型组的性别、初诊白细胞计数、血红蛋白、血小板计数、骨髓原始细胞比例、乳酸脱氢酶差异无统计学意义（P > 0.05）。流式细胞学免疫分型中，RUNX1突变组的AML患者较野生型组更易表达CD36、CD7，但差异无统计学意义（P > 0.05，表 1）。
1.
AML患者琼脂糖凝胶电泳结果
Results of agarose gel electrophoresis of PCR product of RUNX1 in patients with AML.
2.
RUNX1突变的DNA测序图谱
DNA sequencing map of RUNX1 mutation. The nucleotide G at the 237th position is mutated to A, and the overlapping double peak is formed at the nucleotide G, and the Arginine is converted into Lysine.
1.
158例RUNX1基因突变及野生型AML患者的临床特征
Characteristics of 158 AML patients with and without RUNX1 mutations
| Clinical characteristic | Total (n=158) | RUNX1mutated (n=19; 12.0%) | RUNX1wildtype (n=139; 88.0%) | P |
| Male/Female | 158 | 11/8 | 78/61 | 0.883 |
| Median age(year, range) | 55.0 (45.0-65.0) | 66.0 (51.0-75.0) | 53.0 (43.0-63.5) | 0.009 |
| Median WBC count, ×109/L (range) | 15.2 (2.7-68.4) | 15.7 (3.2-89.7) | 14.7 (2.1-67.7) | 0.583 |
| Median Hb, g/L (range) | 76.0 (59.3-89.8) | 72.0 (59.0-86.0) | 76.0 (59.5-90.0) | 0.726 |
| Median Plt, ×109 (range) | 34.5 (19.0-67.0) | 40.0 (19.0-95.0) | 34.0 (18.5-63.5) | 0.247 |
| Median BM blasts (range) | 78.1 (50.5-90.1) | 63.6 (39.5-84.1) | 78.7 (55.7-90.2) | 0.321 |
| LDH | 399.0 (230.0-785.0) | 414.5 (264.8-1046.0) | 399.0 (222-770) | 0.452 |
| FAB subtype (157 analysis) | ||||
| M1 | 13 | 2 | 11 | 1.000 |
| M2 | 62 | 4 | 58 | 0.079 |
| M3 | 15 | 0 | 15 | 0.274 |
| M4 | 29 | 6 | 23 | 0.209 |
| M5 | 37 | 7 | 30 | 0.244 |
| M6 | 1 | 0 | 1 | 1.000* |
| Surface antigen positivity | ||||
| CD34+ | 72 | 10/9 | 62/77 | 0.510 |
| HLA-DR+ | 76 | 12/7 | 64/75 | 0.161 |
| CD117+ | 110 | 11/8 | 99/40 | 0.236 |
| CD13+ | 64 | 9/10 | 55/84 | 0.516 |
| CD33+ | 65 | 6/13 | 59/80 | 0.367 |
| CD38+ | 68 | 10/9 | 58/81 | 0.368 |
| CD64+ | 34 | 5/14 | 29/110 | 0.807 |
| CD36+ | 20 | 5/14 | 15/124 | 0.123 |
| CD7+ | 14 | 4/15 | 10/129 | 0.118 |
2.2. RUNX1基因突变患者的遗传学特征
158例AML患者中，152例有完整核型分析资料，6例因未见核分裂相无法鉴定染色体核型。19例RUNX1突变的AML均有完整的染色体核型，其中12例核型正常，故RUNX1在正常核型中的突变率为16.0%，与野生型组相比差异无统计学意义（P > 0.05）。19例RUNX1突变患者中11例为细胞遗传学中危患者，8例为高危患者，3组突变发生率差异无统计学意义（P > 0.05，表 2）。
2.
