The association of complex genetic background with the prognosis of acute leukemia with ambiguous lineage

Acute leukemia with ambiguous lineage (ALAL) is a rare and highly aggressive malignancy with limited molecular characterization and therapeutic recommendations. In this study, we retrospectively analyzed 1635 acute leukemia cases in our center from January 2012 to June 2018. The diagnose of ALAL was based on either EGIL or 2016 WHO criteria, a total of 39 patients were included. Four patients diagnosed as acute undifferentiated leukemia (AUL) by both classification systems. Among the patients underwent high-throughput sequencing, 89.5% were detected at least one mutation and the median number of gene mutation was 3 (0–8) per sample. The most frequently mutated genes were NRAS (4, 21%), CEBPA (4, 21%), JAK3 (3, 16%), RUNX1 (3, 16%). The mutations detected in mixed-phenotype acute leukemia (MPAL) enriched in genes related to genomic stability and transcriptional regulation; while AUL cases frequently mutated in genes involved in signaling pathway. The survival analysis strongly suggested that mutation burden may play important roles to predict the clinical outcomes of ALAL. In addition, the patients excluded by WHO criteria had even worse clinical outcome than those included. The association of the genetic complexity of blast cells with the clinical outcomes and rationality of the diagnostic criteria of WHO system need to be evaluated by more large-scale prospective clinical studies.

Occurrence of Marek's disease in vaccinated Algerian broiler breeder flocks: A histopathological survey

Background and Aim: Marek's disease (MD) is a lymphoproliferative disease that occurs in chickens. In the absence of control measures, MD causes devastating losses to commercial poultry flocks. Vaccination has enabled dramatic success in the prevention and control of MD. However, the MD vaccination program has failed frequently, and occasional clinical outbreaks have been reported in the vaccinated flocks as well. The present study aimed to describe the clinical and histopathological characteristics of the field cases of MD in broiler breeder flocks. Materials and Methods: A survey on the update of MD occurrence in Algerian broiler breeder flocks was conducted from June 2020 to September 2020. Ten vaccinated broiler breeder flocks located in Central Algeria and having progressive tumors in different visceral organs were evaluated for MD virus infection by conducting a histopathological examination of the birds. Results: The age of the birds affected with MD ranged from 13 to 22 weeks. The mortality rate varied sensitively from 4% to 10%. The clinical symptoms reported in the affected flocks included locomotor, nervous, digestive, and respiratory symptoms. Necropsy of the dead or euthanized birds revealed visceral lymphomatosis in several organs and macroscopic changes in the peripheral nerves (including loss of longitudinal striation, color change [grayish], and volume increase). The histopathological findings included the infiltration and proliferation of lymphocytes and blast cells (lymphoblasts) in various organs of the birds, which are the typical characteristics of MD and, therefore, confirmed the field infection of MD in these birds. Conclusion: The present study provided evidence for the high prevalence of MD in the broiler breeder flocks vaccinated with a bivalent vaccine (turkey herpesvirus+Rispens) at the hatchery. The findings of the present study may indicate highlevel failure of vaccination in these birds.

BMSCs Regulate the Function of NK Cell By CD155/Tigit/CD226 Pathway in MDS Patients

