Cardiovascular sequelae in long-term survivors of young peoples' cancer: a linked cohort study.

BACKGROUND: We aimed to define the incidence and risk of cardiovascular late effects (LEs) identified from inpatient hospital episode statistics (HES) among long-term survivors of cancer in young people by age at diagnosis (0-14 and 15-29 years). METHODS: Records from the Yorkshire Specialist Register of Cancer in Children and Young People (1991-2006) were linked to inpatient HES data (1996-2011) to assess rates of cardiovascular LEs. Rates were compared with the general population in Yorkshire using age-sex-matched HES records for the entire region. RESULTS: Of 3247 survivors of cancer, 3.6% had at least one cardiovascular LE. Overall, cardiovascular hospitalisations for the childhood cohort were threefold higher compared with the general population, but did not differ for young adults. For young adults, increased rates were limited to pericardial disease, cardiomyopathy and heart failure, pulmonary heart disease, hypertension and conduction disorders. CONCLUSIONS: Survivors of childhood and young adult cancer remain at increased risk of cardiovascular LEs compared with the general population.

Population-based demographic study of karyotypes in 1709 patients with adult acute myeloid leukemia.

Few large demographic studies of acute myeloid leukemia (AML) are derived from population-based registries. Demographic and karyotypic data were provided for AML cases from two regional leukemia registry databases in Scotland and the Northern Region of England. A population-based dataset was compiled, comprising 1709 patients aged >16 years (1235 North England/474 Scotland patients). The most common cytogenetic abnormalities involved chromosomes 5 and/or 7 (17%). Patients with the following abnormal chromosome 5/7 combinations: -5, del(5q), -5/-7 and del(5q)/-7 represented a significantly older population (P < 0.01, ANOVA). t(8;21) was the only 'favourable' karyotype found in older age. Karyotypic complexity varied within chromosome 5/7 combination groups; those containing -5, -5/-7, -5/del(7q), del(5q)/-7 or del(5q)/del(7q) combinations were significantly more frequently complex than those containing -7 and del(7q) (P < 0.01, chi2 test). Additional recurring cytogenetic abnormalities within complex karyotypes containing chromosome 5/7 combinations included (in order of frequency), abnormalities of chromosomes 17, 12, 3 and 18. Complex karyotypes not involving chromosomes 5 or 7 represented 30% of all complex karyotypes, occurred in younger patients than those involving chromosomes 5 and 7, and frequently included additional trisomy 8 (26%). In conclusion, we describe subgroups within adverse karyotypes, with different demographics, degree of complexity and additional chromosome abnormalities.

Mutant RAS selectively promotes sensitivity of myeloid leukemia cells to apoptosis by a protein kinase C-dependent process.

RAS mutations arise at high frequency in human malignancy and have been shown to play a role in the disruption of both normal differentiation and proliferation. In addition, RAS influences a number of intracellular signaling pathways, which impinge on proteins that regulate programmed cell death. In this study, we have examined whether this oncogene can influence the activation of the apoptotic process induced by a range of therapeutic agents used to treat leukemia, and we have identified the downstream targets of RAS mediating the observed changes in sensitivity. Using myeloid leukemia cells (P39) retrovirally transduced with mutant H-RAS, we found that the influence of this oncogene was highly dependent on the inducer used: whereas RAS had no significant effect on spontaneous apoptosis or on the response to the cytotoxic drugs (doxorubicin or 1-beta-arabinofuranosylcytosine), P39-RAS cells showed a strongly augmented response to all-trans-retinoic acid (ATRA) in both the induction of apoptosis and differentiation. Because, under some circumstances, RAF has been associated with promoting apoptosis, we examined whether the activation of this kinase by mutant RAS could be responsible for the augmented response to ATRA. However, constitutive activation of RAF did not alter the apoptotic sensitivity of these cells, making it unlikely that RAS promotes apoptosis by stimulating this kinase. Nor did we find that BCL-2 was differentially down-regulated in P39-RAS cells. Rather, we found that the activation of protein kinase C (PKC) by low-dose phorbol ester could almost entirely recapitulate transformation by RAS, in terms of promoting both apoptosis and differentiation after treatment with ATRA. Moreover, the RAS-induced phenotype could be completely abolished by a specific inhibition of PKC under conditions that had no effect on the response of control cells. In conclusion, we have shown that mutant RAS promotes differentiation-associated cell death in P39 cells by stimulating the activity of PKC, which is itself an important regulator of myeloid differentiation. PKC activation, in turn, powerfully synergizes with the PKC-independent action of ATRA. This work identifies a possible explanation for the ability of this oncogene to promote myeloid differentiation of hematopoietic cells. Clinically, it raises the possibility that although leukemias expressing mutant RAS may not show an altered response to cytotoxic agents, they may show enhanced sensitivity to differentiation therapy with ATRA.

