Olga Rios

Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia

BACKGROUND Acquired aplastic anemia results from immune‐mediated destruction of bone marrow. Immunosuppressive therapies are effective, but reduced numbers of residual stem cells may limit their efficacy. In patients with aplastic anemia that was refractory to immunosuppression, eltrombopag, a synthetic thrombopoietin‐receptor agonist, led to clinically significant increases in blood counts in almost half the patients. We combined standard immunosuppressive therapy with eltrombopag in previously untreated patients with severe aplastic anemia. METHODS We enrolled 92 consecutive patients in a prospective phase 1–2 study of immunosuppressive therapy plus eltrombopag. The three consecutively enrolled cohorts differed with regard to the timing of initiation and the duration of the eltrombopag regimen (cohort 1 received eltrombopag from day 14 to 6 months, cohort 2 from day 14 to 3 months, and cohort 3 from day 1 to 6 months). The cohorts were analyzed separately. The primary outcome was complete hematologic response at 6 months. Secondary end points included overall response, survival, relapse, and clonal evolution to myeloid cancer. RESULTS The rate of complete response at 6 months was 33% in cohort 1, 26% in cohort 2, and 58% in cohort 3. The overall response rates at 6 months were 80%, 87%, and 94%, respectively. The complete and overall response rates in the combined cohorts were higher than in our historical cohort, in which the rate of complete response was 10% and the overall response rate was 66%. At a median follow‐up of 2 years, the survival rate was 97%; one patient died during the study from a nonhematologic cause. Marked increases in bone marrow cellularity, CD34+ cell number, and frequency of early hematopoietic progenitors were noted. Rates of relapse and clonal evolution were similar to our historical experience. Severe rashes occurred in two patients, resulting in the early discontinuation of eltrombopag. CONCLUSIONS The addition of eltrombopag to immunosuppressive therapy was associated with markedly higher rates of hematologic response among patients with severe aplastic anemia than in a historical cohort. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT01623167.)

Moderate-dose cyclophosphamide for severe aplastic anemia has significant toxicity and does not prevent relapse and clonal evolution

First-line therapy of severe aplastic anemia (SAA) with high-dose cyclophosphamide causes toxicity and increased short-term mortality. We investigated cyclophosphamide at a lower, more moderate dose in combination with aggressive supportive care to determine whether severe infections might be avoided and hematologic outcomes defined for this regimen. From 2010 to 2012, 22 patients received cyclophosphamide at 120 mg/kg plus cyclosporine and antibacterial, antiviral, and antifungal prophylaxis. Toxicity was considerable, mainly due to prolonged absolute neutropenia, which occurred regardless of pretherapy blood counts, and persisted an average of 2 months. Granulocyte transfusions for uncontrolled infection were required in 5 patients, confirmed fungal infections were documented in 6, and 9 patients died. Nine patients (41%) responded at 6 months. After a median follow-up of 2.2 years, relapse occurred in 2 patients, and cytogenetic abnormalities (including monosomy 7) were observed in 4 patients. Although cyclophosphamide has activity in SAA, its toxicity is not justified when far less dangerous alternatives are available. This trial was registered at www.clinicaltrials.gov as #NCT01193283.

In vivo effects of horse and rabbit antithymocyte globulin in patients with severe aplastic anemia

We recently reported that rabbit antithymocyte globulin was markedly inferior to horse antithymocyte globulin as a primary treatment for severe aplastic anemia. Here we expand on our findings in this unique cohort of patients. Rabbit antithymocyte globulin was detectable in plasma for longer periods than horse antithymocyte globulin; rabbit antithymocyte globulin in plasma retained functional capacity to bind to lymphocytes for up to 1 month, horse antithymocyte globulin for only about 2 weeks. In the first week after treatment there were much lower numbers of neutrophils in patients treated with rabbit antithymocyte globulin than in patients receiving horse antithymocyte globulin. Both antithymocyte globulins induced a “cytokine storm” in the first 2 days after administration. Compared with horse antithymocyte globulin, rabbit antithymocyte globulin was associated with higher levels of chemokine (C-C motif) ligand 4 during the first 3 weeks. Besides a much lower absolute number and a lower relative frequency of CD4+ T cells, rabbit antithymocyte globulin induced higher frequencies of CD4+CD38+, CD3+CD4−CD8− T cells, and B cells than did horse antithymocyte globulin. Serum sickness occurred around 2 weeks after infusion of both types of antithymocyte globulin. Human anti-antithymocyte globulin antibodies, especially of the IgM subtype, correlated with serum sickness, which appeared concurrently with clearance of antithymocyte globulin in blood and with the production of cytokines. In conclusion, rabbit and horse antithymocyte globulins have very different pharmacokinetics and effects on neutrophils, lymphocyte subsets, and cytokine release. These differences may be related to their efficacy in suppressing the immune system and restoring hematopoiesis in bone marrow failure. Clinicaltrials.gov identifier: NCT00260689.

Prolonged cyclosporine administration after antithymocyte globulin delays but does not prevent relapse in severe aplastic anemia.

In severe aplastic anemia, approximately one‐third of responders to standard horse antithymocyte globulin (h‐ATG) plus cyclosporine (CsA) will relapse. Anecdotal experience has suggested that a gradual CsA taper might avoid relapse, but this practice has not been rigorously assessed prospectively. In 2003, we adopted a strategy to taper CsA beyond 6 months, with the intention to reduce hematologic relapse compared with our extensive historical experience. In total, 102 patients received h‐ATG/CsA for 6 months in two sequential clinical protocols: 67 patients (66%) responded and all had the CsA dose tapered per protocol over the subsequent 18 months (total of 2 years). The rate of relapse at 5 years was 33% (95% CI 27–44%), which did not differ from our large historical relapse experience (patients treated before 2003) of 30–40%, in protocols in which CsA was simply discontinued at 6 months. However, time to relapse was prolonged by about 1 year with the longer CsA course. The rates of clonal evolution and overall survival did not differ between the two cohorts. We infer from this large prospective study that CsA taper as implemented delayed but did not prevent relapse. The kinetics of relapse with long course CsA does suggest that a lower long‐term dose might be adequate to maintain patients in remission. Am. J. Hematol. 89:571–574, 2014.

Horse antithymocyte globulin as salvage therapy after rabbit antithymocyte globulin for severe aplastic anemia

The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit antithymocyte globulin (r‐ATG) or cyclophosphamide is not known. We investigated the administration of standard horse ATG (h‐ATG) plus cyclosporine (CsA) in patients who were refractory to initial r‐ATG/CsA (n = 19) or cyclophosphamide/CsA (n = 6) (registered at clinicaltrials.gov as NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as no longer meeting the criteria for severe aplastic anemia. Of the 19 patients who received r‐ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months, and of the 6 patients who received cyclophosphamide, only 1 (17%) responded by 6 months. Among the responders there were no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overall survival for the cohort at 3 years was 68% (95% CI, 50–91%). These results suggest that only a minority can be successfully salvaged after receiving as first therapy either r‐ATG or cyclophosphamide. Although h‐ATG may be utilized in the salvage setting, the overall response rate probably will be lower than when h‐ATG is used as initial treatment. Am. J. Hematol. 89:467–469, 2014.

Alemtuzumab in T-cell large granular lymphocytic leukaemia: interim results from a single-arm, open-label, phase 2 study.

Background T-cell large granular lymphocytic leukemia (T-LGL) is a lymphoproliferative disease presenting with immune-mediated cytopenias and characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes. Methotrexate, cyclosporine, or cyclophosphamide improve cytopenias in 50% of patients as first therapy, but the activity of an anti-CD52 monoclonal antibody, alemtuzumab, is not defined in T-LGL. Methods Twenty-five consecutive subjects with T-LGL were enrolled from October 2006 to March 2015 at the National Institutes of Health (www.clinicaltrials.gov-NCT00345345). Alemtuzumab was administered at 10 mg/day intravenously for 10 days. The primary endpoint was haematologic response at 3 months. Analysis was intention to treat. Here we report the protocol specified interim benchmark of a phase II clinical trial using alemtuzumab in T-LGL. Findings In this heterogeneous, previously treated cohort, 14/25 (56%; 95% CI, 37–73%) subjects had a haematological response at 3 months. In T-LGL cases not associated with myelodysplasia or marrow transplantation, the response rate was 14/19 (74%; 95% CI, 51–86%). First dose infusion reactions were common which improved with symptomatic therapy. EBV and CMV reactivations were common and subclinical. In only 2 patients pre-emptive anti-CMV therapy was instituted. There were no cases of EBV or CMV disease. Alemtuzumab induced sustained reduction of absolute clonal population of T-cytotoxic lymphocytes, as identified by TCRBV-receptor phenotype, but the abnormal clone serendipitously persisted in responders. STAT3 mutations in the SH2 domain, identified in ten subjects, did not correlate with response. When compared with healthy volunteers, T-LGL subjects showed a distinct plasma cytokine and JAK-STAT signature prior to treatment, but neither correlated to response. Interpretation This is the largest and only prospective cohort of T-LGL subjects treated with alemtuzumab yet reported. The high activity with a single course of a lymphocytotoxic agent in a mainly relapsed and refractory suggests that haematologic response outcomes can be accomplished without the need for continued use of oral immunosuppression. Funding This research was supported by the Intramural Research Program of the NIH, National Heart, Lung, and Blood Institute.BACKGROUND T-cell large granular lymphocytic leukaemia (T-LGL) is a lymphoproliferative disease that presents with immune-mediated cytopenias and is characterised by clonal expansion of cytotoxic CD3+ CD8+ lymphocytes. Use of methotrexate, ciclosporin, or cyclophosphamide as first therapy improves cytopenias in 50% of patients, but long-term use of these can lead to toxicity. We aimed to explore the activity and safety of alemtuzumab, an anti-CD52 monoclonal antibody, in patients with T-LGL. METHODS We did this single-arm, phase 2 trial in consecutively enrolled adults with T-LGL referred to the National Institutes of Health in Bethesda, MD, USA. Alemtuzumab was given intravenously at 10 mg per day for 10 days. The primary endpoint was haematological response at 3 months after infusion. A complete response was defined as normalisation of all affected lineages, and a partial response was defined in neutropenic patients as 100% increase in the absolute neutrophil count to more than 5 × 10(8) cells per L, and in those with anaemia, as any increase in haemoglobin of 20 g/L or higher observed in at least two serial measurements 1 week apart and sustained for 1 month or longer without exogenous growth factors support or transfusions. Analysis was by intention to treat. We report results from the first stage of this Simon two-stage design trial; enrolment into the second stage is continuing. This study is registered with ClinicalTrials.gov, number NCT00345345. FINDINGS From Oct 1, 2006, to March 1, 2015, we enrolled 25 patients with T-LGL. 14 patients (56%; 95% CI 35-76) had a haematological response at 3 months. Four patients with associated myelodysplastic syndrome and two who had received haemopoietic stem cell transplantation had either no response or were not evaluable, meaning 14 (74% [49-91]) of the 19 patients with classic T-LGL responded. All patients had an infusion reaction (24 [96%] patients grade 1-2, one [4%] patient grade 3), which improved with symptomatic therapy. All patients developed lymphopenia, with 22 (88%) patients having grade 3 or 4 lymphopenia. The other most common grade 3 and 4 adverse events were leukopenia (eight [32%]) and neutropenic infections (five [20%]). Seven patients died; all were non-responders. INTERPRETATION This is the largest and only prospective study of alemtuzumab in patients with T-LGL. The activity reported with a single course of a lymphocytotoxic drug in patients with mainly relapsed and refractory disease suggests that haematological response can be achieved without continued use of oral immunosuppression. FUNDING National Heart, Lung, and Blood Institute.

Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia

Oligoclonal expansion of CD8+ CD28− lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8+ CD28− cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8+CD28−CD57+ cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8+CD57+ cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1–5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8+ cells from aplastic anemia patients with CD8+CD57+ cell expansion by T-cell receptor deep sequencing, as well as the presence of 1–3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8+CD57+ cells, which also showed decreased diversity compared to total CD4+ and CD8+ cell pools. From analysis of complementarity-determining region 3 sequences in the CD8+ cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8+ T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064)

Circulating exosomal microRNAs in acquired aplastic anemia and myelodysplastic syndromes

Exosomal microRNAs modulate cancer cell metabolism and the immune response. Specific exosomal microRNAs have been reported to be reliable biomarkers of several solid and hematologic malignancies. We examined the possible diagnostic and prognostic values of exosomal microRNAs in two human bone marrow failure diseases: aplastic anemia and myelodysplastic syndromes. After screening 372 microRNAs in a discovery set (n=42) of plasma exosome samples, we constructed a customized PCR plate, including 42 microRNAs, for validation in a larger cohort (n=99). We identified 25 differentially expressed exosomal microRNAs uniquely or frequently present in aplastic anemia and/or myelodysplastic syndromes. These microRNAs could be related to intracellular functions, such as metabolism, cell survival, and proliferation. Clinical parameters and progression-free survival were correlated to microRNA expression levels in aplastic anemia and myelodysplastic syndrome patients before and after six months of immunosuppressive therapy. One microRNA, mir-126-5p, was negatively correlated with a response to therapy in aplastic anemia: patients with higher relative expression of miR-126-5p at diagnosis had the shortest progression-free survival compared to those with lower or normal levels. Our findings suggest utility of exosomal microRNAs in the differential diagnosis of bone marrow failure syndromes. (Registered at clinicaltrials.gov identifiers: 00260689, 00604201, 00378534, 01623167, 00001620, 00001397, 00217594).

Circulating S100A8 and S100A9 protein levels in plasma of patients with acquired aplastic anemia and myelodysplastic syndromes

&NA; The alarmin family members S100A8 and S100A9 are acute phase inflammation proteins, but they also have been proposed as biomarkers in many malignant and non‐malignant diseases. In this study, circulating S100A8 and S100A9 homodimers and S100A8/A9 heterodimers in plasma were systematically investigated by ELISA in aplastic anemia (AA) and myelodysplastic syndromes (MDS). Plasma was obtained from 58 severe AA (SAA) and 30 MDS patients, and from 47 age‐ and sex‐matched healthy donors. In 40 out of the 58 AA subjects, S100A protein levels were measured before and 6 months after immunosuppressive therapy (IST). No differences were observed in AA patients at diagnosis compared to healthy controls for circulating S100A homodimers and heterodimers. After therapy, SAA‐responders showed significantly increased circulating S100A8. Non‐responding patients had significantly higher levels of circulating S100A8/A9 compared to responders and healthy controls, but without variations of S100A8 and S100A9 homodimers. In MDS patients, circulating S100A8 was significantly elevated compared to those of AA and/or healthy controls. By Pearson correlation analysis of protein levels and blood counts, multiple correlations were found. However, as S100A8 and S100A9 are abundantly present in white blood cells and platelets, correlations with blood counts likely mirror the higher number of cells in the blood of some patients. In conclusion, our findings indicate that circulating S100A8 is increased in MDS but not in AA, and that may be useful to distinguish these diseases in the differential diagnosis of bone marrow failure syndromes. Clinicaltrials.gov identifiers: NCT00260689, NCT00604201, NCT01328587, NCT01623167, NCT00001620, NCT00001397.

Aptamer-based proteomics of serum and plasma in acquired aplastic anemia

Single-stranded oligonucleotides containing deoxyuridine are aptamers (SOMAmers) that can bind proteins with high specificity and affinity and slow dissociation rates. SOMAscan, an aptamer-based proteomic technology, allows measurement of more than 1,300 proteins simultaneously for the identification of new disease biomarkers. The aim of the present study was to identify new serum and plasma protein markers for diagnosis of acquired aplastic anemia (AA) and response to immunosuppressive therapies (IST). SOMAscan was used to screen 1,141 serum proteins in 28 AA patients before and after therapy and 1,317 plasma proteins in seven SAA patients treated with standard IST and a thrombopoietin receptor agonist. From our analysis, 19 serum and 28 plasma proteins were identified as possible candidate diagnostic and prognostic markers. A custom immunobead-based multiplex assay with five selected serum proteins (BMP-10, CCL17, DKK1, HGF, and SELL) was used for validation in a verification set (n = 65) of samples obtained before and after IST and in a blinded validation cohort at baseline (n = 16). After technical validation, four biomarkers were employed to predict diagnosis (accuracy, 88%) and long-term response to IST (accuracy, 79%). In conclusion, SOMAscan is a powerful tool for the identification of new biomarkers. We propose further larger studies to validate new candidate serum and plasma diagnostic and prognostic markers of AA.