[Identification of genetic alterations by multiple ligation-dependent probe amplification (MLPA) analysis in oligodendrogliomas]. / Identificación de alteraciones genéticas en oligodendrogliomas mediante amplificación dependiente de ligasa de múltiples sondas (MLPA) (multiple ligation-dependent probe amplification).

Concurrent deletion at 1p/19q is a common signature of oligodendrogliomas, and it may be identified in low-grade tumours (grade II) suggesting it represents an early event in the development of these brain neoplasms. Additional non-random changes primarily involve CDKN2A, PTEN and EGFR. Identification of all of these genetic changes has become an additional parameter in the evaluation of the clinical patients' prognosis, including good response to conventional chemotherapy. Multiple ligation-dependent probe amplification (MLPA) analysis is a new methodology that allows an easy identification of the oligodendrogliomas' abnormalities in a single step. No need of the respective constitutional DNA from each patient is another advantage of this method. We used MLPA kits P088 and P105 to determine the molecular characteristics of a series of 40 oligodendrogliomas. Deletions at l p and 19q were identified in 45% and 65% of cases, respectively. Alterations of EGFR, CDKN2A, ERBB2, PTEN and TP53 were also identified in variable frequencies among 7% to 35% of tumours. These findings demonstrate that MLPA is a reliable technique to the detection of molecular genetic changes in oligodendrogliomas.

DAPK1 promoter hypermethylaiton in brain metastases and peripheral blood.

The DAPK1 gene works as a regulator of apoptosis and is frequently inactivated in cancer by aberrant promoter hypermethylation. Loss of DAPK1 expression is associated with a selective advantage for tumor cells to resist apoptotic stimuli, allowing them to separate from the original tumor; from this point of view, DAPK1 could be considered a tumor metastases inhibitor gene. To verify the participation of DAPK1 silencing in cerebral invasion, we analyzed its promoter methylation status in a series of 28 samples from cerebral metastases using MSP and sequencing of the MSP-product. We have found hypermethylation in 53.6% (15/28) metastatic tumor samples as well as in 27.8% (5/18) of its peripheral blood samples. Our data suggest an important role of DAPK1 for silencing through promoter CpG island hypermethylation in the development of brain metastases from solid tumors. The detection of aberrant hypermethylation on DAPK1 promoter from peripheral blood samples has potential clinical implications as a tumor prognosis marker.

NF2 gene mutations and allelic status of 1p, 14q and 22q in sporadic meningiomas.

Formation of meningiomas and their progression to malignancy may be a multi-step process, implying accumulation of genetic mutations at specific loci. To determine the relationship between early NF2 gene inactivation and the molecular mechanisms that may contribute to meningioma tumor progression, we have performed deletion mapping analysis at chromosomes 1, 14 and 22 in a series of 81 sporadic meningiomas (54 grade I (typical), 25 grade II (atypical) and two grade III (anaplastic)), which were also studied for NF2 gene mutations. Single-strand conformational polymorphism analysis was used to identify 11 mutations in five of the eight exons of the NF2 gene studied. All 11 tumors displayed loss of heterozygosity (LOH) for chromosome 22 markers; this anomaly was also detected in 33 additional tumors. Twenty-nine and 23 cases were characterized by LOH at 1p and 14q, respectively, mostly corresponding to aggressive tumors that also generally displayed LOH 22. All three alterations were detected in association in seven grade II and two grade III meningiomas, corroborating the hypothesis that the formation of aggressive meningiomas follows a multi-step tumor progression model.

[Esthesioneuroblastoma treatment]. / Tratamiento del estesioneuroblastoma. Revisión de la literatura.

INTRODUCTION: Esthesioneuroblastoma is an uncommon malignancy of the olfactory neuroepithelium. The best treatment has yet to be defined. The purpose of this study is to analyze the tumors's behaviour to choose the ideal treatment, the therapeutic strategy and the patterns of failure. MATERIALS AND METHODS: We carry out a revision of the series published between 1994 to 2004. In these series, we found 39 papers with 713 patients. CONCLUSIONS: In this review the 5-years survival rate is 51.2%. Through the analysis of 583 partients found in 34 publications, surgery (alone or combined) is the treatment most used (78%). The commonly management is surgical in combination with radiotherapy (47%).

Six novel mutations in the NF2 tumor suppressor gene.

Six novel mutations were identified in the NF2 tumor suppressor gene in a panel of meningiomas and neurinomas. Screening was performed using a combination of single-strand conformation polymorphism and heteroduplex analyses on polymerase chain reaction-amplified DNA from tumors and matched peripheral blood lymphocytes. Mutations involved exons 2, 7, 11 and 12, and corresponded to three frameshift, one nonsense, one missense and one polymorphism.

Absence of mutation of the p73 gene in astrocytic neoplasms.

In subgroups of astrocytic neoplasms, including glioblastoma (GBM), mutations of the p53 tumour suppressor gene lead to loss of growth-suppressive properties. A p53-related gene termed p73 has recently been identified; its gene product shows structural and functional similarities to p53. After being mapped to chromosome region 1p36, p73 was proposed to act as a tumour suppressor gene, as this region is frequently deleted in a variety of human cancers, including astrocytic tumours. To determine whether p73 is involved in astrocytoma/GBM development, we analysed 10 pilocytic astrocytomas, 15 WHO grade II astrocytomas, 15 WHO grade III anaplastic astrocytomas, and 20 GBM for p73 gene alterations. In parallel, we used six polymorphic markers to determine the allelic status of region 1p36 in this tumour series. Although loss of heterozygosity was evidenced in 12 of 60 cases (20% of samples), PCR-SSCP and direct sequencing failed to detect any gene mutation in the entire coding region and intronic sequences of p73. Eight tumours displayed five distinct polymorphic nucleotide changes, also present in the corresponding normal DNA. These variations consisted of T-->C variation, with no change in Thr173; C-->T transition, with no change in His197; exon 9 simultaneous double change C-->T and T-->C , with no variations in Ala336 and His349, respectively, and C-->T change at exon 9/-24 position of intron 8. These results suggest that, in astrocytic gliomas, p73 may not play a major role as a tumour suppressor, but the relatively high incidence of LOH confirms the presence at 1p36 of an as yet unidentified gene of this category, with a key function in astrocytoma/GBM progression.

A dedicated prosthesis for open thoracostomy.

Due to our dissatisfaction with the mutilation caused by the skin-lined open thoracostomy, we have developed a dedicated prosthesis that is expected to avoid or to substitute for the classic operation. The prosthesis is a corrugated silicone tube with an oval flange at one end (to fix it internally) and a mobile ring on the other (to fix it externally). It is inserted at the bottom of the empyematic cavity after 3 cm of a rib is removed. We have used it in 20 patients whose empyema was secondary to pneumonia (12) or complications of pneumonectomy (4), lobectomy (2), decortication (1), or pleuroscopy (1). Six of those patients have already been cured and their prosthesis removed after 54 to 305 days. In 1 with a persistent postpneumonectomy bronchopleural fistula the device was removed after 299 days and the patient was submitted to a limited thoracoplasty. Six other patients still have unresolved cavities and have been using the prosthesis for 63 to 302 days. Seven patients died of their underlying disease (bilateral pneumonia, 2; acquired immunodeficiency syndrome, 2; mesothelioma, 1; heart failure and pulmonary embolism, 1; unknown, 1) after using the prosthesis for 11 to 160 days. In those patients from whom the prosthesis already has been removed, the scar looks like those commonly seen after removal of an ordinary chest tube. Based on these early favorable results we feel most encouraged to persist in this research. Nevertheless, we are aware that a larger number of patients and a longer follow-up will be necessary before we may make definitive recommendations.

Cytogenetic findings in a human malignant melanoma metastatic to the brain.

Cytogenetic analysis by G-banding of direct and preparations of a malignant melanoma metastatic to the brain in vitro showed a pseudodiploid modal chromosome number, including five marker chromosomes, one of which was an i(6p). These results agree with those recently reported about the preferential involvement of chromosome #6 in malignant melanoma.

C-banding studies in lymphocytes from patients with tumors of the nervous system.

C-banding studies were performed on cultured peripheral blood lymphocytes from 100 patients with tumors of the nervous system and 30 controls. The classification of Patil and Lubs and the heterochromatic index (HI) of Neeley were used to evaluate the heteromorphism. No overall significant differences were found in the frequency of C-band variants when the findings in both tumoral populations and controls were compared, but we did find an excess of 9qh inversions in some groups of tumor patients. An increase in the amount of heterochromatin in patients with pituitary adenomas and high malignancy grade astrocytomas was noted.

Chromosomal involvement secondary to -22 in human meningiomas.

