Microarray and RNA in situ hybridization assay for recurrence risk markers of breast carcinoma and ductal carcinoma in situ: Evidence supporting the use of diverse pathways panels.

Breast tumor stratification by recurrence-risk is critical for deciding patient treatment. Here an approach combining cancer pathways microarray data complemented by RNA in situ hybridization (ISH) was investigated as a means for recurrence marker discovery and visualization in pathology specimens. LncRNA and mRNA expressions in breast carcinomas with low (n = 8) vs intermediate/high (n = 10) recurrence-scores as estimated by 21-gene assay and pathology review were compared by microarray assay. Tissue microarrays were prepared from breast carcinomas (n = 20) and ductal carcinoma in situ (DCIS) specimens (n = 84 patients) with known outcomes. Thirteen RNA ISH assays were performed: lncRNAs (BBC3-1, FER3, RAD21-AS1, ZEB1-2) and mRNAs (GLO1, GLTSCR2, TGFB1, TLR2) (implicated by the microarray data); MKI67; a pooled panel of recurrence-associated proliferation markers (BIRC5, Cyclin B1, MKI67, MYBL2, STK15); a pooled panel of non-proliferation recurrence-associated markers (CEACAM5, HTF9C, NDRG1, TP53, SLC7A5); and lncRNAs H19 and HOTAIR. Seven lncRNAs and 10 mRNAs showed significantly (P < .05) altered upregulation or downregulation by microarray assay: carcinoma RNA ISH staining did not mirror these patterns. HOTAIR staining was associated with a higher breast cancer recurrence score (P = .0152); qualitatively, H19 was massively expressed in a metaplastic triple negative breast carcinoma. Among the DCIS cohort, significant associations with multiple outcome variables were noted for TGFB1 and the non-proliferation panel (P-value range: .0001 to .047); proliferation panel staining showed an association with increasing DCIS grade (P = .0269) but not with outcomes. The findings support recurrence-risk estimation by the use of multi-marker panels that are representative of diverse cellular pathways rather than over-reliance on proliferation targets. H19, HOTAIR, and TGFB1 RNA ISH show potential for selective diagnostics.