Figure 2.
Figure 2. Analyses of clonality between CLL and ALL bone marrow samples. (A-B) NGS assessment was performed by Foundation One Heme, encompassing 406 genes and selected introns of 31 genes that are involved in the gene rearrangement and cancer related at the time of CLL and ALL diagnosis, respectively. DNA was extracted from BM samples (paraffin-embedded tissue) obtained at the diagnosis of CLL and ALL. Tumor mutational burden was measured by the number of somatic protein coding base substitution, insertion, and deletion mutations in the tumor specimen as part of NGS analyses. NGS showed an expansion of TP53L265D mutant clone. (C) For BCR gene rearrangement analysis, polymerase chain reaction (PCR) and capillary gel electrophoresis were adopted. DNA was extracted from paraffin-embedded BM cell clots and used in PCR amplification using Biomed-2 primers targeting all 3 immunoglobulin heavy chain (IgH) frameworks and immunoglobulin κ light chain (InVivoscribe Technologies) in fiver multiplex amplification tubes, each targeting conserved sequence in either framework (FR) 1, 2, or 3 of VH and DH in conjunction with a consensus (JH) primer in case of IgH. The primers for Vκ and Jκ, and Vκ and Kde are used for immunoglobulin κ light chain gene rearrangement. The PCR products were separated and detected by capillary gel electrophoresis on the ABI 3130x1. Blue and green peaks are PCR products amplified in CLL and ALL samples. Red peaks in the background stand for internal standard for molecular weight. BCR gene rearrangement analysis showed an identical monoclonal peak at molecular weight of 147.97 (black arrows). (D) Paraffin-embedded BM cell clots were obtained from the patient at diagnosis of CLL and B-ALL and sent to Adaptive Biotechnologies (Seattle, WA) for immunoglobulin heavy and light chain (VDJ, DJ, IgK) high-throughput sequencing using ClonoSeq assay to compare the sequence differences in clonal BCR gene rearrangements. The frequency of the most common 4 sequences (blue bars) identified on ClonoSeq analyses are presented. They are marked as first, second, third, and fourth in each CLL (upper) and ALL (bottom) sample. ClonoSeq confirmed identical IgH sequences of BCR gene rearrangement segments between CLL and ALL samples. (E) Sequencing of CXCR4 and ALK genes in CLL and ALL showed C-to-T mutation.

Analyses of clonality between CLL and ALL bone marrow samples. (A-B) NGS assessment was performed by Foundation One Heme, encompassing 406 genes and selected introns of 31 genes that are involved in the gene rearrangement and cancer related at the time of CLL and ALL diagnosis, respectively. DNA was extracted from BM samples (paraffin-embedded tissue) obtained at the diagnosis of CLL and ALL. Tumor mutational burden was measured by the number of somatic protein coding base substitution, insertion, and deletion mutations in the tumor specimen as part of NGS analyses. NGS showed an expansion of TP53L265D mutant clone. (C) For BCR gene rearrangement analysis, polymerase chain reaction (PCR) and capillary gel electrophoresis were adopted. DNA was extracted from paraffin-embedded BM cell clots and used in PCR amplification using Biomed-2 primers targeting all 3 immunoglobulin heavy chain (IgH) frameworks and immunoglobulin κ light chain (InVivoscribe Technologies) in fiver multiplex amplification tubes, each targeting conserved sequence in either framework (FR) 1, 2, or 3 of VH and DH in conjunction with a consensus (JH) primer in case of IgH. The primers for Vκ and Jκ, and Vκ and Kde are used for immunoglobulin κ light chain gene rearrangement. The PCR products were separated and detected by capillary gel electrophoresis on the ABI 3130x1. Blue and green peaks are PCR products amplified in CLL and ALL samples. Red peaks in the background stand for internal standard for molecular weight. BCR gene rearrangement analysis showed an identical monoclonal peak at molecular weight of 147.97 (black arrows). (D) Paraffin-embedded BM cell clots were obtained from the patient at diagnosis of CLL and B-ALL and sent to Adaptive Biotechnologies (Seattle, WA) for immunoglobulin heavy and light chain (VDJ, DJ, IgK) high-throughput sequencing using ClonoSeq assay to compare the sequence differences in clonal BCR gene rearrangements. The frequency of the most common 4 sequences (blue bars) identified on ClonoSeq analyses are presented. They are marked as first, second, third, and fourth in each CLL (upper) and ALL (bottom) sample. ClonoSeq confirmed identical IgH sequences of BCR gene rearrangement segments between CLL and ALL samples. (E) Sequencing of CXCR4 and ALK genes in CLL and ALL showed C-to-T mutation.

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