Simple regression for correcting ΔCt bias in RT-qPCR low-density array data normalization.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard for quantifying relative gene expression. Normalization of RT-qPCR data is commonly achieved by subtracting the Ct values of the internal reference genes from the Ct values of the target genes to obtain ΔCt. ΔCt values are then used to derive ΔΔCt when compared to a control group or to conduct further statistical analysis. RESULTS: We examined two rheumatoid arthritis RT-qPCR low density array datasets and found that this normalization method introduces substantial bias due to differences in PCR amplification efficiency among genes. This bias results in undesirable correlations between target genes and reference genes, which affect the estimation of fold changes and the tests for differentially expressed genes. Similar biases were also found in multiple public mRNA and miRNA RT-qPCR array datasets we analysed. We propose to regress the Ct values of the target genes onto those of the reference genes to obtain regression coefficients, which are then used to adjust the reference gene Ct values before calculating ΔCt. CONCLUSIONS: The per-gene regression method effectively removes the ΔCt bias. This method can be applied to both low density RT-qPCR arrays and individual RT-qPCR assays.

Expression of Interferon-γ Receptor Genes in Peripheral Blood Mononuclear Cells Is Associated With Rheumatoid Arthritis and Its Radiographic Severity in African Americans.

OBJECTIVE: The factors responsible for radiographic severity in African American patients with rheumatoid arthritis (RA) are poorly understood. We sought to identify genes whose expression in peripheral blood mononuclear cells is associated with radiographic severity in RA. METHODS: In the first phase of the study, we performed real-time quantitative polymerase chain reaction to analyze the expression of 182 candidate genes in 40 African American RA patients with extremes of radiographic damage (low versus high radiographic scores) and disease duration (≤2 years versus >2 years) and 20 healthy African American control subjects; the genes were selected based on plausible immune pathways. In the second phase, we analyzed the expression of the genes that were shown to be significantly associated with radiographic scores in 576 African American patients with RA and 51 African American control subjects who had not been studied previously, accounting for autoantibody status and disease duration. RESULTS: We observed significant differences in IFNGR1 expression between patients with RA and control subjects (adjusted P [P(adj)] = 6 × 10(-14)) and in IFNGR2 expression between RA patients with erosions and those with no erosions (P(adj) = 0.01 by Wilcoxon's rank sum test). We also observed significant correlations between IFNGR2 expression and radiographic scores (P(adj) = 0.03 for erosions, P(adj) = 0.04 for joint space narrowing, and P(adj) = 0.03 for total radiographic score [zero-inflated negative binomial regression model]) and annualized progression rate (P(adj) = 0.0024 by Spearman's correlation analysis). CONCLUSION: These findings have important implications with respect to the role of interferon-γ (IFNγ) in the pathogenesis of RA and may lead to identification of a biomarker for radiographic damage. Additional studies are needed to define the cell subsets responsible for the association of IFNγ receptor gene expression with radiographic findings, which downstream mechanisms are involved, and generalizability to other RA populations.

Gene expression patterns in peripheral blood cells associated with radiographic severity in African Americans with early rheumatoid arthritis.

Gene expression profiling may be used to stratify patients by disease severity to test the hypothesis that variable disease outcome has a genetic component. In order to define unique expression signatures in African American rheumatoid arthritis (RA) patients with severe erosive disease, we undertook a gene expression study using samples of RNA from peripheral blood mononuclear cells (PBMCs). RNA from baseline PBMC samples of 96 African American RA patients with early RA (<2 years disease duration) was hybridized to cDNA probes of the Illumina Human HT-V3 expression array. Expression analyses were performed using the ca. 25,000 cDNA probes, and then expression levels were compared to the total number of erosions in radiographs of the hands and feet at baseline and 36 months. Using a false discovery rate cutoff of Q = 0.30, 1,138 genes at baseline and 680 genes at 36 months significantly correlated with total erosions. No evidence of a signal differentiating disease progression, or change in erosion scores between baseline and 36 months, was found. Further analyses demonstrated that the differential gene expression signature was localized to the patients with the most erosive disease (>10 erosions). Ingenuity Pathway Analysis demonstrated that genes with fold change greater than 1.5 implicated immune pathways such as CTLA signaling in cytotoxic T lymphocytes. These results demonstrate that CLEAR patients with early RA having the most severe erosive disease, as compared to more mild cases (<10 erosions), may be characterized by a set of differentially expressed genes that represent biological pathways with relevance to autoimmune disease.

