Ann Surg Oncol (2015) 22:662–669 DOI 10.1245/s10434-014-4003-0
ORIGINAL ARTICLE – HEAD AND NECK ONCOLOGY
Cytoplasmic iASPP Expression as a Novel Prognostic Indicator in Oral Cavity Squamous Cell Carcinoma Ji Won Kim, MD1, Jong-Lyel Roh, MD1, Yangsoon Park, MD2, Kyung-Ja Cho, MD2, Seung-Ho Choi, MD1, Soon Yuhl Nam, MD1, and Sang Yoon Kim, MD1,3 1
Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 2Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 3Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea
ABSTRACT Background. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a key inhibitor of tumor suppressor p53 that is overexpressed in several human cancers; however, its role in oral cavity squamous cell carcinomas (OSCC) remains unknown. The present study investigated the prognostic role of iASPP in patients with OSCC. Methods. This study included 186 OSCC patients who underwent curative surgery at our institution between 2000 and 2011. Cytoplasmic and nuclear iASPP expression were examined separately by immunohistochemistry, and dichotomized to low and high. Clinicopathological variables associated with locoregional control (LRC), diseasefree survival (DFS), and overall survival (OS) were identified by univariate and multivariate analyses. Results. Patients were followed-up for a median period of 74 months (range 16–166 months), and 5-year LRC, DFS, and OS was 73.6, 70.2, and 75.3 %, respectively. High iASPP immunostaining reactivity was detected in the cytoplasm and nucleus in 132 (71.0 %) and 93 (50.0 %) patients, respectively. In univariate analysis, pathologic nodal metastasis, advanced overall stage III–IV, lymphovascular invasion, and cytoplasmic iASPP were significantly associated with poor LRC, DFS, and OS (p \ 0.05). High-grade and positive resection margins
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-4003-0) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 27 May 2014; Published Online: 23 August 2014 J.-L. Roh, MD e-mail: [email protected]
were significant factors associated with poor DFS and OS (p \ 0.02). In multivariate analysis, N classification, lymphovascular invasion, and cytoplasmic iASPP expression remained independent variables for LRC, DFS, and OS (p \ 0.05). Conclusion. High iASPP expression in the tumor cell cytoplasm is associated with poor outcomes of OSCC patients in terms of recurrence and survival, suggesting a role for iASPP as a novel biomarker and therapeutic target for OSCC.
Head and neck cancer is the sixth most common malignancy worldwide, and approximately 30 % of head and neck cancers arise in the oral cavity. In 2014, an estimated 42,440 patients will be newly diagnosed with oral cavity cancer in the US, and an estimated 8,390 will die from the disease.1 The majority of oral cavity cancers are squamous cell carcinomas (SCCs); metastasis to draining regional lymph nodes is detected in approximately 50 % of patients at the time of diagnosis and is commonly associated with poor prognosis.2,3 The survival rate of oral cavity SCCs (OSCC) has remained substantially unchanged over the last two decades.4 Pretreatment selection of patients with unfavorable survival outcomes or who require intensified therapy remains difficult, despite the use of prognostic parameters, including clinical stage at diagnosis, tumor size and invasion depth, and histological type.3,5 The identification of novel biomarkers predictive of poor treatment outcome in OSCC is urgent. Inhibitor of apoptosis-stimulating protein of p53 (iASPP), the inhibitory member of the ASPP family, is encoded by PPP1R13L in humans and has been evolutionarily conserved from worms to humans.6 The ASPP family includes three members, namely ASPP1, ASPP2, and iASPP, which
iASPP Expression in OSCC
are specific regulators of p53-, p63-, and p73-mediated apoptosis.7,8 ASPP1 and ASPP2 enhance the apoptotic function of p53, whereas iASPP specifically inhibits p53mediated apoptosis.9 Overexpression of iASPP is associated with resistance to cisplatin-induced apoptosis and radiation therapy.6 iASPP plays a pivotal role in regulating cancer cell proliferation and tumor progression.8–10 Therefore, the restoration of p53 function through the inhibition of iASPP is a potential novel strategy for cancer therapy.10–12 Overexpression of iASPP has been reported in many human cancers, including breast cancer,6 leukemia,13 nonsmall cell lung cancer,14 hepatocellular carcinoma,15 melanoma,12 ovarian,16 endometrial,17 and cervical cancer.18 ASPP1 and ASPP2 are downregulated in tumors, whereas the expression of iASPP is increased in malignant tumors compared with adjacent normal tissues or healthy donor tissues.13,14,18 High expression of iASPP is significantly associated with shorter disease-free survival (DFS) and overall survival (OS) of patients with ovarian and cervical cancer,16,18 and iASPP upregulation has been reported in head and neck SCC (HNSCC) tissues and cell lines, indicating that it may serve as a novel prognostic marker.19 To the best of our knowledge, the prognostic significance of iASPP in OSCC has not been investigated to date. In the present study, we examined the prognostic value of iASPP expression in patients with OSCC who underwent curative surgery. PATIENTS AND METHODS Patients A total of 253 consecutive patients who underwent radical surgery for OSCC at our tertiary referral center between 2000 and 2011 were analyzed. Inclusion criteria was previously untreated OSCC without distant metastasis. Seventy-seven patients were excluded because of a history of previous treatments, palliative treatments for distant metastasis, inadequate clinical follow-up data, or a lack of sufficient specimen to generate a tissue microarray (TMA). The 186 eligible patients were included in the final analyses. Tumor stage was determined according to the American Joint Committee on Cancer (AJCC) TNM staging system (7th edition).20 This study was reviewed and approved by the Institutional Review Board of our institution, and the requirement for informed consent from each patient was waived. All patients underwent wide resection of primary tumors in the oral cavity and combined neck dissection with curative intent. Fifty-seven patients with clinical nodal metastasis underwent modified radical or radical neck dissection involving levels I–V, and 24 patients with
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primary tumors involving the midline and suspected of having contralateral neck metastases had bilateral neck dissection. One-hundred and twenty-nine patients without clinical nodal metastasis (cN0) underwent selective neck dissection involving levels I–III or I–IV. Eighty-eight patients with adverse pathologic features, e.g. positive margins, pT3 or pT4 primary, nodal positivity, perineural invasion, or lymphovascular invasion, were treated with postoperative radiotherapy or chemoradiotherapy, with a median of 60 Gy (range 42–74 Gy) administered in a singly daily fraction. After initial treatment with curative intent, eligible patients were regularly followed by clinical examination and imaging. Patients were scheduled for clinic visits every 1–2 months during the first year, every 2–4 months during the second and third years, every 6 months during the fourth and fifth year, and annually thereafter. The presence of clinical or imaging findings suggestive of recurrence or second primary tumors was confirmed by biopsy and specific additional diagnostic tests. Patients with confirmed recurrence were scheduled for salvage or palliative treatment. Pathology and Immunohistochemical Staining The surgical pathologic results were reviewed for assessment of tumor size, extent, invasion depth, histologic grade, lymphovascular, perineural or other local invasion, positive surgical margin, and regional metastasis. Despite excision with grossly free margins, all surgical specimens were examined microscopically for residual disease on the resection margin. Two board-certified pathologists (YP and K-JC) with [15 years of clinical experience reviewed all the pathologic sections and the TMA results blinded to clinical follow-up data. Primary tumor tissues were obtained from surgical specimens. For immunohistochemical staining, paraffinembedded biopsy specimens were obtained from our pathology archives, and TMAs were constructed from the formalin-fixed tumor blocks. Specific regions from the TMA cores of each primary tumor were selected from the invasive front under microscopy and arranged pairwise in TMA blocks. Formalin-fixed, paraffin-embedded tissue blocks were deparaffinized and used to construct TMAs with a core diameter of 2 mm. Each sample was analyzed in duplicate to decrease sampling error and minimize tissue loss during processing. Immunohistochemical staining for iASPP (ab-34898, dilution 1:200; Abcam, Cambridge, MA, USA) was performed on 4-lm TMA sections using a BenchMark XT automatic immunostaining device (Ventana Medical Systems, Tucson, AZ, USA) with OptiView DAB IHC Detection Kit (Ventana Medical Systems), in accordance
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FIG. 1 Representative images showing immunohistochemical staining of iASPP in oral cavity squamous cell carcinoma. Low iASPP expression (a, d); high cytoplasmic and nuclear iASPP expression (b, e); and predominant iASPP expression in the cytoplasm (c) or the
nucleus (f). iASPP expression was observed in the cytoplasm and nucleus of tumor cells. Original magnification, a–c 9100, d 9400, d– e iASPP inhibitor of apoptosis-stimulating protein of p53
with the manufacturer’s instructions. A sample probed with normal goat serum instead of primary antibody under the same experimental conditions was used as negative control. Immunostaining was graded by the reactivity score (IRS), which measures staining intensity as negative (0), weak (1), moderate (2), or strong (3), and reactivity assessed by the percentage of positively staining cells as 0–5 % (0), 5–25 % (1), 26–50 % (2), 51–75 % (3), and 76–100 % (4). The IRS was calculated by multiplying the grade of staining intensity by that of the staining percentage, giving a minimum-to-maximal score of 0–12. The expression of iASPP in the cytoplasm and nucleus was interpreted separately and dichotomized to low (0–6) and high (7–12) values for outcome analyses.18,19 Representative staining results are shown in Fig. 1.
tumor grade (G1–G2 vs. G3), tumor thickness (B0.7 vs. [0.7 cm), T (T1–2 vs. T3–4), and N (N0 vs. N1–3) classification, overall TNM stage (I–II vs. III–IV), resection margin (not involved vs. involved), perineural invasion, lymphovascular invasion, and cytoplasmic and nuclear iASPP expression (low vs. high). The log-rank test and Cox proportional hazards model were used to examine the significance of differences in recurrence or survival outcomes according to the dichotomized values of the tested variables. Cox proportional hazard regression analyses were performed, including variables with p-values \ 0.05 on univariate analyses. Standard descriptive statistics were calculated as frequency and percentage for categorical data, and median and range for continuous variables. Statistical significance between iASPP expression and clinicopathological parameters was analyzed by the v2test. All tests were twosided, and p-values \ 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS software version 21.0 (IBM Corporation, Armonk, NY, USA).
Statistical Analysis The Kaplan–Meier method was used for locoregional control (LRC), DFS, and OS curve estimations. The time interval for the LRC or DFS endpoint was calculated from the time of surgery or the first day of treatment to the date of recurrence or last follow-up. The time interval for the OS endpoint was calculated from the time of surgery to the date of death from index cancer or any cause, or censored at the date of last follow-up for alive patients. Prognostic factor analyses focusing on LRC, DFS, and OS outcomes were performed by including the dichotomized variables as follows: age (B60 vs. [60 years), sex,
RESULTS Patient Characteristics The 186 eligible patients consisted of 112 men and 74 women, with a median age of 60 years (range 29–95 years). The clinicopathological characteristics of
iASPP Expression in OSCC
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TABLE 1 Patient characteristics Characteristic
N
%
Sex Male
112
60.2
Female
74
39.8
Age [years; median (range)]
60 (29–95)
Smoking
100
53.7
Alcohol drinking
112
60.2
Tongue
139
74.7
Buccal
16
8.6
During a median follow-up of 74 months (range 16–166 months), 64 (34.4 %) patients had recurrences as follows: local (n = 30), regional (n = 42), and distant (n = 24), with mutually overlapping recurrences in part. At the last follow-up, 57 (30.6 %) patients had died of OSCC, 19 (10.2 %) had died of other causes, and three (1.6 %) were alive with disease. The 2-year LRC, DFS, and OS was 89.2, 87.6, and 88.1 %, respectively, and the 5-year LRC, DFS, and OS was 73.6, 70.2, and 75.3 %, respectively.
Primary site
Mouth floor
14
7.5
Gingiva Retromolar trigone
7 5
3.8 2.7
Hard palate
4
2.2
Lip
1
0.5
111/58/17
59.7/31.2/9.1
T1/2
76/80
40.9/43.0
T3/4
9/21
4.8/11.3
N0
102
54.8
N1/N2/N3
33/51/0
17.7/27.4/0
I/II
57/33
30.6/17.7
III/IV
35/61
18.8/32.8
98
52.7
80/8
43.0/4.3
Tumor grade (G1/G2/G3) T classification
N classification
Overall TNM stage
Primary treatment Surgery alone Surgery ? PORT/CRT Follow-up, months Median (range)
74 (16–166)
Last status (NED/DOD/DOC)
98/57/19
52.7/30.6/10.2
Recurrence
64
34.4
CRT concurrent chemoradiation therapy, NED no evidence of disease, DOD died of disease, DOC died of other causes, PORT postoperative radiation therapy, TNM tumor-node-metastasis stage (American Joint Committee on Cancer, 7th edition)
patients are summarized in Table 1. Primary tumors were most commonly found in the oral tongue (74.7 %), and were poorly differentiated (G3) in 17 patients (9.1 %). Thirty (16.1 %) patients had advanced T-stage tumors and 84 (45.2 %) had pathologically positive cervical lymph nodes; 96 (51.6 %) patients were in advanced overall stage III–IV. Median tumor thickness was 0.7 cm (range 0.1–2.5 cm), and 100 (53.8 %) patients had tumors [0.7 cm. The resection margin was involved in 15 (8.1 %) patients. Perineural and lymphovascular invasion were observed in 21 (11.3 %) and 13 (7.0 %) patients, respectively.
