Lineage Acute Lymphoblastic Leukemia Research Articles

WHO-2016 Classification in ALL By Cytogenetics, FISH and Molecular Biology - Experience of Two Reference Centers in Brazil

IntroductionAlmost all neoplasms have their prognosis determined by cytogenetic and molecular factors, which determines a tailored approach for each case. According to the World Health Organization, every case of Acute Lymphoblastic Leukemia (ALL) must be classified not only immunophenotypically but also from a genetic point of view. Over the last few years, it has been recognized the differences in incidence and genetic profile of ALL from Latin America (LA). In Brazil, a large country with a distinctive ethnical background when compared to the rest of LA, few reports on ALL have been published and there is a limited availability of genetic tests for classification, especially in public health service.ObjectiveTo describe cytogenetic (karyotype and fluorescence in situ hybridization, FISH), molecular markers and World Health Organization (WHO) -2016 classification of diagnostic samples sent to two reference centers in Brazil.Material and MethodsThis is a cross-sectional study involving patients diagnosed with ALL from January 2014 to May 2018. Diagnostic bone marrow specimens were analyzed by G band conventional karyotyping (Nomenclature according International System for Human Cytogenomic 2016) (n: 157), molecular markers [(genic fusions by RT-PCR - BCR-ABL1 (n: 96), TCF3-PBX1 (n: 77), ETV6-RUNX1 (n: 83), KMT2A-AFF (n:74)] and B-ALL multi-probe FISH (n:54) were employed according to manufacturer's recommendations and following international guidelines. It was attempted to classify all cases by WHO-2016, as well as by age. Samples with no metaphases, non-conclusive or without molecular tests were considered non-classifiableResultsSamples from 157 ALL patients at diagnosis were analyzed. Median age was 8 years old (1 month - 88 years) [6 (3.8%) cases had less than 12 months; 99 (63%) between 1 to 15 years; 22 (14%) between 16 and 39 years and 30 (19.1%) older than 40 years]. The majority were B cell lineage ALL (93%). Thirty cases were excluded due to missing immunophenotyping, karyotype and/or molecular studies. Alterations were detected in 94 (74%) of 127 cases [88 (76%)/116 in B-ALL and 6 (54%)/11 in T-ALL. It was possible to classify by WHO 2016 B-ALL subtypes 116 cases, all of them stratified by age groups (Graphic 1).ConclusionOur results are consistent with the literature, showing that KMT2A rearrangement predominates in infant ALL, while a pediatric age range from 1 to 15 years old, with a predominance of good prognostic subtypes: hyperdiploidy and ETV6-RUNX1. In adults, there is predominance of the BCR-ABL1 subtype, which seems to be more frequent than non-Latin cohorts. B-ALL NOS was detected in 27.8% of the cases, predominating in the age group of 16 to 39 years, which may correspond to Ph-like ALL, a recently described subtype linked to a worse prognosis and seemingly more common in latinos. DisclosuresNo relevant conflicts of interest to declare.

Excellent Response to Blinatumomab in Children and Young Adults with Refractory B Lineage Acute Lymphoblastic Leukaemia for Persistent MRD or after Debulking Chemotherapy for Higher Disease Burden

Introduction: A recent phase I/II study of blinatumomab in children with relapsed/refractory B cell acute lymphoblastic leukaemia (B-ALL), found 39-51% achieved complete, often MRD negative, remission after two cycles of therapy. Higher responses were observed in patients with less than 50% bone marrow blasts, compared to greater than 50% (55.6% vs 32.7% (95% CI 30.8-78.5 and 20.3-47.1 respectively)1. Furthermore, an adult study showed complete minimal residual disease (MRD) response rates of 78% when blinatumomab was used to treat MRD-positive ALL in haematological remission 2. Hence response rates (and toxicity) to blinatumomab are highly correlated with pre-treatment disease burden. We present preliminary data showing an excellent response to blinatumomab in children and young adults with resistant B-ALL who had persistent MRD, or after debulking chemotherapy to achieve a partial remission.Methods: Eleven patients were identified through a national survey. The mean age of patients was 10 years (range 0.7-22 years, 3 infants and 1 Down syndrome ALL). All patients had B-lineage ALL which was CD19 positive. None had active CNS disease at the point of receiving blinatumomab. Prior to administration of blinatumomab, all patients either had persistent MRD following several courses of intensive chemotherapy or received debulking chemotherapy for heavier marrow infiltrates. Pre-blinatumomab chemotherapy to which the patients had failed to obtain an adequate MRD response included UKALL 2011 Regimens A or C, UKALL R3, Interfant 06 or NOPHO high risk blocks. Patients received 5-15 µg/m2 of blinatumomab for 1-2 cycles prior to definitive therapy.Results: Pre-blinatumomab, all patients except one were in morphological remission with MRD measurable by PCR (0.003-1%), the remaining patient had 9% marrow disease by morphology. After 1-2 cycles of blinatumomab all patients had negative MRD when measured by flow cytometry and/or by PCR, giving a 100% response rate. This was followed by Haemopoietic stem cell transplant (HSCT) in nine patients and the remaining two are awaiting transplant. Further data on patient characteristics, CNS status, relapse and survival outcome are being collected and will be presented at the meeting.Minimal toxicity was observed; of the seven patients in whom toxicity data were available, three had grade 1 CRS, which resolved spontaneously without interruption of therapy or treatment with corticosteroids or Tocilizumab. One patient reported grade 1 neurotoxicity.This preliminary UK experience demonstrates that excellent MRD response is observed with minimal toxicity in children and young adults who receive blinatumomab for persistent MRD or after debulking chemotherapy. This provided a bridge to transplant in patients who would otherwise not have benefited from the procedure because of persistent MRD. We are planning to extend these observations by undertaking a study of this strategy in first high-risk B-ALL relapse.Stackelberg Av, Locatelli F, Zugmaier G, et al. Phase I/Phase II Study of Blinatumomab in Pediatric Patients With Relapsed/Refractory Acute Lymphoblastic Leukemia. Journal of Clinical Oncology. 2016;34(36):4381-4389.Gokbuget N, Dombret H, Bonifacio M, et al. Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia. Blood. 2018;131(14):1522-1531. DisclosuresGhorashian:Celgene: Other: travel support; Novartis: Honoraria. Marks:Novartis: Consultancy; Pfizer: Consultancy; Amgen: Consultancy. Vora:Amgen: Other: Advisory board; Novartis: Other: Advisory board; Jazz: Other: Advisory board; Medac: Other: Advisory board; Pfizer: Other: Advisory board.

Title Point-of-Care Production of CD19 CAR-T Cells in an Automated Closed-System: Report of First Clinical Experience

Introduction CD19 CAR-T cell products were recently approved as therapy for advanced B-cell lineage malignancies. The predominant manufacturing model for this type of therapy is a centralized industrial-type production process. An attractive model of CAR-T cell production and delivery to the patient is via a point-of care-manufacturing process. We report on first cases of implementation of this approach in a setting of compassionate use program.Patients and methods Four patients with relapsed/refractory B-cell lineage malignancies were eligible for compassionate use of CD19 CAR-T cell therapy. Three patients, median age 15 years, had relapsed B-cell lineage acute lymphoblastic leukemia (B-ALL) after haploidentical hematopoietic stem cell transplantation (HSCT). Bone marrow disease burden at therapy start was 12%, 74% and 0,159. The patient with low minimal residual disease (MRD) in the bone marrow had skeletal involvement with multiple lymphomatous mass lesions. One patient had refractory primary mediastinal B-cell lymphoma (PMBCL). The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells from patient-derived leukapheresis product. Automatic production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct, transduction with lentiviral (second generation CD19.4-1BB zeta) vector (Lentigen, Miltenyi Biotec company) and expansion over 12 days in the presence of TexMACS GMP Medium supplemented with MACS GMP IL-7/IL-15 combination. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab 1 hour before CAR-T cell infusion at 8mg/kg.Results All production cycles were successful. Median transduction efficacy achieved was 57%(52-63). Median expansion of T cells was x26(24-43). CD4/CD8 ratio in the final product was 0,750,22-6). All final products passed the in-process and quality controls. The cell products were administered at a dose of 1*106/kg of CAR-T cells. No immediate infusion reactions were reported. In 2 cases mild cytokine release syndrome (CRS) was diagnosed. In one case mild CAR-T cell related encephalopathy developed. In one case additional dose of tocilizumab and one dose of dexamethasone were administered to control CRS and encephalopathy. All patients survived to the point of response evaluation. In 3 cases CAR-T cell expansion was detected to a maximum 25%. MRD-negative responses detected by flow cytometry and PCR were achieved in 2 cases with bone marrow involvement. In two cases with prominent mass lesions an objective response was documented. In the patient with 74% blast in bone marrow at start of therapy, neither CAR-T cell expansion nor leukemia response were documented. Details of therapy and response are summarized in table 1.Conclusion Production of CAR-T cells with the CliniMACS Prodigy TCT process is a feasible and an attractive option that provides a point-of-care manufacturing approach to enable rapid delivery of CAR-T cells to patients in need. Robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy. DisclosuresDropulic:Lentigen, A Miltenyi Biotec company: Employment. Shneider:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen, A Miltenyi Biotec company: Employment. Alex:Miltenyi Biotec: Employment. Essl:Miltenyi Biotec: Employment.

