The impact of total body irradiation on the outcome of patients with follicular lymphoma treated with autologous stem-cell transplantation in the modern era: a retrospective study of the EBMT Lymphoma Working Party.

BACKGROUND: The aim of this study was to investigate the impact of the high-dose regimen on the outcome of patients with follicular lymphoma (FL) having had autologous stem-cell transplantation (ASCT) in a recent time period. PATIENTS: Between 1995 and 2007, 2233 patients with FL had their first ASCT with either a total body irradiation (TBI)-containing regimen or carmustin, etoposide, cytarabine and melphalan (BEAM), of which 47% were autografted in first remission. RESULTS: After a median observation time of 73 months (interquartile range 30-107), 5- and 10-year non-relapse mortality (NRM) was similar (6% and 10% in both groups). No significant NRM differences became evident after multivariate adjustment for confounders. Secondary malignancies were observed in 9.7% and 7.9% of the patients after TBI and BEAM (P = 0.19), which were treatment-related myelodysplastic syndromes/acute myelogenous leukaemia (t-MDS/AML) in 3.4% and 2.8% (P = 0.57). The median time to t-MDS/AML was around 50 months in both groups. Because of a lower relapse incidence, TBI was associated with better event-free survival reaching statistical significance in the patients transplanted in first remission but not in those transplanted beyond first remission. CONCLUSIONS: In patients with FL who received TBI-based ASCT after 1995 increased NRM and t-MDS/AML risks did not emerge compared with BEAM while disease control was at least equivalent.

Long-term survival after treatment for Hodgkin's disease (1973-2002): improved survival with successive 10-year cohorts.

BACKGROUND: The Nottinghamshire Lymphoma Registry contains the details of all the patients diagnosed with lymphoma (since 1 January 1973) within a defined geographical area with a population of 1.1 million. It was therefore possible to study the outcome of treatment for Hodgkin's disease for three 10-year cohorts (1973-1982, 1983-1992 and 1993-2002).The aims of the study were to compare survival time among the three patient cohorts, to identify prognostic factors and to estimate relative survival. METHODS: A total of 745 patients diagnosed between 1973 and 2002 were analysed for survival. Survivorship was estimated by the Kaplan-Meier method and parametric survival models. An accelerated failure-time regression was used for multivariate analysis. RESULTS: Overall, patients were observed for 9.8 (0.3-34.82) years (median(range)), on average. One, five and fifteen-year disease-specific survival was found to be 87% (85-90%), 77% (74-80%) and 70% (67-74%), respectively. For those for diagnosed between 1973 and 1982, the 15-year survival was found to be 57%; for 1983-1992, it was 74% and for 1993-2002, it was 83% (P<0.001). The difference remained significant after adjusting for prognostic factors. The actuarial risk of developing a second malignancy at 20 years was for the 1973-1982 cohort, 12.4%, and for the 1983-1992 cohort, 18.8%. CONCLUSION: Treatment advances and effective management of toxicities of treatment over time, have resulted in a significantly longer survival for patients with Hodgkin's disease diagnosed within a defined population.

IVE (ifosfamide, epirubicin and etoposide) is a more effective stem cell mobilisation regimen than ICE (ifosphamide, carboplatin and etoposide) in the context of salvage therapy for lymphoma.

Two commonly used chemotherapy regimens for lymphoma salvage therapy were compared: ICE (ifosphamide, carboplatin and etoposide) +/- rituximab and IVE (ifosfamide, epirubicin and etoposide) +/- rituximab, for their efficacy in mobilising peripheral blood stem cells for autologous transplantation. Significant differences were observed between the cohorts in terms of number of patients mobilising the stipulated minimum >2 x 10(6) CD34+/kg (99.2% in IVE group versus 83% in ICE group: P = 0.0002) and also in terms of the number of patients achieving the predetermined target of >5 x 10(6) CD34+/kg, both in total and during the first apheresis procedure (72% in IVE versus 51% in ICE group and 49% in IVE versus 7% in ICE group: P = 0.02 and P < 0.0001 respectively). This analysis of two similar groups of patients treated within a single-centre appears to demonstrate that the IVE regimen is a more effective stem cell mobilisation regimen than ICE in the context of salvage therapy for Hodgkin and non-Hodgkin lymphoma, allowing more patients to achieve the target CD34+ cell collection and proceed to high-dose therapy and autologous stem cell transplantation.

Caspase-independent killing of Burkitt lymphoma cell lines by rituximab.

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in "classical" apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.

Donor lymphocyte infusions can result in sustained remissions in patients with residual or relapsed lymphoid malignancy following allogeneic haemopoietic stem cell transplantation.

We treated 17 patients with refractory (n = 7) or relapsed lymphoid malignancy (n = 10) following allogeneic HSCT with donor lymphocyte infusions (DLI). Patients with low-grade disease received DLI alone (n = 7) or following radiotherapy (n = 1). Patients with aggressive disease (n = 9) received prior chemotherapy. Nine out of 15 patients receiving DLI from sibling donors responded after one (n = 6), two (n = 2) and three (n = 1) infusions. Both MUD recipients achieved CR after two and three DLI. In all, 10/17 patients achieved CR including 3/4 patients with chronic lymphatic leukaemia (CLL), 4/4 with mantle cell lymphoma (MCL), 3/4 with follicular NHL but 0/5 with aggressive NHL/Richters. The median CD3 cell dose to achieve CR for siblings was 2 x 10(7)/kg. One patient with CLL had a second transplant following DLI-induced aplasia and is in CR at 14 months giving a final CR rate of 64%. Grade II-IV acute GVHD developed in 45% and chronic GVHD in 8/9 evaluable patients. Of the 11 patients finally achieving CR, one patient with MCL relapsed at 18 months post-DLI but all others remain in remission with a median follow-up of 40 months (range 12-64 months). Low-grade NHL and MCL have a high response rate and sustained remissions following DLI. Aggressive disease responds poorly however, despite pre-DLI chemotherapy.

A randomised multicentre trial of modified CHOP versus MCOP in patients aged 65 years and over with aggressive non-Hodgkin's lymphoma.

BACKGROUND: The aim of this study was to determine in a randomised trial whether there is any significant difference in toxicity between modified CHOP and MCOP chemotherapy in elderly patients with aggressive non-Hodgkin's lymphoma (NHL) and to determine whether this reduced dose chemotherapy can be administered with full dose intensity, low toxicity and produce acceptable survival. PATIENTS AND METHODS: Between 1993 and 2000, 155 eligible patients were randomised into this trial mainly from three centres (Nottingham, Birmingham and Leeds, UK). The patients were newly diagnosed with aggressive NHL and had a median age of 74 years (range 65-91 years). Ninety-six patients (62%) had bulky stage I or II disease; 59 patients (38%) had either stage III or IV disease; 77% had one or more extranodal sites involved at presentation; and 31% showed B symptoms. Seventy-seven patients were randomised to receive six cycles of modified CHOP (cyclophosphamide 600 mg/m(2) i.v., doxorubicin 30 mg/m(2) i.v., vincristine 1 mg i.v. all on day 1 with prednisolone 20 mg bd for days 1-5) every 21 days and 78 patients to MCOP (mitozantrone 10 mg/m(2) i.v. substituted for doxorubicin). Growth factors were not used routinely. After completion of chemotherapy, 39 patients received involved field radiotherapy (35-40 Gy) in 20 fractions. RESULTS: One hundred and one patients (65%) completed all six cycles of chemotherapy. The median course dose intensity was 97%. The median follow-up for 53 surviving patients was 51 months. The median survival was 19 months (95% confidence interval 10-36 months) with an actuarial survival of 47% at 2 years and 42% at 3 years (CHOP versus MCOP, P = 0.79). There was no significant difference in any of the toxicities experienced with either CHOP or MCOP, except for white cell count (46 patients on MCOP and 27 patients on CHOP had grade 3 or 4 toxicity, P = 0.002) and red cell transfusion (37 patients, MCOP; 17 patients, CHOP; P = 0.001). Grade 3 or 4 neutropenia was documented in 75 patients (50%). One patient died from toxicity whilst in remission and seven patients died with septicaemia and persistent NHL. CONCLUSION: This multicentre randomised trial provides further information on the dose intensity achievable with CHOP or MCOP regimens in elderly patients (median age 74 years) with aggressive NHL. These dose-reduced regimens can be given with nearly 100% dose intensity with 65% of patients completing all the treatment. Survival is comparable to that observed with the more intensive regimens given in this age group.

Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

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.

Preliminary experience of allogeneic stem cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH conditioning: an effective regimen with low procedure-related toxicity.

Autologous transplantation has an established role in the treatment of lymphoproliferative disorders, but allogeneic transplantation remains controversial. In an attempt to reduce the high procedure-related mortality reported with allografting in lymphoma, we have used BEAM (BCNU, etoposide, cytarabine and melphalan), a standard conditioning regimen for autologous transplantation. As BEAM may be insufficiently immunosuppressive to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with the anti-CD52 antibody CAMPATH-1G from day -5 to day -1. Twelve patients (median age 46 years) underwent allogeneic transplantation for lymphoma (n = 11) or chronic lymphocytic leukaemia (n = 1) from HLA-identical (n = 9) or mismatched (n = 3) sibling donors. Cyclosporin A and methotrexate were used as graft-versus-host disease (GVHD) prophylaxis. One patient died of progressive lymphoma at day +12, the remaining 11 patients engrafted rapidly, with eight demonstrating full donor chimerism. One patient had an episode of rejection and received a further stem cell infusion with sustained recovery. Only one patient developed GVHD (grade I). The low incidence of acute GVHD may be in part related to persisting levels of in vivo CAMPATH-IG at the time of transplantation. Of 11 evaluable patients, nine achieved complete remission (CR), and a further patient achieved CR after donor lymphocyte infusion at 5 months. Our preliminary experience is that this regimen was well tolerated with a low risk of GVHD and appears no more toxic than a BEAM autograft. Further follow-up is required to see whether the low incidence of GVHD impacts upon relapse risk.

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 x 10(8)/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.

Induction of remission after donor leucocyte infusion for the treatment of relapsed chronic idiopathic myelofibrosis following allogeneic transplantation: evidence for a 'graft vs. myelofibrosis' effect.

A 54-year-old man showed evidence of disease progression and a reduction in donor chimaerism by molecular microsatellite analysis 6 months after an allogeneic peripheral blood stem cell transplant for chronic idiopathic myelofibrosis. He was treated with a single infusion of donor leucocyte infusions (DLI), which led to the development of mild acute graft versus host disease (GVHD) and the rapid restoration of full donor haemopoiesis. This subsequently led to a progressive reduction in marrow fibrosis from grade IV to grade I over the following 6 months. We believe that this is the first report to provide clear evidence for the efficacy of DLI in this setting, which also provides evidence for the existence of a T-cell-mediated 'graft vs. myelofibrosis' effect similar to that seen against other haematological malignancies.

Comparative serial quantitative measurements of chimaerism following unmanipulated allogeneic transplantation of peripheral blood stem cells and bone marrow.

Serial samples were collected from 38 patients following allogeneic transplantation using either unmanipulated peripheral blood stem cells (PBSC) (n = 18) or bone marrow (BM) (n = 20) to assess the incidence of mixed chimaerism using PCR amplification of five VNTR regions. After amplification, products were analysed using the Applied Biosystems ABI PRISM 377 Automated DNA Sequencer and GeneScan software GenoTyper program to determine a quantitative measure of chimaerism. The sensitivity of detection using this method was 0.1%. In the immediate post-transplant period (up to day 30) a significantly lower incidence of mixed chimaerism (MC) occurred in recipients of PBSC compared to BM (P < 0.0005). Between 1 and 6 months there was a significantly lower incidence of low-level MC in patients receiving PBSCT compared to BMT (4/14 v 8/11 respectively, P = 0.04) in patients who had not rejected their grafts or relapsed. Similarly, beyond 6 months 0/9 PBSCT patients compared to 4/9 BMT patients showed MC (P = 0.02). Beyond day 30 13/33 (39%) patients showed intermittent low-level MC, but this was not predictive for subsequent relapse. A rapidly increasing proportion of recipient haemopoiesis was predictive of graft rejection or relapse. Stable continuous MC without relapse was seen in one patient transplanted with PBSC for severe aplastic anaemia. These results suggest that the incidence of intermittent low-level MC is relatively high in the first 6 months following unmanipulated haemopoietic stem cell transplantation but reduces with time and is significantly lower in recipients of PBSC.

