Cell Leukemia Patients Research Articles

Feasibility of tumor‑derived exosome enrichment in the onco‑hematology leukemic model of chronic myeloid leukemia.

Due to the discovery of their role in intra-cellular communications, exosomes, which carry information specific to the cell of origin, have garnered considerable attention in cancer research. Moreover, there is evidence to suggest the possibility of isolating different exosome sub-populations based on target antigens at the cell surface. Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia characterized by the break-point cluster region-proto-oncogene 1 tyrosine-protein kinase (BCR-ABL1) fusion-gene, derived from the t (9;22) translocation. Tyrosine kinase inhibitors (TKIs) target BCR-ABL1 protein and induce major or deep molecular responses in the majority of patients. Despite the fact that several studies have demonstrated the persistence of leukemic cells in the bone marrow niche, even following treatment, TKIs prolong patient survival time and facilitate treatment-free remission. These characteristics render CML a plausible model for investigating the feasibility of tumor-derived exosome fraction enrichment. In the present study, patients in the chronic phase (CP) of CML were treated with TKIs, and the quantification of the BCR-ABL1 exosomal transcript was performed using digital PCR (dPCR). The possibility of tumor-derived exosomes enrichment was confirmed, and for the first time, to the best of our knowledge, the detection of the BCR-ABL1 transcript highlighted the presence of active leukemic cells in patients with CP-CML. According to these findings, tumor-derived exosomes may be considered a novel tool for the identification of active leukemic cells, and for the assessment of innovative monitoring focused on the biological functions of exosomes in CML.

Engineered human platelet-derived microparticles as natural vectors for targeted drug delivery.

Drug targeting has opened a new paradigm in therapeutics with development of delivery vectors like liposomes and polymeric nanoparticles. Although their clinical application is crippled by limited biological adaptability. Off-target toxicity and biocompatibility still remains one of the critical problems in anticancer therapeutics that can be life-threatening. Here we report a quick, simple and facile method of engineering human platelets to generate drug loaded platelet-derived microparticles (PMPs) by top-down approach, which are biocompatible and naturally target leukemia cells. Drug loaded PMPs and cancer cell uptake were characterized by flow cytometry, confocal microscopy, Nanoparticle Tracking Analysis and fluorimetry. Effective drug delivery was tested in cancer cell lines as well as in clinical samples from leukemia patients. We explored that PMPs are capable of carrying multiple drug payloads, have long shelf life and can be harvested in large quantity in short period. Importantly, PMPs exhibited remarkably higher toxicity towards cancer cells than free drug and had lower escape into extravascular spaces. Transfer of drug to cancer cells of leukemia patients was significantly higher than free drug, when delivered through PMPs. Our experiments validated therapeutic application of PMPs as biocompatible drug delivery vector against cancer cells with minimal off-target delivery.

Transplant-related complications are impediments to the success of allogeneic hematopoietic stem cell transplantation for adult T cell leukemia patients in non-complete remission.

Outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with adult T cell leukemia/lymphoma (ATL) are not satisfactory, particularly in patients in non-complete remission at transplantation (Pt-non-CR). We conducted a regional retrospective study in the ATL endemic area of Okinawa, Japan. Of 62 ATL patients, 21 received allo-HSCT in CR and 41 in non-CR. The 3-year overall survival (3yOS) rate and median survival time for the whole cohort was 25.6% and 7.7 months, respectively. The 3yOS of Pt-non-CR was significantly lower than that of patients in CR (Pt-CR) (16.8% vs. 43.6%, P = 0.005). Transplant-related mortality (TRM) was significantly higher in Pt-non-CR than in Pt-CR (46.3% vs. 15.7%, P = 0.025), while there was no significant difference in disease-associated mortality (DAM) between Pt-non-CR and Pt-CR. Multivariable analysis for Pt-non-CR revealed that poor performance status (poor-PS) and higher sIL-2R level (high sIL-2R) adversely affected OS. Poor-PS was associated with higher TRM, but not with higher DAM in Pt-non-CR. High sIL-2R did not affect TRM or DAM in Pt-non-CR. Overall, high TRM rates rather than DAM contribute to the poor outcomes of Pt-non-CR, suggesting that not only disease control but also management of transplant-related complications is required for allo-HSCT in ATL patients.

