Immunogenomics in personalized cancer treatments.

Recent advances in next-generation sequencing technologies have led to significant improvements in cancer genomic research and cancer treatment. Through the use of comprehensive cancer genome data, precision medicine has become more of a reality; albeit, at present, only ~10-15% of patients can benefit from current genomic testing practices. Improvements in cancer genome analyses have contributed to a better understanding of antitumor immunity and have provided solutions for targeting highly cancer-specific neoantigens generated from somatic mutations in individual patients. Since then, numerous studies have demonstrated the importance of neoantigens and neoantigen-reactive T cells in the tumor microenvironment and how their presence influences the beneficial responses associated with various cancer immunotherapies, including immune checkpoint inhibitor therapy. Indeed, cancer immunotherapies that explicitly target neoantigens specific to individual cancer patients would lead to the ultimate form of cancer precision medicine. For this to be realized, several issues would need to be overcome, including the accurate prediction and selection of neoantigens that can induce cytotoxic T cells in individual patients. The precise prediction of target neoantigens will likely accelerate the development of personalized immunotherapy including cancer vaccines and T-cell receptor-engineered T-cell therapy for patients with cancer.

Phase I clinical study of multiple epitope peptide vaccine combined with chemoradiation therapy in esophageal cancer patients.

BACKGROUND: Chemoradiation therapy (CRT) has been widely used for unresectable esophageal squamous cell carcinoma (ESCC) patients. However, many patients develop local recurrence after CRT. In this study, we hypothesized that the immunotherapy by peptide vaccine may be effective for the eradication of minimal residual cancer cells after CRT. This study was conducted as a phase I clinical trial of multiple-peptide vaccine therapy combined with CRT on patients with unresectable ESCC. PATIENTS AND METHODS: HLA-A*2402 positive 11 unresectable chemo-naïve ESCC patients were treated by HLA-A*2402-restricted multi-peptide vaccine combined with CRT. The peptide vaccine included the 5 peptides as follows; TTK protein kinase (TTK), up-regulated lung cancer 10 (URLC10), insulin-like growth factor-II mRNA binding protein 3 (KOC1), vascular endothelial growth factor receptor 1 (VEGFR1) and 2 (VEGFR2). CRT consisted of radiotherapy (60 Gy) with concurrent cisplatin (40 mg/m²) and 5-fluorouracil (400 mg/m²). Peptide vaccines mixed with incomplete Freund's adjuvant were injected subcutaneously once a week on at least 8 occasions combined with CRT. RESULTS: Vaccination with CRT therapy was well-tolerated, and no severe adverse effects were observed. In the case of grade 3 toxicities, leucopenia, neutropenia, anemia and thrombocutopenia occurred in 54.5%, 27.3%, 27.3% and 9.1% of patients, respectively. Grade 1 local skin reactions in the injection sites of vaccination were observed in 81.8% of patients. The expressions of HLA class I, URLC10, TTK, KOC1, VEGFR1 and VEGFR2 antigens were observed in the tumor tissues of all patients. All patients showed peptide-specific cytotoxic T lymphocytes responses in at least one of the 5 kinds of peptide antigens during the vaccination. Six cases of complete response (CR) and 5 cases of progressive disease (PD) were observed after the 8th vaccination. The 4 CR patients who continued the peptide vaccination experienced long consistent CR for 2.0, 2.9 4.5 and 4.6 years. CONCLUSIONS: A combination therapy of multi-peptide vaccine with CRT can successfully be performed with satisfactory levels of safety, and application of this combination therapy may be an effective treatment for patients with unresectable ESCC. TRIAL REGISTRATION: ClinicalTrial.gov, number NCT00632333.

The DNA binding site specificity and antiproliferative property of ternary Pt(II) and Zn(II) complexes of phenanthroline and N,N'-ethylenediaminediacetic acid.

The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

Effect of taurine supplementation on the alterations in amino Acid content in skeletal muscle with exercise in rat.

Taurine included abundantly in skeletal muscle, particularly in the slow-twitch fibers, enhances exercise performance. However, the exact mechanisms for this effect have been unclear. The present study investigated the influence of taurine supplementation on amino acids profile in skeletal muscles as one of mechanisms in the enhancement of exercise performance induced by taurine. In the rats that received taurine solution, amino acids concentrations were comprehensively quantified in two portions with different fiber compositions in the fast-twitch fiber dominant (FFD) gastrocnemius muscle after 2 weeks, and in the gastrocnemius and additional other FFD muscles, liver, and plasma with exhausted exercise after 3 weeks. In the FFD muscles after 2 weeks, a common phenomenon that decreased concentrations of threonine (-16%), serine (-15~-16%), and glycine (-6~-16%) were observed, and they are categorized in the pyruvate precursors for hepatic gluconeogenesis rather than biosynthesis, polar, and side-chain structures. The decreases in the three amino acids were significantly emphasized after an additional week of taurine supplementation in the FFD muscles (p values in three amino acids in these tissues were less than 0.001-0.05), but not in the liver and plasma, accompanied with significantly increase of running time to exhaustion (p <0.05). In contrast, the three amino acids (threonine and serine; p < 0.05, glycine; p < 0.01) and alanine (p < 0.01) in the liver were significantly decreased and increased, respectively, following the exhaustive exercise. In conclusion, the taurine-induced reductions of these amino acids in skeletal muscle might be one of the mechanisms which underpin the enhancement of exercise performance by taurine. Key pointsTaurine ingestion significantly decreased certain amino acids in skeletal muscles accompanied with enhanced exercise performance.The decreased amino acids in common were threonine, serine, and glycine, but not alanine; pyruvate precursor for gluconeogenesis.The alteration of three amino acids in muscles was maintained after exhausted exercise.The muscular alterations of them might be one of taurine-induced roles on exercise performance.

Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma.

Osteosarcoma is the most prevalent bone malignant tumor in children and adolescents, and displays heterogeneous histology and high propensity for distant metastasis. Although adjuvant chemotherapy remarkably improved treatment outcome over the past few decades, prognosis for osteosarcoma patients with pulmonary metastasis is still unsatisfactory. To identify novel therapeutic targets for osteosarcoma, we investigated the gene expression profile of osteosarcomas by cDNA microarray analysis and found transactivation of receptor tyrosine kinase-like orphan receptor 2 (ROR2) expression in the majority of osteosarcoma samples. Treatment of osteosarcoma cell lines with siRNA against ROR2 significantly inhibited cell proliferation and migration. We also identified wingless-type MMTV integration site family, member 5B (WNT5B) as a putative ROR2 ligand and that the physiological interaction of WNT5B and ROR2 could enhance cell migration, indicating the possible roles of ROR2 and WNT5B in the metastatic property of osteosarcoma cells. Taken together, our findings revealed that the WNT5B/ROR2 signaling pathway is a promising therapeutic target for osteosarcoma.

Association of single-nucleotide polymorphisms in MTMR9 gene with obesity.

Genetic factors are clearly involved in the development of obesity, but the genetic background of obesity remains largely unclear. Starting from 62 663 gene-based single-nucleotide polymorphisms (SNPs) in three sequential case-control association studies, we identified a replicated association between the obesity phenotype (BMI > or =30 kg/m(2)) and a SNP (rs2293855) located in the myotublarin-related protein 9 (MTMR9) gene in the chromosomal segment 8p23-p22. P-values (minor allele dominant model) of the first set (93 cases versus 649 controls) and the second set (564 cases versus 562 controls) were 0.008 and 0.0002, respectively. The association was replicated in the third set [394 cases versus 958 controls, P = 0.005, odds ratio (95% CI) =1.40 (1.11-1.78)]. The global P-value was 0.0000005. A multiple regression analysis revealed that gender, age BMI and rs2293855 genotype (minor allele dominant model) were significantly associated with both systolic and diastolic blood pressures. MTMR9 was shown to be the only gene within the haplotype block that contained SNPs associated with obesity. Both the transcript and protein of MTMR9 were detected in the rodent lateral hypothalamic area as well as in the arcuate nucleus, and the protein co-existed with orexin, melanin concentrating hormone, neuropeptide Y and proopiomelanocortin. The levels of MTMR9 transcript in the murine hypothalamic region increased after fasting and were decreased by a high-fat diet. Our data suggested that genetic variations in MTMR9 may confer a predisposition towards obesity and hypertension through regulation of hypothalamic neuropeptides.

Functional single-nucleotide polymorphisms in the secretogranin III (SCG3) gene that form secretory granules with appetite-related neuropeptides are associated with obesity.

CONTEXT: Genetic factors are important for the development of obesity. However, the genetic background of obesity still remains unclear. OBJECTIVE: Our objective was to search for obesity-related genes using a large number of gene-based single-nucleotide polymorphisms (SNPs). DESIGN AND SETTING: We conducted case-control association analyses using 94 obese patients and 658 controls with 62,663 SNPs selected from the SNP database. SNPs that possessed P < or = 0.02 were further analyzed using 796 obese and 711 control subjects. One SNP (rs3764220) in the secretogranin III (SCG3) gene showed the lowest P value (P = 0.0000019). We sequenced an approximately 300-kb genomic region around rs3764220 and discovered SNPs for haplotype analyses. SCG3 was the only gene within a haplotype block that contained rs3764220. The functions of SCG3 were studied. PATIENTS: Obese subjects (body mass index > or = 30 kg/m(2), n = 890) and control subjects (general population; n = 658, body mass index < or = 25 kg/m(2); n = 711) were recruited for this study. RESULTS: Twelve SNPs in the SCG3 gene including rs3764220 were in almost complete linkage disequilibrium and significantly associated with an obesity phenotype. Two SNPs (rs16964465, rs16964476) affected the transcriptional activity of SCG3, and subjects with the minor allele seemed to be resistant to obesity (odds ratio, 9.23; 95% confidence interval, 2.77-30.80; chi(2) = 19.2; P = 0.0000067). SCG3 mRNA and immunoreactivity were detected in the paraventricular nucleus, lateral hypothalamic area, and arcuate nucleus, and the protein coexisted with orexin, melanin-concentrating hormone, neuropeptide Y, and proopiomelanocortin. SCG3 formed a granule-like structure together with these neuropeptides. CONCLUSIONS: Genetic variations in the SCG3 gene may influence the risk of obesity through possible regulation of hypothalamic neuropeptide secretion.

Label-free DNA sensors using ultrasensitive diamond field-effect transistors in solution.

Charge detection biosensors have recently become the focal point of biosensor research, especially field-effect-transistors (FETs) that combine compactness, low cost, high input, and low output impedances, to realize simple and stable in vivo diagnostic systems. However, critical evaluation of the possibility and limitations of charge detection of label-free DNA hybridization using silicon-based ion-sensitive FETs (ISFETs) has been introduced recently. The channel surface of these devices must be covered by relatively thick insulating layers ( SiO2, Si3N4, Al2O3, or Ta2O5) to protect against the invasion of ions from solution. These thick insulating layers are not suitable for charge detection of DNA and miniaturization, as the small capacitance of thick insulating layers restricts translation of the negative DNA charge from the electrolyte to the channel surface. To overcome these difficulties, thin-gate-insulator FET sensors should be developed. Here, we report diamond solution-gate FETs (SGFETs), where the DNA-immobilized channels are exposed directly to the electrolyte solution without gate insulator. These SGFETs operate stably within the large potential window of diamond (>3.0 V). Thus, the channel surface does not need to be covered by thick insulating layers, and DNA is immobilized directly through amine sites, which is a factor of 30 more sensitive than existing Si-ISFET DNA sensors. Diamond SGFETs can rapidly detect complementary, 3-mer mismatched (10 pM) and has a potential for the detection of single-base mismatched oligonucleotide DNA, without biological degradation by cyclically repeated hybridization and denature.

DNA micropatterning on polycrystalline diamond via one-step direct amination.

We report a novel method of one-step direct amination on polycrystalline diamond to produce functionalized surfaces for DNA micropatterning by photolithography. Polycrystalline diamond was exposed to UV irradiation in ammonia gas to generate amine groups directly. After patterning, optical microscopy confirmed that micropatterns covered with an Au mask were regular in size and shape. The regions outside the micropatterns were passivated with fluorine termination by C3F8 plasma, and the chemical changes on the two different surfaces--the amine groups inside the patterned regions by one-step direct amination and fluorine termination outside the patterned regions--were characterized by spatially resolved X-ray photoelectron spectroscopy (XPS). The patterned areas terminated with active amine groups were then immobilized with probe DNA via a bifunctional molecule. The sequence specificity was conducted by hybridizing fluorescently labeled target DNA to both complementary and noncomplementary probe DNA attached inside the micropatterns. The fluorescence micropatterns observed by epifluorescence microscopy corresponded to those imaged by optical microscopy. DNA hybridization and denaturation experiments on a DNA-modified diamond show that the diamond surfaces reveal superior stability. The influence of a different amination time on fluorescence intensity was compared. Different terminations as passivated layers were investigated, and as a result, fluorine termination points to the greatest signal-to-noise ratio.

Effect of Saiko-ka-ryukotsu-borei-to on amylase activity in mice.

The effects of Saiko-ka-ryukotsu-borei-to (SRBT), a Chinese medicinal prescription, on mouse serum amylase activity were investigated in vivo. SRBT was found to not only dose- and/or time-dependently augment amylase activity, but also to increase alpha-amylase protein content and soluble starch metabolic activity. These results provide a rational basis for the clinical use of SRBT that may accompany disease therapy.

Prediction of outcome of advanced cervical cancer to thermoradiotherapy according to expression profiles of 35 genes selected by cDNA microarray analysis.

PURPOSE: To identify a set of genes related to thermoradiosensitivity of cervical carcinoma and to establish a predictive method. METHODS AND MATERIALS: A total of 19 patients with cervical cancer (1 with Stage IIIA, 11 with Stage IIIB, 5 with Stage IVA, and 2 with Stage IVB) who underwent definitive thermoradiotherapy between May 1995 and August 2001 were included in this study. We compared the expression profiles of 8 thermoradiosensitive and 11 thermoradioresistant tumors obtained by punch biopsy before treatment using a cDNA microarray consisting of 23,040 human genes. RESULTS: We selected 35 genes on the basis of a clustering analysis and confirmed the validity of these genes with a cross-validation test. Some of these genes were already known to be associated with apoptosis (BIK, TEGT, SSI-3), hypoxia-inducible genes (HIF1A, CA12), and tumor cell invasion and metastasis (CTSL, CTSB, PLAU, CD44). We developed a "predictive score" system that could clearly separate the thermoradiosensitive group from the thermoradioresistant group. CONCLUSION: These results from the treatment program between May 1995 and August 2001 showed that by using gene-expression profiles we can predict the outcome of thermoradiotherapy for advanced cervical carcinoma. A "predictive score" system was developed that could clearly separate the thermoradiosensitive group from the thermoradioresistant group. These results may eventually lead to the achievement of "personalized therapy" for this disease.

Cystatin 10, a novel chondrocyte-specific protein, may promote the last steps of the chondrocyte differentiation pathway.

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.

Gene-expression profiles of human tumor xenografts in nude mice treated orally with the EGFR tyrosine kinase inhibitor ZD1839.

To date, no single or multiple molecular markers have been successful in predicting sensitivity of individual patients to anti-cancer drugs. As the nature of a specific cancer is considered to be defined by the proteins being expressed in the tumor cells, systematic analysis of gene-expression profiles may provide information reflecting sensitivity of a given tumor to certain drugs. Recent progress in genome technology has enabled us to examine expression profiles of thousands of genes in a single experiment. We used this approach to examine 13 xenografts of human tumors implanted into nude mice for sensitivity to an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), ZD1839 (Iressa). To identify genes that might be associated with sensitivity to this drug we used a cDNA microarray representing 23,040 genes to analyze expression profiles of the 13 xenografts and identified 114 genes whose expression levels correlated significantly with sensitivity of the tumors to ZD1839. We then investigated alteration of expression profiles in response to the ZD1839 treatment in four non-small cell lung cancer (NSCLC) xenografts, of which two (LC6 and LC11) were sensitive and the other two (Lu116 and L27) were resistant to this EGFR-TKI. Systematic analysis of expression at various time points during oral treatment for 14 days, compared with corresponding untreated samples, identified a set of genes whose expression levels changed in the two sensitive tumors but not in the two resistant tumors. The data obtained here should provide useful information on the molecular mechanism underlying clinical responses to EGFR-TKIs, aid the development of novel therapies for lung cancer, and potentially identify predictive molecular markers for sensitivity to ZD1839.

Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro.

Using a culture system that facilitates osteogenic differentiation of bone marrow-derived human mesenchymal stem cells, we analyzed gene-expression profiles during the mineralization process by means of a cDNA microarray system consisting of 23,040 genes. We compared expression profiles of the cells at days 3, 15, and 27 of incubation in media containing either a combination of 0.1 microM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, and 10 mM beta-glycerophosphate, dexamethasone only, ascorbic acid-2-phosphate plus beta-glycerophosphate, or medium without any of these osteogenic supplements. Histochemical analysis revealed osteogenic differentiation of cells incubated in the presence of all three agents, but not in the other cultures. Comparison of the expression profiles disclosed transcriptional stimulation of 55 genes and repression of 82 genes among more than 20,000 examined. A set of differentially expressed genes we report here should contribute to a better understanding of the process of mineralization in the matrix surrounding human mesenchymal stem cells.