pro-B Acute Lymphoblastic Leukemia Research Articles

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

Flow cytometric detection of leukemic blasts in Libyan pediatric patients with acute lymphoblastic leukemia

ABSTRACT The diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

PIWIL1 gene polymorphism and pediatric acute lymphoblastic leukemia relapse susceptibility among Chinese children: a five-center case-control study.

PIWIL1 polymorphisms' role in pediatric acute lymphoblastic leukemia (ALL) relapse susceptibility remains undiscovered. A case-control designed and multiple logistic regression model was performed to evaluate the overall risk of pediatric ALL and five single-nucleotide polymorphisms (SNPs) of PIWIL1 gene (rs35997018 C>T, rs1106042 A>G, rs7957349 C>G, rs10773771 C>T, and rs10848087 A>G) in 785 cases and 1,323 controls, which were genotyped by TaqMan assay. The odds ratio (OR) and its 95% confidence interval (CI) were used to estimate the relationship. Stratified analysis was used to investigate the correlation of rs1106042 and rs10773771 genotypes and pediatric ALL relapse susceptibility in terms of age, sex, number of white blood cells (WBC), immunophenotyping, gene fusion type, karyotype, primitive/naïve lymphocytes, and minimal residual disease (MRD) in bone marrow. Finally, the haplotype analysis was performed to appraise the relationship between inferred haplotypes of PIWIL1 and pediatric ALL risk. Among the five analyzed SNPs, rs1106042 A>G was related to increased ALL risk, and rs10773771 C>T was related to decreased ALL risk. Compared to the GG genotype, the rs1106042 GA/AA had a deleterious effect on children of age <120 months, who were female and male, had high or average number of WBC, pro-B ALL, pre-B ALL, T-ALL, low- and middle-risk ALL, E2A-PBX fusion gene, non-gene fusion, abnormal diploid, high hyperdiploid, hypodiploid, and normal diploid. Moreover, rs1106042 A>G harmfully affected primitive/naïve lymphocytes and MRD on days 15-19, day 33, and week 12. On the contrary, rs10773771 TC/CC exhibited a protective effect on ALL children with the TEL-AML fusion gene. Haplotype analysis demonstrated that haplotypes CAGT, TACC, TACT, and TAGT were significantly associated with increased pediatric ALL relapse susceptibility. PIWIL1 rs1106042 A>G was related to increased ALL risk, and rs10773771 C>T was linked to decreased ALL risk in eastern Chinese children. Rs1106042 GA/AA may predict poor prognosis.

B-389 Validation of One-step In-house RT-qPCR Method for Detecting MLL::AF4 Fusion

Abstract Background Rearrangements of mixed lineage leukemia gene (MLL) with 1 of &amp;gt;130 alternative partner genes is a recurrent cytogenetic finding in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is generally associated with a poor prognosis. Among the most common MLL rearrangement is t(4;11)(q21;q23), forming the MLL::AF4 (also known as KMT2A::AFF1) fusion gene. Unique among MLL rearrangements, MLL::AF4 is almost exclusively associated with pro-B cell ALL and is prototypical of infant ALL, where it carries a very poor prognosis. At least 10 different MLL-AF4 fusion transcripts have been found, due to break-points in different introns, some transcripts being more frequent in either adult or infant ALL. Fast and reliable diagnostic tests represent an important tool in clinical laboratories to detect these rearrangements and . In this context, this work aimed to validate a qualitative method to detect the main MLL::AF4 fusion isoforms. Methods We evaluated the performance of the Hs03043614_ft (e10e4, e9e4 and e9e5) and Hs03024412_ft (e11e4) inventoried TaqMan assays (Thermo Fisher Scientific). Total mRNA from negative and positive samples for each fusion was extracted by the TRIzol reagent according to the manufacturer’s instructions. All assays were performed using TaqMan RNA-to-Ct 1-Step kit (Thermo Fisher Scientific). The inventoried ABL1 assay (Thermo Fisher Scientific) was used as endogenous control. Using positive samples, negative samples and controls of each fusion were performed: sequence verification tests, standardization of RNA input, evaluation of reaction efficiency and determination of the detection limit (LOD). The performance of the assays was evaluated with ABL1 control separately from the fusions. Results Our results showed that both assays worked well for the detection of specific fusions. The best RNA input for the reactions was 200 ng. The efficiencies reactions were: a) with ABL1 in separate reactions: 92.6% and 110.0% for Hs03043614_ft and Hs03024412_ft assays, respectively; The literature recommends the limit of efficiency was between 90% and 110%. The LODs with ABL1 in separate reactions were 10−3 for both assays, respectively. There was no unspecific amplification in negative samples. Conclusion The TaqMan assays with RT one-step protocol evaluated are highly sensitive, fast, and reliable tools for detecting the most frequent MLL::AF4 fusion transcripts.

Generation of a human induced pluripotent stem cell line harboring the infant leukemia-associated fusion gene, KMT2A-AFF1 (IMSUTi002-A-2)

KMT2A-AFF1 (MLL-AF4), formed by chromosomal translocation t(4;11), is observed specifically in infant pro-B acute lymphoblastic leukemia, and the main driver of leukemogenesis. In this study, a human induced pluripotent stem cell (hiPSC) line harboring KMT2A-AFF1 (IMSUTi002-A-2), which has the ability to simultaneously induce the doxycycline-inducible expression of KMT2A-AFF1 and EGFP, was established. This hiPSC can be a powerful model for understanding infant leukemia and risk assessment of leukemogenesis.

Inotuzumab Ozogamicin Induction Followed By Standard Chemotherapy Yields High Remission Rates and Promising Survival in Older (>55 Years) Patients with De Novo B-Lymphoblastic Leukemia (GMALL-Initial1 Trial)

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients (pts) between 55 and 70 years of age. Incorporation of novel antibody-based therapies in the first line induction therapy bears the potential of a significantly increased response rates and survival together with a reduction of treatment toxicity. Methods: This open label phase II study (ClinicalTrials.gov, identifier: NCT03460522) of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Pts aged >55 years with newly diagnosed Ph/BCR-ABL negative B-precursor ALL (at least CD20 positivity of leukemic blasts) were eligible. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Pts achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Results: 43 out of the 45 pts included were analyzed regarding the primary end point. Two pts were excluded per protocol from further analysis, as they received only one cycle of induction or withdraw consent. At diagnosis, median age was 64 years (range 56-80 years, 20 pts ≥65 years and 12 pts ≥70 years), median bone marrow blast count was 80% and median CD22 expression on leukemic blasts was 69% (range 21-99). 38 pts were diagnosed with a common- and 5 with a pro-B ALL. 42/43 pts completed all 3 cycles of induction therapy. All 43 pts achieved a complete remission (CR/CRi), mainly after the 1st induction. Median time between start of 1st induction to start 2nd induction, from 2nd to 3rd induction and from 3rd induction to 1st consolidation therapy was 21 days, 28 days and 30 days, respectively. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 pts, with 23/43 (53%) and 31/42 (74%) pts being MRD negative after 2nd and 3rd induction therapy, respectively. With a median follow-up of 697 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 82-99%) and 81% (95% C.I. 69-94%), respectively. No patient died within the first 6 months after start of induction therapy. 5 events occurred within the first year after study inclusion, 3 deaths in CR and 2 relapsed ALL, resulting in event rate of 12% and a probability of EFS at 1 year (primary endpoint of the study) of 88% (95% CI 79-98). EFS at 2 years was 73% (95% CI 59-88). So far, 7 pts with persisting/reappearance of MRD (2 pts) or relapse (5 pts) were treated with blinatumomab. 9 pts received an aSCT (4 pts in CR1 and 5 pts >CR1), 6 of these pts are alive in CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 74%, 19% and 2% of all cases, respectively), anemia (37%, 5%, 0%), thrombocytopenia (49%, 7%, 2%) and elevation of liver enzymes (14%, 5%, 0%). One patient was reported with suspected veno occlusive disease (after 2nd induction therapy). Classical factors which may be associated with inferior survival outcomes like age, diagnosis of pro-B ALL, disease burden at diagnosis, co-morbidity or no MRD negative CR after induction had no significant impact on OS or EFS. Conclusion: Three cycles of InO as induction therapy for the treatment of older pts with de novo acute B-lymphoblastic leukemia resulted in a very high remission rate. Given that our study met the primary end point (predefined event rate at 1 year ≤40%, observed event rate 12%), these promising study results could be a rationale for integrating InO induction therapy in future treatment recommendations. Ideally, this novel induction therapy should be evaluated in prospective randomized trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Granular B lymphoblastic leukemia.

A 13-year-old Indian boy presented with extreme lethargy and weakness. On examination, there was no hepatosplenomegaly or lymphadenopathy. His blood count showed a white cell count of 18.8 × 109/L, hemoglobin concentration 107 g/L, and platelet count 70 × 109/L. Examination of a blood film led to suspicion of granular lymphoblastic leukemia. There were 60% blast cells with a moderately high nucleocytoplasmic ratio, delicate chromatin, and 1–3 nucleoli. Many of the blast cells contained prominent azurophilic cytoplasmic granules. There were no Auer rods and granulocyte morphology was normal. The suspected lineage was confirmed by flow cytometric immunophenotyping which showed the blast cells to express CD45 (weak), CD34, CD10, CD19, CD20, cytoplasmic(c) CD79a, CD33, and HLA-DR. There was no expression of myeloperoxidase, cCD3, CD7, CD13, CD64, or CD117. Cytogenetic analysis showed a normal male karyotype. Reverse transcriptase for BCR::ABL1 did not detect p210, p190, or p230 transcripts. Granular acute lymphoblastic leukemia (ALL) is uncommon. Making a distinction from acute myeloid and mixed phenotype, acute leukemia is important and requires myeloperoxidase cytochemistry or immunophenotyping. Most cases are pro-B or common ALL but T-lineage cases have also been reported. In addition to B-lineage-specific markers, there can be aberrant expression of CD131 or CD33.2, 3 There is no specific cytogenetic association and the prognostic significance, if any, is uncertain. The authors declare no conflict of interest

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia

AbstractThe fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

Rhodotorula fungemia in a patient with acute lymphoblastic leukemia

Objectives: Rhodotorula is an environmental yeast that belongs to Basidiomycota Phylum. Rhodotorula species are ubiquitous in nature, can be found in soil and freshwater. Immunocompromised patients can develop Rhodotorulosis due to wide-ranging exposure to Rhodotorula in the hospital environment. Case Discussion: The patient was a 3-year-old male with a diagnosis of Pro B-Acute Lymphoblastic Leukemia (ALL). He was admitted to the hospital with complaints of malaise, fatigue, weight loss, and diarrhea between courses of chemotherapy. Rhodotorula was isolated from the patient’s blood culture obtained during the elevation of temperature. After 14 days of amphotericin B treatment, clinical situation of the patient was improved and he was discharged. Conclusion: Rhodotorula spp. as a rare yet emerging pathogen, often presents as fever of unknown etiology resistant to antibacterial treatment and can be associated with fungemia and other severe complications.   

Final Induction Therapy Results of an Open Label Phase II Study Using Inotuzumab Ozogamicin for Induction Therapy, Followed By a Conventional Chemotherapy Based Consolidation and Maintenance Therapy in Patients Aged 56 Years and Older with Acute B-Lymphoblastic Leukemia (INITIAL-1 trial)

