Relapse In Acute Leukemia Patients Research Articles

Abstract A081: Patient-derived xenograft models from hematological malignancies for preclinical drug development and biomarker research

Abstract Background: Acute leukemias and lymphomas encompass a diverse group of hematologic malignancies, posing significant challenges in clinical practice. They often exhibit resistance to standard-of-care (SoC) treatments and have a high recurrence rate. Recent advancements in molecular profiling have identified potential drivers for various leukemia and lymphoma subtypes, offering new therapeutic targets. However, the validation of these targets and the development of drugs rely heavily on preclinical models that faithfully represent the different clinical subtypes. These models play a crucial role in refining target selection and facilitating drug development projects. Methods: AML- and ALL-PDX were derived from bone marrow aspirates or peripheral blood samples, from primary or relapsed acute leukemia patients. Purified cells were transplanted either intravenously (i.v.) and/or subcutaneously (s.c.) into immunodeficient mice. Some mice developed a systemic AML, which was monitored by flow cytometric analysis of blood samples. Non-Hodgkin (NHL) - or Hodgkin lymphoma (HL)-PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice. Results: More than 45 PDX models from AML, ALL, NHL and HL have been successfully established and characterized. Highly individual response rates to SoC and investigational drugs were observed and correlated with mutation and gene expression data. An individual Human Leucocyte Antigen (HLA) profile of each PDX was determined by RNA-seq and a comprehensive HLA matching analysis of the models and peripheral blood mononuclear cell (PBMC) donors was performed to enable personalized, preclinical immuno-oncology studies. Conclusions: Our extensively characterized PDX models derived from hematologic malignancies serve as valuable tools for evaluating novel targeted and immunological therapies. These models provide an exceptional platform for identifying and validating new targets, as well as preclinical screening of compounds and combinations for translational research projects in this field. Citation Format: Antje Siegert, Bernadette Brzezicha, Stephan Fuhrmann, Martin Janz, Clemens Schmitt, Ulrich Keller, Christian Rupp, Wolfgang Walther, Jens Hoffmann. Patient-derived xenograft models from hematological malignancies for preclinical drug development and biomarker research [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A081.

Higher Proportions of Monocytic-Myeloid-Derived Suppressive Cells (mMDSC) before Allotransplant Predict Relapse in Acute Leukemia Patients

Introduction: Myeloid-derived suppressive cells (MDSC) are an heterogeneous group of immature myeloid cells with strong immunosuppressive activities which made them of particular interest in the context of allogeneic stem cell transplant (Allo-SCT). In mice models, MDSC modulate the function of alloreactive T cells and prevent graft versus host disease (GVHD) without impairing graft versus leukemia (GVL) effect. Data are limited in humans, but higher proportions of MDSC in the graft seem to protect against both acute and chronic GVHD. However, the influence of circulating MDSC is not fully explored especially regarding their impact on post-transplant GVL effect and survivals. Here we focused on the role of peripheral levels of monocytic-MDSC (mMDSC) in a cohort of adults with acute (both myeloid or lymphoid) leukemia (AML or ALL) having received Allo-SCT. Methods: This prospective monocentric study aimed at documenting the impact of the percentage of mMDSC before and after allo-SCT in terms of incidence of acute and chronic GVHD, overall (OS) and leukemia-free (LFS) survivals, (grade 3/4 acute or extensive chronic) GVHD-free relapse-free survival (GRFS), relapse and non-relapse mortality (NRM) in a cohort of ALL or AML patients (pts). Inclusion was performed after informed consent at diagnosis as part of a more global study (Abstract ASH 2021 # 3465), pts who were ultimately transplanted being included in this sub-group analysis. mMDSC were defined by the minimal CD14+/CD11b+/CD33+/HLA-DR-/low immunophenotypic pattern, at variance from granulocytic MDSC that lack CD14 and express bright CD15. Cells were assessed in a lysis-no-wash flow cytometry technique using peripheral blood collected on EDTA before conditioning then at day+60 post-transplant. Data acquisition was performed on a Navios® flow cytometer (Beckman Coulter, Miami, FL). mMDSC were expressed as a percentage (%) of total nucleated cells defined as CD45+. The study was registered at the French Commission Nationale de l'Informatique et des Libertés as CNIL 2016-038 and approved by the Ethic Review Board of Nantes University Hospital. Results: Among 73 AML and 14 ALL pts included at diagnosis, 47 and 9, respectively, were ultimately allografted between February 2018 and October 2021. Pts characteristics are provided in Table 1A. The median follow-up for alive pts from Day 0 was 24 months (range: 8-48). Considering the whole cohort, 2-year OS, LFS and GRFS were 61.9+6%, 55.9+7% and 32.4+6%, respectively, while 2-year NRM and Relapse incidence were 11+4% and 29.6+6%. In pre-graft, 46 pts were evaluated at a median of 20 days from Day 0 (range: 6-83). The median % of mMDSC was 0.45% (range: 0-11.85) with no differences between AML and ALL (p=0.32). The best cut-off assessed by ROC analyses was 0.47%. According to this value, the incidence of cytologic relapse at 2 years was significantly higher in pts with >0.47% of mMDSC (14.5+8% vs 44.4+11%, p=0.02, Figure 1) while no impact on survivals, NRM or GVHD was observed. Of note, the incidence of relapse was similar between AML and ALL (29% vs 37.5%, p=0.95). In multivariate analysis (Table 1B), higher pre-graft mMDSC % remained associated with higher risk of relapse (HR: 3.71; 95%CI: 1.05-13.09, p=0.04). At day+60, 42 pts were evaluated. The mMDSC median level was 0.52% (range: 0-5.03) with no differences between AML and ALL (p=0.32). No best cut-off was determined. Survivals were similar between pts with ≤ or >0.52% of mMDSC at this time. It was also the case for incidences of relapse, NRM and chronic GVHD. However, the incidence of severe acute GVHD (grade 3/4) was significantly and paradoxically higher in the group of pts with >0.52% mMDSC (33% vs 5%, p=0.04) suggesting a possible amplification of immunosuppressive mMDSC as a reaction to GVHD since the median time of GVHD grade 2-4 occurrence was 30 days (range: 20-123) post-transplant. Of note, the kinetics (increases or decreases of mMDSC %) between pre-graft and D60 had no influence on outcomes. Conclusion: This prospective study demonstrates a negative impact of higher proportions of mMDSC before allotransplant in leukemic pts. This higher proportion is predicting relapse, suggesting a protective role and tolerance of tumoral cells by mMDSC themselves. This paves the way to therapeutic intervention to decrease the incidence of relapse in such pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Viral Reactivation in Acute Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation in Complete Remission: Stimulation of the Immune System

