Evans Mark Francis, Vacek Pamela Mary, Sprague Brian Lee, Stein Gary Stephen, Stein Janet Lee, Weaver Donald Lee
Department of Pathology & Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont.
University of Vermont Cancer Center, Larner College of Medicine, University of Vermont, Burlington, Vermont.
J Cell Biochem. 2020 Feb;121(2):1736-1746. doi: 10.1002/jcb.29409. Epub 2019 Oct 8.
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.
根据复发风险对乳腺肿瘤进行分层对于确定患者治疗方案至关重要。在此，研究了一种结合癌症通路微阵列数据并辅以RNA原位杂交（ISH）的方法，作为在病理标本中发现复发标志物并进行可视化的手段。通过微阵列分析比较了经21基因检测和病理评估估计的低复发评分（n = 8）与中/高复发评分（n = 10）的乳腺癌中lncRNA和mRNA的表达。从已知预后的乳腺癌（n = 20）和原位导管癌（DCIS，n = 84例患者）标本制备组织微阵列。进行了13种RNA ISH检测：lncRNAs（BBC3-1、FER3、RAD21-AS1、ZEB1-2）和mRNAs（GLO1、GLTSCR2、TGFB1、TLR2）（由微阵列数据表明有牵连）；MKI67；一组复发相关增殖标志物（BIRC5、细胞周期蛋白B1、MKI67、MYBL2、STK15）；一组非增殖性复发相关标志物（CEACAM5、HTF9C、NDRG1、TP53、SLC7A5）；以及lncRNAs H19和HOTAIR。7种lncRNAs和10种mRNAs通过微阵列分析显示出显著（P < 0.05）的上调或下调改变：癌RNA ISH染色并未反映这些模式。HOTAIR染色与较高的乳腺癌复发评分相关（P = 0.0152）；定性地说，H19在化生型三阴性乳腺癌中大量表达。在DCIS队列中，TGFB1和非增殖标志物组与多个结局变量存在显著关联（P值范围：0.0001至0.047）；增殖标志物组染色显示与DCIS分级增加相关（P = 0.0269），但与结局无关。这些发现支持使用代表不同细胞通路的多标志物组进行复发风险评估，而不是过度依赖增殖靶点。H19、HOTAIR和TGFB1 RNA ISH显示出选择性诊断的潜力。
