Phosphorylated Signal Transducer and Activator of Transcription-1 Immunohistochemical Expression Is Associated With Improved Survival in Patients With Oral Squamous Cell Carcinoma

SURGICAL ONCOLOGY AND RECONSTRUCTION

Ma

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Phosphorylated Signal Transducerand Activator of Transcription-1

Immunohistochemical ExpressionIs Associated With Improved Survival

in Patients With Oral SquamousCell Carcinoma

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Elena Pappa, MD, DDS,* Nikolaos Nikitakis, MD, DDS, PhD,yDimitrios Vlachodimitropoulos, MD, PhD,z Dimitrios Avgoustidis, MD, DDS,x

Vlasios Oktseloglou, MD, DDS,k and

Nikolaos Papadogeorgakis, MD, DDS, MSc Lond, PhD{

Purpose: To estimate whether the immunohistochemical (IHC) expression patterns of the tumor suppres-

sor gene signal transducer andactivatorof transcription-1 (STAT1) and its activephosphorylated form(PSTAT1)

serve as potential prognostic and predictive markers in patients with oral squamous cell carcinoma (OSCC).

Materials and Methods: STAT1 and PSTAT1 protein expressions were examined immunohistochemi-

cally in OSCC tumor tissues and adjacent normal mucosa from 49 patients who underwent primary sur-

gery. The IHC scores were correlated with all available clinicopathologic parameters that were obtainedfrom a maximum of 7 years of follow-up, including survival and response to adjuvant therapy treatment.

Results: There was a shift toward lower percentages of cells with STAT1 (P < .014) and PSTAT1 (P < .001)detected in OSCC tumors compared with adjacent normal tissue sites. No association with patients’ clinico-

pathologic characteristicswas shown. However, for the group of patientswho received adjuvant chemother-

apy, increased PSTAT1 intensity of staining in OSCC tumors was strongly associated with better overall

survival (P = .008).

Conclusions: This is the first study to concurrently evaluate STAT1 and PSTAT1 IHC expression patterns

and their prognostic significance in patients with OSCC, highlighting the potential role of PSTAT1 as a bio-

marker in therapeutic decision making. Large prospective studies are needed to verify these findings.

� 2014 American Association of Oral and Maxillofacial Surgeons

J Oral Maxillofac Surg 72:211-221, 2014

nd Maxillofacial Surgeon, Department of Oral and

ial Surgery, ‘‘Evaggelismos’’ Hospital, University of

hens, Greece.

nt Professor, Department of Oral Pathology andMedicine,

ool, University of Athens, Athens, Greece.

nt Professor, Department of Pathology, University of

dical School, Athens, Greece.

nt in Oral and Maxillofacial Surgery, Department of Oral

lofacial Surgery, ‘‘Evaggelismos’’ Hospital, University of

hens, Greece.

nt in Oral and Maxillofacial Surgery, Department of Oral

lofacial Surgery, ‘‘Evaggelismos’’ Hospital, University of

hens, Greece.

{Professor, Department of Oral and Maxillofacial Surgery,

‘‘Evaggelismos’’ Hospital, University of Athens, Athens, Greece.

Address correspondence and reprint requests to Dr Pappa:

Department of Oral and Maxillofacial Surgery, ‘‘Evaggelismos’’ Hospi-

tal, University of Athens, 45-47 Ypsilantou Str, 103-76 Athens,

Greece; e-mail: [email protected]

Received May 16 2013

Accepted June 10 2013

� 2014 American Association of Oral and Maxillofacial Surgeons

0278-2391/13/00803-3$36.00/0

http://dx.doi.org/10.1016/j.joms.2013.06.198

211

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

mailto:[email protected]

http://dx.doi.org/10.1016/j.joms.2013.06.198

212 PSTAT1 AND ORAL SQUAMOUS CELL CARCINOMA

Head and neck squamous cell carcinoma (HNSCC) is

the most common type of malignancy of epithelial or-

igin in the upper aerodigestive truck, encompassing

mainly tumors of the oral cavity, pharynx, and larynx.1

Compared with other anatomic sites, HNSCC is the

sixth most common type of malignancy worldwide,

accounting for approximately 900,000 new cases an-

nually, 139,000 of them in Europe.2,3

Oral squamous cell carcinoma (OSCC), which ac-

counts for 90% of HNSCCs, has long been attributed to

risk factors such as tobacco and alcohol abuse.3-5 In the

past decade, certain types of human papilloma viruses

also have been shown to play an important role in the

pathogenesis of the disease, especially in nonsmokers

and nondrinkers.6,7 Furthermore, oncogenes, tumor

suppressor genes, and gene polymorphisms have beenimplicated in oral cancer.8 However, despite advance-

ments inmultimodality therapy, including surgery, radia-

tion, and chemotherapy, and a better understanding of

themolecular basis of thismalignancy, the 5-year relative

survival rate, which has reached 61.5% for patients of all

stages in the United States, remains largely unsatisfac-

tory.9 This fact underlies the necessity for developing

novel strategies for targetingmultiple molecular compo-nents for more effective prevention and treatment

of OSCC.

Signal transducers and activators of transcription

(STATs) constitute a family of 7 members: STAT1,

STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.10

These proteins remain latent in the cytoplasm in an un-

phosphorylated state (USTATs) until they are activated

through phosphorylation (PSTAT form) by various tyro-sine kinases (TKs), including janus kinases, receptor

TKs, and nonreceptor TKs, such as Rous sarcoma virus

TK and Abelson TK.11 This process is triggered by the

binding of certain extracellular ligands, such as cyto-

kines, growth factors, and hormones, to their receptors

in the cytoplasm.11,12 Activated STATs transduce signals

from the cell surface to the nucleus, where they bind to

DNA of certain genes, leading to their transactivation.12

These genes control proliferation, apoptosis, self-

renewal, and other critical cellular functions.12 There

is also some evidence that USTATs, such as USTAT1,

can bind to DNA.13 However, the binding affinity is

much less than that of PSTAT1.13

Under normal conditions, the activation of STATs is

tightly regulated. In cancer, by contrast, certain STAT

genes, such as STAT3, become activated constitutively,acting as oncogenes and thereby drive the malignant

phenotype of cancer cells.14-16 Conversely, activation

of other STAT genes, such as STAT1, may play a critical

role in tumor cell growth inhibition and in promoting

immunosurveillance, apoptosis, and cell-cycle arrest as

supportedby several studies indifferent typesof tumors,

such as breast cancer,melanoma, papillary thyroid carci-

noma, lymphoma and leukemia, colorectal cancer, and

HNSCC.12,17-23 Others have associated STAT1 with

a better response to adjuvant chemotherapy in

patients with oral cancer.24 These data supported the

idea that STAT1 might function as a tumor suppressor

and that different levels of its expression might affect

the efficiency of various treatment modalities.

The objective of the present studywas to assess pos-sible differences concerning the immunohistochemi-

cal (IHC) expression patterns of STAT1 and PSTAT1

in OSCC tumor cells and their adjacent normal tissues,

obtained from surgical specimens, and their possible

correlation with specific clinicopathologic parameters

of patients, including overall survival. The effect of ad-

juvant treatment modalities on survival in association

with STAT1 and PSTAT1 IHC expression also was in-vestigated.

Materials and Methods

STUDY SAMPLE

The study sample included 49 patients who under-

went primary surgery for OSCC from 2004 through

2007 at the Department of Oral and Maxillofacial Sur-

gery, ‘‘Evaggelismos’’ University Hospital (Athens,

Greece). The surgery involved tumor resection and

neck dissection unilaterally or bilaterally, depending onthe location. Inclusion criteria consisted of the availabil-

ity of follow-up data and tissue specimens from the local

pathology department at the onset of the study. Patients

with tumor recurrence or secondary primarieswere not

included in the study. The patients’ medical records

were reviewed retrospectively to obtain information re-

garding gender, age, site of tumor, histopathologic pa-

rameters, and surgical tumor stage based on the 7thedition of the solid tumors classification system of the

American Joint Committee on Cancer and the Interna-

tional Union for Cancer Control (pTNM), as well as pro-

spectively for adjuvant therapy modalities, disease

recurrence, and survival for a maximum period of 7

years, according to established cancer follow-up proto-

cols.25-27 Microscopic slides of all cases were reviewed

and relevant histopathologic parameters, such astumor grade and status of margins, and perineural and

perivascular invasions were reassessed.

The decision for adjuvant therapy was based on spe-

cific criteria that were individualized for each patient,

including performance status, patient compliance,

and clinicopathologic features. The study was ap-

proved by the ethics committee of ‘‘Evaggelismos’’

Hospital and the University of Athens and informedconsent was obtained from all participants. The study

was conducted in compliance with the ethical princi-

ples for medical research involving human subjects of

the Declaration of Helsinki.

PAPPA ET AL 213

IMMUNOHISTOCHEMISTRY

All tumor samples were fixed in formalin and em-

bedded in paraffin. Four-micron-thick sections were

prepared and fixed on adhesive glass slides. IHC anal-

ysis was performed in an automatic immunostaining

system (BOND-MAX, Leica Biosystems, Buffalo Grove,IL) using rapid polyclonal antibodies for STAT1 and

PSTAT1 (Cell Signaling Technology, Danvers, MA).

Slides were treated with 0.3% H2O2 in deionized water

for 30 minutes to neutralize the endogenous peroxide.

Primary antibody was applied in a concentration of

1:1,000 and incubated overnight at 4�C. Biotinylatedsecondary antibody was applied in a concentration

of 1:2,000 for 1 hour and then peroxide activity wasdeveloped using 3-30-diaminobenzidine. The slides

were counterstained with hematoxylin. Tumors with

known protein expression for STAT1 and PSTAT1

were used as positive controls, and sections with omis-

sion of primary antibody incubation were used as neg-

ative controls.

EVALUATION OF IHC STAINING

The most evenly and heavily labeled areas on the

slides were selected by inspection at low magnifica-

tion and then further analysis was carried out at higher

magnification (�400) to assess the nuclear-stainedepithelial cells. Evaluated parameters included 1) the

intensity of nuclear staining of STAT1 or PSTAT1 and

2) the percentage of epithelial cells with STAT1 or

PSTAT1 nuclear staining with respect to the total num-

ber of epithelial cells counted. The intensity of positive

staining was scored as weak (+), moderate (++), or in-

tense (+++), and the percentage of positively stained

cells was graded low (<35% of cells with STAT1 orPSTAT1 nuclear staining), medium (35-70%), or

high (>70%).

The evaluation involved a pair of areas originating

from cancerous and adjacent morphologically normal

tissue sites. Adjacent normal tissuewas part of themar-

gin of the resected specimen. Only microscopically

normal tissue (ie, without hyperplastic or dysplastic

changes of the epithelium and without inflammationof the connective tissue) was included. In all cases,

multiple slides of the resected specimens were re-

viewed and adjacent normal tissue was selected from

clear margins (ie, without close proximity to the neo-

plastic tissue).

All specimenswere evaluated separately andwithout

clinical information by 2 pathologists (N.N. and D.V.)

and the interobserver variability was very low (<5% ofcases). In cases in which there was initial disagreement,

stainswere re-evaluated by the same investigators using

a multiobserver microscope and discussed until con-

sensus was reached.

STATISTICAL ANALYSIS

The baseline characteristics of patients were sum-

marized as mean and standard deviation for continu-

ous variables and as absolute (number) and relative

(percentage) frequencies for categorical variables.

McNemar test was applied to assess differences inSTAT1 and PSTAT1 IHC parameters between OSCC tu-

mors and their adjacent normal tissues. Two-tailed

Fisher exact test was performed to evaluate possible

associations between STAT1 and PSTAT1 IHC parame-

ters and the various clinical and pathologic features of

patients. Comparisons between continuous variables

were based on 1-way analysis of variance.

Survival curves were calculated according to theKaplan-Meier method and their differences were evalu-

ated using the log-rank test. The significance of various

parameters for overall survival was analyzed by the Cox

proportional hazards model. Statistical analyses were

performed using SPSS 17.0 (SPSS, Inc, Chicago, IL),

with a P value less than .05 as the threshold of

significance.

Results

COHORT CHARACTERISTICS

The frequency distribution of patients’ clinical andpathologic characteristics is presented in Table 1. Of

the 49 patients, 33 were men (67.3%) and 16 were

women (32.7%). The mean age for the entire study co-

hort was 59.2 years (standard deviation, 15.0 yr;

range, 23 to 85 yr). Six patients (12.2%) were classi-

fied as stage I, 8 (16.3%) as stage II, 29 (59.2%) as stage

III, and 6 (12.2%) as stage IV, according to pTNM stag-

ing. Regarding the site of tumor, the tongue was in-volved in 26 cases (53.1%), the floor of the mouth in

4 (8.2%), the lower alveolus and gingiva in 8

(16.3%), the buccal mucosa in 8 (16.3%), and the ret-

romolar region in 3 cases (6.1%). The distribution of

patients according to the histologic grade showed

13 (26.5%), 24 (49.0%), and 12 (24.5%) cases with

well, moderately, and poorly differentiated tumors, re-

spectively. The vast majority of patients (44 of 49;89.8%) received adjuvant therapy. No statistically sig-

nificant difference was observed in the distribution

of the various clinical and pathologic parameters be-

tween men and women.

COMPARISONOF STAT1 AND PSTAT1 IHC STAININGBETWEEN OSCC AND ADJACENT NORMAL TISSUES

Intensity of Staining

STAT1 intensity of staining levels ranged from mod-

erate (++) to intense (+++) in OSCC tumors and their

adjacent normal tissues, whereas PSTAT1 staining in-

volved weak (+) and moderate levels (Table 2).

Table 1. CLINICAL AND PATHOLOGIC FEATURES OF PATIENTS

Characteristics Men Women P* Cohort

Total patients 33 (67.3%) 16 (32.7%) 49 (100.0%)

Age (yr) .157yMean � SD 57.1 � 14.9 63.6 � 14.7 59.2 � 15.0

$55 20 (60.6%) 12 (75.0%) .360 32 (65.3%)

<55 13 (39.4%) 4 (25.0%) 17 (34.7%)

Tumor site .447

Tongue 20 (60.6%) 6 (37.5%) 26 (53.1%)

Buccal 4 (12.1%) 4 (25.0%) 8 (16.3%)

Retromolar 2 (6.1%) 1 (6.3%) 3 (6.1%)

Lower alveolus and gingiva 4 (12.1%) 4 (25.0%) 8 (16.3%)

Floor of mouth 3 (9.1%) 1 (6.3%) 4 (8.2%)

Tumor differentiation .528

Poor 9 (27.3%) 3 (18.8%) 12 (24.5%)

Moderate 17 (51.5%) 7 (43.8%) 24 (49.0%)

Well 7 (21.2%) 6 (37.5%) 13 (26.5%)

Perineural invasion .733

Yes 8 (24.2%) 5 (31.3%) 13 (26.5%)

No 25 (75.8%) 11 (68.8%) 36 (73.5%)

Perivascular invasion .377

Yes 3 (9.1%) 3 (18.8%) 6 (12.2%)

No 30 (90.9%) 13 (81.3%) 43 (87.8%)

Dissection margins .377

Clear ($5 mm) 24 (72.7%) 15 (93.8%) 39 (79.6%)

Close (<5 mm) 6 (18.2%) 1 (6.3%) 7 (14.3%)

Involved 3 (9.1%) 0 (0.0%) 3 (6.1%)

pT .311

1 11 (33.3%) 3 (18.8%) 14 (28.6%)

2 13 (39.4%) 6 (37.5%) 19 (38.8%)

3 7 (21.2%) 3 (18.8%) 10 (20.4%)

