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    Topic review

    NSCLC Concurrent EGFR Genomic Alterations

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    Submitted by: Antonio Russo

    Definition

    Non-small cell lung cancer (NSCLC) accounts for roughly 85–90% of overall cases of lung malignancies and includes different histological subtypes. The treatment landscape of NSCLC has been terrifically changed by the discovery of Epidermal Growth Factor Receptor (EGFR) mutations and their response to the EGFR tyrosine kinase inhibitors (TKIs). EGFR gene aberrations have been defined as oncogenic driver mutations which occurred in 5–17% of lung adenocarcinomas among Caucasian patients, while in approximately 45–55% of the Asian population. Nowadays, EGFR-TKIs are the standard of care for patients affected by advanced EGFR-mutated NSCLC considering their established prolonged progression-free survival (PFS) in comparison to the standard chemotherapy approach. However, TKIs clinical efficacy remains restricted due to the development of resistance, which has been hardly clarified. The recent technological breakthrough and the advent of next-generation sequencing (NGS) platforms have enabled comprehensive profiling of the genome, providing novel evidence of co-existing multiple driver alterations.

    1. Complex EGFR Mutations

    Of note, almost 45% of EGFR gene aberrations are in-frame deletion alterations in exon 19 (19Del) and the p.L858R within exon 21 [1][2]. These activating mutations enhance a better outcome in patients, granting a complete blockade of the EGFR signaling pathway by EGFR-TKIs. Otherwise, EGFR mutations occurring in exons 18 and 20 are correlated with resistance to standard treatments. Uncommonly, complex EGFR alterations could be detected in a single tumor specimen harboring two or more various intra-EGFR mutations [3]. Complex EGFR mutations occur almost in 3–7% of EGFR-mutant patients [4]. Belardinilli et al. described a single clinical case of an NSCLC patient harboring three coexisting aberrations on the EGFR gene, two of which presented on the same allele [2]. In fact, through the use of NGS, the authors detected the simultaneous presence of three missense mutations, a p.L858R and p.L861R both in exon 21 with an allele frequency close to 41%, and a p.R776H in exon 20 with an allele frequency of 67.5%, respectively. Besides, upon therapy with the second-generation EGFR-TKI afatinib, the patient showed a partial response on the target lung lesion with a PFS of eight months. Moreover, a clinical trial conducted by Lee et al. investigated molecular backgrounds of primary resistance to EGFR-TKIs in NSCLC patients harboring sensitive EGFR alterations [5]. The study population included a cohort of 197 patients, out of whom nine individuals had two co-existing EGFR mutations. Additionally, among 11 patients exhibiting de novo resistance to TKI treatment only one patient had a coexisting EGFR complex mutation, particularly p.T790M mutation and 19Del. The authors reported that this patient displayed immediate disease progression involving symptomatic metastasis to the central nervous system (CNS) while receiving EGFR-TKI treatment. Furthermore, a recent analysis by Liang et al. evaluated concomitant alterations in EGFR 19Del/L858R mutation and their correlation with EGFR-TKIs response in a total of 403 NSCLC patients [6]. This trial included two cohorts and comprehensively analyzed the concomitant mutational profiles of EGFR 19Del and p.L858R in TKI naïve patients. The authors assessed that the existence of somatic p.T790M at baseline was similar in 19Del (120, 73.4%) and p.L858R (160, 72.4%) mutations. Furthermore, Zhang et al. screened 187 patients with complex EGFR mutations out of 5898 EGFR-positive NSCLC patients. Fifty-one of these patients were under first-line treatment with first-generation EGFR-TKIs [4]. Namely, 58 patients were found to carry a concurrent alteration in EGFR exon 20 and 21, while 45 patients harbored a concomitant mutation in exon 19 and 21. Considering the genetic aberrations, simultaneous p.T790M and p.L858R were the most common, followed by 19Del and p.L858R. The median PFS was 9.5 months. The overall response rate (ORR) was 52.2% (95% CI 37.2–67.2%), and the disease control rate (DCR) was 71.7% (95% CI, 58.2–85.3%). Additionally, the authors subdivided patients into four groups: A) patients with 19Del and p.L858R; B) patients harboring a 19Del or p.L858R and atypical mutations; C) double atypical mutations; and D) complex mutations with a primary drug-resistant pattern, such as a primary p.T790M mutation or an exon 20 insertion. As reported by the authors, NSCLC patients with exon 19Del and p.L858R exhibited the best ORR and PFS, 75% and 18.2 months, respectively. On the other hand, patients included in group D displaying complex mutations with a primary drug-resistant pattern, such as a primary p.T790M mutation or an exon 20 insertion, have the worst clinical outcomes. Notably, some of these patients carried a sensitizing EGFR alteration (i.e., 19Del/p.L858R/p.L861Q) plus a p.T790M de novo or an exon 20 insertion. Thus, the worst clinical outcomes achieved by these patients could be explained by the fact that they were treated with first and second-generation EGFR-TKIs. Moreover, Benesova et al. described a single case of a patient with complex EGFR alteration [7]. Of note, the patient exhibited partial response under treatment with gefitinib. Otherwise, Sato et al. reported that 6 patients with double EGFR alterations showed a poorer response to gefitinib treatment [8]. De Marchi et al. found 33 patients with double EGFR genomic aberrations in a cohort of 1006 lung cancer patients, with no data being unfortunately available on their clinical outcomes [9]. Li et al. detected 58/5125 EGFR double mutations, with the highest incidence rate of p.T790M and p.L858R [10]. Chen et al. presented 4/36 patients harboring concurrent 19del and p.L858R with a worse response after TKI treatment [11]. Additionally, Chen et al. reported concurrent EGFR complex genomic alterations in 20 patients with the worst outcome in terms of OS [12].

    2. Actionable Concomitant Oncogenic Driver Mutations

    Although actionable oncogenic gene driver mutations in NSCLC were historically considered mutually exclusive, the recent advent of comprehensive genomic profiling in clinical specimens was able to identify a notable number of concurrent alterations in EGFR-mutated NSCLC. Recently, various original research articles and case reports were conducted on this topic, suggesting that some EGFR-mutant NSCLC patients may carry concomitant genetic aberrations in different oncogenic driver genes.

    2.1. ALK

    ALK is a component of the insulin receptor protein-tyrosine kinase superfamily, formerly reported as a nucleophosmin (NPM)-ALK fusion pattern in cell lines of anaplastic large cell lymphoma (ALCL) [13]. In 2007 ALK fusion was described in lung adenocarcinoma for the first time in a limited cohort of Asian individuals [14]. The most common aberration is an inter-chromosomal inversion in the short arm of chromosome 2, which generates a fusion between the echinoderm microtubule-associated protein like-4 (EML4) gene and the ALK gene [15]. Consequently, the fusion EML4-ALK with tyrosine kinase function stimulates proliferation and cell survival [15]. Chromosomal rearrangements in the ALK gene are detected in approximately 5% of NSCLC patients [16]. Moreover, this driver fusion is predominantly estimated mutually exclusive with other genetic mutations, such as EGFR [17]. Notwithstanding, with the advent of novel and powerful technologies like NGS the detection rate of concomitant genetic alterations in EGFR and ALK is systematically increased [17][18]. Liu et al. evaluated the efficacy of TKI treatments on 21 co-altered EGFR and ALK patients with advanced NSCLC [18]. Three out of 21 patients received dual blockade TKI treatment with EGFR- and ALK-TKIs, reaching a PFS of 5.2 months with the combination therapy. Furthermore, analyzing the clinical-pathological features of the concomitant mutation patients the authors found that the double genetic alteration was more likely to occur in young females than in males. Additionally, Hu et al. examined the frequency of concurrent genetic alterations in EGFR-positive patients, evaluating the efficacy of EGFR-TKIs treatment in this setting [19]. Out of 320 patients including in the study population, six patients were found harboring a co-alteration in ALK gene and they achieved a mPFS of five months, shorter compared to those with a single EGFR mutation (mPFS 10.9 months). Namely, four out of six patients with concomitant ALK rearrangement were treated with the first-generation ALK-TKI crizotinib and three obtained partial response according to RECIST criteria. Considering the particular subset of patients, a recent report by Zhuang et al. determined that ALK-TKI therapy for the treatment of 20 patients with a co-alteration in ALK fusion was more active as first-line treatment than in later lines of treatment [20]. Yang et al. assessed that 13/977 NSCLC patients screened harbored a concomitant genetic aberration in EGFR and ALK genes [21]. Out of 13 patients, 10 naïve patients received EGFR-TKIs reaching an ORR of 80% and a mPFS of 11.2 months (95%CI 5.6–16.8). Four patients were treated with crizotinib, and three of them in a second-line setting. Considering the clinical outcomes, two patients appeared to respond to EGFR-TKI, yet not to ALK-TKI; whereas one was sensitive to crizotinib. The only patient who received crizotinib as first-line displayed 15.1 months of PFS, still not show response to consecutive EGFR-TKI treatment. Patients with EGFR and ALK coexisting aberrations seemed to better respond to EGFR-TKIs in the first-line setting. Of note, in order to explain the great heterogeneity of clinical outcomes, the authors suggested that different sensitivities to therapies might be correlated with different levels of EGFR or ALK protein phosphorylation. Fan et al. described a single case of a patient harboring EGFR/ALK alteration, who had partial response under ALK-TKI [22]. Besides, Lee et al. described 12 patients with double EGFR/ALK alteration, 11 of which with a partial response to treatments based on gefitinib, erlotinib or crizotinib [23]. Notably, Miyanaga et al. described a single case where the patient showed response both to first-generation EGFR-TKIs and crizotinib [24]. Sweis et al. presented a case series including four patients treated with erlotinib and crizotinib, achieving a stable disease as the best response [25]. Thumallapally et al. reported a single case harboring an ALK translocation together with an EGFR p.L861Q mutation treated with crizotinib reaching a PFS of 3 weeks [26]. In their exploratory study, Lee et al. found two out of 197 EGFR-positive NSCLC patients with a concurrent genomic alteration in ALK [5]. Notably, the patients were treated with gefitinib and consequently with crizotinib, achieving a partial response. Chang et al. did not report the clinical outcome of their single case [27], as well as Zhu et al. who described two patients out of 139 [28]. Chen et al. described a single case of double EGFR/ALK alteration with poor outcomes [12].

