Quantification of Intraclonal Diversity in Follicular Lymphoma
Janice M Spence, John P Spence, W Richard Burack. University of Rochester School of Medicine and Dentistry, Rochester, NY
Background: All cancers are characterized by genomic instability, and it is hypothesized that tumors with greater clonal diversity are more likely to transform and evolve resistance to chemotherapy. Intra-tumor heterogeneity in follicular lymphoma (FL) has been demonstrated by clone-specific IGH sequence analysis. We have developed a next generation based ultra-deep sequencing approach that allows the quantification of sub-clonal populations within a tumor, detecting subclones as rare as 1 cell in 500. Using FL, we show that this approach can quantify intraclonal diversity.
Design: PCR-based targeted re-sequencing was performed on 12 FL specimens, 3 hyperplastic lymph nodes (NL) and 1 cell line using SOLiD sequencing at ultra-deep levels, averaging > 10,000-fold coverage. Ten regions were analyzed, including promoters from 5 proto-oncogenes (Bcl6, Myc, Pax5, Pim1, RhoH and syk), Bcl2, CD83, and several regions of IGH (Em-J6 and clone-specific IGH Vh-J6), comprising ∼20 kb. Sequencing data was analyzed using a novel filtering algorithm based on both minimum frequency of a sequence and the ability to perform limited sequence assembly.
Results: Combining the 10 non-Vh genes in each FL, a total of 869 mutations were detected (median of 55, range 28-212/case). 11 mutations in were identified in a tetraploid cell line control. The ultra-deep method estimates the relative abundance of cells carrying each mutation. Over 52% of mutations were present in < 25% of cells, the fraction generally used as the lower limit of detection for Sanger sequencing. 141 mutations were present in less than 2% of tumor cells; each FL had >2 mutations indicating one or more subpopulations comprising less than 2% of cells (range 1 – 3). Of the 10 genes assessed, the promoter region of Bcl2 was particularly mutated, accounting for 46% of all non-Vh mutations (median 25, range 4-110). Allele specific sequencing found that the Bcl2 \mutations appear to be restricted to a single allele in each individual. The number of Bcl2 mutations was predictive of total mutations (R2= 0.64).
Conclusions: All cases of FL were shown to include rare sub populations represented by less than 2% of cells. The number of mutations in the Bcl2 promoter correlates with the number detected in all other regions combined. There is significant variability between FL specimens in the number of rare mutations (those mutations present in less than 10% of sequences). These results allow stratification of FL cases into 4 groups based on the total number of mutations.
Monday, March 19, 2012 11:15 AM
Platform Session: Section C, Monday Morning