Translocation and rearrangement of myeloperoxidase gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (subtype M3) is characterized by malignant promyelocytes exhibiting an abundance of abnormally large or aberrant primary granules. Myeloperoxidase (MPO) activity of these azurophilic granules, as assessed by cytochemical staining, is unusually intense. In addition, M3 is universally associated with a chromosomal translocation, t(15;17)(q22;q11.2). In this report, the MPO gene was localized to human chromosome 17 (q12-q21), the region of the breakpoint on chromosome 17 in the t(15;17), by somatic cell hybrid analysis and in situ chromosomal hybridization. By means of MPO complementary DNA clones for in situ hybridization and Southern blot analysis, the effect of this specific translocation on the MPO gene was examined. In all cases of M3 examined, MPO is translocated to chromosome 15. Genomic blot analyses indicate rearrangement of MPO in leukemia cells of two of four cases examined. These findings suggest that MPO may be pivotal in the pathogenesis of acute promyelocytic leukemia.

Erythroleukemia: a review of 15 cases meeting 1985 FAB criteria and survey of the literature.

Erythroleukemia (EL) is a rare form of myelogenous leukemia the classification and definition of which has evolved over the course of its 80-year descriptive history. In 1976 the French American British (FAB) Cooperative Group included EL within the classification system of acute myelogenous leukemias as AML-M6, and agreed on a quantitative standard to be used in the diagnosis of this disorder. The standards were revised in 1985 to the form in use today. We selected a series of 15 cases from our records which specifically fit the FAB criteria for AML-M6. Extensive direct comparison between our series and the old literature is not practical because of the changes which have occurred in classification and definition of the disease. Overall we found a rough correlation between the clinical and laboratory data shown in the old literature on EL and data from our cases. These cases underscore characteristic laboratory features which correspond to what is now defined as AML-M6: these patients present with pancytopenia, frequent peripheral blood nRBCs and no, or few, peripheral blood blasts. In addition, we note the presence of a hybrid myeloid-erythroid blast in the bone marrow in this disease and suggest that this may be characteristic of this type of AML. Old literature on EL has generally shown it to be a disease of the elderly, yet we found a subset of younger patients whose clinical outcome was significantly better than that of the older patients. Finally, EL has historically been viewed as a disease in which patients progress from a prodrome through erythroleukemia to other acute myeloid leukemia (AML) subtypes. Consistent with this idea, half of our 15 patients had been previously diagnosed with myelodysplastic syndrome or received chemotherapy. On the other hand only one of the 15 patients converted to another type of AML during his course.

A variant myelodysplastic syndrome with multilineage Pelgeroid chromatin.

An unusual myelodysplastic syndrome with similar features in two patients is described. The entity is characterized by a maturation arrest at the myelocyte stage, strikingly clumpy chromatin, and a clinical course marked primarily by difficulties caused by anemia and thrombocytopenia. Electron microscopic description of the characteristic abnormal clumpy chromatin cells is included. While the disorder is unquestionably a myelodysplastic syndrome, it is clearly distinct from chronic myelogenous leukemia, chronic myelomonocytic leukemia, and the spectrum of refractory anemias with excess blasts.

A hybrid eosinophilic-basophilic granulocyte in chronic granulocytic leukemia.

Morphologic observation of the peripheral blood smear from a patient with chronic granulocytic leukemia suggested the presence of eosinophilic and basophilic granules in the same individual granulocyte. To unambiguously identify eosinophilic and basophilic granules simultaneously in the same preparation, the authors developed a cytochemical staining procedure using basophil-specific toluidine blue and eosinophil-specific cyanide-resistant peroxidase. Using this new dual stain, they demonstrated that ten out of ten chronic granulocytic leukemia patients they examined had cells that contained both eosinophilic and basophilic granules. The identity of the granules was corroborated by electron microscopic studies. These observations suggest that lineage confusion is common in chronic granulocytic leukemia.

