Sandra Grass

CHOP-like chemotherapy with or without rituximab in young patients with good-prognosis diffuse large-B-cell lymphoma: 6-year results of an open-label randomised study of the MabThera International Trial (MInT) Group

BACKGROUND The MInT study was the first to show improved 3-year outcomes with the addition of rituximab to a CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen in young patients with good-prognosis diffuse large-B-cell lymphoma. Extended follow-up was needed to establish long-term effects. METHODS In the randomised open-label MInT study, patients from 18 countries (aged 18-60 years with none or one risk factor according to the age-adjusted International Prognostic Index [IPI], stage II-IV disease or stage I disease with bulk) were randomly assigned to receive six cycles of a CHOP-like chemotherapy with or without rituximab. Bulky and extranodal sites received additional radiotherapy. Randomisation was done centrally with a computer-based tool and was stratified by centre, bulky disease, age-adjusted IPI, and chemotherapy regimen by use of a modified minimisation algorithm that incorporated a stochastic component. Patients and investigators were not masked to treatment allocation. The primary endpoint was event-free survival. Analyses were by intention to treat. This observational study is a follow-up of the MInT trial, which was stopped in 2003, and is registered at ClinicalTrials.gov, number NCT00400907. FINDINGS The intention-to-treat population included 410 patients assigned to chemotherapy alone and 413 assigned to chemotherapy plus rituximab. After a median follow-up of 72 months (range 0·03-119), 6-year event-free survival was 55·8% (95% CI 50·4-60·9; 166 events) for patients assigned to chemotherapy alone and 74·3% (69·3-78·6; 98 events) for those assigned to chemotherapy plus rituximab (difference between groups 18·5%, 11·5-25·4, log-rank p<0·0001). Multivariable analyses showed that event-free survival was affected by treatment group, presence of bulky disease, and age-adjusted IPI and that overall survival was affected by treatment group and presence of bulky disease only. After chemotherapy and rituximab, a favourable subgroup (IPI=0, no bulk) could be defined from a less favourable subgroup (IPI=1 or bulk, or both; event-free survival 84·3% [95% CI 74·2-90·7] vs 71·0% [65·1-76·1], log-rank p=0·005). 18 (4·4%, 95% CI 2·6-6·9) second malignancies occurred in the chemotherapy-alone group and 16 (3·9%, 2·2-6·2) in the chemotherapy and rituximab group (Fishers exact p=0·730). INTERPRETATION Rituximab added to six cycles of CHOP-like chemotherapy improved long-term outcomes for young patients with good-prognosis diffuse large-B-cell lymphoma. The definition of two prognostic subgroups allows a more refined therapeutic approach to these patients than does assessment by IPI alone. FUNDING Hoffmann-La Roche.

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

BACKGROUND 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. METHODS 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. FINDINGS 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). INTERPRETATION 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. FUNDING Förderverein Krebsforschung Saar-Pfalz-Mosel e.V. (eingetragener Verein: officially registered charity) and HOMFOR (the research programme of the Saarland University Faculty of Medicine).

A frequent target of paraproteins in the sera of patients with multiple myeloma and MGUS

Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins might play a role in the pathogenesis of these neoplasms. We screened a human fetal brain‐derived macroarray with the IgA or IgG containing sera of 192 consecutive MGUS and MM patients. Twenty‐nine of 192 (15.1%) paraproteins reacted with paratarg‐7, a protein of unknown function which is expressed in all human tissues. Paratarg‐7 reactivity was similarly frequent among IgA and IgG paraproteins, but all paratarg‐7 reactive IgG paraproteins belonged to the IgG3 subtype with 24/57 IgG3 (42.1%) paraproteins displaying this specificity. Sequence analysis of paratarg‐7 derived from patients having a paraprotein with specificity for paratarg‐7 revealed no differences to paratarg‐7 derived from patients with paraproteins of other specificities or healthy controls, excluding mutations or polymorphisms as a reason for its autoimmunogenicity. Similarly, Western‐blot analysis showed identical bands for paratarg‐7 derived from patients and controls. The above‐random frequency of paratarg‐7 as a paraprotein target suggests that paratarg‐7 might be involved in the development of the respective clonal proliferations. The identification of paratarg‐7 as an antigenic target enables the more detailed analysis of tumor–host interactions in these patients and their role in the pathogenesis of MM and MGUS.

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

We 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.

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

Paratarg-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 autoantigenic targets of paraproteins is due to inactivation of PP2A

Paratarg-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.

Autosomal-dominant inheritance of hyperphosphorylated paratarg-7.

12 www.thelancet.com/oncology Vol 11 January 2010 Correspondence Autosomal-dominant inheritance of hyperphosphorylated paratarg-7 increase our research content by 50% to an average of six articles per issue. To accommodate this change, the number of reviews per issue will decrease slightly, but other sections of the journal will continue to deliver the usual quantity of news and opinion. We believe this decision keeps the journal in touch with the growing need for evidence-based medicine. I thank you for your continued patronage and I look forward to publishing a lot more of your research in the months ahead.

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. (Cancer Sci 2011; 102: 565–568)

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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC 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.

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

Background: Antigenic targets of paraproteins in monoclonal gammopathy of undetermined significance (MGUS), multiple Myeloma (MM) and Waldenstrom9s 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 Sentrix R 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 > 100kb and an allele frequency > 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 −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.