ADSCs in a fibrin matrix enhance nerve regeneration after epineural suturing in a rat model.

BACKGROUND: Due to their unique properties, adipose derived stem cells (ADSCs) obtain promising potential to enhance nerve regeneration. The aim of this study was to investigate if fibrin-glue embedded ADSCs were a beneficial adjunct to primary coaptation in a rat sciatic nerve model. MATERIALS AND METHODS: Fifty male Lewis rats underwent sciatic nerve transection and subsequent epineural suture repair. The treatment group received ADSCs re-suspended in fibrin glue, while the control group received fibrin glue only. After 7, 21, 35, and 63 days, analysis involved axon count, myelin sheath thickness as well as N- and G-ratios. Additionally, muscle weight quotient (operated vs. non-operated site of the same animal) was calculated and compared between treatment and control groups. For co-detection of vital ADSCs, vessel walls, and Schwann cells, immunolabeling was performed with CM-DiI, SMA, and S-100 antibodies, respectively. RESULTS: ADSCs led to a significant increase of myelinization at day 21 (0.508 ± 0.085 µm vs. 0.381 ± 0.044 µm, P = 0.025) and day 35 (0.872 ± 0.09 µm vs. 0.495 ± 0.078 µm; P = 0.01) after surgery. Axon count was significantly increased at day 21 (420 ± 119 vs. 129 ± 63; P = 0.003) and day 63 (284 ± 137 vs. 111 ± 26; P = 0.046) after surgery. N- and G-ratios were significantly different compared with control indicating enhanced nerve regeneration due to ADSC treatment at each time point (P < 0.05). Muscle weight quotient was significantly higher in the treatment group compared with the control at day 21 (44.01% ± 6.16% vs. 35.03% ± 2.61%; P = 0.014) and day 63 (65.49% ± 2.81% vs. 58.79% ± 4.06%; P = 0.009) after surgery. Co-detection of immunolabeled cells showed vital ADSCs at the neuronal repair site and in close proximity to intraneuronal vessels indicating active participation of ADSCs in the process of nerve regeneration and associated angiogenesis. CONCLUSION: ADSCs embedded in a fibrin matrix can significantly enhance regeneration of peripheral nerve injuries after primary coaptation. © 2015 Wiley Periodicals, Inc. Microsurgery 36:491-500, 2016.

Pulsed acoustic cellular expression (PACE) reduces capsule formation around silicone implants.

UNLABELLED: Capsular contracture remains a major complication after reconstructive or aesthetic breast augmentation. Formation of capsular fibrosis is a multifactorial process. An initial inflammatory reaction appears to be key to the development of capsular contracture. Recent studies have shown that pulsed acoustic cellular expression (PACE) has significant antiinflammatory effects. Thus, this study aimed to determine the potential of PACE to prevent or attenuate capsular contracture around silicone implants in a rodent model. For this study, 36 Lewis rats were divided into two groups, and a textured silicone implant was placed in a dorsal submuscular pocket. One group received PACE treatment, whereas the other group served as the control group and received no treatment. Follow-up evaluations were performed after 10, 35, and 100 days. Capsule thickness, collagen density, myofibroblasts, vascular density, and a semiquantitative real-time polymerase chain reaction that addressed differential gene expression were assessed. The PACE treatment significantly reduced capsule thickness on days 10, 35, and 100 compared with the control group (day 10: 632.9 ± 164.5 vs 932.6 ± 160.8, p < 0.05; day 35: 709.5 ± 175 vs 825.9 ± 313.3, p < 0.0.5; day 100: 736.3 ± 198.1 vs 1,062.3 ± 151.9, p < 0.05). This was accompanied by a significant suppression of proinflammatory genes (cluster of differentiation 68, monocyte chemotactic protein-1, CCL4) and synergistic alterations of pro- and antifibrotic proteins (transforming growth factor-beta 1, matrix metalloproteinase-2). This study showed that the PACE application significantly reduces capsular contracture around silicone implants. A decrease in capsular thickness after PACE treatment seems to be associated with a downregulation of proinflammatory genes and proteins. The study identifies PACE technology as a potential low-cost technique that is easy to use for reduction of capsular contracture after augmentation using silicone implants. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

AKT1E17K mutations cluster with meningothelial and transitional meningiomas and can be detected by SFRP1 immunohistochemistry.

The activating E17K mutation in the AKT1 gene has been detected in several tumor entities. Currently several clinical studies with specific AKT1 inhibitors are under way. To determine whether AKT1 mutations are involved in human tumors of the nervous system, we examined a series of 1,437 tumors including 391 primary intracranial brain tumors and 1,046 tumors of the coverings of the central and peripheral nervous system. AKT1E17K mutations were exclusively seen in meningiomas and occurred in 65 of 958 of these tumors. A strong preponderance was seen in the variant of meningothelial meningioma WHO grade I of basal and spinal localization. In contrast, AKT1E17K mutations were rare in WHO grade II and absent in WHO grade III meningiomas. In order to more effectively detect this mutation, we tested for immunohistochemical markers associated with this alteration. We observed strong up-regulation of SFRP1 expression in all meningiomas with AKT1E17K mutation and in HEK293 cells after transfection with mutant AKT1E17K, but not in meningiomas and HEK293 cells lacking this mutation.

Bed isolation in experimental flap studies in rats: a dispensable procedure.

Review of the literature regarding rodent experimental flap models reveals fundamental differences in applied surgical procedures. Although some authors isolate the flap from its wound bed, others do not. This study was planned to investigate to what extent the insertion of a silicone sheet affects physiological wound healing in experimental flap surgery. An extended epigastric adipocutaneous flap (6 × 10 cm) was raised in 16 male Lewis rats. In the control group (group C), flaps were immediately inset without any intervention. In the experimental group (group M), a silicone sheet barrier was placed between the flap and the wound bed. Mean flap survival area and flap perfusion were evaluated. Microvessel density was visualized by immunohistochemistry, and semiquantitative real-time polymerase chain reaction addressed differential gene expression. All animals were investigated on postoperative day 5. Flap survival area and flap perfusion were found to be similar. Immunohistochemistry, however, demonstrated a significantly increased number of CD31-positive small vessels in group C. The insertion of the silicone sheet barrier (group M) was accompanied by a significantly enhanced expression of proinflammatory genes and a suppression of proangiogenic genes. Our results show that although the silicone membrane has no influence on the surgical outcome in terms of flap survival and perfusion, it does lead to significant molecular alterations in pathways involved in physiological wound healing. These alterations are artificially induced by the foreign body material and conceal the true driving forces of the healing process. As the latter might include relevant therapeutic targets to ameliorate surgical results, we regard wound bed isolation as a dispensable procedure in the study of rodent flap models.

Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.

Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n = 39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis.

Clonal analysis in recurrent astrocytic, oligoastrocytic and oligodendroglial tumors implicates IDH1- mutation as common tumor initiating event.

BACKGROUND: To investigate the dynamics of inter- and intratumoral molecular alterations during tumor progression in recurrent gliomas. METHODOLOGY/PRINCIPAL FINDINGS: To address intertumoral heterogeneity we investigated non-microdissected tumor tissue of 106 gliomas representing 51 recurrent tumors. To address intratumoral heterogeneity a set of 16 gliomas representing 7 tumor pairs with at least one recurrence, and 4 single mixed gliomas were investigated by microdissection of distinct oligodendroglial and astrocytic tumor components. All tumors and tumor components were analyzed for allelic loss of 1p/19q (LOH 1p/19q), for TP53- mutations and for R132 mutations in the IDH1 gene. The investigation of non-microdissected tumor tissue revealed clonality in 75% (38/51). Aberrant molecular alterations upon recurrence were detected in 25% (13/51). 64% (9/14) of these were novel and associated with tumor progression. Loss of previously detected alterations was observed in 36% (5/14). One tumor pair (1/14; 7%) was significant for both. Intratumoral clonality was detected in 57% (4/7) of the microdissected tumor pairs and in 75% (3/4) of single microdissected tumors. 43% (3/7) of tumor pairs and one single tumor (25%) revealed intratumoral heterogeneity. While intratumoral heterogeneity affected both the TP53- mutational status and the LOH1p/19q status, all tumors with intratumoral heterogeneity shared the R132 IDH1- mutation as a common feature in both their microdissected components. CONCLUSIONS/SIGNIFICANCE: The majority of recurrent gliomas are of monoclonal origin. However, the detection of divertive tumor cell clones in morphological distinct tumor components sharing IDH1- mutations as early event may provide insight into the tumorigenesis of true mixed gliomas.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

BACKGROUND: Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. METHODS: We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. RESULTS: Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. CONCLUSION: The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Adipose derived stem cells protect skin flaps against ischemia-reperfusion injury.

BACKGROUND: Advances in the treatment of ischemia- reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. We examined the capability of adipose derived stem cells (ADSCs) to protect tissue against IRI using an extended inferior epigastric artery skin flap as a flap ischemia- reperfusion injury (IRI) model. METHODS: ADSCs were isolated from Lewis rats and cultured in vitro. Twenty- four rats were randomly divided into three groups. Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and group III (ADSCs treatment) underwent 3 h of ischemic insult. During reperfusion group III was treated by intravenous application of ADSCs and group II was left untreated. Five days postoperatively, flap survival and perfusion were assessed. Microvessel density was visualized by immunohistochemistry and semi- quantitative real-time polymerase chain reaction addressed differential gene expression. RESULTS: Treatment with ADSCs significantly increased flap survival (p<0.001) and flap perfusion (p<0.001) when compared to the control group II. Microvessel- density in ADSCs treated group was not significantly increased in any group. No significant differences showed the comparison of the experimental group III and the sham operated control group I. ADSCs treatment (Group III) was accompanied by a significantly enhanced expression of pro-angiogenic and pro-inflammatory genes. CONCLUSION: Overall, our study demonstrates that ADSCs treatment significantly enhances skin flap survival in the aftermath of ischemia to an extent that almost equals surgical results without ischemia. This effect is accompanied with a pronounced and significant angiogenic response and an improved blood perfusion.

Extracorporeal shock wave treatment protects skin flaps against ischemia-reperfusion injury.

Advances in the treatment of ischemia-reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. Using an extended inferior epigastric artery skin flap as a flap ischemia-reperfusion injury (IRI) model, we examined the capability of extracorporeal shock wave treatment (ESWT) to protect tissue against IRI in a rat flap model. Twenty-four rats were used and randomly divided into three groups (n=8 for each group). Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and Group III (ESWT) underwent 3h of ischemic insult. During reperfusion Group III was treated with ESWT and Group II was left untreated. Histological evaluation was made to investigate treatment induced tissue alterations. Survival areas were assessed at 5d postoperatively. Skin flap survival and perfusion improved significantly in the ischemic animals following ESWT (p<0.001, respectively). The tissue protecting effect of ESWT resulted in flap survival areas and perfusion data equal to non-ischemic, sham operated flaps. In line with the observation of better flap perfusion, tissue from ESWT-treated animals (Group III) revealed a significantly increased frequency of CD31-positive vessels compared to both the ischemic (Group II; p=0.003) and the non-ischemic, sham operated control (Group I; p<0.005) and an enhanced expression of pro-angiogenic genes. This was accompanied by a mild suppression of pro-inflammatory genes. Our study suggests that ESWT improves flap survival in IRI by promoting angiogenesis and inhibiting tissue inflammation. The study identifies ESWT as a low-cost and easy to use technique for surgical techniques that aim at reducing ischemia-reperfusion-induced tissue injury.

Accurate classification of metastatic brain tumors using a novel microRNA-based test.

BACKGROUND: Identification of the tissue of origin of a brain metastatic tumor is vital to its management. Carcinoma of unknown primary (CUP) is common in oncology, representing 3%-5% of all invasive malignancies. We aimed to validate a recently developed microRNA-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) test for identifying the tumor tissue of origin, first in a consecutive cohort of metastatic tumors of known origin and then in a cohort of CUP cases resected from the central nervous system (CNS). PATIENTS AND METHODS: One hundred two resected CNS metastatic tumors with known origin, previously classified based on the patient's clinical history and pathological data, as well as a second cohort of resected CNS tumors from 57 patients originally diagnosed as CUP were studied. A qRT-PCR diagnostic assay that measures the expression level of 48 microRNAs was used to classify the tissue of origin of these metastatic tumors. RESULTS: In this blinded study, the test predictions correctly identified the reference diagnosis of the samples of known origin, excluding samples from prostate origin, in 84% of cases. In the second CUP patient cohort, the test prediction was in agreement with the diagnosis that was later confirmed clinically or with pathological evaluation in 80% of cases. CONCLUSION: In a cohort of brain and spinal metastases, a previously developed test based on the expression of 48 microRNAs allowed accurate identification of the tumor tissue of origin in the majority of cases. The high accuracy of this test in identifying the tissue of origin of metastases of unknown primary is demonstrated for the first time and may have broad clinical application.

Epigenetic silencing of death receptor 4 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in gliomas.

PURPOSE: To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy. EXPERIMENTAL DESIGN: We used methylation-sensitive BeadArray technology to identify novel epigenetically regulated genes associated with apoptosis and with potential therapeutic targets in glioma therapy. To elucidate the functional consequences of promoter methylation in the identified target death receptor 4 (DR4), we investigated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated and anti-DR4-mediated apoptosis in glioma cell lines (U373 and A172) with loss of DR4 and one glioma cell line (LN18) with robust DR4 expression. RESULTS: In human astrocytic tumors, we detected DR4 promoter hypermethylation in 60% (n = 5) of diffuse astrocytomas WHO grade 2, in 75% (n = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (n = 33). DR4 is a cell surface protein restricted to glioma cells and is targeted by TRAIL. Glioma cell lines U373 and A172 harbored heavily methylated DR4 promoters, and 5-aza-2-deoxycytidine-mediated demethylation reconstituted DR4 expression in these cell lines. Functional knockdown of DR4 by DR4-specific small interfering RNA in TRAIL-sensitive glioma cell line LN18 significantly mitigated apoptosis induced by an agonistic anti-DR4 antibody. 5-Aza-2-deoxycytidine-mediated demethylation resulted in a functional reconstitution of DR4 on the cell surface of TRAIL-resistant glioma cell line U373 and sensitized U373 to TRAIL-mediated apoptosis. Suppression of DR4 by small interfering RNA in demethylated U373 successfully reestablished the TRAIL-resistant phenotype of U373. CONCLUSIONS: DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies.

RASSF1A, BLU, NORE1A, PTEN and MGMT expression and promoter methylation in gliomas and glioma cell lines and evidence of deregulated expression of de novo DNMTs.

Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5'-aza-2'-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%-57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas.

Down-regulation of the inhibitor of growth 1 (ING1) tumor suppressor sensitizes p53-deficient glioblastoma cells to cisplatin-induced cell death.

Impaired tumor suppressor functions, such as deficient p53, are characteristic for glioblastoma multiforme (GBM) and can cause resistance to DNA-damaging agents like cisplatin. We have recently shown that the INhibitor of Growth 1 (ING1) tumor suppressor is down-regulated in malignant gliomas and that the decrease of ING1 expression correlates with histological grade of malignancy, suggesting a role for ING1 in the pathogenesis and progression of malignant gliomas. Based on this background, the purpose of our current study was to examine the potential impact of ING1 protein levels on DNA-damage response in GBM. Using LN229 GBM cells, which express ING1 proteins and harbor mutant TP53, we are the first to show that DNA damage by cisplatin or ionizing radiation differentially induced the two major ING1 splicing isoforms. The p47 ING1a isoform, that promotes deacetylation of histones, thus formation of heterochromatic regions of DNA, which are less susceptible to DNA damage, was preferentially induced by >50-fold. This might represent a response to protect DNA from damage. Also, ING1 knockdown by siRNA accelerated transit of cells through G1 phase, consistent with ING1 serving a tumor suppressor function, and caused cells to enter apoptosis more rapidly in response to cisplatin. Our results indicate that malignant gliomas may down-regulate ING1 to allow more efficient tumor growth and progression. Also, ING1 down-regulation may sensitize GBM cells with deficient p53 to treatment with cisplatin.

45-year-old male with symptomatic mass in the frontal lobe.

A 45-year-old male patient developed focal seizures in his right arm. Neuroimaging demonstrated a tumor of the left frontal lobe. Tumor classification was undecided after stereotactic biopsy. Neuropathological examination of the open biopsy specimen revealed overlapping morphological features of an oligodendroglioma and a central neurocytoma. Groups of tumor cell featured the typical "fried egg" appearance seen in oligodendroglioma; microcalcifications and a network of branching non-proliferating vessels were present. Neurocytoma-like features included small nucleus-free areas of neuropil and perivascular pseudorosettes. Neuron specific enolase was strongly expressed cytoplasmically in the tumor cells and the "neuropil islands" were found to express synaptophysin. The final diagnosis of an oligodendroglioma with neurocytic differentiation was based on tumor location, clinicopathological findings and diagnostic genotyping. Combined loss of heterozygosity (LOH) on the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q), the "molecular signature" of oligodendrogliomas, was revealed. Besides supporting the diagnosis of an oligodendroglioma, the molecular data allow for additional therapeutic options. These tumors may point to the presence of yet another potential tumor precursor cell similar to the recently discovered "N-O"-cells in the cerebral cortex of rats, capable of differentiation into neurons and oligodendrocytes.

Molecular genetic analysis of oligodendroglial tumors.

Deletions on the short arm of chromosome 1 (1p) and the long arm of chromosome 19 (19q) are molecular hallmark lesions of oligodendroglial tumors. Ever since oligodendroglial tumors with 1p and 19q deletions were shown to respond to chemotherapy, neuropathologists have been facing increasing requests for such molecular analysis. Therefore, there is increasing demand for reliable and simple assays. This brief report compiles information on different technical approaches, including manual and automated loss of heterozygosity analysis, fluorescence in situ hybridization techniques, and quantitative polymerase chain reaction. Herein we present a detailed protocol describing loss of heterozygosity analysis with microsatellite markers currently in use for two multicenter studies.

