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Oncotarget, 2018, Vol. 9, (No. 30), pp: 21156-21165
Research Paper

LpMab-23-recognizing cancer-type podoplanin is a novel
predictor for a poor prognosis of early stage tongue cancer
Akihiro Miyazaki1,*, Hiromi Nakai1,*, Tomoko Sonoda2, Yoshihiko Hirohashi3, Mika
K. Kaneko4, Yukinari Kato4,5, Yoshihiko Sawa6 and Hiroyoshi Hiratsuka1
1

Department of Oral Surgery, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8543, Japan2

Department of Public Health, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan3

Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Japan

4

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi 9808575, Japan

5

New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8575, Japan

6

Deparment of Oral Function & Anatomy, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical
Sciences, Okayama Kita-ku 700-0914, Japan*

These authors have contributed equally to this work

Correspondence to: Yoshihiko Sawa, email: ysawa@okayama-u.ac.jp
Keywords: LpMab-23; predictor; podoplanin; tongue cancer; antibody
Received: October 17, 2017     Accepted: March 12, 2018     Published: April 20, 2018
Copyright: Miyazaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License
3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author andsource are credited.

ABSTRACT
Purpose: We report that the reactivity of a novel monoclonal antibody LpMab-23
for human cancer-type podoplanin (PDPN) is a predictor for a poor prognosis of
tongue cancer.
Patients and Methods: The association between LpMab-23-recognizing cancertype PDPN expression and clinical/pathological features were analyzed on 60 patientswith stage I and II tongue cancer treated with transoral resection of the primarytumor.
Results: In the mode of invasion, the LpMab-23-dull/negative cases weresignificantly larger in cases with low-grade malignancies and without late cervicallymph node metastasis, than in cases with high-grade malignancies and themetastasis. In the high-grade malignant cases, LpMab-23-positive cases weresignificantly larger than LpMab-23-dull/negative cases. The Kaplan–Meier curvesof the five-year metastasis-free survival rate (MFS) were significantly lower in the
LpMab-23 positive patients than in LpMab-23 dull/negative patients. The LpMab-23dull/negative cases showed the highest MFS in all of the clinical/pathological featuresand particularly, the MFS of the LpMab-23 positive cases decreased to less than 60%
in the first year. In the Cox proportional hazard regression models a comparison of thenumbers of LpMab-23 dull/negative with positive cases showed the highest hazard
ratio with statistical significance in all of the clinical/pathological features.
Conclusions: LpMab-23 positive cases may be considered to present a usefulpredictor of poor prognosis for early stage tongue cancer.

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INTRODUCTION

is correlated with a better prognosis, was identified instroma surrounding the tumors in many areas other thanat the invasive front, postulating that PDPN expression on
the stromal fibroblasts may act as a barrier to tumor cell
invasion [28].
From the above, it appears that there are two
conflicting hypotheses for the high expression of PDPN
in cancer: one leading to a better prognosis and the othersuggesting a poorer prognosis. Many sialic acids bind to
PDPN and affect the PDPN conformation by a negative
charge, repelling other molecules. Therefore, it may
be thought that the PDPN conformation is different fordifferent cancer cell types due to the diversity of sialic
acid bindings, and the diversity affects the affinities ofantibodies to PDPN, causing the opposing diagnosis
for the cancer by PDPN immunostaining. A novel mAb
LpMab-23 was recently established for human cancertype PDPN using the cancer-specific mAb (CasMab)technology [29]. Further, the chimeric antibody
chLpMab-23 exhibited antitumor activity via antibodydependent cellular cytotoxicity [30]. The critical epitopeof LpMab-23 is Gly54-Leu64, which is common to bothcancer and normal cells. Importantly, LpMab-23 reacts
with human cancer cells but not with normal cells suchas lymphatic endothelial cells. It may be speculated thatthe cancer-specific reactivity of LpMab-23 could be
due to the cancer specific O-linked glycosylation. The
cancer-type podoplanin has Thr55 and Ser56 aberrantly
O-glycosylated by disialyl-core1 (NeuAc-a2-3Galb1-3(NeuAca2-6) GalNAca1-O-Thr) on an extracellulardomain [30]. There is also the possibility that the different
reactions of LpMab-23 in cancer cells and normal cells
may be a cause of the conformational changes of the
epitope with the loss of the glycosylation on cancer cells,
further studies to determine this are needed. If there aredifferences in the D2-40 reaction to oral cancer, which
has a well-known reactivity to PDPN of both cancer
cells and normal cells, and LpMab-23 with a reactivity
that is specific to the oral cancer-type PDPN, it may bethought that PDPN conformation is different for different
cancers, resulting in the differences in clinical significancefor the PDPN expression. This study aims to investigate
whether the reactivity of LpMAb-23 is a predictor of apoor prognosis for early stage tongue cancer.