Differential expression of breast cancer-associated genes between stage- and age-matched tumor specimens from African- and Caucasian-American Women diagnosed with breast cancer.

BACKGROUND: Recent studies suggest that the poorer breast cancer outcome observed in African-American women (AAW) may, in part, result from underlying molecular factors. The purpose of this study was to investigate gene expression differences between Caucasian-American women (CAW) and AAW that may contribute to this poorer prognosis. METHODS: The expression of 84 genes involved in breast carcinoma prognosis, response to therapy, estrogen signaling, and tumor aggressiveness was assessed in age- and stage-matched CAW and AAW paraffin-embedded breast cancer specimens. The Wilcoxon-Mann-Whitney Test was used to identify genes with a significant difference in expression between CAW and AAW. To determine if the differentially expressed genes could segregate between the CAW and AAW, we performed semi-supervised principal component analysis (SSPCA). RESULTS: Twenty genes were differentially expressed between AAW and CAW. SSPCA incorporating these 20 genes segregated AAW and CAW into two distinct groups. AAW were significantly (p < 0.05) more likely to display aberrations in G(1)/S cell-cycle regulatory genes, decreased expression of cell-adhesion genes, and low to no expression of ESR1, PGR, ERBB2 and estrogen pathway targets. CONCLUSIONS: The gene expression differences identified between AAW and CAW may contribute to more aggressive disease, resistance to therapy, enhanced metastatic potential and poor clinical outcome. These findings support the hypothesis that breast cancer specimens collected from AAW display distinct gene expression differences compared to similar tissues obtained from CAW. Additional population-based studies are necessary to determine if these gene expression variations contribute to the highly aggressive and treatment-resistant breast cancer phenotype frequently observed in AAW.

Kidney injury accelerates cystogenesis via pathways modulated by heme oxygenase and complement.

AKI accelerates cystogenesis. Because cystogenic mutations induce strong transcriptional responses similar to those seen after AKI, these responses may accelerate the progression of cystic renal disease. Here, we modulated the severity of the AKI-like response in Cys1(cpk/cpk) mice, a model that mimics autosomal recessive polycystic kidney disease. Specifically, we induced or inhibited activity of the renoprotective enzyme heme oxygenase (HO) and determined the effects on renal cystogenesis. We found that induction of HO attenuated both renal injury and the rate of cystogenesis, whereas inhibition of HO promoted cystogenesis. HO activity mediated the response of NFκB, which is a hallmark transcriptional feature common to both cystogenesis and AKI. Among the HO-modulated effects we measured, expression of complement component 3 (C3) strongly correlated with cystogenesis, a functionally relevant association as suggested by Cys1(cpk/cpk) mice with genetically induced C3 deficiency. Because both C3 deficiency and HO induction reduce cyst number and cyst areas, these two factors define an injury-stimulated cystogenic pathway that may provide therapeutic targets to slow the formation of new renal cysts and the growth of existing cysts.

Estrogen receptor-α and sex steroid hormones regulate Toll-like receptor-9 expression and invasive function in human breast cancer cells.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor, which is widely expressed in cancer. Synthetic TLR9-ligands induce cancer cell invasion in vitro, but the role of TLR9 in cancer pathophysiology remains unclear. Increased TLR9 expression has been, however, detected in estrogen receptor negative (ER-) breast cancers. In this study, we investigated the effects of ERα expression and sex steroid hormones on TLR9 expression in human ER+ (MCF-7, T47-D) and ER- (MDA-MB-231) breast cancer cell lines in vitro. We also studied TLR9 mRNA expression in archival breast cancer specimens (n = 12) with qRT-PCR, using primer sets that detect only the TLR9A isoform or the isoforms A and B (TLR9A/B). The TLR9 mRNA expression was detected in 10/12 specimens with both primer sets, and in 1/12 with only the TLR9A or the TLR9A/B primer sets. The basal TLR9 mRNA expression levels were significantly lower in the ER+ cell lines as compared with the ER- MDA-MB-231 cells. The transfection of ERα cDNA into MDA-MB-231 cells also resulted in down-regulation of TLR9 expression. While sex steroids had no effect on TLR9 expression in MCF-7 cells, testosterone (10(-8) M) induced TLR9 expression in MDA-MB-231 and T47-D cells. Although bicalutamide blocked this testosterone effect in MDA-MB-231 cells, in T47-D cells bicalutamide increased TLR9 expression and only partially blocked the testosterone effects. Estradiol (10(-8) M) induced TLR9 expression in T47-D cells. The invasive effects of synthetic TLR9-ligands were augmented by testosterone in vitro. This effect was lost in TLR9 siRNA MDA-MB-231 cells and also decreased by over-expression of ERα, which also inhibited NF-κB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERα and sex steroid hormones regulate TLR9 expression and invasive effects in the breast cancer cells. Also, the commonly used hormonal cancer therapy bicalutamide affects TLR9 expression.

Gene expression analysis in radiotherapy patients and C57BL/6 mice as a measure of exposure to ionizing radiation.

Dose assessment after radiological disasters is imperative to decrease mortality through rationally directed medical intervention. Our goal was to identify biomarkers capable of qualitative (nonirradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure. Using real-time quantitative PCR, biodosimetry genes were identified in blood samples from cancer patients undergoing total-body irradiation. Time- (5, 12, 23, 48 h) and dose- (0-8 Gy) dependent changes in gene expression were examined in C57BL/6 mice. A training set was used to derive weighted voting classification algorithms (nonirradiated/irradiated) and continuous regression (dose assessment) models that were tested in a separate validation set of mice. Of eight biodosimetry genes identified in cancer patients ( ACTA2 , BBC3 , CCNG1 , CDKN1A , GADD45A , MDK , SERPINE1 , Tnfrsf10b ), expression of BBC3 , CCNG1 , CDKN1A , SERPINE1 and Tnfrsf10b was significantly (P < 0.05) increased in irradiated mice. CCNG1 and CDKN1A expression segregated irradiated mice from controls with an accuracy, specificity and sensitivity of 96.3, 100.0 and 94.4%, respectively, at 48 h. Multiple linear regression analysis predicted doses for the 0-, 1-, 2-, 4-, 6- and 8-Gy treatment groups as 0.0 ± 0.2, 1.6 ± 1.0, 2.9 ± 1.4, 5.1 ± 2.0, 5.3 ± 0.7 and 10.5 ± 5.6 Gy, respectively. These results suggest that gene expression analysis could be incorporated into biodosimetry protocols for qualitative and quantitative assessment of radiation exposure.

Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.

Activation of the hedgehog (HH) pathway plays a critical role in the development and continued growth of pancreatic adenocarcinoma (PAC). Cyclopamine, a HH pathway inhibitor, has been shown to suppress PAC cell proliferation in vitro and in vivo. However, the molecular basis of response to cyclopamine has not been fully elucidated nor have genes that predict sensitivity to this compound been identified. To better understand these features of HH pathway inhibition, we evaluated the biological and molecular effects of cyclopamine in vitro. The viability of 9 human PAC cell lines following cyclopamine exposure was determined using MTS assay. Proliferation and induction of apoptosis in treated cells were examined by bromo-deoxyuridine incorporation, caspase activation, and mitochondrial membrane potential. Gene expression before and after cyclopamine treatment was determined using Taqman real-time quantitative polymerase chain reaction (RTQ-PCR) and Taqman low-density array (TLDA). Among the cell lines examined, cyclopamine IC50 values ranged from 8.79 to >30 µM. Response to cyclopamine included reduced cell proliferation and induction of apoptosis with and without mitochondrial membrane depolarization. Regression analysis revealed that GLI3 expression significantly correlated with cyclopamine resistance (r = 0.80; p = 0.0102). Knockdown of GLI3 using siRNAs increased sensitivity to cyclopamine. In addition, GLI3 siRNAs decreased PAC cell viability and reduced expression of genes involved in HH signaling (Patched 1 and GLI1) and cell proliferation, similar to cyclopamine. These effects were not observed in PAC cells with undetectable GLI3 expression. These data suggest that Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.

Pyrazolopyridines as potent PDE4B inhibitors: 5-heterocycle SAR.

Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett.2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-α production from isolated human peripheral blood mononuclear cells.