Cytogenetic analyses have been performed on cultures in vitro from 32 human meningiomas, seeking chromosomal anomalies in addition to characteristic monosomy 22. Eight cases showed stem lines with normal karyotype, whereas, monosomy 22 as the only chromosomal deviation characterized the stem line of ten tumors. In 14 samples stem lines or modal numbers displaying numerical deviations (other than -22) and/or structural rearrangements were found. A hyperdiploid modal number was present in three, whereas, it was hypodiploid in the remainder. Numerical deviations in these tumors involved mainly #14 by losses, and also #22; recurrent structural rearrangements involving 1p and 11p were also characteristic features. Thus, these results could imply that involvement of #14, 1p, and 11p would be a form of clonal evolution secondary to monosomy 22 in certain meningiomas.

Cytogenetic follow-up from direct preparation to advanced in vitro passages of a human malignant glioma.

In order to evaluate which cytogenetic evolutionary patterns take place during the in vitro establishment of a permanent glioma cell line, cytogenetic follow-up was performed on direct preparations and primary cultures from a malignant astrocytoma and on serial passages for more than 2 years of in vitro propagation. Sixteen passages were studied, and the presence of a marker chromosome in direct preparations and after in vitro growth permitted us to identify the clonal evolution of the resulting permanent cell line. Near-triploid cells present in the direct study became the main cell population in vitro; chromosomal losses and the acquisition of a few new marker chromosomes were also characteristic features, leading to a stable modal number of around 60 chromosomes in the last passages analyzed. The results provide new evidence of the existence of more than one pattern of chromosomal evolution during the in vitro establishment of human gliomas.

Incidence and origin of dicentric chromosomes in cultured meningiomas.

Based on the cytogenetic findings in 32 human meningiomas, an analysis of dicentric chromosomes, usually present in cultures from meningiomas, has been performed. The incidence and origin of such markers have been analyzed and the chromosomal composition of the stem line in the corresponding sample established (i.e., normal karyotype, -22 as the sole chromosomal deviation, or complex karyotypes in addition to #22 abnormalities). More than 10 dicentric chromosomes were found in 12 of 32 meningiomas (37.5%). Sixty-eight of the markers could be identified individually or as belonging to a chromosome group. Fifty-three percent of the meningiomas characterized by a complex stem line karyotype also displayed dicentric chromosomes in variant cells, whereas only 12.5% of meningiomas with a normal diploid stem line showed such chromosomal aberrations. Chromosomes of groups C and D participated most frequently in the genesis of dicentrics; however, chromosomes 19, 20, 3, 6, and 13 were the most frequently involved. Thus, the existence of a nonrandom pattern of involvement supports the fact that dicentrics might play a biologic role in the progression of human meningiomas.

Chromosomal composition of a series of 22 human low-grade gliomas.

G-banded chromosomal analysis was performed on direct and/or in vitro cultures of 22 low-grade gliomas, including nine grade I-II astrocytomas, nine oligodendrogliomas, one mixed tumor oligodendroglioma-astrocytoma, and three ependymomas. Normal diploid stem lines were present in most astrocytomas and oligodendrogliomas, whereas, all three ependymomas displayed polyploid modal numbers. However, secondary cell lines showed the presence of clonal recurrent numerical abnormalities, mainly polysomy 7, monosomy 10 and 22, and loss of the Y chromosome. Clonal structural rearrangements were present with a low incidence; they mainly involved chromosomes #1 and #7. These patterns of chromosome involvement seem to correlate with the scarce previous cytogenetic banding data available from low-grade gliomas. They are also similar to the chromosome alterations found in high-grade gliomas.

Involvement of 22q12 in a neurofibrosarcoma in neurofibromatosis type 1.

We describe the cytogenetic and molecular genetic findings in a neurofibrosarcoma arising in a patient affected by neurofibromatosis type 1. Multiple chromosomal rearrangements were found but only a few of them were identified as clonal abnormalities, including a deletion of chromosome 22, which at the molecular level proved to be interstitial, mainly involving the 22q12 region. Loss of heterozygosity for markers D22S32 and MB was observed. These findings are in agreement with previous data which suggest a possible involvement of a gene located at 22q11-q13.1 during the neoplastic development of some neurofibromatosis type 1-associated tumors.

Chromosomal patterns in human malignant astrocytomas.

Cytogenetic analysis by direct and/or in vitro preparations was performed on 34 malignant astrocytomas. Thirty tumors showed near-diploid chromosome numbers, whereas, tritetraploid chromosome complements were present in four tumors. The most frequent chromosomal changes implied numerical deviations by a gain of chromosomes #7, #19, and #20, and by losses of #10, #22, and Y. Structural rearrangements were present in stem- or side lines of 24 tumors. Although no common chromosomal rearrangement seems to exist among those tumors, chromosomes #1, #6, #7, and #9 were predominantly involved. Polysomy and structural rearrangements of chromosome #7 could be related to the overexpression of epidermal growth factor gene, previously observed in some malignant gliomas.

