Leukemic Precursors Research Articles

Clofarabine Significantly Increases Eradication of Minimal Residual Disease of B-Precursor ALL Compared to High-Dose Cytarabine in Randomized Trial Coall 08-09

Background Since the FDA approval of clofarabine for the treatment of relapsed or refractory acute lymphoblastic leukemia (ALL) at childhood, several studies have been launched which put clofarabine under scrutiny in combination with other cytostatic drugs as second or third line therapy. As a novel treatment-strategy we introduced the combination of clofarabine with pegylated asparaginase (PEG-ASP) in a randomized fashion in comparison to the standard consolidation course with high dose cytarabine (Hidac) combined with PEG-ASP into the frontline management of ALL within the CoALL 08-09 protocol. The primary objective of the study was to compare the MRD based assessment of the cytotoxic efficacy of the randomized courses in pediatric ALL. Patients and methods CoALL 08-09 was an open, interventional, multi-center, prospective, and randomized clinical trial for patients with newly diagnosed ALL. In March 2010 the trial had been opened for enrollment of patients under 18 years with confirmed diagnosis of acute B- or T-cell precursor leukemia. After a stratified phase I/II, in which patients with high-risk features and high post-induction MRD received the combination of clofarabine and PEG-ASP, the randomized trial started in November 2013. All patients with a measurable MRD at the end of induction (EOI), as measured by RQ-PCR according to EURO-MRD guidelines, were eligible for randomization to receive either the combination of clofarabine 5 x 40 mg/m2 or high dose cytarabine (HIDAC) 4 x 3g/m2 bothin combination with PEG-ASP 2.500 IU/m2 at the beginning of the consolidation phase. Until December 2019 303 study patients were randomized allocating 151 patients towards the clofarabine and 152 patients to the HIDAC arm. Patient characteristics and MRD burden prior to this treatment element were highly comparable. Results The reduction of minimal residual disease was significantly more profound after clofarabine compared to cytarabine (p(Cochran-Armitage)=.01 for BCP-ALL with 93 vs 79 patients reaching MRD negativity (Table 1). MRD status of BCP-ALL after the first randomized consolidation block (day 50) was of prognostic relevance with a statistically significant impact on EFS and relapse rate (Fig. 2a). However, no difference in outcome regarding the event-free and overall survival between the randomized arms was observed (5-year EFS: Clofarabine 85.7, SE=4.1 vs Hidac 84.8, SE=4.7 (p=.96) and OS: 95.7, SE=1.9 vs 92.2, SE=3.2(p=.59) Fig. 2b). This finding was recapitulated independent of gender, age, WBC count at diagnosis, ETV6-RUNX 1 translocation, immunophenotype or overall risk strata). Results for T-ALL patients are in the same range but because of small numbers not statistically significant. No differences in the incidence of severe or persistent toxicity btween randomized treatment elements as well as in the subsequent treatment realization were registered. Conclusion Clofarabine combined with PEG-asparaginase is highly effective and well tolerated in the frontline treatment of ALL. Although the prognostic impact of MRD is still discernible in early consolidation, the greater cytotoxic efficacy of clofarabine reflected by a more frequent eradication of MRD, did not translate into an apparent improvement of outcome likely due to a lack of power in the comparative analysis of randomized patients. Disclosures No relevant conflicts of interest to declare.

Somatic Mutation And Transcriptome Profiling Identifies The E1 Ubiquitin-activating Enzyme Uba1 As A Common Mutation Target In Radiation-induced Lymphomas In p53 Deficient Mouse Models

