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Simple Summary: Dysregulation of noncoding RNAs has been described in numerous types of cancers and it has been associated with oncogenic or tumor suppressor activities. However, the signature of clinically relevant noncoding RNAs in pediatric B-cell acute lymphoblastic leukemia is still poorly understood. In a search for long non-coding RNAs that characterize pediatric B-cell acute lymphoblastic leukemia, we found that the long non-coding RNA AL133346.1 and a neighbouring protein-coding mRNA (CCN2) were significantly over-expressed in leukemia samples compared to healthy bone marrow. Survival analysis showed that patients with high CCN2 expression had a significantly better prognosis. These data suggest that AL133346.1/CCN2 could be useful for discriminating subtypes of leukemia and that CCN2 expression could predict the prognosis of pediatric patients with B-cell acute lymphoblastic leukemia. Abstract: Pediatric acute B-cell lymphoblastic leukemia (B-ALL) constitutes a heterogeneous and aggressive neoplasia in which new targeted therapies are required. Long non-coding RNAs have recently emerged as promising disease-specific biomarkers for the clinic. Here, we identified pediatric B-ALL-specific lncRNAs and associated mRNAs by comparing the transcriptomic signatures of tumoral and non-tumoral samples. We identified 48 lncRNAs that were di erentially expressed between pediatric B-ALL and healthy bone marrow samples. The most relevant lncRNA/mRNA pair was AL133346.1/CCN2 (previously known as RP11-69I8.3/CTGF), whose expression was positively correlated and increased in B-ALL samples. Their di erential expression pattern and their strong correlation were validated in external B-ALL datasets (Therapeutically Applicable Research to Generate E ective Treatments, Cancer Cell Line Encyclopedia). Survival curve analysis demonstrated that patients with “high” expression levels of CCN2 had higher overall survival than those with “low” levels (p = 0.042), and this gene might be an independent prognostic biomarker in pediatric B-ALL. These findings provide one of the first detailed descriptions of lncRNA expression profiles in pediatric B-ALL and indicate that these potential biomarkers could help in the classification of leukemia subtypes and that CCN2 expression could predict the survival outcome of pediatric B-cell acute lymphoblastic leukemia patients., Aula de Investigación sobre la Leucemia infantil: Héroes contra la Leucemia the Ministry of Economy of Spain SAF2015-67919-R, Junta de Andalucía PIGE-0440-2019 Pl-0245-2017 PI-0135-2020, University of Granada PPJIA2019-06 B-CTS-126-UGR18, Spanish Association for Cancer Research (LAB-AECC), Spanish Ministry of Education, Culture and Sports FPU fellowship FPU17/00067 FPU17/01258, PhD "La Caixa Foundation" fellowship LCF/BQ/DE15/10360019, Marie Sklodowska Curie Actions postdoctoral fellowship (H2020-MSCA-IF-2018), "Fundacion Benefica Anticancer Santa Candida y San Francisco Javier" predoctoral fellowship

The development of Next-Generation Sequencing (NGS) has provided useful diagnostic, prognostic, and therapeutic strategies for individualized management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Consequently, NGS is rapidly being established in clinical practice. However, the technology’s complexity, bioinformatics analysis, and the different available options difficult a broad consensus between different laboratories in its daily routine introduction. This collaborative study among Spanish centers was aimed to assess the feasibility, pros, and cons of our customized panel and other commercial alternatives of NGS-targeted approaches. The custom panel was tested in three different sequencing centers. We used the same samples to assess other commercial panels (OncomineTM Childhood Cancer Research Assay, Archer®FusionPlex® ALL, and Human Comprehensive Cancer Panel GeneRead Panel v2®). Overall, the panels showed a good performance in different centers and platforms, but each NGS approach presented some issues, as well as pros and cons. Moreover, a previous consensus on the analysis and reporting following international guidelines would be preferable to improve the concordance in results among centers. Our study shows the challenges posed by NGS methodology and the need to consider several aspects of the chosen NGS-targeted approach and reach a consensus before implementing it in daily practice.

Background: Five-year follow-up of the open-label, phase 3 DASISION study (NCT00481247) demonstrated faster and deeper molecular responses with dasatinib versus imatinib as first-line therapy for CML-CP. NCCN guidelines recommend considering comorbidities when selecting first-line TKI therapy. This exploratory post hoc analysis of DASISION investigates the effect of comorbidities on responses with dasatinib versus imatinib. Design: Patients with newly diagnosed CML-CP were randomized to 100mg dasatinib (n=259) or 400mg imatinib (n=260) QD. Charlson Comorbidity Index (CCI) was retrospectively calculated for each patient at diagnosis; data were retrospectively stratified by CCI categories (2-4, 5-6, and ≥7 [high burden]). Major molecular response (MMR) and MR4.5 were compared between treatments using unstratified Cochran-Mantel-Haenszel tests. Time to response was evaluated using Kaplan-Meier methodology with HRs based on Cox proportional hazards models and P-values from unstratified log-rank tests. Results: Baseline characteristics were mostly balanced between treatments. Fourteen (3%) patients had CCI 2-4, 260 (50%) had 5-6, and 245 (47%) had ≥7. Responses were numerically greater with dasatinib versus imatinib for CCI 2-4 (MMR: 85.7% versus 57.1%, P=0.2542; MR4.5: 71.4% versus 14.3%, P=0.0374) and ≥7 (MMR: 70.8% versus 64.0%, P=0.2552; MR4.5: 45.0% versus 39.2%, P=0.3589), and significantly higher for CCI 5-6 (MMR: 81.1% versus 64.8%, P=0.0033; MR4.5: 42.4% versus 29.7%, P=0.0329). Median time to MMR (months) was significantly shorter with dasatinib than imatinib for CCI 5-6 (15.0 versus 24.0; HR, 1.64; P=0.0006) and ≥7 (12.0 versus 21.4; HR, 1.41; P=0.0279). Grade 3/4 treatment-related adverse events (TRAEs; dasatinib versus imatinib): 0% versus 14% (CCI 2-4), 8% versus 9% (CCI 5-6), 24% versus 13% (CCI ≥7). Frequency of grade 3/4 TRAEs leading to discontinuation was similar: 0% versus 0%, 1% versus 1%, 8% versus 7%. No deaths occurred for CCI 2-4; similar percentage of patients died between treatments for CCI 5-6 and ≥7. Conclusions: Dasatinib was effective across all CCI groups, consistent with previous findings, suggesting that comorbidities do not negatively affect TKI activity. These findings highlight the benefits of first-line dasatinib in patients with CML-CP and elevated comorbidity burden. Funding: Bristol Myers Squibb. Previous presentation: 62nd ASH Annual Meeting, December 5-8, 2020; republished with permission of Elsevier Inc.

Introduction: Primary myelofibrosis (MF) is a rare hematologic disease belonging to the group of Philadelphia-negative chronic myeloproliferative neoplasms. Identification of the Janus Kinase (JAK) gene mutations inaugurated a new era in the targeted therapy of myeloproliferative diseases. Ruxolitinib is the first JAK1/JAK2 inhibitor specifically approved for the treatment of disease-related splenomegaly or symptoms in adult patients with primary myelofibrosis. The objective of this study was to assess the cost-effectiveness of ruxolitinib vs best available therapy (BAT) in MF patients in Spain. Methods: A decision-tree and Markov model were adapted to the Spanish setting to assess the cost-effectiveness of ruxolitinib vs. BAT on a lifetime horizon (≤15 years) from the societal perspective, while healthcare system perspective was included in the one-way sensitivity analysis. The population was assumed to be similar to that of the COMFORT-II clinical trial (CT), which was also the source of treatment efficacy data. BAT composition was derived from the same CT and validated with Spanish experts. Utilities were derived from the COMFORT-I CT. Costs included treatment, management, hospitalizations, emergency and outpatient visits, as well as adverse events and end-of-life costs. Additionally, costs associated to productivity loss were taken into account. Resource use was validated with experts and costs were extracted from Spanish sources. A probabilistic sensitivity analysis was also performed to evaluate the consistency of the results under the uncertainty or variability of the input data. Results: Patients on ruxolitinib accumulated 6.1 life years gained (LYGs), resulting in 73% extra life-years compared to patients treated with BAT (3.5LYs gained). Ruxolitinib provided 4.4 quality-adjusted life years (QALYs), with a 99% improvement compared to BAT (2.2 QALYs). This analysis gave an incremental cost of €47 199 per LYG and an incremental cost of €55 616 per QALY gained from the societal perspective. Conclusions: Ruxolitinib would be cost-effective in Spain according to the end-of-life criteria defined by the NICE and commonly referred for Spain (cost-effectiveness threshold of €61 500/QALY), in line with results published for other European countries.

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the standard of care for patients (pts) with chronic myeloid leukemia in chronic phase (CML-CP). In the 5-year follow-up of the phase 3 DASISION study (NCT00481247), dasatinib was associated with faster and deeper molecular responses than imatinib as first-line therapy for CML-CP (Cortes J et al. J Clin Oncol 2016). The NCCN guidelines recommend that multiple factors independent of risk score should be considered when selecting TKI therapy, including the presence of comorbidities. More than 50% of pts with CML-CP have at least one comorbidity at diagnosis (Hoffmann et al. Leukemia 2015); as such, comorbidities may influence first-line treatment choice for most pts. In this exploratory post hoc analysis of DASISION at 5-years' follow-up, we investigated the effect of comorbidities on response outcomes with dasatinib vs imatinib. Methods: DASISION was a multinational, open-label, phase 3 trial assessing dasatinib vs imatinib for newly diagnosed CML-CP. Pts were randomized to receive 100 mg dasatinib (n = 259) or 400 mg imatinib (n = 260) once daily. Charlson Comorbidity Index (CCI) was retrospectively calculated for each pt at diagnosis. Efficacy and safety data were retrospectively stratified based on CCI score categories 2-4, 5-6, and ≥ 7 (higher scores indicate a higher comorbidity burden). Rates of major molecular response (MMR) and molecular response with a 4.5-log reduction (MR4.5) were compared between treatment groups using unstratified Cochran-Mantel-Haenszel tests. Time to MMR and MR4.5 were evaluated using Kaplan-Meier methodology with hazard ratios (HRs) based on Cox proportional hazards models and P values from unstratified log-rank tests. Confidence intervals (CI) for median time to response were calculated using the Brookmeyer and Crowley method. P values are descriptive and unadjusted for multiple comparisons. Results: In total, 14 (3%) pts had CCI 2-4 (dasatinib 7, imatinib 7), 260 (50%) had CCI 5-6 (dasatinib 132, imatinib 128), and 245 (47%) had CCI ≥ 7 (dasatinib 120, imatinib 125). Baseline characteristics and drug exposure data are shown in the Table. Dose reductions of dasatinib and imatinib occurred in 14% vs 29% (CCI 2-4 group), 23% vs 12% (CCI 5-6 group) and 54% vs 22% (CCI ≥ 7 group) of pts, respectively. In the CCI 2-4 group, the MMR rate was 85.7% with dasatinib and 57.1% with imatinib (P = 0.2542) and the MR4.5 rates were 71.4% and 14.3% (P = 0.0374), respectively. In the CCI 5-6 group, the MMR rate was significantly higher with dasatinib vs imatinib (81.1% vs 64.8%, P = 0.0033), as was the MR4.5 rate (42.4% vs 29.7%, P = 0.0329). Median time to MMR in the CCI 5-6 group was significantly shorter with dasatinib than imatinib (15.0 vs 24.0 months; HR, 1.64; 95% CI, 1.23-2.19; P = 0.0006). In the CCI ≥ 7 group, response rates were numerically higher with dasatinib than imatinib: MMR rates were 70.8% vs 64.0% (P = 0.2552) and MR4.5 rates were 45.0% vs 39.2% (P = 0.3589), respectively. Median time to MMR was significantly shorter in the CCI ≥ 7 group with dasatinib than imatinib (12.0 vs 21.4 months; HR, 1.41; 95% CI, 1.04-1.91; P = 0.0279; Figure). Grade 3-4 treatment-related adverse events (TRAEs) were reported in 0% vs 14% (dasatinib vs imatinib) of pts with CCI 2-4, 8% vs 9% of pts with CCI 5-6, and 24% vs 13% of pts with CCI ≥ 7. Grade 3-4 TRAEs leading to discontinuation did not occur in the CCI 2-4 group, and were reported with similar frequency with dasatinib and imatinib in both CCI 5-6 (1% vs 1%) and CCI ≥ 7 groups (8% vs 7%). No deaths occurred in the CCI 2-4 group; a similar percentage of pts treated with dasatinib and imatinib died in the CCI 5-6 group (8% vs 9%) and CCI ≥ 7 group (13% vs 11%). Conclusions: In this exploratory post hoc analysis, dasatinib was effective across all CCI groups, consistent with previous findings suggesting that comorbidities do not negatively affect TKI activity. Molecular response rates were significantly higher with dasatinib than imatinib in pts with CCI 5-6, and comparable between treatments in pts with CCI ≥ 7. Furthermore, time to response was significantly faster with dasatinib than imatinib in both the CCI 5-6 and CCI ≥ 7 groups, while the frequency of grade 3-4 TRAEs leading to discontinuation was similar between treatments. These findings highlight the benefit of first-line treatment with dasatinib in pts with CML-CP and a high comorbidity burden. Study support: BMS. Figure Disclosures Breccia: Bristol-Myers Squibb/Celgene: Consultancy, Honoraria; Abbvie: Consultancy; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mauro:Sun Pharma/SPARC: Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding. Jabbour:BMS: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding. Saglio:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Incyte: Research Funding; Roche: Research Funding; Ariad: Research Funding; Novartis: Research Funding. le Coutre:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. DeGutis:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Khan:Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Sy:Bristol-Myers Squibb: Current Employment. Swanink:Bristol-Myers Squibb: Current Employment. Cortes:Amphivena Therapeutics: Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Immunogen: Research Funding; Merus: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; BiolineRx: Consultancy, Research Funding; BioPath Holdings: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Telios: Research Funding; Sun Pharma: Research Funding.