    2.2. KRAS

    KRAS alterations are frequently represented by missense mutations occurring in lung adenocarcinomas [29]. Molecular evaluation of KRAS is crucial to predict clinical outcomes and to choose the best therapeutic option, as KRAS-mutant tumors exhibit primary resistance to EGFR-TKIs [29]. Moreover, almost 6–35% of EGFR positive patients harbor a concomitant genetic aberration in the KRAS gene [30]. P.G12C, p.G12V, and p.G12D mutation are the most frequent alteration detected [31]. Several cases have been reported for EGFR and KRAS concurrent alterations. Benesova et al. presented three cases of patients with EGFR mutations combined with KRAS mutation [7]. Despite an initial positive response to EGFR-TKI, the real activity did not last long showing a PFS of three, five, and seven months, respectively. Opposing this report, Zhuang et al. reported a retrospective study involving 3774 patients with concurrent genetic alterations [20]. Namely, 11 patients of the cohort showed a co-alteration in EGFR/KRAS and they were treated EGFR-TKI therapy as first-line treatment, displaying an ORR of 62.5% (5/8). Interestingly, the PFS comparisons between patients with an EGFR/KRAS co-mutation and those carrying a single EGFR mutation were not statistically significant. Ranchiglio et al. identified 14 patients with concurrent EGFR and KRAS mutations, among six with a dominant VAF [32]. Notably, their PFS was significantly shorter compared to EGFR mutations (2.42 months vs. 11.09 months; p = 0.0081), and also the ORR was poorer (16.7% vs. 57.1%). Additionally, Nardo et al. analyzed the prevalence of concurrent KRAS mutations on 106 patients with EGFR-mutant NSCLC focusing on their impact on clinical outcome [33]. Indeed, KRAS co-alterations were detected in 3 patients with a VAF of less than 0.2%, which showed poor clinical outcome to first-line EGFR-TKI, in terms of time to treatment failure (TTF), OS and PFS (five, six and five months, respectively). Lee et al. described six patients with EGFR/KRAS aberration, not reporting their clinical outcomes [23], as Li et al. who reported 30 patients with double alterations out of a cohort of 5125 individuals [10]. Chevallier et al. described a single case [34], as De Marchi et al. [9]. Moreover, Zhang et al. found two out of 120 patients with double concurrent genomic aberrations [4]. Whereas Hu et al. described a single case of EGFR/KRAS out of a cohort including 320 individuals [19], of note the patient showed progression after treatment with erlotinib. Finally, in the trial by Chen et al. [12], seven out of 36 patients displayed a concurrent alteration in EGFR and ALK with poorer PFS after EGFR-TKI treatment.

    2.3. ROS-1

    ROS-1 rearrangements has been detected in almost 1–2% of lung adenocarcinoma [35]. The ALK-TKI crizotinib is highly active in ROS1-rearranged patients [36]. Patients harboring a concomitant mutation in EGFR/ROS-1 are very rare, thus we found little data in the current literature. Zhu et al. described a case of a single patient with concurrent EGFR/ROS-1 alteration [35]. Moreover, in the above-mentioned article by Zhuang et al., two out of 3774 patients harbored a co-alteration in EGFR/KRAS/ROS-1. Namely, one patient showed a progression after second-line treatment with crizotinib and partial response to icotinib as third-line treatment (PFS of 27.5 months), while the second patient had a partial response after first-line treatment with gefitinib (PFS of 12.7 months) [20]. Hu et al. reported one out of 320 patients with double ROS-1/EGFR genomic alteration and a partial response after erlotinib as first-line treatment [11].

    2.4. MET

    Mesenchymal–epithelial transition (MET) encodes a transmembrane tyrosine kinase, which activates downstream signaling pathways by binding to the hepatocyte growth factor. Thusly, it has a crucial role in cell proliferation and survival [37]MET alterations are emerging as important driver aberrations for NSCLCs, particularly MET gene amplification and exon 14 skipping mutations are found with a frequency of 1–11% and up to 4% in lung adenocarcinoma [38]MET amplification is a well-known resistance mechanism against EGFR-TKIs, including the third-generation osimertinib [39][40]. Indeed, MET amplification is accountable for almost 5–22% of secondary resistance to EGFR-TKIs. Particularly, MET amplification induces ErbB3 phosphorylation, hence activating the PI3K/AKT pathway [38]. In line with these data, the treatment combination of EGFR-TKIs and MET-inhibitors has been evaluated in different clinical trials, such as INSIGHT 1 and TATTON [41][42]. Namely, in the phase 1b/2 clinical trial INSIGHT 1, Wu et al. and colleagues evaluated the efficacy of the combination tepotinib/gefitinib in EGFR-mutant patients with MET amplification and secondary resistance to EGFR-TKIs, reporting better mPFS and mOS in this particular subset of patients (16.6 vs. 4.2, HR 0.13; 37.3 vs. 13.1 HR 0.08, respectively) [41]. Additionally, Oxnard et al. examined the safety of osimertinib in combination with selumetinib/savolitinib/durvalumab [40]. Indeed, only three patients harbored MET amplification and p.T790M and they were treated with selumetinib displaying partial response [40]. However, osimertinib combination with savolitinib in patients with MET-driven secondary resistance to EGFR-TKIs is under current evaluation in the ongoing trials SAVANNAH (NCT03778229) and ORCHARD (NCT03944772). Whereas MET exon 14 skipping/EGFR mutations are very rare and poorly explored. In preclinical models MET ex14 decrease sensitivity to EGFR-TKIs [42]. As results of our systematic review of the literature, we found only three papers presenting interesting data on this particular setting. In fact, Chevallier et al. reported 15 patients with EGFR/MET alteration known to be non-pathogenic according to international database [34]. Lee et al. described a single patient with MET amplification >15 gene copies in 17% of tumor cells [5]. Chen et al. reported a single case including in the short PFS group (10% vs. 33% p = 0.018) [12]. Finally, there is a strong rationale for the use of combination of EGFR-TKIs and MET inhibitors in this setting, thus larger studies are warranted.

    2.5. BRAF

    BRAF mutations, both p.V600E and non-p.V600E, are detected in 6–8% of NSCLC cases, inducing downstream activation of the MAPK signaling pathway [43]. Over the decades, several BRAF inhibitors have been developed and the combination of trametinib and dabrafenib was the first treatment approved for advanced BRAF p.V600E-mutant NSCLC [44][45]. Concomitant EGFR/BRAF aberrations are found in approximately 11% of EGFR-positive NSCLC patients, with the BRAF p.V600E mutation most frequently identified [46][47]. Chen et al. retrospectively screened 423 NSCLC patients harboring EGFR 19Del or p.L858R mutations reporting only one patient with concurrent BRAF p.V600E [12]. Of note, the patient showed a poor PFS. Furthermore, Li et al. assessed a comprehensive mutation profiling from 5125 Chinese cohorts and they reported 160 concurrent mutations including two EGFR/BRAF concomitant mutations [10]. Moreover, Rachiglio et al. found hotspot mutation in several genes, including BRAF in 14 patients (21.8%) of their cohort [32]. Zhuang et al. described two cases of concomitant EGFR/BRAF alteration, showing better outcomes with EGFR-TKI than with standard chemotherapy [20].

    2.6. RET

    Rearranged during transfection (RET) gene rearrangements are detected in almost 1% of NSCLC patients [48][49]. Recently, FDA has granted accelerated approval to pralsetinib and selpercatinib for lung cancer patients harboring RET fusion based on ARROW and LIBRETTO-001 clinical trials results [50][51]. In up to 10% of NSCLC patients under osimertinib treatment, oncogenic fusions of RET gene have been considered responsible for acquired resistance [50][52]. Taking into account this, the open-label, multicenter, biomarker-guided, phase 2 clinical trial ORCHARD (NCT03944772) is still recruiting NSCLC patients progressed on 1-L osimertinib therapy, and one cohort includes RET rearranged patients which will receive osimertinib in combination with selpercatinib (LOXO-292) [53][54]. Albeit, the co-presence of EGFR mutation and RET rearrangement is rare, we found a single case report and a research article presenting original data on this particular subset of patients. Hu et al. detected one patient with concurrent EGFR and RET genomic alteration out of a cohort including 320 EGFR positive patients [19]. Particularly, the patient was an Asian young female with lung adenocarcinoma with no history of smoking, treated with gefitinib displaying poor OS and PFS (10.2 and 2.2 months, respectively) and PD as best response. Moreover, Klempner et al. and colleagues reported two patients with secondary acquired RET fusion in Asian EGFR-mutant NSCLC patients, both presenting short survival [55]. Of note, none of the patients reported underwent a combination treatment with EGFR-TKIs and RET-inhibitors. These data available from the literature confirmed the fact that RET fusion is a resistance mechanism in EGFR mutated patients and larger clinical trials are warranted in order to evaluate the potential activity of the combo EGFR-TKIs and RET-inhibitors.

    3. TP53, PTEN, PIK3CA, CDKN2A and RB1

    TP53 gene mutations are identified in 35–55% of NSCLC cases, especially in squamous cell carcinoma (SCC) and in smokers or former smokers [56][57][58]. Inactivating mutations of the TP53 gene affect the normal transcriptional p53 activity leading to tumor susceptibility and hinder patients’ response to chemotherapy treatments [59][60]. Moreover, TP53 alterations might be related to a poor prognosis in NSCLC patients [61]. Almost 55–65% of EGFR-positive NSCLC patients harbor a TP53 coexisting mutation [5][62][63]. Preclinical models have already demonstrated a correlation between TP53 mutation and response to EGFR-TKIs therapy [63][64][65]; namely apoptosis induced by gefitinib is decreased in p53 mutated cells. Mutation in TP53 gene have been divided into disruptive mutations and non-disruptive ones considering the loss of function of p53 protein. Specifically, disruptive mutations produce a complete loss of function of p53, while non-disruptive alterations result in conservative mutations or non-conservative mutations (excepting stop codons) outside the L2–L3 region [64][66][67][68]. Comprehensively, the systematic literature review identified a total of 11 reports evaluating the TP53 status in EGFR-mutant patients with lung adenocarcinoma. Namely, Canale et al. conducted an independent retrospective cohort study on a total of 136 EGFR-mutated NSCLC patients under treatment with first or second-generation TKIs as a first line therapy, in order to assess the role of TP53 gene alterations as predictor of survival and response to EGFR-TKIs therapy [69]. Endpoints of the clinical study were DCR, ORR, PFS and OS. TP53 mutations were detected in 42 (30.9%) out of the 136 patients, indeed according to the classification of TP53 aberrations into disruptive and non-disruptive mutations, the authors observed 11 patients harboring a disruptive TP53 mutation, while most of the patients carried a non-disruptive alteration [68][70]. Thusly, the authors found that TP53 mutations in exon 8 are related to a worse PFS regardless to the EGFR-TKIs treatment. Moreover, after a combined analysis the authors confirmed that the worse clinical outcome was independent from the subtype of EGFR mutations reported. Of note, further analysis was conducted on a sub-cohort of lung adenocarcinoma patients who developed a p.T790M resistance mutation and treated with osimertinib. This broadened analysis confirmed worse PFS and OS. These data were consistent with a previous report by Hou et al. [71]. In fact, this clinical trial examined the impact of TP53 gene alterations on the clinical outcomes in a Chinese cohort of 163 patients with NSCLC. By using NGS to establish the mutational status of EGFR and TP53, 43 EGFR-positive patients were found harboring a concurrent TP53 gene alteration. Considering the treatment outcomes, this subset of patients showed shorter median PFS (6.5 vs. 14.0 months) and median OS (28.0 vs. 52.0 months). Notably, differences in outcomes were particularly meaningful in the subset of patients harboring TP53 gene non-missense mutations, non-disruptive mutations, mutations in exon 6 and in exon 7 and mutations in the non-DNA Binding Domain (DBD) region among all TP53 mutations. Interestingly, these data are consistent with the report by VanderLaan et al. [72] who described 10 patients with TP53 concurrent mutation and worse clinical outcomes. Of note, the authors demonstrated a decreased rate of acquired p.T790M mutation as a mechanism of resistance to gefitinib, erlotinib and afatinib in lung adenocarcinomas with concomitant TP53 mutations. This could be explained as genomic complex tumors might trigger different pathways bypassing EGFR as a target. Additionally, an intriguing retrospective research was reported by Chen et al., who validate the number of concurrent mutation and Tumor Mutational Burden (TMB) in 71 patients with EGFR mutation and under treatment with EGFR-TKIs stratified for PSF [12]. Namely, TMB was defined as somatic, coding, base substitution, and indel mutations per megabase of genome analyzed. No significant differences were assessed between the two groups, yet the shorter PFS subgroup revealed a TMB higher than eight. One could guess that an increased TMB is correlated with the existence of resistance pathways, as previous reports suggested [67]. Furthermore, among overall clinical studies, EGFR-TKIs appeared to have less activity in 67 patients harboring concomitant TP53 gene mutations. A novel treatment option for this particular subset of patients is represented by the combination of EGFR-TKIs and antiangiogenic agents. Indeed, the combination of anlotinib plus icotinib displayed promising activity in the ALTER-L004 clinical trial for EGFR-positive NSCLC patients. Namely, the intention to treat population (ITT) included 14 patients carrying concomitant TP53 alterations, which showed ORR of 78.5% and DCR of 100% [73]. Additionally, in the ACTIVE study, Zhang et al. reported better PFS in the apatinib plus gefitinib group in naïve patients with EGFR mutations and patients harboring TP53 exon 8 mutations showed significant benefit from the dual blockade (HR 0.24 95%CI 0.06–0.91) [74][75]. Rachiglio et al. described 23 EGFR/TP53 mutant cases, exhibiting a mPFS of 12.3 months and mOS of 18.9 months under EGFR-TKI treatment [32]. Interestingly, Sato et al. reported 12 patients (28%) with EGFR/TP53 alteration [8]. Moreover, Zheng et al. demonstrated that 11 patients with co-existing EGFR and TP53 genomic alteration might have a worse prognosis comparing to EGFR-mutant patients [76]. Lee et al. described three cases out of 197 patients [5]. Chevallier et al. reported 15 cases of double mutation, with no difference of survival [34]. Chang et al. found that TP53 was the most common concomitant alteration detected (10/31 patients) [27], as Chen et al. reported in their study [12].

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene and one of the most important negative regulator of the PI3K/AKT signaling pathway [77][78]PTEN is deleted in several types of cancers, such as prostate, endometrial, glioblastoma, breast, melanoma and colon [79][80][81]. Lung cancers are malignant tumors where PTEN deregulation plays a crucial role in tumor cell proliferation, metastasis process, and resistance to treatments. Beyond 40% of NSCLC, cases express loss of PTEN and it is related to poor prognosis, especially for EGFR-positive patients treated with EGFR-TKIs [82]. Various preclinical models have disclosed that PTEN inactivation could alter the pattern of response to EGFR-TKIs [34][83], namely Chevallier et al. reported a retrospective cohort trial of the influence of concurrent mutations on patients with advanced NSCLC treated with TKIs [34]. The authors found five patients harboring a resistance pathogen mutation in PTEN, who showed poor mPFS of 6.8 months. These finding are consistent with a recent report from Huang et al. [73]. Finally, VanderLaan et al. reported 5% (1/19 patients) of PTEN/EGFR altered patients [72].

    It has been already proved that the downstream signaling pathway of the HER family phosphatidylinositol-3-kinase (PI3K) is related to carcinogenesis in lung cancer [11]PIK3CA mutations are detected in almost 3–7% of patients with lung adenocarcinomas and commonly they are located in exons 9 and 20 [84]. These genetic aberrations generate constitutive activation of PI3K, AKT phosphorylation, and mTORC1 downstream which have a crucial role in cell survival and proliferation. In contrast to the mutual exclusivity of various oncogenic aberrations in NSCLCs, the coexistence of PIK3CA mutations with other oncogenic alterations is well established [84][85]. Actually, approximately 3.5% of EGFR-mutant patients harbor PI3KCA gene alterations and this seems to blunt the response to TKIs treatment. In vitro data suggest that EGFR-TKI sensitivity in EGFR-positive NSCLC cell lines has been related to downregulation of the PI3K pathway, and as a matter of fact increased resistance to gefitinib was confirmed after the introduction of the PIK3CA p.E545K mutation into a gefitinib-sensitive lung adenocarcinoma cell line [86]. Eng et al. analyzed the prognostic impact of a concurrent PIK3CA mutation in 13 EGFR-mutant NSCLC patients, finding poor ORR (62% vs. 83%; p = 0.80) and shorter median Time To Progression (TTP) (7.8 vs. 11.1 months; p = 0.84) to EGFR-TKIs [86]. Moreover, Wu et al. examined the significance and the effect of PIK3CA mutations on treatment outcomes to EGFR-TKIs of lung adenocarcinoma [41]. The study population included six PIK3CA mutation-positive patients. In contrast to the analysis by Eng et al., the authors reported similar response (ORR, 66.7 vs. 78.7%; p = 0.476) to EGFR-TKIs as wild-type patients. Notably, PIK3CA-mutant patients displayed a trend toward better PFS (12.0 vs. 8.8 months) and OS (25.1 vs. 21.4 months), still the variations were not statistically significant. Accordingly, Wang et al. investigated a cohort of 1117 NSCLC patients, out of which 17 patients harbored simultaneously a mutation in EGFR and PIK3CA [87]. They found that survival for patients with single PIK3CA mutation was poorer than patients harboring a concurrent double alteration in PIK3CA and EGFR (p = 0.004). Chevallier et al. reported two patients with double EGFR/PIK3CA alteration and poor survival [34]. De Marchi et al. detected 10/1208 individuals concurrent mutated in EGFR and PIK3CA [9], while Rachiglio et al. identified nine patients with double mutations displaying a mPFS of 5.5 months under EGFR-TKIs treatments [32]. Zhang et al. presented four patients harboring concurrent EGFR/PIK3CA genomic alteration [88], whereas Li et al. reported 64 (3.3%) of their 5125 patients [10]. Additionally, Hu et al. described nine out of 320 patients and of note they reported the longest PFS of 7.6 months, while Chen et al. found three out of 36 patients describing lower ORR (43.75% vs. 80.0%; p = 0.024) comparing to the population with a single EGFR alteration [11]. Lammers et al. reported three cases among their study population with poor response to erlotinib treatment [89], whereas Huang et al. recently reported better ORR of 72% among the 18 patients harboring double concurrent genomic alteration under icotinib and anlotinib treatment.

    CDKN2A gene encodes p16, a tumor suppressor which promotes a cell cycle arrest in G1 phase by inhibiting Rb phosphorylation. In NSCLC patients, the inactivation of CDKN2A is one of the most common genomic alterations detected [80], especially through the mechanisms of homozygous deletions (HDs), presented in up to 29–59% of lung adenocarcinomas regardless of the concurrent EGFR mutation [81]. Jiang et al. studied 127 EGFR-positive patients with NSCLC, identifying 31 out of 127 (24.4%) patients with HDs in CDKN2A, who displayed poor ORR to EGFR-TKIs and shorter mPFS. Of note, these results might justify the use of the combo EGFR-TKI and CDK4/6 inhibitors in this particular subset of patients [83]. Moreover, Chang et al. analyzed 31 NSCLC patients with EGFR alteration revealing copy number variation (CNV) loss in CDKN2A gene in seven patients (22.6%) [27]. Notably, four out of seven patients had an intermediate response (six to 12 months of PFS), while the other three patients presented a poor response (<six months). Finally, Skoulidis et al. and colleagues showed 24.6% of CDKN2A alterations in their cohort, concluding that co-alterations in EGFR and CDKN2A were related to EGFR TKIs acquired resistance [90].

    RB1 gene is a regulator of cell cycle and is phosphorylated by CDK4/6 to S-phase entry [91]. The alterations in RB1 pathway have been associated to worse prognosis in NSCLC patients [90]. In their article, Sato et al. and colleagues investigated 43 patients with EGFR mutations revealing 16% (7/43) of RB1 co-alterations [8]. Of note, these patients showed a poor prognosis. Hou et al. examined 71 NSCLC patients with EGFR mutations, of whom seven patients (9.9%) with a concomitant RB1 alteration [92]. Moreover, it is well-established that RB1 loss is a primary event correlated with transformation to Small-Cell Lung Cancer (SCLC) and consequently EGFR-TKIs treatment resistance [93][94]. Additionally, Yu et al. and Kim et al. reported RB1 as one of the most common gene co-altered in NSCLC patients [63]. Particularly, Kim et al. and colleagues identified co-alteration in RB1 as predictor of fast progression to TKI treatment [95].

    4. Methods of Detection

    The mutational analysis should be performed on tissue specimens and the most common methods for EGFR mutation detection with concomitant genomic alterations are reported in Table 1. Generally, the biological material available does not provide an amount of neoplastic cell percentage allowing the use of a Sanger Sequencing method. Conversely, high-sensitivity platforms as digital droplet PCR (ddPCR) (0.1%)[96] , or Amplification Refractory Mutation System (ARMS) with a specificity up to 1%[94]should be able to cleverly detect these pathogenetic variants with a specificity running up to wild-type DNA[94] . Nevertheless, the recent development of NGS accomplishes massive parallel gene mutation analysis and requires low amount of tissue, favoring the identification of several targetable molecular alterations until of 5% of VAF[96] .

    Table 1. Summary of reported demographic characteristics of EGFR-positive NSCLC patients with concomitant genomic alterations.

    Study Study Type Race No. of Pts Concurrent Genomic Alteration Detection Method Sample VAF
    Belardinilli et al. [2] Case Report Caucasian 1 EGFR complex NGS tumor tissue 40.30%
    41.30%
    67.50%
    Benesova et al. [7] Case Series Caucasian 4 EGFR+KRAS
    EGFR complex
    Sanger tumor tissue N/A
    Fan et al. [22] Case Report Asian 1 EGFR+ALK NGS tumor tissue EGFR 15.58%
    ALK 6.42%
    Lammers et al. [89] Case Report Caucasian 1 EGFR+PIK3CA SNapShot PCR tumor tissue N/A
    Lee et al. [23] Case Series Asian 12 EGFR+KRAS
    EGFR+ALK
    Sanger; Real Time PCR after PNA; FISH and IHC tumor tissue N/A
    Miyanaga et al. [24] Case Report Asian 1 EGFR+ALK PNA-LNA PCR clamp method, FISH and IHC tumor tissue N/A
    Sweis et al. [25] Case Series Caucasian 4 EGFR+ALK N/A N/A N/A
    Thumallapally et al. [26] Case Report Caucasian 1 EGFR+ALK FISH, direct sequencing tumor tissue N/A
    Zhu et al. [35] Case Report Asian 1 EGFR+ROS-1 NGS, PCR and FISH tumor tissue N/A
    Yang et al. [21] Case Series Asian 13 EGFR+ALK IHC, FISH, Sanger, RT-PCR and RACE-PCR sequencing tumor tissue N/A
    Hou et al. [71] Retrospective Asian 59 EGFR+TP53
    EGFR+RB1
    NGS tumor tissue N/A
    Zhu et al. [28] Retrospective Asian 2 EGFR+ALK FISH, RT-PCR tumor tissue N/A
    Li et al. [10] Retrospective Asian 149 EGFR+ PIK3CA
    EGFR complex
    EGFR+KRAS
    EGFR+BRAF
    SurPlex®-xTAG70plex-EGFR liquidchip tumor tissue N/A
    Liang et al. [6] Retrospective Asian 403 EGFR complex NGS tumor tissue + plasma N/A
    Liu et al. [97] Retrospective Asian 21 EGFR+ALK NGS tumor tissue + plasma N/A
    Nardo et al. [33] Retrospective Caucasian 3 EGFR+KRAS ddPCR tumor tissue + plasma KRAS <0.2
    Rachiglio et al. [32] Retrospective Caucasian 38 EGFR+KRAS
    EGFR+BRAF
    EGFR+MET
    EGFR+TP53
    EGFR+PIK3CA
    NGS, ddPCR tumor tissue + plasma KRAS 2–38%
    EGFR ≥ 2%
    Sato et al. [8] Retrospective Asian 43 EGFR complex
    EGFR+TP53
    EGFR+RB1
    NGS tumor tissue N/A
    VanderLaan et al. [72] Retrospective Caucasian 19 EGFR+TP53
    EGFR+PIK3CA
    EGFR+PTEN
    NGS, Sanger tumor tissue N/A
    Wu et al. [98] Retrospective Asian 12 EGFR+PIK3CA Sanger, RT-PCR tumor tissue N/A
    Zheng et al. [76] Retrospective Asian 11 EGFR+TP53 NGS tumor tissue N/A
    Zhuang et al. [20] Retrospective Asian 43 EGFR+ALK
    EGFR+ROS-1
    EGFR+KRAS
    EGFR+BRAF
    ARMS tumor tissue N/A
    Huang et al. [73] Prospective Asian 18 EGFR+TP53/PTEN
    EGFR+PIK3CA
    N/A N/A N/A
    Zhang et al. [74] Prospective Asian N/A EGFR+TP53 NGS N/A N/A
    Canale et al. [69] Retrospective Caucasian 136 EGFR+TP53 Sanger, MassARRAY, NGS tumor tissue N/A
    Chang et al. [27] Retrospective Asian 26 EGFR+ALK
    EGFR+TP53
    EGFR+PIK3CA
    EGFR+CDKN2A
    NGS, CNV tumor tissue N/A
    Chen et al. [11] Retrospective Asian 16 EGFR complex
    EGFR+ALK
    EGFR+KRAS
    EGFR+PIK3CA
    EGFR+TP53
    NGS tumor tissue + plasma N/A
    De Marchi et al. [9] Retrospective Caucasian 47 EGFR complex
    EGFR+KRAS
    EGFR+PIK3CA
    NGS, Sanger, SNP array tumor tissue N/A
    Eng et al. [86] Retrospective Caucasian 13 EGFR+PIK3CA mutation hotspot testing, FISH, multiplex sizing assays tumor tissue N/A
    Chevallier et al. [34] Retrospective Caucasian 20 EGFR+TP53
    EGFR+MET
    EGFR+KRAS
    EGFR+PIK3CA
    EGFR+PTEN
    NGS tumor tissue N/A
    Hu et al. [19] Retrospective Asian 21 EGFR+ALK
    EGFR+PIK3CA
    EGFR+KRAS
    EGFR+ROS-1
    EGFR+RET
    EGFR+HER2
    ARMS; adx-RT, mutation detection kit; fusion gene detection kit tumor tissue N/A
    Chen et al. [12] Retrospective Asian 71 EGFR complex
    EGFR+TP53
    EGFR+ALK
    EGFR+BRAF
    EGFR+MET
    NGS, ARMS tumor tissue + plasma N/A
    Lee et al. [5] Retrospective Asian 7 EGFR+ALK
    EGFR+MET
    EGFR+TP53
    EGFR complex
    FISH, NGS, Sanger tumor tissue N/A
    Zhang et al. [88] Retrospective Asian 9 EGFR complex
    EGFR+KRAS
    EGFR+PIK3CA
    FISH, liquid chip platform tumor tissue N/A
    Wang et al. [87] Retrospective Asian 17 EGFR+PIK3CA Sanger, FISH, IHC tumor tissue N/A
    Klempner et al. [55] Case report Asian 2 EGFR+RET NGS tumor tissue 53%
    54%
    62%
    18%

    Abbreviations: No, number; Pts, patients; VAF, variant allele frequency; NGS, next generation sequencing; N/A, not applicable; FISH, fluorescent in situ hybridization; IHC, immunohistochemistry; PCR, polymerase chain reaction; ARMS, amplification refractory mutation system; CNV, copy number variation; SNP, single nucleotide polymorphism; RT-PCR, real time-PCR; ddPCR, digital droplet PCR; RACE-PCR, rapid amplification cDNA ends PCR; PNA-LNA PCR, peptide nucleic acid-locked nucleic acid PCR.

    The entry is from 10.3390/jmp2020016

    References

    1. Gristina, V.; Malapelle, U.; Galvano, A.; Pisapia, P.; Pepe, F.; Rolfo, C.; Tortorici, S.; Bazan, V.; Troncone, G.; Russo, A. The significance of epidermal growth factor receptor uncommon mutations in non-small cell lung cancer: A systematic review and critical appraisal. Cancer Treat. Rev. 2020, 85, 101994.
    2. Belardinilli, F.; Gradilone, A.; Gelibter, A.; Zani, M.; Occhipinti, M.; Ferraro, S.; Nicolazzo, C.; Coppa, A.; Giannini, G. Coexistence of three EGFR mutations in an NSCLC patient: A brief report. Int. J. Biol. Markers 2018, 33, 545–548.
    3. Hsieh, M.-H.; Fang, Y.-F.; Chang, W.-C.; Kuo, H.-P.; Lin, S.-Y.; Liu, H.-P.; Liu, C.-L.; Chen, H.-C.; Ku, Y.-C.; Chen, Y.-T.; et al. Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer. Lung Cancer 2006, 53, 311–322.
    4. Zhang, B.; Wang, S.; Qian, J.; Yang, W.; Qian, F.; Lu, J.; Zhang, Y.; Qiao, R.; Han, B. Complex epidermal growth factor receptor mutations and their responses to tyrosine kinase inhibitors in previously untreated advanced lung adenocarcinomas. Cancer 2018, 124, 2399–2406.
    5. Lee, J.-K.; Shin, J.-Y.; Kim, S.; Lee, S.-H.; Park, C.; Kim, J.-Y.; Koh, Y.; Keam, B.; Min, H.S.; Kim, T.M.; et al. Primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer harboring TKI-sensitive EGFR mutations: An exploratory study. Ann. Oncol. 2013, 24, 2080–2087.
    6. Liang, H.; Li, C.; Zhao, Y.; Zhao, S.; Huang, J.; Cai, X.; Cheng, B.; Xiong, S.; Li, J.; Wang, W.; et al. Concomitant Mutations in EGFR 19Del/L858R Mutation and Their Association with Response to EGFR-TKIs in NSCLC Patients. Cancer Manag. Res. 2020, ume 12, 8653–8662.
    7. Benesova, L.; Minarik, M.; Jancarikova, D.; Belsanova, B.; Pesek, M. Multiplicity of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors. Anticancer. Res. 2010, 30, 1667–1671.
    8. Sato, S.; Nagahashi, M.; Koike, T.; Ichikawa, H.; Shimada, Y.; Watanabe, S.; Kikuchi, T.; Takada, K.; Nakanishi, R.; Oki, E.; et al. Impact of Concurrent Genomic Alterations Detected by Comprehensive Genomic Sequencing on Clinical Outcomes in East-Asian Patients with EGFR-Mutated Lung Adenocarcinoma. Sci. Rep. 2018, 8, 1–10.
    9. De Marchi, F.; Haley, L.; Fryer, H.; Ibrahim, J.; Beierl, K.; Zheng, G.; Gocke, C.D.; Eshleman, J.R.; Belchis, D.; Illei, P.; et al. Clinical Validation of Coexisting Activating Mutations Within EGFR, Mitogen-Activated Protein Kinase, and Phosphatidylinositol 3-Kinase Pathways in Lung Cancers. Arch. Pathol. Lab. Med. 2019, 143, 174–182.
    10. Li, S.; Li, L.; Zhu, Y.; Huang, C.; Qin, Y.; Liu, H.; Renheidenreich, L.; Shi, B.; Ren, H.; Chu, X.; et al. Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: A comprehensive mutation profiling from 5125 Chinese cohorts. Br. J. Cancer 2014, 110, 2812–2820.
    11. Chen, H.; Liu, M.; Dai, Z.; Li, S.; Luo, Y.; Wang, Y.; Su, W.; Cai, W.; Yang, D.; Huang, J.; et al. Concomitant genetic alterations are associated with response to EGFR targeted therapy in patients with lung adenocarcinoma. Transl. Lung Cancer Res. 2020, 9, 1225–1234.
    12. Chen, M.; Xu, Y.; Zhao, J.; Zhong, W.; Zhang, L.; Bi, Y.; Wang, M. Concurrent Driver Gene Mutations as Negative Predictive Factors in Epidermal Growth Factor Receptor-Positive Non-Small Cell Lung Cancer. EBioMedicine 2019, 42, 304–310.
    13. Gristina, V.; La Mantia, M.; Iacono, F.; Galvano, A.; Russo, A.; Bazan, V. The Emerging Therapeutic Landscape of ALK Inhibitors in Non-Small Cell Lung Cancer. Pharmaceuticals 2020, 13, 474.
    14. Russo, A.; Franchina, T.; Ricciardi, G.R.R.; Ferraro, G.; Scimone, A.; Bronte, G.; Russo, A.; Rolfo, C.; Adamo, V. Central nervous system involvement in ALK-rearranged NSCLC: Promising strategies to overcome crizotinib resistance. Expert Rev. Anticancer. Ther. 2016, 16, 615–623.
    15. Hallberg, B.; Palmer, R.H. The role of the ALK receptor in cancer biology. Ann. Oncol. 2016, 27, iii4–iii15.
    16. Khan, M.; Lin, J.; Liao, G.; Tian, Y.; Liang, Y.; Li, R.; Liu, M.; Yuan, Y. ALK Inhibitors in the Treatment of ALK Positive NSCLC. Front. Oncol. 2019, 8, 557.
    17. Gainor, J.F.; Varghese, A.M.; Ou, S.-H.I.; Kabraji, S.; Awad, M.M.; Katayama, R.; Pawlak, A.; Mino-Kenudson, M.; Yeap, B.Y.; Riely, G.J.; et al. ALK Rearrangements Are Mutually Exclusive with Mutations in EGFR or KRAS: An Analysis of 1,683 Patients with Non–Small Cell Lung Cancer. Clin. Cancer Res. 2013, 19, 4273–4281.
    18. Bronte, G.; Incorvaia, L.; Rizzo, S.; Passiglia, F.; Galvano, A.; Rizzo, F.; Rolfo, C.; Fanale, D.; Listì, A.; Natoli, C.; et al. The resistance related to targeted therapy in malignant pleural mesothelioma: Why has not the target been hit yet? Crit. Rev. Oncol. 2016, 107, 20–32.
    19. Hu, W.; Liu, Y.; Chen, J. Concurrent gene alterations with EGFR mutation and treatment efficacy of EGFR-TKIs in Chinese patients with non-small cell lung cancer. Oncotarget 2017, 8, 25046–25054.
    20. Zhuang, X.; Zhao, C.; Li, J.; Su, C.; Chen, X.; Ren, S.; Li, X.; Zhou, C. Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF. Cancer Med. 2019, 8, 2858–2866.
    21. Yang, J.-J.; Zhang, X.-C.; Su, J.; Xu, C.-R.; Zhou, Q.; Tian, H.-X.; Xie, Z.; Chen, H.-J.; Huang, Y.-S.; Jiang, B.-Y.; et al. Lung Cancers with Concomitant EGFR Mutations and ALK Rearrangements: Diverse Responses to EGFR-TKI and Crizotinib in Relation to Diverse Receptors Phosphorylation. Clin. Cancer Res. 2014, 20, 1383–1392.
    22. Fan, J.; Wu, J.; Huang, B.; Zhu, Y.; Shi, H.; Dai, X.; Nie, X. Concomitant EGFR mutation and ALK rearrangement in multifocal lung adenocarcinoma: A case report. Diagn. Pathol. 2020, 15, 42–45.
    23. Lee, T.; Lee, B.; Choi, Y.-L.; Han, J.; Ahn, M.-J.; Um, S.-W. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases. J. Pathol. Transl. Med. 2016, 50, 197–203.
    24. Miyanaga, A.; Shimizu, K.; Noro, R.; Seike, M.; Kitamura, K.; Kosaihira, S.; Minegishi, Y.; Shukuya, T.; Yoshimura, A.; Kawamoto, M.; et al. Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4–ALK-rearranged non–small–cell lung cancer harbored coexisting EGFRmutation. BMC Cancer 2013, 13, 262.
    25. Sweis, R.F.; Thomas, S.; Bank, B.; Fishkin, P.; Mooney, C.; Salgia, R. Concurrent EGFR Mutation and ALK Translocation in Non-Small Cell Lung Cancer. Cureus 2016, 8, 513.
    26. Thumallapally, N.; Yu, H.; Farhan, M.; Ibrahim, U.; Odiami, M. Concomitant Presence of EGFR and ALK Fusion Gene Mutation in Adenocarcinoma of Lung: A Case Report and Review of the Literature. J. Pharm. Pr. 2017, 31, 244–248.
    27. Chang, S.-C.; Lai, Y.-C.; Chang, C.-Y.; Huang, L.-K.; Chen, S.-J.; Tan, K.T.; Yu, P.-N.; Lai, J.-I. Concomitant Genetic Alterations are Associated with Worse Clinical Outcome in EGFR Mutant NSCLC Patients Treated with Tyrosine Kinase Inhibitors. Transl. Oncol. 2019, 12, 1425–1431.
    28. Zhu, J.; Cai, L.; Yang, H.; Wen, Y.; Wang, J.; Rong, T.; Shao, J.; Zhang, L. Echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase rearrangement and epidermal growth factor receptor mutation coexisting in Chinese patients with lung adenocarcinoma. Thorac. Cancer 2014, 5, 411–416.
    29. Devarakonda, S.; Morgensztern, D.; Govindan, R. Genomic alterations in lung adenocarcinoma. Lancet Oncol. 2015, 16, e342–e351.
    30. Zhu, Y.; Liao, X.; Wang, W.; Xu, C.; Zhuang, W.; Wei, J.; Du, K. Dual drive coexistence of EML4-ALK and TPM3-ROS1 fusion in advanced lung adenocarcinoma. Thoracic Cancer 2017, 9, 324–327.
    31. Vasan, N.; Boyer, J.L.; Herbst, R.S. A RAS Renaissance: Emerging Targeted Therapies for KRAS-Mutated Non–Small Cell Lung Cancer. Clin. Cancer Res. 2014, 20, 3921–3930.
    32. Rachiglio, A.M.; Fenizia, F.; Piccirillo, M.C.; Galetta, D.; Crinò, L.; Vincenzi, B.; Barletta, E.; Pinto, C.; Ferraù, F.; Lambiase, M.; et al. The Presence of Concomitant Mutations Affects the Activity of EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small Cell Lung Cancer (NSCLC) Patients. Cancers 2019, 11, 341.
    33. Nardo, G.; Carlet, J.; Marra, L.; Bonanno, L.; Boscolo, A.; Maso, A.D.; Bragadin, A.B.; Indraccolo, S.; Zulato, E. Detection of Low-Frequency KRAS Mutations in cfDNA From EGFR-Mutated NSCLC Patients After First-Line EGFR Tyrosine Kinase Inhibitors. Front. Oncol. 2021, 10, 3055.
    34. Chevallier, M.; Tsantoulis, P.; Addeo, A.; Friedlaender, A. Influence of Concurrent Mutations on Overall Survival in EGFR-mutated Non-small Cell Lung Cancer. Cancer Genom. Proteom. 2020, 17, 597–603.
    35. Xu, C.-W.; Zhu, Y.-C.; Ye, X.-Q.; Yin, M.-X.; Zhang, J.-X.; Du, K.-Q.; Zhang, Z.-H.; Hu, J. Lung cancer with concurrent EGFR mutation and ROS1 rearrangement: A case report and review of the literature. OncoTargets Ther. 2016, ume 9, 4301–4305.
    36. Shaw, A.T.; Solomon, B. Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer. New Engl. J. Med. 2015, 372, 683–684.
    37. Lung, J.; Hung, M.-S.; Lin, Y.-C.; Lee, K.-F.; Jiang, Y.Y.; Huang, S.-L.; Fang, Y.H.; Lu, M.-S.; Lin, C.-K.; Yang, T.-M.; et al. MET exon 14 skipping mutations and gene amplification in a Taiwanese lung cancer population. PLoS ONE 2019, 14, e0220670.
    38. Engelman, J.A.; Zejnullahu, K.; Mitsudomi, T.; Song, Y.; Hyland, C.; Park, J.O.; Lindeman, N.; Gale, C.-M.; Zhao, X.; Christensen, J.; et al. MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling. Science 2007, 316, 1039–1043.
    39. Yu, D.; Li, Y.; Sun, K.D.; Gu, J.; Chen, Z.; Owonikoko, T.K.; Ramalingam, S.S.; Sun, S.-Y. The novel MET inhibitor, HQP8361, possesses single agent activity and enhances therapeutic efficacy of AZD9291 (osimertinib) against AZD9291-resistant NSCLC cells with activated MET. Am. J. Cancer Res 2020, 10, 3316–3327.
    40. Sequist, L.V.; Han, J.-Y.; Ahn, M.-J.; Cho, B.C.; Yu, H.; Kim, S.-W.; Yang, J.C.-H.; Lee, J.S.; Su, W.-C.; Kowalski, D.; et al. Osimertinib plus savolitinib in patients with EGFR mutation-positive, MET-amplified, non-small-cell lung cancer after progression on EGFR tyrosine kinase inhibitors: Interim results from a multicentre, open-label, phase 1b study. Lancet Oncol. 2020, 21, 373–386.
    41. Wu, Y.-L.; Cheng, Y.; Zhou, J.; Lu, S.; Zhang, Y.; Zhao, J.; Kim, D.-W.; Soo, R.A.; Kim, S.-W.; Pan, H.; et al. Tepotinib plus gefitinib in patients with EGFR-mutant non-small-cell lung cancer with MET overexpression or MET amplification and acquired resistance to previous EGFR inhibitor (INSIGHT study): An open-label, phase 1b/2, multicentre, randomised trial. Lancet Respir. Med. 2020, 8, 1132–1143.
    42. Helena, A.Y.; Suzawa, K.; Jordan, E.J.; Zehir, A.; Ni, A.; Kim, H.R.; Kris, M.G.; Hellmann, M.D.; Li, B.T.; Somwar, R.; et al. Concurrent Alterations in EGFR-Mutant Lung Cancers Associated with Resistance to EGFR Kinase Inhibitors and Characterization of MTOR as a Mediator of Resistance. Clin. Cancer Res. 2018, 24, 3108–3118.
    43. Lin, L.; Asthana, S.; Chan, E.; Bandyopadhyay, S.; Martins, M.M.; Olivas, V.; Yan, J.J.; Pham, L.; Wang, M.M.; Bollag, G.; et al. Mapping the molecular determinants of BRAF oncogene dependence in human lung cancer. Proc. Natl. Acad. Sci. 2014, 111, E748–E757.
    44. Planchard, D.; Smit, E.; Groen, H.; Mazieres, J.; Besse, B.; Helland, Å.; Giannone, V.; D’Amelio, A.; Zhang, P.; Mookerjee, B.; et al. Phase 2 trial (BRF113928) of dabrafenib (D) plus trametinib (T) in patients (pts) with previously untreated BRAF V600E–mutant metastatic non-small cell lung cancer (NSCLC). Ann. Oncol. 2017, 28, v637.
    45. Minari, R.; Bordi, P.; La Monica, S.; Squadrilli, A.; Leonetti, A.; Bottarelli, L.; Azzoni, C.; Lagrasta, C.A.M.; Gnetti, L.; Campanini, N.; et al. Concurrent Acquired BRAF V600E Mutation and MET Amplification as Resistance Mechanism of First-Line Osimertinib Treatment in a Patient with EGFR-Mutated NSCLC. J. Thorac. Oncol. 2018, 13, e89–e91.
    46. Meng, P.; Koopman, B.; Kok, K.; ter Elst, A.; Schuuring, E.; van Kempen, L.C.; Timens, W.; Hiltermann, T.J.N.; Groen, H.J.; Berg, A.V.D.; et al. Combined osimertinib, dabrafenib and trametinib treatment for advanced non-small-cell lung cancer patients with an osimertinib-induced BRAF V600E mutation. Lung Cancer 2020, 146, 358–361.
    47. Kohno, T.; Ichikawa, H.; Totoki, Y.; Yasuda, K.; Hiramoto, M.; Nammo, T.; Sakamoto, H.; Tsuta, K.; Furuta, K.; Shimada, Y.; et al. KIF5B-RET fusions in lung adenocarcinoma. Nat. Med. 2012, 18, 375–377.
    48. Lipson, D.; Capelletti, M.; Yelensky, R.; Otto, G.; Parker, A.; Jarosz, M.; A Curran, J.; Balasubramanian, S.; Bloom, T.; Brennan, K.W.; et al. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat. Med. 2012, 18, 382–384.
