A comparative assessment of the curative potential of reduced intensity allografts in acute myeloid leukaemia.

Allogeneic stem cell transplantation (SCT) provides the best mechanism of preventing relapse in acute myeloid leukaemia (AML). However non-relapse mortality (NRM) negates this benefit in older patients. Reduced intensity conditioning (RIC) permits SCT with reduced NRM, but its contribution to cure is uncertain. In the MRC AML15 Trial, patients in remission without favourable risk disease could receive SCT from a matched sibling or unrelated donor (MUD). If aged >45 years, a RIC was recommended and in patients aged 35-44 years, either RIC or myeloablative conditioning was permitted. The aim was to determine which approach improved survival and within which prespecified cytogenetic groups. RIC transplants significantly reduced relapse (adjusted hazard ratio (HR) 0.66 (0.50-0.85), P=0.002) compared to chemotherapy The 5-year overall survival from a sibling RIC (61%) was superior to a MUD RIC (37%; adjusted HR 1.50 (1.01-2.21), P=0.04) due to lower NRM (34 vs 14%, P=0.002) In adjusted analyses, there was a survival benefit for sibling RIC over chemotherapy (59 vs 49%, HR 0.75 (0.57-0.97), P=0.03), with consistent results in intermediate and adverse-risk patients. In patients aged 35-44 years, best outcomes were seen with a sibling RIC transplant, although a comparison with chemotherapy and myeloablative transplant was not significant in adjusted analyses (P=0.3).

EBV-associated post-transplant lymphoproliferative disorder following in vivo T-cell-depleted allogeneic transplantation: clinical features, viral load correlates and prognostic factors in the rituximab era.

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49 300 copies/mL (50-65 200 000 copies/mL) and, notably, 23% and 45% of cases, respectively, had 10 000 and 40 000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12-0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12-2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

Inclusion of chemotherapy in addition to anthracycline in the treatment of acute promyelocytic leukaemia does not improve outcomes: results of the MRC AML15 trial.

Two hundred eighty-five patients, median age 42, with PML-RARα-positive acute promyelocytic leukaemia were randomised to Ara-C-containing 'Medical Research Council (MRC) Chemotherapy'+ATRA (All-trans-retinoic acid) or anthracycline+ATRA (modified 'Spanish') therapy. MRC treatment comprised four courses with ATRA in courses 1-2. Spanish treatment comprised four anthracycline-based courses with ATRA in courses 1-3. In course 3 patients were randomised to gemtuzumab ozogamicin (GO) or not. The Spanish arm received 24-month maintenance. Patients were sequentially molecularly monitored. Quality of life was assessed at baseline, 3, 6, 9, 12, 24 months. Remission rates were similar in both arms (93%): cumulative incidence of haematological relapse (CIHR) was 6% at 5 years; 5 patients relapsed molecularly. Survival post relapse was 80%. There were more deaths in remission in the MRC arm (4% vs 10%: P=0.2). The overall 5-year relapse-free and overall survival was similar between arms (81% vs 82% and 84% vs 83%, respectively). More supportive care and hospitalisation (81.8 vs 63 days, P<0.0001) was required in the MRC arm. GO did not provide benefit. High white blood cell count (>10 × 10(9)/l) was not prognostic overall, or within treatment arms. Both approaches deliver similar results with minor differences in quality of life. MRC treatment required more hospitalisation. This suggests that additional chemotherapy, Ara-C in particular, is not required.

Comparison of gene-expression profiles in parallel bone marrow and peripheral blood samples in acute myeloid leukaemia by real-time polymerase chain reaction.

BACKGROUND: Gene signatures (Indicator genes) in bone marrow that provide more precise prognostication in haematological malignancy have been identified by microarray expression studies. It would be beneficial to measure these diagnostic signatures in peripheral blood. AIMS: To determine the degree of correspondence of gene expression for a set of Indicator genes between bone marrow and peripheral blood in acute myeloid leukaemia (AML). METHODS: Parallel bone marrow aspirate and peripheral blood samples were obtained from 19 patients diagnosed with AML and mononuclear cells isolated from both sample types. mRNA was globally amplified by polyadenylated real-time polymerase chain reaction (polyA RT-PCR); the expression of 15 AML Indicator genes, identified from previous microarray studies, was measured by RT-PCR. All values were normalised to the mean expression of three housekeeping genes (IF2-beta, GAP and RbS9) and were statistically compared using SPSS software. RESULTS: No significant difference in expression between bone marrow and peripheral blood was observed for 10 of the genes (leptin receptor, CD33, adipsin, proteoglycan 1, MB-1, cyclin D3, hSNF2b, proteasome iota, HkrT-1 and E2A), indicating its possible use in monitoring disease activity in peripheral blood samples, whereas c-myb, HOXA9, LYN, cystatin c and LTC4s showed significantly different expression between bone marrow and peripheral blood samples. CONCLUSION: These results indicate a possible use for the method in monitoring AML in peripheral blood by RT-PCR measurement of Indicator genes. In addition, the initial use of polyA PCR facilitates translation to very small clinical samples, including fractionated cell populations, of particular importance for monitoring haematological malignancy.

Elevated levels of WT1 transcripts in bone marrow harvests are associated with a high relapse risk in patients autografted for acute myeloid leukaemia.

Relapse postautograft in acute myeloid leukaemia (AML), may in part arise from leukaemia cells present in the bone marrow (BM) inoculum, and the level of minimal residual disease (MRD) in BM harvests used for autografting may therefore be clinically important. We have used the WT1 transcript as a marker of MRD, which was quantitated by RQ-PCR, in the BM harvests of 24 patients receiving an ABMT for AML. ABL was used as a control gene with WT1 level being normalised to 10(5) copies of ABL per sample. Median WT1 level was 651 copies (range=113-32 700) for the 13 patients with relapse-free survival (RFS) of less than 5 years, and 174 (range=0-1900) for patients with RFS of over 5 years postautograft (P<0.04). The RFS was 10.5 months for patients with WT1 level of >2000 copies (n=5), and has not yet been reached for patients with WT1 level<2000 (n=21), at a median follow-up of 92 months (P<0.05). We show that elevated levels of MRD in BM harvests are associated with a higher relapse risk in patients autografted for AML.

Prior and concurrent administration of recombinant human megakaryocyte growth and development factor in patients receiving consolidation chemotherapy for de novo acute myeloid leukemia--a randomized, placebo-controlled, double-blind safety and efficacy study.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) administered after acute myeloid leukemia (AML) chemotherapy (CT) failed to shorten the time of transfusion-dependent thrombocytopenia in a previous study. In this multicenter, randomized, placebo-controlled, double-blind study we determined the effect of administration of PEG-rHuMGDF prior to CT and of administration prior, concurrent, and 1 day post CT on platelet recovery and transfusion requirements in patients receiving consolidation CT for de novo AML. Patients were randomized to receive either 30 microk/kg PEG-rHuMGDF as a single dose on day -6 ( n=37), placebo as a single dose on day -6 ( n=9), 30 microk/kg PEG-rHuMGDF administered on day -6 followed by 10 microg/kg on days -5 to day 6 (through CT and including the day after CT, n=35), or placebo administered on day -6 to day 6 ( n=9). The median times to transfusion-independent platelet recovery to >20x10(9)/l were 24.5 and 24.0 days in the PEG-rHuMGDF day -6 group and PEG-rHuMGDF day -6 to 6, respectively, compared to 21.0 days in the placebo group. There were no significant differences in the number of days of platelet transfusions between either PEG-rHuMGDF schedule or placebo. The PEG-rHuMGDF day -6 to 6 group had a delayed absolute neutrophil count (ANC) recovery compared to either placebo or PEG-rHuMGDF day -6 treated patients. Thus, alteration of the scheduling of PEG-rHuMGDF in terms of earlier dosing before and during chemotherapy did not improve platelet recovery but rather delayed hematopoietic reconstitution. Although unexpected, these observations may be of major relevance for the design of future clinical trials with recombinant thrombopoietins.

Minimal residual disease in acute myeloid leukaemia.

Relapse remains the main cause of treatment failure in acute myeloid leukaemia (AML). Studies to date suggest that monitoring of minimal residual disease (MRD) in AML is useful in identifying patients at high risk of relapse from those in durable remission. This chapter describes the methodological advances in the detection of MRD and, in particular, focuses on the development of highly sensitive RT-PCR techniques, including real-time, for quantifying MRD. Preliminary results on the clinical utility of MRD monitoring in AML with t(8;21) and inv(16) are promising and provide the basis for further evaluation by quantitative real-time analysis in prospective clinical trials. For AML without a specific fusion transcript, the WT1 gene is an alternative molecular target. The clinical value of quantitative MRD monitoring in AML, however, will need to be confirmed in future studies.

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia.

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFU-Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL-3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant.

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Dual colour FISH in paraffin wax embedded bone trephines for identification of numerical and structural chromosomal abnormalities in acute myeloid leukaemia and myelodysplasia.

AIMS/BACKGROUND: The advent of new treatments for haematological malignancies has led to the need for a correlation between cytogenetic and morphological abnormalities. This study aimed to achieve this by the application of interphase cytogenetics to marrow trephine sections, a technique not previously reported for formalin fixed, paraffin wax embedded trephine biopsies. METHODS: Dual colour fluorescence in situ hybridisation (FISH) was used to detect numerical and structural abnormalities in routinely processed paraffin wax embedded trephine biopsies. Three cases with t(8;21) and three with t(15;17) were analysed, together with a case of trisomy 8. Chromosome specific probes were hybridised with sections and disclosed by fluorescein isothiocyanate and rhodamine/Texas red labelled antidigoxigenin and antibiotin amplification; translocations were identified by colocalisation of probes using a double wavelength bypass filter. RESULTS: A translocation signal was present in 12% and 11.5% of the cells counted in the t(8;21) and t(15;17) cases, respectively, but in none of the normal controls (p < 0.001). In the case of trisomy 8, 9% of the cells counted contained three hybridisation signals for chromosome 8, whereas no cell contained more than two in the normal control (p < 0.001). CONCLUSIONS: This technique is useful for archived routinely processed material, enabling it to be used as a research tool but also, and perhaps more importantly, in clinical practice.

Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RT-PCR method.

Qualitative RT-PCR methods used for monitoring minimal residual disease (MRD) in APL patients fail to predict relapse in up to 25% of patients in remission. We report here the development and evaluation of a highly sensitive (10(-5) and 10(-6) with one round and two rounds of PCR, respectively) competitive RT-PCR method to quantitate the PML-RARalpha fusion transcripts. PML-RARalpha transcript's levels were normalised to 10(5) copies of ABL transcript. Serial BM and PB samples from 16 patients with APL and t(15;17) were examined. Presentation samples from three patients (three BM, one PB) showed levels in the range of 0.7 x 10(6)-3.5 x 10(6) and 1.2 x 10(5) molecules in BM and PB samples respectively. Serial quantitation of MRD in both BM and PB samples showed significantly lower levels of PML-RARalpha transcripts in remission, although the majority of samples remain positive for the PML-RARalpha transcripts even those in long-term remission (up to 94 months). Levels of PML-RARalpha in remission samples were up to 2 x 10(2) and up to 5.2 x 10(1) molecules in BM and PB respectively. BM and PB samples taken from two patients 2-4 months before relapse showed significantly higher levels of PML-RARalpha transcripts (1.2 x 10(4) molecules in BM; 3.5 x 102, 1.2 x 10(2) and 1.2 x 10(3) in PB). The same samples, when tested with a standard qualitative RT-PCR for the amplification of PML-RARalpha (with a sensitivity of 10(-4)) produced negative results. This indicates that the qualitative methods would not have predicted relapse in these patients. Our data show that quantitating PML-RARalpha transcripts with a sensitive method may provide a superior approach for monitoring MRD in APL and identifying patients at high risk of relapse.

Comparison of 'sequential' versus 'standard' chemotherapy as re-induction treatment, with or without cyclosporine, in refractory/relapsed acute myeloid leukaemia (AML): results of the UK Medical Research Council AML-R trial.

This aim of the acute myeloid leukaemia (AML)-R trial was to compare sequential (Seq) ADE (cytarabine, daunorubicin, etoposide) with standard (Std) ADE as remission re-induction treatment and to assess any benefit of cyclosporine (CSA) as a multidrug resistance modulator in refractory/relapsed AML patients. Seq ADE, based on the concept of Timed Sequential Therapy, comprised the same drugs as Std ADE but given at higher doses and in a different sequence. Between 1992 and 1997, 235 patients with relapsed (175) and refractory (60) AML were entered: 170 were randomized between Std versus Seq ADE and 213 between CSA versus no CSA. CSA was initially given at a dose of 5 mg/kg/d and increased to 10 mg/kg/d in the latter part of the trial. Overall, the complete remission (CR) rate was 43%, with Std ADE being significantly better than Seq ADE (54% versus 34%, P = 0.01). CR rates did not differ between the CSA and no CSA arms (41% versus 45%, P = 0.6). Overall, 3 year disease-free survival (DFS) of remitters was 16%, with a relapse risk of 70%. DFS was not significantly different between the chemotherapy or the CSA arms. Overall, 3 year survival was 8%. Survival with Std ADE was significantly better than with Seq ADE (12% versus 6%, P = 0.03). CSA did not affect overall survival, except in patients > or = 60 years, who fared worse on CSA (P = 0.0003). No difference in haematological toxicity between the chemotherapy or CSA arms was seen. Survival was better with longer first CR duration (P < 0.0001). We conclude that Std ADE was superior to Seq ADE for CR achievement and survival, with no benefit with CSA, at the doses used in this study.

Stimulation of autologous proliferative and cytotoxic T-cell responses by "leukemic dendritic cells" derived from blast cells in acute myeloid leukemia.

Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

Achieving pregnancy against the odds: successful implantation of frozen-thawed embryos generated by ICSI using spermatozoa banked prior to chemo/radiotherapy for Hodgkin's disease and acute leukaemia.

Two cases are reported of successful pregnancies following long-term semen banking prior to chemotherapy and radiotherapy for malignancy. With the first case, the patient banked semen at the age of 20 years prior to chemotherapy for Hodgkin's disease; 11 years later the thawed semen was used for IVF with intracytoplasmic sperm injection (ICSI), resulting in twins being born following the transfer of frozen-thawed embryos. In the second case, the patient banked semen at the age of 17 years prior to chemotherapy and radiotherapy for acute myeloid leukaemia; 8 years later it was used for ICSI, resulting in triplets being born following the transfer of frozen-thawed embryos. These cases support long-term semen banking for men whose future fertility may be compromised by suppression of spermatogenesis secondary to administration of chemo/radiotherapy treatment. The advent of successful ICSI combined with embryo cryopreservation has increased the chance of thawed cryopreserved semen achieving fertilization. Banking of a single ejaculate prior to commencement of chemotherapy/radiotherapy treatment may preserve potential fertility without compromising the oncology treatment.

Allogeneic stem-cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH (+/- fludarabine) conditioning combined with post-transplant donor-lymphocyte infusion.

BACKGROUND: We report our updated experience of allogeneic transplantation in lympho-proliferative disorders using a reduced-intensity conditioning regimen combining BEAM (plus fludarabine in three cases) with pre-transplant CAMPATH. Post-transplant donor lymphocytes have been infused for persisting disease or relapse, and both chimerism and minimal residual disease have been monitored utilizing molecular techniques. METHODS: Thirty patients with median age 47.6 years underwent allogeneic transplantation for relapsed or high-risk lymphoproliferative disease using HLA-identical (sibling n = 25, unrelated n = 2) or one antigen mismatched sibling donors (n = 3). Twenty-one had NHL, three had HD and six had CLL/PLL. Stem-cell source was PBSC (n = 24), BM (n = 5) or both (n = 1) with a median CD34 dose of 4.5 x 10(6)/kg. GvHD prophylaxis was with CYA and MTX. RESULTS: Engraftment was prompt in the majority of patients, with a median of 15 days to both ANC > 0.5 and platelets > 20. There have been three transplant-related deaths secondary to viral pneumonitis or bacterial pneumonia. Seven patients developed Grade I-II acute GvHD post-transplant. Of 28 evaluable patients, 18 achieved a CR at assessment 2-3 months post-transplant and a further patient converted from PR to CR following DLI, to give an overall CR rate of 68%. Three patients had early progressive disease and six have relapsed from CR or progressed from PR (two of whom have achieved CR following DLI therapy). Overall survival is 67% and event-free survival 48% at 3 years. With a median follow-up of 1.3 years 57% of patients are currently alive and lymphoma-free. A molecular remission has been achieved in nine of 12 informative patients. DISCUSSION: These encouraging results show that this reduced-intensity conditioning regimen is effective, with a low-toxicity profile compared with conventional TBI-based conditioning, and certainly merits further evaluation in this setting.

Megakaryopoiesis in vitro in myelodysplastic syndromes and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.