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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.

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)].

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.

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.

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.

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.

Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma.

Recent studies have shown the hedgehog and Wnt families of signaling proteins to be associated with tumor initiation, growth, and survival. However, these pathways remain unexplored in ovarian endometrioid adenocarcinoma (OEA). Here, we describe a novel TaqMan low-density array to examine the expression of 26 and 20 genes in the hedgehog and Wnt pathways, respectively, in six matched snap-frozen and formalin-fixed, paraffin-embedded (FPE) OEA specimens.

Antitumor efficacy of capecitabine and celecoxib in irradiated and lead-shielded, contralateral human BxPC-3 pancreatic cancer xenografts: clinical implications of abscopal effects.

Purpose: X-ray therapy (XRT) remains one of the major modalities used to treat patients diagnosed with locally advanced pancreatic adenocarcinoma. However, the effect of XRT on metastatic tumors outside the field of irradiation (abscopal effect) remains largely unknown. In the current study, we examined the effect of XRT alone and in combination with capecitabine and/or celecoxib in both irradiated and lead-shielded contralateral BxPC-3 pancreatic cancer xenografts.

Phase I study of capecitabine with concomitant radiotherapy for patients with locally advanced pancreatic cancer: expression analysis of genes related to outcome.

Purpose: To establish the feasibility of capecitabine with concurrent radiotherapy (XRT) in patients with locally advanced (LA) pancreatic cancer and evaluate the effect of XRT on thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and tumor necrosis factor-alpha (TNF-alpha).

Patients And Methods: Fifteen patients with LA pancreatic cancer received three-dimensional conformal XRT to a dose of 50.4 Gy with capecitabine at escalating doses from 600 to 1,250 mg/m2 bid (Monday through Friday).

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.

Background: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed.

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography.

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy.