Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

INTRODUCTION: The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. METHOD: A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. RESULTS: For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. CONCLUSION: The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR.

Expression of Notch1 and Jagged1 proteins in acute myeloid leukemia cells.

Cell fate of hematopoietic progenitors is regulated by interaction between Notch proteins on progenitors and Notch ligands such as Jagged1 on stromal cells. Since acute myeloid leukemia (AML) originates from dysregulated hematopoietic progenitors, some abnormalities in the Notch-Jagged system may exist in AML cells. As the first step to clarify this, we examined the expression of Notch1 and Jagged1 proteins in eight AML cell lines and 15 fresh AML samples by immunoblotting. In the Notch1 protein, two bands, a 300 kDa band and a 120 kDa band, which appeared to be a full-length protein and a transmembrane fragment, respectively, were recognized in five AML cell lines and six fresh samples. In addition, three of the five cell lines showed a 110 kDa fragment, which appeared to be from an intracellular domain, namely an active form. One cell line showed aberrant sized fragments, which suggested a structural abnormality. Jagged1 protein was recognized in six cell lines and six fresh samples. In four cell lines and four fresh samples, both Notch1 and Jagged1 proteins were observed. In these cells, Notch1 and Jagged1 proteins may interact among themselves. We showed that Notch1 and Jagged1 proteins are widely expressed in AML cells. We hypothesize that some abnormalities in the Notch-Jagged system which cause the excessive self-renewal and the block of differentiation, may be involved in the abnormal proliferation of AML cells.

Human herpesvirus 8 DNA in HIV-negative Japanese patients with multicentric Castleman's disease and related diseases.

Multicentric Castleman's disease (MCD) is a lymphoproliferative disorder characterized by systemic lymphadenopathy and hypergammaglobulinemia. Recently, a French group reported that human herpesvirus 8 (HHV8) DNA was detected in tissue samples of MCD patients. The detection rate was especially high in human immunodeficiency virus (HIV)-positive MCD patients. Thus, HHV8 infection seems to be closely related to HIV infection. In Japan, the HIV infection rate in the general population is very low. To examine whether HHV8 is actually related to MCD in Japan, we performed nested polymerase chain reaction for the HHV8 genome using DNA samples from 7 patients with MCD and 23 patients with related diseases such as POEMS syndrome, amyloidosis, myeloma and lymphoma. They were all HIV-negative Japanese. Three of 7 MCD patients were positive for HHV8. There were no clear differences in clinical characteristics between HHV8-positive patients and negative ones. All other patients were negative for HHV8. Thus, we have shown that some MCD patients in Japan are also infected with HHV8.

[Molecular diagnostic tests in hematologic diseases].

Molecular diagnostic tests are widely performed in managing hematologic malignancies such as leukemia and lymphoma. In this article, we review the present application and problems of the tests. Karyotyping is performed at diagnosis of all kinds of hematologic malignancies. This method needs dividing cells as samples and skilled experts. Fluorescence in situ hybridization(FISH) analysis using cells in interphase is performed, for example, to monitor the effect of interferon on chronic myelogenous leukemia patients. The weak point of this method is that approximately 2% of false-positive cells are inevitable. Southern blot method is used for clonal analysis in some disease, for example, adult T-cell leukemia/lymphoma. Polymerase chain reaction(PCR) method using genomic DNA is performed for limited types of diseases such as lymphoma with bcl-2/IgH fusion gene. Reverse transcription(RT)-PCR method can detect fusion gene transcripts with high sensitivity. This method is useful for detecting minimal residual diseases after chemotherapy or bone marrow transplantation. To perform quantitative analysis, real-time PCR or competitive PCR must be done. In the near future, new technology such as gene expression profiling analysis using DNA microarrays or spectral karyotyping(SKY) method will be used in clinical practice.

Polymerase chain reaction detection of rearranged immunoglobulin heavy chain DNA in plasma samples is useful in the diagnosis of B-cell lymphoma.

