Giuliana Leopardi

Fludarabine-containing regimens severely impair peripheral blood stem cells mobilization and collection in acute myeloid leukaemia patients

We studied the effects of an intensified induction/consolidation treatment containing fludarabine (ICE/FLAN/FLAN) on the mobilization and collection of peripheral blood stem cells (PBSC) in 31 consecutive untreated acute myeloid leukaemia (AML) patients. The complete remission (CR) rate was comparable to classic inductions (68% after ICE; 84% after ICE‐FLAN I). To mobilize PBSC, 19 patients received 10 μg/kg/d of granulocyte‐colony stimulating factor (G‐CSF) starting at day 13 after FLAN, 13 (69%) of whom were found to be nonmobilizers. When a second G‐CSF administration was performed in 10/13 patients mobilization was either not achieved (8/10) or was considered insufficient (<1 × 106 CD34+ cells/kg) (2/10) and all 13 were subsequently submitted to bone marrow harvest. The harvest was considered adequate in 12/13 (92%) patients and autologous BMT (ABMT) has so far been performed in 10/12 cases with a mean of 8.6 × 108/kg nucleated reinfused cells. The median times to neutrophil and platelet recovery after ABMT did not significantly differ from those of two previous series of patients treated with ABMT without fludarabine‐containing regimens. Adequate amounts of PBSC were obtained in 6/19 (31%) patients, who were then reinfused. Median times for platelet recovery were significantly longer than in a previous series of 26 AML cases reinfused with PBSC after treatment with the ICE‐NOVIA induction/consolidation regimen (125 v 20 d to 20 × 109 plt/l, P < 0.02; 218 v 37 d to 50 × 109 plt/l, P < 0.02). In addition, times for platelet recovery after ICE/FLAN/FLAN were not significantly different from those in a previous group treated with ABMT performed after ICE/NOVIA,without fludarabine. We conclude that fludarabine‐containing regimens severely impair mobilization and collection of PBSC in AML patients and seem unsuitable when PBSC autotransplantation is programmed.

Use of peripheral blood stem cells for autologous transplantation in acute myeloid leukemia patients allows faster engraftment and equivalent disease-free survival compared with bone marrow cells

We compared the feasibility and efficacy of autologous bone marrow (ABMT) and peripheral blood progenitor cell transplantation (PBSCT) performed after an identical induction/consolidation in adults with acute myeloid leukemia (AML). From January 1993 to June 1996 91 consecutive AML patients were enrolled in a program consisting of anthracycline-based induction and high-dose cytarabine consolidation (NOVIA). Until May 1994 ABMT was performed; from June 1994, if PBSC collection was adequate, PBSCT was performed. Out of 88 evaluable patients, 73 obtained a complete remission (CR) and 15 were resistant. Allogeneic bone marrow transplantation was performed in 16 patients. Forty-four (50%) were given autologous stem cell transplantation. ABMT was performed in 21 cases; twenty-nine patients were given G-CSF mobilization after NOVIA administration. An adequate number of PBSC was obtained in 23/29 (79%) cases, which were then re-infused. Median times to both neutrophil and platelet recovery from transplant were significantly shorter for the PBSC group (17 vs 36 days to 500 PMN/μl, P < 0.01; 20 vs 150 days to 20000 platelets/μl, P < 0.02; 37 vs 279 days to 50000 platelets/μl, P < 0.03), as were days of hospitalization after the reinfusion (18 vs 33, P < 0.03) and median days to transfusion independence. toxicity was not significant in either group. after a minimum follow-up for live patients of 24 months (longer than the mean time for relapse observed for the pbsc series – 14 months) the percentage of relapses was similar: 11 of 21 (52.4%) and 12 of 23 (52.1%) in the abmt and pbsc groups, respectively. our results indicate that autologous pbsc transplantation, performed after an intensive chemotherapy regimen, is not inferior to abmt in terms of disease-free survival and allows faster recovery times and reduced need for tranfusion support.

SELECTION AND TRANSPLANTATION OF AUTOLOGOUS HEMATOPOIETIC CD34+ CELLS FOR PATIENTS WITH MULTIPLE MYELOMA

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

Selection and transplantation of autologous CD34+ B‐lineage negative cells in advanced‐phase multiple myeloma patients: a pilot study

The feasibility of sequential positive and negative selection of mobilized CD34+ B‐lineage negative cells to achieve tumour‐free autografts in multiple myeloma (MM) patients was evaluated. Peripheral blood stem cells (PBSC) of 14 patients with advanced disease were mobilized. CD34+ cells were enriched in 12 of the patients by the avidin–biotin immunoabsorption technique. Subsequently, CD10+, CD19+, CD20+ and CD56+ cells (B‐lin cells) were removed by immunomagnetic depletion. Minimal residual disease (MRD) was detected by flow cytometry and PCR‐based molecular analysis of the patient specific IgH complementary‐determining region III (CDRIII). Positive selection of stem cells produced a median recovery of 54.7% of the initial content of CD34+ cells (median purity 71.9%). Negative depletion of B‐lineage cells reduced the number of CD34+ cells to 33.3% of the baseline value (median purity 72.7%). However, long‐term culture assays showed the recovery of >60% of primitive haemopoietic progenitor cells after depletion of the B‐lineage‐positive cells. All evaluable patients had detectable disease in PBSC collections. The first step of positive selection of CD34+ cells resulted in >2 logs of tumour cell purging. However, molecular assessment showed the persistence of the disease in 6/7 cases. Immunofluorescence analysis demonstrated 1 additional log of B‐cell purging by negative depletion. More importantly, molecular evaluation of IgH CDRIII region showed the disappearance of myeloma cells in 6/7 patients. 12 patients received a median of 3.9 × 106 CD34+ B‐lin− cells/kg after conditioning with high‐dose melphalan and showed a rapid reconstitution of haemopoiesis. These results were similar to two similar cohorts of patients who received either unmanipulated PBSC or positively selected CD34+ cells after the same conditioning regimen. Severe extrahaematological toxicity was limited to mucositis; no late infections were observed. We concluded that autotransplantation of purified CD34+ B‐lin− cells was associated with a rapid and sustained recovery of haemopoiesis and low peritransplant morbidity. Sequential positive and negative enrichment of stem cells reduced tumour cell contamination in B‐cell malignancies below the lower limit of detection of molecular analysis.

Discrepancy between serological complete remission and concomitant new bone lytic lesions after infusion of escalating low doses of donor lymphocytes in multiple myeloma: a case report.

A graft-versus-myeloma effect has been previously induced by infusing high numbers of donor lymphocytes after allogeneic stem cell transplantation in relapsed/refractory multiple myeloma (MM) patients. A 43-year-old patient with MM refractory to standard chemotherapy and autologous transplantation received an allogeneic HLA-matched T cell-depleted marrow transplant from his sister after conditioning with single dose total-body irradiation, melphalan and cyclophosphamide. Twenty-four months after transplant neither a significant reduction of serum M protein nor evidence of acute or chronic graft-versus-host disease (GVHD) were observed. The patient was then treated with four escalating low doses of donor lymphocyte infusions (DLI) (0.1, 1.0, 5.0 and 5.0 × 106 CD3+ T cells/kg, respectively) over a 13 month period. Following the second infusion a mild liver acute GVHD and a partial, but transient, response occurred. After the last DLI the patient achieved a complete remission and developed extensive chronic GVHD. However, concomitant with the disappearance of clonal plasma cells from the marrow and of serum M protein, two new bone lytic lesions appeared requiring treatment with radiotherapy. In conclusion, escalating low doses of DLI may be effective in MM and may prevent severe acute but not chronic GVHD. However, the efficacy of DLI in extramedullary MM lesions is still unclear.

Cyclophosphamide, pegylated liposomal doxorubicin, vincristine and prednisone (CDOP) plus rituximab is effective and well tolerated in poor performance status elderly patients with non-Hodgkin's lymphoma

