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Abstract Background The cancer genome contains several driver mutations. However, in some cases, no known drivers have been identified; these remaining areas of unmet needs, leading to limited progress in cancer therapy. Whole-genome sequencing (WGS) can identify non-coding alterations associated with the disease. Consequently, exploration of non-coding regions using WGS and other omics data such as ChIP-sequencing (ChIP-seq) to discern novel alterations and mechanisms related to tumorigenesis have been attractive these days. Methods Integrated multi-omics analyses, including WGS, ChIP-seq, DNA methylation, and RNA-sequencing (RNA-seq), were conducted on samples from patients with non-clinically actionable genetic alterations (non-CAGAs) in lung adenocarcinoma (LUAD). Second-level cluster analysis was performed to reinforce the correlations associated with patient survival, as identified by RNA-seq. Subsequent differential gene expression analysis was performed to identify potential druggable targets. Results Differences in H3K27ac marks in non-CAGAs LUAD were found and confirmed by analyzing RNA-seq data, in which mastermind-like transcriptional coactivator 2 (MAML2) was suppressed. The down-regulated genes whose expression was correlated to MAML2 expression were associated with patient prognosis. WGS analysis revealed somatic mutations associated with the H3K27ac marks in the MAML2 region and high levels of DNA methylation in MAML2 were observed in tumor samples. The second-level cluster analysis enabled patient stratification and subsequent analyses identified potential therapeutic target genes and treatment options. Conclusions We overcome the persistent challenges of identifying alterations or driver mutations in coding regions related to tumorigenesis through a novel approach combining multi-omics data with clinical information to reveal the molecular mechanisms underlying non-CAGAs LUAD, stratify patients to improve patient prognosis, and identify potential therapeutic targets. This approach may be applicable to studies of other cancers with unmet needs.

Abstract Background In an extensive genomic analysis of lung adenocarcinomas (LUADs), driver mutations have been recognized as potential targets for molecular therapy. However, there remain cases where target genes are not identified. Super-enhancers and structural variants are frequently identified in several hundred loci per case. Despite this, most cancer research has approached the analysis of these data sets separately, without merging and comparing the data, and there are no examples of integrated analysis in LUAD. Methods We performed an integrated analysis of super-enhancers and structural variants in a cohort of 174 LUAD cases that lacked clinically actionable genetic alterations. To achieve this, we conducted both WGS and H3K27Ac ChIP-seq analyses using samples with driver gene mutations and those without, allowing for a comprehensive investigation of the potential roles of super-enhancer in LUAD cases. Results We demonstrate that most genes situated in these overlapped regions were associated with known and previously unknown driver genes and aberrant expression resulting from the formation of super-enhancers accompanied by genomic structural abnormalities. Hi-C and long-read sequencing data further corroborated this insight. When we employed CRISPR-Cas9 to induce structural abnormalities that mimicked cases with outlier ERBB2 gene expression, we observed an elevation in ERBB2 expression. These abnormalities are associated with a higher risk of recurrence after surgery, irrespective of the presence or absence of driver mutations. Conclusions Our findings suggest that aberrant gene expression linked to structural polymorphisms can significantly impact personalized cancer treatment by facilitating the identification of driver mutations and prognostic factors, contributing to a more comprehensive understanding of LUAD pathogenesis.

Abstract The incidence of vulvar carcinoma varies by race; however, it is a rare disease, and its genomic profiles remain largely unknown. This study examined the characteristics of vulvar squamous cell carcinoma (VSCC) in Japanese patients, focusing on genomic profiles and potential racial disparities. The study included two Japanese groups: the National Cancer Center Hospital (NCCH) group comprised 19 patients diagnosed between 2015 and 2023, and the Center for Cancer Genomics and Advanced Therapeutics group comprised 29 patients diagnosed between 2019 and 2022. Somatic mutations were identified by targeted or panel sequencing, and TP53 was identified as the most common mutation (52–81%), followed by HRAS (7–26%), CDKN2A (21–24%), and PIK3CA (5–10%). The mutation frequencies, except for TP53, were similar to those of Caucasian cohorts. In the NCCH group, 16 patients of HPV-independent tumors were identified by immunohistochemistry and genotyping. Univariate analysis revealed that TP53-mutated patients were associated with a poor prognosis (log-rank test, P = 0.089). Japanese VSCC mutations resembled those of Caucasian vulvar carcinomas, and TP53 mutations predicted prognosis regardless of ethnicity. The present findings suggest potential molecular-targeted therapies for select VSCC patients.

Abstract DNA methylation is an epigenetic modification that results in dynamic changes during ontogenesis and cell differentiation. DNA methylation patterns regulate gene expression and have been widely researched. While tools for DNA methylation analysis have been developed, most of them have focused on intergroup comparative analysis within a dataset; therefore, it is difficult to conduct cross-dataset studies, such as rare disease studies or cross-institutional studies. This study describes a novel method for DNA methylation analysis, namely, methPLIER, which enables interdataset comparative analyses. methPLIER combines Pathway Level Information Extractor (PLIER), which is a non-negative matrix factorization (NMF) method, with regularization by a knowledge matrix and transfer learning. methPLIER can be used to perform intersample and interdataset comparative analysis based on latent feature matrices, which are obtained via matrix factorization of large-scale data, and factor-loading matrices, which are obtained through matrix factorization of the data to be analyzed. We used methPLIER to analyze a lung cancer dataset and confirmed that the data decomposition reflected sample characteristics for recurrence-free survival. Moreover, methPLIER can analyze data obtained via different preprocessing methods, thereby reducing distributional bias among datasets due to preprocessing. Furthermore, methPLIER can be employed for comparative analyses of methylation data obtained from different platforms, thereby reducing bias in data distribution due to platform differences. methPLIER is expected to facilitate cross-sectional DNA methylation data analysis and enhance DNA methylation data resources.

Abstract The mechanism underlying the development of tumors, particularly at early stages, still remains mostly elusive. Here, we report whole-genome long and short read sequencing analysis of 76 lung cancers, focusing on very early-stage lung adenocarcinomas such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma. The obtained data is further integrated with bulk and spatial transcriptomic data and epigenomic data. These analyses reveal key events in lung carcinogenesis. Minimal somatic mutations in pivotal driver mutations and essential proliferative factors are the only detectable somatic mutations in the very early-stage of AIS. These initial events are followed by copy number changes and global DNA hypomethylation. Particularly, drastic changes are initiated at the later AIS stage, i.e., in Noguchi type B tumors, wherein cancer cells are exposed to the surrounding microenvironment. This study sheds light on the pathogenesis of lung adenocarcinoma from integrated pathological and molecular viewpoints.

Abstract Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a subset of ALL that demonstrated a high treatment failure rate. One of the hallmarks of Ph-like ALL is PDGFRB gene fusion, with fusion partner proteins often harboring dimerization domains and enhancing the kinase activity of PDGFRB. We determined a novel oncogenic PDGFRB fusion gene, NRIP1::PDGFRB, from a pediatric patient with ALL, encoding a protein with the carboxy-terminal kinase domain of PDGFRB, without the partner peptide. We confirmed the oncogenic potential of NRIP1::PDGFRB in vitro and the efficacy of all ABL1-specific inhibitor generations, including imatinib, dasatinib, nilotinib, and ponatinib, in suppressing this potential. PDGFRB activation mechanism may include juxtamembrane domain truncation in the predicted peptide. In conclusion, we determined a novel fusion gene pattern in Ph-like ALL.

Lenvatinib, a multitarget tyrosine kinase inhibitor for c-Kit and other kinases, has exhibited promising efficacy in treating advanced or metastatic thymic carcinoma (TC). Here, we present the case of a patient with metastatic TC harboring a KIT exon 11 deletion and amplification. The patient exhibited a remarkable response to lenvatinib but experienced rapid disease progression after discontinuation of lenvatinib, referred to as a “disease flare.” This case report indicates that KIT mutations and amplification can predict lenvatinib response in patients with TC. However, in such cases, there might be a risk of disease flares after lenvatinib discontinuation.

