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Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

Background CPX-351 is approved for the treatment of therapy related acute myeloid leukemia (t-AML) and AML with myelodysplastic related changes (MRC-AML). The benefits of this treatment over standard chemotherapy has not been addressed in well matched cohorts of real-life patients. Methods Retrospective analysis of AML patients treated with CPX-351 as per routine practice. A propensity score matching (PSM) was used to compare their main outcomes with those observed in a matched cohort among 765 historical patients receiving intensive chemotherapy (IC), all of them reported to the PETHEMA epidemiologic registry. Results Median age of 79 patients treated with CPX-351 was 67 years old (interquartile range 62–71), 53 were MRC-AML. The complete remission (CR) rate or CR without recovery (CRi) after 1 or 2 cycles of CPX-351 was 52%, 60-days mortality 18%, measurable residual disease

The familial occurrence of hematological malignancies has been underappreciated. Recent studies suggest that up to 15% of adults with myeloid neoplasms carry germline pathogenic variants in cancer-predisposing genes. This study aimed to identify the underlying germline predisposition variant in patients with a strong family or personal onco-hematological history using whole exome sequencing on sixteen uncharacterized individuals. It was carried out in two groups of patients, one with samples available from two affected relatives (Cohort A) and one with available samples from the index case (Cohort B). In Cohort A, six families were characterized. Two families shared variants in genes associated with DNA damage response and involved in cancer development (CHEK2 and RAD54L). Pathogenic or likely pathogenic germline variants were also found in novel candidate genes (NFATC2 and TC2N). In two families, any relevant pathogenic or likely pathogenic genomic variants were identified. In Cohort B, four additional index cases were analyzed. Three of them harbor clinically relevant variants in genes with a probable role in the development of inherited forms of hematological malignancies (GATA1, MSH4 and PRF1). Overall, whole exome sequencing is a useful approach to achieve a further characterization of these patients and their mutational spectra. Moreover, further investigations may help improve optimization for disease management of affected patients and their families.

Although acute myeloid leukemia (AML) with NPM1 mut /FLT3-ITDneg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1 mut, and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3 D835 variant at diagnosis and a qPCR value of NPM1 mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1 mut/FLT3-ITDneg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.

Pethema Group., [Simple Summary] The use of azacitidine (AZA) and decitabine (DEC) have allowed more elderly acute myeloid leukemia (AML) patients to be treated. However, scarcely any direct comparative data exist between both drugs. This study shows no significant differences in response rates or overall survival (OS) between upfront AZA and DEC treatment in a large retrospective with long-term follow-up cohort of AML patients. However, we identified for the first time the baseline characteristics of patients benefitting from AZA vs. DEC in terms of responses, 120-day mortality and OS. We also show differences in salvage treatment patterns and outcomes after failure to both hypomethylating agents in a real-life setting. Taken together, these findings could help to select the most appropriate hypomethylating agent in monotherapy., The hypomethylating agents, decitabine (DEC) and azacitidine (AZA), allowed more elderly acute myeloid leukemia (AML) patients to be treated. However, there are little direct comparative data on AZA and DEC. This multicenter retrospective study compared the outcomes of AZA and DEC in terms of response and overall survival (OS). Potential predictors associated with response and OS were also evaluated. A total of 626 AML patients were included (487 treated with AZA and 139 with DEC). Response rates were similar in both groups: CR was 18% with AZA vs. 23% with DEC (p = 0.20), CR/CRi was 20.5% vs. 25% (p = 0.27) and ORR was 32% vs. 39.5% (p = 0.12), respectively. Patients with leukocytes < 10 × 109/L, bone marrow blasts < 50% and ECOG ≥ 2 had higher ORR with DEC than with AZA. OS was similar in both groups: 10.4 months (95% CI: 9.2–11.7) vs. 8.8 months (95% CI: 6.7–11.0, p = 0.455), for AZA and DEC, respectively. Age (≥80 years), leukocytes (≥ 10 × 109/L), platelet count (

Quizartinib is a tyrosine kinase inhibitor selectively targeting the FMS-like tyrosine kinase 3 (FLT3) receptor that has been developed for the treatment of acute myeloid leukaemia (AML). The Phase 3 QuANTUM-R study investigated the efficacy of quizartinib monotherapy in patients with relapsed/refractory FLT3-ITD mutation-positive AML. The clinical course of four QuANTUM-R participants exemplifies issues specific to quizartinib treatment and is described here. Patient 1 was FLT3-ITD mutation-negative at AML diagnosis, but became FLT3-ITD mutation-positive during treatment that included several lines of chemotherapy and was therefore a suitable candidate for quizartinib. Because of the clonal shifts of AML during treatment, retesting genetic alterations at each relapse or resistance may help to identify candidates for targeted treatment options. Patient 2 developed QTc prolongation during quizartinib treatment, but the QTc interval normalised after dose reduction, allowing the patient to continue treatment and eventually resume the recommended dose. Patient 3 responded to quizartinib and was scheduled for haematopoietic stem cell transplant (HSCT), but developed febrile neutropenia and invasive aspergillosis during conditioning and subsequently died (to avoid drug-drug interactions, no azole antifungal was administered concomitantly). Care is required when selecting concomitant medications, and if there is potential for interactions (e.g. if prophylactic azole antifungals are required) the quizartinib dose should be reduced to minimise the risk of QTc prolongation. Patient 4 was able to undergo HSCT after responding to quizartinib and experienced a durable response after HSCT while on quizartinib maintenance therapy. Together, these cases illustrate the main issues to be addressed when managing patients under quizartinib, allowing for adequate scheduling and tolerability, bridging to HSCT, and durable remission on maintenance therapy in some patients.

Conventional cytogenetics are the gold standard for the identification of chromosomal alterations recurrent in myeloid neoplasms. Some next-generation sequencing (NGS) panels are designed for the detection of copy number variations (CNV) or translocations, however, their use is far from being widespread. Here we report on the results of a commercial panel including frequent mutations, CNVs and translocations in myeloid neoplasms. Frequent chromosomal alterations were analyzed by NGS in 135 patients with myeloid neoplasms and three with acute lymphoblastic leukemia. NGS analysis was performed using the enrichment-capture Myeloid Neoplasm-GeneSGKit (Sistemas Genómicos, Spain) gene panel including 35 genes for mutational analysis and frequent CNVs and translocations. NGS results were validated with cytogenetics and/or MLPA when possible. A total of 66 frequent alterations included in NGS panel were detected, 48 of them detected by NGS and cytogenetics. Ten of them were observed only by cytogenetics (mainly trisomy 8), and another eight only by NGS (mainly deletion of 12p). Aside from this, 38 secondary CNVs were detected in any of the genes included mainly for mutational analysis. NGS represents a reliable complementary source of information for the analysis of CNVs and translocations. Moreover, NGS could be a useful tool for the detection of alterations not observed by conventional cytogenetics.

