Target Of Paraproteins Research Articles

Saposin C is a frequent target of paraproteins in Gaucher disease-associated MGUS/multiple myeloma.

Patients with Gaucher disease (GD) have an increased risk of monoclonal gammopathies for which antigenic targets might play a role in their pathogenesis. Here we report the identification of saposin C (sapC) as high-titre (1:1000000) target structure of 7/16 GD-associated paraproteins. Anti-sapC immunoglobulin (Ig) showed identity with the paraprotein Ig type and subclass in each patient that showed anti-sapC immunoreactivity. Absorption and depletion studies completely removed the paraprotein from the sera of GD patients. No immunoreactivity against sapC was detected in healthy donors and in other plasma cell dyscrasias, demonstrating that anti-sapC reactivity is highly restricted to GD. Several uncharacterized forms of post-translational modified sapC were detected but their role in the pathogenesis is not clear. We confirm the frequent presence of low-titre (1:250) anti-lysolipid reactivities in the sera of GD patients but we could show that this immunoreactivity is not mediated by the paraprotein and is not restricted to GD patients.

Peripheral B Cells from Patients with MGUS and Multiple Myeloma (MM) Can Be Stimulated by Paraprotein-Target Specific T-Helper Cells to Produce Paraprotein-Identical Monoclonal Antibodies: Rationale for PARs, a Novel Therapeutic Approach with Ultimate Specificity in MGUS/MM

Background: Paratarg-7 (P-7) is the antigenic target of paraproteins(Preuss et al. Int J Cancer 2009;125:656-61) from 15% of European and 37% of African-American MGUS/MM patients, stronlgy supporting a role of P-7 in the pathogenesis of MGUS/MM via chronic auto-antigenic stimulation. All patients with P-7 specific paraproteins are carriers of the hyperphosphorylated version of p-7 (pP-7). We recently identified pP-7 specific T-helper cells which were restricted by certain "permissive" HLA-DR haplotypes (Neumann et al., Int J Cancer 2015; 137:1076-1084). These HLA-DR subtypes are overrepresented among patients with P-7 specific paraproteins compared to the corresponding normal population indicating that there are two prerequisites for the development of MGUS/MM with a P-7 specific paraprotein: 1st carriership of pP-7 and 2nd a permissive HLA-DR subtype. We now investigated the functional role of the pP-7 specific T-helper cells and their interaction with peripheral B cells with cognate specificity.Methods: Three patients with MGUS or MM, respectively, with a P-7 specific paraprotein and pP-7 specific T-helper cells were included in this study so far. In addition, the B cells from one healthy pP-7 carrying son of one of the patients were also analyzed. In vitro stimulation of antigen-specific peripheral B cells by pP-7 specific T-helper cells followed a modified protocol previously described by Lanzavecchia et al. (Eur J Immunol. 1983; 13:733-738). To this end, CD19+ B cells and CD3+ T cells were magnetically isolated from the proband's PBMC. T cells were replaced by corresponding T-helper cell clones.Results: In all patients studied, the autologous pP-7 specific T-helper cells stimulated the peripheral B cells to produce P-7 specific antibodies. The P-7 specific B-cell responses were monoclonal and the immunoglobulin type was the same as the paraprotein of the corresponding patient. In contrast, B-cell stimulation with CMV-pp65 specific T-helper cells used as controls always induced an antigen-specific, yet polyclonal response. When the peripheral B cells of a pP-7 carrying patient's son were also stimulated with pP-7 specific T-helper cells, they induced - in contrast to the mother - a polyclonal P-7 specific antibody response in his B cells, even though mother and son shared a "permissive" HLA-DR haplotype (HLA-DRB1*1301).Conclusion: In patients with MGUS/MM monoclonal B cells are found in the peripheral blood that can be induced to produce monoclonal antibodies identical to the serum paraprotein by T-helper cells with specificity for the antigenic target of the paraprotein. This does not only support an indispensable role of these T-helper cells in the pathogenesis of MGUS/MM via chronic antigenic stimulation, it also proves that precursors of the malignant plasma cells can be found in the peripheral blood that might fuel the development of malignant plasma cells. Cytogenetic and molecular genetic analyses are underway to determine if these precursor B-cells share the malignant genotype of their malignant plasma cells. These B cells can now be targeted by PARs (p araprotein a ntigens for r everse targeting) conjugated to toxins, as parts of bispecific constructs (PAR/CD3 or PAR/CD16) and/or PAR/CAR T cells. Use of PARs can be envisaged prophylactically for carriers of modified autoantigens like pP-7 with a permissive HLA-DR haplotype and a monoclonal B-cell response in vitro or in MM patients achieving a VGPR or CR after treatment for the prevention of relapse. DisclosuresNo relevant conflicts of interest to declare.

CD4⁺ T cells in chronic autoantigenic stimulation in MGUS, multiple myeloma and Waldenström's macroglobulinemia.

