Cytogenetic Subtypes Research Articles

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

Epidemiology, Cytogenetic and Molecular Abnormalities and Outcome of Childhood Acute Myeloid Leukemia- an Israeli Retrospective Multicenter Study

IntroductionAcute myeloid leukemia (AML) contributes to 18% of childhood leukemia cases and about 50% of all deaths of pediatric leukemia in the United States .In high-income countries, intensive therapy has increased survival rates to 60-70% . Contributing factors to the improved survival include refinement in the diagnosis of AML and advancement in therapeutic approaches.In light of the ethnic diversity in the Israeli population, we reviewed patient's data and investigate whether the clinical, biologic and molecular features differ between Israeli Arab and Jewish AML patients.Patients and MethodsWe performed a retrospective epidemiological and clinical study including patients younger than 18 years of age, treated at all six Israeli pediatric oncology centers between 2000-2015. Down syndrome and secondary AML were excluded . The clinical characteristics such as gender, age, subtype of FAB, leukocyte count at diagnosis, cytogenetics and molecular markers were analyzed in correlation with their prognosis between different characteristics groups and different ethnicity. Survival analyses were calculated by Kaplan-Meier survival curves and the log rank test. Multivariate analyses on categorized data were performed using Cox proportional hazards model.ResultsWe analyzed 175 patients, 46% females and 54%, males, 15 % younger than 2 years old, 35% from 2 to 10 years and 50% older than 10 years (median age 9.3±5.6 years ). According to FAB subtype, the majority subtypes were M2 and M4 for 26% and 27% respectively , 40% of patients presented WBC count below 10 X 109/L at diagnosis while 13% of patients higher than 100 X 109/L. The most common cytogenetic abnormalities in these children, including t(8;21)(q22;q22), inv(16)(p13.1q22) and 11q23/MLL-rearranged abnormalities were detected, with the occurrence of 18% , 8% and 13% respectively.Acute promyelocytic leukemia (APL) was diagnosed in 14% of the children. Molecular data such as FLT3 were not evaluated due to small numbers.No statistical difference was observed regarding ethnicity as to age, white blood cell (WBC) and central nervous system (CNS) involvement. However, the Arab patients presented with a significantly higher incidence of M2 (42% vs 23% p=0.035), while the Jewish population demonstrated significantly higher incidence of M4 (33% vs. 10% p=0.011). The high incidence of M3 was common to both ethnic groups. In addition, cytogenetic subtype distribution was similar in both ethnic groups.Out of 175 patients, 114 patients were treated in accordance with the same protocol, namely BFM98 . The demographic characteristics of these 114 pts, as well as their WBC, FAB and cytogenetics were distributed similarly to the whole cohort.Among the 114 AML patients, 105 pts achieved complete remission (CR) (92%) after one course of chemotherapy. Of those who achieved CR after 2 induction cycles, 62 pts are alive and free of disease, 38 pts relapsed, 4 pts died in CR (3 due to bacterial infection and 1 due to post transplant lymphoproliferative disease) , one patient developed myelodysplastic syndrome after treatment for APL . Six patients did not go into remission after two induction phases. Three patients had early death (within six weeks of diagnosis), all due to bleeding- 2 were APL and one with MLL split and hyperleukocytosis.The median follow-up was 6.3 years (0.7y-16y), with a 5-year event free survival (EFS) and overall survival (OS) of 54% and 67% respectively.There was no prognostic significance in EFS and OS related to gender, age and ethnicity.As to risk stratification, there was a statistical difference in EFS and OS in the standard risk (SR) and the high risk (HR) group: 5 year EFS SR group 83% and 39% for HR group (p<0.0001) 5 year OS for SR group 98% and 52% for HR group (p<0.0001).ConclusionsIn this retrospective study of pediatric AML patients in Israel,the clinical, laboratory and cytogenetic characteristics were not significantly different between Jewish and Arab patients, with a similar outcome. We report a 14% incidence of APL - an incidence that is higher than that reported for the European and American populations. It may be of interest to further investigate this increased rate of APL in the Israeli population and possibly identify underlying genetic predisposing factors.The pts treated according to AML-BFM-98 protocol without pCRT have a 5y EFS and OS of 54% and 67%, respectively. These outcomes are similar to those reported for BFM98. DisclosuresNo relevant conflicts of interest to declare.

Distinct Outcome Based on Fusion Transcript in t(16;21) Rearranged Pediatric AML, a Collaborative Retrospective Analysis By the I-BFM SG

Despite the use of of intensive chemotherapy, and allogeneic stem cell transplant (HSCT) in selected cases, children with acute myeloid leukemia (AML) have 5-year overall survival (OS) rates of ~65%. Apart from early clinical response, cytogenetic and molecular aberrations are the most reliable prognostic factors for survival. Identifying prognostic subgroups to stratify patients for treatment in future trials is clinically important. The study of rare AML subtypes can be facilitated by international collaborations. AML with t(16;21) rearrangements includes 2 different translocations: t(16;21)(p11;q22) resulting in the FUS-ERG fusion, and t(16;21)(q24;q22) resulting in the RUNX1-CBFA2T3 fusion.To study outcomes and associated prognostic factors, clinical data from children with t(16;21) AML were collected from 14 study groups participating in the international Berlin-Frankfurt-Munster (I-BFM) network. Patients (age 0-18 years) diagnosed from January 1, 1995, to January 1, 2016, were included. The AML-BFM study group provided data from 1326 unselected patients diagnosed between 1997 and 2013 as a reference cohort. All karyotypes were centrally reviewed by 2 independent cytogeneticists according to the International System for Human Cytogenetic Nomenclature (ISCN 2005). Patients with inconclusive karyotypes were screened by reverse-transcription PCR. Chi-squared and Fisher exact tests were used to compare clinical characteristics. OS and event free survival (EFS) analysis was performed by the Kaplan-Meier method. Cumulative incidence of relapse (CIR) was calculated according to Kalbfleisch and Prentice. The Cox proportional hazards model was used for multivariate analysis, considering as covariables age, white blood cell (WBC) count at diagnosis, cytogenetic risk group and allogeneic hematopoietic stem cell transplantation (HSCT) as time-dependent variable.There were 54 patients with t(16;21): 32 with FUS-ERG and 22 with RUNX1-CBFA2T3 . There were no significant differences in sex and age between patients in the FUS-ERG- or RUNX1-CBFA2T3- groups and the reference cohort. Median WBC at diagnosis was significantly lower for patients with RUNX1-CBFA2T3 (12.5×109/L, range 0.01-185×109/L) than for the reference cohort (19.4×109/L, range 0.01-1900×109/L, P=0.030). Patients with FUS-ERG had no predominant French-American-British (FAB) type, whereas 76.4% of those with RUNX1-CBFA2T3 had a granulocytic FAB type (M1, M2) compared with 42.1% in the reference cohort (P=0.004).The 4-year EFS, OS, and CIR for the reference cohort were 51% [standard error (SE)=1%], 68% (SE=1%), and 32% (SE=1%), respectively. Median follow up for survivors in the t(16;21) cohort was 1.6 years for those with FUS-ERG and 5.0 years for RUNX1-CBFA2T3 . Patients with FUS-ERG had a 4-year EFS of 7% (SE=5%, P <0.0001), 4-year OS of 21% (SE=8%, P <0.0001), and CIR of 74% (SE=8%, P <0.0001). Patients with RUNX1-CBFA2T3 had a 4-year EFS of 77% (SE=9%, P=0.06), OS of 81% (SE=8%, P=0.34) and a CIR of 0%. When patients in the reference cohort were classified in SR patients comprising (inv(16)/t(8;21), and HR patients comprising other cytogenetic subtypes excluding t(16;21), EFS rates were 74% (SE=3%) and 45% (SE=2%), respectively (Fig. 1). Hence, patients with RUNX1-CBFA2T3 had a similar outcomes as SR patients (Figure 1). In multivariate analysis, presence of FUS-ERG and RUNX1-CBFA2T3 were independent risk factors for EFS, with hazard ratios of 1.93 (P <0.0001) and 0.325 (P=0.025), respectively. HSCT was performed in 14 (45.2%) patients with FUS-ERG in first complete remission (CR1). The 4-year EFS for transplanted patients was 15% (SE=15%) compared with 0% (SE=13%) for patients receiving chemotherapy only (p=NS).We identified 2 clinically relevant, distinct subtypes of pediatric AML patients with different t(16;21) rearrangements: FUS-ERG had poor outcomes, whereas RUNX1-CBFA2T3 had favorable outcomes. Patients with RUNX1-CBFA2T3 -rearranged AML might benefit from inclusion in the SR group and receiving less intensive treatment, whereas those with FUS-ERG rearranged AML should be stratified in the HR group. In this study, only patients with FUS-ERG receiving HSCT in CR1 did not relapse. Most current pediatric AML trials consider children with a 5-year OS below 25%-30% for HSCT in CR1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNA Methylation Signatures Predict Cytogenetic Subtype and Outcome in Pediatric Acute Myeloid Leukemia (AML).

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease composed of clinically relevant subtypes defined by recurrent cytogenetic aberrations. The majority of the aberrations used in risk grouping for treatment decisions are extensively studied, but still a large proportion of pediatric AML patients remain cytogenetically undefined and would therefore benefit from additional molecular investigation. As aberrant epigenetic regulation has been widely observed during leukemogenesis, we hypothesized that DNA methylation signatures could be used to predict molecular subtypes and identify signatures with prognostic impact in AML. To study genome-wide DNA methylation, we analyzed 123 diagnostic and 19 relapse AML samples on Illumina 450k DNA methylation arrays. We designed and validated DNA methylation-based classifiers for AML cytogenetic subtype, resulting in an overall test accuracy of 91%. Furthermore, we identified methylation signatures associated with outcome in t(8;21)/RUNX1-RUNX1T1, normal karyotype, and MLL/KMT2A-rearranged subgroups (p < 0.01). Overall, these results further underscore the clinical value of DNA methylation analysis in AML.

Prognostic Relevance of Expression of EMP1, CASP1, and NLRP3 Genes in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

Glucocorticoid (GC), such as prednisolone, is an essential component of multidrug chemotherapy regimen for pediatric acute lymphoblastic leukemia (ALL). Resistance to GC in leukemia cells is associated with disease progression and poor prognosis. Despite the extensive use of GC for many years, molecular mechanisms underlying its resistance in ALL have not been fully uncovered. Recent studies have shown a potential role of EMP1, CASP1, and NLRP3 genes in prednisolone response. In this study on 148 pediatric B-ALL patients, we studied these three genes to assess their association with prednisolone response measured by day 8 blast count after 7 days of induction therapy with prednisolone. Intriguingly, ALL samples exhibited higher expression of EMP1 along with a low expression of CASP1 and NLRP3 compared to disease free normal bone marrow collected from patients with solid tumors. Among the three analyzed genes, only EMP1 was found to be overexpressed in prednisolone poor responders (p=0.015). Further, a comparison of gene expression between cytogenetic subtypes revealed higher expression of EMP1 in BCR-ABL subtype. Expression of EMP1 in multiple gene expression datasets was used for gene set enrichment analysis, which revealed TNF-α, IL-2-STAT5 signaling, inflammatory responses and hypoxia as the major positively associated pathways and E2F targets as negatively associated pathways. Interestingly, the clinical remission rate was higher in CASP1 high patients (p=0.048). In univariate survival analysis, higher EMP1 expression was associated with poor prognostic measures while higher expression of NLRP3 and CASP1 was associated with better prognostic measures in our data. Further, multivariate analysis revealed an independent association of high CASP1 and NLRP3 with a better prognosis. This study strengthens the available evidence that mRNA expression of EMP1, CASP1, and NLRP3 may serve as potential biomarkers for risk stratification of pediatric B-ALL patients.

Profiling 25 Bone Marrow microRNAs in Acute Leukemias and Secondary Nonleukemic Hematopoietic Conditions.

Introduction: The standard treatment of acute leukemias (AL) is becoming more efficacious and more selective toward the mechanisms via which to suppress hematologic cancers. This tendency in hematology imposes additional requirements on the identification of molecular-genetic features of tumor clones. MicroRNA (miRNA, miR) expression levels correlate with cytogenetic and molecular subtypes of acute leukemias recognized by classification systems. The aim of this work is analyzing the miRNA expression profiles in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) and hematopoietic conditions induced by non-tumor pathologies (NTP). Methods: A total of 114 cytological samples obtained by sternal puncture and aspiration biopsy of bone marrow (22 ALLs, 44 AMLs, and 48 NTPs) were analyzed by real-time PCR regarding preselected 25 miRNAs. For the classification of the samples, logistic regression was used with balancing of comparison group weights. Results: Our results indicated potential feasibility of (i) differentiating ALL+AML from a nontumor hematopoietic pathology with 93% sensitivity and 92% specificity using miR-150:miR-21, miR-20a:miR-221, and miR-24:nf3 (where nf3 is a normalization factor calculated from threshold cycle values of miR-103a, miR-191, and miR-378); (ii) diagnosing ALL with 81% sensitivity and 81% specificity using miR-181b:miR-100, miR-223:miR-124, and miR-24:nf3; and (iii) diagnosing AML with 81% sensitivity and 84% specificity using miR-150:miR-221, miR-100:miR-24, and miR-181a:miR-191. Conclusion: The results presented herein allow the miRNA expression profile to de used for differentiation between AL and NTP, no matter what AL subtype.

Dose-Adjusted R-EPOCH Therapy for Aggressive Lymphoma: A single center experience

Objective: When we look at diffuse large B-cell lymphomas, 5-7% are double hit lymphoma, 30-40% are double expressor lymphoma and 5-10% are triple hit lymphomas and they have an aggressive course. In our study, the efficacy of the dose-adjusted-R-EPOCH treatment regimen in the treatment of double hit lymphoma, double expressor lymphoma, triple hit lymphoma; triple expressor lymphoma and primary large B cell lymphomas of the mediastinum are presented.&#x0D; Materials and Methods: Thirty-six patients diagnosed with B-cell lymphoma who received dose-adjusted R-EPOCH treatment were included in the study. The patients were grouped cytogenetically according to the Bcl-2 and Bcl-6 rearrangement by MYC translocation. Response assessment with PET-CT was performed in patients whose planned therapy reached the number of cycles.&#x0D; Results: At the end of the treatment, 61% of the patients had a complete response, 3% had a partial response, and 8% had no response. During the follow-up, 9 of the patients died and while the treatment of 2 patients was still ongoing, the treatment regimen of 1 patient was changed. When the patients were evaluated, 2 had double hit lymphoma (complete response in 2 patients), 9 had double expressive lymphoma (5 patients had complete response, 2 patients had progressed disease, 1 patient died and 1 patient had a change in treatment), and 8 had triple expressor lymphoma (Complete response in 5 patients, death in 2 patients, and progressive disease in 1 patient).&#x0D; Conclusion: The dose-adjusted-R-EPOCH treatment regimen can also provide a high response rate in patients with lymphoma with a triple expressor cytogenetic subtype.

Impact of Race/Ethnicity on Pediatric Core Binding Factor AML Outcomes and Response to Gemtuzumab Ozogamicin

Introduction: Racial disparities in pediatric acute myeloid leukemia (AML) outcomes have consistently demonstrated inferior event-free survival (EFS) and overall survival (OS) in minority populations with the worst outcomes in Black children. However, AML is a heterogenous disease classified by recurrent genetic aberrations, and these disparities have not been thoroughly investigated within specific cytogenetic groups. In addition, race-based response to individual chemotherapy agents in AML has not been explored. Here, we use data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database to describe the effects of race on outcomes and response to gemtuzumab ozogamicin (GO) therapy in core binding factor (CBF) AML, a favorable risk AML subtype. Methods: CBF AML patient data was extracted from the online TARGET database. Race and ethnicity were self-reported. Estimates of 2-year OS and EFS were calculated using the Kaplan-Meier method and analyzed via log-rank statistic. Pearson's Chi-squared test and Fisher's exact test were used to analyze patient variables. Results: The OS in CBF AML differed significantly based on race/ethnicity at 87% (95% CI, 82-93%) for White non-Hispanic (WNH), 77% (95% CI, 67-89%) for Hispanic, and 71% (95% CI, 58-88%) for Black children (p=0.005). EFS was also significantly lower in Black (46%; 95% CI, 32-66%) compared to non-Black children (66%; 95% CI, 60-73%)(p=0.023). When assessed by CBF AML subtype, EFS and OS in t(8;21) AML varied significantly based on ethnicity at 50% (95% CI, 34-75%) and 67% (95% CI, 50-88%) in Black patients, 63% (95% CI, 49-82%) and 69% (95% CI, 55-87%) in Hispanic patients, and 82% (95% CI, 72-92%)(p=0.01) and 90% (95% CI, 93-98%)(p=0.006) in WNH patients. In inv(16) AML, Hispanic patients experienced the highest EFS (71%; 95% CI, 55-92%) followed by WNH (54%; 95% CI, 43-66%) and Black (36%; 95% CI, 17-79%) patients though this did not reach statistical significance (p=0.12). There was no impact of race/ethnicity on OS in inv(16) AML. The use of GO did not alter OS in any subset of CBF AML. EFS appeared to be slightly improved but was not statistically significant. However, Black children with any type of CBF AML had preferential improvement with GO with significantly higher EFS (69% v. 26%, p=0.03) and a trend toward improved OS (81% v. 63%, p=0.19) with GO therapy. These improvements were primarily driven by a significant increase in EFS in inv(16) AML when treated with GO (80% v. 50%, p=0.01) and a similar but non-significant improvement in OS in this group. In Hispanic children there was a trend toward inferior EFS with the use of GO (63% v. 73%, p=0.37) in CBF AML, particularly in t(8;21) patients with a 1-year EFS of 56% (95% CI, 37-87%) compared to 81% (95% CI, 64-100%)(p=0.23). WNH children were more likely to achieve Complete Remission (CR) after course 1 (OR 2.36, 95% CI 1.09-5.10, p=0.03), and Black children were more likely to have persistent Minimal Residual Disease (MRD) after course 2 (OR 9.64, 95% CI 2.48-37.43, p&amp;lt;0.001). No differences were detected in other patient variables. Conclusion: Children with CBF AML represent a favorable cytogenetic risk group with an EFS of 70% and OS of 85%. However, the impact of race/ethnicity on outcome is significant with Black children experiencing the lowest EFS and OS at 46% and 71%, respectively, with this trend persisting across cytogenetic subtypes. Black children were also more likely to remain MRD positive after Induction II and less likely than WNH patients to achieve CR. However, while the addition of GO to standard therapy did not improve outcomes in CBF AML overall, Black children selectively benefited from GO therapy. Black children trended toward improved EFS and OS with GO with the most pronounced effect on EFS in inv(16) which was improved from 50% to 80% at 1 year. These results demonstrate that Black children with CBF AML are disproportionately at risk for poor outcomes compared to other ethnicities, but these risks may be mitigated by the use of GO which increase EFS and OS primarily in this group. Conversely, Hispanic patients trended toward worse EFS with GO, particularly in t(8;21) AML, possibly due to treatment-related toxicity. As CD33 expression and certain ABCB1 SNPs are known to predict response to GO therapy, our ongoing work investigates racial differences in these variables as a potential explanation for the differing responses to GO. Disclosures Rau: Jazz Pharmaceuticals, Inc.: Consultancy, Other: Travel Fees.

Therapy-Related Myeloid Neoplasms in 108 Patients Following Radiation Therapy Only

Background: Exposure to ionizing radiation is associated with the development of therapy-related myeloid neoplasms (t-MNs). Several distinct clinical and cytogenetic subtypes of t-MN have been recognized, and are found to be closely associated with various cytotoxic/genotoxic therapies. However, characteristics of t-MNs that follow radiation therapy (RT) alone are not as clear. We examined clinical, epidemiologic, cytogenetic, and molecular characteristics of 108 cases of t-MN in patients (pts) who received RT alone for an antecedent disease. Methods: Retrospective chart review was done to identify pts in our institution's t-MN database that had received RT alone prior to development of t-MN. Clinical characteristics, indication for RT, latency period between radiation and development of t-MN (LP), cytogenetic features, somatic and germline mutation data, treatment, and overall survival (OS) after development of t-MN were collected. LP and OS were both analyzed using Kaplan-Meier analysis. Results: 108 consecutive pts who developed an RT-related t-MN (48 with t-MDS at diagnosis, 60 with t-AML) were identified from 1971-2020. 99 pts had a primary solid tumor [most common: prostate/testicular (44), breast (30), and gynecological (15)], 4 had a primary lymphoid malignancy, and 5 received radiation for a non-malignant condition. 27 pts had a normal karyotype, 22 had an abnormality of chromosome 5, 11 had an abnormality of chromosome 7, 13 had abnormalities of both 5 and 7, 14 had recurring balanced translocations, and 20 had other clonal abnormalities (Table 1). Median LP for the entire cohort was 6.5 years. The shortest LP was in pts with recurring balanced translocations (median 2.4 years) (Table 1).Treatment course was available for 85 pts; 25 (29%) received intensive remission induction with cytarabine + anthracycline, 35 (44%) received hypomethylating agent-based therapy, 18 (21%) received only supportive care, and 5 (6%) received other therapies. 15 pts (18%) underwent allogeneic hematopoietic cell transplantation. Median OS was 12.7 months; 1-year OS was 53%, and 5-year OS was 23%. 1-year OS and 5-year OS were inferior in pts with an abnormality in chromosome 5 (p&amp;lt;0.01), abnormality in chromosome 7 (p=0.01), and abnormalities in both 5 and 7 (p&amp;lt;0.01) (Figure 1). Pts with chromosome 5 and/or 7 abnormalities had a median OS of 9.1 months. Somatic next-generation sequencing (NGS) data were available for 15 pts at diagnosis of t-MN. The most common somatic mutations were ASXL1 (n=4), IDH2 (3),TET2 (3), U2AF1 (3), TP53 (2), NPM1 (2), FLT3- ITD (2), NRAS (2), STAG2 (2), SRSF2 (2), and CBL (2). Germline testing was available starting in 2011 and was performed in 16 pts. Pathogenic/likely pathogenic variants in CHEK2 p.Ile157Thr (no somatic mutations), DDX41 p.Pro258Leu (somatic ASXL1 mutation), or PALB2 p.Arg170Ilefs*14 (somatic NGS not done) were identified in 3 individuals (18%). All 3 pts had a normal karyotype. Conclusions: RT-related t-MNs have clinical/cytogenetic features, LPs, and outcomes similar to pts who have been exposed to alkylator-based therapy, with abnormalities in chromosome 5 and/or 7 (44%) being the most common subtype, and a median LP of 4.4-6.5 years. OS is poor, despite intensive therapies. Pts with normal karyotype or balanced rearrangements have better OS. Pts with RT-related APL, CBF AML, or normal-karyotype, NPM1-mutated AML have outcomes similar to pts with de novo disease. In addition, 18% of pts who underwent germline testing had a pathogenic variant. Better characterization of pathogenic somatic mutations in these patients and identification of germline predisposition mutations may provide new insights into the etiology of RT-related t-MNs and lead to more successful therapies. Disclosures Godley: UptoDate, Inc.: Honoraria; Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees. Le Beau:American Cancer Society: Membership on an entity's Board of Directors or advisory committees; Varian Medical Systems: Membership on an entity's Board of Directors or advisory committees. Larson:Novartis, Takeda, CVS/Caremark, Celgene, Amgen, Epizyme: Consultancy; Astellas, Celgene, Daiichi Sankyo, Novartis, Rafael Pharmaceuticals, Cellectis, Forty Seven: Research Funding.

