ALBERT

Description: Introduction Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in in the promoter region of cytokine genes have shown to alter their expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 17 (IL-17) is secreted by CD4+ T-cells and has been implicated in the pathogenesis of various autoimmune diseases but its importance in SCT is not well-known. Objective To analyse the influence of IL-17A SNP genotypes on the risk and severity of GvHD and other complications after HLA-identical allo-SCT. Patients and Methods Genomic DNA obtained from peripheral blood samples belonging to 546 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). Genotyping of the polymorphisms of interest, rs8193036 (-737C〉T), rs2275913 (-197G〉A), rs3819024 (-444A〉G), rs4711998 (-877A〉G), were performed by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Results Genotype frequencies are shown in Table 2 and the association between IL-17A genotypes and complications after allo-SCT are shown in Table 3. Patients transplanted from donors harboring genotype CC for the SNP rs8193036 show increased risk of grade III-IV acute GvHD (7/26 vs 47/397, p=0.035) and of grade II-IV acute GvHD (13/26 vs 133/409, p=0.048). Patients transplanted from donors harboring allele A in the SNP rs4711998 show increased risk of extensive chronic GvHD (53/161 vs 43/177, p=0.045). Relapse rate was not related with IL-17A SNP genotypes. Finally a higher risk of toxicity-related mortality (TRM) was observed in patients transplanted from donors harboring allele A for SNP rs2275913 (78/293 vs 46/227, p=0.048), donors harboring allele G for SNP rs3819024 (78/279 vs 46/242, p=0.011) and donors harboring allele A for SNP rs4711998 (68/250 vs 55/229, p=0.044). Conclusions IL-17A SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore, to improve the clinical management of transplanted patients. This results further support the idea of a genetic predisposition to certain complications after allo-SCT. Paper presented on behalf of the GvHD/Immunotherapy committee of the Spanish Group for Hematopoietic Transplantation (GETH). Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4146 Introduction The success of allogeneic stem cell transplantation (allo-SCT) relies on the result of the immune reactions between donor and recipient lymphohemopoietic systems. Chimerism quantification and monitoring post allo-SCT allows to estimate such immune reactions. In recent years, there has been a growing interest in the analysis of circulating cell-free nucleic acids in plasma. Although the ultimate origin of such nucleic acids is unknown, the most plausible hypothesis proposes cell disruption as the cause for the presence of nucleic acids in plasma. Analysis of chimerism in cell-free plasma DNA would complement the results of cell chimerism for a better prediction of different complications (engraftment failure, rejection, graft versus host disease…etc.). Objective Our goal was to quantitatively monitor chimerism in cell-free plasma DNA and to evaluate its usefulness for the prediction/early diagnosis of complications after allo-SCT that would favour early therapeutic intervention. Patients and Methods The study comprised 270 plasma samples from 29 allo-SCT patients. With a median follow up of 45,8 months (14,6– 74,7). Whole blood (WB) (10 mL) was collected in EDTA containing tubes and was processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min and cell-free plasma DNA was extracted according to the blood and body fluid protocol of the QIAamp DNA Blood Mini Kit (Qiagen). Quantitative chimerism analysis was performed by microsatellite PCR (STR; AmpFlSTR SGM plus, Applied Biosystems) in whole blood (WB), T-cells (CD3+) purified using immunomagnetic means (Miltenyi Biotec) and cell-free plasma DNA. Chimerism was monitored at regular intervals post-transplant (+30,+60, +90, +180, +365). Sensitivity of the technique is 1% in WB and cell-free plasma DNA, and 5% in T-cells (derived from a 95% purity of enriched samples). Results During the first year of allo-SCT, the number of patients with mixed chimerism in cell-free plasma DNA was higher than in WB and T-cells (Table 1). Cell-free plasma DNA mixed chimerism was observed for a longer timer after transplant than “cellular” mixed chimerism, with a median of 105 vs. 40 and 27 days for cell-free plasma DNA, T-cells and WB, respectively. Chimerism analysis revealed two groups of patients, one with mixed chimerism in both cell-free plasma DNA and “cellular DNA”, in which the percentage of recipient plasma DNA ranged between 10–30%, probably mostly derived from hematopoietic cells. The other group presented mixed chimerism in cell-free plasma DNA but complete “cellular” chimerism (100% donor) in which cells the percentage of recipient plasma DNA was lower than in the former group (1–10%). A greater number of patients with graft versus host disease (GVHD),mainly acute but also chronic, presented mixed chimerism in cell-free plasma DNA and complete chimerism in WB and T-cells (Table 2). The presence of recipient cell-free plasma DNA could be