Hyperphosphorylated paratarg-7 (pP-7) carrier state is the strongest and most frequent molecular risk factor for MGUS, multiple myeloma (MM) and Waldenström's macroglobulinemia (WM), inherited autosomal-dominantly and, depending on the ethnic background, found in up to one third of patients with MGUS/MM. Since P-7 is the antigenic target of paraproteins that do not distinguish between wtP-7 and pP-7, we investigated CD4(+) T-cell responses in pP-7(+) patients and controls. Peptides spanning amino acids 1-35 or 4-31 containing phosphorylated or nonphosphorylated serine17 were used for stimulation. CD4(+) cells from 9/14 patients (65%) showed a pP-7 specific HLA-DR restricted response. These results demonstrate that pP-7 specific CD4(+) cells can mediate help for pP-7 specific chronic antigenic stimulation of P-7 specific B cells, which might ultimately result in the clonal evolution of a B cell into MGUS/MM/WM producing a P-7 specific paraprotein. Prerequisites for pP-7 specific stimulation of CD4(+) cells appear to be both a pP-7 carrier state and an HLA-DR subtype able to present and recognize pP-7. Our results serve as an explanation for the exclusive autoimmunogenicity of the hyperphosphorylated variant of P-7 and for the different hazard ratios of pP-7 carriers from different ethnic origins to develop MGUS/MM/WM.

Sumoylated HSP90 is a dominantly inherited plasma cell dyscrasias risk factor.

Posttranslationally modified proteins serve as autoimmunogenic targets in a wide spectrum of autoimmune diseases. Here, we identified a posttranslationally modified paraprotein target (paratargs) in monoclonal gammopathies of undetermined significance (MGUS), multiple myelomas (MM), and Waldenstrom's macroglobulinemias (WM) using protein macroarrays that were sumoylated and screened for reactivity with paraproteins from MGUS, MM, and WM patients. We found that paraproteins from a proportion of European, African-American, and Japanese patients specifically reacted with the sumoylated heat-shock protein 90 β isoform-α (HSP90-SUMO1, where SUMO indicates small ubiquitin-like modifier), while no reactivity with HSP90-SUMO1 was detected in over 800 controls. HSP90-SUMO1 was present in blood cells from all patients with HSP90-SUMO1-binding paraproteins. We determined that the HSP90-SUMO1 carrier state is autosomal-dominantly inherited and caused by the inability of SUMO peptidase sentrin/SUMO-specific protease 2 (SENP2) to desumoylate HSP90-SUMO1. HSP90-SUMO1 was detected in a small percentage of healthy individuals from all backgrounds; however, only MGUS, MM, and WM patients who were HSP90-SUMO1 carriers produced HSP90-SUMO1-specific paraproteins, suggesting that sumoylated HSP90 promotes pathogenesis of these diseases through chronic antigenic stimulation. This study demonstrates that harboring HSP90-SUMO1 identifies healthy individuals at risk for plasma cell dyscrasias and that dominant inheritance of posttranslationally modified autoantigenic paratargs is one of the strongest molecular defined risk factors for MGUS, MM, and WM.

Sumoylated HSP-90 Is a Frequent Autosomal-Inherited Target of Paraproteins in Monoclonal Gammopathies

BackgroundChronic antigenic stimulation might have a role in the pathogenesis of monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma. We previously identified a group of 11 autoantigenic paraprotein targets (paratargs) of which paratarg-7 is the most frequent, found in ~15 % of all MGUS/MM/Waldenstrom patients. These paratarg autoantigens all have in common to be 1) recognised by patients paraprotein with an extreme high titer (>1:1011), 2) hyperphosphorylated in patients, and 3) their phosphorylation carrier state is autosomal-dominant inherited in patients families. The finding that all these paratargs were permanently hyperphosphorylated in patients in contrast to healthy controls brought up the assumption that abnormal posttranslational modification of autoantigens might be a frequent finding in those patients.MethodsA RZPD macroarray representing more than 30.000 different human proteins was subjected to in-vitro posttranslational modification using sumoylation-competent recombinant enzymes followed by screening with paraprotein-containing sera at high dilution (1:107). Immunopositive signals were identified and characterized by DNA sequencing, SDS-PAGE, isoelectric focusing, western blotting and ELISA.FindingsTwenty-seven of 226 (11.9%) paraproteins from European, 9/80 (11.2%) from African-American and 9/176 (5.1%) from Japanese patients reacted specifically with the sumoylated heat shock protein-90β isoform-α (HSP90-SUMO). No reactivity was detected in >800 controls. All patients with HSP90-SUMO-binding paraproteins carried HSP90-SUMO. HSP90-SUMO carrier state is autosomal-dominantly inherited and was sown to be caused by the inability of SENP2 to desumoylate HSP90-SUMO. Five of 550 (0.9%) European, 2/100 (2%) African-American and 2/178 (0.8%) Japanese controls carried HSP90-SUMO, resulting in odds ratios of 14.8, 6.2 and 7.4, respectively, of healthy carriers for MGUS/MM/WM. Only, but all MGUS/MM/WM patients who were HSP90-SUMO carriers had HSP90-SUMO-specific paraproteins suggesting that sumoylated HSP-90 is involved in the pathogenesis of MGUS/MM/WM by chronic antigenic stimulation. Demonstration of HSP90-SUMO carriership identifies family members of HSP90-SUMO patients at risk.InterpretationA significant proportion of MGUS/MM/WM are associated with a dominant inheritance of posttranslationally modified autoantigens as shown for members of the paratarg autoantigen family and for HSP90-SUMO, enabling family members at increased risk for MGUS/MMWM to be identified by simple analysis of their peripheral blood. That only patients with MGUS/MM/WM who are carriers of such modified autoantigens have target-specific paraproteins suggests that the posttranslational modification induces auto-immunity and is involved in the pathogenesis of MGUS/MM/WM, for example, by chronic antigenic stimulation.Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. DisclosuresNo relevant conflicts of interest to declare.

