As a disease with a high mortality rate, colorectal cancer (CRC) highlights the importance of comprehending its molecular mechanisms to develop effective therapies. In this study, we conducted RNA sequencing (RNA-seq) analysis on 476 samples consisting of fresh-frozen CRC tissues and adjacent normal tissues obtained from Samsung Medical Center (SMC) in South Korea. By analyzing gene expression differences, we identified that upregulated genes in tumor samples were significantly associated with pathways related to the cell cycle, extracellular matrix (ECM)-receptor interaction, and DNA damage response (DDR). Conversely, genes exhibiting high expression in normal samples were primarily related to metabolic pathways. Tumor samples were found to be enriched in various DDR pathways, prompting an investigation into the enrichment of related genes. Additionally, the analysis focused on the Homologous recombination (HR) pathway, for which the Homologous recombination deficiency (HRD) score was calculated. Furthermore, we categorized colorectal tumor samples into four subgroups using in silico methods to identify the biological and clinical characteristics of each subgroup and compared our results with those of the widely used existing subgrouping methods. Majority of our fibroblast and immune enriched/fibroblast samples aligned with CMS4, characterized by stromal invasion and mesenchymal differentiation, whereas the immune enriched subtype aligned with CMS1, known for its immune-centric profile. To investigate the clinical implications, the composition of the tumor microenvironment (TME) by cell type was examined across four subgroups, along with an immunological analysis. In particular, the calculated HRD score and the expression levels of immune-related genes associated with CRC were analyzed to provide evidence for personalized therapeutic strategies for each subgroup. Our findings provide valuable insights into the molecular mechanisms underlying CRC in Korean patients, particularly in the SMC patient group, and offer opportunities for personalized therapeutic strategies. [BMB Reports 2026; 59(6): 321-328]

Background & Aims:To enhance our understanding of the tumor immune microenvironment (TIME) in hepatocellular carcinoma (HCC), we investigated the heterogeneity of T-cell exhaustion and its association with HBV integrations and direct oncogenic potential in HCC. Methods:We conducted a multi-omics analysis, including single-cell RNA sequencing, whole-exome sequencing, whole-transcriptome sequencing, and next-generation sequencing (NGS)-based HBV integration analysis, in eight patients with virus-related HCC. For validation, bulk RNA sequencing and NGS-based HBV integration analysis were performed in an independent cohort (n = 106). Results:Based on the expression scores of exhaustion markers in effector CD8+ T cells, patients were classified into high (n = 2) and low (n = 6) exhaustion groups (p <0.001). The high-exhaustion group exhibited higher clonal expansion (Gini index: 0.83 vs. 0.48, p = 0.006) and sharing of CD8+ T effector memory and cycling T cells with elevated exhaustion markers. This group also showed increased clonal expansion of CD4+ regulatory T cells and follicular helper T cells (p <0.001) with higher PDCD1 expression. In addition, the high-exhaustion group had higher TP53 mutation rates and signature scores for proliferation subtypes compared with the low-exhaustion group, who predominantly harbored TERT mutations. Moreover, the high-exhaustion group demonstrated more pronounced HBV integrations with elevated intrahepatic covalently closed circular DNA (cccDNA) and pregenomic (pg)RNA levels. Similarly, in the validation cohort, the high-exhaustion group (n = 28) demonstrated stronger proliferation subtype signatures (p <0.001), along with higher HBV integrations, S-fusion transcripts, and an increased intrahepatic viral reservoir (cccDNA/pgRNA) (p <0.05) compared with the low-exhaustion group (n = 78). Conclusions:Our study revealed the heterogeneity in T-cell exhaustion in the TIME of HCC, along with differences in HBV integrations and molecular subtypes. These findings provide insight into the intricate relationship between high exhaustion, proliferation subtype, increased HBV integrations, and enhanced HBV-induced oncogenic potential in virus-related HCC. Impact and implications:This study provides a comprehensive immune landscape of T-cell exhaustion using multi-omics analysis, offering critical insights into T cell heterogeneity in virus-related HCC. It establishes a strong association between higher HBV integration, enhanced oncogenic potential, T-cell exhaustion, and proliferation subtypes in HCC. Our results also establish a basis for personalized therapies tailored to the immune-exhaustion status within the TIME of each patient with HCC.

BACKGROUND:APOBEC plays a crucial role in the mutation process and immune response of non-small cell lung cancer (NSCLC). This study evaluated the role of the APOBEC mutation signature in early-stage epidermal growth factor receptor (EGFR) mutant NSCLC. This study aimed to assess the impact of APOBEC enrichment on recurrence-free survival (RFS), post-recurrence tyrosine kinase inhibitor (TKI) progression-free survival (PFS), and post-recurrence survival (PRS). METHODS:We conducted whole-exome sequencing and whole-transcriptome sequencing in 100 patients diagnosed with pathologic stage II-IIIA non-squamous NSCLC. RESULTS:Among the 100 patients, 18 (18 %) exhibited APOBEC (≥2) enrichment, which did not show a significant association with RFS (P = 0.14). Patients with APOBEC enrichment showed a shorter post-recurrence TKI PFS compared to those without APOBEC enrichment (8.13 months vs. 24.57 months, P = 0.017). In multivariate analysis, poor post-recurrence TKI PFS was associated with the type of EGFR mutation (L858R vs. exon 19 deletion, HR = 1.91, P = 0.04), TP53 mutation (HR = 2.07, P = 0.03), and APOBEC enrichment (≥2 vs. < 2, HR = 2.23, P = 0.02). PRS was 61.10 months and 21.77 months for patients with APOBEC enrichment and patients without APOBEC enrichment, respectively (P = 0.029). In multivariate analysis, poor PRS of EGFR-TKI was associated with the type of EGFR mutation (L858R vs. exon 19 deletion, HR = 2.29, P = 0.04) and APOBEC enrichment (≥2 vs. < 2, HR = 2.71, P = 0.02). CONCLUSIONS:APOBEC enrichment may present at initial diagnosis of early-stage EGFR mutant NSCLC and is associated with poor post-recurrence TKI PFS and PRS rather than RFS.

Early-stage lung adenocarcinomas (LUADs) manifesting as part-solid radiological features have a slowly progressive course to invasive malignancy, but its genomic alterations and tumor microenvironment are poorly understood. We analyzed eleven part-solid type LUADs from female never-smokers. Whole-exome sequencing and whole-transcriptome analysis were conducted on samples from seven patients. Single-cell RNA sequencing was performed on samples from four patients, and spatial transcriptomics was conducted on a sample from one patient. Both the ground-glass and solid components of LUADs exhibited similar characteristics of malignant cells, sharing mutations and showing no significant differences in tumor mutation burden. Compared to the GG component, the solid component showed up-regulation of epithelial-mesenchymal transition (EMT)-related and hypoxia pathways. We identified a cancer-associated secretory (CAS) cell cluster characterized by the expression of the cancer-related gene CEACAM6. CAS cells showed an increasing trend from normal to tumor regions. Interleukin (IL)-1β+ macrophages, a distinct subtype characterized by a pro-inflammatory M1-dominant gene signature, were abundant in solid components. Spatial transcriptomic analysis revealed that IL-1β+ macrophages and CAS cells were co-localized in specific invasive regions of the solid components, which had elevated EMT and hypoxia signatures. We identified that CAS cells and IL-1β+ macrophages are crucial during the progression from the pre-invasive (GG) to invasive adenocarcinoma (solid component). Targeting CAS cells and IL-1β+ macrophages could be preventive or therapeutic strategies for early LUADs. Junsu Choe, Noeul Kang, Jinyeong Lim, Eun Seop Seo, Mina Hwang, Se-Hoon Lee, Myung-Ju Ahn, Yoon-La Choi, Hong Kwan Kim, Woong-Yang Park, Sang-Won Um. Multiomics analysis of lung adenocarcinoma manifesting as radiological part-solid features in female never-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7174.

