Analysis of hematopoietic recovery after autologous transplantation as method of quality control for long-term progenitor cell cryopreservation.

Hematopoietic precursor cells (HPC) are able to restore hematopoiesis after high-dose chemotherapy and their cryopreservation is routinely employed prior to the autologous hematopoietic cell transplantation (AHCT). Although previous studies showed feasibility of long-term HPC storage, concerns remain about possible negative effects on their potency. To study the effects of long-term cryopreservation, we compared time to neutrophil and platelet recovery in 50 patients receiving two AHCT for multiple myeloma at least 2 years apart between 2006 and 2016, using HPC obtained from one mobilization and collection attempt before the first transplant. This product was divided into equivalent fractions allowing a minimum of 2 × 106 CD34+ cells/kg recipient's weight. One fraction was used for the first transplant after median storage of 60 days (range, 17-165) and another fraction was used after median storage of 1448 days (range, 849-3510) at the second AHCT. Neutrophil recovery occurred at 14 days (median; range, 11-21) after the first and 13 days (10-20) after the second AHCT. Platelets recovered at a median of 16 days after both procedures. Considering other factors, such as disease status, conditioning and HPC dose, this single institution data demonstrated no reduction in the potency of HPC after long-term storage.

Recipient/donor HLA and CMV matching in recipients of T-cell-depleted unrelated donor haematopoietic cell transplants.

Improving haematopoietic cell transplantation outcomes by selection of an HLA-matched unrelated donor is best practice; however, donor selection by secondary characteristics is controversial. We studied 1271 recipients with haematological malignancies who underwent T-cell-depleted allografts and had complete data on HLA-matching status for six loci (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) and clinical outcome data. Five-year overall survival was 40.6%. HLA mismatching (at HLA-A, -B, -C, -DRB1, -DQB1) relative risk (RR) 1.22, 95% confidence interval (CI) 1.2-1.5, P=0.033 for 1 mismatch and RR 1.46, 95% CI 1.1-1.9, P=0.009 for >1 mismatch) and CMV mismatching (RR 1.37, 95% CI 1.2-1.6, P<0.001) were significantly associated with inferior survival. Donors aged <30 years showed a trend towards better survival. The multivariate model for mortality, combining CMV and HLA-match status, found an RR of 1.36 (95% CI 1.1-1.7, P=0.003) for HLA matched/CMV mismatched, an RR of 1.22 (95% CI 0.99-1.5, P=0.062) for HLA mismatched/CMV matched and an RR of 1.81 (95% CI 1.4-2.3, P=<0.001) for HLA/ CMV mismatched, compared with the HLA/CMV-matched recipients. These data suggest that HLA and CMV matching status should be considered when selecting unrelated donors and that CMV matching may abrogate the effect of an HLA mismatch.

Sign of the Zodiac as a predictor of survival for recipients of an allogeneic stem cell transplant for chronic myeloid leukaemia (CML): an artificial association.

BACKGROUND: Astrological or Zodiac (star) sign has been shown to be a statistically significant factor in the outcome of a variety of diseases, conditions, and phenomena. METHODS: To investigate its relevance in the context of a stem cell transplant (SCT), we examined its influence in chronic myeloid leukaemia, a disease with well-established prognostic factors. Data were collected on 626 patients who received a first myeloablative allogeneic SCT between 1981 and 2006. Star sign was determined for each patient. RESULTS: Univariate analyses comparing all 12 individual star signs showed considerable variation of 5-year probabilities of survival, 63% for Arians, to 45% for Aquarians, but without significance (P=.65). However, it was possible to pool together star signs likely to provide dichotomous results. Thus, grouping together Aries, Taurus, Gemini, Leo, Scorpio, and Capricorn (group A; n=317) versus others (group B; n=309) resulted in a highly significant difference (58% vs 48%; P=.007). When adjusted for known prognostic factors in a multivariate analysis, group B was associated with an increased risk of mortality when compared with group A (relative risk [RR], 1.37; P=.005). CONCLUSION: In this study, we show that, providing adequate care is taken, a significant relationship between patient star sign and survival post SCT for CML can be observed. This is, however, a completely erroneous result, and is based on the pooling together of observations to artificially create a statistically significant result. Statistical analyses should thus be carried out on a priori hypotheses and not to find a meaningful or significant result.

Factors for graft-versus-host disease after donor lymphocyte infusions with an escalating dose regimen: lack of association with cell dose.

We investigated the risk factors for graft-versus-host disease (GVHD) in 82 patients treated with donor lymphocyte infusions (DLI) using an escalating dose regimen for chronic myeloid leukaemia in relapse following conventional allografting. Two factors emerged as predictors of both acute and chronic GVHD: the infusion of male recipients with lymphocytes from a female donor and the interval between transplant and last DLI, but only the first remained significant at multivariate analysis. Surprisingly, lymphocyte dose did not influence the incidence of GVHD. Our results suggest that DLI can be given in large cell doses without increasing the risk of GVHD.

Response to donor lymphocyte infusions for chronic myeloid leukemia is dose-dependent: the importance of escalating the cell dose to maximize therapeutic efficacy.

Donor lymphocyte infusions (DLI) are an effective treatment for patients with chronic myeloid leukemia (CML) in relapse after allografting but the optimal cell dose has yet to be identified. To address this question, we investigated the factors affecting the dose required to achieve remission (effective cell dose, (ECD)) in 81 patients treated with an escalating dose regimen. The overall proportion of patients who achieved a molecular remission was 88%. The cumulative proportion of remitters increased significantly at each dose level. With a CD3(+) cell dose < or =10(7)/kg, 56% of patients in molecular/cytogenetic relapse obtained molecular remission, whereas only 20% of those in hematologic relapse did so. At the same cell dose, 58% of patients who received lymphocytes from volunteer unrelated donors achieved remission, as compared to 29% of those who received DLI from sibling donors. We conclude that the response to DLI is dose-dependent and that the ECD is influenced by the quantity and phase of CML at relapse and degree of donor/recipient histocompatibility.

Long-term outcome of high-dose melphalan and autologous stem cell transplantation for AL amyloidosis.

Light chain (AL) amyloidosis is the result of a clonal plasma cell expansion, in which amyloidogenic monoclonal light chains deposit in various tissues resulting in organ dysfunction and organ failure. The median survival of patients with AL amyloidosis without therapy is 10-14 months. Several phase II studies report haematological and clinical remission in up to 50% of patients after high-dose melphalan and autologous stem cell transplantation. We analysed retrospectively the long-term outcome of 19 patients treated in this way between August/1996 and December/2001. We observed a relatively high treatment-related mortality of 26%, but 12 patients (63%) were high-risk candidates. Eight patients (42%) surviving longer than 100 days achieved haematological remission and long-term survival, whereas 6 (32%) obtained no clear benefit from high-dose therapy. However, 62% of patients survived beyond 2 years and the median survival from transplant was 48 months (range 0-104 months).

Prognostic factors for patients with chronic myeloid leukaemia in chronic phase treated with imatinib mesylate after failure of interferon alfa.

We assessed clinical results in 145 patients with chronic myeloid leukaemia in chronic phase who satisfied criteria for interferon-alpha failure and were thus eligible for treatment with imatinib at the Hammersmith Hospital. We used univariate and multivariate analyses to develop a risk score based on features defined after treatment for 3 months. We identified a low neutrophil count and poor cytogenetic response (<35% Ph-negative marrow metaphases) at 3 months as principal independent predictive factors and incorporated them into a three-tier prognostic scoring system for individual patients. For patients in the low-, intermediate- and high-risk groups, the probabilities of survival at 24 months were 100, 82 and 40% (P<0.0001) and progression-free survival 100, 66 and 15% (P<0.0001), respectively. This Hammersmith prognostic scoring system was validated with an independent cohort of patients treated at another UK centre.

Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse.

We identified 103 consecutive patients who, 5 years after allogeneic transplantation for chronic myeloid leukaemia (CML), were in molecular remission (MR). The 103 patients were divided into three groups on the basis of reverse transcription-polymerase chain reaction (RT-PCR) studies for BCR-ABL transcripts in the first 5 years post transplant: Group A comprised 63 patients who had been continuously PCR negative; Group B comprised 20 patients with one or more positive PCR result but only at a low level; and Group C comprised 20 patients who had fulfilled the criteria for molecular relapse, been treated with donor lymphocyte infusions (DLI) and had thereafter regained complete MR within the 5-year post-transplant period. The median follow-up for all 103 patients was 8.4 years from transplant (range 5-17.6 years). In group A only one patient relapsed at 9.2 years. In group B eight patients (40%) relapsed: six at molecular, one at cytogenetic and one haematological levels. The actuarial probabilities of survival at 10 years for patients in Groups A, B and C were 97.4%, 92.9% and 100% respectively; the probabilities of relapse were 3%, 54% and 0% respectively. We conclude that molecular studies during the first 5 years post transplant can help to predict long-term leukaemia-free survival and, possibly, cure of CML.

