Adenocarcinomas harbor mutations of genes of EGFR signaling pathway. EGFR belongs to the epidermal growth factor RTK (receptor tyrosine kinase) family. The EGF receptor has an extracellular ligand-binding, a transmembrane and an intracellular domain, the latter having tyrosine kinase activity. The intracellular protein kinase domain contains a small N-terminal lobe and a larger C-terminal lobe. The two parts form the active site cleft that serves as a binding site for ATP. In physiological conditions the extracellular ligand binding (EGF) activates the receptor and the downstream signaling results in cellular transactivation, cell proliferation and survival.

The use of the EGFR receptor as a potential therapeutic target was already suggested in the 1980s by Mendelshon and colleagues [16, 17]. As in many other tumors, EGFR expression has been shown to be increased in lung adenocarcinomas, an early event in carcinogenesis [18]. In 2002, the first data on an EGFR inhibitor treatment in NSCLC were published [19, 20]. The first clinical results showed considerable variability in the response rate [21, 22]. Benefits were mostly observed in non-smokers, women and in the Far Eastern population. The level of EGFR expression detected by immunohistochemistry has not been shown to be of predictive value [23, 24]. Mutations in EGFR were first identified in 2004 and have also been associated with response to therapy [25–27]. All the detected genetic abnormalities were heterozygous, and wild-type EGFR was found in normal lung tissue adjacent to the tumor, suggesting that these mutations are somatic [25]. The EGFR mutation could also be detected in the benign epithelium surrounding the tumor, demonstrating that it is an early event in carcinogenesis [28]. Mutations were demonstrated to affect the kinase domain of the receptor protein causing constitutive activation and downstream signaling in the absence of the receptor ligand. Women and never-smokers were preferentially involved. EGFR mutations show ethnic differences, prevalence ranges 10%–15% in Caucasians and 30%–40% in Asians [29]. Genetic alterations of other major oncogenes, such as KRAS, ALK, ROS1 are mutually exclusive with EGFR mutations. Among adenocarcinomas EGFR mutations are frequently detected in cases with lepidic and papillary growth patterns. The two most common mutations, the so called classical EGFR mutations are i) mutation at codon 858, replacing leucine 858 with arginine (L858R) and ii) the in-frame deletion in exon 19 causing removal of amino-acid residues 746–750 of the expressed protein [30]. These two mutations account for 85%–90% of all EGFR mutations.

Both classical EGFR mutations affect the kinase domain. In the inactive form of the wild-type EGFR molecule an outward rotation of the alphaC helix in the N lobe is provided, which is stabilized by the helical turn of the A-loop. This conformation inhibits the association of amino acids K745 with E762 and the consecutive binding and orientation of ATP. The L858R mutation occurs in the C lobe N-terminal portion of the activation loop resulting the destabilization of the inactive state thereby promoting the conversion in a more active state [31, 32]. The deletion in exon 19 (746ELREA750) occurs immediately before the αC-helix in the N lobe and desrupts the inactive conformation through the shortening of the alpha C loop. The L858R mutant is approximately 50-fold more active than the wild-type enzyme, and the G719S mutant shows about ten times more activity over the wild-type.

In addition to the classic mutations mentioned above (L858R and del19), about 600 other rare EGFR mutations have been described, accounting for about 10%–15% of all EGFR mutations [33]. These include exon 18 E709x, del18, G719x, exon19 insertion, exon20 insertion, and S768I, as well as L861Q affecting exon21. Of these, exon 20 insertions are the most common (4%–10% of all EGFR mutations).

The relative frequency of EGFR mutations based on the COSMIC database is shown in Figure 2 [34].

EGFR mutations show variable sensitivity to EGFR tyrosine kinase inhibition. The specific genotype obtained by DNA sequencing gains special importance as the type of mutation carries the basic potential of resistance to given inhibitor agents. It is of note that resistance or low response to TKI today indicates the consideration of an alternative inhibitor. No response to EGFR TKI treatment has been observed in case of exon 20 insertions [35] except A763-Y764insFQEA [36–38]. For others, the therapeutic effect is less than observed for the classical mutations, e.g., G719x, exon 19 insertions, S768I, L861Q. For these mutations 2nd generation TKI treatment may show improved results.

Germline mutations of the EGFR gene were also described [39], which, if present, increase the incidence of adenocarcinoma, mostly in the presence of additional somatic mutations. The 4 most common germline mutations are T790M (which is one of the most important causes of acquired TKI resistance), V843I, R776H, P848L, all point mutations.

An EGFR activating mutation is a prognostic factor indicating unfavorable outcome. On the other hand, it is a factor predicting the response to tyrosin kinase inhibitor treatment. T790M mutations and mutations in exon 20 are associated with resistance to tyrosin kinase inhibitors.

Although EGFR-TKI treatment has been shown to be effective in most patients with EGFR mutant lung adenocarcinomas, no response is observed in 5%–25% of patients due to intrinsic resistance to these drugs. In addition to the pharmacokinetic complications of the drug, intrinsic resistance is frequently due to some genetic variation. These include most of the exon20 insertions, with the exception of A763-Y764insFQEA- [36–38], T790M, exon 2-7 variant III(vIII) in frame deletion [40] and some other secondary genetic events.

Patients with an initially good therapeutic response may show unfortunate disease progression after 9–19 months of treatment initialization due to acquired resistance (first line TKI: 9–12 months, 19 months first line third generation TKI) [41, 42]. Acquired resistance is often the result of a secondary EGFR mutation, while in many cases it occurs due to activation of an alternative signaling pathway. The most common (49%–63%) acquired mutation is T790M, which results in a threonine-methionine substitution at position 790, the ATP binding site of the receptor protein. This amino acid exchange prevents EGFR-TKI from binding to the kinase domain and results in increased ATP affinity. Further to de novo mutagenesis it is assumed, that a co-existing, drug-resistant EGFR T790M positive subclone has been selected by TKI treatment. In addition to the T790M mutation, EGFR gene amplification and several rare second/third EGFR mutations are considered as resistance mechanisms, including the C797S, L792, L718Q, SV768IL genotypes. The activation of alternative signaling pathways may also occur through a gene amplification, such as MET, HER2 or other rare gene mutations. Resistance may also result from tumor phenotypic alterations, such as transformation towards squamous or small cell carcinoma and epithelial-mesenchymal transition [43, 44]. The most common causes of acquired EGFR TKI resistance and their relative frequency are shown in Figure 3.

