Co-existent cutaneous cryptococcosis of the forearm and cutaneous alternariosis of the leg in patient with metastatic thymoma.

BACKGROUND: Cryptococcosis and alternariosis are rare opportunistic infections often observed in immunocompromised patients. Because Cryptococcus and Alternaria are ubiquitous fungi found in soil, the presence of fungi in the dermis has to be observed on histological examination to confirm a real cutaneous, invasive, infection. PATIENT: We report the first case of concomitant cutaneous cryptococcosis and cutaneous alternariosis, in an immunocompromised patient treated for a metastatic thymoma. CONCLUSION: This observation underlines the fact that the possible co-existence of several rare infections in immunocompromised patients should take into consideration pathogen identification in order to adapt the therapy to individual patient requirements.

Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.

BCR-ABL and v-abl oncogenes induce distinct patterns of thymic lymphoma involving different lymphocyte subsets.

The human BCR-ABL oncogenes encoded by the Philadelphia chromosome (Ph) affect the pathogenesis of diverse types of leukemia and yet are rarely associated with T-lymphoid leukemia. To determine whether BCR-ABL kinases are inefficient in transforming T lymphocytes, BCR-ABL-expressing retroviruses were injected intrathymically into mice. Thymomas that expressed BCR-ABL kinase developed after a relatively long latent period. In most thymomas, deletion of 3' proviral sequences resulted in loss of tk-neo and occasionally caused expression of kinase-active carboxy-terminally truncated BCR-ABL oncoprotein. In contrast, deletion of 3' proviral sequences was not observed in thymomas induced with Abelson murine leukemia virus (A-MuLV). BCR-ABL viruses induced distinct patterns of disease and involved different thymocyte subsets than A-MuLV and Moloney murine leukemia virus (Mo-MuLV). While Mo-MuLV only induced Thy-1+ thymomas, v-abl- and BCR-ABL-induced thymomas often contained mixed populations of B220+ and Thy-1+ lymphocytes in the same tumor. In most v-abl and BCR-ABL tumors, Thy-1+ lymphoid cells expressed CD8 and a continuum of CD4 ranging from negative to positive. Conversely, Mo-MuLV thymomas contained distinct populations of CD4+ cells that were either CD8+ or CD8-. A-MuLV-transformed T-lymphoid cells did not express the CD3/T-cell receptor complex, while BCR-ABL tumors were CD3+. Thus, BCR-ABL viruses preferentially induce somewhat more differentiated T lymphocytes than are transformed by A-MuLV. Furthermore, rare B220+ lymphocytes may represent preferred v-abl and BCR-ABL transformation targets in the thymus.

A direct demonstration of recombination between an injected virus and endogenous viral sequences, resulting in the generation of mink cell focus-inducing viruses in AKR mice.

We analyzed viral recombination events that occur during the preleukemic period in AKR mice. We tagged a molecular chimera between the nonleukemogenic virus Akv and the leukemogenic mink cell focus-inducing (MCF) virus MCF 247 with an amber suppressor tRNA gene, supF. We injected the supF-tagged chimeric virus that contains all of the genes of MCF 247 except the envelope gene, which in turn is derived from Akv, into newborn AKR mice to evaluate its pathogenic potential. Approximately the same percentage of animals developed leukemia with similar latent periods when injected with either the tagged or nontagged virus. DNA from tumors induced in AKR mice by the tagged chimeric virus was analyzed by Southern blotting with the supF gene as a probe. One set of tumors contained the injected supF-tagged virus. Two kinds of supF-tagged proviruses were found in a second set of tumors. One group of supF-tagged viruses had a restriction map consistent with that of the injected virus, while the other group of proviruses had restriction maps that suggested that the proviruses had acquired an MCF virus-like envelope gene by recombination with endogenous viral sequences. These results demonstrate that injected viruses recombine in vivo with endogenous viral sequences. Furthermore, the progression to leukemia was accelerated in mice that develop tumors containing proviruses with an MCF virus env gene, emphasizing the importance of the role of the MCF virus env gene product in transformation.

EBER-1 expression in thymic carcinoma.