Background: Many studies have evaluated the impact of cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (allo-HSCT) showing a significant association with reduced risk of relapse while other studies failed to show the same results. On the other hand, other frequent viral infections or reactivations like Epstein-Barr virus (EBV), Human Herpesvirus 6 (HHV6) and BK virus (BK-V) have not been evaluated in the same context and/or together with CMV. The aim of this study is to evaluate the impact of CMV, EBV, HHV6 and BK-V reactivation in acute leukemia (AL) patients receiving allo-HSCT in complete remission (CR) and evaluate their impact alone or associated on disease relapse. Patients and methods: We evaluated 158 consecutive AL patients who received allo-HSCT at our center between January 2008 and June 2015; 95 (60%) were males, median age was 46 years (range: 18-67), 101 (64%) had acute myeloid leukemia (AML) and 57 (36%) had acute lymphoblastic leukemia (ALL). At transplantation, 114 (72%) patients were in first complete response (CR1) and 44 (28%) were in second CR (CR2). Conditioning regimen was myeloablative for 97 (61%) patients and of reduced intensity in 61 (39%) patients. DNA levels of CMV, EBV, HHV6 and BK-V were detected by quantitative RQ-PCR after weekly monitoring up to 3 months after allo-HSCT. CMV-DNA, EBV-DNA, HHV6-DNA or BK-V-DNA was considered positive when the copies exceeded 1000 copies/ml. Results: Among 158 patients, 45 (28%) patients had CMV reactivation after a median time of 40 days (6-72) after allo-HSCT; 53 (34%) patients had EBV reactivation after a median time of 42 days (18-91) after allo-HSCT; 33 (21%) patients had HHV6 reactivation after a median time of 24 days (6-69) and 30 (19%) patients had BK-V reactivation after a median time of 32 days (6-60) after allo-HSCT. There was 61 (39%) patients who did not have any viral reactivation, 46 (29%) patients who had only one reactivation (no matter the type), and 51 (32%) patients who had at least two or more co-reactivations (group 1). When evaluating relapse incidence, we found that patients who had only one reactivation were not significantly different from those who did not have any reactivation, with a comparable relapse incidence, thus, we analyzed those patients as one group (group 2, n=107). The cumulative incidence of relapse at 6 months for group 1 and group 2 was 2% (95% CI: 0.2-9.4) and 15.9 % (95%CI: 9.7-23.5) respectively, this result remained significantly different when stratifying on the type of conditioning, in RIC: 15.8% (95%CI: 3.7-35.7) vs 21.4% (95%CI: 10.5-35) respectively; and in MAC: 0 % vs 15.4% (95%CI: 7.8-25.3) respectively, p=0.04. This finding was confirmed independently of the presence of GVHD or not. Conclusion: We demonstrate here for the first time, in this homogeneous population, that co-reactivation of multiple viruses after allo-HSCT could lead to a lower incidence of relapse in AL patients in CR; this can be explained as a stimulation of the immune system via both the functional activation and amplification of NK compartment. This could also explain some conflicting results in studies evaluating single viral reactivation alone without consideration of other reactivations. These encouraging results should be validated at a larger multicenter level. Disclosures No relevant conflicts of interest to declare.

Predictive Role of Circulating Immune Cell Subtypes Early after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Leukemia.

Background and ObjectivesCells of innate immunity normally recover in the first weeks to months after allogenenic hematopoietic stem cell transplantation (allo-HSCT). Their relevance in terms of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect is largely unknown. The predictive role of early recovery in the immune cells on acute GVHD and GVL effect after allo-HSCT was investigated in patients with acute leukemia who achieved the first complete remission.MethodsPeripheral blood samples were taken at the median of 14 days (range, 12~29 days) after allo-HSCT. A cohort including 119 samples and characteristics of patients were analyzed. Immune cell populations were identified by flow cytometry.ResultsThe median age was 49.0 years (range, 21~69) at transplantation. Univariate analysis showed that age less than 40 years old, lower frequencies of CD8+ T cells, invariant natural killer T (iNKT) cells, monocytic myeloid derived suppressor cells (M-MDSCs) and higher frequency of immature MDSCs were associated with occurrence of grade III–IV acute GVHD. Multivariate analyses showed that iNKT cells (hazard ratio (HR), 0.453, 95% CI, 0.091~0.844, p=0.024) and M-MDSCs (HR, 0.271, 95% CI, 0.078~0.937, p=0.039) were independent factors. Combination of higher frequencies of both cell subsets was associated with lower incidence of grade III–IV acute GVHD, whereas patients with lower frequency of iNKT cells and higher frequency of M-MDSCs showed significant higher probability of relapse.ConclusionsiNKT cells and M-MDSCs could be relevant cell biomarkers for predicting acute GVHD and/or relapse in acute leukemia patients treated with allo-HSCT.

Abstract 1040: Patient derived xenografts of hematologic malignancies for translational research

Abstract Acute leukemias and lymphomas represent a very heterogeneous group of hematologic malignancies and pose an important challenge in the clinical routine. They frequently develop resistance to the treatment with standard-of-care (SoC) drugs and have a high incidence of disease recurrence. Recent progress in molecular profiling has helped to identify new potential drivers for the different leukemia and lymphoma subtypes, with some of them being potential therapeutic targets. Further target validation and drug development projects are highly dependent on corresponding preclinical models representing the different clinical subtypes. Therefore, we started to establish and characterize new patient derived xenografts (PDX) of AML, ALL and lymphomas for drug development and translational research. AML- and ALL-PDX were derived from bone marrow aspirates or peripheral blood samples, from primary or relapsed acute leukemia patients. Purified cells were transplanted either intravenously (i.v.) and/or subcutaneously (s.c.) into immunodeficient mice. Some mice developed a systemic AML, which was monitored by flow cytometric analysis of blood samples. Non-Hodgkin- or Hodgkin lymphoma-PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, and were usually transplanted subcutaneously into immunodeficient mice. For further characterization, established PDX models were treated with SoC and investigational drugs. In parallel, gene expression profiles as well as mutations were analyzed within the first in vivo passages. More than 20 new PDX models from AML, ALL, NHL and HL have been successfully established and characterized. Highly individual response to the treatments were observed, correlation analyses with mutations and gene expression are ongoing. Our newly and extensively characterized PDX models from hematologic malignancies are suitable tools for preclinical drug development. They provide an exceptional platform for the identification and validation of new targets and allow the preclinical screening of new compounds and combinations for translational research projects. Citation Format: Antje Siegert, Bernadette Brzezicha, Stephan Fuhrmann, Leonid Karawajew, Martin Janz, Clemens Schmitt, Iduna Fichtner, Jens Hoffmann. Patient derived xenografts of hematologic malignancies for translational research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1040.

Efficacy analysis of unrelated cord blood transplantation in the treatment of refractory and relapsed adult acute leukemia

目的探讨非血缘脐血移植（UCBT）挽救治疗难治复发急性白血病（AL）患者的临床疗效和安全性。方法回顾性分析2009年11月至2017年5月22例行UCBT挽救治疗的难治复发成人AL患者的临床资料，全部患者采用清髓性预处理方案，均采用环孢素A/短程霉酚酸酯方案预防GVHD。结果①22例患者中男9例，女13例，中位年龄23（15~44）岁；中位体重52.5（43~82）kg。所有患者回输脐血有核细胞中位数为3.07（1.71~5.30）×107/kg（受者体重），CD34+细胞中位数为1.60（0.63~3.04）×105/kg（受者体重）。②移植后42 d髓系累积植入率为95.5%（95%CI 45.2%~99.7%），中位植入时间为19（13~27）d；移植后120 d血小板累积植入率为81.8%（95%CI 54.2%~93.6%），中位植入时间为42（20~164）d。③Ⅱ~Ⅳ度、Ⅲ~Ⅳ度急性GVHD发生率以及慢性GVHD 2年累积发生率分别为36.4%、13.6%和40.3%。④移植后180 d移植相关死亡率为22.7%；2年累积复发率为18.7%（95%CI 3.6%~42.5%），2年累积无病生存率及累积总生存率分别为53.7%和58.1%。结论对于常规化疗无效的难治复发成人AL患者，初步结果显示采用UCBT安全、有效。

Expression and Clinical Significance of Focal Adhesion Kinase in Patients with Acute Leukemia

To explore the expression of focal adhesion kinase (FAK) in patients with acute leukemia (AL) and its clinical significance. The FAK mRNA levels in leukemic cells of 50 patients with AL and bone marrow mononuclear cells(BMMNC) of 10 healthy controls were measured by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The bone marrow cell chromosomes were prepared using direct and short term culture, and karyotyping was performed by R-banding technique. The expression positive rate of FAK mRNA in leukemic cells of 50 patients with AL was 66.0%, which was significantly higher than that in normal controls (20.0%) (P<0.05). The expression positive rates and levels of FAK mRNA in both ALL and ANLL cells were statistically significantly higher than those in normal controls (P<0.05), but the difference between ALL and ANLL cells was not significant (P>0.05). All the expression- positive rates and levels of FAK mRNA in newly diagnosed AL patients and the relapsed AL patients were significantly higher than those in normal controls (P<0.05), but the difference between the newly diagnosed AL patients and the relapsed AL patients was not significant (P>0.05). Morecover, the expression plsitive rate and levels of FAK mRNA in the remitted patients were lower than those in newly diagnosed and relapsed patients (P<0.05), but were no statistically significantly different from normal controls (P>0.05). The expression- positive rate and level of FAK mRNA in the AL patients with abnormal karyotype were significantly higher than those in the AL patients without abnormal karyotype (P<0.05). FAK mRNA over-express in the leukemia patients, which may be related with the outome of disease, possibly providing novel targeting sites for the treatment of leukemia.

Expression of SHP-1 and SOCS6 in patients with acute leukemia and their clinical implication.

BackgroundTo investigate the expression and clinical relevance of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and suppressor of cytokine signaling 6 (SOCS6) in acute leukemia (AL).Patients and methodsThe enrolled AL patients were divided into three groups (newly diagnosed, relapsed, and complete remission [CR]). Healthy donors were also included as a control group in this study. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to measure messenger RNA (mRNA) expression of SHP-1 and SOCS6. Statistical analysis was conducted to analyze the correlation between mRNA levels of SHP-1 and SOCS6 with patient outcomes.ResultsmRNA expression of SHP-1 was significantly lower in AL patients than that in healthy donors. The newly diagnosed or relapsed AL patients had lower mRNA levels of SHP-1 than the patients in CR. In contrast, SOCS6 mRNA expression was significantly higher in newly diagnosed or relapsed patients than that in patients in CR as well as healthy donors. However, mRNA levels of both SHP-1 and SOCS6 were positively correlated with the patient remission. The chemotherapy-induced remission rate was higher in patients with detectable SHP-1 or SOCS6 expression than in patients with undetectable SHP-1 or SOCS6 expression. Furthermore, the AL patients with detectable SHP-1 mRNA expression had lower incidence rate of invasive fungal infection.ConclusionThe results suggest that expression patterns of SHP-1 and SOCS6 differ in AL patients. Despite the difference, expression of SHP-1 and SOCS6 is associated with favorable outcomes, suggesting an anticancer property of these two genes in AL.

Expression of E-cadherin on bone marrow mononuclear cell surface and in plasma of patients with extra-myeloid leukemia and their clinical significance

Objective To investigate the expression and clinical significance of soluble E-cadherin(sE-cad)and E-cadherin(E-cad)in acute leukemia(AL), and to explore their relationship with the pathogenesis, development and diagnosis of extra-myeloid leukemia. Methods 87 newly diagnosed or relapsed AL patients(19 cases of L1, 29 L2, 14 M2, 20 M3, 4 M4, 1 M5)were collected from hospitalized patients in hematology department of Harbin Medical University Cancer Hospital. The plasma from 20 healthy volunteers was used as control group. The bone marrow was from 15 non-AL patients hospitalized in hematology department(7 cases of thrombocytopenic purpura, 4 iron deficiency anemia, and 4 fever). The expression of sE-cad in the plasma of 47 patients and 20 healthy volunteers was detected by ELISA; the expression of E-cad on the membrane surface of bone marrow MNC in 40 patients and 15 controls was determined by flow cytometry. Results The plasma level of sE-cad in AL group was significantly higher than that in healthy control group[(66.812 ± 52.712)ng/ml vs.(17.976 ± 14.206)ng/ml, P<0.01]. The plasma level of sE-cad in extra-myeloid infiltration AL group was significantly higher than that in no-extra-myeloid infiltration AL group[(83.545 ± 60.759)ng/ml vs.(42.152 ± 22.043)ng/ml, P<0.01]. The plasma level of sE-cad in high leukocytes AL group was higher than that in no-high leukocytes AL group[(85.166 ± 57.828)ng/ml vs.(41.933 ± 32.064)ng/ml, P<0.05]. The percentage of E-cad expression in AL group was significantly lower than that in control group[(13.615 ± 14.038)% vs.(31.700 ± 16.213)%, P<0.01]. The percentage of E-cad expression in no-extra-myeloid infiltration AL group was significantly higher than that in extra-myeloid leukemia infiltration AL group[(18.691± 14.917)% vs.(6.589± 8.959)%,P<0.01]. The percentage of E-cad in no-high-leukocytes AL group was significantly higher in high leukocytes AL group[(20.925±12.081)% vs.(7.446±11.118)%, P<0.01]. Conclusions The expression of E-cad on the membrane surface of bone marrow MNC and the expression of sE-cad in plasma may be closely associated with the occurrence of extra-myeloid leukemia and leukocytosis, which may be one of the important molecular mechanisms of leukemic cell infiltration and leukocytosis. High expression of sE-cad in plasma can be treated as one of index to diagnose extra-myeloid leukemia. Key words: Leukemia, acute; Leukemic infiltration; E-cadherin; Soluble E-cadherin; High leukocytes

