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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.

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.

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.

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., Signaling pathways involved in 65 GIST samples were investigated. Pathways were curated by genetic alterations, including oncogenic mutations and amplification‐dependent expression enhancement for oncogenes, and loss of heterozygosity and expression reduction for tumor suppressor genes. Malignant GIST with metastasis and high‐risk showed mutations, LOH, and aberrant expression dominantly in driver genes associated with signaling pathways of PI3K and the cell cycle.

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). 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. 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. 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.

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.

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.

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., This study describes our analysis of tumors with a low tumor burden level in the Japanese version of The Cancer Genome Atlas (JCGA), a data set of 5020 primary solid tumors. In the ultralow tumor mutational burden tumors, expression analysis showed decreased TP53 inactivation and chromosomal instability. Our analysis focuses on how these tumors can provide insight into tumors with low somatic alteration that are difficult to detect solely by whole exome 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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s11010-017-2977-1) contains supplementary material, which is available to authorized users.

Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell‑type specific, and molecules embedded in exosomes can be useful fluid‑based 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 23‑fold and 20‑fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa‑2 cell line derived from primary tumor cells. Liquid chromatography‑mass spectrometry‑based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis‑derived 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=4.2x10‑4; invasion: r=0.60, P=3.2x10‑2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10‑2; invasion: r=0.51, P=9.2x10‑2). 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.

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.

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.

Next-generation DNA sequencing (NGS) of the genomes of cancer cells is contributing to new discoveries that illuminate the mechanisms of tumorigenesis. To this end, the International Cancer Genome Consortium and The Cancer Genome Atlas are investigating novel alterations of genes that will define the pathways and mechanisms of the development and growth of cancers. These efforts contribute to the development of innovative pharmaceuticals as well as to the introduction of genome sequencing as a component of personalized medicine. In particular, chromosomal translocations that fuse coding sequences serve as important pharmaceutical targets and diagnostic markers given their association with tumorigenesis. Although increasing numbers of fusion genes are being discovered using NGS, the methodology used to identify such fusion genes is complicated, expensive, and requires relatively large samples. Here, to address these problems, we describe the design and development of a panel of 491 fusion genes that performed well in the analysis of cultured human cancer cell lines and 600 clinical tumor specimens.

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.

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.

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.

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.

We analyzed serum ProGRP levels in patients with Ewing sarcoma, and found that 5 out of 9 patients had elevated levels; the values range equally with those of patients with limited disease of small-cell lung carcinoma. Serum ProGRP levels in patients with bone and soft tissue malignancies other than Ewing sarcoma are not elevated. Immunohistochemical studies demonstrated that ProGRP-like immunoreactivities were detected in Ewing sarcoma tissues obtained from 2 patients with elevated serum ProGRP levels, suggesting that ProGRP is a product of tumor cells of Ewing sarcoma. These results indicate that serum ProGRP could serve as a specific tumor marker for Ewing sarcoma. Since ProGRP is a major hormonal product of tumor cells of small-cell lung carcinoma, a typical neuroendocrine carcinoma, it is reasonable to postulate that the present study provides an evidence for Ewing sarcoma to possess neuroendocrine differentiation.

XIAP-associated factor 1 (XAF1) is ubiquitously expressed in normal tissues, but its suppression in cancer cells is strongly associated with tumor progression. Although downregulation of XAF1 is observed in tumors, its expression profile in the peripheral blood of cancer patients has not yet been investigated. Here, we identified a novel XAF1 splice variant in cancer cells and then investigated the expression level of this variant in peripheral blood containing gastric cancer-derived circulating tumor cells (CTCs). To identify splice variants, RT-PCR and DNA sequencing were performed in mRNAs extracted from many cancer cells. We then carried out quantitative RT-PCR to investigate expression in peripheral blood from all 96 gastric cancer patients and 22 healthy volunteers. The XAF1 variant harbored a premature termination codon (PTC) and was differentially expressed in highly metastatic cancer cells versus the parental cells, and that nonsense-mediated mRNA decay (NMD) was suppressed in the variant-expressing cells. Furthermore, splice variants of XAF1 were upregulated in peripheral blood containing CTCs. In XAF1 variant-expressing patients, the expression levels of other NMD-targeted genes also increased, suggesting that the NMD pathway was suppressed in CTCs. Our study identified a novel splice variant of XAF1 in cancer cells. This variant was regulated through the NMD pathway and accumulated in NMD-suppressed metastatic cancer cells and peripheral blood containing CTCs. The presence of XAF1 transcripts harboring the PTC in the peripheral blood may be useful as an indicator of NMD inhibition in CTCs.

The Project HOPE (High-tech Omics-based Patient Evaluation) for cancer medicine aims to evaluate biological characteristics of each cancer tissue as well as diathesis of each patient in around 1,000 consecutive cases per year, who receive operations at the Shizuoka Cancer Center. Cancer tissues are investigated by whole-exome sequencing for 18,835 genes, focusing on 12,776 in-house cancer hotspots from 483 cancer-associated genes. To confirm cancer-specific genetic changes, we analyzed blood cells to collate with data of cancer tissues, and we reevaluate cancer tissues by comprehensive cancer panel for 409 genes. In order to investigate diathesis of the patients, we evaluate 43,015 hotspots associated with non-cancerous diseases. In terms of gene expression profiling, we analyze cancer-specific alterations for 29,833 genes using tumor and adjacent normal tissues. If and when necessary, we investigate tumor and normal tissues by proteomics and metabolomics. The model experiments using glioblastoma cell lines demonstrated that the method is appropriate for clinical application. The Project HOPE makes it possible to implement individualized medicine and to practice preventive and presymptomatic medicine for cancer patients. Furthermore, the project can create important seeds for research and development in cancer medicine.

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.

Background: Recent progression of systematic tumor-molecular profiling study centered on genome sequencing have elucidated molecular signature underlying each tumor type. Tumor molecular profiling enables efficient identification of somatic genetic alterations as potential therapeutic targets. In January 2014, the Shizuoka Cancer Center of Japan launched Project HOPE, which is the first prospective molecular profiling study centered on genome sequencing across multiple tumor types in Japan. Herein, we describe the distributions of the tumor mutation burden (TMB; number of mutations/Mb), genetic alterations frequency, mutational signature across tumor types, and their associations with tissue of origin, histological type, carcinogenic factors, and ethnicity. Methods: Between January 2014 and January 2017, 3,163 tumor samples from 3,011 patients who underwent surgery at the Shizuoka Cancer Center were collected with informed consent and subjected to whole-exome sequencing (WES) with an Ion Proton system. Corresponding peripheral blood samples were also subjected to WES for identification of tumor-specific genetic alterations. Oncogenic fusions were detected by targeted RNA sequencing. Among them, data of 2,126 primary tumor samples obtained from 2,091 patients without neoadjuvant chemotherapy and/or radiation therapy were selected for this study. Results: The principal tumor types of ≥40 primary tumors were as follows (N, TMB): colon adenocarcinoma (COAD; 459, 3.6), rectal adenocarcinoma (READ; 366, 3.1), lung adenocarcinoma (LUAD; 290, 1.7), stomach adenocarcinoma (STAD; 221, 3.0), hepatocellular carcinoma (HCC; 112, 3.6), head and neck squamous cell carcinoma (HNSC; 112, 2.8), breast invasive ductal carcinoma (IDC; 82, 1.1), lung squamous cell carcinoma (LUSC; 81, 5.9). Approximately 8.9% of the samples (154 / 1,723) showed a high mutation burden above the threshold defined by the TMB distribution in each principal tumor type, and the mutation signature was related to a defect of DNA repair and exposure to environmental mutagens. In comparison of distribution of TMB in each tumor type between TCGA and this study, some differences in TMB distribution were observed, including LUAD, LUSC, and IDC. In gene mutations based on OncoKB site (October 2018), level 1 or 2 of somatic gene mutation was frequently observed in glioma (84.6%), gastrointestinal stromal tumor (77.4%), melanoma (40.0%), and LUAD (40.0%). Conclusions: This established catalog of somatic genetic alterations across multiple tumor types in Japanese cancer patients can contribute as benchmark to clinical-trial design with the aim of expanding the number of patients suitable for molecular-targeted therapies in Japan. Based on these data, we are planning clinical trials of molecular-targeted therapies for patients with solid tumor harboring gene alterations. Citation Format: Hirotsugu Kenmotsu, Masakuni Serizawa, Takeshi Nagashima, Keiichi Ohshima, Keiichi Hatakeyama, Yuji Shimoda, Shumpei Ohnami, Koji Maruyama, Tohru Mochizuki, Yasuto Akiyama, Takashi Sugino, Kenichi Urakami, Masatoshi Kusuhara, Ken Yamaguchi. Establishment of a catalog of somatic genetic alterations of Japanese cancer patients across multiple tumor types at Shizuoka Cancer Center [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2721.

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.

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.

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.

The identification of additional therapeutic targets by clinical molecular profiling is necessary to expand the range of molecular-targeted cancer therapeutics. This study aimed to identify novel functional tumor-specific single nucleotide variants (SNVs) in the kinase domain of receptor tyrosine kinases (RTKs), from whole-exome sequencing (WES) data.SNVs were selected from WES data of multiple cancer types using both cancer-related databases and the index reflecting molecular evolution. Immunoblotting and luciferase assay were performed to assess the function of selected SNVs.Among the seven selected SNVs, two, namely neurotrophic receptor tyrosine kinase 1 (NTRK1) V710A and fms related tyrosine kinase 3 (FLT3) K868N, detected in kinase subdomain IX, were investigated. These SNVs inhibited the autophosphorylation of the respective RTKs, thereby reducing the activity of extracellular signal-regulated kinases.RTK subdomain IX is a promising target for the molecular design of kinase inhibitors.

Next-generation DNA sequencing (NGS) of the genomes of cancer cells is contributing to new discoveries that illuminate the mechanisms of tumorigenesis. To this end, the International Cancer Genome Consortium and The Cancer Genome Atlas are investigating novel alterations of genes that will define the pathways and mechanisms of the development and growth of cancers. These efforts contribute to the development of innovative pharmaceuticals as well as to the introduction of genome sequencing as a component of personalized medicine. In particular, chromosomal translocations that fuse coding sequences serve as important pharmaceutical targets and diagnostic markers given their association with tumorigenesis. Although increasing numbers of fusion genes are being discovered using NGS, the methodology used to identify such fusion genes is complicated, expensive, and requires relatively large samples. Here, to address these problems, we describe the design and development of a panel of 491 fusion genes that performed well in the analysis of cultured human cancer cell lines and 600 clinical tumor specimens.

Parathyroid hormone-related protein (PTHrP) contains a nuclear localization signal (NLS) sequence within 87-107. NLS sequences are generally capable of penetrating cellular membranes due to a richness of basic amino acid residues, and thus have been used as cell-penetrating peptides (CPPs) to translocate biologically active peptides/proteins into cells. The NLS sequence of PTHrP is not exception to this finding; however, PTHrP(87-107) contains 2 acidic glutamate residues at 99 and 101 within the basic amino acid stretch, which is not commonly observed in other CPPs such as HIV-1 Tat(48-60). In this study, we indicated structure-function relationship of the PTHrP NLS to understand the effect of acidic glutamate residues on cell permeability and intracellular localization. We chemically synthesized PTHrP(87-107) and its N-terminally truncated analogues. Their intracellular localization pattern was analyzed by microscopy, radioimmunoassay, and fluorescence-activated cell sorting. Although all analogues were translocated into cells, internalization by the cytoplasm and/or nucleus was length-dependent; specifically, PTHrP(97-107), PTHrP(95-107), and PTHrP(93-107) were more frequently localized in the cytoplasm. We assume that reduction in the net positive charge within PTHrP NLS analogues resulted in increased cytoplasm- translocation activity. We propose that PTHrP(97-107) is a useful carrier peptide for delivery and expression of cargo molecules in the cytoplasm.

Background: Cancer genome sequencing enables efficient identification of genetic alterations as potential therapeutic targets, representing a fundamental component of precision oncology to make therapeutic decisions based on individualized genetic signatures. In January 2014, the Shizuoka Cancer Center of Japan launched Project HOPE, which is the first prospective molecular profiling study centered on genome sequencing across multiple tumor types. Herein, we describe the distributions of the tumor mutation burden (TMB; number of mutations/Mb), genetic alterations frequency, mutational signature across tumor types, and their associations with tissue of origin, histological type, and carcinogenic factors. Methods: Between January 2014 and April 2017, 3,174 tumor samples from 3,022 patients who underwent surgery at the Shizuoka Cancer Center were collected with informed consent and subjected to whole-exome sequencing (WES) with an Ion Proton system. Corresponding peripheral blood samples were also subjected to WES for identification of tumor-specific genetic alterations. Oncogenic fusions were detected by targeted RNA sequencing. Samples with estimated tumor purity < 20% were excluded because of the risk of false negatives, leaving 2,899 samples (91%) in the final analysis. Results: Overall, 2,642 samples were derived from the primary tumor, with a median TMB of approximately 2.7. The principal tumor types of ≥40 primary tumors were as follows (N, TMB): colorectal adenocarcinoma (CRAD; 905, 3.3), lung adenocarcinoma (LUAD; 324, 1.6), gastric adenocarcinoma (GAD; 272, 3.0), head and neck squamous cell carcinoma (HNSC; 164, 2.6), breast invasive ductal carcinoma (BIDC; 138, 1.1), hepatocellular carcinoma (HCC; 124, 3.6), lung squamous cell carcinoma (LUSC; 79, 5.6), and gastrointestinal stromal tumor (GIST; 41, 0.7). Approximately 7.8% of the samples (160/2,047) showed a high mutation burden above the threshold defined by the TMB distribution in each principal tumor type, and the mutation signature was related to a defect of DNA repair and exposure to environmental mutagens. Dominant oncogenic pathways based on the profile of genetic alterations in each tumor type were as follows: Wnt (CRAD), receptor tyrosine kinase (LUAD, GIST), TP53 (GAD, HNSC, LUSC), PI3K (BIDC), and chromatin modification (HCC). GIST was the tumor type with the highest proportion of clinically actionable genetic alterations, followed by BIDC and LUAD. Conclusions: This is the first report of a prospective genome sequencing analysis across multiple tumor types in Japan. The resulting mutational profile can provide a landscape of commonalities and differences in genetic profiles among tumor types in Japanese patients with cancer, and can contribute to planning a basket study for expanding the patients suitable for treatment with molecular-targeted drugs. Citation Format: Masakuni Serizawa, Takeshi Nagashima, Keiichi Ohshima, Keiichi Hatakeyama, Yuji Shimoda, Shumpei Ohnami, Kouji Maruyama, Takashi Sugino, Tohru Mochizuki, Yasuto Akiyama, Kenichi Urakami, Masatoshi Kusuhara, Medical staff and experimental staff of the Shizuoka Cancer Center, Ken Yamaguchi. Genomic landscape of Japanese cancer patients across multiple tumor types: Prospective molecular profiling study of 3,022 patients at Shizuoka Cancer Center [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3422.

The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus.

The Project HOPE (High-tech Omics-based Patient Evaluation) for cancer medicine aims to evaluate biological characteristics of each cancer tissue as well as diathesis of each patient in around 1,000 consecutive cases per year, who receive operations at the Shizuoka Cancer Center. Cancer tissues are investigated by whole-exome sequencing for 18,835 genes, focusing on 12,776 in-house cancer hotspots from 483 cancer-associated genes. To confirm cancer-specific genetic changes, we analyzed blood cells to collate with data of cancer tissues, and we reevaluate cancer tissues by comprehensive cancer panel for 409 genes. In order to investigate diathesis of the patients, we evaluate 43,015 hotspots associated with non-cancerous diseases. In terms of gene expression profiling, we analyze cancer-specific alterations for 29,833 genes using tumor and adjacent normal tissues. If and when necessary, we investigate tumor and normal tissues by proteomics and metabolomics. The model experiments using glioblastoma cell lines demonstrated that the method is appropriate for clinical application. The Project HOPE makes it possible to implement individualized medicine and to practice preventive and presymptomatic medicine for cancer patients. Furthermore, the project can create important seeds for research and development in cancer medicine.

Many intestinal hormones are also distributed throughout the central nervous system (CNS). However, the existence of secretin, a typical intestinal hormone, has not been clearly shown in the brain. There have been conflicting data concerning the expression of secretin in the CNS. The aim of this study was to confirm the existence of secretin in the CNS immunochemically and histochemically. Proforms of secretin were detected in porcine cortex extracts by gel filtration chromatography and reverse-phase HPLC with a radioimmunoassay using different specific antibodies raised against secretin and prosecretin (1-41), indicating the production and processing of secretin precursor in the CNS. An immunocytochemical search in several regions of the rat CNS with the two antibodies revealed that secretin-positive neurons were concentrated in a limited area ranging from the corner of the dentate gyrus to the molecular layer in the hippocampus. In situ hybridization also supported this finding. The limited location of secretin-positive cells shown here caused difficulty in detecting secretin in the CNS. The hippocampus is thought to contribute to aging, and secretinergic neurons in this region might play an important role. This study suggests that secretin is produced in the CNS and may act as a neurotransmitter or neuromodulator in the hippocampus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.