Germline genetic context may play a significant role in the development and evolution of cancer, particularly in childhood cancers such as neuroblastoma. We studied the role of putatively functional germline variants (pFGVs) in neuroblastoma, even if they do not directly increase disease risk. Our whole-exome sequencing analysis of 125 patients with neuroblastoma revealed a positive correlation between pFGV burden and somatic mutations. Moreover, patients with higher pFGV burdens exhibited worse outcomes. Similar findings were observed in the separate neuroblastoma cohort. However, contrasting results emerged in adult-onset cancer, emphasizing the importance of germline genetics in neuroblastoma. The enrichment of pFGVs in cancer predisposition genes was evident in neuroblastoma compared to that in healthy and adult-onset cancer populations, and their presence had prognostic significance in neuroblastoma. The combination of germline and clinical risk factors improves survival predictions. Our study highlights the importance of germline variants and their potential implications in pediatric cancer.

We comparatively evaluated the antioxidant properties of key triterpenes from Centella asiatica, including asiatic acid (AA), asiaticoside, madecassic acid, and madecassoside, in several cell types, including skin fibroblasts, macrophages, hepatocytes, and endothelial cells, under conditions promoting oxidative stress. AA conferred the highest viability on Hs68 cells exposed to ultraviolet B (UVB) irradiation. Triterpene pretreatment attenuated the UVB-induced generation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the UVB-induced depletion of glutathione (GSH) in skin fibroblasts. AA most potently inhibited UVB-induced MMP generation, resulting in increased intracellular collagen levels. Pretreatment with triterpenes, particularly AA, significantly improved cell viability and attenuated TBHP-induced levels of ROS, alanine aminotransferase, and aspartate aminotransferase in HepG2 cells. Triterpenes attenuated ROS levels and reduced MDA and GSH expression in EA.hy926 cells. In RAW264.7 macrophages, production of nitric oxide, tumor necrosis factor-α, and interleukin-6 (indicators of LPS-induced oxidative damage) was significantly reduced by treatment with any of the triterpenes. Statistical analyses of triterpene biological activities using principal component analysis and hierarchical clustering revealed that AA exerted the greatest overall influence and showed remarkable activity in Hs68 and HepG2 cells.

Abstract We aimed to predict postoperative prognosis of stage II/III colorectal cancer (CRC). Tumor-like normal microenvironment (tNME) and healthy normal microenvironment (hNME) groups were created using RNA sequencing (RNA-seq) data derived from 273 tumors and 273 paired normal tissues from margins of resected specimens. With a median follow-up of 58.2 months, the tNME group showed poor 5-year recurrence-free survival (54.7 vs. 73.0%, HR = 1.94, P = 0.002) and 5-year overall survival (78.2 vs. 83.0%, HR = 1.76, P = 0.033). Only the tNME group showed similar microbiome diversity between tumor and normal tissues. scRNA-seq revealed a positive co-occurrence of Th17, GZMKhighCD8+T cells, and IL1Bhigh neutrophils in the tumor and normal tissues of tNME. A high proportion of EMP1high epithelial cells was observed in the tNME group, with upregulated epithelial-mesenchymal transition and leukocyte signaling pathways. In conclusion, the microenvironmental status of normal tissues offers a promising biomarker for stage II/III CRC.