RUNX1基因突变与预后核型及完全缓解的相关性
Correlation of RUNX1 mutations with prognostic karyotype and complete remission inAML patients
| Variable | Total | RUNX1mutated | RUNX1wildtype | P |
| Age (year) | 0.005 | |||
| ≥60 | 69 | 14 (20.28) | 55 (79.71) | |
| < 60 | 89 | 5 (5.62) | 84 (94.38) | |
| CR rate (140 analysis) | 71/140 (50.71) | 3/19 (15.79) | 68/121 (56.20) | 0.001 |
| Karyotype (152 analysis) | 0.198 | |||
| Normal | 75 | 12 (16.00) | 63 (84.00) | |
| Abnormal | 77 | 7 (9.09) | 70 (90.91) | |
| Cytogenetic risk group (152 analysis) | 0.069 | |||
| Favorable | 30 | 0 (0.00) | 30 (22.56) | |
| Intermediate | 72 | 11 (57.89) | 61 (45.86) | |
| Adverse | 50 | 8 (42.11) | 42 (31.58) |
2.3. RUNX1基因突变的分子生物学特征
19例RUNX1基因突变的AML患者中，合并FLT3-ITD的患者有7例，占FLT3-ITD基因突变的26.9%，与野生型组比较差异有统计学意义（P < 0.05）；其他基因突变如CEBPA、NPM1、NRAS、IDH2、DNMT3A、ASXL1等均未发现与RUNX1基因突变之间的关联（P > 0.05，表 3）。
3.
RUNX1与细胞遗传学和其他分子突变的相关性
Correlation of RUNX1 with cytogenetic and other molecular mutations
| Variable | RUNX1(+)(n=19) | RUNX1(-)(n=139) | P | |
| ASXL1 | (+) | 4 | 12 | 0.201 |
| (-) | 15 | 127 | ||
| DNMT3A | (+) | 3 | 9 | 0.329 |
| (-) | 16 | 130 | ||
| TET2 | (+) | 3 | 21 | 1.000 |
| (-) | 16 | 118 | ||
| CEBPA (double) | (+) | 0 | 13 | 0.344 |
| (-) | 19 | 126 | ||
| FLT3-ITD | (+) | 7 | 19 | 0.026 |
| (-) | 12 | 120 | ||
| IDH2 | (+) | 4 | 13 | 0.251 |
| (-) | 15 | 126 | ||
| NPM1 | (+) | 2 | 29 | 0.450 |
| (-) | 17 | 110 | ||
| NRAS | (+) | 3 | 13 | 0.641 |
| (-) | 16 | 126 | ||
| C-KIT | (+) | 1 | 13 | 0.874 |
| (-) | 18 | 126 |
2.4. RUNX1基因突变患者对化疗反应CR率的影响
158例AML患者中，140例患者接受了标准剂量的诱导缓解治疗，140例AML总CR率为50.7%。其中RUNX1基因突变患者CR率低于野生型组，差异具有统计学意义（P=0.001，表 2）。对140例AML患者进行随访，随访终点为患者死亡或至2020年6月，RUNX1基因突变组与野生组患者的总生存率差异有统计学意义（P < 0.05，图 3），突变组生存率低于野生组。
3.
RUNX1突变和RUNX1非突变AML分层的KaplanMeier生存曲线
Kaplan-Meier survival curve of the overall survival of AML patients stratified by RUNX1 mutations.
3. 讨论
