Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia.

The treatment of chronic lymphocytic leukemia (CLL) patients with venetoclax-based regimens has demonstrated efficacy and a safety profile, but the emergence of resistant cells and disease progression is a current complication. Therapeutic target of sphingosine kinases (SPHK) 1 and 2 has opened new opportunities in the treatment combinations of cancer patients. We previously reported that the dual SPHK1/2 inhibitor, SKI-II enhanced the in vitro cell death triggered by fludarabine, bendamustine or ibrutinib and reduced the activation and proliferation of chronic lymphocytic leukemia (CLL) cells. Since we previously showed that autologous activated T cells from CLL patients favor the activation of CLL cells and the generation of venetoclax resistance due to the upregulation of BCL-XL and MCL-1, we here aim to determine whether SPHK inhibitors affect this process. To this aim we employed the dual SPHK1/2 inhibitor SKI-II and opaganib, a SPHK2 inhibitor that is being studied in clinical trials. We found that SPHK inhibitors reduce the activation of CLL cells and the generation of venetoclax resistance induced by activated T cells mainly due to a reduced upregulation of BCL-XL. We also found that SPHK2 expression was enhanced in CLL cells by activated T cells of the same patient and the presence of venetoclax selects resistant cells with high levels of SPHK2. Of note, SPHK inhibitors were able to re-sensitize already resistant CLL cells to a second venetoclax treatment. Our results highlight the therapeutic potential of SPHK inhibitors in combination with venetoclax as a promising treatment option for the patients.

Somatic hypermutation profiles in stereotyped IGHV4-34 receptors from South American chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

Venetoclax-resistant CLL cells show a highly activated and proliferative phenotype.

Venetoclax treatment has demonstrated efficacy and a safety profile in chronic lymphocytic leukemia (CLL) patients, however the emergence of resistant cells is a current complication. We and others, previously reported that the activation of CLL cells by signals that mimic microenvironment stimuli favors the upregulation of anti-apoptotic proteins from B cell lymphoma-2 (BCL-2) family that are not targeted by venetoclax, reducing malignant cell sensitivity to the drug. We here studied venetoclax-resistant CLL cells generated in vitro by autologous activated T lymphocytes, and found that they showed an aggressive phenotype characterized by increased expression of activation and proliferation markers. Moreover, surviving cells expressed high levels of B cell lymphoma-extra-large (BCL-XL) and/or myeloid cell leukemia-1 (MCL-1), and a sustained resistance to a second treatment with the drug. Interestingly, the spleen tyrosine kinase (SYK) inhibitor entospletinib, and the phosphoinositide 3-kinase delta (PI3Kδ) inhibitor idelalisib, reduced T cell activation, impaired the generation of leukemic cells with this aggressive phenotype, and were able to restore CLL sensitivity to venetoclax. Our data highlight a novel combination to overcome resistance to venetoclax in CLL.

[Chronic myelogenous leukemia: monitoring and predictors of a favorable response to treatment with imatinib]. / Leucemia mieloide crónica. Monitoreo y factores predictivos de una respuesta favorable en el tratamiento con imatinib.

The expected five-year survival of chronic-phase chronic myeloid leukemia patients treated with tyrosine kinase inhibitors is over 90%. Little data is available regarding the results in the Argentinian population. This information might be of interest as generic imatinib is now available in the region. The aim of this study is to provide information on monitoring and the long-term treatment with imatinib outside of a controlled clinical trial, as well as to analyze the predictive effect of early responses to achieve molecular remission 4.0 (RM 4.0) and the detection of variables that may condition treatment failure. We included 106 patients, who received imatinib 400 mg daily as first-line inhibitor for a median of 8.9 years (IQR 5.8-11.7) between June 2000 and December, 2015. Overall survival was 93%. At latest follow-up 74% of patients continues on initial imatinib. The achievement of response at targeted milestones (6, 12 months) was associated with increased failure-free survival: 87% vs. 56%, p = 0.007; 90% vs. 69% p = 0.01 and was independently associated to RM 4.0: OR 5.6 (95% CI: 1.6-19.0); OR 5.3 (95% CI: 1.4-21.0) p = 0.006. After long-term follow-up, imatinib provided high-rates of response and survival. The prognostic value of response at targeted milestones was confirmed. This study reinforces the importance of molecular monitoring under IS standardization at known timepoints and this must continue to be a target in Argentina.

