Introduction: Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and enable predictions of efficacy of a number of chemo- and immunotherapeutics. We used these expression signatures to develop a robust standardized assay—the Cancer Immunotherapy 2000 (CI2000) panel—that would dissect immunosurveillance mechanisms and discover novel prognostic and predictive immune response biomarkers.
Material and Method: The CI2000 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computation analysis of immunotherapy network model, we predicted key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, adhesion, and apoptosis of cancer cells. This computational analysis produced approximately 400 more targets beyond the core panel. The CI2000 panel also includes a comprehensive set of 1,200 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity. Further, an additional set of housekeeping genes with constant expression between different cancer types was included in the assay for data normalization.
Results and Discussion: The CI2000 assay quantitatively profiles the expression levels of approximately 2,000 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by next-generation HT sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise which often depends on the quality of samples. Control studies have shown that the CI2000 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,000 key cancer immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CI2000 assay.
Conclusions: Comprehensive profiling of tumor-associated immune cells with the CI2000 gene panel provides a sensitive, standardized, and quantitative method to investigate cancer immunoediting mechanisms. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient population responsive to the growing number of immunotherapeutic treatments. Toward this goal, the described CI2000 assay provides a platform for the discovery prognostic and predictive immune response biomarker signatures.
Citation Format: Alex Chenchik, Leonid Iakoubov, Michael Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A010.
- ©2016 American Association for Cancer Research.