Abstract Background Although cervical cancer is often characterized as preventable, its incidence continues to increase in low‐ and middle‐income countries, underscoring the need to develop novel therapeutics for this disease.This study assessed the distribution of fusion genes across cancer types and used an RNA‐based classification to divide cervical cancer patients with a poor prognosis into subgroups. Material and Methods RNA sequencing of 116 patients with cervical cancer was conducted. Fusion genes were extracted using StarFusion program. To identify a high‐risk group for recurrence, 65 patients who received postoperative adjuvant therapy were subjected to non‐negative matrix factorization to identify differentially expressed genes between recurrent and nonrecurrent groups. Results We identified three cases with FGFR3‐TACC3 and one with GOPC‐ROS1 fusion genes as potential targets. A search of publicly available data from cBioPortal (21,789 cases) and the Center for Cancer Genomics and Advanced Therapeutics (32,608 cases) showed that the FGFR3 fusion is present in 1.5% and 0.6% of patients with cervical cancer, respectively. The frequency of the FGFR3 fusion gene was higher in cervical cancer than in other cancers, regardless of ethnicity. Non‐negative matrix factorization identified that the patients were classified into four Basis groups. Pathway enrichment analysis identified more extracellular matrix kinetics dysregulation in Basis 3 and more immune system dysregulation in Basis 4 than in the good prognosis group. CIBERSORT analysis showed that the fraction of M1 macrophages was lower in the poor prognosis group than in the good prognosis group. Conclusions The distribution of FGFR fusion genes in patients with cervical cancer was determined by RNA‐based analysis and used to classify patients into clinically relevant subgroups.

Abstract Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRASG12V expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRASG12V-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRASG12V-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.

Abstract Liver cancer, particularly hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), is more common in Asians than in Caucasians. This is due, at least in part, to regional differences in the prevalence of exogenous factors such as HBV; however, endogenous factors specific to Asia might also play a role. Such endogenous factors include HLA (human leukocyte antigen) genes, which are considered candidates due to their high racial diversity. Here, we performed a pancancer association analysis of 147 alleles of HLA-class I/II genes (HLA-A, B, and C/DRB1, DQA1, DQB1, DPA1, and DPB1) in 31,727 cases of 12 cancer types, including 1684 liver cancer cases and 107,103 controls. HLA alleles comprising a haplotype prevalent in Asia were significantly associated with pancancer risk (e.g., odds ratio [OR] for a DRB1*15:02 allele = 1.12, P = 2.7 × 10–15), and the associations were particularly strong in HBV-related HCC (OR 1.95, P = 2.8 × 10–5). In silico prediction suggested that the DRB1*15:02 molecule encoded by the haplotype does not bind efficiently to HBV-derived peptides. RNA sequencing indicated that HBV-related HCC in carriers of the haplotype shows low infiltration by NK cells. These results indicate that the Asian-prevalent HLA haplotype increases the risk of HBV-related liver cancer risk by attenuating immune activity against HBV infection, and by reducing NK cell infiltration into the tumor.

Abstract Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (Pinteraction = 0.0058). These findings provide new insights into the etiology of lung adenocarcinoma in individuals from East Asian populations, which could be important in developing translational applications.

Abstract Anal squamous cell carcinoma (ASCC) is a rare tumor of the gastrointestinal tract. We aimed to compare the genetic backgrounds and their effect on clinical outcomes between Japanese and Caucasian patients with ASCC. Forty-one patients diagnosed with ASCC at the National Cancer Center Hospital were enrolled and evaluated for clinicopathological features, human papillomavirus (HPV) infection, HPV genotypes, p16 expression, PD-L1, and association of p16 status with the efficacy of concurrent chemoradiotherapy (CCRT). Target sequencing for hotspot mutations in 50 cancer-related genes was performed using genomic DNA from 30 available samples. Of 41 patients, 34 were HPV-positive (among them, HPV 16 was predominant; 73.2%); 38 patients were p16-positive (92.7%); and 39 patients received CCRT, of whom 36 were p16-positive and three p16-negative. p16-positive patients showed better complete response than p16-negative patients. Among 28 samples, 15 showed mutations in PIK3CA, FBXW7, ABL1, TP53, and PTEN; no difference in mutation profiles between the Japanese and Caucasian cohorts was observed. Actionable mutations were detected in both Japanese and Caucasian patients with ASCC. Genetic backgrounds, such as the HPV 16 genotype and PIK3CA mutations, were common regardless of ethnicity. p16 status may be a prognostic biomarker for CCRT in Japanese patients with ASCC.

