Clinical association analysis of ependymomas and pilocytic astrocytomas reveals elevated FGFR3 and FGFR1 expression in aggressive ependymomas.

BACKGROUND: Fibroblast growth factor receptors (FGFRs) are well-known proto-oncogenes in several human malignancies and are currently therapeutically targeted in clinical trials. Among glioma subtypes, activating FGFR1 alterations have been observed in a subpopulation of pilocytic astrocytomas while FGFR3 fusions occur in IDH wild-type diffuse gliomas, resulting in high FGFR3 protein expression. The purpose of this study was to associate FGFR1 and FGFR3 protein levels with clinical features and genetic alterations in ependymoma and pilocytic astrocytoma. METHODS: FGFR1 and FGFR3 expression levels were detected in ependymoma and pilocytic astrocytoma tissues using immunohistochemistry. Selected cases were further analyzed using targeted sequencing. RESULTS: Expression of both FGFR1 and FGFR3 varied within all tumor types. In ependymomas, increased FGFR3 or FGFR1 expression was associated with high tumor grade, cerebral location, young patient age, and poor prognosis. Moderate-to-strong expression of FGFR1 and/or FGFR3 was observed in 76% of cerebral ependymomas. Cases with moderate-to-strong expression of both proteins had poor clinical prognosis. In pilocytic astrocytomas, moderate-to-strong FGFR3 expression was detected predominantly in non-pediatric patients. Targeted sequencing of 12 tumors found no protein-altering mutations or fusions in FGFR1 or FGFR3. CONCLUSIONS: Elevated FGFR3 and FGFR1 protein expression is common in aggressive ependymomas but likely not driven by genetic alterations. Further studies are warranted to evaluate whether ependymoma patients with high FGFR3 and/or FGFR1 expression could benefit from treatment with FGFR inhibitor based therapeutic approaches currently under evaluation in clinical trials.

Polysialic acid is associated with better prognosis and IDH1-mutation in diffusely infiltrating astrocytomas.

BACKGROUND: The aim of the study was to assess the localization of Polysialic acid (polySia) and Neural cell adhesion molecule (NCAM) in grade I-IV astrocytomas by confocal microscopy, and also to clarify and compare their relationship to conventional clinicopathological features in these tumors. METHODS: Study material was stained immunohistochemically for polySia, NCAM and IDH1-R132H point mutation. Confocal microscopy of polySia and NCAM staining was performed on tissue micro-array samples (TMA) of 242 diffusely infiltrating astrocytomas (grade II: 28; grade III: 33; grade IV: 181) and 82 pilocytic astrocytomas. The results were statistically correlated to clinicopathological factors and survival data. RESULTS: PolySia was observed in 45 cases (19%) and NCAM positivity in 92 cases (38%). All 45 tumors with polySia positivity were also positive for NCAM whereas there were 47 tumors which contained positive staining for NCAM but not for polySia. The simultaneous expression was concomitant and colocalized suggesting polysialyated NCAM (polySia-NCAM). PolySia expression was significantly stronger in IDH1 mutated tumors than in IDH1 non-mutated (p = 0.001, chi-square test). There were no significant differences in polySia-NCAM between primary tumors or recurrences (p = n.s., chi-square test). PolySia positivity was associated with longer patient survival in relation to total tumor material (p = 0.020, log-rank test). Furthermore, when only glioblastomas were assessed, patients with positive polySia had significantly better prognosis (p = 0.006, log-rank test). In multivariate survival analysis, polySia was found to be an independent prognostic factor. PolySia was nearly absent in grade I pilocytic astrocytomas (1 immunopositive tumor of 82). CONCLUSIONS: Expression of polySia is common in adult grade II-IV astrocytomas, whereas it is nearly absent in pediatric grade I pilocytic astrocytomas. PolySia positivity is associated with longer survival rates in patients with a grade II-IV astrocytomas and also grade IV glioblastomas assessed separately. The results of this study suggest that IDH1 mutation may be associated with polySia expression pathways in malignant gliomas.

[Diagnosis and prognosis of gliomas--current prospects of molecular diagnostics]. / Glioomien diagnoosi ja ennuste--molekyylidiagnostiikan mahdollisuudet.

Gliomas are tumors of the support cells of the brain and the most common of the primary brain tumors. Treatment of diffuse gliomas is based on surgical excision of the tumor and on radiotherapy and chemotherapy. The diagnosis is made in histopathological examination of the tumor, which today can be complemented with examinations involving molecular diagnostics. The most important new methods predicting the prognosis of glioma patients include demonstrations of the IDH mutation and the 1p/19q co-deletion. Profiling of gliomas may in the future allow tailoring of therapy in a patient-specific manner.

Rapid identification of pediatric brain tumors with differential mobility spectrometry.

Introduction: Brain tumors are a major source of disease burden in pediatric population, with the most common tumor types being pilocytic astrocytoma, ependymoma and medulloblastoma. In every tumor entity, surgery is the cornerstone of treatment, but the importance of gross-total resection and the corresponding patient prognosis is highly variant. However, real-time identification of pediatric CNS malignancies based on the histology of the frozen sections alone is especially troublesome. We propose a novel method based on differential mobility spectrometry (DMS) analysis for rapid identification of pediatric brain tumors. Methods: We prospectively obtained tumor samples from 15 pediatric patients (5 pilocytic astrocytomas, 5 ependymomas and 5 medulloblastomas). The samples were cut into 36 smaller specimens that were analyzed with the DMS. Results: With linear discriminant analysis algorithm, a classification accuracy (CA) of 70% was reached. Additionally, a 75% CA was achieved in a pooled analysis of medulloblastoma vs. gliomas. Discussion: Our results show that the DMS is able to differentiate most common pediatric brain tumor samples, thus making it a promising additional instrument for real-time brain tumor diagnostics.

Aberrant DNA methylation distorts developmental trajectories in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (AT/RTs) are pediatric brain tumors known for their aggressiveness and aberrant but still unresolved epigenetic regulation. To better understand their malignancy, we investigated how AT/RT-specific DNA hypermethylation was associated with gene expression and altered transcription factor binding and how it is linked to upstream regulation. Medulloblastomas, choroid plexus tumors, pluripotent stem cells, and fetal brain were used as references. A part of the genomic regions, which were hypermethylated in AT/RTs similarly as in pluripotent stem cells and demethylated in the fetal brain, were targeted by neural transcriptional regulators. AT/RT-unique DNA hypermethylation was associated with polycomb repressive complex 2 and linked to suppressed genes with a role in neural development and tumorigenesis. Activity of the several NEUROG/NEUROD pioneer factors, which are unable to bind to methylated DNA, was compromised via the suppressed expression or DNA hypermethylation of their target sites, which was also experimentally validated for NEUROD1 in medulloblastomas and AT/RT samples. These results highlight and characterize the role of DNA hypermethylation in AT/RT malignancy and halted neural cell differentiation.

[Childhood brain tumors]. / Lasten aivokasvaimet.

Brain tumors are the second most common pediatric neoplastic disease after leukemias. As causes of mortality and morbidity they add up to the most significant group of tumors. Treatment is based on thorough surgical excision of the tumor. Additional treatment with cytotoxic agents and radiotherapy is applied to malignant tumors. Treatment results have improved so that approximately three children out of four will make complete recovery from brain tumor. Long-term problems are, however, common and often significantly weakening the quality of life.

Impact of Timing of Surgery and Adjuvant Treatment on Survival of Adult IDH-wild-type Glioblastoma: A Single-center Study of 392 Patients.

