BMP4 and PHLDA1 are plausible drug-targetable candidate genes for KRAS G12A-, G12D-, and G12V-driven colorectal cancer.

Despite the frequent detection of KRAS driver mutations in patients with colorectal cancer (CRC), no effective treatments that target mutant KRAS proteins have been introduced into clinical practice. In this study, we identified potential effector molecules, based on differences in gene expression between CRC patients carrying wild-type KRAS (n = 390) and those carrying KRAS mutations in codon 12 (n = 240). CRC patients with wild-type KRAS harboring mutations in HRAS, NRAS, PIK3CA, PIK3CD, PIK3CG, RALGDS, BRAF, or ARAF were excluded from the analysis. At least 11 promising candidate molecules showed greater than two-fold change between the KRAS G12 mutant and wild-type and had a Benjamini-Hochberg-adjusted P value of less than 1E-08, evidence of significantly differential expression between these two groups. Among these 11 genes examined in cell lines transfected with KRAS G12 mutants, BMP4, PHLDA1, and GJB5 showed significantly higher expression level in KRAS G12A, G12D, and G12V transfected cells than in the wild-type transfected cells. We expect that this study will lead to the development of novel treatments that target signaling molecules functioning with KRAS G12-driven CRC.

Japanese version of The Cancer Genome Atlas, JCGA, established using fresh frozen tumors obtained from 5143 cancer patients.

This study aimed to establish the Japanese Cancer Genome Atlas (JCGA) using data from fresh frozen tumor tissues obtained from 5143 Japanese cancer patients, including those with colorectal cancer (31.6%), lung cancer (16.5%), gastric cancer (10.8%) and other cancers (41.1%). The results are part of a single-center study called "High-tech Omics-based Patient Evaluation" or "Project HOPE" conducted at the Shizuoka Cancer Center, Japan. All DNA samples and most RNA samples were analyzed using whole-exome sequencing, cancer gene panel sequencing, fusion gene panel sequencing and microarray gene expression profiling, and the results were annotated using an analysis pipeline termed "Shizuoka Multi-omics Analysis Protocol" developed in-house. Somatic driver alterations were identified in 72.2% of samples in 362 genes (average, 2.3 driver events per sample). Actionable information on drugs that is applicable in the current clinical setting was associated with 11.3% of samples. When including those drugs that are used for investigative purposes, actionable information was assigned to 55.0% of samples. Germline analysis revealed pathogenic mutations in hereditary cancer genes in 9.2% of samples, among which 12.2% were confirmed as pathogenic mutations by confirmatory test. Pathogenic mutations associated with non-cancerous hereditary diseases were detected in 0.4% of samples. Tumor mutation burden (TMB) analysis revealed 5.4% of samples as having the hypermutator phenotype (TMB ≥ 20). Clonal hematopoiesis was observed in 8.4% of samples. Thus, the JCGA dataset and the analytical procedures constitute a fundamental resource for genomic medicine for Japanese cancer patients.

Development of a new method for synthesis of tandem hairpin pyrrole-imidazole polyamide probes targeting human telomeres.

Pyrrole­imidazole (PI) polyamides bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. To visualize telomeres specifically, tandem hairpin PI polyamides conjugated with a fluorescent dye have been synthesized, but the study of telomeres using these PI polyamides has not been reported because of difficulties synthesizing these tandem hairpin PI polyamides. To synthesize tandem hairpin PI polyamides more easily, we have developed new PI polyamide fragments and have used them as units in Fmoc solid-phase peptide synthesis. Using this new method, we synthesized four fluorescent polyamide probes for the human telomeric repeat TTAGGG, and we examined the binding affinities and specificities of the tandem hairpin PI polyamides, the UV­vis absorption and fluorescence spectra of the fluorescent polyamide probes, and telomere staining in mouse MC12 and human HeLa cells. The polyamides synthesized using the new method successfully targeted to human and mouse telomeres under mild conditions and allow easier labeling of telomeres in the cells while maintaining the telomere structure. Using the fluorescent polyamides, we demonstrated that the telomere length at a single telomere level is related to the abundance of TRF1 protein, a shelterin complex component in the telomere.

Whole-genome and Epigenomic Landscapes of Malignant Gastrointestinal Stromal Tumors Harboring KIT Exon 11 557-558 Deletion Mutations.

Gastrointestinal stromal tumors (GIST) with KIT exon 11 deletions involving in codons 557-558 (KIT Δ557-558) exhibit higher proliferation rates and shorter disease-free survival times compared with GISTs with other KIT exon 11 mutations. We analyzed 30 GIST cases and observed genomic instability and global DNA hypomethylation only in high-risk malignant GISTs with KIT Δ557-558. Whole-genome sequencing revealed that the high-risk malignant GISTs with KIT Δ557-558 (12 cases) had more structural variations (SV), single-nucleotide variants, and insertions and deletions compared with the low-risk, less malignant GISTs with KIT Δ557-558 (six cases) and the high-risk (six cases) or low-risk (6 cases) GISTs with other KIT exon 11 mutations. The malignant GISTs with KIT Δ557-558 showed higher frequency and significance in copy number (CN) reduction on chromosome arms 9p and 22q, and 50% of them had LOH or CN-dependent expression reduction in CDKN2A. In addition, SVs with driver potential were detected in 75% of them, in which AKT3 and MGMT were recurrently identified. Genome-wide DNA methylation and gene expression analyses showed global intergenic DNA hypomethylation, SNAI2 upregulation, and higher expression signatures, including p53 inactivation and chromosomal instability, as characteristics of malignant GISTs with KIT Δ557-558 that distinguished them from other GISTs. These genomic and epigenomic profiling results revealed that KIT Δ557-558 mutations are associated with increased genomic instability in malignant GISTs. Significance: We present genomic and epigenomic insights into the malignant progression of GISTs with KIT exon 11 deletions involving in 557-558, demonstrating their unique chromosomal instability and global intergenic DNA hypomethylation.

Development of two 410-cancer-gene panel tests for solid tumors and liquid biopsy based on genome data of 5,143 Japanese cancer patients.

Next-generation sequencing (NGS) is an integral part of precision medicine, and its power for detecting comprehensive genetic alterations may contribute to treatment decisions for patients with advanced, recurrent, or metastatic cancer. An NGS oncology panel developed in the U.S. and Europe, which targets cancer-related genes, has been approved in Japan, and testing is becoming more widespread in clinical oncology practice. However, these panels are based on cancer-related genes selected from cancer databases of Westerners. We aimed to develop an onco-panel for Japanese. We designed two High-tech Omics-based Patient Evaluation (HOPE) onco-panels: HOPE onco-panel Solid for solid tumors and HOPE onco-panel Liquid for liquid biopsy. These were based on genomic information of 5,143 cancer cases in the Japanese Cancer Genome Atlas (JCGA), a database of Japanese cancer cases. Their performance was confirmed using clinical data.

Comparison of genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma in Japanese patients using a novel panel for cancer-related drug-metabolizing enzyme genes.

The differences in genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma remain unclear. We developed a customized, targeted gene sequencing panel for efficient and sensitive identification of germline variants, including whole-gene deletion types for cancer-related drug-metabolizing enzyme genes in lung adenocarcinoma and squamous cell carcinoma. The minor allele frequencies of the variants, confirmed as clinically significant in the Japanese population, did not differ significantly from those of normal participants listed in the public database. Genotype analysis comparing lung adenocarcinoma (n = 559) and squamous cell carcinoma (n = 151) indicated that the variants of DPYD (rs190771411, Fisher's exact test, P = 0.045; rs200562975, P = 0.045) and ALDH2 (rs568781254, P = 0.032) were associated with an increased risk of squamous cell carcinoma compared to adenocarcinoma. Conversely, whole-gene deletion of CYP2A6 was associated with adenocarcinoma but not squamous cell carcinoma. Notably, whole-gene deletion of CYP2A6 was confirmed in 22 patients with lung adenocarcinoma but not in any patients with squamous cell carcinoma. Most patients with whole-gene deletion of CYP2A6 were female non-smokers. The discovery of a whole-gene deletion of CYP2A6 in patients with lung adenocarcinoma may have an important role in clinical practice and advance our understanding of CYP2A6 germline variants and their association with carcinogenesis or their susceptibility to lung adenocarcinoma.

Whole exome sequencing detects variants of genes that mediate response to anticancer drugs.

Certain interindividual differences affecting the efficacy of drug treatment and adverse drug reactions are caused by genetic variants, and their phenotypic effects differ among ethnic groups. In this study, we used whole exome sequencing (WES) systematically to identify germline mutations that influence the activities of drug-metabolizing enzymes, as well as that of a transporter. We analyzed DNA isolated from blood samples from 2,042 Japanese patients with diverse cancers. We identified sequence variants of CYP2B6 (rs3745274), CYP2C9 (rs1057910), CYP2C19 (rs4986893), CYP2C19 (rs4244285), TPMT (rs1142345), NAT2 (rs1799930), NAT2 (rs1799931), UGT1A1 (rs4148323), COMT (rs4680), ABCB1 (rs1045642), and CDA (rs60369023). Wider application of WES will help to determine the effects of mutations on the activities of proteins encoded by drug response genes, and the information gained will accelerate the development of personalized therapies for patients with cancer. Moreover, this knowledge may provide clues for preventing cancer before the onset of symptoms.

Chromosomes without a 30-nm chromatin fiber.

How is a long strand of genomic DNA packaged into a mitotic chromosome or nucleus? The nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fiber, and a further helically folded larger fiber. However, when frozen hydrated human mitotic cells were observed using cryoelectron microscopy, no higher-order structures that included 30-nm chromatin fibers were found. To investigate the bulk structure of mitotic chromosomes further, we performed small-angle X-ray scattering (SAXS), which can detect periodic structures in noncrystalline materials in solution. The results were striking: no structural feature larger than 11 nm was detected, even at a chromosome-diameter scale (~1 µm). We also found a similar scattering pattern in interphase nuclei of HeLa cells in the range up to ~275 nm. Our findings suggest a common structural feature in interphase and mitotic chromatins: compact and irregular folding of nucleosome fibers occurs without a 30-nm chromatin structure.