High Heregulin Expression Is Associated with Activated HER3 and May Define an Actionable Biomarker in Patients with Squamous Cell Carcinomas of the Head and Neck

<div><p>Purpose</p><p>Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level <i>HRG</i> expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level <i>HRG</i> expression defines a subpopulation of SCCHNs with activated HER3.</p> <p>Experimental Design</p><p>qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to <i>HRG</i> expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization.</p> <p>Results</p><p>SCCHN tumors express the highest levels of <i>HRG</i> compared to a diverse collection of other tumor types. We show that high <i>HRG</i> expression is associated with activated HER3, whereas low <i>HRG</i> expression is associated with low HER3 activation in SCCHN tumors. Furthermore, <i>HRG</i> expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens.</p> <p>Conclusions</p><p><i>HRG</i> expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of <i>HRG</i> is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.</p> </div

Expression of <i>HRG</i> in primary vs. recurrent SCCHN. A)

<p>qRTPCR analysis comparing the levels of <i>HRG</i> expression in unmatched primary and recurrent SCCHN specimens. B) qRTPCR analysis comparing the levels of <i>HRG</i> expression in matched primary and recurrent SCCHN. In both cases <i>HRG</i> levels appear to be higher in the recurrent disease setting compared to primary resected SCCHN.</p

Detailed evaluation of <i>HRG</i> and <i>HER3</i> expression in matched therapy naïve and post-chemotherapy SCCHNs.

•<p>RxN corresponds to therapy naïve patients;</p>†<p>PC corresponds to Post-Chemo patients.</p><p>% Autocrine cells were based on tumor cell content only; qRTPCR was performed on macrodissected specimens as outlined in the methods section.</p

Analysis of <i>HRG</i> expression in the indicated epithelial tumors.

<p><b>A)</b> qRTPCR of <i>HRG</i> in the indicated tumor types shows that SCCHNs have the highest median expression of HRG. NSCLC = non-small cell lung cancer; TI–III = pathological stage I–III; CRC = metastatic colorectal carcinoma; Pl/R Ova = platinum refractory ovarian cancers; SCCHN = squamous cell carcinoma of the head and neck; TNB = triple negative breast cancer. B) Statistical analysis of HRG expression in SCCHNs. The two mixture distributions and their joined distribution are depicted in red, green and black lines above. The dotted line indicates the inflection point between the high and low components of expression of HRG in SCCHN, which is set at 0.3689 on the logarithm scale. The sensitivity of identifying HRG overexpressed population using a cutoff of 0.3689 in the logarithm scale (∼1.45 on the linear scale) is 90.8% and the attendant specificity is 93.4%. C) IP-westerns for pHER3 (Y1289), pTyr, and total HER3 in 19 fresh frozen SCCHNs. MCF7 is a negative control; MCF7+ HRG and PCI6A are positive controls. The controls for the pTyr blot were run separately whereas the controls for the pHER3 were run concurrently. D) qRTPCR for HRG in 18/19 SCCHNs (overlap with the IP-western). Black lines below the x-axis indicate the tumors with detectable pHER3 by IP-western.</p

Summary of the Relationship Between <i>HRG</i> Expression and the Extent of Autocrine versus Paracrine Expression in Matched Therapy Naïve Primary Specimens and Post Therapy Biopsies Taken at Recurrence.

*<p>Wilcoxon rank sign test for paired samples.</p

Dual color <i>in situ</i> hybridization for <i>HER3</i> and <i>HRG</i> in benign and malignant tissue shows that <i>HER3</i> and <i>HRG</i> are typically expressed by different cells.

<p>(HER3 = red; HRG = cyan) A) Normal squamous epithelium; B) Normal upper respiratory epithelium; C) well differentiated SCCHN; D) autocrine expression of <i>HRG</i> and <i>HER3</i>. In panels A & B, the yellow arrows point to basal cells and the black arrows point to squamous epithelium or more differentiated cells within the respiratory tract. In panel C, the bracketed structure is a squamous morule, the yellow arrow again points to “basal-like” tumor cells whereas the black arrow identifies spiny cells. Panel D represents anaplastic autocrine cells, characteristic of a poorly differentiated SCCHN. Magnification is 200x for A–C and 400× for part D.</p