Very long-term follow-up of aplastic anemia treated with immunosuppressive therapy or allogeneic hematopoietic cell transplantation

This retrospective cohort study included 302 patients diagnosed with AA who received HCT or IST as first-line therapy from 1973 to 2017 at the University Hospital Basel, Switzerland. Data included demographic information, pre-treatment blood values, date, type, source and protocols for therapy, response to therapy, status at last follow-up, causes of death and type of complications. Given the long observation period, many diagnostic methods had changed over time. Follow up was completed by 2019. Patients were followed at our center. Additional follow-up informations were obtained from referring centers.

Diagnosis and severity were defined by the Camitta Criteria [11]. For severe AA (SAA) the following criteria had to be fulfilled: bone marrow cellularity of < 30% and 2 of 3 of the following criteria: reticulocytes < 20 G/L (manual) or < 60 G/L (automated), absolute neutrophil count (ANC) < 0.5 G/L and platelets < 20 G/L. Patients with a neutrophil count < 0.2 G/L were classified as very severe AA (VSAA). Otherwise non severe AA (NSAA) was diagnosed [12] if there was no evidence for other disease causing marrow failure, hypocellular bone marrow was shown and cytopenias of at least two out of three peripheral blood cell lines below the normal values were documented: ANC < 1.2 G/L and ≥ 0.5 G/L, platelet count < 70 G/L, absolute reticulocyte count < 60 G/L, without fulfilling the criteria for SAA.

A total of 76 (25 %) patients received HCT as first line treatment, the first HCT was performed in 1973. Twenty nine patients (10%) received HCT as a second- or third- line treatment for relapse or non-response to immunosuppression. Treatment protocols and drug preparations changed over time; the majority of patients were conditioned with cyclophosphamide. After 1994 ATG (Thymoglobulin) was added. Fludarabine was given additionally since 2000 for older patients. Irradiation (total body, total lymphoid or thoraco-abdominal) was used in a minority of patients (n=4, 5.3%). GvHD prophylaxis consisted of methotrexate, combined with cyclosporine since 1979. Bone marrow was used as a primary source of stem cells (n=69, 90.8%), less frequently peripheral stem cells (n=5, 6.6%) or cord blood (n=2, 2.6 %). Mainly matched-related donor HCT was performed (n=62, 81.6%). Unrelated donor HCT was mostly second line treatment (n=24); 9 patients had unrelated donor HCT as part of first line treatment (7 of these had a congenital type of marrow failure). A total of 226 (75 %) patients received IST as first line therapy, of which the first course of ATG-based immunosuppression was given in 1973. Treatment protocols and drugs preparations changed over time: From 1976 to 1991 Lymphoser Berna (Swiss Serum and Vaccine Institute, Berne, Switzerland), from 1992 to 2006 Lymphoglobuline Merieux (Merieux, Lyon, France) and since 2007 ATGAM (Pfizer, New York, USA) was used as equine antithymocyte globulin. Besides, thirteen patients were treated with rabbit ATG (Thymoglobulin). From 1973 to 1981 thirty-two patients received haploidentical bone marrow infusion in addition to ATG, as previously described [13]. In none of these patients engraftment occurred and the hematopoetic reconstitution was autologous, thereby confirming that the therapy effect was due to IST alone [2]. These patients are counted among the IST group for the purpose of this analysis. Since 1981 methylprednisolone was given additionally to prevent serum sickness. Cyclosporine was added to the regimen in 1991 and was administered for at least one year. Norethandrolone, an androgen, was given until 1994. Few patients in the nineties were included in studies such as IL-3 or stem cell factor for relapsed disease, in the years 2002-2008 in the EBMT G-CSF trial [14] and since 2016 in the RACE trial (eltrombopag). Of the patients who received immunosuppression, 197 patients were never transplanted regardless of response. All patients received infectious disease prophylaxis and standard supportive care, including transfusions of red blood cells (since 1999 γ-irradiated (30 gy), leucocyte depleted) and platelet concentrates (since 2011 pathogen reduced (Intercept®)), as well as application of broad-spectrum intravenous antibiotics in case of fever and infection. They were either treated in laminar air flow rooms or in single rooms with reverse isolation [10].

Response was classified according to NIH criteria [15]. Complete response (CR) was defined by hemoglobin (Hb) levels > 100g/L, absolute neutrophile count (ANC) > 1.0 G/L, platelets > 100 G/L and no evidence of clonal evolution. Partial response (PR) was defined as transfusion independence, no longer meeting criteria for SAA and peripheral blood counts with Hb > 8 g/dl, ANC > 0.5 G/L and platelets > 20 G/L. Any patient not meeting any of the response criteria for PR or CR was classified as no response (NR).

Relapse in the HCT group is defined as primary non-engraftment or late graft failure. In patients treated by IST relapse is defined as decreasing blood count counts requiring transfusions or administration of another course of IST respectively performing HCT in patients with a prior response (PR or CR) [14].

Patients were evaluated regarding the following complications: clonal evolution to MDS/AML, secondary lymphoma and solid tumor, disease-related complications such as infections (bacterial, viral, fungal or protozoal), hemorrhage and thromboembolic events, therapy-related complications such as iron overload, cardiovascular events, endocrine dysfunctions, aseptic osteonecrosis and GvHD. Acute (aGvHD) and chronic GvHD (cGvHD) were diagnosed and graded according to published criteria [16, 17]. In case of hospital admission due to infection or hemorrhage, the event was classified as severe.

Cause of death was subdivided in six categories: disease-related (infection or hemorrhage), treatment-related (GvHD, graft rejection or death during therapy time), clonal evolution (MDS or AML), secondary malignancy (solid tumor or lymphoma), other (no association to the disease) and unknown.

Median follow up of surviving patients was 17 years (range: 1-41 years). Median age at diagnosis was 24 years (1-80 years), 18 years (1-66 years) in patients treated by HCT compared to 27 years (IQR 1-80 years) in the IST group. Sixty-nine patients (22.8%) were diagnosed with NSAA, 142 patients (47%) with SAA, 79 patients (26.2%) with VSAA and 9 (3%) with inherited bone marrow failure (of these 7 were transplanted upfront, in 2 diagnosis was made after failure of first line IST). Additional patient characteristics are shown in Table 1.

First line treatment was IST in 226 patients (75%), HCT in 76 (25%) of patients. Many patients had second line and subsequent lines of treatment, e.g. IST was second line treatment in 70 (61%) and HCT in 18 (16%); some patients had HCT as third (n=14, 36%) or subsequent line of treatment. Overall, 103 of the 302 patients had a HCT during their treatment course. The third most common second or subsequent line of treatment was the use of eltrombopag (n=13; as part of second or third line treatment). Other treatment included growth factors (n=18), cyclosporine as single agent treatment (n=6) and eculizumab (n=2) to treat clinical paroxysmal nocturnal hemoglobinuria (PNH).

Figure 1 shows response to treatment over time defined as CR, PR and NR as described above. Pie graphs display the proportion of patients in the states of CR, PR and NR at 1 year up to over 25 years in surviving patients, separately for all patients and those with IST and HCT as a first line treatment. PR and NR occurred more frequently after IST as compared to HCT. With long term follow-up most surviving patients were in CR.

Figure 2a outlines OS at 30 years separately for patients with IST and HCT as a first line treatment. EFS is shown in Figure 2b, whereas Figure 2c illustrates cEFS, i.e. the probability of being alive and in remission after several lines of treatment. CEFS is similar to OS indicating that only patients with functioning hematopoiesis remain alive long term. EFS curves show that only few events occur after 10 to 15 years. Table 2 lists OS, EFS, and cEFS at 30 years by first-line treatment, age, decade, and disease severity. There was a major improvement observed after 1980, whereas disease severity did not play an important role. The age distribution was atypical showing a higher mortality in children and adolescents due to higher proportions of children and adolescents being treated in the 1970s.

During follow up 145 patients (48%) died (Figure 2). There were more early deaths in the HCT group and more late deaths in the ATG group. Most deaths in the HCT group occured within the first year after transplant, thereafter death rate remains more stable. Death was mainly therapy-related in the HCT group, whereas disease-related in the IST group (42.5% and 60%, p=0.026). In long-term survivors death was mainly due to malignancy (42.8%) in the HCT group, whereas in post-IST long-term surviving patients the majority of death were caused by typical chronic conditions in the elderly such as heart disease.