探讨儿童急性B前体淋巴细胞白血病(BCP-ALL)铁转出蛋白(Fpn)表达水平与其临床特征和预后的关系。

选择2011年2月至2014年6月四川大学华西第二医院收治的64例BCP-ALL患儿为研究对象，纳入研究组。对其统一按照中国儿童白血病协作组(CCLG)-急性淋巴细胞白血病(ALL) 2008方案进行分型诊断和治疗。采用随机数字表法随机选择同期于本院健康体检的21例健康儿童，纳入对照组。采用荧光定量逆转录-聚合酶链反应(RT-PCR)技术分别检测研究组BCP-ALL患儿初诊时及对照组受试者体检时骨髓和外周血单个核细胞的Fpn表达水平。以Fpn相对表达量(0.18)为界值进行划分，研究组Fpn相对表达量>0.18为Fpn高表达患儿，纳入Fpn高表达亚组(n＝32)，Fpn相对表达量≤0.18为Fpn低表达患儿，纳入Fpn低表达亚组(n＝32)。采用Kaplan-Meier法计算研究组患儿无复发生存(RFS)率、无事件生存(EFS)率和总生存(OS)率。统计学分析Fpn表达水平与研究组BCP-ALL患儿的临床特征、免疫表型、ALL相关融合基因、早期治疗反应、临床危险度及其预后的关系。本研究遵循的程序符合四川大学华西第二医院人体试验委员会制定的伦理学标准，得到该委员会批准。

①研究组与对照组受试者，以及Fpn高、低表达亚组患儿的性别构成比和年龄分布分别比较，差异均无统计学差异(P>0.05)。②研究组Fpn中位相对表达量(0.18)显著低于对照组(2.19)，差异有统计学意义(U＝1 415.0，P<0.001)。③研究组患儿初诊时白细胞计数<50×10⁹/L(47例)和白细胞计数≥50×10⁹/L(17例)患儿的Fpn中位相对表达量分别为0.23和0.04，二者比较，差异亦有统计学意义(U＝399.0，P＝0.02)。分别按照初诊时中位白细胞计数(21.1×10⁹/L)和中位初诊幼稚细胞绝对计数(14.1×10⁹/L)进行划分，研究组初诊时高、低中位白细胞计数和高、低中位幼稚细胞绝对计数患儿的Fpn中位相对表达量均分别为0.09和0.28，差异均有统计学意义(U＝870.0、878.0，P＝0.02、0.03)。研究组患儿中，Fpn高、低表达亚组患儿初诊时中位白细胞计数及中位幼稚细胞绝对计数分别为15.4×10⁹/L和29.3×10⁹/L，8.2×10⁹/L和21.3×10⁹/L，差异亦均有统计学意义(U＝863.5、866.0，P＝0.018、0.019)。Fpn相对表达量与初诊时白细胞计数及幼稚细胞绝对计数均呈显著负相关关系(r_s＝－0.357、－0.366，P＝0.004、0.003)。④Fpn相对表达量与BCP-ALL患儿初诊年龄、性别，以及ALL免疫表型、融合基因类型、糖皮质激素耐药、危险度分组和早期治疗反应均无明确相关关系(P>0.05)。Fpn高、低表达亚组患儿不同临床特征的构成比比较，差异亦无统计学意义(P>0.05)。⑤研究组患儿的中位随访时间为13个月(2～50个月)。Kaplan-Meier生存分析结果显示，Fpn高、低表达亚组患儿的3年RFS率、EFS率、OS率分别为74.4%与61.7%、68.0%与62.4%、85.0%与74.4%，组间比较，差异均无统计学意义(χ²＝0.975、0.102、0.576，P＝0.323、0.749、0.448)。

BCP-ALL细胞Fpn表达水平显著低于正常外周血单个核细胞，并与初诊时白细胞计数和幼稚细胞绝对计数呈显著负相关关系。这高度提示Fpn表达下调有助于细胞内铁的阻滞，满足淋巴白血病细胞旺盛代谢对铁的需求。与乳腺癌等恶性实体肿瘤一样，这可能也是淋巴白血病细胞增殖异常的重要铁代谢调控机制。

To study the expression levels of ferroportin (Fpn) in lymphoblastic leukemia cells in children with precursor B-cell acute lymphoblastic leukemia (BCP-ALL), and to explore the possible correlations with various clinical features and treatment outcomes.

Sixty-four children with newly diagnosed BCP-ALL in West China Second Hospital from February 2011 to June 2014 were enrolled into this study as study group, and were risk-stratified and treated according to the Chinese Children Leukemia Group ALL 2008 (CCLG-ALL 2008) protocol. Twenty-one healthy children for health check-up in our hospital during the same period were chosen by random number table as control group. Levels of Fpn relative media expression levels in bone marrow and peripheral mononuclear cells isolated from leukemic and control group children were determined respectively by fluorescence real-time reverse transcription polymerase chain reaction (RT-PCR). Level of Fpn relative media expression 0.18 was set as the threshold of Fpn relative media expression levels, patients with level of Fpn relative expression >0.18 in study group were enrolled into Fpn high expression levels subgroup (n=32), while patients with level of Fpn relative expression ≤0.18 in study group were enrolled into Fpn low expression levels subgroup (n=32). Prognosis in terms of relapse-free survival (RFS) rate, event-free survival (EFS) rate and overall survival (OS) rate were calculated by Kaplan-Meier method. Correlations between relative median Fpn expression levels and various clinical features, immunophenotype, ALL related fused gene, early treatment response, clinical risk degree and prognosis were analyzed by statistical methods. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of West China Second University Hospital, Sichuan University.

①There were no statistical differences among the gender ratio and age distribution between study group and control group, Fpn high expression levels subgroup and Fpn low expression levels subgroup in study group, respectively (P>0.05). ②The relative median Fpn expression level in study group (0.18) was significantly lower than that in control group (2.19) (U=1 415.0, P<0.001). ③ The relative median Fpn expression levels in patients with initial white blood cell count <50×10⁹/L (47 cases) and those with white blood cell count ≥50×10⁹/L (17 cases) were 0.23 and 0.04 respectively, relative relative and there was statistical difference between them (U=399.0, P=0.02). With the median initial white blood cell count (21.1×10⁹/L) and median absolute blast count (14.1×10⁹/L) as the cutoff points respectively, relative median Fpn expression levels in patient with high and low cell counts were 0.09 and 0.28, respectively (U=870.0, P=0.02), 0.09 and 0.28 (U=878.0, P=0.03), respectively. Similarly, with relative median Fpn expression as the cutoff point, the median white blood cell count and median absolute blast count in Fpn high expression levels subgroup and Fpn low expression levels subgroup were 15.4×10⁹/L and 29.3×10⁹/L (U=863.5, P=0.018), 8.2×10⁹/L and 21.3×10⁹/L (U=866.0, P=0.019) respectively. In addition, Spearman rank correlation analysis showed that Fpn expression level was negatively correlated to both initial white blood cell count and absolute blast counts (r_s=-0.357, -0.366; P=0.004, 0.003). ④No correlations were documented between Fpn expression levels and age at diagnosis, gender, leukemia immunophenotyping, fusion genes, glucocorticoid resistance, risk assignment and early chemotherapy response (P>0.05). No significant differences were disclosed between Fpn high and low expression levels subgroups , in terms of patient proportions with different clinical features (P>0.05). ⑤The median follow-up time was 13 months (2-50 months) in study group. Up to the follow-up endpoint, the rates of 3-year RFS, EFS and OS were 74.4% and 61.7% (χ²=0.975, P=0.323), 68.0% and 62.4% (χ²=0.102, P=0.749), 85.0% and 74.4% (χ²=0.576, P=0.448) in Fpn high and low expression levels subgroups respectively.

Our research findings that Fpn expression level is remarkably down-regulated as compared to normal children, and is negatively correlated to both initial white blood cell count and absolute blast cell count in peripheral blood, which strongly suggest that decreased Fpn expression level in highly proliferating lymphoblastic leukemia cells plays key role in sequestering iron within cells, in order to meet iron demand for enhanced cellular metabolism and proliferation. The dysregulated Hepcidin (Hepc)-Fpn axis might turn out to be an important underlying mechanism of cellular iron modulation in lymphoblastic leukemia cells, as revealed by recent studies in breast cancer cell lines and patients.