Exploratory Analysis of Aminopeptidase A and Pyroglutamyl-Peptidase I Activities in Breast Cancer Sentinel Lymph Nodes Across Categories of Nodal Involvement

Background/Objectives: Breast cancer disseminates through the lymphatic system, and the sentinel lymph node (SLN) is the first nodal station draining the primary tumor. Previous analyses identified changes in nodal redox status associated with disease stage. We explored whether soluble peptidase activities differed according to the extent of nodal involvement, with particular attention to SLNs containing isolated tumor cells (ITCs). Methods: Ninety-two SLNs from women with invasive breast carcinoma were classified as negative (n = 77), ITC-positive (n = 3), micrometastatic (n = 5), or macrometastatic (n = 7) by one-step nucleic acid amplification (OSNA) of cytokeratin 19 mRNA combined with histological and immunohistochemical assessment. Eight soluble peptidase activities (APN, APB, OTXase, ASAP, APA, PGPEP1, IRAP, and EDA) were quantified by spectrofluorometry in nodal homogenates. Groups were compared using the Kruskal–Wallis and Mann–Whitney tests with the Benjamini–Hochberg control of the false discovery rate (FDR). The effect sizes were accompanied by bootstrap confidence intervals, and discrimination was explored by a receiver operating characteristic analysis and logistic regression. Results: None of the eight peptidase activities differed significantly across categories of nodal involvement after FDR correction. APA and PGPEP1 showed the lowest omnibus p values (p = 0.068 and p = 0.074, respectively; q = 0.295 for both). In unadjusted pairwise comparisons, APA and PGPEP1 activities were higher in ITC-positive than in negative nodes (p = 0.038 and p = 0.042, respectively), but these differences did not survive correction within the enzyme family (q = 0.077 and q = 0.125) or across the whole set of 48 comparisons (q = 0.399 for both). The estimated standardized differences were large but highly imprecise (Cohen’s d = 1.47, 95% CI 0.38–3.38; d = 1.49, 95% CI 0.35–3.40), and the nominal differences were sensitive to a single ITC-positive node. When all involved nodes were compared with negative nodes, the area under the curve was 0.46 for both enzymes, and the two-enzyme logistic model did not improve discrimination. Conclusions: Soluble peptidase activity did not differ significantly across categories of nodal involvement. The nominally higher APA and PGPEP1 activities in ITC-positive nodes were exploratory, imprecise, and non-robust, and should not be interpreted as evidence of a distinct ITC-associated enzymatic phenotype. These findings provide a testable hypothesis for future studies and highlight the need for substantially larger numbers of ITC-positive nodes to test it.