Role of p38 in the priming of human neutrophils by peritoneal dialysis effluent.

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight substance that is able to prime human neutrophils for the release of arachidonic acid and superoxide anion. Conventional priming agents, such as tumor necrosis factor alpha (TNF-alpha), are known to signal via mitogen-activated protein (MAP) kinases; at least one possible substrate for MAP kinases is cytosolic phospholipase A(2) (cPLA(2)). Phosphorylation of this enzyme results in arachidonic acid release, and this fatty acid is a potent primer and activator of the human neutrophil NADPH oxidase. Because of the striking similarities between the priming of neutrophils with agents such as TNF-alpha and PDE, we have investigated the signalling pathways evoked by PDE and explored the possibility that cPLA(2) is a target for activated MAP kinases. Our results show that PDE treatment of human neutrophils results in the phosphorylation of the p38 kinase rather than the p42 and p44 kinases. Phosphorylation of p38 is transient with maximal activity being observed 1 min after exposure to PDE. We were unable to demonstrate that activation of p38 resulted in phosphorylation of cPLA(2); furthermore, translocation of this enzyme to a membrane-containing fraction was not enhanced in PDE-treated neutrophils. Taken together, these data suggest that, in a manner similar to that of TNF-alpha, PDE primes human neutrophils by the activation of the p38 kinase. However, unlike the cytokine, the activation of this protein does not result in phosphorylation or activation of cPLA(2).

Early allogeneic transplantation for refractory or relapsed acute leukaemia following remission induction with FLAG.

The prognosis for patients with secondary AML, primary resistant AML or ALL and early (<12 months) relapse of acute leukaemia remains extremely poor with conventional chemotherapy. As part of a strategy to improve the outcome for these patients we have treated 22 consecutive patients (18 AML, four ALL, median age 35 years) with either primary resistant disease (n=3), early relapsed leukaemia (n= 12) or secondary AML (n= 7, four RAEBt, two antecedant ALL and one antecedant Hodgkin's disease) with 'FLAG' induction chemotherapy with the aim of proceeding to early allogeneic transplantation either from sibling or unrelated donors. Eighteen patients achieved CR after one course of FLAG, including five patients who had documented p-glycoprotein-induced multidrug resistance and 10 patients with adverse cytogenetic abnormalities. Eight patients were consolidated with a second course of FLAG prior to transplantation and so far 16 patients have undergone allogeneic transplantation, 10 from unrelated donors and six from sibling donors (one mismatched). By the time of transplant three patients had progressed and were in early relapse and all have relapsed post BMT. Of the remaining 13 patients transplanted in remission, nine remain in CCR at a range of 4-26 months, three have died of transplant-related complications (18%) and one patient has relapsed. We conclude that the use of FLAG induction therapy followed by early allogeneic transplantation from either a sibling or unrelated donor can be an effective strategy for the treatment of this difficult group of young patients with poor risk acute leukaemia and appears to be associated with a low procedure-related risk.

Low incidence of human herpesvirus 8 in stem cell collections from myeloma patients.

Human herpesvirus 8 is a gammaherpesvirus which may be implicated in the pathogenesis of multiple myeloma. Viral DNA sequences have been found in the bone marrow, peripheral blood and leukapheresis products of myeloma patients. These findings have significant implications for the use of leukapheresed cells in the transplantation and immunotherapy of myeloma. The studies suggest the cell which harbours the virus may be dendritic in origin. We have previously reported that dendritic cells cultured for use in the clinical setting do not harbour HHV-8. In this study, we examined the leukapheresis products of a larger cohort of myeloma patients for the presence of HHV-8 using a highly sensitive PCR technique. A strong association between HHV-8 and myeloma was not confirmed, with only 4% of the patient samples positive for viral sequences. While further study is needed, the current use of apheresis cells and their cultured progeny in the treatment of myeloma should not be compromised.

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.