Expanded Low Allele Frequency RAS and BRAF V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial.

PURPOSE: Expanded RAS/BRAF mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. PATIENTS AND METHODS: CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. RESULTS: KRAS, NRAS, and BRAF mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). CONCLUSIONS: We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.

From validity to clinical utility: the influence of circulating tumor DNA on melanoma patient management in a real-world setting.

Melanoma currently lacks a reliable blood-based biomarker of disease activity, although circulating tumor DNA (ctDNA) may fill this role. We investigated the clinical utility (i.e., impact on clinical outcomes and interpretation of radiographic data) of measuring ctDNA in patients with metastatic or high-risk resected melanoma. Patients were prospectively accrued into ≥ 1 of three cohorts, as follows. Cohort A: patients with radiographically measurable metastatic melanoma who underwent comparison of ctDNA measured by a BEAMing digital PCR assay to tissue mutational status and total tumor burden; when appropriate, determinations about initiation of targeted therapy were based on ctDNA data. Cohorts B and C: patients with BRAF- or NRAS-mutant melanoma who had either undergone surgical resection of high-risk disease (cohort B) or were receiving or had received medical therapy for advanced disease (cohort C). Patients were followed longitudinally with serial ctDNA measurements with contemporaneous radiographic imaging to ascertain times to detection of disease activity and progressive disease, respectively. The sensitivity and specificity of the ctDNA assay were 86.8% and 100%, respectively. Higher tumor burden and visceral metastases were found to be associated with detectable ctDNA. In two patients in cohort A, ctDNA test results revealed a targetable mutation where tumor testing had not; both patients experienced a partial response to targeted therapy. In four of 30 patients with advanced melanoma, ctDNA assessments indicated evidence of melanoma activity that predicted radiographic evidence of disease progression by 8, 14, 25, and 38 weeks, respectively. CtDNA was detectable in three of these four patients coincident with radiographic evaluations that alone were interpreted as showing no evidence of neoplastic disease. Our findings provide evidence for the clinical utility of integrating ctDNA data in managing patients with melanoma in a real-world setting.

Compensatory mutations improve general permissiveness to antibiotic resistance plasmids.

Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multidrug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modelling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase ß-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.