Despite the significant advances that have been made in the diagnosis and treatment of non-small cell lung cancer (NSCLC) in the past decades, it remains the leading cause of cancer death worldwide [1]. Among NSCLCs, squamous cell carcinoma (SCC) is one of the most common histological subtypes, accounting for 30%–35% of all NSCLC cases [2].

Lung SCC is strongly associated with tobacco smoking and arises mostly in the central part of the lungs [3]. There is growing evidence that it is more heterogeneous than it is suggested by its three histologic subtypes (keratinizing, non-keratinizing, and basaloid) [4].

While there have been significant breakthroughs in targeted therapies for lung adenocarcinoma, most patients with advanced SCC are treated with immune checkpoint inhibitors (ICIs), platinum-based chemotherapy, or a combination of the two [5]. For SCCs with no PD-L1 expression, platinum-based chemotherapy remains the main treatment option [6]. Therefore, there is an urgent need to develop new targeted therapies for patients with SCC.

Protein complexes mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) are integral components of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. The main functions of mTORC1 include the regulation of cell growth and proliferation, whereas mTORC2 plays essential roles in cytoskeletal reorganization and cell survival [7]. Rictor (encoded by the RICTOR gene) is the scaffold protein of mTORC2 (Figure 1). Amplification of RICTOR has been reported in various cancer types, including approximately 10% of lung SCCs [8–10]. Like PIK3CA copy number gain and PTEN loss, RICTOR amplification affects the PI3K/Akt/mTOR pathway. PIK3CA copy number gain and PTEN loss mostly alter mTORC1 activity, whereas RICTOR amplification primarily results in the alteration of mTORC2 [11].

RICTOR fluorescence in situ hybridization (FISH) is considered the diagnostic standard for the detection of RICTOR amplification [8]. Immunohistochemistry (IHC) is an acceptable, cost-effective alternative to detect certain molecular alterations, including ALK and ROS1 rearrangements [12, 13]. However, Rictor IHC has never been compared to RICTOR FISH in the SCC of the lung.

The purpose of this study was to compare RICTOR amplification as detected by FISH with Rictor expression as detected by IHC in SCC of the lung. Since PD-L1 negative tumors are typically ineligible for immune checkpoint inhibitor therapy, finding a potentially targetable genetic alteration such as RICTOR amplification is especially important for patients with PD-L1 negative advanced SCC. Therefore, we also examined the relationship between RICTOR amplification and PD-L1 expression in these tumors.

This is the first study to compare RICTOR amplification and Rictor expression in human lung SCC. Using FISH, we observed RICTOR amplification in 20% of the cases. This is slightly higher than RICTOR amplification in the Lung Squamous Cell Carcinoma (TGCA, Firehose Legacy) dataset or in previous database analyses, where it has been found in 16% and 7.4%–15% of SCC cases, respectively [19, 20]. The difference among various studies may be due to differences in study populations and methods used to detect RICTOR amplification. An additional explanation for these differences is the high heterogeneity of RICTOR amplification and overexpression, which has been described previously [21] and was also found in our study. Given the potential intratumoral heterogeneity of both Rictor and PD-L1 staining [21–23], we performed both FISH and IHC analysis on whole slides of the tissue blocks, which allows for a higher probability of detecting areas of RICTOR amplification and overexpression of a heterogeneous tumor.

Rictor is the scaffold protein for mTORC2, a protein complex interconnected with the PI3K/Akt/mTOR pathway [24]. The PI3K/Akt/mTOR pathway regulates several essential cellular functions related to environmental signals, including cell proliferation, growth, homeostasis, and metabolism.

Subcellular localization of the PI3K/Akt/mTOR pathway components has been evaluated in various studies. As the first step in the pathway, PI3K generates phosphatidylinositol-3,4,5-triphosphate (PIP3), which binds the pleckstrin homology domain of Akt, recruiting it to the plasma membrane. Once Akt is localized to the plasma membrane, mTORC2 can phosphorylate its Ser473 site, thereby facilitating activation of the PI3K/Akt/mTOR pathway [20, 25, 26]. In contrast, some studies have demonstrated that mTORC2 can also phosphorylate its substrates (e.g., serum/glucocorticoid regulated kinase 1) using intracellular membranes and subcellular spaces such as the endoplasmic reticulum and the perinuclear compartment [25, 26]. Additionally, it is also known that Rictor can interact with integrin-linked kinase (ILK), a protein with a multifunctional role in cell-matrix interactions and that the Rictor-ILK complex may contribute to the phosphorylation of Akt on Ser473 [27, 28]. Consistent with these data, a recent study has described more than 200 proteins that can interact with mTORC2 and possibly regulate its activity and localization [29]. We observed both membrane and cytoplasmic staining for Rictor in our study. Interestingly, in RICTOR amplified cases, Rictor was localized primarily to the plasma membrane, whereas, in non-amplified cases, we found predominantly cytoplasmic staining consistent with intracellular localization.

