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1.  A resampling-based meta-analysis for detection of differential gene expression in breast cancer 
BMC Cancer  2008;8:396.
Background
Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures.
Methods
A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis.
Results
The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results.
Conclusion
The proposed meta-analysis approach has the ability to detect a set of differentially expressed genes with the least amount of within-group variability, thus providing highly stable gene lists for class prediction. Increased statistical power and stringent filtering criteria used in the present study also make identification of novel candidate genes possible and may provide further insight to improve our understanding of breast cancer development.
doi:10.1186/1471-2407-8-396
PMCID: PMC2631593  PMID: 19116033
2.  Gene expression meta-analysis identifies metastatic pathways and transcription factors in breast cancer 
BMC Cancer  2008;8:394.
Background
Metastasis is believed to progress in several steps including different pathways but the determination and understanding of these mechanisms is still fragmentary. Microarray analysis of gene expression patterns in breast tumors has been used to predict outcome in recent studies. Besides classification of outcome, these global expression patterns may reflect biological mechanisms involved in metastasis of breast cancer. Our purpose has been to investigate pathways and transcription factors involved in metastasis by use of gene expression data sets.
Methods
We have analyzed 8 publicly available gene expression data sets. A global approach, "gene set enrichment analysis" as well as an approach focusing on a subset of significantly differently regulated genes, GenMAPP, has been applied to rank pathway gene sets according to differential regulation in metastasizing tumors compared to non-metastasizing tumors. Meta-analysis has been used to determine overrepresentation of pathways and transcription factors targets, concordant deregulated in metastasizing breast tumors, in several data sets.
Results
The major findings are up-regulation of cell cycle pathways and a metabolic shift towards glucose metabolism reflected in several pathways in metastasizing tumors. Growth factor pathways seem to play dual roles; EGF and PDGF pathways are decreased, while VEGF and sex-hormone pathways are increased in tumors that metastasize. Furthermore, migration, proteasome, immune system, angiogenesis, DNA repair and several signal transduction pathways are associated to metastasis. Finally several transcription factors e.g. E2F, NFY, and YY1 are identified as being involved in metastasis.
Conclusion
By pathway meta-analysis many biological mechanisms beyond major characteristics such as proliferation are identified. Transcription factor analysis identifies a number of key factors that support central pathways. Several previously proposed treatment targets are identified and several new pathways that may constitute new targets are identified.
doi:10.1186/1471-2407-8-394
PMCID: PMC2642844  PMID: 19116006
3.  The development of a mini-array for estimating the disease state of gastric adenocarcinoma by array CGH 
BMC Cancer  2008;8:393.
Background
The treatment strategy usually depends on the disease state in the individual patient. However, it is difficult to estimate the disease state before treatment in many patients. The purpose of this study was to develop a BAC (bacterial artificial chromosome) mini-array allowing for the estimation of node metastasis, liver metastasis, peritoneal dissemination and the depth of tumor invasion in gastric cancers.
Methods
Initially, the DNA copy number aberrations (DCNAs) were analyzed by array-based comparative genomic hybridization (aCGH) in 83 gastric adenocarcinomas as a training-sample set. Next, two independent analytical methods were applied to the aCGH data to identify the BAC clones with DNA copy number aberrations that were linked with the disease states. One of the methods, a decision-tree model classifier, identified 6, 4, 4, 4, and 7 clones for estimating lymph node metastasis, liver metastasis, peritoneal dissemination, depth of tumor invasion, and histological type, respectively. In the other method, a clone-by-clone comparison of the frequency of the DNA copy number aberrations selected 26 clones to estimate the disease states.
Results
By spotting these 50 clones together with 26 frequently or rarely involved clones and 62 reference clones, a mini-array was made to estimate the above parameters, and the diagnostic performance of the mini-array was evaluated for an independent set of 30 gastric cancers (blinded – sample set). In comparison to the clinicopathological features, the overall accuracy was 66.7% for node metastasis, 86.7% for liver metastasis, 86.7% for peritoneal dissemination, and 96.7% for depth of tumor invasion. The intratumoral heterogeneity barely affected the diagnostic performance of the mini-array.
Conclusion
These results suggest that the mini-array makes it possible to determine an optimal treatment for each of the patients with gastric adenocarcinoma.
doi:10.1186/1471-2407-8-393
PMCID: PMC2637883  PMID: 19115996
4.  mRNA expression of the DNA replication-initiation proteins in epithelial dysplasia and squamous cell carcinoma of the tongue 
BMC Cancer  2008;8:395.
Background
The tongue squamous cell carcinomas (SCCs) are characterized by high mitotic activity, and early detection is desirable. Overexpression of the DNA replication-initiation proteins has been associated with dysplasia and malignancy. Our aim was to determine whether these proteins are useful biomarkers for assessing the development of tongue SCC.
