ABSTRACT
Alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR assays for reliable detection of 12 AR targets including AR amplification, AR splice variant 7, and 10 AR hotspot mutations, as well as AR and KLK3 gene expression from plasma-derived cell-free DNA and cell-free RNA. The assays demonstrated excellent analytical sensitivity and specificity ranging from 95% to 100% (95% CI, 75% to 100%). Intrarun and interrun variation analyses revealed a high level of repeatability and reproducibility. The developed assays were applied further in peripheral blood samples from 77 patients with advanced prostate cancer to assess their feasibility in a real-world scenario. Optimizing the reverse transcription of RNA increased the yield of plasma-derived cell-free RNA by 30-fold. Among 23 patients with castration-resistant prostate cancer, 6 patients (26.1%) had one or a combination of several AR alterations, whereas only 2 of 54 patients (3.7%) in the hormone-sensitive stage showed AR alterations. These findings were consistent with other studies and suggest that implementation of comprehensive AR status detection in clinical practice is feasible and can support the treatment decision-making process.
Subject(s)
Receptors, Androgen , Humans , Receptors, Androgen/genetics , Male , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Aged , Prostatic Neoplasms/genetics , Prostatic Neoplasms/diagnosis , Reproducibility of Results , Mutation , Sensitivity and Specificity , Middle Aged , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/blood , Kallikreins/blood , Kallikreins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/diagnosis , Prostatic Neoplasms, Castration-Resistant/blood , Multiplex Polymerase Chain Reaction/methodsABSTRACT
Detection of EGFR mutations from blood plasma represents a gentle, non-invasive alternative to rebiopsy and can therefore be used for therapy monitoring of non-small-cell lung cancer (NSCLC) patients. The aim of this project was to investigate whether the Reveal ctDNA™ 28 NGS assay (ArcherDX, Boulder, CO), has a comparable sensitivity and specificity to droplet digital PCR (ddPCR, gold-standard) and is therefore suitable for therapy monitoring of progressing lung cancer patients. First, we validated the NGS assay with a commercially available reference material (SeraCare, Massachusetts, US). Using an input of 22 ng, a sensitivity of 96% and a specificity of 100% could be achieved for variant allele frequencies (VAF) of 0.5%. For variants at a VAF of 0.1% the sensitivity was substantially reduced. Next, 28 plasma samples from 16 patients were analyzed and results were compared to existing ddPCR data. This comparative analysis of patient samples revealed a concordance of 91% between NGS and ddPCR. These results confirm that the Reveal ctDNA™ 28 NGS assay can be used for therapy monitoring of patients under TKI therapy. However, due to the slightly superior sensitivity of ddPCR, a combination of NGS (with broad coverage of a large number of genomic loci) and ddPCR (with targeted highly sensitive detection of specific mutations) might be the ideal approach.
Subject(s)
Carcinoma, Non-Small-Cell Lung/blood , Cell-Free Nucleic Acids/blood , Circulating Tumor DNA/blood , Adult , Aged , Biomarkers, Tumor/blood , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell-Free Nucleic Acids/genetics , Circulating Tumor DNA/genetics , ErbB Receptors/blood , ErbB Receptors/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Liquid Biopsy , Male , Middle Aged , Mutation/geneticsABSTRACT
We here revise the genus Pyropteron Newman, 1832 using molecular and morphological analyses. Our data support the monophyly of Pyropteron with Synansphecia CapuÈe, 1973 (syn. rev.) being its junior subjective synonym. Four taxa are described as new to science, Pyropteron minianiformis xerxes Bartsch, Pühringer, Lingenhöle Kallies ssp. nov., Pyropteron hellenicum Bartsch, Pühringer, Lingenhöle Kallies sp. nov., Pyropteron jordanicum Bartsch, Pühringer, Lingenhöle Kallies sp. nov. and Pyropteron leucomelaena blaesii Bartsch, Pühringer, Lingenhöle Kallies ssp. nov., and 4 species are raised to species rank, Pyropteron nigrobarbata (Rebel, 1916) stat. nov., Pyropteron icteropus (Zeller, 1847) stat. rev., Pyropteron euglossaeformis (Lucas, 1849) stat. rev. and Pyropteron erodiiphaga (Dumont, 1922) stat. rev. To stabilize the taxonomy, we designate neotypes for Pyropteron euglossaeformis stat. rev. and Pyropteron ceriaeformis (Lucas, 1849). Pyropteron pipiziformis (Lederer, 1855) comb. nov., is combined with Pyropteron for the first time. The identity of Pyropteron atlantis (Schwingenschuss, 1935), previously confused with Pyropteron borreyi (Le Cerf, 1922) is fixed. We treat Pyropteron muscaeformis lusohispanica Lastuvka Lastuvka, 2007 syn. nov. as a synonym of Pyropteron koschwitzi (Spatenka, 1992), Pyropteron minianiformis aphrodite Bartsch, 2004 syn. nov. as a synonym of Pyropteron minianiformis destituta (Staudinger, 1894), Pyropteron muscaeformis occidentalis Joannis, 1908 syn. nov. as a synonym of Pyropteron muscaeformis (Esper, 1783), and Sesia lecerfi Oberthür, 1909 syn. nov. is considered a synonym of Bembecia ichneumoniformis ([Denis Schiffermüller], 1775). Finally, we discuss a model whereby frequent switches in hostplant usage drive rapid speciation in the genus Pyropteron.