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

Abstract

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\u2009=\u20098) vs intermediate/high (n\u2009=\u200910) recurrence‐scores as estimated by 21‐gene assay and pathology review were compared by microarray assay. Tissue microarrays were prepared from breast carcinomas (n\u2009=\u200920) and ductal carcinoma in situ (DCIS) specimens (n\u2009=\u200984 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\u2009<\u2009.05) 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\u2009=\u2009.0152); 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\u2009=\u2009.0269) 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.