Previous studies have revealed a positive correlation between RICTOR mRNA expression and DNA copy number in NSCLC [20], which was confirmed with our database analysis. Similarly, we observed a positive correlation between Rictor protein expression and RICTOR gene amplification. However, this correlation was strongly dependent on subcellular localization. Rictor membrane staining showed high specificity (95%) and moderate to high sensitivity (77%) for RICTOR amplification, whereas the specificity and sensitivity of Rictor cytoplasmic staining were much lower (38% and 11%, respectively). Therefore, our recommendation is to use Rictor membrane staining as a surrogate marker for RICTOR gene amplification.

Despite breakthroughs in targeted therapies for lung adenocarcinoma in the past decades, limited progress has been made in the treatment of lung SCC. This seems to be due to the lower frequency of targetable genetic alterations and the less favorable response to small-molecule tyrosine kinase inhibitors [30, 31]. The PI3K/Akt/mTOR pathway represents a potential therapeutic target [32]. The activity of mTORC2 can be inhibited by dual mTOR kinase inhibitors, which affect both mTORC1 and mTORC2 (e.g., vistusertib and sapanisertib). They have been used to treat various types of lung cancer, including SCC of the lung, in early-phase clinical trials [9, 11]. In most of these studies, patients have not been stratified based on the presence of RICTOR amplification. A selective mTORC2 inhibitor (JR-AB2-011), which blocks the interaction between Rictor and mTOR, is already available and has been used with promising results in preclinical trials [33, 34]. Selecting patients with RICTOR-amplified tumors may improve the results.

In advanced cases of SCC of the lung, ICIs represent the only widely used therapeutic option besides radiochemotherapy [6]. Similar to a previous study, which has found no association between gene alterations and PD-L1 expression in advanced lung SCC [35], we observed no association between PD-L1 expression, a predictive marker for the efficacy of ICIs, and RICTOR amplification or Rictor expression. RICTOR amplification occurred in PD-L1 positive and negative cases with similar frequency, suggesting that a significant number of the PD-L1 negative cases can be eligible for mTORC2 inhibitor therapy (Figure 5). Additionally, RICTOR amplification has been speculated to be responsible for the resistance against chemotherapy, tyrosine kinase inhibitors, and ICIs, leading to tumor progression in various types of cancer [36–38]. Therefore, analyzing RICTOR amplification in lung SCC patients who no longer respond to ICIs can reveal a new targetable alteration in this subpopulation of patients.

Several studies have shown a correlation between mTORC2 activity and metastatic disease [33, 39, 40]. This can result in decreased survival in RICTOR-amplified cases [41]. However, similar to our previous observation in small cell lung carcinoma [8], we did not detect a significant correlation between RICTOR amplification and unfavorable clinical outcomes in SCC of the lung. Nevertheless, Rictor membrane staining, which might be directly associated with Akt phosphorylation and thereby mTORC2 activity, did show slightly lower overall survival. These results might be clarified in a study with a larger sample size focused on this particular question.

In conclusion, our findings indicate that a significant proportion of lung SCCs, including PD-L1-negative cases, harbor RICTOR amplification. Despite the initial failures in early-phase clinical trials, one of the key messages of our study is that stratifying patients based on the presence of RICTOR amplification or Rictor overexpression may improve the results. With the advent of next-generation inhibitors that can selectively target mTORC2, the importance of this topic is emerging. Rictor membrane staining can predict RICTOR amplification as detected by FISH with high specificity and sensitivity; therefore, it can potentially be used as a surrogate marker to identify RICTOR-amplified lung SCCs. Analysis of lung SCCs by RICTOR FISH or Rictor IHC, especially in PD-L1-negative cases, can help select patients who may benefit from mTORC2 inhibitor therapy.