Prognosis-related histomorphological and immunohistochemical markers in central nervous system tumors of childhood and adolescence

Brain tumors account for approximately 20% of all childhood cancers, and are the leading cause of cancer morbidity and mortality among children. Although numerous demographic, clinical and therapeutic parameters have been identified over the past few years that have significant prognostic bearing for some pediatric brain tumors, predicting the clinical course and outcome among children with central nervous system tumors is still difficult. A survey of publications on prognosis-related histopathological and immunohistochemical features among pediatric brain tumors revealed 172 series, of which 91 presented statistically significant outcome-associated parameters as defined by a P value of less than 0.05. Most investigations revealing significant prognosis-related markers were performed on medulloblastomas (30 publications), ependymomas (25) and astrocytic tumors (18). In total, 16 cohorts consisted of more than 100 cases (5 on ependymomas, 3 each on medulloblastomas and astrocytic tumors). On the other hand, there were also 13 series with fewer than 20 cases (5 on medulloblastomas). Potentially prognostic histopathological markers vary among different entities and consist of assessment of necroses, mitoses, differentiation, vascular proliferation, and growth pattern, whereas immunohistochemical features include proliferation markers (Ki-67, MIB-1), expression of oncogenes/tumor suppressor genes and their proteins (TP53, c-erbB2), growth factor and hormonal receptors (VEGF, EGFR, HER2, HER4, ErbB-2), cell cycle genes (p27, p14ARF) and cell adhesion molecules, as well as factors potentially related to therapeutic resistance (DNA topoisomerase IIalpha, metallothionein, P-glycoprotein, tenascin). This review discusses the prognostic potential of histopathological and immunohistochemical markers that can be investigated by the practicing neuropathologist as part of the routine diagnostic workload, and scrutinizes their benefit for predicting therapy response and patient outcome among children with brain tumors.This paper is dedicated to Kurt Jellinger, the Grand Master of clinical neuropathology and of scientific publishing     

Histomolecular classification of adult diffuse gliomas: the diagnostic value of immunohistochemical markers

Adult gliomas are most often infiltrative. The World Health Organization (WHO) has classed them into three major groups according to the presomptive cell of origin: astrocytoma, oligodendroglioma and mixed oligoastrocytoma. Depending on the presence or absence of a small number of signs of anaplasia (mitosis, nuclear atypia, cell density, microvascular proliferation and necrosis) the WHO distinguishes grade II (LGG), III (anaplastic), and IV (glioblastomas, GBM). Mutation in the isocitrate deshydrogenase I and II (IDH1 and 2) genes distinguishes grade II, III and secondary GBM from primary GBM. Moreover two additional genetic alterations are recorded in grade II and III gliomas: TP53 mutations that characterize astrocytomas and 1p19q codeletion (as the result of t(1;19)(q10;p10) translocation) recorded in oligodendrogliomas. Mixed gliomas, the most non-reproducible category, share with astrocytomas and oligodendrogliomas the same genetic alterations. Interestingly TP53 mutation (p53+) and 1p19q codeletion (1p19q+) are mutually exclusive and involve IDH mutated (IDH+) glial precursor cells. According to IDH, TP53, and 1p19q status, four major subtypes of LGG are recorded: IDH+/p53−/1p19q−, IDH+/p53+/1p19q−, IDH+/p53−/1p19q+ and triple negative, this last subgroup having the worst prognosis. Interestingly, p53 expression and internexin alpha (INA) expression can replace to some extent TP53 mutation and 1p19 codeletion, respectively. Moreover the antibody directed against the IDH1R132H isoform is highly specific. Because this mutation is the most frequent it is sufficient to assess IDH status in more than 80% of grade II and III gliomas. Taken together these three immunohistochemical markers are contribute greatly to the classification of gliomas and should be tested routinely as diagnostic markers. Finally, although GBM are genetically heterogeneous, the vast majority display EGFR amplification, often associated with EGFR expression, which can be helpful for diagnosis in certain cases.     

