ALBERT

Description: Background:The isolation of cell-free DNA (cfDNA) evolved the concept of liquid biopsy. Several works have analyzed the presence of cfDNA in lymphomas, especially in diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL), however there is limited information regarding the detection of cfDNA in other types of lymphomas or the role of cfDNA detection to monitor disease outcome. Objectives:1.- To analyze the presence of cfDNA at diagnosis of patients with lymphoproliferative malignancies. 2.- To evaluate the change in concentration of cfDNA ([cfDNA]) after treatment in DLBCL patients. Material and Methods:A retrospective study in a single center was performed including 221 adult patients since January 2015 to February 2019 with: DLBCL, HL, marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL) and peripheral T-cell lymphomas (TCL). All patients had plasma samples collected at diagnosis that were stored in the institutional biobank (MarBiobank). The cfDNA were obtained from 1 mL of -80°C frozen stored plasma using the MagMax Cell Free DNA isolation kit (Thermo Fisher Scientific). The cfDNA quantification was performed using the Qubit system with the dsDNA high sensitivitykit (Thermo Fisher) and is expressed as ng/mL of plasma. In addition, patients with DLBCL who were treated with rituximab-CHOP/CHOP-like regimens were evaluated for [cfDNA] analysis, pre and post-treatment, and results were compared with PET-CT scan findings. Results: Stored plasma samples at diagnosis were available in 221 patients: DLBCL 82, HL 22, CLL/LPL 13, FL 36, MZL 37, MCL: 11, TCL 10 and others 10 (B-cell lymphoma unclassified, hairy cell leukemia, Burkitt lymphoma). Successful identification of cfDNA was obtained in 95.9% (212/221) of samples. DLBCL patients showed higher [cfDNA] globally and, DLBCL, HL and FL patients showed a higher concentration than MZL who exhibited the lower concentration of all groups (p=0.009; p=0.013 and p=0.002); see table 1. 50 DLBCL patients with a median follow-up since diagnosis of 25.5 (5-51) months were analyzed. Median age was 67 (19-79) years, males 56% (28). IPI distribution: low-risk 28% (14), low-intermediate 26% (13), high-intermediate 24% (12) and high risk 22% (11) cases. Detection of cfDNA was successful in 100% at diagnosis and in 98% (49) cases post-therapy. The mean [cfDNA] at diagnosis was 2.21 (standard deviation- SD: 1.60) ng/mL with a correlation with LDH concentration (p

Description: Introduction The JAK2V617F allele burden at progenitor levels increases over time in polycythemia vera (PV), with the dominance of the JAK2V617F-positive clone at the CD34+ compartment being an important modifier of the disease phenotype. The relationship between clonal dominance and the evolutionary pattern of JAK2V617F granulocytic load is presently unknown. Objective To analyze the relationship between clonal dominance of CD34+ cells with the evolution of the granulocytic JAK2V617F mutant load in PV patients. Methods A total of 37 patients with PV were included in the study. At time of the study, 30 patients corresponded to the chronic phase of the disease, whereas 7 patients had evolved to myelofibrosis (post-PV MF). Seventeen patients were in early chronic phase (less than 5 years from diagnosis) and 13 patients in late chronic phase (more than 5 years from diagnosis). Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+, CD38-) and progenitors (CD34+, CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F allele burden was measured by quantitative PCR in all three cell fractions. Clonal dominance was defined as an absolute difference ≤ 10% in JAK2V617F between granulocytes and CD34+ populations. Granulocytic JAK2V617F mutant load was measured every year with the evolutionary pattern of the JAK2V617F allele burden being stratified as follows: stable 〈 50%, stable ≥ 50%, progressive increase and unexplained decrease. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results JAK2V617F allele burden in CD34+/CD38- cells was 8%, 24.9% and 71.4% in early chronic phase, late chronic phase and post-PV MF, respectively (p 〈 0.001). Similar results were observed in CD34+/CD38+ cells (JAK2V617F allele burden: 19.1%, 36.1% and 71.8% in early chronic phase, late chronic phase and post-PV MF, respectively, p 〈 0.001). No significant differences were observed among the three groups in granulocyte JAK2V617F allele burden. Six patients (16.2%) presented clonal dominance. According to disease status, clonal dominance was observed in 5 out of 7 (71.4%) patients with post-PV MF and in 1 out of 13 (7.7%) patients in late chronic phase, whereas no patient in early chronic phase showed clonal dominance. JAK2V617F monitoring