Basic characteristics of secondary ALK fusion mutation after EGFR-TKIs resistance
The probability of ALK mutation after EGFR-TKIs resistance is very low. According to previous reports, the calculated probability of occurrence is about 0.01%-0.2%[22] [23] [24]. In our analysis, 4 of 820 patients with acquired drug resistance after EGFR-TKIs developed ALK fusion mutations, with an incidence of 0.49%, which is close with previous reports.
Haiyuan Xu et al. reviewed the NGS genome map data of 3873 patients with EGFR (exon 18-21) mutation and found that 5 new ALK fusion mutations were acquired during EGFR-TKIs treatment[24].Fusion partners of ALK identified in the 5 patients included EML4 (3 cases), STRN(1 case) and CCAAT-enhancer binding protein zeta(CEBPZ) (1 case). All three EML4–ALK fusions emerging after EGFR-TKI resistance were the rare variant E2:A20 (V5)[24]. Other literature reported one case of secondary STRN-ALK fusions in 62 patients with EGFR-mutated metastatic NSCLC with tissue or plasma biopsies at EGFR-TKIs progression[25].Alexa B Schrock et al. Identified a case of Pleckstrin homology domain containing A7 (PLEKHA7)-ALK fusion mutation after application of Erlotinib followed by Osimertinib[26]. A patient with EGFR L858R lung adenocarcinoma developed disease progression after 72.7 months of gefitinib therapy, emerging a novel ALK-R3HDM1 and EML4-ALK dual fusion that might be a delicate mechanism for the acquired resistance of EGFR- TKIs [27].
In our study, 3 EML4-ALK fusion mutations emerged, of which 2 cases were V2 subtype and 1 case was V3 subtype.1 STRN-ALK fusion mutation was detected. Our another study showed that classical EML4-ALK fusion accounted for 94.9% of patients with primary ALK fusion[28]. From this research and other previous literature, non-classical ALK fusions may be more common in acquired EGFR-TKIs resistance.
In this study, EGFR gene mutation was detected negative in 2 of the 4 patients, and the abundance of EGFR-19DEL mutation decreased from 56.98% to 48.34% in 1 case, and from 25.10% to 18.28% in the other. In only four patients, the abundance of EGFR mutation was either reduced or undetectable at the time of resistance.
Analysis of treatment strategies and curative effects of patients:
Previous literature has focused on fusion gene mutations that may be acquired after EGFR-TKIs resistance. Drug resistance mechanisms include B-Raf proto-oncogene(BRAF),ALK,RET,NTRK,fibroblast growth factor receptor 3 (FGFR3) and other mutation types[24] [25] [26]. Few reports have analyzed the therapeutic strategy and efficacy of secondary ALK fusion mutation therapy and there are no relevant recommendations in current guidelines and expert consensus.
Previous reports mostly focused on patients with co-existing of primary EGFR-sensitive mutation and ALK fusion mutation, and the medication strategy for patients with primary double mutations was also controversial. A literature review of 100 patients with primary double mutation found that the Disease control rate(DCR) and Objective response rate(ORR)of first-line use of ALK-TKIs (including Crizotinib and Ceritinib ) were 73.1% and 42.3%, respectively, that were higher than first-line use of EGFR-TKIs (including Gefitinib and Erlotinib) with DCR and ORR of 61.5% and 23.1%, respectively. Therefore, ALK-TKIs may be the first choice for patients with primary double mutation because NSCLC with ALK gene rearrangement tends to be more aggressive. ALK-TKIs seem to be more effective than EGFR-TKIs[29]. ALK inhibitors can be preferentially used in double-positive patients, especially in those with low EGFR mutation abundance[30]. However, there are different views that EGFR-TKIs may be more effective than ALK-TKIs in this group of patients[31] [32], and in patients with ALK/EGFR double mutation, the effectiveness of ALK-TKIs and EGFR-TKIs are worse than those in patients with a single mutation.
Treatment options for ALK fusion after secondary resistance have only been reported in few cases. This study analyzed the diagnoses and treatments of 4 patients with acquired ALK fusion mutation after EGFR-TKIs, and found that the ORR and PFS of ALK-TKIs were similar to those reported in previous literature. In two patients with ALK/EGFR double mutations after resistance, one treated with EGFR-TKI plus ALK-TKI and obtained a long PFS; the other was treated with Ensartinib plus Almonertinib for 2 months, which caused severe systemic edema. The treatment plan was adjusted to Ensartinib every other day plus Osimertinib and showed good tolerance and effect. Previous literature has also reported that the combination therapy of EGFR-TKIs and ALK-TKIs has a good effect[33] [34]. The other two patients in our study used ALK-TKIs alone, Case 1 changed Alectinib to Ensartinib due to a side effect of hemolytic anemia and obtained disease control over 20 months after EGFR-TKI resistance and is still being treated. Case 4 used Alectinib monotherapy and reached PFS of 21 months. The above analysis suggests that AlK-TKIs alone or in combination with EGFR-TKIs are both alternative. It is worth noting that Case 4, after PD of Alectinib, this patient showed Small Cell Lung Cancer (SCLC)transformation in rebiopsy. Chemotherapy achieved certain effects. To our knowledge, this is the first case of ALK fusion followed by acquired SCLC transformation after EGFR-TKIs resistance, which reveals the complexity of resistance and the need for rebiopsy after disease progression. However, due to the small number of patients in this study, more sample size analysis is needed to guide the treatment of patients with acquired ALK fusion mutations.
Tumor origin and tumor heterogeneity of EGFR/ALK co-mutation
There are many studies on the tumor origin of primary EGFR/ALK co-mutation. The basic theory is that EGFR mutation and ALK fusion, both genetic changes, exist from the initial proliferation of tumors[18]. One view is that the changes of these two drivers can develop in the same tumor cell clone and coexist in the process of cancer development through cell line studies[35]. Immunohistochemical analysis of lung cancer tissues from patients with primary EGFR and ALK co-mutation found that EGFR mutation and ALK fusion were co-localized and co-expressed in the same cell group, and the two proteins may play synergistic driving roles in the same cancer cells[18]. Another view is that ALK/EGFR co-mutated lung adenocarcinoma is composed of two tumor cell subpopulations carrying EGFR mutations and ALK fusion mutations, reflecting genetic heterogeneity within the tumor[36] [37]. Cai et al. [38]used laser capture microdissection (LCM) to capture tumor cells with similar or different growth patterns from patients with ALK/EGFR co-mutations, and deduced that the gene changes were divided into four subclones: coexisting with EGFR mutation and ALK rearrangement, single EGFR mutation, single ALK rearrangement, and no gene changes.
Our study found that 2 out of 4 patients had co-existing EGFR and ALK fusion mutations at the time of EGFR-TKIs resistance (case2 had NGS result of metastatic lymph node rebiopsy and case3 had the NGS result of plasma). At present, for secondary ALK fusion gene mutations, it is possible that there was a small number of ALK fusion gene mutations initially. Due to the pressure of EGFR-TKIs therapy and cloning selection, the subsequent ALK fusion gene mutations then could be detected. In the case of primary double mutation, if one of TKIs is selected for first treatment, the remaining uninhibited mutant cell lines of ALK or EGFR will continue to grow[11]. But in the case of secondary EGFR-TKIs resistance, the need for combined application of EGFR-TKIs and ALK-TKIs may be related to the phosphorylation level of downstream proteins activated by the signaling pathway[35]. From our study, it may also be related to the abundance of ALK and EGFR after drug resistance. Therefore, detecting the abundance of EGFR mutation and ALK fusion, as well as the phosphorylation level of downstream proteins, may be beneficial to guide the selection of TKIs in clinical practice.
This study analyzed the clinical characteristics and diagnoses and treatments of four very rare patients with acquired ALK fusion mutation after EGFR-TKIs treatment and only individual cases were reported in the previous literature. Our patients have dynamic results of NGS method detection, indicating the dynamic gene mutation status of these patients. Our study discussed the specific treatment plans of this kind of patients and analyzed the curative effect and benefit of them.