Content uploaded by Evangelos Evangelou
Author content
All content in this area was uploaded by Evangelos Evangelou on Jun 13, 2014
Content may be subject to copyright.
Comparison of the diagnostic accuracy
of lymphatic endothelium markers:
Bayesian approach
Evangelos Evangelou
1
, Panayiotis A Kyzas
1
and Thomas A Trikalinos
1,2
1
Department of Hygiene and Epidemiology, University of Ioannina School of Medicine, Ioannina, Greece
and
2
Institute for Clinical Research and Health Policy Studies, Tufts-New England Medical Center, Boston,
MA, USA
Tumor lymphatic density is evaluated by means of specific lymphatic endothelium markers, and is a potential
predictor of clinically meaningful outcomes. There are many claims on the postulated superiority of some of
these markers to identify lymphatics, always in the absence of quantitative data. We therefore compared the
diagnostic accuracy of the antibody against podoplanin and the commercially available D2-40, employing
Bayesian statistics to account for the absence of a gold standard. We used the pan-endothelial marker CD34 to
identify 23 542 distinct blood and lymphatic vessels in sections from 30 formalin-fixed, paraffin-embedded
archival tissue blocks of head and neck squamous cell carcinoma specimens. We stained two adjacent sections
with podoplanin and D2-40 and identified the continuum of each stained vessel in the sections with a
comprehensive method. Overall, 1864 vessels were stained with both markers, 119 only with podoplanin and
391 only with D2-40. Significantly more vessels with intraluminal red blood cells were stained with D2-40
compared to podoplanin (McNemar’s Po0.0001). Both antibodies had extremely high specificity (99.7% (95%
credible interval (CrI): 99.5–99.9%) and 98.8% (95% CrI: 98.3–99.5%) for podoplanin and D2-40, respectively) and
very high sensitivity (92.6% (95% CrI: 86.1–97.9%) and 97.3% (95% CrI: 94.9–99.2%) for podoplanin and D2-40,
respectively). Inferences were qualitatively similar when we took into account in the analyses the possibility
that the two tests (antibodies) may be correlated. We calculated that 96.3% (95% CrI: 94.2–98.6%) of the vessels
stained with podoplanin and 88.9% (95% CrI: 83.9–95.7%) of the vessels stained with D2-40 were truly
lymphatics. These numbers were in agreement with the observed number of stained vessels without
intraluminal red blood cells. Our results suggest that both antibodies are excellent lymphatic endothelium
markers and that there may be little reason to prefer either of them in most settings.
Modern Pathology (2005) 18, 1490–1497. doi:10.1038/modpathol.3800457; published online 1 July 2005
Keywords: podoplanin; D2-40; specificity; Bayesian inference; lymphangiogenesis
The mechanisms for the metastasis of epithelial
neoplasms via lymphatic vessels have recently
become the focus of intensive research activity.
1–5
Researchers probe into the biology of lymphatics,
the interactions of tumor cells with the lymphatic
vessels, and the mechanisms of lymphangiogenesis.
Central to all these efforts was the discovery of
lymphatic endothelium markers, such as LYVE-1,
the antibody against podoplanin, D2-40, prox-1 and
VEGFR-3.
1,5
It has been postulated that the density
of the lymphatic vessels is a predictor of clinically
meaningful outcomes in a number of malignan-
cies.
6–13
One of the most critical steps in the
assessment of lymphatic vessel density is the
selection of the optimal lymphatic endothelium
marker,
1,2,5,12–23
in order to avoid spurious findings
arising from the low accuracy of the staining.
However, given the absence of a gold standard, the
evaluation of the sensitivity and the specificity of
the lymphatic endothelium markers is a daunting
task.
Antibody against podoplanin is considered by
many researchers to be the most specific lymphatic
endothelium marker available.
1,5,9,14,15,17,21
Such
claims are not based on formal quantitative compar-
isons between different lymphatic endothelium
markers in the same samples, despite the numerous
relative calls in the literature.
