Adele Blackler

High-Throughput Microdissection for Next-Generation Sequencing

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

Abstract 4935: Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers

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

Abstract 449: Targeted proteomic analysis for personalized treatment of muscle invasive bladder cancer

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.

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 3395: Clinical Survey of 19 actionable proteins in multiple indications using multiplex mass spectrometry

Many available oncology therapies are targeted to specific proteins, the most notable examples being therapies targeted to EGFR and Her2. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. As more pathways and proteins are identified as tumor drivers and therapies are developed that target those proteins, and more patient screening tools are needed to efficiently direct patients to correct therapeutic regimens. While chemotherapy regimens are not often considered targeted therapies, protein biomarkers for chemotherapy efficacy have been identified; for example amplification of TOPO2A is known to improve response to anthracycline-based therapy combinations. Though many chemotherapy biomarkers have been identified, screening is not routine, and chemotherapy regimens are not being adjusted for individual tumor biology. To address the growing need for efficient patient screening using minimal tissue, OncoPlex Diagnostics has built a comprehensive protein quantification panel that allows for the simultaneous quantitation of multiple actionable proteins from formalin-fixed, paraffin-embedded patient biopsies using multiplex mass spectrometry. This panel currently quantifies nine proteins that are markers of targeted therapy (including ALK, AR, EGFR, HER2, HER3, MET, MSLN, and PD-L1) and includes the ChemoPlex panel, which quantifies chemotherapy biomarkers (ERCC1, FRalpha, hENT1, RRM1, SPARC, TOPO1, TOPO2A). Since 2013, over 270 biopsies from multiple indications have been analyzed for protein expression in the OncoPlex Diagnostics CAP-qualified, CLIA-certified laboratory, revealing large ranges of expression for many drug targets that are not routinely assayed. Because of the importance of TOPO2A in anthracycline-based therapies, which are commonly prescribed in breast cancer patients, sixty-two breast cancer biopsies were retrospectively reviewed. Of the 62 samples, 41 were positive for TOPO2A expression; ranging from 233-1750amol/ug. Of the primary biopsies, 80% of them expressed TOPO2A; however only two of seven liver metastases were positive for TOPO2A. These data suggest that anthracycline-based therapy might not be as efficacious in metastatic sites due to the lack of TOPO2A. Also of interest, quantification of FRalpha, a biomarker for folate-targeted therapies, showed a 50-fold range of expression in NSCLC cases, and TOPO1, the target of irinotecan and topotecan, showed a 10-fold range of expression in various indications, with 4% of biopsies having no detectable TOPO1. These wide expression ranges of known biomarkers suggest that certain therapies might have vastly different response rates based on biomarker expression. To derive the best therapeutic response to both targeted and chemotherapy regimens, it is necessary to understand each patient tumor biology individually. Citation Format: Fabiola Cecchi, Adele Blackler, Heather Jordan, Marlene Darfler, Todd Hembrough, Michael Stocum, Jon Burrows. Clinical Survey of 19 actionable proteins in multiple indications using multiplex mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2015-3395

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

Abstract 3211: Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection.

Currently, proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue (the most common method of storing tissue specimens) are limited by the overwhelming complexity of the cellular proteome, which makes disease related changes in protein expression difficult to detect. Expression Microdissection (xMD) allows for automated, operator-independent dissection of specific cells or subcellular organelles in FFPE tissue, resulting in the enrichment of specific, selectable protein populations. In xMD, tissue slices that have been stained for specific cellular markers using immunohistochemical (HC) methods are placed under a layer of ethylene vinyl acetate (EVA) polymer film. When the stack is uniformly illuminated (using a rastering laser or a light pulse), the EVA melts and makes contact only over the stained tissue, enabling specific capture and transfer of these areas for further biochemical analysis, while leaving unstained tissue behind. As a proof of principle, we are using the xMD technique to enrich for nuclei in FFPE rat brain tissue sections followed by proteomic analysis. Shotgun proteomic analysis of these samples show a significant enrichment in nuclear localized proteins, with an average 18% of recovered proteins localized to the nucleus-versus 11% of recovered proteins in whole tissue scrapes. Targeted mass spectrometry, using Multiple Reaction Monitoring (MRM), also shows an enrichment of nuclear markers such as histones and DNA-binding proteins, and a de-enrichment of non-nuclear proteins. The enrichment for nuclei allows for the identification of methylated lysines on histones that are not detected in whole tissue scrapes, most likely due to their low abundance. The evolving methodology may allow researchers to profile the sub-cellular proteome of patient samples to identify potential new disease biomarkers and drug targets. This novel technology could also be used to advance our understanding of the biomolecular changes responsible for a specific disease state. Citation Format: Adele R. Blackler, Michael A. Tangrea, Tom Pohida, Nicole Morgan, Robert Bonner, Keshia Mora, John Kakareka, Sanford Markey, Michael R. Emmert-Buck. Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2013-3211