Bcr Gene Rearrangement Research Articles

Non-neoplastic B-cell predominant lymphoid proliferations at the organs exposed to external environment mimicking lymphoma: A potential diagnostic pitfall.

Background: Typically, lymphatic tissue proliferative lesions include either benign lesions or lymphoma. However, not all lymphatic lesions can currently be accurately classified into one category, particularly in mucosal areas that are in contact with the external environment.Aims: To explore the morphology, immunophenotype, and molecular changes of Non-neoplastic B-cell predominant lymphoid proliferations (NBPLP) in pathological areas that are exposed to external surroundings which mimicked lymphoma.Methods and Results: 18 cases of Atypical lymphoid hyperplasia (AtLP) were retrieved in this study. The biopsy samples were mucosal samples obtained from areas exposed to external surroundings, including intestines, urethra, cervix, tonsils, and tongue. Microscopically, there is a different level of B cell hyperplasia accompanied by morphological atypia. We categorized the morphology into 4 groups: type A (7/18), type B (3/18), type C (3/18), type D (5/18). Part of the AtLP was found positive for BCR gene rearrangement (6/15), and TCR gene rearrangement (1/4). The follow-up period ranged from 14.2 to 70 months. No evidence of lymphoma was found. Therefore, we diagnosed all of the presented cases as NBPLP. We illustrated the key differential points and provided valuable diagnostic experience on each subtype.Conclusions: Areas exposed to the external environment are commonly exposed to antigen and easily present with AtLP of NBPLP, accompanying with positive IGH rearrangement. Therefore, a comprehensive evaluation of macroscopic, morphology, immunophenotype, and molecular diagnostics is required to prevent the overdiagnosis of lymphoma.

Alternative end-joining in BCR gene rearrangements and translocations.

Programmed DNA double-strand breaks (DSBs) occur during antigen receptor gene recombination, namely V(D)J recombination in developing B lymphocytes and class switch recombination (CSR) in mature B cells. Repair of these DSBs by classical end-joining (c-NHEJ) enables the generation of diverse BCR repertoires for efficient humoral immunity. Deletion of or mutation in c-NHEJ genes in mice and humans confer various degrees of primary immune deficiency and predisposition to lymphoid malignancies that often harbor oncogenic chromosomal translocations. In the absence of c-NHEJ, alternative end-joining (A-EJ) catalyzes robust CSR and to a much lesser extent, V(D)J recombination, but the mechanisms of A-EJ are only poorly defined. In this review, we introduce recent advances in the understanding of A-EJ in the context of V(D)J recombination and CSR with emphases on DSB end processing, DNA polymerases and ligases, and discuss the implications of A-EJ to lymphoid development and chromosomal translocations.

CD27-IgD- memory B cells are modulated by in vivo interleukin-6 receptor (IL-6R) blockade in rheumatoid arthritis.

IntroductionEnhanced B cell activity, particularly memory B cells have gained interest in evaluating response during therapies with biologics. CD27-IgD- double-negative (DN) B cells lacking the conventional memory marker CD27 are reported to be part of the memory compartment, however, only scarce data is available for rheumatoid arthritis (RA). We therefore focused on DN B cells in RA, studied their isotypes and modulation during interleukin-6 receptor (IL-6R) inhibition by tocilizumab (TCZ).MethodsDN B cells were phenotypically analyzed from 40 RA patients during TCZ at baseline week 12, week 24 and 1 year. A single B cell polymerase chain reaction (PCR) approach was used to study Ig receptors, VH gene rearrangements and specific isotypes.ResultsPhenotypic analysis showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgG-, IgA- and IgM-expressing cells with a clear dominance of IgG+ cells. DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-α) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (P <0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (P <0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (P <0.05) at week 12 and week 24 during TCZ. Patients with a good European League Against Rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (P = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (P = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05 to 2.06).ConclusionsIn RA, the heterogeneous DN B cell compartment is expanded and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0580-y) contains supplementary material, which is available to authorized users.

Disturbed B and T cell homeostasis and neogenesis in patients with ataxia telangiectasia.

Ataxia telangiectasia (AT) is a rare genetic, multi-system disorder characterized by neurodegeneration, chromosome instability, B and T cell immunodeficiency and a predisposition to cancer. We examined immunologic parameters reflecting cell development and proliferation and their relevancy to the clinical phenotype in affected individuals. AT patients from the AT National Clinic in Israel underwent immunological investigation. Their T and B cell workup included lymphocyte subset counts, immunoglobulin levels, responses to mitogenic stimulations, TCR-Vβ families and BCR immunoglobulin heavy chain spectratyping, TCR rearrangement excision circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Thirty-seven AT patients (median age 12.7years, range 4.2-25.1) were evaluated. CD20 B and CD3 T lymphocytes were decreased in 67% and 64% of the patients, respectively, while only 33% of the patients had reduced lymphoproliferative responses. Almost all AT patients displayed extremely low TRECs and KRECs levels, irrespective of their age. Those levels were correlated to one another and to the amounts of CD3+ and CD20+ cells, respectively. Abnormal TCR-Vβ repertoires were found with different degrees of clonality or reduced expression in these AT patients. There was no clear clustering of expansions to specific TCR-Vβ genes. PCR spectratyping analysis of the FR2 IgH BCR gene rearrangements in peripheral blood was abnormal in 50% of the patients. The immunodeficiency associated with AT is combined, remains low over time and not progressive. It is characterized by low TREC and KREC copies suggestive of abnormal T and B cell neogenesis.

ETV6-ABL1 CML Like Disorder with Complex Translocation Involving Chromosome 9, 12 and 14

AbstractAbstract 4438A rare group of myeloproliferative disorders has been described associated with gene rearrangements producing novel tyrosine kinases other than BCR/ABL.A rearrangement involving the ETV6 and ABL1 genes, associated with t(9;12)(q34;p13) translocation, has been detected in Ph-negative chronic myeloproliferative disorders and has been demonstrated to have tyrosine kinase activity in signal transduction pathways similar to the BCR/ABL fusion protein. The ETV6 gene is a member of the E26 transformation-specific family (ETS) of transcription factors located at 12p13. It has been implicated in the rearrangement of over 48 different chromosome bands, ultimately playing a role in leukemogenesis. Thus far, only eleven BCRABL1-negative CML patients with a variant ABL1 gene (9q34) rearrangement, involving fusion to ETV6 aka TEL (12p13) have been reported. ETV6-ABL1 rearrangement have also been implicated in other hematologic diseases including 6 acute B lymphoid leukemia, 1 acute T lymphoid leukemia, 3 acute myeloid leukemia, 1 myelodysplastic syndrome, and 3 chronic myeloproliferative disorders.We present a 62 year old male who sought medical attention for acute coronary syndrome with bone pain and asthenia in May 2011. Physical exam showed a splenomegaly. He was found to have hemoglobin of 9.9g/dL, WBC 90G/L and platelets 105G/L. The peripheral blood differential count revealed 67% segmented neutrophils, 2% eosinophils, 0 basophils, 3% monocyte, 28% immature stages and 5% lymphocytes per 100 white blood cells. Bone marrow biopsy revealed myeloid hyperplasia suggestive of a myeloproliferative disorder. The genetic characteristic of this patient is to present an exceptional chromosomal complex rearrangement according to Pellestor classification, involving 3 chromosomes 9, 14, 12 with five breakpoints: 9q, 12p, 12q, and two different on 14q. More precisely, it probably compounds of a three way translocation t(9;12;14)(q34,p13,q?) associated with a independent translocation between both long arms of chromosomes 12 and 14. FISH analysis using BCR/ABL TC DFusion (Kreatech) shows involvement of ABL1 probe in a translocation with short arm of chromosome 12 and no evidence of rearrangement of BCR gene suggesting the possibility of juxtaposition of ABL1 gene with a gene different than the BCR gene. Moreover whole chromosome 14 painting confirms various involvements of chromosome 14. Molecular biology confirmed the suspected ETV6-ABL1 fusion gene and allows us to follow minimal residual disease. It should be noted that a t(9;12;14)(q34;p13;q22) has already been described as a variant of the t(9;12)(q34;p13) with complex insertions of ETV6 into ABL1. Other chromosome than the 14 has known to be implicated in t(9;12) variant as chromosome 17.First, he was treated with imatinib with progression despite the addition of hydroxyurea. Treatment was switched at 3 months for dasatinib. At 3 months, ETV6-ABL1/ABL1 is 0.32% and at 6 months he obtains a Major Molecular Response (defined as MRD under 0.1% like in BCR-ABL measurement).In conclusion, we present a new case of ETV6-ABL1 myeloproliferative disorder due to complex rearragement which experiments good response to dasatinib suggesting that second generation TKI are a good option for treatment of such disease. Disclosures:No relevant conflicts of interest to declare.

Vasorin Expression During Mouse B cell Development (138.31)

Abstract The stages of B cell development are delineated by stage-specific timing of gene and cell surface marker expression coinciding with exposure to cytokines and growth factors. Transforming growth factor beta (TGF-β) has been previously shown to affect lymphopoesis, as well as more mature processes, such as B-cell isotype switching. Receptors for TGF-β are diverse and widely distributed but display some tissue specificity. Here we describe expression of a Type-I TGF-β membrane receptor, vasorin, previously reported to be almost exclusively expressed by vascular tissue. Using RT-PCR analysis, the message for vasorin was identified in mouse pre-B cell lines prior to BCR gene rearrangement. Thus, vasorin expression may be closely linked to early lymphocyte development at the pro and pre-B stages. To verify that this finding was not an artifact of the cell lines, we looked for vasorin message in primary cell suspensions of spleen, thymus and liver from neonatal mice. Vasorin gene expression is readily detected in the primary repositories of B cell development, the spleen and liver, but not in primary thymocytes. These data suggest that receptor binding of TGF-β may be more vital to B-cell than to T-cell development. Vasorin expression by progenitor B cells may ensure measured proliferation via TGF-β receptor signaling.

Nephrotic syndrome in a patient with IgM myeloma with associated neutrophilia

An unusual case having IgM monoclonal gammopathy with clinical and pathologic features of multiple myeloma (MM) in association with neutrophilia and nephrotic syndrome is reported. The patient showed lytic bone lesions, decreased IgG and IgA levels, Bence-Jones proteinuria, nephrotic proteinuria with edema, and histological plasma cell infiltration typical of MM. Moreover, mature neutrophilic leukocytosis, hepatomegaly, high leukocyte alkaline phosphatase score (LAP), absence of Philadelphia (Ph) chromosome and bcr gene rearrangement were also evidenced, all these features representing findings typical of the recently described plasma cell dyscrasia-associated neutrophilia. After the diagnosis, the patient was treated with melphalan and prednisone, with an excellent response to the treatment. Different from the 30 cases so far reported, this is the first case of plasma-cell dyscrasia with associated neutrophilia due to IgM-producing monoclonal gammopathy. At the same time, this is the first reported case of nephrotic syndrome secondary to IgM myeloma.

Distinguishing between proliferating nodal lymphoid blasts in chronic myelogenous leukemia and non‐Hodgkin lymphoma: Report of three cases and detection of a bcr/abl fusion signal by single‐cell analysis

