Background: Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with oncogenic activities in human cancers; however, the clinical significance of CDC6 in prostate cancer (PCa) remains unclear. Therefore, we investigated whether the CDC6 mRNA expression level is a diagnostic and prognostic marker in PCa. Methods: The study subjects included 121 PCa patients and 66 age-matched benign prostatic hyperplasia (BPH) patients. CDC6 expression was evaluated using real-time polymerase chain reaction and immunohistochemical (IH) staining, and then compared according to the clinicopathological characteristics of PCa. Results: CDC6 mRNA expression was significantly higher in PCa tissues than in BPH control tissues (P = 0.005). In addition, CDC6 expression was significantly higher in patients with elevated prostate-specific antigen (PSA) levels (> 20 ng/mL), a high Gleason score, and advanced stage than in those with low PSA levels, a low Gleason score, and earlier stage, respectively. Multivariate logistic regression analysis showed that high expression of CDC6 was significantly associated with advanced stage (≥ T3b) (odds ratio [OR], 3.005; confidence interval [CI], 1.212-7.450; P = 0.018) and metastasis (OR, 4.192; CI,; P = 0.038). Intense IH staining for CDC6 was significantly associated with a high Gleason score and advanced tumor stage including lymph node metastasis stage (linear-by-linear association, P = 0.044 and P = 0.003, respectively). Conclusion: CDC6 expression is associated with aggressive clinicopathological characteristics in PCa. CDC6 may be a potential diagnostic and prognostic marker in PCa patients.
158 Background: Clinically, CTCs are used primarily in longitudinal monitoring of metastatic disease progression. However, the analysis of specific CTC biomarkers has the potential to optimize patient management by identifying those likely to respond to specific targeted agents. Often, mCRPC is driven by dysregulation of the androgen receptor (AR) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) oncogenic pathways such that blockade of one pathway stimulates the other. These functional deficits in the AR and PI3K/AKT pathways are associated with fusion of the TMPRSS2 and ERG genes and with loss of PTEN protein, respectively. Therefore, we sought to characterize CTCs for the simultaneous presence of both these biomarkers using immunofluorescence (IF) and fluorescence in situ hybridization (FISH). Methods: Nucleated peripheral blood cells from mCRPC patients were attached to slides and examined using CTC technology (Epic Sciences). Cytokeratin-positive/CD45-negative cells with an intact nucleus and a malignancy-consistent morphology were identified as CTCs, and their exact positions on the slides were recorded. The slides were then subjected to multiplex Quantum Dot IF and FISH procedures with anti-AR, -ERG, and -PTEN antibodies and 5’ERG, 3’ERG, PTEN, and Cen10 probes, respectively, on an automated slide-staining platform (Ventana Medical Systems, Inc.). The IF and FISH signals were visualized by spectral imaging (Ventana). Results: The automated IF/FISH staining procedure facilitated multiplex characterization of individual CTCs from patient samples for the protein biomarker targets AR, ERG, and PTEN and the genomic biomarker targets 5’ERG, 3’ERG, PTEN, and Cen10. Conclusions: This method for high-sensitivity, multiplex molecular characterization of critical CTC biomarkers in mCRPC patients might aid oncologists in identifying and stratifying those patients likely to respond to combination therapy with targeted PI3K/AKT inhibitors and anti-androgens/Cyp17 inhibitors. Patient trials examining the clinical utility of this assay in mCRPC are currently underway.
Cervicogenic angina and dyspnea are conditions characterized by chest discomfort and breathing difficulties that resemble angina pectoris and dyspnea of cardiac origin. However, this condition is caused by cervical spine pathology, cervical spondylosis, and radiculopathy. This case study reports a 66-year-old man who presented with cervicogenic angina and dyspnea due to cervical radiculopathy to a chiropractic clinic. The patient underwent a comprehensive diagnostic evaluation, including taking the patient’s history, a physical examination, and radiological investigations, which demonstrated cervical spine involvement consistent with a cervicogenic origin of the pain. The patient's angina-like symptoms and dyspnea improved significantly after chiropractic manipulation of the spine, soft tissue mobilization, and other manual therapies. Accurate diagnosis is essential to minimize unnecessary cardiac interventions and ensure proper therapy for underlying cervical spine problems. This case demonstrates the necessity of conservative management, such as chiropractic care, for patients presenting with cervicogenic angina and dyspnea, particularly when a diagnostic assessment reveals cervical spinal involvement.