Objective To analyze the immune status of MDS patients and evaluate its impact on prognosis.To investigate the specific mechanism of how mesenchymal stromal cells (MSCs) affecting immunity. Methods The study included 84 healthy controls and 84 inpatients with primary MDS, 22 patients with AML who were initially diagnosed in the Department of Hematology, General Hospital of Tianjin Medical University from January 2017 to July 2021.We collected flow cytometry(FCM) reports and clinical data. All MDS patients were stratified by IPSS-R for disease risk. According to IPSS-R classification, Very Low/Low and Int/High/Very high were divided into lower-risk MDS group and higher-risk MDS group, respectively.1. Statistical analysis of lymphocyte subset counts and cytokines in peripheral blood of different types of patients and healthy controls. 2. Correlation analysis between hemoglobin , platelets,neutrophil, blast cells, cytogenetics and lymphocyte counts was performed.3.We generated MSCs in BM samples from MDS patients and HCs.The expression of CD155 in MSCs and TIGIT/CD226 in lymphocytes were detected by FCM. Results 1.Percentage counts of NK cells in the HC group(19.94,95%CI 17.86-22.02，P<0.001)were significantly higher than other groups.The higher-risk group of MDS(11.51,95%CI 9.34-13.68) was obviously lower than the lower-risk group(16.91,95%CI 13.85-19,97)(P=0.006),close to the AML group(9.77,95%CI 6.33-13.20,Figure1).Meanwhile, the level of IFN-Ƴ in serum which is secreted mainly by NK cells was also the highest in the HC group(5.50,95%CI 3.74-7.26), followed by the MDS group and the lowest in the AML group(2.64,95%CI 1.91-3.37,Figure1).In addition, CD8+ T cells and CD19+ cells had similar results as NK cells, but there was no significant difference with CD3+ cells and CD4+ T cells.2.Significantly higher level of NK cells percentage counts were found in patients with hemoglobin ≥ 100 ×10 12/L compared to patients with lower hemoglobin(23.23,95%CI 17.26-29.21 and 15.77,95%CI 13.00-18.55,P=0.010). While the levels of NK cells were higher in patients with blast cells≤5% than in those with higher blast cells.(19.51,95%CI 16.09-22.93 and 14.65,95%CI 11.55-17.74,P=0.027). No significant difference was found between NK cell levels with regard to platelet and neutrophil counts.CD8+ T cells behaved similarly to NK cells. 3.The expression of CD155 on the surface of BMSCs was significantly higher in the MDS group than that in the HC group(P=0.001，Figure3).Moreover,MDS patients had higher expression of TIGIT(P=0.012) and lower expression of CD226(P=0.001,Figure4B) on the surface of NK cells in bone marrow than that in HCs.And CD8+ T cells also have similar results as NK cells.We analyzed the correlation between TIGIT of NK cells and CD155 of BMSCs, and found that they were negatively correlated（P=0.0297）, while CD226 was positively correlated with CD155（P=0.0706，Figure 4C）. Conclusions The decreased immune function in MDS patients is associated with prognosis.BMSCs are responsible for inducing an immune-suppressive microenvironment in MDS through CD155/TIGIT/CD226 pathway, the underlying mechanism remains to be further study. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Flow Cytometric Detection of the Ki-67 Proliferation Index and the Bcl-2 Anti-Apoptotic Index in Myelodysplastic Syndromes and Acute Myeloid Leukemia, and Their Clinical Implications

Introduction: Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry in classification of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To further advance the integration and objectification of flow cytometry for characterization of these malignancies, more dynamic parameters assessing cell behavioral characteristics could prove useful, such as proliferative and (anti-)apoptotic markers. Proliferation and (anti-)apoptosis are processes that are tightly related to the pathogenesis, progression and chemo-/immunotherapy response of cancers. As a result, proliferation and (anti-)apoptotic markers have proven their value as objective parameters in the field of histopathology for diagnostic and prognostic applications in solid tumors and lymphoma. Although use of proliferative and (anti-)apoptotic markers as objective parameters in the diagnostic process of MDS and AML was studied in the past decades, this did not result in the incorporation of these biomarkers in their clinical diagnosis. The recent developments in flow cytometric analyses now allow the quantification of proliferative and (anti-)apoptotic fractions at the level of individual maturing bone marrow cells. Therefore, we aim to determine the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices in maturing bone marrow cells in order to assess whether these parameters could have future clinical implications for the diagnostic work-up of MDS and AML. Methods: Fifty bone marrow aspirates from femoral heads of non-malignant cases, 20 aspirates of MDS patients and 20 aspirates of AML were included in this study. Ten-color flow cytometry in combination with a software-based maturation tool was used for analysis of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices of blast cells and during the erythro-, myelo-, and monopoiesis. Results: Ki-67 proliferative indices of blast cells and immature erythroid, myeloid and monocytic cells were significantly lower in MDS patients compared to the non-malignant cases, while the Bcl-2 anti-apoptotic indices were significantly elevated in these cells. Furthermore, the Bcl-2 anti-apoptotic indices were also increased in mature erythroid, myeloid and monocytic cells of MDS patients. The decreased Ki-67 proliferative indices and increased Bcl-2 anti-apoptotic indices in blast cells and erythroid, myeloid and monocytic cells were even more prominently observed in AML patients. Conclusions: The lowered Ki-67 proliferative indices and elevated Bcl-2 anti-apoptotic indices in blast cells and immature progenitor cells led to a better understanding of the pathophysiology of MDS and AML, and explained the low chemotherapy response of these patients. Side-effects of such therapies can also be explained by the Ki-67 proliferation indices and Bcl-2 anti-apoptotic indices. Moreover, the increase of the Bcl-2 anti-apoptotic fraction is an important factor in the progression of MDS to AML. Future studies on the clinical applications of these parameters for MDS and AML are necessary and can include many applications, such as prediction of chemo-/immunotherapy response, diagnostic and prognostic applications. Disclosures Ramaekers: Nordic-MUbio: Current Employment.

