Journal of Cranio-Maxillofacial Surgery (2008) 36, 89e94

Ó 2007 European Association for Cranio-Maxillofacial Surgery

doi:10.1016/j.jcms.2007.08.006, available online at http://www.sciencedirect.com

Pyruvate kinase isoenzyme M2 is not of diagnostic relevance as a

marker for oral cancer

Juergen ERVENS1, Hendrik FUCHS2, Volker-Till NIEMANN1, Bodo HOFFMEISTER1

1
Department of Maxillofacial & Facial Plastic Surgery (Chairman: Prof. Dr. Dr. B. Hoffmeister),
Charité e Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; 2 Central Department of
Laboratory Medicine and Pathobiochemistry (Chairman: Prof. Dr. R. Tauber), Charité e Universitätsmedizin Berlin,
Campus Benjamin Franklin, Berlin, Germany

SUMMARY. Introduction: Increased aerobic glycolysis is one of the most common abnormalities occurring in the
metabolism of tumour cells with pyruvate kinase as one of the key glycolytic enzymes. The dimeric form of M2-
pyruvate kinase, found predominantly in tumour cells, is designated as tumour M2-pyruvate kinase (TuM2-PK).
The aim of the present, prospective study was to evaluate the diagnostic relevance of TuM2-PK in detecting oral
squamous cell carcinoma (OSCC). Material and methods: TuM2-PK concentration was analysed in ethylene di-
aminetetraacetic acid plasma of 80 untreated patients with histologically confirmed OSCC stages T3 and T4, whilst
90 patients with non-malignant diseases served as controls. The analysis was done using a sandwich enzyme-linked
immunosorbent assay (ScheBoÒ Biotech AG, Gießen, Germany). Immunohistochemical detection of TuM2-PK
was performed by specific mouse monoclonal antibody DF-4. Results: The median TuM2-PK concentration
was 22.92 U/ml in the tumour group and 10.00 U/ml in the control group (p\ 0.001). Using 15 U/ml as a cut-off
yielded a sensitivity of 63% and a specificity of 59%. Moreover, the positive and negative predictive values
were 57% and 64%, respectively. Immunohistochemical staining of tissue sections showed that TuM2-PK
was not selectively expressed in tumour cells but also in non-malignant cells, particularly in more regenerative
ones. Conclusion: Low sensitivity and specificity for stages T3 and T4 OSCC render the TuM2-PK test unsuit-
able as a tool for detecting OSCC, particularly in an early stage. Thus, routine clinical and radiological follow-up is
indispensable after treatment of OSCC. Ó 2007 European Association for Cranio-Maxillofacial Surgery

Keywords: TuM2-PK, pyruvate kinase, oral squamous cell carcinoma, screening test

INTRODUCTION (Mazurek et al., 2005). The inactive dimeric form of M2-

Oral squamous cell carcinoma (OSCC) (most frequent
oral malignancy) is among the 10 most common cancers
in the world, accounting for 10,000 newly diagnosed
cases and approximately 6000 deaths annually in Ger-
many. Early diagnosis of oral cancer continues to be im-
portant because only treatment at an early stage
correlates with a high 5-year-survival rate and a good
clinical and functional outcome (Howaldt et al., 2000).
Although the oral mucosa can be systematically exam-
ined, early detection may be difficult (Field et al., 1995).
Circulating tumour markers is an established method for
detection and monitoring of many malignant tumours
(Perkins et al., 2003). Since cancer cells have high prolif-
erative activity, their metabolic state differs from that of
normal proliferating cells. In particular, tumour cells ex-
hibit increased aerobic glycolysis with pyruvate kinase
as one of the key glycolytic enzymes (Eigenbrodt et al.,
1998; Mazurek et al., 2000). Several isoforms of pyruvate
kinase are tissue-specific: type L in liver and kidney; type
R in erythrocytes; type M1 in muscle, heart, and brain; and
type M2 in lungs and kidneys as well as in undifferentiated
and proliferating tissues. Pyruvate kinase isoenzymes
normally exist as enzymatically highly active tetrameres
pyruvate kinase is expressed by cellular oncogenic coded
kinases during multistep carcinogenesis, with increased
levels in tumour cells and is therefore designated as tumour
M2-pyruvate kinase (TuM2-PK; Ventrucci et al., 2004;
Mazurek et al., 2005). TuM2-PK has been identified not
only in malignant cells of different origin but also in the pe-
ripheral blood and faeces of cancer patients and is under
consideration as a promising new tool in the diagnostic
programme for different malignant tumours (Rijksen
et al., 1988; Varga et al., 2002; Bena-Boupda et al.,
2003; Oremek et al., 2003b; Hardt et al., 2004; Koss
et al., 2004; Ewald et al., 2005; Vogel et al., 2005). How-
ever, TuM2-PK has not been investigated in OSCC so far.
Since early detection of oral cancer is of great impor-
tance for therapy and follow-up, this prospective study
aimed at evaluating the diagnostic relevance of TuM2-
PK for detecting OSCC.
MATERIAL AND METHODS
Patients
The study included 80 untreated patients (25 female, 55
male) with histologically confirmed stages T3 and T4

89
90 Journal of Cranio-Maxillofacial Surgery
OSCC as well as a control group of 90 patients (45 fe-
male, 45 male) with non-malignant diseases (Sobin and
Wittekind, 2002). All patients gave their informed con-
sent. The mean age was 60.5 years in the tumour group
(ranging from 35 to 91 years) and 31.0 years in the con-
trol group (ranging from 14 to 90 years).
Sample collection
Prior to any surgical treatment, peripheral blood samples
were collected in ethylene diaminetetraacetic acid
(EDTA)-supplemented tubes followed by centrifugation
(1500g for 10 min) and removal of the supernatant
plasma. The samples were stored at 20  C until mea-
surement for a maximum of 2 months.
TuM2-PK assay
Quantitative determination of TuM2-PK was performed
in 10 ml EDTA plasma by using a commercially available
sandwich enzyme-linked immunosorbent assay
(ScheBoÒ Biotech AG, Gießen, Germany). TuM2-PK
was adsorbed in the wells of microtiter plates coated
with monoclonal antibodies that were specific for
TuM2-PK and did not cross-react with other isoforms
of pyruvate kinase. The test was performed according
to the manufacturer’s instructions. The colour density
of oxidised 2,20 -azino-bis(3-ethylbenzothiazoline-6-sul-
fonic acid) was photometrically determined for quantita-
tive measurement of TuM2-PK. TuM2-PK concentration
was simultaneously assessed in triplicate; the cut-off
value of 15 U/ml TuM2-PK was taken from the manufac-
turer’s data.
TuM2-PK assay calibration
Calibration of the TuM2-PK assay with the manufactur-
er’s defined concentrations of TuM2-PK (5 U/ml, 15 U/
ml, 40 U/ml, and 100 U/ml) resulted in a standard curve
for calculation of the TuM2-PK concentration of the test
samples. TuM2-PK assay was strictly calibrated for each
new series of measurements. For quantitation of all test
samples, 28 series of measurements with corresponding
28 standard curves were necessary.
Immunohistochemistry
Expression of TuM2-PK in OSCC cells was evaluated
after ablative surgery in all patients. Tissues derived
from resected OSCC were fixed and embedded in Para-
plastÒ for immunohistochemical detection of TuM2-PK.
Tissue sections were stained with the pyruvate kinase-
M2-specific mouse monoclonal antibody DF-4 as descri-
bed by Brinck et al. (1994).
Statistical analysis
Data were statistically analysed by SPSS for windows.
Significance of the difference of the means was calcu-
lated using the ManneWhitney test for non-parametric
distribution. The significance level was set at p \0.05.
RESULTS
First, the plasma TuM2-PK concentration was assessed
in triplicate by sandwich enzyme-linked immunosorbent
assay. In patients suffering from OSCC, the plasma
TuM2-PK concentration ranged from 37 U/ml to
285 U/ml (mean 32.89 U/ml). In contrast, the plasma
TuM2-PK concentration ranged from 74 U/ml to
147 U/ml (mean 12.44 U/ml) in the control group. The
median of the plasma TuM2-PK concentration was
22.92 U/ml in the tumour group and 10.00 U/ml in the
control group. The difference of the medians was found
to be significant (p \0.001), using the ManneWhitney
test (Fig. 1). There were no significant sex- or age-
specific differences within either group.
With a cut-off value of 15 U/ml (manufacturer’s rec-
ommendation), the TuM2-PK test was calculated to
have a sensitivity of 63% and a specificity of 59% in
170 individuals. Its positive and negative predictive
values were 57% and 64%, respectively (Table 1). There
was no significant correlation between the prevalence of
OSCC and a positive TuM2-PK test (data not shown).
Evaluation of the cut-off revealed that the intersection
of sensitivity and specificity was between 15 U/ml and
16 U/ml, and the optimal average at 18 U/ml (Fig. 2),
demonstrating that the cut-off value recommended by
the manufacturer was appropriate. Positive and negative
predictive values intersected between 21 U/ml and
22 U/ml and did not exhibit an optimum but a number
of relative maxima, the most prominent at 34 U/ml.
The TuM2-PK test was also evaluated for intra- and
inter-individuals variability. Using the same test kit at
the same time, triplicates of the plasma TuM2-PK
300
250
mean tumour M2-PK concentration (U/ml)
200
150
100
50
0
-50
-100
N= 90 80
control group tumour group
Fig. 1 e Mean TuM2-PK concentration of the tumour and control
groups. Data shown as boxplots, whiskers indicating the minimum and
maximum, boxes the 25%- and 75%-percentiles, B and * indicating
outlier and extreme values, respectively. The centres of the boxes
indicate the medians. Cut-off line at 15 U/ml. Results of a
ManneWhitney test for non-parametric distribution.
 Pyruvate kinase isoenzyme M2 91

Table 1 e 2  2 Table to discriminate stages T3 and T4 OSCC patients 300

individual tumour M2-PK concentrations (U/ml)

(n ¼ 80) from those with non-malignant diseases (n ¼ 90)

TuM2-PK Tumour group Control group Total

(cut-off # 15 U/ml) 200
Positive 50 37 87
Negative 30 53 83
Total 80 90 170 100

The cut-off value of 15 U/ml TuM2-PK was taken from the manufac-
turer’s data.
0

concentration in a single patient varied up to a maximum

of 118 U/ml in the tumour group and 77 U/ml in the con-
trol group (Fig. 3). In contrast, the inter-individual vari- -100

1
14
27
40
53
66
79
92
10
11
13
14
15
17
ance of the mean plasma TuM2-PK concentration of

the tumour and control group was 322 U/ml and 221
U/ml, respectively (Fig. 3). Additionally, the standard
curves of 28 series of measurements were determined.
Using the manufacturer’s defined concentrations resulted
in a high variability of the single standard curves. Nega-
tive absorbance at low concentrations was not to be ex-
pected but could be explained by the observed high
statistical spread. Nevertheless, the correlation coefficient
of the regression curve of 28 combined series was 0.997
(Fig. 4).
Finally, TuM2-PK expression in OSCC cells was stud-
ied by applying the monoclonal antibody DF-4 in tissues
derived from patients having suffered from OSCC after
ablative surgery. Immunohistochemical staining showed
that all patients had increased TuM2-PK expression in
OSCC cells. TuM2-PK expression was also enhanced
in patients where TuM2-PK test was negative (false neg-
ative patients), as illustrated in Figs. 5 and 6. In order to
detect potential cross-reactions with other isoforms of the
pyruvate kinase, normal non-malignant tissues were im-
munohistochemical stained using the monoclonal anti-
body DF-4. Interestingly, TuM2-PK was not selectively
expressed by OSCC cells but was also found in normal
non-malignant cells, such as those seen in lymph nodes,
salivary glands and muscle. In normal cells, TuM2-PK
expression was particularly enhanced in more regenera-
tive cells, like the secretory acinar cells in salivary glands
5
8
1
4
7
0
individuals
Fig. 3 e Plasma TuM2-PK concentration for each patient of the tumour
group (red) and control group (green). Endpoints of the lines represent
maximum and minimum values. The dashed line indicates the cut-off
level of 15 U/ml.
or follicle cells in lymph nodes (Fig. 7). Even in normal
oral mucosa, the more regenerative basal cells showed
markedly enhanced TuM2-PK expression.
DISCUSSION
Increased aerobic glycolysis is one of the most common
metabolic abnormalities occurring in tumour cells. Prolif-
erating cells and particularly tumour cells express the py-
ruvate kinase isoenzyme type M2, which acts as an
enzymatically highly active tetramere in proliferating
non-tumour cells. In tumour cells, however, it is shifted
by oncogenic coded kinases into the inactive dimeric
form (TuM2-PK) and is always predominant (Eigenbrodt
et al., 1998; Mazurek et al., 2000, 2005). A high plasma
TuM2-PK level was recently shown to correlate with
mean standard curve
0,8
0,7

0,6

0,5

0,4
absorption

0,3

0,2

0,1

0,0

-0,1

-0,2
0 50 100 150
Units
Fig. 2 e Specificity and sensitivity of the TuM2-PK assay dependent on Fig. 4 e Mean standard curve using the standard concentrations for
the cut-off value (U/ml). Squares indicate the arithmetic mean between calibration as defined by the manufacturer. Data shown are the
specificity and sensitivity to depict the optimum. means ^ SD of 28 series of measurements.
92 Journal of Cranio-Maxillofacial Surgery
Fig. 5 e Expression of TuM2-PK in OSCC: enhanced TuM2-PK
expression demonstrated by intensified red staining using monoclonal
antibody DF-4. TF e tumour formation; 120.
various malignant diseases, e.g., renal cell carcinoma,
gastrointestinal cancer, and colon, breast, and lung can-
cers (Oremek et al., 1999; Lueftner et al., 2000; Oremek
et al., 2000; Schneider et al., 2003; Kaura et al., 2004;
Ventrucci et al., 2004; Zhang et al., 2004; Ugurel
et al., 2005). More recently, TuM2-PK has also been
identified as a suitable marker for colorectal cancer
(Hardt et al., 2004; Ewald et al., 2005; Murphy et al.,
2006). Since so many malignant epithelial tumours corre-
late with a high plasma TuM2-PK level, this prospective
study was initiated to evaluate the diagnostic relevance of
TuM2-PK for detecting OSCC, which has not been in-
vestigated to date.
In the present study on OSCC, the median TuM2-PK
concentration of the tumour group was 22.92 U/ml com-
pared with 10.00 U/ml in the control group (Fig. 1),
which is in accordance with results previously reported
by various authors for other malignancies (Varga et al.,
2002; Roigas et al., 2003; Ventrucci et al., 2004; Zhang
et al., 2004). The test failed to discriminate between the
two groups despite the highly significant difference be-
tween the median TuM2-PK concentration in the tumour
and control groups (p\ 0.001). This is demonstrated by
the overlapping boxes in Fig. 1. Thus the test results were
without clinical relevance despite being statistically sig-
nificant due to insufficient discrimination. Similar results
were reported for renal cell carcinoma patients and pa-
Fig. 6 e Tissue section of OSCC derived from patient with false
negative TuM2-PK test after ablative surgery: enhanced TuM2-PK
expression demonstrated by intensified red staining using monoclonal
antibody DF-4. KPF e keratin pearl formation; TF; 60.
tients suffering from haematological malignancies
(Varga et al., 2002; Staib et al., 2006).
The results presented here are in agreement with Ore-
mek et al. (2000), who found no significant differences in
the TuM2-PK level between Robson stages III and IV re-
nal cell carcinoma patients. They also revealed no statis-
tically significant difference in the TuM2-PK level
between stages T3 and T4 OSCC patients. Further inves-
tigations should focus on the question of whether a de-
creasing postoperative TuM2-PK level in patients with
a high preoperative level indicates tumour regression.
The majority of available tumour markers are of lim-
ited value, since low sensitivity and specificity prevent
them from discriminating adequately between cancer
and non-cancer patients and thus render them inapplica-
ble for monitoring and particularly for screening (Perkins
et al., 2003). On the other hand, to be assessed as having
sufficient sensitivity and specificity, the TuM2-PK test
would be expected to detect nearly all tumour patients
above and nearly all non-tumour patients below the
cut-off of 15 U/ml (manufacturer’s recommendation;
Fig. 3). However, the present study resulted in a sensitiv-
ity of only 63% and a specificity of 59% in the test pa-
tients (Table 1 and Fig. 2). This is in contrast to higher
sensitivities and specificities reported by others for differ-
ent malignancies, especially in patients with metastatic
tumours (Lueftner et al., 2000; Kaura et al., 2004; Zhang

Fig. 7 e Expression of TuM2-PK in normal lymph node using
monoclonal antibody DF-4. Note enhanced staining of active follicle
cells; 60.
et al., 2004). Furthermore, combining the TuM2-PK test
with conventional tumour markers like CEA, CA 19-9,
CA 27.29 and S100b has increased its diagnostic effi-
ciency (Hardt et al., 2000; Schneider et al., 2000;
Schulze, 2000; Schneider and Schulze, 2003; Ugurel
et al., 2005). The small tumour mass of OSCC, even in
stage T4, could be a possible explanation for the findings
of this study. However, the results are in agreement with
Varga et al. (2002), Hegele et al. (2003), Oremek et al.
(2003b), Roigas et al. (2003) and Staib et al. (2006)
who have found similar sensitivities and specificities
for other malignancies and suggested that the TuM2-
PK test was unsuitable for monitoring and early detection
of cancer.
The TuM2-PK test has shown high variability of the
single standard curves with negative absorbance at low
concentrations (Fig. 4) as well as a high inter- and in-
tra-individual variability (Fig. 3). The TuM2-PK concen-
tration ranged from 37 U/ml to 285 U/ml in the tumour
group and from 74 U/ml to 147 U/ml in the control
group, respectively. This is consistent with previous stud-
ies reporting a mean interassay coefficient of variance
ranging up to 17.5% and confirms the specific problems
involved in the measurement of TuM2-PK (Varga et al.,
2002; Hegele et al., 2003). Negative absorbance at low
concentrations may probably be attributed to statistical
spread as corroborated by the standard deviation (SD)
Pyruvate kinase isoenzyme M2 93
of the standard concentrations of the test kits, or by ham-
pering effects of particular serum components in some
patients. Intra-individual variability in measurement of
TuM2-PK concentration in triplicate varied using differ-
ent batches of the test kits and could be caused by min-
imal differences in production (Fig. 3).
In this study, the plasma TuM2-PK concentration was
abnormally high in patients with non-malignant diseases.
With a rate of 41%, the TuM2-PK test leads to false pos-
itive values (Table 1 and Fig. 3). Since all patients of the
control group were healthy volunteers without any in-
flammatory or general diseases in history, the increased
TuM2-PK concentrations cannot be explained. The
plasma TuM2-PK concentration was also found to be ab-
normally high in various diseases such as diabetic ne-
phropathy (Oremek et al., 2003c), acute and chronic
pancreatitis (Ventrucci et al., 2004), rheumatic disease
(Oremek et al., 2003a) and chronic cardiac failure (McDo-
well et al., 2004). Furthermore, even lipaemic or icteric
serum affects the plasma TuM2-PK concentration and
leads to false positive values (Oremek et al., 2003a). Since
various non-malignant diseases can affect the TuM2-PK
concentration, it must be assumed that increased aerobic
glycolysis in general can affect the TuM2-PK test by
cross-reaction with other isoforms of the pyruvate kinase.
Thus, it seems that TuM2-PK is not a tumour-specific
marker but more likely a marker for aerobic glycolysis,
which is indoubtedly increased in malignancies.
Finally, TuM2-PK expression in OSCC cells was im-
munohistochemically evaluated using the monoclonal
antibody DF-4. OSCC cells showed significant TuM2-
PK expression in all tumour patients (Figs. 5 and 6).
TuM2-PK expression was immunohistochemically dem-
onstrated for renal cell carcinoma and lung cancer in pre-
vious studies by Brinck et al. (1994) and Schneider et al.
(2003). Surprisingly, even patients with negative TuM2-
PK tests (false negative patients) had clearly detectable
expression of TuM2-PK, as demonstrated by the intensi-
fied red staining in Fig. 6. This may be attributed to dif-
ferent epitope-dependent selectivity of DF-4 compared
with the ScheBoÒ antibody or to different release of
TuM2-PK into serum, thus varying among the individual
patients. It is also notable that TuM2-PK was not selec-
tively expressed by OSCC cells but was also found in
normal non-malignant cells such as those in muscle, sal-
ivary glands, and lymph nodes. Furthermore, TuM2-PK
expression was particularly more strongly expressed in
cells with high proliferative activity, like the more regen-
erative basal cells in normal oral mucosa or the follicle
cells in lymph nodes (Fig. 7). These findings suggest
that either the monoclonal antibody DF-4 does not selec-
tively detect the dimeric form of M2-pyruvate kinase but
cross-reacts non-specifically with other isoforms of the
pyruvate kinase or that TuM2-PK is not selectively ex-
pressed in cancer cells. The latter assumption is corrobo-
rated by the results of the ScheBoÒ assay.
CONCLUSION
The TuM2-PK test is not selective for the inactive di-
meric form of M2-pyruvate kinase but also detects other
94 Journal of Cranio-Maxillofacial Surgery
isoforms of the pyruvate kinase by non-specific cross-
reactions. Immunohistochemical staining of tissue sec-
tions was not tumour-selective but proved to be increased
in all highly regenerative tissues. Low sensitivity and
specificity even for stages T3 and T4 OSCC render the
TuM2-PK test as unsuitable for detecting OSCC, partic-
ularly in an early stage. Thus, routine clinical and radio-
logical follow-up is still indispensable after oncologic
treatment of OSCC and the search for other detecting
and monitoring markers of OSCC needs to be continued.
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Dr. Med. Dr. Med. Dent. Juergen ERVENS
Department of Maxillofacial & Facial Plastic Surgery
Charité e Universitätsmedizin Berlin
Campus Benjamin Franklin
Hindenburgdamm 30
D-12200 Berlin
Germany
Tel.: +49 30 8445 2474
Fax: +49 30 8445 4458
E-mail: juergen.ervens@charite.de
Paper received 13 July 2006
Accepted 29 August 2007