was performed in 29 patients. According to their granulocytic JAK2V617F evolutionary patterns, 9 patients remained with a stable JAK2V617F below 50%, 13 patients showed a stable JAK2V617F ≥ 50%, 6 patients experienced a progressive increase of the JAK2V617F mutant load and 1 case presented an unexplained decrease of JAK2V617F. Clonal dominance was observed in 5 out of 20 (25%) patients with a stable JAK2V617F 〉50% or a changing profile in comparison to 0 out of 9 patients with stable JAK2V617F 〈 50% (p=0.05). Conclusion Clonal dominance correlates with JAK2V617F allele burden evolutionary pattern.This may be a factor to be taken into account in the therapeutic strategy of PV patients and supports the need to monitor JAK2V617F burden during clinical follow-up. Funding this study was supported by grants from the Ministry of Education and Science of Spain and Instituto de Salud Carlos III FEDER (EC10-136, FIS PI10/018087, RD09/0076/00036 and RD12/0036/0010), 2009SGR929 and by grant from Asociación Española Contra el Cáncer Catalunya. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND Somatic mutations in the myeloid differentiation primary response gene 88 (MYD88) have been described in B-cell lymphomas, including DLBCL. Our group has confirmed the remarkable site-specific occurrence of MYD88 mutations at some immune-privileged locations and, in addition, has described that DLBCL patients are at high risk of progression and death after first-line treatment, with independence of other well-known clinical prognostic factors (Leukemia 2014). AIMS To further analyze the clinical responsiveness and outcome after second-line treatment in DLBCL patients carrying MYD88 L265P. METHODS A series of 175 patients with DLBCL diagnosed at our institution between 2000 and 2013 were included. Inclusion criteria were: full clinical data available, treatment with remission intention, enough material for DNA extraction and absence of HIV infection. The presence of MYD88 L265P mutation was assessed by allele-specific oligonucleotides (ASO)-PCR in DNA samples extracted from FFPE tissue. RESULTS In 129 patients (74%) treatment consisted on rituximab plus CHOP or CHOP-like schedules, while the remaining cases received other treatments depending on their clinical requirements. With a median follow-up time of 57 months, progression free survival (PFS) at 5 years after first-line treatment was 57%. Twelve of these patients (9.3%) had MYD88 L265P mutation, and these patients had a significantly worse PFS that patients who did not (18% vs 59%, p=0.018). Overall survival (OS) at 5 years after first line treatment was 65% (17% vs 72%, with vs without MYD88 mutation, respectively; p=0.001). We further analyzed the outcome after second-line treatment in 32 cases: 6 patients had refractory disease (0 with MYD88 mutation) and 26 patients were in first relapse. Three out of 5 patients carrying MYD88 mutation and 25 out of 27 patients without MYD88 mutation were treated with chemotherapy. Only one out of 5 cases (20%) with MYD88 mutation responded to second-line therapy whereas response was observed in 16 out of 27 cases (59%) without MYD88 mutation(p=0.106). PFS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 34% in those without MYD88 mutation (p=0.092). Moreover, OS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 37% in those without MYD88 mutation (p=0.019)(Figure 1). CONCLUSION Our study shows that MYD88 L265P in DLBCL patients is not only associated to worse respond after first -line therapy, but also to a very poor response to second-line therapy, and consequently to a dismal outcome. New treatments are urgently needed for these patients. Acknowledgments: This study was supported in part by 2014SGR567 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background MicroRNAs (miRNAs) act as negative regulators of expression of genes that participate in cellular proliferation, apoptosis and/or carcinogenesis. MiRNAs have been shown to play a role in both solid and hematological tumors. Interestingly, several miRNA profiles have been differentially related to the mutational status of multiple tumors. A mutation in the JAK2 kinase (V617F) has been associated with essential thrombocythemia (ET) in around 40–50% of cases. However, the biological basis of ET in patients lacking the JAK2V617F mutation is still unknown. miRNAs may be crucial to the development of ET in these patients, yet their role in JAK2V617F-negative ET remains to be elucidated. Aim To characterize the expression pattern of miRNAs involved in JAK2V617F-negative ET and to identify the targets of these miRNAs. Methods Total RNA was extracted from isolated platelets from 19 ET patients (10 