Cancer cells exhibit a high proliferative activity and are therefore strongly dependent on the process of glycolysis requiring a specialized isoenzyme distribution and activity. One of the key enzymes involved in glycolysis is the pyruvate kinase (PK). This enzyme is known to appear in various isoforms called L, R and M, which are expressed in a tissue-specific manner.1). The M-type of the pyruvate kinase can be further subclassified in M1, which is predominantly expressed in cells of skeletal muscles, heart and brain, and M2, which is found in lung and kidney, as well as in tissues of low differentiation or high proliferative activity. In benign proliferating cells the M2-type of the pyruvate kinase (M2-PK) is found in a highly active tetrameric configuration, whereas the nearly inactive dimeric variant of M2-PK, called Tumor M2-PK (TuM2-PK), is found predominantly in malignant cells.2 The tetramer:dimer ratio of M2-PK determines whether glucose carbons are converted into lactate under the production of energy (tetrameric form) or channeled into synthetic processes such as DNA, phospholipid and amino acid synthesis (dimeric form).
TuM2-PK molecules have been detected not only in malignant cells of different origin2, 3 but also in the peripheral blood4, 5, 6, 7 and stool8 of cancer patients. For TuM2-PK quantification in the peripheral blood, plasma was shown to provide higher stability and therefore higher data reproducibility compared to serum.9 Provided that TuM2-PK is predominantly expressed in malignant cells, the TuM2-PK plasma concentration might be a useful marker of the tumor load in cancer patients.
In our study, we measured the concentration of TuM2-PK in plasma samples from 300 melanoma patients and 53 healthy controls. In addition, serum samples obtained in parallel from the same patients were analyzed for their concentration of the currently best established tumor marker of melanoma, S100β.
Patients and methods
After informed consent, 300 patients with histologically confirmed malignant melanoma were included into our study in chronological order of their presentation at the Skin Cancer Unit. Disease stage, tumor load and current treatment of the patients were documented. Disease staging was performed according to the revised staging criteria of the American Joint Committee on Cancer (AJCC).10 Briefly, stage I and II included patients with primary melanoma, stage III included patients with regional lymph node, satellite and/or in-transit metastases and stage IV included patients with distant metastases. Tumor load was measured by physical examination, X-ray or CT of the chest, ultrasound or CT of the abdomen and lymph nodes as well as MRI of the brain. A time distance of at least 2 weeks following any surgical procedure was required for inclusion. According to their disease stage the patients received different therapeutic regimens enclosing cytotoxic drugs (dacarbacine, cisplatinum, temozolomide, vindesine and fotemustine) as well as immunomodulatory agents (interferon(IFN)-α, vaccines) in various combinations and schedules. Follow-up examinations were performed at least in 3 months intervals including the above-mentioned techniques for the measurement of tumor load as well as blood chemistry. Detailed clinical characteristics of the patients are summarized in Table I. Blood samples of 53 control volunteers matched in age and gender were kindly provided by the Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Wuerttemberg/Hessen, Germany. All controls were healthy blood donors undergoing regular physical and laboratory examinations. Blood samples and clinical data of patients and controls were collected with Institutional Review Board approval.
Table I. Patients' Characteristics
Number of cases
TuM2-PK (U/ml) median (25%/75%)
S100β (μg/l) median (25%/75%)
Data for TuM2-PK and S100β are represented as median (25% quartile; 75% quartile). Statistical analyses were performed using the Wilcoxon rank sum test (patients vs. controls; males vs. females; tumor-bearing vs. tumor-free), the Jonckheere test (disease stages) and the Kruskal-Wallis-test combined with the multiple comparision procedure of Dunn (patients under current therapy vs. untreated patients). p < 0.05
Blood was drawn once from each patient and healthy volunteer into uncoated serum tubes (8 ml) and ethylenediaminetetraacetic acid (EDTA)-containing plasma tubes (8 ml), respectively. After a resting period of at least 30 min and a maximum of 60 min the tubes were centrifugated at 2500g for 10 min. Serum and plasma was harvested and subsequently aliquoted and stored at −20°C until usage.