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of elderly patients with ALL remains dismal, even after moderately intensive chemotherapy. Incorporation of antibody-based therapies in the first line induction therapy bears the potential of a significant increased response rate and survival with the reduction of treatment toxicity.Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Patients aged >55 years with newly diagnosed Ph/BCR-ABL negative acute B-precursor ALL were eligible. Leukemic blasts had CD22 surface expression of at least 20%. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Patients achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis (registered with ClinicalTrials.gov, identifier: NCT03460522).Results: As of July 2021, 45 patients were included, with induction results available for 43 patients. Two patients were excluded per protocol from further analysis, as they received only one induction: 1 prolonged toxicity (CR after induction), 1 withdrawn consent. Median age at initial diagnosis was 64 years (range 56-80 years). 38 patients were diagnosed with a common- and 5 with a pro-B ALL. Median CD22 expression on leukemic blasts was 69% (range 21-99%). Due to suspected therapy related liver toxicities, a single patient received only 2 induction cycles (complete remission after 1st induction). All other patients completed 3 cycles of induction therapy and achieved complete remission (CR/CRi), mainly after the 1st induction. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 patients, with 23/43 (53%) and 31/42 (74%) patients being MRD negative after 2nd and 3rd induction therapy, respectively. Four patients with MRD failure (≥10 -4) after the 3rd induction therapy are still in CR (2 were treated with conventional chemotherapy per this protocol, one proceeded to allogeneic stem cell transplantation (aSCT), one received blinatumomab). MRD below the threshold of 10 -4 was detected in 15 patients after the 2nd induction (no relapse occurred so far). 8 of these 15 patients were MRD negative after the 3rd induction. So far, 7 events have been reported (during year 1: 3 deaths in CR and 1 relapsed ALL; during year 2: 1 death in CR and 2 patients with relapse). With a median follow-up of 422 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 80-100%) and 77% (95% C.I. 57-96%), respectively. 4 patients received an aSCT in 1st CR, 1 patient in 2nd CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 60%, 12% and 3% of all cases, respectively), anemia (28%, 2%, 0%), thrombocytopenia (35%, 7%, 3%) and elevation of liver enzymes (14%, 5%, 0%). So far, one patient has been reported with suspected veno occlusive disease (after 2nd induction therapy).Conclusion: Three cycles of InO as induction therapy for the treatment of elderly patients with acute B-cell ALL results in very high remission rates with MRD-response in up to 90% (MRD negativity or MRD-levels <10 -4). The promising EFS and survival rates of these patients warrant further investigation of this novel induction therapy in a prospective randomized trial. [Display omitted] DisclosuresStelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Medac: Speakers Bureau; MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Wäsch: Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Nachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Haenel: Jazz: Consultancy, Honoraria; GSK: Consultancy; Bayer Vital: Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Goekbuget: Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Erytech: Consultancy; Cellestia: Consultancy; Incyte: Other: Research funding for Institution; Jazz Pharmaceuticals: Other: Research funding for institution; Novartis: Consultancy, Other: Research funding for Institution; Gilead/Kite: Consultancy; Morphosys: Consultancy; Pfizer: Consultancy, Other: Research funding for institution; Servier: Consultancy, Other; Abbvie: Other. OffLabel Disclosure:Inotuzumab Ozogamicin

Synergistic Activity of BH3-Mimetics By Combined Targeting of Anti-Apoptotic Regulators in B-Cell Precursor ALL

In acute lymphoblastic leukemia (ALL), the most frequent malignancy in children and adolescents, deregulated cell death pathways contribute to leukemia development and therapy failure. Apoptosis (programmed cell death) is controlled at the mitochondrial level by different pro- and anti-apoptotic regulators. Molecules of the BCL-2 family are key in regulating intrinsic apoptosis signaling. Pro-apoptotic BH3-only proteins such as BIM and BID activate pro-death proteins like BAX and BAK leading to cell death, while anti-apoptotic BCL-2 family members including BCL-2, MCL-1 and BCL-XL sequester pro-apoptotic molecules, thereby preventing pro-death protein activation and apoptosis induction. Small molecule inhibitors have been developed, which bind to the anti-apoptotic molecules BCL-2, MCL-1 and BCL-XL leading to cell death induction. In particular inhibition of BCL-2, a key regulator of survival in B-cell malignancies including BCP-ALL, by the specific inhibitor venetoclax (VEN) has shown substantial, clinical anti-tumor activity. However, in BCP-ALL heterogeneous sensitivities for VEN have been described suggesting that other BCL-2 family members like MCL-1 and BCL-XL interfere with BCL-2 inhibition thereby counteracting VEN activity leading to poor treatment response.In this study, we compared the effects of inhibition of the anti-apoptotic BCL-2 family members BCL-2 (venetoclax, VEN), MCL-1 (S63845) and BCL-XL (A-1331852), investigated molecular mechanisms and determinants of inhibitor sensitivity, and addressed potential synergistic activity upon simultaneous blockage of BCL-2 together with MCL-1 or BCL-XL in BCP-ALL.First, we investigated the activity of the single inhibitors to induce cell death (positive staining for propidium iodide) in 7 BCP-ALL cell lines and a series of 27 BCP-ALL patient-derived xenograft samples (PDX). Titrating increasing concentrations, we estimated half-maximal effective concentrations (EC 50) for each inhibitor showing heterogenous responses of individual samples to the different inhibitors. Importantly, sensitivities to either inhibitor were not associated with leukemia characteristics including recurrent genetic alterations described in BCP-ALL. We also did not observe similar sensitivities to the inhibitors of individual samples, however some VEN insensitive samples showed sensitivity to MCL-1 and/or BCL-XL-inhibition, suggesting functional substitution of the anti-apoptotic regulators.Next, we investigated anti-apoptotic addictions of BCP-ALL cells to BCL-2 family proteins upon VEN treatment. Analyzing apoptosis signaling after exposure to VEN and BH3-peptides, we identified that ALL cells adapt their anti-apoptotic addiction to MCL-1 and BCL-XL as an escape strategy from VEN-induced cell death. Analyzing protein complexes by co-immunoprecipitation, we found that exposure of ALL cells to VEN rapidly led to reduced BIM/BCL-2 and compensatory increased BIM/MCL-1 complexes. Conversely, S63845 reduced BIM/MCL-1 complexes and led to increased BIM/BCL-2 binding. Importantly, both protein complexes could be effectively disrupted by combination treatment with VEN and S63845, which resulted in release of BIM and promotion of apoptosis signaling.Based on our mechanistic findings, we evaluated BH3-mimetic combinations for cell death induction using multi-dose matrix assays to calculate synergy metrics based on the Bliss independence model. Analyzing combination effects in seven BCP-ALL cell lines and four PDX samples, we identified positive mean synergy scores of VEN with S63845 and A-1331852 in all cases. However, the most synergistic area of the multi-dose-response matrices differed among different leukemias, indicating different extents of addictions and thereby synergies at different drug concentrations.Finally, combined BCL-2 and MCL-1 inhibition was evaluated in vivo in a PDX model of KMT2A-ENL positive pro-B ALL. Combination treatment of VEN with S63845 led to reduced leukemia loads in spleen, bone marrow and CNS as compared to single agent treatment.Taken together, we found heterogeneous responses of BCP-ALL samples to BH3-mimetics antagonizing BCL-2, MCL-1 and BCL-XL. The ability of leukemia cells to adapt their anti-apoptotic dependency from BCL-2 to MCL-1 or BCL-XL can be used as target for combination therapy, demonstrating synergistic activity in PDX samples ex vivo and in vivo. DisclosuresNo relevant conflicts of interest to declare.

Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia

Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse.Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target.We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene.The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse.To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression.To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed.In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. DisclosuresCario: Novartis: Other: Lecture Fee.