The Epstein-Barr virus-encoded small nuclear RNA, EBER-1, has been shown to be a suitable target for the in situ hybridization detection of EBV in routinely processed tissue specimens. We evaluated the presence of EBV in thymic carcinoma and invasive thymoma using EBER-1 in situ hybridization on formalin-fixed paraffin-embedded tissue sections. EBER-1 expression was demonstrated in a case of lymphoepithelioma-like thymic carcinoma, but was not detectable in other thymic carcinomas including six squamous cell carcinomas, a clear cell carcinoma and seven invasive thymomas. As reported in three previous cases of EBV-associated thymic carcinoma, lymphoepithelioma-like thymic carcinoma was shown to be closely associated with EBV in our series.

Patterns of responsiveness of T cell lines and thymocytes reveal waves of specific activity in the post-natal murine thymus.

Antibody binding of CD3, CD4, or CD8 molecules can induce cytoplasmic calcium mobilization in T lymphocytes, usually interpreted as indicating signal transduction. Using such assays, in a CD4+ CD8+ thymocyte line and its single positive progeny we have identified characteristic patterns of responsiveness that are reproducible in vivo in a subpopulation of newborn 'double positive' thymocytes but virtually absent in adult thymuses. In particular, these cells appear to be high responders to the binding of anti-CD3 F(ab)'2 fragments. We have followed the presence of such highly responsive thymocytes in the perinatal period and the first 15 days of life. Intriguingly, these cells populate the newborn thymus in three distinct waves. Such patterns of responsiveness may define early 'selectable' thymocytes.

Determinants of thymotropism in Kaplan radiation leukemia virus and nucleotide sequence of its envelope region.

Radiation leukemia viruses (RadLVs) are a group of murine leukemia viruses which are induced by radiation and cause T-cell leukemia. Viral clones isolated from the BL/VL3 lymphoid cell line derived from a thymoma show variable tropism and leukemogenic potential. We have constructed chimeric viruses by in vitro recombination between two viruses, a RadLV that is thymotropic and an endogenous ecotropic virus that is nonthymotropic. We show here that, in contrast to thymotropism determinants identified previously, which lie in the long terminal repeat (LTR), it is the envelope region that is responsible for the thymotropism of BL/VL3 RadLV. The nonthymotropic virus which we have rendered thymotropic by transfer of the env region of RadLV in the present study has been shown previously to become thymotropic when the LTR of another thymotropic virus is inserted in its genome. Thus, the LTR and envelope gene may be involved in complementary action to lead to thymotropism.

Radiation leukemia virus (RadLV)-induced leukemogenesis is associated with an increased number and activity of thymic macrophages.

The radiation leukemia virus (RadLV) is a chronic leukemia retrovirus that induces thymic lymphomas in C57BL/6 mice after a latency of 3 to 6 months. During the pre-leukemic (PL) period, the number of thymic macrophages gradually increased up to 100 fold. Of the cells in a RadLV-induced lymphoma, 0.3% were large macrophages packed with infected lymphoma cells. These thymic lymphoma macrophages (TLM) also ingested RadLV-induced lymphoma cells in vitro. Cultured RadLV-induced lymphoma lines could activate and fix C3 fragments through the alternative complement pathway (ACP). C3-bound lymphoma cells elicited an oxidative burst (OB) response in TLM but not in bone-marrow macrophages (BMM). However, IL4 treatment of BMM rendered them capable of responding with an OB following triggering by C3-opsonized cells. Thymic macrophages (TM) responded moderately with OB to C3-opsonized cells and this response was elevated if the TMs were treated by rIL4. The OB reaction of the TLMs could be partially inhibited by anti-LFA-I or anti-MALA-2 antibodies, and was completely inhibited by anti-CR3 antibodies. These results suggest that IL4 can prime macrophages for triggering an OB reaction and that the interaction between C3-opsonized cells and IL4-primed macrophages is mediated primarily through CR3.

Thymic targets for Abelson murine leukemia virus are early gamma/delta T lymphocytes.

Molecular analysis has shown that the majority of Abelson murine leukemia virus (Ab-MuLV)-induced primary thymomas represent transformed gamma/delta thymocytes. Many of these thymomas are of monoclonal origin as judged by provirus integration pattern and contain rearranged genes encoding T-cell receptor (TCR) gamma and delta chains but germ-line immunoglobulin heavy-chain genes. Some of the monoclonal tumors contain multiple rearranged alleles encoding TCR gamma, delta, and beta chains. Further, one Ab-MuLV thymoma cell line contained germ-line-configuration TCR gamma- and delta-chain genes, which became rearranged after in vitro propagation. Clones of this cell line were observed to rearrange these genes after intrathymic passage. Also, some subclones of this cell line underwent rearrangement of their immunoglobulin heavy-chain genes in culture. These observations suggest that the thymic targets for Ab-MuLV transformation are early gamma/delta thymocytes, some of which continue to rearrange their TCR gamma- and delta-chain genes.

