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.     

Treatment of poor-risk myelodysplastic syndromes and acute myeloid leukemia with a combination of 5-azacytidine and valproic acid

5-azacytidine (AZA) has become standard treatment for patients with higher-risk myelodysplastic syndrome (MDS). Response rate is about 50% and response duration is limited. Histone deactylase (HDAC) inhibitors are attractive partners for epigenetic combination therapy. We treated 24 patients with AZA (100?mg/m(2), 5?days) plus valproate (VPA; continuous dosing, trough serum level 80-110?μg/ml). According to WHO classification, 5 patients had MDS, 2 had MDS/MPD, and 17 had acute myeloid leukemia (AML). Seven patients (29%) had previously received intensive chemotherapy, and five had previous HDAC inhibitor treatment. The overall response rate was 37% in the entire cohort but significantly higher (57%) in previously untreated patients, especially those with MDS (64%). Seven (29%) patients achieved CR (29%) and two PR (8%), respectively. Hematological CR was accompanied by complete cytogenetic remission according to conventional cytogenetics in all evaluable cases. Some patients also showed complete remission according to FISH on bone marrow mononuclear cells and CD34(+) peripheral blood cells, as well as by follow-up of somatic mitochondrial DNA mutations. Four additional patients achieved at least marrow remissions. Factors influencing response were AML (vs. MDS), marrow blast count, pretreatment, transfusion dependency, concomitant medication with hydroxyurea, and valproic acid (VPA) serum level. This trial is the first to assess the combination of AZA plus VPA without additional ATRA. A comparatively good CR rate, relatively short time to response, and the influence of VPA serum levels on response suggest that VPA provided substantial additional benefit. However, the importance of HDAC inhibitors in epigenetic combination therapy can only be proven by randomized trials.     

Chromosomes in myelodysplastic syndrome: structural abnormalities of chromosome 11. Czechoslovak MDS Cooperative Group

In a series of 121 consecutive patients with a myelodysplastic syndrome (MDS), studied in two laboratories, of which 87 (71.9%) had abnormal karyotypes, twelve had a structural abnormality of the long arm of chromosome 11 (13.8%). There were six deletions, one ring chromosome and five reciprocal translocations, all involving a chromosome band 11q23. Of these twelve patients, five had a refractory anemia (RA) and seven a refractory anemia with excess of blasts (RAEB). RA was associated more frequently with 11q deletions as the sole abnormality, while translocations or multiple chromosome abnormalities were commonly associated with RAEB. The study shows that the 11q aberrations represent frequent structural chromosome rearrangements in MDS.     

The peculiarities of cytogenetic changes in different types of myelodysplastic syndrome

A cytogenetic study of bone marrow aspirate from 32 patients with different types of myelodysplastic syndrome (MDS) has been carried out. The patients were from eight regions of Ukraine. Chromosome deletions prevailed in the spectrum of karyotype changes. The largest number of chromosome abnormalities was revealed in patients with a refractory anemia with an excess of blasts (66.6% of cases). Chromosomal changes that involved three or more chromosomes occurred among 27% of all karyotype changes examined by us. Transformation of myelodysplastic syndrome to acute myeloid leukemia (AML) was found in 5 patients (45.4% of the cases) among 11 patients with abnormal karyotypes. We propose that cytogenetic confirmation of increased apoptosis in the bone marrow from the myelodysplastic syndrome patients is a phenomenon of chromosome fragmentation. The risk of transformation of myelodysplastic syndrome to acute myeloid leukemia was measured with the use of a new international score system, IPSS.     

Detection of submicroscopic deletions in band 17p13 in patients with the Miller-Dieker syndrome

The Miller-Dieker syndrome (MDS), a syndrome with lissencephaly, distinctive craniofacial features, growth impairment, and profound developmental failure, has been associated with a deletion of the distal part of chromosome band 17p13. A minority of patients with the syndrome do not have a deletion detectable with current cytogenetic techniques. Using three highly polymorphic DNA probes (pYNZ22, pYNH37.3, and p144D6) we have detected microdeletions in three MDS patients, two of whom had no visible abnormalities of chromosome 17. Loci defined by two of the DNA probes, pYNZ22 and pYNH37.3, were deleted in all three patients. The most distal locus, defined by p144D6, was present in one MDS patient, possibly defining the distal limits of the MDS region in band 17p13.3. None of these loci were absent in one case of lissencephaly without MDS.     

SNP array karyotyping allows for the detection of uniparental disomy and cryptic chromosomal abnormalities in MDS/MPD-U and MPD

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder.     

Chromosomal instability correlates with poor outcome in patients with myelodysplastic syndromes irrespectively of the cytogenetic risk group

Chromosomal instability (CIN), defined by an elevated frequency of the occurrence of novel chromosomal aberrations, is strongly implicated in the generation of aneuploidy, one of the hallmarks of human cancers. As for aneuploidy itself, the role of CIN in the evolution and progression of malignancy is a matter still open to debate. We investigated numerical as well as structural CIN in primary CD34‐positive cells by determining the cell‐to‐cell variability of the chromosome content using fluorescence‐in situ‐hybridization (FISH). Thereby, CIN was measured in 65 patients with myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and control subjects. Among MDS patients, a subgroup with elevated levels of CIN was identified. At a median follow‐up of 17.2 months, all patients within this ‘high CIN’ subgroup had died or progressed to AML, while 80% of MDS patients with normal CIN levels had stable disease (P < 0.001). Notably, there was no statistically significant difference between ‘normal CIN’ and ‘high CIN’ MDS patients regarding established risk factors. Hence, elevated CIN levels were associated with poor outcome, and our method provided additional prognostic information beyond conventional cytogenetics. Furthermore, in all three MDS patients for whom serial measurements were available, development of AML was preceded by increasing CIN levels. In conclusion, elevated CIN levels may be valuable as an early indicator of poor prognosis in MDS, hence corroborating the concept of CIN as a driving force in tumour progression.     

Acute Myeloid Leukemia (AML) with Erythroid Predominance Exhibits Clinical and Molecular Characteristics that Differ from Other Types of AML

The clinical importance of erythroid predominance in bone marrow of patients with acute myeloid leukemia (AML) is controversial. These cases represent a heterogeneous group of diseases that historically have been classified into different categories. We studied 313 AML patients and specifically compared the clinical, cytogenetic, and molecular features of cases of AML with erythroid predominance, arbitrarily defined as ≥50% erythroid precursors, to AML cases without erythroid predominance. We also assessed 51 patients with a high-grade myelodysplastic syndrome (MDS), refractory anemia with excess blasts (RAEB). All neoplasms were classified according to the World Health Organization classification. With the exception of therapy-related AML/MDS, the presence of erythroid predominance in variously classified categories of AML was associated with a survival advantage. In addition, AML with erythroid predominance had a lower frequency of cytogenetic abnormalities as well as a lower frequency of mutations involving NPM1, NRAS and FLT3 as compared with AML without erythroid predominance. We conclude that the clinical, cytogenetic, and molecular features of AML with erythroid predominance in the non-therapy-related setting are much closer to those of a high-grade myelodysplastic syndrome than they are to other types of AML.     

Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization.

Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13) from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed in a blinded comparison of three MDS patients with submicroscopic deletions and in four normal relatives used as controls. The controls showed both chromosome 17 homologues labeled in 85%-95% of cells, while each patient showed only one homologue labeled in 75%-80% of cells. Two MDS patients with cryptic translocations were also studied. In one case, a patient and her mother had the same der(17) (p+), but the reciprocal product of the translocation could not be identified in the mother by G-banding (i.e., it was a "half-cryptic" translocation). FISH revealed a 3q;17p translocation. The other case involved a patient with apparently normal karyotype. Because a large molecular deletion was found, a translocation involving two G-negative telomeres (i.e., a "full-cryptic" translocation) was postulated. FISH studies on her father and normal brother showed an 8q;17p translocation. These studies demonstrate that in situ hybridization is an efficient method for deletion detection in Miller-Dieker syndrome. More important, parental studies by FISH on patients demonstrating molecular deletions and a normal karyotype may identify cryptic translocation events, which cannot be detected by other molecular genetic strategies. Similar in situ strategies for deletion detection can be developed for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or DiGeorge syndrome.     

Molecular mechanism of the schedule-dependent synergistic interaction in EGFR-mutant non-small cell lung cancer cell lines treated with paclitaxel and gefitinib

The regimen of cytarabine, aclarubicin and G-CSF (CAG) has been widely used in China and Japan for treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We searched literature on CAG between 1995 and 2010 and performed a meta-analysis to determine its overall efficacy using a random-effects or fixed-effects model. Thirty five trials with a total of 1029 AML (n = 814) and MDS (n = 215) patients were included for analysis. The CR rate of AML (57.9%) was significantly higher than that of MDS (45.7%) (p < 0.01). No difference in CR was noted between the new (56.7%) and relapsed/refractory AML (60.1%) (p > 0.05). The CR rate was also significantly higher in patients with favorable (64.5%) and intermediate (69.6%) karyotypes than those with unfavorable one (29.5%) (p < 0.05). Remarkably, the CR rate of CAG was significantly higher than those of non-CAG regimens (odds ratio 2.43). CAG regimen was well tolerated, with cardiotoxicity in 2.3% and early death in 5.2% of the cases. In conclusion, CAG regimen was an effective and safe regimen for the treatment of AML, and may be more effective than non-CAG regimens. Randomized controlled trials are strongly recommended to evaluate its efficacy and safety in comparison with the current standard treatment.     