It is surprising that, despite the wealth of data that has been accumulated on the incidence and therapeutic significance of EGFR mutations, very little is known about the origin and causes of EGFR mutations. Few publications correlate different etiological factors and EGFR mutations. It appears that air pollution, including exposure to microparticles, can induce EGFR mutations [45]. In addition, exposure to radon in the residential environment may also have a potential EGFR mutation-inducing effect [46].

EGFR genotyping is generally based on sequence analysis of tumor DNA isolated from biopsy samples. As a fast alternative approach to predict the efficacy of EGFR inhibitory treatment, the expression of total EGFR protein was first attempted for immunohistochemistry, but as already mentioned, this remained inconclusive [23, 47] Mutation-specific monoclonal antibodies against exon 19 E746-A750del and exon 21 L858R mutations are available, which represent approx. 90% of all EGFR mutations (clone 6B6 for exon19 deletions; and clone 43B2 against point mutation of exon21 L858R, Cell Signaling Technology) [48]. Tests with these antibodies have shown relatively high sensitivity and specificity for the two mutations indicated, especially for L858R. The lower sensitivity for exon19 deletions is mostly explained by the fact that the exon19 antibody only detects the most common deletion of exon19 (deletion E746-A750). This deletion is of 15 base pairs and represents 50%–65% of all exon19 deletions. However, a variety of deletions, including 9, 12, 16, 18, and 24base-pairs variants have been identified, each producing slightly different protein and antigen epitope structure not detectable by the commercial exon19 antibody [49]. According to the literature the EGFR mutation-specific antibodies have a fair sensitivity and high specificity in identifying lung adenocarcinomas with classic EGFR mutations, while they do not recognise uncommon EGFR mutations. They did not provide sufficient sensitivity (about 40%–60%) or specificity (70%) for the detection of all EGFR mutations, compared to gold standard sequencing methods [50–53]. Sanger DNA sequencing has been widely used, but its disadvantages, primarily its low sensitivity (requirement of 40%–50% mutant DNA in samples) [54], has before led to the development of more sensitive detecting methods including real-time quantitative PCR (RT-PCR). However, this method is relatively expensive, time consuming, and not incorporated in routine diagnostic procedures in many departments of pathology. In contrast, immunohistochemistry has lower costs, shorter turnaround time, and is available in the majority of laboratories. IHC is a rapid, cheap and well-known assay that does not require huge tumor cell content and performs quite well even in degraded tissue (e.g., decalcified bone tissue) or cytology samples. EGFR-mutant-specific antibodies cannot replace conventional molecular methodologies, but they could be very helpful in small tumor samples with poor material [53].

KRAS is one of the longest known oncogenes and activating mutations play a crucial role in the early oncogenesis of several types of tumors, such as pancreatic ductal adenocarcinoma, colon cancer and lung cancer [55]. HRAS and NRAS are the other two members of the RAS gene family with clinical impact [56]. The human homologue of the RAS gene, HRAS, located on the short arm of chromosome 11 at position 11p15.1-11p15.3, was first described in the early 1980s in a human bladder cancer cell line. A short time later KRAS, a gene showing homologue features was detected in lung adenocarcinoma, located on the short arm of chromosome 12 at position 12p11.1-12p12.1. The NRAS gene is located on chromosome 1 [57]. All three RAS genes have 4 exons and broadly similar structures [58]. The KRAS gene encodes two protein isoforms composed of 188 and 189 amino acids (KRAS-4B and 4A), the single amino acid difference is a result of alternative splicing [59]. The KRAS protein is a cell membrane-associated G protein with GTP-ase activity [60,61], coupling cell surface growth factor receptors such as EGFR to various intracellular (mitogenic) signaling pathways. The most common signaling pathways involved are the mitogen activated protein kinase (MAPK) [62, 63] and phosphatidylinositol 3-kinase (PI3K) pathways [64]. The active RAS protein binds GTP, in which guanine nucleotide exchange factors (GEFs) participate through GTP-GDP exchange. GTPase activating proteins (GAPs) inactivate RAS by enhancing the GTPase activity of the RAS [60, 61, 65]. RAS gene mutations at specific sites result in spontaneous activation of the RAS protein without mitogen signaling, leading to uncontrolled cell proliferation and cell survival. As these play an important role from the earliest phase of carcinogenesis they are called oncogenic, or hot-spot mutations [55, 66] In the case of KRAS G12C or G12V mutations, intracellular levels of RAS-related proteins (RALs) are elevated and AKT phosphorylation is reduced [67].

The earliest and most frequent mutations in lung adenocarcinoma occurs in the KRAS gene and affects the EGFR/RAS/RAF signaling pathway [68–70]. The prevalence of the KRAS mutation is around 30% in the Western population, compared to around 10% in the Far East. In lung cancer, mutations in NRAS or HRAS are rare, with a prevalence of less than 1% each. The vast majority of KRAS mutations (about 80%) affect codon 12 within exon 2 of the gene and is close to the section encoding the nucleotide binding site of the KRAS protein. The most common change of codon 12 mutations is a G>T transversion, which results in a glycine-cysteine (G12C) or glycine-valine (G12V) substitution at the protein level. Another type of codon 12 mutation is G>A transition, replacing amino acid glycine by aspartic acid (G12D). Less frequent codon 12 mutations are also known: G12A, G12S, G12R, G12F. Occasionally, mutations can be detected in codon 13 (G13C) or codon 61 (Q61H) [71].

