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Heterogeneity of Ki-67 and p53 Expression in Glioblastoma

Heterogeneity of Ki-67 and p53 Expression in Glioblastoma 10.2478/chilat-2014-0102 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) ORIGINAL ARTICLE Heterogeneity of Ki-67 and p53 Expression in Glioblastoma Arvids Jakovlevs*, Andrejs Vanags**, Dainis Balodis*, Janis Gardovskis**, Ilze Strumfa* * Department of Pathology, Riga Stradins University, Latvia ** Department of Surgery, Riga Stradins University, Latvia SUMMARY Introduction. Heterogeneity is a characteristic feature of malignant tumours. It challenges the treatment regimens as well as can impair the diagnostic accuracy. Glioblastoma multiforme (GBM), a high-grade malignant glial tumour, is known for the extreme morphological heterogeneity giving rise also to the term itself. Aim of the study was to evaluate heterogeneity of pathogenetically and diagnostically important cardinal tumour features, namely, cellular proliferation and tumour suppressor protein expression in GBMs. Material and methods. The study group comprised 101 GBMs, retrospectively identified by archive search. The inclusion criteria comprised validated diagnosis (by World Health Organisation criteria) and lack of prior treatment. Recurrent GBMs as well as other glial and non-glial tumours were excluded from the study. Insufficient tissue materials comprising stereotactic biopsies and tissues affected by widespread necrosis (exceeding 90%) were also excluded. Proliferation activity (by Ki-67) and expression of aberrant p53 protein was detected by immunohistochemical investigation (IHC) of formalin-fixed, paraplast-embedded tumour samples. Polymeric visualisation system was used to detect bound primary antibodies. The expression of each antigen was measured by computed morphometry in at least 200 cells of hot and cold spots in each tumour. The data were expressed as the relative value. Heterogeneity was estimated as the mathematical difference between the highest and lowest expression value in each tumour. Descriptive statistics was applied. The 95% confidence intervals (CI) were determined as well. Results. The highest proliferation activity ranged 15 – 95%; mean 43.9% [95% CI = 40.3 – 47.6]. The lowest proliferation activity ranged 2 – 95%, mean 20.1% [16.8 – 23.4]. The mean proliferation heterogeneity was 23.8% [21.5 – 26.2]; range 0 – 67%. The mean heterogeneity of p53 protein expression was 11.7% [8.9 – 14.6], ranging 0 – 75%. Conclusions. GBM is characterized by marked heterogeneity regarding proliferation rate and expression of p53 protein that may affect diagnostic accuracy and grading of gliomas in small samples of tissue material as well as survival in case of small residual tumour after surgical treatment. Key words: glioblastoma, heterogeneity, immunohistochemistry, proliferation INTRODUCTION investigation and sensitive visualisation systems. The The issue of intratumoural heterogeneity (ITH) is a implementation of immunohistochemistry as a practical well-known feature of malignant tumours. The tumour surrogate for molecular analysis also has to be evaluated clonal evolution model described by P. Nowell in 1976 in sufficiently large tumour groups as such substitution explained the ITH on Darwinian selection over time, has showed high efficiency in certain epithelial tumours generated by genetic instability (Nowell, 1976). This (Bhargava et al., 2010). selection results in origination of different cellular Amplification of oncogenes and inactivation of subpopulations with distinct mutations and protein tumour suppressor genes are frequently found genetic expression profiles leading to various biological features abnormalities in human cancers including GBMs (Nowell, 1976). Such heterogeneity provides the (Ohgaki and Kleihues, 2007; Ng et al., 2012). TP53 tumour with several advantages promoting tumour is a well-known tumour suppressor gene that has growth and progression as well as resistance to therapy, been widely studied over these past decades in many but it can also lead to erroneous diagnosis. cancers. The gene encodes p53 protein that is involved ITH is described in many neoplasms including in cell cycle regulation and prevents the proliferation of glioblastoma (GBM), which is an extremely genetically damaged cells. TP53 is frequently mutated heterogeneous tumour in many aspects. ITH in glial in GBMs, including 28% of primary and 65% of tumours has also been described regarding cellular secondary GBM (Ohgaki and Kleihues, 2007). p53 over- proliferation and p53 protein expression (Coons and expression is frequently seen in many TP53 mutations Johnson, 1993; Dalrymple et al., 1994; Ren et al., 2007; thus immunohistochemistry can be used as surrogate Soussi and Lozano, 2005). However, some of the studies method for detection TP53 mutation (Milinkovic et al., are small, comprising only 11 gliomas (Ren et al., 2007). 2012). In addition, the ongoing technological progress both Uncontrolled cellular proliferation is the hallmark of in diagnostic evaluation and treatment necessitates the neoplastic process. It directly represents tumour integrated evaluation, including high-affinity behaviour. Thus, proliferation activity, expressed also 11 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) as proliferation index can be used as an auxiliary tool primary antibodies were detected by high-sensitivity polymeric visualisation system EnVision linked with in evaluation of grade in glial tumours together with morphological features (Louis et al., 2007). However, horseradish peroxidase. For the development of colour regional heterogeneity of proliferative activity and p53 signal, 3,3’-diaminobenzidine was used as chromogen. All immunohistochemistry reagents were produced by protein expression is an important factor that influences Dako, Glostrup, Denmark. accurate estimation of tumour and its malignancy grade. Evaluation of immunohistochemical staining and data AIM OF THE STUDY analysis Computed morphometry was performed using The aim of this study was to evaluate the heterogeneity specialised software Kappa Image Base (KAPPA opto- of p53 protein expression and proliferation activity by electronics Inc., United States of America). The optical Ki-67 performing immunohistochemical investigation system was represented by Axiolab (Zeiss, Oberkochen, of glioblastoma tissue material. Germany) microscope, and Kappa CF 11 DSP camera was used for image capture. Hot and cold spots were MATERIAL AND METHODS identified at scanning magnification (100x). The scoring Patients and tissue specimens was performed in at least 200 tumour cells using high- In this study, formalin-fixed paraffin-embedded tissues power magnification of 400x. Only nuclear expression from 101 glioblastoma patients were investigated. of markers was scored. The expression of Ki-67 and The consecutive cases were identified by retrospective p53 was evaluated quantitatively as the relative value: archive search in a single university hospital. Only percentage of positive tumour cells over total tumour primarily diagnosed, histologically proven cases of GBMs cells (%). An evaluation of highest and lowest relative which unequivocally met the criteria of classification values of expression (%) for Ki-67 and p53 were of tumours of the central nervous system, issued by measured in GBM specimens. The heterogeneity was World Health Organisation (Louis et al., 2007) were calculated as a difference between highest and lowest included in the study. The inclusion criteria comprised finding in the particular tumour. Descriptive statistical also the lack of prior treatment. Recurrent GBMs as analysis was performed including calculation of 95% well as other glial and non-glial tumours were excluded confidence interval (CI) by CIA software as described by from the study. Insufficient tissue materials comprising Altman et al., 2000. stereotactic biopsies and tissues affected by widespread necrosis (exceeding 90%) were also excluded. RESULTS Immunohistochemical visualization The study group comprised 101 GBM patients (52 males, Immunohistochemical visualisation of p53 and Ki- 49 females) with the mean age of 61.2 years [95% CI = 67 proteins was performed. Briefly, 3 μm-thick tissue 58.8 – 63.5]. sections were cut on electrostatic slides, deparaffinized in Expression of p53 and Ki-67 was confined to the xylene and hydrated in a series of graded ethanol. Heat nuclei of neoplastic cells (Figure 1). The staining was induced antigen retrieval was performed in a microwave heterogeneous in distribution (Table 1) and varied in oven using basic TEG buffer (pH 9.0). After blocking of different fields. Thus, the highest proliferation activity endogenous peroxidase, the sections were incubated ranged 15 – 95%, resulting in mean value 43.9% [40.3 – with primary antibodies at room temperature for 60 min. 47.6]. The lowest proliferation activity ranged 2 – 95%, The characteristics of primary antibodies were following: mean 20.1% [16.8 – 23.4]. The mean proliferation anti-Ki-67, monoclonal mouse antibody against human heterogeneity by the difference between the highest antigen, clone MIB-1; anti-p53, monoclonal mouse and lowest proliferation activity was 23.8% [21.5 – antibody against human antigen, clone DO-7. The bound 26.2]; range 0 – 67%. Fig. 1. Diverse p53 protein expression and proliferation activity in glioblastoma. A. Intense p53 expression in tumour cells. Note the characteristic nuclear reactivity. Immunoperoxidase (IP), anti-p53, original magnification (OM) 100x. B. Lack of p53 expression in tumour cells. IP, anti-p53, OM 100x. C. High proliferation activity in tumour cells. IP, anti-Ki-67, original magnification (OM) 100x. 12 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) Table 1. Proliferation activity by Ki-67 and p53 Abbreviations in the Figure: Ki67Max, the highest expression values in glioblastoma proliferation activity; Ki67Min, the lowest pro- liferation activity; p53Max, the highest relative Highest Lowest Hetero- Parameter expression of p53 protein; p53Min, the lowest value, % value,% geneity, % relative expression of p53 protein Ki-67 Range 15 – 95 2 – 95 0 – 67 Mean 43.9 20.1 23.8 DISCUSSION 95% CI 40.3 – 47.6 16.8 – 23.4 21.5 – 26.2 This study demonstrated that GBMs show significant ITH of cell proliferation and p53 protein expression. Such p53 Range 0 – 100 0 – 100 0 – 75 heterogeneity may pose a problem in the examination Mean 34.1 22.3 11.7 of small tissue material. 95% CI 26.4 – 41.2 15.2 – 29.5 8.9 – 14.6 There are very few studies regarding p53 expression heterogeneity in GBMs. We found a single one by Ren Abbreviations in the Table: CI, confidence interval for et al., 2007. They performed microdissection in different the mean areas of 11 gliomas, including 8 GBMs, followed by TP53 gene mutation analysis and p53 protein Both highest and lowest expression of p53 protein immunohistochemistry in the dissected neoplastic reached extreme values: 0 – 100%. Aberrant p53 tissues. Seven gliomas had p53 gene mutations and protein expression was absent (0%) in 23 GBMs. five from these were positive for p53 protein. The Only rare tumour cell nuclei (1 – 5%) expressed p53 authors noticed the presence of different TP53 gene in 8 GBMs. Moderate p53 expression (6 – 50%) was mutations in distinct areas within the same tumour. found in 40 GBMs. However, 30 GBMs showed strong Coexistence of wild-type and mutated TP53 was also expression of p53 in more than 50% of nuclei. The found in different areas from the same GBM (Ren et mean heterogeneity by the difference between the al., 2007). Such findings indicate high rate of p53 gene highest and lowest relative expression was 11.7% [8.9 – heterogeneity in GBMs. p53 protein expression by 14.6], ranging 0 – 75%. immunohistochemistry seems to be more influenced ki67Max from heterogeneity because not all mutations result in Ki67Min 2A p53Max 95 p53 protein over-expression. The present study confirms p53Min also that immunohistochemistry can be successfully applied as surrogate method in GBM research. Thus, larger tumour group comprising more than hundred cases could be investigated. The examination can also be included in clinical diagnostic pathway to characterise the individual tumour heterogeneity. Proliferation activity, known also as proliferation fraction or Ki-67 proliferation index is a powerful 24 prognostic indicator in a variety of neoplasms. There 15 16 is strong correlation with the grade of malignancy and 8 proliferation index in glial neoplasms (Louis et al., 2007). Proliferation index in diffuse astrocytomas (grade 2) is ki67Max Ki67Min p53Max p53Min usually less than 4%, with the mean 2.5 %, anaplastic ki67Max astrocytomas (grade 3) show indices in the range of 5 – Ki67Min 10%. For GBMs (grade 4) mean values of 15 – 20% 2B p53Max have been reported (Louis et al., 2007). However, p53Min considerable overlap of values among different