Virological events leading to spontaneous AKR thymomas.

The spontaneous leukemias of AKR mice are caused by mink cell focus-forming (MCF) viruses. These viruses are generated by recombination between several endogenous murine retroviruses. The virological events leading to the generation of the leukemogenic agent were investigated by using an oligonucleotide specific for the U3 region of the leukemogenic virus and env-reactive oligonucleotide probes specific for the different classes of endogenous murine leukemia virus. It was shown that (i) the leukemogenic MCF virus is formed by recombination between at least three different endogenous sequences; (ii) the U3 donor for the leukemogenic virus is the inducible xenotropic virus Bxv-1; (iii) all spontaneous tumors contain viruses with duplicated enhancer regions in their long terminal repeats; (iv) enhancer duplication is a somatic event, since Bxv-1 contains only one copy; (v) the first recombinant virus detectable in mass populations of thymocytes by Southern hybridization analysis contains all structural features of the ultimate leukemogenic virus; and (vi) the multiple novel viruses in a given tumor represent progeny of the same unique recombination events. On the basis of these results, an analysis of the virological events leading to AKR thymomas is presented.

T-cell lymphoma lines derived from rat thymomas induced by Moloney murine leukemia virus: phenotypic diversity and its implications.

The phenotype of 27 Moloney murine leukemia virus-induced rat thymic lymphomas and 36 cell lines derived from these tumors was determined by using 18 monoclonal antibodies directed against hematopoietic cell surface determinants. The cell lines and the primary tumors from which they were derived were clonally related as determined by the pattern of provirus integration and the pattern of rearrangement of the T-cell receptor beta and delta and Igh loci. The differentiation phenotype of the primary tumors and the cell lines derived from them were related. The differences observed between the primary tumors and the cell lines could be explained either by the selection of subpopulations of tumor cells during establishment in culture or by the phenotypic instability of the tumor cells. One cell line (LE3Sp) underwent the transition from a CD4+ CD8+ to a CD4+ CD8- phenotype following exposure to interleukin-2 in culture. Both the primary tumors and the cell lines derived from them express a wide range of phenotypes which correspond to multiple stages in T-cell development. This observation suggests that the pleiomorphism of retrovirus-induced lymphomas, which had been suggested previously from the analysis of mouse tumors, is an intrinsic property of the process of oncogenesis and is not due to the transformation of different types of cells by spontaneously arising leukemogenic variants of the inoculated virus. The wide spectrum of phenotypes expressed by these tumors suggests that Moloney murine leukemia virus may infect and transform T cells at various stages of development. Alternatively, the target cells may be immature T-cell precursors which, following transformation, continue to differentiate. A host of early findings, suggesting that the repertoire of target cells is restricted to poorly differentiated hematopoietic progenitors, and the ability of the LE3Sp cell line to differentiate in culture indicate that the latter possibility may be more likely. The data in this report address the extent and mechanism of the phenotypic variability of retrovirus-induced rodent T-cell lymphomas. In addition, they demonstrate the potential usefulness of the T-cell lymphoma lines we have established in studies of oncogenesis and T-cell differentiation.

Western thymomas lack Epstein-Barr virus by Southern blotting analysis and by polymerase chain reaction.

The authors investigated 16 western thymomas, 9 from the United States and 7 from Europe, for the presence of Epstein-Barr virus (EBV) DNA sequences by both Southern blot hybridization analysis and polymerase chain reaction using EBV-specific DNA probes that detect the long internal repeat and terminal repeat regions and the EBNA-1 gene. None of the 16 thymomas contained evidence of the EBV genome, even though we could detect EBV by Southern blotting when EBV DNA represents less than or equal to 1% of the total DNA and by polymerase chain reaction when a single EBV-positive cell is present among 10(5) EBV-negative cells. These results fail to demonstrate EBV genome in western thymomas and stand in contrast to those of McGuire et al (Am J Pathol 1988, 131:385) who previously reported that the EBV genome is present in thymomas occurring in southern Chinese patients. Therefore EBV does not appear to be implicated in the pathogenesis of all thymomas. The presence of EBV in eastern thymomas, regions where EBV is endemic may be due to epidemiologic factors and/or genetic predispositions.

Retrovirus-like particles in human thymomas.

The pathogenesis of thymoma is unclear. In this study retrovirus-like particles in human thymomas were detected by electron microscopy. Forty-two thymomas; 25 without complications and 17 associated with autoimmune disorders such as myasthenia gravis (13), systemic lupus erythematosus (1), polymyositis (1), Sjögren's syndrome (1), and pure red cell anemia (1), were examined. Thymic tissues from 9 infants suffering from congenital heart diseases and 7 hyperplastic thymuses obtained from myasthenic patients served as controls. The retrovirus-like particles were observed in 37.0% of thymomas without complications; 50.0% of thymomas associated with myasthenia gravis and other autoimmune disorders; 62.5% of thymuses associated with myasthenia gravis; and 33.3% of thymuses from infants with heart disease. The envelopes, including the central cores of the retrovirus-like particles, had diameters ranging from 70 to 460 nm, depending on the source of the specimen. The retrovirus-like particles were located in the cytoplasm, vacuoles, vesicles and lumens of the endoplasmic reticula of epithelial and/or plasma cells. Some retrovirus-like particles were seen budding from plasma membranes. These observations suggest that the retrovirus-like particles in thymomas might be an activated form of retrovirus originating in normal thymic tissue.

Unique role for the T cell receptor in retrovirus binding by the C6VL thymoma.

Binding of cognate Radiation leukemia virus (RadLV) by the C6VL/1 thymoma involves a subset of TCR molecules in association with CD4 molecules expressed by that cell line. A CD4- variant of C6VL/1 has now been isolated which also has RadLV binding capacity. Stable expression of the TCR, class I, and CD5 molecules but not Thy1.2 and CD4 molecules has been demonstrated, and the C6VL/1 origin of this cell has been confirmed by Southern blot analysis using probes specific for the TCR beta chain gene. This cell line has maintained binding capacity for RadLV/C6VL prepared as an immunoabsorbent matrix, but unlike the parent C6VL/1 cell line, binds significantly less well to the related RadLV/VL3 isolate. Binding of the variant cell line to RadLV/C6VL can be completely inhibited by anti-clonotypic antibody to the TCR but only weakly by anti-H-2Kb antibody used at the same concentration. These data suggest that the TCR on C6VL/1 can interact with RadLV in the absence of any co-receptor function of CD4 and implicates the TCR as a sufficient receptor for retrovirus.

Increased incidence of thymoma in Chinese myasthenia gravis: possible relationship with Epstein-Barr virus.

Thymectomy was carried out for treatment of myasthenia gravis in 27 unselected Chinese patients and thymoma was found in 13 of them. This 48% incidence of thymomas is two to three times greater than in Japanese and European patients, respectively. The reason for the higher incidence of thymomas observed in Chinese patients may be related to the presence of the Epstein-Barr virus genome in thymoma. Furthermore, all of the thymomas in our patients were lymphoepithelial and histologically resemble nasopharyngeal carcinoma and undifferentiated carcinoma of the salivary gland. Both these tumours are closely linked to the Epstein-Barr virus and in Hong Kong, nasopharyngeal carcinoma is the third commonest cause of death from malignancy. We recommend early thymectomy for patients with myasthenia gravis particularly in geographical areas where there is a high incidence of nasopharyngeal carcinoma and undifferentiated carcinoma of the salivary gland.

Measurement of binding specificity between T cell lymphomas and murine leukemia viruses.

We have previously reported the presence of receptors on radiation leukemia virus (RadLV)-induced thymomas and malignant thymocytes from AKR mice which specifically bind retrovirus produced by these T cell clones. These receptors have been shown to have specificity for virus reminiscent of an immune-specific receptor. Previous studies on T cell lymphoma binding to retroviruses have involved measurement of the interaction of labelled virus with cells using fluorescence-activated cell sorter (FACS) analysis (McGrath et al., J. Virol. (1978) 25, 923; McGrath and Weissman, Cell (1979) 17, 65; Weissman and McGrath, Curr. Top. Microbiol. Immunol. (1982) 98, 103). Here we report development of an assay for measuring lymphoma binding to virus, prepared as an immunoabsorbent adhered to a microtiter plate. Using this assay, we have shown that only T and not B cell lymphomas can bind to T cell-tropic viruses, and some cell lines have greatest specificity for homologous virus. The AKR-derived T cell lymphomas, SL3 and KKT-2, show greater specificity for leukemogenic AKR viruses, than for an AKR xenotropic virus or the recombinant AKR virus, MCF247. The RadLV-induced T cell lines, C6VL/1 and BL/VL3, have been found to bind cross-reactively to several different murine leukemia viruses (MuLVs). RadLV-induced T cell lymphomas do have greater specificity for their cognate retroviruses since free, homologous retrovirus can best block the interaction between cells and virus adhered to the wells of a microtiter plate. Cross-reactive interactions are more easily demonstrated by this assay, probably because low avidity interactions are stabilized as a result of the mode of virus presentation. Binding specificity for retroviral envelope determinants has been demonstrated using a rat anti-retroviral antiserum prepared as an F(ab)1 fragment. This antiserum can inhibit the interaction between the C6VL/1 thymoma and its RadLV virus. Specificity of this antibody for a gp70-like protein was confirmed by NaDodSO4-polyacrylamide gel electrophoresis (PAGE) and by loss of this activity after absorption of antibody on virus. Antibodies specific for RadLV/VL3 gp70 determinants can inhibit the interaction of C6VL/1 with RadLV/VL3 suggesting that cross-reactive binding to heterologous virus is also specific for a gp70 viral env determinant.

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.

The Mlvi-1 locus involved in the induction of rat T-cell lymphomas and the pvt-1/Mis-1 locus are identical.

Mlvi-1 defines a locus of proviral integration in rat thymomas induced by Moloney murine leukemia virus. pvt-1/Mis-1 represents an independently identified locus which becomes rearranged either by chromosomal translocation in murine plasmacytomas or by provirus insertion in retrovirus-induced murine and rat thymic lymphomas. Although it had been claimed that pvt-1/Mis-1 and Mlvi-1 represent two different loci, we present here evidence showing that they are identical. This finding demonstrates the need for rigorous characterization of any newly identified common regions of integration in retrovirus-induced neoplasms.

Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA.

Abelson murine leukemia virus induces oligoclonal pre-B lymphoma in mice. The expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor induction in several mouse strains, and additional genetic events are thought to be required. We postulated that the helper Moloney murine leukemia virus might induce these events, and its potential role as an insertional mutagen was assessed by the search of a common helper provirus integration site in Abelson murine leukemia virus lymphomas. Molecular cloning of cellular sequences adjacent to Moloney proviruses enabled us to identify a cellular region, designated Ahi-1, which was found occupied by the helper proviruses in 16% of Abelson pre-B-cell lymphomas. All proviruses for which the precise integration site within Ahi-1 could be mapped were found to be in the same orientation. Ahi-1 has been mapped to mouse chromosome 10 and represents a new common proviral integration site. These data suggest that the helper virus contributes to the induction of secondary genetic events which may be important for the development of Abelson murine leukemia virus-induced pre-B-cell lymphoma.

Recombination between two integrated proviruses, one of which was inserted near c-myc in a retrovirus-induced rat thymoma: implications for tumor progression.

Of 17 Moloney murine leukemia virus (MoMuLV)-induced rat thymomas, 2 contained rearrangements in c-myc. In one of these tumors the observed rearrangement was not due to the insertion of an intact MoMuLV provirus. The rearranged c-myc DNA fragment from this thymoma was cloned and examined by restriction endonuclease mapping, hybridization to MoMuLV proviral DNA probes, and DNA sequence analysis. These analyses revealed that the c-myc rearrangement in this tumor was due to the presence of a partially duplicated MoMuLV long terminal repeat (LTR) 5' to c-myc exon 1. The orientation of this LTR structure was opposite to the transcriptional orientation of c-myc. The sequences at the 3' flanking side of the LTR structure were derived from a cellular DNA region which maps to the same chromosome as c-myc (chromosome 7), although to a site distant from this proto-oncogene. These findings present evidence for a homologous recombination event occurring between sequences of two proviruses integrated on the same chromosome, one of which was inserted near the c-myc proto-oncogene. The recombination product contains three copies of the MoMuLV LTR 72-base-pair direct repeat and is associated with a high level of c-myc expression. The reciprocal product of this recombination was not detected. We propose that recombination between homologous sequences may play a significant role in the generation of chromosomal rearrangements and therefore in tumor induction and progression.