SARS-CoV-2 infection can impact survival of patients with acute myeloid leukemia (AML). International experts recommend considering delaying or stopping AML treatment, test patients who need intensive induction and s prioritizing outpatient treatment. However there is little published evidence in AML. Objective To analyze the clinical futures and outcome of SARS-CoV-2 infection in AML patients. Methods and patients Observational multicenter study between March and May 2020; 117 patients reported from 47 Spanish centers, but 13 had no PCR or antibody test documented, finally including 104 patients from 45 hospitals. Results The median age was 68 years, men (56.7% vs 43.3%), and the median time from AML diagnosis to SARS-CoV-2 was 4 months. The mean of comorbidities was 1.2, high blood pressure (40.4%), heart disease (17.3%), diabetes (13.5%), smoking (8.8%), chronic obstructive pulmonary disease or emphysema (7.7%), renal failure (6.7%) and liver dysfunction (1.9%). Cytogenetic risk was low in 16.9%, intermediate in 57.1% and high in 26.0%; 55.7% had active disease, 39.2% complete remission and 5.1% partial response. 29.4% were off-therapy and 70.6% under antileukemic treatment at the time of SARS-CoV-2: induction chemotherapy (25.3%), hypomethylating (19.3%), clinical trial (17.0%), consolidation chemotherapy (14.8%), venetoclax (3.4%), FLT3 inhibitors (3.4%) and/or maintenance (1.1%). Overall 3.7% were newly diagnosed (no prior therapy), 77.8% had received one line of treatment, 14.8% two and 3.7% four. 15.4% had prior allogeneic transplantation. Only 4.0% of the patients were asymptomatic, while the main signs and symptoms were fever (77.8%), pneumonia (75.0%), cough (65.3%), dyspnea (52.0%), diarrhea (20.4%), nausea and/or vomiting (12.2%), rhinorrhea (10.2%) and headache (7.4%). Analytical parameters were: neutrophils 3112 cells/µL (1900-7300), lymphocytes 1090 cells/µL (1000-3000), interleukin 6 118 pg/mL (0-100), ferritin 4505 ng/mL (15-150) and D-dimer 2823 ng/mL (20-500), with liver enzymes altered in 23.9% of cases. 84.2% received specific treatment for coronavirus infection: chloroquine or hydroxychloroquine (82.2%), lopinavir/ritonavir (54.0%), corticosteroids (39.6%), azithromycin (33.0%), tocilizumab (15.8%), plasma convalescent (3.0%), clinical trial medication (3.0%), remdesivir (2.0%) and/or anakinra (1.0%). The course was mild in 14.7% (no hospitalization), moderate in 32.0% and severe in 53.3%. The implementation of intensive measures was assessed in 48.2%(14.9% admitted to the ICU and the remaining 33.3% rejected). The mean time to negativization was 20.5 days, duration of symptoms 17.6 days and the hospital stay 11.1 days. In 48.1% of the cases treatment for AML was maintained, in 26.6% delayed and in 25.3% modified due to coronavirus disease.47.5% died, establishing an association between mortality and age over 60 years (58.3% vs 36.4%, p=0.043), ≥2 lines of treatment (72.7% vs 44.3%, p=0.020), active disease (62.5% vs 29.4%, p=0.002) and pneumonia (61.2% versus 22.7%, p=0.002). Overall 47.5% overcame the infection, and in 5.0% SARS-CoV-2 genetic material was still detected at the time of analysis. A non-significant lower mortality rate was observed among: previous transplantation (45.7% vs 64.3%, p=0.19), neutrophil >1900 cells/µL (41.1% vs 60.0%, p=0.09), lymphocyte >1000 cells/µL (42.9% vs 63.6%, p = 0.09) and hydroxychloroquine/chloroquine plus azithromycin (35.3% vs 60.0%, p=0.10). Conclusions SARS-CoV-2 infection produces high mortality among AML patients. Mortality was correlated with age, active disease and pneumonia. Disclosures Martinez-Lopez: Janssen-cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Novartis: Consultancy; Janssen: Consultancy, Honoraria; Incyte: Consultancy, Research Funding.

It has been suggested that 1–2% of acute promyelocytic leukemia (APL) patients present variant rearrangements of retinoic acid receptor alpha (RARα) fusion gene, with the promyelocytic leukaemia zinc finger (PLZF)/RARα being the most frequent. Resistance to all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) has been suggested in PLZF/RARα and other variant APLs. Herein, we analyze the incidence, characteristics, and outcomes of variant APLs reported to the multinational PETHEMA (Programa para el Tratamiento de Hemopatias Malignas) registry, and we perform a systematic review in order to shed light on strategies to improve management of these extremely rare diseases. Of 2895 patients with genetically confirmed APL in the PETHEMA registry, 11 had variant APL (0.4%) (9 PLZF-RARα and 2 NPM1-RARα), 9 were men, with median age of 44.6 years (3 months to 76 years), median leucocytes (WBC) 16.8 × 109/L, and frequent coagulopathy. Eight patients were treated with ATRA plus chemotherapy-based regimens, and 3 with chemotherapy-based. As compared to previous reports, complete remission and survival was slightly better in our cohort, with 73% complete remission (CR) and 73% survival despite a high relapse rate (43%). After analyzing our series and performing a comprehensive and critical review of the literature, strong recommendations on appropriate management of variant APL are not possible due to the low number and heterogeneity of patients reported so far.

Development of de novo hematologic malignancies in donor cells after allogeneic stem cell transplantation (allo-SCT) provides a useful in vivo model to study the process of leukemogenesis. A systematic analysis of the cases reported in the literature was performed to identify risk factors and mechanisms involved in the pathogenesis of donor cell–derived hematologic neoplasms (DCHN) and leukemogenic transformation. Relevant data were extracted from 137 cases. Cases of DCHN show a wide heterogeneity with regard to recipient/donor age, sex mismatch, and conditioning regimen. Some characteristics, such as the type of primary disease, the type of hematologic malignancy of the DCHN, and the stem cell source used in the transplant procedure, differ from those expected. Mechanisms involved in the pathogenesis of DCHN are complex, and several hypotheses have been proposed, such as pre-existing hematologic neoplasms or premalignant clones in the donor, decreased immune surveillance, and damage to bone marrow microenvironment in the recipient. Most likely several if not all these mechanisms play a role in DCHN development. Novel approaches, such as next-generation sequencing to study consecutive samples after allo-SCT in these patients, appear to be promising to decipher the mechanisms of leukemogenesis.

BACKGROUND The revised genetic risk classification established by the European Leukemia Net (ELN) in 2017 stratifies patients diagnosed with acute myeloid leukemia (AML) into 3 prognostic categories (favourable, intermediate, and adverse) based on cytogenetic and molecular characteristics.The ELN classification is widely accepted in AML patients despite the fact that validation studies were performed in participants who received exclusively first-line treatment with intensive chemotherapy. For this reason, it is not well established whether the ELN risk groups are applicable to patients on non-intensive first-line treatment. OBJECTIVES - To describe and compare baseline characteristics at diagnosis between patients with AML treated with intensive and non-intensive therapy. - To assess whether the ELN prognostic classification is applicable in these subgroups of patients. METHODS We retrospectively analysed patients with newly diagnosed AML admitted to our center between 2007 and 2020. Patients with acute promyelocytic leukemia (M3), patients younger than 18 years old and/or patients who received exclusively supportive treatment were excluded. Demographic and clinical data, disease characteristics at diagnosis and first-line treatment were collected. Cytogenetic and molecular characteristics were used to classify patients in ELN risk groups. RESULTS Of the total of patients (n=218), one hundred and fifty-six (71.6%) received intensive chemotherapy treatment, while 62 (28.4%) were treated with non-intensive strategies. Idarubicin and cytarabine based schemes regimens (IA) were administered in most patients (98.6%) who received intensive treatment while the rest received fludarabine based regimens. One patient (0.6%) was treated with danurubicin and cytarabine liposome (CPX-351). Fifty-four (87%) patients treated with non-intensive regimens received hypomethylating agents, mostly azacitidine. Five patients (8%) were treated with venetoclax in combination with a hypomethylating agent. Table 1 shows the characteristics at diagnosis in both groups of patients. Patients who received intensive chemotherapy were younger and had higher leukocyte count, LDH values and a higher percentage of blasts in peripheral blood and bone marrow with a median of 40% and 62% blasts respectively. On the other hand, patients under non-intensive treatment more frequently presented a past history of hemopathy and a higher percentage of bone marrow dysplasia. Regarding ELN stratification significant differences were found between both groups. Patients who received aggressive chemotherapy vs patients who did not, were classified in low (28% vs. 7%), intermediate (36% vs. 58%) and high risk (36% vs. 35%) respectively (Figure 1). At the end of the follow-up, 41% of the patients who had received intensive therapy were alive while only 6.5% of the patients who had received non-intensive treatment were alive. Significant differences in survival were observed between both groups (p In the intensive chemotherapy group, significant differences in survival were observed according to ELN risk stratification (p However, in patients receiving non-intensive therapies, there were no significant differences in survival among different prognostic categories (p=0.06). In this group, 1-year OS was 25%, 57.6% and 40.7% and median OS was 2.1, 14.8 and 10.1 months for low, intermediate and high-risk groups respectively. See Figure 2. CONCLUSIONS: As validated in previous trials, ELN classification constitutes an adequate prognostic marker for patients with newly diagnosed AML treated with intensive chemotherapy. In our series, this classification does not appear to be a good predictor of survival for patients diagnosed with AML who initiated non-intensive treatments. Further validation in prospective studies are needed to better classify this growing subgroup of patients in clinical practice. Figure 1 Figure 1. Disclosures Martín-Rojas: Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Font Lopez: Pfizer: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees; CELGENE-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kwon: Novartis, Celgene, Gilead, Pfizer: Consultancy, Honoraria.

INTRODUCTION Several studies have shown that morphological remission at day 14 is a predictor of post-induction response in patients with acute myeloid leukemia (AML) undergoing an intensive treatment. However, the role of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) at day 14 remains unknown. The aim of our study is to explore the role of MRD at day 14 and its association with outcomes of patients with AML undergoing an intensive treatment. METHODS We conducted a retrospective study in adult patients with newly a diagnosed AML in our center between 2007 and 2020. Adult patients who received intensive chemotherapy, excluding those with an acute promyelocytic leukemia, were included. Bone marrow aspiration was performed at day 14 after induction to assess morphological response and MRD by MFC. Early blast clearance (EBC) was defined as RESULTS A total of 131 patients were analyzed. Median age was 55.6 years (IQR 42.3-64.2). The most frequent AML subtype was AML with myelodysplasia-related changes (34.4%), followed by NPM1-mutated AML (32.1%). The most commonly used induction regimen was "7+3" (96.2%) (Table 1). On day 14 bone marrow aspiration, median cellularity was 0.5/5 (IQR 0.5-1). 107 patients (81.7%) showed a blast reduction >50% compared to diagnosis and 87 patients (66.4%) had less than 5% of blasts. In this latter group, 28.6% of patients had a positive MRD and 71.4% had a negative MRD. NPM1-mutated AML showed the highest EBC rates while AML with myelodysplasia-related changes had the lowest rates (83.3% versus 55.5%; p=0.04). Furthermore, there were statistically significant differences in EBC rates based on the 2017 European Leukemia Net risk stratification, with 80% of EBC in low risk, 66.6% in intermediate risk and 53.4% in high risk AML (p=0.038). No differences were observed in MRD at day 14 based on AML subtypes or risk stratification. We subsequently analyzed the negative (NPV) and positive predictive values (PPV) of day 14 bone marrow aspiration results by morphology and MFC to predict post-induction results. As a predictor of post-induction CR, day 14 EBC had a NPV of 82% and a PPV of 69%, while day 14 MRD had a NPV of 86% and a PPV of 49%. However, for predicting post-induction MRD, day 14 EBC had a NPV of 49% and a PPV of 15%, while day 14 MRD had a NPV of 71% and PPV of 74%. The correlation between day 14 and post-induction bone marrow aspiration is shown in Table 2. Bivariate analysis showed that achieving CR with negative MRD in post-induction bone marrow aspiration was associated with EBC (p CONCLUSION Patients showing EBC with negative MRD on day 14 bone marrow aspiration are more likely to achieve post-induction CR with negative MRD, with day 14 MRD by MFC being the only independent factor able to predict post-induction CR with negative MRD in our cohort. However, further prospective studies are needed to confirm our findings. Figure 1 Figure 1. Disclosures Martín-Rojas: Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Kwon: Novartis, Celgene, Gilead, Pfizer: Consultancy, Honoraria.

