Brain And Acute Leukemia, Cytoplasmic Research Articles

IMMU-28. THE INTRA-TUMORAL SPATIAL HETEROGENEITY OF T CELL ANTIGENS IN GLIOBLASTOMA: AN INTEGRATED MULTI-OMICS APPROACH

Abstract This study investigates the intratumoral heterogeneity of T cell antigen presentation in 15 newly diagnosed glioblastoma patients. Our multi-omics approach includes mass spectrometry-based immunopeptidome analysis, next-generation sequencing (whole-exome and RNA sequencing), and imaging mass cytometry performed on biopsies deriving from the necrotic core (NEC), the gadolinium contrast-enhancing region (T1), and the peritumoral infiltrating zone (INF, 5-ALA positive). A total of 24493 unique HLA class I and 17394 unique HLA class II peptides were identified. Comparative profiling of peptides from our study and a benign tissue database (in-house; n = 429 donors combined with HLA ligand atlas (https://hla-ligand-atlas.org)) revealed that 20% of HLA class I ligands are glioblastoma exclusive. Of these ligands, 21%, 21%, and 15% were exclusively presented in the INF, T1, or NEC zone, respectively. Here, we focused on the INF-specific antigens as this zone is likely to remain after tumor resection and gives rise to glioblastoma recurrence. Interestingly, two INF-specific HLA ligands showed a frequency presentation of 45% in our patient cohort. One ligand originated from BAALC (Brain and acute leukemia cytoplasmic protein) and one from NCAN (Neurocan core protein), which are both known to be glioblastoma-associated proteins. Immunogenicity of pre-selected candidate antigens was assessed with autologous expanded T cells and revealed a set of novel promising targets for immunotherapy. Integrated RNA/DNA sequencing also enabled the identification of neoepitopes deriving from tumor- and region-specific mutations. Furthermore, imaging mass cytometry contributed to investigating the immune compartment of the tumor microenvironment in high dimensionality and spatial resolution. In conclusion, our approach characterized the intra-tumoral regional heterogeneity of both infiltrating immune cells and tumor antigens. This multi-omics spatial atlas of the immune landscape can be used for the informed design of immunotherapy strategies against glioblastoma.

Over-Expression of ERG Predicts Poor Outcome in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) treatment aims at the eradication of all leukemic cells to achieve cure. Available treatments produce complete remission (CR) in up-to 80% of patients. However, ~60% of them will relapse. Various prognostic factors such as baseline white blood cell count (WBC), cytogenetics and mutations of FLT3 , NPM1 , CEBPA have been shown to affect the survival. The high expression of brain and acute leukemia cytoplasmic (BAALC) and ETS- related gene (ERG) has been associated with poor prognosis but due to limited prospective studies, their role as prognostic factors is still unclear. We aim to substantiate the prognostic value of BAALC and ERG expression in adult AML patients.Materials and methods: All patients included in this prospective study (N=149) between March 2012 and February 2015 underwent marrow for following investigations: (i) Cytogenetic analyses- by standard techniques of chromosome banding and FISH (ii) Mutation profiling of NPM1 , FLT3 -ITD and CEBPA by capillary electrophoresis and direct DNA sequencing. Long range PCR was done to detect KMT2A-PTD (MLL -PTD) (iii) BAALC and ERG expressions by quantitative real time PCR assays. Abelson (ABL ) gene was used as an internal control. According to the baseline median values of BAALC and ERG expression, patients were divided into high and low expressers.Patients received standard induction chemotherapy with daunorubicin 60 mg/m2 for 3 days and cytosine arabinoside 100 mg/m2 for 7 days. Marrow was done 21-28 days after start of chemotherapy. If marrow was in remission, then patients received 3 courses of consolidation therapy with 12-18 gm/m2 of cytosine arabinoside (HiDAC). Patients underwent allogeneic transplant if feasible according to molecular and cytogenetic risk.Statistical analysis was done using SPSS-21. Overall survival (OS) was defined as time from start of induction therapy to time to last follow up or death. Relapse free survival (RFS) was defined as time from achieving CR to time to relapse, death or last follow up if in CR. Results of the expression studies, cytogenetic and molecular groups were analyzed for their impact on OS and RFS. Variables known to have prognostic implications such as baseline WBC, cytogenetics risk groups and molecular markers were grouped according to high/low BAALC and ERG expressers. Comparison of these prognostic factors within high/low expressers was carried out by Chi-square method. Survival was calculated by Kaplan-Meier method and prognostic factors were compared by log-rank test. Multivariate analysis was carried out using the Cox proportional hazards model. All covariates whose univariate models reflected a P value less than 0.2 were considered for inclusion in the model.Results: A total of 149 patients were accrued. The median age of the cohort was 27 years (range 15-58 ) with male predominance (M:F =98:51). Of 149 patients, 105 achieved CR after 1 or 2 cycles of chemotherapy. Twenty-seven patients were refractory of which 18 were lost to follow up and hence excluded from OS analysis. Seventeen patients died during induction. The OS of 131 patients was 36.5% at 2 years. The OS and RFS of 105 patients who achieved CR was 54% and 43.8% respectively. The median value (60 BAALC copies /ABL and 58 ERG copies /ABL ) was used to distinguish high expressers from low expressers. At baseline, copy numbers of 149 patients ranged from 11.58 to 242.23 for BAALC and 16.2 to 328.2 for ERG. For BAALC , 69/149(46.3%) were high expressers as compared to 80/149 (53.7%) as low expressers. Concerning ERG , 76/149(51%) were high and 73/149(49%) were low expressers. The OS and RFS of high BAALC versus low BAALC expressers at 2 years was 13.8% vs 59.8% (p<0001) and 17.6% vs 72.1% (p<0001) respectively. Similarly OS and RFS of high ERG vs low ERG expressers at 2 years was 5.3% vs 66.4% (p<0001) and 5% vs 71.2% (p<0001) respectively (Figure).The median baseline WBC count was significantly higher in high BAALC/ERG expressers. There was no difference in distribution of FLT3 -ITD, NPM1, CEBPA, MLL -PTD and cytogenetic risk groups in high versus low BAALC and ERG expressers. Multivariate analysis showed that ERG overexpression was only prognostic factor affecting OS and RFS compared to high BAALC expressers, MLL -PTD mutation, cytogenetic risk groups and baseline WBC.Conclusion: This study shows that high ERG expression adversely affects prognosis of AML patients independent of other well known prognostic factors [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BAALC Is a Key Mediator of Leukemia Development in Congenital Neutropenia

