Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas.

AIMS: Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor-κB (NF-κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF-κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL. METHODS: We conducted the systematic sequencing of 21 genes relevant to the NF-κB signalling network for 71 PCNSLs as well as the pyrosequencing of CD79B and MYD88 mutation hotspots in a further 35 PCNSLs and 46 glioblastomas (GBMs) for validation. RESULTS: The results showed that 68 out of 71 PCNSLs had mutations in the NF-κB gene network, most commonly affecting CD79B (83%), MYD88 (76%), TBL1XR1 (23%), PRDM1 (20%) and CREBBP1 (20%). These mutations, particularly CD79B and MYD88, frequently coincided within each tumour in various combinations, simultaneously affecting diverse pathways within the network. No GBMs had hotspot mutation of CD79B Y196 and MYD88 L265. CONCLUSIONS: The prevalence of CD79B and MYD88 mutations in PCNSLs was considerably higher than reported in systemic diffuse large B-cell lymphomas. This observation could reflect the paucity of antigen stimuli from the immune system in the central nervous system (CNS) and the necessity to substitute them by the constitutive activation of CD79B and MYD88 that would initiate the signalling cascades. These hotspot mutations may serve as a genetic hallmark for PCNSL serving as a genetic marker for diagnose and potential targets for molecular therapy.

Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma.

Pilocytic astrocytomas (PAs), WHO malignancy grade I, are the most frequently occurring central nervous system tumour in 5- to 19-year-olds. Recent reports have highlighted the importance of MAPK pathway activation in PAs, particularly through a tandem duplication leading to an oncogenic BRAF fusion gene. Here, we report two alternative mechanisms resulting in MAPK activation in PAs. Firstly, in striking similarity to the common BRAF fusion, tandem duplication at 3p25 was observed, which produces an in-frame oncogenic fusion between SRGAP3 and RAF1. This fusion includes the Raf1 kinase domain, and shows elevated kinase activity when compared with wild-type Raf1. Secondly, a novel 3 bp insertion at codon 598 in BRAF mimics the hotspot V600E mutation to produce a transforming, constitutively active BRaf kinase. Although these two alterations are not common, they bring the number of cases with an identified 'hit' on the Ras/Raf-signalling pathway to 36 from our series of 44 (82%), confirming its central importance to the development of pilocytic astrocytomas.

Expression of the human NOV gene in first trimester fetal tissues.

NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.

Expression and synthesis of insulin-like growth factor (IGF)-I, -II and their receptors in human glioma cell lines.

Insulin-like growth factor (IGF)-I and -II are involved in the regulation of brain development and are thought to play a pivotal role in the proliferation of gliomas. Expression of IGF-I, IGF-II, the type I and type II IGF receptor were studied in a panel of thirty glioma cell lines by Northern blotting and PCR analysis. IGF-II mRNA expression with transcripts of 4.8 and 6.0 kb was shown only in one glioma cell line (NCE-G96) and no transcripts for IGF-I, IGF-I-R and IGF-II-R could be detected by Northern analysis in total RNA. However, PCR analysis revealed signals in 19/28 cell lines for IGF-I, 27/30 for ICE-II, 19/28 for IGF-I-R and 22/28 glioma cell lines for IGF-II-R. Additional IGF-I, IGF-II, IGF-I-R and IGF-II-R PCR products were detected which might represent alternative splicing products or variants. In addition, the secretion of IGF-I and IGF-II peptides was measured by radioimmunoassay. IGF receptor status and binding characteristics were established by Scatchard analysis. Proliferation assays showed different effects of IGFs and IGF analogues on the proliferation of these cell lines. Des-(1-3)IGF-I showed an unexpected inhibitory activity on glioma cell proliferation. This may have either been due to a direct effect of the ligand for the induction of a more differentiated state refractory to its action.

The distribution of the deleted in colon cancer (DCC) protein in human tissues.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins and is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticuloendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithelium, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer.