Mutations generally inhibit the interaction of KRAS with GAPs and the hydrolysis of GTP bound by KRAS, thereby keeping the KRAS protein in an active conformation [72]. Different mutations activate different intracellular signaling pathways to different degrees. In KRAS G12C mutant tumors, the classical mitogenic signaling pathway (RAS-RAF-MEK-ERK) is activated, whereas the PI3K-AKT-mTOR pathway is dominant for the other mutant genotypes. This may be due to a different RAF affinity in each mutation type [73] The G>T transversions resulting in G12C exchange are supposedly associated with polycyclic aromatic hydrocarbons (PAH) exposure in tobacco smoke. On the other hand, G>A mutations resulting in KRAS G12D are more common in non-smokers. The prevalence of KRAS mutations is 40% in heavy smokers, 30% in former current non-smokers and 20% in never-smokers [74]. The most common type is G12C, which accounts for 40% of all KRAS mutations, while G12V and G12D account for 19% and 11%, respectively. The average relative frequency of KRAS mutation types, based on large databases, is shown in Figure 4.

There are also significant geographical and social differences in the prevalence of KRAS G12C, with a higher prevalence in Western countries and a lower prevalence in the Far East (8.9%–19.5% and 1.4%–3%, respectively), which may be related to smoking habits. Our own observation also suggests this, as KRAS G12C is more common in Eastern Hungary, the care area of our centre, than in the better developed western part of the country, which may be explained by the smoking habits of the population here (unpublished data). Although KRAS mutations, especially G12C, are strongly associated with smoking, KRAS mutations are surprisingly unfrequent in small cell lung cancer, which occurs almost exclusively in heavy smokers [74, 75]. In squamous cell carcinomas, which are also strongly associated with smoking, the occurrence of KRAS mutations is rare or only occurs in mixed tumors, such as adenosquamous carcinoma [71]. The frequency of KRAS mutations also varies in the histological subtypes of adenocarcinoma. KRAS mutations are most often detected with mucinous morphology, but different genotypes show variable frequencies (G12C rarely, whereas G12V and G12D more often) [76]. Among mucinous tumors, KRAS mutations are almost exclusively found in invasive mucinous adenocarcinomas (69%), with no KRAS mutations detected in adenocarcinoma in situ and colloid carcinoma. However, mucinous carcinomas do not harbour EGFR mutations [77]. Interestingly, the KRAS mutation is more common in women [78]. The metastatic pattern of the tumor also differs with the mutant genotype, with G12C tumors having a higher incidence of intrapulmonary metastasis (38% vs. 21%) and a lower incidence of pleural metastasis (4% vs. 39%). It was also observed that brain metastasis is less frequent in KRAS mutations (33% vs. 40%), but the frequency of brain metastasis is similar for each KRAS mutation type [79, 80].

The presence of EGFR and KRAS mutations are mutually exclusive. In theory they affect the same signaling pathway and the presence of one mutation is sufficient for tumourigenesis. Despite this generally accepted view, KRAS and EGFR mutations can rarely occur within the same tumor [76, 81]. A recent large retrospective study of data from 3,774 patients found the concomitant presence of two or three driver mutations in 1.7% of cases, most commonly EGFR/KRAS mutations (in 0.53% of cases). It should be noted that this study examined a patient population from the Far East, where the prevalence of EGFR mutations is inherently high, 43% in this study [82].

The particular role of KRAS in tumorigenesis is also suggested by the observation that in one of the most common congenital pulmonary malformations, congenital pulmonary airway malformation type I (CPAM), KRAS mutations (G12V or G12D) are often detected, predominantly in intracystic mucinous cell clusters. In fact, these mutations can be identified in nonmucinous cystic regions as well, but not in healthy lung tissue. In these patients, KRAS mutant adenocarcinoma of mucinous character frequently appears later, indicating to the oncogenic nature of the mutation [83–85].

The prognostic role of KRAS mutations in lung adenocarcinoma has long been the subject of intensive studies. Although many data have been published on this issue, its prognostic significance is still not clearly understood. Investigations of prognostic significance are complicated by the fact that the term “KRAS mutation” itself is not uniform, mutation types, ethnic, gender and histological differences as well as treatment mode should also be considered. In addition, other genetic abnormalities, such as EGFR mutations, modify the behavior of KRAS wild-type cases and affect prognosis [86]. However, most of the large case-control studies published in recent years support the hypothesis that the presence of KRAS mutations, particularly KRAS G12C mutations, has a negative prognostic significance, and reduce both OS and PFS [87–90]. There is also evidence that increased mutated KRAS levels in circulating tumour DNA also have a negative prognostic significance [91].

The predictive significance of the KRAS mutation has also been extensively studied. Many reports suggest that mutant KRAS is not a negative predictor of conventional chemotherapy [92] However, as with prognostic significance, the question is more nuanced, with many factors to consider, such as the type of KRAS mutation, the patient population studied and treatment characteristics. In early-stage resected NSCLC patients, no significant predictive value of KRAS mutation status was found for adjuvant treatment [93], and similar results were obtained in the neoadjuvant setting [94]. A more recent study showed that KRAS mutation is a negative predictor of cytotoxic chemotherapy in advanced NSCLC [95].

Another important question is the impact of KRAS mutations on targeted therapies, especially EGFR targeting. Many conflicting observations have been reported on this topic, too. Most studies have shown that the presence of a KRAS mutation has a negative predictive effect in this respect, EGFR TKI treatment in these patients having a worse objective response rate. However, no difference in survival has been found between KRAS mutant/EGFR wild-type and KRAS wild-type/EGFR wild-type patients, therefore, the significance of KRAS mutation to select patients for EGFR TKI treatment appeared to be limited [96]. The results of individual studies are significantly affected by the type of KRAS mutation present: while poor treatment efficacy is seen with G12C and G12V, better response rates are seen with G12D and G12S [97].

Testing for KRAS mutations is possible from tumor DNA isolated from tumor tissue, bronchial brush smears, plasma or pleural fluid (cfDNA analysis), using either a single-gene PCR-based approaches, classical or a next-generation sequencing [98, 99].

BRAF is a member of the rapidly accelerated fibrosarcoma (RAF) kinase family. Its role is signal transduction from the RAS protein to the mitogen-activated protein kinase cascade (MAPK) [100]. The RAF protein is composed of three main domains: CR1, CR2, CR3. CR1 functions as an auto-inhibitor of the kinase CR3 domain and is also responsible for RAS-GTP binding. The CR2 region forms a flexible link between the CR1 and CR3 domains. Upon activation the RAS-GTP binds to the RAS-GTP binding site (RBD) of the CR1 domain. BRAF is then phosphorylated on amino acids T599 and S602, which results in a protein conformational change. This active form homo- or heterodimerizes with other RAF family proteins, also contributing to the stabilization of the active conformation. The results is the activated BRAF kinase domain, which phosphorylates MEK1, the downstream member of the MAPK signaling pathway [101].

BRAF is one of the most frequently mutated genes in human tumors. Mutations are most frequently detected in melanoma (40%–50% of cases), but are also common in papillary thyroid cancer, colorectal cancer and NSCLC [102–105].

(It is interesting to note that Davies [102] was the first to report the BRAF mutation, but in her publication she uses a different nomenclature, V599E, to refer to the mutation he detected - now called V600E-because the sequence of the protein had previously been misinterpreted, A31 G32 A33 was mistaken for R31 P32. Because A33 was missing from earlier sequences, some studies incorrectly assigned wrong numbers to coding mutations and amino acids.)

BRAF mutations most commonly occur at codon 600 in exon 15 of the gene, resulting in the exchange of amino acid valine to glutamate (V600E) of the protein. Other substitutions, such as V600D/K/R, can be also rarely seen at this site. These mutations are also known as class I mutations [106]. The V600E mutation results in a marked increase in BRAF kinase activity (up to 500-700-fold compared to wild-type BRAF), with a consequent stimulation of the MAPK signaling pathway. The V600E mutation results in the conformational change of the monomer BRAF protein already possessing with an active kinase function that would otherwise gain through dimerization of the wild-type conformation [107]. In melanoma, V600E is the most common BRAF mutation, whereas in NSCLC only half of the mutations affecting BRAF belong to this group. NonV600E mutations form a heterogeneous group and can be further classified into class II and class III [101].

BRAF mutations play an important role in lung carcinogenesis, as also demonstrated experimentally in vivo [108]. Data from large case-control studies suggest that BRAF mutations occur in 2.2%–4.9% of NSCLC. Owsley found 772 BRAF mutations (4.1%) out of 18,944 NSCLC cases, of which 30.7% (237 cases) were V600E mutations [103]. In Villaruz’s study, 21 cases (2.2%) of 951 adenocarcinomas proved to be BRAF mutant, 81% of which were V600E [109]. V600E mutations occur mainly in women, non-smokers, are associated with micropapillary morphology and have a worse prognosis than wild type [110]. Non-V600E mutations are exclusively detected in smokers, in equal proportions in men and women, and are not associated with a prognosis worse than the wild type (Marchetti, 36 BRAF mutations detected in 739 adenocarcinomas- 4.9%- of which 56.8% were V600E) [111]. The results of individual studies are contradictory regarding the prognostic role. Villaruz et al. observed a better outcome for V600E, while others did not observe any difference in prognosis [109, 112] The contradictory results are likely explained by the fact that BRAF mutations are relatively rare and only small cohorts of patients could be examined despite extended studies. Co-mutations are relatively common, mainly mutations in KRAS and PI3K, while in nonV600E, mutations in p53 and STK11 are common [106].

The conformational change through the mutation enables the differentiation of the BRAF protein from the wild-type form. Thus, the BRAF status can also be assessed by immunohistochemistry for V600E mutations. The commercially available monoclonal diagnostic antibody was raised against a synthetic version of the V600E-encoded protein fragment located around the amino acid affected by the mutation [113]. This antibody detects the BRAF V600E mutant epitope with sufficient sensitivity and specificity, as has been demonstrated in several tumor types such as colorectal carcinoma, papillary thyroid cancer and melanoma [114–118] However, it is not applicable to V600D/K/R or class II and III non-V600E mutations. Since almost half of the BRAF mutations in NSCLC are nonV600E, the IHC test is of limited use to identify tumors harbouring BRAF mutations. However, according to current NCCN recommendations, specific TKI inhibitor treatment should be used for V600E mutation positive tumors [119]. Thus, the IHC methodology may be considered as a screening test for the identification of these patients in histological conditions. In addition, the same guidelines (and updated version also) recommend an NGS-based methodology to determine a comprehensive BRAF status [120].

The ALK gene (anaplastic lymphoma kinase) was discovered as a result of genetic studies in anaplastic large cell lymphoma. The gene is located on the short arm of chromosome 2, in the 2p23 region. As a member of the insulin receptor superfamily, ALK encodes a tyrosine kinase-activated transmembrane receptor protein whose function is only partially understood. In humans, ALK expression is detected intermittently during neural development, with a decline in expression during postnatal life. In adults, it is expressed only scattered in some neurons, endothelium and in pericytes of the brain. The ALK protein contains an extracellular ligand-binding, a transmembrane, and a cytoplasmic kinase domain [122]. The ligand(s) for the ALK receptor have not been unequivocally identified. The role of pleiotrophin, midkine was hypothesized [123], followed by other candidates, including heparin, FAM150A and FAM150B [124].

The main types of ALK gene alterations include rearrangements (fusions), gene amplification, and point mutations [125].

Fusions of the ALK gene, like the amplification of the gene leads to constitutive activation. Amplification of ALK has been detected in neuroblastoma [126] breast cancer, anaplastic large cell lymphoma and pulmonary sarcomatoid carcinoma [127]. On the contrary, point mutations are the most common causes of resistance to ALK TKI treatment. Known resistance mutations are G1269A, C1156Y, L1196M and several other point mutations [128, 129].

The 2p chromosomal region is sensitive to genotoxic effects, favoring the breakage of the ALK gene, with the result of gene fusions and increased expression of the kinase domain of the ALK protein. In 1994, a t [2, 5] translocation was first described in anaplastic large cell lymphoma, resulting in an NPM (nucleophosmin)-ALK fusion gene, an event that is detected in 60%–80% of ALCL [130]. It has subsequently been described in additional tumors: e.g., inflammatory myofibroblastic tumor [131], colorectal and breast cancer, and esophageal squamous cell carcinoma [132]. Further fusion partners were later identified. The setup of the rearrangements is common in that the breakpoint leaves the entire ALK tyrosine kinase domain intact, while the promoter region always comes from the fusion partner. The fusion partner also contains an oligomerization domain, the presence of which allows ligand-independent constitutive activation of the receptor protein.

