Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

Validation of a new prognostic index for patients with chronic lymphocytic leukemia.

BACKGROUND: The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. A prognostic index based on widely available clinical and laboratory features was recently developed to predict survival among patients with previously untreated CLL. This index requires validation in an independent series of patients before widespread use can be recommended. METHODS: The Mayo Clinic CLL database was used to evaluate the validity and reproducibility of the new prognostic index. RESULTS: A total of 440 patients with newly diagnosed CLL who were seen at the Mayo Clinic within 12 months of diagnosis and for whom data were available with which to calculate index score were identified. Patients were classified as low, intermediate, or high risk using the prognostic index. The estimated median survival times were: not reached for low risk, 10.1 years for intermediate risk, and 7.2 years for high risk. The estimated median and 5-year survival by prognostic index risk category were similar to those originally reported. The prognostic index risk category added predictive value beyond that of Rai risk alone (P=.004). The prognostic index risk category remained a predictor of survival when analysis was limited to Rai stage 0 (P=.03) and nonreferred patients (P<.0001) and also predicted time to treatment (P<.0001). CONCLUSIONS: The results of the current study confirm the ability of a newly developed prognostic index to predict survival among patients with previously untreated CLL. The study also extended the utility of the index by demonstrating that it is useful at diagnosis, retains prognostic value when applied exclusively to Rai stage 0 patients, is effective in nonreferred patients, and predicts time to treatment.

Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup Phase III Trial E2997.

PURPOSE: Genomic features including unmutated immunoglobulin variable region heavy chain (IgVH) genes, del(11q22.3), del(17p13.1), and p53 mutations have been reported to predict the clinical course and overall survival of patients with chronic lymphocytic leukemia (CLL). In addition, ZAP-70 and Bcl-2 family proteins have been explored as predictors of outcome. PATIENTS AND METHODS: We prospectively evaluated the prognostic significance of a comprehensive panel of laboratory factors on both response and progression-free survival (PFS) using samples and data from 235 patients enrolled onto a therapeutic trial. Patients received either fludarabine (FL; n = 113) or fludarabine plus cyclophosphamide (FC; n = 122) as part of a US Intergroup randomized trial for previously untreated CLL patients. RESULTS: Complete response (CR) rates were 24.6% for patients receiving FC and 5.3% for patients receiving FL (P = .00004). PFS was statistically significantly longer in patients receiving FC (median, 33.5 months for patients receiving FC and 19.9 months for patients receiving FL; P < .0001). The occurrence of del(17p13.1) (hazard ratio, 3.428; P = .0002) or del(11q22.3) (hazard ratio, 1.904; P = .006) was associated with reduced PFS. CR and overall response rates were not significantly different based on cytogenetics, IgVH mutational status, CD38 expression, or p53 mutational status. Expression of ZAP-70, Bcl-2, Bax, Mcl-1, XIAP, Caspase-3, and Traf-1 was not associated with either clinical response or PFS. CONCLUSION: These results support the use of interphase cytogenetic analysis, but not IgVH, CD38 expression, or ZAP-70 status, to predict outcome of FL-based chemotherapy. Patients with high-risk cytogenetic features should be considered for alternative therapies.