Catherine Nissen

Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT* SAA Working Party

This is an analysis of 509 patients with severe aplastic anaemia (SAA) treated in Europe between 1981 and 1986; 218 patients were treated by allogeneic bone marrow transplantation (BMT) from HLA identical sibling donors and 291 with immunosuppressive therapy (IS) with antilymphocyte globulin (ALG). The overall actuarial survival was 63% after BMT and 61% after IS therapy at 6 years. All patients fulfilled the criteria of SAA; however, most patients with a neutrophil count of <0.2 × 109/l also had infections and haemorrhages. Therefore a further subclassification was defined by pretreatment peripheral blood neutrophil count: very severe aplastic anaemia (vSAA) (<0.2 × 109/l neutrophils) and moderately severe aplastic anaemia (mSAA) (0.2‐0.5 × 109/l neutrophils).

Late haematological complications in severe aplastic anaemia

137 patients with severe aplastic anaemia (SAA) were treated in Basel from 1976 to 1986. 34 underwent bone marrow transplantation (BMT) and 103 received antilymphocyte globulin (ALG) therapy. We have analysed the incidence of late haematological complications in both groups of patients.

Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin

Summary. 468 severe aplastic anaemia (SAA) patients registered in the EBMT‐SAA registry who did not undergo bone marrow transplantation and were treated with immunosuppressive therapy (IS; 96% of patients received ATG) were evaluated. Their median age was 23 years (range 1–73) at initial IS therapy, 59% were males; in 69% the aetiology of SAA was idiopathic. Of these 468 patients, 245 had a follow‐up of <2 years after IS 166/245 died, 71/245 are still alive, 8/245 are lost to follow‐up. Of 223 patients who survived ≥2 years (LTS long‐term survivors), 191 are alive, 21 died >2 years and 11 are lost. Median follow‐up of 223 LTS was 4.1 years (range 2.0–10.9). Comparison of 166 patients who died <2 years and 223 LTS revealed no difference at time of initial IS therapy as regards sex, duration of AA, or its aetiology, but the age distribution and, in particular, severity of SAA differed significantly: more LTS were between 21 and 40 years old (44%v. 32%, P<0.02), less LTS had reticulocytes <20 × 109/l (63%v. 80%, P<0.001), polymorphonuclear granulocytes (PMN) <0.2 × 109/l (30%v. 57%. P<0.001), haemorrhages (58%v. 79%, P<0.002) and infection (30%v. 49%, P<0.005) at time of IS. A gradual improvement of blood counts was seen in patients alive ≥ 2 years after IS. At 2 years after IS 80% had a normal haemoglobin and PMN >0.5 × 109/l, but only after 5 years 80% of cases had platelets > 50 × 109/l. Development of clonal disease was reported of 31 LTS: 19 developed paroxysmal nocturnal haemoglobinuria (PNH), one acute leukaemia, 11 myelodysplastic syndromes and of these 11 five subsequently acute leukaemia. The majority of these patients (23/31) are still alive. Actuarial mortality of LTS is 22% at 8 years, but so far no plateau was achieved. It is concluded that SAA patients who become LTS following IS, show an improvement in haematological status but are probably not cured and are prone to develop clonal (malignant) disease.

Relapse of aplastic anaemia after immunosuppressive treatment: a report from the European Bone Marrow Transplantation Group SAA Working Party

Summary This study was designed to determine the incidence of relapse and factors predictive for relapse in 719 patients with severe aplastic anaemia (SAA) after immunosuppressive treatment (IS). Patients developing myelodysplasia or acute leukaemia after IS, and patients receiving a transplant, were excluded from this analysis. Response was defined as reaching complete independence from transfusions, relapse was defined as becoming again transfusion dependent. This criteria was validated by similar figures when using other ‘relapse criteria’ such as drop in neutrophil or platelet counts.

