Feasibility of peripheral blood progenitor cell mobilization and harvest to support chemotherapy intensification in elderly patients with poor prognosis: non-Hodgkin's lymphoma.

Mobilized peripheral blood progenitor cells (PBPC) are widely employed in the management of adult patients with high-risk non-Hodgkin's lymphoma (NHL), though their use in the elderly has received little attention. This study was mounted to assess the feasibility of the mobilization, harvesting, and reinfusion of PBPC in NHL patients aged >60. Twenty patients (median age: 67, range: 61-80) with poor-prognosis NHL entered the pilot study: nine others were discarded for various reasons. Thus, the program was applicable to 69% of potential candidates. Fourteen patients were at onset and six were being treated for refractory disease or relapses. Mobilization was induced with cyclophosphamide 4 g/m(2), followed by 5 micro g/kg per day granulocyte-colony stimulating factor (G-CSF) s.c. until PBPC collection or hemopoietic recovery. Sixteen patients (80%) displayed some signs of mobilization (CD34+: >5/ micro l). Maximum mobilization varied with median circulating CD34+ cells and colony forming units-granulocyte/macrophage (CFU-GM) peaks of 17.2/ micro l (range: 8.1-210) and 1,650/ml (range: 540-62,900), respectively. A median of two leukaphereses resulted in the harvesting of a median of 6.7x10(6) (range: 0.3-33.6) CD34+/kg and 21.1x10(4) (range: 1.2-209) CFU-GM/kg. Intensified therapy with intermediate-dose melphalan, associated or not with mitoxantrone, was delivered with autologous PBPC support to 13 patients and always resulted in rapid and stable hemopoietic reconstitution. The program was well tolerated and no treatment-related deaths occurred. Twelve patients are still alive with a 5-year survival projection of 59%. In conclusion, the results demonstrate the feasibility of using autologous PBPC to support therapy intensification even in elderly patients.

Growth advantage of chronic myeloid leukemia CFU-GM in vitro: survival to growth factor deprivation, possibly related to autocrine stimulation, is a more common feature than hypersensitivity to GM-CSF/IL3 and is efficiently counteracted by retinoids +- alpha-interferon.

Bcr/abl fusion gene, in experimental models, induces survival to growth factor deprivation and hypersensitivity to IL3. However, conflicting data were reported about chronic myeloid leukemia (CML) progenitors. We investigated the responsiveness of purified CML CFU-GM to GM-CSF/IL3 and their survival to growth factor deprivation. CFU-GM hypersensitivity to IL3 and/or GM-CSF was found in 3/11 CML cases only. CML CFU-GM survived well in stroma-free 'mass' culture (5 x 10(4) cells/ml) without cytokine addition, up to day 11, average recovery being around 95% in medium + 10% fetal bovine serum and 67-81% in serum-free medium. Conversely, normal progenitors declined steadily, particularly after extensive purification (18 +/- 10% recovery at the 7th day), and in serum-free medium (4 +/- 6% recovery). By contrast, normal and CML CFU-GM declined in a similar way in limiting dilution cultures (1-10 cells/50 microl). We also investigated the effects of retinoic acid and alpha-interferon on CFU-GM survival. Both all-trans- and 13-cis retinoic acid, particularly in combination with alpha-interferon, reduced CML CFU-GM recovery down to normal progenitors' values. In conclusion, hypersensitivity to CSFs is rare in CML, whereas resistance to growth factor deprivation has been confirmed in mass, but not in limiting, dilution cultures. Both stereoisomers of retinoic acid, at therapeutic concentrations and in combination with alpha-interferon, can overcome the survival advantage of CML progenitors.

Multiple myeloma: the number of reinfused plasma cells does not influence outcome of patients treated with intensified chemotherapy and PBPC support.

Multiple myeloma (MM) is characterized by the expansion of tumor plasma cells in bone marrow (BM), but neoplastic cells have been consistently detected in peripheral blood (PB). Peripheral blood progenitor cell (PBPC) collections have been widely used to support high-dose therapy for MM patients. A flow cytometric technique has been used to detect plasma cells in PB and PBPC harvests. High CD38 expression identified these cells, and their nature was confirmed by the coexpression of specific antigens, such as CD138 and cytoplasmic immunoglobulins. Malignant plasma cell reinfusion could negatively affect response rate and survival, as demonstrated in other hematological malignancies. To address this issue, the relationship between the number of reinfused plasma cells, response to chemotherapy and event-free survival (EFS) have been analyzed. Sixty-four MM patients were treated with intensified chemotherapy at diagnosis. They were mobilized with cyclophosphamide and G-CSF, and then treated with melphalan 100 mg/m2 (MEL100) followed by PBPC support. A second course was given after 2 months, and a third to patients not in complete remission. There was no correlation between the number of reinfused plasma cells and response rate after this intensified chemotherapy: patients attaining complete remission received 3.6 x 106/kg CD38+ cells, while those with a partial or no response received 5.6 and 2.9 x 106/kg CD38+ cells. Similarly, there was no correlation between the number of reinfused plasma cells and EFS. Patients receiving less than 4.85 x 106/kg CD38+ cells experienced a median EFS of 34.2 months as opposed to 36.4 months for those receiving more than 4.85 x 106/kg CD38+ cells (P = 0.7). Recurrence of the disease is consistently observed in MM: our data suggest that in vivo residual tumor cells, rather than reinfused plasma cells are more likely to be responsible for relapse. Bone Marrow Transplantation (2000) 25, 25-29.

Negative immunomagnetic ex vivo purging combined with high-dose chemotherapy with peripheral blood progenitor cell autograft in follicular lymphoma patients: evidence for long-term clinical and molecular remissions.

The feasibility and efficacy of a novel immunomagnetic ex vivo negative purging method was evaluated on peripheral blood progenitor cells (PBPC) from 13 non-Hodgkin's lymphoma patients (eight follicular, FL; three mantle cell, MCL; two FL with histologic transformation). A peculiar feature of the study was the collection of PBPC after prolonged tumor debulking. Our method included a stem cell enrichment phase followed by cell incubation with anti-B cell MoAbs (anti-CD19, CD20, CD22, CD23), addition of immunobeads, and then positive cell removal by passage on a Max-Sep (Baxter Immunotherapy) cell separator. Engraftment was rapid and stable. Hematological values were assessed 1 and 2 years after the autograft. Purging efficacy was molecularly assessed in a panel of 11 patients who showed persistence of PCR-detectable lymphoma cells on PBPC harvests despite intensified chemotherapeutic debulking. PCR-negativity was obtained in vitro and persisted in vivo after autograft in three FL patients; five more FL patients, whose purged PBPC were PCR+, converted to stable (3 patients) or fluctuating (two patients) PCR negativity after autograft. MCL patients never reached PCR negativity. Thus, ex vivo purging may have a role for FL patients harvesting PCR-positive PBPC after intensified chemotherapy. In contrast, the addition of ex vivo purging seems to be of little if any benefit for MCL patients.

In vitro growth and quantification of early (CD33-/CD38-) myeloid progenitor cells: stem cell factor requirement and effects of previous chemotherapy.

BACKGROUND AND OBJECTIVE: All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application. The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells. DESIGN AND METHODS: Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E. Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3). RESULTS: The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture. SCF, but not IL3, was essential for that growth. The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension. An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM. In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission. INTERPRETATION AND CONCLUSIONS: CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time. The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.

Multiple myeloma: reduced plasma cell contamination in peripheral blood progenitor cell collections performed after repeated high-dose chemotherapy courses.

The possibility of reducing tumour cell contamination by cytotoxic drug courses prior to peripheral blood progenitor cell (PBPC) collection was evaluated in two consecutives groups of multiple myeloma (MM) patient candidates for autograft. All patients were at disease onset and received two VAD (vincristine, doxorubicin and dexamethasone) courses as initial debulking. In the first group (44 patients), mobilization and harvest were performed 'upfront', after a single cyclophosphamide (CY) administration of 4 g/m2; in the second group (17 patients), PBPC were collected at the end of a high-dose sequential chemotherapy programme, including: CY 5 g/m2, etoposide (VP16) 2 g/m2, a chemotherapy-free interval with three courses of high-dose dexamethasone, a final mobilizing CY at 7 g/m2. G-CSF was given following each high-dose cytotoxic drug. Cytofluorimetric analysis was performed to quantify progenitors (CD34+ cells) and plasma cells, identified by the high CD38 expression and/or CD38 and CD138 coexpression. Large amounts of PBPC were collected in either group (median harvested CD34+/kg: 15.8 x 10(6) and 13.4 x 10(6), respectively; P=0.9). Circulating plasma cells were significantly higher in patients mobilized 'upfront' compared to those who received the high-dose sequence (median peak values of CD38bright/microl: 39 and 10, respectively; P=0.02); a similar difference was observed in the amount of contaminating plasma cells in the harvest products (median CD38bright/kg: 7.4 x 10(6) and 1.3 x 10(6), respectively; P=0.02). The results demonstrate that an in vivo purging approach is feasible in myeloma patients through repeated high-dose chemotherapy courses; this may provide less-contaminated material suitable for further in vitro purging procedures.

Peripheral blood progenitor cell mobilization in patients with primary refractory lymphoma or at first relapse: comparison with patients at diagnosis and impact on clinical outcome.

Peripheral blood progenitor cell (PBPC) mobilization was evaluated in 53 patients receiving the high-dose sequential (HDS) regimen: 27 had non-Hodgkin's lymphoma or Hodgkin's disease, primary refractory or at first relapse, 26 had non-Hodgkin's lymphoma at diagnosis. Mobilization was assessed following either 7 g/m2 cyclophosphamide (48 patients) or 2 g/m2 etoposide, both followed by G-CSF (filgrastim) at 5 microg/kg/d. PBPC mobilization was significantly higher in patients at diagnosis compared to refractory/relapsed patients (median peak values of circulating CFU-GM: 25,209/ml v 4270/ml, P < 0.0001 and CD34+ cells: 286/microl v 47/microl, P < 0.0001). All patients receiving HDS as up-front treatment mobilized enough PBPC for an autograft, often requiring a single leukapheresis; whereas only 15 patients under salvage treatment with HDS were able to complete PBPC autograft. Bone marrow (BM) cells, alone or with PBPC, were needed in six patients, and autograft could not be performed in six patients. Among refractory/relapsed patients, those having a high PBPC mobilization experienced a significantly longer EFS compared to those who had not; autograft completion also significantly enhanced EFS. Thus, the use of an effective mobilizing protocol does not ensure adequate PBPC mobilization in moderately pretreated patients; low mobilization must be considered as an early sign of poor outcome in patients receiving a high-dose salvage programme.

Expression of antigens associated with the individual stages of the inflammatory response in child and adult as a possible distinctive method for recurrent and chronic tonsillitis.

Monoclonal antibodies (MoAbs) specific for the antigens associated with each stage of an inflammatory response were assayed with tonsillar mononuclear cells (TMNG). MoAbs BMA 27 E 10 and BMA 4 D 10 were used as markers for the early stages, BMA RM 3/1 for the intermediate stage, BMA 25 F 9 for the late stage, and BMA G 16/1 for the chronic stage. TMNC were obtained from patients operated for (1) recurrent tonsillitis with hypertrophy caused by common flora (children); (2) an indication for surgery for chronic tonsillitis in adults; (3) patients who were 'warm' tonsillectomized for a second peri-tonsillar phlegmon. Our results are presented and discussed in the light of their possible clinical significance. Our findings indicate that clinical chronic tonsillitis in the adult really is such. In the adults studied there was a high expression of antigens which is associated with the chronic stages, while the low expression of antigens is associated with the intermediate stage and an even lower antigen expression indicates the acute stage. In children what is considered to be chronic tonsillitis may perhaps be more correctly regarded as an expression of recurrent inflammation.

Expansion of T cells expressing low CD4 or CD8 levels in B-cell chronic lymphocytic leukemia: correlation with disease status and neoplastic phenotype.

Peripheral blood (PB) T cells from 56 patients with B-cell chronic lymphocytic leukemia (B-CLL) were analyzed by two- and three-color immunofluorescence (IF) to determine the expansion of distinct T-cell subsets and their relationship with the clinical and biological features of the disease. We detected the expansion of an unusual T-cell subpopulation expressing lower CD4 or CD8 levels (CD4lo, CD8lo) than classic T cells (CD4hi, CD8hi). This subpopulation also expressed low levels of the CD3/TCR alpha/beta complex and was CD19-CD13-CD14-. A phenotypic analysis probing the activation level of CD4lo, CD8lo, CD4hi, and CD8hi cells showed that they comprised increased counts of HLA-DR+, CD11b+, CD45R0+, and CD45RA+ cells. Subset expansion ranged from 2.1- to 13.6-fold. Statistical analysis showed that the size of some of these subsets was correlated to intrinsic features of the tumor. First, CD4loHLA-DR+ cell counts were higher in patients with stage A than those with stages B and C disease. Second, CD8loHLA-DR+ cell counts were higher in patients in stable remission than in those at diagnosis. Third, CD4loHLA-DR+, CD4loCD45R0+, CD4loCD45RA+, and CD4hiCD11b+ cell counts were higher in patients whose tumor cells expressed high levels of surface immunoglobulin (sIg) than in those expressing low levels. The involvement of CD4lo and CD8lo cells in most of these correlations suggests that they may be tumor-reactive cells. Similar cells described in human and murine autoimmune disease have been shown to be autoreactive anergic cells, which may derive from nonclassic pathways of T-cell development.