CytogeneticsTissue Fixation Significantly Impacts MLH1 Promoter Methylation Analysis: A Quality Improvement Study

Abstract

Abstract MLH1 promoter methylation (MPM) analysis is an invaluable tool for distinguishing sporadic from inherited colorectal and endometrial cancers. At our institutional clinical genomics lab, we observed an unusually high failure rate of MPM testing in endometrial (EM) cancers. This triggered a quality improvement (QI) project to identify the underlying cause and improve the success rate of our MPM assay. We retrospectively identified all MPM cases (EM and non-endometrial (NEM) cancers) between January 2019 and October 2022 through querying our departmental database. The failure rates of these two groups were juxtaposed with the following variables: DNA concentration, performing technologists, operating surgeons, and specimen collection day (Monday through Saturday). A subsequent fishbone analysis was performed to evaluate the impact of specimen handling, fixation, and embedding procedure/timeline in the gross room and histology labs. We identified one-hundred thirty-one EM cases and eighty-two NEM cases that underwent MPM testing. Thirty-one EM cases (24%) and seven NEM cases (9%) failed, re-demonstrating the higher failure rate (X2=7.9, P-value=0.005) of EM cases. There was no significant difference in DNA concentration, performing technologists, and operating surgeons when compared to passed cases, however, a highly significant difference was seen concerning day of specimen collection (X2=53, P-value<0.0001). Most notably, all failed EM (n=31) and NEM cases (n=7) were collected on either Thursday or Friday (failure rate of 26% and 59%, respectively, 44% jointly). Our fishbone analysis revealed that specimens accessioned Thursday afternoon and Friday were fixed throughout the weekend before embedding on Monday, resulting in prolonged fixation (one extra day). This is consistent with previous studies’ conclusions showing DNA quality (but not quantity) significantly worsened with longer formalin fixation. Upon presenting these data to lab personnel, workflow changes were implemented. Cancer tissue that would have originally fixed over the weekend was instead processed on Saturday starting December 2022. This QI project revealed that prolonged formalin fixation of specimens over the weekend dramatically reduces the success rate of MPM analysis of EM cancer cases. Subsequently, we expanded our investigation to include other molecular assays at our institution that have returned similar results. Since the implementation of the revised workflow in December 2022, a preliminary prospective analysis showed the MLH1 failure rate of EM cancer tissues collected on Thursday and Friday has decreased from 44% to 11% in the past three months (December 2022 – February 2023). The current findings have important implications for the use of FFPE specimens in genetic analysis and highlight the need for careful consideration of fixation workflows, especially over the weekend, in sample processing protocols.