S-1 and irinotecan plus bevacizumab versus mFOLFOX6 or CapeOX plus bevacizumab as first-line treatment in patients with metastatic colorectal cancer (TRICOLORE): a randomized, open-label, phase III, noninferiority trial.

Background: Combination therapy with oral fluoropyrimidine and irinotecan has not yet been established as first-line treatment of metastatic colorectal cancer (mCRC). We carried out a randomized, open-label, phase III trial to determine whether S-1 and irinotecan plus bevacizumab is noninferior to mFOLFOX6 or CapeOX plus bevacizumab in terms of progression-free survival (PFS). Patients and methods: Patients from 53 institutions who had previously untreated mCRC were randomly assigned (1 : 1) to receive either mFOLFOX6 or CapeOX plus bevacizumab (control group) or S-1 and irinotecan plus bevacizumab (experimental group; a 3-week regimen: intravenous infusions of irinotecan 150 mg/m2 and bevacizumab 7.5 mg/kg on day 1, oral S-1 80 mg/m2 twice daily for 2 weeks, followed by a 1-week rest; or a 4-week regimen: irinotecan 100 mg/m2 and bevacizumab 5 mg/kg on days 1 and 15, S-1 80 mg/m2 twice daily for 2 weeks, followed by a 2-week rest). The primary end point was PFS. The noninferiority margin was 1.25; noninferiority would be established if the upper limit of the 95% confidence interval (CI) for the hazard ratio (HR) of the control group versus the experimental group was less than this margin. Result: Between June 2012 and September 2014, 487 patients underwent randomization. Two hundred and forty-three patients assigned to the control group and 241 assigned to the experimental group were included in the primary analysis. Median PFS was 10.8 months (95% CI 9.6-11.6) in the control group and 14.0 months (95% CI 12.4-15.5) in the experimental group (HR 0.84, 95% CI 0.70-1.02; P < 0.0001 for noninferiority, P = 0.0815 for superiority). One hundred and fifty-seven patients (64.9%) in the control group and 140 (58.6%) in the experimental group had adverse events of grade 3 or higher. Conclusion: S-1 and irinotecan plus bevacizumab is noninferior to mFOLFOX6 or CapeOX plus bevacizumab with respect to PFS as first-line treatment of mCRC and could be a new standard treatment. Clinical trials number: UMIN000007834.

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Phase I/II study of sequential therapy with irinotecan and S-1 for metastatic colorectal cancer.

BACKGROUND: Both irinotecan (CPT-11) and S-1 are active against colorectal cancer; however, as S-1 is a prodrug of 5-fluorouracil (5-FU), 5-FU and its metabolites might inhibit the antitumour effect of CPT-11. Therefore, we designed a sequential combination, in which CPT-11 infusion was given on day 1 and S-1 was given orally at 80 mg m(-2) per day on days 3-16 every 3 weeks. METHODS: Twelve patients entered the phase I study, and the recommended doses were determined as a CPT-11 dose of 150 mg m(-2) and an S-1 dose of 80 mg m(-2). RESULTS: In all, 36 patients entered the phase II study, of whom 4 and 16 had complete and partial responses. The overall response rate was 55.6% (95% confidence interval, 38.1-72.1%), and median progression-free survival was 7.7 months (95% confidence interval, 4.8-12.6 months). Grade 3 neutropenia was the most common haematological toxicity and occurred in 6.5% of 215 treatment courses. Grade 3 non-haematological toxicities included anorexia (1.4%) and diarrhoea (0.9%). There was no grade 4 toxicity of any kind. CONCLUSION: Our results suggest that this regimen is convenient, safe and promising, compared with conventional regimens for patients with metastatic colorectal cancer.

Comparison of adrenal functions in kidney transplant recipients with different long-term immunosuppressive treatments--prednisolone and azathioprine versus prednisolone and cyclosporine.

Adverse effects of cyclosporine on the adrenal cortex have been documented in animal experiments, but nothing has been reported in human subjects. Endogenous cortisol in peripheral blood was monitored for three years after transplantation, with 30 kidney recipients on two different immunosuppressive treatments. In the azathioprine group, 16 patients were treated with coadministration of prednisolone at an initial dose of 120 mg/day. In the cyclosporine group, 14 patients were also treated with prednisolone, using an initial dose of 60 mg per day. Short ACTH stimulation tests were performed to reconfirm the results obtained by basal cortisol monitoring. During the first year following transplant, cortisol concentrations in the cyclosporine group were higher, though not significantly so, than those in the azathioprine group, in accordance with cumulative amounts of prednisolone administered. At three years, however, the mean cortisol concentrations in the azathioprine group were 2-3 times higher than those in the cyclosporine group (P < 0.05). All patients in the azathioprine group responded well to ACTH, whereas 4 patients out of 14 in the cyclosporine group showed continuous severe suppression without considerable response to ACTH (P < 0.01). In conclusion, we would like to suggest that adrenocortical toxicity of long-term cyclosporine use may appear one year after transplant, resulting in chronic suppression of the adrenal cortex, and, accordingly, difficulty in further reduction of prednisolone use.

Enhancement of anticoagulant action by warfarin-benzbromarone interaction.

To investigate the interaction between warfarin potassium and benzbromarone, administration of benzbromarone to patients receiving long-term treatment with both drugs was discontinued for 1 week and then resumed, and the resulting changes in the coagulation system were examined. Thrombotest value, activity of coagulation factors II and VIII, concentration of protein induced by vitamin K absence or antagonist-II (PIVKA-II), total plasma concentration of warfarin, and free warfarin concentration were measured during the period of concurrent administration of the two drugs, 1 week after discontinuation of benzbromarone, and after resumption of benzbromarone administration. After administration of benzbromarone had been discontinued for 1 week, the thrombotest value and factor II activity rose significantly whereas PIVKA-II activity dropped significantly compared with corresponding levels before discontinuation, but these parameters tended to revert to the previously maintained levels after resumption of benzbromarone treatment. Activity of the vitamin K-independent factor VIII displayed almost no changes, however. Total plasma warfarin concentration also decreased significantly, and free warfarin concentration was nearly unchanged. These results verified that the anticoagulant action of warfarin is enhanced by concurrent administration of benzbromarone. Accordingly, adequate consideration must be devoted to the prevention of grave hemorrhagic tendencies when these two drugs are administered concurrently.

Enhancement of chemotherapeutic effects with focused shock waves: extracorporeal shock wave chemotherapy (ESWC).

The effects of shock waves in combination with various anti-cancer agents i.e. Bleomycin (BLM), Cisplatin (CDDP) and 5-fluorouracil (5-FU) on tumor cells suspended in media containing these agents were examined. GCIY cells derived from human gastric cancer and LS 174T and SW480 cells derived from human colon cancers were used for in vitro experiments; GCIY and SW480 cells were also transplanted into nude mice for in vivo study. It was only with BLM that enhancement was evident in all three cell lines, with a degree of chemotherapeutic enhancement proportional to the amount of shock wave energy applied. Ladder formation of DNA in GCIY cells was observed only when treated with both BLM and shock waves in combination. When SW480 and GCIY cells transplanted into the backs of nude mice were treated with a combination of intravenously (i.v.) injected BLM and regional exposure to shock waves, a significant enhancement of chemotherapeutic effects was observed in terms of the tumor growth curve.

A new combination chemotherapy with cis-diammine-glycolatoplatinum (Nedaplatin) and 5-fluorouracil for advanced esophageal cancers.

OBJECTIVE: The efficacy of a new chemotherapeutic combination consisting of Cis-diammineglycolatoplatinum (Nedaplatin), a derivative of cisplatin (CDDP), and 5-fluorouracil (5FU) was evaluated in patients with advanced esophageal carcinomas. METHODS: Nedaplatin was administered at a dose of 80 or 100 mg/m2 with 500 ml of saline by slow drip infusion for 120 minutes on day 1. 5FU at a dose of 350 or 500 mg/m2 was mixed with 1,000 ml of saline and administered by continuous infusion for 24 hours on days 1 to 5. PATIENTS OR MATERIALS: This combination chemotherapy was tried in 17 patients with metastatic, recurrent, or bulky unresectable esophageal cancers. Of these, 15 evaluable patients received at least two courses of chemotherapy. RESULTS: The response rates in assessable and all patients were 60% and 52.9%, respectively. Cases with lymph node and liver metastases, as well as primary lesions, showed excellent response to the therapy with positive response rates of 54.5% (6/11), 100% (5/5) and 58.4% (7/12), respectively. The median response duration was 7 (range 3 to 37+) months for patients who achieved a partial response. Adverse drug reactions were limited to three cases of grade 3 toxicity, including allergy, and decreased hemoglobin and platelets, which were well tolerated by the patients. CONCLUSION: The present study thus indicated the combination chemotherapy of Nedaplatin and 5FU to be safe and efficacious for advanced esophageal cancer. Further investigations are clearly warranted.

[Functional screening of p53 status in tumor cells using Saccharomyces cerevisiae].

We have detected both germ-line and somatic p53 mutations in lymphocytes, cell lines and tumor tissues using a functional analysis of p53 tumor suppressor gene based on yeast transcription assay. Through our screening projects of the p53 gene, a number of missense p53 mutations were identified as loss-of-function mutations. This method, previously termed FASAY, is rapid, sensitive, less-expensive and can be automated for screening both somatic and germ-line p53 mutations.

[Genetic alterations of human colorectal cancer].

Genetic alterations of several oncogenes and tumor suppressor genes are associated with human colorectal carcinogenesis. Especially in mutations, the K-ras, p53, APC and DCC gene frequently occurred, and these gene alterations seem to have important roles in colorectal carcinogenesis. We investigated 28 human colon cancer specimens obtained from surgery and five human colon cancer cell lines by PCR-SSCP assay, PCR-OSH assay, RT-PCR or sequencing method. Forty percent of cancers from surgical specimens had Ki-ras 2 (codon 12/13), p53 (Exon 5-8), APC (MCR) gene mutations, and fifty-seven percent of them had lower expression of DCC gene that of normal matched colon mucosa of the same patient. G to A transition was the most frequent in K-ras mutational spectrum in this case; 25% of patients had both k-ras and p53 gene point mutations. Form the results, we concluded that it in colorectal carcinogenesis for both K-ras and p53 gene point mutations might not necessary occur.

CONSEL: for assessing the confidence of phylogenetic tree selection.

UNLABELLED: CONSEL is a program to assess the confidence of the tree selection by giving the p-values for the trees. The main thrust of the program is to calculate the p-value of the Approximately Unbiased (AU) test using the multi-scale bootstrap technique. This p-value is less biased than the other conventional p-values such as the Bootstrap Probability (BP), the Kishino-Hasegawa (KH) test, the Shimodaira-Hasegawa (SH) test, and the Weighted Shimodaira-Hasegawa (WSH) test. CONSEL calculates all these p-values from the output of the phylogeny program packages such as Molphy, PAML, and PAUP*. Furthermore, CONSEL is applicable to a wide class of problems where the BPs are available. AVAILABILITY: The programs are written in C language. The source code for Unix and the executable binary for DOS are found at http://www.ism.ac.jp/~shimo/ CONTACT: shimo@ism.ac.jp

Effects of phosphorus-containing calcium preparation (bone meal powder) and calcium carbonate on serum calcium and phosphorus in young and old healthy volunteers: a double-blinded crossover study.

To evaluate the effects of bone meal powder (BEC) on calcium and phosphorus metabolism, a calcium absorption test was conducted using a preparation of calcium carbonate (CAC) as the control drug. A total of 12 healthy volunteers, consisting of 6 younger (aged 20-29 years, 3 men and 3 women) and 6 older (aged 60-69 years, 3 men and 3 women) persons, were subjected to a double-blinded crossover study. Serum calcium (s-Ca) level significantly increased to 105.3% +/- 1.9% (P < 0.01 vs the basal value; mean +/- SD) from the basal value in the BEC group and to 104.4% +/- 2.7% (P < 0.01) in the CAC group at 3h post load. Urinary excretions of calcium (u-Ca/glomerular filtration rate, u-Ca/GF) after BEC and CAC load rose to 226.6% +/- 154.5% (P < 0.05) and 211.1% +/- 148.0% (P < 0.05), respectively. Serum phosphorus (s-P) levels after BEC load increased to 110.0% +/- 15.1% (P < 0.05), whereas that after CAC load showed no significant change (99.3% +/- 7.9%). On the other hand, urinary excretion of phosphorus (u-P/GF) after CAC load decreased to 60.0% +/- 32.4% (P < 0.01) and that in the BEC group showed no significant change (92.5% +/- 49.5%). The increase in s-Ca led to decrease in serum intact parathyroid hormone (i-PTH) level [77.3% +/- 33.4% (P < 0.05) for BEC and 69.5% +/- 20.3% (P < 0.01) for CAC] although s-P was increased by the BEC load. The responses to BEC and CAC administration were compared in the younger and the older groups. The responses in the younger and the older group showed fundamentally the same trends and to the same extent. However, the changes in serum ionized calcium (i-Ca) and i-PTH levels at 1.5 h post load were significantly smaller in the older group than in the younger group (P < 0.01; P < 0.05). The increment in s-P level after BEC load in the older group was larger than that in the younger group. In conclusion, BEC can modulate not only calcium but also phosphorus metabolism in both younger and older subjects. Further investigations are required to evaluate the effects of BEC on bone density and safety for renal function in long-term observations.

Appropriate likelihood ratio tests and marginal distributions for evolutionary tree models with constraints on parameters.

We show how to make appropriate likelihood ratio tests for evolutionary tree models when parameters such as edge (internodes or branches) lengths have nonnegativity constraints. In such cases, under the null model of an edge length being zero, the marginal distribution of this parameter is proven to be a "half-normal", that is, 50% zero values and 50% the positive half of a normal distribution. Other constrained parameters, such as the proportion of invariant sites, give similar results. To make likelihood ratio tests between nested models, e.g., H(0): homogeneous site rates, and H(1): site rates follow a gamma distribution with variance 1/k, then asymptotically as sequence length increases, the distribution under H(0) becomes a mixture of chi distributions, in this case 50% chi(0), and 50% chi(1) (where the subscript denotes degrees of freedom, i.e. , not the usually assumed 100% chi(1); which leads to a conservative test). Such mixtures are sometimes called distributions. Simulations show that even with sequences as short as 125 sites, some parameters, including the proportion of invariant sites, fit asymptotic distributions closely.

Suppression of endogenous cortisol for evaluating pharmacodynamics of prednisolone in early allograft rejection in renal transplantation.

Concentrations of endogenous cortisol were examined in 34 kidney-transplant recipients by improved "high-performance" liquid chromatography. Ten recipients were treated with prednisolone and azathioprine, the others with prednisolone and cyclosporine. Peripheral serum samples were collected just before transplantation, daily for two weeks after the transplant, weekly until discharge for about two months, and then monthly or occasionally. Mean (+/- SD) cortisol concentrations, initially 145 +/- 87 micrograms/L, decreased immediately to 5.93 +/- 5.11 micrograms/L after transplant, remained at almost these same values for two months, and then swiftly increased to 51 +/- 59 micrograms/L by 1000 days. Cortisol concentrations within the period characterized by a cumulative dose of prednisolone at 300-700 mg were correlated significantly with the presence or absence of acute allograft rejection; patients with cortisol greater than 4 micrograms/L had a higher risk of rejection. The majority of stable patients showed cortisol concentrations between 1 and 4 micrograms/L throughout the cumulative prednisolone period characterized above. Concentrations less than 1 microgram/L after high-dose administration of methylprednisolone were accompanied by severe lung infection. We conclude that suppressed concentrations of endogenous cortisol, as assessed by highly specific HPLC, might provide a basis for predicting the therapeutic efficacy and adverse effects of prednisolone.

Evaluating hypotheses on the origin and evolution of the New Zealand alpine cicadas (Maoricicada) using multiple-comparison tests of tree topology.

The statistical testing of alternative phylogenetic trees is central to evaluating competing evolutionary hypotheses. Fleming proposed that the New Zealand cicada species Maoricicada iolanthe is the sister species to the major radiation of both low-altitude and montane Maoricicada species. However, using 1,520 bp of mitochondrial DNA sequence data from the cytochrome oxidase subunit I, tRNA aspartic acid, and the ATPase subunit 6 and 8 genes, we inferred that both M. iolanthe and another low-altitude species, Maoricicada campbelli, are nested within the montane Maoricicada radiation. Therefore, we examined the stability of the inferred phylogenetic placement of these two species using the newly developed Shimodaira-Hasegawa test (SH test) implemented in a maximum-likelihood framework. The SH test has two advantages over the more commonly used Kishino-Hasegawa (KH) and Templeton tests. First, the SH test simultaneously compares multiple topologies and corrects the corresponding P: values to accommodate the multiplicity of testing. Second, the SH test is correct when applied to a posteriori hypotheses, unlike the KH test, because it readjusts the expectation of the null hypothesis (that two trees are not different) accordingly. The comparison of P: values estimated under the assumptions of both the KH test and the SH test clearly demonstrate that the KH test has the potential to be misleading when the issue of comparing of a posteriori hypotheses is ignored and when multiple comparisons are not taken into account. The SH test, in combination with a variety of character-weighting schemes applied to our data, reveals a surprising amount of ambiguity in the phylogenetic placement of M. iolanthe and M. campbelli.

Consistency of SINE insertion topology and flanking sequence tree: quantifying relationships among cetartiodactyls.

Short interspersed nuclear elements (SINEs) have been used to generate unambiguous phylogenetic topologies relating eukaryotic taxa. The irreversible nature of SINE retroposition is supported by a large body of comparative genome data and is a fundamental assumption inherent in the value of this qualitative method of inference. Here, we assess the key assumption of unidirectional SINE insertion by comparing the SINE insertion-derived topology and the phylogenetic tree based on seven independent loci of five taxa in the order Cetartiodactyla (Cetacea + Artiodactyla). The data sets and analyses were largely independent, but the loci were, by definition, linked, and thus their consistency supported an irreversible pattern of SINE retroposition. Moreover, our analyses of the flanking sequences provided estimates of divergence times among cetartiodactyl lineages unavailable from SINE insertion analysis alone. Unexpected rate heterogeneity among sites of SINE-flanking sequences and other noncoding DNA sequences were observed. Sequence simulations suggest that this rate heterogeneity may be an artifact resulting from the inaccuracies of the substitution model used.

Detection of APC mutations by a yeast-based protein truncation test (YPTT).

APC gene mutations play a role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) and non-FAP patients. Almost all of the APC mutations are nonsense or frameshift mutations, which truncate the APC protein and are thought to inactivate normal APC function. We show a novel method for detecting nonsense and frameshift APC gene mutations by using Saccharomyces cerevisiae. Polymerase chain reaction (PCR)-amplified APC fragments are cloned directly into yeast expression vectors in vivo, and the yeast expresses a hemagglutinin epitope (HA)-tagged APC peptide. When an APC fragment contains a nonsense or frameshift mutation, HA-tagged truncating APC peptide can be detected by Western blotting using an anti-HA antibody. We identified both germ-line and somatic APC mutations in patients with FAP and non-FAP colorectal tumors, respectively. This method, called the yeast-based protein truncation test (YPTT), is simple and fairly cheap, and it can be applied to any genes that are inactivated by protein truncating mutations.

Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast.

The detection of inactivating mutations in tumor suppressor genes is critical to their characterization, as well as to the development of diagnostic testing. Most approaches for mutational screening of germ-line specimens are complicated by the fact that mutations are heterozygous and that missense mutations are difficult to interpret in the absence of information about protein function. We describe a novel method using Saccharomyces cerevisiae for detecting protein-truncating mutations in any gene of interest. The PCR-amplified coding sequence is inserted by homologous recombination into a yeast URA3 fusion protein, and transformants are assayed for growth in the absence of uracil. The high efficiency of homologous recombination in yeast ensures that both alleles are represented among transformants and achieves separation of alleles, which facilitates subsequent nucleotide sequencing of the mutated transcript. The specificity of translational initiation of the URA3 gene leads to minimal enzymatic activity in transformants harboring an inserted stop codon, and hence to reliable distinction between specimens with wild-type alleles and those with a heterozygous truncating mutation. This yeast-based stop codon assay accurately detects heterozygous truncating mutations in the BRCA1 gene in patients with early onset of breast cancer and in the APC gene in patients with familial adenomatous polyposis. This approach offers a rapid and reliable method for genetic diagnosis in individuals at high risk for germ-line mutations in cancer susceptibility genes.

Oligomerization is not essential for growth suppression by p53 in p53-deficient osteosarcoma Saos-2 cells.

The carboxy-terminal portion of the p53 protein contains the tetramerization domain, and the introduction of multiple missense mutations in this domain disrupts the formation of p53 tetramers, resulting in the production of dimeric or monomeric forms of p53. It has recently been shown that a single missense or nonsense mutation in this domain affects the functional properties of p53 both in yeast and in mammalian cells. In this study, we tested the oligomerization of p53 with mutations in the oligomerization domain, when expressed in a human osteosarcoma cell line, Saos-2, in vivo. We found that single point mutations, including two missense and two nonsense mutations, in the alpha-helix of the oligomerization domain disrupted the oligomerization of p53, but that p53 still retained its ability to inhibit colony formation of cells to some degree. These results suggest that oligomerization and the carboxy-terminal basic domain are not prerequisite for p53-dependent tumor suppression, and this may explain why few of the tumor-derived p53 mutations that have been examined so far are carboxy-terminal mutations.