RUNX1基因是造血功能的关键调节因子，参与造血干细胞的出现和调节，而RUNX1基因突变被认为是经典的“二次打击学说”中的Ⅱ类突变，通过干扰细胞的正常分化而参与AML的发生发展[6]。有研究证实了在细胞遗传学正常的AML患者中RUNX1中出现了体细胞点突变[7]，其中RUNX蛋白由3个主要结构域即RUNX同源结构域、转录激活结构域和抑制结构域组成[8]，而RUNX1基因突变分布在整个RUNX1蛋白中，主要分为两类：N-末端Runx-同源结构域中的错义突变和导致C-末端截短移码或无义突变[9-10]。RUNX1突变导致造血干细胞和祖细胞生长缓慢，生物合成低，细胞表型小，核糖体生物发生明显减少，影响其扩增以及导致髓细胞分化减少，从而作为一种白血病前期状态参与AML的发生发展[11-14]。
本研究发现RUNX1基因在AML患者中的总突变率为12.0%，这与国外报道一致[15-17]。但在RUNX1基因突变组中≥60岁患者和 < 60岁患者的突变率为分别为63.2%、36.8%，均高于国外报道的突变率[20-21]。Khan等[22]发现RUNX1突变在年轻患者中相对比较少见，但是随着年龄的增长，与具有复杂细胞遗传学异常的患者相比，在中等风险细胞遗传学（特别是具有正常核型的那些）的老年AML患者中RUNX1突变频繁地发生。不仅如此，国外有报道称在AML中RUNX1基因突变与患者年龄、性别、低乳酸脱氢酶相关[23-24]，但本研究仅发现RUNX1基因突变更易发生在老年患者组，而与初诊白细胞计数、性别、乳酸脱氢酶水平无明显相关性，推测原因可能与样本量及标本检测方法不同有关，具体需扩大样本量进一步验证。
本研究发现RUNX1基因突变组更易表达CD36、CD7，不易表达CD64、CD117，但差异无统计学意义（P > 0.05），未发现RUNX1基因突变与FAB亚型之间的关联。有研究发现RUNX1突变易发生在M0和特定的染色体畸变如21三体和13三体中[25]。除此之外，国外一项大型多中心的病例分析报道，RUNX1基因突变与FAB分型中的M0亚型、免疫分型中的CD34、HLA-DR密切相关，与CD33负相关[8]。但本研究未发现RUNX1基因突变组与野生型组在这些方面的差异，可能与样本量少以及突变检测方法有关。种族和地区差异所导致的患者亚型分布不同是否造成突变率差异亦有待进一步研究。本研究发现RUNX1基因突变在细胞遗传学中等和不利风险组中比在良好核型AML患者中更易发生，与国外许多报道一致[8, 26]。本文与国外报道一致认为RUNX1基因突变与FLT3-ITD基因突变呈正相关，但不同的是我们亦发现RUNX1突变与ASXL1突变不相关，但有国外报道称RUNX1基因突变与NPM1负相关，与ASXL1密切相关，这种差异可能与本研究样本例数少及基因检测方法有关，但不完全排除种族差异，本研究和他人研究均未发现RUNX1和NRAS基因突变之间的关联[16-19]。
最近有研究表明，突变型RUNX1是某些类型白血病细胞生长和存活所必需的，通过在小鼠和造血细胞中探讨了RUNX1是否直接参与造血细胞对细胞毒性剂的反应，发现RUNX1在转录后被细胞毒性剂上调，在体外用阿糖胞苷处理后，在原发性AML细胞中也观察到上调, 说明在AML、骨髓增生异常综合征和T细胞急性淋巴细胞白血病患者中，RUNX1突变明显与化疗耐药和预后不良有关[4]。本研究进一步证实了RUNX1基因突变组患者经过标准剂量的诱导缓解治疗CR率较野生型组低，且差异有统计学意义，与国外的研究结果具有一致性[17, 27-29]。有研究提出RUNX1突变在骨髓增生异常综合征中的突变率为40%，其中30%~40%发生转化为AML[26]。Kohlmann等[30]提出RUNX1突变作为临床上有用的生物标志物，可以追踪从骨髓增生异常综合征到继发性AML以及白血病微小残留病监测。但有文献报道称RUNX1可作为肿瘤抑制因子，RUNX1功能受损可促进白血病的发展[31]。尽管如此，通过小鼠模型发现RUNX1本身的缺失或显性失活对于白血病的发展是不够的，而需要额外的协作事件。然而，RUNX1功能受损导致随后遗传改变的机制尚不完全清楚，有学者则认为RUNX1活性越低，白血病发生率越高[32]。
综上所述，本研究对RUNX1基因突变在中国人群中初发AML的发病率、临床特征、分子生物学特征以及对化疗的反应进行了报道，其中RUNX1基因突变的统计描述进一步佐证了其作为不利预后的分子生物学地位。在以后的研究中可以把伴随RUNX1突变的AML和无RUNX1突变的AML合理的区别开来，并且为根据是否伴随RUNX1突变而选择更合适的治疗方案提供一定的参考。但RUNX1突变在AML中的作用机制，以及如何利用这些机制为这些患者开发潜在的靶向疗法有待进一步深入研究。
Biography
杨艳丽，主任医师，副教授，硕士生导师，E-mail: yangyanli0702@126.com
Funding Statement
安徽省高校自然科学研究重点项目（KJ2019A0375）
References
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