Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison

Abstract

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Figure 1

Comparison of platforms and primers using 35 validation chronic lymphocytic leukemia/small lymphocytic lymphoma samples. A: Summary of sequencing results by framework region (FR) 1 PGM, FR1 MiSeq, leader MiSeq, and the reference method. The bottom panel shows color-coded qualitative results as explained in the key. The top panel shows bar graphs corresponding to the percentage of total reads for each clonal sequence detected. B: The detection of clonal sequences using the FR1 primer set with PGM versus MiSeq platforms was compared. C: The MiSeq platform was used to compare output by the FR1 and leader primer sets (excluding case 1, which failed sequencing by leader). Major discordant results were seen when there was a failure by one assay and not others (case 1 failed sequencing by leader based on the total reads), when the dominant sequence did not meet quantitative criteria above background for unequivocal clonotype reporting, or if the identified clonotype showed significant qualitative differences [productive versus unproductive or mutated (+) versus unmutated (0)]. Exact results were seen when the sequence identity and the number of sequences detected that fulfilled criteria for clonality were identical. Cases with minor discordance showed additional minor clonal sequences that met clonality criteria by one primer set or platform and not the other, and these are designated as detected extra sequences. F, failure; ND, not detected; NR, not reported.

Figure 2

Validation study: comparison of mutation rates by framework region (FR) 1 and leader primer sets for all matching clonotypes (n = 46) identified by sequencing on the MiSeq platform. A: Scatterplot showing the correlation and linear regression analysis of mutation rates (r = 0.9918; 95% CI, 0.9852 to 0.9955; R² = 0.9837; P < 0.0001). The best fit linear regression line (black line) and 95% CIs (red dashed lines) are showns. B: Bland-Altman plot showing a difference versus mean analysis of the mutation rates identified by the two primer sets (FR1 versus leader) with a mean ± bias of 0.326 ± 0.6644 and 95% limits of agreement of −0.79 to 1.44 (red dotted lines). The green line represents if the difference between the assays was zero (0). C: Histogram showing frequency distribution of mutation rates grouped by bins: 0%, 0.1% to 1.4%, 1.5% to 1.9%, 2.0% to 3.0%, and >3.1%. The two sets of bars correspond to results obtained by leader (black bars) and FR1 (gray bars) primer sets. D: Clinical implementation: histogram showing frequency distribution of mutation rates grouped by bins as in C. The mutation rate bins of all productive rearrangements detected in samples by leader or FR1 sequencing (435 productive clonotypes in 423 samples) are shown. Among the 228 productive rearrangements by leader and 207 by FR1 primer sets, the distribution of the mutation rates showed no statistically significant differences (χ²). Clonotypes with borderline somatic hypermutation rate (2.0% to 3.0%) account for a small proportion of the total productive rearrangements [overall, 3.4% (n = 15/435)], detected at similar rates by both primer sets: 2.6% (n = 6/228) for leader and 3.4% (n = 7/207) for FR1.

Figure 3

Flowchart of the analysis strategy of the 423 clinical chronic lymphocytic leukemia/small lymphocytic lymphoma samples evaluated by the framework region (FR) 1 and leader primer sets on the MiSeq platform. Thirty-six challenging samples were evaluated by both primer sets and based on the success of the analysis as outlined above; cases were reassigned to the alternate primer set when appropriate. The bottom row indicates whether successful identification of at least one productive clonal sequence per case was observed. Asterisk includes one case (case 98 in Figure 4) in which the reflexed leader analysis was not informative because it did not detect a major clone (polyclonality) and was discordant from FR1 results.

Figure 4

Thirty-six cases from the clinical implementation cohort evaluated by both framework region (FR) 1 and leader primer sets. Summary of sequencing results by FR1 MiSeq and leader MiSeq. The bottom panel shows color-coded qualitative results as explained in the key. The top panels show bar graphs corresponding to the percentage of total reads for each clonal sequence detected by the two primer sets. The numbers at the top of each column correspond to the unique sample identifier. F, failure; ND, not detected; NR, not reported.