Immunohistochemical appearance of HNE-protein conjugates in human astrocytomas

Gliomas are tumors originating from astrocytes, oligodendrocytes or ependimal cells. Those of astrocytic origin are the most widespread of primary brain tumors and account for more then 60% of all CNS neoplasms. The current state of knowledge on the associations between tumor etiology and oxidative stress suggests that environmental factors that cause oxidative stress could also induce and promote cancer, especially in case of hereditary predisposition. Among mediators of oxidative stress, lipid peroxidation product 4-hydroxynonenal (HNE) is of particular relevance in oncology, as it is known to act as a growth-regulating factor and a signaling molecule. The aim of present study was to investigate by immunohistochemistry the presence of HNE-modified proteins in different types of astrocytoma. Our study comprised 45 astrocytic tumors. These tumors were graded in accordance with the WHO classification as diffuse astrocytomas (DA), anaplastic astrocytomas (AA) and glioblastomas (GB), while each group comprised 15 tumors. Slides of paraffin-embedded tumor tissue were stained with hematoxylin-eosin or were prepared for immunohistochemistry with monoclonal antibodies to HNE-histidine conjugate. Positive immunohistochemical reaction to HNE was analyzed semi-quantitatively. HNE positivity was proportional with malignancy of astrocytomas. The weakest presence of HNE-histidine adducts was found in DA, followed by AA and GB. Lowest intensity of HNE immunopositivity was present in tumor cells of almost all DA, predominantly around blood vessels. In malignant variants of astrocytoma, AA and GB, HNE positivity was moderate to strong, and diffusely distributed in all tumors.     

Real-time quantification of the proliferative state in astrocytomas

OBJECTIVE: To evaluate proliferative activity in a set of gliomas and to compare the quantitative data obtained by a real-time processor with the labelling index (LI) and mitotic index (MI). STUDY DESIGN: Ki-67 immunostaining was performed on paraffin-embedded specimens from 42 cases of glioblastomas, 17 cases of anaplastic astrocytomas and 14 cases of low grade astrocytomas. Nuclear positivity was calculated as LI and by a real-time image processor for quantitative evaluation. MI was also calculated at 10 high-power fields. The data obtained from glioblastomas were compared with those from anaplastic and low grade astrocytomas. To all the data was applied the Pearson test to verify the correlation between counting and quantitative values and between proliferative markers and survival. RESULTS: A positive trend from low grade astrocytomas to glioblastomas was found for Ki-67 (LI and quantitative values) and MI, with highly significant differences between the three grades of gliomas considered. A good correlation between LI and quantitative values of Ki-67 was found. Very little relationship resulted between survival and Ki-67 LI. No relationship was found between survival and quantitative values of Ki-67. CONCLUSION: Ki-67 allowed effective separation of astrocytic tumors with different grades of malignancy. Quantitative evaluation of color information by means of a real-time processor proved to be a useful, objective and fast way to obtain readings, useful for grading purposes but not for prognostic evaluation.     

Immunohistochemical detection of lactoferrin in human astrocytomas and multiforme glioblastomas

The presence of lactoferrin in astrocytomas, anaplastic astrocytomas and multiforme glioblastomas was determined by immunohistochemistry; the staining intensity and the percentage of neoplastic stained cells were graded and statistical analysis was performed by non-parametric methods. A moderate to strong diffuse immunoreactivity for lactoferrin was shown in glial elements of astrocytomas, while the positivity was progressively reduced in anaplastic astrocytomas and in multiforme glioblastomas, some of which were unstained; a highly significant difference was found between scores relative to astrocytomas and glioblastomas. We suggest that the lactoferrin may be produced by neoplastic astrocytes which permits a greater availability of iron for metabolic cellular processes. Alternatively, the cytoplasmic localization of lactoferrin in neoplastic astrocytes may be the consequence of defective or functionally impaired lactoferrin receptors at the cellular surface.     

Decidualized and pre-decidualized normal endometrial stromal cells produce more O-linked N-acetylglucosamine containing epitope H than non-decidualized normal endometrial stromal cells

The epitope H contains an O-linked N-acetylglucosamine residue in a specific conformation and/or environment recognized by the monoclonal antibody H (mAbH). mAbH stains two bands with Mr x10(-3) of 209 and 62 in lysates of cultured rat astrocytes. In addition, in extracts of cultured MCF-7 breast carcinoma cell line cells it stains cytokeratin 8 and five polypeptides originating from Triton X-100-soluble (Mr x10(-3) of 232, 67 and 37) and from the Triton X-100-insoluble (Mr x10(-3) of 51 and 50) fractions, respectively. In our previous studies we used the mAbH to investigate by immunostaining the expression of the epitope H in normal human brains, human brains with a variety of lesions, astrocytic tumors, infiltrating ductal breast carcinomas, fibroadenomas, and mitochondria-rich normal, metaplastic and neoplastic cells. In order to gain further insight into the expression patterns of the epitope H in human tissues we used the mAbH to investigate the immunohistochemical expression of the epitope H in normal human endometrium, including 30 cases of proliferative endometrium, 30 cases of early secretory endometrium, 30 cases of mid secretory endometrium, 30 cases of late secretory endometrium and 30 cases of decidual tissues. The main results were the following: 1) The decidual stromal cells presented in all cases high cytoplasmic expression of the epitope H; 2) The pre-decidual stromal cells presented in all cases of late secretory endometrium significant cytoplasmic expression of the epitope H ranging from moderate to high expression; 3) The non pre-decidual stromal cells of the functional endometrial layer presented in all cases insignificant cytoplasmic expression of the epitope H ranging from null to low expression; 4) The stromal cells of the basal layer of the endometrium and decidua did not express the epitope H in any case; 5) The endometrial stromal granulocytes did not express the epitope H in any case and 6) The blood vessel wall cells (endothelial and smooth muscle) of the endometrium through the whole duration of the menstrual cycle and of the decidua presented high cytoplasmic expression of the epitope H. It is concluded that decidualized and pre-decidualized human normal endometrial stromal cells show increased expression of the O-linked N-acetylglucosamine containing epitope H compared to non-decidualized endometrial stromal cells. These findings suggest that the expression of the epitope H may be under positive progesteronic control in normal human endometrium. Further investigation of the antigens bearing the epitope H might help to gain further insight into the histophysiology and the pathology of human endometrium.     

Lectins as differentiation markers of human gliomas.

The lectins Concanavalin A (Con A), Ricinus communis agglutinin (RCA-I), Peanut agglutinin (PNA) and Wheat germ agglutinin (WGA) as well as the immunomarkers for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used in a series of 21 glial tumors (4 pylocytic astrocytomas, 5 grade II astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas and 5 oligodendrogliomas). ConA binds to all tumoral astrocytes in low grade astrocytomas, as well as to well differentiated tumoral astrocytes in anaplastic astrocytomas and glioblastomas. RCA-I has a similar behaviour. PNA, and to a lesser degree WGA, binds selectively to the oligodendroglial plasma membrane in well differentiated oligodendrogliomas. The results suggest that these lectins are markers of differentiation in gliomas rather than of malignancy.     

Expression of the small heat shock protein (hsp) 27 in human astrocytomas correlates with histologic grades and tumor growth fractions

Summary 1. Cellular expression and distribution of the stress response small heat shock protein 27 (hsp27) in 39 high-grade astrocytomas (27 glioblastoma multiformes, 12 anaplastic astrocytomas) and in 27 low-grade astrocytomas (grade I–II) were analyzed immunohistochemically.2. The correlation between hsp27 expression and tumor growth fractions of the astrocytomas was examined following Ki-67 immunostaining.3. The hsp27 staining was cell cytoplasmic. The hsp27 immunopositive rate was significantly higher in high-grade astrocytomas; the rates were 74% for glioblastomas, 58% for anaplastic astrocytomas, and 37% for low-grade astrocytomas. The small and large tumor cells, especially in glioblastomas, multinucleated tumor giant cells, tumor cells in the pseudopalisading and necrotic areas, cells of the microvascular endothelial proliferations, and tumor vascular smooth muscles were usually hsp27 positive. The mean percentage of hsp27-positive cells was significantly higher in the glioblastomas alone and in the combined high-grade astrocytomas, compared to the low-grade, and in recurrent rather than in primary high-grade astrocytomas.4. The high-grade astrocytomas had a highly statistical significant Ki-67 labeling index. The Ki-67 labeling indices were significantly higher in the hsp27-positive than the hsp27-negative astrocytomas, irrespective of the histological grade. In the high-grade astrocytomas with a Ki-67 labeling index of five and above, 81% of those tumors were hsp27 positive.5. Thus, a large number of human astrocytomas express hsp27, and hsp27 expression correlates with histological grades of astrocytoma and with tumor growth fractions. This being the case, hsp27 is likely to have a role in the growth of human astrocytomas.     

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.     

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.     