Changing etiology of macrocytosis. Zidovudine as a frequent causative factor.

Macrocytosis is most commonly associated with vitamin B12 and folate deficiencies, followed by alcoholism, liver disease, and malignant neoplasms. Many laboratories have observed that in recent years macrocytosis increasingly has been associated with zidovudine treatment of acquired immune deficiency syndrome. One hundred consecutive inpatients in a large metropolitan urban hospital with mean corpuscular volumes greater than 110 fL were studied; 44% were patients with acquired immune deficiency syndrome being treated with zidovudine, 19% were alcoholics, and 12% had malignant neoplasms. Only 3% were folate deficient and just 4% were vitamin B12 deficient. This study suggests that zidovudine has become the most common cause of macrocytosis in the hospitalized urban patient population and that vitamin B12 and folate deficiencies have decreased in proportion.

Multiple methods for platelet enumeration. Observation of a newly introduced bias.

After the introduction of Coulter S-Cal, a bias became apparent between platelet counts obtained from instruments calibrated with this material and those obtained from a Clay Adams Ultra-Flo. Statistical methods were used to compare platelet counts obtained from the Coulter S-Plus IV, Ortho ELT-800, Clay Adams Ultra-Flo 100, and phase microscopy. At a P value of 0.01, paired t analysis revealed statistically significant biases between the Ultra-Flo and each of the other methods. Significant biases were also found between phase microscopy and each of the other methods, although these were of a smaller magnitude. The results indicate the necessity for users of multiple platelet counting methods to conduct comprehensive interinstrument evaluations, particularly when altering methods of calibration.

Melanin inclusions in the peripheral blood leukocytes of a patient with malignant melanoma.

Dark pigmented inclusions were found in the monocytes and polymorphonuclear leukocytes of a patient with widely metastatic malignant melanoma. Special stains demonstrated the granules were composed of melanin. The authors are not aware of any previous case report of patients with metastatic malignant melanoma that had leukocytic melanin inclusions. Prospective search for this phenomenon in malignant melanoma patients may allow such a finding to be used as a diagnostic tool.

Polymerase chain reaction in the diagnosis of chromosomal breakpoints.

Polymerase chain reaction-based methods for the detection of translocation-induced gene rearrangements are now widely used for diagnostics and patient monitoring. This article concentrates on two of the best studied chromosome translocations resulting in specific gene rearrangements and oncogene activation: the Philadelphia translocation of chronic myelogenous leukemia and acute leukemias, and the t(14;18) translocation of follicular lymphomas.

Minimal residual disease in acute promyelocytic leukemia.

In the last decade our understanding of acute promyelocytic leukemia (APL) has advanced tremendously. The recognition of all-trans retinoic acid (ATRA) as a powerful therapeutic agent paralleled the cloning of the t(15;17) breakpoint. RtPCR for the PML-RARA hybrid mRNA has become the hallmark of molecular diagnosis and molecular monitoring in APL. Current techniques are useful in predicting complete remission and a possible cure in many patients who repeatedly test negative by PCR. Standardizing techniques and improving the sensitivity of the assay are important. Doing this in a way so that clinically relevant minimal residual disease can be distinguished from "indolent disease" remains among the future challenges in APL.

Expression of granule protein mRNAs in acute promyelocytic leukemia.

The granule proteins are among the most abundant and characteristic proteins of myeloid cells. They are essential for the antimicrobial activity of these cells and they provide important markers for the differentiation stage of the myeloid series and for the diagnosis of myeloid leukemias. In acute promyelocytic leukemia (APL) there is high production of myeloperoxidase, and its cytochemical detection as well as the t(15;17) chromosomal translocation are important markers in the diagnosis of this acute myelogenous disease. The expression of other granule protein genes in APL has not been systematically determined. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) method to determine the pattern of expression of granule protein genes at the mRNA level in APL cells. We have examined the expression of the primary granule proteins defensin, myeloperoxidase, elastase, and cathepsin G; the secondary granule proteins lactoferrin, collagenase, and transcobalamin; as well as lysozyme, a protein reportedly found in both primary and secondary granules. mRNAs for all of these granule proteins were present in normal bone marrow mononuclear cells. We found that APL cells from three patients contain, in addition to myeloperoxidase mRNA, mRNAs for elastase, cathepsin G, and lysozyme. One patient had faint but detectable lactoferrin mRNA signal, but collagenase and transcobalamin mRNAs were not detectable in this patient. Defensin mRNA was found in one of the three APL patients, and all the primary granule protein mRNAs measured were found to be expressed in the APL cell line NB4. None of the secondary granule protein mRNAs measured were detectable in NB4 cells. After treatment with retinoic acid (RA), which induces neutrophil maturation of these cells, weak induction of lactoferrin and collagenase but not transcobalamin was observed. However, in view of the weak transcobalamin signal observed in normal bone marrow, the absence of transcobalamin in RA-induced NB4 cells must be interpreted with caution. Interestingly, elastase and cathepsin G mRNA disappeared after RA induction, whereas defensin and myeloperoxidase mRNAs remained present. These findings indicate that granule protein mRNAs are regulated separately and differently, and that only minimal expression of secondary granule protein genes can occur in APL cells.

A novel interferon-inducible gene expressed during myeloid differentiation.

The acute promyelocytic leukemia cell line, NB4, can be induced to differentiate to mature granulocytes by retinoic acid treatment. A novel retinoic acid-inducible cDNA clone, designated RI58, was isolated from a cDNA library constructed from retinoic acid-treated NB4 cells by differential hybridization. RI58 cDNA encodes a protein of 58kDa which has a similarity in its amino acids sequence to interferon (IFN)-inducible proteins. In addition, RI58 was induced by recombinant human IFN-alpha (rhIFN-alpha) in NB4 cells. RI58 was detectable within 4 hours post-stimulation with rhIFN-alpha, while it took as long as 1day after retinoic acid stimulation. Culture supernatant from retinoic acid-treated NB4 cells also induced RI58 expression similarly as rhIFN-alpha. This activity in culture supernatant was inhibited by anti-leukocyte IFN antiserum which showed specific reactivity to rhIFN-alpha. These results indicate that RI58 is induced by retinoic acid stimulation through autocrinally secreted IFN-alpha from NB4 cells. In the retinoic acid-treated NB4 cells, the expression of RI58 was increased along the process of differentiation. On the other hand, it was expressed constitutively in untreated non-hematopoietic cell lines and mature hematopoietic cell lines.

Carbonic anhydrase: a marker for the erythroid phenotype in acute nonlymphocytic leukemia.

Protein markers are often used to corroborate the morphological subtyping of hematopoietic malignancy. Most commonly, surface markers are used for the phenotyping of hematopoietic cells; however, internal proteins have also been used as markers. Glycophorin, hemoglobin A, hemoglobin F, and transferrin have all been used as markers for the erythroid phenotype. We have recently shown that carbonic anhydrase is constitutively and aberrantly expressed in two erythroleukemic cell lines. We here show that it is also present in high levels in primary erythroleukemic blasts and that it is a useful marker for the M6 phenotype when classifying acute nonlymphocytic leukemia.

Changes in tRNA levels during the induction of hemoglobin synthesis in Friend leukemia cells.

There is a three- to four-fold decrease in the content/cell of tRNAs for ten different amino acids four days after the induction of erythroid differentiation in Friend leukemia cells, consistent with the decrease in cell volume that occurs. Surprisingly, there is an approximately two-fold increase in the cellular content of each of these tRNAs between day 4 and day 6 after induction, indicating the net synthesis of tRNA late in induction. The tRNA changes affect all species and do not result in tRNA specialization for hemoglobin synthesis, as occurs in normal erythroid development. The tRNA content of imidazole-treated cells, which do not synthesize hemoglobin although they undergo other changes of erythroid differentiation, decreases initially as described above, but shows no increase from day 4 to day 6.

