Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis.

AIMS: It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status. METHODS: MGMT protein expression was quantified in tumour cells in 171 GBMs from the population-based Region of Southern Denmark (RSD)-cohort using a double immunofluorescence approach. Pyrosequencing was performed in 157 patients. For validation we used GBM-patients from a Nordic Study (NS) investigating the effect of radiotherapy and different TMZ schedules. RESULTS: When divided at the median, patients with low expression of MGMT protein (AF-low) had the best prognosis (HR = 1.5, P = 0.01). Similar results were observed in the subgroup of patients receiving the Stupp regimen (HR = 2.0, P = 0.001). In the NS-cohort a trend towards superior survival (HR = 1.6, P = 0.08) was seen in patients with AF-low. Including MGMT promoter methylation status, we found for both cohorts that patients with methylated MGMT promoter and AF-low had the best outcome; median OS 23.1 and 20.0 months, respectively. CONCLUSION: Our data indicate that MGMT protein expression in tumour cells has an independent prognostic significance. Exclusion of nontumour cells contributed to a more exact analysis of tumour-specific MGMT protein expression. This should be incorporated in future studies evaluating MGMT status before potential integration into clinical practice.

Age and surgical outcome of low-grade glioma in Sweden.

BACKGROUND: Low-grade gliomas (LGG) are slow-growing primary brain tumors that typically affect young adults. Advanced age is widely recognized as a poor prognostic factor in LGG. The impact of age on postoperative outcome in this patient group has not been systemically studied. METHODS: We performed a nationwide register-based study with data from the Swedish Brain Tumor Registry (SBTR) for all adults diagnosed with a supratentorial LGG (WHO grade II astrocytoma, oligoastrocytoma, or oligodendroglioma) during 2005-2015. Patient- and tumor-related characteristics, postoperative complications, and survival were compared between three different age groups (18-39 years, 40-59 years, and ≥60 years). RESULTS: We identified 548 patients; 204 patients (37.2%) aged 18-39 years, 227 patients (41.4%) aged 40-59 years, and 117 patients (21.4%) ≥60 years of age. Unfavorable preoperative prognostic factors (eg, functional status and neurological deficit) were more common with increased age (P < .001). In addition, overall survival was significantly impaired in those 60 years and above (P < .001). We observed a clear dose-response for age with separation of survival curves at 50 years. Biopsy was more common in patients ≥60 years (P < .001). Subgroup analysis of patients with resection revealed a higher amount of postoperative neurological deficits in older patients (P = .029). CONCLUSION: In general, older patients with LGG have several unfavorable prognostic factors compared with younger patients but seem to tolerate surgery in a comparable fashion. However, more neurological deficits were observed following resections in elderly. Our data further support a cutoff at 50 years rather than 40 years for selection of high-risk patients.

The prognostic value of pre-treatment thrombocytosis in two cohorts of patients with non-small cell lung cancer treated with curatively intended chemoradiotherapy.

Chemoradiotherapy is the standard of care for inoperable stage III non-small cell lung cancer (NSCLC). This treatment, however, offers only a small chance of cure and is associated with many side effects. Little research has been made concerning which patients benefit most/least from the treatment. The present study evaluates the prognostic value of anemia, leukocytosis and thrombocytosis at diagnosis in this treatment setting. In the present study, data were collected retrospectively for 222 patients from two different phase II studies conducted between 2002-2007 in Sweden with patients treated with chemoradiotherapy for stage IIIA-IIIB NSCLC. Clinical data and the serum values of hemoglobin (Hgb), White blood cells (WBC) and Platelets (Plt) at enrollment were collected for all patients and studied in relation to overall survival using Kaplan-Meier product-limit estimates and a multivariate Cox proportional hazards regression model. The results showed that patients with thrombocytosis (Plt > 350 x 109/L) had a shorter median overall survival (14.5 months) than patients with normal Plt at baseline (23.7 months). Patients with leukocytosis (WBC > 9 x 109/L) had a shorter median survival (14.9 months) than patients with a normal WBC at baseline (22.5 months). However, in a multivariate model including all lab parameters and clinical factors, only thrombocytosis and performance status displayed a prognostic significance. In Conclusion, thrombocytosis showed to be an independent prognostic marker associated with shorter overall survival in stage III NSCLC treated with curatively intended chemoradiotherapy. This knowledge can potentially be used together with established prognostic factors, such as performance status when choosing the optimal therapy for the individual patient in this clinical setting.

Expression of LRIG1 is associated with good prognosis and human papillomavirus status in oropharyngeal cancer.

BACKGROUND: The incidence of human papillomavirus (HPV)-associated oropharyngeal cancer has increased rapidly during the past decades. HPV is typically associated with a favourable outcome; however, a need exists for new and more effective prognostic and predictive markers for this disease. Leucine-rich repeats and immunoglobulin-like domains (LRIG)-1 is a tumour suppressor protein that belongs to the LRIG family. LRIG1 expression has prognostic significance in various human cancers, including cervical cancer, where HPV is a key aetiological agent. METHODS: The prognostic value of LRIG1 and LRIG2 immunoreactivity was investigated in tumour specimens from a Swedish cohort of patients with tonsillar and base of tongue oropharyngeal cancers, including 278 patients. RESULTS: LRIG1 immunoreactivity correlated with disease-free survival and overall survival in univariate and multivariate analyses. Notably, patients with HPV-positive tumours with high LRIG1 staining intensity or a high percentage of LRIG1-positive cells showed a very good prognosis. Furthermore, LRIG1 expression correlated with HPV status, whereas LRIG2 expression inversely correlated with HPV status. CONCLUSIONS: Taken together, the results suggest that LRIG1 immunoreactivity could be a clinically important prognostic marker in HPV-associated oropharyngeal cancer.

The dynorphin/κ-opioid receptor system and its role in psychiatric disorders.

The dynorphin/κ-opioid receptor system has been implicated in the pathogenesis and pathophysiology of several psychiatric disorders. In the present review, we present evidence indicating a key role for this system in modulating neurotransmission in brain circuits that subserve mood, motivation, and cognitive function. We overview the pharmacology, signaling, post-translational, post-transcriptional, transcriptional, epigenetic and cis regulation of the dynorphin/κ-opioid receptor system, and critically review functional neuroanatomical, neurochemical, and pharmacological evidence, suggesting that alterations in this system may contribute to affective disorders, drug addiction, and schizophrenia. We also overview the dynorphin/κ-opioid receptor system in the genetics of psychiatric disorders and discuss implications of the reviewed material for therapeutics development.

Genetic variations in VEGF and VEGFR2 and glioblastoma outcome.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) are central components in the development and progression of glioblastoma. To investigate if genetic variation in VEGF and VEGFR2 is associated with glioblastoma prognosis, we examined blood samples from 154 glioblastoma cases collected in Sweden and Denmark between 2000 and 2004. Seventeen tagging single nucleotide polymorphisms (SNPs) in VEGF and 27 in VEGFR2 were genotyped and analysed, covering 90% of the genetic variability within the genes. In VEGF, we found no SNPs associated with survival. In VEGFR2, we found two SNPs significantly associated to survival, namely rs2071559 and rs12502008. However, these results are likely to be false positives due to multiple testing and could not be confirmed in a separate dataset. Overall, this study provides little evidence that VEGF and VEGFR2 polymorphisms are important for glioblastoma survival.

PET response and tumor stabilization under erlotinib and bevacizumab treatment of an intracranial lesion non-invasively diagnosed as likely chordoma.

INTRODUCTION: Chordoma is a rare and a slow-growing tumor originating from the notochord and commonly localized in the skull base. Surgery and occasionally radiotherapy have emerged as the treatments of choice. In the relapsed situations available treatment options are strictly limited; however, recently molecularly targeted agents have been proposed to be of potential beneficial value. THE CASE: A 63-year-old male presenting with seizures and an extradural mass in the left brain hemisphere. An attempt to resect the tumor was followed by severe bradycardia when manipulating with the dura and therefore discontinued. It was considered too hazardous even to take a biopsy specimen. The tumor was considered radiologically and macroscopically as a chordoma. As the tumor progressed after radiotherapy, chemotherapy with erlotinib in combination with cetuximab was initiated. This treatment was interrupted due to progressive disease and toxicity. However, combination treatment with erlotinib and bevacizumab normalized the uptake of [11C]methionine PET signal and resulted in a slight tumor shrinkage on MRI. The patient is still (March 2011) free of symptoms, without cranial nerve deficits or seizures. DISCUSSION: This report shows that erlotinib and bevacizumab in combination may completely quench the transport of the essential amino acid methionine to a treatment refractory intracranial tumor bearing radiological and clinical characteristics of a chordoma. Further studies are necessary to establish this strategy as a treatment option for this indication.

Expression of leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins in human ependymoma relates to tumor location, WHO grade, and patient age.

Three human leucine-rich repeats and immunoglobulin-like domains (LRIG1-3) genes and proteins have recently been characterized. LRIG1 has been shown to be a suppressor of tumor growth by counteracting the signaling of epidermal growth factor receptor (EGFR) family members, including EGFR (ERBB1). Expression of LRIG proteins seems to be of importance in the pathogenesis of astrocytic tumors. In this study, the expression of LRIG1-3 was evaluated in 51 human ependymomas by immunohistochemistry. LRIG proteins were detected in all ependymomas analyzed, however, with a pronounced heterogeneity in expression and subcellular localization. Higher cytoplasmic immunoreactivity of LRIG1 correlated with older patient age and higher LRIG1 nuclear immunoreactivity with lower WHO Grade. LRIG1 displayed a stronger immunoreactivity in the cytoplasm and nuclei in spinal ependymomas than in the posterior fossa or supratentorial ependymomas, while perinuclear LRIG3 was more highly expressed in supratentorial than in infratentorial ependymomas. The indications that expression and subcellular localization of LRIG proteins could be pathogenetically associated with specific clinicopathological features of ependymoma tumors might be of importance in the carcinogeneses and tumor progression of human ependymomas.

LRIG1 and squamous epithelial uterine cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking.

The aim is to evaluate LRIG1 as a prognosis predictor and correlations to cofactors in squamous cell cervical cancer. LRIG1 expression was studied in 128 cervical carcinomas and was compared with expression of nine other tumor markers. Smoking history was registered and pretreatment serum estradiol and progesterone levels were evaluated in 79 women. At clinical stage IB, 58% of the tumors showed LRIG1 expression, but there was a decline by increasing stage (33% in stage IV). Ninety percent of women with stage IB cancer and LRIG1 positivity survived, as compared to 64% without expression (P = 0.02). LRIG1 expression did not predict prognosis in advanced stages, but in stage IIA there was a marked relative difference, with 75% survival in tumors expressing LRIG1, as compared to 43% in those without. No correlation was found between LRIG1 and the other nine tumor markers studied. A high serum progesterone and smoking correlated to absent LRIG1 expression. We conclude that LRIG1 appears to be a significant prognosis predictor in early-stage cervical cancer, independent of the other tumor markers that were studied. Diminished expression in advanced stages and the inverse correlation to serum progesterone and smoking indicates that LRIG1 is a tumor suppressor in cervix.

Induction of apoptosis by intracellular potassium ion depletion: using the fluorescent dye PBFI in a 96-well plate method in cultured lung cancer cells.

Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, activate caspases and induce DNA fragmentation, events which are features of apoptosis. Here we describe a 96-well plate method using the cell permeable form of K+ binding benzofuran isophtalate (PBFI-AM) to measure intracellular K+ content in relation to untreated control. Cultured human pulmonary mesothelioma cells (P31) and small-cell lung cancer cells (U1690) were treated with K+ flux modulators in order to deprive the cells of intracellular K+. The combination of K+ influx inhibition with 10 micromol/L bumetanide plus 10 micromol/L ouabain and K+ efflux stimulation with 3 mg/L amphotericin B or 5 micromol/L nigericin efficiently reduced the intracellular K+ content after 3 h. Manipulation of K+ fluxes with subsequent intracellular K+ depletion induced apoptosis of lung cancer cells, as detected by caspase-3 activity after 3 h K+ depletion followed by 24 h proliferation and TUNEL positive staining after 48 h proliferation. We concluded that the PBFI-AM assay was a useful tool to determine intracellular K+ content in relation to untreated control, and that intracellular K+ depletion of lung cancer cells by clinically used drugs of relevant concentrations induced apoptosis. These findings may lead to novel therapeutic strategies in the treatment of lung cancer.

Effects of smoking and irradiated volume on inflammatory response in the lung of irradiated breast cancer patients evaluated with bronchoalveolar lavage.

Quantitative measurements of the effects of irradiation on normal tissues in humans have been hard to obtain because most tissues are inaccessible and/or direct responses are difficult to quantify in a nondestructive manner. Pneumonitis and fibrotic lung disease are adverse effects seen in varying intensity in patients treated with radiotherapy for carcinomas of the thorax, e.g., breast cancer. In the present study the aim was to evaluate the inflammatory reaction in the underlying parenchyma following postoperative irradiation with bronchoalveolar lavage technique. Twenty-one patients (11 smokers, 10 nonsmokers) with breast cancer stage T1N0M0 received radiotherapy with photons to a target dose of 56 Gy following breast conservative surgery. Nineteen healthy controls were also included. The results showed a clear elevation of neutrophils, mast cells, eosinophils, and lymphocytes in the total irradiated groups, compared to controls. When subclassifying the material according to smoking habit, it was obvious that the smokers displayed a significantly decreased inflammatory reaction, i.e., reduced levels of mast cells and lymphocytes, compared to both nonsmoking controls and patients. Eosinophils were seen in an elevated number in all irradiated patients. Radiological signs of pneumonitis were observed in three patients, all in the nonsmoking group. No correlation was found between the volume of lung irradiated and the inflammatory response. It is concluded that bronchoalveolar lavage is a suitable and sensitive method for investigating radiotherapy-induced reactions in the human lung. Furthermore, ongoing smoking during the treatment depressed the inflammatory response in the lung parenchyma induced by irradiation. The present study as well as earlier observations justify further studies concerning the possibility of interaction of smoking with cancer treatment, both from the view of therapeutic failures and reduced adverse effects.

Estramustine-binding protein and specific binding of the anti-mitotic compound estramustine in astrocytoma.

Estramustine-binding protein (EMBP) is a M(r) 46,000 heterodimeric protein originally isolated from prostatic tissue. It has a demonstrated high affinity for, and selective binding of, estramustine, which is a derivative of 17 beta-estradiol and nornitrogen mustard with antimitotic activity. In this study, we have analysed the expression of an EMBP-like protein in astrocytoma specimens. Immunohistochemistry revealed a pronounced reactivity for EMBP in astrocytoma grades III-IV as well as in metastatic prostatic adenocarcinoma used as positive control. In astrocytoma grades I-II, the expression was weak. The EMBP-like protein was quantified by radioimmunoassay in astrocytoma tumor tissue with higher concentrations in malignant astrocytoma, grades III-IV, compared to grades I-II tumors. Western immunoblotting of immunoaffinity purified EMBP-like protein under nonreducing conditions revealed an immunoreactivity corresponding to M(r) 138,000 and 200,000, indicating a different structure of EMBP in astrocytoma compared to prostatic tissue. Specific binding and the presence of saturable binding sites for 3H-labeled estramustine were demonstrated in astrocytoma tissues expressing EMBP-like protein. Scatchard plot analysis showed a Kd at approximately 30 nM, which suggests a binding affinity for estramustine in the same range as previously reported for EMBP in the prostate. Moreover, the number of estramustine binding sites/g tumor as calculated from the Scatchard plots was well correlated with the EMBP levels determined in the radioimmunoassay. In conclusion, an EMBP-like protein is expressed in astrocytoma. This protein may be responsible for the specific binding of estramustine in the tumor tissue. Whether this specific binding of estramustine is of importance for the cytotoxic effect in glioma cells remains to be evaluated.

The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer.

Wild-type p53 protein has been shown to inhibit angiogenesis through thrombospondin in the preclinical setting. Here, we determined the associations between the expression of the angiogenic factor vascular endothelial growth factor (VEGF) and the p53 status, including different mutation sites and types, in primary breast cancer. Cytosols from 224 primary breast cancer patients were analyzed with an enzyme immunoassay for determination of human VEGF165 protein content. p53 status was determined by cDNA-based sequencing of the entire coding region, by immunohistochemistry (IHC), and by a p53 luminometric immunoassay (LIA) method. Statistically significant associations was found between higher VEGF content and non-wild-type p53 status for all methods; sequence-based data (P = 0.0019), IHC data (P = 0.0068), and the LIA method (r = 0.427; P > 0.001). Highest VEGF values were detected in tumors with p53 insertions, deletions, and stop codon mutations (P = 0.0043). Combining p53 status and VEGF content resulted in additional prognostic information, relapse-free survival (RFS; P = 0.0377), overall survival (OS; P = 0.0319), and breast cancer corrected survival (BCCS; P = 0.0292). In multivariate analysis, the relative hazard increased when the VEGF data were added to the p53 status, with a relative hazard of 1.7 for RFS and 3.0 for BCCS, compared with 1.1 for RFS and 1.4 for BCCS among the patients with either high VEGF content or p53 mutation. Higher VEGF content was statistically significantly correlated with a worse outcome for patients with estrogen receptor-positive tumors receiving adjuvant tamoxifen: RFS (P = 0.0471), OS (P = 0.0134), BCCS (P = 0.0064), as well as in multivariate analysis with point estimates of 3.4 and 2.1 for BCCS and RFS, respectively. VEGF expression is related to p53 status in human breast cancer patients. Combining VEGF with p53 status resulted in better prognostic prediction.

Effects of sucralfate on acute and late bowel discomfort following radiotherapy of pelvic cancer.

PURPOSE: Radiotherapy, a cornerstone in the management of pelvic cancer, is accompanied by intestinal reactions. Therefore, we investigated the possible effects of sucralfate, an aluminium hydroxide complex of sulfated sucrose used in the treatment of gastric ulcer, in preventing radiation-induced diarrhea and bowel discomfort in patients treated with curative intention for pelvic cancer with external radiotherapy. PATIENTS AND METHODS: The study was double-blind and placebo-controlled and included 70 patients with carcinoma in the prostate or urinary bladder without distant metastases (T1-4No1xMo) and a performance status of greater than or equal to 90% on the Karnofsky scale. Radiotherapy was conventionally delivered with high-energy photons (four-field technique, the total dose 64 Gy, 2 Gy daily, total treatment time 5 to 6 weeks). Dose granules of sucralfate or placebo were dispensed to each patient 2 weeks after radiation started and continued for 6 weeks. All analyses were performed blindly. RESULTS: The frequency of defecation and stool consistency were significantly improved by sucralfate. Fourteen patients in the placebo group and three in the sucralfate group required symptomatic therapy with loperamide. One year later, the patients in the sucralfate group displayed significantly less problems with frequency of defecation, mucus, and blood in the stools compared with the placebo group. There was also a lower intake of loperamide and the weight decrease was less pronounced in the sucralfate group. There was no evidence of adverse effects associated with the use of sucralfate. CONCLUSION: It is suggested that sucralfate can be of beneficial value in diminishing bowel discomfort during treatment and, most importantly, sucralfate also reduces the late bowel disturbances that follow radiotherapeutic treatment of pelvic malignancies. The earlier proposed mechanisms of action (eg, protection of denuded mucosa, cytoprotective properties, binding bile acids) seem adequate to explain the present effects of sucralfate.

Vascular endothelial growth factor is of high prognostic value in node-negative breast carcinoma.

PURPOSE: The prognostic value of vascular endothelial growth factor (VEGF) protein, known to stimulate endothelial growth and angiogenesis, was evaluated in node-negative breast carcinoma (NNBC) and compared with established prognostic factors. PATIENTS AND METHODS: In 525 consecutive patients with primary invasive NNBC (T1-2N0M0; tumor, node, metastasis stage), of whom 500 patients did not receive any systemic therapy, the cytosolic levels of VEGF165 were measured by using a quantitative enzyme-linked immunosorbent assay. The median follow-up was 46 months. Univariate and multivariate analyses were performed. RESULTS: VEGF level was significantly inversely correlated with estrogen receptor (ER) positivity but positively associated with tumor size and histologic grade. Patients with VEGF levels above the median value (2.40 pg/microg of DNA) showed a significantly shorter survival time (P=.0012) than patients with levels less than the median value, also when analyzed as a continuous variable (P=.0277). Tumor size, grade, and ER expression were all statistically significant for overall survival in univariate analyses (P=.0069, P=.014, and P < .001, respectively). Multivariate analysis showed that VEGF level was the strongest predictor of overall survival (P=.0199). Histologic grade was also an independent predictor of survival (P=.0477). Among the 381 patients with ER-positive tumors, a group in general considered to have a good prognosis, we found a significant reduction in survival for those with levels of VEGF greater than the median value (P=.0009). CONCLUSION: The results suggest that the level of VEGF165 protein is an independent, strong prognostic factor for survival in patients with NNBC, especially in the subgroup of patients with ER positivity. Thus, cytosolic VEGF165 might be useful to select patients for adjuvant systemic therapy.

