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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

Models of clonal evolution developed by the CNV analysis.FL and t-FL samples are represented in white and dark grey ovals respectively. The CNVs occurring at each stage are shown below the ovals. (A) In pt1 two additional samples (AD1548 FL and 1787 t-FL) that were not investigated during the 454 deep sequencing experiments were analyzed and are shown as dotted ovals. We observed a model of clonal selection, where more primitive ancestor cells not detected in the first biopsy (depicted in light grey) later become the R0012 t-FL and R2005 FL dominant clones. The time course between clinical episodes (bottom) indicates that these precursor cells can persist for many months before disease presents clinically. The dotted arrows represent the evolution from an ancestor subclone into a more mutated tumor cell, whilst the bold arrows show the emergence of an ancestor cell, by clonal selection, identical to the R0012 t-FL and R2005 FL respectively. (B) In pt3 we observe a model of divergent evolution. The more primitive progenitor cell (light grey oval) has already acquired 2 CNV and subsequently acquires the loss of 6q and the loss of 15q arms captured within the R8403 FL1 and R9129 FL2 tumors respectively.
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pone.0134833.g005: Models of clonal evolution developed by the CNV analysis.FL and t-FL samples are represented in white and dark grey ovals respectively. The CNVs occurring at each stage are shown below the ovals. (A) In pt1 two additional samples (AD1548 FL and 1787 t-FL) that were not investigated during the 454 deep sequencing experiments were analyzed and are shown as dotted ovals. We observed a model of clonal selection, where more primitive ancestor cells not detected in the first biopsy (depicted in light grey) later become the R0012 t-FL and R2005 FL dominant clones. The time course between clinical episodes (bottom) indicates that these precursor cells can persist for many months before disease presents clinically. The dotted arrows represent the evolution from an ancestor subclone into a more mutated tumor cell, whilst the bold arrows show the emergence of an ancestor cell, by clonal selection, identical to the R0012 t-FL and R2005 FL respectively. (B) In pt3 we observe a model of divergent evolution. The more primitive progenitor cell (light grey oval) has already acquired 2 CNV and subsequently acquires the loss of 6q and the loss of 15q arms captured within the R8403 FL1 and R9129 FL2 tumors respectively.

Mentions: The analysis of genomic copy number variation (CNV) represents an alternative method that our group [16] and others [17] have used to trace the evolution of tumor clones associated with FL at relapse or transformation. To test whether the CNV pattern was concordant with the profile detected by IgH-VH deep sequencing and in order to identify genomic events characteristic of the FL/t-FL samples, a genome wide analysis using the Affymetrix SNP 6.0 array was performed on the FL/t-FL samples from pt1 and pt3. For pt1 two additional samples (AD1548 and 1787) were also included (Figs 1A and 5A). In pt1, who displayed a pattern compatible with a direct evolution, we observed two samples (t-FL R0012 and FL R2005) with fewer CN changes as compared to the immediate preceding biopsy and identical to those of predicted progenitor clones (Fig 5A); this non-linear CPC pattern is consistent with a model of clonal selection and assumes the presence of subclones that persist over time and may become dominant under certain selection pressures such as therapy. Instead, the SNP 6.0 array results of pt3 confirm the existence of a CPC with 2 CNV gains of the entire chromosome 12 and loss of 18p (Fig 5B). This CPC subsequently acquired a further loss of 6q detected in the R8403 FL biopsy and a loss of 15q in the R9129 FL biopsy.


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)

Models of clonal evolution developed by the CNV analysis.FL and t-FL samples are represented in white and dark grey ovals respectively. The CNVs occurring at each stage are shown below the ovals. (A) In pt1 two additional samples (AD1548 FL and 1787 t-FL) that were not investigated during the 454 deep sequencing experiments were analyzed and are shown as dotted ovals. We observed a model of clonal selection, where more primitive ancestor cells not detected in the first biopsy (depicted in light grey) later become the R0012 t-FL and R2005 FL dominant clones. The time course between clinical episodes (bottom) indicates that these precursor cells can persist for many months before disease presents clinically. The dotted arrows represent the evolution from an ancestor subclone into a more mutated tumor cell, whilst the bold arrows show the emergence of an ancestor cell, by clonal selection, identical to the R0012 t-FL and R2005 FL respectively. (B) In pt3 we observe a model of divergent evolution. The more primitive progenitor cell (light grey oval) has already acquired 2 CNV and subsequently acquires the loss of 6q and the loss of 15q arms captured within the R8403 FL1 and R9129 FL2 tumors respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556522&req=5

pone.0134833.g005: Models of clonal evolution developed by the CNV analysis.FL and t-FL samples are represented in white and dark grey ovals respectively. The CNVs occurring at each stage are shown below the ovals. (A) In pt1 two additional samples (AD1548 FL and 1787 t-FL) that were not investigated during the 454 deep sequencing experiments were analyzed and are shown as dotted ovals. We observed a model of clonal selection, where more primitive ancestor cells not detected in the first biopsy (depicted in light grey) later become the R0012 t-FL and R2005 FL dominant clones. The time course between clinical episodes (bottom) indicates that these precursor cells can persist for many months before disease presents clinically. The dotted arrows represent the evolution from an ancestor subclone into a more mutated tumor cell, whilst the bold arrows show the emergence of an ancestor cell, by clonal selection, identical to the R0012 t-FL and R2005 FL respectively. (B) In pt3 we observe a model of divergent evolution. The more primitive progenitor cell (light grey oval) has already acquired 2 CNV and subsequently acquires the loss of 6q and the loss of 15q arms captured within the R8403 FL1 and R9129 FL2 tumors respectively.
Mentions: The analysis of genomic copy number variation (CNV) represents an alternative method that our group [16] and others [17] have used to trace the evolution of tumor clones associated with FL at relapse or transformation. To test whether the CNV pattern was concordant with the profile detected by IgH-VH deep sequencing and in order to identify genomic events characteristic of the FL/t-FL samples, a genome wide analysis using the Affymetrix SNP 6.0 array was performed on the FL/t-FL samples from pt1 and pt3. For pt1 two additional samples (AD1548 and 1787) were also included (Figs 1A and 5A). In pt1, who displayed a pattern compatible with a direct evolution, we observed two samples (t-FL R0012 and FL R2005) with fewer CN changes as compared to the immediate preceding biopsy and identical to those of predicted progenitor clones (Fig 5A); this non-linear CPC pattern is consistent with a model of clonal selection and assumes the presence of subclones that persist over time and may become dominant under certain selection pressures such as therapy. Instead, the SNP 6.0 array results of pt3 confirm the existence of a CPC with 2 CNV gains of the entire chromosome 12 and loss of 18p (Fig 5B). This CPC subsequently acquired a further loss of 6q detected in the R8403 FL biopsy and a loss of 15q in the R9129 FL biopsy.

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