<p>Supplementary Tables 1-7. Supplementary Table 1. SBT-EOC cases used for molecular analyses Supplementary Table 2. Mutations Screened on the Sequenom/OncoMap3 Platform Supplementary Table 3. Primer sequences for the indicated exons of TP53, ERBB2 and NRAS Supplementary Table 4. Clinico-pathologic characteristics of the subset of SBT-EOC used for molecular analysis compared to the entire SBT-EOC cohort Supplementary Table 5A. Somatic mutations found in borderline and invasive ovarian tumors Supplementary Table 5B. Genes in which somatic mutations were found according to grade and co-existing serous borderline regions Supplementary Table 6. Genes differentially expressed between paired borderline and invasive regions of SBTEOC (p<0.01, not corrected for multiple comparison). Supplementary Table 7. Clinico-pathological characteristics of SBTEOC cases used for progesterone receptor (PGR) immunostaining</p>
IntroductionNovel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described.MethodsAnalysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays.ResultsAfter a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs.ConclusionsRET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.
Abstract In this phase II, single arm trial (ACTRN12617000720314), we investigate if alternating osimertinib and gefitinib would delay the development of resistance to osimertinib in advanced, non-small cell lung cancer (NSCLC) with the epidermal growth factor receptor ( EGFR) T790M mutation ( n = 47) by modulating selective pressure on resistant clones. The primary endpoint is progression free-survival (PFS) rate at 12 months, and secondary endpoints include: feasibility of alternating therapy, overall response rate (ORR), overall survival (OS), and safety. The 12-month PFS rate is 38% (95% CI 27.5–55), not meeting the pre-specified primary endpoint. Serial circulating tumor DNA (ctDNA) analysis reveals decrease and clearance of the original activating EGFR and EGFR -T790M mutations which are prognostic of clinical outcomes. In 73% of participants, loss of T790M ctDNA is observed at progression and no participants have evidence of the EGFR C797S resistance mutation following the alternating regimen. These findings highlight the challenges of treatment strategies designed to modulate clonal evolution and the clinical importance of resistance mechanisms beyond suppression of selected genetic mutations in driving therapeutic escape to highly potent targeted therapies.
BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts. METHODS We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs. RESULTS Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the BACKGROUND sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil. CONCLUSIONS UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.
e14523 Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log 2 ratio ≥0.15 and losses < -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.
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