INTEGRATIVE ANALYSIS OF GENE MUTATIONS AND CHROMOSOMAL ABNORMALITIES BY NEXT-GENERATION SEQUENCING AND FISH IN HEMATOPOIETIC PROGENITORS OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Hernandez Sanchez M. 06/10/16; 135148; S115
Maria Hernandez Sanchez
Contributions
Contributions
Abstract
Abstract: S115
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Auditorium 2
Background
Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent FISH and Next-Generation Sequencing (NGS) studies have shown that the genetic alterations may appear in CD34+ early hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.
Aims
To analyze both chromosomal abnormalities and gene mutations in CD34+ hematopoietic progenitors from CLL patients to elucidate whether these genetic events appear previously during the B-cell differentiation process.
Methods
NGS and FISH studies were carried out in isolated CD19+ mature B cells from 57 CLL bone marrow samples to assess the presence of gene mutations and/or cytogenetic aberrations, respectively. Amplicon-based deep NGS (454 Life Sciences, Roche) was performed to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88 and XPO1. The presence of genetic lesions on 11q, 12, 13q, 14q and 17p was studied by FISH. All cases with any mutations in CD19+ cells were also sequenced in their corresponding CD34+ early progenitors (coverage=1159X). Moreover, FISH was also performed in the CD34+ fraction of a subset of CLL patients with cytogenetic changes (n=9).
Results
NGS studies revealed a total of 28 mutations in 24 CLL patients (24/57, 42.1%). NOTCH1 was the most frequently mutated gene (22.8%), followed by XPO1 (8.8%), SF3B1 (7%), FBXW7 (5.3%), TP53 (3.5%) and MYD88 (1.8%). CLLs with mutations in any of these genes, except to MYD88, were associated with an unmutated IGHV status (P=0.001). Most of the patients showed the same mutations in their corresponding CD34+ cells (21/24, 87.5%). Comparing the mutations in both cell populations, two different patterns could be identified. In one group, the mutational burden remained similar between both cell fractions. Mutations in NOTCH1 (11/13) and XPO1 (5/5) were predominant in this group. By contrast, the other group showed a high decrease or even absence of these mutations in the CD34+ cells than the corresponding B lymphocytes being mutations in TP53 (2/2), FBXW7 (2/3) and SF3B1 (3/4) the most recurrent in this group. FISH analysis revealed that 5 out of 9 CLL cases with cytogenetic alterations in B mature lymphocytes presented also the same aberrations on the CD34+ cells. Of note, IGH alterations did not appear in any of the CD34+ cases (0/2), whereas 11q- (2/2) and 13q- (3/5) were present in the CD34+ early progenitors. Gathering both techniques, 2 out of 9 CLLs had both chromosomal aberrations and mutations in their mature lymphocytes. Noteworthy, one of these cases showed an IGH alteration and NOTCH1 mutation (altered cells=80% and 51%, respectively) on the CD19+ population. However, only NOTCH1 mutation (31%) was found on the CD34+ cells. The other patient presented 13q- (93%), 11q- (64%) and XPO1 mutation (39%) on the CD19+ B cells. The same alterations were also present in hematopoietic progenitors (72%, 49% and 31%, respectively).
Conclusion
Our data confirmed that chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes, but also in the CD34+ early progenitors of CLL patients. Moreover, our findings shed light about the hierarchy existing in the appearance of these events: 11q-, 13q-, NOTCH1 and XPO1 mutations could be early hits on CLL pathogenesis, whereas IGH alterations, TP53, SF3B1 and FBXW7 mutations seemed to show in a later maturational level. Funding: PI15/01471; Junta de Castilla y León (MHS)
Session topic: CLL natural history and progression
Keyword(s): CD34+ cells, Chronic lymphocytic leukemia, Fluorescence in situ hybridization, Mutation analysis
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Auditorium 2
Background
Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent FISH and Next-Generation Sequencing (NGS) studies have shown that the genetic alterations may appear in CD34+ early hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.
Aims
To analyze both chromosomal abnormalities and gene mutations in CD34+ hematopoietic progenitors from CLL patients to elucidate whether these genetic events appear previously during the B-cell differentiation process.
Methods
NGS and FISH studies were carried out in isolated CD19+ mature B cells from 57 CLL bone marrow samples to assess the presence of gene mutations and/or cytogenetic aberrations, respectively. Amplicon-based deep NGS (454 Life Sciences, Roche) was performed to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88 and XPO1. The presence of genetic lesions on 11q, 12, 13q, 14q and 17p was studied by FISH. All cases with any mutations in CD19+ cells were also sequenced in their corresponding CD34+ early progenitors (coverage=1159X). Moreover, FISH was also performed in the CD34+ fraction of a subset of CLL patients with cytogenetic changes (n=9).