Overexpression of iASPP in Oral Cavity Squamous Cell Carcinoma iASPP immunoreactivity was predominant in the cytoplasm of HNSCC tumor cells, although nuclear staining was also observed (Fig. 1). Of 186 study patients, 53 (29.0 %) and 65 (34.9 %) showed a staining intensity of grade 3 in the cytoplasm and nucleus, respectively. High-intensity staining of iASPP was found in both the cytoplasm and nucleus of tumor tissues, and high staining intensity ([grade 2) in the cytoplasm was significantly correlated with that in the nucleus (p \ 0.001). Grade 4 positive staining in the cytoplasm and nucleus was observed in 154 (82.7 %) and 73 (39.2 %) patients, respectively. High-intensity iASPP staining in the cytoplasm was also significantly correlated with that in the nucleus (p \ 0.001). Overall, high IRS scores ([6) in the cytoplasm and nucleus were found in 132 (71.0 %) and 93 (50.0 %) patients, respectively, and were positively correlated with each other (p \ 0.001) [electronic supplementary Table S1]. Univariate and Multivariable Analyses for Recurrence and Survival The results of univariate analyses of factors affecting patient outcome are shown in Table 2. Pathologic nodal metastasis, advanced overall stage III–IV, and lymphovascular invasion were significantly associated with poor LRC, DFS, and OS (p \ 0.005). High-grade tumors and positive resection margins were also significant factors associated with poor DFS and OS (p \ 0.02). Tumor thickness ([0.7 cm) was a significant factor for OS (p = 0.008) but not for LRC or DFS (p [ 0.05). Cytoplasmic iASPP expression was significantly associated with LRC, DFS, and OS (p \ 0.05), whereas nuclear iASPP expression was not (p [ 0.1) [Fig. 2]. In addition, the subgroup analyses of LRC, DFS, and OS between high cytoplasmic/low nuclear iASPP versus high cytoplasmic/high nuclear iASPP showed no significant differences (p [ 0.3). In multivariate analyses, N classification, lymphovascular invasion, and cytoplasmic iASPP expression remained independent variables for LRC, DFS, and OS (p \ 0.05) [Table 3].
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TABLE 2 Univariate analysis of factors affecting locoregional control, disease-free survival, and overall survival Locoregional control HR
a
95 % CI
Disease-free survival p value
a
HR
a
95 % CI
Overall survival p value
a
HRa
95 % CI
p valuea
Age (years) B60
1
[60
1.298
1 0.753–2.237
0.348
0.605–1.744
0.922
1.401
1 0.847–2.317
0.185
0.667–1.800
0.719
1.702
0.988–2.934
0.050
0.605–1.744
0.922
0.540–1.971
0.923
0.553–2.011
0.873
1.155–4.814
0.018
1.223–3.741
0.008
0.580–2.430
0.638
2.217–6.940
\0.001
1.814–5.809
\0.001
1.626–6.397
0.001
0.829–3.453
0.149
Sex Male
1
Female
1.027
Smoking No Yes
1
1 1.045
1.096
1
1 0.590–1.852
0.880
2.48
0.630–2.311
0.579
1.06
0.075
1
1.027 1
0.231–0.322
0.442
1.03
0.523–1.662
0.936
1.381–5.358
0.004
0.933–2.577
0.091
0.561–2.176
0.774
1.188
1.560–4.333
\0.001
3.923
1.381–3.925
0.002
Alcohol drinking No
1
Yes
1.201
1
1 1.05
Tumor grade G1–G2
1
G3
2.060
0.929–4.566
2.721
1 2.358
Tumor thickness (cm) B0.7
1
[0.7
1.138
1 0.664–1.952
0.638
1.551
0.580–2.444
0.635
1.105
1.190–3.535
0.010
2.600
1.161–3.556
0.013
1 2.139
pT classification T1–T2
1
T3–T4
1.190
pN classification N0 Any N?
1
1 2.051
1
1
1
Overall stage I–II
1
III–IV
2.032
1 2.329
1 3.246
RM Not involved
1
Involved
2.173
1 0.979–4.822
0.056
2.720
0.575–2.825
1.275
1.415
2.080–8.910
\0.001
1.325–6.024
0.007
0.801–2.365
0.248
1 1.381–5.357
0.004
3.225
0.698–2.867
0.336
1.692
4.091
2.061–8.121
\0.001
4.913
2.469–9.778
\0.001
1 1.906
1.032–3.520
0.039
1 1.901
1.012–3.408
0.046
0.861–2.330
0.170
0.907–2.620
0.110
PNI No
1
Yes
1.275
1
1
LVI No
1
Yes
4.305
1
1
Cytoplasmic iASPP expression Low High
1 2.826
Nuclear iASPP expression Low
1
High
1.376
1 1.417
1 1.541
Bold values are statistically significant (p \ 0.05) CI confidence interval, HR hazard ratio, PNI perineural invasion, LVI lymphovascular invasion, RM resection margin, iASPP inhibitor of apoptosis-stimulating protein of p53 a
Cox proportional hazards model was performed on univariate analysis
0.8
Low
0.6
High
0.4 0.2
B
1.0 0.8
C Low
Overall survival
1.0
667
Disease-free survival
A Locoregional control
iASPP Expression in OSCC
0.6
High 0.4 0.2
0
24
48
72
96
120
Low
0.6
High
0.4
P = 0.047 0.0
0.0
0.0
0.8
0.2
P = 0.036
P = 0.005
1.0
0
24
Follow-up (months)
48
72
96
120
0
Follow-up (months)
24
48
72
96
120
Follow-up (months)
FIG. 2 Kaplan–Meier curves comparing locoregional control (a), disease-free survival (b) and overall survival (c), according to cytoplasmic iASPP expression. Log-rank test, p \ 0.05 TABLE 3 Multivariate analyses of factors affecting locoregional control, disease-free survival, and overall survival Locoregional control HR
a
Disease-free survival
95 % CI
p value
1.072–3.302
0.028
a
HR
a
Overall survival
95 % CI
p value
1.435–4.055
0.001
a
HRa
95 % CI
p valuea
1.676–5.497
\0.001
1.960–8.431
\0.001
1.144–4.273
0.018
pN classification N0
1
Any N?