Survival Pattern of Hairy Cell Leukemia Patients in the Last 20 Years in Lebanon

Survival Pattern of Hairy Cell Leukemia Patients in the Last 20 Years in Lebanon

Oxidative phosphorylation inhibition induces anticancerous changes in therapy-resistant-acute myeloid leukemia patient cells.

Treatment of acute myeloid leukemia (AML) is still a challenge because of common relapses or resistance to treatment. Therefore, the development of new therapeutic approaches is necessary. Various studies have shown that certain cancers, including some chemoresistant AML subsets, have upregulated oxidative phosphorylation. In this study, we aimed to assess treatment-resistant AML patients' cell modulation using oxidative phosphorylation inhibitors metformin and atovaquone alone and in various combinations with cytosine analog cytarabine and apoptosis inducer venetoclax. Metabolic activity analysis using Agilent Seahorse XF Extracellular Flux Analyzer revealed that peripheral blood mononuclear cells' metabolic state was different among treatment-resistant AML patients. We demonstrated that metformin decreased therapy-resistant-AML cell oxidative phosphorylation ex vivo, cotreatment with cytarabine and venetoclax slightly increased the effect. However, treatment with atovaquone did not have a marked effect in our experiment. Cell treatment had a slight effect on cell proliferation inhibition; combination of metformin, cytarabine, and venetoclax had the strongest effect. Moreover, a slightly higher effect on cell proliferation and cell cycle regulation was demonstrated in the cells with higher initial oxidative phosphorylation rate as demonstrated by gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Proteomic analysis by liquid chromatography-mass spectrometry demonstrated that chemoresistant AML cell treatment with metformin modulated metabolic pathways, while metformin combination with cytarabine and venetoclax boosted the effect. We suggest that oxidative phosphorylation inhibition is effective but not sufficient for chemoresistant AML treatment. Indeed, it causes anticancerous changes that might have an important additive role in combinatory treatment.

Moxetumomab pasudotox for the treatment of relapsed and/or refractory hairy cell leukemia

ABSTRACTIntroduction: Hairy cell leukemia is a rare indolent B-cell malignancy, characterized by pancytopenia, recurrent infections, and splenomegaly. After initial therapy with purine nucleoside analogs, up to 50% of patients relapse after several years of remission. The number of relapsed patients is increasing and, until recently, there was no approved therapy with durable responses for hairy cell leukemia patients in the relapsed setting, thus the need for new non-chemotherapy approach with significant efficacy and less myelosuppression.Areas covered: Moxetumomab pasudotox is a recombinant immunotoxin containing a Fv fragment of an anti-CD22 monoclonal antibody and truncated Pseudomonas exotoxin (PE38). The authors reviewed pre-clinical and clinical studies that led to the FDA approval of the drug in patients with relapsed and/or refractory hairy cell leukemia, who received at least two prior therapies, including at least one purine nucleoside analog.Expert opinion: Moxetumomab pasudotox demonstrated a durable complete remission rate of 30% in heavily pretreated patients with hairy cell leukemia, and MRD eradication in 85% of responding patients. Moxetumomab pasudotox got a global FDA approval in September 2018. The US prescribing information carries boxed warnings regarding the risk of capillary leak syndrome and hemolytic uremic syndrome. Long-term follow-up of the pivotal study is ongoing (NCT01829711).

