Impact of somatic mutations and transcriptomic alterations on cancer aneuploidy.

Aneuploidy has been recognized as one of hallmark of tumorigenesis since the early 20th century. Recent developments in structural variation analysis in the human genome have revealed the diversity of aneuploidy in cancer. However, the effects of gene mutation and expression in tumors on aneuploidy remain poorly understood. Here, we performed whole exome analysis of over 5,000 Japanese cancer cases and investigated the impact of somatic mutations and gene expression alterations on aneuploidy. First, we evaluated tumor content and genomic alterations that could influence aneuploidy. Next, we compared the aneuploidy frequency in 18 cancer types and observed that TP53 mutations were associated with the aneuploidy on specific chromosomes in colorectal and gastric cancers. Finally, we used expression analysis to isolate pathways involved in aneuploidy accumulation from tumors without TP53 mutations. Chromosomal instability and cell cycle aberration were associated with aneuploidy in TP53 wild-type tumors, and 26 commonly upregulated genes were identified in aneuploidy-high solid tumors without TP53 mutations. Among them, two cancer-related genes (CENPA and PBK) were involved in aneuploidy. Our integrated analysis revealed that both TP53 mutations and transcriptomic alterations independent of somatic mutations affect aneuploidy accumulation. Our findings will facilitate further understanding of diverse aneuploidies in the tumorigenesis.

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.

Tumor cell enrichment by tissue suspension enables detection of mutations with low variant allele frequency and estimation of germline mutations.

Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome. However, accurate sequencing cannot be performed in cancers harboring diffuse tumor cells because of low tumor content. We performed tumor cell enrichment using tissue suspension of formalin-fixed, paraffin-embedded (FFPE) tissue sections with low tumor cell content. The enriched fractions were used to efficiently identify mutations by sequencing a target panel of cancer-related genes. Tumor-enriched and residual fractions were isolated from FFPE tissue sections of intestinal and diffuse gastric cancers harboring diffuse tumor cells and DNA of suitable quality was isolated for next-generation sequencing. Sequencing of a target panel of cancer-related genes using the tumor-enriched fraction increased the number of detectable mutations and variant allele frequency. Furthermore, mutation analysis of DNA isolated from tumor-enriched and residual fractions allowed us to estimate germline mutations without a blood reference. This approach of tumor cell enrichment will not only enhance the success rate of target panel sequencing, but can also improve the accuracy of detection of somatic mutations in archived specimens.

Mutational concordance analysis provides supportive information for double cancer diagnosis.

BACKGROUND: Mutation analysis using next-generation sequencing highlights the features of tumors with somatic alterations. However, the mutation profile of double cancer remains unclear. Here, we analyzed tumors derived from the same patient using whole exome sequencing (WES) to investigate the coherence of somatic mutations in double cancer. METHODS: First, the tumor mutational burden (TMB) was investigated using WES of 5521 tumor specimens from a Japanese pan-cancer cohort. The frequencies of mutation concordance were then compared in these cancers. Finally, we calculated the expected value of mutational concordance fitting a Poisson distribution to determine the relationship between double and metastatic cancers. RESULTS: In all, 44, 58, and 121 paired samples were diagnosed as double cancer, multifocal lesions (derived from identical tissues), and metastasis, respectively. Our analysis revealed that common somatic mutations were almost entirely absent in double cancer, whereas primary tumors and metastatic foci harbored several identical alterations. Concordance of the mutation profile in the same patient reflects the tumor origin and development, suggesting the potential for identifying double cancer based on common somatic mutations. Furthermore, according to a Poisson distribution, double cancer could be discriminated based on paired samples from the same patient. The probability of double cancer with more than 10 mutations was ≤1 part-per-billion (ppb, 10- 9). In multifocal lesions, 74% of tumor pairs accumulated ≤10 common mutations, implying a difference in tumor origin within identical tissues. CONCLUSIONS: These findings indicate that counting common somatic mutations can indicate the differences in origin between tumors derived from the same patient. Our mutation coherence analysis can thus provide beneficial information for diagnosing double cancer.