Renal CD14 expression correlates with the progression of cystic kidney disease.

Monocyte and macrophage markers are among the most highly overexpressed genes in cpk mouse kidneys with severely progressive renal cystic disease. We show here that one of these markers, CD14, is abnormally transcribed, activated and shed in cystic kidneys. However, these abnormalities were not associated with an increased number of interstitial CD14-positive mononuclear cells. Instead, we found that most non-cystic and cystic renal tubular epithelia were CD14-positive; even distal nephron-derived principal cells. Cd14 was significantly overexpressed in the kidneys of 5-day-old cpk mice and further increased as the disease progressed. In the cpk model with variable rates of cystic kidney enlargement (due to an intercross of two distinct genetic backgrounds), Cd14 expression positively correlated with kidney volume, exceeding the correlation with MCP-1, an established marker of autosomal-dominant polycystic kidney disease (ADPKD). In 16 patients with ADPKD, the baseline urinary CD14 level showed some tendency to correlate with the 2-year change in total kidney volume; however, the tendency was not statistically significant. But the association was significant when the analysis was confined to males. Clearly more studies need to be done to evaluate the utility of CD14 as a marker for outcomes in ADPKD.

Rationally designed pharmacogenomic treatment using concurrent capecitabine and radiotherapy for glioblastoma; gene expression profiles associated with outcome.

PURPOSE: Previous preclinical studies suggested that concurrent capecitabine and radiation could be an effective new treatment modality for glioblastoma (GBM). In the current study, we investigate toxicity and response to this regimen and explore associations between gene expression and patient outcome. EXPERIMENTAL DESIGN: Eighteen newly diagnosed GBM patients received concurrent capecitabine at 625 mg/m2 BID (25% escalation) and irradiation (60 Gy total) for 6 weeks followed by 4 weeks of capecitabine only. Maintenance capecitabine was administered for 14 days every 3 weeks until progression or unacceptable toxicity. Expression analysis of 94 genes involved in capecitabine metabolism and radiation response was done on tissues obtained before therapy. The relationship of gene expression with time-to-progression (TTP) and overall survival (OS) was investigated using univariate Cox proportional hazards regression, semi-supervised principle component analysis, and class prediction modeling. RESULTS: The maximum tolerated dose of capecitabine was 625 mg/m2 BID. Median patient TTP and OS were 247 and 367 days, respectively. Cox regression identified 24 genes significantly (P<0.025) associated with patient outcome. Semi-supervised principle component analysis identified two patient populations significantly different in both TTP (P=0.005) and OS (P=0.015). Class prediction modeling determined that eight genes (RAD54B, MTOR, DCTD, APEX2, TK1, RRM2, SLC29A1, and ERCC6) could collectively classify patients into outcome subgroups with 100% accuracy and precision. CONCLUSIONS: Capecitabine and concurrent radiation for newly diagnosed GBM seems to be well tolerated and comparable to temozolomide and radiation. A gene expression profile predictive of patient outcome that may be useful in patient stratification for therapy was also elucidated.

Gli1 promotes cell survival and is predictive of a poor outcome in ERalpha-negative breast cancer.

Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERalpha mRNA was strongly correlated in ERalpha-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERalpha-positive cell lines; this increase was prevented by treatment with the ERalpha-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERalpha-negative cell lines, but caused only a modest reduction in ERalpha-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERalpha (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERalpha-negative, including triple negative, cancers (P = 0.005), but not ERalpha-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERalpha; however, our data indicate a greater functional effect of Gli1 in ERalpha-negative cancers.

Validation of endogenous internal real-time PCR controls in renal tissues.

BACKGROUND: Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. METHODS: To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. RESULTS: Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. CONCLUSION: A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity.

A shift from nuclear to cytoplasmic breast cancer metastasis suppressor 1 expression is associated with highly proliferative estrogen receptor-negative breast cancers.

