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High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Carlotti E, Wrench D, Rosignoli G, Marzec J, Sangaralingam A, Hazanov L, Michaeli M, Hallam S, Chaplin T, Iqbal S, Calaminici M, Young B, Mehr R, Campbell P, Fitzgibbon J, Gribben JG - PLoS ONE (2015)

Bottom Line: The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs.By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate.The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

View Article: PubMed Central - PubMed

Affiliation: Centre for Haemato-Oncology, Barts Cancer Institute - a CR-UK Centre Of Excellence, Queen Mary University of London, London, United Kingdom.

ABSTRACT
Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

No MeSH data available.


Related in: MedlinePlus

Flow-sorting of the 4 different B cell sub-populations.Lymph nodes cell suspensions from sequential biopsies obtained from patients with FL/t-FL were stained and sorted in 4 different populations according to the expression of IgD, CD38, CD10 and CXCR4. ME: IgD-CD38-; PGC: IgD+CD38+, CB: IgD-CD38+CD10+CXCR4+; CC: IgD-CD38+CD10+CXCR4-. Flow-cytometric identification of the 4 sub-populations in the sample R8403, patient 3 (top), sample R0012, patient 1 (middle) and tonsils (bottom). In the 2 patients samples the numbers indicate the percentage of the gated and sorted subsets whilst in the tonsil example the numbers are representative of 8 independent experiments on as many different biopsies.
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pone.0134833.g002: Flow-sorting of the 4 different B cell sub-populations.Lymph nodes cell suspensions from sequential biopsies obtained from patients with FL/t-FL were stained and sorted in 4 different populations according to the expression of IgD, CD38, CD10 and CXCR4. ME: IgD-CD38-; PGC: IgD+CD38+, CB: IgD-CD38+CD10+CXCR4+; CC: IgD-CD38+CD10+CXCR4-. Flow-cytometric identification of the 4 sub-populations in the sample R8403, patient 3 (top), sample R0012, patient 1 (middle) and tonsils (bottom). In the 2 patients samples the numbers indicate the percentage of the gated and sorted subsets whilst in the tonsil example the numbers are representative of 8 independent experiments on as many different biopsies.

Mentions: Four different sub-populations were flow-sorted from reactive tonsils and FL biopsies cryopreserved cell suspensions and fluorescently labeled using the monoclonal antibodies: CD38-PE-Cy7 (clone HB7), CD10-PE (clone HI10a), CXCR4 PE-Cy5 (clone 12G5), IgD biotin (clone IA6-2), (Becton Dickinson; San Jose, CA, USA), CD77 FITC (clone 5B5) and anti-biotin APC (clone Bio3-18E7) (Miltenyi Biotec). Non-viable cells were excluded by simultaneously staining with DAPI (Sigma-Aldrich). Gates to distinguish the different subsets were set using cells suspensions from tonsils and the fluorescence minus one control. By flow sorting using a FACS Aria II (Becton Dickinson) fluorescently labeled cells were separated into four different subsets (Fig 2). Data acquisition and analysis was performed using DIVA software, version 6.13 (Becton Dickinson). The average purity in each sub-population was >90%. A further characterization of these populations was performed by staining with CD27 APC (clone M-T271) and CD20 FITC (clone 2H7) antibodies (Becton Dickinson; San Jose, CA, USA), cell cycle analysis, and Real-Time PCR analysis of AICDA and mir155h6 expression (S1A–S1E Fig). Briefly, cell cycle analysis was carried out on frozen cell suspensions from tonsils, using the fixation and permeabilization buffers (eBioscience) and DAPI staining according to the commercial protocol (cells were re-suspended in 1.5mL of 2% FBS/PBS and 3μl of DAPI). RNA was extracted from tonsils and primary FL cell suspensions and the lymphoblastoid cell line NcNc, using the commercial kit RNAeasy Mini Kit (Qiagen). Standard cDNA synthesis was performed using the SuperscriptII Reverse Transcriptase and Random Primers (Invitrogen), according to the manufacturer's instructions and using 100 ng of RNA per reaction. Predesigned TaqMan Gene Expression assays for AICDA, mir155h6 and 18S genes were obtained from Applied Biosystem. 18S was used as endogenous control. Both the target and the control genes were run in duplicate. The quantitative PCR was performed using the Taqman 7900HT Fast Real Time PCR System and the recommended manufacturer’s conditions. Gene expression values were then calculated using the ΔΔCt method.


