ALBERT

Description: Introduction: Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with acute myeloid leukemia (AML). Mutated IDH enzymes produce in excess the D stereoisomer 2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. Total serum 2-HG (D and L stereoisomers) 〉2µM and ratio D/L 2-HG 〉2.5 are strong predictors of the presence of IDH1/2 mutations at diagnosis (Janin et al; JCO 2014). We measured 2-HG levels in serial samples of 47 patients with IDH1/2 mutations during induction or first salvage therapy to determine whether 2HG can serve as a surrogate marker of treatment efficacy. Methods: Between 2007 and 2015, 47 AML patients with IDH1 or IDH2 mutation received intensive chemotherapy as induction therapy (n=42) or as first salvage regimen (n=5) in our center (Table 1). Genomic DNA was extracted from BM samples using conventional procedures. IDH1-(codon R132) and IDH2- targeted (codons R140 and R172) regions were amplified by PCR with primers containing Ion Torrent adapters and unique barcodes to generate libraries. Pooled amplicons libraries were clonally amplified on Ion Spheres using the Ion Xpress Template 200 Kit. All available serum samples (n=394) prior and during induction/salvage chemotherapy were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. We studied the serum 2-HG values (D-2-HG and ratio D/L 2-HG) evolution during IC according to complete remission (CR) evaluated at the end of IC. When a patient had a missing value for 2-HG, the missing value was replaced by the last available observation (up to 7 days before the missing value). At each day we tested the difference between the dosage values of the responders and the non-responders with non-parametric Wilcoxon 2-sided tests. The analyses were done with SAS software (version 9.3; SAS Institute, Cary, NC). Table 1. Patients and treatment characteristics: AML at diagnosis / RR AML (N) 42 / 5 Median age (years) 58.2 (23.1 - 75.7) Sex ratio (M/F) 23/24 Conventional cytogenetic: - Normal karyotype - Trisomy 8 - Monosomy 7 39 (83%) 3 (6%) 2 (4%) Associated gene mutations: - NPM1- FLT3-ITD- FLT3-TKD 24 (51%) 10 (21%) 5 (11%) IDH mutation: - IDH1 R132 (N) - IDH2 R140 (N) - IDH2 R172 (N) 8 (17%) 32 (65%) 7 (15%) Intensive treatment: - "3+7" regimen - Intensified induction chemotherapy - High-dose cytarabine based chemotherapy 24 (51%) 19 (40%) 4 (8%) Results: Complete remission (CR) 1 was obtained in 35/42 patients (83%) and CR2 in 3/5 (60%) after IC. All (8/8) IDH1 -R132 mutated patients achieved CR1, 25/28 (89%) IDH2 -R140 mutated patients and 3/4 achieved CR1 and CR2 respectively; 2/6 (33%) IDH2 -R172 mutated patients and 0/1 achieved CR1 and CR2, respectively. Median serum 2-HG values before IC were as follow: total 2-HG 10.9 µM (range: 2.1-135.5); D-2-HG 10.7 µM (range: 1.7-132.6) and ratio D/L 36.9 (range: 3.9-131.2). Median number of serum 2-HG values per patient was 7 (range: 5-10). Serum D-2-HG and ratio D/L 2-HG evolution according to CR status after IC began to differ significantly at day 5 after IC (Wilcoxon test P=0.02, Figure 1). The difference between responders and non-responders increased overtime (Wilcoxon test P

Description: Thymic-independent peripheral expansion of CD8+ cells derived from the graft in the initial stage of post-HSCT immune recovery is a well-known physiological event. Nevertheless, the description of symptomatic LGL leukemias and aggressive malignant cases in this setting may generate uncertainty, mostly in those cases in which the cytotoxic T lymphocyte expansion CTLe persists beyond the early transplantation period. We aimed to assess the nature of CTLe in adults during the post-alloHSCT period in a series of 154 patients with a long term surveillance. We studied the longitudinal kinetics of those expansions, their relation to clinical events, and their phenotypic and molecular features, including recently reported CTL leukemia-STAT3 mutations. In our study, trying to adhere to the WHO annotation of T-LGL, we considered two definitions for a CTL expansion: an absolute increase (≥ 2000 x109/L), and a relative expansion (a CD8/CD4 ratio ≥ 1.5), persisting more than six months in both cases. Persistent relative CTLe cases are frequent (49%) and related with timoglobulin