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Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G

Simple Summary Currently, response rates in multiple myeloma (MM) have increased dramatically, with more than 50% otablef those who respond satisfying complete response criteria. Achieving frequent deep responses has necessarily led to test conceptual advantages for assessing and treating MM patients with only minimal residual disease (MRD). In this review, we present and discuss the clinical relevance, methodology, and challenges for measuring MRD in MM. Abstract The game-changing outcome effect, due to the generalized use of novel agents in MM, has cre-ated a paradigm shift. Achieving frequent deep responses has placed MM among those neoplasms where the rationale for assessing MRD is fulfilled. However, its implementation in MM has raised specific questions: how might we weight standard measures against deep MRD in the emerging CAR-T setting? Which high sensitivity method to choose? Are current response criteria still useful? In this work, we address lessons learned from the use of MRD in other neoplasms, the steps followed for the harmonization of current methods for comprehensively measuring MRD, and the challenges that new therapies and concepts pose in the MM clinical field.

Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro. Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor. Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. Figure 1 Figure 1. Disclosures Jerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival.

Background Despite the longer survival achieved in multiple myeloma (MM) patients due to new therapy strategies, a concern is emerging regarding an increased risk of secondary primary malignancies (SPMs) and how to characterize those patients at risk. We performed a retrospective study covering a 28-year follow-up period (1991-2018) in a tertiary single institution. Material and methods Data of 403 MM patients were recorded and compared with the epidemiologic register of the population area covered by our centre, calculating the standardize incidence ratio (SIR) for the different types of SPMs diagnosed in the MM cohort. Fine and Gray regression models were used to identify risk factors for SPMs. Results Out of the 403 MM patients, 23 (5.7%) developed SPMs: 13 therapy-related myeloid (TRM) malignancies (10 of them (77%) myelodysplastic syndrome (MDS), 1 acute lymphoid leukaemia and 9 solid neoplasms. In the MM cohort, the relative risk of MDS was significantly higher than in the general population. Survival of patients with TRM malignancies was poor with a median of 4 months from the diagnosis, and most of them showed complex karyotype. Within the MM subset, multivariable analysis showed a higher risk of TRM malignancies in patients that previously received prolonged treatment with lenalidomide (>18 months). Conclusions Though the improvement in MM outcome during the last decades is an unprecedented achievement, it has been accompanied by the rise in TRM malignancies with complex cytogenetic profile and poor prognosis that are in the need of an improved biologic and therapeutic approach.

Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34(+) bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down-regulation to be specific to CMML compared with other related disorders. Chromatin-regulator mutated cases showed decreased BAP1 dosage. We validated a significant over-expression of the double strand break-fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.

Bone marrow infiltration (BMI), categorized as an extra-nodal site, affects stage and is associated with poor prognosis in newly diagnosed lymphoma patients. We have evaluated the accuracy of PET/CT and bone marrow biopsy (BMB) to assess BMI in 372 lymphoma patients [140 Hodgkin Lymphoma (HL) and 232 High Grade B-cell non-Hodgkin Lymphoma (HG B-NHL), among them 155 Diffuse Large B-Cell Lymphoma (DLCL)]. For HL cases, and taking into account PET/CT, sensitivity, negative predictive value (NPV) and accuracy were 96.7, 99.3, and 99.3% while those of BMB were 32.3, 83.8, and 85%, respectively. For HG B-NHL and considering PET/CT, sensitivity, NPV, and accuracy were 52.7, 81.7, and 84.1%, while those of BMB were 77.6, 90.2, and 90.7%, respectively. In the HG B-NHL group, 25 patients would have been under-staged without BMB. These results lead us to recommend PET/CT and the avoidance of BMB to assess BMI in HL. In the case of HG B-NHL, bone marrow status should be assessed firstly by means of PET/CT; only in either focal or diffuse PET/CT with low borderline SUV max values or in negative cases, should BMB be carried out afterwards. In the HG B-NHL setting and at the present moment, both techniques are complementary.

INTRODUCTION The Revised International Prognostic Scoring System (IPSS-R) for myelodysplastic syndromes (MDS) has established an intermediate category where a disease-modifying intervention is a matter of debate. Flow cytometry allows us to determine a fraction of immature myeloid cells in a semiautomated procedure. The aim of this study, mirroring IPSS-R study inclusion criteria, was to test whether bone marrow (BM) CD34+My percentage has independent prognostic value in the MDS setting. METHODS BM CD34+My cells were quantified, at diagnosis, selecting CD34+/CD45+/CD11b±/CD13+. Patients were excluded when receiving treatment for altering the natural course of the disease and when IPSS-R could not be calculated due to the lack of metaphases. Finally, Cox analyses were performed, on a series of 260 patients, for overall survival (OS) and time to acute myeloid leukemia (AML) transformation. RESULTS By analyzing ROC curves, the most accurate prognostic variable, regarding blasts by cytology and CD34+ by cytometry, was the percentage of blasts by microscopy. The percentage of CD34+My in BM showed an AUC of 0.767 and 0.576 for time to AML transformation and OS, respectively. When performing a multivariate regression including the IPSS-R and the percentage of BM CD34+My cells >1%, both factors predicted for a shorter time to AML transformation. In addition, CD34+My percentage successfully stratified the intermediate IPSS-R category into two prognostic groups with a relative risk of 5.73 (95% CI [1.2-27.8]; P = .03). CONCLUSION We found that BM CD34+My percentage has an independent value concerning the IPSS-R, especially relevant for the prediction of transformation to AML and within the intermediate group.

Deletion of 13q14 as the sole abnormality is a good prognostic marker in chronic lymphocytic leukemia (CLL). Nonetheless, the prognostic value of reciprocal 13q14 translocations [t(13q)] with related 13q losses has not been fully elucidated. We described clinical and biological characteristics of 25 CLL patients with t(13q), and compared with 62 patients carrying interstitial del(13q) by conventional G-banding cytogenetics (CGC) [i-del(13q)] and 295 patients with del(13q) only detected by fluorescence in situ hybridization (FISH) [F-del(13q)]. Besides from the CLL FISH panel (D13S319, CEP12, ATM, TP53), we studied RB1 deletions in all t(13q) cases and a representative group of i-del(13q) and F-del(13q). We analyzed NOTCH1, SF3B1, and MYD88 mutations in t(13q) cases by Sanger sequencing. In all, 25 distinct t(13q) were described. All these cases showed D13S319 deletion while 32% also lost RB1. The median percentage of 13q-deleted nuclei did not differ from i-del(13q) patients (73% vs. 64%), but both were significantly higher than F-del(13q) (52%, P

Background and Aim: Chronic lymphocytic leukemia (CLL) is an incurable neoplasm with considerable clinical heterogeneity that is underpinned by distinct genetic changes. This poses a great challenge to the development of effective treatment, as responses to novel therapies may vary depending upon the biological subtype of disease. NOTCH1 has emerged as the most commonly mutated gene in CLL at diagnosis, its frequency rises with progression, and the variant c.7541_7542delCT accounts for approximately 80% of cases. NOTCH1-mutated (NOTCH1mut) CLL subset represents a subtype that responds poorly to anti-CD20 drugs, has worse outcomes, and a proliferative profile. We aimed, among the proliferative signals, to study the D-type cyclins that govern cell cycle entry in a NOTCH1mut subset which lacks an accurate targeted therapy. Methods: We performed targeted sequencing on 289 CLL cases with a TruSeq Custom Amplicon panel from Illumina, containing 13 genes recurrently mutated in CLL. Paired-end sequencing reached a mean depth of 938 reads/base. Out of 30 NOTCH1mut patients, RNA from the CD19+ fraction was available in 25 cases. Cyclins D1, D2, D3 and cyclin dependent kinases CDK4 and CDK6 expression was measured by RT-qPCR in 25 NOTCH1mut and 57 NOTCH1wild type (NOTCH1wt). CD19+ B-CLL cells from 7 NOTCH wt and 2 NOTCH1mut patients were cultured in Expansion Human Media supplemented with CD40L, IL-4 antibiotics and inactivated BFS. On day ten 2,5x105 B cells were added to a combination of ON-TARGETplus SMART pool siRNA NOTCH1, pool siRNA CCND1, SMART pool siRNA CCND2, or ON-TARGETplus siCONTROL as negative control. Electroporation was performed with Neon Transfection System in 10 ul tips using settings of 1400 volts, 10ms width, and 3 pulses. Transfected cells were double stained with Annexin V-FITC conjugate and PI in isotonic solution, and then analyzed by FC. Viable cells were defined as double negative cells. Results: A total of 82 CLL patients, 25 NOTCH1mut and 57 NOTCH1wt were included in the RT-qPCR study. Figure 1a shows fold change expression and p value comparing NOTCH1mutvs.NOTCH1wt of CDK4 (FC= 11,1; p Next we transfected cultured primary cells from two c.7541_7542delCTNOTCH1mut and 5 NOTCH1wt patients. Figure 1b shows the cell viability assay after 48 h of the transfection, highlighting a marked reduction in viable cells in NOTCH1mut cases in each silencing experiment, (siRNA_Control: 77% NOTCH1sirna: 71 %, CCND2siRNA: 73% and CCND1: 72%) which was not paralleled in NOTCH1wt cases (siRNA_Control: 81% NOTCH1sirna: 80 %, CCND2siRNA: 84% and CCND1: 78 %). Figure 1C shows how the apoptosis index increased in every silencing experiment compared with NOTCH1mut siRNA pool control. However, for therapeutic purposes the accurate approach should increase apoptosis, leading the cells to a controlled death, and reduce necrosis, which is responsible of inflammation and collateral damage. The optimal apoptosis/necrosis ratio in our experiments, it is shown by the silencing of CCDN2 in NOTCH1mut (ratio A/N=5,15) where percentage of necrotic cells was markedly reduced (fig.1d). Conclusions: We found a marked upregulation of cyclins governing the transition from G1 to S phase of the cell cycle in patients with CLL and NOTCH1 mutation compared with those wild type cases for this lesion. In addition, silencing of cyclin D2 in the NOTCH1mut CLL subset was able to reach a similar degree of apoptosis than silencing NOTCH1, with a more favourable reduction of necrosis, emerging as a suitable candidate for a therapeutic target. Disclosures Jerez: Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background and Aim: The entity defined by the WHO 2017 classification as myeloid neoplasms with germinal predisposition without preexisting disorder or organ dysfunction is particularly interesting within myelodysplastic syndromes (MDS) for three main reasons: i) in myeloid disease derived from congenital bone marrow failure, therapeutic strategies (e.g., type of conditioning regimen) are defined; it is not the case within the group of young patients with MDS harbouring germinal variants, actually a group candidate for allogeneic transplantation of hematopoietic progenitors, ii) its incidence exceeds the cases secondary to congenital bone marrow failure, and iii) the implications of genetic counseling to patients and relatives are yet to be defined and have not been addressed at the time of diagnosis of MDS. Methods: Whole exome sequencing (WES) was performed on 118 tumour and 73 paired germinal DNA from patients of 16 Spanish Group of MDS (GESMD) centers, diagnosed with de novo MDS between 16-60 years old without previous organ dysfunction. WES libraries were sequenced on a HiSeq4000-NovaSeq6000-Illumina platform, mean number of reads per sample was 144,429,985 with a Phred Quality Score >30 in 94% of bases and a 100x average depth. To identify potential germline-predisposing mutations, a selection tool incorporating 279 genes associated with cause or predisposition to bone marrow failure or cancer was implemented. The analysis of the variants was carried out by means of an in house pipeline: filtering out intronic, synonymous, and those variants with minor allele frequency (MAF) in the general population >1% (ExAC, 1000 Genomes-phase3, TOPmed), and requiring the presence both in tumour and germline DNA with a VAF>37%. In 45 cases without germline material the last requirement was substituted by not being reported as somatic in COSMIC in any cancer. To determine pathogenicity we followed conservative criteria: a CADD Phred score ≥20 and to be considered deleterious in, at least, three out of six used algorithm. Results: In 118 patients, the median age at diagnosis was 47 years with the following WHO 2017 diagnoses: 12% MDS-SLD, 9% MDS-RS, 34% MDS-MLD, 30% MDS-EB, 3% MDS-del(5q), 1%MDS-U, 11% CMML. We found deleterious/pathogenic germ variants in 68 of 118 patients. Strikingly, we found a higher frequency than expected, for this specific subset, in genes not yet considered in the category of myeloid neoplasms with germline predisposition: MSH6 (n=5;4.2%), ATR (n=5;4.2%), ERCC6L2 (n=4;3.4%), PMS2 (n=2; 1.6%), MLH1 (n=3;3.5%), HCLS1 (n=2;1.7%), ITGB3 (n=2;1.7%), LYST (n=4; 3.4%), SAMD9 (n=1;0.8%), MSH2(n=1;0.8%). In genes already considered in the category of myeloid neoplasms with germline predisposition: MPL (n=2;1.7%), DDX41 (n= 2;1.7%), RUNX1 (n=1; 0.8%), ANKRD26 (n=1;0.8%). We also detected 10 cases with deleterious germline variants in genes related to Fanconi anemia (BRIP1, FANCC, FANCD2, FANCG, FANCM, SLX4, XRCC2, RAD51C and BRCA2), 2 cases with a germline variant in a Shwachman-Diamond gene DNAJC21 (1.7%), and 3 cases with germline variant in a telomere biology gene (RTEL1, CTC1 and TERT). We then focused on the characterization of the variants found in 5 genes not considered to date as predisposing to MDS: MSH6, MSH2, MLH1, ATR and PMS2 involved in the instability of microsatellites and whose alteration determines genomic instability and predisposition to a different number of solid tumors. The frequency of patients carrying these variants (13.5%) is much higher than the frequency of DDX41 (1.7%) or CEBPA (not found in our cohort), the two genes currently considered in the category with germline predisposition without a preexisting disorder or organ dysfunction. The sixteen patients carrying these mutations were characterized by a median age of 47 (16-60) years with the following diagnoses, 33% MDS-MLD, 27% MDS-EB1 and 40% CMML, presenting up to 40% with decrease cellularity in the bone marrow. Conclusions: We describe, for the first time, a high frequency of germinal variants in genes that drive genomic instability by modulating the microsatellite pathway, in young adults diagnosed with MDS without previous organ dysfunction. Their frequency and high pathogenicity index warrant functional validation experiments and pose them as potential candidates to be included in future classifications and to be considered in clinical, therapeutic and genetic counseling strategies. Disclosures Díez-Campelo: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sanz:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Healthcare: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffman - La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen - Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease resulting from mutations in the LYST/CHS1 gene, which encodes for a 429 kDa protein, CHS1/LYST, that regulates vesicle trafficking and determines the size of lysosomes and other organelles. To date, 60 different mutations have been characterized, and a reasonably straightforward phenotype-genotype correlation has been suggested. We describe two patients on opposite ends of the CHS clinical spectrum with novel missense mutations. We characterized these patients in terms of their mutations, protein localization and expression, mRNA stability, and electrostatic potential. Patient 1 is the first report of a severe early-onset CHS with a homozygous missense mutation (c.11362 G>A, p.G3725R) in the LYST/CHS1 gene. This molecular change results in a reduction at the CHS1 protein level, not due to an mRNA effect, but maybe a consequence of both, a change in the structure of the protein and most likely attributable to the remarkable serious perturbation in the electrostatic potential. Patient 2, who exhibited the adolescence form of the disease, was found to be homozygous for a novel missense mutation c.961 T>C, p.C258R, which seemed to have minor effect on the structure of the CHS1/LYST protein. Reexamining accepted premises of missense mutant alleles being reported among patients with clinically mild forms of the disorder should be carried out, and attempts to link genotype and clinical phenotype require identifying the actual molecular effect of the mutation. Early and accurate diagnosis of the severity of the disease is extremely important to early differentiate patients who would benefit from premature enrollment into a transplantation protocol.

