**3. Genetic alterations in NSCLC associated with brain metastasis**

Targeted therapies that successfully combat tumor cells outside the brain may fail to be effective behind the BBB. There are several reasons for this, one of which are differences in genetic alterations between the primary tumors and their metastases [23]. Patients suffering from NSCLC have been classified according to the genetic changes in the primary tumor, which include epidermal growth factor receptor (EGFR), Kirsten rat sarcoma (KRAS), and anaplastic lymphoma kinase (ALK). NSCLC brain metastasis-specific mutations can be detected in the cerebrospinal fluid (CSF) and can also be used to evaluate the presence of disease and response to therapy [24].

#### **3.1 EGFR mutations**

EGFR is a receptor for extracellular growth factors such as epithelial growth factor (EGF) and tumor growth factor-α (TGFα). Binding of these factors causes a structural change and activation of the receptor complex, resulting in the activation of signaling pathways that promote cell proliferation, motility, and survival. Dysregulation of the receptor is associated with various human cancers. The prevalence of EGFR mutations is dependent on a variety of factors, including ethnicity, gender, smoking, tumor heterogeneity, and tumor progression. EGFR is often overexpressed in NSCLC and the two most frequent EGFR mutations encountered involve exon 19 (deletions) and 21 (L858R mutations) [25–27]. There is data supporting that CNS metastases of NSCLC are promoted by EGFR-activated mesenchymal–epithelial transition (MET) through mitogen-activated protein kinases (MAPK) signaling. EGFR activates signal transducer and activator of transcription 3 (STAT3) via the expression of interleukin-6 (IL-6) which would increase the risk of BM [28]. NSCLC patients with EGFR mutations at the time of diagnosis or in the early stages of the disease seem to have two times higher risk of brain metastasis [29–31]. In a series of 30 primary tumor/ metastasis series, there was discordance between EGFR status as measured by IHC of one-third of sample pairs and a little less by FISH [32]. In 14 out of 54 paired samples of lung adenocarcinomas, EGFR alterations of EGFR were restricted to the brain metastases [33]. In a recent paper by Haim et al., the EGFR mutational status of brain metastasis could be predicted with an accuracy of almost 90% by using clinical, radiological, and molecular data for deep learning strategies [34]. Obviously, the presence of CNS metastases leads to poorer outcomes (viz., 11.6 months vs 18.7 months) as shown in a study on 101 EGFR positive metastatic NSCLC previously treated with either combination chemotherapy or oral TKI [35]. The progression of the cerebral lesions is also relatively high during treatment in these patients and there is a connection between the EGFR mutations and EMT-related tumor invasion [36, 37].

#### **3.2 KRAS mutations**

The K-Ras protein is encoded by the KRAS gene and is part of the RAS/MAPK pathway, where it transfers signals to proliferate and divide from extracellular into the nucleus. A single substitution of a nucleotide may serve as an activator of the signaling pathway turning tissue hyperplasia into invasive cancers. Although it is believed

that KRAS and EGFR mutations are mutually exclusive [38, 39], yet cases of simultaneous occurrence were found [40–42]. Nearly 15–30% of NSCLCs have activating mutations in the KRAS gene that are associated with adenocarcinoma initiation and clinical aggressiveness [38, 43, 44]. There is a clear connection between KRAS mutations and smoking history [45, 46]. In a study of 482 lung adenocarcinomas (LADC), it was found that KRAS mutations also occur in patients who had never smoked, but the mutations differ from those in the tumors of smokers. For instance, transition mutations (G > A) prevail in those who never smoked while transversion mutations (G > T or G > C) are typical for NSCLCs in smokers [47]. The relation between KRAS mutations in NSCLC and propensity for brain metastasis is still unknown and need to be further studied [36, 42]. Approximately 25% of brain metastatic tumors with KRAS mutations were observed in smokers [44]. Other mutations, including ROS proto-oncogene 1, liver kinase B1 (LKB1), and hepatocyte growth factor receptor (HGFR), were associated with the development of lung carcinoma [48–50], but their relations with brain metastasis of lung cancer is also unknown. LKB1 is inactivated in nearly 30% of all NSCLCs [46] and its effects are synergistic with those of KRAS mutation on the progression of lung cancer and the development of metastases in general [51, 52]. In a study of 154 patients with NSCLC, Zhao et al. concluded that KRAS mutations in combination with low LKB1 copy numbers (CNs) are related to a 20-fold increase in brain metastasis [53]. So far, therapeutic KRAS targeting has been unsuccessful.