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

Abstract 4214: A microfluidic platform to capture and detect cancer cells in Leukemia patients

Abstract Introduction : BioFluidica has developed a platform, The Liquid Scan™ to analyze circulating cancer cells in whole blood. The Liquid Scan is unique in its performance metrics compared to existing platforms by employing fully automated instrumentation in conjunction with a highly sensitive and a specific cell detection microfluidic chip. This technology can be applied to a broad variety of tumors, including liquid tumors such as leukemias and lymphomas. The unique design of the microfluidic chip permits the capture and analysis of live circulating leukemic cells (CLCs) from whole blood, even when these cells appear undetectable by other methods. The Liquid Scan has been applied to capture CLCs from patients suffering from acute myeloid, as well as acute lymphoblastic leukemia (AML and ALL, respectively). These leukemias are acute diseases of the blood and bone marrow, with mutational changes and clonal expansion of abnormal myeloid and lymphoblastic precursors, respectively claiming a great number of lives every year. Bone marrow biopsies/aspirations have been the standard methods for reliably diagnosis and monitoring these diseases. These procedures are extremely painful for patients, necessitate specialized centers, and trained personnel, which increases the overall cost of treatment. Methods: We have applied the Liquid Scan™ to capture CLCs and characterization, using specific antibodies to capture known clones of leukemic blasts. As a model, we use Kasumi-1, KG-1 and HL-60 cells for AML, and SupB15 cells for ALL, spiked in healthy whole blood. Results: We characterized the ability of the The Liquid Scan™ to capture and release circulating leukemic cells from whole blood, by using cell line models spiked-in whole healthy donor blood and applied to the microfluidic chip. Our results are directly applicable to patient in different stages of the disease, both as an initial diagnostic tool, and as a way to monitor recovery and diagnose relapse. Conclusions: Precision medicine (PM) is significantly changing the treatment of cancer and is bringing new hope for patients. Diagnostic testing for circulating tumor and leukemic cells are promising prognostic and predictive tools for selecting optimal therapies based on the patient’s genetic content. Highly sensitive, and selective BioFluidica’s system is highly sensitive and selective with the capability of rapidly delivering purified and concentrated circulating cancer cells compatible with downstream molecular diagnostics. Citation Format: Rolf Muller, Rachel Toughiri, Alena Bartakova, Paul Diaz, Craig Carson, Paul Armistead, George Fedoriw, Mathew Foster, Maryam Zomorrodi, Jennifer Barber-Singh, Veronica Cheung, Emily Mirkin, Roksolana Melnychuk, Elizabeth Fabio, Sangeetha Purushotham, Judy Muller-Cohn. A microfluidic platform to capture and detect cancer cells in Leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4214.

Abstract 1741: Identification of microsatellite instability in hematologic malignant cell lines

Abstract Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt’s lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies. Citation Format: Priyanjali Bhattacharya, Naveed Anjum Chikan, Arati Sharma, Dhimant Desai, Charyguly Annageldiyev, Pinaki Patel, Trupti N. Patel. Identification of microsatellite instability in hematologic malignant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1741.

S104 THE BRAF INHIBITOR VEMURAFENIB COMBINED WITH RITUXIMAB PRODUCES A HIGH RATE OF DEEP AND DURABLE REMISSIONS IN RELAPSED OR REFRACTORY HAIRY CELL LEUKEMIA: UPDATED RESULTS OF A PHASE‐2 TRIAL