Lymphomas in elderly patients require special attention, not only for possible important co-morbidities and poor performance status, but also for diminished organ functions and altered drug metabolism, which modify the pharmacokinetics of the treatment [1]. Thus, first-line elderly non-Hodgkin’s lymphoma (NHL) patients are frequently treated with a consequent dose reduction [2,3]. Similarly, the treatment outcome of salvage therapy for relapsed/ refractory NHL patients is limited by multidrug resistance, low performance status and the high toxicity of secondand third-line regimens. Anthracycline-based regimens are extremely effective in NHL; the main disadvantage is cardiotoxicity. Pegylated liposomal doxorubicin has been shown to determine lower toxicity if compared with conventional doxorubicin, while having similar efficacy in patients with Kaposi’s sarcoma and solid tumors. Due to the prolonged half-life (nearly 43 h), it offers the opportunity of obtaining prolonged exposure of tumor cells to active concentrations, with possible effects on the disease. A recent study concluded that an improvement in the survival of elderly patients with lymphoma is related to an optimized toxicity chemotherapy schedule [4,5]. In addition, cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) plus rituximab has been demonstrated to be superior to CHOP alone in elderly patients with diffuse large cell lymphoma [6,7]. We thus tested the safety and efficacy of a regimen [cyclophosphamide, pegylated liposomal doxorubicin, vincristine and prednisone (CDOP) plus rituximab] using a pegylated liposomal doxorubicin instead of conventional doxorubicin, in order to reduce the toxic profile and preserve the dose intensity, administered as follows: day 1, cyclophosphamide 750 mg/m, pegylated liposomal doxorubicin (Caelyx, Schering Plough S.A., Italy) 40 mg/m, vincristine 1.4 mg/m, prednisone 100 mg p.o. days 1 – 5, rituximab 375 mg/ m on day 15 of every cycle; granulocyte colonystimulating factor (G-CSF) from day 10 to day 14. Therapy was repeated every 21 days for 6 cycles. Complete and partial remission were assessed as the overall response. Toxicity was evaluated according to the WHO criteria. Thirteen patients were treated (Table I); 12 (92%) completed the 6 courses and were assessable for response (Table II). A dose reduction (25%) was carried out in 2 cases (1 frail, 1 relapsed-refractory), due to their extremely altered clinical condition. Seven patients (53%) obtained complete remission, 5 (31%) partial remission, for an overall response rate of 84%. No major toxicity (WHO grade III/IV) occurred. Only 1 patient delayed therapy for grade II hematological toxicity. After a median follow-up of 18 months (range 8 – 22 months), 7 patients are in continuous complete remission (53%), 3 (23%) in partial remission, 1 in stable disease, whereas only 2 patients progressed. In our opinion, one of the reasons for the high overall response rate (84%) and of the maintenance of the response, even in this category of very fragile patients, is the respect of the doses, as well as of the

Clinical Relevance of Acute Pancreatitis in Allogeneic Hemopoietic Stem Cell (Bone Marrow or Peripheral Blood) Transplants

This study investigated the clinical relevanceof acute pancreatitis in allogeneic hemopoietic stemcell (bone marrow or peripheral blood) transplants(BMT). We studied 26 patients undergoing BMT. Thepreparative regimen was busulfan and cyclophosphamide in 17patients and total body irradiation and cyclophosphamidein 9 patients. Graft-versus-host disease (GVHD)prophylaxis consisted of cyclosporin A and short-term methotrexate in all 26 patients. The pancreaswas studied using amylase and lipase serum levels,abdominal contrast-enhanced tomography, and/orultrasound. Clinical and laboratory signs of acutepancreatitis were found in two patients with acutehepatointestinal GVHD, and in one patient with acutehepatic GVHD and cytomegalovirus infection. This patientdied of multiorgan failure, with interstitial acutepancreatitis at autopsy; the other two patients recoveredwith general supportive care and GVHD therapy. Wesuggest that in the patients with complications afterBMT, particularly acute hepatic/hepatointestinal GVHD, and cytomegalovirus infection, the possibilityof acute pancreatitis should be considered.

Pegfilgrastim effectively mobilizes PBSC in a poor mobilizer multiple myeloma patient.

Abstract:  Studies performed on mice and healthy human volunteers have shown that a single dose of pegfilgrastim (Peg‐GCSF) is effective in stimulating peripheral blood stem cells (PBSC) mobilization. This prompted us to try the stimulation with pegfilgrastim in a patient previously non‐mobilizing with a combination of chemotherapy and filgrastim. In December 2003, a 65‐yr‐old man was diagnosed as having stage III A IgG/k multiple myeloma. He received three courses of polichemotherapy (DC‐IE) obtaining a stable response. Afterwards, the patient was treated with high‐dose cyclophosphamide (CPM; 7 g/sqm) plus daily 10 mcg/kg filgrastim in order to mobilize PBSC, without success. After 2 months off therapy, the disease progressed and the patient received alternate cycles VAD (vincristine, dexamethasone, adriblastine)/high‐dose dexamethasone. A second attempt to mobilize PBSC, using daily 10 mcg/kg filgrastim after the second and third VAD cycle, failed. In a further attempt to mobilize PBSC, we administered a single dose of 12 mg pegfilgrastim on day 5 after a fourth VAD course. Daily evaluation of circulatory CD34+ cells was started from day 8 after the end of chemotherapy. On day +10 postchemotherapy the CD34+ cell count was 24/μL and two aphaeresis were performed, harvesting 1.6 × 106 and 0.89 × 106 CD34+ cells/kg respectively (total 2.49 × 106 cells/kg). The only side effect was moderate skeletal pain. The patient underwent successful transplantation. The median times necessary to recover 0.5 × 109 PMN/L and 20 × 109 platelets/L after PBSC reinfusion were 9 and 12 d respectively. The patient did not need red blood cell or platelet transfusions. He experienced a sustained engraftment and maintains complete remission 9 months after the reinfusion. In conclusion, a single dose of pegfilgrastim was able to mobilize a sufficient number of CD34+ in a multiple myeloma patient not responsive to two previous attempts with high or standard dose chemotherapy followed by filgrastim. This approach, if confirmed on larger series and other diseases, could open new opportunities in stem cell mobilization for poor or non‐mobilizers.

Autologous transplantation of chemotherapy-purged PBSC collections from high-risk leukemia patients: a pilot study

We have recently demonstrated that the combination of the alkylating agent nitrogen mustard (NM) and etoposide (VP-16) is capable of eliminating, ex vivo, leukemic cells contaminating PBSC collections and this is associated with a significant recovery of primitive and committed hematopoietic progenitor cells. Based on these data a pilot study on autologous transplantation of NM/VP-16 purged PBSC for high-risk leukemic patients was recently initiated. Twelve patients (seven females and five males) with a median age of 46 years (range 18–57) have been treated. Two patients had acute myeloblastic leukemia (AML) resistant to conventional induction treatment, four patients had secondary AML in I complete remission (CR), one patient was in II CR after failing a previous autologous BM transplantation, while two additional AML individuals were in I CR achieved after three or more cycles of induction treatment. Two patients with high-risk acute lymphoblastic leukemia (ALL) in I CR and one patient with mantle cell lymphoma and leukemic dissemination were also included. Eight patients showed karyotypic abnormalities associated with a poor clinical outcome. The mobilizing regimens included cytosine arabinoside and mitoxantrone with (n = 6) or without fludarabine (n = 3) followed by subcutaneous administration of G-CSF (5 μg/kg/day until the completion of PBSC collection) and G-CSF alone (n = 3) (15 μg/kg/day). A median of two aphereses (range 1–3) allowed the collection of 7.2 × 108 TNC/kg (range 3.4–11.5), 5 × 106 CD34+ cells/kg (range 2.1–15.3) and 9.2 × 104 CFU-GM/kg (0.3–236). PBSC were treated with a constant dose of 20 μg of VP-16/ml and a median individual-adjusted dose (survival ⩽5% of steady-state BM CFU-GM) of NM of 0.7 μg/ml (range 0.25–1.25). Eleven patients were reinfused after busulfan (16 mg/kg) and Cy (120 mg/kg) conditioning with a median residual dose of 0.3 × 104 CFU-GM/kg (0–11.5). The median time to neutrophil engraftment (>0.5 × 109/l) for evaluable patients was 25 days (range 12–59); the median time to platelet transfusion independence (>20 and >50 × 109/l) was 40 days (18–95) and 69 days (29–235), respectively. Hospital discharge occurred at a median of 25 days (18–58) after stem cell reinfusion. Four individuals are alive in CR (n = 3) or with residual nodal disease (n = 1 lymphoma patient) with a follow-up of 32, 26, 3 and 14 months, respectively. Seven patients died due to disease progression or relapse (n = 5) or extrahematological transplant toxicity (n = 2). Our data suggest that pharmacological purging of leukapheresis collections of leukemic patients at high-risk of relapse is feasible and ex vivo treated cells reconstitute autologous hematopoiesis.

An observational study of once weekly intravenous ganciclovir as CMV prophylaxis in heavily pre-treated chronic lymphocytic leukemia patients receiving subcutaneous alemtuzumab

Fifteen consecutive resistant/relapsed chronic lymphocytic leukemia (CLL) patients (median age: 65 years) received alemtuzumab for 16 consecutive weeks. All patients had negative CMV anti genemia at baseline. Five patients received oral acyclovir 800 mg twice a day for CMV prophylaxis and 10 patients got intravenous (iv) ganciclovir 7.5 mg/kg once a week. A total of five CMV reactivations occurred, four in the acyclovir and one in the ganciclovir prophylaxis group. Alemtuzumab was then discontinued and all patients were treated with iv ganciclovir 7.5 mg/kg per day. All patients achieved negative CMV anti genemia after a median of 15 days of therapy. Weekly iv ganciclovir prophylaxis and alemtuzumab treatment were then restarted without any further CMV reactivations. In conclusion, weekly iv ganciclovir appears feasible and effective in preventing CMV reactivation and disease in this setting of high-risk immunocompromised patients, allowing an easier management of a therapy otherwise difficult to be routinely used.