VACOP-B versus VACOP-B plus autologous bone marrow transplantation for advanced diffuse non-Hodgkin's lymphoma: results of a prospective randomized trial by the non-Hodgkin's Lymphoma Cooperative Study Group.

PURPOSE: The aim of this multicenter randomized study was to compare conventional therapy with conventional plus high-dose therapy (HDT) and autologous bone marrow transplantation (ABMT) as front-line treatment for poor-prognosis non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between October 1991 and June 1995, 124 patients, aged 15 to 60 years, with diffuse intermediate- to high-grade NHL (Working Formulation criteria), stages II bulky (> or = 10 cm), III, or IV were enrolled. Sixty-one patients were randomized to receive etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (VACOP-B) for 12 weeks and cisplatin, cytarabine, and dexamethasone (DHAP) as a salvage regimen (arm A), and 63 to receive VACOP-B for 12 weeks plus HDT and ABMT (Arm B). RESULTS: There was no significant difference in terms of complete remissions (CRS) in the two groups: 75% in arm A, and 73% in arm B. The median follow-up observation time was 42 months. The 6-year survival probability was 65% in both arms. There was no difference in disease-free survival (DFS) or progression-free survival (PFS) between the two groups. DFS was 60% and 80% (P = .1) and PFS was 48% and 60% (P = .4) for arms A and B, respectively. Procedure feasibility was the major problem. In arm B, 29% of enrolled patients did not undergo HDT and ABMT. A statistical improvement in terms of DFS (P = .008) and a favorable trend in terms of PFS (P = .08) for intermediate-/high- plus high-risk group patients assigned to HDT and ABMT was observed. CONCLUSION: In this study, conventional chemotherapy followed by HDT and ABMT as front-line therapy seems no more successful than conventional treatment in terms of overall results. However, our results suggest that controlled studies of HDT plus ABMT should be proposed for higher risk patients.

Mobilization/transplantation of peripheral blood progenitor cells for aggressive non-Hodgkin's lymphoma with marrow involvement.

Thirty-five aggressive non-Hodgkin's lymphomas (NHL) with marrow involvement received high-dose cyclophosphamide (7 g/m2) and G-CSF in order to collect peripheral blood progenitor cells (PBPC). Fourteen patients were in partial remission, 16 patients were in relapse ('sensitive', 12; 'resistant', 4) and five patients were refractory to conventional treatment. A good yield of PBPC was obtained in 30 patients, while a low number of CD34+ cells and of CFU-GM was seen in two cases. Two patients entered progression and one patient died. Thirty patients underwent PBPC autografting. Twenty-nine out of 35 (83%) patients entered complete remission (CR). Two patients died in CR of infection following marrow aplasia 3 and 6 months after autografting. At 3 years the probability of survival and disease-free survival (DFS) are 62 and 51%, respectively.

Modified in vitro conditions for cord blood-derived long-term culture-initiating cells.

OBJECTIVE: The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. MATERIALS AND METHODS: We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4). RESULTS: The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively). CONCLUSIONS: Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

Autografting with Ph-negative progenitors in patients at diagnosis of chronic myeloid leukemia induces a prolonged prevalence of Ph-negative hemopoiesis.

OBJECTIVE: In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myeloreductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. MATERIALS AND METHODS: We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%-100% Ph-negative PBPC for a median time of 28 months (range 18-50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). RESULTS: Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%-100% to 0%-25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21-43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term-culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p < 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p < 0.03). CONCLUSION: We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.

Very primitive hemopoietic cells (LTC-IC) are present in Philadelphia negative cytaphereses collected during early recovery after chemotherapy for chronic myeloid leukemia (CML).