Correlation of vascular endothelial growth factor content with recurrences, survival, and first relapse site in primary node-positive breast carcinoma after adjuvant treatment.

PURPOSE: To determine the predictive value of vascular endothelial growth factor (VEGF) for relapse-free survival (RFS) and overall survival (OS) in primary node-positive breast cancer (NPBC) after adjuvant endocrine treatment or adjuvant chemotherapy. MATERIALS AND METHODS: VEGF was quantitatively measured in tumor cytosols from 362 consecutive patients with primary NPBC using an enzyme immunoassay for human VEGF(165). Adjuvant treatment was given to all patients, either as endocrine therapy (n = 250) or chemotherapy (n = 112). The median follow-up time was 56 months. RESULTS: Univariate analysis showed VEGF to be a significant predictor of RFS (P =.0289) and OS (P =.0004) in the total patient population and in patients who received adjuvant endocrine treatment (RFS, P =.0238; OS, P =.0121). In the group of patients who received adjuvant chemotherapy, no significant difference was seen in RFS, but a difference was seen in OS (P =.0235). Patients with bone recurrences tended to have lower VEGF expression (median, 2.17 pg/microg DNA) than patients with visceral metastasis (4.41 pg/microg), brain metastasis (8.29 pg/microg), or soft tissue recurrences (3.16 pg/microg). Multivariate analysis showed nodal status (P =.0004), estrogen receptor (ER) status (P <.0001), and tumor size (P =.0085) to be independent predictors of RFS. VEGF was found to be an independent predictor of OS (P =.0170; relative risk [RR] = 1.82), as were ER (P <.0001; RR = 5.19) and nodal status (P =.0002; RR = 2.58). For patients receiving adjuvant endocrine treatment, multivariate analysis showed VEGF content to be an independent predictor of OS (P =.0420; RR = 1.90) but not of RFS. CONCLUSION: The results suggest that VEGF(165) content in tumor cytosols is a predictor of RFS and OS in primary NPBC. VEGF content might also predict outcome after adjuvant endocrine treatment, but further studies in a prospective setting with homologous treatments are required.

Apoptotic tumor cell death induced by estramustine in patients with malignant glioma.

Estramustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, is metabolized and exerts specific cytotoxic effects in malignant glioma in vitro and in vivo. In the present study, we have evaluated the cytotoxic effect of EMP in the clinical situation with regard to appearance of DNA damage and its correlation to the uptake of estramustine (EaM) in human malignant astrocytoma tissue. Ten patients were given 280 mg of EMP p.o. 12 h before surgery. Specimens from brain tumor tissue were collected during surgery and used for detection of fragmented DNA, a hallmark of apoptosis, with in situ end labeling (ISEL) and agarose gel techniques. The main metabolite of EMP in glioma tissue, EaM, was analyzed with gas chromatography. It was demonstrated that EMP induced clusters of ISEL-positive tumor cells and fragmentation of DNA on agarose gels in patients treated with EMP. In the same patients, a significant uptake of EaM in tumor tissue was demonstrated. In control patients, who were not treated with EMP, and in two EMP-treated patients with no uptake of EaM, no signs of fragmented DNA and only a few scattered ISEL-positive cells were seen in the tumor tissue. Signs of apoptosis were also seen in two different experimental models, i.e., in vitro cell cultures of rat glioma cells and an in vivo rat glioma model. It is suggested that EaM can induce apoptosis by a direct effect on a subpopulation of glioma cells in human brain tumors in the clinical situation.