Using DNA extracted from plasma samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients, we attempted to detect the monoclonal rearrangement of immunoglobulin heavy chain gene by amplifying complementarity-determining region 3 (CDR3) by semi-nested polymerase chain reaction (PCR) method (plasma PCR). In 19 of 37 (51%) cases, clonal DNA was detected. With the same PCR method using DNA extracted from peripheral blood mononuclear cells, clonal DNA was detected in 8 of the 37 cases (22%). These 8 were in advanced stages with bone marrow (BM) invasion mostly. On the other hand, the 19 positive cases by plasma PCR included those in early stages without BM invasion or with normal soluble interleukin-2 receptor (sIL-2R) and lactate dehydrogenase (LDH) values. In 15 healthy volunteers, plasma PCR showed no clonal DNA. In cases in which tumor biopsy was difficult to perform, plasma PCR was helpful for determining whether or not the tumor was B-NHL. Plasma PCR is simple and has high specificity, although its sensitivity is insufficient.

[DNA diagnosis of hematopoietic malignancies].

The recent advances in molecular biology and gene engineering have contributed considerably to the diagnosis and treatment of hematopoietic malignancies, such as leukemia and malignant lymphoma. These advances also made possible precise determination of the clonal origin of malignant cells, the subtype of leukemia or lymphoma, and the clinical prognosis in each patient. Furthermore, minimal residual malignant cells in leukemia or lymphoma patients after achieving complete remission could be detected by DNA analysis. Based on these analyses, treatment can theoretically be tailored for each patient. We discuss in the present paper the usefulness of DNA or gene analyses of immunoglobulin heavy chain gene in clonal assessment of lymphocytic malignancies and in detecting minimal residual disease in the patient.

Establishment of a double Philadelphia chromosome-positive acute lymphoblastic leukemia-derived cell line, TMD5: effects of cytokines and differentiation inducers on growth of the cells.

A double Philadelphia chromosome (Ph)-positive leukemia cell line with common-B cell phenotype, designated TMD5, was established from the blast cells of a patient with double Ph-positive acute lymphoblastic leukemia. TMD5 cells expressed 190 kDa BCR/ABL chimeric protein and 145 kDa ABL protein. The cells proliferated without added growth factors. Autocrine growth mechanism was not recognized. The addition of growth factors such as G-CSF, GM-CSF, IL-3, IL-6, or Stem Cell Factor did not affect the growth. Herbimycin A suppressed the growth of TMD5 cells at the low concentration that did not affect Ph-negative cells. It suppressed tyrosine phosphorylation of intracellular proteins in TMD5 cells. Dexamethasone and dibutyryl cyclic AMP also suppressed the growth. They, however, did not affect the phosphorylation significantly. Neither all-trans retinoic acid nor interferon-alpha affected the growth. TMD5 cells, characterized minutely here and rare in that they have double Ph chromosomes, will be a useful tool for the study of Ph-positive leukemia.

A case of atypical chronic myeloid leukemia regarded as MDS with myeloproliferative features.

We report a case of atypical chronic myeloid leukemia (aCML) who showed marked neutrophilia without dysplastic features, basophilia or monocytosis. These findings diverged somewhat from the FAB criteria for aCML. The patient's erythroid cells and megakaryocytes were dysplastic. His marrow cells formed no spontaneous colonies, as shown by cell culture. The cells formed many small-sized neutrophil colonies with G-CSF stimulation. Interestingly, they formed mainly neutrophil colonies with GM-CSF stimulation. These findings were different from those of chronic myelomonocytic leukemia cells and chronic granulocytic leukemia cells. This aCML case showed the cytological features of myelodysplastic syndrome.

Effect of vesnarinone, a quinolinone derivative, on the growth of leukemic blasts in acute myelogenous leukemia.

We studied the effects of vesnarinone, a quinolinone derivative used clinically for the treatment of chronic congestive heart failure, on the leukemic blast progenitors in acute myelogenous leukemia (AML) patients and on the normal hematopoietic precursors, colony-forming unit in culture (CFU-C), and colony-forming unit erythroid (CFU-E). Leukemic blast progenitors made blast colonies in the presence of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3). Blast colony-formation was suppressed by vesnarinone in a dose-responsive manner regardless of growth factor added. Vesnarinone suppressed the primary (PE1) and secondary (PE2) colony-formation of leukemic blast progenitors in six AML patients tested. The suppression by vesnarinone did not significantly differ between PE1 and PE2. This finding suggests that vesnarinone exerts an almost equivalent effects on the terminal divisions and the self-renewal of leukemic blast progenitors. Furthermore, this drug suppressed the growth of clonogenic cells of five AML cell lines, OCI/AML1a, OCI/AML2, OCI/AML3, OCI/AML5, and OCI/AML6. Normal hematopoietic precursors CFU-C and CFU-E were also suppressed by vesnarinone, although vesnarinone was less toxic to normal hematopoietic than to leukemic blast progenitors. The possible usefulness of vesnarinone as a new approach to the treatment of AML patients is discussed.