Background:Up to 50% of hairy cell leukemia (HCL) patients relapse after purine analogs. We identified the BRAF‐V600E kinase mutation as the genetic cause of HCL (Tiacci et al., NEJM 2011). We then documented, in 26 relapsed/refractory patients treated with the oral BRAF inhibitor vemurafenib for a median of 16 weeks, 96% of overall responses (including 35% complete remissions ‐ CR), obtained after a median of 8 weeks (Tiacci et al., NEJM 2015). However, residual bone marrow (BM) HCL cells persisted even in CR patients (5–10% cells) and the median relapse‐free survival among all responding patients was 9 months.Aims:Since HCL strongly expresses CD20, rituximab could improve the efficacy of BRAF inhibition by targeting leukemic cells resistant to vemurafenib.Methods:In this academic, phase‐2, single‐arm, single‐center trial (EudraCT 2014‐003046‐27), relapsed/refractory HCL patients received vemurafenib (960 mg b.i.d.) for 8 weeks and concomitant rituximab (375 mg/m2 i.v.) every 2 weeks. Rituximab was then given as consolidation 4 times every 2 weeks after the end of vemurafenib. CR required normal blood counts, no palpable splenomegaly, and no leukemic cells at morphologic analysis of BM biopsies and blood smears.Results:We enrolled 31 patients (median age: 59 years) with a median of 3 previous therapies, including 8 primary refractory cases (26%). Toxicity was mostly of grade 1‐2, without myelosuppression, and overlapped that of either drug when used alone.Strikingly, a CR was achieved by 26/27 (96%) patients evaluable for efficacy, including 2 patients with incomplete platelet recovery at the end of treatment who however normalized the platelet count soon after; and 2 patients with incomplete resolution of splenomegaly who nonetheless remain in otherwise continuous CR at 22.5 and 25 months from the end of treatment. Among the 26 CR cases, all previously treated with purine analogs, there were also some patients previously refractory to rituximab (n = 5) and/or who had relapsed after a prior BRAF inhibitor (n = 7; 5 obtained a short‐lived PR and 2 a CR after treatment with the BRAF inhibitor). Notably, a CR was obtained after just 4 weeks of vemurafenib and two concomitant doses of rituximab in 15/24 evaluable patients (63%). Minimal residual disease (MRD) by allele‐specific PCR (sensitivity: 0.05% BRAF‐V600E alleles) was absent in the BM of 17/26 (65%) patients; in 8/17 patients (47%), MRD clearing was obtained before rituximab consolidation.Progression‐free survival (PFS) in the 29 evaluable patients was 83% at a median follow‐up of 29.5 months (range: 2–45) (Fig. 1A). Progressions (n = 5) always occurred in cases who were MRD‐positive, including 3/5 patients whose immediate prior treatment was a BRAF inhibitor (leading to PR in all 3 cases). Indeed, PFS was significantly longer in MRD‐negative CR cases (100% in 17 patients at 30.5 months of median follow‐up) than in MRD‐positive CR cases (44% in 9 patients at a median follow‐up of 24.5 months) (p = 0.001; Fig. 1B). In subsequent BM evaluations, 16/17 (94%) CR patients maintained a MRD‐negative status after a median of 22.5 months (range 12.5–36.5); in the remaining patient, MRD reappeared 12.5 months later but he remains in continuous CR at 38 months after the end of treatment.Summary/Conclusion:Vemurafenib plus rituximab is a brief, safe and non‐myelotoxic regimen that induces MRD‐negative durable responses in the majority of relapsed/refractory HCL patients. Randomized testing of this regimen against the chemotherapy‐based standard of care in the frontline setting is warranted.image