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

Tisagenlecleucel-T for the treatment of acute lymphocytic leukemia

ABSTRACTIntroduction: Cellular immunotherapy with autologous or allogeneic T cells, genetically engineered to express chimeric antigen receptors (CARs) or T-cell receptors, in order to redirect their cytotoxic specificity toward malignant cells, is emerging as a promising new treatment modality. The most advanced approach in clinical development is the use of anti-CD19 CAR T-cells for the treatment of CD19+ B-cell malignancies, including acute lymphocytic leukemia (ALL).Areas covered: Recently, the Food and Drug Administration (FDA) approved the first anti-CD19 CAR T-cell product, tisagenlecleucel, for the treatment of pediatric and young adult patients with relapsed/refractory ALL. In this overview, we described the advances in the field, including a summary of clinical trials with tisagenlecleucel in ALL published to date.Expert opinion: CAR T-cell therapy has been developed in the context of small clinical studies and very few centers have had to deal with the challenges of managing CAR T-cells administration. However, this approach is likely to become a standard option for patients with relapsed/refractory B-cell lineage ALL.

Abstract 4129: RUNX2 as a regulator of high-risk T-ALL

Abstract For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is <10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p <0.0005) and those who failed induction therapy (p <0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p < 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p < 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p < 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p <0.001) and bone marrow (p <0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival. Citation Format: Nitesh Devinarayan Sharma, Christian K. Nickl, Stuart S. Winter, Huining Kang, Ksenia Matlawska Wasowska. RUNX2 as a regulator of high-risk T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4129.

Issue Highlights-May 2018 (94B3).

Issue Highlights-May 2018 (94B3).

Ikaros regulates epigenetic and transcriptional programs in progenitor B cell leukemia

Abstract Hematopoietic cell development is tightly controlled by a network of transcriptional and epigenetic regulators, many of which are mutated or have altered expression in hematological malignancies. Ikaros is a transcription factor that is critical for the proper development of several hematopoietic lineages, and essential for the B-cell lineage. It is recognized as a critical tumor suppressor in precursor B-cell lineage acute lymphoblastic leukemia (pre-B ALL), and is emerging to play roles also in other hematopoietic malignancies. In pre-B ALL, Ikaros mutations are particularly prevalent in the Ph+ (BCR-ABL1+) and ‘Ph-like’ subgroups of leukemia, and Ikaros mutations correlate with poor prognosis. To study the mechanisms of Ikaros tumor suppressor function, we developed a mouse model and a human model system with selective perturbation of Ikaros in Ph+ pre-B ALL cells. This has revealed conserved deregulated genes and pathways, and underscored the role of Ikaros in regulating progenitor-restricted gene programs. Furthermore, our recent studies have highlighted the role of Ikaros in regulation of chromatin structure, and revealed a novel role in epigenetic regulation. It is challenging to therapeutically target mutations in a tumor suppressor factor. It is therefore important to understand the mechanism of action and downstream targets to elucidate targetable vulnerabilities of the Ikaros-mutant leukemic cells. Investigating the tumor suppressor role of Ikaros in developing B cells also sheds light on its role in normal B cell development. We are using our newly established models of Ikaros-mutated pre-B ALL to study the underlying molecular mechanisms and downstream targets, and will present our results to-date at the meeting.

Overexpression of dihydrofolate reductase is a factor of poor survival in acute lymphoblastic leukemia.

Dihydrofolate reductase (DHFR) has an important function in DNA synthesis and is a target of methotrexate, which is a crucial treatment option for acute lymphoblastic leukemia (ALL). However, the number of studies conducted to date on DHFR expression in childhood ALL is limited. The aim of the present study was to determine whether the expression of DHFR is associated with survival in childhood ALL. The expression of DHFR in 96 children with ALL and 100 control individuals was determined using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the expression of DHFR mRNA in children with ALL was significantly increased (P<0.001), compared with that in the control group. In addition, increased levels of DHFR mRNA were observed in patients with B-cell lineage, compared with patients with T-cell lineage ALL (P<0.05). The Kaplan-Meier estimator analysis revealed that children with ALL who exhibited increased levels of DHFR mRNA had a decreased overall survival time (P<0.05). It was observed that certain patient prognostic features (including age, sex, white blood cell count and high DHFR expression), are associated with poor survival (log-rank test, P<0.05). Therefore, the results of the present study indicated that DHFR upregulation is a factor for poor survival in ALL.

