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Adult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy., Significance: True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies., (©2024 The Authors; Published by the American Association for Cancer Research.)

The new ASCO/CAP guidelines on hormone receptor testing in breast cancer recommends standard operating procedures (SOPs) established to confirm or adjudicate estrogen receptor (ER) results with weak or ≤10% staining, and the status of internal controls (ICs) reported for cases with 0% to 10% staining. The aim of this study is to determine the frequency of ER testing with weak or ≤10% staining that may require additional steps following SOPs and to identify any correlation between hormone receptor status of the tumor and the likelihood of finding IC. Breast cancer cases between January 2014 and April 2019 were included to identify negative, low-positive and weak-positive cases. The presence/absence of IC was correlated to tumor type. Following ASCO/CAP guidelines, 29.8% of cases (374/1261) will need additional steps to confirm/adjudicate results due to negative, low, or weak positive ER status. The probability of finding IC is ~50% lower in cases of ER and progesterone receptor (PgR) negative tumors. Repeat testing may be warranted in 13.1% (92/700) of all cases due to lack of IC. In conclusion, the new ASCO/CAP guidelines recommend laboratories to establish and follow SOP to confirm or adjudicate ER results for about 30% of the cases before reporting hormone receptors status. Over 40% of cases with <10% tumor ER positivity lacked IC that may need a comment per the guidelines indicating a repeat testing may be warranted. However, the presence/absence of IC may be related to the subtype of breast cancer and should not necessarily bring into question the validity of the test., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)