In vitro growth of myeloid and erythroid progenitor cells from myelodysplastic patients in response to recombinant human granulocyte-macrophage colony-stimulating factor.

Marrow progenitor cells from 14 myelodysplastic (MDS) patients and 17 normal donors were assayed in semisolid cultures supplemented with increasing doses of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or medium conditioned by 5637 bladder carcinoma cells (5637CM). At doses of supplements shown to be optimal for colony formation in cultures of normal marrow, myeloid (day 14) colony numbers were subnormal in 10 of 14 MDS marrows cultured in 5637CM and in 8 of 14 cultures containing rhGM-CSF (2.5 ng/ml). However, a high dose of rhGM-CSF (20 ng/ml) raised myeloid colony numbers in cultures of many MDS marrows, so that 9 of 14 now yielded colonies within the normal range; increased levels of 5637CM failed to do this. Erythroid colony growth was poor in 13 of 14 MDS marrow cultures supplemented with erythropoietin in addition to 5637CM or rhGM-CSF. High concentrations of rhGM-CSF did not increase erythroid growth. These data suggest that myeloid progenitors from the MDS clone may have a decreased responsiveness to hemopoietins which can be overcome at high concentrations of growth factors.

Estimation of effective and total erythropoiesis in myelodysplasia using serum transferrin receptor and erythropoietin concentrations, with automated reticulocyte parameters.

The erythroid abnormality in patients with myelodysplasia (MDS) is multifactorial, with ineffective erythropoiesis and poor in vitro progenitor response to erythropoietin (EPO). Serum EPO concentration is variable among patients for a given haemoglobin concentration. We studied 19 non-transfusion-dependent patients with MDS, and 13 healthy elderly control subjects in an attempt to define the factors governing variability in serum EPO and to further characterise the anaemia of MDS. Serum EPO concentration was appropriate for the degree of anaemia in 15/19 MDS patients, and was positively related to mean cell volume (MCV), mean cell haemoglobin (MCH), and percentage highly fluorescent reticulocytes (% HFR), but not to absolute or percentage reticulocyte count. Although the observed/predicted ratio for serum transferrin receptor (TfR) concentration was low in 12 of 19 MDS subjects, no relationship to haemoglobin concentration, reticulocytes or serum EPO was seen. Serum TfR was positively correlated with WBC and platelet counts. Serum TfR was higher in patients with sideroblastic anaemia than refractory anaemia. Standardized in vivo p50 was positively correlated to red cell 2,3 diphosphoglycerate concentration, although this was not the only factor influencing the oxygen dissociation curve. We conclude that effective erythroid output responsive to endogenous EPO drive in MDS is positively related to MCV, MCH and % HFR. Serum TfR may not represent effective output as precisely as % HFR, but may be proportional to total marrow erythropoietic activity.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

Functional disturbance of marrow stromal microenvironment in the myelodysplastic syndromes.

The potential contribution of abnormal marrow stromal function to ineffective haemopoiesis in the myelodysplastic syndromes is unclear. We have compared the ability of stromal layers from normal (n = 7) and myelodysplastic (n = 9) marrow to alter proliferation and survival of the granulocyte-macrophage colony-stimulating factor/interleukin-3-dependent cell line F-36P. Co-cultures for 72 h in the absence of exogenous cytokines were either in direct contact with stroma or separated by transwell inserts. On normal stromal layers, the ratio of adherent F-36P cells relative to stromal cells increased from a mean of 0.2 +/- 0.01 (s.d.) at 4 h of co-culture to 0.34 +/- 0.08 after 72 h (n = 7). Corresponding values on myelodysplastic stroma (0.2 +/- 0.02 at 4 h and 0.35 +/- 0.05 at 72 h; n = 9) indicated that the ability of myelodysplastic stromal layers to regulate short-term proliferation of F-36P cells may be similar to normal. Apoptosis of F-36P cells was quantified after co-culture with normal or myelodysplastic stroma: results from myelodysplastic co-cultures were standardized as a fraction of values from co-cultures with paired normal stroma (apoptotic ratio). Augmented apoptosis of F-36P cells was detected in 8/9 co-cultures with myelodysplastic stroma (mean = 15.7 +/- 9.7%, n = 9), compared with corresponding normal stroma (mean = 12.4 +/- 4.6%, n = 7, P < 0.05) with a mean apoptotic ratio of 1.4 +/- 0.5 (P < 0.05). There was no correlation between stroma-related apoptosis and FAB type, tumour necrosis factor-alpha concentrations in the culture supernatant or numbers of stromal macrophages, and no evidence of involvement of the Fas pathway. Increased apoptosis was detected in cells grown in transwell inserts over stroma (23.8 +/- 3%, n = 5) compared to adherent cells in cultures with normal stromal layers, but this survival difference was not observed in co-cultures with myelodysplastic stroma. These results suggest that abnormal stromal function in patients with myelodysplastic syndromes may contribute to increased apoptosis of haemopoietic cells within the marrow microenvironment. The effect appears to be dependent on close cellular contact, rather than the release of soluble factors, but the exact mechanism remains unclear.