1.881
1 2.412
1 3.036
LVI No
1
Yes
3.248
1 1.533–6.882
0.002
2.415
1 1.168–4.993
0.017
1.046–3.598
0.036
4.066
Cytoplasmic iASPP expression Low
1
High
2.847
1 1.330–6.096
0.007
1.940
1 2.211
Bold values are statistically significant (p \ 0.05) CI confidence interval, HR hazard ratio, LVI lymphovascular invasion, iASPP inhibitor of apoptosis-stimulating protein of p53 a
Cox proportional hazard regression analyses were performed with backward elimination, including variables with p-values \ 0.05 on univariate analyses
DISCUSSION The present study is the first report showing a significant correlation between overexpression of iASPP and treatment outcomes in human OSCC. Our results showed a statistically significant relationship between high expression of cytoplasmic iASPP and increased disease recurrence rates and poor survival outcome of OSCC patients who underwent curative surgery with or without postoperative radiotherapy or chemoradiotherapy. These findings indicate that iASPP plays a role in OSCC and could serve as a novel prognostic biomarker predictive of treatment outcome in OSCC. The results of the present study were in accordance with those of previous studies in other human malignancies6,12–18 showing that iASPP overexpression in tumor tissues is significantly correlated with tumor aggressiveness, as indicated by increased metastasis, radio- or
chemotherapy resistance, and post-treatment recurrence. In the present study, positive iASPP immunostaining was detected in most OSCC tissue samples and predominantly in the cytoplasm, although it was also present in the nucleus, consistent with a recent report on iASPP in HNSCC.19 The diffuse reactivity in the cytoplasm and nucleus could be attributed to the broad specificity of the anti-iASPP antibody used in our study, which shows high affinity for both isoform 1 (located predominantly in the cytoplasm) and isoform 2 (located predominantly in the nucleus) of iASPP. The cellular location of iASPP expression varies among tumor types. Exclusively, cytoplasmic expression of iASPP was reported in non-small cell lung cancer,14 hepatocellular carcinoma,15 and prostate cancer,21 whereas both cytoplasmic and nuclear expression was found in melanoma,12 cervical cancer,18 and HNSCC.19 Although iASPP immunoreactivity was observed in approximately 50 % of
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the cases analyzed, the present study showed that overexpression of iASPP in the cytoplasm, but not in the nucleus, is an important prognostic indicator in OSCC. This might imply a potentially different biological/prognostic nature between cytoplasmic and nuclear iASPP expression, which requires further investigation. In cervical cancer, increased expression of nuclear iASPP is correlated with shorter OS and DFS, whereas cytoplasmic iASPP expression is not, and nuclear iASPP overexpression is correlated with chemo- and radioresistance.18 In HNSCC, overexpression of iASPP in the cytoplasm and nucleus is an independent prognostic indicator for OS and DFS.19 Further investigation is necessary to understand the relation between iASPP cellular localization and its prognostic significance. In the present study, cytoplasmic and nuclear iASPP expression levels were not significantly associated with lymph node metastasis (p [ 0.1). This differs from previous findings showing that nuclear iASPP is enriched in metastatic melanomas and significantly correlated with poor OS.12 As a key p53 inhibitor, iASPP plays an important role in apoptosis regulation. In keratinocytes, iASPP was recently shown to participate in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion, suggesting iASPP is a key regulator of epithelial homeostasis.22 Epithelial-mesenchymal transition (EMT), which is critical in normal epithelial cells and for the metastatic process, may be activated by the upregulation of iASPP.12,23 The role of iASPP in increased metastatic propensity requires further molecular and clinical investigations. Liu et al. 19 examined the clinical relevance of iASPP expression as a determinant of prognosis in 109 patients with laryngeal (n = 99) or hypopharyngeal (n = 10) SCC who underwent partial or total laryngectomy. These authors showed that cytoplasmic and nuclear overexpression of iASPP is significantly associated with advanced T or clinical stage, lymph node metastasis, and recurrence (p \ 0.005), and significantly correlated with shorter DFS and OS (both p \ 0.001 for cytoplasmic and nuclear expression). Our results confirmed the independent prognostic value of iASPP in patients with OSCC in addition to pathologic factors such as lymph node metastasis and lymphovascular invasion. Furthermore, inhibition of iASPP activity in cancer cells had a negative effect on tumorigenesis and improved radio-/chemosensitivity, as reported previously.15,16 Therefore, iASPP may serve as a potential therapeutic target in OSCC and HNSCC. Our study had several limitations inherent to its retrospective design. However, potential bias was reduced through the inclusion of patients with a single pathology of SCC arising in the oral cavity who underwent uniform treatments, including initial curative resection and neck dissection. Another limitation of the study is that iASPP expression was
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not examined in all tissues of primary tumors and metastatic lymph nodes in the neck. Assessment of iASPP expression in metastatic lymph nodes may provide additional information regarding its prognostic significance. CONCLUSIONS The present study showed that iASPP is overexpressed in OSCC tissues, and increased cytoplasmic iASPP is correlated with recurrence and poor survival outcomes in OSCC patients, suggesting iASPP as a novel biomarker and therapeutic target for OSCC. Further study is necessary to validate the present findings. ACKNOWLEDGMENT This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, which is funded by the Ministry of Education, Science and Technology (Grant No. NRF-2012R1A1A2002039), Seoul, Korea (Jong-Lyel Roh). DISCLOSURE Ji Won Kim, Jong-Lyel Roh, Yangsoon Park, Kyung-Ja Cho, Seung-Ho Choi, Soon Yuhl Nam, Sang Yoon Kim have declared no conflicts of interest.
REFERENCES 1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. 2. Tankere F, Camproux A, Barry B, Guedon C, Depondt J, Gehanno P. Prognostic value of lymph node involvement in oral cancers: a study of 137 cases. Laryngoscope. 2000;110:2061–5. 3. Sano D, Myers JN. Metastasis of squamous cell carcinoma of the oral tongue. Cancer Metastasis Rev. 2007;26:645–62. 4. Murthy V, Agarwal JP, Laskar SG, et al. Analysis of prognostic factors in 1180 patients with oral cavity primary cancer treated with definitive or adjuvant radiotherapy. J Cancer Res Ther 2010;6:282–9. 5. Funk GF, Karnell LH, Robinson RA, Zhen WK, Trask DK, Hoffman HT. Presentation, treatment, and outcome of oral cavity cancer: a national cancer data base report. Head Neck. 2002;24:165–80. 6. Bergamaschi D, Samuels Y, O’Neil NJ, et al. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet. 2003;33:162–7. 7. Trigiante G, Lu X. ASPP and cancer. Nat Rev Cancer. 2006;6: 217–26. 8. Cai Y, Qui S, Gao X, Gu SZ, Liu ZJ. iASPP inhibits p53-independent apoptosis by inhibiting transcriptional activity of p63/p73 on promoters of proapoptotic genes. Apoptosis. 2012;17:777–83. 9. Samuels-Lev Y, O’Connor DJ, Bergamaschi D, et al. ASPP proteins specifically stimulate the apoptotic function of p53. Mol Cell. 2001;8:781–94. 10. Alsafadi S, Tourpin S, Andre´ F, Vassal G, Ahomadegbe JC. p53 family: at the crossroads in cancer therapy. Curr Med Chem. 2009; 16:4328–44. 11. Bell HS, Ryan KM. iASPP inhibition: increased options in targeting the p53 family for cancer therapy. Cancer Res. 2008;68: 4959–62. 12. Lu M, Breyssens H, Salter V, et al. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell. 201;23:618–33.
