Case Report: Application of ex-vivo drug sensitivity testing to identify personalized treatment options for an adolescent with diffuse midline glioma

Abstract

Diffuse midline glioma (DMG) is a pediatric brain cancer that has a dismal prognosis with limited treatment options. We present the treatment course and outcome of an adolescent male diagnosed with a thalamic DMG carrying a histone H3.3 K27M (H3K27M) alteration. Tumor biopsies were taken at diagnosis for histological analysis, molecular profiling, and ex vivo drug sensitivity testing (DST). Seven months after diagnosis, the patient had recurrent/progressive disease after radiotherapy and an ineffective molecular-guided therapy based on tumor molecular profiling. The patient then started a novel functional precision medicine (FPM)-guided two-drug combination of disulfiram, based on the DST results of this drug on the patient's tumor cells obtained at diagnosis, and ONC 201, the only drug that has advanced to a phase III clinical trial for H3K27M-DMG. Neuroimaging demonstrated a treatment response, and the patient lived for fifteen months after starting this personalized therapy. Disulfiram was discontinued after three months due to significant peripheral neuropathy. Our case describes the feasibility and limitations of using DST of patient-derived tumor cells to identify potentially effective personalized and novel therapies for DMG, which should be evaluated for efficacy and safety in formal N-of-1 clinical trials settings. We discuss the benefits and risks of this approach, particularly considering its use in children, adolescents, and young adults with pediatric brain cancers.

Keywords: diffuse midline glioma; drug sensitivity testing; functional precision medicine; molecular guided therapy; pediatric brain cancer.

Figure 1

Summary of DST results for drugs used by the patient and for marizomib. DST curves are shown for marizomib (A), two other drugs with high potency that the patient used [disulfiram (B) and panobinostat (C)], and for three drugs with low potency that the patient used [ONC201 (D), everolimus (E), and temozolomide (F)]. Briefly, the freshly biopsied tumor cells were dispersed by enzymatic digestion, shipped on ice to a CLIA laboratory at the University of Washington (16), and on the next day were plated with each drug on opaque 6x384-well plates at 2000 cells per well. After a 3-day incubation, cell viability was measured using CellTiter Glo 2.0 (Promega Corp., Madison, WI, USA). Each drug was assayed at five concentrations in duplicate. XLFit from IBDS was used to generate dose-response curves using the 4-parameter logistic dose response model, tumor cell killing potency of each drug at the half-maximal concentration in micromolar, or IC₅₀, and percentage of viable cells at the maximal concentration of each drug (%VIA at [Max]) as a measure of drug efficacy; the drug potency and efficacy values are shown below each dose response curve. Cu⁺, copper in the form of copper gluconate.

Figure 2

Treatment course and thalamic DMG tumor area measurements showing tumor regression with radiation therapy and with the FPM therapy of disulfiram and ONC201. The graph plots the measured area of the primary thalamic DMG tumor from each MRI. Above the graph, the time periods for the different treatments are shown in black boxes. Disulfiram was used at 250 mg QD, except during a time period at 250 mg BID (gray box). Surgeries are indicated by diamonds: blue diamond, external shunt placement and septostomy (day 1); yellow diamond, external shunt removal and third ventriculostomy (day 7); red diamond, internal shunt placement and septostomy (day 226). The lower left of the graph shows a timeline from tumor biopsy on the day 7 surgery to profiling including DST. The time period of the peripheral neuropathy is indicated as an open box. Standard of care radiation therapy led to tumor regression, observed as a reduction in tumor area on days 88 and 149. Following radiation therapy, an ineffective molecular-guided therapy with the drug combination temozolomide, panobinostat, everolimus, and hydroxychloroquine led to tumor progression, observed as an increase in tumor area on days 203 and 225. The FPM therapy of disulfiram alone, from days 210-224, disulfiram and ONC201, from days 258-333, and ONC201 alone led to a dramatic decrease in tumor area. The patient died on day 677 from treatment-resistant secondary tumors that were first visualized on day 255.

Figure 3

MRI images showing primary DMG tumor regression during and after FPM therapy with disulfiram and ONC201. T1-post gadolinium axial images are shown from the following days: (A) day 2; (B) day 88; (C) day 149; (D) day 203; (E) day 225; (F) day 255; (G) day 288; (H) day 351; (I) day 409; and (J) day 469. In J, the location of the primary tumor is within the yellow box, and secondary tumors are evident above the location of the primary tumor.