derived by cell disruption in GVHD target organs. Discussion Quantitative follow-up of chimerism in cell-free plasma DNA, does not reproduce the results of “cellular” chimerism, as reported previously, and therefore, could not replace it for post-SCT follow-up. On the other hand, chimerism analysis in cell-free plasma DNA could be combined with the conventional follow-up of “cellular” chimerism for an improved diagnosis of life-threatening complications such as acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Post-hematopoietic stem cell transplantation (SCT) chimerism monitoring is important to assess engraftment, anticipate relapse and provide information on the development of graft versus host disease, facilitating therapeutic intervention. The aim of this study was to test the technical efficacy and clinical utility of a novel quantitative PCR approach targeting insertion/deletion polymorphisms (indel-PCR). Materials (or patients) and methods This study included 157 samples (81 bone marrow, 60 peripheral blood (PB), 11 T-cells, 2 myeloid cells, 2 CD34-cells, 1 NK-cells purified using immunomagnetic technology) of 24 patients who underwent SCT for haematological malignancies. Additionally, 2 sets of 11 artificial mixtures were created using PB leukocytes of two healthy subjects (a male and a female) with known percentages of male leukocytes (putative recipient): 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0.01, 0. Chimerism analysis was performed by the gold-standard STR-PCR (AmpFlSTR SGM Plus®, Life Technologies, USA) and by indel-PCR (Mentype® DIPscreen, Mentype® DIPquant, Biotype, Germany). Concordance between both methods was calculated with SPSS using intraclass correlation coefficient. Results The number of informative loci identified with indel-PCR (〉3/patient) was higher than with STR-PCR for all patients. Concordance between both methods for the 157 patient samples and 11 artificial mixtures was "very good" (intraclass correlation coefficient=0.96). Of note, analysis of artificial mixtures provides evidence of significantly (≥2 logs) higher sensitivity by indel-PCR (0.01%) than by STR-PCR (1%, Table 1). Moreover, indel-PCR shows unprecedented quantification capacity (Table 1). Out of the 168 samples analyzed, 32 were positive and 15 negative by both methods, while 121 were positive only by indel-PCR (95% with

Description: Introduction. Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in cytokine genes have shown to influence gene expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 1 (IL1) is a proinflammatory cytokine that induces the production of cytokines and chemokines and plays an important role in the inflammatory processes. IL1 is involved in various cellular functions, including cell proliferation, differentiation, and apoptosis. The IL1 family consists of two biologically actives forms (IL1A and IL1B). Several polymorphisms of these genes have been implicated in the pathogenesis of autoimmune diseases, however, their importance in SCT is not well-known. Objective To investigate the relationship between 4 SNPs in IL1A and IL1B genes and the susceptibility to the development of GvHD and other complications after HLA-identical allo-SCT. Patients and methods Genomic DNA obtained from peripheral blood samples belonging to 509 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). One SNP, rs1800587 (-889 C〉T), in the promoter region of the IL1A gene and three SNPs in the IL1B gene (two in the promoter region rs16944 (-511C〉T), rs1143627 (-31 T〉C) and one in exon 5 rs1143634 (+3954 C〉T) were genotyped by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Univariate and multivariate regression analysis were performed using Cox regression in the presence of competing risks except for chronic GvHD for which we used logistic regression model. All variables with p≤0.10 according to univariate analysis were included in the multivariate analysis. For multivariate analyses, p values were two sided and the outcomes were considered to be significant for p

Description: Introduction Graft versus host disease (GVHD) is the main cause of morbi-mortality after allogeneic stem cell transplantation (allo-SCT). Despite considerable advances in our understanding of the pathophysiology, nowdays anticipation of GVHD is an unresolved matter. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have shown to be associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. In the present study, we propose a novel predictive model based on both clinical and genetic (SNP) variables applying an innovative estimation linear regression model, the least absolute shrinkage and selection operator (LASSO), in a large cohort of HLA-identical sibling donor allo-SCT. Patients and Methods The study evaluated 25 SNPs in 12 genes (Table 1) in genomic DNA obtained from PB samples from 273 patients with available acute GVHD (aGVHD) data and 213 patients with chronic GVHD (cGVHD) data included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH) and their HLA-identical sibling donors. Each SNP was assessed for different models of transmission (recessive, dominant, co-dominant and additive), producing 25 SNPs x 4 models = 100 variables. Clinical variables known to influence the development of GVHD were also considered (Table 1). Univariate regression analysis was performed using Cox regression (data not shown). Multivariant analysis was made with LASSO, an innovative estimation method for linear regression models which is able to select a set of optimal predictors from a large set of potential predictor variables and was considered as a variables selection method under the estimation of a Logit regression model. In this model, the strength of the penalty term is controlled by a smoothing parameter (λ), which is chosen by maximizing the area under ROC curve (AUC) and the correct classification rate (CCR). The statistical model was fitted (goodness-of-fit assessment) by randomly selecting the 85% of the data (the so-called "training set"), and the predictive ability was computed with the remaining 15% (the so-called "testing set"). In order to evaluate the performance and the prediction ability of each model, training and testing samples were randomly selected a total of 100 times. The distribution of the CCR and the AUC over the 100 samplings, were shown by means of box plots and statistical summary in the results data. Finally, for prediction purposes, we considered a cut-off value according to the proportion of Y=1 in the sample (0.28 for grades II-IV aGVHD, 0.11 for grades III-IV aGVHD and 0.30 for extensive cGVHD). Results The best model to predict aGVHD II-IV included 11 genetic variables and no clinical variables with a CCR for patients who developed (CCR1) aGVHD II-IV of 63.6% (Figure 2). The best model to predict aGVHD III-IV included 20 genetic and 7 clinical variables with a CCR1 for aGVHD III-IV of 100%. The best model to predict extensive chronic GVHD included 10 genetic and 3 clinical variables with a CCR1 for extensive cGVHD of 80%. On the other hand, predictive models with only clinical variables showed a poorer CCR1 for patients who developed aGVHD II-IV, aGVHD III-IV and extensive cGVHD (55.6%, 50% and 66.7% respectively; Figure 1). Based on the results from LASSO multivariate analyses, a risk score was calculated for grades II-IV and III-IV aGVHD as well as for cGVHD and extensive cGVHD. Patients were categorized into two groups: low risk (below the cut-off value) and high risk (above the cut-off). Such risk model was able to stratify patients who develop grades II-IV aGVHD (p

Description: Background: Polymorphisms in regulatory sequences of cytokine genes are known to influence their expression and, therefore, the intensity of the immune response. Some of such polymorphisms have been associated with the outcome of allogeneic stem cell transplantation (SCT). Objective: To evaluate the association between donor (D) and recipient (R) genotype for the IL-10 gene -1082 SNP (single nucleotide polymorphism) and -1064 STR (short tandem repeat) polymorphism with the dynamics of chimerism and the development of complications after SCT. Patients and methods: 24 HLA-matched conventional SCT. IL-10 genotypes were determined in an -1082 SNP allele-specific PCR (A vs G) including the -1064 STR in the product which is revealed by capillary electrophoresis. Results were analyzed using Fisher’s exact test due to the reduced sample size. Results: The frequency of alloreactive genotypes (AA or A) for the SNP was 37,5% AA and 79,2% A in the D and 33,3 AA and 95,8% A in the R. No association was observed between the homozygous or heterozygous presence in the D or R of the alloreactive allele for the SNP genotype and the dynamics of chimerism or complications post-SCT. Alleles 4 to 10 for the STR genotype were found in contrast to previous reports (alleles 7–16). The frequency of alloreactive alleles (homozygous, +/+, or heterozygous, +), with greater number of dinucleotide repeats (alleles 8–10 in the present series) for the STR was 8,3% +/+ and 62,5% + in the D and 8,3% +/+ and 50% + in the R. The presence of alloreactive alleles (heterozygous) in the donor was significantly associated with the development of extense chronic GVHD (EcGVHD, Table 1). In patients transplanted from such donors, the incidence of mixed chimerism (MC) in peripheral blood during the first month post-SCT was lower although without statistical significance (Table 1). The higher alloreactivity of these donors favors the establishment of complete chimerism and therefore, increases the incidence of EcGVHD. Conclusions: The present study shows an association between the presence in the donor of alloreactive alleles for the IL-10 -1064 STR polymorphism and the development of EcGVHD, in a chimerism mediated fashion, after HLA- identical SCT. The analysis of a larger number of patients as well as further cytokines will eventually allow to confirm these observations and to establish this type of studies as a means for an improved management of transplanted patients. Table 1. Influence of alloreactive alleles (8–10) for the IL-10-1064 STR polymorphism on chimerism and complications post-STC.

Description: Abstract 3051 INTRODUCTION The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+, Tregs) which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation (allo-SCT), Tregs are known to mitigate graft versus host disease (GVHD) while maintaining a graft versus leukemia effect (GVL). Allele (GT)15 for the functional (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene is associated with a higher expression of FOXP3 and production of a greater amount of Tregs. However, its impact in the allo-SCT setting has not been analyzed. OBJECTIVE To analyze the impact of the (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene on the development of complications and ultimately on the success of conventional HLA-identical allo-SCT. MATERIALS AND METHODS The study includes 33 patients with hematological malignancies, treated with myeloablative HLA-identical peripheral blood allo-SCT (Table 1). Diagnosis, classification and grading of GVHD were made by clinical criteria and confirmed when necessary by pathological examination of histological samples from gut, skin, liver or lung, according to international consensus criteria. Donor and recipient genomic DNA was purified from EDTA anticoagulated peripheral blood before allo-SCT and using QIAamp Blood DNA extraction kit (Qiagen). Genotyping of the (GT)n microsatellite polymorphism in the FOXP3 gene was performed by a fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) method (GeneAmp 7900; Applied Biosystems) and sized by capillary electrophoresis (POP7 - ABI PRISM 3130 xL Genetic Analyzer; Applied Biosystems) followed by fragment analysis (GeneMapper 4.0 Software; Applied Biosystems) as previously described [Bassuny WM, et al. Immunogenetics. 2003;55 :149–56]. RESULTS The median follow-up time for the cohort was 34 months (range 9.5–110). Allelic frequencies observed were similar to those previously reported (50.5% (GT)15, 41% (GT)16 and 7% (GT)17; no (GT)14 or (GT)18 alleles were found). Patients transplanted from donors harboring allele (GT)15 showed a lower incidence of grades II-IV acute GVHD (29% vs 67%; p =0.049). These patients also showed a trend to a lower incidence of severe (grades III-IV) GVHD (12% vs 33%; p =0.167) as well as chronic GVHD (75% vs 100%; p =0.143; Table 1, Figure 1). No statistically differences were found between patients transplanted from (GT)15 and non-(GT)15 donors in terms of relapse rate (38% vs 33%; p =0.825; Table 1) or cumulative incidence of relapse (CIR at 2 years 35.3% vs 37.5%, Figure 2). Finally, survival analysis did not show statistically significant differences between the two groups of patients in terms of median event (relapse) free survival (EFS, 15.6 months vs 4.5 months, p =0.686) or overall survival (OS, 29 months vs not reached, p =0.610). CONCLUSIONS Tregs are known to modulate the allotolerance-alloreactivity balance between donor and recipient in the allo-SCT setting, mitigating GVHD while preserving the anti-tumor effect (GVL) of the donor graft. In the present study, the presence of allele (GT)15 in the donor, which promotes a higher expression of FOXP3 and greater amount of Tregs, affected allo-SCT outcome by decreasing grades II-IV acute GVHD and chronic GVHD, without affecting GVL (no differences in CIR and OS). Analysis of this polymorphism can help in appropriate donor selection and, more importantly, drive a tailored management of patients submitted to allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The current gold standard for chimerism quantification after allo-stem cell transplantation (SCT) is based on the polymerase chain reaction of DNA short tandem repeats (STR-PCR). This methodology is sex-independent, highly informative and quantitatively accurate. However, this method has a moderate sensitivity (1–3%). Objective: To test the usefulness of increasing the assay sensitivity by 3 different approaches in order to detect recipient residual cells. Material and Methods: Two sets of 20 samples, belonging to artificial mixtures of male and female peripheral blood (PB) cells in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0) were prepared to test the assay sensitivity and reproducibility. Additionally 58 samples from 10 selected patients showing different clinical evolution post-SCT were retrospectively studied. Chimerism was analyzed on genomic DNA (purified QIAamp DNA blood kit, Qiagen) by multiplex STR-PCR (AmpFlSTR SGM Plus revealed on capillary electrophoresis in an ABI Prism 3100 automated DNA sequencer; Applied Biosystems). Three different approaches aimed to increase the sensitivity of STR-PCR were tested: (1) Increasing DNA concentration in the PCR (1x, 2.5x, 5x), (2) Increasing the concentration of the PCR product for the capillary electrophoresis (1x, 2.5x, 5x) and (3) Increasing the sample injection time into the capillary for the electrophoresis (10s, standard time, 30s and 50s). Results: The standard STR-PCR showed a sensitivity of 1% in both sets of male-female PB artificial mixtures. Both approaches increasing DNA concentration in the PCR and the PCR product in the capillary electrophoresis did not achieved better sensitivity. Moreover, appropriate analysis of the samples was frequently hampered due to fluorescence saturation. However, longer injection times (30s, 50s) showed that currently underrepresented DNA fragments were conspicuously overestimated, giving a one-log increase in the sensitivity of the STR-PCR assay, which reached a 0.1%. This approach was used to re-analyze the 23 SCT patient samples previously identified as in complete chimerism by standard STR-PCR. The 0.1% sensitivity methodological approach allowed to detect residual/reappearing recipient cells in three of the 23 analyzed samples. Therefore this new approach would have allowed to anticipate the detection of graft rejection and disease relapse in 2/10 patients, 7 and 77 days respectively. Conclusion: Long injection times (30s and 50s) in the capillary electrophoresis after STR-PCR result in a one log sensitivity increase (from 1% to 0.1%). This would allow earlier diagnosis of adverse events after SCT favoring a prompt introduction of therapeutic interventions.