Decreased Expression of Nucleophosmin/B23 Increases Drug Sensitivity of Adriamycin-Resistant Lymphoblastic Leukemia Molt-4 Cells through mdr-1 Regulation

Nucleophosmin /B23 (NPM, also known as B23, numatrin, or N038) is regularly identified as multifunctional nuclear protein, not only an important playerin ribosome biogenesis but also a potential regulator for cell proliferation. Aberrant expression of NPM, such as mutation, deletion, over-expression or rearrangement, could lead to malignant transformation in tumor cells. It is reported that over-expression of NPM in solid tumor had been detected as a poor prognostic factor and related to drug-resistance development. In adult de novo acute myeloid leukemia (AML), mutated NPM without FLT3-ITD showed a good response to chemotherapy in AML. Although a great deal of knowledge of NPM has been accumulated, little information on the role of NPM in drug resistance in leukemia is available. According to our previous investigation, NPM levels are up regulated in drug-resistant cell lines and primary leukemic cells. High expressions of NPM could be observed in refractory or relapsed acute leukemia patients, which significantlycorrelated with poor prognosis. In this study, we wanted to evaluate whether NPM is involved in drug resistance in lymphocytic leukemia cells. We generated an adriamycin (ADM)-resistant lymphoblastic cell line Molt-4/ADR (MAR). Cell proliferation, sensitivity to chemotherapy agents and expressions of drug resistance related molecules were assessed. The IC50 of Molt-4 cells were 0.584±0.113umol/L and of MAR cells were 22.56±1.94 umol/L, meaning MAR cells were 38.63 fold resistant to ADM. The confocal microscopy assay showed that MAR cells had very low levels of ADM fluorescence,however, when treated with the P-gp inhibitor Verapamil Hydrochloride, ADM accumulation in MAR cells increased greatly. These cells were not only resistant to other MDR agents like daunorubicin and vincristine, but also slightly resistant to a panel of non-MDR agents, such as VP-16 and cyclophosphamide. Furthermore, MAR cells gained an expression of mdr-1(P-gp) and a higher expression of NPM compared to Molt-4 cells. Knockdown of NPM by RNA interference (RNAi) suppressed the viability of both Molt-4 and MAR cells. In both Molt-4 and MAR cells, IC 50 of NPM knockdown groups decreased sharply. IC50 of Molt-4 cells was reduced from 0.582±0.112 μmol/L before NPM RNAi to 0.186±0.019μmol/L after NPM RNAi. IC50 of MAR was reduced from 22.56±1.94μmol/L before NPM RNAi to 3.83±0.381μmol/L after NPM RNAi. Knocking down NPM not only increased Molt-4 cells’ drug sensitivity but also reverse resistance of MAR towards Adriamycin. With an increase of drug sensitivity, we found the down-regulation of P-gp and Akt /mTOR signaling in MAR cells after NPM RNAi. To further understanding the relationship between NPM and mdr-1, we applied RNAi to down-regulate mdr-1 in MAR cells. Knockdown of mdr-1 could also reverse the drug resistance. NPM expression revealed no significantly fluctuation after mdr-1 silencing . It could be concluded that knockdown of NPM reversed the drug resistance by down-regulating P-gp and Akt /mTOR signal pathway, indicating that NPM may serve as a potential modulator in drug resistance. DisclosuresNo relevant conflicts of interest to declare.