Primary acquired sideroblastic erythropoiesis in non-anaemic and minimally anaemic subjects.

Six patients had primary sideroblastic erythropoiesis together with a haemoglobin concentration of 12.0 g/dl or higher. In four cases this was associated with macrocytosis. Other abnormalities included failure of erythroid progenitor growth from peripheral blood in three cases and occasional dysplastic appearances in neutrophils and megakaryocytes. Sideroblastic erythropoiesis seems to be an early manifestation of the myelodysplastic syndrome and may present clinically at a pre-anaemic stage.

Identification of disease- and therapy-associated proteome changes in the sera of patients with myelodysplastic syndromes and del(5q).

Using ProteinChip array technology, which is based on the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed proteomic analyses on sera from myelodysplastic syndromes (MDSs) patients with an interstitial deletion of the long arm of chromosome 5 (del(5q)) and those from control individuals. One analysis with 80 samples from 29 patients and 51 control subjects resulted in the detection of 61 peak differences. Another analysis with 36 paired-samples from 18 patients collected before and after the treatment with lenalidomide (Revlimid) identified 19 differential peak features. We also observed differential profiles between the pre-treatment samples from the responders and those from the non-responders reflected by eight peak differences. On the basis of these data we developed two classification models that could distinguish between the diseased and the control subjects or between the responders and the non-responders. Efforts were made to purify and identify a range of differential peak proteins. We conclude that inter-α trypsin inhibitor, heavy chain H4 (fragments), serum transferrin, transthyretin (variants), haemoglobin and a protein peak at m/z 2791 could be potential disease-associated markers for del(5q) MDS. Platelet factor 4 (PF-4) and a peak at m/z 8559 may serve as therapy-associated markers and be potentially useful for monitoring and predicting the response to therapy.

Evolving trends in the treatment of low-risk myelodysplastic syndromes: immunomodulation and beyond--9th European Hematology Association Congress Geneva, Switzerland, 10-13 June 2004.

The 9th European Hematology Association Congress, held in Geneva, Switzerland, from 10 through 13 June 2004, offered a number of educational programmes that focused on myelodysplastic syndromes (MDS). This report will summarize the material presented at the educational symposium entitled 'Evolving Trends in the Treatment of Low-Risk MDS: Immunomodulation and Beyond'. The overview of the presentations includes a comparative review of the classification systems for MDS; a discussion of treatment strategies and management issues for patients in lower risk disease categories; a description of a novel class of immunomodulators, the IMiDs((R)), and a presentation of updated data from clinical trials of the IMiD compound, lenalidomide, in the treatment of MDS.

Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia.

Mutations in the receptor tyrosine kinase (RTK/RAS) signalling pathway frequently provide a proliferative signal in myeloid malignancies. However, the role of RASSF1A, SHP-1 and SOCS-1, negative regulators of RTK/RAS signalling, has not been extensively investigated in the myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). This study employed methylation-specific polymerase chain reaction (MS-PCR) to determine if aberrant promotor methylation of RASSF1A, SHP-1 and SOCS-1 is involved in the pathogenesis of myeloid malignancies. Patients with MDS (n = 107), AML (n = 154) and juvenile myelomonocytic leukaemia (JMML, n = 5) were investigated, together with 15 normal controls. Primers were located in the promotor region of each gene as well as within exon 2 of SOCS-1. Methylation of RASSF1A was found in five of 55 (9%) MDS cases, but not in any of 57 AML cases studied. RASSF1A methylation was present in one case (20%) of JMML. SHP-1 methylation was present in 13 of 121 (11%) AML cases but was not found in MDS or JMML. SOCS-1 promoter methylation was present in eight of 74 (11%) MDS patients but was not seen in JMML or AML. Importantly, RAS mutations and RASSF1A and SOCS-1 methylation were mutually exclusive indicating that approximately 30% of MDS cases had a defect of the RTK/RAS pathway and its negative regulation. Finally, SOCS-1 exon 2 methylation may not be pathogenetically relevant, since it was detected in samples from normal individuals and did not correlate with promotor methylation.

Colony-cluster ratio and cluster number in cultures of circulating myeloid progenitors as indicators of high-risk myelodysplasia.

Circulating myeloid progenitors were assayed in 172 normal subjects and 147 patients with myelodysplastic syndrome (MDS). Patients whose cultures had colony/cluster ratios (CCR) less than 0.3 had significantly shorter survival periods than comparable patients with CCR greater than 0.3. A second prognostic indicator, which complemented CCR, was identified in patients with less than 5% blasts. Median survival was significantly reduced in patients with greater than 15 clusters/ml blood despite colony and cluster numbers being predominantly within the normal range. Characteristic differences were found in three FAB groups large enough to allow statistical analysis. Survival amongst patients with refractory anaemia with excess of blasts (RAEB) was related to CCR and was independent of cluster number. Amongst sideroblastic patients (SA) survival related only to cluster number. Refractory anaemia (RA) patients included individuals in both high-risk groups with only three patients out of 64 showing both features. Amongst all the MDS patients, those with CCR greater than 0.3 and less than 15 clusters/ml blood formed a low-risk group (n = 60) with a relatively good prognosis of whom 85% survived the study period (median duration 938 d) including 94% of those in this group with less than 5% marrow blast cells.

Growth response to cytokines of circulating myeloid progenitors from myelodysplastic patients at diagnosis and more than 600 days after diagnosis.

Myeloid colony growth from the peripheral blood of myelodysplastic (MDS) patients was assessed for abnormal in vitro response to haemopoietic growth factors (granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-3 (IL-3)). Abnormal colony growth, increased or reduced, was observed with each of the factors. No specific growth pattern was related to any of the French-American-British classification (FAB) types of disease. MDS patients who had survived greater than 600 days after diagnosis (n = 34) showed significantly fewer abnormalities than patients assayed at the time of diagnosis (n = 37), the major difference being less frequent stimulation of colony growth. These findings indicate that the time of sampling relative to diagnosis needs to be considered when interpreting the in vitro response to growth factors of myeloid colonies from MDS patients.

Pathogenesis, etiology and epidemiology of myelodysplastic syndromes.

BACKGROUND AND OBJECTIVE: The myelodysplastic syndromes are common hematological malignancies affecting predominantly elderly people. Patients usually present with chronic cytopenias which gradually worsen due to progressive bone marrow failure or transformation into acute myeloid leukemia. Disease prevention is more cost-effective than therapeutic intervention and the establishment of the etiology and pathogenesis of MDS therefore assumes considerable importance. This review will outline current concepts of the pathobiology of MDS, putative etiological insults and the mechanisms of disease initiation as well as recent contributions to the descriptive epidemiology of these disorders. EVIDENCE AND INFORMATION SOURCES: The authors of the present review have a long-standing interest in the pathogenesis, etiology and epidemiology of MDS. Journal articles covered by the Science Citation Index and Medline have been reviewed and personal experience and discussion with international experts collated. STATE OF THE ART AND PERSPECTIVES: The initiation processes for the development of MDS remain unknown. A poorly defined transforming event affects a pluripotent or multipotent progenitor cell in the bone marrow, conferring a growth advantage upon it and eventually establishing clonal hematopoiesis. An important pathogenetic mechanism in MDS is premature intramedullary cell death via excessive apoptosis, explaining the apparent paradox of a cellular marrow in combination with peripheral cytopenias (ineffective hematopoiesis). Therapy-related MDS/AML following exposure to alkylating agents is the only clear etiological factor thus identified. Increasing evidence for exposure to benzene and radiation and the development of MDS is emerging. Benzene hematotoxicity is mediated via both genotoxic and non-genotoxic mechanisms, leading to aplasia, apoptosis and initiation (via genetic mutation) of clonal disorders such as MDS. Further studies of benzene hematotoxicity and therapy-related MDS should provide models for the elucidation of initiation events in MDS pathogenesis. The importance of such studies is emphasized by the rising frequency of MDS which largely reflects improved diagnostic criteria, increased physician awareness and extended use of diagnostic procedures in the elderly. Demographic changes will lead to a marked increase in MDS over the next few decades.