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.

Emanuela Carlotti,Jude Fitzgibbon,Maria Calaminici,Lena Hazanov,Peter Campbell,Ajanthah Sangaralingam,Guglielmo Rosignoli,Simon Hallam,Sameena Iqbal,Miri Michaeli,Bryan Young,Ramit Mehr,John G Gribben,Tracy Chaplin,David Wrench,Jacek Marzec,Jose Angel Martinez Climent

doi:10.1371/journal.pone.0134833

Emanuela Carlotti, Jude Fitzgibbon + Show 15 more

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https://doi.org/10.1371/journal.pone.0134833

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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.

Highlights

Follicular lymphoma (FL) is an indolent disease characterized by interspersed episodes of remission and relapse, associated with a decreased sensitivity to therapeutic agents [1]
We examined the immunoglobulin heavy chain gene (IgH-VH) repertoire in 7 lymph node biopsies, from three patients selected as showing patterns of direct evolution and common progenitor cell (CPC) (Fig 1A and 1B)
In pt3 we identified in 3/5 libraries from the FL1 R8403 samples (PGC, ME and not sorted (NS)) an identical clone to FL2 R9129 major tumor clone (MC) and one with 1 additional somatic hypermutation (SHM) at a frequency between 0.0003 and 0.002

Summary

Introduction

Follicular lymphoma (FL) is an indolent disease characterized by interspersed episodes of remission and relapse, associated with a decreased sensitivity to therapeutic agents [1]. It is well established that the t(14;18) translocation is the founder genetic aberration of this disease and it is observed in about 90% of patients at diagnosis. This rearrangement provides a survival advantage to the B cell clone but it is not sufficient to initiate lymphoma [4, 5]. Generation sequencing studies have identified early driver mutations in chromatin regulator genes alongside genes that regulate the interaction of the tumor with its microenvironment [6]. These observations suggest that the second hit responsible for switching a long living B cell into a lymphoma cell could reside either in an altered epigenetic program or in a deviated interaction with other populations [6,7,8,9]

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