Pure Red Cell Aplasia Patients Research Articles

Exploration of B and T Cell Receptor Repertoires Reveals Distinct Mechanisms in Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and Aplastic Anemia

Background: Pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA) and aplastic anemia (AA) are immune-mediated diseases that mainly attack erythrocyte or erythroid progenitor cells. This study aimed to investigate changes related to autoimmunity in the B cell receptor (BCR) and T cell receptor (TCR) repertoires in these diseases. Methods: Patients with newly diagnosed primary PRCA, AIHA, and AA and healthy individuals with matched age and sex (normal controls, NC) were recruited. Peripheral blood was collected, and BCR and TCR repertoires were sequenced by next-generation immunosequencing. The diversity and clonal expansion of the repertoires, gene and gene combination usage, somatic hypermutation (SHM) of BCR, and the sequence length, hydrophobicity, motifs of the most diverse epitope encoded (complementary-determining region 3, CDR3), T cell clustering, and BCR light chain and TCRα chain were analyzed. Results: 10 PRCA, 10 AIHA, 10 AA, and 7 NC were finally enrolled. For the broad repertoire metrics analysis, only the TCR repertoire of the AA group was more diverse compared with NC (p＜0.05). For BCR and TCR repertoires, PRCA, AIHA, and AA showed uniform characteristics in gene usage. The preferential gene usage of PRCA immunoglobulin heavy (IGH) chains such as IGHV3-23, IGHV3-74 were correlated with memory cells and red blood cell antigen recognition; the higher usage of IGHV4-31 in the AIHA group was associated with secretion of auto-antibodies; whereas AA patients had more gene usage of neutralizing antibodies (p＜0.05). In the gene usage in T cell receptor β (TRB) chains, PRCA patients had skewed usage of genes associated with T cell dysregulation and hyperinflammation such as TRBV1 and TRBV11-2; the increased gene usage in AIHA and AA were also found in other autoimmune diseases, which correlated with abnormal antigen recognition. PRCA and AA also had specific TRBV-J gene combination usage. Only PRCA had higher BCR SHM compared with NC (p＜0.05). The length of CDR3 was similar but the hydrophobicity was different among different disease groups. 12, 28, and 22 motifs were found respectively in BCR of PRCA, AIHA, and AA; and 13, 6, and 6 motifs were found in TCR. Compared with NC, PRCA, AIHA, and AA showed a considerable lack of physiology T cell clusters, and 147, 98, and 141 disease-specific T cell clusters in each disease was found, respectively. For light chain of BCR, PRCA and AIHA had the tendency of more usage of κ chains, whereas AA had more usage of λ chains. For TCRα chains, the three diseases used more genes related to hypersensitivity reactions (p＜0.05). Conclusion: PRCA, AIHA, and AA had different BCR and TCR repertoire characteristics in gene and gene combination usage, hydrophobicity and motifs of CDR3, and T cell clustering, which might contribute to autoimmune antigen recognition. The abnormalities were mainly T cell-related for PRCA, B cell-related for AIHA and both for AA. Keywords: pure red cell aplasia; autoimmune hemolytic anemia; aplastic anemia; BCR; TCR

STK10 Mutation Block Erythropoiesis in Acquired Pure Red Cell Aplasia Via Down-Regulated the Ribosome Biosynthesis

