Matherly L H, Taub J W, Ravindranath Y, Proefke S A, Wong S C, Gimotty P, Buck S, Wright J E, Rosowsky A
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
Blood. 1995 Jan 15;85(2):500-9.
A retrospective study of clinical resistance to methotrexate (MTX) was performed on 29 archival specimens of frozen lymphoblasts obtained from children with acute lymphoblastic leukemia (ALL), including 19 at initial presentation and 10 at first relapse. Blasts were assayed for dihydrofolate reductase and MTX transport by flow cytometry using the fluorescent methotrexate analog, PT430 (Rosowsky et al, J Biol Chem 257:14162, 1982). In contrast to tissue culture cells, patient blasts were often heterogeneous for dihydrofolate reductase content. Of the 19 specimens at initial diagnosis, 7 exhibited dual blast populations, characterized by threefold to 10-fold differences in relative dihydrofolate reductase; the dihydrofolate reductase-overproducing populations comprised 12% to 68% of the total blasts for these specimens. Remission duration intervals for patients exhibiting dual blast populations were notably shorter than for patients expressing a single blast population with lower dihydrofolate reductase ( < or = 9 months v > or = 15 months, respectively), a difference that was statistically significant (P = .045). There was no apparent correlation between expression of increased dihydrofolate reductase at diagnosis and known patient and disease prognostic features (immunophenotype, age, sex, and white blood count). For the relapsed patients, 4 of 10 exhibited dual lymphoblast populations with elevated dihydrofolate reductase. The majority of the patient lymphoblast specimens were entirely competent for MTX transport and, likewise, expressed immunoreactive reduced folate carriers by indirect immunofluorescence staining with specific antiserum to the transporter. Three patients (2 at relapse and 1 at diagnosis) exhibited heterogeneous expression of imparied MTX transport (14% to 73% of blasts). In only 1 of these patients did the majority of the lymphoblasts (73%) show impaired MTX transport and for this specimen, immunoreactive carrier proteins were virtually undetectable. These results suggest that heterogeneous expression of elevated dihydrofolate reductase and impaired MTX transport are important modes of resistance in childhood ALL patients undergoing chemotherapy with MTX and that these parameters may serve as predictive indices of clinical response to MTX.
对29份取自急性淋巴细胞白血病（ALL）患儿的冷冻淋巴母细胞存档标本进行了甲氨蝶呤（MTX）临床耐药性的回顾性研究，其中19份取自初诊时，10份取自首次复发时。使用荧光甲氨蝶呤类似物PT430，通过流式细胞术检测淋巴母细胞中的二氢叶酸还原酶和MTX转运情况（Rosowsky等人，《生物化学杂志》257:14162，1982年）。与组织培养细胞不同，患者的淋巴母细胞中二氢叶酸还原酶含量往往存在异质性。在初诊的19份标本中，7份呈现双淋巴母细胞群体，其相对二氢叶酸还原酶存在3倍至10倍的差异；这些标本中，二氢叶酸还原酶过度产生的群体占总淋巴母细胞的12%至68%。出现双淋巴母细胞群体的患者缓解持续时间明显短于二氢叶酸还原酶水平较低的单一淋巴母细胞群体的患者（分别为≤9个月对≥15个月），这一差异具有统计学意义（P = 0.045）。诊断时二氢叶酸还原酶表达增加与已知的患者和疾病预后特征（免疫表型、年龄、性别和白细胞计数）之间没有明显相关性。对于复发患者，10例中有4例呈现二氢叶酸还原酶升高的双淋巴母细胞群体。大多数患者的淋巴母细胞标本完全具备MTX转运能力，同样，通过用针对转运蛋白的特异性抗血清进行间接免疫荧光染色，也表达免疫反应性还原叶酸载体。3例患者（2例复发，1例初诊）呈现MTX转运受损的异质性表达（占淋巴母细胞的14%至73%）。在这些患者中，只有1例大多数淋巴母细胞（73%）显示MTX转运受损，并且对于该标本，几乎检测不到免疫反应性载体蛋白。这些结果表明，二氢叶酸还原酶升高和MTX转运受损的异质性表达是接受MTX化疗的儿童ALL患者耐药的重要模式，并且这些参数可能作为对MTX临床反应的预测指标。
