In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer

Abstract

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

Keywords: drug screen; patient-derived xenograft; pediatric cancer; precision medicine.

Figure 1. Development of preclinical models from 46 fresh samples

1. Flow diagram of sample allocation for molecular profiling (M), primary culture, high‐throughput drug screening (HTS), and patient‐derived xenograft (PDX) drug studies.

2. Outcome of in vitro expansion in 46 fresh samples.

3. Outcome of PDX establishment in 46 fresh samples.

4. Outcome of in vitro and in vivo drug testing attempts in 46 fresh samples.

Figure 2. Overview of drug hits identified by high‐throughput drug screening in 13 patient‐derived samples

1. Z score for area under the dose–response curve (AUC) and IC₅₀ of 37 different drugs (shown along the horizontal axis) identified as hits in 13 of 17 samples screened. A drug hit is defined as z score of less than −2 for both AUC and IC₅₀. Each dot in a column represents a sample screened for that drug. The size of the dot corresponds to the IC₅₀ z score for that sample (the larger the dot, the smaller the IC₅₀). Dots below the black horizontal line represent sample with AUC z score of less than −2. Dots are color coded for drug hit types. All color dots below the black line represent a hit for the corresponding drug.

2. Plots of AUC z score against IC₅₀ z score for each of the drugs screened in the 13 samples with drug hits.

Source data are available online for this figure.

Figure 3. In vivo drug efficacy studies in patient‐derived xenografts

1. A

   Treatment response in 16 hematologic malignancy (HM) and non‐CNS (central nervous system) solid patient‐derived xenograft (PDX) models. Objective responses including maintained complete response (MCR), complete response (CR), and partial response (PR) were observed in 10 of 16 models. Drugs are indicated as chemotherapy (Ch), targeted agent (T), or combination treatment (C).

2. B–D

   Event‐free survival (EFS) and percentage of human CD45⁺ leukocytes in peripheral blood in three acute lymphoblastic leukemia (ALL) orthotopic models. An event is defined as human CD45 cells above 25% in the peripheral and is represented by the dotted line.

3. E–K

   EFS and tumor volume in seven non‐CNS subcutaneous PDX models which demonstrated objective response in one or more treatments.

4. L

   EFS in a CNS orthotopic model in which drug sensitivity was observed. EFS is time of inoculation of tumor cells to event (defined by neurologic symptoms or weight loss).

Data information: Survival curves were estimated for each treatment group using the Kaplan–Meier method and compared with the untreated control group in each PDX model statistically using log rank test. P value for log rank test for comparison of EFS: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. The exact P values are provided in Appendix Table S6. ALCL, anaplastic large cell lymphoma; Cyclo, cyclophosphamide; IRN, irinotecan; PD, progressive disease; SD, stable disease; Topo, topotecan; TMZ, temozolomide; VXL, vincristine/dexamethasone/L‐asparaginase. Source data are available online for this figure.

Figure 4. Individualized therapeutic options in 56 pediatric high‐risk cancers

1. A

   Overview of each patient’s precision oncology platform results. The highest tier of therapy options for each patient is shown. A total of 55 recommendations were made in 39 patients.

2. B

   Tier of therapy and related molecular alterations. Structural variant (SV), single‐nucleotide variant (SNV) with loss of heterozygosity (LOH) in a tumor suppressor gene, copy number variant (CNV).

3. C, D

   Treatment response by therapy tier. Fourteen of 29 patients with molecular‐based therapeutic options received the treatment.

4. E

   Tests contributing to the identification of treatment by tier.

Figure EV1. Molecular aberrations in 55 pediatric high‐risk cancers

1. Genes with somatic and germline DNA mutations (single‐nucleotide variant (SNV) and indel) considered to be pathogenic or likely pathogenic by whole genome sequencing (WGS) and/or panel sequencing. Thirty of 55 samples were found to have 1 or more pathogenic or likely pathogenic mutations. The cohort consists of 27 central nervous system (CNS) tumors, 8 hematologic malignancies (HMs), and 20 non‐CNS solid tumors. Targetable aberrations are indicated by asterisks.

2. Tumor mutation burden (TMB) derived from WGS in 23 samples obtained at diagnosis and 24 samples at refractory/relapse.

3. Structural variants (SVs) detected by WGS and/or RNA‐seq in 55 samples. Seventeen reportable SVs included 13 fusions, 2 oncogenic activating (ACT) SVs, and 2 tumor suppressor (TS) loss‐of‐function SV.

4. Reportable copy number variations (CNVs) included amplifications (≥ 6 copies), loss of heterozygosity (LOH) associated with a loss‐of‐function mutation in a tumor suppressor gene (TSG LOH) and homozygous deletion (HOMDEL) of TSG. Twenty‐four samples were found to have 1 or more reportable CNV. Targetable aberrations are indicated by asterisks.