Pharmacogenomics, Race, and Treatment Outcome in Pediatric Acute Myeloid Leukemia.

Importance: Disparities in outcomes exist between Black and White patients with acute myeloid leukemia (AML), with Black patients experiencing poorer prognosis compared with their White counterparts. Objective: To assess whether varying intensity of induction therapy to treat pediatric AML is associated with reduced disparities in treatment outcome by race. Design, Setting, and Participants: A comparative effectiveness analysis was conducted of 86 Black and 359 White patients with newly diagnosed AML who were enrolled in the AML02 trial from 2002 to 2008 or the AML08 trial from 2008 to 2017. Statistical analysis was conducted from July 2023 through January 2024. Interventions: Patients in AML02 were randomly assigned to receive standard low-dose cytarabine-based induction therapy or augmented high-dose cytarabine-based induction therapy, whereas patients in AML08 received high-dose cytarabine-based therapy. Main Outcomes and Measures: Cytarabine pharmacogenomic 10-single-nucleotide variant (ACS10) scores were evaluated for association with outcome according to race and treatment arm. Results: This analysis included 86 Black patients (mean [SD] age, 8.8 [6.5] years; 54 boys [62.8%]; mean [SD] leukocyte count, 52 600 [74 000] cells/µL) and 359 White patients (mean [SD] age, 9.1 [6.2] years; 189 boys [52.6%]; mean [SD] leukocyte count, 54 500 [91 800] cells/µL); 70 individuals with other or unknown racial and ethnic backgrounds were not included. Among all patients without core binding factor AML who received standard induction therapy, Black patients had significantly worse outcomes compared with White patients (5-year event-free survival rate, 25% [95% CI, 9%-67%] compared with 56% [95% CI, 46%-70%]; P = .03). By contrast, among all patients who received augmented induction therapy, there were no differences in outcome according to race (5-year event-free survival rate, Black patients, 50% [95% CI, 38%-67%]; White patients, 48% [95% CI, 42%-55%]; P = .78). Among patients who received standard induction therapy, those with low ACS10 scores had a significantly worse 5-year event-free survival rate compared with those with high scores (42.4% [95% CI, 25.6%-59.3%] and 70.0% [95% CI, 56.6%-83.1%]; P = .004); however, among patients who received augmented induction therapy, there were no differences in 5-year event-free survival rates according to ACS10 score (low score, 60.6% [95% CI, 50.9%-70.2%] and high score, 54.8% [95% CI, 47.1%-62.5%]; P = .43). Conclusions and Relevance: In this comparative effectiveness study of pediatric patients with AML treated in 2 consecutive clinical trials, Black patients had worse outcomes compared with White patients after treatment with standard induction therapy, but this disparity was eliminated by treatment with augmented induction therapy. When accounting for ACS10 scores, no outcome disparities were seen between Black and White patients. Our results suggest that using pharmacogenomics parameters to tailor induction regimens for both Black and White patients may narrow the racial disparity gap in patients with AML.

The Tomato Brown Rugose Fruit Virus Movement Protein Gene Is a Novel Microbial Source Tracking Marker.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples.

Intrinsic suppression of type I interferon production underlies the therapeutic efficacy of IL-15-producing natural killer cells in B-cell acute lymphoblastic leukemia.

BACKGROUND: Type I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown. METHODS: Using high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL. RESULTS: We find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChigh human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15. CONCLUSION: We find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.

Tomato brown rugose fruit virus Mo gene is a novel microbial source tracking marker.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker. Importance: Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.

Identification and in vitro validation of neoantigens for immune activation against high-risk pediatric leukemia cells.

There is experimental and clinical data to indicate the contribution of immune-escape mechanisms in relapsed/refractory pediatric leukemia. Studies have shown the accumulation of mutations that translate to peptides containing tumor-specific epitopes (neoantigens). The effectiveness of neoantigen-based vaccines has been shown in several clinical trials in adults. Though the initial results are encouraging, this knowledge must be developed to account for the uniqueness of pediatric cancer biology. We have completed the initial proof-of-concept analysis on a high-risk pediatric leukemia specimen and identified usable neoantigen sequences. We describe this approach, including the bioinformatics method and experimental model to verify their function that can be further broadened for personalized neoantigen prediction and testing for the generation of anticancer vaccines against high-risk pediatric leukemias.

