Citations

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at ≈2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

Dihydropyrimidine dehydrogenase (DPD) is one of the factors that determine the efficacy and toxicity of 5-fluorouracil. Variations in DPD activity may result from alterations at the transcriptional level of the DPYD gene. Heterogeneity in DPYD expression has been reported, but the molecular mechanisms responsible for this remain unclear. We investigated methylation of the DPYD promoter as a mechanism for transcriptional regulation of DPYD in the RKO colorectal cancer cell line. We demonstrate that the active transcription machinery for DPYD is present in RKO cells, but promoter binding of Sp1, a transactivator of DPYD, was inhibited, which on subsequent examination was shown to be associated with dense promoter methylation. Treatment with 5-aza-2′-deoxycytidine alone or the combination of 5-aza-2′-deoxycytidine and trichostatin A induced demethylation of the promoter and markedly increased the DPYD mRNA level in RKO cells but not in unmethylated WiDr cells. Furthermore, in vitro methylation of the DPYD promoter decreased promoter activity. These data suggest an important role for methylation in DPYD suppression. The transcriptional suppression of DPYD by methylation may be responsible for the increased 5-fluorouracil sensitivity observed in some patients. This may also provide insight into the mechanism underlying the downregulation of DPYD in some colorectal cancers.

Centrin is a calcium-binding centrosome/basal body–associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.

Abstract The use of tyrosine kinase inhibitors (TKI) has yielded great success in treatment of lung adenocarcinomas. However, patients who develop resistance to TKI treatment often acquire a somatic resistance mutation (T790M) located in the catalytic cleft of the epidermal growth factor receptor (EGFR) enzyme. Recently, a report describing EGFR-T790M as a germ-line mutation suggested that this mutation may be associated with inherited susceptibility to lung cancer. Contrary to previous reports, our analysis indicates that the T790M mutation confers increased Y992 and Y1068 phosphorylation levels. In a human bronchial epithelial cell line, overexpression of EGFR-T790M displayed a growth advantage over wild-type (WT) EGFR. We also screened 237 lung cancer family probands, in addition to 45 bronchoalveolar tumors, and found that none of them contained the EGFR-T790M mutation. Our observations show that EGFR-T790M provides a proliferative advantage with respect to WT EGFR and suggest that the enhanced kinase activity of this mutant is the basis for rare cases of inherited susceptibility to lung cancer. [Cancer Res 2007;67(10):4665–70]

Abstract Two common single nucleotide polymorphisms in immunoregulatory genes (TNF G308A, rs1800629 and IL10 T3575A, rs1800890) have been recently reported as risk factors for non–Hodgkin lymphoma (NHL) in a large pooled analysis. We systematically investigated the effects of other established NHL risk factors in relation to the tumor necrosis factor (TNF) G308A or interleukin 10 (IL10) T3575A genotypes. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) from 1,172 cases and 982 population-based controls in a U.S. multicenter study. We investigated NHL overall and two common subtypes [diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma]. NHL risks were increased among those with both an autoimmune condition and the TNF G308A GA/AA (ORNHL, 2.1; 95% CI, 1.0–4.2) or the IL10 T3575A TA/AA genotype (ORNHL, 1.6; 95% CI, 0.9–2.6) compared with individuals without an autoimmune condition and with the common TNF G308A GG or IL10 T3575A TT genotype, respectively; results were similar for DLBCL and follicular lymphoma. We found that elevated DLBCL risk associated with last-born status was more pronounced among those with TNF G308A GA/AA (ORDLBCL, 2.7; 95% CI, 1.1–6.4) or IL10 T3575A TA/AA (ORDLBCL, 2.9; 95% CI, 1.6–5.2). Similarly, elevated DLBCL risk associated with obesity (body mass index, ≥35 versus &lt;25 kg/m2) was observed only among those with TNF G308A GA/AA (ORDLBCL, 2.5; 95% CI, 1.1–5.7) or IL10 T3575A TA/AA genotypes (ORDLBCL, 2.0; 95% CI, 1.1–3.5). These exploratory results require replication but provide evidence that autoimmune conditions, late birth order, and obesity act partly through a common inflammatory pathway, posing a greater risk to individuals with variant TNF and IL10 genotypes than those with wild-type alleles. [Cancer Res 2007;67(10):5042–54]

AbstractPrevious studies suggest that enzymes involved in the androgen metabolic pathway are susceptibility factors for prostate cancer. Estrogen metabolites functioning as genotoxins have also been proposed as risk factors. In this study, we systematically tested the hypothesis that common genetic variations for those enzymes involved in the androgen and estrogen metabolic pathways increase risk for sporadic and familial prostate cancer. From these two pathways, 46 polymorphisms (34 single nucleotide polymorphisms, 10 short tandem repeat polymorphisms, and 2 null alleles) in 25 genes were tested for possible associations. Those genes tested included PRL, LHB, CYP11A1, HSD3B1, HSD3B2, HSD17B2, CYP17, SRD5A2, AKR1C3, UGT2B15, AR, SHBG, and KLK3 from the androgen pathway and CYP19, HSD17B1, CYP1A1, CYP1A2, CYP1B1, COMT, GSTP1, GSTT1, GSTM1, NQO1, ESR1, and ESR2 from the estrogen pathway. A case-control study design was used with two sets of cases: familial cases with a strong prostate cancer family history (n = 438 from 178 families) and sporadic cases with a negative prostate cancer family history (n = 499). The controls (n = 493) were derived from a population-based collection. Our results provide suggestive findings for an association with either familial or sporadic prostate cancer with polymorphisms in four genes: AKR1C3, HSD17B1, NQO1, and GSTT1. Additional suggestive findings for an association with clinical variables (disease stage, grade, and/or node status) were observed for single nucleotide polymorphisms in eight genes: HSD3B2, SRD5A2, SHBG, ESR1, CYP1A1, CYP1B1, GSTT1, and NQO1. However, none of the findings were statistically significant after appropriate corrections for multiple comparisons. Given that the point estimates for the odds ratio for each of these polymorphisms are &lt;2.0, much larger sample sizes will be required for confirmation. (Cancer Epidemiol Biomarkers Prev 2007;16(5):969–78)

