Quantitative proteomic analysis of HER2 protein expression in PDAC tumors

Abstract

Metastatic pancreatic adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, with a 5-year survival rate of only 11%, necessitating identification of novel treatment paradigms. Tumor tissue specimens from patients with PDAC, breast cancer, and other solid tumor malignancies were collected and tumor cells were enriched using laser microdissection (LMD). Reverse phase protein array (RPPA) analysis was performed on enriched tumor cell lysates to quantify a 32-protein/phosphoprotein biomarker panel comprising known anticancer drug targets and/or cancer-related total and phosphorylated proteins, including HER2^Total, HER2^Y1248, and HER3^Y1289. RPPA analysis revealed significant levels of HER2^Total in PDAC patients at abundances comparable to HER2-positive (IHC 3+) and HER2-low (IHC 1+ /2+ , FISH-) breast cancer tissues, for which HER2 screening is routinely performed. These data support a critical unmet need for routine clinical evaluation of HER2 expression in PDAC patients and examination of the utility of HER2-directed antibody-drug conjugates in these patients.

Keywords: HER2; Laser microdissection; Pancreatic adenocarcinoma; Proteomics.

Fig. 1

Overview of study design. A Formalin-fixed, paraffin-embedded (FFPE) primary and/or metastatic tumor surgical biopsy specimens were prospectively obtained from patients with PDAC, breast cancer, and other solid tumor malignancies as part of an IRB-approved Molecular Tumor Board (MTB) study at the Inova Schar Cancer Institute (ISCI). B Workflow diagram depicting tissue specimen analysis. Tumor epithelium (5–10 µm²) from tissue specimens was harvested via LMD at a ratio of 1 mm² LMD area per 2.5 µl buffer into a lysis buffer containing 50 mM Bond-Breaker TCEP Solution, 225 mM Tris–HCl, 4% v/v sodium dodecyl sulfate (SDS), 10% v/v glycerol, in MilliQ Type I water. RPPA analysis of the LMD enriched tissue lysates was performed by Theralink Technologies, Inc., as previously described [32, 33]. C Representative micrographs of tissue thin sections stained with hematoxylin and eosin (H&E) mounted onto a glass slide histopathological assessment (left, 5 µm), and onto a slide containing a polyethylene naphthalate (PEN) membrane after LMD harvest of tumor epithelium (right, 8 µm). Clinical NGS was performed by a commercial sequencing laboratory

Fig. 2

RPPA quantification of HER2^Total, pHER2^Y1248, pHER3^Y1289 and correlation with CLIA-approved clinical IHC scoring. Fisher's Exact tests were performed using SAS software (v9.4) to compare the RPPA abundances of HER2^Total, pHER2^Y1248, and pHER3^Y1289 between groups. An asterisk (*) indicates p < 0.05. NS  not significant