Leucemia mieloide crónica: Monitoreo y factores predictivos de una respuesta favorable en el tratamiento con imatinib / Chronic myelogenous leukemia: monitoring and predictors of a favorable response to treatment with imatinib

La supervivencia a cinco años de los pacientes con leucemia mieloide crónica en fase crónica tratados con inhibidores de tirosina quinasa es superior al 90%. Existen escasos datos a nivel local. Esta información puede ser de interés, ya que el imatinib genérico se encuentra disponible en la región. El objetivo del presente estudio es proporcionar información del monitoreo y los resultados a largo plazo del tratamiento con imatinib fuera de un ensayo clínico controlado, así como analizar el valor predictivo de respuestas tempranas para el logro de respuesta molecular 4.0 y la detección de variables que puedan condicionar falla al tratamiento. Se incluyeron 106 pacientes tratados con imatinib 400 mg diarios como inhibidor de primera línea durante una mediana de 8.9 años IQR (5.8-11.7) entre junio del 2000 y diciembre del 2015. La supervivencia global fue de 93%. En la última evaluación, 74% de los pacientes continuaba recibiendo el imatinib inicial. La obtención de respuesta en los objetivos temporales específicos (6, 12 meses) se asoció con mayor supervivencia libre de falla: 87% vs. 56%, p = 0.007; 90% vs. 69% p = 0.01 y mayor adquisición de respuesta molecular 4.0: OR 5.6 (IC 95% 1.6-19.0) p = 0.003; OR 5.3 (IC 95% 1.4-21.0) p = 0.006. Luego del prolongado seguimiento, el imatinib proporcionó altas tasas de respuesta y supervivencia. Se confirmó el valor pronóstico de la respuesta en momentos temporales específicos. Este estudio refuerza la importancia del monitoreo estandarizado en los puntos temporales conocidos, que debe continuar siendo un objetivo en Argentina.

The expected five-year survival of chronic-phase chronic myeloid leukemia patients treated with tyrosine kinase inhibitors is over 90%. Little data is available regarding the results in the Argentinian population. This information might be of interest as generic imatinib is now available in the region. The aim of this study is to provide information on monitoring and the long-term treatment with imatinib outside of a controlled clinical trial, as well as to analyze the predictive effect of early responses to achieve molecular remission 4.0 (RM 4.0) and the detection of variables that may condition treatment failure. We included 106 patients, who received imatinib 400 mg daily as first-line inhibitor for a median of 8.9 years (IQR 5.8-11.7) between June 2000 and December, 2015. Overall survival was 93%. At latest follow-up 74% of patients continues on initial imatinib. The achievement of response at targeted milestones (6, 12 months) was associated with increased failure-free survival: 87% vs. 56%, p = 0.007; 90% vs. 69% p = 0.01 and was independently associated to RM 4.0: OR 5.6 (95% CI: 1.6-19.0); OR 5.3 (95% CI: 1.4-21.0) p = 0.006. After long-term follow-up, imatinib provided high-rates of response and survival. The prognostic value of response at targeted milestones was confirmed. This study reinforces the importance of molecular monitoring under IS standardization at known timepoints and this must continue to be a target in Argentina.

BCR-ABL Mutations in Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors and Impact on Survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

BCR-ABL mutations in chronic myeloid leukemia treated with tyrosine kinase inhibitors and impact on survival.

This is the largest Latin American study of BCR-ABL mutations in chronic myeloid leukemia (CML) patients, resistant to imatinib (IM). In 195/467 (41%) patients, mutations were detected. The most frequent mutation was T315I (n = 31, 16%). Progression-free (PFS) and overall survival (OS) at 5 years were lower in patients with BCR-ABL mutations (43% vs. 65%, p = 0.07 and 47% vs. 72%, p = 0.03, respectively) and in those with the T315I mutation (p = 0.003 and p = 0.03). OS and PFS were superior in subgroup who switched to second generation inhibitors (SGIs) after IM failure (OS: 50% vs. 39% p = 0.01; PFS: 48% vs. 30% p = 0.02). BCR-ABL mutations conferred a significant poor prognosis in CML patients.

Molecular monitoring of imatinib in chronic myeloid leukemia patients in complete cytogenetic remission: does achievement of a stable major molecular response at any time point identify a privileged group of patients? A multicenter experience in Argentina and Uruguay.

BACKGROUND: Monitoring minimal residual disease (MRD) by real-time quantitative polymerase chain reaction (RT-PCR) in chronic myeloid leukemia (CML) patients is mandatory in the era of tyrosine kinase inhibitors. Achieving a major molecular response (MMR) at 12 and 18 months predicts a better progression and event-free survival. PATIENTS AND METHODS: The objective of this prospective, multicentric study was to evaluate MRD by standardized RT-PCR in 178 patients with chronic-phase CML who were treated with imatinib at different institutions in Argentina and Uruguay and to determine if achievement of a stable MMR (BCR-ABL transcript levels < 0.1%) identifies a low-risk cytogenetic relapse group. The median age of the patients was 50 years, and 55% of them had received imatinib as first-line therapy. BCR-ABL transcript levels were measured after achievement of complete cytogenetic remission (CCyR) and at 6-month intervals. RESULTS: MMR was detected in 44% patients at the start of the study. This value increased to 79% at month 36 of evaluation. Complete molecular response (CMR) also increased from 24% to 52% of patients. Not achieving a stable MMR determined a higher risk of cytogenetic relapse (9% of MMR patients not achieving an MMR vs. 1% of patients who achieved MMR). Patients with sustained MMR had a significantly better cytogenetic relapse-free survival at 48 months (97% vs. 87%; P = .008) but showed no differences in overall survival. Patients who did not remain in CCyR changed treatment. CONCLUSIONS: A stable MMR is a strong predictor for a durable CCyR. Standardized molecular monitoring could replace cytogenetic analysis once CCyR is obtained. These results emphasize the validity and feasibility of molecular monitoring in all standardized medical centers of the world.

Risk-adapted therapy with three or six cycles of doxorubicin/bleomycin/vinblastine/dacarbazine plus involved-field radiation therapy in Hodgkin lymphoma, based on prognosis at diagnosis and early response: results from the GATLA study.

BACKGROUND: Doxorubicin/bleomycin/vinblastine/dacarbazine (ABVD) plus involved-field radiation therapy (IFRT) is the gold-standard treatment for early and advanced stages of Hodgkin lymphoma (HL). We evaluated the outcomes of patients according to prognosis at diagnosis and over time to determine who achieved complete remission (CR). PATIENTS AND METHODS: Treatment-naive patients under the age of 75 years at all stages of HL were eligible. The favorable group (FG) contained patients with stage IA-IIIA disease without bulky areas who achieved CR after the third cycle of ABVD. They received only IFRT at 25 Gy. Patients in the unfavorable group (UG) exhibited stages IIIB and IV HL. The UG also included all patients with bulky disease and the subset of the FG without CR after 3 cycles of ABVD, ie, slow responders (FGSR). The UG received 6 cycles of ABVD plus IFRT at 30 Gy to bulky areas at diagnosis or to those areas remaining positive after the third cycle of ABVD. RESULTS: In total, 584 patients were evaluable: 285 of them belonged to the FG, and 299 to the UG. Rates of CR were 98% and 85% for the FG and the UG, respectively (P < .001). Sixty patients in the FG received 6 cycles of ABVD because they had not achieved CR after 3 cycles (ie, the FGSR subgroup). The 5-year event-free survival rate was 89% for the FG, 66% for the FGSR, and 72% for the UG (P < .001). The overall survival at 5 years was significantly better for the FG (98%) than for the FGSR (87%) and the UG (88%; P < .001). CONCLUSION: Patients from the FG demonstrated excellent outcomes compared with those from the FGSR and UG, despite receiving less chemotherapy and fewer doses of IFRT.

Is assessment of surface CD38 expression worthwhile as a prognostic factor in chronic lymphocytic leukemia patients?

We studied the clinical impact of CD38 expression in 226 chronic lymphocytic leukemia patients (CLL) at disease presentation and during follow up to determine its prognostic significance, progression free survival (PFS) and overall survival (OS), and to verify whether this parameter changed over time. Various patients' characteristics were studied including gender, Rai and Binet stages, immunoglobulin light chain expression, lymphocyte doubling time and CD38 expression. After a median follow up of 53 months (range 6-282), 62% CD38 positive(+) patients required therapy. PFS and OS at 84 months were significantly lower for CD38(+) patients: 20 and 71% respectively, compared to CD38 negative(-): 70 and 96%. At multivariate analysis CD38(+) showed to be the best factor for predicting progression: HR 3.3, 95%CI 2.10-5.14, p = 0.000. Its expression did not change in 98% re-evaluated patients. We confirm that CD38(+) is a stable parameter for the identification of CLL patients with a more aggressive disease course.

Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasia.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. METHODS: We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). RESULTS: Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P < or = 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). CONCLUSION: Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases.