Year : 2010 | Volume
: 53 | Issue : 1 | Page : 20--23
Proliferating cell nuclear antigen, p53 and micro vessel density: Grade II vs. Grade III astrocytoma
Priya Malhan, Nuzhat Husain, Shalini Bhalla, Rakesh K Gupta, Mazhar Husain
Department of Pathology, CSMMU, Lucknow, India
Department of Pathology, CSM Medical University, Lucknow
Histological classification and grading are prime procedures in the management of patients with astrocytoma, providing vital data for therapeutic decision making and prognostication. However, it has limitations in assessing biological tumor behavior. This can be overcome by using newer immunohistochemical techniques. This study was carried out to compare proliferative indices using proliferating cell nuclear antigen (PCNA), extent of p53 expression and micro vessel morphometric parameters in patients with low grade and anaplastic astrocytoma. Twenty-five patients, each of grade II and grade III astrocytoma were evaluated using monoclonal antibodies to PCNA, p53 protein and factor VIII related antigen. PCNA, p53-labeling indices were calculated along with micro vessel morphometric analysis using Biovis Image plus Software. Patients with grade III astrocytoma had higher PCNA and p53 labeling indices as compared with grade II astrocytoma (29.14 plus/minus 9.87% vs. 16.84 plus/minus 6.57%, p 0.001; 18.18 plus/minus 6.14% vs. 6.14 plus/minus 7.23%, p 0.001, respectively). Micro vessel percentage area of patients with grade III astrocytoma was also (4.26 plus/minus 3.70 vs. 1.05 plus/minus 0.56, p 0.001), higher along with other micro vessel morphometric parameters. Discordance between histology and one or more IHC parameters was seen in 5/25 (20%) of patients with grade III astrocytoma and 9/25 (36%) of patients with grade II disease. PCNA and p53 labeling indices were positively correlated with Pearson«SQ»s correlation, p less than 0.001 for both). Increased proliferative fraction, genetic alterations and neovascularization mark biological aggressiveness in astrocytoma. Immunohistochemical evaluation scores over meet the challenge of accurate prognostication of this potentially fatal malignancy.
|How to cite this article:|
Malhan P, Husain N, Bhalla S, Gupta RK, Husain M. Proliferating cell nuclear antigen, p53 and micro vessel density: Grade II vs. Grade III astrocytoma.Indian J Pathol Microbiol 2010;53:20-23
|How to cite this URL:|
Malhan P, Husain N, Bhalla S, Gupta RK, Husain M. Proliferating cell nuclear antigen, p53 and micro vessel density: Grade II vs. Grade III astrocytoma. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Jan 18 ];53:20-23
Available from: https://www.ijpmonline.org/text.asp?2010/53/1/20/59177
Diffuse nonpilocytic astrocytoma refers to a significant proportion of primary brain tumor and extends over a continuous morphologic spectrum of differentiation and tumor grade.  WHO grading system, used to classify these tumors into prognostically meaningful grades. However, the correlation between histological grade and prognosis does not hold well for some cases. , This suggests that within histologically identical groups, there exist tumor subtypes with different prognostic behavior.
The current study was formulated to evaluate the role of proliferative indices using PCNA-LI, p-53 expression and micro vessel morphometry in predicting the behavior of astrocytoma and their correlation with WHO histological grades.
Material and Methods
This study looked into 50 cases including 25 of WHO grade II astrocytoma and 25 of WHO grade III astrocytoma. Cases were selected according to study material available in tissue blocks and to get comparable age and sex matched groups for data analysis. A detailed clinical evaluation was recorded and histopathological examination of biopsies was done.
The specimens were fixed in 10% formalin after which they were processed into paraffin blocks. Sections with 3-5 micron thickness were cut and fixed at 56 0 C for two hours. Dewaxing and hydration followed this. Antigen retrieval was done in citrate buffer (pH 6.0) using microwave technique. After blocking for endogenous peroxidase, the sections were incubated overnight with primary antibody at 4 0 C. The PC-10 monoclonal antibody to PCNA (Dakopatt, Denmark) diluted 1:100, Do-7 monoclonal antibody to p53 protein (Dakopatt, Denmark) diluted 1:100 and anti factor VIII related antigen (Dakopatt, Denmark) diluted 1:100 were used. The antibody attachment was visualized using LSAB2 kit (Dakopatt, Denmark) using the standard protocol. Sections were counter stained with hematoxylin. Known positive controls and negative controls were included in each batch. Each slide for PCNA and p-53 immunostaining was evaluated manually for counting immunopositive nuclei (brown) and immunonegative tumor nuclei (blue) in 10 microscopic fields (1000X magnification). Selection of microscopic fields was done by choosing the representative fields for proliferative activity in tumor tissue, which is expressing quantitatively highest number of immune-positive nuclei. Hemorrhagic areas, infiltrative edges of the tumors and section borders were avoided.
