The endocrine disrupting alkylphenols and 4,4'-DDT interfere with estrogen conversion and clearance by mouse liver cytosol.

Endocrine disrupting chemicals (EDCs) are ubiquitous compounds known for negative impacts on reproductive functions and for increasing cancer risk. EDCs are believed to cause the harmful effects in part through their inappropriate low-affinity binding to steroid receptors and other possible non-receptor mediated paradigms, however there is a need to further elucidate other mechanisms involving the direct and indirect impact of EDCs on reproductive functions. We examined the metabolism of 17ß-estradiol (E2) and estrone (E1) by cell-free hepatic cytosol in the presence of alkylphenols (nonylphenol/NP and 4-tert-octylphenol/tOP), Dichlorodiphenyltrichloroethane (4,4'-DDT) and other EDCs. Tandem liquid chromatography mass spectrometry was utilized to quantitatively assess the impact of each EDC on estrogen clearance, inter-conversions and downstream metabolism by mouse liver cytosol. The results revealed that NP and tOP (0.1-3µg/mL) significantly reduced the hepatic cytosol clearance and biotransformation of estrogens with inclination for accumulating E2, the stronger estrogen form, than E1. Alkylphenols also caused up to a 34-fold increase in the E2/E1 ratio possibly by suppressing the hepatic E2→E1 conversion by 17ß-hydroxysteroid dehydrogenase (17ßHSD) types 2, 4 while displaying a weaker inhibition of E1→E2 conversion by type 1, 17ßHSD. On the other hand, the pesticide 4,4'-DDT was a weaker inhibitor of clearance of estrogens by the cytosol preparations when compared to alkylphenols, whereas chemicals such as phthalates and atrazine were ineffective. Our data suggest that exposure to NP, tOP and DDT can indirectly increase the estrogenic load by suppressing the hepatic clearance of estrogens and by elevating the E2/1 ratio and could therefore increase the risk of reproductive lesions.

A study of parabens and bisphenol A in surface water and fish brain tissue from the Greater Pittsburgh Area.

Pollution from xenoestrogens has been discovered in the aquatic environment of the Greater Pittsburgh Area and is suspected to be caused by the failing sewer system. Personal care products and plasticizers have the potential to enter the water supply though treated and untreated sewage. Many of these compounds are suspected xenoestrogens. Paraben detection in surface waters was as follows: methyl paraben ranged between 2.2 to 17.3 ppt; ethyl paraben was not detectable; propyl paraben was detected at 9.2 and 12.0 ppt; butyl paraben was detected at 0.2 ppt. BPA was detected between 0.6 and 15.4 ppt. Estrogenic potential of extracts from fish brain tissue was tested via Bromodeoxyuridine MCF-7 analysis and paired with HPLC-MS to investigate the presence of xenoestrogens. All samples were non-detectable for parabens. BPA was detected in 44 of the 58 samples, with a range from non-detectable to 120 pg/g. BCFs were calculated. Results were statistically significant for location of capture (p < 0.05) and correlation existed between estrogenicity and BPA.

Efficacy of signal pathway inhibitors alone and in combination with Cisplatin varies between human non-small cell lung cancer lines.

BACKGROUND: Signal pathway inhibitors (SPI) are designed to act synergistically with conventional cytotoxic drugs to control cancer progression. The objective of this study was to evaluate the effect of various SPI, both alone and in combination with cisplatin, on three different non-small cell lung cancer (NSCLC) cell lines. MATERIALS AND METHODS: Cell lines (A549, 201T, 273T) representing NSCLC were treated for 72 h in the presence or absence of inhibitors to PI3K (LY-294002; Tocris Bioscience, Ellisville, MO), BCL-2 (Gossypol; Sigma-Aldrich, St. Louis, MO), Cox-2 (NS-398 [Sigma-Aldrich] or Celecoxib [Pfizer]), MAPK (U0126 [Sigma-Aldrich]), and EGFR (Iressa; AstraZeneca, Macclesfield, United Kingdom) both alone and in combination with 10 or 30 mum cisplatin (Sigma-Aldrich) (18 possible regimens for each cell line). MTT assay (Trevigen, Gaithersburg, MD) was used to measure cytotoxicity. Controls were represented by cells with either a pure culture medium (monotherapy regimen control) or culture medium with the corresponding dose of cisplatin (combination regimen control). Unpaired t-test was used to classify response to therapy as highly sensitive (P < 0.01), sensitive (0.01 < or = P < 0.05), or resistant (P > or = 0.05). Concordance was defined as a similar response category between cell lines. RESULTS: The concordance rate was 50% between each of the three cell lines when SPI were used as monotherapy. In combination regimens, the concordance rates were 33% (A549 and 273T), 17% (A549 and 201T), and 75% (273T and 201T). The 273T cells were most susceptible to therapy, having 11 (61%) highly sensitive responses, whereas A549 cells were least susceptible with 14 (78%) resistant responses. CONCLUSIONS: There is a substantial degree of variability between NSCLC cell lines in response to SPI, both alone and in combination with cisplatin.

Use of novel autoantibody and cancer-related protein arrays for the detection of esophageal adenocarcinoma in serum.

OBJECTIVE: The aim of this study was to evaluate the feasibility of using novel autoantibody and cancer-related protein arrays to identify potential biomarkers for the early detection of esophageal adenocarcinoma in serum. METHODS: Sera from 18 patients with esophageal adenocarcinoma and 14 with gastroesophageal reflux disease were added to microarrays designed to detect circulating autoantibodies to 51 tumor-associated antigens. Sera from the same patients were also added to a 53-plex assay for various cancer-related proteins. Cutoff values at 3 standard deviations above the mean expression of gastroesophageal reflux disease were used as a boundary for positivity. RESULTS: Nine proteins and 11 autoantibodies were able to individually segregate at least 1 esophageal adenocarcinoma sample from gastroesophageal reflux disease by means of cutoff values. The most discriminative marker was Fas ligand in the protein array, which was associated with 83.3% sensitivity and 100% specificity. The best performing autoantibody, NY-ESO-1, detected 3 esophageal adenocarcinoma samples. When both of these markers were combined, a sensitivity of 88.9% and specificity of 100% were attained. CONCLUSIONS: Cancer-related protein and autoantibody arrays provide a technically simple and rapid method of identifying potential biomarkers for the detection of esophageal adenocarcinoma in serum. Furthermore, combining these platforms improves the diagnostic power of either platform alone. Integrating technologies that detect the expression of multiple proteins and autoantibodies in serum may provide a noninvasive and accurate method of detecting early esophageal adenocarcinoma.

Cross-reactive CD4+ T cells against one immunodominant tumor-derived epitope in melanoma patients.

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.

Optimal markers for real-time quantitative reverse transcription PCR detection of circulating tumor cells from melanoma, breast, colon, esophageal, head and neck, and lung cancers.

BACKGROUND: The detection of circulating tumor cells (CTCs) may prove useful for screening, prognostication, and monitoring of response to therapy. However, given the large background of circulating cells, it is probably necessary to detect 1 cancer cell in >10(6) leukocytes. Although reverse transcription (RT)-PCR is potentially sensitive and specific enough to achieve this goal, success will require the use of appropriate mRNA markers. The goal of this study was to identify optimal marker combinations for detection of CTCs. METHODS: An extensive literature and internet database survey was conducted to identify potential markers. We then used real-time quantitative RT-PCR to test for expression of selected potential markers in tissue samples from primary tumors of breast, colon, esophagus, head and neck, lung, and melanoma and normal blood samples. Markers with high expression in tumors and a median 1000-fold lower expression in normal blood were considered potentially useful for CTC detection and were tested further in an expanded sample set. RESULTS: A total of 52 potential markers were screened, and 3-8 potentially useful markers were identified for each tumor type. The mRNAs for all but 2 markers were found in normal blood. Marker combinations were identified for each tumor type that had a minimum 1000-fold higher expression in tumors than in normal blood. CONCLUSIONS: Several mRNA markers may be useful for RT-PCR-based detection of CTCs from each of 6 cancer types. Quantification of these mRNAs is essential to distinguish normal expression in blood from that due to the presence of CTCs. Few markers provide adequate sensitivity individually, but combinations of markers may produce good sensitivity for detection of the presence of these 6 neoplasms.

Autoantibody profiles reveal ubiquilin 1 as a humoral immune response target in lung adenocarcinoma.

There is considerable evidence that the presence of cancer can elicit a humoral immune response to specific proteins in the host, and these resulting autoantibodies may have potential as noninvasive biomarkers. To characterize the autoantibody repertoire present in the sera of patients with lung adenocarcinoma, we developed a high-density peptide microarray derived from biopanning a lung cancer phage display library. Using a 2,304-element microarray, we interrogated a total of 250 sera from Michigan lung cancer patients and noncancer controls to develop an "autoantibody profile" of lung adenocarcinoma. A set of 22 discriminating peptides derived from a training set of 125 serum samples from lung adenocarcinoma patients and control subjects was found to predict cancer status with 85% sensitivity and 86% specificity in an independent test set of 125 sera. Sequencing of the immunoreactive phage-peptide clones identified candidate humoral immune response targets in lung adenocarcinoma, including ubiquilin 1, a protein that regulates the degradation of several ubiquitin-dependent proteasome substrates. An independent validation set of 122 serum samples from Pittsburgh was examined using two overlapping clones of ubiquilin 1 that showed 0.79 and 0.74 of the area under the receiver operating characteristics curve, respectively. Significantly increased levels of both ubiquilin 1 mRNA and protein, as well as reduced levels of the phosphorylated form of this protein, were detected in lung tumors. Immunofluorescence using anti-ubiquilin 1 antibodies confirmed intracellular expression within tumors cells. These studies indicate that autoantibody profiles, as well as individual candidates, may be useful for the noninvasive detection of lung adenocarcinoma.

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

A combination of molecular markers accurately detects lymph node metastasis in non-small cell lung cancer patients.

Occult lymph node metastasis (micrometastasis) is a good prognostic indicator in non-small cell lung cancer (NSCLC) and could be used to direct adjuvant chemotherapy in stage I patients. This study was designed to evaluate molecular markers for detection of occult lymph node metastasis in NSCLC, define the best marker or marker combination to distinguish positive from benign lymph nodes, and evaluate these markers in lymph nodes from pathologically node-negative (pN(0)) NSCLC patients. Potential markers were identified through literature and database searches and all markers were analyzed by quantitative reverse transcription-PCR in a primary screen of six NSCLC specimens and 10 benign nodes. Selected markers were further evaluated on 21 primary NSCLC specimens, 21 positive nodes, and 21 benign nodes, and the best individual markers and combinations were identified. A combination of three markers was further validated on an independent set of 32 benign lymph nodes, 38 histologically positive lymph nodes, and 462 lymph nodes from 68 pN(0) NSCLC patients. Forty-two markers were evaluated in the primary screen and eight promising markers were selected for further analysis. A combination of three markers (SFTPB, TACSTD1, and PVA) was identified that provided perfect classification of benign and positive nodes in all sample sets. PVA and SFTPB are particularly powerful in tumors of squamous and adenocarcinoma histologies, respectively, whereas TACSTD1 is a good general marker for NSCLC metastasis. The combination of these genes identified 32 of 462 (7%) lymph nodes from 20 of 68 (29%) patients as potentially positive for occult metastasis. Long-term follow-up will determine the clinical relevance of these findings.

The HGF receptor c-Met is overexpressed in esophageal adenocarcinoma.

The hepatocyte growth factor (HGF) receptor, Met, has established oncogenic properties; however, its expression and function in esophageal adenocarcinoma (EA) remain poorly understood. We aimed to determine the expression and potential alterations in Met expression in EA. Met expression was investigated in surgical specimens of EA, Barrett's esophagus (BE), and normal esophagus (NE) using immunohistochemistry (IHC) and quantitative reverse transcriptase polymerase chain reaction. Met expression, phosphorylation, and the effect of COX-2 inhibition on expression were examined in EA cell lines. IHC demonstrated intense Met immunoreactivity in all (100%) EA and dysplastic BE specimens. In contrast, minimal immunostaining was observed in BE without dysplasia or NE specimens. Met mRNA and protein levels were increased in three EA cell lines, and Met protein was phosphorylated in the absence of serum. Sequence analysis found the kinase domain of c-met to be wild type in all three EA cell lines. HGF mRNA expression was identified in two EA cell lines. In COX-2-overexpressing cells, COX-2 inhibition decreased Met expression. Met is consistently overexpressed in EA surgical specimens and in three EA cell lines. Met dysregulation occurs early in Barrett's dysplasia to adenocarcinoma sequence. Future study of Met inhibition as a potential biologic therapy for EA is warranted.

Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies.

BACKGROUND: Increased plasma DNA has been found in cancer patients and may have potential as a tumor marker. The objectives of this study were to develop a controlled, quantitative PCR (QPCR) assay to measure plasma DNA and then evaluate plasma DNA concentrations as a tumor marker in patients with thoracic malignancies. METHODS: We developed a QPCR assay for DNA, using the human beta-actin gene. Plasma samples were analyzed from 58 patients with esophageal cancer (EC; 20 banked samples and 38 prospectively collected samples) and 25 patients with lung cancer (LC; all prospectively collected). Control groups consisting of 51 patients with gastroesophageal reflux disease (GERD; 23 banked samples and 28 prospectively collected) and 11 healthy volunteers were also analyzed. RESULTS: The assay had an experimental variability <4%. In our banked samples, the mean concentration of plasma DNA in EC was 819.0 microg/L (range, 46.2-4738.0 microg/L) vs 432.0 microg/L (6.0-2888.0 microg/L) in GERD (P = 0.02). However, the prospectively collected samples had lower DNA concentrations, and there was no difference between cancer patients and controls. The mean DNA concentration was 10.6 microg/L (range, 7.0-14.0 microg/L) in healthy volunteers and 10.5 microg/L (range, 4.0-23.5 microg/L) in GERD controls vs 13.0 microg/L (range, 4.5-46.5 microg/L) in EC and 14.6 microg/L (range, 3.0-30.0 microg/L) in LC. CONCLUSIONS: Our data indicate that plasma DNA concentrations are of limited diagnostic value when samples are prospectively collected and uniformly handled. This is in contrast to previously published results. Qualitative analysis of DNA may be needed if plasma nucleic acids are to be used as a diagnostic tool in cancer screening.

Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics.

BACKGROUND: Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection. METHODS: We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma. RESULTS: We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 microm filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent. CONCLUSIONS: Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis.

Temperature-controlled primer limit for multiplexing of rapid, quantitative reverse transcription-PCR assays: application to intraoperative cancer diagnostics.

BACKGROUND: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate. METHODS: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples. RESULTS: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1-2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples. CONCLUSION: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.