A Comprehensive Circulating Tumor DNA Assay for Detection of Translocation and Copy-Number Changes in Pediatric Sarcomas.

Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.

Activated natural killer cells predict poor clinical prognosis in high-risk B- and T-cell acute lymphoblastic leukemia.

B- and T-cell acute lymphoblastic leukemia (B/T-ALL) may be refractory or recur after therapy by suppressing host anticancer immune surveillance mediated specifically by natural killer (NK) cells. We delineated the phenotypic and functional defects in NK cells from high-risk patients with B/T-ALL using mass cytometry, flow cytometry, and in silico cytometry, with the goal of further elucidating the role of NK cells in sustaining acute lymphoblastic leukemia (ALL) regression. We found that, compared with their normal counterparts, NK cells from patients with B/T-ALL are less cytotoxic but exhibit an activated signature that is characterized by high CD56, high CD69, production of activated NK cell-origin cytokines, and calcium (Ca2+) signaling. We demonstrated that defective maturation of NK cells into cytotoxic effectors prevents NK cells from ALL from lysing NK cell-sensitive targets as efficiently as do normal NK cells. Additionally, we showed that NK cells in ALL are exhausted, which is likely caused by their chronic activation. We found that increased frequencies of activated cytokine-producing NK cells are associated with increased disease severity and independently predict poor clinical outcome in patients with ALL. Our studies highlight the benefits of developing NK cell profiling as a diagnostic tool to predict clinical outcome in patients with ALL and underscore the clinical potential of allogeneic NK cell infusions to prevent ALL recurrence.

Venetoclax and Navitoclax in Combination with Chemotherapy in Patients with Relapsed or Refractory Acute Lymphoblastic Leukemia and Lymphoblastic Lymphoma.

Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-XL/BCL2 inhibitor, may allow targeting of both BCL2 and BCL-XL without dose-limiting thrombocytopenia associated with navitoclax monotherapy. The safety and preliminary efficacy of venetoclax with low-dose navitoclax and chemotherapy was assessed in this phase I dose-escalation study (NCT03181126) in pediatric and adult patients with relapsed/refractory (R/R) acute lymphoblastic leukemia or lymphoblastic lymphoma. Forty-seven patients received treatment. A recommended phase II dose of 50 mg navitoclax for adults and 25 mg for patients <45 kg with 400 mg adult-equivalent venetoclax was identified. Delayed hematopoietic recovery was the primary safety finding. The complete remission rate was 60%, including responses in patients who had previously received hematopoietic cell transplantation or immunotherapy. Thirteen patients (28%) proceeded to transplantation or CAR T-cell therapy on study. Venetoclax with navitoclax and chemotherapy was well tolerated and had promising efficacy in this heavily pretreated patient population. SIGNIFICANCE: In this phase I study, venetoclax with low-dose navitoclax and chemotherapy was well tolerated and had promising efficacy in patients with relapsed/refractory acute lymphoblastic leukemia or lymphoblastic lymphoma. Responses were observed in patients across histologic and genomic subtypes and in those who failed available therapies including stem cell transplant.See related commentary by Larkin and Byrd, p. 1324.This article is highlighted in the In This Issue feature, p. 1307.

Safety, pharmacokinetics, and pharmacodynamics of panobinostat in children, adolescents, and young adults with relapsed acute myeloid leukemia.

BACKGROUND: Novel therapies are urgently needed for pediatric patients with relapsed acute myeloid leukemia (AML). METHODS: To determine whether the histone deacetylase inhibitor panobinostat could be safely given in combination with intensive chemotherapy, a phase 1 trial was performed in which 17 pediatric patients with relapsed or refractory AML received panobinostat (10, 15, or 20 mg/m2 ) before and in combination with fludarabine and cytarabine. RESULTS: All dose levels were tolerated, with no dose-limiting toxicities observed at any dose level. Pharmacokinetic studies demonstrated that exposure to panobinostat was proportional to the dose given, with no associations between pharmacokinetic parameters and age, weight, or body surface area. Among the 9 patients who had sufficient (>2%) circulating blasts on which histone acetylation studies could be performed, 7 demonstrated at least 1.5-fold increases in acetylation. Although no patients had a decrease in circulating blasts after single-agent panobinostat, 8 of the 17 patients (47%), including 5 of the 6 patients treated at dose level 3, achieved complete remission. Among the 8 complete responders, 6 (75%) attained negative minimal residual disease status. CONCLUSIONS: Panobinostat can be safely administered with chemotherapy and results in increased blast histone acetylation. This suggests that it should be further studied in AML.

RSK inhibitor BI-D1870 inhibits acute myeloid leukemia cell proliferation by targeting mitotic exit.

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.

Venetoclax in combination with cytarabine with or without idarubicin in children with relapsed or refractory acute myeloid leukaemia: a phase 1, dose-escalation study.

BACKGROUND: Outcomes for children with relapsed or refractory acute myeloid leukaemia remain poor. The BCL-2 inhibitor, venetoclax, has shown promising activity in combination with hypomethylating agents and low-dose cytarabine in older adults for whom chemotherapy is not suitable with newly diagnosed acute myeloid leukaemia. We aimed to determine the safety and explore the activity of venetoclax in combination with standard and high-dose chemotherapy in paediatric patients with relapsed or refractory acute myeloid leukaemia. METHODS: We did a phase 1, dose-escalation study at three research hospitals in the USA. Eligible patients were aged 2-22 years with relapsed or refractory acute myeloid leukaemia or acute leukaemia of ambiguous lineage with adequate organ function and performance status. During dose escalation, participants received venetoclax orally once per day in continuous 28-day cycles at either 240 mg/m2 or 360 mg/m2, in combination with cytarabine received intravenously every 12 h at either 100 mg/m2 for 20 doses or 1000 mg/m2 for eight doses, with or without intravenous idarubicin (12 mg/m2) as a single dose, using a rolling-6 accrual strategy. The primary endpoint was the recommended phase 2 dose of venetoclax plus chemotherapy and the secondary endpoint was the proportion of patients treated at the recommended phase 2 dose who achieved complete remission or complete remission with incomplete haematological recovery. Analyses were done on patients who received combination therapy. The study is registered with ClinicalTrials.gov (NCT03194932) and is now enrolling to address secondary and exploratory objectives. FINDINGS: Between July 1, 2017, and July 2, 2019, 38 patients were enrolled (aged 3-22 years; median 10 [IQR 7-13]), 36 of whom received combination therapy with dose escalation, with a median follow-up of 7·1 months (IQR 5·1-11·2). The recommended phase 2 dose of venetoclax was found to be 360 mg/m2 (maximum 600 mg) combined with cytarabine (1000 mg/m2 per dose for eight doses), with or without idarubicin (12 mg/m2 as a single dose). Overall responses were observed in 24 (69%) of the 35 patients who were evaluable after cycle 1. Among the 20 patients treated at the recommended phase 2 dose, 14 (70%, 95% CI 46-88) showed complete response with or without complete haematological recovery, and two (10%) showed partial response. The most common grade 3-4 adverse events were febrile neutropenia (22 [66%]), bloodstream infections (six [16%]), and invasive fungal infections (six [16%]). Treatment-related death occurred in one patient due to colitis and sepsis. INTERPRETATION: The safety and activity of venetoclax plus chemotherapy in paediatric patients with heavily relapsed and refractory acute myeloid leukaemia suggests that this combination should be tested in newly diagnosed paediatric patients with high-risk acute myeloid leukaemia. FUNDING: US National Institutes of Health, American Lebanese Syrian Associated Charities, AbbVie, and Gateway for Cancer Research.

