Mutations in ras genes in myelocytic leukemias and myelodysplastic syndromes

Acute myelocytic leukemias (AML) are characterized by a remarkably high incidence (approximately 30%) of point mutations affecting codons 12, 13, or 61 within ras genes. A predominant involvement of N-ras sequences has been established. Neither Philadelphia chromosome-positive chronic myelocytic leukemia nor other chronic myeloproliferative disorders show a similar frequency. However, a proportion of myelodysplastic syndromes, namely, the chronic myelomonocytic subtype (CMML) also show this molecular feature. The following is a brief discussion of the possible biologic and clinical implications of these observations.     

Human megakaryocytopoiesis--normal and abnormal.

Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management.     

Molecular genetics of chronic neutrophilic leukemia,chronic myelomonocytic leukemia and atypical chronic myeloid leukemia

According to the 2008 World Health Organization classification, chronic neutrophilic leukemia, chronic myelomonocytic leukemia and atypical chronic myeloid leukemia are rare diseases. The remarkable progress in our understanding of the molecular genetics of myeloproliferative neoplasms and myelodysplastic/myeloproliferative neoplasms has made it clear that there are some specific genetic abnormalities in these 3 rare diseases. At the same time, there is considerable overlap among these disorders at the molecular level. The various combinations of genetic abnormalities indicate a multi-step pathogenesis, which likely contributes to the marked clinical heterogeneity of these disorders. This review focuses on the current knowledge and challenges related to the molecular pathogenesis of chronic neutrophilic leukemia, chronic myelomonocytic leukemia and atypical chronic myeloid leukemia and relationships between molecular findings, clinical features and prognosis.     

Myelodysplastic syndromes.

The myelodysplastic syndromes are a heterogeneous group of hematopoietic stem cell disorders characterized by dysplastic and ineffective hematopoiesis and a varying risk of transformation to acute leukemia. Although the natural history of these syndromes is variable, several factors appear to be of prognostic importance, including the French-American-British classification, the karyotype, in vitro colony formation, and others. The pathogenesis of the myelodysplastic syndrome is not known, but recent evidence suggests that alterations of cellular oncogenes may be a causative factor. There is no standard therapy for myelodysplasia, and thus novel approaches to patient management are warranted.     

Identification of 3q21q26 syndrome by "multipoint" interphase FISH analyses in childhood myeloid leukemia

Cytogenetic syndrome involving bands 3q21 and 3q26, known as "3q21q26 syndrome" has been observed in adult patients with acute myelogenous leukemia (0.5-2%), chronic myelogenous leukemia in blast crisis (20%), myelodysplastic syndromes and myeloproliferative disorders. In the present study bone marrow samples from two boys (12 and 16 years), diagnosed with CML and AML respectively, were investigated using conventional cytogenetic methods, interphase "multipoint" fluorescence in situ hybridization (FISH), dual color-FISH and multiplex FISH. The "multipoint" FISH analysis identified in de novo childhood AML case an inv(3)(q21q26) and a complex 3q rearrangement including inversion and duplication in the CML case. The "3q21q26 syndrome" is associated with normal or elevated platelet counts with marked abnormalities of megakaryocytopoiesis, involvement of multiple hematopoietic lineages. The affected patients were resistant to conventional chemotherapy and had a short survival. This syndrome is very rare in de novo childhood AML, and simultaneous presence of 3q inversion and duplication, to our knowledge, has not yet been identified in hematological malignancies. The results of our study emphasize the importance of classical and modern cytogenetic analysis in the diagnosis of hematologic malignancies, because in the majority of cases they can provide additional diagnostic information for the clinicians in deciding the best therapeutic approach, precise classification and prognosis of the disease.     

Case report: A large extramedullary granulocytic sarcoma as the initial presenting feature of chronic myeloid leukemia

Granulocytic sarcomas (chloromas) are rare extramedullary tumors consisting of primitive granulocytic cells. They arise de novo, or are associated with other hematologic disorders such as acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative disorders. We report here on a case of a 62-year-old woman who presented with a large swelling in her right groin and leg. The mass was confirmed by biopsy to be a granulocytic sarcoma. Bone marrow examination showed mild hypercellularity but no evidence of increase in blast count. However, cytogenetic examination of the marrow showed t(9;22), indicating an unexpected diagnosis of chronic myeloid leukemia.     

Chronic neutrophilic leukemia with marked myelodysplasia terminating in blast crisis

A case of chronic neutrophilic leukemia (CNL) is reported. The patient had a history of bleeding, and showed sustained mature neutrophilic leukocytosis, hepatosplenomegaly, a high leukocyte-alkaline phosphatase score, elevated serum vitamin B12 and uric acid, and the presence of D?hle bodies in the neutrophils. In addition to the above typical features, marked myelodysplastic changes in the erythroid and megakaryocytic series were observed in the bone marrow. Three months after the diagnosis of CNL had been established, the hematological picture evolved into a blast crisis of monocytic type. Such findings give support to the statement of CNL as a distinct entity belonging to the spectrum of the myeloproliferative disorders.     

Lectin histochemistry of human bone marrow: investigation of trephine biopsy specimens in normal and reactive states and neoplastic disorders

Summary The lectin binding pattern of bone marrow cells in normal and reactive states and in various neoplastic disorders was investigated using trephine biopsy specimens taken from the iliac crest. The tissue samples were routinely processed (fixed in formalin and embedded in paraffin wax) and subjected to mild decalcification with EDTA. The following results were obtained. (1) More than half of the 23 fluoresceinated lectins used reacted with normal blood cells and/or their neoplastic derivatives. Inhibition tests with the appropriate sugars confirmed the specificity of binding for the majority, but not all, of the lectins. (2) WGA, Con A, PSA, STA and RCA₆₀ and RCA₁₂₀ produced a particularly intense reaction with normal, reactive and neoplastic myeloid cells. Erythroblasts exhibited weak staining in a few cases by a few lectins (WGA producing the strongest staining), while megakaryocytes nearly always remained unstained. Neoplastic lymphoid cells in various lymphoproliferative disorders and plasmacytoma cells generally reacted with the same lectins as the myeloid cells. (3) Since neoplastic myeloid cells in various myelodysplastic and myeloproliferative disorders exhibited a lectin binding pattern similar to that of myeloid cells in normal and reactive bone marrow, it is unlikely that lectin histochemistry of the bone marrow will prove of great value in the diagnosis of myelodysplastic—myeloproliferative disorders.     

积分系统参与MDS诊断的可行性研究

目的:骨髓增生异常综合征(MDS)是一种起源于造血干细胞,以高风险向急性髓系白血病转化为特点的难治性血细胞质、量异常的异质性疾病。对不明原因血细胞减少或者有巨幼红细胞血症的老年人,如果得不到明确的诊断就不会获得有效的治疗。探讨对伴有不明原因血细胞减少或大红细胞血症患者采用一种积分系统来预测诊断为骨髓增生异常综合征(MDS)的可行性。方法:应用四个因子即年龄≥65,红细胞平均体积(MCV),红细胞分布宽度(RDW),和乳酸脱氢酶(LDH)的一个积分系统来计算诊断MDS的概率。结果:调查了303个病人,他们都因为不明原因的血细胞减少或者大红细胞血症在过去三年(2010~2012)进行了骨髓检查,最终三分之二患者被诊断为MDS,9%为疑似MDS,采用积分系统前后的MDS诊断率分别为12%和48%。结论:此四个因子的积分系统可以用于指导MDS患者的诊断。提高MDS的诊断率,减少不必要的骨髓穿刺,减轻病人的疼痛和医疗费用。     

骨髓增生异常综合征合并非霍奇金淋巴瘤一例病例报道并文献复习

目的:探讨骨髓增生异常综合征合并非霍奇金淋巴瘤的发病机制、诊断及治疗,并判断其预后情况。方法:回顾我院收治的一例骨髓增生异常综合征(MDS)患者同时诊断间变大细胞淋巴瘤(ALCL)患者的临床资料,总结其诊断及治疗经过,对其预后进行评价。结果:该患者诊断间变大细胞淋巴瘤后,经化疗对症治疗病情无显著改善,且患者ALK阳性,考虑预后较差,建议造血干细胞移植。结论:骨髓增生异常综合征合并非霍奇金淋巴瘤的病例在国内外十分罕见,治疗方法目前尚需进一步研究。     

G-CSF and GM-CSF in clinical trials.

Hematopoietic growth factors have now been purified, cloned, and produced in bacteria and yeast. Those that are currently in clinical study include erythropoietin, GM-CSF, G-CSF, M-CSF (also called CSF-1), and multi-CSF (also called interleukin 3). Growth factor appear likely to enhance the recovery and function of circulating white cells after standard-dose cancer therapy and high-bone-dose cancer therapy with marrow transplant and to restore leukocyte numbers and competence in the acquired immune deficiency syndromes and myelodysplastic syndromes. Phase I, II trials in AIDS, in cancer patients receiving chemotherapy, in cases of myeloproliferative disease, and after bone marrow transplant have been published. The results of phase III studies are just becoming available.     

