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Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

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Most patients with KRASG12C–mutant non–small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRASG12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRASG12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRASG12C colorectal cancer.Significance:The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079

Supplementary figures and tables

Triple-negative breast cancer (TNBC) represents the most lethal subtype of breast cancer due to its aggressive clinical features and the lack of effective therapeutic targets. To identify novel approaches for targeting TNBC, we examined the role of protein phosphatases in TNBC progression and chemoresistance. Protein phosphatase 1 regulatory subunit 14B (PPP1R14B), a poorly defined member of the protein phosphatase 1 regulatory subunits, was aberrantly upregulated in TNBC tissues and predicted poor prognosis. PPP1R14B was degraded mainly through the ubiquitin–proteasome pathway. RPS27A recruited deubiquitinase USP9X to deubiquitinate and stabilize PPP1R14B, resulting in overexpression of PPP1R14B in TNBC tissues. Gain- and loss-of-function assays demonstrated that PPP1R14B promoted TNBC cell proliferation, colony formation, migration, invasion, and resistance to paclitaxel in vitro. PPP1R14B also induced xenograft tumor growth, lung metastasis, and paclitaxel resistance in vivo. Mechanistic investigations revealed that PPP1R14B maintained phosphorylation and stability of oncoprotein stathmin 1 (STMN1), a microtubule-destabilizing phosphoprotein critically involved in cancer progression and paclitaxel resistance, which was dependent on PP1 catalytic subunits α and γ. Importantly, the tumor-suppressive effects of PPP1R14B deficiency could be partially rescued by ectopic expression of wild-type but not phosphorylation-deficient STMN1. Moreover, PPP1R14B decreased STMN1-mediated α-tubulin acetylation, microtubule stability, and promoted cell-cycle progression, leading to resistance of TNBC cells to paclitaxel. Collectively, these findings uncover a functional and mechanistic role of PPP1R14B in TNBC progression and paclitaxel resistance, indicating PPP1R14B is a potential therapeutic target for TNBC.Significance:PPP1R14B upregulation induced by RPS27A/USP9X in TNBC increases STMN1 activity, leading to cancer progression and paclitaxel resistance.

Introduction: Prostate cancer therapy involves the use of androgen deprivation therapy, chemotherapy, targeted therapy and immunotherapy. Chemotherapy involves the use of anti-tubulins (docetaxel, cabazitaxel), platinum salts, and topoisomerase inhibitors (TOPO1, TOPO2A). There is no biomarker of chemotherapy that is routinely used. We examined 87 prostate cancer samples using targeted proteomics for biomarkers of response or resistance to chemotherapy agents. Biomarkers of resistance include ERCC1 (Platinum), TUBB3 (taxanes), ALDH1A1 (cyclophosphamide), while response biomarkers include TOPO1 (irinotecan, topotecan), TOPO2A (doxorubicin, epirubicin), and hENT1 (Gemcitabine). We also measured markers for several antibody-drug conjugates targets (Her2, Her3, Trop2) in our clinical proteomics (CLIA) platform and several ADC/CAR-T proteins (PSMA, STEAP1, Nectin4, Claudin 18.2. in our research platform Methods: Tumor areas from Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from clinical samples of prostate cancer received at our CLIA certified laboratory were microdissected and a quantitative proteomic analysis of 72 biomarkers were conducted using selected reaction monitoring mass spectrometry (SRM-MS). Discussion:[WL1] Androgen receptor was detected in majority of the samples (83%) with a 19x range of distribution (329 amol/ug - 24063 amol/µg). Majority of prostate cancer samples expressed a range of resistance markers for anti-tubulin inhibitors (TUBB3: 69% detection with a 14x range) indicating that the high expressors is likely to be resistant to docetaxel/cabazitaxel based regimens. ERCC1, a marker for resistance to platinum-based agents, was not detected in 26% of cases, potentially enabling a cisplatin/carboplatin-based regimen in 1/4th of prostate cancer patients. Cyclophosphamide could be effective in 10% of cases where ALDH1A1 was not detected. TOPO1, a marker for irinotecan-based therapy was observed in 94% of cases with a range of ~6x (488 - 2760 amol/µg). Other chemotherapy agents that are not routinely used in prostate cancer was observed in select groups. These include doxorubicin biomarker TOPO2A in 34% of cases with 10x range, gemcitabine biomarker hENT1 in 36% of cases (4x range), temozolomide biomarker MGMT (ND in 12% of cases). ADC biomarker analysis revealed HER2 which was observed in 59% of the cases with range of expression from 301 - 1255 amol/µg including a significant population of low HER2 (84%, Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Amanda Strasbaugh, Marya Melkie, Negin Ghafourian, Xuefeng B. Ling. Quantitative proteomics of antibody-drug conjugates and chemotherapy targets in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2160.

Introduction: Trop2 is overexpressed in many cancers and currently Trop2 ADC is approved in TNBC. In ADC drug design, it is imperative to assess not only the levels of the receptor but also the payload biomarkers. We have developed a multiplex mass spectrometry method to quantitate 72 actionable proteins from FFPE samples that requires minimal tissue (2-3 sections). This panel includes chemotherapy, targeted therapy and immunotherapy agents. The test is run in our CAP, CLIA, and NYSDOH approved laboratory. For this study, we examined a subset of samples run in the clinical lab for the levels of Trop2 (target biomarker) and payload biomarkers (Topo1, TUBB3). Topo1 is a chemosensitive marker for irinotecan, while TUBB3 is a chemoresistance marker for tubulin inhibitors. Methods: FFPE tissue sections from 1140 clinical samples from a variety of cancers were examined. These include breast (n=318), colorectal (n=228), ovarian (n=199), GBM (n=69), NSCLC (n=169), HNSCC (n=91), and Gastric cancer (n=66). Two sections (10 µ) of FFPE tissue were cut on DIRECTOR slides and only the tumor areas were laser microdissected for downstream analysis which resulted in tryptic peptides. 1µg of peptides (~4000 cells) along with heavy peptides was injected into a triple quad mass spec and 72 biomarkers were quantitated concurrently. Results: Trop2 showed a wide range of expression in various cancers. Almost all (95%)breast cancer samples expressed Trop2 which exhibited a wide range (93x; 157 - 14650 amol/µg). Topo1 and TUBB3 were expressed in 93% and 60% of samples respectively. 1/4th of NSCLC does not express Trop2 and there is a 106x difference in expression of Trop2 in NSCLC. Topo1 was expressed in almost all samples while TUBB3 was expressed in 80% of NSCLC. Majority of ovarian cancer samples (85%), HNSCC (89%), Gastric cancer (88%) samples expressed Trop2 with a 113x, 134x, 47x difference in expression. Chemosensitive biomarker Topo1 was expressed in almost all ovarian (96%), HNSCC (91%) and Gastric (99%) cancer samples. Chemoresistant marker TUBB3 was expressed in 66% of ovarian cancer, 44% of HNSCC and 45% of gastric cancer samples. In contrast to above cancers, only 10% of Glioblastoma samples express Trop2 and only 3/4th of GBMs express Topo1. Discussion: In a randomly selected group of cancers, we have found Trop2 is expressed in majority of Breast, Ovarian, Lung, HNSCC and gastric cancers and minimal expression in GBM. Given the range of expression of anti-tubulin resistance marker in many solid tumors, a payload biomarker guided clinical trial is highly recommended in ADCs that employ anti-tubulin inhibitors. In contrast, wide expression of chemosensitive biomarker for Topo1 payload makes it a promising candidate for many solid tumors. Further studies are warranted to determine the level of target and payload biomarkers that will be required for a clinical response. Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Guolin Zhang, Amanda Strasbaugh, Marya Melkie, Xuefeng B. Ling. Clinical survey of Trop2 antibody drug conjugate target and payload biomarkers in multiple cancer indications using multiplex mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4099.

About 15% of breast cancers are HER2 over-expressing (HER2 IHC 3+ or IHC 2+/ISH+)), but another 45% have low levels of HER2 (HER2 IHC 2+/ISH- or IHC 1+), and these patients are not currently approved for treatment with trastuzumab. Recently, a new HER2 ADC, DS-8201 showed anti-tumor activity, not only in patients with HER2 over-expressing breast cancer but also in HER2 low expressing tumors in whom to date, there are no effective anti-HER2 therapies indicated. FDA-approved HER2 in vitro diagnostic tests have recognized several limitations including effects of pre-analytical variable (fixation affects antibody sensitivity), limited dynamic range of chromogen-based IHC, and subjectivity in interpretation of the HER2 score. Additionally, the cut-off values (percentage of cells to be positive) defining HER2 positive have been changing over time. Therefore, more accurate, sensitive, precise and objective assays to better identify patients who may benefit from anti-Her2 treatment therapies (e.g DS-8201) are needed. To address this gap, we evaluated upcoming technologies targeted MS and QRT-PCR and aim to compare expression with current diagnostic HER2 tests in FFPE samples Using selected reaction monitoring mass spectrometry (SRM-MS), we quantified proteins from formalin-fixed, paraffin-embedded tissue samples that had been classified as HER2 0, 1+, 2+ or 3+ by IHC (n=107). HER2 protein concentration measured by SRM-MS was compared between patients in different HER2 IHC classifications using an ANOVA, adjusting for multiple comparisons. HER2 concentration (measured by SRM-MS) was progressively increased according to HER2 IHC grouping (i.e. lowest concentration in HER2 0 samples, highest in HER2 3+ samples). HER2 levels were significantly elevated in 2+ vs. 0 (2.2-fold increase, p < 0.05) samples, and trended higher in 2+ vs. 1+ (1.6-fold increase, p = 0.07) and in 1+ vs. 0 (1.4-fold increase, p = 0.17) samples. About 73% of samples scored as IHC0 had detectable Her2 by SRM-MS (from 168 to 623 amol/µg). Among HER2 IHC 0 samples, ~15% (7/47) had HER2 concentrations above the median levels for the 1+ group. Similarly, 19% (3/16) 1+ samples had HER2 levels above the median for the 2+ group. About 20% of samples co-expressed either ERBB1 and/or ERBB3. Simultaneously from FFPE sections we quantified protein level of payload response and resistance markers (MDR, MRP1, Topo1 and SLFN11). We used an objective multiplex non-antibody-based method to quantify multiple targets from FFPE tissue. SRM-MS revealed a range of HER2 expression over 100 orders of magnitude and identify markers of payload response or resistance in the same assay. The differences seen in payload markers expression could affect therapeutic efficacy and may suggest differing responses to Her2-targeted ADC, depending on tumor biology. Multiplexed quantitative MS could be used to accurately predict which patients will derive the most benefit from Her2-ADC therapy based on the specific biology of their tumor. These studies are ongoing. Citation Format: Fabiola Cecchi, Mark Gustavson, Danielle Carroll, Sriram Sridhar, Steven Coats, Anuja Bhalkikar, Sheeno Thyparambil, Wei-Li Liao, Todd Hembrough. Quantitative mass spectrometry of HER2 protein levels reveals high variability within HER2 IHC grades [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5128.

Chemotherapy is the mainstay for the treatment of ovarian cancer. Taxanes, platinum salts, 5-FU, anthracyclines, gemcitabine are used extensively in ovarian cancer, however, there is no biomarker of chemotherapy that is routinely used. We examined 169 ovarian cancer samples using targeted proteomics for biomarkers of response or resistance to chemotherapy agents. Biomarkers of resistance include ERCC1 (Platinum), TUBB3 (taxanes), ALDH1A1 (cyclophosphamide) while response biomarkers include TOPO1 (irinotecan, topotecan), TOPO2A (doxorubicin, epirubicin), hENT1 (Gemcitabine).We also measure markers for several antibody-drug conjugates (FR-alpha, Her2, Trop2, gPNMB, MSLN). Methods: Tumor areas from Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from clinical samples of ovarian cancer that were received at our CLIA certified laboratory were microdissected and a selected reaction monitoring mass spectrometry (SRM-MS) quantitative proteomic analysis of 72 biomarkers were conducted. Discussion: The majority of ovarian cancer samples expressed a range of resistance markers for cyclophosphamide (ALDH1A1: 87% positive ranging from 227-10766 amol/µg), platinum agents (70% positive) and taxanes (71%positive). However, they also expressed a range of response biomarkers for chemotherapies that are conventionally used to treat ovarian cancer. These include irinotecan/topotecan (TOPO1: 97% positive ranging from 459 -3299), doxorubicin (TOPO2A: 50% positive ranging from 402-3825 amol/µg), gemcitabine (hENT1/RRM1: 42% positive). Novel chemotherapy agents that could potentially be used include temozolomide (40% of patients did not express MGMT, resistance marker for temozolomide). The vast majority (78%) of ovarian cancer samples did not express any drug efflux pump proteins MRP and MDR1. Examination of potential ADC markers revealed 74% positivity for the antibody target FR-alpha with a 42 fold range of expression (585 -25000 amol/µg) and 71% positivity for the payload resistance marker TUBB3. Similarly, Trop2 (56% positivity) exhibited a wide dynamic range (222-12778 amol/µg). Another ADC target mesothelin was expressed in 66% of the cases with a 35x range of expression (302 - 10,700 amol/µg). While 56% of ovarian cancer expressed Her2 (262 -5011 amol/µg), only 4% expressed high levels of Her2 (>750 amol/µg), making them suitable for current anti-Her2 therapy. Others could potentially benefit from clinical trials targeting low Her2 expression. The ability to multiplex 72 protein biomarkers from 2-3 FFPE sections provides immense actionable information on clinical treatment or for patient stratification for clinical trials. Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Eunkyung An, Anuja Bhalkikar, Thomas Guiel, Robert Heaton. Molecular profiling of ovarian cancer by targeted proteomics to inform personalized therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4156.

Background: Antibody-Drug Conjugates (ADCs) are poised to become an extremely important class of therapeutics in oncology. By conjugating cytotoxic payloads to antibodies that target proteins found primarily on cancer cells, ADCs represent a novel mechanism for directing extremely toxic small molecules specifically to tumor cells. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also screening for the presence of any markers of resistance or response to the payload. Several proteins, such as multi-drug effluxers and tubulin-beta 3, have been implicated in resistance to small molecule cytotoxins and microtubule inhibitor drugs. OncoPlex Diagnostics has built a multiplexed ADC biomarker panel that simultaneously quantifies the levels of the antibody target and putative resistance markers for several known payloads, such as maytansinoids, auristatins and taxanes, as well as response markers for the topoisomerase inhibitor payloads SN-38 and doxorubicin. Methods: Liquid Tissue-Selected Reaction Monitoring (LT-SRM) is a multiplexed, quantitative method that uses mass spectrometry to quantify proteins based on a unique sequence of amino acids, and thus does not have the same limitations as traditional antibody-based, semi-quantitative protein detection methods, such as immunohistochemistry. We developed a LT-SRM assay to quantify protein levels of EGFR, FRalpha, Her2, CD30 and Mesothelin (antibody targets) and MCL1, MDR, MRP1, tubulin-beta3, Topo1 and Topo2a (payload response and resistance markers) simultaneously from FFPE biopsies. Calibration curves for all the proteins in the ADC panel are linear over 5-orders of magnitude, with limits of detection for each analyte between 25 and 400 amol/ug of tissue. Results: Analysis of FFPE tumor tissues show a broad range of expression for the ADC proteins, with some tissues showing no detectable levels of some payload markers. Clinical analysis of FRalpha showed a range of expression from Conclusions: The OncoPlexDx ADC panel can determine of a cutoff for expression levels of the antibody-target protein necessary for ADC response as well as identify markers of payload response or resistance to further understand how these markers affect therapeutic efficacy. This panel can be used to predict which patients will derive the most benefit from ADC therapy based on the specific biology of their tumor. Citation Format: Adele Blackler, Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Fabiola Cecchi, Marlene Darfler, Todd Hembrough, Jon Burrows. Development of a mass spectrometry based antibody-drug conjugate biomarker panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3398. doi:10.1158/1538-7445.AM2015-3398

BackgroundImmune check point proteins play a pivotal role in immune evasion by the tumor. Recent trials involving inhibitors of the immune checkpoint protein pairs, PD-1 and PD-L1 have demonstrated anti-tumor activity. Measuring the levels of immune check point proteins and other members of the immunological synapse will help clinicians personalize therapy. Currently, immunohistochemistry (IHC) is the preferred diagnostic to assess PD-L1 status; however, PD-L1 positivity varies based on the antibody that is used. Additionally, PD-L1 negative patients by IHC have responded to anti-PD-L1 therapy implicating disconnect between PD-L1 diagnostics and response. We have developed and clinically validated a quantitative mass spectrometric technique that not only quantitates PD-L1 in formalin fixed paraffin embedded (FFPE) tissue but can concurrently quantitate other members (B7H3, B7.1, B7.2, OX40L) of the immunological synapse using the same tissue section. MethodRecombinant PD-L1 protein was used to identify optimal quantitative peptides for PD-L1 assay. Standard curves were generated using labeled and unlabeled peptides. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was then run on archived FFPE sections from in 9 normal tissues, 21 early staged (stage 1 and 2) and 4 advanced staged (stage 3) NSCLC patients. In addition PD-L1 was also assayed in bladder, breast and gastric cancer. Results PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis demonstrated moderate correlation (R2 = 0.8894). Normal lung tissue did not express PD-L1; ∼24% of early stage (5/21) and 50% of advanced stage NSCLC (2/4) expressed measurable PD-L1 protein. PD-L1 was detected more frequently in squamous cell carcinoma than adenocarcinoma. We are currently assessing the levels of PD-L1 and other targets of the immunological synapse using multiplex mass spectrometry and comparing it with IHC in 100 cholangiocarcinoma and possible inclusion of PD-L1diagnostics in clinical trials. DiscussionThe need to characterize expression levels of druggable targets in small biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Initial PD-L1 screening using clinical NSCLC samples suggests that more advanced NSCLC patients are more likely to be PD-L1 positive compared to early stage NSCLC patients. Laser microdissection (LMD) can be used to specifically microdissect the immunological synapse. Additional quantitative assays for both lymphocyte (CD8, CD68) and immunotargets (B7-H3,B.1, B7.2 etc) have been developed for assessing the ‘immune profile’ in tumor associated stroma via LMD. This immuno-proteomic assay of the key immunological synapse members within tumor and/or stroma may lead to improved personalized immunotherapy. Citation Format: Sheeno P. Thyparambil, Fabiola Cecchi, Eunkyung An, Wei-Li Liao, Jon Burrows, Todd Hembrough, Daniel Catenacci. Development and clinical validation of a quantitative mass spectrometric assay for immuno-oncology targets in FFPE samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4255. doi:10.1158/1538-7445.AM2015-4255

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 (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. Citation Format: Fabiola Cecchi, Wei-Li Liao, Sheeno Thyparambil, Marlene Darfler, David Krizman, Todd Hembrough, Jon Burrows, Don Bottaro, Daniel V.T. Catenacci. A novel clinical tool that provides quantitative and accurate measurement of Met protein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3397. doi:10.1158/1538-7445.AM2015-3397

Background: Given the prevelance of chromosomal instability and gene amplification (amp+) as driver mechanisms in gastroesophageal adenocarcinoma (GEC), we assessed the incidence of KRAS gene amp+ and gene copy number (GCN) along with protein expression, at baseline and after molecular targeted therapy in GEC cell lines/tissues. KRAS undergoes activating mutation in various cancers. KRAS mutation is rare (80%). We observed diminished KRAS amp+ growth/proliferation both in vitro and in vivo using combined MEK and AKT pathway inhibitors over either alone, and compared to various inhibitor controls (anti-EGFR, anti-MET, anti-HER2). Conclusions: In this series, we observed KRAS wild type gene amp+ to be present in a subset (16%) of GEC patients at diagnosis, correlating with very high protein expression. KRAS amp+ was present after treatment with trastuzumab in HER2+ patients, and also after anti-MET therapy. These data suggest that KRAS amp+ represents a molecular subset with advanced disease at diagnosis. The observation of acquired KRAS amp+ after targeted therapies may be a resistance mechanism to anti-HER and anti-MET inhibitors. Inhibition using combined MEK/AKT pathway inhibitors, and proof-of-principle siRNA, warrants further investigation for KRAS amp+ tumors. Citation Format: Les Henderson, Peng Xu, Brittany Rambo, Wei-Li Liao, Todd Hembrough, Daniel Catenacci. KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A55. doi: 10.1158/1557-3125.RASONC14-A55

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

Introduction: Translocations in ALK, ROS1 and RET have been shown to be oncogenic in NSCLC. Lung cancers having ALK or ROS1 rearrangements represent unique subpopulations that are seen in only 2-5% and 1-2% of NSCLC, respectively. ALK fusions lead to constitutive activation of ALK signaling involved in cell proliferation. Crizotinib has significant anti-tumor activity in ALK rearranged NSCLC and break-apart FISH is the approved diagnostic test to determine treatment eligibility. However, FISH is laborious, expensive and low throughput, and thus is not ideal for the detection of oncogenic drivers of low frequencies. In patients with advanced disease, a small tissue biopsy is often the only material available so yielding as much information as possible from a limited sample is necessary. The aim of this study was to develop a multiplexed quantitative Liquid-Tissue-selected reaction monitoring (LT-SRM) assay for assessing ALK, ROS1, and RET expression within our “Lung OncoPlex” MS test. The LT-SRM platform quantitates these translocation markers along with several diagnostic and potentially targetable biomarkers, e.g. TTF1, K7, p63, K5, EGFR, HER2, HER3, MET, KRAS and IGF1R, in NSCLC. Methods: We used trypsin digestion mapping of recombinant proteins specific for ALK, ROS1, and RET to identify optimal quantitative peptides. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in Pyrococcus complex matrix to determine LOD, LLOQ, accuracy, precision and linearity of the assays. The ALK assay was pre-clinically validated in an EML4-ALK rearrangement positive cell line-H3122. ALK and ROS1 were screened in 87 archived FFPE sections from NSCLC. Results: We identified at least two optimal peptides for each target. At least one peptide from each protein had acceptable technical assay performance and was used for assay development. H3122 cell expressed 396 amol ALK/ug cell protein, while 11 ALK translocation positive NSCLC tissues expressed ALK from 107 to 437 amol/ug protein. ALK peptides were not detected in ALK negative control NSCLC tissues or in a single ALK translocation positive case. ROS1 was detected in 2 of 87 NSCLC samples at levels of 659 amol/ug in a case of unknown translocation status and 377 amol/ug in a ROS1 translocation positive case. Finally, the Lung OncoPlex assay successfully subtyped lung adenocarcinoma and quantified the other potentially targetable biomarkers. Conclusions: The Lung OncoPlex assay was able to detect ALK protein in 11/12 ALK rearranged samples. In the one proteomically negative/FISH+ case, we are performing ALK IHC to assess ALK protein expression, as well as DNA sequencing to evaluate for potential mutations within the MS targeted peptides. Of the two cases positive for ROS1 by the MS assay, one is known to be FISH positive and the other is undergoing FISH verification. RET protein expression has not yet been assessed in any known RET translocation positive cases; however, the RET technical performance suggests this is a promising assay and we are continuing to screen for RET positive control samples. While additional studies are needed to validate the clinically utility of the ALK, ROS1, and RET assay; multiplexed proteomic screening of patient tissue could be performed at the time of initial biopsy, allowing for simultaneous assessment of multiple clinically actionable gene rearrangements and biomarker targets. Citation Format: Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Christopher P. Hartley, patrick Ma, Jaime Rodriguez, Ignacio Wistuba, Jon Burrows, Todd Hembrough, Laura J. Tafe. Multiplexed mass spectrometry-based assay to quantify translocation markers from non-small cell lung cancer (NSCLC) FFPE tissue. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B09.

Introduction: Aberrant over-expression of receptor tyrosine kinases, including the MET, HER, FGFR, and IGFR families along with other critical downstream oncogenic mediators including KRAS, BRAF, PI3 Kinase and SRC are known drivers of colorectal cancer (CRC), subdividing the disease into distinct molecular subsets. Inter-patient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care. Moreover, intra-patient tumor heterogeneity from primary tumor to metastatic disease is likely to influence biomarker prediction of response to specific targeted agents. 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. Other protein quantification methods (ELISA, ECL) would require non-standard tissue processing for analysis. We present a quantitative mass spectrometric (MS) assay for CRC utilizing Liquid Tissue - Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of relevant oncoproteins in a cohort of CRC paired primary and metastatic tumor tissues. Methods: 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 ‘CRC-plex’ MS assay with multiplexed SRM quantification of Met, RON, EGFR, HER2, HER3, IGF1R, FGFR2, KRAS and cSRC on 42 primary human CRC cancer tissues, with paired metastases when available obtained from core biopsy or metastatectomy, using laser capture microdissection of the target material from a single unstained 10μm thick section per sample. Results: Validation of the CRC-plex SRM assay on cell lines and FFPE tissues revealed very high concordance when compared to IB and IHC. Multiplex oncogene quantification of all tissues, to the attomole/microgram level, will be presented, highlighting inter-patient and intra-patient (from primary to metastasis) heterogeneity of samples. Conclusions: Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure relevant actionable oncoproteins in FFPE clinical samples. The CRC-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/or post-treatment assessment. Citation Format: Todd Hembrough, Wei-Li Liao, Les Henderson, Peng Xu, Sheeno Thyparambil, Jon Burrows, Daniel V. Catenacci. Development of a quantitative colorectal cancer SRM 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 41. doi:10.1158/1538-7445.AM2013-41

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

Introduction: RON and Met, members of the Met family of tyrosine kinases, are implicated as mediators of tumor progression and metastasis in cancer. Over-expression of each receptor is prognostic of poor survival in resected and metastatic cancers, and expression of RON/Met in preclinical models and early phase clinical trials predicts response to RON/Met specific inhibitors. RON is expressed as a number of alternate splice variants/transcripts, complicating the quantification of the receptor by standard clinical methods. Inter/intrapatient tumor heterogeneity suggests that an expedient, reliable, medium throughput oncogene protein expression profiling will provide vital information to better personalize cancer care, with emphasis on serial biopsies to assess acquired treatment resistance mechanisms. 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. Other protein quantification methods (ELISA, ECL) would require non-standard tissue processing for analysis. We sought to develop a quantitative mass spectrometric (MS) assay for RON utilizing Liquid Tissue – Selected Reaction Monitoring (SRM), with subsequent multiplex quantification of RON, Met, and other previously validated proteins in a panel of gastroesophageal cancer (GEC) cell lines and tissues. Methods: Using trypsin digestion mapping of recombinant RON, 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. The final assay was validated and the N-terminal RON SRM demonstrated an LOD/LOQ of 62/125. Alternate peptides were chosen to quantify differences in RON splice variants/transcripts. We then tested the RON MS assay using a panel of FF GEC cell lines previously characterized by immunoblot (IB) and IHC FFPE pellet. In addition to RON, we multiplexed SRM quantification of Met, EGFR, HER2, HER3, IGF1R, and cSRC. We evaluated 15 GEC lines including three AGS lines: wild type (AGS-WT), scrambled shRNA (AGS-SC) and RON shRNA knockdown (AGS-KD) to assess ‘post-treatment’ changes in oncogene expression profiles. We then evaluated 20 GEC human cancer tissues and 5 paraneoplastic normal tissues using laser capture microdissection of the target material from a single unstained 10μm thick section per sample. Results: In the initial analysis, 4/10 cell lines (HCC827, Colo205, HT29, A431) expressed N-terminal RON (∼250 amol/μg cell protein). Validation of the RON 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 N-terminal RON (284/323 amol/ug cell protein), while RON was not detected in AGS-KD cells, as expected. Correlation of IB with RON intracellular/extracellular MS assay data will be presented. Measurement of RON in the GEC tissues correlated well with IHC. RON expression was seen in 75% of GEC tissues, and was lower/undetectable in adjacent normal tissues. 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. Conclusions: Taken together, these data demonstrate a sensitive, accurate, and quantitative assay to measure RON and its variants in FF cells. Multiplexed oncogene expression of these tumors was feasible and expedient using limited tissue, and is a novel clinically applicable approach for tumor characterization for baseline and post-treatment assessment.

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

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

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.

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