tumour grades have been described (Torp, 2002). Thus, Ki-67 cannot be used alone as a diagnostic tool. Despite this limitation, evaluation of Ki-67 is a useful adjunct in morphological grading of astrocytomas in combination with histopathological features as well as clinical data and radiologic findings. It can also help in some problematic cases, for example, evaluating neoplasm with low- grade histology in the available tissue material if other factors indicate high-grade tumour. On the other hand, regional heterogeneity has been demonstrated in cell proliferation in different areas within the same tumour ki67Max Ki67Min p53Max p53Min (Coons and Johnson, 1993). The highest proliferation in GBMs has been shown in those cells located at the solid Fig. 2. Distribution of the highest and lowest Ki- tumour-infiltrated parenchyma interface (Dalrymple et 67 proliferation indices and p53 expression values al., 1994). in glioblastoma by box-plot (2A) and point scatter (2B) analysis. 13 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) The heterogeneity can unpredictably influence the 5. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. survival after surgical treatment leading to small residual Astrocytic tumors // Bosman FT, Jaffe ES, Lakhani tumour volume. Regarding the diagnostic accuracy, the RS, Ohgaki H. WHO classification of tumours of the highest impact of heterogeneity can be expected in small central nervous system. 3rd edition. Lyon: IARC, tissue samples such as stereotactic biopsy specimens 2007; 13 – 52 because it may not include areas with the highest 6. Milinkovic V, Bankovic J, Rakic M, Milosevic N, proliferation activity or sufficient histopathological Stankovic T, Jokovic M, Milosevic Z, Skender- features of the neoplasm we are dealing with. Thus, we Gazibara M, Podolski-Renic A, Pesic M, Ruzdijic S, should be aware of diagnostic accuracy limitations in Tanic N. Genomic instability and p53 alterations in small tissue samples. patients with malignant glioma // Exp Mol Pathol, 2012; 93:200 – 206 CONCLUSIONS 7. Ng K, Kim R, Kesari S, Carter B, Chen CC. GBM is characterized by extreme heterogeneity regar- Genomic profiling of glioblastoma: convergence of ding proliferation rate and expression of tumour fundamental biologic tenets and novel insights // J suppressor proteins. The identified heterogeneity can Neurooncol, 2012; 107:1 – 12 affect diagnostic accuracy and grading of gliomas in 8. Nowell PC. The clonal evolution of tumor cell small samples of tissue material as well as the outcome populations // Science, 1976; 194:23 – 28 after surgical treatment. 9. Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma // Am J Pathol, 2007; Conflict of interest: None 170:1445 – 1453 10. Ren ZP, Olofsson T, Qu M, Hesselager G, Soussi T, REFERENCES Kalimo H, Smits A, Nistér M. Molecular genetic 1. Altman DG, Machin D, Bryant TN, Gardner RJ. analysis of p53 intratumoral heterogeneity in Statistics with confidence. 2nd ed. Bristol: BMJ human astrocytic brain tumors // J Neuropathol Books; 2000 Exp Neurol, 2007; 66:944 – 954 2. Bhargava R, Beriwal S, Dabbs DJ, Ozbek U, So- 11. Soussi T, Lozano G. p53 mutation heterogeneity in ron A, Johnson RR, Brafsky AM, Lembersky BC, cancer // Biochem Biophys Res Commun, 2005; Ahrendt GM. Immunohistochemical surrogate 331:834 – 842 markers of breast cancer molecular classes predicts 12. Torp SH. Diagnostic and prognostic role of Ki67 response to neoadjuvant chemotherapy: a single immunostaining in human astrocytomas using institutional experience with 359 cases // Cancer, four different antibodies // Clin Neuropathol, 2002; 2010; 116:1431 – 1439 21:252 – 257 3. Coons SW, Johnson PC. Regional heterogeneity in the proliferative activity of human gliomas Address: as measured by the Ki-67 labeling index // J Arvids Jakovlevs, Neuropathol Exp Neurol, 1993; 52:609 – 618 Department of Pathology, Riga Stradins University 4. Dalrymple SJ, Parisi JE, Roche PC, Ziesmer SC, Dzirciema Street 16, Riga, LV-1007, Latvia Scheithauer BW, Kelly PJ. Changes in proliferat- E-mail: Arvids.Jakovlevs@rsu.lv ing cell nuclear antigen expression in glioblastoma multiforme cells along a stereotactic biopsy trajec- tory // Neurosurgery, 1994; 35:1036 – 1045 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Chirurgica Latviensis de Gruyter

