Analysis of preclinical studies using human tumors xenografted into rodents is commonly performed with Tumor Growth Index (TGI) and Tumor Growth Delay index (TGDi). To circumvent the limitations of these parameters, two new parameters, Time To Relapse (TTR) and Tumor Growth Speed (TGS), were developed using a mathematical modeling approach based on an exponential tumor growth. TTR is similar to progression free survival used in human clinical trials and TGS characterizes the pattern of tumor cell proliferation. Parameters were estimated for each rodent by the maximum likelihood method and statistical analyses were performed using ANOVA. These criteria can be used when tumor growths are assessed by repeated measures of their volume. As an example, we used data from a previously published study, which aimed to evaluate the relationship between histology, genetic parameters, and response to alkylating agents in a series of twelve gliomas. The group treated with temozolomide was reanalyzed using our criteria. This group presented a significantly longer TTR than the control group. TTR was also different according to tumor type: oligodendrogliomas relapsed later than glioblastomas. The TGS was different according to the tumor type. Loss of heterozygosity (LOH) 1p ± 19q, LOH 10p ± 10q, telomerase activity, PTEN mutation, and EGFR amplification were related to temozolomide efficacy. Our criteria provide additional information to those given by TGI and TGDi. Due to statistical properties of TTR and TGS, some relations between the parameters such as tumor type or genetic alterations can be studied with TTR and TGS and not with TGI or TGDi.

Background ASPM ( Abnormal Spindle-like Microcephaly associated ) over-expression was recently implicated in the development of malignant gliomas. Results To better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo . Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models. Conclusion These data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme.

Purpose: Retinoblastoma is the most common primary intraocular tumor in children. In industrialized countries, 95% of patients are cured by chemotherapy and conservative treatments. However, these treatments can increase the risk of secondary tumors in patients with a constitutional alteration of the RB1 gene.Photodynamic therapy represents a nonmutagenic therapeutic approach, and may reduce the incidence of secondary tumors.To study the in vivo efficacy of photodynamic therapy, human retinoblastoma xenografts were established in nude mice.Methods: Three xenografted cell lines, RB102-FER, RB109-LAK and RB111-MIL, were characterized and used for therapeutic evaluation.Mice were randomly divided into control and treatment groups with 5-8 mice in each group. Treatment groups received irradiation alone, photosensitizer alone or both in 2 of the 3 models and in the third model, photosensitizer plus irradiation was compared to untreated controls. mTHPC was injected intraperitoneally at a dose of 0.6 mg/kg and verteporfin intravenously at a dose of 1 mg/kg. Illuminations were performed 24h after mTHPC and 1 h after verteporfin injections.Results: A transient but significant response to mTHPC was observed for RB102-FER (p = 0.03) and a significant response to mTHPC for RB111-MIL (p < 10(-4)) with partial regression maintained for more than 60 days. No significant difference between the different groups was observed for RB109-LAK, except in the verteporfin plus laser group (p = 0.01).Conclusions: The studies confirmed the suitability of the three xenograft models for the evaluation of photodynamic therapy in retinoblastoma. Our findings suggest that PDT may represent an alternative conservative treatment for these tumors. (C) 2010 Elsevier B.V. All rights reserved.

Abstract Purpose: Epidermal growth factor receptor (EGFR) signal transduction pathways are implicated in malignant glioma aggressiveness and promote tumor cell invasion, proliferation, and angiogenesis. Nevertheless, response to EGFR tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) has been disappointing in clinical trials. One potential explanation may come from the diversity of molecular alterations seen in gliomas. To validate that hypothesis, we have investigated responses to gefitinib on various tumor parameters in human malignant gliomas that exhibited different molecular alterations. Experimental Design: We used a panel of six human malignant gliomas from established xenografts characterized for their genetic (EGFR, PTEN, TP53, and CDKN2A) and molecular (EGFR, PTEN, ERK, and Akt) alterations. Tumors were treated with gefitinib (1 or 10 μmol/L) for prolonged periods (8 or 16 days) in an organotypic brain slice model that allowed quantification of invasion, proliferation, and angiogenesis. Results: In nontreated tumors, EGFR amplification was associated with profuse tumor cell invasion. After treatment, invasion was inhibited in tumors with EGFR amplification in a dose-dependent manner. Treatment had only antiproliferative effect in two of three tumors with EGFR amplification. Tumors with PTEN loss were resistant to treatment. We did not observe shrinkage of the tumors after treatment. None of the tumors had mutations of the EGFR kinase domain. Gefitinib had similar antiangiogenic effect in all of the tumors. Conclusions: Gefitinib reduces cell invasion in EGFR amplified tumors. PTEN loss of expression seems to be a determinant of resistance. Interestingly, inhibition of angiogenesis by gefitinib seems independent on the EGFR genetic status of the tumors.

Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.

