Chao Gong (Presenter)
NantOmics
Bio: Research Scientist at NantOmics
Authorship: Chao Gong, Kathleen Bengali, Alexi Drilea, David Krizman, Marlene Darfler, Wei-li Liao, Sheeno Thyparambil, Todd Hembrough
Research & Development, NantOmics, 9600 Medical Center Drive, Suite 300, Rockville, Maryland, USA
| Short Abstract Many oncology therapies target specific proteins; therefore, technologies to quantify tumor-specific protein targets are required. Our current platform uses laser-assisted microdissection to isolate and collect tumor cells for analysis from FFPE tissues mounted on DIRECTOR® slides. However, sections cut onto glass microscope slides are often the only materials available, limiting the utility of high resolution laser microdissection. In this study, we present the evaluation of a tissue mesodissection platform to effectively dissect tumor cells standard glass slides, followed by Liquid Tissue® SRM assays to simultaneously quantify 26 protein biomarkers. Quantitative results from mesodissection and laser microdissection are compared. |
Long Abstract
Introduction: Targeted cancer therapy is becoming the main focus of current oncology drug development. To achieve the desired effect of targeted therapy, it is imperative to develop tumor-specific diagnostic assays that can detect and quantitate clinically relevant biomarkers directly in patient tumor tissue. Formalin-fixed and paraffin-embedded (FFPE) tissues are valuable resources that can be analyzed for protein biomarkers and provide key information to assist patient therapy decisions. We have previously developed a multiplexed Liquid Tissue® SRM assay to measure the abundance (amol/µg) of 26 target proteins in FFPE patient tumor tissue. For this platform, only a small amount of tissue is needed and sample collection is routinely performed using laser-assisted microdissection for a single 10 µm thick tissue section on the DIRECTOR® slide. The DIRECTOR® slide has a patented energy transfer coating on which tissue sections are directly mounted. When the laser hits this coating, light energy is converted to kinetic energy to transfer cells off the slide and capture in a collection tube. The accuracy and precision of this technique ensures that only tumor cells of interest are collected while stromal and other normal cells are excluded. However, normal pathology practice for FFPE tissue samples is to be cut directly onto standard glass microscope slides. In many cases, tumor sections on glass slides are the only materials available. Mesodissection technology had been recently developed that allows for direct dissection of tumor tissue from standard glass microscope slides. For circumstances where tissue sectioning on DIRECTOR® slides is not a viable option, it may provide an alternative way to isolate tumor cells of interest for molecular analysis. In this work, we evaluated the feasibility of using the mesodissection method in our clinical mass spectrometry platform, and compared it to laser microdissection for targeted protein quantitation.
Methods: Six patient tumor tissue blocks with various levels of cellular heterogeneity were obtained from multiple tumor types: 2 breast, 2 lung, 1 colon and 1 abdominal metastasis from a primary colon. Seven serial tissue sections were cut for each block at 5 µm thickness each. The first tissue section was placed on a standard glass slide and stained with haematoxylin and eosin (H&E). The remaining six sections were serially sectioned and alternately placed on 3 glass slides and 3 DIRECTOR® slides to minimize histological variability from section to section. Using the digital image of the H&E stained slide as a reference, a pathologist identified and marked areas of interest that contain tumor cells to be dissected. Dissection of the areas of interest was performed on glass slides using the MilliSect instrument for mesodissection method while the MMI instrument was utilized to perform laser microdissection from the DIRECTOR® slides. The same areas of interest were dissected from serial sections using the 2 different methods based on the marked tumor cell locations. The dissected tumor pellets were processed by Liquid Tissue® SRM assay workflow for targeted protein analysis. Quantitative levels of 26 biomarker proteins were used for the comparability analysis between the two tissue dissection methods. Additionally, actin and tubulin were measured for quality control purposes.
Results and conclusions: Analysis of the 26 protein biomarkers across 6 tumor samples showed a broad range of expression levels of the analytes: from ND (not detected) to ~ 8000 amol per µg total protein. For actin and tubulin, quantities ranged from ~ 100 fmol to ~ 500 fmol per µg total protein analyzed. Quantitation results from mesodissection and laser microdissection platforms were compared for each analyte. For the 20 proteins that were detected and quantitated, values from mesodissection (average of three replicates per tissue block) are all within ±15% DEV from the mean value of both dissection methods. On average, the mesodissection experimental value is −1.5% of mean value from both dissection experiments. The analytical variance for each dissection method followed by Liquid Tissue® SRM assay were also evaluated. For each analyte, the CV% from three experimental replicates ranged from 0.8% to 14.9% for mesodissected tissue and 0.4% to 23.2% for laser microdissected tissue. On average, CV% from mesodissection is 7.6% while laser microdissection is 7.5%. Overall, equivalency between the two dissection methods was demonstrated for collecting tumor cells from heterogeneous tissue sections. Based on this study, we conclude that mesodissection of FFPE tissue on standard glass slides using the MilliSect instrument can also be routinely used on our platform for clinical diagnostic protein quantitation.
References & Acknowledgements:
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| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Receive salary from NantOmics |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | expense will be reimbursed by NantOmics |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |