上皮生長因子受器抑制劑原發或後天性抗藥性的探索

Acquired and Primary Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

吳尚俊(Shang-Gin Wu)

指導教授 ： 施金元 ； 楊泮池

國立臺灣大學/醫學院/臨床醫學研究所/博士(2016年)

https://doi.org/10.6342/NTU201700068

摘要

緒論：精準醫療已經越來越受重視。病人腫瘤帶有上皮生長因子受器基因突變(EGFR mutation)對上皮生長因子受體激酶抑制劑（EGFR TKI）有著很好的治療反應。但後天抗藥性終究會產生。後天性的上皮生長因子受器基因突變，T790M，可以在一半對上皮生長因子受體激酶抑制劑產生抗藥性的病人腫瘤中偵測到，但依然有很多我們未知後天性抗藥性機制。除了後天性抗藥性的產生，在臨床試驗中，大約有4~10%帶有上皮生長因子受器基因突變的病人，對上皮生長因子受體激酶抑制劑產生了原發性抗藥性，其中，上皮生長因子受器在exon 20的基因突變，或是原發性T790M的存在，都是可能的因素，另外，上皮生長因子受體下游的PIK3和BIM都可能有關係，而BIM和細胞凋亡有關係，而上皮生長因子受體激酶抑制劑就是透過細胞凋亡造成腫瘤縮小，因此我們開始了一連串的上皮生長因子受體激酶抑制劑抗藥性研究。
病患及研究方法：我們收集肺腺癌組織標本，萃取RNA或DNA，用以進一步以RT-PCR的方式以及基因定序的方式，針對相關的致癌驅動基因的突變進行偵測，另外也收集後天性抗藥性產生後的檢體，進行基因突變檢測外，也加入了病理學的詳細觀察，另外，根據上皮生長因子受體激酶抑制劑藥物使用的狀況以及治療反應，結合臨床特徵以及存活分析，進而分析影響 上皮生長因子受體激酶抑制劑產生原發性或後天性抗藥性的機制預後因子。
另外，我們也以微陣列分析的方式，去搜尋可能的抗藥性基因突變，進而進行細胞學的實驗驗證，並且藉由在抗藥性細胞進行專一性基因剔除，來確認預測的基因的功能以及引起抗藥性機制的研究，最後再收集臨床檢體，對腫瘤切片進行免疫化學染色或是測定病人血中基因表現量，來分析此搜尋到的基因是否真的在臨床上會造成上皮生長因子受體激酶抑制劑抗藥性。
結果：首先，我們探究引起第二代上皮生長因子受體激酶抑制劑，afatinib，的後天性抗藥性機轉。在42個對afatinib產生後天性抗藥性後取得的組織標本綜合分析，後天性T790M產生的比例為一半，至於其他的致癌基因突變是沒有偵測到；也沒有發現有病裡組織學型態的轉變，接著探究EGFR下游的因子，PIK3CA基因突變，在抗藥性上的角色，我們收集大量的肺腺癌組織標本用以偵測PIK3CA基因突變，我們從760個藥物治療前的檢體發現，發現肺腺癌的PIK3CA基因突變率為1.8%，同時，有很高比率的病人(85.7%）是同時有PIK3CA和上皮生長因子受器基因突變，而且PIK3CA基因突變並未對上皮生長因子受體激酶抑制劑的治療效果和無疾病惡化存活期有影響。比較上皮生長因子受體激酶抑制劑治療前和產生後天性抗藥性後(2.9%)取得的組織標本，兩者的PIK3CA基因突變率並無統計學上的差別(p = 0.344)。除了PIK3CA，透過比較56個對上皮生長因子受體激酶抑制劑原發性抗藥性與271個有治療部分反應的患者，當中有52個病人(15.9%)有BIM的刪除多型性，我們並沒有發現BIM刪除的多型性會對晚期腺癌患者的治療反應有影響(16.1% 對 15.9%；p = 0.970)，而且對藥物的無疾病惡化存活期也沒有差別(10.5個月對 8.5個月；p = 0.340)。即使選擇經典型上皮生長因子受體基因突變的病人，結果一樣。
在基礎研究方面，我們使用 cDNA 微陣列分析對上皮生長因子受體激酶抑制劑敏感及抗藥性的細胞株，篩選出對上皮生長因子受體激酶抑制劑抗藥性的細胞有較高 IGFBP-7 之表現，收集臨床病人的惡性胸水去驗證 IGFBP-7 在使用上皮生長因子受體激酶抑制劑前、後的表現，也發現在產生後天性抗藥性後，胸水中腫瘤細胞的 IGFBP-7 有明顯的上升。之後，我們在有抗藥性的細胞做專一性基因剔除IGFBP-7，除了重新對上皮生長因子受體激酶抑制劑恢復感受性，並且透過對 BIM 的抑制增加了上皮生長因子受體激酶抑制劑治療後的細胞凋亡，此抗藥性的訊號傳遞是藉由抑制 IGF-1R 形成。藉基因轉殖建立了有過量表達IGFBP-7 的穩定細胞株，相對於控制組，在經過 gefitinib 治療後，可見較不易因 gefitinib而細胞凋亡，但對於腫瘤在 gefitinib 下的存活，兩組並無明顯的差距。接著，我們收集臨床檢體進行進一步的驗證IGFBP-7對於上皮生長因子受體激酶抑制劑抗藥性的角色，在41個第一線使用 gefitinib 的病人檢體做免疫化學染色，可以見到有較強 IHC 表現的病人，有較短的腫瘤無疾病惡化存活期。從75個病人的血液中以ELISA測量IGFBP-7的表現，有較高血中濃度的IGFBP-7的病人，同樣有較短的腫瘤無疾病惡化存活期。
結論： T790M依然是對afatinib最主要的後天性抗藥性機制，我們並沒有發現其他的後天性致癌基因突變或病理學型態的轉變。而我們從藥物治療前的檢體發現，發現肺腺癌的PIK3CA基因突變率很低，同時，有很高比率的病人是同時有PIK3CA和上皮生長因子受器基因突變。比較上皮生長因子受體激酶抑制劑治療前和產生後天性抗藥性後取得的組織標本，兩者的PIK3CA基因突變率很類似。除此之外，根據我們一連串的研究，PIK3CA基因突變和BIM的刪除多型性都不會對上皮生長因子受體激酶抑制劑產生原發性抗藥性影響。而抑制了IGFBP-7會藉由抑制 IGF-1R 的磷酸化，恢復抗藥性細胞對上皮生長因子受體激酶抑制劑的感受性，IGFBP-7是一個充分但非必要的抗藥性機制。
我們根據一連串對上皮生長因子受體激酶抑制劑抗藥性的研究的結果可以使我們更瞭解相關的抗藥性機制。藉此，希望能提出的一種新的治療肺癌策略，帶來一個可以克服上皮生長因子受體激酶抑制劑抗藥性新的時代。

