Monica Soracco

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-Hodgkins 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.

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

Summary: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 accele-rated/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 (γGT). 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 (⩾1 × 108/kg), whereas patients in group B (n = 10) received escalating numbers of cells (2 × 105 up to 2 × 108/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 ⩾1 × 108, 2 × 107/kg, 2 × 106/kg, and 2 × 105/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 × 108/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.

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 1321/105 cells, P=0.003) and a significantly lower risk of exceeding 1000 EBV copies per 105cells (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.

Rituximab Treatment for Epstein-Barr Virus DNAemia after Alternative-Donor Hematopoietic Stem Cell Transplantation

We report 55 patients undergoing an alternative-donor hematopoietic stem cell transplantation (HSCT) who developed Epstein-Barr virus (EBV) DNAemia, with >1000 EBV copies/10(5) peripheral blood mononuclear cells (PBMCs), and were treated with rituximab (375 mg/m(2)). The median patient age was 47 years (range, 20-65 years), and graft sources were mismatched family members (n = 4), unrelated donors (n = 46), and unrelated cord blood (n = 5). The conditioning regimen included antithymocyte globulin (ATG) in all patients. The median time to development of EBV DNAemia was day 27 post-HSCT (range, day 5 to day 242), with a median of 60 EBV copies/10(5) PBMCs (range, 1-5770 copies/10(5) PBMCs). The number of EBV copies was reduced to <1000/10(5) PBMCs on day +7 after initiation of rituximab therapy in 51% of the patients, on day +14 in 73% of the patients, and on +21 in 92% of the patients. Overall, 50 of 55 patients (91%) cleared EBV after one dose (n = 25) or more than one dose (n = 25) of rituximab. Factors predicting transplantation-related mortality (TRM) in multivariate analysis were a reduction to <1000 EBV copies/10(5) PBMCs by day +7 of treatment (relative risk [RR], 0.2; P = .01) and disease phase in remission (RR, 0.3; P = .05). TRM was 23% in the 40 patients with none or one of the negative predictors and 60% in the 15 patients with both negative predictors (P = .001). Of these latter 15 patients, 3 developed clinical posttransplantation lymphoproliferative disorder (PTLD). All 3 of these patients had a high EBV load on day +7 of rituximab therapy. This study confirms the effectiveness of rituximab in controlling EBV DNAemia in patients undergoing allogeneic HSCT. Patients with increasing EBV copies despite rituximab therapy are at high risk for EBV PTLD and may be considered for alternative therapies.

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.

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 × 106/kg, with 0.55 × 106/kg CD3+ cells. Nine patients received cyclosporin for graft‐versus‐host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 109/l with a median platelet count of 60 × 109/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.

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.

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.