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Risk of Japanese carriers of hyperphosphorylated paratarg-7, the first autosomal-dominantly inherited risk factor for hematological neoplasms, to develop monoclonal gammopathy of undetermined significance and multiple myeloma

Authors


To whom correspondence should be addressed. E-mail: michael.pfreundschuh@uks.eu

Abstract

Hyperphosphorylated paratarg-7 (pP-7) is a frequent target of paraproteins in German patients with monoclonal gammopathy of undetermined significance (MGUS)/multiple myeloma (MM). The frequency of MGUS/MM is lower in Japan than in Europe. As pP-7, the first molecularly defined autosomal-dominant risk factor for any hematological neoplasm, is inherited in a dominant fashion, we determined the incidence of the pP-7 carrier state in a Japanese population, and compared the frequency of pP-7-specific paraproteins and the pP-7 carrier state in Japanese and German patients with MGUS/MM. Peripheral blood from 111 Japanese patients with MGUS/MM and 278 healthy blood donors was analyzed for the pP-7 carrier state by isoelectric focusing and for pP-7-specific antibodies by ELISA. The Japanese group was compared with 252 German MGUS/MM patients and 200 healthy controls. Five of 111 (4.5%) Japanese and 35/252 (13.9%) German IgA/IgG MGUS/MM patients had a pP-7-specific paraprotein (P = 0.009). The prevalence of healthy pP-7 carriers in the Japanese study group was 1/278 (0.36%), whereas it was 4/200 in the German group (P = 0.166). The relative risk for pP-7 carriers developing MGUS/MM had an odds ratio of 13.1 in the Japanese and 7.9 in the German group. In conclusion, the fraction of pP-7 carriers with a pP-7-specific paraprotein is lower among Japanese than in German patients with MGUS/MM, but pP-7 carriers in both ethnic groups have a high risk of developing MGUS/MM. (Cancer Sci 2011; 102: 565–568)

Multiple myeloma (MM) is a B-lymphocyte-derived malignancy characterized by a monoclonal proliferation of plasma cells that produce a clonal immunoglobulin. Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic precursor condition commonly preceding MM.(1) Recent studies show that family members of MGUS and MM patients have a two to threefold higher risk of developing MGUS/MM.(2,3) Environmental influences, chance occurrence, and inherited factors might all contribute to familial clusters. Also, the incidence of MM and the prevalence of MGUS are reportedly two to threefold higher among Black people than White people,(4–6) and lower in Asians.(6,7) Until now, the pathogenesis of MGUS/MM has remained obscure. A causal relationship between MGUS/MM and chronic antigenic stimulation has been suggested by the results of several studies,(8–12) hence the identification of the antigenic stimuli of B-cell neoplasms might be of considerable importance. Antigenic targets of paraproteins were discovered accidentally due to clinical symptoms caused by the paraprotein (e.g. chronic cold agglutinin disease or cryoglobulinemia(13) or bleeding disorders(14)), because of interference of the paraprotein with laboratory tests ordered for the clinical work-up of the patient (e.g. HIV-1 p24 antigen in an HIV-infected patient with myeloma),(15) and/or by screening paraproteins against pre-defined antigens (e.g. anti-streptolysin, anti-DNA, or anti-IgG(13)). The first systematic studies covering a broad spectrum of potential antigens used serological identification of antigens by expression cloning (SEREX), which allows for the systematic screening of putative antibody–antigen interactions, even if neither the antigen nor the antibody are known.(16) cDNA libraries derived from human testis, lung, and breast cancer, bovine and porcine muscle, and wheat germ were expressed in Escherichia coli and investigated by SEREX for reactivity with paraproteins from the sera of 114 patients with MGUS or MM. More than 6 × 108 paraprotein–antigen interactions were probed, resulting in the identification of only four antigens, each recognized by the paraprotein of only one patient.(17,18) In a complementary approach using a human fetal brain-derived macroarray and IgA or IgG paraprotein-containing sera, the paraproteins of 29 (15.1%) consecutive MGUS and MM patients reacted with paratarg-7 (P-7).(19) Paratarg-7 is identical to STOML2 (stomatin [EPB72]-like), also known as HSPC108 or stomatin-like protein and SLP-2,(20) that has also been reported to be expressed in all human tissues and overexpressed(21,22) in several cancers.(23) Other investigators have reported that P-7 modulates T cell activation,(24) and in a recent publication it was claimed that SLP-2 is required for stress-induced mitochondrial hyperfusion.(25) In an extension of our earlier study, the high frequency of P-7-specific paraproteins in the sera of MGUS/MM patients (35/252; 14%) was confirmed in a subsequent study.(26) Moreover, it was shown that all patients with P-7-specific paraproteins were carriers of a hyperphosphorylated version of the protein (pP-7) and that this hyperphosphorylation is inherited in a dominant fashion.(26,27) As only 2% of healthy Germans are carriers of pP-7, the pP-7 carrier state is associated with an increased risk (odds ratio, 7.9) to develop MGUS/MM. Thus, pP-7 is the first molecularly defined inherited risk factor known for any hematological neoplasm. Because of the autosomal-dominant inheritance of pP-7, it is of interest to determine the prevalence of the pP-7 carrier state and the frequency of pP-7-specific paraproteins in other ethnic groups. As the incidence of MGUS and MM is lower in Asians than in Europeans,(6,7) it was the aim of this study to compare a German population with a Japanese population with respect to the prevalence of the pP-7 carrier state and the incidence of P-7-specific paraproteins in MGUS and MM.

