Genetically identical twin transplantation for chronic lymphocytic leukemia.

We identified 19 persons with B-cell chronic lymphocytic leukemia (CLL) who received genetically identical twin blood cell or bone marrow transplants after high-dose conditioning. Ten are alive (eight disease-free) with a median follow-up of 89 months (range, 31-171 months); 5-year relapse rate was 50% (95% confidence interval (CI), 26-73%). Estimated 5-year survival and disease-free survival were 61% (95% CI, 37-82%) and 45% (95% CI, 23-68%). In two of four patients tested at 12 and 21 months by polymerase chain reaction no evidence of residual CLL was detected post-transplant. In one recipient who relapsed at 6 years, molecular studies showed a different CLL clone from that detected pretransplant. This clone was subsequently identified in the donor suggesting transfer of occult leukemia at the time of transplant. Genetically identical twin transplants can result in long-term disease-free survival and molecular remissions, these data suggest the potential for CLL control in the absence of allogeneic graft-versus-leukemia effect. The case of leukemia transfer indicates the need for careful evaluation of donors prior to graft collection.

Collection of hematopoietic stem cells from patients with autoimmune diseases.

We reviewed data from 24 transplant centers in Asia, Australia, Europe, and North America to determine the outcomes of stem cell collection including methods used, cell yields, effects on disease activity, and complications in patients with autoimmune diseases. Twenty-one unprimed bone marrow harvests and 174 peripheral blood stem cell mobilizations were performed on 187 patients. Disease indications were multiple sclerosis (76 patients), rheumatoid arthritis (37 patients), scleroderma (26 patients), systemic lupus erythematosus (19 patients), juvenile chronic arthritis (13 patients), idiopathic autoimmune thrombocytopenia (8 patients), Behcet's disease (3 patients), undifferentiated vasculitis (3 patients), polychondritis (1 patient) and polymyositis (1 patient). Bone marrow harvests were used in the Peoples Republic of China and preferred worldwide for children. PBSC mobilization was the preferred technique for adult stem cell collection in America, Australia, and Europe. Methods of PBSC mobilization included G-CSF (5, 10, or 16 microg/kg/day) or cyclophosphamide (2 or 4 g/m2) with either G-CSF (5 or 10 microg/kg/day) or GM-CSF (5 microg/kg/day). Bone marrow harvests were without complications and did not affect disease activity. A combination of cyclophosphamide and G-CSF was more likely to ameliorate disease activity than G-CSF alone (P < 0.001). g-csf alone was more likely to cause disease exacerbation than the combination of cyclophosphamide and g-csf (P = 0.003). Three patients died as a result of cyclophosphamide-based stem cell collection (2.6% of patients mobilized with cyclophosphamide). When corrected for patient weight and apheresis volume, progenitor cell yields tended to vary by underlying disease, prior medication history and mobilization regimen. Trends in the approaches to, and results of, progenitor cell mobilization are suggested by this survey. While cytokine-based mobilization appears less toxic, it is more likely to result in disease reactivation. Optimization with regard to cell yields and safety are likely to be disease-specific and prospective disease-specific studies of mobilization procedures appear warranted.

Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout.

BACKGROUND: Many gene therapy applications require the co-ordinated delivery of more than one reading frame. We wished to systematically compare IRES in the context of a retroviral vector to determine which was the most effective for protein production and viral titre. To do this we monitored expression of IL-12, as co-ordinated expression of both p35 and p40 subunits is required for production of the active heterodimer. METHODS: Retroviral vectors were constructed to express human IL-12 in which an IRES initiates translation of the p40 subunit, with the IRES optimally aligned to the initiation codon of p40. Vectors containing an IRES from either polio virus (PV), encephalomyocarditis virus (EMCV), foot and mouth disease virus (FMDV) or murine leukaemia virus (MLV) were compared with a vector expressing IL-12 as a single protein (Flexi-12; in which the two IL-12 subunits are linked by a peptide). RESULTS: All vectors produced high titre virus and directed synthesis of IL-12 in target cells. The bicistronic vectors containing the IRES from EMCV and PV were the most effective in infected 3T3 cells, producing up to 40 ng IL-12/10(6) cells/48 h, similar to the 50 ng IL-12/10(6) cells/48 h obtained with Flexi-12. The IRES from PV was the most efficient in human melanoma cells. CONCLUSIONS: Bicistronic retroviral vectors have been constructed that effectively transduce target cells and produce high levels of protein. Target cell specificity of IRES function was observed. The combination of Flexi-12 and the IRES from PV will be useful in the generation of vectors expressing IL-12 with a second protein such as IL-2 for transduction of melanoma cells.

Allogeneic bone marrow transplantation for acute leukemia in patients over the age of 40 years. Acute Leukemia Working Party of the European Group for Bone Marrow Transplantation (EBMT).

