Search

INTRODUCTION Allogeneic hematopoietic stem cell transplantation (alloHSCT) has been established as a powerful treatment modality for patients with hematological malignancies. The graft-versus-leukemia effect, however, is strongly associated with the occurrence of graft-versus-host disease (GVHD) and subsequent transplant-related mortality (TRM). Several strategies are applied in order to prevent GVHD, the post-transplant cyclophosphamide (PT-Cy) is one of the most used in Haploidentical HSCT (Haplo-HSCT). Cyclophosphamide (CY) is an alkylating agent with antineoplastic and immunosuppressive activities. CY is metabolized by highly polymorphic enzymes to produce phosphoramide mustard which is a bifunctional DNA alkylating agent, is the therapeutically active metabolite. Thus, the aim of our study is to identity polymorphisms in the genes of the CY metabolism and correlated with complications post-HSCT (GVHD, TRM, veno-occlusive disease (VOD) or hemorrhagic cystitis (HC)). METHODS We selected 182 consecutive patients who received an Haplo-HSCT from 2007 to 2019. Eleven genes related to CY metabolism were analyzed (Table 1). The genotyping was performed in peripheral blood samples for recipient using an enrichment-capture custom gene panels (IDT probes) in a MiSeq platform (Illumina, USA). The bioinformatic analysis was carried out with BaseSpace software (Illumina, USA). Variants located in coding region and splicing sites were analyzed. We selected polymorphisms corresponding to read depth ≥30X in the canonical isoform with an allele frequency ≥0.4 and represented in at least 5% in our cohort. The collected clinical variables were age/gender recipient and donor, pathology, pretransplant status, conditioning regimen, total body irradiation, basal ferritin and CMV reactivation. Fisher test was used to compare the differences among groups. Statistical Package for the Social Sciences (SPSS, Chicago, USA) was used for statistical test. RESULTS The cumulative incidence rates for aGVHD of II-IV and III-IV grades at 100 days was 39% and 12% respectively. The cumulative incidence rates for cGVHD, moderate or severe cGVHD and TRM at 1000 days were 37%, 19% and 29%, respectively. Among the cases analyzed, 9% developed VOD and 25% HC. Patients who received ablative conditioning regimen presented a higher incidence of TRM (p=0.005). No other clinical data was associated with complications post HSCT. Forty polymorphisms were detected in 9 genes by bioinformatic analysis. The variants that presents some correlation (p Overall, polymorphisms related with decrease activity of enzymes that active cyclophosphamide (level lor active metabolite) were correlated with higher aGVHD, cGVHD, TRM and VOD (Table 1). On the other hand, polymorphisms associated with low activity in detoxification enzymes were correlated with higher toxicity (TRM). As described in bibliography, GSTM1 null allele were correlated with higher probability of developing VOD. CONCLUSIONS Genetic analysis of CY metabolism genes correlated with several post HSCT complications. The analyses of this variants before transplant could facilitate personalized risk and clinical management of patients undergoing Haplo HSCT. Results must validated in others cohorts of patients. Disclosures Kwon: Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Jazz: Consultancy, Honoraria.

Introduction: Non-Hodgkin B lymphomas (NHL-B) are a large group of heterogeneous diseases, with well-defined, histological and clinical features. Currently, the lymph node biopsy is essential for diagnosis, often obtained from hard to reach areas. Our aims with this study was to analyze genetics variants at diagnosis trying to discriminate between different NHL-B histologic subtypes using formalin fixed paraffin embedded (FFPE) tissue sections and circulating tumor free DNA (ctDNA) samples. Material and Methods: This is a retrospective, cross-sectional, single-center study. Sixty patients were selected with a diagnosis of different NHL-B subtypes: Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal lymphoma (ML), mantle cell lymphoma (MCL) and plasmablastic lymphoma (PL). Sixty FFPE tissue samples and 23 ctDNA specimens obtained at diagnosis were selected. We performed an enrichment panel of 54 genes recurrently mutated in lymphomas (Lymphoma Solution; Sophia Genetics) by next generation sequencing (NGS; NextSeq; Illumina). The depth of 90% of the readings was greater than 2600x. Quantitative variables were expressed as median and range. Categorical variables were expressed as frequency and percentage. The Fisher exact test was used to compare the distribution of categorical variables. The Mann-Whitney test was used to compare differences between quantitative variables. Statistical significance was set at p < 0.05. Results: Our study shows that 97% (58/60) of patients presented any variant. The most frequently mutated genes were KMT2D, EP300 and SOCS1. Exclusive variants were detected in FL in the genes CCND1, PAX5, CREBBP, BCL2 and REL genes. In the same way, in DLCBL the variants detected in the BRAF, TCF3, XPO1, PIM1, CHD2, ID3 and BCL6 genes were exclusive of this subtype. Furthermore, the genes BCL2 (p=0.0021), CREBBP (p=0.0004), NOTCH2 (p=0.03), PAX5 (p=0.01) and TNFRSF14 (p=0.04) are more frequently altered in patients with FL, ATM (p=0.02) gene