Immunosuppressive Therapy Response Research Articles

HLA Class I Allele Loss and Bone Marrow Transplantation Outcomes in Immune Aplastic Anemia

In patients with immune-mediated acquired aplastic anemia (AA), HLA class I alleles often disappear from the surface of hematopoietic progenitor cells, potentially enabling evasion from cytotoxic T lymphocyte-mediated pathogenesis. Although HLA class I allele loss has been studied in AA patients treated with immunosuppressive therapy (IST), its impact on allogeneic bone marrow transplantation (BMT) has not been thoroughly investigated. The purpose of this study was to evaluate the clinical implications of HLA class I allele loss in patients with acquired AA undergoing allogeneic BMT. The study enrolled acquired AA patients who underwent initial BMT from unrelated donors through the Japan Marrow Donor Program between 1993 and 2011. The presence of HLA class I allele loss due to loss of heterozygosity (HLA-LOH) was assessed using pretransplantation blood DNA and correlated with clinical data obtained from the Japanese Transplant Registry Unified Management Program. A total of 432 patients with acquired AA were included in the study, and HLA-LOH was detected in 20 of the 178 patients (11%) available for analysis. Patients with HLA-LOH typically presented with more severe AA at diagnosis (P=.017) and underwent BMT earlier (P < .0001) compared to those without HLA-LOH. They also showed a slight but significant recovery in platelet count from the time of diagnosis to BMT (P=.00085). However, HLA-LOH status had no significant effect on survival, engraftment, graft failure, chimerism status, graft-versus-host disease, or other complications following BMT, even when the 20 HLA-LOH+ patients were compared with the 40 propensity score-matched HLA-LOH- patients. Nevertheless, patients lacking HLA-A*02:06 or HLA-B*40:02, the alleles most frequently lost and associated with a better IST response, showed higher survival rates compared to those lacking other alleles, with estimated 5-year overall survival (OS) rates of 100% and 44%, respectively (P=.0042). In addition, in a specific subset of HLA-LOH- patients showing clinical features similar to HLA-LOH+ patients, the HLA-A*02:06 and HLA-B*40:02 allele genotypes correlated with better survival rates compared with other allele genotypes, with estimated 5-year OS rates of 100% and 43%, respectively (P=.0096). However, this genotype correlation did not extend to all patients, suggesting that immunopathogenic mechanisms linked to the loss of certain HLA alleles, rather than the HLA genotypes themselves, influence survival outcomes. The survival benefit associated with the loss of these two alleles was confirmed in a multivariable Cox regression model. The observed correlations between HLA loss and the pretransplantation clinical manifestations and between loss of specific HLA class I alleles and survival outcomes in AA patients may improve patient selection for unrelated BMT and facilitate further investigations into the immune pathophysiology of the disease.

Single-cell RNA Sequencing Reveals Novel Cellular Factors for Response to Immunosuppressive Therapy in Aplastic Anemia.

Aplastic anemia (AA) is a lethal hematological disorder; however, its pathogenesis is not fully understood. Although immunosuppressive therapy (IST) is a major treatment option for AA, one-third of patients do not respond to IST and its resistance mechanism remains elusive. To understand AA pathogenesis and IST resistance, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) from healthy controls and patients with AA at diagnosis. We found that CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells were significantly depleted in AA, which suggests that the depletion of CD34+ early-stage erythroid precursor cells and PROM1+ hematopoietic stem cells might be one of the major mechanisms for AA pathogenesis related with BM-cell hypoplasia. More importantly, we observed the significant enrichment of CD8+ T cells and T cell-activating intercellular interactions in IST responders, indicating the association between the expansion and activation of T cells and the positive response of IST in AA. Taken together, our findings represent a valuable resource offering novel insights into the cellular heterogeneity in the BM of AA and reveal potential biomarkers for IST, building the foundation for future precision therapies in AA.

Whole-exome sequencing identifies FANC heterozygous germline mutation as an adverse factor for immunosuppressive therapy in Chinese aplastic anemia patients aged 40 or younger: a single-center retrospective study

Acquired aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia, and immunosuppressive therapy (IST) is the optional first-line management. Several studies identified the influencing factors on IST response; however, there are still a considerable number of patients suffering from poor prognoses. In this study, we enrolled 61 AA patients aged ≤ 40 years old, and whole-exome sequencing (WES) found unexpected high FANC heterozygous germline mutations (28/61, 45.9%). Patients with FANC mutations have a significantly lower absolute reticulocyte count and CD34+ % in the bone marrow and also lower 3-, 6-, and 9-month IST response than that without mutation, which were 0% vs. 25% (P = 0.017), 26.3% vs. 42.1% (P = 0.495), and 29.4% vs. 72.2% (P = 0.011), especially in anti-thymocyte globulin combined with the cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P = 0.143), 25% vs.83.3% (P = 0.103), and 25% vs. 100% (P = 0.003), respectively. The event-free survival in the FANCwt group was also better than that in the FANCmut group (P = 0.016) and also showed in patients who received ATG + CsA treatment (P = 0.045). In addition, all the adverse effects of FANC germline mutation were not significant in stem cell–transplanted group. Our result indicated that the WES-based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. This study was registered at chictr.org.cn (# ChiCTR2100054992).

HLA Class I Allele-Specific Pathology Defines Clinical Manifestations of Immune Aplastic Anemia

Leukocytes that have lost the cell surface expression of an HLA class I allele due to somatic HLA gene mutations or 6p loss of heterozygosity (LOH) are often present in patients with immune aplastic anemia (AA), consistent with T cell-mediated bone marrow cell destruction. A recent NIH study suggested that the clinical significance and underlying pathology of HLA loss may differ according to the type of HLA allele affected (Zaimoku et al. Blood 2021) because (1) the significant correlation of HLA loss with clonal evolution was primarily attributed to patients who lost HLA-B*14:02 and A*02:01; (2) all patients who lost B*40:02 and A*02:06 achieved a hematologic response to immunosuppressive therapy (IST), though HLA loss in general was not correlated with the IST response; (3) loss of B*07:02 and B*40:01 was associated with older age of onset; (4) patients who retained B*14:02, B*07:02, and B*40:01 also showed clinical features of patients who lost respective HLA alleles. We aimed to confirm the HLA class I allele-related clinical manifestations in Japanese AA patients. HLA loss was assessed by SNP array, HLA flow cytometry, digital PCR, or deep nucleotide sequencing. An HLA allele was classified as "affected by loss" in individual patients when the single allele was lost or inactivated by somatic mutations or when both HLA-A and HLA-B alleles in a haplotype were lost due to 6p LOH with no evidence of loss or inactivation of a single HLA allele. Clinical characteristics were compared according to the affected HLA alleles. In 496 Japanese patients with AA (severe, n = 368; non-severe, n = 128; median age, 59 [range, 1-89] years), there were 146 (29%) patients with HLA loss. HLA-B*40:02, A*02:06, A*31:01, and B*54:01 were more frequently affected in our cohort than in the NIH cohort, where B*14:02, A*02:01, B*08:01, and B*07:02 held the majority of lost alleles (Figure 1). The median age of patients with B*40:01 loss (75 years) was highest among the groups of patients with HLA allele loss, consistent with the NIH study. The prevalence of ≥0.003% paroxysmal nocturnal hemoglobinuria (PNH) clones was significantly reduced in patients with B*40:02 loss but not in others. Sex and severity of AA were not markedly different across the groups of patients with HLA allele loss. Outcomes after anti-thymocyte globulin (ATG)-based IST were evaluable in 263 of the 496 patients. Although HLA loss was correlated with the IST response at 6 months (as reported by our group), loss of HLA-A*02:01 did not contribute to the high response, consistent with the NIH study. Loss of frequently affected HLA alleles in Japanese patients (B*40:02, A*02:06, B*54:01, etc.) was generally correlated with the IST-response. Further, patients who carried 5 HLA-B alleles (B*40:01, B*40:02, B*44:03, B*54:01, and B*56:01) without loss still displayed higher response rates than other patients. In contrast, the favorable effect of A*02:06 loss was not seen in patients who carried A*02:06 without loss. A*02:06 loss, the presence of the 5 HLA-B alleles (irrespective of loss), and previously reported HLA class II alleles (DRB1*13 and DRB1*15) were independently correlated with the IST response. The incidence of clonal evolution to myeloid neoplasms after ATG-based IST was significantly higher in patients with HLA-A*02:01 loss than other patients (Figure 2), as observed in the NIH study; A*02:01 was affected in 4 of 7 patients showing both HLA loss and clonal evolution in our cohort; B*40:02, B*44:02, and B*52:01 were affected in other 3 patients. In summary, our study in Japanese AA patients broadly confirmed the associations of loss of specific HLA alleles with clinical manifestations observed in American patients, including older age of onset in patients with HLA-B*40:01 loss, high IST response rates in patients who lost B*40:02 and A*02:06 but not in patients with A*02:01 loss, and frequent clonal evolution in patients with A*02:01 loss. We also revealed the correlation of 5 HLA-B alleles with IST response, irrespective of loss, similar to the relationship of B*14:02 to clonal evolution in the United States, and the reduced prevalence of PNH clones in patients with B*40:02 loss. These findings strongly suggest that the heterogeneous pathology of AA can be classified by affected HLA alleles. This should be useful for studies to unravel the pathophysiology and improve the outcomes of patients with immune AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Whole-Exome Sequencing Identifies Fanc Heterozygous Germline Mutation As an Adverse Factor for Immunosuppressive Therapy in Chinese Aplastic Anemia Patients Aged 40 or Younger: A Single-Center Retrospective Study

Aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia. Currently, immunosuppressive therapy (IST) using cyclosporine (CsA) with or without anti-thymoglobulin (ATG) are the standard first-line management for AA patients not eligible for allogeneic stem cell transplantation. Several studies identified the influencing factors on IST response and survival, however, there are still a considerable number of patients suffering from poor prognosis even if they have achieved workable treatment, which encourage us to further identify additional prognosis / response predictor. The whole-exome sequencing (WES) is an accurate molecular technique to rule out congenital hematopoietic failures in young patients with pancytopenia. Up to date, several reports showed a relatively higher FANC heterozygous germline mutation rate was found in AA/MDS patients than general population, which may contribute to hematopoietic failure and confer congenital susceptibility to myeloid malignancies. Mostly, immunosuppressive therapy (IST) rescues hematopoiesis stem cell from cytotoxic T lymphocyte mediated bone marrow failure, but may exert limited effect in reconstruct gene deficit induced hematopoietic dysfunction. At present, few study focus on exploring the impact of FANC mutation on the response of AA to IST treatment, as well as its long-term prognosis. Herein, we respectively reviewed the AA patients who have the WES result in our cohort. A total of 61 patients aged ≤40 years old diagnosed with AA and underwent WES analysis were enrolled in this study, and result showed unexpected high FANC heterozygous germline mutations in our cohort (28/61, 45.9%) (Fig. A). Patients with FANC mutations have a significantly lower absolute reticulocyte count, and CD34+ % in bone marrow, and also lower 3, 6, and 9-month IST response than that without mutation, which were 0% vs. 25% (P=0.017), 26.3% vs. 42.1% (P=0.495), and 29.4% vs. 72.2% (P=0.011), especially in anti-thymocyte globulin combined with Cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P=0.143), 25% vs.83.3% (P=0.103), and 25% vs. 100% (P=0.003), respectively (Fig. B-E). While the efficiencies of patients received HSCT were all 100% at 3rd, 6th, and 9th months evaluation. The overall survival (OS) didn't have a difference based on FANC status as well as treatment strategy (IST vs. HSCT), only one patient in the FANCmut group transformed into acute myeloid leukemia (AML) 49 months after the failure of CsA single treatment and died due to severe infection (Fig F, G). The event-free survival (EFS) in the FANCwt group was better than that in the FANCmut group (P=0.016) (Fig H), and also showed in patients received ATG + CsA treatment (P=0.045) (Fig I). We further explored the possible factors that may affect the EFS, and the FANC mutation was found to be the only predictor associated with poorer EFS [odds ratio (OR)=5.576, 95% confidence interval (CI): 1.169-26.590, P=0.031] in patients received IST (Fig J). In summary, WES based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. For patients younger than 40-year-old without FANC germline mutation, ATG + CsA can exert a curative effect that was comparable to HSCT, while for cases with FANC mutation, HSCT may be a better choice. FANC germline mutation can negatively impact the IST response for Chinese patients with AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of ATG-Based Immunosuppressive Treatment and Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Transfusion-Dependent Non-Severe Aplastic Anemia: A Single Center, Retrospective Analysis

Transfusion-dependent non-severe aplastic anemia (TD-NSAA) refers to aplastic anemia (AA) that is dependent on blood transfusion but does not fulfilling the criteria for severe AA (SAA). It has been clear that allogeneic hematopoietic stem cell transplantation (HSCT) and anti-thymocyte globulin (ATG)-based immunosuppressive therapy (IST) is the front line for the treatment of SAA, but there is no consensus on the treatment timing and strategy for TD-NSAA. The natural history observation reported that the progress-free survival (PFS) of TD-NSAA at 60 months after diagnosis is around 62%, but decreased significantly to 22% at 120 months. In China, the practical guideline recommends cyclosporine-based IST with or without hematopoietic stimulating regimes (such as androgens) as initial treatment, and further treated as SAA if no response was observed after 6 months management. As reported, the hematopoietic response of ATG-based IST was much better than CsA-based IST (75% Vs. 55.8%, 6-month) for TD-NSAA, however, the relapse and clonal expansion are still the unavoidable events. For HSCT, the long-term PFS is much better than IST for SAA, but the complications (including graft versus host disease, various infection, etc.), transplantation related mortality, as well as donor availability still limited its application. At present, there is limited data compared the efficiency of HSCT and IST for TD-NSAA. With the development of haplo-identical transplantation in China, most TD-NSAA own a donor, and we also performed HSCT for those young patients after informed consent. Herein, we respectively compared the efficiency of ATG-based IST and HSCT for TD-NSAA, and also analyzed the risk factors that contribute to the overall survival (OS) as well as event free survival (EFS). During Jul 2011 to Dec 2019, 97 TD-NSAA patients were diagnosed in our cohort, among them, 5 only received supportive treatment, 30 received CsA-based IST with or without androgen, and the other 62 patients received HSCT or ATG-based IST therapy. We further analyzed the efficiency and prognosis of 55 patients aged 16-60 years old who received HSCT or ATG-based IST therapy. Among them, 27 (49.09%) patients received ATG-based IST, and the others performed HSCT (including 14 from sibling donor, 7 from haplo-identical donor and 7 from unrelated donor) (the baseline information showed in Table1). Results showed that the 12-month overall response rate (ORR) of ATG-based IST and HSCT were 48.1% (with 18.52% CR) and 78.57% (with 60.71% CR), respectively (P=0.04, Fig A). The median time of achieving transfusion-free was 94(35-205) and 24(11-139) days, respectively in ATG-based IST and HSCT group (P<0.001, Fig B). The 5-year OS and EFS rates were 54.9±13.3% and 34.5±12.1% in ATG-based IST group respectively, and 70.0±9.0% and 62.6±9.6% in HSCT group, while no statistically differences was observed (Fig C and D). To explore the possible influencing factors of 5-year OS and EFS, we performed COX regression analysis based on age, disease duration, baseline absolute neutrophil count (ANC), hemoglobin (HB), platelet count (PLT), serum ferritin (SF), lymphocyte percentage, and CD4/CD8 ratio. Univariate analysis showed that disease duration, baseline HB, SF, and ANC may affect OS and EFS of patients in ATG-based IST group (Table 2), and multivariate analysis showed that SF≧1000ng/ml was an independent risk factor for OS in ATG-based IST group. While in the HSCT group, patients with disease duration≧20 months had a higher risk of events (Table 3). The subgroup analysis showed that in HSCT group, patients with disease duration of 6 to 20 months had a lower risk of events (HR=0.196, 95%CI: 0.046-0.832); the HR of the group <6 months and ≥20 months were not statistically significant (Fig E). TD-NSAA patients with disease duration between 6 months and 20 months had a higher EFS rate in HSCT group than that in ATG-based IST group (Fig F). The subgroup analysis of mortality was not statistically significant (Fig G). To conclusion, TD-NSAA patients should be actively treated, patients with shorter disease duration and no iron overload may achieve a better response. The 12-month ORR in the HSCT group was higher than that in the IST group, but there was no significant difference in OS rate and EFS rate, even there is a great difference tendency. For TD-NSAA patients with a disease duration between 6 to 20 months, HSCT has a higher EFS rate than IST, which may be a better choice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apolipoprotein-a Is a Potential Prognosis Biomarker for Severe Aplastic Anemia Patients Treated with ATG-Based Immunosuppressive Therapy: A Single-Center Retrospective Study

Aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia with a risk of hemorrhage and infection. AA can be further divided into severe AA (SAA) and non-severe AA (NSAA) based on the severity of the disease. Anti-thymoglobulin (ATG)-based immunosuppressive therapy (IST) is the standard first-line management for SAA and very severe AA (VSAA) patients not eligible for hematopoietic stem cell transplantation (HSCT). Several studies identified the influencing factors on response and survival, including patient age, disease severity, absolute reticulocyte count and the interval between diagnosis and treatment. However, the prognosis cannot be improved if the patient exhibits poor response indicators when ATG-based IST is the only appropriate method. Adipocytes occupy the bone marrow of SAA, but the function of these cells is not yet clarified. Herein, we conducted this study aimed to explore the serum lipid changes during the ATG-based IST and identify the putative indicators that may predict the prognosis, thereby implying them as potential therapeutic target. A total of 61 newly diagnosed SAA/VSAA patients aged between 10 and 60 years who received ATG-based IST were enrolled, and divided into IST-response (IST-R, n=40) and IST-non-response (IST-NR, n=21) groups based on the 9-month response effect. Differences were observed in blood lipids level, immunoglobulins and complements among groups after ATG-based IST. Significantly higher serum total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipoprotein A (Apo-A) levels were detected in the IST-NR group than the IST-R group pretreatment (all P<0.05). After ATG-treatment, the Apo-A levels increased in the IST-R group while decreased drastically in the IST-NR group (Fig. A-F). Both groups showed decreased IgA after 3 months after IST, and the level in the IST-R group was much lower than that in the IST-NR group (P<0.05) (Fig. G-K). In order to predict the 9-month response early, we conducted a correlation analysis and found that the 9-month IST response had a moderate negative correlation with IgA level at 3 months (Spearman's r=-0.516, P=0.006) and a negative correlation to pretreatment Apo-A (Spearman's r=-0.380, P=0.004). Considering the dramatic changes between the IST-R and IST-NR groups, enrolled patients were respectively divided into two subgroup based on the cutoff value of Apo-A and IgA by receiver operating characteristic (ROC), and found that patients with lower Apo-A (<1.205 g/L) level pretreatment had a better event-free survival (EFS) (P=0.008)，but no differences were observed on OS (Fig. L-M). The patients with IgA level ≥1.72 g/L showed poorer EFS compared to those with IgA <1.72 g/L, although no statistically significant difference was noted (Fig. N-O). In conclusion, the current study found that serum Apo-A is a potential prognosis biomarker for the newly diagnosed <60-year-old SAA/VSAA patients who received ATG-based IST, and the level of 3-month IgA can also be an indicator for 9-month IST response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Natural killer cells in peripheral blood at diagnosis predict response to immunosuppressive therapy in severe aplastic anemia.

Immunosuppressive therapy (IST) consisting of antihuman thymocyte globulin and cyclosporine A is the first-line therapy for patients with severe aplastic anemia (AA) who are ineligible for undergoing bone marrow transplantation. The aim of the study was to evaluate the correlation between natural killer (NK) cells and response to IST in SAA patients. We retrospectively included 93 AA patients and detected NK cells in peripheral blood by flow cytometry. Both the proportion and absolute number of NK cells in newly diagnosed SAA patients were significantly lower than in controls, while the proportion and absolute number of NK cells in complete remission patients treated with IST were remarkably increased compared with treatment-naïve SAA patients. Additionally, the absolute number of NK cells at diagnosis was positively correlated with initial blood counts. For SAA patients receiving IST, the proportion of NK cells at baseline and 6months was significantly higher in responders than in non-responders. Unexpectedly, we found that the increase in the proportion of NK cells at 6months after IST was closely related to the recovery of hematopoiesis. ROC curve identified 7.3% of NK cells proportion at diagnosis as the cutoff value to predict response to IST. The response rate was higher in NK proportion high group than in NK proportion low group. Multivariate logistic regression analysis further confirmed the independent predictive value of NK cells proportion in assessing IST response. The proportion of NK cells at diagnosis may serve as a promising predictor of response to IST in patients with SAA.

Response To Immunosuppressive Therapy In Patients Of Acquired Aplastic Anaemia: A Single Center Experience From A Developing Country.

Aplastic Anaemia (AA) is characterized by pancytopenia and hypocellular marrow. Immunosuppressive therapy (IST) SHOWS impressive haematological response; however, risk of relapse and clonal evolution persists. The objective of the study is to assess response to IST in patients with aplastic anaemia. A retrospective single centre study at AFBMTC / NIBMT for patients of acquired AA was conducted from January 2005 to December 2019.Inclusion criteria included diagnosed cases of acquired AA receiving IST for at least 12 weeks and age >2 years. IST included cyclosporine (CsA) alone, CsA + androgens, CsA + rabbit anti thymocyte globulin (rATG), CsA + horse anti thymocyte globulin (hATG). Primary outcome measure was response to IST; secondary outcome measure was overall survival (OS). A total of 513 patients received IST. Median age was 23 years (range 2-97 years). In study cohort, 155 (30.2%) patients responded to the IST, 63 (12.3%) achieved complete response (CR) while 92 (17.9%) achieved partial response (PR). The ORR of CsA in NSAA, SAA and VSAA was 52.6%, 28.10% and 10% respectively; whereas ORR of CsA + rATG in NSAA, SAA and VSAA was 50%, 35.1% and 22.5% respectively. OS was 38% at a median follow up of 36 months. There was a significant difference in the survival distributions of different treatment modalities (p=0.016). Median survival time 60 months (CsA), 9 months (CsA+ androgens) and 39 months (CsA+ rATG/hATG.) . In resource constrained settings, single agent CsA remains a reasonable alternative with modest activity and acceptable side effect profile.

Hypercalciuria may predict better response to immunosuppressive therapyin renal sarcoidosis: a case series.

Renal sarcoidosis is a rare cause of tubulointerstitial nephritits(TIN). The clinical and pathological characteristics, as well as outcomes, of renal sarcoidosis remain unclear. This single-center study retrospectively analyzed 18 patients affected bysarcoidosis with tubulointerstitial nephritis (TIN) and 53 patients with tubulointerstitial nephritisnot related to sarcoidosis.Patientswere further stratified into the granulomatous (12 sarcoidosis and 6 non-sarcoidosis) and non-granulomatous (6 sarcoidosis and 47 non-sarcoidosis) TIN groups. Half of the patients with renal sarcoidosis had signs ofacute kidneyinjury at kidneybiopsy, 94% of whom presented with extra-renal involvement. The prevalence of hypercalcemia, hypercalciuria, and elevated serum angiotensin-converting enzyme levels was 27.6%, 33.3%, and 31.3%, respectively. Renal sarcoidosis patients with eGFR < 30mL/min/1.73 m2 scored higher for total chronic tubulointerstitial injury (p = 0.044) and glomerular sclerosis (p = 0.027). Compared to non-sarcoidosispatients, higher urinary calcium levels (for patients with GFR [Formula: see text] 40mL/min/1.73 m2, p = 0.034), lower scores of acute tubular injury (p = 0.008), and more prominent glomerular sclerosis were observed in renal sarcoidosis. Similar characteristics of chronicity and hypercalciuria were also identified in granulomatous interstitial nephritis; however, interstitial inflammation was obvious (p = 0.001). Patients with renal sarcoidosis were initially treated with corticosteroids. Five patients receiving immunosuppressive agents showed better long-term renal recovery. High 24-h urine calcium (adjusted by weight) was identified as a factor associated with long-term remission. Renal sarcoidosis is a systemic disease of insidious onset and chronic progression, sharing similar features of chronicity and hypercalciuria with granulomatous interstitial nephritisof other cause. Hypercalciuria may predict a better response toimmunosuppressive therapy, presumablyindicating active interstitial inflammation; thus, strengthened immunosuppression might be considered.

First-Line Choice for Severe Aplastic Anemia in Children: Transplantation from a Haplo-Identical Donor Versus Immunosuppressive Therapy

Background: Severe aplastic anemia (SAA) is a fatal disorder in children if treated inappropriately. According to the current algorithm, transplantation from a matched related donor (MRD) is recommended as the first-line option in children with SAA, if a MRD is unavailable, IST with a combination of anti-thymocyte globulin (ATG) and cyclosporine (CsA) represents the first-line choice. However, numerous limitations of immunosuppressive therapy (IST) exists. IST children may have incomplete recovery, and appear to be associated with an increased risk of clonal evolution and subsequent relapse. Additionally, the unavailability of horse-ATG in China also make the response of IST at a discount greatly. At the same time, great progresses in recent years in haploidentical transplantation might also promote changes in the algorithm of SAA treatment.Methods: We retrospectively compared the outcomes of children with SAA who received IST or who underwent hematopoietic stem cell transplantation (HSCT) from a haplo-identical donor (HID) as first-line choice between 2007 and 2016. The conditioning regimen for SCT from a haploidentical donor consisted of intravenous busulfan, cyclophosphamide and rabbit ATG. All of children were administered with granulocyte colonystimulating factor (G-CSF)-primed bone marrow (BM) combined with G-CSF-primed peripheral blood (PB) hematopoietic stem cells.Results: A total of 52 SAA children under the age of 17 years were initially treated with IST (n=24) or haplo-identical HSCT (n=28) as first-line choice. The last follow-up for all surviving patients was June 1, 2017. In IST group, by 6 months, 15 of the 21 subjects (71.4%) improved with first-line IST and achieved a partial response (n=10) or complete response (n=5). In SCT group, 27 patients (96.4%) achieved primary engraftment at a median of 12 (range, 10-21) days except one suffered from primary graft failure. The cumulative incidences (CI) of grade II-IV and of grade III-IV acute graft versus host disease (GVHD) were 48.1±1.0% and 11.1±0.4%, respectively. The 3-year CI of extensive chronic GVHD was 3.7±0.1%. The estimated 10-year overall survival were 73.4±12.6% and 89.3±5.8% in patients treated with IST or HID-HSCT (P=0.806). However, the failure-free survival was significantly inferior in patients receiving IST than in those undergoing transplantation from a HID [52.6±10.5% versus 89.3±5.8, P=0.008]. In univariate analysis, the choice of first-line immunosuppressive therapy was the only adverse predictor for failure-free survival. At the last follow-up, completely normal blood count was observed in 11 of 20 (55.0%) and 24 of 25 (96.0%) alive cases in IST and HID-HSCT cohort (P=0.003).Conclusions: Based on the superior FFS, high rate of engraftment and acceptable incidence of GVHD, the choice of HID HSCT for SAA children without a matched related donor as a first choice option seems reasonable. These suggest that SCT from a haplo-identical donor could be considered as first-line choice in children who lack a matched related donor, especially in experienced transplantation centers. DisclosuresNo relevant conflicts of interest to declare.

Relationship between plasma rabbit anti-thymocyte globulin concentration and immunosuppressive therapy response in patients with severe aplastic anemia.

Patients with acquired aplastic anemia (AA) without HLA-matched sibling donors or aged >40years receive immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG). We investigated the relationship between plasma rabbit ATG (r-ATG) concentration and IST response. From May 2012 to October 2017, 81 patients with severe AA who required initial IST were included. A 1:1 block randomization was employed for 2.5 and 3.5mg/kg doses of r-ATG. No significant difference in response rates was observed between the 2.5 and 3.5mg/kg groups (63% vs. 58%, P=.894). Median r-ATG concentrations on days 14 and 28 after IST were 15.2 (0.0-97.7) and 1.8 (0.0-74.9µg/mL), respectively. According to r-ATG concentration, response rates were significantly higher in the group with higher r-ATG concentration than in those with lower r-ATG concentration (day 14, 88% vs. 52%; P=.006 and day 28, 79% vs. 46%; P=.005). In multivariate analysis, higher r-ATG concentrations at day 28 were independent predictors of favorable response to IST at 6months (odds ratio, 0.29; 95% confidence interval, 0.09-0.93; P=.037). The present data indicate that higher r-ATG concentration at day 28 resulted in improved IST response.

Early Immunosuppressive Therapy for Idiopathic Pneumonia Syndrome Prior to Bronchoscopy Is Associated with Favourable Outcomes

Introduction: Idiopathic pneumonia syndrome (IPS) is a dangerous complication following haematopoietic stem cell transplantation (HSCT). Immunosuppressive therapy (IST) using high-dose methylprednisone and etanercept produces complete response (CR) in the majority of IPS cases. However, exclusion of respiratory pathogens by bronchoscopy prior to IST commencement carries procedural risks that may worsen respiratory failure and IPS severity, with potential negative impact upon IST response and survival. Given this, we perform bronchoscopy post-IST commencement only in those patients with only mild (nasal oxygen) or severe (already intubated) respiratory failure. We commence IST within 24 hours of clinical and radiological suspicion of IPS, and cease IST if alternative aetiologies are subsequently proven. Aim: We aimed to evaluate the safety and efficacy of our local early-intervention IST strategy in patients with new-onset suspected IPS following HSCT. Methods: We performed a retrospective, single-centre descriptive cohort study of patients who received IST for IPS between 2014-2019. Details collected included: patient demographics, IPS diagnostic details/severity, IST response, and survival. The primary study objectives were the incidence of complete response (CR), defined by cessation of supplemental oxygen for at least 48 hours, and overall survival (OS) following IST. IST comprised high-dose corticosteroids (methylprednisolone 2mg/kg/day or equivalent) and etanercept (25mg twice weekly for 4 weeks, then weekly to complete 12 total doses). Results: Of the 480 HSCT performed during the study period, 17 patients developed suspected IPS and received IST. At IPS diagnosis, 10 (59%) required intensive care admission. Non-invasive (NIV) and invasive (IV) ventilation was required in 4 (24%) and 5 (29%) patients, respectively. All patients met clinical and radiological American Thoracic Society (ATS) criteria for IPS. Bronchoscopy was only performed in 9 (53%), comprising all 5 receiving IV and 4 who were only requiring supplemental nasal oxygen. All 4 patients receiving NIV were considered too high-risk for post-bronchoscopy intubation, so did not proceed. Of those who underwent bronchoscopy, none experienced worsening respiratory failure, however 1 received a revised diagnosis of bacterial pneumonia and ceased IST within 3 days of commencement, and survived. In the remaining 16 patients, CR to IST was achieved in 14 (88%) patients, with 1-year OS of 44%. However, at a mean 17 months follow-up, OS for the whole cohort was 31% (Figure 1), reflecting a high incidence of non-relapse mortality (NRM) at 63% (Table 1). There was no significant difference in response or survival between patients who did or did not undergo bronchoscopy. Conclusion: Our early-intervention IST strategy for IPS appears to produce similar response rates and survival as those reported in other prospective and retrospective series, without any obvious safety concerns. Although adherence to ATS diagnostic criteria and exclusion of occult infectious aetiologies is preferred, bronchoscopy may be safely delayed or omitted in a subset of patients. Early intervention may arrest the respiratory failure and ultimately avoid mechanical ventilation. Disclosures No relevant conflicts of interest to declare.

Deficit of circulating CD19+ CD24hi CD38hi regulatory B cells in severe aplastic anaemia.

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24hiCD38hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL‐10). We investigated circulating B‐cell subpopulations, including CD24hiCD38hi Bregs, as well as total B cells, CD4+ T cells, CD8+ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24hiCD38hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL‐10; total B‐cell counts and the other B‐cell subpopulations were similar to those of healthy individuals. CD24hiCD38hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B‐cell counts were unexpectedly associated with IST response. Markedly reduced CD24hiCD38hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.

Deep Immunoprofiling By Flow Cytometry and NGS Reveals Distinct T Cell Profile in Pediatric Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is a rare variant of bone marrow (BM) failure that is typically diagnosed 2-3 months after an acute attack of hepatitis in patients that are seronegative for known hepatitis viruses. Although very little is known about its etiology, an autoimmune mechanism is presumed based on clinical response to immunosuppressive therapy (IST) and reports of oligoclonal T-lymphocyte expansion and skewing of CDR3 region length. However due to the rareness of this disease, the published evidence is still limited. We analyzed 18 pediatric patients consecutively diagnosed with HAAA in the Czech Republic between 2004-2019. Based on BM biopsy examination, 14 patients had aplastic anemia (AA) and interestingly 4 patients were histologically consistent with refractory cytopenia of childhood (RCC). Median age of these patients was 6.6 years (range 1-18) with a male to female ratio of 2:1. The prevalence of HAAA in our cohort of pediatric patients with BM failure (n=107; excluding patients with known genetic cause) was 17%. The presence of a paroxysmal nocturnal hemoglobinuria clone was excluded and cytogenetic evaluation showed no abnormalities. We examined both the diagnostic BM and peripheral blood by flow cytometry in all patients (with the exception of one peripheral blood sample) and performed deep immunophenotyping of T cells in 7 of them. Ten patients were selected for T cell receptor beta (TRB) repertoire and whole-exome sequencing (WES) based on the availability of DNA samples isolated from the BM before the start of the treatment. TRB sequencing was performed following the SOP developed by the EuroClonality-NGS working group with DNA input normalized to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data. Bioinformatic analysis was performed with ARResT/Interrogate. WES was performed on Illumina NextSeq 500 and libraries prepared using the Agilent SureSelectXT Human All Exon V6+UTRs kit. Vertebrate virus detection was performed using the VirCapSeq-VERT probe capture enrichment set and massive parallel sequencing from 8 BM DNA samples taken at the time of diagnosis. We found significant activation of CD8 positive T cells based on the expression of HLA-DR compared to non-HAAA RCC and AA patients (n=20 and n=21 respectively, Mann-Whitney p&lt;0.0001). Three out of 7 patients with deep T cell immunophenotype available showed a decrease of naive CD27pos45RAposCD8pos T cells. From TRB repertoire analysis we were able to identify a total of 5 private immunodominant clones in 3 patients with relative abundance of up to 11% from all productive TRB sequences. These clones were not found in any previous publications or clonotype databases based on their CDR3 sequence. We detected 5 clones shared by 3 or more patients; these were previously published in other cohorts and are not specific for HAAA. WES analysis did not reveal any previously published variant in genes associated with immune dysregulation or immunodeficiency, or any candidate variant for functional validation. No relevant vertebrate virus signal was detected in the 8 samples. Out of all 18 patients, 3 patients underwent MSD-HSCT without preceding immunotherapy. Fifteen patients were treated according to standard European Working Group on MDS/SAA protocols with anti-thymocyte globulin (horse n=6; rabbit n=9), corticosteroids and cyclosporin A. In total, 7 patients reached complete remission on IST by day 120 and 7 patients with insufficient response underwent MUD-HSCT. One patient died shortly after MUD-HSCT because of toxicity. We did not observe any correlation of T cell populations at diagnosis or day 120 of IST (activated and exhausted T cells) and clinical response. Similarly, the presence of an immunodominant clone or oligoclonal TRB repertoire with lower diversity at diagnosis had no significant impact on the outcome of these patients in our cohort. In conclusion, we present the biggest cohort of pediatric patients with HAAA analyzed by next-generation sequencing and flow cytometry so far. Even though our results show significantly increased activation of CD8 positive T-cells and presence of expanded clones, we did not confirm that these factors would be useful and significant for the prediction of treatment outcome. Large number of our patients did not fully respond to IST, requiring HSCT. Supported by AZV 18-07-00430, 16-32568A and PRIMUS/17/MED/11. Disclosures No relevant conflicts of interest to declare.

Polyclonal Immune Response in T-LGL Leads to Clonal Expansions Preceding Occurrence of STAT3 Mutations Further Solidifying Clonal Dominance

T cell large granular lymphocyte leukemia (T-LGLL) characterized by excessive clonal cytotoxic T cell proliferation, is often accompanied by cytopenias and is thought to result from immune-mediated suppression, either by misguided immune antigenic recognition or mimicry triggered by auto-, tumor or viral antigens. Irrespective of the mode of evolution, T-LGLL progresses via: i) purely reactive clonal outgrowth in the context of a polyclonal immune response or: ii) a transforming event such as a STAT3 mutation (STAT3MT) within the immunodominant clone.Studies of TCR rearrangement using DNA based NGS of the TCR Vβ complementarity-determining region 3 (TCR VB CDR3) facilitate analyses of T cell clonality, as the CDR3-based “biological barcodes” allow for clonal quantification. We used serial CDR3 clonotyping accompanied by clonal STAT3 mutant burden measurements to recapitulate the pathological cascade in the course of T-LGLL. We asked if a polyclonal/oligoclonal immune response is the primary process that is clonally “imprinted” by STAT3MT, which would likely arise with the most proliferative clone. We were interested in the dynamics of underlying clonal behavior during treatment.Our initial cohort included 207 well characterized LGL patients. Most presented with anemia (46%), and/or neutropenia (46%). VB expansion was present in 94% of cases, with an average LGL count of 2317k/uL .STAT3MT were found by targeted deep sequencing in 38% of patients in 4 common hotspots: 42% Y640F, 34% D661Y, 11% D661V, and 8% N647I, with an average clonal burden of 28%. Multiple STAT3MT variants were found in some patients. From this cohort, serial samples obtained at presentation and throughout clinical course were obtained from 20 representative cases (10 STAT3MT and 10 STAT3WT), and subjected to analysis of clonal dynamics, including simultaneous deep TCR VB and STAT3 NGS. Patients were sequenced at an average of 4 time points (range 2-8). At least one major clonotype was identified in all patients, and multiple major clonotypes were identified in half.Analyses of clonal architecture revealed that STAT3 clones arose with VB expanded clones. In all cases, the TCR clonal burden was greater than that of STAT3MT demonstrating for the first time that STAT3MT is not the ancestral event for clonal expansion; rather, it evolves within the pre-expanded immunodominant clone.More than half of the patients were treated with immunosuppressive therapy (IST) achieving an approximate 40% response rate. Distinct patterns of clonal dynamics were seen following treatment (see Figure). In some patients, both the STAT3 (if present) and the major TCR clone decreased upon successful therapy. A correlation between a specific IST treatment and a clonal burden decrease was not found. In a subset of patients, the clones persisted despite a hematologic response, suggesting the major clonotype was functionally silenced. We also observed a common phenomenon of TCR “clonotype switching”, whereby therapy contracts one major clonotype, while another previously “minor” clonotype emerges. These newly expanded clones did not harbor STAT3MT, and most patients with “switching” were resistant to IST therapy. Multiple clonotypes were present at initial sampling in a few patients without STAT3MT and persisted at the same rate in subsequent samplings, precluding identification of a clear immunodominant clonotype. A stable or increasing clonal burden of both STAT3MT and VB CDR3 was seen in IST non-responders.In sum, STAT3MT were found to be a secondary event to the clonal expansion of the TCR VB clonotype, as a response within an already expanded clonotype and not the initiator of clonal expansion. The dynamics of both the STAT3MT and the TCR VB clonotype can be assessed over disease course and treatment regimens and demonstrate additional clinical ultility when applied to larger prospective clinical trials. The difficulty in finding a direct correlation between response to specific IST and a decrease in TCR VB clonal burden may be due to variable time frames between samplings, heterogeneity of IST regimens and response assessment, or large asymptomatic clonotypes. Prior to this study, the association of remission and elimination of immunodominant clonotypes was unclear. Our results suggest that clonal elimination is not necessary for complete clinical response; rather, the clone can be contracted to a manageable clonal burden or silenced. DisclosuresMustjoki:Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Eltrombopag Combined with G-CSF and Cyclosporine Could Effect for Severe Acquired Aplastic Anemia

BackgroudAcquired aplastic anemia (AA) is a potential life-threatening hematopoietic stem cell (HSC) disorder resulting in cytopenia. The first line therapy for AA is HSC transplantation for young patients who have suitable donors and immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporine for the remaining patients. However, about 30% of patients are refractory to IST or relapse after IST. IST with antithymocyte globulin and cyclosporine result in severe complication and mortality infection. To reduce the mortality infection and increase the response of IST for AA is still problem. Eltrombopag, a thrombopoietin mimetic, demonstrated efficacy in restoring trilineage hematopoiesis, has recently emerged as an encouraging and promising agent for patients with refractory AA. To explore the effect of eltrombopag for severe acquired aplastic anemia, we treated seven severe AA patients with eltrombopag combined with cyclosporine and G-CSF. Herein we report initial results of the eltrombopag combined with cyclosporine and G-CSF for severe AA.MethodsThe diagnostic of AA patient consisted of a complete blood count, a bone marrow biopsy, bone marrow karyotype analysis and assessment of a paroxysmal nocturnal hemoglobinuria (PNH) clone. Patients with SAA aged ≥18 years old who without suitable donors received eltrombopag 75mg/d, cyclosporine 6mg/kg by oral, and G-CSF 300ug/d by subcutaneous injection from diagnosis. Red blood was infused to maintained HB more than 60g/L. Platelet were infused to maintained PLT more than 20x109/L. G-CSF was administered until neutrophil count more than 1.0x109/L. Vale concentration of cyclosporine were maintained more than 100ug/ml in blood plasm and maintained two years. Eltrombopag was taper down when platelet was more than 100x109/L. Eltrombopag was given at least three months. Antibacterial was administered when patient was high fever. Posaconazole were given for fungal infections prophylaxsis. Hematologic improvements were assessed by the National Institutes of Health (NIH) response criteria for AA.ResultsThe median age of 7 patients with SAA was 44 years old (range 19-68 yr). Full hematologic improvements were achieved in 3 patients. All patients achieved platelet and RBC infusion independence. The median time from the first eltrombopag therapy to platelet infusion independence was 35 days (range 33-46d). The median time from the first eltrombopag therapy to RBC infusion independence was 40 days (range 30-50d). Median 6 units (1200ml) (range 3-10U, 600ml -2000ml) RBC and 7 units (2.5x109/unit) platelet were infused. With median 8 months follow-up (3-12 months),3 patients are still full hematologic improvements and 4 platelet and RBC infusion independence. No severe fugual infection was observed in this group patients. ALT slightly elevate in one patient. No other severe adverse effect was observed.ConclusionsTreatment of SAA patients with G-CSF、cyclosporine combined with eltrombopag is feasible and effect. Our results deserve further research and confirmation in larger samples. DisclosuresNo relevant conflicts of interest to declare.

Clinical outcomes in adult patients with aplastic anemia: A single institution experience.

Newer treatment modalities are being investigated to improve upon historical outcomes with standard immunosuppressive therapy (IST) in aplastic anemia (AA). We analyzed outcomes of adult patients with AA treated with various combinatorial anti-thymoglobulin-based IST regimens in frontline and relapsed/refractory (R/R) settings. Pretreatment and on-treatment clinical characteristics were analyzed for relationships to response and outcome. Among 126 patients reviewed, 95 were treatment-naïve (TN) and 63, R/R (including 32 from the TN cohort); median ages were 49 and 50 years, respectively. Overall survival (OS) was superior in IST responders (P < .001). Partial response to IST was associated with shorter relapse-free survival (RFS), as compared with complete response (P = .03). By multivariate analysis, baseline platelet and lymphocyte count predicted for IST response at 3 and 6 months, respectively. While additional growth factor interventions led to faster count recovery, there were no statistically significant differences in RFS or OS across the various frontline IST regimens (i.e., with/without G-CSF or eltrombopag). While marrow cellularity did not correlate with peripheral-blood counts at 3 months, cytomorphological assessment revealed dyspoietic changes in all nonresponders with hypercellular-marrow indices. Covert dysplasia, identified through early bone marrow assessment, has implications on future therapy choices after IST failure. Salvage IST response depended upon prior response to ATG: prior responders (46%) vs. primary refractory (0%) (P < .01). In the R/R setting, there was no survival difference between IST and allogeneic stem cell transplant groups, with a trend toward superior OS in the former. Transplant benefits in the R/R setting may be underrealized due to transplant-related mortality.

Long-Term Outcome after Immunosuppressive Therapy with Rabbit ΑΤG for Pediatric Aplastic Anemia: A Single-Center Retrospective Study in China

▪Background: Antithymocyte globulin (ATG)-based immunosuppressive therapy (IST) has been successfully used as the first-line treatment for severe / very severe aplastic anemia (SAA/VSAA) patients if no HLA-matched sibling donor was eligible for HSCT as a first choice. It was reported rabbit ATG (rATG) produced more profound immunosuppressive activity compared to horse ATG (hATG). However, recent clinical studies indicated that the stronger lympholytic activity did not mean that rATG was more effective. Most experiences from adult SAA/VSAA implied the efficacy of rATG was worse than hATG. However, susceptibility of children to intensive IST might not be exactly the same as adult patients, long-term efficacy of rATG in historic studies for children with SAA/VSAA was still elusive.Purpose: This study includes the largest cohort of pediatric AA patients treated with first-line rATG+CSA regimen published to date after a median follow-up of 69 months, aiming to assess the long-term outcome of rATG for children, and to identify the significant prior factors in clinical decision making.Methods: We reviewed 231 SAA/VSAA patients under 18 years old assigned to rATG+CSA from February 2000 to May 2014 in Department of Pediatrics, the Blood Diseases Hospital & Institute of Hematology, CAMS & PUMC. Response was evaluated 3, 6, 9, 12 24, 36 and 60 months after IST. We separately defined SAA-II as a specific type of gradually progressed SAA from a NSAA status within a longer period for at least 6 months. Multivariate logistic regression models were used to evaluate the effects of variables on the responses at different time points. Multivariate Cox model analysis of overall survival (OS) and failure-free survival (FFS) was calculated for variables with a log rank P value less than 0.1 in Kaplan-Meier analysis.Results: Of the overall patients, the total responded patients were 79(34.3%), 110(51.6%), and 129 (60.6%) at 6, 9, 12 months following IST, respectively. Intriguingly, 22 patients achieved delayed response between 12 months and 24months after IST, which increased the overall response rate by 10.2%, afterwards the rate reached a plateau by 3 years with the best response rate of 74.6% (Figure 1). Differences in baseline clinical parameters pre-IST were associated with response to IST. Absolute neutrophil count (ANC) less than 0.1*109/L was associated with an unfavorable early response rate at 6 months (P=0.009); absolute lymphocyte count (ALC) less than 1.6*109/L was a significant predictor for better response by 6 months and 12 months in multivariate analysis [6 months, P=0.033 vs. 12 months, P=0.021]. Lower absolute reticulocyte count (ARC no more than 18.5*109/L) predicted worse late IST response by 2 years and 3 years. In our large series of cohort, 5-year OS and FFS were 82.7% and 61.9%. Patients with VSAA as a significantly unfavorable prognostic factor had a much lower probability of 5-year survival when compared to patients diagnosed with SAA (76.4% vs. 87.2%, P<0.001, Figure2A). In multivariate analysis, SAA-II (P=0.021, Figure2B), and a pretreatment lower ARC (P=0.020, Figure2C) were independent unfavorable prognostic factors for FFS, but moderate PNH clone size (more than 5%) was verified as a good predictor for FFS (P=0.006, Figure 2D). At the last follow-up, twelve of the 135 responders relapsed after IST, meanwhile eight patients in responders and seven patients in non-responders experienced clonal evolution after IST, corresponding to cumulative incidences at 5.2% of relapse and 6.5% of evolution, which were obviously lower than previous reports.Conclusions: The combination of rATG and CSA was confirmed as an effective first-line therapy for children with SAA/VSAA in our cohort. We discerned a protracted recovery but an ultimately comparable long-term outcome of rATG. Baseline blood parameters (ANC, ALC, ARC) were predictive factors of response rate. Intensive supportive care may be necessarily pivotal to survival in cases of VSAA. Importantly, moderate PNH clone might be beneficial to FFS. Besides, for those who experienced gradually progressed disease course, early HSCT might be a more preferable option than receiving IST although further validation remains to be done. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hepatitis-associated aplastic anaemia: clinical characteristics and immunosuppressive therapy outcomes

目的分析肝炎相关再生障性贫血（HAAA）临床特征，评价其免疫抑制治疗（IST）疗效及生存状况。方法回顾性分析944例接受IST的重型／极重型AA（SAA/VSAA）患者，比较41例HAAA患者与年龄、造血衰竭程度相匹配的123例特发性AA（IAA）临床特征、血液学反应率、长期生存率及克隆性演变情况。结果944例SAA/VSAA患者中HAAA 41例（4.34%），HAAA患者中VSAA所占比例明显高于IAA患者（65.9％对39.4%，P=0.001）。HAAA与匹配的IAA比较，患者感染发生率差异无统计学意义，但感染控制所需时间明显延长[21 (4~100) d对13 (3~139) d，P=0.048]。HAAA患者CD3+、CD3+CD4+、CD3+CD8+ T淋巴细胞绝对值及CD4+/CD8+细胞比值均明显低于IAA患者，而CD3+ CD8+ T淋巴细胞比例明显高于IAA患者，差异均有统计学意义。HAAA与IAA患者IST后3个月（34.1％对34.1%，P=1.000）、6个月（56.1％对53.7%，P=0.787）及12个月（73.2％对68.3%，P=0.558）血液学反应率差异无统计学意义，两组患者预期5年总生存（OS）率、无事件生存（EFS）率比较差异均无统计学意义（OS率：90.0％对87.1%，P=0.700；EFS率：71.9％对62.4%，P=0.450）。结论HAAA少见，造血衰竭更为严重，感染相对难以控制，采用标准IST方案治疗可获得与IAA患者相当的疗效。