Abstract Lymph node metastasis (LNM) is a well-established prognostic factor in endometrial cancer (EC). We aimed to construct a model that predicts LNM and prognosis using preoperative factors such as myometrial invasion (MI), enlarged lymph nodes (LNs), histological grade determined by endometrial biopsy, and serum cancer antigen 125 (CA125) level using two independent cohorts consisting of 254 EC patients. The area under the receiver operating characteristic curve (AUC) of the constructed model was 0.80 regardless of the machine learning techniques. Enlarged LNs and higher serum CA125 levels were more significant in patients with low-grade EC (LGEC) and LNM than in patients without LNM, whereas deep MI and higher CA125 levels were more significant in patients with high-grade EC (HGEC) and LNM than in patients without LNM. The predictive performance of LNM in the HGEC group was higher than that in the LGEC group (AUC = 0.84 and 0.75, respectively). Patients in the group without postoperative pathological LNM and positive LNM prediction had significantly worse relapse-free and overall survival than patients with negative LNM prediction (log-rank test, P

The nature of microRNA (miRNA) dysfunction in carcinogenesis remains controversial because of the complex connection between miRNA structural diversity and biological processes. Here, we found that oncofetal IGF2BP3 regulates the selective production of a subset of 3'-isoforms (3'-isomiRs), including miR-21-5p and Let-7 family, which induces significant changes in their cellular seed occupancy and structural components, establishing a cancer-specific gene expression profile. The D-score, reflecting dominant production of a representative miR-21-5p+C (a 3'-isomiR), discriminated between clinical early-stage lung adenocarcinoma (LUAD) cases with low and high recurrence risks, and was associated with molecular features of cell cycle progression, epithelial-mesenchymal transition pressure, and immune evasion. We found that IGF2BP3 controls the production of miR-21-5p+C by directing the nuclear Drosha complex to select the cleavage site. IGF2BP3 was also involved in the production of 3'-isomiRs of miR-425-5p and miR-454-3p. IGF2BP3-regulated these three miRNAs are suggested to be associated with the regulation of p53, TGF-β, and TNF pathways in LUAD. Knockdown of IGF2BP3 also induced a selective upregulation of Let-7 3'-isomiRs, leading to increased cellular Let-7 seed occupancy and broad repression of its target genes encoding cell cycle regulators. The D-score is an index that reflects this cellular situation. Our results suggest that the aberrant regulation of miRNA structural diversity is a critical component for controlling cellular networks, thus supporting the establishment of a malignant gene expression profile in early stage LUAD. [ABSTRACT FROM AUTHOR]

Abstract Targeted sequencing for somatic mutations across the hotspots of 50 cancer-related genes was performed using biopsy specimens to investigate whether clinicopathological factors and genomic alterations correlated with prognosis in locally advanced cervical cancer. Seventy patients diagnosed with International Federation of Obstetrics and Gynecology (FIGO) stage III to IVA cervical cancer underwent radiotherapy or concurrent chemoradiotherapy at the National Cancer Center Hospital between January 2008 and December 2017. Mutations were detected in 47 of 70 [67% of cases; frequency of genetic alterations was as follows: PIK3CA (51%), FBXW7 (10%), PTEN (7.1%), and TP53 (5.7%)]. The Cancer Genome Atlas (TCGA) datasets showed a similar distribution of somatic mutations, but PIK3CA mutation frequency was significantly higher in our cohort than in TCGA datasets (P = 0.028). Patients with TP53 mutation were significantly related to poor progression-free survival (PFS) (hazard ratio [HR] = 3.53, P = 0.042). Patients with tumor diameters > 70 mm were associated with poor prognosis (HR = 2.96, P = 0.0048). Patients with non-HPV16/18 genotypes had worse prognosis than those with HPV16/18 genotypes (HR = 2.15, P = 0.030). Hence, patients with locally advanced cervical cancer, TP53 mutation, large tumor diameter, and non-HPV16/18 genotype were independently correlated with poor PFS, despite concurrent chemoradiotherapy.

