Lung cancer has been the leading cause of cancer-related deaths worldwide. Globally, it accounted for 2.2 million new cases and 1.8 million deaths in 2020 (1). Adenocarcinoma is the dominant histologic subtype, accounting for 60% of non-small cell lung cancers (NSCLCs) (2). Although recent advances in treatment modalities, including targeted therapy and immunotherapy, have demonstrated better clinical outcomes than conventional chemotherapy in patients with advanced lung adenocarcinoma (2, 3), the mortality of this lethal disease is still high, with a 5-year survival rate of only 24% (4), suggesting the unmet clinical need for personalized therapeutic approaches to improve prognosis.

Reactive oxygen species modulator 1 (Romo1) was first identified in 2006 in a patient with head and neck cancer who was resistant to chemotherapy after recurrence (5). Located in the mitochondrial membrane, this novel protein is a major regulator of the production of intracellular reactive oxygen species (ROS), and Romo1-induced ROS play a significant role in cell proliferation in both cancer and normal cells (6, 7). In addition, Romo1-induced ROS are related to the drug resistance of 5-fluorouracil (5-FU), and Romo1 overexpression induces invasive activity in various cancer cells (8–10). These findings suggest that Romo1 may not only be involved in carcinogenesis but may also influence the response to anticancer treatment. This concept has been proven using recent clinical data (9, 11–13). Chung et al. demonstrated that tumor Romo1 overexpression was significantly associated with poor survival and vascular invasion in patients with hepatocellular carcinoma who underwent curative resection (9). Lee et al. demonstrated that high Romo1 expression was significantly associated with early recurrence and poor survival after surgical resection in patients with NSCLC (11). Romo1 overexpression in tumor tissue was significantly associated with poor response and clinical outcomes in patients with advanced NSCLC treated with platinum doublets (12). In addition, Kim et al. reported that elevated Romo1 expression in curatively resected colorectal cancer was significantly associated with poor survival outcomes and frequent lymph node metastasis (13). These data suggest that Romo1 is a promising biomarker for malignancies. However, the clinical usefulness of this protein has never been explored in patients with cancer harboring driver genetic alterations.

In this study, we aimed to determine the clinical implications of Romo1 expression in patients with epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma treated with first-line tyrosine kinase inhibitors (TKIs). We investigated whether Romo1 expression is related with the response to treatment, survival rate, and the development of secondary T790M mutations after TKI failure.

Our study demonstrated that high Romo1 expression was significantly associated with poor PFS and OS in patients with EGFR-mutant lung adenocarcinoma treated with frontline targeted therapy. In addition, it was associated with lower rate of secondary T790M mutations after TKI use. To the best of our knowledge, this is the first study to suggest the predictive and prognostic value of Romo1 in oncogene-driven malignancies. The current data highlight that high Romo1 expression might confer an aggressive phenotype, requiring tailored therapeutic approaches among EGFR-mutant tumors.

EGFR mutations are major driving genetic alterations that account for 15–50% of patients diagnosed with lung adenocarcinoma (14, 15). EGFR-TKIs are recommended as the first-line treatment in patients harboring these mutations as they improve both PFS and OS compared with those only treated with chemotherapy (16). However, the efficacy of EGFR-TKIs is still suboptimal, mainly because of the primary and secondary resistance, which is one of the major challenges in the management of these patients (17–19). Identification of the mechanisms leading to TKI resistance or prediction of treatment response before TKI use is critical to improve the clinical outcomes in this patient population. Although the resistant mechanism for EGFR-TKIs has been widely studied, it is not fully understood; T790M mutation, concurrent genetic alterations, MET amplification, epithelial-mesenchymal transition (EMT), and histological transformation are considered to be contributing factors (16, 17).

Cancer cells are characterized by increased ROS levels compared with normal cells, and the upregulated ROS are involved in various biological processes of carcinogenesis, including cell signaling, proliferation, and metastasis, as well as chemoresistance (18–20). Cumulating evidence indicates that ROS play a critical role in EGFR-TKI resistance. Increased oxidative stress induces TKI resistance of EGFR-mutant cells by activating phosphorylation and impairing the dimerization of EGFR (21, 22). In addition, ROS play a critical role in EMT engagement (23, 24) and interferon gamma-induced Src activation via the Raf/ERK signaling pathway (25). Moreover, chronic treatment with EGFR-TKIs enhances intracellular oxidative stress through NOX4, and morphologic changes and dysfunction of mitochondria (26, 27). Antioxidants can alleviate TKI-resistance by inhibiting ROS-induced aberrant phosphorylation of EGFR and AKT (26, 28). Taken together, ROS are involved in both primary and secondary resistance to EGFR-TKIs via various mechanisms, including abnormal EGFR phosphorylation, EMT, and Sarc activation.

Romo1 is a major regulator of intracellular ROS production, and Romo1-induced ROS are indispensable for the proliferation of normal and cancer cells (6, 7). Studies have consistently demonstrated the potential value of Romo1 as a novel biomarker for various lung cancer treatment (11, 12, 29, 30). Table 6 summarizes these previous studies and the present data, evaluating the clinical implications of Romo1 in lung cancer patients. Different studies have demonstrated different cutoffs from each other; however, the low H score cutoffs in two studies (11, 30) can be attributed to the early stage population enrolled in those studies. The present data suggest that Romo1 levels seem to be abundant in advanced disease. Based on the critical association between Romo1 and ROS production and the contribution of ROS in EGFR-TKI resistance, we hypothesized that Romo1 may be a novel biomarker in patients treated with targeted therapy. In addition to the predictive and prognostic values of Romo1, our present data suggest the possibility of this novel protein as a potential treatment target for EGFR-mutant NSCLC, considering the crucial association between ROS and TKI resistance. To address this issue, we have just started a preclinical study on the regulation mechanism of Romo1, combinational effect of Romo1 inhibition and EGFR-TKIs, and drug screening for its inhibitors.

Recent studies have demonstrated the clinical usefulness of Romo1 gene expression and the novel association between micro RNA and Romo1. Alizamir et al. demonstrated that Romo1 mRNA expression increased in bladder cancer tissues and that Romo1 overexpression was associated with advanced tumor (31). Similarly, Afshar et al. showed that Romo1 mRNA was overexpressed in gastric cancer tissues and that its overexpression was associated with unfavorable prognosis in patients with gastric cancer (32). Moreover, Wang et al. reported that LINC00319, a long non coding RNA, is correlated with early recurrence of bladder cancer, and that LINC00319 is a sponge for miR-4492, which directly targets Romo1 expression. In that study, the miR-4492/Romo1 axis was shown to regulate proliferation, migration, and tumor invasion of bladder cancer cells (33). These findings suggest that evaluating Romo1 gene expression could be more useful than evaluating protein expression using IHC because the former can be less variable than the latter. In addition, the potential role of micro RNA as a regulator of Romo1 in various human malignancies should be investigated in future studies.

Secondary T790M mutation is a major resistance mechanism and has been identified in approximately half of patients who progressed after EGFR-TKI therapy (34). Detection of this mutation is important for the management of EGFR-mutant patients because T790M-positive tumors are susceptible to third-generation EGFR-TKIs, such as osimertinib. Although mutational analysis using tumor tissue or body fluid is recommended, it is often limited by factors, such as the invasiveness of tissue acquisition and the insufficiency or inaccessibility of tissue samples. Previous studies have demonstrated that the frequent T790M mutation is associated with 19del and long-term exposure to EGFR-TKIs (35–37). Our present data confirmed these previous findings, and additionally suggest that high Romo1 expression could be a negative predictor of T790M development after frontline TKI. The reason that Romo1 is associated with the emergence of the T790M mutation is unclear; however, it may be attributed to the different durations of treatment with EGFR-TKI according to different Romo1 expression. As suggested in the previous studies, T790M-positive cells undergo selection and are enriched during EGFR-TKI treatment (38–40). In addition, time is required for the development of T790M-dominant tumor because cells harboring the mutation grow slowly compared with T790M-negative counterparts (41). Based on these findings, we hypothesize that shorter treatment duration may be the cause of lower frequency of the T790M mutation in high Romo1 population. Our hypothesis should be elucidated through further investigations.

The present data suggest that Romo1 overexpression confers a distinct aggressive phenotype that may require different treatment strategies. Because TKI monotherapy could be less effective in patients with high Romo1 expression, other therapeutic options, such as combination with chemotherapy or immunotherapy, can be considered. A recent study showed that pemetrexed-based doublet combined with gefitinib showed better RR (84% vs. 67%, p < 0.001), longer PFS (20.9 vs. 11.9 months, HR = 0.49; p < 0.001), and OS (50.9 vs. 38.8 months, HR = 0.722; p = 0.021) compared with gefitinib monotherapy in EGFR-mutant tumors (42). To prove our hypothesis and determine the optimal treatment strategy for those with different Romo1 expression levels, large-scale prospective investigations are required.

This study had several limitations. First, it is a relatively small retrospective study performed at a single institute, and the slides were reviewed by a single pathologist. Second, we did not include nutritional status or comorbidities as clinical characteristics, which might influence the survival outcome of our patient population. To overcome these potential caveats, we aimed to enhance the validity of our data by long-term follow-up. Third, tumors harboring other driver genetic alterations, such as ROS1 and ALK fusions, were excluded because of their low frequency in our patient cohort. Fourth, the interval changes in Romo1 expression in patients with progressive disease after TKI failure were not investigated. As mentioned earlier, EGFR-TKIs can induce ROS production, and thus, Romo1 expression can be affected by previous treatments. The predicted value or impact of the dynamics of Romo1 expression on subsequent treatment could be an interesting topic for future studies. Finally, we did not simultaneously explore other factors that could be associated with TKI resistance, including immune phenotypes or co-existing genetic alterations. We are currently working on a possible association between Romo1 expression and immunologic signatures or concurrent genetic alterations in EGFR-mutant populations.

The present data demonstrate that high Romo1 expression is associated with unfavorable clinical outcomes, not only due to the poor response to first-line TKI treatment but also because of the less emergence of secondary T790M mutation. These findings highlight that Romo1 overexpression might confer a distinct aggressive phenotype among EGFR-mutant tumors, requiring a different therapeutic approach, although further studies are needed to verify our results. In addition, future studies should focus on whether Romo1 has similar clinical implications in other malignancies harboring driver genetic alterations, and optimal treatment strategy for the Romo1-overexpressed, EGFR-mutant tumors. Large prospective studies validating our data might facilitate the clinical use of Romo1 expression for the risk stratification and prediction of prognosis in those patient population.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Kyung Hee University Hospital. The patients/participants provided their written informed consent to participate in this study.

SHL conceived and designed the study. WGK, J-YS, and SHL collected, analyzed, and interpreted the data, and drafted the article. SHL revised the paper critically. All authors contributed to the article and approved the submitted version.

This research was supported by a grant from the Basic Research Program through the National Research Foundation funded by the Ministry of Science and ICT (2019R1F1A1041812), and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare (HI18C1944) of the Republic of Korea.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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