Common Acute Lymphoblastic Leukemia Research Articles

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but <0.1% MRD, and slow responders with >0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin

Formalin, a common laboratory fixative, is a type 1 carcinogen; a biohazard with risks, environmental, disposal, and legal costs; and a chemical modifier of protein epitopes in tissues. A less-toxic tissue preservation method is therefore badly needed. We have developed a novel tissue preservation medium, Amber, composed of low-potassium dextran glucose, 10% honey, and 1% coconut oil. This study investigates Amber as compared with formalin with respect to the following aspects: (1) histologic preservation, (2) epitope integrity with immunohistochemistry (IHC) and immunofluorescence (IF), and (3) integrity of tissue RNA. Rat and human lung, liver, kidney, and heart tissues were collected and stored for 24 hours at 4 °C in Amber or formalin. The tissues were evaluated with hematoxylin and eosin; IHC: thyroid transcription factor, muscle-specific actin, hepatocyte-specific antigen, and common acute lymphoblastic leukemia antigen; and IF: VE-cadherin, vimentin, and muscle-specific actin. RNA quality upon extraction was also assessed. Amber demonstrated superior and/or noninferior performance in rat and human tissue evaluation with respect to standard techniques of histology, IHC, IF, and extracted RNA quality. Amber maintains high-quality morphology without compromising the ability to perform IHC and nucleic acid extraction. As such, Amber could be a safer and superior substitute to formalin for clinical tissue preservation for contemporary pathological examination.

Retrospective Study of B Lymphoblastic Leukemia to Assess the Prevalence of TEL/AML1 in South India: A Study of 214 Cases and Review of Literature

Abstract Introduction Translocation t(12;21)(p13;q22), a recurrent and an invisible chromosomal abnormality, resulting in TEL/AML1 gene fusion, associated with good prognosis, has been described to be a common abnormality, in children with B-acute lymphoblastic leukemia (B-ALL). Objectives The initial observation of very few TEL/AML1 positive patients at this center on testing by fluorescence in situ hybridization (FISH) led to study the prevalence of the abnormality, compare with the global distribution, and evaluate clinical, pathological, molecular, and cytogenetic features in TEL/AML1 positive patients. Materials and Methods A retrospective study of all B-ALL patients tested for TEL/AML1 gene fusion during the period January 2009 to November 2020 was undertaken. Clinicopathological, molecular, cytogenetic, treatment, and follow-up details were collected. All publications dealing with TEL/AML1 gene rearrangement were reviewed post Google and PubMed search. Results TEL/AML1gene rearrangement was assessed by FISH in 178 patients and by reverse transcription polymerase chain reaction in 36 patients and detected as the sole abnormality in 8.4% patients with additional genetic abnormalities noted on FISH evaluation. Normal karyotype was noted in 14/18 (77.7%) of these patients and 2 had complex karyotype. Complete blood count revealed hemoglobin to range from 35 to 116 g/L (median: 74 g/L), white blood count: 1.01–110×109/L (median: 7.8×109/L), platelet counts: 10–115×109/L (median: 42×109/L), blast count in peripheral smear: 0–98% (median: 41%). Immunophenotyping demonstrated 94.4% were CD34 positive, common acute lymphoblastic leukemia associated antigen (CALLA) positive with aberrant expression of CD13, CD33, CD56, singly or in combination in 58.8%. Conclusion TEL/AML1 fusion is rare in Indian patients with B-ALL and appears to be much rarer in our region. The detection of relevant specific abnormalities is of fundamental importance in B-ALL patients and these geographic variations can be used in defining management policies.

MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X–like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

The Detailed Study of CD10: A Stromal Marker in Breast Carcinoma

Breast carcinoma is one of the most frequently occurring cancers among women with one million cases. Breast cancer is the primary cause of death in women around the world. It is one of the major concerns in public health due to its high occurrence and growing tendency. This complex functional structure develops from a highly modified apocrine sweat gland in the female, but remains rudimentary in male. Breast develops embryological into two lines along milk lines extending from axilla to groin. CD-10 is a 90- to 110-kDa cell surface zinc dependent metalloproteinase which is known as “Common Acute Lymphoblastic Leukaemia Antigen” (CALLA). CD-10 acts as a stem cell regulator in the breast and prevents uncontrolled proliferation on stem cells The present study is designed to study the expression of CD-10, a breast carcinoma stromal marker and its correlation with ER, PR and HER2/neu status in breast carcinoma.

Distribution of Antigens Defined by OKB Monoclonal Antibodies on Benign and Malignant Lymphoid Cells and on Nonlymphoid Tissues

AbstractMonoclonal antibodies OKB1, OKB2, OKB4 and OKB7 have been previously shown to detect distinctive antigens displayed on B, but not on T, lymphocytes. Benign and malignant lymphoid cells were investigated for their reactivity with these antibodies in cell suspension by indirect immunofluorescence and in cryostat tissue sections by the avidin-biotin complex immunoperoxidase technique. Fetal liver pre-B cells and pre-B and common type acute lymphoblastic leukemia cells isolated from 15 patients were OKB1− OKB2 OKB4+ OKB7−. All mature lymphoid tissue B cells and the neoplastic cell surface immunoglobulin-positive (SIg+) B cells isolated from each of 47 B cell neoplasms were OKB2+. OKB1 and OKB7 were expressed by interfollicular, follicular center, and many, but not all, mantle zone B cells. OKB4 was expressed by follicular center cells, but not by mantle zone or interfollicular B cells. The neoplastic SIg+ B cells isolated from 45 of 47 B cell malignancies were OKB1+ OKB4+, and those isolated from 45 of 46 B cell malignancies were OKB7+. The neoplastic B cells of one mantle zone lymphoma were OKB1−, of one small lymphocytic cell lymphoma were OKB7−, of one large cell lymphoma were OKB4−, and of one small lymphocytic cell lymphoma with a monoclonal gammopathy were OKB1− OKB4−. Normal and myeloma plasma cells were OKB−. The malignant T cells isolated from 12 T cell neoplasms were OKB2− OKB4−, but were OKB1+ and/ or OKB7+ in 3 cases. Thus, the OKB antibodies appear to detect distinctive antigens that may be expressed at different stages of B cell differentiation. In addition, OKB4 reacted with selected renal and respiratory epithelium, and OKB2 reacted with a wide range of epithelial tissues. The OKB antibodies should prove useful in the investigation of B cell differentiation and may aid in the identification and characterization of lymphoproliferative malignancies with significant therapeutic and prognostic differences not identifiable by conventional histopathologic and immunologic methods.

Ia Antigen Expression by Human Malignant Lymphomas: Correlation With Conventional Lymphoid Markers

The Ia (p23,30) antigens are useful in determining the B- or T-cell origin of normal peripheral blood lymphocytes. However, exceptions to the preferential B-cell expression of Ia antigens exist, i.e., many plasma cells are Ia– and T cells are rarely Ia+. Therefore, we investigated Ia expression (assayed by direct immunofluorescence using heteroantisera) by cells isolated from 33 malignant lymphomas and 7 benign lymph nodes. This was compared with surface immunoglobulin (SIg) and sheep erythrocyte (SRBC) receptor (E rosette) expression. Results with normal lymph nodes were similar to those described for peripheral blood. Most lymph node B cells were Ia+SIg+. Occasional Ia+SIg– B cells and rare Ia+ T cells were present. Ia antigens were expressed in parallel with SIg in 15 of 19 B-cell lymphomas. In 4 B-cell lymphomas, substantial numbers of neoplastic Ia+SIg– cells were also present, suggesting the presence of neoplastic cells at varying stages of differentiation. Heterogeneity was also seen in those lymphomas associated with monoclonal proteins, as small numbers of Ia+ and Ia– plasma cells were present. Seven cases expressed the T-cell (la–SIg–E+) phenotype. The common acute lymphoblastic leukemia phenotype (Ia+SIg–E–) was rarely expressed and may be uncommon among lymphomas. Three lymphomas expressing the Ia+E+ phenotype were shown to be B-cell malignancies whose SIg had SRBC specificity. One Ia 4 T-cell lymphoproliferative disorder was found. Thus, Ia antigens are useful B-cell markers in most instances. Furthermore, their enumeration allows the demonstration of phenotype heterogeneity in B-cell malignancies, analogous to that described for T-cell malignancies.

