Citations

Abstract The rationale behind gene-disease association studies is that genetic variants (polymorphisms) result in alterations in intermediate phenotypes. However, genotype-phenotype correlations have not been established for most polymorphisms. In this study, we correlated genotype data of genes involved in the nucleotide excision repair pathway with mutagen sensitivity phenotype, quantified by benzo(a)pyrene diol epoxide (BPDE)-induced chromatid breaks in peripheral blood lymphocytes in 422 healthy subjects recruited into a twin study that included 138 pairs of monozygotic twins, 51 pairs of dizygotic twins, and 44 siblings. Among a panel of single nucleotide polymorphisms examined, we found that BPDE sensitivity was modified by individual polymorphisms in XPC, RAD23B, and XPA genes. Specific haplotypes and diplotypes of XPC also modified BPDE sensitivity profiles. In addition, a more consistent and stronger correlation was observed between mutagen sensitivity phenotype and the combination of multiple polymorphisms in the nucleotide excision repair pathway. Specifically, when XPC-PAT, XPC Lys939Gln, XPA A23G, and RAD23B Val249Ala were analyzed together, we observed a significant dose-response relationship between increasing mutagen sensitivity with increasing number of adverse alleles: mutagen sensitivity for those carrying zero to two, three to five, and six or more adverse alleles were 0.64, 0.68, and 1.06, respectively (P for trend = 0.008), and the results remained significant after adjusting for multiple comparisons. Using individuals carrying zero to two adverse alleles as the reference group, the risks of being mutagen sensitive (mutagen sensitivity values greater than the median) were 1.05 (95% confidence interval, 0.68-1.64) and 4.48 (95% confidence interval, 1.21-16.61) for those carrying three to five and six or more adverse alleles, respectively. Analyses of the effects of genotype combinations yielded similar results. These findings underscore the importance of assessing the collective effects of a panel of polymorphisms in the same pathway in modulating mutagen sensitivity. As risk assessment for cancer risk is moving toward a multigenic pathway-based approach, future genotype-phenotype correlation studies should also investigate the combined effects of multiple genetic variants. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2065–71)

OBJECTIVE—Triglyceride-rich lipoproteins, primarily chylomicrons, can contribute to plasma free fatty acid (FFA) concentrations via spillover of fatty acids during intravascular hydrolysis into the venous effluent of some tissues. The present study was undertaken to determine whether spillover occurs in the splanchnic bed of humans. RESEARCH DESIGN AND METHODS—Arterial and hepatic venous blood was sampled in postabsorptive (n = 6; study A) and postprandial (n = 5; study B) obese humans during infusion of carbon-labeled (14C or 13C) oleate and 3H triolein, the latter incorporated into a lipid emulsion as a surrogate for chylomicrons. Spillover was determined by measuring production of 3H oleate. RESULTS—Splanchnic spillover was higher than nonsplanchnic systemic spillover in both study A (60 ± 7 vs. 24 ± 6%; P < 0.01) and study B (54 ± 3 vs. 16 ± 5%; P < 0.005). Because portal vein sampling is not feasible in humans, assumptions regarding actual spillover in nonhepatic splanchnic tissues were required for the spillover calculation. A mathematical model was developed and demonstrated that nonhepatic splanchnic spillover rates in study A and study B of 69 and 80%, respectively, provided the best fit with the data. There was preferential splanchnic uptake of triglyceride fatty acids compared with FFAs in study B (fractional extraction 61 ± 3 vs. 33 ± 2%; P < 0.005). CONCLUSIONS—These data confirm previous studies indicating that the transport of FFAs and triglyceride fatty acids are partitioned in tissues and indicate that splanchnic spillover from triglyceride-rich lipoproteins may be a significant source of both portal venous and systemic FFAs.

Purpose The traditional end point for colon adjuvant clinical trials is overall survival (OS). We previously validated disease-free survival (DFS) after 3-year follow-up as an excellent predictor of 5-year OS results. Here we explore shorter term DFS and OS end points, as well as stage dependency. Methods Individual patient data from 18 phase III trials including 43 arms and 20,898 patients were pooled. Association measures included correlation of event rates within arms, correlation of hazard ratios (HRs) between arms, trial level significance comparisons (via log-rank testing), and a formal surrogacy model. Results DFS at earlier times was less accurate in predicting OS than 3-year DFS, but 2-year DFS remained a strong predictor. DFS with 1-year minimum follow-up demonstrated perfect negative predicted value; all trials negative at 1 year for DFS were negative for 5-year OS. OS with 3-year minimum follow-up was also an excellent predictor for 5-year OS; OS at earlier time points provided inaccurate prediction. The association between 3-year DFS and 5-year OS was greater for stage III patients; correlation of HR within trials was 0.92 (95% CI, 0.85 to 0.95) for stage III patients and 0.70 (95% CI, 0.44 to 0.80) for stage II patients. Conclusion DFS outcomes after 2- or 3-year median follow-up are excellent predictors of 5-year OS. DFS outcomes are appropriate for trials in which the majority of patients are stage III. DFS after 2- or 3-year median follow-up should be considered as the primary end point in future colon adjuvant trials.

Abstract Increased mammographic density (MD), the proportion of dense tissue visible on a mammogram, is a strong risk factor for breast cancer, common in the population and clusters in families. We conducted the first genome-wide linkage scan to identify genes influencing MD. DNA was obtained from 889 relatives (756 women, 133 men) from 89 families. Percent MD was estimated on 618 (82%) female family members using a validated computer-assisted thresholding method. The genome-wide scan included 403 microsatellite DNA markers with an average spacing of 9 cM. Fine mapping of a region of chromosome 5p (5p13.1-5p15.1) was done using 21 additional closely spaced DNA markers. Linkage analyses were conducted to quantify the evidence for a gene responsible for MD across the genome. The maximum log odds for linkage (LOD) score from the genome-wide scan was on chromosome 5p (LOD = 2.9, supporting linkage by a factor of 102.9 or 794 to 1) with a 1-LOD interval spanning 28.6 cM. Two suggestive regions for linkage were also identified on chromosome 12 (LOD = 2.6, 1-LOD interval of 14.8 cM; and LOD = 2.5, 1-LOD interval of 17.2 cM). Finer mapping of the region surrounding the maximum LOD on chromosome 5p resulted in stronger and statistically significant evidence for linkage (LOD = 4.2) and a narrowed 1-LOD interval (13.4 cM). The putative locus on chromosome 5p is likely to account for up to 22% of variation in MD. Hence, 1 or more of the 45 candidate genes in this region could explain a large proportion of MD and, potentially, breast cancer. [Cancer Res 2007;67(17):8412–8]

Purpose We update Radiation Therapy Oncology Group trial 8911 (USA Intergroup 113), a comparison of chemotherapy plus surgery versus surgery alone for patients with localized esophageal cancer. The relationship between resection type and between tumor response and outcome were also analyzed. Patients and Methods The chemotherapy group received preoperative cisplatin plus fluorouracil. Outcome based on the type of resection (R0, R1, R2, or no resection) was evaluated. The main end point was overall survival. Disease-free survival, relapse pattern, the influence of postoperative treatment, and the relationship between response to preoperative chemotherapy and outcome were also evaluated. Results Two hundred sixteen patients received preoperative chemotherapy, 227 underwent immediate surgery. Fifty-nine percent of surgery only and 63% of chemotherapy plus surgery patients underwent R0 resections (P = .5137). Patients undergoing less than an R0 resection had an ominous prognosis; 32% of patients with R0 resections were alive and free of disease at 5 years, only 5% of patients undergoing an R1 resection survived for longer than 5 years. The median survival rates for patients with R1, R2, or no resections were not significantly different. While, as initially reported, there was no difference in overall survival for patients receiving perioperative chemotherapy compared with the surgery only group, patients with objective tumor regression after preoperative chemotherapy had improved survival. Conclusion For patients with localized esophageal cancer, whether or not preoperative chemotherapy is administered, only an R0 resection results in substantial long-term survival. Even microscopically positive margins are an ominous prognostic factor. After a R1 resection, postoperative chemoradiotherapy therapy offers the possibility of long-term disease-free survival to a small percentage of patients.

Abstract Purpose: Incidence and mortality rates for renal cell carcinoma (RCC) have been rising for decades. Unfortunately, the molecular events that support RCC carcinogenesis remain poorly understood. In an effort to gain a better understanding of signaling events in clear cell RCC (cRCC), we investigated the antitumor activity of secreted frizzled-related protein 1 (sFRP1), a negative regulator of Wnt signaling. Experimental Design: Genomic profiling of cRCC tumors and patient-matched normal tissues was done and confirmed using quantitative PCR and immunohistochemistry. Methylation-specific PCR was done on patient samples to evaluate the mechanism responsible for sFRP1 loss. sFRP1 expression was restored in cRCC cells and the effects on tumor phenotype were characterized. Results: Genomic profiling, quantitative PCR, and immunohistochemistry indicated that loss of sFRP1 occurred in cRCC and papillary RCC patient tissues. Twelve Wnt-regulated genes were up-regulated in cRCC tissues, including c-myc and cyclin D1, potentiators of cell proliferation and survival. Methylation of the sFRP1 gene was one mechanism identified for attenuation of sFRP1 mRNA. Stable reexpression of sFRP1 in cRCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. Conclusions: To our knowledge, this is the first report to show that stable restoration of sFRP1 expression in cRCC cells attenuates the cRCC tumor phenotype. Our data support a role for sFRP1 as a tumor suppressor in cRCC and that perhaps loss of sFRP1 is an early, aberrant molecular event in renal cell carcinogenesis.

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK 1 R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK 1 R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK 1 R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK 1 R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK 3 R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP C ), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrP C are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP C in the proteolytic processing of APP. Cellular overexpression of PrP C inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrP C in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP C on the β-secretase cleavage of APP required the localization of PrP C to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP C via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrP C and may have implications for both Alzheimer's and prion diseases.

Purpose The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status. Patients and Methods The expression of EGFRvIII was assessed by immunohistochemistry in 649 patients with newly diagnosed GBM. These data were then examined in conjunction with the expression of phospho-intermediates (in a subset of these patients) of downstream AKT and Ras pathways and YKL-40 as well as with known clinical risk factors, including the Radiation Therapy Oncology Group's recursive partitioning analysis (RTOG-RPA) class. Results The RTOG-RPA class was highly predictive of survival in EGFRvIII-negative patients but much less predictive in EGFRvIII-positive patients. These findings were seen in both an initial test set (n = 268) and a larger validation set (n = 381). Similarly, activation of the AKT/MAPK pathways and YKL-40 positivity were predictive of poor outcome in EGFRvIII-negative patients but not in EGFRvIII-positive patients. Pair-wise combinations of markers identified EGFRvIII and YKL-40 as prognostically important. In particular, outcome in patients with EGFRvIII-negative/YKL-40–negative tumors was significantly better than the outcome in patients with the other three combinations of these two markers. Conclusion Established prognostic factors in GBM were not predictive of outcome in the EGFRvIII-positive subset, although this requires confirmation in independent data sets. GBMs negative for both EGFRvIII and YKL-40 show less aggressive behavior.