Immunophenotype of a transient myeloproliferative disorder in a newborn with trisomy 21

Cytologic, immunologic, and cytogenetic studies were performed on the blast cells of a newborn with Down syndrome and transient myeloproliferative disease. This hematologic disorder is uncommon, and occurs primarily in infants with Down syndrome. This boy presented with a high white blood cell count and a high percentage of blast cells, without anemia or thrombocytopenia. Chromosome analysis showed a constitutional trisomy 21 without any other clonal abnormality. A three-color flow cytometric analysis was performed and revealed two different CD45 dim, CD34(+), CD117(+), CD56(+) immature subpopulations: the normal immature myeloid precursor and an immature blast cell population that expressed CD41, CD42, CD61, CD36, CD13, CD1a, and CD2. We postulate that this population could be the leukemic precursor involved in the acute megakaryoblastic leukemia frequently observed in children with Down syndrome.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Leukocyte mean corpuscular volume in the differential diagnosis of acute leukemias

The leukocyte mean corpuscular volume was measured in 61 cases of acute leukemia. The volume of blast cells allows a clear distinction between lymphoblastic and other varieties of acute leukemia. There seems to be good correlation between patients'' age, leukocyte corpuscular volume, cytological and cytochemical findings. Furthermore, myeloblastic leukemias.Leukemic cells can be characterized by their volume, which is close to that of normal cells of the same line. This observation supports the clonal theory of their origin and concurs with the classical nomenclature which separates them into lymphoblastic, myeloblastic and monoblastic types.     

Genomics in myeloid leukemias: an array of possibilities

Myeloid leukemias are clonal hematopoietic stem cell disorders characterized either by proliferation of one or more of the myeloid lineages (chronic myelogenous leukemia) or by clonal expansion of myeloid blasts (acute myeloid leukemia). Over the past several years our knowledge of these hematologic malignancies has increased tremendously. The result is a classification that incorporates morphologic, immunophenotypic, genetic and clinical features in an attempt to define biologically and clinically relevant entities. Nevertheless, in many tumor subtypes the pathogenic event is still unknown. Furthermore, well-defined leukemia subgroups exhibit considerable heterogeneity, arousing the suspicion that several molecularly distinct subtypes might exist within the same cytogenetic category. Therefore, an ideal classification system would ultimately be based on the underlying molecular pathogenesis, but such knowledge is not yet available. However, by surveying the expression levels of thousands of genes in parallel, DNA microarrays have recently contributed to an increasingly refined molecular taxonomy of myeloid disorders. This powerful technology is becoming well established and has been used to diagnosis cancer and predict clinical outcome, to discover novel tumor subclasses, to gain insights into pathogenesis, and to identify new therapeutic targets. While many challenges remain ahead, genomic technologies have already demonstrated tremendous potential. We expect whole genome approaches will significantly contribute to a better understanding of the pathogenesis and result in a refined molecular classification of myeloid leukemias.     

Biphenotypic acute leukemia: a case report

We describe an uncommon case of acute leukemia in which leukemic blasts expressed myeloid antigens and cyCD79alpha molecule. In this 49-year old male patient, two distinct blast populations were detected in peripheral blood and bone marrow samples: one of small size resembling lymphoblasts and another with pink cytoplasmic granules resembling myeloblasts. Cytochemical reaction for myeloperoxidase was negative in both cell types. Conventional cytogenetic analysis showed a normal karyotype (46 XY) in all metaphases studied, while gene rearrangement analysis by seminested PCR of the immunoglobulin heavy chain (Ig-H) and T-cell-gamma chain (TCR-gamma) receptor, showed a germline configuration of the TCR and clonal rearrangement of Ig-H chain genes. Multicolour cytofluorimetric analysis showed that bone marrow and peripheral blood blasts expressed CD19, CD79alpha bright, CD22 and terminal deoxynucleotidyl transferase (TdT) as lymphoid markers, CD13, CD117, CD15 as myeloid markers, CD34, HLA-DR as stem cell markers. CD33 myeloid antigen was expressed by 50% of the blastic population. No differences in the immunophenotypic profile were detected in the two blast populations which were identified by morphology. According to EGIL (European Group of Immunological Classification of Leukemias) and WHO (World Health Organization) criteria, a diagnosis of biphenotypic acute leukemia (BAL) was made. The patient was treated with AML induction therapy followed by autologous stem cell transplantation, but relapse free survival was 6 months. The patient died a few weeks later due to unresponsiveness to salvage chemotherapy regimens. We conclude that patients with BAL should have a risk stratification with treatment tailored to their immunophenotype and gene rearrangement profiles.     

A new approach to the evolution of the blastic crisis from chronic myelocytic leukemia: dynamic interplay of cellular alterations and a changing microenvironment.

The mechanisms responsible for the massive hyperplasia and for the blastic crisis in chronic myelocytic leukemia are poorly understood. The most generally accepted hypothesis proposes that this progression is due to the development of genetic instability in the leukemic cells. In particular, the two phases of the disease are believed to reflect different, discrete genetic events. Such events remain undefined as yet, and the causal significance of observed genetic aberrations is not clear. An alternative hypothesis is presented here. It is assumed that the feedback interactions adjust the relative probabilities of maturation and replication of the 'committed' as well as the pluripotent cells, and further that mitotic cells at all stages possess considerable phenotypic adaptability; in particular their self-renewal capacity can vary in response to changes in the cellular composition of the tissue even within a conventionally defined compartment. On this basis, it is shown that chronic leukemia can arise and evolve into the blastic crisis from a progressive decline in a single clonal characteristic--inducibility to maturation. It is shown, with the help of mathematical considerations, how an initial hereditable event in an early hemopoietic cell can cause a disturbance of the tissue which feeds back onto the individual members of the clone, resulting in a cascade of dynamic changes which can lead to blast cell dominance.     

Restoration of energy metabolism in leukemic mice treated by a siddha drug--Semecarpus anacardium Linn. nut milk extract

Chronic myeloid leukemia (CML) is a clonal disorder characterized by proliferation of hematopoietic cells that possess the BCR-ABL fusion gene resulting in the production of a 210 kDa chimeric tyrosine kinase protein. CML, when left untreated, progresses to a blast phase during which the disease turns aggressive and shows poor response to known treatment regimens. We have studied a Siddha herbal agent, Semecarpus anacardium Linn. nut milk extract (SA) for its antileukemic activity and its effect on the changes in energy metabolism in leukemic mice. Leukemia was induced in BALB/c mice by tail vein injection of BCR-ABL(+) 12B1 murine leukemia cell line. This resulted in an aggressive leukemia, similar to CML in blast crisis, myeloid subtype, confirmed by histopathological study and RT-PCR for the p210 mRNA in the peripheral blood, spleen and liver. Leukemia-bearing mice showed a significant increase in lipid peroxides, glycolytic enzymes, a decrease in gluconeogenic enzymes and significant decrease in the activities of TCA cycle and respiratory chain enzymes as compared to control animals. SA treatment was compared with standard drug imatinib mesylate. SA administration to leukemic animals resulted in clearance of the leukemic cells from the bone marrow and internal organs on histopathological examination and this was confirmed by RT-PCR for the p210 mRNA. Treatment with SA significantly reversed the changes seen in the levels of the lipid peroxides, the glycolytic enzymes, the gluconeogenic enzymes and the mitochondrial enzymes. These effects are probably due to the flavonoids, polyphenols and other compounds present in SA which result in total regression of leukemia and correction of the alterations in energy metabolism. Study of animals treated with SA alone did not reveal any adverse effects. On the basis of the observed results, SA can be considered as a readily accessible, promising and novel antileukemic chemotherapeutic agent.     

The prognostic significance of cytological, histological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts. A follow up study

Two independent observers performed a double review of cytological and histological bone marrow material obtained at diagnosis and during follow up in 34 patients with the myelodysplastic syndrome (MDS), subtypes refractory anaemia (RA) and RA with ringed sideroblasts (RA-S) (26 and 8 patients, respectively). Average values were used for the analyses. Data obtained at diagnosis confirmed earlier observations that a worse prognosis was indicated by high blast cell counts (P less than 0.01), presence of blast foci and clonal cytogenetic abnormalities (P = 0.08). Data obtained during follow up, in addition, showed that an increased probability of progression to FAB-subtype RA with an excess of blasts was related to both the occurrence of blast foci (P less than 0.05) and the occurrence of new or additional clonal abnormalities (karyotype shift) (P less than 0.01). The relationship between parameters investigated at diagnosis, during follow up, and in the pooled material, points to RA-S being a separate entity having a better prognosis than RA, and further substantiates an earlier observed relationship between blast cell accumulation and the frequency of cytogenetically abnormal metaphases.     

Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre-B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.     

The immunophenotypic and cytogenetic characteristics of the blast cells in subvariants of acute myeloid leukemia

The results of study of morpho-cytochemical peculiarities, antigenic profile and peripheric blood and/or bone marrow blast cell karyotype of 21 patients with acute myeloid leukemia are presented. Hemopoietic cell immunophenotyping was carried out with the use of cytofluorometer FACScan and chromosome cytogenetic analysis with the use of analyzer "Metascan". It has been shown that in the AML M1 blast cell plasmatic membrane carries pan-myeloid CD33 and CD13 antigens, the last having high density of expression, and the CD38 antigen, which is a myeloid cell-precursor marker. In these patients tetraploidy, being the testimony of karyotype change, has been ascertained. It has been found out that the AML M2 blasts, except pan-myeloid antigens, express the cell proliferation CD71 marker. Blast cell karyotype peculiarities typical for this leukemia sub-variant have been revealed. In patients with the AML M4 in 3 of 6 cases an anomalous karyotype has been found. It has been also shown that the CD14 antigen, and rather its percentage in blast total population, is the differential-diagnostic criterion for the AML M4 and M5.     

Prognostic significance of some clinical, morphological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts

In 27 patients initially diagnosed as refractory anaemia (RA) or RA with sideroblasts (RA-S) according to the FAB-classification a number of clinical, morphological and cytogenetic parameters were correlated for prognostic significance. From these correlations it emerged that severe cytopenia is centrally positioned with regard to clinical course in RA and RA-S. Positive correlations were found to initial diagnosis, clonal cytogenetic abnormalities, progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia (AML), the percentage of bone marrow blast cells and prolonged half life for radioactively labeled iron. The degree of peripheral blood granulocytopenia, alone, was correlated to bone marrow hypoplasia. Moreover, the frequency of abnormal karyotypes was inversely correlated to bone marrow cellularity and proportional to the frequency of bone marrow blast cells. From these relationships it may be proposed that chromosome abnormalities are associated with prolonged blast cell generation times and inhibition of blast cell maturation resulting in reduced marrow cellularity and blast cell accumulation, and, in the peripheral blood, falling percentages of neutrophil granulocytes. With the blast cell accumulation the bone marrow cellularity again becomes hyperplastic and the preleukaemic condition is transformed into RAEB or AML.     

Thymocyte subsets transformed by Abelson murine leukemia virus.

The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.     

Clonal growth and self-renewal of human myeloid leukemia cell lines (BRM and DD) in serum-free semi-solid culture

Primary and secondary colony formation of two new human myeloid leukemia cell lines (BRM and DD) were studied in serum-free semisolid cultures. The results indicate that bovine serum albumin and transferrin were essential for clonal growth in chemically defined medium. Insulin contributed only moderately beneficial effects. Initial cell density was also a major modulator of plating efficiency. Positive cooperation between the leukemia cells was shown by using autologous conditioned media. This is the first serum-free culture method that allows self-renewal of human myeloid leukemia cell lines in terms of secondary colony formation in methylcellulose cultures.     

Tumors as clonal proliferation.

Cytogenetic studies indicate that most tumors are clonal (i.e. unicellular in origin) and have karyotypic alterations. These are not consistent, but non-random abnormalities are being increasingly identified by banding techniques, pointing to the sites on human chromosomes where genes important in neoplastic development are located. It is postulated that tumor progression occurs as a result of genetic lability within the neoplastic clone, leading to emergence of increasingly mutant subpopulations (often recognizable cytogenetically) with more malignant properties. In the context of this hypothesis, acute leukemia, chronic leukemia, and preleukemia can be viewed as differing only in the rate at which an abnormal hemic clone is expanding, with progression to a more aggressive phase (e.g. the "blast crisis" of chronic granulocytic leukemia) reflecting emergence of a new predominant subpopulation as the result of an additional genetic change. These concepts, and the cytogenetic data from which they have been derived, may help our understanding of basic tumor biology, and have some practical applications in the diagnosis of human neoplasms.