iASPP Expression in OSCC 13. Zhang X, Wang M, Zhou C, Chen S, Wang J. The expression of iASPP in acute leukemias. Leuk Res. 2005;29:179–83. 14. Chen J, Xie F, Zhang L, Jiang WG. iASPP is over-expressed in human non-small cell lung cancer and regulates the proliferation of lung cancer cells through a p53 associated pathway. BMC Cancer. 2010;10:694. 15. Lu B, Guo H, Zhao J, et al. Increased expression of iASPP, regulated by hepatitis B virus X protein-mediated NF-kappaB activation, in hepatocellular carcinoma. Gastroenterology. 2010; 139:2183–94. 16. Jiang L, Siu MK, Wong OG, et al. iASPP and chemoresistance in ovarian cancers: effects on paclitaxel mediated mitotic catastrophe. Clin Cancer Res. 2011;17:6924–33. 17. Liu WK, Jiang XY, Ren JK, Zhang ZX. Expression pattern of the ASPP family members in endometrial endometrioid adenocarcinoma. Onkologie. 2010;33(10):500–3. 18. Cao L, Huang Q, He J, Lu J, Xiong Y. Elevated expression of iASPP correlates with poor prognosis and chemoresistance/ radioresistance in FIGO Ib1-IIa squamous cell cervical cancer. Cell Tissue Res. 2013;352:361–9.
669 19. Liu Z, Zhang X, Huang D, et al. Elevated expression of iASPP in head and neck squamous cell carcinoma and its clinical significance. Med Oncol. 2012;29:3381–8. 20. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. AJCC Cancer Staging Manual. 7th ed. New York: SpringerVerlag; 2010:29–40. 21. Zhang B, Xiao HJ, Chen J, Tao X, Cai LH. Inhibitory member of the apoptosis-stimulating protein of p53 (ASPP) family promotes growth and tumorigenesis in human p53-deficient prostate cancer cells. Prostate Cancer Prostatic Dis. 2011;14:219–24. 22. Chikh A, Matin RN, Senatore V, et al. iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia. EMBO J. 2011;30:4261–73. 23. Zhao WH, Wu SQ, Zhang YD. Downregulation of miR-124 promotes the growth and invasiveness of glioblastoma cells involving upregulation of PPP1R13L. Int J Mol Med. 2013;32: 101–7.
ORIGINAL ARTICLE – HEAD AND NECK ONCOLOGY
Cytoplasmic iASPP Expression as a Novel Prognostic Indicator in Oral Cavity Squamous Cell Carcinoma Ji Won Kim, MD1, Jong-Lyel Roh, MD1, Yangsoon Park, MD2, Kyung-Ja Cho, MD2, Seung-Ho Choi, MD1, Soon Yuhl Nam, MD1, and Sang Yoon Kim, MD1,3 1
Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 2Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 3Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea
ABSTRACT Background. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a key inhibitor of tumor suppressor p53 that is overexpressed in several human cancers; however, its role in oral cavity squamous cell carcinomas (OSCC) remains unknown. The present study investigated the prognostic role of iASPP in patients with OSCC. Methods. This study included 186 OSCC patients who underwent curative surgery at our institution between 2000 and 2011. Cytoplasmic and nuclear iASPP expression were examined separately by immunohistochemistry, and dichotomized to low and high. Clinicopathological variables associated with locoregional control (LRC), diseasefree survival (DFS), and overall survival (OS) were identified by univariate and multivariate analyses. Results. Patients were followed-up for a median period of 74 months (range 16–166 months), and 5-year LRC, DFS, and OS was 73.6, 70.2, and 75.3 %, respectively. High iASPP immunostaining reactivity was detected in the cytoplasm and nucleus in 132 (71.0 %) and 93 (50.0 %) patients, respectively. In univariate analysis, pathologic nodal metastasis, advanced overall stage III–IV, lymphovascular invasion, and cytoplasmic iASPP were significantly associated with poor LRC, DFS, and OS (p \ 0.05). High-grade and positive resection margins
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-4003-0) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 27 May 2014; Published Online: 23 August 2014 J.-L. Roh, MD e-mail: [email protected]
were significant factors associated with poor DFS and OS (p \ 0.02). In multivariate analysis, N classification, lymphovascular invasion, and cytoplasmic iASPP expression remained independent variables for LRC, DFS, and OS (p \ 0.05). Conclusion. High iASPP expression in the tumor cell cytoplasm is associated with poor outcomes of OSCC patients in terms of recurrence and survival, suggesting a role for iASPP as a novel biomarker and therapeutic target for OSCC.
Head and neck cancer is the sixth most common malignancy worldwide, and approximately 30 % of head and neck cancers arise in the oral cavity. In 2014, an estimated 42,440 patients will be newly diagnosed with oral cavity cancer in the US, and an estimated 8,390 will die from the disease.1 The majority of oral cavity cancers are squamous cell carcinomas (SCCs); metastasis to draining regional lymph nodes is detected in approximately 50 % of patients at the time of diagnosis and is commonly associated with poor prognosis.2,3 The survival rate of oral cavity SCCs (OSCC) has remained substantially unchanged over the last two decades.4 Pretreatment selection of patients with unfavorable survival outcomes or who require intensified therapy remains difficult, despite the use of prognostic parameters, including clinical stage at diagnosis, tumor size and invasion depth, and histological type.3,5 The identification of novel biomarkers predictive of poor treatment outcome in OSCC is urgent. Inhibitor of apoptosis-stimulating protein of p53 (iASPP), the inhibitory member of the ASPP family, is encoded by PPP1R13L in humans and has been evolutionarily conserved from worms to humans.6 The ASPP family includes three members, namely ASPP1, ASPP2, and iASPP, which
iASPP Expression in OSCC
are specific regulators of p53-, p63-, and p73-mediated apoptosis.7,8 ASPP1 and ASPP2 enhance the apoptotic function of p53, whereas iASPP specifically inhibits p53mediated apoptosis.9 Overexpression of iASPP is associated with resistance to cisplatin-induced apoptosis and radiation therapy.6 iASPP plays a pivotal role in regulating cancer cell proliferation and tumor progression.8–10 Therefore, the restoration of p53 function through the inhibition of iASPP is a potential novel strategy for cancer therapy.10–12 Overexpression of iASPP has been reported in many human cancers, including breast cancer,6 leukemia,13 nonsmall cell lung cancer,14 hepatocellular carcinoma,15 melanoma,12 ovarian,16 endometrial,17 and cervical cancer.18 ASPP1 and ASPP2 are downregulated in tumors, whereas the expression of iASPP is increased in malignant tumors compared with adjacent normal tissues or healthy donor tissues.13,14,18 High expression of iASPP is significantly associated with shorter disease-free survival (DFS) and overall survival (OS) of patients with ovarian and cervical cancer,16,18 and iASPP upregulation has been reported in head and neck SCC (HNSCC) tissues and cell lines, indicating that it may serve as a novel prognostic marker.19 To the best of our knowledge, the prognostic significance of iASPP in OSCC has not been investigated to date. In the present study, we examined the prognostic value of iASPP expression in patients with OSCC who underwent curative surgery. PATIENTS AND METHODS Patients A total of 253 consecutive patients who underwent radical surgery for OSCC at our tertiary referral center between 2000 and 2011 were analyzed. Inclusion criteria was previously untreated OSCC without distant metastasis. Seventy-seven patients were excluded because of a history of previous treatments, palliative treatments for distant metastasis, inadequate clinical follow-up data, or a lack of sufficient specimen to generate a tissue microarray (TMA). The 186 eligible patients were included in the final analyses. Tumor stage was determined according to the American Joint Committee on Cancer (AJCC) TNM staging system (7th edition).20 This study was reviewed and approved by the Institutional Review Board of our institution, and the requirement for informed consent from each patient was waived. All patients underwent wide resection of primary tumors in the oral cavity and combined neck dissection with curative intent. Fifty-seven patients with clinical nodal metastasis underwent modified radical or radical neck dissection involving levels I–V, and 24 patients with