In a number of human cancers, a high level ofexpression of podoplanin (PDPN) limited to the invasion
front of the cancer has been reported in squamous cell
carcinomas of the upper digestive tract, from the oralcavity to the esophagus [1–5]. The PDPN expression
has also been shown in osteosarcoma, mesothelioma,
basal cell carcinoma of the skin, follicular dendritic celltumors, germ cell tumors of the ovary and testis, andhemangioblastoma, and brain tumors such as astrocytomas
or glioblastomas [6–13]. It appears that in some cases oftumors, the high expression of PDPN is closely correlatedwith the severity of clinical deterioration: invasion,
metastasis into regional lymph nodes, recurrences, andshorter survival times [14–18]. There are two explanations
for the significance of the high expression of PDPN. One
is as an induction of the transdifferentiation of epithelial
cells into motile mesenchymal cells, a process known
as epithelial–mesenchymal transition (EMT) promotion
with E-cadherin downregulation [19]; the other is notconcerned with EMT [14], but both of two papers referto a high expression of PDPN as being related to theenhancement of a high metastatic potential. Different fromthese studies, however, there are also reports that a low
expression of PDPN in uterine cervix cancers as correlatedwith poorer outcomes: lymphatic metastasis and poorer
survival rates [20, 21].
Further, a high expression of PDPN has also
been observed in cancer-associated fibroblasts (CAFs)
of the stroma surrounding tumors [22–24]. There aresome reports that the high expression of PDPN in
CAFs is implicated in a poorer prognosis of invasive
adenocarcinomas of the lung and pancreas [22–24].
Invasive ductal carcinoma of the pancreas has desmoplasia
with abundant fibrous connective tissue. The PDPN
is a lymphatic vessel marker (clone D2-40; one of theanti-humanPDPN monoclonal antibodies: mAbs) and
expression of PDPN by stromal CAFs has been reported
to be a prognostic indicator in various types of cancer.
The PDPN-expressing CAFs enhances the progressionof invasive ductal carcinomas of the pancreas, and ahigh ratio of PDPN-expressing CAFs is an independent
predictor of poor outcomes. In these cases, PDPN may
enhance the tumor-promoting effects of CAFs due toincreased RhoA activity [19, 25]. The PDPN plays a keyrole in the cell process elongation by the actin cytoskeleton
rearrangement dependent on the binding activity witha cytoplasmic linker protein ezrin via RhoA family
signaling. The PDPN up-regulates a Rho-GTPase activity
resulting in ezrin phosphorylation and phosphorylated
ezrin mediates the connection of PDPN to F-actin. The
PDPN induces the formation of membrane-actin structuresand promotes tumor cell invasion via plasma membrane
extensions [26, 27]. However, there is also a case wherecolorectal CAFs with high PDPN expression, whichwww.oncotarget.com