Mutation analysis of DKK1 and in vivo evidence of predominant p53-independent DKK1 function in gliomas.

DKK1 protein belongs to a family of inhibitors of the Wnt/beta1-catenin signaling pathway. Sporadic mutations affecting almost each major player of the Wnt/beta1-catenin pathway have been described in a variety of human carcinomas. DKK1 translation can be induced by p53, thereby linking TP53 and Wnt/beta1-catenin signaling pathways. These findings raise questions in regard to human gliomas, which similar to carcinomas carry a high rate of mutations in TP53. To analyze DKK1 for its role in initiation or progression, we screened a series of 73 brain tumors for structural alterations in the entire coding sequence by single-strand conformation polymorphism and direct sequencing. While several sequence variants were detected, there were no obvious mutations affecting DKK1. Further, we analyzed the prevalence of mRNA from TP53, DKK1 and CTNNB1 and of p53 and beta1-catenin protein in a series of human gliomas with and without mutations in TP53. Transcription and expression of CTNNB1/beta1-catenin and DKK1 proved to be independent of TP53/p53. These data support in vivo function of DKK1, independent of p53, in human gliomas with no major impact on their pathogenesis.

Somatic mutations in peroxisome proliferator-activated receptor-gamma are rare events in human cancer cells.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor activated after binding a lipophilic ligand, such as naturally occurring 15dPGJ2. There is striking evidence that PPAR-gamma activation leads not only to an increase in insulin sensitivity, but also to tumor cell apoptosis and cell cycle arrest. A growing number of diabetes mellitus II patients currently benefit from treatment with synthetic PPAR-gamma agonists, the thiazolidinediones (TZDs), which sensitize peripheral cells towards insulin. Furthermore, some TZDs are undergoing clinical investigations for the treatment of malignant diseases. Therefore, detailed information on the frequency of genetic alterations of PPAR-gamma in malignant tumor cells is necessary. MATERIAL/METHODS: PPAR-gamma DNA of 33 histologically different tumor cells was isolated, purified, and all coding regions were separately amplified by PCR. The coding exons were then analyzed by single-stranded conformational polymorphism (SSCP) and bidirectional DNA sequencing. RESULTS: In five breast cancer brain metastasis samples from patients and 28 cancer cell lines derived from lymphoma, glioblastoma, and breast carcinoma we found only one coding region shift in exon 5b of the glioblastoma U373 DNA. This silent mutation does not lead to a change in amino acid alignment. No further polymorphisms, including those which have already been described, could be detected in any other sample. CONCLUSIONS: We conclude that somatic mutations in the PPAR gene are exceedingly rare events in malignant tumor cells. This makes PPAR-gamma more unlikely to act as a tumor suppressor gene, making it a stable and suitable target for TZD biological cancer therapy.

No ING1 mutations in human brain tumours but reduced expression in high malignancy grades of astrocytoma.

The ING1 family of proteins has been shown to have regulatory functions in oncogenesis, apoptosis, DNA repair and cell cycle regulation. Here we present the first report on LOH analysis of the ING1 locus, mutation analysis of the complete coding sequence including intron-exon boundaries and expression analysis of the different ING1 splice products and protein isoforms in primary brain tumours. No somatic ING1 mutations were detected. Semi-quantitative analysis revealed higher levels of p33ING1b RNA in benign than in malignant lesions. This correlation was significant in a subset of 37 astrocytic tumours WHO grades I to IV. ING1 protein isoforms p47ING1a, p33ING1b and p24ING1c were found to be expressed variably in this series. Our findings support a regulatory contribution of ING1 to the development or progression of brain tumours.

No preferential loss of paternal 19q alleles in oligodendroglial tumors.

Recently, exclusive loss of paternal 19q alleles in six of six oligodendrogliomas has been reported, indicating that parental imprinting plays a role in these tumors. We examined a series of 10 oligodendrogliomas and 3 oligoastrocytomas with allelic losses on 1p and 19q for the parental origin of the lost alleles. Ten cases lost paternal 1p alleles and 3 cases lost maternal alleles. For 19q, six cases had loss of paternal alleles and seven cases of had loss of maternal alleles. These random distributions do not support the hypothesis that parental imprinting accounts for inactivation of the putative oligodendroglioma tumor suppressor genes.