PB1696 EVALUATION OF TUMOR CELL SENSITIVITY TO DAUNORUBICIN USING WST1 TEST IN ACUTE MYELOBLASTIC LEUKEMIA PATIENTS

Background:In recent years the MTT test for the assessment of the sensitivity of tumor cells to cytotoxic drugs in leukemia patients were used. In present study, the possibility of the WST1 test as a screening method of drug sensitivity of tumor cells of leukemia patients using peripheral blood and bone marrow samples was evaluated.Aims:The aim of this work was to study the effectiveness of the WST1 test for the initial estimation of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the treatment response of patients with acute myeloblastic leukemia.Methods:The study included 42 patients with acute myeloblastic leukemia (AML). The average age of the patients was 53.3 ± 12.7 years. Leukemia cells were obtained from peripheral blood and bone marrow of patients. The sensitivity of tumor cells of patients with AML to daunorubicin in vitro using the WST1 test was made prior to initiating of cytotoxic therapy. Confirmation of multidrug resistance by evaluation of MDR1 mRNA expression levels in tumor cells of AML patients was performed on bone marrow and/or peripheral blood samples using quantitative real‐time reverse transcription‐polymerase chain reaction (real‐time qPCR).In all patients, the diagnosis was confirmed by the study of myelogram, flow cytofluorimetry, immunocytochemistry, cytogenetic analysis, as well as standard laboratory and instrumental studies that are included in the standard examination of patients with leukemia. Based on immunopenotyping and cytogenetic analysis, each patient was assigned to a risk group and prognosis. The obtained results were compared with the clinical and hematological response of these patients to cytotoxic therapy, as well as with the MDR1 mRNA expression level.Also we evaluated the survival of leukemia patients depending on the sensitivity to daunorubicin and MDR1 mRNA expression levels in tumor cells of AML patients.All statistical analyses were done with computer software MS Excel, OriginPro 7.5, Statistica 10.0.Results:Regardless of the class of chemotherapeutic drugs administered to the patients, the survival rate was higher in the group of patients with high sensitivity to daunorubicin and low expression levels of the MDR1 mRNA in tumor cells. Patients received daunorubicin within the “7 + 3” regimen (7 days of cytarabine and 3 days of daunorubicin) showed a high correlations (r = 0.8, p <0.05) between the expression levels of MDR1 mRNA and sensitivity to daunorubicin, moderate correlations (r = 0.63, p < 0.05) between the response to chemotherapy and sensitivity to daunorubicin, and a moderate correlations between the prediction (r = 0.48, p < 0.05) and the risk group (r = 0.49, p < 0.05) and sensitivity to daunorubicin. It is important to note that a high correlations (r = 0.86, p < 0.05) was found between the expression levels of MDR1 mRNA and the response to therapy.Summary/Conclusion:The obtained data demonstrate that the WST1 test can be effectively used as a screening method for the initial assessment of the sensitivity of tumor cells to daunorubicin in vitro and prediction of the response to treatment of AML patients.

Expression Difference of RhoH Gene in Leukemia and Its Clinical Significance

To investigate the expression and clinical significance of RhoH gene in bone marrow cells of leukemia patients. 31 cases of leukemia and 15 cases of non-tumor as controls were collected. The expression of RhoH in bone marrow cells was detected by real-time quantitative PCR (RQ-PCR). The median expression level of RhoH was used as the cut-off value. The newly diagnosed patients were divided into RhoH high expression group and low expression group. The relationship of different RhoH expression levels with clinical features and prognosis of newly diagnosed patients was analyzed. The mRNA expression of RhoH in the bone marrow cells of 31 cases of leukemia was significantly lower than that in the control group, mRNA expression of RhoH in the ALL group was significantly lower than that in AML group (P＜0.05). Compared with the RhoH high expression group, the proportion of bone marraw blasts and LDH level in the RhoH low expression group was significantly increased (P＜0.05), but there were no significant differences in clinical features such as age, white blood cell count, hemoglobin level, platelets count, PCT and CRP level (P＞0.05). In AML, the recurrence rate after standard chemotherapy in RhoH low expression group was higher than that in high expression group, while the expression of RhoH not correlated with other prognostic genes of AML. In ALL, the recurrence rate in RhoH low expression group was not statistically significant different from that in high expression group. RhoH may be involved in the genesis of acute leukemia. In AML, RhoH expression negatively correlates with recurrence rate, which can be used as a prognostic indicator independently. In ALL, RhoH may participate in the disease process through other mechanism.

Correlation of minimal residual disease negativity (MRD-) with prognosis in high-risk myeloma (HRM).

