Ali Mukherjee

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.

HER3, p95HER2, and HER2 protein expression levels define multiple subtypes of HER2-positive metastatic breast cancer.

Trastuzumab is effective in the treatment of HER2/neu over-expressing breast cancer, but not all patients benefit from it. In vitro data suggest a role for HER3 in the initiation of signaling activity involving the AKT–mTOR pathway leading to trastuzumab insensitivity. We sought to investigate the potential of HER3 alone and in the context of p95HER2 (p95), a trastuzumab resistance marker, as biomarkers of trastuzumab escape. Using the VeraTag® assay platform, we developed a dual antibody proximity-based assay for the precise quantitation of HER3 total protein (H3T) from formalin-fixed paraffin-embedded (FFPE) breast tumors. We then measured H3T in 89 patients with metastatic breast cancer treated with trastuzumab-based therapy, and correlated the results with progression-free survival and overall survival using Kaplan–Meier and decision tree analyses that also included HER2 total (H2T) and p95 expression levels. Within the sub-population of patients that over-expressed HER2, high levels of HER3 and/or p95 protein expression were significantly associated with poor clinical outcomes on trastuzumab-based therapy. Based on quantitative H3T, p95, and H2T measurements, multiple subtypes of HER2-positive breast cancer were identified that differ in their outcome following trastuzumab therapy. These data suggest that HER3 and p95 are informative biomarkers of clinical outcomes on trastuzumab therapy, and that multiple subtypes of HER2-positive breast cancer may be defined by quantitative measurements of H3T, p95, and H2T.

Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

Background The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples. Methodology and Principal Findings Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation. Significance This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.

Proximity-based assays for the detection of activated HER3, HER2/HER3 heterodimers and HER3/PI3K complexes in formalin-fixed, paraffin-embedded cell line controls and tumors.

Abstract #4040 Although kinase-defective, the role of the HER3 receptor in driving tumor growth through dimerization with other HER family members is well recognized. There is increasing interest in developing therapeutic strategies to interfere with signaling through this receptor, either directly or indirectly. At the same time, there is recognition that the activation of HER3 could be an important biomarker of drug response and resistance. In particular the HER2/HER3 heterodimer has been extensively studied and, among all the possible homo and heterodimers formed by HER receptors, is considered the most mitogenic because of the ability of HER3 to effectively couple to the pro-survival PI3K/Akt pathway.
 Using the VeraTag™ technology, we have developed a suite of assays designed to detect quantitatively the activation status of the HER3 receptor by measuring HER3-specific phosphorylation and formation of HER3 signaling complexes in formalin-fixed, paraffin-embedded (FFPE) cell line controls and tissue samples.These assays are dual antibody-based and measure complexes of proteins in close association since signal is generated only when the antibodies are bound to target-specific epitope sites proximal to one another. A capillary electrophoresis instrument equipped with laser-induced fluorescence provides a highly sensitive and quantitative read-out of the assay. We report data on assays which detect a ligand-induced, 2-5-fold change in HER2/HER3 heterodimer formation, a 10-20 fold change in phosphorylated HER3 signal and as high as a 10 fold change in HER3/PI3K complex formation in a series of cell line controls, spanning a wide range of HER2 and HER3 receptor levels. FFPE data were verified with results from co-immunoprecipitation using antibodies identical to those used in the VeraTag assay. The influence of the HER2 and HER3 levels on the formation of HER2/HER3 heterodimers and correlation with HER3 phosphorylation and formation of the PI3K adaptor complex will be discussed.
 In addition, we have used these assays to landscape HER3 activation in a number of tissues, including breast and ovarian tumors, focusing particularly on HER2 positive breast cancer, where the HER2/HER3 heterodimer is thought to play a particularly significant role. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4040.

Multiple Subtypes of HER-2/Neu-Positive Metastatic Breast Cancer.

Background: Using IHC or FISH to select patients for trastuzumab-based therapy, only half of HER2-positive patients show evidence of response. In vitro data implicate HER2:HER3 heterodimers and p95HER2 (p95), the truncated 95-kilodalton C-terminal fragment of HER-2 lacking the trastuzumab binding site, as mediators of resistance to trastuzumab at the receptor level. We have previously reported that central FISH-positive patients with low HER2 protein expression by VeraTag had significantly reduced response to trastuzumab compared to patients who had FISH-positive tumors with high HER2 protein expression (Lipton, SABCS 2008). Adding quantitative measurements of HER3 and p95, we offer evidence for the existence of multiple sub-types of HER2-positive tumors that respond differently to trastuzumab. Methods: Using the VeraTag assay, quantitative protein measurements of HER2, HER3, and p95 were made in FFPE specimens from a cohort of patients with metastatic breast cancer (MBC) and correlated with time to progression (TTP) and overall survival (OS) following treatment with first-line trastuzumab using Kaplan-Meier (KM) and Cox proportional hazards regression analyses. Results: Measurements of HER2 (H2T), HER3 (H3T) and p95 were made in FFPE tumor samples from 95 patients treated with first-line trastuzumab for metastatic breast cancer. Within the group that overexpressed HER2 by the VeraTag Assay (n=60), a group with highly overexpressed HER2 (n=15) had shorter TTP and OS than those that had moderate HER2 overexpression (median TTP 4.6 vs. 12 mos, HR=2.1; p=0.011; median OS 29 vs. 40 mos, HR=2.0; p=0.047). Within the subgroup with moderate H2T overexpression (n=45), bivariate Cox analyses demonstrated that p95 and H3T were independent predictors of TTP (p95 HR=2.1; p=0.031; H3T HR=3.5; p=0.0037). For OS, p95 was significant and H3T showed a strong trend (p95 HR=2.5; p=0.025, H3T HR=2.2; p=0.089). Univariate KM analysis with the p95 + and H3T + groups combined, gives the results in the table below. These data suggest that HER2-positive breast cancer patients can be classified into at least 4 sub-groups with different outcomes following trastuzumab treatment. Conclusions: These data suggest the existence of multiple subgroups of HER2-positive patients expressing varying HER2, p95, and HER3 levels that experience different clinical outcomes following treatment with trastuzumab. Furthermore, the association of HER3 and p95 overexpression with poor response to trastuzumab in otherwise HER2-positive tumors suggests possible treatment approaches with combinations of targeted therapies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2030.

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.