Deep Learning Technology-based Model to Identify Benign and Pro-B Acute Lymphoblastic Leukemia (ALL): Xception + LIME

Leukemia is a type of cancer that occurs when abnormal blood cells take place in the bone marrow. Leukemia can either be acute (fastly growing) or chronic (slowly growing) and it is considered as one of the most commonly diagnosed cancers for children younger than the age of 15 or adults older than the age of 55. Leukemia can be diagnosed through various types of tests and depending on the aggressiveness of the disease, the treatment may differ. To provide a low-cost, time-efficient solution, this study employs the deep learning technique to train the Xception, VGG16, VGG19, and MobileNet models to optimize the accuracy of medical image detection. Through medical imaging, the trained model is able to detect anomalies in the dataset and identify whether the given data is a benign acute lymphoblastic leukemia (ALL) or a Pro-B ALL. Overall, this VGG16 showed the most optimal performance in terms of accuracy and precision, producing a 98.5% accuracy in detecting abnormal regions from the dataset. This study also further used XAI technique and a deep convolutional neural network to visualize the results of anomalies. As a result, this paper concluded that both deep learning and machine learning techniques are yet to replace human resources and intelligence as the heatmap and the LIME portrayal identified different regions as abnormal parts, therefore proving the inconsistency of deep learning technology.

MiR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia

Abstractt(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4–driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

Challenges in Care of Children with Acute Leukemia in a Government Hospital in India: A Retrospective Analysis

Abstract Introduction Acute leukemia (AL) is among the most common treatable cancers in childhood but many children are forced to abandon therapy. Objective We have explored reasons for treatment abandonment through this study. Materials and Methods Retrospective analysis from an ongoing registry data of childhood AL patients, where all newly diagnosed AL patients of ≤18 years of age (June 2014–November 2017) were included. Patients &gt;18 years of age, having any history of treatment of AL outside our institute, and/or patients with relapsed AL were excluded. The primary outcome of the study was treatment abandonment rate. Results A total of 710 AL patients were included in the study, average distance traversed to reach the hospital being 161.66 km. Most children were aged 1 to 10 years (49.4%, n = 351), followed by &gt;10 to 18 years (46.6%, n = 331), and ≤1 year age (3.9%, n = 28). The commonest symptoms were fever (67.4%), pallor (38.6%), bleeding (11.5%), bone pain (13.8%), neck swellings (14.9%), and, rarely, testicular swellings or Superior vena cava (SVC) syndrome (1.1%). A high abandonment rate was noted prior to leukemia subtyping (35.2%, n = 250) mostly among males 62% (n = 155) and the 1- to 10-year group 55.6% (n = 139). A total of 460 (64.8%) patients were subsequently subcategorized by immunophenotyping. Precursor B-cell (Pre-B) Acute Lymphoblastic Leukemia (ALL) in 307 (43.2%) patients was the commonest subtype, followed by early Pre-B ALL (Pro-B ALL) in 10 (1.4%), T-cell ALL (T-ALL) in 51 (7.1%), Acute Myeloid Leukemia (AML) in 45 (6.3%), Acute Promyelocytic Leukemia (APML) in 28 (3.9%), and Mixed Phenotypic Acute Leukemia (MPAL) in 19 (2.6%). Conclusion The most common group of patients was aged 1 to 10 years (median age: 5 years). An abandonment rate of 35.2% was seen prior to complete diagnostic workup. The reason for this high abandonment, despite good disease prognosis, is a relevant social and health issue, and needs further evaluation. The problems discussed in this study are relevant to lower-income families and areas where health care is not easily accessible. The government agencies, nongovernment organizations and society would need to work together to overcome these issues.

Genome-wide interference of ZNF423 with B-lineage transcriptional circuitries in acute lymphoblastic leukemia

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9–mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis.

Akut Lenfoblastik Lösemide Flow Sitometri ile İmmünofenotiplemenin Prognoz Üzerine Etkileri

