Prognostic factors and follow-up parameters in patients with paroxysmal nocturnal hemoglobinuria (PNH): experience of the Austrian PNH network.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease characterized by a deregulated complement system, chronic Coombs-negative, intravascular hemolysis, and a variable clinical course with substantial risk to develop thromboembolic events. We analyzed diagnostic and prognostic parameters as well as clinical endpoints in 59 adult patients suffering from PNH in 5 hematology centers in Austria (observation period: 1978-2015). Median follow-up time was 5.6 years. The median clone size at diagnosis amounted to 55% and was higher in patients with classical PNH (81%) compared to patients with PNH associated with aplastic anemia (AA) or myelodysplastic syndromes (MDS) (50%). The clone size also correlated with lactate dehydrogenase (LDH) levels. In one patient, anemia improved spontaneously and disappeared with complete normalization of LDH after 16 years. Seventeen patients received therapy with eculizumab. The rate of thromboembolic events was higher in the pre-eculizumab era compared with eculizumab-treated patients but did not correlate with the presence of age-related clonal hematopoiesis or any other clinical or laboratory parameters. Peripheral blood colony-forming progenitor cell counts were lower in PNH patients compared with healthy controls. Only two patients with classical PNH developed MDS. Overall, 7/59 patients died after 0.5-32 years. Causes of death were acute pulmonary hypertension, Budd-Chiari syndrome, and septicemia. Overall survival (OS) was mainly influenced by age and was similar to OS measured in an age-matched healthy Austrian control cohort. Together, compared with previous times, the clinical course and OS in PNH are favorable, which may be due to better diagnosis, early recognition, and eculizumab therapy.

Therapy-related myelodysplastic syndromes deserve specific diagnostic sub-classification and risk-stratification-an approach to classification of patients with t-MDS.

In the current World Health Organization (WHO)-classification, therapy-related myelodysplastic syndromes (t-MDS) are categorized together with therapy-related acute myeloid leukemia (AML) and t-myelodysplastic/myeloproliferative neoplasms into one subgroup independent of morphologic or prognostic features. Analyzing data of 2087 t-MDS patients from different international MDS groups to evaluate classification and prognostication tools we found that applying the WHO classification for p-MDS successfully predicts time to transformation and survival (both p < 0.001). The results regarding carefully reviewed cytogenetic data, classifications, and prognostic scores confirmed that t-MDS are similarly heterogeneous as p-MDS and therefore deserve the same careful differentiation regarding risk. As reference, these results were compared with 4593 primary MDS (p-MDS) patients represented in the International Working Group for Prognosis in MDS database (IWG-PM). Although a less favorable clinical outcome occurred in each t-MDS subset compared with p-MDS subgroups, FAB and WHO-classification, IPSS-R, and WPSS-R separated t-MDS patients into differing risk groups effectively, indicating that all established risk factors for p-MDS maintained relevance in t-MDS, with cytogenetic features having enhanced predictive power. These data strongly argue to classify t-MDS as a separate entity distinct from other WHO-classified t-myeloid neoplasms, which would enhance treatment decisions and facilitate the inclusion of t-MDS patients into clinical studies.

Comorbidity as prognostic variable in MDS: comparative evaluation of the HCT-CI and CCI in a core dataset of 419 patients of the Austrian MDS Study Group.

BACKGROUND: The evaluation of comorbidity is of increasing importance in patients with hematologic disorders. PATIENTS AND METHODS: In the present study, the influence of comorbidity on survival and acute myeloid leukemia (AML) evolution was analyzed retrospectively in 419 patients with de novo myelodysplastic syndromes (MDS) (observation period: 1985-2007). The median age was 71 years (range 24-91 years). Two different scoring systems, the hematopoietic stem-cell transplantation-specific comorbidity index (HCT-CI) and the Charlson comorbidity index (CCI) were applied. RESULTS: The HCT-CI was found to be a significant prognostic factor for overall survival (OS, P < 0.05) as well as event-free survival (EFS, P < 0.05) in our patients, whereas the CCI was of prognostic significance for OS (P < 0.05), but not for EFS. For AML-free survival, neither the HCT-CI nor the CCI were of predictive value. A multivariate analysis including age, lactate dehydrogenase, ferritin, karyotype, number of cytopenias, French-American-British groups, and comorbidity was applied. Comorbidity was found to be an independent prognostic factor in patients with low- or int-1-risk MDS (P < 0.05) regarding OS and EFS. CONCLUSIONS: Together, our data show that comorbidity is an important risk factor for OS and EFS in patients with MDS.

