Next-generation sequencing of tyrosine kinase inhibitor-resistant non-small-cell lung cancers in patients harboring epidermal growth factor-activating mutations

In this study we analyzed biopsy specimens of patients who underwent second biopsy after treatment failure with the first generation EGFR-TKIs. There was a significant difference between the frequency of EGFR T790M and other EGFR-activating mutations, with significant variability among cases (4.8–41.3 %). The existence of EGFR and other mutations within the same tumor sample identified by NGS highlights the importance of this type of analysis in guiding appropriate cancer therapy.

High-throughput sequencing was able to detect T790M mutation in a number of cases with the same accuracy of conventional highly sensitive conventional PCR methods, such as PNA-LNA PCR clamp [16] and Cycleave real-time PCR [15]. While high sensitivity and specificity of these methods is well established [19‐27], the use of NGS provides important advantages with clarifying activating mutation rate in tumor sample as well as greater detection of rare mutations outside of target areas [28‐31]. In addition, to emphasize the power of NGS in clinical practice, we should also try to develop its applications and usages such as challenging specimens or testing processes, such as peripheral blood in the future.

NGS is also able to overcome issue of germ-line DNA contamination, similar to that of new PCR methods, such as digital PCR [32]. This tolerance of germ-line DNA contamination allows for more streamlined sample preparation techniques, without need for time-consuming procedures such as macro- or micro-dissection. In this study, all samples were extracted from FFPE biopsy specimens, highlighting both versatility and potential use of NGS in clinical settings. Furthermore NGS is able to quantify gene mutations within a tumor sample. Due to the unpredictablity of PCR amplification and germ line DNA contamination, observed mutations does not always reflect the penetrance of a mutation within a sample. While most highly sensitive detection methods provide only categorical results such as positive and negative, our analysis was able to identify the degree of EGFR T790M and other EGFR-activating mutations within a sample that could not be explained by germ-line DNA contamination and/or PCR efficacy. These results are consistent with previous reports detailing T790M allelic frequency in terms of both intra-tumor heterogeneity in localized lung adenocarcinomas [33] and allelic imbalances [34]. Our analysis was able to identify the degree of EGFR T790M and other EGFR-activating mutations within a sample that could not be explained by germ-line DNA contamination and/or PCR efficacy. Future treatment with next-generation EGFR-TKIs targeting T790M is likely to be informed by such analyses, as patients should be treated based upon their EGFR acquired mutation [35].

In addition to EGFR mutations, we also evaluated another 50 oncogenes thought to have an important role in cancer pathogenesis (Table 4). A large number of mutations were identified in this analysis. However, how much extent these genes affect tumorigenicity, tumor progression, and resistance to EGFR-TKIs is difficult to assess, as some mutations may represent only passive alterations (passenger mutations). Although many of these mutations were identified in a single patient, a series of mutations including TP53 P72R, KDR Q472R, and KIT M541L were detected in more than two cases, suggesting a role in disease progression.

TP53 P72R was the most common mutation, detected in 13 of 15 cases (86.7 %). In human populations, TP53 codon 72 is encoded by the nucleotide sequence CCC, which encodes proline, or CGC, which encodes arginine. While proline is the most common amino acid found at this residue, comparative sequence analyses have detected a high degree (>50 %) of TP53-R72 variants among certain populations [36]. The current understanding of TP53 biology is that TP53-R72 is more effective at inducing apoptosis and protecting stressed cells from neoplastic development than the more common TP53-P72 [37]. However, it is not yet understood how these functional differences might translate between in vitro and in vivo settings [38, 39], making it difficult to assess the role of this sequence variant of EGFR-TKI resistance.

KDR (kinase insert domain receptor, also known as VEGFR2) is an important factor in tumor development and progression due to its pro-angiogenic effects [40]. KDR Q472H mutations were detected in 5 of 15 cases (33.3 %), making it the second most common gene variant observed outside of EGFR. In human populations, codon 472 of KDR is encoded by the nucleotide sequence CAA, which encodes glutamine, or CAT, which encodes histidine. The Q472H variant is thought to affect protein function due to increased phosphorylation after vascular endothelial growth factor (VEGF)-A stimulation, along with increased binding efficiency for VEGF-A165 [41]. The effect of Q472H on microvessel density is thought to occur as a result of increased phosphorylation of VEGFR2 [42]. Here, increased microvessel density may have contributed to EGFR-TKI resistance, suggesting that VEGFR2 inhibition may inhibition may become an important therapeutic option in patients with documented EGFR-TKI resistance.

V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (KIT) M541L substitutions were detected in 2 of 15 cases (13.3 %). c-KIT is one of the primary targets of imatinib, and mutations in KIT are predictive of the efficacy of the drug in gastrointestinal stromal tumors (GIST) [43]. Several case reports have suggested a potential role of the KIT M541L variant in the sensitivity of Imatinib for aggressive fibromatosis [44‐46]. Furthermore, a wide array of in vitro analyses support a role for the L541 variant in tumorigenesis. FDC-P1 cells transfected with KIT-L541 showed an enhanced proliferative response, while KIT-L541 cells were more sensitive to imatinib than those expressing wild-type KIT [47]. Inokuchi, et al. observed a higher frequency of L541 variants among patients with chronic myelogenous leukemia (CML), which is consistent with increased tyrosine kinase activation and proliferative responses in KIT-L541 cells relative to wild-type controls [48]. From the view point of EGFR-TKI resistance, these data suggest a causative role for the KIT L541 variant in recurrence and drug resistance of NSCLC. Suppression of KIT with drugs like Imatinib may be a useful therapeutic choice in patients with KIT-variant tumors.

Five (cases 3, 4, 5, 12 and 13) out of six NSCLC patients that are negative for EGFR-T790M mutation harbored “compound mutations” (a rare EGFR mutation in combination with a more frequent activating mutation). On the other hand, all T790M-positive tumors (cases 1, 6, 7, 8, 9, 10 and 11) lack an additional rare mutation apart from the presence of a frequent inhibitor-sensitive EGFR mutation. Among these compound mutations (specifically rare mutations), tumors harboring S768I in exon 20 is known as resistant to EGFR-TKIs. On the contrary, tumors harboring point mutations in exon 18 and dual mutation of exon 19 deletion and S768I are reported to possible response to EGFR-TKIs. There have been limited data in other compounds mutations. So a role of these mutations in causing drug resistance in T790M-negative patients is uncertain and need to be evaluated [49].