Aim. We investigated the efficacy and safety of durvalumab (D) with or without tremelimumab (T) in addition to single -agent chemotherapy (CT) in patients with platinum -resistant recurrent ovarian cancer (PROC) lacking homologous recombination repair (HRR) gene mutations. Patients and methods. KGOG 3045 was an open -label, investigator -initiated phase II umbrella trial. Patients with PROC without HRR gene mutations who had received >= 2 prior lines of therapy were enrolled. Patients with high PD -L1 expression (TPS >= 25%) were assigned to arm A (D + CT), whereas those with low PD -L1 expression were assigned to arm B (D + T75 + CT). After completing arm B recruitment, patients were sequentially assigned to arms C (D + T300 + CT) and D (D + CT). Results. Overall, 58 patients were enrolled (5,18, 17, and 18 patients in arms A, B, C, and D, respectively). The objective response rates were 20.0, 33.3, 29.4, and 22.2%, respectively. Grade 3-4 treatment -related adverse events were observed in 20.0, 66.7, 47.1, and 66.7 of patients, respectively, but were effectively managed. Multivariable analysis demonstrated that adding T to D + CT improved progression -free survival (adjusted HR, 0.435; 95% CI, 0.229-0.824; P = 0.011). Favorable response to chemoimmunotherapy was associated with MUC16 mutation (P = 0.0214), high EPCAM expression (P = 0.020), high matrix remodeling gene signature score (P = 0.017), and low FOXP3 expression (P = 0.047). Patients showing favorable responses to D + T + CT exhibited significantly higher EPCAM expression levels (P = 0.008) and matrix remodeling gene signature scores (P = 0.031) than those receiving D + CT. Conclusions. Dual immunotherapy with chemotherapy showed acceptable response rates and tolerable safety in HRR non -mutated PROC, warranting continued clinical investigation. (c) 2024 Elsevier Inc. All rights reserved.

3564 Background: Approximately 2-4% of patients with metastatic colorectal cancer (mCRC) harbor HER2 amplification, a subset notorious for poor response rates following conventional treatments. The TRIUMPH trial introduces anti-HER2 therapy, combining trastuzumab and pertuzumab. Despite its potential, the observed response rate stands at 30%, accompanied by an early recurrence within 3.5 months with acquired resistance, which emphasizes the need for further investigations into resistance mechanisms within this patient population. Here, we delineate the dynamic changes in both the tumor and its microenvironment in response to treatment and mechanisms of acquired resistance using spatial transcriptomics. Methods: Within the TRIUMPH trial, a total of 20 formalin-fixed paraffin-embedded (FFPE) biopsy slides from 13 patients were collected before and after treatment or at one time. Utilizing these FFPE slides, we employed imaging-based single-cell spatial transcriptomics, using a custom panel tailored for the tumor and its microenvironment. This panel includes gene sets related to tumor growth, drug resistance, and immune response. Results: To comprehend the complexities of the tumor and its surroundings, we categorized tumor tissue into distinct sections, encompassing tumor, stromal, and immune-enriched areas based on their neighbor cells. Remarkably, we noted a substantial rise in the infiltration of T cells and myeloid cells into tumor-enriched regions post-treatment. T cells increased from a median of 1.5% to 2.2%, and myeloid cells from 4.3% to 7.4%, particularly in patients showing a response. Additionally, ERBB2 expression in tumor cells was high, whereas the non-response group exhibited lower expression levels. Prior to treatment, fibroblast and endothelial cells were intricately intertwined with tumor cells. However, subsequent to treatment, these cells segregated into stromal regions, exhibiting an increase in their proportions. Notably, angiogenic tumor-associated macrophage (angio-TAM), expressing SPP1 and VEGFA, were localized at the interface of tumors and stromal cells and proliferated following disease progression. This spatial reorganization signifies dynamic alterations within the tumor microenvironment following treatment. Conclusions: Our study on HER2-amplified metastatic colorectal cancer in the TRIUMPH trial uncovers intriguing spatial changes post anti-HER2 therapy. Notably, dynamic reorganization of fibroblast and endothelial cells into stromal regions suggest complex treatment responses. Identification of angio-TAM expressing SPP1 and VEGFA adds depth to understanding the contribution of tumor microenvironment to the resistance mechanism. These findings highlight the intricate tumor microenvironment shifts, urging further exploration for optimized therapeutic strategies.