Background: The identification of immunophenotype subgroups is very important for the diagnosis and prognosis of acute lymphoblastic leukemia(ALL). Material and Methods: The study included 105 children with ALL(65 males, 40 females; mean age 5.9±3.8 years) who were treated TR-ALL 2000(modified) BFM treatment protocol. Results: The distributions of EGIL classification were pro-B ALL(n=1), common B ALL(n=46), pre-B ALL(n=40), pre-T ALL(n=8), cortical T ALL(n=6), and mature T ALL(n=4). Leukocyte≥100,000/mm³, lymphadenopathy≥2 cm, mediastinal involvement were commonly identified in T ALL group. T ALL had a poor response to chemotherapy according to 8th-day peripheral circulation blast counts and 15th-day bone marrow aspiration(BMA) blast counts. The recurrence, mortality, and death rate in the induction period of treatment were frequently detected in T ALL group. The variables that had prognostic potential, as indicated by univariate analyses, were leukocyte count, hepatomegaly, splenomegaly, and lymphadenopathy at the time of diagnosis, 8th-day steroid response, 15th-day BMA response, risk group, recurrence, and immunophenotyping. Multivariate Cox regression analysis demonstrated that only the leukocyte count(HR 2.51, p &amp;lt; 0.001) was a predictor of prognosis. Conclusion: Immunophenotyping may be effective in the diagnosis and prognosis of ALL, identification of risk groups, and in risk-based treatment planning. T ALL had a poor prognosis.

Immunophenotypic and Genotypic Features, Clinical Characteristics, and Treatment Outcome of Adult Pro-B Acute Lymphoblastic Leukemia: Results of the German Multicenter Trials GMALL 03/87 and 04/89

In contrast to childhood acute lymphoblastic leukemia (ALL), the cell-biological features, clinical characteristics, and treatment outcome of CD10− pro-B ALL have not yet been determined in larger series of adult patients. Therefore, we studied 57 adult patients with newly diagnosed pro-B ALL (median age, 30 years) enrolled in two consecutive German multicenter ALL studies (03/87 and 04/89). Extensive immunophenotypic characterization of leukemic blasts could be performed on all patients, whereas adequate cytogenetic data were available in 33 cases and molecular studies in 18 cases, using reverse transcription-polymerase chain reaction to detect MLL-AF-4 transcripts. Twenty-two patients demonstrated a t(4;11)(q21;q23) and/or MLL-AF-4 rearrangements, and 6 patients had other structural abnormalities, including a t(9;22)(q34;q11) (N = 2). Nine patients had a normal karyotype. Patients with 11q23 abnormalities tended to be younger (median age, 29 years) and were characterized by male predominance (64%), hyperleukocytosis (median leukocyte count, 168 × 109/L), and a frequent coexpression of CD65s (64%) as compared with patients with other cytogenetic abnormalities or a normal karyotype. Twelve of 16 (75%) pro-B ALL patients in study 03/87 and 30 of 41 (73%) in study 04/89 achieved a complete remission (CR). Sixteen of 30 patients in study 04/89 remain in continuous CR (CCR) in contrast to only 2 of 12 patients in study 03/87. Interestingly, all 7 patients treated with high-dose cytarabine and mitoxantrone as consolidation in study 04/89 remain alive and leukemia-free. One patient in study 03/87 and 8 in study 04/89 underwent autologous (N = 2) or allogeneic (N = 7) bone marrow transplantation (BMT). The median remission duration was 420 days for patients in study 03/87 and has not yet been reached in study 04/89. The median survival time of all pro-B ALL patients was 571 days in study 03/87 and 747 days in study 04/89. Among the 22 patients with a t(4;11) and/or MLL-AF-4 rearrangements, 17 achieved a CR and 8 are still in CCR, of whom 4 underwent an allogeneic BMT. Remission duration and overall survival did not differ significantly between pro-B ALL patients with 11q23 abnormalities and those with a normal karyotype or other structural abnormalities. These data indicate that intensification of postremission treatment may improve the prognosis of adult pro-B ALL, including patients with a t(4;11).© 1998 by The American Society of Hematology.

HDAC7 is a major contributor in the pathogenesis of infant t(4;11) proB acute lymphoblastic leukemia.

HDAC7 is a major contributor in the pathogenesis of infant t(4;11) proB acute lymphoblastic leukemia.

Immature Immunoglobulin Gene Rearrangements Are Recurrent in B Precursor Adult Acute Lymphoblastic Leukemia Carrying TP53 Molecular Alterations.

Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.