    49. Hu, M.; Subbiah, V.; Wirth, L.; Schuler, M.; Mansfield, A.; Brose, M.; Curigliano, G.; Leboulleux, S.; Zhu, V.; Keam, B.; et al. 1913O Results from the registrational phase I/II ARROW trial of pralsetinib (BLU-667) in patients (pts) with advanced RET mutation-positive medullary thyroid cancer (RET+ MTC). Ann. Oncol. 2020, 31, S1084.
    50. Drilon, A.; Oxnard, G.R.; Tan, D.S.; Loong, H.H.; Johnson, M.; Gainor, J.; McCoach, C.E.; Gautschi, O.; Besse, B.; Cho, B.C.; et al. Efficacy of Selpercatinib in RET Fusion–Positive Non–Small-Cell Lung Cancer. New Engl. J. Med. 2020, 383, 813–824.
    51. Piotrowska, Z.; Hazar-Rethinam, M.; Rizzo, C.; Nadres, B.; Van Seventer, E.E.; Shahzade, H.A.; Lennes, I.T.; Iafrate, A.J.; Dias-Santagata, D.; Leshchiner, I.; et al. Heterogeneity and Coexistence of T790M and T790 Wild-Type Resistant Subclones Drive Mixed Response to Third-Generation Epidermal Growth Factor Receptor Inhibitors in Lung Cancer. JCO Precis. Oncol. 2018, 2, 1–15.
    52. Le, X.; Puri, S.; Negrao, M.V.; Nilsson, M.B.; Robichaux, J.P.; A Boyle, T.; Hicks, J.K.; Lovinger, K.L.; Roarty, E.B.; Rinsurongkawong, W.; et al. Landscape of EGFR-Dependent and -Independent Resistance Mechanisms to Osimertinib and Continuation Therapy Beyond Progression in EGFR-Mutant NSCLC. Clin. Cancer Res. 2018, 24, 6195–6203.
    53. ClinicalTrials.gov. Search Results 05/12/2021. Available online: (accessed on 25 April 2021).
    54. Mogi, A.; Kuwano, H. TP53 Mutations in Nonsmall Cell Lung Cancer. J. Biomed. Biotechnol. 2011, 2011, 1–9.
    55. Klempner, S.J.; Bazhenova, L.A.; Braiteh, F.S.; Nikolinakos, P.G.; Gowen, K.; Cervantes, C.M.; Chmielecki, J.; Greenbowe, J.R.; Ross, J.S.; Stephens, P.J.; et al. Emergence of RET rearrangement co-existing with activated EGFR mutation in EGFR -mutated NSCLC patients who had progressed on first- or second-generation EGFR TKI. Lung Cancer 2015, 89, 357–359.
    56. Deben, C.; Deschoolmeester, V.; Lardon, F.; Rolfo, C.; Pauwels, P. TP53 and MDM2 genetic alterations in non-small cell lung cancer: Evaluating their prognostic and predictive value. Crit. Rev. Oncol. 2016, 99, 63–73.
    57. Viktorsson, K.; De Petris, L.; Lewensohn, R. The role of p53 in treatment responses of lung cancer. Biochem. Biophys. Res. Commun. 2005, 331, 868–880.
    58. Ma, X.; Le Teuff, G.; Lacas, B.; Tsao, M.; Graziano, S.; Pignon, J.-P.; Douillard, J.-Y.; Le Chevalier, T.; Seymour, L.; Filipits, M.; et al. Prognostic and Predictive Effect of TP53 Mutations in Patients with Non–Small Cell Lung Cancer from Adjuvant Cisplatin–Based Therapy Randomized Trials: A LACE-Bio Pooled Analysis. J. Thorac. Oncol. 2016, 11, 850–861.
    59. Bronte, G.; Cicero, G.; Cusenza, S.; Galvano, A.; Musso, E.; Rizzo, S.; Sortino, G.; Roselli, M.; Bazan, V.; Fiorentino, E.; et al. Monoclonal antibodies in gastrointestinal cancers. Expert Opin. Biol. Ther. 2013, 13, 889–900.
    60. Ahrendt, S.A.; Hu, Y.; Buta, M.; McDermott, M.P.; Benoit, N.; Yang, S.C.; Wu, L.; Sidransky, D. p53 Mutations and Survival in Stage I Non-Small-Cell Lung Cancer: Results of a Prospective Study. J. Natl. Cancer Inst. 2003, 95, 961–970.
    61. Nahar, R.; Zhai, W.; Zhang, T.; Takano, A.; Khng, A.J.; Lee, Y.Y.; Liu, X.; Lim, C.H.; Koh, T.P.T.; Aung, Z.W.; et al. Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing. Nat. Commun. 2018, 9, 1–11.
    62. Galvano, A.; Peri, M.; Guarini, A.A.; Castiglia, M.; Grassadonia, A.; De Tursi, M.; Irtelli, L.; Rizzo, S.; Bertani, A.; Gristina, V.; et al. Analysis of systemic inflammatory biomarkers in neuroendocrine carcinomas of the lung: Prognostic and predictive significance of NLR, LDH, ALI, and LIPI score. Ther. Adv. Med Oncol. 2020, 12, 1758835920942378.
    63. Rho, J.K.; Choi, Y.J.; Ryoo, B.-Y.; Na, I.I.; Yang, S.H.; Kim, C.H.; Lee, J.C. p53 Enhances Gefitinib-Induced Growth Inhibition and Apoptosis by Regulation of Fas in Non–Small Cell Lung Cancer. Cancer Res. 2007, 67, 1163–1169.
    64. Molinavila, M.A.; Bertran-Alamillo, J.; Gascó, A.; Mayo-De-Las-Casas, C.; Sánchez-Ronco, M.; Pujantell-Pastor, L.; Bonanno, L.; Favaretto, A.G.; Cardona, A.F.; Vergnenègre, A.; et al. Nondisruptive p53 Mutations Are Associated with Shorter Survival in Patients with Advanced Non–Small Cell Lung Cancer. Clin. Cancer Res. 2014, 20, 4647–4659.
    65. Brunetti, O.; Derakhshani, A.; Baradaran, B.; Galvano, A.; Russo, A.; Silvestris, N. COVID-19 Infection in Cancer Patients: How Can Oncologists Deal With These Patients? Front. Oncol. 2020, 10, 734.
    66. Riley, T.; Sontag, E.; Chen, P.; Levine, A. Transcriptional control of human p53-regulated genes. Nat. Rev. Mol. Cell Biol. 2008, 9, 402–412.
    67. Huang, M.; Jin, J.; Zhang, F.; Wu, Y.; Xu, C.; Ying, L.; Su, D. Non-disruptive mutation in TP53 DNA-binding domain is a beneficial factor of esophageal squamous cell carcinoma. Ann. Transl. Med. 2020, 8, 316.
    68. Dutta, S.; Pregartner, G.; Rücker, F.G.; Heitzer, E.; Zebisch, A.; Bullinger, L.; Berghold, A.; Döhner, K.; Sill, H. Functional Classification of TP53 Mutations in Acute Myeloid Leukemia. Cancers 2020, 12, 637.
    69. Canale, M.; Petracci, E.; Delmonte, A.; Bronte, G.; Chiadini, E.; Ludovini, V.; Dubini, A.; Papi, M.; Baglivo, S.; De Luigi, N.; et al. Concomitant TP53 Mutation Confers Worse Prognosis in EGFR-Mutated Non-Small Cell Lung Cancer Patients Treated with TKIs. J. Clin. Med. 2020, 9, 1047.
    70. Offin, M.; Rizvi, H.; Tenet, M.; Ni, A.; Sanchez-Vega, F.; Li, B.T.; Drilon, A.; Kris, M.G.; Rudin, C.M.; Schultz, N.; et al. Tumor Mutation Burden and Efficacy of EGFR-Tyrosine Kinase Inhibitors in Patients with EGFR-Mutant Lung Cancers. Clin. Cancer Res. 2019, 25, 1063–1069.
    71. Hou, H.; Qin, K.; Liang, Y.; Zhang, C.; Liu, D.; Jiang, H.; Liu, K.; Zhu, J.; Lv, H.; Li, T.; et al. Concurrent TP53 mutations predict poor outcomes of EGFR-TKI treatments in Chinese patients with advanced NSCLC. Cancer Manag. Res. 2019, ume 11, 5665–5675.
    72. VanderLaan, P.A.; Rangachari, D.; Mockus, S.M.; Spotlow, V.; Reddi, H.V.; Malcolm, J.; Huberman, M.S.; Joseph, L.J.; Kobayashi, S.S.; Costa, D.B. Mutations in TP53, PIK3CA, PTEN and other genes in EGFR mutated lung cancers: Correlation with clinical outcomes. Lung Cancer 2017, 106, 17–21.
    73. Huang, D.; Zhong, D.; Zhang, C.; Zhang, Y.; Shang, Y.; Wang, L. Study of anlotinib combined with icotinib as the first-line treatment in non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations (ALTER-L004). J. Clin. Oncol. 2020, 38, 9573.
    74. Zhang, L.; Zhao, H.; Zhang, Z.; Yao, W.; Min, X.; Gu, K.; Yu, G.; Cheng, C.; Cui, J.; Miao, L.; et al. LBA50 ACTIVE: Apatinib plus gefitinib versus placebo plus gefitinib as first-line treatment for advanced epidermal growth factor receptor-mutant (EGFRm) non-small-cell lung cancer (NSCLC): A multicentered, randomized, double-blind, placebo-controlled phase III trial (CTONG1706). Ann. Oncol. 2020, 31, S1181.