Materials and Methods

Patients and controls.  This study was approved by the local German ethical review board (Ethikkommission der Ärztekammer des Saarlandes, Saarbr¨cken, Germany) and the internal review board of Nagoya City University and Nagoya City Midori Municipal Hospital (Nagoya, Japan). Serum samples were taken from 111 Japanese patients with MGUS/MM treated at Nagoya City University Hospital or Nagoya City Midori Municipal Hospital and 252 German patients treated at Saarland University Medical School (Homburg/Saar, Germany). Serum protein electrophoresis identified a monoclonal spike in these samples, which was shown to contain a monoclonal IgA, IgD, or IgG paraprotein by immunofixation. The German control group consisted of 200 healthy employees of Saarland University Medical School and the Japanese control group consisted of 278 Japanese healthy blood donors. “Healthy” was defined as being healthy in the pre-donation medical check-up and having no monoclonal immunoglobulin by serum electrophoresis and immunofixation. In addition, the healthy donors who were identified by isoelectric focusing (IEF) as pP-7 carriers were contacted and they confirmed that they were without any major health problems and explicitly without malignant disease, in particular.

The same criteria were applied in our previous study to the German population of 252 MGUS/MM patients. The German control group was the same as in the previous study. There was no difference with respect to age, gender (all patients), progression to MM, or stage of the disease between patients with a P-7-specific paraprotein. Patients were listed under MGUS if the first blood sample was obtained while they had still MGUS, even if they later progressed into MM. Whenever possible, human materials were obtained during routine diagnostic or therapeutic procedures and stored at −80°C until use.

Western blot sample preparation.  For Western blot analyses, blood samples were centrifuged and washed with PBS. The pellet was resuspended in 8 M urea, 0.1 M phosphate, 10 mM Tris–HCl (pH 8.0), and 0.1% NP-40 and incubated at 20°C for 15 min.

Isoelectric focusing for determination of pP-7 carrier state.  Blood samples were centrifuged and washed with PBS followed by lysis in lysis buffer containing 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris–HCl, and 0.1% NP40 (15 min, 20°C) and stored at −20°C until use. Equal volumes of sample and loading buffer were mixed. Samples were analysed by IEF on a gel with a fixed pH gradient (pH 3.0–10.0) according to the manufacturer’s instructions (Novex pH 3.0–10.0; Invitrogen, Karlsruhe, Germany) followed by an immunoblot screening.

Immunoblot staining.  After lysates from whole peripheral blood were separated by IEF or SDS-PAGE under reducing conditions, the proteins were transferred to an Immobilon-P PVDF membrane (Immobilon; Millipore, Eschborn, Germany) by semi-dry blotting. The membrane was blocked overnight at 4°C in TBST/milk buffer (10% [v/v] milk in 10 mM Tris–HCl [pH 7.5], 150 mM NaCl, and 0.1% [v/v] Tween 20), washed and incubated for 1 h at room temperature with serum in TBST (paraprotein-containing serum from patients at a dilution of 1:108 and from controls at a dilution of 1:103). After three washings in TBST, the membranes were incubated for 1 h at room temperature with mouse anti-human IgG–POX antibody (BioRad, Dreieich, Germany) diluted 1:3000 in TBST. The membranes were then washed in TBST, followed by detection using Pharmacia’s ECL (enhanced chemiluminescence) system (General Electric Healthcare, Fairfield, CT, USA).

Paratarg-7 ELISA for detection of paraproteins with specificity to P-7.  To determine the antigen specificity of the paraproteins, the P-7 ELISA using full-length recombinant paratarg-7 was carried out as described previously.(19)

Results

Thirty-five of 252 (13.9%) paraproteins from the German MGUS/MM patients were shown by ELISA to react with paratarg-7. In contrast, only 5/111 Japanese patients (4.5%) had a paraprotein specific for paratarg-7 (Table 1) and were carriers of pP-7 (Fig. 1). This difference is significant with a P-value of 0.009 (chi-square test). The anti-paratarg-7 reactivity of these sera had titres ranging from 1:108 to 1:1010. None of the sera from healthy controls reacted at a dilution of ≤1:102. Lower serum dilutions were not tested because they cause too much background in the P-7 ELISA. As was the case with the German IgG paraproteins with specificity for P-7,(19) all Japanese P-7 reactive IgG paraproteins (4/78) belonged to the IgG3 subtype, with 4/38 IgG3 (10.5%) paraproteins displaying this specificity. The prevalence of healthy pP-7 carriers in the healthy Japanese population, as determined by IEF (Fig. 1) followed by immunoblot staining, was lower (1/278 or 0.36%) than in the German (4/200 or 2.0%) population. This is a strong trend but, due to the low prevalence of a pP-7 carrier state in healthy controls, did not reach significance (P = 0.166; Fisher’s exact test). Nevertheless, the relative risk for pP-7 carriers to develop MGUS/MM is significant in both ethnic groups with an odds ratio of 13.1 (95% confidence interval, 1.5–113.1; = 0.020) in the Japanese and 7.9 (95% confidence interval, 2.8–22.6; = 0.0001) in the German population.

Table 1.   Detection of paratarg-7-specific paraproteins in 252 German and 111 Japanese consecutive patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM)
 MGUS (%)MM (%)Total (%)
  1. †All pP-7 reactive IgG paraproteins were of the IgG3 subclass.

Japanese patients
 IgA 0/3 (0) 1/22 (4.5) 1/25 (4.0)
 IgD 0/0 (0) 0/8 (0) 0/8 (0)
 IgG 0/8 (0) 4/70 (5.3)† 4/78 (5.1)†
 Total 0/11 (0) 5/100 (5.0) 5/111 (4.5)
German patients
 IgA 2/24 (8.3) 4/21 (19.0) 6/45 (13.3)
 IgD 0/0 (0) 0/0 (0) 0/0 (0)
 IgG15/117 (12.8)†14/90 (15.5)†29/207 (14.0)†
 Total17/141 (12.1)18/111 (16.2)35/252 (13.9)
Figure 1.

 Western blot analysis after isoelectric focusing of paratarg-7 (P-7) derived from Japanese and German patients with P-7-reactive paraproteins. Bands of whole peripheral blood lysates from two Japanese patients with multiple myeloma (J1, J2) with anti-P-7 paraproteins, one German patient with a P-7-specific paraprotein (G2) and one German patient with a non-P-7-specific paraprotein (G1) migrated differently.

Discussion

This is the first study to show that pP-7, which was identified as the first dominantly inherited risk factor for any hematological neoplasm in White people, is also found in Japanese patients with MGUS/MM and is a strong and highly significant risk factor for developing MGUS/MM in both Japanese and German pP-7 carriers. A causal relationship between MGUS/MM and chronic antigenic stimulation has been suggested by the results of several studies; however, results have generally been inconsistent.(4,5,10,11,28–37) The specificity of the P-7 binding paraproteins was extensively discussed in our previous published study(19) and has recently been confirmed by cloning the B-cell receptors of two patients with a P-7-specific paraprotein.(38)

The MGUS/MM patients in this study carrying pP-7, and those with paraproteins that did not bind to P-7, showed no significant difference with respect to age, sex, or course of disease (data not shown). Even though the frequency of P-7 as a paraprotein target is only 4.5% in Japanese patients, it is still much higher than expected by chance and suggests a direct or indirect role of pP-7 in the pathogenesis of these diseases in both the Japanese and German populations. All IgG paraproteins with specificity for P-7 belonged to the IgG3 subclass, both in the Japanese patients (4/38 or 10.5%) and in the German patients (24/57 or 42.1%).(19) The reason for this is unknown, but indicates that additional factors might be necessary for the recognition of pP-7 as an auto-antigen by the autologous immune system. Recent results from our laboratory show that the difference between “wild-type” and pP-7 is due to a phosphorylation at a single site (serine 17 of the molecule; unpublished data). Because of the dominant inheritance of P-7 hyperphosphorylation, a polymorphism in one of the plethora of kinases is more likely to be responsible for the phosphorylation differences than a deficiency or decreased activity of a phosphatase, which should be compensated by the second allele.

Several reports suggest that gene mutations or genetic polymorphisms might be associated with the risk of MM.(39–41) However, results have been inconsistent and significant findings have not been replicated convincingly.(42) Hyperphosphorylated P-7 is the first molecularly characterized structure that provides a plausible explanation for the familial clustering of cases of MGUS/MM, at least in cases with a P-7-specific paraprotein. Indeed, we observed two pedigrees with familiar MGUS/MM, and all affected members in these two families were carriers of pP-7.(43) It is now possible to investigate whether previously reported cases of familial MGUS/MM(2,3,5,35) can also be explained by the carrier state of pP-7.

The frequency of the carrier state of pP-7 among patients with MGUS/MM and in healthy controls reveals a 13.1-fold increased risk to develop MGUS/MM for Japanese carriers and a 7.9-fold increased risk for German carriers. These are, to the best of our knowledge, the highest odds ratios for an MGUS/MM risk factor reported to date in either ethnic group.(2,4,5,44) The number of families with MGUS/MM patients carrying pP-7 is still too small to estimate the risk of a family member carrying pP-7; it is at least 13.1 and 7.9 in the Japanese and German population, respectively, but is probably much higher, because other, yet unidentified, genetic factors shared between family members might further increase the risk for MGUS/MM among family members.

The odds ratios for carriers of pP-7 to develop MGUS/MM was significant for the German study group (4/200 vs 35/252; = 0.0001; chi-square test) and Japanese study group (1/278 vs 5/111; = 0.008; Fisher’s exact test). Testing of family members for the pP-7 carrier state by simple IEF enables the identification of family members who are at increased risk of developing MGUS/MM.

In contrast to the carrier state of pP-7, which is under exclusive genetic control, the nature of the immune response against pP-7 is complex and might involve both genetic and environmental factors. The fact that genetic factors are relevant is suggested by the previous findings that all IgG paraproteins with specificity for P-7, analyzed to date, are of the IgG3 subclass and 42.1% of all IgG3 paraproteins react with pP-7.(19) The frequency of pP-7 as an antigenic target and/or stimulus for paraprotein-producing clones and the availability of many families with MGUS/MM patients with the pP-7 carrier state now allow for the analysis of tumor-host interactions in the presence and absence of the antigen in the respective patients and family members, and to study more specifically the role of environmental factors and immunoregulatory deficiencies, such as the recently reported dysfunction of regulatory T cells(45) in patients with MGUS and MM.

The fact that pP-7 functions as the antigenic target of the paraproteins of all MGUS/MM patients with pP-7, suggests that the hyperphosphorylated protein plays a role in the development of sporadic and familial MGUS/MM. The hyperphosphorylation of P-7 appears to be the most obvious likely reason for its autoimmunogenicity. Whether pP-7 induces the development of MGUS/MM by chronic antigenic stimulation or whether it is only a marker or an epiphenomenon of another dominantly inherited susceptibility to develop MGUS/MM can now be investigated in the respective patients and their (not yet) affected relatives.

Acknowledgments

This work was supported by Förderverein Krebsforschung Saar-Pfalz-Mosel, HOMFOR (the research program of the Saarland University Faculty of Medicine, Homburg/Saar, Germany), Wilhelm Sander-Stiftung (Munich, Germany), and Grants-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare, Japan (16-17, 21-8-5). We thank all Japanese and German patients for participating in the study.

Disclosure Statement

KDP and MP have applied for a relevant patent. None of the other authors have a conflict of interest.

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