Session: 641. Chronic Lymphocytic Leukemias: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Lymphoid Leukemias, CLL, assays, Diseases, Lymphoid Malignancies, Technology and Procedures, molecular testing
Methods: Subjects included 51 CLL patients and 12 healthy donors to obtain controls B-cell repertoires. BIOMED-2 PCR was performed according to the recommendation of the ERIC group. ARTISAN PCR was performed as earlier described by our group (Koning et al.). BIOMED-2 amplicons were sequenced by Sanger and ONT. ARTISAN PCR amplicons were sequenced by ONT. Internal NGS validation was performed by comparing ONT vs PacBio sequencing. A bioinformatic pipeline was developed for mutational status, clonal space and non-clonal B-cell repertoire characterization.
Results: We performed a pre-validation step in 10 samples by comparing BIOMED-2 amplicons subject to Sanger sequencing vs ARTISAN amplicons measured by ONT sequencing. There was a 100% agreement in VDJ identification and mutational status classification. The VH genes coverage was significantly higher with Nanopore vs Sanger sequencing (mean coverage= 99.3% vs 88.4%) with an error rate of 2.5 variants/kb.
We then compared the ARTISAN-ONT performance with BIOMED-2 Sanger in an expansion cohort. ARTISAN-ONT performed with an equivalent mutational status classification in 96% of the cases (49/51). The correlation of IGHV homology was r = 0.85, p <0.0001. The sensitivity of ARTISAN-Nanopore was 91% and the specificity was 100%. In the discordant cases both methods lead to the identification of distinct clonal IGHV rearrangements. By ONT sequencing these cases had a low read count that could account for this discrepancy.
Since ARTISAN-Nanopore allows the characterization of the full B-cell repertoire, we further analyzed its characteristics contrasting with the repertoire of healthy donors.
ARTISAN-Nanopore allowed the unequivocal detection of overexpressed clonal reads in all cases, healthy donors showed no evidence of clonal expansion. The Clonal Space (CS, i.e., proportion of clonal reads/non-clonal reads) was 0.66 for M-CLL and 0.71 for UM-CLL. One sample showed bi-clonality with two overexpressed clonal rearrangements.
Healthy donors showed an average repertoire richness of 1178 vs 2806 in M-CLL and 2817 in UM-CLL as assessed by 0D Hill Index. CLL showed a significant lower number of common rearrangements: 63 in UM-CLL, 31 in M-CLL as compared with 1076 in healthy donors. On the other hand, CLL cases showed higher mean aminoacidic charge and larger CDR3 mean length on non-clonal B-cells (charge 0.75 in M-CLL, 0.58 in UM-CLL vs 0.15 in healthy donors; CDR3 length 18.2 aa in M-CLL, 19.55 in UM-CLL and 15.8 in healthy donors). This data indicates disturbances in the diversity and composition of the remaining non-clonal B-cell repertoire in CLL.
Conclusion: Here we present an amplicon NGS-based strategy to determine the IGHV mutational status and the size of the occupied clonal space by the tumor clone, which also enables the characterization of the non-clonal B-cell repertoire in CLL patients. The strategy it fast, reliable, shows high specificity and sensitivity. This method relies on ONT sequencing, thus implies a relatively low investment being suitable for small laboratories around the world. This new methodology represents a cost-effective approach that should facilitate molecular testing availability in CLL.
Disclosures: No relevant conflicts of interest to declare.
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