Abstract Background: The APOBEC family of zinc-coordinating enzymes converts cytosines to uracils in single-strand DNA. APOBEC plays a crucial role in the mutation process of non-small cell lung cancer (NSCLC) and is essential for both adaptive and innate immune responses. This study aims to evaluate the role of the APOBEC mutation signature in early-stage non-small cell lung cancer (NSCLC). Methods: We conducted whole-exome sequencing and whole-transcriptome sequencing using fresh tissue or formalin-fixed paraffin-embedded samples from 100 patients diagnosed with pathologic stage II-IIIA non-squamous NSCLC, all of whom underwent complete resection between January 2014 and December 2020 at Samsung Medical Center. This study aimed to assess the impact of APOBEC enrichment on disease-free survival (DFS), progression-free survival (PFS) of EGFR-TKI, and overall survival following recurrence. Results: Median follow-up duration was 58.2 months (range, 11.3-141.1). Of the patients, 74% were never-smokers, and 52% had stage III disease. Among the 100 patients, 18 (18%) exhibited APOBEC enrichment (≥ 2). The median RFS was 25.2 months (95% CI, 17.3-33.2). Notably, APOBEC enrichment did not show a significant association with DFS (P=0.14). Among the 76 patients who experienced radiological recurrence, 69 received EGFR-TKI as first-line treatment. The median PFS for EGFR-TKI was 22.9 months (95% CI, 13.0-32.8). However, within this group, patients with APOBEC enrichment showed a shorter PFS compared to those without APOBEC enrichment (8.1 months vs. 24.6 months, P=0.014). In multivariate analysis, poor PFS of EGFR-TKI was associated with the type of EGFR mutation (L858R vs. exon 19 deletion, HR=1.9, P=0.04), TP53 mutation (mutation vs. wild type, HR=2.1, P=0.03), and APOBEC enrichment (≥ 2 vs. <2, HR=2.2, P=0.02). Overall survival from the starting EGFR-TKI was 61.1 months and 21.7 months for patients with APOBEC enrichment or patients without APOBEC enrichment, respectively (P=0.027). Conclusions: APOBEC mutation signature may be presented at initial diagnosis of early-stage EGFR mutant NSCLC and it was associated with poor PFS of EGFR-TKI and overall survival. Citation Format: Hyun Ae Jung, Jinyeong Lim, Yoon-La Choi, Sehhoon Park, Jong-Mu Sun, Myung-Ju Ahn, Se-Hoon Lee. The role of APOBEC in early stage-EGFR mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2503.

Manganese porphyrins reportedly exhibit synergic effects when combined with irradiation. However, an in-depth understanding of intratumoral heterogeneity and immune pathways, as affected by Mn porphyrins, remains limited. Here, we explored the mechanisms underlying immunomodulation of a clinical candidate, MnTnBuOE-2-PyP5+ (BMX-001, MnBuOE), using single-cell analysis in a murine carcinoma model. Mice bearing 4T1 tumors were divided into four groups: control, MnBuOE, radiotherapy (RT), and combined MnBuOE and radiotherapy (MnBuOE/RT). In epithelial cells, the epithelial–mesenchymal transition, TNF-α signaling via NF-кB, angiogenesis, and hypoxia-related genes were significantly downregulated in the MnBuOE/RT group compared with the RT group. All subtypes of cancer-associated fibroblasts (CAFs) were clearly reduced in MnBuOE and MnBuOE/RT. Inhibitory receptor–ligand interactions, in which epithelial cells and CAFs interacted with CD8+ T cells, were significantly lower in the MnBuOE/RT group than in the RT group. Trajectory analysis showed that dendritic cells maturation-associated markers were increased in MnBuOE/RT. M1 macrophages were significantly increased in the MnBuOE/RT group compared with the RT group, whereas myeloid-derived suppressor cells were decreased. CellChat analysis showed that the number of cell–cell communications was the lowest in the MnBuOE/RT group. Our study is the first to provide evidence for the combined radiotherapy with a novel Mn porphyrin clinical candidate, BMX-001, from the perspective of each cell type within the tumor microenvironment.

