Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

BACKGROUND: In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. METHODS: Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). RESULTS & CONCLUSIONS: We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

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.

Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC).

Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p <0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p <0.0001) and a shorter survival in MM patients with active disease requiring treatment (p ≤ 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM.

Two novel variants of the ABCG5 gene cause xanthelasmas and macrothrombocytopenia: a brief review of hematologic abnormalities of sitosterolemia.

Essentials Diagnosis of sitosterolemia, a rare recessive or syndromic disorder, is usually delayed. Peripheral blood smear is extremely useful for establishing the suspicion of sitosterolemia. High-throughput sequencing technology enables the molecular diagnosis of inherited thrombocytopenias. Accurate characterization of sitosterolemia helps us determine appropriate management. SUMMARY: Background Sitosterolemia (STSL) is a recessive inherited disorder caused by pathogenic variants in the ABCG5 and ABCG8 genes. Increased levels of plasma plant sterols (PSs) usually result in xanthomas and premature coronary atherosclerosis, although hematologic abnormalities may occasionally be present. This clinical picture is unfamiliar to many physicians, and patients may be at high risk of misdiagnosis. Objectives To report two novel ABCG5 variants causing STSL in a Spanish patient, and review the clinical and mutational landscape of STSL. Patient/Methods A 46-year-old female was referred to us with lifelong macrothrombocytopenia. She showed familial hypercholesterolemia-related xanthomas. Molecular analysis was performed with high-throughput sequencing. Plasma PS levels were evaluated with gas-liquid chromatography. The STSL landscape was reviewed with respect to specific online databases and all reports published since 1974. Results A blood smear revealed giant platelets and stomatocytes. Novel compound heterozygous variants were detected in exons 7 (c.914C>G) and 13 (c.1890delT) of ABCG5. The patient showed an increased plasma level of sitosterol. These findings support the diagnosis of STSL. In our review, we identified only 25 unrelated STLS patients who presented with hematologic abnormalities including macrothrombocytopenia. It remains unknown why only some patients develop hematologic abnormalities. Conclusions This is the first Spanish STSL patient to be reported and molecularly characterized. The early diagnosis of STLS is strongly supported by the presence of stomatocytes in blood smears. The definitive diagnosis of STSL by measurement of serum PS levels and molecular analyses prompted the use of ezetimibe therapy.

Results of allogeneic stem cell transplantation in the Spanish MDS registry: prognostic factors for low risk patients.

Although new agents have been approved for the treatment of MDS, the only curative approach is allogeneic hematopoietic stem cell transplantation (HSCT) and thus, in particular circumstances this procedure has been proposed as a treatment option for low risk patients. We have retrospectively analyzed the results of HSCT in 291 patients from the Spanish MDS registry with special attention to low risk MDS (LR-MDS) in order to define the variables that could impact their clinical evolution after transplantation. At 2 years OS was 51% and EFS was 50% (95% CI 0.7-4.5 years for OS and 95% CI 0.1-3.9 years for EFS). Among 43 LR-MDS, transplant-related mortality was 28%. At 3 years, OS was 67% (95% CI 264.7-8927.2 days for OS) and EFS was 64% (95% CI 0-9697.2 days for EFS). In the multivariate analysis only cytogenetics retained statistical significant effect on both OS (p=.047) and EFS (p=.046). Conditioning regimen could improve outcome among this subset of patients (OS 86% and RFS 100% for patients receiving RIC regimen). The present study confirms that specific disease characteristic as well as transplant characteristics have a significant impact on transplant outcome. Regarding low risk patients a non-myeloablative conditioning would be preferable especially in cases without high-risk cytogenetics.

Diagnostic utility of bone marrow examination for the assessment of patients with fever of unknown origin: a 10-year single-centre experience.

