Jason N. Berman

Myeloid leukemia of Down syndrome (ML-DS) is a distinct form of pediatric acute myeloid leukemia (AML) that responds to reduced-intensity chemotherapy, as compared with non-DS AML that requires intensive chemotherapy and often stem cell transplant. While most patients with ML-DS have a favorable prognosis, outcomes for those with refractory or relapsed disease are dismal. Children’s Oncology Group study AAML1531 introduced the use of measurable residual disease by multiparameter flow cytometry at the end of the first course of induction therapy (EOI-1 MRD) for risk stratification of treatment intensity. Of 280 patients with ML-DS who were enrolled, 41 were classified as high risk (HR) due to positive EOI-1 MRD, and treated with intensified chemotherapy similar to that used for pediatric non-DS AML. Treatment intensification did not improve the 2-year event-free survival compared with patients who were MRD-positive treated with reduced-intensity therapy in the predecessor study AAML0431 (80.5% ± 12.4% vs 76%; P = .247) or overall survival (80.5% ± 12.4% vs 76.2% ± 18.6%; P = .819), but significantly increased the frequency of febrile neutropenia and sepsis events. While stratification of treatment intensity based on MRD was not beneficial, molecular markers of relapse risk proposed by the Japan Children’s Cancer Group for ML-DS (alterations of CDKN2A, ZBTB7A, JAK2, TP53) proved prognostic. Relapse risk was 50% in patients who were HR from AAML1531 with any high-risk molecular marker compared with 6.7% in those without. Similar relapse results were obtained in the MRD-negative AAML1531 group, suggesting molecular risk markers can predict outcome and thus be used to stratify therapy in ML-DS. This trial was registered at www.clinicaltrials.gov as #NCT02521493.

Despite advances in precision medicine, 30% of high-risk pediatric cancers lack an actionable molecular target, hindering effective treatment and affecting survival outcomes. Although mouse patient-derived xenograft (PDX) models offer additional insights into clinical drug responses, delivering findings from these models within a clinically actionable time frame remains challenging. This international collaboration between two national precision medicine programs demonstrates proof-of-principle that individualized larval zebrafish PDXs can robustly and rapidly assess clinical responses in high-risk pediatric cancers. Retrospective zebrafish PDX testing was performed on tumor samples from 10 pediatric patients with high-risk cancers. Drug responses in zebrafish models were correlated with clinical responses for each patient and directly compared with responses in cognate mouse PDX models. Responses to conventional and targeted therapies, administered as single agents or in combinations, were assessed. Zebrafish PDXs were successfully established from all 10 patients and provided robust drug response data in every case, including from three patients whose tumor samples could not be engrafted in mice. Remarkably, zebrafish models accurately recapitulated patient responses for 11 of 12 treatment regimens. These findings highlight the potential of larval zebrafish PDX models to provide real-time, clinically relevant drug response data, supporting their potential use in prospective precision medicine studies.Significance:This proof-of-principle study is the first to compare drug responses in larval zebrafish and mouse PDX models with patient outcomes in pediatric precision oncology, showing high concordance. Results highlight the potential of zebrafish PDX models to predict drug responses in high-risk cancers more accurately, rapidly, and cost-effectively in prospective studies.

Mouse and larval zebrafish PDX drug efficacy for nonresponsive patients. A to C, Patient information, alongside mouse PDX waterfall plot of maximum tumor regression, and larval zebrafish tumor cell numbers for each therapy for neuroblastoma zccs373 (A), sarcoma zccs262 (B), and osteosarcoma zccs43 (C). For mouse PDX data, colored columns represent individual mice. For larval zebrafish data, error bars represent mean ± SEM, each colored dot represents an individual larva, and a threshold is based on SEM of 1 dpi. Statistical analysis was conducted using a one-way ANOVA with the Dunnett multiple comparisons test, comparing the mean of each treatment group with control (4 dpi), P values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001). mut, mutant; dpi, days post-injection.

Features of mouse PDX, larval zebrafish PDX and in vitro models

CONSORT flow diagram for patients included in the study and the criteria not met for those excluded

Patient cohort details, including molecular targets, clinical response to therapy, and mouse PDX development

Larval zebrafish drug efficacy studies for patients with no available mouse PDX. A to C, Patient information, alongside larval zebrafish tumor cell numbers for each therapy for rhabdomyosarcoma zccs170 (A), gastrointestinal stromal tumor zccs15 (B), and Ewing sarcoma zccs276 (C). For larval zebrafish data, error bars represent mean ± SEM, each colored dot represents an individual larva, and a threshold is based on SEM of 1 dpi. Statistical analysis was conducted using a one-way ANOVA with the Dunnett multiple comparisons test, comparing the mean of each treatment group with control (4 dpi), P values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001. D, Representative image of a five microns sectioned control larval zebrafish xenograft for patient zccs170. Deep Red–labeled patient cells present in the YS 4 dpi and labeled with DAPI to confirm viability. White circle in the images point to zccs170 tumor cells. Images were taken with 10× and 20× magnification. The scale bar is 100 µm. dpi, days post-injection.

Single agent and combination doses established in the larval zebrafish PDX at 35°C for 3 to 7 days post fertilization zebrafish for 72-hour treatment.

Mouse PDX tumor growth curves and Kaplan-Meier survival curves

Consistency in drug response between patients and their cognate larval zebrafish and mouse PDX models. Evaluable response to therapy was compared for each patient and their cognate larval zebrafish PDX and mouse PDX models. Patient and mouse responses are indicated as OR criteria. Larval zebrafish PDX models are indicated as response (inhibition of cell growth), SD (cell number maintained), or PD (cell growth in the presence of drug). Maintained CR, CR, and PR for patient and mouse are considered equivalent to R (response) in the larval zebrafish model. The concordance of response for each model type with patient response is summarized in the legends on the right. ***For zccs373 (nonresponder to triple combination), mouse PDX responded to dual combination. MCR, maintained complete response; NT, not treated; zf, zebrafish.