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VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma
Published on: December 28, 2015
Molecular analysis of immunoglobulin variable genes supports a germinal center experienced normal counterpart in
Anne Pham-Ledard1, Martina Prochazkova-Carlotti2, Mélanie Deveza3
1INSERM U1053, Team 3 Oncogenesis of Cutaneous Lymphomas, Univ. Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France; Dermatology Department, CHU Bordeaux,1 avenue Jean Burguet, 33000 Bordeaux, France.
Insights
Primary cutaneous diffuse large B-cell lymphoma, leg-type originates from germinal center-experienced B cells. These cells are selected by superantigens, indicating a stage between germinal center B cells and plasma cells.
Area of Science:
- Hematology
- Oncology
- Immunology
Background:
- Primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) exhibits an immunophenotype consistent with germinal center-experienced B lymphocytes.
- The BCL2+ MUM1+ BCL6+/- markers suggest a specific B-cell maturation stage.
Purpose of the Study:
- To determine the cell of origin for PCLBCL-LT by analyzing immunoglobulin gene sequences, surface immunoglobulin expression, and class switching features.
- Investigating the imprinted maturation history within immunoglobulin gene sequences.
Main Methods:
- Clonality analysis using the BIOMED2 protocol and VH leader primers on DNA from 14 patient skin biopsies.
- Alignment and comparison of clonal immunoglobulin heavy variable (IGHV) sequences to germline sequences to calculate homology.
- Evaluation of superantigen binding sites and selection pressure using the multinomial Lossos model.
Main Results:
- Functional monoclonal immunoglobulin sequences were identified in all 14 cases (IGHV, IGLV, or IGKV).
- High immunoglobulin variable (IGV) mutation rates (median 15.5%) with conserved superantigen binding sites were observed.
- Features of selection pressure were identified in 11/12 cases, predominantly negative selection (75%).
- Intraclonal variation was detected in 3/8 specimens.
- Surface immunoglobulin was IgM in 12/12 cases, with heterozygous IGHM gene deletion in half of the cases, indicating class switching events on a non-functional allele.
Conclusions:
- The cell of origin for PCLBCL-LT is a germinal center-experienced B cell.
- Superantigen-driven selection plays a role in the pathogenesis of PCLBCL-LT.
- The tumor cells represent a stage between germinal center B cells and plasma cells.
Background:
Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-).
Objectives:
As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin.
Methods:
Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model.
Results:
A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch μ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression.
Conclusion:
These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell.
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