Comparison of SKIP expression in malignant pleural mesotheliomas with Ki-67 proliferation index and prognostic parameters

Introduction

Malignant mesothelioma is a cancer of mesothelial cells mainly originating from the pleura, although the pericardium, peritoneum or tunica vaginalis may also be affected. Malignant pleural mesothelioma (MPM) is related to environmental and occupational asbestos contact and typically has a poor prognosis. Although the most significant etiological agent is asbestos, radiotherapy and inhalation of other silicates, such as thorium dioxide and erionite, are other etiological factors [1, 2].
Despite the therapeutic applications including chemotherapy, radiotherapy, immunotherapy and radical surgery performed in some patients, MPM still has a poor prognosis, with a reported average survival of approximately 12 months [3, 4, 5].
Different scoring systems have been used to estimate the prognosis of MPM. In these systems, performance conditions, disease subtypes, age, gender and basic laboratory parameters of the patients were used [6, 7]. However, these systems have not been routinely used because of difficulties in evaluating multiple parameters and in applying them to clinical settings. The most important prognostic factors are accepted as histological type, performance condition of the patient and weight loss. Epithelial histology, early tumor stage, high performance score and absence of weight loss are factors positively affecting survival [6, 7].
Various studies have been performed to determine the correlation of MPM progression with markers such as survivin, fibulin-3 and mesothelin [8, 9, 10]. Although a relationship with prognosis and diagnosis was found for some of these markers, it was not found in some others. However, an immunohistochemically detectable marker that accurately defines the prognosis has not been determined yet. Therefore, a cheap, repeatable, effective and easy parameter is required to accurately predict the MPM prognosis.
Ki-67 is a nuclear non-histone protein that can be used as a proliferation marker because its activity increases during mitosis. It may also be used to evaluate the division speed of tumor cells [11]. Ki-67 is a monoclonal antibody developed against a nuclear antigen only available in proliferated cells. Determining Ki-67 expression levels has been demonstrated to be helpful in predicting the prognosis of breast cancer, prostate cancer and many other malignancies such as malign mesothelioma [12, 13, 14].
Ski-interacting protein (SKIP) is a transcriptional cofactor effective in oncogenesis, controlling many signaling pathways that have a role in cell multiplication and differentiation [15, 16]. In the early stages of cancer formation, up-regulation of SKIP protein has been detected. Increased SKIP expression in breast cancer, hepatocellular cancer (HCC) and bladder cancer was found to correlate with a poor prognosis; however, there have so far been no studies investigating the correlation between SKIP expression and the prognosis of MPM [17, 18, 19].
In the present study we aimed to identify the presence of SKIP expression in MPM and to evaluate its relationship with the Ki-67 proliferation index and prognostic parameters.

Material and methods

Local ethical approval was obtained from the Dicle University Medical Faculty Ethics Committee. The medical records and the pathologic blocks of the patients with MPM between 2006 and 2012 were retrospectively evaluated. If the patients met the following criteria, they were excluded from the study: 1) the blocks were taken from the archives for consultation in another centre; 2) insufficient tissue quality and/or quantity in available blocks; 3) patients who were lost to follow-up. Fifty-two formalin-fixed paraffin-embedded tumor tissue samples and slides were retrieved in total from the archives of the Pathology Department, with relevant clinical data (gender, age at diagnosis and follow-up data) obtained from the patient records.
Diagnostic re-evaluation of the slides of each case was made according to the World Health Organization classification guidelines of MPM (epithelioid, sarcomatoid, desmoplastic, or biphasic). The clinical records and histopathological diagnosis of all patients were fully documented.
All cases were clinically staged in accordance with the tumor, node, metastasis (TNM) classification of MPM.
Age (≤ 60 or > 60 years old), gender, histopathological subtype (epithelial or non-epithelial), stage of the disease (stage I-II or stage III-IV), low (score of 1) or high (score of 2 or 3) SKIP expression and Ki-67 proliferation index (≤ 30% or > 30%) were determined as evaluation criteria.

Immunohistochemistry

Ki-67 (pre-diluted, ready-to-use; PRM 325 AA, rabbit monoclonal, Biocare, Concord, USA) and anti-SKIP antibodies (1 : 200 dilution; ab154575, rabbit polyclonal, Abcam, Cambridge, UK) were used for immunohistochemical staining. After the archive case slides were re-examined by light microscopy, 4-µm cross-sections were made from the paraffin blocks. The tissue sections were stained by standard immunohistochemical techniques using a Ventana BenchMark Ultra Automated Immunostainer (BenchMark Ultra; Ventana Medical Systems, Tucson, AZ, USA), using heat-mediated antigen retrieval with citrate buffer at pH 6 and a standard diaminobenzidine detection kit (Ventana). Evaluations were made by light microscopy (Olympus BX53, Tokyo, Japan).
Immunostained sections were examined by two investigators (G.T and U.A.) who were blind to patient characteristics. Nuclear staining was accepted as Ki-67 positivity. Approximately 500 cells were counted on every slide, and the evaluation of proliferative activity (proliferation index) was made according to the study by Deraco et al. [20].
The SKIP expression was assessed by semi-quantitative (manual) scoring. Only cytoplasmic staining was adopted. The presence of SKIP staining in 10% or more of tumor cells was accepted as SKIP positivity. Intensity of staining was graded as weak (score of 1), moderate (score of 2), or strong (score of 3).

Statistical analysis

SPSS version 11.5 (Statistical Package for the Social Sciences) was used for statistical analyses. The correlation of SKIP expression with the patients’ clinical and pathological characteristics was analyzed using the chi-square test. The effects of clinical and pathological characteristics and SKIP expression status on general survival were evaluated by the Kaplan–Meier method in univariate analysis and by the Cox regression method in multivariate analysis. Results were considered statistically significant at a p value less than 0.05.

Results

We reviewed the medical records of 89 patients with MPM. Of them, 52 patients were eligible for the study. The mean age of the patients was 62.3 ±12.2 years (40-83 years); 36 (69.2%) of them were male and 16 (30.8%) were female. The distribution of MPM histological types was as follows: 45 epithelioid (86.5%), 4 sarcomatoid (7.7%) and 3 biphasic (5.8%).

Immunohistochemistry

SKIP and Ki-67 expression was observed in all MPM tissues in our study (Figs. 1 and 2). SKIP staining was observed in a cytoplasmic expression pattern in these tissues. Nuclear staining was observed with Ki-67. The scoring of SKIP expression showed high levels of positivity in 21 cases and low levels of it in 31 cases. The Ki-67 proliferation index was greater than 30% in 18 cases and equal to or lower than 30% in 34 cases.
There were significant differences in the expression of SKIP and the Ki-67 proliferation index in the epithelioid and non-epithelioid histological types (Table I).

Survival

The overall median survival of the patients was 8.5 months (95% CI: 9.37-15.13).
Clinical and pathological characteristics were evaluated by univariate and multivariate analyses. According to the univariate log rank test, the survival period was found to be significantly shorter (p < 0.05) in relation with the Ki-67 proliferation index being over 30%, increased SKIP expression intensity, presence of non-epithelial histological type of MPM and the disease stage being III or IV during diagnosis (Table I). Figures 3 and 4 show the Kaplan–Meier survival curves of the patients compared with SKIP and Ki-67 expression. There was no effect of age or gender on the prognosis of MPM. In the multivariate analysis, while it was determined that SKIP expression prevalence, Ki-67 proliferation index being over 30% and non-epithelial histological type had an independent negative effect on survival according to the multivariable Cox regression analysis results, there could not be detected any effect of advanced stage disease as an independent factor on survival. When the effects on survival were examined in Cox regression analysis, it was observed that high SKIP expression intensity, a Ki-67 proliferation index over 30% and non-epithelioid histological type of MPM reduced survival by two-fold, four-fold and five-fold respectively (Table II).
A Ki-67 proliferation index below 30% was found 15.5 ±1.9 years, being over 30% was found 6.2 ±0.9 years, 12.3 ±1.4 years in total, in those with lower SKIP expression 20.5 ±2.2 years, in those with high SKIP expression 6.6 ±1.0 years, 12.3 ±1.4 years, in epithelioid type 13.3 ±1.6 years, in non-epithelioid type as 5.7 ±1.7 years and 12.3 ±1.4 years in total.
A moderate positive correlation was observed between the Ki-67 proliferation index and SKIP expression (p = 0.011, r = 0.352).

Discussion

To the best of our knowledge, this is the first study to evaluate the effect of SKIP expression on prognosis in MPM patients. We found that SKIP expression may be an indicator of poor prognosis in MPM patients. However, this study was a preliminary investigation, with a small sample size; therefore, a larger study is warranted to confirm the results.
Malignant mesothelioma has a poor prognosis and occurs due to environmental factors. Like other types of malignancy, it is a disease with a complex, heterogeneous genetic and biochemical background. The exact mechanisms of MPM pathogenesis are currently unclear [1, 2, 3, 4, 5]. The multifarious biological and clinical features of MPM have prompted the search for more useful prognostic and predictive markers for use in diagnosis and therapy.
Notch signaling proteins, SKIP, tumor growth factor- signaling proteins, CBF-1, Smad2 and E7 oncoprotein are transcriptional cofactors that play roles in many signaling pathways that control cell multiplication and differentiation; thus, they may also be involved in oncogenesis [15, 16, 21]. Increased SKIP protein is detected in early cancer formation, and SKIP expression has been described as a promising factor for predicting prognosis in HCC, breast cancers and bladder cancers [17, 18, 19]. The present study also indicates SKIP expression to be useful for predicting poor outcome in patients with MPM. Biomarkers such as survivin, fibulin-3 and mesothelin have also been suggested as prognostic factors to predict poor outcome in MPM, but they are not sufficient for use as prognostic biomarkers [8, 9, 10].
It is known that the Ki-67 proliferation index increases in MPM patients and that it is related to poor prognosis [9, 14]. In HCC, alterations in SKIP expression and the Ki-67 proliferation index were found to be correlated, and it was reported that the increase of both of them is a short survey indicator in HCC patients [18]. In our study, an increase in SKIP expression and the Ki-67 proliferation index was detected in MPM patients, and it was observed that the increase in these values was related to poor prognoses. It was demonstrated that the increase in Cox regression analysis reduced the survival rate two-fold.
In HCC patients, it was found that SKIP was effective in proliferation assessment similar to Ki-67. It has been reported that proliferation of SKIP expressing cells in HCC increases in G1 and S phases of the cell cycle [18]. It was also found that SKIP expressed by the retinoblastoma gene directly affects pRb protein. It was suggested that SKIP could advance tumorigenesis by being effective in cell proliferation [16]. The presence of a correlation between both values in our study suggests that SKIP is effective in proliferation assessment in MPM patients, similarly to the case in HCC.
Many factors were defined regarding the prognosis of MPM patients. These included age, gender, non-epithelioid histological type, thrombocytosis and different biological agents [2, 22]. We did not find any effect of age or gender on the prognosis of MPM. In the multivariate Cox regression analysis, it was found that the factors affecting survival were SKIP expression intensity, Ki-67 proliferation index and non-epithelioid histological type. In previous studies, it was reported that the Ki-67 proliferation index was higher in MPM cases and that survival periods were shorter [9, 14]. Similarly, the Ki-67 proliferation index was higher in non-epithelioid cases in our study.
SKIP expression has not been studied before in MPM cases, but it has been reported as a prognostic factor in HCC, cancer of the bladder or breast [17, 18, 19]. It has been reported that in high-grade urothelial carcinoma, SKIP mRNA levels increase much more than those in low-grade urothelial carcinoma and that this is associated with a decrease in the survival rate [19]. In breast cancer, it was reported that the increase in SKIP expression was correlated with an increase in the Ki-67 proliferative index [17]. An increase in SKIP expression in the G1 and S phases was also reported. Additionally, it was observed that the increase in SKIP expression caused a decrease in p27 values in MCF-7 and MDA-MB-231 cell lines, which resulted in oncogenic potential. In breast cancer cases, it was detected that the increase in SKIP value was related to lower patient survival [16, 17, 18].
In conclusion, we observed that SKIP expression was increased in MPM patients. The increase in SKIP expression was found to correlate with the Ki-67 proliferative index, and it is an effective factor predicting the patient survival period. Although this was a preliminary study, we identified SKIP expression as a useful predictor of poor prognosis. Clinical studies investigating this approach will be required to confirm our results.

The authors declare no conflict of interest.

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Address for correspondence

Gül Türkcü
Dicle Üniversitesi TIp Fakültesi,
Sur/DiyarbakIr, Turkey
tel. 90 412 2488001
fax 90 412 2488523
e-mail: gaturkcu@gmail.com