Methods
We analyzed the mRNA expression of CDC6, CDT1, MCM2 and CDC45 in formalin-fixed, paraffin-embedded benign and malignant tongue tissues using quantitative real-time PCR followed by statistical analysis.
Results
We found that the expression levels are significantly higher in malignant SCC than mild precancerous epithelial dysplasia, and the expression levels in general increase with increasing grade of precancerous lesions from mild, moderate to severe epithelial dysplasia. CDC6 and CDC45 expression is dependent of the dysplasia grade and lymph node status. CDT1 expression is higher in severe dysplasia than in mild and moderate dysplasia. MCM2 expression is dependent of the dysplasia grade, lymph node status and clinical stage. The expression of the four genes is independent of tumor size or histological grade. A simple linear regression analysis revealed a linear increase in the mRNA levels of the four genes from the mild to severe dysplasia and SCC. A strong association was established between CDC6 and CDT1, and between MCM2 and CDC45 expression. The nonparametric receiver operating characteristic analysis suggested that MCM2 and CDC45 had a higher accuracy than CDC6 and CDT1 for distinguishing dysplasia from tongue SCC.
Conclusion
These proteins can be used as biomarkers to distinguish precancerous dysplasia from SCC and are useful for early detection and diagnosis of SCC as an adjunct to clinicopathological parameters.
doi:10.1186/1471-2407-8-395
PMCID: PMC2648984  PMID: 19116018
5.  Quantification of SLIT-ROBO transcripts in hepatocellular carcinoma reveals two groups of genes with coordinate expression 
BMC Cancer  2008;8:392.
Background
SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver.
Methods
Expression of SLIT-ROBO family members was quantified by real-time qRT-PCR in 14 HCC cell lines, 8 normal and 35 tumor tissues from the liver. ANOVA and Pearson's correlation analyses were performed in R environment, and different clinicopathological subgroups were pairwise compared in Minitab. Gene expression matrices of cell lines and tissues were analyzed by Mantel's association test.
Results
Genewise hierarchical clustering revealed two subgroups with coordinate expression pattern in both the HCC cell lines and tissues: ROBO1, ROBO2, SLIT1 in one cluster, and ROBO4, SLIT2, SLIT3 in the other, respectively. Moreover, SLIT-ROBO expression predicted AFP-dependent subgrouping of HCC cell lines, but not that of liver tissues. ROBO1 and ROBO2 were significantly up-regulated, whereas SLIT3 was significantly down-regulated in cell lines with high-AFP background. When compared to normal liver tissue, ROBO1 was found to be significantly overexpressed, while ROBO4 was down-regulated in HCC. We also observed that ROBO1 and SLIT2 differentiated histopathological subgroups of liver tissues depending on both tumor staging and differentiation status. However, ROBO4 could discriminate poorly differentiated HCC from other subgroups.
Conclusion
The present study is the first in comprehensive and quantitative evaluation of SLIT-ROBO family gene expression in HCC, and suggests that the expression of SLIT-ROBO genes is regulated in hepatocarcinogenesis. Our results implicate that SLIT-ROBO transcription profile is bi-modular in nature, and that each module shows intrinsic variability. We also provide quantitative evidence for potential use of ROBO1, ROBO4 and SLIT2 for prediction of tumor stage and differentiation status.
doi:10.1186/1471-2407-8-392
PMCID: PMC2632672  PMID: 19114000
6.  Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol 
BMC Cancer  2008;8:391.
Background
Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers.
Methods
In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models.
Results
Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo.
Conclusion
Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.
doi:10.1186/1471-2407-8-391
PMCID: PMC2639606  PMID: 19113992
7.  Aberrant allele-specific replication, independent of parental origin, in blood cells of cancer patients 
BMC Cancer  2008;8:390.
Background
Allelic counterparts of biallelically expressed genes display an epigenetic symmetry normally manifested by synchronous replication, different from genes subjected to monoallelic expression, which normally are characterized by an asynchronous mode of replication (well exemplified by the SNRPN imprinted locus). Malignancy was documented to be associated with gross modifications in the inherent replication-timing coordination between allelic counterparts of imprinted genes as well as of biallelically expressed loci. The cancer-related allelic replication timing aberrations are non-disease specific and appear in peripheral blood cells of cancer patients, including those with solid tumors. As such they offer potential blood markers for non-invasive cancer test. The present study was aimed to gain some insight into the mechanism leading to the replication timing alterations of genes in blood lymphocytes of cancer patients.
Methods
Peripheral blood samples derived from patients with prostate cancer were chosen to represent the cancerous status, and samples taken from patients with no cancer but with benign prostate hyperplasia were used to portray the normal status. Fluorescence In Situ Hybridization (FISH) replication assay, applied to phytohemagglutinin (PHA)-stimulated blood lymphocytes, was used to evaluate the temporal order (either synchronous or asynchronous) of genes in the patients' cells.
