Therapy-related Leukemia Research Articles

Human CREBBP acetyltransferase is impaired by etoposide quinone, an oxidative and leukemogenic metabolite of the anticancer drug etoposide through modification of redox-sensitive zinc-finger cysteine residues

Etoposide is an extensively prescribed anticancer drug that, unfortunately, causes therapy-related leukemia. The mechanisms by which etoposide induces secondary hematopoietic malignancies are poorly documented. However, etoposide-related leukemogenesis is known to depend on oxidative metabolites of etoposide, notably etoposide quinone, that can react with protein cysteine residues such as in topoisomerases II. CREBBP is a major histone acetyltransferase that functions mainly as a transcriptional co-activator. This epigenetic enzyme is considered as a tumor suppressor that plays a major role in hematopoiesis. Genetic alterations affecting CREBBP activity are highly common in hematopoietic malignancies. We report here that CREBBP is impaired by etoposide quinone. Molecular and kinetic analyses show that this inhibition occurs through the rapid and covalent (kinhib = 16.102 M-1. s−1) adduction of etoposide quinone with redox sensitive cysteine residues within the RING and PHD Zn2+-fingers of CREBBP catalytic core leading to subsequent release of Zn2+. In agreement with these findings, experiments conducted in cells and in mice treated with etoposide showed irreversible inhibition of endogenous CREBBP activity and decreased H3K18 and H3K27 acetylation. As shown for topoisomerases II, our work thus suggests that the leukemogenic metabolite etoposide quinone can impair the epigenetic CREBBP acetyltransferase through reaction with redox sensitive cysteine residues.

Therapy-related B-cell acute lymphoblastic leukemia in adults has unique genetic profile with frequent loss of TP53 and inferior outcome.

Therapy-related B-cell acute lymphoblastic leukemia in adults has unique genetic profile with frequent loss of TP53 and inferior outcome.

Therapy-Related Pure Erythroid Leukaemia with Complex Karyotype Following Successful Treatment for Acute Promyelocytic Leukaemia

Therapy-Related Pure Erythroid Leukaemia with Complex Karyotype Following Successful Treatment for Acute Promyelocytic Leukaemia

Allogeneic Hematopoietic Stem Cell Transplantation in Therapy Related Acute Leukemia.

Therapy related acute leukemia consists of a unique subset of acute leukemia with an increased frequency of high risk cytogenetic and molecular abnormalities, dismal response to therapy, higher relapse rates and poor overall survival. Therapy related acute myeloid leukemia (t-AML) is a better defined disease entity than therapy related acute lymphoid leukemia (t-ALL). However, in recent times, t-ALL is also being increasingly recognized and extensively studied. Therapy related acute myeloid leukemia is usually classified together with therapy related myelodysplastic syndrome. However, the management of these two diseases maybe different regarding needs of induction chemotherapy and eligibility for upfront allogeneic hematopoietic stem cell transplantation (Allo HSCT). There is also evidence regarding differences in prognosis and outcomes between these two entities. Allo HSCT offers a potential for cure in t-AML and t-ALL. However, existing literature on the same is confounded by inclusion of t-MDS and secondary acute leukemias. Here we review the current evidence on the outcomes and predictors of outcomes of Allo HSCT in the management of therapy related acute leukemias. We also shed light into the under-representation of therapy related leukemias in clinical trials. This stresses the need for prospective trials incorporating measurable residual disease monitoring and sequential next generation sequencing based genomic data for accurate prognostication and management of therapy related acute leukemia.

56. Therapy-Related B-Lymphoblastic Leukemia in adults is associated with frequent TP53 alterations and inferior survival

56. Therapy-Related B-Lymphoblastic Leukemia in adults is associated with frequent TP53 alterations and inferior survival

Clinical impact of clonal hematopoiesis in patients with lymphoma undergoing ASCT: a national population-based cohort study.