Characterization of tumors with ultralow tumor mutational burden in Japanese cancer patients.

Tumor mutational burden analysis using whole-exome sequencing highlights features of tumors with various mutations or known driver alterations. Cancers with few changes in the exon regions have unclear characteristics, even though low-mutated tumors are often detected in pan-cancer analysis. In the present study, we analyzed tumors with low tumor mutational burden listed in the Japanese version of The Cancer Genome Atlas, a data set of 5020 primary solid tumors. Our analysis revealed that detection rates of known driver mutations and copy number variation were decreased in samples with tumor mutational burden below 1.0 (ultralow tumor), compared with those in samples with low tumor mutational burden (≤5 mutations/Mb). This trend was also observed in The Cancer Genome Atlas data set. In the ultralow tumor mutational burden tumors, expression analysis showed decreased TP53 inactivation and chromosomal instability. TP53 inactivation frequently correlated with PI3K/mTOR-related gene expression, implying suppression of the PI3K/mTOR pathway in ultralow tumor mutational burden tumors. In common with mutational burden, the T cell-inflamed gene expression profiling signature was a potential marker for prediction of an immune checkpoint inhibitor response, and some ultralow tumor mutational burden tumor populations highly expressed this signature. Our analysis focused on how these tumors could provide insight into tumors with low somatic alteration that are difficult to detect solely using whole-exome sequencing.

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.

Driver gene alterations and activated signaling pathways toward malignant progression of gastrointestinal stromal tumors.

Mutually exclusive KIT and PDGFRA mutations are considered to be the earliest events in gastrointestinal stromal tumors (GIST), but insufficient for their malignant progression. Herein, we aimed to identify driver genes and signaling pathways relevant to GIST progression. We investigated genetic profiles of 707 driver genes, including mutations, gene fusions, copy number gain or loss, and gene expression for 65 clinical specimens of surgically dissected GIST, consisting of six metastatic tumors and 59 primary tumors from stomach, small intestine, rectum, and esophagus. Genetic alterations included oncogenic mutations and amplification-dependent expression enhancement for oncogenes (OG), and loss of heterozygosity (LOH) and expression reduction for tumor suppressor genes (TSG). We assigned activated OG and inactivated TSG to 27 signaling pathways, the activation of which was compared between malignant GIST (metastasis and high-risk GIST) and less malignant GIST (low- and very low-risk GIST). Integrative molecular profiling indicated that a greater incidence of genetic alterations of driver genes was detected in malignant GIST (96%, 22 of 23) than in less malignant GIST (73%, 24 of 33). Malignant GIST samples groups showed mutations, LOH, and aberrant expression dominantly in driver genes associated with signaling pathways of PI3K (PIK3CA, AKT1, and PTEN) and the cell cycle (RB1, CDK4, and CDKN1B). Additionally, we identified potential PI3K-related genes, the expression of which was upregulated (SNAI1 and TPX2) or downregulated (BANK1) in malignant GIST. Based on our observations, we propose that inhibition of PI3K pathway signals might potentially be an effective therapeutic strategy against malignant progression of GIST.

Pro-gastrin-releasing peptide as a marker for the Ewing sarcoma family of tumors.

BACKGROUND: Pro-gastrin-releasing peptide (ProGRP) is an established tumor marker of small cell lung cancer. The purpose of this study was to determine if ProGRP could serve as a tumor marker for the Ewing sarcoma family of tumors (ESFTs). METHODS: Sixteen patients with ESFTs (mean age 32 years) were included in this study. As a control group, 42 patients with other tumor types that clinically or pathologically mimic ESFTs were also analyzed. Pre-treatment serum ProGRP and neuron-specific enolase (NSE) levels, the relationships between these levels, and tumor volume were investigated. In addition, serial changes in the serum or plasma ProGRP (6 patients) and NSE levels (5 patients) were measured over the course of treatment. RESULTS: Pre-treatment serum ProGRP levels were higher than the normal range in 8 of 16 patients; for these eight patients, ProGRP levels positively correlated with tumor volume (R = 0.99). In the control group, ProGRP levels were within the normal range, except for the two patients. Changes in ProGRP levels during treatment were consistent with tumor volume. Serum NSE levels were elevated in 14 of 16 patients with ESFTs and 8 of 42 patients with other tumor types. The range of NSE elevation was much smaller compared to that of ProGRP. Our data indicate that ProGRP is superior to NSE in terms of specificity. CONCLUSIONS: Serum ProGRP levels were elevated in half of the patients with ESFTs and reflected therapeutic response. ProGRP is a reliable tumor marker for the diagnosis of ESFTs and evaluation of treatment response.

Mutational burden and signatures in 4000 Japanese cancers provide insights into tumorigenesis and response to therapy.

Tumor mutational burden (TMB) and mutational signatures reflect the process of mutation accumulation in cancer. However, the significance of these emerging characteristics remains unclear. In the present study, we used whole-exome sequencing to analyze the TMB and mutational signature in solid tumors of 4046 Japanese patients. Eight predominant signatures-microsatellite instability, smoking, POLE, APOBEC, UV, mismatch repair, double-strand break repair, and Signature 16-were observed in tumors with TMB higher than 1.0 mutation/Mb, whereas POLE and UV signatures only showed moderate correlation with TMB, suggesting the extensive accumulation of mutations due to defective POLE and UV exposure. The contribution ratio of Signature 16, which is associated with hepatocellular carcinoma in drinkers, was increased in hypopharynx cancer. Tumors with predominant microsatellite instability signature were potential candidates for treatment with immune checkpoint inhibitors such as pembrolizumab and were found in 2.8% of cases. Furthermore, based on microarray analysis, tumors with predominant signatures were classified into 2 subgroups depending on the expression of immune-related genes reflecting differences in the immune context of the tumor microenvironment. Tumor subpopulations differing in the content of infiltrating immune cells might respond differently to immunotherapeutics. An understanding of cancer characteristics based on TMB and mutational signatures could provide new insights into mutation-driven tumorigenesis.

Identification of a neoantigen epitope in a melanoma patient with good response to anti-PD-1 antibody therapy.

Recent advances in next-generation sequencing have enabled rapid and efficient evaluation of the mutational landscape of cancers. As a result, many cancer-specific neoantigens, which can generate antitumor cytotoxic T-cells inside tumors, have been identified. Previously, we reported a metastatic melanoma case with high tumor mutation burden, who obtained complete remission after anti-PD-1 therapy and surgical resection. The rib metastatic lesion, which was used for whole-exome sequencing and gene expression profiling in the HOPE project, showed upregulated expression of PD-L1 mRNA and a high single-nucleotide variants number of 2712. In the current study, we focused on a metastatic melanoma case and candidate epitopes among nonsynonymous mutant neoantigens of 1348 variants were investigated using a peptide-HLA binding algorithm, in vitro cytotoxic T-cell induction assay and HLA tetramer staining. Specifically, from mutant neoantigen data, a total of 21,066 9-mer mutant epitope candidates including a mutated amino acid anywhere in the sequence were applied to the NetMHC binding prediction algorithm. From in silico data, we identified the top 26 mutant epitopes with strong-binding capacity. A cytotoxic T-cell induction assay using 5 cancer patient-derived PBMCs revealed that the mutant ARMT1 peptide sequence (FYGKTILWF) with HLA-A*2402 restriction was an efficient neoantigen, which was detected at a frequency of approximately 0.04% in the HLA-A24 tetramer stain. The present success in identifying a novel mutant antigen epitope might be applied to clinical neoantigen screening in the context of an NGS-equipped medical facility for the development of the next-generation neoantigen cancer vaccines.