Acute lymphoblastic leukemia in a patient with longstanding polycythemia vera: cytogenetic analysis reveals two distinct abnormal clones.

A 68-year-old female patient is described in whom acute lymphoblastic leukemia followed a long course of polycythemia vera. Chromosomal analysis of a peripheral blood specimen at the time of blastic transformation revealed two distinct clones: one characterized by a chromosomal abnormality frequently noted in polycythemia vera and the other by a rearrangement characteristically observed in lymphoid malignancies. These findings suggest the existence of two independent hematologic diseases: this phenomenon would not support the speculation from previous reports that lymphoproliferative disorders in patients with polycythemia vera arise from clonal evolution.

Complete resolution of pure red cell aplasia in a patient with chronic lymphocytic leukemia following antithymocyte globulin therapy.

Pure red cell aplasia has been reported to be associated with chronic lymphocytic leukemia. It has been proposed that this complication may be a result of T-cell populations that suppress erythropoiesis. It has been postulated that antithymocyte globulin might reverse this abnormality by eliminating the population of suppressor T cells responsible for this inhibition. We treated a 74-year-old man who had B-cell chronic lymphocytic leukemia and pure red cell aplasia that was refractory to cytotoxic and corticosteroid therapy with equine antithymocyte globulin and methylprednisolone sodium succinate. This therapy resulted in a durable complete remission of both the chronic lymphocytic leukemia and the pure red cell aplasia and was associated with normalization of helper/suppressor T-cell ratios in the bone marrow. Antithymocyte globulin should be investigated further as a therapeutic modality for patients with pure red cell aplasia associated with chronic lymphocytic leukemia.

Carbonic anhydrase is aberrantly and constitutively expressed in both human and murine erythroleukemia cells.

The levels of the erythrocyte proteins carbonic anhydrase (CA) and hemoglobin (Hb) change coordinately during human ontogeny. To further probe the coordinate gene expression of these two proteins in vitro, we used an immunoblotting technique to measure their levels during erythroid differentiation in normal human and murine erythroid progenitors, in human and murine erythroleukemia cells, and in normal murine erythroid progenitors infected with Friend virus. Levels of CA and Hb seem to gradually increase in normal differentiating stem cells. In contrast, both human and murine erythroleukemia cells show high levels of CA, but not of Hb, prior to induction of differentiation. Friend virus infection of normal murine progenitors appears to stimulate CA synthesis as an initial and integral step in transformation. In addition, both the erythroleukemia cells and the erythroid progenitors transformed with Friend virus seem to contain much higher levels of CA than Hb during the early stages of differentiation. This relationship is in marked contrast to normal erythroid differentiation, in which Hb levels are always higher than CA levels. Thus, neoplastic transformation seems to be associated with aberrant production of CA that does not correspond to a maturation arrest of the normal differentiation sequence.

Expression of the c-fos protooncogene by human and murine erythroblasts.

The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.

Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation.

During the course of differentiation of early human myeloid cells toward monocytes and granulocytes, cell surface expression of the cell adhesion molecule, CD11b/CD18 (Mo1) increases dramatically and expression of myeloperoxidase (MPO), a bacteriocidal enzyme, decreases markedly. Using the inducible promyelocytic cell line HL-60 as a model, we studied the mRNA expression of these genes. Differentiation of these cells along both a monocytic and a granulocytic pathway demonstrated that the mRNA levels of the two subunits of CD11b/CD18 increased in a pattern temporally and quantitatively similar to the increase in cell surface expression of this heterodimer. In contrast, the expression of MPO mRNA decreased in a temporal and quantitative pattern similar to the known decrease in MPO protein during differentiation, suggesting that regulation of these myeloid-specific proteins may occur at the level of mRNA expression. These findings have important implications with regard to the nature of the block in differentiation in acute nonlymphocytic leukemia and the regulation of myeloid gene expression.