A series of 12 human gliomas was established as xenografts in nude mice and used to evaluate the relationship between histology, genetic parameters, and response to alkylating agents. Eight were high-grade oligodendroglial tumors, and four were glioblastoma. They were characterized for their genetic alterations, including those considered as "early" alterations, namely loss of chromosome 1 +/- loss of chromosome 19q, TP53 mutation, and those considered as "late" alterations, namely loss of chromosome 10, loss of chromosome 9p, EGFR genomic amplification, PTEN mutation, CDKN2A homozygous deletion, and telomerase reactivation. Chemosensitivity of xenografts to four alkylating agents, temozolomide (42 mg/kg, days 1-5, p.o.), 1,3-bis(2-chloroethyl)-1-nitrosourea (5 mg/kg, day 1, i.p.), Ifosfamide (90 mg/kg, days 1-3, i.p.), and carboplatin (66 mg/kg, day 1, i.p.) was tested by administration of drugs to tumor-bearing mice. Although each tumor presented an individual response pattern, glioblastoma had a lower chemosensitivity than oligodendrogliomas, and temozolomide was the most effective drug. Deletion of 1p +/- 19q was associated with higher chemosensitivity, whereas late molecular alterations, particularly EGFR amplification, were associated with chemoresistance. These results suggest that the combined use of histology and molecular markers should eventually be helpful selecting the most appropriate agents for treatment of malignant oligodendrogliomas and astrocytomas.

OBJECT:The goal of this study was to examine allelic losses and telomerase activity in meningiomas to determine whether they could be used to predict disease recurrence.METHODS:To identify predictive markers of recurrence, a cohort of high-grade (24 World Health Organization [WHO] Grade II and six WHO Grade III) and low-grade (21 WHO Grade I) meningiomas was investigated for losses of heterozygosity (LOHs) on chromosomes 1p, 9p, 10q, 14q, and 22q, a deletion of CDKN2A, and telomerase activity. Results of molecular analyses were compared with radiological and histological findings and progression-free survival (PFS). Losses of heterozygosity on chromosomes 22q, 1p, and 10q, as well as telomerase activity were related to the WHO histological grades of the lesions (p < 0.01, p < 10(-5), p < 10(-4), and p = 0.002, respectively). In the absence of an LOH on 22q, the other alterations were found infrequently. Overall, the number of molecular alterations was closely related to the histological grades of the lesions (p < 10(-6)). An LOH on 22q occurred much more frequently in convexity or falx (33 [87%] of 38 lesions) than in skull base or spinal meningiomas (four [31%] of 13 lesions) (p < 0.001). The histological grade; Simpson grade; an LOH on chromosome 1p, 9p, or 10q; and telomerase activity were correlated with a shorter PFS time (p < 10(-4), p = 0.02, p = 0.000365, p = 0.022, p = 0.00027, and p = 0.000512, respectively).CONCLUSIONS:On the basis of these data the authors suggest that LOH analysis and a telomerase activity assay could be useful to determine molecular predictors of outcome in patients with meningiomas.

In order to develop preclinical models of malignant astrocytomas and oligodendrogliomas, a series of 54 resected gliomas (37 from oligodendroglial lineage and 17 from astrocytic lineage) were xenografted subcutaneously into nude mice. Molecular alterations commonly observed in gliomas subtypes, including LOH 1p and 1q, LOH 19q, LOH 10p and 10q, LOH 9p, TP53 and PTEN mutations, EGFR amplification, CDKN2A homozygous deletion and telomerase reactivation were systematically screened in the original and xenografted tumours. In all, 23 gliomas grew in nude mice. The most anaplastic tumours were selected as shown by pathological and molecular studies of the original tumour as well as shorter survival in patients whose tumours were successfully grafted. Comparison between the two growth profiles showed that 10q LOH and EGFR amplification gave a tumorigenic advantage. With a few exceptions, the genetic pattern was remarkably stable before and after growth in nude mice.These results suggest that subcutaneous xenografts are useful and reproducible models to analyse the molecular profile of malignant astrocytoma and oligodendroglioma. This represents the first step to improve our understanding of the correlations between molecular alterations and response to standard or experimental therapies.

An inhibitor of telomerase activity maps to chromosome 10p15.1. A series of 51 high-grade gliomas was analyzed for loss of heterozygosity (LOH) on chromosome 10 and for telomerase activity. In univariate analysis, LOH10p (59%) and LOH10q (61%) were associated with telomerase activity (55%; p < 0.0001 and p = 0.0006). In multivariate analysis, only LOH10p remained statistically related to telomerase activity, suggesting that the telomerase repressor gene located on 10p15.1 is inactivated in high-grade gliomas.

Object. Chromosomal deletions of 10q and candidate genes such as PTEN and DMBT1 have been thoroughly investigated in glioblastomas but few data specifically address oligodendrogliomas. Methods. In this study, 39 pure oligodendrogliomas were investigated for loss of heterozygosity (LOH) on 10q, PTEN mutations, and DMBT1 homozygous deletions. The LOH on 10q was found in 19 (48%) of 39 oligodendrogliomas and was closely related to anaplasia (p = 0.02), shorter time to progression (p = 0.0005), and poorer survival (p = 0.035). The DMBT1 homozygous deletions were found in 10 (26%) of 39 oligodendrogliomas but only one PTEN mutation was detected. The LOH on 10q is a strong predictor of survival and could be a valuable prognostic marker in oligodendrogliomas. Conclusions. Frequent inactivation of DMBT1 contrasting with rare mutations of PTEN may indicate that DMBT1 is preferentially involved in oligodendrogliomas. Nevertheless, the absence of a correlation with survival makes the role of DMBT1 in tumorigenesis still questionable and warrants further investigation.