Abstract B51: Adaptation to oncogene-induced metabolic stress by MondoA (MLXIP) drives common acute lymphoblastic leukemia (cALL) malignancy

Abstract MondoA (also known as MLXIP, MAX-like protein X interacting protein) is a metabolic stress sensor and a proglycolytic transcription factor potentially involved in metabolic addiction features of leukemia and the Warburg effect. MondoA dimerizes with MLX within the MYC interactome and promotes longevity in C. elegans (Johnson et al., 2014). The MYC interactome comprises the MYC/MAX/MNT/MLX/MLXIP transcription factor network: Its key players MYC, MNT and MLXIP differentially mediate proliferation, differentiation, or metabolism by heterodimerization with MAX or MLX. We previously described MondoA to promote malignancy of common precursor B-cell acute lymphoblastic leukemia (cALL). MondoA knockdown (MKD) in cALL cell lines in xenografted mice reduced the number of leukemic blasts (Sipol, 2014). Here we report that MondoA high expression was observed in ALL subtypes with no MYC overexpression. RNA-sequencing data of 132 primary ALL bone marrow samples confirmed the inverse correlation of MYC and MondoA. Interestingly, in subgroups of ALL with low MYC expression and high MondoA (cALL with BCR-ABL, cALL with TCF3-PBX, cALL with ETV6-RUNX1, and cALL with hyperdiploid karyotype), metabolic gene sets did not appear as upregulated. In contrast, cALL samples with high MYC expression and low MondoA (proB-ALL with MLL rearrangements and B-ALL with IGH-MYC fusion gene) demonstrated upregulation of pathways for oxidative phosphorylation and fatty acid metabolism in addition to targets of E2F, G2M checkpoints, and MYC targets. Using CRISPR/CAS9-mediated knockout (MKO), we demonstrate that MondoA dials down MYC-induced metabolic stress and increases leukemia stress resistance. By limiting mitochondrial pyruvate dehydrogenase (PDH) activity in PDHK1 (pyruvate dehydrogenase kinase 1)-dependent manner, MondoA decreases oxidative phosphorylation. In line with limiting effect of MondoA on oxidative phosphorylation, we observed that MondoA decreases ROS generation in B cells. In conclusion, MondoA is restricting MYC-target gene expression to promote leukemia cell survival by facilitating glycolysis and adaption to oxidative stress. MondoA limits pyruvate availability for the TCA cycle by decreasing PDH activity, thus ensuring consistent glycolytic flux, mediating the Warburg effect, and insuring integrity of leukemia metabolism and ROS balancing in response to oncogene activation. Citation Format: Alexandra Sipol, Erik Hameister, Andreas Petry, Agnes Görlach, Jürgen Ruland, Guenther Richter, Stefan Burdach, Poul Sorensen. Adaptation to oncogene-induced metabolic stress by MondoA (MLXIP) drives common acute lymphoblastic leukemia (cALL) malignancy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B51.

Development of Novel Anti-CD10 Target Modules for Redirection of Universal CAR T Cells Against CD10-Positive Malignancies