Several studies have reported uneven results when evaluating the prognostic value of bone marrow biopsy (BMB) and PET/CT as part of the staging of diffuse large B‐cell lymphoma (DLBCL). The heterogeneity of the inclusion criteria and not taking into account selection and collinearity biases in the analysis models might explain part of these discrepancies. To address this issue we have carried a retrospective multicenter study including 268 DLBCL patients with a BMB and a PET/CT available at diagnosis where we estimated both the prognosis impact and the diagnostic accuracy of each technique. Only patients treated with R‐CHOP/21 as first line (n = 203) were included in the survival analysis. With a median follow‐up of 25 months the estimated 3‐year progression‐free survival (PFS) and overall survival (OS) were 76.3% and 82.7% respectively. In a multivariate analysis designed to avoid a collinearity bias with IPI categories, BMB‐BMI [bone marrow involvement](+) (HR: 3.6) and ECOG PS > 1 (HR: 2.9) were independently associated with a shorter PFS and three factors, age >60 years old (HR: 2.4), ECOG PS >1 (HR: 2.4), and abnormally elevated B2‐microglobulin levels (HR: 2.2) were independently associated with a shorter OS. In our DLBCL cohort, treated with a uniform first‐line chemotherapy regimen, BMI by BMB complemented performance status in predicting those patients with a higher risk for relapse or progression. In this cohort BMI by PET/CT could not independently predict a shorter PFS and/or OS.