JAK2V617F-positive and 9 JAK2V617F-negative) and 10 healthy controls. The expression of 670 mature miRNAs was determined by TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT real time PCR system. miRNA expression data was analyzed by the 2-ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software v 2.13.To identify molecular pathways potentially altered by the expression of multiple miRNAs, we used Diana-mirPath, which performs an enrichment analysis of multiple miRNA target genes, comparing each set of miRNA targets to all known KEGG pathways. The mRNA expression of identified putative targets was analyzed by TaqMan gene expression assays (Applied Biosystems) and correlated with miRNA expression in order to select candidate genes for further target validation by Renilla-Luciferase assay and Western Blot. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in K562 together with 1μM of modified psicheck2 vector containing the 3’UTR region to be validated. Renilla luciferase levels were measured at 24h post-tranfection. The miRNAs identified in the Renilla-Luciferase assay were further confirmed by Western Blot. 100nM of pre-miR/pre-miR negative control were transfected in the HL60 cell line and protein levels were measured at 24h post-transfection. Anti-SOCS1 (ab119954) and Anti-SOCS3 (ab62584) primary antibody (abcam) were used. Results Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic miRNA signature (p

Description: The complex interplay between bone marrow-derived mesenchymal stromal cells (BM-MSC) and neoplastic hematopoietic cells is involved in the progression of myeloproliferative neoplastic (MPN) diseases. Extracellular vesicles (EV) have emerged as a complex cell-to-cell communication system within the neoplastic microenvironment. EV are able to reprogram recipient cells by transferring proteins, mRNA and microRNA from their cell of origin. We aimed to analyze the microRNA content of EV obtained from MPN BM-MSC, as well as the changes induced when these EV are incorporated into hematopoietic progenitor cells (HPC). EV were isolated from BM-MSC of MPN patients (n=22) and healthy donors (HD) (n=19) by ultracentrifugation. Characterization of EV by transmission electron microscopy (TEM), immunoblot, multiparametric flow cytometry (MFC) and NanoSight analysis revealed vesicles with a typical bilayer-membrane characteristic morphology with a size inferior to 500 nm, which were positive for various EV markers as CD63 and CD81, and for MSC markers as CD73, CD90 and CD44 (Figure 1). MicroRNA profiling by 384-well microfluidic cards (TaqMan® MicroRNA Array A) showed an overall increase in the microRNA expression in the MPN-MSC-derived EV, when compared to EV from donor MSC. Using RT-PCR, we observed that miR-155 was selectively enriched in EV released by MPN-MSC. An overexpression of this microRNA was observed in EV (p=0.032), while a downregulation was observed in BM-MSC (p=0.0078) (Figure 2). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+ cells obtained by immunomagnetic selection) were co-cultured with Vybrant Dil-labeled EV. For functional studies apoptosis and clonogenic assays (CFU-GM) were performed. We observed an increase in CD34+ cell viability after incorporating EV from BM-MSC (HD and MPN). Moreover, an increase (p=0.04) in miR-155 expression was observed when HD HPC incorporated EV from MPN-MSC. When neoplastic CD34+ cells incorporated the EV derived from MPN-MSC an increase of CFU-GM number was also observed. We suggest that EV released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and hematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases. Funding: GRS 1034/A/14 (C. Sanidad, JCYL) and FCT (SFRH/BD/86451/2012) Figure 1 EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 1. EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 2 Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Figure 2. Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Disclosures Sánchez-Guijo: Bristol-Myers-Squib: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction Mesenchymal stem cells (MSC) are a key component of the hematopoietic niche. In the allogeneic hematopoietic stem cell transplantation (allo-HSCT) setting, the bone marrow stroma, and thus, their MSC remain of host origin. In a preliminary study we observed that, some patients at the early post allo-HSCT period, presented senescent bone marrow (BM) MSC, a finding that has not been previously described nor studied. The aims of our current study were: a) To multiparametrically characterize BM MSC at the early post allo-HSCT period (day +21). b) To confirm senescence of MSC and correlate with clinical and biological parameters (including biomarkers). c) To