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Multiple mutations in the EGFR gene in lung cancer: a systematic review

Background Lung cancer is a public health problem worldwide. Currently, identifying genetic mutations in the epidermal growth factor receptor (EGFR) has brought significant changes in diagnosing and managing patients with lung cancer. The presence of multiple mutations, defined as the presence of more than one EGFR mutation, has been reported in a few studies. Therefore, we carried out this systematic review to describe the most common multiple mutations in the EGFR gene. Methods We conduct a systematic review of descriptive studies, cohorts, and clinical trials published in Scopus, PubMed, Scielo, and Virtual Health Library literature. The inclusion criteria for the systematic review were descriptive studies, cohorts, and clinical trials with the presence of multiple mutations in the EGFR gene. It was followed the Preferred Reporting Items for Systematic Meta-Analyses (PRISMA) guidelines. Results In the systematic review, 41 articles were included. Four hundred and forty-six cases with multiple mutations in the EGFR gene were found (0.95% of the patients included in the studies). The most prevalent dual mutations observed were T790M + L858R and deletions in exon 19 + T790M. Triple mutations were found in 9 cases (2.017%). According to reports, the presence of T790M mutation in the multiple mutations has been associated with poor clinical outcomes. Discussion The presence of multiple mutations in the EGFR gene is rare. It is of great importance to consider the T790M mutation since it generates resistance to pharmacological management and has worse outcomes. The most important limitation was that clinical information data and follow-up could not be collected in a large percentage of patients. Therefore, future work should be focused on clinical characteristics, follow-up and repercussions in the treatment of patients with multiple mutations.

In recent years, identifying genetic mutations in the epidermal growth factor receptor (EGFR) has brought significant changes in diagnosing and managing patients with lung cancer (6). The EGFR gene is located on chromosome 7p11.2 and contains 28 exons. The protein it encodes has an extracellular domain, a transmembrane domain, and a cytoplasmic domain (7). The cytoplasmic domain (also called the tyrosine kinase domain) is responsible for the phosphorylation of its downstream targets and self-regulation. The impact of mutations in the EGFR gene falls on cell proliferation and differentiation regulation, hence its association with cancer (8,9).
The incidence of EGFR mutations differs significantly according to ethnicity, with incidences of 10-15% reported in European and North American populations, 26% in Latin America (10), and up to 62% in Asian people (11). In a recent study published by Suda et al. (12), in which 13,951 samples were obtained from patients with lung cancer, it was shown that the rate of smoking was higher in patients with the mutation in the EGFR gene (P=0.02). Deletion of exon 19 and the missense mutations in exon 21 (L858R) are the most common mutations of the EGFR gene (80-90%). They are considered common mutations sensitive to treatment with tyrosine kinase inhibitors (TKIs) (13). The use of EGFR TKIs has been first-line therapy in treating NSCLC with EGFR mutations (14). Patients with advanced NSCLC with TKIs-sensitive EGFR mutations may develop progressive disease after 12 months, mainly from secondary EGFR mutations leading to TKIs resistance (15). The main mutation after TKIs management is the substitution of the threonine residue at position 790 with methionine (T790M) within exon 20 of the EGFR gene, corresponding to approximately half of the cases of acquired resistance to TKIs (16,17).
The presence of multiple mutations, defined as the presence of more than one EGFR mutation, has not been much studied, and the clinical impact is uncertain. Therefore, we carried out this systematic review to describe the most common multiple mutations in the EGFR gene with clinical data collection. We present the following article according to the Preferred Reporting Items for Systematic Meta-Analyses (PRISMA) reporting checklist (available at https://tlcr.amegroups.com/article/ view/10.21037/tlcr-22-235/rc).

Search strategy
We conduct a systematic review of descriptive studies, cohorts, and clinical trials published in Scopus, PubMed, Scielo, and Virtual Health Library literature. No limits were placed on the language or publication period in the search. The search was performed with the following terms: lung AND EGFR AND ("dual" or "multiple" AND "mutation").
The PRISMA guidelines were followed for data extraction, analysis, and reporting (18). The search was carried out in December 2021. We consider papers available in the following languages: English and Spanish.