Avidity-Engineered CD3 Engaging DARPin ® Targeting Three Tumor Associated Antigens Induce Strong and Specific T Cell Dependent Killing of AML Cells with Potential for Improved Safety

AML is driven by leukemic stem cells (LSC) that resist conventional chemotherapies and remain unaffected in their niche, continually replenishing circulating blast cells. We postulated that an avidity-engineered CD3 engaging DARPin ® (Designed Ankyrin Repeat Protein) able to simultaneously target LSC-specific CD70 as well as CD123 and CD33 could allow highly efficient and specific T cell-mediated killing of AML LSCs and circulating blast cells while preserving a therapeutic window towards healthy cells. Moreover, this simultaneous targeting of three different tumor associated antigens (TAAs) has the potential to address tumor heterogeneity, allowing targeting of AML cells with different co-expression patterns and/or expression levels of each single TAA. To achieve this ambitious goal we used our DARPin ® platform to build a novel class of triple targeting CD3 engaging molecules. Our DARPin ® libraries contain trillions of molecules allowing the generation of highly diverse binders against target proteins that can be easily combined into multi-specific DARPins ® to elicit desired biological effects. We leveraged this proprietary platform to screen multi-specific CD3 engaging DARPin ® molecules, including serum albumin binding DARPins ® for systemic half-life extension, and identify the optimal target affinity and molecular architecture to ensure potent avidity-driven T cell-mediated killing of AML cells while sparing healthy cells. This approach allowed the generation of CD3 engaging DARPins ® able to target simultaneously CD33, CD123, and CD70. Such DARPins ® demonstrated, in both allogenic and autologous setting, single digit pM potency against AML cell lines and primary cells expressing any combination of at least 2 of the 3 targeted TAAs, while showing low activity against single TAA-expressing cells, the latter representing cells of the healthy compartment. Higher expression of the selected TAAs on LSCs vs normal hematopoietic stem cells (HSC) can further enhance the selectivity of such an avidity driven molecule, leading to the preferential killing of LSC over HSC. Moreover, our multi-specific T cell engager (TCE) format resulted in a significant decrease in cytokine release in a whole blood test system for cytokine release syndrome (CRS) when compared to other mono-targeting TCE therapies, confirming its specificity and the potential for an improved safety profile within the normal hematopoietic system. Additionally, while showing similar anti-tumor efficacy in a mouse xenograft model using Molm-13 cell line and human PBMCs, CRS measured in serum 4 h after the initial injection of our multi-specific DARPin ® molecule was drastically reduced compared to a reference CD33 TCE, further strengthening the evidence that our multi targeting DARPins might also exhibit a good safety profile in humans. In conclusion, we were able to generate multi-specific CD3 engaging DARPin ® molecules with tailored affinities towards different TAAs showing exceptional efficacy and with the potential for superior safety over mono-specific TCE approaches, including systemic half-life extension to avoid a continuous intravenous infusion-based therapy. Disclosures Bianchi: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reschke: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Reichen: Molecular Partners AG (MAG): Current holder of stock options in a privately-held company. Fischer: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Grübler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Eggenschwiler: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Krieg: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ioannou: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ragusa: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Looser: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Spitzli: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Herzog: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Villemagne: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kaufmann: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Matzner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Auge: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Hänggi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ali: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Franchini: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Kirkin: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Schlereth: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Luethi: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Ochsenbein: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Riether: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Steiner: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company. Goubier: Molecular Partners AG (MAG): Other: Owns stock options and/or shares of the company.

