A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood

Introduction: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. Methods: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. Results: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. Conclusions: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible

The AIDS-Like Disease of CD4C/Human Immunodeficiency Virus Transgenic Mice Is Associated with Accumulation of Immature CD11b(Hi) Dendritic Cells

CD4C/human immunodeficiency virus (HIV) transgenic mice develop an AIDS-like disease. We used this model to study the effects of HIV-1 on dendritic cells (DC). We found a progressive decrease in total DC numbers in the lymph nodes, with a significant accumulation of CD11b(Hi) DC. In the thymus, the recovery of transgenic CD8α(+) DC had a tendency to be lower. Spleen DC were augmented in the marginal zone. Transgenic DC showed a decreased capacity to present antigen in vitro, consistent with their reduced major histocompatibility complex class II expression and impaired maturation profile. The accumulation of immature DC may contribute to disease and may reflect an adaptive advantage for the virus by favoring its replication and preventing the generation of fully functional antiviral responses

CD4(+) T Cells from CD4C/HIV(Nef) Transgenic Mice Show Enhanced Activation In Vivo with Impaired Proliferation In Vitro but Are Dispensable for the Development of a Severe AIDS-Like Organ Disease

The cellular and molecular mechanisms of dysfunction and depletion of CD4(+) T lymphocytes over the course of human immunodeficiency virus type 1 (HIV-1) infection are still incompletely understood, but chronic immune activation is thought to play an important role in disease progression. We studied CD4(+) T-cell biology in CD4C/HIV transgenic (Tg) mice, in which Nef expression is sufficient to induce a severe AIDS-like disease including a preferential decrease of CD4(+) T cells. We show here that Nef-expressing Tg CD4(+) T cells exhibit an activated/memory-like phenotype which appears to be independent of antigenic stimulation, as documented in experiments involving breeding with AD10 TcR Tg mice. In addition, in vivo bromodeoxyuridine incorporation showed that a larger proportion of Tg than non-Tg CD4(+) T cells entered the S phase. However, in vitro, Tg CD4(+) T cells were found to have a very limited capacity to divide in response to stimulation with anti-CD3 and anti-CD28 or in allogeneic mixed leukocyte reactions. Interestingly, despite these observations, the deletion of Tg CD4(+) T cells had little impact on the development of other AIDS-like organ phenotypes. Thus, the Nef-induced chronic activation of CD4(+) T cells may exhaust the T-cell pool and may contribute to the thymic atrophy and the low number of CD4(+) T cells observed in these Tg mice

Upfront <i>DPYD</i> Genotyping and Toxicity Associated with Fluoropyrimidine-Based Concurrent Chemoradiotherapy for Oropharyngeal Carcinomas: A Work in Progress

Background: 5-FU-based chemoradiotherapy (CRT) could be associated with severe treatment-related toxicities in patients harboring at-risk DPYD polymorphisms. Methods: The studied population included consecutive patients with locoregionally advanced oropharyngeal carcinoma treated with carboplatin and 5-FU-based CRT one year before and after the implementation of upfront DPYD*2A genotyping. We aimed to determine the effect of DPYD genotyping on grade ≥3 toxicities. Results: 181 patients were analyzed (87 patients before and 94 patients following DPYD*2A screening). Of the patients, 91% (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of six additional patients with alternative DPYD variants (two c.2846A>T and four c.1236G>A mutations). Grade ≥3 toxicities occurred in 71% of the patients before DPYD*2A screening versus 62% following upfront genotyping (p = 0.18). When retrospectively analyzing additional non-DPYD*2A variants, the relative risks for mucositis (RR 2.36 [1.39–2.13], p = 0.0063), dysphagia (RR 2.89 [1.20–5.11], p = 0.019), and aspiration pneumonia (RR 13 [2.42–61.5)], p = 0.00065) were all significantly increased. Conclusion: The DPYD*2A, c.2846A>T, and c.1236G>A polymorphisms are associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can identify patients in whom 5-FU-related toxicity should be avoided

Prevalence of K-RAS Codons 12 and 13 Mutations in Locally Advanced Head and Neck Squamous Cell Carcinoma and Impact on Clinical Outcomes

Background. RAS gene mutations have an impact on treatment response and overall prognosis for certain types of cancer. Objectives. To determine the prevalence and impact of K-RAS codons 12 and 13 mutations in patients with locally advanced HNSCC treated with primary or adjuvant chemo-radiation. Methods. 428 consecutive patients were treated with chemo-radiation therapy and followed for a median of 37 months. From these, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was isolated and analyzed for K-RAS mutational status. Results. DNA extraction was successful in 197 samples. Of the 197 specimens, 3.5% presented K-RAS codon 12 mutations. For mutated cases and non-mutated cases, complete initial response to chemoradiation therapy was 71 and 73% (P=0.32). LRC was respectively 32 and 83% (P=0.03), DFS was 27 and 68% (P=0.12), distant metastasis-free survival was 100 and 81% (P=0.30) and OS was 57 and 65% (P=0.14) at three years. K-Ras codon 13 analysis revealed no mutation. Conclusion. K-RAS codon 12 mutational status, although not associated with a difference in response rate, may influence the failure pattern and the type of therapy offered to patients with HNSCC. Our study did not reveal any mutation of K-RAS codon 13