BACKGROUND: The purpose of our study was to analyze the impact of time interval from referral to surgery and from surgery to adjuvant treatment on survival of adult isocitrate dehydrogenase-wild-type (IDH-wt) glioblastomas. METHODS: Data on 392 IDH-wt glioblastomas diagnosed at the Tampere University Hospital in 2004-2016 were obtained from the electronic patient record system. Piecewise Cox regression was used to calculate hazard ratios for different time intervals between referral and surgery, as well as between surgery and adjuvant treatments. RESULTS: The median survival time from primary surgery was 9.5 months (interquartile range: 3.8-16.0). Survival among patients with an interval exceeding 4 weeks from referral to surgery was no worse compared to <2 weeks (hazard ratio: 0.78, 95% confidence interval: 0.54-1.14). We found indications of poorer outcome when the interval from surgery to radiotherapy exceeded 30 days (hazard ratio: 1.42, 95% confidence interval: 0.91-2.21 for 31-44 days; and 1.59, 0.94-2.67 for over 45 days). CONCLUSIONS: Interval from referral to surgery in the range of 4-10 weeks was not associated with decreased survivals in IDH-wt glioblastomas. In contrast, delay exceeding 30 days from surgery to adjuvant treatment may decrease long-term survival.

Motor dysfunction as a primary symptom predicts poor outcome: multicenter study of glioma symptoms.

Background and objectives: The objectives of this study were to investigate the prognostic value of primary symptoms and leading symptoms in adult patients with diffuse infiltrating glioma and to provide a clinical perspective for evaluating survival. Methods: This study included a retrospective cohort from two tertiary university hospitals (n = 604, 2006-2013, Tampere University Hospital and Turku University Hospital) and a prospective cohort (n = 156, 2014-2018, Tampere University Hospital). Preoperative symptoms were divided into primary and leading symptoms. Results were validated with the newer WHO 2021 classification criteria. Results: The most common primary symptoms were epileptic seizure (30.8% retrospective, 28.2% prospective), cognitive disorder (13.2% retrospective, 16.0% prospective), headache (8.6% retrospective, 12.8% prospective), and motor paresis (7.0% retrospective, 7.1% prospective). Symptoms that predicted better survival were epileptic seizure and visual or other sense-affecting symptom in the retrospective cohort and epileptic seizure and headache in the prospective cohort. Predictors of poor survival were cognitive disorder, motor dysfunction, sensory symptom, tumor hemorrhage, speech disorder and dizziness in the retrospective cohort and cognitive disorder, motor dysfunction, sensory symptom, and dizziness in the prospective cohort. Motor dysfunction served as an independent predictor of survival in a multivariate model (OR = 1.636). Conclusion: Primary and leading symptoms in diffuse gliomas are associated with prognoses in retrospective and prospective settings. Motor paresis was an independent prognostic factor for poor survival in multivariate analysis for grade 2-4 diffuse gliomas, especially in glioblastomas.

Method for the Intraoperative Detection of IDH Mutation in Gliomas with Differential Mobility Spectrometry.

Isocitrate dehydrogenase (IDH) mutation status is an important factor for surgical decision-making: patients with IDH-mutated tumors are more likely to have a good long-term prognosis, and thus favor aggressive resection with more survival benefit to gain. Patients with IDH wild-type tumors have generally poorer prognosis and, therefore, conservative resection to avoid neurological deficit is favored. Current histopathological analysis with frozen sections is unable to identify IDH mutation status intraoperatively, and more advanced methods are therefore needed. We examined a novel method suitable for intraoperative IDH mutation identification that is based on the differential mobility spectrometry (DMS) analysis of the tumor. We prospectively obtained tumor samples from 22 patients, including 11 IDH-mutated and 11 IDH wild-type tumors. The tumors were cut in 88 smaller specimens that were analyzed with DMS. With a linear discriminant analysis (LDA) algorithm, the DMS was able to classify tumor samples with 86% classification accuracy, 86% sensitivity, and 85% specificity. Our results show that DMS is able to differentiate IDH-mutated and IDH wild-type tumors with good accuracy in a setting suitable for intraoperative use, which makes it a promising novel solution for neurosurgical practice.

The tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a group of medulloblastomas and supratentorial primitive neuroectodermal tumours: an association of CA IX with poor prognosis.

BACKGROUND: Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumours (PNETs) are the most common highly aggressive paediatric brain tumours. In spite of extensive research on these tumours, there are only few known biomarkers or therapeutic target proteins, and the prognosis of patients with these tumours remains poor. Our aim was to investigate whether carbonic anhydrases (CAs), enzymes commonly overexpressed in various tumours including glioblastomas and oligodendrogliomas, are present in MBs and PNETs, and whether their expression can be correlated with patient prognosis. METHODS: We determined the expression of the tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a series of MB/PNET specimens (n = 39) using immunohistochemistry. RESULTS: Endothelial CA II, cytoplasmic CA II, CA IX and CA XII were expressed in 49%, 73%, 23% and 11% of the tumours, respectively. CA II was detected in the neovessel endothelium and the tumour cell cytoplasm. CA IX was mainly expressed in the tumour cells located in perinecrotic areas. CA XII showed the most homogenous distribution within the tumours. Importantly, CA IX expression predicted poor prognosis in both univariate (p = 0.041) and multivariate analyses (p = 0.016). CONCLUSIONS: We suggest that CA IX should be considered a potential prognostic and therapeutic target in MBs and PNETs.

Amplification and overexpression of KIT, PDGFRA, and VEGFR2 in medulloblastomas and primitive neuroectodermal tumors.

Medulloblastomas (MB) and primitive neuroectodermal tumors (PNET) are the most common malignant brain tumors in children. These two tumor types are histologically similar, but have different genetic backgrounds and clinical outcomes. Other brain tumors, such as gliomas, frequently have coamplification and overexpression of receptor tyrosine kinases KIT, platelet-derived growth factor receptor alpha (PDGFRA), and vascular endothelial growth factor receptor 2 (VEGFR2). We investigated protein expression and gene copy numbers of KIT, PDGFRA, and VEGFR2 in 41 MB and 11 PNET samples by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). KIT and PDGFRA expression was detected in both MBs and PNETs, whereas VEGFR2 expression was weak in these tumors. KIT, PDGFRA, and VEGFR2 amplifications were all present in 4% of MBs/PNETs, and KIT amplification was associated with concurrent PDGFRA and VEGFR2 amplifications (P

The Expression of Carbonic Anhydrases II, IX and XII in Brain Tumors.

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that participate in the regulation of pH homeostasis in addition to many other important physiological functions. Importantly, CAs have been associated with neoplastic processes and cancer. Brain tumors represent a heterogeneous group of diseases with a frequently dismal prognosis, and new treatment options are urgently needed. In this review article, we summarize the previously published literature about CAs in brain tumors, especially on CA II and hypoxia-inducible CA IX and CA XII. We review here their role in tumorigenesis and potential value in predicting prognosis of brain tumors, including astrocytomas, oligodendrogliomas, ependymomas, medulloblastomas, meningiomas, and craniopharyngiomas. We also introduce both already completed and ongoing studies focusing on CA inhibition as a potential anti-cancer strategy.

Moderate-to-strong expression of FGFR3 and TP53 alterations in a subpopulation of choroid plexus tumors.

Deregulation of fibroblast growth factor receptor (FGFR) signaling is tightly associated with numerous human malignancies, including cancer. Indeed, FGFR inhibitors are being tested as anti-tumor drugs in clinical trials. Among gliomas, FGFR3 fusions occur in IDH wild-type diffuse gliomas leading to high FGFR3 protein expression and both, FGFR3 and FGFR1, show elevated expression in aggressive ependymomas. The aim of this study was to uncover the expression of FGFR1 and FGFR3 proteins in choroid plexus tumors and to further characterize FGFR-related as well as other genetic alterations in FGFR3 expressing tumors. Expression levels of FGFR1 and FGFR3 were detected in 15 choroid plexus tumor tissues using immunohistochemistry of tissue microarrays and 6 samples were subjected to whole mount FGFR3 staining. Targeted sequencing was used for deeper molecular analysis of two FGFR3 positive cases. Moderate expression of FGFR1 or FGFR3 was evidenced in one third of the studied choroid plexus tumors. Targeted sequencing of a choroid plexus carcinoma and an atypical choroid plexus papilloma, both with moderate-to-strong FGFR3 expression, revealed lack of protein-altering mutations or fusions in FGFR1 or FGFR3, but TP53 was altered in both tumors. FGFR3 and FGFR1 proteins are expressed in a subpopulation of choroid plexus tumors. Further studies using larger cohorts of patients will allow identification of the clinicopathological implications of FGFR1 and FGFR3 expression in choroid plexus tumors.

MK2 Inhibition Induces p53-Dependent Senescence in Glioblastoma Cells.

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While TP53 deletion is rare in glioblastomas, these tumors often carry TP53 mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53wt stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact TP53 gene. However, targeting MK2 in tumors with TP53 mutations may accelerate disease progression. These findings are highly relevant since TP53 mutations occur in over 50% of all cancers.

Identifying brain tumors by differential mobility spectrometry analysis of diathermy smoke.

OBJECTIVE: There is a need for real-time, intraoperative tissue identification technology in neurosurgery. Several solutions are under development for that purpose, but their adaptability for standard clinical use has been hindered by high cost and impracticality issues. The authors tested and preliminarily validated a method for brain tumor identification that is based on the analysis of diathermy smoke using differential mobility spectrometry (DMS). METHODS: A DMS connected to a special smoke sampling system was used to discriminate brain tumors and control samples ex vivo in samples from 28 patients who had undergone neurosurgical operations. They included meningiomas (WHO grade I), pilocytic astrocytomas (grade I), other low-grade gliomas (grade II), glioblastomas (grade IV), CNS metastases, and hemorrhagic or traumatically damaged brain tissue as control samples. Original samples were cut into 694 smaller specimens in total. RESULTS: An overall classification accuracy (CA) of 50% (vs 14% by chance) was achieved in 7-class classification. The CA improved significantly (up to 83%) when the samples originally preserved in Tissue-Tek conservation medium were excluded from the analysis. The CA further improved when fewer classes were used. The highest binary classification accuracy, 94%, was obtained in low-grade glioma (grade II) versus control. CONCLUSIONS: The authors' results show that surgical smoke from various brain tumors has distinct DMS profiles and the DMS analyzer connected to a special sampling system can differentiate between tumorous and nontumorous tissue and also between different tumor types ex vivo.

Identification of an alternatively spliced isoform of carbonic anhydrase XII in diffusely infiltrating astrocytic gliomas.

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.

NRF2, DJ1 and SNRX1 and their prognostic impact in astrocytic gliomas.

Nuclear factor erythroid 2-related factor 2 (NRF2), DJ1 and sulfiredoxin 1 (SRXN1) are transcription factors which protect cells from the oxidative damage caused by reactive oxygen species and, on the other hand, are associated with resistance to cancer treatments. The immunohistochemical expression of NRF2, DJ1 and SRNX1 was assessed in human grade II-IV astrocytic gliomas. Their association to clinicopathologic and essential molecular factors was evaluated. The RNA expression levels and genetic alterations were analyzed from publicly available datasets. All studied molecules were commonly expressed. The cytoplasmic NRF2 expression was higher in tumors with a higher malignancy grade, whereas the nuclear and cytoplasmic DJ1 expression was associated with a lower grade. The presence of the isocitrate dehyrdogenase 1 mutation (IDH1) was associated with an increasing cytoplasmic and nuclear expression of NRF2 and a nuclear DJ1 expression. When primary grade IV astrocytomas were compared to secondary glioblastomas, nuclear DJ1 was associated with secondary tumors. In grade II-IV tumors, the cytoplasmic NRF2 expression was associated with a poor prognosis, whereas nuclear NRF2 and both cytoplasmic and nuclear DJ1 were associated with a better patient prognosis. Recurrent homozygous deletions of DJ1 were observed, especially in the IDH wild-type samples. When only the glioblastomas were evaluated, nuclear NRF2 and SRNX1 predicted better survival. As a conclusion, NRF2, DJ1 and SNXR1 can be used as prognosticators in gliomas.

Whole-exome sequencing identifies germline mutation in TP53 and ATRX in a child with genomically aberrant AT/RT and her mother with anaplastic astrocytoma.

