Acute Lymphoblastic Leukemia Blast Cells Research Articles

An Intelligent Telediagnosis of Acute Lymphoblastic Leukemia using Histopathological Deep Learning

Leukemia, a global health challenge characterized by malignant blood cell proliferation, demands innovative diagnostic techniques due to its increasing incidence. Among leukemia types, Acute Lymphoblastic Leukemia (ALL) emerges as a particularly aggressive form affecting diverse age groups. This study proposes an advanced mechanized system utilizing Deep Neural Networks for detecting ALL blast cells in microscopic blood smear images. Achieving a remarkable accuracy of 97% using MobileNetV2, our system demonstrates high sensitivity and specificity in identifying multiple ALL sub-types. Furthermore, we introduce cutting-edge telediagnosis software facilitating real-time support for clinicians in promptly and accurately diagnosing various ALL subtypes from microscopic blood smear images. This research aims to enhance leukemia diagnosis efficiency, which is crucial for the timely intervention and managing this life-threatening condition.

Fuzzy clustering of Acute Lymphoblastic Leukemia images assisted by Eagle strategy and morphological reconstruction

Patients with Acute Lymphoblastic Leukemia (ALL) require prompt diagnosis since it has the chance to become fatal if neglected for a few weeks. The microscopic image of lymphocyte cells creates doubts even for expert pathologists because normal lymphocytes cells and ALL blast cells are both very smooth. Therefore, proper segmentation of White Blood Cells (WBC) is a very crucial aspect of the task. Consequently, the focus of the study is on segmenting the WBCs in Acute Lymphoblastic Leukemia (ALL) images utilizing classical crisp and fuzzy clustering approaches like K-means (KM) and Fuzzy C-means (FCM). But these classical clustering approaches are very sensitive to noise and initial cluster center initialization and hence trapped in local optima. As a result, these techniques may produce incorrect cluster centers. Firstly, researchers are employing Nature-Inspired Optimization Algorithms (NIOAs) as an alternate methodology for both crisp and fuzzy clustering problems to solve the initial cluster center initialization issue. Therefore, using a two-stage Eagle Strategy based on Stochastic Fractal Search (SFS) method, this research proposes a fuzzy clustering methodology. Secondly, morphological reconstruction has been employed for filtering the membership matrix to guarantee noise-immunity. A scrupulous parallel study is performed among the proposed eagle strategy based fuzzy clustering depending on morphological reconstruction with some well-known NIOA based fuzzy clustering and crisp clustering approaches, and classical clustering methodologies like KM and FCM in view of a collection of color ALL images and regular performance metrics. Experimental results demonstrate that recommended ES-SFS based fuzzy clustering technique with morphological reconstruction surpasses the most of utilized approaches in words of computation effort, quality metrics, and robustness. Additionally, to get rid of the random effect in the achieved numerical results, a non-parametric strategy is used for statistical validation.

Results from an Ongoing Phase 1 Study Indicate ACDT-301 (Camidanlumab Tesirine) Is Well-Tolerated in Patients with Relapsed or Refractory CD25-Positive Acute Leukemia

Introduction: There is a significant need for improved therapeutics for older patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Five-year survival rates dip sharply as patient age increases, falling to ~40-45% for patients with ALL over 40 years of age and ~10% for adults with AML over 65 years of age. Cell surface expression of CD25 on AML and ALL blast cells is associated with adverse outcomes, including induction failure, relapse, and shorter overall survival. ADCT-301 (camidanlumab tesirine) is an antibody drug conjugate composed of a human CD25-targeting monoclonal antibody conjugated to a pyrrolobenzodiazepine dimer cytotoxin. Camidanlumab tesirine (Cami-T) has demonstrated anti-tumor efficacy in mouse xenograft models of CD25-expressing hematologic malignancies. We present interim data from a Phase 1 study of Cami-T treatment in patients with relapsed or refractory (R/R) CD25-positive acute leukemia.Methods: A Phase 1, open-label, dose-escalation (part 1) and expansion (part 2) multicenter, US study is enrolling patients with R/R CD25-positive acute leukemia. The primary objective is to assess the tolerability and safety of Cami-T and determine the maximum tolerated dose (MTD). Investigator-assessed anti-tumor activity, pharmacokinetics (PK), and the presence of anti-drug antibodies are also being evaluated. Eligible patients are ≥18 years of age, have CD25-positive AML or ALL without extramedullary disease, have failed or are intolerant to established therapy, and have an ECOG performance status of 0-2. CD25-positive AML or ALL is defined as CD25 expression on ≥5% of leukemic cells within bone marrow aspirate or biopsy. In Part 1, patients are assigned to treatment using a 3+3 dose-escalation design, based on assessment of dose-limiting toxicities (DLTs) during Cycle 1, to determine the MTD. Cami-T is given as an intravenous infusion on Day 1 of each cycle. The first cohort started treatment on a 21-day (q3w) cycle at a starting dose of 3 μg/kg. Dose frequency in subsequent cohorts may increase to once-weekly (qw) in the absence of DLTs. Part 2 will further evaluate safety, tolerability, PK and clinical activity at the dose recommended from Part 1.Results: As of June 26, 2017, 29 patients (20 male, 9 female) with AML have been treated with Cami-T (median 2 cycles, range: 1-7). The median age is 67 years [range 22-82] and patients had received a median of 3 (range: 1-9) previous chemotherapies. Five (17%) patients had undergone prior allogeneic stem cell transplantation. No DLTs were observed up to the highest evaluated q3w dose of 92 µg/kg. After switching to weekly dosing, one DLT was reported in the 30 μg/kg dose group. A total of 250 treatment-emergent adverse events (TEAEs) were reported in 28/29 (97%) patients. The most common TEAEs were fatigue and nausea (both n=7) followed by diarrhea, pneumonia and pyrexia (all n=5; Table). Grade ≥3 TEAEs were reported by 22/29 (76%) patients, of which fatigue (n=4), febrile neutropenia (n=4) and pneumonia (n=3) were the most common. Five deaths from TEAEs were recorded (one case each of blood disorder, cardiac arrest and pneumonia and two cases of disease progression). Two patients experienced infusion-related reactions. Three patients experienced TEAEs leading to a dose delay or reduction (one case each of skin rash, pericarditis and supraventricular tachycardia), and one patient each in the 52 μg/kg q3w and 30 μg/kg qw dose groups discontinued treatment due to Grade 3 skin rash. Transient CD25+ blast clearance in 2 patients who received 1 and 7 cycles, respectively, of Cami-T 32 μg/kg q3w, was observed, supporting an on-target activity of Cami-T. No responses or remissions have been obtained to date. PK data show increasing Cami-T total and PBD-conjugated antibody exposure with dose. Rapid systemic clearance of the drug with levels below limits of quantification suggest that q3w dosing may be insufficient for therapeutic efficacy, prompting exploration of a qw schedule.Conclusions:In this ongoing Phase 1 study in patients with CD25-positive R/R AML, single-agent Cami-T has shown an acceptable safety profile thus far. Additional patients with AML and ALL are being enrolled and dose escalation will continue until the MTD is identified, with further results expected later this year.Study sponsored by ADC Therapeutics. https://clinicaltrials.gov/ct2/show/NCT02588092 [Display omitted] DisclosuresGoldberg:Pfizer: Research Funding; ADC Therapeutics: Research Funding; Genentech: Research Funding. Solh:ADC Therapeutics: Research Funding; Amgen: Speakers Bureau; Celgene: Speakers Bureau. Ungar:ADC Therapeutics: Employment, Other: Stock option interest. Rizzieri:Erytech: Research Funding; Shire: Research Funding. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding. Spira:ADC Therapeutics: Research Funding. Chung:ADC Therapeutics: Research Funding. Stock:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy. He:ADC Therapeutics: Employment, Other: Potential equity interest. Boni:ADC Therapeutics: Employment, Other: Potential equity interest. Atallah:ADC Therapeutics: Research Funding.

Development and Validation of a Sensitive UHPLC-MS/MS-Based Method for the Analysis of Folylpolyglutamate Synthetase Enzymatic Activity in Peripheral Blood Mononuclear Cells: Application in Rheumatoid Arthritis and Leukemia Patients.

Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs such as methotrexate (MTX), which is commonly used for the treatment of (pediatric) leukemia and the anchor drug in rheumatoid arthritis (RA) treatment. To date, assessment of FPGS catalytic activity relies on assays using radioactive substrates that are labor-intensive and require relatively large numbers of cells. Here, we describe a nonradioactive, ultra-high-performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS)-based method allowing for sensitive and accurate measurements of FPGS activity in low cell numbers (ie, 1-2 × 10) of biological specimens, including leukemic blast cells of acute lymphoblastic leukemia patients and peripheral blood mononuclear cells of patients with RA. The UHPLC-MS/MS assay was validated with 2 CCRF-CEM human leukemia cells, one proficient and one deficient in FPGS activity. Linearity of time and protein input were tested by measuring FPGS activity at 30-180 minutes of incubation time and 10-300 mcg protein extract. In addition, FPGS enzyme kinetic parameters were assessed. The FPGS enzymatic assay showed a linear relation between FPGS activity and protein input (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed for evaluation of FPGS enzyme kinetic parameters revealing Km values for the substrates MTX and L-glutamic acid of 64 µmol/L and 2.2 mmol/L, respectively. The mean FPGS activity of acute lymphoblastic leukemia blast cells (n = 4) was 3-fold higher than that of CCRF-CEM cells and 44-fold and 88-fold higher than that of peripheral blood mononuclear cells from MTX-naive (n = 9) and MTX-treated RA patients (n = 6), respectively. Collectively, given its sensitivity with low cell numbers and avoidance of radioactive substrates, UHPLC-MS/MS-based analysis of FPGS activity may be eligible for routine therapeutic drug monitoring of MTX in RA and leukemia for therapy (non)response evaluations.

Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogeny-related developmental programs

AbstractHuman lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid–restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid–restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.

PF194 EVALUATION OF TELOMERE LENGTH AND TELOMERASE ACTIVITY AS PROGNOSTIC MARKERS IN A PEDIATRIC PATIENT COHORT WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Telomere length maintenance is tightly regulated with cell proliferation and senescence. To overcome physiologic telomere attrition with cell division, long‐term repopulating cells like stem and progenitor cells upregulate telomerase. Pathophysiologically, over 90% of cancer cells reactivate telomerase enabling replicative immortality. Acute lymphoblastic leukemia (ALL) is the most common type of acute leukemias in pediatric patients (pts) and a neoplastic disease with a rapid clonal expansion.Aims:We aimed to assess the mitotic history of the malignant ALL clone by telomere length (TL) and the potential of telomerase activity (TA) as a prognostic marker.Methods:Peripheral blood samples collected before treatment initiation from 9 pediatric pts (4 females/ 5 males) with newly diagnosed pre‐B cell ALL according to WHO 2016 criteria were analyzed for TL and TA. All pts were treated according to the international collaborative protocol (AIEOP‐BFM ALL 2009 Registry) for children and adolescents with ALL. Based on (cyto)genetics and treatment response, 6 pts are in the intermediate and 3 pts are in the high‐risk prognostic group. TL was measured in subsets of leukocytes by automated multicolor flow‐FISH and compared to TL percentiles of the reference range obtained from over 400 healthy individuals. TA in leukocytes was analyzed by Telomere Repeat Amplification Protocol.Results:The median age of the patient (pt) cohort was 10.8 years (range: 2.8–17.8) at diagnosis and study entry. The median leukocyte count was 9.8 G/L (1.4–38.6) with a median lymphocyte count of 2.0 G/L (0.6–8.1) and a median lymphoblast count of 6.37 G/L (0.2–31.27). The median TL value in blasts was with 5.2 kb (1.9–8.3) significantly (p = 0.016) lower than 7.5 kb (6.9–9.3) for the B‐cell subset. In 6/9 pt samples the TL values for blasts were below the 1st percentile of the TL reference range for lymphocytes whereas 3 pts had values between the 1st and 50th percentile. The median age‐adjusted difference in TL (dTL = difference of TL to the 50th percentile for respective age) was −4.5 kb (−7.1–1.1) in blasts which was significantly higher than median dTL values in lymphocytes (−2.1 kb; range −6.2–0) and B‐cells (−1.9 kb, range −2.6–0.4). Median TA in the mononuclear cell fraction containing blasts was 2.4 telomere‐elongated products (TP)/cell (0.3–11.1), whereas no TA was detected in leukocytes from healthy donors. Notably, pediatric ALL pts with a high risk score (n = 3) had a higher median TA of 5.5 TP/cell (2.2–11.1) than those with an intermediate risk score (n = 6) with 1.8 TP/cell (0.3–3.3).Summary/Conclusion:The significantly lower TL values in ALL blast cells from newly diagnosed pediatric pts reflect the mitotic history of the malignant clone with a high number of cell divisions. Despite measurable TA in ALL blasts, attrition of TL seemed not compensated and therefore, the extent of clonal proliferation might even be underestimated. Importantly, the higher median TA in blast cells of pts in the high‐risk prognostic group indicates that TA could be an attractive prognostic marker. Further data on TL and TA on serial pt samples will identify if TA could also serve as predictive marker and therapeutic target.