We have previously demonstrated that Philadelphia negative (Ph-ve) hemopoietic cells can be collected by leukaphereses after an acute leukemia-like chemotherapy during the early hemopoietic recovery in patients with chronic myeloid leukemia (CML). In this study we have evaluated whether these collections contain very primitive hemopoietic cells defined as 'long-term culture initiating cells' (LTC-IC) and whether these cells belong to the Ph-positive or Ph-negative population. Twenty-eight out of 76 cytaphereses collected in 15 patients with CML proved to contain Ph-ve cells only (six patients), 21 showed only Ph+ve cells (five patients), and 27 a mixture of Ph+ve and Ph-ve cells (four patients). In cytaphereses containing Ph-ve cells only, we found variable numbers of LTC-ICs, more consistently when we mobilized patients in the first 3 months from diagnosis. In three cases cytogenetic analysis on LTC-ICs and CFU-GM confirmed results obtained on fresh samples. Ph-positive collections were devoid of LTC-ICs except for 2/21 samples. However, their cytogenetic analysis revealed a small number of Ph-negative progenitors. LTC-ICs were randomly detected in mixed (Ph+ve and Ph-ve) collections. In conclusion these data indicate that, in a consistent proportion of chronic myeloid leukemia patients, intensive chemotherapy is able to recruit Ph-ve LTC-ICs in to the peripheral blood. Moreover these data provide the biological basis for developing autografting programs with Ph-negative cells.

CMV-antigenemia after allogeneic bone marrow transplantation: correlation of CMV-antigen positive cell numbers with transplant-related mortality.

One hundred and thirty-four consecutive patients undergoing HLA-identical BMT were prospectively followed on a weekly basis for the development of CMV antigenemia (CMVAg-emia). End-points of the study were (1) incidence, (2) risk factors, and (3) predictive effect on transplant-related mortality (TRM). Fifty-six patients developed CMVAg-emia between day 8-366 (median 40) with an overall actuarial risk of 43%. The median number of positive cells a diagnosis was 4 (range 1-48) the median maximum number was 6.5 (range 1-435). Positive cells are expressed as number/2.5 x 10(5) cells. In multivariate analysis, T cell depletion (TCD) (RR 2.9, P = 0.0009) and acute graft-versus-host disease (RR 2.1, P = 0.01) were the two risk factors predictive for CMVAg-emia. The risk of developing CMV-IP was significantly higher in patients with, as compared to patients without, CMVAg-emia (P = 0.0005) and occurred mostly in patients who received TCD marrow (P = 0.0009) despite treatment with gancyclovir or foscarnet at the time of CMVAg-emia. TRM was 24% in patients not developing CMVAg-emia; it was 21 and 47% in patients with "4" positive cells at diagnosis of CMV (P = 0.008), and 12 vs 54% for patients with "6" positive cells during infection (P = 0.0003). Both were predictive of TRM in multivariate analysis (P = 0.04 and P = 0.002). In conclusion, the risk of developing CMVAg-emia post-allo BMT is influenced by the marrow T cell content and by the occurrence of acute GVHD. High numbers of CMV antigen positive cells are associated with considerable transplant-related mortality, and may therefore identify patients eligible for early aggressive therapy.

Relapse after allogeneic BMT for chronic myeloid leukemia (CML) may be sustained by a small number of leukemic 'stem cells': a hypothesis.

'.the leukemic stem line is a small minority within the total cell mass;. when the leukemic stem line is not exceeding the normal stem cell numbers, its proliferation may still be under partial control.' LG Lajtha, Blood Cells 1981; 7: 45-62 We performed cytogenetic analysis on fresh bone marrow cells and on progenitor cell colonies in a patient who relapsed after allogeneic BMT for CML and was subsequently treated with donor lymphocyte infusions (DLI). Two Philadelphia-positive clones were identified at relapse. One clone displayed an additional chromosomal abnormality most likely induced by radio-chemotherapy and therefore arising in a single cell. This cell displays the characteristics of a stem cell, since it was able to support 20% of Ph-positive hemopoiesis for 5 months. If the progeny of a single Ph-positive stem cell account for 20% of hemopoiesis, a very low number of leukemic stem cells may sustain relapse after allogeneic BMT. This is in keeping with two observations: (1) at relapse, long-term culture initiating cells (LTC-IC) were all donor-derived and Ph-negative; (2) on average, the pace of the disease is very slow after relapse following allogeneic-BMT. Therefore, we hypothesize that a small number of leukemic stem cells may be involved in the initial events of relapse following BMT for CML.

Early predictors of transplant-related mortality (TRM) after allogeneic bone marrow transplants (BMT): blood urea nitrogen (BUN) and bilirubin.

Transplant-related mortality (TRM) following allo- geneic bone marrow transplantation (BMT) remains a major concern and early identification of patients at risk may be clinically relevant. In this study we describe a predictive score based on bilirubin and blood urea nitrogen (BUN) levels on day +7 after BMT. The patient population consisted of 309 consecutive patients who underwent BMT from sibling (n = 263) or unrelated donors (n = 46) for hematologic disorders between December 1990 and December 1996. Of 27 laboratory tests taken on day +7 after BMT, serum bilirubin (P = 0.02) and BUN (P = 0.007) were found to be independent predictors of TRM in multivariate analysis. The median levels of bilirubin (0.9 mg/dl) and of BUN (21 mg/dl) were then used as a cut-off and a score of 1 was given for values equal/greater than the median. There were 216 patients with scores 0-1 (low risk) on day +7 (bilirubin <0.9 and/or BUN <21) and 93 patients with score 2 (high risk) (bilirubin >/=0.9 and BUN >/=21): the latter had more grade III-IV acute graft-versus-host disease (P = 0.03), slower neutrophil (P = 0.02) and slower platelet engraftment (P = 0.002). The actuarial 5 year TRM is 22% for low risk vs44% for high risk patients (P = 0.0003). For HLA-identical siblings TRM is 20% vs35% (P = 0.01), for unrelated donors it is 20% vs 65% (P = 0.01). Day +7 score was highly predictive of TRM on multivariate analysis (hazard ratio 1.9, P < 0.01), after adjustment for year of transplant (P < 0.00001), unrelated vs sibling donors (P = 0.001), patient age (P = 0.01) and diagnosis (P = 0.01). These results were validated on an independent group of 82 allogeneic BMT recipients in a pediatric Unit who showed an actuarial TRM of 16% for low risk vs 46% for high risk patients (P = 0.002). This study suggests that it may be possible to identify patients with different risks of TRM on day +7 after BMT: high risk patients could be eligible for programs designed to intensify prophylaxis of post-transplant complications.

Interferon-alpha protects Philadelphia-negative progenitors from exhaustion in chronic myeloid leukemia patients with cytogenetic response.

INTRODUCTION: Normal immature hematopoietic progenitors are relatively well preserved in most patients newly diagnosed with chronic myeloid leukemia, but tend to decline rapidly with time. Such exhaustion could reflect a suppressive effect of the Philadelphia positive clone expansion and/or be induced by Interferon-alpha treatment. MATERIALS AND METHODS: A total of 51 CML patients were classified into three groups. Newly diagnosed untreated patients were group A (n=30). Of the 21 treated individuals with Interferon-alpha, for at least 12 months, 15 showed no cytogenetic response (group B) while six showed persisting major/complete response (group C). Patients belonging to groups A and B were mobilized with chemotherapy plus G-CSF while patients of group C received a short course of G-CSF only. RESULTS: Patients responding to IFN-alpha (group C) showed comparable numbers of bone marrow Ph- long-term culture initiating cells to those of newly diagnosed individuals (group A): 8.5 (<1-65)/10(6) MNC vs 10.5 (<1-30), while non-responders had markedly lower numbers: <1 (<1-5). The amount of Ph- LTC-IC collected was significantly lower in patients of group B 1.8 (0-325)x10(2)/kg than in patients of either group A 31.3 (0-952)x10(2)/kg (P<0.002) or group C 109 (8-259)x10(2)/kg (P<0.01). Interestingly, five patients of group B who had 100% Ph+ metaphases, but Ph- progenitors in their bone marrow, mobilized normal amounts of Ph(-) progenitors. CONCLUSION: These findings suggest that the decline of normal hematopoietic progenitors, currently observed in the majority of CML patients, is not induced by IFN-alpha treatment, but it is likely due to the expanding leukemic clone. They also indicate that normal hematopoietic reservoir is consistently preserved in patients given IFN-alpha early after diagnosis and achieving a stable cytogenetic response.

Factors predicting response and graft-versus-host disease after donor lymphocyte infusions: a study on 593 infusions.

In the present study, we analyze factors predicting graft-versus-host disease (GvHD) and response after donor lymphocyte infusions (DLI). A total of 100 patients received 593 DLI between June 1990 and December 2000 in a bulk dose (n=14) or in escalating dose infusions (n=86). Patients were analyzed after stratification for type of relapse: (1). molecular relapse (n=6), (2). cytogenetic relapse (n=20), (3). chronic phase of chronic myeloid leukemia (CML) or complete remission of other disease post chemotherapy (n=24), (4). CML in accelerated/blastic phase (n=14), (5). resistant disease not responding to chemotherapy (n=36). The proportion of responders to DLI in these five groups was 100, 90, 75, 36 and 0% (P<0.0001). Factors predicting response by multivariate analysis were type of relapse (P<0.0001), post-DLI GvHD (P=0.005), pancytopenia (P=0.008), and a diagnosis of CML (P=0.04). Acute GvHD (grades II-IV) occurred in 21 patients (21%), and correlated in multivariate analysis with pancytopenia and less than four DLI. Other predictors of GvHD were the number of CD3+cells/infusion and serum levels of gamma-glutamyl transferase (gammaGT). The actuarial probability of treatment-related mortality was 9% for HLA identical siblings and 44% for alternative donor transplants (P=0.006). Response to DLI is predicted by tumor burden and is associated with GvHD and pancytopenia.

Donor lymphocyte infusions (DLI) in patients with chronic myeloid leukemia following allogeneic bone marrow transplantation.

Donor lymphocyte infusions (DLI) were given between June 1990 and March 1996 to 18 patients with chronic myeloid leukemia (CML) for the treatment of cytogenetic (n = 6) or hematologic relapse (n = 12) following an allogeneic bone marrow transplant (BMT). Patients were divided in two groups: patients in group A (n = 8) received a large dose of donor lymphocytes (> or = 1 x 10(8)/kg), whereas patients in group B (n = 10) received escalating numbers of cells (2 x 10(5) up to 2 x 10(8)/kg). The median number of DLI in group A was 2 (range 1-3); the median number of infusions in group B was 7 (range 3-9). Acute GVHD occurred in 12 patients (grades I-III) and was a major cause of death in two. The risk of developing GVHD correlated with the number of cells infused: 37%, 14%, 5% and 0% for DLI with cells > or = 1 x 10(8), 2 x 10(7)/kg, 2 x 10(6)/kg, and 2 x 10(5)/kg, respectively (P = 0.01). Median transaminase levels were found to be significantly increased in patients with, as compared to patients without, acute GVHD (GPT 412 vs 28 IU/l; P = 0.03). Severe aplasia occurred in four and was a contributing cause of death in two patients. Overall, four patients died as a consequence of DLI and all received > 1 x 10(8)/kg cells: the actuarial risk was 38% in group A and 14% in group B (P = 0.1). There were 10 complete and three partial cytogenetic responses: the actuarial probability at 5 years of being Ph negative was 69%: it was 46% for group A and 85% for group B (P = 0.1). The longest patient is now 6 years post-DLI, Ph negative, BCR-ABL negative. The actuarial 3 year survival is 38% in group A and 86% in group B (P = 0.06). The study confirms that DLI post-BMT is not innocuous and that there is a definite long-lasting antileukemic effect in patients with CML. It also suggests that: (1) the risk of developing GVHD correlates with the number of infused cells; (2) that significant elevations of serum GPT levels are associated with GVHD; and (3) that the use of escalating doses of cells may allow the identification of side-effects and discontinuation of infusions before life-threatening GVHD has developed.

High-dose chemo-radiotherapy followed by autologous Philadelphia chromosome-negative blood progenitor cell transplantation in patients with chronic myelogenous leukemia.

Twenty-three patients with chronic myelogenous leukemia in early chronic phase (ECP) and not previously treated with alpha-interferon (IFN-alpha) (10 patients), in ECP but pretreated with IFN-alpha (<12 months) (seven patients) and in late chronic phase (LCP) pretreated with IFN-alpha (>12 months) (six patients) underwent autografting with Philadelphia (Ph) chromosome-negative blood progenitor cells (BPCs) (20 patients), or partially/totally Ph-positive BPCs (three patients), previously mobilized during the early phase of recovery after aplasia induced by intensive chemotherapy. The conditioning regimen consisted of high-dose chemotherapy alone or followed by total body irradiation (TBI). Recombinant G-CSF was given after BPCs infusion on day +8. All patients in ECP not pretreated with IFN-alpha are alive and five of them are Ph-negative in the marrow after autografting. Six of seven patients autografted with Ph-negative BPCs in the group of ECP pretreated with IFN-alpha (<12 months) are alive and two of them are still Ph-negative in the marrow. In the same group, the only patient transplanted with partially Ph-positive BPCs, died of blastic transformation 2 months after reinfusion. Three patients (two patients autografted with Ph-negative BPCs and one patient with Ph-positive BPC) in the group of LCP pretreated with IFN-alpha >12 months are alive but Ph-positive after autografting. The other three patients of the same group died of procedure-related toxicity (two patients) and blastic transformation (one patient). Seventeen patients (10/10 ECP not pretreated with IFN-alpha; 5/7 ECP pretreated with IFN-alpha and 2/6 LCP pretreated with IFN-alpha) of 23 autografted patients were treated with IFN-alpha +/- IL-2. Toxicities after autografting were mostly related to myelosuppression, particularly thrombocytopenia. All patients of the two groups pretreated with IFN-alpha developed febrile episodes during the aplastic phase following BPCs reinfusion. No patient autografted in ECP and those not pretreated with IFN-alpha developed febrile episodes. This is also probably due to the use of i.v. antibiotic and antimicotic prophylaxis when neutrophils were < or = 1 x 10(9)/l after autografting. Greater toxicity was observed in patients pretreated with IFN-alpha, being lethal in two cases in LCF. In conclusion, the "in vivo' manipulation approach employed in our institution is a safe procedure and it results in a high collection of Ph-negative cells in the blood if the cells are harvested: (1) in early chronic phase; (2) in early phase of recovery after chemotherapy-inducing aplasia; (3) in patients not extensively pretreated with IFN-alpha. The data presented here have shown encouraging trends in chronic phase of CML and offer new perspective for patients without an HLA-identical donor or for patients who do not respond to IFN-alpha.

High-dose cyclophosphamide followed by autografting can improve the outcome of relapsed or resistant non-Hodgkin's lymphomas with involved or hypoplastic bone marrow.

We report our experience of high-dose cyclophosphamide (HDCY) followed by high-dose therapy (HDT) and peripheral blood progenitor cell (PBPC) autografting in patients with diffuse, intermediate and high-grade non-Hodgkin's lymphomas who have failed conventional treatment. From 1991 to 1996, 54 consecutive patients pre-treated with a median of two chemotherapy lines entered the study. Eighteen patients (33%) were still responders to conventional chemotherapy (sensitive relapse), and 20 patients (37%) were in partial response (PR) after chemotherapy (CT). Sixteen patients (30%) were resistant to conventional CT either at presentation (non responder) or in relapse (resistant relapse). Thirty-nine patients had bone marrow involved by disease and fifteen had an hypoplastic marrow following conventional treatment. Patients received HDCY (7gr/m2) and G-CSF or GM-CSF in order to collect PBPC. Median collected CD34+ cells was 12.3 x 10(6)/Kg (range 0.7-197). After HDT (BEAM or Melphalan + TBI) 50 patients underwent PBPC autografting. According to intention to treat, 44 (81%) of 54 patients achieved complete remission (CR) (50% after HDCY and 31% after HDT). Procedure related death occurred in 6 patients (11%), one after HDCY and 5 after autografting. Twenty-nine (66%) of 44 patients are still in CR, 7 to 63 months (median 27 months) after the procedure. Three-year probability of survival, disease-free survival and progression-free survival are 63%, 64% and 52% respectively. In conclusion, HDCY is an effective procedure not only in mobilizing PBPC, but also in reducing tumour burden. HDT with PBPC support may further improve the outcome in this category of high-risk non-Hodgkin's lymphomas.

Mobilization/transplantation of peripheral blood progenitor cells for aggressive non-Hodgkin's lymphoma with marrow involvement.

Thirty-five aggressive non-Hodgkin's lymphomas (NHL) with marrow involvement received high-dose cyclophosphamide (7 g/m2) and G-CSF in order to collect peripheral blood progenitor cells (PBPC). Fourteen patients were in partial remission, 16 patients were in relapse ("sensitive", 12; "resistant", 4) and 5 patients were in refractory to conventional treatment. A good yield of PBPC was obtained in 30 patients, while a low number of CD34+ cells and of CFU-GM was seen in two cases. Two patients entered progression and one patient died. Thirty patients underwent PBPC autografting. Twenty-nine out of 35 (83%) patients entered complete remission (CR). Two patients died in CR of infection following marrow aplasia 3 and 6 months after autografting. At 3 years the probability of survival and disease-free survival (DFS) are of 62% and 51% respectively.

In vivo B-cell depletion with rituximab for alternative donor hemopoietic SCT.

We retrospectively analyzed 55 patients given a fixed dose of rituximab (200 mg) on day+5 after an alternative donor transplant, to prevent EBV DNA-emia; 68 alternative transplants who did not receive prophylactic rituximab served as controls. The two groups were comparable for donor type, and all patients received anti-thymocyte globulin in the conditioning regimen. Rituximab patients had a significantly lower rate of EBV DNA-emia 56 vs 85% (P=0.0004), a lower number of maximum median EBV copies (91 vs 1321/10(5) cells, P=0.003) and a significantly lower risk of exceeding 1000 EBV copies per 10(5)cells (14 vs 49%, P=0.0001). Leukocyte and lymphocyte counts were lower on day +50 and+100 in rituximab patients, whereas Ig levels were comparable. The cumulative incidence of grade II-IV acute GvHD was significantly reduced in rituximab patients (20 vs 38%, P=0.02). Chronic GvHD was comparable. There was a trend for a survival advantage for patients receiving rituximab (46 vs 40%, P=0.1), mainly because of lower transplant mortality (25 vs 37%, P=0.1). Despite the drawback of a retrospective study, these data suggest that a fixed dose of rituximab on day +5 reduces the risk of a high EBV load, and also reduces acute GvHD.

Idarubicin, intermediate-dose cytarabine, etoposide, and granulocyte-colony-stimulating factor are able to recruit CD34+/HLA-DR- cells during early hematopoietic recovery in accelerated and chronic phases of chronic myeloid leukemia.

A group of 46 patients with chronic myelogenous leukemia (CML) [chronic phase (CP), 24 patients; accelerated phase (AP), 22 patients] ineligible for allogeneic bone marrow transplantation were given an intensive chemotherapy regimen consisting of idarubicin, intermediate-dose cytarabine, and etoposide. All patients had previously received interferon-alpha and only 2 had shown a partial cytogenetic response. During early recovery from chemotherapy-induced aplasia, peripheral blood progenitor cells (PBPC) were harvested by leukapheresis. All metaphases were found to be Philadelphia chromosome (Ph) negative in the collection from 17 of 46 (37%) patients [CP, 12 of 24 (50%); AP, 5 of 22 (23%)], and a decrease to less than 50% Ph-positive metaphases was seen in an additional 6 (CP, 3 patients; AP, 3 patients). The percentage of patients showing complete Ph disappearance was 64% in those receiving this procedure within the first year of diagnosis. In vitro studies were performed to assess the behavior of the Ph-negative PBPC. In clonogenic cultures they responded to stem cell factor and were able to grow as mixed colonies. Moreover, long-term culture initiating cells (LTCIC) were present in many Ph-negative collections but rarely in Ph-positive PBPC. In 4 females, clonality was studied by analyzing X chromosome inactivation and methylation patterns of the DXS255 locus with the probe M27 beta. Hematopoiesis was polyclonal in all 4 patients tested. Thus far, the Ph-negative collections have been used for autografting in 16 patients (CP, 11 patients; PA, 5 patients) after conditioning with total-body irradiation, etoposide, and cyclophosphamide or idarubicin.(ABSTRACT TRUNCATED AT 250 WORDS)

Philadelphia-chromosome-negative peripheral blood stem cells can be mobilized in the early phase of recovery after a myelosuppressive chemotherapy in Philadelphia-chromosome-positive acute lymphoblastic leukaemia.