8024 Background: MRD is a standard measurement for response assessment in MM. Despite new treatments, HRM patients continue to have poor prognosis. We evaluated the impact of MRD- in high vs standard (SRM) risk patients. Methods: We retrospectively evaluated all consecutive MM/plasma cell leukemia patients who had routine MRD testing by 1-tube 8-color advanced flow cytometry with 2,000,000 events and sensitivity level 10-5 at our center from 2015-2018 after initial therapy. Kaplan-Meier and log-rank test were used to assess survival estimates and differences between study groups. Results: 136 patients with MRD testing after initial therapy/autologous transplant (ASCT) were identified. Patient and disease characteristics are included in Table. At a median follow-up of 14 months (1-36), PFS and OS were significantly worse in HRM vs SRM. During the study period, 50% of HRM had progressed (relapse and/or death) vs 20% in SRM (p=0.0006). No patients with SRM died, but 4 (14%) in the HRM group did (p=0.0007). Regardless of MRD status, HRM patients had statistically significant worse PFS than SRM; at median f/u 10% SRM MRD-; 20% SRM MRD+; 40%HRM MRD-; 45% HRM MRD+ had either relapsed or died (p=0.0041). MRD status did not impact OS in either group (p=0.0914), however longer follow up is needed to assess survival. Conclusions: Genetic abnormalities (FISH/GEP) remain a powerful prognostic indicator for myeloma regardless of MRD status. For newly diagnosed myeloma patients treated with novel triple initial therapy and frontline ASCT, achieving MRD negative status didn’t mitigate the poor prognosis outcomes of HRM. [Table: see text]

Retrospective Review of the Use of High-Dose Cyclophosphamide, Bortezomib, Doxorubicin, and Dexamethasone for the Treatment of Multiple Myeloma and Plasma Cell Leukemia

BackgroundMultiple myeloma (MM) usually follows a clinical course leading to refractoriness and limited treatment options in advanced stages, which might need bridge therapies to either autologous stem cell transplantation or novel therapies. We report our experience with the high-dose chemotherapy mCBAD (modified cyclophosphamide, bortezomib, doxorubicin, and dexamethasone) regimen in newly diagnosed MM (NDMM), relapsed/refractory MM (RRMM), and plasma cell leukemia (PCL) patients. Patients and MethodsWe searched our electronic records database for MM patients who received mCBAD from 2010 to 2016 for 28-day cycles of cyclophosphamide 350 mg/m2 intravenously (I.V.) twice daily with mesna 400 mg/m2 I.V. daily (days 1-4), bortezomib 1.3 mg/m2 subcutaneously/I.V. (days 1, 4, 8, 11), doxorubicin 9 mg/m2 daily continuous infusion (days 1-4), dexamethasone 40 mg orally daily (on days 1-4, 9-12, 17-20). International Myeloma Working Group (IMWG) criteria were used for response assessment and diagnosis. Descriptive statistics, Fisher exact test, χ2, Wilcoxon rank sum, and Kaplan–Meier were used for statistical purposes. ResultsOne hundred forty patients met the inclusion criteria. A median of 2 cycles of therapy was administered. The overall response rate was 85% in patients with RRMM (n = 116) and 100% in NDMM (n = 13) and PCL (n = 11) patients. Respective median progression-free survival (mPFS) for NDMM, PCL, and RRMM were 19.61 months (95% confidence interval [CI], 5.26 to not applicable [NA]), 7.56 months (95% CI, 4.7 to NA), and 4.64 months (95% CI, 3.75-6.73). Patients with RRMM who used mCBAD as a bridge to autologous transplant (36.2%) had mPFS (11.48 months; 95% CI, 7.52-15.9 months) compared with those who did not (mPFS: 3.19 months; 95% CI, 2.4-3.75 months). Cytopenias occurred in more than 90% of patients, and febrile neutropenia was noted in 26%. All cases of treatment-related mortality (8%) occurred in patients with RRMM, except for 1 patient with PCL. ConclusionmCBAD results in high response rates in myeloma and PCL, however, with high treatment-related mortality. Its use in RRMM should be limited to patients who have immediate need for therapy without other treatment options and who have good performance status (score of 0-1) or NDMM if novel agents are not available depending on practice setting. mCBAD can be a treatment option for patients with PCL.

