BACKGROUND: This report and a companion report describe a validation of the ability of serum proteomic profiling via SELDI-TOF mass spectrometry to detect prostatic cancer. Details of this 3-stage process have been described. This report describes the development of the algorithm and results of the blinded test for stage 1.

Background: The analysis of bodily fluids using SELDI-TOF MS has been reported to identify signatures of spectral peaks that can be used to differentiate patients with a specific disease from normal or control patients. This report is the 2nd of 2 companion articles describing a validation study of a SELDI-TOF MS approach with IMAC surface sample processing to identify prostatic adenocarcinoma. Methods: We sought to derive a decision algorithm for classification of prostate cancer from SELDI-TOF MS spectral data from a new retrospective sample cohort of 400 specimens. This new cohort was selected to minimize possible confounders identified in the previous study described in the companion paper. Results: The resulting new classifier failed to separate patients with prostate cancer from biopsy-negative controls; nor did it separate patients with prostate cancer with Gleason scores <7 from those with Gleason scores ≥7. Conclusions: In this, the 2nd stage of our planned validation process, the SELDI-TOF MS–based protein expression profiling approach did not perform well enough to advance to the 3rd (prospective study) stage. We conclude that the results from our previous studies—in which differentiation between prostate cancer and noncancer was demonstrated—are not generalizable. Earlier study samples likely had biases in sample selection that upon removal, as in the present study, resulted in inability of the technique to discriminate cancer from noncancer cases.

These results suggest that increased expression and changes in the subcellular distribution of both p160erbB-3 and p185erbB-2 represent early events in the development and progression of prostatic adenocarcinomas. The high expression and distribution of both p160erbB-3 and p185erbB-2 are retained both in advanced-stage primary and metastatic tumors.

Jab1 (Jun activation domain binding protein 1), integrated into COP9 signalosome complex (CSN), induces protein instability of many tumor suppressors and cell cycle regulators and is therefore a novel target in cancer therapy. Curcumin, an inhibitor of Jab1/CSN-associated kinase(s), has been reported to suppress tumor growth; however, curcumin is highly hydrophobic, and this feature prevents its usage as an antitumor drug. To increase the solubility and targeted delivery, we generated a water-soluble polyethylene glycol (PEG)-conjugated curcumin system, in which curcumin is covalently linked to PEG 35kD . PEGylated curcumin showed much greater reduction of cell growth than free curcumin in pancreatic cancer cells. Cells treated with PEGylated curcumin had increased arrest at the mitotic phase with the formation of abnormal multinucleated cells, indicating that this compound affects cell cycle progression, which may contribute to cell growth inhibition. The stabilities of Jab1 target proteins were also examined. PEGylated curcumin increased protein stability of these proteins in pancreatic cancer cells and directly inhibited the activity of Jab1/ CSN-associated kinases. Moreover, the inhibitory effect of PEGylated curcumin on cell proliferation was blunted in pancreatic cancer cells with Jab1 knockdown. The results suggest that PEGylated curcumin inhibits cell proliferation through suppression of Jab1/CSN activity. More importantly, the new compound sensitized pancreatic cancer cells to gemcitabine-induced apoptosis and cell proliferation inhibitory effects. Collectively, the PEGylated curcumin conjugate has much more potent effects on pancreatic cancer cell growth inhibition than free curcumin. The current study provides a biologic rationale to treat patients with pancreatic adenocarcinoma with the nontoxic phytochemical conjugated to PEG for systemic delivery.Pancreatic ductal adenocarcinoma represents greater than 80% of all pancreatic neoplasms with a death/incidence ratio of approximately 0.99 (Farrow and Evers, 2004;Brand and Mahr, 2005). Although gemcitabine currently is the most commonly used drug for treatment of pancreatic cancer (Burris et al

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/β-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 μM WNT974 ± 100 μM carboplatin. Viability was evaluated with the ATPlite assay. The IC 50 was calculated using a dose-response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥ 30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P = 0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/β-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

Multiple studies have reported that surface enhanced laser desorption/ionization time of flight mass spectroscopy (SELDI-

It is accepted that the aldehyde-based fixation of cells can affect the immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We therefore investigated the effects of aldehyde-based fixation and the individual steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. The immunohistochemical detection of Ki67/MIB-1 and PCNA was evaluated after various times of fixation in 10% neutral-buffered formalin (NBF) plus after each of the individual cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that, in addition to fixation, each of the different steps in tissue processing has effects on immunorecognition of the epitopes recognized by these antibodies. The extensive dehydration through ethanols to absolute ethanol had only modest effects except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, the establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. Antigen retrieval was able to compensate for most, but not all of the losses in staining following fixation and exposure to xylene; however, AR gave very consistent results for most steps of tissue processing, suggesting that AR should also be used in staining for PCNA. The cellular variations that were noted indicate that the effects of fixation and other steps of tissue processing may depend upon how antigens are packaged by specific cells.

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