Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC)

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM) was analyzed pre-transplant and 3–4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.     

Bone marrow purging with mafosfamide--a critical survey

Autologous bone marrow transplantation (ABMT) is increasingly used to consolidate remissions, primarily in hematological disease. Various purging strategies have been developed to minimize the risk of reimplantation of tumor cells with the bone marrow autotransplant. Pharmacological purging with the oxazaphosphorine derivative mafosfamide has been studied extensively, and recent clinical data suggest that purging with mafosfamide may translate into superior remission duration if compared to nonpurged ABMT in acute leukemia. Chemical and experimental data relevant to mafosfamide-purging and clinical results are reviewed, with special emphasis on safety aspects.     

Autologous bone marrow transplantation (ABMT) using unpurged marrow as intensification for first complete remission in acute leukaemia (AL)

In order to assess the clinical advantage of autologous bone marrow transplantation (ABMT) without ex vivo purging, the results in 26 patients (10 AML, 16 ALL) in 1. CR were analyzed retrospectively. All patients received 3 consolidation cycles "in-vivo purging" before marrow harvesting. Beside relapses infections and cardiac failure were the most frequent complications. After 1 to 12.5 months 11 cases relapsed with a higher probability in patients who had a longer period of induction and between CR and ABMT. 12 patients became relapse-free survivors 6 to 53 months after ABMT with a stable plateau after 12.5 months for 8 patients. In conclusion, ABMT following "in-vivo purging" as the strongest one-step postremission therapy in patients with acute leukaemias may be a way for better long-term results in these patients.     

Poor prognosis of acute lymphoblastic leukemia with translocation (1;19) in childhood: potential interest of allogeneic bone marrow transplantation

We report a new case of childhood acute lymphoblastic leukemia with translocation (1;19). At the time of diagnosis, the only adverse prognosis factor was the existence of this translocation. Under conventional chemotherapy, the girl experienced early marrow relapse (duration of first remission was 2 months). She received allogeneic bone marrow transplantation during the second remission and is alive in continuous complete remission 20 months after transplant. Several earlier reports have suggested that children with the (1;19) have a poor prognosis; If this poor response to conventional therapy is confirmed, an allogeneic bone marrow transplantation should be considered during the first remission.     

Immune recovery after allogeneic stem cell transplantation: study of 19 patients

The aim of this study was to access average delays for novogeneration of myeloid and lymphoid cells after allogeneic bone marrow transplantation (BMT) outcome and factors affecting this organization. A prospective analysis over 2 years (01/01/07 to 31/12/08) enrolling 19 children treated with allogeneic intrafamilial bone marrow transplantation. Indications for bone marrow transplantation were: aplastic anemia (3 cases), bemoglobinopathies (9 cases), myelodysplastic syndrome (1 case) and primary immunodeficiency (6 cases). Different conditioning regiments were used according to the indication. The study of immune reconstitution was based on the quantitative determination of immunoglobulin and lymphocyte subpopulation. These tests were routinely requested to 1 month, 2 months, 3 months, 6 months, 9 months and 12 months. The average time of engraftment was 18 days (12-24). A rate of CD4+T lymphocytes>200/mm3 was provided within an average of 2,5 months (1-7). The average time to obtain CD8+T lymphocytes>200/mm3 was 2 months (1-5). The humoral immune reconstitution was made within an average of 2 months (1-4). A report of CD4+/CD8+T lymphocytes>I was obtained within 10 months and a half (1-24). Univaried analysis showed a correlation between the bone marrow sex matched and the faster reorganization of CD8+T cells (p=0.042). A quantity of CD34+>6 10(6)/kg was significantly associated with the recapture of a formula lymphocyte CD4+/CD8+T>1 (p=0.03) Immune recovery post bone marrow transplantation in children begins with myeloid lineage then lymphoid B then lymphoid T The inversion of the report CD4+/CD8+T lymphocytes, seems to be influenced by the high contain of CD34+cells in the graft as well as the type of conditioning.     