HAP1 loss confers l-asparaginase resistance in ALL by downregulating the calpain-1-Bid-caspase-3/12 pathway

Abstractl-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3. Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase–induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.

Retrospective Analysis of Hairy Cell Leukemia Patients Treated with Different Modalities as First Line: Real-Life Experience Over 20years.

We aimed to analyze the characteristics and response rates of different treatment modalities in hairy cell leukemia patients over 20 diagnosed as hairy cell leukemia (HCL). Clinical data, response rates and survival outcome of the patients who were diagnosed with HCL were retrospectively analyzed. Fifty-two patients with a median age of 50 (28-87) years were enrolled in the study. 38 patients (73%) were male and male to female ratio was 2.7. First line therapy was cladrabine in 36 patients (69.2%). The overall response rate was 97%. CR and PR rates were 86.1% and 11.1%, respectively. Interferon was used in 10(19.2%) patients who were diagnosed before 2000syears. CR and PR rates were 70% and 30%, respectively. Although the CR rates were lower in IFN group, this difference could not be reached statistically significance (p = 0.24). The median follow up was 48months (12-252). The median OS was not reached and median PFS was 150months (95% CI, 116-214). The OS at 36 and 48months were 95.9% and 92.3%, respectively and the PFS at 36 and 48months were 90.2% and 83.4%, respectively. After the introduction of purine analogues, the fate of the HCL patients have been changed. Cladrabin achieved very high response rates in both young and older patients, in our study. Although relapse still constitutes a problem, another single dose of cladrabine results in good response rates.

Clinical and Prognostic Significance of Cell Sensitivity to Chemotherapy Detected in vitro on Treatment Response and Survival of Leukemia Patients.

Multidrug resistance (MDR) is a major challenge in leukemia treatment. The objective of this study was to identity predictors of MDR to allow for rapid and economical assessment of the efficacy of planned antitumor therapy for leukemia patients. The study included 113 patients with acute and chronic leukemias. Prior to antitumor therapy, we measured the sensitivity of tumor cells of patients to the panel of chemotherapeutic drugs, together with MDR1 mRNA and P-glycoprotein (P-gp) expression as one of the mechanisms of MDR, and compared these data with the response to therapy. The scales for leukemia patients according to therapy response, drug sensitivity of tumor cells, MDR1 mRNA and P-gp levels, and the presence of unfavorable immunological and cytogenetic markers were introduced for subsequent correlation analysis. We show that the drug resistance of tumor cells of leukemia patients estimated in vitro at diagnosis correlates with a poor response to chemotherapy and is usually combined with aberrant and immature immunological markers, cytogenetic abnormalities, and a high expression of MDR1 mRNA and P-gp. All together, these factors indicate unfavorable prognosis and low survival of leukemia patients. Thus, the sensitivity of tumor cells to chemotherapeutic drugs measured in vitro at diagnosis may have prognostic value for individual types of leukemia.

Ibrutinib Therapy Releases Leukemic Surface IgM from Antigen Drive in Chronic Lymphocytic Leukemia Patients.

In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated in vivo and recover in vitro, suggesting a reversible influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM. We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL. At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture in vitro. The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway. This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy.See related commentary by Burger, p. 2372.

HSR19-086: Healthcare Costs and Resource Utilization Associated With Adverse Events Among Hairy Cell Leukemia Patients Treated With Purine Nucleoside Analogs

Background: Purine nucleoside analogs (PNAs) are highly effective for first-line treatment of hairy cell leukemia (HCL). In clinical trials of single PNAs, several adverse events (AEs) were reported; however, little is known regarding the costs and healthcare resource utilization (HRU) resulting from AEs in HCL patients (pts) treated with PNAs in non-clinical trial settings. Objective: Determine the costs and HRU of high incident and clinically important AEs associated with PNA therapy in HCL pts in the Truven MarketScan database. Methods: Adults (aged ≥18 years) with ≥2 HCL diagnosis codes ≥30 days apart during January 1, 2006–December 31, 2015 were included. Pts had ≥1 prescription claim for a PNA (cladribine or pentostatin ± rituximab) after HCL diagnosis date. First PNA claim date was defined as the index date. Pts had continuous health plan enrollment for ≥6 months at baseline and ≥12-months follow-up with no PNA in the baseline period. Pts were placed into cohorts based on the occurrence of myelosuppression (MSPN) and opportunistic infections (OI) as these were highest incident and clinically important AEs observed. Generalized linear models were used to compare outcomes during the 12-month follow-up. Results: Of the 219 pts with no history of MSPN, 101 developed MSPN (incidence [I]: 461 per 1000 pt-years) and of 619 pts with no history of OI, 26 developed OI (I: 42 per 1000 pt-years). Demographics were similar between pts with and without MSPN and OI. Pts who developed OI or MSPN had significantly higher inpatient admissions and costs (Table 1). Conclusions: PNA-treated HCL pts who developed MSPN or OI incurred higher HRU than those who did not develop either condition. This indicates the need for new therapeutic strategies to reduce HCL-treatment-associated toxicities.

Abstract A024: Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting

Abstract Our aim was to identify a T-cell receptor (TCR) that recognizes a leukemia-associated self-antigen that is highly expressed in acute myeloid leukemia and that is restricted by HLA-A2 (expressed in approximately 50% of Caucasians) for future adoptive T-cell therapy. Tumor-associated self-antigens (self-TAA) make attractive targets for adoptive T-cell therapy in cancer. However, autologous T-cells are tolerant to self-TAA expressed on self-HLA if antigen expression is sufficiently high. In contrast, T-cells are not tolerant to self-antigens presented on non-self HLA. By identifying alloreactive T-cells reactive to self-peptide in complex with foreign HLA and subsequently cloning their TCR, it is possible to redirect patient T-cells against self-TAA. The advantage of targeting self-antigens is the potential application of TCR-engineered T-cells in all cancer patients with a certain cancer type and expressing the restricting HLA molecule. We identified a protein that is selectively expressed in normal myeloid cells and highly expressed by leukemic cells in patients with acute myeloid leukemia (AML). HLA-A2 negative CD8 T-cells from healthy donors were stimulated with HLA-A2 positive dendritic cells that were expressing the full-length target antigen. CD8 T-cells recognizing peptides from the target antigen in complex with HLA-A2 were subsequently identified and sorted using fluorescently labeled HLA-A2-peptide-multimers. T-cell clones obtained from sorted multimer-positive T-cells responded to HLA-A2 positive patient leukemia cells and cell lines expressing the antigen, whereas no response was seen to HLA-A2 positive antigen negative targeT-cells, unless loaded with the relevant peptide. Responses were measured as secretion of interferon gamma, expression of the T-cell activation marker CD137 or expression of the degranulation marker CD107. The TCR sequences were identified, and we next evaluated specificity and functionality of TCR-engineered cells. Indeed, TCR-transduced healthy donor T-cells were able to selectively kill HLA-A2 positive AML patient-derived leukemic cells as well as antigen positive cell lines in vitro. We also performed a comprehensive peptide scan to determine potential cross reactivity of the obtained TCR. For this scan, the amino acids (AA) in each position of the target peptide-epitope were individually replaced by every other natural amino acid (19 AA for each position x 9 postions = 171 peptides). In addition, potential responses to other variants of the 9mer target peptide based on the natural antigen protein sequence were evaluated (8mer, 10mers, 11mers and 12mers). Based on the results obtained with TCR-transduced T-cells (interferon gamma ELISA), candidate peptides naturally occurring in the proteome to which our TCR could cross-react were not identified. These data warrant further evaluation of the TCR for in vivo efficacy and specificity. Citation Format: Maxi-Lu Böschen, Weiwen Yang, Erlend Strønen, Johanna Olweus. Identifying a T-cell receptor for immunotherapy against a leukemia-associated self-antigen in an allogeneic setting [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A024.