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 [
8,
17]. 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 [
53]. Complex
EGFR mutations occur almost in 3–7% of
EGFR-mutant patients [
54]. 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 [
17]. 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 [
49]. 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 [
30]. 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 [
54]. 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 [
18]. 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 [
34]. 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 [
44]. Li et al. detected 58/5125
EGFR double mutations, with the highest incidence rate of p.T790M and p.L858R [
29]. Chen et al. presented 4/36 patients harboring concurrent 19del and p.L858R with a worse response after TKI treatment [
43]. Additionally, Chen et al. reported concurrent
EGFR complex genomic alterations in 20 patients with the worst outcome in terms of OS [
48].
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 [
83,
84,
85]. Inactivating mutations of the
TP53 gene affect the normal transcriptional p53 activity leading to tumor susceptibility and hinder patients’ response to chemotherapy treatments [
86,
87]. Moreover,
TP53 alterations might be related to a poor prognosis in NSCLC patients [
88]. Almost 55–65% of
EGFR-positive NSCLC patients harbor a
TP53 coexisting mutation [
49,
89,
90]. Preclinical models have already demonstrated a correlation between
TP53 mutation and response to EGFR-TKIs therapy [
90,
91,
92]; 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 [
91,
93,
94,
95]. 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 [
41]. 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 [
95,
96]. 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. [
27]. 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. [
35] 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 [
48]. 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 [
94]. 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% [
39]. 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) [
40,
97]. 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 [
33]. Interestingly, Sato et al. reported 12 patients (28%) with
EGFR/TP53 alteration [
34]. 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 [
37]. Lee et al. described three cases out of 197 patients [
49]. Chevallier et al. reported 15 cases of double mutation, with no difference of survival [
46]. Chang et al. found that
TP53 was the most common concomitant alteration detected (10/31 patients) [
42], as Chen et al. reported in their study [
48].
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 [
98,
99].
PTEN is deleted in several types of cancers, such as prostate, endometrial, glioblastoma, breast, melanoma and colon [
100,
101,
102]. 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 [
103]. Various preclinical models have disclosed that
PTEN inactivation could alter the pattern of response to EGFR-TKIs [
46,
104], namely Chevallier et al. reported a retrospective cohort trial of the influence of concurrent mutations on patients with advanced NSCLC treated with TKIs [
46]. 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. [
39]. Finally, VanderLaan et al. reported 5% (1/19 patients) of
PTEN/EGFR altered patients [
35].
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 [
43].
PIK3CA mutations are detected in almost 3–7% of patients with lung adenocarcinomas and commonly they are located in exons 9 and 20 [
105]. 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 [
105,
106]. 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 [
45]. 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 [
45]. Moreover, Wu et al. examined the significance and the effect of
PIK3CA mutations on treatment outcomes to EGFR-TKIs of lung adenocarcinoma [
69]. 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 [
51]. 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 [
46]. De Marchi et al. detected 10/1208 individuals concurrent mutated in
EGFR and
PIK3CA [
44], while Rachiglio et al. identified nine patients with double mutations displaying a mPFS of 5.5 months under EGFR-TKIs treatments [
33]. Zhang et al. presented four patients harboring concurrent
EGFR/PIK3CA genomic alteration [
50], whereas Li et al. reported 64 (3.3%) of their 5125 patients [
29]. 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 [
43]. Lammers et al. reported three cases among their study population with poor response to erlotinib treatment [
20], 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 [
101], especially through the mechanisms of homozygous deletions (HDs), presented in up to 29–59% of lung adenocarcinomas regardless of the concurrent
EGFR mutation [
102]. 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 [
104]. 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%) [
42]. 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 [
107].
RB1 gene is a regulator of cell cycle and is phosphorylated by CDK4/6 to S-phase entry [
108]. The alterations in RB1 pathway have been associated to worse prognosis in NSCLC patients [
107]. In their article, Sato et al. and colleagues investigated 43 patients with
EGFR mutations revealing 16% (7/43) of
RB1 co-alterations [
34]. 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 [
109]. 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 [
110,
111]. Additionally, Yu et al. and Kim et al. reported
RB1 as one of the most common gene co-altered in NSCLC patients [
90]. Particularly, Kim et al. and colleagues identified co-alteration in
RB1 as predictor of fast progression to TKI treatment [
112].