Lymph node biopsies were analyzed from three patients with chronic myelogenous leukemia (CML) showing nodal blast proliferation. Immunohistochemically, the blasts from all three patients had an immature marker profile with a T-blast population (cCD3+, CD4-, CD7+, CD8-, CD99+, terminal deoxynucleotidyl transferase +) and a hematopoietic progenitor cell marker (CD34). In two patients, the blasts also expressed myeloid lineage specificity (naphthol AS-D chloroacetate esterase activity and myeloperoxidase positivity). However, it was difficult to distinguish between blast proliferation in CML and non-Hodgkin lymphoma from these immunohistopathological findings alone. Subsequently, bcr gene rearrangement and bcr/abl mRNA expression were detected by Southern blot and reverse transcription-polymerase chain reaction analysis of the lymph nodes. Fluorescence in situ hybridization (FISH) analysis of lymph node touch smears also disclosed bcr/abl gene fusion signals in the blasts of all patients, confirming that the blasts were derived from Philadelphia chromosome-positive CML. Accurate discrimination between the proliferating nodal blasts of CML and non-Hodgkin lymphoma is essential for determining subsequent therapy. FISH analysis of bcr/abl in single-cell blast preparations is an efficient tool that allows rapid, accurate cytopathological diagnosis of extramedullary blast-phase CML and its discrimination from non-Hodgkin lymphoma.

Concurrent Mediastinal B Cell Lymphoma and Chronic Myeloid Leukemia with an Unusually Favorable Response to Chemotherapy

A 67-year-old Chinese woman presented with mediastinal B cell lymphoma in 1992 with incidental leukocytosis. Bone marrow and peripheral blood findings confirmed the diagnosis of chronic myeloid leukemia (CML). After combination chemotherapy and radiotherapy for lymphoma, her peripheral blood counts remained normal, and she refused further treatment for nearly six years. Frank hematologic relapse occurred in 1998 and low dose hydroxyurea was used, which was stopped after six months owing to cytopenia. She remained well without treatment at 12-year follow up. Retrospective Southern blot analysis confirmed BCR gene rearrangement in marrow in 1992 and 1998, but not in the lymphoma or the latest peripheral blood. Fluorescence in-situ hybridzation analysis showed no Philadelphia chromosome positive (Ph + ) cells in the peripheral blood at last (FISH) follow-up, but BCR/ABL remained detectable. The relevance of the concomitant occurrence of CML and lymphoma and the unusually favorable response of CML to chemotherapy to the pathogenesis of CML is discussed.

Value of molecular monitoring during the treatment of chronic myeloid leukemia: a Cancer and Leukemia Group B study.

Disappearance of the Philadelphia chromosome during treatment for chronic myeloid leukemia (CML) has become an important therapeutic end point. To determine the additional value of molecular monitoring during treatment for CML, we performed a prospective, sequential analysis using quantitative Southern blot monitoring of BCR gene rearrangements of blood and marrow samples from Cancer and Leukemia Group B (CALGB) study 8761. Sixty-four previously untreated adults with chronic-phase CML who were enrolled onto CALGB 8761, a molecular-monitoring companion study to a treatment study for adults with chronic-phase CML (CALGB 9013). Treatment consisted of repetitive cycles of interferon alfa and low-dose subcutaneous cytarabine. Blood and marrow Southern blot quantitation of BCR gene rearrangements was compared with marrow cytogenetic analysis before the initiation of treatment and of specified points during therapy. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to detect residual disease in patients who achieved a complete response by Southern blot or cytogenetic analysis. Quantitative molecular monitoring by Southern blot analysis of blood samples was found to be equivalent to marrow monitoring at all time points. Twelve of 62 (19%) follow-up samples studied by Southern blot analysis had a complete loss of BCR gene rearrangement in matched marrow and blood specimens. Southern blot monitoring of blood samples was also found to be highly correlated to marrow cytogenetic evaluation at all points, although there were four discordant cases in which Southern blot analysis of blood showed no BCR gene rearrangement, yet demonstrated from 12% to 20% Philadelphia chromosome-positive metaphase cells in the marrow. RT-PCR analysis detected residual disease in five of six patients in whom no malignant cells were detected using Southern blot or cytogenetic analyses. Quantitative Southern blot analysis of blood samples may be substituted for bone marrow to monitor the response to therapy in CML and results in the need for fewer bone marrow examinations. To avoid overestimating the degree of response, marrow cytogenetic analysis should be performed when patients achieve a complete response by Southern blot monitoring. This approach provides a rational, cost-effective strategy to monitor the effect of treatment of individual patients, as well as to analyze large clinical trials in CML.

