Comparison of array comparative genomic hybridization (aCGH) to FISH and cytogenetics in prognostic evaluation of chronic lymphocytic leukemia.

INTRODUCTION: High-resolution array comparative genomic hybridization (aCGH) is a method of evaluating chromosomal alterations over the entire genome. We compared aCGH with routine cytogenetics and FISH in detecting genetic alterations in chronic lymphocytic leukemia (CLL). METHODS: Array comparative genomic hybridization testing was performed on 55 cases of CLL in addition to a standard panel of FISH probes (ATM on 11q22, trisomy 12, 13q14, p53 on 17p13). The frequency of detecting abnormalities was compared, and discordant results between methodologies were compared. RESULTS: Fifty-five CLL cases [male to female ratio of 2.2:1 and a mean age of 71 (52-90)] were analyzed by both aCGH and FISH. This group of CLL cases showed genetic abnormalities by FISH (60%; 27/45). In contrast to FISH, aCGH detected genetic abnormalities in 82% (45/55) of CLL cases; aCGH identified genetic abnormalities not detected by FISH studies in 16% (7/45) of cases, whereas FISH identified abnormalities not detected by aCGH in only 7% (3/45) of cases. Rare recurring genetic alterations were detected by aCGH including losses in 6q, 8p, 10q, 14q32, and 18q and gains in 10q. DISCUSSION: Our findings suggest aCGH is an effective technique for evaluating recurring genetic abnormalities in CLL and improves on standard FISH in detecting genetic abnormalities in CLL.

KIT activation is a ubiquitous feature of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract, and they are generally resistant to chemotherapy and radiation therapy. Most GISTs express the KIT receptor tyrosine kinase protein, and a subset of GISTs contain activating mutations within the KIT juxtamembrane region. We evaluated 48 GISTs, including 10 benign, 10 borderline, and 28 malignant cases, to determine whether KIT expression and activation are general properties of these tumors. Immunohistochemical KIT expression was demonstrated in each case. Somatic KIT mutations were found in 44 tumors (92%), of which 34 (71%) had juxtamembrane region mutations. Other GISTs had KIT mutations in the extracellular region (n = 6) and in two different regions in the tyrosine kinase domain (n = 4). Contrary to previous reports, KIT mutations were not identified preferentially in higher-grade tumors: indeed, they were found in each of 10 histologically benign GISTs. Notably, mutations in all KIT domains were associated with high-level KIT activation/phosphorylation, and KIT activation was also demonstrated in the four GISTs that lacked detectable KIT genomic and cDNA mutations. These studies underscore the role of KIT activation in GIST pathogenesis, and they suggest that activated KIT might represent a universal therapeutic target in GISTs.

PLAG1 alterations in lipoblastoma: involvement in varied mesenchymal cell types and evidence for alternative oncogenic mechanisms.

Lipoblastomas are rare soft tissue tumors that occur primarily in young children. They typically contain variably differentiated adipocytes, primitive mesenchymal cells, myxoid matrix, and fibrous trabeculae. Abnormalities in chromosome 8, leading to rearrangements of the PLAG1 gene, were demonstrated recently in four lipoblastomas. In the present report, we determine the frequency of PLAG1 alterations in 16 lipoblastomas from children aged 13 years or younger, and we also evaluate the stages of lipoblastoma differentiation at which PLAG1 genomic alterations are found. Eleven lipoblastomas (69%), including those with either classic or lipoma-like histology, had rearrangements of the 8q12 PLAG1 region. Another three lipoblastomas had polysomy for chromosome 8 in the absence of PLAG1 rearrangement. Only two cases (13%) lacked a chromosome 8 abnormality. Notably, the lipoblastomas with chromosome 8 polysomy had up to five copies of chromosome 8 as an isolated cytogenetic finding in an otherwise diploid cell. We also demonstrate that PLAG1 alterations are found in a spectrum of mesenchymal cell types in lipoblastomas, including lipoblasts, mature adipocytes, primitive mesenchymal cells, and fibroblast-like cells. This finding is consistent with neoplastic origin in a primitive mesenchymal precursor and with variable differentiation to a mature adipocyte end-point. Hence, our studies provide biological validation for the clinical observation that lipoblastomas can evolve into mature, lipoma-like, lesions. They also suggest that PLAG1 dosage alterations caused by polysomy 8 might represent an alternative oncogenic mechanism in lipoblastoma.

PLAG1 fusion oncogenes in lipoblastoma.

Lipoblastomas are pediatric neoplasms resulting from transformation of adipocytes. These benign tumors are typically composed of adipose cells in different stages of maturation within a variably myxoid matrix, and they contain clonal rearrangements of chromosome band 8q12. Because lipoblastomas resemble embryonic adipose tissue, characterization of their transforming mechanisms might reveal biological pathways in physiological adipogenesis. Herein, we demonstrate that lipoblastoma chromosome 8q12 rearrangements bring about promoter-swapping events in the PLAG1 oncqgene. We show that the hyaluronic acid synthase 2 (HAS2) or collagen 1 alpha 2 (COL1A2) gene promoter regions are fused to the entire PLAG1 coding sequence in each of four lipoblastomas. PLAG1 is a developmentally regulated zinc finger gene whose tumorigenic function has been shown previously only in epithelial salivary gland cells. Our findings reveal that PLAG1 activation, presumably resulting from transcriptional up-regulation, is a central oncogenic event in lipoblastoma.

TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumors (IMTs) are neoplastic mesenchymal proliferations featuring an inflammatory infiltrate composed primarily of lymphocytes and plasma cells. The myofibroblastic cells in some IMTs contain chromosomal rearrangements involving the ALK receptor tyrosine-kinase locus region (chromosome band 2p23). ALK-which is normally restricted in its expression to neural tissues-is expressed strikingly in the IMT cells with 2p23 rearrangements. We now report a recurrent oncogenic mechanism, in IMTs, in which tropomyosin (TPM) N-terminal coiled-coil domains are fused to the ALK C-terminal kinase domain. We have cloned two ALK fusion genes, TPM4-ALK and TPM3-ALK, which encode approximately 95-kd fusion oncoproteins characterized by constitutive kinase activity and tyrosylphosphorylation. Immunohistochemical and molecular correlations, in other IMTs, implicate non-TPM ALK oncoproteins that are predominantly cytoplasmic or pre- dominantly nuclear, presumably depending on the subcellular localization of the ALK fusion partner. Notably, a TPM3-ALK oncogene was reported recently in anaplastic lymphoma, and TPM3-ALK is thereby the first known fusion oncogene that transforms, in vivo, both mesenchymal and lymphoid human cell lineages.

Replication timing of CD4 and CD8 in single-positive peripheral blood lymphocytes.

The regulatory events leading to the mutually exclusive expression of CD4 and CD8 on peripheral lymphocytes are not fully understood. In particular, the association between DNA replication timing and transcriptional activity of these genes has not been previously investigated. Here, the replication kinetics of the CD4 and CD8 loci in mature single-positive T-cell populations have been examined using a novel approach to the separation of CD4(+) or CD8(+) lymphocytes into discrete cell cycle fractions and a competitive PCR replication timing assay. While the timing of replication of each of these loci is independent of their expression in mature CD4 or CD8 single positive T-cells, the replication of CD8, but not of CD4, shifts to a later time in S phase in transcriptionally silent HS68 fibroblast cells. These findings suggest that changes in DNA replication timing are associated with the developmentally regulated but not with the tissue-specific expression of CD4 and CD8.

The herpes simplex virus type 1 regulatory protein ICP27 coimmunoprecipitates with anti-Sm antiserum, and the C terminus appears to be required for this interaction.

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP27 is required for the inhibition of host cell splicing during viral infection and for the reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs). To determine what effect ICP27 had on splicing proteins that might cause their redistribution, we looked at proteins that were immunoprecipitated with anti-Sm antisera. No significant changes were seen in the migration or amounts of several snRNP common and snRNP-specific proteins from infected cells labeled with [35S]methionine, suggesting that the synthesis of these proteins was not altered by viral infection. However, when cells were labeled with 32Pi, differences were seen in the phosphorylation of at least two proteins depending on whether ICP27 was expressed. One protein, which had an apparent molecular mass of about 85 kDa, was highly phosphorylated during wild-type HSV-1 infection but much less so during infection with an ICP27 null mutant. The other protein, which migrated at the position of the U1 70-kDa protein and was precipitated with U1-specific antiserum, was also more highly phosphorylated when ICP27 was expressed during infection. Furthermore, a phosphoprotein with an apparent molecular mass of 63 kDa was found to coimmunoprecipitate with anti-Sm antisera during wild-type HSV-1 infection. ICP27 has an apparent molecular mass of 63 kDa, and immunoblot analysis confirmed that ICP27 coimmunoprecipitated with snRNPs. Analysis of mutations throughout the ICP27 protein demonstrated that the region that was required for this interaction was the C terminus of the protein, which includes a cysteine-histidine-rich region that resembles a zinc-finger-like motif. These data suggest that ICP27 interacts with snRNPs during infection and that it fosters changes in the phosphorylation state of at least two proteins that immunoprecipitate with snRNPs, although these studies do not demonstrate whether it does so directly or indirectly.

The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.

Herpes simplex virus type 1 infection results in a reorganization of antigens associated with the small nuclear ribonucleoprotein particles (snRNPs), resulting in the formation of prominent clusters near the nuclear periphery. In this study, we show that the immediate-early protein ICP27, which is involved in the impairment of host cell splicing and in the changes in the distribution of snRNPs, is also required for reassorting the SR domain splicing factor SC35. Other viral processes, such as adsorption and penetration, shutoff of host protein synthesis, early and late gene expression, and DNA replication, do not appear to play a role in changing the staining pattern of splicing antigens. Furthermore, the C-terminal repressor region of ICP27, which is required for the inhibitory effects on splicing, also is involved in redistributing the snRNPs and SC35. During infection or transfection with five different repressor mutants, the speckled staining pattern characteristic of uninfected cells was seen and the level of a spliced target mRNA was not reduced. Infections in the presence of activator mutants showed a redistributed snRNP pattern and a decreased accumulation of spliced target mRNA. Moreover, two arginine-rich regions in the N-terminal half of ICP27 were not required for the redistribution of snRNPs or SC35. Substitution of these regions with a lysine-rich sequence from simian virus 40 large-T antigen resulted in a redistribution of splicing antigens. Unexpectedly, a repressor mutant with a ts phenotype showed a redistributed staining pattern like that seen with wild-type infected cells. During infections with this ts mutant, splicing was not inhibited, as shown in this and previous studies, confirming its repressor phenotype. Furthermore, both the mutant and the wild-type protein colocalized with snRNPs. Therefore, the redistribution of snRNPs and SC35 correlates with ICP27-mediated impairment of host cell splicing, but these alterations are not sufficient to fully inhibit splicing. This indicates that active splicing complexes are still present even after dramatic changes in the organization of the snRNPs.

Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.