Limin Liu

Prevalence of Activated & Total p95HER2 and Other Receptor Tyrosine Kinases in Breast Cancer.

Background: HER2-overexpressing breast cancer has poor prognosis and is often resistant to HER2 targeted monoclonal antibody therapy. One of the mechanisms of de novo or acquired resistance is expression of p95HER2, various forms of truncated HER2 receptors with missing amino-terminal extra cellular domains. Methods for profiling various forms of HER2 receptors and other receptor tyrosine kinases (RTKs) with transactivation potential in primary and metastatic tumors may provide valuable insight into the shifting disease pathogenesis.Methods: A novel technology capable of specifically detecting phosphorylation events in ErbB family RTKs has been developed. This multiplexed protein microarray platform utilizes the formation of a unique immuno-complex requiring the co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes (glucose oxidase & horse radish peroxidase) in proximity enabled the profiling of the RTK with extreme sensitivity. The analytical specificity is greatly enhanced given the requirement for simultaneous binding of three different antibodies. Different configuration of detector antibodies allowed differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). Here we report successful profiling of signal transduction pathway molecules for their expression and activation using 240 FNA samples collected from breast cancer patients (stage II to IV) with various ER/PR/HER2 status.Results: The FNA samples collected using G23 gauge needles were analyzed for expression and activation status for various RTKs and downstream signal transduction molecules including p95HER2, HER2, HER1, HER3, IGF-1R, PI3K and Shc. We found:O 100% concordance between primary HER2-IHC status and HER2 expression analysis based on proximity mediated immuno-microarray system O presence of varying degree of p95HER2 in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients O ∼50% of p95HER2 expressors had activated p95HER2 O 25% of HER2-positive samples also had some levels of HER1 and HER3 activation O HER2-negative (HER2: 2+ with FISH-, 1+ and 0) samples also had 100% concordant HER2 expression, but number of them showed low but significant levels of HER2, HER3 and HER 1 activation.These findings may have implication on selection of appropriate targeted therapeutics.Discussion: The multiplexed-immuno microarray was utilized to detect the expression and phosphorylation of p95HER2, HER2, other RTKs (including HER3, HER1, IGF-1R, Shc, and PI3K) in 240 FNA samples collected from unique breast cancer patients with various ER/PR/HER2 status. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options as disease-profile changes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3053.

Abstract 1217: Detection of HER2 expression and phosphorylation in breast cancer CTC samples by CEER™

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Survival rates of metastatic breast cancers (mBCAs) are considerably low. Often, tumor cells at the primary site may not reflect the profile of the tumor cell population in recurrent disease. Circulating tumor cells (CTCs) isolated from peripheral blood offer a non-invasive disease monitoring modality. Identification of reliable molecular markers within (CTCs) from patients with recurrent disease may further improve breast cancer survival. Hence, we have developed a multiplexed immunoassay for monitoring activated HER2 protein and their expression levels using a multiplexed immuno-microarray platform. Collaborative Enzyme Enhanced Reactive-immunoassay (CEER™) was utilized to analyze HER2 profiles in CTCs isolated from 76 BCA patients (stages III to IV) with HER2 negative primary disease. The CEER technology utilizes the formation of a unique immuno-complex requiring co-localization of two detector antibodies around capture antibodies immobilized on immuno-array to profile HER2 protein expression and activation. The collaboration between two channeling-enzymes conjugated on two detection antibodies in proximity, enables the profiling of the target proteins with extreme sensitivity and specificity. Approximately 25% of HER2 negative BCA patients in this cohort showed varying levels of HER2 activation in CTCs isolated from the recurrent disease. About 8% of patients with HER2 activation in CTCs also showed significant HER2 over-expression. This suggests mechanisms of HER2 activation either through formation of receptor heterodimer (in samples without HER2 over-expression) or homodimer (in samples with HER2 overexpression) formation. The distinct HER2 discordance between primary tumor and recurrent disease demonstrates an urgent need for routine monitoring of HER2 status in CTCs found in mBCA patients. Incidence of HER2 alterations in CTCs should be considered in selecting effective treatment regimens for BCA patients with relapsed disease. Furthermore, CEER can be used for profiling other druggable targets for guiding effective clinical strategies including rational targeted agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1217. doi:1538-7445.AM2012-1217

Abstract 4552: Utilization of pathway activation and somatic mutation analysis in combination in fine needle aspirates to identify candidate drugs for effective treatment of breast cancer

A critical hurdle in implementing prognostic and predictive biomarkers in clinical trials is our inability to perform an efficient molecular characterization of the limited amounts of available clinical samples. Here we report a comprehensive molecular analysis of fine needle aspirate (FNA) samples collected from metastatic sites of 58 breast cancer (BCA) patients. The analysis included interrogation of key receptor tyrosine kinases and their downstream signaling molecules including HER1, HER2, p95HER2, HER3, cMET, IGF1R, PI3K, Shc, AKT and ERK as well as oncogenic somatic mutational profiling. A multiplexed immunoarray CEER™ (Collaborative Enzyme Enhanced Reactive-immunoassay) platform was utilized to determine the levels of pathway protein expression and phosphorylation. All FNA collections provided sufficient materials for the combined analysis of multiplexed pathway expression/activation and somatic mutation analysis. All the FNA from metastatic sites (mFNA) collected from HER2 positive primary tumors (determined by IHC) were also CEER-HER2 positive. Over-expression of HER2 was found in 10% of mFNAs collected from BCA patients with HER2 negative primary tumors. PIK3CA mutations were found in 20% of the samples in this cohort. Statistically significant higher levels of phosphorylated AKT, ERK (downstream effector targets) as well as HER1, HER2, HER3 and IGF1R (PI3K membrane recruiter proteins) were found in mFNAs with PIK3CA mutations. Subset of PIK3CA wildtype patients also showed robust pathway signature. Hence, evaluation of biomarkers in BCA patients should include both pathway proteins as well as mutation analysis. A comprehensive disease profiling can provide insightful information on efficacy of specific agents on its intended target protein inhibition. As CEER requires minimal amount of specimen, performance of routine pathway profiling can be achieved in clinical settings. A multiplexed pathway analysis can also provide valuable information about potential drug resistance mechanisms. Furthermore, a combined mutational and pathway activation profiling can guide therapeutic strategies in clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4552. doi:1538-7445.AM2012-4552

Abstract 3179: Functional profiling of multiple signal pathway proteins in breast cancer patients

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., full length-HER2). We applied COPIA to investigate the levels of expression and activation of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other targets in signal transduction pathways. Here, we report the functional pathway signatures for multiple proteins in 250 frozen tissues obtained from BCA patients with various primary histology and from 50 fine needle aspirate (FNA) samples collected from metastatic sites (mFNA) in advanced BCA patients with various ER/PR/HER2 status. There was a high concordance between primary HER2-IHC status and COPIA-HER2 expression analysis. Significant levels of p95HER2 were observed in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients, and low but detectable levels in some sample tissues with IHC-HER2 negative (2+ with FISH - / 1+ /0) were also observed. Over 50% of p95HER2-expressors had activated p95HER2, and over 25% of HER2-positive samples also had HER1, HER3, IGF1-R and other RTKs and transduction protein expression and/or activation. As the disease profile often shifts in recurrent breast cancer, our unique assay format can be utilized to provide valuable clinical information on limited samples obtained from evolving disease to help oncologists adjust their disease treatment options for each patient according to their ‘personal’ cancer profile-shift. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options accordingly. 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 3179.

Abstract 4019: Functional profiling of signal transduction pathway proteins in gastric cancer patients

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Although gastrectomy is the only curative treatment in gastric cancer (GCA) patients, a high recurrence rate ranging from 40 ∼ 60% following curative surgery still accounts for poor overall survival. In order to further improve survival, there is an urgent need to identify reliable molecular prognostic markers for survival or recurrence following adjuvant chemoradiation therapy, which will evolve to the development of patient-tailored treatment strategies. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting molecules critical for cancer proliferation. As the transduction pathway proteins function in cell signaling and transmit signals regulating growth, differentiation, adhesion, migration, and apoptosis, they have become targets of various therapeutic agents. Hence, we have developed a multiplexed immunoassay platform for functional profiling of the transduction pathway proteins. The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). We applied COPIA to investigate the levels of expression and activation of signaling proteins in frozen tissues collected from GCA patients. Here we report the prevalence of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other signal transduction pathway protein expression and their levels of activation in GCA patients. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting specific markers identified by functional profiling. As the disease profile often shifts in recurrent cancers and under different therapeutic pressure, clinical information obtained by analyzing samples (often with limited availability) obtained from ‘evolving / heterogeneous disease’ can help clinicians adjust their disease treatment options for each patient according to ‘personal’ cancer profile-shift. 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 4019.

Therapeutic Implications of Detection of Amplification and Activation of HER2 and Other Receptor Tyrosine Kinases (RTKs) in Circulating Tumor Cells (CTCs) in Recurrent Breast Cancer.

Background: HER2 is one of four trans-membrane RTKs in epidermal growth factor receptor family, and HER2-positive phenotype has been associated with aggressive subtype of breast cancer with HER2 gene amplification. Approximately 15-20% of breast cancers are considered HER2-positive by IHC or FISH analysis. Recently, changes in HER2 expression status between primary tumor and CTCs found in recurrent metastatic disease have been reported to occur at a significant frequency. Methods for detecting HER2 expression and phosphorylation in serially collected CTCs may provide valuable insight into the overall disease profile shift, and therefore lead to better selection of therapy for each patient. Methods: A triple-antibody-enzyme-channeling multiplexed protein microarray platform has been developed to detect the phosphorylation on target molecules. Extremely high assay specificity was achieved by immuno-complex formation via co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes in proximity enabled profiling of the RTKs with a single-cell level sensitivity. In order to validate the method on clinical samples, CTCs from 77 breast cancer patients on different therapy regimens were analyzed at various time points along their course of therapy. Results: Whole blood of 77 metastatic cancer patients and 60 healthy volunteers were analyzed for CTC-HER2 expression and activation. We observed significant HER2 status conversion with recurrent disease. 29% of patients with negative HER2 expression in the primary tumor showed HER2-amplification in isolated CTCs. Phosphorylated HER2 receptors were found in 52% of patients with primary HER2 negative disease. The enhancement of assay sensitivity and specificity using proximity mediated immuno-assay made the detection of HER2 activation (even without amplification) possible when isolated CTCs were stimulated with ligands to other RTKs with transactivation potential. Discussion: The multiplexed-proximity mediated immunoassay successfully detected the expression of HER2 RTKs and their degree of activation in CTCs isolated from recurrent breast cancer patients. As we hypothesize that CTCs found in metastatic stage represent the most aggressive and invasive cell population, serial CTC-profiling can lead to better therapy selection/adjustment and disease/treatment monitoring as there are available options to choose appropriate kinase inhibitors for RTK-targeted therapies. While significant number of patients acquired HER2-amplification in their CTCs, substantially higher rate of CTC-HER2 activation was found in relapsed metastatic disease. The unique triple-antibody mediated immuno-microarray analysis allowed a single cell level profiling of the CTC-HER2. The ability to profile serially collected CTCs will provide valuable information on changes occurring in tumor cells as a function of time and treatment. This method can provide guidance, not only for initial selection of targeted therapeutics, but also in subsequent monitoring for rapidly 9evolving9 disease in individual patient. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3010.