Contributions
Abstract: EP626
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Mutational status of the IGHV genes, IGHV gene family usage, and B-cell receptor stereotype are the key prognostic factors for CLL. However, the routinely used Sanger sequencing technique may fail for the cases with an expansion of more than one tumor B-cell clones, especially with rearranged genes of the same IGHV family (up to 4% CLL cases)
Aims
To evaluate the robustness of NGS technology for IGHV analysis in complicated CLL cases.
Methods
DNA samples from CLL patients (N=26) for whom we previously failed to obtain IGHV rearrangements data by Sanger sequencing included in the study. IGHV loci were PCR amplified as described in BIOMED-2 protocol and by Campbell et al. Sequencing libraries were prepared with Nextera XT kit (Illumina, USA) according to the manufacturer's recommendations and were analyzed with MiSeq Genome Analyzer (Illumina, USA). Sequencing data were processed via Mixcr software (Bolotin et al. https://github.com/milaboratory/mixcr). The mutational status and receptor stereotype were identified using IMGT and ARRest databases according to ERIC recommendations.
Results
In 20 cases two clonal rearrangements were identified; in 18 cases IGHV genes of the same family were used in both clones (IGHV3 for 14 patients and IGHV1 for 4). One patient had IGHV1-69 и IGHV7-4 genes rearrangements (these two families are usually amplified with the same primer). One more patient had IGHV1-69 and IGHV3-73. In 19 cases only one rearrangement was productive, and one patient had two productive IGHV rearrangements. Herewith the mutational status did not differ within the rearrangement pairs. Thus, we determined U-CLL for 16 patients and M-CLL for 4 patients. Besides, two patients with stereotyped receptor CLL#2 and one patient with CLL#1 were found in U-CLL group. Three patients were found with only one productive clonal rearrangement. The heterogeneity of Sanger sequencing in these cases was presumably related to an insufficient amount of tumor cells in the sample comparing with a high polyclonal background. Three patients had three clonal rearrangements. In two cases all rearrangements were productive (IGHV3 and IGHV4 families) and highly mutated. One of these patients had two malignancies - CLL and mantle cell lymphoma. Both these two cases were classified as M-CLL. The third case demonstrated the major productive rearrangement (91% clones) with unmutated IGHV3-53 gene and two small (<5%) unmutated clones with IGHV1-69 (productive) and IGHV3-74 (unproductive).
Conclusion
Using NGS approach we have shown that in the cases with two IGHV gene rearrangements from the same VH gene family, variants with IGHV3 family are the most common (77,7%). U-CLL variants were also prevalent. Though Sanger sequencing is still more suitable for routine IGHV gene analysis in CLL, a significant number of complicated cases might benefit from NGS technology, therefore, allow to obtain definite diagnosis and assign appropriate treatment.
Keyword(s): IgH rearrangment, Mutation status, Prognosis
Abstract: EP626
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Mutational status of the IGHV genes, IGHV gene family usage, and B-cell receptor stereotype are the key prognostic factors for CLL. However, the routinely used Sanger sequencing technique may fail for the cases with an expansion of more than one tumor B-cell clones, especially with rearranged genes of the same IGHV family (up to 4% CLL cases)
Aims
To evaluate the robustness of NGS technology for IGHV analysis in complicated CLL cases.
Methods
DNA samples from CLL patients (N=26) for whom we previously failed to obtain IGHV rearrangements data by Sanger sequencing included in the study. IGHV loci were PCR amplified as described in BIOMED-2 protocol and by Campbell et al. Sequencing libraries were prepared with Nextera XT kit (Illumina, USA) according to the manufacturer's recommendations and were analyzed with MiSeq Genome Analyzer (Illumina, USA). Sequencing data were processed via Mixcr software (Bolotin et al. https://github.com/milaboratory/mixcr). The mutational status and receptor stereotype were identified using IMGT and ARRest databases according to ERIC recommendations.
Results
In 20 cases two clonal rearrangements were identified; in 18 cases IGHV genes of the same family were used in both clones (IGHV3 for 14 patients and IGHV1 for 4). One patient had IGHV1-69 и IGHV7-4 genes rearrangements (these two families are usually amplified with the same primer). One more patient had IGHV1-69 and IGHV3-73. In 19 cases only one rearrangement was productive, and one patient had two productive IGHV rearrangements. Herewith the mutational status did not differ within the rearrangement pairs. Thus, we determined U-CLL for 16 patients and M-CLL for 4 patients. Besides, two patients with stereotyped receptor CLL#2 and one patient with CLL#1 were found in U-CLL group. Three patients were found with only one productive clonal rearrangement. The heterogeneity of Sanger sequencing in these cases was presumably related to an insufficient amount of tumor cells in the sample comparing with a high polyclonal background. Three patients had three clonal rearrangements. In two cases all rearrangements were productive (IGHV3 and IGHV4 families) and highly mutated. One of these patients had two malignancies - CLL and mantle cell lymphoma. Both these two cases were classified as M-CLL. The third case demonstrated the major productive rearrangement (91% clones) with unmutated IGHV3-53 gene and two small (<5%) unmutated clones with IGHV1-69 (productive) and IGHV3-74 (unproductive).
Conclusion
Using NGS approach we have shown that in the cases with two IGHV gene rearrangements from the same VH gene family, variants with IGHV3 family are the most common (77,7%). U-CLL variants were also prevalent. Though Sanger sequencing is still more suitable for routine IGHV gene analysis in CLL, a significant number of complicated cases might benefit from NGS technology, therefore, allow to obtain definite diagnosis and assign appropriate treatment.
Keyword(s): IgH rearrangment, Mutation status, Prognosis