John Winslow

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.

Quantitation of p95HER2 in Paraffin Sections by Using a p95-Specific Antibody and Correlation with Outcome in a Cohort of Trastuzumab-Treated Breast Cancer Patients

Purpose: p95HER2 is an NH2-terminally truncated form of HER2 that lacks the trastuzumab binding site and is therefore thought to confer resistance to trastuzumab treatment. In this report, we introduce a new antibody that has enabled the first direct quantitative measurement of p95HER2 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. We sought to show that quantitative p95HER2 levels would correlate with outcome in trastuzumab-treated HER2-positive metastatic breast cancer. Experimental Design: The novel p95HER2 antibody used here was characterized for sensitivity, specificity, and selectivity over full-length HER2. Quantitative p95HER2 levels were measured in 93 metastatic breast tumors using a VeraTag FFPE assay to determine the correlation of p95HER2 levels with outcomes. Results: Within a cohort of trastuzumab-treated metastatic breast cancer patients, high levels of p95HER2 were found to correlate with shorter progression-free survival [hazard ratio (HR), 1.9; P = 0.017] and overall survival (HR, 2.2; P = 0.012) in patients with tumors selected to be HER2 positive by the VeraTag HER2 assay. For those with tumors found to be fluorescence in situ hybridization positive, elevated p95HER2 correlated similarly with shorter progression-free survival (HR, 1.8; P = 0.022) and overall survival (HR, 2.2; P = 0.009). Conclusions: We have successfully generated an antibody that can specifically detect p95HER2, and developed an assay to quantify expression in FFPE tumor specimens. Using this novel assay, we have identified a group of HER2-positive patients expressing p95HER2 that have a worse outcome while on trastuzumab. As p95HER2 retains sensitivity to kinase inhibitors, measurement of p95HER2 in breast tumor sections may be useful in guiding treatment for patients with HER2-positive breast cancer. Clin Cancer Res; 16(16); 4226–35. ©2010 AACR.

Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action

IntroductionLigand-bound and phosphorylated ErbB/HER heterodimers are potent signaling forms of this receptor family, and quantitative measurements of these active receptors may be predictive of patient response to targeted therapies. Using VeraTag™ technology, we developed and characterized quantitative assays measuring epidermal growth factor (EGF)-dependent increases in activated HER receptors in tumor cell line lysates and formalin-fixed, paraffin-embedded (FFPE) tumor sections. We demonstrated the ability of the assays to quantitatively measure changes in activated HER1 and HER2 receptor levels in cell lines following treatment with 2C4, erlotinib, and lapatinib. We utilized these assays to determine the prevalence and distribution of activated HER1, HER2, and HER1-HER2 heterodimers in 43 HER2-positive breast tumors.MethodsAssays for activated HER1 and HER2 receptors in FFPE and cell lysate formats were developed using VeraTag™ technology, which requires the proximity of an antibody pair for light-dependent release of a fluorescently labeled tag, followed by capillary electrophoresis-based quantitation.ResultsLigand-dependent and independent HER1-HER2 heterodimer levels measured by lysate and FFPE VeraTag™ assays trended with HER1 and HER2 expression levels in tumor cell lines, which was confirmed by co-immunoprecipitation. The formation of EGF-dependent HER1-HER2 heterodimers were inhibited by the HER2-targeted monoclonal antibody 2C4 and stabilized by the HER1 tyrosine kinase inhibitor (TKI) erlotinib. EGF-dependent HER1 and HER2 phosphorylation was inhibited by lapatinib and erlotinib. Further, we observed that dominant receptor signaling patterns may switch between HER1-HER1 and HER1-HER2, depending on drug mechanism of action and relative levels of HER receptors. In FFPE breast tumors that expressed both HER1 and HER2, HER1-HER2 heterodimers were detected in 25 to 50% of tumors, depending on detection method. The levels of activated phospho-HER1-HER2 heterodimers correlated with HER1 or HER2 levels in an analysis of 43 HER2-positive breast tumors.ConclusionsVeraTag™ lysate assays can be used as a tool for understanding the mechanism of action of targeted HER-family inhibitors in the preclinical setting, while VeraTag™ FFPE assays of activated HER receptors combined with total HER2 measurements (HERmark®) in tumor samples may provide a more accurate prediction of clinical response to both HER1 and HER2 targeted therapies.

Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH.

The selection of patients with HER2-positive breast cancer for treatment with trastuzumab is based on the measurement of HER2 protein expression by immunohistochemistry, or the presence of HER2 gene amplification by fluorescence in situ hybridization (FISH). By using multivariate analyses, we investigate the relationship between quantitative measurements of HER2 expression or HER2:HER2 dimers and objective response (Response Evaluation Criteria in Solid Tumors), time to progression, and breast cancer survival after trastuzumab treatment in a cohort of patients with metastatic breast cancer who were primarily selected for treatment by FISH. The VeraTag assay, a proximity-based assay designed to quantitate protein expression and dimerization in formalin-fixed, paraffin-embedded tissue specimens, was used to measure HER2 protein expression and HER2:HER2 dimer levels. In a Cox proportional hazards analysis, higher HER2 expression or HER2:HER2 dimer levels were both correlated with longer survival (P=0.0058 and P=0.016, respectively) after treatment with trastuzumab in a population of patients that were either FISH-positive (90%) or immunohistochemistry 3+ (10%). Patients with higher levels of HER2 expression or HER2:HER2 dimers seemed to derive little benefit from the addition of concomitant chemotherapy to trastuzumab, whereas those with lower levels benefited significantly [interaction test P=0.43 (HER2 expression), P=0.27 (HER2:HER2 dimers)]. These data suggest that more quantitative or functional measurements of HER2 status may facilitate the development of more personalized treatment strategies for patients with metastatic breast cancer.

High HER2 Expression Correlates with Response to the Combination of Lapatinib and Trastuzumab

Purpose: Expression of p95HER2 has been associated with resistance to trastuzumab-based therapy in patients with metastatic breast cancer. Conversely, high levels of HER2 have been linked with increased clinical benefit from anti-HER2 therapy. In this work, we aimed to investigate whether the levels of p95HER2 and HER2 can predict response to anti-HER2 therapy in patients with breast cancer. Experimental Design: We measured p95HER2 and HER2 by VeraTag and HERmark, respectively, in primary tumors of patients enrolled in the neoadjuvant phase III study NeoALTTO and correlated these variables with pathologic complete response (pCR) and progression-free survival (PFS) following lapatinib (L), trastuzumab (T), or the combination of both agents (L+T). Results: A positive correlation between p95HER2 and HER2 levels was found in the 274 cases (60%) in which quantification of both markers was possible. High levels of these markers were predictive for pCR, especially in the hormone receptor (HR)–positive subset of patients. High HER2 expression was associated with increased pCR rate upon L+T irrespective of the HR status. To examine whether the levels of either p95HER2 or HER2 could predict for PFS in patients treated with lapatinib, trastuzumab or L+T, we fit to the PFS data in Cox models containing log2(p95HER2) or log2(HER2). Both variables correlated with longer PFS. Conclusions: Increasing HER2 protein expression correlated with increased benefit of adding lapatinib to trastuzumab. HER2 expression is a stronger predictor of pCR and PFS than p95HER2 for response to lapatinib, trastuzumab and, more significantly, L+T. Clin Cancer Res; 21(3); 569–76. ©2014 AACR.

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.

Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab

BACKGROUND Patients with metastatic breast cancer (MBC) overexpressing HER2 (human epidermal growth factor receptor 2) are currently selected for treatment with trastuzumab, but not all patients respond. PATIENTS AND METHODS Using a novel assay, HER2 protein expression (H2T) was measured in formalin-fixed, paraffin-embedded primary breast tumors from 98 women treated with trastuzumab-based therapy for MBC. Using subpopulation treatment effect pattern plots, the population was divided into H2T low (H2T < 13.8), H2T high (H2T ≥ 68.5), and H2T intermediate (13.8 ≤ H2T < 68.5) subgroups. Kaplan-Meier (KM) analyses were carried out comparing the groups for time to progression (TTP) and overall survival (OS). Cox multivariate analyses were carried out to identify correlates of clinical outcome. Bootstrapping analyses were carried out to test the robustness of the results. RESULTS TTP improved with increasing H2T until, at the highest levels of H2T, an abrupt decrease in the TTP was observed. KM analyses demonstrated that patients with H2T low tumors [median TTP 4.2 months, hazard ratio (HR) = 3.7, P < 0.0001] or H2T high tumors (median TTP 4.6 months, HR = 2.7, P = 0.008) had significantly shorter TTP than patients whose tumors were H2T intermediate (median TTP 12 months). OS analyses yielded similar results. CONCLUSIONS MBC patients with very high levels of H2T may represent a subgroup with de novo resistance to trastuzumab. These results are preliminary and require confirmation in larger controlled clinical cohorts.

Quantitative Measurements of Tumoral p95HER2 Protein Expression in Metastatic Breast Cancer Patients Treated with Trastuzumab: Independent Validation of the p95HER2 Clinical Cutoff

Purpose: P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay (Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients. Experimental Design: In the current study, the predictive value of the p95 VeraTag assay cutoff established in the training set was retrospectively validated for PFS and OS in an independent series of 240 trastuzumab-treated MBC patients from multiple institutions. Results: In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with shorter PFS (HR = 1.43; P = 0.039) and shorter OS (HR = 1.94; P = 0.0055) where both outcomes were stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS (HR = 2.41; P = 0.0003) and OS (HR = 2.57; P = 0.0025) in the hormone receptor-positive subgroup of patients (N = 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95 VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes. Conclusions: The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients. Clin Cancer Res; 20(10); 2805–13. ©2014 AACR.

Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer

BackgroundWe have recently described the correlation between quantitative measures of HER2 expression or HER2 homodimers by the HERmark assay and objective response (RR), time-to progression (TTP), and overall survival (OS) in an expanded access cohort of trastuzumab-treated HER2-positive patients with metastatic breast cancer (MBC) who were stringently selected by fluorescence in situ hybridization (FISH). Multivariate analyses suggested a continuum of HER2 expression that correlated with outcome following trastuzumab. Here we investigate the relationship between HER2 expression or HER2 homodimers and OS in a clinic-based population of patients with MBC selected primarily by IHC.MethodsHERmark, a proximity-based assay designed to detect and quantitate protein expression and dimerization in formalin-fixed paraffin-embedded (FFPE) tissues, was used to measure HER2 expression and HER2 homodimers in FFPE samples from patients with MBC. Assay results were correlated with OS using univariate Kaplan-Meier, hazard function plots, and multivariate Cox regression analyses.ResultsInitial analyses revealed a parabolic relationship between continuous measures of HER2 expression and risk of death, suggesting that the assumption of linearity for the HER2 expression measurements may be inappropriate in subsequent multivariate analyses. Cox regression analyses using the categorized variable of HER2 expression level demonstrated that higher HER2 levels predicted better survival outcomes following trastuzumab treatment in the high HER2-expressing group.ConclusionsThese data suggest that the quantitative amount of HER2 expression measured by Hermark may be a new useful marker to identify a more relevant target population for trastuzumab treatment in patients with MBC.

HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant

BACKGROUND Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa. MATERIALS AND METHODS With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000 and 2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression. RESULTS We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8RF/mm2 was observed in 53% (n=54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels (p<0.001). CONCLUSIONS These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa.