Abstract
We present a cohort of 41 patients with osimertinib resistance biopsies, including 2 with an acquired CCDC6–RET fusion. Although RET fusions have been identified in resistant EGFR-mutant non–small cell lung cancer (NSCLC), their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6–RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6–RET was sufficient to confer resistance to EGFR tyrosine kinase inhibitors (TKI). The selective RET inhibitors BLU-667 and cabozantinib resensitized CCDC6–RET-expressing cells to EGFR inhibition. Finally, we treated 2 patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well tolerated and led to rapid radiographic response in both patients. This study provides proof of concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients.
Significance: The role of RET fusions in resistant EGFR-mutant cancers is unknown. We report that RET fusions mediate resistance to EGFR inhibitors and demonstrate that this bypass track can be effectively targeted with a selective RET inhibitor (BLU-667) in the clinic.
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Introduction
Osimertinib is a highly selective, central nervous system–penetrant, third-generation EGFR tyrosine kinase inhibitor (TKI) which nearly doubles progression-free survival compared with first-generation EGFR TKIs and is now the standard front-line therapy for EGFR-mutant non–small cell lung cancer (NSCLC; ref. 1). In addition, osimertinib remains the preferred second-line therapy for T790M-mediated resistance to first- and second-generation EGFR TKIs (2). Despite high initial response rates, however, patients typically develop acquired resistance after about 1 to 2 years of treatment.
Mechanisms of osimertinib resistance are under active investigation but thus far have primarily been studied in the second-line, T790M-positive setting because front-line use represents a more recent shift in the treatment paradigm. Prior studies demonstrated overlap between resistance mechanisms to osimertinib and to first- or second-generation EGFR TKIs, including bypass pathway activation (e.g., MET amplification) and histologic transformation seen upon progression on all classes of EGFR inhibitors (3–6). One notable exception is the EGFR T790M mutation, which develops in 50% to 60% of patients progressing on the older drugs, whereas for osimertinib, T790M is a marker of sensitivity. Furthermore, EGFR C797S is recurrently observed in osimertinib resistance, but not in resistance to first-generation drugs, as expected based on the drug-receptor binding characteristics (7–9). However, the number of osimertinib-resistant cases reported to date remains limited, and a significant proportion of osimertinib-resistant cases lack a clearly identified pathway driving resistance (4).
Acquired fusions, including those involving RET, have recently been reported in a small number of patients progressing on osimertinib and other EGFR TKIs (4, 10–13). Historically, EGFR TKI resistance studies had not identified RET fusions, but this may have been due to the use of limited genotyping platforms that likely did not include RET. Fusions involving RET, a recently described driver oncogene in NSCLC, can be difficult to detect using standard next-generation sequencing (NGS) platforms. The functional role of RET and other fusions in EGFR TKI acquired resistance and the potential impact of RET-directed inhibitors in this population are unknown.
To characterize osimertinib resistance mechanisms including acquired fusion alterations, we analyzed tumor tissue or circulating tumor DNA (ctDNA) from a cohort of patients progressing on osimertinib. We also assessed the functional implications of RET fusions in EGFR-mutant cell line models and treated 3 patients with EGFR-mutant NSCLC and acquired RET fusions with combined EGFR and RET inhibition.
Results
Osimertinib Resistance Cohort
Our study began as a survey of osimertinib resistance mechanisms among patients at Massachusetts General Hospital (MGH). A total of 41 patients with EGFR-mutant NSCLC were treated with single-agent osimertinib and underwent resistance assessment at progression between July 2014 and August 2018 (Table 1). There were 26 women and 15 men, with median age of 64 (range, 40–87). One patient received first-line osimertinib, 16 were treated in the second-line setting, and 24 were treated as third-line or later. All had T790M-positive disease before osimertinib except the front-line patient. Fifteen patients had received another third-generation EGFR TKI before osimertinib [rociletinib (12 patients), nazartinib (2 patients), and ASP8273 (1 patient)]. The median duration of osimertinib treatment was 11.6 months (range, 1–32.7). To assess osimertinib resistance mechanisms, 17 patients had both a tissue biopsy and ctDNA analysis, 15 had tissue only, and 9 had ctDNA only at clinical progression. Three patients had two distinct metastases sampled at osimertinib resistance.
Characteristics of the osimertinib-resistant cohort and the patients with fusion-positive EGFR-mutant NSCLCs
Observed Osimertinib Resistance Mechanisms
A total of 35 tissue biopsies among 32 osimertinib-resistant patients were analyzed (Fig. 1). All had adenocarcinoma histology prior to osimertinib; two transformed to small cell lung cancer and one to squamous cell histology after progression on osimertinib. Molecular testing was performed on all cases, with the founder EGFR mutation detected in each specimen. Six (19%) patients had acquired EGFR C797S, each in cis configuration with T790M; 7 (22%) developed MET amplification (defined as MET: centromere 7 ratio ≥ 2.2 by FISH). In 12 (38%) cases, T790M was not identified (11 previously T790M-positive) and no other resistance driver was detected, whereas in 3 (9%) cases, T790M was maintained without an identified resistance mechanism.
Summary of anatomic and molecular pathology findings from osimertinib-resistant cohort. This heat map summarizes the findings of tissue (top) and ctDNA (bottom) analysis obtained at the time of clinical progression on osimertinib. Key resistance mechanisms are highlighted (see legend). Note that for patients with multiple tissue biopsies (4A/B, 5A/B, and 14A/B), the same plasma results are shown below each tissue biopsy result.
Among 26 patients with ctDNA analysis at osimertinib resistance, the founder EGFR mutation was detected in 22 samples; the remaining 4 lacked detectable EGFR and therefore were uninformative for resistance mechanisms, which were also likely below the limit of detection (Fig. 1). Resistance mechanisms detected via ctDNA were similar in spectrum to tissue samples with 7 (32%) C797S and 5 (23%) MET amplifications (defined as mean plasma copy number ≥ 2.1). The number of samples with both tissue and informative ctDNA was too small for meaningful concordance analysis.
We observed intertumoral heterogeneity in all 3 patients who had two distinct metastatic foci biopsied. Two patients had C797S detected at one metastatic site, whereas the other was C797 wild-type; the third had MET amplification detected within a pleural fluid cell block but had normal MET copy number in a coincident lung biopsy. In each case, no other putative resistance mechanism was identified in the second biopsy site.
One patient with plasma-only osimertinib resistance analysis (#33) had both CCDC6–RET [mutant allele frequency (MAF) 1.9%] and TPM3–NTRK1 fusion (MAF 0.1%) detected in ctDNA (EGFR del19; MAF 14.2%). Given this finding, we used the MGH Solid Fusion Assay (SFA), an RNA-based anchored multiplex polymerase chain reaction (AMP), developed to identify fusion events in tissue biopsies, and found that 24 of 34 (71%) osimertinib-resistant tissue biopsies had sufficient tissue for analysis (14). Among these, we detected a CCDC6–RET fusion in a progressing pleural metastasis in patient 1 and a PCBP2–BRAF fusion in a new liver metastasis which developed on osimertinib in patient 2 (Fig. 1; Table 1). In addition, patient 3 in our osimertinib-resistant cohort underwent NGS of a growing omental nodule at Foundation Medicine, and an AGK–BRAF fusion was observed. In each case, there was concurrent T790M “loss,” and no other resistance mechanisms were identified in the tissue.
To broaden our cohort of patients with EGFR-mutant NSCLC with acquired fusion events, we retrospectively ran the SFA on a subset of EGFR-mutant tissue biopsies obtained at MGH over the past 10 years (Table 1). Many of these older biopsies were obtained upon progression on erlotinib, afatinib, and gefitinib and did not originally undergo SFA. Among them, we identified 1 additional afatinib-resistant patient who had a CCDC6–RET fusion (#42; described in more detail below) and 1 patient (#43) with a BAIAP2L1–BRAF fusion detected after progression on chemotherapy/osimertinib. We also included 1 patient from the University of California, Irvine (UCI), who acquired an NCOA4–RET fusion on FoundationOne NGS tissue testing obtained upon progression on first-line afatinib/cetuximab therapy (#44; described in further detail below).
CCDC6–RET Expression in EGFR-Mutant NSCLC Cell Lines Confers Resistance to EGFR Inhibitors.
Next, we sought to determine whether gene fusions observed in the patients described above are sufficient to cause acquired drug resistance. We initially focused on the CCDC6–RET fusion gene. CCDC6–RET-expressing cell lines were generated by lentiviral infection of PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells (Supplementary Fig. S1). Cells expressing CCDC6–RET grew similarly to parental cells in the absence of EGFR inhibitor. When treated with osimertinib, PC9CCDC6–RET and MGH134CCDC6–RET cells continued to proliferate, in contrast to parental cells which showed a net decrease in cell viability (Fig. 2A). Of note, the proliferation rate of CCDC6–RET-expressing cells decreased in osimertinib, suggesting that RET activation does not fully compensate for EGFR signaling loss, although it is sufficient to drive acquired resistance.
The CCDC6–RET fusion is sufficient for conferring resistance to EGFR TKIs and can be overcome by combined EGFR and RET inhibition. A, PC9 and MGH134 cells expressing the CCDC6–RET gene fusion or empty vector (EV) were treated with 1 μmol/L osimertinib (OSI) or vehicle (VEH) and cell proliferation determined over the course of 5 days (ratio compared with the beginning of treatment). Data shown are the mean ± SEM of three independent biological replicates. B, PC9EV and PC9CCDC6–RET cells were treated with 100 nmol/L afatinib, 1 μmol/L osimertinib, BLU-667, or combinations for 6 hours and harvested for western blotting with the indicated antibodies. The arrow indicates the phospho-RET band. C, PC9EV and PC9CCDC6–RET cells were treated with BLU-667, or afatinib or osimertinib in the absence or presence of 1 μmol/L BLU-667, and cell viability was determined after 72 hours. The same BLU-667 data are replotted in both panels for comparison purposes. Data are shown as a percentage of vehicle-treated control and are the mean ± SEM of three independent biological replicates.
We next examined the consequences of CCDC6–RET expression on downstream signaling pathway activation in PC9 and MGH134 cells. Compared with parental cells, which did not express detectable RET protein, phosphorylated RET was detected in both PC9CCDC6–RET and MGH134CCDC6–RET cells (Fig. 2B; Supplementary Fig. S2A). CCDC6–RET expression alone did not lead to increased activation of downstream MAPK (phospho-ERK1/2) or PI3K (phospho-AKT) signaling at baseline; however RET, ERK1/2, and AKT phosphorylation was retained in the presence of afatinib or osimertinib in both PC9CCDC6–RET and MGH134CCDC6–RET cells (Fig. 2B; Supplementary Fig. S2A). Thus, expression of the CCDC6–RET fusion is sufficient to confer resistance to EGFR TKIs in EGFR-mutant NSCLCs.
Acquired Resistance Resulting from CCDC6–RET Expression Can Be Overcome by EGFR plus RET Inhibition.
Acquired resistance resulting from activation of other bypass signaling pathways can be overcome via dual pathway suppression (15, 16). To determine whether a similar strategy might overcome CCDC6–RET-mediated acquired resistance, we treated PC9CCDC6–RET cells with the selective RET inhibitor BLU-667 (17) in the absence or presence of EGFR TKIs. Treatment with BLU-667 alone suppressed RET phosphorylation but did not decrease downstream ERK or AKT phosphorylation (Fig. 2B). Combined treatment with BLU-667 and either osimertinib or afatinib completely suppressed both phospho-ERK and phospho-AKT and decreased cell viability to a similar level as parental cells treated with EGFR TKI (Fig. 2C). Similar results were observed in MGH134CCDC6–RET cells (Supplementary Fig. S2). In addition, PC9CCDC6–RET and MGH134CCDC6–RET cells were sensitive to EGFR TKI + cabozantinib, a multikinase inhibitor with RET activity (Supplementary Figs. S2, S3A, and S3B). Taken together, these data demonstrate that acquired resistance resulting from the CCDC6–RET fusions can be overcome by dual EGFR plus RET blockade.
MEK but Not BRAF Inhibitors Overcome Acquired Resistance Resulting from PCBP2–BRAF Fusion.
To expand our investigation beyond the CCDC6–RET fusion, we examined whether the novel PCBP2–BRAF fusion observed in patient 2 was driving resistance. We established a cell line (MGH845-1) from a core needle liver biopsy of the patient (Supplementary Fig. S4A and S4B) and confirmed the presence of the PCBP2–BRAF fusion gene and EGFR T790M loss (Supplementary Fig. S4C and S4D). Knockdown of BRAF in MGH845-1 using siRNAs targeting the BRAF coding sequence retained within the PCBP2–BRAF fusion had a modest effect on cell viability and further sensitized cells to osimertinib (Supplementary Fig. S5A and S5B). Consistent with a prior report examining de novo BRAF fusions in melanoma (18), the MGH845-1 cells were sensitive to the MEK inhibitor trametinib but not to the RAF inhibitors dabrafenib and LXH245 (Supplementary Fig. S5C).
Treatment of EGFR-Mutant Acquired RET Fusion–Positive Patients with EGFR plus RET Inhibition.
The preclinical results showing that combining EGFR and RET inhibitors can overcome resistance conferred by CCDC6–RET were sufficiently compelling to suggest patient treatment should be explored. The first MGH patient identified with an acquired RET fusion (Table 1; patient 42) was a 44-year-old man with del19 EGFR-mutant advanced NSCLC who received front-line cisplatin/pemetrexed and second-line afatinib (1 year), and then underwent a bronchoscopic biopsy of a growing lung lesion showing a CCDC6–RET fusion by SFA. Baseline tissue was not available for RET testing. He was treated with erlotinib 150 mg daily combined with off-label cabozantinib 60 mg daily. Scans after 1 month showed stable disease (RECIST 1.1), but subsequent scans after 2.5 months showed disease progression and prompted treatment discontinuation (19). He had grade 1 diarrhea, rash, and aspartate aminotransferase elevation.
A 60-year-old woman with del19 EGFR-mutant advanced NSCLC (patient 1) received front-line afatinib (1 year), acquired T790M, and was treated with osimertinib (18 months). She then underwent a pleural biopsy revealing a CCDC6–RET fusion via SFA. Baseline tissue was insufficient for SFA, but RET FISH was negative, suggesting the CCDC6–RET fusion was indeed acquired. Given the suboptimal response the first patient had using the multitargeted TKI cabozantinib and the successful experience with the selective RET TKI BLU-667 in NSCLCs harboring RET fusions as the primary oncogenic driver, we wrote an individual patient investigational new drug (IND) protocol for osimertinib plus BLU-667 (17). She began osimertinib 80 mg daily and BLU-667 200 mg daily, and then increased BLU-667 to 300 mg after 2 weeks of treatment. Her dyspnea improved within days of therapy initiation. Scans after 8 weeks revealed a marked response with RECIST tumor shrinkage of 78% (Fig. 3A). The combination was well tolerated with only grade 1 toxicities including fatigue, leukopenia, hypertension, xerostomia, and transaminitis. Treatment is ongoing at the time of this writing (3.5 months on treatment).
Responses observed in the 2 patients treated with osimertinib and BLU-667. A, Treatment response of patient 1 to osimertinib and BLU-667. Serial coronal contrast-enhanced computed-tomography images of the thorax demonstrate a right bottom lobe lung mass and pleural nodularity (red arrows) seen at baseline (left) with partial response after 8 weeks of treatment with BLU-667 and osimertinib (right). B, Treatment response of patient 44 to osimertinib and BLU-667, with significant improvement in left top and left bottom lobe pulmonary opacities (right; circled) compared with baseline (left).
Finally, we collaborated with colleagues at UCI who identified a similar patient (Table 1, patient 44). A 67-year-old woman underwent surgery and adjuvant cisplatin/pemetrexed for a stage IIIA del19 EGFR-mutant lung adenocarcinoma, with subsequent recurrence. She received afatinib/cetuximab (2 years) and then underwent a lung biopsy, which demonstrated an acquired NCOA4–RET fusion by FoundationOne NGS testing (not present in the pretreatment biopsy). An individual IND protocol was again utilized. She took osimertinib 80 mg daily and BLU-667 200 mg daily for 2 weeks, then 300 mg daily for 2 weeks, and then ultimately escalated to 400 mg daily. Scans after 8 weeks also revealed a marked response with RECIST tumor shrinkage of 78% (Fig. 3B). Grade 1 toxicities including fatigue, diarrhea, anemia, thrombocytopenia, and dysguesia, and grade 2 leukopenia and neutropenia were observed. Treatment is ongoing at the time of this writing (4 months on treatment).
Discussion
Here we examine mechanisms of acquired resistance to osimertinib with a focus on RET fusions, demonstrating in engineered cell lines that they can mediate acquired resistance to EGFR TKIs and providing proof-of-principle clinical data that targeting this bypass track with a selective RET inhibitor like BLU-667 can be highly effective in patients. Both patients treated with osimertinib plus BLU-667 had rapid and impressive improvements in their cancer. This has immediate clinical implications for EGFR-mutant patients and suggests that testing for RET fusions should become part of standard panels used upon acquired EGFR resistance. Importantly, osimertinib and BLU-667 were well tolerated in these 2 patients, and further study of this combination in additional patients is warranted.
The paradigm of testing for bypass track activation at acquired resistance to EGFR TKIs has precedence in MET amplification, a resistance mechanism first described in 2007 (15). Ten years later, the clinical validity of inhibiting EGFR plus MET in patients with MET amplification–driven resistance was demonstrated through the combination of osimertinib and the MET inhibitor savolitinib (20). Prior EGFR plus MET TKI combinations were tested, but success was limited, likely due to trial designs lacking a focus on true MET amplification as the resistance driver, as well as the poor tolerability of prior regimens built primarily on an erlotinib backbone (21–23). Just as osimertinib, a well-tolerated third-generation EGFR TKI, has led to better-tolerated combinations with MET inhibitors, our experience suggests that we may see similar ease of building combination regimens for RET-mediated acquired resistance. The high RET selectivity of BLU-667 may also be a contributing factor to the tolerability of this combination. BLU-667 has been shown to be >15 times more potent on RET than any other kinase and >10 times more potent on RET than approved multitargeted kinase inhibitors like cabozantinib (17). The overall tolerability of osimertinib plus BLU-667 in both of our patients is an early sign of the high selectivity of BLU-667 and the feasibility of combining the two agents.
Preclinical modeling demonstrated that CCDC6–RET fusion expression resulted in sustained MAPK and PI3K signaling in the presence of EGFR inhibition and, in both models tested, was sufficient to cause EGFR TKI resistance. However, in both PC9CCDC6–RET and MGH134CCDC6–RET cells, EGFR TKIs exhibited partial activity in suppressing downstream signaling and slowing cell proliferation. Although we cannot rule out the possibility that differences in expression levels of the CCDC6–RET fusion may contribute, these results suggest that CCDC6–RET may not fully recapitulate EGFR signaling such that resistant cells harboring this fusion retain partial dependency on EGFR signaling.
Other groups have also found RET fusions in EGFR-mutant patients with TKI resistance (4, 10–13). Reckamp and colleagues studied nearly 33,000 samples undergoing clinical plasma ctDNA testing at Guardant Health and identified 116 patients with NSCLC with RET fusions, including 17 with co-occurring EGFR mutations (10). Five EGFR mutants had available information about their clinical course, and all 5 had received prior first- or second-generation TKIs, whereas three had also received osimertinib before the RET fusion was identified. Schrock and colleagues assessed over 3,500 EGFR-mutant patients undergoing tissue sampling at Foundation Medicine for fusions and identified 19 patients with a RET fusion, including one afatinib-resistant L858R EGFR-mutant patient with an NCOA4–RET fusion, who had stable disease for 7 months on cabozantinib plus afatinib (11). This patient anecdote is especially interesting in the context of the 3 patients treated with EGFR plus RET inhibitors we present here, as there are now at least two reported cases treated with cabozantinib that had stable disease as a best response, in stark contrast with two reported cases treated with BLU-667 and osimertinib that had dramatic and rapid responses.
With broad NGS panels steadily gaining popularity, we believe it is feasible for the oncology community to start testing for RET and other oncogene fusions in postresistance EGFR-mutant biopsies. However, there are some noteworthy caveats. Translocation breakpoints may be present at any point in the genomic DNA and often occur in intronic regions; thus, focused NGS panels that examine only exons may miss these aberrations. Larger NGS libraries and alignment tools allowing mapping of DNA sequences to two different genomic sites can help overcome this obstacle. At MGH, our molecular pathology group has developed an RNA AMP technology to identify gene rearrangements without prior knowledge of the fusion partner (14). This SFA can detect chimeric transcripts at the RNA level which also enables prediction of the involved (transcribed) exons, typically fused at exon–intron junctions. In addition, SFA technology is compatible with the often short and fragmented nucleic acids input from formalin-fixed paraffin-embedded specimens. We acknowledge that, although the SFA can identify RET fusion partners by sequence, other technologies with specific advantages also exist. For example, FISH preserves the tissue context and enables gene fusion assessment on very small samples.
Our cohort adds to the growing body of knowledge about osimertinib acquired resistance. Acquired RET fusions should be considered a potentially actionable finding at osimertinib resistance, but treatment options remain unclear for acquired BRAF fusions, which will require more detailed mechanistic studies to unravel the complexities of RAF signaling in these patients. In addition to the fusion cases discussed, we observed C797S in 27% of patients, consistent with other experiences (4). Because all cases were found in cis with T790M, there is not currently a targeted treatment strategy clinically available for these patients, though preclinical concepts are emerging (24–26). In addition, we saw MET amplification in 24% of patients, which is encouraging given the promising treatment strategies available now for these patients (20).
Our study is limited by its assessment of osimertinib primarily in the second-line (or beyond) T790M-positive setting; we acknowledge that our findings may not be directly applicable to patients who receive osimertinib for newly diagnosed EGFR-mutant NSCLC. However, the patients we and others have identified with RET fusions after first- or second-generation EGFR TKIs lead us to believe that RET fusions will likely be recurrent findings after front-line osimertinib. Small numbers, especially only 2 patients treated with the osimertinib plus BLU-667, also limit our study. Further study of osimertinib plus BLU-667 will be needed to define clinical activity in a larger cohort of patients. Finally, 8 of the patients in our cohort were on osimertinib for less than 6 months prior to undergoing progression biopsies, and hence the findings in those cases may reflect an intrinsic resistance clone.
In conclusion, RET fusions are a bona fide acquired resistance mechanism among EGFR-mutant cancers, and treatment with osimertinib plus BLU-667 may be a well-tolerated and effective therapy for this group.
Methods
Patients
All sequential patients with EGFR-mutant NSCLC seen at MGH who underwent a tissue biopsy and/or ctDNA analysis after clinical progression on osimertinib and had sufficient tissue for molecular analysis were included. The sites of biopsy were selected by the treating physician; progressing lesions were biopsied whenever feasible. We identified additional patients with EGFR-mutant NSCLC and fusions detected by SFA, regardless of prior therapy. All patients provided signed informed consent under an Institutional Review Board (IRB)–approved protocol which allows chart review for research, NGS, and exploratory research on tissue biopsies. The study was conducted in accordance with the principles of the Declaration of Helsinki.
Molecular Testing of Tissue Biopsies
All osimertinib-resistant tissue biopsies were analyzed by Clinical Laboratory Improvement Amendments–certified assays performed in the MGH Center for Integrated Diagnostics or Foundation Medicine using methods which have been described previously, including the MGH SNaPshot NGS panel, MGH SFA, FoundationOne NGS panel, and FISH for MET and EGFR amplification (14, 27). SNaPshot uses AMP to detect single-nucleotide variants, insertions/deletions, and copy-number alterations in genomic DNA using the ArcherDX platform and Illumina NextSeq NGS. During this project, the SNaPshot assay platform was broadened from a 39-gene panel (NGS-V1) to a 91-gene panel (NGS-V2). The SFA is an AMP-based platform for targeted fusion transcript detection using NGS. The list of genes covered by each assay is provided in Supplementary Table S1. Tissue MET and EGFR amplification was tested by FISH, with amplification defined as a ratio of MET or EGFR to centromere 7 of > 2.2.
Plasma ctDNA Testing
All plasma samples were analyzed by the Guardant360 NGS platform (Guardant Health) as described previously (28). Further details of the Guardant platform are available upon request.
Treatment with Osimertinib plus BLU-667
Study of the osimertinib plus BLU-667 combination was conducted via single patient IND and clinical protocol (Supplementary Data) that was reviewed and approved by the FDA and the local IRB of each site. Prior to treatment, written informed consent was obtained from each patient.
Cell Culture
The PC9 and MGH134 cell lines have been previously described (29). MGH845-1 cells were generated from a core needle biopsy of a liver metastasis from a patient progressing on osimertinib using methods that have been previously described (16).
Generation of CCDC6–RET-Expressing Cell Lines
A CCDC6–RET fusion construct was synthesized by GenScript and ligated into the pLENTI6/V5-D-TOPO vector using the ViraPower Lentiviral Directional TOPO Expression Kit (Life Technologies). Lentivirus was generated by transfecting the pLENTI6 constructs and packaging plasmids into 293FT cells (Life Technologies). Virus production, collection, and infection were completed following the manufacturer's protocol. Transduced cells were selected in blasticidin (10–20 mg/mL) for 1 week.
Cell Viability Assay
For drug dose–response assays, cells were seeded into 96-well plates 24 hours before addition of drug. Cell proliferation was determined by CellTiter-Glo assay (Promega) 72 to 120 hours after adding drug, using standard protocols. For time-course experiments, multiple plates were seeded and drugged in identical fashion. At the indicated time points, plates were frozen at −80°C. All plates in an experiment were developed with CellTiter-Glo simultaneously. Luminescence was measured with SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices).
Please see Supplementary Methods for additional information.
Disclosure of Potential Conflicts of Interest
Z. Piotrowska is a consultant/advisory board member for AstraZeneca, Ariad/Takeda, Novartis, AbbVie, and Spectrum. J.F. Gainor reports receiving a commercial research grant from Genentech and is a consultant/advisory board member for Bristol-Myers Squibb, Novartis, Loxo, Array Biopharma, Theravance, Pfizer, Merck, Roche, Ariad/Takeda, Amgen, Agios, Regeneron, and Oncorus. V.W. Zhu has received honoraria from the speakers bureaus of AstraZeneca, Roche-Foundation Medicine, and Roche/Genentech; has ownership interest (including stock, patents, etc.) in TP Therapeutics; and is a consultant/advisory board member for TP Therapeutics. J.J. Lin has received honoraria from the speakers bureaus of Boehringer Ingelheim and Chugai. R.J. Nagy has ownership interest (including stock, patents, etc.) in Guardant Health. R.B. Lanman is Chief