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

EXPLOITING CBF DEPENDENCY IN B-ACUTE LYMPHOBLASTIC LEUKAEMIA AS A NOVEL THERAPEUTIC APPROACH

Leukaemia is the most common childhood cancer and acute lymphoblastic leukaemia (ALL) is responsible for a third of all childhood cancer deaths. Despite the overall good prognosis, failure to eradicate the disease leads to relapse in ∼20% of patients and survival rates for infants are less than 50%. There is therefore a clear unmet need for less toxic and more effective targeted treatments. ETV6-RUNX1 translocation is the first-hit event in ∼25% of B-ALLs, initiating a clinically silent pre-leukaemia in utero. The translocation fuses almost the entire RUNX1 protein to the repressor domain of ETV6 and the chimeric product is proposed to mediate a block in B cell differentiation by repressing RUNX1 targets. Interestingly, the remaining RUNX1 allele is not mutated, but is instead often amplified suggesting an onco-supportive role of the endogenous RUNX1. Using genetic and pharmacological inhibition, we have discovered that RUNX1/CBF is essential for the growth and survival of B-ALL cells in vitro and in vivo. Global RNA-Seq on CBF-depleted B-ALL cells revealed significant downregulation of E2F and Myc signature genes along with a marked increase of p53 targets, consistent with these cells accumulating in G0 and subsequently becoming apoptotic. Notably, RNA-Seq and mass cytometry analyses of pro-B cells derived from an hiPSC ETV6-RUNX1 knockin model reveal a similar cell cycle trend indicating that ETV6-RUNX1-mediated inhibition of RUNX1 targets may induce a quiescence-like pre-leukaemic state while continued RUNX1 expression is essential for the survival of overt leukaemia. We propose that CBF-dependency is an Achilles heel of ALL cells that could be exploited by targeting either CBF itself or its key downstream nodes. By combining clinically tested compounds targeting multiple CBF effectors we aim to improve efficacy of potential treatments and circumvent resistance.

Role of CD10 Marker in Differentiating Malignant Thyroid Neoplasms from Benign Thyroid Lesions (Immunohistochemical & Histopathological Study).

BACKGROUND:CD10 was initially recognised as a cell–surface antigen expressed by acute lymphoblastic leukaemias, and hence it’s early designation as Common Acute Lymphoblastic Leukemia Antigen (CALLA). Also, it has been proven to be reactive in various non-lymphoid cells and tissue and different types of neoplasms.AIM:To evaluate the immunohistochemical expression of CD10 in malignant thyroid neoplasms and different benign lesions and to assess whether CD10 can be used as a malignancy marker in thyroid pathology or not.MATERIAL AND METHODS:A total of 83 archived, formalin fixed, paraffin embedded tissue blocks of 83 cases of malignant thyroid neoplasms and different benign lesions. The samples were immunohistochemically analysed for CD10 expression. A p-value of less than 0.05 was considered statistically significant.RESULTS:CD10 was expressed in 91% of the studied malignant thyroid neoplasms and 58% of benign thyroid lesions. It was expressed in 26 of 28 (92.9%) conventional papillary carcinomas, ten of 10 (100%) follicular variants of papillary carcinoma, seven of nine (77.8%) minimally invasive follicular carcinomas, two of three (66.7%) widely invasive follicular carcinomas, and seven of 7 (100%) undifferentiated carcinomas, seven of 11 (66.7%) adenomatous nodules and eight of 15 (53.3%) follicular adenomas. No statistically significant correlations were detected between CD10 expression and patients’ age, sex, lymph node metastasis, tumour stage and capsular invasion.CONCLUSION:CD10 shows strong sensitivity (91.2%) and moderate specificity (42.3%) in the diagnosis of malignancy overall and shows strong sensitivity (86.4%) and moderate specificity (42.3%) in the diagnosis of malignancy in the follicular-patterned lesions. So, CD10 might be useful in differentiating malignant from benign thyroid lesions (good positive test) and in the diagnosis of follicular variant of papillary carcinoma.

Adaptation to Metabolic Stress By Mondoα in Common B-Cell Acute Lymphoblastic Leukemia

Malignant cells evolve adaptive mechanisms to survive metabolic stress to drive progression. We previously described the stress sensor MondoA as promoting malignancy of common acute lymphoblastic leukemia (cALL). MondoA knockdown (MKD) in cALL cell lines xenografted into mice reduced the number of leukemic blasts compared to control cells (Sipol 2014). Next we investigated potential mechanisms of MondoA mediated malignancy. Microarray analyses of MKD cALL lines revealed an induction of glycolytic and hypoxia response associated gene sets. To validate and expand these results we generated cALL lines with MondoA knockout (MKO) via CRISPR/cas9 gene editing. Colony forming assays and direct cell counting demonstrated reduced proliferation of MKO cells under normoxic conditions compared to controls. However, under hypoxia, there was a diminished MondoA dependent growth advantage. This suggests that MondoA proficient cells may have a selective growth advantage in the presence of oxygen, and that MondoA might confer the ability to utilize oxidative phosphorylation and thus increase metabolic activity. We therefore measured mitochondrial respiration by determining oxygen consumption rates (OCR) in control and MKO cells using an extracellular flux analyzer. Surprisingly, MKO cells displayed a higher basal OCR as well as significantly increased extracellular acidification rate (ECAR) and glycolysis. This suggested that MondoA might actually limit oxidative phosphorylation and glycolysis in response to glucose. We therefore hypothesized that MondoA might remodel cALL metabolism towards an alternative energy source. Indeed, Kegg pathway analysis of microarray data showed upregulation of fatty acid synthesis (FAS) and fatty acid oxidation (FAO) genes by MondoA. This suggested that MondoA might facilitate sustained fatty acid metabolism to maintain high proliferative rates. Key regulators of citrate to oleate conversion, fatty acid elongation and cholesterol synthesis (ACLY, ELOVL5, ELOVL6, ELOVL1, FASN, HMGCR, HMGCS1) were stimulated by MondoA. Colorimetric assays demonstrated decreased NADPH and Acetyl-CoA levels corresponding to disregulated fatty acid metabolism by MKO. In addition, ROS measurements by electron paramagnetic resonance (EPR) using the spin trap CMH in the presence or absence of NADPH oxidase NOX2 inhibitory peptide gp91ds-Tat, a readout of NOX activity, demonstrated that MKO cells increase cytoplasmic ROS production, mediated by NOX2. Moreover, MondoA depleted leukemia cells generate more ROS in glutamine-restricted media. In summary, (1) MondoA increases leukemic cell proliferation under both normoxia and hypoxia; (2) MondoA increases metabolic activity, in particular fatty acid metabolism; and (3) MondoA decreases cytoplasmic ROS production in glutamine-deprived conditions. We conclude that MondoA has multiple functions in supporting malignancy of cALL, most likely by increasing fatty acid metabolism while simultaneously providing adaptation to oxidative stress. DisclosuresNo relevant conflicts of interest to declare.

TLE1 as an indicator of adverse prognosis in pediatric acute lymphoblastic leukemia

PurposeAcute lymphoblastic leukemia (ALL) is the most common type of cancer in children, and despite the high rate of cure (over 80%) it still has a big impact on morbidity and mortality. The Transducin-like enhancer of split 1 (TLE1), a transcriptional corepressor, has been described as dysregulated and recently emerged as a tumor marker in several cancer types, including hematologic malignancies. MethodsIn the present study TLE1 gene expression was evaluated by RT-qPCR. A total of 60 consecutive pathological ALL samples and 8 normal bone marrow samples were included. Associations between TLE1 levels and clinicopathological features were estimated using Mann-Whitney tests. ResultsTLE1 mRNA levels were significantly diminished in ALL samples when compared to normal counterparts (fold change -1.45, p-value 0.039). Lower TLE1 expression levels were associated with poorer prognostic features such as age at diagnosis (<1 or >9 years-old), absence of the Common Acute Lymphoblastic Leukemia Antigen (CALLA) and high white cell count. Considering immunophenotype, decreased expression of TLE1 was only evident for T-cell ALL, what was validated using gene expression profiling data available in public repositories. No associations with event or overall survival were observed. However, TLE1 expression was statistically different between patients who achieved complete clinical remission (CCR) from those that relapsed or died. ConclusionThese data are of particular interest and give support for a plausible role of TLE1 as a tumor suppressor in T-cell ALL. Moreover, the prognostic value of this corepressor may assist ALL treatment stratification and suggest the need of alternative regimens.

Targeting FLT3 Signaling in Childhood Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the second most common leukemia of childhood and is associated with high rates of chemotherapy resistance and relapse. Clinical outcomes for children with AML treated with maximally intensive multi-agent chemotherapy lag far behind those of children with the more common acute lymphoblastic leukemia, demonstrating continued need for new therapeutic approaches to decrease relapse risk and improve long-term survival. Mutations in the FMS-like tyrosine kinase-3 receptor gene (FLT3) occur in approximately 25% of children and adults with AML and are associated with particularly poor prognoses. Identification and development of targeted FLT3 inhibitors represents a major precision medicine paradigm shift in the treatment of patients with AML. While further development of many first-generation FLT3 inhibitors was hampered by limited potency and significant toxicity due to effects upon other kinases, the more selective second- and third-generation FLT3 inhibitors have demonstrated excellent tolerability and remarkable efficacy in the relapsed/refractory and now de novo FLT3-mutated AML settings. While these newest and most promising inhibitors have largely been studied in the adult population, pediatric investigation of FLT3 inhibitors with chemotherapy is relatively recently ongoing or planned. Successful development of FLT3 inhibitor-based therapies will be essential to improve outcomes in children with this high-risk subtype of AML.

Abstract 4515: Metabolic stress sensor MondoA mediates in vivo aggressiveness of common ALL by induction of HIF1α

Abstract Tumor cells evolve adaptive mechanisms to survive hypoxia, nutrient deprivation, oxidative or genotoxic stress to ultimately drive tumor progression (Sorensen et al. 2015). We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Lymphocytes, in particular B Lymphocytes are adapted to hypoxic environments from their very beginning. They are destined to travel from the hypoxic bone marrow via normoxic peripheral blood to hypoxic lymph nodes. They thus are specialized in glycolysis to compensate the lack of oxidative phosphorylation under hypoxic conditions. Leukemic counterparts of B-lymphocytes exploit these features for their survival. Glucose-derived metabolites control the nuclear activity of the transcription factor MondoA. Here we report on the expression of MondoA in common B-cell acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Methods. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA. Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. Results. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rhabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in mice after MondoA KD. Further microarray analysis revealed an enrichment of glycolytic and hypoxia response gene sets by MondoA. Moreover, HIF1a induction under hypoxia required MondoA. We demonstrate that hypoxia facilitates vincristine resistance of cALL. MondoA is induced under hypoxia and confers cALL cells chemotherapeutic resistance. Tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response, in particular by induction of HIF1a. Citation Format: Alexandra A. Sipol, Thomas G. Grunewald, Juliane Schmaeh, Monique L. den Boer, Rebeca Alba Rubío, Michaela Baldauf, Caroline Wernicke, Martin Horstmann, Gunnar Cario, Günther Richter, Stefan Burdach. Metabolic stress sensor MondoA mediates in vivo aggressiveness of common ALL by induction of HIF1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2017-4515

Isolation and characterization of adult mammary stem cells from breast cancer-adjacent tissues.

Normal adult mammary stem cells (AMSCs) are promising sources for breast reconstruction, particularly following the resection of breast tumors. However, carcinogenic events can potentially convert normal AMSCs to cancer stem cells, posing a safety concern for the use of AMSCs for clinical tissue regeneration. In the present study, AMSCs and autologous primary breast cancer cells were isolated and compared for their ability to differentiate, their gene expression profile, and their potential to form tumors in vivo. AMSCs were isolated from normal tissue surrounding primary breast tumors by immunomagnetic sorting. The pluripotency of these cells was investigated by differentiation analysis, and gene expression profiles were compared with microarrays. Differentially expressed candidate genes were confirmed by reverse transcription-polymerase chain reaction and western blot analyses. The in vivo tumorigenicity of these cells, compared with low-malignancy MCF-7 cells, was also investigated by xenograft tumor formation analysis. The results revealed that AMSCs isolated from normal tissues surrounding primary breast tumors were positive for the stem cell markers epithelial-specific antigen and keratin-19. When stimulated with basic fibroblast growth factor, a differentiation agent, these AMSCs formed lobuloalveolar structures with myoepithelia that were positive for common acute lymphoblastic leukemia antigen. The gene expression profiles revealed that, compared with cancer cells, AMSCs expressed low levels of oncogenes, including MYC, RAS and ErbB receptor tyrosine kinase 2, and high levels of tumor suppressor genes, including RB transcriptional corepressor 1, phosphatase and tensin homolog, and cyclin-dependent kinase inhibitor 2A. When injected into nude non-obese diabetic/severe combined immunodeficiency-type mice, the AMSCs did not form tumors, and regular mammary ductal structures were generated. The AMSCs isolated from normal tissue adjacent to primary breast tumors had the normal phenotype of mammary stem cells, and therefore may be promising candidates for mammary reconstruction subsequent to breast tumor resection.