We recently reported high frequency of cooperating acquired CSF3R and RUNX1 mutations in severe congenital neutropenia (CN) patients that developed AML or MDS. CN is a pre-leukemic bone marrow failure syndrome (BMFS) and longitudinal retrospective analysis of CN bone marrow samples allows identification of the pathological intracellular events that lead to leukemogenic transformation.In the present study, we report the identification of a key mechanism of leukemogenesis in CN. We made use of the induced pluripotent stem cell (iPSC) model and embryoid body (EB)-based in vitro hematopoietic differentiation of iPSCs generated from a CN patient harboring ELANE mutation before (CN-iPSCs) and after (CN/AML-iPSCs) overt AML. CN/AML-iPSCs clones revealed CSF3R and RUNX1 mutations and trisomy 21. EB-based hematopoietic differentiation revealed that CN-iPSCs produced similar amounts of CD34+ cells, but approximately 2-fold reduction of mature granulocytes and CFU-G colonies in CFU-assay, as compared to healthy donor-derived iPSCs. CN/AML-iPSCs produced CD34+ cells but did not differentiate into mature granulocytes at all.Analysis of the gene expression profiles of five CN/AML patients including our index patient, revealed that genes upregulated in early hematopoietic stem/progenitor cells (HSPCs), such as Brain And Acute Leukemia, Cytoplasmic (BAALC), Hematopoietic Prostaglandin D Synthase (HPGDS), Natriuretic Peptide Receptor 3 (NPR3) and CD109 were markedly increased in CN/AML blasts harboring both RUNX1 and CSF3R mutations, as compared to hematopoietic cells prior to leukemia. This genetic signature suggests transformation of hematopoietic cells into more primitive hematopoietic progenitors after acquisition of CSF3R and RUNX1 mutations. We were able to confirm strong increase of BAALC, HPGDS, NPR3 and CD109 mRNA levels in CD34+ cells derived from CN/AML-iPSCs, as compared to CN-iPSCs, suggesting that our iPSC-model is reliable to study leukemogenesis.In order to evaluate which of these candidate genes (BAALC, HPGDS, NPR3 and CD109)is responsible for the leukemogenic transformation in CN, we generated CN/AML-iPSCs clones deficient for one selected genes. We established CRISPR-Cas9-mediated gene knockout using transfection of CN/AML-iPSCs with pSpCas9(BB)-2A-GFP vector expressing Cas9 and sgRNA, that were designed to specifically target first exon of each candidate gene. CN/AML-iPSCs transfected with specific sgRNAs were sorted and single cell clones were analyzed further.Knockout of the selected genes did not affect survival and pluripotency of CN/AML-iPSCs and we were able to study EB-based hematopoietic differentiation of gene-edited CN/AML-iPSCs clones. Interestingly, knockout of HPGDS, NPR3 or CD109 did not correct defective granulocytic differentiation of CN/AML-iPSCs, whereas knockout of BAALC in CN/AML-iPSCs resulted in the tremendous increase of the granulocytic differentiation, as assessed by FACS, CFU assay, morphological analysis of cytospin preparations and nitroblue tetrazolium (NBT) assay. Granulocytic differentiation of BAALC -deficient CN/AML-iPSCs was comparable to CN-iPSCs, but was still reduced in comparison to healthy donor-derived iPSCs.These data clearly showed that knockout of BAALC in CN/AML-iPSCs overcomes differentiation block at the early hematopoietic progenitor cell stage, demonstrating a fundamental role of BAALC in leukemia development in CN. BAALC, HPGDS and CD109 are known to be markedly up-regulated in RUNX1 -mutated de novo AML and qRT-PCR of CD34+ cells generated from BAALC -knockout CN/AML-iPSCs revealed a decrease of mRNA expression levels of BAALC, HPGDS and CD109 . At the same time, knockout of RUNX1 in CN/AML-iPSCs using CRISPR/Cas9-editing revealed even higher decrease of mRNA levels of BAALC, NPR3 and CD109 . These data suggest that HPGDS and CD109 up-regulation is downstream of RUNX1 -mediated BAALC induction in AML.Taken together, we established a reliable in vitro model to study leukemogenesis in the pre-leukemic BMFS, using CN as an example. With the help of this model, we identified BAALC up-regulation as a key “leukemogenic” factor downstream of RUNX1 and CSF3R mutations. Based on these observations, we conclude that inhibition of BAALC in hematopoietic cells of CN patients may prevent leukemogenic transformation of hematopoiesis or eliminate AML blasts in CN-AML. DisclosuresNo relevant conflicts of interest to declare.

A Novel Role for Brain and Acute Leukemia Cytoplasmic (BAALC) in Human Breast Cancer Metastasis.

Brain and Acute Leukemia, Cytoplasmic (BAALC) is a protein that controls leukemia cell proliferation, differentiation, and survival and is overexpressed in several cancer types. The gene is located in the chromosomal region 8q22.3, an area commonly amplified in breast cancer and associated with poor prognosis. However, the expression and potential role of BAALC in breast cancer has not widely been examined. This study investigates BAALC expression in human breast cancers with the aim of determining if it plays a role in the pathogenesis of the disease. BAALC protein expression was examined by immunohistochemistry in breast cancer, and matched lymph node and normal breast tissue samples. The effect of gene expression on overall survival (OS), disease-free and distant metastasis free survival (DMFS) was assessed in silico using the Kaplan-Meier Plotter (n=3,935), the TCGA invasive breast carcinoma (n=960) and GOBO (n=821) data sets. Functional effects of BAALC expression on breast cancer proliferation, migration and invasion were determined in vitro. We demonstrate herein that BAALC expression is progressively increased in primary and breast cancer metastases when compared to normal breast tissue. Increased BAALC mRNA is associated with a reduction in DMFS and disease-free survival, but not OS, in breast cancer patients, even when corrected for tumor grade. We show that overexpression of BAALC in MCF-7 breast cancer cells increases the proliferation, anchorage-independent growth, invasion, and migration capacity of these cells. Conversely, siRNA knockdown of BAALC expression in Hs578T breast cancer cells decreases proliferation, invasion and migration. We identify that this BAALC associated migration and invasion is mediated by focal adhesion kinase (FAK)-dependent signaling and is accompanied by an increase in matrix metalloproteinase (MMP)-9 but not MMP-2 activity in vitro. Our data demonstrate a novel function for BAALC in the control of breast cancer metastasis, offering a potential target for the generation of anti-cancer drugs to prevent breast cancer metastasis.

Optimal time-points for detecting expression levels of BAALC, EVI1, and WT1 genes in patients with acute myeloid leukemia: a meta-analysis

ABSTRACT Objectives This meta-analysis examined the prognostic role of brain and acute leukemia, cytoplasmic (BAALC), Ecotropic virus integration site-1 (EVI1) and Wilms’ tumor 1 (WT1) genes at different time-points during conventional chemotherapy. Methods A systematic search of publications indexed in the electronic databases from January 1988 to October 2020 was performed. Over 7525 cases of AML from 25 studies were involved. Results At diagnosis, overexpression of either BAALC or EVI1 had a negative impact on complete remission achievement (Summary Odds ratios [SORs] for BAALC = 0.32; SORs for EVI1 = 0.49) and survival outcome. The summary hazard ratios of overall survival (OS) and disease-free survival (DFS) were 1.97 and 2.04 for BAALC and 1.33 and 1.86 for EVI1, respectively. The prognostic value of pretreatment WT1 levels was heterogeneous while subgroup analyses unveiled that overexpressed WT1 may correlate with a favorable outcome (summary hazard ratio [SHR] for OS = 0.42). Both WT1 and BAALC played a role in prognosis assessment at post-induction and the diagnostic performance of WT1 transcript reduction was superior to the absolute WT1 level. Post-consolidation WT1 overexpression consistently indicated an increased risk of relapse, while the combined HR for RFS was statistically insignificant (SHR = 4.22). Conclusion These findings confirm the application of BAALC and EVI1 at diagnosis, WT1 after induction chemotherapy in AML patients throughout conventional chemotherapy.

Prognostic significance of combined BAALC and MN1 gene expression level in acute myeloid leukemia with normal karyotype.

Acute myeloid leukemia with normal karyotype (AML-NK) is the largest group of AML patients with very heterogeneous disease outcome. In order to ensure more precise risk stratification new molecular markers have been introduced, like expression level for BAALC (Brain and Acute Leukemia, Cytoplasmic) and MN1 (Meningioma 1) genes. In this study, we investigated expression level of both genes in 111 adult AML-NK at diagnosis and examined their prognostic potential. BAALC and MN1 expression were detected in about one third of the patients, and positive correlation between these two genes was found. The BAALC+ /or MN1+ status was not associated with the presence of FLT3-ITD mutations, but exhibited strong correlation with NPM1wt status (P<.001). Therefore, among BAALC+ /or MN1+ patients the most frequent ones were FLT3-ITD- /NPM1- double negative patients with intermediate prognosis. When BAALC+ /or MN1+ patients were divided into BAALChigh /BAALClow (21/21) and MN1high /MN1low (21/22) groups, we detected that BAALChigh /or MN1high patients had a tendency toward lower complete remission rate. Also, survival analysis showed that BAALChigh /or MN1high patients had shorter disease-free survival and overall survival (OS). The most pronounced influence on prognosis was detected in FLT3-ITD- /NPM1- group of patients that are lacking reliable prognostic markers, where OS in BAALChigh /or MN1high was only 5months vs 25months in BAALClow /or MN1low . These findings indicate that BAALC and MN1 expression level could be used for more precise risk stratification of AML-NK patients and especially FLT3-ITD- /NPM1- patients, transforming this intermediate-risk group, into a group with an adverse prognosis.

WT-1, BAALC, and ERG Expressions in Iranian Patients with Acute Myeloid Leukemia Pre- and Post-chemotherapy.

Purpose: Acute myeloid leukemia (AML) is the most prevalent acute leukemia in adults. It possesses different cytogenetic and molecular features. The expression of Wilms tumor-1 (WT1), brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) might be considered as prognostic factors in AML patients. The aim of this study was to determine the mRNA expressions of WT-1, BAALC and ERG genes in bone marrow of mononuclear cells and their effects on complete remission in the Iranian AML patients, pre- and post- chemotherapy. Methods: Forty AML patients with normal karyotype were evaluated. The mRNA gene expressions were measured with quantitative real-time PCR in bone marrow of mononuclear cells of AML patients at the baseline and after chemotherapy. The subtypes of AML and flow cytometry panel were also assessed. Complete remission (CR) after the treatment was addressed for all patients. Results: The mRNA expressions of WT-1, BAALC and ERG were significantly decreased after the treatment (p = 0.001, 0.017, 0.036). WT-1 mRNA expression was inversely correlated with CR after chemotherapy (P =0.024). There was also significant correlation between baseline expression of BAALC and CR (P =0.046). No significant correlation was observed between ERG and CR pre- and post- chemotherapy (P =0.464 and 0.781). There was also significant correlation between BAALC mRNA expression and CD34+ (P <0.001). Conclusion: The present study showed that WT-1 decreased significantly after standard chemotherapy which could have favorable effects on CR. Also, the high expression of BAALC could have a poor prognostic role in AML patients. The identification of these gene expressions can be an efficient approach in targeted therapy among AML patients.

Brain and Acute Leukemia, Cytoplasmic Gene Overexpression as a Prognostic Factor in Egyptian De novo Adult Acute Myeloid Leukemia Patients

Abstract Background: Brain and acute leukemia, cytoplasmic (BAALC) gene is identified on chromosome 8q22.3 and implicated in normal hematopoiesis. BAALC gene overexpression is associated with poor outcome. Methods: We aimed to evaluate BAALC expression in de novo Egyptian acute myeloid leukemia (AML) cases and determine its prognostic value. We recruited 70 patients with de novo AML diagnosed and treated at clinical pathology and medical oncology departments, fulfilling inclusion criteria in our prospective study and evaluated BAALC expression level. Patients received induction therapy. The Institutional Review Board approved our study. Results: The mean age was 39.2 years ± 11.87, (18–60) with a male/female ratio of 3/2. The cutoff value of BAALC as a prognostic factor was 2.11 with sensitivity (86.1%), specificity (80%), positive predictive value (88.6%), and negative predictive value (76.2%.) (P &lt; 0.001), 43 (61.4%) patients had high BAALC expression. Seventy-two percent of patients in the low BAALC group achieved complete remission (CR) compared to 42.1% in high BAALC expression group (P = 0.03). Patients with low BAALC (123.1 ± 4.9) had longer mean survival time than high BAALC group (45.85 ± 5.1) (P = 0.000). Conclusion: High-BAALC expression is an adverse prognostic factor, with a higher risk of relapse, lower CR rates, and lower survival in Egyptian de novo AML patients.

Over expression of brain and acute leukemia, cytoplasmic and ETS-related gene is associated with poor outcome in acute myeloid leukemia.

The high expression of brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) has been reported to influence the outcome in acute myeloid leukemia (AML), but due to limited prospective studies, their role as prognostic factors is unclear. At diagnosis, the prognostic value of BAALC and ERG expression with respect to other cytogenetic and molecular markers was analyzed in 149 AML patients. Patients were divided into quartiles which resulted in the formation of four groups (G1-G4) based on expression values of BAALC and ERG and clinical response defined across groups. Groups with similar survival probabilities were merged together and categorized subsequently as high versus low expressers. Patients with high BAALC and ERG expression had significantly lower overall survival (OS; BAALC: p = 0.001 at 5 years 29.4% vs. 69.8%; ERG: p < 0.0001 at 5 years 4% vs. 50.4%) and disease-free survival (BAALC: p = 0.001 at 5 years 19.5% vs. 69.8%; ERG: p < 0.0001 at 5 years 4.2% vs. 47%). Patients were further stratified combining BAALC and ERG expression in an integrative prognostic risk score (IPRS). After a median follow-up of 54 months (95% CI 45-63 months) among survivors, IPRS for high versus low expressers was a significant predictor for OS (BAALC + ERG: 4% vs. 71.6%, p < 0.0001) and DFS (BAALC + ERG: 4.5% vs. 74.1%, p < 0.0001). In a multivariate model, IPRS of BAALC + ERG expression retained prognostic significance for OS (hazard ratio [HR] 2.96, 95%CI 1.91-4.59, p < 0.001) and DFS (HR 3.61, 95%CI 2.26-5.76, p < 0.001).

Allogeneic stem cell transplantation mitigates the adverse prognostic impact of high diagnostic BAALC and MN1 expression in AML

For most acute myeloid leukemia (AML) patients, an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of sustained remissions and long-term survival. At diagnosis, high expression of the AML-associated genes BAALC (brain and acute leukemia, cytoplasmic) and MN1 (meningioma-1) were repeatedly linked to inferior outcomes in patients consolidated with chemotherapy while data for patients receiving HSCT remain limited. Using clinically applicable digital droplet PCR assays, we analyzed the diagnostic BAALC/ABL1 and MN1/ABL1 copy numbers in 302 AML patients. High BAALC/ABL1 and MN1/ABL1 copy numbers associated with common adverse prognostic factors at diagnosis. However, while high diagnostic copy numbers of both genes associated with shorter event free survival (EFS) and overall survival (OS) in patients receiving chemotherapy, there was no prognostic impact in patients undergoing HSCT. Our data suggests that the adverse prognostic impact of high BAALC and MN1 expression are mitigated by allogeneic HSCT. But preHSCT BAALC/ABL1 and MN1/ABL1 assessed in remission prior to HSCT remained prognosticators for EFS and OS independent of the diagnostic expression status. Whether allogeneic HSCT may improve survival for AML patients with high diagnostic BAALC or MN1 expression should be investigated prospectively and may improve informed decisions towards individualized consolidation options in AML.