Context In the treatment of CML-CP, early molecular responses to tyrosine kinase inhibitors (TKIs) are associated with improved long-term outcomes, and major molecular response (MMR) at 18 months is an important goal. Obesity may elevate the risk of CML (Strom 2009), and high BMI at diagnosis can delay time to response and reduce MMR rate in patients treated with first-line imatinib (Breccia 2013). In the phase 3 DASISION study ( NCT00481247 ), dasatinib was associated with earlier, more durable, and deeper responses than imatinib (Cortes 2016). Objective Investigate the relationship between BMI and response to first-line TKIs. Design DASISION was an open-label multinational study assessing dasatinib versus imatinib for newly diagnosed CML-CP. Patients were randomized to 100mg dasatinib or 400mg imatinib once daily. In this exploratory analysis, patients were stratified according to high (≥25kg/m2) or normal BMI ( Results Collectively, 259 patients received dasatinib (high BMI, n=109; normal BMI, n=147; unknown, n=3) and 260 received imatinib (high BMI, n=107; normal BMI, n=147; unknown, n=6). Baseline characteristics were balanced within BMI subgroups. High BMI subgroup: significant improvements for dasatinib versus imatinib in median time to MMR (9.2 vs 27.6 months; P Conclusions Improved outcomes were seen in patients with high BMI treated with dasatinib versus imatinib; however, these differences were not seen with normal BMI. These findings need further validation to ascertain the role of BMI in predicting treatment responses in patients with CML-CP. Funding BMS. Previous presentation ASH 2019, HOPA 2020.

BackgroundThe analysis of molecular haematopoietic chimerisms (HC) has become a well-established method to monitor the transplant evolution and to assess the risk of relapse after allogeneic stem cells transplantation (allo-STC). Different techniques and molecular markers are being used for chimerism surveillance after transplantation, including quantitative real-time PCR (qPCR) and the recently developed digital PCR (dPCR). This study aims to compare the sensitivity and accuracy of both methods to quantify HC and predict early relapse.MethodologyHC was evaluated using custom PCR systems for the specific detection of the Y-chromosome, null alleles and insertion-deletion polymorphisms. A total of 281 samples from 28 adult patients who underwent an allo-SCT were studied. Increasing mixed chimerism was detected prior to relapse in 100% of patients (18 relapses).ResultsCompared with conventional qPCR amplification, dPCR predicted relapse with a median anticipation period of 63 days versus 45.5 days by qPCR. Overall, 56% of the relapses were predicted earlier with dPCR whereas 38% of the relapses where detected simultaneously using both techniques and only in 1 case, relapse was predicted earlier with qPCR.ConclusionsIn conclusion, chimerism determination by dPCR constitutes a suitable technique for the follow-up of patients with haematological pathologies after allo-STC, showing greater sensitivity to predict an early relapse.

Programmed death 1 (PD-1) activation triggers an immune checkpoint resulting in inhibition of T cells that leads to peripheral tolerance. Some PD-1 polymorphisms have been described and associated with the development of autoimmune diseases or cancer predisposition, but there are few data concerning the relevance of such polymorphisms on the clinical outcome after allogeneic hematopoietic stem cell transplant (alloHSCT). We analyzed the distribution of the SNPs PD-1.1G/A (rs36084323) and PD-1.3G/A (rs11568821) genotypes of the donor in a cohort of 1485 alloHSCT from HLA-identical sibling donors. We found an increased risk of grades II to IV graft-versus-host disease (GvHD) in patients receiving grafts from donors homozygous for the G allele at the rs36084323 SNP (P = 0.033; hazard ratio [HR] 2.2; 95% confidence interval [CI] 1.1 to 4.8) and also from donors homozygous for the A allele at the rs11568821 position (P

Background Expression of the pro-apoptotic BCL-2-interacting mediator ( BIM ) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1 + cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established. Design and methods We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2’-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed. Results We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression ( p BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2′-deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis. Conclusion Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM .

MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34+ cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML. (Mol Cancer Res 2008;6(12):1830–40)

The clinical significance of aberrant promoter methylation of the canonical Wnt pathway antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5, Wif1, Dkk3, and Hdpr1) and also putative tumor-suppressor gene Wnt5a, belonging to the non-canonical Wnt signaling pathway, was investigated in a large series of 75 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia by methylationspecific polymerase chain reaction. At least one methylated gene was observed in cells from 66% (49/75) of patients (methylated group). Disease-free survival and overall survival at 9 years were 51 and 40%, respectively, for the unmethylated group and 3 and 2%, respectively, for the methylated group (both P < 0.0001). Multivariate analysis demonstrated that the Wnt methylation profile was an independent prognostic factor predicting disease-free survival (P = 0.007) and overall survival (P = 0.039). Abnormal DNA methylation of promoter-associated CpG islands in the Wnt signaling pathway is very common in Philadelphia chromosome-positive acute lymphoblastic leukemia and potentially defines subgroups with distinct clinical characteristics.

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.

Journal Article; BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P¼0.03) and T-ALL vs B-ALL (50 vs 9%, P¼0.001). Relapse rate (62 vs 44%, P¼0.05) and mortality (59 vs 43%, P¼0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (Po0.001 y Po0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation o

The PARK2 gene, previously identified as a mutated target in patients with autosomal recessive juvenile parkinsonism (ARJP), has recently been found to be a candidate tumor suppressor gene in ovarian, breast, lung and hepatocellular carcinoma that maps to the third common fragile site (CFS) FRA6E. PARK2 is linked to a novel described PACRG gene by a bidirectional promoter containing a defined CpG island in its common promoter region. We have studied the role of promoter hypermethylation in the regulation of PARK2 and PACRG expression in different tumor cell lines and primary patient samples. Abnormal methylation of the common promoter of PARK2 and PACRG was observed in 26% of patients with acute lymphoblastic leukemia and 20% of patients with chronic myelogenous leukemia (CML) in lymphoid blast crisis, but not in ovarian, breast, lung, neuroblastoma, astrocytoma or colon cancer cells. Abnormal methylation resulted in downregulation of PARK2 and PACRG gene expression, while demethylation of ALL cells resulted in demethylation of the promoter and upregulation of PARK2 and PACRG expression. By FISH, we demonstrated that a lack of PARK2 and PACRG expression was due to biallelic hypermethylation and not to deletion of either PARK2 or PACRG in ALL. In conclusion, our results demonstrate for the first time that the candidate tumor suppressor genes PARK2 and PACRG are epigenetically regulated in human leukemia, suggesting that abnormal methylation and regulation of PARK2 and PACRG may play a role in the pathogenesis and development of this hematological neoplasm.

In this study, we report the case of a Philadelphia (Ph) positive chronic myelogenous leukemia (CML) patient with the presence of p190 and p210 BCR-ABL1 mRNA fusion transcripts derived from e1a2 and b3a2 BCR-ABL1 genomic rearrangements, respectively. The presence of e1a2 BCR-ABL1 genomic rearrangement was seen in 2 different clones, one with the rearrangement and another one with the rearrangement and deletion of the BCR gene of the non-rearranged chromosome 22. After treatment with imatinib, the p210 transcript could not be detected, whereas p190 was still present 6 months after initiation of imatinib therapy and progression to blast phase. The absence of p210 transcript post treatment indicates that the clone with b3a2 responded to imatinib and that the observed resistance was associated to cells harboring the e1a2 genomic rearrangement. Despite resistance of this patient to imatinib, no evidence of mutations in the kinase domain of ABL1 was found. Loss of normal BCR in one cell clone may contribute to the resistance to imatinib due to the lack of BCR mediated inhibition of BCR-ABL1.

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

Allo-SCT has a strong curative potential for AML patients mainly due to a GVL effect. Unfortunately, GvL and GVHD are intimately linked. IFN regulatory factor-3 (IRF3), by modulating innate immune reactions, could impact on the incidence and intensity of GVL and GVHD. We analyzed two gene variants in IRF3 (rs7251 and rs2304205) on the clinical outcome of 249 AML patients submitted to HLA-identical sibling allo-SCT. Patients with a donor carrying the dominant GG gene variant in rs7251 had, as compared with GC and CC variants, a lower acute GVHD (aGVHD) III-IV incidence (4% vs 11% vs 27%; P = 0.0078), a higher relapse incidence (49% vs 35% vs 26%; P = 0.018), and lower TRM (7% vs 24% vs 18%; P = 0.0065). In functional studies, the GG variant was associated with lower production of IFN-gamma, decreased lymphocyte proliferation after antigen presentation by DCs, and lower cytotoxic response of mature natural killer cells. Patients carrying the AA dominant variant in rs2304205 had higher relapse incidence (50% vs 39% vs 18%, P = 0.0068). The presence of both variants (GG in rs7251 and AA in rs2304205) in donors and patients resulted in a stronger clinical impact.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

Additional file 1 Expression of 250 miRNAs in resistant and responder CML patients. The data provided represent the expression of microRNA. The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML. Yes

Background Granulocyte colony-stimulating factor mobilized peripheral blood stem cells are increasingly used instead of bone marrow as a stem cell source for transplantation. Whereas this change is almost complete for autologous transplantation, there are some concerns when considering allogeneic transplants. Design and Methods We performed a retrospective case-control study including 820 adult patients who had received an allogeneic stem cell transplant from an HLA-identical sibling donor. Quality of life (QoL) was assessed in 150 patients using the EORTC Quality of Life Questionnaire C30 (QLQ-00). Results There were no statistically significant differences in overall survival at ten years (bone marrow: 48.9% vs. peripheral blood stem cells: 39.8%; p=0.621), transplant-related mortality (bone marrow: 28.9% vs. peripheral blood stem cells: 34.4%; p=0.682) or relapse incidence at 9 years (29.4% vs. 35.2%, respectively; p=0.688). Similar outcomes were maintained independently of the phase of the disease. However, multivariate analysis identified a higher incidence of acute graft-versus-host disease grades II-IV (p: 0.023; Hazard ratio [HR]: 1.41; 95% confidence interval [CI]: 1.05-1.89) and grades III-IV (p: 0.006; HR: 1.89; 95% CI: 1.20-2.98), in the peripheral blood stem cells-stem cell transplant group. As previously described, extensive chronic graft-versus-host disease was also more frequent in the peripheral blood stem cells group (28% vs. 15.6%; p

