Recent advances in the diagnosis and classification of myeloid neoplasms--comments on the 2008 WHO classification.

The fourth edition of the World Health Organization (WHO) classification of myeloid neoplasms refined the criteria for some previously described myeloid neoplasms and recognized several new entities based on recent elucidation of molecular pathogenesis, identification of new diagnostic and prognostic markers, and progress in clinical management. Protein tyrosine kinase abnormalities, including translocations or mutations involving ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB, and FGFR1, have been used as the basis for classifying myeloproliferative neoplasms (MPN). Two new entities - refractory cytopenia with unilineage dysplasia and refractory cytopenia of childhood have been added to the group of myelodysplastic syndromes (MDS), and 'refractory anemia with excess blasts-1' has been redefined to emphasize the prognostic significance of increased blasts in the peripheral blood. A list of cytogenetic abnormalities has been introduced as presumptive evidence of MDS in cases with refractory cytopenia but without morphologic evidence of dysplasia. The subgroup 'acute myeloid leukemia (AML) with recurrent genetic abnormalities' has been expanded to include more molecular genetic aberrations. The entity 'AML with multilineage dysplasia' specified in the 2001 WHO classification has been renamed 'AML with myelodysplasia-related changes' to include not only cases with significant multilineage dysplasia but also patients with a history of MDS or myelodysplasia-related cytogenetic abnormalities. The term 'therapy-related myeloid neoplasms' is used to cover the spectrum of disorders previously known as t-AML, t-MDS, or t-MDS/MPN occurring as complications of cytotoxic chemotherapy and/or radiation therapy. In this review, we summarize many of these important changes and discuss some of the diagnostic challenges that remain.

Analysis of prognostic factors in patients with refractory anemia with excess of blasts (RAEB) reclassified according to WHO proposal.

The WHO classification subdivides the FAB RAEB category into RAEB-1 (bone marrow (BM) blasts <10%, peripheral blasts <5%) and RAEB-2 (bone marrow blasts >10% and peripheral blasts >5%). We reclassified according to WHO criteria 228 RAEB patients and analysed them in terms of haematological, karyotypic and prognostic features. We used the database of 680 MDS patients referred to our Institution from 1990 to 2000. Clinical features at presentation, such as sex, age, leukocyte count, polymorphonuclear cell count (PMN), platelet count, haemoglobin level, presence of one or more lineage dysplasia were tested in univariate and multivariate analysis in the two groups of RAEB-1 and RAEB-2 reclassified patients. In multivariate analysis we identified prognostic significant factors in the two patient groups, which consisted of age >70 years and platelet count <100 x 10(9)l(-1) for RAEB-1 category, while for RAEB-2 group parameters negatively influencing survival and risk of progression were haemoglobin <10g/dl, platelet count <100 x 10(9)l(-1), bone marrow blastosis >15% and complex karyotype. We also found differences in cytogenetic data (more balanced translocations and complex karyotypes in RAEB-2 group, p=0.02), and in survival (23.3 months in RAEB-1 vs. 16.1 months in RAEB-2 group, p=0.001). WHO classification provides valuable prognostic information for RAEB patient population, and can identify those subjects with more unfavourable prognosis who should be offered alternative therapeutic strategies.

Refractory anaemia with excess of blasts in transformation re-evaluated with the WHO criteria: identification of subgroups with different survival.

One of the major changes suggested by the World Health Organization (WHO) classification with respect to the French-American-British (FAB) proposal for myelodysplastic syndromes (MDS) was to lower the bone marrow (BM) blast count from 30 to 20%, thus eliminating the refractory anaemia with excess of blasts in transformation (RAEB-t) category. However, a general consensus has not been reached, and several authors still retain RAEB-t as an MDS sub-entity. We re-evaluated our series of 74 patients classified as RAEB-t according to the FAB criteria by stratifying them into two subsets: patients with at least 5% peripheral blast (PB) cells but with BM blasts <20% (group I) and patients with BM blastosis between 20 and 30% and PBs <5% (group II). We found differences among the two groups regarding sex, haematological parameters at presentation (white blood cell and neutrophil counts, haemoglobin level) and frequency of infectious episodes during the course of disease. We did not find differences as to the frequency of acute myeloid leukaemia transformation, but a significant difference was evidenced as to survival (9.3 vs. 16 months in group I vs. group II, respectively; p = 0.02). Furthermore, at our institution, we compared the RAEB-t group I patients who, based on >5% PBs, should be included in the RAEB-II category according to the WHO criteria, with a group of 98 patients who were diagnosed as RAEB-II according to the WHO criteria. The findings showed that the aggregation of these two subsets appeared inappropriate, because patients of the two groups showed different clinical features and rates of acute transformation. In conclusion, the RAEB-t entity according to the FAB criteria, although including heterogeneous clinical patient subsets, should more likely be considered as an advanced stage of MDS, rather than a true acute myeloid leukaemia.