in MCL; NFKBIE (p=0.03), CIITA (p=0.02), MYC (p=0.009) and PIM1 (p=0.04) in DLCBL, among which it was found that high-grade lymphomas are more frequently associated with mutations in CHD2 (p=0.02), MYC (p=0.03) and MYD88 (p=0.02; data not shown). In the subgroup of 23 patients with paired samples (FFPE/ctDNA), 95% (22/23) of patients had mutations in any of the genes on the panel in FFPE tissue samples and 82% (19/23) in ctDNA specimens. The number of variants detected was different depending on the type of sample analysed, 52% of the variants were detected in both specimens, 39 only in FFPE tissue and 9% in ctDNA; Figure 1). In variants detected in both samples, the average value of the VAF was higher that when is detected only in one of the samples (FFPE: 28.1 vs 5.8 (p We did not find significant differences in the number of variants depending on the stage, classification, tumour burden or bulky mass (Table 1). Conclusion: In our study, we were able to identify genetic variables that could characterize different histological groups of NHL-B in FFPE tissue and ctDNA samples, by using a commercial gene panel of NGS. The mutational profile obtained from ctDNA and FFPE tissue is comparable in all histological subtypes, regardless tumour burden, aggressiveness or stage, throwing each one of them complemental information. These results support the hypothesis that could be possible to distinguish different histologies through non-invasive strategies, specially oriented to lesions where obtaining a tissue sample is difficult. We are working on the development of clinical-genetic algorithms that allow us to achieve our goal. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Allogeneic haematopoietic stem cell transplant (allo-HSCT) could be the only curative therapy for patients with hematological malignancies due to graft effect against tumor. However, approximately 40% of patients develop post-transplantation complications such as acute graft-versus-host disease (aGVHD). Cytokines and their receptors are involved in regulatory and inflammatory processes that occur during GVHD. Therefore, the analysis of polymorphisms (SNPs) that affect the expression or activity of these genes could be used as biomarkers to predict the development of these complication. The aim of this study was to select new polymorphisms in cytokine genes (interleukins, chemokines and their receptors) to build models to predict the development of aGVHD after allo-HSCT from an HLA-identical sibling donor. METHODS We retrospectively selected 88 patients with hematological malignancies who received an allo-HSCT from an HLA-identical sibling donor from 2000 to 2015. A total of 176 pre-transplant recipient (R) and donor (D) peripheral blood samples were collected. The genotyping was performed using an enrichment-capture gene panels (include 132 genes (73 interleukins, 59 chemokines) in a HiSeq platform (Illumina, USA). The bioinformatic analysis was carried out with the GeneSystemssoftware (Sistemas Genómicos, Spain). Variants located in coding region, splicing sites, and UTR, 5'upstream and 3'downstream zones (+ 200pb) were analyzed. We selected polymorphisms corresponding to non-synonymous variants, represented in at least 5% of our cohort, with readings ≥30X in the canonical isoformwith an allele frequency ≥ 0.4. Fisher test was used to compare the differences among groups. Multiple logistic regression models were performed using combination of interleukins and chemokines polymorphisms selected previously that could be applied to clinical practice to predict aGVHD. The models with the highest AUC value, sensitivity and specificity value and the lowest number of genetic variants used were selected.Statistical Package for the Social Sciences (SPSS, Chicago, USA) was used for statistical test. RESULTS The cumulative incidence rates for aGVHD of grades II-IV and III-IV at 100 days after transplantation were 48.93% and 18.08%, respectively. The clinical variables (age, gender, pathology, stem cell source and previous transplantation) were not correlated with II-IV or III-IV aGVHD. However, patients who received ablative conditioning regimen presented a lower incidence of III-IV aGVHD (p= 0.041). Using filters defined previously, we detected 481 polymorphisms (350 coincident, 68 specific R and 63 for D) in the interleukins group. On the other hand, we detected 339 polymorphisms (267 coincident, 29 specific R and 43 for D) in the group of chemokines. Finally, 17 polymorphisms were selected in the interleukin group and 10 in the chemokine group for its correlation with the aGVHD (p We developed multiple logistic regression models for II-IV and III-IV aGVHD in interleukins and chemokines genes (Table 3). The identification of these 15 polymorphisms could help us to prevent the developing II-IV and III-IV aGVHD. CONCLUSIONS We have identified new genetic polymorphisms correlate with the risk of developing aGVHD after allo-HSCT from an HLA-identical sibling donor. Based on these data, we have developed a genetic score that encompasses polymorphisms of greater relevance. In this way, patients at greatest risk for developing this type of post-transplantation complication who could benefit from personalized management through immunosuppression and other drugs. Disclosures No relevant conflicts of interest to declare.

Introduction Donor cell derived myeloid neoplasm (DCMN), defined as the development of de novohematological malignancies from cells of donor origin,is a late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We report on seven cases of DCMN in which whole-exome sequencing (WES) at different time-points after allo-HSCT, as well as in a sample from each donor, was performed. The ultimate objective was to accurately described the clonal architecture, spatial heterogeneity and identify somatic mutations that are induced in the process of leukemogenesis and clonal evolution of myeloid neoplasm. Donors were also analyzed to detect underlying condition predisposing to the development of DCMN. Patient and Methods Seven patients with a confirmed diagnosis of DCMN and their donors were recruited from different Spanish institutions. This cohort included a total of 32 BM samples at different time points after allo-HSCTand one PB sample from each donor (one case received dual allo-HSCT, CB and PB from both donors were obtained), which provided a total of 40 samples. Genomic DNA samples were prepared according to Agilent SureSelect-XT Human exon 50Mb enrichment kit (Agilent Technlogies, Santa Clara, CA) preparation guide and libraries were sequenced on Illumina HiSeq platform (Illumina, San Diego, CA). DNA sequencing data from recipient post-transplant BM samples, were matched against their donor PB sample and previous post-transplant BM samples to identify the acquisition of mutations along the post allo-HSCT period.Germline variants in donors were studied in order to detect mutations that predisposed to the development of a myeloid neoplasm.The research protocol was approved by the Ethic Committee of Gregorio Marañón General University Hospital. Patients´ and donors´ information was collected from their medical records. Results Clinical and biological characteristics of the 7 patients with DCMN and their donors are shown in Table 1. Mutational profiles obtained from the follow-up samples at different time-points post-HSCT demonstrated high intra-tumor genetic heterogeneity and clonal dynamic for all cases. The number of variants are increased over time and at the moment of DCMN diagnosis, the median number of variants was 28, ranging from 18 to 92 variants (Figure 1). WES identified in DCMN patients gene mutations commonly seen in adult AML or MDS, such as in SETBP1, DNMT3A, TET2, RUNX1, CSF3R, EP300and IDH2.In addition, others non-silent variants were acquired in all cases. Among the additional novel alterated genes, we found 23 strong candidateswith oncogenic potential. LUC7L2, NOP14, LAMA5, SKOR2, EML1, SNX13, RHPN2, IRS1, MTG2, TENM2, MEFV, GSE1, NOTCH4, DTX1, CNOT4, PNKP, GRB7,SENP7,TAF1L, ZKSCAN2, ZBTB20, ZNF461 and MEGF10. Analysis of CNVs revealed numerical alterations across the post allo-HSCT samples in patients 1, 2, 3, 4, 5 and 7. The most common chromosomal alterations in DCMN were monosomy 7 and other chromosome 7 abnormalities, which detected in the 86% (6/7) of the patients. Although none of the donors developed a myeloid neoplasm at the moment of diagnosis of DCMN in recipient, donor 1 revealed an abnormal karyotype (45,XY,-7) at the moment of the allo-HSCT. All other donors harbored at least one pathogenic or probably-pathogenic variants, most probably of germline origin, in genes involved in hematological or solid tumor predisposition. Conclusions The development of DCMN involves dynamic genomic processes that begin months before the clinical onset. In this study, our integrated multi-step analysis revealed the intra-tumor heterogeneity and evolutionary history of seven DCMN. The present study reveals a process of sequential clonal expansions promoted by the acquisition of somatic mutations in donor hematopoietic cells. Detection of heritable or acquired gene mutations in donors associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-HSCT. Leukemic transformation ofdonor hematopoietic stem cells provides a useful in vivomodel to study the mechanisms involved in leukemogenesis. Novel approaches based on high-depth next generation sequencing to study consecutive samples from post-transplant period in these patients, appear promising to discover new genes involved in myeloid neoplasm and to decipher the mechanisms of leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Introduction: Lymphomas continue to be a leading cause of