Abstract HER2-17: HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer

Abstract

Background: The HER2 antibody drug conjugate (ADC) fam-trastuzumab deruxtecan-nxki (T-DXd) significantly improves outcomes over standard chemotherapy in pts with HER2-LOW (IHC 1+ and IHC 2+ FISH-) metastatic breast cancer (MBC). Data from the DAISY (NCT04132960) trial in pts with HER2 IHC 0 or “ultra low” MBC revealed median progression-free survival (PFS) of 4.2 mos vs 6.7 mos in HER2-LOW and 11.1 mos in HER2 3+ pts (Diéras V, et al. Cancer Res. 2022;82(4_Suppl):PD8-02). There is an urgent need to develop methods to accurately discern HER2 LOW expression versus HER2 IHC 0/ultra-low expression so appropriate pts may receive T-DXd. We have developed a quantitative HER2 assay using reverse phase protein array (RPPA) technology with laser capture microdissection (LCM) enrichment of tumor epithelium that measures HER2 expression over a 65-fold dynamic range in HER2 IHC 0 to 3+ breast cancers. In I-SPY 1 and 2 trials we found that RPPA-assessed quantitative HER2 and activated/phosphorylated HER2 expression predicted for pathologic complete response in pts with HER2 IHC 0, HER2-LOW and HER2 3+ disease with various HER2 and other targeted agents. We then developed and validated the first CLIA/CAP-accredited quantitative HER2 protein expression and activation assay, and now explore quantitative HER2 expression in HER2 IHC 0/ultra low disease. Given the recent approval of sacituzumab govitecan-hziy in TNBC and the results of the TROPiCS-02 (NCT03901339), we also evaluated quantitative RPPA-based TROP-2 expression levels in HER2 IHC 0 breast cancer. Methods: LCM-enriched tumor epithelium was obtained from freshly cut FFPE core needle and resected breast cancer samples from 175 pts with pathology-determined HER2 IHC 0 status (N=68 ER+ and N=107 ER-). Quantitative HER2 output is generated as a relative intensity unit (RU) that is interpolated to an internal calibrator curve and a population referent comprised of known HER2+ (IHC 3+ and 2+/FISH+), HER2-LOW and HER2 IHC 0/ultra-low tumors to generate population-based cut-points as a referent. RPPA-based quantitative HER2 expression are reported as one of four levels of expression across the HER2 dynamic range observed: “non/extremely low”, “modest“, “moderate”, and “high”. Quantitative TROP2 protein expression levels were also measured for each case by the RPPA assay on the same lysate using the same adjectival determinants of relative expression as HER2. Results: For the HER2 IHC 0 ER+ cohort, we observed HER2 protein expression over a 25-fold dynamic range and that 57% (N=39) had HER2 non/extremely low, 37% (N=25) modest, and 4 (6%) moderate/high HER2 expression by RPPA. For the HER2 IHC 0 ER- cohort, we observed HER2 protein expression over a 15-fold dynamic range and that 71% (N=76) had non/extremely low, and 31% (N=29%) modest HER2 expression. We observed TROP-2 protein expression over a 35-fold dynamic range across all tumors measured, and it was found to be at least modestly expressed in 95% (37/39) ER+ and 95% (72/76) ER- of the IHC 0/ultra-low tumors that were also found to be HER2 non/extremely low by RPPA. Conclusions: LCM-RPPA quantitative assessment of HER2 expression showed that nearly 40% of ER+ IHC 0 and 30% of ER- IHC 0 “ultra-low” breast cancers actually had modest to moderate HER2 expression. Interestingly, this frequency approximates the response rate of 30% seen with T-DXd in HER2 IHC 0 pts in the DAISY trial. Quantitatively measured TROP2 is at least modestly expressed in the vast majority of RPPA-assessed HER2 IHC 0 cancers regardless of ER status, making a TROP2-directed ADC an attractive therapeutic option for these pts. If validated in larger studies, LCM-RPPA-based HER2 expression could provide a better understanding of the potential for therapeutic efficacy with T-DXd in patients with HER2 IHC “ultra-low” disease, and may better define true ultra-low HER2 expression in HER2 IHC 0 tumor biopsies at baseline and refine the lower limit of the HER2-LOW designation. Citation Format: Emanuel F. Petricoin, Brian A. Corgiat, Joyce O’Shaughnessy, Patricia LoRusso, Kris Weinberg, Justin Davis, Chelsea Gawryletz. HER2-17 Novel Quantitative HER2 Assay for Determining Dynamic HER2 Expression in the HER2 IHC 0 “Ultra-Low” Setting: Implications for Precision Therapy in HER2- Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-17.

Quantitative proteomic analysis of HER2 protein expression in PDAC tumors

“…HER2 IHC analysis was independently performed for 23 patients using the same specimens as were used for the LMD-RPPA analysis (Additional file 1: Table S3). The HER2 Total abundances quantified by IHC and RPPA were not correlated, consistent with previous findings [ 7 ]. Further, RPPA-based quantification of HER2 Total and pHER2 Y1248 were not correlated in PDAC or breast cancer tumors.…”

“…Tumor epithelium was harvested via LMD for RPPA quantification of total HER2 (HER2 Total ), phosphorylated (p)HER2 Y1248 , and pHER3 Y1289 as part of a 32-protein/phosphoprotein biomarker, research use only (RUO) panel in a commercial CLIA/CAP-accredited laboratory (Theralink Technologies, Inc.) examining the abundances of targets with known relevance in solid tumors, as previously described (Fig. 1 B, C) [ 7 , 16 – 18 , 28 , 29 , 32 , 33 ]. Protein and phosphoprotein-level expression was determined by comparing the RPPA-derived quantitative values from each patient LMD tumor sample in our cohort against an existing and extensively validated RPPA reference dataset derived from LMD enriched tumor epithelium from 400 breast cancer specimens with centrally-determined HER2-positive/amplified (IHC 3+ /2+ , FISH-positive) and HER2-negative/unamplified (IHC 0/1+ /2+ , FISH-negative) status.…”

“…The CLIA RPPA HER2 assay (Theralink Technologies, Inc.) uses the Invitrogen SP3 clone (rabbit recombinant antibody, (catalog MA5-14509, dilution 1:400). However, while the RPPA and IHC assays use different clones, the reference population used by the RPPA CLIA laboratory to qualify the RPPA assay results were derived from 400 breast tumors whose HER2 IHC was previously determined using the 4B5 clone and the results were > 95% correlated [ 7 ].…”

“…Historically, HER2-directed therapy was only indicated for HER2-positive breast cancer patients, defined as those with HER2 immunohistochemistry (IHC) 3+ , or IHC 2+ and positive fluorescence in situ hybridization (FISH; HER2/CEP17 ratio ≥ 2.0 and HER2 copy number (CN) signals/cell ≥ 4). Recent studies have shown the analytical and technical limitations of IHC-based quantitation of HER2 protein expression in HER2 unamplified/negative tumors due to the limitations and variability of interpretation by pathologists and the lack of accuracy and precision of HER2 protein quantitation using current HER2 IHC-based assays in the HER2 unamplified setting [ 5 – 7 ]. Moreover, recent data has found that approximately 60% of patients who were previously classified as HER2-negative could be considered as HER2-low, defined as IHC 1+ /2+ and FISH-negative [ 8 , 9 ].…”

“…Selective harvest of discrete cellular subpopulations from the complex milieu of the tumor microenvironment (TME) via laser microdissection (LMD) prior to reverse phase protein array (RPPA) analysis allows for the quantitative measurement and functional assessment of the activation state of protein drug targets and other known cancer-related proteins directly from enriched populations of microdissected tumor cells [ 28 , 29 ]. IHC-based determinations of HER2 expression in the HER2-low (IHC 1+ /2+ , FISH-negative) or HER2-ultra low (IHC 0) were not analytically accurate when compared to a CLIA quantitative RPPA-based HER2 assay to determine protein expression [ 7 ]. Nearly 40% of estrogen receptor (ER)-positive HER2 IHC 0 breast cancer tumors, and 30% of ER-negative HER2 IHC 0 breast cancer tumors expressed modest to moderate amounts of HER2 by RPPA, which approximates a 30% response rate seen with T-DXd in HER2-low patients in the DAISY trial [ 11 ].…”