Deletion 3p in two lung adenocarcinomas metastatic to the brain.

Cytogenetic analysis by direct and in vitro preparation of brain metastatic lesions from two lung adenocarcinomas have shown the presence of a recurrent chromosomal abnormality, del(3p). These results add new evidence about the presence of 3p- marker chromosomes in adenocarcinoma of the lung.

Molecular analysis of genomic abnormalities in human gliomas.

A series of 57 malignant gliomas, including 27 astrocytomas grade III-IV (glioblastoma multiforme), 15 astrocytomas grade I-II, and 15 tumors with major oligodendroglial component, was examined to detect molecular abnormalities of loci at specific chromosome regions. At the cytogenetic level, these regions have been shown to be nonrandomly involved in neoplastic development of these histologic subtypes of tumor. We used a panel of 24 polymorphic DNA probes to analyze loss of heterozygosity (LOH) at loci on chromosomes 7, 9, 10, 13, 17p, and 22q. In addition, the retinoblastoma (RB1) oncosuppressor gene, the platelet-derived growth factor A (PDGFA) gene, and the epidermal growth factor receptor (EGFR) gene were analyzed directly. Loss of genetic information on the short arm of chromosome 17 was observed in both low- and high-grade astrocytomas, whereas no oligodendroglial tumor was characterized by this type of aberration. LOH for chromosome 10, mainly compatible with loss of the entire chromosome, was primarily evidenced in the more malignant forms and in isolated cases diagnosed as low-grade astrocytomas. Again, no oligodendroglial tumor displayed losses of chromosome 10. In contrast, four tumors with major oligodendroglial component showed losses involving 9p markers, primarily interferon A and B (IFNA, IFNB); this feature was also observed in two low-grade astrocytomas and in 11 high-grade tumors. Isolated cases displayed LOH for markers on chromosomes 13 and 22, whereas EGFR amplification was almost exclusively evidenced in the more malignant forms which, in most instances, also presented LOH for chromosome 10. In general, the samples with lower malignancy stage displayed a lesser grade of abnormalities, mainly restricted to losses at 17p and chromosome 10 in astrocytomas grade I-II and at 9p in oligodendrogliomas. In contrast, about 50% of the high-grade tumor samples analyzed included abnormalities at two or more loci, with a recurrent association of EGFR amplification and LOH for chromosome 10; this association was evident in 26% of the high-grade astrocytomas.

A case of pituitary adenoma with 58 chromosomes.

Chromosome analysis was performed on a growth hormone secreting pituitary adenoma. G-banding analysis showed a stem line with 58 chromosomes including numerical deviations of pairs #3, #5, #7, #9, #11, #12, #13, #17, #19, #20, and gonosomes was found. These findings add new evidence of the nonrandom involvement of C and F group chromosomes in numerical deviations in this tumor type.

Cytogenetic clones in a recurrent neurofibroma.

Chromosome studies were performed on a plexiform neurofibroma arising in a probable von Recklinghausen's disease patient, who also showed a de novo constitutional reciprocal translocation, t(1;22)(p32;q11). Banding analysis of the metaphases obtained from two primary cultures in vitro showed the presence of five cytogenetic clones, characterized by different chromosomal rearrangements. In addition to t(1;22), marker chromosomes involved pairs 1, 2, 3, 5, 8, 9, 10, 12, 16, and X. These findings suggest a possible polyclonal evolution in this neurofibroma.

High-resolution analysis of chromosome arm 1p alterations in meningioma.

Loss of heterozygosity (LOH) for loci on chromosome arm 1p is a relatively common event in human meningioma, and this anomaly has been proposed to be associated with the development of grade II or grade III forms (atypical and anaplastic meningiomas). Nevertheless, the limited data available do not allow the establishment of the frequency and the extent of the affected 1p regions. To determine the status of chromosome 1p in meningiomas, we have performed a comprehensive analysis of LOH on 1p in 100 meningiomas using a high density of 1p-marker loci. Allelic loss was found in 35% of tumors, most corresponding to nontypical meningiomas that also displayed losses for loci on chromosome 22. Although some tumors displayed complex rearrangements leading to distinct 1p deletions, the patterns of loss indicated two main target regions: 1p36 and 1p34-p32, which represent the most frequently involved regions, whereas 1p22 and 1p21.1-1p13 regions appeared deleted in some tumors. These results suggest that there may be several putative tumor suppressor genes on 1p, the inactivation of which may be important in the pathogenesis of meningiomas, as well as in other tumor types.