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primary tumors involving the midline and suspected of having contralateral neck metastases had bilateral neck dissection. One-hundred and twenty-nine patients without clinical nodal metastasis (cN0) underwent selective neck dissection involving levels I–III or I–IV. Eighty-eight patients with adverse pathologic features, e.g. positive margins, pT3 or pT4 primary, nodal positivity, perineural invasion, or lymphovascular invasion, were treated with postoperative radiotherapy or chemoradiotherapy, with a median of 60 Gy (range 42–74 Gy) administered in a singly daily fraction. After initial treatment with curative intent, eligible patients were regularly followed by clinical examination and imaging. Patients were scheduled for clinic visits every 1–2 months during the first year, every 2–4 months during the second and third years, every 6 months during the fourth and fifth year, and annually thereafter. The presence of clinical or imaging findings suggestive of recurrence or second primary tumors was confirmed by biopsy and specific additional diagnostic tests. Patients with confirmed recurrence were scheduled for salvage or palliative treatment. Pathology and Immunohistochemical Staining The surgical pathologic results were reviewed for assessment of tumor size, extent, invasion depth, histologic grade, lymphovascular, perineural or other local invasion, positive surgical margin, and regional metastasis. Despite excision with grossly free margins, all surgical specimens were examined microscopically for residual disease on the resection margin. Two board-certified pathologists (YP and K-JC) with [15 years of clinical experience reviewed all the pathologic sections and the TMA results blinded to clinical follow-up data. Primary tumor tissues were obtained from surgical specimens. For immunohistochemical staining, paraffinembedded biopsy specimens were obtained from our pathology archives, and TMAs were constructed from the formalin-fixed tumor blocks. Specific regions from the TMA cores of each primary tumor were selected from the invasive front under microscopy and arranged pairwise in TMA blocks. Formalin-fixed, paraffin-embedded tissue blocks were deparaffinized and used to construct TMAs with a core diameter of 2 mm. Each sample was analyzed in duplicate to decrease sampling error and minimize tissue loss during processing. Immunohistochemical staining for iASPP (ab-34898, dilution 1:200; Abcam, Cambridge, MA, USA) was performed on 4-lm TMA sections using a BenchMark XT automatic immunostaining device (Ventana Medical Systems, Tucson, AZ, USA) with OptiView DAB IHC Detection Kit (Ventana Medical Systems), in accordance
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FIG. 1 Representative images showing immunohistochemical staining of iASPP in oral cavity squamous cell carcinoma. Low iASPP expression (a, d); high cytoplasmic and nuclear iASPP expression (b, e); and predominant iASPP expression in the cytoplasm (c) or the
nucleus (f). iASPP expression was observed in the cytoplasm and nucleus of tumor cells. Original magnification, a–c 9100, d 9400, d– e iASPP inhibitor of apoptosis-stimulating protein of p53
with the manufacturer’s instructions. A sample probed with normal goat serum instead of primary antibody under the same experimental conditions was used as negative control. Immunostaining was graded by the reactivity score (IRS), which measures staining intensity as negative (0), weak (1), moderate (2), or strong (3), and reactivity assessed by the percentage of positively staining cells as 0–5 % (0), 5–25 % (1), 26–50 % (2), 51–75 % (3), and 76–100 % (4). The IRS was calculated by multiplying the grade of staining intensity by that of the staining percentage, giving a minimum-to-maximal score of 0–12. The expression of iASPP in the cytoplasm and nucleus was interpreted separately and dichotomized to low (0–6) and high (7–12) values for outcome analyses.18,19 Representative staining results are shown in Fig. 1.
tumor grade (G1–G2 vs. G3), tumor thickness (B0.7 vs. [0.7 cm), T (T1–2 vs. T3–4), and N (N0 vs. N1–3) classification, overall TNM stage (I–II vs. III–IV), resection margin (not involved vs. involved), perineural invasion, lymphovascular invasion, and cytoplasmic and nuclear iASPP expression (low vs. high). The log-rank test and Cox proportional hazards model were used to examine the significance of differences in recurrence or survival outcomes according to the dichotomized values of the tested variables. Cox proportional hazard regression analyses were performed, including variables with p-values \ 0.05 on univariate analyses. Standard descriptive statistics were calculated as frequency and percentage for categorical data, and median and range for continuous variables. Statistical significance between iASPP expression and clinicopathological parameters was analyzed by the v2test. All tests were twosided, and p-values \ 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS software version 21.0 (IBM Corporation, Armonk, NY, USA).
Statistical Analysis The Kaplan–Meier method was used for locoregional control (LRC), DFS, and OS curve estimations. The time interval for the LRC or DFS endpoint was calculated from the time of surgery or the first day of treatment to the date of recurrence or last follow-up. The time interval for the OS endpoint was calculated from the time of surgery to the date of death from index cancer or any cause, or censored at the date of last follow-up for alive patients. Prognostic factor analyses focusing on LRC, DFS, and OS outcomes were performed by including the dichotomized variables as follows: age (B60 vs. [60 years), sex,
RESULTS Patient Characteristics The 186 eligible patients consisted of 112 men and 74 women, with a median age of 60 years (range 29–95 years). The clinicopathological characteristics of
iASPP Expression in OSCC
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TABLE 1 Patient characteristics Characteristic
N
%
Sex Male
112
60.2
Female
74
39.8
Age [years; median (range)]
60 (29–95)
Smoking
100
53.7
Alcohol drinking
112
60.2
Tongue
139
74.7
Buccal
16
8.6
During a median follow-up of 74 months (range 16–166 months), 64 (34.4 %) patients had recurrences as follows: local (n = 30), regional (n = 42), and distant (n = 24), with mutually overlapping recurrences in part. At the last follow-up, 57 (30.6 %) patients had died of OSCC, 19 (10.2 %) had died of other causes, and three (1.6 %) were alive with disease. The 2-year LRC, DFS, and OS was 89.2, 87.6, and 88.1 %, respectively, and the 5-year LRC, DFS, and OS was 73.6, 70.2, and 75.3 %, respectively.
