NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas

The chromosomal aberration t(2:5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1+ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin's disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRdelta-chain gene rearranged (Ddelta2-Ddelta3 and Vdelta1-Jdelta1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRdelta-chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH.     

Intubation dacryocystography in patients with a clinical diagnosis of chronic canaliculitis ("streptothrix")

Magnification dacryocystography in 18 patients with chronic infective canaliculitis showed dilatation and irregularity of the affected lacrimal canaliculi, with prominent filling defects in almost all cases. Filling defects are uncommon in other diseases of the canaliculi and serve to confirm the diagnosis.     

Accumulation of hypointense lesions ("black holes") on T1 spin-echo MRI correlates with disease progression in multiple sclerosis

MRI findings are increasingly used as outcome measures in therapeutic trials in MS. The discrepancy between the extent of the lesions on conventional T2 images and the clinical condition of the patient is one of the problems encountered in such studies. This clinical-radiological paradox prevents the use of MRI data as surrogate markers of disability in MS. A recent pilot study suggested a relationship between hypointense lesions on T1 MRI and disability. To assess in more detail the correlation of changes in hypointense lesion load on T1-weighted spin-echo MR images ("black holes") with changes in disability in MS, we studied 46 patients with clinically definite MS at baseline and after a median follow-up of 40 months. There was a significant correlation between baseline disability and hypointense lesion load (Spearman rank correlation coefficient [SRCC] = 0.46, p = 0.001). In secondary progressive patients, the rate of accumulation of these "black holes" was significantly related to progression rate (SRCC = 0.81, p < 0.0001). We speculate that the appearance of hypointense lesions is the MRI equivalent of a failure of remission. Overall, T1 lesion load measurements correlated better with clinical assessments than T2 lesion load measurements. Quantification of hypointense lesion load on T1-weighted spin-echo MRI helps to resolve the clinical-radiological paradox between disability and MRI and has the potential to be a surrogate marker of disability in MS.     

The morphologic spectrum of non-Hodgkin's lymphomas with BCL1/cyclin D1 gene rearrangements

BCL1/PRAD1 gene rearrangements involving the cyclin D1 gene are a feature of about 70% of centrocytic/mantle-cell lymphomas (CC/MCL) but are identified in only a small proportion of other B-cell non-Hodgkin's lymphomas. Of 37 lymphomas found to have BCL1/cyclin D1 (PRAD1, CCND1) gene rearrangements, 30 fit the morphologic and immunophenotypic criteria for typical CC/MCL. Seven cases with morphologic features atypical for CC/MCL were identified. CD5+ monoclonal B cells were documented in all these cases. Six cases were subsequently stained for cyclin D1 protein, and all showed nuclear positivity. Five cases had variably sized foci of cells with moderately abundant pale cytoplasm resembling parafollicular/monocytoid B cells, marginal zone cells, hairy cells, or even proliferation centers. Transformed-appearing cells were also present in some lymphomas. In one case, striking follicular colonization created a markedly nodular growth pattern mimicking a follicular lymphoma. A sixth case had a marked predominance of small, round lymphocytes at some sites, mimicking a small lymphocytic lymphoma. Five of these six cases also had areas more typical of CC/MCL. The seventh case was a CD5-positive splenic marginal zone-like lymphoma (SMZL) with plasmacytic differentiation and circulating villous lymphocytes consistent with a splenic lymphoma with villous lymphocytes (SLVL). These cases illustrate the morphologic spectrum of small B-cell lymphoid neoplasms that have BCL1/cyclin D1 gene rearrangements and overexpression of cyclin D1. Despite the BCL1 translocation and cyclin D1 overexpression, the splenic lymphoma with plasmacytic differentiation was definitely not a CC/MCL and fit the clinicopathologic entity of SMZL/SLVL. The other six cases are best considered CC/MCL variants based on a combined morphologic, immunophenotypic, and genotypic evaluation. Genotypic or immunophenotypic studies to identify cyclin D1 rearrangements and overexpression, although not pathognomonic, are useful in recognizing these variant CC/MCL cases, which can mimic almost any of the other well-described but more indolent low-grade B-cell lymphomas and leukemias. Some of the variant CC/MCL cases had features in common with the CD5+ cyclin D1+ SMZL/SLVL, suggesting a possible relationship between these two otherwise distinct entities.     

VACTERL with hydrocephalus. A distinct entity with a variable spectrum of multiple congenital anomalies

In this report we describe the experience and follow-up data in 4 patients presenting the "VACTERL-hydrocephaly association". A review of the literature and the present data show that the inheritance pattern of this association is not clear at the present time and that data on the long-term prognosis are scarse.     

Identification of a novel growth-promoting factor with a wide target cell spectrum from various tumor cells as catalase

We have previously reported the purification from human erythrocyte extracts of a novel growth-promoting factor with a wide target cell spectrum. The factor has been identified as catalase. As cell extracts from a variety of tumor cell types exhibited both growth-promoting and catalase activities, the relationship between the two activities was examined using cell extracts from three different cell types, human myeloid cells (U937), human melanoma cells (A375-C6), and human B cells (Daudi). The growth-promoting and catalase activities were well correlated in these cell extracts. The antibody against human catalase absorbed not only catalase activity, but also the growth-promoting activity of extracts from these cell types. Treatment of the cell extracts from these cells with an irreversible catalase inhibitor, aminotriazole, abolished both the catalase and growth-promoting activities. In contrast, glutathione peroxidase (GSH-Px) activity was neither absorbed with the anti-catalase antibody, nor inhibited by aminotriazole. In addition, GSH-Px exhibited growth-promoting activity only in the presence of glutathione (GSH). These results, in conjunction with the effect of aminotriazole on the growth-promoting activity of catalase, suggest that catalase is the major growth-promoting molecule in the cell extracts, and H2O2-decomposing activity is important. Northern blot analysis revealed that these cells contained authentic catalase mRNA, and the mRNA level was compatible with the catalase and growth-promoting activities in the cell extracts. These results suggest that the growth-promoting activity in the tumor cell extracts is due to catalase.     

Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells

The murine proliferin gene family, which has been shown to respond consistently to tumor promoters and other cellular pro-oxidant agents in C3H/10T1/2 cells, was used to monitor responses after treatment of these cell cultures with toxic, pro-oxidant asbestos fibres. Proliferin mRNA levels were increased by amosite, crocidolite or chrysotile asbestos fibres, especially in the presence of fresh serum and at low cell densities. Promotion of morphological transformation was confirmed in two-stage focus formation assays using crocidolite at a fibre density that induced proliferin expression. Asbestos-induced gene expression was inhibited by millimolar levels of N-acetylcysteine (NAC), supporting a linkage between: (i) induced oxidant stress that was sufficient to promote morphological transformation; (ii) induction of proliferin expression. Other anti-oxidant compounds (dithiothreitol and pyrrolidine dithiocarbamate) or enzymes (superoxide dismutase and catalase) did not inhibit induced expression. Non-fibrous powders (titanium dioxide, quartz or silica gel) were also effective inducers of proliferin mRNA accumulation. Latex beads and activated charcoal were effective at higher particle densities, implying that ubiquitous particle-induced surface membrane effects can lead to an NAC-reversible step necessary for proliferin induction. The results showed that asbestos resembled all other promoters of morphological transformation in C3H/10T1/2 cells in that an antioxidant-sensitive induction of the proliferin gene family occurred following treatment.     

Loss of p16/INK4A protein expression in non-Hodgkin's lymphomas is a frequent finding associated with tumor progression

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.     

Differential expression of BCL-6, CD138/syndecan-1, and Epstein-Barr virus-encoded latent membrane protein-1 identifies distinct histogenetic subsets of acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas

This study was aimed at defining the histogenesis of the pathologic spectrum of acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas (AIDS-NHL), including AIDS-related small noncleaved cell lymphoma (AIDS-SNCCL), AIDS-related large noncleaved cell lymphoma (AIDS-LNCCL), AIDS-related large cell immunoblastic lymphoma plasmacytoid (AIDS-IBLP), and AIDS-related primary effusion lymphoma (AIDS-PEL). Forty-six cases of AIDS-NHL were investigated for the expression pattern of BCL-6, a protein specifically expressed by germinal center (GC) B-cells, and CD138/syndecan-1 (syn-1), a marker of post-GC B-cell differentiation. Expression of BCL-6 and syn-1 segregated two major phenotypic patterns among AIDS-NHL: (1) the BCL-6+/syn-1- pattern associated with AIDS-SNCCL and AIDS-LNCCL; (2) the BCL-6-/syn-1+ pattern associated with AIDS-IBLP and AIDS-PEL. Among systemic AIDS-NHL infected by Epstein-Barr virus (EBV), expression of the EBV-encoded latent membrane protein-1 (LMP-1) preferentially associated with the BCL-6-/syn-1+ profile. Analysis of nonneoplastic lymph nodes showed that the two phenotypic patterns detected in AIDS-NHL correspond to physiologic stages of B-cell development, i.e., GC B-cells (BCL-6+/syn-1-) and preterminally differentiated post-GC B-cells (BCL-6-/syn-1+). Thus, BCL-6+/syn-1- AIDS-NHL reflects a GC stage of differentiation, whereas AIDS-NHL which are BCL-6-/syn-1+, and LMP-1+ when infected by EBV, derive from B cells that have entered post-GC plasmacell differentiation. These findings are relevant for the pathogenesis and differential diagnosis of AIDS-NHL.     

