Leukemic Population Research Articles

The Role of Hypoxic Bone Marrow Microenvironment in Acute Myeloid Leukemia and Future Therapeutic Opportunities.

Acute myeloid leukemia (AML) is a hematologic malignancy caused by a wide range of alterations responsible for a high grade of heterogeneity among patients. Several studies have demonstrated that the hypoxic bone marrow microenvironment (BMM) plays a crucial role in AML pathogenesis and therapy response. This review article summarizes the current literature regarding the effects of the dynamic crosstalk between leukemic stem cells (LSCs) and hypoxic BMM. The interaction between LSCs and hypoxic BMM regulates fundamental cell fate decisions, including survival, self-renewal, and proliferation capacity as a consequence of genetic, transcriptional, and metabolic adaptation of LSCs mediated by hypoxia-inducible factors (HIFs). HIF-1α and some of their targets have been associated with poor prognosis in AML. It has been demonstrated that the hypoxic BMM creates a protective niche that mediates resistance to therapy. Therefore, we also highlight how hypoxia hallmarks might be targeted in the future to hit the leukemic population to improve AML patient outcomes.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.

Multiplex technologies for the assessment of minimal residual disease and low-level mutation detection in leukaemia: mass spectrometry versus next-generation sequencing.

With the focus of leukaemia management shifting to the implications of low-level disease burden, increasing attention is being paid on the development of highly sensitive methodologies required for detection. There are various techniques capable of identification of measurable residual disease (MRD) either evidencing as relevant mutation detection [e.g. nucleophosmin 1 (NPM1) mutation] or trace levels of leukaemic clonal populations. The vast majority of these methods only permit detection of a single clone or mutation. However, mass spectrometry and next-generation sequencing enable the interrogation of multiple genes simultaneously, facilitating a more complete genomic profile. In the present review, we explore the methodologies of both techniques in conjunction with the important advantages and limitations associated with each assay. We also highlight the evidence and the various instances where either technique has been used and propose future strategies for MRD detection.

How I treat chronic lymphocytic leukemia after venetoclax

AbstractVenetoclax-based regimens have expanded the therapeutic options for patients with chronic lymphocytic leukemia (CLL), frequently achieving remissions with undetectable measurable residual disease and facilitating time-limited treatment without chemotherapy. Although response rates are high and durable disease control is common, longer-term follow-up of patients with relapsed and refractory disease, especially in the presence of TP53 aberrations, demonstrates frequent disease resistance and progression. Although the understanding of venetoclax resistance remains incomplete, progressive disease is typified by oligoclonal leukemic populations with distinct resistance mechanisms, including BCL2 mutations, upregulation of alternative BCL2 family proteins, and genomic instability. Although most commonly observed in heavily pretreated patients with disease refractory to fludarabine and harboring complex karyotype, Richter transformation presents a distinct and challenging manifestation of venetoclax resistance. For patients with progressive CLL after venetoclax, treatment options include B-cell receptor pathway inhibitors, allogeneic stem cell transplantation, chimeric antigen receptor T cells, and venetoclax retreatment for those with disease relapsing after time-limited therapy. However, data to inform clinical decisions for these patients are limited. We review the biology of venetoclax resistance and outline an approach to the common clinical scenarios encountered after venetoclax-based therapy that will increasingly confront practicing clinicians.

Immunophenotypic characterization of pediatric acute myeloid leukemia with inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFb-MYH11

The aim of this study was to describe the immunophenotype of leukemic cells in acute myeloid leukemia (AML) with inv(16) (p13.1q22)/CBFb-MYH11 and t(16;16)(p13.1;q22)/CBFb-MYH11 in children. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. We investigated bone marrow samples from 36 pediatric patients with initially diagnosed AML with inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFb-MYH11. Immunophenotypic profile of leukemic cells was very heterogeneous: cells expressed antigens of early stages of differentiation (CD34, CD117, CD123) as well as markers of mature monocytes (CD11c, CD14, CD64) and neutrophils (CD65, CD15). Moreover, in 55.6% of cases lymphoid coexpressions were noticed (CD2 – the most frequent one). Furthermore, in 83.3% of cases we detected the separation of leukemic cells population into two parts: more “immature” – myeloblastic, which expressed early markers of differentiation (CD34, CD117), and more “mature” part, expressing monocytic antigens (CD11b, CD14, CD33). There was no clear separation between these parts of population. Despite the immunophenotypic similarity between monocytic part of leukemic population and normal monocytes, in 87.5% of studied cases there were same lymphoid coexpressions on these cells as on leukemic myeloblasts. Moreover, we showed that levels of CBFb-MYH11 expression in leukemic monocytes and myeloblasts were comparable. Presence of these characteristics in monocytes allows to consider them as part of leukemic cells population and take into consideration during the total immunophenotype reporting.

The spleen as a sanctuary site for residual leukemic cells following ABT-199 monotherapy in ETP-ALL

AbstractB-cell lymphoma 2 (BCL-2) has recently emerged as a therapeutic target for early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), a high-risk subtype of human T-cell ALL. The major clinical challenge with targeted therapeutics, such as the BCL-2 inhibitor ABT-199, is the development of acquired resistance. We assessed the in vivo response of luciferase-positive LOUCY cells to ABT-199 monotherapy and observed specific residual disease in the splenic microenvironment. Of note, these results were confirmed by using a primary ETP-ALL patient-derived xenograft. Splenomegaly has previously been associated with poor prognosis in diverse types of leukemia. However, the exact mechanism by which the splenic microenvironment alters responses to specific targeted therapies remains largely unexplored. We show that residual LOUCY cells isolated from the spleen microenvironment displayed reduced BCL-2 dependence, which was accompanied by decreased BCL-2 expression levels. Notably, this phenotype of reduced BCL-2 dependence could be recapitulated by using human splenic fibroblast coculture experiments and was confirmed in an in vitro chronic ABT-199 resistance model of LOUCY. Finally, single-cell RNA-sequencing was used to show that ABT-199 triggers transcriptional changes in T-cell differentiation genes in leukemic cells obtained from the spleen microenvironment. Of note, increased expression of CD1a and sCD3 was also observed in ABT199-resistant LOUCY clones, further reinforcing the idea that a more differentiated leukemic population might display decreased sensitivity toward BCL-2 inhibition. Overall, our data reveal the spleen as a site of residual disease for ABT-199 treatment in ETP-ALL and provide evidence for plasticity in T-cell differentiation as a mechanism of therapy resistance.

Clinical significance of CD34+CD117dim/CD34+CD117bri myeloblast-associated gene expression in t(8;21) acute myeloid leukemia.

t(8;21)(q22;q22) acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy with a high relapse rate in China. Two leukemic myeloblast populations (CD34+CD117dim and CD34+CD117bri) were previously identified in t(8;21) AML, and CD34+CD117dim cell proportion was determined as an independent factor for this disease outcome. Here, we examined the impact of CD34+CD117dim/CD34+CD117bri myeloblast-associated gene expression on t(8;21) AML clinical prognosis. In this study, 85 patients with t(8;21) AML were enrolled. The mRNA expression levels of CD34+CD117dim-associated genes (LGALS1, EMP3, and CRIP1) and CD34+CD117bri-associated genes (TRH, PLAC8, and IGLL1) were measured using quantitative reverse transcription PCR. Associations between gene expression and clinical outcomes were determined using Cox regression models. Results showed that patients with high LGALS1, EMP3, or CRIP1 expression had significantly inferior overall survival (OS), whereas those with high TRH or PLAC8 expression showed relatively favorable prognosis. Univariate analysis revealed that CD19, CD34+CD117dim proportion, KIT mutation, minimal residual disease (MRD), and expression levels of LGALS1, EMP3, CRIP1, TRH and PLAC8 were associated with OS. Multivariate analysis indicated that KIT mutation, MRD and CRIP1 and TRH expression levels were independent prognostic variables for OS. Identifying the clinical relevance of CD34+CD117dim/CD34+CD117bri myeloblast-associated gene expression may provide new clinically prognostic markers for t(8;21) AML.

Cytogenetically Aberrant Cells in the Stem Cell Compartment (CD34+lin) in Acute Myeloid Leukemia

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.

How I Diagnose Minimal/Measurable Residual Disease in B Lymphoblastic Leukemia/Lymphoma by Flow Cytometry.

Assessment for minimal/measurable residual disease (MRD) is a powerful prognostic factor in B lymphoblastic leukemia/lymphoma (B-LL/L) that is quickly becoming standard of care in assessing patients with B-LL/L posttherapy. MRD can be assessed using methodologies including flow cytometry and molecular genetics, with the former being rapid, relatively inexpensive, and widely applicable in many hematopathology/flow cytometry laboratories. This article presents an approach to MRD detection in B-LL/L by flow cytometry through case presentations with illustration of several potential pitfalls. We review normal maturation patterns, antigens used for assessment, flow panels that can be utilized, considerations to be made during therapy, and clinical impact. The benefits and drawbacks when using the "different from normal" and "leukemia associated phenotype" approaches are considered. Evaluation for MRD in B-LL/L by flow cytometry relies on a knowledge of normal immunophenotypic patterns associated with B-cell maturation in states of rest and marrow regeneration so that one can identify patterns of antigen expression that differentiate abnormal, leukemic populations from regenerating hematogones or B-cell precursors. The nature of therapy can affect normal patterns, a phenomenon especially important to take into consideration given the increased use of targeted therapies in the treatment of B-LL/L. Flow cytometry is widely available in many laboratories and is a cost-effective way to evaluate for B-LL/L MRD. However, panel validation and interpreter education are crucial for accurate assessment.

The evolution of relapse of adult T cell acute lymphoblastic leukemia

BackgroundAdult T cell acute lymphoblastic leukemia (T-ALL) is a rare disease that affects less than 10 individuals in one million. It has been less studied than its cognate pediatric malignancy, which is more prevalent. A higher percentage of the adult patients relapse, compared to children. It is thus essential to study the mechanisms of relapse of adult T-ALL cases.ResultsWe profile whole-genome somatic mutations of 19 primary T-ALLs from adult patients and the corresponding relapse malignancies and analyze their evolution upon treatment in comparison with 238 pediatric and young adult ALL cases. We compare the mutational processes and driver mutations active in primary and relapse adult T-ALLs with those of pediatric patients. A precise estimation of clock-like mutations in leukemic cells shows that the emergence of the relapse clone occurs several months before the diagnosis of the primary T-ALL. Specifically, through the doubling time of the leukemic population, we find that in at least 14 out of the 19 patients, the population of relapse leukemia present at the moment of diagnosis comprises more than one but fewer than 108 blasts. Using simulations, we show that in all patients the relapse appears to be driven by genetic mutations.ConclusionsThe early appearance of a population of leukemic cells with genetic mechanisms of resistance across adult T-ALL cases constitutes a challenge for treatment. Improving early detection of the malignancy is thus key to prevent its relapse.

Reduction of Clostridium Difficile Infection in an Academic Medical Center Blood and Marrow Transplant Population

Background/Aim: Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea among hospitalized patients. Blood and Marrow Transplant patients appear to be one of the most vulnerable populations due to previous antimicrobial exposure, graft vs. host disease and prolonged hospitalizations. CDI in cancer patients is associated with higher mortality rates and prolonged hospitalization. CDI affects the course of the disease by delaying treatments such as chemotherapy. Furthermore, the estimated CDI infection-related costs are 4.8 billion dollars for acute care facilities alone. Methods: In 2015, an increase in house-wide CDI at Robert Wood Johnson University Hospital occurred and triggered an extensive analysis of these cases with Blood and Marrow Transplant and leukemic patients totaling 30% of all CDI in the hospital in 2019. This increase occurred despite the previous implementation of Tru D ultraviolet radiation for all CDI rooms and all Blood and Marrow Transplant unit rooms. Several other initiatives including decreased use of proton pump inhibitors, decreased utilization of piperacillin-tazobactam and house-wide monitoring of hand hygiene did not significantly decrease the percentage of CDI comprised by Oncology patients. In 2018, various other CDI prevention initiatives included: daily Chlorhexidine bathing for adults, implementation of a nursing specimen collection sheet with CDI Criteria; cleaning with OxyCide sporicidal hospital disinfectant, and ongoing hand hygiene champion training with the implementation of an electronic hand hygiene system. Although this resulted in a house-wide CDI SIR &amp;lt; 1 for 3 out of 4 Quarters of 2018, which was a 13% decrease from 2017, there was still no change in the rates in oncology population comprising 25% of all CDI cases. In 2019, CDI orders were updated to increase the denial of testing if patients received laxatives within the last 48 hours and work began with nursing to startscreening upon admission for diarrhea within the past 48 triggering a CDI order. Again, the 2019 house-wide CDI SIR decreased (0.903) but Blood and Marrow Transplant and leukemic patients CDI increased, making up 30% of all CDI cases. A retrospective review of all oncology CDI cases was conducted, noting that all tested PCR positive. A Blood and Marrow Transplant- specific action plan was developed which included the initiation of prophylactic oral Vancomycin on admission for all allogeneic and autologous patients. Results: After implementation of prophylactic oral vancomycin, there have been 9 CDI in the first 7 months of 2020 in the oncology population and only 1 CDI in the Blood and Marrow Transplant and leukemic population during this timeframe. This is an 89% decrease from the same period of 2019 for the Blood and Marrow/leukemic population. In addition, only 3.7% of the house-wide CDI is from the Blood and Marrow Transplant and leukemic population; 23% of the house-wide CDI is from the Oncology population. As of yet, np untoward effects have been seen after this implementation. The incidence of documented bacteremia has remained constant at approximately 1.5 patients/ quarter; acute GVHD in allogeneic patients has remained constant at approximately 4 patients/ quarter. Conclusions: The utilization of vancomycin prophylactically upon admission for all autologous and allogeneic patients may have a significant impact on CDI rates in the oncology population. More time is needed to evaluate the effects of this at Robert Wood Johnson University Hospital but this is the first and only intervention that has helped to decrease the CDI rates in Oncology in 5 years. Next steps include participating in a national collaborative on CDI prevention with several other similar institutions to drive actions for improvement. Goals of this collaboration include strengthening our antibiotic stewardship committee, to have a dedicated staff person to coordinate CDI prevention activities including nurse and physician champions and to increase adherence to use of hand hygiene/ gowns/ gloves for families and visitors. Disclosures No relevant conflicts of interest to declare.

The Transmembrane Protein CD37 Is Not a Promising Therapeutic Target in Acute Myeloid Leukaemia

Acute myeloid leukaemia is a disease of the haematopoietic stem and progenitor cells distinguished by an impairment of proliferation, propagation and differentiation of the haematopoietic lineage[1]. AML is a heterogenous disease, where patient stratification and personalised therapeutic strategies are of importance[2]. The transmembrane protein CD37 has been associated with regulation of cell to cell interaction and proliferation in both B and T cells. Mice knockout studies have suggested that CD37 is not crucial for B cell development, however, it is highly expressed in malignant and suboptimal conditions and thus represent a good target for B-cell malignancies[3]. Pereira et al. reported that their anti-CD37 antibody-drug conjugate was a potential therapeutic in AML[4]. Their study revealed anti-leukemic effect in primary AML material and cell lines bothin vivoandin vitro. An investigation of CD37 expression in AML patients is needed to understand the relevance of CD37 immunotherapy. To investigate the relevance of targeted therapy of CD37 the chimeric mAb NNV003 anti-CD37 antibody was evaluated in AML samples. Single-cell time-of-flight mass cytometry (CyTOF), an established tool to analyse the immune signalling in bone marrow and peripheral blood, was used to investigate CD37 expression in 59 AML patient samples. A panel was designed to categorise hierarchical landscape of AML and the leukemic stem cell population (LSCs). CyTOF identified CD37 expression exclusively in the B-cell population of our 59 patients, whereas the AML blast population was negative for CD37 expression. Flow cytometry, however; identified 5/59 patients with CD37 expression in the leukemic blast population. Although previous studies show that an anti-CD37 drug conjugate showed anti-leukemic effectin vivoandin vitro, our results do not support that it is a promising therapeutic biomarker. We have uncovered that malignant expression of CD37 is not a feature of the AML patients investigated. We do not consider CD37 to be a promising and good target for precision therapy of AML. Kayser, S. and M.J. Levis, Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol, 2018. 180(4): p. 484-500.Patel, J.P., et al., Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012. 366(12): p. 1079-89.Payandeh, Z., et al., Anti-CD37 targeted immunotherapy of B-Cell malignancies. Biotechnol Lett, 2018. 40(11-12): p. 1459-1466.Pereira, D.S., et al., AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Mol Cancer Ther, 2015. 14(7): p. 1650-60. Disclosures Heyerdahl: Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Dahle:Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Gjertsen:Novartis:Consultancy;KinN Therapeutics AS:Current equity holder in private company;Pfizer Inc:Consultancy;BerGenBio AS:Consultancy, Research Funding;Alden Cancer Therapy AS:Current equity holder in private company.Mccormack:KinN Therapeutics AS:Current equity holder in private company.