Refractory anaemia with excess of blasts (RAEB): analysis of reclassification according to the WHO proposals.

The French-American-British (FAB) classification assigns patients with myelodysplastic syndromes to the category of refractory anaemia with excess blasts (RAEB) if they have a medullary blast count of 5-20%, and/or a peripheral blast count of 2-5%. The new World Health Organization (WHO) classification subdivides RAEB into RAEB I with a medullary blast count < or =10% and a peripheral blast count < or =5% and RAEB II with >10% medullary and/or >5% peripheral blasts. RAEB II is also diagnosed if Auer rods are present. In 558 patients, we analysed these subtypes of RAEB in terms of haematological characteristics, karyotype anomalies and prognosis. RAEB I was diagnosed in 256 and RAEB II in 302 patients. In the RAEB II group, 22% of patients had >5% peripheral blasts or the presence of Auer rods. The median survival was 16 months for RAEB I as compared with 9 months for RAEB II. Patients with Auer rods, regardless of their medullary and peripheral blast count, had no worse prognosis. No significant differences were identified between the RAEB subtypes with respect to clinical, morphological, haematological and cytogenetic parameters. The survival data support the WHO reclassification of RAEB based on peripheral and medullary blast counts and Auer rods. The WHO classification is useful for diagnosis and provides risk stratification, supported by cytogenetic data for clinical decision making, identifying those RAEB patients with an unfavourable prognosis who should be offered chemotherapy or stem cell transplantation.

Apoptosis susceptibility and cell-cycle distribution in cells from myelodysplastic syndrome patients: modulatory in-vitro effects of G-CSF and interferon-alpha.

Susceptibility to apoptosis varies in different forms of myelodysplastic syndromes (MDS). Our in vitro study aimed at better defining the cell kinetic profile by investigating whether G-CSF and interferon-alpha (IFNalpha) were capable of controling apoptotic/proliferative mechanisms in RAEB as well as in RAEB-t forms. Apoptosis and cell-cycle distribution were measured in mononuclear and in CD34+ cells from bone marrow samples of 27 MDS patients with RAEB (n = 15) and RAEB-t (n = 12). In selected samples, the in vitro influence of G-CSF and lymphoblastoid (Ly)-IFNalpha on the apoptotic susceptibility and on the cell kinetics of the above MDS populations was evaluated. RAEB samples showed a significantly greater apoptosis than RAEB-t ones, both in mononuclear cells (14.76%+/-8.73 vs. 5.95%+/-3.88, P= 0.0058) and in CD34+ cells (24.66%+/-16.08 vs. 3.96%+/-2.57, P = 0.0007). Short-term cell culture in the presence of G-CSF reduced apoptosis in CD34+ cells in all 4 RAEB samples tested (39.1%+/-40.7 vs. 21.0%+/-23.5, P = n.s.); the percentage of cells in S-phase significantly increased in 3/4 samples (19.90%+/-4.40 vs. 32.40%+/-7.85, P = 0.03). Ly-IFNalpha protected CD34+ cells from apoptosis in 3/4 RAEB samples (25.7%+/-8.06 vs. 10.9%+/-8.8, P = n.s.), but did not modulate cell-cycle distribution. G-CSF and Ly-IFNalpha failed to affect apoptosis and proliferation in RAEB-t. These observations indicate that in RAEB forms increased apoptosis can be efficiently counteracted in most of the samples by both G-CSF and Ly-IFNalpha, suggesting that only in these forms a retained regulatory mechanism on the apoptotic/ proliferative balance may allow therapeutic intervention with apoptotic regulators.

Factors influencing survival in myelodysplastic syndromes in a Brazilian population: comparison of FAB and WHO classifications.

The WHO classification for myelodysplastic syndromes (MDS) has introduced new categories with prognostic relevance. Our aim was to examine the predictive value of the WHO and the FAB classification compared to parameters of peripheral blood, bone marrow and IPSS. Clinical data, peripheral blood counts, bone marrow (BM) cytology and histology and survival were analyzed in consecutive newly diagnosed adult patients with MDS. All cases were diagnosed according to FAB criteria and reclassified by the WHO proposal. Among 150 patients entering the study median age was 58 years (12-90). According to FAB, 90 patients had refractory anemia (RA), 18 sideroblastic anemia, 34 refractory anemia with excess of blasts (RAEB), three RAEB-t and five chronic myelomonocytic leukemia. Using the WHO proposal, one half of the patients with RA changed category. One patient had the 5q-syndrome. There were 25 cases with refractory cytopenias with multilineage dysplasia (RCMD) and 23 WHO "unclassified". These last patients presented few cell atypias, favorable IPSS and a good survival as has been described for refractory cytopenias in pediatric MDS. Hypocellular BM was found in 24% of the patients. Karyotype was available in only 85 cases. In the univariate analysis, both classifications, hemoglobin values, hypercellular bone marrow and IPSS had an influence on survival. Using the bootstrap resampling as stability test for the model created by the multivariate analysis, the WHO classification entered the model in 73%, FAB in 38% and IPSS in only 7%. Therefore, in a setting with a high number of low-risk MDS, the WHO classification is the best predictor of survival of the patients.

Prognostic indicators and scoring systems for predicting outcome in patients with myelodysplastic syndromes.

A number of clinical, laboratory, morphological and genetic factors are useful to predict the natural course of Myelodysplastic syndromes (MDS). The identification of these factors resulted in the development of scoring systems that aid to differentiate high risk patients from those with a better prognosis. At the initial approach towards a patient with MDS the clinician will take into account the individual's age and performance score, and the morphological characteristics of the peripheral blood and bone marrow, including number of dysplastic lineages and blast count, as proposed by the new World Health Organization classification. Some laboratory features like the neutrophil and platelet count and the lactate dehydrogenase levels are of additional independent prognostic importance. Finally, the karyotype of the malignant hematopoietic cells is a very strong prognostic variable and therefore mandatory in the assessment of patients with MDS. By using part of the above-mentioned factors, the International Prognostic Scoring System has proven reliable in grouping MDS patients into one of four risk categories and can be used in the stratification of patients in therapeutic trials. With the avenue of more sophisticated molecular techniques like gene expression profiling, it might become possible not only to predict the natural course of the disease more precisely, but also to identify patient populations that are prone to respond to specific drugs especially designed for specific genetic lesions.

Refractory anemia with excess of blasts in transformation: analysis of reclassification according to the WHO proposals.

The category of "refractory anemia with excess blasts in transformation" (RAEB-T) has been abandoned in the new WHO-classification of myelodysplastic syndromes (MDS). The majority of patients previously belonging to this category are now classified as acute myeloid leukaemia (AML). In the FAB-classification, patients had been assigned to the RAEB-T category if they had either (1) a medullary blast count between 20 and 30% or (2) a peripheral blast count of at least 5%, or (3) Auer rods detectable, irrespective of the blast count. We analyzed these subtypes of RAEB-T in terms of hematological characteristics, karyotype anomalies, and prognosis. Patients with more than 20% medullary blasts and patients with at least 5% peripheral blasts as the sole defining parameter for RAEB-T had a median survival of 6 months, as compared to 11 months in patients with Auer rods as the sole defining parameter. The presence of Auer rods therefore does not convey a particularly bad prognosis and does not justify placing patients in a high-risk category of MDS or even classifying them as AML. This finding supports the elimination of Auer rods as a parameter for classification in the new WHO system. On the other hand, the reclassification into RAEB II (according to WHO proposals) of previous RAEB-T patients with a peripheral blast count of at least 5% is problematic, because this feature predicts a median survival not different from that of AML patients.

Erythroleukemia: a need for a new definition.

The new WHO classification abolishes the frontier between RAEB-t with 20% of blasts and leukemia with 30% of blasts. We review the definitions of erythroleukemia and discuss the relationship between FAB AML6, RAEB-t and AML6 variant. We ask whether secondary erythroleukemias are the same entity as RAEB-t on survival, karyotype and cytologic characteristics. We suggest that 'AML6 variant' with pure erythroid lineage proliferation would be the real de novo erythroleukemia. Current FAB AML6 entity will probably be classified in either subgroup (1) multilineage dysplasia; (2) therapy-related leukemia; or (3) acute erythroid leukemia subdivided into erythroleukemia (erythroid/myeloid) and pure erythroid leukemia, in the WHO classification - a classification which highlights the importance of clinical and cytogenetic prognostic factors.

Re-evaluation of refractory anemia with excess blasts in transformation.