death in HIV-positive patients (HIV+ pts) in the cART era. The aim of this study was to review our 15-year single institution experience in performing Autologous Stem Cell Transplantation (ASCT) for HIV-infected and non-HIV patients with high-risk or relapsed lymphomas, focusing on infectious complications. Patients and Methods: We retrospectively evaluated our cohort of 28 HIV+ pts who underwent an ASCT between 2000-2015, and compared it with a well-matched control group of 39 HIV-negative pts. Patient and ASCT characteristics are described in Table 1. All HIV+ pts were on cART. Chemomobilization was used in 60% of the HIV cohort and 84% of the controls. BEAM conditioning regimen was the most common. All transplants were performed in the same tertiary hospital JACIE-accredited SCT unit. The primary end points were first-year cumulative incidence (CI) of infection, total infectious episodes and infection-related mortality. For the analysis, we defined 3 different time frames: 1st Pre-engraftment; 2nd from engraftment to day +100 and 3rd from day +100 until 1 year after SCT. Events occurring during the first 2 periods were considered early infections as compared to late. Results: All patients received antiviral and anti-PJP prophylaxis, but a significantly higher proportion of HIV+ pts were given antibacterial and antifungal prophylaxis (2/3 vs 1/3 approximately, Table 1). G-CSF support was initiated in all HIV recipients and 66% of controls, and the median days of use was longer for the HIV group (7 vs 4 days, p= 0.04). Median time to neutrophil engraftment was similar in both groups (13 vs 11 days, p=0.55); 93% of HIV pts and all control pts reached ANC > 500c/uL by day +30. Infectious episodes (IE) are described in Table 2, divided by pathogen subtype and time frame. Globally, all infection subtypes were more common in the HIV-infected cohort at some point. The most significant findings from the analysis are as follows. CI of early global infections: HIV 75% vs non-HIV 25% (p= 0.04), Figure 1. Median number of global infectious events: HIV 65 vs non-HIV 39 (p= 0.002; OR 1.8 [1.2-2.8]). Bacteria: CI of pre-engraftment and early bacterial infections were not different among groups (42% vs 28%, p= 0.47), but the median number of bacterial episodes was clearly different: HIV 17 vs non-HIV 12 (p= 0.08; OR 2.16 [0.96-4.8]). Fungi: CI of early fungal infections: HIV 10.7% vs non-HIV 0% (p= 0.03); minor infections were not considered. Viruses: CI of early viral infections: HIV 46% vs non-HIV 15% (p= 0.004). Median nº of early viral IE: HIV 19 vs non-HIV 6 (p= 0.007; OR 4.16 [1.43-12]). CI of late viral infections: HIV 30% vs non-HIV 11.7% (p= 0.04). CMV reactivations were by far more common in the HIV cohort (p=0,01). HIV viral load bleeps were documented in 35% of the HIV patients (most commonly in the day +30 control) and one post-transplant virological failure was diagnosed, forcing HAART substitution. Of note, 1st year CI of infection-related mortality was 14% in the HIV group vs 0% in the non-HIV group (p= 0.01), Figure 2. Three HIV+ pts suffered early fulminant septic episodes (1 E. coli + Enterococcus, 1 Rothiamucilaginosa, 1 non-clarified - possible Stenotrophomonasmaltophilia) and a 29-year old woman in CR after a 1st line for a stage IVsB Burkitt-like lymphoma died due to a severe influenza A pneumonia. Length of admission was also significantly longer for the HIV+ pts (median days 34 vs 28, p=0.041). Regarding long-term outcome, median follow up as of July 2016 is 82 months for the HIV+ group and 70 months for the control group: 57% and 61% of the pts in each cohort are still alive, respectively. One HIV-infected pt and 3 controls have been lost to follow-up. EFS: 1 year 71.4% vs 81.9% (ns); 5 years: 63.9% vs 66.5% (ns). Overall Survival: 1 year 75% vs 84% (ns); 5 years 66.3% vs 74.6% (ns). Conclusion: Autologous stem cell transplantation has been proven to be feasible and effective in HIV-related lymphomas, but in our experience and despite great advances in cART and virological control, HIV+ patients are at high risk of infection and this might influence post-ASCT short-term survival. It is mandatory to focus on prophylactic and supportive measures and to choose carefully the optimal timing for transplantation. Table 2 Table 2. Disclosures No relevant conflicts of interest to declare.

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