In 2007, Soda and colleagues detected the EML4 (echinoderm microtubule associated protein-like 4)-ALK gene rearrangement in NSCLC. This gene rearrangement is caused by an inversion of the chromosome region 2p21-23. The extracellular and transmembrane regions of ALK are replaced by EML4. There are various EML4 breakpoints and therefore, several variants of the fusion gene are known [133]. The EML4-ALK gene rearrangement results in constitutive activation of ALK RTK, an oncogenic pathway in NSCLC. The resulting EML4-ALK fusion gene product represents a novel molecular target for the treatment of non-small cell lung cancer. ALK gene rearrangement occurs in 3%–6% of all NSCLC. It is typically associated with adenocarcinoma morphology (including papillary, mucinous, and squamous cell variants) [134]. Detected mainly in non-smokers or light smokers and typically in young patients. ALK gene rearrangement, EGFR and KRAS mutation are mutually exclusive events [135].

The demonstration of EML4-ALK gene rearrangements was challenging due different variants of the fusion, requiring multiplex testing in the PCR era. FISH-based detection could be adopted with satisfying efficacy since the 3′and 5′ends of the ALK gene get separated due to the rearrangement, and their labelling with separate fluorescent probes result in the characteristic split signal. Currently, the FISH test is the gold standard for the detection of ALK rearrangements in clinical samples, requiring specific probes, fluorescence equipment and properly experienced pathologist.

Because of the above drawbacks, an immunohistochemical alternative for the detection of ALK gene involvement has been attempted. This assumes that ALK protein is not expressed in normal lung tissue, but gene fusion and ALK gene activation result in moderately increased expression of ALK protein. However, the detection of the protein underwent an evolution. A “traditional” diagnostic antibody (ALK1) previously used for anaplastic lymphoma was not sufficiently sensitive (sensitivity 67%, specificity 97%). However, the release of new antibody clones promptly followed [5A4, D5F3, anti-ALK (1A4)] and the use of highly sensitive amplification systems allowed to achieve adequate sensitivity and specificity. The advantage of IHC testing is its low cost, wide availability and rapid turnaround time. In immunohistochemistry, the ALK fusion protein shows granular cytoplasmic staining. In signet ring cells (a morphology often seen in ALK-positive adenocarcinoma), staining is present along the membrane in a thin rim that can be difficult to distinguish from background staining. Several studies have demonstrated that the use of properly validated antibody and immunohistochemical platform, together with an external control (e.g., appendix with intense ALK positivity in the ganglion cells of the wall), provides a highly reproducible result. The study of Mino-Kenudson et al. in 2010 (n = 153, sensitivity 100%, specificity 99.0%) using clone D5F3 [136] and that of Paik et al. in 2011 (n = 735, sensitivity 100%, specificity 96.2%) using clone 5A4 both showed high concordance between Ventana IHC and FISH results [137].

The high sensitivity of IHC to detect ALK aberrations is today generally accepted. Despite the rare IHC negative but FISH positive cases published in the literature, the current recommendations accept the use of IHC methodology without FISH confirmatory testing in histological specimens [138] as well as in cell block specimens prepared from malignant pleural effusions [139]. However, the IHC methodology is neither perfectly applicable nor validated in large series on bronchoscopic brush cytology specimens. For cytology preparations the FISH break apart test should primarily be chosen.

The ROS1 gene is located on chromosome 6 in the 6q22 region. The gene was originally discovered in the 1980s during the study of avian sarcoma viruses. The human ROS gene is homologous to the v-ros proto-oncogene of the UR2 sarcoma virus [140, 141]. The protein contains an extracellular and an intracellular domain, the latter having tyrosine kinase activity with structural similarity to the ALK protein. The physiological role of the ROS1 gene and protein is poorly understood, but it is thought to be involved in differentiation signaling pathways of various epithelial tissues during embryonic development [142]. ROS1 protein expression is observed in the kidney, the cerebellum, the peripheral nerves, various parts of the alimentary canal, but is not expressed in lung tissue under normal conditions [143]. The physiological ligand of the receptor is still debated. The structure of the extracellular domain suggests that cell adhesion plays a role in its activation [142] The intracellular signaling pathways activated by ROS1 are also not well understood, but induction of MAPK and PI3K pathways is hypothesized [144]. Its oncogenic relevance was first demonstrated in the early 2000s in glioblastoma [145]. In lung tumors, rearrangements affecting ROS1 gene were first described in 2007 [146]. ROS1 involvement was observed in 1%–2% of NSCLC [147]. As a result of the rearrangement, various breakpoints in the ROS1 gene may evolve, including exons 32, 34-36, or introns 31 or 33. The fusion gene commonly contains the tyrosine kinase domain of ROS. There are 9 different fusion partners known in lung cancer, such as FIG, CD74, SLC34A2 and SDC4, EZR and the list is growing [148, 149]. The oncogenic mechanism of gene rearrangement is not understood. ROS1 rearrangements typically occur in lung adenocarcinomas, rarely in adenosquamous carcinoma. These adenocarcinomas generally show a solid pattern, frequently of signet ring cell type. Younger patients and non-smokers are more frequently affected [150]. It is often detected at an advanced stage and brain metastasis is common [151]. Interestingly, lung carcinoma patients with ROS1 gene rearrangements have a higher incidence of paraneoplastic thromboembolic events [152].

Due to the larger set of fusion variants the ROS1 rearrangement can be most effectively demonstrated by FISH analysis, using ROS1 specific DNA-probes and the detection strategy of the ALK testing. Sequencing by different NGS platforms is also applicable if tumor tissue and appropriate amounts of DNA or RNA are provided. Rearrangement of the gene is associated with overexpression of the ROS-protein, allowing the use of ROS1-specific antibodies. For IHC-based diagnostic testing, the use of the D4D6 ROS1 antibody clones is recommended. In positive cases, fine granular cytoplasmic staining is observed. Fusion variants show a different staining pattern, which may be the result of the intracellular function and localization of the fusion partner. For CD74-ROS1, a globular pattern with randomly arranged cytoplasmic granules of 6–8 mm diameter and weaker background cytoplasmic staining was described, explained by the fact that CD74 is associated with intracellular membrane systems. Membranous staining was seen in the presence of EZR-ROS1 fusion, presumably due to the ezrin protein binding to plasma membrane and actin cytoskeleton [153] A uniform scoring system for the ROS1 IHC reaction is still missing, most studies use the H-score calculated from the staining intensity and the proportion of positive tumor cells. With appropriate preanalytical and analytical standards, this antibody can achieve high sensitivity (95%–100%) but relatively poor specificity (63%–90%) [154–157]. The low specificity may potentially originate from a moderate ROS1 expression by macrophages, reactive alveolar epithelium, or even by tumors without ROS rearrangement. A higher cut-off value results in a higher specificity. Overall, the IHC test has a high negative predictive value and is therefore suitable as a screening test, with a negative IHC result virtually ruling out the presence of ROS1 fusion. A positive IHC result requires confirmation by genetic means, such as FISH or NGS technology [158, 159]. Since ROS1 rearrangements are rare, the relatively simple IHC staining is highly effective and avoids the mass need for expensive molecular testing [138, 160].

The RET oncogene was identified in the 1980s by transfection of DNA extracted from a human T-cell lymphoma cell line [161]. The RET gene is located on chromosome 10 at position 10q11.22 [162] and encodes a transmembrane tyrosine kinase growth factor receptor. Its extracellular domain contains 4 cadherin-like structures [163]. Its ligand is glial cell line-derived neurotrophic factor [164, 165]. Ligand binding results in dimerization and activation of the receptor, which then activates several intracellular signaling pathways such as PI3K/AKT, RAS/RAF/MEK/ERK or JAK2/STAT3. RET activity is important for kidney and nervous system development, gene expression is precisely regulated in space and time during embryogenesis [165]. RET is required for the proper development of the enteric nervous system, in particular for the migration of neural crest cells and enteric neurons into the wall of the alimentary canal. The absence of RET expression or activity is associated with the development of Hirschsprung disease (segmental aganglionosis of the colon) [166].

Various genetic defects within the RET gene are also related with carcinogenesis. CCDC6-RET fusions have been detected in thyroid cancer as early as in 1990 [167]. Further to oncogenic fusions, activating point mutations of the RET gene are also known. They are involved in the development of medullary thyroid carcinoma and MEN2A syndrome, among others [168, 169]. In NSCLC, RET gene rearrangement was first reported in a Korean non-smoking male patient in 2012 [170–173]. Several large studies have reported that RET rearrangement is present in 1%–2% of NSCLC (Takeuchi 0.9% in 1482 NSCLC, Qiu 1.4% in 1587 NSCLC) [149, 173]. Following the summary of data from 4857 NSCLC patients from previous studies, the prevalence of RET rearrangement proved to be 1.4%, while in the adenocarcinoma group of 3,576 patients 1.8% had RET fusions [174]. KIF5B-RET fusion was observed with the highest prevalence (52%), this type was typical for women, while CCDC6-RET was mostly observed in men with a prevalence of 26% in total. In addition, several rare fusion partners have been described (MIR392, ZBTB41, ITGA8, SLC39A8 [149]. Along with the RET rearrangements, several other genetic events may be detected; [175]. It is known that RET fusions are responsible for acquired therapy resistance during EGFR TKI inhibitor treatment in 1%–2% of the cases, mainly involving CCDC6 as the fusion partner; [176]. In most RET rearrangements, the transmembrane domain is lost, resulting in a chimeric cytosolic protein that exerts its oncogenic effect through constitutive activation of the kinase domain [161] The majority of RET rearrangements, like ALK and ROS1 fusions, occur in young, non-smoker or mild smoker women with lung adenocarcinoma diagnosis; [173, 177]. This patient group has a significantly higher incidence of brain metastases both at diagnosis (27%) and overall during the course of the disease than the RET wild-type group [178].

The demonstration of RET gene fusions in clinical samples is of great importance, as there are several FDA-approved small-molecule inhibitors promising effective treatment. Initially, non-specific multikinase inhibitors were used, more recently followed by RET-selective inhibitors [179–182].

RET rearrangements can be demonstrated by several alternative methodologies. Although immunohistochemistry is a widely available method and has proven useful in detecting ALK and ROS1 rearrangements, its value in detecting RET fusions is unfortunately limited. The IHC methodology using RET specific antibodies had low sensitivity and specificity, with a false positive rate of 62% and a false negative rate of 46%, in other words, RET rearranged samples could not be equivocally identified. These results have been unanimously confirmed by several studies, and therefore the use of IHC is not recommended for the diagnosis of RET gene rearrangement [183, 184].

On the contrary, the FISH methodology has been shown to be successful in detecting a substantial amount of gene rearrangements. Using RET gene region specific probes FISH had a high sensitivity of 100% for the chimeric proteins KIF5B and CCDC6 but less than 100% for the other partners. On the other hand, a surprisingly poor specificity of 45%–60% was measured, therefore, the currently available DNA probes have not been recommended for routine diagnostics of RET gene fusions [184, 185]. Considering all these issues NGS remains the optimal tool for general RET testing. DNA-based NGS showed a sensitivity of 87.2%–100% for detecting RET fusions, while its specificity was also highly satisfying (98.1%–100%) [186].