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

Patient: Male, 21 Final Diagnosis: Methotrexate induced neurotoxicity Symptoms: Dysarthria • hemiparesis Medication: Methotrexate Clinical Procedure: — Specialty: Hematology Objective: Challenging differential diagnosis Background: Intraventricular administration of methotrexate (MTX) using an Ommaya reservoir is a useful therapeutic maneuver for malignant CNS involvement in patients with hematological malignancies. MTX-induced subacute neurotoxicity is a rare complication that typically progresses with involvement of the basal ganglia. Local toxicity due to misplaced catheters has been described, although the impact of normally positioned catheters on toxicity is not clear. Case Report: We report the case of a 21-year-old man diagnosed with stage IV diffuse large B-cell lymphoma who experienced a central nervous system relapse. While receiving intraventricular MTX using an Ommaya reservoir and systemic MTX, he experienced sudden left-side hemiparesis. All diagnostic tests were negative except for altered MRI findings with FLAIR hyperintensity in the basal ganglia and restricted diffusion in the same location that followed the track of the Ommaya catheter. The syndrome resolved after administration of high-dose steroids, and the patient received subsequent MTX courses without recurrence. Conclusions: MTX-induced neurotoxicity is a rare adverse event related to systemic and intrathecal administration of the drug. Many cases of Ommaya-related CNS symptoms have been described, although most were related to misplaced or malfunctioning catheters. Here we present a case of subacute MTX toxicity affecting the area around a correctly positioned catheter, suggesting that the catheter track could be more susceptible to MTX-induced toxicity.

Mould-active prophylaxis is affecting the epidemiology of invasive mycoses in the form of a shift toward less common entities such as fusariosis. We analyze the characteristics of invasive fusariosis and its association to antifungal prophylaxis in a retrospective cohort (2004–2017) from a tertiary hospital in Madrid, Spain. Epidemiological, clinical, microbiological, and antifungal consumption data were retrieved. Isolates were identified to molecular level, and antifungal susceptibility was tested. Eight cases of invasive fusariosis were diagnosed. Three periods were identified according to incidence: 2014 (five cases). All except one case involved breakthrough fusariosis. During the earliest period, the episodes occurred while the patient was taking itraconazole (two) or fluconazole (one); more recently, while on micafungin (three) or posaconazole (one). Early cases involved acute leukemia at induction/consolidation, recent cases relapsed/refractory disease (P = .029). Main risk factor for fusariosis (62.5%) was prolonged neutropenia (median 44 days). Galactomannan and beta-D-glucan were positive in 37.5% and 100% of cases, respectively. All isolates except F. proliferatum presented high minimal inhibitory concentrations (MICs) against the azoles and lower MIC to amphotericin B. Most patients received combined therapy. Mortality at 42 days was 62.5%. Resolution of neutropenia was associated with survival (P = .048). Invasive fusariosis occurs as breakthrough infection in patients with hematologic malignancy, prolonged neutropenia, and positive fungal biomarkers. Recent cases were diagnosed in a period of predominant micafungin use in patients who had more advanced disease and protracted neutropenia and for whom mortality was extremely high. Resolution of neutropenia was a favorable prognostic factor.

We present our experience in patients with hematologic malignancy and Pseudomonas aeruginosa infection treated with ceftolozane-tazobactam. We performed a single-center case-control study comparing patients with hematologic malignancy and P. aeruginosa infection treated with ceftolozane-tazobactam (study group) with similar patients not treated with ceftolozane-tazobactam (control group) to assess safety and efficacy. Nineteen cases and 38 controls were analyzed. Cases were younger (45.6 years versus 57.6 years; P = 0.012) and less frequently had bacteremia (52.6% versus 86.8%; P = 0.008). They also had worse Multinational Association for Supportive Care in Cancer (MASCC) scores (10.2 versus 16.1; P = 0.0001), more hospital-acquired infections (78.9% versus 47.4%; P = 0.013), and more extremely drug-resistant (XDR) P. aeruginosa infections (47.4% versus 21.1%; P = 0.015). Cases received a median of 14 days (7 to 18 days) of ceftolozane-tazobactam (monotherapy in 11 cases [57.9.6%]). Ceftolozane-tazobactam was mostly used as targeted therapy (16 cases; 84.2%) because of resistance (9 cases; 47.4%), failure (4 cases; 21.1%), and toxicity (3 cases; 15.8%). Ten cases had bacteremia (52.6%). The sources were pneumonia (26.3%), catheter-related bacteremia (21.1%), primary bacteremia (21.1%), and perianal/genital (15.7%), urinary (10.5%), and skin/soft tissue (5.3%) infection. No toxicity was attributed to ceftolozane-tazobactam. More than 60% had neutropenia, and 15.8% fulfilled the criteria for sepsis. There were no significant differences in clinical cure at day 14 (89.5% versus 71.1%; P = 0.183) or recurrence (15.8% versus 10.5%; P = 0.675). Thirty-day mortality was lower among cases (5.3% versus 28.9%; P = 0.045). Ceftolozane-tazobactam was well tolerated and at least as effective as other alternatives for P. aeruginosa infection in patients with hematologic malignancy, including neutropenic patients with sepsis caused by XDR strains.

Acute myeloid leukemia is a clonal but heterogeneous disease differing in molecular pathogenesis, clinical features and response to chemotherapy. This latter frequently consists of a combination of cytarabine and anthracyclines, although etoposide, demethylating agents, and other drugs are also used. Unfortunately, chemoresistance is a common and serious problem. Multiple mechanisms account for impaired effectiveness of drugs and reduced levels of active agents in target cells. The latter can be due to lower drug uptake, increased export or decreased intracellular proportion of active/inactive agent due to changes in the expression/function of enzymes responsible for the activation of pro-drugs and the inactivation of active agents. Characterization of the "resistome", or profile of expressed genes accounting for multi-drug resistance (MDR) phenotype, would permit to predict the lack of response to chemotherapy and would help in the selection of the best pharmacological regime for each patient and moment, and to develop strategies of chemosensitization.

