Detection of circulating tumor cells in breast cancer patients using multiplex reverse transcription-polymerase chain reaction and specific primers for MGB, PTHRP and KRT19 correlation with clinicopathological features.

AIM: The aim of this study was to correlate the clinicopathological features of breast cancer patients with the positive detection of parathyroid hormone-related protein (PTHRP), cytokeratin protein 19 (KRT19) and mammaglobin (MGB) using a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay developed to detect circulating tumor cells (CTCs) in peripheral blood of patients with breast cancer. PATIENTS AND METHODS: Peripheral blood samples were collected from 54 breast cancer patients and 20 healthy blood donors. Subsequently, the samples were processed for RNA extraction and analyzed for the expression of PTHRP, KRT19 and MGB using specific primers and multiplex RT-PCR. RESULTS: The positive detection rates in breast cancer patients for PTHRP, KRT19 and MGB were 68.5%, 63% and 22.2% and for healthy donors 10%, 0% and 10%, respectively. The statistical analysis revealed that PTHRP- and KRT19-positive detections correlated with the diagnosis of breast cancer while the combined positive detections of PTHRP-plus-KRT19 correlated with the presence of distant metastasis, especially with bone metastasis. Moreover, positive detections of KRT19 correlated with high proliferation rate of breast cancer tumors. MGB-positive detections did not add any diagnostic advantage in such analysis. CONCLUSION: Multiplex-PCR based detection of CTCs using PTHRP and KRT19 primers can provide useful information for the disease.

Expression of the insulin-like growth factor 1 (IGF-1) and type I IGF receptor mRNAs in human HLE-B3 lens epithelial cells.

BACKGROUND/AIM: The E peptide of the IGF-1Ec transcript has been documented to stimulate the growth of different cell lines, via a type I IGF-1 receptor (IGF-1R)-independent mechanism. The aim of the present study was to determine the implication of the IGF-1Ec isoform into the posterior capsule opacification process in human lens epithelium. MATERIALS AND METHODS: The expression of the IGF-1 system was characterized in human HLE-B3 lens epithelium cells and the mitogenic activity of IGF-1 and synthetic E peptide and the effects of growth hormone (GH) and dihydrotestosterone (DHT) were examined, using qualitative real-time PCR, RT-PCR, Western blot analysis and trypan blue exclusion assays in wild-type and IGF-1R knock-out HLE-B3 cells. RESULTS: The data showed that HLE-B3 cells express only the IGF-1Ea and IGF-1R transcripts. GH increased the expression of IGF-1Ea and of the previously undetectable IGF-1Eb mRNA. Finally, IGF-1 did not present any activity in the knock-out cells. CONCLUSION: The IGF-1Ea isoform is the main source for the formation of mature IGF-1 in HLE-B3 cells. The effects of exogenous IGF-1 depend on the existence of IGF-1R. IGF-1 Ec is not expressed even in the presence of GH or DHT nor has it any effect on cell proliferation.

Methods of detection of circulating melanoma cells: a comparative overview.

Disease dissemination is the major cause of melanoma-related death. A crucial step in the metastatic process is the intravascular invasion and circulation of melanoma cells in the bloodstream with subsequent development of distant micrometastases that is initially clinically undetectable and will eventually progress into clinically apparent metastasis. Therefore, the use of molecular methods to detect circulating melanoma cells may be of value in risk stratification and clinical management of such patients. Herein, we review the currently applied techniques for the detection, isolation, enrichment and further characterization of circulating melanoma cells from peripheral blood samples in melanoma patients. Furthermore, we provide a brief overview of the various molecular markers currently being evaluated as prognostic indicators of melanoma progression.

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients.

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

Detection of the circulating tumor cells in cancer patients.

As the presence of tumor cells circulating in the blood is associated with systemic disease and shortened survival, the establishment of a method to detect circulating tumor cells (CTCs) is of critical importance for a more concise staging and follow-up of cancer patients. Recently, the most robust strategies for the determination of CTCs are the PCR-based methods and the CellSearch® system that exploits the immunofluorescent characterization and isolation of cancer cells. Herein, we analyzed the experimental strategies used for determining CTCs with respect to accuracy, sensitivity and reproducibility in cancers of the breast, colon, prostate and melanoma.

uPA, uPAR and TGFß1 expression during early and late post myocardial infarction period in rat myocardium.

The expression patterns of transforming growth factor beta 1 (TGFß1), urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) were analysed after artery ligation-induced myocardial infarction (MI) in the rat myocardium. uPA and uPAR expressions were significantly increased both at transcriptional and protein level during early phase post MI period (uPA at 1 hour and uPAR at 24 hours post infarction). TGFß1 mRNA expression profile revealed a significant increase of TGFß1 expression from day 4 up to 8 weeks post infarction. These data suggest that the need for an increasing TGFß1 bioavailability during the post-infarction period in rat myocardium is achieved in the early post MI period by an increased expression of uPA/uPAR proteolytic system (indirect activation of latent TGFß1) and in the late post MI period by direct regulation of TGFß1 expression. It is therefore concluded that differential regulation of the TGFß1 bioavailability may be a crucial step of the repair mechanisms during the post MI infarction period in the rat myocardium.

The glutamatergic system expression in human PC-3 and LNCaP prostate cancer cells.

BACKGROUND: The glutamatergic system (Glu system) comprises the Glu receptors (GluRs), the Glu transporters (GluTs) and glutamine synthetase (GS). MATERIALS AND METHODS: Using PCR-based detection and Western blot analysis, the expression of Glu system components was assessed in human androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells. RESULTS: iGluRs, such as NR1, NR2A, NR2C, NR2D and NR3B; mGLuRs such as mGluR1, mGluR2, mGluR3, mGluR4 and mGluR5; GluTs such as EAAT1, EAAT2, EAAT3 and EAATS; and GS mRNA were steadily expressed in both cell lines. In addition, NR3A, mGluR6, mGluR8 and EAAT4 mRNA were differentially expressed in PC-3 and LNCaP cells, mGluR7 and EAAT4 mRNA expression was induced and mGluR8 was silenced by dihydrotestosterone (DHT) treatment in LNCaP cells. GS, EAAT1 and mGLuR5 were also detected at the protein level in both PC-3 and LNCAP cells. CONCLUSION: These data suggest that the Glu system could be an important regulator of prostate cancer cell biology.

Molecular markers detecting circulating melanoma cells by reverse transcription polymerase chain reaction: methodological pitfalls and clinical relevance.

Herein, we expound the theory of circulating melanoma cells (CMCs) and their detection with reverse transcription polymerase chain reaction as a molecular staging approach. We discuss the molecular markers that have been used for CMC detection focusing on the use of these markers for multiplex detection analysis. Finally, we comment on the contradictory data of CMC detection studies in the literature and we propose possible solutions which may contribute to the clinical significance of CMC detection in patient management.

Detection of circulating tumor cells in prostate cancer patients: methodological pitfalls and clinical relevance.

Disseminated malignancy is the major cause of prostate cancer-related mortality. Circulating tumor cells (CTCs) are essential for the establishment of metastasis. Various contemporary and molecular methods using prostate-specific biomarkers have been applied to detect extraprostatic disease that is undetectable by conventional imaging techniques, assessing the risk for disease recurrence after therapy of curative intent. However, the clinical relevance of CTC detection is still controversial. We review current literature regarding molecular methods used for the detection of CTCs in the peripheral blood and bone marrow biopsies of patients with prostate cancer, and we discuss the methodological pitfalls that influence the clinical significance of molecular staging.

The kisspeptin (KiSS-1)/GPR54 system in cancer biology.

Kisspeptin (KiSS-1) gene, initially described as a melanoma metastasis suppressor gene, encodes a number of peptides (kp-54, kp-14, kp-13, kp-10), which are endogenous ligands to a G protein-coupled receptor, referred as hOT7T175 or AXOR12 or GPR54. So far intensive investigation has provided substantiate evidence supporting the role of KiSS-1/GPR54 system in cancer biology as well as in the regulation of the reproductive function and trophoblast invasion. The precise mechanism by which KiSS-1/GPR54 system is affecting cancer cell growth and metastasis includes complex endocrine, paracrine and autocrine actions. Nevertheless, the detail mechanism of such actions is still under intensive investigation. Herein we review the evidence which support the role of KiSS-1/GPR54 system in cancer biology.