AbstractPurpose: Colorectal carcinoma (CRC) can be divided into two nonoverlapping groups: those that are chromosomally unstable but microsatellite stable (MSS CIN+) and those that are chromosomally stable but microsatellite unstable (MSI CIN−). However, a third group with neither chromosome nor microsatellite instability (MSS CIN−) makes a substantial contribution to the total CRC burden. The clinicopathologic features of MSS CIN− CRC are not well delineated. We assessed the relationship between age and chromosomal instability (CIN) status as measured by ploidy and allelic imbalance in a series of MSS tumors.Experimental Design: We studied a prospectively collected series of CRC patients at Mayo Clinic Rochester. A total of 84 samples of MSS CRC in patients ≤50 years old were identified between 1994 and 1997. A consecutive series of 90 MSS CRC in patients ≥65 years old served as a comparison group. CIN status was assessed using two techniques: ploidy analysis by flow cytometry and small chromosome changes as measured by genomewide fractional allelic imbalance.Results: CRC in the young-onset group was more likely to involve the rectum and to be high stage. MSS tumors in the young-onset group were more often diploid (46%) than those in older patients (26%; P = 0.006). This difference was maintained in the subset of MSS CRC that were high stage (42% versus 18%; P = 0.02) and in rectal cancers (50% versus 23%; P = 0.04).Conclusion: A greater proportion of young patients with MSS CRC has diploid tumors than patients who develop MSS CRC over age 65.

Abstract Two variants (rs1447295/DG8S737) of chromosome 8q24 were recently reported to be associated with increased risk of prostate cancer (PC). To confirm this finding, we genotyped and compared the frequencies of these polymorphisms among 1,121 Caucasian men with PC (435 men with familial PC, 491 men with sporadic PC, and 195 men with aggressive PC) to 545 population-based controls. For the single nucleotide polymorphism marker rs1447295, frequencies of the minor allele (A) were 10.3% in controls, 11.9% in sporadic cases, 16.7% in familial cases, and 17.2% in aggressive cases. Compared with controls, the A allele was significantly more common in both familial PC [odds ratios (OR), 1.93; 95% confidence intervals (95% CI), 1.37–2.72; P = 0.0004] and aggressive PC (OR, 1.87; 95% CI, 1.28–2.74; P = 0.0005) but not for sporadic PC (OR, 1.16; 95% CI, 0.85–1.58; P = 0.25). Although the A allele was more frequent in aggressive PC cases when compared with controls, the allele frequencies were similar among cases with high- and low-grade PC (Gleason grades &lt;7 and ≥7, respectively). For the microsatellite marker DG8S737, the −8 allele was significantly more frequent in familial PC (OR, 1.68; 95% CI, 1.09–2.60; P = 0.031), whereas the −10 allele was more frequent in aggressive PC (OR, 2.85; 95% CI, 1.52–5.36; P = 0.0004). Haplotype analysis showed significant differences in haplotype frequencies between the familial PC (P = 0.006) and aggressive PC (P = 0.005) cases versus controls. The −8/A haplotype showed the strongest association with familial PC (P = 0.008), whereas the −10/A haplotype was most strongly associated with aggressive PC (P = 0.00005). These results further confirm the importance of these two polymorphic variants (rs1447295 and DG8S737) as risk factors for PC. However, the mechanism explaining this increased risk has not yet been established. [Cancer Res 2007;67(7):2944–50]

The congenital myasthenic syndromes include end-plate (EP) acetylcholinesterase deficiency, presynaptic abnormalities affecting the evoked release or size of transmitter quanta, and acetylcholine (ACh) receptor (AChR) channelopathies stemming from a kinetic abnormality and/or deficiency of AChR. A kinetic abnor mality predicts, and AChR deficiency may predict, one or more mutations in an AChR subunit gene. These clues have led to the identification of 53 mutations in different subunits of AChR in 55 kinships of the congenital myasthenic syndromes. The mutations either increase or decrease the response to ACh, produce AChR deficiency, or both. In the slow-channel syndromes, prolonged opening episodes of AChR cause cationic overloading of the EP and an EP myopathy; the mutations occur in different subunits and different domains of the subunits and have dominant positive effects. The M1 and M2 mutations slow channel closure, increase apparent affinity for ACh, and variably enhance desensitization, and the extracellular αG153S enhances affinity for ACh, promoting reopening of the diliganded receptor. In the low-affinity fast-channel syndrome, εP121L reduces affinity for ACh and reopening of the diliganded receptor, resulting in a de creased response to ACh and shorter burst durations. Severe EP AChR deficiency results from heterozy gous or homozygous mutations that terminate translation prematurely; these are concentrated in the ε subunit, probably because substitution of the fetal γ for the adult ε subunit can rescue the phenotype from fatal null mutations in ε. Variable AChR deficiency and variable functional abnormalities stem from hetero allelic nonsense and missense mutations in AChR subunit genes. NEUROSCIENTIST 4:185-194, 1998