Download Prognostic markers in stage I oral cavity squamous cell carcinoma

The Laryngoscope
C 2013 The American Laryngological,
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Rhinological and Otological Society, Inc.
Prognostic Markers in Stage I Oral Cavity Squamous Cell Carcinoma
Johannes Dunkel, MD; Samuli Vaittinen, PhD, MD; Reidar Grenman, PhD, MD; Ilpo Kinnunen, PhD, MD;
Heikki Irjala, PhD, MD
Objectives/Hypothesis: Early-stage oral squamous cell carcinoma (OSCC) treatment is based on anatomic location, clinical TNM staging, and histological grade. It is a heterogeneous disease group. Classification of patients with OSCC by immunohistochemical analysis of established oncoproteins and evaluate disease course was our primary objective. Characterization of
stage I OSCC patients in Southwest Finland was our secondary objective.
Study Design: Immunohistochemical analysis of tumor specimens and retrospective analysis of patient data of the
patient treated in Turku University Hospital for T1N0M0 OSCC during the years 2000-2004.
Methods: Paraffin-embedded tumor specimens from 35 OSCC patients were collected and analyzed for HIF-1a, CD44,
p16, Ki67, and podoplanin by immunohistochemistry and correlated with clinical findings.
Results: Tumoral CD44 and HIF1-a expression levels, in combination, predicted 5-year disease-free survival. Reduced
expression of CD44 and elevated expression of HIF1-a is associated with the lowest probability of disease-free survival compared to the population as a whole (P < .001 in Kaplan-Meier analysis). Patients with grade I tumors demonstrated improved
disease-specific survival compared to those with grade II tumors (P ¼ .027). No association was seen between p16 expression, Ki67 labeling index, or podoplanin expression and prognosis in our 35 specimens.
Conclusions: HIF-1a and CD44 immunohistochemical detection could potentially serve as a prognostic tool in therapy
selection for early-stage OSCC.
Key Words: HIF-1a, podoplanin, p16, CD44, immunohistochemistry, head and neck cancer, head and neck squamous
cell carcinoma, biomarker, prognosis.
Level of Evidence: 2b.
Laryngoscope, 123:2435–2441, 2013
INTRODUCTION
Approximately 275,000 new patients are diagnosed
with oral cavity cancer yearly, and it is estimated to
cause 1.7% of all cancer-related deaths worldwide.1,2 The
clinical behavior of early-stage oral squamous cell carcinoma (OSCC) is highly variable. Treatment of early
stage OSCC has varied from combined surgery with or
without neck dissection to different combinations of
radiotherapy. Local surgery, usually laser assisted, combined with sentinel lymph node biopsy or selective neck
dissection is the modality currently used by many centers. However, when our samples were collected,
sentinel lymph node biopsies had yet to become general
practice. A recent Cochrane review on surgical treatFrom the Medicity Research Laboratory (J.D., H.I.), Department of
Pathology (S.V.) and Turku Doctoral Programme of Biomedical Sciences
(J.D.), University of Turku; and the Department of Pathology (S.V.) and
Department of Otorhinolaryngology–Head and Neck Surgery (R.G., I.K.,
H.I.), Turku University Hospital, Turku, Finland.
Editor’s Note: This Manuscript was accepted for publication
October 22, 2012.
This work was supported by Turku Doctoral Programme of Biomedical
Sciences, The Finnish Medical Foundation, Turku University Foundation,
The Finnish Otolaryngology Foundation, Turku University Hospital, and
Kirsti and Tor Johansson Cancer and Heart Foundation.
The authors have no other funding, financial relationships, or conflicts of interest to disclose.
Send correspondence to Reidar Gr
enman, PhD, MD, Department
of Otorhinolaryngology, Turku University Hospital, Kiinamyllynkatu 4-8,
P.O. Box 52, 20521 Turku, Finland. E-mail: [email protected]
ment of oral and oropharyngeal cancer showed no
advantage in elective neck dissection in the clinically
negative neck compared to therapeutic neck dissection
when indicated.3 In general, local surgery is a sufficient
treatment, but occasionally the disease behaves aggressively and currently no reliable markers predicting the
course of the disease exist.
