ALBERT

Description: Background: After long-term treatment with tyrosine kinase inhibitors (TKIs), an important proportion of patients with chronic myeloid leukemia (CML) achieve and maintain a deep molecular response (DMR) that allows them to stop treatment indefinitely. However, approximately 50% of these patients on treatment-free remission (TFR) experience relapse by undetermined reasons. It has been described that TKIs may induce a potent antileukemic response that conditions the outcome of the discontinuation. Our objective was to analyze different immune parameters that could be used as biomarkers of safer treatment discontinuation. Objectives: To determine the modulation of immune biomarkers that could be related to current treatment with TKIs on patients with CML ("On TKI") or to successful TFR in patients that maintain DMR after treatment withdrawal ("Off TKI") or to relapse after TFR on patients that lost DMR. Materials & methods: We analyzed by flow cytometry the peripheral blood mononuclear cells (PBMCs) from 45 patients with CML "On TKI" for at least 9 months (imatinib (11), nilotinib (9), dasatinib (20) or bosutinib (5)), 17 patients "Off TKI" for at least 7 months who kept DMR at the moment of sampling (last TKI before TFR: imatinib (7), nilotinib (6), dasatinib(4)), 7 patients "Off TKI" for at least 1 year and 4 months who relapsed during TFR (samples from 3 patients were taken previous to TKI reintroduction (on relapse); samples from 4 patients were taken after they restarted treatment with TKIs ("On TKI" relapse), 4 patients with recent diagnosis of CML still untreated, and 20 healthy donors as basal control. Results: 1) Patients characteristics are shown in table 1. 2) Treatment with TKIs induced an increase of 8.6±1.2% ((p4%; CD86+

Description: Introduction: The diagnostic criteria for polycythemia vera (PV) has recently been updated by the World Health Organization (WHO). The criterion for erythrocytosis has been modified downwards: hemoglobin (hb)〉 16.5 g/dL or hematocrit (hto)〉 49% in men and hb〉 16 g/dL or hto〉 48% in women. This reduction increases the potential number of patients that would be test for JAK2 V617F mutation if PV is suspected. The V617F mutation in the JAK2 gene is present in 95% of cases of PV. It is estimated that the prevalence of this mutation in the general population is around 0.2%. Our aims are to determine the prevalence of JAK2 V617F in individuals with erythrocytosis according to WHO2016 criteria and to find prognostic factors that could help to identify patients with PV. Methods: We prospectively studied all hemograms performed in our laboratory during 7 nonconsecutive days. Variables studied were hb, hto, leukocytes, neutrophils, platelets, MCV, MCH, MCHC and RDW. JAK2 V617F mutation was studied in all males that had hb〉 16.5 g/dl or hto〉 49% or females that had hb〉 16 g/dl or hto〉 48%. JAK2 V617F mutation was studied by PCR assay in which an amplification control fragment and the JAK2 mutant allele were simultaneously amplified. All positive samples were confirmed by quantitative real-time PCR in a reference laboratory. Positive results were considered when the JAK2 V617F allele ratio was ≥ 0.7. The variables collected were correlated with the result of the JAK2 test in a univariate way. The T-Student test was used for the quantitative variables and the Chi-square test for the categorical variables. For the cell count variables, the Mann-Whitney U test was used. Results: A total of 15366 HG were analyzed. 1271 (8.3%) met the inclusion criteria for erythrocytosis. JAK2 V617F was performed on 1001 samples (270 samples were not suitable for the PCR assay due to low quality). Twelve samples (1.2%) were positive for JAK2 V617F mutation. However, 5 samples were excluded due to a known diagnosis of myeloproliferative neoplasm. Therefore, finally prevalence of JAK2 V617 mutation in 996 patients that met WHO erythrocytosis criteria was 0.8% (8/996). Medians for all parameter studied for each group are shown in table 1. In order to find out parameters that could increase the incidence probabilities to identify patients with JAK2 V617F we performed an univariate analysis of the variables included, according to JAK2 mutational status. We found that patients with JAK2 V617F had higher levels of leukocytes, neutrophils, platelets and RDW than patients with negative JAK2 (p

Description: Background: Bone marrow (BM) examination is essential in the staging of diffuse large B-cell lymphoma (DLBCL). The assessment of BM involvement includes both histology (gold-standard) and flow cytometry (FC), but few studies have compared BM biopsy (BMB) histologic findings with results of FC analysis of BM aspirate. Discordance between both techniques generates debate about the staging and the prognostic significance in these cases. Methods: We performed a retrospective single-center analysis of patients with DLBCL, not otherwise specified (NOS) diagnosed during a 4-year period (2014-2017). Patients were divided in three groups according to BM findings of BMB and FC at diagnosis. Standard FC was performed by 4-color flow panel until 2016 and by 8-color FC since then. We described main characteristics of each group at diagnosis and analyzed survival outcomes. We applied means of descriptive statistics and Pearson's chi-squared test, and analyzed survival outcomes according to Kaplan-Meier, using Cox regression for comparisons. Results: We analyzed 59 cases, which were divided in three groups: 40 cases (67.8%) presented both negative histology and FC (BMB-/FC-), 10 (16.9%) showed BM involvement using both histology and FC (BMB+/FC+) and 9 cases (15.3%) presented discordant results, all of them with negative histology and positive FC (BMB-/FC+). Clinical and biological characteristics of each group at diagnosis are presented in Table 1. Median infiltration by FC analysis of the BMB-/FC+ group was 0.8% (0.1-2.9) and 3/9 patients presented discordant immunophenotype of lymphoma cells between BM and node biopsy. If we considered BM infiltration as positive in all BMB-/FC+ cases, 4/9 (6.8% of all patients) would be upstaged. First-line treatment was homogeneous in all patients. With a median observation time of 18 months, progression-free survival (PFS) after 2 years was 67%, 22% and 22% with BMB-/FC-, BMB-/FC+ and BMB+/FC+, respectively (Figure 1A), with a multivariate hazard ratio (HR) of 1.9 (95% CI 1.2-2.9, p=0.004) and an univariate HR for FC+ (BMB-/FC+ and BMB+/FC+) vs FC- (BMB-/FC-) of 3.3 (95% CI 1.5-7.3, p=0.003). Two-year overall survival (OS) was 68%, 41% and 33% with BMB-/FC-, BMB-/FC+ and BMB+/FC+, respectively (Figure 1B); multivariate HR was 1.6 (95% CI 1.1-2.6, p=0.042) and univariate HR for FC+ vs FC- was 2.5 (95% CI 1.1-5.9, p=0.035). We found no significant difference between BMB-/FC+ and BMB+/FC+ in survival outcomes. Conclusions: In our series, the group with discordant BM infiltration (BMB-/FC+) presented worsen survival outcomes than BMB-/FC-. Such results should be validated in prospective studies because published series are retrospective and not focused specifically on DLBCL. BM infiltration detected by FC analysis but not by BMB could be considered as extranodal involvement at DLBCL NOS diagnosis. Disclosures García Gutiérrez: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding, Speakers Bureau.

