Eunkyung An

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

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

Abstract 3398: Development of a mass spectrometry based antibody-drug conjugate biomarker panel

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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 <LOD to 13500 amol/ug. An examination of Her2 positive tissue showed a wide range of Her2 expression (over 20-orders of magnitude), as well as a wide range of expression for the payload markers. The differences seen in the payload markers suggest differing responses to Her2-targeted ADCs, independent of Her2 levels, and indicate that different patient populations might respond better to different payloads, depending on tumor biology. 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

Abstract 566: ChemoPlex SRM assay predicts response to specific chemotherapeutic agents in NSCLC

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 and these proteins have been identified as predictive biomarkers of response for many chemotherapy agents. While it has been demonstrated that expression levels of these biomarkers can predict chemotherapy response, chemotherapy in both the adjuvant and metastatic settings for NSCLC are not based on biomarker analysis. We have developed a quantitative, multiplex, mass spectrometry based diagnostic assay that can identify multiple biomarkers associated with chemotherapy benefit. At the time of abstract submission, the expression levels of each biomarker were surveyed in 23 NSCLC patients, and the biomarker level for each chemotherapy agent (ERCC1 and XRCC1 for Platinum based chemotherapy; FR alpha and GART for pemetrexed; hENT1 and RRM1 for gemcitabine; and TUBB3 for taxane) was evaluated with clinical outcome. We are extending these analyses with a population of 100 NSCLC tumors with associated outcome. Our pilot study shows a wide range of expression for each biomarker in patient biopsies. Among these biomarkers, FR alpha in NSCLC shows over 4 orders of magnitude in expression range. Also as expected, each biomarker level was found to be correlated with clinical outcome. For example, patients with high level of FR alpha showed improved response to pemetrexed; patients with lower level of TUBB3 showed improved response to taxane and patients with lower level of hENT1 showed poorer response to gemcitabine. Our preliminary retrospective data analysis suggests that the ChemoPlex SRM assay can help predict response to specific chemotherapeutic agents. This test can be used to characterize individual tumor biology and identify actionable targets that can better inform patient care. Citation Format: Eunkyung An, Tae-Jung Kim, Manish Monga, Kathleen Bengali, Alexi Drilea, Joseph Reilly, Marlene Darfler, Jon Burrows, Todd Hembrough. ChemoPlex SRM assay predicts response to specific chemotherapeutic agents in NSCLC. [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 566. doi:10.1158/1538-7445.AM2015-566

Abstract B09: Multiplexed mass spectrometry-based assay to quantify translocation markers from non-small cell lung cancer (NSCLC) FFPE tissue.

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.

Abstract A24: Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples.

Background: Binding of PD-L1 expressed on tumor cells to the PD1 on T lymphocytes transduces immuno-inhibitory signals which cripples the T cell9s ability to combat the tumor. Several anti-PD-L1 and anti-PD1 agents are in clinical trials and both regimens have reported promising preliminary results in NSCLC patients. (Brahmer et al., 2012 and Inman, 2013).These studies suggest that tumor expression of PD-L1 is associated with a response to either anti-PD-L1 and anti-PD1 treatment. Immunohistochemistry (IHC) is the current method to assess PD-L1 expression in FFPE tissue; however, PD-L1 IHC has yielded mixed results; some studies showed high false positive by IHC while another study showed that 13% of the PD-L1 negative patients responded to treatment. Moreover, IHC is low throughput and assessing multiple druggable targets by IHC is tissue consuming. As such, there is an urgent need to develop quantitative and highly multiplexed tests to assess biomarker expression. We have developed and clinically validated a quantitative mass spectrometric assay to measure PD-L1 protein expression in FFPE tissue biopsies. Method: We used trypsin digestion mapping of recombinant PD-L1 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 assay. 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. We also used Lung OncoPlex assay to sub-classify NSCLC samples to adenocarcinoma and squamous cell carcinoma. All of the samples were screened in replicates and multiple machines were used to check technical variability. Results: A 10 point calibration curve using five replicates was used to determine the LOD (75 amol) and LOQ (100 amol) for the PD-L1 assay. Fourteen (14) cell lines were assayed for PD-L1 expression by LT-SRM. PD-L1 protein expression was detected in 7 out of 14 cell lines The regression analysis between SRM and mRNA analysis (Broad Institute) demonstrated excellent correlation (R2=0.8894). The NSCLC cell line HCC827 and breast cancer cell line MDA-MB-231 had the highest levels of PD-L1, 374.78 and 298.27 amol/μg protein, respectively. Our initial clinical analysis of NSCLC tissue shows that while no normal lung tissue expresses detectable levels of PD-L1, ~24 % of early stage NSCLC (5/21) and 50 % of advanced stage NSCLC (2/4) express measurable PD-L1 protein. Interestingly, in this initial cohort, all of the PD-L1 positive early staged NSCLC were squamous cell carcinoma while in a small set of advanced staged NSCLC, PD-L1 expression was seen in both squamous cell carcinoma (1/3) and adenocarcinoma (1/1). Characterization of larger cohorts of NSCLC tissue is currently underway and will be presented. Discussion: The need to characterize expression levels of druggable targets in small NSCLC biopsies is becoming ever more critical as new drug targets and biomarkers are identified. Here we describe the development and initial clinical validation of a quantitative proteomic PD-L1 assay which accurately measures PD-L1 expression levels in FFPE tumor tissue. 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. Additionally, patients with squamous cell carcinoma are very likely to express PD-L1. Interestingly, Soria et al. (2013) has recently shown a high response rate to PD-L1 therapy in smokers with squamous cell carcinoma. We are currently expanding this initial clinical validation to assess PD-L1 expression levels in larger cohorts, including both adeno and squamous carcinoma. Additional quantitative assays for both lymphocyte (CD3, CD8, CD68) and immunotargets (PD1, B7-H3) are under development. This proteomic assay promises to be a critical component of our multiplexed biomarker analysis, and will allow more accurate identification of potential candidates for PD-L1 or PD1 targeted therapies. Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Jaime Rodriguez, Ravi Salgia, Ignacio I. Wistuba, Jon Burrows, Todd Hembrough. Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples. [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 A24.

Abstract 918: Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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<20%) for FR alpha, hENT1, TOPO1, TOPO2A, and RRM1, respectively. Fourteen cell lines were assayed for the Chemo-target expressions by LT-SRM, and regression analysis between protein and mRNA analysis for each target demonstrated varying correlations (R2=0.91(FR alpha); 0.78 (hENT1); 0.16 (TOPO1); 0.56 (TOPO2A); 0.59 (RRM1)) suggesting that RT-PCR measurements of mRNA levels would not be representative of cellular protein levels and therefore not useful for biomarker analysis for physicians. Our initial clinical analysis shows that FR alpha was detectable only in certain lung tumors (especially adenocarcinoma) and ovarian tumors; breast cancer tumors were found to have a wide range of hENT1 expression (LOD -1,284 amol/ug) while hENT1 expression in other tissues ranged from 90 to 377 amol/ug. TOPO1 had fairly ubiquitous expression (359 -1,300 amol/ug) except for one GEC and one breast cancer tissue. TOPO2A was identified in all tissue types (227-1,057 amol/ug). All samples except 1 GEC tissue express RRM1 (160 - 958 amol/ug) 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