Brain tumors typically arise sporadically and do not affect several family members simultaneously. In the present study, we describe clinical and genetic data from two patients, a mother and her daughter, with familial brain tumors. Exome sequencing revealed a germline missense mutation in the TP53 and ATRX genes in both cases, and a somatic copy-neutral loss of heterozygosity (LOH) in TP53 in both atypical teratoid/rhabdoid tumor (AT/RT) and astrocytoma tumors. ATRX mutation was associated with the loss of ATRX protein expression. In the astrocytoma case, R132C missense mutation was found in the known hotspot site in isocitrate dehydrogenase 1 (IDH1) and LOH was detected in TP53 The mother carried few other somatic alterations, suggesting that the IDH1 mutation and LOH in TP53 were sufficient to drive tumor development. The genome in the AT/RT tumor was atypically aneuploid: Most chromosomes had experienced copy-neutral LOH or whole-chromosome gains. Only Chromosome 18 had normal diploid status. INI1/hSNF5/SMARCB1 was homozygously deleted in the AT/RT tumor. This report provides further information about tumor development in a predisposed genetic background and describes two special Li-Fraumeni cases with a familial brain tumor.

Carbonic anhydrase II in the endothelium of glial tumors: a potential target for therapy.

Carbonic anhydrase isozyme II (CA II) is a cytosolic enzyme that is highly expressed in most organs, including the brain, where it is mainly located in the oligodendrocytes. Recent studies have shown that its expression is induced in the endothelium of neovessels in melanoma and esophageal, renal, and lung cancer. Immunological studies further indicate that CA II represents a major target antigen stimulating an autoantibody response in melanoma patients. These results prompted us to investigate endothelial CA II expression in two types of brain cancer: oligodendrogliomas and astrocytomas. A series of 255 astrocytoma and 71 oligodendroglial tumor specimens was immunostained for CA II. The staining results were correlated with a number of different clinicopathological factors and survival data. CA II showed weak or no expression in low-grade tumors, while grade 3 mixed oligoastrocytoma and glioblastoma multiforme were the most positively stained tumor types. Survival analysis indicated that endothelial CA II staining is significantly associated with a poor prognosis in patients with astrocytomas. About 17% of patients with CA II-negative tumors (weak or no endothelial signal) were still alive at the end of the follow-up period of five years. The presence of CA II in the tumor endothelium suggests that it may play an important functional role in tumor metabolism. From a clinical perspective, the results also open new avenues for selecting tumor types for dendritic cell therapy trials.

Expression of carbonic anhydrase IX in astrocytic tumors predicts poor prognosis.

PURPOSE: Carbonic anhydrase IX (CA IX) is a hypoxia-inducible enzyme, which is associated with neoplastic growth. Ectopic CA IX expression has been observed in several tumors, whose normal counterparts do not express this enzyme. Normal human brain tissue shows only slight or no expression of CA IX. EXPERIMENTAL DESIGN: We describe CA IX expression in human diffusely infiltrating astrocytomas. The association of CA IX is evaluated with clinicopathologic and molecular factors including cell proliferation and apoptosis as well as the expression of p53 and epidermal growth factor receptor. RESULTS: CA IX immunopositivity was observed in 284 cases of 362 (78%) tumors. The positive areas were often located in close proximity to necrotic regions (P < 0.001). The CA IX immunoreactivity showed strong association with tumor malignancy grades (P < 0.0001). CA IX showed no association with p53 expression nor did it correlate with epidermal growth factor receptor-amplification, apoptosis, or cell proliferation. CA IX intensity had significant prognostic value in univariate (P=0.0011, log-rank test) and multivariate survival analysis (P = 0.038, Cox analysis). CONCLUSIONS: CA IX expression is common in diffusely infiltrating high-grade astrocytomas. Our results suggest that CA IX is a useful biomarker for predicting poor prognosis of astrocytic tumors. It may also be a promising target molecule for the improvement of therapeutic interventions in astrocytomas.