[Plasma cell leukemia with amyloid deposition and osteogenetic change at the site of an extramedullary plasmacytoma].

A 49-year-old man was admitted with swelling in the left lower extremity, and a mass in the left lower abdomen. Laboratory findings showed an increased WBC of 15,000/microliter with 41% plasma cells, and immunoglobulin (Ig) A of 2,557mg/dl with a monoclonal component. A roentgenogram and computed tomograph of the abdomen revealed that a 5 x 10 cm mass with calcification located in the iliopsoas muscle. Plasma cell leukemia with extramedullary plasmacytoma was diagnosed, and the patient was treated with high-dose dexamethasone (40 mg/day for 4 days), resulting in a good response with the disappearance of plasma cells in peripheral blood and a marked decrease in serum Ig A. However, the patient's condition deteriorated in spite of various treatments, and he died of heart failure 5 months after admission. With informed consent from relatives, a necropsy was performed and infiltration of plasma cells in the mass in the iliopsoas muscle was noted. We reported this case because plasma cell leukemia with amyloid deposition and osteogenesis at the site of extramedullary plasmacytoma is very rare.

Phosphorylation of signaling proteins in factor-independent myeloid leukemia cell lines.

We investigated the mechanisms of factor-independent growth of four human myeloid leukemia cell lines. The autocrine mechanisms were ruled out by RT-PCR method examining growth factor mRNA. The immunoblotting method showed that many proteins were tyrosine phosphorylated irrespective of the stimulation with growth factors (G-CSF and GM-CSF) in factor-independent cell lines while the phosphorylation was induced stimulation dependently in a factor-dependent cell line. MAP kinase was constitutively phosphorylated in factor-independent cell lines. JAK2 protein was not tyrosine phosphorylated before the stimulation. It was significantly phosphorylated after the stimulation in three factor-independent cell lines although the stimulation did not affect their growth. JAK1 protein was not phosphorylated either before or after the stimulation. In conclusion, constitutive phosphorylation of signaling proteins seemed to be related to factor-independent growth. MAP kinase was involved in the phosphorylation, while JAK1 and JAK2 were not.

Clonal involvement of eosinophils in therapy-related myelodysplastic syndrome with eosinophilia, translocation t(1;7) and lung cancer.

We report a therapy-related MDS (RAEB) patient with eosinophilia, unbalanced translocation der(7)t(1;7) (q12;q22) and lung cancer. We observed no increase in cytokine levels in serum or in the conditioned medium (CM) of peripheral T cells cultured with or without IL-2. When bone marrow (BM) cells were cultured with GM-CSF, IL-3 and SCF in a semisolid system, the colonies were exclusively eosinophilic. Cytogenetic analysis of the colony cells identified the same chromosome abnormality in all metaphases to that of BM cells. Suspension and clonogenic colony assay of BM cells cultured with various cytokines showed predominant eosinophilic growth and differentiation with GM-CSF, but not with the other cytokines examined. These findings, together with mild morphological abnormalities of eosinophils, indicate clonal involvement of eosinophils in the myelodysplastic syndrome (MDS) clone, and that the eosinophilia was derived from the neoplastic clone with the translocation and was not associated with the patient's lung cancer.

[Three cases of drug-induced hemolytic uremic syndrome].

We report three cases of drug-induced hemolytic uremic syndrome (HUS). Three patients with advanced gastrointestinal cancer underwent a curative operation and adjuvant chemotherapy with Mitomycin C (MMC), 5FU and Ara-C. Later, progressive anemia, thrombocytopenia, renal dysfunction and elevation of serum LDH were recognized. A diagnosis of HUS was made. As they had no symptoms of infectious diseases or relapse of cancer, the cause of HUS was thought to be MMC. Treatment with antiplatelet drugs and fresh frozen plasma was effective for two patients. However, one patient died of pulmonary edema.

