Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN.

Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN.

Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.

The 2;5 chromosomal translocation occurs in most anaplastic large-cell non-Hodgkin's lymphomas arising from activated T lymphocytes. This rearrangement was shown to fuse the NPM nucleolar phosphoprotein gene on chromosome 5q35 to a previously unidentified protein tyrosine kinase gene, ALK, on chromosome 2p23. In the predicted hybrid protein, the amino terminus of nucleophosmin (NPM) is linked to the catalytic domain of anaplastic lymphoma kinase (ALK). Expressed in the small intestine, testis, and brain but not in normal lymphoid cells, ALK shows greatest sequence similarity to the insulin receptor subfamily of kinases. Unscheduled expression of the truncated ALK may contribute to malignant transformation in these lymphomas.

Lack of correlation of N-myc gene amplification with prognosis in localized neuroblastoma: a Pediatric Oncology Group study.

Multiple copies of N-myc proto-oncogene are only rarely detected in localized neuroblastomas (NBs), and the prognostic relevance of amplification in this subset of patients is not clear. We analyzed a series of 850 children with NB admitted to a Pediatric Oncology Group NB Biology Study and identified six patients with localized NBs harboring N-myc gene amplification. Three patients whose tumors showed favorable histology by Shimada classification and low-risk histological features according to the Joshi classification have remained disease-free, whereas two of three patients with unfavorable histology tumors have developed recurrent disease. Although earlier studies have indicated that N-myc amplification is associated with diploid DNA content, flow cytometric analysis revealed that only two of the localized tumors contained stem lines with diploid DNA content. Loss of chromosome 1p was not detected by fluorescence in situ hybridization in the two tumors examined. N-myc protein was detected by immunohistochemical studies in four of the five NBs analyzed. However, N-myc protein was not visualized in one of the tumors with stroma-rich histology, and Western blot analysis revealed only low levels of N-myc protein expression in another NB with favorable histology. These studies indicate that the presence of N-myc amplification in localized NBs does not necessarily portend an adverse outcome. Furthermore, the biological features of this subset of N-myc-amplified NBs appear to differ from those of more advanced N-myc-amplified tumors.

Extensive genomic abnormalities in childhood medulloblastoma by comparative genomic hybridization.

We analyzed 27 samples of primary medulloblastoma, using comparative genomic hybridization and a novel statistical approach to evaluate chromosomal regions for significant gain or loss of genomic DNA. An array of nonrandom changes was found in most samples. Two discrete regions of high-level DNA amplification of chromosome bands 5p15.3 and 11q22.3 were observed in 3 of 27 tumors. Nonrandom genomic losses were most frequent in regions on chromosomes 10q (41% of samples), 11 (41%), 16q (37%), 17p (37%), and 8p (33%). Regions of DNA gain most often involved chromosomes 17q (48%) and 7 (44%). These findings suggest a greater degree of genomic imbalance in medulloblastoma than has been recognized previously and highlight chromosomal loci likely to contain oncogenes or tumor suppressor genes that may contribute to the molecular pathogenesis of this tumor.

The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1.

A t(3;5)(q25.1;q34) chromosomal translocation associated with myelodysplastic syndrome and acute myeloid leukemia (AML) was found to rearrange part of the nucleophosmin (NPM) gene on chromosome 5 with sequences from a novel gene on chromosome 3. Chimeric transcripts expressed by these cells contain 5' NPM coding sequences fused in-frame to those of the new gene, which we named myelodysplasia/myeloid leukemia factor 1 (MLF1). RNA-based polymerase chain reaction analysis revealed identical NPM-MLF1 mRNA fusions in each of the three t(3;5)-positive cases of AML examined. The predicted MLF1 amino acid sequence lacked homology to previously characterized proteins and did not contain known functional motifs. Normal MLF1 transcripts were expressed in a variety of tissues, most abundantly in testis, ovary, skeletal muscle, heart, kidney and colon. Anti-MLF1 antibodies detected the wild-type 31 kDa protein in K562 and HEL erythroleukemia cell lines, but not in HL-60, U937 or KG-1 myeloid leukemia lines. By contrast, t(3;5)-positive leukemia cells expressed a 54 kDa NPM-MLF1 protein, but not normal MLF1. Immunostaining experiments indicated that MLF1 is normally located in the cytoplasm, whereas NPM-MLF1 is targeted to the nucleus, with highest levels in the nucleolus. The nuclear/nucleolar localization of NPM-MLF1 mirrors that of NPM, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in myeloid leukemia cells.

His-1 and His-2: identification and chromosomal mapping of two commonly rearranged sites of viral integration in a myeloid leukemia.

