Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

Area of Science:

• Oncology
• Biochemistry
• Molecular Biology

Background:

• Truncated HER2 isoforms (t-erbB2) and proteolysis fragments are linked to drug resistance and poor prognosis in HER2-positive breast cancer.
• Distinguishing t-erbB2 from full-length HER2 is crucial for accurate diagnosis and treatment planning.

Purpose of the Study:

• To develop a high-selectivity cytopathology assay for differentiating t-erbB2 from full-length HER2 without isoform-specific antibodies.
• To analyze the clinical relevance of t-erbB2 expression and its associated signaling pathways in breast tumors.

Main Methods:

• A microfluidic, single-cell western blot assay was developed using electrophoretic separation and a pan-HER2 antibody.
• The assay resolves and detects both full-length HER2 and smaller t-erbB2 isoforms in single-cell lysates.
• Breast tumor biopsies were analyzed, followed by target multiplexing and clustering analyses for signaling scrutiny.

Main Results:

• The assay successfully distinguished t-erbB2 from full-length HER2 in single-cell lysates.
• Two out of eight breast tumor biopsies showed significant t-erbB2 expression (15% and 40% of cells).
• Statistically significant differences in t-erbB2 to full-length HER2 ratios and associated signaling (e.g., ribosomal S6) were observed.

Conclusions:

• Cytometric assays capable of profiling protein isoforms and signaling states can offer novel cancer classification methods.
• This approach provides a precise way to describe drug resistance mechanisms involving oncoprotein isoforms and fragments.
• The developed assay has the potential to advance clinical pathology and inform treatment decisions for HER2-positive breast cancer patients.