In humans, three neurotrophic tyrosine receptor kinase (NTRK) genes are known, encoding the transmembrane neurotrophin receptors TrkA, TrkB and TrkC. These Trk receptors are involved in embryonic development of the central and peripheral nervous system [187]. In adults, they are expressed only in neural tissue and skeletal muscle [188]. Ligand-dependent activation of Trk receptors activate several biochemical pathways, including MAPK and PI3K signaling [189]. Chromosomal rearrangements of NTRK genes result in increased expression and/or activation of Trk receptors [190]. The occurrence of NTRK gene fusions is characteristic for some rare tumor types, such as mammary analogue secretory carcinoma of the salivary gland or congenital infantile fibrosarcoma. ETV6-NTRK3 rearrangements are detected in 90% of these cases [191, 192]. Although NTRK gene rearrangements are generally rare, they have been detected in a broad range of common solid tumor types. NSCLC, colorectal carcinoma, GIST, papillary thyroid carcinoma, melanoma, pancreatic adenocarcinoma and gliomas were reported with very rare NTRK involvement of less than 1% of cases [193]. In NSCLC, the incidence rate was only 0.1%–0.3%, an order of magnitude lower than the frequency of ALK or ROS1 gene rearrangements [194]. In two very large NSCLC case-control studies, the rates of NTrk fusions were 0.1% (Gatalica, 4,073 lung adenocarcinomas [195]) and 0.23% (Solomon, 3,993 lung adenocarcinomas [196]). NTRK1 fusion could be demonstrated most (68%), followed by NTRK3 (24%) and NTRK3 fusion as the least common change. Because of the rarity of occurrence, even the largest studies had limitations defining detailed clinical characteristics of patients with NTRK fusions. They suggest equal distribution in both women and men, with a wide age range. The majority of those carrying the fusion are non-smokers, but heavy smokers were not excluded. Most NTRK-positive tumors proved to be adenocarcinomas with mucinous or poorly differentiated morphology, but fusions have also been detected in neuroendocrine carcinoma and even squamous cell carcinoma [197].

Although rare, the identifications of these tumors opens the way for NTRK-targeted TKI therapy, that is available in the last couple of years promising a favorable therapeutic response in patients with NTRK gene fusion [198, 199]. In a 2023 study, 51 patients with advanced NSCLC harboring an NTRK fusion had an ORR of 62.7% following entrectinib treatment, while the PFS and OS was 28.0 and 41.5 months, respectively [200]. Predictive testing of NTRK gene fusion-due to the altogether 3 TrK genes and numerous fusion partner genes-is quite cumbersome, even by the classic FISH arrangement. Due to the 3 independent NTRK gene regions potentially involved, three FISH assays and tests would be required. However, while the detection of fusions involving the NTRK3 gene by FISH had good sensitivity, too many false negative cases were reported for NTRK1 fusion detection. This may be due to intrachromosomal rearrangements involving a short segment, allowing only limited signal separation in the break-apart probe assay, causing interpretation difficulties. Another problem appeared as FISH probes could not detect rearrangements with some fusion partners. Because of these drawbacks, NTRK FISH were not recommended for routine diagnostics [201]. As an alternative, RNA-based massively parallel sequencing (MPS) is considered as a favorable methodology. However, this technique is not widely available, not mentioning the turnaround time and costs of the test.

Taking everything together, immunohistochemical detection of Trk proteins as a screening test should be considered. Immunohistochemistry is available in virtually all pathology departments, is relatively rapid and inexpensive and sufficiently works with small amounts of tumor tissue. Commercially available diagnostic antibodies detect all Trk proteins. The staining pattern is variable: membrane, cytoplasmic and nuclear positivity can all be present. Unfortunately, a validated scoring system is not available at this time. A positive tumor is defined as one with at least 1% of tumor cell positivity, any kind of positivity more intense than background should be satisfactory, regardless of the staining pattern. Confirmatory testing of positive cases by MPS seems to be necessary for proper interpretation.

According to the literature the sensitivity of the IHC method ranges from 75% to 97% and the specificity is remarkably high, reaching 98%–100% [202–204]. Previous large studies have shown that the sensitivity of IHC is not uniform for the three NTRK gene fusions, 96.2%, 100% and 79.4% sensitivity rates were measured for NTRK1, -2, and -3, respectively, while the specificity was 81.1%. More specifically for the lung adenocarcinoma patient group, IHC sensitivity was 87.5% and specificity 100% [196]. The above large studies all used the Abcam EPR17341 antibody clone. However, in a more recent study, 133 (14.8%) out of 1068 NSCLC cases were panTrk IHC positive, but only 2 cases could be confirmed by RNA-based testing, resulting a positive predictive value of 1.5% for the IHC test applied [205]. Unlike in previous studies, in this work the C17F1 antibody clone was used and any staining was accepted as positive. In conclusion, predictive IHC testing of NTRK involvement also should be performed with care. Sensitivity and specificity rates may be strongly influenced by the type of the diagnostic antibody used. However, sensitivity of IHC supposed to be relatively lower for NTRK3 fusions, the reason of which is not known in detail. If uncovered, low sensitivity of the IHC screening could drop out patients of an effective treatment opportunity.

The mesenchymal epithelial transition (MET) proto-oncogene is located in t4.1he chromosomal region 7q21-q31 and encodes a transmembrane receptor tyrosine kinase protein [206, 207]. The MET protein is expressed in diverse cells of epithelial origin, and is further expressed in liver cells, endothelial cells and neurons. The ligand for this receptor is hepatocyte growth factor (HGF), which is mainly produced by mesenchymal cells, such as fibroblasts [208]. The extracellular part of the receptor protein is responsible for ligand binding and includes domains like the semaphoring and the immunoglobulin-plexin transcription factor domain. The intracellular part is composed of the juxtamembrane domain and the catalytic domain [209, 210]. Ligand binding activates the protein by causing homodimerization, which then leads to autophosphorylation of tyrosine residues in the catalytic domain. Activated MET induces several intracellular activation pathways through MAPK, PI3K, Nf-kB and signal transducer and activator of transcription3 (STAT) signaling. HGF/MET activation plays a key role in epithelial-to-mesenchymal transitions (EMT) by regulating extracellular matrix adhesion and cytoskeletal changes [211, 212]. The deactivation mechanism of the activated signaling pathway deserves attention, as changes in this process play a key role in the carcinogenic effect of MET [213]. After ligand binding, homodimerisation and intracellular signaling the active receptor protein is internalized by clathrin-mediated endocytosis, it is partially degraded but recycling and return to the cell membrane is possible. This process is controlled by ubiquitin ligase casitas B-lineage lymphoma (CBL), which recognizes the Tyr1003 residue encoded in exon 14 of the MET gene and the ubiquitinated MET is degraded by the endosome system [214, 215].

