Programmed death-1 (PD-1, PDCD1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CTLA4) play central roles in immune checkpoint pathways. Single nucleotide polymorphisms (SNPs) of PDCD1 and CTLA4 have been reported to be associated with susceptibility to some autoimmune diseases. However, the potential association between SNPs in these immune checkpoint genes and risk of chronic immune thrombocytopenia (cITP) remain controversial and obscure. The aims of this study were to clarify the influence of PDCD1 and CTLA4 SNPs on the risk of developing cITP and its clinical features. We obtained genomic DNA from 119 patients with cITP and 223 healthy controls; their genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Patients with cITP had a significantly higher frequency of the PDCD1 +7209 TT genotype compared with healthy controls. The CTLA4 -1577 GG genotype and CT60 GG genotype showed higher frequencies of platelet count <5 × 10 /l at diagnosis, minimum platelet count <5 × 10 /l, and bleeding symptoms. Moreover, the PDCD1 -606 AA genotype and +63379 TT genotype were significantly associated with a lower number of patients who achieved a complete response to prednisolone treatment. Our results suggest that the immune checkpoint polymorphisms may affect the susceptibility to the clinical features of cITP, and treatment response of the affected patients.
Interleukin-10 (IL-10) and IL-10 receptor (IL-10R) single nucleotide polymorphisms have been implicated in the pathogenesis of many cancers. We investigated the influence of IL-10 -592C/A, IL-10RA I224V, and IL-10RB K47E on the risk of developing multiple myeloma (MM) and the clinical features of MM. We extracted the genomic DNA from 128 MM patients and 202 healthy controls and used polymerase chain reaction-restriction fragment length polymorphism method to detect IL-10 promoter -592C/A (rs1800872), IL-10RA (rs2228055), and IL-10RB K47E (rs2834167) genotypes. Overall survival (OS) was defined as the interval from the date of diagnosis to the date of death or last clinical appointment. No statistically significant difference was observed in the genotype and allele frequencies of IL-10 -592C/A, IL-10RA I224V, and IL-10RB K47E between MM patients and healthy controls. IL-10RA II genotype was significantly associated with a hemoglobin level lower than that of IV and VV genotypes (mean ± standard deviation, 9.21 ± 2.46 vs 10.3 ± 2.33 g/dL; P = .021). IL-10 -592 AA genotype was significantly associated with OS better than that of CA and CC genotypes (median OS, 74.5 vs 46.3 months; P = .047). We observed significant differences in survival between patients treated with thalidomide and/or bortezomib and those treated with conventional treatments (median OS, 74.5 vs 38.2 months; P = .021). Therefore, we also examined the effect of IL-10 and IL-10R polymorphisms on the clinical variables and OS of patients treated with thalidomide and/or bortezomib. In addition, IL-10RB EE genotype was significantly associated with poorer survival than KK and KE genotypes (median OS, 46.3 vs 78.8 months; P = .015). Our findings indicate that IL-10 and IL-10R gene polymorphisms may not contribute to the susceptibility to MM but may be associated with the severity and prognosis of MM. In particular, IL-10RB K47E polymorphism may contribute to the poor prognosis of MM patients treated with thalidomide and/or bortezomib.
Background and Aims: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes, such as proliferation, apoptosis, and cellular damage response. Long non-coding RNA (lnc RNA), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNA are deregulated in diverse human cancers and associated with disease progression, however little is available in multiple myeloma (MM). We have previously shown that lnc RNA MALAT1 was a stress response gene associated with MM progression. We found that lnc RNA NEAT1 is also highly expressed in MM cells by transcriptome analysis with next generation sequencer (NGS). NEAT1 is recently revealed to play an important role on DNA damage response (DDR) as downstream of p53, and thereby involves in carcinogenesis. However its exact role in cancers is still in controversy. In this study, we tried to elucidate role and regulation mechanism of NEAT1 during MM progression. Materials and Methods: Total 119 MM, 47 MGUS patients and 15 controls and 9 MM cell lines are subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. NEAT1 and its longer isoform NEAT1_2 RNA expressions were determined by RQ-PCR. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. The expression levels were normalized with ACTB and calculated with delta Ct value. Whole transcriptome analysis was performed in part of the samples by using Illumina Next Seq 500. MM cell lines transfected with tet-on p53 overexpression vector or tet-on sh-RNA HSF1 were used. RNase H-activating LNA™ GapmeR antisense oligonucleotides were used to knockdown lnc RNA in vitro. Results: The expression level of NEAT1 was significantly higher in MM (median 0.97) than MGUS (median 0.31) (p<0.0001). NEAT1 level did not differ in between control (median 0.38) and MGUS (p=0.97). Although the median level was not statistically different (0.046 in MM; 0.031 in MGUS; 0.127 in control), substantial number of MM cases showed very high level of NEAT1_2. In MM samples, both NEAT1 and NEAT1_2 expression did not differ according to ISS (p=0.52, p=0.29) and cytogenetic risk group (p=0.49, p=0.203). NEAT1 and MALAT1 expression was positively correlated (r=0.632, p<0.0001 in all samples, r=0.62, p<0.0001 in MM only). NEAT1_2 was also positively correlated with MALAT1 expression (r=0.49, p<0.0001 in MM), and NEAT1 (r=0.35, p<0.0001 in MM). NEAT1 expression level and RNA structure were confirmed by transcriptome analysis with NGS. Since p53 promotes NEAT1/NEAT1_2 expression, we checked correlation in between these two genes expression levels. NEAT1 expression were positively correlated with both p53 and p21 (r=0.30, p<0.0001, r=0.41, p<0.0001). Positive correlations were also found in between NEAT1 and HSP90s (r=0.29, p=0.029 with HSP90AA1, r=0.29, p=0.029 with HSP90AB1, r=0.411, p=0.0018 with HSP90B1). NEAT1 was upregulated by MDM2 inhibitor nutlin3A in p53 wild type cell lines and by tet-on p53 overexpression in p53 null KMS11. Interestingly bortezomib and doxorubicin significantly increased NEAT1 and NEAT1_2 by 5-10 folds in MM cell lines even in p53 null KMS11. HSP90 inhibitors did not affect NEAT1/NEAT1_2 expression, but inhibition of HSF1, which is upstream transcription factor of HSP90, by either HSF1 inhibitor KNK437 or tet-on sh-HSF1 attenuated NEAT1/NEAT1_2 expression induced by bortezomib. NEAT1 knockdown by GapmeR did not affect cell growth. Overall survival and progression free survival of the newly diagnosed MM patients did not differ in between high and low NEAT1/NEAT1_2 expression. Conclusion: Our results revealed that NEAT1/NEAT1_2 are regulated by heat shock pathway in addition to p53 pathway. Positive correlations of NEAT1 expression level with HSP90s level and existence of heat shock element in NEAT1 promoter region support this model. Considering the role of NEAT1/NEAT1_2 in DDR, our result suggests that this lncRNA may involve MM progression via damage response. Further studies elucidating roles of NEAT1 and other lncRNAs in MM contributes to development of novel therapy as well as to understand MM pathogenesis. Disclosures Tsukamoto: Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.
[Introduction] Immune thrombocytopenia (ITP) is an autoimmune disorder showing T helper type1 (Th1) cytokine polarization. Second-line treatment for ITP is generally used for patients with persistent thrombocytopenia and bleeding tendency after steroid treatment. We have recently shown Th1/Th2 cytokine polymorphisms affect the risk of chronic ITP. However, no previous study has demonstrated the association between Th1/Th2 cytokine polymorphisms and treatment response for chronic ITP. We explored the influence of Th1/Th2 cytokine polymorphisms to the clinical features and treatment response in patients with chronic ITP. [Patients and Methods] The present study included 126 Japanese patients (92 female and 34 male, median age: 47.7, range: 2.4-82.3 years) diagnosed with chronic ITP according to the criteria of ITP International Working Group. The patient characteristics were summarized in Table1. We examined Th1/Th2 cytokine polymorphisms: INF-g +874T/A, INF-gR -611G/A, IL-4 -590C/T, IL-4Ra Q576R, IL-10 -592C/A, IL-10RA I224V, and IL-10RB K47E using by PCR based method and direct sequencing. Clinical characteristics, including age, gender, platelet number, bleeding tendency, treatment response were also investigated. Second-line treatment was defined as additional therapy after steroid treatment. This study was approved by the Institutional Review Board of Gunma University Hospital. [Results] We divided chronic ITP patients (n=126) into 2 groups: the patients who received second-line treatment (SL group, n=36) and the patients who did not receive second-line treatment (non-SL group, n=90). In SL-group, the splenectomy was performed in 19 patients (51.4%) and thrombopoietin receptor agonists were given in only 7 patients (19.4%). The clinical characteristics and Th1/Th2 cytokine genotype frequencies of SL group and non-SL group were shown in Table1. The patients in SL group showed severer thrombocytopenia at diagnosis (SL group: median 12.0~109/L vs. non-SL group: median 31.0~109/L, p value=0.001). However, no significant difference was observed between SL group and non-SL group in gender, age, or bleeding tendency. As compared to non-SL group, SL group had significantly higher frequencies of IL-4 -590 CC genotype (low expression type) (SL group: 25.0% vs. non-SL group: 8.9%, p=0.02) and IL-10RB K47E non-EE genotype (low expression type) (SL group: 86.1% vs. non-SL group: 66.7%, p=0.03). Multivariate analysis of requirement for second-line treatment showed an independent significance of IL-4 -590C/T CC genotype (p=0.01) and severe thrombocytopenia (p<0.05). Furthermore, IL-10RB K47E non-EE genotype was associated with thrombocytopenia during the clinical course and requirement for second-line treatments in all patients with ITP. [Conclusion] Our data suggest that Th2 cytokine polymorphisms, IL-4 -590 CC genotype and IL-10RB K47E, increase the requirement of second-line treatment for chronic ITP. Disclosures No relevant conflicts of interest to declare.
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