Biological Difference between L858R and Exon 19 Deletion Contributes to Recurrence-Free Survival of Resected Non-Small Cell Lung Cancer

Introduction: The differences in biological characteristics among different genotypes of classical EGFR mutations have not been clarified. This study aimed to clarify the clinical and biological differences between L858R and 19 deletion in NSCLC. Methods: We analyzed a cohort of 191 consecutive cases of surgically resected NSCLC harboring EGFR driver mutations (L858R or 19 deletion) in which curative resection was performed in Aichi Cancer Center Hospital, Nagoya, Japan, from January 2006 to September 2021 and in which recurrence subsequently developed. We also subjected 61 surgically resected NSCLC specimens harboring EGFR driver mutations (L858R or 19 deletion) to an RNA sequencing analysis. Results: In patients with stage I disease, the median time to recurrence did not differ to a statistically significant extent between the types of EGFR mutations; however, among those with stage II and III disease, the median time to recurrence in patients with the L858R genotype tended to be shorter in comparison to those with 19 deletion (log-rank test, p = 0.47 and 0.46, respectively). In comparison to 19 deletion tumors, L858R tumors had higher cytological malignancy (e.g., mitotic ability) and showed stronger immunogenicity. Conclusion: L858R and 19 deletion tumors are likely to have a slight difference in the time to recurrence. They suggest that even in EGFR driver tumors, which are treated as the same disease category, the biological characteristics of the tumors are different, which may leave room for innovations in postoperative treatment and treatment at recurrence.


Introduction
The development of molecular diagnostics has made a great contribution to the diversification of therapeutic strategies for advanced NSCLCs, especially in EGFRmutant NSCLC, which is the most common type of driver mutation, the standard treatment, including molecular targeted agents has matured, resulting in a dramatic improvement of the prognosis [1]. Because of the significant genomic diversity in EGFR mutations (even the classical mutations, L858R and 19 deletion, are treated as the same Masago [1], it is also an area where further improvement in treatment strategies, such as combination drugs with EGFR-tyrosine kinase inhibitors (TKIs) and indications for immune checkpoint inhibitor, is desired.
There are few reports on the biological differences between the two classical mutations; however, the results of a proteomic analysis have been reported. According to this report, 19 deletion tumors showed the involve-ment of SUMOylation, EMT, ERK/MAP kinase, and Hippo signaling, while various pathways related to cancer cell survival were identified in L858R tumors [2]. There have been several reports on tumor grade using recurrence-free survival as an indicator, but the results have been controversial, and moreover, many of them were only observational studies with no biological considerations [3]. In fact, for estimating tumor grade, the analysis of time to recurrence for each postoperative stage in an uncensored model may reduce biases (e.g., the presence of a residual tumor and the amount of residual tumor). In addition, the RNA sequences of tumors and the analysis of differences in their expression may reveal differences in background biological characteristics.
This study aimed to clarify the clinical outcome of resected EGFR-mutant NSCLCs with classical EGFR mutations (L858R and 19 deletion) and reveal the biological difference between tumors with L858R and 19 deletion using an RNA expression analysis.

Patients
We used a cohort of 191 consecutive patients who underwent surgical resection of NSCLC in Aichi Cancer Center Hospital, Nagoya, Japan, from January 2006 to September 2021. The inclusion criteria of this cohort were patients with primary NSCLC harboring EGFR driver mutations (L858R or 19 deletion) who underwent curative resection and who subsequently experienced recurrence. We also used 61 surgically resected NSCLC specimens harboring EGFR driver mutations (L858R or 19 deletion) for an RNA sequencing analysis. This retrospective study was approved by the Institutional Review Board of Aichi Cancer Center (No: 2018-2-20). The study was conducted in accordance with the principles of the Declaration of Helsinki.

Conventional Gene Test
EGFR genes were analyzed as previously described [4].

RNA Sequence
For RNA sequencing (n = 61), an RNA sequence library was prepared using the NEBNext ® Ultra TM RNA Library Prep Kit for Illumina ® (New England Biolabs, Ipswich, MA, USA) in accordance with the manufacturer's protocols. The enriched libraries were sequenced as 150-bp paired-end reads using NovaSeq (Illumina, San Diego, CA, USA) at Veritas Genetics (Danvers, MA, USA). RNA-seq data were analyzed using the CLC Genomics Workbench 20.1 software program (Qiagen). Control sequencing data were obtained from the ENA database (https://www.ebi. ac.uk/ena/browser/home). A Gene Set Enrichment Analysis was performed at RaNA-seq (https://ranaseq.eu/).

Statistics
To evaluate the risk factors associated with the time to recurrence and the prognosis, a Cox proportional hazards regression model with a step-down procedure was used. The time to recurrence and survival curves were determined using the Kaplan-Meier method. A log-rank test was performed to evaluate differences between the survival curves. All statistical analyses were performed using the JMP 12 software program (SAS Institute, Cary, NC, USA).

Results
The clinicopathological findings of patients with EGFR-mutated NSCLC who received curative surgery and who developed the recurrent disease are listed in Table 1 Among patients with pathological stage I disease, the median time to recurrence of patients with the two types of EGFR mutations did not differ to a statistically significant extent (Fig. 1a, b); however, among those with stage II and III disease, the median time to recurrence in patients with the p.L858R genotype tended to be shorter in comparison to those with the 19 deletion (log-rank test, p = 0.47 and 0.46, respectively) (Fig. 1c).
The results of clustering according to the EGFR genotypes are shown in Figure 2a. A volcano plot of the gene expression according to EGFR genotypes is shown in Figure 2b. The difference in the gene expression between tumors with L858R and 19 deletion is shown in Figure 3. The gene expression of amphiregulin (AREG) was higher in L858R tumors than in 19 deletion tumors. On the other hand, the expression of neuronal pentraxin 1 (NPTX1), proline-rich 4 (PRR4), and adenylate cyclase 1 (ADCY1) in L858R tumors was lower than those  in 19 deletion tumors. The expression of HLA-C, HLA-DRA, and HLA-DPB1, which may reflect the immune environment around the tumor, was higher in 19 deletion tumors (Table 3).
GSEA was carried out to explore the biological differentiation between L858R and 19 deletion tumors. The pathways and gene ontology (GO) were analyzed in our study. Pathway enrichment demonstrated that L858R tumors were mainly involved in the cell cycle and cell cycle checkpoint pathways (Fig. 4a). GO enrichment demonstrated that L858R tumors were involved in cell cycle activity and synapse assembly (Fig. 4b).

Discussion
Among early-stage resected EGFR-mutant NSCLCs, the time to recurrence in patients with L858R tumors tended to be shorter than those with 19 deletion tumors. However, we could not draw a clear conclusion because of the small number of cases that did not receive adjuvant chemotherapy to exclude its influence on this cohort (Fig. 1d-f). In comparison to 19 deletion tumors, L858R tumors had higher cytological malignancy, which refers to the overall nature of the tumor such as metastasis, proliferation, and invasive potential, (e.g., mitotic ability), and showed stronger immunogenicity, which may cause the short recurrence-free time in early-stage NSCLCs.
Among patients with completely resected NSCLC, patients with 19 deletion tumors had better recurrencefree survival than those with L858R tumors [3]. Diseasefree survival in patients with pN1-pN2 19 deletion tumors was better than in those with pN1-pN2 L858R tumors [3]. Another study reported that 19 deletion tumors were associated with better disease-free survival in comparison to L858R tumors among stage III lung cancer patients who received lung resection [5]. In terms of advanced stage, EGFR-TKIs are reported to be more beneficial for patients with 19 deletion tumors than those with L858R tumors [6]. These previously reported data are compatible with our results, and a gene expression analysis of our data may explain the background of these phenomena.
The GSEA demonstrated that L858R tumors have high AREG, which is one of the ligands of EGFR, expression levels, and it is possible that the growth signal is activated in a ligand-dependent manner, which may explain why EGFR-TKIs are less effective for L858R tumors [6].
Other genes that are more highly expressed in L858R are described below. GO annotations related to myosinbinding protein H (MYBPH) include structural constituents of muscle. Among BRAF V600E-positive papillary thyroid carcinoma cases with brain metastasis, MYB-PH was confirmed by a microarray analysis as one of the proteins that was elevated [7]. GO annotations related to solute carrier family 8 member A2 (SLC8A2) include calmodulin binding and calcium:sodium antiporter activity. In the tumor environment of advanced colorectal cancer, SCL28A2 has been identified as one of the proteins that increase Th17 cells and cause immunosuppression. SCL28A2 has been identified as one of these proteins [8]. Villin1 (VIL1) represents a dominant part of the brush border cytoskeleton, which functions in the capping, severing, and bundling of actin filaments. In advanced colorectal cancer, lymph node metastasis and elevated VIL1 have been reported based on the results of an mRNA expression analysis by RT-PCR [9].
In the environment around the 19 deletion tumor, the expression of MHC class 1 and 2 is increased, and the acquired immune activity of both class 1 and 2 may be high, which may reflect the high immunogenicity. In other words, this immunogenicity may be due to the efficacy in suppressing recurrence. It was also reported that more myeloid dendritic cells were present in patients with EGFR 19 deletion than in those with L858R mutation, which suggests the TIME of EGFR L858R NSCLC was more immunosuppressive [6].
Other genes that are more highly expressed in 19 deletion tumors are as follows. ADCY2 is insensitive to Ca (2+)/calmodulin and is stimulated by the G protein beta and gamma subunit complex. It has been reported that ADCY was decreased in patients with distant metastasis of rectal cancer in comparison to those without distant metastasis [10]. PRR4 may have a protective function in the eye. It has been reported that suppression of circulating RNA PRH1-PRR4 in NSCLC cell lines increased cell malignancy and promoted apoptosis [11]. NPTX1 is a member of the neuronal pentraxin gene family. It has been reported that NPTX1 is decreased in a hepatocellular carcinoma cell line and that blocking AKT1 increases NPTX1 and suppresses cell growth [12]. Immediate early response 3 (IER3) functions in the protection of cells from Fas-induced or tumor necrosis factor type alpha-induced apoptosis. It has been reported that the knockout of IER3 in a tongue cancer cell line reduced the level of VEGF-C and decreased the invasive capacity [13].
On the other hand, in the function enrichment analysis, it was suggested that L858R tumors have MHC class 2 (antigen processing pathway is elevated) and that interferon gamma-dependent immune activity is increased, so that there is immune tolerance in the surrounding environment. It is suggested that the effect of immune checkpoint inhibitors, especially CTLA4 inhibitor, which is administered based on its effect on dendritic cells, may be effective for L858R tumors [14].
In addition, GSEA showed that the cell cycle was more active in L858R, suggesting the activation of RhoGT-Pase by formin and the activation of neurotransmitters in the tumor, and the biological grade may be higher in L858R [5], although these events need to be confirmed using in vitro experiments. The formin family and Daams are expressed in tissues known to require Wnt and are consistent with Daams being effectors of Wnt signaling during vertebrate development [15]. The ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion [16]. Particularly under chronic stress, the continuous release of neurotransmitters from the neuroendocrine system can have a highly profound impact on the occurrence and prognosis of breast cancer [17].
In high pathological stages, L858R tumors may benefit from combination treatment with EGFR-TKIs, such as anti-CTLA, or in combination with an anti-EGFR antibody. In addition, regarding postoperative EGFR-TKI treatment, it may be better to treat L858R tumors more aggressively. Based on the increased activity of EGFR ligands, the therapeutic effect may be improved by devising targeted therapy, such as anti-EGFR antibody treatment. The results of the GE analysis also suggest that 19 deletion tumors may benefit from combined anti-VEGF therapy.