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Additional file 1: Supplementary Table 1. Custom set of primers for mutations in exons 16 and 18 for sanger sequencing. Supplementary Table 2. Custom set of probes and primers for mutation Y646N for RT-qPCR. Supplementary Table 3. Custom set of probes and LNA primers for mutations Y646C and Y646S for RT-qPCR. Supplementary Table 4. Custom set of probes and LNA primers for mutation A692V for RT-qPCR. Supplementary Table 5. Clinical characteristics, immunohistochemical and molecular markers available in patients treated with R-Benda VS. R-CHOP.

Post-transplant cyclophosphamide (PTCY) effectively prevents graft-versus-host disease after unmanipulated HLA-haploidentical HSCT. The use of PTCY in the unrelated donor HSCT setting is less explored. We conducted a retrospective study of 132 consecutive patients undergoing a matched or 9/10 mismatched unrelated donor HSCT in 4 centers in Spain, 60 with anti-thymocyte globulin (ATG)-based prophylaxis combined with MTX-CsA, and 72 using a PTCY-based regimen. Peripheral blood stem cells were used as graft in most patients (111 patients, 84%); mMUD donors were balanced between groups. Cumulative incidences of grades II-IV and III-IV acute GVHD at 100 days were lower in the PTCy group (46% vs. 67%, p = 0.008; 3% vs. 34%, p = 0.003), without statistically significant differences in the 2-year cumulative incidence of chronic moderate-severe GVHD. At 2 years, no significant differences were observed in overall survival, event-free survival, cumulative incidence of relapse, and non-relapse mortality. GVHD was the most frequent cause of NRM in the ATG group. No differences were observed between groups in the composite endpoint of GVHD-free and relapse-free survival. In this study, PTCy combined with additional immunosuppression after MUD/mMUD HSCT showed a reduction of aGVHD rate with safety results comparable to those obtained with the ATG-based prophylaxis.

Despite considerable advances in our understanding of the pathophysiology of graft-versus-host disease (GVHD), its prediction remains unresolved and depends mainly on clinical data. The aim of this study is to build a predictive model based on clinical variables and cytokine gene polymorphism for predicting acute GVHD (aGVHD) and chronic GVHD (cGVHD) from the analysis of a large cohort of HLA-identical sibling donor allogeneic stem cell transplant (allo-SCT) patients. A total of 25 SNPs in 12 cytokine genes were evaluated in 509 patients. Data were analyzed using a linear regression model and the least absolute shrinkage and selection operator (LASSO). The statistical model was constructed by randomly selecting 85% of cases (training set), and the predictive ability was confirmed based on the remaining 15% of cases (test set). Models including clinical and genetic variables (CG-M) predicted severe aGVHD significantly better than models including only clinical variables (C-M) or only genetic variables (G-M). For grades 3-4 aGVHD, the correct classification rates (CCR1) were: 100% for CG-M, 88% for G-M, and 50% for C-M. On the other hand, CG-M and G-M predicted extensive cGVHD better than C-M (CCR1: 80% vs. 66.7%, respectively). A risk score was calculated based on LASSO multivariate analyses. It was able to correctly stratify patients who developed grades 3-4 aGVHD (P < .001) and extensive cGVHD (P < .001). The novel predictive models proposed here improve the prediction of severe GVHD after allo-SCT. This approach could facilitate personalized risk-adapted clinical management of patients undergoing allo-SCT.

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein a (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

To determine and quantify the differences produced in chromatin structure after selective in situ DNA digestion with restriction endonuclease (RE).Chromatin fiber structure from a murine cell line was analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-based method entails the generation of a binary image and a skeleton characteristic of the chromatin fiber. Morphologic features (surface, perimeter, shape, number of triple points and Euler number) of the chromatin structure can then be quantified from the digitized images. The results of these experiments were compared with those obtained from direct digestion of naked DNA using the same endonucleases in an attempt to correlate the distribution of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestion.Homologous chromatin fiber regions are differentially affected by the action of each RE, although they are indistinguishable under light microscopy.The proposed analytical routine constitutes a valuable tool for uncovering subtle alterations in the structure of chromatin fiber that has been modified under different experimental conditions.

Human lymphocyte chromatin either extended or condensed in interphase nuclei and chromosomes was in situ digested by the restriction endonuclease AluI and then hybridized with alphoid probes specific for chromosome 1 (D1Z5 locus), for chromosome X (DXZ1 locus), and with a classical satellite DNA probe specific for chromosome 9 (D9Z1 locus). Fluorescent hybridization signals were quantified by digital analysis of high-resolution images obtained by a Photo-CD system in an attempt to analyze the differential DNA removal produced by AluI in specific repetitive DNA sequences with known restriction site frequency and distribution. The analysis of area and average pixel grey count of hybridization signals suggests that the greater the degree of chromatin stretching, the higher the accessibility of the probe and/or reporter molecules to the target. Nevertheless, this greater hybridization efficiency does not result in a higher fluorescence intensity due to dispersion of individual signals. Specific repetitive DNA at D9Z1 locus (classical) remained impervious to digestion, while that at DXZ1 (alphoid) was extensively removed, according to the frequency and distribution of restriction sites. Nevertheless, though the restriction sites were at least as frequent as at the DXZ1 locus, DNA at the D1Z5 locus (alphoid) was only partially removed. This indicates that chromatin organization within the C-band partially prevents extraction of alphoid sequences, supporting the hypothesis that alphoid DNA sequences are differentially organized among chromosomes. Overall, the same results were obtained from condensed and extended chromatin, suggesting that higher-order chromatin organization does not influence the in situ DNA cleavage and removal by AluI.