prophylaxis (p≤0.001), acute graft versus host disease (GVHD, p=0.02), reduced intensity conditioning (p=0.04) and fungal and viral infections in the early post-HSCT. No differences in the number of serious infectious events from day 180 was found. Absolute CTLe are scarce (9%), related with chronic GVHD and absence of relapses. TCR rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. We studied in a cross sectional manner with an extended immunophenotypic panel 17 patients: 5 patients with an absolute CTLe and 12 cases with a relative CTLe. A similar cytotoxic T αβ-effector phenotype was observed in all cases, with slight differences in the expression of CD25, CD16 and 1a. One patient with a relative CTLe expressed CD56 intensely: his ratio normalized at day 730 and no immune-related events were recorded. DNA stored during the post-alloHSCT setting was available from 68/75 relative CTLe patients (14/14 absolute CTLe cases). All of them went through molecular TCR rearrangement and STAT3 exon 21 mutations determination. In the relative CTLe cohort, TCR rearrangement was described as clonal, oligoclonal or polyclonal in 77%, 16% and 7%, respectively. Regarding absolute CTLe patients, TCR rearrangement was described as clonal in all the patients (n=14) of this subset. To increase the sensibility of the Sanger PCR, it was performed on DNA from CD3+ sorted cells in 54 out of 68 cases. No STAT3 mutation could be found in the CD3+ sorted fraction of relative or absolute defined CTLe. Not using an absolute threshold would establish a diagnosis of a persistent CTL expansion in 49% of our cohort of allo-transplanted patients. Additional diagnostic tools, as an effector phenotype, the presence of a NK marker or a monoclonal TCR rearrangement would not reduce significantly that percentage: CD57 was invariably expressed in CTLe cases, and 80% of our patients with expansions showed a TCR monoclonal pattern. STAT3 mutations resulting in persistent proliferation of CTL clones are a frequent event in large granular lymphocytic leukemia, and those clones have also been described in autoimmunity-driven disorders as acquired aplastic anemia and hypocellular myelodysplastic syndromes. We establish in this study the absence of exon 21 STAT3 mutations in the persistent CTL expansions found in a large series of patients with a long-term post-alloHSCT surveillance. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period, supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4545 Introduction: Allogeneic transplantation is the only curative option in the treatment of multiple high risk hematologic neoplasms. Only 25–30% of patients have an HLA identical sibling donor and searching for a compatible unrelated donor or cord blood renders satisfactory results in around 60–70%. Haploidentical transplantation (HAPLO) offers a therapeutic alternative to more than 95% of such patients with the advantages of quick availability, easy programming and a committed donor. Patients and Methods: We evaluate the results of HAPLO with a reduced intensity conditioning regimen (Fludarabine 30 mg/m2 ×5 days (-6 to -2), Cyclophosphamide 14,5 mg/kg ×2 days (-6 and -5), IV Busulfan 3,2 mg/kg × 1–3 days (BUX, days -4 to -2) employing high doses of Cyclophosphamide post graft infussion (50 mg/kg days +3 and +4) as GVHD prophylaxis together with standard doses of cyclosporine and mycophenolate from day +5. Results: From Dec-2007, we have done 26 HAPLO in 4 spanish centers. Median age was 38 years (16–57), 20 were male and all were in advanced phases of their diseases (12 Hodgkin′s, 6 AML, 3 ALL, 2 MM, 1 MDS, 1 MF y 1 NHL). Previous autologous HSCT has been employed in 13 and allogeneic HSCT in 6 (2 MURD and 4 UCB). Disease status at HAPLO was CR in 8, PR in 14 and refractory in 4. Bone marrow was used in 16 and unmodified peripheral blood in 10. The haploidentical donor was patient′s mother (8), father (3), siblings (11) or other relatives (4). BUX was used in 1 dose (15), 2 doses (8) or 3 doses (2) and TBI 200 cGy in 1 case. Mean neutrophils engraftment was achieved on day +18 (13–26) and platelets 〉50K on day +27 (17–150) in all but 2 cases of graft failure (7.7%) due to progression (MF) or relapse (M7-AML). Main toxicities