ALBERT

Description: Chimerism refers to the relative proportion of donor and recipient DNA after hematopoietic stem cell transplantation (HSCT) and its quantitative follow-up is of great clinical utility in this setting. PCR of short tandem repeats (STR-PCR) constitutes the gold standard method for chimerism quantification, although more sensitive PCR techniques (such as qPCR) have recently arisen. We compared the sensitivity and the quantification capacity of both techniques in patient samples and artificial mixtures and demonstrated adequate performance of both methods, with higher sensitivity of qPCR and better quantification skills of STR-PCR. By qPCR, we then prospectively followed up 57 patients that were in complete chimerism (CC) by STR-PCR. Twenty-seven patients (59%) showed 0.1–1% recipient DNA in the bone marrow. Only 4 patients presented 0.1–1% recipient DNA in peripheral blood (PB), and one of them relapsed. Finally, by qPCR, we retrospectively studied the last sample that showed CC by STR-PCR prior to relapse in 8 relapsed patients. At a median of 59 days prior to relapse, six patients presented mixed chimerism by qPCR in PB. Since both approaches have complementary characteristics, we conclude that different techniques should be applied in different clinical settings and therefore propose a methodological algorithm for chimerism follow-up after HSCT.

Description: INTRODUCTION: In chronic myeloid leukemia (CML) patients in chronic phase (CML-CP), BCR-ABL levels ≤10% at 3 months measured by RT-qPCR (IS) has been consistently correlated with probabilities to obtain an optimal response at 12 months. Monitoring molecular response with automated cartridge-based detection system GeneXpert BCR-ABL (Cepheid®) method has shown an optimal correlation with standardized BCR-ABL (IS) EUTOS method in patients with complete cytogenetic response (CCyR). However, is not known if both methods are also equivalent when measuring BCR-ABL levels above 1%, and therefore, the utility of GeneXpert in order to evaluate response at 3 months must be confirmed. AIMS: To validate the predictive value of molecular response at 3 months with GeneXpert method METHODS: We have studied 125 new consecutive CML-CP patients treated with tyrosine kinase inhibitors (TKIs) followed in 13 centers. Median age at diagnosed was 55 years. The percentage of low, intermediate and high risk Sokal groups were 42%, 40% and 18% . First line treatment was imatinib (IM), nilotinib (NI), dasatinib (DA) or bosutinib (BO) in 58%, 28%, 13% and 1% of the patients, respectively. BCR-ABL level was measured by GeneXpert platform, where all necessary steps to measure BCR-ABL levels are automatically performed. ABL was used as gene control. The study was approved by the Ethics Committee. RESULTS: Median follow up was 43 months. The proportion of patients that achieved CCyR by 12 months, analyzed by intention to treat, was 84% (108/123). Probabilities for each specific TKI were 78%, 93%, 100% and 100% for IM, NI, DA and BO respectively. 23% (96/125) of patients required treatment changed due to resistance or intolerance. Treatment discontinuation probabilities were 32%, 11%, 5% and 0% for IM, NI, DA and BO respectively. Only 4% (5/125) did not achieve an optimal response at 3 months (BCR-ABL ≤10%), which is significant lower compare to results obtain with historical series when using EUTOS IS method. 10% cut-off at 3 month was unable to identify patients that achieved an optimal response in further evaluations. By 12 months, this cutoff did not correlate with probabilities to obtain CCyR (50% vs 86% (p=0.1) or major molecular response (MMR) (60% vs 79% (p=0.21)). In order to find a cutoff that could correlate with optimal response at 12 months, we used a receiver operating characteristic curve to identify the optimal cutoff in transcript level that would allow us to classify the patients as high risk or low risk with maximal sensitivity and specificity for each individual outcome. At 3 months, patients with transcript levels ≤ 1.6% had significantly better probabilities to obtain an optimal response by 12 months, with 81% and 94% sensitivity and specificity for CCyR. With this new cutoff, probabilities for CCyR and MMR at 12 months were 98% vs 54% (p

Description: Introduction: Immune reconstitution (IR) has a significant impact in HSCT outcome with a role against opportunistic infections and in disease control. In the setting of unmanipulated haploidentical transplantation (Haplo-HSCT), some groups have identified the absolute leukocyte count on day +30 (ALC30) as an independent prognostic factor in terms of overall survival (OS), disease free survival (DFS) and infection related mortality (IM). The aim of this study was to evaluate the impact of early IR on different HSCT outcomes in patients who underwent Haplo-HSCT with postransplant cyclophosphamide (PTCy) at our institution. Patients and methods: Eighty-eight patients received a Haplo-HSCT from 2011 to 2016. Thirty-six percent of the patients received myeloablative conditioning regimen and 64% received reduced intensity regimen. Graft-versus-host disease (GVHD) prophylaxis was based on PTCy, cyclosporine and mycophenolate mofetil. Early IR was assessed through the analysis of different lymphocyte subpopulations at days +30 and +90 after transplantation, including ALC30 (cellular analyzer DXH, Beckman Coulter®); CD3+ lymphocyte count and their different subpopulations (CD4+ and CD8+ lymphocytes, naive and memory T cells) and NK cells count. Lymphocytes subpopulations were determined by multiparametric flow cytometry (FC500 and Navios, Beckman Coulter®). ROC curves were used to determine the optimal cut-off values for each of the studied variables. Results: Eighty-one patients were studied, excluding 7 who died before day +30. Median follow-up was 26 months (10-43). Patient´s characteristics are shown in Table 1. CMV reactivation was documented in 76% (62) of the patients, 4% (3) developed a proven invasive fungal infection, and 31% (25) presented hemorrhagic cystitis. Median OS and DFS were 26 months (10-43) and 24 months (9-39), respectively. IM rate and NRM rate were 10% and 24%, respectively, at the end of follow up. Median lymphocyte populations counts at day +30 are shown in Table2. ALC30 below 100 cells/uL (p= 0.027) and CD4+ naïve lymphocytes below 12 cells/uL (p=0.033) (both corresponding to the 25 percentile) were associated with lower OS compared to patients with higher counts at day +30 (Figure 1). Patients with ALC30 lower than 300 cells/uL (p=0.026) showed significantly higher NRM; CD8+ count lower than 20 cells/uL (p=0.022) also showed higher NRM. NK cells counts at day +30 lower than 14 cells/uL (p=0.014), near to percentile 25, predicted higher IM (62.5% vs 37.5%). We did not identify any lymphocyte subpopulation that could predict DFS. Patients with acute GVHD grades II-IV showed values of ALC30 lower than 200 cells/uL (p=0.051), although non-statistically significant. No relationship was found between lymphocytes subpopulations at day +90 and HaploSCT outcomes. Conclusions: Our study supports the prognostic significance of early IR after unmanipulated haploidentical transplantation with PTCy, as previously described by other groups. ALC30, CD4+ naïve lymphocytes, and CD8+ lymphocyte count at day +30 may be good early predictors for OS and NRM in this setting. On the other hand, low NK cells counts (lower than percentile 25) predicted higher IM. Patients with very low lymphocyte counts should be monitored closely as they are at high risk for infectious complications, NRM and OS. Disclosures No relevant conflicts of interest to declare.