High EVI1 Expression Predict Adverse Outcome in Children with Acute Myeloid Leukemia: A Multicenter, Nonrandomized Clinical Study in China

Backgroud and purpose In clinical studies in adult acute myeloid leukemia (AML), high ecotropic virus integration-1 (EVI1) gene expression has shown to be an independent prognostic factor. However, the predictive value of high EVI1 expression (EVI1+) in children with AML is little known. The purpose of this study was to investigate frequency and prognostic significance of high EVI1 expression in children with AML. Patients and methods Total of 403 newly diagnosed childhood AML patients from 7 centers of south of China from November 2014 to May 2020 were included in the study. A diagnostic assay detecting multiple EVI1 splice variants was developed to determine the relative EVI1 expression by single real-time quantitative polymerase chain reaction in the 403 newly diagnosed pediatirc patients with AML younger than 14 years. Patients were treated with C-HUANAN-AML15 protocol. In the C-HUANAN-AML15 protocol, tandem 2 courses of FLAG-IDA regimen, or sequential DAE(3+5+10) and DAE(3+5+8) were applied as induction chemotherapy. One course of Homoharringtonine (substitution of Amsacrine in MRC-AML 15 protocol)/ Cytarabine/ Etoposide and one course of Mitoxantrone/ Cytarabine in consolidation chemotherapy were uniform in both groups. 75 patients selected hematopoietic stem cell transplantation (HSCT) in first CR. The median follow-up period was 20 (0.8 to 67.7) months up to July 2020. The similarity of clinical data was analyzed by the chi square test and COX proportional hazard function model. Complete remission (CR) rates, event-free survival (EFS) and overall survival (OS) were compared by Log-Rank chi square test. Results EVI1+ was found in 36/403 (8.9%) of children with de novo AML. The clinical features of the patients with EVI1+(n=36) compared with EVI1-(n=367) AML were summarized in Table 1. No significant differences in sex, white blood cell count, and age were noted between EVI1+and EVI1- AML. When studying the relationship between EVI1+ and conventional classification criteria such as morphology (FAB classification) and cytogenetic data, EVI1+ was predominantly found in FAB M7, t(9;11) and other t(v;11q23), as well as complex karyotype, but mutually exclusive with t(8;21), inv(16)/t(16;16), CEBPA, NPM1 or C-KIT mutations. Furthermore, EVI1+ was significantly associated with unfavoralbe cytogenetic risk. Twenty-five patients (22 cases in the EVI1-group and 3 cases in the EVI1+ group) who gave up treatment or transferred to other hospital when treatment was not completed were excluded, and the remaining 378 AML children with standard treatment were included in the survival analysis. Univariate analysis showed that initial white blood cell count (≥50×109/L), RUNX1-RUNX1T1 fusion gene, FLT3-ITD mutation and EVI1+ affected 5-year EFS (P=0.000, 0.001, 0.016, 0.011). Multivariate prognostic analysis with COX proportional hazard function model for EFS identified EVI1+ as an independent prognosis factor (HR0.531, 95%CI 0.298-0.945, P=0.032). Patients with EVI1+ AML (n=12) who received HSCT in first CR had better 5-year EFS, but the difference was not statistically significant (46.103% vs. 56.250%, P=0.33). Conclusion It could be concluded that EVI1+ can be found in ~10% of pediatric AML in China. It is predominantly found in intermediate to unfavorable cytogenetic subtypes and predicts adverse outcome. EVI1 screening at diagnosis should be included in risk stratification of pediatric AML. Whether pediatric patients with EVI1+ AML can benefit from HSCT in first CR needs further reasearch. Disclosures No relevant conflicts of interest to declare.

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL

AbstractMost relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.