Description: INTRODUCTION: The 2016 update of the World Health Organization (WHO) classification criteria for Polycythemia Vera (PV) included a decrease of the hemoglobin threshold. This modification increases exponentially the individuals with potential PV. International groups have proposed diagnostic algorithms for the screening of individuals with potential PV. However, these algorithms are not based on the prevalence of JAK2V617F in this population, and to our concern has not been prospectively validated. Our aim is to develop and validate an algorithm based on previously reported prevalence of JAK2V617F in individuals with potential PV in our environment. METHODS: This study was designed in two phases: a first phase for the development of the algorithm and a second phase for the validation of the algorithm. Phase 1: from previous data collected from our group of JAK2 V617F prevalence in individuals with erythrocytosis according to WHO criteria we performed an area under the curve (ROC) analysis with optimal cutoff point in order to maximize sensibility and specificity of the collected variables. The thresholds for platelets and neutrophils were selected for being clinically relevant and with an AUC 〉0.8. With these two variables and their optimal cutoff point, a sequential 2-step-algorithm was designed (figure 1). Phase 2: for the validation of the algorithm, hemograms from outpatient individuals were selected, and the "2-step-algorithm" was applied. Therefore, if samples met the criteria for step 1 (hemoglobin 〉 16.5 g/dl for men or 16 g/dl for women or hematocrit 〉49 % for men or 48 % for women) then, the step 2 was applied (platelets 〉 248x103/ml or neutrophils〉5.98x103/ml). Finally, all samples that passed the 2-step-algorithm, were tested for JAK2 V617F. With the chosen samples a JAK2 V617F qualitative PCR with a sensibility of at least 〉0.1% was performed. The positive results were validated and quantified in our reference laboratory, and those with an allele burden 〉1% were considered positive. RESULTS: A total of 15298 hemograms were initially selected. Of those, 1595 (10.4%) met the erythrocytosis criterion (step 1). 581 (36%) of these met the step 2 criteria and the JAK2 V617F PCR was performed in 501 samples. A total of 7 positive cases (1.4%) were found, none of which was previously diagnosed of myeloproliferative neoplasm. The median of hemoglobin and hematocrit was 16.3 g/dl and 50.3% respectively. Of the selected samples, 2 of them met the platelet criterion, 4 of them the neutrophils criterion and 1 of them met both. After reviewing clinical records, 43.8% of them were found to have cardiovascular events history history: 1 patient had atherosclerosis of the lower limbs, 1 had acute coronary syndrome and 1 had an ischemic stroke. Characteristics of JAK2V617F individuals are detailed in table 1. Comparing to step 1, the addition of step 2 increases the efficiency of the algorithm 1.75 times. In order to know the prevalence of JAK2 V617F clonal hematopoiesis in our environment, 300 unselected samples were tested. We found 1 positive result. However, after reviewing clinical records, this sample was found to belong to a patient with known myeloproliferative neoplasm. Thus, JAK2 V617F prevalence in our environment was considered

Description: There is a demand to understand B-cell lymphoma pathogenesis better, to identify new markers, and to define multiple lymphoproliferative disorders more accurately. MicroRNAs (miRNAs) are regulators of protein translation, comprising a group of more than 1500 short noncoding single-strand RNA molecules of approximately 22 nucleotides in length. They are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum. Expression of individual miRNAs and miRNA signatures allows specific cell-differentiation stages to be identified, and is a powerful diagnostic and prognostic method. Here we review what is known about the pathogenic relevance of miRNAs, and use of miRNAs for the diagnosis and prognosis of B-cell lymphomas. Most of the published data concern chronic lymphocytic lymphoma and diffuse large B-cell lymphoma, and implicate miRNAs in the pathogenesis of these diseases. They identify miRNAs that could be used for diagnosis, prognosis, or prediction of response to specific therapies.

Description: Introduction: Cytogenetic and molecular landscape at diagnosis and the depth of response to induction therapy are the most powerful prognostic tools available in patients with Acute Myeloid Leukemia (AML). Among the tests to measure Minimal Residual Disease (MRD), one of the most commonly used is flow cytometry. Nowadays, there is no consensus about optimal time for measurement and the threshold above which has greater prognostic value in AML, as well as its involvement in therapeutic management. Material and methods: We performed a single-center retrospective analysis of 62 patients diagnosed with AML between 2015 and 2019 that reached complete remission after first induction. Patients were stratified according to the European Leukemia Net (ELN) risk classification. MRD measurement was made in bone marrow samples with an 8-color flow cytometer (sensitivity 10-5), cut-off 0.1%. We divided our cohort in two groups according to MRD after induction: negative MRD (MRD-) and positive MRD (MRD+). The baseline characteristics of each group were compared using the Chi2 test. The survival analysis was performed through Kaplan-Meier method and the risk was calculated with Cox regression. The Overall Survival (OS) was defined as the period of time from diagnosis to death and the leukemia-free state (LFS) as the period of time from CR to either relapse or death. Statistical analysis were carried out using SPSS version 19.0. P 0.1% after first induction by flow cytometry has shown in our population the identification of a AML subgroup of high risk, specially relevant in the intermediate risk group of ELN classification. MRD+ leads to higher risk of relapse, and these patients benefit from more aggressive therapeutic strategies, including allogeneic HSCT. However, MRD+ group has more risk of MRD persistence prior to HSCT, the last being a knowing factor of relapse after allogeneic HSCT, what would justify more aggressive strategies after HSCT in these patients. Disclosures Piris-Villaespesa: Novartis: Honoraria, Other: Advisory Boards. García Gutiérrez:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

Description: Introduction: Patients with Acute Myeloid Leukemia (AML) and positive Minimal Residual Disease (MRD) prior to allogeneic transplant are currently considered to be a group at high risk of relapse. Multiparameter flow cytometry is a standard technique to measure MRD, and generally we use a 0.1% threshold for positivity. The clinical significance of those patients with an MRD levels 〉0% but 0% but 0% but 0.1% is especially relevant in the IR and AR groups of the European LeukemiaNet risk classification. In the AR subgroup even any detectable level of positive MRD could identify patients with unfavorable post-transplant OS and RFS outcomes. We must establish post-transplant strategies in these patients to improve survival. Disclosures Garcia-Gutiérrez: Pfizer: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Incyte: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Bristol-Myers Squibb: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding; Novartis: Consultancy, Other: Travel, Accommodation, Expenses, Research Funding.

Description: Chronic Lymphocytic Lymphoma (CLL) is a heterogeneous disease in which many important factors for its prognosis have been identified. The normal functioning of p53 is one of the most critical barriers against cancer; therefore, if it has a deletion and/or mutation, it is a robust biomarker for the therapeutic response in CLL. The possibility was raised that some germline single - nucleotide polymorphisms of TP53 in healthy populations may also affect p53 function. One of the most studied polymorphisms of the TP53 gene is codon 72 in exon 4, a CGC to CCC transition (R72P), due to its potential effect on cancer risk. As with many types of cancer, its association with a worse prognosis in CLL is unclear. We analyzed the relationship of the genotypes of the TP53 codon 72 polymorphism in a large cohort of patients with CLL, to demonstrate the association of codon 72 with the evolution of the disease. Using the IDIPHIM patient database, 558 patients with a diagnosis of CLL were included, with clinical data, immunophenotype studies, FISH, IgHV, and karyotype, at the time of diagnosis and during follow-up. The TP53 codon 72 Arg/Arg, Arg/Pro, and Pro/Pro genotypes were analyzed using RT-PCR and Sanger sequencing techniques. After analyzing the sample of patients, 321 patients with the Arg/Arg genotype, 202 with the Arg/Pro genotype, and 35 with the Pro/Pro genotype were found. In the comparative analysis of the three groups, the patients with the Pro/Pro genotype had a higher number of patients in advanced stages B and C. The latter had a significant association with Binet staging (p = 0.002) compared to the other groups. Likewise, patients with the Pro/Pro genotype had a higher incidence of Richter transformation, whose association was significant (p = 0.013). Also, the patients who were within the Pro/Pro genotype group showed a significant association (p = 0.030) with the Time to the first treatment (TFT), also observing that the group of patients with the Arg/Pro genotype had a more considerable time until your first treatment. 