Abstract Hairy cell leukemia (HCL) cells express an immunoglobulin (Ig) heavy chain variable (VH) gene with different degrees of somatic mutation. We recently reported that of 82 patients with HCL, 14 expressed VH4-34 and that these patients had a poorer outcome compared to the other 68 patients. Moreover, poor prognosis in the VH4-34 patients was independent of the known poor-prognosis variant of HCL, called HCLv. To confirm these findings, and to more extensively characterize the somatic mutations occurring in the non-VH4-34 patients, we studied mutations from these and an additional 32 patients. A total of 118 rearranged VH sequences from 114 HCL patients were analyzed. Four patients had 2 rearrangements each, none of them VH4-34. Seventeen (15%) of the 114 patients had VH4-34 rearrangements, comprising 10 (38%) of 26 HCLv and 7 (8%) of 88 classic HCL patients (p=0.0005). Sixteen (94%) of 17 VH4-34 rearrangements were unmutated (>98% homologous to germline), compared to 15 (15%) of the other 101 (p=2 × 10-10). Median homology to germline was 99.6% in VH4-34 vs 95.4% in the other rearrangements (p<0.0001). Previous clinical findings that VH4-34+ HCL is associated with high white blood cell count at diagnosis, inferior response and progression-free survival after initial cladribine therapy, and poor overall survival from diagnosis independent of HCLv status, were confirmed. Characterization of the locations of somatic VH mutations in lymphocytic leukemias is rare. One published study reported that in 172 chronic lymphocytic leukemia (CLL) rearrangements, mutations were concentrated in the complementarity determining (CDR) regions, with higher replacement/silent (R/S) mutation ratios for CDRs (4.60) vs framework (FR) regions (1.72) (Blood, 103:3490-3495, 2004). We found that in our 118 HCL rearrangements containing 1385 mutations, R/S was 2.40 overall, 4.29 for CDRs, and 1.83 for FRs, nearly identical to the reported CLL data. Other features reported in CLL, including mutations preferentially in RGYW hot spots and consisting predominantly of transitions rather than transversions, were also observed in HCL. These data suggest that like CLL, VH mutations in HCL result from canonical somatic hypermutation and select for stable antigen receptors, similar to the mutation process taking place in the normal germinal center. However, the origin of VH4-34+ HCL appears different and requires further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2125.