Wei-Li Liao (Presenter)
NantOmics, LLC
Authorship: Wei-Li Liao, Fabiola Cecchi, Adele Blackler, Sheeno Thyparambil, Eunkyung An, Zhichang Yang, Yuan Tian, Marlene Darfler, David Krizman, Todd Hembrough
NantOmics, LLC
| Short Abstract Many available oncology therapies target specific proteins. Additionally, protein biomarkers affecting chemotherapy efficacy have been identified. For targeted therapies to have maximal efficacy, it is necessary to identify patients whose tumors express the target protein. In this study we present a multiplexed Liquid Tissue-Selected Reaction Monitoring assay to simultaneously quantify 26 clinically-actionable proteins from a single microgram of FFPE patient tissue. The assay’s performance and tumor expression levels of these biomarkers in over 300 clinical biopsies will be presented. By providing absolute quantitation of multiple actionable proteins from patient biopsies, clinical mass spectrometry is delivering personalized cancer care. |
Long Abstract
Introduction: Targeted cancer therapies designed to disrupt proteins in tumor signaling pathways are the current focus of cancer drug development. Additionally, expression levels for multiple biomarkers have been shown to correlate with efficacy of chemotherapy, indicating that measuring tumor expression of these proteins could allow chemotherapy to be treated as targeted-therapy. Immunohistochemistry (IHC) is the standard method to detect proteins in formalin-fixed-paraffin-embedded (FFPE) tissues, however, there is a clinical need to characterize multiple biomarkers in patient tissue and testing multiple proteins by IHC is tissue and time-consuming. Our multiplexed Liquid Tissue-Selected Reaction Monitoring (LT-SRM) assay simultaneously quantifies 26 clinically-actionable proteins from a single microgram of FFPE patient tissue. This assay is run routinely in a CAP/CLIA lab to guide patient care.
Methods: Using trypsin digestion mapping of recombinant proteins, we identified unique peptides and built quantitative mass spectrometry-based assays for receptor tyrosine kinase targets (EGFR, HER2, HER3, MET, IGF1R, RON, AXL, FGFR2), proteins involved in immune checkpoints (PD-L1), histology markers (K7, TTF, K5, p63), clinically-actionable gene rearrangements (ALK, ROS1) and the predictive/prognostic protein markers for chemotherapy agents (TOPO1, TOPO2A, hENT1, RRM1, FRalpha, SPARC, ERCC1). These assays were multiplexed into a single SRM analysis of 1ug of tumor protein. Assays were clinically validated on FFPE tumor tissues using laser microdissected tumor tissue from a 1-2 10um thick section per sample. We quantified expression levels of these biomarkers in over 300 clinical biopsies from multiple tumor indications in our CLIA-certified-CAP-accredited laboratory; and demonstrated reproducibility of the assay over years.
Results: Calibration curves for all targets demonstrated linear range up to 25fmol on column, with LOD for each analyte between 75 and 400amol and %CV<20% for each concentration point. Repeat analysis of tissue over a 1 year period demonstrates incredible reproducibility of the assay.
In 69 breast cancer biopsies, TOPO2A, a biomarker for routinely prescribed anthracycline-based therapies, was detected in 74% cases ranging from 266-1975amol/ug. Interestingly, while 79% of the primary breast biopsies (49/62) expressed TOPO2A, only two of seven liver metastases were positive for TOPO2A. Similarly, SRM detected TOPO2A in 75.8% primary GEC biopsies (47/62) ranging from 318-22550amol/ug. Currently, biomarker screening is not done before initiation of anthracycline-based therapies. These data suggest that TOPO2a expression should be routinely determined prior to beginning therapy, to identify patient who will not respond.
We have previously shown that LT-SRM analysis of HER2 can identify patients that will have an enhanced response to anti-HER2 therapies, unlike IHC or gene amplification. Analysis of patient samples identified 69.6%(48/69) breast cancer and 67.7%(42/62) gastroesphageal cancer cases that were HER2 positive by SRM, ranging from 212-9400amol/ug and 185-9600amol/ug, respectively. 4.4% (3/69) of breast cancer and 12.9% (8/62) of GEC showed HER2-SRM>750amol/ug, a cutoff consistent with gene amplification; suggesting these patients would be candidates for HER2-targeted therapies.
Interestingly, ALK and ROS1 proteins, targets of crizotinib/ceritinib, were seen in 1% (1/95) and 2.1% (2/95) of NSCLC tested, respectively. Proteomics analysis of these low-prevalence oncogenic-drivers as part of a multiplexed screening of patients’ initial biopsies saves time and tissues.
Conclusions: This clinical mass spectrometry assay could conceivably quantify upwards of 100 actionable proteins in a single analysis, and new analytes are routinely being added to our menu. LT-SRM analysis provides clinicians with valuable diagnostic information and ensures that all patients whose cancers express clinically-actionable markers have the opportunity to receive treatment.
By providing absolute quantitation of multiple actionable proteins from patient biopsies, clinical mass spectrometry is delivering personalized cancer care.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | NantOmics, LLC |
| Board Member | no | |
| Stock | yes | NantOmics, LLC |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |