以RNA進行非小細胞肺癌標靶治療的基因檢驗：應用於惡性肋膜積水與支氣管鏡刷取等細胞學檢體

Abstract

標靶治療的發展是非小細胞肺癌治療的重大進展。近來一些重要的臨床試驗顯示腫瘤細胞的特定基因變異，是決定某些非小細胞肺癌標靶藥物效果的重要預測因子。據此，以臨床檢體進行基因檢測，已成為晚期非小細胞肺癌患者診治的重要環節。然而由於考慮標靶治療的患者處於無法手術治療的疾病晚期，因而診斷檢體常侷限於小的生檢切片或細胞學檢體，而這些檢體往往不足以進行分子檢驗。因應這樣的挑戰，發展能以微小或非組織學檢體檢測腫瘤基因變異的方法，將有助於篩選更多能對標靶治療產生反應的患者。 惡性肋膜積水是非小細胞肺癌常見的併發症，而積水的採檢相對容易、較不具侵襲性、而且可以反覆進行。然而，以惡性肋膜積水的細胞基因體DNA進行Sanger氏定序，已被證實無法靈敏的檢測EGFR基因突變。事實上，由於非腫瘤細胞的干擾，以異質性檢體進行直接定序以偵測腫瘤細胞基因變異並不靈敏。由於惡性肋膜積水中的非腫瘤細胞不表現EGFR基因，或僅有低度表現，我們因而假設以細胞RNA作為初始模版的EGFR基因定序，可能可以減少非腫瘤細胞的干擾。在我們的研究中，我們平行比較了三種方法（以細胞的RNA進行定序、以及以基因體DNA進行定序或質譜儀分析），用於肺腺癌惡性肋膜積水檢體的EGFR基因突變檢測。其結果顯示以RNA進行EGFR基因定序，可以大幅改善從惡性肋膜積水檢體偵測EGFR基因突變的靈敏度。而由於RNA 定序的突變偵測率較佳，伴隨著對第一線EGFR酪氨酸激酶抑制劑臨床療效的預測也較準確。對於發生後天抗藥性的患者，以RNA進行的EGFR基因定序對續發性T790M突變的偵測結果也令人滿意。 雖然我們證實RNA定序適用於惡性肋膜積水檢體的EGFR基因突變檢測，然而由於 RNA 本質不穩定，以及廣泛存在於環境的核糖核酸酶，因此應用此技術必須審慎的進行檢體的處理，也因此限制其在臨床實務的普及性與用途。免疫細胞化學染色是已經確立的技術，而且經常做為惡性肋膜積水的輔助評估方法。最近已發展出對兩種主要突變形式（L858R 點突變與第19外顯子的E746-A750剔除）的EGFR蛋白質具有專一性結合，可以應用於免疫染色的兔子單株抗體。我們的研究顯示，以這兩種突變專一性抗體進行惡性肋膜積水的免疫細胞染色，對設定檢測的EGFR基因突變，具有相當不錯的偵測靈敏度與精確性。相對於患者的臨床特性，積水的免疫細胞染色對第一線EGFR酪氨酸激酶抑制劑的腫瘤治療反應與無進展存活期，也提供了較好的預測。 過去十年來，微型圓徑探頭支氣管內超音波的發展，已大幅改進以軟性支氣管鏡檢查評估周邊型肺癌的成效。這個進展帶來的一個新的議題，亦即這項技術提供的檢體是否也能夠用於分子檢驗。由於我們證實以RNA為模版的Sanger氏定序，非常適用於細胞學檢體的突變分析。在本研究中，我們也探討利用支氣管內超音波輔助取得的剩餘刷取檢體，評估以RNA的Sanger氏定序進行多基因（EGFR基因、KRAS基因、與EML4-ALK融合基因）分析之可行性。研究結果顯示實施此多基因檢測方式的成功率極高，而且突變的偵測結果也相當好。結合肺癌診斷中日益普及的高階支氣管鏡檢查技術，與此一有效檢測腫瘤基因變異的方法，將可有效的篩選更多的晚期非小細胞肺癌患者，依據其腫瘤細胞的分子特性，決定個別化標靶藥物的治療。 總結而言，我們的研究針主要著重如何使非小細胞肺癌標靶治療的預測性基因變異檢測能更有效與廣泛的應用。要根據腫瘤基因的變異篩選非小細胞肺癌的標靶治療，首先需要實際與有效的取得可用的檢體，並發展適合這些類型臨床檢體的基因檢測方法。值得注意的是，我們的研究顯示簡單的以細胞的RNA取代基因體DNA作為分析的初始模版，可以讓傳統的Sanger氏定序適用於以異質性檢體檢測標靶治療預測性的基因變異。除了改善Sanger氏定序的靈敏度，此種基因分析方式的其他特徵，如增加微小檢體的核酸量、對帶有多重外顯子的基因可以減少所需的的複製與定序反應次數、以及可以用相同的方法分析基因突變與重組等，使其進一步適用於多基因的檢測。

The development of targeted therapy represents an important advance in the treatment of non-small cell lung cancer (NSCLC). Recent pivotal trials have demonstrated that the presence of specific genetic alterations in tumor cells is an important factor for individualizing treatment with some targeted agents in NSCLC. As a result, genetic assay in clinical samples has become an integral part of care for advanced NSCLC patients. However, as the cases considered for targeted therapy present at advanced stages, diagnostic materials are often limited to small biopsies or cytological specimens, which may be insufficient for molecular analysis. To this end, alternative approaches that can detect genetic alterations from minute or cytological samples must be explored to select more patients who are likely to respond to targeted therapy. Malignant pleural effusion (MPE) is a common complication of NSCLC, and effusion sampling is easy, relatively non-invasive and repeatable. However, Sanger sequencing of cell-derived genomic DNA from MPE samples was found not sensitive for EGFR-mutation detection. It is known that direct sequencing is not exquisitely sensitive in heterogeneous samples because of the interference from non-tumor cells. As contaminated non-tumor cells within MPE may have no or lower EGFR expression, we hypothesized that EGFR sequencing using cell-derived RNA as the starting template could be less prone to interference from non-tumor cells. In the study, we compared three methods (sequencing from cell-derived RNA versus sequencing and mass-spectrometric analysis from genomic DNA) parallelly for EGFR-mutation detection from MPE samples of lung adenocarcinoma. The results demonstrated that EGFR sequencing using RNA as template greatly improves sensitivity for EGFR-mutation detection from samples of MPE. The better mutation-detection yield of sequencing from RNA was coupled with the superior prediction of efficacy to first-line EGFR tyrosine kinase inhibitors. In patients with acquired resistance, EGFR sequencing from RNA provided satisfactory detection of secondry T790M mutation. Despite the promise of using RNA for EGFR-mutation detection from MPE samples, the inherently labile nature of RNA, as well as the ubiquitous presence of RNase, warrants the requisite of prudent sample processing and limits its popular application in clinical practice. Immunocytochemistry is a well-established technique and frequently applied as an adjuvant method for the evaluation of MPE. Recently, two rabbit monoclonal antibodies binding specifically to the two major forms of mutant EGFR, L858R point mutation and E746-A750 deletion in exon 19, have been developed for immunostaining. In the study, we demonstrated that effusion immunocytochemistry with these two mutant-specific antibodies exhibited satisfactory sensitivity and specificity for identifying predefined EGFR mutations. Effusion immunocytochemistry also provided a superior prediction of tumor response and progression-free survival to first-line EGFR tyrosine kinase inhibitors than clinical characteristics. In the past decade, the advent of miniature radial-probe endobronchial ultrasound (EBUS) has greatly improved the capacity of flexible bronchoscopy in evaluating peripheral lung cancer. An issue accompanying with this advance is whether the specimens offered by this technique promise for molecular testing. In the study, we evaluated the feasibility of multi-gene analyses (EGFR, KRAS and EML4-ALK fusion) alternately from waste brushing content obtained by EBUS-assisted bronchoscopy, utilizing RNA-based Sanger sequencing which was found promising for mutational analyses from cytological samples. We demonstrated that multi-gene analysis could be implemented from cytology-proven brushing samples with a very high successful rate, and the yields for detecting these mutations were satisfactory. Coupled with the expansion use of advanced bronchoscopic technologies in the diagnosis of lung cancer, this approach could effectively recruit more patients to receive individualized targeted therapy according to the molecular characteristics of tumors cells. In summary, our research is directed at optimizing implication of genetic testing that is predictive for targeted therapy in advanced NSCLC. We reinforce that selection of targeted therapy in NSCLC based on tumor genetic characteristics requires practical system for obtaining clinical samples, along with exploring appropriate methods of genetic testing for the corresponding samples. Notably, our studies feature that simple substitution of cell-derived RNA for genomic DNA as the starting template could enable conventional Sanger sequencing as a suitable method for identifying predictive mutations from heterogeneous samples. Besides improving sensitivity with Sanger sequencing, other characterics, including more plenty of genetic content in small samples, fewer rounds of amplification/sequencing required for genes with multiple exons and detecting mutations and translocations in a similar manner, further enable RNA-based approach feasible for multi-gene analysis.