High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Carlotti E, Wrench D, Rosignoli G, Marzec J, Sangaralingam A, Hazanov L, Michaeli M, Hallam S, Chaplin T, Iqbal S, Calaminici M, Young B, Mehr R, Campbell P, Fitzgibbon J, Gribben JG - PLoS ONE (2015)

Flow-sorting of the 4 different B cell sub-populations.Lymph nodes cell suspensions from sequential biopsies obtained from patients with FL/t-FL were stained and sorted in 4 different populations according to the expression of IgD, CD38, CD10 and CXCR4. ME: IgD-CD38-; PGC: IgD+CD38+, CB: IgD-CD38+CD10+CXCR4+; CC: IgD-CD38+CD10+CXCR4-. Flow-cytometric identification of the 4 sub-populations in the sample R8403, patient 3 (top), sample R0012, patient 1 (middle) and tonsils (bottom). In the 2 patients samples the numbers indicate the percentage of the gated and sorted subsets whilst in the tonsil example the numbers are representative of 8 independent experiments on as many different biopsies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556522&req=5

pone.0134833.g002: Flow-sorting of the 4 different B cell sub-populations.Lymph nodes cell suspensions from sequential biopsies obtained from patients with FL/t-FL were stained and sorted in 4 different populations according to the expression of IgD, CD38, CD10 and CXCR4. ME: IgD-CD38-; PGC: IgD+CD38+, CB: IgD-CD38+CD10+CXCR4+; CC: IgD-CD38+CD10+CXCR4-. Flow-cytometric identification of the 4 sub-populations in the sample R8403, patient 3 (top), sample R0012, patient 1 (middle) and tonsils (bottom). In the 2 patients samples the numbers indicate the percentage of the gated and sorted subsets whilst in the tonsil example the numbers are representative of 8 independent experiments on as many different biopsies.
Mentions: Four different sub-populations were flow-sorted from reactive tonsils and FL biopsies cryopreserved cell suspensions and fluorescently labeled using the monoclonal antibodies: CD38-PE-Cy7 (clone HB7), CD10-PE (clone HI10a), CXCR4 PE-Cy5 (clone 12G5), IgD biotin (clone IA6-2), (Becton Dickinson; San Jose, CA, USA), CD77 FITC (clone 5B5) and anti-biotin APC (clone Bio3-18E7) (Miltenyi Biotec). Non-viable cells were excluded by simultaneously staining with DAPI (Sigma-Aldrich). Gates to distinguish the different subsets were set using cells suspensions from tonsils and the fluorescence minus one control. By flow sorting using a FACS Aria II (Becton Dickinson) fluorescently labeled cells were separated into four different subsets (Fig 2). Data acquisition and analysis was performed using DIVA software, version 6.13 (Becton Dickinson). The average purity in each sub-population was >90%. A further characterization of these populations was performed by staining with CD27 APC (clone M-T271) and CD20 FITC (clone 2H7) antibodies (Becton Dickinson; San Jose, CA, USA), cell cycle analysis, and Real-Time PCR analysis of AICDA and mir155h6 expression (S1A–S1E Fig). Briefly, cell cycle analysis was carried out on frozen cell suspensions from tonsils, using the fixation and permeabilization buffers (eBioscience) and DAPI staining according to the commercial protocol (cells were re-suspended in 1.5mL of 2% FBS/PBS and 3μl of DAPI). RNA was extracted from tonsils and primary FL cell suspensions and the lymphoblastoid cell line NcNc, using the commercial kit RNAeasy Mini Kit (Qiagen). Standard cDNA synthesis was performed using the SuperscriptII Reverse Transcriptase and Random Primers (Invitrogen), according to the manufacturer's instructions and using 100 ng of RNA per reaction. Predesigned TaqMan Gene Expression assays for AICDA, mir155h6 and 18S genes were obtained from Applied Biosystem. 18S was used as endogenous control. Both the target and the control genes were run in duplicate. The quantitative PCR was performed using the Taqman 7900HT Fast Real Time PCR System and the recommended manufacturer’s conditions. Gene expression values were then calculated using the ΔΔCt method.

Bottom Line: The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs.By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate.The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

View Article: PubMed Central - PubMed

Affiliation: Centre for Haemato-Oncology, Barts Cancer Institute - a CR-UK Centre Of Excellence, Queen Mary University of London, London, United Kingdom.

ABSTRACT
Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

No MeSH data available.


Related in: MedlinePlus