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 2510 Relapse remains the main cause of treatment failure in patients with acute myeloid leukemia (AML) in first remission (CR1) after allogeneic hematopoietic stem cell transplantation (SCT). The detection of minimal residual disease (MRD) in AML has improved in the past years with multiparametric flow cytometry (MFC) and molecular analysis (RT-PCR). However the prognostic impact of pre-transplantation MRD and the outcome after SCT has not been well studied. The aim of this study was to evaluate pre-transplantation MRD in patients in first remission undergoing myeloablative allogeneic SCT. We retrospectively studied 35 consecutive patients receiving myeloablative SCT for AML in first cytologic remission after intensive chemotherapy with available MRD determination before transplant. MRD was studied by 4-color MFC on bone marrow aspirates, and quantitative RT-PCR (NPM1, WT1, MLL) on bone marrow and/or peripheral blood samples obtained within thirty days before transplant. Thirty-five patients consecutively transplanted in our institution between 1999 and 2012, and for which pre-transplant MRD data was available were analyzed (Table 1). Eighteen showed negative MRD pre-transplant whereas 17 showed positive values. Characteristics of patients were homogeneous between both groups, including number of chemotherapy cycles received before transplantation (Table 1). Within the MRD-negative group, 17 patients showed negative MRD by MFC (12 of them showed negative values also by PCR) and 1 patient showed negative MRD by PCR (MFC not available). Within the MRD-positive group, 9/17 (52%) patients showed MRD-positive values by MFC: in 5 cases MRD was also detected by PCR, only 1 showed negative PCR and in the remaining 3 cases, PCR was not available. On the other hand, in 8/17 (47%) patients MRD was not detected by MFC, however, PCR detected MRD in all of the cases in bone marrow (2), peripheral blood (4) or both samples (2). With a median follow-up of the whole series of 23 months, 2-years estimates of overall survival were 82% (95% CI, 97–55) and 30% (95% CI, 3–71) for MRD-negative and MRD-positive patients (p=0.045), respectively. Cumulative incidence of relapse were 21% (95% CI, 5–48) and 56% (95% CI 10–73) for MRD-negative and MRD-positive patients (p=0.11). In the MRD-negative group, cause of death was toxicity in 11% of the cases and relapse in 11%, while in the MRD-positive group, 17% of patients died due to toxicity and 23% due to relapse. Conclusions: Our data shows that the presence of MRD before allogeneic SCT in patients with AML in CR1 is associated with a significant worse OS rate compared to patients with negative MRD, as well as a tendency towards a higher risk of relapse. The detection of MRD by MFC correlates with the detection by PCR in most of the cases. However, in a significant group of patients, MRD was detected only by PCR. This could be related to differences in sensitivity between both methods. Further studies including larger series are needed to confirm these observations. Table 1 Patient and Disease Characteristics by MRD status Pre-transplant MRD MRD-negative MRD-positive N=35 18 17 Age at transplant (median, R) 36 (19-62) 42 (19-68) Gender (male/female) 8/10 12/5 Cytogenetics and molecular markers     Normal/Intermediate risk 77% 83%     FLT3+ 28% 27%     NPM1+/FLT3- 6% 7%     FLT3-/NPM1- 28% 40%     High-risk 22% 17% Secondary AML 16% 6% Cycles pre-transplantation (median, R) 3 (2-5) 3 (2-5) MRD detection     MFC only 27% 18%     RT-PCR only 5% 0%     Both 68% 82% Donor*     HLA-identical sibling 50% 35%     HLA-matched unrelated 22% 29%     SCU-dual 22% 24%     HLA-haploidentical related 6% 12% acute GVHD II-IV (n°/patients at risk) 44% (8/18) 24% (4/17) chronic GVHD lim/ext (n°/patients at risk) 53% (8/15) 57% (8/14) MRD: minimal residual disease, MFC: multiparametric flow cytometry, RT-PCR: real time PCR, GVHD: graft vs host disease, SCU-dual: single cord blood with co-infusion of selected CD34+ cells from a third party HLA-mismatched donor. *The MRD negative group includes 2 MAC SCU-dual cases with primary graft failure rescued immediatly by a second graft (1 Dual and 1 Haplo) using a RIC regimen. The MRD positive group includes 1 MAC SCU-dual case rescued immediatly by a second graft (1 Haplo). Disclosures: No relevant conflicts of interest to declare.