Large-scale sequencing studies have revolutionized our understanding of cancer genomes, revealing a catalogue of somatically-acquired cancer-driving mutations that are the major focus of therapeutic drug development. However, the impact of inherited germline variants – an essential component of individual precision medicine for cancer - has not been well-studied. Deficiency of the tumor-suppressor gene TP53 is known to play a central role in development of radiation induced malignancies. Studies in mice show that some driver mutations exclusively or preferentially occur on one parental allele, indicating the strong effects of inherited polymorphisms in shaping tumor genomic landscapes. We hypothesize that different biological pathways underlie the development of radiation-induced lymphomas, and that these differences may offer opportunities for therapeutic intervention. We exposed a well characterized genetically heterogeneous mouse model of T cell lymphoma (129/SvImJ x Mus spretus backcross) that are either germline p53+/- or p53-/- to a single dose of 4 Gy total body gamma radiation (RT). We performed both whole exome sequencing to avg. on-target read depth of 74 +/- 14 on a subset of 32 lymphomas (16 of each p53 genotype); microarray gene expression analysis (Agilent) was performed for matched samples and an additional 12 samples for comparison. Standard pipelines including BWA/PICARD/GATK/MuTect/PinDel/CNVKit and R packages affy, limma, or custom scripts were used. Human cancer mutation statistics were derived from COSMIC, TCGA, cBioPortal and manually from published T-ALL sequencing studies All animals developed aggressive lymphomas <12 months after RT. The p53-/- animals had shorter tumor latencies (107+/-36 days) compared to p53+/- mice (222+/-57 days, p = 2.36x10-10, log-rank test), and had 2-fold the point mutations compared to lymphomas from p53+/- mice (8 vs 4 mutations; p = 0.03, Wilcoxon test). Recurrent p53-dependent and/or strain-specific point and structural mutations were identified at loci with known homologs in human cancer such as Notch1, Pten, and Ikzf1. One particularly gene of interest is Uba1 (E1 ubiquitin activating enzyme), in which point mutations were exclusively seen in lymphomas from p53-/- mice. These mutations map to the ubiquitin-activating and E2 interacting domains of Uba1, suggesting that the disruption of the ubiquitin pathway is a driver of RT-induced lymphomas. Integrating these findings with expression network and differential expression analysis, we confirmed functional loss of Uba1 and identified unique signatures for RT-induced Early T-cell Precursor (ETP) leukemia, a clinically aggressive lymphoma subtype. Our data demonstrate the profound influence of both germline strain-specific polymorphisms and the timing of p53 loss on the overall pathways to tumorigenesis, as well as on selection of specific mutations in potential drug target genes, such as Uba1.

Comparison of Early T-Cell Precursor and Non-ETP Subtypes Among 122 Chinese Adults With Acute Lymphoblastic Leukemia

Background: Adult T-cell acute lymphoblastic leukemia (T-ALL) is a rare hematological malignancy and significantly linked to poor outcomes. Early T-cell precursor (ETP) leukemia is a unique subtype of T-ALL. The aim of this study is to compare the differences between ETP and non-ETP ALLs in China.Methods: We retrospectively analyzed the records of 122 adult T-ALL patients diagnosed and treated at our center between January 2014 and June 2019. All the patients enrolled were categorized into ETP and non-ETP ALL by immunophenotype, and further statistical analyses about clinical data and prognostic factors were performed.Results: Among the 122 cases, the male-to-female ratio was 2.8:1, and the median age is 29 (range, 16–82) years. Except for 10 patients with insufficient immunophenotyping results, 47.3% (53/112) are ETP and 52.7% (59/112) are non-ETP. Compared with non-ETP patients, ETP-ALL patients had lower white blood cell counts and lactate dehydrogenase levels, while they were older and had higher platelet counts and fibrinogen levels (all p < 0.05). Complete remission (CR) was achieved in 68.0% (83/122) of patients, 64.2 and 76.3% in ETP and non-ETP, respectively (p = 0.160). In total, 44.6% (37/83) of patients relapsed. Allogeneic stem cell transplantation (allo-SCT) was successfully performed in 36.1% (44/122) of patients, of which 79.5% (35/44) were in CR1. With a median follow-up of 9.1 (range, 0.5–70.3) months, the estimated 2-year overall survival (OS) and relapse-free survival (RFS) rates for the cohort were 38.0 ± 5.1 and 39.1 ± 6.3%, respectively. In the ETP group, the 2-year OS rate was 40.7 ± 8.2% and the RFS rate was 47.2 ± 10.7%, while in the non-ETP group, the 2-year OS rate was 37.9 ± 7.0% and the RFS rate was 39.2 ± 8.3% (both p > 0.05). In the landmark analysis of CR1 patients who had a survival of more than 6 months, the allo-SCT group had significantly better survival outcomes than the chemotherapy group, and the 2-year OS rates and RFS rates were 80.1 ± 7.3 vs. 28.4 ± 8.4% and 68.9 ± 8.8 vs. 12.8 ± 7.2%, respectively (both p < 0.0001). A multivariate analysis suggests that allo-SCT acts as an independent prognostic factor for both OS and RFS.Conclusions: Our results revealed that ETP accounted for a high proportion of T-ALL in Chinese. There are no CR rates and prognosis differences between ETP and non-ETP. Allo-SCT in CR1 can significantly improve patients' survival.

Abstract 3247: Development of CAR-T cell therapy for B-ALL using a point-of-care approach

Abstract Introduction: CD19-targeted CAR-T cell immunotherapy is now an approved treatment for B cell leukemias and lymphomas. Current protocols for producing CAR-T cells use viral vectors for genetic modification and take 12-15 days to expand sufficient numbers of T cells, constituting an important bottleneck for the widespread use of this therapy. The development of non-viral, plasmid-based systems like Sleeping Beauty (SB) transposons, coupled with the emergence of large scale, closed electroporation devices, present the opportunity of generating high numbers of CAR-T cells in a short period of time and transforming this in a point-of-care (POC) therapy. Methods and Results: In this work, mononuclear cells were isolated using Ficoll and electroporated using Nucleofector IIb electroporator combined with plasmids encoding 19BBz CAR (in the pT3 SB transposon backbone) and SB100x transposase. Cells were rested for 24h and used for in vitro and in vivo experiments. The phenotype was assessed by flow cytometry. The in vitro cytotoxicity assay was performed using Calcein-AM dye on target cells incubated with different ratios of effector cells. 8-12-week-old-female-NSG were injected iv. 5 × 106 RS4;11 GFP or 105 Nalm-6 GFP and after 3 days were treated with different doses of recently electroporated CAR-T cells. CAR expression on day 1 following electroporation ranged between 5-15% and in vitro cytotoxic activity against RS4;11 or Nalm-6 CD19+ B cell precursor leukemia lines was low or absent. NSG mice engrafted with RS4;11 and treated with 1 × 105 19BBz+ cells showed improved survival when compared to mice treated with mock electroporated cells. POC approach was also efficient against Nalm-6, where 19BBz+CAR-T cells (7 × 105 per mice) improved the survival of mice. In both models, decreased tumor burden in blood and spleen was observed in mice treated with 19BBz. Head to head comparison of 19BBz cells used in POC approach or expanded for 8-12 days in vitro showed similar antitumor activity in vivo against RS4;11 cells, leading to equivalent improvements in mice survival. Conclusion: In summary, we show that the POC strategy is a viable approach for the use of CAR-T cells, obviating the need for T cell expansion while having the potential to greatly decrease the total cost and time of CAR-T cell therapy manufacturing. Citation Format: Luiza Abdo, Luciana Rodrigues Barros, Mariana Saldanha Viegas, Luisa Vieira Marques, Priscila de Sousa Ferreira, Leonardo Chicaybam, Martín Hernán Bonamino. Development of CAR-T cell therapy for B-ALL using a point-of-care approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3247.

Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line.

In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia.

Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL. To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents. Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine. Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

Assessment of the Impact of Wild Stinkhorn Mushroom Extracts on Different Cancer Cell Proliferation and Study of Primary Metabolites

Objective: Present study aims to evaluate the efficacy of methanolic and ethyl acetate extracts of wild mushroom Phallus sp. on cell proliferation of both normal and cancer cells. This study also looked at anti-oxidant potentiality of methanolic extract and also unravels the phytochemical profiling of both extracts. Methods: Anti-proliferative activity was assessed by MTT assay on different human cancer cell lines such as MCF-7, MOLT-4, REH and Peripheral Blood Mononuclear Cells or PBMC isolated from a healthy donor. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was used for comparative assessment of phytochemical constituents of both extracts. The anti-oxidant profile of methanolic extract was also evaluated by DPPH and ABTS•+ assays. Results: Results indicated that the both methanolic and ethyl acetate extracts of Phallus sp. showed appreciable anti-proliferative activity against breast cancer cell line MCF-7 with IC50 of 8.544±2.812 μg/mL and 35.279±2.863 μg/mL respectively. Both of the extracts also showed its moderate impact on human B cell precursor leukemia cell line (REH) with IC50 of 25.987±2.696 μg/mL for methanol and 51.484±1.480 μg/mL for ethyl acetate extract respectively. No effect was observed in MOLT-4 cell line. Methanolic extract was selected as better anti cancer extract over ethyl acetate extract. No significant anti-proliferative activity was observed in normal PBMC by both extracts. GC-MS analysis indicated that 43 and 114 compounds were identified from methanolic and ethyl acetate extracts respectively. Among them nine compounds shared its existence in both of the extracts. Different derivatives of ergosterol and several fatty acid esters ware identified as major components from both of the extracts. Methanolic extracts of the Phallus sp. showed its effectiveness on both of DPPH and ABTS•+ free radical, and result indicated that it contain more flavonoid content than phenol. Conclusion: The methanolic extract of Phallus sp. show very specific anti-proliferative effect on MCF-7 with moderate anti-oxidant activity and holds a great promise for isolation of bio molecules for treating Breast Cancer. Several derivatives of ergosterol identified as probable anti-cancer compound.

Assessment of Serum IL-15 in Adult Acute Leukemia Patients

Abstract Background Interleukin 15 (IL-15), a proinflammatory cytokine, regulates the functions of the immune system and controls the differentiation of hematopoietic cells. However, it may also promote the development of leukemia through its role in enhancing the survival, proliferation and differentiation of leukemic precursors. Aim of the work To assess expression of IL-15 level in adult Egyptian acute leukemia patients, its correlation with disease free survival, overall survival and relapse rate, as well as its possible correlation with other prognostic parameters. Patients and methods Serum IL-15 level was measured using enzyme-linked immunosorbent assay in 30 newly diagnosed acute lymphoblastic leukemia (ALL) patients, 30 newly diagnosed acute myeloid leukemia (AML) patients and 30 age and sex-matched healthy control subjects. The patients were recruited from clinical hematology department at Ain shams university hospital over the period from April 2017 to October 2018. Results In ALL patients: The serum IL-15 level was higher in ALL patients compared to control subjects (P = 0.015). Patients with ALL with high serum IL 15 level had shorter survival compared to those with low or average serum IL 15 level (P = 0.005). In AML patients: The serum IL-15 level was higher in AML patients compared to control subjects (P = 0.010). Patients with AML with high serum IL 15 level had shorter survival compared to those with low or average serum IL 15 level (P &amp;lt; 0.001). Serum IL15 level has negative correlation with the date of death in patients with AML (P = 0.001). Conclusion Interleukin 15 (IL-15) is a useful poor prognostic marker in patients with newly diagnosed acute leukemia, also it can be used as a predictor for survival. Abbreviations AML (acute myeloid leukemia), ALL (acute lymphoblastic leukemia), IL-15 (Interleukin 15).

Differential Depletion of Bone Marrow Resident B-ALL after Systemic Administration of Endosomal TLR Agonists.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. While frontline chemotherapy regimens are generally very effective, the prognosis for patients whose leukemia returns remains poor. The presence of measurable residual disease (MRD) in bone marrow at the completion of induction therapy is the strongest predictor of relapse, suggesting that strategies to eliminate the residual leukemic blasts from this niche could reduce the incidence of recurrence. We have previously reported that toll-like receptor (TLR) agonists achieve durable T cell-mediated protection in transplantable cell line-based models of B cell precursor leukemia (B-ALL). However, the successful application of TLR agonist therapy in an MRD setting would require the induction of anti-leukemic immune activity specifically in the bone marrow, a site of the chemotherapy-resistant leukemic blasts. In this study, we compare the organ-specific depletion of human and mouse primary B-ALL cells after systemic administration of endosomal TLR agonists. Despite comparable splenic responses, only the TLR9 agonist induced strong innate immune responses in the bone marrow and achieved a near-complete elimination of B-ALL cells. This pattern of response was associated with the most significantly prolonged disease-free survival. Overall, our findings identify innate immune activity in the bone marrow that is associated with durable TLR-induced protection against B-ALL outgrowth.

Assessment of serum interleukin-15 in adult acute leukemia patients (Egyptian sample)

Background Interleukin 15 (IL-15), proinflammatory cytokine, regulates immune system functions and controls hematopoietic cell differentiation. It promotes leukemia development through enhancing survival, proliferation, and differentiation of leukemic precursors. Aim The aim was to assess the expression of IL-15 level in adult Egyptian acute leukemia patients, its correlation with disease-free survival, overall survival and relapse rate, and its possible correlation with other prognostic parameters. Patients and methods Serum IL-15 was measured using ELISA in 30 newly diagnosed acute lymphoblastic leukemia (ALL) patients, 30 newly diagnosed acute myeloid leukemia (AML) patients, and 30 healthy controls recruited from Ain Shams University Hospital from 2017 to 2018. Results In ALL patients IL-15 was higher in patients compared with the control. IL-15 was higher in patients with non-high-risk cytogenetics compared with those with high-risk cytogenetics. Patients with high IL-15 who achieved first complete response (CR) were less than those with low/average IL-15. Patients with high IL-15 who achieved minimal residual disease (MRD) negativity were less than those with low/average IL-15. Patients with high IL-15 had shorter survival compared with those with low/average IL-15 level. Optimal cutoff value for IL-15 in predicting patient survival in ALL patients was 200 ng/l. In AML patients: IL-15 was higher in patients compared with the control. IL-15 was higher in patients with high-risk features compared with those without. Patients with high IL-15 achieved first CR less than those with average IL-15. Patients with high IL-15 achieved MRD negativity less than those with average IL-15. Patients with high IL-15 had shorter survival compared with those with low/average IL-15. Optimal cutoff value for IL-15 in predicting patient survival in AML patients was 190 ng/l. IL-15 has negative correlation with death date in AML patients. Conclusion IL-15 is a useful poor prognostic marker in newly diagnosed acute leukemia patients’ also it can be used as a predictor for CR, MRD, and survival.

A case of an unusual lineage switch in late relapse ALL—is it actually a secondary leukemia?

Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid precursors. According to immunophenotype, it is further subdivided into precursor B cell ALL and precursor T cell ALL, with precursor B cell ALL being much more common both in children and adults. Lineage switch from one lymphoid lineage to another during the course of the disease is extremely rarely reported. Here, we describe a case of a child who initially presented as a precursor B-ALL but 15 years later and after two successfully treated relapses of the original ALL presented with early T cell precursor leukemia. Although it was considered as a relapse, it could be interpreted as a case of secondary leukemia, which can be explained as a consequence of treatment as well as a constitutional feature of an individual. Also, it draws attention to the possibility that hematopoietic cells, and in that context also leukemic cells, are much more plastic and capable of reprogramming than previously thought.

Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (&lt;20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (&gt;80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment. Figure Disclosures No relevant conflicts of interest to declare.

Contribution of Myeloid-Drived Suppressor Cells to Leukemia Relapse in Patients with B-ALL Who Underwent Allo-HSCT

Background:B cell acute lymphoblastic leukemia (B-ALL) is a clonal hematopoietic stem cell neoplasm. Despite advances in its therapeutic strategies, including induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT), relapse remains the major barrier to the improvement of survival. Unfortunately, the underlying mechanism of relapse in B-ALL is largely unknown. Myeloid-derived suppressor cell (MDSC), a relatively novel population of immunomodulatory cells, has been identified to be associated with the onset of solid tumor and poor prognosis. However, the role of MDSCs played in prompting B-ALL relapse is vague. Aims:The present study was performed to explore the association between the percentage of MDSCs in bone marrow (BM) and B-ALL relapse and to investigate the effects of MDSCs on relapse of B-ALL. Methods:Firstly, twenty relapsed B-ALL patients and 10 non-relapsed patients after allo-HSCT as well as 14 healthy donors were enrolled. The percentage of CD33+HLA-DR-CD11b+ MDSCs in BM was analyzed by flow cytometry (FCM). Secondly, we co-cultured GFP+ Nalm-6 cells (a B cell precursor leukemia cell line initiated from an adolescent male) with MDSCs depleted bone marrow mononuclear cells (BMMCs) or replete of MDSCs from relapsed B-ALL patients post-transplantation. Then, we tested the direct effects of MDSCs on leukemia cells proliferation by co-culturing Nalm-6 cells with MDSCs. In addition, the effects of MDSCs on the functions of T cells and natural killer (NK) cells were also investigated. Briefly, CD3+ T cells or CD3-CD56+ NK cells purified from BMMCs via FCM were co-cultured with the same donor's CD11b+HLA-DR-CD33+ MDSCs using anti-CD3/CD28 beads, PMA or K562 cells as activators. The functional indexes of T cells or NK cells, including proliferation, IFN-γ, CD107a, Granzyme B, and perforin were detected by FCM. Furthermore, we analyzed the expression levels of immune response regulating proteins, including Foxp3 and PD-1 in T cells co-cultured with MDSCs. Finally, we detected the cytokines regulating MDSCs proliferation and activation in the sera of BM from relapsed B-ALL patients after allo-HSCT . Results:We demonstrated an increased percentage of MDSCs in BM of relapsed patients compared with paired non-relapsed cases (n=10 vs. 10, 28.76% vs. 5.18%, P=0.0068) and healthy donors (n=20 vs. 14, 22.76% vs. 6.57%, P=0.0007). In vitro experiments showed that the MDSCs sorted from relapsed patients' BM enhanced the proliferation of tumor cells (n=3, the mean number of GFP+Nalm-6 cells in co-culture with MDSCs group 2.2×104, vs. without MDSCs group 1.2×104, P=0.0447) in a BMMCs condition rather than a co-culture of tumor cells with MDSCs directly. MDSCs obtained from relapsed B-ALL patients' BM could suppress T cell proliferation and IFN-γ production in a cell dose-dependent manner. The percentages of proliferated CD4+T cells were 89.5%, 61.2%, 49.7%, 7.1% at a ratio of T: MDSC 1:0, 1:0.5, 1:1, 1:2 respectively (n=3, P&lt;0.05); The percentages of proliferated CD8+T cells were 74.7%, 57.6%, 37.8%, 10.6% at a ratio of T: MDSC 1:0, 1:0.5, 1:1, 1:2 respectively (n=3, P&lt;0.05). MDSCs from relapsed patients suppressed the cytotoxicity of NK cells. The percentages of IFN-γ+,CD107a+ andGranzyme B+ cells among NK cells were significantly lower in co-culture with MDSCs group than in without MDSCs group (n=4, IFN-γ+ cells, mean 0.6 fold, P=0.0237; CD107a+ cells, mean 0.8 fold, P=0.0242; Granzyme B+ cells, mean 0.8 fold, P=0.0326). We revealed that MDSCs sorted from relapsed patients' BMs induced PD-1 expression on T cells. The percentages of PD-1+ cells among CD4+ cells and CD8+ cells were both significantly higher in co-culture with MDSCs group than in without MDSCs group (n=6, among CD4+ cells, mean 1.8 fold, P=0.0342; among CD8+ cells, mean 1.4 fold, P=0.0041), which may contribute to the dysfunction of T cells. In addition, we found that the mean levels (ng/L) of IL-17A, IL-21, and IL-22 were significantly higher in the sera of BM from relapsed patients than in non-relapsed patients (n=5 vs. 4, IL-17A, 46.9 vs. 6.1, P=0.0077; IL-21, 31.8 vs. 5.6, P=0.0078;IL-22, 132.7 vs. 20.7, P=0.0050). Conclusions:Our results suggest the increased MDSCs might contribute to the relapse of B-ALL via promoting leukemia proliferation, suppressing the function of T cells and NK cells. Our findings suggest that MDSC could be served as a potential therapeutic target for this disease. Disclosures No relevant conflicts of interest to declare.

Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Coagulation FXIII-A Protein Expression Defines Three Novel Sub-populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized by Distinct Gene Expression Signatures.

Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the “B-other” genetic category and had an unfavorable disease outcome.Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR.Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of “B-other” samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the “B-other” subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL.Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

The source of HLA molecules on platelets: Does platelets adsorb soluble HLA molecules from their environment?

Background: The origin and function of human leukocyte antigen (HLA) class I molecules on platelets are still highly arguable. Given the differences in the results of the previous studies in this regard, the lack of research in recent years, and the clinical importance of HLA class I molecules, the absorption capacity of platelets for soluble HLA class I molecules was studied in this investigation. Materials and Methods: In this experimental study, HLA-A2 antigen was purified from a B cell precursor leukemia cell line (Nalm-6) by cell membrane protein solubilization and usage of HLA-A2 affinity column. Platelet concentrates (PCs) were received from Tehran Blood Transfusion Center. Eighteen bags of HLA-A2-negative PCs were prepared randomly and treated with various concentrations of the purified HLA antigen (100, 500, and 1000 ng/ml) for 48 to 72 hours. Subsequently, the HLA-A2 levels were evaluated on platelets by flow cytometery technique. Data were evaluated using repeated measure ANOVA.P-values less than 0.05 were considered significant. Results: The results of this study showed that the purified protein was an HLA molecule (HLA-A2). After the treatment of platelets and HLA molecules, platelets inability was shown for the attracting of HLA molecules. This finding was true in both media of RPMI and plasma. The differences between the case (HLA-treated platelets) and control (untreated platelets) were not significant (p-values&gt; 0.05). Conclusion: Platelets were unable to significantly adsorb exogenous HLA antigens from their environment. Further studies are needed to unravel the nature and origin of HLA molecules on platelets.

Decitabine-containing G-CSF priming regimen overcomes resistance of primary mediastinal neoplasm from early T-cell precursors to conventional chemotherapy: a case report.

Early T-cell precursor (ETP) leukemia represents a new subtype of T-lymphoblastic leukemia/lymphoma with unique immunophenotypes expressing T-cell and one or more of the myeloid/stem cell markers. Here, we report a young patient who had primary mediastinal mass and pleural effusion without bone marrow involvement. A CT-guided mediastinal biopsy and flow cytometry analysis of the pleural effusion revealed the blast cells to have complicated immunophenotypes: strongly expressed T-cell antigen CD7, myeloid-lineage antigens CD33 and CD13 and stem cell markers cTdT, CD34, and HLA-DR; dimly expressed myeloid-lineage specific antigen cMPO and B-cell antigen cCD79a; but did not express T-cell specific antigen cytoplasmic CD3 and B-cell specific antigen CD19. Clonal T-cell receptor rearrangement eventually determined the cell of origin from ETPs, not myeloblasts. The patient showed primary resistance to lymphoid and myeloid-directed induction therapy. Finally, low-dose decitabine combined with modified-CAG regimen induced a complete remission and allogeneic stem cell transplantation was performed as consolidation. The case indicates a primary mediastinal neoplasm from ETP with distinctive immunophenotype from leukemia type. Low-dose decitabine and modified-CAG regimen in combination with allogeneic stem cell transplantation may improve the outcome of patient.

Outcome of 126 adolescent and adult T-cell acute leukemia/lymphoma patients and the prognostic significance of early T-cell precursor leukemia subtype

目的分析我国急性T淋巴细胞白血病/淋巴瘤（T-ALL/LBL）患者的临床特征，探究急性早期前体T淋巴细胞白血病/淋巴瘤（ETP-ALL/LBL）分型的预后意义。方法回顾性分析2008年1月至2014年12月间在四川大学华西医院就诊的126例T-ALL/LBL患者临床资料，基于白血病细胞的免疫表型将其分为三组：ETP-ALL/LBL（CD1a−，CD8−，CD5−/dim以及一种或多种干细胞和髓系相关抗原表达）、近似ETP-ALL/LBL（除CD5+外其他同ETP-ALL表型特征）及非ETP-ALL/LBL（non-ETP-ALL/LBL）组，对患者的实验室指标及预后相关因素进行分析。结果126例T-ALL/LBL患者中男女比例为2.5∶1，中位年龄为25（14~77）岁，ETP-ALL/LBL亚型的比例高达47.6%。T-ALL患者首次化疗完全缓解（CR1）率显著高于T-LBL患者（64.4%对30.8%，P=0.032）；初诊外周血WBC>50×109/L的患者CR1率显著高于WBC ≤ 50×109/L的患者（78.4%对50.9%，P=0.010）。相较于non-ETP-ALL/LBL组，ETP-ALL/LBL组患者发病年龄更大（P<0.001）、外周血WBC更低（P<0.001）、病程中中枢神经系统浸润率更低（10.0%对30.2%，P=0.009）。ETP-ALL/LBL组患者CR1率显著低于non-ETP-ALL/LBL组（37.3%对84.6%，P<0.001）；ETP-ALL/LBL组患者较non-ETP-ALL/LBL组患者总生存期短，但差异无统计学意义（P=0.073）。T系抗原CD1a+组、CD8+组、CD4+组的CR1率均比相应的阴性组高（P值分别为0.002、0.000、0.001），而髓系抗原CD33+组、CD56+组CR1率均比相应的阴性组低（P值分别为0.035、0.035）。结论中国青少年和成人T-ALL患者中ETP-ALL/LBL亚型比例高，ETP-ALL/LBL属于成人ALL的高危亚型，需要更精确的诊断及新的治疗策略来改善预后。

Synthesis of New N1Arylpiperazine Substituted Xanthine Derivatives and Evaluation of their Antioxidant and Cytotoxic Effects.

Cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases in 2012, with most of the clinically used drugs being ineffective. Methylxanthines have raised more interest in research on modifying their structure because of their diverse biological activity. In addition, the piperazine nucleus is one of the most important heterocycles exhibiting remarkable pharmacological activities. The structure of the obtained compounds was characterized and elucidated by IR, 1H and 13C NMR and LCMS spectral analysis. The purity of the substances was proven by corresponding melting points and elemental analysis. The antioxidant activity was evaluated by four common methods - DPPH, ABTS, FRAP and lipid peroxidation assay. The cytotoxic effects of the tested series were evaluated using the standard MTT-dye reduction assay on three tumour cell lines. A series of new xanthine derivatives comprising an arylpiperazine moiety at N1 were synthesized. The cytotoxicity against human T-cell leukemia cell SKW-3, human acute myeloid leukemia HL-60 and human Bcell precursor leukemia cell REH was evaluated. The relationship between the structure and citotoxicity of the compounds was investigated by quantitative structure-activity relationship (QSAR) analysis and the important structural parameters were drawn. The highest antioxidant activity was demonstrated by compound 6c. The highest cytotoxic effect was observed for compound 6f. It was found that cytotoxicity against SKW-3 depends on the electron density distribution in the structures. Branching of the molecular skeleton and introduction of heteroatoms like fluorine and sulfur in the structures also significantly improved the antiproliferative activity of the compounds.