Pregnancy outcomes after peripheral blood or bone marrow transplantation: a retrospective survey.

BACKGROUND: Some patients treated by transplantation of haemopoietic stem cells (peripheral blood or bone marrow) become permanently infertile, but others retain or recover fertility. We assessed the outcome of conception in women, and partners of men previously treated by autologous or allogeneic stem cell transplantation (SCT). METHODS: We sent questionnaires to 229 centres of the European Group for Blood and Marrow Transplantation. We sought details about the original disease, transplant procedure, and outcome of conception for both male and female patients. FINDINGS: 199 centres gave information relating to 19412 allogeneic and 17950 autologous transplant patients. 232 (0.6%) patients conceived after SCT. Crude annual birth rate for 4-month survivors of SCT was lower than the national average for England and Wales at 1.7 per 1000 patients. 312 conceptions were reported in 113 patients (74 allograft) and partners of 119 patients (93 allograft). Most pregnancies were uncomplicated and resulted in 271 livebirths. 28 (42%) of 67 allograft recipients had caesarean section compared with 16% in the normal population (difference =26% [95% CI 15-38]), 12 (20%) of 59 had preterm delivery compared with a normal rate of 6% (14% [4-24]), and 12 (23%) of 52 had low birthweight singleton offspring compared with a normal rate of 6% (17% [6-29]). INTERPRETATION: Pregnancy after SCT is likely to have a successful outcome. Pregnancies in allograft patients who have received total body irradiation should be treated as high risk for maternal and fetal complications.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation.

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

Cytomegalovirus seropositivity adversely influences outcome after T-depleted unrelated donor transplant in patients with chronic myeloid leukaemia: the case for tailored graft-versus-host disease prophylaxis.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after haematopoietic stem cell transplantation from matched unrelated donors (MUD). The role of T-cell depletion (TCD) as a strategy to prevent GVHD is controversial because of the associated increased risk of leukaemic relapse, graft failure and delayed immune reconstitution. The demonstration that donor lymphocyte infusion (DLI) is effective salvage therapy if patients relapse after transplantation for chronic myeloid leukaemia (CML) prompted us to examine the proposal that TCD may be a form of GVHD prophylaxis particularly suited to this disease in patients undergoing MUD transplantation. We analysed the outcome of 106 consecutive patients with CML in first chronic phase who underwent MUD transplantation. Patients were conditioned with cyclophosphamide and total body irradiation (TBI), and received in vivo TCD, using CD52 monoclonal antibody, as GVHD prophylaxis. Donor lymphocytes were infused at the time of leukaemic relapse. The projected survival at 5 years for all patients was 52.6%. The probability of developing severe acute GVHD (grade 3 or 4) was 14.5%. The only significant predictor of overall survival in univariate and multivariate analysis was patient cytomegalovirus (CMV) serostatus: in CMV-negative patients survival at 5 years was 60% vs. 42% in CMV-positive patients (P = 0.006). The use of TCD was associated with an increased risk of relapse (62% probability at 5 years after transplant), but 80% of patients who received DLI achieved molecular remission that was durable in all but two cases. In vivo TCD, in conjunction with DLI at relapse, is a valuable GVHD prophylactic regimen in CMV-seronegative recipients of MUD allografts, but in CMV-seropositive patients this approach is associated with an increased non-relapse mortality. Consequently, GVHD prophylactic regimens in MUD transplantation should be tailored according to the patient and donor pretransplant characteristics.

Estimation of current leukaemia-free survival following donor lymphocyte infusion therapy for patients with leukaemia who relapse after allografting: application of a multistate model.