PCNA-LI was calculated as a percentage of total number of immunopositive tumor nuclei.th
The p-53 expression was assessed similarly and expressed as a percentage of immunopositive (brown) tumor nuclei. Cases with p53 LI greater than 10% were taken as p53 positive and cases with less than 10% were taken as p53 negative. 
Quantification of angiogenesis was done in digitalized images using an image analysis system (Biovis plus Expert Vision, Mumbai India). Fields showing highest vascularity (i.e. hot spots) were chosen to determine the micro vessel density excluding those with muscular wall. In each case 10 microscopic fields were photographed with a digital camera (at 400 X magnification). Morphometric analysis included the mean vessel area, mean perimeter, mean diameter, mean major and minor axis length and percentage area. The entire enclosed area of these profiles was used for calculation of the vascular parameters using Biovis Image plus Software.
The mean and standard deviation of PCNA-LI, p-53-LI and micro vessel parameters were calculated for different grades of astrocytoma. The correlation between the different proliferation variables was evaluated using Pearson correlation coefficient. All the statistical analysis was performed using SPSS for windows software.
Characteristics of Patients
The average age of patients with grade II was 31.6 years (range 4-65) and Grade III astrocytoma was 33.7 (range 10-70) years. Maximum number of cases of grade III astrocytoma were located in the frontal region (28%) followed by parietal and frontoparietal region (16% each). While cases of grade II astrocytoma were located in the frontal and frontotemporal region (16% each) followed by frontoparietal, temporal and temporoparietal regions (12% each).
Proliferative Indices by Immunohistochemistry
PCNA-LI ranged from 4.47% - 46.77% in patients with Grade III astrocytoma and from 4.30 - 33.74% in Grade II patients. [Figure 1]
Comparison of mean PCNA-LI of grade III and II astrocytoma cohorts by unpaired 't' test was found to be statistically significant (p less than 0.001). The median of the study population was found 21.7% and accordingly the subjects were divided into two groups; high and low PCNA-LI having greater than 21.7% and less than 21.7% labeling indices respectively. Grade III patients the labeling indices were high in 80% (n is equal to 20) patients, the values ranging from 22.64 - 46.77%. While 20% patients (n is equal to 5) the values ranged from 4.47 - 19.20%. In grade II astrocytoma the results were vice versa with only five patients with high LI (range 22.22 - 33.74%). p53 -LI ranged from 8.20 - 32.80% in grade III while it ranged between 0.64 - 22.92% in grade II patients. [Figure 1] With labeling indices greater than 10% taken as p53 positive, the study showed 88% of grade III and 24% of grade II patients as p53 positive. [Figure 2] a,b
For micro vessel morphometric analysis 10 fields rich in vascularity were selected [Figure 2] c,d and vessels outlined by Biovis Image plus Software. All six parameters were done in the study population. [Table 1] On comparing the mean of all six parameters in both cohorts using unpaired student 't' test, the values were found to be statistically significant (p less than 0.001). The median percentage micro vessel area of the study population (n is equal to 50) was found to be 1.71%. Accordingly the subjects were divided into two groups; high and low area greater than 1.71% and less than 1.71% respectively. Amongst the Grade III astrocytoma, 88% subjects had a high micro vessel area with values ranging from 1.79% to 16.50% while 88% subjects with Grade II astrocytoma had a low micro vessel area (range 0.14 - 1.64%).
Discordance between histology and immunohistochemistry in grade III astrocytoma was seen with respect to all three IHC parameters in two patients (eight per cent); two IHC parameters in two patients (eight per cent) and one IHC parameter in one patient (four per cent) i.e. a total of five patients (20%) showed discordance in this cohort. The grade II astrocytoma showed discordance with respect to all three IHC parameters in one patient (4%); two IHC parameters in three patients (12%) and one IHC parameter in five patients (20%).
Although progress has been made in the understanding of glioma tumorigenesis and the ability to predict tumor behavior and progression, there has been little progress in extending the survival or quality of life for these patients and meaningful therapy to the individual patient still remains limited. As therapeutic options expand accurate patient stratification becomes critical for optimal patient management. With the advent of molecular and immunohistochemical techniques useful in paraffin embedded material, additional characterization of brain gliomas is possible.
