Rajiv Dua

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.

Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels of c-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens.

Abstract LB-323: Subclassification of squamous cell carcinomas of the head and neck based on HER/ErbB and c-MET receptor protein expression and activation profiles

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The response rates of HER1 antibody monotherapy (∼13%) are comparable to the added response rates of HER1 antibody therapy when used in combination with chemotherapy (∼16%) in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). Measures of HER1/ErbB1/EGFR protein levels by immunohistochemistry (IHC) or gene copy number by fluorescent in-situ hybridization (FISH) do not correlate with response, therefore, alternative gene or protein expression signatures are needed to identify patients most likely to respond to HER1 targeted therapies. Previously, quantitative VeraTag proximity immunoassays performed on formalin-fixed, paraffin imbedded SCCHN tumor tissue identified an activated HER1 signature (HER1-HER1 homodimers; HER1pY1173) that was significantly more pronounced in SCCHN compared to other carcinoma of the head and neck. In this study, we analyzed the expression of HER1, 2, 3 and c-MET along with the activation of HER1 and HER3 in 56 SCCHN tumors. Our results identified two distinct protein expression profiles: an activated HER1 profile, and a second profile characterized by the different expression levels of HER2, HER3, phosphorylated HER3, and c-MET relative to activated HER1. Our working hypothesis is that the first profile correlates with sensitivity to HER1 targeted therapies while the second profile correlates with either sensitivity or resistance to HER1 targeted therapies. HER1 protein levels measured by VeraTag assay varied over a ∼20-fold dynamic range, and significantly correlated with HER1 IHC staining and mRNA levels determined by qRT-PCR. HER2 and HER3 protein expression spanned a 15- to 20-fold range, and correlated with mRNA levels. Several of the highest HER1 expressing tumors have increased gene copy number; however, similar high values were measured in tumors without amplification, indicating that additional mechanisms of high HER1 expression exist in SCCHN. Although high HER1 activation measured by HER1-HER1 dimer and HER1pY1173 correlates with HER1 levels (r=∼0.6, p<0.05), and are associated with the highest levels of HER1 expression measured by multiple methods, not all high HER1 expressing tumors displayed highly activated HER1. These observations may explain why total HER1 expression in SCCHN tumors, determined by IHC or FISH, does not correlate with response to targeted therapies. On the other hand, approximately 16% of the SCCHN tumors studied here (9/56) exhibit a highly activated HER1 profile; similar in magnitude to the response rate seen with HER1 targeted antibody therapy. Future studies are designed to further validate the HER1 activation signature as a predictor of response to HER1 targeted therapies and/or the attenuation of response due to HER2, HER3 or c-MET expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2011-LB-323

Abstract LB-66: Quantitative assessment of HER/erbB receptor protein expression and activation status in FFPE tumor samples identifies an activated HER1 signature in squamous cell carcinomas of the head and neck

The oncogenesis and maintenance of several solid tumor types appears dependent on HER1/erbB1 activation status through several mechanisms including gene mutations, gene amplification/overexpression, and ligand availability. However, it has been difficult to correlate HER1 protein levels or activation status in squamous cell carcinoma of the head and neck (SCCHN) to the 10-30% targeted-drug response rates due to the lack of robust, sensitive, and quantitative assays in formalin-fixed, paraffin embedded (FFPE) tumor tissues. Toward the goal of having improved predictive assays for anti-EGFR based therapy we used the novel VeraTag TM technology to develop sensitive and quantitative assays to measure the total HER1, HER2, and HER3 protein levels and the activated forms represented by HER1-HER1 homodimers, phosphorylated HER1, and HER1-HER2 heterodimers in SCCHN and other head and neck (HN mean=34-fold) and other HN mean=25-fold). In SCCHN samples, HER1 VeraTag protein measurements significantly correlated with HER1 protein as measured by IHC (Spearman9s r =0.587, p=0.0083, n=19), HER1 DNA levels as measured by FISH (r =0.688, p=0.0016, n=19), and HER1 mRNA levels measured by qPCR (r =-0.649, p=0.0036, n=18). HER2 and HER3 VeraTag protein levels were generally low, with approximately half of the SCCHN tumors having below detectable levels. HER3 VeraTag protein levels correlated with HER3 mRNA (qPCR) levels (r =-0.574, p=0.016) whereas the HER2 protein and mRNA (qPCR) measurements did not significantly correlate. Active HER1 was notable in approximately 50% of SCCHN tumors as measured by a signal to background (S/B) >2- to 3-fold for HER1-HER1 homodimers, or HER1 phosphotyrosine (pY) (pY1173 and pan-pY), or HER1-HER2 heterodimers. Although the distribution of HER1 protein expression levels was similar between the SCCHN and other HN the SCCHN tumors had dramatically greater HER1 activation as seen with increased relative levels of HER1-HER1 homodimers (p=0.0055) and HER1pY1173 (p=0.0036). The SCCHN tumor set could be divided into several subgroups based on signatures characterized by HER1 VeraTag protein levels, activation profile, and c-Met protein levels. Two notable signatures include tumors having medium to high HER1 protein levels/active HER1 with co-expression of either relatively low or high c-Met levels, represented by 2/19 and 5/19 tumors, respectively. The association of these signatures with prognosis and targeted drug responses will be evaluated in future studies with clinical samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-66.

Abstract B177: Detection and quantification of ligand‐receptor interactions in formalin‐fixed paraffin embedded samples by a novel proximity‐based assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin‐fixed paraffin embedded (FFPE) sections, a format used to preserve human tissue samples. This is the only format for readily available tissues from clinical trials and patient disease management. However, phosphorylation is generally less stable and consequently, less reproducible which can, in a clinical setting lead to ambiguous data. Here, we describe an alternative, novel approach to detect and quantify activation of receptors in FFPE samples. We used the ligand/receptor, HGF/c‐MET as our model system. HGF is the only known ligand of c‐MET and ligand binding causes activation of the c‐MET receptor. Using, the VeraTag technology, a proximity‐based assay, validated antibodies were used to detect and quantify total c‐MET and total HGF in FFPE samples as well as to detect and quantify ligand stimulated and endogenous HGF/c‐MET interactions in FFPE cell line pellets. The detection was validated in human non‐small cell lung and stomach carcinoma FFPE samples. Our data demonstrate a novel approach to detect and quantify ligand‐receptor interactions by VeraTag proximity‐based assays that can be widely applied to measure activation of receptors in archived FFPE human tissue samples. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B177.