Primary site
Mouth floor
14
7.5
Gingiva Retromolar trigone
7 5
3.8 2.7
Hard palate
4
2.2
Lip
1
0.5
111/58/17
59.7/31.2/9.1
T1/2
76/80
40.9/43.0
T3/4
9/21
4.8/11.3
N0
102
54.8
N1/N2/N3
33/51/0
17.7/27.4/0
I/II
57/33
30.6/17.7
III/IV
35/61
18.8/32.8
98
52.7
80/8
43.0/4.3
Tumor grade (G1/G2/G3) T classification
N classification
Overall TNM stage
Primary treatment Surgery alone Surgery ? PORT/CRT Follow-up, months Median (range)
74 (16–166)
Last status (NED/DOD/DOC)
98/57/19
52.7/30.6/10.2
Recurrence
64
34.4
CRT concurrent chemoradiation therapy, NED no evidence of disease, DOD died of disease, DOC died of other causes, PORT postoperative radiation therapy, TNM tumor-node-metastasis stage (American Joint Committee on Cancer, 7th edition)
patients are summarized in Table 1. Primary tumors were most commonly found in the oral tongue (74.7 %), and were poorly differentiated (G3) in 17 patients (9.1 %). Thirty (16.1 %) patients had advanced T-stage tumors and 84 (45.2 %) had pathologically positive cervical lymph nodes; 96 (51.6 %) patients were in advanced overall stage III–IV. Median tumor thickness was 0.7 cm (range 0.1–2.5 cm), and 100 (53.8 %) patients had tumors [0.7 cm. The resection margin was involved in 15 (8.1 %) patients. Perineural and lymphovascular invasion were observed in 21 (11.3 %) and 13 (7.0 %) patients, respectively.
Overexpression of iASPP in Oral Cavity Squamous Cell Carcinoma iASPP immunoreactivity was predominant in the cytoplasm of HNSCC tumor cells, although nuclear staining was also observed (Fig. 1). Of 186 study patients, 53 (29.0 %) and 65 (34.9 %) showed a staining intensity of grade 3 in the cytoplasm and nucleus, respectively. High-intensity staining of iASPP was found in both the cytoplasm and nucleus of tumor tissues, and high staining intensity ([grade 2) in the cytoplasm was significantly correlated with that in the nucleus (p \ 0.001). Grade 4 positive staining in the cytoplasm and nucleus was observed in 154 (82.7 %) and 73 (39.2 %) patients, respectively. High-intensity iASPP staining in the cytoplasm was also significantly correlated with that in the nucleus (p \ 0.001). Overall, high IRS scores ([6) in the cytoplasm and nucleus were found in 132 (71.0 %) and 93 (50.0 %) patients, respectively, and were positively correlated with each other (p \ 0.001) [electronic supplementary Table S1]. Univariate and Multivariable Analyses for Recurrence and Survival The results of univariate analyses of factors affecting patient outcome are shown in Table 2. Pathologic nodal metastasis, advanced overall stage III–IV, and lymphovascular invasion were significantly associated with poor LRC, DFS, and OS (p \ 0.005). High-grade tumors and positive resection margins were also significant factors associated with poor DFS and OS (p \ 0.02). Tumor thickness ([0.7 cm) was a significant factor for OS (p = 0.008) but not for LRC or DFS (p [ 0.05). Cytoplasmic iASPP expression was significantly associated with LRC, DFS, and OS (p \ 0.05), whereas nuclear iASPP expression was not (p [ 0.1) [Fig. 2]. In addition, the subgroup analyses of LRC, DFS, and OS between high cytoplasmic/low nuclear iASPP versus high cytoplasmic/high nuclear iASPP showed no significant differences (p [ 0.3). In multivariate analyses, N classification, lymphovascular invasion, and cytoplasmic iASPP expression remained independent variables for LRC, DFS, and OS (p \ 0.05) [Table 3].
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TABLE 2 Univariate analysis of factors affecting locoregional control, disease-free survival, and overall survival Locoregional control HR
a
95 % CI
Disease-free survival p value
a
HR
a
95 % CI
Overall survival p value
a
HRa
95 % CI
p valuea
Age (years) B60
1
[60
1.298
1 0.753–2.237
0.348
0.605–1.744
0.922
1.401
1 0.847–2.317
0.185
0.667–1.800
0.719
1.702
0.988–2.934
0.050
0.605–1.744
0.922
0.540–1.971
0.923
0.553–2.011
0.873
1.155–4.814
0.018
1.223–3.741
0.008
0.580–2.430
0.638
2.217–6.940
\0.001
1.814–5.809
\0.001
1.626–6.397
0.001
0.829–3.453
0.149
Sex Male
1
Female
1.027
Smoking No Yes
1
1 1.045
1.096
1
1 0.590–1.852
0.880
2.48
0.630–2.311
0.579
1.06
0.075
1
1.027 1
0.231–0.322
0.442
1.03
0.523–1.662
0.936
1.381–5.358
0.004
0.933–2.577
0.091
0.561–2.176
0.774
1.188
1.560–4.333
\0.001
3.923
1.381–3.925
0.002
Alcohol drinking No
1
Yes
1.201
1
1 1.05
Tumor grade G1–G2
1
G3
2.060
0.929–4.566
2.721
1 2.358
Tumor thickness (cm) B0.7
1
[0.7
1.138
1 0.664–1.952
0.638
1.551
0.580–2.444
0.635
1.105
1.190–3.535
0.010
2.600
1.161–3.556
0.013
1 2.139
pT classification T1–T2
1
T3–T4
1.190
pN classification N0 Any N?
