Abstract
Molecular targeting therapy for non-small cell lung cancer (NSCLC) has clarified the importance of mutation testing when selecting treatment regimens. As a result, multiple-gene mutation tests are urgently needed. We developed a next-generation sequencer (NGS)-based, multi-gene test named the MINtS for investigating driver mutations in both cytological specimens and snap-frozen tissue samples. The MINtS was used to investigate the EGFR, KRAS, BRAF genes from DNA, and the ERBB2, and the ALK, ROS1, and RET fusion genes from RNA. We focused on high specificity and sensitivity (≥0.99) and even included samples with a cancer cell content of 1%. The MINtS enables testing of more than 100 samples in a single run, making it possible to process a large number of samples submitted to a central laboratory, and reducing the cost for a single sample. We investigated 96 cytological samples and 190 surgically resected tissues, both of which are isolated in daily clinical practice. With the cytological samples, we compared the results for the EGFR mutation between the MINtS and the PNA-LNA PCR clamp test, and their results were 99% consistent. In the snap-frozen tissue samples, 188/190 (99%) samples were successfully analyzed for all genes investigated using both DNA and RNA. Then, we used 200 cytological samples that were serially isolated in clinical practice to assess RNA quality. Using our procedure, 196 samples (98%) provided high-quality RNA suitable for analysis with the MINtS. We concluded that the MINtS test system is feasible for analyzing “druggable” genes using cytological samples and snap-frozen tissue samples. The MINtS will fill a needs for patients for whom only cytological specimens are available for genetic testing.
Highlights
Recent progress in molecular targeting therapy for non-small cell lung cancer (NSCLC) has clarified the importance of mutation testing when selecting treatment regimens [1,2,3]
Using DNA, the MINtS was used to investigates epidermal growth factor receptor (EGFR), the Kirsten rat sarcoma viral oncogene homolog gene (KRAS) [18], the v–raf murine sarcoma viral oncogene homolog B1 gene (BRAF) [19,20,21], and the erb-b2 receptor tyrosine kinase 2 gene (ERBB2) [22,23,24]; the target regions were amplified by multiplex PCR
Using RNA, the MINtS was used to investigate the anaplastic lymphoma receptor kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) [25, 26], and ret proto-oncogene (RET) fusion genes [25, 27] as well as the ornithine decarboxylase antizyme 1 (OAZ1) housekeeping gene; the targets were amplified by multiplex RT-PCR
Summary
Recent progress in molecular targeting therapy for non-small cell lung cancer (NSCLC) has clarified the importance of mutation testing when selecting treatment regimens [1,2,3]. The types of NSCLC specimens submitted to genetic testing differ among counties. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens are utilized in many counties, whereas cytological specimens account for a significant portion of samples in others. Japan is one of the counties where cytological samples isolated during endoscopic examination represent a significant portion of samples, accounting for 1/3 of the NSCLC samples submitted to genetic testing [9]. The disadvantage of using cytological samples include that the specimens contain fewer the cancer cells than FFPE samples and the histological type is not always evident. Their advantages include that the presence of cancer cells is quickly confirmed and the samples can be readily submitted to genetic testing
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