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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.

Protein-targeted therapies are expected to selectively kill tumor cells that express the targeted protein biomarker. Although a tumor mass may initially respond to targeted therapies based on expression of the targeted protein, all cells within a tumor may not express the targeted protein above a critical threshold level; therefore, those cells that do not express, or that downregulate expression of, the targeted protein may not be responsive to therapy. The ability to monitor the dynamic expression of these protein biomarkers throughout the course of therapy may allow for treatment to be personalized in real-time in response to the evolving nature of the tumor. This report demonstrates, by monitoring a single patient through multiple therapies, how targeted mass spectrometry is an effective, quantitative method that provides real-time analysis of multiple therapeutically associated targeted proteins that can be used to personalize a patient's treatment strategy throughout the course of care.

Background Combined targeted strategy with letrozole (Le) and lapatinib (La) improves progression-free survival (PFS) in patients with metastatic breast cancer (MBC) co-expressing hormone receptor-positive (HR+) and HER2+ but not in HR+/HER2-negative (HER-) disease (Johnston J Clin Oncol 2009). However, among HER2+ tumors, quantitative levels of HER2 are heterogeneous with a broad dynamic range corresponding to approximately 163.7 to 17446.7 amol/µg as previously reported (Nuciforo SABCS 2014). In addition, within HER2- tumors, quantitative measurement of HER family proteins may identify those patients most likely to benefit from the addition of La to Le. In this retrospective study, we tested the prognostic and predictive ability of HER proteins quantification in clinically HER2+ tumor samples from the EGF30008 study. Methods Formalin-fixed paraffin-embedded primary tumor tissues sections from HER2+ MBC population were used. After laser microdissection, tissue lysates were prepared for selected reaction monitoring mass spectrometry (SRM-MS) analysis. Absolute quantitation was accomplished through simultaneous detection of endogenous target and synthetic labeled heavy peptide identical to analytical targets (EGFR, HER2, HER3). HER2 protein levels were correlated with PAM50 molecular subtypes, ERBB2 and ESR1 genes by nCounter. PFS and overall survival (OS) were analyzed by Kaplan–Meier and log-rank test. To determine whether HER2 protein levels were predictive of La benefit, we tested the interaction term of HER2 protein as a continuous variable by treatment arm in a Cox model. Results Within the HER2+ study cohort (n=219), 107 had an available tumor block; 84 cases had sufficient material for HER expression measurement by SRM-MS. Average HER2 levels were 2321.1 amol/ug (median, 817.6). HER2 levels were lower in Le+La (n=43; mean, 1761 amol/ug) compared to Le (n=41; mean, 2908 amol/ug) arms, although the difference was non-significant (p=0.108). No expression of EGFR and HER3 was observed. HER2 protein levels were significantly different among PAM50 subtypes with HER2-enriched (HER2E) tumors showing the highest expression followed by Basal-like, Luminal A, Luminal B, and Normal-like (p Conclusions Levels of HER2 protein in HER2+ MBC are extremely heterogeneous. An association between HER2 protein and gene expression by nCounter was observed. HER2E tumors by PAM50 showed the highest levels of HER2 protein. Within the group of HER2+ MBC by standard IHC/FISH, tumors with high HER2 protein had a statistically non-significant worse outcome and do not seem to benefit from La. Further validation of these findings is warranted. Citation Format: Nuciforo P, Thyparambil S, Galván P, Vilaro M, Jimenez J, Liao W-L, Cecchi F, Blackler A, Press MF, Gagnon R, Ellis C, Hembrough T, Johnston S, Prat A. Quantitative HER family proteins assessment as prognostic and predictive biomarkers in the EGF30008 clinical trial. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-08.

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

Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.

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: Standard treatment for muscle-invasive bladder cancer (MIBC) includes chemotherapy with either gemcitabine-cisplatin (GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC). These regimens have similar clinical complete response rates of approximately 30%. While no targeted treatment has been approved for bladder cancer, clinical trials have identified biochemical markers that predict the chemoresponsiveness of MIBC tumors to specific chemotherapeutic agents. For example, patients with high hENT1 and low RRM1 expression responded better to GC-based chemotherapy than their counterparts, and HER2 overexpression predicted resistance to cisplatin-based therapy. To quantify targets that are known indicators of tumor behavior, we used targeted proteomic analysis to assess 30 different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) MIBC tumor tissue. Methods: Twelve FFPE MIBC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area from 1 or 2 slides. Following laser microdissection of the marked areas, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins in each patient sample. Results: Of the 12 patient samples, 7 (58%) expressed high levels of hENT1 and 11 (92%) expressed low levels of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. A single patient expressed high levels of RRM1, an indication for non-gemcitabine based therapy. All 12 patients expressed TUBB3, a marker of resistance to taxane (vinblastine) and 10 patients (83%) lacked expression of FR-alpha, a marker of sensitivity to methotrexate. The majority of patients expressed a marker of sensitivity to anthracycline (TOPO2A) and did not express a resistance biomarker for platinum therapy (ERCC1). Of 3 patients whose tumors expressed HER2, 2 had overexpression (>750 amol/ug) and would thus be eligible for HER2 basket trials. Further, multiplex-targeted proteomics discovered patients expressing FGFR1 (17%), cMET (33%), Axl (17%) and IDO1 (25%), which would make them eligible for clinical trials of targeted or immunotherapies. Conclusion: MIBC is heterogeneous and expresses a wide range of proteins. Yet, it continues to be treated with only 2 chemotherapeutic regimens. Multiplexed proteomics is currently being used in clinical practice to inform personalized patient care decisions with identification and the relative quantities of actionable proteins known to predict tumor behavior. Citation Format: Fabiola Cecchi, Sheeno Thyparambil, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Henry Frierson, Daniel Theodorescu. Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 449.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Well differentiated HCC can sometimes be extremely difficult to be distinguished from other hepatocellular mass lesions such as hepatocellular adenoma and dysplastic liver nodule due to considerable morphologic overlaps. Immunohistochemical markers may have a limited utility, especially on core biopsy. Choice of treatment depends on the extent and location of the cancer, and the overall health of the patient. For patients who are healthy enough to undergo surgery and who have early-stage cancer confined to the liver, treatment may involve hepatic resection; however many patients develop a cancer recurrence, which is the main cause of death in long-term evaluations. Recurrence rates after treatment with resection are high, highlighting the importance of finding effective adjuvant treatments and markers that aid in determining differences between hepatocellular lesions. To explore differences between hepatocellular lesions that could be indicators of tumor behavior, we used targeted proteomic analysis to assess different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) HCC tumor tissue Twenty-two FFPE HCC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area. Following laser microdissection, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins. As anticipated, well differentiated HCC, hepatocellular adenoma and dysplastic nodule expressed high rates of P-glycoprotein and the majority expressed multi-drug resistance gene protein (MDR1). Such markers may account in part for the chemotherapy refractory nature of HCC. All 22 patients expressed high levels of hENT1 and lacked expression of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. All 22 patients lacked expression of marker of sensitivity to anthracycline (TOPO2A) and the majority of patients did not express a marker of resistance to platinum therapy (ERCC1). Of the 22 patients whose tumors expressed EGFR, 5 had expression above the 75%ile and would thus be eligible for EGFR small molecule inhibitor therapy. Dysplastic liver nodule patients did not expressed significant level of EGFR. Further, multiplex-targeted proteomics discovered patients expressing cMET and IDO1, which indicate eligibility for clinical trials of targeted therapies or immunotherapies. 60% of dysplastic liver nodule patients did not expressed cMET at any level This study retrospectively evaluates HCC patients in an attempt to identify predictors of tumor behavior. Proteomic and genomic screening should be performed to identify differences between various hepatocellular lesions. Prospective evaluation of molecular and genetic profile is warranted in HCC patients to be distinguished from other hepatocellular mass lesions. Citation Format: Fabiola Cecchi, Nam Ku, Hanlin Wang, Adele Blackler, Todd Hembrough, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jiaoti Huang. Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4935.

e18242Background: Tumor molecular profiling is increasingly being used to develop personalized treatment strategies for cancer patients (pts). The OncoPlex Dx (OPDx) test (NantOmics) uses a quantit...

There are few reports on the high-affinity 5-HT(2A) agonist (4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).Here we provide the first behavioral and neurophysiological profile of TCB-2 in C57BL/6J mice, with direct comparisons to the 5-HT(2A/2C) agonist (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), in addition to determinations of 5-HT(2A) mediation via pretreatment with the selective 5-HT(2A) antagonist MDL 11,939.In a dose-dependent manner, TCB-2 induced head twitches, decreased food consumption in food-deprived mice, induced hypothermia, and increased corticosterone levels, with no effects on locomotor activity or anxiety-like behaviors in the open field. Similar effects were observed in side-by-side dose-response comparisons with DOI; although at the highest dose tested (5.0 mg/kg), TCB-2 induced significantly fewer head twitches, and a significantly enhanced hypothermic response, versus DOI. Pretreatment with MDL 11,939 blocked head twitches and temperature change following TCB-2 and DOI, confirming 5-HT(2A) mediation of these responses. Although MDL 11,939 pretreatment blocked DOI-induced suppression of feeding, MDL 11,939 had no effect on TCB-2-induced suppression of feeding. Previous studies show that 5-HT(2A) function is altered by changes in serotonin transporter (SERT) expression and function. In SERT knockout (-/-) mice, TCB-2-induced head twitches and hypothermia were greatly diminished compared to SERT wild-type (+/+) mice.The current studies are important, as they are the first to assess the effects of TCB-2 in mice, and are among the first to report the behavioral and neurophysiological effects of this conformationally restricted phenethylamine analog compound, which has 65-fold greater effects on signaling via the phosphoinositide versus arachidonic acid pathways.

74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.

Laser-based microdissection technologies precisely recover histological regions or cells from cytology preparations and tissue sections. Yet, due to the high-cost and time-intensiveness of these methods, coarse manual microdissection is the most commonly used tool for laboratory-based studies and clinical applications, enabling rapid procurement but at the expense of reduced cell purity. We present a low-cost, do-it-yourself expression microdissection (DIY xMD) kit that combines the speed and ease-of-use of manual methods with the precision of laser-based instruments. Using DIY xMD, we isolated cell targets including ER+ nuclei and GMS-stained aspergillis fungi from clinical specimens. We also demonstrate the advantage of cellular enrichment in a cytology-based study where BRAF and KRAS mutations were masked by background cells in a manual dissection, yet identifiable after cells were recovered by DIY xMD. The new system makes high-throughput, efficient microdissection accessible to investigators and clinicians throughout the world. Citation Format: Michael A. Tangrea, Avi Z. Rosenberg, Patricia A. Fetsch, Michael D. Armani, Liqiang Xi, Mark Raffeld, Tina Thu Pham, Yun Chen, Neil O'Flaherty, Rebecca Stussman, Adele R. Blackler, Qiang Du, Jeffrey C. Hanson, Mark J. Roth, Armando C. Filie, Michael H. Roh, Jason D. Hipp, Michael R. Emmert-Buck. Do-It-Yourself expression microdissection (DIY xMD): A low-cost, high-throughput cell and organelle isolation system. [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 230. doi:10.1158/1538-7445.AM2015-230

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

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example amplification of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies nine proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-qualified, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. Because of the importance of TOPO2A in anthracycline-based therapies, which are commonly prescribed in breast cancer patients, sixty-two breast cancer biopsies were retrospectively reviewed. Of the 62 samples, 41 were positive for TOPO2A expression; ranging from 233-1750amol/ug. Of the primary biopsies, 80% of them expressed TOPO2A; however only two of seven liver metastases were positive for TOPO2A. These data suggest that anthracycline-based therapy might not be as efficacious in metastatic sites due to the lack of TOPO2A. Also of interest, quantification of FRalpha, a biomarker for folate-targeted therapies, showed a 50-fold range of expression in NSCLC cases, and TOPO1, the target of irinotecan and topotecan, showed a 10-fold range of expression in various indications, with 4% of biopsies having no detectable TOPO1. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually. Citation Format: Fabiola Cecchi, Adele Blackler, Heather Jordan, Marlene Darfler, Todd Hembrough, Michael Stocum, Jon Burrows. Clinical Survey of 19 actionable proteins in multiple indications using multiplex mass spectrometry. [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 3395. doi:10.1158/1538-7445.AM2015-3395

1045 Background: The folate pathway is a critical nucleotide biosynthetic pathway in many tumor cells, and blockade of this pathway with antifolates has demonstrated clinical utility in NSCLC and m...

4050 Background: Trastuzumab-based chemotherapy is standard treatment for HER2-positive advanced gastric cancer (AGC). Although increased HER2 gene amplification by fluorescent in situ hybridizatio...

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example high expressive of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-accredited, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually.

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

Currently, proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue (the most common method of storing tissue specimens) are limited by the overwhelming complexity of the cellular proteome, which makes disease related changes in protein expression difficult to detect. Expression Microdissection (xMD) allows for automated, operator-independent dissection of specific cells or subcellular organelles in FFPE tissue, resulting in the enrichment of specific, selectable protein populations. In xMD, tissue slices that have been stained for specific cellular markers using immunohistochemical (HC) methods are placed under a layer of ethylene vinyl acetate (EVA) polymer film. When the stack is uniformly illuminated (using a rastering laser or a light pulse), the EVA melts and makes contact only over the stained tissue, enabling specific capture and transfer of these areas for further biochemical analysis, while leaving unstained tissue behind. As a proof of principle, we are using the xMD technique to enrich for nuclei in FFPE rat brain tissue sections followed by proteomic analysis. Shotgun proteomic analysis of these samples show a significant enrichment in nuclear localized proteins, with an average 18% of recovered proteins localized to the nucleus-versus 11% of recovered proteins in whole tissue scrapes. Targeted mass spectrometry, using Multiple Reaction Monitoring (MRM), also shows an enrichment of nuclear markers such as histones and DNA-binding proteins, and a de-enrichment of non-nuclear proteins. The enrichment for nuclei allows for the identification of methylated lysines on histones that are not detected in whole tissue scrapes, most likely due to their low abundance. The evolving methodology may allow researchers to profile the sub-cellular proteome of patient samples to identify potential new disease biomarkers and drug targets. This novel technology could also be used to advance our understanding of the biomolecular changes responsible for a specific disease state. Citation Format: Adele R. Blackler, Michael A. Tangrea, Tom Pohida, Nicole Morgan, Robert Bonner, Keshia Mora, John Kakareka, Sanford Markey, Michael R. Emmert-Buck. Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection. [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 3211. doi:10.1158/1538-7445.AM2013-3211