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Highly Sensitive Detection of ALK Resistance Mutations in Plasma

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Highly Sensitive Detection of ALK Resistance Mutations in Plasma Yoshida et al. BMC Cancer (2018) 18:1136 https://doi.org/10.1186/s12885-018-5031-0 RESEARCH ARTICLE Open Access Highly sensitive detection of ALK resistance mutations in plasma using droplet digital PCR Ryohei Yoshida1*, Takaaki Sasaki1, Yasuhiro Umekage1, Sachie Tanno1, Yusuke Ono2, Munehiko Ogata2, Shinichi Chiba3, Yusuke Mizukami2,4 and Yoshinobu Ohsaki1 Abstract Background: On-target resistance mechanisms found in one-third of patients receiving anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) are secondary ALK mutations in ALK-rearranged non-small cell lung cancer (NSCLC). There are large variations in the resistant mutations, unlike the epithelial growth factor receptor (EGFR) T790 M seen with the use of EGFR-TKIs. Liquid biopsy approaches using cell-free DNA (cfDNA) are used for screening and monitoring of mutations in NSCLC. However, feasible protocol for the simultaneous detection of multiple secondary ALK mutations using droplet digital PCR (ddPCR) has not been developed. An efficient strategy using cfDNA in cancer diagnostics, the development of more accurate and cost-effective tools to identify informative multiple secondary ALK mutations is clinically required. Methods: To establish a feasible assay to monitor ALK-TKI resistance mutations, we first evaluated the feasibility of ddPCR-based screening for cfDNA mutation detection of 10 distinct secondary ALK mutations. Positive samples were then re-analyzed using mutation-specific probes to track the growth of mutation clones with a high sensitivity. Results: Blood samples from seven ALK-positive patients were analyzed using the ddPCR protocol. Secondary G1202R ALK mutations were identified in 2 of 7 patients by the screening assay. Using the mutation-specific probes, monitoring the resistant clone during the clinical course of the disease was well demonstrated in each of the patients. Conclusion: The protocol for ddPCR-based liquid biopsy has a feasibility for the screening of secondary ALK-TKI resistance mutations and offers a tool for a cost-effective monitoring of progression in NSCLC. Keywords: Resistance, ALK inhibitors, Liquid biopsy, Cell-free DNA (cfDNA), Droplet digital PCR (ddPCR) Background been identified in 3–7% of patients with NSCLC (herein In 2007, anaplastic lymphoma kinase (ALK) was discov- referred to as “ALK-positive” lung cancer) [2]. ered as a potential target in non-small cell lung cancer In patients with ALK-positive NSCLC, the ALK inhibi- (NSCLC) [1]. In a small subset of NSCLC tumors, a tor crizotinib resulted in significant higher rates of re- chromosomal inversion event leads to fusion of a por- sponse with an objective response rate (ORR) of 74% tion of the ALK gene with the echinoderm microtubule– and a median progression-free survival (PFS) of associated protein-like 4 (EML4) gene. The resulting 10.9 months than chemotherapy [3]. Multiple mecha- EML4-ALK fusion protein is constitutively active and nisms of crizotinib resistance have been described in transforming, leading to a state of oncogene addiction. ALK-positive NSCLC, with secondary mutations in the ALK rearrangements included EML4-ALK fusions have ALK kinase domain having been detected in approxi- mately 20% of patients, most commonly L1196 M (the “gate-keeper” mutation) and G1269A [4–6]. Although next-generation ALK inhibitors such as alec- * Correspondence: [email protected] 1Respiratory Center, Asahikawa Medical University, 2-1-1-1 tinib, ceritinib, and brigatinib are now available for pre- Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan clinical and clinical testing, little is known about the Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoshida et al. BMC Cancer (2018) 18:1136 Page 2 of 8 potential mechanisms of resistance to these agents. In practice is limited. Among the new technologies for one small study, some patients who relapsed on ceritinib quantifying cfDNA, droplet digital PCR (ddPCR) provides were found to have acquired new tumor mutations in the highest sensitivity for detecting and tracking action- the ALK kinase domain, either at position G1202 or at able mutations at frequencies as low as 0.05–0.01% [16– position F1174 [7]. The fact that the more commonly 18]. In the current study, we sought to establish a liquid occurring crizotinib-resistant ALK mutations (i.e., biopsy protocol for ddPCR-based detection of multiple L1196 M and G1269A) were not observed in ceritinib- TKI-induced secondary mutations in ALK-positive resistant samples, suggesting that different patterns of NSCLC patients. We also describe clinical course of pa- resistance will emerge depending on the selective pres- tients with G1202R-positive NSCLC in details. sure of the individual ALK inhibitors for deriving resist- ant tumor subclones. Methods Preclinical studies have demonstrated that alectinib is Patients active against the crizotinib-resistant ALK mutations Seven patients with ALK-positive NSCLC, previously L1196 M, C1156Y, and F1174 L, implying that alectinib treated or undergoing treatment, were examined be- may be effective in patients who have developed a resist- tween January 2015 and December 2016 at the Asahi- ant to crizotinib through other mechanisms [8, 9]. Gai- kawa Medical University Hospital, Asahikawa, Japan. All nor et al. reported the spectrum and frequency of the patients were diagnosed with ALK-positive NSCLC by numerous known secondary mutations of ALK inhibi- fluorescence in situ hybridization (FISH) or immunohis- tors in 103 patients with ALK-positive NSCLC [6], show- tochemistry (IHC). Patients were treated with daily cri- ing that a high frequency of G1202R mutation was a zotinib or alectinib. The Standard Response Evaluation common feature of each ALK inhibitor. Recently, lorlati- Criteria in Solid Tumors (RECIST 1.0) was used to nib as a 3rd-generation ALK inhibitor has been devel- evaluate treatment responses. We obtained written in- oped and is expected to have a therapeutic effect on formed consents from all study participants, and the secondary mutations, including G1202R [10]. Although study was approved by the Institute of Biomedical Re- treatment strategies using multiple ALK inhibitors search and Innovation Research Ethics Committee. against secondary mutations is rapidly developing, the methods for detecting the actual causal genes in the Sample collection and DNA extraction clinic have not been established. Blood collection was performed every 3 months during Tissue biopsy is a gold-standard for the molecular ALK inhibitor treatment as long as blood samples were diagnosis of lung cancer; however, serial sampling is not scored as being positive in the first screening step. Blood feasible as a routine assessment for the recurrent tumor. samples (limited to < 16 mL) were collected in 8 mL Genomic (g) DNA shed from tumors into the general tubes containing EDTA-2 K (SPM-L1008EMS; Sekisui circulation has been intensively studied for monitoring Medical, Co., Ltd.), with plasma isolated within 2 h. tumor genetics and offers opportunities to trace the gen- Tubes were centrifuged at 1,100 g for 10 min at room omic evolution of cancer systematically [11, 12] . Some- temperature. After the first centrifugation, tubes were times referred to as “liquid biopsy”, genotyping of centrifuged at 18,000 g for 10 min at 4 °C (Fig. 1). The cell-free DNA (cfDNA) isolated from plasma and other supernatant, containing cell-free plasma, was collected body fluid represents an attractive non-invasive tool to in 2 mL serum tubes and stored at − 80 °C until purifica- monitor cancer progression and response to treatments tion. For the purification process, cfDNA was isolated (e.g. resistance). Given advances in sequencing technol- from cell-free plasma in a volume of 2–4 mL with the ogy, recent studies have demonstrated that the muta- QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, tions detected by the cfDNA genotyping are highly Germany) according to the manufacture’s protocol. Fi- concordant to those obtained by tumor biopsy [13, 14]. nally, cfDNA was extracted with elution buffer in vol- In addition, such an approach may overcome the prob- ume of 100 μL. After purification, cfDNA aliquots were lems associated with intratumor or intra-lesion genetic quantified with the Qubit dsDNA HS Assay Kit (Thermo heterogeneity since the liquid biopsy using next-gener- Fisher Scientific, Waltham, MA, USA) and a Qubit 3.0 ation sequencing (NGS) will be able to cover the whole Fluorometer (Thermo Fisher Scientific). genetic landscape of tumor in all its complexity in near future [15]. Detection of ALK resistance mutations in cfDNA An efficient strategy using cfDNA in cancer diagnos- To detect ALK inhibitor resistance mutations in cfDNA, tics, the development of more accurate and cost-effec- we used a ddPCR genotyping assay in conjunction with the tive tools to identify informative mutations is clinically QX200 Droplet Digital PCR System (Bio-Rad, Hercules, required. The analysis of cfDNA using NGS is still ex- USA). The cfDNA was analyzed in two steps. In the first pensive, time-consuming, and its value in clinical (screening) step, cfDNA was analyzed with the multiplex Yoshida et al. BMC Cancer (2018) 18:1136 Page 3 of 8 Fig. 1 Schematic outlining the protocol used to detect recurrent ALK mutations from plasma-derived cfDNA. Firstly, blood collection from patients with ALK-positive NSCLC was performed, cell-free plasma was extracted. The cfDNA was isolated in the purification process and quantified using Qubit. We used a ddPCR genotyping assay in conjunction with the QX200 Droplet Digital PCR System.
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