Medical Officer at Guardant Health, Inc.; has ownership interest (including stock, patents, etc.) in Guardant Health, Inc., Biolase, Inc., and Forward Medical, Inc.; and is a consultant/advisory board member for Forward Medical, Inc. M. Mino-Kenudson is a consultant/advisory board member for Merrimack Pharmaceuticals and H3 Biomedicine. A.J. Iafrate reports receiving a commercial research grant from Blueprint Medicines and has ownership interest (including stock, patents, etc.) in ArcherDx. R.S. Heist is a consultant/advisory board member for Boehringer Ingelheim, Tarveda, and Novartis. A.T. Shaw is a consultant/advisory board member for Blueprint Medicines, Loxo, KSQ Therapeutics, Ignyta, Takeda, Ariad, Daiichi-sankyo, Taiho, Pfizer, Genentech, Roche, Novartis, Chugai, Guardant, Foundation Medicine, and Natera. E.K. Evans has ownership interest (including stock, patents, etc.) in Blueprint Medicines. S.-H.I. Ou has received honoraria from the speakers bureaus of Roche/Genentech, Pfizer, AstraZeneca, Takeda, and Foundation Medicine; has ownership interest (including stock, patents, etc.) in TP Therapeutics; and is a consultant/advisory board member for Roche, AstraZeneca, and Takeda. B. Wolf has ownership interest (including stock, patents, etc.) in Blueprint Medicines Corporation. A.N. Hata reports receiving commercial research grants from Novartis, Amgen, and Relay Therapeutics. L.V. Sequist reports receiving commercial research support from Novartis, Boehringer Ingelheim, and Merrimack Pharmaceuticals, and is a consultant/advisory board member for AstraZeneca, Blueprint Medicines, Pfizer, Merrimack Pharmaceuticals, and Genentech. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: Z. Piotrowska, H. Isozaki, J.F. Gainor, A.T. Shaw, E.K. Evans, S.-H.I. Ou, B. Wolf, A.N. Hata, L.V. Sequist
Development of methodology: Z. Piotrowska, H. Isozaki, J.K. Lennerz, S. Yoda, R.B. Lanman, A.J. Iafrate, B. Wolf, A.N. Hata, L.V. Sequist
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Piotrowska, H. Isozaki, J.K. Lennerz, J.F. Gainor, I.T. Lennes, V.W. Zhu, N. Marcoux, A.K. Riley, V. Nangia, R.J. Nagy, D. Dias-Santagata, M. Mino-Kenudson, A.J. Iafrate, R.S. Heist, E.K. Evans, C. Clifford, S.-H.I. Ou, A.N. Hata, L.V. Sequist
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Piotrowska, H. Isozaki, J.K. Lennerz, V.W. Zhu, S.R. Digumarthy, D. Dias-Santagata, A.J. Iafrate, R.S. Heist, A.T. Shaw, S.-H.I. Ou, B. Wolf, A.N. Hata, L.V. Sequist
Writing, review, and/or revision of the manuscript: Z. Piotrowska, H. Isozaki, J.K. Lennerz, J.F. Gainor, I.T. Lennes, V.W. Zhu, N. Marcoux, S.R. Digumarthy, S. Yoda, J.J. Lin, R.J. Nagy, R.B. Lanman, M. Mino-Kenudson, A.J. Iafrate, R.S. Heist, A.T. Shaw, E.K. Evans, S.-H.I. Ou, B. Wolf, A.N. Hata, L.V. Sequist
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Z. Piotrowska, H. Isozaki, J.K. Lennerz, M.K. Banwait, S.R. Digumarthy, W. Su, R.J. Nagy, C. Clifford, L.V. Sequist
Study supervision: Z. Piotrowska, C. Clifford, B. Wolf, A.N. Hata, L.V. Sequist
Acknowledgments
This study was supported by NCI grants 2R01CA137008 (L.V. Sequist) and K08CA197389 (A.N. Hata), and funding from the American Cancer Society (Z. Piotrowska) and LUNGevity (Z. Piotrowska). Additional support was provided by Lung Strong, the Kevin Hoffman family, the Susanne E. Coyne fund, Targeting a Cure for Lung Cancer, and Be a Piece of the Solution. Blueprint Medicines provided BLU-667 for both preclinical and clinical experiments, and ctDNA testing was supported by Guardant Health.
Footnotes
Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).
- Received August 31, 2018.
- Revision received September 21, 2018.
- Accepted September 24, 2018.
- Published first September 26, 2018.
- ©2018 American Association for Cancer Research.
References
Volume 8, Issue 12