Results
We demonstrated that: (i) the aberrant epigenetic profile, as delineated by the cancer status, is a reversible modification, evidenced by our ability to restore the normal patterns of replication in three unrelated loci (CEN15, SNRPN and RB1) by introducing an archetypical demethylating agent, 5-azacytidine; (ii) following the rehabilitating effect of demethylation, an imprinted gene (SNRPN) retains its original parental imprint; and (iii) the choice of an allele between early or late replication in the aberrant asynchronous replication, delineated by the cancer status, is not random but is independent of the parental origin.
Conclusion
The non-disease specific aberrant epigenetic profile displayed in peripheral blood cells of patients with a solid tumour (unlike genetic aberrations) can be reversed, by an epigenetic drug applied in vitro, to the normal. It appears that the cancerous status differentiates between two allelic counterparts in a non-random manner, but independent of the parental origin
doi:10.1186/1471-2407-8-390
PMCID: PMC2629776  PMID: 19109880
8.  Haptoglobin phenotype is not a predictor of recurrence free survival in high-risk primary breast cancer patients 
BMC Cancer  2008;8:389.
Background
Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective follow-up study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence free survival.
Methods
Two sample sets of high-risk primary breast cancer patients participating in a randomised national trial investigating the effectiveness of high-dose chemotherapy were analysed. Sera in set I (n = 63) were analysed by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker finding. Initial results were validated by analysis of sample set II (n = 371), using one-dimensional gel-electrophoresis.
Results
In sample set I, the expression of a peak at mass-to-charge ratio 9198 (relative intensity ≤ 20 or > 20), identified as haptoglobin (Hp) alpha-1 chain, was strongly associated with recurrence free survival (global Log-rank test; p = 0.0014). Haptoglobin is present in three distinct phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), of which only individuals with phenotype Hp 1-1 or Hp 2-1 express the haptoglobin alpha-1 chain. As the expression of the haptoglobin alpha-1 chain, determined by SELDI-TOF MS, corresponds to the phenotype, initial results were validated by haptoglobin phenotyping of the independent sample set II by native one-dimensional gel-electrophoresis. With the Hp 1-1 phenotype as the reference category, the univariate hazard ratio for recurrence was 0.87 (95% CI: 0.56 – 1.34, p = 0.5221) and 1.03 (95% CI: 0.65 – 1.64, p = 0.8966) for the Hp 2-1 and Hp 2-2 phenotypes, respectively, in sample set II.
Conclusion
In contrast to our initial results, the haptoglobin phenotype was not identified as a predictor of recurrence free survival in high-risk primary breast cancer in our validation set. Our initial observation in the discovery set was probably the result of a type I error (i.e. false positive). This study illustrates the importance of validation in obtaining the true clinical applicability of a potential biomarker.
doi:10.1186/1471-2407-8-389
PMCID: PMC2627917  PMID: 19108738
9.  Smoking, environmental tobacco smoke, and risk of renal cell cancer: a population-based case-control study 
BMC Cancer  2008;8:387.
Background
Kidney and renal pelvis cancers account for 4% of all new cancer cases in the United States, among which 85% are renal cell carcinomas (RCC). While cigarette smoking is an established risk factor for RCC, little is known about the contribution of environmental tobacco smoke (ETS) to RCC incidence. This study assesses the role of smoking and ETS on RCC incidence using a population-based case-control design in Florida and Georgia.
Methods
Incident cases (n = 335) were identified from hospital records and the Florida cancer registry, and population controls (n = 337) frequency-matched by age (+/- 5 years), gender, and race were identified through random-digit dialing. In-person interviews assessed smoking history and lifetime exposure to ETS at home, work, and public spaces. Home ETS was measured in both years and hours of exposure. Odds ratios and 95% confidence intervals were calculated using logistic regression, controlled for age, gender, race, and BMI.
Results
Cases were more likely to have smoked 20 or more pack-years, compared with never-smokers (OR: 1.35, 95% CI: 0.93 – 1.95). A protective effect was found for smoking cessation, beginning with 11–20 years of cessation (OR: 0.39, 95% CI: 0.18–0.85) and ending with 51 or more years of cessation (OR: 0.11, 95% CI: 0.03–0.39) in comparison with those having quit for 1–10 years. Among never-smokers, cases were more likely to report home ETS exposure of greater than 20 years, compared with those never exposed to home ETS (OR: 2.18; 95% CI: 1.14–4.18). Home ETS associations were comparable when measured in lifetime hours of exposure, with cases more likely to report 30,000 or more hours of home ETS exposure (OR: 2.37; 95% CI: 1.20–4.69). Highest quartiles of combined home/work ETS exposure among never-smokers, especially with public ETS exposure, increased RCC risk by 2 to 4 times.
