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Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results. We developed a targeted proteomic assay based on selected reaction monitoring mass spectrometry (SRM-MS) and quantified levels (amol/μg) of Her2-SRM protein in cell lines (n = 27) and GEC tissues (n = 139). We compared Her2-SRM protein expression with IHC/FISH, seeking to determine optimal SRM protein expression cutoffs in order to identify HER2 gene amplification. After demonstrating assay development, precision, and stability, Her2-SRM protein measurement was observed to be highly concordant with the HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM expression of the covariates Met, Egfr, Her3, and HER2 heterogeneity, as well as their interactions (cell lines r 2 = 0.9842; FFPE r 2 = 0.7643). In GEC tissues, Her2-SRM protein was detected at any level in 71.2 % of cases. ROC curves demonstrated that Her2-SRM protein levels have a high specificity (100 %) at an upper-level cutoff of >750 amol/µg and sensitivity of 75 % at a lower-level cutoff of

Background: Trastuzumab showed survival benefit for Her2-positive gastroesophageal cancers (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, both assays are low throughput with various limitations. Methods: We developed a selected reaction monitoring mass spectrometric (SRM-MS) assay and quantified levels (amol/ug) of Her2-SRM in cell lines (n=27) and GEC tissues (n=139). We compared Her2-SRM expression with IHC/FISH, seeking to determine optimal SRM expression cut-offs to identify HER2 amplification. Results: After demonstrating assay development, precision, and stability, Her2-SRM measurement was observed to be highly concordant with HER2/CEP17 ratio, particularly in a multivariate regression model adjusted for SRM-expression of Met, Egfr, Her3, and HER2-heterogeneity covariates, and their interactions (cell lines r2=0.9842; FFPE r2=0.7643). In GEC tissues, Her2-SRM was detected in 71.2% of cases, and 12.3% were identified as ‘HER2+’. ROC curves demonstrated HER2-SRM levels to have high specificity (100%) at an upper-level cut-off of >750 amol/µg and sensitivity (75%) at lower-level cut-off of

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 (

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Background: Current cancer treatment regimens rely on the use of chemotherapy agents that inhibit DNA replication and repair machinery. Several proteins are involved in this mechanism, such as TOPO1, TOPO2A, RRM1, FR-alpha and hENT1. The expression levels and activities of these proteins can greatly affect the success of chemotherapy; however current treatment indications are not based on tumor expression levels of these proteins. We have developed a quantitative, multiplexed ChemoPlex SRM method to evaluate these markers in a host of solid tumors from a limited amount of FFPE biopsy tissue using our Liquid Tissue®-SRM (LT-SRM) platform. Use of this method will enable a physician to understand individual tumor molecular machinery and ultimately could lead to individualized treatment decisions leading to better patient care. Methods: We used trypsin digestion mapping of recombinant proteins to identify optimal quantitative peptides for the ChemoPlex SRM assay. Standard curves were generated to determine the LOD, LOQ, accuracy, precision and linearity of the assay. The assay was pre-clinically validated on 14 cell lines with known expression levels of these Chemo-targets, and the assay was then run on microdissected archived FFPE human tissue samples from lung, gastro-esophageal cancer (GEC), breast, liver, colorectal, and ovarian tumors. Results: The peptides chosen for the 5 Chemo-Plex targets had LOD values of 150, 50, 300, 200, and 100 amol (CV Discussion: We describe the development and initial clinical validation of a quantitative proteomic ChemoPlex SRM assay which accurately measures the expression of five chemotherapy targets in FFPE tumor tissue. When multiplexed along with other druggable biomarkers, the ChemoPlex SRM assay will allow more accurate identification of patients that are likely to benefit from the combination of chemotherapy and targeted therapies. Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Adele Blackler, Jamar Uzzell, Kathleen Bengali, Marlene Darfler, Jon Burrows, Todd Hembrough. Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2014-918

Two receptors for interleukin 8 (IL-8), IL-8rA and IL-8rB, have been cloned. Previous studies of neutrophils indicated that the two C-X-C chemokines, IL-8 and NAP-2, bind to IL-8rB with high affinity but that only IL-8 binds to IL-8rA with high affinity. In this study, human kidney embryonal 293 cells were transfected to express solely IL-8rA or IL-8rB (the cells are designated IL-8rA/293 and IL-8rB/293, respectively). The authors show that NAP-2 bound both IL-8rA and IL-9rB specifically. While NAP-2 and IL-8 bound IL-8rB with comparable high affinity (2.9 +/- 0.5 and 2.8 +/- 0.8 nM, respectively), NAP-2 showed a lower binding affinity to IL-8rA (9 +/- 2 nM) compared with IL-8 (1.3 +/- 0.5 nM). A lower number of binding sites was detected for NAP-2 than for IL-8 on IL-8rA/293 cells as well on IL-8rB/293 cells. On both cell types (IL-8rA/293 and IL-8rb/293), NAP-2 and IL-8 could completely inhibit [125I]NAP-2 binding, while unlabelled NAP-2 could only partially compete for [125I]IL-8 binding. Functional assays revealed that although NAP-2 is chemotactic for both IL-8rA/293 and IL-8rB/293 cells, it is less potent than IL-8. While NAP-2 induced chemotaxis of IL-8rB/293 cells at the same optimal concentrations as IL-8 (10-100 ng/ml), the induction of optimal migratory response of IL-8rA/293 cells required much higher concentrations of NAP-2 than IL-8 (1000-3000 ng/ml and 10-100 ng/ml, respectively). The dose-response curve of the IL-8rB/293 cells to IL-8 was bell shaped, while the response to NAP-2 was sustained at a plateau level even at concentrations as high as 3000 ng/ml. It is likely that tertiary structural differences between NAP-2 and IL-8 account for their divergent abilities to bind and chemoattract 293 cells transfected with either IL-8 receptor type A or type B.