    75. Zhang, Z.; Luo, F.; Zhang, Y.; Ma, Y.; Hong, S.; Yang, Y.; Fang, W.; Huang, Y.; Zhang, L.; Zhao, H. The ACTIVE study protocol: Apatinib or placebo plus gefitinib as first-line treatment for patients with EGFR-mutant advanced non-small cell lung cancer (CTONG1706). Cancer Commun. 2019, 39, 1–8.
    76. Zheng, C.; Li, X.; Ren, Y.; Yin, Z.; Zhou, B. Coexisting EGFR and TP53 Mutations in Lung Adenocarcinoma Patients Are Associated With COMP and ITGB8 Upregulation and Poor Prognosis. Front. Mol. Biosci. 2020, 7, 30.
    77. Abdulkareem, I.H.; Blair, M. Phosphatase and tensin homologue deleted on chromosome 10. Niger. Med J. 2013, 54, 79–86.
    78. Emilella, M.; Efalcone, I.; Econciatori, F.; Incani, U.E.; DEL Curatolo, A.; Einzerilli, N.; Nuzzo, C.C.; Evaccaro, V.; Evari, S.; Cognetti, F.; et al. PTEN: Multiple Functions in Human Malignant Tumors. Front. Oncol. 2015, 5, 24.
    79. Incorvaia, L.; Fanale, D.; Bono, M.; Calò, V.; Fiorino, A.; Brando, C.; Corsini, L.R.; Cutaia, S.; Cancelliere, D.; Pivetti, A.; et al. BRCA1/2 pathogenic variants in triple-negative versus luminal-like breast cancers: Genotype–phenotype correlation in a cohort of 531 patients. Ther. Adv. Med Oncol. 2020, 12, 1758835920975326.
    80. Xiao, J.; Hu, C.-P.; He, B.-X.; Chen, X.; Lu, X.-X.; Xie, M.-X.; Li, W.; He, S.-Y.; You, S.-J.; Chen, Q. PTEN expression is a prognostic marker for patients with non-small cell lung cancer: A systematic review and meta-analysis of the literature. Oncotarget 2016, 7, 57832–57840.
    81. McGranahan, N.; Favero, F.; De Bruin, E.C.; Birkbak, N.J.; Szallasi, Z.; Swanton, C. Clonal status of actionable driver events and the timing of mutational processes in cancer evolution. Sci. Transl. Med. 2015, 7, 283ra54.
    82. Lukas, J.; Parry, D.; Aagaard, L.; Mann, D.J.; Bartkova, J.; Strauss, M.; Peters, G.; Bartek, J. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nat. Cell Biol. 1995, 375, 503–506.
    83. Iwakawa, R.; Kohno, T.; Anami, Y.; Noguchi, M.; Suzuki, K.; Matsuno, Y.; Mishima, K.; Nishikawa, R.; Tashiro, F.; Yokota, J. Association of p16 Homozygous Deletions with Clinicopathologic Characteristics and EGFR/KRAS/p53 Mutations in Lung Adenocarcinoma. Clin. Cancer Res. 2008, 14, 3746–3753.
    84. Oliveira, M.; Ruiz-Pace, F.; Matito, J.; Perez-Lopez, R.; Suñol, A.; Bellet, M.; Escriva-De-Romani, S.; Zamora, E.; Gomez, P.; Garrigós, L.; et al. Determinants of concordance in clinically relevant genes (CRG) from synchronously acquired tumor biopsies (tBx) and ctDNA in metastatic breast cancer (MBC). J. Clin. Oncol. 2019, 37, 1075.
    85. Augereau, P.; Patsouris, A.; Bourbouloux, E.; Gourmelon, C.; Lacourtoisie, S.A.; Rigaud, D.B.; Soulié, P.; Frenel, J.S.; Campone, M. Hormonoresistance in advanced breast cancer: A new revolution in endocrine therapy. Ther. Adv. Med Oncol. 2017, 9, 335–346.
    86. Eng, J.; Woo, K.M.; Sima, C.S.; Plodkowski, A.; Hellmann, M.D.; Chaft, J.E.; Kris, M.; Arcila, M.E.; Ladanyi, M.; Drilon, A. Impact of Concurrent PIK3CA Mutations on Response to EGFR Tyrosine Kinase Inhibition in EGFR-Mutant Lung Cancers and on Prognosis in Oncogene-Driven Lung Adenocarcinomas. J. Thorac. Oncol. 2015, 10, 1713–1719.
    87. Wang, L.; Hu, H.; Pan, Y.; Wang, R.; Li, Y.; Shen, L.; Yu, Y.; Li, H.; Cai, D.; Sun, Y.; et al. PIK3CA Mutations Frequently Coexist with EGFR/KRAS Mutations in Non-Small Cell Lung Cancer and Suggest Poor Prognosis in EGFR/KRAS Wildtype Subgroup. PLoS ONE 2014, 9, e88291.
    88. Zhang, Q.; Sun, T.; Kang, P.; Qian, K.; Deng, B.; Zhou, J.; Wang, R.; Jiang, B.; Li, K.; Liu, F.; et al. Combined analysis of rearrangement of ALK, ROS1, somatic mutation of EGFR, KRAS, BRAF, PIK3CA, and mRNA expression of ERCC1, TYMS, RRM1, TUBB3, EGFR in patients with non-small cell lung cancer and their clinical significance. Cancer Chemother. Pharmacol. 2016, 77, 583–593.
    89. Lammers, P.E.; Lovly, C.; Horn, L. A patient with metastatic lung adenocarcinoma harboring concurrent EGFR L858R, EGFR germline T790M, and PIK3CA mutations: The challenge of interpreting results of comprehensive mutational testing in lung cancer. J. Natl. Compr. Cancer Netw. 2014, 12, 6–11.
    90. Skoulidis, F.; Heymach, J.V. Co-occurring genomic alterations in non-small-cell lung cancer biology and therapy. Nat. Rev. Cancer 2019, 19, 495–509.
    91. Jiang, J.; Gu, Y.; Liu, J.; Wu, R.; Fu, L.; Zhao, J.; Guan, Y. Coexistence of p16/CDKN2A homozygous deletions and activating EGFR mutations in lung adenocarcinoma patients signifies a poor response to EGFR-TKIs. Lung Cancer 2016, 102, 101–107.
    92. Wu, Y.-L.; Zhou, C.; Hu, C.-P.; Feng, J.; Lu, S.; Huang, Y.; Li, W.; Hou, M.; Shi, J.H.; Lee, K.Y.; et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): An open-label, randomised phase 3 trial. Lancet Oncol. 2014, 15, 213–222.
    93. Thunnissen, E.; Borczuk, A.C.; Flieder, D.B.; Witte, B.; Beasley, M.B.; Chung, J.-H.; Dacic, S.; Lantuejoul, S.; Russell, P.A.; Bakker, M.D.; et al. The Use of Immunohistochemistry Improves the Diagnosis of Small Cell Lung Cancer and Its Differential Diagnosis. An International Reproducibility Study in a Demanding Set of Cases. J. Thorac. Oncol. 2017, 12, 334–346.
    94. Musgrove, E.A.; Caldon, C.E.; Barraclough, J.; Stone, A.; Sutherland, R.L. Cyclin D as a therapeutic target in cancer. Nat. Rev. Cancer 2011, 11, 558–572.
    95. Kim, Y.; Lee, B.; Shim, J.H.; Lee, S.-H.; Park, W.-Y.; Choi, Y.-L.; Sun, J.-M.; Ahn, J.S.; Ahn, M.-J.; Park, K. Concurrent Genetic Alterations Predict the Progression to Target Therapy in EGFR-Mutated Advanced NSCLC. J. Thorac. Oncol. 2019, 14, 193–202.
    96. Peifer, M.; Fernández-Cuesta, L.; Sos, M.L.; George, J.; Seidel, D.; Kasper, L.H.; Plenker, D.; Leenders, F.; Sun, R.; Zander, T.; et al. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nat. Genet. 2012, 44, 1104–1110.
    97. Liu, J.; Mu, Z.; Liu, L.; Li, K.; Jiang, R.; Chen, P.; Zhou, Q.; Jin, M.; Ma, Y.; Xie, Y.; et al. Frequency, clinical features and differential response to therapy of concurrent ALK/EGFR alterations in Chinese lung cancer patients. Drug Des. Dev. Ther. 2019, ume 13, 1809–1817.
    98. Wu, S.-G.; Chang, Y.-L.; Yu, C.-J.; Yang, P.-C.; Shih, J.-Y. The Role of PIK3CA Mutations among Lung Adenocarcinoma Patients with Primary and Acquired Resistance to EGFR Tyrosine Kinase Inhibition. Sci. Rep. 2016, 6, 35249.
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