Furthermore, Ingenuity Pathway Analysis (S4 Fig in S1 File) showed that the top 5 pathways enriched in the HDB samples spiked with 50 and 500 SKBR3 cells compared with the sham-spiked HDB samples were ovarian cancer signaling, hereditary breast cancer signaling, mismatch repair in eukaryotes, endometrial cancer signaling, and glioma signaling (all P < 0.5 × 10−9), and the top upstream regulators included ERBB2, CDKN1A, estrogen receptor, and TP53 (S4 Fig in S1 File). Here, ESR1 is linked to central nervous system cancer.