Evaluation of Patient Factors and Sample Pre-Analytics on Predictive Multiplex Immunohistochemical Assays in Immuno-Oncology Patients

Project Summary/Abstract

Therapies against immunologic checkpoint proteins, such as PD-L1, have revolutionized the treatment of multiple

malignancies, such as malignant melanoma, lung cancer, bladder cancer, or gastroesophageal cancer. All of these

therapies depend on predictive biomarker assays for PD-L1 protein expression in tumor tissue to identify patients who

will most likely respond. PD-L1 assays face two significant challenges: (i) they often need to be performed on small tissue

biopsies where tumor tissue is limited and (ii) the threshold for positivity by immunohistochemistry (1%) is very small,

highlighting crucial dependence on utmost precision and pre-analytical sample validity.

To address the issue of small and limited samples in biopsies, we have developed a chromogenic multiplexed

immunohistochemical assay that combines PD-L1 assessment with tissue and cell type-specific markers to provide a

combined diagnostic and predictive assay on a single histologic slide. To move multiplexed assays around PD-L1 into the

clinic, there exists an important knowledge gap: what are the patient-specific factors and specimen-related pre-analytical

variables that can influence the readout of PD-L1 positivity? Very limited knowledge is available about these variables.

Importantly, because the PD-L1 positivity threshold is so low and requires the reliable separation of two very small

numbers (5,000 patients. Aim 2: We will test how specimen-related pre-analytical variables affect the assay using

a wide variety of fresh, frozen, and formalin fixed tissue types and sizes. Aim 3: Once we have defined optimal patient-

specific and specimen-related procedures, we will validate our multiplex assay in a prospective cohort of immunotherapy

patients at MSKCC.

Significance: This project will yield abundant data about the pre-analytical variables that influence a PD-L1 multiplex

immunohistochemistry assay. These data will inform optimal specimen acquisition and handling and strategies for

avoiding or mitigating inaccurate assay results. Innovation: This will be the first study to systematically explore both

patient context factors and specimen pre-analytics in multiplexed immunohistochemical testing for immuno-oncology.

Our close collaboration with commercial test developers as part of our research team will accelerate the translation of

our scientific findings into optimized assays for the direct benefit of patients.