Results
NGS studies revealed a total of 28 mutations in 24 CLL patients (24/57, 42.1%). NOTCH1 was the most frequently mutated gene (22.8%), followed by XPO1 (8.8%), SF3B1 (7%), FBXW7 (5.3%), TP53 (3.5%) and MYD88 (1.8%). CLLs with mutations in any of these genes, except to MYD88, were associated with an unmutated IGHV status (P=0.001). Most of the patients showed the same mutations in their corresponding CD34+ cells (21/24, 87.5%). Comparing the mutations in both cell populations, two different patterns could be identified. In one group, the mutational burden remained similar between both cell fractions. Mutations in NOTCH1 (11/13) and XPO1 (5/5) were predominant in this group. By contrast, the other group showed a high decrease or even absence of these mutations in the CD34+ cells than the corresponding B lymphocytes being mutations in TP53 (2/2), FBXW7 (2/3) and SF3B1 (3/4) the most recurrent in this group. FISH analysis revealed that 5 out of 9 CLL cases with cytogenetic alterations in B mature lymphocytes presented also the same aberrations on the CD34+ cells. Of note, IGH alterations did not appear in any of the CD34+ cases (0/2), whereas 11q- (2/2) and 13q- (3/5) were present in the CD34+ early progenitors. Gathering both techniques, 2 out of 9 CLLs had both chromosomal aberrations and mutations in their mature lymphocytes. Noteworthy, one of these cases showed an IGH alteration and NOTCH1 mutation (altered cells=80% and 51%, respectively) on the CD19+ population. However, only NOTCH1 mutation (31%) was found on the CD34+ cells. The other patient presented 13q- (93%), 11q- (64%) and XPO1 mutation (39%) on the CD19+ B cells. The same alterations were also present in hematopoietic progenitors (72%, 49% and 31%, respectively).
Conclusion
Our data confirmed that chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes, but also in the CD34+ early progenitors of CLL patients. Moreover, our findings shed light about the hierarchy existing in the appearance of these events: 11q-, 13q-, NOTCH1 and XPO1 mutations could be early hits on CLL pathogenesis, whereas IGH alterations, TP53, SF3B1 and FBXW7 mutations seemed to show in a later maturational level. Funding: PI15/01471; Junta de Castilla y León (MHS)
Session topic: CLL natural history and progression
Keyword(s): CD34+ cells, Chronic lymphocytic leukemia, Fluorescence in situ hybridization, Mutation analysis
Abstract: S115
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Auditorium 2
Background
Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent FISH and Next-Generation Sequencing (NGS) studies have shown that the genetic alterations may appear in CD34+ early hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.
Aims
To analyze both chromosomal abnormalities and gene mutations in CD34+ hematopoietic progenitors from CLL patients to elucidate whether these genetic events appear previously during the B-cell differentiation process.
Methods
NGS and FISH studies were carried out in isolated CD19+ mature B cells from 57 CLL bone marrow samples to assess the presence of gene mutations and/or cytogenetic aberrations, respectively. Amplicon-based deep NGS (454 Life Sciences, Roche) was performed to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88 and XPO1. The presence of genetic lesions on 11q, 12, 13q, 14q and 17p was studied by FISH. All cases with any mutations in CD19+ cells were also sequenced in their corresponding CD34+ early progenitors (coverage=1159X). Moreover, FISH was also performed in the CD34+ fraction of a subset of CLL patients with cytogenetic changes (n=9).
Results
NGS studies revealed a total of 28 mutations in 24 CLL patients (24/57, 42.1%). NOTCH1 was the most frequently mutated gene (22.8%), followed by XPO1 (8.8%), SF3B1 (7%), FBXW7 (5.3%), TP53 (3.5%) and MYD88 (1.8%). CLLs with mutations in any of these genes, except to MYD88, were associated with an unmutated IGHV status (P=0.001). Most of the patients showed the same mutations in their corresponding CD34+ cells (21/24, 87.5%). Comparing the mutations in both cell populations, two different patterns could be identified. In one group, the mutational burden remained similar between both cell fractions. Mutations in NOTCH1 (11/13) and XPO1 (5/5) were predominant in this group. By contrast, the other group showed a high decrease or even absence of these mutations in the CD34+ cells than the corresponding B lymphocytes being mutations in TP53 (2/2), FBXW7 (2/3) and SF3B1 (3/4) the most recurrent in this group. FISH analysis revealed that 5 out of 9 CLL cases with cytogenetic alterations in B mature lymphocytes presented also the same aberrations on the CD34+ cells. Of note, IGH alterations did not appear in any of the CD34+ cases (0/2), whereas 11q- (2/2) and 13q- (3/5) were present in the CD34+ early progenitors. Gathering both techniques, 2 out of 9 CLLs had both chromosomal aberrations and mutations in their mature lymphocytes. Noteworthy, one of these cases showed an IGH alteration and NOTCH1 mutation (altered cells=80% and 51%, respectively) on the CD19+ population. However, only NOTCH1 mutation (31%) was found on the CD34+ cells. The other patient presented 13q- (93%), 11q- (64%) and XPO1 mutation (39%) on the CD19+ B cells. The same alterations were also present in hematopoietic progenitors (72%, 49% and 31%, respectively).
Conclusion
Our data confirmed that chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes, but also in the CD34+ early progenitors of CLL patients. Moreover, our findings shed light about the hierarchy existing in the appearance of these events: 11q-, 13q-, NOTCH1 and XPO1 mutations could be early hits on CLL pathogenesis, whereas IGH alterations, TP53, SF3B1 and FBXW7 mutations seemed to show in a later maturational level. Funding: PI15/01471; Junta de Castilla y León (MHS)
Session topic: CLL natural history and progression
Keyword(s): CD34+ cells, Chronic lymphocytic leukemia, Fluorescence in situ hybridization, Mutation analysis
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Auditorium 2
Background
Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent FISH and Next-Generation Sequencing (NGS) studies have shown that the genetic alterations may appear in CD34+ early hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.
Aims
To analyze both chromosomal abnormalities and gene mutations in CD34+ hematopoietic progenitors from CLL patients to elucidate whether these genetic events appear previously during the B-cell differentiation process.
Methods
NGS and FISH studies were carried out in isolated CD19+ mature B cells from 57 CLL bone marrow samples to assess the presence of gene mutations and/or cytogenetic aberrations, respectively. Amplicon-based deep NGS (454 Life Sciences, Roche) was performed to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88 and XPO1. The presence of genetic lesions on 11q, 12, 13q, 14q and 17p was studied by FISH. All cases with any mutations in CD19+ cells were also sequenced in their corresponding CD34+ early progenitors (coverage=1159X). Moreover, FISH was also performed in the CD34+ fraction of a subset of CLL patients with cytogenetic changes (n=9).
Results
NGS studies revealed a total of 28 mutations in 24 CLL patients (24/57, 42.1%). NOTCH1 was the most frequently mutated gene (22.8%), followed by XPO1 (8.8%), SF3B1 (7%), FBXW7 (5.3%), TP53 (3.5%) and MYD88 (1.8%). CLLs with mutations in any of these genes, except to MYD88, were associated with an unmutated IGHV status (P=0.001). Most of the patients showed the same mutations in their corresponding CD34+ cells (21/24, 87.5%). Comparing the mutations in both cell populations, two different patterns could be identified. In one group, the mutational burden remained similar between both cell fractions. Mutations in NOTCH1 (11/13) and XPO1 (5/5) were predominant in this group. By contrast, the other group showed a high decrease or even absence of these mutations in the CD34+ cells than the corresponding B lymphocytes being mutations in TP53 (2/2), FBXW7 (2/3) and SF3B1 (3/4) the most recurrent in this group. FISH analysis revealed that 5 out of 9 CLL cases with cytogenetic alterations in B mature lymphocytes presented also the same aberrations on the CD34+ cells. Of note, IGH alterations did not appear in any of the CD34+ cases (0/2), whereas 11q- (2/2) and 13q- (3/5) were present in the CD34+ early progenitors. Gathering both techniques, 2 out of 9 CLLs had both chromosomal aberrations and mutations in their mature lymphocytes. Noteworthy, one of these cases showed an IGH alteration and NOTCH1 mutation (altered cells=80% and 51%, respectively) on the CD19+ population. However, only NOTCH1 mutation (31%) was found on the CD34+ cells. The other patient presented 13q- (93%), 11q- (64%) and XPO1 mutation (39%) on the CD19+ B cells. The same alterations were also present in hematopoietic progenitors (72%, 49% and 31%, respectively).
Conclusion
Our data confirmed that chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes, but also in the CD34+ early progenitors of CLL patients. Moreover, our findings shed light about the hierarchy existing in the appearance of these events: 11q-, 13q-, NOTCH1 and XPO1 mutations could be early hits on CLL pathogenesis, whereas IGH alterations, TP53, SF3B1 and FBXW7 mutations seemed to show in a later maturational level. Funding: PI15/01471; Junta de Castilla y León (MHS)
Session topic: CLL natural history and progression
Keyword(s): CD34+ cells, Chronic lymphocytic leukemia, Fluorescence in situ hybridization, Mutation analysis
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