Ten patients in first or second relapse with Philadelphia chromosome acute lymphoblastic leukaemia, ineligible for allogeneic sibling marrow transplantation, were treated with an intensive chemotherapy regimen including idarubicin, intermediate-dose arabinosylcytosine, etoposide and G-CSF. Peripheral blood stem cells were collected by leukapheresis during initial early WBC recovery from chemotherapy-induced aplasia. In 5/10 patients all metaphases in leukapheresis products were found to be Philadelphia-chromosome-negative and they have been used as autotransplants after conditioning with TBI/etoposide/cyclophosphamide (or idarubicin) and G-CSF. All five patients showed sustained engraftment and one of them is alive and well Philadelphia-chromosome-negative 18 months after transplant. These preliminary results suggest that it is possible to recover Philadelphia-chromosome-negative blood stem cells after intensive chemotherapy, even in advanced patients, and to perform autografting with these cells.

Low transplant mortality in allogeneic bone marrow transplantation for acute myeloid leukemia: a randomized study of low-dose cyclosporin versus low-dose cyclosporin and low-dose methotrexate.

Sixty patients undergoing allogeneic bone marrow transplant for acute myeloid leukemia (AML) in first remission (CR1; n = 49) or more advanced phase (n = 11) were entered in a prospective trial of graft-versus-host disease (GvHD) prophylaxis: low-dose cyclosporin A (IdCSA; 1 mg/kg/d from day -1 to +20 day; n = 28) or IdCSA plus low-dose methotrexate (IdMTX; 10 mg/m2 for day +1, 8 mg/m2 for days +3, +6, and +11; n = 32). Primary end points were acute GvHD (aGvHD) and transplant-related mortality (TRM); secondary end points were relapse and survival. The conditioning regimen consisted of cyclophosphamide (120 mg/kg) and fractionated total body irradiation (3.3 Gy/d for 3 consecutive days). The actuarial risk of developing aGvHD grade II-III was 61% for IdCSA alone and 34% for IdCSA + IdMTX (P = .02). The actuarial risk of TRM at 1 year was 11% versus 13%, respectively, and older patients (>/= 29 years) had higher TRM than younger patients (22% v 5%, P = .01). The age effect was significant in the IdCSA group (P = .04) but not in the IdCSA + IdMTX group (P = .1). The median follow-up is 4.4 years, with an overall actuarial survival of 78% for CR1 patients and 36% for patients with advanced disease. For patients in CR1 the outcome of the two regimens was as follows: survival 77% versus 80% (P = .6), relapse 20% versus 9% (P = .1), and TRM 13% versus 17% (P = .6). This study suggests that TRM can be reduced in AML patients undergoing allogeneic marrow transplants with a mild conditioning regimen and low-dose immunosuppression, and this translates in a 78% 5-year survival for CR1 patients. Beyond CR1 the major obstacle remains leukemia relapse, which is not prevented by low-dose in vivo immunosuppression.

Transplantation of HLA-mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications.

This trial was designed to test the use of CD34+ selected haemopoietic stem cells (HSC) in HLA-mismatched donor-recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single-dose total-body irradiation (sTBI). 10 patients aged 16-50 with advanced malignancies and a two- or three-antigen mismatched family donor entered this study. Donor marrow and G-CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34+ cells were 5.66 x 10(6)/ kg, with 0.55 x 10(6)/kg CD3+ cells. Nine patients received cyclosporin for graft-versus-host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 x 10(9)/l with a median platelet count of 60 x 10(9)/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0-I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20-144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) (n = 5), renal failure (n = 1). GvHD (n = 1) and Aspergillus meningitis (n = 1). Two patients are alive 365-495 d post transplant, one in remission and one in relapse. This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post-transplant immunodeficiency leads to a high risk of lethal infectious complications.