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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

Background: Human epidermal growth factor receptor 2 (HER2) status is an important predictive biomarker in breast cancer (BC). Tumor heterogeneity has been described, with changes in HER2 expression levels between lesions and over the disease course. HER2 expression is assessed on tissue biopsies, at primary diagnosis and in metastatic lesions. A whole-body imaging technique such as PET/CT could help understand expression levels in different lesions. A 68Ga-labeled single domain antibody (sdAb) targeting the HER2 receptor has been developed and proven safe (Keyaerts et al., 2016). Imaging is performed at 90 min post-injection (pi). We report results of a phase II trial to assess the repeatability of the technique in 20 patients and the correlation of tracer uptake with HER2 tissue expression of the lesions present at the time of imaging. Methods: Twenty patients (pts) with a locally advanced or metastatic BC with at least one lesion of minimum 12 mm were included. Pts were injected intravenously with a typical protein mass of 100 µg and a radioactive dose ranging from 98-168 MBq 68GaNOTA-anti-HER2 sdAb. PET/CT images were obtained at 90 min pi. A second tracer injection followed by PET/CT was done with a maximal interval of 8 days. To assess repeatability, up to 5 lesions per pt were selected, with no more than 2 in a single organ. Peak Standard Uptake Values (SUVpeak) of the lesions were measured on both scans and compared with a t-test and Bland-Altman Plots. Images were compared to other available medical or imaging data and interpreted considering the subject’s disease course. Serum and plasma samples were collected before injection and between 60 and 365 days pi and stored for future detection of anti-drug antibodies (ADA) and liquid biopsies analysis for the presence of HER2 amplification. Tissue samples were assessed by central labs using mass spectrometry, immunohistochemistry and in fluorescence situ hybridization. Results: Twenty women with BC (6 HER2+, 14 HER2-) with a mean age of 58.6 y (37-81) were included. Three pts were scanned only once (2 due to withdrawal of consent, 1 due to covid pandemic). Repeatability of the technique was visually scored as excellent. For quantification, 50 lesions were compared on both scans in 17 pts without significant differences between the two measurements (p=0.40). The repeatability coefficient (RC) was 38.2%. The mean absolute percentage difference (MAPD) was 13.6%, comparable to repeat values reported for 18F-FDG. In 3 out of 6 HER2-positive (HER2+) patients, lesions showed high uptake, even better visible than using 18F-FDG in 2 of them. In 2 HER2+ subjects with a negative scan, lesions were confirmed to be true negatives: one patient did not relapse from BC but had tuberculosis; the other was confirmed to have a radiopneumonitis after radiotherapy and no relapse. In 1 HER2+ patient, the uptake was unexpectedly low. However, the HER2 status was also not reconfirmed in the metastatic setting for this subject. In 1 HER2-negative patient, the tumor HER2 status was changed from negative to positive based on a subsequent image-guided biopsy performed in this study. High tracer uptake was also seen in many of the patients presenting with HER2-low BC (IHC 1+ or 2+), indicating the potential of the tracer to detect low-level HER2 expression. Additional correlation to centrally performed tissue and blood analysis is ongoing. Conclusion: 68GaNOTA-Anti-HER2 PET/CT shows high uptake in HER2-expressing BC lesions but also in HER2-low lesions. The technique shows good repeatability and, in some cases, even better sensitivity than 18F-FDG PET/CT. Specificity was confirmed in relapse-free lesions such as tuberculosis and radiopneumonitis. Its sensitivity makes it a promising technique to assess HER2+ and HER2-low lesions in BC patients. Citation Format: Odrade Gondry, Catarina Xavier, Wim Waelput, Omar Al Dabssi, Marian Vanhoeij, Sandrine Aspeslagh, Sofie Joris, Christel Fontaine, Guy Verfaillie, Jacques De Grève, Katrien Glorieus, Ine Luyten, Frederik Vandenbroucke, Sophie Bourgeois, Laurens Raes, Sheeno Thyparambil, Nick Devoogdt, Ilse Vaneycken, Julie Cousaert, Vicky Caveliers, Hendrik Everaert, Tony Lahoutte, Marleen Keyaerts. Assessment of repeatability and uptake quantification of 68GaNOTA-anti-HER2 sdAb PET/CT in patients with locally advanced or metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-02-05.

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.

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.

Introduction Approximately 20% of breast cancer patients overexpress HER2 and are treated with anti-HER2 therapies. However, there is a great deal of disparity of HER2 levels in the patients that are classified as HER2 positive (IHC3+). Techniques like FISH or IHC do not allow for HER2 quantification and a significant proportion of patients are wrongly classified as HER2 positive. Liquid Tissue-Selected reaction monitoring (LT-SRM) is a multiplexed mass spectrometric technique that can objectively quantify levels of Her2 and other targets from formalin fixed paraffin embedded (FFPE) sections. Given the different available anti-HER2 therapies (trastuzumab, TDM1, lapatinib and pertuzumab) with different modes of action, it would be beneficial for a clinician to understand the levels EGFR and HER3 so as to personalize the therapy. In this work, we have used LT-SRM to quantitate HER2, EGFR and HER3 from FFPE samples (one slide) of patients treated with anti-HER2 agents and correlated the levels of these proteins to clinical outcome. Methods FFPE sections from 60 HER2 positive (IHC3+) primary breast cancer patients were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue® buffer. Of the 60 samples, 24 were from metastatic setting and 36 from adjuvant setting. After trypsin digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. In addition to LT-SRM, FISH for HER2 was also conducted. Results HER2 quantitation by LT-SRM revealed receptor level ranges from 283 to 14938 amol/µg. ROC analysis was conducted and a cut-off of 2758.75 amol/µg gave the optimal sensitivity and specificity. Survival analysis revealed statistically significant DFS (4.40 years vs 3.38 years; p=0.013) and OS (4.43 years vs 4.03 years; p= 0039) in patients expressing ≥ 2758.75 amol/µg in the adjuvant setting and also statistically significant OS (5.51 years vs 3.37 years; p=0.037) in the metastatic setting. Correlation of HER2 FISH and levels of HER2 is ongoing. Approximately 41% of samples expressed EGFR (range 45 to 2317 amol/µg) and similarly 51% of the samples expressed HER3 (range 84 to 360 amol/µg) with 18% of samples expressing all three targets. Correlation of EGFR/HER3 expression with clinical outcome is ongoing. Conclusion We used an objective multiplex non-antibody based method to quantify multiple targets from FFPE tissue. Clinical correlation analysis of HER2 revealed improved OS and DFS in samples with high HER2 protein levels. Currently, we are expanding these studies to a larger set of samples and taking into account also the expression of other markers such as EGFR and HER3. This approach can potentially identify those tumors that are more dependent on these receptors for survival and also those patients that are exquisitely sensitive to anti-HER2 therapy. Citation Format: Paolo Nuciforo, Sheeno Thyparambil, Claudia Aura, Ana Garrido-Castro, Vicente Peg, Jose Jimenez, William Hoos, Jon Burrows, Todd Hembrough, Jose Perez-Garcia, Javier Cortes, Maurizio Scaltriti. Quantitative measurement of HER2 levels by multiplexed mass spectrometry from FFPE tissue predicts survival in patients treated with anti-HER2 based therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-29.

The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and HER2), a third (HER3) currently under investigation for its possible role in the acquisition of multidrug resistance and a fourth (HER4), the role of which is still matter of debate. Drugs inhibiting EGFR or HER2 show significant antitumor activity in the clinic, however, acquisition of resistance is a hallmark of these and most targeted therapies. In the case of EGFR and HER2, one of the emerging resistance mechanisms is the co-expression of HER3. Indeed, recent reports show that inhibition of the PI3K pathway leads to upregulation of HER3, and subsequent resistance. Clinical analysis of protein levels in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative and requires significant amounts of tissue. Moreover, the vast majority of research groups consider specific HER3 staining by IHC particularly challenging. However, accurate measurement of these targets is critical both for properly defining treatment groups and predicting patterns of resistance. In order to address these issues, we used trypsin digestion mapping and stable isotope labeled peptides to develop a panel of quantitative mass spectrometric (MS) assays to measure the levels of EGFR, HER2, HER3 and other clinically relevant targets in FFPE breast cancer tissue. These quantitative MS assays were then multiplexed to analyze 1μg of tumor protein. In this study, we multiplexed HER family analysis on 31 HER2 positive breast cancers. Tumor tissue was microdissected from FFPE sections, and subjected to quantitative MS analysis of EGFR, HER2, HER3 as well as IGF1R and cMET. Quantitation of HER2 showed a broad range of HER2 expression in these tissues. The highest expresser measured 26 fmol/ug tumor tissue, representing amplification and massive protein over expression. In contrast, five tissues showed low levels of HER2 expression, below 1 fmol/ug, similar to HER2 non-amplified cell lines. This suggests that MS quantitation can identify patients with low expression of HER2 who are unlikely to respond to trastuzumab therapy. As a matter of fact, 3 of these 5 low expressing patients had outcome data and showed no response to trastuzumab treatment. In 28 of 31 patient tissue samples, HER3 showed low levels of expression (100–300 amol/ug tumor tissue) similar to HER3 expression in cell lines, and comparable to low expressing EGFR and HER2 cell lines. The remaining 3 patients had no detectable HER3. This study demonstrates the feasibility of measuring HER3 in multiplex in FFPE breast cancer tissue. Based on the low but widespread expression of HER3 in this cohort, it may be most useful to assess HER3 expression after initial treatment as a marker of potential resistance to targeted therapies. Taken together, these data demonstrate that a sensitive and quantitative assay to measure oncoproteins in FFPE clinical samples may help stratify patients with variable expression of these targets. Our quantitation of oncogene expression from clinical samples uses a small amount of tissue, is clinically applicable and alleviates the problem of scoring either positive or negative for the expression of a given protein. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-19.

Background: Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands accounting for 10% of salivary gland malignancies and 1% of head and neck cancers. About 1,200 new cases are diagnosed annually in the United States. ACC is typically chemoresistant and clinical trials of multiple targeted agents found few responders. Studies of ACC oncogenesis have described frequent fusion of MYB-NFIB genes and other infrequent genomic mutations. ACC has not been proteomically characterized. We hypothesized that proteogenomic analysis of ACC tumor tissues would identify clinically relevant molecular differences between ACC and other tumor types. The objective of this study was to identify altered disease pathways and potential drug targets in ACC. Methods: Clinical tumor samples of ACC and squamous cell carcinoma (SCC) of the head and neck were analyzed using the GPS Cancer diagnostic test, which includes whole genome and transcriptome sequencing, and proteomic expression analysis using mass spectrometry. To quantify proteins, tumor areas of formalin-fixed paraffin-embedded tissue sections were marked by a pathologist, microdissected and solubilized. The resulting lysate was analyzed with mass spectrometry to quantitate 30 clinically relevant proteins. Samples were further analyzed with a global proteomics platform intended to discover targetable protein biomarkers. Proteins that were overexpressed or underexpressed (defined as a 1.5-fold difference between ACC and SCC) were subjected to pathway analysis to identify perturbed pathways and potential drug targets. Finally, the mRNA expression in ACC tumors was compared with RNA-seq data from various solid tumor types using the k-nearest neighbors algorithm. Results: In 14 tumor samples, unsupervised hierarchical clustering analysis of 4,002 proteins revealed a clear separation between ACC (n=8) and SCC (n=6) tissues. Four of 8 ACC samples harbored an MYB-NFIB fusion and single samples had fusions of MYBL-NFIB or AHI1-NFIB. Mutational burden in ACC and SCC samples was 1.35 and 3.53 mutations per megabase, respectively. Pathway analysis found enrichment of ACC genes in essential cell cycle processes. CDK6 protein expression was 4-fold higher in ACC samples than SCC samples by mass spectrometry (p=0.0036) and 3-fold higher at the mRNA level. Expression of p16 protein was 3-fold lower in ACC than in SCC (p=0.0289) and corresponding p16 mRNA levels were 17-fold lower in ACC than SCC. All ACC samples harbored intact retinoblastoma (RB1) gene. Expression of mRNA in 15 ACC samples was compared with that of 25 different tumor types from TCGA and the authors' clinical laboratory; breast cancer was found to be the nearest neighbor to ACC. Conclusions: Proteogenomic analysis revealed CDK6 overexpression in ACC. The combination of CDK6 overexpression, p16 underexpression and RB1 proficiency suggests that ACC tumors may respond to treatment with CDK6 inhibitors. Citation Format: Sheeno P. Thyparambil, Yeoun Jin Kim, Andrew G. Chambers, Dongyao Yan, Shankar Sellappan, Andrew J. Sedgewick, Yulia Newton, J Zachary Sanborn, Charles J. Vaske, Stephen C. Benz, Fabiola Cecchi, Hyunseok Kang, Todd A. Hembrough. Potential drug targets for adenoid cystic carcinoma elucidated by proteogenomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 778.

Background: Chemotherapy represents the cornerstone of treatment for patients with advanced sarcomas. Anthracyclines are the first line of treatment; however, other agents such as gemcitabine, topotecan, taxanes and temozolomide have shown clinical activity in sarcoma patients. While no biomarker for chemotherapy has been approved for clinical use, a growing body of literature has identified tumor molecular characteristics predictive of response or resistance to chemotherapy. These include expression of the proteins ERCC1 (platinum), TUBB3 (taxane), TOP2A (anthracyclines), TOP1 (irinotecan, topotecan), hENT1/RRM1 (gemcitabine) and MGMT (temozolomide). Our goal was to assess the landscape of chemotherapy biomarkers in several sarcoma subtypes to identify potentially beneficial treatment regimens. Methods: Formalin-fixed, paraffin-embedded tumor tissues from clinical samples of sarcoma representing 5 subtypes were microdissected and assayed with the GPS Cancer molecular test. The test combines whole-genome sequencing, RNA-seq, and proteomic expression analysis of 30 biomarker proteins using mass spectrometry. Results: Seventy-five sarcoma samples were characterized (Table 1). Expression of protein biomarkers varied by sarcoma subtype. Of interest, approximately two-thirds of leiomyosarcomas lacked resistance markers for platinum (ERCC1) and taxanes (TUBB3). Of osteosarcomas, 93% expressed the response marker for gemcitabine (hENT1), while only 14% expressed the protein marker of gemcitabine resistance (RRM1). The vast majority of Ewing's sarcomas expressed MGMT, a marker of temozolomide resistance. Conclusions: In clinical sarcoma samples, proteomic analysis can identify distinct patterns of expression that are predictive of response or resistance to chemotherapies. Additional analysis is in progress; correlation between expression of chemotherapeutic biomarkers and clinical outcomes in a subset of sarcoma patients will be presented. Table 1. Proportions of sarcoma patients (N=75) with tumor expression of chemotherapeutic biomarker proteinsMarkers of response (agent)Markers of resistance (agent)Sarcoma subtypeTOP1(topotecan, irinotecan)TOP2A(doxorubicin)hENT1(gemcitabine)ERCC1(platinum)TUBB3(taxane)RRM1(gemcitabine)MGMT(temozolomide)Leiomyosarcoma (n=25)4133228321658Liposarcoma (n=13)1504654621667Ewing's sarcoma (n=12)3383383504288Osteosarcoma (n=14)779346571467Rhabdomyosarcoma (n=11)36278264456488Tumor expression is defined as quantitated protein above the threshold determined to be indicative of clinical response/resistance. The thresholds (in attomoles per microgram of total tumor protein) are as follows: TOP1: 1340, TOP2A: 1570, hENT1: 100, ERCC1: 75, TUBB3: 850, RRM1: 700, MGMT: 200. Citation Format: Sheeno P. Thyparambil, Shankar Sellappan, Dongyao Yan, Fabiola Cecchi, Antoine Italiano, Todd A. Hembrough. The molecular landscape of sarcoma can inform selection of personalized chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1628.

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.

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: 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

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.

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

The epidermal growth factor receptor (EGFR) is a drug target for both small molecule and antibody therapeutics and has been approved in non small-cell lung carcinoma (NSCLC) and colorectal carcinoma (CRC) among other indications. These drugs block receptor signaling though blockade of the tyrosine kinase domain, or through inhibition of ligand binding. Current genomic tests measure receptor amplification, RNA levels, the mutation status of receptor or pathway molecules (EGFR or kRAS mutations) but no current assay can directly assess the activation state of the EGFR or its downstream signaling pathway components. Indeed, the EGFR mutation positive NSCLC tumors (thought to be constitutively active) show a high response rate to TKI therapy, but the many non responders (50% or more) demonstrate the limitation of genomic analysis. Since activation of EGFR is necessary for the response to these targeted agents, it is critical to measure what levels of receptor activation and downstream signaling determines tumor responsiveness to EGFR targeted therapies in these patients. For this reason, we have developed a panel of new diagnostic assays which measure the activation of the EGFR and key downstream signaling proteins through quantitation of the phosphorylation state of these proteins. 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. We preclinically validated the multiplexed Liquid Tissue® phospho-SRM assay on formalin fixed EGF stimulated A431 cells. We followed up these in vitro studies with phospho-SRM analysis of FFPE NSCLC xenograft explants where extensive independent histopathologic and molecular characterization had been performed, allowing us to benchmark our phospho-SRM analysis with standard diagnostic analyses. We have now extended these quantitation studies by measuring the expression of EGFR and phospho-EGFR in FFPE tissues obtained from relevant human clinical trial cohorts – Gefitinib treated NSCLC and Cetuximab treated CRC. It is hoped that we will be able to correlate EGFR expression, activation and signaling in these tumors with responsiveness to EGFR targeted therapy, and to validate this assay for use as a companion diagnostic to guide therapy in both NSCLC and CRC. 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 4919. doi:10.1158/1538-7445.AM2011-4919

Lung cancer is the leading cause of cancer death worldwide. Non-small cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. The most prevalent subtypes of NSCLC are adenocarcinoma (ADC) and squamous cell carcinoma (SCC), which combined account for approximately 90%. Ten resected NSCLC patient tumors (5 ADC and 5 SCC) were directly introduced into severely immune deficient (NOD-SCID) mice, and the resulting xenograft tumors (XT) were analyzed by standard histology and immunohistochemistry (IHC) and by proteomics profiling. Mass spectrometry (MS) methods involving 1- and 2-dimensional LC-MS/MS, and multiplexed selective reaction monitoring (mSRM, or MRM), were applied to identify and quantify the xenograft proteomes. Hierarchical clustering of protein profiles distinguished between the ADC and SCC subtypes. The differential expression of >175 proteins was found to constitute a distinctive proteomic signature associated with NSCLC subtype, and an mSRM method was developed that provided relative quantification of a subset of highly differentially expressed proteins (i.e. >10-fold; p Expression Pathology Inc. 9620 Medical Center Dr. Rockville, MD 20850 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 5097. doi:10.1158/1538-7445.AM2011-5097

The IGF-1R signal pathway is activated in many cancer types which has led to active clinical development of both antibody and small molecule IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF-1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify and select patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R- specific SRM assay was developed and preclinically validated on cell lines expressing a range of IGF1R protein. The final assay has a LOD of 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 2211. doi:10.1158/1538-7445.AM2011-2211

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

The epidermal growth factor receptor (EGFR) is a drug target for both small molecule and antibody therapeutic approaches for several cancers; however, current methods of selecting patients that will most likely respond to anti-EGR therapy are not effective. Better methods for patient stratification are needed. To this end we have developed an approach which can determine both absolute EGFR levels and the phosphorylation status of EGFR directly in formalin-fixed paraffin-embedded (FFPE) patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform, a combination of tissue microdissection, Liquid Tissue® processing which turns dissected tissue to a complete solubilized tryptic digest, and mass spectrometry-based selected reaction monitoring (SRM). This approach was used to measure the EGFR protein and its phosphorylation status in formalin fixed tissue culture cells, xenograft tumors, and patient tumor tissue. For assay development, 3 distinct tryptic peptides were assessed for absolute protein quantitation and multiple peptides where specific residues are known to become phosphorylated (pT693 and pY1197) were assessed for assaying the phosphorylation status of the EGFR protein. We demonstrate the ability to detect and quantify the EGFR protein and to monitor its phosphorylation status directly in patient tumor tissue. This approach offers a dynamic range and quantification which is superior to traditional IHC methods, and that could be used to identify and stratify patients most likely to benefit from anti-EGFR therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3753.