4 2 (6.1%) 4 (25.0%) 6 (12.2%)

Tumor size (cm) .481yMean � SD 2.9 � 1.5 3.2 � 1.1 3.0 � 1.4

$2 21 (63.6%) 13 (81.3%) .324 34 (69.4%)

<2 12 (36.4%) 3 (18.8%) 15 (30.6%)

pN .459

0 11 (33.3%) 5 (31.3%) 16 (32.7%)

1 22 (66.7%) 10 (62.5%) 32 (65.3%)

2 0 (0.0%) 0 (0.0%) 0 (0.0%)

3 0 (0.0%) 1 (6.3%) 1 (2.0%)

Stage .116

I 6 (18.2%) 0 (0.0%) 6 (12.2%)

II 6 (18.2%) 2 (12.5%) 8 (16.3%)

III 19 (57.6%) 10 (62.5%) 29 (59.2%)

IV 2 (6.1%) 4 (25.0%) 6 (12.2%)

Disease recurrence .364

Yes 15 (45.5%) 10 (62.5%) 25 (51.0%)

No 18 (54.5%) 6 (37.5%) 24 (49.0%)

Adjuvant therapy .696

Radiation therapy only 14 (42.4%) 10 (62.5%) 24 (49.0%)

Chemotherapy only 1 (3.0%) 0 (0.0%) 1 (2.0%)

Chemotherapy and radiation therapy 14 (42.4%) 5 (31.3%) 19 (38.8%)

None 4 (12.1%) 1 (6.3%) 5 (10.2%)

Abbreviation: SD, standard deviation.* Two-tailed Fisher exact test.y One-way analysis of variance.

Pappa et al. PSTAT1 and Oral Squamous Cell Carcinoma. J Oral Maxillofac Surg 2014.


Table 2. COMPARISON OF FREQUENCY DISTRIBUTION OF STAT1 AND PSTAT1 IHC PARAMETERS BETWEEN OSCCTUMORS AND THEIR ADJACENT NORMAL TISSUES

IHC Parameters OSCC Tumors (n = 49) P*

STAT1 Adjacent normal tissues

Intensity of staining intense moderate .092

Intense 4 (8.2%) 3 (6.1%)

Moderate 10 (20.4%) 32 (65.3%)

Percentage of cells high medium .014

High 7 (14.3%) 19 (38.8%)

Medium 3 (6.1%) 20 (40.8%)

PSTAT1 Adjacent normal tissues

Intensity of staining weak moderate .180

Weak 38 (77.6%) 2 (4.1%)

Moderate 7 (14.3%) 2 (4.1%)

Percentage of cells low high or medium <.001

Low 9 (18.4%) 2 (4.1%)

High or medium 30 (61.2%) 8 (16.3%)

Abbreviations: IHC, immunohistochemical; OSCC, oral squamous cell carcinoma; PSTAT1, phosphorylated signal transducerand activator of transcription-1; STAT1, signal transducer and activator of transcription-1.* McNemar test.


PAPPA ET AL 215

Differences concerning STAT1 intensity of staining be-tween OSCC and adjacent normal tissue sites were

noted in 26.5% of patients (13 cases), leading to

a 14.3% increment in the frequency distribution of in-

tense STAT1 staining in OSCC. Conversely, PSTAT1 in-

tensity of staining differed in only 18.4% of patients (9

cases), involving a 10.2% increment in the frequency

distribution of weak as opposed to moderate PSTAT1

staining in OSCC tissues. However, none of these dif-ferences reached a statistically significant

level (Table 2).

Percentages of Cells

The percentages of cells with STAT1 staining in

OSCC and the adjacent normal tissues ranged fromme-

dium to high, whereas the percentages of cells with

PSTAT1 staining were extended into 3 categories(low, medium, and high). In almost half of patients

(44.9%; 22 of 49 cases), the percentages of cells with

STAT1 staining differed between OSCC tumors and

their adjacent normal tissues. This reflects a decrease

by 32.7% in the frequency of high percentages of cells

with STAT1 staining in OSCC tissues and a shift toward

medium percentages that reached a statistically signif-

icant level (P = .014; Table 2). Analogous differencesconcerning the percentages of cells with PSTAT1 stain-

ing among the aforementioned sites were seen in the

vast majority of patients (65.3%; 32 of 49 cases), lead-

ing to a decrease by 57.1% in the frequency of high and

medium percentages of cells with PSTAT1 staining and

a shift toward low percentages in OSCC tumors. These

changes reached a high level of statistical significance

(P < .001; Table 2).

Representative examples for the IHC parameters oftumors with PSTAT1 staining are shown in Figure 1.

ASSOCIATION OF PSTAT1 AND STAT1 IHCPARAMETERS IN OSCC TUMORS WITH CLINICALAND PATHOLOGIC DATA

Clinical and pathologic data of the study population

were segregated by STAT1 and PSTAT1 intensity of

staining levels and categories of percentages of cells

in OSCC tumors, and statistical comparisons were

performed. Patient characteristics involved gender

(women vs men), age ($55 vs <55 yr), OSCC site

(tongue vs other), tumor differentiation (poor andmoderately vs well differentiated), perivascular and

perineural invasion, pT score (1 or 2 vs 3 or 4), tumor

size ($2 vs <2 cm), pN score (pN0 vs pN+), pTNM

staging (stages I and II vs III and IV), and disease recur-

rence. Data are presented in Tables 3 and 4 for STAT1

and PSTAT1, accordingly.

Concerning STAT1 intensity, the vast majority of pa-

tients (71.4%; 35 of 49 cases) showed moderate stain-ing, whereas the remaining patients (28.6%; 14 cases)

showed intense STAT1 staining (Table 3). None of the

frequency distribution comparisons of STAT1 inten-

sity of staining levels in OSCC tumors per patient char-

acteristic reached a statistically significant level of

difference. Relevant comparisons also were per-

formed between the percentages of cells with

STAT1 staining in OSCC tumors and patient character-istics, without leading to any significant association.

More specifically, medium percentages of cells with

STAT1 staining accounted for 39 patients (79.6%),

whereas high percentages accounted only for 10

FIGURE 1. Representative examples of phosphorylated signal transducer and activator of transcription-1 immunohistochemical parameters inoral squamous cell carcinoma tumors. A, B,Oral squamous cell carcinoma tumor with moderate intensity of staining for phosphorylated signaltransducer andactivator of transcription-1 andmediumpercentageof cellswith phosphorylated signal transducer andactivator of transcription-1expression (A,originalmagnification,�100;B,originalmagnification,�400).C,D,Oral squamous cell carcinoma tumor withweak intensity ofstaining for phosphorylated signal transducer and activator of transcription-1 and smaller percentage of cells with phosphorylated signal trans-ducer and activator of transcription-1 expression (C, original magnification, �100; D, original magnification, �400).



(20.4%) and the frequencies of medium percentages

outweighed the frequency of high percentages in all

comparisons.

In contrast, PSTAT1 intensity of staining levels in

OSCC tumors ranged from weak to moderate levels,accounting for 45 patients (91.8%) and only 4 patients

(8.2%), respectively (Table 4). Weak PSTAT1 staining

was the prevalent staining level category in all compar-

isonsperformed, regardless of thepatient characteristic

under study. There was a tendency toward a higher

frequency of moderate PSTAT1 staining for well-

differentiated tumors (P = .052) and pTNM early stage

tumors (P = .065). Moreover, the percentages of cellswith PSTAT1 staining in OSCC tumors ranged among

the low, medium, and high categories (39 patients

[79.6%], 6 patients [12.2%], and 4 patients [8.2%],

respectively). The frequency distribution of the vari-

ous categories of percentages of cells with PSTAT1

staining according to patient characteristics resulted

in no statistically significant association.

EVALUATION OF SURVIVAL

The median overall survival for the study cohort was52.4 months (range, 0.9 to 95.2 months; Table 1). The

followingparameterswere significantpredictors for sur-

vival with the application of the Cox proportional haz-

ards model: perineural invasion (P = .004; hazard ratio

[HR] = 3.20; 95% confidence interval [CI], 1.44-7.14),

pT score (P= .049;HR=2.35; 95%CI, 1.00-4.68), pNsta-

tus (P = .013; HR = 3.47; 95% CI, 1.30-9.24), and pTNM

stages (P= .013;HR=3.87; 95%CI, 1.33-11.29).Data arepresented in Table 5. None of the STAT1 and PSTAT1

IHC characteristics served as a significant predictor

for overall survival in the entire patient group

(Table 6).

Table 3. CLINICAL AND PATHOLOGICCHARACTERISTICS OF PATIENTS ACCORDING TOSTAT1 INTENSITY OF STAINING LEVELS IN OSCCTUMORS

Characteristics

STAT1 Intensity of

Staining in OSCC

Tumors

P*

Intense

(+++)

Moderate

(++)

Total patients 14 (28.6%) 35 (71.4%)

Gender .501

Women 6 (37.5%) 10 (62.5%)

Men 8 (24.2%) 25 (75.8%)

Age (yr) 1.000

$55 9 (28.1%) 23 (71.9%)

<55 5 (29.4%) 12 (70.6%)

OSCC site .205

Tongue 5 (19.2%) 21 (80.8%)

Other 9 (39.1%) 14 (60.9%)


Well 3 (23.1%) 10 (76.9%)

Low and moderate 11 (30.6%) 25 (69.4%)


Yes 5 (38.5%) 8 (61.5%)

No 9 (25.0%) 27 (75.0%)

Perivascular invasion .334

Yes 3 (50.0%) 3 (50.0%)

No 11 (25.6%) 32 (74.4%)

pT score .104

1 or 2 12 (36.4%) 21 (63.6%)

3 or 4 2 (12.5%) 14 (87.5%)

Tumor size (cm) .735

$2 9 (26.5%) 25 (73.5%)

<2 5 (33.3%) 10 (66.7%)

pN score .501

pN0 6 (37.5%) 10 (62.5%)

pN+ 8 (24.2%) 25 (75.8%)

Stage .503

Early (I and II) 5 (35.7%) 9 (64.3%)

Late (III and IV) 9 (25.7%) 26 (74.3%)


Yes 5 (20.0%) 20 (80.0%)

No 9 (37.5%) 15 (62.5%)

Abbreviations: OSCC, oral squamous cell carcinoma; STAT1,signal transducer and activator of transcription-1.* Two-tailed Fisher’s exact test P values.

Pappa et al. PSTAT1 and Oral Squamous Cell Carcinoma. J Oral

Maxillofac Surg 2014.

Table 4. CLINICAL AND PATHOLOGICCHARACTERISTICS OF PATIENTS ACCORDINGTO PSTAT1 INTENSITY OF STAINING LEVELS INOSCC TUMORS

Characteristics

PSTAT1 Intensity of

Staining in OSCC

Tumors

P*

Moderate

(++) Weak (+)

Total patients 4 (8.2%) 45 (91.8%)

Gender 1.000

Women 1 (6.3%) 15 (93.8%)

Men 3 (9.1%) 30 (90.9%)

Age (yr) .602

$55 2 (6.3%) 30 (93.8%)

<55 2 (11.8%) 15 (88.2%)

OSCC site .612

Tongue 3 (11.5%) 23 (88.5%)

Other 1 (4.3%) 22 (95.7%)


Well 3 (23.1%) 10 (76.9%)

Poor and moderate 1 (2.8%) 35 (97.2%)


Yes 0 (0.0%) 13 (100.0%)

No 4 (11.1%) 32 (88.9%)

Perivascular invasion 1.000

Yes 0 (0.0%) 6 (100.0%)

No 4 (9.3%) 39 (90.7%)

pT score .289

1 or 2 4 (12.1%) 29 (87.9%)

3 or 4 0 (0.0%) 16 (100.0%)

Tumor size (cm) .427

$2 1 (2.9%) 33 (97.1%)

<2 3 (20.0%) 12 (80.0%)

pN score .096

pN0 3 (18.8%) 13 (81.3%)

pN+ 1 (3.0%) 32 (97.0%)

Stage .065

Early (I and II) 3 (21.4%) 11 (78.6%)

Late (III and IV) 1 (2.9%) 34 (97.1%)


Yes 1 (4.0%) 24 (96.0%)

No 3 (12.5%) 21 (87.5%)

Abbreviations: OSCC, oral squamous cell carcinoma;PSTAT1, phosphorylated signal transducer and activator oftranscription-1.* Two-tailed Fisher’s exact test P values.



PAPPA ET AL 217

Kaplan-Meier survival curves were calculated for all

patients after taking each of the abovementioned

parameters into account. Statistically significant differ-ences were noted among the various survival distribu-

tions according to perineural invasion (P = .003), pT

score (P = .043), pN evaluation (P = .008), and

pTNM staging (P = .007) when the log-rank test was

applied. The median survival time was calculated for

the following groups of patients: with perineural inva-

sion (52.4 months), with pT score 3 or 4 (20.4months), with pN+ (24.4 months), with pTNM late

staging (24.4 months), and with moderate PSTAT1

staining levels in OSCC tumors (43.9 months).

Proceeding to the multivariate analysis, a series of

Cox regression models was calculated with the

Table 5. EFFECT OF DEMOGRAPHIC ANDCLINICOPATHOLOGIC CHARACTERISTICS OFPATIENTS ON SURVIVAL, BASED ON COXPROPORTIONAL HAZARDS CALCULATION

Characteristics P HR (95% CI)

Gender

Female .189 1.69 (0.77-3.71)

Age (years)

$55 .376 1.46 (0.63-3.34)

OSCC site

Tongue .444 0.74 (0.35-1.59)

Tumor grade

Poor .103 0.41 (0.14-1.20)

Perineural invasion

Yes .004 3.20 (1.44-7.14)

Perivascular invasion

Yes .060 2.57 (0.96-6.83)

pT score

3 or 4 .049 2.35 (1.00-4.68)

pN score

pN+ .013 3.47 (1.30-9.24)

Tumor margins

Involved .229 2.13 (0.62-7.27)

Stage

Late (III and IV) .013 3.87 (1.33-11.29)

Abbreviations: CI, confidence interval; HR, hazard ratio;OSCC, oral squamous cell carcinoma.



Table 6. STAT1ANDPSTAT1 IHC CHARACTERISTICS INOSCC TUMORS AS PREDICTORS FOR SURVIVAL,BASED ON COX PROPORTIONAL HAZARDSCALCULATION

IHC Characteristics P HR (95% CI)

STAT1

Intensity of staining

Intense vs moderate .380 0.67 (0.27-1.65)

Percentage of cells

High vs medium .093 2.03 (0.89-4.65)

PSTAT1

Intensity of staining

Moderate vs weak .224 0.29 (0.04-2.13)

Percentage of cells

High or medium vs low .701 1.20 (0.48-2.97)

Abbreviations: CI, confidence interval; HR, hazard ratio; IHC,immunohistochemical; OSCC, oral squamous cell carci-noma; PSTAT1, phosphorylated signal transducer and activa-tor of transcription-1; STAT1, signal transducer and activatorof transcription-1.




forward stepwise method, taking into account the

abovementioned parameters as covariates: perineural

invasion versus no invasion, pT score 3 or 4 versus

pT score 1 or 2, pN+ versus pN0, and pTNM late ver-

sus early staging. In the final model, only pTNM staging(P = .038; HR = 3.18; 95% CI, 1.06-9.53) and perineural

invasion (P = .028; HR = 2.50; 95% CI, 1.10-5.68) were

statistically significant predictors for overall survival in

the entire patient group.

EVALUATION OF SURVIVAL BASED ON ADJUVANTTREATMENT MODALITY

All adjuvant treatment modalities were segregatedinto 3 groups: radiation therapy only, chemotherapy

only or in combination with radiation therapy, and

no treatment. The adjuvant treatment modality deci-

sion was associated with the pN status (P < .001)

and pTNM staging (P = .001) of patients. The median

survival time per adjuvant treatment modality was cal-

culated only for the group of patients with chemother-

apy alone or in combination with radiation therapy,which reached 24.4 months.

Kaplan-Meier survival curves were calculated ac-

cording to STAT1 and PSTAT1 intensity of staining

levels and percentages of cells with STAT1 and PSTAT1

staining per adjuvant treatment modality group. Ap-

parently, the application of the log-rank test resulted

in a statistically significant difference concerning the

survival distributions based on PSTAT1 intensity of

staining levels (P = .008). This result suggests that

moderate PSTAT1 intensity of staining in OSCC tumors

is associated with a better response to adjuvant che-motherapy compared with weak staining (Fig 2).

Discussion

To the authors’ knowledge, the present study is the

first to examine PSTAT1 and STAT1 IHC expression pat-terns inOSCC tumors and their adjacent normal tissues

concurrently and to correlate them with the clinico-

pathologic parameters of patients, including overall

survival and response to adjuvant treatment modali-

ties. Thus far, only some studies have investigated the

role of STAT1 signaling in head and neck cancer with-

out focusing on its activated form (PSTAT1), with the

exception of Leibowitz et al.28,29 Laimer et al24 studiedSTAT1 activation in patients with OSCC tumors and its

role as a predictive biomarker for survival when associ-

ated with adjuvant chemotherapy and used a different

study design thatwas based exclusively on the percent-

ages of cells with STAT1 staining.

Concerning OSCC tumors and their adjacent normal

tissues in the present study cohort, the obtained data

showed that STAT1 intensity of staining involved in-tense and moderate levels, whereas PSTAT1 displayed

moderate and weak levels. Moreover, the percentages

of cells with STAT1 staining ranged from high to me-

dium levels and the percentages of cells with PSTAT1

FIGURE 2. PSTAT1 intensity of staining as a predictor for adjuvant chemotherapy response. Kaplan-Meier survival curves for patients treatedwith chemotherapy only or with chemotherapy and radiation therapy (Chemotherapy+) according to PSTAT1 intensity of staining levels inOSCC tumors (moderate vs weak). Moderate PSTAT1 staining was associated with better survival in this group of patients. PSTAT1, phosphor-ylated signal transducer and activator of transcription-1; OSCC, oral squamous cell carcinoma.


PAPPA ET AL 219

staining extended among low, medium, and high cate-

gories. In fact, the last category was met in only 4 cases

(8.2%). These differences can be explained by the fact

that STAT1 antibody detects endogenous levels of the

STAT1 a (91 kDa) and b (84 kDa) isoforms, regardless

of the STAT1 phosphorylation status, whereas the

PSTAT1 (tyrosine 701) antibody detects endogenouslevels of STAT1 protein only when phosphorylated at

tyrosine 701.13 Therefore, higher ranges concerning

the IHC parameters of STAT1 are attributed to the inac-

tive form of STAT1 reserve (USTAT1) in the nuclei of

the cells and to the larger number of cells with USTAT1

protein expression.

When OSCC tumors were compared with their adja-

cent normal tissues, a decrease by 32.7% in the fre-quency of high percentages of cells with STAT1

staining was noted (P = .014). In addition, an ever

greater decrease that reached 57.1%, concerning the

frequency of high and medium percentages of cells

with PSTAT1 staining, also was noted (P < .001). This

observation reflects the fact that STAT1 expression is

downregulated in oral cancer, leading to a smaller num-

ber of cells with USTAT1 reserve. The even smallernumber of cancer cells with PSTAT1 staining indicates

that, in addition to a decrease in the availability of

STAT1 molecules, the mechanisms responsible for

STAT1 activation are deficient in cancer. Because the

percentages of cells with total STAT1 protein expres-

sion reached the medium range for most tumors and

the percentages of cells with PSTAT1 protein expres-

sion reached the low range, it is logical to assume that

the ratio of USTAT1 to PSTAT1 increases in OSCC tu-

mors. Although it has been shown that USTAT1 mono-

mers can bind to DNA on their own, their affinity is

much less than that for PSTAT1 homodimers.13 Thus,it is reasonable to suggest that the decrease in the num-

ber of cells with PSTAT1 protein expression weakens

the tumor suppressor activities of STAT1 protein in

OSCC tumors.

These findings are consistent with those of Yim

et al23 who used a similar methodology. They compared

STAT1 protein expression between papillary thyroid

carcinomas and their adjacent normal sites and con-cluded that STAT1 DNA-binding activity in tumors was

significantly lower than that in surrounding normal thy-

roid tissues. In contrast, the study of Xi et al19 in patients

with HNSCC resulted in no differences concerning this

type of comparison, although STAT1 protein expres-

sion was lower in HNSCC tumors and adjacent normal

tissue sites compared with oropharyngeal tissues from

healthy controls.The tumor suppressor role of STAT1, which is con-

ducted by triggering apoptosis and growth arrest,

has been associated mainly with one of the following

mechanisms: 1) direct activation of apoptotic genes,

2) interaction with p53 and induction of apoptosis


by enhancing the expression of p53-responsive genes,

and 3) inhibition of the activity of the MDM2 gene and,

therefore, p53-associated apoptosis enhancement.30

Activation of STAT1 within the tumor can be triggered

by cell endogenous signaling pathways or by autocrine

or paracrine growth factors. Moreover, STAT1 can be

activated by immune cells that secrete interferons

(IFNs), thus resulting in antitumor immunosurveil-lance action.28,29,31 Recently, it has been shown that

B-RAF mutations are associated with lower STAT1

activity in papillary thyroid carcinomas, explaining

further the molecular mechanisms of dysregulation

of STAT1 expression in cancer.23

Focusing on the active form of STAT1 (PSTAT1) has

led to more interesting findings concerning the role of

the STAT1 gene in cancer progression and differentia-tion, as in the present results. When PSTAT1 intensity

of staining in OSCC tumors was compared with the pa-

tients’ clinicopathologic parameters, higher PSTAT1

staining showed a tendency for an association with

well-differentiated tumors (P = .052) and pTNM early

stage tumors (P = .065). However, a statistically signifi-

cant level was not reached, probably owing to the small

number of patients sharing these histopathologic fea-tures. Conversely, PSTAT1 intensity of staining in

OSCC tumors was not associated with survival in the

entire study group. A report by Widschwendter et al18

in breast cancer associated the increased expression

of PSTAT1 with a significant improvement in overall

and disease-free survival. Simpson et al20 concluded

that the presence of high levels of nuclear STAT1 corre-

lated with improved survival in patients with colorectalcancer, whereas Laimer et al24 associated STAT1 activa-

tion in patients with OSCC and only a negative lymph

node status and not with overall survival.20,24 These

discrepancies among the various researchers may be

attributed to the fact that IHC parameter analysis was

based on different study designs, which are not

directly comparable. Moreover, patient characteristics

were different in number, treatment modalities, andtumor sites.

Several other factors were shown to be independent

predictors for overall survival in the present study sam-

ple, including perineural invasion, lymph node status,

tumor size, andpTNMstaging.However, in thefinalmul-

tivariate Cox regression model, only pTNM staging and

perineural invasionwere significant. The prognostic im-

portance of TNM staging for overall survival has beenrecognized for many decades.32,33 Perineural invasion

has been associated with an increased risk for local

recurrence, a higher rate of metastasis, and decreased

survival in patients with HNSCC in many studies.34

Regarding treatment modalities, the present results

suggest that patients who retained higher intensity

levels of PSTAT1 staining in OSCC tumors (moderate

vs weak) responded better to adjuvant radiation ther-

apy and chemotherapy. Because this effect was not

found in patients who were treated with adjuvant radi-

ation therapy only and those with no adjuvant treat-

ment, it is attributed to chemotherapy. This finding

cannot be directly compared with the study of Laimer

et al24 who reported a better response to adjuvant che-

motherapy in patients with OSCC and ‘‘higher STAT1activation’’—implying larger percentages of cells that

expressed STAT1 by these researchers—because the

2 studies differed drastically in design. However, the

2 findings support the role of STAT1 proteins as poten-

tial markers for chemotherapy in patients with

OSCC tumors.24

Thus far, certain molecular mechanisms that could

explain the enhanced chemosensitivity of STAT1-positive cancer cells have been reported: 1) synergism

between some chemotherapeutic agents that engage

with an antitumor immune response, which also impli-

cates expression of the STAT1 gene: it has been shown

that PSTAT1 is necessary for the HLA class I antigen

processing machinery component in HNSCC cells, the

downregulationofwhichmay lead toanescapeofmalig-

nant cells from the immune response29,35; 2) synergismbetween fluorouracil or doxorubicin and various IFNs

on STAT1 activation, inducing proapoptotic signaling

pathways in breast cancer cell lines and hepatocellular

carcinoma cells36,37; 3) the ataxia telangiectasia

mutated protein cell-cycle checkpoint pathway, which

is another site of interactionbetweenSTAT1andchemo-

therapeutic agents, inducing DNA damage and geno-

toxic stress and resulting in cell growth arrest andinhibition of DNA synthesis30,38,39; and 4) sensitization

of tumor cells to chemotherapeutic agent induced

apoptosis through methylation inhibitor treatment in

HNSCC, because STAT1 upregulation enhances apo-

ptosis, whereas the promoter methylation is the

primary mechanism of STAT1 downregulation in head

and neck carcinogenesis.19

In conclusion, the present research has shown thatthe percentages of cells with STAT1 and PSTAT1 stain-

ing are decreased in OSCC tumors compared with ad-

jacent normal tissue sites and that a strong association

for higher PSTAT1 intensity of staining levels with

overall survival has emerged in chemotherapy-

treated patients. Although a limitation of the present

study may be the relatively small sample, the present

findings are intriguing and reinforced by the reportof Laimer et al,24 which is the sole study on STAT1 ac-

tivation and chemotherapy response in patients with

OSCC tumors thus far, despite great differences in

methodology.24 Prospective randomized clinical trials

are needed to establish the role of PSTAT1 as a predic-

tive biomarker for the clinical outcome of patients

with OSCC.

PAPPA ET AL 221

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Phosphorylated Signal Transducer and Activator of Transcription-1 Immunohistochemical Expression Is Associated With Improved Survival in Patients With Oral Squamous Cell Carcinoma