Anne Kuller

A novel proteomics-based clinical diagnostics technology identifies heterogeneity in activated signaling pathways in gastric cancers.

Purpose The aim of this study was to utilize the proteomics-based Collaborative Enzyme Enhanced Reactive (CEER) immunoassay to investigate protein tyrosine phosphorylations as diagnostic markers in gastric cancers (GCs). Experimental Design Protein lysates from fresh-frozen 434 advanced stage GCs were analyzed for phosphorylation of HER1, HER2, p95HER2, HER3, cMET, IGF1R and PI3K. The pathway activation patterns were segregated based on the tumor HER2 status. Hierarchical clustering was utilized to determine pathway coactivations in GCs. Prognostic value of pathway activation patterns was determined by correlating disease-free survival times of the various GC subgroups using Kaplan-Meier survival analysis. CEER was also used to determine the presence of tyrosine phosphorylated signaling cascades in circulating tumor cells (CTCs) and ascites tumor cells (ATCs). Results Utilizing a novel diagnostics immunoassay, CEER, we demonstrate the presence of p95HER2 and concomitantly activated signaling pathways in GC tumor tissues, CTCs and ATCs isolated from GC patients for the first time. p95HER2 is expressed in ∼77% of HER2(+) GCs. Approximately 54% of GCs have an activated HER1, HER2, HER3, cMET or IGF1R and demonstrate a poorer prognosis than those where these receptor tyrosine kinases (RTKs) are not activated. Hierarchical clustering of RTKs reveals co-clustering of phosphorylated HER1:cMET, HER2:HER3 and IGF1R-PI3K. Coactivation of HER1 with cMET renders GCs with a shorter disease-free survival as compared to only cMET activated GCs. Conclusions Our study highlights the utility of a novel companion diagnostics technology, CEER that has strong implications for drug development and therapeutic monitoring. CEER is used to provide an increased understanding of activated signaling pathways in advanced GCs that can significantly improve their clinical management through accurate patient selection for targeted therapeutics.

Activated cMET and IGF1R-Driven PI3K Signaling Predicts Poor Survival in Colorectal Cancers Independent of KRAS Mutational Status

Background Oncogenic mutational analysis provides predictive guidance for therapeutics such as anti-EGFR antibodies, but it is successful only for a subset of colorectal cancer (CRC) patients. Method A comprehensive molecular profiling of 120 CRC patients, including 116 primary, 15 liver metastasis, and 1 peritoneal seeding tissue samples was performed to identify the relationship between v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) WT and mutant CRC tumors and clinical outcomes. This included determination of the protein activation patterns of human epidermal receptor 1 (HER1), HER2, HER3, c-MET, insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositide 3-kinase (PI3K), Src homology 2 domain containing (Shc), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) kinases using multiplexed collaborative enzyme enhanced reactive (CEER) immunoassay. Results KRAS WT and mutated CRCs were not different with respect to the expression of the various signaling molecules. Poor prognosis in terms of early relapse (<2 years) and shorter disease-free survival (DFS) correlated with enhanced activation of PI3K signaling relative to the HER kinase pathway signaling, but not with the KRAS mutational status. KRAS WT CRCs were identified as a mixed prognosis population depending on their level of PI3K signaling. KRAS WT CRCs with high HER1/c-MET index ratio demonstrated a better DFS post-surgery. c-MET and IGF1R activities relative to HER axis activity were considerably higher in early relapse CRCs, suggesting a role for these alternative receptor tyrosine kinases (RTKs) in driving high PI3K signaling. Conclusions The presented data subclassified CRCs based on their activated signaling pathways and identify a role for c-MET and IGF1R-driven PI3K signaling in CRCs, which is superior to KRAS mutational tests alone. The results from this study can be utilized to identify aggressive CRCs, explain failure of currently approved therapeutics in specific CRC subsets, and, most importantly, generate hypotheses for pathway-guided therapeutic strategies that can be tested clinically.

Abstract 4146: MultiOmyx multiplexed tumor infiltrating lymphocyte panel provides comprehensive immunophenotyping from a single FFPE slide

Immune checkpoint therapies target immune regulatory pathways to enhance anti-tumor immune response. These therapies have contributed to important clinical advances, and are a promising approach to combat cancer. Development of effective immune checkpoint therapies requires an understanding of the host immune response within the tumor microenvironment. GE Healthcare, through its affiliate Clarient Diagnostics Service Inc., has developed a multiplexed Tumor Infiltrating Lymphocyte (TIL) Panel* consisting of 12 key cancer immune markers: CD3, CD4, CD8, CD20, CD45RO, CD56, CD68, CTLA4, FOXP3, PD1, PD-L1 and Pan-CK. This MultiOmyx TIL panel identifies individual Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), Tregulatory (CD3+CD4+FoxP3), memory T-cells (CD3+CD4+CD45RO), anergic T-cells (PD1+CD8+), natural killer cells (CD56+), macrophages (CD68+), and B-cells (CD20+) within the tumor and the stromal regions. MultiOmyx multiplexed immunofluorescence technology enables qualitative and quantitative analysis of these 12 biomarkers’ expression and co-localization from a single formalin-fixed, paraffin-embedded (FFPE) slide. MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. Herein, we report an analysis of immune response in the tumor microenvironment within solid tumors including breast cancer, lung cancer, colorectal cancer, esophageal cancer, prostate cancer, and melanoma utilizing the MultiOmyx TIL Panel. The results revealed two distinct immunologic phenotypes, high TIL tumors and Low TIL tumors. The high TIL tumors showed enhanced T cell population within the tumor and in the peritumoral stroma including CD8+ cytotoxic T cells, CD4+ helper T cells and CD45RO+ memory T cells. Increased expression of immune checkpoints markers such as CTLA4 and PD-1 were also observed. PD-1 was predominantly expressed in CD8+ cytotoxic T cells while CTLA4 was mostly found on CD4+ FOXP3+ regulatory T cells. PD-L1 expression was also induced, mainly on Pan-CK+ tumor cells and CD68+ macrophages. High density of PD-L1+ |CD68+ macrophages was localized in the stroma surrounding the tumor region. Conversely, many of the immune markers were not expressed in low-TIL tumors. Immunophenotyping analysis offered by the MultiOmyx TIL panel may facilitate the identification of appropriate immunotherapeutic approach. Tumor with high TIL profiling may be effectively treated with single-agent immune checkpoint therapy, while tumor with low TIL profiling may require combination therapy with an agent that enhances endogenous antitumor response. *MultiOmyx TIL panel is currently research use only tool. Citation Format: Qingyan Au, Kathy Nguyen, Raghav Padmanabhan, Anne Kuller, Eddie Moler, Nicholas Hoe. MultiOmyx multiplexed tumor infiltrating lymphocyte panel provides comprehensive immunophenotyping from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4146.

Abstract 3678: Combination analysis of oncogenic mutations and signaling pathway proteins identifies effective therapeutic targets in colorectal cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite overall improvements in colorectal cancer (CRC) therapies, an understanding of reasons for therapeutic response/non-response as well as disease recurrence remains poor. Currently, the gold standard for CRC prognosis remains the clinicopathological staging of tumors. Addition of molecular markers for predicting prognosis and therapeutic outcomes can significantly enhance the clinical managements of CRCs. A comprehensive molecular characterization of 116 stages I to IV Asian CRC patients that also included 2 paired normal samples and 19 hepatic metastases of which 10 were from paired primary tumor samples (total 136 specimens) was performed. Somatic mutational analysis scored for 12 different mutations resident within the KRAS, BRAF and PIK3CA oncogenes alongside a parallel determination of kinase activation profiles for HER1, HER2, HER3, c-MET, IGF1R, PI3K, Shc, AKT and ERK using the multiplexed Collaborative Enzyme Enhanced Reactive (CEER™) immunoassay. CEER is a highly sensitive and specific proximity assay that relies on the formation of a triple antibody complex surrounding the target protein. Various mutations of KRAS and BRAF were found in 41% (48/116) and 4% (5/116) of patients, respectively. Incidence of PIK3CA mutations were 11% (13/116) in this cohort. Overall, KRAS G12D and G13D mutations were the most frequent in this cohort. Several primary as well metastatic CRCs with KRAS mutations demonstrated a second mutation in the PIK3CA gene. While both ErbB-driven and non-ErbB-driven pathway signatures were observed in this cohort, there was no apparent correlation found between the KRAS mutational status of the tumors and the activation status of the downstream/upstream pathway signals. Molecular characterization of CRC samples for oncogenic mutations should be complemented with functional pathway profiling to pinpoint potential therapeutic target(s). The tumors dependence on specific pathway cannot be predicted based on mutations alone hence it is critical to also evaluate pathway activation for selecting more effective clinical strategy. Our study also demonstrated modulation in signaling pathways concomitant with disease recurrence. Such clinical validations and comprehensive molecular profiles of CRC tumors can allow for the implementation of these companion biomarkers in CRC therapeutic clinical trials. 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 3678. doi:1538-7445.AM2012-3678

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.