Bone marrow (BM) examination is included in the diagnostic algorithm of fever of unknown origin (FUO), although its role is not clearly determined. The purpose of this study was to assess the role of BM studies in patients with FUO. We retrospectively reviewed 45 consecutive patients (25% human immunodeficiency virus-positive) with FUO who underwent a BM study in the University Hospital of Salamanca from 2000 to 2010. We analysed the diagnostic role of BM smears, multiparameter flow cytometry analysis, histology and microbiological cultures. Five patients (11%) were finally diagnosed by BM study (three had an infectious disease and two were found to have haematological malignancies), all of whom were immunocompetent patients. Histology was the most useful study (diagnosis was obtained in 4/5 patients), while BM cultures did not establish the final diagnosis in any patient. Flow cytometry established the diagnosis in one patient, although this patient was also diagnosed by histology. In conclusion, BM study is useful for establishing the aetiology of FUO. BM biopsy for histological examination should be always mandatory if a BM examination is performed.

Risk factors for thrombotic microangiopathy in allogeneic hematopoietic stem cell recipients receiving GVHD prophylaxis with tacrolimus plus MTX or sirolimus.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a feared complication of allogeneic hematopoietic SCT (HSCT) owing to its high mortality rate. The use of calcineurin inhibitors or sirolimus (SIR) for GVHD prophylaxis has been suggested as a potential risk factor. However, the impact of tacrolimus (TAC) and SIR combinations on the increased risk of TA-TMA is currently not well defined. We retrospectively analyzed the incidence of TA-TMA in 102 allogeneic HSCT recipients who consecutively received TAC plus SIR (TAC/SIR) (n=68) or plus MTX (TAC/MTX)±ATG (n=34) for GVHD prophylaxis. No significant differences were observed in the incidence of TA-TMA between patients receiving TAC/SIR vs TAC/MTX±ATG (7.4% vs 8.8%, P=0.8). Only grade III-IV acute GVHD, previous HSCT and serum levels of TAC >25 ng/mL were associated with a greater risk of TA-TMA. Patients developing TA-TMA have significantly poorer survival (P<0.001); however, TA-TMA ceased to be an independent prognostic factor when it was included in a multivariate model. In conclusion, the combination of TAC/SIR does not appear to pose a higher risk of TA-TMA. By contrast, we identified three different risk groups for developing TA-TMA.

Genome-wide profiling of methylation identifies novel targets with aberrant hypermethylation and reduced expression in low-risk myelodysplastic syndromes.

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

Optimisation of mesenchymal stromal cells karyotyping analysis: implications for clinical use.

PURPOSE: The aim of this study was to optimise the yield of metaphases in mesenchymal stromal cells (MSC) in vitro cultures and to study the karyotype of MSC expanded in good manufacturing practice (GMP) conditions for clinical use. BACKGROUND: MSC are being increasingly used in clinical trials for a number of diseases. Biosafety demonstration in all cases is mandatory. Unfortunately, current standard karyotyping methods fail to obtain enough number of evaluable metaphases. METHODS AND MATERIALS: In the present work, to optimise the yield of metaphases in MSC expanded in vitro, we have tested several conditions by modifying colcemid concentration (we have tested 0.01, 0.05 and 0.1 µg mL(-1) ) and exposure time (during 5, 15 and 24 h). We further applied these optimised conditions to 61 MSC expansions in GMP conditions for clinical use. RESULTS: Our results show that the highest number of metaphases was obtained when MSC were incubated with 0.05 µg mL(-1) of colcemid overnight (15 h), compared to the remaining experimental conditions. In most cases (59/61 cases) enough number of metaphases was obtained. And what is more relevant, only in one case a karyotypic abnormality was found (trisomy of chromosome 10), and cells were subsequently discarded for clinical use. CONCLUSION: We describe here an optimal method to obtain enough number of metaphases for karyotype analysis of in vitro expanded MSCs, what is essential for their clinical use in cell therapy programmes.

Monoclonal free light chains can be found in heavy chain diseases.

Heavy chain diseases (HCDs) are rare B-cell lymphoproliferative neoplasias characterized by the production of a monoclonal component consisting of a truncated monoclonal Ig heavy chain without the associated light chain. Among them, patients with gamma-HCD are so rare that no more than 150 cases can be found in the literature. In this paper, we report one additional case: an 83-year-old man with a gamma-HCD, in whom a kappa light chain component was detected in the serum by using the serum free light-chain assessment and in addition monoclonal kappa cytoplasmic expression was detected in bone marrow plasma cells by flow cytometric analysis. In the work-up of the patient, the underlying anatomopathological lymphoproliferative disease corresponded to a lymphoplasmacytic lymphoma, as it is stated in the current World Health Organization classification (2008), with both lymphadenopathic and bone marrow infiltration. As in other cases, several autoimmune manifestations (antiphospholipidic syndrome and immune thrombocytopenia) were present during the course of the disease in this patient. This case report illustrates a new case of gamma-HCD, in which serum free light-chain analysis and flow cytometry represented a valuable tool for diagnosis, a finding that could be very important for the future management of these patients.

Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q- syndrome.

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS-MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype: CD45(-)/CD73(++)/CD34(-)/CD271(++). They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinical-biological data an unsupervised hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5q- syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5q- syndrome.

Oral beclomethasone dipropionate for the treatment of gastrointestinal acute graft-versus-host disease (GVHD).

Acute graft-versus-host disease (aGVHD) remains one of the most severe complications after allogeneic transplantation; in particular, the presence of gut involvement has been related to increased mortality and poorer response. The use of systemic steroids remains the standard for first-line treatment despite its severe secondary effects. Beclomethasone dipropionate (BDP) is a topically active corticosteroid with low absorption, thereby avoiding many of the deleterious side effects associated with systemic steroids. In the present study we analyzed the efficacy of BDP in a series of 26 patients who were diagnosed with grade 1 and 2 gastrointestinal aGVHD. Twenty patients (77%) responded to BDP treatment, 17 (65.5%) reached complete remission (CR), and 3 (11.5%) showed partial response. Among those patients who reached CR, 5 relapsed, although 1 of them reached second CR after a second course of BDP; therefore, 13 (50%) of the 26 patients did not require systemic steroids to treat gastrointestinal aGVHD. CR rates in those showing gastrointestinal symptoms were 68% for patients with persistent nausea, 50% for those with vomiting, and 54% for those with diarrhea (P=.2). No patient included in the study developed any symptom related to adrenal axis suppression. Thirteen patients (50%) developed >or=1 infectious episode during the first 100 days after transplantation. Transplant-related mortality was 0% at 100 days, and overall transplant-related mortality was 30%, with only 2 patients dying due to infectious complications. Therefore, our study shows that monotherapy with oral BDP is an effective initial therapeutic approach for mild to moderate intestinal GVHD, which avoids complications related to systemic steroids.

CD34 + cell dose and outcome of patients undergoing reduced-intensity-conditioning allogeneic peripheral blood stem cell transplantation.

While in bone marrow allogeneic transplantation the infusion of high doses of progenitor stem cells has a favourable impact on outcome, due to a faster hematopoietic and immune recovery, in the peripheral blood allo-setting the infusion of a high number of CD34 cells increases the risk of extensive chronic graft vs. host disease (cGVHD). This higher incidence of extensive cGVHD has an adverse impact on outcome due to a higher transplant related mortality, specially among patients receiving T-cell depleted allogeneic transplantation with myeloablative conditioning. By contrast, patients undergoing reduced intensity conditioning regimen may benefit from increasing higher CD34 + cell doses, especially those categorized as high risk according to disease status at transplant. Thus, the source of progenitors cells, type of conditioning and GVHD prophylaxis, among other factors, may influence the effect of the progenitor cell dose on outcome after allogeneic transplant.

Quality of life assessment in patients undergoing reduced intensity conditioning allogeneic as compared to autologous transplantation: results of a prospective study.

The aim was to analyze quality-of-life (QOL) during the first year post transplant in 47 patients undergoing reduced-intensity conditioning (RIC) allotransplantation, and to compare these with a similar subgroup of patients receiving autologous stem cell transplantation (ASCT). We used self-reported questionnaires. Each answer scored from 0 (not at all) to 4 (very much), with higher scores indicating worse functioning. Mean value of physical categories among RIC transplants ranged between 1.23 and 0.77 indicating that patients scored very low for physical symptoms. Patients undergoing ASCT had higher scores in questionnaires performed early after transplant and then gradually improved (P < 0.001). Overall, when we compared physical functioning scores, allo-RIC did significantly better (P = 0.049). Nevertheless, while allo-RIC scores were significantly better for the first three questionnaires, ASCT patients did better in the last two questionnaires. These findings are in accordance with the toxicities observed in both subgroups which are lower in the RIC group early after transplant. No significant differences were observed between either subgroup for any of the functional, social/ family, psychological distress and satisfaction with doctor/nurse relationship items. We have observed similar QOL among patients undergoing RIC-allo as compared to ASCT although GVHD remains an important 'event' in QOL.

Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation.

Since graft-versus-leukemia (GVL) is the main weapon for disease eradication after reduced intensity conditioning (RIC) allogeneic SCT, the availability of sensitive and specific techniques to monitor changes in tumor load after transplant are especially helpful. These minimal residual disease techniques would allow an early intervention in the event of low tumor burden, for which immunotherapy is highly effective. Some authors have found an association between persistence of MRD, mixed chimerism and risk of relapse. Nevertheless, data from the literature remain contradictory and further correlations should be established, especially in RIC transplants. In this study we have analyzed the impact of MRD and chimerism monitoring on the outcome of 34 patients undergoing RIC allogeneic SCT who were considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions. At day +100 25 (75%) patients reached complete remission (CR), there were five (15%) partial responses and three patients progressed. Incidence of grade 2-4 aGVHD and extensive cGVHD were 35% and 58%, respectively. Sixteen percent of patients developing aGVHD relapsed as compared to 47% in those without aGVHD (P = 0.03) and also 10% of patients developing cGVHD relapsed as compared to 50% relapses in those without cGHVD (P = 0.03). Four patients (12%) died due to early (n = 1) and late (n = 3) transplant-related mortality. After a median follow-up of 15 months, 24 out of the 34 patients remain alive. Projected overall survival and disease-free survival at 3 years are 68% and 63%, respectively. Early chimerism analysis showed 67% of patients with complete chimerism (CC) in bone marrow (BM), 86% in peripheral blood (PB), 89% in granulocytes and 68% in T lymphocytes. On day +100, these figures were 68%, 79%, 90% and 73%, respectively, and on day +180 there were 83% patients with CC in BM, 100% in PB, 100% in granulocytes and 100% in T lymphocytes. We observed a trend to a higher incidence of relapse in patients with mixed chimerism (MC) as compared to patients with CC. MRD monitoring by flow cytometry and/or RT-PCR analysis was performed in 23 patients. MRD assessment on days +21 to +56 after transplant allowed identification of patients at risk of relapse. In this sense, seven out of 12 patients (58.3%) who had positive MRD on days +21 to +56 relapsed as compared to none out of 11 patients who had negative MRD (P = 0.002). Of the seven patients with criteria to monitor MRD who relapsed after transplant, all but one remained MRD positive until relapse. By contrast, 10 patients remained MRD negative and all of them are in continuous CR. In nine additional patients, persistence of MRD or mixed chimerism was observed after transplant and withdrawal of cyclosporin with or without DLI was performed. Only two out of these nine patients relapsed. MRD clearance was preceded by CC and GVHD. In conclusion, in our study we found that RIC allogeneic transplantation can be used in patients considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions with both low toxicity and low transplant-related mortality. Simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing RIC transplants and so can be helpful for individualizing treatment strategies after transplant.