Circulating tumor cells in colorectal cancer: detection methods and clinical significance.

Colorectal cancer is one of the most frequently diagnosed malignancies in both men and women. Although curative resection is the major treatment option, approximately half of all patients eventually develop distant metastases. Thus, the need for early detection of occult metastases has led to extensive investigation with regard to the detection of disseminated tumor cells in biological fluids, including peripheral blood or bone marrow of cancer patients. In this review, we summarize the methods currently implemented for disseminated tumor cell detection in colorectal cancer. In addition, we discuss the pitfalls of each method and the future perspectives in the development of an easily applied, quick and inexpensive method which will enable the reliable detection of circulating tumor cells with optimal sensitivity and specificity.

Combined androgen blockade therapy can convert RT-PCR detection of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) transcripts from positive to negative in the peripheral blood of patients with clinically localized prostate cancer and increase biochemical failure-free survival after curative therapy.

BACKGROUND: The clinical relevance of positive molecular staging as defined by reverse transcriptase-polymerase chain reaction (RT-PCR) detections of both prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) transcripts in the peripheral blood (PB) of patients with prostate cancer is still debatable. METHODS: We analyzed the biochemical failure-free survival (bFFS) of prostate cancer patients with positive molecular staging who underwent immediate curative therapy (Group I, n=39) compared to prostate cancer patients who did convert their positive molecular staging by the administration of combined androgen blockade (CAB) for 12 months prior to curative treatment (Group II, n=15). RESULTS: The median bFFS for Group I was 9 months (95% CI 5-13 months) and was significantly lower compared to Group II (>36 months, p<0.001). In Group I, the median time for PSA values of >2.0 ng/mL was 18 months (95% CI 12-21 months, range 12-36 months). Notably, only one patient from Group II reached PSA values>2.0 ng/mL at 36 months post-curative treatment. CONCLUSIONS: In patients with clinically localized prostate cancer and positive RT-PCR detection of PSA and PSMA transcripts in PB, CAB can convert positive molecular staging status to negative and by doing so it modifies the post-curative therapy bFFS of patients with clinically localized prostate cancer.

Molecular detection of tyrosinase transcripts in peripheral blood from patients with malignant melanoma: correlation of PCR sensitivity threshold with clinical and pathologic disease characteristics.

BACKGROUND: Positive molecular detection of tyrosinase transcripts (TYR mRNA) in RNA extracts of peripheral blood (PB) samples from patients with malignant melanoma provides evidence of disease dissemination. METHODS: Total RNA extracted from PB was quantified and subjected to RT-PCR under ultra-sensitive and reduced-sensitivity PCR conditions using SSRT-II. Positive TYR mRNA detection in 78 melanoma patients and 40 healthy volunteers was correlated with clinical stage, Breslow's evaluation of tumor thickness, Clark's assessment of tumor invasion, the location of the primary tumor site, and tumor histology. The assay sensitivity was evaluated by spiking PB with the melanoma cell line SK-MEL-28. RESULTS: Using ultra-sensitive PCR conditions, eight out of 40 RNA (20%) samples from healthy volunteers and 50 out of 78 RNA (64.1%) samples from melanoma patients tested positive. Using reduced-sensitivity PCR conditions, we found only two positives in 40 RNA samples from healthy subjects and 20 positives in 78 RNA samples (25.6%) from melanoma patients. Only positive PCR samples for the reduced-sensitivity PCR assay correlated significantly with stage IV (metastatic) disease (p=0.0395). There was no significant correlation between positive TYR mRNA samples for either PCR condition (ultra-sensitive and reduced-sensitivity) with Breslow's classification of tumor thickness, Clark's assessment of tumor invasion, location of the primary tumor site, and type of tumor histology. CONCLUSIONS: We conclude that reduced-sensitivity rather than ultra-sensitive PCR conditions correlate with clinical stage in melanoma patients.