Retinoblastoma gene deletions in human glioblastomas

The retinoblastoma susceptibility gene, RB, is the best characterised of the tumour suppressor genes, or 'anti-oncogenes'. Abnormal function of the RB protein is thought to result in loss of an inhibitory effect on cell growth, and thus contribute towards the development of certain human cancers. One group of human cancers of particular interest in relationship to retinoblastoma gene function are the gliomas, which are central nervous system tumours thought to originate from the neuroectoderm, the embryological tissue which also gives rise to retinoblastomas. We have therefore examined a group of benign and malignant gliomas for evidence of structural alterations of the RB gene. Four out of nine (44%) glioblastomas, the most malignant gliomas, showed loss of heterozygosity of a locus within this gene. In addition, one of these hemizygous tumours showed deletion of part of the RB protein-coding region, and this abnormality was also present in cells cultured from the tumour. These findings suggest that RB gene abnormalities may contribute to the development of glioblastomas.     

Polyreactivity of monoclonal antibodies made against human erythrocyte membranes with various pathogenic bacteria

Glycophorins comprise the major sialoglycoproteins of the human erythrocyte membrane. Several years ago we described a murine monoclonal antibody (MAb), designated 124,D-7 (IgM), developed by in vitro immunization with human erythrocyte membranes as antigen. We found the MAb reacted with a neuraminidase-dependent epitope on glycophorin A. Recent findings using ELISA with various bacteria as coating antigens have demonstrated strong cross-reactions of MAb 124,D-7 with some bacteria like Legionella and no reaction with bacteria such as Streptococcus pneumoniae. A second MAb, 130,E-4 (IgM), generated by the in vitro immunization technique, agglutinated human red cells irrespective of blood groups. This MAb showed strong cross-reactions with bacteria different from those being positive with MAb 124,D-7. The broad cross-reactivities of the two MAbs suggested that they are polyreactive antibodies. Sequencing of the V(H) and V(L) genes of MAb 124,D-7 showed germ-like sequences characteristic of polyreactive antibodies. The nucleotide sequences of the V(H) and V(L) genes of MAb 124,D-7 matched sequences coding for antibodies against CD34 and cross-reacting streptococcal antibodies. For Legionella pneumophila, the main interacting band on immunoblots was identified as the major outer membrane protein by mass spectrometry after separation by isoelectric focusing followed by SDS-PAGE. Flow cytometry showed that the epitope for MAb 124,D-7 was not displayed on live L. pneumophila but became exposed after heat treatment. Studies with one of the control MAbs, 145,F-2, directed against phosphorylcholine, which is known to be present on erythrocytes and some bacteria, showed that the epitope is deeply buried in the human erythrocyte membrane as neither neuraminidase nor papain exposed the epitope. The positive control MAb 3/1 directed against an epitope on LPS of L. pneumophila revealed weak cross-reactions with Pseudomonas aeruginosa.     

Immunostaining of colorectal cancer with monoclonal anti-CEA antibodies compared to serum and tumor CEA content

Carcinoembryonic antigen was measured in serum and in extracts from 37 colorectal tumors and we found a poor correlation between circulating and tumor CEA. Monoclonal antiCEA antibodies were used in indirect immunoperoxidase staining of the corresponding formalin fixed tissue sections. We found that serum CEA measurement had a sensitivity of only 41.9% as compared to 90.3% for the immunohistochemical staining. The positive and negative predictive values for immunostaining were respectively 96.6% and 72.7%. Immunohistochemical staining of tissue sections with monoclonal anti CEA coupled to other biochemical or immunological assays could be a useful adjunct for the diagnosis of premalignant or slightly modified tissues.     

PCNA (cyclin) autoantibodies and monoclonal antibodies reveal similar patterns of cyclin (PCNA) antigen staining in human cultured cells

Double immunofluorescence and [3H]thymidine autoradiographic analysis of the same field of transformed human amnion cells (AMA) reacted with proliferating cell nuclear antigen (PCNA) autoantibodies and a monoclonal antibody (mAb 19F4) specific for cyclin (PCNA) revealed similar patterns and sequence of cyclin (PCNA) antigen staining during S-phase. These results suggest that immunofluorescence patterns obtained with PCNA autoantibodies reflect in fact patterns of cyclin (PCNA) antigen staining.     

Predictive and prognostic markers in human glioblastomas

Opinion statement Glioblastomas (GBMs) are among the most aggressive of all known human tumors. The median survival times remain in the 12- to 15-month range despite aggressive surgery, radiation, and chemotherapy. Through molecular and genetic profiling efforts, underlying mechanisms of resistance to these therapies are becoming better understood. The present standard of care has been shaped by the recently reported phase III study by the European Organisation for Research and Treatment of Cancer and the National Cancer Institute of Canada, which found that the addition of temozolomide (TMZ) to radiation therapy significantly improved outcome compared with radiation alone. However, careful examination of these data reveals that not all GBM patients benefited from the addition of TMZ to radiation therapy. A companion correlative study found that GBM patients with tumors with MGMT promoter methylation appeared to derive the greatest benefit from the addition of TMZ to radiation therapy. Although this finding is provocative, it should be kept in mind that this study was performed retrospectively and that prospective validation is required before MGMT methylation can be used for clinical stratification purposes. However, this study does show promise for the tailoring of future treatments according to the molecular and genetic profiles of an individual's tumor rather than using the “one-glove-fits-all≓ approach that is currently being followed. As more effective “smart drugs≓ are developed, molecular and genetic profiling will assume even greater importance in this regard.     

EGFR gene amplification - rearrangement in human glioblastomas

Immunostaining using an affinity-purified rabbit polyclonal antibody against the extracellular domain of the epidermal-growth-factor receptor (EGFR) showed over-expression occurring in a fraction of tumor cells in 17 out of 18 human glioblastomas and in a majority of cells in 7 of the 18. Southern-blotting technique using a full-length EGFR cDNA probe showed a variable degree of amplification in 10 of the 17 glioblastomas, which was associated with EGFR over-expression in each case. In 2 of the glioblastomas with EGFR gene amplification, a rearrangement of the gene affecting the extracellular domain of the receptor was identified and DNA sequence analyses revealed an identical deletion-rearrangement of 801 base pairs between exons 2 to 7, resulting in an in-frame fusion of exons I and 8. © 1995 Wiley-Liss, Inc.     

Characterization of the 20S proteasome in human glioblastomas

Proteasomes are multisubunit proteases involved in many cellular processes, including tumorigenesis and immune surveillance. In their catalytic core, the 20S proteasome, the beta1, beta2 and beta5 subunits show peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, respectively. By IFN-gamma and TNFalpha stimulus, these subunits are replaced by their counterparts LMP2, MECL-1 and LMP7, defined inducible subunits, thus originating the immunoproteasome, and expression of the proteasome activator PA28 is enhanced. These modifications strengthen MHC-class I restricted peptide generation. The 20S proteasome has been detected immunohistochemically in formalin-fixed samples purified from fresh surgical specimens of 18 tumors (G20S) and from 8 samples of normal peritumoral tissue. The G20S, LMP2, MECL-1 and LMP7 increased in only 12 cases, along with unvaried trypsin-like and decreased PGPH and chymotrypsin-like activities; PA28 was unvaried in all 18 samples. The immunoproteasome alterations may represent an anomalous immunological attitude of glioblastomas.     

IL-6 gene amplification and expression in human glioblastomas

The aggressiveness of human gliomas appears to be correlated with the upregulation of interleukin 6 (IL-6) gene. Using quantitative PCR methods, we detected amplification and expression of the IL-6 gene in 5 of 5 primary glioblastoma samples and in 4 of 5 glioblastoma cell lines. This finding suggests that the amplification of IL-6 gene may be a common feature in glioblastomas and may contribute to the IL-6 over-expression.     

Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S-phase in cultured cells--some applications of PCNA antibodies

Immunofluorescence studies using PCNA autoantibodies specific for the proliferation-sensitive protein cyclin have revealed dramatic changes in the nuclear distribution of this protein during the S-phase of normal and transformed cells. Patterns of cyclin antigen distribution subdivide S-phase and have provided new cell cycle landmarks. Some of these (nucleolar exclusion or staining), mimic topographical patterns of DNA synthesis thus arguing for a role of this protein in some specific aspect of DNA replication. Cells outside S-phase (G0 included) stain only weakly with PCNA antibodies, stressing the usefulness of this reagent for identifying proliferating cells (S-phase cells) of both normal and malignant origins.     

Genetic analysis of human glioblastomas using a genomic microarray system

Genomic microarray systems can simultaneously provide substantial genetic and chromosomal information in a relatively short time. We have analyzed genomic DNA from frozen sections of 30 cases of primary glioblastomas by GenoSensor Array 300 in order to characterize gene amplifications, gene deletions, and chromosomal information in the whole genome. Genes that were frequently amplified includedPFC2/CYLN2 (63.3%),EGFR (53.3%),IL6 (53.3%),ABCB1 (MDR1) (36.7%), andPDGFRA (26.7%). Genes that were frequently deleted includedFGFR2 (66.7%),MTAP (60.0%),DMBT1 (56.7%),CDKN2A (p16)/MTAP (50.0%),PIK3CA (43.3%), andEGR2 (43.3%), but deletion ofRB1 orTP53 was rarely detected. Chromosomal gains were observed frequently for 7q (33.3%), 7p (20.0%), and 17q (13.3%). Loss of the 10q was frequently detected in 13 of 30 cases (46.7%). Loss of the entire chromosome 10 was seen in 9 of 30 cases (30.0%), and was often accompanied byEGFR amplification (7 cases, 77.8%). The GenoSensor Array 300 proved to be useful for identification of genome-wide molecular changes in glioblastomas. The obtained microarray profile can also yield valuable insight into the molecular events underlying carcinogenesis of brain tumors and may provide clues about clinical correlations, including response to treatment.     

Immunohistological and functional analyses of lymphoid infiltrates in human glioblastomas

An immunohistological analysis of tumor tissue obtained from seven patients with malignant gliomas demonstrated varying levels of lymphoid cell infiltration. The tumor infiltrating lymphocytes obtained from each sample were cultured in vitro by a limiting dilution technique. In three of the cases studied many tumor infiltrating lymphocyte microcultures selectively lysed autologous glioblastoma cells but did not lyse allogeneic gliomas, natural killer-resistant fresh melanoma cells or K562 target cells. These cultures were found to consist of CD 3+ cells. In six cases studied a variable number of microcultures lysed both autologous tumor and K562 target cells only. A minority of the microcultures studied were cytolytic for allogeneic glioma cells and fresh melanoma target cells. The cytolytic activity expressed by tumor infiltrating lymphocytes against autologous tumor cells was significantly greater (P less than 0.001) than that obtained by the corresponding peripheral blood lymphocytes cultured in a similar manner. The present immunohistological and functional studies suggest that there is an immune response to human glioblastomas in vivo with an accumulation of cells with antitumor activity at the tumor site.     

Immunostaining of ductal breast carcinomas with the monoclonal-antibody-h

Twenty cases of infiltrating ductal breast carcinomas were stained with the mouse monoclonal antibody H, an IgM, in order to estimate the distribution of the epitope (a carbohydrate moiety) recognized by this antibody in the malignant cells. The results showed that in all cases examined, the epitope was present in the cytoplasm of the neoplastic cells arranged mainly in a diffuse pattern. To identify the polypeptides that carry this epitope, we used extracts from the MCF-7 human breast carcinoma eel line in immunoblotting experiments. In addition to cytokeratin 8 this epitope was also found on five polypeptides originating from Triton X-100-soluble (M(r)x10(-3) of 232, 67, and 37) and from the Triton X-100-insoluble (M(r)x10(-3) of 51, and 50) fractions, respectively. The contribution, if any, of these polypeptides to the staining patterns is not known. These data reveal the existence of a common epitope between cytokeratin 8 and other cellular polypeptides. They also serve as a note of caution in the interpretation of immunohistochemical staining when anti-cytokeratin antibodies are used.     

Gene expression profiles of human glioblastomas are associated with both tumor cytogenetics and histopathology

Despite the increasing knowledge about the genetic alterations and molecular pathways involved in gliomas, few studies have investigated the association between the gene expression profiles (GEP) and both cytogenetics and histopathology of gliomas. Here, we analyzed the GEP (U133Plus2.0 chip) of 40 gliomas (35 astrocytic tumors, 3 oligodendrogliomas, and 2 mixed tumors) and their association with tumor cytogenetics and histopathology. Unsupervised and supervised analyses showed significantly different GEP in low- vs high-grade gliomas, the most discriminating genes including genes involved in the regulation of cell proliferation, apoptosis, DNA repair, and signal transduction. In turn, among glioblastoma multiforme (GBM), 3 subgroups of tumors were identified according to their GEP, which were closely associated with the cytogenetic profile of their ancestral tumor cell clones: (i) EGFR amplification, (ii) isolated trisomy 7, and (iii) more complex karyotypes. In summary, our results show a clear association between the GEP of gliomas and tumor histopathology; additionally, among grade IV astrocytoma, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone. Further studies in larger series of patients are necessary to confirm our observations.     

Invasiveness in vitro and biological markers in human primary glioblastomas

Invasion of spheroids from 20 human primary glioblastomas into precultured fetal rat brain tissue in culture has been studied and quantified. Between 30 and 98 percent of the normal brain tissue was destroyed by invading glioma cells within 4 days. The degree of invasion did not correlate with patient survival. A slightly higher invasiveness and shorter survival was seen in tumors with EGF receptor overexpression, and the opposite pattern was found for tumors with a TP53 mutation.The degree of invasiveness in vitro was far higher than would be expected from the dynamics of clinically observed tumor spread. This suggests that mechanisms suppressing invasion may be operative in the normal brain; alternatively the differences may be due to a higher permissiveness of the fetal brain tissue for invasion in vitro.     

Histopathological correlates with survival in reoperated glioblastomas

The addition of concomitant and adjuvant chemotherapy to radiation therapy after surgical resection has increased significantly the survival of patients with glioblastoma (GB). In conjunction, there has been an increasing fraction of patients who present with new enlarged areas of contrast enhancement and edema on post-treatment imaging that improve without further treatment. It remains to be established how this phenomenon, commonly termed pseudoprogression, can be distinguished from true tumor recurrence defined as the histological presence of active high-grade tumor, as well as its prognostic significance. Data for over 500 patients undergoing surgery for recurrent GB were reviewed. Pathological specimens were categorized as those that contained active high-grade glioma in any amount, and those that did not. Patient survival was compared between these two groups, and independent associations were assessed using Cox proportionate hazards regression analysis. 59 patients met the study criteria including complete pathological and follow-up data. Mean age was 53 ± 11 years. Median survival from suspected recurrence and initial diagnosis were 8 [5–14] and 20 [12–30] months. Seventeen patients (29 %) had no evidence of active high-grade tumor and 42 (71 %) had at least focal active high-grade glioma. Pathologic pseudoprogression at re-operation (p = 0.03) and gross total resection (p = 0.01) were independently associated with survival. The histopathological features defined here and used to assess the tumor at reoperation were independently associated with survival. These findings may be important in designing treatment strategies and clinical trial endpoints for patients with GB.     

Microglial/macrophage expression of interleukin 10 in human glioblastomas.

Interleukin 10 (IL-10) expression has been found to be correlated with the extent of malignancy in gliomas. In vitro, IL-10 increases proliferation and migratory capacity in human glioma cell lines. In this study, we localized the site of IL-10 synthesis in gliomas to cells of microglial origin. Biopsy specimens from 11 patients with malignant glioma were processed on native tissues and at early cell culture passages (0-4). IL-10 mRNA was analyzed by RT-PCR and in situ hybridization. Protein was quantitatively assessed by ELISA in cell culture supernatants, and cells expressing IL-10 were determined by a combination of immunohistochemistry for CD68 (specific for microglia/macrophage lineage) and IL-10 in situ hybridization. IL-10 mRNA decreased from passage 0 to 4 in all samples and was undetectable beyond passage 5. Such downregulation of mRNA leads to a steep decrease of IL-10 protein in culture supernatants (below detection level, 0.05 ng/ml, beyond passage 1). The combination of in situ hybridization for IL-10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL-10 mRNA. Our results identify microglia/macrophage cells as the major source of IL-10 expression in gliomas which decreases markedly during early passages of primary cultures of human gliomas due to a progressive reduction of microglia/macrophages present.