Description: Introduction P210 BCR-ABL translocation resulting from rearrangements within the major breakpoint cluster region (M-BCR), either e13a2 or e14a2, is the molecular hallmark of chronic myeloid leukemia (CML). However, some CML patients may harbor atypical BCR-ABL rearrangements such e1a2 P190 BCR-ABL which involves the minor breakpoint cluster region (m-BCR). Response to therapy with tyrosine kinase inhibitors (TKI) and outcome of such atypical patients is not well defined. Objective To evaluate response to TKI therapy of CML patients with the atypical e1a2 P190 BCR-ABL translocation. Patients and Methods Since 2009, 4 patients with CML in chronic phase and with atypical e1a2 P190 BCR-ABL rearrangement have been recruited in various institutions belonging to the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH). Patient characteristics, treatments administered and response to therapy for the 4 patients is shown in Table 1. BCR-ABL transcripts were revealed at diagnosis by quantitative PCR followed by conventional agarose electrophoresis of PCR products. Molecular follow-up of BCR-ABL transcripts throughout treatment was performed by quantitative PCR following the guidelines of the European Leukemia Net. Results One patient received treatment (HU and INF+araC) prior to TKI (Pat. 1; Table 1). All 4 patients received Imatinib as initial TKI treatment. Two of the patients treated with Imatinib (Pat. 1,2) obtained a complete molecular response (CMR) and the other 2 (Pat. 3,4) only achieved a complete hematological response (CHR) as best response (Table 1). All patients had to switch to a second generation TKI (3 Nilotinib and 1 Dasatinib) due to intolerance to Imatinib (n=1; Pat. 1) or resistance (n=3; Pat. 2-4). The patient who received Dasatinib as second line TKI (Pat. 3) only achieved a partial hematologic response (PHR) and was changed to Nilotinib as third line TKI, achieving CHR after which the patient entered in blast crisis and died 36 months after diagnosis (Table 1). Overall, only 1 (Pat. 1) out of the 4 patients included in the present study achieved a sustained molecular response with Imatinib. At last follow-up, among the 4 patients included in the study, all 4 had needed a change of TKI, 1 had died due to disease progression (Pat. 3) and only 2 of them retained a molecular response (Pat. 1,2). Conclusion CML patients harboring atypical e1a2 P190 BCR-ABL transcripts show a poor response and short-lived responses to TKI therapy and therefore should be identified as high-risk patients at diagnosis. These patients must be closely monitored during therapy with TKI and should be treated upfront with a second generation TKI or even be considered for allogeneic SCT in the early phase of the disease. Paper presented on behalf of the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH). AJ-V and IB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Allogeneic transplantation is the only curative option for patients with high risk leukemias or MDS. Only one third of them have an HLA identical sibling donor and around 60-70% will find an unrelated donor, that´s why haploidentical stem cell transplantation (HAPLO-HSCT) offers a therapeutic option to most of these patients. Myeloablative conditioning (MAC) produce better disease control than reduced intensity conditioning regimens (RIC), but with higher toxicity, rendering long term similar results. Patients and methods: We retrospectively evaluated the results of our MAC-HAPLO regimens in patients diagnosed with high risk leukemias or MDS (Fludarabine 30 mg/m2 x5 days, Cyclophosphamide14,5 mg/kg x2 days, IV Busulfan 3,2 mg/kg x 3 days (BUX3), or Fludarabine 40 mg/m2 x4 days and IV Busulfan 3,2 mg/kg x 4 days (BUX4)) with GVHD prophylaxis based on PT-CY (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5, performed in GETH centers (Spanish Group for Hematopoietic Transplantation). Results: From Feb-2012, 65 MAC-HAPLO HSCT have been reported by 14 centers. Median age was 41 years (15-67), 66% were males and all were in advanced disease phase or presented high risk features (AML 47/ALL 8/MDS 5/ Others 5). Previous HSCT had been employed in 12% (autologous in 5, allogeneic in 3), and in 88% the HAPLO-HSCT was their first transplant. Disease status at HAPLO-HSCT was morphologic CR in 80%, but disease persisted in 52% (MRD+ by flow or molecular markers 32%, morphologic disease 20%). Their disease risk index (DRI) was high or very high in 65%, and the comorbidity index (HCT-CI) was higher than 2 in 18%. PBSC was the graft source in 56 (86%), non T-cell depleted in all cases. The haploidentical donor was the patient´s mother (21.5%), father (12%), siblings (43%) or offspring (23%). MAC regimen was BUX3 in 25 (38.5%) and BUX4 in 40 patients (61.5%). Median infused CD34+ cells were 5.31 x10e6/kg (2.75-11.42). There were no graft failures. Median neutrophils engraftment was reached at day +17 (12-29) and platelets 〉20K at day +26 (11-150). Complete chimerism was obtained at a median of 28 days (13-135) in 60 evaluated patients. Cumulative incidence (CI) of non-relapse mortality (NRM) was 12.5% at day +100 and 19% at 1 year. CI of grade II-IV acute GVHD was 28.5% at day +100, and grade III-IV was 6.5%. CI of chronic GVHD at 2 years was 28%, being extensive in 8% . No differences in acute or chronic GvHD CI were seen when comparing BUX3 against BUX4. After a median follow-up of 17 months (5-50), estimated 24-months event-free survival (EFS) and overall survival (OS) were 58,5% and 60% respectively. CI of relapse or progression was 21%. No significant differences in NRM, EFS, OS and relapse incidence were detected between BUX3 and BUX4. The impact of CR prior to MAC-HAPLO, the DRI or chronic GvHD in the disease control have not been apropiately demostrated probably due to the limited number of events in our series. Conclusions: IV Busulfan based MAC-HAPLO with PT-CY in the treatment of high risk leukemias and MDS offers good disease control with manageable toxicity, with either BUX3 or BUX4. These efective MAC regimens combined with peripheral blood HAPLO donors could control high risk hematologic diseases in the long term. Severe acute or chronic GvHD are low frecuency events, but relapses persist as the main problem in this patients population. Disclosures No relevant conflicts of interest to declare.

Description: Basis. Abnormalities in chromosome 8 (8p-/8q+) are observed in 2-5% of CLL patients. Microarray studies have revealed up to 30-40% of 8 alterations in del(17p) patients and an independent association with poor outcome. Large series assessing CLL patients with 8p-/8q+ are scarce. Aims. 1. To describe the frequency of 8q gains (8q+) and 8p losses (8p-) in CLL patients with del(17p); 2. To compare cytogenetic and clinical characteristics between patients with 8p-/8q+ (Alt-chr8) and those with normal chromosome 8 (N-chr8); 3. To assess their prognostic value. Patients and methods. From 2,249 patients included in the Spanish CLL database, 75 del(17p) cases were selected. Gains of MYC (8q24) and losses of LPL (8p22) were studied by FISH. Clinical and cytogenetic data of Alt-chr8 and N-chr8 were compared. Results. 