N. H. Russell

The effect of the serotherapy regimen used and the marrow cell dose received on rejection, graft-versus-host disease and outcome following unrelated donor bone marrow transplantation for leukaemia.

unrelated donor (ud) transplantation is the only potentially curative therapy for many leukaemia patients but is associated with a high mortality and morbidity. we sought to identify factors that could be optimised to improve outcome following ud transplantation in adults. data was retrospectively analysed on 55 patients sequentially receiving ud transplants for cml or acute leukaemia (al), all of whom received serotherapy for the prevention of gvhd and rejection. all patients received standard conditioning regimens. the first 28 patients transplanted also received combined pre- and post-transplant serotherapy with campath 1g (days −5 to +5) and standard dose csa plus mtx as gvhd prophylaxis (protocol 1). the subsequent 27 patients received a 5-day course of pre-transplant serotherapy alone either with atg (cml patients) or campath 1g (al patients) on days −5 to −1 inclusive, with high-dose csa plus mtx (protocol 2). the incidence of acute gvhd was low with no patient receiving either protocol developing >grade 2 disease. The use of protocol 2 and the administration of a bone marrow cell dose above the median (2.17 × 108/kg) were the most important factors predicting engraftment (P = 0.03 and P = 0.001, respectively) but this only remained significant for cell dose in multivariate analysis (P = 0.03). Overall survival for the group was 45% at 3 years and was influenced by both age (P = 0.02) and disease status at transplantation (P = 0.001). Receiving a cell dose above the median was also associated with a trend towards better survival (P = 0.08), due primarily to a reduction in the TRM to 8.2% compared with 54.5% in those receiving a lower cell dose (P = 0.002). We conclude that pre-transplant serotherapy alone is highly effective at preventing acute GVHD following UD BMT and that additional post-transplant serotherapy does not confer any benefit. Furthermore, a high marrow cell dose infused has a major effect in reducing transplant-related mortality following UD BMT. Bone Marrow Transplantation (2000) 25, 411–417.

A study to determine the safety profile and maximum tolerated dose of micafungin (FK463) in patients undergoing haematopoietic stem cell transplantation

This open-label, dose-escalation study assessed the maximum tolerated dose (MTD) of the new antifungal micafungin in patients undergoing haematopoietic stem cell transplantation (HSCT). Participants received 3, 4, 6 or 8 mg/kg/day micafungin intravenously from 7 days to a maximum of 28 days or until neutropaenia resolved. The MTD was defined as the highest dose not causing the same Grade 3 or 4 adverse event in three or more patients. All 36 participants received ⩾8 days treatment for a median of 18 days (range: 8–28); 1 patient withdrew consent and a further 11 discontinued to receive another systemic antifungal agent for a suspected infection. No case of confirmed invasive fungal infection occurred. Adverse events were those expected for patients undergoing HSCT and showed no evidence of dose-related toxicity. Criteria for MTD were not met; no patient had a Grade 3 or 4 adverse event considered causally related to micafungin. Thus, the MTD of micafungin can be inferred to be 8 mg/kg/day or higher.

Stem cell mobilisation in lymphoproliferative diseases

A number of different regimens have evolved for the mobilisation of peripheral blood stem cells for autologous transplantation in patients with lymphoma or myeloma. A successful regimen could be defined as one which consistently resulted in the collection of an optimal number of CD34+ cells with a minimum number of apheresis procedures with minimal toxicity. Initial protocols, which used chemotherapy alone as a mobilising agent, have now been replaced by regimens involving the use of haematopoietic growth factors either alone or in combination with variable doses of cyclophosphamide. Although there is good evidence that high-dose cyclophosphamide (6–7 g/m2) is an effective mobilising agent it is associated with significant toxicity and many groups have now utilised lower doses of cyclophosphamide with reduced toxicity which have still proven to be effective in the majority of patients. More recently a number of ‘second generation’ combined salvage chemotherapy and mobilisation regimens have been reported for use in the lymphomas which have the advantage of avoiding a specific stem cell mobilisation step and at the same time appear more consistently effective at mobilising stem cells than cyclophosphamide and G-CSF. These regimens are associated with fewer ‘poor-mobilisers’ and indeed some patients who have failed previous mobilisation with cyclophosphamide and G-CSF have been successfully re-mobilised. It is clear that in both lymphoma and myeloma patients the success of PBSC mobilisation is affected by the amount and type of previous chemotherapy and radiotherapy and probably other pre-treatment factors as exemplified by variability seen in normal donors mobilised with G-CSF alone. In myeloma most groups have utilised cyclophosphamide in variable doses in combination with G-CSF or GM-CSF. However, recent randomised studies have confirmed that G-CSF alone is an effective and non-toxic alternative although it appears that the efficacy of G-CSF as a single agent is related to the dosage used with daily doses of 16 μg/kg/day or greater being most effective. Thus, disease-specific mobilisation strategies appear to be emerging and these will undoubtedly be modified further as more is understood concerning the biology of blood stem cell mobilisation.

Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia

We have used a combination of flow cytometric assays to define multidrug resistance (MDR) positive and negative blasts in cryopreserved samples from 47 MRC trial patients with acute myeloblastic leukaemia (AML). Our primary test is a standardized assay for daunorubicin accumulation. Confirmatory assays for MDR comprised the cyclosporin modulation assay for rhodamine‐123 uptake as a measure of functional P‐glycoprotein and the measurement of lung resistance protein and multidrug resistance associated protein (with LRP‐56 and MRPr1 respectively).

Matched unrelated donor stem cell transplant in 131 patients with follicular lymphoma: an analysis from the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation.

Matched unrelated donor stem cell transplantation (MUD‐SCT) provides the only curative option for patients with follicular lymphoma (FL) who fail conventional therapies and do not have a sibling donor. The purpose of this study was to analyse the outcome of patients with FL treated with MUD‐SCT included in the European Group for Blood and Marrow Transplantation registry. 131 patients treated with reduced‐intensity conditioning (RIC, n = 87) or conventional myeloablative (CONV, n = 44) MUD‐SCT between 2000 and 2005 were included. Median time from diagnosis to MUD‐SCT was 47 months and the median number of previous therapeutic regimens was 4 (previous autograft: 47%). RIC recipients were significantly older, with a longer interval from diagnosis to MUD‐SCT and had failed a previous autograft more frequently than CONV recipients. Non‐relapse mortality (NRM) was 24% and 30% at 100‐d and 1‐year, respectively. After a median follow‐up of 36 months, 17% of the patients developed disease progression, the 3‐year progression‐free survival (PFS) being 47%. Three‐year overall survival (OS) for the whole series was 51%. On multivariate analysis, RIC regimens were associated with at lower NRM and a significantly longer PFS and OS. This retrospective study demonstrated that MUD‐SCT results, even in heavily pre‐treated populations, in a meaningful PFS and OS.

Adjuvant α-interferon improves complete remission rates following allogeneic transplantation for multiple myeloma

Allogeneic transplantation may be curative in a proportion of patients with multiple myeloma (MM), but relapse is a major cause of treatment failure. We sought to improve complete remission (CR) rates by the use of α-interferon (α-IFN) in patients not in CR when evaluated 4 months post-transplant. We report five of 13 evaluable patients undergoing allogeneic sibling BM or PBSC transplantation for MM between 1990 and 1997 who met the criteria for adjuvant α-IFN therapy. A starting dose of 3 MU × 3/week was commenced at median time of day +126 (range day +112–224) post-transplant and was well-tolerated. In contrast to other reports we observed no increased toxicity in terms of GVHD compared to those patients not receiving α-IFN therapy and only one patient treated with α-IFN has developed chronic GVHD. Durable CRs were achieved in two patients within 8 weeks of starting therapy whilst two other patients required a longer course of α-IFN to achieve CR (36 weeks and 30 weeks, respectively). One patient whose paraprotein was rapidly rising at the time of α-IFN therapy clinically relapsed despite 6 months of treatment. None of the patients who achieved CR following -IFN therapy have relapsed and we conclude that α-IFN is a safe and effective adjuvant treatment for some patients in the achievement of CR following allogeneic transplantation for myeloma.

Mobilisation of peripheral blood stem cells with IVE and G-CSF improves CD34+ cell yields and engraftment in patients with non-Hodgkin's lymphomas and Hodgkin's disease.

The transplantation of mobilised peripheral blood stem cells is associated with more rapid engraftment than marrow transplantation. We have previously reported that G-IVE (G-CSF, ifosphamide, VP-16, epirubicin) improves the yield of CD34+ cells mobilised in patients with lymphoproliferative disorders compared with cyclophosphamide 3 g/m2 and G-CSF (G/CYCLO). In this study we have extended these observations to a larger series of patients including different lymphoma subtypes. Ninety-seven patients undergoing stem cell mobilisation were studied. Forty-two patients with lymphoproliferative disorders received G-IVE for mobilisation and 55 patients G/CYCLO. The median number of mobilised CD34+cells per leucapheresis was significantly higher for those patients receiving G-IVE: 5.82 × 106/kg (0.19–36) compared with 1.2 × 106/kg (0.04–17), P < 0.001 which resulted in a significantly reduced number of leucapheresis procedures performed in the g-ive group. when patients were analysed dependent on underlying disease g-ive mobilised significantly more cd34+cells per leucapheresis for all lymphoma types reaching 8.41 × 106/kg (0.2–32) compared to 1.32 × 106/kg (0.06–17) for patients with high-grade NHL mobilised with G-IVE and C-GCSF respectively (P = 0.012). For patients with low-grade NHL 3.12 × 106/kg (0.10–24.39) compared to 1.08 × 106/kg (0.04–9.74) were collected (P = 0.04) and for patients with Hodgkin’s disease 3.02 × 106/kg (1.48–36) and 1.04 × 106/kg (0.1–12.3) (P = 0.001). Mobilisation with G-IVE resulted in the achievement of clinically significant CD34+ cell thresholds in a significantly higher proportion of patients compared to cyclophosphamide and G-CSF reaching >2.5 × 106/kg CD34+ cells in 88% vs 62% (P = 0.004), >5 x 106/kg in 67% vs18% (P = 0.001) and >10 × 106/kg in 31% vs 14.5% (P = 0.05). Furthermore, an analysis of engraftment demonstrated that there was a significant reduction in the time to achieve platelet counts of >20 and >50 × 109/l in patients receiving each incremental dose of CD34+ cells. We conclude that G-IVE mobilises significantly more CD34+cells than G/CYCLO in patients with lymphoproliferative disorders. This effect is consistent in patients with high-grade NHL, low-grade NHL and HD and results in fewer failed stem collections and increased CD34+ cells available for transplantation which results in significantly accelerated platelet engraftment post transplant.

A novel BCR-ABL fusion gene (e2/1a) in a patient with Philadelphia-positive chronic myelogenous leukaemia and an aggressive clinical course

A novel variant BCR‐ABL mRNA transcript was detected by reverse transcription polymerase chain reaction (RT‐PCR) in a patient with Philadelphia positive (Ph+ve) chronic myelogenous leukaemia (CML) who had an aggressive clinical course. Sequence analysis of the amplified cDNA fragment revealed an in‐frame fusion with part of BCR exon e2 joined to part of ABL exon 1a. PCR of genomic DNA from this patient demonstrated that this unusual chimaeric mRNA resulted from breakpoints that fell within each of these exons. This is the first report of breakpoints occurring within both ABL and BCR exons and the first fusion to involve ABL exon 1a.

Low incidence of acute graft-versus-host disease and recurrent leukaemia in patients undergoing allogeneic haemopoietic stem cell transplantation from sibling donors with methotrexate and dose-monitored cyclosporin A prophylaxis

One of the major aims of allogeneic haemopoietic stem cell transplantation has been the effective suppression of graft-versus-host disease (GVHD) without loss of a graft-versus-leukaemia effect. For GVHD suppression, one of the most frequently used regimens has been the combination of cyclosporin (CsA) and a short course of methotrexate (MTX) although the optimal usage of these agents remains unclear. Here, we report the results of 55 patients with standard risk leukaemia who have undergone allogeneic transplantation using either bone marrow (n = 48) or G-CSF mobilised peripheral blood stem cells (n = 7) using CsA and MTX for GVHD prophylaxis where the dosage of CsA was regularly adjusted to maintain a trough whole blood level of 95–205 ng/ml for the first 50 days post-transplant. To achieve this level of CsA in the immediate post-transplant period, over 40% of patients required dose adjustments of CsA as a result of sub-therapeutic levels on day +1 post-transplant. The achievement of CsA levels within the therapeutic range was expedited following the introduction of a sliding scale for dose adjustment. With this regimen we have observed a low incidence of acute GVHD with only 11% of patients developing ⩾grade II disease. With a median follow-up of 66 months (range 8–132) the probability of relapse is only 6.6%. The disease-free survival probability for all patients was 72% at 5 years. These results demonstrate that effective GVHD prevention with CsA and MTX can be achieved without a high risk of recurrent leukaemia provided that rapid attainment of therapeutic CsA levels is achieved and maintenance within a low therapeutic range may help to maximise this effect.

Contrasting in vitro effects for the combination of fludarabine, cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (FLAG) compared with daunorubicin and Ara-C in P-glycoprotein-positive and P-glycoprotein-negative acute myeloblastic leukaemia

It has been suggested that the FLAG remission induction regimen comprising fludarabine (F‐ara), cytosine arabinoside (Ara‐C) and granulocyte colony‐stimulating factor (G‐CSF) may be capable of overcoming P‐glycoprotein (P‐gp)‐related multidrug resistance (MDR) in patients with acute myeloblastic leukaemia (AML). We have investigated the in vitro response of P‐gp‐positive and ‐negative AML clones to FLAG and compared this with their response to treatment with Ara‐C and daunorubicin (DNR). Twenty‐four cryopreserved samples from patients with AML were studied using a flow cytometric technique for the enumeration of viable (7‐amino actinomycin D negative) cells. Samples consisted of 12 P‐gp‐positive and 12 P‐gp‐negative cases, as measured by the MRK16 antibody. The results were analysed by calculating the comparative drug resistance (CDR), i.e. the percentage cell death caused by Ara‐C + DNR subtracted from the percentage cell death, caused by FLAG after 48 h incubation in suspension culture. P‐gp‐positive clones were shown to have a significantly higher CDR than P‐gp‐negative clones (P = 0·001). Furthermore, a significant positive correlation (r2 = 0·40, P < 0·01) was found between P‐gp protein expression and CDR. However, P‐gp function, measured using cyclosporin modulation of rhodamine 123 (R123) uptake, was not associated with the CDR, demonstrating that there are other properties of P‐gp, besides its role in drug efflux, that modulate the responsiveness of AML blasts to chemotherapy. These results are consistent with a potential benefit for FLAG in P‐gp‐positive AML, but not P‐gp‐negative AML, compared with standard anthracycline and Ara‐C therapy.