Comparative proteomic analysis identifies exosomal Eps8 protein as a potential metastatic biomarker for pancreatic cancer.

Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell­type specific, and molecules embedded in exosomes can be useful fluid­based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23­fold and 20­fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa­2 cell line derived from primary tumor cells. Liquid chromatography­mass spectrometry­based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis­derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2x10­4; invasion: r=0.60, P=3.2x10­2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10­2; invasion: r=0.51, P=9.2x10­2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.

Molecular profiling and sequential somatic mutation shift in hypermutator tumours harbouring POLE mutations.

Defective DNA polymerase ε (POLE) proofreading leads to extensive somatic mutations that exhibit biased mutational properties; however, the characteristics of POLE-mutated tumours remain unclear. In the present study, we describe a molecular profile using whole exome sequencing based on the transition of somatic mutations in 10 POLE-mutated solid tumours that were obtained from 2,042 Japanese patients. The bias of accumulated variations in these mutants was quantified to follow a pattern of somatic mutations, thereby classifying the sequential mutation shift into three periods. During the period prior to occurrence of the aberrant POLE, bare accumulation of mutations in cancer-related genes was observed, whereas PTEN was highly mutated in conjunction with or subsequent to the event, suggesting that POLE and PTEN mutations were responsible for the development of POLE-mutated tumours. Furthermore, homologous recombination was restored following the occurrence of PTEN mutations. Our strategy for estimation of the footprint of somatic mutations may provide new insight towards the understanding of mutation-driven tumourigenesis.

Tumor mutational burden analysis of 2,000 Japanese cancer genomes using whole exome and targeted gene panel sequencing.

Tumor mutational burden (TMB) is an emerging characteristic in cancer and has been associated with microsatellite instability, defective DNA replication/repair, and response to PD-1 and PD-L1 blockade immunotherapy. When estimating TMB, targeted panel sequencing is performed using a few hundred genes; however, a comparison of TMB results obtained with this platform and with whole exome sequencing (WES) has not been performed for various cancer types. In the present study, we compared TMB results using the above two platforms in 2,908 solid tumors that were obtained from Japanese patients. For next-generation sequencing, we used fresh-frozen tissue specimens. The Ion Proton System was employed to detect somatic mutations in the coding genome and to sequence an available cancer panel that targeted 409 genes. We then selected 2,040 samples with sufficient tumor cellularity for TMB analysis. In tumors with TMB-high (TMB ≥ 20 mutations/Mb), TMB derived from WES correlated well with the estimated TMB (eTMB) based on panel sequencing, whereas TMB in the remaining tumors showed a weak correlation. In particular, eTMB was overestimated in tumors with low-frequency mutations, resulting in the accumulation of EGFR mutations not being discriminated as a feature of lung cancer with low-frequency mutations. The eTMB in tumors harboring POLE mutations and microsatellite instability was not overestimated, suggesting that panel sequencing could accurately estimate TMB in tumors with high-frequency mutations such as hypermutator tumors. These results may provide helpful information for interpreting TMB results based on clinical sequencing using a targeted gene panel.

Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors.

Identification of driver genes contributes to the understanding of cancer etiology and is imperative for the development of individualized therapies. Gene amplification is a major event in oncogenesis. Driver genes with tumor-specific amplification-dependent overexpression can be therapeutic targets. In this study, we aimed to identify amplification-dependent driver genes in 1,454 solid tumors, across more than 15 cancer types, by integrative analysis of gene expression and copy number. Amplification-dependent overexpression of 64 known driver oncogenes were found in 587 tumors (40%); genes frequently observed were MYC (25%) and MET (18%) in colorectal cancer; SKP2 (21%) in lung squamous cell carcinoma; HIST1H3B (19%) and MYCN (13%) in liver cancer; KIT (57%) in gastrointestinal stromal tumors; and FOXL2 (12%) in squamous cell carcinoma across tissues. Genomic aberrations in 138 known cancer driver genes and 491 established fusion genes were found in 1,127 tumors (78%). Further analyses of 820 cancer-related genes revealed 16 as potential driver genes, with amplification-dependent overexpression restricted to the remaining 22% of samples (327 tumors) initially undetermined genetic drivers. Among them, AXL, which encodes a receptor tyrosine kinase, was recurrently overexpressed and amplified in sarcomas. Our studies of amplification-dependent overexpression identified potential drug targets in individual tumors.

Multi-omics Profiling of Patients with Melanoma Treated with Nivolumab in Project HOPE.

BACKGROUND: Project HOPE (High-tech Omics-based Patient Evaluation) has been in progress since 2014 and uses whole-exome sequencing (WES) and gene expression profiling (GEP). Among a total of 1,685 patients with cancer, 13 with melanoma were registered and characterized using multi-omics analyses to investigate specific biomarkers in responders to programmed cell death-1 (PD-1) blockade. MATERIALS AND METHODS: The patients with melanoma comprised of six males and seven females, and their mean age was 68 years. Five patients were treated with nivolumab, and two were responders. RESULTS: GEP analysis demonstrated that PD-L1 expression was positive in for cases, and melanoma-associated antigens and tumor signaling-associated genes were up-regulated in tumor compared with normal tissues. Additionally, WES analysis indicated more single nucleotide variants (SNVs) per melanoma tumor compared to other tumor types. Remarkably, a case of complete remission after nivolumab therapy showed high expression of PD-L1 protein and the highest number of SNVs. CONCLUSION: The novel approach used in Project HOPE might be an efficient tool that facilitates identifying specific biomarkers predictive of good responders to anti-PD-1 therapy.

Comprehensive characterization of genes associated with the TP53 signal transduction pathway in various tumors.

The TP53 signal transduction pathway is an attractive target for cancer treatments. In this study, we conducted a comprehensive molecular evaluation of 907 patients with cancer in Japan to identify genomic alterations in the TP53 pathway. TP53 mutations were frequently detected in many cancers, except melanoma, thymic tumors, gastrointestinal stromal tumors, and renal cancers. The frequencies of non-synonymous single nucleotide variants (SNVs) in the TP53 family members TP63 and TP73 were relatively low, although genes with increased frequencies of SNVs were as follows: PTEN (11.7%) in breast cancer, CDKN2A (11.1 and 9.6%) in pancreas and head and neck cancers, and ATM (18.0 and 11.1%) in liver and esophageal cancers. MDM2 expression was decreased or increased in patients with mutant or wild-type TP53, respectively. CDKN1A expression was increased with mutant TP53 in head and neck cancers. Moreover, TP63 overexpression was characteristically observed in squamous cell carcinomas of the lung, esophagus, and head and neck region. Additionally, overexpression of TP63 and TP73 was frequently observed in thymomas. Our results reveal a spectrum of genomic alterations in the TP53 pathway that is characteristic of many tumor types, and these data may be useful in the trials of targeted therapies.

Optimizing an ion semiconductor sequencing data analysis method to identify somatic mutations in the genomes of cancer cells in clinical tissue samples.

Identification of causal genomic alterations is an indispensable step in the implementation of personalized cancer medicine. Analytical methods play a central role in identifying such changes because of the vast amount of data produced by next generation sequencer. Most analytical techniques are designed for the Illumina platform and are therefore suboptimal for analyzing datasets generated by whole exome sequencing (WES) using the Ion Proton System. Accurate identification of somatic mutations requires the characterization of platform-dependent error profiles and genomic properties that affect the accuracy of sequence data as well as platform-oriented optimization of the pipeline. Therefore, we used the Ion Proton System to perform WES of DNAs isolated from tumor and matched control tissues of 1,058 patients with cancer who were treated at the Shizuoka Cancer Center Hospital. Among the initially identified candidate somatic single-nucleotide variants (SNVs), 10,279 were validated by manual inspection of the WES data followed by Sanger sequencing. These validated SNVs were used as an objective standard to determine an optimum cutoff value to improve the pipeline. Using this optimized pipeline analysis, 189,381 SNVs were identified in 1,101 samples. The analytical technique presented here is a useful resource for conducting clinical WES, particularly using semiconductor-based sequencing technology.

Integrated next-generation sequencing analysis of whole exome and 409 cancer-related genes.

The use of next-generation sequencing (NGS) techniques to analyze the genomes of cancer cells has identified numerous genomic alterations, including single-base substitutions, small insertions and deletions, amplification, recombination, and epigenetic modifications. NGS contributes to the clinical management of patients as well as new discoveries that identify the mechanisms of tumorigenesis. Moreover, analysis of gene panels targeting actionable mutations enhances efforts to optimize the selection of chemotherapeutic regimens. However, whole genome sequencing takes several days and costs at least $10,000, depending on sequence coverage. Therefore, laboratories with relatively limited resources must employ a more economical approach. For this purpose, we conducted an integrated nucleotide sequence analysis of a panel of 409-cancer related genes (409-CRG) combined with whole exome sequencing (WES). Analysis of the 409-CRG panel detected low-frequency variants with high sensitivity, and WES identified moderate and high frequency somatic variants as well as germline variants.

Immune response-associated gene analysis of 1,000 cancer patients using whole-exome sequencing and gene expression profiling-Project HOPE.

Project HOPE (High-tech Omics-based Patient Evaluation) has been progressing since its implementation in 2014 using whole-exome sequencing (WES) and gene expression profiling (GEP). With the aim of evaluating immune status in cancer patients, a gene panel consisting of 164 immune response-associated genes (56 antigen-presenting cell and T-cell-associated genes, 34 cytokine- and metabolism-associated genes, 47 TNF and TNF receptor superfamily genes, and 27 regulatory T-cell-associated genes) was established, and its expression and mutation status were investigated using 1,000 cancer patient-derived tumors. Regarding WES, sequencing and variant calling were performed using the Ion Proton system. The average number of single-nucleotide variants (SNVs) detected per sample was 183 ± 507, and the number of hypermutators with more than 500 total SNVs was 51 cases. Regarding GEP, seven immune response-associated genes (VTCN1, IL2RA, ULBP2, TREM1, MSR1, TNFSF9 and TNFRSF12A) were more than 2-fold overexpressed compared with normal tissues in more than 2 organs. Specifically, the positive rate of PD-L1 expression in all patients was 25.8%, and PD-L1 expression was significantly upregulated in hypermutators. The simultaneous analyses of WES and GEP based on immune response-associated genes are very intriguing tools to screen cancer patients suitable for immune checkpoint antibody therapy.

Prevalence of low-penetrant germline TP53 D49H mutation in Japanese cancer patients.

Using whole exome sequencing data obtained from 1,685 Japanese cancer patients, we examined genetic variations of germline TP53 and found 10 types of non-synonymous single nucleotide variants. In the present study, we focused on 6 patients with germline D49H mutation located in the transactivation domain 2 of p53 protein, since the mutation seemed to be prevalent in cancer patients and to be pathogenic. According to the initial survey for family history of the proband with the germline TP53 D49H mutation, one osteosarcoma patient and his pedigree fulfill the criteria for Li-Fraumeni-like syndrome and the 2009 Chompret criteria for germline TP53 mutation screening. Since this patient possesses double germline mutations of TP53 D49H and A159D, further studies are required to evaluate contribution of the D49H mutation in this morbidity. The remaining 5 patients had family histories of cancer, but none fulfills the criteria either for the Li-Fraumeni/Li-Fraumeni-like syndromes or the 2009 Chompret criteria for germline TP53 mutation screening. It is possible to postulate that the germline TP53 D49H mutation is likely to be low-penetrant in some pedigrees. The present study also indicates that the survey for the germline TP53 mutation plays an important role in clinical practice as it will prevent mistaking cancer patients with unusual heredities for sporadic cases.