Genetic events may affect MET protein function resulting in oncogenic effects. MET gene amplification results in increased expression and constitutive activation of the kinase protein. This mechanism is supposed to be responsible for acquired resistance during EGFR TKI treatment in 3%–4% of [42, 216, 217]. Various point mutations have also been detected in several tumours, including lung carcinoma, but their oncogenic role remains unclear [218]. Moreover, some gene fusions have also been described, such as KIF5B-MET, which have potential oncogenic effects and serving as therapeutic targets [219].

Exon 14 mutations are the best known MET alterations with pathogenetic and apparently, clinical significance. This specific mutation type is a result of a two base pair insertion in intron 13. The insertion represents an alternative mRNA splicing site with the consequence of the “skipping” of the entire exon 14 during translation for protein synthesis. Therefore, the functional molecule lacks the juxtamembrane segment containing the Tyr1003 residue responsible for the internalisation of the activated receptor. Thus, the exon 14 skipping mutation enhances the stability of activated MET on the surface of the cell, resulting in a prolonged activity of the receptor signaling [220, 221]. According to one of the first large case-control studies, this mutation is present in about 3% (131/4,402) of NSCLC cases [222]. According to a recent large meta-analysis, exon14 skipping mutations can be detected at a rate of 2% in NSCLC, no significant geographical differences are reported. The prevalence proved to be 12% in non-smokers and 2% in smokers, with a similar overall prevalence of MET14 skipping mutation positive patients with a history of smoking and non-smoking. It is more common in women and at older age (average is 73 years). It is noteworthy that while the histological type of adenocarcinoma has a prevalence of 2.4%, it is detected in 12% of sarcomatoid carcinomas [223]. Association with major driver mutations (EGFR, KRAS, BRAF and ALK, ROS1 gene fusions) appears to be rare, but some other genetic events, such as MET, EGFR amplification, or PI3K mutations may co-occur [222]. Data to date suggest that MET exon 14 mutations in NSCLC are associated with a poor prognosis.

The therapeutic targeting of MET kinase is relevant with multiple TKI drugs (e.g., tepotinib, capmatinib, savolitinib) resulting in good therapeutic response [224, 225]. This is true for the exon-skipping alteration but also for the Y1003N mutation of the juxtamembrane domain, which also inhibits CBL-mediated degradation [222].

Due to the heterogeneity of Met exon 14 aberrations, their detection is another challenge for diagnostic pathology laboratories. Attempts have also been made to detect MET alterations by immunohistochemistry. While overexpression of MET protein can be detected in many tumor types in many cases, MET overexpression can be detected in 35%–72% of NSCLC by immunohistochemistry [226, 227]. Unfortunately, in the majority of IHC positive cases, there is no Met exon 14 skipping or MET amplification. As shown in a recent study, 71 tumors in 181 NSCLC cases had MET overexpression detectable, but only 1% of IHC positive cases had amplification and 3% had Met exon 14 skipping. These results also show that IHC-based detection is not a suitable screening test for MET alterations [228]. RNA-based assays have the highest sensitivity and can detect exon 14 skipping independent of the underlying diverse genetic alterations by detecting fusions of exons 13 and 15 following mRNA transcription. The disadvantage of the RNA-based methodology is its sensitivity to RNA degradation [229]. Unfortunately, amplicon-based DNA NGS tests have a detection rate of only 63% [230], whereas hybrid-capture NGS methodology can achieve better results, but requires larger amounts of sample DNA, which is frequently not provided from small biopsy samples [231]. To overcome this issue circulating free DNA (liquid biopsy) should have lower sensitivity but a high positive predictive value [232].

HER2 (ERBB2) is a member of the HER growth factor receptor family. This family also includes EGFR, EGFR3 and EGFR4. The HER2 gene is located in the 17q11.2-q12 region. The encoded receptor protein has an extracellular ligand-binding, a transmembrane and an intracellular tyrosine kinase domain, like other growth factor receptors of the family [233]. It is unique compared to other members of the HER family in that it has no known ligand but has an intrinsic tyrosine kinase activity and is specifically prone to homo- or heterodimerization, which results in its activation. A frequent heterodimerization partner is HER3. Activated HER2 can activate several intracellular signaling pathways such as MAPK, PI3K, and STAT [234].

Several alterations may occur in the HER2 gene, which have oncogenic effects. Gene amplification of HER2 is well known in breast cancer, one of the oldest known therapeutic targets [235], but is also common in gastric [236] and ovarian cancer. HER2 alterations can also occur in lung carcinoma, but gene amplification is relatively rare. However, HER2 amplification in NSCLC may be a potential cause of acquired resistance during EGFR TKI treatment [42]. HER2 amplification is most easily detected by FISH, which is analogous to the common testing practice in breast carcinoma, with a HER2/CEP17 hybridization signal ratio greater than 2. Importantly, HER2 overexpression is often detected by immunohistochemistry, which is usually due to a balanced increase of copy number (polysomy). In this case the HER2/CEP17 ratio does not exceed 2 determined the FISH analysis [237]. Various mutations in the HER2 gene may also occur in the coding regions of all three domains with a rate of 2%–4% of NSCLC. In the first study, published in 2004, HER2 mutations were presented in 10% of the lung adenocarcinomas [238] Subsequently, several studies have reported higher case numbers, with lower frequencies (1.6% testing 671 NSCLC cases) [239]. The most common types of mutations proved to be in-frame insertions in the kinase domain coding region in exon 20. These mutations change the protein conformation and increase kinase activity, thereby activating intracellular signaling [240]. HER2 exon 20 insertions are like exon 20 insertions detected in the EGFR gene [241]. Many of these insertions have been described, the most common being the YVMA insertion, which was detected in 68% of the 98 HER2 mutant tumors detected in a study of altogether 2,788 patients [242]. HER2 mutations occur mainly in women, non-smokers, are associated with adenocarcinoma histology and brain metastasis is common in these patients [243]. Another large study reported HER2 mutations in 24 of 920 patients (3%), 71% non-smokers, 58% women, mean age was 62 years [244]. The co-occurrence of HER2 mutations with other classical driver mutations is virtually excluded [239, 245]. However, some HER2 mutations develop in about 1% of cases of acquired resistance to EGFR TKI treatment [246]. Neither immunohistochemistry nor FISH is suitable for HER2 mutation detection. Since many types of these mutations are known, NGS sequencing methodology is the only effective way of testing [247].