Cytogenetic study of 20 cases of refractory anemia

The myelodysplastic syndromes are a group of hematological disorders not yet clearly defined. The authors describe the chromosomal aspects of 20 cases observed in the region of Clermont-Ferrand in comparison with the bibliographical data.     

Mitochondrial diseases in children including Leigh syndrome--biochemical and molecular background

Mitochondrial diseases in children are more frequently caused by mutations in nuclear DNA then in mtDNA. Special clinical phenotypes are associated with the mutations in SURF1 gene, in SCO2 gene and with mtDNA depletion syndromes. Leigh syndrome is the most common clinical presentation of various mitochondrial disorders during childhood. Elevation of lactate in blood, cerebrospinal fluid and urine is a simple biochemical marker of mitochondrial disorders but its specificity and sensitivity are low. Biochemical investigation of muscle biopsy and search for mitochondrial mutations remain a gold standard in the diagnosis. The standarized diagnostic criteria to establish level of diagnostic certainty (possible, probable, definite) are proposed to be used in practice; these include clinical features, neuroimaging and muscle biopsy investigations. Further research directions to improve our understanding of mitochondrial pathologies in children are suggested.     

Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are clonal hematologic malignancies characterized by pancytopenia, dysplastic hematopoiesis, and a propensity to leukemic transformation. Increased apoptosis has been noted in MDS as a possible explanation for ineffective hematopoiesis, with lower levels in progression to and in de novo acute leukemia. Apoptosis can be measured by binding of Annexin V to exposed membrane phosphatidylserine. We postulated that the apoptotic index would aid in the differential diagnosis of MDS versus other hematopoietic diseases. We examined 33 bone marrow aspirates suspected of hematopoietic malignancy for apoptotic index by Annexin V analysis using a Becton Dickinson FACStar+ flow cytometer. The apoptotic index was expressed as the percentage of Annexin V-positive cells divided by total mononuclear cells in the gate. By standard morphologic analysis, 16 cases were diagnosed as MDS (9 refractory anemia [RA], 2 refractory anemia with ringed sideroblasts [RARS], 1 refractory anemia with excess of blasts [RAEB], 3 chronic myelomonocytic leukemia [CMML], and 1 unclassified), 11 as acute leukemia (AL), 6 as myeloproliferative disorders (MPD). Eight cases (uninvolved marrow of five patients with lymphoproliferative disorders [LPD], one patient with multiple myeloma, and two patients with anemia of chronic disease) served as nonneoplastic controls. A higher degree of apoptosis was observed in MDS (mean = 44.7%; range = 29.5--60%) compared with MPD (mean = 8.2%; range = 2.3--15.4%), AL (mean = 16.1%; range = 5.1--29.4%), and control marrow samples (mean = 11.6%; range = 1.5--21%). Additionally, the apoptotic index was significantly higher in MDS compared with MPD (P < 0.0001). In conclusion, a high apoptotic index occurs in MDS, supporting previous reports and suggesting that Annexin V analysis can be used as an adjunct in the diagnosis of MDS versus MPD. This would be particularly useful for the often-difficult distinction between early MDS and early MPD cases with equivocal morphology.     

Megakaryocytic features useful for the diagnosis of myeloproliferative disorders can be obtained by a novel unsupervised software analysis

An unsupervised method for megakaryocyte detection and analysis is proposed, in order to validate supplementary tools which can be of help in supporting the pathologist in the classification of Philadelphia negative chronic myeloproliferative disorders with thrombocytosis. The experiment was conducted on high power magnification photomicrographs taken from hematoxylin-and-eosin 3 micrometer thick sections of formalin fixed, paraffin embedded bone marrow biopsies from patients with reactive thrombocytosis or chronic myeloproliferative disorders. Each megakaryocyte has been isolated in the photos through an image segmentation process, mainly based on mathematical morphology and wavelet analysis. A set of features (e.g. area, perimeter and fractal dimension of the cell and its nucleus, shape complexity via elliptic Fourier transform, and so on) is used to characterize the disorders and discriminate between essential thrombocythemia and idiopathic myelofibrosis. Features related to the general contour of the cell like cytoplasmic area and perimeter are good markers in distinguishing between normal or reactive and pathologic megakaryocytes while nuclear features and global circularity are helpful in the differential diagnosis between ET and prefibrotic IMF. The method proposed should be considered as a fast preprocessing tool for the diagnostic phase and its use can be extended to solve different object recognition problems.     

