Long-term immune recovery after CD34+ immunoselected and unselected peripheral blood progenitor cell transplantation: a case-control study.

BACKGROUND AND OBJECTIVE: CD34+ stem cell selection induces extensive T-cell depletion as a consequence of ex vivo manipulation. The impact of T-cell depletion on long-term immunologic recovery after autologous CD34+ peripheral blood progenitor cell transplantation (CD34+ PBPCT) is not well characterized. We compared the long term immunologic recovery in two groups of patients submitted to CD34+ PBPCT or unselected autologous peripheral blood progenitor cell transplantation (uPBPCT). DESIGN AND METHODS: Eight patients in both groups were closely matched for diagnosis, age, disease status at transplantation and conditioning regimen and lymphocyte phenotype was prospectively evaluated during long-term post-transplantation follow-up. RESULTS: At a median of 18 months after transplantation, CD3+ lymphocyte subset remained below the normal range in both groups. CD19+ B lymphocytes subset after CD34+ PBPCT was within the normal range in both groups. CD4+ lymphocytes were depressed while the CD8+ lymphocyte subset was increased in group A and in the normal range in group B. As a result, inversion of CD4/CD8 ratio was documented in both groups. T-activated lymphocytes (CD3DR+) and natural killer (CD16/56+) cells were increased in both groups. INTERPRETATION AND CONCLUSIONS: Long-term immune recovery appears to be unaffected by extensive ex vivo manipulation in this adult population when compared to recovery after unmanipulated PBPCT. CD34+ selection, although causes an extensive depletion of T lymphocytes in the graft does not represent a risk factor for delayed CD4+ recovery late after transplantation. Elevated numbers of NK cells and activated T-cells, which have antineoplastic activity, are maintained late after autologous CD34+ transplantation.

The application of two different blood cell separators to harvest CD34+ cells in patients suffering from non Hodgkin's lymphoma.

From January 1996 until now, thirty-eight PBSC procedures were carried out on 20 patients suffering from NHL, mobilized by polichemotherapy regimens plus recombinant human Granulocyte-Growth Factor (rhG-CSF). Patients were enrolled in PBSC procedures using Dideco Excel (group A) and Cobe Spectra v.4.7 (group B) blood cell separators. Twelve patients were enrolled in group A (6 males and 6 females, median age 33) and 9 patients in group B (5 males and 4 females, median age 55). The mean White Blood Cell (WBC) and Mononuclear Cells Fraction (MNC) peripheral blood counts were not statistically different in either group and neither were blood CD34+ cell peripheral counts. CD34+ cell peripheral value was predictive of the CD34+ yield while mean values of harvested CD34+ cells were not significantly different. CD34+ cell efficiencies were statistically the same. The CD34+ cell purity of the apheresis harvest was statistically different between the two groups (group A = 3.0+/-2.2%; group B = 1+/-0.9%) p = 0.001. High CD34+ cell yields were observed in both groups which confirms that both blood cell separators are able to harvest hematopoietic progenitor cells from peripheral blood.

Long-lasting complete remission in plasma cell leukemia after aggressive chemotherapy and CD34-selected autologous peripheral blood progenitor cell transplant: molecular follow-up of minimal residual disease.

Plasma cell leukemia is a rare disease associated with very poor survival with standard treatment. We report a patient affected by plasma cell leukemia treated with aggressive chemotherapy and autologous CD34-selected PBPC who achieved a complete remission now lasting more than 2 years. Molecular studies confirmed the presence of minimal residual disease (MRD) despite the absence of disease activity. High-dose chemotherapy with stem cell rescue may be applied to selected patients considering the impact of the treatment on survival. The meaning of molecular MRD in this setting is unclear.

Highly fluorescent reticulocytes after CD34+ peripheral blood progenitor cell transplantation.

Highly fluorescent reticulocyte (HFR) counts were evaluated in 13 consecutive patients affected by hematological malignancies and submitted to autologous selected CD34+ peripheral blood progenitor cell (PBPC) transplantation. Results were compared with a historical group of patients comparable for age, disease and conditioning regimen submitted to unfractionated PBPC transplantation. HFR counts of the CD34+ group declined to an undetectable level from day +4 to day +10 when they became detectable and reached 5% of total reticulocyte count by day +12. In the historical group, the nadir was identical but the recovery was faster (day +9). Total reticulocyte count > 1% was achieved at days +17 and +11, respectively. The absolute neutrophil count (ANC) recovery was identical in both groups, achieving a value > 0.5 x 10(9)/l at day +13 after reinfusion. Hence, in the historical group, HFR count gave advance notice of complete and stable hemopoietic engraftment while in the CD34+ group HFR and ANC count showed almost simultaneous recovery.