compare these cells with those from healthy donors. Methods We obtained BM samples on the day +21 post allo-HSCT from 136 patients. MSC were isolated, ex-vivo expanded and characterized, according to the criteria of the International Society for Cellular Therapy. We also obtained samples from peripheral blood at same day +21, for the study of biomarkers, which were analyzed by Luminex technique. The data were correlated with information from complete blood counts (CBC), and the morphological study of the bone marrow the same day. MSC from healthy donors were used as control. Results Patient baseline and transplant related characteristics are detailed in table 1. MSC were expanded ex-vivo showing normal growth, which were cryopreserved in passage 3 in the 33% (n=45/136) of the patients (Group-MSC-N). On the other hand, the remaining 67% (n=91/136), MSC showed premature signs of senescence, thus, not reaching to passage 1 (Group-MSC-S). Full BM chimerism on day+21 was seen in both groups (p=0.03). Concerning acute graft versus-host disease (aGVHD), in the Group-MSC-S, the incidence was 73% compared to 44% in the Group-MSC-N (p=0.001). The median time of the onset of aGVHD was 43 and 37 days respectively (p=0.04). The majority of cases presented with grades I-II in both groups. Plasma levels of biomarkers showed increased levels of Fas Ligand in the Group-MSC-S (p=0.009). There were no statistically significant differences regarding mortality nor relapse rates. Conclusions Bone marrow Mesenchymal stem cells may be severely damaged in some allo-HSCT recipients early after transplantation (day+21). This fact strongly correlates with the risk of development of acute GVHD. Disclosures Díez-Campelo: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Introduction. Genetic studies in patients with Ph-negative myeloproliferative neoplasms (MPNs) are essential to establish a correct diagnosis and to optimize their management. Recently, it has been demonstrated that it is possible to detect molecular alterations present in solid tumors and hematologic neoplasms by the analysis of circulating tumor DNA in plasma samples, which is known as liquid biopsy. It has been reported that most of the circulating cell-free DNA (cfDNA) has its origin in immature hematopoietic and bone marrow cells; however, there is limited information about liquid biopsy applications in MPNs. Objective. To analyze the molecular profile of circulating tumor DNA in patients with MPNs. Patients and methods. Peripheral blood samples from 75 patients with MPNs were collected at the time of diagnosis: 21 polycythemia vera (PV), 42 essential thrombocythemia (ET), 10 primary myelofibrosis (PMF) and two non-classifiable MPNs. Cellular DNA was extracted from the granulocytic fraction isolated by density gradient centrifugation and cfDNA was obtained from 1-3ml of plasma (MagMAX Cell-Free DNA Isolation Kit, Thermo Fisher Scientific). cfDNA purity was ascertained by capillary electrophoresis (4200 TapeStation system, Agilent). Molecular characterization was performed in paired samples of granulocytes DNA and cfDNA by next generation sequencing (NGS). Libraries were prepared using a custom panel that covered the whole codifying region of 25 myeloid-associated genes (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology (Miseq, Nextseq) with a 3000x minimum coverage. Results. The amount of total cfDNA/mL in plasma was significantly higher in PMF (mean 97 ng/ml) than in PV and ET (mean 18 and 23g/ml, respectively) (p = 0.003, Kruskal-Wallis). Overall, 144 mutations in driver (JAK2, CALR, MPL) and non-driver genes were detected in the granulocytic fraction with similar frequencies to what has been described for PV, ET and PMF. The most frequently mutated non-driver genes where ASXL1 (18.7%), TET2 (17.3%), DNMT3A (6.7%), SRSF2 (6.7%) and IDH2 (5.3%). Sequencing of cfDNA showed a total of 146 mutations. All mutations detected in the granulocytic fraction were also detected in the paired cfDNA sample (100% concordance); two additional mutations in MPL and ASXL1 were detected in plasma in one case. The median variant allele frequency (VAF) present in cfDNA was 29% (range 0.86 - 91.73%), which is far superior to what has been described in solid neoplasms or lymphomas (median 0.41%, range 0.03% - 97.6%). A strong correlation was observed between the VAFs of granulocytic DNA and cfDNA (r = 0.875, p 〈 0.001, Spearman) (Figure 1). The mutation VAFs detected in cfDNA were significantly higher than VAFs detected in granulocytes (p 〈 0.001, Wilcoxon). In particular, MPL mutations presented 2.5 higher VAF in cfDNA than in granulocytes (p = 0.018, Wilcoxon). This finding was confirmed and quantified by digital PCR. Interestingly, in one PMF patient the p.W515L MPL driver mutation was originally only detectable by NGS in cfDNA, but not in granulocytes. This mutation was identified by ultra-sensitive digital PCR in both cfDNA (VAF 2.30%) and granulocytes (VAF 0.16%). Conclusions. The analysis of circulating tumor DNA allows the characterization of the molecular abnormalities of patients with Ph negative myeloproliferative neoplasms. The sensitivity for mutation detection in driver and non-driver genes was equal or even superior to that obtained when studying the isolated granulocytic population. Disclosures Salar: Roche: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy. Besses:Gilead: Research Funding. Bellosillo:TermoFisher Scientific: Consultancy, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Description: BACKGROUND Current treatment choices are based on generalized outcome data from clinical trials, but not all MM patients respond the same and a considerable percentage of them do not achieve a desirable treatment endpoint. Contradictory results based on the rate of descent of the monoclonal component (M-component) to predict long-term outcome have been published, but no to identify patients with insufficient response to a therapeutic regimen. The ability to identify treatment early on resistance could accelerate the introduction of another (and more effective) line of therapy. AIM Develop and validate a rule based on the rate of descent of the M-component, to predict the probability of disease resistance to reach a complete remission (CR) in MM patients. METHODS Three studies were conducted: exploratory, confirmatory, and clinical validation. A total of 87 patients treated between July 2014 and September 2018, were included for the first two. Patients who were unable to complete the planned treatment due to toxicity or comorbidities, and those treated for palliative purposes only, were excluded. A therapeutic regimen was considered to be effective if CR was achieved with it. Conversely, a therapeutic regimen was considered to be ineffective if disease progression was observed during treatment, or if CR was not achieved with it. The percentage of the daily decrease in the M-component achieved by each treatment cycle is calculated dividing the percentage of the decrease during the cycle by the number of days elapsed (n.nn% /day). Timing between measurement of the M-component do not differed more than 15% from the scheduled cycle time. In the exploratory study using the receiver operating characteristic (ROC) curve through SPSS v24, the ability to discriminate between effective and ineffective therapeutic regimen was investigated in 99 cycles, to identify the optimal cutoff, and to desing a rule; followed by a confirmatory study of the rule in 52 cycles different from those of the first study. A third clinical validation study was carried out with 62 patients, 31 with treatment guided by response speed rule (RSR-guided) and 31 not (unguided). RESULTS In the exploratory study it was observed that the area under the ROC curve was 0.971 (CI 95%: 0.93 - 1.00) (p 1.40% /day in a first cycle it does not indicate efficacy. In the confirmatory study it was observed for the RSR a sensitivity 100% (CI 95%: 92 - 100), specificity 100% (CI 95%: 66 - 100), reliability 100% (CI 95%: 93 - 100). In the clinical validation study the most common treatments in 1st, 2nd, and 3rd line were VBCMP/VBAD, VCD, and KRd for candidates, and VCD, Rd, DRd or KRd in non-candidates respectively. No significant differences were observed in the type of treatment used between RSR-guided and unguided patients. The median (months) to reach the CR in the RSR-guided patients was lower (8.5 vs 12.1 months; p = 0.003). RS-guided patients need fewer cycles to achieve CR (5.29 vs. 10.84; p = 0.002). The CR rate at 18 months was 94.3% (CI 95%: 84.1 - 100) and 74.2% (CI 95%: 58.4 - 90.0) for RSR-guided and unguided patients respectively, although in unguided patients the rate rose to 96.8% (CI 95%: 90.4 - 100.0) continuing treatment until 30 months. No significant differences were found in the number of lines for CR (1.96 vs 2.42; p = 0.054). CONCLUSIONS The Response Speed Rule (RSR), defined as a speed of M-component descent of ≤ 1.40% /day in two successive cycles, predicted with great accuracy the current ineffectiveness of a therapeutic regimen to deliver CR. During the clinical validation of this cutoff, it was shown that CR is achieved in less time and with fewer cycles using RSR. This is a simple metric that can be used broadly and accelerate the introduction of another (and more effective) line of therapy. Figure Disclosures No relevant conflicts of interest to declare.