Carmen Franco-Hernandez

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity.

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q allelic constitution in a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification of the 1p/19q allelic deletions on these brain neoplasms.

Genomic deletions at 1p and 14q are associated with an abnormal cDNA microarray gene expression pattern in meningiomas but not in schwannomas

The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5-69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas.

Biología molecular de los glioblastomas

La formacion de los glioblastomas es muy diversa, pudiendo presentarse de ?novo? o provenir de recidivas de astrocitomas que van progresando hacia mayores grados de malignidad. La alteracion molecular mas frecuente que se encuentra en estos tipos tumorales es la perdida de heterocigocidad del cromosoma 10 en el que se han identificado varios genes supresores de tumores. Las vias geneticas TP53/MDM2/ P14arf y CDK4/RB1/P16ink4 implicadas en division celular, se encuentran desreguladas en la mayoria de los gliomas asi como los genes que promueven la division celular, entre ellos EGFR. Por ultimo el aumento de factores de crecimiento y angiogenicos tambien esta involucrado en el desarrollo de estos tipos tumorales. Uno de los objetivos de la biologia molecular en tumores de estirpe glial es intentar encontrar marcadores o alteraciones geneticas que permitan abordar mejor la clasificacion de los glioblastomas, su evolucion y pronostico asi como su tratamiento. La diversidad y la cantidad de las alteraciones moleculares presentes en glioblastomas probablemente sea el motivo por el que todavia no se han encontrado farmacos efectivos para combatirlos. En la actualidad, con la aparicion de nuevas tecnicas de biologia molecular, se puede intentar individualizar y clasificar a los pacientes en funcion de su expresion genica. Esto abre una ventana esperanzadora a la aparicion de nuevos farmacos que tengan como diana exclusiva a los genes y/o proteinas alterados de las celulas tumorales en funcion de su patron de expresion genica individualizado para cada tumor. En este articulo revisamos los mecanismos moleculares mas frecuentes en la patogenesis de los glioblastomas.

Mutational analysis of the CITED4 gene in glioblastomas

Allelic losses at 1p appear as a characteristic feature of glial tumors (glioblastoma, astrocytoma, oligodendroglioma, and mixed forms) and, together with 19q losses, represent a prognostic parameter predictive for chemosensitivity and survival primarily in anaplastic oligodendrogliomas [1e4]. Although several genes located at 1p ( p73, CAMTA1, p18, hRAD54, Patched2, Riz1, KIF1b) have been analyzed previously for inactivating mutations related to glioma development, no specific candidate genes have been identified [5e10]. CITED4 (CREB-binding protein/ p300-interacting transactivator with E/D-rich tail 4) is located at 1p34wp35 and encodes a 184eamino acid protein [11]. It is a member of the CITED family, which includes four identified genes, although only three of them are present in mammals (including humans): CITED1, 2, and 4 [12]. All family members share the presence of the CITED domain, which is able to interact with CBP (CREB-binding protein) and p300 [13]. These proteins are transcriptional co-activators that act in two ways to increase gene transcription: (1) by binding transcription factors with RNA polymerase II and (2) by acting like acetyltransferases [14]. Proteins CBP and p300 are able to act on the nucleosome [15]. CITED4 generally has a nuclear location, but cytoplasmatic translocation or loss of nuclear expression has been observed in breast cancer development, in which case it might represent a prognostic marker [13]. Therefore, CITED4 has been proposed as a candidate gene involved in neoplasms characterized by 1p loss. Tews and co-workers [16] recently reported on mutational (in 45 samples) and methylation (in 62 samples) studies of this gene. This glioma series primarily included tumors with major oligodendroglial components, and 15 different CITED4 polymorphisms, mostly single nucleotide exchanges, but no mutations were identified. In parallel, aberrant methylation of the CITED4-associated CpG island was primarily found in oligodendrogliomas with 1p/19q losses that generally showed at least 50% CITED4 reduced expression [16]. Although less frequent than in oligodendroglial tumors, allelic losses at 1p have also been described in astrocytomas (lowgrade and anaplastic) and glioblastomas [1,17], the most malignant form of glial neoplasms. Thus, we performed a mutational study of CITED4 in a series of 24 glial tumors (22 primary glioblastomas, 1 low-grade astrocytoma, and its recurrent secondary glioblastoma) using polymerase chain reaction/single-strand conformation polymorphism

Brain Metastases: Gene Amplification Using Quantitative Real-Time Polymerase Chain Reaction Analysis

Brain metastasis is the most common intracranial malignancy in adults, occurring in about 10 to 30% of adult cancer patients. Tumor metastasis is a complex process requiring cell motility, invasion, platelet aggregation ability, angiogenesis, and avoidance of host immune responses. Increasing gene dosage through oncogene copy gain (extra copies) or amplification is a common genetic mechanism for up-regulation of gene expression; this mechanism participates in the development of solid tumors and metastases by stimulation of cell division or by inhibition of cell death or cell cycle arrest. Using quantitative real time polymerase chain reaction we determined gene dosage of eight tumor-related genes: ELF3, MDM4, LRRN2, PDGFRA, EGFR, MYC, MDM2, and ERBB2, in a series of 18 brain metastases originated from lung cancer (six cases), breast carcinoma (three cases), melanoma (three samples), ovarian carcinoma (two cases), and one each from colon, bladder, kidney and undifferentiated carcinoma. Copy gains, in at least one gene, were identified in ten cases (55%) whereas amplification could be determined in six samples (33%). Concurrent alterations were observed in three lesions derived from two lung carcinoma and from one ovarian carcinoma: one of the lung carcinoma metastases accumulated copy gains of LRRN2, MDM2, PDGFRA, and ERBB2, whereas the second displayed amplification of LRNN2 and ERBB2. The lesion derived from the ovarian carcinoma accumulated amplification of MYC and EGFR. Differential amplification levels could be determined for the intracellular / extracellular domains of genes EGFR and/or ERBB2 in four cases. These data suggest that copy gain/gene amplification appears associated with greater tumor aggressiveness and that quantitative real time polymerase chain reaction is an alternative methodology to identify differential intragenic amplification levels.

Detection of gene amplification and copy gains in brain metastases of solid tumors using quantitative real-time polymerase chain reaction

Metastases represent w5e15% of malignant brain tumors, and lung, breast, and colon carcinomas and melanoma represent the most frequent primary tumors causing brain metastases [1]. Tumor metastasis is a complex process, requiring cell motility, invasion, platelet aggregating ability, angiogenesis, and avoidance of host immune responses [2]. Thus, the metastatic mechanism most likely represents a multistep process combining both genetic and epigenetic changes that activate or inactivate tumor-related genes [2e5]. Increased gene dosage through oncogene copy gains (overdose) or amplification is a common genetic mechanism for upregulation of gene expression and is involved in the development of solid tumors by stimulation of cell division or inhibition of cell death or cell-cycle arrest [2,3,5]. Using quantitative real-time polymerase chain reaction (PCR) analysis, we recently identified not only amplification (O5-fold) but also gene overdose (low-level 1 to 5-fold gene amplification) involving EGFR in brain metastases [6]. Accordingly, we used this methodology to determine gene dosage of seven tumor-related genes with proven involvement in primary neoplasms or with location in or near chromosomal regions frequently involved in brain metastases [7]. The genes studied were LRRN2 (leucine rich repeat neuronal 2; alias glioma amplified on chromosome 1, GAC1), MDM4 (transformed 3T3 cell double minute 4), and ELF3 (E74-like factor 3) (all three located at 1q32); MDM2 (transformed 3T3 cell double minute 2) (at 12q13wq14) and PDGFRA (platelet-derived growth factor receptor alpha polypeptide) (4q11wq13); MYC (v-myc myelocytomatosis viral oncogene homolog; alias c-MYC ) (8q24.12wq24.13); and ERBB2 (erythroblastic leukemia viral oncogene homolog 2) (17q21.1). These genes participate in the control of key cellular functions such as signal transduction control, regulation of cell proliferation, or encoding of transcriptional factors [2,8]. We analyzed 18 brain metastases originating from melanoma (3 cases), lung cancer (6 cases), breast carcinoma (3 cases), ovarian carcinoma (2 cases), colon (1 case), kidney (1 case), and bladder carcinoma (1 case); 1 metastatic lesion was diagnosed as an undifferentiated carcinoma for which primary tumor origin could not be determined. To verify the amplification status of these genes, quantitative real-time PCR was performed using the Light Cycler