Abstract 5419: A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features
Authors: Berrino Enrico , Aquilano Maria Costanza , Valtorta Emanuele , Amodio Vito , Germano Giovanni , Gusmini Marco , Sarotto Ivana , Sapino Anna , Marsoni Silvia , Sartore-Bianchi Andrea , Bardelli Alberto , Siena Salvatore , Bonoldi Emanuela , Marchio Caterina 
Published: June 2022

Abstract
Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism.
Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&E slides and on IHC sections stained with antibodies raised against CD8.
Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes.
Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined.
Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5419.