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Supplementary Data from Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway via c-jun-NH2 Terminal Kinase Activation and PTEN Upregulation

CCR Translation for This Article from Induction of B-Chronic Lymphocytic Leukemia Cell Apoptosis by Arsenic Trioxide Involves Suppression of the Phosphoinositide 3-Kinase/Akt Survival Pathway via c-jun-NH2 Terminal Kinase Activation and PTEN Upregulation

Purpose: Arsenic trioxide (ATO) induces B-cell chronic lymphocytic leukemia (B-CLL) cell apoptosis in vitro. We sought to study the mechanism involved in this effect and whether ATO is suitable for combination therapies with protein kinase inhibitors.Experimental Design: B-CLL cells were isolated from the peripheral blood of 28 patients. Cell viability studies with ATO alone or in combination with kinase inhibitors were done by flow cytometry, Western blotting, and immunofluorescence analyses.Results: After 48 hours, 3 μmol/L ATO induced apoptosis (average 75%) in all B-CLL samples studied and with minimal effect on normal peripheral blood lymphocytes. Apoptosis entailed Akt and NF-κB inactivation, XIAP downregulation, and PTEN upregulation, thus implying inhibition of the phosphoinositide 3-kinase (PI3K) survival pathway. Indeed, the combination of ATO and PI3K inhibitors increased the apoptotic effect of either agent alone. ATO also induced c-jun-NH2 terminal kinase (JNK) activation, and this was crucial and required for subsequent apoptotic events, as inhibiting JNK activity by either gene silencing or specific inhibitors prevented Akt and NF-κB inactivation, caspase activation, and mitochondrial damage. Moreover, JNK activation was the earliest response to ATO, preceding and determining reactive oxygen species production.Conclusions: We identified the mechanism involved in ATO action on B-CLL cells and show that the combination of low doses of ATO and PI3K inhibitors efficiently induces B-CLL cell death. ATO may therefore constitute an efficient treatment for B-CLL, particularly in combined therapies. Clin Cancer Res; 16(17); 4382–91. ©2010 AACR.

Background: Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to COVID-19 related poor outcomes, including thrombosis and death, due to the advanced age, the presence of comorbidities, and the disease and treatment-related immune deficiency. In this retrospective multicenter study, conducted by ERIC, the European Research Initiative on CLL, we assessed the risk of thrombosis and bleeding in patients with CLL affected by severe COVID-19.Methods: The study included patients from 79 centers across 22 countries. Data collection was conducted between April and May 2021.Results: A total of 793 patients from 79 centers were included in the study with 593 being hospitalized (74.8%). Among these, 518 were defined as having severe COVID: 162 were admitted to the ICU while 356 received oxygen supplementation outside the ICU. Most patients (90%) were receiving thromboprophylaxis. During COVID-19 treatment, 8.8% developed a thromboembolic event, while 4.8% experienced bleeding. Thrombosis developed in 20.5% of patients who were not receiving thromboprophylaxis, but only in 8.1% of patients who were on thromboprophylaxis. Bleeding episodes were more frequent in patients receiving intermediate/therapeutic versus prophylactic doses of low-molecular-weight heparin (LWMH) (11.1% vs. 4.2%, respectively) and in elderly. In multivariate analysis, peak D-dimer level was a poor prognostic factor for thrombosis occurrence (OR=1.020, 95%CI 1.006‒1.033), while thromboprophylaxis use was protective (OR=0.194, 95%CI 0.061‒0.614). Age and LMWH intermediate/therapeutic dose administration were prognostic factors in multivariate model for bleeding (OR=1.055, 95%CI 1.013-1.103 and OR=2.490, 95%CI 1.044-5.935, respectively). Conclusions: Patients with CLL affected by severe COVID-19 are at a high risk of thrombosis if thromboprophylaxis is not used, but also at increased risk of bleeding under the LMWH intermediate/therapeutic dose administration.

Patients with chronic lymphocytic leukemia (CLL) may be more susceptible to Coronavirus disease 2019 (COVID-19) due to age, disease, and treatment-related immunosuppression. We aimed to assess risk factors of outcome and elucidate the impact of CLL-directed treatments on the course of COVID-19. We conducted a retrospective, international study, collectively including 941 patients with CLL and confirmed COVID-19. Data from the beginning of the pandemic until March 16, 2021, were collected from 91 centers. The risk factors of case fatality rate (CFR), disease severity, and overall survival (OS) were investigated. OS analysis was restricted to patients with severe COVID-19 (definition: hospitalization with need of oxygen or admission into an intensive care unit). CFR in patients with severe COVID-19 was 38.4%. OS was inferior for patients in all treatment categories compared to untreated (p

This is a randomized phase-2 trial aimed to compare consolidation vs. maintenance in untreated patients with follicular lymphoma (FL) responding to induction. 146 patients were enrolled from 25 Spanish institutions (ZAR2007; ClinicalTrials.gov #NCT00662948). Patients in PR or CR/CR[u] after R-CHOP were randomized 1:1 to

Progressió clínica; Evasió immune Progresión clínica; Evasión inmune Clinical progression; Immune evasion Background Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention. This work was supported by the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI17/00950, M.C., PI18/01392, P.A. and PI17/00943, F.B.) and co-financed by the European Regional Development Fund (ERDF) and Fundación Asociación Española Contra el Cáncer (M.C. and P.A.), Gilead Fellowships (GLD16/00144, GLD18/00047, F.B.) and Fundació la Marató de TV3 (201905–30-31 F.B). S.B. is the recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero. R.V-M. is supported by a Torres Quevedo fellowship from the Spanish Ministry of Science and Innovation (PTQ-16-08623). A.E-C. is funded by ISCIII/MINECO (PT17/0009/0019) which is co-funded by FEDER. M.C. holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-2018).