The activation of a specific cellular immune response against human cytomegalovirus (CMV) is an important key factor to solving CMV infection after bone marrow transplantation (BMT). In the present study, our purpose was to identify the HLA-A24-restricted cytotoxic T cell (CTL) epitope from the CMV immunogenic matrix protein pp65. We selected five CMV pp65 peptides with HLA-A24 binding motif from the HLA peptide binding predictions web site. Peptide binding assay was performed using biotinylated HLA-A24-restricted MAGE-1 peptide as a reference peptide and transporter associated with antigen processing (TAP)-deficient T2-A24 cells expressing high level of HLA-A24 protein as target cells. After co-incubation of biotinylated MAGE-1 and titrated amounts of competitor peptides with T2-A24 cells, the binding of each peptide was analyzed on a flow cytometer. Peptide binding assay showed that three out of five peptides derived from CMV pp65 bound to T2-A24 cells with various affinity levels. CTL induction assay using peptide-pulsed DC derived from eight HLA-A24(+) donors revealed that the peptide (QYDPVAALF) with the highest affinity was able to elicit potent CTLs which killed peptide-pulsed TISI cells. These CTLs were found to show the killing activity against human fibroblast cells transduced with both HLA-A*2402 and CMV pp65 cDNAs, and CMV-infected HLA-A24(+) fibroblast cells. These results suggested that the peptide (QYDPVAALF) is one of HLA-A24-restricted CTL epitope derived from CMV pp65 protein and may be of therapeutic value in peptide-based vaccines against CMV infection in BMT patients.

Mass propagation of the Japanese angelica tree (Aralia elata seem.)‘Zaoh’ line No.2 was established through somatic embryos. Petioles of leaflets were cultured for induction of calli on an MS medium containing 1mgl-1 of 2, 4-D in combination with 0.5mgl-1 BA. The initiated calli were moved onto MS medium supplemented with glutamine 450mgl-1 and asparagine 300mgl-1. An embryogenic callus developed after 5months and was moved onto regulator-free MS medium. Numerous plantlets were regenerated from this embryogenetic callus, and rooted plantlets were potted after acclimation and planted to the field. The plants had the same characteristics in morphology in the field, except for the number of thorns per internode. The plants also had the same resistance to Phytophthora disease as was in the original genotype ‘Zaoh’ line No.2. This study provided a method for mass propagation from petioles of leaflets and proves that regenerated plants maintain the morphogenetic characteristics and disease resistance of the original genotype.

Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 μg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.

Chromogranin A (CgA) is a member of a family of highly acidic proteins, chromogranins, which are co-stored in the adrenergic neurons and paraneurons and co-released with adrenaline and noradrenaline (NAd) in response to adequate stimulation. The present study provides novel evidence that CgA-like immunoreactivity (IR) is stored in the exocrine cells in the granular convoluted tubule, and is secreted into saliva by stimulation with NAd and acetylcholine (ACh) in the isolated and perfused rat submandibular gland. NAd at 1 microM produced maximum secretion of CgA-like IR (<< 0.9 mM) and a marked increase in salivary flow. Further increases in NAd concentration (10 or 100 microM) yielded concentration-dependent decreases in both responses. ACh at 1 microM produced maximum salivary flow and a slight elevation of CgA-like IR secretion (6 microM); 100 microM ACh decreased the salivary flow but increased the CgA-like IR secretion (0.6 mM). Electron microscopic examination showed vigorous compound exocytosis of secretory granules in the cells of the granular convoluted tubule when the submandibular gland was stimulated with 1 microM NAd. These results provide an experimental basis for the view that the salivary CgA-like IR secretion may be a sensitive and quantitative index of the activity of the sympathetic nervous system innervating the gland.

Complete amino acid sequences of two novel bioactive polypeptides, each containing 66 amino acid residues, BmK AS and BmK AS-1 purified from the venom of Chinese scorpion Buthus martensi Karsch, have been determined by Edman sequencing and mass spectrometry on native proteins, reduced and S -carboxymethylated proteins and their peptides obtained after cleavage with proteolytic enzymes. Sequence analysis showed 86.4% structural identity between BmK AS and BmK AS-1 and also a high sequence similarity between BmK ASs and AaH IT4, a unique anti-insect toxin and a ligand of Na + channels obtained from Sahara scorpion A. australis Hector, but poor sequence homology between BmK ASs and those of the known α-, β-type and long-chain insect-selective type scorpion neurotoxins. The positions of four disulfide bridges in BmK AS-1 were established as Cys-12 and Cys-62, Cys-16 and Cys-37, Cys-23 and Cys-44, and Cys-27 and Cys-46, which are the same as those in α- and β-scorpion neurotoxins. These results suggest that BmK ASs and AaH IT4 may form a new group sharing similar structural and functional properties in the family of scorpion neurotoxic polypeptides.