1H, 13C, and 15N Backbone assignments of the human brain and acute leukemia cytoplasmic (BAALC) protein

The brain and acute leukemia cytoplasmic (BAALC; UniProt entry Q8WXS3) is a 180-residue-long human protein having six known isoforms. BAALC is expressed in either hematopoietic or neuroectodermal cells and its specific function is still to be revealed. However, as a presumably membrane-anchored protein at the cytoplasmic side it is speculated that BAALC exerts its function at the postsynaptic densities of certain neurons and might play a role in developing cytogenetically normal acute myeloid leukemia (CN-AML) when it is highly overexpressed by myeloid or lymphoid progenitor cells. In order to better understand the physiological role of BAALC and to provide the basis for a further molecular characterization of BAALC, we report here the 1H, 13C, and 15N resonance assignments for the backbone nuclei of its longest hematopoietic isoform (isoform 1). In addition, we present a 1HN and 15NH chemical shift comparison of BAALC with its shortest, neuroectodermal isoform (isoform 6) which shows only minor changes in the 1H and 15N chemical shifts.

The Prognostic Significance of the BMI-1 and BAALC Genes in Adult Patients with Acute Myeloid Leukemia.

The aim of this work is to investigate the different expression patterns of B cell-specifics moloney murine Leukemia virus integration site-1 (BMI-1) and brain and acute leukemia, cytoplasmic (BAALC) genes, their prognostic and clinical significance in newly diagnosed cytogenetically heterogenous adult acute myeloid leukemia patients. BMI-1 and BAALC expression was detected in the bone marrow of patients using quantitative real-time reverse transcription polymerase chain reaction with cut off value set at 50th percentile for both genes. BMI-1 and BAALC overexpression was detected in 50% of cases which suggest their potential as molecular markers. A statistical significant correlation was found between BMI-1 expression with hepatomegaly (P value = 0.007), hemoglobin level-grouped (P value = 0.047) and cytogenetic risk groups (P value = 0.036). There was a statistically significant correlation between BAALC and age (P value = 0.015), lymphadenopathy (P value = 0.043), CD34 expression (P value = 0.003) and near statistical significance with FAB sub-groups (P value = 0.054). No statistical significance was noted for other hematological findings and response to treatment except for BAALC gene and treatment response (P value = 0.014). No statistical significance in overall survival and disease free survival for both genes was found. Their prospective screening in combination with other molecular markers can help refine myeloid leukemia staging and prognosis toward optimizing therapeutic interventions.

Prognostic Impact of Blood MN1 Copy Numbers Before Allogeneic Stem Cell Transplantation in Patients With Acute Myeloid Leukemia.

High expression of the leukemia-associated gene meningioma-1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation- and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.

Mechanism of BAALC-Mediated Leukemogenesis Downstream of RUNX1-Mutations in Severe Congenital Neutropenia

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. We recently reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. To study the mechanism of leukemia development in CN, we established a model for step-wise leukemia progression in CN using iPSC-based hematopoietic differentiation in combination with CRISPR/Cas9-mediated gene editing of iPSCs. Using this model, we confirmed that co-acquisition of CSF3R and RUNX1 mutations is necessary and sufficient to induce leukemia in CN. We also identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event downstream of RUNX1 and CSF3R mutations. BAALC mRNA was upregulated in CN/AML blasts (n = 5) and in CD34+ HSPCs generated from CN/AML iPSCs of two patients. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs reversed defective myeloid differentiation of CN/AML blasts to the levels observed in healthy donor hematopoietic stem cells.We further investigate the mechanism of BAALC up-regulation. In silico analysis of the BAALC gene promoter in combination to publicly available ChIP-Seq data revealed three putative RUNX1 binding sites that were validated using ChIP assay in lysates of NB4 cells. Interestingly, transduction of healthy donor CD34+ cells with lentiviral constructs expressing WT RUNX1 led to inhibition of BAALC mRNA expression, whereas transduction with two RUNX1 RUNT domain mutants resulted in the marked BAALC up-regulation, as compared to the control BFP transduced cells. These data suggest that mutated RUNX1 failed to inhibit BAALC expression in CD34+ HSPCs.To evaluate the mechanism of leukemogenic transformation in CN, we performed RNA-Seq analysis of CD34+ cells derived from CN and CN/AML iPSC clones. GSEA revealed that changes in gene expression between CN- and CN/AML-HSPCs were strongly correlated with gene expression signatures of “Wierenga STAT5 targets” and “reactome ATF4 targets”, an observation in line with the markedly elevated levels of STAT5 and ATF4 in CN/AML-HSPCs. Importantly, gene expression differences between CN/AML-HSPCs and CN-HSPCs were correlated to “Valk AML” targets in GSEA, suggesting that HSPCs generated from CN/AML-iPSCs possess characteristics of AML cells. Strong support for the leukemogenic role of upregulated BAALC in CN/AML was provided by further GSEA analysis of the BAALC KO CN/AML-HSPCs. We observed a reversal in the expression of a majority of genes in the studied leukemia-associated pathways in CN/AML-HSPCs after BAALC knockout compared with CN/AML-HSPCs.Since there are no direct inhibitors for BAALC available and protein structure is not solved yet, BAALC effects can be targeted only indirectly. Morita et al., Leukemia, 2015 showed that BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and Klf4. We treated CD45+ cells generated from CN/AML or healthy donor (HD) iPSCs with MEK1/2- or MEK1-specific inhibitors or vehicle control and evaluated cell proliferation and differentiation (CFU assay). We were able to induce ~ 40-60 % cell death of CN/AML cells upon treatment with each of inhibitors, whereas HD CD45+ cells were unaffected. Moreover, treatment of CN/AML cells with MEK1/2 inhibitor led to an increase in CFU-G formation, as compared to vehicle control cells.Using connectivity Map analysis of RNA-Seq data of CD34+ cells generated from CN/AML iPSCs vs CN/AML BAALC KO iPSCs, we identified small molecule p38/MAPK14 inhibitor that could possibly reverse BAALC-mediated leukemogenic gene expression signature. We treated CN/AML iPSC-generated CD34+ cells for 7 days with this inhibitor and subsequently performed CFU assay. We found an increase in CFU-GM formation.In summary, using CN/AML-iPSC-model, we confirmed the major role of BAALC in leukemia development downstream of CSF3R and RUNX1 mutations in CN. Inhibition of MAPK/ERK-pathway downstream of BAALC reduced proliferation and partially induced myeloid differentiation of CN/AML-derived hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Monitoring ESAM Expression Levels Reveals Autonomous Fluctuation of Leukemia Stem Cells By Autocrine/Paracrine Cytokine Signaling

Recently , we showed that hematopoietic stem cells (HSCs) fluctuate in a dynamic trajectory for self-renewal and lineage commitment, which regulates HSC heterogeneity (Cell Rep 2018). Although the significance of heterogeneity is difficult to interpret, it might enable HSCs to maintain a dynamic equilibrium of homeostatic hematopoiesis and to adapt promptly to stress-induced hematopoiesis based on physiological demands. We hypothesized that similar to HSCs, leukemia stem cells (LSCs) are heterogeneous with active fluctuation, which contributes to treatment resistance. Hence, this study aimed to show the fluctuation of LSCs and determine the underlying molecular mechanisms.We previously reported that the endothelial-cell selective adhesion molecule (ESAM) is useful as a life-long marker for HSCs in humans and mice (Blood 2009, Exp Hematol 2013). ESAM expression levels precisely indicated the activation status of HSCs (J Immunol 2012). Furthermore, approximately two-thirds of patients with acute myelogenous leukemia (AML) were positive for ESAM expression on leukemia cells in a heterogeneous manner. (Exp Hematol 2013). We exploited human AML lines as LSC-like cells and monitored expression levels of ESAM. Flow cytometry analyses revealed wide-ranging ESAM expression levels on KG1a and CMK lines. In KG1a cells, ESAM-high cells were concentrated in the CD34+CD38- fraction with high growth capability, whereas ESAM-low cells were less proliferative. Notably, when sorted into ESAM-high or ESAM-low subgroups, both groups reconstituted the parental heterogeneous distribution of ESAM levels in culture. Furthermore, single self-renewing LSC-like clones also reproduced the same heterogeneous population regardless of their original ESAM levels. These results suggested that LSC-like clones developed the heterogeneous AML cell population by fluctuating, which could be monitored with ESAM levels.The transcriptomes of ESAM-high and ESAM-low LSC-like cells in KG1a line were compared to obtain comprehensive information of key molecules involved in fluctuation and heterogeneity. We found that expression of T-cell immunoglobulin and mucin domain 3, reported as a reliable LSC marker for human AML (Cell Stem Cell 2010), was variable in parallel with ESAM. In addition, network analysis demonstrated that “genes related to hematopoietic progenitor cells” were activated whereas “genes related to blood cell maturation” were downregulated in ESAM-high cells. These results suggested that ESAM-high LSC-like cells were more identical to the conventional concept of LSCs than ESAM-low cells. However, expression levels of BAALC, SPP1, and ITGB7 were elevated in ESAM-low cells. BAALC encodes “brain and acute leukemia cytoplasmic (BAALC)” protein, which inhibits drug-induced apoptosis in KG1a cells (Hematology 2012). SPP1 encodes “secreted phosphoprotein 1 (SPP-1), also known as osteopontin, which induces chemo-resistance in AML cells (Life sci 2017). Furthermore, a recent paper reported that the active form of integrin □7 can be a therapeutic target of myeloma stem cells (Nat Med 2018). Thus, we infer that fluctuating to the ESAM-low stage may contribute to the refractory nature of LSCs in AML.We performed an up-streaming analysis on the transcriptome data to determine the signaling pathways involved in the fluctuation. The tumor necrosis factor alpha (TNFα) and the transforming growth factor beta (TGFβ) signaling pathways were found to affect ESAM expression levels. When TNFα or TGFβ1 was added to culture medium, the proliferation of KG1a cells was suppressed in a dose-dependent manner. Furthermore, addition of TNFα or TGFβ1 significantly suppressed ESAM expression levels on a subset of KG1a cells, which enhanced their heterogeneity. When cultured in serum-free cytokine-free medium, KG1a cells became uniform in terms of ESAM expression levels, with the ESAM-low population disappearing . Intriguingly, we found that KG1a cells produced TNFα and TGFβ1 autonomously, which induced the cell heterogeneity in an autocrine/paracrine manner. Surprisingly, blocking these signaling pathways by receptor inhibitors induced apoptosis in KG1a cells. These results suggest that autocrine/paracrine cytokine signaling plays important roles in promoting the variable and heterogeneous nature of LSCs, the inhibition of which might be a strategy for therapeutic intervention in refractory AML. DisclosuresYokota:Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Jansen Pharmaceutical K.K: Honoraria; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

BAALC and ERG expression levels at diagnosis have no prognosis impact on acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell transplantation.