Conclusion
These findings confirm known associations between smoking and RCC and establish a potential etiologic role for ETS, particularly in the home. Differences in methods of retrospective measurement of lifetime smoking and ETS exposure may contribute to discrepancies in measures of associations across studies, and should be addressed in future research.
doi:10.1186/1471-2407-8-387
PMCID: PMC2633310  PMID: 19108730
10.  Histone H1x is highly expressed in human neuroendocrine cells and tumours 
BMC Cancer  2008;8:388.
Background
Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation.
Methods
Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study.
Results
We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues.
Conclusion
We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate.
doi:10.1186/1471-2407-8-388
PMCID: PMC2631592  PMID: 19108733
11.  Genetic analysis of the vitamin D receptor gene in two epithelial cancers: melanoma and breast cancer case-control studies 
BMC Cancer  2008;8:385.
Background
Vitamin D serum levels have been found to be related to sun exposure and diet, together with cell differentiation, growth control and consequently, cancer risk. Vitamin D receptor (VDR) genotypes may influence cancer risk; however, no epidemiological studies in sporadic breast cancer (BC) or malignant melanoma (MM) have been performed in a southern European population. In this study, the VDR gene has been evaluated in two epithelial cancers BC and MM.
Methods
We have conducted an analysis in 549 consecutive and non-related sporadic BC cases and 556 controls, all from the Spanish population, and 283 MM cases and 245 controls. Genotyping analyses were carried out on four putatively functional SNPs within the VDR gene.
Results
An association with the minor allele A of the non-synonymous SNP rs2228570 (rs10735810, FokI, Met1Thr) was observed for BC, with an estimated odds ratio (OR) of 1.26 (95% CI = 1.02–1.57; p = 0.036). The synonymous variant rs731236 (TaqI) appeared to be associated with protection from BC (OR = 0.80, 95%CI = 0.64–0.99; p = 0.047). No statistically significant associations with MM were observed for any SNP. Nevertheless, sub-group analyses revealed an association between rs2228570 (FokI) and absence of childhood sunburns (OR = 0.65, p = 0.003), between the 3'utr SNP rs739837 (BglI) and fair skin (OR = 1.31, p = 0.048), and between the promoter SNP rs4516035 and the more aggressive tumour location in head-neck and trunk (OR = 1.54, p = 0.020).
Conclusion
In summary, we observed associations between SNPs in the VDR gene and BC risk, and a comprehensive analysis using clinical and tumour characteristics as outcome variables has revealed potential associations with MM. These associations required confirmation in independent studies.
doi:10.1186/1471-2407-8-385
PMCID: PMC2639605  PMID: 19105801
12.  Thymidylate synthase, dihydropyrimidine dehydrogenase, ERCC1, and thymidine phosphorylase gene expression in primary and metastatic gastrointestinal adenocarcinoma tissue in patients treated on a phase I trial of oxaliplatin and capecitabine 
BMC Cancer  2008;8:386.
Background
Over-expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in tumor tissue is associated with insensitivity to 5-fluorouracil (5-FU). Over-expression of ERCC1 correlates with insensitivity to oxaliplatin (OX) therapy, while high thymidine phosphorylase (TP) levels predict for increased sensitivity to capecitabine (Xel).
Methods
Biopsies of metastatic tumor were taken before OX (130 mg/m2 day 1) given with Xel (1200–3000 mg/m2 in two divided doses days 1–5 and 8–12) every 3-weeks. Micro-dissected metastatic and primary tumors were analyzed for relative gene expression by real-time quantitative polymerase chain reaction. The clinical protocol prospectively identified the molecular targets of interest that would be tested. Endpoints for the molecular analyses were correlation of median, first and third quartiles for relative gene expression of each target with response, time to treatment failure (TTF), and survival.
Results
Among 91 patients participating in this trial; 97% had colorectal cancer. The median number of prior chemotherapy regimens was 2, and most had prior 5-FU and irinotecan. In paired samples, median mRNA levels were significantly higher in metastatic versus primary tumor (-fold): TS (1.9), DPD (3.8), ERCC1 (2.1) and TP (1.6). A strong positive correlation was noted between DPD and TP mRNA levels in both primary (r = 0.693, p < 0.0005) and metastatic tissue (r = 0.697, p < 0.00001). There was an association between TS gene expression and responsive and stable disease: patients whose intratumoral TS mRNA levels were above the median value had significantly greater risk of early disease progression (43% vs 17%), but this did not translate into a significant difference in TTF. ERCC1 gene expression above the third quartile was associated with a shorter TTF (median 85 vs 162 days, p = 0.046). Patients whose TS mRNA levels in metastatic tumor tissue were below the median had a longer overall survival (median 417 vs 294 days, p = 0.042).
Conclusion
Target gene expression in primary tumor was significantly lower than that in paired metastatic tissue. High ERCC1 mRNA levels in metastatic tumor was associated with a shorter TTF. Lower expression of TS mRNA correlated with a lower chance of early PD with XelOX therapy and improved overall survival.
doi:10.1186/1471-2407-8-386
PMCID: PMC2637882  PMID: 19105824
13.  Expression of centromere protein F (CENP-F) associated with higher FDG uptake on PET/CT, detected by cDNA microarray, predicts high-risk patients with primary breast cancer 
BMC Cancer  2008;8:384.
Background
Higher standardized uptake value (SUV) detected by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) correlates with proliferation of primary breast cancer. The purpose of this study is to identify specific molecules upregulated in primary breast cancers with a high SUV and to examine their clinical significance.
Methods
We compared mRNA expression profiles between 14 tumors with low SUVs and 24 tumors with high SUVs by cDNA microarray. We identified centromere protein F (CENP-F) and CDC6 were upregulated in tumors with high SUVs. RT-PCR and immunohistochemical analyses were performed to validate these data. Clinical implication of CENP-F and CDC6 was examined for 253 archival breast cancers by the tissue microarray.
Results
The relative ratios of CENP-F and CDC6 expression levels to β-actin were confirmed to be significantly higher in high SUV tumors than in low SUV tumors (p = 0.027 and 0.025, respectively) by RT-PCR. In immunohistochemical analysis of 47 node-negative tumors, the CENP-F expression was significantly higher in the high SUV tumors (74%) than the low SUV tumors (45%) (p = 0.04), but membranous and cytoplasmic CDC6 expressions did not significantly differ between both groups (p = 0.9 each). By the tissue microarray, CENP-F (HR = 2.94) as well as tumor size (HR = 4.49), nodal positivity (HR = 4.1), and Ki67 (HR = 2.05) showed independent impact on the patients' prognosis.
Conclusion
High CENP-F expression, correlated with high SUV, was the prognostic indicators of primary breast cancer. Tumoral SUV levels may serve as a pretherapeutic indicator of aggressiveness of breast cancer.
doi:10.1186/1471-2407-8-384
PMCID: PMC2631591  PMID: 19102762
14.  Antioxidant intervention of smoking-induced lung tumor in mice by vitamin E and quercetin 
BMC Cancer  2008;8:383.
Background
Epidemiological and in vitro studies suggest that antioxidants such as quercetin and vitamin E (VE) can prevent lung tumor caused by smoking; however, there is limited evidence from animal studies.
Methods
In the present study, Swiss mouse was used to examine the potential of quercetin and VE for prevention lung tumor induced by smoking.
Results
Our results suggest that the incidence of lung tumor and tumor multiplicity were 43.5% and 1.00 ± 0.29 in smoking group; Quercetin has limited effects on lung tumor prevention in this in vivo model, as measured by assays for free radical scavenging, reduction of smoke-induced DNA damage and inhibition of apoptosis. On the other hand, vitamin E drastically decreased the incidence of lung tumor and tumor multiplicity which were 17.0% and 0.32 ± 0.16, respectively (p < 0.05); and demonstrated prominent antioxidant effects, reduction of DNA damage and decreased cell apoptosis (p < 0.05). Combined treatment with quercetin and VE in this animal model did not demonstrate any effect greater than that due to vitamin E alone. In addition, gender differences in the occurrence of smoke induced-lung tumor and antioxidant intervention were also observed.
Conclusion
We conclude that VE might prevent lung tumor induced by smoking in Swiss mice.
doi:10.1186/1471-2407-8-383
PMCID: PMC2625366  PMID: 19099597
15.  Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer 
BMC Cancer  2008;8:382.
Background
Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk.
Methods
A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area.
Results
Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09–2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09–2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms.
Conclusion
Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.
doi:10.1186/1471-2407-8-382
PMCID: PMC2626602  PMID: 19099590
16.  Aberrant Expression of ID2 protein and its correlation with EBV-LMP1 and P16(INK4A) in Classical Hodgkin Lymphoma in China 
BMC Cancer  2008;8:379.
Background
The relationships between the expression of ID2, EBV-LMP1 and P16(INK4A) in Chinese classical Hodgkin lymphoma are unknown and need exploring.
Methods
Samples of classical Hodgkin lymphoma from 60 Chinese patients were analyzed for the expression of ID2, EBV-LMP1 and p16(INK4A) proteins by immunohistochemistry.
Results
ID2 protein was expressed in 83.3% of this group of classical Hodgkin lymphoma, staining strongly in both cytoplasm and nucleus of the Hodgkin and Reed-Sternberg (HRS) cells. EBV-LMP1 and P16(INK4A) were overexpressed in 85.0% and 71.7% of Hodgkin lymphoma, respectively. EBV-LMP1 was noted in the cytoplasm, membrane and nucleus of HRS cells; P16(INK4A) was in the nucleus and cytoplasm. Microscopically, ID2, EBV-LMP1 and P16(INK4A) staining distinguished the HRS cells from the complex background of lymphocytes. ID2 was positively correlated with EBV-LMP1(P < 0.01), but P16(INK4A) was inversely related to EBV-LMP1 (P < 0.05).
Conclusion
It is suggested that ID2, EBV-LMP1 and P16(INK4A) could play an important role in the evolution of classical Hodgkin lymphoma, and be considered as potential adjunct markers to identify HRS cells in diagnosis.
doi:10.1186/1471-2407-8-379
PMCID: PMC2625365  PMID: 19099554
17.  Polymorphism +17 C/G in Matrix Metalloprotease MMP8 decreases lung cancer risk 
BMC Cancer  2008;8:378.
Background
Matrix metalloproteases (MMPs) constitute a family of enzymes capable of degrading different components of the extracellular matrix and are implicated in the invasion of tumor cells through the basement membrane. Polymorphisms in MMP genes may result in changes in the expression of MMPs being associated with the development and progression of cancer. We have investigated the association between three polymorphisms (-1607 1G/2G, +17 C/G and -77 A/G) in the human collagenases MMP1, MMP8 and MMP13 and the risk of development or progression of lung cancer.
Methods
A hospital-based case-control study was designed including 501 lung cancer patients and 510 controls matched. Genotypes were determined by PCR-RFLP. Results were analyzed using unconditional logistic regression, Cox's proportional hazard regression, and the Kaplan-Meier method.
Results
The MMP1 and MMP13 promoter polymorphisms were not associated with lung cancer risk, while the C/G polymorphism in MMP8 was associated with a statistically significant decreased risk of developing lung cancer (ORadj = 0.65; 95%CI = 0.45–0.93). The Kaplan-Meier analysis showed that the polymorphisms in MMP1, MMP8 and MMP13 not seem to modify the overall survival. Multivariate analysis revealed that MMP1, MMP8 and MMP13 polymorphisms are not independent prognostic factors for overall survival.
Conclusion
This study suggests that the polymorphism in MMP8 is associated with a decreased lung cancer risk, which can be used as a prognostic marker in lung cancer.
doi:10.1186/1471-2407-8-378
PMCID: PMC2628929  PMID: 19094243
18.  Role of cAMP in the promotion of colorectal cancer cell growth by Prostaglandin E2 
BMC Cancer  2008;8:380.
Background
Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) reaction, stimulates the growth of colonic epithelial cells. It is inferred that the abrogation of prostaglandins' growth-promoting effects as a result of COX inhibition underlies the advantageous effects of non-steroidal anti-inflammatory drugs in colorectal carcinoma (CRC). Despite this appreciation, the underlying molecular mechanisms remain obscure since cell culture studies have yielded discrepant results regarding PGE2's mitogenicity.
Methods
We have employed several alternative approaches to score cell proliferation and apoptosis of 4 CRC cell lines exposed to PGE2 under various conditions. To investigate the role of cAMP in PGE2's functions, activation of the cAMP pathway was assessed at different levels (changes in cAMP levels and PKA activity) in cells subjected to specific manipulations including the use of specific inhibitors or prostanoid receptor-selective agonists/antagonists.
Results
Our data document that the dose-response curve to PGE2 is 'bell-shaped', with nano molar concentrations of PGE2 being more mitogenic than micro molar doses. Remarkably, mitogenicity inversely correlates with the ability of PGE2 doses to raise cAMP levels. Consistent with a major role for cAMP, cAMP raising agents and pertussis toxin revert the mitogenic response to PGE2. Accordingly, use of prostanoid receptor-selective agonists argues for the involvement of the EP3 receptor and serum deprivation of HT29 CRC cells specifically raises the levels of Gi-coupled EP3 splice variants.
Conclusion
The present data indicate that the mitogenic action of low PGE2 doses in CRC cells is mediated via Gi-proteins, most likely through the EP3 receptor subtype, and is superimposed by a second, cAMP-dependent anti-proliferative effect at higher PGE2 doses. We discuss how these findings contribute to rationalize conflictive literature data on the proliferative action of PGE2.
doi:10.1186/1471-2407-8-380
PMCID: PMC2615781  PMID: 19099561
19.  Class I histone deacetylases 1, 2 and 3 are highly expressed in renal cell cancer 
BMC Cancer  2008;8:381.
Background
Enhanced activity of histone deacetylases (HDAC) is associated with more aggressive tumour behaviour and tumour progression in various solid tumours. The over-expression of these proteins and their known functions in malignant neoplasms has led to the development of HDAC inhibitors (HDI) as new anti-neoplastic drugs. However, little is known about HDAC expression in renal cell cancer.
Methods
We investigated the expression of HDAC 1, 2 and 3 in 106 renal cell carcinomas and corresponding normal renal tissue by immunohistochemistry on tissue micro arrays and correlated expression data with clinico-pathological parameters including patient survival.