RESULTS
Subjects
Tissue blocks from 60 patients from 33 to 92 yearsof age (male [n=33], female [n=27]) who had undergone
partial glossectomy without elective neck dissection(under 66 years of age [n=33]; and above 66 years of age[n=27]) were tested. All cancers were T1 (tumor 2cm orless in the greatest dimension) or T2 (tumor more than
2cm but not more than 4 cm in the greatest dimension)
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of the T-classification and Grade 1 (well differentiated)(n=29), Grade 2 (moderately differentiated), or Grade 3(poorly differentiated)(n=31). For the mode of invasion
according to the YK classification, 42 subjects belonged
to YK-1 (well defined borderline), YK-2 (cords, lessmarked borderline), or YK-3 (groups of cells, no distinct
borderline) while 14 subjects belonged to YK-4C (diffuse
invasion, cord-like type invasion) and 4 belonged to YK4D (diffuse invasion, diffuse type invasion). There were
three subjects with local recurrence and 14 subjects withlate cervical lymph node metastasis.

of LpMab-23 were predominantly more positive thandull/negative (P<0.01). In the late cervical lymph nodemetastasis, significantly more LpMab-23-dull/negative
reactions were determined in cancers without metastasis
while with metastasis, the reactions of LpMab-23 werepredominantly more positive than dull/negative (P<0.01).
There were no statistically significant correlations between
the LpMab-23 reactivity and gender, age, T-classification,
histological grading, and local recurrence (Table 1).
There were however statistically significant
differences in the age, mode of invasion, and LpMab-23
reactivity for the five-year metastasis-free survival rate
(MFS)(Table 2). The survival rate was higher in patients
under 66 years of age than in patients older than 66 years(p<0.05), and higher in patients with the mode of invasion
YK-1/YK-2/YK-3 than in YK-4C and YK-4D patients(p<0.01). Notably, the survival rate was lower in patientswith LpMab-23 positive than in patients with dull/negative
reactivity (Table 2).

Immunohistochemical analysis for the reactionof D2-40 and LpMab-23
In the study, it was found that it is possible todistinguish 5 cases of combinations of reactions with D240 and LpMab-23 to the tongue squamous cell carcinoma:
D2-40-positive and LpMab-23-negative; D2-40 and
LpMab-23-double positive; D2-40-dull and LpMab-23positive; D2-40-dull and LpMab-23-strongly positive; D240-negative and LpMab-23-positive (Figure 1, Figure 2).
Further, reaction of LpMab-23 to cancer cells was foundon the cell membrane and cytoplasm (Figure 1). The D240 reacted with lymphatic vessels and the marginal cellsof cancer, while LpMab-23 did not react with lymphatic
vessels in any of the tissue samples tested (Figure 1).
There was no cancer tissue without reaction to either
D2-40 or LpMab-23. In cancer cells with low malignant
potential, D2-40 is positive on the cell membrane but
LpMab-23 is more commonly negative (Figure 1). In
the D2-40 and LpMab-23-double positive cancer, there
were cells which have cytoplasm with reaction products
to LpMab-23 (Figure 1). In the D2-40-dull cancers, thereaction to LpMab-23 was more pronounced (Figure 2).
It was found that the cancer tissue where almost all cells
are LpMab-23 positive has many lymphocyte infiltrations
and was composed of cancer cells with cytoplasm showing
diffusion of reaction products to LpMab-23 (Figure 2).
In cancer tissue with high malignant potential, the cells
were commonly D2-40 dull/negative and LpMab-23
positive/strongly positive (Figure 2). Furthermore, active
lymphangiogenesis was most commonly found among
LpMab-23 positive-cancer cells with no reaction to D2-40(Figure 2).

Association between LpMab-23 reactivity andprognosis
The Kaplan–Meier curves of MFS were significantly
lower in LpMab-23 positive patients (59.4%), in patientswith the mode of invasion YK-4C (57.1%) and YK4D (25.0%), and patients older than 66 years (63.0%)
compared with LpMab-23 negative patients (96.4%),
patients with the mode of invasion YK-1/YK-2/YK-3(88.1%), and patients under 66 years of age (87.9%). The
MFS of LpMab-23 positive and YK-4C or 4D patientswas lower, decreasing below 60% in the first year of the
60 months of follow-up (long-rank)(Figure 3). In the Cox
proportional hazard regression models the LpMab-23
positive vs dull/negative showed the highest hazard ratio
and a statistically significantly difference (Table 3). In theunivariate analysis, age, mode of invasion, and LpMab-23
reactivity are associated with late cervical lymph nodemetastasis: significantly different for patients under 66
years vs patients aged 66 years or older, for YK-1/YK-2/
YK-3 vs YK-4C vs YK-4D, LpMab-23 dull/negative vspositive (p<0.05). In the multivariate analysis among age,mode of invasion, and LpMab-23 reactivity, LpMab-23
reactivity is only identified as an independent factor;
significantly different for LpMab-23 dull/negative vspositive (p<0.05).

Association between LpMab-23 reactivity andclinical featuresDISCUSSION

There were statistically significant differences in themode of invasion and late cervical lymph node metastasis
among the significant LpMab-23 reactivity vs clinicaland pathological parameters (Table 1). In the mode ofinvasion, significantly more LpMab-23-dull or negative
reactions were determined in the YK-1/YK-2/YK-3
group; in the YK-4C and YK-4D groups, the reactions
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The aim of this study is to investigate the usefulnessof LpMab-23, a unique mAb for cancer-type PDPN, as apredictor for a poor prognosis of early stage tongue cancer.
The PDPN is a mucin-type highly glycosylated protein
identified in podocytes [31], osteocytes of rodent bones[32], type I alveolar cells [33], and lymphatic endothelial
cells [3]. The PDPN is also expressed in the choroid plexus
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Figure 1: D2-40-positive and LpMab-23-negative/positive early stage tongue squamous cell carcinoma. These sections

are visualized by DAB staining for reaction products with D2-40 to normal PDPN and with LpMab-23 for the cancer-specific PDPN with
Hematoxylin staining of nuclei (blue). The top row shows panels of a D2-40-positive (middle panel) and LpMab-23-negative (right panel)
tongue carcinoma (65 year old female, Grade 1, YK-2, no recurrence, no metastasis). Lymphatic vessels are reacted with D2-40 (arrows) andcells of the cancer at the margins are also D2-40 positive on the cell membrane (arrowheads) while in the adjacent section, neither cancer
cells nor lymphatic vessels are LpMab-23 negative (arrowheads). Bars: 100 μm. The middle row shows panels of a D2-40 and LpMab-23double positive tongue carcinoma (57 year old female, Grade 1, YK-4C, no recurrence, with metastasis). Lymphatic vessels are reacted with
D2-40 (arrows) but not with LpMab-23. Almost all of the invasive cancer cells are D2-40 positive with many lymphocyte infiltrations similar
to LpMab-23 (arrowheads). Bars: 100 μm. The bottom row shows magnified views of the box highlighted in each panel of the middle row.
Almost all cancer cells have both membrane and cytoplasm with the diffusion of LpMab-23 reaction products. Bars: 20 μm.

Figure 2: D2-40 dull/negative and LpMab-23 positive early stage tongue squamous cell carcinoma. The sections