1
1 2.051
1
1
1
Overall stage I–II
1
III–IV
2.032
1 2.329
1 3.246
RM Not involved
1
Involved
2.173
1 0.979–4.822
0.056
2.720
0.575–2.825
1.275
1.415
2.080–8.910
\0.001
1.325–6.024
0.007
0.801–2.365
0.248
1 1.381–5.357
0.004
3.225
0.698–2.867
0.336
1.692
4.091
2.061–8.121
\0.001
4.913
2.469–9.778
\0.001
1 1.906
1.032–3.520
0.039
1 1.901
1.012–3.408
0.046
0.861–2.330
0.170
0.907–2.620
0.110
PNI No
1
Yes
1.275
1
1
LVI No
1
Yes
4.305
1
1
Cytoplasmic iASPP expression Low High
1 2.826
Nuclear iASPP expression Low
1
High
1.376
1 1.417
1 1.541
Bold values are statistically significant (p \ 0.05) CI confidence interval, HR hazard ratio, PNI perineural invasion, LVI lymphovascular invasion, RM resection margin, iASPP inhibitor of apoptosis-stimulating protein of p53 a
Cox proportional hazards model was performed on univariate analysis
0.8
Low
0.6
High
0.4 0.2
B
1.0 0.8
C Low
Overall survival
1.0
667
Disease-free survival
A Locoregional control
iASPP Expression in OSCC
0.6
High 0.4 0.2
0
24
48
72
96
120
Low
0.6
High
0.4
P = 0.047 0.0
0.0
0.0
0.8
0.2
P = 0.036
P = 0.005
1.0
0
24
Follow-up (months)
48
72
96
120
0
Follow-up (months)
24
48
72
96
120
Follow-up (months)
FIG. 2 Kaplan–Meier curves comparing locoregional control (a), disease-free survival (b) and overall survival (c), according to cytoplasmic iASPP expression. Log-rank test, p \ 0.05 TABLE 3 Multivariate analyses of factors affecting locoregional control, disease-free survival, and overall survival Locoregional control HR
a
Disease-free survival
95 % CI
p value
1.072–3.302
0.028
a
HR
a
Overall survival
95 % CI
p value
1.435–4.055
0.001
a
HRa
95 % CI
p valuea
1.676–5.497
\0.001
1.960–8.431
\0.001
1.144–4.273
0.018
pN classification N0
1
Any N?
1.881
1 2.412
1 3.036
LVI No
1
Yes
3.248
1 1.533–6.882
0.002
2.415
1 1.168–4.993
0.017
1.046–3.598
0.036
4.066
Cytoplasmic iASPP expression Low
1
High
2.847
1 1.330–6.096
0.007
1.940
1 2.211
Bold values are statistically significant (p \ 0.05) CI confidence interval, HR hazard ratio, LVI lymphovascular invasion, iASPP inhibitor of apoptosis-stimulating protein of p53 a
Cox proportional hazard regression analyses were performed with backward elimination, including variables with p-values \ 0.05 on univariate analyses
DISCUSSION The present study is the first report showing a significant correlation between overexpression of iASPP and treatment outcomes in human OSCC. Our results showed a statistically significant relationship between high expression of cytoplasmic iASPP and increased disease recurrence rates and poor survival outcome of OSCC patients who underwent curative surgery with or without postoperative radiotherapy or chemoradiotherapy. These findings indicate that iASPP plays a role in OSCC and could serve as a novel prognostic biomarker predictive of treatment outcome in OSCC. The results of the present study were in accordance with those of previous studies in other human malignancies6,12–18 showing that iASPP overexpression in tumor tissues is significantly correlated with tumor aggressiveness, as indicated by increased metastasis, radio- or
chemotherapy resistance, and post-treatment recurrence. In the present study, positive iASPP immunostaining was detected in most OSCC tissue samples and predominantly in the cytoplasm, although it was also present in the nucleus, consistent with a recent report on iASPP in HNSCC.19 The diffuse reactivity in the cytoplasm and nucleus could be attributed to the broad specificity of the anti-iASPP antibody used in our study, which shows high affinity for both isoform 1 (located predominantly in the cytoplasm) and isoform 2 (located predominantly in the nucleus) of iASPP. The cellular location of iASPP expression varies among tumor types. Exclusively, cytoplasmic expression of iASPP was reported in non-small cell lung cancer,14 hepatocellular carcinoma,15 and prostate cancer,21 whereas both cytoplasmic and nuclear expression was found in melanoma,12 cervical cancer,18 and HNSCC.19 Although iASPP immunoreactivity was observed in approximately 50 % of
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the cases analyzed, the present study showed that overexpression of iASPP in the cytoplasm, but not in the nucleus, is an important prognostic indicator in OSCC. This might imply a potentially different biological/prognostic nature between cytoplasmic and nuclear iASPP expression, which requires further investigation. In cervical cancer, increased expression of nuclear iASPP is correlated with shorter OS and DFS, whereas cytoplasmic iASPP expression is not, and nuclear iASPP overexpression is correlated with chemo- and radioresistance.18 In HNSCC, overexpression of iASPP in the cytoplasm and nucleus is an independent prognostic indicator for OS and DFS.19 Further investigation is necessary to understand the relation between iASPP cellular localization and its prognostic significance. In the present study, cytoplasmic and nuclear iASPP expression levels were not significantly associated with lymph node metastasis (p [ 0.1). This differs from previous findings showing that nuclear iASPP is enriched in metastatic melanomas and significantly correlated with poor OS.12 As a key p53 inhibitor, iASPP plays an important role in apoptosis regulation. In keratinocytes, iASPP was recently shown to participate in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion, suggesting iASPP is a key regulator of epithelial homeostasis.22 Epithelial-mesenchymal transition (EMT), which is critical in normal epithelial cells and for the metastatic process, may be activated by the upregulation of iASPP.12,23 The role of iASPP in increased metastatic propensity requires further molecular and clinical investigations. Liu et al. 19 examined the clinical relevance of iASPP expression as a determinant of prognosis in 109 patients with laryngeal (n = 99) or hypopharyngeal (n = 10) SCC who underwent partial or total laryngectomy. These authors showed that cytoplasmic and nuclear overexpression of iASPP is significantly associated with advanced T or clinical stage, lymph node metastasis, and recurrence (p \ 0.005), and significantly correlated with shorter DFS and OS (both p \ 0.001 for cytoplasmic and nuclear expression). Our results confirmed the independent prognostic value of iASPP in patients with OSCC in addition to pathologic factors such as lymph node metastasis and lymphovascular invasion. Furthermore, inhibition of iASPP activity in cancer cells had a negative effect on tumorigenesis and improved radio-/chemosensitivity, as reported previously.15,16 Therefore, iASPP may serve as a potential therapeutic target in OSCC and HNSCC. Our study had several limitations inherent to its retrospective design. However, potential bias was reduced through the inclusion of patients with a single pathology of SCC arising in the oral cavity who underwent uniform treatments, including initial curative resection and neck dissection. Another limitation of the study is that iASPP expression was
J. W. Kim et al.
not examined in all tissues of primary tumors and metastatic lymph nodes in the neck. Assessment of iASPP expression in metastatic lymph nodes may provide additional information regarding its prognostic significance. CONCLUSIONS The present study showed that iASPP is overexpressed in OSCC tissues, and increased cytoplasmic iASPP is correlated with recurrence and poor survival outcomes in OSCC patients, suggesting iASPP as a novel biomarker and therapeutic target for OSCC. Further study is necessary to validate the present findings. ACKNOWLEDGMENT This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, which is funded by the Ministry of Education, Science and Technology (Grant No. NRF-2012R1A1A2002039), Seoul, Korea (Jong-Lyel Roh). DISCLOSURE Ji Won Kim, Jong-Lyel Roh, Yangsoon Park, Kyung-Ja Cho, Seung-Ho Choi, Soon Yuhl Nam, Sang Yoon Kim have declared no conflicts of interest.
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