An empirically derived approach to the classification and diagnosis of mood disorders

This article describes a system for diagnosing mood disorders that is empirically derived and designed for its clinical utility in everyday practice. A ran-dom national sample of psychiatrists and clinical psychologists described a randomly selected current patient with a measure designed for clinically ex-perienced informants, the Mood Disorder Diagnostic Questionnaire (MDDQ), and completed additional research forms. We applied factor analysis to the MDDQ to identify naturally occurring diagnostic groupings within the patient sample. The analysis yielded three clinically distinct mood disorder dimen-sions or spectra, consistent with the major mood disturbances included in the DSM and ICD over successive editions (major depression, dysthymia, and mania), along with a suicide risk index. Diagnostic criteria were determined strictly empirically. Initial data using diagnostic efficiency statistics sup-ported the accuracy of the dimensions in discriminating DSM-IV diagnoses; regression analyses supported the discriminant validity of the MDDQ scales; and correlational analysis demonstrated coherent patterns of association with family history of mood disorders and functional outcomes, supporting va-lidity. Perhaps most importantly, the MDDQ diagnostic scales demonstrated incremental validity in predicting adaptive functioning and psychiatric his-tory over and above DSM-IV diagnosis. The empirically derived syndromes can be used to diagnose mood syndromes dimensionally without complex di-agnostic algorithms or can be combined into diagnostic prototypes that eliminate the need for ever-expanding categories of mood disorders that are clini-cally unwieldy.     

Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation. MLL-CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL-CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal gamma-irradiation or N-ethyl-N-nitrosourea (ENU), MLL-CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy-induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL-CBP therapy-related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.     

Analysis of human dysplastic haematopoiesis in long-term bone marrow culture

In this paper we have analysed the behaviour of myelodysplastic marrow in a long-term bone marrow liquid culture system (LTBMC) from eleven patients with myelodysplastic syndromes with regard to cellularity, day-7 and day-14 CFU-GM growth, cluster formation, adherent cells and CFU-F formation. An altered CFU-GM pattern was found in 64% of cases at diagnosis, while normal growth was seen in the remaining cases, all of which were affected by refractory anaemia. The levels of CFU-GM, as well as cellularity, were reduced in myelodysplastic marrows compared to normal controls over the whole duration of LTBMCs. Cases with a normal CFU-GM level at diagnosis also showed pathological behaviour when examined in LTBMC. The duration of dysplastic haematopoiesis was significantly shorter than that of controls. The proliferative ability of CFU-F was reduced in 50% of cases as shown by replating experiments. In conclusion, myelodysplastic marrow shows an abnormal behaviour in LTBMC, even in those cases which present normal CFU-GM growth at diagnosis.     

Molecular mechanisms that produce secondary MDS/AML by RUNX1/AML1 point mutations

RUNX1/AML1 point mutations have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. A heterozygous germline mutation of the RUNX1 gene causes a familial platelet disorder with a predisposition to AML. RUNX1 mutations have also been detected with high frequency in minimally differentiated AML M0 subtypes and myelodysplastic/myeloproliferative neoplasms. Here we propose a new disease category of myelodysplastic neoplasms (MDN) consisting of MDS refractory anemia with excess blasts and AML with myelodysplasia-related changes, including therapy-related cases. RUNX1 mutations have been detected in about 20% of patients with "MDN". Among the MDN cases, histories of radiation exposure, therapy-related myeloid neoplasms after successful treatment for acute promyelocytic leukemia, and leukemic transformation of myeloproliferative neoplasms have been reported to have a strong association with RUNX1 mutations. The mutations occur in a normal, a receptive, or a disease-committed hematopoietic stem cell. It is suspected that the "MDN" phenotypes are defined by the RUNX1 mutations in addition to some other abnormalities.     

Myelodysplastic syndromes: their history, evolution and relation to acute myeloid leukaemia

The myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal disorders arising from a multipotent haemopoietic progenitor which share a leukaemic propensity, 30% of cases culminating in acute myeloid leukaemia (AML). Their pathogenesis probably entails multiple steps, phenotypic progression being determined by either expansion or evolution of the abnormal clone. The clonal origin of certain cases of de novo AML is analogous to that of MDS and evidence that they share a common pathogenesis and distinct biological characteristics is beginning to emerge.