Mycoplasma infection induces a scleroderma-like centrosome autoantibody response in mice.

Development of autoantibodies to intracellular molecules is a universal feature of autoimmune diseases and parallels onset of chronic inflammatory pathology. Initiating antigens of disease-specific autoantibody responses are unknown. We previously showed that the major targets of autoantibodies in scleroderma are centrosomes, organelles involved in mitotic spindle organization. Here we show that centrosome autoantibodies are induced in mice by mycoplasma infection. The centrosome-specific antibody response involves class switching of preexisting IgM to IgG isotypes, suggesting a T cell-dependent mechanism. The antibody response spreads to include additional intracellular targets, with newly recruited autoantibody specificities arising as IgM isotypes. Antibiotic treatment of mice prevents autoantibody development. Centrosome autoantibodies may provide an aetiological link between infection and human autoimmunity and suggest novel therapeutic strategies in these disorders.

Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia.

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.

BARCODE-ALL: accelerated and cost-effective genetic risk stratification in acute leukemia using spectrally addressable liquid bead microarrays.

An increasing number of risk-stratifying genetic lesions in acute leukemia are being discovered and characterized. To translate this important and increasing volume of information from the research laboratory into effective clinical care, however, new, fast and comprehensive assays are needed. Toward this end, we have developed a two-stage multiplexing assay of broad applicability, which combines multiplex polymerase chain reaction with multiplex detection on spectrally addressable liquid bead microarrays. Using pediatric lymphoblastic leukemia as a model system, we demonstrate that all seven of the fusion transcripts resulting from risk-stratifying chromosomal translocations can be assayed in a single well of a 96-well multiplate with 100% specificity and sensitivity, within 6 h of specimen collection. The assay is automatic and high throughput and represents a significant improvement over previously available assays targeting the same genetic changes. We conclude that user-defined assays that multiplex both target selection and detection may have broad applicability in the management of hematological malignancies.

P504S: a new molecular marker for the detection of prostate carcinoma.

The ability to diagnose prostate carcinoma would be improved by the detection of a tumor-associated antigen. P504S, a cytoplasmic protein, was recently identified by cDNA library subtraction in conjunction with high throughput microarray screening from prostate carcinoma. The aim of this study was to establish the pattern of expression of P504S in prostate carcinoma and benign prostatic tissue. A total of 207 cases, including 137 cases of prostate carcinoma and 70 cases of benign prostate, from prostatectomies (n = 77), prostate needle biopsies (n = 112), and transurethral prostate resections (n = 18) were examined by immunocytochemistry for P504S. P504S showed strong cytoplasmic granular staining in 100% of prostate carcinomas regardless of Gleason scores and diffuse (>75% of tumor) staining in 92% of cases. In contrast, 171 of 194 (88%) of benign prostates, including 56 of 67 (84%) benign prostate cases and 115 of 127 (91%) cases of benign glands adjacent to cancers were negative for P504S. The remainders of benign prostates were focally and weakly positive for P504S. The staining pattern of these normal glands was different and easily distinguishable from that observed in prostate carcinoma. Expression of P504S was not found in basal cell hyperplasia, urothelial cells/metaplasia and small atrophic glands that may mimic prostate carcinoma. Our findings indicate that P504S is a highly sensitive and specific positive marker for prostate carcinoma.

Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression.

Factors that determine the biological and clinical behavior of prostate cancer are largely unknown. Prostate tumor progression is characterized by changes in cellular architecture, glandular organization, and genomic composition. These features are reflected in the Gleason grade of the tumor and in the development of aneuploidy. Cellular architecture and genomic stability are controlled in part by centrosomes, organelles that organize microtubule arrays including mitotic spindles. Here we demonstrate that centrosomes are structurally and numerically abnormal in the majority of prostate carcinomas. Centrosome abnormalities increase with increasing Gleason grade and with increasing levels of genomic instability. Selective induction of centrosome abnormalities by elevating levels of the centrosome protein pericentrin in prostate epithelial cell lines reproduces many of the phenotypic characteristics of high-grade prostate carcinoma. Cells that transiently or permanently express pericentrin exhibit severe centrosome and spindle defects, cellular disorganization, genomic instability, and enhanced growth in soft agar. On the basis of these observations, we propose a model in which centrosome dysfunction contributes to the progressive loss of cellular and glandular architecture and increasing genomic instability that accompany prostate cancer progression, dissemination, and lethality.

Expression of translation initiation factors elF-4E and elF-2alpha and a potential physiologic role of continuous protein synthesis in human platelets.

It is generally believed that platelets do not have a functionally significant protein synthetic machinery. However, our analysis demonstrated that normal bone marrow megakaryocytes express high levels of translation initiation factors eIF-4E and eIF-2alpha and the expression of these protein synthesis initiation factors is continued in platelets (as determined by immunohistochemistry and Western blot analysis). Both eIF-4E and eIF-2alpha are key regulators of protein synthesis. The eIF-4E is a rate-limiting part of a multisubunit complex, eIF-4F, that binds to the 5' cap structure present in virtually all eukaryotic mRNAs, and carries out transfer of mRNAs to ribosomes for translation. Translation initiation factor eIF-2alpha is also a rate-limiting protein which associates with two other proteins to form an eIF-2 initiation factor complex responsible for the transfer of initiator methionyl-tRNA to the 40S ribosomal subunit. We confirm that expression of eIF-4E and eIF-2alpha is biologically relevant in that platelets continue protein synthesis, albeit at a 16 times lower rate than WBC (as determined by 35S-labeled amino acid incorporation, SDS-PAGE and scintillation counting). Finally, we determined that protein synthesis inhibitors (puromycin and emetine) attenuate the platelet aggregation response to a combination of ADP and epinephrine, but potentiate the response to collagen. Our data are consistent with the existence of different signal transducing pathways mediating the response to ADP/epinephrine and collagen. We suggest that the ADP/epinephrine response is positively affected by continuously synthesized proteins, while the response to collagen is modulated by continuously produced inhibitory proteins. Taken together, our results suggest that continuous protein synthesis is important for platelet function and its role in platelet physiology and pathophysiology deserves further study.

The mitotic machinery as a source of genetic instability in cancer.

Development and growth of all organisms involves the faithful reproduction of cells and requires that the genome be accurately replicated and equally partitioned between two cellular progeny. In human cells, faithful segregation of the genome is accomplished by an elaborate macromolecular machine, the mitotic spindle. It is not difficult to envision how defects in components of this complex machine molecules that control its organization and function and regulators that temporally couple spindle operation to other cell cycle events could lead to chromosome missegregation. Recent evidence indicates that the persistent missegregation of chromosomes result in gains and losses of chromosomes and may be an important cause of aneuploidy. This form of chromosome instability may contribute to tumor development and progression by facilitating loss of heterozygocity (LOH) and the phenotypic expression of mutated tumor suppressor genes, and by favoring polysomy of chromosomes that harbor oncogenes. In this review, we will discuss mitotic defects that cause chromosome missegregation, examine components and regulatory mechanisms of the mitotic machine implicated in cancer, and explore mechanisms by which chromosome missegregation could lead to cancer.

Expression of the eukaryotic translation initiation factors 4E and 2alpha in non-Hodgkin's lymphomas.

Transition of cells from quiescence to proliferation requires an increase in the rate of protein synthesis, which is regulated in part by two key translation initiation factors, 4E and 2alpha. The expression and activity of both factors are increased transiently when normal resting cells are stimulated to proliferate. They are constitutively elevated in oncogene transformed cultured cells, and overexpression of either initiation factor in rodent cells makes them tumorigenic. In this study we investigate an association between the expression of translation initiation factors and lymphomagenesis. We have analyzed the expression of the protein synthesis initiation factors 4E and 2alpha by immunohistochemistry in reactive lymph nodes and several types of non-Hodgkin's lymphoma representing a wide range of clinical behaviors based on the Revised European-American Lymphoma behavioral classification. The study included 7 benign lymph nodes with follicular hyperplasia, 26 indolent lymphomas (6 marginal zone lymphomas, 7 small lymphocytic lymphomas, and 13 follicular lymphomas, grades 1 and 2), 16 moderately aggressive lymphomas (8 mantle cell lymphomas and 8 follicular lymphomas, grade 3), 24 aggressive lymphomas (14 large-B-cell lymphomas and 10 anaplastic large-cell lymphomas), and 15 highly aggressive lymphomas (7 lymphoblastic lymphomas and 8 Burkitt's lymphomas). Strong expression of initiation factors 4E and 2alpha was demonstrated in the germinal centers of reactive follicles. Minimal or no expression was seen in the mantle zones and surrounding paracortices, indicating that high expression of initiation factors 4E and 2alpha is associated with the active proliferation of lymphocytes. Most cases of aggressive and highly aggressive lymphomas showed strong expression of initiation factors 4E and 2alpha, in contrast to the cases of indolent and moderately aggressive lymphoma, in which their expression was intermediate between the germinal centers and the mantles of reactive follicles. A positive correlation was found between the expression of both initiation factors 4E and 2alpha and the Revised European-American Lymphoma behavior classification (P < 0.05). Thus, constitutively increased expression of initiation factors 4E and 2alpha may play an important role in the development of lymphomas and is correlated with their biological aggressiveness.

