Identification of Clonal Immunoglobulin λ Light-Chain Gene Rearrangements in AL Amyloidosis using Next Generation Sequencing

[Introduction] AL amyloidosis is caused by the deposition of abnormally folded clonal immunoglobulin (IG) light chains (LCs, λ:κ = 3:1) made by malignant plasma cells in the bone marrow (BM), which leads to multi-organ dysfunction, often involving the heart, kidney, liver, skin, and nerves. However, little is known about what regulates organ tropism of amyloid deposition in this disease. In addition, no study has analyzed the repertoire of IG germlines of plasma cells in the BM in AL amyloidosis using next generation sequencing (NGS). In this study, we aimed to identify the clonal composition of IG λ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using NGS. [Material and method] BM cells were obtained at diagnosis from 38 patients with AL amyloidosis and those with other plasma cell disorders: multiple myeloma (MM, n = 7), and monoclonal gammopathy of undetermined significance (MGUS, n = 11) with λ-type monoclonal paraprotein. Seven normal control (NC) patients had either immune thrombocytopenia or malignant lymphoma without BM invasion. Genomic DNA was extracted from the BM mononuclear cells preserved in LABO Banker1 or BM clots in O.C.T compound using QIAamp DNA Blood Mini kit. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction using a 5′ primer for the IGLV1/2 framework 3 (FR3) region and 3′ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were performed using an Illumina MiSeq sequencer and then analyzed by an open-source program called Vidjil. All subjects provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of the Chiba University Graduate School of Medicine and Japanese Red Cross Medical Center. [Results] Clinical and laboratory features of 38 patients with AL amyloidosis were as follows: primary AL amyloidosis (n = 31); 15 and 20 patients had cardiac and renal dysfunctions, respectively, and secondary AL amyloidosis with MM (n = 7); 4 and 1 patient had cardiac and renal dysfunctions, respectively. In patients with AL amyloidosis, the median plasma cell count in BM aspirate smears was 3.3% (0.1%-50.4%), and the median difference in involved and uninvolved light chains (dFLC) was 104.5 mg/L (28.5mg/L -2673.3mg/L). Representative results of the Vidjil analysis in NC, MGUS, AL amyloidosis, and MM are shown in Figure 1. The most abundant IGLV gene accounted for not >1% of the reads, and there was no dominant germline in NC samples. Therefore, we defined the dominant clone as >1% of IG germlines in plasma cells. According to this definition, clonal IG germlines were found in 27 of 31 patients with primary AL amyloidosis (87%), 5 of 7 with secondary AL amyloidosis (71%), 7 of 7 with MM (100%), and 8 of 11 with MGUS (73%). However, the size of clones in AL amyloidosis (median 3.1%, 0.38%-14.3%) was significantly smaller than that in MM (median 17.8%, 2.2%-17.9%) (P<0.001), and similar to that in MGUS (median 3.8%, 0.4%-32.0%). Importantly, in patients with AL amyloidosis, the dFLC and involved/uninvolved FLC ratio was not correlated with the clonal size of plasma cells in our repertoire analysis using NGS, suggesting that small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Regarding IGLV germline usage, IGLV1-51 was the most frequent repertoire in AL amyloidosis with heart dysfunction (7 of 16 cases) and renal dysfunction (7 of 21 cases). No relationship between the IGLV germlines and organ tropisms was observed. [Conclusion] We successfully identified the clonal composition of IGLV genes in the BM of most patients with AL amyloidosis using NGS, according to the differences in the V and J region recombination and CDR3 sequences using the Vidjil program. In AL amyloidosis, the clonal size of plasma cells in the BM is small and small malignant clones of plasma cells may secret FLC and cause LC depositions in AL amyloidosis. Figure 1 Disclosures Suzuki: Ono: Research Funding; BMS: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

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Clonal Immunoglobulin λ Light-Chain Gene Rearrangements Detected By Next Generation Sequencing in POEMS Syndrome

Blood ◽

10.1182/blood.v128.22.4405.4405 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4405-4405

Author(s):

Chika Kawajiri-Manako ◽

Naoya Mimura ◽

Masaki Fukuyo ◽

Hiroe Namba-Fukuyo ◽

Bahityar Rahmutulla ◽

...

Keyword(s):

Light Chain ◽

Plasma Cells ◽

Disease Status ◽

Poems Syndrome ◽

Chain Gene ◽

Gene Rearrangements ◽

Clone Size ◽

Light Chain Gene ◽

Vegf Level ◽

Generation Sequencing

Abstract [Backgrounds] POEMS syndrome is a rare plasma cell dyscrasia and its pathogenesis including the significance of monoclonal plasma cells in disease progression is poorly understood. Monoclonal plasma cells only produce λ immunoglobulins, and genes encoding immunoglobulin λ light chain (IGL) V regions are derived from IGLV1-44 or IGLV1-40 germline sequences. Here we analyzed the clonality in IGLV gene rearrangements using next generation sequencing (NGS) to evaluate the significance of monoclonal plasma cell clone size and follow its changes in clinical course to understand the pathogenesis of POEMS syndrome. [Methods] Patients who were diagnosed with POEMS syndrome between November 2006 and October 2015 at Chiba University Hospital were included in the study. As positive controls, 3 multiple myeloma (MM) patients with λ-type monoclonal light chain, and 9 negative control patients were also analyzed. NGS libraries were constructed from genomic DNA samples, extracted from bone marrow mononuclear cells. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction (PCR) using a 5' primer for the IGLV1/2 framework 3 (FR3) region and 3ʹ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were carried out using an Illumina MiSeq sequencer. The closest matched germline sequences were determined using the ImMunoGeneTics database. Subsequently, frequencies of each clonotype that were characterized by a unique V-J rearrangement, conserved complementarity determining region 3 (CDR3) anchors and a unique CDR3 amino acid sequence, were calculated. [Results] Twenty-eight patients with POEMS syndrome were enrolled. All the patients had λ-type M protein. The median follow up time of the patients was 24.4 months (range, 3.7 - 113.9). Firstly we analyzed the usage of IGLV germline genes in each case. In 8 cases, the POEMS syndrome-specific germline sequences, IGLV1-40 or IGLV1-44, were dominant; accounting for more than 40% of all germline sequence usage. However, other samples showed minimal or no differences from controls, indicating that the clonal expansion of monoclonal plasma cells is generally low in patients with POEMS syndrome. Analyzing frequencies of the most dominant rearrangement in each germline, the clonal IGLV gene rearrangements of POEMS syndrome-specific germline sequences were significantly increased in 10 POEMS patients (35.7%; IGLV1-44: n = 8, IGLV1-40: n = 2). Significant increase of clone sizes were not directly linked to the initial disease status (vascular endothelial growth factor [VEGF] level and percentage of plasma cells in the bone marrow), overall survival and progression-free survival of POEMS patients. In 12 patients that we were able to follow their clinical courses, the clone size of IGLVgene rearrangements correlated with disease course assessed with serum VEGF level in most cases (n = 8), as they decreased with serum VEGF levels in disease remission and increased with re-elevation of serum VEGF in relapse cases. Clone sizes without significant increase at diagnosis were constantly flat even after achieving disease remission (n = 3). In one case, clone size with significant increase at diagnosis was unchanged even after serum VEGF level decreased (n = 1). Further observation is needed in this case. [Discussion] Considering the cases with significant increase of IGLV rearrangement clones, it was confirmed that clonal light chain gene expression is restricted to the IGLV1-44 and IGLV1-40 germline sequences, as previously reported. IGLV gene rearrangement clone was not detected as significant increase in some cases. Therefore, we speculated that there are certain numbers of patients with POEMS syndrome with extremely low frequency of clone cells. On the other hand, significant increase of clone size did not reflect disease status, suggesting that disease status is not regulated by tumor burden alone. By contrast, in cases with significantly increased clones, clone sizes changed dependiing on the disease status. These data do demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome. [Conclusions] Our analysis of IGLV gene rearrangements has demonstrated the association between the size of IGLV gene rearrangement clones and the clinical courses in POEMS syndrome. Disclosures No relevant conflicts of interest to declare.

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Faculty Opinions recommendation of Detection of gene rearrangements in targeted clinical next-generation sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727206223.793527628 ◽

2017 ◽

Author(s):

Alberto Falchetti

Keyword(s):

Next Generation Sequencing ◽

Gene Rearrangements ◽

Next Generation ◽

Generation Sequencing

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Structure of extrachromosomal circular DNAs generated by immunoglobulin light chain gene rearrangements

Immunology Letters ◽

10.1016/0165-2478(91)90238-6 ◽

1991 ◽

Vol 27 (1) ◽

pp. 19-23 ◽

Cited By ~ 6

Author(s):

Toshiyasu Hirama ◽

Sunao Takeshita ◽

Yataroh Yoshida ◽

Hideo Yamagishi

Keyword(s):

Light Chain ◽

Chain Gene ◽

Gene Rearrangements ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Circular Dnas

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Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4970-4976.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4970-4976

Author(s):

J Y Zhang ◽

W Bargmann ◽

H R Bose

Keyword(s):

Cell Lines ◽

Light Chain ◽

Lymphoid Cells ◽

Chain Gene ◽

Gene Rearrangements ◽

B Cell Differentiation ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

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Immunoglobulin light chain gene rearrangements display hierarchy in absence of selection for functionality in precursor-B-ALL

Leukemia ◽

10.1038/sj.leu.2402548 ◽

2002 ◽

Vol 16 (8) ◽

pp. 1448-1453 ◽

Cited By ~ 17

Author(s):

M van der Burg ◽

BH Barendregt ◽

T Szczepañski ◽

ER van Wering ◽

AW Langerak ◽

...

Keyword(s):

Light Chain ◽

Chain Gene ◽

Gene Rearrangements ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Selection For

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Organization and genomic complexity of bovine λ-light chain gene locus

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2009.12.012 ◽

2010 ◽

Vol 135 (3-4) ◽

pp. 306-313 ◽

Cited By ~ 18

Author(s):

Yfke Pasman ◽

Surinder S. Saini ◽

Elspeth Smith ◽

Azad K. Kaushik

Keyword(s):

Light Chain ◽

Gene Locus ◽

Chain Gene ◽

Light Chain Gene ◽

Genomic Complexity ◽

Λ Light Chain

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Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4970 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4970-4976 ◽

Cited By ~ 7

Author(s):

J Y Zhang ◽

W Bargmann ◽

H R Bose

Keyword(s):

Cell Lines ◽

Light Chain ◽

Lymphoid Cells ◽

Chain Gene ◽

Gene Rearrangements ◽

B Cell Differentiation ◽

Immunoglobulin Light Chain ◽

Light Chain Gene ◽

Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

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