Cataract Formation after Bone Marrow Transplantation

Bone marrow transplantation is an established therapy for many patients with hematologic disorders [1]. Because many of these patients survive for long periods, quality of life and the possibility of late complications are increasingly important [2, 3]. Patient outcome depends on the results of the treatment used as preparation for bone marrow transplantation, the type and severity of the underlying disease, and the extent of acute and chronic graft-versus-host disease (GVHD) [4-7]. Cataract formation has been recognized in bone marrow transplant recipients as one of the first and most frequently occurring late complications of total-body irradiation (TBI) [8-10]. Single-dose TBI of 10 Gy or more causes cataract formation in all patients [11, 12]. Fractionation of the TBI dose to reduce this effect on the lens resulted in cataract formation in fewer than 20% of the patients after 4 years [11]. However, this therapy must be studied in a group of patients with a longer follow-up period. The minimal dose to allow lens opacification to develop in experimental models [13] and long-term observation of Hiroshima survivors [14] suggest that the risk after fractionated TBI may be understated. Therefore, we tried to determine the incidence of cataract formation resulting from different regimens used as preparation for bone marrow transplant and to evaluate the influence of other risk factors. Methods Study Design This study was part of a prospective, controlled cohort study to evaluate late effects of bone marrow transplantation. Some results involving ischemic microvascular lesions of the ocular fundus [15] and chronic cyclosporine-associated nephrotoxicity [16] have been reported. All patients were examined according to a standardized protocol before bone marrow transplantation; 3, 6, and 12 months after transplantation; and once per year thereafter. The examination included full clinical and ophthalmologic evaluations by a senior-level ophthalmologist, which consisted of a visual acuity test and slit-lamp and fundus examinations after pupillary dilatation. The dates of first manifestations of cataracts and of operations were recorded. Patients Participants included all patients who had bone marrow transplantations at the Kantonsspital, Basel, Switzerland, between 1979 and December 1991 for malignant hematologic diseases or severe aplastic anemia. During this time, 290 patients received either allogeneic (n = 256), autologous (n = 27), or syngeneic (n = 7) bone marrow transplants. All patients with a follow-up period of at least 180 days were included in this study of cataract formation. Preparative Regimens and Prevention of Graft-versus-Host Disease The basic bone marrow transplant protocol remained the same throughout the entire study period and has been reported in detail [17, 18]. Briefly, patients with a malignant hematologic disease were treated with cyclophosphamide, 60 mg/kg body weight per day on 2 consecutive days, followed by TBI. Starting in April 1986, this treatment was preceded by etoposide (VP-16), 30 mg/kg, given on day 9 before transplant. Patients with severe aplastic anemia received cyclophosphamide alone, 50 mg/kg per day, for a period of 4 days. Patients with a malignant disease and previous irradiation were given preparatory doses of cyclophosphamide and busulfan. For prevention of GVHD, patients were given cyclosporine A alone and, starting in 1991, cyclosporine A combined with methotrexate. From December 1985 to December 1989, the bone marrow of patients older than 25 years with malignant diseases was depleted of T cells by counterflow elutriation [19]. Patients with autologous or syngeneic bone marrow transplants received no treatment to prevent GVHD. Starting in 1982, high-dose methylprednisolone was applied as the first line of treatment for GVHD of grade II or more: This therapy included 1000 mg on the first day and 500 mg on the second day followed by 0.5 mg/kg per day. This treatment was repeated as many as three times if necessary. Patients with chronic GVHD were treated with cyclosporine A alone or in combination with prednisone in an initial dose of 1 mg/kg per day. Irradiation Protocol Total-body irradiation was applied by a linear accelerator (Philips SL 75-5, 4.2 MeV; Crawley, United Kingdom) throughout the entire study period. Radiation parameters were adapted as required. From 1979 to September 1985, TBI was given in a single dose calculated as a maximal lung dose of 10 Gy and at a rate of 7.5 cGy/min. In the first 18 patients, the lung was shielded after a dose of 8 Gy. Thereafter shielding was discontinued. Since October 1985, TBI was fractionated in 6 doses of 2 Gy on 3 consecutive days before bone marrow transplantation [12]. The first 16 patients were irradiated at a dose rate of 20 cGy/min. Thereafter, this dose rate was reduced to 3.5 cGy/min. The applied dose was calculated to be the maximal lung dose. No shielding of the eyes was ever applied. Statistical Analyses All medians and ranges of numeric variables were calculated by the NCSS statistical program and compared using the chi-square test. Analyses of the risk for cataract formation and the probability of operation were done according to the method of Kaplan and Meier [20]. For the univariate analysis, the log-rank test with two-sided significance levels was used to compare risk groups for cataract formation [21]. Proportional-hazards regression analysis was used to relate potential risk factors to the development of cataracts [22, 23]. A forced-entry regression of all tested variables, such as age, sex, TBI, type of irradiation, chronic GVHD, as well as treatment with prednisone for more than 3 months after bone marrow transplantation, was used. Results Patients One hundred ninety-seven of 290 (68%) patients had follow-up periods of at least 180 days after bone marrow transplantation and could be evaluated for cataract formation. Their primary characteristics are listed in Table 1. There were 105 male and 92 female patients between 2 and 50 years old (median age, 25 years). Of 164 patients treated with TBI, 74 received single-dose TBI and 90 received fractionated TBI. Thirty-three patients were given chemotherapy alone as preparatory treatment. Table 1. Characteristics of Patients Evaluated for Cataract Formation As expected from the treatment protocols, the three groups of patients differed with respect to type of bone marrow transplant, disease, and median follow-up period. In addition, more women were prepared with single-dose TBI, but fewer with fractionated TBI or chemotherapy alone. Allogeneic bone marrow transplant predominated in all treatment groups. In preparation for autologous bone marrow transplantation, most patients received either fractionated TBI or chemotherapy alone. Most patients with hematologic cancers received TBI, whereas all patients with severe aplastic anemia were given preparatory chemotherapy alone. Furthermore, during the study, new indications for bone marrow transplant were tested. Thus, myelodysplastic syndromes or lymphoma has been treated by bone marrow transplantation only since 1986. At this time, all patients received fractionated TBI. Survival As of 1 January 1992, the actuarial probability of survival according to Kaplan and Meier analysis for the 290 patients treated during this study was 43% (95% CI, 35% to 51%) at 15 years. Of 197 patients who survived the first 180 days, 134 (68%) were alive 0.5 to 16 years after transplantation (median survival, 3.4 years), 110 of them after a preparatory regimen of TBI (45 received single-dose TBI and 65 fractionated TBI) and 24 after chemotherapy alone. Cataract Formation No patient showed lens opacification in pretransplant ophthalmologic evaluations. After bone marrow transplantation, cataracts developed in 70 of the 197 patients (36%) within 3 to 63 months (median, 38.4 months). Forty-six patients (23%) needed surgical correction of their cataracts. There were important differences based on the type of preparative regimen used before transplant (Table 2). Table 2. Incidence of Cataract Formation and Surgical Repair after Bone Marrow Transplantation according to the Type of Total-Body Irradiation* Single-Dose, Total-Body Irradiation After single-dose TBI, cataracts developed in 51 of the 74 (69%) patients (Table 2). Lens opacification started as early as 3 months after bone marrow transplantation and was observed in all 51 patients (CI, 93% to 100%) alive 3.5 years after surgery (Figure 1). Forty-four (59%) patients needed surgical repair of cataracts, which included simple lens extraction in 6 patients and simultaneous implant of new lenses in 34 patients. In 4 patients, the operation was done at another institution and the type of surgery was unknown. The probability of being operated on was 85% (CI, 75% to 95%) 6 years after bone marrow transplant. After 9 years, all patients alive needed surgical repair of their lenses (Figure 2). The cataract surgery was done as early as 2 years after bone marrow transplantation in some patients. The median time to surgery, however, was 4.1 years after transplantation. The interval between the onset of cataracts and surgical treatment was about 1.5 years. Twenty-four of the 44 (55%) patients having surgery showed residual lens opacifications called after-cataract, 16 (36%) of them requiring yag-laser therapy. Visual acuity improved after treatment in most patients. However, 4 of the 44 (10%) patients treated with surgery have residual visual damage that interferes with normal daily activity despite surgical correction. Figure 1. The probability of cataract development in 197 patients who were given preparatory single-dose, total-body irradiation, fractionated irradiation, or chemotherapy alone (no irradiation) and then bone marrow transplantation. Figure 2. The probability of requiring cataract surgery in 197 patients who were given preparatory single-dose or fractionated total-body irradiation

Late Clonal Complications in Severe Aplastic Anemia

One hundred and seventy patients with severe aplastic anemia (SAA) were treated in Basel, from 1976 to 1992. Forty one underwent bone marrow transplantation (BMT) and 129 antilymphocyte globulin (ALG) therapy. As of January 1, 1993, 99 of the 170 patients are alive (58% +/- 7%) and the probability to be alive at 15 years is 54% +/- 4%. Until now, 29 patients have developed a clonal complication. All occurred within the ALG group. Nine patients developed a myelodysplastic syndrome (MDS), 16 patients paroxysmal nocturnal hemoglobinuria (PNH) and 4 patients both, PNH and MDS. The cumulative risk of developing a clonal complication after ALG-therapy is 42% +/- 13% at 15 years; for MDS this risk is 26% +/- 8% and for PNH 25% +/- 5%. The development of a clonal disease directly affects long term prognosis. The survival of the patients with stable disease is 81% +/- 10% and 36% +/- 13% for those with clonal evolution (p = 0.001). The most important risk factor is the type of treatment. In contrast to patients treated with ALG, none of the patients treated with BMT developed MDS or PNH (p < 0.001). No other clinical parameter, such as age, sex, etiology of SAA, severity of the disease and splenectomy correlate with an increased risk of developing this complication. In contrast, morphological parameters at the time of diagnosis, during bone marrow regeneration and at remission are indications in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

Umbilical cord blood from preterm human fetuses is rich in committed and primitive hematopoietic progenitors with high proliferative and self-renewal capacity

Human umbilical cord blood (CB) has been recognized as a source of hematopoietic stem cells for transplantation. While hematopoietic properties of neonatal CB from full-term pregnancies have been well characterized, little is known about CB from early gestational ages. We analyzed the content and the growth properties of primitive and committed hematopoietic progenitors in preterm CB from second trimester (week 16-28; n = 17) and early third trimester (week 29-34; n = 17) in comparison with term CB (n = 18). The frequency of CD34+ and CD34+CD38- cells was significantly higher in preterm than in term CB (mean, 2.51% and 0.56% vs 0.88% and 0.13%;p < 0.002). The number of colony forming units (CFU) in preterm CB was about twofold higher (230 +/- 6 vs 133 +/- 14/ 10(5) mononuclear cells; p < 0.05) and correlated with the content of CD34+ progenitors (r = 0.73). Long-term culture initiating cells (LTC-IC) were enriched about 2.5-fold (6.7 +/- 2.9 vs 2.6 +/- 1.2/10(5) cells; p < 0.05). Progenitors from preterm CB could be expanded in stroma-free liquid cultures supplemented with hematopoietic growth factors as efficiently as progenitors from term neonates. In short-term cultures containing erythropoietin (Epo), interleukin (IL)-1, IL-3, and IL-6, or granulocyte- (G-) and granulocyte-macrophage colony-stimulating factor (GM-CSF) together with stem cell factor (SCF) or Flt3 ligand (FL), expansion of CFUs was six- to eightfold at week 1. In long-term cultures containing thrombopoietin (TPO) and FL, an approximately 1000-fold expansion of multilineage progenitors was observed at week 10. In summary, we show that preterm CB compared with term CB is richer in hematopoietic progenitors, and that precursors from preterm CB can be extensively expanded ex vivo. This may have implications for the development of transplantation and gene transfer strategies targeting circulating fetal stem cells.