Implementation of prognostic biomarkers associated
with head and neck squamous cell carcinoma (HNSCC)
is needed. The TNM classification system is the most
used and reliable factor predicting prognosis in HNSCC.
Tumor grade may not hold prognostic value according to
previous studies.4 Several biomarkers have been studied,
but none are routinely used in clinical practice.
Candidate biomarkers exist. Ki67 is a proliferation
marker expressed by cells that have entered the cell
cycle. It is widely used in breast cancer, but its prognostic role in HNSCC has remained controversial.5,6
Hypoxia-related markers, such as HIF-1a, have been
investigated, but their roles in the prognoses of HNSCC
remain disputable.7 The hyaluronic acid receptor, CD44,
is a cell adhesion and homing receptor known to play
multiple roles in cancer development.8 Podoplanin is a
lymphatic endothelial marker also expressed by the basal membrane of the epithelium. It has been linked to
poor outcome in HNSCC.9 The cell cycle progression inhibitor, p16, is a protein indicating human papilloma
virus (HPV) infection in HNSCC.10,11
DOI: 10.1002/lary.23888
Laryngoscope 123: October 2013
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
2435
In Finland, OSCC is often diagnosed relatively
early due to a comprehensive local basic health care system.12 In the absence of prognostic biomarkers, patients
in most centers undergo diagnostic imaging and surgical
treatment, including a sentinel lymph node biopsy. To
investigate efficient methods of predicting disease
course, we screened candidate prognostic markers by
immunohistochemistry in OSCC biopsy samples. Additionally, we wanted to elucidate the incidence and
natural course of stage I oral carcinoma in patients
treated with only local surgery in an indiscriminate population of 700,000 in Southwestern Finland.
MATERIALS AND METHODS
Patients
This population-based study was conducted in the area of
Turku University Hospital with a population base of approximately 700,000. OSCC samples were collected in the Department
of Otorhinolaryngology–Head and Neck Surgery at Turku University Hospital during the years 2000 to 2004. Diagnoses were
confirmed from hematoxylin and eosin-stained sections by the
same experienced pathologist at Turku University Hospital.
TNM status was based on clinical examination, including panendoscopy and a contrast-enhanced computed tomography scan of
the head and neck. All samples were collected prior to treatment,
and the primary treatment consisted exclusively of surgical
resection of the tumor. Clinical outcome was retrospectively
traced from the hospital’s records. Local recurrent disease, cervical lymph node metastasis, distant metastasis, and diseaserelated death were considered as end points.
Antibodies
Monoclonal primary antibodies used in this study were
Hermes-3 for CD4413 at 0.20 lg/mL, D2-40 for podoplanin
(Covance, Princeton, NJ) 1:100; Ki-67 MIB-1 clone (Dako,
Glostrup, Denmark) 1:25; HIF-1a (Thermo Fisher Scientific,
Waltham, MA), 1:100; p16 (BD Pharmingen, Franklin Lakes,
NJ), 1:25; and 3G614, an antibody targeting a chicken T-cell
antigen, serving as a negative control. All of the antibodies
were diluted in phosphate-buffered saline (PBS) at the concentration indicated above, and they were of the mouse antihuman immunoglobulin G (IgG) subtype.
Microscopic Evaluation
D2-40 staining was graded as follows: grade 1, positive
reaction in the mucosal epithelial cells limited to only basal
cells; grade 2, positive in the basal and upper cell layers, but
lower than that in the fourth layer; grade 3: positive from the
basal to the fifth layer and beyond. Intra- and peritumoral D240þ vessels were counted with a 0.0625-mm2 grid under a magnification of 400. The CD44 staining was graded as follows:
grade 1, <10% of the tumor cells stained positively; grade 2,
10% to 50% of the cells stained positively; grade 3, >50% of the
cells stained positively. The Ki67 labeling index was evaluated
by counting the percentage of positive cells in five different
locations within the tumor. Both HIF-1a staining and p16 staining were evaluated as low, moderate, or strong in the area of
the highest grade within each tumor.
All of the slides were evaluated by a single blind approach
by two independent observers (J.D., H.I.) under an Olympus BX40 (Olympus, Tokyo, Japan) microscope. In cases where the
examiners’ results differed, the samples were re-examined until
consensus was reached.
Statistical Analysis
Relationships among clinicopathological variables, immunohistochemical staining patterns, and prognoses were
evaluated. To study the combined effect of CD44 and HIF-1a,
CD44 expression of above 50% was considered as high and that
of below 50% as low. Moderate and strong HIF-1a staining patterns were both considered as strong. These two variables were
then cross-tabulated to divide patients into three groups (CD44
low/HIF-1a high; CD44 low/HIF-1a low or CD44 high/HIF-1a
high; and CD44 high/HIF-1a low).
Kaplan-Meier survival curves were constructed to compare
both disease-specific 5-year survival as well as 5-year disease-free
survival, where disease-related death was used as an end point in
overall survival and recurrent disease or metastasis as an end
point in disease-free survival; significance levels were calculated
with the log-rank test. Correlation coefficients were determined by
Spearman’s rho. Incidences of end points among groups were
cross-tabulated. Significance values were calculated with the
Mann-Whitney U test or Fisher exact test where applicable.
IBM SPSS Statistics version 19.0 (IBM, Armonk, NY) was
used for all statistical analyses.
RESULTS
Patient Data
Immunohistochemistry
Samples were fixed in formalin, embedded in paraffin, and
cut into 5 lm sections. Deparaffinization was performed with
xylene and rehydration with descending alcohol. Boiling in 0.01%
citric acid at a pH of 8.0 in a microwave oven for 6 minutes was
done for antigen retrieval, and samples were cooled down. Hydrogen peroxide (0.3%) in PBS was used to block endogenous
peroxidase activity. Samples were washed, and unspecific binding
was blocked with 1.5% normal serum in PBS for 60 minutes. Primary antibody was added to the samples and incubated at room
temperature for 60 minutes. Slides were washed in PBS, and secondary antibody (Elite anti-mouse IgG ABC kit; Vector
Laboratories, Burlingame, CA) was used according to the manufacturer’s recommendations. Diaminobenzidine was used for
visualization for 3 minutes at room temperature, and Mayer’s hematoxylin was used for background staining.
Laryngoscope 123: October 2013
2436
The material consists of 44 clinical T1N0M0 previously untreated OSCCs diagnosed during the selected 5year period of 2000 to 2004. Sixteen (36%) of the
patients were males. The ages ranged from 34 to 87
years, with a mean age of 65 years and median age of 63
years. Eight patients were smokers, six had smoked earlier, and 30 had never smoked. Thirty-four of the tumors
were located in the mobile tongue. Other sites were floor
of the mouth (n ¼ 6), mandibular gingiva (n ¼ 3), and
maxillary gingiva (n ¼ 1). Six were excluded from immunohistochemical analyses because we were not able to
obtain the primary surgical sample and three because
the samples available were deemed insufficient. Thus,
35 samples of the 44 were immunohistochemically
analyzed.
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
had no statistical significance in clinical outcome (Table
1) or immunohistochemical staining patterns (Table 2).
Eleven patients had a normal tissue margin of <10 mm
in the primary surgical specimen after primary treatment. A close follow-up only was chosen for eight of
these patients, mostly due to other severe diseases
affecting the general health of the patient, two were
reoperated, and one received postoperative radiotherapy.
Of these 11 patients, two were diagnosed with a locally
recurrent disease, two with local recurrences and neck
metastases, one metastatic disease, and one patient had
a second primary tumor 71 months postoperatively. The
lack of sufficient margins had no impact on disease-specific or disease-free survival in this patient material.
Follow-Up of the Patients
In this retrospective study, 11 (25%) patients
relapsed and six (14%) died of the disease within the follow-up time of 5 years (Fig. 1A). The mean follow-up
time until recurrence was 14 months after primary
treatment. Local recurrence occurred in eight patients
(18%), five of whom had additional metastases (supplementary data). Exclusively local diseases were treated
with surgery alone or in combination with radiotherapy;
two patients are alive and one died of a disease-related
cause. Three patients with metastases, in addition to local
recurrence, were reoperated, and two patients only
received palliative treatment; all of these patients died of a
disease-related cause. Three patients had neck metastases,
one of whom had additional distant metastases during the
follow-up. One of these was reoperated successfully, one
died despite surgical treatment and radiotherapy, and one
received only palliative treatment. Four patients had a second primary disease more than 5 years after primary
treatment. Three of these patients with second primary
carcinomas were operated, one of whom received additional
radiotherapy, and one received radiotherapy alone. The 5year disease-specific survival for all patients was 86%, and
5-year disease-free survival was 75%.
Fig. 1. Kaplan-Meier curves for disease-free survival during the 5year follow-up. (A) All patients pooled. (B) Patients grouped by
CD44 and HIF1-a status.
Results After Primary Surgery
Local surgery was the selected curative treatment
modality for all patients in this patient population. Histologically, the tumors were of squamous cell origin and
the mean size was 15 mm (4 mm–35 mm). Although
clinically T1, the histological examination revealed five
of the tumors to be over 20 millimeters in size (2035mm), indicating a pT2 disease. The tumor size had no
prognostic significance. Twenty-nine tumors were grade
I, 10 were grade II, and five were not originally classified. Higher tumor grade predicted poorer diseasespecific survival (P ¼ .027), and a strong tendency was
seen in disease-free survival (P ¼ .059). Smoking history
Laryngoscope 123: October 2013
Role of Human Papillomavirus in the
Pathogenesis of OSCC
Two (5.7%) of the 35 tumors analyzed stained positive for p16 indicating a human papillomavirus (HPV)
infection in the tumor (Fig. 2A). Expression of p16 had
no impact on disease-specific survival in this limited material of 35 samples.
Course of the Disease in Relation to Podoplanin
Expression and Ki67 Labeling Index
Podoplanin staining was analyzed on the basal cell
layer of the tumor. Podoplanin-positive lymphatic vessels
were counted both intratumorally and in the peritumoral
area (Fig. 2B). None of these correlated with the course of
the disease (Table 1). The proliferation marker, Ki67, was
stained, and the proportion of the positive cells in the tumor was counted (Fig. 2C). The Ki67 labeling index did
not correlate with the course of the disease (Table 1).
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
2437
Fig. 2. Examples of images of the immunostained tumor samples for all markers. (A) p16 staining. (B) Podoplanin staining. (C) Ki67 staining.
(D) CD44 staining, low expression. (E) CD44 staining, high expression. (F) HIF1-a staining, low expression. (G) HIF1-a staining, high expression. (H) Negative control.
Laryngoscope 123: October 2013
2438
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
TABLE 1.
lll.
Disease-related death within follow-up
Yes
No
Local recurrence or metastasis within follow-up
Yes
No
p-value
Yes
No
p-value
1
5
7
25
n.s.
1
9
7
21
n.s.
Earlier
0
6
1
5
Male
Female
3
3
13
25
n.s.
3
8
13
20
1
1
28
<0.001*
5
24
<0.05*
2
Yes
4
1
6
1
<0.03**
n.s.
5
1
5
1
n.s.**
n.s.
No
29
4
9
24
<25%
25%
2
4
19
13
n.s.
4
7
17
10
n.s.
<10%
0
3
n.s.
2
1
n.s.
10-50%
>50%
4
1
12
15
6
2
10
14
Low
1
18
2
17
<0.02*
Intermediate
High
2
2
6
6
5
3
3
5
<0.03**
Low
1
4
1
4
n.s.
Intermediate
High
3
1
17
9
6
3
14
7
Neither
0
10
<0.004*
0
10
<0.002*
One of two
Both
2
5
15
3
n.s.**
4
6
13
2
<0.001**
n.s.
n.s.
n.s.
p-values were considered statistically significant if p<0.05.
*p-values from cross tabulatation of events over five years, calculated with Fisher’s exact test.
**p-values from Kaplan-Meier curves, calculated with the log rank test.
Course of the Disease in Relation to HIF-1a and
CD44 Expression
The tumors were stained for the hypoxia-related
marker HIF-1a and a cell adhesion protein CD44 (Fig.
2D–G). Strong HIF-1a expression correlated negatively
with the 5-year disease-free survival (P ¼ .022) in the
Kaplan-Meier analysis. A negative trend was seen in the
connection between HIF-1a expression and 5-year disease-specific survival, but the differences among groups
failed to reach statistical significance (P ¼ .29). Positive
trends were seen between CD44 expression and both disease-specific and disease-free survival in Kaplan-Meier
analyses. The combination of high CD44 expression and
low HIF-1a expression had a strong positive correlation
with the 5-year disease-free survival in the KaplanMeier analysis (P < .001, Fig. 1B). None of the 10
patients with low HIF-1a- and high CD44-expressing
tumors had disease recurrence in contrast to 4/17 (24%)
for patients with one and 6/8 (75%) with both risk factors (P < .002, Table 1). The same trend appeared in
disease-specific survival.
DISCUSSION
Due to the difficulty in predicting the outcome of
oral cavity cancer, currently all patients receive surgical
Laryngoscope 123: October 2013
treatments combined with sentinel lymph node biopsy,
radiotherapy, or both. Our objective was to discover an
efficient method of selecting patients needing local
resection alone, through detection of biomarkers in the
cancer biopsy. Patients with low expression levels of
HIF-1a and high expression levels of CD44 all stayed in
remission, whereas those with high expression levels of
HIF-1a and low expression levels of CD44 had a 75% recurrence
rate
during
their
5-year
follow-up.
Independently, these markers are weak predictors of 5year disease-free survival. With the combination of these
markers we were able to reliably predict disease course
even in a reasonably small sample size of 35 OSCC samples. Our findings could suggest that using CD44 and
HIF-1a immunohistochemistry, in combination, would be
an effective way to select patients to be potentially
treated by one treatment modality alone to reduce
morbidity.
The extent of the disease and patient-related factors
are the key elements influencing the choice of treatment
modality in OSCC today. Surgery ranges from limited
surgery to extensive operations requiring major tissue
reconstruction. Radiotherapy, especially when combined
with chemotherapy, increases the risk of adverse events
(e.g., xerostomia, osteoradionecrosis, and difficulties in
swallowing and speech). Nevertheless, even T1 tumors,
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
2439
TABLE 2.
xxxx.
Smoking
Ki67 LI
<25%
25%
Yes
No
Earlier
p-value
2
5
15
10
4
2
n.s.
HPV infection
Yes
1
1
0
n.s.
Podoplanin expression at the basal membrane
No
Low
5
0
22
5
6
0
n.s.
Intermediate
3
12
5
High
Low
2
4
7
11
1
4
Intermediate
0
8
0
High
<10 %
1
1
5
2
2
0
10-50 %
2
11
3
>50 %
Neither
2
3
11
5
3
2
One of two
1
12
4
Both risk factors
1
7
0
HIF-1a grade
CD44 grade
Combination of high HIF-1a and low CD44 expression
n.s.
n.s.
n.s.
p-values were considered statistically significant if p<0.05.
p-values were calculated with Fisher’s exact test.
which have been resected with sufficient margins, may
recur. Thus, the means of selecting patients requiring
more than one treatment modality are needed. Our data
show that local resection alone can yield a 25% recurrence rate of small local tumors during the routine
follow-up time of 5 years. This is unacceptable by the
current high standards of oral cancer treatment. This
would obviously indicate that either all oral cancer
patients need extensive cancer therapy or that the
means of selecting patients for less extensive therapy
are needed.
The significance of CD44 in OSCC remains unclear.
Overexpression has previously been linked to poor outcome in squamous cell carcinomas of the oropharynx,
hypopharynx, and larynx, whereas evidence concerning
the oral cavity is slightly less assuring.15 Elevated
expression of CD44 has also been linked to poor outcome
in other malignancies such as hematological malignancies,16 colorectal cancer,17 and gastric cancer.18 It has
previously been speculated that CD44 can be used as a
cancer stem cell marker, meaning that tumor cells
expressing CD44 are capable of forming new tumors.19
Hypoxic tumors have poorer response to radiotherapy as reviewed by Silva et al.20 HIF-1a plays a role in
cancer development by activating the transcription of
several different genes under hypoxic conditions.21 Many
of these genes are crucial in angiogenesis, apoptosis, and
cell metabolism. The same genes have been shown to be
upregulated by hypoxia and HIF-1a in vitro.22
Interestingly, taking into account the combined
expression levels of CD44 and HIF-1a proved to be effective in predicting disease-free survival of OSCC patients.
This combination is effective because both molecules
participate in a broad spectrum of cellular functions.
Thus, evaluating both of their expression levels gives us
Laryngoscope 123: October 2013
2440
information of several aspects of the tumor
microenvironment.
There is emerging evidence that podoplanin could
serve as a potential prognostic marker in oral cancer as
podoplanin is overexpressed by metastatic tumors.9 The
tumors we analyzed were stage I tumors, which have
not metastasized. The density of lymphatic vasculature
has been shown to correlate positively with lymph node
metastases at the time of diagnosis of HNSCC.23 Our
study showed no significance between either podoplanin
expression of the basal membrane or lymphatic vasculature and clinical outcome in early stage OSCC.
The biomarker p16 is a valid biomarker in screening for a transcriptionally active HPV infection.24
Tumors of the oropharynx can be screened for HPV by
p16 immunohistochemistry to find patients with
improved survival, which is due to higher radiosensitivity.25,26 The role of HPV infection in oral cavity cancer
initiation and progression remains disputable, and we
were not able to address this issue with only two HPVpositive samples.
The Ki67 labeling index had no impact on prognosis
in these early-stage forms of disease, even though some
studies have indicated otherwise in HNSCC.27
Demographically, the early-stage patients analyzed
here did not have the typical profile or risk factors for
HNSCC: 64% were female and 68% were life-long nonsmokers. However, increasing numbers of nonsmoking
female patients with oral cancer have been previously
reported.28 The median age of 63 years corresponded
well with previously reported oral cancer patients.
Evaluating the prognoses of HNSCC patients is difficult. HNSCC may have different characteristics and
behave in different ways according to the site of origin.29–31 This suggests that OSCC is in fact a
Dunkel et al.: Prognostic Markers in Oral Cavity Cancer
heterogeneous group of malignancies occurring in the
same region. Our findings may simplify the categorization process.
CONCLUSION
The outcome data we collected of 44 patients show
that treating even low-stage OSCC aggressively is
required if no method of grouping the patients is used.
We think that developing an efficient clinical chemistry
method that assesses CD44 and HIF-1a coexpression
patterns in OSCC biopsies could provide us with a
method of predicting the disease course. This method
could serve as an interesting yet easy and cost-effective
way to select the patients for whom one treatment modality would be sufficient, and thereby reduce the
morbidity caused by the therapy.
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