Repetitive elements are heavily methylated in normal tissues, but hypomethylated in malignant tissues, driving the global genomic hypomethylation found in cancer. This hypomethylation results in chromosomal instability, a well-characterized feature of the advanced phases of chronic myeloid leukemia (CML). We investigated methylation changes of DNA repetitive elements (LINE1, Alu, Satellite-alpha and Satellite-2) during the progression of CML from chronic phase (CP) to blast crisis (BC). CP-CML samples were significantly more hypomethylated for all repetitive sequences compared with normal samples. Furthermore, a more profound level of hypomethylation was observed among BC samples compared with CP samples. Our data suggest that repetitive DNA hypomethylation are closely associated with CML progression.

Tumor associated antigens (TAA) provide attractive targets for cancer-specific immunotherapy. PRAME is a TAA gene up-regulated in advanced phases of chronic myeloid leukemia (CML). To date, molecular mechanisms for the expression of PRAME have never been studied. We found that some Ph’-positive cell lines did not express PRAME. The expression of PRAMEwas restored in these cell lines by treatment with 5 -aza-2 -deoxycytidine, suggesting that the expression of PRAME is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the CpG sites of the PRAME exon 2 in these cancer cell lines revealed a close relationship between the methylation status of the PRAME gene and its expression. A methylation-specific PCR analysis demonstrated that hypomethylation of PRAME was significantly more frequent in CML blast crisis (70%) than in chronic phase (36%) (P = 0.01) and was correlated with high expression levels of PRAME transcripts (P < 0.0001). These results suggest that hypomethylation of PRAME up-regulates its expression in CML and might play a significant role in the progression of the disease.

Purpose: To examine cancer genes undergoing epigenetic inactivation in a set of ETV6/RUNX1-positive acute lymphoblastic leukemias in order to define the CpG island methylator phenotype (CIMP) in the disease and evaluate its relationship with clinical features and outcome. Experimental Design: Methylation-specific PCR was used to analyze the methylation status of 38 genes involved in cell immortalization and transformation in 54 ETV6/RUNX1-positive samples in comparison with 190 ETV6/RUNX1-negative samples. Results: ETV6/RUNX1-positive samples had at least one gene methylated in 89% of the cases. According to the number of methylated genes observed in each individual sample, 20 patients (37%) were included in the CIMP− group (0-2 methylated genes) and 34 (67%) in the CIMP+ group (>2 methylated genes). Remission rate did not differ significantly among either group of patients. Estimated disease-free survival and overall survival at 9 years were 92% and 100% for the CIMP− group and 33% and 73% for the CIMP+ group (P = 0.002 and P = 0.04, respectively). Multivariate analysis showed that methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.01) and overall survival (P = 0.05). A group of four genes (DKK3, sFRP2, PTEN, and P73) showed specificity for ETV6/RUNX1-positive subset of samples. Conclusion: Our results suggest that methylation profile may be a potential new biomarker of risk prediction in ETV6/RUNX1-positive acute lymphoblastic leukemias.

Purpose To examine cancer genes undergoing epigenetic inactivation in a set of T-cell acute lymphoblastic leukemias (T-ALLs) to obtain the CpG island methylator phenotype (CIMP) in the disease and its possible correlation with clinical features and outcome of the patients. Patients and Methods Methylation-specific polymerase chain reaction was used to analyze methylation of the ADAMTS-1, ADAMTS-5, APAF-1, ASPP-1, CDH1, CDH13, DAPK, DIABLO, DKK-3, LATS-1, LATS-2, NES-1, p14, p15, p16, p57, p73, PARK-2, PTEN, sFRP1/2/4/5, SHP-1, SYK, TMS-1, and WIF-1 genes in samples from 50 consecutive T-ALL patients (19 children and 31 adults). Results were compared with results obtained in 286 B-cell acute lymphoblastic leukemias (B-ALLs). Results A total of 88% of the T-ALL samples had at least one gene methylated. According to the number of methylated genes observed in each individual sample, 12 patients (24%) were included in the CIMP− group (zero to two methylated genes), and 38 patients (76%) were included in the CIMP+ group (> two methylated genes). Clinical features and remission rate did not differ significantly among both groups of patients. Estimated disease-free survival (DFS) rate at 12 years and overall survival (OS) rate at 13 years were 100% and 91% for the CIMP− group and 20% and 17% for the CIMP+ group, respectively (P = .0006 and P = .003, respectively). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor in predicting DFS (P = .05) and OS (P = .02). A group of five genes (SYK-1, ASPP-1, sFRP-2, sFRP-5, and WIF-1) showed specificity for T-ALL compared with B-ALL. Conclusion Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in T-ALL.

Downregulation of the large tumor suppressor 2 (LATS2/KPM) gene is associated with poor prognosis in acute lymphoblastic leukemia

Promoter hypermethylation plays an important role in the inactivation of cancerrelated genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). To determine the extent of hypermethylation in ALL, we analyzed the methylation status of the CDH1, p73, p16, p15, p57, NES-1, DKK-3, CDH13, p14, TMS-1, APAF-1, DAPK, PARKIN, LATS-1, and PTEN genes in 251 consecutive ALL patients.Atotal of 77.3% of samples had at least 1 gene methylated, whereas 35.9% of cases had 4 or more genes methylated. Clinical features and complete remission rate did not differ among patients without methylated genes, patients with 1 to 3 methylated genes (methylated group A), or patients with more than 3 methylated genes (methylated group B). Estimated disease-free survival (DFS) and overall survival (OS) at 11 years were 75.5% and 66.1%, respectively, for the nonmethylated group; 37.2% and 45.5% for methylated group A; and 9.4% and 7.8% for methylated group B (P < .0001 and P .0004, respectively). Multivariate analysis demonstrated that the methylation profile was an independent prognostic factor in predicting DFS (P < .0001) and OS (P .003). Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in ALL

Dkk-3 is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a ‘hot spot’ for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P¼0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P¼0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.

