Part I: Tisssue placenta growth factor mRNA
2.1.1 Patients and oral cancer specimens
This study has been reviewed and approved by the Institutional Review Board and an informed
consent was obtained from each patient before collection of surgical samples. Sixty-three OSCC
patients (59 men and 4 women; mean age 56 years; range 33-81 years) were included in this study.
Paired surgical samples were collected from OSCC and adjacent normal-looking oral mucosal
(non-OSCC) tissues. All tissues were freshly embedded in OCT compound, snap frozen, and kept at
-80˚C until use. PlGF mRNA levels in both OSCC and non-OSCC tissues were measured by
quantitative RT-PCR. Diagnosis of OSCC was based on histological examination of hematoxylin
and eosin-stained tissue sections.
All OSCC patients received total surgical excision of their tumors plus either selective (39
patients) or radical neck dissection (24 patients) at the Department of Oral and Maxillofacial
Surgery, National Taiwan University Hospital, Taipei, Taiwan during the period from January 2008
to December 2011. Follow-up duration was defined as the period between the operation date and day of the last visit, according to the patient’s chart. If involved surgical margin, perineural invasion
or lymphovascular permeation of OSCC, or extracapsular spread of metastatic cervical lymph node
49
were detected histologically, postoperative concurrent chemoradiation therapies were also included
in the treatment protocol. In this study, 4 patients received adjuvant radiotherapy and another 33
patients received both radiotherapy and chemotherapy after surgery. None of the patients had
received any form of tumor-specific therapy before total surgical excision of the lesion. Of the 63
cases of OSCC, 23 were buccal mucosa, 19 tongue, 10 gingiva, 6 palate, and 5 floor of the mouth
cancers. Histological features of OSCC were further classified into three different types (well-,
moderately-, and poorly-differentiated OSCC). Of the 63 OSCC cases, there were 59 (94%) well-
and 4 (6%) moderately-differentiated OSCCs. Clinical staging and TNM status of OSCCs at initial
presentation of the tumor were determined by clinical palpation, head-and-neck magnetic resonance
imaging (MRI), chest X-ray, abdominal sonography, and the whole body bone scan according to the
guidelines from AJCC (Sobin et al., 2002).
2.1.2 Patients’ oral habits
Details of patients’ oral habits, including daily/weekly consumption of areca quid, cigarette,
and alcohol as well as the duration of these habits were recorded. OSCC patients were defined as
areca quid chewers when they chewed 2 or more areca quids daily for at least one year, as cigarette
smokers when they smoked every day for at least one year and consumed more than 50 packs of
cigarettes per year, and as alcohol drinkers when they drank more than four days and consumed
more than 20 g of pure alcohol per week for at least one year. According to these definitions, 56
50
(88%) patients were areca quid chewers, 56 (88%) were smokers, and 51 (81%) were drinkers.
Furthermore, all of our OSCC patients stopped chewing areca quids after surgery, some of them
stopped smoking completely after surgery, and some of them continued to smoke with a reduced
number of cigarettes (< 5 cigarettes per day) after surgery according to the inquisition from the
patients and their family members.
2.1.3 Quantitative real-time reverse transcription-polymerase chain reaction
Total cellular RNA was isolated using an RNA extraction kit (Qiagen Inc., Alameda, CA, USA)
from tissue homogenized with Trizol reagent (Invitrogen Inc., Carlsbad, CA, USA) as
recommended by the manufacturers. The mRNA expression levels after each treatment were
determined by quantitative RT-PCR using the TaqMan® Gene Expression Assays (Applied
Biosystem, Foster City, CA, USA) for PlGF (ID: Hs00182176_m1) and glyceraldehyde
3-phosphate dehydrogenase (GAPDH; ID: Hs99999905_m1) on an ABI Prism real-time PCR
system (Applied Biosystem) as previously described (Wei et al., 2005). Threshold cycle (CT) is the
fractional cycle number at which the fluorescence generated by cleavage of the probe exceeds a
fixed level above baseline. For a chosen threshold, a smaller starting copy number results in a
higher CT value. In this study, we chose GAPDH mRNA as an internal control. The relative amount
of tissue PlGF mRNA, standardized against the amount of GAPDH mRNA, was expressed as -∆CT
= – (PlGF CT – GAPDH CT). Then, the relative expression level of tissue PlGF mRNA in each
51
OSCC patients was expressed as -∆CT = – (OSCC CT – non-OSCC CT).