// Rocio I.R. Macias 1, 2 , Anabel Sanchez-Martin 1 , Gabriela Rodriguez-Macias 1, 3 , Luis I. Sanchez-Abarca 4 , Elisa Lozano 1, 2 , Elisa Herraez 1, 2 , Maria D. Odero 5 , Jose L. Diez-Martin 3, 6 , Jose J.G. Marin 1, 2, * and Oscar Briz 1, 2, * 1 Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), University of Salamanca, IBSAL, Salamanca, Spain 2 National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Madrid, Spain 3 Department of Hematology-BMT Unit, Hospital General Universitario Gregorio Maranon, Madrid, Spain 4 Department of Hematology, University Hospital of Salamanca, IBSAL, Salamanca, Spain 5 Department of Biochemistry and Genetics and CIMA, University of Navarra, Pamplona, CIBERONC, Spain 6 Gregorio Maranon Institute for Health Research (IISGM), Madrid, Spain * These authors have contributed equally to this work Correspondence to: Rocio I.R. Macias, email: rociorm@usal.es Keywords: AML; cancer; chemoresistance; chemotherapy; tyrosine kinase inhibitor Received: December 21, 2017 Accepted: April 24, 2018 Published: June 19, 2018 ABSTRACT Background : Chemoresistance often limits the success of the pharmacological treatment in acute myeloid leukemia (AML) patients. Although positive results have been obtained with tyrosine kinase inhibitors (TKIs), such as sorafenib, especially in patients with Fms-like tyrosine kinase 3 (FLT3)-positive AML, the success of chemotherapy is very heterogeneous. Here we have investigated in vitro whether the transportome (set of expressed plasma membrane transporters) is involved in the differential response of AML to sorafenib. Methods : The sensitivity to sorafenib-induced cell death (MTT test and anexin V/7-AAD method) was evaluated in five different cell lines: MOLM-13, OCI-AML2, HL-60, HEL and K-562. The transportome was characterized by measuring mRNA using RT-qPCR. Drug uptake/efflux was determined by flow cytometry using specific substrates and inhibitors. Results : The cytostatic response to sorafenib was: MOLM-13>>OCI-AML2>HL-60>HEL≈K-562. Regarding efflux pumps, MDR1 was highly expressed in HEL>K-562≈MOLM-13, but not in OCI-AML2 and HL-60. BCRP and MPR3 expression was low in all cell lines, whereas MRP4 and MRP5 expression was from moderate to high. Flow cytometry studies demonstrated that MRP4, but not MRP5, was functional. The expression of the organic cation transporter 1 (OCT1), involved in sorafenib uptake, was MOLM-13>OCI-AML2≈HL-60 and non detectable in HEL and K-562. Transfection of HEL cells with OCT1 increased the sensitivity of these cells to sorafenib, whereas inactive genetic variants failed to induce this change. Conclusion: Together with changes in the expression/function of receptors targeted by TKIs, the expression of plasma membrane transporters involved in sorafenib uptake/efflux may affect the response of leukemia cells to this drug.

We have estimated the pharmacological sensitivity and synergism of 125 individual patient samples for all drugs and combination treatments for acute myeloid leukemia in the context of the overall patient population. Each ex vivo pharmacological profile identifies drugs and treatments for which the patient's malignant cells are particularly sensitive or resistant, assisting in the selection of individualized treatments. Background: We have evaluated the ex vivo pharmacology of single drugs and drug combinations in malignant cells of bone marrow samples from 125 patients with acute myeloid leukemia using a novel automated flow cytometry-based platform (ExviTech). We have improved previous ex vivo drug testing with 4 innovations: identifying individual leukemic cells, using intact whole blood during the incubation, using an automated platform that escalates reliably data, and performing analyses pharmacodynamic population models. Patients and Methods: Samples were sent from 24 hospitals to a central laboratory and incubated for 48 hours in whole blood, after which drug activity was measured in terms of depletion of leukemic cells. Results: The sensitivity of single drugs is assessed for standard efficacy (E-MAX) and potency (EC50) variables, ranked as percentiles within the population. The sensitivity of drug-combination treatments is assessed for the synergism achieved in each patient sample. We found a large variability among patient samples in the dose-response curves to a single drug or combination treatment. Conclusion: We hypothesize that the use of the individual patient ex vivo pharmacological profiles may help to guide a personalized treatment selection.

Introduction Donor cell leukemia (DCL) is a rare complication of allogeneic stem cell transplantation (allo-SCT). The leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. The objective of the present study is to study the dynamics of emergence of mutations that precede the development of DCL. Material and methods We report the case of a female patient diagnosed of acute lymphoblastic leukemia with t(1;19), who developed normal karyotype acute myeloid leukemia (AML) of donor origin 16 months after unrelated cord blood transplantation (UCBT). Whole exome sequencing(SureSelect-XT Human exon capture 50Mb Agilent Technologies) was performed by next generation sequencing (Hiseq 2000 Illumina) on bone marrow samples post allo-SCT, obtained in the days +98, +189, +350, +486 (DCL diagnosis) and + 569 (DCL relapse) as well as on the UCB unit used in SCT. The exome of bone marrow samples post allo-SCT were aligned to the human reference genome (NCBI build 37/hg19), non-synonymous variants in the coding regions of genes related to leukemia were selected and matched to the UCB exome to identify the acquired variants. Variants meeting such criteria were evaluated with three softwares (SIFT, Polyphen and Mutation Taster) to predict their functional effects. The UCB exome was aligned to the human reference genome (NCBI build 37/hg19). Results In silico variants analysis revealed progressive emergence of multiple mutations related to the development of leukemia in bone marrow samples post allo-SCT (Figure 1). Interestingly three of the mutated genes (PTPN11, NRAS, MAP2K1) are part of the same cellular pathway (RAS-MAPK). In addition, mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones (Figure 2). Finally a mutation in SH2B3 gene, a gene that encodes for a regulatory protein of the JAK-STAT pathway, which links strongly to JAK2 inhibiting the activation of the pathway, was detected in the CBU. Conclusions The present study reveals the acquisition of additional somatic mutations in donor cells during leukemogenesis, including mutations not previously described in AML initiation (POU2F2, CA9, POU4F1, FMN2, TLR9, ELF5). Although the cause of DCL onset seems to be multifactorial, the infusion of a CBU with pre-leukemic potential due to mutation in SH2B3 in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in the case here reported. The study of a greater number of DCL cases by next generation sequencing could help to understand the process of leukemogenesis. Figure 1 Mutations related to the onset of donor cell leukemia after hematopoietic transplantation. Figure 1. Mutations related to the onset of donor cell leukemia after hematopoietic transplantation. Figure 2 Dynamic of mutation acquisitions in leukemic clones.(DCL: Donor cell leukemia, CBU: Cord blood unit, CBT: Cord blood transplantation, Quim: Quimerism, CQ: Complete quimerism) Figure 2. Dynamic of mutation acquisitions in leukemic clones.(DCL: Donor cell leukemia, CBU: Cord blood unit, CBT: Cord blood transplantation, Quim: Quimerism, CQ: Complete quimerism) Disclosures No relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a well-established therapeutic option for hematological malignancies. Combination antiretroviral therapy (cART) has enabled the treatment of medical conditions in patients infected with the human immunodeficiency virus (HIV) in the same way as in the general population. Moreover, improvements in supportive care have allowed HIV-infected patients with life-threatening hematological disorders to be treated with Allo-HSCT. We report on four HIV-infected patients with hematological malignancies receiving an Allo-HSCT in our institution, and on the use of donor lymphocyte infusions to successfully treat post-Allo-HSCT relapse. Of note, one of them is the first HIV(+) patient to receive a "dual transplant" (unrelated umbilical cord blood stem cells combined with mobilized T cell-depleted CD34(+) stem cells from a mismatched third party donor). cART drugs interactions were satisfactorily managed. This approach provided long-term control of the hematological disease. Nevertheless, despite adequate immune reconstitution, infections were the main cause of morbidity and mortality after Allo-HSCT.

e18510Background: The aim of this study is to determine the ability of Vivia's novel test (based on studying the ex-vivo sensitivity to drugs) to predict the complete remission (CR) rates after ind...

Background: We have overcome the limitations of 40 years of ex vivo testing. The aim of this study is to determine the ability of Vivia's novel test to predict the complete remission (CR) rates after induction chemotherapy with cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML. Material and Methods: Bone marrow samples from adult patients diagnosed with de novo AML in Spanish centers from the PETHEMA group were included. Whole marrow samples maintaining their Native Environment were incubated for 48h in well plates containing Ara-C, Ida, or their combination. Pharmacological responses are calculated using population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders and the remaining as resistant. Results: 390 patient samples were used to calculate the dose response (DR) curves for Ara-C alone, Ida alone, and their synergism. For clinical correlation we used 142 patients with median 56 years. The strongest clinical predictor was the Area Under the Curve (AUC) of the DR of Ara-C, and the AUC of IDA. The GAM models revealed a significant relationship between the AUC of the concentration-effect curves of both, idarubicin and, particularly, Ara-C, with greater values associated to higher probabilities of post-induction resistance. The fitted Generalized Additive Method predictions of expected values for each patient were in turn related to overall survival when a discrimination value to define positive and negative test results that prioritized specificity over sensitivity was chosen based on equaling positive and negative predictive values (Fig 1A). Prioritizing specificity over sensitivity reflects the higher cost of false positive over false negative decisions: only in very rare instances, an effective treatment would be erroneously negated to a sensitive patient at the expense of overlooking a number of resistant patients. However, the later patients could take their chances on re-induction therapy. While for diagnostics sensitivity and specificity should both be optimized, for Personalized Medicine the positive and negative predictive values should be optimized preferentially because they define the patient response correlation. Fig 1B shows a table illustrating the correlation between clinical outcome (columns) and the test predictions (lines). From a diagnostic criteria (columns), clinically resistant patients (1st column) are not well predicted with a Sensitivity of 51%, while clinically sensitive patients (2nd column) are very well predicted with a Specificity of 94%. From a Precision Medicine criteria (Lines), patients predicted resistant (1st line) and well predicted with 80% positive predictive value, similar to patients predicted sensitive (2nd line) well predicted with 79% Negative Predictive Value. The test does not properly identify 23/142 that are clinically resistant and the test predicts as sensitive (bottom left quadrant right panel). This mismatched subgroup mimics the problems from molecular markers where a resistant clone present in a minority of leukemic cells cannot be detected yet drives the patient response. However, this group mismatch does not prevent a good correlation with the test predicted outcomes. Flow cytometry identified 2 clones in 75% of these 23 samples, and we revised all samples analyzing each of 2 clones separately whenever they were present. Results did not change by this clonal analysis, suggesting flow cytometry may not identify resistant clones. Future improvements of the test adding 16 concentrations to the dose response curves may be able to detect the presence and parameters of these resistant clones driving patient response. Conclusions: This novel test is able to predict the clinical response to Ida + Ara-C induction with overall correlation and predictive values of 80%, higher than ever achieved. It is significantly higher than the current clinical response rate of 66.7%. Thus this novel test may be valuable information to guide 1st line patient therapy. Figure 1. ROC curve and clinical correlation Figure 1. ROC curve and clinical correlation Disclosures Ballesteros: Vivia Biotech: Employment. Cordoba:Celgene: Research Funding. Ramos:GlaxoSmithKline: Honoraria; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria. Gaspar:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Gomez:Vivia Biotech: Employment. Hernández:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment.

Haploidentical bone marrow transplantation with post-grafting cyclophosphamide: multicenter experience with an alternative salvage strategy

INTRODUCTION AND AIMS Almost seventy percent of Acute Myeloid Leukemia (AML) patients will relapse. Fludarabine, Ara-C, Idarrubicine, G-CSF (FLAG-Ida) and FLAG-Ida and Gentuzumab-Ozogamicin (FLAGO-Ida) is frequently used before allogenic stem cell transplant (ASCT). There are several studies analyzing the risk factors for survival in relapsed/refractory patients, but none of them were performed exclusively in patients treated with FLAG-Ida. We analyzed the results of in the Spanish trials conducted by PETHEMA. We also present a predictive score system of survival prognosis based on the data of the patients enrolled METHODS This is a retrospective, multicentre study of the PETHEMA AML epidemiologic Registry. In PETHEMA protocols 98,99,2007,2010 and 2011, FLAG-Ida was included as salvage treatment in patients relapsed or resistant after a 2ndinduction based in 3+7. In PETHEMA trial 2007, FLAG-Ida (or FLAGO-Ida) was administered if complete remission (CR) was not attained in the first induction cycle. The use of FLGO in PETHEMA 2007 trial depended on the availability of GO for each centre. We use overall survival (OS) as final end point of the analysis. Univariate survival analysis was performed by Long-Rank test. Stepwise backward variable selection and Cox proportional hazard multivariable analysis was performed (covariables selected if p RESULTS We analyzed 259 patients consecutively registered between 1999 and 2012. The median age was 52 (16-76) and the median follow-up of the patients still alive was 23 months. Only 38 (14%) patients received FLAGO-Ida as salvage treatment. Favorable karyotype (inv 16) and t(8;21) was present in 13 (6%) and 11 (5%) respectively; intermediate in 145 (62%) and adverse in 62 (21%). Previous ASCT was performed in 86 patients (33%). The number of patients who achieved CR or CR without recovery of peripheral counts (CRi) was 132 (52%), PR in 23 (9%). Resistance accounted for 75 (29%) and death for 24 (9%). For the purposes of the current study, we decided to include patients with t(8;21) in the group of adverse karyotype group due to the similar median OS compared with MRC adverse karyotype (Median OS,0,42 vs 0,45 years). In univariate analysis karytotype (p=0.0001), FLT3-ITD positive (p=0.005), previous transplant (comparing no transplant, autologous and allogenic, p=0.012) and time to relapse (comparing refractory vs 12 months; p=0.0002) were significant risk factors. Contrary to other studies previous ASCT was associated to better outcome (median OS=2.3years, p=0.012). In multivariate analyses, the following variables remained as independent risk factors for survival: TTT (p=0.003), cytogenetic risk (p12 months: 0, refractory: 2, relapsed8 points, 44.6% of patients, median OS 0.4 years). CONCLUSIONS: This retrospective study based on an Epidemiologic Registry, shows that in primary refractory and first relapsed AML patients, the use FLAG-Ida or FLAGO-Ida salvage regimens lead to 52% of second CR. In addition, we have developed a scoring system using FLT3-ITD, refined cytogenetics, previous ASCT and TTT that can predict survival, identifying before administering FLAG-IDA a good prognosis population representing 27% of patients in which FLAG-IDA (followed by ASCT) can be an optimal therapy. This prognostic index should be validated in an independent cohort. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The population of elderly patients with hematologic malignancies is increasing and so will the activity of stem cell transplantation (SCT) in this population. The aim of this study was to analyze the toxicity and survival of allogeneic SCT in patients 50 years and older (elderly group), and compare the results with a standard adult population (young group).Thirty-two elderly patients (median age 52.5, range 50-59 years) and 97 young patients (median 32, range 20-40) received a myeloablative, allogeneic SCT from HLA-identical siblings at a single institution, and formed the basis of this retrospective study. The majority of transplants in both groups were performed with non-T-cell-depleted bone marrow, conditioned with busulfan + cyclophosphamide and received cyclosporine + methotrexate as graft-versus-host disease (GVHD) prophylaxis. The percentage of high-risk patients was nearly double in the elderly group (41% vs. 23%, p = 0.06).We observed a low incidence of toxicities in the elderly group, including veno-occlusive disease, acute and chronic GVHD, transplant-related mortality, time to engraftment, and relapse incidence, without significant differences compared within the young group. The 3-year survival rates were not statistically different between the elderly and young groups: 51% vs. 55% for all patients; 87% vs. 69% in chronic myeloid leukemia; 79% vs. 62% in standard risk patients and 13% vs. 31% in high risk ones. In multivariate analyses no significant difference in overall survival was found between age groups.According to our experience, age alone (between 50-59), should not be considered a contraindication to a conventional HLA identical sibling transplant.

Abstract 4557 BACKGROUND: Natural killer (NK) cells are innate immune effectors that directly lyse virally infected or malignant cells. There are 2 different subsets of NK cells with distinct phenotypic and functional characteristics: the CD56dim subset, which composes 90% of peripheral blood NK cells and has a cytotoxic function, and the CD56bright subset, which cooperates with dendritic cells and T cells in lymph nodes to secrete interferon and promote adaptive immune responses. NK cells are the first donor-derived lymphocyte subset to reconstitute after hematopoietic stem cell transplantation, reaching normal levels after 1 month. Nearly all phenotyping studies of NK subsets after haploidentical hematopoietic stem cell transplantation (HHSCT) reveal a rapid reconstitution of NK cells towards the CD56bright subset. In addition, Y.-J. Chang et al found the highest 2-year survival in patients with a high number of CD56bright NK cells after unmanipulated HHSCT. We analyzed reconstitution of the NK compartment between days 90 and 180 after unmanipulated bone marrow HHSCT with reduced intensity conditioning (RIC). METHODS: Six adults received unmanipulated bone marrow HHSCT after RIC (fludarabine 30 mg/m2 [day –6 to –2], cyclophosphamide 14.5 mg/kg [day –6 and –5], and busulfan i.v. 3.2mg/kg [day –3]) at our institution between July 2007 and July 2010. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of cyclophosphamide 50mg/kg (days +3 and +4) and cyclosporine A and mycophenolate mofetil from day +5 onwards. We monitored the reconstitution kinetics of circulating NK cells (CD56+, CD3–), and the CD56bright and CD56dim subsets by multiparametric flow cytometry (FC 500 Beckman® Coulter) at day +90 and day +180 after transplantation. Patient characteristics and clinical outcomes are shown in Table 1. 6 patients who underwent allogeneic HLA-identical sibling HSCT with RIC during the same period were used as controls. RESULTS: After HHSCT, NK cells reached normal levels in all patients but one at day +90, with a median number of NK cells of 111/mm3 (range, 25–195/mm3). At day +180 the median number of NK cells was 92/mm3 (range, 4–272/mm3). When we analyzed the absolute number of CD56bright and CD56dim subsets at day +90, we observed 2 patterns: Two patients showed skewed NK cell reconstitution towards CD56bright (Patient no. 3: 54 CD56bright/mm3; 11 CD56dim/mm3. Patient no. 4: 70 CD56bright/mm3; 17 CD56dim/mm3). Three patients reconstituted with a CD56dim/CD56bright ratio towards the CD56dim cell subset, similar to that of healthy adults (Patient no. 1: 17 CD56bright/mm3; 178 CD56dim/mm3. Patient no. 5: 9 CD56brigh/mm3; 135 CD56dim/mm3. Patient no. 6: 20 CD56bright/mm3; 116 CD56dim/mm3). One patient did not achieve adequate NK cell reconstitution (Patient no. 2: 15 CD56bright/mm3; 10 CD56dim/mm3). In contrast, in the control group, an increase in the CD56bright NK cell subset was not observed in any of the patients at any point. It is worth noting that 2 of the 3 patients with better clinical outcome (no GvHD, no relapse), namely patients no. 3 and no. 4 were the ones with skewed NK cell reconstitution towards the CD56bright NK cell subset. The other patient with a better clinical outcome (patient no. 6) had a normal CD56dim/CD56bright ratio at day +90. However, he showed an early CD56bright reconstitution (363 CD56bright/mm3; 34 CD56dim/mm3) in an additional determination on day +30. NK cell subsets reconstitution kinetics is shown in Figure 1. CONCLUSIONS: In our experience, NK cell reconstitution is adequate after RIC unmanipulated bone marrow HHSCT. Some patients recovered with a high proportion of CD56bright NK cells, as previously reported in other studies on HHSCT. Although limited by the sample size, our results are consistent with the previously observed survival advantage of patients with high early levels of CD56bright NK cells after unmanipulated haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4825 Introduction: The majority of patients with classical Hodgkinxs Lymphoma (cHL) are cured with primary treatment. However, a significant proportion of patients will not achieve a complete response (CR) or relapse after completion of initial therapy and need to be rescued with high-dose chemotherapy and stem cell transplantation (SCT): autologous (aSCT) and/or alogeneic (Alo-SCT). The identification of clinical and biological characteristics of these patients at diagnosis is still a challenge and the most prognostic systems used to date fail to identify a proportion of patients with worse prognosis. The best understanding of the biology of cHL could help to identify these patients and different groups works in the use of biological marker as determinants of clinical outcome. Bcl2 over-expression has been described in cHL and seems to be an independent marker associated to bad prognosis in probably relation with alterations in apoptosis regulatory molecules. Objetive: To retrospectively analyze the frequency of Bcl2 protein over-expression in tissue biopsies of patients diagnosed with cHL, which required intensive treatment in our centre. Patients and Methods: We revised clinical data and samples at diagnosis of patients with cHL who received SCT (aSCT or/and Allo-SCT) due to partial remission, relapse or progression and we determined Bcl2 expression protein by immunohistochemistry. All patients received at least 2 lines of treatment previous to intensive treatment with SCT. We analyzed the expression of Bcl2 according to each patient histology: Nodular Sclerosis (NE), Mixed cellularity (MC), Lymphocyte-rich (LR), and Lymphocyte-depleted (LD). Results: Between September 1997 and May 2010, 39 patients with cHL required intensive treatment in our center: 32 aSCT and 7 Allo-SCT due to partial remission, refractory, relapse or progression after first line of treatment. Average age was 32 years (range: 18–64), males: 25/females 14. The characteristics of patients and remission status at transplant are described in table 1. We had available samples at diagnosis from 31 out of 39 patients (80%): 20 NE, 5 MC, 6 LR and we found over-expression of Bcl2 in 17/20 (85%) NE; 5/5(100%) MC and 2/6 LR (33%). Conclusions: Our results suggest that over-expression of Bcl2 is frequent in patients with cHL which needed intensive treatment after failure of the first line of therapy (particularly with NE and MC). Further studies are needed to confirm the worse prognosis of Bcl2 expression in cHL and if may be useful at diagnosis in association with other clinical parameters to identify patients with poor prognosis. Table 1 . CLASSICAL HODGKIN LYMPHOMA (c HL) NODULAR SCLEROSIS cHL MIXED CELLULARITY cHL LYMPHOCYTE-RICH cHL LYMPHOCYTE-DEPLETED cHL N (39) 23 8 7 1 Sex (F/M) 11/12 8/0 5/2 1/0 Age, Years: Md(R) 30(18-64) 37(31-62) 36(19-64) 54 Stage: I, II/III, IV 11/12 3/5 5/2 0/1 Bulky disease 14YES/9NO 1YES/7NO 2YES/5NO 1NO B Symptoms: (N) YES/NO (13) YES/(10) NO (3) YES/(5) NO (6) YES/(1) NO (1) YES Intensive treatment 5Alo-SCT/18aSCT 2AloSCT/6aSCT 7aSCT 1aSCT N:type of trasplant (status at transplant) 18: aSCT(11CR/5PR/2RF); 5:AloSCT(2PR with previous aSCT/1PR/1CR/1RF) 6: aSCT (4CR/2PR) 7:aSCT (6CR/1PR) 1:aSCT (in CR) 2: Alo-SCT(1CR/1PR with previus aSCT) Sample revised: N 20 (23 total NE) 5 (8 total MC) 6 (7 total LR) 0/1 Bcl2 expression: P/Nv (%) 17P/3Nv (85%) 5P/0Nv (100%) 2P/4Nv (33%) Not aplicable Alo-SCT: Alogeneic stem cell transplant; aSCT: autologous stem cell transplant; CR: Complete Remission; F: female; LD: Lymphocyte-deplete; LR: Lymphocyte rich; M: male; MC: mixed cellularity Md: median; n: number; NE: nodular sclerosis; Nv: negative; P: positive; PR: Parcial remision R: range; RF: refractory Disclosures: No relevant conflicts of interest to declare.