Introduction. Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in cytokine genes have shown to influence gene expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 1 (IL1) is a proinflammatory cytokine that induces the production of cytokines and chemokines and plays an important role in the inflammatory processes. IL1 is involved in various cellular functions, including cell proliferation, differentiation, and apoptosis. The IL1 family consists of two biologically actives forms (IL1A and IL1B). Several polymorphisms of these genes have been implicated in the pathogenesis of autoimmune diseases, however, their importance in SCT is not well-known. Objective To investigate the relationship between 4 SNPs in IL1A and IL1B genes and the susceptibility to the development of GvHD and other complications after HLA-identical allo-SCT. Patients and methods Genomic DNA obtained from peripheral blood samples belonging to 509 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). One SNP, rs1800587 (-889 C>T), in the promoter region of the IL1A gene and three SNPs in the IL1B gene (two in the promoter region rs16944 (-511C>T), rs1143627 (-31 T>C) and one in exon 5 rs1143634 (+3954 C>T) were genotyped by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Univariate and multivariate regression analysis were performed using Cox regression in the presence of competing risks except for chronic GvHD for which we used logistic regression model. All variables with p≤0.10 according to univariate analysis were included in the multivariate analysis. For multivariate analyses, p values were two sided and the outcomes were considered to be significant for p Results Genotypic frequencies observed for the 4 SNPs analyzed were in accordance with the Hardy-Weinberg equilibrium and were similar to those previously reported. Results of the multivariate analysisof the association between IL1A and IL1B genotypes and complications after allo-SCT are shown in Table 2. No association between the SNPs analyzed and complications other than acute GVHD were found. Patients with the alloreactive TT genotype for the SNP rs1143627 show increased risk of grade II-IV and III-IV acute GvHD (HR=6.87 and HR=19.19 respectively). In addition the presence of the rs16944 alloreactive A allele in the recipient was also associated with a significantly higher incidence of grade II-IV and mainly III-IV acute GvHD (HR=11.85 for grade II-IV and HR=53.48 for grade III-IV) Finally a higher risk of grades III-IV acute GvHD was observed in patients harboring alloreactive TT genotype for SNP rs1800587 (HR=3.12). Conclusions These results further support the idea of a genetic predisposition to certain complications after allo-SCT especially GvHD. IL1A and IL1B SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore to tailor the use of immunomodulatory approaches and, ultimately, to improve the clinical management of transplanted patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Philadelphia positive acute lymphoblastic leukemia (Ph + ALL) accounts for approximately 20% -30% of all adult ALL. The prognosis of patients with Phi + ALL is unfavorable when treated with standard chemotherapy schemes, presenting a long-term survival of 15% -20%. Since the introduction of Imatinib (IM) to treatment regimens the survival of these patients has improved, although allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option. We conducted a retrospective analysis of Ph + ALL patients before and after IM became available in order to analyze the impact of IM on survival in adult Phi + ALL. Patients and methods Between April 1997 and April 2013 we diagnosed 120 over 15 year old patients with ALL (B and T lineage), 31 (25.8%) of which were Phi +, all B lineage. Of these 31 cases, 30 were treated with protocols from Spanish group PETHEMA with curative intent. 14 of them (47%) were treated with chemotherapy and Imatinib (IM cohort) and 16 (53%) with chemotherapy (pre-IM cohort). In 17 of the 30 cases allogeneic HSCT was performed, 7 in the pre-IM cohort and 10 in the IM cohort. In the post-transplant period, two patients were treated with Dasatinib due to positive minimal residual disease ( BCR-ABL1 positive). The probabilities of overall survival (OS) (death) and event free survival (EFS) (no response, relapse or death) were estimated using the Kaplan-Meier product limit method. Differences between groups were tested using the X2 test. Univariate analysis was performed using Cox regression models or log-rank test. Multivariate analysis was performed using Cox proportional hazards regression model. The study was conducted in accordance with the Declaration of Helsinki. Results The median age was 38 years (range, 15-66 years), 17 patients were males and 13 females. The whole series survival was 32.4 ± 9.2%. The OS mean of the pre-IM cohort was 3.1 years (CI 95%, 0.5-5.7) and 6.9 years (CI 95%, 4.4-9.4) in the IM cohort ([figure 1][1]). The main characteristics of both groups are reflected in Table 1. When we analyzed the EFS, the variables that influenced it were being treated with IM (48% in the IM cohort versus 12.5% in the pre-IM cohort, p = 0.03), having received an allogeneic HSCT (45% versus 8%, p = 0.004) and being in first complete remission before allogeneic HSCT (51% versus 0%, p

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

Abstract 1302 Introduction: Interleukin 17 (IL-17) is a pro-inflammatory cytokine secreted by CD4+ helper T (TH) cells, TH17 cells. IL-17F is the most recently discovered member of the IL-17 cytokine family. IL-17F induces several cell types to express cytokines, chemokines, growth factors, and adhesion molecules, which are crucial molecules in leukocyte recruitment and activation. On this basis, IL-17F has been associated with the pathogenesis of multiple autoimmune diseases. Allele G for the polymorphism A7488G seems to play a protective role for the development of such diseases. However, no data is available about the possible impact of this polymorphism on the development of complications after allogeneic stem cell transplantation (allo-SCT). Objective: To analyze the influence of the donor (Dn) and recipient (Rc) genotype for the polymorphism A7488G in the IL-17F gene on the outcome of HLA-identical related allo-SCT. Material and Methods: The study comprised 143 allo-SCT patients and their donors (286 samples) included in the Spanish Group for Hematopoietic Stem cell Transplantation (GETH) DNA bank. Genomic DNA was purified from peripheral blood samples obtained, after written informed consent, from patients pre-SCT and donors. The A7488G polymorphism of the IL-17F gene was analyzed by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) following the procedure developed by Tahara et al. (J Clin Immunol 2008, 28:44-49). Results were analyzed using the Pearson's Chi-square Test. Results: In accordance with previous reports, allele G was present in a reduced number of individuals, 30/286 (10.5%) corresponding to 19/143 Allo-SCT (13.3%). The genotypes of the Dn were 129/143 (90.2%) AA and 14/143 (9.8%) AG, while those of the Rc were 127/143 (88.8%) AA and 16/143 (11.2%) AG. No Dn or Rc with GG genotype was found. Dn and Rc genotype combinations were as follows: DnAA-RcAA, 124/143 (86.7%); DnAG-RcAG, 11/143 (7.7%), DnAA-RcAG, 5/143 (3.5%), DnAG-RcAA, 3/143 (2.1%). The genotype of the donor influenced transplant outcome in terms of relapse (35/129 (27.1%) when the donor was AA vs. 1/14 (7.1%) when the donor was AG; p=0.119) and survival (exitus 64/129 (49.6%) when the donor was AA vs. 3/14 (21.4%) when the donor was AG; p=0,045). No association between Dn or Rc genotype and graft versus host disease (acute or chronic) or other complications was observed. The differences observed in relapse and survival according to the genotype of the donor, were especially true for AG genotype patients (no significant differences were observed in AA genotype patients). In fact, relapse (3/5 (60%) when the Dn was AA vs. 0/11 (0%) when the Dn was AG; p=0.004) and survival (exitus 5/5 (100%) when the Dn was AA vs. 1/11 (9,1%) when the Dn was AG; p Conclusions: The polymorphism A7488G of the IL-17F gene, which causes a His-to-Argsubstitution at amino acid 161 (H161R), influences the outcome of HLA-identical related allo-SCT. The presence of allele G in the Dn had a favourable effect on transplant outcome, in terms of lower relapse rate and better survival. Analysis of a larger set of Rc/Dn pairs is ongoing which will allow to increase the number of individuals harboring allele G and, ultimately, to confirm the results shown here. In such case, genotyping for the polymorphism A7488G of the IL-17F gene could aid in donor selection or drive a risk-adapted management of transplanted patients. Disclosures: No relevant conflicts of interest to declare.

TO THE EDITOR: Baccarani et al, on behalf of European LeukemiaNet, recently published the new recommendations for the management and monitoring of patients with chronic myeloid leukemia (CML). These recommendations, which are based mainly on the greater experience obtained during the past few years with tyrosine kinase inhibitors, update and improve the previous recommendations, published in 2006. One of the main proposals consists of the definition of optimal and suboptimal response and therapeutic failure after 3 months of imatinib treatment. The latest recommendations define the optimal response at 3 months as a complete hematologic response (CHR) with at least a minor cytogenetic response (CgR), which means that the number of Ph metaphases is equal to or lower than 65%. A suboptimal response is defined as a CHR associated with failure in the CgR (meaning that more than 95% of metaphases will be Ph ) and therapeutic failure is considered when CHR response is not achieved. Depending on the group in which the patient is included, subsequent clinical and therapeutic management will differ. However, these new recommendations result in a group of patients remaining unclassified. Patients with CHR and minimal CgR (between 66% and 95% Ph metaphases) at 3 months of imatinib treatment cannot be included in any of the three definitions mentioned (ie, optimal, suboptimal, or failure). Consequently, no recommendations currently exist for this group of patients with CML. The frequency of minimal CgR at 3 months becomes difficult to estimate, given that the IRIS (International Randomized IFN versus STI571) study did not publish results, though data suggest that the number of patients belonging to this group could be relevant. de Lavallade et al performed cytogenetic analyses on 203 patients at 3 months and found that 110 (54%) had between 36% and 95% Ph metaphases. Although this study did not distinguish between patients with minor or minimal CgR, the likelihood of the whole group obtaining a complete CgR at 5 years reached 96.4%. In contrast, Baccarani et al, in the previous European LeukemiaNet recommendations, showed that the group with minimal CgR at 3 months in combination with the group of null CgR could still obtain a complete CgR of 50% at 2 years. Unfortunately, neither of these studies analyzed separately the minimal CgR group, and the response or survival estimations therefore remain speculative. Given that the approach to the group in question could be very open, with several acceptable alternatives, we consider that the authors should clarify this point in order to manage more accurately and uniformly our patients with CML.