Genetically distinct astrocytic and oligodendroglial components in oligoastrocytomas

Oligoastrocytomas are glial tumours consisting of a mixture of neoplastic astrocytic and oligodendroglial cells. Genetic alterations of oligoastrocytomas include loss of heterozygosity of chromosomes 1p and/or 19q (LOH 1p/19q), typically occurring in oligodendrogliomas, and mutations of TP53, frequently occurring in astrocytomas. To investigate whether these neoplastic cell types in oligoastrocytomas have different genetic profiles, we examined the two different components of oligoastrocytomas in comparison with the histological diagnosis of the specific tumour area for LOH 1p/19q and TP53 mutations by using microdissection technique. We found a variety of lost markers for 1p and 19q, and the presence of two different TP53 mutations in the tumour samples. In the majority of cases (9/11), the oligodendroglial and astrocytic components of an individual oligoastrocytoma displayed the same genotype. We present two cases of biphasic oligoastrocytomas with aberrant findings, suggesting the coexistence of genetically and morphologically distinct tumour cell clones in these tumours. In one case, the oligodendroglial part of the tumour showed LOH19q, whereas the astrocytic part showed TP53 mutation (codon 273). In another case, we found LOH 1p/19q in the oligodendroglial component, but two retained areas on chromosome 1p in the astrocytic component of the tumour. No evidence was found for the coexistence of tumour cells with the two genotypical changes within the same morphological region of one individual tumour. The two cases of biphasic oligoastrocytomas in our sample that display a different genotype in the astrocytic and oligodendroglial part of the tumour show that different components of an oligoastrocytoma may be derived from different cell clones during neoplastic transformation.     

Role of temozolomide in pediatric brain tumors

Features of temozolomide Temozolomide (TMZ) belongs to the imidazotetrazine class and it is a DNA-methylating agent that has a good antitumor activity. Despite of dacarbazine, TMZ is spontaneously converted into its active metabolite 5-(3-methyltriazen-l-yl)imidazole-4-carboxamide at physiologic pH, so it is not required in enzymatic demethylation in the liver. TMZ is able to cross the blood brain barrier and is stable at gastric acid pH so it has almost 100% oral bioavailability and is rapidly absorbed after it is taken orally.Temozolomide in cancer patients On the basis of the relatively safe toxicity and the findings achieved in adult malignant gliomas, phase I and II clinical trials were set up to evaluate the opportunity of using this novel drug in pediatric cancer, too. In this review, we evaluate the antitumor activity of TMZ against high-grade gliomas, low-grade-gliomas, and medulloblastoma/primitive neuroectodermal tumors analyzing several phases I and II clinical trials in children.Conclusions In spite of the poor activity of TMZ against pediatric brain tumors, the use of the drug in combination with other compounds should be evaluated in phases I and II clinical trials. Moreover, the evaluation of the methylation status of the O6-methylguanine DNA methyltransferase promoter in glioblastoma biopsy specimens could be assayed as a predictive factor of TMZ efficacy.     

Single photon emission computerized tomography in childhood hydrocephalus

Single photon emission computerized tomography (SPECT) is now widely used as one of the tools in evaluating cerebral blood flow (CBF). The authors report the CBF changes in childhood hydrocephalus. Five pediatric cases studied by ¹²³I-IM SPECT in children are presented. The authors counted radioactivities both in early and delayed images in each patient, and calculated the reabsorption ratio (RR). Two negative-RR cases and three positive-RR cases were found. All of the negative-RR patients had a poor prognosis, while all of the positive-RR patients had a favorable outcome.     

