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

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.

Germline Polymorphisms in the Nuclear Receptors PXR and VDR as Novel Prognostic Markers in Metastatic Colorectal Cancer Patients Treated With FOLFIRI.

Nuclear receptors act as mediators of cancer-related inflammation and gene expression. They have a regulatory effect on genes encoding proteins related to drug adsorption, distribution, metabolism, and excretion. The aim of the present study was to highlight novel prognostic markers among polymorphisms in genes encoding for nuclear receptor proteins and inflammation-related cytokines in patients treated with a FOLFIRI regimen. This study included two independent cohorts comprising a total of 337 mCRC patients homogeneously treated with first-line FOLFIRI. Genotyping of 246 haplotype-tagging polymorphisms in 22 genes was performed using bead array technology. The NR1I2 (PXR)-rs1054190 and VDR-rs7299460 polymorphisms were significantly associated with patient overall survival (OS). A detrimental effect of the NR1I2 rs1054190-TT genotype on OS was observed in both the discovery and replication cohorts (HR = 6.84, P = 0.0021, q-value = 0.1278 and HR = 3.56, P = 0.0414, respectively). Patients harboring the NR1I2 rs1054190-TT genotype had a median OS of 9 months vs. 21 months in patients with C-allele (P < 0.0001 log-rank test). VDR rs7299460-T was consistently associated with a longer OS in both cohorts (discovery: HR = 0.61, P = 0.0075, q-value = 0.1535; replication: HR = 0.57, P = 0.0477). Patients with the VDR rs7299460-T allele had a median OS of 23 months compared to 18 months in those with the CC genotype (P = 0.0489, log-rank test). The NR1I2-rs1054190 polymorphism also had an effect on the duration of progression-free survival, consistent with the effect observed on OS. Two novel prognostic markers for mCRC treated with FOLFIRI were described and, if validated by prospective trials, have a potential application in the management of these patients.

Clonal Evolution of TP53 c.375+1G>A Mutation in Pre- and Post- Neo-Adjuvant Chemotherapy (NACT) Tumor Samples in High-Grade Serous Ovarian Cancer (HGSOC).

Carboplatin/paclitaxel is the reference regimen in the treatment of advanced high-grade serous ovarian cancer (HGSOC) in neo-adjuvant chemotherapy (NACT) before interval debulking surgery (IDS). To identify new genetic markers of platinum-resistance, next-generation sequencing (NGS) analysis of 26 cancer-genes was performed on paired matched pre- and post-NACT tumor and blood samples in a patient with stage IV HGSOC treated with NACT-IDS, showing platinum-refractory/resistance and poor prognosis. Only the TP53 c.375+1G>A somatic mutation was identified in both tumor samples. This variant, associated with aberrant splicing, was in trans configuration with the 72Arg allele of the known germline polymorphism TP53 c.215C>G (p. Pro72Arg). In the post-NACT tumor sample we observed the complete expansion of the TP53 c.375+1G>A driver mutant clone with somatic loss of the treatment-sensitive 72Arg allele. NGS results were confirmed with Sanger method and immunostaining for p53, BRCA1, p16, WT1, and Ki-67 markers were evaluated. This study showed that (i) the splice mutation in TP53 was present as an early driver mutation at diagnosis; (ii) the mutational profile was shared in pre- and post-NACT tumor samples; (iii) the complete expansion of a single dominant mutant clone through loss of heterozygosity (LOH) had occurred, suggesting a possible mechanism of platinum-resistance in HGSOC under the pressure of NACT.

rs4143815-PDL1, a New Potential Immunogenetic Biomarker of Biochemical Recurrence in Locally Advanced Prostate Cancer after Radiotherapy.

Up to 30-50% of patients with locally advanced prostate cancer (PCa) undergoing radiotherapy (RT) experience biochemical recurrence (BCR). The immune system affects the RT response. Immunogenetics could define new biomarkers for personalization of PCa patients' treatment. The aim of this study is to define the immunogenetic biomarkers of 10 year BCR (primary aim), 10 year overall survival (OS) and 5 year BCR (secondary aims). In this mono-institutional retrospective study, 549 Caucasian patients (a discovery set n = 418; a replication set n = 131) were affected by locally advanced PCa and homogeneously treated with RT. In the training set, associations were made between 447 SNPs in 77 genes of the immune system; and 10 year BCR and 10 year OS were tested through a multivariate Cox proportional hazard model. Significant SNPs (p-value < 0.05, q-value < 0.15) were analyzed in the replication set. Replicated SNPs were tested for 5 year BCR in both sets of patients. A polymorphism in the PDL1 gene (rs4143815) was the unique potential genetic variant of 10 year BCR (training set: p = 0.003, HR (95% CI) = 0.58 (0.41-0.83); replication set: p = 0.063, HR (95% CI) = 0.52 (0.26-1.04)) that was significantly associated with 5 year BCR (training set: p = 0.009, HR (95% CI) = 0.59 (0.40-0.88); replication set: p = 0.036, HR (95% CI) = 0.39 (0.16-0.94)). No biomarkers of OS were replicated. rs4143815-PDL1 arose as a new immunogenetic biomarker of BCR in PCa, giving new insights into the RT/immune system interaction, which could be potentially useful in new approaches using anti-PDL1 therapies for PCa.

A Clinical-Genetic Score to Identify Surgically Resected Colorectal Cancer Patients Benefiting From an Adjuvant Fluoropyrimidine-Based Therapy.

There are clinical challenges related to adjuvant treatment in colorectal cancer (CRC) and novel molecular markers are needed for better risk stratification of patients. Our aim was to integrate our previously reported clinical-genetic prognostic score with new immunogenetic markers of 5-year disease-free survival (DFS) to evaluate the recurrence risk stratification before fluoropyrimidine (FL)-based adjuvant therapy. The study population included a total of 270 stage II-III CRC patients treated with adjuvant FL with (FL + OXA, n = 119) or without oxaliplatin (FL, n = 151). Patients were genotyped for a panel of 192 tagging polymorphisms in 34 immune-related genes. The IFNG-rs1861494 polymorphism was associated with worse DFS in the FL + OXA (HR = 2.14, 95%CI 1.13-4.08; P = 0.020, q-value = 0.249) and FL (HR = 1.97, 95%CI 1.00-3.86; P = 0.049) cohorts, according to a dominant model. The integration of IFNG-rs1861494 in our previous clinical genetic multiparametric score of DFS improved the patients' risk stratification (Log-rank P = 0.0026 in the pooled population). These findings could improve the discrimination of patients who would benefit from adjuvant treatment. In addition, the results may help better elucidate the interplay between the immune system and chemotherapeutics and help determine the efficacy of anti-tumor strategies.

Identification of Novel Somatic TP53 Mutations in Patients with High-Grade Serous Ovarian Cancer (HGSOC) Using Next-Generation Sequencing (NGS).

Somatic mutations in TP53 are a hallmark of high-grade serous ovarian cancer (HGSOC), although their prognostic and predictive value as markers is not well defined. Next-generation sequencing (NGS) can identify novel mutations with high sensitivity, that may be repurposed as potential druggable anti-cancer targets and aid in therapeutic decisions. Here, a commercial NGS cancer panel comprising 26 genes, including TP53, was used to identify new genetic markers of platinum resistance and patient prognosis in a retrospective set of patients diagnosed with epithelial ovarian cancer. Six novel TP53 somatic mutations in untreated tumors from six distinct patients diagnosed with HGSOC were identified: TP53 c.728_739delTGGGCGGCATGA (p.Met243_Met247del, in-frame insertion or deletion (INDEL); TP53 c.795_809delGGGACGGAACAGCTT (p.Gly266_Phe270del, in-frame INDEL); TP53 c.826_827GC>AT (p.Ala276Ile, missense); TP53 c.1022insT (p.Arg342Profs*5, frameshift INDEL); TP53 c.1180delT (p.Ter394Aspfs*28, frameshift INDEL); and TP53 c.573insT (p.Gln192Serfs*17, frameshift INDEL). Novel TP53 variants were validated by classical sequencing methods and their impact on protein expression in tumors explored by immunohistochemistry. Further insights into the potential functional effect of the mutations were obtained by different in silico approaches, bioinformatics tools, and structural modeling. This discovery of previously unreported TP53 somatic mutations provides an opportunity to translate NGS technology into personalized medicine and identify new potential targets for therapeutic applications.