Genetic variation in the renin-angiotensin-aldosterone system is associated with cardiovascular risk factors and early mortality in established coronary heart disease.

This study examined renin-angiotensin-aldosterone (RAAS) system gene variants for associations with cardiovascular risk factors and outcomes in coronary heart disease. Coronary disease patients (n=1186) were genotyped for 21 single-nucleotide polymorphisms (SNPs) within angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensin-II type-1 receptor (AGTR1) and aldosterone synthase (CYP11B2). Associations with all-cause mortality and cardiovascular readmissions were assessed over a median of 3.0 years. The AGT M235T 'T' allele was associated with a younger age of clinical coronary disease onset (P=0.006), and the AGT rs2478545 minor allele was associated with lower circulating natriuretic peptides (P=0.0001-P=0.001) and E/E(1) (P=0.018). Minor alleles of AGT SNPs rs1926723 and rs11122576 were associated with more frequent history of renal disease (P0.04) and type-2 diabetes (P0.02), higher body mass index (P0.02) and greater mortality (P0.007). AGT rs11568054 minor allele carriers had more frequent history of renal disease (P=0.04) and higher plasma creatinine (P=0.033). AGT rs6687360 minor allele carriers exhibited worse survival (P=0.02). ACE rs4267385 was associated with older clinical coronary disease onset (P=0.008) and hypertension (P=0.013) onset, increased plasma creatinine (P=0.01), yet greater mortality (P=0.044). Less history of hypertension was observed with the AGTR1 rs12685977 minor allele (P=0.039). Genetic variation within the RAAS was associated with cardiovascular risk factors and accordingly poorer survival.

The atypical Rho GTPase RhoBTB2 is required for expression of the chemokine CXCL14 in normal and cancerous epithelial cells.

The Rho family of small GTPases control cell migration, cell invasion and cell cycle. Many of these processes are perturbed in cancer and several family members show altered expression in a number of tumor types. RhoBTB2/DBC2 is an atypical member of this family of signaling proteins, containing two BTB domains in addition to its conserved Rho GTPase domain. RhoBTB2 is mutated, deleted or silenced in a large percentage of breast and lung cancers; however, the functional consequences of this loss are unclear. Here we use RNA interference in primary human epithelial cells to mimic the loss of RhoBTB2 seen in cancer cells. Through microarray analysis of global gene expression, we show that loss of RhoBTB2 results in downregulation of CXCL14-a chemokine that controls leukocyte migration and angiogenesis, and whose expression is lost through unknown mechanisms in a wide range of epithelial cancers. Loss of RhoBTB2 expression correlates with loss of CXCL14 secretion by head and neck squamous cell carcinoma cell lines, whereas reintroduction of RhoBTB2 restores CXCL14 secretion. Our studies identify CXCL14 as a gene target of RhoBTB2 and support downregulation of CXCL14 as a functional outcome of RhoBTB2 loss in cancer.

Variation in RNA expression and genomic DNA content acquired during cell culture.

Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three. Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations. These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer. This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous.

The design and synthesis of water-soluble analogues of CB30865, a quinazolin-4-one-based antitumor agent.

4-[N-[7-Bromo-2-methyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl]-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide (CB30865) is a quinazolin-4-one antitumor agent whose high growth-inhibitory activity (W1L2 IC(50) = 2.8 +/- 0.50 nM) is believed to have a folate-independent locus of action. In addition, CB30865 represents a class of compounds with unique biochemical characteristics such as a delayed, non-phase specific, cell-cycle arrest. The low aqueous solubility of CB30865 prompted a search for more water-soluble analogues for in vivo evaluation of this class of compounds. It was thought that aqueous solubility could be increased by the introduction of amino functionalities at the 2-position of the quinazolin-4-one ring. A variety of compounds (5a-j, 31a-c, 32, and 33) were synthesized in a linear fashion starting from 3-chloro-4-methylaniline. Most of these compounds (e.g., 5a, 5b, 5g) were significantly more water-soluble than CB30865 (636 microM for 5a at pH 6 and 992 microM for 5g at pH 6). In addition, some of them were up to 6-fold more cytotoxic than CB30865 (e.g., for 5a, W1L2 IC(50) = 0.49 +/- 0.24 nM) and retained its novel biochemical characteristics.

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study.

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab' cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab' maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab')(2). Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab' maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab' maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab')(2) used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab' maleimide over murine versions of this antibody suggesting that humanised divalent-Fab' maleimide should be a useful vehicle for repeated therapies.

Comparative expressed sequence hybridization to chromosomes for tumor classification and identification of genomic regions of differential gene expression.

Altered expression of genes can have phenotypic consequences in cancer development and treatment, developmental abnormalities, and differentiation processes. Here we describe a rapid approach, termed comparative expressed sequence hybridization (CESH), which gives a genome-wide view of relative expression patterns within tissues according to chromosomal location. No prior knowledge of genes or cloning is required, and minimal amounts of tissue can be used. Expression profiles are achieved in a manner similar to the identification of chromosomal imbalances by comparative genomic hybridization analysis. The approach is demonstrated to indicate a chromosomal region that harbors overexpressed genes that may be associated with a drug-resistant phenotype. In addition, known and new regions of differential gene expression in both normal tissues and tumor samples from the soft tissue sarcoma group of rhabdomyosarcoma (RMS) are indicated. These regions included 2p24; overexpression of MYCN at 2p24 was confirmed by quantitative reverse transcription-PCR for all of the alveolar RMS cases and did not necessarily correspond to genomic amplification. Evidence including region specific microarray analysis indicated that overexpression of several genes from a region may be required for detection by CESH. This evidence is consistent with clusters of functionally related genes and mechanisms that affect the expression of a number of genes at a particular genomic location. The distinctive CESH profiles demonstrated in different subtypes of RMS show potential for tumor classification.

Late results of surveillance of clinical stage I nonseminoma germ cell testicular tumours: 17 years' experience in a national study in New Zealand.

OBJECTIVE: To re-evaluate a national prospective study in New Zealand after 17 years to define whether orchidectomy alone and surveillance for nonseminoma germ cell testicular tumour (NSGCTT) is a sound policy and matches the results achieved by other treatment protocols. PATIENTS AND METHODS: Between 1980 and 1997, 248 men with stage I NSGCTT, from six New Zealand centres, were managed by orchidectomy alone and surveillance, with treatment of relapses using combination chemotherapy. RESULTS: Seventy of the 248 patients (28%) relapsed; 42 of 92 (46%) with vascular and/or lymphatic invasion (VLI) in the primary tumour relapsed, whereas only 26 of 151 (17%) without this feature relapsed (P<0.001). VLI was the only identifiable risk factor for relapse in this series. Only one relapse occurred >28 months after orchidectomy. Despite poor compliance in some patients (12%) their survival was not prejudiced. Three patients died from disease despite chemotherapy at relapse. At 17 years and a median follow-up of 53 months, 242 of the 248 men are disease-free and the disease-specific survival rate is 98%. CONCLUSIONS: This study shows that orchidectomy alone and treatment of relapses produces excellent long-term results without the adverse effects associated with retroperitoneal node dissection or elective chemotherapy for high-risk cases.

A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus.

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.

A novel, orally administered nucleoside analogue, OGT 719, inhibits the liver invasive growth of a human colorectal tumor, C170HM2.

OGT 719 is a novel p.o. bioavailable nucleoside analogue in which galactose is incorporated onto the fluoropyrimidine moiety of the cytotoxic agent 5-fluorouracil (5-FU). OGT 719 has been designed to reduce the systemic toxicity normally associated with 5-FU while retaining activity against disease localized in the liver, in which it may be preferentially localized through the asialoglycoprotein receptor (ASGP-R). We report studies confirming the activity of OGT 719 in inhibiting growth of metastatic human colorectal tumors in the liver of nude mice. The human colorectal cancer cell line C170HM2 readily forms liver metastases in vivo. Oral administration of 1500 mg/kg/day OGT 719 inhibited liver tumor burden by 95% compared with vehicle control, without any observable signs of toxicity. When the tumor burden was increased and the same OGT 719 treatment was compared with a standard clinical dose regimen of 25 mg/kg/day 5-FU/leucovorin given i.v., both treatments were equally efficacious, although 5-FU/leucovorin treatment started 7 days earlier. In contrast to 5-FU, OGT 719 is p.o. bioavailable and has a plasma half-life between 1.5 and 3 h. Several colorectal cancer cell lines express the asialoglycoprotein receptor, although no significant levels can be detected in C170HM2 cells, consistent with the observation that OGT 719 is approximately 3 log orders of magnitude less potent in vitro than 5-FU. Flux through thymidylate synthase, as measured by 3H release from [3H]dUrd, was inhibited by OGT 719 at 4 h. The notable difference in the potency of OGT 719 efficacy on C170HM2 cells in vitro and in vivo supports our model of liver-specific activation of OGT 719. As our data suggest, OGT 719 may significantly inhibit growth of metastatic colorectal tumors in the liver in vivo. This hypothesis is presently being explored in clinical trials for primary hepatocellular carcinoma and colorectal liver metastases.

A molecular cytogenetic approach to studying platinum resistance.

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.

Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action.

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound.

Use of body mass index to monitor treatment of obese adolescents.

PURPOSE: Absolute weight loss may not always be the best measure of adherence and response to therapy in obese adolescents if weight gain owing to linear growth is not considered. We wished to compare short-term absolute weight and height changes with changes in body mass index (BMI) in a group of severely obese adolescents to determine the most meaningful measure of treatment response. METHODS: We analyzed weight, height, and BMI in 27 adolescents, 10-18 years of age, referred for management of severe obesity. Subjects attended clinic on at least three occasions within a 6-24-month period. Detailed profiles of usual daily food intakes, physical activities, and family and environmental structure/activities were obtained, and specific goals to achieve weight control were negotiated with adolescents and their families at each visit. Weight, height, and BMI at the initial visit and at the most recent visit were compared. RESULTS: While 48% of our population actually lost weight, 78% either had no change or a decrease in BMI during the observation period. Differences between initial and most recent heights and BMIs were statistically significant, but weight changes were not significant. CONCLUSIONS: In addition to weight, BMI should be routinely used and reported when monitoring the response to specific interventions in growing adolescents. Evaluation of weight alone may underestimate the adolescent's adherence to treatment goals.

Bilateral germ cell testicular tumors in New Zealand: experience in Auckland and Christchurch 1978-1994.

PURPOSE: The incidence of germ cell testicular tumors (GCTTs) is increasing world wide, and with effective treatment, the majority of patients are being cured. Thus, the clinical and social impact of a second testicular tumor is becoming more important. The frequency, cumulative risk, and relative risk of developing a second testicular cancer in New Zealand have been documented and compared with other reports. PATIENTS AND METHODS: The records of 741 men presenting with germ cell testicular cancer in Auckland and Christchurch between 1978 and 1994 have been reviewed, and these data have been compared with data from other published studies. Cumulative risk was assessed by the Kaplan-Meier method. RESULTS: Over 2% of the study population developed a second germ cell testicular cancer. The cumulative risk was 5.2% over 15 years. The relative risk of developing a contralateral testicular tumor is 27.5 times higher than age-matched New Zealand peers. These results match the only comparable report in the literature. Five of the 16 bilateral tumors (31%) were synchronous, which is a higher incidence than in any other reported series. There was no concordance of histology in the first and second tumors. Prior exposure to cisplatin combination chemotherapy did not prevent the development of a second tumor. CONCLUSION: Men who are cured of a germ cell testicular cancer have a greatly increased risk of developing a second testicular cancer. Such patients should be informed of this risk and ideally kept under long-term surveillance.

Effect of hammerhead ribozyme against human thymidylate synthase on the cytotoxicity of thymidylate synthase inhibitors.

One of the resistance mechanisms to folate-based thymidylate synthase (TS) inhibitors is the increase in TS activity in tumor cells. Human B lymphoblastoid cell line (W1L2) was made resistant to a lipophilic non-polyglutamatable TS inhibitor (ZM249148), and the subline (W1L2:R179) showed a 20-fold increase in TS enzyme activity with concomitant overexpression of TS mRNA. To overcome the resistance, we designed a ribozyme that can cleave the CUC sequences in a triple tandemly repeated sequence of TS mRNA. Expression of this ribozyme in W1L2:R179 cells transfected with Epstein Barr virus-based expression vector resulted in sensitization to TS inhibitors concomitantly with a decrease of TS expression. The ribozyme expressed in transfectants was shown to be functional in cleaving artificial TS RNA in vitro.

Results of the surveillance policy of stage I non-seminomatous germ cell testicular tumours.

A series of 115 patients with clinical Stage I non-seminomatous germ cell testicular tumours were managed with orchiectomy and close surveillance (median follow-up 36 months, range 3-119); 34 (29.5%) relapsed, 21 within 6 months, 29 within a year and the latest at 28 months. At relapse all patients were treated with platinum or analogue-based drug combinations, supplemented in 7 by retroperitoneal node dissection; 30 patients achieved durable remissions and 2 have had further relapses successfully treated. Two died; both had malignant teratoma intermediate with primary stage T1 and vascular and/or lymphatic invasion of primary tumour. At a median follow-up time of 36 months, the survivors (98.3%) demonstrate no evidence of disease, these results matching the outcome of other methods of management. Vascular and/or lymphatic invasion was associated with an enhanced relapse rate but specific histology, T stage of the primary and pre-orchiectomy serum alpha-fetoprotein status did not appear to favour relapse. The first sign of relapse was tumour marker alone in 10 patients, radiological features alone in 12, or both in 10 patients. However, in 2 cases the relapse was first detected clinically. Furthermore, pre-orchiectomy and relapse marker status did not correlate well. These points emphasise the importance of all aspects of follow-up, none of which can be safely omitted.

Production of the word-final English /t/-/d/ contrast by native speakers of English, Mandarin, and Spanish.

The primary aim of this study was to determine if adults whose native language permits neither voiced nor voiceless stops to occur in word-final position can master the English word-final /t/-/d/ contrast. Native English-speaking listeners identified the voicing feature in word-final stops produced by talkers in five groups: native speakers of English, experienced and inexperienced native Spanish speakers of English, and experienced and inexperienced native Mandarin speakers of English. Contrary to hypothesis, the experienced second language (L2) learners' stops were not identified significantly better than stops produced by the inexperienced L2 learners; and their stops were correctly identified significantly less often than stops produced by the native English speakers. Acoustic analyses revealed that the native English speakers made vowels significantly longer before /d/ than /t/, produced /t/-final words with a higher F1 offset frequency than /d/-final words, produced more closure voicing in /d/ than /t/, and sustained closure longer for /t/ than /d/. The L2 learners produced the same kinds of acoustic differences between /t/ and /d/, but theirs were usually of significantly smaller magnitude. Taken together, the results suggest that only a few of the 40 L2 learners examined in the present study had mastered the English word-final /t/-/d/ contrast. Several possible explanations for this negative finding are presented. Multiple regression analyses revealed that the native English listeners made perceptual use of the small, albeit significant, vowel duration differences produced in minimal pairs by the nonnative speakers. A significantly stronger correlation existed between vowel duration differences and the listeners' identifications of final stops in minimal pairs when the perceptual judgments were obtained in an "edited" condition (where post-vocalic cues were removed) than in a "full cue" condition. This suggested that listeners may modify their identification of stops based on the availability of acoustic cues.