Restriction endonuclease (RE) in situ digestion (REISD) of human metaphase chromosomes and interphase nuclei may uncover cryptic polymorphisms. This technique can be applied to identify the individual origin of cells and thus analyze the hemopoietic chimerism that eventually results in leukemic patients after allogeneic bone marrow transplantation (BMT). In the current study, results of REISD with different REs are shown. In particular, the use of Sau 3A reveals a polymorphism for constitutive heterochromatin of chromosome 3 and may differentiate BMT donor (D) and recipient (R) cells. Once pre-BMT characterization shows a different Sau 3A digestion pattern of D and R cells, it is possible to monitor the development of hematopoietic cell populations in the R bone marrow after BMT. A panel of 24 patients who underwent BMT and their Ds were analyzed. The method presented here allowed cells from D and R to be distinguished, and therefore to quantify the post-BMT hemopoletic chimerism, in 6 (25%) of the cases. This quantitative and sex-independent genetic approach to the study of hemopoietic chimerism has already shown itself to be useful in patients with leukemia who require a BMT, but could also be extended to other transplant situations.

Restriction endonuclease in situ digestion (REISD) with Sau3A of human metaphase chromosomes and interphase nuclei produces a conspicuous banding pattern involving pericentromeric regions of chromosomes 9 and 3. Constitutive heterochromatin of chromosome 9 is never digested by this enzyme while that of chromosome 3 is polymorphic, giving rise to three possible karyotypes: homozygous digested (3--), homozygous undigested (3++) or heterozygous individuals (3+-). Discrimination of this polymorphism between donor and recipient cells constitutes a rapid sex-independent method to monitor quantitatively the chimaerism achieved after bone marrow transplantation. An image processing and analysis (IPA)-assisted procedure which resolves residual fluorescent regions in metaphase chromosomes or interphase nuclei after REISD has been developed. IPA-REISD has interesting advantages over the basic REISD method by allowing a rapid, objective and precise discrimination of the polymorphism in large cell samples.

Fully quantitative cytological techniques for the analysis of hemopoietic chimerism are very limited and largely restricted to sex-chromosome detection after sex-mismatched bone marrow transplants (BMTs). The aim of the present investigation was to assess the usefulness of autosomal polymorphisms for the size of heterochromatic regions in the identification of donor and recipient cells and therefore in the quantification of the hemopoietic chimerism after sex-matched BMT.Hemopoietic chimerism was followed up in 3 transplanted patients targeting a polymorphism for the size of the pericentromeric heterochromatin (PCH) of chromosome 9, uncovered by restriction endonuclease (RE) in situ digestion (REISD) with the RE Sau3A, to differentiate donor and recipient cells on conventional bone marrow chromosome preparations.The polymorphism for the size of the PCH of chromosome 9 allowed differentiation of donor and recipient cells targeting both metaphase and interphase nuclei. The misidentification error for the polymorphism for the size of HPC of chromosome 9 was estimated as 1% for metaphases and 6-11% for interphases. The 3 cases studied showed complete chimerism in the first post-BMT sample analyzed, which was maintained in 2 of them. One patient relapsed and showed transient mixed chimerism. One month later, this patient achieved a second complete remission, showing complete chimerism again. In this patient, who received a sex-mismatched BMT, chimerism was also quantified by sex-chromosome identification using established methods, such as conventional cytogenetics and FISH, and the results obtained were similar to those rendered by Sau3A-REISD.The polymorphism for the size of the PCH of chromosome 9 uncovered by Sau3A-REISD allows accurate quantification of the hemopoietic chimerism after sex-matched BMT.

Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse.FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears.Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission.Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (, This work was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS grants PI08/1463, PI11/00708, PI14-01731 and RD12/0036/0061, co-financed by ERDF (FEDER) Funds from the European Commission, as well as grants from the Fundacion LAIR and Asociacion Madrilena de Hematologia y Hemoterapia (AMHH). Sequencer 3130xl Genetic Analyzer was partially supported by ISCIII-FIS grants PI01-3624, PI08-36173. VN and CML were partially supported by a Post-Residency Research Fellowship from the Instituto de Investigacion Sanitaria Gregorio Maranon (IiSGM).