Allogeneic bone marrow transplantation has become routine treatment for selected patients with leukaemia and other haematological disorders. A standard measure of the efficacy of this treatment for patients is assessment of leukaemia-free survival, that is, the probability of being alive and in remission at different time intervals after transplant. During the last 10 years a new approach to managing patients who relapse after allografting was developed - namely infusion of lymphocytes (white blood cells) collected from the original donor. Such donor lymphocyte infusions (DLI) are highly effective in restoring complete remission and it appears that these remissions are durable. Consequently estimates of long-term success of allografting must consider salvage of initial relapses with DLI. To do so a new estimator of treatment efficacy, called 'current leukaemia-free survival' is sometimes used in the medical literature. This curve attempts to estimate the probability that a patient is alive in an original remission or in subsequent remission after treatment with DLI at a given time after transplant. Here we show that this estimator does not actually estimate the probability of interest and we use a multi-stage model to develop a new and more appropriate estimator for 'current leukaemia-free survival'.

Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia.

An analysis was performed of the response to treatment with donor lymphocyte infusions (DLI) and the survival in 66 consecutive patients who relapsed after primary treatment by allogeneic stem cell transplantation for BCR-ABL-positive chronic myeloid leukemia. The transplant donor was an HLA-identical sibling (n = 35) or a "matched" unrelated volunteer (n = 31). Fifty-seven patients were transplanted in chronic phase, eight in accelerated phase, and one in second chronic phase. The recognition of relapse was based on precise molecular, cytogenetic, or hematologic criteria. The median interval from transplant to relapse was 12 months (range 3-85). The median interval from relapse to initiation of DLI was 9.4 months (range 1-70). Patients received DLI from their original transplant donors on a bulk-dose (n = 34) or on an escalating-dose (n = 32) regimen. Patients were monitored serially by hematologic, cytogenetic, and molecular criteria. Molecular remission was defined by the finding of negative results by nested primer reverse transcriptase polymerase chain reaction (RT-PCR) for BCR-ABL transcripts on two consecutive occasions, subject to satisfactory controls. Forty-four patients (67%) achieved molecular remission. Patients who had relapsed to advanced phase disease and patients with short intervals between transplant and relapse had significantly lower probabilities of achieving molecular remission. Of the 44 patients who achieved molecular remission, 4 reverted to a PCR-positive status at 15, 18, 37, and 87 weeks after remission. The probability of survival for patients who achieved molecular remission was significantly better than for those who failed to do so (95% versus 53% at 3 years post-DLI, P = .0001). We conclude that the majority of molecular remissions after DLI are durable, and thus the majority of responding patients may prove to have been cured. (Blood. 2000;96:2712-2716)

Cytokine secretion in mixed lymphocyte culture: a prognostic indicator of renal allograft rejection in addition to HLA mismatching.

We have previously demonstrated significant inter-individual variations in cytokine protein secretion between normal individuals and patients prior to renal transplantation. In this study, pre-transplant patient vs. donor mixed lymphocyte cultures (MLC) were set up between 57 renal allograft patient/donor pairs, and secretion of cytokine protein (IL-2, IL-4, IL-6, IL-10 and IFN-gamma) into the culture supernatant measured by ELISA. Significant inter-individual variations in protein secretion in MLC were observed for all cytokines studied. Univariate analysis demonstrated that high levels of IFN-gamma and IL-10 in MLC and spontaneous IL-4, together with female donor sex and a high degree of HLA mismatching (especially HLA-DR) were significantly associated with rejection. However, multivariate analysis revealed the greatest risk of rejection (RR = 25.5, P = 0.003) was associated with a combination of high IL-10 secretion in MLC and mismatching for at least four HLA antigens (HLA-A, -B and -DR). It remains to be determined whether cytokine secretion in MLC is linked to cytokine gene polymorphisms. In future, assays for measuring either cytokine secretion or genetic polymorphisms may prove to be useful in aiding donor selection and tailoring immunosuppressive therapy.

Estimating leukemia-free survival after allografting for chronic myeloid leukemia: a new method that takes into account patients who relapse and are restored to complete remission.

A significant number of patients who relapse after allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML) will achieve sustained remissions after treatment with interferon-alpha, second transplants, or donor lymphocyte infusions (DLI) from the original stem cell donor. Because leukemia-free survival (LFS) is at present defined as survival without evidence of relapse at any time posttransplant, patients who relapse but are then restored to complete remission are treated as failures when estimating LFS. We have established a new category of LFS, termed current LFS (CLFS), which we define as survival without evidence of leukemia at the time of most recent assessment. To gauge the contribution of treatment for relapse to the efficacy of allogeneic SCT in the management of CML in chronic phase, we compared conventional LFS and CLFS in 189 consecutive patients who underwent SCT over a 7-year period with a minimum follow-up of 3 years. Patients with sibling donors (n = 111) received cyclosporine and methotrexate as prophylaxis for graft versus host disease; patients with unrelated donors (n = 78) also received Campath-1G or 1H as intravenous T-cell depletion. The 5-year LFS defined conventionally was 36% (CI: 29% to 43%) versus a 5-year CLFS of 49% (CI: 36% to 62%). This new method of defining LFS confirms the view that appropriate "salvage" therapy, principally DLI, makes a major contribution to the capacity of allogeneic SCT to produce long-term LFS in patients who receive SCT for CML and emphasizes the importance of redefining LFS to take account of successful treatment of relapse.

Summary curves for patients transplanted for chronic myeloid leukaemia salvaged by a donor lymphocyte infusion: the current leukaemia-free survival curve.

A significant number of patients who relapse after allogeneic stem cell transplantation (SCT) for chronic myeloid leukaemia (CML) will achieve sustained molecular remissions after treatment with donor lymphocyte infusions (DLI) from the original stem cell donor. Leukaemia-free survival, defined as survival without evidence of relapse at any time after transplant does not account for patients who are successfully treated with DLI. To summarize adequately the response to treatment, a new summary probability, called the current leukaemia-free survival (CLFS), is proposed. This quantity is defined as the probability that a patient is alive and in remission at a given time after transplant. We discuss two statistical methods for estimating CLFS. The first is based on a multistate modelling approach. The second is based on an estimate constructed by looking at appropriate differences between Kaplan-Meier estimates. We compare these estimates using data on 189 consecutive patients who underwent SCT over a 7-year period.

Cytogenetic status pre-transplant as a predictor of outcome post bone marrow transplantation for chronic myelogenous leukaemia.

We have analysed pre-transplant cytogenetic findings in 418 patients with CML in pre-blastic phase who underwent allogeneic BMT between February 1981 and January 1998. Five different patient groups were identified: A = Philadelphia (Ph)+; B = Ph-, BCR-ABL+; C = variant Ph (VPh); D = Ph chromosome plus at least one of: trisomy 8, +Ph, chromosome 17 abnormalities and E = other abnormalities in addition to the Ph chromosome. There were two principal conclusions. Firstly, Ph- patients showed a better outcome, and VPh patients a worse outcome, than those with a standard Ph, both in terms of leukaemia-free survival (LFS) (76.9%, 22.1% and 31.9%) and the risk of treatment failure relative to those with a standard Ph (relative risks of 0.49 and 1.92, respectively). One contributing factor may be relapse: no Ph- patients relapsed, whereas all other groups showed similar probabilities of relapse at 5 years (range 33.0-44. 0%). Secondly, those with the additional changes of +8, +Ph and i(17q) did not show a worse outcome than those with no additional changes (5 year survival of 44.7% vs 51.8%; 5 year LFS of 40.6% vs 31.9%), whereas those with other additional changes may fare worst of all (40.4% and 16.0%, respectively). Bone Marrow Transplantation (2000) 25, 143-146.

Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia.

Donor lymphocyte infusion (DLI) was originally administered as a single, relatively large dose of lymphocytes called a bulk dose regimen (BDR). It has since been suggested that the use of an escalating dose regimen (EDR) may be equally effective against leukemia while it induces less graft-versus-host disease (GVHD). We therefore compared the efficacy and incidence of complications in a nonrandomized sequential study of the 2 regimens in 48 consecutive patients who had relapses with cytogenetic or hematologic evidence of chronic myeloid leukemia after allogeneic stem cell transplantation. Twenty-eight patients were treated on a BDR (August 1990 to November 1995) and 20 were treated on an EDR (December 1995 to January 1998). Although the probability of achieving cytogenetic remission within 2 years of starting DLI did not differ significantly between the 2 groups (EDR, 91% [CI, 63%-98%] vs. BDR, 67% [CI,49%-83%], P =.70), the incidence of GVHD was much lower using EDR (10% vs. 44%, P =.011). When we considered only subsets of patients treated by BDR or EDR who had received comparable total lymphoid cell doses, the incidence and severity of acute and chronic GVHD were both significantly lower for recipients treated by EDR than for recipients treated by BDR (P =.005 and P =.031, respectively). These findings suggest that the incidence of GVHD associated with the EDR is low, not because the final cell dose is small, but because lymphocytes are administered over a considerable number of months. (Blood. 2000;95:67-71)