Epigenetic Targeting of TERT-Associated Gene Expression Signature in Human Neuroblastoma with TERT Overexpression.

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy. SIGNIFICANCE: Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.

Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.

AIM: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs. METHODS: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute. RESULTS: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance. CONCLUSION: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.

Clofarabine Can Replace Anthracyclines and Etoposide in Remission Induction Therapy for Childhood Acute Myeloid Leukemia: The AML08 Multicenter, Randomized Phase III Trial.

PURPOSE: To identify effective and less toxic therapy for children with acute myeloid leukemia, we introduced clofarabine into the first course of remission induction to reduce exposure to daunorubicin and etoposide. PATIENTS AND METHODS: From 2008 through 2017, 285 patients were enrolled at eight centers; 262 were randomly assigned to receive clofarabine and cytarabine (Clo+AraC, n = 129) or high-dose cytarabine, daunorubicin, and etoposide (HD-ADE, n = 133) as induction I. Induction II consisted of low-dose ADE given alone or combined with sorafenib or vorinostat. Consolidation therapy comprised two or three additional courses of chemotherapy or hematopoietic cell transplantation. Genetic abnormalities and the level of minimal residual disease (MRD) at day 22 of initial remission induction determined final risk classification. The primary end point was MRD at day 22. RESULTS: Complete remission was induced after two courses of therapy in 263 (92.3%) of the 285 patients; induction failures included four early deaths and 15 cases of resistant leukemia. Day 22 MRD was positive in 57 of 121 randomly assigned evaluable patients (47%) who received Clo+AraC and 42 of 121 patients (35%) who received HD-ADE (odds ratio, 1.86; 95% CI, 1.03 to 3.41; P = .04). Despite this result, the 3-year event-free survival rate (52.9% [44.6% to 62.8%] for Clo+AraC v 52.4% [44.0% to 62.4%] for HD-ADE, P = .94) and overall survival rate (74.8% [67.1% to 83.3%] for Clo+AraC v 64.6% [56.2% to 74.2%] for HD-ADE, P = .1) did not differ significantly across the two arms. CONCLUSION: Our findings suggest that the use of clofarabine with cytarabine during remission induction might reduce the need for anthracycline and etoposide in pediatric patients with acute myeloid leukemia and may reduce rates of cardiomyopathy and treatment-related cancer.

A phase II clinical trial of adoptive transfer of haploidentical natural killer cells for consolidation therapy of pediatric acute myeloid leukemia.

Consolidation therapies for children with intermediate- or high-risk acute myeloid leukemia (AML) are urgently needed to achieve higher cure rates while limiting therapy-related toxicities. We determined if adoptive transfer of natural killer (NK) cells from haploidentical killer immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA)-mismatched donors may prolong event-free survival in children with intermediate-risk AML who were in first complete remission after chemotherapy. Patients received cyclophosphamide (Day - 7), fludarabine (Days - 6 through - 2), and subcutaneous interleukin-2 (Days - 1, 1, 3, 5, 7, and 9). Purified, unmanipulated NK cells were infused on Day 0, and NK cell chimerism and phenotyping from peripheral blood were performed on Days 7, 14, 21, and 28. As primary endpoint, the event-free survival was compared to a cohort of 55 patients who completed chemotherapy and were in first complete remission but did not receive NK cells. Donor NK cell kinetics were determined as secondary endpoints. Twenty-one patients (median age at diagnosis, 6.0 years [range, 0.1-15.3 years]) received a median of 12.5 × 106 NK cells/kg (range, 3.6-62.2 × 106 cells/kg) without major side effects. All but 3 demonstrated transient engraftment with donor NK cells (median peak donor chimerism, 4% [range, 0-43%]). KIR-HLA-mismatched NK cells expanded in 17 patients (81%) and contracted in 4 (19%). However, adoptive transfer of NK cells did not decrease the cumulative incidence of relapse (0.393 [95% confidence interval: 0.182-0.599] vs. 0.35 [0.209-0.495]; P = .556) and did not improve event-free (60.7 ± 10.9% vs. 69.1 ± 6.8%; P = .553) or overall survival (84.2 ± 8.5% vs. 79.1 ± 6.6%; P = .663) over chemotherapy alone. The lack of benefit may result from insufficient numbers and limited persistence of alloreactive donor NK cells but does not preclude its potential usefulness during other phases of therapy, or in combination with other immunotherapeutic agents. TRIAL REGISTRATION: www.clinicaltrials.gov , NCT00703820 . Registered 24 June 2008.