Mutations in the p53 gene are not limited to classic 'hot spots' and are not predictive of p53 protein expression in high-grade non-Hodgkin's lymphoma.

We have investigated the relationship between the p53 genotype and phenotype in a series of 22 high-grade non-Hodgkin's lymphomas (NHLs) in which we sequenced the p53 gene open reading frame (exons 2-11). Immunostaining for p53 was already available for these cases. Mutations were found in 10/22 cases (45%) and 3/10 were in exons 4 or 10 outside the classic 'hot spot' regions (exons 5-8). Comparison with immunostaining indicated that, besides cases with the 'expected' patterns (in which gene mutation and protein detection were either both present or both absent) there were also cases in which p53 protein was detected in the absence of any mutation and those with a mutant gene in which the protein was undetectable. These data show that: (1) in high-grade NHLs mutations frequently occur outside the classic hot spot regions and (2) staining for p53 is not predictive of the status of the gene, i.e. whether or not a mutation is present. Therefore in order to document p53 involvement in lymphoid tumours it is necessary both to sequence at least the whole translated open reading frame of the gene and to show evidence of protein expression by immunostaining.

Antibody for detecting p53 protein by immunohistochemistry in normal tissues.

AIMS: To establish whether PAb248 recognises human p53 as well as murine p53 and if so, to determine its distribution in normal tissues. METHODS: The ability of PAb248 to recognise human p53 was established by analysis of the human osteosarcoma derived Saos-2 cell line, which lacks the p53 gene, before and after transfection with p53 cDNA, using western blotting and immunoprecipitation. Immunostaining on normal tissues and cell lines was carried out using an immunoperoxidase technique. The two anti-p53 antibodies PAb 240 and DO-7 were used as controls. RESULTS: The anti-p53 PAb248 monoclonal antibody stained the Saos-2 cell line after, but not before, transfection with p53 cDNA. Both western blots and immunoprecipitations performed with this antibody revealed a 53,000 molecular weight band. With immunostaining, this antibody detects p53 protein in most lymphoid and human epithelial cells in a cytoplasmic-perinuclear localisation that has not been described before. In the same tissues nuclear staining could be seen in a few scattered cells using the PAb240 antibody. The topographical distribution of wild type p53 was not related to proliferating areas but, rather, to short-lived populations of cells. CONCLUSIONS: Immunostaining of wild type p53 is demonstrable not only in its nuclear form using antibody PAb240 but also in it common cytoplasmic-perinuclear localisation in normal tissues using the PAb248 monoclonal antibody. This opens up new possibilities for its study in both physiological and pathological conditions.

Studies on relationships between metastatic and non-metastatic tumor cell populations using lineages labeled with dominant selectable genetic markers.

The relationships between metastatic and non-metastatic cell populations co-existing in composite neoplasms have been studied using cell lineages marked with a dominant selectable marker (neomycin resistance), by transfection. The experimental circumstances were arranged so that the lineages were known to be genotypically distinct (i.e. not merely phenotypic variants of the same lineage) and so that a single metastatic clone was each time combined with a mixed polyclonal non-metastatic population and both partners were distinctly and recognizably marked. This made it possible to ascertain the fates of clones with different metastatic capabilities during tumor progression and metastasis and evaluate their relative contributions to the clinical extent of disease. It was found that metastatic and non-metastatic cell lineages co-existed in most of the late-stage primary tumors examined and that a cell lineage that is invariably non-metastatic, when growing on its own, can with surprising frequency be found thriving in distant metastatic deposits, when it grows to form a primary tumor in combination with a metastatic partner. In fact, occasional metastases from such tumors contained no detectable cells of the metastatic lineage. The endowment of a tumor cell lineage with a new, clinically significant, capability which it convincingly and reproducibly did not manifest before, by another coexisting cell population raises several new questions about the contribution of such phenomena to the overall debilitating properties of the neoplasm and the geometric progression of its impact on the host.