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 m 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

1a,25-Dihydroxyvitamin D 3 (1,25D 3 ) 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 D 3 -24-hydroxylase), the enzyme that catabolizes 1,25D 3 , 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 patientmatched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D 3 . The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS) assay for 1,25D 3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D 3 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 1,25D 3 and increased 1,25D 3 half-life. Furthermore, combination of 1,25D 3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D 3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D 3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment. ' 2006 Wiley-Liss, Inc.Key words: 1a,25-dihydroxyvitamin D 3 ; 25-hydroxyvitamin D 3 -24 hydroxylase; CYP24; nonsmall cell lung cancer Lung cancer is a significant health problem in the United States; it now kills more women than does breast cancer, and more men than do prostate and colon cancers combined.1 Major advances in treatment have been limited, and the prognosis for lung cancer patients remains poor, with overall 5-year survival rates less than 15%.2 Therefore, there is a tremendous need to develop effective therapeutic approaches for this disease. 1a,25-Dihydroxyvitamin D 3 (1,25D 3 ), the biologically active form of vitamin D, has antiproliferative activity in a variety of tumor model systems in vitro and in vivo and is currently under evaluation in clinical trials in cancer (reviewed by Beer and Myrthue 3 ). We initiated studies to explore whether the antiproliferative effects of 1,25D 3 in NSCLC could be increased by inhibiting its metabolism.

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

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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. [Cancer Res 2007;67(7):3461-7]