Human gammadelta T-lymphocytes expressing Vgamma2Vdelta2 T-cell receptors (TCRs) can be stimulated by aminobisphosphonates and can kill certain tumor cells. Although germline-encoded lysine residues ...on the Jgamma1.2 segment have been demonstrated to be essential for the recognition of nonpeptide antigens by human gammadelta T-cells, the role of the junctional sequences of the TCR delta chain in the recognition of aminobisphosphonates by Vgamma2Vdelta2+ T-cells remains unknown. We examined the structure of complementarity-determining region 3 (CDR3) of Vdelta2 chains expressed by aminobisphosphonate-stimulated human gammadelta T-cells. CDR3 size-spectratyping analysis of Vdelta2 chains revealed that risedronate did not induce a CDR3 size distribution pattern of Vdelta2 cells that was distinct from that of Vdelta2 cells cultured without risedronate. The clonality of risedronate-expanded Vdelta2 T-cells was also determined by sequencing analysis, with the result that no particular consensus sequences were observed. However, most Vdelta2 T-cells freshly isolated from peripheral blood carried a distinctive junctional motif consisting of a hydrophobic amino acid residue (valine, leucine, or isoleucine Val/Leu/Ile) at conserved position 97, and this feature was not altered by risedronate stimulation. A significant proportion of Vdelta1 T-cells from the same individual had Leu at position 97, but Vdelta1 T-cells did not expand in response to risedronate. Moreover, Vdelta2 T-cells from the nonresponder against risedronate also carried a Val/Leu/Ile amino acid residue at position 97. These results suggest that the presence of a hydrophobic amino acid residue at position 97 in CDR3 of the TCR delta chain is not sufficient to account for the recognition of aminobisphosphonate by human gammadelta T-cells.
Summary
Immunosuppressive therapy has been employed as the initial treatment for acquired chronic pure red cell aplasia (PRCA), such as idiopathic, thymoma‐associated, or large granular lymphocyte ...(LGL) leukaemia‐associated PRCA, which is thought to be immune‐mediated. To explore the overall long‐term outcome following immunosuppression and to identify the risk factors for death in these disorders, we conducted nationwide surveys in Japan 2004 and 2006, and identified a total of 185 patients with acquired chronic PRCA, including 72 idiopathic, 41 thymoma‐associated and 14 LGL leukaemia‐associated cases of PRCA for whom data was available. The
present study evaluated 127 patients with these three subsets of PRCA. The median overall survival has not yet been reached in idiopathic PRCA. The estimated median overall survival times in patients with thymoma‐associated and LGL leukaemia‐associated PRCA were 142·1 and 147·8 months, respectively. Twenty‐two deaths were reported, and the response to induction therapy and relapse of anaemia were found to be associated with death. The major causes of death were infection in seven patients and organ failure in another seven patients. The results suggest that maintenance therapy and the management of infectious complications are crucial for improving the prognosis of chronic PRCA.
Background. Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by anti-platelet autoantibodies. The underlying mechanism is thought to involve the production of IgG ...autoantibodies specific for platelet membrane antigens such as glycoprotein (GP) IIb/IIIa and GPIb/IX, although the anti-platelet autoantibody testing is less sensitive. The ASH and the IWG guidelines for the management of ITP do not recommend routine testing of anti-platelet autoantibodies for the diagnosis of ITP, and thus diagnostic biomarkers for ITP remain to be developed. Although the principal pathophysiology of ITP is believed to be an IgG-mediated autoimmune disease, there are few evidence for the B-cell receptor (BCR) repertoires which are associated or related to this disorder.
Objectives. In order to identify the characteristics of IgG-BCRs that could be a potential candidate for a biomarker for primary ITP, we have investigated the comprehensive and precise repertoires of IgG-BCRs of peripheral blood B cells by using the next-generation BCR repertoire analysis comprised of high-throughput DNA sequencing and bioinformatics.
Materials and Methods. Eleven adult ITP patients and nine volunteer donors without any hematologic disorders were included in this study. Diagnostic criteria for ITP were based on the ASH and the IWG guidelines and secondary ITP was excluded. Written informed consent was obtained before drawing blood samples. Heparinized peripheral blood was taken and mononuclear cells were isolated by density gradient centrifugation. Total RNA extraction and cDNA synthesis were done by standard protocols. Unbiased amplifications of IgG-BCRs were performed by adaptor-ligation PCR and the amplicons were sequenced by next-generation sequencing. Each sequence read was analyzed by the bioinformatics software created by Repertoire Genesis Incorporation (Ibaraki, Japan), and the usage of IGHV, IGHD, IGHJ and IGHC and CDR3 sequences were determined, according to the previously reported (Iwamoto M, et al. ASH 2016, #4542). This study was approved by the institutional review board.
