Identification of neoepitope reactive T-cell receptors guided by HLA-A*03:01 and HLA-A*11:01 immunopeptidomics.

BACKGROUND: Tumor-specific mutated proteins can create immunogenic non-self, mutation-containing 'neoepitopes' that are attractive targets for adoptive T-cell therapies. To avoid the complexity of defining patient-specific, private neoepitopes, there has been major interest in targeting common shared mutations in driver genes using off-the-shelf T-cell receptors (TCRs) engineered into autologous lymphocytes. However, identifying the precise naturally processed neoepitopes to pursue is a complex and challenging process. One method to definitively demonstrate whether an epitope is presented at the cell surface is to elute peptides bound to a specific major histocompatibility complex (MHC) allele and analyze them by mass spectrometry (MS). These MS data can then be prospectively applied to isolate TCRs specific to the neoepitope. METHODS: We created mono-allelic cell lines expressing one class I HLA allele and one common mutated oncogene in order to eliminate HLA deconvolution requirements and increase the signal of recovered peptides. MHC-bound peptides on the surface of these cell lines were immunoprecipitated, purified, and analyzed using liquid chromatography-tandem mass spectrometry, producing a list of mutation-containing minimal epitopes. To validate the immunogenicity of these neoepitopes, HLA-transgenic mice were vaccinated using the minimal peptides identified by MS in order to generate neoepitope-reactive TCRs. Specificity of these candidate TCRs was confirmed by peptide titration and recognition of transduced targets. RESULTS: We identified precise neoepitopes derived from mutated isoforms of KRAS, EGFR, BRAF, and PIK3CA presented by HLA-A*03:01 and/or HLA-A*11:01 across multiple biological replicates. From our MS data, we were able to successfully isolate murine TCRs that specifically recognize four HLA-A*11:01 restricted neoepitopes (KRAS G13D, PIK3CA E545K, EGFR L858R and BRAF V600E) and three HLA-A*03:01 restricted neoepitopes (KRAS G12V, EGFR L858R and BRAF V600E). CONCLUSIONS: Our data show that an MS approach can be used to demonstrate which shared oncogene-derived neoepitopes are processed and presented by common HLA alleles, and those MS data can rapidly be used to develop TCRs against these common tumor-specific antigens. Although further characterization of these neoepitope-specific murine TCRs is required, ultimately, they have the potential to be used clinically for adoptive cell therapy.

Preoperative BRAF(V600E) mutation screening is unlikely to alter initial surgical treatment of patients with indeterminate thyroid nodules: a prospective case series of 960 patients.

BACKGROUND: Preoperative B-type Raf kinase Val600Glu mutation, or BRAF(V600E), analysis has been proposed as a tool to guide initial surgery for indeterminate thyroid nodules. This study sought to determine if cytologic markers of malignancy are associated with the BRAF(V600E) mutation and if preoperative BRAF(V600E) testing would alter the initial management of patients with indeterminate nodules. METHODS: Patients who underwent surgery for a thyroid nodule between 2003 and 2012 at a tertiary care center were prospectively enrolled. Stored nodule samples were retrospectively genotyped for the BRAF(V600E) mutation. BRAF(V600E) status, demographics, cytologic and histopathologic findings, and choice of initial surgery were examined. RESULTS: A total of 960 patients were enrolled, of which 310 (32%) had an indeterminate nodule. The BRAF(V600E) mutation was identified in 13 patients (4%), 12 of whom had either cytologic atypia or were Bethesda category V. Three percent of Bethesda category III or IV nodules that were malignant harbored the mutation compared with 42% of Bethesda category V malignancies. Nuclear grooves (P = .030), pseudoinclusions (P < .001), and oval nuclei (P = .022) were all more common among BRAF(V600E) mutants. The sensitivities of using BRAF testing alone, cytologic atypia/Bethesda category V classification, or both, were 15%, 73%, and 76%, respectively. Twelve of the 13 BRAF(V600E) mutants had total thyroidectomies initially due to worrisome cytologic features, and therefore the initial management of only one patient would have been altered if BRAF(V600E) testing had been performed preoperatively. CONCLUSIONS: Preoperative mutation screening for BRAF(V600E) does not meaningfully improve risk stratification and is unlikely to alter the initial management of patients with indeterminate nodules.

Early referral for 24-h esophageal pH monitoring may prevent unnecessary treatment with acid-reducing medications.

BACKGROUND: Gastroesophageal reflux disease (GERD) affects nearly 25 % of adults; however, an objective diagnosis is rarely established. We hypothesized that patients' symptoms and response to acid-reducing therapy are poor predictors of the outcome of 24-h esophageal pH monitoring. METHODS: A review of 24-h esophageal pH monitoring studies performed at an ambulatory tertiary care center between 2004 and 2011 was performed. Demographics, type of GERD symptoms, and duration and response to acid-reducing medications before referral for pH monitoring were collected. DeMeester score, symptom sensitivity index (SSI), and symptom index (SI) were tabulated and compared with the patients' symptoms and response to medical therapy. RESULTS: One hundred patients were included. Of all reported symptoms, only heartburn was more common in patients with positive DeMeester scores, but there were no correlations between any symptoms and SSI or SI scores. Sixty-nine percent of patients with esophageal symptoms had a positive DeMeester score compared with only 29 % of patients with extraesophageal symptoms (P < 0.01). Esophageal symptoms and endoscopic evidence of GERD significantly increased the likelihood of having a positive DeMeester score, but they had no influence on SSI or SI scores. There was no correlation between response to acid-reducing medications and DeMeester, SSI, or SI scores. A total of 536 person-years of acid-reducing medications were prescribed to the study population, of which 151 (28 %) were prescribed to patients who had a negative pH study. CONCLUSIONS: Extraesophageal symptoms and response to empiric trials of acid-reducing medications are poor predictors of the presence of GERD and the DeMeester score is more likely to identify GERD in patients who met other empiric diagnostic criteria than SSI or SI. Early referral for 24-h esophageal pH monitoring may avoid lengthy periods of unnecessary medical therapy.