RNA-based Genetic Testing for Targeted Therapy in Non-small Cell Lung Cancer: Application to Cytological Specimens of Malignant Pleural Effusion and Bronchoscopic Brushing
Date Issued
2015
Date
2015
Author(s)
Tsai, Tzu-Hsiu
Abstract
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.
Subjects
non-small cell lung cancer
targeted therapy
tyrosine kinase inhibitor
EGFR mutation
EML4-ALK fusion
predictive biomarker
molecular diagnostics
Sanger sequencing
reverse-transcription polymerase chain reaction
Type
thesis
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