Panel of Methylation-based PCRs for Separating Mucinous Lung Adenocarcinomas from Gastrointestinal Metastases Involving the Lung.

Abstract

The primary sites-of-origin of mucinous adenocarcinomas are often difficult to determine, as there are no reliable immunohistochemical markers. The aim of our project is to use a panel of 4 methylation-based droplet digital PCRs (methyl-ddPCRs) to distinguish pulmonary mucinous adenocarcinomas from gastrointestinal (GI) metastases to the lung. The 4 PCRs developed use methylation-independent primers for amplification and methylation-dependent probes to measure signals at 4 individual CpG sites, predicted to be differentially methylated across lung (LUAD), gastroesophageal (GEAD), pancreatic (PAAD), and/or colorectal (CORE) adenocarcinomas based on The Cancer Genome Atlas methylation array data. The PCRs were tested on an internal cohort of 35 formalin-fixed paraffin-embedded lung adenocarcinomas tumors, which were mostly mucinous tumors, and 22 COREs, GEADs, and PAADs that metastasized to the lung. Decision trees using PCR data were applied to predict the diagnoses of these 57 references and 12 additional challenging cases. Decision tree predictions were found to be concordant with the primary versus metastatic assignments for 83-90% of reference cases across multiple analyses. For difficult/challenging cases, decision tree predictions were concordant with the favored impressions for 5/10 cases that underwent subsequent clinicopathologic review, and suggested diagnoses for 2 remaining cases with insufficient data. Preliminary assessments also support the application of these PCRs to smaller specimens. Our experiences suggest that methyl-ddPCRs can be viable solutions for many difficult differentials in surgical pathology.