No prognostic significance of immunophenotypic changes at the end of remission induction therapy in children with B-lineage acute lymphoblastic leukemia

Detection of aberrant antigen expression in acute lymphoblastic leukemia (ALL) by flow cytometric is proposed for the quantification of minimal residual disease (MRD). There are few studies that investigate the stability of the antigen expression in children with B lineage ALL at the end of remission induction therapy and determine its prognostic impact. Between 2010 and 2015, 691 bone marrow specimens of childhood ALL were sent at diagnosis for immunophenotypic characterization, and follow-up samples for MRD were analyzed on day 33. Of these, 155 patients with MRD more than or equal to 0.01% were eligible for the study. Immunophenotypic studies were performed by multiparametric flow cytometry using four-colour monoclonal antibody combinations. Overall, 86 of 155 (55.5%) cases showed phenotype shifts at least one marker. CD19 was the most stable markers. By contrast, CD20 was significantly different between diagnosis and day 33 in nearly one third of the cases. Shifts of antigen expression was not significantly associated with EFS, RFS or OS (P > 0.05). Multivariate analysis showed that WBC and BCR-ABL have independent prognostic value in childhood ALL. Changes in antigen expressions were commonly occurred at the end of induction and not associated with prognostic value in patients whose MRD were positive on day 33.

Cyclin dependent kinase inhibitor 2A/B gene deletions are markers of poor prognosis in Indian children with acute lymphoblastic leukemia.

Cyclin dependent kinase inhibitor 2A/B (CDKN2A/B) genes are implicated in many malignancies including acute lymphoblastic leukemia (ALL). These tumor suppressor genes, with a key regulatory role in cell cycle are located on chromosome 9p21.3. Previous studies involving CDKN2A/B gene deletions have shown mixed associations with survival outcome in childhood ALL. Hundred and four newly diagnosed children with ALL (1-14 years) were enrolled in this study. Genomic DNA from pretreatment bone marrow/peripheral blood samples of these children was investigated for copy number alterations in CDKN2A/B genes using multiplex ligation dependent probe amplification assay. Immunophenotype subtyping and cytogenetic and molecular analysis of ALL was performed at start of induction chemotherapy in all children. Children were monitored for response to prednisolone (Day 8), complete morphological remission, and minimal residual disease at the end of induction. The minimum postinduction follow-up period was 6 months. CDKN2A/B deletions were seen in 19.8% (18/91) of B lineage acute lymphoblastic leukemia (B-ALL) and 38.5% (5/13) of T lineage acute lymphoblastic leukemia (T-ALL). Monoallelic CDKN2A/B deletions were found in 61.1% of total deletions in B-ALL while all the children with T-ALL harbored biallelic deletions. The prevalence of CDKN2A/B gene deletions was found to be significantly higher in older children (P=0.002), in those with higher leukocyte count (P=0.037), and in National Cancer Institute high risk group patients (P=0.001) in the B-ALL subgroup. Hazard ratio was significantly high for children with CDKN2A/B deletions in total cohort (P=0.004). Children with CDKN2A/B deletion had significantly lesser event free survival (P=0.03). CDKN2A/B deletions were significantly more prevalent in T-ALL subgroup and were found to have higher hazard ratio and lesser event free survival in total cohort in our study.