Cytogenetic survey of 117 Tunisian patients with de novo myelodysplastic syndrome

Cytogenetic studies were performed on 117 Tunisian patients with de novo myelodysplastic syndromes (MDS). According to the French-American-British (FAB) criteria 40 patients presented with refractory anaemia (RA, 34%), eight with refractory anaemia with ringed sideroblasts (RARAS, 7%), 19 with refractory anaemia with excess of blasts (RAEB, 16%), 16 with refractory anaemia with excess of blasts in transformation (RAEB-t, 14%), 18 had chronic myelomonocytic leukaemia (CMML, 15%) and 16 unclassifiable MDS (14%). Seventy-five were men and forty-two were women. Five were children and 112 were adults with a median age of 58 years. Fifty-five per cent of the patients presented clonal chromosome abnormalities. Rates of abnormality varied from one FAB subtype to the other: 55% in RA, 75% in RARAS, 63% in RAEB, 75% in RAEB-t and 28% in CMML. The most frequent chromosome abnormalities were del(5q) (22 cases), monosomy 7 (12 cases), del(12p) (6 cases), and trisomy 8 (5 cases). Rare abnormalities were also found: ring of chromosome 12 and trisomy 15. Conventional cytogenetics remains the basic technique in identifying chromosomal abnormalities associated with MDS.     

Geographic disparities in cardiovascular mortality among patients with myelodysplastic syndromes: A population-based analysis.

IntroductionClonal hematopoiesis, a precursor to myelodysplastic syndromes (MDS), constitutes a novel cardiovascular disease (CVD) risk factor, causing growing interest in cardiovascular outcomes in MDS. Rurality is associated with increased CVD but studies on cardiovascular geographic disparities in MDS are lacking.MethodsUsing the U.S. Surveillance, Epidemiology, and End Results (SEER) registry, we identified 52,750 MDS patients between 2001 and 2016. Rurality was defined using Rural-Urban Continuum Codes. Cox regression estimated the association of rurality and cardiovascular death.ResultsMDS incidence was equal in urban and rural populations (6.7 per 100,000). Crude probability of cardiovascular death was higher among rural MDS patients. Adjusting for age, sex, race/ethnicity, marital status, insurance, and MDS risk (defined from histology), rural patients had 12% increased risk of CVD death compared to urban patients (HR=1.12, 95%CI 1.03–1.21). HR for CVD death was 1.22 (95%CI 1.01–1.5) in patients from the most rural areas (less than 2500 urban population). Among MDS patients younger than 65 years, rurality was associated with 25% increased risk of CVD death (HR=1.25, 95%CI 1.01–1.59).DiscussionThis population-based analysis suggests that rural residence is linked to higher burden of cardiovascular death in patients with MDS. The disparity is not explained by demographic factors or MDS risk. Interventions targeting CVD may improve outcomes in rural MDS patients.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Identification of a metabolic gene panel to predict the prognosis of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is clonal disease featured by ineffective haematopoiesis and potential progression into acute myeloid leukaemia (AML). At present, the risk stratification and prognosis of MDS need to be further optimized. A prognostic model was constructed by the least absolute shrinkage and selection operator (LASSO) regression analysis for MDS patients based on the identified metabolic gene panel in training cohort, followed by external validation in an independent cohort. The patients with lower risk had better prognosis than patients with higher risk. The constructed model was verified as an independent prognostic factor for MDS patients with hazard ratios of 3.721 (1.814-7.630) and 2.047 (1.013-4.138) in the training cohort and validation cohort, respectively. The AUC of 3-year overall survival was 0.846 and 0.743 in the training cohort and validation cohort, respectively. The high-risk score was significantly related to other clinical prognostic characteristics, including higher bone marrow blast cells and lower absolute neutrophil count. Moreover, gene set enrichment analyses (GSEA) showed several significantly enriched pathways, with potential indication of the pathogenesis. In this study, we identified a novel stable metabolic panel, which might not only reveal the dysregulated metabolic microenvironment, but can be used to predict the prognosis of MDS.     

Distinct Clinical and Experimental Characteristics in the Patients Younger than 60 Years Old with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) mainly occur in elderly individuals in Western countries. However, MDS is commonly found in young individuals (<60 years) in Asia. The reason for the high incidence in younger individuals is still unclear, and the differences in disease features between young and elderly patients with MDS have been not well recognized. To explore these issues, in this study, we analyzed the clinical and experimental characteristics of MDS in the patients younger and older than 60 years old and characterized the potential age-associated differences. The results showed that over half of the patients with MDS (61.9%) were younger than 60 years old upon the first diagnosis. The younger patients were more likely to be female, who have lower risk and less advanced MDS. The occurrence of trisomy 8 and bone marrow failure were more frequent in the younger patients than the older ones. The marrow CD34+ cells in the younger patients showed lower proliferation and higher apoptosis in comparison with that in the older ones. Obvious amplification of T cells and low CFU formation could be found in the younger patients. CFU formation was significantly increased in the younger patients after the removal of activated T cells. In addition, the younger patients had a lower frequency of p15^INK4B methylation, longer survival expectancy and less AML transformation. In summary, the younger patients with MDS in China may show more benign disease features than the older ones. Enhanced immunological response may be involved in the pathogenesis of MDS in the patients younger than 60 years.     