Abstract 1328 Introduction: CD4+CD25+ regulatory T-cells (Treg) play an important role in inducing and maintaining allogeneic tolerance and can inhibit graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). However, the dynamics of donor and recipient Treg cell populations after Allo-SCT has not been studied yet. Objective: To analyze the dynamics of chimerism in Tregs and compare it to that of T lymphocytes (TL; CD3+) and whole blood leukocytes (WBL). Material and Methods: The study includes 53 patients subjected to Allo-SCT (myeloablative and non-myeloablative). PB samples were obtained weekly during the first month and every 14 days after day +30 until complete chimerism (CC) was achieved, and at fixed time-points (+30, +60, +90, +180, +365) thereafter. TL and Tregs were purified from PB until CC was achieved using inmunomagnetic technology (Miltenyi Biotec; CD3+ Microbeads and CD4+/CD25+ Treg Isolation Kit, respectively). Chimerism analysis was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on genomic DNA obtained from WBL as well as on cell lysates obtained from purified TL and Tregs. Complete chimerism was considered in samples with percentage of recipient cells Results: Median follow up for the whole cohort of patients was 338 days. CC was spontaneously achieved in WBL, TL and Tregs in 45/53 patients (85%) in a median time of 35.41, 38.8 and 42.5 days respectively. 5/3 patients (9%) suffered from graft failure/rejection showing mixed chimerism (MC) with increasing percentages of recipient cells both in WBL and leukocyte lineages. 3/53 patients (6%) maintained mixed chimerism (MC) in WBL and leukocyte lineages at day +180, with no signs of graft rejection or disease relapse. Analysis of the chimerism dynamics of those patients who spontaneously achieve CC revealed two different groups: Group 1 included 25/45 patients who achieved CC at the same time in WBL, TL and Tregs. Group 2 included 20/45 patients who achieved CC in WBL while maintaining MC in TL and Tregs. Interestingly enough, 9/20 patients from Group 2 maintained MC in Tregs 7–75 days after achieving CC in both WBL and TL (Figure 1). In a preliminary analysis, the small sample size precluded from obtaining statistically significant associations between the dynamics of chimerism in Tregs and the development of complications such as relapse or GVHD after SCT. Conclusions: To our knowledge, this is the first study dealing with Treg chimerism after SCT. We have shown it is feasible and can be performed on a routine basis together with standard lineage specific chimerism follow up. Although there is an association between chimerism dynamics in Tregs and TL, this is not absolute and a percentage of patients maintain residual Tregs of recipient origin after WTL and TL have become of complete donor origin. In this small cohort, Treg chimerism did not influence the development of post-SCT complications. Analysis of a larger and more homogeneous cohort would allow establishing the usefulness of Treg chimerism testing for the management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.