Topometric analysis of diffuse astrocytomas

OBJECTIVE: To investigate differences between astrocytomas of WHO grade 2 and anaplastic astrocytomas of WHO grade 3 in terms of topometric variables characterizing individual tumor cell nuclei. STUDY DESIGN: Paraffin sections from surgical specimens from 25 astrocytomas (grade 2, n = 11; grade 3, n = 14) were analyzed by means of an image analysis system. At least 300 tumor cell nuclei were measured in the region with the highest Ki-67 proliferation index. Three different kinds of topometric variables were determined for each tumor cell nucleus: (1) several distances; (2) the variable Angle 2/1, the angle between the straight lines representing the distance to the nearest and second-nearest nucleus; and (3) the number of neighbors according to our mathematical definition. RESULTS: Most topometric variables showed distinct differences between the 2 tumor grades (multivariate analysis of variance), with 88% cases correctly reclassified by means of cross-validated discriminant analysis. The variables with the highest discriminatory power were the SD of Angle 2/1 and the ratio between the distance to the second-nearest and nearest tumor cell nucleus, with lower values for these variables in anaplastic astrocytomas. Even variables concerning neighborhood relationships showed significant differences. CONCLUSION: The results of this pilot study show that this first set of topometric variables is sufficient to detect differences between topologic characteristics of tumor cell nuclei in astrocytomas grade 2 and grade 3. Topometry seems to be an important tool for grading astrocytomas.     

Ploidy of endothelium in high-grade astrocytomas

To determine the ploidy and proliferative activity of the endothelium in high-grade astrocytomas, the nuclear DNA content of 11 high-grade astrocytomas, including two gliosarcomas, was measured by cytophotometry. This technique allowed comparison of the endothelial population with the astrocytic population. In all cases, the endothelium was diploid, with an average of 24.6% of cells in the S + G2M phases of the cell cycle. In contrast, the astrocytic population displayed marked DNA abnormalities. The two gliosarcomas had a marked difference in proliferative activity of the endothelium with 4% and 40% of cells, respectively, in the S + G2M phases. These data indicate that the vast majority of endothelial cells compromising the vascular hypercellularity observed in high-grade astrocytomas are in the normal cell cycle whereas in many cases the malignant astrocytes are not. The nuclear DNA content of gliosarcomas appears to be similar to other high-grade astrocytomas.     

Genetic analysis to complement histopathological diagnosis of brain tumors

Gliomas, the most frequent tumors originating in the human nervous system, are divided into various subtypes. Currently, microscopic examination alone is insufficient for classification and grading so that genetic profiles are increasingly being emphasized in recognition of the emerging role of molecular diagnostic approaches to glioma classification. Glioblastomas (WHO grade IV) may develop de novo (primary glioblastomas) or through progression from lower-grade astrocytomas (secondary glioblastomas), while both glioblastomas show similar histological features. In contrast, they do constitute distinct disease entities that evolve through different genetic pathways, and are likely to differ in prognosis and response to therapy. Oligodendrogliomas (WHO grade II) account for 2.7% of brain tumors and 5-18% of all gliomas. Since this tumor is recognized as a particular subtype of glioma that shows remarkable responses to chemotherapy, a correct diagnosis is of prime importance. The difficulty is that histological differentiation of oligodendrogliomas from diffuse astrocytomas is highly subjective in cases without typical morphological features and there is a lack of reliable immunohistochemical markers. While histological distinction of low-grade gliomas from reactive astrocytes is also often difficult, reactive astrocytes usually lack genetic alterations. More biological and molecular approaches to glioma classification thus appear warranted to provide improved means to achieve correct diagnoses.     

Apolipoprotein D expression in primary brain tumors: analysis by quantitative RT-PCR in formalin-fixed, paraffin-embedded tissue.

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category (p<0.05 for each comparison) but not from each other (p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas (p<0.05) but did not vary from other infiltrating tumors (p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue (p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas.     

Relationship between EGFR overexpression and gene amplification status in central nervous system gliomas

OBJECTIVE: To correlate epidermal growth factor receptor (EGFR) protein overexpression, as assessed by immunohistochemistry, with EGFR gene amplification determined by fluorescence in situ hybridization in a series of gliomas. STUDY DESIGN: Forty-seven central nervous system gliomas, including 34 cases of glioblastoma multiforme, 3 oligodendrogliomas, 4 juvenile pilocytic astrocytomas and 5 low grade astrocytomas, were obtained from the files of the University of Utah Pathology Department. In each case a representative paraffin block was selected, and EGFR protein expression was quantified using immunohistochemistry. EGFR gene amplification status was determined by fluorescence in situ hybridization. RESULTS: EGFR protein overexpression was detected in 9 cases of glioblastoma multiforme. EGFR gene amplification was present in 7 of these cases. Both nonamplified glioblastomas demonstrated only 2+ overexpression of EGFR protein. None of the low grade, pilocytic or anaplastic astrocytomas demonstrated either EGFR protein overexpression or gene amplification. CONCLUSION: EGFR protein overexpression is closely associated with gene amplification. Seventy-eight percent of cases showing protein overexpression demonstrated gene amplification. All cases of central nervous system neoplasms showing protein overexpression but lacking gene amplification were associated with only 2+ protein overexpression. All central nervous system neoplasms demonstrating gene amplification and/or overexpression were high grade neoplasms.     

Characteristics of tumour vessels in cytological squash smears of astrocytic tumours

S. Sato, Y. Sato, K. Marutsuka, H. Takeshima and Y. Asada Characteristics of tumour vessels in cytological squash smears of astrocytic tumours Objective: Smear preparations are useful tools from which to diagnose brain tumours intraoperatively. Although vascular proliferation is histologically a key feature of high‐grade astrocytoma, the characteristics of tumour vessels in smear preparations have not been determined. Methods: We examined the density and morphological parameters (area, width, nuclear layer and branches of vessel wall) of tumour vessels in squash smears of 43 primary astrocytomas (grade II diffuse astrocytomas, n = 9; grade III anaplastic astrocytomas, n = 13; grade IV glioblastomas, n = 21) and normal brain tissues (n = 11). Results: Vessel density and all morphological parameters were significantly higher in grade IV than in the other grades of tumours and in normal brain tissue. Vessel area, width and nuclear layer were greater in grade III than in normal brain tissue. The sensitivity and specificity of these vessel parameters for astrocytomas were 75–100% and 82–100%, respectively. Conclusions: Tumour vessel evaluations from squash smears provide useful information for the intraoperative diagnosis and grading of astrocytic tumours.     

CD9 expression in solid non-neuroepithelial tumors and infiltrative astrocytic tumors.

The tetraspan membrane protein CD9 is normally expressed in the mature myelin sheath and is believed to suppress the metastatic potential of certain human tumors. In this study we identified CD9 in a variety of brain tumors by immunohistochemical (IHC) and immunoblotting analyses. We examined 96 tumor samples and three glioma cell lines in addition to a murine brain tumor model of transplanted glioma cells in CD9-deficient mice and control mice. CD9 was expressed not only in solid non-neuroepithelial tumors but also in infiltrative malignant neuroepithelial tumors. Among the neuroepithelial tumors, high-grade astrocytic tumors, including glioblastomas and anaplastic astrocytomas, showed higher immunoreactivity than low-grade cerebral astrocytomas. Thus, CD9 expression in astrocytic tumors correlated with their malignancy. In the murine brain tumor model, transplanted glioma cells were shown to grow and spread through myelinated areas irrespective of the presence or absence of CD9 expression in the recipient's brain. These results indicate that the CD9 expression of astrocytic tumors plays a significant role in the malignancy independent of CD9 expression in the surrounding tissue. This might be explained by the observation that the CD9 molecule is associated with a mitogenic factor, membrane-anchored heparin-binding epidermal growth factor, which is known to be upregulated in malignant gliomas.     

Surface proteins localized in ruffling membranes and filopodia of human glioma cells detected using Mab S-11E10

We prepared mouse monoclonal antibody (Mab S-11E10) for the surface antigen specifically distributed in ruffling membranes and filopodia of cultured human glioma cells. The antibody reacted to the specified structures of other glioma cells (U251MG, KNS 60) as well as those of KNS 42 cells, which were the immunizing source. The antigens, identified by Mab S-11E10, had molecular weights of 65 and 66 kDa. Immunohistochemically, the antibody reacted only to astrocytes in the human brain, but the cross-reactivity was noted in extraneural tissues such as lymphocytes and epithelium of the bowel ducts. In 5 out of 6 low grade astrocytomas, most of the tumor cells were strongly stained, while tumor cells of highly cellular areas of anaplastic astrocytomas and glioblastomas were either not stained or only weakly stained. The specific localization of 65-66 kDa doublet proteins may suggest relation to spreading and locomotion of cells, and may correlate to astrocytic differentiation and/or function.     