Study selection and data extraction
The inclusion criteria for the systematic review were descriptive studies, cohorts, and clinical trials with the presence of multiple mutations in the EGFR gene. Two reviewers (JJC and JPC) screened all the titles and abstracts from the publications and performed an eligibility assessment. Retrieved articles were rejected if the eligibility criteria were not met. A third reviewer (RPM) was consulted when eligibility criteria were unclear. Hand searches were performed from the papers that seemed to be relevant.
The extracted data from each article, when possible, were age, gender, smoking status, staging of the tumor, pathologic description of the tumor, kind of pathological sample, molecular method of diagnosis, history of TKIs use and resistance, and survival. All cases were analyzed individually and organized according to the exon with the lowest denomination present in the multiple mutations.

Data synthesis and analysis
Univariate analysis was applied to determine the distribution of clinical and pathological findings.

Multiple mutations data
Four hundred and forty-six cases with multiple mutations in the EGFR gene were found out of 46,679 samples included in the systematic review (0.95% of the total) ( Tables 1,2). Although not all the articles mentioned the histological type of cancer, according to the report's information, it was possible to collect 125 cases of adenocarcinoma, 7 cases of adenosquamous carcinomas, 13 cases described as non-adenocarcinoma, and 1 case as bronchoalveolar carcinoma (previous terminology). The majority of samples were obtained after surgical (7 articles) or needle biopsy procedures (7 articles).
From the data of the patients included with follow-up, it was possible to obtain that the average progression-free survival (PFS) was 8 months (n=29 patients), and the overall survival (OS) was 19.3 months (n=24 patients).
In 181 cases (  Figure 1 Flow chart of the systematic literature review.           patients in the group with any EGFR gene mutation (except deletion in exon 19, L858R, and de novo T790M) plus a de novo mutation T790M in the EGFR gene, 60 cases from the group with the presence of a deletion in exon 19 or L858R, and any EGFR gene mutation (except deletion in exon 19, L858R, and de novo T790M), 12 cases from the group of a combination of any EGFR gene mutation (except deletion in exon 19, L858R, and de novo T790M) and two cases of combination of deletion in exon 19 and L858R mutation. In 140 of the 175 patients, the T790M mutation of exon 20 was present, of which 100 cases were de novo, and 40 patients were acquired after TKI treatment. In the group of de novo T790M mutations, it can be established from the cases with clinical information that the mean age was 55.18 years, none was previously managed with TKIs, and the PFS and OS were 8.45 months (1 article, 2 patients) and 24.02 months (2 articles, 3 patients), respectively. However, survival data is limited to a small number of cases. In the case of patients with T790M acquired mutation, the average age was 61.69 years; 90% of the subjects had previous management with TKIs, where the majority were first-generation TKIs. In this group of patients, more information on patients was available, and the PFS was 3.62 months (5 articles, 17 patients) and OS 8.03 months (3 articles, 10 patients).
Triple mutations were found in 9 cases (2.017%). In 5 of them, the dual T790M/L858R mutations were associated with a third variable mutation. Interestingly, in two instances, triple mutations were composed of the most frequent mutations found in our study: del19/T790M/ L858R. The cases are described in greater detail in Table 4.