are visualized by DAB staining for reaction products with D2-40 to normal PDPN and LpMab-23 for the cancer-specific PDPN with
Hematoxylin staining of nuclei (blue). The top row shows panels of a D2-40 dull (middle) and LpMab-23-positive (right) tongue carcinoma(77 year old female, Grade 2, YK-4C, no recurrence, with metastasis). The cells of the cancer at the margins are weakly reacted with D2-40(arrows) while in the adjacent section, the reaction with LpMab-23 (arrows) is stronger than that with D2-40. Bars: 100 μm. The middle
row shows panels of a D2-40 dull and LpMab-23-positive tongue carcinoma (79 year old female, Grade 1, YK-4C, no recurrence, withmetastasis). The invasive cancer cells showed few reactions with D2-40 (arrows) while in the adjacent section the cells showed strong
reaction with LpMab-23 (arrows). Bars: 100 μm. The bottom row shows panels of a D2-40 negative and LpMab-23 positive-tongue
carcinoma (77 year old female, Grade 2, YK-4C, no recurrence, with lymph node metastasis). The invasive cancer cells show no reactions
with D2-40 while there is lymphangiogenesis with much D2-40-positive lymphatic vasculature found among cancer cells (arrowheads). In
the adjacent section the cancer cells show a reaction with LpMab-23 (arrows). Bars: 100 μm.
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Table 1: Relevance between LpMab-23 reactivity and clinical/pathological features
LpMab-23 reactivity

Clinical/pathological features
Dull and Negative

Positive

P-value

No.

%

No.

%

29

48.3

31

51.7

Male

16

26.7

17

28.3

Female

13

21.7

14

23.3

<66

18

30

15

25

>66

13

21.7

14

23.3

T1

11

18.3

18

30

T2

18

30

13

21.7

Grade 1

15

25

14

23.3

Grade 2, 3

14

23.3

17

28.3

YK-1, 2, 3

27

45

15

25

YK-4C

2

3.3

12

20

YK-4D

0

0

4

6.7

No

26

43.3

31

51.7

Yes

3

5

0

0

No

28

46.7

18

30

Yes

1

1.7

13

21.7

All patientsNS

GenderNS

AgeNS

T-classification
NS

Histological grading
NS

Mode of invasion
P<0.01

Local recurrenceNS

Late cervical lymph node metastasis
P<0.01

NS, not significant. Ratio was rounded off to the 1st decimal place.
[34], mesothelial cells [35], epidermal basal layer cells,
tooth germ epithelial cells, salivary gland myoepithelium[36], thymus type I epithelial cells, follicular dendritic
cells, and in the central nervous system and perineurium[37]. The D2-40 is a well-known antibody to human
PDPN of normal tissue described above while LpMab-23
of which the epitope is Gly54-Leu64 of cancer-type
PDPN does not react with PDPN of normal cells [29,30]. In the immunohistochemical analysis of differences
between D2-40 and LpMab-23 in tongue cancer tissue, thereaction to lymphatic vessels was observed in D2-40 but
not in LpMab-23 (Figure 1), suggesting that LpMab-23 is
specific for cancer-type PDPN. There was no cancer tissue
without reaction to either D2-40 or LpMab-23. In LpMab23-positive tissue, it was observed that cells reacted
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with LpMab-23 on the cell membrane and cytoplasm(Figure 1, 2). There was diversity in the reactions of D240 and LpMab-23 to cancer cells, and there were cases
where the reactions to the same subject were similar ordifferent (Figure 1, 2), suggesting that early stage tongue
cancer cells produce both normal PDPN and LpMab-23recognizing cancer-type PDPN at several extents.
The prognosis of oral squamous cell carcinomas
depends on cervical lymph node metastasis. It is
generally accepted that patients with clinically negative
cervical lymph nodes will have a good prognosis, though
the prognosis of patients with lymph node metastasis
occurring after surgery or radiotherapy of the primarytumor is poor. However, some early stage patients withclinically negative cervical lymph nodes have a poor
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Table 2: Relevance between clinical/pathological features including LpMab-23 reactivity and five-year metastasisfree survival rate (MFS)
Clinical/pathological features

Number of patients

MFS (%)