Inactivation of protein‐phosphatase 2A causing hyperphosphorylation of autoantigenic paraprotein targets in MGUS/MM is due to an exchange of its regulatory subunits

Hyperphosphorylated paratarg-7 (pP-7) carrier state is the strongest molecularly defined risk factor for monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM). pP-7 is inherited as autosomal-dominant trait and depending on the ethnic background is found in over one-third of MGUS/MM patients. P-7, which is the antigenic paraprotein target in these patients, is hyperphosphorylated at serine17. P-7 hyperphosphorylation can be induced in wild-type P-7 (wtP-7) carriers by PKCζ and reverted by protein-phosphatase 2A (PP2A). Here we show that dephosphorylation of pP-7 is defective in pP-7 carriers due to inactivation of the PP2A by substitution of the regulatory B55δ subunit with B56γ3. In lymphoblastoid cell lines from pP-7 carriers, transfection of recombinant B55δ or treatment with ceramide led to a partial reconstitution of PP2A activity and dephosphorylation of pP-7 to wtP7. Similar results were observed with other previously reported autoantigenic paraproteins targets. In conclusion, the mechanisms responsible for the defective dephosphorylation and maintaining the hyperphosphorylated state of P-7 and other autoantigenic paraprotein targets have been elucidated, facilitating the identification of the genetic basis underlying this phenomenon which is obviously common in the pathogenesis of MGUS/MM/WM and not restricted to pP-7 cases.

Inherited predisposition to multiple myeloma

Multiple myeloma (MM) is the second most common hematologic malignancy in the United States, after non-Hodgkin lymphoma. Family pedigree analyses of high-risk families, case-control studies and racial disparities in disease incidence all point to a potential inherited predisposition to MM. Genome-wide association studies (GWASs) have identified susceptibility loci in a number of cancers and such studies are currently underway in MM. To date, GWASs in MM have identified several potential regions of interest for further study on chromosomes 3p22, 7p15.3, 8q24 and 2p23.3. In addition, several targets of paraproteins (so called 'paratargs') in MM have been identified. Hyperphosphorylation of the paratarg protein, which is inherited in an autosomal dominant manner, appears a common mechanism underlying the antigenicity of these proteins. One particular protein, hyperphosphorylated paratarg-7 (pP-7) is a common target in persons with myeloma and has also been identified in affected members of several high-risk MM families. It appears that the frequency of pP-7 as an antigenic target may be particularly high in African American patients with MM, which could be part of the explanation for observed racial disparities in the incidence of MM. In this review we focus on available data in the area of inherited predisposition to MM, and highlight future research directions.

New Insights in the Pathogenesis of MGUS, Multiple Myeloma (MM) and Waldenström‘s Macroglobulinemia (WM): Chronic Antigenic Stimulation by Autoantigenic Targets of Paraproteins Is Mediated by CD4+ t-Cells

Abstract 472Hyperphosphorylated paratarg-7 (pP-7) is the target of paraproteins in 15% of Caucasian, 4.5% of Asian and 28% of Afro-American patients with MGUS/MM. Carriers of pP-7 have an 8 to 13 times increased risk to develop IgA/IgG-MGUS and multiple myeloma (Grass et al., Lancet Oncology 2009; 10:950–956) and a 6.5 times increased risk to develop IgM-MGUS and Waldenstrom‘s macroglobulinemia (Grass et al., Blood. 2011 Mar 10;117(10):2918–23.). Analysis of affected families showed that pP-7 is inherited in a dominant fashion, explaining cases of familial MGUS/MM/WM. Since paratarg-7 is hyperphosphorylated and inherited in a dominant fashion, we extended our studies to investigate the prevalence of further antigenic targets of paraproteins and their phosphorylation state. All (8/8) autoantigenic paraprotein target (paratargs no. 2, 5, 6, 7, 8, 9, 10, and 11) moleculary defined to date were shown to be hyperphosphorylated in the respective patients compared to normal controls, and the hyperphosphorylation state of the respective paraproteins targets was shown to be inherited in a dominant fashion for all autoantigenic paraprotein targets for which enough family members were available for such an analysis. The consistent finding of hyperphosphorylation of autoantigenic paraprotein targets suggests a common underlying mechanism associated with the pathogenesis of MGUS/MM, and implies a significant role of the hyperphosphorylation state in the pathogenesis of these neoplasms. Co-immunoprecipitation identified PKCzeta as the kinase responsible for the phosphorylation of most and PP2A as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. SNPs or mutations of PKCzeta and PP2A as the reason for the abnormal phosphorylation of the paraproteins targets were excluded. However, PP2A was shown to be inactivated by phosphorylation of its catalytic subunit at Y307. To determine the SNP or mutation which is associated with the inactivation of PP2A we carried out a genome-wide linkage analysis. We found strong evidence for a parametric linkage (full penetrant autosomal dominant model; disease marker allele frequency=0.02) with a LOD score of 5.9 on chromosome 4q35.For pP-7 hyperphosphorylation we demonstrated that the hyperphosphrylation is due to an single additional phosphorylation at serine 17, which is located in the paraprotein-binding epitope of pP-7. Analysis of the T-cell response against normo- and hyperphosphorylated paratarg-7 derived peptides in affected and non-affected family members demonstrated that pP-7, but not wild-type p-7 induces a strong and specific CD4+ T-cell response in the patients with pP-7, which was shown to be HLA-DQ and HLA-DR restricted. This suggests that pP-7 confers a chronic antigenic stimulation which is mediated by CD4+ T-cells and that these pP-7 specific CD4+ T-cells stimulate a B-cell clone with the same specificity, eventually resulting in the clonal evolution of this B-cell clone. In summary, our results support the hypothesis of a role of chronic autoantigenic stimulation in the pathogenesis of MGUS/MM/WM. Indviduals carrying pP-7 are at particular risk, if their MHC-II haplotype allows the recognition of pP-7 derived antigenic peptides by their CD4+ T cells. We suggest that the different prevalence of pP-7 carriers and defined MHC-II haplotypes explain the different incidence rates of MGUS/MM/WM in different ethnic groups. Disclosures:Preuss:Ludwig Institute for Cancer Research: Patents & Royalties. Pfreundschuh:Ludwig Institute for Cancer Research: Patents & Royalties.