Abstract 1154 Poster Board I-176 INTRODUCTION Glutathione-S-transferase (GST) M1 and T1 are multifunctional enzymes involved in the metabolism of environmental carcinogens and chemotherapeutic drugs. The immunogenic activity of these enzymes and their association with graft rejection has been widely proved in solid organ transplantation. In this study we examined whether genetic variability (absence versus presence of the gene in donor/recipient pairs) could be related to acute graft versus host disease (aGvHD) in patients undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloHSCT) from related donor (RD). PATIENTS AND METHODS We evaluated 64 patients with acute leukemia (34 AML and 30 ALL) undergoing alloSCT and receiving myeloablative conditioning (MAC), with a minimum follow-up of 100 days. Donors were HLA-identical siblings in all cases, except in 2 presenting only a single HLA disparity. As source of HSCs, bone marrow progenitors were used in 25 patients (40%) and peripheral blood progenitors in 39 (60%). Total body irradiation (TBI) was used for conditioning treatment in 18 cases (28%). GvHD prophylaxis consisted of a short-term combination of Cyclosporine and Methotrexate. Presence in homozygous or heterozygous (positivity) or absence (negativity) of GSTM1/T1 genes were determined by real-time PCR. A Chi-square test was used to evaluate qualitative variables and non-parametric tests for quantitative variables. The Cox proportional-hazard model was applied to multivariate analysis. RESULTS GSTT1 and GSTM1 positivity was observed in 73 and 47% of patients, respectively, and in 72 and 46% of donors, respectively. Nineteen of 64 patients (30%) presented grade II-IV aGvHD. The incidence of grade II-IV aGVHD in GSTM1-positive patients was 79% versus 21% in GSTM1-negative patients. In univariate analysis, only GSTM1 positive patients developed grade II-IV aGvHD (p=0.001). In multivariate analysis, GSTM1 positivity was the only variable significantly associated with the appearance of grade II-IV aGvHD (p=0.002). No significant association was detected between GSTT1 genetic variability and the incidence of aGvHD. We found no significant difference in patients overall survival in relation to GSTM1 and GSTT1 genetic variability. CONCLUSIONS GSTM1-positive recipients develop more likely grade II-IV aGvHD, independently of other known risk factors. GSTM1 gene determination (homozygous or heterozygous) could be used to assess the aGvHD risk in allogeneic hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Background and objetives: Acute graft versus host disease (aGVHD) represents one of the main causes of death in patients who receive allogeneic stem cells transplantation (SCT). Quantification of hematopoietic chimerism by real-time PCR allows the detection of residual receptor cells with a high sensitivity (0.01%). Since aGVHD is supposed to be produced by reactivity of donor cells against host cells, this study was designed to relate the appearance of aGVHD with the amount of receptor cells remaining early after SCT. Patients and methods: Since January 2003, twenty seven patients who underwent myeloablative allogeneic SCT for acute leukemia (48% myeloid) could be included in the study (a minimum of 100 days follow-up was required). A sibling donor was used in 18 cases (67%) and bone marrow was the source of stem cells in 15 transplants (only in 2 cases the source was umbilical cord blood whereas the remaining were collected from peripheral blood). Conditioning with total body irradiation (TBI) was performed in 9 cases (33%). All the patients received the same GVHD prophylaxis with cyclosporine and methotrexate. Chimerism was determined by quantitative real-time PCR amplification of null alleles or insertion/deletion polymorphisms as previously described (Leukemia2005; 19: 336–43). Granulocytic engraftment, defined by the presence of more than 0.5 ×10e9/L granulocytes in two consecutive determinations, appeared at a median of 14 days (11–27) in our series. At least one chimerism sample had to be collected in the next week after granulocytic engraftment. Risk factors related to aGVHD were recorded and analyzed using Chi square for qualitative variables and non parametric tests for quantitative variables. Multivariate analysis was performed using Cox regression. Results: Eleven patients (40%) presented aGVHD (grade II–IV) in our series. Median receptor chimerism at the time of engraftment was 0.27% (0.01–7), showing significantly lower levels in patients who developed aGVHD (0.10 vs 0.57%, p=0.001 Mann-Whitney test). A chimerism value of 0.1% at the time of granulocytic engraftment was used to classify patients. COR curves were plotted previously for a better selection of the cut-off point. At 0.1% or lower, 8 of 9 patients (89%) developed aGVHD, whereas only 3 of 18 patients above this value presented aGVHD signs (p Interpretation and conclusions: Our data show that early monitoring of chimerism may be useful for identification of patients at risk of developing aGVHD after SCT. With the quantitative method used, a low chimerism receptor level at the time of engraftment (0.1% or lower) predicts the onset of aGVHD and can anticipate it in more than one week. This could allow the use of measures destined to reduce aGVHD in higher risk patients.

Abstract 468 AML is worldwide the most frequent indication for allo-SCT. This is most likely due to the curative potential of the graft versus leukemia (GVL) effect associated with the procedure. Unfortunately, GVL and GVHD are intimately linked. Thus, it is important to identify markers predictive of severe GVHD, to balance the risk of this complication and the risk of relapse in a particular AML patient. The innate immune system, as the initial regulator of the inflammatory response, mediates an important role in these reactions. After conditioning regimen, toll-like receptors initiate the innate inflammatory response by activating intracellular signaling cascades that converge on the activation of NF-κB and interferon regulatory factor-3 (IRF3). IRF3 activation in bone marrow derived dendritic cells (DCs) results in natural killer (NK) cell activation inducing an anti-tumor NK response. We hypothesized that genetic variability in IRF3 in either the donor or the recipient could impact on the degree of the inflammatory response and on GVHD and GVL effect allo-SCT. We analysed the effect of two frequent single nucleotide polymorphisms (SNPs) in IRF3 (rs7251 and rs2304205), which are inherited in a haplotype, on the GVHD and GVL in 249 patients diagnosed with AML and submitted to HLA-identical sibling allo-SCT. Those patients with a donor carrying dominant GG gene variant in rs7251 (45% of the donors) had, as compared to GC (44%) and CC (11%) variants, lower aGVHD III-IV incidence (4% vs 11% vs 27%; p=0.0078) (Figure 1A), higher relapse incidence (49% vs 35% vs 26%; p=0.018) (Figure 1B), and lower TRM (7% vs 24% vs 18%; p=0.0065). This clinical impact on severe aGVHD, relapse, and TRM was retained at multivariate analysis. Further, GG gene variant in rs7251 when present in the patient was also associated with lower aGVHD III-IV incidence (4% vs 13% vs 24%; p=0.009), higher relapse incidence (50% vs 34% vs 31%; p=0.014), and with a trend for a lower TRM (9% vs 19% vs 23%; p=0.064). Patients carrying AA dominant gene variant in rs2304205 in IRF3 presented a higher relapse incidence than the rest of genotypes (50% vs 39% vs 18%, p=0.0068). However, this impact was not retained at multivariate analysis. Functional studies in 180 healthy individuals showed that after stimulation of peripheral blood with cytomegalovirus (CMV) peptides, GG gene variant in rs7251 presented lower IFN-γ serum production than the rest of individuals, and GG gene variant was associated with lower number of IFN-γ producing mature NK cells, lower number of cytotoxic NK cells against K562 cell line and lower proliferation of T cells after antigen presentation by DCs. In conclusion, we show that a particular gene variant in IRF3 in the donors is associated with a low incidence of severe aGVHD and high incidence of relapse in AML patients submitted to allo-SCT. This finding could be explained by its effect in the inflammatory and adaptive immune response, with lower IFN-g production, lower lymphocyte proliferation after antigen presentation by DCs and lower mature NK cell response. Thus, these results suggest that, if possible, when transplanting an AML patient with a low risk of relapse it might be preferable to select a donor harbouring GG in rs7251 in IRF3, and when transplanting an AML patient with a high risk of relapse after the transplant it might be preferable to consider select a donor GC or CC in rs7251 in IRF3. Disclosures: No relevant conflicts of interest to declare.

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