3,12,16,20–22
We set out to
compare the diagnostic accuracy of podoplanin and
Received 11 April 2005; revised 31 May 2005; accepted 1 June
2005; published online 1 July 2005
Correspondence: Dr PA Kyzas, DDS, Department of Hygiene
and Epidemiology, University of Ioannina School of Medical,
Ioannina 45 110, Greece.
E-mail: md03791@cc.uoi.gr
Modern Pathology (2005) 18, 1490– 1497
&
2005 USCAP, Inc All rights reserved 0893-3952/05
$30.00
www.modernpathology.org
D2-40 in the same samples, employing a Bayesian
approach to account for the absence of a gold
standard.
Materials and methods
Tissue Samples
We used 4-mm-thick sections from a sample of 30
formalin-fixed, paraffin-embedded archival tissue
blocks of head and neck squamous cell carcinoma
specimens. In all, 25 of these were biopsy speci-
mens, while five were taken from excised tumors.
We counted the total number of vessels (both blood
and lymphatic) using the pan-endothelial marker
CD34.
24,25
A monoclonal antibody raised against
podoplanin, as described previously
26,27
(kindly
provided by Dr Geleff, Department of Pathology,
University of Vienna, Austria), and the commercial
monoclonal antibody D2-40 (SIG-730, Signet Labo-
ratories, Dedham, MA, USA, dilution 1:40) were
used to stain lymphatic vessels in two adjacent
sections. Immunohistochemistry was performed
using the EnVision System (DAKO, Copenhagen,
Denmark), as previously described.
13
Evaluation of Staining Patterns
We assessed the expression of the antibody against
podoplanin and D2-40 in each section using an
Olympus BX-51 microscope at 400 magnification.
We aimed to identify whether each single vessel in
any section was stained with both antibodies, only
with podoplanin or only with D2-40. For this
reason, we recognized the continuity of each vessel
in the adjacent sections. We decided on a set of a
priori formulated rules to evaluate the staining.
First, we specified that vessels containing red blood
cells were most likely blood vessels.
28
We therefore
considered all stained vessels with intraluminal
erythrocytes as potential ‘false-positives’. We re-
corded these potential ‘false-positives’ in order to
check the plausibility of our calculations. Second,
we considered vessels, which appeared joined in
one section and separated in the other, as one in
both sections. Third, we excluded any vessels that
were identified only in one section (stained by
either antibody) but could not be identified in the
adjacent section. Hence, we only counted vessels
that could be identified in both sections stained or
unstained by the lymphatic markers. Finally, to
ensure that no double counting occurred, we
photographed areas with high lymphatic vessel
density and compared them in the neighboring
sections with image processing software. Counts of
positively stained vessels with either antibody, as
well as potential ‘false-positive’ counts were tabu-
lated in 2 2 contingency tables. We compared
whether the two antibodies had any difference
in the number of identified lymphatic vessels
and in their potential ‘false-positive’ counts using
McNemar’s test.
Assessment of Diagnostic Accuracy
The two antibodies may be conceived as two
diagnostic tests to identify lymphatics. In order to
assess their accuracy, one has to calculate their
sensitivity (the proportion of positively stained
vessels that are indeed lymphatic vessels) and their
specificity (the proportion of the unstained vessels
that are not lymphatic vessels).
29
In the absence of a
gold standard, it is not possible to use ‘classical’,
frequentist approaches to estimate these quanti-
ties.
30
In simple terms, there are five unknown
parameters (the sensitivities and the specificities of
the two antibodies as well as the true prevalence of
the lymphatic vessels in the examined sections),
which cannot be estimated directly from the data.
However, such problems can be addressed with
nonfrequentist, Bayesian approaches, using infor-
mation on staining combinations with both anti-
bodies, as previously proposed.