Antibody-based therapies in patients with acute lymphoblastic leukemia.

The use of multiagent combination chemotherapy regimens results in cure rates of >90% for children and ∼40% for adults with acute lymphoblastic leukemia (ALL) but is associated with extensive toxicity and disappointingly low efficacy in relapsed patients. ALL blast cells express several surface antigens, including CD20, CD22, and CD19, which represent valuable targets for immunotherapy. Monoclonal antibodies, antibody-drug conjugates, and bispecific T-cell-engaging antibodies targeting these antigens offer novel mechanisms of action. Within the last several years, the anti-CD20 antibody rituximab has been added to chemotherapy for newly diagnosed patients <60 years with CD20+ pre-B ALL and significantly improved the 2-year event-free survival from 52% to 65%. In adults with relapsed or refractory CD22+ ALL, the antibody-drug conjugate inotuzumab ozogamicin resulted in a complete response rate of 81% and median overall survival of 7.7 months with reduced toxicity compared with standard chemotherapy. Similarly, the bispecific T-cell-engaging antibody blinatumomab yielded a complete response rate of 44% and a median overall survival of 7.7 months in an extensively treated ALL population. Moreover, ∼80% of ALL patients in complete remission with evidence of minimal residual disease (MRD) achieved a complete MRD response following treatment with blinatumomab. These results highlight the tremendous promise of antibody-based treatment approaches for ALL. Ongoing and future research is critical to further define the role of the various immunotherapies in the frontline treatment of ALL. Additional challenges include the optimal sequencing of the available antibodies in the relapsed setting as well as their integration with stem cell transplant and chimeric antigen receptor T-cell therapy.

Comparison of CD22 Expression between Baseline, End of Treatment, and Relapse Among Patients Treated with Inotuzumab Ozogamicin Who Responded and Subsequently Relapsed in Two Clinical Trials

Introduction: Downregulation or loss of B-cell ALL surface antigens (most notably CD19) in patients with acute lymphoblastic leukemia (ALL) targeted by various therapeutic modalities including CART and bi-specific T-cell engagers (BiTEs) has been implicated in acquired resistance to those therapies. Inotuzumab Ozogamicin (InO) is a calicheamicin-conjugated antibody targeting CD22 on ALL blast cells. In the Phase 3 INO-VATE trial, patients with refractory (R/R) ALL who received InO vs standard of care chemotherapy (SC) achieved greater remission, MRD negativity, and improved survival. This study reports CD22 expression levels at baseline, end of treatment (EOT) and relapse in R/R ALL patients who received InO or SC as salvage therapy and subsequently relapsed; the goal of this analysis was to understand if relapse was associated with changes in CD22 expression.Methods: The study population consisted of R/R ALL patients who were treated with InO or SC as part of two clinical trials: Phase 1/2 Study 1010 (NCT01363297; Phase 1, Expansion Phase, + Phase 2 at a dosage of 1.8 mg/m2) and Phase 3 Study 1022 (NCT01564784). This analysis incorporated CD22 expression levels at baseline, at end of treatment, and at relapse. Central laboratory flow cytometry was used to assess CD22 expression, which was quantified as % CD22-positive leukemic blasts and as Molecules of Equivalent Soluble Fluorochrome (MESF), a quantitative measure of CD22 density on leukemic blasts. Outcomes are reported in InO (Study 1022 or 1010) and SC patients who responded to treatment (patients who achieved complete remission/complete remission with incomplete hematologic recovery [CR/CRi]) and subsequently relapsed. CR was defined by <5% marrow blasts and the absence of peripheral blood leukemic blasts; with recovery of hematopoiesis.Results: 25 InO patients (Study 1022, n=19; Study 1010, n=6) and 11 SC patients who responded to treatment and subsequently relapsed were evaluable for central-lab CD22 analysis at baseline. Among InO patients who responded to treatment, the majority had leukemic blasts that were ≥90% CD22-positive at baseline (Study 1022, 66.7%; Study 1010, 66.7%); of the evaluable patients at EOT (Study 1022, n=4; Study 1010, n=0), leukemic blast CD22-positivity was >0-<70%. At relapse, all evaluable InO patients had leukemic blasts with detectable CD22-positivity; with blasts that were predominately <90% CD22 positive (Study 1022, n=6/7; Study 1010, n=5/6 [Table 1]). In contrast, the majority of evaluable subjects who received SC and achieved CR/CRi exhibited CD22 positivity that remained ≥90% at EOT and at relapse When CD22 expression was quantified as MESF, a similar trend was evident (Table 2). In Study 1022, InO patients had a median CD22 MESF of 4085.0 at baseline, which declined to 94.0 at EOT and 418.0 at relapse. In contrast, the median CD22 MESF at EOT and at relapse (3738.0 and 3738.0, respectively) was similar to baseline (4065.0) for SC patients.Conclusions: Among R/R ALL patients who responded to InO treatment and subsequently relapsed, a decrease in CD22 positivity and receptor density was evident from baseline to EOT and relapse, but emergent CD22 negativity was not evident. These results suggest that recurrent disease is associated with decreased CD22 expression, but is not generally attributable to the outgrowth of CD22-negative clones. [Display omitted] DisclosuresStock:Jazz Pharmaceuticals: Consultancy. Cassaday:Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Incyte: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Kite Pharma: Research Funding; Adaptive Biotechnologies: Consultancy; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. DeAngelo:Pfizer Inc: Consultancy, Honoraria; BMS: Consultancy; Glycomimetics: Research Funding; Shire: Honoraria; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Amgen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria; Takeda: Honoraria; Incyte: Consultancy, Honoraria. Jabbour:Novartis: Research Funding; Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. O'Brien:Pharmacyclics: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Alexion: Consultancy; Sunesis: Consultancy, Research Funding; Regeneron: Research Funding; TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Janssen: Consultancy; Gilead: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; Vaniam Group LLC: Consultancy; Pfizer: Consultancy, Research Funding; Amgen: Consultancy; Acerta: Research Funding; Abbvie: Consultancy; Astellas: Consultancy; Kite Pharma: Research Funding. Stelljes:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; MSD: Consultancy; JAZZ: Honoraria. Wang:Pfizer: Employment, Equity Ownership. Liau:Pfizer: Employment, Equity Ownership. Nguyen:Navigate BioPharma Services, Inc., a Novartis Subsidiary: Employment. Sleight:Pfizer Inc: Employment, Equity Ownership. Vandendries:Pfizer: Employment, Equity Ownership. Neuhof:Pfizer: Employment, Equity Ownership. Laird:Pfizer: Employment, Equity Ownership. Advani:Novartis: Consultancy; Amgen: Research Funding; Glycomimetics: Consultancy; Pfizer: Honoraria, Research Funding.

