Calibration of cell-free DNA measurements by next-generation sequencing.

OBJECTIVES: Accurate monitoring of disease burden depends on accurate disease marker quantification. Although next-generation sequencing (NGS) is a promising technology for noninvasive monitoring, plasma cell-free DNA levels are often reported in misleading units that are confounded by non-disease-related factors. We proposed a novel strategy for calibrating NGS assays using spiked normalizers to improve precision and to promote standardization and harmonization of analyte concentrations. METHODS: In this study, we refined our NGS protocol to calculate absolute analyte concentrations to (1) adjust for assay efficiency, as judged by recovery of spiked synthetic normalizer DNAs, and (2) calibrate NGS values against droplet digital polymerase chain reaction (ddPCR). As a model target, we chose the Epstein-Barr virus (EBV) genome. In patient (n = 12) and mock (n = 12) plasmas, NGS and 2 EBV ddPCR assays were used to report EBV load in copies per mL of plasma. RESULTS: Next-generation sequencing was equally sensitive to ddPCR, with improved linearity when NGS values were normalized for spiked DNA read counts (R2 = 0.95 for normalized vs 0.91 for raw read concentrations). Linearity permitted NGS calibration to each ddPCR assay, achieving equivalent concentrations (copies/mL). CONCLUSIONS: Our novel strategy for calibrating NGS assays suggests potential for a universal reference material to overcome biological and preanalytical variables hindering traditional NGS strategies for quantifying disease burden.

Less Is More: Evaluation of Gross Examination Protocol for Cystectomy Specimens Following Neoadjuvant Chemotherapy

OBJECTIVES: Neoadjuvant chemotherapy (NAC) confers a survival advantage for muscle-invasive bladder cancer and is now recommended for chemotherapy-eligible patients. NAC may result in absent gross tumor, and current cystectomy gross examination protocols do not specify approach for these cases. METHODS: We included cystectomies performed from 2010 to 2018, capturing a period pre- and post-NAC recommendations. Gross descriptions were reviewed and slides of patients who received NAC were evaluated for microscopic tumor, number of blocks with tumor, and location of those blocks. RESULTS: We identified 239 radical cystectomies for bladder cancer (147 NAC, 92 non-NAC). Gross lesions were not identified for 91 cases. NAC cases had more total blocks submitted (mean, 17.5) compared with non-NAC cases (mean, 16.6). More NAC cases had additional blocks submitted (20 cases) compared with non-NAC cases (2), which were more frequently additional random sections. Of 108 NAC cases with residual carcinoma, only 2 (1.9%) were upstaged on additional random sections. CONCLUSIONS: At our institution, NAC and non-NAC cases are grossed with similar numbers of initial blocks; however, NAC cases are more likely to submit additional sections of gross lesions and random bladder without significant changes in stage. Our data suggest current gross examination protocols are sufficient for NAC cystectomies.

Carbol fuchsin stain enhances detection of poly-(d, l-lactide-co-glycolide) microspheres in exenatide extended-release cutaneous injection-site foreign body reaction.

Injection of high-viscosity fluids into subcutaneous tissues may lead to a granulomatous reaction called sclerosing lipogranuloma (SL). Poly-(d,l-lactide-co-glycolide) (PLG or PLGA) microspheres are used as vehicles for extended-release drugs. Here we describe the histopathologic features of a case of SL induced by exenatide extended-release injections, and the staining pattern of PLG microspheres and microsphere remnants with carbol fuchsin.