Tracking Cell Transplants in Femoral Osteonecrosis with Magnetic Resonance Imaging: A Proof-of-Concept Study in Patients.

PURPOSE: Osteonecrosis is a devastating complication of high-dose corticosteroid therapy in patients with cancer. Core decompression for prevention of bone collapse has been recently combined with the delivery of autologous concentrated bone marrow aspirates. The purpose of our study was to develop an imaging test for the detection of transplanted bone marrow cells in osteonecrosis lesions. EXPERIMENTAL DESIGN: In a prospective proof-of-concept clinical trial (NCT02893293), we performed serial MRI studies of nine hip joints of 7 patients with osteonecrosis before and after core decompression. Twenty-four to 48 hours prior to the surgery, we injected ferumoxytol nanoparticles intravenously to label cells in normal bone marrow with iron oxides. During the surgery, iron-labeled bone marrow cells were aspirated from the iliac crest, concentrated, and then injected into the decompression track. Following surgery, patients received follow-up MRI up to 6 months after bone marrow cell transplantation. RESULTS: Iron-labeled cells could be detected in the access canal by a dark (negative) signal on T2-weighted MR images. T2* relaxation times of iron-labeled cell transplants were significantly lower compared with unlabeled cell transplants of control patients who were not injected with ferumoxytol (P = 0.02). Clinical outcomes of patients who received ferumoxytol-labeled or unlabeled cell transplants were not significantly different (P = 1), suggesting that the added ferumoxytol administration did not negatively affect bone repair. CONCLUSIONS: This immediately clinically applicable imaging test could become a powerful new tool to monitor the effect of therapeutic cells on bone repair outcomes after corticosteroid-induced osteonecrosis.

Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse.

Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.

Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways.

CREB (cAMP Response Element Binding protein) is a transcription factor that is overexpressed in primary acute myeloid leukemia (AML) cells and associated with a decreased event-free survival and increased risk of relapse. We recently reported a small molecule inhibitor of CREB, XX-650-23, which inhibits CREB activity in AML cells. Structure-activity relationship analysis for chemical compounds with structures similar to XX-650-23 led to the identification of the anthelminthic drug niclosamide as a potent anti-leukemic agent that suppresses cell viability of AML cell lines and primary AML cells without a significant decrease in colony forming activity of normal bone marrow cells. Niclosamide significantly inhibited CREB function and CREB-mediated gene expression in cells, leading to apoptosis and G1/S cell cycle arrest with reduced phosphorylated CREB levels. CREB knockdown protected cells from niclosamide treatment-mediated cytotoxic effects. Furthermore, treatment with a combination of niclosamide and CREB inhibitor XX-650-23 showed an additive anti-proliferative effect, consistent with the hypothesis that niclosamide and XX-650-23 regulate the same targets or pathways to inhibit proliferation and survival of AML cells. Niclosamide significantly inhibited the progression of disease in AML patient-derived xenograft (PDX) mice, and prolonged survival of PDX mice. Niclosamide also showed synergistic effects with chemotherapy drugs to inhibit AML cell proliferation. While chemotherapy antagonized the cytotoxic potential of niclosamide, pretreatment with niclosamide sensitized cells to chemotherapeutic drugs, cytarabine, daunorubicin, and vincristine. Therefore, our results demonstrate niclosamide as a potential drug to treat AML by inducing apoptosis and cell cycle arrest through inhibition of CREB-dependent pathways in AML cells.