Abstract A55: Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities

Abstract Introduction: Nearly 35% of children with acute myeloid leukemia (AML) die, despite aggressive but toxic and nonspecific therapies. New approaches to find and drug pediatric AML targets are clearly needed. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the RARA agonist tamibarotene. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by RARA regulation. Methods: Four AML/APL cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes, with an over-representation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters. Some of the highly correlated, SE-associated genes in these clusters encode proteins involved in inflammation. Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes. Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples reduced cell viability and increased apoptosis (detected by annexin V+ and activated caspase 3/7), with no effect in RARA SE- pAML samples. In the RARA SE+ samples, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) and DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial). Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and have seen a high frequency of a RARA SE in pAML. Tamibarotene has antiproliferative, proapoptotic, and prodifferentiation effects in RARA SE+ pAML. We are confirming these results in a RARA SE+ patient-derived xenograft mouse model, evaluating combinations, and interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Citation Format: Monika Perez, Alfred Daramola, Oscar Sias-Garcia, Helen Wei, Nikitha Cherayil, Charles Y. Lin, Joanna S. Yi. Defining the transcriptional regulation of pediatric AML as a new strategy to find potential druggable vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A55.

Sporadic oncocytic tumors with features intermediate between oncocytoma and chromophobe renal cell carcinoma: comprehensive clinicopathological and genomic profiling

Morphology, clinical behavior, and genomic profiles of renal oncocytoma (RO) and its malignant counterpart chromophobe renal cell carcinoma (ChRCC) are distinctly different. However, there is a substantial group of sporadic oncocytic tumors with peculiar hybrid phenotypes as well as a perplexing degree of morphologic and immunohistochemical overlap between classic RO and ChRCC with eosinophilic cytoplasm. The aim of this study is to provide detailed characterization of these hybrid tumors.Thirty-eight sporadic oncocytic neoplasms with ambiguous morphology from two institutions were reviewed by 4 pathologists. CKIT positivity was used as a selection criterion. We correlated CK7 and S100A1 immunostaining and detailed morphologic features with cytogenetic profiles. DNA from the formalin-fixed paraffin-embedded tissues was extracted and analyzed using cytogenomic microarray analysis (CMA) to evaluate copy number alterations (CNA) and ploidy. CMA categorized cases into 3 groups: RO (N=21), RO variant (N=7), and ChRCC (N=10). Cytogenetic RO had either no CNA (48%) or loss of chromosome 1p, X, or Y (52%). RO variant had additional chromosomal losses [-9q, -14 (n=2), -13] and chromosomal gains [+1q (n=2), +4, +7 (n=2), +13, +19, +20, and +22]. ChRCCs were either hypodiploid with numerous monosomies (40%) or hypotetraploid with multiple relative losses (60%). RO, RO variant, and ChRCC groups differed significantly in tumor architecture (p<0.01), stroma (p=0.013), presence of nuclear wrinkling, perinuclear halos, and well-defined cell borders in >5% of cells (p<0.01), focal cell clearing (p=0.048) and CK7 expression (p<0.02). Pathologic prediction of the cytogenetic subtype using only two categories (benign RO or malignant ChRCC) would overcall or undercall up to 40% of tumors that were ChRCC based on cytogenetics. This finding provides the rationale for an intermediate diagnostic category of the so-called hybrid tumors (hybrid oncocytic/chromophobe tumor [HOCT]). HOCT was a heterogeneous group enriched for cytogenetic RO variant. Other HOCTs have a profile of either RO or ChRCC. The genomic profile allows classification of oncocytic tumors with ambiguous morphology into RO, RO variant, and ChRCC. Several architectural and cytologic features combined with CK7 expression are significantly associated with cytogenetic RO, RO variant, or ChRCC tumors. Doubled hypodiploidy by whole-genome endoduplication is a common phenomenon in eosinophilic ChRCC.

Prognostic Role of Lymphocyte/Monocyte Ratio in Chronic Lymphocytic Leukemia

BackgroundChronic lymphocytic leukemia (CLL) is a B-lymphoproliferative disease with varying clinical characteristics, which occurs mostly in older ages. In studies from literature, we see that different parameters are examined to determine the prognosis of CLL. The main purpose of our study is to determine the relationship of lymphocyte/monocyte ratio (LMR) value in CLL, which has been previously shown to be a prognostic factor in various solid organ tumors and some hematological malignancies.MethodsA total of 173 patients who were followed up between 2005 and 2019 were retrospectively analyzed. The diagnostic age, gender, laboratory, absolute lymphocyte and monocyte count, LMR and overall survival (OS), treatment and responses, recurrence, cytogenetic subtype and mortality rates were examined.ResultsThe median LMR was 26.7 and it was considered as cut-off value of 26. A positive correlation was found between LMR and Rai Stage. LMR was significantly higher in patients who have an indication for treatment or who died.ConclusionsIn our study, in CLL, LMR has been shown to be over 26 in advanced stages, in relapse or with indication of a treatment. With the increase of LMR, it was found that survival and disease-free gap decreased.