BACKGROUND/AIMS: To determine breast cancer metastasis suppressor 1 (BRMS1) expression in breast cancers and the efficacy of BRMS1 as a prognostic indicator, BRMS1 expression was assessed in two sets of breast cancer tissues. METHODS: Epithelial cells from 36 frozen samples of breast cancers and corresponding normal breast were collected by laser capture microdissection and assessed for BRMS1 by quantitative RT-PCR and immunohistochemistry. BRMS1 was also evaluated by immunohistochemistry in a tissue microarray of 209 breast cancers and correlated with indicators of prognosis [estrogen receptor (ER), progesterone receptor (PR), ErbB2, p53, p27(Kip1), Bcl2 and Ki-67]. RESULTS: BRMS1 mRNA and protein were higher in 94 and 81%, respectively, of breast cancers than in corresponding normal epithelium. BRMS1 localization was predominantly nuclear, but 60-70% of cancers also exhibited cytoplasmic immunostaining. Breast cancers with lower nuclear than cytoplasmic BRMS1 (nuclear score - cytoplasmic score < or =0; 11% of cancers) had lower ER, lower PR and higher Ki-67 expression. There was also a trend toward poorer overall survival in this group of cancers, but this was only of borderline significance (p = 0.073). In Cox proportional hazards models, loss of nuclear BRMS1 was not a significant predictor of overall survival. CONCLUSIONS: Loss of nuclear BRMS1 was associated with ER-negative cancers and a high rate of proliferation, but was not an independent indicator of prognosis.

Hedgehog pathway expression in heterogeneous pancreatic adenocarcinoma: implications for the molecular analysis of clinically available biopsies.

Recent studies suggest that hedgehog (HH)-pathway signaling is required for the initiation and continued growth of pancreatic adenocarcinoma (PAC). Definitive gene expression analysis of PAC remains difficult, owing to the host desmoplastic stromal interaction and subsequent tumor heterogeneity. The primary goal of this study was to evaluate the effect of heterogeneity within a series (n=5) of matched clinical PAC biopsies [snap-frozen, formalin-fixed paraffin-embedded (FPE), endoscopic ultrasound-guided fine-needle aspirate (EUS-FNA)]. Differential expressions, specific to tumor cells, were evaluated by comparisons of uninvolved pancreas (n=9), EUS-FNA (n=14), and macrodissected (tumor-cell-enriched) biopsies (n=16). To determine whether treatment modulates gene expression, a unique (independent) set of synchronous EUS-FNA samples (n=4) was obtained before, and 2 weeks after, chemoradiation. mRNA levels were evaluated using real-time quantitative polymerase chain reaction formatted in a TaqMan low-density array, which was capable of simultaneously quantifying 46 independent genes in the HH pathway. Protein levels for Patched, Smoothened, and glioma-associated oncogene 1 (Gli-1) in FPE tissues were determined, using immunohistochemistry. A significant concordance (P<0.0001) was observed in the HH-pathway mRNA levels between matched surgically resected (both snap-frozen and FPE) and EUS-FNA biopsies. HH-pathway mRNA levels changed (increased) only after macrodissection, suggesting localization to tumor cells. Immunohistochemical staining for Patched, Smoothened, and Gli-1 confirmed the increased (P<0.001) levels of protein in the PAC cells, compared with cells from uninvolved pancreas. EUS-FNA biopsies that were obtained before and during chemoradiation demonstrated no significant changes in HH-pathway gene expression. Collectively, these studies demonstrate presence of HH-pathway expression in all the clinical PAC biopsies examined, suggesting that this is a significant tumor-associated target and offering the possibility that specific molecular profiling might be attempted from these heterogeneous tissues.

Variation in the relative copy number of the TLR7 gene in patients with systemic lupus erythematosus and healthy control subjects.

OBJECTIVE: To determine whether there is an increase in the number of TLR7 gene copies in patients diagnosed as having systemic lupus erythematosus (SLE) and whether gene amplification influences the autoantibody profiles in SLE patients, as has recently been reported in the BXSB/Yaa mouse model of lupus. METHODS: We used a modified real-time quantitative polymerase chain reaction protocol to calculate the relative TLR7 gene copy number according to the comparative 2(-DeltaDeltaC(t) ) method in 99 SLE patients and 91 healthy controls matched for sex and ethnicity. Autoantibody profiles were determined by standard methods. RESULTS: The relative number of TLR7 gene copies in SLE patients and healthy controls varied; however, no significant concordance between the number of relative gene copies and the SLE phenotype was found. There was also no difference in variation by ethnic group. Comparison of the relative gene copy numbers according to the presence or absence of antinuclear antibodies (ANAs), the ANA staining patterns, and the presence or absence of anti-RNA-associated antigen antibody showed no statistically significant difference in the SLE patients. CONCLUSION: We determined that although the relative gene copy number of TLR7 varied in both SLE patients and healthy controls, it was not significantly increased among our SLE patients as compared with our controls. We found no detectable trend for an association between the relative gene copy number and the autoantibody profile in SLE patients.

