Immunohistochemical expression of Ki 67 , EGFR and TRKC and their correlation with prognostic factors in medulloblastoma

First submission on 22/01/14; last submission on 16/06/14; accepted for publication on 16/06/14; published on 20/08/14 1. MSc of Health Sciences-School of Medicine of Pontifícia Universidade Católica do Paraná (PUC-PR); professor of Pathology at School of Medicine-PUC-PR. 2. Resident Doctor at Hospital Nossa Senhora das Graças, Curitiba-PR. 3. Resident Doctor at School of Medicine-PUC-PR. 4. PhD student in Pahology, Universidade Federal do Paraná (UFPR), assistant professor, UFPR. 5. PhD in Pathology-UFPR; head of Anatomical Pathology Service at Hospital Infantil Pequeno Príncipe. 6. PhD in Pathology-UFPR; full professor of the postgraduate course in Health Sciences-PUC-PR. ABStrACt

introDuCtion Medulloblastoma is the most common malignant tumor of the nervous system in childhood.It is an invasive embryonal tumor of the cerebellum with tendency to metastasis by subarachnoid space (13) .It shows a neuroepithelial histological pattern from the cerebellum, consisting of small-round-cells, with hyperchromatic nuclei, immersed in delicate fibrillary matrix (neuropil).Furthermore, medulloblastoma represents 20% of intracranial tumors in children, and its peak incidence occurs around 8 years of age, and only 30% of cases occur in adults (12) .
The prognostic factors already established for cervical cancer are: (1) age of patient at diagnosis; (2) the presence of residual tumor postoperatively; and (3) presence of metastasis.These three factors divide the medulloblastoma patients in at high or low risk.Patients at high risk, usually, are those under three years of age at diagnosis, and/or with metastasis, and/or presence of residual tumor postoperatively, and generally low five-years survival rate (8,12) .Patients upper than 3 years of age at diagnosis, without residual tumor after surgery, and without metastasis, are at low risk and therefore tend to have better fiveyear survival rate (2,8,13) .
originAL ArtiCLE J Bras Patol Med Lab, v. 50, n. 4, p. 290-295, agosto 2014 10.5935/1676-2444.20140030The classification of embryonal tumors based only in clinical and histopathological criteria tends to be limited.However, with the advancement of molecular techniques, the discovery of genetic protein disorders has bringing on new therapeutic and prognostic perspectives in several pediatric tumors.However, the molecular knowledge in medulloblastoma are still scarce, and studies based on molecular analysis, gene expression, and protein expression may provide valuable information that would help in understanding of the pathogenesis of these lesions, in choosing the therapeutic targets, and in the best prognostic stratification in risk groups.Thus, the stratification of this neoplasia in at-risk groups would include biomarkers identification as gene and/or protein modifications, which sometimes may be due to a more aggressive subtype (3,13) .
Several studies conducted with groups of children's tumor, also of embryonal origin, are also using new biomarkers of cellular growth and differentiation, which better stratify these tumors in more accurate prognostic groups, in order to implement more aggressive therapies in the groups that really need it.There are several biomarkers involved in pathways of cellular growth and differentiation that are still under study and appear to have promising results in understanding the biology of medulloblastoma and in its stratification in more reliable prognostic groups, including specially neutrophins and growth factors such as tropomyosin receptor kinase (TRK) and epidermal growth factor receptor (EGFR), pro-apoptotic and anti-apoptotic proteins (B-cell lymphoma 2 [Bcl-2]) and proliferative proteins (Cyclin-D1) (3)(4)(5)(6)15) .

oBJECtivE
The search for new biomarkers useful in routine diagnosis, prognosis, or in the therapy of this neoplasia motivated this study to observe the immunohistochemical expression of Ki67, receptor neurotrophin-3 (TRKC), EGFR, Bcl-2, and ciclina-D1 biomarkers in medulloblastoma, and to evaluate the relationship of these biomarkers with the clinicopathological variables with prognostic value (age, presence of residual tumor, presence of metastases, and risk group).The review of histological slides was performed with hematoxylin-eosin (HE), and the histological subtype of samples were classified according to World Health Organization (WHO) classification of 2007, so that only one case was desmoplastic subtype and other cases were classic subtype.

MAtEriAL AnD MEthoD
From the histological slides, two representative areas of the tumor were selected and five tissue microarrays (TMAs) were created.Areas non-neoplastic of cerebellum were also included in these TMAs.
The immunohistochemical expression of Ki67, Bcl-2, and cyclin-D1 proteins were evaluated by counting the number of positive tumor cells in a 100 tumor cells count per high power field, it was counted four high power fields for each tumor (two fields in each of the to samples).These counts were compiled, and the average values of each neoplasia now represent the immunohistochemical expression of each protein analyzed by high power field.
In the evaluation of immunohistochemical expression of TRKC and EGFR markers, we used the morphometry of colors of the Image Proplus ® application software applicative, in Dell ® computer connected to a Dino Eye ® camera and to an Olympus BX40 microscope.A normal cerebellum sample was used to standardize the immunohistochemical expression of these two antibodies.In this normal cerebellum sample, an image by high power field (400×) was obtained, it showed immunopositive areas for each one of the two antibodies; they were supplied to the software that stored them as a mask.To determine the immunopositive average area per high power field of these two antibodies in medulloblastoma; for each studied case, four images of 40× objective were obtained.The normal cerebellum image (mask) was superposed over each one of these images using the software, which identifies the areas of color similar to the mask, therefore immunopositive, and provides the measure, in square millimeters (mm 2 ), of total immunopositive are per high power field.
The following clinical data were obtained from medical records review of 29 patients: age at diagnosis, risk group, type of surgical resection (complete or not), occurrence or not of postoperative tumor recurrence and time for these recurrence, presence or absences of metastases at diagnosis, realization or not of postoperative adjuvant chemotherapy, and occurrence or not of post-treatment deaths, and time for death.The time for this study follow-up ranged from two to thirteen years.Only 29 from 35 cases had suitable records data (six records were not found or could not be found).
The variable normality condition was evaluated by the Kolmogorov-Smirnov test.To evaluate the correlation of two quantitative variables, the Spearman's correlation coefficient was estimated.For the comparison between two groups in relation to the quantitative variables, the nonparametric Mann-Whitney test was used.The p < 0.05 values indicate statistical significance.Data were analyzed with the Statistica v.8.0 software.

rESuLtS
Considering the 29 studied patients, the average age of patients at diagnosis was 4.8 years, the youngest patient was a month of age and the oldest was 15 years.Twenty patients were at high risk (age under 3 years, presence of residual tumor after surgery and/or presence of metastasis at diagnosis).All patients were subjected to surgical treatment, as recommended by the literature, which 15 of them still had residual lesion after surgery.None of the 29 patients in this study had metastasis at diagnosis.The presence of postoperative recurrence was observed in nine of the 29 patients and the average time for these recurrences was 1.7 years.Twenty-six patients required postoperative chemotherapy.Among the 29 studied patients, three deaths were observed, the average time between the diagnosis and the final outcome was 32.7 days (Table 1).
The average immunoexpression for Ki67, TRKC, and EGFR were compared to the study clinical variations (age, age group, if older or younger than three years of age, risk group, whether   between TRKC and Ki67 (p = 0.027).These analyses were not performed with Bcl-2 neither cyclin-D1, because only six and four cases were positive for these antibodies, respectively.

DiSCuSSion
The medulloblastoma diagnosis is primarily based on morphological criteria; however, occasionally immunohistochemistry may be used, with anti-synaptophysin antibodies (SYN) and anti-glial fibrillary acidic protein (GFAP).Other prognostic biomarker have been proposed for medulloblastoma, Ki67 proliferative protein has been extensively studied, besides having a correlation with the prognosis of these lesions, since higher proliferative indices seems to be correlated with poor prognosis (9,10,14) .
The result analysis showed that older patients had higher expression of TRKC (p = 0.033) (Table 3).A higher expression of these biomarkers seems to be associated with a better prognosis, a fact already recorded in the literature, which also considers the age group over three years as at low risk (9) .We also observed a statistical trend toward Ki67 is inversely correlated with age (p = 0.088), i.e., the higher the age, the lower proliferative rate.This also seems to be in agreement with the literature, since children over 3 years of age are considered at low risk, and, consequently would have lower proliferative rates and better prognosis (Table 3) (10) .
TRKC represents the pathway of growth factors receptors.The main factors found in this group are TRKC, EGFR, platelet derived growth factor receptor (PDGFR), and insulinlike growth factor (IGF) receptor.TRKC, when activated in high or low, presence of complete resection after surgery, presence of postoperative recurrence, presence of postoperative chemotherapy, and occurrence of deaths), data were shown in Tables 2 and 3. Furthermore, the average immunoexpressions of biomarkers were correlated to each other (Table 3).The older patients showed TRKC higher expression (p = 0.033).There was inversely proportional and statistically significant correlation medulloblastoma, stimulate apoptosis.The apoptosis degree is directly proportional to TRKC expression, so, the higher the concentration, the better the prognosis (3,11,12) .
The study showed an inversely proportional and statistically significant correlation between TRKC and Ki67 (p = 0.027), and the greater the Ki67, the lower the TRKC expression.This seems to be in agreement with literature since TRKC is a biomarker of apoptosis of good prognosis, and Ki67 is biomarker of cellular proliferation and poor prognosis, when presenting high levels (9,10) .We also observed a trend (p = 0.071) to a directly proportional correlation between TRKC and EGFR, so that when the first is increasing the latter increases also, which seems to be an antagonistic effect, since TRKC stimulate the apoptosis and EGFR promotes cell growth.
The immunohistochemical expression of Ki67 protein is a marker widely used to assess the rate of cell proliferation in tumors.Medulloblastoma are rapidly proliferating tumors of and have high Ki67 expression (1,7) .The inverse correlation between Ki67 and TRKC indicate, therefore, a better prognosis.

tABLE 1 -
Shows the clinicopathologic variables, risk group and mortality rate of studied patients (n = 29) and the immunostaining of evaluated biomarkers (n = 35)

tABLE 3 -
Shows the correlation between the average immunoexpression of biomarkers and the age of patients (n = 29) at diagnosis, and the correlation of biomarker with each other (n = 35)

tABLE 2 -
Shows the correlation between the average immunoexpression of biomarkers and the clinicopathologic variables of studied patients (n = 29) TRKC and EGFR immunoexpression in square millimeters (mm 2 ); Ki67 immunoexpression in number of positive nuclei in 100 nuclei counted in high power field (HPF = 400×) SD: Standard deviation; TRKC: neurotrophin-3 receptor; EGFR: epidermal growth factor receptor; HPF: high power field.