P027 DIFFERENTIAL BINDING OF RBPJ AND CUX1 TO IBD CAUSAL SNP RS1887428 MODIFIES EXPRESSION OF JAK2

Recently, genetic variants conferring risk for IBD were fine-mapped to single variant resolution. Eight of these causal variants resulted in amino acid substitutions while ten were non-coding. The mechanism by which these non-coding causal variants confer disease risk is unknown, though presumably they influence gene expression by differential binding of transcription factors. SNP rs1887428 is located in the promoter region of Janus kinase 2 (JAK2), an enzyme that interacts with STAT family transcription factors and cytokine receptors to regulate hematopoiesis and the immune response. We constructed oligodeoxynucleotide baits containing risk or protective alleles of rs1887428 and the surrounding sequence. A nuclear extract was made from the T cell acute lymphoblastic leukemia line Jurkat. The baits were used in affinity purification of DNA-binding transcription factors from the extract and the recovered proteins were identified by mass spectrometry. In vitro assays using affinity purification, electrophoresis mobility shift (EMSA), and dual luciferase were used to confirm the allelic preference of the identified proteins. We found that transcription factor RBPJ, the downstream effector of the Notch signaling pathway, binds preferentially to the risk allele. The homeodomain transcriptional repressor CUX1 binds exclusively to the protective allele, which was confirmed by EMSA. Co-expression of the intracellular domain of NOTCH1, partner of RBPJ, increased expression of the JAK luciferase reporter while co-expression of CUX1 decreased expression. Conversely, knock-down of CUX1 with siRNA increased expression. We have shown that a causal SNP in the promoter of JAK2 is regulated by transcriptional activators and repressors. These results suggest that the mechanism of IBD causal SNPs may not be reducible to a canonical binding site but may involve cooperative and competitive interactions of multiple nuclear proteins.

Abstract 3874: Epigenetic deregulation of SOCS5 expression in T cell lineage acute lymphoblastic leukemia

Abstract For children, adolescents and young adults with T-lineage acute lymphoblastic leukemia, event free survival following relapse is &amp;lt;10%. A variety of hematologic malignancies activate JAK-STAT signaling through activating mutations in JAK1-3, IL7R and downregulation of negative regulators. SOCS5 belongs to the suppressor of the cytokine signaling (SOCS) family, which are known cytokine-inducible negative regulators of JAK-STAT and other signaling pathways. SOCS5 is involved in negative regulation of Th2 development. However, the roles of SOCS5 in blood cancer, in particular T-ALL have not been elucidated so far. Using Affymetrix U133 Plus 2.0 microarray we assessed SOCS5 expression levels in a cohort of 100 T-ALL samples derived from AALL0434 COG study. SOCS5 was deregulated in T-ALL cells, and its expression was lowered in cases harboring MLL gene rearrangements (MLL-R) compared to the remaining cases (P &amp;lt; 0.0001). Because we did not identify any mutations in SOCS5 gene in the cohort of 100 cases, we hypothesized that SOCS5 expression is regulated by aberrant DNA methylation in T-ALL. Using methyl-specific PCR (MS-PCR) and bisulfite sequencing we found that SOCS5 expression is regulated by promoter/1st exon CpG island methylation in T-ALL. CCRF-CEM and ALL-SIL cells, and selected primary T-ALL samples showed hypermethylation in promoter/1st exon region corresponding to lower levels of SOCS5 expression. In contrary, Koptk1 and PF382 cells, and T-ALL samples with higher levels of SOCS5 had hypomethylated SOCS5 promoter/1st region. SOCS5 transcript and protein expression were induced by DNA demethylating agent, 5-azacitidine in ALL-SIL and CCRF-CEM cells (P &amp;lt; 0.0001). The increase in SOCS5 expression was correlated with its promoter demethylation as shown by MS-PCR. SOCS5 mRNA and protein levels were also increased in ALL-SIL cells transduced with DNMT1 shRNA compared to negative control (P &amp;lt; 0.0001) indicating that inhibition of DNA methyltransferase activity restores SOCS5 expression in T-ALL. To assess whether histone deacetylation is involved in epigenetic regulation of SOCS5 expression, we treated CCRF-CEM and ALL-SIL cells with deacetylase inhibitor Trichostatin A (TSA). Treatment with TSA restored histone acetylation and caused upregulation of SOCS5 (P &amp;lt; 0.0001). We next examined the effects of SOCS5 on leukemogenesis by shRNA-mediated knock down of SOCS5 in KoptK1 and PF382 cells. Silencing of SOCS5 increased cellular proliferation as shown by an increase in T-ALL cell number (P &amp;lt; 0.0001). Reduced expression of SOCS5 led to an increase in MYC, IL4, IL4Rα, IL7R expression and STAT6 activation. These results indicate that SOCS5 is epigenetically deregulated in T-ALL. Future studies are required to evaluate the tumor suppressor roles of SOCS5 in a subset of T-ALL, in particular, in MLL-R cases via the regulatory effect on cytokine and growth factor receptor signaling. Citation Format: Nitesh Sharma, Huining Kang, Christian C. Nickl, Scott A. Ness, Stuart S. Winter, Ksenia Matlawska-Wasowska. Epigenetic deregulation of SOCS5 expression in T cell lineage acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3874. doi:10.1158/1538-7445.AM2017-3874

Characteristics and clinical outcomes of elderly patients with acute lymphoblastic leukemia