Phase II study of capecitabine with concomitant radiotherapy for patients with locally advanced pancreatic cancer: up-regulation of thymidine phosphorylase.

PURPOSE: The objectives of this phase II study were to evaluate the effect of radiation (XRT) on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-alpha (TNF-alpha) and the efficacy of capecitabine-XRT in patients with locally advanced pancreatic cancer. PATIENTS AND METHODS: Twenty patients received 50.4 Gy XRT with capecitabine 1,600 mg/m(2) on Monday through Friday for 6 weeks determined from our phase I study (Saif MW, Eloubeidi MA, Russo S, et al. J Clin Oncol. 2005;23:8679-8687). After capecitabine-XRT, stable and responding patients received capecitabine 2,000 mg/m(2) for 14 days every 3 weeks till progression. Restaging was performed every 9 weeks. Tumor specimens were procured with endoscopic ultrasound-fine needle aspiration before and at week 2 after capecitabine-XRT was started to evaluate the effect of XRT on TP, DPD, and TNF-alpha mRNA levels, determined by reverse transcriptase-polymerase chain reaction. RESULTS: Among 20 patients, 4 (20%) had a partial response and 13 (65%) had stable disease. Two patients underwent surgical resection (10%). The 6-month survival rate was 84%, and the 1-year survival was 58%. Grade > or =3 toxicities included nausea/vomiting (5%), thrombosis (5%), hyperbilirubinemia (5%), and grade 3 gastrointestinal bleeding (5%). TP was elevated during week 2 when compared with the pre-XRT TP (P = 0.01). However, no such effect of XRT was found either on DPD (P = 0.22) or on TNF-alpha (P = 0.6). No correlation between TP and TNF-alpha was noticed. Also, no association between TP/DPD ratio and efficacy of capecitabine was identified. CONCLUSIONS: This phase II study further confirms our phase I results and suggests that capecitabine-XRT is an effective, tolerable, and convenient alternative to an infusional 5-fluorouracil regimen for patients with pancreatic cancer. Although results support the use of capecitabine-XRT and TP was up-regulated, there appears to be additional genes associated with the response to capecitabine.

Palmar-plantar erythrodysesthesia in patients receiving capecitabine and intratumor thymidine phosphorylase and dihydropyrimidine dehydrogenase: is there a pharmacologic explanation?

BACKGROUND: Palmar-plantar erythordysesthesia (PPE) is the most common toxicity of capecitabine. Preclinical studies have shown that radiation therapy (RT) upregulates thymidine phosphorylase (TP), which could in turn increase the efficacy of capecitabine. Capecitabine is degraded by dihydropyrimidine dehydrogenase (DPD), and a deficiency in this enzyme can increase the toxicity of capecitabine. However, the effect of radiation therapy on frequency of PPE and association with TP and DPD has not been fully characterized. PATIENTS AND METHODS: Toxicity data were collected prospectively in 33 patients and retrospectively in 25 patients with locally advanced pancreatic cancer enrolled in 3 clinical trials who received capecitabine/RT followed by capecitabine alone. Tumor specimens on 33 patients were procured via endoscopic ultrasonography standards 1 week befoer RT and 2 weeks after RT to evaluate TP and DPD messenger RNA levels. Roche grading was used to assess PPE. RESULTS: Among 58 patients, 14 (24%) developed PPE. The capecitabine group tended to have a higher incidence of PPE than the capecitabine/RT group (17.2% vs. 10.3%; P = .12). Grade 2/3 PPE, was observed in 15.5% of patients receiving capecitabine and 1.7% of patients receiving capecitabine/RT (P = .0078; overall: 17.2%). Median cumulative dose of capecitabine for first development of PPE was 235,000 mg/m2 in the capecitabine/RT group, and 3,185,000 mg/m2 in the capecitabine group, with the relative frequency of an event occurring in the capecitabine/RT arm versus capecitabine of 0.59. Time to occurrence of first episode of PPE was a median of 5 weeks with capecitabine/RT and 6 weeks with capecitabine. Log-rank test showed that neither age, sex, performance status, nor ethnicity were associated with development of PPE. There was no clear difference in tumor responses of patients who had PPE versus those who did not have PPE. Mean tumor TP level was higher among patients with versus without PPE (275.77 vs. 215.29; P = .32). Mean tumor DPD level was lower among patients with versus without PPE (55.18 vs. 63.58; P = .49). Mean TP-DPD ratio was higher among patients with versus without PPE (10.29 vs. 3.04; P = .31). CONCLUSION: No significant association of PPE with higher tumor thymidine phosphorylase or lower tumor DPD levels was found. There was no difference in the incidence, severity, or time to occurrence of PPE with capecitabine/RT versus capecitabine, indicating no effect of RT. Further studies to evaluate the underlying mechanism of PPE are required.

Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer.

The hedgehog pathway regulates epithelial-mesenchymal interactions, differentiation, proliferation and survival during development. Stimulation of hedgehog signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. Using real-time, quantitative PCR, laser capture microdissection, and immunohistochemistry, distinctive patterns of expression of the hedgehog pathway members patched 1 (PTCH1), smoothened, GLI1, GLI2 and the 3 hedgehog ligands were identified for epithelial cells and stromal fibroblasts in benign breast and breast cancer. Hedgehog ligands were expressed at higher levels in some cancer epithelial cell lines compared to noncancerous epithelial cells. Correspondingly, expression of GLI1, a transcription factor and transcriptional product of hedgehog signaling, was increased 8-fold in cancer epithelial cell lines; however, PTCH1, also a transcriptional target of hedgehog signaling in many cell types, was not increased. GLI1 protein and mRNA, and PTCH1 and sonic hedgehog (SHH) proteins were elevated in 3 of 10 breast cancers; however, PTCH1 transcripts were not consistently increased. Hedgehog-mediated transcription, as indicated by a reporter of GLI-dependent promoter activity and by expression of GLI1 transcripts, was reduced by the hedgehog pathway inhibitor cyclopamine in both MDA-MB-435 cancer epithelial cells and MCF10AT epithelial cells, a cell line derived from benign breast. However, cyclopamine reduced viability of cancer epithelial cell lines, including MDA-MB-435, but did not specifically affect fibroblasts or epithelial cells from benign breast, including MCF10AT. Treatment with sonic hedgehog ligand diminished the cyclopamine-induced reduction in GLI-dependent promoter activity in MCF10AT and MDA-MB-435 and viability of MDA-MB-435. These results demonstrate modulation of GLI-mediated transcription in both cancer and benign-derived epithelial cells by cyclopamine and sonic hedgehog, and further suggest that hedgehog signaling contributes to the survival of only the cancer epithelial cells. Determination as to whether the increase in GLI1 and SHH expression in breast cancer indicates a significant increase in hedgehog signaling will require further evaluation.

Increased expression of thymidylate synthetase (TS), ubiquitin specific protease 10 (USP10) and survivin is associated with poor survival in glioblastoma multiforme (GBM).

BACKGROUND: The limited success of empirically designed treatment paradigms for patients diagnosed with glioblastoma multiforme (GBM) emphasizes the need for rationally designed treatment strategies based on the molecular profile of tumor samples and their correlation to clinical parameters. METHODS: In the current study, we utilize a novel real-time quantitative low density array (RTQ-LDA) to identify differentially expressed genes in de novo GBM tissues obtained from patients with distinctly different clinical outcomes. Total RNA was isolated from a cohort of 21 GBM specimens obtained from patients with either good (long-term survival (LTS) >36 months post surgery, n = 8) or poor (died of the disease (DOD) <24 months post surgery, n = 13) prognosis. Non-neoplastic brain tissue (n = 5) was obtained from patients who underwent surgery for refractory epilepsy. Demographic data was assessed for correlation with survival using Cox proportional hazards models. Sufficient RNA was available to use RTQ-LDA to quantify the expression of 93 independent genes in 5 LTS, 4 DOD, and 5 non-neoplastic brain samples. The eight differentially expressed genes identified by RTQ-LDA in LTS versus DOD (P