2.1.4 Statistical analysis
The mean PlGF mRNA levels between OSCC and non-OSCC tissues were compared by paired
t-test. The correlation between the PlGF mRNA levels in OSCC samples and clinicopathological parameters of OSCC patients was analyzed by Student’s t-test. Cumulative survival was analyzed
with the Kaplan-Meier product-limit method. The duration of recurrence-free survival was
measured from the beginning of surgery to the time of recurrence (complete) or the last follow-up
(censored). Comparison of cumulative survival between groups was performed using the log-rank
test with the Statistica program 7.0 (StatSoft Inc., Tulsa, OK, USA). Univariate and multivariate
survival analyses were performed with the Cox proportional hazard regression model to assess
additional prognostic values of the different variables using SAS 9.1 (SAS Institute Inc., Morrisville,
NC, USA). A P-value of less than 0.05 was considered statistically significant.
Part II: Serum placenta growth factor level
2.2.1 Patients and serum samples
This study has been reviewed and approved by the Institutional Review Board and an informed
consent was obtained from each patient before collection of serum samples. Serum samples were
collected from 72 patients (64 men and 8 women; mean age 54 years; range 28-77 years) with
52
OSCC before and 3 months after surgical removal of OSCCs and from 30 healthy control subjects
(26 men and 4 women; mean age 29 years, range 23-50 years) before extraction of impacted
mandibular third molars. Diagnosis of OSCC was based on histological examination of hematoxylin
and eosin-stained tissue sections. All OSCC patients received total surgical excision of their tumors
plus either selective (39 patients) or radical neck dissection (33 patients) at the Department of Oral
and Maxillofacial Surgery, National Taiwan University Hospital, Taipei, Taiwan during the period
from January 2007 to December 2010. If involved surgical margin, perineural invasion or
lymphovascular permeation of OSCC, or extracapsular spread of metastatic cervical lymph node
were detected histologically, postoperative concurrent chemoradiation therapies were also included
in the treatment protocol. In this study, 5 patients received adjuvant radiotherapy and another 34
patients received both radiotherapy and chemotherapy after surgery. None of the patients had
received any form of tumor-specific therapy before total surgical excision of the lesion. Of the 72
cases of OSCC, 30 were buccal mucosa, 16 tongue, 16 gingiva, 5 palate, and 5 floor of the mouth
cancers. Histological features of OSCC were further classified into three different types (well-,
moderately-, and poorly-differentiated OSCC). Of the 72 OSCC cases, there were 64 (89%) well-
and 8 (11%) moderately-differentiated OSCCs. Clinical staging and TNM status of OSCCs at initial
presentation of the tumor were determined by clinical palpation, head-and-neck magnetic resonance
imaging (MRI), chest X-ray, abdominal sonography, and the whole body bone scan according to the
guidelines from AJCC (Sobn et al., 2002).
53
2.2.2 Patients’ oral habits
Details of patients’ oral habits, including daily/weekly consumption of areca quid, cigarette,
and alcohol as well as the duration of these habits, were recorded. OSCC patients were defined as
areca quid chewers when they chewed 2 or more areca quids daily for at least one year, as cigarette
smokers when they smoked every day for at least one year and consumed more than 50 packs of
cigarettes per year, and as alcohol drinkers when they drank more than four days and consumed
more than 20 g of pure alcohol per week for at least one year. According to these definitions, 60
(83%) patients were areca quid chewers, 59 (82%) were smokers, and 58 (81%) were drinkers.
Furthermore, all of our OSCC patients stopped chewing areca quids after surgery, some of them
stopped smoking completely after surgery, and some of them continued to smoke with a reduced
number of cigarettes (< 5 cigarettes per day) after surgery according to the inquisition from the
patients and their family members. In addition, all the subjects in the control group were selected
according to strict criteria and none of them have areca quid chewing and cigarette smoking habits.
2.2.3 Enzyme-linked immunosorbent assay (ELISA)
Serum PlGF protein levels were measured by enzyme-linked immunosorbent assay (ELISA,
R&D Systems, Minneapolis, MN, USA) in triplicate according to the manufacturer’s protocol.
Serum concentrations of PlGF were expressed as pg/ml of protein.
54
2.2.4 Statistical analysis
The mean pre- and post-surgery serum PlGF protein levels of OSCC patients and mean serum PlGF
protein levels of normal control subjects were compared first by analysis of variance (ANOVA)
among groups and then by Student’s t-test or paired t-test between any two groups, where
appropriate. The correlation between the serum PlGF protein levels and clinicopathological parameters of OSCC patients was analyzed by Student’s t-test. Cumulative survival was analyzed
with the Kaplan-Meier product-limit method. The duration of recurrence-free survival was
measured from the beginning of surgery to the time of recurrence (complete) or the last follow-up
(censored). Comparison of cumulative survival between groups was performed using the log-rank
test with the Statisca program (StatSoft Inc., Tulsa, OK, USA). Univariate and multivariate survival
analyses were performed with the Cox proportional hazard regression model to assess additional
prognostic values of the different variables using SAS 9.1 (SAS Institute Inc., Morrisville, NC,
USA). A P-value of less than 0.05 was considered statistically significant.
55
III. Results
Part I: Increased placenta growth factor mRNA level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
3.1.1 PlGF mRNA levels in oral cancer tissues
The mean PlGF mRNA levels in the paired OSCC and non-OSCC tissues of 63 OSCC patients
are presented in Table 1.1. The lower CT value is interpreted as having higher initial copy numbers
of PlGF mRNA in tissues. We found that the mean PlGF mRNA CT valuewas significantly lower in
OSCC (7.2 ± 0.3) than in control counterpart of non-OSCC tissues (9.1 ± 0.6, P < 0.001) (Table
1.1).
3.1.2 Correlation between the PlGF mRNA levels in OSCC samples and clinicopathological
parameters of OSCC patients
In this study, the higher the -∆CT, the greater the copy number of PlGF mRNA in tissues.
Correlation between the mean PlGF mRNA -∆CT value in 63 paired surgical samples and
clinicopathological parameters of 63 OSCC patients is shown in Table 1.2. We found that the mean
PlGF mRNA -∆CT value was significantly higher in OSCC patients with larger tumor size (P =
56
0.030), positive lymph node metastasis (P = 0.003), more advanced clinical stages (P = 0.013), and
the presence of loco-regional recurrence (P = 0.039) (Table 1.2). No significant correlation was
found between the mean PlGF mRNA -∆CT value and patients’ age, gender, cancer location, and
histology of OSCC. The PlGF mRNA -∆CT value was also not associated with the areca quid
chewing, cigarette smoking, or alcohol drinking habit (data not shown).
3.1.3 Survival analyses
Univariate analysis performed by Cox proportional hazard regression model identified larger
tumor size (P = 0.027), positive lymph node metastasis (P = 0.003), advanced clinical stage (P =
0.032), and PlGF mRNA -∆CT value > 2 (P = 0.008, the median PlGF mRNA -∆CT value was 1.9)
as correlating with poorer survival of OSCC patients. Multivariate analyses with Cox regression
model further identified positive lymph node metastasis (P = 0.019) and PlGF mRNA -∆CT value >
2 (P = 0.016) as two independent unfavorable prognosis factors (Table 1.3). The Kaplan-Meier
curve showed that OSCC patients with a PlGF mRNA -∆CT value > 2 had a significantly poorer
cumulative recurrence-free survival than those with a PlGF mRNA -∆CT value ≦2 (P = 0.017,
log-rank test, Figure 1.1). In addition, the Kaplan-Meier curve showed that OSCC patients with
larger tumor size, nodal metastasis, and advanced clinical stage also had a significantly poorer
cumulative recurrence-free survival (Figures 1.2~1.4).
57
Part II: Increased serum placenta growth factor level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
3.2.1 Serum PlGF levels in normal and cancer patients
The mean serum PlGF protein levels in 30 normal control subjects and in 72 OSCC patients
before and 3 months after surgical excision of the OSCC are presented in Table 2.1. The mean
serum PlGF protein level was significantly higher in pre-surgery OSCC patients (19.1 ± 10.7 pg/ml)
than in normal control individuals (10.1 ± 4.5 pg/ml, P < 0.001). Three months after total surgical
removal of the tumors, the mean serum PlGF protein level dropped to 11.0 ± 6.6 pg/ml which was
significantly lower than the mean pre-surgery serum PlGF protein level (P < 0.001) but had no
significant difference to the normal control serum PlGF protein level (P = 0.519) (Table 2.1).
3.2.2 Correlation between the serum PlGF protein levels in pre-surgery and clinicopathological parameters of OSCC patients
Correlation between the mean serum PlGF protein levels in pre-surgery OSCC patients and
clinicopathological parameters of OSCC patients is shown in Table 2.2. We found that the mean
serum PlGF protein level was significantly higher in pre-surgery OSCC patients with larger tumor
size (P = 0.015), positive lymph node metastasis (P = 0.001), more advanced clinical stages (P =
0.002), and loco-regional recurrence (P = 0.037). No significant correlation was found between the
58
mean serum PlGF protein level and patients’ age, gender, cancer location, and histology of OSCC.
The serum PlGF protein level was also not associated with the areca quid chewing, cigarette
smoking, or alcohol drinking habit (Table 2.2).
3.2.3 Survival analysis
Univariate analysis performed by Cox proportional hazard regression model identified larger
tumor size (P = 0.029), positive lymph node metastasis (P = 0.004), advanced clinical stage (P =
0.031), and serum PlGF protein level > 19.1 pg/ml (P = 0.007) as correlating with poorer survival
of OSCC patients. Multivariate analysis with Cox regression model further identified positive
lymph node metastasis (P = 0.018) and high serum PlGF protein level (> 19.1 pg/ml, P = 0.014) as
independent unfavorable prognosis factors (Table 2.3). The Kaplan-Meier curve showed that OSCC
patients with a serum PlGF protein level > 19.1 pg/ml had a significantly poorer cumulative
recurrence-free survival than those with a serum PlGF protein levels ≦ 19.1 pg/ml (P = 0.009,
log-rank test, Figure 2.1). In addition, the Kaplan-Meier curve showed that OSCC patients with
larger tumor size, positive nodal metastasis, more advanced clinical stage had a significantly poorer
cumulative recurrence-free survival (Figures 2.2~2.4).
To further investigate whether the serum PlGF protein level could be a biomarker for
prediction of the recurrence of OSCC, we chose the serum PlGF protein level greater than 19.1
pg/ml (which was equal to the mean normal control value plus 2 standard deviations) as a cutoff
59
point for identification of the presence of cancer recurrence. By this definition, the sensitivity,
specificity, and positive predictive value for tumor recurrence was 80%, 56%, ad 78%, respectively.
This work was published by Oral Oncology and the article is included as appendix B1. Other
publication related to this dissertation is included as appendix B2.
60
IV. Discussion
Part I: Increased placenta growth factor mRNA level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
In this study, we measured the PlGF mRNA level in 63 paired OSCC and non-OSCC tissue samples.
The relatively lower mean PlGF mRNA CT value in OSCC than in non-OSCC tissues indicated an
increased PlGF mRNA expression in OSCC tissues than in non-OSCC tissues. The finding of PlGF
mRNA expression in normal-looking oral mucosal (non-OSCC) tissue adjacent to OSCC also
suggests that the PlGF mRNA expression is an early event in oral carcinogenesis. Takahashi et al.
(1994) found a significantly higher PlGF mRNA expression in hypervascular renal cell carcinoma
tissues than in adjacent normal kidney tissues. Chen et al. (2004) reported a significantly higher
PlGF protein level in gastric cancer tissues than in the corresponding non-cancerous mucosal tissues.
Parr et al. (2005) also demonstrated that the PlGF protein expression is dramatically increased in
breast cancer tissues compared with normal breast tissues. Actually, overexpression of PlGF mRNA
or protein has been demonstrated in a variety of human carcinomas including breast (Parr et al.,
2005), gastric (Chen et al., 2004),lung (Zhang et al., 2005),colorectal (Wei et al., 2005), renal cell
(Takahashi et al., 1994),uterine cervical (Kodama et al., 1997), and hepatocellular carcinomas (Ho
et al., 2007).
61
A significant association of higher PlGF mRNA and protein levels with regional nodal
metastasis of OSCC was shown in the present and our previous studies (Cheng et al., 2010). The
similar findings were also demonstrated in human breast cancer (Parr et al., 2005). The reasons why
PlGF increased cancer metastasis could be explained as follows. First, angiogenesis is a pivotal
factor for tumor growth and metastasis (Carmeliet et al., 2000). PlGF can promote vessel growth
and maturation directly by affecting endothelial and mural cells, as well as indirectly by recruiting
pro-angiogenic cell types. PlGF can also increase the expression of VEGFA which is a potent
angiogenic factor (Fischer et al., 2008). Furthermore, PlGF displaces VEGFA from FLT1, which
liberates VEGFA and allows it to activate FLK1 (also known as VEGFR2) and augment
VEGF-driven angiogenesis (Park et al., 1994). PlGF also activates and attracts macrophages that
release angiogenic and lymphangiogenic molecules (Selvaraj et al., 2003). In addition, PlGF can
promote tumor angiogenesis, lymphangiogenesis, and the formation of the premetastatic niche
(Fischer et al., 2008). Second, PlGF promotes the growth, survival and migration of metastatic
tumor cells. Third, PlGF enhances the expression of matrix metalloproteinase 9 (MMP9) which
facilitates the cancer cell invasion and metastasis (Fischer et al., 2008). Fourth, PlGF directly
regulates the motility of human non-small cell lung cancer cells (Chen et al., 2008) and also
stimulates in vitro motility and invasion of the human breast tumor cell lines (Taylor et al., 2007).
