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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.

Background: Biomarkers to predict the recurrence risk are required to optimize perioperative treatment. Adjuvant chemotherapy for patients with human epidermal growth factor 2-positive (HER2-positive) early breast cancer is decided by pathological responses of neoadjuvant chemotherapy (NAC). However, whether pathological responses are appropriate biomarkers is unclear. Currently, there are several studies using minimal residual disease (MRD) as a predictor of prognosis in solid tumors. However, there is no standard method for detecting MRD. Objectives: This study aimed at prospectively evaluating the relationship between MRD detection and recurrence in Asian patients with HER2-positive early breast cancer. Design: Prospective, observational, single-group, and exploratory. This study will include 60 patients from 2 institutions in Japan and the Philippines. The invasive disease-free survival (IDFS) rates of the MRD-positive and MRD-negative groups are compared in patients with HER2-positive early breast cancer who undergo surgery after receiving NAC. Methods and analysis: Circulating tumor DNA (ctDNA) levels of patients will be evaluated 6 times: before NAC, after NAC, after surgery, and annually after surgery for 3 years. We will analyze the genetic profile of blood and tissue samples using the Todai OncoPanel (TOP) and the methylation level of DNA. The primary endpoint is IDFS. Secondary endpoints include overall survival (OS) and disease-free survival (DFS). Patient enrollment began in June 2022, and new participants are still being recruited. Ethics: This study has been approved by the National Cancer Center Hospital Certified Review Board in March 2022 and has been approved by the Research Ethics Board of the participating center. Discussion: Our findings will contribute to determining whether MRD detection using TOP is useful for predicting the recurrence of HER2-positive early breast cancer. If this is proven, MRD detected by TOP could be used in the future as a biomarker to assist in the de-/escalation of treatment strategies in the next interventional trial, thereby avoiding overtreatment in patients at low risk, and in the addition of intensive treatment modalities for those in patients at high risk.

Objective: Oncological feasibility of segmentectomy for internal non–small cell lung cancer (NSCLC) has not been assessed adequately. We assessed the oncological feasibility of segmentectomy for inner-located NSCLC by investigating surgical margins and patient prognosis after undergoing the procedure. Methods: Of the 3555 patients who underwent resection for lung cancer between 2013 and 2019 at our institution, 659 patients who underwent segmentectomy for clinical stage 0 to stage1A NSCLC were included in this study. Patients were separated into 2 groups according to whether the tumor was in the inner or outer third of the lung area. Clinical characteristics and prognoses were retrospectively compared between the groups. Results: Of the included 659 cases, 183 (27.8%) were inner-located, and 476 (72.2%) had outer-located NSCLC. The surgical margin was significantly shorter in the inner-located group than in the outer group (median, 16 vs 25 mm; P

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.

The journey to implement cancer genomic medicine (CGM) in oncology practice began in the 1980s, which is considered the dawn of genetic and genomic cancer research. At the time, a variety of activating oncogenic alterations and their functional significance were unveiled in cancer cells, which led to the development of molecular targeted therapies in the 2000s and beyond. Although CGM is still a relatively new discipline and it is difficult to predict to what extent CGM will benefit the diverse pool of cancer patients, the National Cancer Center (NCC) of Japan has already contributed considerably to CGM advancement for the conquest of cancer. Looking back at these past achievements of the NCC, we predict that the future of CGM will involve the following: 1) A biobank of paired cancerous and non-cancerous tissues and cells from various cancer types and stages will be developed. The quantity and quality of these samples will be compatible with omics analyses. All biobank samples will be linked to longitudinal clinical information. 2) New technologies, such as whole-genome sequencing and artificial intelligence, will be introduced and new bioresources for functional and pharmacologic analyses (e.g., a patient-derived xenograft library) will be systematically deployed. 3) Fast and bidirectional translational research (bench-to-bedside and bedside-to-bench) performed by basic researchers and clinical investigators, preferably working alongside each other at the same institution, will be implemented; 4) Close collaborations between academia, industry, regulatory bodies, and funding agencies will be established. 5) There will be an investment in the other branch of CGM, personalized preventive medicine, based on the individual’s genetic predisposition to cancer.

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.

EUS–TA in unresectable pancreatic cancer requires not only a tissue diagnosis but also tissue collection in anticipation of comprehensive genomic profiling. However, the optimal puncture target remains controversial. Therefore, the Primary and Metastatic Lesions in Pancreatic Cancer (PRIMATE) study was designed to clarify the optimal target by comparing the success rates for meeting OncoGuide NCC Oncopanel (NOP) analysis criteria on pre-check primary and metastatic lesion specimens obtained during the same EUS–TA session in patients with invasive pancreatic ductal adenocarcinoma. In this ongoing prospective study, two specimens, each from primary and metastatic lesions, are obtained by EUS–TA (typically using a 19G fine-needle biopsy needle) in patients with invasive pancreatic ductal adenocarcinoma. The primary endpoint is the proportion of EUS–TA specimens that meet NOP analysis criteria during pre-check (i.e., tumor cellularity of ≥20% and a tissue area of ≥4 mm2), which are then compared between primary and metastatic lesions. This study has been approved by the National Cancer Center Institutional Review Board (Research No. 2022-168). The results of this study will be reported at an international conference and published in an international peer-reviewed journal. The trial registration number is UMIN 000048966.

Abstract The incidence of second primary neoplasms arising in the skin reconstructive flap (SNAF) is increasing because of the increase in head and neck flap reconstruction and cancer survival. Prognosis, optimal treatment, and their clinicopathological-genetic features are under debate and are difficult to diagnose. We retrospectively reviewed SNAFs based on a single center’s experience over 20 years. Medical records and specimens of 21 patients with SNAF who underwent biopsies between April 2000 and April 2020 at our institute were retrospectively analyzed. Definite squamous cell carcinoma and the remaining neoplastic lesions were subclassified as flap cancer (FC) and precancerous lesions (PLs), respectively. Immunohistochemical studies focused on p53 and p16. TP53 sequencing was conducted using next-generation sequencing. Seven and 14 patients had definite FC and PL, respectively. The mean number of biopsies/latency intervals was 2.0 times/114 months and 2.5 times/108 months for FC and PL, respectively. All lesions were grossly exophytic and accompanied by inflamed stroma. In FC and PL, the incidences of altered p53 types were 43% and 29%, respectively, and those of positive p16 stains were 57% and 64%, respectively. Mutation of TP53 in FC and PL were 17% and 29%, respectively. All except one patient with FC under long-term immunosuppressive therapy survived in this study. SNAFs are grossly exophytic tumors with an inflammatory background and show a relatively low altered p53 and TP53 rate and a high p16 positivity rate. They are slow-growing neoplasms with good prognoses. Diagnosis is often difficult; therefore, repeated or excisional biopsy of the lesion may be desirable.

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.

Cutaneous adnexal tumors are neoplasms that arise from skin appendages. Their morphologic diversity and phenotypic variability with rare progression to malignancy make them difficult to diagnose and classify, and there is currently no established treatment strategy. To overcome these difficulties, this study investigated the transcription factor SOX9 expression, morphology, and genetics of skin adnexal tumors for understanding their biology, especially their histogenesis. We showed that cutaneous adnexal tumors and their nontumor counterparts of skin and appendages exhibit expression patterns similar to that of SOX9. Its expression intensity and pattern, as well as histopathologic evaluation of tumors, were analyzed using digital images of 69 normal skin adnexal 9-type organs and 185 skin adnexal 29-type tumors as references. It was possible to distinguish basal cell carcinoma from squamous cell carcinoma, sebaceous carcinoma, and pilomatrixoma with significant differences, along with porocarcinoma from squamous cell carcinoma. Furthermore, unsupervised machine learning “computational pathology” was used to derive a multiregion whole-exome sequencing fusion method termed “genocomputed pathology.” The genocomputed pathology of three representable adnexal carcinomas (porocarcinoma, hidradenocarcinoma, and spiradenocarcinoma) was evaluated for total nine cases. We showed that there was more heterogeneity than expected within the tumors as well as the coexistence of components lacking driver fusion genes. The presence or absence of potential driver genes, such as PIK3CA, YAP1, and PTEN, in each region was identified, highlighting a therapeutic strategy for cutaneous adnexal carcinoma encompassing heterogeneous tumors.

Objective: We aimed to visualize complicated patterns of lymph node metastases in surgically resected non–small cell lung cancer by applying a data mining technique. Methods: In this retrospective study, 783 patients underwent lobectomy or pneumonectomy with systematic mediastinal lymph node dissection for non–small cell lung cancer between January 2010 and December 2018. Surgically resected lymph nodes were classified according to the International Association for the Study of Lung Cancer lymph node map. Network analysis generated patterns of lymph node metastases from stations 1 to 14, and the degree of connection between 2 lymph node stations was assessed. Results: The median number of lymph nodes examined per patient was 20, and the pathological N category was pN0 in 428 cases, pN1 in 132, pN2 in 221, and pN3 in 2. N1 lymph node stations had strong associations with superior mediastinal lymph node stations for patients with primary tumors in the upper lobes and with station 7 for the lower lobes. There was also a connection from the N1 lymph node stations to superior mediastinal lymph node stations in the lower lobes. In the right middle lobe, an even distribution from station 12m toward stations 2R, 4R, and 7 was noted. We released an interactive web application to visualize these data: http://www.canexapp.com. Conclusions: Lymph node metastasis patterns differed according to the lobe bearing the tumor. Our results support the need for clinical trials to further investigate selective mediastinal lymph node dissection.

Abstract Since U.S. President Barack Obama announced the Precision Medicine Initiative in his New Year’s State of the Union address in 2015, the establishment of a precision medicine system has been emphasized worldwide, particularly in the field of oncology. With the advent of next-generation sequencers specifically, genome analysis technology has made remarkable progress, and there are active efforts to apply genome information to diagnosis and treatment. Generally, in the process of feeding back the results of next-generation sequencing analysis to patients, a molecular tumor board (MTB), consisting of experts in clinical oncology, genetic medicine, etc., is established to discuss the results. On the other hand, an MTB currently involves a large amount of work, with humans searching through vast databases and literature, selecting the best drug candidates, and manually confirming the status of available clinical trials. In addition, as personalized medicine advances, the burden on MTB members is expected to increase in the future. Under these circumstances, introducing cutting-edge artificial intelligence (AI) technology and information and communication technology to MTBs while reducing the burden on MTB members and building a platform that enables more accurate and personalized medical care would be of great benefit to patients. In this review, we introduced the latest status of elemental technologies that have potential for AI utilization in MTB, and discussed issues that may arise in the future as we progress with AI implementation.

Abstract Malignant melanoma is the most aggressive skin cancer that originates from melanocytes. Primary or metastatic pleural melanoma shares clinical and imaging characteristics with primary pleural tumors, such as pleural mesothelioma. Identification of the primary site can be challenging to distinguish between primary and secondary melanomas. We report a case of a 46‐year‐old woman with metastatic, rapidly progressing pleural melanoma mimicking primary pleural tumor. The metastatic pleural tumor from a primary cutaneous melanoma was diagnosed by reevaluating a previous surgical specimen. When evaluating patients with pleural melanoma, the primary site should be reevaluated to distinguish between primary and secondary melanomas.

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 To implement precision oncology, analytical validity as well as clinical validity and utility are important. However, proficiency testing (PT) to assess validity has not yet been systematically performed in Japan. To investigate the quality of next-generation sequencing (NGS) platforms and cancer genome testing prevalent in laboratories, we performed pilot PT using patient samples. We prepared genomic DNA from the cancer tissue and peripheral blood of 5 cancer patients and distributed these to 15 laboratories. Most participating laboratories successfully identified the pathogenic variants, except for two closely located KRAS variants and 25 bp delins in EGFR. Conversely, the EGFR L858R variant was successfully identified, and the allele frequency was similar for all the laboratories. A high DNA integrity number led to excellent depth and reliable NGS results. By conducting this pilot study using patient samples, we were able to obtain a glimpse of the current status of cancer genome testing at participating laboratories. To enhance domestic cancer genome testing, it is important to conduct local PT and to involve the parties concerned as organizers and participants.

Masachika Ikegami and Shinji Kohsaka et al. develop MicroSEC, a computational pipeline to filter sequencing artifacts from archival formalin-fixed and paraffin-embedded samples. Given that archival FFPE tissue is of great interest for genomic analysis, but difficult to reliably analyze, this tool may improve the ability of researchers to probe sequencing data from these samples.

Abstract Purpose We aimed to examine the characteristics of imaging findings of adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) in the lungs of smokers compared with those of non-smokers. Materials and methods We included seven cases of AIS and 20 cases of MIA in lungs of smokers (pack-years ≥ 20) and the same number of cases of AIS and MIA in lungs of non-smokers (pack-years = 0). We compared the diameter of the entire lesion and solid component measured on computed tomography (CT) images, pathological size and invasive component diameter measured from pathological specimens, and CT values of the entire lesion and ground-glass opacity (GGO) portions between the smoker and non-smoker groups. Results The diameters of AIS and MIA on CT images and pathological specimens of the smoker group were significantly larger than those of the non-smoker group (p = 0.036 and 0.008, respectively), whereas there was no significant difference in the diameter of the solid component on CT images or invasive component of pathological specimens between the two groups. Additionally, mean CT values of the entire lesion and GGO component of the lesions in the smoker group were significantly lower than those in the non-smoker group (p = 0.036 and 0.040, respectively). Conclusion AIS and MIA in smoker’s lung tended to have larger lesion diameter and lower internal CT values compared with lesions in non-smoker’s lung. This study calls an attention on smoking status in CT-based diagnosis for early stage adenocarcinoma.

PurposePatients with advanced/relapsed rare cancers have few treatment options. Analysis of circulating tumor DNA in plasma may identify actionable genomic biomarkers using a non-invasive approach.Patients and MethodsRare cancer patients underwent prospective plasma-based NGS testing. Tissue NGS to test concordance was also conducted. Plasma DNA alterations were assessed for incidence, functional impact, therapeutic implications, correlation to survival, and comparison with tissue NGS.ResultsNinety-eight patients were analyzed. Diseases included soft-tissue sarcoma, ovarian carcinoma, and others. Mean turn-around-time for results was 9.5 days. Seventy-six patients had detectable gene alterations in plasma, with a median of 2.8 alterations/patient. Sixty patients had a likely pathogenic alteration. Five received matched-therapy based on plasma NGS results. Two developed known resistance mutations while on targeted therapy. Patients with an alteration having VAF ≥5% had a significantly shorter survival compared to those of lower VAF. Tissue NGS results from eleven of 22 patients showed complete or partial concordance with plasma NGS.ConclusionPlasma NGS testing is less invasive and capable of identifying alterations in advanced rare cancers in a clinically meaningful timeframe. It should be further studied as a prospective enrollment assay in interventional studies for patients with rare advanced stage cancers.Clinical Registration[https://www.umin.ac.jp/ctr/index-j.htm], identifier UMIN000034394.