The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR (HER1) and HER2), and two receptors (HER3 and HER4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and HER2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other members of the EGFR superfamily. It was recently shown that HER2 co-expression mediates resistance in EGFR inhibitor (cetuximab) treated head and neck cancer (Sci Transl Med 7(3)99). Similarly, much attention has been paid to HER3 both as a bona fide drug target as well as a resistance mechanism (Oncogene 27, 3944). Finally, HER4, though less well studied may play a role in drug response (Breast Cancer Research 11:R50). We have previously build HER1 and HER2 specific SRM assays. HER3 is usually expressed at much lower levels than EGFR and HER2 (often 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 4567. doi:1538-7445.AM2012-4567

The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and Her2), and two receptors (Her3 and Her4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and Her2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other members of the EGFR superfamily. It was recently shown that Her2 co-expression mediates resistance in cetuximab treated head and neck cancer (Sci Transl Med 7(3)99). Similarly, much attention has been paid to Her3 both as a bona fide drug target as well as a resistance mechanism. Finally, Her4, though less well studied appears to be another resistance mechanism. Her3 is usually expressed at much lower levels than EGFR and Her2 (often By using trypsin digestion mapping, we identified multiple unique peptide sequences from Her3 which were quantitated by SRM Mass spectrometry. Our assay was preclinically validated using the single most sensitive peptide, quantitating Her3 expression in multiple different cell lines, and human NSCLC primary tumor xenografts. These preclinical studies were then extended by assessing the expression levels of Her3 in two cohorts of clinical tumor tissue which had been treated with Her family antagonists. First, we measured Her3 expression in a set of neoadjuvant gefitinib treated NSCLC tumors. In this cohort, 12/15 tumor showed low but measurable levels of Her3 expression, ranging from 50–100 amol/ug tumor tissue. In a second tissue set, we measured Her3 expression in a cohort of advanced (Stage III-IV) breast cancer tissues which had undergone post resection adjuvant treatment with trastuzumab. These breast cancer samples demonstrated a higher level of expression of Her3, ranging from 50 − 250amol/ug tumor tissue, and 15/18 tumors were Her3 positive. In both studies, the relationship between Her family expression and response to either gefitinib or trastuzumab is currently under study. It is critically important to understand mechanisms of resistance in patients undergoing targeted therapies, and Liquid Tissue-SRM promises to be a platform which can deliver extremely high sensitivity, absolute specificity as well as multiplexing capabilities to assess the four HER family targets in unison. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A152.

The observation that cancer cells can undergo aerobic glycolosis (the Warburg Effect) has been the target of a great deal of research in recent years, specifically directed to development of novel agents which can disrupt this pathway and potentially retard cancer growth. Recent studies, by Lisanti and colleagues, have suggested that the critical site of aerobic glycolysis may not be the tumor epithelial cells, but instead the ‘Warburg Effect’ is occurring in the tumor associated fibroblasts and other stromal cells. In order to better define the importance of ‘tumor’ vs. ‘stroma’ components of the Warburg effect, we have developed a quantitative multiplexed assay which can be performed directly in formalin fixed paraffin embedded (FFPE) patient tissue. This multiplex assay is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which renders dissected tissue to a completely solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). For mass spectrometric analysis, we developed 9 different quantitative assays for each of the Warburg Effect components (LDHA, PKM2, FBA(a), PGKI, LDHA, PMI, EnolaseA, and TPI) as well as Caveolin-1 which was identified as a marker of active aerobic glycolysis in tumor fibroblasts. We then quantitated all of these proteins in multiplex on matched tumor and stromal tissue laser microdissected from 10 different FFPE breast carcinoma tumor samples. This analysis demonstrated broadly divergent levels of glycolytic enzyme expression across the set of tumor samples, and in most cases, much higher enzyme expression in tumor cells as opposed to tumor associated stroma cells. However there were several cases where the stromal level of Warburg protein expression was elevated above matched tumor epithelial cell expression. These results demonstrate that by using the Liquid Tissue® technology, we can separately microdissect tumor and stroma in FFPE tumor sections and characterize the specific tumor/stroma components, be they pharmacodynamic analyses, drug target analysis, or seed vs. soil studies. Second, this technology reinforces the capability of SRM based mass spectrometry studies to function in massively multiplexed fashion, where 20 to 40 or more specific analytes can be quantitated by analysis of 0.5ug of protein lysate from a single FFPE tumor section. Finally, we are in the process of clinically validating this Warburg Effect multiplex assay and will then perform the assay on a larger, completely annotated tumor cohort in order to extend our knowledge of how tumor vs. stroma expression of Warburg effect components impacts tumor progression and therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2011-1514

Many of the current targeted therapies in oncology target the activity of either receptor tyrosine kinases (Her2, EGFR, IGF1R, cMet, FGFR, PGDFR) or cytoplasmic tyrosine kinases (cSrc, AurA, PI3K, MEK, Raf, Akt). The current slate of clinically useful diagnostic tests measure target expression either directly by immunohistochemistry or indirectly by extrapolation from the level of gene or mRNA expression/amplification. These assays are limited by lack of quantitation, and no assay can directly assess the activation state of the signaling pathway components. The lack of information regarding target activation and downstream signal transduction can be overcome using the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of multiple proteins and their phosphorylation status directly in formalin fixed tissue. In order to fill this diagnostic ‘gap’ we have used this platform to develop a quantitative multiplexed phospho-target assay format which measures the specific phosphorylation state of many clinically relevant oncogenic kinases (EGFR, cMet, Her3, Erk, cSrc, Mek, Akt, p70S6K) directly in FFPE tumor tissue. This phosphopeptide multiplex assay was initially preclinically validated on the A431 tumor cell line which harbors an amplification of the EGFR gene. These cells were stimulated with a dose range of EGF (50-200ng/ml) or in a time course study (EGF 50ng/ml for 5-30min). Confluent, EGF stimulated cells were then formalin fixed, subjected to Liquid Tissue® processing, and then phospho-enriched using TiO2 magnetic beads. The resulting enriched phosphopeptides were then analyzed by mass spectrometry. This method demonstrated the feasibility, and reproducibility of this method for quantitating EGFR pY1197, EGFRpT693, AKT pS473, p-p70S6K pS447, ERK pT202/pY204. We extended this study by performing phosphoenrichment and mass spectrometric analysis of human tumor xenografts, primary human tumor NSCLC explants and clinical trial tissue from EGFR inhibitor treated NSCLC and colorectal cancer patients. In each case we were able to enrich and measure the phosphorylation of a large set of important oncogenic kinases. Our intention is to develop this diagnostic tool to provide a multiplex assay format in formalin fixed tissue that can be applied from preclinical to clinical studies that will impact both targeted drug development and patient stratification needs in this era of personalized healthcare. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2011-5129

ENMD-981693 is a novel, orally-active antimitotic and antiangiogenic molecule that was discovered through a screening effort directed towards Aurora kinases. In addition to activity against Aurora A (AurA), ENMD-981693 is a potent inhibitor of several tyrosine kinases which drive the neo-vascularization of growing tumors, namely JAK2, VEGFR2 (KDR), PDGFRα and the FGF receptor family members FGFR1, R2, and R3. Multiple myeloma (MM) is a disease in which AurA and angiogenesis have been shown to play a role in disease progression. In addition, up to 15% of MM patients have a t(4;14) translocation, which leads to ectopic overexpression of the FGFR3, and is associated with a poor clinical outcome. In preclinical models, inhibitors of FGFR3 have exhibited antitumor activity towards t(4;14) bearing MM tumors. In in vitro proliferation assays, ENMD-981693 demonstrated an IC50 of ∼2 μM towards several MM cell lines, including H929, U266 and RPMI-8226. In immunoprecipitation studies, ENMD-981693 inhibits the kinase activity of both FGFR3 (IC50 ∼0.8 μM) and JAK2 (IC50 ∼0.5 μM) in the MM cell lines U266 and H929. These activities are in accord with the inhibitory activity of ENMD-981693 on the purified kinase domains of FGFR3 (IC50=0.5 μM) and JAK2 (IC50=0.12 μM). ENMD-981693 also inhibits activation of JAK2 activity in the HEL cell line which expresses the polycythemia vera mutated form of JAK2 (V617F). Mice implanted with H929 tumor xenografts were treated for 4 days with 75 – 225 mg/kg/d of the tartrate salt of ENMD-981693 (ENMD-2076). The amount of phosphorylated (and thus activated) FGFR3 immunoprecipitated from lysates of H929 tumors showed a significant decrease following drug treatment with no change in total FGFR3. In complementary immunoprecipitation studies, a similar decrease in JAK2 activity was seen in these H929 tumors treated with ENMD-981693 tartrate. Daily, oral treatment of RPMI-8226 xenografts with ENMD-981693 tartrate (225 mg/kg) resulted in a significant (67%) reduction in tumor burden. Follow up studies will assess the activity of ENMD-981693 tartrate on MM tumor xenografts which harbor the t(4;14) translocation, and whose growth is largely driven by FGFR3. Taken together, ENMD-981693 has been identified as a single agent that targets multiple critical mediators of MM growth, and angiogenesis. This suggests that ENMD-981693 has the potential for clinical activity in MM, particularly in patients with the t(4;14) translocation and in polycythemia vera patients with mutated JAK2. An IND filing is planned for ENMD-981693 in 2007.

Background Analysis 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. Methods A 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. Results The 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. Conclusions This 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.