Laura E. Hogan

Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia

Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5′-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

Integrated genomic analysis of relapsed childhood acute lymphoblastic leukemia reveals therapeutic strategies.

Despite an increase in survival for children with acute lymphoblastic leukemia (ALL), the outcome after relapse is poor. To understand the genetic events that contribute to relapse and chemoresistance and identify novel targets of therapy, 3 high-throughput assays were used to identify genetic and epigenetic changes at relapse. Using matched diagnosis/relapse bone marrow samples from children with relapsed B-precursor ALL, we evaluated gene expression, copy number abnormalities (CNAs), and DNA methylation. Gene expression analysis revealed a signature of differentially expressed genes from diagnosis to relapse that is different for early (< 36 months) and late (≥ 36 months) relapse. CNA analysis discovered CNAs that were shared at diagnosis and relapse and others that were new lesions acquired at relapse. DNA methylation analysis found increased promoter methylation at relapse. There were many genetic alterations that evolved from diagnosis to relapse, and in some cases these genes had previously been associated with chemoresistance. Integration of the results from all 3 platforms identified genes of potential interest, including CDKN2A, COL6A2, PTPRO, and CSMD1. Although our results indicate that a diversity of genetic changes are seen at relapse, integration of gene expression, CNA, and methylation data suggest a possible convergence on the WNT and mitogen-activated protein kinase pathways.

Endogenous knockdown of survivin improves chemotherapeutic response in ALL models

Although the cure rate of newly diagnosed acute lymphoblastic leukemia (ALL) has improved over the past four decades, the outcome for patients who relapse remains poor. New therapies are needed for these patients. Our previous global gene expression analysis in a series of paired diagnosis-relapse pediatric patient samples revealed that the antiapoptotic gene survivin was consistently upregulated upon disease relapse. In this study, we demonstrate a link between survivin expression and drug resistance and test the efficacy of a novel antisense agent in promoting apoptosis when combined with chemotherapy. Gene-silencing experiments targeting survivin mRNA using either short-hairpin RNA (shRNA) or a locked antisense oligonucleotide (LNA-ON) specifically reduced gene expression and induced apoptosis in leukemia cell lines. When used in combination with chemotherapy, the survivin shRNA and LNA-ON potentiated the chemotherapeutic antileukemia effect. Moreover, in a mouse primary xenograft model of relapse ALL, the survivin LNA-ON decreased survivin expression in a subset of animals, and produced a statistically significant decrease in tumor progression. Taken together, these findings suggest that targeting endogenous levels of survivin mRNA by LNA-ON methods may augment the response to standard chemotherapy by sensitizing otherwise resistant tumor cells to chemotherapy.

Treating Persistent Distress and Anxiety in Parents of Children With Cancer An Initial Feasibility Trial

Persistent anxiety is common among parents of children with cancer and may affect the family’s well-being and adjustment. The goals of this pilot study are to determine the feasibility and potential efficacy of a brief cognitive-behavioral parent intervention aimed at reducing parental distress and anxiety related to their child’s cancer diagnosis. Parents of children with cancer, at least 1 month postdiagnosis, were screened at an outpatient oncology clinic, and those reporting elevated levels of distress were offered a 4-session cognitive–behavioral intervention based on a modified version of the Surviving Cancer Competently Intervention Program–Newly Diagnosed. Five parents reporting persistent distress received the intervention. Results revealed decreases in parents’ distress, state anxiety, and depressive symptoms, as well as in parents’ feelings of burden associated with their children’s cancer. This initial study suggests that identification of parents with prolonged heightened psychological distress is feasible and acceptable and that offering them a brief intervention within a pediatric oncology setting may be beneficial.

New targeted therapies for relapsed pediatric acute lymphoblastic leukemia

ABSTRACT Introduction: The improvement in outcomes for children with acute lymphoblastic leukemia (ALL) is one of the greatest success stories of modern oncology however the prognosis for patients who relapse remains dismal. Recent discoveries by high resolution genomic technologies have characterized the biology of relapsed leukemia, most notably pathways leading to the drug resistant phenotype. These observations open the possibility of targeting such pathways to prevent and/or treat relapse. Likewise, early experiences with new immunotherapeutic approaches have shown great promise. Areas covered: We performed a literature search on PubMed and recent meeting abstracts using the keywords below. We focused on the biology and clonal evolution of relapsed disease highlighting potential new targets of therapy. We further summarized the results of early trials of the three most prominent immunotherapy agents currently under investigation. Expert commentary: Discovery of targetable pathways that lead to drug resistance and recent breakthroughs in immunotherapy show great promise towards treating this aggressive disease. The best way to treat relapse, however, is to prevent it which makes incorporation of these new approaches into frontline therapy the best approach. Challenges remain to balance efficacy with toxicity and to prevent the emergence of resistant subclones which is why combining these newer agents with conventional chemotherapy will likely become standard of care.

Anti-CD19 chimeric antigen receptor targeting of CD19 + acute myeloid leukemia

Aberrant expression of CD19 in acute myeloid leukemia (AML) is commonly associated with t(8;21)(q22;q22), although AML cases lacking this translocation occasionally express CD19. Mixed-phenotype acute leukemia also frequently expresses CD19. Chimeric antigen receptor (CAR) technology is a major breakthrough for cancer treatment, with the recent approval of CD19-directed CAR (CD19CAR) for treating B-cell malignancies. However, little information exists on using CD19CAR for other CD19 positive neoplasms such as AML. Our findings indicate that CD19CAR therapy can potentially be used for those with mixed phenotype leukemia and a subset of AML cases.

Dying to predict outcome

Early treatment response is one of the most robust outcome predictors in childhood acute lymphoblastic leukemia (ALL). Intuitively, it is not unexpected that this factor would be an essential prognostic determinant, as it encompasses aspects of the complex interrelationship between disease, host and therapy. Early response can be measured in many ways, including morphologic reduction of blasts in the blood or bone marrow, or by more sensitive measures such as the detection of minimal residual disease (MRD) using flowcytometry or molecular techniques at time points during the initial weeks of treatment. In all cases, regardless of response measure and methodology, studies have demonstrated the favorable prognostic impact of rapid early responses to treatment. In fact, on the recently completed Pediatric Oncology Group protocol P9900, endinduction marrow MRD was the most important prognostic variable in multivariate analysis with 5-year event-free survival (EFS) rates of 88 ± 1%, 59 ± 5%, 49 ± 6%, and 30% ± 8% for those patients who were MRD negative, or MRD positive in the ranges of 0.01– 0.1%, 0.1–1%, and >1%, respectively [1]. Moreover, when used in conjunction with favorable clinical and cytogenetic features, the absence of peripheral blood MRD on day 8 combined with negative day 29 MRD, identified a subgroup of 12% of patients with a 97% ± 1% 5-year EFS [1]. Given the power of early response as a prognostic tool, numerous studies have been undertaken to dissect the factors that are essential in mediating response, with the premise that a better understanding of these factors early on in treatment could potentially provide an opportunity to modify therapy to overcome resistance. Since one of the essential ways that chemotherapeutic agents exert their effect is by inducing apoptosis, defects in apoptotic signaling have been postulated to play a key role in the acquisition of cellular drug resistance. In this issue of Pediatric Blood & Cancer, Mata et al. examined the expression profile of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 in association with several key clinical and biological variables in children with ALL. Apoptosis is an evolutionarily conserved, active, energy dependent mechanism of cellular death and is regulated by the relative levels of both proand anti-apoptotic proteins as reviewed by Hengartner [2]. Lower expression levels of pro-apoptotic CASP3, CASP8, and FAS were associated with a poor day 7 response to treatment (white blood cell count >5,000/mm3) among 139 children ages 1–17 years with newly diagnosed ALL, treated according to the Brazilian Childhood Leukemia Treatment Group protocol. In addition, lower FAS expression was associated with lower 5-year event-free survival. It is noteworthy that while CASP3, CASP8, and FAS levels were associated with day 7 response, only FAS expression also correlated with event-free survival. The authors concluded that there is an association between lower expression of levels of pro-apoptotic genes and a poor response to therapy and poor prognosis. Somewhat counterintuitively, they found that higher expression of BCL2, an anti-apoptotic gene, was associated with a lower white count and low risk group classification; however, this finding is not inconsistent with several past studies that have failed to demonstrate an inverse correlation between BCL-2 levels at diagnosis and early response and outcome [3-5]. Despite the essential role of apoptosis in mediating response to chemotherapy, several other recent studies have also highlighted some of the challenges in defining the prognostic role of expression of individual components of the apoptotic cascade. Using gene expression microarray analyses, Flotho et al. [6] have previously shown that low expression of caspase-8 associated protein 2 (CASP8AP2), a gene that interacts with CASP8 in mediating apoptosis and which has been show to be a determinant of glucocorticoid signaling, was associated with MRD positivity on days 19 and 46 of therapy in children with ALL treated on St. Jude Total Therapy Studies. In this series, CASP8AP2 expression was lower in patients less than 1 year of age and higher in patients with hyperdiploidy, a favorable cytogenetic feature, but was not associated with other known outcome predictors. Validating these findings in an independent cohort, the authors observed that lower CASP8AP2 levels at diagnosis were a strong predictor of relapse, but were no better than age (older than 10 years) in predicting outcome. Cario et al. [7] defined gene expression profiles predictive of MRD of 10−3 or greater at week 12 of therapy, a very high risk patient subgroup with relapse-free survival rates of only 19%, and found low expression of several apoptosis-related genes, however, not members of the caspase family or FAS. More recently, Bhojwani et al. [8] identified a 24-probe set signature that was highly predictive of day 7 marrow status and a 47-probe set signature predictive of relapse among a cohort of 99 children with high risk ALL who were treated on the Children’s Oncology Group study 1961. While two of the six genes predictive of a rapid early response on day 7 were pro-apoptotic, they were distinct from the genes predictive of relapse and were not members of the caspase family or FAS. Furthermore, while the outcome