Minimal Residual Disease and IKZF1 As Predictors of Relapse, and Increased Treatment Related Mortality in Down Syndrome Acute Lymphoblastic Leukemia: A Unique and Large International Matched Case-Control Study

INTRODUCTION Down syndrome patients with acute lymphoblastic leukemia (DS ALL) are less likely to have a favorable (cyto)genetic subtype and are at higher risk of relapse and treatment-related mortality (TRM) than non-DS ALL (Buitenkamp et al., Blood 2014). At present, the independent predictive value of minimal residual disease (MRD) is unclear and may be biased by an unequal distribution of (cyto)genetic risk groups among DS ALL and non-DS ALL patients. This study was aimed to decipher the prognostic implications of MRD and IKZF1 deletions and the frequency of TRM in a matched cohort of DS ALL cases and non-DS ALL controls. METHODS Each DS ALL patient was matched to 3 non-DS ALL patients based on treatment protocol, induction treatment, cytogenetic subtype, IKZF1 status, age (cutoff at 10 years), and white blood cell count (cutoff at 50,000 cells/µl). For MRD analysis, matching was only on induction treatment and excluded the MRD-guided treatment arm; for survival analyses, matching included the MRD-guided treatment arm, thus resulting in two separately matched cohorts. Patients who died during induction were excluded from matching. Absolute MRD levels were measured with RQ-PCR, log-transformed and analyzed with a multilevel mixed-effects linear regression model. Matched Cox proportional hazard regression models were used to analyze event-free survival (EFS), overall-survival (OS), relapse-free survival (RFS) and mortality in remission as surrogate for TRM. RESULTS Patients treated between 2002 and 2018 on Dutch DCOG-ALL10/11 trials, Australian ANZCHOG-ALL8 and AIEOP-BFM-ALL2009 trials, and UKALL2003 trial were included, resulting in 160 DS ALL and 5313 non-DS ALL patients. Out of these 160 DS ALL patients, 13 died during induction versus 42/5313 non-DS ALL patients (8.1% versus 0.8%, p&amp;lt;0.0001). Exclusion of induction deaths and patients with missing IKZF1 or MRD data, resulted in 110 DS ALL matched to study MRD differences (1 DS ALL could not be matched and was excluded), and 93 DS ALL matched to analyze survival (ALL11 was excluded due to short follow-up; 3 DS ALL could not be matched and were excluded). In the matched cohort, 22% (24/110) had favorable cytogenetics, 19% (21/110) had an IKZF1 deletion, 26% (29/110) were ≥10 years, and 17% (19/110) had a WBC ≥50,000 cells/µl. Only 3 cases (2 DS, 1 non-DS) did not achieve complete remission (excluding induction deaths). Median follow-up time of survivors was 7.1 years. The MRD levels did not differ between DS ALL and matched non-DS ALL patients at end of induction (p=0.96) nor when analyzed over time (p=0.43). In accordance, the 5-yr RFS did not differ between DS ALL and matched non-DS ALL patients (85 ± 4% versus 89 ± 2%, p=0.09). The 5-yr TRM of patients in remission was higher in DS ALL compared to non-DS ALL (11 ± 3% versus 3 ± 1%, p=0.001). In line, 5-yr EFS was lower in DS ALL compared to non-DS ALL patients (74 ± 5% versus 86 ± 2%, p=0.0007), as was 5-yr OS (77 ± 5% versus 93 ± 2%, p=0.0001). Multivariable analysis revealed that IKZF1 deletion can discriminate DS ALL patients at high risk of relapse (RFS: HR&amp;gt;3, 95% CI=1-12, p=0.05). The effect of IKZF1 deletion was stronger in DS ALL patients than in the non-DS ALL patients in our cohort. CONCLUSION The MRD levels did not differ between DS ALL and non-DS ALL patients when matched for (cyto)genetics and other risk factors. In accordance, the overall relapse rate of DS ALL patients did not differ from that of matched non-DS ALL patients. Similar to non-DS ALL, IKZF1 deletion is an adverse risk factor for DS-ALL, indicating the need for treatment aimed at reducing the high relapse risk. DS ALL patients suffer more frequently from death in induction and from treatment while in remission, which jeopardizes treatment intensification. Therefore, the efficacy of targeted, less toxic therapies such as immunotherapies should be assessed in DS ALL. Disclosures van der Velden: Jansen: Research Funding; BD Biosciences: Research Funding; Amgen: Honoraria, Research Funding. Pieters:jazz farmaceuticals: Consultancy; medac: Consultancy. Zwaan:Celgene: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding; Janssen: Consultancy; Servier: Consultancy; Roche: Consultancy; Incyte: Consultancy; Sanofi: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Daiichi Sankyo: Consultancy.

A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD &lt; 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

Rara Is a Druggable Super-Enhancer Regulated Dependency in Pediatric AML

Introduction: New approaches to find and then drug pediatric acute myeloid leukemia (AML)-specific targets are clearly needed to help the nearly 35% of patients who still die from the disease. While RARA is a known druggable target in acute promyelocytic leukemia (APL), the utility of using retinoic acid agonists in non-APL AML has not proven consistently beneficial. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult non-APL AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was detected at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the second-generation retinoic acid agonist tamibarotene which has led to a phase II clinical trial. This study confirms that characterization of the chromatin-defined dependencies in specific cancers can pinpoint targets which can be drugged. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by strong RARA dependence in a majority of pAML. Methods: Three AML cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. For in vivo assessment, 200,000 cells of a RARA SE+ pAML patient sample were injected into each NSGS mouse by tail-vein injection. One week after injection, tamibarotene (6mg/kg) or vehicle treatment was initiated by gavage (n=7 each arm). Peripheral blood monitoring of leukemia burden was determined flow cytometry. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes (Fig 1a), with an overrepresentation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters (Fig 1b). Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes (Fig 1c). Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples suppressed proliferation and increased apoptosis (detected by annexin V+), with minimal effect in Kasumi, a pAML cell line without a RARA SE (Fig 2a). In the RARA SE+ cell lines and samples only, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) (Fig 2b). High RARA mRNA levels confirmed the SE assignments in both cell lines and patient samples (Fig 2c). Tamibarotene induced the transcription of DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial) (Fig 2d). Tamibarotene suppressed colony formation ability in RARA SE+ cell lines. An ongoing RARA SE+ patient-derived pAML xenograft confirmed tamibarotene markedly suppressed disease progression (Fig 3), with vehicle mice requiring euthanasia 42 days after tail-vein injection for significant disease burden, while the tamibarotene treated mice continued to be well-appearing at the same timepoint. Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and seen a high frequency of a RARA SE in pAML. Tamibarotene has anti-proliferative, proapoptotic, and on-target pro-differentiation effects in RARA SE+ pAML in vitro and marked anti-leukemia activity in vivo. Given these positive findings, we are evaluating combinations of other AML-active agents with tamibarotene. Additionally, as there is a range of sensitivity to tamibarotene in RARA SE+ samples, we are also interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Disclosures Wei: NHI NHLBI Grant: Other: received funding . Lin:Syros Pharmaceuticals: Equity Ownership, Patents &amp; Royalties.

Outcomes of Patients with Acute Myeloid Leukemia (AML) with Myelodysplasia Related Changes (AML-MRC) Are Dependent on Diagnostic Criteria and Therapy

Outcomes of Patients with Acute Myeloid Leukemia (AML) with Myelodysplasia Related Changes (AML-MRC) Are Dependent on Diagnostic Criteria and Therapy

Outcomes of Patients with Acute Myeloid Leukemia with Myelodysplastic Are Dependent on Diagnostic Criteria and Therapy

Outcomes of Patients with Acute Myeloid Leukemia with Myelodysplastic Are Dependent on Diagnostic Criteria and Therapy

Sirpαfc Treatment Targets Human Acute Myeloid Leukemia Stem Cells

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and is the most common type of acute leukemia in adults. Although a majority of patients achieve remission following cytotoxic chemotherapy, most will relapse and ultimately die. Therapy resistance and relapse are driven by leukemia stem cells (LSC). Evidence of genetic and functional heterogeneity in the LSC compartment underscores the importance of developing therapeutic strategies that will target all subclones effectively. We previously showed that LSCs in AML depend on CD47-SIRPα interaction to evade immune surveillance (Theocharides et al, JEM 2012). CD47 acts as a "do not eat me" signal that binds to the inhibitory receptor SIRPα on macrophages and masks cancer cells from macrophage-mediated phagocytosis. TTI-621 (Trillium Therapeutics Inc., Ontario, Canada) is a human SIRPαFc protein formed by fusing the IgV doman of human SIRPα to a human IgG1-Fc moiety; it is designed to bind CD47 on leukemia cells and disrupt its interaction with SIRPα on host macrophages. Our previous studies in AML cell lines and a small number of primary AML samples demonstrated increased phagocytosis in vitro and decreased engraftment in xenotransplant models following SIRPαFc treatment (Theocharides et al, JEM 2012, Petrova et al, Clin Cancer Res 2017). Here, we tested the efficacy of TTI-621 against a broad panel of primary AML samples in xenotransplantation models to determine efficacy and response rates in this heterogeneous disease. Bulk cells obtained from the peripheral blood of 30 AML patients representing a broad range of cytogenetic and molecular subtypes were transplanted intrafemorally into sublethally-irradiated NSG mice. After a 2-week engraftment period, mice were treated with either SIRPαFc or control IgG by intraperitoneal injection 3×/week for 4 weeks, following which leukemic engraftment was determined by flow cytometry. In all but 1 sample, a significant reduction in AML engraftment was seen in SIRPαFc-treated mice compared to controls. For 23 samples defined as good responders, SIRPαFc treatment resulted in 91% (range 53-100%, p&lt;0.0001) and 98% (range 88-100%, p&lt;0.0001) reduction of leukemic engraftment in the injected femur and in non-injected bones, respectively, compared to controls. Six samples demonstrated a lesser response that was largely observed in the non-injected bones, with relative reduction of 69% (range 43-93%, p=0.03); these samples were defined as partial responders. The majority of samples from patients with unfavorable features such as age &gt;60, adverse cytogenetic risk, and secondary AML, as well as samples obtained from relapsed/resistant patients, were classified as good responders. Notably, 20 of 23 good responders had a high LSC17 score, which we have shown is associated with poor initial therapy response and short survival following standard treatments (Ng et al, Nature 2016). To determine whether SIRPαFc treatment killed LSCs, we transplanted leukemia cells harvested from primary treated mice into untreated secondary recipients at limiting dilution. For four independent samples, including three partial responders and the one non-responder, we observed a significantly lower LSC frequency (3.9-10.3 fold, p=0.002-0.024) in mice transplanted with SIRPαFc-treated cells compared to controls, indicating that SIRPαFc treatment reduced LSC numbers in primary mice, despite partial or no reduction of bulk disease. Our data demonstrate that SIRPαFc effectively targets LSCs in a human AML xenotransplantion model with high response rates across a heterogeneous cohort of primary AML samples, including samples with unfavorable risk features. SIRPαFc may be most effective in the remission setting as maintenance therapy for patients with detectable residual disease, to eradicate residual LSCs and prevent relapse. Disclosures Jin: Trillium Therapeutics: Other: licensing agreement. Wong:Trillium Therapetuics: Employment. Uger:Trillium Therapetuics: Employment. Minden:Trillium Therapetuics: Other: licensing agreement. Danska:Trillium Therapeutics: Other: licensing agreement, Research Funding. Wang:Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; NanoString: Other: Travel and accommodation.