were grade 1–2 mucositis in 50%, febrile neutropenia in 75% and CMV reactivations in 58% with a 100 days NRM of 3.8% (1/26, VOD and MOF) and 10% NRM at 6 months (2/20). Grade II-IV acute GVHD appeared in 10/23 patients at risk (43%) and grade III-IV in 4/23 (17%). Chronic GVHD affected to 4/15 (27%), being extensive in 1/15 (6.7%). With a median follow-up of 9 months (1–38), 13/22 (59%) are alive in CR, progression or relapse has ocurred in 6/24 (25%). Immune reconstitution seems fast and complete in those evaluated. Conclusions: HAPLO with high-dose cyclophosphamide as GVHD prophylaxis is a useful alternative in the treatment of high risk hematologic tumours, with low toxicity, acceptable GVHD incidence and severity, long lasting remissions, and fast immunological reconstitution. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3025 Introduction: Although allogeneic stem cell transplantation (allo-SCT) is the only curative treatment for MM, it is associated to a high morbility and mortality. Moreover, relapses are common after allo-RIC. Accordingly, new strategies are required to reduce both the risk of relapse and the toxicity of the procedure. As we have previously demonstrated, Bz induces a selective depletion of alloreactive T-cells and has immunomodulatory properties which might be of potential benefit for GVHD control. The primary end point of this study was to evaluate the efficacy of allo-RIC in terms of response when Bz was added as part of a reduced intensity conditioning prior to allo-SCT. Secondary end points included incidence of GVHD and analysis of the toxicity of the procedure when Bz is also administered post-infussion as part of the GVHD prophylaxis. Method: Prior to allo-RIC, patients received two cycles of Bz plus dexamethasone. Conditioning consisted of fludarabine (30 mg/m2 intravenously on days -9 to -5) and melphalan (70 mg/m2 intravenously on days -4, -3) plus Bz 1, 3mg/m2 on day - 11 and -2. GVHD prophylaxis included cyclosporine (CsA) and methotrexate for the first 9 patients and CsA plus MTX and Bz on days +3 and +7 for the remaining 7 patients. From day +50 post allo-RIC 7 cycles of Bz (+1, +8, +15) were administered, the first two cycles every 28 days and the rest every 56. Results: 16 patients from the Twenty-one initially enrolled, were evaluable. All 16 patients had received at least 2 lines of therapy including autologous-SCT. Disease status was CR or nCR in 4 patients, 9 had PR and the remaining 3 patients had relapsed / progressive disease. 15 patients maintained or improved status at transplant including all × patients with active disease at transplant. Eight patients (50%) relapsed, four with extramedullary involvement. No patient developed grade 4 aGVHD.Grades 2–3 aGVHD occurred in 6 patients (37%). Interestingly, two out of the nine (29%) patients who received Bz on days +3 and 7 developed grades 2–3 acute GVHD as compared to four of the nine (44%) who did not receive it. In terms of toxicity, one patient did not achieve platelet engraftment and 2 patients developed peripheral neuropathy requiring treatment withdrawal. 8 patients died, four of them due to relapse (MRT: 25%). With a median follow-up of 457 days overall survival was 46%. Conclusions: The current trial is the first evaluating the efficacy and safety of Bz as part of a reduced intensity conditioning regimen among patients with high risk MM undergoing allogeneic transplantation. Regarding the efficacy of the procedure all but one patient improved disease status post-alloRIC although relapse rate was still high in this heavily pretreated population. In addition, Bz post-alloSCT is well tolerated and may decrease the incidence of GVHD. Disclosures: Perez-Simón: Janssen-Cilag: Patents & Royalties. Off Label Use: This study evaluates the efficacy of Bortezomib as part of a reduced intensity conditioning regimen among patients with high risk MM undergoing allogeneic transplantation. Rosiñol:Celgene: Honoraria; Janssen: Honoraria. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction Allogeneic transplantation is the only curative option for patients with high risk hematologic malignancies. HAPLO-HSCT offers a therapeutic option to most of these patients with the advantages of quick availability, easy programation and logistics, and a committed donor. This procedure has shown promissing results in patients diagnosed with relapsed or refractory Hodgkin´s disease (Burroughs LM et al. Biol Blood Marrow Transplant 2008; 14:1279-1287). Patients and Methods We retrospectively evaluate the results of HAPLO-HSCT with RIC regimens (Fludarabine 30 mg/m2 x5 days (-6 to -2), Cyclophosphamide14,5 mg/kg x2 days (-6 to -5), Busulfan IV 3,2 mg/kg x 1-2 days (BUX, days -3 to -2) or 200 cGy TBI on day -1) and GVHD prophylaxis based on HD-CY (50 mg/kg on days +3 and +4) and a calcineurin inhibitor plus mycophenolate from day +5 performed in GETH centers to patients diagnosed with relapsed or refractory Hodgkin´s disease. Results From March-2009, 29 HAPLO-HSCT have been performed in patients diagnosed with relapsed or refractory Hodgkin´s disease in 11 GETH centers. Median age was 31 years (18-53), 19 were males and all were in advanced phases of their disease. Autologous HSCT was previously employed in 90% of them, and allogeneic HSCT in 10%. Disease status at HAPLO-HSCT evaluated by PET was complete remission in 8 (28%) and persistent disease in 21 (72%). Bone marrow was the stem cell source in 15 (52%) and peripheral blood in 14 (48%), without T-cell depletion in all cases. The haploidentical donor was the patient´s mother (13), father (2), brother (8), sister (5) or daughter (1). The RIC regimens employed included 1 dose BUX (11), 2 doses BUX (14) or 200cGy TBI (4). Median neutrophils engraftment was day +17 (11-44) and platelets 〉20K was day +26 (11-150). Main toxic complications were grade II-III muchositis in 50%, febrile neutropenia in 75% and CMV reactivations in 58% with a transplant related mortality rate of 7% (2/29) at day +100 and 17% (5/29) at 6 months post-transplant. Acute GVHD grade II-IV affected to 7/28 patients at risk (25%), with grade III-IV in 3/28 (11%). Chronic GVHD was present in 3/19 (16%), being extensive in 1/19 (5%). After a median follow-up of 9 months (0.3-49), 13/22 (59%) remain alive and in complete remission. Relapse or progression occured in 6/28 (21%). Immune reconstitution was fast and complete in those evaluated. Conclusions HAPLO-HSCT with HD-CY is a useful tool in the treatment of patients with relapsed or refractory Hodgkin´s disease, rendering long-lasting remissions with limited toxicity, low GVHD incidence and early immune reconstitution. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Allogeneic haematopoietic stem cell transplantation (alloHCT) is a potentially curative approach for patients (pts) with multiple myeloma (MM). The high transplant related mortality (TRM) rate with myeloablative conditioning has resulted in a shift to reduced intensity conditioning regimens (RIC). However, most MM pts who receive an alloRIC ultimately relapse and their treatment remains a challenge. Since alloHCT can modify the biology of the disease, including the immune environment, responses after alloHCT to rescue therapies previously used before alloHCT could be improved. Our main objective was to evaluate the efficacy of regimens including new drugs in MM pts relapsing after alloHCT comparing the efficacy achieved before and after alloHCT. Material and Methods: We report a retrospective multicenter analysis of 126 consecutive pts that underwent alloHCT for MM from 2010 to 2013 at 8 Spanish centers. Results: Baseline pts and transplant characteristics are shown in Table I. The median of prior therapies prior to alloHCT was 3 (1-9), 83% had received previous autologous HCT and 26% had high risk cytogenetic. 71 (56%) and 48 pts (38%) had been treated with regimens containing proteasome inhibitors (PI) and immunomodulatory drugs (IMIDs) before transplantation, respectively. Disease status at transplant was complete remission (CR) in 16 (13%) pts, partial response (PR) or very good PR in 86 (68%) and 24 (18.5%) had relapsed/progressive disease. 35 pts (28%) had active extramedullary disease at transplant. The majority of pts received peripheral blood HCT (90%), RIC (90%) with fludarabine plus melphalan based conditioning regimen (61%) and calcineurin inhibitor plus MTX as GVHD prophylaxis (68%). 