Clonal hematopoiesis of indeterminate potential (CHIP) is suspected of being a risk factor for patients with cancer. This study aimed to assess the clinical consequences of CHIP in patients with lymphoma intended for high-dose chemotherapy and autologous stem-cell transplantation (ASCT) in a population-based setting. We identified 892 lymphoma patients who had undergone stem cell harvest at all transplant centers in Denmark. A total of 565 patients had an available harvest sample, which was analysed for CHIP by next-generation sequencing, and the median follow-up was 9.1 years. Of the patients who were intended for immediate ASCT, 25.5% (112/440) carried at least one CHIP mutation. In contrast to previous single-center studies CHIP was not associated with inferior overall survival (OS) in multivariate analyses. However, patients with mutations in genes of the DNA repair pathway (PPM1D, TP53, RAD21, BRCC3) had a significant inferior OS (HR after 1 year of follow-up 2.79, 95% confidence interval 1.71-4.56; p < 0.0001), which also was evident in multivariate analysis (p = 0.00067). These patients had also increased rates of therapy-related leukemia and admission to intensive care. Furthermore, in patients who did not undergo immediate ASCT, a significant inferior OS of individuals with DNA repair mutations was also identified (p = 0.003).

Outcomes of therapy-related acute lymphoblastic leukemia in adults after allogeneic stem cell transplantation.

Therapy-related acute lymphoblastic leukemia (t-ALL) is an increasingly recognized subset of therapy-related acute leukemia. There are limited data on the role of allogeneic hematopoietic stem cell transplantation (HSCT) in t-ALL. Recent reports suggest comparable outcomes of t-ALL with de novo ALL after HSCT. We retrospectively reviewed all patients of t-ALL who underwent HSCT at our center. Patients were analyzed for prior malignancy, therapy, time to diagnosis of t-ALL, clinical, laboratory characteristics, transplant details, relapse-free survival (RFS), and overall survival (OS). Eighteen patients (M:F ratio 1:1; Median age 44years) underwent HSCT for t-ALL. Median latent period from primary malignancy to t-ALL was 44.8months. 11q23 rearrangement and t(9;22) were present in 33.3% and 22.2% patients, respectively. Stem cell donors were matched related, matched unrelated, and haploidentical in 27.8% (n=5), 55.6% (n=10), and 16.7% (n=3) patients, respectively. Five patients died before D+ 100 (27.8%). Estimated 2-year RFS and OS were 47.1% and 51.8%, respectively. We did not find any pretransplant and post-transplant risk factors that were predictive of improved OS or RFS after multivariate analysis. Allogeneic HSCT outcomes in t-ALL were comparable to HSCT outcomes in de novo ALL. Multicenter studies with more patients and longer follow-up may provide factors affecting outcome and survival.

Secondary therapy-related acute megakaryocytic leukemia in childhood acute lymphocytic leukemia: report of one case and review of literature

Objective To explore the laboratory characteristics and diagnostic methods for therapy-related acute megakaryocytic leukemia (t-AMKL). Methods The data of one child with acute lymphoblastic leukemia (ALL) in the Blood Disease Hospital of Chinese Academy of Medical Sciences & Peking Union Medical College in September 2014 was retrospectively analyzed. After inducing remission for more than 43 months, the child was diagnosed as t-AMKL. Results After the diagnosis of ALL, the child was given chemotherapy with standard childhood ALL regimen. After 43 months, t-AMKL was diagnosed by comprehensive morphology, cytogenetics, and molecular biology. Bone marrow morphology showed that the proportion of primitive cells was 0.44; flow cytometry showed the phenotype was abnormal myeloid primitive cells; the pathology result showed that the abnormal cells weakly expressed CD42b and CD61; the electron microscopy showed platelet peroxidase (PPO)-positive and myeloperoxidase (MPO)-negative; the bone marrow immunohistochemistry showed the positive rate of CD41 was 34%; the child had a complex karyotype. After reviewing his medical history, he was diagnosed as t-AMKL. Conclusion The t-AMKL is relatively rare, and it is helpful to improve the prognosis of patients by completing the relevant examinations for early diagnosis. Key words: Leukemia, lymphoid; Child; Leukemia, megakaryoblastic, acute; Neoplasms, second primary; Diagnosis

Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Therapy-related acute lymphoblastic leukemia is a distinct entity with adverse genetic features and clinical outcomes

