晚期非小细胞肺癌EGFR蛋白磷酸化、基因突变与EGFR-TKI疗效相关性的研究

Relationship between EGFR protein phosphorylation,EGFR mutation and EGFR-TKI efficacy in advanced non-small cell lung cancer

背景与目的：近年来以吉非替尼和厄洛替尼为代表的表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitors，EGFR-TKI），因其在晚期非小细胞肺癌（advanced non-small cell lung cancer，NSCLC）治疗中独特的临床疗效和较低的不良反应而备受关注。尽管EGFR基因突变是目前认为最确切的预测EGFR-TKI疗效的指标，但与临床疗效间并非“全或无”的关系，提示仍有其他机制参与其中。本研究旨在探讨晚期NSCLC组织标本中EGFR磷酸化酪氨酸1068（EGFR-pTyr1068）、1173（EGFR-pTyr1173）表达与EGFR基因突变的关系，及其在EGFR-TKI治疗中的疗效预测价值。方法：采用变性高效液相色谱法（denaturing high performance liquid chromatography，DHPLC）检测205例晚期NSCLC患者组织中EGFR基因突变（19、21外显子突变）情况；并采用免疫组化方法检测其EGFR-pTyr1068、EGFR-pTyr1173表达。结果：晚期NSCLC患者组织中EGFR-pTyr1068和1173表达阳性率分别为80.0%（164/205）、57.6%（95/165）；其表达与临床病理特征（年龄、性别、病理类型、吸烟状态、疾病分期）无相关性。全组EGFR基因突变率为44.9%（92/205），与吸烟状态有关（P=0.024），而与其他临床病理特征（性别、年龄、病理类型、疾病分期）无关。EGFR基因突变与EGFR-pTyr1068表达呈弱相关性（P〈0.001），与EGFR-pTyr1173无相关性（P=0.297）。EGFR基因突变型患者EGFR-TKI治疗的客观缓解率（objective response rate，ORR）、疾病控制率（disease control rate，DCR）和中位无进展生存期（progress free survival，PFS）分别为48.3%（43/89）、80.9%（72/89）和8.8个月（95%CI：6.11～11.42），均明显高于EGFR基因野生型患者[16.2%（17/105）、56.2%（59/105）和2.1个月，95%CI：0.89～3.24]，差异有统计学意义（P〈0.001，P〈0.001，P=0.024）；EGFR-pTyr1068表达阳性患者ORR和DCR分别为37.7%（58/154）和74.7%（115/154），均明显高于表达阴性患者[5.0%（2/40）和40.0%（16/40）]，差异有统计学意义（P〈0.001）。EGFR-pTyr1068表达阳性患者中位PFS为7.0个月，较表达阴性患者（1.2个月）明显延长，差异有统计学意义（P〈0.001）。而EGFR-pTyr1173表达与EGFR-TKI疗效呈负相关性，EGFR-pTyr1173阳性者ORR、DCR和PFS分别为27.8%（25/90）、64.4%（58/90）和4.8个月，显著低于阴性患者[37.9%（25/66）、83.3%（55/66）和7.7个月，P=0.123，P=0.007，P=0.016]。以EGFR基因突变状态分层进行亚组分析显示，在EGFR基因野生型患者中，EGFR-pTyr1068表达阳性率为69.0%（69/100）， EGFR-pTyr1068表达阳性和阴性患者ORR分别为23.2%（16/69）和3.2%（1/31），DCR分别为69.6%（48/69）和35.5%（11/31），差异均有统计学意义（P=0.010，P=0.001）；EGFR-pTyr1068表达阳性患者中位PFS为3.6个月，较表达阴性患者（1.2个月）明显延长，差异有统计学意义（P〈0.001）。16例EGFR-pTyr1068阳性表达且对EGFR-TKI有效患者，中位PFS为15.6个月（95%CI：7.28～23.9）。多因素分析显示，EGFR-pTyr1068是EGFR基因野生型患者EGFR-TKI治疗的独立疗效预测因子（OR=0.24，95%CI：0.16～0.37，P〈0.001）。结论：EGFR-pTyr1068可作为晚期NSCLC患者接受EGFR-TKI治疗的有效预测因子，尤其对从EGFR基因野生型患者中筛选EGFR-TKI治疗有效者具有重要作用。