To identify genes that contribute to myeloid leukemogenesis we have cloned viral integration sites from a CasBrM-MuLV-induced interleukin 3-independent myeloid leukemia cell line. Genomic probes derived from cellular sequences flanking two integrated proviruses were used to screen restriction digests of DNAs from a panel of 52 hematopoietic cell lines, 30 of which were established from CasBrM-MuLV- or MoMuLV-induced mouse leukemias. Probes from one integration site (His-1) defined a region that was rearranged in 3/52 cell lines, and probes from a second integration site (His-2) identified a rearrangement in 2/52 cell lines. Both cases of His-2 rearrangements occurred in concert with viral insertions in the His-1 locus. Genetic mapping of these loci using interspecific backcross analysis assigned the His-1 locus to mouse chromosome 2 and the His-2 locus to mouse chromosome 19. In situ hybridization with a probe from the human homologous region mapped the His-1 locus to human chromosome 2q14-q21. No recombinants were observed between His-2 and Gin-1, a common site of provirus integration in Gross passage A MuLV-induced T-cell leukemias, in 131 backcross animals, suggesting that these loci are tightly linked. The His-1 locus maps to mouse chromosome 2 distinct from any known oncogene or common site of integration but near the proximal breakpoint for a deletion that is observed in over 90% of radiation-induced leukemias.

Clinicopathologic manifestations and breakpoints of the t(3;5) in patients with acute nonlymphocytic leukemia.

Acquired chromosomal rearrangements in acute nonlymphocytic leukemia (ANLL) have been linked to specific clinicopathologic features that suggest new disease subtypes. In this collaborative study, we report five patients with ANLL and a t(3;5) in their leukemic cells. At diagnosis, four of the patients had a t(3;5) as their sole karyotypic anomaly; the remaining patient had additional structural and numerical abnormalities. Careful cytogenetic analysis indicated that the breakpoints of this rearrangement are 3q25.1 and 5q34, in contrast to the various breakpoints reported in earlier studies (3q21----3q25 and 5q31----5q35). The karyotypic, morphologic, and clinical characteristics of this group, as well as those of 14 previously reported patients with the t(3;5), were compared to identify any features that might warrant consideration of a specific syndrome. The available information indicates a worldwide distribution and a nearly equal male:female ratio for patients with this translocation. The median age of the group, 37 years, was younger than that of all patients with ANLL, 49 years. A preceding myelodysplastic syndrome was observed in three patients. The limited numbers of observations on leukocyte count, hemoglobin level, and platelet count precluded meaningful comparison with data for ANLL patients in general. Although each FAB morphologic subtype, except M3, occurred in patients with a t(3;5), the frequency of M6 was much greater than expected. Bone marrows from each of the five patients we report showed increased numbers of megakaryocytes; trilineage dysplasia was observed in the marrow of each of the four patients for whom it could be assessed. Taken together, these findings suggest that the t(3;5) may affect cells capable of differentiation into multiple lineages.

p27KIP1 deletions in childhood acute lymphoblastic leukemia.

The p27KIP1 gene, which encodes a cyclin-dependent kinase (CDK) inhibitor, has been assigned to chromosome band 12p12, a region often affected by cytogenetically apparent deletions or translocations in childhood acute lymphoblastic leukemia (ALL). As described here, fluorescence in situ hybridization (FISH) analysis of 35 primary ALL samples with cytogenetic evidence of 12p abnormalities revealed hemizygous deletions of p27KIP1 in 29 cases. Further analysis of 19 of these cases with two additional gene-specific probes from the 12p region (hematopoietic cell phosphatase, HCP and cyclin D2, CCND2) showed that p27KIP1 is located more proximally on the short arm of chromosome 12 and is deleted more frequently than either HCP or CCND2. Of 16 of these cases with hemizygous deletion of p27KIP1, only eight showed loss of HCP or CCND2, whereas loss of either of the latter two loci was uniformly associated with loss of p27KIP1. Missense mutations or mutations leading to premature termination codons were not detected in the coding sequences of the retained p27KIP1 alleles in any of the 16 ALL cases examined, indicating a lack of homozygous inactivation. By Southern blot analysis, one case of primary T-cell ALL had hemizygous loss of a single p27KIP1 allele and a 34.5-kb deletion, including the second coding exon of the other allele. Despite homozygous inactivation of p27KP1 in this case, our data suggest that haploinsufficiency for p27KIP1 is the primary consequence of 12p chromosomal deletions in childhood ALL. The oncogenic role of reduced, but not absent, levels of p27KIP1 is supported by recent studies in murine models and evidence that this protein not only inhibits the activity of complexes containing CDK2 and cyclin E, but also promotes the assembly and catalytic activity of CDK4 or CDK6 in complexes with cyclin D.

Lack of homozygously inactivated p73 in single-copy MYCN primary neuroblastomas and neuroblastoma cell lines.

We examined 18 neuroblastoma cell lines and 32 primary single-copy MYCN tumor specimens to determine whether mutations of p73, a novel p53-related gene located in chromosome band 1p36.33, contribute to the genesis or progression of childhood neuroblastoma. By fluorescence in situ hybridization, 16 of the 18 cell lines, but only 3 of the 32 primary tumors, had evidence of a deleted p73 allele. Sequence analysis of the p73 coding region in the mRNAs expressed by these cell lines and tumors did not reveal inactivating mutations, suggesting that p73 is not homozygously inactivated in neuroblastoma. However, several novel splice forms of p73 mRNAs were identified, including one without exon 11 that predominated in multiple MYCN-amplified cell lines. Its encoded p73 protein differed from other splice forms in that the C-terminus was derived from an alternative reading frame. Further study of the functional properties of the protein encoded by this splice form of p73 will be needed to determine whether it contributes to the pathogenesis of childhood neuroblastoma with MYCN gene amplification.

Detection of MYCN gene amplification in neuroblastoma by fluorescence in situ hybridization: a pediatric oncology group study.

To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (< or =30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization.

Loss of heterozygosity (LOH) for markers on chromosome arm 16q in Wilms tumor has been linked to an increased risk of treatment failure. We therefore postulated that fluorescence in situ hybridization (FISH) with probes from this region might enhance current strategies for identifying high-risk patients at diagnosis. In a blinded comparative pilot study of 19 Wilms tumor samples from 18 patients with favorable histology, FISH and DNA polymorphism analysis yielded concordant results in 14 cases, either retention (n = 6) or loss (n = 8) of chromosome arm 16q markers. Discordant findings in 4 of the 5 remaining cases resulted from detection of LOH, but no loss by FISH. Two of these cases, directly comparable at marker D16S422, appeared to have tumor-specific uniparental disomy, in that 2 copies of D16S422 and the 16 centromere were evident, despite LOH. In 2 other cases, the discrepancies could be explained by LOH confined to loci distal to the D16S422 locus. In the fifth case, FISH detected 2 distinct populations of tumor cells, one characterized by normal diploidy and the other by monosomy 16, whereas DNA polymorphism analysis failed to indicate LOH altogether. Thus, FISH confirmed the presence of allelic loss (hence, the possible location of biologically important tumor suppressor genes) on the distal long arm of chromosome 16 in cases of favorable-histology Wilms tumor, with the advantages of technical simplicity, successful analysis of samples that were otherwise uninformative by analysis of DNA polymorphisms, and the addition of internal controls for chromosomal aneusomy. We suggest that combined analysis of the chromosome 16q region in Wilms tumor by FISH and DNA polymorphism analysis would improve evaluations to identify high-risk patients who might benefit from alternative therapy.

Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma.

BACKGROUND/PURPOSE: Deletion of the short arm of chromosome 1 (1p) is one of the poor prognostic factors in human neuroblastomas. Recent studies have suggested that one or more of the neuroblastoma tumor suppressor genes reside in this region and have identified the shortest region of overlap (SRO) on 1p36. The purpose of this study was to examine deletions of 1p in human neuroblastomas by fluorescence in situ hybridization (FISH). METHODS: Two-color FISH analysis was performed to detect chromosome 1p36 abnormalities in 42 MYCN-amplified neuroblastomas. Four different probes from the 1p36 region, the E2F2, NPPA, D1S160, and CDC2L1 loci were used for detection of 1p abnormalities. A repeat sequence probe, which is specific for the heterochromatic region of chromosome 1 (pUC1.77), was used as a control. RESULTS: Large deletions of 1p36 were observed in 31 (73.8%) of 42 tumors, whereas the remaining 11 (26.2%) showed no deletion. In these 11 tumors, a translocation of 1p was found in one and a duplication of 1p was detected in another. CONCLUSIONS: A strong correlation between 1p abnormalities and MYCN amplification was found in this study. MYCN-amplified neuroblastomas were found to show large deletions of 1p encompassing the SRO. FISH provided a rapid and reliable method to detect hemizygous deletions of 1p.

Mental health consumer participation on boards and committees: barriers and strategies.

The article provides an overview of factors related to the participation of consumers of mental health services on boards and committees. Six primary barriers to effective consumer participation are addressed: incongruency between stated values and actual practice, tokenism, lack of representativeness, role strain, poor communication and economic factors. Strategies are proposed to increase the potential for effective implementation of consumer participation. The article concludes with a broad statement affirming interdependence and the potential for increasing the effectiveness of boards and committees and exerting a positive influence on the system.

Localization of the gene for uridine monophosphate synthase to human chromosome region 3q13 by in situ hybridization.