The serum concentration of S100β was measured using a sandwich immunoluminometric assay (LIA-mat Sangtec 100, Sangtec Medical, Bromma, Sweden) following the manufacturer's instructions. Briefly, serum samples were thawed, diluted (1:2) and thereafter subjected to polystyrene tubes precoated with an MAb recognizing the β-subunit of the S100 protein. After washing, captured S100β was incubated with a second MAb conjugated to an isoluminol derivative and thereafter detected by a light reaction quantified using a luminometer (Berthold ACL, Pforzheim, Germany). The lowest measurable S100β concentration was determined as 0.02 μg/l; the intra- and inter-assay variations were < 10%.
The plasma concentration of TuM2-PK was assessed using a sandwich enzyme-linked immunosorbent assay (ELISA) system described previously (ScheBo Biotech, Giessen, Germany).9 Briefly, plasma samples were thawed, diluted (1:100) and thereafter subjected to 96-well multititer plates coated with a monoclonal antibody (clone I, ScheBo Biotech) specifically recognizing the dimeric form of M2-PK. After 60 min of incubation, plates were washed and thereafter incubated with a second, biotin-conjugated anti-M2-PK monoclonal antibody (clone II, ScheBo Biotech) for 30 min. After washing, bound TuM2-PK molecules were detected with a streptavidin-coupled horseradish peroxidase reaction and subsequently quantified by optical density measurement at 405 nm using a microtiter plate reader. The standard curve revealed linearity for TuM2-PK concentrations between 5 and 100 U/ml. The intra- and inter-assay variations were 3.5% and 5.3%, respectively.
Tissue samples from cutaneous or subcutaneous melanoma metastases as well as healthy skin were obtained from melanoma patients after informed consent and with Institutional Review Board approval. Immediately after surgery the samples were cleared from fatty tissue, subsequently snap frozen using liquid nitrogen and thereafter stored at −80°C until further use. Frozen tissue specimen were thawed and extracted with a lysis buffer containing 10 mM Tris, 1 mM NaF and 1 mM mercaptoethanol (Merck, Darmstadt, Germany), pH 7.4 as described previously.3 From this lysates, pyruvate kinase activities (Vmax) were measured according to;11 concentrations of TuM2-PK were quantified using a sandwich ELISA (ScheBo Biotech). For Western blotting, the tissue lysates were pre-diluted with lysis buffer to a final protein concentration of 2 μg/μl and thereafter diluted (1:2) with SDS sample buffer. A lysate of the breast cancer cell line MDA-MB-453 known to express TuM2-PK12 was used as positive control. The lysates were applied to a 10% SDS-polyacrylamide gel (tissue lysates at 20 μg protein/slot, MDA-MB-453 lysate with 5 μg protein/slot). After separation, the proteins were transferred onto a nitrocellulose membrane by electroblotting. The mouse monoclonal anti-TuM2-PK antibody DF4 (50 ng/ml; ScheBo Biotech) was used for detection. Visualization of bound probes was performed by a diaminobenzidine/H2O2 redox reaction.
Normality of the data was tested using the d'Agostino-Pearson-test, revealing the serum concentration of S100β as well as the plasma concentration of TuM2-PK as skewed data. All data are therefore presented as median (25% percentile; 75% percentile). For statistical comparisons the Wilcoxon rank-sum test (patients vs. controls; males vs. females; tumor-bearing vs. tumor-free), the Jonckheere test (disease stages) and the Kruskal-Wallis-test (different modes of therapy vs. no therapy, melanoma tissue vs. healthy skin) were used. Probabilities of overall survival and progression-free survival were analyzed using the Kaplan-Meier method in combination with the log-rank test; end points were death from melanoma and progress or relapse of melanoma disease, respectively. A cut-off value at which the most pronounced deterioration in the prediction of tumor load and prognosis takes place was determined for plasma TuM2-PK using the critlevel analysis.13 Multivariate analysis was performed using the proportional hazard model of Cox. For all statistical tests, differences with a p value < 0.05 were considered significant.