58 p.-8 fig.-6 tab. supl., We previously showed that MMP-9 contributes to CLL pathology by regulating cell survival and migration and that, when present at high levels, MMP-9 induces cell arrest. To further explore the latter function, we studied whether MMP-9 influences the gene-expression profile in CLL. Microarray analyses rendered 131 differentially expressed genes in MEC-1 cells stably transfected with MMP-9 (MMP-9-cells) versus cells transfected with empty vector (Mock-cells). Ten out of twelve selected genes were also differentially expressed in MEC-1 cells expressing the catalytically inactive MMP-9MutE mutant (MMP-9MutE-cells). Incubation of primary CLL cells with MMP-9 or MMP-9MutE also regulated gene and protein expression, including CD99, CD226, CD52, and CD274. Because CD99 is involved in leukocyte transendothelial migration, we selected CD99 for functional and mechanistic studies. The link between MMP-9 and CD99 was reinforced with MMP-9 gene silencing studies, which resulted in CD99 upregulation. CD99 gene silencing significantly reduced CLL cell adhesion, chemotaxis and transendothelial migration, while CD99 overexpression increased cell migration. Mechanistic analyses indicated that MMP-9 downregulated CD99 via binding to α4β1 integrin and subsequent inactivation of the Sp1 transcription factor. This MMP-9-induced mechanism is active in CLL lymphoid tissues, since CD99 expression and Sp1 phosphorylation was lower in bone marrow-derived CLL cells than in their peripheral blood counterparts. Our study establishes a new gene regulatory function for MMP-9 in CLL. It also identifies CD99 as an MMP-9 target and a novel contributor to CLL cell adhesion, migration and arrest. CD99 thus constitutes a new therapeutic target in CLL, complementary to MMP-9., This work was supported by Grant SAF2015-69180R and Red Temática de Investigación Cooperativa en Cáncer Grant RD12/0036/0061 from the Ministry of Economy and Competitivity (Spain) (to AGP); S2010/BMD-2314 (to AGP) from the Comunidad de Madrid/European Union; and by the Concerted Research Actions (KU Leuven C1 Grant C16/17/010) and the Research Foundation of Flanders (FWO-Vlaanderen, to EUB, PEVdS, and GO).

PURPOSE Among Bruton's tyrosine kinase inhibitors, acalabrutinib has greater selectivity than ibrutinib, which we hypothesized would improve continuous therapy tolerability. We conducted an open-label, randomized, noninferiority, phase III trial comparing acalabrutinib and ibrutinib in patients with chronic lymphocytic leukemia (CLL). METHODS Patients with previously treated CLL with centrally confirmed del(17)(p13.1) or del(11)(q22.3) were randomly assigned to oral acalabrutinib 100 mg twice daily or ibrutinib 420 mg once daily until progression or unacceptable toxicity. The primary end point was independent review committee–assessed noninferiority of progression-free survival (PFS). RESULTS Overall, 533 patients (acalabrutinib, n = 268; ibrutinib, n = 265) were randomly assigned. At the data cutoff, 124 (46.3%) acalabrutinib patients and 109 (41.1%) ibrutinib patients remained on treatment. After a median follow-up of 40.9 months, acalabrutinib was determined to be noninferior to ibrutinib with a median PFS of 38.4 months in both arms (95% CI acalabrutinib, 33.0 to 38.6 and ibrutinib, 33.0 to 41.6; hazard ratio: 1.00; 95% CI, 0.79 to 1.27). All-grade atrial fibrillation/atrial flutter incidence was significantly lower with acalabrutinib versus ibrutinib (9.4% v 16.0%; P = .02); among other selected secondary end points, grade 3 or higher infections (30.8% v 30.0%) and Richter transformations (3.8% v 4.9%) were comparable between groups and median overall survival was not reached in either arm (hazard ratio, 0.82; 95% CI, 0.59 to 1.15), with 63 (23.5%) deaths with acalabrutinib and 73 (27.5%) with ibrutinib. Treatment discontinuations because of adverse events occurred in 14.7% of acalabrutinib-treated patients and 21.3% of ibrutinib-treated patients. CONCLUSION In this first direct comparison of less versus more selective Bruton's tyrosine kinase inhibitors in CLL, acalabrutinib demonstrated noninferior PFS with fewer cardiovascular adverse events.

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment.

There is a Blood Commentary on this article in this issue., Key Points Both watch-and-wait and treated CLL patients have high mortality rates when admitted for COVID-19. Receiving a BTKi for CLL at COVID-19 diagnosis severe enough to require hospitalization did not influence case fatality rate in this study., Given advanced age, comorbidities, and immune dysfunction, chronic lymphocytic leukemia (CLL) patients may be at particularly high risk of infection and poor outcomes related to coronavirus disease 2019 (COVID-19). Robust analysis of outcomes for CLL patients, particularly examining effects of baseline characteristics and CLL-directed therapy, is critical to optimally manage CLL patients through this evolving pandemic. CLL patients diagnosed with symptomatic COVID-19 across 43 international centers (n = 198) were included. Hospital admission occurred in 90%. Median age at COVID-19 diagnosis was 70.5 years. Median Cumulative Illness Rating Scale score was 8 (range, 4-32). Thirty-nine percent were treatment naive (“watch and wait”), while 61% had received ≥1 CLL-directed therapy (median, 2; range, 1-8). Ninety patients (45%) were receiving active CLL therapy at COVID-19 diagnosis, most commonly Bruton tyrosine kinase inhibitors (BTKi’s; n = 68/90 [76%]). At a median follow-up of 16 days, the overall case fatality rate was 33%, though 25% remain admitted. Watch-and-wait and treated cohorts had similar rates of admission (89% vs 90%), intensive care unit admission (35% vs 36%), intubation (33% vs 25%), and mortality (37% vs 32%). CLL-directed treatment with BTKi’s at COVID-19 diagnosis did not impact survival (case fatality rate, 34% vs 35%), though the BTKi was held during the COVID-19 course for most patients. These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Future epidemiologic studies are needed to assess severe acute respiratory syndrome coronavirus 2 infection risk, these data should be validated independently, and randomized studies of BTKi’s in COVID-19 are needed to provide definitive evidence of benefit., Visual Abstract