The bifunctional enzyme uridine monophosphate (UMP) synthase catalyzes the last two steps in de novo pyrimidine biosynthesis. A genetic deficiency in the activity of this enzyme causes the inherited human disease orotic aciduria. We used a human cDNA probe to localize the gene for UMP synthase to human chromosome region 3q13 by the technique of in situ hybridization.

Reassignment of the human ARH9 RAS-related gene to chromosome 1p13-p21.

The human ARH9 gene (originally rhoC), a member of the RAS gene superfamily, was initially isolated on the basis of cross-hybridization with a RAS-related cDNA from the marine snail Aplysia. The ARH9 gene locus was previously assigned to the telomeric region of chromosome 5q by isotopic chromosomal in situ hybridization and Southern analysis of somatic cell hybrid DNAs; the gene was noted to cosegregate with the CSF1 gene locus in human-rodent somatic cell hybrids carrying partial chromosomes 5, together with other human chromosomes. With the recent reassignment of the human CSF1 locus to chromosome 1, region p13-p21, it seemed important to reexamine the localization of the ARH9 gene, since it segregates 100% concordantly with the CSF1 locus in hybrid cells. Results of our investigation demonstrate that the ARH9 locus is also present in hybrids retaining chromosome 1, but not 5. Using hybrids carrying partial 1p, we mapped the ARH9 locus relative to other 1p loci, localizing the gene to the region 1p13-p31. Fluorescence in situ hybridization to metaphase chromosomes with a genomic ARH9 clone refined the gene's localization to chromosome 1, bands p13-p21.

Deletion of 1p36 in childhood endodermal sinus tumors by two-color fluorescence in situ hybridization: a pediatric oncology group study.

Childhood endodermal sinus tumors (CESTs) are a unique category of germ cell tumors involving the testis and extragonadal region in children less than 4 years of age. Recent studies of CEST have shown recurrent cytogenetic abnormalities involving the short arm of chromosome 1, most commonly, a deletion of distal 1p. Experience with neuroblastomas has shown that cytogenetic analyses may underestimate the frequency of 1p deletion. To determine the frequency of deletion of Ip in CEST and to verify that 1p is, in fact, deleted and not translocated, we analyzed ten tumors by two-color fluorescence in situ hybridization on single-cell suspensions of interphase nuclei by using a cosmid probe from the PITSLRE kinase (p58) locus (previously mapped to 1p36) cohybridized with plasmid probe pUC1.77 (which recognizes the 1q heterochromatic region) to determine the copy number of chromosome 1. Eight of the ten tumors examined showed evidence of deletion of 1p36. Five of the eight tumors exhibited multiple subdones, and all subdones showed deletion of at least one copy of 1p36, indicating that the deletion probably occurred before the development of chromosome 1 aneusomy. We conclude that deletions of the short arm of chromosome 1, specifically 1p36, do occur in CEST and probably occur at a, higher incidence than that found in neuroblastoma Further studies are needed to determine the degree of overlap of the common area of deletion in CEST with that of neuroblastoma and to determine whether 1p deletion in CEST has prognostic significance.

Inactivation of the NF1 gene in human melanoma and neuroblastoma cell lines without impaired regulation of GTP.Ras.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, encodes neurofibromin, a protein whose GTPase-activating function can negatively regulate GTP-Ras by accelerating its conversion to inactive GDP-Ras. In schwannoma cell lines from patients with neurofibromatosis, loss of neurofibromin was previously shown to be associated with impaired regulation of GTP-Ras. Our analysis of other neural crest-derived tumor cell lines has shown that some melanoma and neuroblastoma cell lines established from tumors occurring in patients without neurofibromatosis contain reduced or undetectable levels of neurofibromin, with concomitant genetic abnormalities of the NF1 locus. In contrast to the schwannoma cell lines, GTP-Ras was appropriately regulated in the melanoma and neuroblastoma lines that were deficient in neurofibromin, even when c-H-ras was overexpressed in the lines. These results demonstrate that some neural crest tumors not associated with neurofibromatosis have acquired somatically inactivated NF1 genes and suggest a tumor-suppressor function for neurofibromin that is independent of Ras GTPase activation.

Amplification of the E2F1 transcription factor gene in the HEL erythroleukemia cell line.

The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies, we isolated genomic clones encompassing the human E2F1 gene. We then used fluorescence in situ hybridization to localize E2F1 to human chromosome 20q11, telomeric to the p107 locus, a gene whose product is related to the retinoblastoma gene product (pRb). This finding contrasts with the 1p36 and 6q22 chromosomal locations previously assigned E2F2 and E2F3, two additional members of the E2F family. Although deletions or structural rearrangements of E2F1 were not detected in 14 primary acute leukemia or myelodysplasia samples with structural abnormalities of chromosome 20q11, the gene was amplified and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb.