LAPTM4B与肺癌多基因突变状态及EGFR-TKIs耐药关系的分析

Relationship between LAPTM4B and multigene mutation status and EGFR-TKIs resistance in lung cancer

目的观察溶酶体相关跨膜蛋白4B(LAPTM4B)在非小细胞肺癌(non-small cell lung cancer,NSCLC)不同基因突变状态下的表达,探究LAPTM4B在EGFR-TKIs耐药中的作用。方法收集464例细胞学标本,其中31例为EGFR-TKIs靶向治疗后继发耐药基因T790M突变的标本,离心沉淀制成细胞块,经HE及免疫细胞化学染色确诊为NSCLC,应用实时荧光定量PCR法联合检测多基因(EGFR、ALK、ROS1、RET、KRAS、BRAF、HER-2、NRAS、PIK3CA、c-MET)突变状态;免疫细胞化学染色检测不同基因改变状态下LAPTM4B的表达。收集EGFR-TKIs治疗前的31例标本及配对的EGFR-TKIs治疗后继发耐药基因T790M突变标本,对比分析继发耐药基因T790M突变前后LAPTM4B的表达变化。结果464例NSCLC细胞学标本进行多基因突变联合检测,结果示单基因突变365例(78.7%)、双基因伴随突变14例(3%)、野生型85例(18.3%),阳性率为81.7%。单基因EGFR、ALK、ROS1、RET、KRAS、BRAF、HER-2、NRAS、c-MET突变率分别为59%、6%、3%、2%、4%、2%、2%、0.2%、1%;双基因伴随EGFR+PIK3CA、ALK+KRAS、EGFR+BRAF、EGFR+HER-2、EGFR+RET、EGFR+KRAS、RET+HER-2突变率分别为1%、0.4%、0.4%、0.4%、0.4%、0.2%、0.2%。LAPTM4B蛋白表达与患者性别、年龄无关,与肿瘤分化程度呈正相关(P<0.001),差异有统计学意义。LAPTM4B蛋白表达与基因突变及EGFR基因突变密切相关,差异有统计学意义(P<0.05);与K-RAS等基因突变无关。基因突变型病例相对于基因野生型病例,LAPTM4B蛋白表达增高的风险增加(OR=2.94,95%CI:1.804~4.791)。31例EGFR-TKIs继发耐药基因T790M突变NSCLC标本的LAPTM4B蛋白表达水平高于EGFR-TKIs治疗前标本,差异有统计学意义(P<0.05)。结论LAPTM4B蛋白表达水平与NSCLC肿瘤分化程度及EGFR基因突变相关。LAPTM4B在EGFR-TKIs继发耐药基因T790M突变时表达增加,可能是EGFR-TKIs继发耐药产生的机制。

Purpose To investigate the relationship between lysosomal-associated transmembrane protein 4B(LAPTM4B)and multigene mutations in non-small cell lung cancer(NSCLC),and to explore the role of LAPTM4B in resistance of EGFR-TKIs.Methods A total of 464 cytological specimens were collected,including 31 cases with secondary drug resistance gene T790M mutation after EGFR-TKIs targeted therapy.All the samples were centrifuged to retain residual precipitation for cell block.All cases were diagnosed as NSCLC by HE staining and immunocytochemical staining(ICC).The mutation status of multiple genes(EGFR,ALK,ROS1,RET,KRAS,BRAF,HER-2,NRAS,PIK3CA,c-MET)was detected by real-time fluorescence PCR assay.The differences in the expression of LAPTM4B were studied to explore the relationship between LAPTM4B and multiple gene mutations by ICC.31 samples without secondary drug resistance gene T790M mutation before EGFR-TKIs treatment were also collected.The different expression levels of LAPTM4B with or without secondary T790M mutation were compared analyzed.Results Among 464 NSCLC cytological specimens which were tested for multigene mutations,365 cases(78.7%)presented with single gene mutation,14 cases(3%)with two concomitant mutations and 85 cases(18.3%)with wild-type.The positive rate was 81.7%.Single gene EGFR,ALK,ROS1,RET,KRAS,BRAF,HER-2,NRAS,or c-MET mutations were detected in 59%、6%、3%、2%、4%、2%、2%、0.2%、1%cases respectively.3%harboring two concomitant mutations were found as follows:1%cases with EGFR and PIK3CA mutations,0.4%with ALK and KRAS mutations,0.4%with EGFR and BRAF mutations,0.4%with EGFR and HER-2 mutations,0.4%with EGFR and RET mutations,0.2%with EGFR and KRAS mutations,0.2%with RET and HER-2 mutations.The expression levels of LAPTM4B were positively correlated with tumor differentiation,which was statistically significant(P<0.001).However,no statistically significant correlations were identified between the LAPTM4B levels and gender or age.The expression levels of LAPTM4B were significantly correlated with gene mutations and EGFR gene mutation,and the difference was statistically significant(P<0.05).But,it was not associated with other genes mutations such as KRAS and so on.The increased risk of LAPTM4B expression level in mutated genotype was higher than that in wild-type(OR=2.94,95%CI:1.804-4.791).In addition,LAPTM4B protein expression in 31 cases of NSCLC samples with secondary drug resistance gene T790M mutation was dramatically increased compared with EGFR-TKIs sensitive cases,and the difference was statistically significant(P<0.05).Conclusion The expression levels of LAPTM4B are significantly correlated with tumor differentiation and EGFR gene mutations.The expression of LAPTM4B is increased in secondary drug resistance of EGFR-TKIs with T790M mutation,which may be the mechanism of EGFR-TKIs drug resistance.