AbstractPatients with therapy-related acute lymphoblastic leukemia (t-ALL) represent a small subset of acute lymphoblastic leukemia (ALL) patients who received genotoxic therapy (ie, chemotherapy or radiation) for a prior malignancy. These patients should be distinguished from patients with de novo ALL (dn-ALL) and ALL patients who have a history of prior malignancy but have not received cytotoxic therapies in the past (acute lymphoblastic leukemia with prior malignancy [pm-ALL]). We report a retrospective multi-institutional study of patients with t-ALL (n = 116), dn-ALL (n = 100), and pm-ALL (n = 20) to investigate the impact of prior cytotoxic therapies on clinical outcomes. Compared with patients with pm-ALL, t-ALL patients had a significantly shorter interval between the first malignancy and ALL diagnosis and a higher frequency of poor-risk cytogenetic features, including KMT2A rearrangements and myelodysplastic syndrome-like abnormalities (eg, monosomal karyotype). We observed a variety of mutations among t-ALL patients, with the majority of patients exhibiting mutations that were more common with myeloid malignancies (eg, DNMT3A, RUNX1, ASXL1), whereas others had ALL-type mutations (eg, CDKN2A, IKZF1). Median overall survival was significantly shorter in the t-ALL cohort compared with patients with dn-ALL or pm-ALL. Patients who were eligible for hematopoietic cell transplantation had improved long-term survival. Collectively, our results support t-ALL as a distinct entity based on its biologic and clinical features.

Characteristics and Outcomes of Therapy-Related Versus De Novo Acute Myeloid Leukemia with Normal Karyotype

Characteristics and Outcomes of Therapy-Related Versus De Novo Acute Myeloid Leukemia with Normal Karyotype

Mitochondrial dysfunction and oxidative stress in bone marrow stromal cells induced by daunorubicin leads to DNA damage in hematopoietic cells

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism, some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study, we investigated the damage to BM stromal cells induced by a chemotherapy drug, daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence, generation of intracellular reactive oxygen species, production of cytokines and chemokines, and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function, and negative regulators of reactive oxygen species. Surprisingly, it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore, coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2′-deoxyguanosine in hematopoietic cells. Overall, our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells.

1399P - Hematologic malignancies in temozolomide-treated metastatic pancreatic neuroendocrine tumours

BackgroundMetastatic pancreatic neuroendocrine tumors (PNET) are generally not curable; however, some patients may have prolonged survival. Until recently, metastatic PNET were primarily managed with somatostatin-analogs. New developments demonstrating therapeutic value of temozolomide (TMZ) in these patients resulted in its off-label use in National Comprehensive Cancer Network guidelines. Given alone, or in combination with other therapies, TMZ is associated with improved clinical outcomes. However, serious hematologic adverse events (AEs) like agranulocytosis, lymphopenia and aplastic anemia are not uncommon. At the University of Kansas Cancer Center (KUCC), 3 patients with history of TMZ-treated metastatic PNET developed hematologic malignancies. The purpose of this study is to determine the incidence of secondary malignancies (SM) in this patient population. MethodsA systematic review of all known clinical trials, case reports, and other relevant literature regarding PNET and TMZ published before September 2017 was conducted using PubMed, Embase, Cochrane Library, and the FDA Adverse Event Reporting System (FAERS). ResultsWe analyzed 38 publications and 8,215 cases reported from FAERS. AEs ranged from agranulocytosis to myelodysplastic syndrome. No publications reported any SM. The 3 patients identified at KUCC are as follows: 1) 29-year-old female with TMZ-treated metastatic PNET developed acute myeloid leukemia with cytogenetics consistent with therapy-related leukemia. 2) 80-year-old male with TMZ-treated metastatic PNET developed diffuse large B-cell lymphoma. 3) 12-year-old male with TMZ-treated metastatic PNET developed high-grade T-cell lymphoblastic lymphoma. All these patients succumbed to their hematologic malignancies, and not the underlying PNET. ConclusionsAlthough we observed 3 cases at KUCC, this retrospective review did not find any cases of SM in TMZ-treated metastatic PNET. We believe that the leukemogenic potential of TMZ is underreported. It is important for treatment guidelines to address this risk in the decision to pursue TMZ treatment. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Abstract 1233: Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity

Abstract In cancer, suppression of host immunity is related, in part, to immature myeloid-derived suppressor cells (MDSCs) expanded in response to various tumor- or stroma-derived factors. Here, the expression and activity of myeloperoxidase (MPO) was examined in both granulocytic (PMN) and monocytic (M) MDSC subsets recovered from the spleens of 4T1 tumor-bearing mice, as well as interferon regulatory factor-8 (IRF8) knockout mice (IRF8-/-) which develop high levels of MDSCs due to IRF8-deficiency. Phenotypically identical cells from non-tumor-bearing mice served as controls. In addition, the role of MPO in PMN-MDSCs as a determinant of DNA damage induced by the anticancer agents etoposide (VP-16), camptothecin (CPT) and parthenolide (PTL), was evaluated for potential immunomodulatory effects. MPO protein content was ~4-fold greater in PMN-MDSCs from tumor-bearing compared to non-tumor-bearing mice, while MPO specific activity was equivalent in these two populations. MPO protein content in PMN-MDSCs from IRF8-/- mice was at least comparable to levels found in tumor-bearing hosts. In both IRF8-/- and tumor-bearing mice, PMN-MDSCs contained 5-10-fold greater MPO protein and overall enzyme activity compared to isolated M-MDSCs. VP-16 (5 µM)- and PTL (100 µM)-induced DNA strand breaks were 4-fold greater in PMN- compared to M-MDSCs from tumor-bearing mice. In addition, the heme-synthesis inhibitor, succinylacetone (SA) reduced MPO activity 2-3-fold in PMN-MDSCs from tumor-bearing mice with a corresponding decrease in drug-induced DNA damage for both VP-16 and PTL with no effect on CPT-induced DNA damage. The antioxidant dehydroascorbate decreased VP-16-induced DNA damage in PMN-MDSCs containing MPO activity, but not in those cells pre-incubated with SA to deplete MPO. These results indicate that MPO-dependent activation of VP-16 and PTL enhance DNA damage. Antioxidant protection against VP-16-induced DNA damage is consistent with MPO-mediated oxidative activation of VP-16 to a redox-cycling phenoxyl radical (Mol. Pharmacol. 79: 479-487, 2011). Overall, these results suggest that: 1) PTL may be useful to eliminate PMN-MDSCs; 2) MPO activation of VP-16 in PMN-MDSCs may be deleterious via oxidative DNA damage resulting in mixed-lineage leukemia (MLL) gene rearrangements known to be causal for therapy-related myeloid leukemias. Support: CA172105; CA090787; CA140622 Citation Format: Jack C. Yalowich, Ragu Kanagasabai, Colleen S. Netherby, Michelle N. Messmer, Scott I. Abrams. Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1233.

Therapy-related acute leukemia with mixed phenotype initially misdiagnosed as therapy-related all

Therapy-related acute leukemia with mixed phenotype initially misdiagnosed as therapy-related all

Late relapse in acute myeloid leukemia (AML): clonal evolution or therapy-related leukemia?

Late relapse, defined as relapse arising after at least 5 years of remission, is rare and occurs in 1–3% of patients with acute myeloid leukemia (AML). The underlying mechanisms of late relapse remain poorly understood. We identified patients with AML who achieved remission with standard induction chemotherapy and relapsed after at least five years of remission (n = 15). Whole exome sequencing was performed in available bone marrow samples obtained at diagnosis (n = 10), remission (n = 6), and first relapse (n = 10). A total of 41 driver mutations were identified, of which 11 were primary tumor-specific, 17 relapse-specific, and 13 shared (detected both in primary and relapsed tumor samples). We demonstrated that 12 of 13 shared mutations were in epigenetic modifier and spliceosome genes. Longitudinal genomic characterization revealed that in eight of 10 patients the founder leukemic clone persisted after chemotherapy and established the basis of relapse years later. Understanding the mechanisms of such quiescence in leukemic cells may help designing future strategies aimed at increasing remission duration in patients with AML.

Therapy-Related Myeloid Neoplasms with Balanced Chromosome Rearrangements Frequently Arise from Pre-Existing Clonal Haematopoiesis

IntroductionTherapy-related myeloid neoplasms (tMN) are an increasing healthcare problem resulting from rising long-term survival from primary cancers. Approximately 80% of cases are associated with an adverse karyotype and have a dismal prognosis; pre-existing clonal haematopoiesis (CH) appears to be a predisposing factor and mutations in genes including TP53, PPM1D and DNMT3A have been detected prior to chemo- or radiotherapy exposure. In contrast ~20% of tMN are characterised by balanced chromosome rearrangements and have a relatively favourable outcome; chemotherapy agents targeting topoisomerase II have been implicated in generating these rearrangements however the involvement of CH has not previously been described.Methods and resultsWe performed whole exome sequencing (WES) of samples taken at diagnosis (D) and molecular complete remission (mCR) followed by targeted capture and error-corrected deep sequencing (ECS) on ≥2 mCR samples for 11 patients with therapy-related acute promyelocytic leukemia (tAPL) and 20 patients with de novo APL (dnAPL). All patients had t(15;17) / PML-RARA at diagnosis with no additional cytogenetic abnormality.The median age of patients with tAPL was 46.7y (range 30-78); primary cancer types were breast (4), colorectal (2), lymphoma (3), CNS (1) and testicular (1). Ten patients had received chemotherapy and 4 radiotherapy. The median latency between primary cancer treatment and tAPL diagnosis was 3.9y (range 2.2-6.1). Patients received a mixture of chemotherapy- and arsenic-based treatments.The median age of patients with dnAPL was 40.3y (range 18-69); all patients were treated with the AIDA regimen and none developed tMN subsequently.There were no significant differences between the number or type of mutations between dnAPL and tAPL however disease associated somatic mutations were detectable in mCR samples by ECS in 4/11 tAPL samples compared to 0/20 dnAPL samples (p=0.04).Mutations detected in mCR were UPN1: PPM1D exon (e) 6 1bp deletion (del, variant allele fraction, VAF, D 32% mCR 11.3%); UPN2: DNMT3A e8 1bp del (VAF D 40.2% mCR 0.48%); UPN3: DNMT3A e10 1bp del (VAF D 35.4% mCR 2.9%); UPN4: MYCN e3 6bp insertion (ins, VAF D 38% mCR 0.37%).We screened samples from these patients and a further 39 dnAPL patients for a panel of genes with known CH associated mutations (CH-M) using ECS and detected additional mutations in 2 tAPL patients (UPN4, DNMT3A G104R, VAF D 0% mCR 3% and UPN5 DNMT3A R693H VAF D 2% mCR 25%, who subsequently developed tMN with complex karyotype). We did not detect CH-M in diagnostic samples from any patient with dnAPL and publicly available NGS datasets encompassing 220 patients only showed 1 APL case with a CH-M (DNMT3A e8 ins, prior cytotoxic exposure unknown). We detected treatment-emergent CH clones in mCR by ECS in 6/59 dnAPL patients treated with AIDA (DNMT3A n=3, PPM1D n=2, TP53 n=1, SF3B1 n=1).Applying the same techniques to six patients with therapy-related core-binding factor AML, we identified a persistent CH-M in mCR in one (DNMT3A e15 del, VAF D 38%, mCR 11.8%).DNA samples taken at the time of primary cancer diagnosis were available from UPN 1-3. In UPN1 we detected the PPM1D mutation in a lymph node (LN) involved with T-cell lymphoma (LN) (VAF 2.1%) and an uninvolved staging bone marrow (VAF 3.3%). In UPN3 the DNMT3A mutation was detected in a breast biopsy (VAF 0.7%) and LN involved with carcinoma (VAF 0.9%). In UPN2 the DNMT3A mutation was not detected in the LN biopsy diagnostic for Hodgkin lymphoma.We used FACS to isolate T, B, monocyte, granulocyte and CD34+ cells from complete remission samples from UPN1-4 with >99% purity and detected the persistent mutations in each cell compartment e.g. UPN3 VAF: T cell 1.3%, B cell 8.6%, monocyte 4%, granulocyte 3.7%, CD34+ 3.9%.Diagnostic material from UPN1 and 4 was injected into irradiated NSG mice. After 12 weeks we detected multilineage human engraftment in both samples by FACS in 1/3 mice from UPN1 and 2/3 mice from UPN4. We detected the PPM1D and MYCN mutations respectively in bone marrow samples from each engrafting mouse.ConclusionsTogether these findings indicate that tMN with balanced chromosome rearrangements can occur on a background of non-malignant CH. Using ECS we observed this phenomenon in 5/17 (29%) patients with therapy-related APL or CBF AML. This has important implications for planning curative therapy, notably for tAPL where effective cytotoxic-free regimens are available. DisclosuresRussell:Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau; Daiichi Sankyo: Consultancy. Hills:Daiichi Sankyo: Consultancy, Honoraria.

Bone Marrow T Cell Changes By Multiplex IHC after Treatment with Flotetuzumab, a CD123 x CD3 Bispecific Dart® Protein, in a Primary Refractory t-AML Patient

Therapy-related acute myelogenous leukemia (t-AML) is associated with adverse genetic lesions, complex karyotype, and TP53 mutation; it is challenging to treat and confers a poor prognosis. We describe a 74-year-old female patient with AML that developed 9 years after receiving 6 cycles of cytotoxic (FOLFOX) chemotherapy as adjuvant treatment of colorectal carcinoma. At diagnosis, the bone marrow (BM) biopsy revealed blast count of 35%, with normal karyotype, 5q loss and AML1 (RUNX1) locus (21q22) amplification by fluorescence in situ hybridization (FISH). Initial treatment with 5 cycles of azacitidine (AZA) failed to induce a response. The patient was subsequently treated on a Phase 1 trial of flotetuzumab (MGD006/S80880) (FLZ), a novel T-cell redirecting (CD123 x CD3) DART protein [NCT02152956]. Prior to FLZ, BM blasts demonstrated clonal evolution with a complex karyotype (92,XXXX, t(14;21)(q22;q22)) and new IDH1, and TET2 mutations.Patient received a total of 3 cycles (28 days/cycle) of FLZ. After one FLZ cycle, the patient achieved a (complete response) CR, with normal cytogenetics, and residual IDH1 and TET2 mutations. After 2 additional cycles of FLZ consolidation, the CR was maintained, with loss of the IDH1 mutation, but persistence of the TET2 mutation. CR was maintained with no additional therapy for approximately 7 months. Serial BM samples were evaluated for T cells (CD3, CD4, CD8, FOXP3 and PD-L1) using multiplex IHC (Fig 1). BM T cells were unchanged during AZA treatment. At the initial bone marrow on FLZ (CR), significant increases in CD3+, CD3+CD8-, CD3+ CD8+, and FOXP3-positive T cells and PD-L1 expression compared to baseline were noted. CD3+CD8+ T cells persisted in the BM 1 month beyond completion of the additional 2 cycles of consolidation with FLZ, while other T cells subsets and PD-L1 expression returned to baseline (Fig. 1) Three months after the last FLZ treatment, all T cell subsets had returned to baseline, with an early increase in leukemic blasts (8%), but normal cytogenetics. Five months after FLZ treatment blasts were unchanged, but a new abnormal cytogenetic clone, with additional mutations, accompanied by a rise in PD-L1 expression cells was observed. Seven months after FLZ treatment, frank leukemia with 50% blasts developed, and all T cell subsets and PD-L1 expression had returned to pre-treatment levels.Consistent with its proposed mechanism of action, these data highlight for the first time the induction of an increase in T-cell infiltration, which persist beyond FLZ, in the bone marrow microenvironment of an AML patient that also achieved a complete and durable anti-leukemic response after treatment with FLZ. [Display omitted] DisclosuresLelièvre:Servier: Employment. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment. Fox:MacroGenics: Research Funding.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

Characteristics and Predictors for Secondary Leukemia and Myelodysplastic Syndrome in Ewing and Osteosarcoma Survivors

Long-term survivors of Ewing sarcoma (ES) and osteosarcoma may be at risk for therapy-related acute leukemia or myelodysplastic syndrome (t-AL/MDS). We retrospectively reviewed the clinicopathologic characteristics of 1071 patients with osteosarcoma (n=757) and ES (n=314) who were treated between 1985 and 2014. Multivariable competing risk analysis was used to analyze predictors of t-AL/MDS, including a radiation dose (≥55.8Gy vs <55.8Gy)×disease site (pelvis/spine vs other) interaction term. A supplemental nested case-control study was conducted to assess the association between cumulative chemotherapy dose and t-AL/MDS. The median follow-up for surviving patients was 97months (range, 0.03-380). Twenty patients developed t-AL/MDS, all of whom received chemotherapy and 15 of whom were treated with radiation therapy. Radiation therapy to ≥55.8Gy was associated with development of t-AL/MDS (adjusted hazard ratio, 2.89; 95% confidence interval [CI], 1.23-6.80; P=.015), and there was a significant radiation dose×disease site interaction term (adjusted hazard ratio, 6.70; 95% CI, 2.71-16.53; Pinteraction<.001). The 5-year cumulative incidence of t-AL/MDS in patients receiving ≥55.8Gy radiation therapy to the pelvis or spine was 5.0% (95% CI,0.9-14.9) for osteosarcoma and 10.7% for ES (95% CI, 3.3-23.2). In our nested case-control study, cumulative doses of ifosfamide and etoposide were associated with development of t-AL/MDS. Patients with osteosarcoma and ES receiving ≥55.8Gy of radiation therapy to the pelvis or spine appear to be at increased risk for t-AL/MDS. Treatment with high cumulative doses of chemotherapy may further augment this risk.