Chromosomal instability (CIN) is associated with poor prognosis in human cancer. However, in certain animal tumor models elevated CIN negatively impacts upon organism fitness, and is poorly tolerated by cancer cells. To better understand this seemingly contradictory relationship between CIN and cancer cell biological fitness and its relationship with clinical outcome, we applied the CIN70 expression signature, which correlates with DNA-based measures of structural chromosomal complexity and numerical CIN in vivo, to gene expression profiles of 2,125 breast tumors from 13 published cohorts. Tumors with extreme CIN, defined as the highest quartile CIN70 score, were predominantly of the estrogen receptor negative (ER⁻), basal-like phenotype and displayed the highest chromosomal structural complexity and chromosomal numerical instability. We found that the extreme CIN/ER⁻ tumors were associated with improved prognosis relative to tumors with intermediate CIN70 scores in the third quartile. We also observed this paradoxical relationship between CIN and prognosis in ovarian, gastric, and non–small cell lung cancer, with poorest outcome in tumors with intermediate, rather than extreme, CIN70 scores. These results suggest a nonmonotonic relationship between gene signature expression and HR for survival outcome, which may explain the difficulties encountered in the identification of prognostic expression signatures in ER⁻ breast cancer. Furthermore, the data are consistent with the intolerance of excessive CIN in carcinomas and provide a plausible strategy to define distinct prognostic patient cohorts with ER⁻ breast cancer. Inclusion of a surrogate measurement of CIN may improve cancer risk stratification and future therapeutic approaches. Cancer Res; 71(10); 3447–52. ©2011 AACR.