Simple SummaryDespite p53 aberration, as a prognostic biomarker, still remaining a matter of debate for gastroesophageal adenocarcinoma (GEA), the characterization of p53/TP53is routinely performed to assign chromosomal instability (CIN). The current gold standard for p53 assessment is immunohistochemistry (IHC). However, several studies with other tumors have demonstrated that “low” IHC staining levels should be considered as aberrant as “strong” staining due to mutated p53. We investigated the potential of molecular assays, such as droplet digital PCR and next-generation sequencing, for the implementation of IHC. The results suggest that molecular approaches in solid and liquid biopsies could improve the characterization of “low” IHC cases, revealing that the majority harbor a deletion of one allele and a mutation of the other one. Redefining the current IHC model through adequate recognition of the p53 “low-level” phenotype as aberrant might be helpful in better understanding the prognostic role of p53 and possibly, in the future, correctly assigning target treatment.AbstractChromosomal instability (CIN) is very frequent in gastroesophageal adenocarcinoma (GEA) and it is characterized byTP53deletions/mutations resulting in p53 nuclear accumulation, as revealed by immunohistochemistry (IHC), which considers the cases with “high” staining levels to be positive. Aiming to improve aberrantTP53detection, droplet digital PCR (ddPCR) was used to evaluateTP53deletion in formalin-fixed, paraffin-embedded DNA (FFPE-DNA) and cell-free DNA (cfDNA). To further investigate the mutationalTP53profile, next-generation sequencing (NGS) was performed in a subset of FFPE samples. After combining “low” and “high” IHC staining level groups, the proportion of deletion events was significantly higher compared to the “intermediate” group (72.9% vs. 47.5%,p-value = 0.002). The ddPCRTP53deletion assay was feasible for cfDNA but only had good agreement (72.7%, Cohen’s kappa = 0.48) with the assay performed with FFPE-DNA of the “low-level” group. NGS analysis confirmed that, in the “low-level” group, a high percentage (66.7%) of cases were aberrant, with disruptive mutations that probably led to p53 loss. Data suggested that p53 IHC alone underestimates the CIN phenotype in GEA and that molecular analysis in both solid and liquid biopsies could be integrated with it; in particular, in cases of completely negative staining.