Inotuzumab ozogamicin (InO) treatment in patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL): Analysis from INO-VATE by bone marrow blast percentage (BMB%).

7028Background: InO is a calicheamicin-conjugated antibody targeting CD22 on ALL blast cells. Here we report outcomes in R/R ALL patients (pts) receiving InO or standard of care chemotherapy (SC) i...

Evaluation of Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia Using EuroFlow Approaches

Background &amp; Aims. Evaluation of the minimal residual disease (MRD) at different stages of chemotherapy is one of key prognostic factors and a factor of stratification of patients into risk groups in acute lymphoblastic leukemia (ALL). The MRD detection on Day 15 and at later stages is based on identifying blast cells with a leukemia-associated immune phenotype. The aim is to assess the potential of 8-color standardized EuroFlow panels and to detect individual criteria for MRD monitoring during primary diagnosis. Materials &amp; Methods. The analysis included data on the primary immune phenotype and MRD assessment during chemotherapy in 10 adults and 35 children with a confirmed diagnosis of B-cell precursors ALL. Results. The ALL phenotype characteristics at the stage of primary diagnosis permit to make the most complete description of the of 8-color standardized EuroFlow panels. This gives an opportunity to select the most informative antigen combinations for further MRD monitoring. Combinations with CD58/CD38, CD81/CD9 antigen expression, as well as assessment of pan-myeloid CD13, CD33 antigen co-expression may be recommended as the most frequent aberrant immune phenotypes of blast cells in ALL. As for B-lineage progenitor cells in children on Day 15 of the induction therapy, a detection of TdT+ сyCD22&lt;sup&gt;+&lt;/sup&gt; cell population is necessary in addition to the quantification of CD10+ and/or CD34&lt;sup&gt;+&lt;/sup&gt; В-lineage progenitor cells. Conclusion. Therefore, the 8-color standardized EuroFlow panels permit not only to characterize the primary ALL immune phenotype in details, but may also be widely used for MRD evaluation at all stages of chemotherapy.

The use of oligonucleotide aptamers in cancer therapy

Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers - particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.

Future of Therapy in Acute Lymphoblastic Leukemia (ALL)--Potential Role of Immune-Based Therapies.

The prognosis of acute lymphoblastic leukemia (ALL) in adults remains poor, and novel treatment options are needed to improve outcomes. Immunotherapeutic strategies have been a recent focus in this area, targeting specific surface antigens on the ALL blast cells. Our review concentrates on four major class of antibody therapies, namely, (1) naked and unconjugated antibodies, (2) immunoconjugates and immunotoxins, (3) bi-specific T cell engaging (BiTE) therapy, and (4) chimeric antigen receptor (CAR) expressing T cells. We review preclinical and clinical data, and update on the latest advances in this exciting field, and suggest how these therapies can be incorporated into the treatment algorithm for ALL.

Tenovin-6-mediated inhibition of SIRT1/2 induces apoptosis in acute lymphoblastic leukemia (ALL) cells and eliminates ALL stem/progenitor cells.

BackgroundAcute lymphoblastic leukemia (ALL) is a heterogeneous group of malignant disorders derived from B- or T-cell lymphoid progenitor cells. ALL often is refractory to or relapses after treatment; thus, novel targeted therapy for ALL is urgently needed. In the present study, we initially found that the level of SIRT1, a class III histone deacetylase, was higher in primary ALL cells from patients than in peripheral blood mononuclear cells from healthy individuals. But it is not clear whether inhibition of SIRT1 by its selective small molecule inhibitor Tenovin-6 is effective against ALL cells.MethodsWe tested the effect of Tenovin-6 on ALL cell lines (REH and NALM-6) and primary cells from 41 children with ALL and 2 adult patients with ALL. The effects of Tenovin-6 on cell viability were determined by MTS assay; colony-forming assays were determined by soft agar in ALL cell lines and methylcellulose medium in normal bone marrow cells and primary ALL blast cells; cell apoptosis and cell cycling were examined by flow cytometry; the signaling pathway was determined by Western blotting; ALL stem/progenitor cells were seperated by using MACS MicroBead kit.ResultsThe results showed that Tenovin-6 treatment activated p53, potently inhibited the growth of pre-B ALL cells and primary ALL cells, and sensitized ALL cells to frontline chemotherapeutic agents etoposide and cytarabine. Tenovin-6 induced apoptosis in REH and NALM-6 cells and primary ALL cells and diminished expression of Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP) in such cells. Furthermore, inhibition of SIRT1 by Tenovin-6 inhibited the Wnt/β-catenin signaling pathway and eliminated ALL stem/progenitor (CD133 + CD19-) cells.ConclusionOur results indicate that Tenovin-6 may be a promising targeted therapy for ALL and clinical trials are warranted to investigate its efficacy in ALL patients.

Expanded Natural Killer Cells For The Control Of Minimal Residual Disease In Ph+ Acute Lymphoblastic Leukemia Patients

The management of Ph+ acute lymphoblastic leukemia (ALL) patients has profoundly changed during the last decade. Virtually all adult Ph+ ALL patients, including the elderly where the abnormality accounts for over 50% of cases, can obtain a complete remission (CR) with the use of TK inhibitors (and steroids) without systemic chemotherapy (Vignetti M et al. Blood 2007;109:3676; Foà R et al. Blood 2011;118:6521). Most patients remain, however, minimal residual disease (MRD)-positive. The possibility of targeting MRD via an immune-mediated control is particularly appealing in these patients. Previous studies have shown that natural killer (NK) cells with killing activity against autologous blasts may be expanded from ALL patients in CR (Torelli GF et al. Haematologica 2005;90:785). NK cell recognition of malignant targets is regulated by activating and inhibitory receptors. The major receptors with activating functions are NKG2D, DNAM-1 and the natural cytotoxicity receptors (NCRs) (NKp30, NKp44 and NKp46). MIC-A/B and ULBPs are ligands for NKG2D, PVR and Nec-2 for DNAM-1, while NCRs are orphan receptors. The pathways of NK-ALL recognition are unclear. Possible differences in NK cell killing susceptibility and in the expression of NK cell activating ligands among subgroups of ALL patients have been suggested.The aims of this study were: 1) to analyze the pathways of NK-ALL recognition, with particular attention to Ph+ samples; and 2) to verify whether differences in NK cell activating receptor ligand expression among molecularly-defined subgroups of patients correlate with the susceptibility to recognition and killing by NK cells activated and expanded under GMP conditions.PBMCs were collected from 23 healthy donors and 3 adult Ph+ ALL patients in CR. NK cells were enriched and cultured for 14 days in the presence of irradiated autologous feeder cells, autologous plasma, IL-2 and IL-15. The expression of the activating receptors NKG2D, DNAM-1 and NCRs was then analyzed. Samples from 46 newly diagnosed adult ALLs, median age 34 years (18-74), were also investigated: 39 patients had B-ALL - 15 BCR-ABL+, 7 MLL-AF4+, 2 E2A-PBX1+, 15 negative - and 7 had T-ALL. The expression of the NKG2D and DNAM-1 ligands on ALL blasts was analyzed. The cytotoxic activity of ex vivo expanded NK cells against primary ALL blasts was determined in a 51Cr release assay. For blocking experiments, NK cells were pre-treated with the anti-NKG2D or anti-DNAM-1 neutralizing mAbs.NK cells from healthy donors and from Ph+ ALL patients could be expanded respectively 33.2±15.2 and 39.1±19.3 fold. Expanded NK cells were represented by a homogenous population displaying a high expression of CD56 and CD16, in the absence of CD3. DNAM-1, NKG2D, NKp30 and NKp44 activating receptors presented a significantly increased expression after expansion from healthy donors (DNAM-1 p=.0007; NKG2D p=.0004; NKp30 p=.05; NKp44 p=.001). DNAM-1 and NKG2D showed a significantly increased expression after expansion also from Ph+ ALL patients (DNAM-1 p=.0012; NKG2D p=.045), while the expression of NCRs was not tested on these samples. The phenotypic analysis performed within molecularly-defined subgroups of ALL revealed that Ph+ cases presented an overall high surface expression of NKG2D and DNAM1 ligands. In particular, when compared to ALLs carrying no known molecular markers, Ph+ samples showed significantly higher levels of ULBP-1, ULBP-3 and MIC-B (p=.008, p=.026 and p=.033, respectively). In line with the phenotypic results, primary blasts from Ph+ ALL (n=5) appeared significantly more susceptible to NK-dependent lysis than B-ALL without molecular aberrations (n=6) (p=.007). When cytotoxic assays were performed in the presence of neutralizing mAbs, the NK cell killing potential was significantly inhibited by anti-DNAM-1 (p=.006), suggesting a pathway of recognition of ALL blast cells in the setting of the Nec-2/DNAM-1 interaction.The high expression of ligands for activating receptors in Ph+ ALL cases, together with the highest levels of susceptibility to NK cell-mediated lysis by this subgroup of ALL, point to a possible therapeutic use of autologous NK cells activated and expanded ex vivo. Management of Ph+ ALL patients - particularly the elderly - with TK inhibitors plus an immune-based strategy aimed at controlling/eradicating MRD without the use of systemic chemotherapy and/or transplant program is worthy of being investigated. Disclosures:No relevant conflicts of interest to declare.

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

AbstractWe report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL.

Novel Antibody-Based Therapies For Acute Lymphoblastic Leukemia

A major breakthrough in the treatment of acute lymphoblastic leukemia (ALL) was the availability of targeted therapies targeting either specific transcripts, such as bcr-abl fusion protein by tyrosine kinase inhibitors (TKIs), or specific antigens by mAbs. ALL blast cells express a variety of specific antigens (eg, CD19, CD20, CD22, CD33, and CD52) that serve as targets for mAbs. To date, the most data are available for anti-CD20 (rituximab), which has been combined with chemotherapy for the treatment of mature B-ALL/Burkitt lymphoma. Studies with rituximab have also been completed in B-precursor ALL. Another antigen, CD19, is of great interest due to a very high rate of expression in ALL. It can be targeted by a bispecific mAb, blinatumomab, directed against CD19 and CD3. Smaller studies or case reports are also available for the anti-CD52 (alemtuzumab), anti-CD22 (epratuzumab), and anti-CD33 (gemtuzumab) mAbs. Available data demonstrate that mAb therapy in ALL is a highly promising treatment approach. However, several details for an optimal treatment approach, such as the required level of antigen expression, timing, schedule, dosage, and stage of disease, still need to be defined.

Acute Lymphoblastic Leukemia Blast Cells Stimulate the Autocrine Production of CCL2 and IL-8 in Bone Marrow Stromal Cells

The interactions of Acute Lymphoblastic Leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival and resistance to chemotherapy. ALL stimulates BM stromal cells, which reciprocally promotes leukemia cell survival. To identify molecules critically involved in leukemia–microenvironment crosstalk, we performed gene expression profiling analyses of primary BM endothelial cells (BMEC) and BM mesenchymal stem cells (BMMSC) following stimulation by primary ALL cells. Leukemia stimulation of BM stromal cells upregulates the expression of several inflammatory chemokines, including CCL2 and IL-8/CXCL8. Secretion of these molecules was confirmed by ELISA assays of in vitro co-culture experiments and in BM plasma samples from pediatric ALL patients. Most primary ALL samples were found to express mRNA for CCR2 and CXCR1/CXCR2, which are the cognate receptors for CCL2 and IL-8, respectively. Primary ALL cells expressing at least one myeloid marker (CD13, CD15 or CD33) exhibited increased mRNA expression of CCR2 (p = 0.02). Leukemia cells from most patients express CCL2 and IL-8 chemokines (ELISA test) but at lower levels than that of BMEC and BMMSC. In vitro functional studies revealed that the proliferation, survival and migration of primary ALL cells co-cultured with BM stromal cells were not affected by addition of CCL2, IL-8 or of neutralizing antibodies to these chemokines. On the other hand, both chemokines were found to enhance BMEC and BMMSC survival in serum-free medium and to increase their proliferation in serum-starved conditions. Interestingly, CCL2 and IL-8 affected endothelial morphogenesis as shown in Matrigel assays. Since CCL2 and IL-8 have suppressive effects in normal hematopoiesis but do not seem to affect primary ALL cells, it is possible that these chemokines may contribute to the establishment of survival/proliferative selective advantage for ALL cells in the leukemic BM microenvironment. In addition, CCL2 and IL-8 seems indirectly to contribute to ALL cell survival by stimulating the supporting BM stromal cells. Finally, preliminary results showed that standard risk pediatric ALL patients with BM plasma levels below 577pg/ml have better survival rates than those with higher CCL2 levels (p = 0.08). In conclusion, this work suggests a significant role for the chemokines CCL2 and IL-8 in the leukemia/microenvironment crosstalk in human ALL, and suggests that these molecules may represent valuable targets for therapeutic intervention in this cancer. Supported by: CNPq, FAPESP.

Tumor Lysis Syndrome Induced by Dexamethasone

Tumor Lysis Syndrome Induced by Dexamethasone

Effects of granulocyte-macrophage colony-stimulating factor and interleukin 6 on the growth of leukemic blasts in suspension culture.

We examined the stimulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL)-6 on the in vitro proliferation of leukemic blast cells from patients with acute leukemia. Bone marrow or peripheral blood leukemic blast cells were obtained from 21 patients, including 14 cases of acute myeloblastic leukemia (AML), four cases of acute lymphoblastic leukemia (ALL), two cases of acute undifferentiated leukemia, and one case of acute mixed-lineage leukemia. The proliferation of leukemic blast cells was evaluated by measuring the incorporation of 3H-thymidine into cells incubated with various concentrations of cytokines for 3 days. GM-CSF stimulated the DNA synthesis (with greater than 2.0 stimulation index) of blast cells in 9 of 14 (64%) AML cases, two cases of acute undifferentiated leukemia and one case of acute mixed-lineage leukemia. Only two cases of AML blasts responded to IL-6 to grow in the short-term suspension cultures. GM-CSF and IL-6 did not display a synergistic effect on the growth of leukemic cells. Moreover, GM-CSF and IL-6 did not stimulate the proliferation of ALL blast cells. Binding study also revealed the specific binding of GM-CSF on the blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia. Our results indicated that leukemic blast cells of acute undifferentiated leukemia and acute mixed-lineage leukemia possessed functional GM-CSF receptors.

Prognostic implication of Ecto-5′-nucleotidase activity in acute lymphoblastic leukemia

Ecto-5'-nucleotidase (5'-N) activity was determined in 191 patients (71 children and 120 adults) with acute leukemia. Elevated values for 5'-N were registered in common acute lymphoblastic leukemia (ALL), but blast cells of T-cell ALL (T-ALL) and common ALL antigen-negative non-T-ALL had low enzyme activity comparable with the values of acute non-lymphocytic leukemia. Dependence of remission duration on 5'-N activity was analyzed in 74 adults with ALL, treated similarly in a prospective multicenter trial. The remission curves for ALL patients with 5'-N activity lower than 10 nmol/h x 10(6) cells were substantially and significantly better than those of patients with high activity (greater than 10 nmol/h x 10(6) cells). This difference was also evident in the immunologic subclass common ALL. Statistical evaluation showed that an interaction between immunologic subtype of the blast cells and their 5'-N activity had prognostic significance for remission duration. In addition to the independent factor, initial age, this interaction was also prognostic for survival.