Background and Aim: Azacitidine have shown clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs, encoding for immunogenic proteins which are normally expressed in the testicles and trophoblastic cells of the ovary, have been shown to undergo derepression after the use of demethylating agents in cancer cell line models. We took advantage of the unique model of Aza-treated high risk MDS to in vivocharacterize candidates for immunotherapy following hypometilating therapy. Methods:Targeted RNA-Seq (tRNA-Seq) designed to capture 214 CTA genes was performed in 19 MDS or CMML patients at day 0 and at day +28 after first AZA cycle. In 10 patients the analysis was extended to third and/or four cycles. Sequencing, coverage, mapping and alignment was performed using the Ion Torrent Browser Suite. We used LIMMA package or empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes. A homogeneous treatment schedule with AZA (75 mg/m2/day x 7 days) was received by each patient.The response criteria were from IWG 2006, including a bone marrow and cytogenetic reevaluation after 6 cycles. The main candidates selected from de tRNA-seq experiment were characterized at protein level in cell lysate and/or plasma by Western Blot. Primary antibodies used from Thermo Fisher were: ADAM29, DDX53, PRAME, B-TUBULINA, FAM46D, B-ACT, TMPRSS12, MAGEB4, GAPDH, MAGEA1, MAGEA2, TSPY2, Cxorf48, DNAJB8, NY-ES0-1, CT83/Cxorf61 and TFDP3 from Santa Cruz Biotechnology. We used either cell lysate or plasma to study the protein levels in non-reducing SDS-PAGE at different acrylamide concentration depending on the protein size. ImageJ was used to quantify protein levels. Results:The median age of the cohort was 69 (range 48-81 years). The cohort consisted of 16 MDS and 3 CMML patients. MDS Patients were stratified based on IPSS as low or intermediate-1 (n=14) and intermediate-2 or high (n=2) risk groups and CMML patients based on a CPSS of intermediate-2 (n=3). Five patients were classified as complete responders achieving a complete cytogenetic and/or marrow remission (CR). Regarding the tRNA-Seq, on average, we achieved 3.888.308 mapped reads per sample (p25-p75, 1.2x106 - 5.9x106) which represents a sufficient depth for digital gene expression profiling of 214 genes. CTAs re-expressed significantly after one AZA cycle in complete responders were TFDP3 (FC=6,4), ZNF645 (FC=2), MAGEB4 (FC=3,1), MAGEA5 (FC=2), DDX53 (FC=2), VENTPX1 (FC=5,5), TSPY2(FC=3,4) and TSYP3 (FC=3,5). Other potential candidates with significantly re-expression in any responders vs. non responder were:SLCO6A1, ADAM29, DDX53, PRAME, FAM46D, TMPRSS12, MAGEA1, MAGEA2, Cxorf48, DANJB8, NYESO-1 and CT83. In the western-blot experiments, protein expression, longitudinally measured in peripheral blood lysate and/or plasma showed a parallel dynamic to gene expression in the case of TFDP3and DDX53. Conclusions:In this study, targeted RNA-seq is shown as effective and accurate tool to detect weakly expressed transcripts as CTAs. In this work, TFDP3 and DDX53, validated at proteomic level, emerge as most promising candidates for immunotherapy in combination with hypomethylating agents in MDS patients. 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.

Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure. Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score >30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded. Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%). Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. Table. Table. Disclosures Díez-Campelo: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background Among patients with B-cell chronic lymphoid leukemia, those with 13q14 deletion have a favorable outcome. However, whether the percentage of cells with 13q- influences the prognosis or the biological characteristics of this disease is unknown. We analyzed the clinico-biological characteristics and outcome of patients with B-cell chronic lymphoid leukemia with loss of 13q as the sole cytogenetic aberration. Design and Methods Three hundred and fifty patients with B-cell chronic lymphoid leukemia were studied. Clinical data were collected and fluorescence in situ hybridization and molecular studies were carried out. In addition, a gene expression profile was obtained by microarray-based analysis. Results In 109 out of the 350 cases (31.1%) loss of 13q was the sole cytogenetic aberration at diagnosis. In the subgroup of patients with 80% or more of cells with loss of 13q (18 cases), the overall survival was 56 months compared with not reached in the 91 cases in whom less than 80% of cells had loss of 13q ( p < 0.0001). The variables included in the multivariate analysis for overall survival were the percentage of losses of 13q14 ( p =0.001) and B symptoms ( p =0.007). The time to first therapy in the group with 80% or more vs. less than 80% of losses was 38 months vs. 87 months, respectively ( p =0.05). In the multivariate analysis the variables selected were unmutated status of IgVH ( p =0.001) and a high level of β2microglobulin ( p =0.003). Interestingly, these differences regarding overall survival and time to first therapy were also present when other cut-offs were considered. The gene expression profile of patients with a high number of losses in 13q14 showed a high proliferation rate, downregulation of apoptosis-related genes, and dysregulation of genes related to mitochondrial functions. Conclusions Patients with B-cell chronic lymphoid leukemia with a high number of losses in 13q14 as the sole cytogenetic aberration at diagnosis display different clinical and biological features: short overall survival and time to first therapy as well as more proliferation and less apoptosis. A quantification of the number of cells showing a genetic abnormality should, therefore, be included in the study of the prognostic factors of B-cell chronic lymphoid leukemia.

Monoclonal gammopathy of undetermined significance (MGUS) progresses to plasma cell dyscrasia at a rate of 1% per year. A high prevalence of MGUS has been noted in series of patients with immune disorders or chronic infections.Retrospective cohort and a cross-sectional study to analyze the prognostic value of aberrant (CD38(+ +)CD138(+) CD19(-)CD45(weak)) to normal phenotype (CD38(+ +)CD138(+) CD19(+)CD45(+)) bone-marrow plasma cells ratio (A/N ratio) for the development of a plasma cell dyscrasia and the association with the presence of a chronic immune disorder.A total of 322 patients were included with a median follow-up of 46 months. Analysis for progression revealed an increased A/N ratio as the main independent prognostic variable. A significant association between a reduced A/N ratio and the diagnosis of a chronic immune condition was found. Using receiver-operating characteristic analysis we created an A/N ratio range from 4 to 0.20. Values of 4 or higher define a group at high risk of progression (OR 10.7). A/N values of 0.20 or lower are associated with immune disorders or chronic infections (OR 20.9).Extreme values of the A/N ratio seem to be related with two different conditions: high risk of progression, and immune condition-related MGUS.

[Figure][1] A 38-year-old man was admitted because of cough, fever, headache, and malaise. He was pale but otherwise in good general condition. The hemogram showed: white blood cell count, 1.7 × 109/L; hemoglobin, 97 g/L (mean corpuscular volume, 99 fL); and platelets, 7 × 109/L. A blood

An increasing numbers of patients are being diagnosed with asymptomatic early-stage chronic lymphocytic leukemia (CLL), with no treatment indication at baseline. We applied a high-throughput deep-targeted analysis, especially designed for covering widely TP53 and ATM genes, in 180 patients with inactive disease at diagnosis, to test the independent prognostic value of CLL somatic recurrent mutations. We found that 40/180 patients harbored at least one acquired variant with ATM (n=17, 9.4%), NOTCH1 (n=14, 7.7%), TP53 (n=14, 7.7%) and SF3B1 (n=10, 5.5%) as most prevalent mutated genes. Harboring one ‘sub-Sanger’ TP53 mutation granted an independent 3.5-fold increase of probability of needing treatment. Those patients with a double-hit ATM lesion (mutation+11q deletion) had the shorter median time to first treatment (17 months). We found that a genomic variable: TP53 mutations, most of them under the sensitivity of conventional techniques; a cell phenotypic factor: CD38-positive expression; and a classical marker as β2-microglobulin, remained as the unique independent predictors of outcome. The high-throughput determination of TP53 status, particularly in this set of patients frequently lacking high-risk chromosomal aberrations, emerges as a key step, not only for prediction modeling, but also for exploring mutation-specific therapeutic approaches and minimal residual disease monitoring.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

[Figure][1] A 43-year-old woman was admitted due to a 6-week history of asthenia. She was pale; no other findings were observed in the physical exam. Blood count was 2.4 × 109/L, (56N, 33L, 9M, 1Eo), hemoglobin was 69 g/L, and platelet count was 15 × 109/L; 3% blast cells were counted in

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been widely used after autologous peripheral blood stem cell transplant (APBSCT) in an attempt to reduce the duration of neutropenia, but whether this treatment has any influence on long-term engraftment remains unknown. We have retrospectively analyzed data from breast cancer patients to compare post-APBSCT rhG-CSF administration in terms of the short-term benefit and myeloid marrow regeneration after 1 year. Group A included 10 patients not treated with post-APBSCT rhG-CSF, while groups B and C comprised 15 and 13 patients treated with this drug from days +1 and +6, respectively. No differences among the three groups were found in age, diagnosis, previous chemo-radiotherapy, CD34+/CD71- cell concentration in pre-transplant bone marrow (BM), mobilization schedule, CD34+ cell yield, conditioning regimen and post-transplant radiotherapy. Post-APBSCT rhG-CSF was shown to accelerate neutrophil recovery, but there were no significant differences in platelet recovery, transfusion requirements, days of fever, antibiotic administration or inhospital stay. With regard to BM hematopoietic precursors 1 year after APBSCT, significantly lower concentrations of total CD34+ cells, committed CD34+/CD33+ subsets, and more immature CD34+/CD71- cells were found in both groups B and C compared with patients not having received the cytokine (group A). Thus, post-APBSCT rhG-CSF administration does not appear to beneficially affect procedure outcome, and might even impair long-term marrow hematopoiesis.

We assessed the mobilization capacity of taxol with rhG-CSF, both as a single chemotherapeutic agent and in the presence of cyclophosphamide (CY), and compared the effect with yields achieved when mobilization was performed solely with rhG-CSF. Fifteen patients with breast cancer received taxol 170 mg/m2 (continuous infusion, day 1) and rhG-CSF (8 microg/kg/day, from day 2 until the end of apheresis) (T-G group), while seven breast cancer patients were additionally treated with CY (4 g/m2) on day 2, followed by rhG-CSF starting at similar doses on day 3 (T-CY-G group). The PBSC collections after taxol with/without CY were compared with those of 30 breast cancer patients who had received rhG-CSF (8 microg/kg/day) for mobilization. No differences were found in the characteristics of patients included in any of the three mobilization groups. The median yield of CD34+ cells from all patients included in taxol containing schedules was 9 x 106/kg (range 2-26) collected with a median of one apheresis procedure (range 1-4). Leukaphereses began earlier in the T-G group (median day 8, range 7-10) than in the T-CY-G group (median day 13, range 11-17). In most patients (20 out of 22) who received taxol containing regimens, more than 2.5 x 106 CD34+ cells/kg, a threshold considered to be sufficient for hematopoietic reconstitution, were collected with a single apheresis. Those patients in the T-G group experienced less neutropenic and thrombocytopenic days, with all neutropenic fever episodes developing in patients treated with the T-CY-G schedule (43%). When considering priming with rhG-CSF alone in our historical cohort of 30 breast cancer patients, a significant detrimental effect was observed in comparison with taxol mobilizing schedules, in the number of aphereses performed, in the total yield CD34+cells and in the number of patients who achieved the target dose of 2.5 x 106/kg CD34+ cells within the first collection procedure. We conclude that taxol containing schedules are effective in mobilizing PBSC and facilitate the collection of high yields of CD34+ cells (usually more than 5 x 106/kg recipient body weight) with a reduced number of apheresis procedures. Taxol, as a single agent with rhG-CSF, exhibits less hematological toxicity than the combination chemotherapy mobilization regimen including CY. Bone Marrow Transplantation (2000) 25, 231-235.

The frequency of monoclonal gammopathy of undetermined significance (MGUS) is higher in patients with type I Gaucher disease (GD I) than in the general population. Although enzyme replacement therapy is effective in the control of the disease, its effect on MGUS is still controversial. We present the case of a 65-year-old woman with extensive GD I associated with IgM MGUS, in whom enzyme replacement therapy succeeded in eradicating the monoclonal component. This observation further supports the idea that enzyme replacement therapy decreases the chronic antigenic stimulation responsible for gammopathies in Gaucher disease.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.

Acute graft-versus-host disease (aGVHD) after autologous progenitor cell transplantation has been associated with blood transfusion or cyclosporine. Mild aGVHD grades I-II, identified as autoaggression or engraftment syndrome, has recently been described in autologous progenitor transplantation. Here, we report the first case of pathologically documented grade IV aGVHD after autologous peripheral blood progenitor cell transplantation in a patient with breast cancer. The allogeneic origin was excluded by molecular techniques, and no cyclosporine or cytokines were administered.

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

[Figure][1] A 16-year-old girl was admitted for fever and severe malaise. Multiple ecchymoses and asymmetry of labia majora were observed. The hemogram showed a white blood cell count of 8.7 × 109/L, hemoglobin 82 g/L, and platelets 33 × 109/L. A blood smear showed 3% blasts. Fibrinogen

We describe a transient bone marrow heterogeneous B-cell lymphoproliferation with a complex spectrum of cytological, phenotypic, molecular, and immune features in the setting of adult patients with acute myeloid leukemia following umbilical cord blood transplantation procedure. In our view, the lymphoproliferation described herein could add another element to the differential diagnosis of post-transplant lymphoproliferative disorders.