A Bispecific Antibody Targeting CD117 and CD3 Enables T Cell Mediated Killing of CD117-Expressing Healthy and Malignant Hematopoietic Cells

INTRODUCTION: Hematopoietic stem and progenitor cells (HSPCs) support life-long hematopoiesis. A single HSPC can also be at the origin of hematological malignancies, such as Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). Allogeneic HSCT with the intent to eliminate recipient AML or MDS and at the same time replace recipient HSPC with donor-HSPC and immune cells is a life-saving therapeutic option for many patients. However, chemotherapy (and sometimes in addition gamma-irradiation based conditioning regiments) prior to HSCT are associated with substantial toxicity. Thus, due to benefit-outweighing treatment-related toxicity and mortality, frail, multi-morbid and elderly patients are usually excluded from potentially curative allo-HSCT approaches. For these reasons, more selective preconditioning strategies, leading to residual AML/MDS elimination and creating "space" for incoming HSPCs, are required. Selective targeting of CD117 with monoclonal antibodies has been proposed as a strategy to remove endogenous HSPCs, enabling an effective but mild preconditioning. However, specific conditioning of AML and MDS patients, prior to HSCT, might require a more potent effector cell type. We hypothesized that a CD117 and CD3 binding, T cell engaging and activating antibody construct (CD117xCD3 TEA) with a short half-life might be an ideal means to selectively eliminate CD117-expressing healthy HSPCs and residual CD117-expressing AML or MDS cells prior to allo-HSCT. METHODS: We cloned and expressed CD117xCD3 TEA in tandem scFv format and produced it by transient gene expression in Chinese hamster ovary cells (CHO-S). The fusion proteins were purified to homogeneity by protein A affinity chromatography. We derived target cell lines with varying surface levels of CD117 (high, medium and low) from CD117 negative parental cell lines HL-60 and MOLM-14 (Myburgh et al., Leukemia, 2020). To assess T cell mediated killing of target cells, we mixed them with human T cells (purified and enriched after negative selection) at varying Effector-to-Target (E:T) cell ratios and added CD117xCD3 TEA at different concentrations. The mixture was incubated and specific killing was quantified via flow cytometry at different time-points. RESULTS: In order to characterize the biocidal properties of CD117xCD3 TEA, we performed in vitro killing experiments against cell lines, HSCPs from healthy donors and blast cells from AML patients. A dose-dependent in vitro killing of the cell lines was observed in the presence of various concentrations of CD117xCD3 TEA and of human T cells at an E:T cell ratio of 10:1 after 24h. The HL60 CD117 high cell line was efficiently lysed (~90%) at 100 ng/ml of CD117xCD3 TEA, corresponding to ~1.8 nM. In similar experiments with different E:T cell ratios, we observed that both HL60 CD117 high and CD117 medium cells could be quantitatively killed at E:T ratios as low as 1:1, while the killing of CD117 low cells required a higher density of T cells. The biocidal effect on non-transduced HL60 cells was negligibly low, confirming the requirement of a simultaneous engagement of CD117 and CD3 for specific killing. We repeated the same experiment with an engineered MOLM14 cell line, which also expressed CD117 at comparable high levels, incubating the target cell line with human T cells at an E:T of 1:1 for 24, 48 or 72, 120 or 192 hours. Complete killing of the target cell line was achieved at 120 and 192 hours and after supplemental addition of T cells and CD117xCD3 TEA at 72 hours (see example figure). Experiments with primary cells (HSPCs from healthy donors or blast cells from AML patients) at an E:T of 1:1 confirmed specific killing of target cells in an antigen-density- and concentration-dependent manner after 48h. CONCLUSIONS: We have generated a novel bispecific antibody, which binds to human CD117 (expressed on HSCPs and AML/MDS blast cells) and to CD3 (expressed on T cells), which we term CD117xCD3 TEA. The antibody induces selective T cell-mediated killing of cell lines with different surface levels of CD117, as well as of healthy HSPCs and primary human AML cells. Thus, the newly generated CD117xCD3 TEA might be developed clinically in order to erradicate residual AML/MDS and at the same time serve as a milder preconditioning approach prior to allo-HSCT in frail AML/MDS patients. Figure 1 Figure 1. Disclosures Kiefer: ETH Zurich: Current Employment, Patents & Royalties: CD117xCD3 TEA. Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Guggisberg: F. Hoffmann-La Roche AG: Current Employment. Abdelmotaleb: F. Hoffmann-La Roche AG: Current Employment. Mock: Philogen S.p.A.: Current Employment. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: University of Zurich: Patents & Royalties: CD117xCD3 TEA; CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company.

Myeloid Sarcoma Reveals a Unique Tumor Micro-Environment and Differential Prognosis Based on Transplant Status and Type of Treatment Received

Background Myeloid sarcoma (MS) is the rare extramedullary proliferation of myeloid blasts that disrupts the normal architecture of various tissues in patients with Acute myeloid leukemia (AML). MS can present concurrently with untreated AML or present as relapsed AML. Based on our clinical observations and emerging immune checkpoint inhibitor (ICI) studies in AML (Davids et al NEJM 2016), MS appears to behave differently based on transplant status and tends to have better ICI sensitivity. One of the prominent mechanisms of relapse post-transplant is immune escape via the loss of leukemia recognition by the graft. This is mediated by down regulation of HLA expression. We hypothesize that the immune signatures in the bone marrow (BM) microenvironment are distinct from those from the local MS microenvironment. Thus, we aimed to comprehensively explore the MS microenvironment at single-cell level, providing clues for more effective therapies including ICI, and insights into the molecular pathogenesis, clinicopathological features and outcomes of MS. Methods This is an investigator-initiated study where we collected paired fresh MS tissues and BM samples from patients with MS as well as retrospective collection of paired MS and BM samples that are already sectioned and paraffin-embedded. Eligible patients were consentable adults > 18 years old and have MS lesions whether at diagnosis or at relapse of their AML disease. We excluded patients who have multiple malignancies or patients who are unable to provide samples. Accepted samples are 10-millimeter tissue or 1 million cells per milliliter. The fresh samples were cryopreserved and used for single cell RNA sequencing and ex vivo immune cellular assays. The sectioned tissues were used for multiplex immunohistochemical staining (mIHC) that tested a panel of 6 antibodies (CD3, CD68, CD4, CD33, PDL-1 and HLA-DR). We sought to further perform spatial transcriptome analysis (Visium) that complements mIHC for in-depth characterization of the MS microenvironment and immunopathogenesis. The clinical and genomic annotations for these samples were abstracted from the patients' charts. We have utilized Next generation sequencing (NGS) panels that interrogated 24- 180 oncogenes, including FLT3, IDH, CALR, NPM1, KRAS, NRAS, and TP53. Survival estimates using K-M curves and multivariate analysis were performed via SPSS. Results Our study recruited 30 patients so far. Mean age at diagnosis was (57.6 ± 14 years); 63.3% of the patients were males (n=19) and 80% of the patients were Caucasian (n=24). In our cohort, 46.6% of the patient had MS diagnosed as the cause of relapse after stem cell transplant (n=14) while the rest had MS at the initial diagnosis of AML. The most common cytogenetic abnormality in our cohort was complex karyotype in 30% of the patients. Molecular landscape of MS at diagnosis and relapse was done in 90% of the patients and shown in figure 1A,B. MS conferred a poor prognosis if diagnosed on initial presentation vs a new presentation at relapse post stem cell transplant with a median OS (4 months vs 25 months p 0.003) (HR 3.9 - 95% CI 1.4-11.1) figure 2. Interestingly, we have noticed that patients who received Venetolcax based therapies have poor prognosis compared to other therapies (IDH/FLT3 inhibitors, conventional chemotherapies, or ICI); median OS (9.3 months vs 24 months p 0.02) (HR 3.9 - 95% CI 1.2-12.6) figure 3. In a prelim analysis using validated mIHC, we have observed abundant HLA-DR expression in both blast cells and nonmalignant stromal cells of the MS microenvironment (n=4) figure-4. By contrast, PD-L1 was expressed mainly on a number of stromal cells rather than blast cells in the MS microenvironment. Further special localization analysis of tumor immune infiltrates (TILs) is being conducted to disclose clinical outcomes and biological significance of PD-L1, HLA-DR and TILs in MS. Spital transcriptomics are in process and will be presented. Conclusion MS diagnosed concurrently with untreated AML appears to confer a poor prognosis and behave differently than MS diagnosed as relapse post-transplant. MS appears to be resistant to Venetoclax based therapies and patients tends to do worse on this therapy. In contrast to BM microenvironment, blast cells within MS appears to have abundant expression of HLA-DR in both neoplastic tumor cells and nonmalignant stromal cells promoting immune recognition which might explain their sensitivities to ICI. Figure 1 Figure 1. Disclosures Abaza: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinner: Pfizer: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria. Altman: Glycomimetics: Other: data monitoring committee; ALX Oncology Inc.: Research Funding; Loxo: Research Funding; ImmunoGen: Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Research Funding; Aprea: Research Funding; Astellas: Honoraria, Research Funding; Aptos: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Research Funding; Biosight: Membership on an entity's Board of Directors or advisory committees, Other: reimbursement for travel, Research Funding; Kura Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kartos Therapeutics: Research Funding.

Identification of Protein Biomarker Signatures for Acute Myeloid Leukemia (AML) Using Both Nontargeted and Targeted Approaches

Acute myeloid leukemia (AML) is characterized by an increasing number of clonal myeloid blast cells which are incapable of differentiating into mature leukocytes. AML risk stratification is based on genetic background, which also serves as a means to identify the optimal treatment of individual patients. However, constant refinements are needed, and the inclusion of significant measurements, based on the various omics approaches that are currently available to researchers/clinicians, have the potential to increase overall accuracy with respect to patient management. Using both nontargeted (label-free mass spectrometry) and targeted (multiplex immunoassays) proteomics, a range of proteins were found to be significantly changed in AML patients with different genetic backgrounds. The inclusion of validated proteomic biomarker panels could be an important factor in the prognostic classification of AML patients. The ability to measure both cellular and secreted analytes, at diagnosis and during the course of treatment, has advantages in identifying transforming biological mechanisms in patients, assisting important clinical management decisions.

A Novel Method for the Evaluation of Bone Marrow Samples from Patients with Pediatric B-Cell Acute Lymphoblastic Leukemia—Multidimensional Flow Cytometry

Multicolor flow cytometry (FC) evaluation has a key role in the diagnosis and prognostic stratification of ALL. Our aim was to create new analyzing protocols using multidimensional dot-plots. Seventy-two pediatric patients with ALL were included in this single-center study. Data of a normal BM sample and three BM samples of patients with BCP-ALL were merged, then all B cell populations of the four samples were presented in a single radar dot-plot, and those parameters and locations were selected in which the normal and pathological cell populations differed from each other the most. The integrated profile of immunophenotype resulted in a simple, rapid, and accurate method. There were no significant differences between the percentages of lymphoblasts in the detection of minimal residual disease (MRD) by multidimensional or conventional FC method (p = 0.903 at Day 15 and p = 0.155 at Day 33). Furthermore, we found associations between the position and the number of clusters of blast cells in the radar plots and cytogenetic properties (p = 0.002 and p < 0.0001 by the position and p = 0.02 by the number of subclones). FC analysis based on multidimensional dot-plots is not only a rapid, easy-to-use method, but can also provide additional information to screen cases which require detailed genetic examination.

Chemotherapy-Responsive Acute Myeloid Leukemia in a Veiled Chameleon (Chamaeleo calyptratus)

A 4-year-old captive-bred male veiled chameleon (Chamaeleo calyptratus) presented with anorexia, weight loss, and stomatitis. Complete blood count revealed pancytopenia and a marked leukocytosis (197 x103/µL) composed of blast cells (195 x103/µL) that had oval to irregular nuclei with finely stippled chromatin and occasional nucleoli. The diagnosis was acute leukemia of presumptive myeloid origin. Treatment with prednisone (1.5 mg/kg once daily orally) and cytosine arabinoside (300 mg/m2 subcutaneously) was initiated. Post-treatment hematologic analysis revealed decreased blast count (88.5 x103/µL) and improved mentation. Additional doses of cytosine arabinoside were given two and three weeks after the initial diagnosis with marked improvement in circulating blast concentration (15.3 x103/µL). During the course of treatment, which included the chemotherapeutics, fluid therapy, and oral supportive care, the chameleon’s weight increased 32.5% (199 g to 295 g). Unfortunately, the animal died 33 days after presentation. Histopathologic evaluation revealed hypocellular bone marrow with rare blast-like cells within vessels and mycotic granulomatous hepatitis with intralesional hyphae and fructiferous bodies. The blast cells expressed Iba1 but not CD3, CD79a, or lysozyme, suggesting a myeloid origin. Cell morphology further reinforced an acute myeloid leukemia. The authors surmised that the chameleon was responding to treatment, but ultimately succumbed to the mycotic hepatitis. This report describes the first case of acute myeloid leukemia with response to chemotherapeutic intervention in a veiled chameleon.