Portions of chromosomes 1p and 19q, which are frequently deleted in oligodendrogliomas, are subject to genomic imprinting, suggesting that the putative tumor suppressor genes could be monoallelically expressed. The parental origins of 1p and 19q allele losses were determined in 6 cases of pure oligodendroglioma. An equilibrated parental loss (3 maternal and 3 paternal) was found for 1p deletions. In contrast, 19q deletions always occurred on the paternal copy (p = 0.015). In this setting, a cloning strategy based on a search for homozygous deletion or mutation of the remaining allele would be appropriate for identifying the tumor suppressor gene located on 1p but inappropriate for identifying the presumably monoallelically expressed tumor suppressor gene located on 19q.

Glioblastoma (GBM) is considered by the WHO classification to represent the most malignant grade of the astrocytic tumors. However, a subset of GBM includes recognizable areas with oligodendroglial features, suggesting that some GBM may also have an oligodendroglial origin. The aim of this study was to analyze the molecular profile of GBM associated with an oligodendroglial component (GBMO). We analyzed a series of 25 GBMO. Loss of heterozygosity (LOH) on 1p and 19q, known as common markers of oligodendroglial tumors, were observed in 40% and 60% of cases, respectively; 72% of the tumors displayed one or both of these markers. All but 4 tumors (84%) showed alterations known to be preferentially involved in the progression of astrocytic tumors to GBM, such as EGFR amplification (44%), P16 deletion (48%), LOH on 10q (64%), PTEN (20%), and TP53 (24%) mutations. Therefore, GBMO displayed all the genetic aberrations found in "standard" GBM with a comparable incidence, but differed from GBM by having a higher rate of LOH on 1p and 19q. These results suggest that GBMO might represent a subgroup of tumors of oligodendroglial origin that is distinct from the "standard" GBM in terms of tumorigenesis pathway.

OLIG2 is a recently identified transcription factor involved in the specification of cells in the oligodendroglial lineage. We investigated the expression of OLIG2 by in-situ hybridisation in 21 brain tumours: nine grade II and III oligodendrogliomas, three grade II oligoastrocytomas, and nine non-oligodendroglial tumours (four grade IV astrocytomas, two meningiomas, a dysembryoplastic neuroepithelial tumour, and two metastases). OLIG2-positive cells corresponding to neoplastic oligodendrocytes were present in all oligodendrogliomas and oligoastrocytomas. By contrast, OLIG2 expression was not detected in the non-oligodendroglial tumours. Thus, oligodendroglioma probably arise from oligodendrocyte precursor cells. OLIG2 should prove a useful marker for the diagnosis of oligodendroglial tumours.

OBJECTIVE:To identify different genetic molecular profiles in oligodendrogliomas and to evaluate their prognostic significance.METHODS:The main genetic alterations reported in glial tumors were investigated in 26 oligodendrogliomas (10 World Health Organization grade II and 16 World Health Organization grade III). Correlation between identified molecular changes and pathologic grade or clinical course was subsequently analyzed using univariate and multivariate statistical analyses.RESULTS:Loss of heterozygosity (LOH) on chromosome 1p, 19q, and 10; P16/CDKN2A homozygous deletion; EGFR (epidermal growth factor receptor) amplification; and TP53 and PTEN mutations were observed in 14 (54%), 15 (58%), 9 (35%), 7 (27%), 5 (19%), 1 (4%), and 0 cases. LOH 1p and 19q were tightly associated (p < 0.0001). A mutual exclusion was found between LOH 1p/19q and EGFR amplification (p = 0.01), P16/CDKN2A deletions (p = 0.001), or LOH on 10q (p = 0.03), suggesting the existence of distinct genetic subsets in oligodendrogliomas. On univariate analysis, age <50 years (p = 0.002) and LOH 1p (p = 0.01) were associated with a longer progression-free survival (PFS) whereas LOH 10q (p = 0.03) and EGFR amplification (p = 0.007) were associated with a worse PFS. In multivariate analyses, age <50 years (p = 0.001) and LOH 1p (p = 0.006) remained independent predictive factors for PFS.CONCLUSION:These results provide evidence for two alternative molecular pathways of progression in oligodendrogliomas. The first one is associated with loss of 1p and 19q and the second one with P16/CDKN2A deletion, 10q loss, and EGFR amplification. The findings confirm the value of loss of 1p as predictor of longer progression-free survival; in addition, the study demonstrates the unfavorable impact of 10q loss and EGFR amplification on the prognosis.