Objective To explore the differences in clinical and laboratory parameters between elderly and non-elderly patients with acute lymphoblastic leukemia (ALL). Poor prognostic factors in elderly patients were explored to guide the individualized treatment. Methods Two hundred and seventy-nine ALL patients were divided into two groups: elderly group with their age more than 60 years (60-79 years) and non-elderly group with their age less than 60 years (14-59 years). The differences in clinical and laboratory parameters, abnormal molecular genetics on related genes, including IKZF1, PAX5, NOTCH1, PHF6, SH2B3, LEF1, and JAK1, as well as the correlations with treatment response and clinical outcome were compared between the two groups. Results Males accounted for a smaller part in elderly group [42.9% (21/49) vs. 61.7%(142/230), P=0.015]. The percentage of B cell lineage ALL (B-ALL), Philadelphia chromosome positive (Ph +) and CD33 positive rate were higher in elderly group compared with those in non-elderly group [87.8% (43/49) vs. 70.4% (162/230), P=0.009; 47.8% (22/49) vs. 27.4% (58/230), P=0.007; 56.8% (21/49) vs. 39.0% (64/230), P=0.049, respectively]. While both lymphodenopathy and total complete remission (CR) rate gained the upper hand in non-elderly group [38.9% (81/230) vs. 20.0% (9/49), P=0.016; 91.3% (178/195) vs. 68.3% (28/41), P 0.05). Conclusions Compared with non-elderly ALL patients, elderly ones harbor their intrinsic characteristics which might give rise to inferior outcomes. As a consequence, more attention should be poured into treating this particular group of ALL patients to improve their prognosis. Key words: Elderly; Leukemia, lymphoblastic, acute; Philadelphia chromosome; Mutation

Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome.

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease.

Minimal residual disease detection in Tunisian B-acute lymphoblastic leukemia based on immunoglobulin gene rearrangements.

IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.

Bone Relapse in T Lineage Acute Lymphoblastic Leukemia in a Child: A Case Report and Review of Literature

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. T-cell ALL accounts for 10% to 15% of cases. Acute lymphoblastic leukemia can rarely relapse in unusual extra medullary sites like bone. Hereby, we report a case of 7-years old male child presented with left arm swelling, initially confused with bone tumor but later found to be infiltration by leukemic blasts. We reviewed all previous cases and suggest that in a patient of ALL presenting with a bone swelling during or after completion of therapy, one should suspect of bone relapse.

Response to Prednisone Window Treatment Is a Universal Early Prognostic Marker in Chinese Children with T Lineage ALL Despite Outcome Disparity: A Multi-Center Experience

BackgroundApproximately 15% of children newly diagnosed for acute lymphoblastic leukemia (ALL) present with the T cell lineage (T-ALL), which historically has had inferior outcomes compared to the children with precursor-B cell ALL. In this study, we analyzed the efficacy of the Chinese Children Leukemia Group 2008 (CCLG-ALL 2008) protocol on pediatric T-ALL patients enrolled from 4 centers located at north, east, and southwest of China, to identify specific prognostic factors.Patients and methodsPatients enrolled on the CCLG-ALL 2008 Protocol came from four hospitals in China: two hospitals in the southwest, one in the east, and one in the north, with 33, 37, 34 and 48 patients respectively. The study was approved by the hospitals' Ethical Committee, with informed consent obtained from patients' patents or legal guardians.Definitions: Failures of relapse-free survival (RFS) included relapse and death of any cause. M1: Blast <5%, M2: 5%≤Blast ≤25%, M3: Blast >25%.ResultsOutcome disparity is observed across the four hospitals (Table 1 and Figure 1). Response to prednisone window treatment (Good vs. Poor) was highly prognostic and its prognostic value held universally across the centers despite the outcome disparity across the centers and abandonment: log-rank test stratified by centers (hospitals) P value = 0.0311, and by abandonment (Y vs. N) P = 0.0004. Multivariable Cox regression analysis showed that response to prednisone window treatment was an independent early (before end of remission induction) prognostic factor (Table 2). The hazard ratio of prednisone response (Poor vs. Good) was 2.6 (95% CI 1.49 to 4.57) after accounting for RFS disparities across centers and abandonment. When further adjusted for post-induction prognostic markers by Day-35 and Month-three bone marrow status (M1, M2 or M3), response to predisone window remained boarder-line significant (P=0.0683). Prednisone response was only significantly associated with Day-15 bone-marrow status, with poor responders tended to likely have M2 and M3 marrow status at Day 15 (Table 3). Day-15 and Day-35 marrow status was not significantly associated with RFS, but patients with Day-35 M2 and M3 status tended to fail early. Marrow status before intensification (Month 3) seemed highly associated with RFS, with all 4 M3 patients failed within 1 year.The percentage of abandonment in the southwest 1, the southwest 2, the east, and the north hospital was 21.6%, 33.3%, 8.8% and 25% respectively; the difference was not significant (P=0.1078). Nonetheless the east hospital seemed to have distinctly lower percentage of abandonment. The percentage of non-urban patients in the southwest1, the southwest2, the east and the north is 52.8%, 75.8%, 61.8% and 68.8% respectively; this difference was not significant (P=0.2171), although the percentage seemed higher in southwest. Patients from urban area tended to fair better in RFS but the difference was not statistically significant (P=0.1785). There was a weak trend that abandonment rate tended to be higher among non-urban patients, but not statistically significant (P=0.3361). Patients' Day-35 bone marrow status (with M2 and M3 marrow related to higher likelihood of abandonment), abandonment and socio-economic status might be relevant factors for the inter-center disparity in RFS. With the current data however, we are unable to identify any strong factor that can explain the inter-center disparity of treatment outcome.ConclusionResponse to prednisone window treatment is a universal early prognostic marker in Chinese children with T lineage ALL despite outcome disparity. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease Assessment of Remission after Induction Therapy Is Superior to Morphologic Assessment for Risk Stratification in Childhood Acute Lymphoblastic Leukemia: A Report from the Children's Oncology Group (COG)

▪Background: Minimal residual disease (MRD) assessment after initial therapy is integral to modern risk stratification in both precursor B and T lineage acute lymphoblastic leukemia (B-ALL and T-ALL). While MRD is used to determine depth of remission, remission is still defined, both in clinical practice and clinical trials, according to morphological assessment. We aimed to determine the outcomes of children, adolescents and young adults with discordant assessments of remission by morphology vs. by MRD, and in doing so, the extent to which morphologic assessment of remission contributes to risk assessment in this population.Methods: We identified a cohort of patients age 1-30.99 years enrolled on frontline COG trials for B-ALL [standard risk (SR): AALL0331; high risk (HR) AALL0232] and T-ALL (AALL0434) that underwent bone marrow assessment of remission at the end of induction therapy (Day 29). Morphologic response was assessed by local centers and was categorized according to traditional criteria: M1 (<5% leukemic blasts - remission) vs. M2 (5-25%) vs. M3 (>25%). MRD was measured by flow cytometry at one of two central laboratories. We determined predictors of MRD discordance and compared event free survival (EFS) between those with discordant vs. concordant morphology/MRD remission assessments.Results: Day 29 remission assessments and central MRD data were available on 9,350 patients, 7,857 (84%) with B-ALL (AALL0331: N=5049; AALL0232: N=2808) and 1,493 (16%) with T-ALL. Table 1 shows the distribution of end induction marrow morphology vs. flow cytometry results. Few patients with M2/M3 marrows had discordant low MRD values. For example, of 84 patients with M3 morphology, only 2 (2.4%) had MRD <5%. Of 202 patients with M2/M3 morphology, 23 (11.4%) had MRD<1% and 9 (4.5%) had MRD<0.1%. Subsequent analyses of discordance were thus restricted to patients with M1 morphology but flow cytometry consistent with failure to achieve remission (MRD>=5%). Using this definition, discordance was uncommon among subjects with B-ALL (66/7,748; 0.9%) but significantly more common in T-ALL (97/1,400; 6.9%; p<0.0001). Among subjects with B-ALL and M1 morphology, significant predictors of discordance (MRD>=5%) in multivariable regression included variables traditionally associated with poor response: age >=10 years [odds ratio (OR)=1.7, 95th percentile confidence interval (CI) 1.1-2.8; p=0.03), presenting white blood cell count >=50,000/microliter (OR=2.1, CI 1.3-3.6; p=0.004), and unfavorable compared to favorable cytogenetics (OR=31, CI 8.9-109; p<0.0001). In B-ALL, subjects with end induction M1 morphology but discordant MRD (>=5%) had modestly superior 5-year EFS when compared to those with M2 morphology and MRD >=5% (33.1%±6.2% vs. 22.0%±6.9%; p=0.03), but EFS was significantly inferior to those with M1 morphology and concordant MRD (<5%) (33.1%±6.2% vs. 86.8%±0.4%; p<0.0001) (Figure 1). In T-ALL, the 5-year EFS of subjects with M1 morphology/discordant MRD was not significantly different from those with M2 morphology and MRD >=5% (80.3%±7.3% vs. 62.7%±13.5%; p=0.13); outcomes of both groups were superior to their equivalents with B-ALL, in keeping with known slower disease clearance kinetics in T-ALL.Conclusions: Patients in morphologically defined remission but with MRD >=5% have outcomes similar to those who fail to achieve morphological remission. These results suggest that, in addition to measuring depth of remission, MRD should replace morphology in defining remission in subjects with ALL, with consequent implications for risk stratification, treatment assignment and eligibility for experimental agents. [Display omitted] [Display omitted] DisclosuresLoh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Borowitz:HTG Molecular: Consultancy; Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding; BD Biosciences: Research Funding. Wood:Juno: Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement.

Resistant T-Cell Acute Lymphoblastic Leukemias That Emerge after In Vivo Treatment with Dexamethasone Frequently Down-Regulate Glucocorticoid Receptor Protein Expression

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with T lineage ALL (T-ALL) accounting for approximately 20% of cases. Many T-ALL patients present with high risk clinical features, and receive aggressive multi-agent genotoxic therapies that pose a substantial risk of long-term adverse health effects. Glucocorticoids (GCs) are an integral part of current treatment strategies for T-ALL and other lymphoid cancers. Despite decades of clinical use, the molecular mechanisms underlying GC efficacy and resistance are incompletely understood. Limited initial response to GCs correlates with poor outcome, and secondary GC resistance is frequently observed in relapsed patients. Thus, identifying drugs that either enhance initial responses or prevent resistance to GCs could significantly improve the treatment of T-ALL and other lymphoid malignancies. Our laboratory performed retroviral insertional mutagenesis (RIM) in wild-type (KrasWT) and KrasG12D "knock-in" mutant mice to generate a panel of primary transplantable T-ALLs that recapitulate the genetic heterogeneity found in human cancers. We used these reagents to assess the efficacy of treatment with the GC dexamethasone (DEX) alone and in combination with the PI3 kinase (PI3K) inhibitor GDC-0941. We observed a robust and significant overall response to 15 mg/kg/day DEX in cohorts of recipient mice transplanted with 10 independent parental KrasWT (n=5) and KrasG12D (n=5) T-ALLs, which was modestly enhanced by combined treatment with 125 mg/kg/day GDC-0941. Prolonged in vivo treatment resulted in outgrowth of 65 independent relapsed T-ALLs, many of which harbor novel retroviral integrations. We verified intrinsic drug resistance in a number of these relapsed leukemias by transplanting them into secondary recipients and treating these mice with 15 mg/kg/day DEX. Intriguingly, 23 (35%) of the T-ALLs that relapsed after an initial response to treatment exhibited markedly reduced GC receptor (GR) protein expression, suggesting a novel and common mechanism for evading GC-induced cell death. Similarly, analysis of T-ALL patient samples showed that low GR expression is rare in diagnostic specimens, but enriched in relapsed/refractory cases. We have identified two likely mechanisms of GR down-regulation in relapsed mouse T-ALLs including a nonsense mutation in the gene encoding GR and a retroviral integration in a putative distal GR promoter region that greatly reduces mRNA expression. We also performed transcriptome (RNA-seq) analysis in one sensitive parental and corresponding resistant KrasWT T-ALL after short-term in vivo DEX treatment, and observed a dramatic reduction in the number of differentially expressed genes in the resistant versus parental leukemia. We recently generated transcriptomes from this panel of 10 primary RIM-induced T-ALLs with the goal of identifying a GC response signature, and also to assess the ability of resistant cells that have lost or retained GR expression to activate this program. Finally, in contrast to previous studies in this genetically accurate in vivo preclinical model (Dail et al. Nature 2014), very few relapsed T-ALLs showed reduced Notch intracellular domain (NICD) expression relative to the corresponding parental leukemia suggesting that loss of Notch1 activation is not a major mechanism contributing to DEX resistance. Taken together, these data reveal an unexpected and common putative mechanism of GC resistance in T-ALL that can inform the development of treatment strategies for relapsed/refractory patients. We are currently focusing on analyzing transcriptome data and performing whole exome sequencing in order to uncover additional mechanisms of resistance to DEX, and then using these data to identify and investigate targeted inhibitors that might overcome GC resistance in vivo. DisclosuresDail:Genentech, Inc.: Employment. Sampath:Genentech: Employment.