Myeloperoxidase mRNA analysis in acute lymphoblastic leukemia.

Expression of the myeloperoxidase (MPO) gene at the mRNA level is a better lineage marker than enzymatic activity in early myeloid precursors and their leukemic counterparts. Its diagnostic use depends on the specificity of expression for myeloblasts and its absence in blasts of lymphoid lineage. The present study investigates MPO mRNA expression in adult acute lymphoblastic leukemia (ALL), using reverse transcription-polymerase chain reaction. Of a total of 13 cases, six were found to have blasts positive for MPO mRNA; in all of these cases, the blasts were cytochemically negative for MPO. This unexpected finding of MPO mRNA positivity in six of 13 cases was further investigated at the molecular level. Bcr gene rearrangement analysis was positive in all six cases for the bcr breakpoint diagnostic of chronic myelogenous leukemia (CML). Only three of these six cases were cytogenetically positive for a Philadelphia (Ph) chromosome. Based on molecular analysis, these cases are considered as CML presenting in blast crisis of lymphoid lineage, as opposed to de novo ALL. The remaining seven cases were Ph negative at the cytogenetic and molecular levels; the leukemic blasts were MPO mRNA negative, confirming the lack of MPO gene expression in de novo ALL.

Unusual expression of mRNA typical of Philadelphia positive acute lymphoblastic leukemia detected in chronic myeloid leukemia

The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of “p190” CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia. © 1996 Wiley-Liss, Inc.

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearranged bcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. The bcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, and bcr rearrangement analyses.

Efficacy of in vitro treatment of chronic myelogenous leukemia cell line, K562 cells, using 4-hydroperoxycyclophosphamide, alpha-interferon and gamma-interferon.

Alpha-interferon (alpha-IFN) and gamma-interferon (gamma-IFN) are highly effective biologic agents in the treatment of chronic myelogenous leukemia (CML). 4-hydroperoxycyclophosphamide (4-HC) is the most commonly used chemotherapeutic drug when purging bone marrow contaminated with malignant cells. To investigate the efficacy of alpha-IFN, gamma-IFN and 4-HC as purging agents in CML patients, K562 cells, a human CML cell line, were treated in vitro with these drugs singly or in combination. The cytotoxic effect was evaluated by MTT assay. Data were analyzed for synergism by the median effect principle of Chou and Talalay. Using Southern blot analysis, the effect of purging on the suppression of bcr gene rearrangement was also evaluated. Alpha-IFN, gamma-IFN and 4-HC all showed good concentration-dependent cytotoxic effect. Median cytotoxic doses of alpha-IFN gamma-IFN and 4-HC were 0.03 microM 0.20 microM and 1.97 microM respectively, and molar ratios of median doses of alpha-IFN to 4-HC, gamma-IFN to 4-HC and alpha-IFN to gamma-IFN were 1:80, 1:10 and 1:8, respectively. The combination treatment of alpha-IFN and 4-HC, and gamma-IFN and 4-HC showed significant synergistic cytotoxic activities, but combination of alpha-IFN and gamma-IFN was antagonistic. Status of bcr gene rearrangements in K562 cells was not changed after the treatments. Our results appear to provide an efficient method for removing CML cells from the bone marrow of CML patients.