Mixed-phenotype acute leukemia consisting of five heterogeneous leukemic populations without the expression of CD34.

Mixed-phenotype acute leukemia consisting of five heterogeneous leukemic populations without the expression of CD34.

AML-354: High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Patients with Relapsed/Refractory AML

We designed a 51-parameter mass cytometry panel and interrogated acute myeloid leukemia (AML) heterogeneity to assess cellular hierarchy, anti-apoptotic molecules, exhaustion markers and targets for CAR T-cell therapy in R/R AML. We generated two-dimensional t-SNE maps to visualize distinct populations and observed significant heterogeneity among patients with regards to leukemic compartment composition. Notably, the leukemic bone marrow compartment harbored leukemia stem cells (CD34+CD38-), immature leukemic cells (CD34+CD38+), more differentiated leukemic blasts (CD33+CD34-) and major immune subsets. We initially assessed the distribution of anti-apoptotic molecules across leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and leukemic blasts with monocytic differentiation had significantly low levels of Bcl-2. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemic cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors resulted in highly synergistic effects in AML PDX models (Carter et.al 2018, 2019). Of interest, Bcl-xl was ubiquitously expressed across leukemic and immune cell clusters. Next, we assessed expression patterns of CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns for all CAR T-cell therapy targets in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 expression was the most promising marker and expressed across almost all leukemia compartments. Nevertheless, CLL-1 expression on leukemic cells was not ubiquitous in a small group of patients. In line with leukemic cell heterogeneity, we found a significant degree of variation in immune cell composition. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively. Single-cell profiling of R/R AML using mass cytometry reveals significant heterogeneous expression patterns of molecules targeted by CAR T-cells, immunotherapeutic approaches and targeted therapies. This approach provides a rationale to adopt combinatorial therapeutic approaches to target distinct leukemic populations with widely varying target expression.

Describing the evolution of myeloid-derived leucocytes in treated B-lineage paediatric acute lymphoblastic leukaemia with a data-driven granulocyte-monocyte-blast model.

Acute lymphoblastic leukaemia (ALL) is associated with a compromised myeloid system. Understanding the state of granulopoiesis in a patient during treatment, places the clinician in an advantageous position. Mathematical models are aids able to present the clinician with insight into the behaviour of myeloid-derived leucocytes. The main objective of this investigation was to determine whether a proposed model of ALL during induction therapy would be a usable descriptor of the system. The model assumes the co-occurrence of the independent leukaemic and normal marrow populations. It is comprised of four delay-differential equations, capturing the fundamental characteristics of the blood and bone marrow myeloid leucocytes and B-lineage lymphoblasts. The effect of treatment was presumed to amplify cell loss within both populations. Clinical data was used to inform the construction, calibration and examination of the model. The model is promising-presenting a good foundation for the development of a clinical supportive tool. The predicted parameters and forecasts aligned with clinical expectations. The starting assumptions were also found to be sound. A comparative investigation highlighted the differing responses of similarly diagnosed patients during treatment and further testing on patient data emphasized patient specificity. Model examination recommended the explicit consideration of the suppressive effects of treatment on the normal population production. Additionally, patient-related factors that could have resulted in such different responses between patients need to be considered. The parameter estimates require refinement to incorporate the action of treatment. Furthermore, the myeloid populations require separate consideration. Despite the model providing explanation, incorporating these recommendations would enhance both model usability and predictive capacity.

