Antigone Sourla

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.

IGF-1 Expression in Infarcted Myocardium and MGF E Peptide Actions in Rat Cardiomyocytes in Vitro

Insulinlike growth factor-1 (IGF-1) expression is implicated in myocardial pathophysiology, and two IGF-1 mRNA splice variants have been detected in rodents, IGF-1Ea and mechano-growth factor (MGF). We investigated the expression pattern of IGF-1 gene transcripts in rat myocardium from 1 h up to 8 wks after myocardial infarction induced by left anterior descending coronary artery ligation. In addition, we characterized IGF-1 and MGF E peptide action and their respective signaling in H9C2 myocardial-like cells in vitro. IGF-1Ea and MGF expression were significantly increased, both at transcriptional and translational levels, during the late postinfarction period (4 and 8 wks) in infarcted rat myocardium. Measurements of serum IGF-1 levels in infarcted rats were initially decreased (24 h up to 1 wk) but remained unaltered throughout the late experimental phase (4 to 8 wks) compared with sham-operated rats. Furthermore, specific anti-IGF-1R neutralizing antibody failed to block the synthetic MGF E peptide action, whereas it completely blocked IGF-1 action on the proliferation of H9C2 cells. Moreover, this synthetic MGF E peptide did not activate Akt phosphorylation, whereas it activated ERK1/2 in H9C2 rat myocardial cells. These data support the role of IGF-1 expression in the myocardial repair process and suggest that synthetic MGF E peptide actions may be mediated via an IGF-1R independent pathway in rat myocardial cells, as suggested by our in vitro experiments.

Insulin-like growth factor I and urokinase-type plasminogen activator bioregulation system as a survival mechanism of prostate cancer cells in osteoblastic metastases: development of anti-survival factor therapy for hormone-refractory prostate cancer.

Androgen ablation therapy is the first-line therapy in metastatic prostate cancer. It initially offers an objective clinical response, but eventually is stalled by the ominous development of refractoriness to hormonal therapy (stage D3). This ominous event signifies poor prognosis, uncontrolled tumor growth and curtailed survival to a median of only 10 months (1). Salvage chemotherapy does not improve the overall survival of stage D3 patients (2,3). The bone is the most prevalent site for metastases of prostate cancer (4). These lesions are associated with a potent local osteoblastic reaction, in contrast to the overwhelming majority of other solid tumors, whose bony metastases are generally associated with osteolysis (5,6). Osteoblastic metastases almost always represent the first and, frequently, the exclusive site of disease progression to stage D3 (3,7–9). The number of skeletal metastatic foci is the most powerful independent prognostic factor associated with limited response to hormone ablation therapy and poor survival of advanced prostate cancer (7–9). Disease progression to hormone-refractoriness frequently occurs only in the osteoblastic metastases, even though hormonal therapy may still provide adequate and sustained control of disease at the primary site (7–12). Consequently, the bone may not be just a passive, innocent bystander that suffers the consequences of metastatic tumor growth, but it may constitute the favorable microenvironment for the homing of metastatic prostate cancer cells (13–15). This may be secondary to interactions of prostate cancer cells with osteoblasts, to promote both the growth of metastatic prostate cancer cells and the osteoblastic reaction (13–17). Recently, bone-derived growth factors were reported to protect metastatic prostate cancer cells from chemotherapy-induced apoptosis, introducing the novel concept of bone-derived survival factors (18–20). We reviewed the current knowledge on osteoblast-derived survival factors, their relationship with local factors, such as the insulin-like growth factor I (IGF-I) and urokinase-type plasminogen activator (uPA) bioregulation system. These factors are implicated in the osteoblastic reaction and the benchto-bedside development of an anti-survival factor therapy, which has provided encouraging preliminary data in a phase II clinical trial with terminally ill, hormone-refractory and chemotherapy-resistant prostate cancer patients.

Preferential expression of IGF-1Ec (MGF) transcript in cancerous tissues of human prostate: evidence for a novel and autonomous growth factor activity of MGF E peptide in human prostate cancer cells.

By alternative splicing the IGF‐1 gene produces several different transcripts, including IGF‐1Ec (MGF). The latter has been mainly associated with muscle regeneration processes.

Systemic cytokine response following exercise-induced muscle damage in humans

Abstract Background: Muscle adaptation which occurs following eccentric exercise-induced muscle damage has been associated with an acute inflammatory response. The purpose of this study was to investigate serum interleukin-6 (IL-6), osteoprotegerin and receptor activator of nuclear factor kB ligand (OPG/RANKL) concentrations following muscle damage. We measured changes for several days following muscle damage. Methods: Ten healthy young males performed an eccentric exercise protocol using their quadriceps. Blood samples were withdrawn before and at 6 h, 2 days, 5 days and 16 days post-exercise. Functional and clinical measurements were performed before, and on days 1, 2, 5, 8, 12 and 16 post-exercise. Results: The exercise protocol resulted in muscle damage, indicated by changes in biochemical markers. An increase in IL-6 and OPG, and a decrease in RANKL concentrations were seen at 6 h and on day 2 post-exercise; the OPG:RANKL ratio was increased at 6 h post-exercise (p<0.05). Conclusions: Changes in IL-6 and OPG/RANKL system may represent systemic responses in muscle inflammation and repair processes. However, further studies are needed to elucidate a potential systemic and/or local role of the OPG/RANKL system in skeletal muscle repair. Clin Chem Lab Med 2009;47:777–82.

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

Abstract 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. Clin Chem Lab Med 2009;47:1–11.

Detection of circulating tumor cells in bladder cancer patients

The methods employed for the detection of circulating bladder cancer cells (CBCs) and their use as a molecular staging tool in clinical settings are thoroughly reviewed. CBC isolation and enrichment methods are discussed according to their advantages and pitfalls along with the clinical data of PCR-based techniques used for CBC detection. In addition, we review the specificity of molecular markers that have been proposed so far for CBC identification, and we comment on the controversial clinical data, proposing laboratory approaches which may improve the clinical significance of CBC detection in bladder cancer.

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.

Insulinlike growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro.

The transcription of the insulinlike growth factor 1 (igf-1) gene generates three mRNA isoforms, namely IGF-1Ea, IGF-1Eb and IGF-1Ec (or MGF [mechano growth factor]). Herein, we analyzed the expression of IGF-1 isoforms in eutopic and ectopic endometrium (red lesions and endometriotic cysts) of women with endometriosis, and we characterized the actions of a synthetic MGF E-peptide on KLE cells. Our data documented that all three igf-1 gene transcripts are expressed in the stromal cells of the eutopic and ectopic endometrium; however, endometriotic cysts contained significantly lower IGF-1 isoform expression, both at the mRNA and protein level, as was shown using semiquantitative PCR and immunohistochemical methods. In addition, the glandular cells of the eutopic endometrium did not express any of the IGF-1 isoforms; however, the glandular cells of the ectopic endometrium (red lesions) did express the IGF-1Ec at mRNA and protein level. Furthermore, synthetic MGF E-peptide, which comprised the last 24 amino acids of the MGF, stimulated the growth of the KLE cells. Experimental silencing of the type 1 IGF receptor (IGF-1R) and insulin receptor expression of KLE cells (siRNA knock-out methods) did not alter the mitogenic action of the synthetic MGF E-peptide, revealing that MGF E-peptide stimulates the growth of KLE cells via an IGF-1R-independent and insulin receptor-independent mechanism. These data suggest that the IGF-1Ec transcript might generate, apart from mature IGF-1 peptide, another posttrans-lational bioactive product that may have an important role in endometriosis pathophysiology.

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.