關鍵字

肺癌 ； 肺腺癌 ； 標靶治療 ； 精準治療 ； 上皮生長因子受體酪氨酸激酶抑製劑 ； 後天性抗藥性 ； 原發姓抗藥性 ； T790M ； BIM ； 多形性 ； PIK3CA ； IGFBP-7

並列摘要

Introduction: Precision medicine plays an important role for lung cancer treatment. The epidermal growth factor receptor (EGFR) mutation positive patients of lung adenocarcinoma had favored response to EGFR tyrosine kinase inhibitors (TKIs). However, acquired drug resistance developed invariably. Secondary T790M formation is the major acquired resistance mechanism. In addition to T790M, there were still some unknown mechanisms of acquired resistance to EGFR TKI. In addition, 4-10% EGFR mutation-positive patients suffered from primary (intrinsic) resistance to EGFR TKIs. Exon-20 insertion or de novo T790M had been considered the causes of primary resistance. The downstream factors, PI3KCA mutation and BIM deletion polymorphism may also have impact on treatment effectiveness. We want to explore the primary and acquired resistance of EGFR TKIs.
Material and Methods: We collected lung adenocarcinoma tissue specimens, including: lung biopsy, malignant pleural effusion and surgical excision tumors. Oncogenic mutation analysis by RT-PCR. PCR and fragment length analysis was used to detect BIM polymorphism. Primary resistance was defined as clinical disease progression within 3 months or the first CT examination following EGFR TKI treatment showed objective disease progression. Acquired EGFR TKI resistance was defined according to Jackman’s clinical criteria. Patients’ clinical characteristics, EGFR TKI treatment response, and progression-free survival were analyzed. Statistical analysis was performed using Chi-square test and Kaplan-Meier method.
In addition, we looked for the putative gene according to microarray data in comparison with TKI-sensitive and TKI-resistance lung adenocarcinoma cells. The function and expression of the putative gene was validated by knockdown or overexpression of the putative gene in vitro. We collected surgical specimens and peripheral blood to detect the expression level of the putative gene to analysis the correlation between the gene and treatment efficacy of EGFR TKI.
Results: First, we explored the mechanism of acquired resistance to afatinib. Forty-two patients had tissue specimens taken after acquiring resistance to afatinib. The sensitizing EGFR mutation were all consistent between pre- and post-afatinib tissues. Twenty patients (47.6%) had acquired T790M mutation. T790M rate was not different between first-generation EGFR TKI-naïve patients (50%) and first-generation EGFR TKI-treated patients (46.4%) (p = 0.827). No clinical characteristics or EGFR mutation types were associated with the development of acquired T790M. No other second-site EGFR mutations were detected. There were no small cell or squamous cell lung cancer transformation. Other genetic mutations were not identified in PIK3CA, BRAF, HER2, KRAS, NRAS, MEK1, AKT2, LKB1 and JAK2.
In addition to EGFR mutation, variable oncogenic driver mutation had been studied. Some driver mutations had impact on EGFR TKI treatment efficacy and also cause primary or acquired resistance. PIK3CA was the one of the oncogenic driver mutations in lung adenocarcinoma. To understand the impact of PIK3CA mutations on clinical characteristics and treatment response to EGFR TKIs of lung adenocarcinoma, we examined PIK3CA and EGFR mutations in lung adenocarcinoma patients, and analyzed their clinical outcomes. Surgically excised tumor, bronchoscopy biopsy/brushing specimens and pleural effusions were prospectively collected from 1029 patients. PIK3CA and EGFR mutations were analyzed by RT-PCR and direct sequencing. This study showed that the PIK3CA mutation could be detected in a small proportion (1.8%) of lung adenocarcinomas, but with high concomitant EGFR mutations. PIK3CA mutation did not confer primary resistance to EGFR TKIs, nor was it associated with a shorter PFS. The PIK3CA mutation rates were similar between tissues that are EGFR TKI-naïve (1.8%) and those with acquired resistance to EGFR TKI (2.9%). According to the paired tissue specimens between EGFR TKI-naïve and acquired resistance to EGFR TKI, the acquired PIK3CA (E545K) mutation can be detected in only one of 74 patients (1.4%).
We also studied whether the BIM deletion polymorphism caused primary resistance of EGFR TKIs. During June 2005 to December 2012, we enrolled the 327 EGFR mutation-positive patients, 56 patients experienced primary resistance and 271 patient had partial response to EGFR TKI. Fifty-two patients (15.9%) had tumors with BIM deletion polymorphism. The BIM deletion frequency was not different between the two groups (16.1% vs. 15.9%; p = 0.970). BIM deletion polymorphism was also unrelated to EGFR mutation types (p = 0.449). BIM deletion polymorphism did not confer a shorter PFS (p = 0.335), even in lung adenocarcinoma patients with classical EGFR mutations (p = 0.386). The frequency of BIM deletion polymorphism in EGFR mutation-positive patients was similar between those with primary resistance and those with partial response to EGFR TKI, even in patients harboring tumors with classical EGFR mutations. In addition, there was no correlation between BIM polymorphism and PFS of EGFR TKI in EGFR-mutation positive patients. Therefore, BIM deletion polymorphism does not account for intrinsic resistance to EGFR TKI.
In addition to clinical study, we used cDNA microarray to screen differentially expressed genes between EGFR TKI-sensitive and acquired EGFR TKIs-resistance cell lines. The expression levels of the screened genes were validated by RT-PCR and Western blot. IGFBP-7 is one of the genes associated with the development of acquired resistance to gefitinib in lung adenocarcinoma cell line.
IGFBP-7 is over-expressed in gefitinib-resistant cells. The IGFBP-7 mRNA expression in the malignant pleural effusion of patients with acquired resistance to EGFR TKI was significant higher than that in the treatment-naïve patients. Knockdown IGFBP-7 reverses gefitinib resistance in PC9/IR cells. Knockdown IGFBP-7 could increase gefitinib induced-apoptosis. IGFBP-7 induced EGFR TKI resistance is associated with anti-apoptosis by suppression of BIM. IGFBP-7 affected the mechanism of EGFR TKI resistance resulted from IGF-IR. Although overexpression IGFBP-7 could decrease gefitinib induced apoptosis, cell viability assay did not showed difference after treating gefitinib.
In clinical tissue samples, low IGFBP-7 serum level is associated with longer progression free survival of EGFR TKI as the first line treatment in lung adenocarcinoma patients. For early stage lung adenocarcinoma patients, lower IGFBP-7 level of resected tumor predicts a longer 5-year tumor relapse-free survival. Positive IGFBP-7 immunohistochemical stain could predict a shorter PFS of the first-line EGFR TKI treatment.
Conclusion: Acquired T790M mutation is still the most common mechanism of acquired resistance to afatinib, a second-generation EGFR TKI. The prevalence of acquired T790M were similar in patients with and without prior exposure to first-generation EGFR TKI. Besides, PIK3CA mutation may not be associated with primary resistance to EGFR TKI among lung adenocarcinoma patients. Acquired PIK3CA mutation related to EGFR TKI treatment is rare. BIM deletion polymorphism also does not confer primary resistance to EGFR TKI. IGFBP-7 may be a sufficient, but not necessary factor to confer resistance to the EGFR TKI through inhibition of IGF-1R.
We understood the resistance mechanism of EGFR TKIs according to the result of the serial studies. This will bring a new era to the overcoming resistance to EGFR TKI and suggest a new treatment strategy of lung cancer.

並列關鍵字

lung cancer ； adenocarcinoma ； target therapy ； precision medicine ； EGFR ； tyrosine kinase inhibitor ； acquired resistance ； primary resistance ； T790M ； BIM ； polymorphism ； PIK3CA ； IGFBP-7

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