Genomic Analysis of Cellular Hierarchy in Acute Myeloid Leukemia Using Ultrasensitive LC-FACS-Seq

Genomic Analysis of Cellular Hierarchy in Acute Myeloid Leukemia Using Ultrasensitive LC-FACS-Seq

Clinical Characteristics and Outcome of Biphenotypic Acute Leukemia: 10 Cases Report and Literature Review

Background: Based on the cells' antigen differentiation expression patterns, most cases of acute leukemia (AL) are classified as either myeloid or lymphoid lineage. However, there are patients with leukemic blast population that co-express both lymphoid and myeloid characteristics, known as biphenotypic acute leukemia (BAL) or mixed-phenotype acute leukemia (MPAL). BAL is a rare subgroup of acute leukemia with a poor prognosis. Currently, a standard chemotherapy treatment has yet to be established. Aims: This study aims to retrospectively investigate the incidence, pathological characteristics, and clinical outcome of BAL patients from the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China, between January, 2014 and June 2019. Methods: From January 2014 to June 2019, the medical records of newly diagnosed BAL based on the EGIL criteria, or ALAL based on the 2008/2016 WHO criteria and who were admitted at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) were retrospectively reviewed. The diagnostic workup of BAL was based on initial morphological, cytochemical, and immunophenotypic evaluation of the BM. Using the EGIL scoring system and 2008/2016 WHO criteria, treatment methods and outcome data, including induction chemotherapy, complete remission (CR), relapse, and death, were collected and reviewed. This study was an observational, retrospective, and descriptive study of the clinical aspects of BAL, which was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Results: Among a total of 6100 newly diagnosed patients with AL, 10 (0.16%) patients satisfied the definition of BAL based on the EGIL criteria, or MPAL based on the WHO criteria, including 7 males and 3 females. The median age of these patients at diagnosis was 19 years (range 3-67 years). One patient (Pt #1) had extramedullary invasion, including neck, mediastinum (area 8), posterior septal group, left axillary, peritoneal and retroperitoneal lymph nodes. Another patient (Pt #7) had extramedullary invasion with central nervous system leukemia. Immunophenotypic characteristics showed that among 10 BAL patients, 4 cases carried B/Myeloid phenotype, 4 cases carried T/Myeloid phenotype and 2 case carried T/B phenotype. For 8 patients with myeloid lineage differentiation, MPO was positive in 6 (75%), CD13 in 4 (50%), CD33 in 4 (50%), CD38 in 6 (75%), CD58 in 3 (37.5%), CD117 in 4 (50%) patients. In 5 patients with B-lymphoid lineage differentiation, CD19 was positive in 4 (80%), CD79a in 5 (100%) patients. The most frequently T-lymphoid lineage positive markers were CD7 and cCD3, which were positive in 4 of 5 (80%) patients. The stem cell markers HLA-DR and CD34 were both positive in 7 (7/10, 70%) patients, while CD117 was positive in 4 (4/10, 40%) patients. Cytogenetic analysis results showed that 7 of 10 patients had normal karyotypes (Pt#1, 2, 6, 7, 8, 9,10) while the other 3 patients had clonal abnormalities. Pt#4 had 46, xx, t(9;22)(q34;q11) aberration; Pt#6 had 45, XY, -7/46, idem,+8 aberration; and Pt#7 had 45, X,-Y, del(7)q32, t(8,21) (q22;q22) aberration. Two patients (Pt# 3, 5) had RUNX1 gene mutation, one patient (Pt#4) had BCR/ABL fusion gene mutation, and one patient (Pt#9) had JAK1, JAK3, FBXW7 mutation. Six patients received ALL-directed induction therapy (VDLP), whereas two patients received AML-directed induction therapy (MEA and IA regimens). Overall, 4 of 8 (50%) patients with chemotherapy achieved complete remission (CR) after initial induction therapy. In the AML-directed therapy group, 1 of 2 (50%) patients achieved CR. Meanwhile, 3 of 6 (50%) patients achieved CR after ALL-directed induction chemotherapy. Two patients received HSCT after initial CRs, one patient (Pt#7) died two months after transplantation due to the infection, and another patient (Pt#5) is still alive. With an average of 14.3 (4.0-42.0) months follow-up, the median survival time was 7 months. Conclusion: We reported 10 cases of BAL, including 4 cases of B/Myeloid phenotype, 4 cases of T/Myeloid phenotype and 2 case of T/B phenotype. 4 of 8 patients achieved CR (50%) after initial chemotherapy. Although many patients achieved CR after initial chemotherapy, but the relapse rate was very high and the CR rate after relapse was very low. Our results confirmed that BAL is a rare malignancy with a very poor prognosis. Disclosures No relevant conflicts of interest to declare.

Efficient Elimination of Acute Myeloid Leukemia Cells through Inhibition of KDM4A in Combination with PARP Inhibition

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 &amp; IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation &gt; 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (&gt;15% reduction) and increased apoptosis (&gt;20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.

Targeted Killing of AML Cells By Bispecific Antibody Armed Activated T Cells

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of hematopoietic stem/progenitor cells that poses a significant therapeutic challenge. Recurrence of the disease is attributed to leukemia-initiating cells, also referred to as leukemic stem cells (LSCs), which are thought to be spared from chemotherapy and capable of reinitiating the disease. Thus, novel non-toxic therapeutic strategies to target and eradicate LSCs and AML blasts are urgently needed. Immunotherapy (IT) is an attractive strategy to improve outcomes for patients with AML, as it does not rely on the cytotoxic mechanisms employed by conventional chemotherapies. Methods: Bispecific antibody was produced by chemical hetero-conjugation of anti-CD33 monoclonal antibody (gemtuzumab ozogamicin [GO]) linked to calicheamicin to anti-CD3 monoclonal antibody (anti-CD3 x GO bispecific antibody, CD33Bi) or anti-CD3 and anti-CD123 (CD123Bi). We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD33Bi and CD123Bi armed ATC using flow cytometry-based cytotoxicity assays. Cytokines and chemokines released during CD33Bi or CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. A xenogeneic NOD/SCID/gamma chain KO (NSG) mouse model was used for the evaluation of in vivo activity of Bispecific antibody (50ng/million cells) Armed activated T cells (BATs). Engraftment of AML cells-KG1 was tracked by quantifying the human CD45+ AML cells using flow cytometry. Two patient samples were analyzed for blast and LSC population before and after incubation with CD123-BATs. Results: In a non-radioactive quantitative flow cytometry-based cytotoxicity assay Bispecific antibody (50ng/million cells) Armed activated T cells (BATs) show substantial killing by both CD33-BATs and CD123-BATs against AML cell lines (EOL1, KG1, NoMo1 and TF-1) at 1:1 and 2:1 E/T ratios ranging from 60-90% cytotoxicity and produced increased levels of Th1 cytokines (TNF-alpha, GM-CSF and IFN-gamma). The KG1 engrafted 6- to 8-week-old NSG mice (n=8 mice/group) injected IV with 20 x 106 CD33-BATs (3x/week for 4 weeks; 100ml/injection) was able to inhibit leukemic cell expansion significantly compared to 0.06 mg/kg gemtuzumab ozogamicin [GO] antibody (p&lt;0.0006); likewise significant inhibition of leukemic cell expansion was observed with CD123-BATs compared to PBS control mice (p&lt;0.009). One patient sample incubated with CD123-BATs showed significant reduction in blast after CD123-BATs treatment compared to before treatment (13.2% vs. 30%) or CD34+CD38- LSC after CD123-BATs treatment (5.2%) compared to before treatment (20%). Second patient sample also showed 50% reduction in blast population, no LSC were detected in this sample. Conclusions: ATC armed with CD33Bi or CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations, able to inhibit leukemic cell expansion significantly compared to the control mice. This approach may provide a potent and non-toxic strategy to target leukemic blasts and leukemic stem cells. Disclosures Huang: TransTarget Inc.: Other: Co-Founder.

Lineage Tracing in Chronic Lymphocytic Leukemia Reveals Clones with Stable Gene Expression States That Differentially Respond to Therapy

Cancer's ability to evolve and adapt is a major challenge to therapeutic success. Fueling this evolution is vast tumor heterogeneity, with constituent clones varying in their genetics, epigenetics and response to therapy. As a field, however, we have yet to couple the genetic alterations present within individual clones to their transcriptional or functional outputs. Here, we applied a novel adaptation of clone tracing that integrates DNA barcoding with single-cell RNA sequencing (scRNA-seq) to HG3, a CLL cell line harboring del(13q) and no other known cancer drivers, to model in vitro responses to front-line chemotherapy with fludarabine and cyclophosphamide at clone-level resolution. To generate a high-diversity barcode library compatible with scRNA-seq, a random pool of 20 base pair DNA barcodes was introduced into the 3'UTR of a reporter gene in a lentiviral expression vector. This viral barcode library was transduced into HG3 cells (at MOI 0.1 to minimize multiple barcode-tagging of cells) and 1.2x106 barcoded cells were sorted and expanded to establish the parental barcoded HG3 population. We subsequently treated this barcoded population with an LD95 combined dose of fludarabine and mafosfamide (the in vitro analog of cyclophosphamide) in 8 parallel experiments. Cell barcodes were sequenced prior to treatment (TP1) and following outgrowth from treatment (TP2) for analysis of clonal composition. 10,000 cells each from TP1 and from 2 of 8 parallel replicates at TP2 were processed for scRNA-seq. We observed a massive decrease in viability across all 8 replicates, with regrowth occurring at 20 days post-treatment. Barcode analysis revealed a marked decrease in clonal diversity from TP1 to TP2 (11,827 to 2,622 ± 380, n=8; or ~78%), and clones that survived treatment did so consistently such that 94% of surviving cells in each replicate had a clonal identity that was present in all 8 replicates. Analysis of clonally-resolved transcriptional profiles revealed that clones consistently fell into one of two stable gene expression states (clusters) prior to treatment, with nominal intermixing between populations. Treatment predominantly selected for clones comprising the smaller of these two clusters (TP1-'high tolerance'), with only a minimal number of resistant clones originating from the larger cluster at TP1 (TP1-'low tolerance'). Pathway and gene set enrichment analysis of these two TP1 clusters demonstrated that TP1-high tolerance had a stark upregulation of common CLL signaling pathways (i.e. WNT and CXCR4, an inflammatory/migratory phenotype) and a reliance on chromatin modification pathways. TP1-low tolerance, on the other hand, exhibited upregulated type 2 antigen presentation and prostaglandin biosynthesis/metabolism which has a known role in driving inflammation and migration in adjacent cells (Wang et al, BMJ 2006). These gene expression states remained stable after treatment, but with the added upregulation of well-described mechanisms of resistance to cyclophosphamide, with TP1-high tolerance exhibiting upregulation of GSTP1, a glutathione S-transferase that is a main mediator of cyclophosphamide metabolism, and TP1-low tolerance exhibiting upregulation of members of the ALDH family thought to ameliorate toxicity from chemotherapy-induced ROS (Andersson et al, Acta Oncologica 1995). Through this work, we resolved the underlying clonal composition of a CLL cell line and observed that the constituent clones exhibit stable and discrete gene expression states that differentially respond to chemotherapy. We noted two different axes of resistance - a more successful avenue that relies on WNT and CXCR4 signaling as well as cyclophosphamide clearance for resistance, and a less-successful avenue that involves clearance of reactive oxygen species for survival. The intersection of these two critical CLL pathways in in vitro resistance to first-line CLL therapy is of particular interest given the FAT1 (WNT regulator) mutations and CXCR4 upregulation frequently seen in chemo-refractory CLL (Messina et al, Blood 2014; Burger et al, Blood 2006), and future efforts will assess the stability and interplay of these two pathways in patient samples collected upon relapse to fludarabine and cyclophosphamide. Further, our approach provides a template for the high-resolution study of tens of thousands of clones and their respective phenotypes in a mixed leukemic population. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.