Digital Assessment of CMYC and Ki67 in Diffuse Large B-Cell Lymphoma

Abstract

CMYC expression by immunohistochemistry has been shown to be an independent prognostic factor in DLBCL, although manual scoring methods suffer from low reproducibility and both intra- and interobserver variation. The critical cut-off value of 40% CMYC expression is often difficult to determine, due to variability in nuclear stain intensity in tumor cells. We sought to determine if digital image analysis demonstrates utility in CMYC quantification and prognostic impact, in new diagnostic samples of DLBCL. The pathology case files at our institution were reviewed for patients with a new diagnosis of DLBCL, obtained between 2010 and 2023. 30 such patients were selected, and CMYC immunohistochemical stains were scanned into the Aperio CS2 digital pathology scanning system (Leica Biosystems). Tumor areas were defined by manual region of interest selection, after review by 3 board certified hematopathologists. A customized image analysis algorithm was derived from Aperio ImageScope software’s preset nuclear macro algorithm, by setting a score of 1+/3 (weak nuclear staining) at a threshold of 220. Percentages of weak (1+/3), moderate (2+/3), and strong (3+/3) tumor nuclei were computed, and Hscores were calculated. Digitally quantified CMYC expression percentages varied widely in the 30 samples (mean 32.8%, standard deviation 12.9%, range 13.5 – 66.7%). The calculated H-scores were similarly variable (average 53.1, standard deviation 26.8, range 21.6 - 131.8). H-scores demonstrated a negative correlation with overall survival (r=0.27). When an H-score cut-off value of 75 was selected, the group with H-scores > 75 demonstrated lower overall survival (average 961 days, standard deviation 824 days) than the group with Hscores < 75 (average 2049 days, standard deviation 818 days); the relationship is statistically significant (p<0.05). Digital image analysis of CMYC expression demonstrated diagnostic utility in a group of 30 patients with newly diagnosed DLBCL. Calculated H-scores correlated inversely with patient survival, and an H-score cut-off value of 75 separated patients into longer and shorter survival groups.