Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

Chi-Chih Kang,Courtney Schiffman,Jessica Bockhorn,Haiyan Huang,Toby M Ward,Amy E Herr,Mark D Pegram

doi:10.1038/s41698-018-0052-3

Chi-Chih Kang, Courtney Schiffman + Show 5 more

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https://doi.org/10.1038/s41698-018-0052-3

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In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated.

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Oncoproteins and their truncated protein forms are implicated in tumor progression, metastasis, and drug resistance.[1,2,3] Human epidermal growth factor receptor 2 (HER2, a.k.a. erbB2, UniprotP04626) can be expressed as the full-length receptor or as truncated forms (t-erbB2s).[1]
Pertuzumab, nor T-DM1 can bind to t-erbB2s as the HER2 isoforms lack the extracellular domain of full-length HER2, which includes the therapeutic antibody-binding epitopes
The single-cell resolution western blot (scWB) device (Fig. 1a) is a microscope slide coated with a thin layer of photoactive polyacrylamide gel (PAG)

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Introduction

Oncoproteins and their truncated protein forms are implicated in tumor progression, metastasis, and drug resistance.[1,2,3] Human epidermal growth factor receptor 2 (HER2, a.k.a. erbB2, UniprotP04626) can be expressed as the full-length receptor or as truncated forms (t-erbB2s).[1]. The canonical full-length protein is targeted by trastuzumab, pertuzumab—both humanized IgG1 monoclonal antibodies, and by the antibody-drug conjugate ado-trastuzumab emtansine (T-DM1).[8]. No FDA-approved antibody-based therapies against HER2 target the truncated HER2 isoforms or CTFs.[9,10] Neither trastuzumab, pertuzumab, nor T-DM1 can bind to t-erbB2s as the HER2 isoforms lack the extracellular domain of full-length HER2, which includes the therapeutic antibody-binding epitopes. The expression of t-erbB2 proteins (p95, p110, or Δ16) in cancer cells suggests one possible resistance mechanism against antibodybased anti-HER2 therapies.[1,7] Some, though not all,[11] clinical studies have shown that metastatic HER2-positive patients expressing t-erbB2s have worse clinical outcomes when treated with trastuzumab,[12] as evidenced by shorter progression-free survival rates.[13,14] Comparison was made to patients expressing only full-length HER2. Expression of t-erbB2s has been associated with lymph node and brain metastases.[12,15,16] While patients with t-erbB2s may have worse progression-free survival under anti-HER2 trastuzumab therapy, these patients can benefit from other treatments, such as erbB2-selective tyrosine kinase inhibitors.[17,18] precise tumor classifications that include information about expression of truncated oncoprotein isoforms and CTFs— rare tumor markers—hold promise in guiding treatment decisions for specific patients.[19]

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