Antilymphocyte globulin for myelodysplastic syndrome

Bennett, J.M., Catovsky, D., Daniel, M-T., Flandrin. G., Galton, D.A.G., Gralnick, H.R. & Sultan, C. (1980) A variant form of hypergranular promyelocytic leukaemia (M3). British Journal of Haematologg. 44, 169-1 70. Lemei. P. (1985) Cytochemical examinations of cultivated unclassified leukaemic cells. Histochemical Journal, 17, 1377-1 380. Li, C.Y., Lam, K.W. & Yarn. L.T. (1973) Esterases in human leukocytes. Journal of Histochemistry and Cgtochemistrg, 2 1, 1-1 2. Pegoraro, L.. Abrahm. J., Cooper, R.A., Levis, A.. Lange, B.. Meo. P. & Rovera. G. ( 1 980) Differentiation of human leukemias in response to 12-0-tetradecanoylphorbol-13-acetate in vitro. Blood, 55,859862. Tomonaga. M., Yoshida. Y.. Tagawa. M.. Jinnai, I.. Kuriyama. K.. Amenomori, T., Yoshioka, A.. Matsuo. T.. Nonaka, H. &Ichimaru. M. (1985) Cytochemistry of acute promyelocytic leukemia (M3): leukemic promyelocytes exhibit heterogeneous patterns in cellular differentiation. Blood, 66, 3 50-3 57. neity of AML M3 was pointed out. Strong positivity of BE in ‘faggot cells’ was not described. 12-0-tetradecanoylphorbol-13-acetate (TPA) induced a monocyte/macrophage differentiation in leukaemic promyelocytes in AML M3 (Pegoraro et al, 1980; Lemei, 1985). ‘Faggot cells’ cultivated with TPA for 3 d became firmly adherent to the bottom of a culture dish and acquired macrophage-like morphology with several Auer rods retained (unpublished results). The reported case represents AML M3 because of the hypergranular cell morphology, the ‘faggot cells’, the typical haemostatic findings, and the course of the disease. Being a monocytic differentiation marker the strong BE positivity in AML M3 cells might cause problems in differentiating of promyelocytic and myelomonocytic leukaemias. The ‘faggot cells’ as well as normal promyelocytes have the capability to differentiate into monocytoid cells (strong BE) in vivo and in vitro: from this point of view there is no reason to speak about a differentiation infidelity (Tomonaga et al, 1985) in such AML M3 cases.

Immunostimulatory effects of different antilymphocyte globulin preparations: a possible clue to their clinical effect

Summary Antilymphocyte globulin (ALG) and antithymocyte globulin (ATG) have an established role in the treatment of severe aplastic anaemia. The response rate ranges from 40% to 80%. Its mode of action is believed to be complement dependent lysis of immunocompetent cells which inhibit haemopoietic maturation. This might not be the sole mechanism. We have tested four different preparations of ALG/ATG for their mitogenic effect on normal peripheral blood cells and on enriched T‐cells in vitro by 3H‐thymidine incorporation. We found marked differences between the four preparations. One was strongly mitogenic and able to induce profound release of haemopoietic growth factors. This mitogenic effect could be detected in the serum of patients during ALG treatment. Clinical response rates of this preparation are about 80%. Three other preparations were of lower or no stimulatory effect. Clinical response rates with these preparations vary between 40% and 60%. From our results, we postulate that the beneficial effect of ALG could be partially due to its ability to stimulate release of haemopoietic growth factors. The mitogenicity of different ALG/ATG preparations should be tested as an in vitro parameter of clinical efficacy.

STAT3-mutations indicate the presence of subclinical T cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.