19% receiving allo-HCT from an unrelated donor (91% 10/10 HLA matched). All pts engrafted. Grade II-IV acute GVHD occurred in 54% pts (grade IV 8%) and chronic GVHD in 45% (moderate 15%, severe 12%). TRM within the first 100 days after transplant was 6% (overall TRM 28%). 60% pts improved their pre-transplant response, with an overall response rate of 74% (56% CR). After a median follow-up of 92 months for pts alive (22-197), the OS was 51 and 43% at 2 and 5 years respectively. 75 pts (59.5%) relapsed after alloHCT, 57 of them with extramedullary involvement. Median time to relapse was 8 months post-transplant (1-141). The cumulative incidence of relapse was 79% at 3 years. Median OS after relapse was 22 months (8-33). Seventeen out of 75 pts who relapsed received IP both in the pre-transplant and in the post-transplant period. Sixteen pts out of 17 who received IP achieved at least PR pre-transplant while 10 out of these 16 pts responded again to PI post-HCT. Moreover, 1 patient reached a deeper response (CR) post as compared to pre alloHCT (PR) and 1 patient who was refractory pre-alloHCT did respond post-alloHCT. In addition, 6 out of 7 pts who did not respond to IP post-transplant reached stable disease with time to progression (TTP) lasting 4 to 12 months. Twelve out of 75 pts who relapsed received IMIDs both pre and post-alloHCT. Ten pts out of 12 who received IMID pre-alloHCT achieved at least PR, and 8 out of these 10 pts responded again to IMIDs post-alloHCT. Moreover, 1 patient who had been refractory to IMIDs in the pre-transplant period reached CR after alloHCT. In addition, 2 out of 4 pts who were refractory to IMIDs in the post-transplant period reached stable disease with TTP of 8 to 13 months. Remarkably, among pts who respond both in the pre and the post-transplant period to IP or IMIDs, the time to response (TTR) and time to progression (TTP) was similar despite the regimens used in the pre-transplant setting were more aggressive (TTR 3 vs 3.5 and TTP 9 vs 7 months before and after alloHCT for IP, and TTR 4 both before and after alloHCT and TTP 10 vs 9.5 months before and after alloHCT for IMIDs). All but 2 pts received first generation IP pre and post-alloHCT (two pts received carfilzomib) and all but 5 received first generation IMIDs (5 pts were treated with pomalidomide). Conclusions: MM pts relapsing after alloHCT should be considered candidates to receive new drugs, as they can achieve response rates at least in a similar proportion and durability to those observed in the pre-transplant setting. This finding is in contrast to the usual course of the disease outside the alloHCT setting, where response rates and TTP decreases with consecutive lines of treatment. Disclosures Mateos: Janssen: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Description: Abstract 1412 Over the last decade, numerous new drugs have been incorporated in the treatment armamentarium of multiple myeloma (MM). However, there is not much information on the role of single nucleotide polymorphisms (SNPs) regarding toxicity and efficacy of the new myeloma therapies. Aims: to analyze the influence of genetic polymorphisms on toxicity and outcome of induction therapy on patients included in the trial of the Spanish PETHEMA/GEM 05 for newly diagnosed MM elderly patients (age 65 or more, “GEM05mas65”). (Lancet Oncol 2010; 11: 934). Patients and Methods: Between March, 2006, and October, 2008, 260 patients with untreated multiple myeloma, 65 years and older, from 63 Spanish centres, were randomly assigned to receive six cycles of botezomib plus melphalan and prednisone VMP (n=130) or bortezomib plus thalidomide and prednisone (VTP; n=130) as induction therapy. Genetic studies were performed in blood samples from 169 patients among the 260 included in the original trial (VMP: 84 patients; VTP: 85 patients). Toxicity and outcome parameters were analyzed and related to the genotype of polymorphisms in genes involved in bortezomib (CYP1A2 *1C, CYP1A2 *1F, CYP3A4), thalidomide (CYP2C19 *2, CYP2C19 *17) and melphalan metabolism (GSTP1 I105V) as well as bortezomib transport (MDR1 −3435C〉T) and drug target (PSMB5 1042G〉A). Results: Clinical results in our cohort reproduced those of the 260 patients of the original trial already published. The most frequent non haematological toxicity was peripheral neuropathy, similar in both arms. Among the 169 patients included in our study CYP2C19 *17 T carriers had worse overall response (p=0.033). Likewise, MRD1 −3435TT carriers presented less incidence of grade 3–4 neutropenia (p=0.041) meanwhile patients AA for CYP2C19 *2 genotype presented more grade 3–4 thrombopenia (p=0.009). The impact of the different genotypes among the two arms and inside each one was also analyzed. Wild type patients for MRD1 −3435T SNP displayed higher rate of severe neutropenia only in the VMP arm and in a genetic load depending manner (CC=71%, CT=38%, TT=22%; p= 0.012). Conclusions: Our results suggest that polymorphisms in genes involved in the metabolism of thalidomide, (CYP2C19 *17 y *2) or bortezomib transport (MDR1 −3435C〉T) may result in a thalidomide modified metabolism rate or in a lower efficacy in the bortezomib transport, suggesting a potential influence in the haematological toxicity (neutropenia and thrombopenia) and overall response rates in MM patients treated with VMP or VTP. These results together with the relative elevated frequency of these SNPs in this population (〉20%) justifies the interest of studying the genetic profile since it can become a step forward on the individualized management of MM patients. Disclosures: Mateos: Celgene: Honoraria; Janssen: Honoraria. Lahuerta:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria.

Description: Abstract 2997 Introduction. The identification of genetic variants predictive of response to G-CSF mobilization might be useful in deciding the best strategy to obtain haematopoietic progenitors (HP) in patients scheduled for autologous peripheral blood progenitor cell transplant. Recently, one study has demonstrated the relationship among polymorphisms in genes implicated in trafficking and homing of CD34+ cells and the degree of mobilization after G-CSF therapy among healthy donors (Haematologica 2011; 96: 102–109). Aims. To evaluate if polymorphisms in five genes (CD44 rs13347 C〉T, CSF3R rs3917924 A〉G, CXCR4 rs2680880 A〉T, CXCL12 rs1801157 G〉A, and VCAM1 rs1041163 T〉C) previously associated with the number of G-CSF mobilized CD34+ cells in healthy donors, can also predict the mobilization efficacy in a group of patients with hematological malignancies. Patients and Methods. We retrospectively evaluated 183 patients who were treated with s.c. G-CSF at 10 mcg/kg during 4 days. HP collection was initiated or not at day 5 according to the CD34+ number in peripheral blood (PB). Patients were selected among two groups: (1) poor mobilizers (n=109), who failed a mobilization attempt, presenting with 2 ×106CD34+ cells/kg in a first and only apheresis. The genetic variants were genotyped by allelic discrimination polymerase chain reaction (PCR) assays using TaqMan®Genotyping Assays (Applied Biosystems). Results. Patients diagnosed with lymphoma, myeloma and acute leukaemia were 40%, 38% and 21% of poor mobilizers, and 38%, 46% and 16% of good mobilizers, respectively. On the overall group, the genetic variant TT rs1801157 in CXCL12 was significantly associated with poor mobilization (p=0.040). Among lymphoma patients, the presence of the C allele in VCAM1 was significantly associated with mobilization rate (49% vs 19% among poor and good mobilizers respectively, p=0.011). In this lymphoma group, a trend towards poor mobilization was also observed in relation with homocygosis for the T allele of CXCL12 (12% vs 0% in poor and good mobilizers, respectively, p=0.066). The analyzed variables had no impact on the mobilization capacity in patients with myeloma or acute leukaemia. Discussion. Genetic variants in VCAM and CXCL12 seem to be related with the mobilization yield after G-CSF, particularly in lymphoma patients. Other polymorphisms in adhesion molecules related to the degree of CD34+ cell mobilization in healthy donors have not shown a relevant role in patients with hematological malignancies, probably reflecting the predominant effect of disease biology and/or of previous treatments. Funding. This study was supported in part by a research grants 04515/GERM '2f 06; RECAVA RD06/0014/0039, and FIS 10/02594 Disclosures: No relevant conflicts of interest to declare.

Description: Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Description: Abstract 4050 Introduction. We and others have described the efficacy of paclitaxel-based chemotherapy in mobilizing large amounts of hematopoietic progenitors (HP) both in patients with solid tumors,(Bone Marrow Transplant 2000;25:231–5), and with hematological malignancies (Haematologica 2008; 93:161–3). Like most drugs, the taxanes are not controlled by the actions of one gene, but believed to be dependent on several polymorphic proteins. Single nucleotide polymorphisms (SNPs) in the ABCB1 gene, which encodes the transport protein P-glycoprotein, or in in metabolic enzymes, such as CYP2C8, have been suggested to influence the pharmacokinetics and clinical response to paclitaxel. Aim. To retrospectively evaluate the effects of five known allelic variants in the CYP2C8, and ABCB1genes on the mobilization ability and toxicity of the anticancer agent paclitaxel. Patients and Methods. 107 patients with hematological malignancies (43 lymphoma, 41 myeloma, 23 acute leukemia) received paclitaxel 170 mg/m2 i.v. by continuous infusion for 24 hours (day 1) followed by 8 mg/kg s.c G-CSF daily until the last apheresis. 77% received this treatment after failure of mobilization with G-CSF, and the rest as first line therapy because of risk factors for failure to achieve successful mobilization. The genetic variants (ABCB1 rs1045642 A 〉G, ABCB1 rs2032582 C〉A, ABCB1 rs2032582 C〉T, CYP2C8 rs10509681 C〉T, and CYP2C8 rs11572080 A〉G), were genotyped by allelic discrimination polymerase chain reaction (PCR) assays using TaqMan®Genotyping Assays (Applied Biosystems). The effect of genotypes on mobilization efficacy and on maximal hematological toxicity (according to the NCI version 3) was retrospectively assessed. Results. Allelic frequencies for rs1045642 A〉G, rs2032582 C〉A, rs2032582 C〉T, rs10509681 C〉T, and rs11572080 A〉G variants, were 0.46, 0.40, 0.8, 0.13, and 0.13, respectively. Successful mobilization (〉2 106/kg CD34+ cells) was achieved in 57% of patients. No reproducible significant associations between genotype and outcome (evaluated as number of CD34+ cells/kg in the first and total apheresis, number of collections performed, and on the mobilization success [p〉0.05]), nor on myeloid or platelet toxicity (p〉0.05) were found for any of the SNPs analyzed. Discussion. This study on a well-defined patient population suggests that the presently evaluated variant alleles in CYP2C8 and ABCB1 genes do not explain the substantial interindividual variability in the outcome or hematological toxicity of the mobilization schedule using paclitaxel and G-CSF. Funding. This study was supported in part by a research grants 04515/GERM/06; RECAVA RD06/0014/0039, and FIS 10/02594. Disclosures: No relevant conflicts of interest to declare.