Results
Almost 60% of renal cell carcinomas expressed the HDAC isoforms 1 and 2. In contrast, HDAC 3 was only detected in 13% of all renal tumours, with particular low expression rates in the clear cell subtype. HDAC 3 was significantly higher expressed in pT1/2 tumours in comparison to pT3/4 tumours. Expression of class I HDAC isoforms correlated with each other and with the proliferative activity of the tumours. We found no prognostic value of the expression of any of the HDAC isoforms in this tumour entity.
Conclusion
Class I HDAC isoforms 1 and 2 are highly expressed in renal cell cancer, while HDAC 3 shows low, histology dependent expression rates. These unexpected differences in the expression patterns suggests alternative regulatory mechanisms of class I HDACs in renal cell cancer and should be taken into account when trials with isoform selective HDI are being planned. Whether HDAC expression in renal cancers is predictive of responsiveness for HDI will have to be tested in further studies.
doi:10.1186/1471-2407-8-381
PMCID: PMC2631013  PMID: 19099586
20.  tabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO) 
BMC Cancer  2008;8:377.
Background
The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.
Methods
Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.
Results
Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.
Conclusion
The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.
doi:10.1186/1471-2407-8-377
PMCID: PMC2626601  PMID: 19094209
21.  Increased p38-MAPK is responsible for chemotherapy resistance in human gastric cancer cells 
BMC Cancer  2008;8:375.
Background
Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with Multidrug resistance (MDR). Many different mechanisms have been suggested to explain the development of an MDR phenotype in cancer cells. One of the most studied mechanisms is the overexpression of P-glycoprotein (P-gp), which is a product of the MDR1 gene. Tumor cells often acquire the drug-resistance phenotype due to upregulation of the MDR1 gene. Overexpression of MDR1 gene has often been reported in primary gastric adenocarcinoma.
Methods
This study investigated the role of p38-MAPK signal pathway in vincristine-resistant SGC7901/VCR cells. P-gp and MDR1 RNA were detected by Western blot analysis and RT-PCR amplification. Mitgen-activated protein kinases and function of P-gp were demonstrated by Western blot and FACS Aria cytometer analysis. Ap-1 activity and cell apoptosis were detected by Dual-Luciferase Reporter Assay and annexin V-PI dual staining.
Results
The vincristine-resistant SGC7901/VCR cells with increased expression of the multidrug-resistance 1 (MDR1) gene were resistant to P-gp-related drug and P-gp-unrelated drugs. Constitutive increases of phosphorylated p38-MAPK and AP-1 activities were also found in the drug-resistant cells. Inhibition of p38-MAPK by SB202190 reduced activator protein-1 (AP-1) activity and MDR1 expression levels and increased the sensitivity of SGC7901/VCR cells to chemotherapy.
Conclusion
Activation of the p38-MAPK pathway might be responsible for the modulation of P-glycoprotein-mediated and P-glycoprotein-unmediated multidrug resistance in the SGC7901/VCR cell line.
doi:10.1186/1471-2407-8-375
PMCID: PMC2628930  PMID: 19091131
22.  Gemcitabine-based versus fluoropyrimidine-based chemotherapy with or without platinum in unresectable biliary tract cancer: a retrospective study 
BMC Cancer  2008;8:374.
Background
There is no standard palliative chemotherapy regimen in biliary tract cancers (BTC). Fluoropyrimidine or gemcitabine, with or without platinum, are most frequently used. We conducted this study to clarify the efficacy of palliative chemotherapy in BTC.
Methods
Patients with unresectable BTC treated with palliative chemotherapy between Oct 2001 and Aug 2006 at Seoul National University Hospital were reviewed retrospectively. Histologically confirmed cases of intrahepatic cholangiocarcinoma, gallbladder cancer, extrahepatic bile duct cancer, and ampulla of Vater carcinoma were enrolled. We analyzed the efficacy of regimens: gemcitabine (G) versus fluoropyrimidine (F) and with or without platinum (P).
Results
A total of 243 patients were enrolled. 159 patients (65%) were male and the median age of the patients was 60 years (range 26–81). Intrahepatic cholangiocarcinoma, gallbladder cancer, extrahepatic bile duct cancer, and ampulla of Vater carcinoma were 92, 72, 58, and 21 cases, respectively. The median progression free survival (PFS) was 4.3 months (95% CI, 3.7–4.9) and median overall survival (OS) was 8.7 months (95% CI, 7.4–10.0). Ninety-nine patients received G-based chemotherapy (94 GP, 5 G alone), and 144 patients received F-based chemotherapy (83 FP, 61 F alone). The response rate (RR), disease control rate (DCR), PFS and OS of G-based chemotherapy versus F-based chemotherapy were 16.7% vs. 19.5% (P = 0.591), 52.8% vs. 58.9% (P = 0.372), 4.0 months vs. 4.3 months (P = 0.816), and 7.8 months vs. 9.1 months (P = 0.848), respectively. Sixty-six patients received F or G without P, and 177 patients received F or G with P. The RR, DCR, PFS and OS of chemotherapy without P versus chemotherapy including P were 12.7% vs. 20.6% (P = 0.169), 46.0% vs. 60.6% (P = 0.049), 3.3 months vs. 4.4 months (P = 0.887), and 10.6 months vs. 8.1 months (P = 0.257), respectively.