Brain and acute leukemia, cytoplasmic (BAALC) and ETS-related gene (ERG) expression levels are independent prognostic factors for acute myeloid leukemia (AML); however, their prognostic impacts on AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) require further investigation. We studied 71 de novo AML patients treated with allo-HSCT and defined low and high expressers according to the median expression levels of BAALC and ERG at diagnosis respectively. High BAALC expression was associated with wild-type NPM1 (P = 0.000) and RUNX1 mutations (P = 0.027). High ERG expression was associated with FLT3-ITD absence (P = 0.003) and wild-type NPM1 (P = 0.001). BAALC and ERG expression levels were significantly correlated with each other (P = 0.001). Survival analyses including Kaplan-Meier curves and univariate and multivariate analysis consistently reported that there were no significant differences for both event-free survival (EFS) and overall survival (OS) (all P > 0.1), between high versus low BAALC and ERG expressers. Our study suggested that despite of their well-known adverse role in prognosis of AML, neither BAALC nor ERG expression levels at diagnosis had effect on survival of AML patients who underwent allo-HSCT.

Clinical impact of BAALC expression in high-risk acute promyelocytic leukemia.

Although overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is associated with primary resistant disease and shorter relapse-free, disease-free, and overall survival in different subsets of acute myeloid leukemia (AML), little is known about its clinical impact in acute promyelocytic leukemia (APL). Using real-time reverse transcriptase polymerase chain reaction, we showed that BAALC expression is significantly lower in APL compared with other subsets of AML (P < .001). We also demonstrated that BAALC overexpression was associated with shorter disease-free survival (DFS) (hazard ratio [HR], 4.43; 95% confidence interval [CI], 1.29-15.2; P = .018) in 221 consecutive patients (median age, 35 years; range, 18-82 years) with newly diagnosed APL homogeneously treated with all-trans retinoic acid and anthracycline-based chemotherapy. Cox proportional hazard modeling showed that BAALC overexpression was independently associated with shorter DFS in the total cohort (HR, 5.26; 95% CI, 1.52-18.2; P = .009) and in patients with high-risk disease (ie, those with initial leukocyte counts >10 × 109/L) (HR, 5.3; 95% CI, 1.14-24.5; P = .033). We conclude that BAALC expression could be useful for refining risk stratification in APL, although this needs to be confirmed in independent cohorts.

Validation of a Molecular Risk Score for Prognosis of Patients with Acute Promyelocytic Leukemia

Introduction: Risk stratification in acute promyelocytic leukemia (APL) is based on clinical parameters, namely leukocyte and platelet counts at initial diagnosis as combined in the Sanz Score. However, during the last years the influence of additional molecular genetic markers on prognosis of APL patients has been postulated. In 2015 we published the results of a molecular risk score integrating expression data of the genes brain and acute leukemia, cytoplasmic (BAALC), ets' related gene (ERG) and Wilms' Tumor 1 (WT1) with strong independent influence on outcome and relapse risk of APL patients treated in the German AMLCG studies (Hecht et al., Leuk Res 2015). The aim of our study was to validate our data in an independent patient cohort.Methods: In cooperation with the German SAL (Study Alliance Leukemia) group we obtained a validation set of samples of mononuclear cells derived from the bone marrow of 76 patients with confirmed diagnosis of APL prior to therapy. The validation cohort consisted of 37 female and 39 male patients with a median age of 50 years (range 20-82 years; Table 1). Patients were diagnosed and treated between 2000 and 2014 mostly with an ATRA plus Idarubicin based induction therapy followed by Sanz score-dependent consolidation and maintenance chemotherapy. RNA was extracted using the AllPrep DNA/RNA Mini Kit and cDNA was synthesized using 500µg of RNA with a QuantiTect Reverse Transcription Kit (both Qiagen, Hilden, Germany). Expression levels of BAALC, ERG and WT1 were then analyzed using quantitative real-time RT-PCR with the same conditions and primers as used in the original test cohort and the same calibrator cDNA from cell lines was used for the analysis. The following gene expression levels were defined as negative risk factors in preceding studies: BAALC expression ≥25th percentile (BAALChigh), ERG expression ≥75th percentile (ERGhigh) and WT1 expression ≤25th percentile or ≥75th percentile (WT1low or high). The integrative risk score was calculated as described before: For the presence of one of the mentioned risk factors, one scoring point was assigned to a respective patient, i.e. a maximum of 3 points (one point for BAALChigh, ERGhigh and WT1low or high, respectively) and a minimum of 0 points (i.e. presenting with none of the aforementioned risk factors). Overall survival (OS), relapse free survival (RFS) and cumulative incidence of relapse (CIR) were calculated using the Kaplan-Meier method.Results: The expression of the three genes BAALC, ERG and WT1 compared to healthy controls were exactly the same as in the original cohort. Application of the molecular risk score on APL patients of the validation cohort clearly discriminated patients according to their risk comparable to the original APL cohort (Figure 1). Patients with 0 points had a very good prognosis with no deaths or relapses, whereas patients with 3 points (i.e. concurrent presence of all three risk factors) had a very poor outcome with an OS of 51%, a RFS of 50% and a CIR of 38%. The differences between patients with 1 and 2 points were less pronounced in the validation cohort. However, the statistical analyses in the validation cohort did not yield significance. As the main reason we assume that the validation cohort altogether showed a significantly improved OS and RFS compared to the original cohort which was treated a decade earlier, so differences between the subgroups could not be that pronounced. For both cohorts stratification by the Sanz Score did not show significant differences in outcome (analysis for RFS p=0.46 in original cohort and p=0.44 in validation cohort).Conclusion: A validated molecular-based integrative risk score was able to define subgroups of APL patients with different outcome independently of the Sanz score. The validation of the strong influence of the combination of molecular risk factors is particularly interesting as the new analyses of the three genes BAALC, ERG and WT1 separately did not confirm the strong results of the single analyses of each gene. The discrimination of patients according to the risk score is evident though. This argues for their integration in future studies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Blood BAALC Copy Numbers Determined By Digital Droplet PCR at Timepoint of Allogeneic Transplantation in Complete Remission Predicts Relapse in Patients with Acute Myeloid Leukemia

Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC (Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALC copy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR.We identified 82 AML pts with PB in first (60%) or second CR (23%) or CRi (17%) up to 28 days prior to HSCT available. Median age at HSCT was 63.9 (range 50.8-76.2) years (y). All pts received non-myeloablative (NMA) conditioning (fludarabine 3x30 mg & 2 Gy total body irradiation). At diagnosis, mutation status (mut) of the NPM1, CEBPA, IDH1, IDH2,&DNMT3A gene & presence of FLT3-ITD or FLT3-TKD were assessed. In pre-HSCT PB, absolute quantification of BAALC copy numbers was performed by ddPCR & results were normalized to ABL1 copy numbers.Additionally, absolute BAALC copy numbers wereassessedin PB of healthy controls (n=7) with a median age of 62.7 (range 39.6-82.0) y.Pts were grouped according to the European LeukemiaNet (ELN) classification in 21% favorable, 23% intermediate-I, 24% intermediate-II, 23% adverse & 9% unknown. Pts & healthy control were evenly matched in age (P=1) & sex (P=1). BAALC/ABL1 copy numbers did not differ between AML pts at HSCT (median 0.03 [range 0.01-2.48]) & the healthy controls (median 0.04 [range 0.03-0.10], P=.34, Figure 1).A cut-off point of 0.14absolute BAALC/ABL1 copies was determined using the R package ’OptimalCutpoints’ & used to define pts with high (26%) & low (74%) pre-HSCT BAALC/ABL1 copy numbers. The copy number at this cut-off point was higher than the two-fold standard deviation over the median of the healthy controls (0.10 BAALC/ABL1).Pts with high & low pre-HSCT BAALC/ABL1 copy numbers did not differ significantly in pre-treatment characteristics (i.e. hemoglobin, white blood count, platelets, blasts in bone marrow or PB, ELN genetic group, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 mut) or remission status at HSCT (CR1 vs. CR2 vs. CRi). However, pts with high pre-HSCT BAALC/ABL1 copy numbers had a significantly higher cumulative incidence of relapse (CIR, P=.02, Figure 2a) & shorter overall survival (OS, P=.02, Figure 2b). High pre-HSCT BAALC/ABL1 copy numbers especially impacted on CIR when we restricted our analysis to pts with normal cytogenetics (P=.003). In multivariate analysis for the entire cohort, high pre-HSCT BAALC/ABL1 copy numbers retained the prognostic impact on CIR (Hazard Ratio [HR] 3.6, Confidence Interval [CI] 1.6-8.2, P=.002) after adjustment for disease status at HSCT (P=.006) & the prognostic impact on OS (HR 2.2, CI 1.1-4.3, P=.02).In conclusion, ddPCR is a feasible method for absolute quantification of BAALC copy numbers in PB, which may indicate residual disease burden in AML pts. High PB BAALC/ABL1 copy numbers (>0.14) in AML pts in hematologic CR at HSCT associated with higher CIR & shorter OS in univariate & multivariate models. AML pts with high PB BAALC/ABL1 copy numbers at HSCT should be closely monitored for relapse in the post-transplant period. In the future prospective studies will be required to validate the absolute PB BAALC/ABL1 copy number cut-off point & to evaluate whether AML pts with high BAALC/ABL1 copy numbersmight benefit from additional treatment before HSCT. [Display omitted] [Display omitted] DisclosuresPoenisch:Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

BAALC and ERG Expression in Egyptian Patients with Acute Myeloid Leukemia, Relation to Survival and Response to Treatment

AIM:Aim was to detect Brain and Acute Leukemia, Cytoplasmic (BAALC) and ETS-related gene (ERG) expression in patients with acute myeloid leukemia (AML) as well as to study their biologic and prognostic impact on the disease outcome and survival.PATIENTS AND METHODS:The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.RESULTS:The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression. BAALC was expressed in 36 (81.82%) of AML cases versus 10 (22.72%) of the control group which was highly statistically significant (P < 0.001). While ERG was positive in 39(88.64%) of cases and 8(18.18 %) of controls and that was also highly statistically significant (P < 0.001).CONCLUSION:Further researches still needed to clarify the role of BAALC and ERG in the pathogenesis of leukemia and their importance as targets for treatment of AML.