Analysis of the BRAFV600E Mutation in Central Nervous System Tumors

BRAF^V600E mutations are involved in the development of melanoma, colon cancer, and papillary thyroid carcinoma. These mutations are also found in primary brain tumors at low to moderate frequencies. In this study, we investigated a series of brain tumors to determine the prevalence and associated clinicopathologic features of BRAF^V600E mutations. By direct sequencing, we analyzed 223 brain tumors, including 51 gangliogliomas (GGs), 45 pilocytic astrocytomas (PAs), 12 pleomorphic xanthoastrocytomas (PXAs), 35 glioblastomas (GBs), 28 anaplastic astrocytomas (AAs), 44 oligodendroglial tumors (ODGs), 3 anaplastic oligoastrocytomas, and 5 diffuse astrocytomas. Thirty-six cases (16.1%) exhibited the BRAF^V600E mutation, including 66.7% of PXAs, 23.5% of GGs, 15.6% of PAs, and 9.7% of the malignant gliomas; the latter included 14.3% of AAs, 8.6% of GBs, and 4.5% of ODGs. Copy number aberration at the 7q34 (BRAF) locus was found in 73.1% of PAs and 50% of PXAs. 9p Homozygous deletion was found in 66.7% of PXAs, but it was not correlated with the BRAF^V600E mutation. Patients' age, sex, histologic grade, and progression-free survival were also not correlated with the BRAF^V600E mutation. The BRAF^V600E mutation in brain tumors did not have prognostic value but is certainly a diagnostic marker and therapeutic target, not only for pediatric low-grade gliomas but also for malignant gliomas, even though the rate of mutation was not high. These results should be verified in a larger study with more cases and a longer follow-up period to overcome the limitation of small sample size.     

Grade classification of neuroepithelial tumors using high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and pattern recognition

Clinical data have shown that survival rates vary considerably among brain tumor patients, according to the type and grade of the tumor. Metabolite profiles of intact tumor tissues measured with high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy (HRMAS (1)H NMRS) can provide important information on tumor biology and metabolism. These metabolic fingerprints can then be used for tumor classification and grading, with great potential value for tumor diagnosis. We studied the metabolic characteristics of 30 neuroepithelial tumor biopsies, including two astrocytomas (grade I), 12 astrocytomas (grade II), eight anaplastic astrocytomas (grade III), three glioblastomas (grade IV) and five medulloblastomas (grade IV) from 30 patients using HRMAS (1)H NMRS. The results were correlated with pathological features using multivariate data analysis, including principal component analysis (PCA). There were significant differences in the levels of N-acetyl-aspartate (NAA), creatine, myo-inositol, glycine and lactate between tumors of different grades (P<0.05). There were also significant differences in the ratios of NAA/creatine, lactate/creatine, myo-inositol/creatine, glycine/creatine, scyllo-inositol/creatine and alanine/creatine (P<0.05). A soft independent modeling of class analogy model produced a predictive accuracy of 87% for high-grade (grade III-IV) brain tumors with a sensitivity of 87% and a specificity of 93%. HRMAS (1)H NMR spectroscopy in conjunction with pattern recognition thus provides a potentially useful tool for the rapid and accurate classification of human brain tumor grades.     

Quantitative analysis of vessels with smooth muscle layer in astrocytic tumors: correlation with histological grade and prognostic significance

Angiogenesis plays an important role in the progression of astrocytic tumors and its evaluation is a major prognostic factor. Although the form of proliferating vessels ranges from fine capillaries to well-developed vascular structures with a smooth muscle layer, the characteristics of vascular smooth muscle cells (SMCs) are not understood in detail. We therefore examined the density, size and shape of tumor vessels, as well as CD34-immunoreactive (CD34-Vs) or α-smooth muscle actin-immunoreactive (SMA-Vs) vessels in 46 primary astrocytomas (grade II diffuse astrocytomas, n=11, grade III anaplastic astrocytomas, n=15, grade IV glioblastomas, n=20) and in normal brain tissues from 10 autopsies. We also examined the expression of high molecular weight caldesmon (h-CD, a marker of the contractile phenotype of smooth muscle) and of platelet-derived growth factor receptor β (PDGFR-β). The SMA-Vs were significantly more dense and larger in grade IV than grade III, whereas those of CD34-Vs did not differ between grade III and IV. Changes in the shape of CD34-Vs and SMA-Vs correlated with histological grading. The expression of h-CD was reduced, whereas that of PFGFR-β was increased in high grade-astrocytomas. Kaplan-Meier analysis indicated that the density of SMA-Vs, the size of both CD34 and SMA-Vs and PDGFR-β expression were significant prognostic factors. These findings suggest that SMA-Vs are significantly associated with the progression of astrocytomas and that these vessels provide useful information for the histological diagnosis and survival of patients with these types of brain tumors.     

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.