Temporal and spatial expression of distinct troponin T genes in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only < 60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.     

Subcutaneous panniculitis-like T-cell lymphoma: further evidence for a distinct neoplasm originating from large granular lymphocytes of T/NK phenotype

We report the case of a 20 year-old caucasian woman who presented a primary subcutaneous panniculitis-like T-cell lymphoma (SPTCL) as an invasive tumor of the chest wall. Herein, the neoplastic cells were found to express a CD3+CD8+ phenotype but also displayed variably the natural killer (NK)-associated antigens CD56 and CD57 as well as granzyme B. On cytological examination, these cells showed a large granular lymphocyte (LGL)-like morphology with presence of azurophilic granules in their cytoplasm. Electron dense and membrane bound granules like those found in cytotoxic T lymphocytes (CTL) were also demonstrated by electron microscopy. Neither rearrangement of the T-cell receptor subunits nor Epstein-Barr virus (EBV) genome was observed at the molecular level. The LGL-like features of the neoplastic cells found in this case and the presence of NK-associated antigens provide additional support to the cytotoxic derivation of most SPTCL.     

Antitumor effect of a weak superlow frequency stochastic magnetic field with 1/f spectrum

Antiblastomic effect of low strength low frequency fluctuating magnetic field with 1/f power spectrum (1/f MF) analogous to spectra of normal biological fluctuations is studied experimentally. For the first time it is demonstrated that stabilization of cell-cell surface interactions as well as suppression of mitotic activity of malignant cells exposed to 1/f MF may result in considerable inhibition of cancer growth in animals. These effects were shown to be absent in weak low frequency magnetic fields with spectra other than 1/f.     

An MHC-compatible allogeneic bone marrow donor with a distinct role of T cell subsets in graft-versus-leukemia effect and lethal graft-versus-host disease

Our previous results in a murine model indicated that the GVL effect against radiation-induced leukemias could be induced in not only MHC-incompatible but also MHC-compatible allogeneic BMT, and that the intensity of the GVL effect induced in MHC-compatible allogeneic BMT varied among different leukemias and the donor/host strain combinations used. With the use of a radiation-induced T cell leukemia which followed the induction of the GVL effect in both MHC-compatible and -incompatible, allogeneic BMT, the role of T cell subsets in the development of the GVL effect and GVHD was studied. The results indicated that Lyt2+ T cells contaminating donor BM were consistently critical for the induction of the GVL effect in MHC-incompatible (B10) and -compatible (B10.BR and AKR) allogeneic BMT of leukemia-bearing C3H mice, but the depletion of L3T4+ T cells had no effect. In contrast, lethal GVHD induced by AKR donor lymph node cells was totally dependent on L3T4+ T cells, but the depletion of Lyt2+ T cells had no effect. On the other hand, both T cell subsets could cause lethal GVHD induced by MHC-incompatible (B10) and -compatible (B10.BR) allogeneic donors. The distinct roles of T cell subsets of AKR donors were confirmed by the preferential induction of the GVL effect with the AKR donor bone marrow mixed with lymph node cells which had been depleted of L3T4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and expression of palmitoyl-protein thioesterase 2 (PPT2), a homolog of lysosomal palmitoyl-protein thioesterase with a distinct substrate specificity

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.     

Expression of a CD3 epsilon transgene in CD3 epsilon(null) mice does not restore CD3 gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.     

Tumors with reduced expression of a cytotoxic T lymphocyte recognized antigen lack immunogenicity but retain sensitivity to lysis by cytotoxic T lymphocytes

A murine solid tumor was transfected to express various levels of an allogeneic major histocompatibility complex class I gene (K216), in order to test the effect of the level of antigen expression on immunogenicity and sensitivity to lysis by cytotoxic T lymphocytes (CTL). The growth rates of clones of tumor cells expressing different levels of the transfected gene were similar in vitro and in nude mice. Although all tumor cells, including cells freshly isolated from growing tumors, were equally sensitive to lysis by specific CTL, only tumor cells expressing the highest level of the K216 antigen stimulated CTL and were rejected by normal mice. In contrast, tumor cells expressing lower levels of antigen failed to immunize for CTL and grew progressively in normal mice, despite retaining expression of the transfected gene and remaining fully sensitive to CTL-mediated lysis; thus, the threshold of antigen needed to stimulate CTL responses was considerably higher than that needed to lyse tumor cells. Reduction of K216 antigen expression from 100-fold to 40-fold above background, impaired significantly the ability of the tumor cells to induce a K216-specific immune response, while tumor cells expressing K216 at levels 2-fold above background were as susceptible to CTL-mediated lysis as tumor cells expressing 50-fold more antigen. The important implication of these findings is that some tumors occurring in nature may not be immunogenic but nevertheless express antigens which are potential targets for immune therapy.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.     

Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.