Tumor growth depends upon multiple factors including cell proliferation, genetic alterations or deletion of negative regulatory elements, oncogene amplification and angiogenesis. In the present study, three parameters were studied in two comparable cohorts of patients with WHO histological grade III and grade II astrocytoma.
Assessment of proliferation activity provides important information about tumor behavior in astrocytoma.  Studies have been done using proliferative markers like Ki-67 expression and the use of new antibodies in the survival prognostication in astrocytoma. , Several studies have emphasized the association of PCNA-LI with tumor malignancy grade and to have prognostic significance in number of malignancies. , Expression of Ki 67, TP53, p27, and p21 was examined using immunohistochemical analysis by Yue et al.  in three groups of grade II astrocytoma including group 1 with recurrent malignant disease, group 2 with recurrent low grade tumor and group three with no recurrence. Ki 67 labeling index expression was significantly higher in group 1 (even though it was similar between initial and recurrent tumors) than that of group 3 (p less than 0.05). However, there was no difference between group 2 (both initial and recurrent tumors) and group 3. The TP53 protein accumulation was also higher in group 1 than in group 2 or 3 (p  Our study found an association of PCNA-LI with histological grade to be statistically significant (p less than 0.001) implying that this marker could assist in discriminating between grade II and grade III astrocytoma. Kirkegaard  found the mean percentage positive nuclear area by image cytometric quantitation of PCNA in grade III and II astrocytoma which were akin to the results of the present study. Sallinen et al,  also observed PCNA-LI to be strongly associated with the grades of malignancy in astrocytoma.
Molecular determinants of glioma progression suggest it to be a multistep process  Mutation of p53 gene is thought to represent an early event in the tumorigenesis of diffuse astrocytoma with mutation detectable in 30-40% of all grades. Nevertheless, immunopositivity does not always equate with gene mutation. Ono et al. estimated that one-fourth of their astrocytomas were p53 immunoreactive without an associated gene mutation. Secondary glioblastoma multiforme (GBM) subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF (platelet derived growth factor) and TP53 events.  In our study of patients with grade III astrocytoma p53 expression (LI greater than 10%) was demonstrable in 88% and 24% in grade II patients. Ellison et al. demonstrated immunoreactivity in 74% Grade III astrocytoma and 66% of Grade II astrocytoma. 32% of Grade III astrocytoma patients had LI greater than 60% while only eight per cent of grade II astrocytoma. Although there is a biological heterogeneity within the astrocytic tumors, the p53 expression does show positive correlation with grade of malignancy. , Bronisker et al. (2007) analyzed clinical and malignant characteristics of low grade gliomas in children. They observed that TP53 over expression was more common after malignant transformation. 
Angiogenesis plays an important role in the growth and progression of tumor.  Brain tumors exhibit marked and aberrant blood vessel formation indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vessels are used for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players.  Gliomas are very rich in micro vessels, studies on evaluating endothelial cell proliferation and angiogenesis in these tumors is significant. The usual method of evaluating angiogenic potential is to measure micro vessel density (MVD) in hot spots. In our study we found significantly higher values for all six morphologic parameter, that is, mean area, mean perimeter, mean diameter, mean major and minor axis length and mean percentage area in grade III astrocytoma as compared to grade II malignancy (p less than 0.001). Previous studies have also shown higher mean vessel area for grade III astrocytoma. , Among our cases, 88% of grade III astrocytoma and 12% of grade II astrocytoma had high micro vessel percentage area (greater than 1.71%). Previous studies have suggested that MVD can serve as an early prognostic marker for low grade astrocytoma which can aid in the prediction of their future malignant transformation and hence patients ultimate survival.  Inhibition of angiogenesis could be a very useful therapeutic strategy for malignant gliomas to develop antiangiogenic therapies. 
The immunohistochemical results of 20% grade III astrocytoma and 36% grade II astrocytoma did not show concordance with histology in our study thus implying that histological typing may over or under rate the actual biological behavior of astrocytoma. Thus immunohistochemical evaluation may prove to be a vital prognostic parameter in astrocytoma as has been well documented in previous survival analysis by different authors. Paucity of follow up in terms of survival remains a limitation of the present study. The issue highlighted through results of our study is that all histological low grade astrocytoma may not actually be biologically indolent and it is this particular subset of patients in which immunohistochemistry scores over histological grading. This category of patients with biologically aggressive behavior merits close clinical follow-up and serial neuro imaging to detect early recurrence and malignant transformation.
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