Abstract LB-230: Quantitative immunohistochemistry for melanocortin 1 receptor using phosphor integrated dots

Visual scoring of biomarkers by immunohistochemistry (IHC) on formalin-fixed paraffin embedded tissues constitutes a major part of histopathologic diagnosis and staging of several cancers. To improve sensitivity and linearity of IHC methods, we have developed a quantitative IHC technique using streptavidin coated phosphor integrated dot (PID) fluorescent nanoparticles (Gonda et al, Scientific Reports, 2017). In retrospective analysis of clinically established biomarkers (i.e., PD-L1, HER2, CSF-1R), IHC-PID has provided high-sensitivity quantitative detection of target proteins with a broad dynamic range. The present study examines the utility of IHC-PID in the biomarker discovery and validation process by evaluating melanocortin subtype 1 receptor (MC1R) in cell lines and clinical biopsy samples. MC1R is currently under investigation as a potential diagnostic and therapeutic target for malignant melanoma, a lethal cancer with poor long-term prognosis. MC1R is an endocytic cell-surface G-protein coupled receptor that exhibits relatively high expression in malignant melanoma tumors vs. normal tissues. Notably, current melanoma therapies, including MAPKi (e.g., vemurafenib, dabrafenib) increase MC1R expression pharmacologically on melanoma cells (unpublished results). MC1R expression was determined in cell lines by standard radio-ligand binding assay (125I-NDP-α-MSH). The SK-Mel-28 cell line showed 5-6 fold increased expression of endogenous MC1R when compared to the A375 cell line. Formalin fixed pellets of these two cell lines were used for analytical validation of PID-IHC. By utilizing dedicated image-processing algorithms, the readout of average PID score/cell was determined to be 285 and 98, for SK-Mel-28 and A375 cell lines, respectively. To confirm the increase in sensitivity with the PID technology, enabling quantitative IHC and improved detection of MC1R, de-identified melanoma samples from varying sites were collected by surgical biopsy at The University of Iowa Hospitals and Clinics and scored by a pathologist. Sister sections were stained by conventional IHC or IHC-PID. For IHC-PID, sections were counter stained by hematoxylin; tumor regions were hand annotated by a pathologist; and fluorescent images in 5 microscopic fields were captured and analyzed. Background PID threshold was determined by using no-primary antibody as controls. IHC-PID was performed on MS0759 (T1b N0 M0), MS0045 (T3a N1a M0) and MS0868 (T4b N3 M1a) samples and showed average PID score/cell of 217±38, 68±12, and 160±21, respectively. Ongoing studies are expanding on IHC-PID use to assess MC1R expression on a clinical melanoma panel (n=20) in late stage patients undergoing pharmacological interventions including MAPKi. The overall goal of this project is to develop PID-based quantitative IHC to support the development of MC1R as an imaging biomarker for patient stratification and monitoring clinical response to MC1R-targeted radionuclide therapy for malignant melanoma. Note: This abstract was not presented at the meeting. Citation Format: Apollina Goel, Kenji Nishikawa, Etsuko Futaya, Ildiko Poylak, Robert Holt, Mengshi Li, Edwin Sagastum, Brenna Marks, Aaron D. Bossler, Frances L. Johnson, Michael K. Schultz, Hideki Goda, Hisatake Okada, Kelly Orcutt, George Abe, Kenneth Bloom. Quantitative immunohistochemistry for melanocortin 1 receptor using phosphor integrated dots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-230.