30
The basic idea
behind any Bayesian assessment of diagnostic
accuracy is to utilize external information in order
to bypass the restrictions posed by the unknown
quantities. Prior knowledge (knowledge not stem-
ming from the data) can be used to describe the
unknown quantities mathematically, as distribu-
tions of probable values. The available data from
the current study are summarized in the table of
positively stained vessels with either antibody; the
potential ‘false-positive’ counts have not been taken
into account in the assessment of the diagnostic
accuracy. Finally, the information conveyed by the
data is combined with the prior information using
Bayes’ theorem, to yield the final, ‘posterior’ know-
ledge on the specificities and sensitivities of the
two antibodies. One can then easily draw inferences
on the diagnostic accuracy of the two tests.
The first step in any Bayesian analysis is to obtain
reasonable prior distributions, based on sound
judgments. In our case, a priori very little was
known about the true number of the lymphatic
vessels in the sections. We approximated this
uncertainty by using a uniform prior distribution
for the prevalence of lymphatics (the proportion of
true lymphatic vessels among all vessels) in the
sections. This distribution considers all prevalence
values between 0 and 100% as equally probable, and
thus is completely noninformative. Limited infor-
mation was also available on the sensitivity of each
antibody. We assigned a broad range of probable
values between 60 and 100% for the sensitivities
of both antibodies (Figure 1). This was based on
observations from electron microscopy studies,
26
where all small lymphatic vessels were intensely
stained with podoplanin and larger lymphatics were
not intensely stained. Since the vast majority of the
lymphatic vessels are small, this prior seems to be
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1491
Modern Pathology (2005) 18, 1490–1497
valid. We specified the same prior for D2-40. These
monoclonal antibodies are considered to be highly
specific.
1,5,9,14,15,17,21,26
Therefore, we assigned to
both antibodies high specificities. Also, prior
beliefs regarded the antibody against podoplanin
as the most specific lymphatic endothelium
marker;
1,5,9,14,15,17,21
hence, we set priors between
90 and 100% for the specificity of the antibody
against podoplanin, and between 80 and 100% for
D2-40 (Figure 1). In secondary analyses, we exam-
ined if the results changed when we assigned the
same priors between 80 and 100% to the specificity
of both antibodies. Various other vague (noninfor-
mative) prior distributions were also used. A first set
of analyses considered the two tests (antibodies)
statistically independent. This assumption may not
hold if D2-40 recognizes podoplanin protein.
31
This
would mean that the two tests are correlated (not
statistically independent), and should be taken into
account mathematically.
32,33
Thus, in a second set
of analyses, we allowed for possible correlation
between the two tests. We specified a prior for
this correlation that ranged between negligible
and very high. (Details on the technicalities of
the approaches, as well as the results of sensitivity
analyses, are provided as online supplementary
information.)
Based on the estimates of diagnostic accuracy, we
calculated how many of the positively stained
vessels would truly be lymphatics in different
tissues, where lymphatics may be commonly or
sparsely found. This proportion is otherwise called
the positive predictive value of each antibody and is
dependent on the prevalence of the lymphatics in a
section. Finally, we compared the predicted number
of false positively stained vessels in our samples
with the number of stained vessels with intra-
luminal erythrocytes in order to assess the plausi-
bility of our findings. Given that the counts of the
potential ‘false-positive vessels’ were not taken into
account in the estimation of the diagnostic accura-
cies, this may serve as an external validation of the
calculations.
Analyses were conducted in R 2.0.1 (R Founda-
tion for Statistical Computing, Vienna), JAGS 0.80
(Martyn Plummer, 2005) and Intercooled Stata 8.2
(Stata Corp., College Station, TX, USA). Results are
presented in the form of posterior medians and 95%
equally tailed posterior credible intervals (CrI) (95%
CrI). CrIs are analogues of the usually encountered
(frequentist) 95% confidence intervals. McNemar
test P-values are two tailed, and are considered
significant for Po0.05.
Results
Immunostaining
Typical staining patterns with both antibodies for
lymphatic vessels are shown in Figure 2. Generally,
positively stained vessels with either lymphatic
marker were thin walled and irregularly shaped,
and were often comprised by two to three endothe-
lial cells. Overall, we identified 23 542 different
blood vessels and lymphatics in the 30 specimens
we examined. Of these, 1983 and 2255 were stained
with the antibody against podoplanin and with
D2-40, respectively (Table 1). The distribution of
the vessel counts in the 2 2 tables across the 30
specimens did not differ (Breslow-Day P¼1.00).
Significantly more vessels were positive for D2-40
(Po0.001). There were 394 vessels that were
positive either for podoplanin or D2-40, but had
intraluminal red blood cells and were considered as
potential ‘false-positives’ (Table 2). These potential
‘false-positives’ were significantly more common
with D2-40 compared to podoplanin (Po0.001).
Diagnostic Accuracy
Under the assumption that the two tests are
statistically independent, both antibodies were
found to be extremely specific for the identification
of lymphatic vessels. The posterior estimates of the
specificities of the two antibodies were clearly
separated (Figure 3a), although their absolute dif-
ference was very small. Specificities were 99.7%
(95% CrI: 99.5–99.9%) and 98.8% (95% CrI: 98.3–
99.5%) for the antibody against podoplanin and
D2-40, respectively. Podoplanin had lower sensitiv-
ity (92.6% (95% CrI: 86.1–97.9%)) compared to
Figure 1 Prior distributions for the unknown quantities. Bayesian
approaches borrow strength from prior information (information
not stemming from the data) on the five unknown parameters in
the problem. Prior information is expressed as distributions of
probable values. The same Beta(12,3) prior was set for the
sensitivities of the two tests (thin solid line). A Beta(71.25,3.75)
prior was used for the specificity of the antibody against
podoplanin (thick solid line) and a Beta(31.5,3.5) for the
specificity of D2-40 (thick dashed line). The prior for the
prevalence (thin dashed line) was a uniform distribution. All
distributions extend from 0 to 100%. Narrow distributions imply
that a narrow range of values is more probable (has a higher
probability density).
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1492
Modern Pathology (2005) 18, 1490–1497
Figure 2 (a) Adjacent sections from the same tumor sample (laryngeal squamous cell carcinoma, SCC), in which the same vessels
(indicated by arrows of the same color) are positively stained with both podoplanin and D2-40 (magnification 400). Vessels appearing
joined in one section and separated in the other were counted as one, for both the sections. (b) Adjacent sections from the same tumor
sample (laryngeal SCC), in which a vessel with luminal red blood cells (potential ‘false-positive’) is negative for podoplanin and positive
for D2-40 (magnification 1000). Red arrows indicate red blood cells. (c) Adjacent sections from the same tumor sample (SCC of the floor
of the mouth), in which a vessel with luminal red blood cells (potential ‘false-positive’) is positively stained with both podoplanin and
D2-40 (magnification 1000). Notice the vessel close to the positive one, which has also luminal red blood cells (red arrow), but is
negative for both the antibodies. (d) Adjacent sections from the same tumor sample (laryngeal SCC), in which both true- and false-
positive vessels can clearly be seen (magnification 400). All of these vessels are positive for both markers. Red arrows indicate red
blood cells; arrows of the same color indicate the same vessels in the two sections. (e) Adjacent sections from the same tumor sample
(SCC of the tongue), in which a vessel with luminal red blood cells (potential ‘false-positive’) is negative for podoplanin and positive for
D2-40 (blue arrow, magnification 4000). There is also a vessel without red blood cells, which is positively stained with both markers
(green arrow). (f) Adjacent sections from the same tumor sample (SCC of the lower lip), showing a vessel without luminal red blood cells,
which is podoplanin negative and D2-40 positive (magnification 1000). (g) Adjacent sections from the same tumor sample (laryngeal
SCC), showing a vessel filled with lymphocytes, without red blood cells, which is positively stained with both markers.
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1493
Modern Pathology (2005) 18, 1490–1497
D2-40 (97.3% (95% CrI: 94.9–99.2%)) as shown in
Figure 3b. Similar inferences were drawn when we
used a different set of priors.
The estimated prevalence of the lymphatic vessels
was 8.8% (95% CrI: 8.1–9.5%). We calculated that
the proportion of positively stained vessels (positive
predictive values) that are truly lymphatics in our
samples was 96.3% (95% CrI: 94.2–98.6%) and
88.9% (95% CrI: 83.9–95.7%) for podoplanin and
D2-40, respectively. As evident from Figure 4,
although the two antibodies had very similar
diagnostic accuracy, the positive predictive values
may differ, especially when lymphatics are sparse.
Based on these estimates, the predicted proportion
of falsely positively stained vessels would be 3.7
and 11.1% for the two antibodies, respectively.
The corresponding observed proportions of stained
vessels with intraluminal erythrocytes were 4.9 and
13.2% (Table 2).
The second set of analyses that allowed for
correlation between the two tests yielded qualita-
tively similar results. Podoplanin and D2-40 were
estimated to have specificities 95.6% (95% CrI:
92.0–98.9%) and 94.8% (95% CrI: 90.9–98.3%) and
sensitivities 82.6% (95% CrI: 62.7–94.7%) and
Table 1 Counts of stained or unstained vessels with either
antibody
D2-40
Stained Unstained
Podoplanin Stained 1864 119
Unstained 391 21 168
Figure 3 Posterior distributions assuming that the two antibodies
do not yield statistically correlated results. The combination of
the prior knowledge and the data yields the final estimates of the
analysis (posterior knowledge). (a) Posterior distributions of the
specificity of the antibody against podoplanin (thick solid line)
and D2-40 (thick dashed line). For comparative reasons, the
prior distributions for the specificities of the antibody against
podoplanin (thin solid line) and D2-40 (thin dashed line) are
also shown. (b) Posterior distributions of the sensitivity of the
antibody against podoplanin (thick solid line), and of D2-40 (thick
dashed line). The thin solid line is the common prior for the
sensitivities of the two antibodies.
Figure 4 Positive predictive values for different prevalences of
lymphatic vessels assuming that the two antibodies do not yield
statistically correlated results. The proportion of the stained
vessels that are truly lymphatic vessels (positive predictive value)
for the antibody against podoplanin (solid line) and for D2-40
(dashed line) is plotted against the prevalence of the lymphatic
vessels in a tissue section. When lymphatics are sparse, the
difference in the positive predictive value increases. The dots
represent the calculated values for our samples, where the
estimated prevalence of the true lymphatics was 8.8%.
Table 2 Counts of vessels with intraluminal erythrocytes with
either antibody
D2-40
Stained Unstained
Podoplanin Stained 82 15
Unstained 215 0
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1494
Modern Pathology (2005) 18, 1490–1497
90.0% (95% CrI: 74.0–97.6%), respectively. The
positive predictive value was 50.9% for podoplanin
and 48.9% for D2-40.
Discussion
In the current study, we compare two lymphatic
endothelium markers, and provide handy numerical
estimates for their diagnostic accuracy. Our results
suggest that both markers have extremely high
specificity and sensitivity for the identification of
lymphatic vessels, and have comparable positive
predictive values in most settings.
Up to date, experts’ opinions favored the antibody
against podoplanin as the most reliable and specific
marker at hand.
1,5,9,14,15,17,21
Nevertheless, scientists
emphasize the necessity for a quantitative evalua-
tion of the accuracy of the so-called ‘specific
lymphatic endothelium markers’.
1,5,19–22
To our
knowledge, our work is the first report that pursues
direct comparisons between two lymphatic endothe-
lium markers and provides quantitative estimates
for their specificity, sensitivity and positive pre-
dictive value. Contrary to prior beliefs, we found
that both antibodies are essentially equally specific,
in all sets of analyses. Thus, there may be little
reason to prefer either one of the antibodies in most
settings.
In our sections, 5% of the vessels stained by the
antibody against podoplanin and 13% of those
stained by D2-40 contained intraluminal red blood
cells. These counts were very close to those
calculated based on the positive predictive values
of the two antibodies (approximately 4 and 11%,
respectively) under the assumption that the tests
are statistically independent. This may be seen as
an indirect support in favor of these findings,
since the potential ‘false-positive’ counts were
not taken into account in the Bayesian analyses.
On the contrary, allowing for correlation between
the two tests, one would calculate that almost half
of the stained vessels were not lymphatics. Analyses
allowing for correlation between the two antibodies
are heavily dependent on the used prior distri-
butions, and this might be a reasonable explanation
for the high number of expected ‘false-positives’.
That high proportions of ‘falsely positively’ stained
vessels would be incompatible with electron
microscopic immunohistology findings,
26
where all
examined podoplanin-stained vessels in normal
skin were found to be lymphatics, and reciprocally,
all lymphatics were stained with podoplanin. Tak-
ing all the above into account, we lend more
credence to the inferences drawn from the model
that assumes that the two tests are statistically
independent.
The approach we used may yield reliable esti-
mates for the prevalence
30
of the lymphatic vessels
in a tissue section. One of the main drawbacks of the
assessment of the lymphatic vessel density in
histological sections is the very high interobserver
variability in the identification of the areas with the
highest vessel concentration, the so-called ‘hot-
spots’.
34
On the contrary, the prevalence of the
lymphatics in a section is not so subjective, since
the assessor simply counts everything. These pre-
valence estimates might be equally good or better
predictors of clinical outcomes compared to the
assessment of the lymphatic vessel density. More-
over, the whole procedure could be automated
through specialized software, using a PC connected
to a light microscope.
35
Some limitations of our study should be dis-
cussed. First, we estimated the total number of
vessels (blood and lymphatics) using CD34. It has
been reported that CD34 is expressed both in blood
vessels and lymphatics, but blood vessels stain more
intensely.
24,25,36
Thus, the total number of vessels we
counted may be an underestimation of the true
value. However, this did not bias our findings, as
evident from sensitivity analyses. Second, we did
not perform double immunostaining, as this would
be technically more difficult. Nevertheless, given
our elaborate methodology for the evaluation of the
staining patterns, large misclassification is unlikely.
Third, there is an inherent subjectivity in Bayesian
approaches, expressed by the morphology of the
prior distributions. The choice of the prior distri-
butions mostly affects inferences based on the
assumption that the two antibodies are statistically
dependent. However, the used priors are based
on the available knowledge and a comprehensive
literature search.
1,5,9,14,15,17,21,26
Finally, we consid-
ered stained vessels with intraluminal red blood
cells as potential ‘false-positives’. Intraluminal
erythrocytes might be a histologic artifact resulting
from pressure-induced leakage of a neighboring
blood vessel in a true lymphatic vessel.
26
This
phenomenon is observed in lymphangiomas and
might also happen in rapidly growing tissues, like
malignant tumors.
26
However, we believe that the
majority of the potential ‘false-positives’ are indeed
blood vessels; otherwise, the two antibodies ought
to yield roughly the same number of potential ‘false-
positive’ vessels.
Allowing for these caveats, both antibodies can
be regarded as reference standards for the identifica-
tion of lymphatic vessels in most settings. Similar
approaches could be employed for the evaluation
of diagnostic accuracy of other markers, such as
the remaining lymphatic endothelium markers,
antibodies detecting blood vessels or antibodies
from different manufacturers detecting the same
antigen.
Acknowledgement
We thank Silvana Geleff for kindly providing us the
antibody against podoplanin, and for her useful
comments.
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1495
Modern Pathology (2005) 18, 1490–1497
References
1 Saharinen P, Tammela T, Karkkainen MJ, et al.
Lymphatic vasculature: development, molecular regu-
lation and role in tumor metastasis and inflammation.
Trends Immunol 2004;25:387–395.
2 Padera TP, Kadambi A, di Tomaso E, et al. Lymphatic
metastasis in the absence of functional intratumor
lymphatics. Science 2002;296:1883–1886 (Epub 2002
April 25).
3 Reis-Filho JS, Schmitt FC. Lymphangiogenesis in
tumors: what do we know? Microsc Res Technol 2003;
60:171–180.
4 Jain RK, Fenton BT. Intratumoral lymphatic vessels: a
case of mistaken identity or malfunction? J Natl Cancer
Inst 2002;94:417–421.
5 Stacker SA, Achen MG, Jussila L, et al. Lymphangio-
genesis and cancer metastasis. Nat Rev Cancer 2002;2:
573–583.
6 Beasley NJ, Prevo R, Banerji S, et al. Intratumoral
lymphangiogenesis and lymph node metastasis in
head and neck cancer. Cancer Res 2002;62:1315–1320.
7 Straume O, Jackson DG, Akslen LA. Independent
prognostic impact of lymphatic vessel density and
presence of low-grade lymphangiogenesis in cuta-
neous melanoma. Clin Cancer Res 2003;9:250–256.
8 Schoppmann SF, Bayer G, Aumayr K, et al. Austrian
Breast and Colorectal Cancer Study Group. Prognostic
value of lymphangiogenesis and lymphovascular in-
vasion in invasive breast cancer. Ann Surg 2004;240:
306–312.
9 Munoz-Guerra MF, Marazuela EG, Martin-Villar E, et
al. Prognostic significance of intratumoral lymphan-
giogenesis in squamous cell carcinoma of the oral
cavity. Cancer 2004;100:553–560.
10 Maula SM, Luukkaa M, Grenman R, et al. Intratumoral
lymphatics are essential for the metastatic spread and
prognosis in squamous cell carcinomas of the head and
neck region. Cancer Res 2003;63:1920–1926.
11 Laura R-B, Benoit T, Emile G, et al. Lymphatic vessel
density and vascular endothelial growth factor-C
expression correlate with malignant behavior in hu-
man pancreatic endocrine tumors. Clin Cancer Res
2004;10:6919–6928.
12 Franchi A, Gallo O, Massi D, et al. Tumor lymphan-
giogenesis in head and neck squamous cell carcinoma:
a morphometric study with clinical correlations.
Cancer 2004;101:973–978.
13 Kyzas PA, Geleff S, Batistatou A, et al. Lymphangio-
genesis in head and neck squamous cell carcinoma:
Evidence and prognostic implications. J Pathol 2005;
206:170–177.
14 Matsui K, Nagy-Bojarsky K, Laakkonen P, et al.
Lymphatic microvessels in the rat remnant kidney
model of renal fibrosis: aminopeptidase p and podo-
planin are discriminatory markers for endothelial cells
of blood and lymphatic vessels. J Am Soc Nephrol
2003;14:1981–1989 (Erratum in J Am Soc Nephrol
2003; 14(10): following table of contents).
15 Birner P, Obermair A, Schindl M, et al. Selective
immunohistochemical staining of blood and lymphatic
vessels reveals independent prognostic influence of
blood and lymphatic vessel invasion in early-stage
cervical cancer. Clin Cancer Res 2001;7:93–97.
16 Valencak J, Heere-Ress E, Kopp T, et al. Selective
immunohistochemical staining shows significant
prognostic influence of lymphatic and blood vessels
in patients with malignant melanoma. Eur J Cancer
2004;40:358–364.
17 Clarijs R, Ruiter DJ, de Waal RM. Lymphangiogenesis
in malignant tumours: does it occur? J Pathol 2001;
193:143–146.
18 Dumoff KL, Chu C, Xu X, et al. Low D2-40 immuno-
reactivity correlates with lymphatic invasion and
nodal metastasis in early-stage squamous cell carci-
noma of the uterine cervix. Mod Pathol 2005;18:
97–104.
19 Chu AY, Litzky LA, Pasha TL, et al. Utility of
D2-40, a novel mesothelial marker, in the diagnosis
of malignant mesothelioma. Mod Pathol 2005;18:
105–110.
20 Choi WW, Lewis MM, Lawson D, et al. Angiogenic and
lymphangiogenic microvessel density in breast carci-
noma: correlation with clinicopathologic parameters
and VEGF-family gene expression. Mod Pathol 2005;
18:143–152.
21 Cunnick GH, Jiang WG, Gomez KF, et al. Lymphangio-
genesis and breast cancer metastasis. Histol Histo-
pathol 2002;17:863–870.
22 Pepper MS. Lymphangiogenesis and tumor metastasis:
myth or reality? Clin Cancer Res 2001;7:462–468.
23 Kahn HJ, Marks A. A new monoclonal antibody, D2-40,
for detection of lymphatic invasion in primary tumors.
Lab Invest 2002;82:1255–1257.
24 Tsuji T, Sasaki Y, Tanaka M, et al. Microvessel
morphology and vascular endothelial growth factor
expression in human colonic carcinoma with or with-
out metastasis. Lab Invest 2002;82:555–562.
25 Saad RS, Liu YL, Nathan G, et al. Endoglin (CD105)
and vascular endothelial growth factor as prognostic
markers in colorectal cancer. Mod Pathol 2004;17:
197–203.
26 Breiteneder-Geleff S, Soleiman A, Kowalski H, et al.
Angiosarcomas express mixed endothelial phenotypes
of blood and lymphatic capillaries: podoplanin as
a specific marker for lymphatic endothelium. Am
J Pathol 1999;154:385–394.
27 Breiteneder-Geleff S, Matsui K, Soleiman A, et al.
Podoplanin, novel 43-kd membrane protein of glome-
rular epithelial cells, is down-regulated in puromycin
nephrosis. Am J Pathol 1997;151:1141–1152.
28 Karkkainen MJ, Makinen T, Alitalo K. Lymphatic
endothelium: a new frontier of metastasis research.
Nat Cell Biol 2002;4:2–5.
29 Loong TW. Understanding sensitivity and specificity
with the right side of the brain. BMJ 2003;327:
716–719.
30 Joseph L, Gyorkos TW, Coupal L. Bayesian estimation
of disease prevalence and the parameters of diagnostic
tests in the absence of a gold standard. Am J Epidemiol
1995;141:263–272.
31 Schacht V, Dadras SS, Johnson LA, et al. Up-regulation
of the lymphatic marker podoplanin, a mucin-type
transmembrane glycoprotein, in human squamous cell
carcinomas and germ cell tumors. Am J Pathol
2005;166:913–921.
32 Georgiadis MP, Johnson WO, Gardner IA, et al.
Correlation-adjusted estimation of sensitivity and
specificity of two diagnostic tests. J R Stat Soc Ser C
2003;52:63–76.
33 Dendukuri N, Lawrence J. Bayesian approaches
to modeling the conditional dependence between
multiple diagnostic tests. Biometrics 2001;57:158–
167.
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1496
Modern Pathology (2005) 18, 1490–1497
34 Vermeulen PB, Gasparini G, Fox SB, et al. Second
international consensus on the methodology and
criteria of evaluation of angiogenesis quantifica-
tion in solid human tumours. Eur J Cancer 2002;38:
1564–1579.
35 Hannen EJ, van der Laak JA, Manni JJ, et al. Com-
puter assisted analysis of the microvasculature in
metastasized and nonmetastasized squamous cell
carcinomas of the tongue. Head Neck 2002;24:
643–650.
36 Schmid K, Birner P, Gravenhorst V, et al. Prognostic
value of lymphatic and blood vessel invasion in
neuroendocrine tumors of the lung. Am J Surg Pathol
2005;29:324–328.
Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/
modpathol)
Diagnostic accuracy of podoplanin and D2-40
E Evangelou et al
1497
Modern Pathology (2005) 18, 1490–1497