Brain tumors of childhood: nosological and diagnostic problems

Brain tumors containing undifferentiated cells were selected from a series of 504 childhood brain tumors; 117 were analyzed. Most tumors were medulloblastomas, followed by cerebral neuroblastomas, pineocytomas-blastomas, ependymoblastomas, and polar spongioblastomas. Of each oncotype, the main histological features were evaluated, including differentiation and the most important prognostic factors. The terminology and different tumor entities are discussed in light of the recent PNET system. The usefulness of its application is evaluated in relation to prognosis.Supported by grant no. 87.01446.44 from the Italian National Research Council (CNR), Special Project Oncology, Rome, and from the Italian Association for Cancer Research (AIRC), MilanPresented at the 11th Meeting of the European Society for Paediatric Neurosurgery, Naples 1988     

儿童恶性脑胶质瘤P53与细胞增殖核抗原表达的预后价值

目的探讨Ｐ５３与细胞增殖核抗原（ＰＣＮＡ）在儿童恶性脑胶质瘤表达的预后价值。方法采用ＡＢＣ免疫组化方法对３３例儿童恶性脑胶质瘤Ｐ５３与ＰＣＮＡ表达进行回顾性研究。结果３３例儿童恶性脑胶质瘤中Ｐ５３表达阳性１５例（４５％），ＰＣＮＡ表达阳性２９例（８８％）。间变性星形细胞瘤、胶质母细胞瘤及髓母细胞瘤Ｐ５３蛋白表达的阳性比例分别为５／１２、９／１６、１／５，肿瘤Ｐ５３蛋白阴性者其细胞增殖活性均较阴性者高（Ｐ＜００５）；ＰＣＮＡ标记指数与肿瘤的恶性程度呈正相关（Ｐｅａｒｓｏｎ列联系数＝０．５６，Ｐ＜００１）。Ｐ５３或ＰＣＮＡ表达阳性者存活率分别显著低于Ｐ５３或ＰＣＮＡ表达阴性者（Ｐ＜００５，Ｐ＜００１）。结论Ｐ５３基因突变以及由此导致的细胞异常增殖与儿童恶性脑胶质瘤的发生和发展有关；ＰＣＮＡ能较好地反映胶质瘤的恶性程度，其表达检测对临床预后判定有重要参考价值。     

p53 Expression in four human medulloblastoma-derived cell lines

p53 is a tumor suppressor gene found on the short arm of chromosome 17. Loss of onep53 allele and alteration of the other is found in a variety of tumors, including highgrade glial tumors. Point mutations in the remaining allele occur in a highly conserved region of the gene encompassing exons 5–8. Although 40–50% of medulloblastomas lose sequences on the short arm of chromosome 17, alteration ofp53 in these tumors is infrequent. To further characterize genetic alteration ofp53 in medulloblastoma, we performed a mutational analysis of four medulloblastoma-derived cell lines established by our laboratory. Using two variable-number tandem repeat markers which map distally top53, we found evidence indicating loss of sequences on the distal end of chromosome 17 p in all four lines. However, no gross alterations of thep53 gene were detected. Northern analysis revealed expression of equivalent amounts of full-lengthp53 messenger RNA in each cell line. Using the polymerase chain reaction to amplify exons 5–8 of thep53 gene, we directly sequenced the amplified fragments and detected no mutations in any of the cell lines. Our results demonstrate that loss ofp53 function through gene deletion and/or recesive mutation is not required for growth in our cell lines.     

脑肿瘤中P53基因突变与突变型P53蛋白表达的研究

采用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）法对４１例脑肿瘤进行Ｐ５３基因突变的检测，并与抗突变型Ｐ５３蛋白单抗的免疫组化染色结果相对照，结果显示脑肿瘤中Ｐ５３基因突变率为３４．１％（１４／４１），胶质瘤为２８％（１０／３６）。Ｐ５３基因突变与突变型Ｐ５３蛋白表达具有高度一致性，且二者均与脑肿瘤的分化程度有关。Ｐ５３基因突变及蛋白的表达不仅出现在高恶度胶质瘤及转移癌，而且可出现在低恶度胶质瘤中，表明Ｐ５３基因的突变在脑瘤的发生、发展过程中起重要作用。     

Role of Peripheral Adenosine A1 Receptors in the Regulation of Sleep Apneas in Rats

