Annette Juul Vangsted

Variants in ELL2 influencing immunoglobulin levels associate with multiple myeloma

Multiple myeloma (MM) is characterized by an uninhibited, clonal growth of plasma cells. While first-degree relatives of patients with MM show an increased risk of MM, the genetic basis of inherited MM susceptibility is incompletely understood. Here we report a genome-wide association study in the Nordic region identifying a novel MM risk locus at ELL2 (rs56219066T; odds ratio (OR)=1.25; P=9.6 × 10−10). This gene encodes a stoichiometrically limiting component of the super-elongation complex that drives secretory-specific immunoglobulin mRNA production and transcriptional regulation in plasma cells. We find that the MM risk allele harbours a Thr298Ala missense variant in an ELL2 domain required for transcription elongation. Consistent with a hypomorphic effect, we find that the MM risk allele also associates with reduced levels of immunoglobulin A (IgA) and G (IgG) in healthy subjects (P=8.6 × 10−9 and P=6.4 × 10−3, respectively) and, potentially, with an increased risk of bacterial meningitis (OR=1.30; P=0.0024).

Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T‐cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid‐derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA‐DR−/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

Effect of pamidronate 30 mg versus 90 mg on physical function in patients with newly diagnosed multiple myeloma (Nordic Myeloma Study Group): a double-blind, randomised controlled trial

BACKGROUND Compared with placebo, prophylactic treatment with bisphosphonates reduces risk of skeletal events in patients with multiple myeloma. However, because of toxicity associated with long-term bisphosphonate treatment, establishing the lowest effective dose is important. This study compared the effect of two doses of pamidronate on health-related quality of life and skeletal morbidity in patients with newly diagnosed multiple myeloma. METHODS This double-blind, randomised, phase 3 trial was undertaken at 37 clinics in Denmark, Norway, and Sweden. Patients with multiple myeloma who were starting antimyeloma treatment were randomly assigned in a 1:1 ratio to receive one of two doses of pamidronate (30 mg or 90 mg) given by intravenous infusion once a month for at least 3 years. Randomisation was done by use of a central, computerised minimisation system. Primary outcome was physical function after 12 months estimated by the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (scale 0-100). All patients who returned questionnaires at 12 months and were still on study treatment were included in the analysis of the primary endpoint. This study is registered with ClinicalTrials.gov, number NCT00376883. FINDINGS From January, 2001, until August, 2005, 504 patients were randomly assigned to pamidronate 30 mg or 90 mg (252 in each group). 157 patients in the 90 mg group and 156 in the 30 mg group were included in the primary analysis. Mean physical function at 12 months was 66 points (95% CI 62·9-70·0) in the 90 mg group and 68 points (64·6-71·4) in the 30 mg group (95% CI of difference -6·6 to 3·3; p=0·52). Median time to first skeletal-related event in patients who had such an event was 9·2 months (8·1-10·7) in the 90 mg group and 10·2 months (7·3-14·0) in the 30 mg group (p=0·63). In a retrospective analysis, eight patients in the pamidronate 90 mg group developed osteonecrosis of the jaw compared with two patients in the 30 mg group. INTERPRETATION Monthly infusion of pamidronate 30 mg should be the recommended dose for prevention of bone disease in patients with multiple myeloma. FUNDING Nordic Cancer Union and Novartis Healthcare.

Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation

Individual variations in the ability to cope with DNA damage by DNA repair may be essential for the response to chemotherapy, since cancer cells from patients with an effective DNA repair may survive treatment. We have studied the effect on time to treatment failure (TTF) and overall survival (OS) of polymorphism in the DNA repair genes ERCC1, ERCC2 and XRCC3, and in the apoptotic genes PPP1R13L and CD3EAP in 348 patients with multiple myeloma undergoing autologous bone marrow transplantation. Carriers of the variant C‐allele of ERCC2 K751Q, the variant T‐allele of XRCC3 T241M and the variant A‐allele of CD3EAP G‐21A had a 1.3‐fold, 1.8‐fold and 1.9‐fold longer TTF, respectively, than homozygous wild type carriers (p = 0.006, p = 0.004, p < 0.001). The polymorphism CD3EAP G‐21A also had significant effect on OS (p < 0.045). The polymorphism ERCC2 K751Q may to be related to sex, since the prolonged TTF was only seen in women (p = 0.001). Carriers of the combination of variant alleles of ERCC2 K751Q and XRCC3 T241M had 2.8‐fold longer TTF (p = 0.0002). This indicates that suboptimal repair of both DNA mechanisms favors prolonged TTF and that polymorphism in ERCC2, XRCC3 and CD3EAP predicts the outcome for patients treated with autologous stem cell transplantation.

No influence of the polymorphisms CYP2C19 and CYP2D6 on the efficacy of cyclophosphamide, thalidomide, and bortezomib in patients with Multiple Myeloma.

BackgroundThe response to treatment varies among patients with multiple myeloma and markers for prediction of treatment outcome are highly needed. Bioactivation of cyclophosphamide and thalidomide, and biodegradation of bortezomib, is dependent on cytochrome P450 metabolism. We explored the potential influence of different polymorphisms in the CYP enzymes on the outcome of treatment.MethodsData was analyzed from 348 patients undergoing high-dose treatment and stem cell support in Denmark in 1994 to 2004. Clinical information on relapse treatment in 243 individual patients was collected. The patients were genotyped for the non-functional alleles CYP2C19*2 and CYP2D6*3, *4, *5 (gene deletion), *6, and CYP2D6 gene duplication.ResultsIn patients who were treated with bortezomib and were carriers of one or two defective CYP2D6 alleles there was a trend towards a better time-to-next treatment. We found no association between the number of functional CYP2C19 and CYP2D6 alleles and outcome of treatment with cyclophosphamide or thalidomide. Neither was the number of functional CYP2C19 and CYP2D6 alleles associated with neurological adverse reactions to thalidomide and bortezomib.ConclusionThere was no association between functional CYP2C19 and CYP2D6 alleles and treatment outcome in multiple myeloma patients treated with cyclophosphamide, thalidomide or bortezomib. A larger number of patients treated with bortezomib are needed to determine the role of CYP2D6 alleles in treatment outcome.

The polymorphism IL-1beta T-31C is associated with a longer overall survival in patients with multiple myeloma undergoing Auto-SCT

Proinflammatory cytokines are suspected to play a role in the pathogenesis of multiple myeloma (MM). Therefore, it is possible that inborn genetic variations leading to a modified expression of these cytokines will influence the outcome for these patients. We investigated 348 MM patients undergoing high-dose melphalan treatment followed by Auto-SCT and examined the influence of single nucleotide polymorphisms (SNPs) in genes involved in the inflammatory response. We found that the polymorphism IL-1β T-31C significantly influenced overall survival (OS; P=0.02) and that carriers of the variant C-allele had a significantly longer survival than homozygous wild-type allele TT-carriers (relative risk 0.6 (95% CI=0.5–0.9); P=0.008). The polymorphisms IL-6 G-174C, IL-10 C592A, PPARγ2 Pro12Ala, COX-2 A-1195G, COX-2 T8473C and NFKB1 ins/del did not influence the OS in this group of patients. Furthermore, homozygous carriers of the variant allele of IL-1β T-31C were at 1.37-fold (CI=1.05–1.80) increased risk of MM as compared with population-based controls (P=0.02). Our results indicate that IL-1β is involved in the pathogenesis of MM.

A polymorphism in NFKB1 is associated with improved effect of interferon-α maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support

Interferon-alpha as maintenance therapy after high-dose therapy for multiple myeloma has been intensively debated during the last 30 years because several clinical studies have been published with conflicting results. In the present study, the authors addressed the question, of whether inborn variation in genes involved in inflammation influence treatment outcome in multiple myeloma patients receiving interferon-alpha as maintenance therapy. The findings suggest that a polymorphism in NFKB1 may be associated with improved effect of interferon-α in multiple myeloma patients, with homozygous carriers of the wild type allele having longer survival. Background Maintenance therapy with interferon-α after high-dose treatment with stem cell support in multiple myeloma has been intensively debated. In this study, we evaluated the response to treatment with interferon-α in relation to genetic variation in genes related to inflammation. Design and Methods In a retrospective study of 296 patients with multiple myelom undergoing high-dose therapy between 1994 and 2004, 146 patients were treated with interferon-α as maintenance therapy. We tested the polymorphisms IL1B T-31C, IL6 G-174C, NFKB1-94ins/delATTG, CD3EAP G-21A and PPP1R13L IVS1 A4364G for associations with time to treatment failure and overall survival with and without interferon-α treatment. Results The wild type ins-allele of polymorphism NFKB1-94 ins/delATTG was, by multivariate Cox analysis, associated with longer time to treatment failure (p=0.01) and overall survival (p=0.0084) when tested between treatment arms and in the subgroup of patients treated with interferon-α the wild type ins-allele was associated with longer overall survival (p=0.002). In the absence of interferon-α treatment, there was no association between the polymorphisms and treatment outcome, except for patients homozygous for the wild type G allele of IL6 G-174C who survived longer (p= 0.0074) than variant allele carriers. There was no association between the polymorphisms IL1B T-31C, CD3EAP G-21A and PPP1R13L IVS1 A4364G and treatment outcome for interferon-α. Conclusions Patients who are homozygous carriers of the wild type ins-allele of the NFKB1 -94ins/delATTG polymorphism may benefit from treatment with interferon-α, in contrast to patients carrying the variant allele. This result may indicate that the effect of interferon-α treatment is dependent on the availability of nuclear factor-κB and the polymorphism in NFKB1 may, therefore, be a good prognostic marker for multiple myeloma patients on maintenance treatment with interferon-α after high-dose therapy. A prospective study of interferon-α treatment in relation to NFKB1 -94ins/delATTG is highly warranted.

Genetic variations in multiple myeloma II: association with effect of treatment.

Association studies on genetic variation to treatment effect may serve as a predictive marker for effect of treatment and can also uncover biological pathways behind drug effect. Single‐nucleotide polymorphisms (SNPs) have been studied in relation to high‐dose treatment (HDT), thalidomide‐ and bortezomib‐based therapy, maintenance treatment with interferon‐α and in relation to therapy‐related adverse effects caused by treatment. Candidate genes for prediction of effect of HDT include DNA repair genes, CYP genes and genes involved in inflammation and apoptosis such as IL1B and RAI. In thalidomide‐ and bortezomid‐based therapy, candidate genes include TNFA and genes involved in the nuclear factor kappa B pathway (NFKB2 and TRAF3), respectively. In maintenance treatment with interferon‐α, a polymorphism in gene NFKB1 is a candidate gene for prediction for effect. Adverse effect includes infection, osteonecrosis of the jaw (ONJ), venous thrombotic events (VTE) and peripheral neuropathy (PN). A SNP in MBL2 and MPO gene was associated with septicemia and a SNP in the gene CYP2C8 was strongly associated with ONJ. Several SNPs in genes encoding DNA repair, apoptosis, inflammation and genes involved in function of the nervous system have been associated with VTE induced by thalidomide and with PN induced by bortezomib. SNP analysis is simple and can be performed, e.g., on blood and buccal cells. Further analysis of SNPs in clinical trials is needed, and collaboration between scientific groups will be an advantage because SNP analysis required large number of patients.

Single nucleotide polymorphisms in the promoter region of the IL1B gene influence outcome in multiple myeloma patients treated with high-dose chemotherapy independently of relapse treatment with thalidomide and bortezomib

Little is known on the impact of polymorphisms in the IL1B gene on outcome in multiple myeloma. In a population-based study of 348 Danish myeloma patients treated with high-dose treatment (HDT), 146 patients treated with INF-α maintenance treatment, and in 243 patients with relapse, we analysed the impact on outcome of HDT, INF-α maintenance treatment, and treatment with thalidomide and bortezomib at relapse, in relation to the major identified functional polymorphisms in the promoter region of IL1B. The wild-type C-allele of IL1B C-3737T and non-carriage of the IL1B promoter haplotype TGT (−3737T, −1464G and −31T), giving high IL1B promoter activity, were associated with longer time-to-treatment failure (TTF) (HR, 1.4 (1.0–1.9) and 1.5 (1.1–2.0)) and overall survival (HR, 1.8 (1.2–2.6) and 1.6 (1.1–2.3)) after HDT. Among INF-α treated patients, a trend towards better TTF was found in patients carrying the wild-type C-allele of IL1B C-3737T (HR, 1.6 (1.1–2.4)). Furthermore, among INF-α treated patients, gene–gene interaction studies on IL1B C-3737T and NFКB1-94ins/del ATTG revealed a fourfold increase in TTF for homozygous carriers of wild-type alleles at both loci as compared to variant allele carriers at both loci. No relation to genotype and outcome was found for relapse patients treated with thalidomide or bortezomib. Our results indicate that a subpopulation of myeloma patients carrying the wild-type C-allele of IL1B C-3737T and non-carriers of the promoter haplotype TGT (−3737T, −1464G and −31T) benefit from a better outcome of HDT and INF-α treatment, an effect that may be related to the NF-κB pathway.

Smoldering multiple myeloma risk factors for progression: a Danish population‐based cohort study

Several risk scores for disease progression in patients with smoldering multiple myeloma (SMM) have been proposed; however, all have been developed using single‐center registries. To examine risk factors for time to progression (TTP) to multiple myeloma (MM) for SMM, we analyzed a nationwide population‐based cohort of 321 patients with newly diagnosed SMM registered within the Danish Multiple Myeloma Registry between 2005 and 2014. Significant univariable risk factors for TTP were selected for multivariable Cox regression analyses. We found that both an M‐protein ≥30 g/L and immunoparesis significantly influenced TTP (HR 2.7, 95%CI (1.5;4.7), P = 0.001, and HR 3.3, 95%CI (1.4;7.8), P = 0.002, respectively). High free light chain (FLC) ratio did not significantly influence TTP in our cohort. Therefore, our data do not support recent IMWG proposal of identifying patients with FLC ratio above 100 as having ultra high‐risk of transformation to MM. Using only immunoparesis and M‐protein ≥30 g/L, we created a scoring system to identify low‐, intermediate‐, and high‐risk SMM. This first population‐based study of patients with SMM confirms that an M‐protein ≥30 g/L and immunoparesis remain important risk factors for progression to MM.