Background
Traditionally, level of neoplastic differentiation is a crucial index of adjuvant chemotherapy and prognosis. Retinoblastoma (RB), the most common primary malignant intraocular tumor in childhood, has different pathological phenotypes from undifferentiated tumor cells, Homer-Wright rosettes (HWR) or Flexner-Winterstein rosettes(FWR) to fleurettes. These phenotypes reflect to some extent the level of tumor differentiation which is a key factor in grade of retinoblastoma and prediction of its prognosis [
1],[
2]. For example, retinocytoma (RC), also called retinoma, has been proposed to be the benign variant of RB since it is largely composed of benign-appearing cells with elongated eosinophilic fleurettes similar to the inner segment of photoreceptor cells and because it has better prognosis than RB in general [
3]-[
5]. However, these components with various degree of differentiation have been a challenge to be morphologically identified by researchers, sometimes even by experienced pathologists. It would therefore be very useful to find out a panel of molecular markers to distinguish them and to predict progression of RB.
p16
INK4a, a tumor suppressor protein, has played an important role in cell cycle control, cell senescence and tumor development. It has been extensively studied in both benign and malignant lesions with very different results, ranging from its loss to its clear overexpression [
6]. For instance, in benign lesions such as nevi and neurofibroma, p16
INK4a overexpression was associated with senescence [
7],[
8]; whereas in malignancy such as HPV-positive cervical cancer, breast cancer and colorectal adenocarcinoma, it appeared to be associated with high-grade tumors along with RB gene alterations [
9]-[
11]. In recent years, p16
INK4a has been used as a diagnostic marker to distinguish between benign and malignant lesions, and some evidence suggests that it also plays a prognostic role in tumors that overexpress p16
INK4a[
12].
p16
INK4a expression in RB remains controversial, especially in regarding of its expression patterns in histological phenotypes of RB [
4],[
13],[
14]. Dimaras and colleagues reported that p16
INK4awas expressed in fleurette regions but negative in undifferentiated regions [
4]. Conversely, SE Coupland et al. described a reverse expression pattern of p16
INK4a that was positive in undifferentiated areas of 23 RB tumors [
14]. To better understand p16
INK4aexpression and its potential role in RB, we evaluated the expression patterns of p16
INK4a, Ki67 and CRX through a large cohort of 65 RB tumors. CRX is required for the terminal differentiation and maintenance of photoreceptors [
15] and has been shown to be a differentiation marker of RB [
16],[
17]. Our results demonstrated that p16
INK4a expression increased with the dedifferentiation of RB, showing strongly positive in undifferentiated cells and HWR, weakly to moderately positive in FWR, but negative in well-differentiated cells with fleurettes. This expression trend of p16
INK4a was consistent with that of Ki67, but reverse to that of CRX in RB tumors. The results indicated that p16
INK4a would be a valuable molecular marker of RB in distinguishing histological phenotypes and in serving as a predictor of its prognosis.
Methods
Tumor samples
Formalin-fixed, paraffin-embedded retinoblastoma blocks were retrieved from the archives of ocular pathology department at Zhongshan Ophthalmic Center of Sun Yat-sen University, China during a 3-year period (2008 to 2010). The cases with chemotherapy or radiotherapy prior to enucleation were excluded. Serial sections of 4 μm thick were cut, and subjected to hematoxylin and eosin (HE) staining. The tumor samples were divided into four groups according to the level of cell differentiation: undifferentiated (UD) group predominantly consisting of undifferentiated tumor cells and pseudorosettes; HWR or FWR group in which rosettes accounted for over 70% of the lesion according to the predominant rosette phenotype; and RC group in which fleurettes characteristic of photoreceptor differentiation accounted for over 60% of the lesion [
1],[
2]. The study was approved by Medical Ethical Committee of Zhongshan Ophthalmic Center, Sun Yat-Sen University.
Slide preparation and immunohistochemistry
Sections were deparaffinized in xylene, re-hydrated and incubated in 0.3% hydrogen peroxide (H2O2, Sigma, St. Louis, USA) for 30 min to block endogenous peroxidase activity. Antigen retrieval was achieved using 0.01 M citrate buffer, pH 6, in a pressure cooker for 45 s. For immunohistochemistry, the sections were stained with rabbit anti-CRX (1:200) (C7498, Sigma, St. Louis, USA), mouse anti-human p16INK4a (ZJ11, Maixin-bio, China) and rabbit anti-Ki67 (1:200) (sc-15402, Santa Cruz, California, USA) respectively. Signals were detected using the Dako-Cytomation EnVision+ anti-rabbit or anti-mouse secondary system. The slides were then incubated in DAB solution (Vector Laboratories DAB substrate kit for peroxidase) for approximately 1 min, then washed in PBS and counterstained briefly in Harris hematoxylin. Images were taken using a BX51 microscope (Olympus, Japan). The primary antibody was replaced by dilution buffer as a negative control. The residual retinal tissue adjacent to tumor was considered as an internal control.
The sections were semi-quantitatively evaluated for the expression level of proteins according to reference with modifications [
18]. Briefly, the intensity of CRX and p16
INK4a expression was scored on a scale of 0–2: 0, negative, 1, weak, and 2, strong; whereas the extent of both p16
INK4a and Ki67 expressions was scored as followings: grade 0, no staining; grade 1, < 40%; grade 2, 40-80%; and grade 3, 80–100% of tumor cells in the respective lesion. To analyze the association of p16
INK4a with clinicopathological features, both grade 0 and 1 were considered as low expression, both grade 2 and 3 as high expression. The high-risk feature evaluation included the presence and extent of optic nerve invasion and the massive choroidal invasion [
19]. All specimens were screened and evaluated by two experienced pathologists blinded to the clinical information.
Statistical methods
Spearman’s rank test and the Kruskal-Wallis test were used to analyze the progressive increase of p16INK4a expression in various pathological types of RB. The association between the expression measured via immunohistochemistry and high-risk features was analyzed using chi-squared tests and Kruskal-Wallis tests. In all analyses, p < 0.05 was accepted as statistically significant. These analyses were performed using the SPSS 16.0 software package (SPSS Inc., Chicago, Illinois, USA).
Discussion
The development of RB involves sequential genetic lesions, and the loss of RB protein (pRB) is the first step and the basis of other biological alterations. In 1998, Schwartz found that pRB dysregulation resulted in increased p16
INK4a expression in colon cancer, due to positive feedback [
21]. In recent years, the progressive increase of p16
INK4a expression accompanying pRB dysfunction had been described in many tumors [
22]. In 2011, Witkiewicz summarized two complementary models for the appearance of tumors with high levels of p16
INK4a[
6]. In the first model, p16
INK4a was elevated in some precancerous lesions due to oncogenic stress. If the RB1 gene was functional, the cells became senescent; otherwise, they became cancerous. In the second model, p16
INK4a increased via a feedback loop in response to pRB dysfunction and was associated with highly malignant tumors. The results of the present study were consistent with the second mechanism described by Witkiewicz.
Immunohistochemistry investigation of p16
INK4a in RB tumor samples had been reported with controversial results. Dimaras et al. described p16
INK4a expression in 8 RC cases, showing p16
INK4a positive in the benign RC region but negative in the RB area [
4]. However, SE Coupland and colleagues reported the reverse expression pattern that p16
INK4a was negative in RC area but positive in poorly and moderately differentiated areas in their 23 RB tumors [
14]. Similarly, Indovina et al. described that 6 out of 11 RB cases were 100% positive for p16
INK4a and that other cases were 5%-40% positive [
13], implying that undifferentiated tumor cells were positive. In our 65 cases, the expression pattern of p16
INK4a in RB was closer to what SE Coupland described in a way that the level of p16
INK4a was increased from negative expression in well-differentiated fleurette structures to highly strong expression in poorly differentiated area. This pattern of p16
INK4a in RB was similar to that of Ki67, but reverse to that of CRX. Therefore, our study indicated that p16
INK4a could serve as a potential molecular marker to distinguish the level of cell differentiation in RB tumors.
The classic function attributed to p16
INK4a has been cell cycle regulation in the nucleus; however, p16
INK4a overexpression could occur in both the cytoplasm and nucleus of malignant cells. For example, in colorectal and breast cancers, p16
INK4a exhibited strong nuclear/cytoplasmic positivity in primary or metastatic carcinomas, whereas negativity or low nuclear expression was observed in normal mucosa and benign fibroadenoma [
23],[
24]. Previous studies had shown that p16
INK4a appeared in the cytoplasm and/or nucleus in RB tumor cells [
4],[
13],[
14]. Similarly, in our RB cases, the location of p16
INK4a expression largely depended on the degree of tumor differentiation, from staining in both the cytoplasm and nucleus of poorly differentiated cells to primary cytoplasm of moderately differentiated cells. Moreover, negative expression of p16
INK4a was seen in both the residual retina adjacent to the tumor and normal human retinal tissue. Nevertheless, the significance of different sublocations of p16
INK4a expression in malignant cells has not been clearly clarified yet,which may be associated with the prognosis of the malignant tumor. Many efforts have been made to understand the regulation mechanisms underlying p16
INK4a expression location within the tumor cells and its possible therapeutic value by re-locating the dislocated p16
INK4a to the optimal subcellular site in the malignancy [
25]-[
27]. More work is needed to answer these questions in the RB cases.
To date, the prognostic factors of RB remained a subject of intense discussion among ophthalmologists. The only consensus was that some high-risk histopathological features, such as the presence and extent of optic nerve invasion and the location and extent of uveal invasion [
28], may be predictive. Grade of tumor differentiation has also been served as a key predictor of RB prognosis for a long time. In our 65 RB cases, both RC and FWR groups could be classified into the low grade of RB tumors, where most of tumors expressed low level of p16
INK4a. However, the other two groups HWR and UD could be taken as the high grade of RBs, where most of tumors expressed high level of p16
INK4a. Our results also demonstrated tumors with high expression of p16
INK4a had higher risk features with the optic nerve invasion and uveal invasion. These data suggested that the overexpression of p16
INK4a might be a risk predictor of the poor prognosis of RB tumors.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YL and YPL designed the experiment. YL and XZ analyzed the data and wrote the manuscript. YL, STW, WXZ and JXL carried out the immunohistochemical examination. PZ and YPL evaluated clinicopathological data. YPL provided the funding support. All authors read and approved the final manuscript.