The effects of administration of N⁶-p-sulfophenyladenosine (p-SPA), a peripheral adenosine A₁ receptor agonist, and 8-(p-sulfophenyl)theophylline (p-SPT), a peripheral adenosine A₁ receptor blocker, on spontaneous apneas were studied in 10 adult Sprague–Dawley rats by monitoring respiration, sleep, and blood pressure for 6 h. Intraperitoneal injection of p-SPA (1 mg/kg) to rats suppressed spontaneous central apneas during non-rapid eye movement sleep by 50% in comparison to control recordings (p = 0.03). This effect was blocked by pretreatment with an equimolar dose of p-SPT (0.67 mg/kg) indicating that p-SPA suppression of apneas was receptor mediated in the peripheral nervous system. Administration of p-SPA did not affect apnea expression in rapid eye movement sleep and had no effect on sleep or blood pressure at the dose tested. Administration of p-SPT (0.67, 6.7, and 30 mg/kg) to rats had no effect on apneas, sleep, or blood pressure. The lack of p-SPT effect on sleep apneas argues against a physiologic role for endogenous adenosine in the peripheral nervous system as a modulator of sleep apnea expression under baseline conditions.     

Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer's disease in Poland

Mutations in three causative genes have been identified in patients with an autosomal-dominant form of early-onset Alzheimer's disease (EOAD). To determine the spectrum of mutations in a group consisting of 40 Polish patients with clinically diagnosed familial EOAD and 1 patient with mild cognitive impairment (MCI) and family history of AD, we performed a screening for mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP) genes. Four previously recognized pathogenic mutations in PSEN1 gene (H163R, M139V) and APP gene (T714A, V715A), and three novel putative mutations in PSEN1 gene (P117R and I213F) and PSEN2 gene (Q228L) were identified. The 34 patients with no mutations detected were older than the patients with mutations. A frequency of APOE4 allele was higher in this group. Frequency of mutations is relatively low (17%), possibly due to used operational definition of a patient with familial EOAD (a patient having at least one relative with early-onset dementia). It could be concluded that screening for mutations in the three genes could be included in a diagnostic program directed at patients with a positive family history or age of onset before 55 years.     

The effects of N-methyl-N-nitrosourea and azoxymethane on focal cerebral infarction and the expression of p53, p21 proteins

If the activity of pro-apoptotic genes can be down-regulated by certain chemicals, cells may be protected from apoptosis. To test this hypothesis in a cerebral infarction model, we used N-methyl-N-nitrosourea (MNU) and azoxymethane (AOM), which were approved gene-modulating chemicals. A focal cerebral infarction was created by coagulation of the right middle cerebral artery and ipsilateral common carotid artery (CCA) and simultaneous transient occlusion of the contralateral CCA for 30 min in 25 adult Sprague-Dawley rats that were sacrificed 24 h later. In one group (n=7), MNU (5 mg/kg) was injected intravenously 30 min before initiation of ischemia. In another group (n=7), AOM (15 mg/kg) was administered intraperitoneally before 24 h of ischemia. The infarction volumes were checked and the brains were stained for p53 and p21 proteins. The width in micrometers of the peri-infarct area containing p53 or p21 protein-positive cells, and the number of p53 or p21 protein-positive cells (cells/HPF) were measured at an adjacent peri-infarct area. The AOM-treated group showed a significantly reduced infarction volume (by 42.5%, p<0.001), a significantly greater number of p53 positive cells (by 12.0%, p<0. 05), and a significantly wider p53 protein-positive area (by 15.6%, p<0.01) than the untreated group. AOM did not show any influence on the expression pattern of the p21 protein. MNU had no effect in the expression of p53 or p21 proteins. As a result, we concluded that AOM revealed a protective effect in ischemia by suppressing the pro-apoptotic activity of the p53 gene. Safer chemicals that can modulate apoptotic genes, if any, will provide a new therapeutic modality for cerebral infarction.     

90K/Mac-2 BP在人脑星形细胞瘤中的表达

目的 分析90K/ Mac-2 BP(Mac-2 binding protein)在人脑星形细胞瘤中的表达.方法 RT-PCR和WB(Western blot)检测90K蛋白在人脑星形细胞瘤以及正常脑组织中的表达.结果 RT-PCR发现90K mRNA在正常脑组织中微量表达,相对表达量为0.116±0.017;而在人脑星形细胞瘤中高表达,相对表达量为0.407±0.151,两组相比有显著差异(t=6.065,P＜0.05).低级别组(WHOⅠ-Ⅱ级)与高级别组(WHO Ⅲ-Ⅳ级)相对表达量分别为0.295±0.067和0.516±0.128,两组相比有显著差异(=8.138,P＜0.05).WB发现90K蛋白在正常脑组织中微量表达,相对表达量为0.291±0.064,星形细胞瘤中90K蛋白相对表达量为1.163±0.391,两组相比有显著差异(t=15.68,P＜0.05).低级别组与高级别组90K蛋白相对表达量分别为0.902±0.272和1.415±0.318,两组相比有显著差异(t=6.539,P＜0.05).WB结果表明90K蛋白在星形细胞瘤中的表达情况与RT-PCR结果表明90K mRNA在星形细胞瘤中的表达情况一致.结论 90K在星形细胞瘤中的表达显著升高,高级别星形细胞瘤中的表达较低级别更高,提示90K可能在星形细胞瘤的发生发展过程中发挥了重要作用,90K蛋白可能是星形细胞瘤相关抗原,在今后的免疫治疗中可能作为一种目标抗原.     

Apoptosis in cerebral astrocytic tumours and its relationship to expression of the bcl-2 and p53 proteins

Apoptosis is an important determinant of tumour growth which can be regulated by the bcl-2 and p53 genes. This study examines the relationship between apoptosis, growth fraction (Ki-67 immunolabelling index), and accumulation of the bcl-2 and p53 proteins in a spectrum of cerebral astrocytic tumours (n=81). including fibrillary astrocytomas (n=16), anaplastic astrocytomas (n=19), and glioblastomas (n=46). Median apoptosis indices (AIs) increased across this spectrum of tumours, and a significant (P<0.0001) correlation was demonstrated between A1 and Ki-67 labelling index (LI). Immunolabelling with the bcl-2 antibody was found in 44% of fibrillary astrocytomas, 42% of anaplastic astrocytomas, and 28% of glioblastomas. It was also found in the vascular endothelial proliferation typically seen in glioblastomas, and in the giant, multinucleated cells of some glioblastomas. No clear relationship between AI and bcl- 2 accumulation was evident. Immunolabelling with the p53 antibody was found in 56% of fibrillary astrocytomas, 79% of anaplastic astrocytomas, and 50% of glioblastomas. No clear relationship between AI and patterns of p53 immunolabelling was evident. Equal proportions of p53-positive tumours were bcl- 2 positive and bcl- 2 negative, but a small proportion of p53-negative tumours was bcl- 2 positive. The correlation between A1 and Ki-67 LI is in line with findings in other malignant tumours. We suggest that the regulation of apoptosis in astrocytic tumours is too complex for a clear association between A1 and bcl- 2 and p53 protein expression to be demonstrated.     

Activation of neuroblast proliferation in explant culture of the Drosophila larval CNS

The developmental control of neuroblast proliferation is absolutely required for the assembly and function of the central nervous system. A lethal mutation in trol results in the failure of quiescent neuroblasts to begin division at the appropriate time. I have established a culture system in which quiescent neuroblasts in explants of Drosophila larval CNSs initiate cell division in vitro to normal in vivo levels. This activation requires removal of the CNS for culture after a specific developmental stage and the presence of fetal calf serum or a larval extract in the medium. Either supplement is effective when heat-treated. Substitution of the steroid hormone ecdysone or the non-steroidal ecdysone analog RH5992 for either fetal calf serum or larval extract also results in activation of neuroblast proliferation. Culture of trol^sd CNSs with wildtype larval extract or ecdysone results in the defective neuroblast proliferation phenotype observed in trol mutants in vivo, while culture of wildtype CNSs with trol^sd extract produces normal neuroblast proliferation.     

Evaluation of p53 protein expression and proliferative potential based on the MIB-1 positive index in astrocytic gliomas

Fifty-seven patients with infiltrative astrocytic gliomas, comprising 29 cases of glioblastoma, 13 cases of anaplastic astrocytoma and 15 cases of low-grade astrocytoma, were examined. In 12 cases of astrocytic glioma, the p53 gene mutation was investigated by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). p53 overexpression was observed in 20 (35%) of the 57 patients with astrocytic gliomas. The occurrence of p53 overexpression was apparently correlated with the histological grading of the astrocytic gliomas. Of the 20 patients with glioblastoma who were less than 60 years old (mean age ± SD, 39.2 ± 15.0 years), 50% demonstrated p53 overexpression, whereas only 2 (22%) patients with glioblastoma who were more than 60 years old (mean age ± SD, 65.1 ± 5.8 years) had p53 overexpression. A substantial difference in survival associated with glioblastoma was noted between the p53-positive group (mean survival 28.9 months) and the p53-negative group (mean survival 11.3 months) based on Kaplan-Meier survival curves (P= 0.01). Even among patients with glioblastoma who were less than 60 years old, a better survival rate was recognized in those with p53 overexpression than in those without p53 (P= 0.03). The MIB-1 indices tended to increase with tumor malignancy, and a poor survival time was significantly associated with elevated values for the MIB-1 index. A substantial difference in survival between the two groups of patients who had MIB-1 indices of above 5% and below 5% was evident from the Kaplan-Meier survival curves (P= 0.04). The group that had no p53, and MIB-1 indices of above 5%, was found to show the malignant gliomas most frequently. Although most of the gliomas with p53 overexpression demonstrated MIB-1 indices of above 5%, there was no significant correlation between p53 protein expression and the MIB-1 index. Of the 12 patients with astrocytic glioma who were examined by PCR-SSCP, 3 (one glioblastoma and 2 anaplastic astrocytomas) revealed p53 gene mutation correlated with p53 (DO-7) overexpression.     

Brain tumors in childhood: Statistical analysis of cases from the brain tumor registry of Japan

This paper attempts to summarize and statistically analyze 2,361 pediatric brain tumors out of a total of 20,192 human brain tumors from about 250 institutes in Japan during the period 1969–1978 in order to obtain an idea of their incidence and results of treatment. This report uses the classification and nomenclature adopted by UICC (Unio Internationalis Contra Cancrum) in 1965. Survival rate was computed as relative survival rate by the method reported by Cutler in 1964 [3]. Interestingly, germinomas (7.8%) are still frequent tumors in Japan. Astrocytoma (19.7%), medulloblastoma (16.6%), craniopharyngioma (12.5%), germinoma (7.8%) and ependymoma (6.6%) occurred in that order of frequency. Five-year survival rate of medulloblastoma was 35.7% in subtotal and 55.3% in total removal. Radiotherapy was an effective therapeutic adjunct for the treatment of medulloblastoma. The germinoma prognosis was good, with a 5-year survival rate of 42.4% without and 69.1% with radiation therapy, revealing radiotherapy as a statistically significant treatment. Radiotherapy, with or without shunt, is thus the treatment of choice. The survival rate of patients with craniopharyngiomas was also high, with a 5-year survival of approximately 75% in partial and 76.3% in total removal. Most of the craniopharyngiomas were treated by partial removal of the tumor, with or without radiotherapy. Patients treated with radiotherapy survived a little longer than patients without.A part of this paper was presented at the 13th Annual Meeting of the International Society for Pediatric Neurosurgery