Prognostic impact of age and gender in 897 untreated patients with primary myelodysplastic syndromes.

BACKGROUND: The International Prognostic Scoring System (IPSS) is the golden standard to assess prognosis in myelodysplastic syndromes (MDS). The aim of this analysis was to study age and gender as interacting variables for individualized prognostication. PATIENTS AND METHODS: In all, 897 patients with primary MDS treated with supportive care only were examined in a retrospective multicenter study. A Cox model was developed to determine the prognostic impact of age and gender on survival and to examine their modulating influence on IPSS results. Based on main effects and interactions of these variables, we established an individualized age- and gender-adapted scoring system to improve prognostication in MDS. RESULTS: While the risk of a patient in the IPSS is best represented by the values 0 (low), +1 (intermediate-1), +2 (intermediate-2), and +3 (high), these values were found to vary between -1.9 and +3.5 in the same patients when including age and gender. Whereas in low-risk MDS, male patients were found to have a less favorable survival, a particularly high risk (+3.5) was found in younger (< or = 66 years) high-risk female patients. CONCLUSION: The inclusion of age and gender and their respective interactions contribute to improved and individualized prognostication in MDS.

Deferasirox induces regression of iron overload in patients with myelodysplastic syndromes.

BACKGROUND: Transfusion-related morbidity is an emerging problem in chronically transfused patients with myelodysplastic syndromes (MDS). Although several iron-chelating drugs are available, the optimal way of treatment of iron-overload remains uncertain. A major disadvantage of deferoxamine is that the drug has to be applied as continuous subcutaneous infusion. Therefore, novel oral agents have been developed. One of these drugs is deferasirox (Exjade). MATERIALS AND METHODS: We report on 14 MDS patients who were treated with deferasirox (500-1500 mg daily) for up to 24 months. In these patients, treatment responses were recorded by determining serum ferritin levels before and during therapy and by applying recently established response criteria. RESULTS: In all patients except one, ferritin levels decreased during therapy. Four patients showed a complete response, one a minor response and five a stable iron load. In the responding patients, initially elevated liver enzymes decreased substantially. No substantial change in transferrin saturation or transfusion frequency was recorded. Side effects were mild and tolerable in most patients. In one patient, treatment with deferasirox was stopped because of impaired kidney function. CONCLUSION: Our data show that treatment with deferasirox is a reasonable approach to counteract iron overload in patients with MDS.

Iron overload in myelodysplastic syndromes (MDS) - diagnosis, management, and response criteria: a proposal of the Austrian MDS platform.

Transfusion-related morbidity is an emerging challenge in chronically transfused patients with low-risk myelodysplastic syndromes (MDS). In these patients, transfusion-induced iron overload may represent a leading medical problem. However, although iron-chelating drugs are available, little is known about optimal diagnostic tools, predisposing factors, and the optimal management of these patients. In the current article, we provide recommendations for the diagnosis, prevention and treatment of iron overload in MDS and propose treatment response criteria. Consensus criteria and resulting recommendations were discussed and formulated by members of the MDS platform of the Austrian Society of Haematology and Oncology in a series of meetings and conferences in 2006 and 2007. These recommendations should facilitate and assist in recognition of iron overload, selection of patients, timing of treatment, drug selection and the measurement of treatment responses.

Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML). PATIENTS AND METHODS: We treated six patients with imatinib-resistant CML in haematological relapse (leukocytes > 20,000 microL(-1)) with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target rapamycin serum concentration of 10-20 pg mL(-1). RESULTS: A major leukocyte response with decrease to less than 10,000 microL(-1) was obtained in two patients, and a minor transient response was seen in two other patients. In responding patients, we also observed a decrease in vascular endothelial growth factor (VEGF) mRNA levels in circulating leukaemic cells. Side effects during rapamycin treatment were mild in most patients. In one patient, pneumonia developed. Rapamycin was also found to counteract growth of CML cells in vitro as determined by (3)H-thymidine incorporation. Moreover, rapamycin inhibited the growth of Ba/F3 cells exhibiting various imatinib-resistant mutants of BCR/ABL, including the T315I variant that exhibits resistance against most currently available BCR/ABL kinase inhibitors. CONCLUSIONS: Rapamycin shows antileukaemic effects in imatinib-resistant CML in vitro and in vivo. Larger trials with rapamycin or rapamycin-derivatives in combination with other targeted drugs are warranted to further determine clinical efficacy in CML.

Cytogenetic patterns in multiple myeloma after a phase of preceding MGUS.

BACKGROUND: Presenting the same histological diagnosis, multiple myeloma (MM) shows a large genomic variety, resulting in variable times of overall survival. MATERIALS AND METHODS: To investigate major cytogenetic categories (any 14q-translocation, t(11;14), t(4;14), 13q-deletions, 17p-deletions) and their clinical consequences in MM after a pre-existing monoclonal gammopathy (MM post-MGUS), we performed a comparative analysis of 41 patients with MM post-MGUS and 287 patients with unknown prior history MM (U-MM). RESULTS: In MM post-MGUS, a t(11;14) was found to be more frequent than in U-MM (24% vs. 14%) and it was associated with significantly shortened survival (24 months vs. 70 months in U-MM; P = 0.01). MM post-MGUS was further characterized by a higher frequency of 13q-deletions only (absence of all other specific abnormalities; 28% vs. 12% in U-MM; P = 0.02). A 13q-deletion only was an indicator of long survival in MM post-MGUS (median not yet reached) as opposed to U-MM (median survival, 29 months; P = 0.001). 17p-deletions were infrequent in MM post-MGUS (3% vs. 16% in U-MM; P = 0.04). Survival times for patients with t(4;14) and/or 17p-deletions and other abnormalities were similar in both MM patient cohorts. CONCLUSIONS: Our data suggest that t(11;14) and 13q-deletions have distinct prognostic implications in the context of MM post-MGUS.

The IPSS-R has prognostic impact in untreated patients with MDS del(5q).

The IPSS-R proved to be a powerful tool for the assessment of prognosis in MDS patients. We aimed at a validation of the IPSS-R for patients with MDS harboring deletion (5q) isolated or accompanied by additional aberrations. The study was based on 444 MDS patients from MDS centers in Europe. 67% of the patients were female, median age was 69 years. 43.5% had MDS del(5q), 5.9% were diagnosed with RCUD, 2.0% RARS, 18.4% RCMD, 14.6% RAEB-I and 15.5% RAEB-II. According to the IPSS-R, there were 9.9% very low, 39.6% low, 16.6% intermediate, 12.8% high, 20.9% very high risk patients. For very low risk patients survival was 7.5 years, low 9.0 years, intermediate 6.5 years, high 1.5 years and very high 0.7 years (p < 0.001). For low and intermediate risk, the probability of AML evolution was significantly different (p = 0.03) as well as for high versus very high risk groups (p = 0.002). The IPSS-R proved to be an appropriate prognostic tool for MDS with del(5q).

Refinement of the international prognostic scoring system (IPSS) by including LDH as an additional prognostic variable to improve risk assessment in patients with primary myelodysplastic syndromes (MDS).

The international prognostic scoring system (IPSS) is considered the gold standard for risk assessment in primary myelodysplastic syndromes (MDS). This score includes several prognostic factors except serum lactate dehydrogenase (LDH). We evaluated the prognostic power of LDH as an additional variable in IPSS-based risk assessment. For this purpose, a total of 892 patients with primary MDS registered by the Austrian-German cooperative MDS study group was analyzed retrospectively. Multivariate analysis confirmed the value of established parameters such as medullary blasts, karyotype and peripheral cell counts and showed that elevated LDH was associated with decreased overall survival (P<0.00005) and increased risk of AML development (P<0.00005), independent of the system used to classify MDS (FAB or WHO). Moreover, elevated LDH was found to be a significant predictor of poor survival within each IPSS risk group and within each FAB group except RAEB-T. To exploit these results for refined prognostication, each IPSS risk group was split into two separate categories (A=normal LDH vs B=elevated LDH). Using this LDH-assisted approach, it was possible to identify MDS patients with unfavorable prognosis within the low and intermediate IPSS risk groups. We propose that the IPSS+LDH score should improve clinical decision-making and facilitate proper risk stratification in clinical trials.

Association of human T-cell leukemia virus and myelodysplastic syndrome in a central European population.

In addition to a few disorders such as acute T-cell leukemia that are typically associated with the human T-cell leukemia virus (HTLV) 1 in endemic regions, this virus may also play a role in some other hematological diseases. Here, we examine the incidence of HTLV in hematological diseases from a nonendemic region in central Europe. Data obtained by PCR and/or serological techniques from a total of 730 cases showed that besides the expected presence of HTLV-1 in T-lymphoid diseases (2 of 27 cases), HTLV-1 was only detected in myelodysplastic syndrome (MDS), in which an incidence of 17% (11 of 65 cases) was found. A correlation with a history of multiple transfusions or treatment with blood products in the HTLV-1-positive MDS could not be ascertained. Cytogenetics detected the presence of del(5)(q) in six HTLV-positive cases (five MDS and one T-cell acute lymphocytic leukemia) but in only one HTLV-negative case. These data indicate that allelic deletions of a series of 5q-located genes that typically occur in MDS may be associated with HTLV infections in central Europe.

In vivo and in vitro effects of cytokines and the hemoregulatory peptide dimer (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 on G alpha16-positive hematopoiesis.

G proteins play an important role in signal transduction from cytokine receptors to intracellular effectors via different pathways, eg involving tyrosine kinases. In our previous studies, we demonstrated that mRNA expression of the hematopoiesis-specific G protein alpha-subunit G alpha16 is a sensitive marker indicating the appearance of early myeloid and lymphoid progenitors. This study was designed to investigate cytokine effects on hematopoiesis in vivo and in vitro as reflected by G alpha16 expression and sensitivity to the hemoregulatory peptide (pEEDCK)2 which harbors a structural homology to the effector domain of G alpha16. Investigations on blood samples from lymphoma patients undergoing salvage therapy with different cytokine support showed that monitoring of the expression of G alpha16 mRNA which appears to play a role in cytokine signalling via tyrosine kinases was a valuable complementation to CD34 screening for analyzing hematopoietic recovery after chemotherapy. We demonstrated that in contrast to CD34 which is only expressed in quiescent cells, G alpha16 transcription occurs independently of cell cycle state. In vitro, we could show that G alpha16 was also a valuable marker for confirming the immature state of ex vivo expanded blood stem cells from patients. A further part of the study was focused on the response of G alpha16 and CD34 expressing cells to the granulocyte-derived hemoregulatory peptide (pyroGlu-Glu-Asp-Cys-Lys)2 = (pEEDCK)2 which harbors a G alpha16-homologous sequence motif. Results obtained from in vitro assays which involved estimation of colony outgrowth from CD34-positive cells showed that the effect of (pEEDCK)2 on CD34 cells enhanced the effect of IL-3 or SCF. These data indicate that G alpha16 may co-operate with (pEEDCK)2 in triggering the cytokine response of immature hematopoietic cells.

Both IGH translocations and chromosome 13q deletions are early events in monoclonal gammopathy of undetermined significance and do not evolve during transition to multiple myeloma.

Molecular and genetic events associated with the transition from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) are still poorly characterized. We investigated serial bone marrow specimens from 11 patients with MGUS who eventually progressed to MM (MM post-MGUS) by interphase fluorescence in situ hybridization for immunoglobulin heavy-chain gene (IgH) translocations and chromosome 13q deletions (del(13q)). In nine patients, IgH translocations were present both in MGUS and MM post-MGUS plasma cells, including three t(11;14)(q13;q32) and one t(4;14)(p16;q32), which was observed already 92 months prior to MM. Similarly, all five MM patients with del(13q) had this aberration already at the MGUS stage. Two patients without IgH translocation and del(13q) had chromosomal gains suggesting hyperdiploidy, but IgH translocations and/or del(13q) did not emerge at MM post-MGUS. IgH translocations and del(13q) are early genetic events in monoclonal gammopathies, suggesting that additional events are required for the transition from stable MGUS to progressive MM.

Expression of G alpha 16, a G-protein alpha subunit specific for hematopoiesis in acute leukemia.

G-proteins are essential in signal transduction pathways. A G-protein alpha subunit termed G alpha 16 was found to be exclusively expressed in hematopoietic cell lines. In cells derived from patients, G alpha 16 expression has been detected in progenitor- and pre-B ALL cells and also in peripheral blood stem cells (PBSC). In this study, we analyzed G alpha 16 expression using a RT-PCR technique by testing elutriated blood cells from normal donors, PBSC from breast cancer patients and bone marrow or peripheral blood cells from acute leukemia patients. Both of two ALL patients and 15/16 AML patients expressed G alpha 16. In elutriation experiments, G alpha 16 expression was found in fractions containing the highest number of precursor cells but was absent in mature T and B cell fractions. In addition, CD34-enriched PBSC were positive for G alpha 16 expression. Further in vitro experiments using the cell line KG1 showed that G alpha 16 expression was not affected by the growth inhibiting hemoregulatory peptide pEEDCK which has a sequence homology present within G alpha 16. Taken together, these data demonstrate that G alpha 16 is expressed in various normal and malignant hematopietic progenitors but not in their differentiated counterparts. G alpha 16 could play a vital role in signal transduction pathways controlling proliferation in early normal and malignant hematopoiesis.

FMS hemizygosity in myeloid dysplasia and acute myeloid leukemia with chromosomal aberration del(5)(q) demonstrated by polymerase chain reaction.

Interstitial deletions of the long arm of chromosome 5 del(5)(q), are recurring aberrations in the myelodysplastic syndrome and acute myeloid leukemia. Several genes located in region (5)(q23-34) have been implicated as being of pathogenic importance. In this study seven samples of six patients with myelodysplastic syndrome and acute myeloid leukemia who have the del(5)(q) aberration were analyzed by polymerase chain reaction (PCR) and Southern blot technique. FMS hemizygosity was demonstrated in all patients. PCR analysis from peripheral blood samples confirmed the observations of this aberration found by semiquantitative Southern blot. PCR-based analysis can be used for primary diagnosis in addition to cytogenetic evaluation and for follow-up in patients with del(5)(q) aberration.

Validation of WHO classification-based Prognostic Scoring System (WPSS) for myelodysplastic syndromes and comparison with the revised International Prognostic Scoring System (IPSS-R). A study of the International Working Group for Prognosis in Myelodysplasia (IWG-PM).

A risk-adapted treatment strategy is mandatory for myelodysplastic syndromes (MDS). We refined the World Health Organization (WHO)-classification-based Prognostic Scoring System (WPSS) by determining the impact of the newer clinical and cytogenetic features, and we compared its prognostic power to that of the revised International Prognostic Scoring System (IPSS-R). A population of 5326 untreated MDS was considered. We analyzed single WPSS parameters and confirmed that the WHO classification and severe anemia provide important prognostic information in MDS. A strong correlation was found between the WPSS including the new cytogenetic risk stratification and WPSS adopting original criteria. We then compared WPSS with the IPSS-R prognostic system. A highly significant correlation was found between the WPSS and IPSS-R risk classifications. Discrepancies did occur among lower-risk patients in whom the number of dysplastic hematopoietic lineages as assessed by morphology did not reflect the severity of peripheral blood cytopenias and/or increased marrow blast count. Moreover, severe anemia has higher prognostic weight in the WPSS versus IPSS-R model. Overall, both systems well represent the prognostic risk of MDS patients defined by WHO morphologic criteria. This study provides relevant in formation for the implementation of risk-adapted strategies in MDS.

Dysplastic versus proliferative CMML--a retrospective analysis of 91 patients from a single institution.

In chronic myelomonocytic leukemia (CMML) segregation of two subtypes has been suggested depending on WBC count-myelodysplastic (MD-CMML) and myeloproliferative (MP-CMML). In a retrospective analysis of 91 (60/31) previously untreated CMML patients, we compared the presenting clinical, haematological, laboratory and bone marrow features and examined the clinical impact of this reclassification. LDH values and bone marrow cellularity were significantly increased in MP-CMML. Median survival was significantly longer for patients with MD-CMML, progression rate was higher for MP-CMML. Patients with MD-CMML had longer median preleukemic duration; after transition to AML, MP-CMML patients had longer median survival. In MDS phase anemia was more common in MP-CMML and thrombocytopenia more common in MD-CMML whereas transfusion rates showed no difference. Evaluation of prognostic scoring systems for both groups confirmed that patients' characteristics and outcome could be well compared. Our data suggest that segregation into MD-CMML and MP-CMML is justified.

Analysis of peripheral blood progenitor cells demonstrates limitations of minimal residual disease diagnosis in a case of acute promyelocytic leukemia.

Detection of minimal residual disease (MRD) by analysis of the PML-RAR alpha fusion transcript using the RT-PCR method is routinely carried out on peripheral blood and bone marrow of patients with APL (AML, FAB:M3). Therapy aims to achieve repeated negative results in these patients thus confirming clinical complete remission. We report a case of APL in second complete remission in which no leukemic cells had been detected in BM and PB for 20 months, and in which PBPC-pheresis was carried out for future transplantation. In two of five pheresis PML-RAR alpha fusion transcripts were detected. This shows that the residual leukemic population may only reach detection level after enrichment by PBPC-pheresis.

Thymidine kinase expression. A marker for malignant cells.

The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to discriminate between normal growing cells and virally transformed cells or lines derived from tumours. In material (blood and bone marrow) taken from leukaemia patients, we identified the leukaemic cells in a surplus of normal leucocytes. From cell cultures representing a tumour progression model, only the later, and malignant, stages showed enhanced fluorescence, whereas benign tumour cells looked normal.