Results. Total 2,009,943 in-frame and 315,469 unique reads were obtained from twenty blood samples, and 29,049 to 160, 013 reads (100,497 reads in average) from each sample. Diversity of IgG BCRs was not different between the ITP patients and controls based on inverse Simpson and evenness Pielou's indexes. Comparisons of the IGHV repertoires covering fifty-nine IGHV subfamilies between the patients and controls revealed an increased usage of IGHV4-28 (0.053% vs. 0.005%, p=0.006) and a less usage of the IGHV3-15 (1.28% vs. 3.63%, p=0.04) segment in ITP patients (Fig. 1). The 220 distinct IGHV4-28-carrying sequences were identified in ITP patients and 135 out of these clones used the IGHJ4 gene segment (Fig. 2). These IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Only eight B-cell clones bearing IGHV4-28/IGHJ4 were identified in control donors. However, no identical IGHV4-28/IGHJ4-bearing B-cell clones were found across different ITP patients.
Discussion and Conclusion. The present study has demonstrated the preferential usage of the rearranged IGHV4-28/IGHJ4 gene by circulating IgG B cells in ITP. Roark et al. have previously reported the usage of IGHV3-30 by the anti-platelet immunoglobulin from two patients with ITP, demonstrated by the Fab/phage display libraries (Blood 2002;100:1388). Discordant results may be explained by the limited numbers of the patients examined or different HLA backgrounds. Although the antigen specificity of BCRs encoded by the IGHV4-28/IGHJ4 gene in the present patient cohort remains to be elucidated, our findings may contribute to the development of genetic testing for the diagnosis of ITP.
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Kitaura:Repertoire Genesis Incorporation: Employment. Suzuki:Repertoire Genesis Incorporation: Equity Ownership.
Background. Idiopathic pure red cell aplasia (PRCA) and secondary PRCA not responding to the treatment of the underlying diseases are generally thought be immune-mediated and treated by ...immunosuppressive therapy. We previously conducted the PRCA2004/2006 study and reported that poor response to induction therapy and relapse of anemia were associated with death. Principal causes of death were infections and organ failure. Based on the literatures, idiopathic PRCA may represents the prodrome to myelodysplastic syndromes. Theoretically, there are two potential mechanisms of unresponsiveness to immunosuppression; the clonal hematopoiesis by the stem/progenitor cells that have undergone somatic mutations during disease progression of PRCA and the clonal changes of auto-aggressive lymphocytes reacting against erythroid progenitors.
Objectives. In this study, we investigated the somatic mutations of myeloid malignancy-associated genes in acquired PRCA in order to determine how often clonal hematopoiesis is detected in this disorder.
Materials and Methods. This study included 23 patients with chronic acquired PRCA (12 idiopathic, 7 thymoma-, 2 LGL leukemia- and 2 systemic lupus erythematosus-associated PRCA) with a median age of 62 (range: 40-62). Disease status was varying. After obtaining informed consent, heparinized blood was drawn and mononuclear cells were separated by density gradient centrifugation. Extracted genomic DNA samples were subjected to targeted sequencing for 54 myeloid malignancy-associated genes using a TruSight Myeloid Sequencing Panel kit according to the manufacturer's instruction (Illumina). Criteria for the significant somatic mutations of myeloid malignancy-associated genes in the present study were as follows: potential functional consequences such as missense, nonsense or frameshift mutations; exclusion of previously reported SNPs; being recurrently detected in two sequencing runs; variant allele frequency (VAF) exceeding 0.02 and less than 0.40. The institutional review board approved the experimental protocol.
Results. We detected some mutations of the targeted genes in 20 out of 23 patients, and the somatic mutations defined by the criteria mentioned above were found in 10 patients including 6 idiopathic, 3 thymoma-associated and one LGL leukemia-associated PRCA (Fig. 1). These 10 patients had 38 distinct mutations in 20 genes. Variant allele frequencies were 0.02 to 0.37 (median, 0.04; average, 0.06, Fig. 2). Four patients had more than one mutated genes and multiple genes were mutated in some patients (Fig. 1). The most frequently mutated gene was CUX1 that was found in four patients, and STAG2, DNMT3A, KDM6A, SMC3A, ASXL1, TET2 and TP53 were mutated in more than one patient.
Discussion/Conclusion. This study demonstrated that myeloid malignancy-associated genes were somatically mutated in 43% of acquired chronic PRCA patients. This figure appears to exceed the prevalence rate of clonal hematopoiesis of indeterminate potential (CHIP) in the general population with the age of 60s. These mutations were presumably carried by monocytes, because DNA samples were prepared from PBMCs in this study cohort. Profiles of mutated genes in PRCA appear to be different from those of aplastic anemia that were previously reported by other groups. It is yet to be known whether this could result from the different nature of both diseases, or the difference in the experimental protocols. Our findings strongly encourage conducting a prospective study to confirm our observation and clarify the diagnostic and predictive values of somatic mutations of myeloid malignancy-associated genes in acquired PRCA. This project is ongoing in collaboration with the prospective cohort study PRCA2016 being conducted in Japan.
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Nakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Matsuda:GlaxoSmithKline K.K.: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co, Ltd.: Honoraria; Kyowa Hakko Kirin Co, Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Sanofi K.K.: Honoraria; Beckman Coulter K.K.: Honoraria. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Bristol-Myesr Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Chugai: Research Funding; Astellas: Research Funding; Sumitomo Dainippon: Research Funding; Novartis: Research Funding; Toyama Chemical: Research Funding.
A 19-year-old man with systemic sclerosis (SSc) was hospitalized for autologous peripheral blood stem cell transplantation (auto-PBSCT) due to progressive scleroderma and cardiac involvement despite ...conventional treatment. During the administration of cyclophosphamide (60 mg/kg/day for 2 days) for mobilization and collection of CD34+ selected peripheral blood stem cells, he developed congestive heart failure. Echocardiogram showed hypokinetic asynergy from the septum to posterior wall, which might indicate underlying cardiac damage. We were also concerned about the risk of high-dose cyclophosphamide-induced cardiotoxicity. Since the dose-limiting toxicity of thiotepa, an alkylating agent, is myelosuppression, and cardiac toxicity due to thiotepa is less common, we used a conditioning regimen consisting of thiotepa (10 mg/kg/day, day −5) and low-dose cyclophosphamide (50 mg/kg/day, days −3 and −2), instead of the conventional high-dose cyclophosphamide (50 mg/kg/day × 4 days/course). The post-transplant course was uneventful, and the modified Rodnan skin thickness score improved from 32 to 15. The present case report demonstrates that thiotepa can be employed as a conditioning regimen for auto-PBSCT in SSc patients with cardiac involvement in order to reduce cyclophosphamide-induced cardiotoxicity.
Pure red cell aplasia (PRCA) is characterized by normocytic normochromic anemia associated with reticulocytopenia and erythroid hypoplasia in otherwise normal bone marrow. The acquired chronic PRCA ...may present as a primary hematological disorder in the absence of any other diseases or develop associated with certain conditions such as thymoma, lymphoproliferative disorders, infections, collagen vascular diseases, or after exposure to a wide variety of drugs. Idiopathic PRCA and secondary PRCA not responding to the treatment of the underlying diseases are generally treated by immunosuppressive therapy and the most patients require long-term treatment. Glucocorticoids, cyclosporine and cyclophosphamide are commonly used for initial treatment as monotherapy or combination therapy and majority of the patients respond to immunosuppression. However, most patients treated with these medications relapse during tapering of the treatment. Because of the extreme rarity of this disease, the long-term outcome of chronic PRCA following immunosuppression remains unclear and the adverse risk factors for survival have to be elucidated.
In order to identify the adverse risk factors for survival in acquired chronic PRCA following immunosuppression and improve the prognosis, we have conducted nationwide surveys for adult acquired chronic PRCA in Japan. We conducted the first study for adult chronic PRCA in japan in 2004 and 2006 that had been diagnosed between 1990 and 2006 across 109 institutions, and collected the data on a total of 185 patients (PRCA2004/2006 study). We have also started another cohort study in 2016 in collaboration with the Japanese Society of Hematology (JSH) (PRCA2016 study) that was approved by the institutional review board and the ethical committee of the JSH.
In PRCA2004/2006 study 73 patients (39%) were classified as having idiopathic PRCA and 112 patients (61%) as having secondary PRCA, including 42 thymoma- and 14 large granular lymphocyte leukemia (LGL)-associated PRCA. The details on idiopathic, thymoma-associated and LGL-leukemia-associated PRCA have been previously reported (Sawada K, et al. Haematologica 2007;92:1021; Hirokawa M, et al. Haematologica 2008;93:27; Fujishima N, et al. Haematologica 2008;93:1555). The last questionnaires for the patients with these three subtypes of PRCA were sent to the institutions in 2012 for collecting the data on relapse, subsequent treatment and efficacy and outcome.
The PRCA2004/2006 study revealed that the median overall survival (OS) has not yet been reached in idiopathic PRCA following immunosuppression and the estimated median OS times in thymoma-associated and LGL leukemia-associated PRCA were 142 and 148 months, respectively. Twenty-two deaths were reported and the major causes of death were infection and organ failure (Hirokawa M, et al. Brit J Haematol 2015;169:879). Poor response to induction therapy and relapse of anemia were demonstrated to be associated with death and this was also confirmed by the Mantel-Byar analysis (p<0.001) (Fig. 1). The efficacy of re-induction therapy for relapsing PRCA was inferior to the initial inductive therapy (p<0.001) (Fig. 2).
With the results of this study, we have proposed the clinical guidelines for management of PRCA in 2011 and we conducted another cohort study in 2016 in collaboration with the Japanese Society of Hematology (JSH) (PRCA2016 study). The primary endpoint of this study is the OS in chronic PRCA following immunosuppression and the principal aim is to learn whether or not the prognosis would be improved. Between 2006 and 2014, 554 PRCA patients were registered in the JSH hematological disorder registry and we asked the physicians to participate in this study (Fig. 3). The potential size of patient cohort is 181, including 116 idiopathic and 65 secondary PRCA and these patients will be followed up during the next ten years. The PRCA2016 cohort study is expected to reveal the unbiased results regarding the long-term survival and quality of life in PRCA following immunosuppression.
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Fujisawa:Bristol myers Squibb: Honoraria; Beckman Coulter: Honoraria; Eisai: Honoraria; Janssen Pharmaceutical: Honoraria; Novartis: Honoraria; Astellas: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Otsuka Pharmaceutical: Honoraria, Research Funding; SHIONOGI CO, LTD: Honoraria, Research Funding; Shire plc: Honoraria; Alexion Pharmaceutical: Honoraria; Pfizer: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; MSD: Research Funding. Yonemura:Alexion Pharma GK: Honoraria, Research Funding. Kurokawa:Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding.
In the original publication of this article, “Conflict of interest” was published incorrectly. The corrected “Conflict of interest” is given below for your reading.
Plasma removal by washing is an effective approach to prevent transfusion reactions by platelet concentrates (PCs). Recently, washed PCs were released by the Japanese Red Cross Society (JRCS).
This ...retrospective multicenter study evaluated the efficacy and safety of released washed PCs (RWPCs) between September 2016 and January 2017 in Japan. The RWPCs were prepared by washing leukoreduced apheresis PCs with the platelet additive solution, BRS-A, using automated cell processors.
Clinical data were obtained from 91 patients and 1210 RWPC transfusions at 50 institutions. The median number of RWPC transfusions per patient was 8 (range, 1–91). RWPCs were used in 94.5% of the patients with a history of recurrent or severe transfusion reactions for preventing such reactions. Responses of RWPCs were evaluated as complete response (91.6%), partial response (8.2%), no-change (0.2%), and progression (0%) and overall response was equal across subgroups divided by patients’ profiles. The median corrected count increment (CCI) at 1 and 24 h post-transfusion were 13.5 (range, 1.9–35.4) × 109/L and 3.5 (range, −13 to 53.6) × 109/L, respectively, and median CCI at 24 h was 5.5 (range, −13 to 53.6) × 109/L in patients without risk factors associated with platelet transfusion refractoriness. Transfusion reactions to RWPCs were observed in only nine transfusions (0.7%), all of which were mild allergic reactions.
This study demonstrated that RWPCs were effective and safe in patients with a history of transfusion reactions. Further prospective studies on efficacy together with cost-benefit analysis in RWPCs are needed.
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