19.7% (110/558) had a second neoplasm, having a significantly higher association with the homozygous groups (Arg/Arg and Pro/Pro) than with the Arg/Pro group, which on the contrary, had fewer second neoplasms (p = 0.016) (see Table 1). Regarding the type of tumors, we found 14.5% of the bladder, 14.5% of the skin, 14.5% of the colon, 13.6% of the prostate, and 12.7% of the lung. No associations were found between Codon 72 and CD38+, ZAP70+, complex karyotype, IgHV, NOTCH-1, del 11q, 12+, p53, del 13q, TP53 mutation. Still, when forming a group between the p53 deletion and TP53 mutation, if significant differences were found (p = 0.023), Pro / Pro group had the highest percentage. The overall survival was 156.32 months (139.92 - 172.72), showing that patients with the Arg/Pro genotype live 40 months more significantly than the other groups (p = 0.028) (see Figure 1). Finally, in the multivariate analysis, age, complex karyotype, 11q deletion, p53 deletion, unmutated IgHV, and Pro/Pro genotype at codon 72 were identified as independent variables associated with an increased risk of death (see Table 2). In conclusion, the Pro/Pro genotype of TP53 Codon 72 has a potential role in the progression and the higher mortality of patients with CLL. Conversely, the Arg / Pro genotype was associated with a lower incidence of second malignancies and higher overall survival. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Bone marrow (BM) examination at diffuse large B-cell lymphoma (DLBCL) diagnosis is essential in staging and has prognostic implication. According to the last recommendations (Cheson, et al. JCO 2014) BM biopsy (BMB) is only needed for those patients with a negative BM infiltration by positron emission tomography (PET) for whom identification of occult discordant histology - whose biological and prognostic implications are unknown - is clinically important. Despite its greater sensitivity, flow cytometry (FC) is secondary in BM assessment. Our aim was to compare PET, BMB and FC in the study of BM infiltration at DLBCL diagnosis. Methods Retrospective study in two hospitals in Madrid of patients diagnosed with DLBCL NOS from January 2014 to January 2020. A complete BM assessment including PET, BMB and FC was performed in all included patients. The hole series (n=102) was analysed separately according with BM infiltration by each technique, differences between biological, clinical and laboratory variables were studied applying descriptive statistics tests when appropriate (Fisher's exact test, chi-square test, Student's T test and Mann-Witney U test). Event-free survival (EFS) and overall survival (OS) were analysed with Kaplan-Meier estimator according to BM infiltration positive vs negative for each technique, using Cox proportional-hazard model for comparisons. Results BM infiltration was not assessed in 2 patients by BMB and in 4 patients by FC due to technical reasons. Analysing separately the series according to BM infiltration by each technique (PET+ 25 vs PET- 77, BMB+ 15 vs BMB- 85 and FC+ 16 vs FC- 82) the basal characteristics were comparable between groups, except from extranodal sites ≥2, Ann Arbor III-IV and elevated LDH level in groups with positive BM infiltration. The variables associated with worsen EFS in univariate analysis were age ≥80 years (HR 2.31; CI 95% 1.1-5.1), cell-of-origin (COO) non-GCB (HR 2.33; CI 95% 1.1-4.9), extranodal sites ≥2 (HR 2.39; CI 95% 1.2-4.7), Ann Arbor III-IV (HR 4.55; CI 95% 2.0-10.5), and elevated LDH level (HR 2.32; CI 95% 1.1-4.7). The variables statistically related with worsen OS were COO non-GCB (HR 2.91; CI 95% 1.2-6.8), extranodal sites ≥2 (HR 2.61; CI 95% 1.2-5.5), Ann Arbor III-IV (HR 5.97; CI 95% 2.1-17.3), elevated LDH level (HR 2.36; CI 95% 1.1-5.4), and elevated beta-2 microglobulin level (HR 3.82; CI 95% 1.1-12.9). Double-expressor phenotype did not demonstrated association with EFS or OS. Median infiltration by FC analysis was 0.9% (0.05-27). The series distribution among BM infiltration is presented in Figure 1. Median follow-up was 25 months (0.3-90). Survival curves according to BM infiltration by PET, BMB and FC are presented in Figure 2. Univariate analysis among the type of infiltration by each technique are presented in Table 1. Multivariate analysis included age ≥80 years, COO non-GCB, BM FC+ and IPI score 3-5; BM infiltration by FC demonstrated no association with EFS (HR 2.2; CI 95% 0.9-5.3) or OS (HR 2.5; CI 95% 0.9-6.5). Conclusions BM infiltration by PET at DLBCL NOS diagnosis has not survival implication, contrary to infiltration demonstrated by BMB or FC. Cases with positive infiltration by PET but negative by BMB and FC could be false positive in PET or false negative in BMB/FC. According to our results the patients with discordant lymphoproliferative disorder BM infiltration presented worse prognosis and FC is probably the most important technique in this regard. Disclosures No relevant conflicts of interest to declare.