Osvaldo Figari

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.

Lymphocyte subsets recovery following allogeneic bone marrow transplantation (BMT): CD4+ cell count and transplant-related mortality

To assess the kinetics of lymphocyte subset recovery, 758 allografted patients were monitored by surface markers (CD3, CD4, CD8, CD56), with a 5-year follow-up. The donor was a matched sibling donor (MSD) (n=502) or an alternative donor (family mismatched or unrelated, AD) (n=256). The stem cell source was bone marrow for all patients. CD4+ cell recovery was influenced—in univariate analysis—by three factors: donor type, patient age and GvHD. This was not the case for CD8+ and CD56+ cells. The median CD4+ cell count on day +35 after HSCT was 86/μl. Patients achieving this CD4+ cell count had significantly lower transplant-related mortality (TRM) compared to patients who did not achieve this CD4+ cell count (20 vs 39%, P=0.00001), due to a lower risk of lethal infections (24 vs 47%, P=0.0003). In multivariate analysis MSD (RR 3.45, P=0.0001) and recipient age less than 16 years (RR 3.23, P=0.003) were significantly associated with a better CD4+ cell recovery. CD4+ counts on day +35 was predicted TRM (RR=1.97, P=0.0017) together with acute GvHD grade II–IV (RR 1.59, P=0.0097). No difference of TRM was observed for CD8+ and CD56+ cell counts.

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.

Cyclic ADP-ribose generation by CD38 improves human hemopoietic stem cell engraftment into NOD/SCID mice.

Cyclic ADP‐ribose (cADPR) is a potent and universal intracellular calcium mobilizer, recently shown to behave as a new hemopoietic cytokine stimulating the in vitro proliferation of both committed and uncommitted human hemopoietic progenitors (HP). Here, we investigated the effects of cADPR on engraftment of hemopoietic stem cells (HSC) into irradiated NOD/SCID mice. Two different protocols were used: i) a 24 h in vitro priming of cord blood‐derived mononuclear cells (MNC) with micromolar cADPR, followed by their infusion into irradiated mice (both primary and secondary transplants); and ii) co‐infusion of MNC with CD38‐transfected, cADPR‐generating, irradiated murine 3T3 fibroblasts. We demonstrated a dual effect of cADPR on human HP in vivo: i) enhanced proliferation of committed progenitors, responsible for improvement of short‐term engraftment; ii) expansion of HSC, with increased long‐term human engraftment into secondary recipients and a significantly higher expansion factor of CD34+ progenitors in mice co‐infused with MNC and CD38+ 3T3 fibroblasts. These results hold promise for the possible therapeutic use of cADPR, and of cADPR‐producing stroma, to achieve long‐term expansion of human HSC, that is, those HP capable of self‐renewal and responsible for repopulation of the bone marrow.

Foscarnet prophylaxis of cytomegalovirus infections in patients undergoing allogeneic bone marrow transplantation (BMT): a dose-finding study

This is a dose-finding study using foscarnet for CMV prophylaxis after allogeneic bone marrow transplantation (BMT) in 20 high risk patients (unrelated donors, or T cell depleted, and/or advanced disease). Foscarnet was started on day +1 after BMT and continued until day +100. We explored four different dose levels, patients being entered at the lowest dose level until one patient experiences CMV-reactivation, identified as two consecutive positive CMV antigenemias (CMVAg-emia). The four dose levels expressed as mg/kg/day between days 1 and 30 (induction) and between days 31 and 100 (maintenance) were respectively: dose level I = 60/30 (n = 5); dose level II = 120/60 (n = 4); dose level III = 120/90 (n = 5) and dose level IV = 120/120 (n = 6). All patients showed engraftment: PMN ≥0.5 × 109/l at a median interval of 16, 21, 17, 15 days after BMT, and Plt ⩾30 × 109/l on days 19, 16, 17, 17 respectively. CMVAg-emia was seen in 10 patients at a median interval of 53 days post-BMT (range 33–89) with a median of 10 CMV antigen+ cells (range 1–16). There was a dose effect of foscarnet on CMVAg-emia: respectively 4/5 patients (80%), 2/4 (50%), 3/5 (60%) and 1/6 (18%) at dose levels I, II, III, IV (P = 0.1). CMV disease was seen in 3/9 (33%) at dose levels I, II and 0/11 at dose levels III, IV (P = 0.07). The median number of CMV antigen-positive cells at diagnosis of CMV infection was different: 13 in dose levels I–II and two in dose levels III–IV (P = 0.01). Increased creatininine was seen in 15 patients with a mean of 1.8 mg% (range 1.5–5.7) and was the cause of discontinuation in nine patients (45%). Renal toxicity was reversible in all nine patients. Overall actuarial TRM at 2 years was 31%: 47% for patients at dose levels I–II and 19% for patients at dose levels III–IV. In conclusion, foscarnet exhibits a dose-dependent prophylactic effect on CMVAg-emia, CMV disease and transplant-related mortality with acceptable and reversible renal toxicity. Bone Marrow Transplantation (2000) 26, 23–29.

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.

Normal primitive haemopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time

We carried out studies to quantify Ph‐negative progenitors both in steady state and during regeneration after chemotherapy and G‐CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady‐state bone marrow, Ph‐negative long‐term culture initiating cells (LTC‐IC) and Ph‐negative colony‐forming‐cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P < 0.001 and P < 0.02, respectively). The absolute number of mobilized Ph‐negative progenitors was markedly higher in group A versus group B (P < 0.02 for LTC‐IC, P < 0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph‐negative LTC‐IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph‐negative LTC‐IC in the bone marrow predicted the yield of Ph‐negative LTC‐IC mobilized into peripheral blood (P < 0.001). The bone marrow frequency of Ph‐positive LTC‐IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph‐negative circulating progenitors would appear to be soon after diagnosis.

PBSC collection from G-CSF primed donors

Peripheral blood stems cells (PBSCs) have been used in autologous transplantation as an alternative to bone marrow-derived cells. Recently, PBSCs have been collected from healthy donors after priming with G-CSF and used for allogeneic transplantation. We have a comparatively large experience with PBSC collection in autologous and allogeneic settings. The five cell separators we employ are: the CS3000 plus, AS 104, Excel, Cobe Spectra and MCS 3p. These machines appear to have different efficacies but no studies have been carried out on this topic. In a prospective study we have randomly assigned donors to different cell separators to evaluate their efficiency. Twenty-five donors underwent the procedure and 50 leukaphereses were carried out. Donors were given 5 micrograms kg-1 d-1 of recombinant human G-CSF for 3 days and 10 micrograms kg-1 d-1 for 4 days subcutaneously. Leukaphereses were performed on days 6 and 7 of G-CSF administration. The results of our study show that a total value of CD34+ cells ranging from 48.44 x 10(6) to 270.37 x 10(6) can be collected from donors with a white cell count ranging from 40.50 x 10(3) microL-1 to 51.34 x 10(3) microL-1 and mononuclear cells ranging from 16.42 to 20.37%. The Excel and the MCS 3p seem to differ from the other machines in terms of higher CD34+ cell collection efficiency. The Excel appears to be even more efficient than the MCS 3p, but this may not reflect reality because the Excel processes 12 L of blood while the MCS 3p employs an 8 L procedure. All the machines showed satisfactory results in terms of yield and quality of the harvests.

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft.

OBJECTIVE An important step in successful autografting of patients with chronic myelogenous leukemia is the delivery of a leukemia-free graft. We conducted this study to determine whether the cytogenetic response after autografting was correlated with the number of BCR ABL-positive cells present within the stem cell grafts. MATERIALS AND METHODS By BCR-ABL mRNA quantification, we studied the serial pheresis products from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplantation. We correlated the residual disease within the graft reinfused with the cytogenetic response following transplantation, taking into consideration those responses that lasted 12 months or more. RESULTS Thirty-two patients received a graft with 0-35% Ph-metaphases and 19 received a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months, and 14 of them (78%) received a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients experienced short-lived responses or had >35% Ph-positive cells in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between a BCR-ABL/ABL ratio less than or =0.01 and the achievement of complete or major cytogenetic remission after autografting (chi(2) test, p = 0.0001). Patients reinfused with grafts contaminated at low levels with leukemic cells also showed a longer duration of the response (log-rank test, p = 0.0009). Eleven patients were reinfused with the lowest level of contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients experienced prolonged neutropenia or thrombocytopenia following stem cell reinfusion and nine of them had long-lasting complete or major cytogenetic responses after transplant. CONCLUSION This study demonstrates that the number of BCR-ABL positive cells present in a stem cell graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting. [corrected]

Progenitor cells trapped in marrow filters can reduce GvHD and transplant mortality

A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filter-discarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001–2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997–2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II–IV graft-versus-host disease (GvHD) (42 vs 15%, P=0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P=0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P=0.15). This study suggests that a significant proportion of LTC-IC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM.