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Study Shows Potential of Drug Sensitivity Profiling With Sequencing for Pediatric Cancers

NEW YORK – Results from a two-year pilot study published this week in NPJ Precision Oncology demonstrated that ex vivo drug sensitivity profiling in a real-world setting is feasible and could enhance the value of next-generation sequencing-based molecular diagnostics in pediatric cancers.

Researchers from the Hopp Children's Cancer Center Heidelberg in Germany developed a patient-derived ex vivo 3D fresh culture format for drug sensitivity profiling, which they used to identify drug sensitivities in a high proportion of patients within the Individualized Therapy For Relapsed Malignancies in Childhood (INFORM) pediatric precision oncology program, including some lacking actionable genetic alterations.

INFORM enrolls relapsed or refractory pediatric cancer patients for comprehensive molecular analysis, including whole exome sequencing, RNA sequencing, and DNA methylation profiling and matches them to targeted treatments. A 2021 report on long-term outcomes found that among patients with tumors harboring genetic alterations with a high level of evidence — such as alterations affecting ALK, BRAF, and NTRK — progression-free survival times doubled after two years of follow-up. However, for patients with lower-evidence alterations, progression-free survival did not improve with molecularly matched targeted treatments.

That study suggested that pediatric precision oncology programs could benefit from adding additional layers of molecular and functional analyses beyond NGS such as liquid biopsy, proteomics, and drug sensitivity profiling. In the current study, the investigators sought to evaluate the usefulness of drug sensitivity profiling in the context of the INFORM program.

The drug sensitivity profiling platform developed by the researchers involved three steps. First, viable tumor tissue was collected and shipped, then tumor tissue was processed into spheroid cultures in 384-well plates and screened for drug sensitivity. Lastly, individual drug sensitivity and resistance profiles were generated and reported to the INFORM molecular tumor board. The study included 132 tumor samples from 35 pediatric oncology centers in seven countries which were screened against a library of 75 to 78 clinically relevant drugs.

Out of 65 screened samples, 47 had at least one drug hit and some of the samples had 10 or more hits. No hits were identified for 18 of the samples. In a subset of 14 cases with strong genetic driver alterations identified by molecular profiling, nine had at least one drug sensitivity hit corresponding to the tumor-driver alteration. The authors noted that in 81 percent, or 38 out of 47 cases, a drug hit was identified in samples in which whole-exome sequencing and RNA-seq did not identify a strongly actionable target.

Several included case reports' illustrated correlation of ex vivo drug sensitivity profiles with the clinical courses of the patients. In one case, a 7-year-old patient with a BCR/NTRK2 fusion identified by NGS was enrolled in a trial of Bayer's NTRK inhibitor Vitrakvi (larotrectinib) and, after relapsing, a trial of Bayer's next-generation NTRK inhibitor, selitrectinib. After yet another progression, the patient had a biopsy showing the tumor sample was resistant against all NTRK inhibitors in the library, but had high sensitivity to MET-targeting inhibitors. Subsequent sequencing analysis turned up a MET amplification as the likely cause of resistance to NTRK inhibition, reflecting other reports in the literature on NTRK resistance. The patient's sample also had unexpected drug hits, including Pfizer's ALK inhibitor Lorbrena (lorlatinib) and the JAK1/2 inhibitor ruxolitinib.

Two other case studies of patients with EWSR1:FLI1-positive Ewing sarcoma and a central nervous system high-grade neuroepithelial tumor with MN1 alterations demonstrated close correspondence between the clinical course of disease and predictions by the drug sensitivity profiling platform.

"Overall, our INFORM drug sensitivity pipeline can identify drug hits, matching corresponding molecular driver alterations and, more importantly, can identify unexpected drug sensitivities in a high proportion of pediatric solid and brain tumors lacking clinically relevant molecular targets," the researchers wrote in their paper.

In addition to confirming the utility of drug sensitivity profiling as a supplement to sequencing in the INFORM population, the study also laid out a protocol for collecting and processing the tumor samples that would enable small centers without specialized laboratory facilities to participate in functional precision oncology programs.