Abstract Somatic genetic alteration analysis was performed for post-hysterectomy high-risk early-stage uterine cervical cancer patients who underwent post-operative radiation therapy. Post-operative radiation therapy was performed for patients with pathological features of pelvic lymph node metastasis, parametrium invasion, or positive vaginal margin, which corresponded to the post-operative high-risk category. DNA was extracted from paraffin-embedded surgical specimens, and 50 somatic hotspot genetic alternations were detected using Ion AmpliSeq Cancer Hotspot Panel. The existence of actionable mutation was assessed based on OncoKB evidence level > 3A. Between January 2008 and November 2019, 89 patients who underwent abdominal radical hysterectomy followed by post-operative radiation therapy were identified. The follow-up period for living patients was 82.3 months (range 9.3–153.9), and the 5-year relapse-free survival and overall survival rates were 72.6% and 85.9%, respectively. The most frequently detected somatic mutation was PIK3CA (26 [29.2%] patients); however, no prognostic somatic genetic alterations were identified. Actionable mutations were detected in 30 (33.7%) patients. Actionable mutations were detected in approximately one-third of patients, suggesting that precision medicine can be offered to patients with post-operative high-risk uterine cervical cancer in the near future.

Malignant struma ovarii presenting with follicular carcinoma is extremely rare, and its mechanism of tumorigenesis remains unknown. Here, we present a case of malignant struma ovarii with peritoneal dissemination of follicular carcinoma, for which a molecular analysis for major oncogenic gene alterations in follicular thyroid carcinoma was performed. A 39-year-old nulliparous woman was referred with a diagnosis of highly differentiated follicular carcinoma of ovarian origin. Primary thyroid cancer was not diagnosed, and she had a normal thyroid function. 123I scintigraphy revealed multiple peritoneal dissemination that was surgically resected. Histologically, the tumor consisted of numerous follicles without nuclear features of papillary thyroid carcinoma. Tumor samples were investigated for 50 cancer-related genes, including RAS, BRAF, and p53, and PPARg-PAX8 gene fusion by targeted DNA sequencing and fluorescence in situ hybridization, respectively. No major oncogenic gene alterations were detected. These negative findings suggest a different mechanism of tumorigenesis from that of adult-type follicular thyroid carcinoma. Keywords: Malignant struma ovarii, Follicular carcinoma, RAS, PPARgamma, BRAF

Drug-induced interstitial lung disease (DIILD) is a serious side effect of chemotherapy in cancer patients with an extremely high mortality rate. In this study, to identify genetic variants with greater risk of DIILD, we carried out whole genome sequencing (WGS) of germline DNA samples from 26 patients who developed DIILD, and conducted a case-control association study between these 26 cases and general Japanese population controls registered in the integrative Japanese Genome Variation Database (iJGVD) as a screening study. The associations of 42 single nucleotide variants (SNVs) showing P < 0.0001 were further validated using an independent cohort of 18 DIILD cases as a replication study. A further combined analysis of the screening and replication studies showed a possible association of two SNVs, rs35198919 in intron 1 of the chromosome 22 open reading frame 34 (C22orf34) and rs12625311 in intron 1 of the teashirt zinc finger homeobox 2 (TSHZ2), with DIILD (Pcombined = 1.87 × 10-5 and 5.16 × 10-5, respectively). Furthermore, in a subgroup analysis of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-induced interstitial lung disease (ILD), we observed seven candidate SNVs that were possibly associated with ILD (P < 0.00001). This is the first study to identify genetic markers for the risk of DIILD using WGS. Collectively, our novel findings indicate that these SNVs may be applicable for predicting the risk of DIILD in patients receiving chemotherapy.