Discussion
Multiple mutations in the EGFR gene are a rare event. We found 446 of 46,679 patients with multiple mutations in the present review. These mutations were reported mainly in exons 18 to 21. Only one case reported mutation in exon 16 (48). We included patients from different ethnicity, predominantly Asian and European. The most common multiple mutation was the L858R mutation in exon 21 with the T790M in exon 20 (de novo and acquired). In contrast to Kate et al. (51), the most common multiple mutations were a deletion in exon 19 with the T790M mutation in exon 20. In our study, there were 158 patients with the L858R and T790M mutations, from which detailed information could be obtained in some of them. Gao et al. (52) reported 63 cases, of which 37 (58.7%) patients were older than 60 years, 37 cases were women (58.7%), most of these patients had no history of smoking (80.9%), and in 49 cases (77.7%) were adenocarcinomas.
The most common single mutation associated with dual mutations was the mutation in exon 20 (T790M) (de novo and acquired), with 305 cases in total. In the patients with the T790M mutation that we were able to collect data from (138 patients), we found that patients with the acquired T790M mutation are more frequent with the subgroup of patients with a deletion in exon 19. In contrast, de novo mutations are more abundant in patients with the L858R mutation. Liang et al. (58) analyzed 25 studies including 1,770 patients; they observed that T790M was more frequent in exon 19 deletion than in L858R among patients with acquired resistance to TKIs.
According to previous data, around 73% of lung cancers with the T790M mutation have a second mutation in the EGFR gene (59). In our study, the T790M mutation (including de novo and acquired cases) was present in 69.05% (n=310/446) of all multiple mutations. This mutation is usually detected in NSCLC patients previously exposed to TKIs, where approximately half of the cases developed resistance to this type of drug (39). The study by Singh et al. (60). showed that the coexistence of the de novo EGFR T790M mutation with any other EGFR mutation had primary resistance to first-and second-generation EGFR TKIs. A hypothesis suggests that the T790M mutation by itself could be a weak oncogene, requiring a secondary mutation to enhance cancer development (59). However, clinical follow-up data are limited to a few patients. We found that the PFS and OS from patients with acquired T790M mutation plus other mutations (PFS: 3.6 months, OS: 8.2 months) are lower than patients with de novo T790M mutation plus other mutations (PFS: 8.4 months, OS: 24 months). In the study by Lin et al. (61), it was shown  (64).
In vitro studies have observed that dual mutations reduce the response to TKIs compared with one mutation (65,66). A recent systematic review by Attili et al. (67) described that the objective response rate (ORR) and the PFS are different in patients with dual common and uncommon mutations treated with first-generation or second-generation TKIs. They defined common mutation as the deletion in exon 19 or L858R mutation; and uncommon mutation as any mutation but L858R, exon 19 deletions, or de novo T790M mutation. They found that patients with uncommon dual mutations had less ORR and PFS than common dual mutations. They concluded that in patients with common dual mutations and dual mutations of common and uncommon, the similar overall response and survival outcomes with any TKIs are similar to a single common EGFR mutation. At the same time, the patients with uncommon mutations have higher benefits with second and third-generation TKIs.
The nine cases with triple mutations in the world literature are interesting. It was possible only to obtain information from four patients from our analysis. It is striking to see that a large percentage of the mutations involved are the most common mutations, including exon 19 deletions, L858R and T790M; which raises the hypothesis that with the development of last generation TKIs, the tumor biologically must give "third hits" to maintain its oncogenic activity. Especially, third-generation TKIs have shown better efficacy and safety profiles than other inhibitors. However, their resistance mechanism is more heterogeneous than that of other inhibitors, and in many cases, the resistance mechanism has not yet been identified (68).
This article has limitations. The most important was that clinical information data could not be collected in a large percentage of patients. In addition, where data were available, the type of information was not uniform, which prevented the extraction of accurate epidemiological data, especially regarding patient follow-up. Also, in general, when studying multiple EGFR gene mutations in patients with lung neoplasms, there is a risk that a mutation may be missed because molecular screening techniques are used in many medical centers and not specific genotyping methods (69). In our study, we mainly analyzed patients with deletions in exon 19; however, according to a recent study by Chen et al. (70), there are a large number of variants in this type of mutation (different starting locations and with or without amino acid insertion/substitution) that may have clinical repercussions, for which we consider it essential to take this into account for future studies.

Conclusions
According to our analysis, multiple mutations in the EGFR gene are found in less than 1% of the samples studied in patients with lung neoplasms. The most frequent dual mutations were T790M with L858R and exon 19 deletions with T790M. The most common single mutation associated with dual mutations was the mutation in exon 20 (T790M) (de novo and acquired). Acquired T790M mutations are more frequent in the subgroup of patients with a deletion in exon 19, whereas de novo mutations are more abundant in patients with the L858R mutation. The presence of T790M mutation showed poor outcomes in the follow-up of patients.
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