Clonal Selection of a Novel Deleterious TP53 Somatic Mutation Discovered in ctDNA of a KIT/PDGFRA Wild-Type Gastrointestinal Stromal Tumor Resistant to Imatinib

Abstract

The standard of care for the first-line treatment of advanced gastrointestinal stromal tumor (GIST) is represented by imatinib, which is given daily at a standard dosage until tumor progression. Resistance to imatinib commonly occurs through the clonal selection of genetic mutations in the tumor DNA, and an increase in imatinib dosage was demonstrated to be efficacious to overcome imatinib resistance. Wild-type GISTs, which do not display KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations, are usually primarily insensitive to imatinib and tend to rapidly relapse in course of treatment. Here we report the case of a 53-year-old male patient with gastric GIST who primarily did not respond to imatinib and that, despite the administration of an increased imatinib dose, led to patient death. By using a deep next-generation sequencing barcode-aware approach, we analyzed a panel of actionable cancer-related genes in the patient cfDNA to investigate somatic changes responsible for imatinib resistance. We identified, in two serial circulating tumor DNA (ctDNA) samples, a sharp increase in the allele frequency of a never described TP53 mutation (c.560-7_560-2delCTCTTAinsT) located in a splice acceptor site and responsible for a protein loss of function. The same TP53 mutation was retrospectively identified in the primary tumor by digital droplet PCR at a subclonal frequency (0.1%). The mutation was detected at a very high allelic frequency (99%) in the metastatic hepatic lesion, suggesting a rapid clonal selection of the mutation during tumor progression. Imatinib plasma concentration at steady state was above the threshold of 760 ng/ml reported in the literature for the minimum efficacious concentration. The de novo TP53 (c.560-7_560-2delCTCTTAinsT) mutation was in silico predicted to be associated with an aberrant RNA splicing and with an aggressive phenotype which might have contributed to a rapid disease spread despite the administration of an increased imatinib dosage. This result underlies the need of a better investigation upon the role of TP53 in the pathogenesis of GISTs and sustains the use of next-generation sequencing (NGS) in cfDNA for the identification of novel genetic markers in wild-type GISTs.

Keywords: TP53; circulating tumor DNA; gastrointestinal stromal tumor; imatinib; liquid biopsy.

Figure 1

(A) Immunhistochemical staining for CD117 (c-KIT) and (B) tumor composition of spindle cells and eosinophilic cytoplasm (hematoxylin and eosin) on the metastatic hepatic tissue.

Figure 2

(A) Course of disease from the time of diagnosis, treatment administered, PET/CT images, and allele frequency of TP53 indel are shown. The mutated allele frequency in tumor tissue (black columns) is reported for the primary tumor [minor allele frequency (MAF) 0.1%, ddPCR] and for the metastatic lesion (MAF 99%, ddPCR) at the time of surgery and biopsic sampling, respectively. The ctDNA fraction (gray columns) is reported for the sample IM_21.1 [MAF 2.7%, next-generation sequencing (NGS)] and sample IM_21.2 (MAF 9.7%, NGS). PET/CT scans reporting the diameter of target lesions and performed in concomitance to blood sampling are shown as well. On the bottom of the plot, the imatinib dosage administered is indicated. (B) ddPCR plots reporting the signal generated from the wild-type (green dots) and the mutated (blue dots) sequence are shown. In chronological order are reported the primary tumor DNA, the metastatic DNA, and the IM_21.1 cfDNA.

Figure 3

In the figure are reported (A) the normal sequence and exon splicing of TP53 pre-mRNA (exons 5–6) and (B) aberrant splicing caused by the c.560-7_560-2delCTCTTAinsT (red) likely to generate an in-frame deletion of 30 nucleotides from mRNA due to the activation of a cryptic splice site (blu).