Conclusion
In unresectable BTC, F-based and G-based chemotherapy showed similar efficacy in terms of RR, DCR, PFS and OS. The benefit of adding P to F or G was not significant except for DCR. Further prospective studies which define the efficacy of various chemotherapeutic regimens in BTC are warranted.
doi:10.1186/1471-2407-8-374
PMCID: PMC2615782  PMID: 19091129
23.  Cimetidine inhibits salivary gland tumor cell adhesion to neural cells and induces apoptosis by blocking NCAM expression 
BMC Cancer  2008;8:376.
Background
Cimetidine, a histamine type-2 receptor antagonist, has been reported to inhibit the growth of glandular tumors such as colorectal cancer, however the mechanism of action underlying this effect is unknown. Adenoid cystic carcinoma is well known as a malignant salivary gland tumor which preferentially invades neural tissues. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express neural cell adhesion molecule (NCAM), that HSG cell proliferation may be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. We further demonstrated that cimetidine inhibited NCAM expression and induced apoptosis in HSG cells. Here, we investigated the effects of cimetidine on growth and perineural/neural invasion of salivary gland tumor cells.
Methods
In this study, we have examined the effect of cimetidine on cancer cell adhesion to neural cells in vitro, one of the critical steps of cancer invasion and metastasis. We have also used an in vivo carcinogenesis model to confirm the effect of cimetidine.
Results
We have demonstrated for the first time that cimetidine can block the adhesion of HSG cells to neural cell monolayers and that it can also induce significant apoptosis in the tumor mass in a nude mouse model. We also demonstrated that these apoptotic effects of cimetidine might occur through down-regulation of the cell surface expression of NCAM on HSG cells. Cimetidine-mediated down-regulation of NCAM involved suppression of the nuclear translocation of NF-κB, a transcriptional activator of NCAM gene expression.
Conclusion
These findings suggest that growth and perineural/neural invasion of salivary gland tumors can be blocked by administration of cimetidine via induction of apoptosis and in which NCAM plays a role.
doi:10.1186/1471-2407-8-376
PMCID: PMC2635382  PMID: 19091137
24.  Down-regulation of GRP78 is associated with the sensitivity of chemotherapy to VP-16 in small cell lung cancer NCI-H446 cells 
BMC Cancer  2008;8:372.
Background
Chemotherapy resistance remains a major obstacle for the treatment of small cell lung cancer (SCLC). Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum chaperone, plays a critical role in chemotherapy resistance in some cancers. However, whether the suppression of the chaperone can enhance the sensitivity of chemotherapy in SCLC is still unclear.
Methods
The SCLC NCI-H446 cells were divided into three groups: BAPTA-AM→A23187-treated group, A23187-treated group and control-group. Immunofluorescence, western blot and RT-PCR were used to assess the expression of GRP78 at both protein and mRNA levels. Cell apoptosis and the cell cycle distributions of the cells were analyzed by flow cytometry in order to evaluate the therapeutic sensitivity to VP-16.
Results
The expression of GRP78 at both protein and mRNA levels in the BAPTA-AM→A23187-treated cells dramatically decreased as compared to that in both A23187-treated and control groups. After treatment by VP-16, the percentage of apoptotic cells in BAPTA-AM→A23187-treated cells were: 33.4 ± 1.01%, 48.2 ± 1.77%, 53.0 ± 1.43%, 56.5 ± 2.13%, respectively, corresponding to the concentrations of BAPTA-AM 10, 15, 25, 40 μM, which was statistically significant high in comparison with the A23187-treated group and untreated-group (7.18 ± 1.03% and 27.8 ± 1.45%, respectively, p < 0.05). The results from analysis of cell cycle distribution showed that there was a significantly decreased in G1 phase and a dramatically increased in S phase for the BAPTA-AM→A23187-treated cells as compared with the untreated cells.
Conclusion
BAPTA-AM is a strong inhibitor of GRP78 in the NCI-H446 cell line, the down-regulation of GRP78 can significantly increase the sensitivity to VP-16. The suppression of GRP78 may offer a new surrogated therapeutic approach to the clinical management of lung cancer.
doi:10.1186/1471-2407-8-372
PMCID: PMC2628931  PMID: 19087354
25.  Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer 
BMC Cancer  2008;8:373.
Background
In a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.
Methods
The expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.
Results
The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.
Conclusion
A reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.
doi:10.1186/1471-2407-8-373
PMCID: PMC2628383  PMID: 19091121

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