Genetic polymorphisms in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a family of clonal disorders characterized by dyshematopoiesis and susceptibility to acute myelogenous leukemia. Tumor necrosis factor-a (TNF-alpha) and transforming growth factor-beta (TGF-beta) are cytokines that play key roles in the pathogenesis of MDS. There have been several reports on the presence of genetic polymorphisms in the DNA sequence encoding the leader sequence of the TGF-beta protein, and in the -308 promoter region of TNF-alpha. The association between TNF-alpha and TGF-beta1 gene polymorphism and the susceptibility to MDS and the progression of the disease was investigated. As compared with healthy control subjects (n = 74), patients with MDS (n = 55) showed no significant deviations in genotype or allele frequencies of TNF-alpha. Similarly, there were no differences in the distribution of TNF-alpha genotypes between the MDS patients with only anemia (mild group) and those with bi- or pancytopenia (severe group). On the other hand the TT homozygosity at codon 10 in exon 1 of TGF-beta1 gene was associated with a severe degree of cytopenia [95% CI OR = 4.889, p = 0.0071]. These findings suggest that the investigated genetic polymorphisms do not predispose to the development of MDS, but that TGF-beta1 gene polymorphism may affect the disease progression.     

Bmi-1基因表达在骨髓增生异常综合征中的临床意义

目的:探讨Bmi-1基因表达在骨髓增生异常综合征(MDS)中的临床意义。方法:选择2011年1月~2012年12月我院收治的MDS患者41例为研究组,另选取非恶性血液病患者20例为对照组,检测Bmi-1的表达水平,并检测其骨髓白血病干细胞免疫表型(CD34+CD38-CD123+),然后分析患者Bmi-1的表达水平与骨髓原始细胞比例及骨髓染色体核型的关系。结果:Bmi-1表达水平研究组患者明显高于对照组,且研究组中RA患者明显低于RAEB患者,但RA患者及RAEB患者均高于对照组(P0.01);Bmi-1基因高表达组白血病干细胞免疫表型CD34+CD38-CD123+/CD34+明显高于Bmi-1基因低表达组患者(P0.01);Bmi-1基因高表达组中,骨髓原始细胞5%的病例数及染色体不良核型发生率均高于低表达组。结论:Bmi-1基因的表达可以作为MDS患者在分子水平上的恶性程度标志之一。     

Flow cytometric evaluation of proliferative activity and ploidy in myelodysplastic syndromes and acute leukemias

Propidium iodide (PI) DNA distribution of bone marrow (BM) cells was studied by flow cytometry (FCM) in 36 patients without hematologic or malignant disease (normal BM) and in 172 patients with anemias (36 pts), myelodysplastic syndromes (MDS) (33 pts) and acute leukemia (AL) at diagnosis (60 pts), remission (24 pts) and relapse (19 pts). White blood cells from normal male subjects were used as an external diploid reference standard (median CV = 3.8). Patients with normal BM, anemias, MDS and acute leukemia at diagnosis had tritiated thymidine labeling index (LI) and most with MDS and AL had also evaluable cytogenetics performed on the same BM sample used for FCM. In normal BM, median aliquot of cells with PI-DNA content intermediate between the diploid and the tetraploid value (2n-4n cells %) was 15.7. The ratio between the fluorescence intensity of the G0/1 peak of normal BM cells and the fluorescence intensity of the G0/1 peak of the reference standard (FI ratio) ranged from 93 to 1.05 (mean +/- 2SD). The 2n-4n cell % was higher than normal in anemias (p less than .001), lower in leukemias (p less than .001) and widely scattered in MDS. A linear correlation was found between 2n-4n cell % and LI, with 2n-4n cell % value higher than LI. The FI ratio was lower than normal in anemias (p less than .05), higher in AL with normal cytogenetics (p less than .02) and broadly scattered in MDS with normal cytogenetics. From our experience, PI-DNA-FCM is a simple and adequate method to evaluate proliferative activity in hematologic diseases. Nevertheless, caution must be taken in attributing small changes in FI ratio to aneuploidy, since they are found in anemias and in MDS and AL with normal cytogenetics, possibly due to differences in PI uptake by different cell types.     

U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.