Simonetta Rizzi

The addition of plerixafor is safe and allows adequate PBSC collection in multiple myeloma and lymphoma patients poor mobilizers after chemotherapy and G-CSF

We report 13 multiple myeloma (MM) or lymphoma patients who were failing PBSC mobilization after disease-specific chemotherapy and granulocyte-CSF (G-CSF), and received plerixafor to successfully collect PBSCs. Patients were considered poor mobilizers when the concentration of PB CD34+ cells was always lower than 10 cells/μL, during the recovery phase after chemotherapy and/or were predicted to have inadequate PBSC collection to proceed to autologous transplantation. Plerixafor (0.24 mg/kg) was administered subcutaneously for up to three consecutive days, while continuing G-CSF, 10–11 h before the planned leukapheresis. Plerixafor administration was safe and no significant adverse events were recorded. We observed a 4.7 median fold-increase in the number of circulating CD34+ cells after plerixafor as compared with baseline CD34+ cell concentration (from a median of 6.2 (range 1–12) to 21.5 (range 9–88) cells/μL). All patients collected >2 × 106 CD34+ cells/kg in 1–3 leukaphereses. In all, 5/13 patients have already undergone autograft with plerixafor-mobilized PBSCs, showing a rapid and durable hematological recovery. Our results suggest that the pre-emptive addition of plerixafor to G-CSF after chemotherapy is safe and may allow the rescue of lymphoma and MM patients, who need autologous transplantation but are failing PBSC mobilization.

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.

Safety of autologous hematopoietic stem cell transplantation in patients with multiple myeloma and chronic renal failure

Patients with multiple myeloma (MM) and chronic renal failure have generally been excluded from myeloablative therapy programs followed by hematopoietic stem cell support because of the potential increase in transplant-related morbidity and mortality. We here report our experience treating six MM patients with moderate to severe renal insufficiency, with autologous stem cell transplantation. One of these patients required chronic hemodialysis since the diagnosis of MM was made. Peripheral blood stem cell collection was performed with either cyclophosphamide 5.5–7 g/m2 + G-CSF, 5 μg/kg/day (patients 1–3, 5 and 6) or G-CSF, 15 μg/kg/day alone (patient No. 4). Four patients (Nos 1–4) received autotransplant as front-line therapy, while the last two patients were treated in relapse, which occurred following prior autologous stem cell transplantation in support of melphalan, 200 mg/m2 (No. 5) or maintainance therapy with alpha-interferon (No. 6). High-dose chemotherapy administered as preparation to transplant included busulfan 12 mg/kg + melphalan 80 mg/m2 (patients 1–3 and 6) or melphalan 80 mg/m2 alone (patients 4 and 5) in order to reduce mucosal damage. Following transplant, prompt and sustained recovery of hematopoiesis was documented in all the patients; 500 PMN/μl and 20000 platelets/μl were reached after a median of 13 and 14 days, respectively. None of the patients suffered from WHO grade 3–4 infectious complications. Transplant-related toxicity included grade 3–4 oral mucositis (patients 1, 4 and 5) and veno-occlusive disease (patient No. 3). Renal function either improved or remained stable throughout the transplant period. All the patients but one responded to therapy, three of them are progression free after 2, 15 and 26 months; two relapsed after 16 and 4 months and one died from cholangiocarcinoma 7 months after transplant, while still in remission. Although our experience is limited so far, these results appear promising and support the investigational use of myeloablative therapy in MM patients with chronic renal failure.

High-dose busulfan and cyclophosphamide are an effective conditioning regimen for allogeneic bone marrow transplantation in chemosensitive multiple myeloma

The present clinical trial was undertaken to investigate the toxicity and antimyeloma activity of busulfan (BU) and cyclophosphamide (CY) at the maximum tolerated doses of, respectively, 16 mg/kg and 200 mg/kg (BU-CY 4) as conditioning therapy for allogeneic bone marrow transplantation (BMT) in 19 consecutive patients with multiple myeloma (MM). Twelve (63%) had failed to respond to prior chemotherapy, while the remaining 37% had chemosensitive disease. No life-threatening or fatal regimen-related complications were observed. The incidence of veno-occlusive disease of the liver was zero according to Jones’ criteria and 21% according to McDonald’s system. Transplant-related mortality was 37%. Using stringent criteria, the frequency of complete remission (CR) was 42% among all patients and 53% among those who could be evaluated. With a median follow-up of 21 months for all patients and 66 months for survivors, the actuarial probability of survival and event-free survival at 4 years from BMT was 26% (95% CI: 7–46) and 21% (95% CI: 3–39), respectively. A more favorable outcome of transplantation was observed in the subgroup of patients with chemosensitive disease who had a transplant-related mortality of 14%, an overall CR rate of 86% (95% CI: 49–97) and a 4-year projected probability of event-free survival of 57% (95% CI: 20–93). Four of these patients are currently alive in continuous CR after 54, 66, 80 and 94 months, respectively. It is concluded that BU-CY 4 as conditioning for allogeneic transplantation for MM is associated with acceptable morbidity and relatively low mortality. This regimen exerts substantial antimyeloma activity, resulting in a high CR rate and durable responses, especially in patients with chemosensitive disease. Long-lasting remission and probable cure is possible following allogeneic stem cell transplantation for MM.

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.

Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti‐idiotype vaccination

Summary. Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti‐idiotype vaccination were labelled with clinical‐grade anti‐CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal‐calf‐serum‐free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte–macrophage colony stimulating factor plus interleukin 4 (IL‐4), followed by either tumour necrosis factor‐α (TNF‐α) or a cocktail of IL‐1β, IL‐6, TNF‐α and prostaglandin‐E2. The CD14+ cell yield was increased from 17·6 ± 6·5% to 93·8 ± 6·3% (recovery 64·4 ± 15·4%, viability > 97%). After cell culture, phenotypic analysis showed that 86·7 ± 6·8% of the cells were DC: 2·27 ± 0·9 × 108 DC/leukapheresis were obtained, which represented 20·7 ± 4·6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28·6 ± 3% of initial CD14+ cells) of DC than TNF‐α alone, with secretion of larger amounts of IL‐12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 ± 10%. Finally, interferon‐α‐2b was at least as efficient as IL‐4 in inducing the differentiation of mature, functional DC from monocytes.

Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients

Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53–73) received NK cells from haploidentical KIR-ligand–mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6–68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3–51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03). Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells. Clin Cancer Res; 22(8); 1914–21. ©2016 AACR. See related commentary by Muntasell and López-Botet, p. 1831

Short- and long-term haematological surveillance of healthy donors of allogeneic peripheral haematopoietic progenitors mobilized with G-CSF: a single institution prospective study

Summary:Healthy allogeneic donors, who were treated with G-CSF and underwent peripheral blood haematopoietic precursor collection at our Institution, were enrolled in a short- and long-term haematological surveillance protocol for a 5–7-year period. To date, 94 donors have been assessed with a mean follow-up of 30 months (4–84); for 30 subjects, the follow-up is ⩾48 months. During G-CSF administration, 23/94 donors showed a significant platelet count decrease from the baseline. Pre-apheresis platelet decrement correlated with the total G-CSF dose administered, baseline platelet level and donor age. Normal platelet counts returned within 4–8 months. PMN and/or lymphocyte lower values were observed in 55/94 donors 2 weeks after G-CSF administration, with mean drops from the baseline of 40 and 36% for PMN and lymphocytes, respectively. The PMN decrease correlated inversely with donor age, as younger donors were more affected than older ones, whereas the lymphocyte decrease correlated directly with the total blood volumes processed in the apheresis courses, in particular for donors subjected to large volume leukaphereses. Long-term observation showed moderate neutrophil reduction (25% count drop from the baseline) in four of the 30 donors observed for four years or more. 14 donors showed persistent, slight lymphocytopenia (mean drop of 13%) until the third year, with recovery in the fourth year of follow-up.

An immunotoxin containing momordin suitable for bone marrow purging in multiple myeloma patients.

Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.

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.