Strong expression of TuM2-PK in melanoma tissue
Nine tissue samples derived from cutaneous or subcutaneous melanoma metastases and 3 specimens from healthy skin were analyzed for TuM2-PK expression and PK activity (Vmax). TuM2-PK was stronger expressed in melanoma specimens compared to healthy skin as shown by immunostaining (Fig. 1). Tissue samples from melanoma metastases revealed significantly higher concentrations of TuM2-PK (p = 0.0126, Kruskal-Wallis-test) and higher Vmax activities (p = 0.0124) than samples from healthy skin (Table II).
Table II. TuM2-PK and PK Activity in Melanoma Lesions and Healthy Skin1
TuM2-PK (Units/g wet weight)
PK activity (Vmax) (U/g wet weight)
Tissue samples from cutaneous or subcutaneous melanoma metastases as well as healthy skin were snap frozen immediately after surgery. TuM2-PK concentration and PK activity (Vmax) were measured as described in Patients and Methods. Melanoma specimens reveal a significantly higher TuM2-PK concentration (p = 0.0126, Kruskal-Wallis-test) and PK Vmax activity (p = 0.0124) compared to healthy skin samples.
High concentrations of TuM2-PK in plasma from melanoma patients
Plasma as well as corresponding serum samples were analyzed for TuM2-PK and S100β concentrations, respectively, from 300 melanoma patients with a mean age of 55.7 ± 13.0 years. The median follow-up time was 15.6 months.
As shown in Table I, the median TuM2-PK plasma concentration was significantly increased in melanoma patients compared to healthy controls (p = 0.0036; Wilcoxon rank sum test). There was no significant correlation between gender or age of the patients and plasma TuM2-PK or serum S100β, respectively. In corresponding blood samples, the TuM2-PK plasma concentration was positively correlated with the serum concentration of S100β (p = 0.00006; rank correlation test of Kendall).
Plasma TuM2-PK and serum S100β concentrations correlate with disease stage and tumor load
The Jonckheere test revealed an association of increased plasma concentrations of TuM2-PK with advanced disease stages of melanoma patients (p < 0.0005; Table I). Elevated serum S100β concentrations were also found to correlate with the stage of disease (p < 0.0005; Table I). In regard to the tumor load, tumor-bearing patients showed significantly higher TuM2-PK plasma concentrations as well as S100β serum concentrations than tumor-free patients (both p < 0.0005; Wilcoxon rank sum test; Table I).
Plasma TuM2-PK and serum S100β concentrations under therapy
To study a potential effect of therapeutic modalities on plasma TuM2-PK as well as serum S100β, the patients were grouped as follows: 1) patients receiving cytotoxic therapy, 2) patients treated with IFN-α, 3) patients under therapy regimens other than the both foregoing, enclosing patients under biochemotherapy, vaccination or protein-kinase inhibitory treatment and 4) patients without any therapy during the last 8 weeks before blood withdrawal (Table I). The Kruskal-Wallis-test combined with Dunn's multiple comparison procedure revealed that the plasma level of TuM2-PK was significantly enhanced in patients under current treatment with cytotoxics (p = 0.0014; Table I). Serum S100β was strongly elevated in patients currently treated with cytotoxics as well as to a lower extent in patients under immunotherapy with IFN-α (p = 0.00001; Table I). The exclusion of patients under current treatment within the last 8 weeks prior to blood withdrawal (n = 67) from our data analysis resulted in lower but still statistically significant correlations of plasma TuM2-PK/serum S100β with tumor load (p = 0.045/0.0029) and disease stage (p = 0.016/0.018), respectively, in the remaining 233 patients.
Correlation of plasma TuM2-PK and serum S100β with overall and progression-free survival
During the median follow-up time of 15.6 months, 48 patients experienced a disease progression and 38 patients died from melanoma; 12/48 progressions and 5/38 deaths occurred in the group of patients who were tumor-free at the time of study inclusion (n = 243). To analyze the prognostic impact of the TuM2-PK plasma concentration, the patients were grouped according to their plasma level of TuM2-PK utilizing a cut-off value of 15.0 U/ml as determined by critlevel analysis. The same was done according to serum concentrations of S100β using a cut-off value of 0.12 μg/l as recommended by the assay's manufacturer. Using the Kaplan-Meier method combined with the log-rank test, we found a strong association of elevated TuM2-PK plasma concentration with a reduced probability of overall (p < 0.000005; Fig. 2a) and progression-free (p = 0.023; data not shown) survival. Likewise, elevated serum levels of S100β showed a strong correlation with a poor overall (p < 0.000005; Fig. 2b) and progression-free (p < 0.00005; data not shown) survival. The date of blood withdrawal was used as the starting point for survival analyses for both markers. The same analysis considering the time point of first diagnosis of melanoma revealed similar results (data not shown).
Using a cut-off value of 15 U/ml, plasma TuM2-PK was able to predict a disease progression with a sensitivity of 27.1% (13/48 patients) and a specificity of 83.7% (211/252 patients); a patient's death with a sensitivity of 44.7% (17/38) and a specificity of 84.7% (222/262 patients). For disease progression the positive predictive value was 0.241 and the negative predictive value was 0.858. For a patient's death the positive predictive value was 0.298 and the negative predictive value was 0.914.
Using a cut-off value of 0.12 μg/l, serum S100β predicted a disease progression with a sensitivity of 45.8% (22/48 patients) and a specificity of 84.9% (214/252 patients); a patients' death with a sensitivity of 78.9% (30/38) and a specificity of 88.5% (232/262 patients). For disease progression the positive predictive value was 0.367 and the negative predictive value was 0.892. For a patient's death the positive predictive value was 0.500 and the negative predictive value was 0.967.
A multivariate analysis of data obtained from the subgroup of patients with metastasized disease (AJCC stage III and IV; n = 110) revealed serum S100β as the strongest independent predictor of overall survival (p = 0.00001; proportional hazards model of Cox), followed by tumor load (p = 0.004) and plasma TuM2-PK (p = 0.028). The stage of disease could not be ascertained as an independent predictive factor of survival (p = 0.541). In regard to progression-free survival, the tumor load resulted as the only independent prognostic factor (p = 0.00001), whereas disease stage (p = 0.49), plasma TuM2-PK (p = 0.66) as well as serum S100β (p = 0.92) were of no independent predictive value.
Analysing the subgroup of patients without detectable tumor load (n = 243), we found neither plasma TuM2-PK nor serum S100β as significant predictors of disease progression and survival (data not shown). Additionally, we performed a subgroup analysis of nonmetastasized patients (AJCC stage I and II; n = 190). In this subgroup only 6 disease progressions and no deaths occurred during the follow-up period. Thus, no predictors of survival or disease progression could be determined. Only 1 out of the 6 progressors was measured with elevated concentrations of serum S100β and plasma TuM2-PK; the other 5 progressors were negative for both markers.
Combination of serum S100β and plasma TuM2-PK
Accounting for a combination of both markers, plasma TuM2-PK and serum S100β, we obtained 4 subgroups of patients with different probabilities in regard to overall and progression-free survival (Fig. 3). Subdivision of patients with low serum S100β (= 0.12 μg/l) into 2 groups (a and b) according to their plasma TuM2-PK concentration revealed significantly heterogenous estimation curves for progression-free survival (p < 0.0005; Fig. 3a). In regard to overall survival, the difference between these curves (a and b) scantily miss statistical significance (p = 0.055). Here, patients with high serum S100β can be further subdivided into 2 groups (c and d) of significantly different probability of survival (p = 0.0025; Fig. 3b).
Malignant melanoma is a disease of increasing incidence with only few available therapeutic options of sufficient efficacy. In the case of early detection and surgical excision of the primary tumor, melanoma patients have a high probability of long-term cure. After the occurrence of metastases, the patient's prognosis is strongly impaired with a 5-year survival rate between 5 and 10% in patients with distant metastases.10
Marker molecules originating from melanoma cells and thereafter becoming detectable in the patient's peripheral blood are important tools for the detection of a disease progression and for the prediction of the patient's prognosis. To serve as a useful tumor marker, the concentration of these molecules should correlate with the patient's tumor load.
In melanoma, S100β currently is the most widely used tumor marker in the clinical routine. Serving as a marker of an acute damage of the central nervous system in serum and cerebrospinal fluid for nearly a decade,14 it was recently described as a reliable indicator of tumor load, disease progression and therapy response in patients with advanced stages of melanoma.15, 16, 17, 18 In these patients, S100β is a useful prognostic marker with elevated serum levels of S100β indicating a poor overall survival compared to patients with serum concentrations within normal ranges.19 In tumor-free patients, e.g., after surgical excision of their primary tumor or lymph node metastases, tumor markers like S100β correlating with the tumor load are of no prognostic use.19
A variety of different proteins of the peripheral blood, in particular in serum, have been investigated for their potential relevance as tumor markers in melanoma revealing similar characteristics as known from S100β.20, 21, 22 However, clinical studies comparing a potential new marker with the well-established marker S100β, or even combining both markers, the new and the familiar one, have been rarely performed up to now.
TuM2-PK has been proven to be overexpressed in different malignant entities like gastric carcinoma,23 lung cancer24 and renal cell carcinoma.3 Moreover, TuM2-PK could be detected in the peripheral blood of cancer patients and might hereby serve as a tumor marker.4, 5, 6, 7
In our study, we show that TuM2-PK is overexpressed in tissue from metastatic melanoma lesions compared to healthy skin. To evaluate TuM2-PK as an useful tumor marker in the peripheral blood of melanoma patients, we analyzed TuM2-PK and S100β in plasma and serum samples, respectively, obtained from the same blood withdrawal in each patient. This procedure provided the possibility to compare the sensitivity and specificity of both markers as well as combine them in order to evaluate their potential to serve as markers of the patient's tumor load, disease stage and prognosis.
We observed significantly increased TuM2-PK plasma levels in patients with melanoma compared to healthy individuals. Moreover, TuM2-PK plasma concentrations were found to correlate with the patients' tumor loads and disease stages. In melanoma patients with distant metastases, patients with high plasma concentrations of TuM2-PK showed a reduced overall survival and time to progression compared to patients with low plasma levels. Despite the finding that plasma TuM2-PK is an independent predictive factor of overall survival, its predictive value is not superior to that of serum S100β. However, the combination of both markers significantly improved the estimation of patients' progression-free and overall survival, even in the subgroup of S100β-negative patients.
In regard to current therapeutic interventions at the time point of the investigative blood withdrawal, we found a significant increase of TuM2-PK in plasma samples from patients under chemotherapy. This might be due to an enhanced tumor cell death and subsequent release of TuM2-PK molecules into the circulation after exposure to cytotoxic drugs. Alternatively, this observation could also be explained by the high tumor load of the patients in advanced disease stages, which are much more frequent in the cytotoxic treatment group than in all of the other treatment groups. The same effect became visible in a higher extent (p = 0.00001 vs. p = 0.0014) for S100β displayed by an increase in its serum concentration in patients receiving chemotherapy.
In conclusion, our results show that a combined use of the plasma marker TuM2-PK and the serum marker S100β improves the prediction of progression-free and overall survival in metastasized melanoma patients compared to the use of S100β alone. Similar observations have been made for other cancer entities.25 However, according to the results of this study neither plasma TuM2-PK nor serum S100β proved to be reliable prognostic indicators in tumor-free melanoma patients. Further investigations are needed to find suitable predictive markers for this particular subgroup of patients.
We are gratefully obliged to S. Mazurek, Institute for Biochemistry and Endocrinology, Veterinary Faculty, University of Giessen, for kindly providing her expertise in the detection and quantification of M2-PK in tissue samples. Moreover, we thank K. Decker from ScheBo Biotech, Giessen, for her helpful support as well as F. Stötzer and H. Eichler from the Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Wuerttemberg/Hessen, Mannheim, for the kind supply with blood samples of healthy donors.