Jon Burrows

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue.

BackgroundAnalysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies.MethodsA mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel.ResultsThe assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg.ConclusionsThis report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.

Absolute Quantitation of Met Using Mass Spectrometry for Clinical Application: Assay Precision, Stability, and Correlation with MET Gene Amplification in FFPE Tumor Tissue

Background Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69–1) and 100% specific (95% CI 0.92–1) for MET amplification. Conclusions The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

High HER2 protein levels correlate with increased survival in breast cancer patients treated with anti‐HER2 therapy

Introduction: Current methods to determine HER2 (human epidermal growth factor receptor 2) status are affected by reproducibility issues and do not reliably predict benefit from anti‐HER2 therapy. Quantitative measurement of HER2 may more accurately identify breast cancer (BC) patients who will respond to anti‐HER2 treatments.

Quantitative proteomic analysis of HER2 expression in the selection of gastric cancer patients for trastuzumab treatment

Abstract Background A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies. Patients and methods GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics. Results Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy. Conclusions Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.BACKGROUND A wide range of response rates have been reported in HER2-positive gastric cancer (GC) patients treated with trastuzumab. Other HER2-targeted therapies for GC have yet to show efficacy in clinical trials. These findings raise question about the ability of standard HER2 diagnostics to accurately distinguish between GC patients who would and would not benefit from anti-HER2 therapies. PATIENTS AND METHODS GC patients (n = 237), including a subset from the Trastuzumab in GC (ToGA) trial were divided into three groups based on HER2 status and history of treatment with standard chemotherapy or chemotherapy plus trastuzumab. We applied mass spectrometry-based proteomic analysis to quantify HER2 protein expression in formalin-fixed tumor samples. Using HER2 expression as a continuous variable, we defined a predictive protein level cutoff to identify which patients would benefit from trastuzumab. We compared quantitated protein level with clinical outcome and HER2 status as determined by conventional HER2 diagnostics. RESULTS Quantitative proteomics detected a 115-fold range of HER2 protein expression among patients diagnosed as HER2 positive by standard methods. A protein level of 1825 amol/µg was predicted to determine benefit from the addition of trastuzumab to chemotherapy. Trastuzumab treated patients with HER2 protein levels above this cutoff had twice the median overall survival (OS) of their counterparts below the cutoff (35.0 versus 17.5 months, P = 0.011). Conversely, trastuzumab-treated patients with HER2 levels below the cutoff had outcomes similar to HER2-positive patients treated with chemotherapy. (Progression-free survival = 7.0 versus 6.5 months: P = 0.504; OS = 17.5 versus 12.6 months: P = 0.520). HER2 levels were not prognostic for response to chemotherapy. CONCLUSIONS Proteomic analysis of HER2 expression demonstrated a quantitative cutoff that improves selection of GC patients for trastuzumab as compared with current diagnostic methods.

Use of formalin-fixed, paraffin-embedded tissue for proteomic biomarker discovery.

Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.

Quantification of Anaplastic Lymphoma Kinase Protein Expression in Non–Small Cell Lung Cancer Tissues from Patients Treated with Crizotinib

BACKGROUND Crizotinib has antitumor activity in ALK (anaplastic lymphoma receptor tyrosine kinase)-rearranged non-small cell lung cancer (NSCLC). The current diagnostic test for ALK rearrangement is breakapart fluorescence in situ hybridization (FISH), but FISH has low throughput and is not always reflective of protein concentrations. The emergence of multiple clinically relevant biomarkers in NSCLC necessitates efficient testing of scarce tissue samples. We developed an anaplastic lymphoma kinase (ALK) protein assay that uses multiplexed selected reaction monitoring (SRM) to quantify absolute amounts of ALK in formalin-fixed paraffin-embedded (FFPE) tumor tissue. METHODS After validation in formalin-fixed cell lines, the SRM assay was used to quantify concentrations of ALK in 18 FFPE NSCLC samples that had been tested for ALK by FISH and immunohistochemistry. Results were correlated with patient response to crizotinib. RESULTS We detected ALK in 11 of 14 NSCLC samples with known ALK rearrangements by FISH. Absolute ALK concentrations correlated with clinical response in 5 of 8 patients treated with crizotinib. The SRM assay did not detect ALK in 3 FISH-positive patients who had not responded to crizotinib. In 1 of these cases, DNA sequencing revealed a point mutation that predicts a nonfunctional ALK fusion protein. The SRM assay did not detect ALK in any tumor tissue with a negative ALK status by FISH or immunohistochemistry. CONCLUSIONS ALK concentrations measured by SRM correlate with crizotinib response in NSCLC patients. The ALK SRM proteomic assay, which may be multiplexed with other clinically relevant proteins, allows for rapid identification of patients potentially eligible for targeted therapies.

Abstract 1207: Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues.

Aberrant over-expression of receptor tyrosine kinases, (e.g. MET, HER, FGFR, and IGFR) as well as other oncogenic mediators (e.g. KRAS, PI3 Kinase and SRC) are known drivers of gastroesophageal adenocarcinoma (GEC), subdividing the disease into distinct molecular subsets. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. To date, clinical quantification of protein in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative at best. Moreover, IHC of multiple proteins of interest is laborious, time consuming, wasteful of scarce tissue, and costly. We present a quantitative mass spectrometric (MS) assay for FFPE GEC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a panel of gastroesophageal cancer (GEC) cell lines and tissues. Using trypsin digestion mapping of recombinant oncoproteins, we identified unique peptide sequences, and built quantitative MS assays which could be multiplexed into a single SRM analysis of 1μg of tumor protein. Assays were preclinically validated on 10 different formalin fixed (FF) cell lines. We then tested the GEC-plex assay on a panel of FFPE GEC cell lines characterized by immunoblot (IB), IHC, and gene copy number by FISH. In addition to RON, we multiplexed SRM quantification of Met, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC. We evaluated 17 GEC lines including AGS wild type, scrambled shRNA (AGS-SC) and RON shRNA knockdown (AGS-KD) to assess ‘post-treatment’ changes in oncogene expression. We then evaluated 100 GEC human cancer tissues with paired peritoneal metastases when available and select paraneoplastic normal tissues using laser microdissection of tumor tissue from a single unstained 10μm thick section. Validation of the GEC-plex SRM assay on GEC cell lines revealed very high concordance when compared to IB and IHC measurement. The AGS-WT/SC cells showed comparable levels of RON (284/323 amol/μg cell protein), while RON was not detected in AGS-KD cells, as expected. Measurement of oncoproteins in GEC cell lines and tissues correlated well with IHC and FISH data. Multiplex oncogene quantification of all cell lines and tissues, along with expression profile changes in the AGS RON KD line compared to AGS-WT/SC will be presented. Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FF cells. The GEC-plex multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue from clinical samples, and is a novel clinically applicable approach for tumor characterization for baseline and post-treatment assessment. Citation Format: Daniel V. Catenacci, Peng Xu, Les Henderson, Wei-Li Liao, Sheeno Thyparambil, Jon Burrows, Todd Hembrough. Development of a quantitative gastroesophageal cancer selected reaction monitoring mass Spectrometric Multiplex Assay for use in FFPE tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2013-1207

Abstract 5537: Quantitative multiplexed SRM analysis of oncogenic receptors in FFPE colorectal carcinoma tissue

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Multiple receptor tyrosine kinases are the target of either approved drugs (Her2, EGFR) or robust clinical development efforts (cMet, IGF1R, Her3, etc). In many cases initial drug response is followed by resistance and tumor progression. There are many mechanisms which have been proposed for this including co-expression of alternate RTKs that continue to stimulate tumor growth in the presence of EGFR/Her2 inhibition. Because of the potential that co-expression of oncogenic receptors mediates resistance in these clinical trials, we have developed a panel of new SRM assays which measure the expression of these critical RTKs in multiplex from FFPE tissues. These assays are based on the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed paraffin embedded (FFPE) tissue. Here we describe the quantitative multiplexed analysis of EGFR, IGF1R and cMet expression in a cohort of 75 archival FFPE colorectal cancer tumors from Cetuximab treated patients. This analysis identified 7 different fingerprints of expression of these three targets. Since each of these targets has either approved inhibitors, or highly active clinical development programs, we are hopeful that this analytical approach can help to identify patients who are most likely to respond to single or combination therapies of RTK inhibitors Follow up studies are underway to expand the RTK- multiplex, repeating EGFR, IGF1R and cMet and adding Her2, Her3, Her4, DR5, cSrc, and other drug targets into the multiplex. Our intention is to use this broader multiplex to re-analyze these patients and attempt to correlate the multiplexed expression of these targets with drug response data. 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 5537. doi:1538-7445.AM2012-5537

Abstract 25: Whole genome sequencing and quantitative proteomics reveal HPV integration and HER2 overexpression in a patient with cervical cancer: Comprehensive omics analysis driving clinical treatment decisions

Introduction: Selection of drugs to treat patients with cancer is typically based on the anatomical site in which the tumor is located. Here we report a treatment decision for a patient with relapsed, advanced cervical cancer that was based on a comprehensive omics analysis using whole genome sequencing (WGS) combined with quantitative proteomics. Methods: The patient was a 44-year-old female whose disease had progressed following surgery and more than 4 lines of chemotherapy. WGS was performed on the patient9s formalin-fixed, paraffin-embedded (FFPE) metastatic tumor sample and a matched-normal reference sample. Quantitative proteomics was performed on the FFPE tumor sample by Selected Reaction Monitoring Mass Spectrometry and was quantitated at the atomolar level. Results: WGS found somatic mutations and rearrangements and reads mapping to human papillomavirus type 18 (HPV 18). Mutations more commonly found in breast cancer (ERBB2, CDH1, and CLTCL1) were noted. The HPV 18 genome was integrated into chromosome 17 in close proximity to a 7-fold amplification of the ERBB2 gene. Proteomic analysis of the FFPE tumor validated and quantitated overexpression of HER2 protein resulting from ERBB2 gene amplification, with 11,322 amol/μg of tissue protein. Clinically observed ranges for breast or gastric cancer are 150-500 amol/μg, with levels above 750 amol/μg correlating with FISH-positive amplification and clinical efficacy of trastuzumab (unpublished observation). Based on these comprehensive omic findings, trastuzumab, a therapy approved for breast and gastric cancer, was administered. The patient experienced a reduction in the size of her tumor (by CT/PET) and stabilization of her disease for 5 months. Conclusion: WGS and proteomic profiling of this patient9s disease identified, confirmed, and quantitated an appropriate target for pharmaceutical intervention. The patient presented with cervical cancer; however, the WGS analysis pointed towards a potentially causative integration of the HPV 18 genome resulting in ERBB2 amplification along with genomic mutations more commonly found in breast cancer. Proteomic analysis further validated and quantitated the HER2 expression resulting from ERBB2 gene amplification, leading to the patient9s treatment with trastuzumab. Our findings argue for the use of comprehensive omics analysis to guide decision support for personalized management of cancer care with therapies determined based on a quantitative proteomic signature, independent of anatomical tumor type. Citation Format: Stephen Benz, J Zackary Sanborn, Nicole S. Hensley, Todd Hembrough, Charles J. Vaske, Jon Burrows, Shahrooz Rabizadeh, Ivor Royston, Patrick Soon-Shiong. Whole genome sequencing and quantitative proteomics reveal HPV integration and HER2 overexpression in a patient with cervical cancer: Comprehensive omics analysis driving clinical treatment decisions. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 25.

Her2 expression in gastroesophageal cancer (GEC) FFPE tissue using mass spectrometry (MS) and correlation with HER2 gene amplification.

82 Background: HER2+ GEC derived benefit from trastuzumab. Her2 IHC is semi-quantitative, subjective, and sensitive to antigen instability; HER2 FISH is laborious, expensive, and subjective. False positivity/negativity have been described. Also, these are low throughput assays; there is known molecular heterogeneity, with several putative biomarkers, and only scarce tissue to assess for each. We sought to evaluate the association of Her2 MS expression with HER2 FISH, along with other markers within the ‘GEC-plex’. Methods: We utilized a previously described unique Her2 peptide and quantification method (Hembrough et al J Clin Oncol 32,2014(suppl 3;abstr17)). The assay was run on 27 cell lines, in parallel with HER2:CEP17 FISH. Her2 expression thresholds were established for HER2 amplification using ROC curves. We adjusted for Her3, Egfr, and Met MS expression levels and sample HER2:CEP17 ratio heterogeneity in a multiple linear regression model. The model/cut-offs were then validated prospectively on GEC ...