Restrictive usage of monoclonal immunoglobulin λ light chain germline in POEMS syndrome

POEMS syndrome is a rare plasma cell disorder characterized by peripheral neuropathy, monoclonal gammopathy, and other paraneoplastic features, including organomegaly, endocrinopathy, skin changes, edema, and effusions.^1-3  The pathogenesis of this disease is not well understood, but overproduction of vascular endothelial growth factor (VEGF), presumably secreted by plasmacytoma cells or platelets, is considered to be responsible for the characteristic symptoms.^4-6  Treatment for POEMS syndrome includes surgical resection or radiation for plasmacytoma, and chemotherapy, especially high-dose melphalan, followed by autologous peripheral blood stem cell transplantation.^7-9  Recently, thalidomide,^10,11  lenalidomide,¹²  and anti-VEGF monoclonal antibodies have also been used.^13-15 

A unique feature of this syndrome is that the proliferating monoclonal plasma cells are essentially λ-restricted in most cases, and κ type is extremely rare.¹⁶  However, the pathogenetic role of λ-type M protein in POEMS syndrome is unclear. Here we determined complete nucleotide sequences of monoclonal immunoglobulin λ light chain (IGL) variable regions in patients with POEMS syndrome and found that Vλ germline usage is highly restricted to the Vλ1 subfamily and very limited germlines.

This study was approved by the Institutional Review Board of Chiba University Hospital.

A total of 15 patients (9 men and 6 women; median age at diagnosis, 54.7 years) were included in the study (Table 1). All patients met the criteria for the diagnosis of POEMS syndrome¹⁷  and had the 5 distinctive features of POEMS. The serum monoclonal immunoglobulin light chain was λ-type in all patients. The serum monoclonal heavy chain component was IgG in 7 patients and IgA in 8 patients. Informed consent was obtained from the patients in accordance with the Declaration of Helsinki.

Total RNA was extracted from bone marrow mononuclear cells of patients with POEMS syndrome using TRIzol (Invitrogen, Carlsbad, CA), and single-stranded cDNA was synthesized using Superscript II (Invitrogen). The V-J region of the IGL gene was amplified by reverse-transcribed polymerase chain reaction (PCR) using 5′ degenerate primers for the Vλ1-Vλ2-Vλ3 consensus leader lesion (5′-ATGGCCKGSWYYSYTCTCCTC-3′) and 3′ primers matching the consensus upstream part of the cλ exon (5′-CTCCCGGGTAGAGAAGTCACT-3′) as previously described.¹⁸  To identify the amount of monoclonal PCR products, we used heteroduplex analysis in which PCR products are denatured at 95°C for 5 minutes and subsequently renatured at 4°C for 1 hour to induce homoduplex or heteroduplex formation.¹⁹  When sufficient monoclonal PCR product was available for direct sequencing, PCR products were subjected to cycle sequencing using the aforementioned primers. If the amount was small, homoduplex bands on the polyacrylamide gel after heteroduplex analysis were excised and monoclonal PCR fragments were eluted from the solubilized gel slice using QIAEX II (Qiagen, Valencia, CA) and subjected to cycle sequencing.

Sequence data were analyzed using the database of the International ImMunoGeneTics information system (Montpellier, France), and mutations were identified by comparison with the germline sequence. The average mutation rate of the IGL gene was calculated as pre-viously described.²⁰ 

Of 15 patients with POEMS syndrome who had λ-type M protein, nucleotide sequencing of the IGL gene was successful in 11 (Table 1). The remaining 4 patients' samples had none or too little of the monoclonal band to sequence in heteroduplex analysis. Sequence analysis revealed that the V-region of the monoclonal Igλ gene of all 11 patients was restricted to the Vλ1 subfamily. Searching for homologies with IGL germlines revealed that they were derived exclusively from IGLV1-44*01 (9 patients) or IGLV1-40*01 (2 patients). Complete nucleotide and amino acid sequences are shown in Figure 1. IGLV rearrangements were mutated with homologies to germline ranging from 81.5% to 96.1% (average homology, 91.1%). In the IGL-J region, the IGLJ3*2 gene was found in 11 of 11 patients. The rearrangements were mutated with homologies to germline ranging from 84.1% to 100% (average homology, 92.2%). All complementarity-determining region 3 (CDR3) were composed of 11 amino acids, with identical acidic isoelectric point values of 13.0 and similar molecular weights ranging from 1441.6.7 to 1552.8 (median, 1524.5). The average nucleotide mutation rate was 8.6% with 7.5% in the framework regions (FWRs) and 11.8% in CDRs. The ratio of replacement/silent (R/S) mutations was higher in the CDRs than in the FWRs.

In this study, the monoclonal IGL gene could not be analyzed in 4 patients' samples. Two cases had very small amounts of monoclonal band, and the other 2 had no visible monoclonal band at amplification by Vλ1-3 degenerate primers. The latter 2 cases might have monoclonal IGL genes belonging to a Vλ subfamily other than Vλ1-3.

Analyses of monoclonal light chain genes have been extensively carried out in plasma cell dyscrasias by others. González et al reviewed 4 reports of IGL gene usage in Ig-λ type multiple myeloma and showed that mainly the Vλ1 (27%), Vλ2 (28%), and Vλ3 (41%) subfamilies were used to a similar extent in λ-type myeloma and that Vλ4 through Vλ10 subfamily usage was extremely rare (4%).²¹  Within these families, no clear preference for individual gene segments was observed. Comenzo et al reported the results of IGL germline gene analysis in 39 patients with AL amyloidosis.²²  The clonal AL gene subfamily belonged to different subfamilies, including Vλ1 (26%), Vλ2 (13%), Vλ3 (15%), Vλ6 (28%), and Vκ1 (18%). Other germline genes were used, of which the IGLV6S1 germline of the Vλ6 subfamily was relatively dominant. We found that the monoclonal IGLV gene of all 11 POEMS patients belonged to the Vλ1 subfamily with only 2 germlines of Vλ1 represented. There has been only one other report of IGLV gene analysis in POEMS syndrome by a French group.¹⁸  In that study, Soubrier et al¹⁸  found that both of the 2 cases analyzed were derived from the Vλ1-IGLV1-40 germline, the same as in our study (Figure 1A,C). Taken together with our results, currently available data on 13 cases indicate that IGLV genes are restricted to the 2 Vλ1 germlines: IGLV1-44 in 9 and IGLV1-40 in 4 across 2 ethnically different populations.

Why is the λ light chain germline repertoire so restrictive and what links exist between germline gene use and VEGF in POEMS syndrome? Perfetti et al reported that amyloid V regions were highly mutated in AL amyloidosis and amyloidogenic light chains undergo antigen-driven selection.²³  Because the average nucleotide mutation rate and the R/S mutation ratio were higher in the CDRs than in the FWRs, restricted light chains might undergo antigen-driven selection in POEMS. These restricted λ chains might interact with VEGF-related protein and play a role in VEGF secretion. In myeloma, interactions between light-chain CDR3 sites and proteins, such as Tamm-Horsfall proteins, have been previously described.²⁴  However, the higher R/S mutation ratio in the CDRs and retention of cysteine residues in FR1 and FR3, which were critical to structure, suggest that these specific light chains might interact with VEGF-related protein at FRWs but not CDRs like B-cell superantigen.²⁵ 

In conclusion, IGL-M protein in POEMS syndrome belongs to the Vλ1 subfamily and is markedly restricted to a very limited number of germlines. Although exact mechanisms responsible for this syndrome are still unclear, these data suggest that the restricted use of IGL plays an important role in pathogenesis and may represent a new clinical entity. Further studies are required to clarify the relationship between high VEGF levels and restricted usage of Vλ genes in POEMS syndrome.

The authors thank Ms Megumi Naito for technical assistance, Ms Kimie Shibuya for secretarial assistance, and Dr Takeshi Tokuhisa and Dr Akemi Sakamoto of the Department of Developmental Genetics, Chiba University Graduate School of Medicine, for helpful discussions.

Contribution: D.A., C.N., M.T., H.T., C.O., and Y.S. designed the research; D.A. and C.N. performed research, analyzed data, and wrote the paper; E.S., Y.T., K.O., S.O., N.S., S. Masuda, R.C., and M.N. designed the research and analyzed data; S. Misawa, and S.K. organized collection of patient samples; and all authors approved the final version of the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Chiaki Nakaseko, Division of Hematology, Department of Clinical Cell Biology, Chiba University Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba City, Chiba, 260-8670, Japan; e-mail: chiaki-nakaseko@faculty.chiba-u.jp.