The identification of patients who will benefit from therapy is one of the more difficult questions in prostate cancer disease management. Approximately 60% of men with diagnosed prostate cancer will have an ETS rearranged tumor foci. The prostate cancer gene fusions identified thus far are characterized by the fusion of 5’ genomic regulatory elements (commonly androgen regulated), such as TMPRSS2, with the ETS family of transcription factors which include ERG, ETV1, ETV4, and ETV5. About 40-50% of prostate cancers have the TMPRSS2/ERG gene fusion which leads to the over expression of oncogenic transcription factors. The goal of this study was to develop a novel, integrated diagnostic testing method that accounts for tissue heterogeneity including multiple gene re-arrangements in single transformed nuclei. We selected a method that sequentially reflexes from an initial H&E to ERG immunohistochemistry (IHC), and finally to a quantum dot (Life Technologies) based FISH probes for the detection of multiple gene rearrangements in prostate cancer. To this end, probes specific to the ETS gene rearrangements including, 3’ and 5’ ERG, TMPRSS2, NDRG1, ETV1, and ETV4, were detected with up to four different quantum dot bioconjugates simultaneously in single nuclei. We have shown sensitive and specific detection of gene rearrangements with this testing method in the xenograft models, VCaP, H660, and LNCaP, as well as in samples from prostate needle biopsies and radical prostatectomies. In 6 of 88 cases, the ERG IHC was diagnostic of PIN (prostatic intraepithelial neoplasia) that was missed on initial examination of the H&E stain. Further, the four color quantum dot FISH assay inclusive of ERG gene rearrangements was confirmatory of ERG IHC reactivity. In addition, we also clearly demonstrated instances of multiple gene rearrangements in the 5’ and 3’ ends on TMPRSS2 and NDRG1 in the same cancer nuclei, but not in benign nuclei from the same tissue. The signal patterns associated with various genomic events assessed were: no rearrangement (normal), translocation through insertion, and translocation through deletion. In summary, we propose that the ERG antibody is likely to be a key component in a diagnostic PIN IHC cocktail, that follows the initial H&E, and may be reflexed to a multiplexed quantum dot FISH assay that incorporates the detection of prevalent gene rearrangements. This method may be useful as an aid in detecting clinically relevant diagnostic markers, since multiple gene rearrangements in the same cell may be identified early in prostate cancer progression. Studies are in progress to evaluate this testing strategy for prognostic value in assessing prostate cancer progression in selected prostate cancer and biopsy cohorts. At time of submission, assay is not approved for use in the US. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2218. doi:10.1158/1538-7445.AM2011-2218
39 Background: ERG gene rearrangements lead to overexpression of the ERG oncogene in 50% of localized and metastatic prostate cancers. Recent studies have indicated that this gene fusion correlates with androgen sensitivity in prostate cancer patients and an association with increased DNA Repair activity (PARP1). The PTEN tumor suppressor has also been shown to regulate the AKT pathway, and related to increased PARP1 activity. We hypothesized that future PARP1 inhibitors and other targeted therapies for mCRPC will likely require the development of a companion diagnostic assay for measuring ERG rearrangement and PTEN deletion status in CTCs. Methods: We isolated CTCs utilizing the CellSearch system from mCRPC patients for enumeration. Following enumeration, the cells were transferred to glass slides and interrogated utilizing the automated Benchmark staining platform with multiplexed Quantum Dot FISH inclusive of probes for 5’ERG, 3’ERG, PTEN, and Cen 10. FISH signals were visualized utilizing spectral imaging. Results: Successful transfer of the CTCs from the CellSearch to a glass slide were achieved In model cell line systems, in cultured cells (LnCaP and VCaP) either directly or after spiking to PBL and five out of 6 times we were able to successfully demonstrate simultaneous single copy 5’ and 3’ ERG and PTEN/Cen 10 labeled probes. Studies are in progress to extend the clinical utility of this assay to isolated CTC from cases with metastatic prostate cancer. Conclusions: CTCs are likely to be utilized clinically for monitoring disease progression mCRPC patients as measures for therapeutic efficacy. As more therapeutics are approved for use in mCRPC patients, the ability for oncologists to molecularly characterize the type of CTCs driving tumor burden is likely to play a significant role in optimizing patient management by determining the type and the sequence of future targeted therapies. We have for the first time demonstrated a platform to molecularly characterize patient CTCs on an automated platform that is amenable to standard clinical use.
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