Extension of allogeneic transplants to older patients has been limited by a high risk of transplant-related death and graft-versus-host disease. To evaluate the feasibility in older patients, a retrospective analysis of the procedure was performed for first remission acute leukemia in 192 patients aged over 40 years and compared with a group of 1119 recipients aged from 16 to 40 years reported to the EBMT from 1986 to 1992. Patient-, disease-, and treatment-related variables were compared between the two age groups using the chi2 statistical method for categorical variables. Variables differing significantly or recognized as potential prognostic factors were included in a multivariate analysis. Leukemia-free survival and relapse were comparable among the age groups in the two types of leukemias. Incidence of graft-versus-host disease was higher in the older group of ALL patients. Older patients with AML in first remission had a higher treatment-related mortality incidence, with no influence on survival. A pair-matched analysis of AML patients did not show any statistical difference in the probability of LFS, RI, TRM, and survival for the two age cohorts of patients. These results suggest that BMT should be considered for patients over 40 years of age.

Relapse of acute promyelocytic leukemia follows serial negative RT-PCR assays: a cautionary tale.

Six patients with acute promyelocytic leukaemia (APL) and t(15;17) were investigated by cytogenetics and by reverse transcriptase polymerase chain reaction (RT-PCR) for the fusion transcript PML-RARA. The clone was detected in remission following all-trans retinoic acid and chemotherapy by cytogenetics and/or by RT-PCR up to 2 months from diagnosis. Thereafter the PML-RARA transcript was not seen in 20/21 first remission samples in five cases studied. Remission was maintained in two patients, one following bone marrow transplantation (BMT). Despite serial negative RT-PCR results, PML-RARA reemerged at or prior to relapse following BMT in the remaining three cases. Frequent molecular monitoring and caution in the interpretation of negative RT-PCR results are indicated in APL.

Immunisations after bone marrow transplantation: results of a European survey and recommendations from the infectious diseases working party of the European Group for Blood and Marrow Transplantation.

Increased numbers of recipients of BMTs and autologous BMTs are becoming long-term survivors. Existing data support that loss of protective immunity to agents such as tetanus and poliovirus is common in patients who received BMTs and autologous BMTs. Thus, a reimmunisation programme is needed to ensure immunity. A questionnaire concerning immunisation practices was sent to EBMT member centres. The immunisation practices varied extensively in the 59 responding BMT and 48 responding autologous BMT centres. Sixty five per cent of responding centres routinely immunise patients who received allogeneic BMTs whereas 37% were routinely immunising recipients of autologous BMTs. Tetanus toxoid and inactivated poliovirus vaccine were the most frequently used vaccines in both BMT and autologous BMT centres. Immunisations of recipients of both BMTs and autologous BMTs with tetanus toxoid, diphtheria toxoid, inactivated poliovirus vaccine and influenza vaccine are recommended. Other vaccines, and in particular live attenuated vaccines, may be considered on an individual basis.

Prognostic factors in autologous stem cell transplantation for multiple myeloma: an EBMT Registry Study. European Group for Bone Marrow Transplantation.

Autologous bone marrow- and blood progenitor cell transplantation was performed in 130 patients with multiple myeloma in 16 European centers between 1986 and 1993. At the time of follow-up, 77 patients were alive and 53 were dead. Complete remission after transplantation was obtained in 47% of all patients. The actuarial survival at 65 months was 28%. The median duration of relapse-free survival among patients who were in complete remission after transplantation was 29 months. The following factors were predictive for longer survival and freedom of progression in a univariate analysis: Male sex, age less than 45 years, a low serum-beta-2-microglobulin value at diagnosis, prior administration of only one treatment regimen, response on conventional chemotherapy immediately pretransplant and the use of a preparative regimen including melphalan. The last factor, in addition to stage I disease at diagnosis, male sex and responsive disease immediately pretransplant, were also demonstrated as independent predictive variables for longer survival in a multivariate analysis. Progression-free survival was significantly better for patients who were in complete remission after transplantation, as compared to those with persisting signs of disease. We conclude that high-dose chemo-radiotherapy with autologous stem cell transplantation can induce long-term responses, primarily in younger, male patients with chemotherapy-responsive early disease. High-dose melphalan, as single drug or in combination, appeared to be superior to other regimens. The chance of being persistently disease-free seemed to be greatest for patients being in complete remission already before the transplantation.

European survey of bone marrow transplantation in acute promyelocytic leukemia (M3). Working Party on Acute Leukemia of the European Cooperative Group for Bone Marrow Transplantation (EMBT).

Acute promyelocytic leukemia (M3) is a distinct subtype of AML considered to have better response to chemotherapy and a higher cure rate than other subtypes. We analyzed the outcome for 362 M3 patients transplanted in Europe from November 1979 to December 1992 and reported to the acute leukemia registry of the European Cooperative Group for Bone Marrow Transplantation (EMBT). Of these 362 patients, 187 received an autograft, 129 in first remission (CR1) and 58 in second remission (CR2), and 175 an allograft, 142 in CR1 and 33 in CR2. Patients autografted in CR1 had at 7 years a leukemia-free survival (LFS) of 48 +/- 5%, a relapse rate (RR) of 41 +/- 5% and a probability of transplant-related mortality (TRM) of 18 +/- 6%. Patients allografted in CR1 had a LFS of 42 +/- 6%, a RR of 28 +/- 5% and a TRM probability of 42 +/- 8%. For patients transplanted in CR2, the respective figures after auto and allotransplantation were: LFS: 31 +/- 7% and 22 +/- 8%, RR: 54 +/- 8% and 64 +/- 11%, TRM: 23 +/- 9% and 40 +/- 9%. These data, which do not permit comparison between autologous and allogeneic BMT, indicate that roughly 45% of M3 patients achieving CR1 may be cured by a marrow transplant. Since the recent use of transretinoic acid-containing induction regimens has increased early control for patients with AML M3, it will be important to find out how these results affect outcome following allogeneic or autologous BMT.

Multiple chromosome abnormalities in a drug resistant TdT positive B-cell leukemia.

A 17-year old caucasian male presented with B-cell acute leukemia which proved aggressive and refractory to treatment. Cytogenic investigation showed a single clone with a complex karyotype 49,XY,del(2)(p13),+4,del(4)(p11),-6,+i(6)(p),+7,+8, t(8;14), (q24;q32),del(17)(p11). This includes the Burkitt's translocation and a deletion at the site of the immunoglobulin kappa light chain gene. Clonal evolution included tetraploidy, duplication of the derived chromosomes and, terminally, trisomy 1q. Immunological investigation revealed a monoclonal population of B-cell blasts, expressing the kappa light chain, and with an extremely rare combination of SIg and TdT positivity. Immunoglobulin gene rearrangement confirmed monoclonalility. Tetraploidy of the clone and del(17)(p11) have been previously described only in a cell line or at end stage disease in B-ALL. It is suggested that the chromosomal abnormalities present at diagnosis were directly related to the refractory nature of this leukemia.

Marrow autotransplantation accelerates haematological recovery in patients with malignant melanoma treated with high-dose melphalan.

In a Phase I study, melphalan 140 mg/m2 was administered to 8 patients with disseminated malignant melanoma. Marrow was removed from the patients immediately before melphalan administration and returned i.v. 8 h later. Studies on marrow culture and melphalan pharmacokinetics predicted that this was a safe time to administer non-cryopreserved marrow. Four patients received lower doses of i.v. melphalan without autologous marrow. In the group receiving autologous marrow the time for recovery of peripheral-blood granulocytes to 800/mm2 or greater was significantly less (P = 0.01) than in those not receiving marrow. In 7 patients the tumour showed evidence of response to the drug and there was 1 complete remission. This treatment deserves investigation in patients with tumours more sensitive to drugs than melanoma.

Relation of "lymphoid" phenotype and response to chemotherapy incorporating vincristine-prednisolone in the acute phase of Ph1 positive leukemia.

Forty-four patients with Ph positive leukemia (36 developing blast crisis after chronic phase and eight presenting in acute leukemia) were classified into subgroups on the basis of reactivity of blasts with an anti-serum made against non-T,non-B acute lymphoid leukemia (ALL+), levels of terminal transferase enzyme (TdT+) and morphology. Positivity with anti-ALL serum was the most sensitive and reliable marker, and TdT was an important aid. The presence of "lymphoid" blasts in blast crisis of CML was related to the response to chemotherapy incorporating Vincristine and Prednisolone (VP). Patients with ALL+ blasts frequently (14 of 15 cases) responded to therapy while 21 of 25 patients who had no ALL+ blasts failed to respond. The clinical course of the ALL+ patients was variable: eight patients remitted with return to the appearances of the chronic phase; four patients demonstrated elimination of the Ph1 positive clone with hypoplasia and this was followed by normal (Ph1 negative) marrow regeneration in two. Subsequent relapse was of either the ALL+ "lymphoid" or the ALL-myeloid type. A regimen incorporating VP should be the treatment of choice in "lymphoid" blast crisis of CML.

Lymphoid blast crisis in chronic myeloid leukaemia and Philadelphia positive acute lymphoid leukaemia.

Membrane markers (anti-ALL and anti-Ia antisera) and an enzyme marker (terminal transferase) have been used to define an L-type or "lymphoid" type of acute transformation in chronic myeloid leukaemia and Ph1 positive acute leukaemia. Patients with L-type ("lymphoid") blasts responded to regimens including vincristine and prednisolone (VP). The markers showed better correlation with survival than did the morphology of blasts. The clinical course of patients was variable; elimination Ph1 positive clone (and hypoplasia), return to the chronic phase and relapses (including meningeal leukaemia) were observed. In contrast, patients with myeloid blasts ("M" type of blast crisis) failed to respond to vincristine and prednisolone.