Six1 expression correlates with poor prognosis in luminal breast cancer and is high in luminal breast cancer TICs. (A) Association of Six1 with intrinsic subtype. Box-and-whisker plots are shown for two independent datasets, 779 tumor dataset and UNC311 dataset. P values were calculated with ANOVA. Individual P-values comparing expression of Six1 in luminal B tumors to other subtypes are as follows: 779 dataset versus Basal = 0.0004, Claudin = 1.43E-6, HER2 = 0.07, LumA = 0.03. UNC311 dataset versus Basal = 0.0089, Claudin = 0.0046, HER2 = 0.4561, LumA = 0.0388. (B) Relapse free survival curve of Kaplan-Meier analyses with a combined 779 breast tumor data set. Kaplan Meier curves are shown for all breast cancer subtypes, Luminal A tumors, and Luminal B tumors. (C) Six1 mRNA expression was determined by RT-PCR, and normalized to Cyclophilin B mRNA. Luminal B patient xenografts PE4, PK12 and PK15 were injected in NOD-scid IL2Rgnull mice, after which the tumors were excised, CD24+CD44- and CD24low CD44+ populations isolated, and Six1 mRNA levels determined. PE4; ER+(90%)PR+(75%)HER2-, PT12; ER+(93%)PR+(15%)HER2-, and PT15; ER+(8%)PR-HER2-. ANOVA, analysis of variance; RT-PCR, reverse transcriptase-polymerase chain reaction.