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, characterized by immunodeficiency. Hence, patients with CLL might be considered more susceptible to severe complications from COVID-19. We undertook this retrospective international multicenter study to characterize the course of COVID-19 in patients with CLL and identify potential predictors of outcome. Of 190 patients with CLL and confirmed COVID-19 diagnosed between 28/03/2020 and 22/05/2020, 151 (79%) presented with severe COVID-19 (need of oxygen and/or intensive care admission). Severe COVID-19 was associated with more advanced age (≥65 years) (odds ratio 3.72 [95% CI 1.79–7.71]). Only 60 patients (39.7%) with severe COVID-19 were receiving or had recent (≤12 months) treatment for CLL at the time of COVID-19 versus 30/39 (76.9%) patients with mild disease. Hospitalization rate for severe COVID-19 was lower (p < 0.05) for patients on ibrutinib versus those on other regimens or off treatment. Of 151 patients with severe disease, 55 (36.4%) succumbed versus only 1/38 (2.6%) with mild disease; age and comorbidities did not impact on mortality. In CLL, (1) COVID-19 severity increases with age; (2) antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect; (3) age and comorbidities did not impact on mortality, alluding to a relevant role of CLL and immunodeficiency.

Resumen La evaluacion de la enfermedad minima residual (EMR) es un objetivo importante en el tratamiento de la leucemia linfocitica cronica (LLC). Su obtencion es predictiva de una supervivencia libre de progresion (SLP) y una supervivencia global prolongadas y podria considerarse una variable subrogada de la SLP en el contexto del tratamiento con quimioinmunoterapia. La evaluacion de la EMR mediante citometria de flujo o tecnicas moleculares en la era de los nuevos inhibidores de BCR o Bcl-2 podria identificar la secuencia de tratamiento mas coste-efectiva y la duracion de la misma. Una aproximacion terapeutica guiada por el nivel de EMR tambien podria determinar que pacientes se beneficiarian de un tratamiento de consolidacion o de una finalizacion precoz del mismo. En esta revision discutimos los diferentes metodos de analisis de la EMR, que muestras deben ser analizadas, el papel futuro de la deteccion del ADN tumoral circulante y el papel potencial de la negatividad de la EMR en la practica clinica en la era moderna del tratamiento de la LLC.

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression.

Introduction: The BCL-2 inhibitor venetoclax (Ven) has been approved on monotherapy or combined with rituximab in relapsed/refractory CLL patients (pts) and combined with obinutuzumab in previously untreated CLL pts. However, evidence from clinical trials can be difficult to generalize to real-world patient populations. The VENARES study assesses the real-world use of Ven following approval to inform of subpopulations underrepresented in clinical trials. Methods: This is Spanish non-interventional retrospective, multicenter post-marketing observational study. The main objective was to evaluate the effectiveness of Ven in adult CLL pts by the overall response rate (ORR) at 9 months (mo) after the first Ven dose administration. Secondary objective was to evaluate the effectiveness for the Ven monotherapy and the Ven combined with rituximab subpopulations. Consecutive adult pts with diagnosis of CLL who have initiated Ven at least 9 mo before the inclusion in the study were included. Data of pts are retrospectively reviewed until the date of last follow-up or death. Results: 125 pts diagnosed with CLL and who met the eligibility criteria were analyzed. The median age was 72 years (67 - 77) with 76.8% being older than 65 years. Most patients were male (68.8%), had a concurrent disease (65.6%). ECOG PS was recorded in 76 pts: 40 pts (32%) had PS 0, 30 pts (24%) PS 1 and 6 pts (4.8%) PS 2. Pts had received a median of 4 prior lines of therapy (range 1-13 lines). At baseline, among the 92 pts with known Binet stage, 31 (33.7%) had stage C and 38 (41.3%) had stage B; bulky nodes ≥ 5 cm were present in 20 of 87 pts; 49 pts (39.2%) had an absolute lymphocyte count ≥ 25 x 10 9/L and 33 of 54 pts (61%) baseline beta-2 microglobulin value above of 3500 ng/mL. In total, 29 of 90 patients (32%) assessed had Cr 17p deletion, 28 of 86 patients (32%) tested had TP53 mutations, and 46 of 56 patients (82%) who were tested had unmutated immunoglobulin heavy-chain variable (IGHV) status. Ven was administered as monotherapy in 71 pts (57.6%), combined with rituximab in 36 pts (28.8%), combined with obinutuzumab in 5 pts (4%) and combined with other drugs in 13 pts (10.4%). 83 of 125 patients included were evaluable for the primary objective of the study: the ORR at 9 mo was 84.3% (70 patients): CR/CRi in 44 (53%) pts, PR/nPR in 26 pts (31.3%), SD in 9 pts (10.8%) and PD in 4 pts (4.8%). By treatment, in the evaluable patients, ORR at 9 months were 79.2% (38 of 48 patients) in the Ven monotherapy group, with 45% of CR/CRi, and 92.3% (24 of 26 patients) in the Ven combined with rituximab, with 61% CR/Cri. The median duration of PFS was not reached at the time of the analysis (1-June-2021). Kaplan-Meier estimates of the probability of PFS at 24 mo was 75.4% (95% CI, 58.2 - 86.3). Disease progression occurred in 21 pts. Assessment of minimal residual disease (MRD) was available for 32 patients (25.6%) on the basis of peripheral-blood samples, bone marrow or both. Best undetectable MRD was reached in fourteen patients (43.8%). uMRD was more common in pts treated with Ven combined with R (83.3%, 5 of 6 pts) than in pts treated with Ven monotherapy (33.3%, 7 of 21 pts). Adverse events (AEs) were reported during Ven therapy in all 125 patients, 93 of these pts reported AEs related to Ven. Related to Ven, 67 patients (53.6%) experienced at least one AE: 52 pts (41.6%) had neutropenia being grade 3 and 4 in 22 (42.3%) and 9 (17.3%) pts, respectively. 9 pts (7.2%) had febrile neutropenia. Thrombocytopenia and anemia were less common occurring in 5.6% and 2.4%, respectively. Tumor lysis syndrome (TLS) occurred in 4 of 125 pts during ramp-up (3 laboratory and 1 clinical), 2 of them were related to Ven both lab TLS. None of the pts discontinued therapy due to TLS. Richter transformation was observed in 6 pts (4.8%). Other common AEs was diarrhea (10.4%), but most cases were mild. Conclusions: Our first real-world data show that Ven monotherapy or combined with rituximab is effective in highly pre-treated CLL patients, ORR at 9 mo was 84.3% in all population and PFS estimate at 24 mo was 75.4% with similar outcomes to those in the pivotal clinical trials. The safety profile of Ven was consistent with prior experience of Ven in monotherapy or combined with rituximab and no new safety signals were detected. Disclosures Baltasar: Janssen, Abbvie: Consultancy. Terol: Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel; BMS: Consultancy; Roche: Consultancy; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Hospital Clinico Valencia: Current Employment. Moreno: Janssen, Abbvie: Research Funding; Abbvie, Janssen, AstraZeneca: Speakers Bureau; Abbvie, Janssen, AstraZeneca, Beigene: Membership on an entity's Board of Directors or advisory committees. Osorio: Janssen, Abbvie, Roche: Consultancy. De la Cruz: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Beigene: Membership on an entity's Board of Directors or advisory committees; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees; JANSSEN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirkin: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. de la Serna: AbbVie, AstraZeneca, Roche: Speakers Bureau; ABBVIE, ASTRAZENECA,ROCHE: Research Funding; AbbVie, AstraZeneca, Beigene, Gilead, GSK, Janssen, Jazzpharma, Novartis, Roche: Consultancy. Arguiñano: Takeda, Sanofi, Janssen, BMS-Celgene, Abbvie: Speakers Bureau; Takeda, Sanofi, Janssen, BMS-Celgene, Abbvie: Consultancy. Loscertales: Janssen, Abbvie, Roche, Gilead: Speakers Bureau; Janssen, Abbvie, Astra-Zeneca, Beigene, Roche, Gilead: Consultancy. García: Janssen, Roche, Gilead, Celgene: Consultancy; Janssen, AbbVie: Research Funding; Janssen, Roche, Gilead, AbbVie, Celgene: Other: medical meetings funding. Pérez Persona: BMS/Celgene: Consultancy, Other: Support for attending meetings and/or travel, Speakers Bureau; Amgen: Consultancy, Other: Support for attending meetings and/or travel, Speakers Bureau; Takeda: Speakers Bureau; AbbVie: Other: Support for attending meetings and/or travel, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; GSK: Consultancy; Incyte: Consultancy. Pérez-Encinas: Janssen: Consultancy. Caballero: Celgene, Janssen, Novartis, Abbvie: Speakers Bureau; Celgene, Janssen, Amgen: Consultancy. Ruiz-Zorrilla: Abbvie: Current Employment. Moreno: abbvie: Current Employment. Ferrà: Janssen, Roche, Gilead, Takeda, Abbvie: Consultancy; Janssen, Roche, Gilead, AbbVie: Other: medical meetings funding.

Context: Increased selectivity of the Bruton tyrosine kinase inhibitor (BTKi) acalabrutinib versus ibrutinib may improve tolerability. Objective: To compare acalabrutinib versus ibrutinib in patients with chronic lymphocytic leukemia (CLL). Design: Open-label, randomized, noninferiority, phase 3 trial. Patients: Previously treated CLL patients with del(17p) or del(11q) by central lab. Interventions: Oral acalabrutinib 100 mg twice daily (BID) or ibrutinib 420 mg once daily (QD) (stratified by del(17p) status, Eastern Cooperative Oncology Group performance status [2 vs ≤ 1], and number of prior therapies [1–3 vs ≥4]) until progression or unacceptable toxicity. Main Outcome Measures: Primary endpoint was progression-free survival (PFS), as assessed by independent review committee; secondary endpoints of all-grade atrial fibrillation (AF), grade ≥3 infection, Richter transformation, and overall survival (OS) were assessed in hierarchical order. Results: 533 patients (acalabrutinib, n=268; ibrutinib, n=265) were randomized (median age 66 years; median 2 prior therapies; del(17p) 45.2%; del(11q) 64.2%). At a median follow-up of 40.9 months (range 0.0–59.1), acalabrutinib was noninferior to ibrutinib with a median PFS of 38.4 months in both arms (HR: 1.00; 95% CI: 0.79–1.27). Acalabrutinib was statistically superior to ibrutinib in all-grade AF incidence (9.4% vs 16.0%; P=0.023). Among other secondary endpoints, incidences of grade ≥3 infection (acalabrutinib: 30.8%, ibrutinib: 30.0%) and Richter transformation (acalabrutinib: 3.8%, ibrutinib: 4.9%) were comparable between arms. Median OS was not reached in either arm (HR: 0.82; 95% CI: 0.59–1.15), with 63 (23.5%) deaths in the acalabrutinib arm and 73 (27.5%) in the ibrutinib arm. Among any-grade adverse events (AEs) in ≥20% of patients in either arm, acalabrutinib was associated with lower incidence of hypertension (9.4%, 23.2%), arthralgia (15.8%, 22.8%), and diarrhea (34.6%, 46.0%) but a higher incidence of headache (34.6%, 20.2%) and cough (28.9%, 21.3%). AEs led to treatment discontinuation in 14.7% of acalabrutinib- versus 21.3% of ibrutinib-treated patients. Any-grade AEs of clinical interest that were less frequent with acalabrutinib included cardiac (24.1% vs 30.0%), hypertension (above), and bleeding events (38.0% vs 51.3%). Conclusions: In this first head-to-head trial of BTKis in CLL, acalabrutinib demonstrated noninferior PFS with less cardiotoxicity and fewer discontinuations due to AEs versus ibrutinib.

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.

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.

Summary Otlertuzumab (TRU-016) is a humanized anti-CD37 protein therapeutic that triggers direct caspase-independent apoptosis of malignant B cells and induces antibody-dependent cell-mediated cytotoxicity. Patients with relapsed chronic lymphocytic leukaemia (CLL) received either otlertuzumab (20 mg/kg) weekly by IV infusion for two 28-day cycles then every 14 days for four 28-day cycles and IV bendamustine (70 mg/m2) on Days 1 and 2 of each cycle for up to six 28-day cycles or bendamustine alone. Thirty-two patients were treated with otlertuzumab and bendamustine and 33 with bendamustine alone. Overall response rate according to the International Workshop on Chronic Lymphocytic Leukaemia criteria was 69% in the otlertuzumab and bendamustine arm and 39% in the bendamustine alone arm (P = 0·025). Median progression-free survival (PFS) was 15·9 months in the otlertuzumab and bendamustine arm and 10·2 months in the bendamustine alone arm (P = 0·0192). There was a higher incidence of pyrexia (34% vs. 12%) and neutropenia (59% vs. 39%) with the combination but this did not result in a higher incidence of severe (grade 3/4) infections (13% vs. 27%). This combination significantly increased the response rate and prolonged the PFS over single agent bendamustine in patients with relapsed or refractory CLL.

19 p.-9 fig.-1 tab., CLL remains an incurable disease in spite of the many new compounds being tested. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the gene expression profile induced by ATO in CLL cells. ATO modulated many genes, largely involved in oxidative stress, being HMOX1 the most upregulated gene, also induced at the protein level. ATO also increased MMP-9, as we previously observed, both at the mRNA and protein level. Using specific inhibitors, qPCR analyses, and gene silencing approaches we demonstrate that upregulation of MMP-9 by ATO involved activation of the p38 MAPK/AP-1 signaling pathway. Moreover, gene silencing HMOX1 or inhibiting HMOX1 activity enhanced p38 MAPK phosphorylation and c-jun expression/activation, resulting in transcriptional upregulation of MMP-9. Overexpression of HMOX1 or enhancement of its activity, had the opposite effect. Cell viability analyses upon modulation of HMOX1 expression or activity demonstrated that HMOX1 had a proapoptotic role and enhanced the cytotoxic effect of ATO in CLL cells. We have therefore identified a new mechanism in which HMOX1 plays a central role in the response of CLL cells to ATO and in the regulation of the anti-apoptotic protein MMP-9. Thus, HMOX1 arises as a new therapeutic target in CLL and the combination of HMOX1 modulators with ATO may constitute an efficient therapeutic strategy in CLL., This work was supported by grants SAF2012-31613 and SAF2015-69180R (AGP) and RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0061 (AGP), from the Ministry of Economy and Competitiveness (MINECO), Spain; S2010/BMD-2314-Neoplasbim (AGP) from the Comunidad de Madrid/European Union; and a grant from the Fundación para la Investigación Biomédica Hospital Universitario Puerta de Hierro, Madrid (JAGM).

27 p.-3 fig.-2 tab., Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4β1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology., This work was supported by grants SAF2015-69180R from the Ministerio de Economía y Competitividad (Spain)/Fondo Europeo de Desarrollo Regional (FEDER), European Union; S2010/BMD-2314 from the Comunidad de Madrid/European Union (AGP.); and by the Concerted Research Actions (KU Leuven C1 Grant C16/17/010) and the Research Foundation of Flanders (FWO-Vlaanderen, to EUB, PEVdS, and GO).

5 p.-2 fig., In chronic lymphocytic leukemia (CLL), α4β1 integrin (the main α4 heterodimer in CLL) and vascular endothelial growth factor (VEGF) share several pathological properties, including involvement in cell migration and survival.Accordingly, elevated expression of α4 integrin (≥30% cells) constitutes an adverse prognostic marker and VEGF serum levels increase with CLL progression and could also have prognostic value. CLL cells express VEGF receptor 1 (VEGFR1), VEGFR2, and VEGFR3 receptors.VEGFR2 is the main signaling receptor in response to VEGF165 and its elevated expression correlates with CLL aggressivenes. Previous studies in endothelial cells have demonstrated interactions and functional cross talk between integrins (αvβ3, α9β1, α3β1, α5β1) and the VEGF/VEGFR2 system, including integrin-mediated adhesion to VEGF and VEGF-induced integrin activation.Moreover, shedding of syndecan-1 in endothelial cells induced coupling of α4β1 integrin and VEGFR2, VEGFR2 activation, and angiogenesis. It is not known whether α4β1 interacts with VEGF/VEGFR2 or/and contributes to VEGF functions in CLL and we have addressed this in the present study., This work was supported by grants SAF2015-69180R from the Ministry of Economy and Competitivity (Spain) and S2010/BMD-2314 from the Comunidad de Madrid/European Union (A.G.-P.).