Direct Transplantation of Chronic Myelogenous Leukemia Cells into Nude Mice and Establishment of a Leukemic Stem Cell (Ph1+, CD34+) Line Dependent on Mouse Bone Marrow Stromal Cells in vitro

Peripheral blood cells from a female patient with Ph1‐positive chronic myelogenous leukemia (CML) in blast crisis were serially transplanted in BALB/c nude mice for 16 passages. This in vivo cell line, designated CML‐N‐1, had Ph1chromosome abnormality and BCR gene rearrangement. The cells expressed CD11b, CD13, CD33, CD34, CD38, and HLA‐DR antigens until the 11th passage and subcutaneous tumors produced by these passages were composed of admixtures of immature and maturing cells that differentiated to basophils when cultured in vitro. From the 12th passage on, the tumors became composed mainly of immature cells expressing CD13, CD34, and HLA‐DR, and no longer differentiated to basophils even upon in vitro culture. In contrast to the vigorous proliferation in vivo, CML‐N‐1 cells from any passage failed to proliferate in vitro under standard liquid culture conditions with or without growth factors, such as granulocyte‐macrophage colony‐stimulating factor, granulocyte colony‐stimulating factor, monocyte colony‐stimulating factor, interleukin 3, interleukin 6 and stem cell factor. However, a continuously growing cell line, designated CML‐C‐1, was established by culturing CML‐N‐1 cells on feeder layers of mouse bone marrow stromal cells. This mouse bone marrow stromal cell‐dependent cell line showed immature cell morphology and expressed early myeloid phenotype positive for CD13, CD34, and HLA‐DR. These results indicate that mouse bone marrow stromal cells provide a certain growth factor(s) active on human leukemia cells.

T-Lymphoid Extramedullary (Lymphadenopathic) Blast Crisis In Cml.

T-cells are only rarely involved in chronic myeloid leukemia. We report an unusual case of T-lymphoid extramedullary (lymphadenopathic) blast crisis in a 66-year-old patient with Philadelphia positive chronic myeloid leukemia. The lymph node morphological picture resembled that of a peripheral T-cell lymphoma. Immunohistochemical stainings showed most lymph node blasts to have T-phenotype (positivity for CD-2, CD-3 and CD-5). Flow cytometric phenotyping confirmed T-phenotype, with positivity for CD-7, CD-38 and TdT. Cytogenetic analysis of lymph node cells revealed Philadelphia chromosome with an additional chromosomal abnormality (add(6p)). The BCR-gene rearrangement in the lymph node blasts was identical to that in the bone marrow. This case adds to the growing evidence that CML may be a disorder of the common stem cell from which T-, B- and myeloid progenitors originate.

Establishment and Characterization of a New Ph1-Positive ALL Cell Line (ALL/MIK) PresentingbcrGene Rearrangement onbcr-2and ALL-Typebcr/ablTranscript: Suggestion ofin vitroDifferentiation to Monocytoid Lineage

A new Ph1-positive acute lymphoblastic leukemia cell line, designated as ALL/MIK, has been developed from a patient with Ph1-positive acute leukemia. The ALL/MIK cells showed an immunophenotype of common ALL with rearranged JH and Jk genes. The ALL/MIK cells showed no M-bcr rearrangement using Southern blot analysis with either 3' or 5' M-bcr probes, but had the bcr gene rearrangement on bcr-2 within the first intron of the bcr gene. Consistent with this result, the reverse transcriptase-dependent polymerase chain reaction (RT-PCR) assay revealed that the ALL/MIK cells contained the transcript derived fusion of the first exon of bcr gene and the second exon of abl gene. Although the ALL/MIK cells were defined as early pre-B cells by immunophenotypical and genotypical analyses, they were capable of differentiating into monocytoid lineage by when cultured with TPA. Furthermore, another Ph1-positive ALL cell line, (TOM-1), was investigated for its ability to differentiate to monocytoid lineage. TOM-1 was also induced to monocytoid lineage by TPA. Thus, the present study suggested that the leukemic transformation in some Ph1-positive ALL may occur at the level of multipotential hematopoietic cells capable of differentiating towards lymphoid and myelo-monocytoid lineage.

DNA analysis to aid in the diagnosis of chronic myeloproliferative disorders.