The category 'refractory anemia with excess blasts in transformation (RAEBt)' consists of two sub-sets; one group is categorized based on the percentage of blasts in the marrow (> or =20%) and other is based on the percentage of blasts in the peripheral blood (> or =5%). We separated RAEBt patients based on these two criteria and compared hematologic and clinical relevance to assess the reasonable basis for the new classification. All RAEBt patients showing peripheral blood (PB) blasts of > or =5% were re-classified as RAEB by the WHO classification. This subset of RAEBt patients had lower percentages of bone marrow (BM) blasts, and notably they showed frequent complex cytogenetic abnormalities, including -5/5q- and/or -7/7q-. Moreover, the RAEBt patients of this group had shorter survivals compared to RAEBt patients with BM blasts between 20 and 30%. We next assessed hematologic and clinical relevance between refractory anemia with excess blasts (RAEB) and RAEBt patients with PB blasts of > or =5%. Except for the percentage of blasts in the PB (P=0.0037) and BM (P=0.0073), there was no significant difference in hematologic or clinical features between RAEB patients with BM blasts of > or =11% and RAEBt patients with PB blasts of > or =5%. When MDS patients with PB blasts of > or =5% (RAEBt by the FAB classification) were included as RAEB-II based on the "MDS 2000 classification', there was a high frequency of patients with complex chromosome changes, involving 5q and 7q, with significant poorer outcome compared to those with RAEB-I. Although it is still controversial whether MDS patients with BM blasts 20% or more should be considered as acute leukemia, the utilization of the 'MDS 2000 classification' might be useful to designate MDS patients diagnosed based on the percentage of blasts in the peripheral blood.

A variant form of myelodysplastic syndrome with Ph- minor-BCR/ABL transcript.

This study concerns a patient with minor (m)-BCR/ABL transcript-positive and Philadelphia (Ph) chromosome-negative myelodysplastic syndrome (MDS). The patient was a 78-year-old man whose condition was diagnosed as refractory anemia with excess of blasts in transformation. Molecular genetic studies, using reverse transcriptase polymerase chain reaction analysis detected m-BCR/ABL messenger RNA. We used spectral karyotyping to analyze metaphase cells but could not detect a Ph chromosome. Fluorescence in situ hybridization, however, revealed fusion signals of BCR and ABL probes on an apparently normal chromosome 22.

The myelodysplastic syndromes: towards a functional classification.

The French-American-British classification of myelodysplastic syndromes (MDS) has contributed greatly to better communication and conduct of clinical trials. However, the advent of novel cytogenetic, immunological and molecular techniques in recent years warrant some alterations to this purely morphological classification. This review aims at highlighting the advances which reflect more closely the unique biological and clinical features of various subtypes of MDS. We propose a comprehensive classification of MDS, to include the newly defined categories, as well as those not included in previous classifications, such as the therapy-induced and hereditary MDS. We hope that this classification will help in focusing attention on the biological features of MDS, the understanding of which will be crucial to combat this disease.

Fas/Apo-1 (CD95) expression and apoptosis in patients with myelodysplastic syndromes.

Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% +/- 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% +/- 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.

Mixed myelodysplastic and myeloproliferative syndromes.

Aplastic anemia, myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (MPD) are stem cell disorders. There is no clear-cut demarcation of them. Hypoplastic MDS displays features of aplastic anemia and MDS, on the other side mixed myelodysplastic and myeloproliferative syndromes (MDS-MPS) develop. In our collection of 566 MDS patients, features of myelodysplasia as well as myeloproliferation, MDS-MPS, were present in 25 patients (4.4%). Twelve patients had at the time of diagnosis megakaryocytic proliferation and thrombocythemia beside signs of MDS, and seven had myelodysplasia with granulocytic proliferation and leukocytosis. In another six patients, MDS was the first diagnosis and the proliferative phase developed later during the course of the disease. These patients can be characterized as MDS-MPS in evolution. All subjects had a variable degree of anemia. While the level of thrombocythemia has been relatively stable, the number of leukocytes has been progressive, but rarely extended beyond 100 x 10(9)/l. Ring-sideroblasts and myelofibrosis were frequent findings. Two more homogeneous MDS-MPS groups emerged in our analysis: sideroblastic anemia with thrombocythemia and a group fulfilling the criteria of Philadelphia chromosome negative and bcr-abl negative "atypical chronic myeloid leukemia (aCML)'. One patient with thrombocythemia and three with leukocytosis (23%) transformed to acute myeloid leukemia (AML). Men prevailed (12/13) in patients with leukocytosis and MDS-MPS in evolution. Of the 46% MDS-MPS patients with chromosomal aberrations, del(20)(q) is of interest.