Alterations in DNA methylation play an important pathophysiological role in the development and progression of colorectal cancer. We comprehensively profiled DNA methylation alterations in 165 Korean patients with colorectal cancer (CRC), and conducted an in-depth investigation of cancer-specific methylation patterns. Our analysis of the tumor samples revealed a significant presence of hypomethylated probes, primarily within the gene body regions; few hypermethylated sites were observed, which were mostly enriched in promoter-like and CpG island regions. The CpG Island Methylator PhenotypeHigh (CIMP-H) exhibited notable enrichment of microsatellite instability-high (MSI-H). Additionally, our findings indicated a significant correlation between methylation of the MLH1 gene and MSI-H status. Furthermore, we found that the CIMP-H had a higher tendency to affect the right-side of the colon tissues and was slightly more prevalent among older patients. Through our methylome profile analysis, we successfully verified the methylation patterns and clinical characteristics of Korean patients with CRC. This valuable dataset lays a strong foundation for exploring novel molecular insights and potential therapeutic targets for the treatment of CRC. [BMB Reports 2024; 57(2): 110-115]

Abnormalities in glucose metabolism that precede the onset of type 2 diabetes (T2D) activate immune cells, leading to elevated inflammatory factors and chronic inflammation. However, no single-cell RNA sequencing (scRNA-seq) studies have characterized the properties and networks of individual immune cells in T2D. Here, we analyzed peripheral blood mononuclear cells (PBMCs) from non-diabetes and T2D patients by scRNA-seq. We found that CD14 monocytes in T2D patients were in a pro-inflammatory state and intermediate monocytes expressed more MHC class II genes. In T2D patients, cytotoxic CD4 T cells, effector memory CD8 T cells, and γδ T cells have increased cytotoxicity and clonal expansion. B cells were characterized by increased differentiation into intermediate B cells, plasma cells, and isotype class switching with increased expression of soluble antibody genes. These results suggest that monocytes, T cells, and B cells could interact to induce chronic inflammation in T2D patients with pro-inflammatory characteristics.

Lack of diversity and proportionate representation in genomics datasets and databases contributes to inequity in healthcare outcomes globally[1][1],[2][2]. The relationships of human diversity with biological and biomedical phenotypes are pervasive[3][3], yet remain understudied, particularly in a single-cell genomics context. Here we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA-sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 healthy donors and 6 controls, spanning 7 population groups across 5 countries. AIDA is one of the largest healthy blood datasets in terms of number of cells, and also the most diverse in terms of number of population groups. Though population groups are frequently compared at the continental level, we identified a pervasive impact of sub-continental diversity on cellular and molecular properties of immune cells. These included cell populations and genes implicated in disease risk and pathogenesis as well as those relevant for diagnostics. We detected single-cell signatures of human diversity not apparent at the level of cell types, as well as modulation of the effects of age and sex by self-reported ethnicity. We discovered functional genetic variants influencing cell type-specific gene expression, including context-dependent effects, which were under-represented in analyses of non-Asian population groups, and which helped contextualise disease-associated variants. We validated our findings using multiple independent datasets and cohorts. AIDA provides fundamental insights into the relationships of human diversity with immune cell phenotypes, enables analyses of multi-ancestry disease datasets, and facilitates the development of precision medicine efforts in Asia and beyond. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

PurposeBiliary tract cancers (BTCs) are rare and show a dismal prognosis with limited treatment options. To improve our understanding of these heterogeneous tumors and develop effective therapeutic agents, suitable preclinical models reflecting diverse tumor characteristics are needed. We established and characterized new patient-derived cancer cell cultures and patient-derived xenograft (PDX) models using malignant ascites from five patients with BTC.Materials and MethodsFive patient-derived cancer cell cultures and three PDX models derived from malignant ascites of five patients with BTC, AMCBTC-01, -02, -03, -04, and -05, were established. To characterize the models histogenetically and confirm whether characteristics of the primary tumor were maintained, targeted sequencing and histopathological comparison between primary tissue and xenograft tumors were performed.ResultsFrom malignant ascites of five BTC patients, five patient-derived cancer cell cultures (100% success rate), and three PDXs (60% success rate) were established. The morphological characteristics of three primary xenograft tumors were compared with those of matched primary tumors, and they displayed a similar morphology. The mutated genes in samples (models, primary tumor tissue, or both) from more than one patient were TP53 (n=2), KRAS (n=2), and STK11 (n=2). Overall, the pattern of commonly mutated genes in BTC cell cultures was different from that in commercially available BTC cell lines.ConclusionWe successfully established the patient-derived cancer cell cultures and xenograft models derived from malignant ascites in BTC patients. These models accompanied by different genetic characteristics from commercially available models will help better understand BTC biology.

e20571 Background: In a previous pilot study, we classified early-stage EGFR-mutant NSCLC according to two transcriptomic subtypes (i.e., terminal respiratory unit [TRU], which was related with the downregulation of a proliferative-related pathway; and non-TRU, which was related with the activation of a proliferative-related pathway) and the presence of a co-existing TP53 mutation. In turn, the clinical significance of the APOBEC mutation signature has not been elucidated clearly owing to the small sample size of the previous pilot study. Thus, this study aimed to validate the predictive value of transcriptomic subtype, TP53 mutation status, and APOBEC mutation signature, to expand our pilot study via the use of additional samples and a long-term follow-up. Methods: We performed whole-exome sequencing and whole-transcriptome sequencing using fresh tissue or FFPE from 101 patients with pathological stage II–IIIA non-squamous EGFR-mutant NSCLC who underwent complete resection between January 2014 and December 2020 at the Samsung Medical Center. This study evaluated the recurrence-free survival (RFS) and progression-free survival (PFS) afforded by EGFR-TKI therapy after recurrence according to transcriptomic subtype, TP53 mutation status, and APOBEC mutation signature. Results: The median duration of the follow-up period was 54.2 months (range, 11.3–107.7 months). Six patients were excluded because of failure of the initial quality control; thus, 95 patients were included in the final analysis. The median age was 61.9 years (range, 44.7–91.0 years), and 75.8% of the patients were never-smokers. Moreover, 51.6% of the patients had stage III disease. Among the 95 patients, 47 (49.5%) patients were in the non-TRU group and 44 (46.3%) patients carried a TP53 mutation. The median RFS was 25.8 months (95% CI, 17.6–34.0 months). Patients with non-TRU and/or TP53 mutation had an inferior RFS compared with patients with TRU and wild-type TP53, regardless of the pathological stage (hazard ratio, 1.9; 95% CI, 1.4–2.7). The APOBEC mutation signature was not associated with RFS ( P = 0.17). Among the 65 patients who experienced radiological recurrence, 59 patients received EGFR-TKI therapy as the first-line treatment. The median PFS of EGFR-TKI therapy was 20.7 months (95% CI, 13.6–26.6 months). However, among these patients, those with the APOBEC mutation signature had a shorter PFS than those without this mutation signature (8.2 vs. 23.5 months, P < 0.05). Conclusions: This study showed consistently that the transcriptomic subtype and TP53 mutation status were predictive factors of RFS, regardless of the pathological stage, in patients with early-stage EGFR-mutant NSCLC. Additionally, the APOBEC mutation signature may be present at the initial diagnosis of early-stage EGFR-mutant NSCLC and was associated with a poor PFS after EGFR-TKI therapy.