Diagnosing chronic myeloproliferative disorders (CMPD) can be difficult because of overlap and possible transitions between the different conditions and their similarity to reactive myeloproliferations. DNA analysis was applied to improve differentiation of CMPDs. All subtypes of CMPD analyzed, including chronic myeloid leukemia, agnogenic myeloid metaplasia, polycythemia vera, and essential thrombocythemia, had in common that granulocytes and bone marrow cells were clonal in origin, as shown by X chromosome-linked DNA polymorphism in conjunction with methylation patterns (n = 32). Reactive myeloproliferations, by contrast, showed polyclonal inactivation patterns. Clonality could not distinguish CMPD from cases of myelodysplastic syndrome because the latter (n = 7) also exhibited clonal hematopoiesis. Because of their clonal origin, peripheral granulocytes were used in all cases (n = 201) to detect bcr gene rearrangement. Despite possible morphologic overlap between different types of CMPD, bcr gene rearrangement was specific for chronic myeloid leukemia and could be applied to differentiate chronic myeloid leukemia from other CMPDs in cases of equivocal morphologic diagnosis. Chronic myeloproliferative disorders represent clonal hemopoietic diseases that probably have specific underlying genetic defects. Thus DNA analysis can aid substantially in the differential diagnosis of CMPD.

Marrow fibrosis associated with a Philadelphia chromosome

Three patients had marked marrow fibrosis and an apparent Philadelphia (Ph) chromosome. Hematologic, cytogenetic, and molecular studies demonstrated the heterogeneity of such cases, including the first example of clinically typical myelofibrosis (MF) associated with a bcr gene rearrangement characteristic of chronic myelogenous leukemia (CML).

Diminished A-LAK cytotoxicity and proliferation accompany disease progression in chronic myelogenous leukemia

We have compared the proliferative and cytotoxic capacities of a highly purified population of recombinant interleukin-2 (rIL-2)-activated peripheral blood mononuclear cells (PBMNC), termed adherent lymphokine- activated killer cells (A-LAK), in 15 chronic phase (CP) and 10 advanced disease (AD) Ph-positive chronic myelogenous leukemia (CML) patients. The selective enrichment of CML A-LAK cells depended on their propensity to adhere to plastic and to proliferate when cultured in the presence of rIL-2 for 14 days. In both CP and AD patients, 14-day culture resulted in growth of a uniform population of large granular lymphocytes. While less than 10% of the A-LAK cells were CD56-/CD3+ (mature T lymphocytes), 82% +/- 12% of A-LAK cells from early CP patients (diagnosed less than 1 year from study), 84% +/- 3% of A-LAK cells from late CP patients (studied greater than 1 year after diagnosis), and 87% +/- 3% of A-LAK cells from AD patients were CD56+/CD3- (activated natural killer [NK] cells). No bcr gene rearrangement could be found in A-LAK cells from 13 CP and six AD CML patients studied. A-LAK cells from seven early CP CML patients displayed similar cytotoxicity against K562 (80% +/- 7% lysis at effector:target ratio of 20:1) and against Raji (80% +/- 12% lysis) compared with A-LAK from 17 normal individuals (72% +/- 3% K562 lysis, P = .21; 74% +/- 5% Raji lysis, P = .39). However, the cytotoxicity of A-LAK cells from eight late CP patients (59% +/- 5% K562 lysis, P = .02; 52% +/- 8% Raji lysis, P = .02) and that of 10 AD patients studied at any point after diagnosis (31% +/- 3% K562 lysis, P less than .001; 25% +/- 6% Raji lysis, P less than .001) was significantly lower than that of seven early CP CML patients and 17 normals. The proliferative potential of A-LAK cells from seven early CP CML patients (291 +/- 191- fold) was significantly greater than that of A-LAK cells from 17 normal individuals (23 +/- 3-fold, P = .03), eight late CP patients (46 +/- 17- fold, P = .02), and 10 AD patients (5.4 +/- 1.9-fold, P = .01). In contrast to CML A-LAK, K562 cytotoxicity of unstimulated mature peripheral blood NK cells was significantly lower in early CP CML patients than in normals and remained low at all stages of disease.