Bacteremia in patients with hematological malignancies. Analysis of risk factors, etiological agents and prognostic indicators.

BACKGROUND AND OBJECTIVE: Patients with hematological malignancies are at increased risk for developing bacteremia. No previous study has investigated the risk and prognostic indicators of bacteremia in such patients using a statistical approach. METHODS: A case-control study was performed in 106 patients with hematological malignancies (group A). Two hundred and twelve patients were included as controls and divided into two groups: 106 patients with hematological malignancy without bacteremia (group B) and 106 HIV-infected patients with bacteremia (group C). RESULTS: At univariate analysis, bacteremia risk factors in group A were: neutropenia for more than six days (p = 0.03 vs. group B), central venous catheter usage (p = 0.04) and absence of antibiotic prophylaxis (p = 0.03). At multivariate analysis, the use of CVC and neutropenia were independent bacteremia risk factors. The most frequent etiological agents were: Staphylococcus epidermidis and Pseudomonas aeruginosa. Comparing groups A and C, the distribution of Staphylococcus spp. was different, with S. epidermidis being prevalent in hematological patients only. As regards gram-negative organisms, it is of note that no episode of NT-Salmonella bacteremia was observed in group A, unlike group C, where they represent the second leading etiological agents. In group A, 14% of the patients died. Persistent neutropenia (p = 0.01) and the presence of relapsed neoplasm (p = 0.04) were prognostic indicators of bacteremia. INTERPRETATION AND CONCLUSIONS: Our findings suggest that bacteremia in patients with hematological malignancies strictly correlates with the intensity and length of neutropenia and CVC usage. Although we observed a low mortality rate, we stress that this clinical condition requires special attention from the physician, who must recognize and treat it promptly.

Granulocyte colony-stimulating factor-primed leukocyte transfusions in candida tropicalis fungemia in neutropenic patients.

Optimal management of fungemia in neutropenic patients is still controversial. Several reports have already stressed the poor prognosis in invasive candidiasis (80% mortality in several reports). Therefore granulocyte transfusions would appear to be useful in the management of these infections. We report the use of rhG-CSF-primed granulocyte transfusions plus amphotericin B in two neutropenic patients who developed life-threatening systemic fungal infections. This approach was successful and both patients fully recovered from the infection.

RhG-CSF-mobilized CD34+ peripheral blood progenitors are myeloperoxidase-negative and noncycling irrespective of CD33 or CD13 coexpression.

Cycling status, myeloperoxidase expression, informative surface markers, and proliferative potential of peripheral blood hemopoietic progenitors (PBHP) were evaluated by flow cytometry in 10 patients affected by resistant lymphoma, and submitted to stem cell mobilization with combination chemotherapy (MiCMA) followed by recombinant human granulocyte colony-stimulating factor (rhG-CSF, 5 micrograms/kg/day). CD34+ PBHP coexpressed myeloid-associated and activation antigens, i.e., CD33 (96%, range 85-99), CD13 (99.5%, range 99.4-99.8), HLA-DR (99%, range 96.5-99.8), and CD38 (98%, range 90-100), lacked intracytoplasmic myeloperoxidase (MPO, < 3%), and resided in the Gzero/G1 phase of the cell cycle (96.5%, range 81-99.5, compared with 70%, range 49-78 bone marrow HP; p = 0.0007), independently of surface membrane phenotype; S-phase percentages of sorted CD34+ CD33(+)/-, CD34+CD38(+)/-, CD34+HLA-DR(+)/-, CD34+CD45RA(+)/-, CD34+CD45RO(+)/-, and CD34+CD41a(+)/- subpopulations were always negligible. In colony assays, 5-week-old long-term cultures seeded with CD34+CD33- cells yielded as many colonies as did CD34+CD33+ cells. In conclusion, rhG-CSF-mobilized CD34+ PBHPs contain noncycling, highly immature progenitors in which the expression of myeloid-associated antigens, i.e., CD33 or CD13, might not be indicative of myeloid commitment.

Separation of chemotherapy plus G-CSF-mobilized peripheral blood mononuclear cells by counterflow centrifugal elutriation: in vitro characterization of two different CD34+ cell populations.

Counterflow centrifugal elutriation (CCE) has been extensively employed in T cell depletion of bone marrow cells for allografting. Nevertheless very little is known about CCE properties of mobilized hematopoietic progenitors. In this study five leukapheresis products collected after chemotherapy and G-CSF from patients with non-Hodgkin's lymphoma were elutriated. Two mononuclear cell fractions were obtained containing smaller and less dense cells (lymphocyte fraction) and larger and denser cells (monocyte fraction), respectively. The presence of immature CD34+ progenitor cells, not co-expressing CD33, CD38 and HLA-DR antigens, was demonstrated in both cell fractions. CD34+ cells were isolated from each fraction and grown in various culture conditions (CFU-GM and BFU-E assay, blast cell colony assay, cytokine supplemented liquid culture). CD34+ cells isolated from the monocyte fraction showed a longer lasting expansion in liquid culture and a higher number of blast cell colonies than CD34+ cells selected from the lymphocyte fraction. Moreover a significant reduction of T cell number was obtained in the monocyte fraction. These data suggest that chemotherapy plus G-CSF-mobilized progenitor cells show a characteristic behavior when subjected to CCE, allowing an efficient T cell depletion without losing more immature progenitors.

rhG-CSF in healthy donors: mobilization of peripheral hemopoietic progenitors and effect on peripheral blood leukocytes.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16 micrograms/kg/day was given to 9 healthy donors to recruit hemopoietic progenitors (HP) for allogeneic transplantation or donor leukocyte infusion. rhG-CSF was administered s.c. for 5 days. No side effects were encountered except for moderate bone pain and lumbago. Mobilization was effective, reaching a peak median value of 187 x 10(3) CD34+ cells/ml (range 51.2-1127) and 2170 x 10(3) colony-forming units-granulocyte macrophage (CFU-GM)/ml (range 1138-4190). Peak values were obtained at a median of 4 days of rhG-CSF and represented, respectively, a 13-fold and a 37-fold increase from baseline values (p = 0.0007 and p = 0.006). White blood cell (WBC) counts increased 6-fold from baseline values (p < 0.0007) and reached a median peak of 34 x 10(6)/ml (23.5-59). Polymorphonuclear (PMN), and mononuclear (MNC) cells increased 10-fold and 2-fold, respectively (p = 0.0039 and p = 0.0026) and reached a median peak of 32.1 x 10(6)/ml (18.2-52) and 4.42 x 10(6)/ml (3.14-12.42). Absolute lymphocyte and monocyte counts increased at peak day in all donors 1.5-fold and 5.7-fold from baseline values (p = 0.0017 and p = 0.0018). In 7 of 9 donors, lymphocyte subsets were analyzed in detail. CD3+ and CD19+ lymphocytes increased 1.5-fold and 3-fold, respectively (p = 0.032 for both). NK and activated T lymphocytes doubled at a median of 4 days of rhG-CSF (p = 0.032 and p = NS, respectively). Similar changes were observed in lymphocytes collected in leukapheresis product. T helper and T suppressor subsets displayed a similar increase. Thus, besides the anticipated priming effect on HP and PMN, rhG-CSF in healthy donors produced an unexpected and still unexplained modification of lymphocyte subsets in peripheral blood.

Adjuvant therapy with rhGM-CSF for the treatment of Blastoschizomyces capitatus systemic infection in a patient with acute myeloid leukemia.

We report a patient with acute myeloid leukemia and Blastoschizomyces capitatus sepsis who developed multiple abscesses when neutrophils recovered. The patient did not respond to antifungal therapy and her clinical condition showed an improvement only after rhGM-CSF was added to the treatment.

Salvage chemotherapy with pentostatin in prolymphocytic leukemia.

We report 4 cases of prolymphocytic leukemia (PLL) resistant to conventional chemotherapy that were treated with pentostatin. We obtained 1 complete remission (CR) and 2 partial remissions (PR). Our data confirm the effectiveness of this drug in the treatment of prolymphocytic leukemia.

In vitro expansion of CD34+ cells mobilized with chemotherapy and G-CSF.

Hemopoietic CD34+ progenitors were isolated by immunomagnetic method from normal bone marrow (BM) or from peripheral blood (PB) of patients with non-Hodgkin's lymphoma treated with chemotherapy and granulocyte colony-stimulating factor (GCSF). Aliquots were seeded in long-term cultures (LTC) on bone marrow-derived stromal layers; non-adherent and adherent clonogenic content of the cultures was assayed weekly. The final recovery and the clonogenic efficiency of the CD34+ cells were slightly higher in PB samples than in BM controls. In long term cultures PB cells sustained hemopoiesis as much as BM cells; at week 3 and 4 PB total mononuclear cells and CD34+ cells showed a non-adherent cell recovery higher than the respective BM controls. Furthermore, PB CD34+ cells were expanded in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF alone or combined with interleukin 3 (IL3), stem cell factor (SCF), interleukin 1 (IL1), interleukin 6 (IL6). The combination of GM-CSF, IL3, SCF, IL1 and IL6 produced the maximum increase of both mononuclear cells (30-fold) and granulocyte-macrophage colony forming units (CFU-GM) (4.6-fold) after 7 days of cultures; yet after 14 days a strong decrease of the CFU-GM occurred. These data suggest that G-CSF following chemotherapy mobilizes both early and committed hemopoietic progenitors.