Effect of six weekly transfusions on canine marrow grafts: tests for sensitization and abrogation of sensitization by procarbazine and antithymocyte serum.

We have previously shown that blood transfusions can immunize a dog and lead to rejection of a subsequent marrow graft despite lethal total body irradiation (TBI). Sensitization to histocompatibility antigens induced by two prior transfusions of whole blood could be overcome by a regimen of procarbazine and anti-thymocyte serum (ATS) preceding TBI. The current study investigated a) whether this regimen could abrogate sensitization induced by six weekly transfusions given from days --50 to --15 preceding a marrow graft, and b) whether platelet survival studies and two in vitro tests of immunity could predict marrow graft rejection. All donor-recipient pairs were histoincompatible, unrelated, and of different breed. Twenty-two recipients received platelet concentrate transfusions and eight received whole blood transfusions. Recipients were given 1200 R TBI and a graft of marrow and peripheral blood leukocytes from the transfusion donor on day 0. Three of 15 recipients (20%) given procarbazine, 12.5 mg/kg i.v. on days --8, --6, and --4, and ATS, 0.6 ml/kg subcutaneously on days --7, --5 and --3, Rejected their grafts, whereas 11 of 15 dogs (73%) not given procarbazine and ATS rejected their grafts (p less than 0.01). Serum lymphocytotoxic antibodies, peripheral leukocyte migration inhibition, and in vivo donor platelet recovery and survival were studied in those recipients receiving six weekly transfusions and in 18 other recipients receiving a single donor transfusion 3 months before marrow grafting. No significant correlation was found among these in vitro and in vivo tests of sensitization. Sensitization to marrow grafts was not reliably detected by the presence of cytotoxic antibodies or leukocyte migration inhibition. Platelet survival, however, was positively correlated with the results of marrow grafting in 12 of 15 (80%) evaluable recipients (p approximately 0.15).     

Linked unresponsiveness: early cytokine gene expression profiles in cardiac allografts following pretreatment of recipients with bone marrow cells expressing donor MHC alloantigen

Linked unresponsiveness operates to induce specific unresponsiveness to fully mismatched vascularized allografts in recipients pretreated with anti-CD4 antibody and syngeneic bone marrow cells expressing a single donor MHC class I alloantigen. The aim of the study was to evaluate early post transplant cytokine expression in allografts where linked unresponsiveness was required for long term graft survival. CBA (H2(k)) mice were pretreated with CBK (H2(k)+K(b)) bone marrow cells under the cover of anti-CD4 antibody 28 days before transplantation of a CBK or a C57BL/10 (H2(b)) cardiac allograft. In both cases graft survival was prolonged (MST=100 days). Intragraft expression for interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12(p40), IL-18, iNOS, transforming growth factor (TGF)-beta(1) and C-beta was determined on day 1.5, 3, 7 and 14 after transplantation. Whereas rejecting allografts displayed a sharp peak in IFN-gamma, IL-2, IL-4 and IL-10 expression, non-rejecting allografts were characterized by an initial TGF-beta(1) and IFN-gamma production. An increasing IL-4 expression towards day 14 was a unique feature of linked unresponsiveness. All non-rejecting allografts were characterized by an increasing IL-12(p40) production towards day 14. In summary, the early cytokine expression pattern in allografts after bone marrow induced operational tolerance is influenced by the quantity of donor alloantigens expressed on the graft as well as on the bone marrow inoculum.     

Status of allogeneic bone marrow transplantation in childhood in the GDR

Of 25 HLA-identical, MLC negative transplants 10 patients had acute lymphoblastic leukaemia (ALL), 8 acute nonlymphoblastic leukaemia (ANLL), 3 severe aplastic anaemia, 2 malignant histiocytosis, 1 patients neuroblastoma and 1 Fanconi anaemia. 3 HLA nonidentical, MLC positive transplants were performed, two children had malignant infantile osteopetrosis and 1 child had a severe combined immunodeficiency disease. Patients with ALL and ANLL received cyclophosphamide and single dose total body irradiation. 3 patients received fractionated TBI. The results for the allogeneic group overall indicate that the actuarial disease free survival rate is 0.62. 16 of 25 patients are in continuous complete remission (CCR) periods of 3-78 months posttransplant. All three transplanted children with severe aplastic anaemia alive disease-free for periods of 21-81 months. 10 patients with ALL were transplanted (2 in first remission for high risk ALL, 8 in second remission). 7 of 10 patients are alive and disease-free (CCR rate 0.67). 8 patients underwent BMT for ANNL while in first remission in 7 patients and in third partial remission in 1 patient. 4 of 8 patients are alive and disease-free for periods of 25-56 months (CCR rate 0.50). 1 patient with neuroblastoma stage IV survives 24 months, 1 child with Fanconi anemia died on day +25 of GVHD and septicaemia. 1 of the 2 patients transplanted for malignant histiocytosis relapsed 3 months posttransplant, 1 patient is alive and disease-free 5 months posttransplant. In none of the HLA-nonidentical and MLC positive transplantations T-cell depleted marrow engrafted.     

Late marrow recurrences in childhood acute lymphoblastic leukaemia.

Thirty children with acute lymphoblastic leukemia had a recurrence in the bone marrow after treatment was stopped electively. A second haematological remission was achieved in 27 (90%), and the median duration of remission was shortest (six months) in those relapsing within six months of stopping treatment. Four of six children relapsing over one year after stopping treatment remained in second haematological remission. Leukaemic infiltration of the central nervous system developed in four patients remaining in marrow remission. It is concluded that conventional chemotherapy is unlikely to be effective in children with acute lymphoblastic leukaemia who relapse soon after stopping treatment, that "reprophylaxis" of the central nervous system probably with long-term intrathecal chemotherapy is essential, and that some patients relapsing after prolonged unmaintained remission may achieve long-term leukaemia-free survival.     

Transplantation of bone marrow with constitutional chromosomal anomalies. Cytogenetic studies and clinical implications

We report on two cases of transplantation of bone marrow with constitutional chromosomal anomalies. A female patient with acute myelocytic leukemia (FAB, M 3) in first complete remission received a bone marrow graft from her sister with the karyotype 47 XXX (triple-X-syndrome). A male patient with Ph-positive CML and a constitutional Robertsonian t(14; 15) received HLA and MLC loci compatible bone marrow from his sister who was also a carrier of the Robertsonian t(14; 15). Our findings indicate that transplantation of marrow from donors with balanced chromosomal translocation is possible, although no conclusion can be made regarding long term results as both recipients died early from infectious complications.     

Proliferation of peripheral lymphocytes to interleukin-2 and interleukin-4 after marrow transplantation.

Defects in the interleukin-2 (IL-2)-mediated T-lymphocyte activation/proliferation pathway have been implicated as contributing to the compromised immune function observed in patients following bone marrow transplantation (BMT). Since interleukin-4 (IL-4) is also involved in T-lymphocyte function, we have examined whether phytohemagglutinin (PHA)- or anti-CD3 (OKT3)-activated lymphocytes obtained from patients after allogeneic or autologous BMT are capable of proliferating in response to human recombinant IL-4, and compared these results to those obtained using human recombinant IL-2. Peripheral blood lymphocytes from marrow graft recipients were initially cultured for 3 days in the presence of PHA or OKT3. Such mitogen-activated lymphocytes exhibited little or no proliferation (as assessed by incorporation of [3H]-thymidine) following culture for an additional 3 days in the presence of IL-4 or IL-2. Results were similar for lymphocytes obtained from patients early (less than 4 months) after marrow grafting and those obtained from long-term marrow graft recipients with chronic graft-vs-host disease at the time of testing. In contrast, lymphocytes obtained from healthy individuals proliferated in response to IL-4, as well as to IL-2, following initial activation with PHA or OKT3. Immunofluorescence analysis showed that in normals equal numbers of CD4 and CD8 cells proliferated after stimulation with anti-CD3 antibody and IL-2. However, in BMT patients there was a predominant proliferation of CD8 cells using the same stimulator. These results indicate that defects in the IL-4-mediated T-lymphocyte activation/proliferation pathway may also contribute to the immunodeficiency observed following BMT.     

Interleukin-2 induces chemotactic deficiency in patients with onco hematologic malignancies and autologous bone marrow transplantation.

Unusual gram positive bacteremia has been reported in non granulopenic patients receiving recombinant human interleukin-2 (IL-2) suggesting a beneficial effect of anti gram positive prophylaxis in such patients. We report here studies on granulocyte functions examined during the course of high dose IL-2 therapy (16 to 24 million IU/m2/days for 11 to 18 days) administered during a period of 35 days in 14 patients including 4 solid tumors, 5 chronic myeloid leukemias, 4 recipients of autologous bone marrow transplant (ABMT) and 1 recipient of syngeneic bone marrow transplant. Neutrophils functions were studied before IL-2 administration (d 0), after the first cycle (d 8) and after the third cycle (d 36). Nylon fiber adherence, superoxide production, random migration, phagocytosis, nitroblue tetrazolium reduction, lysozyme and elastase release were not impaired significantly throughout therapy. However N-Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) stimulated chemotaxis of granulocytes, normal before therapy, was significantly impaired as early at d 8 and severely inhibited at d 36 (p less than 0.001). Three septicemia, one corynebacteria parvum septicemia and two gram-negative septicemia despite normal neutrophil counts and oxacillin or Penicillin G plus Pefloxacin prophylaxis, occurred among the 14 patients studied. Although neutrophil functions were not more depressed in transplanted patients than in the other non transplanted patients, special attention should be paid to such patients in whom delayed immune reconstitution could increase the risk of sepsis.     

Allogeneic bone marrow transplantation for high risk non-Hodgkin's lymphoma during first complete remission

Allogeneic bone marrow transplantation from histocompatible sibling donors was performed in six patients with extranodal involvement of high grade lymphoma during first complete remission. Five patients had lymphoblastic lymphoma and one had diffuse undifferentiated lymphoma. The cytoreductive/immunosuppressive regimen consisted of total body irradiation and high dose cyclophosphamide. Four patients are alive in complete remission at 8 months, 14 months, 21 months and 47 months post transplantation. One patient who relapsed 7 months after his initial transplantation underwent a second transplantation but another relapse 17 months later led to his death. One patient died of chronic graft-versus-host disease and at autopsy there was no evidence of lymphoma. These data demonstrate that allogeneic bone marrow transplantation can produce durable remissions in patients with high grade lymphoma who present with bone marrow, central nervous system and/or skin involvement.     

Laminar air flow versus barrier nursing in marrow transplant recipients

Forty-eight patients with acute leukaemia in relapse (n = 14), acute leukaemia in complete remission (n = 19), chronic myeloid leukaemia (n = 8) or severe aplastic anaemia (n = 7) received a marrow transplant. The first 26 patients were nursed in laminar-air-flow plastic isolators while the next 22 patients were treated in barrier nursing rooms. Gnotobiotic parameters and morbidity in the 2 groups are compared. Good decontamination of the gastro-intestinal tract was obtained using either of the 2 isolation techniques. The incidence of bacterial and mycotic infections, as well as the supportive care required by the patients was almost equal in both groups. Our results also suggest that the incidence of graft versus host disease may decrease with efficient decontamination of the patients.     

Graft-versus-host disease following interleukin-2/lymphokine-activated killer (LAK) cell immunotherapy in a patient with acute myelogenous leukaemia in second complete remission: autologous LAK cells following allogeneic bone marrow transplantation are donor-derived

A 48-year-old man was treated by allogeneic bone marrow transplantation (BMT) in first remission of M4 acute myelogenous leukaemia (AML). He experienced no graft-versus-host disease (GVHD) and 7 months later he relapsed. Following further chemotherapy, he entered a second complete remission; however, he refused a further allogeneic or autologous BMT but agreed to immunotherapy with interleukin-2 and autologous lymphokine-activated killer (LAK) cells. He tolerated this treatment well but went on to develop grade II skin GVHD. Polymerase chain reaction studies of DNA microsatellites of the autologous LAK cells showed that they were of donor origin. The patient remained well for 9 months until, immediately following the introduction of prednisolone for his persistent GVHD, he relapsed. He declined further active treatment and died 5 months later. The case shows that IL-2/LAK cells can be safely given to patients who have experienced no GVHD following allo-BMT and are likely to be effective through an ongoing graft-versus-leukaemia effect.     

In vitro activity of folic acid on the proliferation dynamics of human bone marrow CFU-GM in propyphenazone-induced granulocytopenia]

The in vitro action of folic acid was tested on the proliferation of bone marrow granulocyte-macrophage progenitor cells from a patient with drug-induced (propyphenazone) neutropenia in remission 20 days after the drug had been suspended. Various bone marrow cultures were prepared with standard stimulant, adding, respectively: folic acid, propyphenazone and both folic acid and propyphenazone together. Growth was tested on day 7, 12 and 19 of incubation. Under baseline culture with standard stimulant, CFU-GM growth was characterized by successive proliferation waves of various entity: the first on day 7 was very high, the second, on day 12 was rather low, and the third, on day 19 was intermediate. This behaviour is different from what is usually observed in normal subjects in steady-state, whose first (AC-A+AC-B) and second (AC-C) proliferation period are of similar entity. The prevalence of the first proliferation period in our case is interpreted as the result of a renewed granulocytopoietic activity after drug-induced bone marrow suppression. This indicates a maintained integrity of the negative-feedback mechanism of homeostatic regulation on granulocytopoietic activity. The sole addition of propyphenazone on the in vitro bone marrow cell cultures of our patient produced a reduction of those CFU-GM that had grown during the first period whereas the growth during the second and third period remained unvaried. Thus the growth peak in cultures treated with propyphenazone occurred on day 19, which seems to correspond with the necessary time for a spontaneous remission from neutropenia, clinically observed to be 20 days after suspension of the propyphenazone-containing drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Report of two cases of acute myelogenous leukemia immunized with autologous leukemia-derived hybrid cells

Reports that immunizations with leukemia-derived hybrid cells prolonged the survival of leukemic mice led us to attempt an analogous approach in two adult patients with acute myeloid leukemia (AML). Hybrid cells were prepared from the pretreatment marrows of the newly-diagnosed patients with D98OR cells, in the first case, and with KR12 cells, in the second case. (D98OR and KR12 cells are human cell-lines.) Hybrids formed with KR12 cells expressed HLA antigens of both parental sources and some of the clonal isolates expressed myeloid-associated determinants. The immunizations were performed during the first complete clinical remission; the patients were demonstrably immunocompetent. Positive delayed type hypersensitivity responses to both (X-irradiated) hybrid cells and to (X-irradiated) autologous pretreatment marrow were observed following the immunizations. Mixed lymphocyte reactions toward autologous marrow were positive in one of the patients. In both, relapse occurred approximately two months after the first immunization and eight months after first diagnosis. The first patient remained in complete remission for two and one-half years following reinduction chemotherapy; reinduction chemotherapy was unsuccessful in the second patient.     

Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells

Composite-tissue (e.g., hand allograft) allotransplantation is currently limited by the need for immunosuppression to prevent graft rejection. Inducing a state of tolerance in the recipient could potentially eliminate the need for immunosuppression but requires reprogramming of the immunological repertoire of the recipient. Skin is the most antigenic tissue in the body and is consistently refractory to tolerance induction regimens using bone marrow transplantation alone. It was hypothesized that tolerance to skin allografts could be induced in rats by injecting epidermal cells with bone marrow cells during the first 24 hours of life of the recipients. Brown Norway rats (RT1n) served as donors for the epidermal cells, bone marrow cells, and skin grafts. Epidermal cells were injected intraperitoneally and bone marrow cells were injected intravenously into Lewis (RT1l) newborn recipient rats. In control groups, recipients received saline solution with no cells (group I, n = 12), bone marrow cells only (group II, n = 15), or epidermal cells only (group III, n = 15). In the experimental group (group IV, n = 18), recipients received epidermal and bone marrow cells simultaneously. Skin grafts were transplanted from Brown Norway (RT1n) rats to the Lewis (RT1l) rats 8 weeks after cell injections. Skin grafts survived an average of 8.5 days in group I (10 grafts), 9.2 days in group II (12 grafts), and 12 days in group III (14 grafts). Grafts survived 15.5 days (8 to 26 days) in group IV (15 grafts). The difference was statistically significant (p < 0.05). Hair growth was observed in some accepted grafts in group IV but never in the control groups. This is the first report of prolonged survival of skin allografts in a rat model after epidermal and bone marrow cell injections. Survival prolongation was achieved across a major immunological barrier, without irradiation, myeloablation, or immunosuppression. It is concluded that the presentation of skin-specific antigens generated a temporary state of tolerance to the skin in the recipients that could have delayed the rejection of skin allografts.     

Alterations of glycosphingolipid-based blood group antigen expression on erythrocytes and in plasma studied on consecutive samples after a blood group O to A bone marrow transplantation.

A blood group A1Le(a-b+) individual with chronic myeloid leukaemia had received a bone marrow graft from an HLA-identical OLe(a+b-) donor. Twelve months after bone marrow transplantation (BMT), the red blood cells of the patient became agglutinable with anti-A blood group reagents. To elucidate whether the blood group A antigen expression was of plasma or of bone marrow origin, total non-acid glycosphingolipid fractions were prepared from red blood cells and plasma collected 17 months after BMT, and from plasma collected 13, 15 and 19 weeks after BMT. The glycolipid fractions were analysed by thin-layer chromatography and immunostained with monoclonal A-antibodies, and permethylated and permethylated-reduced derivatives of selected plasma samples were analysed by mass spectrometry. The results strongly indicate the presence of host bone marrow-produced blood group A red blood cells. Furthermore, the presence of a blood group H active pentaglycosylceramide type 1 (H-5-1) (Table I), characteristic for an OLe(a-b-) secretor, was seen in plasma 3-4 weeks before clinical chronic graft versus host disease (GVHD). After treatment of chronic GVHD, this expression disappeared. The blood group ALeb (A-7-1) antigen produced by the recipient seems to be present and to increase with time in all plasma samples. This also seems to be the case for the Leb and A-6-1 antigens.     

Treatment results of nine patients with Burkitt's lymphoma

From 6/79 until 2/86, 9 patients (median age 39) with Burkitt's lymphoma were treated. Stage D disease was seen in 7 cases, stage C in two and stage A in one. The main symptom was abdominal pain or a rapidly progressing abdominal tumor. Three patients had bone marrow involvement and two had a Burkitt's leukemia. Three had typical chromosomal aberrations. Therapy consisted of a variety of chemotherapy regimens plus additional radiotherapy and/or bulk surgery. Two patients achieved complete remissions (of 6 and 20+ months duration), and 4 partial remissions were obtained. The remaining patients had either progressive, drug resistant disease or died early. One patient is currently alive and in complete remission at 20+ months. A second patient is alive at 20+ months in partial remission with traces of IgM-paraprotein still detectable. The main causes of death were tumor-lysis syndrome (4 patients) and therapy related sepsis with progressive tumor (3 patients). This poor outcome is probably due to a high proportion of high-risk patients and suboptimal therapy for this rapidly proliferating tumor.