Aruna Raghavachar

Treatment of Aplastic Anemia with Antilymphocyte Globulin and Methylprednisolone with or without Cyclosporine

BACKGROUND AND METHODS Immunosuppression is the most effective treatment for patients with aplastic anemia, except for bone marrow transplantation. The best results are achieved with antilymphocyte globulin or cyclosporine. Patients have been treated successfully with a combination of both agents, but there has been no controlled evaluation of its efficacy. We conducted a randomized, multicenter trial in 84 patients not eligible for bone marrow transplantation, comparing treatment with antilymphocyte globulin and methylprednisolone (41 patients--the control group) with antilymphocyte globulin, methylprednisolone, and cyclosporine (43 patients--the cyclosporine group). RESULTS At three months significantly more patients in the cyclosporine group had a complete or partial remission in response to treatment than did patients in the control group (65 percent vs. 39 percent, P less than 0.03); this difference was confirmed at six months (70 percent vs. 46 percent, P less than 0.05). The superior results of the regimen including cyclosporine were most evident in the patients with severe or very severe aplastic anemia, whose response rate at six months was 65 percent, as compared with 31 percent of such patients in the control group (P less than 0.02). Granulocyte and hemoglobin levels became normal in most patients who responded, but platelet counts continued to be subnormal in 61 percent of the patients. Ten of 52 patients with responses (3 in the cyclosporine group and 7 in the control group) relapsed 4 to 37 months after treatment. The actuarial survival of all patients at 41 months is 64 percent in the cyclosporine group and 58 percent in the control group (P = 0.16); among the patients with severe or very severe disease, survival is 80 percent and 44 percent, respectively (P = 0.077). Cyclosporine had substantial but reversible side effects. CONCLUSIONS Immunosuppressive treatment of aplastic anemia with antilymphocyte globulin, methylprednisolone, and cyclosporine appears to be more effective than a regimen of antilymphocyte globulin and methylprednisolone without cyclosporine and may thus represent a treatment of choice for patients who are not eligible for bone marrow transplantation.

Effectiveness of immunosuppressive therapy in older patients with aplastic anemia

Aplastic anemia is defined as peripheral blood pancytopenia associated with unexplained hypocellularity of the bone marrow without excess of blast cells. If aplastic anemia goes untreated, patients die of bleeding or infections caused by aplasia. Bone marrow transplantation and immunosuppressive treatment have improved outcome, with remission rates of 60% to 80% (1-4). The decision between immunosuppressive therapy and bone marrow transplantation depends largely on the availability of a bone marrow donor. In many centers, the upper age limit for allogeneic bone marrow transplantation in patients with aplastic anemia has been set at 40 to 55 years (5-8). This limit is traditionally less stringent for immunosuppressive treatment (2, 9-14). However, data are scarce, and no study has specifically addressed outcomes in older patients with aplastic anemia. After immunosuppressive treatment, hematologic recovery is slow and often incomplete (15), and clonal transformations, such as myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, or solid tumor, may occur (16-21). As a consequence, fear of life-threatening complications during prolonged aplasia as well as concerns about increased risk for clonal transformations, particularly in older patients, prevail. We sought to 1) determine the outcome of patients 50 years of age or older receiving immunosuppression therapy for aplastic anemia and 2) investigate the response and complication rate among these patients compared with that of younger patients. Methods Design This retrospective cohort study used data from 56 centers reporting to the European Group for Blood and Marrow Transplantation (EBMT) Severe Aplastic Anaemia Working Party between 1974 and 1997. Collected data included demographic information, pretreatment blood values, type of immunosuppressive therapy, date and number of courses of immunosuppressive therapy, response to therapy, date of last known vital status, cause of death, and type and date of late complications for every patient. Follow-up was completed by June 1997. Patients We included 810 patients from the EBMT Registry in whom acquired severe aplastic anemia was diagnosed according to current criteria (22), adequate immunosuppressive therapy (antilymphocyte globulin, cyclosporine, or both) was instituted, and bone marrow transplantation was not performed as second-line treatment. For the purpose of this analysis, patients were separated into three age groups: 50 to 59 years of age (n=115), 60 years of age or older (n=127), and 20 to 49 years of age (n=568). The latter patients served as the reference group. The Severe Aplastic Anaemia Working Party regularly stipulates the definitions for disease and late complications to its participants. No central slide review was performed. Table 1. Characteristics of 810 Patients with Aplastic Anemia Outcome Measures Outcome measures analyzed were overall survival, causes of death, response to immunosuppressive therapy, rate of relapse in responders, and late complications. Late complications were defined as secondary development of a myelodysplastic syndrome, leukemia, paroxysmal nocturnal hemoglobinuria, or solid tumor. Cause of death was classified as related to aplastic anemia or its treatment (bleeding or infection), secondary to late complications, unrelated to aplastic anemia, or unknown. Response to immunosuppressive therapy was defined as reaching complete independence from transfusions. Relapse was defined as dependence on transfusions after 3 months of independence from transfusions. Statistical Analysis Group differences were analyzed by using the Kruskal-Wallis test for continuous variables and the Fisher exact test for categorical variables. Survival probabilities were calculated by using the Kaplan-Meier estimator. Time at risk started at the date of first treatment and ended at the date of an event (response to immunosuppressive therapy, relapse, complication, or death) or the date of last known vital status, whichever came first. We calculated 95% CIs of survival probabilities according to the method of Rothman and Boice. Variables significantly associated with the risk for death were assessed by univariate and multivariate analysis. A two-sided log-rank test was used for comparisons. Because patients were treated at 56 centers, the possibility that center-specific differences in supportive care and patient selection would bias the results cannot be excluded. Therefore, univariate survival comparisons between age groups were made by using the log-rank test stratified on center. Sixty-eight percent of patients were treated at 6 centers (39 to 191 patients per center); 32% of patients were treated at 50 centers (1 to 24 patients per center). Grouping the latter patients led to 7 centers that were used for stratification. Proportional hazards regression analysis was used to assess the effect of known risk factors on survival. Variables considered were sex, age group, disease severity (reflected by neutrophil count at diagnosis), type of treatment (antilymphocyte globulin, cyclosporine, or both), and calendar year (1974 to 1979, 1980 to 1989, or 1990 to 1997). A backward stepwise procedure was used to eliminate nonsignificant variables (cut-off value, P>0.2). To adjust for the inherently increased risk for death with older age, the number of deaths observed after immunosuppressive treatment for aplastic anemia was compared with the expected number of deaths in a general European population matched for sex and age. The standardized mortality ratio (observed deaths/expected deaths) was calculated for each year after treatment. The 95% CI of the standardized mortality ratio was calculated by assuming a Poisson distribution of the number of observed deaths. The changes in risk for events over time were computed for the whole study sample as well as for the three groups separately. Statistical analysis was performed by using the SPSS statistical program (SPSS for Windows, release 6.1, SPSS, Inc., Chicago, Illinois). Role of the Funding Source The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Patients Demographic and disease characteristics of all 810 patients are listed in Table 1. Significant differences were seen among age groups. More female patients were 60 years of age or older, and more men were in the reference group (P<0.001). Fewer cases of viral-associated aplastic anemia were seen in patients 50 to 59 years of age, and no cases were seen in those 60 years of age or older (P=0.008). More patients 60 years of age or older received cyclosporine alone, and more patients in the reference group received antilymphocyte globulin (P<0.001). The proportion of older patients increased continuously over time: Until 1979, 13% of the patients were 50 years of age or older; this proportion increased to 27% from 1980 to 1989 and to 38% since 1990. Hence, the median follow-up was 47 months for surviving patients 50 to 59 years of age and 31 months for patients 60 years of age or older compared with 44 months in reference patients (P<0.001). Severity of disease, as reflected by neutrophil counts at diagnosis and number of courses of immunosuppressive treatment, did not significantly differ among the groups. Multiple interrelations were found among calendar year, type of immunosuppressive therapy, neutrophil count at diagnosis, and age. Calendar year was significantly associated with type of treatment (Table 1). Before 1980, all patients received antilymphocyte globulin alone; since 1990, 51% patients received antilymphocyte globulin and cyclosporine, 31% received antilymphocyte globulin alone, and 18% received cyclosporine alone (P<0.001). Since 1990, however, the type of immunosuppressive therapy was not equally distributed among the age groups (Table 1). Patients 60 years of age or older more often received cyclosporine alone (32%), whereas most patients in the reference group received a combination of antilymphocyte globulin and cyclosporine (61%) (P<0.001). Finally, disease severity was associated with age and calendar year. Before 1980, neutrophil counts less than 0.2 109 cells/L were encountered in 44% of patients in the reference group, 20% of patients 50 to 59 years of age, and none of the patients 60 years of age or older. Since 1990, low neutrophil counts were observed in 19% of patients in the reference group, 15% of patients 50 to 59 years of age, and 55% of patients 60 years of age or older. Survival At the time of last follow-up, 552 of 810 patients (68%) were alive and 258 (32%) had died (survival rate at 5 years, 67% [95% CI, 65% to 69%]). Survival was influenced by age: The older patients were at diagnosis, the lower the survival rate (Table 2). Survival also improved over time; the 5-year survival rate was 52% (CI, 39% to 64%) in patients treated before 1980 compared with 65% (CI, 60% to 69%) in patients treated from 1980 to 1989 and 73% (CI, 66% to 88%) in patients treated since 1990 (P<0.001). This improvement was most pronounced in the reference group (P<0.001) and was not statistically significant in patients 60 years of age or older (Table 2). Table 2. Five-Year Survival by Univariate Analysis Causes of Death Two hundred fifty-eight patients (32%) died. The mortality rate was 39% (45 of 115) for patients 50 to 59 years of age and 43% (55 of 127) for patients 60 years of age or older compared with 28% (158 of 568) for patients in the reference group. The main causes of death were directly related to aplastic anemia: Bleeding or infection occurred in 205 of 258 patients (79%). Thirty of 258 patients (12%) died of late complications, and 23 of 258 patients (9%) died of causes unrelated to aplastic anemia or its treatment. In all three age groups, bleeding or infection was the main cause of death (Table 3). The excess of mortality in older patients was due to aplasia-related d

Antilymphocyte immunoglobulins stimulate peripheral blood lymphocytes to proliferate and release lymphokines

Abstract: Five different preparations of antilymphocyte immunoglobulins (ATG) and antithymocyte immunoglobulins (ALG) with good or little clinical response were compared for their hematopoietic and immunological activities. All ATG/ALG lots demonstrated complement‐mediated cytotoxicity on peripheral blood mononuclear cells. They had different titers of antibody specificities against lymphocyte cell membrane antigens. Neither clinically effective nor ineffective lots demonstrated any apparent colony stimulating activity on bone marrow mononuclear cells. Purified Natural Killer cells failed to be stimulated by ATG/ALG in liquid culture. ATG/ALG demonstrated potent T‐cell stimulating activity comparable to phytohemagglutinin. This stimulation was blocked by anti‐IL‐2 receptor monoclonal antibodies, and was inhibited dose‐dependently by cyclosporin‐A. Some clinically ineffective ATG/ALG lots also stimulated T cells to release lymphokines. The differences in these characteristics among ATG/ALG lots provide some clues to guide further efforts to elucidate a key mechanism of therapeutic effectiveness.

A phase I/II trial of recombinant human interleukin-6 in patients with aplastic anaemia

Summary. In a phase I/II study, 11 patients with marrow failure (10 with acquired aplastic anaemia and one with pancytopenic Fanconi anaemia) were treated with recombinant human interleukin‐6 (rhIL‐6) to assess the safety and tolerability of rhIL‐6 and its effects on peripheral blood counts, bleeding complications and transfusion requirements. All patients with acquired aplastic anaemia were refractory to immunosuppressive treatment or had relapsed after immunosuppressive therapy and were not bone marrow transplantation candidates. Recombinant hIL‐6 was to be given as a once‐daily subcutaneous injection for 28 d at doses ranging from 0‐5 to 5‐0βg/kg. After an observation period of 2 weeks, five patients received a second treatment course of 28 d.

Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission

In an attempt to evaluate the role of thrombopoietin (TPO) in the pathobiology of aplastic anaemia (AA), we have examined TPO levels in sera from 54 AA patients and 119 healthy controls. A total of 92 samples were collected from AA patients: 43 samples were harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples when patients were in partial (n = 10) or complete remission (n = 16) following immunosuppressive treatment. TPO serum levels were assessed by a sandwich‐antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. Serum samples from normal donors revealed a mean TPO level of 95.3 ± 54.0 pg/ml (standard deviation). Mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728 ± 1074 pg/ml (P < 0.001 compared to normal controls; mean platelet count at that time: 27 × 109/l). TPO serum levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P < 0.001). However, despite normal platelet counts (mean 167 × 109/l), TPO levels remained significantly elevated in complete remission (mean TPO 1009 ± 590 pg/ml, P < 0.001 compared to normal controls). There was a significant inverse correlation between serum TPO levels and platelet counts in AA patients who were not transfused for at least 2 weeks prior to sample collection (coefficient of correlation (r) = −0.70, P < 0.0001).

Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron‐deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron‐deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti‐thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre‐treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.

High levels of thrombopoietin in sera of patients with essential thrombocythemia : Cause or consequence of abnormal platelet production?

Abstract Thrombopoietin (TPO) is the most important regulator of megakaryocyte development and platelet production. Platelet production is thought to be regulated by a negative regulatory feed back loop. In an attempt to evaluate the role of TPO in the pathobiology of essential thrombocythemia (ET), we have examined levels of TPO and other cytokines with thrombopoietic activity (interleukin-6 and interleukin-11) in sera obtained from 25 patients with ET (ten treated, 15 untreated) and 117 healthy control subjects. TPO serum levels were assessed using a sandwich-antibody ELISA that utilizes a polyclonal rabbit antiserum for both capture and signal. The mean serum TPO level in 25 ET patients was significantly elevated (545±853 pg/ml) as compared with that in healthy controls (95.3±54.0 pg/ml,p<0.001). The difference in TPO serum levels between ten treated (781±1229 pg/ml) and 15 untreated ET patients (388±458 pg/ml) did not reach statistical significance (p=0.09). We conclude that either consumption or production of TPO is altered in ET. Failure of appropriate feedback regulation and continued megakaryocyte stimulation by an elevated TPO may play an important role in the pathobiology of ET.

Paroxysmal Nocturnal Haemoglobinuria: A Replacement of Haematopoietic Tissue?

Acquired somatic mutations of the PIG-A gene lead to deficient expression of glycosyl-phosphatidyl-inositol-anchored proteins (GPI-AP) by haematopoietic cells and play a causative role in the pathogenesis of paroxysmal nocturnal haemoglobinuria (PNH). However, PIG-A mutations do not explain how the defective PNH clone can expand. It was hypothesized that a selection process conferring a relative advantage to the GPI-AP-deficient population is required. Since GPI-AP-deficient cells are also detectable in a substantial proportion of patients with otherwise typical aplastic anaemia (AA), the mechanisms inducing bone marrow failure might selectively spare the GPI-deficient cells. In order to examine the growth characteristics of GPI-AP-deficient cells in more detail, we performed repeated analyses of GPI-AP expression on peripheral blood cells in 41 patients with AA. We observed four patterns of the course of GPI-AP-deficient populations: (1) 13 patients showed normal expression of GPI-AP in the first analysis and in at least two follow-up studies at a median time of 709 days after the first analysis. (2) Secondary evolution of a GPI-AP-deficient population was a rare event. Only 4 patients with initially normal GPI-AP expression developed a GPI-AP-deficient population during follow up after immunosuppressive treatment. (3) Persistence of GPI-AP-deficient cells was observed in 16 patients during a median follow-up time of 774 days. However, in some patients, the size of the GPI-AP-deficient population increased substantially. (4) Disappearance of a GPI-AP-deficient population was observed in 8 patients. The time course of GPI-AP expression in relation to the treatment suggests that therapeutic interventions might modulate the ratio of normal versus GPI-AP-deficient haematopoiesis. Overall, these data argue against an ‘absolute growth advantage’ of GPI-AP-deficient cells. Our data are consistent with the hypothesis that haematopoietic failure caused by damage to normal haematopoiesis allows the outgrowth of a GPI-AP-deficient population. Thus, in at least some patients GPI-AP-deficient cells might pre-exist at a very low percentage and replace haematopoiesis after an insult to the normal cells.

Association between structural and numerical chromosomal aberrations in acute myeloblastic leukemia: a study by RT-PCR and FISH in 447 patients with de-novo AML

Abstract We analyzed 447 patients with de novo AML using α-satellite probes for the chromosomes 7, 8, X, and Y and RT-PCR for t(8;21), t(15;17) and inv(16). In 130/447 patients (29%) chromosomal aberrations were found. Thirty-three patients (7%) had a t(8;21); 11 of these had the additional loss of a sex chromosome (p<0.001) and two a trisomy 8. Twenty-nine patients (6%) had a t(15;17); four of these had a trisomy 8. Sixteen patients (4%) displayed an inv(16); four of these had a trisomy 8. Twenty-two patients (5%) had a sole trisomy 8 and one patient the combination of trisomy 8 and trisomy X. Five patients (1%) displayed the loss of a Y-chromosome as the sole abnormality and two patients had a sole trisomy X. In 22 patients (5%) a monosomy 7 was found, and in none of these patients were additional chromosomal aberrations detected by RT-PCR (p<0.05). In conclusion, trisomy 8 and the loss of a gonosome are frequently associated with structural chromosomal aberrations with a significant association of –X/Y and t(8;21). The absence of these genomic lesions in AMLs with monosomy 7 suggests that the monosomy 7 has a specific role in the development of these leukemias and their clinical course.

Induction of myeloperoxidase in five cases of acute unclassified leukaemia

Summary. For further analysis of their lineage affinity the differentiation capacity of five cases of acute unclassified leukaemias were studied in vitro using 5 nm 12‐O‐tetradecanoyl‐phorbol‐13‐acetate as an inducing agent. After 4 d in vitro a co‐expression of both myeloid and early B‐lymphoid or myeloid and early T‐lymphoid antigens on the same population of cells was found. While ultrastructural analysis of the blasts prior to culture revealed the morphology of undifferentiated blasts without any specific endogenous peroxidase activity, the cultured blasts displayed monoblastic features and myeloperoxidase activity after 4 d in vitro. Since this type of peroxidase is highly specific for the myelomonocytic lineage the data indicate myelomonocytoid differentiation of all five cases studied. The co‐expression of lymphoid as well as myeloid antigens and myeloperoxidase by the same blasts after culture could be indicative of bilineage differentiation capacity characterizing the original blasts as bipotential progenitor cells.