== Expressions of p16INK4a, CRX and Ki67 in a representative RB case from UD group (40). and subjected to hematoxylin and eosin staining. Immunohistochemical staining was further done with antibodies p16INK4a, CRX and Ki67. The correlation of p16INK4aexpression with CRX and Ki67 and clinicopathological features of RB were analyzed. == Results == RB tumor histologically consists of various differentiation components including undifferentiated (UD) cells, Homer-Wright rosettes (HWR) or Flexner-Winterstein Thapsigargin rosettes (FWR) and fleurettes characteristic of photoreceptor differentiation or Retinocytoma (RC). p16INK4aexpression was negative in both fleurette region and the residual retinal tissue adjacent to the tumor, weakly to moderately positive in FWR, strongly positive in both HWR and UD region. However , CRX had the reverse expression patterns in comparison with p16INK4a. It was strongly positive in photoreceptor cells within the residual retina and fleurettes, but weakly to moderately positive in UD area. Together with Ki67 staining, high p16INK4aexpression was associated with poor histological differentiation of RB tumors, which had higher risk features Rabbit Polyclonal to MRPL54 with the optic nerve invasion and uveal invasion. == Conclusions == p16INK4aexpression increased with the decreasing level of cell differentiation of RBs. RB tumors extensively expressing Thapsigargin p16INK4atended to have higher risk features with poor prognosis. This study suggested that p16INK4awould be a valuable molecular marker of RB to distinguish its histological phenotypes and to serve as a predictor of its prognosis. == Virtual Slides == The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_180 Keywords: Retinoblastoma, Differentiation, p16INK4a, Immunohistochemistry == Background == Traditionally, level of neoplastic differentiation is a crucial index of adjunct chemotherapy and prognosis. Retinoblastoma (RB), the most common primary malignant intraocular Thapsigargin 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. p16INK4a, 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, p16INK4aoverexpression was associated Thapsigargin 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, p16INK4ahas 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 p16INK4a[12]. p16INK4aexpression in RB remains controversial, especially in regarding of its expression patterns in histological phenotypes of RB [4, 13, 14]. Dimaras and colleagues reported that p16INK4awas expressed in fleurette regions but negative in undifferentiated regions [4]. Conversely, SE Coupland et al. described a reverse expression pattern of p16INK4athat was positive in undifferentiated areas of 23 RB tumors [14]. To better understand p16INK4aexpression and its potential role in RB, we evaluated the expression patterns of p16INK4a, 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 p16INK4aexpression 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 p16INK4awas consistent with that of Ki67, but reverse to that of CRX in RB tumors. The results indicated that p16INK4awould 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.