Heterogeneity of Ki-67 and p53 Expression in Glioblastoma

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Abstract

10.2478/chilat-2014-0102 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) ORIGINAL ARTICLE Heterogeneity of Ki-67 and p53 Expression in Glioblastoma Arvids Jakovlevs*, Andrejs Vanags**, Dainis Balodis*, Janis Gardovskis**, Ilze Strumfa* * Department of Pathology, Riga Stradins University, Latvia ** Department of Surgery, Riga Stradins University, Latvia SUMMARY Introduction. Heterogeneity is a characteristic feature of malignant tumours. It challenges the treatment regimens as well as can impair the diagnostic accuracy. Glioblastoma multiforme (GBM), a high-grade malignant glial tumour, is known for the extreme morphological heterogeneity giving rise also to the term itself. Aim of the study was to evaluate heterogeneity of pathogenetically and diagnostically important cardinal tumour features, namely, cellular proliferation and tumour suppressor protein expression in GBMs. Material and methods. The study group comprised 101 GBMs, retrospectively identified by archive search. The inclusion criteria comprised validated diagnosis (by World Health Organisation criteria) and lack of prior treatment. Recurrent GBMs as well as other glial and non-glial tumours were excluded from the study. Insufficient tissue materials comprising stereotactic biopsies and tissues affected by widespread necrosis (exceeding 90%) were also excluded. Proliferation activity (by Ki-67) and expression of aberrant p53 protein was detected by immunohistochemical investigation (IHC) of formalin-fixed, paraplast-embedded tumour samples. Polymeric visualisation system was used to detect bound primary antibodies. The expression of each antigen was measured by computed morphometry in at least 200 cells of hot and cold spots in each tumour. The data were expressed as the relative value. Heterogeneity was estimated as the mathematical difference between the highest and lowest expression value in each tumour. Descriptive statistics was applied. The 95% confidence intervals (CI) were determined as well. Results. The highest proliferation activity ranged 15 – 95%; mean 43.9% [95% CI = 40.3 – 47.6]. The lowest proliferation activity ranged 2 – 95%, mean 20.1% [16.8 – 23.4]. The mean proliferation heterogeneity was 23.8% [21.5 – 26.2]; range 0 – 67%. The mean heterogeneity of p53 protein expression was 11.7% [8.9 – 14.6], ranging 0 – 75%. Conclusions. GBM is characterized by marked heterogeneity regarding proliferation rate and expression of p53 protein that may affect diagnostic accuracy and grading of gliomas in small samples of tissue material as well as survival in case of small residual tumour after surgical treatment. Key words: glioblastoma, heterogeneity, immunohistochemistry, proliferation INTRODUCTION investigation and sensitive visualisation systems. The The issue of intratumoural heterogeneity (ITH) is a implementation of immunohistochemistry as a practical well-known feature of malignant tumours. The tumour surrogate for molecular analysis also has to be evaluated clonal evolution model described by P. Nowell in 1976 in sufficiently large tumour groups as such substitution explained the ITH on Darwinian selection over time, has showed high efficiency in certain epithelial tumours generated by genetic instability (Nowell, 1976). This (Bhargava et al., 2010). selection results in origination of different cellular Amplification of oncogenes and inactivation of subpopulations with distinct mutations and protein tumour suppressor genes are frequently found genetic expression profiles leading to various biological features abnormalities in human cancers including GBMs (Nowell, 1976). Such heterogeneity provides the (Ohgaki and Kleihues, 2007; Ng et al., 2012). TP53 tumour with several advantages promoting tumour is a well-known tumour suppressor gene that has growth and progression as well as resistance to therapy, been widely studied over these past decades in many but it can also lead to erroneous diagnosis. cancers. The gene encodes p53 protein that is involved ITH is described in many neoplasms including in cell cycle regulation and prevents the proliferation of glioblastoma (GBM), which is an extremely genetically damaged cells. TP53 is frequently mutated heterogeneous tumour in many aspects. ITH in glial in GBMs, including 28% of primary and 65% of tumours has also been described regarding cellular secondary GBM (Ohgaki and Kleihues, 2007). p53 over- proliferation and p53 protein expression (Coons and expression is frequently seen in many TP53 mutations Johnson, 1993; Dalrymple et al., 1994; Ren et al., 2007; thus immunohistochemistry can be used as surrogate Soussi and Lozano, 2005). However, some of the studies method for detection TP53 mutation (Milinkovic et al., are small, comprising only 11 gliomas (Ren et al., 2007). 