Relation of protein kinase A and protein kinase C to signaling pathways of hematopoietic factors in leukemia cell lines.

In terms of growth, differentiation, and signaling pathways of hematopoietic factors, the effects of protein kinase C (PKC) activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase A(PKA) activator, dibutyryl cyclic adenosine monophosphate (dbcAMP) were examined in vitro using three factor-responsive myeloid leukemia cell lines. TPA suppressed the growth of OCI/AML-1 and OCI/AML-5 cells but stimulated the proliferation of OCI/AML-4 cells. TPA differentiated OCI/AML-4 and OCI/AML-5 cells to macrophage-like cells. dbcAMP suppressed the growth of the three without differentiation. The stimulation of TPA induced tyrosine phosphorylation of some proteins in OCI/AML-4 and OCI/AML-5 cells. Their molecular weights were the same as those phosphorylated by hematopoietic factors. The patterns of phosphorylated proteins were different between the two. TPA induced the phosphorylation of MAP kinase, but not that of JAK2 protein in three cell lines. The stimulation of dbcAMP did not induce tyrosine phosphorylation in three cell lines. Overnight exposure of TPA inhibited the tyrosine phosphorylation induced by hematopoietic factors, although that of dbcAMP did not. We suggest a close relation of PKC to signaling pathways of hematopoietic factors, however, the ways of relation were diverse among the cell lines and the clear mechanism explaining its effects on growth and differentiation was not elucidated.

The effect of basic and acidic fibroblast growth factors (bFGF and aFGF) on the growth of leukemic blast progenitors in acute myelogenous leukemia.

The effect of basic and acidic fibroblast growth factors on leukemic blast progenitors was studied in 14 patients with acute myelogenous leukemia and in one patient with chronic myelocytic leukemia in myeloid crisis. bFGF and aFGF stimulated blast-colony formation by leukemic blast progenitors cultured in methylcellulose in two patients. In the other 13 patients, no significant effect of either FGF on blast-colony formation was noted. The combination of bFGF or a FGF and G-CSF, GM-CSF, interleukin-3, or stem cell factor (SCF) had a synergistic effect on blast-colony formation in three patients. In the other patients, however, synergism between FGF and CSF was not detected. In fact, bFGF was found to suppress the stimulation of blast-colony formation due to GM-CSF in one of 10 patients and that due to SCF in four of eight patients. aFGF suppressed the stimulation of blast-colony formation due to GM-CSF in two of 11 patients and that due to SCF in four of eight patients. The results show that bFGF and aFGF do not directly play a major role in leukemic hematopoiesis but that they may modulate the cytokine network affecting leukemic cell growth.

Diversity and similarity in signaling pathways of hematopoietic growth factors in human leukemia cell lines.

Diversity and similarity in signaling pathways of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and stem cell factor (SCF) in five human factor-responsive leukemia cell lines were investigated by immunoblotting to detect tyrosine phosphorylation of intracellular proteins. G-CSF induced tyrosine phosphorylation of a set of proteins with few different components according to the cell lines. IL-3 also induced phosphorylation of several proteins. In a lymphoid cell line, phosphorylation patterns induced by IL-3 were somewhat different from that in myeloid cell lines. Phosphorylation patterns by G-CSF and those by IL-3 were similar in myeloid cell lines. In a cell line which responded to both IL-3 and SCF, almost similar sets of proteins were phosphorylated by each, although phosphorylation of a 92-KDa protein was specific to IL-3 and that of a 140-200-KDa protein was specific to SCF. Taken together, proliferative growth factors induced tyrosine phosphorylation of similar sets of proteins with little difference according to each growth factor and each target cell line.

All-trans retinoic acid upregulates thrombomodulin and downregulates tissue-factor expression in acute promyelocytic leukemia cells: distinct expression of thrombomodulin and tissue factor in human leukemic cells.

The expressions of thrombomodulin (TM) and tissue factor (TF) by all-trans retinoic acid (ATRA) were studied in human leukemic cell lines including NB4 (acute promyelocytic leukemia) and U937 (monoblastic leukemia). ATRA remarkably upregulated TM antigen expression in cell lysates as well as TM cofactor activity on the cell surfaces of NB4. The level of TM mRNA in NB4 cells was increased by ATRA. Inherently procoagulant NB4 cells contained markedly higher content of TF, which was efficiently reduced by ATRA. Modest increase of TM and decrease of TF were observed when NB4 cells were treated with dibutyryl cyclic adenosine monophosphate (dbcAMP). On the other hand, both ATRA and dbcAMP showed dramatic increase of TM antigen level and modest decrease of TF antigen in U937 cells. These results suggest that ATRA regulates expressions of TM and TF antigens and activity in NB4 and U937 cell lines, and provide evidence for a potential efficiency of ATRA as a preventive and therapeutic agent for disseminated intravascular coagulation in promyelocytic and monocytic leukemia.

Modulation of growth factor receptors on acute myeloblastic leukemia cells by retinoic acid.

The effects of retinoic acid (RA) on the proliferation of acute myeloblastic leukemia (AML) cells were studied. AML samples were divided into three groups. Namely, RA stimulated blast colony formation by AML samples in group A and inhibited that by the samples in group B, regardless of added growth factors. For the samples in group C, RA inhibited the colonies formed by granulocyte colony-stimulating factor (G-CSF) but stimulated those by granulocyte macrophage CSF (GM-CSF). To investigate the mechanism involved, the effects of RA on growth factor receptors on AML cells were examined by flow cytometry using fluorolabeled ligands. For the samples in groups A and B, RA affected neither G-CSF receptor (GR) nor GM-CSF receptor (GMR). For the samples in group C, exposure to 10(-7) M RA for 1 day clearly increased GMR, but did not affect GR. This finding supports the hypothesis that the increase of GMR is one of the causes of the stimulative effects of RA on cells cultured with GM-CSF in group C.

The effects of retinoic Acid analogs on the blast cells of acute myeloblastic-leukemia in culture.

The effects of retinoic acid (RA) analogues, Am80 and Ch55, on acute myeloblastic leukemia (AML) cells, acute promyelocytic leukemia (APL) cells, and normal bone marrow (BM) cells, were studied in vitro. These analogues have different binding affinity to RA receptors and cellular retinoic acid binding proteins (CRABP). All-trans RA (ATRA) showed various effects on the proliferation of AML cells and BM cells. These analogues showed similar effects. Regarding the differentiation of APL cells into neutrophils, Ch55 and Am80 were more potent than ATRA. Ch55, which does not bind to CRABP, may overcome the clinical problem of retinoid-resistance due to the induction of CRABP.

Growth potentiating activity of endogenous production of interleukin-1 and tumor necrosis factor alpha in blast cells of acute myeloblastic leukemia.

For the optimal growth of clonogenic cells in acute myeloblastic leukemia (AML), several cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 are required in addition to colony-stimulating factor (CSF), which may be produced by blast cells themselves. In the present study, we addressed the potential role of endogenous production of TNF-alpha and/or IL-1 in the in vitro growth of AML clonogenic cells supported by IL-3. Addition of a specific neutralizing antibody against TNF-alpha (anti-TNF-alpha) to the culture significantly reduced the growth-stimulating effect of IL-3 on the cells in 11 of 14 patients. Simultaneous addition of anti-IL-1 alpha and anti-IL-1 beta also partly affected the growth, although to a much lesser extent when compared to the effect observed with anti-TNF-alpha. In 3 patients, the growth-stimulating effect of IL-3 was completely abrogated by anti-TNF-alpha or a combination of all three antibodies. Constitutive TNF-alpha transcript was observed in 5 patients and TNF-alpha protein was present in culture supernatant. Following in vitro culture, a transient but profound increase in c-fos, c-jun, TNF-alpha and IL-1 beta mRNA levels was observed. Anti-TNF-alpha inhibited the accumulation of TNF-alpha transcript, suggesting that membrane-integrated TNF-alpha may be partly responsible for the induction of TNF-alpha mRNA. It seems likely that the accumulation of these genes occurs through a protein kinase C-independent signaling pathway.