Introduction. Despite the high proportion of prolonged remissions obtained with ibrutinib in patients with CLL, complete responses (CR) are rarely observed. For the purpose of increasing the deepness of response, ibrutinib has been tested in combination with other drugs that exert a different mechanism of action. Thus, monoclonal antibodies (mAbs) have been concomitantly combined with ibrutinib in untreated or R/R CLLs. Nonetheless, several data derived from both in vitro and clinical studies do not support a synergistic effect of the concomitant administration of ibrutinib with anti-CD20 mAbs. Herein, we present the preliminary results of a multi-center, non-randomized phase 2 study aimed to determine the efficacy and safety of the sequential treatment of CLL patients with ibrutinib followed, in those not attaining CR, by a consolidation phase with ofatumumab (GELLC-7, EudraCT number 2016-004937-26). Patients and methods. Patients aged ≥18 years, physically fit (CIRS score < 6) with treatment-naïve CLL were enrolled in this study. Patients received an induction phase consisting of 12 cycles (28-day) of ibrutinib in monotherapy at 420 mg once daily. Patients attaining a CR after this induction phase were kept on ibrutinib until progression. In contrast, patients not obtaining a CR also continued on ibrutinib but received a consolidation treatment with 7 doses of ofatumumab (300 mg D1 and 1000mg D8 of C13, 1000 mg D1 of C14-C18). The primary endpoint of the study was the CR rate assessed after 20 cycles of treatment (2 months after completing ofatumumab consolidation). Results. 84 patients with a median age 69 years (range 38-84 yrs), 71% male, were included in this study. At inclusion, 83.3% had Binet stage B/C, 61% unmutated IGHV status, and 19% high risk genetic aberrations (7.6% 17p deletion and/or TP53mut, and 11.4% 11q deletion). At the interim data cut-off (June 2019), 7 patients had discontinued the study (progression to Richter transformation, n=1; patient withdrawal, n=3; adverse events [AE], n=3, including one G5 AE), 5 of them during the first 12 cycles of treatment. Sixty-seven patients received the induction phase with 12 cycles of ibrutinib, whereas 22 patients completed 20 cycles of treatment and were evaluable for the primary endpoint of the study. After 12 cycles of ibrutinib, 3 patients (4.5%) were in CR, 54 patients (80.5%) in PR, 6 patients (9%) in PR with lymphocytosis, and 4 patients (6%) in SD. In 20 patients receiving the consolidation with ofatumumab an improvement in response was observed, with 8/20 patients (40%) attaining a CR (7 patients converted PR to CR, and one patient SD to CR), whereas the remaining 12 patients were classified as PR. Two patients that were already in CR at cycle 12 maintained the CR under ibrutinib monotherapy. MRD was undetectable in blood ( Grade ≥3 adverse events (AEs) were experienced by 26 patients (31%), whilst 22 serious AEs were observed in 16 patients (19%) (14 infections, 1 febrile neutropenia, 3 dyspnoea, 1 anemia, 1 edema/pleural effusion, 1 renal insufficiency, 1 squamous carcinoma). The most common G3/4 AEs were hematological toxicity (neutropenia [7.1%], anemia [4.5%], thrombocytopenia [2.4%]) and infections (8.5%). The Gr 5 AE consisted of a severe peripheral edema and pleural effusion leading to death. The great majority of SAEs (67%) and G3/4 AEs (66%) were observed during the first 12 cycles of treatment with ibrutinib monotherapy. Conclusions. The preliminary analysis of the GELLC7 trial showed that the addition of consolidation with ofatumumab after 12 cycles of prior treatment with ibrutinib was well tolerated and elicited a deeper response. These results support the potential role of a sequential therapeutic strategy in CLL, where the addition of a consolidation with mAbs in patients with low tumor burden might improve the quality of the response. Finally, more mature results will be further presented at the meeting. Disclosures Abrisqueta: Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. González-Barca:Kiowa: Consultancy; Celgene: Consultancy; Takeda: Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celtrion: Consultancy. Terol:Roche: Consultancy; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; Astra Zeneca: Consultancy; Gilead: Research Funding. Baltasar Tello:GILEAD: Honoraria; JANSSEN: Consultancy, Honoraria; ABBVIE: Honoraria; ROCHE: Honoraria. de la Serna:Roche, AbbVie, Gilead, Janssen, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche, AbbVie, Janssen, Gilead: Speakers Bureau. Ramirez Payer:GILEAD SCIENCES: Research Funding. Cordoba:Janssen: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Kyowa-Kirin: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Roche: Honoraria, Speakers Bureau; FUNDACION JIMENEZ DIAZ UNIVERSITY HOSPITAL: Employment; Celgene: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: 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; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Ibrutinib followed by Ofatumumab Consolidation

The prognostic impact of biallelic ATM abnormalities (ATM mutation and concurrent 11q deletion) remains unknown. We studied ATM, BIRC3, SF3B1, and NOTCH1 genes in 118 treatment-naïve CLL patients at diagnosis. Patients with biallelic ATM alteration had a similar time to first treatment (TTFT) and shorter overall survival (OS) compared with patients with isolated 11q deletion and shorter TTFT and OS when compared to patients with wild-type ATM. Furthermore, biallelic ATM alteration (HR: 6.4; p ≤ 0.007) was significantly associated with an increased risk of death similar to p53 deletion (HR: 6.1; p ≤ 0.004), superior to 11q deletion alone (HR: 2.8; p ≤ 0.022) and independent of other significant parameters such as age, advanced clinical stage, and complex karyotype. Our results suggest the identification of ATM mutations in CLL patients with 11q deletion at diagnosis is clinically relevant and predicts disease progression, poor response to the treatment, and reduced OS independent of other molecular prognostic factors.

CLL remains an incurable disease in spite of the many new compounds being studied. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the influence of stromal cells, key components of the CLL microenvironment, on the response of CLL cells to ATO. Bone marrow stromal cells induced CLL cell resistance to 2 μM ATO and led to activation of Lyn, ERK, PI3K and PKC, as well as NF-κB and STAT3. Mcl-1, Bcl-xL, and Bfl-1 were also upregulated after the co-culture. Inhibition experiments indicated that PI3K and PKC were involved in the resistance to ATO induced by stroma. Moreover, idelalisib and sotrastaurin, specific inhibitors for PI3Kδ and PKCβ, respectively, inhibited Akt phosphorylation, NF-κB/STAT3 activation and Mcl-1 upregulation, and rendered cells sensitive to ATO. Mcl-1 was central to the mechanism of resistance to ATO, since: 1) Mcl-1 levels correlated with the CLL cell response to ATO, and 2) blocking Mcl-1 expression or function with specific siRNAs or inhibitors overcame the protecting effect of stroma. We have therefore identified the mechanism involved in the CLL cell resistance to ATO induced by bone marrow stroma and show that idelalisib or sotrastaurin block this mechanism and restore sensibility to ATO. Combination of ATO with these inhibitors may thus constitute an efficient treatment for CLL.

Resumen El linfoma de celulas del manto (LM) es una entidad clinica heterogenea con un comportamiento clinico agresivo y una supervivencia corta en algunos pacientes, mientras que en otros sigue un curso clinico indolente. Los recientes avances en la biologia del LM han demostrado la existencia de genes implicados en la desregulacion de vias relacionadas con el ciclo celular, asi como la presencia de poblaciones subclonales con mutaciones recurrentes (p53 , ATM , NOTCH2) con impacto en la progresion clinica y refractariedad al tratamiento. La estratificacion pronostica ayuda a distinguir entre formas indolentes y agresivas del LM. Actualmente, los pacientes jovenes se benefician de quimioterapia alternante y consolidacion con trasplante autologo en primera linea, y los pacientes ancianos se tratan con regimenes estandar y mantenimiento con rituximab. Los pacientes en recaida se benefician de regimenes que incluyen bortezomib y lenalidomida. Ademas, el empleo de inhibidores de tirosincinasas (ibrutinib, idelalisib) esta ofreciendo resultados clinicos muy prometedores.

Mantle cell lymphoma (MCL) is a clinically heterogeneous non-Hodgkin lymphoma with an aggressive clinical behaviour and short survival in some cases and an indolent course in others. Advances in the biology and pathogenesis of MCL have unveiled several genes involved in deregulation of cell cycle checkpoints and the finding of subclonal populations with specific recurrent mutations (p53, ATM, NOTCH2) with an impact on disease progression and refractoriness to treatment. Prognostic stratification helps to distinguish between indolent and aggressive forms of MCL. Currently, younger fit patients benefit from more intensive front line chemotherapy regimens and consolidation with autologous transplantation, while older or frail patients are treated with less intensive regimens and rituximab maintenance. For relapsing disease, the introduction of bortezomib and lenalidomide containing regimens and B-cell receptor pathway inhibitors such as ibrutinib and idelalisib in combination with immunochemotherapy have emerged as therapeutic agents with promising clinical outcomes.

This prospective multi-institutional phase II study was designed to assess the efficacy and safety of dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) plus rituximab (DA-EPOCH-R) in untreated patients with poor prognosis large B-cell lymphomas. Eighty-one patients diagnosed with diffuse large B-cell lymphoma (DLBCL, n = 68), primary mediastinal DLBCL (n = 6) and follicular lymphoma Grade 3b (n = 7), with an age-adjusted International Prognostic Index >1, were eligible for analysis. Median age was 60 years (range: 21-77). Sixty-five patients (80·2%) achieved complete response. After a median follow-up time of 64 months, 10-year event-free survival and overall survival (OS) were 47·8% and 63·6%, respectively. None of the studied clinical and biological characteristics were associated with poorer outcome. Interestingly, patients with BCL6 rearrangement achieved a 10-year OS of 100%, while patients with BCL2 rearrangement exhibited a poorer outcome compared to activated B-cell tumours and germinal centre B-cell without BCL2 rearranged tumours. Results achieved with DA-EPOCH-R showed a good long-term outcome and a tolerable toxicity profile in high-risk large B cell lymphoma patients. Outcome was not affected by tumour cell proliferation or by cell of origin, highlighting the requirement of new biological markers for patient subclassification of high-risk DLBCL patients.

Among the strategies to optimize engraftment of cord blood (CB) stem cell transplantation (SCT), single CB with the coinfusion of CD34+ stem cells from an HLA-mismatched auxiliary donor (haplo-cord) provides a valid alternative for adult patients without a suitable donor. A total of 132 high-risk adult patients with hematological malignancies from 3 Spanish institutions underwent myeloablative haplo-cord SCT. The median age was 37 years and median weight was 70 kg; 37% had active disease. The median number of postprocessing CB total nucleated and CD34+ cells was 2.4 × 107/kg (interquartile range [IQR], 1.8 to 2.9) and 1.4 × 105/kg (IQR, .9 to 2), respectively. Neutrophil engraftment occurred in a median of 11.5 days (IQR, 10.5 to 16.5) and platelet engraftment at 36 days (IQR, 25.5 to 77). Graft failure was 2% overall and only 9% for CB. Cumulative incidence of acute graft-versus-host disease (GHVD) grades II to IV was 21% and cumulative incidence of chronic GVHD was 21%. Median follow-up was 60 months (range, 3.5 to 163). Overall survival was 43.5%, event-free survival was 38.3%, nonrelapse mortality was 35%, and relapse was 20% at 5 years. Myeloablative haplo-cord SCT results in fast engraftment of neutrophils and platelets, low incidences of acute and chronic GVHD, and favorable long-term outcomes using single CB units with relatively low cell content. Moreover, CB cell dose had no impact on CB engraftment and survival in this study. Therefore, haplo-cord SCT expands donor availability while reducing CB cell dose requirements.

Recent research suggests that growth differentiation factor 11 (GDF11) could reverse age-related diseases and that its blood concentration decreases with age. This poses plasma from young donors as a therapeutic GDF11 source to treat age-related diseases. In addition, the tissue source of circulating GDF11 remains unknown. We analysed GDF11 levels in paired samples of serum, plasma and platelet lysate (PL) from 23 volunteers. Plasma and PL were collected by plateletpheresis. Here, we show that GDF11 is highly concentrated in platelets and that the circulating levels reported in previous studies could be biased as a result of serum sample manipulation.

Minimal residual disease (MRD) assessment is an important endpoint in the treatment of chronic lymphocytic leukaemia (CLL). It is highly predictive of prolonged progression-free survival (PFS) and overall survival and could be considered a surrogate for PFS in the context of chemoimmunotherapy based treatment. Evaluation of MRD level by flow cytometry or molecular techniques in the era of the new BCR and Bcl-2 targeted inhibitors could identify the most cost-effective and durable treatment sequencing. A therapeutic approach guided by the level of MRD might also determine which patients would benefit from an early stop or consolidation therapy. In this review, we discuss the different MRD methods of analysis, which source of tumour samples must be analysed, the future role of the detection of circulating tumour DNA, and the potential role of MRD negativity in clinical practice in the modern era of CLL therapy.

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. All our funders are public or charitable organizations. A grant from Fondation de France (0057921) to MV and DS-M. SUDOE to AA, JP, MV and CV. SAF2011-25390 and SAF2014-54763-C2-1-R to JP. Fondo Social Europeo (FSE) to JP and AA. JP is supported by Fundacion ARAID.

The effectiveness of rituximab maintenance therapy in the treatment of chronic lymphocytic leukemia has been investigated in a phase 2 clinical trial that included an initial treatment with rituximab 500 mg/m2 on day 1 (375 mg/m2 the first cycle), fludarabine 25 mg/m2 on days 1 to 3, cyclophosphamide 200 mg/m2 on days 1 to 3, and mitoxantrone 6 mg/m2 on day 1 (R-FCM), for 6 cycles, followed by a maintenance phase with rituximab 375 mg/m2 every 3 months for 2 years. Sixty-seven patients having achieved complete response (CR) or partial response (PR) with R-FCM were given maintenance therapy. At the end of maintenance, 40.6% of patients were in CR with negative minimal residual disease (MRD), 40.6% were in CR MRD-positive, 4.8% remained in PR, and 14% were considered failures. Six of 29 patients (21%) who were in CR MRD-positive or in PR after R-FCM improved their response upon rituximab maintenance. The 4-year progression-free survival (PFS) and overall survival rates were 74.8% and 93.7%, respectively. MRD status after R-FCM induction was the strongest predictor of PFS. Maintenance with rituximab after R-FCM improved the quality of the response, particularly in patients MRD-positive after initial treatment, and obtained a prolonged PFS. This trial was registered at www.clinicaltrialsregister.eu as identifier #2005-001569-33.

INTRODUCTION The use of new drugs in chronic lymphocytic leukemia (CLL) has allowed to revert the poor prognosis conferred by both the alterations of TP53 (deletion and mutation) and the absence of somatic hypermutation (HMS) of the heavy chain gene of immunoglobulins (IGHV). These alterations are part of the essential pre-treatment tests of the CLL according to the International Workshop on CLL Guidelines (iwCLL, Hallek et al, 2018). Three independent methodologies are required for these analyses, consequently time- and cost-consuming. Next-generation sequencing (NGS) could integrate all three types of studies into one. AIM To design a NGS panel for the detection of genetic alterations with diagnostic, prognostic and predictive value in CLL, as well as other alterations with clinical relevance. PATIENTS AND METHODS A capture panel (SureSelect QXT, Agilent) of 170Kb was designed covering the coding region of 17 genes, 11 regions of copy number alteration (CNAs), 2 translocations and the HMS-IGHV. A total of 83 samples of patients with CLL from three centers were sequenced. The effectiveness of the panel to detect already known alterations was evaluated in two groups of samples: 46 samples that presented any essential alteration of the iwCLL, including mutations of TP53, del (11q), +12, del (13q), del (17p), and the HMS status of the IGHV (group 1), and in 19 other samples with ≥1 different known cytogenetic / molecular alteration (group 2). Four healthy individuals were included as control. The samples were sequenced in a MiSeq (Illumina, average coverage of 600x, paired-end reading of 126bp). The analysis was carried out using a specific algorithm developed by Dreamgenics bioinformaticians, confirming the alterations with Integrative Genome Viewer (IGV). RESULTS The NGS panel allowed the identification of 214 mutations, 92 CNA regions and 83 IGHV genes. The concordance of results between the NGS panel and conventional methods was 100% (78/78) for mutations, 92% for CNAs (172/187) and 90% for use of IGHV (57/63). Considering the alterations of group 1, the agreement was 100% in mutations of TP53 (26/26), 100% of del (11q) (41/41), 98% +12 (41/42), 98% of del (17p) (42/43), and 79% of del (13q) (33/42). Two of the discrepancies in CNAs were cases identified by NGS but FISH negative [del (17p) and del13q)], probably because they are small deletions ( CONCLUSIONS This panel of NGS allows to detect> 93% of the essential genetic alterations of the iwCLL, and> 94% of the total genetic alterations analyzed. The results are preliminary but promising, although validation of the methodology is required. Disclosures No relevant conflicts of interest to declare.