P-value

Male

33

84.8

NS

Female

27

66.7

<66

33

87.9

>66

27

63.0

T1

29

79.3

T2

31

74.2

Grade 129

86.2

Grade 2, 3

31

67.7

YK-1, 2, 3

42

88.1

YK-4C

14

57.1

YK-4D

4

25.0

dull and negative

28

96.4

positive

32

59.4

GenderAge
P<0.05T-classification
NS

Histological grading
NS

Mode of invasion
P<0.01

LpMab-23 reactivity
P<0.01

NS, not significant. Ratio was rounded off to the 1st decimal place.
Table 3: Cox proportional hazard regression models
Clinical/pathological features

Hazard ratio

95% CI

P-value

Gender (female vs male)

2.33

0.78 to 6.95

0.13

Age (>66 vs <66)

3.54

1.11 to 11.32

0.03

T-classification (T1 vs T2)

1.23

0.43 to 3.55

0.70

Histological grading (Grade 1 vs Grade 2, 3)

2.57

0.81 to 8.21

0.11

Mode of invasion (YK-1, 2, 3 vs YK-4C vs YK-4D)

2.92

1.53 to 5.56

<0.01

LpMab-23 (dull/negative vs positive)

14.95

1.95 to 114.65<0.01

Univariate analysis

Multivariate analysis (age, mode of invasion, LpMab-23 reactivity)
Age (>66 vs <66)

2.47

0.76 to 8.07

0.13

Mode of invasion (YK-1, 2, 3 vs YK-4C vs YK-4D)

1.59

0.78 to 3.23

0.20

LpMab-23 reactivity (dull/negative vs positive)

9.33

1.11 to 78.51

0.04

Ratio was rounded off to the 2nd decimal place.

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prognosis because of developing late cervical lymph
nodes [38]. Much research has been attempting to identify
potential molecular and morphological predictors for latecervical lymph node metastasis, but so far none have madean impact on routine clinical procedures [39]. The YKclassification focused on the shape of tumor-cell cords at
the tumor-host interface has been used for pathologicalgrading of malignancies. It is well known that the mode ofinvasion classified by the YK classification is a potentially
important predictor of cervical lymph node metastasis
and patient outcomes in patients with oral squamouscell carcinomas [40, 41]. Importantly, by the mode ofinvasion, the number of LpMab-23-dull/negative cases
was significantly more numerous in the YK-1/YK-2/YK-3
group than in the YK-4C and YK-4D groups with highrisk for late cervical lymph node metastasis (Figure 1, 2,
Table 1). In the YK-4C and YK-4D group, the numberof LpMab-23-positive cases was larger than the numberof LpMab-23-dull/negative cases. Further, there weresignificantly more of LpMab-23-dull/negative cases inpatients without late cervical lymph node metastasis thanin the cases with metastasis (Figure 1, 2, Table 1). Active
lymphangiogenesis was more common among LpMab-23
positive cases with no reaction to D2-40 (Figure 2). These
differences suggest that LpMab-23-dull/negative findings
are linked to good prognostic factors and long lymphatic
metastasis-free interval for tongue cancer patients.
There were also a statistically significantly
differences in LpMab-23, as well as in age and modeof invasion for the five-year metastasis-free survival
rate (MFS) and the Kaplan–Meier curves (Table 2). The
Kaplan–Meier curves of MFS were significantly lower inthe patients with LpMab-23-positive than in the patientswith LpMab-23 dull/negative (Figure 3). The LpMab-23dull/negative cases showed the highest MFS in all of theclinical/pathological features tested and particularly, MFSof the cases with LpMab-23 positive and with YK-4C andYK-4D decreased to less than 60% in the first year (Figure3). These suggests that LpMab-23-positive patients may
be considered a high-risk group for late cervical lymph
node metastasis, like patients aged 66 years or older
and with YK-4C and YK-4D in the mode of invasion.

Figure 3: Five-year metastasis-free survival curves (MFS). The Kaplan–Meier curves of MFS were significantly lower in patients

aged 66 years or older (n=27) than in the patients with under 66 years of age (n=33)(p=0.02), lower in the patients with mode of invasion
YK-4C (n=14) and 4D (n=4) than in the patients with mode of invasion YK-1/2/3 (n=42)(p=0.001), lower in the patients with LpMab-23
positive (n=32) than in the patients with LpMab-23 negative (n=28)(p=0.001). Particularly, the survival rate of patients with LpMab-23
positive decreased to less than 60% in the first year during 60 months of follow-up (long-rank) as well as the patients with the mode ofinvasion YK-4C and 4D.
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showed the highest hazard ratio and statistical significanceas an independent predictor in the multivariate analysis
among age, mode of invasion, and LpMab-23 reactivity,
as well as in the univariate analysis among all of the allclinical and pathological characteristics tested (Table3). These results suggest that LpMab-23-positive may
be considered a very useful predictor of poor prognosis
for early stage tongue cancer. A limitation of this study
is the small number of patients with early stage tongue
cancer a result of the study being retrospective in a single
institution. Despite this, we still believe that it would be
useful to define the utility of the LpMab-23 antibody in theclinical field soonest possible. Further studies involving
advanced tongue cancer would be able to provide more
relevant information for establishing evaluation systemsof potential cervical lymph nodes.

sections were rinsed by 10 mM PBS and then immersed
for 10 minutes in methanol containing 3% hydrogen
peroxide, followed by incubation in 0.1% goat serum for
30 minutes at room temperature. The sections were thenincubated with a 1 μg/ml D2-40 (Agilent Technologies Inc.,
Santa Clara, CA, USA) and 20 μg/ml LpMab-23 which has
been established as a cancer specific mAb [26] diluted into
the blocking solution at 4°C overnight followed by signal
development processes using the EnVision Detection
System (Agilent Technologies, Inc., Santa Clara, CA,
USA). The sections were counterstained with Mayer’s
hematoxylin (Agilent Technologies). All procedures were
performed after washing with 10 mM PBS for 30 min
at room temperature. After the treatment with primary
antibodies the sections were washed three times in PBS for
10 min, immunostained for 0.5 hr at 20°C with 0.1 μg/ml
of peroxidase-conjugated goat anti-mouse and goat anti-rat
IgGs (Agilent Technologies), and visualized with a DAB
peroxide substrate kit (Agilent Technologies). Expressionof PDPN in lymphatic endothelial cells in the stroma served
as an internal positive control. The immunostained sections
were mounted in 50% polyvinylpyrrolidone solution andexamined by microscopy. The immunoreactivity of D2-40
and LpMab-23 to the cancer cells were categorized into twogroups: clearly positive and dull (seems to be weakly or
false-positive)/negative. The assessment was performed by
all co-authors.

PATIENTS AND METHODS
Subjects
This study was conducted with sixty patients withstage I and II tongue cancer who had undergone partial
glossectomy without elective neck dissection for the earlystage tongue cancer (aged 33-92 years; median age 66
years). Specimens were taken from tissue obtained at thesurgery. The clinical pathology parameters and the followup data were obtained from the clinical database, chart
review, and an interview. The extent of the tumor and thehistopathological grading were classified according to theseventh edition of the UICC/TNM Classification [42].
The mode of invasion was determined according to the
Yamamoto-Kohama (YK) classification for oral squamouscell carcinomas with the assessment of the invasion at
the tumor host interface corresponding to an infiltrative
growth pattern (INF) of digestive tract cancer [43, 44].
The study was approved by the institutional review boardof Sapporo Medical University School of Medicine, withwritten informed consent obtained from all patients.

Statistical analysis
The correlation between the immunoreactivity of
LpMab-23 and the clinical/pathological parameters wereanalyzed using the Pearson Chi-square test. Clinicaloutcomes (metastasis-free survival; MFS) were assessedby the Kaplan–Meier method and the differences between
the survival curves were analyzed using the log-rank
test. To evaluate the correlations between the survival
rate and clinical/pathological parameters, univariate andmultivariate regression analyses according to the Cox
proportional hazards regression models were used, and thehazard ratios with 95% CI and P-values were determined.
All tests were two-sided, and P-values below 0.05 wereconsidered statistically significant. Four co-authors
evaluated samples, independently. Ratios were rounded
off to the 1st (Table 1, 2) or 2nd decimal places (Table 3).

Immunostaining
The specimens were 2-μm thick paraffinembedded sections cut using a rotary microtome,
mounted on 3-aminopropyltriethoxysilane-coated slides,
deparaffinized in changes of xylene, rehydrated indecreasing concentrations of ethanol, and rinsed with
deionized distilled water for 10 min. The slides were
immersed in 10 mM sodium citrate buffer (pH 6.0) andboiled in a microwave oven for 10 min to break formalinformed protein cross-links masking the PDPN antigen.
Immunohistochemistry was performed using the avidinbiotin peroxidase complex technique. After the boiling, thewww.oncotarget.com

Author contributions
Conception and design: Miyazaki, Sawa.
Financial support: Miyazaki, Kato, Sawa.
Administrative support: Miyazaki, Kato, Sawa,

Hiratsuka.
Provision of study materials or patients: Miyazaki,
Nakai, Kaneko, Kato, Hiratsuka.
Collection and assembly of data: Miyazaki, Nakai.

21163

Oncotargetathological evaluation: Hirohashi, Miyazaki,
P
Nakai, Sawa.
Data analysis and interpretation: Miyazaki, Nakai,
Sonoda.
Manuscript writing: Miyazaki, Sawa
Final approval of manuscript: Sawa.

from embryonal carcinoma in testicular germ cell tumors.
Oncogene. 2004; 23:8552–56.
7. Ordóñez NG. D2-40 and podoplanin are highly specificand sensitive immunohistochemical markers of epithelioid
malignant mesothelioma. Hum Pathol. 2005; 36:372–80.
8. Roy S, Chu A, Trojanowski JQ, Zhang PJ. D2-40, anovel monoclonal antibody against the M2A antigen as amarker to distinguish hemangioblastomas from renal cell
carcinomas. Acta Neuropathol. 2005; 109:497–502.

ACKNOWLEDGMENTS
This work was supported in part by Grant-inAid for Scientific Research (C) 16K11729 (Miyazaki,A.), Grant-in-Aid for Scientific Research (B) 15H05060(Sawa, Y.), and Grant-in-Aid for Challenging Research(Exploratory) 17K19777 (Sawa, Y.) from the Japan Society
for the Promotion of Science, and from the Basic Scienceand Platform Technology Program for Innovative Biological
Medicine from the Japan Agency for Medical Research anddevelopment, AMED (Kato, Y.) and by the Platform Projectfor Supporting Drug Discovery and Life Science Research(Basis for Supporting Innovative Drug Discovery and Life
Science Research [BINDS]) from AMED (Kato, Y.).

9. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose
T, Matsutani M. Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol. 2006; 111:483–88.
10. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, Matsutani M.
Podoplanin expression in primary central nervous system germ
cell tumors: a useful histological marker for the diagnosis ofgerminoma. Acta Neuropathol. 2006; 111:563–68.
11. Yu H, Gibson JA, Pinkus GS, Hornick JL. Podoplanin (D240) is a novel marker for follicular dendritic cell tumors.
Am J Clin Pathol. 2007; 128:776–82.
12. Ishida M, Kushima R, Okabe H. Immunohistochemical
demonstration of D2-40 in basal cell carcinomas of the skin.
J Cutan Pathol. 2008; 35:926–30.

CONFLICTS OF INTEREST
The authors indicate no potential conflicts ofinterest.

13. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE,
Fukayama M. Podoplanin is regulated by AP-1 andpromotes platelet aggregation and cell migration in
osteosarcoma. Am J Pathol. 2011; 179:1041–49.

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