Prognostic Impact of V_H Gene Mutational Status and CD38 Expression in Japanese CLL Patients.

In the western countries, it has been well established that both immunoglobulin VH gene mutational status and CD38 expression are useful prognostic markers in chronic lymphocytic leukemia (CLL). However, it is not clear whether it is also true in other regions, especially such as Japan where CLL incidence is not so frequent as other countries. Therefore, we investigated the prognostic impact of VH gene mutational status and CD38 expression in Japanese B-CLL. The subjects of this study were 44 patients (29 males and 15 females) referred to our institutions between March 1999 and March 2004. The median age at the time of diagnosis was 68 years (37–92 years). The diagnosis was based on immunophenotypic analysis and cell morphology analyzed on Wright’s–stained peripheral blood and bone marrow smears. Median follow-up period of these patients was 4.0 years (0.5–34.2 years).

cDNA mainly prepared from peripheral blood samples of the CLL patients was amplified using V_H family-specific framework region primers or leader primers, and C_H primers. PCR products were sequenced directly or after TA-cloning using the BigDye Terminator Cycle Sequencing FS Ready Reaction kit on a 310 Genetic Analyzer. Nucleotide sequences were compared to the nearest germ line V_H genes in databases: IMGT, V-QUEST or IgBLAST. Of 44 B-CLL patients, IgH variable region genes could be sequenced from their cDNA in 43 patients; no amplified band was obtained in 1 patient. The usage of the seven V_H gene families in the 43 B-CLL patients were as following: V_H 1, 4/43 (9.3%); V_H 2, 2/43 (4.6%); V_H 3, 23/43 (53.5%); V_H 4, 12/43 (27.9%); V_H 5, 1/43 (2.3%); V_H 6, 1/43 (2.3%); V_H 7, 0/43 (0%). Eighteen cases (41.9%) displayed unmutated V _H genes, defined as the sequences having more than or equal to 98% homology with nearest germ line gene, and 25 cases (58.1%) showed somatically mutated, defined as less than 98% homology. The proportion of unmutated cases in this study was almost comparable to previous reports, which showed a range of 30% to 50%. It have been uniformly reported that prognostic difference is apparent between unmutated (bad) and mutated (good) groups. Also in this study, the overall survival, defined as the time from diagnosis to death from any cause or to last contact, was significantly shorter for unmutated cases compared to mutated cases estimated by the Kaplan-Meier method as previous reports: predicted 50% survival rate for the unmutated cases was 9.1 years, but that for mutated cases did not reach the median survival. The difference was significant (p=0.029, log-rank test).

Cell surface CD38 expression, which has been reported to correlate with a poor prognosis, was analyzed by flow cytometry in all patients. With the cut off level of 30% in CD19+CD5+ lymphocytes, 13 patients (29.5%) were estimated to be CD38 positive and 31 patients (70.5 %) to be negative. There was no significant difference in overall survival between those 2 groups (p=0.519, log-rank test).

In conclusion, V_H gene mutational status is a strong prognostic indicator whereas CD38 expression is not in our B-CLL cohort.