Moreover, an antagonistic PlGF/FLT1 peptide can inhibit the growth and metastasis of human
breast cancer xenografts (Taylor et al., 2007). Fifth, PlGF inhibits the differentiation of dendritic
62
cells and in turn suppresses the antigen recognition and anti-tumor immune defense (Fischer et al.,
2008). In summary, PlGF may promote lymph node metastases through multiple mechanisms such
as an increase in tumor angiogenesis and lymphangiogenesis, an increase in tumor cell survival,
motility, migration and invasion, an elevated expression of MMP9, and an inhibition of the immune
surveillance by dendritic cells.
This study showed a significant association of PlGF mRNA overexpression in OSCCs with
larger tumor size and positive lymph node metastasis. Because higher T and N statuses always
result in more advanced clinical stages of OSCC, it is not difficult to explain why OSCCs with the
higher expression of PlGF mRNA are prone to have the more advanced clinical stages. Indeed, high
expression of PlGF mRNA or protein is significantly associated with an advanced clinical stage of
gastric (Chen et al., 2004), lung (Zhang et al., 2005),and colorectal cancers (Wei et al., 2005).
A significant correlation between the higher PlGF mRNA expression (higher -∆CT value) in
OSCCs and the poorer recurrence-free survival in OSCC patients was demonstrated in this study. In
addition, PlGF mRNA -∆CT value > 2 was identified as an independent unfavorable prognosis
factor by multivariate analyses with Cox regression model. Previous study also showed a significant
association of higher level of PlGF mRNA with a shorter survival in patients with colorectal
carcinoma (Wei et al., 2005). In addition, an increased level of PlGF protein is significantly related
to poor prognosis in patients with breast (Parr et al., 2005) or gastric carcinomas (Chen et al., 2004).
Furthermore, high PlGF protein or mRNA level is significantly associated with recurrence of breast
63
cancer (Parr et al., 2005).PlGF protein or mRNA level can also predict the early recurrence after
radical resection of hepatocellular carcinoma (Ho et al., 2007).The above findings indicate that
PlGF protein or mRNA level may be an important prognostic indicator for patients with certain
types of human carcinomas including OSCC.
Our results showed a significant increase in the PlGF mRNA expression in OSCC tissues
compared to that in non-OSCC tissues. We also found that the PlGF mRNA level in OSCC samples
was significantly correlated with tumor size, N status, clinical staging, and loco-regional recurrence
of OSCCs. Moreover, OSCC patients with a higher PlGF mRNA expression had a poorer
cumulative recurrence-free survival than those with lower PlGF mRNA levels. PlGF mRNA -∆CT
value > 2 was also identified as an independent unfavorable prognosis factors for OSCCs by
multivariate analyses. These results indicate that the PlGF mRNA level may be a biomarker for
prediction of the progression of OSCCs and the prognosis of OSCC patients in Taiwan.
Part II: Increased serum placenta growth factor level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
We recently found that the higher PlGF protein expression in OSCC tissues is significantly
associated with the more advanced tumor progression and shorter patient survival. Because PlGF is
64
a soluble peptide, its serum level may indicate the angiogenic activity of the tumor and may serve as
a predictive biomarker for cancer progression, recurrence and prognosis. In this study, we measured
the pre-surgery and post-surgery serum PlGF protein levels in 72 OSCC patients and found that the
higher serum PlGF protein level could return to near normal control value 3 months after total
excision of the cancer. The prominent rise of serum PlGF protein value in pre-surgery OSCC
patients and the significant fall of serum PlGF protein value after surgical removal of tumor tissue
indicate that the OSCC tissue is the major source of PlGF proteins. Our recent study showed the
expression of PlGF in OSCC tumor cells with the mean labeling index of 51 ± 19 (Cheng et al.,
2010).Thus, at least part of the cancer cell-produced PlGF proteins may secrete into stromal tissue,
diffuse into adjacent vascular or lymphatic vessels, and drain into systemic circulation, finally
diffuse into adjacent vascular or lymphatic vessels, and drain into systemic circulation, finally