Background and purpose：EGFR-TKI （EGFR-tyrosine kinase inhibitors）, represented by geiftinib and erlotinib, have exhibited signiifcant antiproliferative effects against non-small cell lung cancer （NSCLC） with low toxicity.EGFR gene mutation was discovered to be a predictive biomarker for EGFR-TKI treatment. Although the efifcacy of EGFR-TKI is limited toEGFR wild-type patients, it is still noticeable suggesting that some other mechanisms are responsible for it. The current study is aimed at evaluating the expression of phosphorylated EGFR in advanced NSCLC, investigating its relationship withEGFR mutations and EGFR-TKI efifcacy.Methods：EGFR gene mutations were detected by denaturing high performance liquid chromatography （DHPLC） in 205 stageⅢB-ⅣNSCLC patients. The expressions of phosphorylated tyrosine 1068 （pTyr1068） and 1173 （pTyr1173） were detected by immunohistochemistry.Results：The positive expressions of pTyr1068 and pTyr1173 were 80.0% （164/205） and 57.6% （95/165） respectively. None of them were related to clinical pathological characteristics （age, gender, pathological type, smoking status, disease stage）.EGFR gene mutation rate was 44.9% （92/205）, which was only related to smoking status （P=0.024） compared to other clinical pathological characteristics.EGFR gene mutations were poorly related to pTyr1068 expression （P〈0.001） and not related to pTyr1173 expression （P=0.297）. The objective response rate （ORR）,disease control rate （DCR）, and progressive free survival （PFS） of EGFR-TKI treatment in patients withEGFR mutations were 48.3% （43/89）, 80.9% （72/89） and 8 months （95%CI： 6.11-11.42） respectively, which were signiifcantly higher than that ofEGFR wild-type patients [ORR=16.2% （17/105,P〈0.001）; DCR=56.2%（59/105,P〈0.001）; Median PFS： 2.1 months, （95%CI： 0.89-3.24;P=0.001）]. Superior ORR： DCR and PFS appeared in patients with pTyr1068 positive expression compared to negative [ORR： 37.7% （58/154）vs 5.0% （2/40,P〈0.001）; DCR： 74.7% （115/154）vs 40.0% （16/40,P〈0.001）; Median PFS： 7.0 monthsvs 1.2 months,P〈0.001）]. Inversely, the patients with pTyr1173 positive expression had lower ORR, DCR and shorter PFS [ORR： 27.8% （25/90）vs 37.9%（25/66,P=0.123）; DCR： 64.4% （58/90）vs 83.3% （55/66,P=0.007）; Median PFS： 4.8 monthsvs 7.7 months （P=0.016）]. In subgroup ofEGFR wild-type patients, positive expression of pTyr1068 was 69.0% （69/100）.EGFR wild-type patients with pTyr1068 positive expression had a prolonged PFS and elevated ORR and DCR compared to negative [median PFS： 3.6 monthsvs 1.2 months （P〈0.001）; ORR： 23.2%vs 3.2% （P=0.010）; DCR： 69.6%vs 35.5% （P=0.001）]. Sixteen patients with pTyr1068 positive expression who responded to EGFR-TKI treatment in this subgroup had a remarkable PFS [median PFS： 15.6 months （95%CI：7.28-23.9）]. Multiple factor analysis showed that the expression of pTyr1068 was an independence predictor factor for EGFR-TKI treatment （OR=0.24, 95%CI： 0.16～0.37,P〈0.001）. Conclusion：Phosphorylation at Tyr1068 of EGFR might be a potential predictive factor for clinical response and survival of EGFR-TKI treatment in patients with advanced NSCLC, especially inEGFR wild-type patients.