Blood Level of Phosphoglycerate Kinase Does not Correlate with Presence or Extent of Tumor

It is generally recognized that tumor growth is dependent on tumor neovascularization, or angiogenesis (1). There are several activators and inhibitors that balance the angiogenic process (2). One of the angiogenesis inhibitors is an internal fragment of plasminogen called angiostatin (3). Plasminogen is a plasma zymogen that contains five consecutive triple disulfide-linked domains (called kringles) followed by a serine proteinase module. Urokinaseor tissue-plasminogen activator convert plasminogen to plasmin by hydrolysis of a peptide bond in the serine proteinase module. Plasmin is processed by tumor cells to give angiostatin fragments containing variable numbers of kringle domains (1-41/2, 1-4 and 1-3). Plasmin processing occurs in three stages (reviewed in 4). Two disulfide bonds in the 5th kringle domain are cleaved, which leads to hydrolysis of peptide bonds by a serine proteinase and release of kringle 1-41/2 fragments. These fragments are then trimmed by matrix metalloproteinases to produce either kringle 1-4 or 1-3. The precipitating event in angiostatin formation is cleavage of the kringle 5 disulfides, a process that is facilitated in the extracellular space by the glycolytic enzyme, phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) (5). Transformed cells in culture secrete abundant PGK, as much as 2 μg from a million cells in 24 hours. In contrast, primary cells secrete barely detectable levels. In addition, the blood of mice bearing transplanted human fibrosarcoma tumors contained several-fold more PGK than that of mice without tumors and administration of PGK to tumor-bearing mice caused an increase in plasma levels of angiostatin and a decrease in tumor vascularity and rate of tumor growth (5). These findings imply that relatively small amounts of PGK are secreted from normal tissues and that elevated blood levels of PGK may indicate the presence of tumor. This notion is supported by studies of transcript and protein profiles associated with survival in lung adenocarcinoma. Using oligonucleotide microarrays, Bhattacharjee et al (6) analyzed mRNA expression levels of 12,600 transcripts in 186 lung tumor samples, including 139 lung adenocarcinomas. They found that a high level of the PGK transcript is significantly associated with poor survival. Using quantitative two-dimensional polyacrylamide gel electrophoresis analysis, Chen et al (7) quantified 682 individual protein spots in 90 lung adenocarcinomas. Like PGK message, elevated PGK protein was significantly correlated with poor outcome. We have measured plasma PGK levels in 82 patients with different cancers, 27 patients with non-cancer-related conditions and 22 healthy volunteers. Serum PGK levels in a validation set of 107 patients with lung adenocarcinoma were also measured. Murine monoclonal and sheep polyclonal anti-PGK antibodies were used to develop a sandwich-type ELISA for PGK as described previously (7). There was a linear relationship between readout and PGK concentration up to 100 ng.mL. The intraassay variability was 11%, while the interassay variability was 27%. Plasma and serum levels of PGK were measured in this study. Plasma PGK levels were not affected by the type of anticoagulant used in its preparation: EDTA, citrate or heparin. For convenience, EDTA was used as the anticoagulant for preparation of plasma. There was also no appreciable effect of the time between collection and processing of blood samples (up to 24 hours’ delay at room temperature) on the plasma level of PGK. Serum PGK levels were approximately twice the plasma levels in healthy volunteers. Plasma PGK levels in 82 patients with breast (11), colorectal (23), ovarian (13), lung (10), leukemia (7) or other cancers (16), 27 patients with non-cancer-related conditions and 22 healthy volunteers were measured. The cancer patients were not evaluated for stage or extent of tumor. Serum PGK levels in a validation set of 107 patients with lung adenocarcinoma (66 stage I, 15 stage II, 20 stage III and 6 stage IV) (7) and 48 patients with squamous lung carcinoma (31 stage I, 11 stage II and 6 stage III) were also measured. Consent was received from all patients and the project was approved by the respective Institutional Review Boards. The data was coded to protect patient confidentiality. The International Journal of Biological Markers, Vol. 19 no. 2, pp. 170-172 © 2004 Wichtig Editore