Abstract P4-01-05: Cell free DNA analysis identifies actionable ERBB2 amplifications in patients with HER2 equivocal breast cancer
Authors: Rich TA , Raymond VM , Ahn ER , Banks KC , Brufsky A , Lee C , Lippman M , Pluard TJ , Schwab RB , Lanman RB 
Published: May 2019

Abstract
Background:
Determination of ERBB2 (HER2) expression or amplification informs eligibility of HER2-targeted therapies. ASCO and NCCN guidelines recommend evaluation of HER2 status on primary invasive breast cancers and on a metastatic site if stage IV, where possible, as treatment is based on the status of the metastasis. Reassessment of HER2 status should also be considered in patients with disease recurrence as initially HER2-negative tumors may acquire HER2 amplification at progression. HER2 status can be complicated by equivocal results from in situ hybridization (ISH) and/or immunohistochemistry (IHC). Clarification requires reflex testing on the same tissue specimen or repeat testing on a new specimen, however some patients' tissue status remains equivocal. Furthermore, metastases to bone, lung, or brain may be difficult to re-biopsy or of low DNA quality. Rapid and non-invasive blood-based cell-free DNA (cfDNA) NGS may facilitate identification of HER2 targetable disease in advanced breast cancer.
Methods:
We assessed the frequency of ERBB2 amplification detectable by a blood-based cell-free DNA (cfDNA) assay among patients with metastatic breast cancer with equivocal HER2 results in tissue. cfDNA samples were ordered as part of routine clinical care using an assay validated for the detection of copy number amplification in ERBB2 (tests run between 03/2014-04/2017 by Guardant Health, Redwood City, CA). Submitted pathology reports were reviewed for HER2 status which was categorized as positive, negative, or equivocal based on the interpretation issued by the reading pathologist at the time the test was ordered. Patients were included if they had an equivocal result on IHC and/or ISH unless both assays were performed on the same specimen and one provided a definitive negative or positive HER2 result. Additionally, 4 patients with equivocal IHC or ISH results were excluded as biopsy of another tumor site revealed a positive HER2 result around the same time as the equivocal test. For the 349 patients with multiple cfDNA samples, the earliest pathology report was referenced.
Results:
Tissue HER2 status was available for 1,853 unique patients (98.8% female, median age at testing was 58y, range 26-91y). 141 patients (7.6%) had equivocal HER2 results in tissue; 99 by IHC alone, 14 by ISH alone, and 28 were equivocal by both assays. Among these, 126 patients (89.4%) had at least one sample with ctDNA detected. 12/126 (9.5%) had amplification of ERBB2 detected in at least one cfDNA sample. Samples were drawn a median of 267 days after tissue collection (range 4 days – 11.5 years). Frequency of ERBB2 amplification was similar regardless of time between tissue and blood collection but was higher among patients with ISH results alone (4/14, 36.4%) compared to those with IHC alone (6/89, 6.7%) or both assays (6/26, 7.6%; p=0.006).
Conclusion:
cfDNA testing identifies a significant number of patients with HER2-targetable advanced breast cancer whose tissue was HER2 equivocal. cfDNA testing may supplement tissue-based methods to help clarify HER2 status in metastatic disease as well as identify patients who may acquire HER2 amplification subsequent to their initial biopsy.
Citation Format: Rich TA, Raymond VM, Ahn ER, Banks KC, Brufsky A, Lee C, Lippman M, Pluard TJ, Schwab RB, Lanman RB. Cell free DNA analysis identifies actionable ERBB2 amplifications in patients with HER2 equivocal breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-05.