Pharmacokinetic/Pharmacodynamic Modeling to Support the Re-approval of Gemtuzumab Ozogamicin.

Gemtuzumab ozogamicin (Mylotarg; Pfizer, New York, NY) was the first antibody-drug conjugate to be approved for CD33-positive acute myeloid leukemia (AML). However, it was voluntarily withdrawn from the US market due to lack of clinical benefit in the confirmatory phase III trial. In 2012, several investigator cooperative studies using a different dosing regimen showed efficacy, but pharmacokinetic (PK) data were not collected in these trials. Through simulation of expected concentrations for new dosing regimens, PK/pharmacodynamic modeling was able to support the safety and efficacy of these regimens. Significant exposure-response relationships were found for the attainment of complete remission with and without platelet recovery, attainment of blast-free status, the time course of myelosuppression, several grade ≥ 3 hepatic adverse events, and veno-occlusive disease. Gemtuzumab ozogamicin received full approval by the US Food and Drug Administration (FDA) in September 2017 for newly diagnosed and relapsed AML in adult patients and relapsed AML in pediatric patients aged 2-17 years.

The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003). AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

Immunohistochemical characterization of CD33 expression on microglia in Nasu-Hakola disease brains.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by formation of multifocal bone cysts and development of leukoencephalopathy, caused by genetic mutations of either DNAX-activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). Although increasing evidence suggests a defect in microglial TREM2/DAP12 function in NHD, the molecular mechanism underlying leukoencephalopathy with relevance to microglial dysfunction remains unknown. TREM2, by transmitting signals via the immunoreceptor tyrosine-based activation motif (ITAM) of DAP12, stimulates phagocytic activity of microglia, and ITAM signaling is counterbalanced by sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs)-mediated immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling. To investigate a role of CD33, a member of the Siglecs family acting as a negative regulator of microglia activation, in the pathology of NHD, we studied CD33 expression patterns in five NHD brains and 11 controls by immunohistochemistry. In NHD brains, CD33 was identified exclusively on ramified and amoeboid microglia accumulated in demyelinated white matter lesions but not expressed in astrocytes, oligodendrocytes, or neurons. However, the number of CD33-immunoreactive microglia showed great variability from case to case and from lesion to lesion without significant differences between NHD and control brains. These results do not support the view that CD33-expressing microglia play a central role in the development of leukoencephalopathy in NHD brains.

Perspectives for therapeutic targeting of gene mutations in acute myeloid leukaemia with normal cytogenetics.

The acute myeloid leukaemia (AML) genome contains more than 20 driver recurrent mutations. Here, we review the potential for therapeutic targeting of the most common mutations associated with normal cytogenetics AML, focusing on those affecting the FLT3, NPM1 and epigenetic modifier genes (DNMT3A, IDH1/2, TET2). As compared to early compounds, second generation FLT3 inhibitors are more specific and have better pharmacokinetics. They also show higher anti-leukaemic activity, leading to about 50% of composite complete remissions in refractory/relapsed FLT3-internal tandem duplication-mutated AML. However, rapid relapses invariably occur due to various mechanisms of resistance to FLT3 inhibitors. This issue and the best way for using FLT3 inhibitors in combination with other therapeutic modalities are discussed. Potential approaches for therapeutic targeting of NPM1-mutated AML include: (i) reverting the aberrant nuclear export of NPM1 mutant using exportin-1 inhibitors; (ii) disruption of the nucleolus with drugs blocking the oligomerization of wild-type nucleophosmin or inducing nucleolar stress; and (iii) immunotherapeutic targeting of highly expressed CD33 and IL3RA (CD123) antigens. Finally, we discuss the role of demethylating agents (decitabine and azacitidine) and IDH1/2 inhibitors in the treatment of AML patients carrying mutations of genes (DNMT3A, IDH1/2 and TET2) involved in the epigenetic regulation of transcription.

Influence of aberrant myeloid expression on acute lymphoblastic leukemia in children and adolescents from Maranhão, Brazil.

The aim of this study was to evaluate myeloid expression in acute lymphoblastic leukemia (ALL) in children and adolescents who had been referred to the Oncology Department in a hospital in the State of Maranhão based on demographic, laboratory, and clinical data. Myeloid expression was evaluated in 65 patients under 18 years of age who were diagnosed with morphological, cytochemical, and immunophenotypes of ALL. Demographic, laboratory (hemogram), and clinical variables were obtained from medical records. The sample was divided into groups with and without anomalous myeloid expression to analyze the variables. Myeloid expression was observed in 49.2% of the sample. Platelet count was significantly lower in the group of children without aberrant myeloid expression (33,627 platelets/mm(3), P = 0.01). A total of 88.9% of children with B-cell ALL without myeloid expression showed less than 50,000 platelets/mm(3) (P = 0.01). Thus, platelet count may be an important parameter in the diagnosis of children with ALL without myeloid aberrant expression and may indicate a greater risk of bleeding during treatment in this group.

Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia.

Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.

Microglial CD33-related Siglec-E inhibits neurotoxicity by preventing the phagocytosis-associated oxidative burst.

Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia. Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris. Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues. Increased neurotoxicity of microglia after knockdown of Siglece mRNA was neutralized by the reactive oxygen species scavenger Trolox. Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.

Clinical importance of myeloid antigen expression in Moroccan patients with adult B-lineage acute lymphoblastic leukemia.

The prognostic significance of myeloid antigen (MyAg) expression in acute lymphoblastic leukemias (ALL), especially in adult patients, is still controversial. In the present report, frequency and clinical significance of MyAg (CD13 and/or CD33) in blast cells were assessed in 80 consecutive adult (≥18 years) patients with B-lineage acute lymphoblastic leukemia (B-ALL), representing 66.7% of 120 patients diagnosed as having ALL during the study period. Immunophenotyping was used to classify leukemic cells as Bor Tlymphoblasts and to identify the aberrant expression of myeloid-associated antigens. MyAg expression was documented in 52.5% of the 80 B-ALL cases analyzed. CD13 was the most commonly antigen expressed (36.3%) followed by CD33 (28.8%). No significant associations were found between the expression of MyAg and the presence of known adverse prognostic features (eg: age>30 years, male gender, high WBC count and Philadelphia positivity). Also, we failed to observe any statistically significant difference between MyAg-positive and MyAg-negative patients in terms of achievement of complete remission and overall survival at 3 years. This study demonstrates that the presence of MyAg on lymphoblastic cells lacks prognostic value In Moroccan patients with adult B-ALL.

Anti-CD33-antibodies labelled with the alpha-emitter Bismuth-213 kill CD33-positive acute myeloid leukaemia cells specifically by activation of caspases and break radio- and chemoresistance by inhibition of the anti-apoptotic proteins X-linked inhibitor of apoptosis protein and B-cell lymphoma-extra large.

AIM: The emerging interest in radioimmunotherapies employing alpha-emitters for cancer treatment like high risk-leukaemia leads to the question of how these radionuclides exhibit their cytotoxicity. To clarify the molecular mechanisms of cell death induction, we investigated the molecular effects of the alpha-emitter Bismuth-213 (Bi-213) bound to a monoclonal anti-CD33-antibody ([Bi-213]anti-CD33) on the cell cycle and on apoptosis induction in sensitive as well as in beta- and gamma-radiation-resistant CD33-positive acute myeloid leukaemia (AML) cells. METHODS: The cytotoxic potential of the radioimmunoconjugate [Bi-213]anti-CD33 was analysed in the CD33-expressing human AML cell line HL-60 and in radiation- and chemoresistant HL-60-derived cell lines. Cell cycle and apoptosis induction analyses were performed via flow cytometry. Activation of apoptosis pathways was determined by immunodetection. RESULTS: [Bi-213]anti-CD33 induced apoptotic cell death in CD33-positive AML cells specifically. Molecular analyses revealed that the intrinsic mitochondrial pathway of apoptosis was activated resulting in caspase-9 activation. In the apoptotic executioner cascade caspase-3 was activated and its substrate poly (ADP-ribose) polymerase (PARP) was cleaved. Notably, [Bi-213]anti-CD33 overcame radio- and chemoresistance by reversing deficient activation of apoptosis pathways in resistant CD33-positive AML cells and by the downregulation of inhibitors of apoptosis B-cell lymphoma-extra large (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) involved in leukaemia resistance. CONCLUSION: [Bi-213]anti-CD33 exhibits its cytotoxic effects specifically in CD33-expressing AML cells via induction of the intrinsic, mitochondrial pathway of apoptosis. The abrogation of chemo- and radioresistances and the reactivation of apoptotic pathways seem to be promising for the treatment of patients with so far untreatable resistant AML and underline the importance of this emerging therapeutic approach of targeted alpha-therapies.

TGF-ß1-dependent L1CAM expression has an essential role in macrophage-induced apoptosis resistance and cell migration of human intestinal epithelial cells.

Patients with chronic inflammatory bowel disease (IBD) have an increased risk to develop colorectal cancer (CRC) particularly after long duration of the disease. Chronic inflammation of the intestinal mucosa is characterized by a marked enrichment of immune cells such as macrophages as well as by high expression of cytokines and growth factors including transforming growth factor-beta 1 (TGF-ß1). The adhesion molecule L1CAM mediates chemoresistance and migration of tumor cells and is elevated in CRC tissues being associated with metastatic spread and poor prognosis for the patients. In this study, we examine the role of TGF-ß1-induced L1CAM expression and macrophages in malignant transformation of intestinal epithelial cells. We demonstrate that TGF-ß1 stimulation leads to a Slug-dependent upregulation of L1CAM expression already in the colonic intestinal epithelial cell line NCM460 thereby enhancing cell motility and apoptosis resistance. Accordingly, NCM460 cells acquired a migratory and apoptosis-resistant phenotype if transfected with L1CAM. Immunohistochemistry of colonic biopsies revealed considerable L1CAM expression in intestinal epithelial cells in tissues from IBD patients but not in normal colonic tissues. Moreover, L1CAM expression increased with duration of disease being associated with the presence of CD33+ macrophages. Coculture with macrophages generated from monocyte colony-stimulating factor (MCSF)-treated monocytes led to the upregulation of Slug and L1CAM in NCM460 cells thereby elevating cell motility and apoptosis resistance. Pharmacological inhibition of TGF-ß1 signalling abolished expression of Slug and L1CAM in cocultured NCM460 cells resulting in decreased cell migration and apoptosis resistance. In conclusion, these data provide new insights into the mechanisms by which IBD promotes malignant transformation of intestinal epithelial cells and underscore the role of L1CAM and macrophages in this scenario.

Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (~0.001%-0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ~50% in purified episome-expressing cells. Lineage-committed CD33(+)CD45(+)CD34(-) myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG(+)TRA-1-81(+) hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC.

Hyper-eosinophilia in granular acute B-cell lymphoblastic leukemia with myeloid antigen expression.

Acute lymphoblastic leukemia with eosinophilia (ALLEo) is a rare but a distinctive clinical entity. Clinical features of idiopathic hyper-eosinophilic syndrome (HES) can be seen in patients with ALLEo. We report a 10-year-old girl, in whom HES was initially suspected but further investigation confirmed the diagnosis of acute B-cell lymphoblastic leukemia with myeloid antigen expression. Clinical response to chemotherapy was excellent with achievement of complete remission for 4 years. Serum interleukin-3 and -5 were elevated at presentation and normalized with disappearance of eosinophilia after induction therapy, supporting the reactive nature of eosinophilia in ALLEo. Hematologic malignancy should be considered in patients with hyper-eosinophilia, before attributing it to HES.