2012). In addition, the ongoing technological progress both Uncontrolled cellular proliferation is the hallmark of in diagnostic evaluation and treatment necessitates the neoplastic process. It directly represents tumour integrated evaluation, including high-affinity behaviour. Thus, proliferation activity, expressed also 11 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) as proliferation index can be used as an auxiliary tool primary antibodies were detected by high-sensitivity polymeric visualisation system EnVision linked with in evaluation of grade in glial tumours together with morphological features (Louis et al., 2007). However, horseradish peroxidase. For the development of colour regional heterogeneity of proliferative activity and p53 signal, 3,3’-diaminobenzidine was used as chromogen. All immunohistochemistry reagents were produced by protein expression is an important factor that influences Dako, Glostrup, Denmark. accurate estimation of tumour and its malignancy grade. Evaluation of immunohistochemical staining and data AIM OF THE STUDY analysis Computed morphometry was performed using The aim of this study was to evaluate the heterogeneity specialised software Kappa Image Base (KAPPA opto- of p53 protein expression and proliferation activity by electronics Inc., United States of America). The optical Ki-67 performing immunohistochemical investigation system was represented by Axiolab (Zeiss, Oberkochen, of glioblastoma tissue material. Germany) microscope, and Kappa CF 11 DSP camera was used for image capture. Hot and cold spots were MATERIAL AND METHODS identified at scanning magnification (100x). The scoring Patients and tissue specimens was performed in at least 200 tumour cells using high- In this study, formalin-fixed paraffin-embedded tissues power magnification of 400x. Only nuclear expression from 101 glioblastoma patients were investigated. of markers was scored. The expression of Ki-67 and The consecutive cases were identified by retrospective p53 was evaluated quantitatively as the relative value: archive search in a single university hospital. Only percentage of positive tumour cells over total tumour primarily diagnosed, histologically proven cases of GBMs cells (%). An evaluation of highest and lowest relative which unequivocally met the criteria of classification values of expression (%) for Ki-67 and p53 were of tumours of the central nervous system, issued by measured in GBM specimens. The heterogeneity was World Health Organisation (Louis et al., 2007) were calculated as a difference between highest and lowest included in the study. The inclusion criteria comprised finding in the particular tumour. Descriptive statistical also the lack of prior treatment. Recurrent GBMs as analysis was performed including calculation of 95% well as other glial and non-glial tumours were excluded confidence interval (CI) by CIA software as described by from the study. Insufficient tissue materials comprising Altman et al., 2000. stereotactic biopsies and tissues affected by widespread necrosis (exceeding 90%) were also excluded. RESULTS Immunohistochemical visualization The study group comprised 101 GBM patients (52 males, Immunohistochemical visualisation of p53 and Ki- 49 females) with the mean age of 61.2 years [95% CI = 67 proteins was performed. Briefly, 3 μm-thick tissue 58.8 – 63.5]. sections were cut on electrostatic slides, deparaffinized in Expression of p53 and Ki-67 was confined to the xylene and hydrated in a series of graded ethanol. Heat nuclei of neoplastic cells (Figure 1). The staining was induced antigen retrieval was performed in a microwave heterogeneous in distribution (Table 1) and varied in oven using basic TEG buffer (pH 9.0). After blocking of different fields. Thus, the highest proliferation activity endogenous peroxidase, the sections were incubated ranged 15 – 95%, resulting in mean value 43.9% [40.3 – with primary antibodies at room temperature for 60 min. 47.6]. The lowest proliferation activity ranged 2 – 95%, The characteristics of primary antibodies were following: mean 20.1% [16.8 – 23.4]. The mean proliferation anti-Ki-67, monoclonal mouse antibody against human heterogeneity by the difference between the highest antigen, clone MIB-1; anti-p53, monoclonal mouse and lowest proliferation activity was 23.8% [21.5 – antibody against human antigen, clone DO-7. The bound 26.2]; range 0 – 67%. Fig. 1. Diverse p53 protein expression and proliferation activity in glioblastoma. A. Intense p53 expression in tumour cells. Note the characteristic nuclear reactivity. Immunoperoxidase (IP), anti-p53, original magnification (OM) 100x. B. Lack of p53 expression in tumour cells. IP, anti-p53, OM 100x. C. High proliferation activity in tumour cells. IP, anti-Ki-67, original magnification (OM) 100x. 12 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) Table 1. Proliferation activity by Ki-67 and p53 Abbreviations in the Figure: Ki67Max, the highest expression values in glioblastoma proliferation activity; Ki67Min, the lowest pro- liferation activity; p53Max, the highest relative Highest Lowest Hetero- Parameter expression of p53 protein; p53Min, the lowest value, % value,% geneity, % relative expression of p53 protein Ki-67 Range 15 – 95 2 – 95 0 – 67 Mean 43.9 20.1 23.8 DISCUSSION 95% CI 40.3 – 47.6 16.8 – 23.4 21.5 – 26.2 This study demonstrated that GBMs show significant ITH of cell proliferation and p53 protein expression. Such p53 Range 0 – 100 0 – 100 0 – 75 heterogeneity may pose a problem in the examination Mean 34.1 22.3 11.7 of small tissue material. 95% CI 26.4 – 41.2 15.2 – 29.5 8.9 – 14.6 There are very few studies regarding p53 expression heterogeneity in GBMs. We found a single one by Ren Abbreviations in the Table: CI, confidence interval for et al., 2007. They performed microdissection in different the mean areas of 11 gliomas, including 8 GBMs, followed by TP53 gene mutation analysis and p53 protein Both highest and lowest expression of p53 protein immunohistochemistry in the dissected neoplastic reached extreme values: 0 – 100%. Aberrant p53 tissues. Seven gliomas had p53 gene mutations and protein expression was absent (0%) in 23 GBMs. five from these were positive for p53 protein. The Only rare tumour cell nuclei (1 – 5%) expressed p53 authors noticed the presence of different TP53 gene in 8 GBMs. Moderate p53 expression (6 – 50%) was mutations in distinct areas within the same tumour. found in 40 GBMs. However, 30 GBMs showed strong Coexistence of wild-type and mutated TP53 was also expression of p53 in more than 50% of nuclei. The found in different areas from the same GBM (Ren et mean heterogeneity by the difference between the al., 2007). Such findings indicate high rate of p53 gene highest and lowest relative expression was 11.7% [8.9 – heterogeneity in GBMs. p53 protein expression by 14.6], ranging 0 – 75%. immunohistochemistry seems to be more influenced ki67Max from heterogeneity because not all mutations result in Ki67Min 2A p53Max 95 p53 protein over-expression. The present study confirms p53Min also that immunohistochemistry can be successfully applied as surrogate method in GBM research. Thus, larger tumour group comprising more than hundred cases could be investigated. The examination can also be included in clinical diagnostic pathway to characterise the individual tumour heterogeneity. Proliferation activity, known also as proliferation fraction or Ki-67 proliferation index is a powerful 24 prognostic indicator in a variety of neoplasms. There 15 16 is strong correlation with the grade of malignancy and 8 proliferation index in glial neoplasms (Louis et al., 2007). Proliferation index in diffuse astrocytomas (grade 2) is ki67Max Ki67Min p53Max p53Min usually less than 4%, with the mean 2.5 %, anaplastic ki67Max astrocytomas (grade 3) show indices in the range of 5 – Ki67Min 10%. For GBMs (grade 4) mean values of 15 – 20% 2B p53Max have been reported (Louis et al., 2007). However, p53Min considerable overlap of values among different tumour grades have been described (Torp, 2002). Thus, Ki-67 cannot be used alone as a diagnostic tool. Despite this limitation, evaluation of Ki-67 is a useful adjunct in morphological grading of astrocytomas in combination with histopathological features as well as clinical data and radiologic findings. It can also help in some problematic cases, for example, evaluating neoplasm with low- grade histology in the available tissue material if other factors indicate high-grade tumour. On the other hand, regional heterogeneity has been demonstrated in cell proliferation in different areas within the same tumour ki67Max Ki67Min p53Max p53Min (Coons and Johnson, 1993). The highest proliferation in GBMs has been shown in those cells located at the solid Fig. 2. Distribution of the highest and lowest Ki- tumour-infiltrated parenchyma interface (Dalrymple et 67 proliferation indices and p53 expression values al., 1994). in glioblastoma by box-plot (2A) and point scatter (2B) analysis. 13 ACTA CHIRURGICA LATVIENSIS • 2014 (14/1) The heterogeneity can unpredictably influence the 5. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. survival after surgical treatment leading to small residual Astrocytic tumors // Bosman FT, Jaffe ES, Lakhani tumour volume. Regarding the diagnostic accuracy, the RS, Ohgaki H. WHO classification of tumours of the highest impact of heterogeneity can be expected in small central nervous system. 3rd edition. Lyon: IARC, tissue samples such as stereotactic biopsy specimens 2007; 13 – 52 because it may not include areas with the highest 6. Milinkovic V, Bankovic J, Rakic M, Milosevic N, proliferation activity or sufficient histopathological Stankovic T, Jokovic M, Milosevic Z, Skender- features of the neoplasm we are dealing with. Thus, we Gazibara M, Podolski-Renic A, Pesic M, Ruzdijic S, should be aware of diagnostic accuracy limitations in Tanic N. Genomic instability and p53 alterations in small tissue samples. patients with malignant glioma // Exp Mol Pathol, 2012; 93:200 – 206 CONCLUSIONS 7. Ng K, Kim R, Kesari S, Carter B, Chen CC. GBM is characterized by extreme heterogeneity regar- Genomic profiling of glioblastoma: convergence of ding proliferation rate and expression of tumour fundamental biologic tenets and novel insights // J suppressor proteins. The identified heterogeneity can Neurooncol, 2012; 107:1 – 12 affect diagnostic accuracy and grading of gliomas in 8. Nowell PC. The clonal evolution of tumor cell small samples of tissue material as well as the outcome populations // Science, 1976; 194:23 – 28 after surgical treatment. 9. Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma // Am J Pathol, 2007; Conflict of interest: None 170:1445 – 1453 10. Ren ZP, Olofsson T, Qu M, Hesselager G, Soussi T, REFERENCES Kalimo H, Smits A, Nistér M. Molecular genetic 1. Altman DG, Machin D, Bryant TN, Gardner RJ. analysis of p53 intratumoral heterogeneity in Statistics with confidence. 2nd ed. Bristol: BMJ human astrocytic brain tumors // J Neuropathol Books; 2000 Exp Neurol, 2007; 66:944 – 954 2. Bhargava R, Beriwal S, Dabbs DJ, Ozbek U, So- 11. Soussi T, Lozano G. p53 mutation heterogeneity in ron A, Johnson RR, Brafsky AM, Lembersky BC, cancer // Biochem Biophys Res Commun, 2005; Ahrendt GM. Immunohistochemical surrogate 331:834 – 842 markers of breast cancer molecular classes predicts 12. Torp SH. Diagnostic and prognostic role of Ki67 response to neoadjuvant chemotherapy: a single immunostaining in human astrocytomas using institutional experience with 359 cases // Cancer, four different antibodies // Clin Neuropathol, 2002; 2010; 116:1431 – 1439 21:252 – 257 3. Coons SW, Johnson PC. Regional heterogeneity in the proliferative activity of human gliomas Address: as measured by the Ki-67 labeling index // J Arvids Jakovlevs, Neuropathol Exp Neurol, 1993; 52:609 – 618 Department of Pathology, Riga Stradins University 4. Dalrymple SJ, Parisi JE, Roche PC, Ziesmer SC, Dzirciema Street 16, Riga, LV-1007, Latvia Scheithauer BW, Kelly PJ. Changes in proliferat- E-mail: Arvids.Jakovlevs@rsu.lv ing cell nuclear antigen expression in glioblastoma multiforme cells along a stereotactic biopsy trajec- tory // Neurosurgery, 1994; 35:1036 – 1045

Journal

Acta Chirurgica Latviensisde Gruyter

Published: Nov 24, 2014

Keywords: Medicine; Clinical Medicine; Surgery; Surgery, other

References