Mature CD10+ and immature CD10− neutrophils present in G-CSF–treated donors display opposite effects on T cells

AbstractThe identification of discrete neutrophil populations, as well as the characterization of their immunoregulatory properties, is an emerging topic under extensive investigation. In such regard, the presence of circulating CD66b+ neutrophil populations, exerting either immunosuppressive or proinflammatory functions, has been described in several acute and chronic inflammatory conditions. However, due to the lack of specific markers, the precise phenotype and maturation status of these neutrophil populations remain unclear. Herein, we report that CD10, also known as common acute lymphoblastic leukemia antigen, neutral endopeptidase, or enkephalinase, can be used as a marker that, within heterogeneous populations of circulating CD66b+ neutrophils present in inflammatory conditions, clearly distinguishes the mature from the immature ones. Accordingly, we observed that the previously described immunosuppressive neutrophil population that appears in the circulation of granulocyte colony-stimulating factor (G-CSF)–treated donors (GDs) consists of mature CD66b+CD10+ neutrophils displaying an activated phenotype. These neutrophils inhibit proliferation and interferon γ (IFNγ) production by T cells via a CD18-mediated contact-dependent arginase 1 release. By contrast, we found that immature CD66b+CD10− neutrophils, also present in GDs, display an immature morphology, promote T-cell survival, and enhance proliferation and IFNγ production by T cells. Altogether, our findings uncover that in GDs, circulating mature and immature neutrophils, distinguished by their differential CD10 expression, exert opposite immunoregulatory properties. Therefore, CD10 might be used as a phenotypic marker discriminating mature neutrophils from immature neutrophil populations present in patients with acute or chronic inflammatory conditions, as well as facilitating their isolation, to better define their specific immunoregulatory properties.

Cluster of differentiation 97 as a biomarker for the detection of minimal residual disease in common acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disorder. Clinical parameters, immunophenotype, cytogenetic factors, and minimal residual disease (MRD) are among currently used factors in risk stratification and therapy determination of ALL patients. MRD is gaining importance nowadays for therapy efficacy, follow-up, and relapse risk estimation. Recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. Cluster of differentiation (CD) 97 is one of the markers that show overexpression in pediatric ALL. In this study, we aimed to assess the value of CD97 as a biomarker for MRD detection in pediatric ALL. Patients and methods This cohort study was conducted on 30 newly diagnosed patients with B-ALL. There were 16 male and 14 female patients with a mean age of 8.38±4.21. Twenty patients were in the low-risk group and 10 patients were in the high-risk group and were treated according to modified CCG 1991. A panel of monoclonal antibodies was used, with special emphasis on CD10, CD19, CD34, and CD97 at diagnosis and at day 14 postinduction of chemotherapy for MRD detection. Results The percentage of CD19/CD97, CD34/CD97, and CD10/CD97 at day 0 was 57.15±21.74, 57.73±21.20, and 57.87±20.77, whereas at day 14 it was 6.09±2.50, 10.67±8.89, and 5.97±2.44, respectively (P<0.001). CD97 was expressed in 81.5% of patients at diagnosis and was not detected at day 14 (P<0.001). One patient had blast counts more than 5% by light microscopy, whereas 29 patients had MRD more than 0.1 by flow cytometry at day 14 (P<0.001). Conclusion CD97 can be used for MRD tracing in pediatric ALL.

Identification of neprilysin as a potential target of arteannuin using computational drug repositioning

The discovery of arteannuin (qinghaosu) in the 20th Century was a major advance for medicine. Besides functioning as a malaria therapy, arteannuin is a pharmacological agent in a range of other diseases, but its mechanism of action remains obscure. In this study, the reverse docking server PharmMapper was used to identify potential targets of arteannuin. The results were checked using the chemical-protein interactome servers DRAR-CPI and DDI-CPI, and verified by AutoDock Vina. The results showed that neprilysin (also known as CD10), a common acute lymphoblastic leukaemia antigen, was the top disease-related target of arteannuin. The chemical-protein interactome and docking results agreed with those of PharmMapper, further implicating neprilysin as a potential target. Although experimental verification is required, this study provides guidance for future pharmacological investigations into novel clinical applications for arteannuin.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.