Centrosome defects and genetic instability in malignant tumors.

Genetic instability is a common feature of many human cancers. This condition is frequently characterized by an abnormal number of chromosomes, although little is known about the mechanism that generates this altered genetic state. One possibility is that chromosomes are missegregated during mitosis due to the assembly of dysfunctional mitotic spindles. Because centrosomes are involved in spindle assembly, they could contribute to chromosome missegregation through the organization of aberrant spindles. As an initial test of this idea, we examined malignant tumors for centrosome abnormalities using antibodies to the centrosome protein pericentrin. We found that centrosomes in nearly all tumors and tumor-derived cell lines were atypical in shape, size, and composition and were often present in multiple copies. In addition, virtually all pericentrin-staining structures in tumor cells nucleated microtubules, and they participated in formation of disorganized mitotic spindles, upon which chromosomes were missegregated. All tumor cell lines had both centrosome defects and abnormal chromosome numbers, whereas neither was observed in nontumor cells. These results indicate that centrosome defects are a common feature of malignant tumors and suggest that they may contribute to genetic instability in cancer.

Intracranial leiomyosarcoma: a neuro-oncological consequence of acquired immunodeficiency syndrome.

Leiomyosarcoma has recently been identified as an AIDS-related tumor, usually occurring in children in the chest or abdomen. We describe a case of intracranial leiomyosarcoma in an adult with AIDS. An enhancing mass adjacent to the right occipital cortical surface was diagnosed during a work-up for left arm weakness. Imaging characteristics were suggestive of a malignant neoplasm versus meningioma. The patient had a craniotomy for total excision of the lesion. Pathology showed a leiomyosarcoma. Metastatic work-up was negative. The patient refused radiation therapy, but is well without evidence of recurrence at 8 months follow-up. The differential diagnosis for intracranial lesions in AIDS should be expanded to include leiomyosarcoma. Surgical resection of the lesion is recommended.

Selective outgrowth of a posttransplant B-immunoblastic lymphoma expressing a latent membrane protein-1 deletion variant.

BACKGROUND: Posttransplant lymphoproliferative disorders are generally associated with Epstein-Barr virus (EBV) and are of B cell origin. We report the case of a B-immunoblastic lymphoma that developed in a pretransplantation EBV-seronegative woman 4 months after kidney transplant from her HLA-haploidentical brother. The patient successfully underwent immunotoxin therapy for lymphoma and has been in remission for 36 months. METHODS: Latent EBV genomes were identified by polymerase chain reaction, and the purified amplification products were directly sequenced with [35S]dATP. RESULTS: Molecular analysis of the latent membrane protein (LMP)1 oncogene of EBV, which was expressed in most tumor cells, revealed a 30-base pair deletion. No wild-type LMP1 sequences were found. Analysis of peripheral blood mononuclear cells from the EBV-seropositive donor showed the presence of both the LMP1 deletion variant and the wild-type sequence. The LMP1 deletion variant and the wild-type sequence were also identified within peripheral blood mononuclear cells of the EBV-seroconverted kidney recipient 20 months after lymphoma therapy. CONCLUSION: This pattern is consistent with a natural growth advantage of B cells expressing the LMP1 deletion variant in the immunocompromised host.

Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells.

Granulocyte colony-stimulating factor (G-CSF) has been used to improve granulocyte count in chronic neutropenia and myelodysplasia, to minimize the incidence and duration of neutropenia during conventional chemotherapy, and to mobilize peripheral blood stem cells prior to leukapheresis for use in autologous and allogeneic marrow transplantation. The most common toxicity is bone pain, and other reactions such as inflammation at the site of injection have also occurred. In patients with chronic neutropenia, splenomegaly has been described with long-term use, and extramedullary hematopoiesis has also been reported. However, thus far, no life-threatening sequelae of these effects are found in the literature. We now describe a case of spontaneous splenic rupture four days following a six-day course of G-CSF therapy in an allogeneic donor of peripheral blood stem cells.

Deletion variants within the NF-kappa B activation domain of the LMP1 oncogene prevail in acquired immunodeficiency syndrome-related large cell lymphomas and human immunodeficiency virus-negative atypical lymphoproliferations.

This sequencing study of 17 acquired immunodeficiency syndrome-related lymphomas (9 primary brain, 8 systemic) and 8 human immunodeficiency virus-negative atypical lymphoproliferations expressing large amounts of the latent membrane protein 1 (LMP1) of Epstein-Barr virus was performed to characterize the carboxy terminal NF-kappa B activation domain of LMP1 at the molecular level in an immunocompromised host. In-frame deletions within the NF-kappa B activation domain were identified in all but 2 primary brain lymphomas, 4 systemic lymphomas, and 4 atypical lymphoproliferations, ie, in 60% of cases. In addition, non silent point mutations (range 1 to 5, mean 3.3) were detected in all cases. Although all changes occurred within the first 100 nucleotides of the carboxy terminal NF-kappa B activation domain--a critical sequence for the protein half-life--not a single point mutation was found in the remaining 62 nucleotides, necessary for malignant transformation. Such a clustering of nonrandom sequence variations, associated with a high oncoprotein expression in immunocompromised hosts, suggests that this part of the LMP1 oncogene behaves as a hypervariable region with natural selection of growth-promoting variants through prolongation of the protein half-life.

Leukemia with megakaryocytic differentiation following essential thrombocythemia and myelofibrosis. Case report and review of the literature.

Leukemias of megakaryocytic lineage are rare and heterogeneous clinical entities. The nomenclature published in the literature is confusing and perhaps inappropriate to designate these primary myeloproliferative disorders. We describe a patient with essential thrombocythemia who evolved through myelofibrosis and myeloid metaplasia to a final picture of leukemia with megakaryocytic differentiation in the peripheral blood. This case illustrates different aspects of a chronic myeloproliferative disorder where myelofibrosis and myeloid metaplasia are frequent but secondary events. We have reviewed the literature focusing on the role of clonal megakaryocytic proliferation in myelofibrosis and on the clinical characterization of leukemia with megakaryocytic phenotype. We also present our interpretation of the literature which indicates that a formal review of the nomenclature is urgently needed.

PHA induces IL-2 receptors on B-CLL cells and is a potential biological response modifier for the LIL-2-diphtheria toxin, DAB486IL-2.

DAB486IL-2 is an IL-2-diphtheria toxin conjugate which was developed to be specifically cytotoxic to cells bearing high affinity IL-2 receptors. The high affinity IL-2 receptor is a heterodimer comprising p55 and p75 subunits. While the p75 subunit appears to be ubiquitously expressed among the common North American leukemias and lymphomas, the p55 subunit is more restricted in its expression. To broaden the therapeutic relevance of the DAB486IL-2 we have sought physiologically feasible inducers of the p55 IL-2 receptor subunit. This report describes that PHA, in vitro, induces the p55 IL-2 receptor subunit on initially p55-negative B-CLL cells and converts toxin-insensitive leukemia cells to a toxin-sensitive state.

Parvovirus B19-associated hemophagocytic syndrome.

Parvovirus B19 is a recently described pathogen, associated with an increasing spectrum of clinical manifestations. We present the first reported case, to our knowledge, of parvovirus B19-associated hemophagocytic syndrome, in which the diagnosis of parvovirus infection was documented by the presence of B19-specific IgM and IgG antibodies. Pancytopenia resolved immediately following splenectomy and the patient recovered completely.

Immunophenotypic analysis of cells isolated from bone marrow biopsies in patients with failed bone marrow aspiration ('dry tap').

A method for the immunophenotypic analysis of bone marrow cells in cases of failed bone marrow aspiration is described. Cell suspensions are obtained by mechanical disaggregation of bone marrow core biopsies. The isolated cells are stained with the appropriate antibodies and analyzed by flow cytometry. The usefulness of the method is illustrated by presenting immunophenotypic data obtained in eight consecutive cases accessioned by the authors' laboratory. The method is simple and reproducible. It allows for parallel morphologic examination with Romanovski-type stains and is capable of generating multivariate, quantitative, immunophenotypic data useful in the diagnosis of leukemia and lymphoma.