ARTICLE ABSTRACTPurpose: The specific role of chromosomal instability (CIN) in tumorigenesis has been a matter of conjecture. In part, this is due to the challenge of directly observing chromosome mis-segregation events as well as the inability to distinguish the role of CIN, which consists of increased rates of chromosome mis-segregation, from that of aneuploidy, which is a state of nondiploid chromosome number.Experimental Design: Here, we examine the contribution of CIN to the prognosis of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) by directly surveying tumor cells, fixed while undergoing anaphase, for evidence of chromosome mis-segregation. Hematoxylin and eosin–stained samples from a cohort of 54 patients were used to examine the relationship between frequencies of chromosome mis-segregation and patient prognosis, overall survival, and response to treatment.Results: We show that a two-fold increase in the frequency of chromosome mis-segregation led to a 24% decrease in overall survival and 48% decrease in relapse-free survival after treatment. The HR of death in patients with increased chromosome mis-segregation was 2.31 and these patients were more likely to present with higher tumor stage, exhibit tumor bone marrow involvement, and receive a higher International Prognostic Index score.Conclusions: Increased rates of chromosome mis-segregation in DLBCL substantiate inferior outcome and poor prognosis. This is likely due to increased heterogeneity of tumor cells leading to a larger predilection for adaptation in response to external pressures such as metastasis and drug treatments. We propose that targeting CIN would yield superior prognosis and improved response to chemotherapeutic drugs. Clin Cancer Res; 17(24); 7704–11. ©2011 AACR.