8p- and/or 8q+ were found in 21/75 patients (28%). In the Alt-chr8 group, 8q+ was more frequent than 8p- (71% vs. 52%) and 29% showed concomitance of both abnormalities, suggesting the presence of i(8q). Six different FISH patterns were identified, some of them coexisting in the same patient (Table 1). Conventional cytogenetics data were available in 47 cases (15 Alt-chr8 and 32 N-chr8). Alt-chr8 group showed a higher median number of alterations and frequency of complex karyotypes (P=0.048 and P=0.013). In the Alt-chr8 group, the karyotype revealed 8p-/8q+ in 3 patients and in 9 cases with abnormal karyotype, the presence of marker chromosomes, added material and/or cryptic alterations would explain the FISH results (Table 1). From 66 cases, routine FISH data (13q, 12 and 11q) were available and no significant differences were detected among Alt-chr8 and N-chr8, as with other clinical and analytical parameters at diagnosis. Of note, shorter Overall Survival (OS) was observed for Alt-chr8, although differences were only significant for patients with 8p- (P=0.012, Figure 1). Interestingly, for 3 patients of Alt-chr8 group, previous non-del(17p) samples already presented 8p-/8q+. Conclusions. 1. In CLL patients with del(17p), detection of 8p- and/or 8q+ is associated with an increased karyotypic complexity and a worse outcome; 2. 8p-/8q+ could act as a primary event that trigger del(17p). More cases are required to confirm this hypothesis. Acknowledgments.PI11/01621; RD12/0036/0044, RD12/0036/0069; 2014/SGR585; Fundació La Caixa. Table 1. Karyotypes and FISH results of patients with del(17p) and Alt-chr8. Conventional Cytogenetics FISH ID Karyotype % del(17p) Chromosome 8 alteration % Pattern* 1 46,XX,del(8)(p21),add(10)(q26),add(17)(p13),+2ac[5]/47,XX,+12,add(17)(p13),del(18)(q21),add(22)(q13)[3] 80 20 1O2G 8p- 2 - 95 75 3 46,XX,add(6)(q24),add(14)(q32,3),i(17)(q10)[6]/46,XX[8] 95 75 4 - 76 50 5 45,XY,-5,-9,-15,add(17)(p13),+18,-21,+2mar[13]/46,XY[37] 70 17 6 46,X,der(X),add(8)(p23),del(13)(q12q22),add(17)(p13)[11]/46,XX[13] 10 32 1O3G 8p- and 8q+ 7 - 95 64 8 45,XY,add(3)(q29),del(4)(q26q35),der(7)(1p36-1p32::7p22-7q32::15q22-15q26), -8,der(9),del(13)(q21q34),-15,-17,-18,+19,add(19)(p13),+2mar,+ac[17]/46,XY[3] 78 34/21 1O3G/2O3G 9 44,X,-X,-6,der(13;15)(q10;q10),add(17)(p13),-20,+mar[13]/46,XX[7] 95 10/23 1O3G/1O2G 10 - 95 66/31 11 45,XY,add(6)(q22),del(11)(q11q22),-17[15]/44,XY,add(6)(q22),del(11)(q11q22),-17,-20,-22,+mar[2] 87 40/24 12 46,XX,del(13)(q14q21)[2]/45,X,-X,del(13)(q14q21)[3]/45,XX,add(3)(q27), t(9;10)(q21;q22),+12,der(12)t(12;17)(q11;p11),del(13)(q14q21),-14,-17[7]/46,XX[8] 70 62 2O3G 8q+ 13 46,XY[30] 14 88 14 46,XY[13] 80 82 15 47,XY,+12[8]/46,XY,add(1)(p34),add(2)(q34),t(11;22)(p14;q11),+12,-22[15]/46,XY[11] 75 18 16 43,X,-X,del(2)(p15),+4,-7,add(11)(q21),-12,-13,add(14)(q32),add(17)(p11)[6]/46,XX[9] 19 14 17 45,XY,del(6)(q?),-9,add(14)(q32),-22,+mar[9]/ 46,XY,del(6)(q?),add(17)(p13),add(19)(q13)[21] 55 23 18 45,XY,add(6)(p11),-22[13]/46,XY,i(17)(q10)[5]/46,XY[16] 16 57 19 - 70 66 2O4G 20 - 90 81 2OnG 21 46,XY[11] 43 60 4O4G Tetraploid *O: LPL signal in orange, G: MYC signal in green. Figure 1. Kaplan Meier plots for OS and (A) 8p- and/or 8q+, (B) 8p- or (C) 8q+. Figure 1. Kaplan Meier plots for OS and (A) 8p- and/or 8q+, (B) 8p- or (C) 8q+. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Calreticulin (CALR) is a multifunctional protein regulated by calcium that is located in the endoplasmic reticulum. Recently, mutations in the calreticulin gene have been described in patients with the diagnosis of essential thrombocytemia (ET) and primary myelofibrosis (PMF), mainly in JAK2-negative cases. CALR mutations are localized to exon 9 and generate deletions or insertions that lead to a frameshift change resulting in a mutant protein. The detection of these mutations helps in the actual diagnosis of JAK2 mieloproliferative syndromes (MPN). Our aim is to assess the utility of the determination of these mutations in the management of patients with diagnosis of MPN in our center. Patients and methods: This study includes 94 patients with diagnosis of JAK2-negative MPN retrospectively selected following clinical and analytical criteria between 2008 and 2014 in our center (Table 1, 2). CALR mutations were performed with the use of fluorescent PCR following the methods described by Klampf et al. (NEJM, 2013). Results: 94 patients were analyzed, 77 of them had the diagnosis of TE, 8 of PMF and 9 of others disorders of myelodisplastic/mieloproliferative. 22% of the cases of ET had mutations in CALR (Table 1). In these mutations, a total of 53% were type I mutations (52-bp deletion) and 47% were type II mutations (5-bp insertion). Only one mutation was infrequent, a 46-pb deletion. We have found statistical correlation in the number of platelets depending on the presence of the mutation and in the largest number of platelets in type II mutations. 33% of the cases of PMF had mutations in CALR, all of them type I. Among other diseases not included in MPN, one of them had a type I mutation (data not shown). Conclusions: Our results are close to recently published results regarding the frequency of mutation and as the largest number of platelets in type II mutations with respect to mutation type I. This study confirms the importance of CALR mutations determination in the diagnosis of JAK2-negative ET and PMF with high clinical suspicion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: It´s well known that NK cells exert an important role in postransplant immune reconstitution. Their maturation process through the contact with recipient´s bone marrow stromal cells is crucial for the development of their functional capacities. Previous studies showed the relevance of NK alloreactivity in relapse prevention and the influence of NK subpopulations and expression of activating receptors on CMV infection control, antitumor effects, and graft versus host disease (GVHD). Unmanipulated Haploidentical (Haplo) transplantation with postrasplant cyclophosphamide (Cy) is an emerging alternative for patients (pts) with no identical sibling donor with encouraging results. We previously reported preliminary data of NK cell reconstitution in 7 pts in Haplo setting. Our aim is to contrast our previous data in 22 pts comparing with conventional HLA-identical (HLAid) Stem Cell Transplantation (SCT) and to analyse the relationship between NK reconstitution and CMV reactivation and GVHD. Methods: 22 pts received an Haplo SCT and 7 pts an HLAid SCT in Gregorio Marañon Hospital between January 2013 and April 2014. Peripheral blood was used in all cases. Conditioning regimen comprised fludarabine (Flu), Cy and busulfan (Bux) for Haplo and Flu/Bux or Flu/Melphalan for HLAid SCT. Prophylaxis for GVHD consisted of high dose Cy on +3, +4, cyclosporine (CsA) A and mycophenolatemofetil for Haplo and CsA/Methotrexate for HLAid. Table1 show patients characteristics. For analysis of NK reconstitution and receptor expression multi-colour flow cytometry on FC500 and Navios Beckman Coulter® was used. Total NK cells, CD56 intensity and expression of NKG2A, NKp30, Nkp46 and NKG2D was studied at +15, +30, +60, and +90. For comparison between the two groups Mann–Whitney U-test was used. Results: Median NK cells/mm3 on +15 and +30 were significantly lower on Haplo than HLAid group ( 0 (0-5.5) and 28.5 (12.2-96.5) vs 69.5 (55-134) and 276 (151-422); p