Table 1.
Summary of CLL patient characteristics a.
Table 2.
Frequencies of CLL patients grouped by number of dominant IGH rearrangements.
Fig 1.
Flow chart for molecular analysis of CLL patients.
(1) CDR3 analysis was initially performed to screen for patients exhibiting more than one dominant CDR3 peak. (2) Clonotypic sequences corresponding to dominant CDR3 peaks were characterized. Primers were designed based on unique sequences on the CDRs and were tested for specificity. (3) CLL cells were sorted in aliquots of 1, 10 or 100 cells and clonal analysis was performed by nested PCR using clone-specific primers. (4) Selected genomic DNA samples were subjected to next-generation IGH sequencing.
Fig 2.
SCA identifies clonal origin of multiple rearranged heavy chain genes.
(A) A representative result of biallelic rearrangements derived from single B-cell clone is shown in patient CLL-2. Each column represents nested PCR results of the same single cells. (B) Detection of clonal specific sequences in non-overlapping B-cell populations characterized biclonality or multiple clones (multiclonality). Biclonal or multiclonal B-cells comprised both monoallelic and biallelic clones. The number of single cells analyzed in each patient is larger than the number shown here. Analyses of 10-cell aliquots are shown in CLL-129 and CLL-105 to demonstrate and confirm the existence of clones that were infrequent. SC, single cell.
Table 3.
Characterization of rearranged IGHV-IGHD-IGHJ genes in U-CLL patients having two dominant IGH rearrangements.
Table 4.
Characterization of rearranged IGHV-IGHD-IGHJ genes in M-CLL patients having two or more dominant IGH rearrangements and corresponding B-cell clones.
Table 5.
Longitudinal analysis of IGH biclonality in U-CLL, as determined by SCA.
Table 6.
Longitudinal analysis of IGH biclonal and multiclonal diversity in M-CLL, as determined by SCA.
Fig 3.
Multiclonality is frequently observed in M-CLL.
IGH sequence frequencies were characterized by next-generation IGH sequencing and are plotted on log scale from 0.1–100%. Samples included a) seven CLL patients characterized as having more than one clone by SCA (U-CLL: CLL-67, CLL-100; M-CLL: CLL-43, CLL-105, CLL-112, CLL129, CLL200), b) six typical CLL with single B-cell clone (U-CLL: CLL-102, CLL-106, CLL-110; M-CLL: CLL108, CLL-127, CLL-184), c) three healthy donors (N1, N2, N3), and d) three MM and two WM patients in whom two B-cell clones were previously reported (MM_PT3, MM_PT4, MM_PT5, WM1-09 and WM1-19) [19, 20]. For sample N3, top frequencies were ≤0.035%, thus were placed outside of the y-axis for reference only, not to scale. An arbitrary cutoff line was drawn at the highest frequency found in HD. Dominant clones in CLL are defined as those with frequencies above the cutoff line. The number of dominant clones for each sample is shown on the right. Closed circle, clone identified by both ImmunoSEQ and SCA; open circle, clone identified only by ImmunoSEQ.
Table 7.
Comparison of clonal frequencies estimated by SCA and ImmunoSEQ NGS.