Hyperphosphorylation of autoantigenic targets of paraproteins is due to inactivation of PP2A

AbstractParatarg-7, a frequent autoantigenic target, and all other autoantigenic targets of human paraproteins molecularly defined to date are hyperphosphorylated in the respective patients compared with healthy controls, suggesting that hyperphosphorylation of autoantigenic paraprotein targets is a general mechanism underlying the pathogenesis of these paraproteins. We now show that hyperphosphorylation of paratarg-7 occurs because of an additional phosphorylation of Ser17, which is located within the paraprotein-binding epitope. Coimmunoprecipitation identified phosphokinase C ζ (PKCζ) as the kinase responsible for the phosphorylation of most, and phosphatase 2A (PP2A) as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. Single-nucleotide polymorphisms (SNPs) or mutations of PKCζ and PP2A were excluded. However, PP2A was inactivated by phosphorylation of its catalytic subunit at Y307. Stimulation of T cells from healthy carriers of wild-type paratarg-7 induced a partial and transient hyperphosphorylation between days 4 and 18, which was maintained by incubation with inhibitors of PP2A, again indicating that an inactivation of PP2A is responsible for the hyperphosphorylation of autoantigenic paraprotein targets. We conclude that the genetic defect underlying the dominantly inherited hyperphosphorylation of autoantigenic paraprotein targets is not in the PP2A itself, but in genes or proteins controlling PP2A activity by phosphorylation of its catalytic subunit.

Paraproteins of familial MGUS/multiple myeloma target family-typical antigens: hyperphosphorylation of autoantigens is a consistent finding in familial and sporadic MGUS/MM

AbstractParatarg-7 (P-7) is a frequent paraprotein target in monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), and Waldenström macroglobulinemia. Patients with P-7-specific paraproteins carry a hyperphosphorylated paratarg-7 (pP-7). Because pP-7 carrier state is dominantly inherited, we determined the paraprotein targets in 4 families with familial MGUS/MM. No antigenic target was identified for the paraproteins from 2 members of one family. Paraproteins from affected members of 2 other families targeted P-7, and paraproteins from 4 affected members of a fourth family targeted P-8, which is encoded by the ATG13 gene. P-8 was hyperphosphorylated in the affected family members (pP-8) and pP-8 carrier state is inherited in a dominant fashion. Six additional autoantigenic nonfamilial paraprotein targets were also hyperphosphorylated in the respective patients compared with normal controls. We conclude that paraproteins of affected members with familial MGUS/MM share family-typical hyperphosphorylated antigens and hyperphosphorylation of paraprotein targets might be a general mechanism underlying the pathogenesis of MGUS/MM.

Hyperphosphorylation of auto-antigen targets of paraproteins in MGUS, multiple myeloma (MM), and Waldentrom macroglobulinemia (WM) is a consistent finding and suggests chronic antigenic stimulation by post-translationally modified auto-antigens as a common mechanism playing a role in the pathogenesis of MGUS/MM/WM.

e18573 Background: Paratarg-7 (P-7) is a frequent target of paraproteins from patients with MGUS, multiple myeloma (MM) and Waldenstroem's macroglobulinemia (WM). Patients with paratarg-7 specific paraproteins are carriers of a hyperphosphorylated paratarg-7 (pP-7). pP-7 carrier state is inherited in an autosomal-dominant fashion, explaining cases of familial MGUS/MM/WM. The aim of this study was to determine whether other auto-antigenic targets of human paraproteins are also hyperphosphorylated. Methods: Moleculary defined auto-antigenic targets of paraproteins were subjected to DNA sequencing, gel electrophoresis and isoelectric focussing. Results: Of 11 molecularly defined antigenic targets of paraproteins, 9 were auto-antigens. Of these 9 cases, autologous tissues from the patients whose paraprotein targeted the respective auto-antigen were available from 8 patients. These autoantigens were: paratarg-2= TTP2, paratarg-5 = microtubulus-associated protein, paratarg-6 = LAPTM5, paratarg-7 = SLP-2, paratarg 8 = ATG-13, paratarg 9 = RSP16, paratarg-10 = SPAG7 and paratarg 11 = SIVA. SNPS or mutations in the autologous patient were excluded by sequencing the genes coding for the respective auto-antigens in the patients and in healthy controls. However, isoelectric focussing before and after phosphatase treatment revealed that all autoantigens tested were hyperphosphorylated in the respective patients compared to healthy controls. For two antigenic targets (paratarg-7 and paratarg-8) an autosomal-dominant inheritance could be shown. Conclusions: The consistent finding of hyperphosphorylated autoantigens suggests that these posttranslationally modified autoantigens entertain a chronic auto-immune response. Hyperphosphorylation of antigenic targets of paraproteins represents a common underlying mechanism associated with the pathogenesis of MGUS/MM, the elucidation of which can now be addressed using specific molecular tools.

Hyperphosphorylated paratarg-7: a new molecularly defined risk factor for monoclonal gammopathy of undetermined significance of the IgM type and Waldenström macroglobulinemia

AbstractWe recently described paratarg-7 (P-7), a protein of unknown function, as the target of 15% of immunoglobulin A (IgA) and IgG paraproteins in monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma. To determine the frequency of P-7 as a paraprotein target in IgM-MGUS and Waldenström macroglobulinemia (WM), sera from patients with IgM-MGUS/WM were tested for reactivity with recombinant P-7 by enzyme-linked immunoabsorbent assay. The specificity of the paraprotein-mediated reaction was shown by absorption studies and cloning of the respective B-cell receptor. The paraproteins of 18 (9 WM and 9 IgM-MGUS) of 161 patients (11%) reacted with P-7. Isoelectric focusing and phosphatase treatment showed that P-7 was hyperphosphorylated (pP-7) in all patients with an anti–P-7-specific IgM paraprotein tested. Because only 4 of 200 healthy controls (2%) were carriers of pP-7, pP-7 carrier state is associated with a significantly increased risk (odds ratio = 6.2; P = .001) for developing IgM-MGUS/MW. Family analyses showed that the pP-7 carrier state is inherited as a dominant trait. After IgA/IgG-MGUS and multiple myeloma, IgM-MGUS/WM is the second neoplasia associated with pP-7 carrier state. The dominant inheritance of pP-7 explains cases of familial IgM-MGUS/WM and enables the identification of family members at increased risk.

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

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.

Hyperphosphorylation of Antigenic Targets of Paraproteins In MGUS and Multiple Myeloma (MM) Is a Consistent Finding: Implications for the Pathogenesis of MGUS/MM

AbstractAbstract 4085 Background:Hyperphosphorylated paratarg-7 (pP-7) is the target of paraproteins in 15% of patients with MGUS/MM (Preuss et al. Int J Cancer 2009; 125:656-61). Carriers of pP-7 have an 8-times increased risk to develop IgA/IgG-MGUS and multiple myeloma (Grass et al., Lancet Oncology 2009; 10:950-956) and a 6.5 times increased risk to develop IgM-MGUS and Waldenström′s macroglobulinemia (Grass et al., Blood 2009;114:1513s). Analysis of affected families showed that pP-7 is inherited in a dominant fashion. Thus, pP-7 is the first molecularly defined autosomal-dominant risk factor for any hematological neoplasm. Objective: Since paratarg-7, a frequent target of paraproteins in patients with MGUS/MM is hyperphosphorylated and inherited in a dominant fashion we extended our studies to investigate the prevalence of further antigenic targets and their phosphorylation state. Methods:Sera of MGUS/MM patients and relatives of affected families were tested for antibody reactivity against antigens represented in a fetal brain derived protein macroarray using a modified SEREX approach (Preuss et al. International J. Cancer 2009;125, 656–661). Lysates of peripheral blood from patients, family-members and controls were tested by gel electrophoresis and isoelectric focusing before and after phosphatase treatment. Results:Analysis of 3 families with multiple members affected by MGUS and MM revealed that the antigenic targets of paraproteins from affected members of a given family are identical: 2 families shared paratarg-7 and 1 family shared paratarg-8 as the family-typic paraprotein antigen. Paratarg-8 is encoded by the ATG13 gene, a member of the „autophagy regulatory complex“ family of genes. Paratarg-8 showed no mutations or polymorphisms in patients compared to controls. However, IEF before and after phosphatase treatment showed that paratarg-8 is hyperphosphorylated (pP-8) in the affected family members compared to healthy controls and that pP-8 carrier state is also inherited in a dominant fashion. This finding prompted us to check previously identified paraprotein targets for hyperphosphorylation, which, in addition to paratarg-7 and paratarg-8 was possible for paratarg 2, 5, 6, 9, 10, 11. In all 8/8 cases, IEF before and after phosphatase treatment revealed that the patients were carriers of a hyperphosphorylated version of their paraprotein target compared to healthy controls. Conclusions:The paraproteins of members affected by familial MGUS/MM are directed against family-specific antigenic targets, suggesting that the genetic background shared by these patients contains a chronic auto-immune response. Paratarg-7 and paratarg-8 are the first family-typic antigens that have been molecularly defined to date. The fact that all antigenic targets of paraproteins molecularly defined to date are hyperphosphorylated in patients compared to healthy controls implies a significant role of the hyperphosphorylation state in these neoplasms. Analysis of the T-cell response against normo- and hyperphosphorylated paraprotein targets in affected and non-affected family members is underway as are genome-wide association to determine the SNP or mutation which is associated with the hyperphosphorylation of paraprotein targets. Disclosures:Lynch:State of Nebraska LB595 support: Research Funding; Charles F. and Mary C. Heider Chair at Creighton University.: Research Funding.

Association of hyperphosphorylated paratarg-7, the first autosomal-dominantly inherited risk factor for hematological neoplasms, with MGUS and multiple myeloma in different ethnic groups.

8111 Background: Hyperphosphorylated paratarg-7 (pP-7) is a frequent target of paraproteins in Caucasian patients with MGUS/MM (Preuss et al. Int J Cancer. 2009; 125:656-61). Carriers of pP-7 have an 8-times increased risk to develop IgA/IgG-MGUS and multiple myeloma (Grass et al., Lancet Oncology. 2009; 10:950-956) and a 6.5 times increased risk to develop IgM-MGUS and Waldenström's macroglobulinemia (Grass et al., Blood. 2009;114:1513s). Analysis of affected families showed that pP-7 is inherited in a dominant fashion. Thus pP-7 is the first molecularly defined autosomal-dominant risk factor for any hematological neoplasm. Because of its autosomal-dominant inheritance we investigated the prevalence of pP-7 carrier state in a Japanese population where the frequency of MGUS/MM is lower than in Europe, and compared it with that of a Caucasian population of MGUS/MM patients and healthy controls. Methods: Peripheral blood from 252 Caucasian patients with MGUS/MM and 200 healthy blood donors was analyzed for pP-7 carrier state by isoelectric focussing and for pP-7 specific antibodies by ELISA and compared with 59 Japanese MGUS/MM patients and 178 healthy Japanese controls. Results: 35/252 (13.9%) of German, but only 2/59 (3.4%) of Japanese IgA/IgG MGUS/MM patients had a pP-7 specific paraprotein (p=0.025; c2-test). The prevalence of healthy pP-7 carriers in the German population was 4/200 (2.0%), while it was only 1/178 (0.6%) in the Japanese population (p=0.376; Fisher's exact test). The relative risk for pP-7 carriers to develop MGUS/MM has an odds ratio of 7.9 (p<0.001) in the Caucasian population, while it is 6.2-fold (p=0.139) in the Japanese population. Conclusions: The fraction of pP-7 carriers with a pP-7 specific paraprotein among the patients with MGUS/MM is higher among Caucasians than Japanese patients. Nevertheless, pP-7 carrier state is associated with a similarly increased risk to develop MGUS/MM in both populations. pP-7 is the risk factor with by far the highest odds ratio to develop IgA/IgG-MGUS and multiple myeloma known to date in both ethnic groups. No significant financial relationships to disclose.

Abstract 1169: Paratarg-7, the first autosomal-dominantly inherited risk factor for hematological malignancies: single nucelotide poylmorphism- and microsatellite-based genome-wide linkage analysis

Abstract Background: Antigenic targets of paraproteins in monoclonal gammopathy of undetermined significance (MGUS), multiple Myeloma (MM) and Waldenstrom's macroglobulinemia (WM) might play a role in the pathogenesis of these neoplasms by chronic antigenic stimulation, but very few have been identified, of which most were specific for one individual paraprotein only. In contrast, we recently described paratarg-7, a protein of unknown function which is expressed in all human tissues as the target of 15% of IgA and IgG paraproteins in MGUS and MM. Mutations or polymorphisms of paratarg-7 were not found, but hyperphosphorylation was detected in 35/252 (14%) of MGUS/MM patients and in 18/161 (11%) of WM patients, all of whom had an anti-paratarg-7 specific paraprotein. Analysis of 16 families demonstrated that hyperphosphorylated paratarg-7 is inherited in a dominant fashion and carriers of hyperphosphorylated paratarg-7 have an increased risk of developing MGUS/MM/WM (odds ratio 7.9; 95% CI: 2.8-22.6, p=0.0001). The aim of this study was to identify chromosome regions linked to hyperphosphorylated paratarg-7, a frequent antigenic target of paraproteins in MGUS/MM/WM. Methods: Serum and peripheral blood cells were obtained from 90 relatives of 16 families of patients with MGUS/MM/WM. We used a high density, single nucleotide polymorphism genotyping assay, the Illumina SentrixR BeadChip Array, the HumanHap300-Duo Bead Chip, comprising 318.000 tag single nucleotide polymorphisms to perform a genome-wide linkage analysis of 48 relatives of 4 families by filtering 23211 SNP markers with a spacing of &amp;gt; 100kb and an allele frequency &amp;gt; 0.15. Additionally, we carried out non-parametric as well as parametric analyses based on the genotypes of different microsatellite markers. Results: We found strong evidence for parametric linkage (full penetrant autosomal dominant model; disease marker allele frequency=0.02; LOD= 5.9) and non-parametric linkage (Zmean= 5.1; P&amp;lt;10−5) on chromosome 4q35. Conclusions: Hyperphosphorylated paratarg-7 is a highly significant risk factor for MGUS, WM and MM, with the highest odds ratio of any risk factor reported to date for these diseases. We report the first genome-wide linkage study of a new molecularly defined risk factor of MGUS//MM/WM and novel loci on 4q35 linked to hyperphosphorylated paratarg-7. Identification of novel causative genes for MGUS/MM/WM will now advance our understanding of the pathogenesis in these diseases. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1169.

Abstract 1924: The paraproteins of patients with familial MGUS / multiple myeloma (MM) target family-specific antigens: Experience with paratarg-7 and paratarg-8

Abstract Background: Antigenic targets of paraproteins in monoclonal gammopathy of undetermined significance (MGUS) and multiple Myeloma (MM) might play a role in the pathogenesis of these neoplasms by chronic antigenic stimulation. However, very few antigens have been identified, of which most were specific for one individual paraprotein only. In contrast, we recently described paratarg-7 (P-7), a protein of unknown function which is expressed in all human tissues as the target of 15% of IgA and IgG paraproteins in MGUS and MM (Grass et al. Lancet Oncology 2009; 10: 950-956). MGUS/MM patients with P-7 specific paraproteins all carry a hyperphosphorylated variant of P-7 (pP-7), which is inherited in a dominant fashion, explaining pP7-posistive cases of familial MGUS/MM. pP-7 is the first molecularly defined autosomal-dominant risk factor for MGUS/MM and is associated with the highest odds ratio (7.9) of all risk factors for MGUS/MM known to date. Since not all cases of familial MGUS/MM have a P-7 specific paraprotein, we set out to identify the antigenic targets of paraproteins in cases of familial MGUS/MM that are not carriers of pP-7. Methods: The sera of 28 members of a family with familial MGUS/MM (Lynch et al. N Engl J Med 2008;359:152-157) were tested for antibody reactivity against antigens represented in a fetal brain derived protein macroarray using a modified SEREX approach (Preuss et al. International J. Cancer 2009;125, 656-661). Results: Sera from 28 family members were analyzed. The sera of all 6 family members affected by MGUS (n=3) and MM (n=3) reacted with paratarg-8, which is encoded by the ATG13 gene, a member of the “autophagy regulatory complex” family of genes. The titers ranged from 1:107 to 1:108. No antibody reactivity was detected in the sera of non-affected family members nor in the paraproteins from &amp;gt;300 other patients tested. Conclusions: The paraproteins of members affected by familial MGUS/MM are directed against family-specific antigenic targets, suggesting that the genetic background shared by these patients entertains a chronic auto-immune response. Paratarg-7 and paratarg-8 are the first family-specific antigens that have been molecularly defined to date. Whether the antigen/paraprotein interaction is an epiphenomeon or invovled in the pathogenesis of familial cases of MGUS/MM can now be addressed using specific molecular tools. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1924.

Abstract 4772: Hyperphosphorylated Paratarg-7, the first autosomal-dominantly inherited risk factor for hematological neoplasias: hyperphosphorylation by PKCzeta

Abstract Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins might play a role in the pathogenesis of these neoplasms. By screening a commercially available recombinant human cDNA expression macroarray we identified paratarg-7 as the antigenic target of the paraprotein from 14% (35/252) of all patients analyzed (Preuss et al. Int J Cancer 2009). The high frequency of hyperphosphorylated paratarg-7 as the antigenic target of paraproteins from MM/MGUS patients suggests that the modified protein is involved in the development of sporadic and familial MGUS/MM by acting as a chronic antigenic stimulus for the respective B-cell clones. With a relative risk of 7.9, the carrier state of hyperphosphorylated paratarg-7 (pP-7) is the strongest risk factor for the development of MGUS/MM. Moreover, the analysis of consanguineous relatives of MM/MGUS patients with an anti-paratarg-7 specific paraprotein showed that pP7 is inherited in a dominant fashion (Grass et al., Lancet Oncology, 2009). Here we present data showing that the hyperphosphorylation of paratarg-7 is due to an additional phosphorylation of this protein at the identical phosphorylation site when compared to normal paratarg-7. We identified protein kinase C zeta as responsible for this hyperphosphorylation which specifically occurs on Ser17 which is part of the 15 amino acid region identified as the paraprotein-binding epitope in all paratarg-7-immunopositive patients. In additional experiments no DNA modification of PKCzeta was detected. As shown by Western blot experiments and quantitative PCR no overexpression or copy number variation of PKCzeta was shown, suggesting that other not yet identified enzyme(s) must be involved in the process of paratarg-7 hyperphosphorylation or the maintenance of the hyperphosphorylated state. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4772.

Hyperphosphorylated Paratarg-7 Is a Frequent Antigenic Target of IgM Paraproteins, Is Dominantly Inherited and Represents a Highly Significant Risk Factor for Monoclonal Gammopathy of Undetermined Significance of the IgM Type (IgM-MGUS) and Waldenstrom's Macroglobulinemia (WM), Allowing for the Identification of Family Members at Risk in Cases of Familial IgM-MGUS and WM.

Abstract 3935Poster Board III-871 BackgroundAntigenic targets of paraproteins in MGUS, multiple Myeloma (MM) and WM might play a role in the pathogenesis of these neoplasms by chronic antigenic stimulation, but very few have been identified, of which most were specific for one individual paraprotein only. In contrast, we recently described paratarg-7, a protein of unknown function which is expressed in all human tissues as the target of 15% of IgA and IgG paraproteins in MGUS and MM (Grass et al.; Lancet Oncology 2009 in press). MethodsTo determine if and how frequently paratarg-7 functions as the antigenic target of the IgM paraproteins in MGUS/WM, sera from patients with IgM-MGUS/WM were tested for reactivity with recombinant paratarg-7 by ELISA. The specificity of the paraprotein-mediated reaction was demonstrated by absorption studies with recombinant paratarg-7 and by cloning the B-cell receptor from bone marrow cells of patients with a paratarg-7 specific paraprotein. Lysates of peripheral blood from patients and controls were tested by gel electrophoresis and isoelectric focusing before and after phosphatase treatment. Moreover, paratarg-7 cDNA was sequenced to exclude SNPs and mutations. ResultsThe paraproteins of 18 (9 WM and 9 IgM-MGUS) of 161 (11%) sera from patients in Germany, USA and Greece reacted specifically with paratarg-7, proving paratarg-7 as the first antigen identified as a paraprotein target in a significant proportion of patients with IgM-MGUS/WM. Mutations or polymorphisms of paratarg-7 were not found. However, 2D-gelelectrophoresis, isoelectric focusing and phosphatase treatment revealed that paratarg-7 was hyperphosphorylated in all patients with an anti-paratarg-7 specific IgM-paraprotein tested. In contrast, only 4 of 200 (2%) healthy blood donors were carriers of hyperphosphorylated paratarg-7. Thus, carriers of hyperphosphorylated paratarg-7 have a significantly increased risk (odds ratio= 6.5; 95%-CI: 2.1-19.6; p=0.001) for developing IgM-MGUS/MW. Moreover, family analyses of relatives of IgM-MGUS/WM patients with an anti-paratarg-7 specific paraprotein revealed that the hyperphosphorylated state of this protein is inherited as a dominant trait. The results obtained in IgM-MGUS/WM are similar to recent observations made in 252 patients with IgG- or IgA-MGUS/MM where hyperphosphorylated paratarg-7 was also associated with a significantly increased risk of developing IgG- and IgA-MGUS (odds ratio: 7.9; 95% CI, 2.8-22.6; p=0.0001). ConclusionsHyperphosphorylated paratarg-7 is a highly significant risk factor for MGUS, WM and MM, with the highest odds ratio of any risk factor reported to date for these diseases. Hyperphosphorylated paratarg-7 is the first molecularly defined and dominantly inherited risk factor identified for any hematological neoplasm reported to date. The carrier state of hyperphosphorylated paratarg-7 explains cases with familial MGUS, MM and WM and enables the identification of family members of patients at increased risk for MGUS/WM/MM. That only MGUS/WM/MM patients who are carriers of hyperphosphorylated paratarg-7 had a paratarg-7 specific paraprotein suggests that the hyperphosphorylation of paratarg-7 induces auto-immunity and is involved in the pathogenesis of these diseases, e.g. by chronic antigenic stimulation. The identification of paratarg-7 as a frequent antigenic target enables the more detailed analysis of tumor-host interactions in these patients and its role in the pathogenesis of these diseases. Moreover, its dominant inheritance and the identification of familial cases with MGUS/MM/WM and hyperphosphorylated paratarg-7 carrier state facilitate genome-wide screens for the identification of the SNP responsible for hyperphosphorylation of this molecule. Disclosures:No relevant conflicts of interest to declare.

Association of a dominantly inherited hyperphosphorylated paraprotein target with sporadic and familial multiple myeloma and monoclonal gammopathy of undetermined significance: a case–control study

Chronic antigenic stimulation might have a role in the pathogenesis of monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma. The aim of this study was to search for factors underlying the autoimmunogenicity of paratarg-7, a frequent antigenic target of paraproteins in MGUS and multiple myeloma. Between January, 2005, and February, 2009, serum and peripheral blood cells were obtained from consecutive patients with MGUS or multiple myeloma and healthy blood donors, and paratarg-7 was analysed by DNA sequencing, SDS-PAGE, isoelectric focusing, and western blotting. Mutations or polymorphisms of paratarg-7 were not noted, but hyperphosphorylation was detected in 35 (13.9%) of 252 patients with MGUS or multiple myeloma, all of whom had an anti-paratarg-7-specific paraprotein. Analysis of eight families showed that hyperphosphorylated paratarg-7 is inherited in a dominant fashion, and that carriers of hyperphosphorylated paratarg-7 have an increased risk of developing MGUS and multiple myeloma (odds ratio [OR] 7.9, 95% CI 2.8-22.6; p=0.0001). Familial MGUS and multiple myeloma were associated with a dominant inheritance of hyperphosphorylated paratarg-7, enabling family members at increased risk for MGUS or multiple myeloma to be identified. That only patients with MGUS or multiple myeloma who are carriers of hyperphosphorylated paratarg-7 had a paratarg-7-specific paraprotein suggests that the hyperphosphorylation of paratarg-7 induces auto-immunity and is involved in the pathogenesis of MGUS and multiple myeloma; for example, by chronic antigenic stimulation. Förderverein Krebsforschung Saar-Pfalz-Mosel e.V. (eingetragener Verein: officially registered charity) and HOMFOR (the research programme of the Saarland University Faculty of Medicine).