Journal of Experimental & Clinical Cancer Research 2009, 28:120 http://www.jeccr.com/content/28/1/120 Background Testicular cancer is a clinically, epidemiologically, and histologically heterogeneous group of neoplasms that represents 1% of malignant tumors in males. Germ cell testicular cancer is the most common type of tumor in males between 15 and 40 years of age, comprising approx- imately 98% of all testicular cancers, with an annual inci- dence of 7.5 per 100,000 inhabitants [1-3]. Germ cell testicular tumors are classified into two major sub-groups based on histological findings: seminomas and non-sem- inomas, each comprising approximately 50% of cases. This malignancy possesses a high cure rate in its early and even in its metastatic stages, reaching 10-year survival rates between 90 and 100% [4,5]. However, there remains a sub-group of patients with poor prognosis with approx- imately 40% of 10-year mortality, regardless of treatment. In addition, 20–30% of germ cell tumors show recurrence that frequently exhibits refractoriness to multi-agent chemotherapy. Human chorionic gonadotropin (hCG), alpha-fetopro- tein (AFP), and lactate dehydrogenase (LDH) are serum tumor markers (STMs) that play a clear role in diagnosis, staging, risk classification, and clinical management of testicular germ cell tumors. Elevation of one or more markers is associated with disease progression and adverse prognosis [6,7]. Seminoma tumors do not increase AFP levels, and occasionally increase hCG [8]. One main feature of cancer is marked angiogenesis, which is essential for tumor growth and metastasis, exerting an impact on outcome and survival rates, including those of germ cell testicular tumors. The most important ang- iogenic stimulatory factor is vascular endothelial growth factor (VEGF), a mitogen specific for vascular endothelial cells [9]. VEGF is known for its ability to induce vascular permeability, to promote endothelial proliferation as well as migration, and to act as a critical survival factor for endothelial cells [10]. VEGF mRNA and protein expres- sion is significantly higher in germ cell testicular tumors than in normal testis, and this expression correlates with microvascular density within the tumor [11]. Moreover, it has been shown that VEGF expression is correlated with metastases in these tumors [12]. hCG is a well-characterized hormone primarily produced by placenta and by other normal and tumor tissues in small amounts [13]. It has been described not only as an important peptide hormone during implantation [14], but also as an angiogenic factor for uterine endothelial cells [15]. It has been found that hCG possesses a role in the angiogenic process in vivo and in vitro by increasing capillary formation and endothelial cell migration in a direct association with the quantity of hCG administered; also, hCG-induced neovascularization was similar to that produced by VEGF and basic fibroblastic growth factor (bFGF) [16]. In addition, it has been proposed that hCG could induce VEGF production in tissues such as placenta [17] and granulosa cells [18,19]. Elevated hCG expression in serum, urine, or tumor tissue is usually a sign of aggressive disease and poor prognosis in germ cell tumors [8]. It is found in 40–60% of non- seminomatous germ cell tumors and in 30% of seminoma germ cell tumors [20]. However, no direct association has been reported between hCG and angiogenesis in cancer. The objective of this study was to determine the relation- ship between hCG serum levels, angiogenesis, and VEGF expression in germ cell testicular tumors. Methods Experimental design and patients With previous Institutional Research and Ethics Board approval, we conducted a retrospective analytical study at the Instituto Nacional de Cancerología in Mexico City. We studied the tumor tissue of 101 patients with a diagnosis of germ cell testicular cancer that underwent surgery between 1992 and 2002. AFP (normal range: 0–8.5 ng/mL), hCG (normal range: 0–4 mIU/mL), and LDH (normal range: 119–213 UI/L) serum levels were performed in all patients prior to sur- gery and before receiving chemotherapy, for risk stratifica- tion and follow-up. These markers were determined by using routine automated analyzers in the Department of Clinical Chemistry and Serum Markers, Instituto Nacional de Cancerología. The hCG was measured using the SIEMENS IMMULITE 2000 which is a highly specific, solid-phase, two-site chemiluminiscent immunometric assay that measures intact hCG without nicked forms and free subunits (Siemens; Los Angeles, CA, USA). AFP was measured with SIEMENS IMMULITE 2000 (Siemens; Los Angeles, CA, USA) and LDH with SYNCHRON LX20 (Beckman Coulter; Fullerton, CA, USA). Abdominal com- puted tomography scan and conventional chest x-ray were performed for disease staging according to the AJCC sys- tem. A database was made containing the clinical varia- bles of all patients including IGCCCG risk status classification. Patients who received chemotherapy, radio- therapy, or both previous to surgery were excluded. Tissue retrieval and immunohistochemistry assays Initial diagnostic biopsies were fixed in 10% neutral buff- ered formalin and embedded in paraffin. Morphologic evaluation was made in 3-μm tissue sections stained by the standard hematoxylin-eosin method. Sections 3-μm in thickness were mounted on slides and subsequently deparaffinized and rehydrated. Antigen was retrieved with 10 mM sodium citrate solution (pH 6.0) preheated to Page 2 of 7 (page number not for citation purposes)