Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health.
Flow Cytometry Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health.
Chest. 2014 Jan;145(1):108-112. doi: 10.1378/chest.13-1071.
Lymphangioleiomyomatosis (LAM), sporadic or in women with tuberous sclerosis complex (TSC), is characterized by cystic lung destruction, lymphatic involvement (eg, chylous pleural effusions, lymphangioleiomyomas), and renal angiomyolipomas (AMLs). The multisystem manifestations of LAM appear to result from metastatic dissemination of LAM cells bearing inactivating mutations or having loss of heterozygosity (LOH) of the tumor suppressor genes TSC1 or TSC2, which leads to hyperactivation of the mammalian target of rapamycin. Sirolimus slows the decline of lung function, reduces chylous effusions, and shrinks the size of AMLs. The purpose of this study was to determine the effect of sirolimus on circulating LAM cells.
Cells from blood were isolated by a density-gradient fractionation system and from urine and chylous effusions by centrifugation. Blood cells were incubated with anti-CD45-fluorescein isothiocyanate (FITC) and anti-CD235a-R-phycoerythrin (PE) antibodies, and urine and chylous effusion cells were incubated with anti-CD44v6-FITC and anti-CD9-R-PE antibodies. Cells were sorted and analyzed for TSC2 LOH.
LAM cells with TSC2 LOH were identified in 100% of blood specimens and 75% of urine samples from patients before therapy. Over a mean duration of 2.2 ± 0.4 years of sirolimus therapy, detection rates of LAM cells were significantly decreased to 25% in blood (P < .001) and 8% in urine (P = .003). Following therapy, a greater loss of circulating LAM cells was seen in postmenopausal patients (P = .025).
Patients receiving sirolimus had a progressive loss of circulating LAM cells that depended on time of treatment and menopausal status.
淋巴管平滑肌瘤病（LAM），散发性或在结节性硬化症复合征（TSC）女性中，其特征为囊性肺破坏、淋巴累及（例如，乳糜性胸腔积液、淋巴管平滑肌瘤）和肾血管平滑肌脂肪瘤（AML）。LAM 的多系统表现似乎是由于携带失活突变或 TSC1 或 TSC2 肿瘤抑制基因杂合性丢失（LOH）的 LAM 细胞的转移播散所致，导致哺乳动物雷帕霉素靶蛋白（mTOR）的过度激活。西罗莫司可减缓肺功能下降、减少乳糜性渗出液并缩小 AML 的大小。本研究的目的是确定西罗莫司对循环 LAM 细胞的影响。
通过密度梯度分级分离系统从血液中分离细胞，通过离心从尿液和乳糜性渗出液中分离细胞。用抗 CD45-异硫氰酸荧光素（FITC）和抗 CD235a-R-藻红蛋白（PE）抗体孵育血细胞，用抗 CD44v6-FITC 和抗 CD9-R-PE 抗体孵育尿液和乳糜性渗出液细胞。对细胞进行分选并分析 TSC2 LOH。
在治疗前，100%的血液标本和 75%的尿液标本中鉴定出具有 TSC2 LOH 的 LAM 细胞。在接受西罗莫司治疗的平均 2.2 ± 0.4 年期间，LAM 细胞的检出率在血液中显著下降至 25%（P <.001），在尿液中下降至 8%（P =.003）。治疗后，绝经后患者的循环 LAM 细胞丢失更大（P =.025）。
接受西罗莫司治疗的患者存在循环 LAM 细胞的进行性丢失，这取决于治疗时间和绝经状态。