Background: Acquired pure red cell aplasia (PRCA) is anemia associated with the absence of erythroblasts and characterized by persistent and easy recurrence. However, the underline mechanisms of acquired PRCA remain obscure, and the role of gene mutation in the pathogenesis of acquired PRCA was not elucidated yet. Aims: To identify gene mutations in acquired PRCA patients and their possible effects on the origin of the disease. Methods: Blood and buccal genome DNA extracted from thirty newly diagnosed patients with acquired PRCA were detected with whole exome sequencing. The candidate genes with high frequency in acquired PRCA but the low frequency in 1000 normal genomes which may affect protein function were selected. The pGreen-CMV-puro system was employed to generate lentivirus expressing short hairpin RNAs to target the candidate genes, and stable transfected K562 cell lines with silenced candidate genes were constructed. The erythroid and megakaryocytic differentiation was evaluated for the transfected K562 cell lines either by benzidine staining hemoglobin and CD235a expression or CD41 expression on the cell surface. STK10 gene was selected which affects the erythropoiesis. The influence of the STK10 gene on the expression of p53 mRNA/ protein was also detected. The RNA sequencing in STK10 silenced K562 cells was performed and KEGG enrichment was analyzed. Next, ribosome RNA synthesis was detected, and ribosome proteins and p53 signaling pathway were also detected by western blotting. Results: Through whole exome sequencing in patients with acquired PRCA, we confirmed that STK10 gene mutation is common in acquired PRCA patients, the mRNA/protein expression of STK10 was reduced and p53 increased in the bone marrow of the patients in which the gene mutated. Furthermore, the silence of the STK10 gene through the lentiviral vector harboring short hairpin RNAs in K562 cells could inhibit the erythropoiesis after being induced by Hemin. Whereas, CD41 expression was similar in STK10 silenced K562 cells to control K562 cells after being inducted by phorbol 12-myristate 13-acetate. We performed RNA sequencing in STK10 silenced K562 cells. KEGG enrichment analysis of differentially expressed ribosome biosynthesis pathway and p53 signaling pathway was affected. 28S and 18S in ribosome RNA synthesis impaired in these STK10 silenced K562 cells through RNA electrophoresis. Further, through the western blotting test in STK10 silenced K562 cells, we found ribosome proteins expression down-regulated and p53, phospho-p53 and p21 expression up-regulated due to STK10 mutated. Conclusion: STK10 gene mutation is common in patients with acquired PRCA and causes the reduction of mRNAs and protein expression in the mutated patients. The underlying research revealed STK10 gene mutation could affect the ribosome biosynthesis pathway and down-regulated the ribosome protein level, contributing to the abnormal erythropoiesis. This research elucidated the pathogenesis of PRCA, which may provide evidence for precise diagnosis and exploring potential therapeutic targets in patients with acquired PRCA. Keywords: pure red cell aplasia, mutation, erythropoiesis, STK10, ribosome biosynthesis

High TOX expression on CD8+ T cells in pure red cell aplasia.

Thymocyte selection-associated high-mobility group box protein (TOX) is an important molecule regulating the development and exhaustion of T lymphocytes. Our aim is to investigate the role of TOX in the immune pathogenesis of pure red cell aplasia (PRCA). TOX expression of CD8+ lymphocytes from the peripheral blood of patients with PRCA was detected by flow cytometry. Additionally, the expression of immune checkpoint molecules PD1 and LAG3 and cytotoxic molecules perforin and granzyme B of CD8+ lymphocytes was measured. The quantity of CD4+CD25+CD127low T cells was analyzed. TOX expression on CD8+ T lymphocytes in PRCA patients was significantly increased (40.73 [Formula: see text] 16.03 vs. 28.38 [Formula: see text] 12.20). The expression levels of PD1 and LAG3 on CD8+ T lymphocytes in PCRA patients were significantly higher than those in the control group (34.18 [Formula: see text] 13.26 vs. 21.76 [Formula: see text] 9.22 and 14.17 [Formula: see text] 13.74 vs. 7.24 [Formula: see text] 5.44, respectively). The levels of perforin and granzyme in CD8+ T lymphocytes of PRCA patients were 48.60 [Formula: see text] 19.02 and 46.66 [Formula: see text] 25.49, respectively, which were significantly higher than those of the control group (31.46 [Formula: see text] 7.82 and 16.17 [Formula: see text] 4.84, respectively). The number of CD4+CD25+CD127low Treg cells in PRCA patients was significantly decreased (4.30 [Formula: see text] 1.27 vs. 1.75 [Formula: see text] 1.22). In PRCA patients, CD8+ T cells were activated and exhibited overexpression of TOX, PD1, LAG3, perforin, and granzyme B, while regulatory T cells decreased. These findings suggest that T cell abnormality plays a critical role in the pathogenesis of PRCA.

Clinical Significance of a Miniscule GPI(-) Granulocyte Population in Patients with Bone Marrow Failure

[Background] Small (0.003-1.0%) populations of glycosylphosphatidylinositol (GPI)-deficient granulocytes (Grs) (sPNH-GPs) are often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes, and are thought to represent a marker of immune-mediated bone marrow (BM) failure. On the other hand, our recent studies revealed that miniscule (<0.003%) GPI(-) Gr populations (mPNH-GPs), which are derived from mono- or oligo-clonal PIGA-mutated hematopoietic stem cells (HSCs), are detectable in the peripheral blood (PB) of 80% of healthy individuals (HIs) and some cord blood (CB) samples (ASH 2021, #2181). The high prevalence of mPNH-GPs in HIs and the similarly high (60%) prevalence of PNH-phenotype Grs in AA patients suggest that the proliferation of PIGA-mutated HSCs may inevitably occur in immune-mediated AA. However, our earlier study failed to detect mPNH-GPs in some AA patients in long-term remission who possessed HLA-class I allele-lacking (HLA[-]) Grs after immunosuppressive therapy (IST) (unpublished data). In patients with long-standing AA, the prevalence of mPNH-GPs may be lower than that in HIs due to the replacement of mPNH-GPs by other aberrant Grs. [Methods] To test our hypothesis, we examined PB samples from 40 patients who developed AA 1-43 (median 11) years previously and were all negative for sPNH-GPs for the presence of mPNH-GPs. At the time of sampling, 28 including 6 patients possessing HLA(-) Grs were in remission after IST (n=8), thrombopoietin-receptor agonists (TPO-RAs, n=14), anabolic steroids (AS) (n=3) after failing IST, or spontaneous resolution (n=3). Eight patients were refractory to all therapies, 3 of which were transfusion dependent. The remaining 4 with non-severe AA had not been treated. Four patients with pure red cell aplasia (PRCA) who had a 12-32 year history of anemia responding to cyclosporine (CsA) were included in this analysis. PB samples from 93 HIs and 22 CB served as controls, and samples from a patient with chronic myeloid leukemia (CML) were used as a positive control for clonal hematopoiesis. mPNH-GPs were deemed positive when 4 or more CD11bhigh dots that formed tight clusters were present in the FLAER- fraction of Grs that were enriched using microbeads coated with PE-labeled anti-CD55 and anti-CD59 antibodies. [Results] We first determined the prevalence of mPNH-GPs in HIs (n=93) at different ages. The prevalence was approximately 80% in all different age groups with the exception of the CB, which showed a prevalence of 40.9% (Figure 1). In contrast, mPNH-GPs (0.101-1.998x10-3%) were detected only in 7 (17.5%) of the 40 patients with a long history of AA (Figure 1). These 7 patients were refractory to IST but were in remission at the time of sampling after responding to TPO-RAs (n=3, Figure 2) and AS (n=1); while 3 experienced spontaneous resolution of AA after cessation of treatment. Of the 33 AA patients who were negative for mPNH-GPs, only 8 were in remission, and 5 of the 8 possessed HLA(-) Grs that accounted for 75%-99% (median 96%) of the total Grs (Figure 2). All 4 patients with PRCA who were negative for sPNH-GPs possessed mPNH-GPs, despite the fact that they responded to CsA. The lack of mPNH-GPs in AA patients with complete clonal hematopoiesis with HLA(-) Grs prompted us to examine a CML patient whose Grs were 100% BCR-ABL-positive. mPNH-GPs were undetectable at the diagnosis, but became detectable 2 weeks after the initiation of nilotinib therapy, and increased after 6 months when the patient achieved cytogenetic remission (Figure 2). [Discussion] The persistence of mPNH-GPs in AA patients who failed to respond to IST suggests that immune mechanisms may have been not involved in the patients in whom GPI-deficient Grs remained miniscule. Given that mPNH-GPs were undetectable in AA patients possessing HLA(-) Grs and in many patients with long-standing AA refractory to IST, the presence of mPNH-GPs may be a marker of oligo- or poly-clonal hematopoiesis, which can be eliminated by the clonal expansion of HLA(-) HSCs or some other HSCs with intrinsic abnormalities in patients with long-standing AA, findings that are compatible with the absence of mPNH-GPs at the diagnosis in a CML patient. Lastly, the presence of mPNH-GPs without expansion in PRCA patients responding to CsA suggests that immunopathogenesis of PRCA is distinct from that of CsA-responsive AA, which turns mPNH-GPs into sPNH-GPs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T cell clonal expansion and STAT3 mutations: a characteristic feature of acquired chronic T cell-mediated pure red cell aplasia

Acquired chronic pure red cell aplasia (PRCA) develops idiopathically or in association with other medical conditions, including T cell large granular lymphocytic leukemia (T-LGLL) and thymoma. T cell dysregulation is considered a cardinal pathogenesis of PRCA, but genetic-phenotypic associations in T cell abnormalities are largely unclear. We evaluated an extended cohort of 90 patients with acquired PRCA, including 26 with idiopathic, 36 with T-LGLL-associated and 15 with thymoma-associated PRCA, for their T cell immuno-phenotypes, clonalities and STAT3 mutations. TCR repertoire skewing of CD8+ T cells was detected in 37.5% of idiopathic, 66.7% of T-LGLL-associated and 25% of thymoma-associated PRCA patients, and restriction to Vβ1 was most prominent (41%). Clonalities of TCRβ or γ chain and STAT3 mutational status were statistically associated (P = 0.0398), and they were detected in all three subtypes. The overall response rate to cyclosporin A was 73.9%, without significant difference by subtypes nor STAT3 mutational status. The T cell dysregulations, such as TCR repertoire skewing with predominant Vβ1 usage, clonality and STAT3 mutations, were frequently found across the subtypes, and the close associations between them suggest that these T cell derangements reflect a common pathophysiological mechanism among these PRCA subtypes.

Isolated anemia in patients with large granular lymphocytic leukemia (LGLL)

Patients with large granular lymphocytic leukemia (LGLL) frequently present with neutropenia. When present, anemia is usually accompanied by neutropenia and/or thrombocytopenia and isolated anemia is uncommon. We evaluated a cohort of 244 LGLL patients spanning 15 years and herein report the clinicopathologic features of 34 (14%) with isolated anemia. The patients with isolated anemia showed a significantly male predominance (p = 0.001), a lower level of hemoglobulin (p < 0.0001) and higher MCV (p = 0.017) and were less likely to have rheumatoid arthritis (p = 0.023) compared to the remaining 210 patients. Of the 34 LGLL patients with isolated anemia, 13 (38%) presented with pure red cell aplasia (PRCA), markedly decreased reticulocyte count and erythroid precursors, and more transfusion-dependence when compared to non-PRCA patients. There was no other significant clinicopathologic difference between PRCA and non-PRCA patients. 32 patients were followed for a median duration of 51 months (6–199). 24 patients were treated (11/11 PRCA and 13/21 non-PRCA patients, p < 0.02). The overall response rate to first-line therapy was 83% [8/11 (72.7%) for PRCA, 12/13 (92.3%) for non-PRCA], including 14 showing complete response and 6 showing partial response with a median response duration of 48 months (12–129). Half of non-PRCA patients who were observed experienced progressive anemia. During follow-up, no patients developed neutropenia; however, 5/27 (18.5%) patients developed thrombocytopenia. No significant difference in overall survival was noted between PRCA and non-PRCA patients. In summary, this study demonstrates the unique features of LGLL with isolated anemia and underscores the importance of recognizing LGLL as a potential cause of isolated anemia, which may benefit from disease-specific treatment. LGLL patients with PRCA were more likely to require treatment but demonstrated similar clinicopathologic features, therapeutic responses, and overall survival compared to isolated anemia without PRCA, suggesting PRCA and non-PRCA of T-LGLL belong to a common disease spectrum.

Clinical and pathophysiological features of acquired pure red cell aplasia: based on the concept of T-cell dysregulations

Acquired pure red cell aplasia (PRCA) develops in a variety of contexts and thus, should be regarded as a syndrome. The three major subtypes of acquired PRCA are idiopathic PRCA, T cell large granular lymphocytic leukemia (T-LGLL)-associated PRCA, and thymoma-associated PRCA. Although the Japanese National Research Group on Idiopathic Bone Marrow Failure Syndromes of the Ministry of Health, Labor and Welfare of Japan has made significant contributions to our understanding of PRCA, details of its clinical characteristics, and pathophysiological mechanisms remain largely unknown. A recent epidemiological analysis using the JSH Hematologic Disease Registry revealed that approximately 100 new cases with acquired PRCA were diagnosed annually in Japan, which was higher than previously thought to be. The median age of the patients was 73 years. A prospective observational study on chronic PRCA (PRCA2016) is currently ongoing, and it may provide new clinical insights into acquired PRCA. Dysregulation of T cells has been shown to play a central role in PRCA. We studied T cell clonalities and STAT3 mutations in 90 PRCA patients and discovered that clonal T cell expansions were frequently recognized and closely associated with STAT3 mutations in the three major types of PRCA.

Identification of Adverse Risk Factors for Survival in Acquired Pure Red Cell Aplasia Receiving Immunosuppressive Therapy By the Japan PRCA2004/2006 Study and Introduction to the New Cohort Study PRCA2016

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. [Display omitted] DisclosuresFujisawa: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.

Clonal hematopoiesis in adult pure red cell aplasia

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.

Gene mutation profile in patients with acquired pure red cell aplasia.

Acquired pure red cell aplasia (PRCA) is a disorder characterized by normocytic anemia associated with reticulocytopenia and an absence of erythroblasts. The gene mutation profile in acquired PRCA is not defined yet. In this study, we aimed to identify the gene mutation spectrum of patients with acquired PRCA and the correlation between gene mutations and response to immunosuppressive therapy (IST). Thirty newly diagnosed acquired PRCA patients were enrolled in this study, and then whole-exome sequencing were performed among these patients and a panel with 93 candidate genes which associated with other bone marrow failure for the following analysis. Subsequently patients were treated with IST for at least 2years. When treated with IST, there were thirteen complete response, ten partial response (ORR 76.7%), and seven no response at a medium of 8 (6-10)months. Totally twenty-three mutations in fifteen genes were detected in sixteen patients (53%). The mutated genes were associated with transcription, signal transduction, and epigenetic regulation pathways. The most frequent transitions in the point mutations were C > T. Age, gender, hemoglobin level at diagnosis, and gene mutation or not did not influence the response to IST. However, although patients with BCOR or BCORL1 mutations had a similar response to IST compared with those without mutation (P = 0.235), they had a better response than those with other gene mutations (P = 0.0193). In conclusion, patients with acquired PRCA may have clonal gene mutations. The patients with BCOR and BCORL1 mutations may suggest a better response to IST compared with those with other mutations.

Immunosuppression therapy is effective for both acquired tumor-associated and primary pure red cell aplasia: a match pair case-control study.

No agreement had been reached on the treatment of patients with pure red cell aplasia (PRCA) secondary to indolent malignancies. Data was collected from patients with acquired PRCA from May, 2014 to May, 2018 in Peking Union Medical College Hospital. Tumor-associated PRCA and primary PRCA patients were matched at a ratio of 1:2 with compatible baseline characteristics. All patients had been treated with CsA or sirolimus for at least 6 months with the efficacy and adverse events recorded. Twelve tumor-associated PRCA patients (3 thymoma, 8 lymphoproliferative disorders, and 1 smoldering multiple myeloma) with stable underling disease and 24 acquired primary PRCA patients were selected. 83.3% tumor-associated PRCA patients and 100% primary PRCA patients (P = 0.436) responded to immunosuppression therapy (IST) at a median of 2.5 and 3.5 months (P = 0.137), respectively. No different was found in side effects. The ORR at the end of a median of 21.5-month follow-up was 75% and 70.8% (P = 0.795), respectively. No tumor progression was reported except one secondary patient had lymphoma relapse after 2 years of IST and was given chemotherapy again. These results suggested IST had similar effect, safety on patients with tumor-associated, and primary PRCA patients when the tumors were stable.

Etiologies and Treatment Burden in Adult Patients with Pure Red Cell Aplasia: A Single-Center Experience and Review of Literature.

Background Pure red cell aplasia (PRCA) is less common blood disorder; the causes and the treatments of PRCA are varied. Methods We conducted a retrospective study during January 2010–December 2017, to explore the etiologies and to evaluate the response and treatment burden in adult patients with PRCA. Results Of 32 PRCA patients, median age was 57 years (18–90 years). Median hemoglobin level and reticulocyte count at the time of diagnosis were 5.6 g/dL (3.3–7.3 g/dL) and 0.3% (0.1–0.7%), respectively. Median time to hematologic recovery was 12 weeks (3–72 weeks), and median number of red blood cell transfusion (RBC) was 20 units (4–100 units). Causes of PRCA were erythropoiesis-stimulating agent (ESA) (47%), parvovirus B19 infection (19%), thymoma (13%), zidovudine (6%), primary autoimmune PRCA (6%), Kaposi's sarcoma (3%), systemic lupus erythematosus (3%), and ABO-mismatched stem cell transplantation (3%). Only 9 out of 24 treated patients achieved hematologic response within 8 weeks of treatment. Intravenous immunoglobulin therapy provided 100% response rate in patients with parvovirus B19-associated PRCA and primary autoimmune PRCA. Low response rate was found in patients receiving immunosuppressants and chemotherapy for the treatment of ESA and thymoma-associated PRCA, respectively. Conclusions Treatment outcome of PRCA depended upon the causes and the types of treatment, and the burden of RBC transfusion was very high in patients with ESA and thymoma-associated PRCA.

Successful sirolimus treatment of patients with pure red cell aplasia complicated with renal insufficiency.

For patients with pure red cell aplasia (PRCA), cyclosporine (CsA) is the first line therapy. Occasionally, some patients who suffer from renal insufficiency cannot tolerate CsA. To explore the efficacy and tolerance of sirolimus treatment for those patients, twelve PRCA patients with renal insufficiency from May 2014 to May 2018 in Peking Union Medical College Hospital were enrolled, treated with sirolimus, and followed up at the median time of 16 (10-50) months. Eleven patients (91.7%) responded to sirolimus, with 58.3% complete response (CR) and 41.7% partial response (PR). The median time to achieve the optimum effect was 4 (1-7) months. The serum creatinine level remained stable or even reduced during the treatment period for eleven patients. Seven patients (58.3%) reported adverse events during sirolimus therapy, including increased blood glucose, infection, skin rash, elevated triglyceride or total cholesterol, and elevated serum creatinine compared with baseline. No treatment-related death was noticed during the follow-up time. Three patients relapsed with an overall response rate of 75.0% at 1year. These results suggested that sirolimus was effective and tolerable for patients with PRCA complicated with renal insufficiency.

Recent progress in the diagnosis and management of acquired pure red cell aplasia

Acquired pure red cell aplasia (PRCA) is characterized by normocytic anemia, reticulocytopenia, and a marked decrease in erythroid cell count in the bone marrow. PRCA develops in the context of various backgrounds, including recently recognized immune checkpoint inhibitor-associated PRCA, that need careful differential diagnoses. Besides humoral abnormalities such as major ABO-incompatible allogeneic hematopoietic stem cell transplantation-related PRCA, dysregulations of T cells have been shown. STAT3 gene mutations of cytotoxic T cells were identified in 40% of PRCA patients, which might suggest their use as novel molecular markers for PRCA. As initial management options for PRCA, red blood cell transfusion and immunosuppressive therapy (IST) drugs, such as cyclosporin, are usually selected. Roughly 80% of patients respond to IST; however, some relapse afterward or are refractory to IST. When patients with PRCA become refractory to two or three lines of IST, allogeneic hematopoietic stem cell transplantation (HCT) would become an appropriate choice, although the optimal procedures for allogeneic HCT have not been determined. A prospective study of PRCA in Japan has been ongoing since 2016 to solve the myriad clinical issues of PRCA.

Frequent STAT3 Mutations in CD8+ T Cells from Patients with Pure Red Cell Aplasia

Background: Dysregulation of T-cell mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure (BMF) remains unknown.Methods: Patients with AA, AA-paroxysmal nocturnal hemoglobinuria (AA-PNH) syndrome, MDS or PRCA were enrolled in this study. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells using an allele-specific PCR (AsPCR) to detect STAT3 Y640F or D661Y mutations and amplicon sequencing using primers covering entire coding region of STAT3. CD4+ T cells, CD8+ T cells or granulocytes were sorted, if possible, and also subjected to the analyses. The T-cell receptor (TCR) Vβ repertoire was analyzed using whole blood samples by flow cytometry.Results: A total of 124 patients including PRCA (n=42), AA (n=54), AA-PNH (n=7) and MDS (n=21) were enrolled. The subtypes of PRCA were as follows: idiopathic, n= 15, T-LGLL-associated, n=13; thymoma or thymic cancer-associated, n=7; autoimmune disease-associated, n=5; adverse drug reactions, n=1; and human parvovirus B19 infection complicated by T-LGLL, n=1. As an initial step in the STAT3 mutational analysis, we screened all patients with an AsPCR. Among the 42 patients with PRCA, 3 (10%) of 29 patients without T-LGLL and 6 (46%) of 13 patients with T-LGLL were positive for mutations. In contrast, none of the patients with AA, MDS or AA-PNH were positive (P value= 0.000031). Then, we examined MNC-derived DNA from 73 patients (PRCA, n=42 and AA, n=31) using amplicon sequencing. In this sequencing analysis, the median depth of coverage was 6,219x (range; 1,065-14,188). No STAT3 mutations were detected in 31 AA patients. In contrast, 15 (36%) PRCA patients possessed STAT3 mutations. The variant allele frequency of STAT3 mutations ranged from 0.0057 to 0.489. In all of the 7 patients studied, the STAT3 mutations were restricted to sorted CD8+ T cells. Three patients were negative for STAT3 mutations in MNCs but found to be positive when sorted CD8+ T cells were analyzed. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder-associated and T-LGLL-associated PRCA was 33% (5/15), 29% (2/7), 20% (1/5), and 77% (10/13), respectively. In total, STAT3 mutations were detected in 8 of 29 (28%) PRCA patients without T-LGLL and 10 of 13 (77%) PRCA patients with T-LGLL. When TCRVβ repertoires of CD8+ T cells sorted from 3 STAT3 mutation(+) PRCA patients without T-LGLL were analyzed, skewed TCRVb repertoires were evident in all patients, and STAT3 mutations were detected in skewed TCRVβ fractions from 2 patients whose samples were available for cell sorting. The STAT3-mutation(+) patients were younger (median age of 63 years vs 73 years, P= 0.026) and less responsive to cyclosporine (CsA) (46% [6/13] vs 100% [8/8], P= 0.0092) in comparison to STAT3-mutation(-) patients. Of note, 4 of 8 STAT3 mutation(+) patients who had been refractory to CsA were treated with CY and all of them responded well, whereas none of 8 STAT3 mutation(-) patients required secondary treatment with CY owing to a sustained response to CsA.Discussion: This study is the first to reveal frequent STAT3 mutations in PRCA patients, even in thouse without T-LGLL, using a large cohort of patients. The presence of STAT3-mutated CD8+ T cells may be unique background of PRCA, irrespective of disease etiology. Poor response to CsA in STAT3 mutation(+) patients suggests that STAT3-mutated CD8+ T cells may be less sensitive to the inhibitory effects of CsA than non-mutated CD8+ T cells. In contrast, our analyses failed to detect STAT3 mutations in any of 52 AA patients, suggesting that STAT3 mutation(+) T cells had little impact on the development of AA in Japanese patients.Conclusion:STAT3 mutations are frequently detected in the CD8+ T cells of PRCA patients, regardless of the presence of T-LGLL. The identification of STAT3 mutations may be useful for appropriately managing patients with PRCA. DisclosuresNakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Somatic Mutations of Myeloid Malignancy-Associated Genes in Acquired Pure Red Cell Aplasia in Adults

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. [Display omitted] DisclosuresNakao: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.

Frequent STAT3 mutations in CD8+ T cells from patients with pure red cell aplasia

AbstractDysregulation of T-cell–mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and which is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure remains unknown. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells from PRCA patients (n = 42), AA (n = 54), AA–paroxysmal nocturnal hemoglobinuria (AA-PNH; n = 7), and MDS (n = 21) using an allele-specific polymerase chain reaction and amplicon sequencing. STAT3 mutations were not detected in any of the 82 patients with AA/PNH/MDS but were detected in 43% of the 42 PRCA patients. In all 7 STAT3-mutation–positive patients who were studied, the STAT3 mutations were restricted to sorted CD8+ T cells. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder–associated, and T-LGLL–associated PRCA was 33% (5 of 15), 29% (2 of 7), 20% (1 of 5), and 77% (10 of 13), respectively. The STAT3-mutation–positive patients were younger (median age, 63 vs 73 years; P= .026) and less responsive to cyclosporine (46% [6 of 13] vs 100% [8 of 8]; P= .0092) in comparison with STAT3-mutation–negative patients. The data suggest that STAT3-mutated CD8+ T cells may be closely involved in the selective inhibition of erythroid progenitors in PRCA patients.

Pure red cell aplasia associated with thymoma: a report of a single-center experience.

Pure red cell aplasia (PRCA) associated with thymoma is relatively rare, and relevant reports are limited. We investigated the clinical features and outcomes of PRCA associated with thymoma in this study. A retrospective review of all PRCA patients who underwent surgical resection of thymoma from April 1, 2004, to December 31, 2015, was performed. We experienced eight patients with PRCA among 146 patients who underwent surgical resection of thymoma. Extended thymectomy (n=4) and thymectomy (n=4) were performed for thymoma. Regarding the WHO classification of thymoma, the subtypes were type B2 or B3 in seven patients, and the stage of thymoma was advanced in seven patients. Complete resection was achieved macroscopically in only five patients. Recurrence of thymoma occurred in four patients who underwent complete resection. PRCA was diagnosed after surgical resection of thymoma in six patients (range 1-101 months, median 56.5 months). Cyclosporine was used for PRCA in six patients. Pneumonia of treatment-related complications due to cyclosporine occurred in all patients. The follow-up period ranged from 13-147 months (median 54.5 months) after the PRCA diagnosis. Three patients obtained complete remission of anemia by cyclosporine. Although one patient was able to stop taking cyclosporine because of complete remission of anemia, transfusion was needed due to relapse of PRCA. Five patients died, with the main causes of death diagnosed as pneumonia (n=4) and cardiac failure (n=1). PRCA associated with thymoma was diagnosed postoperatively in three-quarter of patients. We should be alert for the occurrence of PRCA even after resection of thymoma, especially in patients with incomplete resection or advanced disease. Cyclosporine was effective for PRCA, but treatment-related complications occurred, particularly pneumonia. As treatment for PRCA associated with thymoma and its complications were combined in a complex manner, treating PRCA associated with thymoma can be quite difficult.

The clinical characteristics and therapy response of patients with acquired pure red cell aplasia

ABSTRACT Objective: To summarize the clinical characteristics of acquired pure red cell aplasia (PRCA) patients diagnosed in our hospital in the last 10 years. Method: The clinical features, immune state and treatment response of acquired PRCA patients diagnosed in our hospital from January 2007 to January 2017 were retrospectively analyzed. Results: The results showed that thymoma (13.21%) and parvovirus B19 (11.32%) were the most common causes for secondary PRCA. Ferritin (Fer) levels and erythropoietin (EPO) levels were increased in PRCA patients. The total CR and PR rate of immunosuppressive therapy in our studies was 68.29% and 12.20%, respectively. Patients with EPO level >400 U/L and Fer level >200 ng/ml had significantly lower CR rate than others. The patients with EPO level >400 U/L also had longer hemoglobin recovery time than patients with EPO level ≤400 U/L. Patients treated with corticosteroids (CS) + cyclosporine A (CsA) had lower relapse rate compared to the CS group (29.17% vs. 80.00%, P < .05). Conclusion: Our data showed that patients with PRCA had high EPO and Fer levels. Thymoma and viral infections are the most common causes for secondary PRCA. The CS+ CsA group had lower relapse rate than CS group although response rate was similar. Increased EPO and Fer levels might be the negative factors for prognosis of acquired PRCA.

Effect of sirolimus on erythropoiesis of K562 cell line and patients with pure red cell aplasia in vitro

目的明确西罗莫司体外对K562细胞系、获得性纯红细胞再生障碍（PRCA）患者骨髓单个核细胞向红细胞分化的影响。方法以K562细胞系及治疗前PRCA患者、正常对照的骨髓单个核细胞为研究对象，采用红系甲基纤维素培养基诱导其向红系分化，观察10、100、1 000 nmol/L西罗莫司对红细胞分化的影响。并进一步在PRCA原代细胞的培养体系中加入患者自身血清，观察各组红系爆式集落形成单位（BFU-E）的变化。结果不同浓度梯度西罗莫司对K562细胞系血红蛋白生成及红系表面抗原表达无明显影响；无论在正常对照还是PRCA患者骨髓单个核细胞中，西罗莫司处理组与未加药物的对照组比较，BFU-E差异无统计学意义；PRCA患者细胞加入自身血清后，其BFU-E较未加血清者明显减少[（76.40±22.48）个对（136.33±12.58）个，t=−4.329，P=0.001]，且患者细胞+自身血清+1 000 nmol/L西罗莫司组BFU-E[（97.14±15.83）个]明显高于患者细胞+自身血清组（P=0.038）。结论西罗莫司可能无直接刺激红细胞生长和分化作用，但可以拮抗患者血清对红细胞的抑制作用，可能通过抑制血清中的成分而起作用。