ALBERT

Description: T-cell depletion (TCD) of allogeneic stem cells transplantation (HSCT) is the most powerful approach to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. However, the prolonged immune deficiency resulting from TCD significantly impairs the outcome of HSCT from a haploidentical family donor (haplo-HSCT). In a phase I-II clinical trial (protocol MMTK007), we showed that the add-back of suicide-gene transduced donor lymphocytes (TK+ cells) provides early immune-reconstitution (IR), crucial to avoid opportunistic infections and disease relapse. While the achievement of an early and sustained IR significantly reduced the incidence of infectious complications and mortality, the kinetic of abatement of viral reactivations differed in different patients, suggesting variability in the repertoire, function and persistence of viral-specific T cells. To gain insights on the activity of the post-transplant immune system, we focused on immune responses to cytomegalovirus (CMV) and Ebstein Barr virus (EBV) as clinical paradigm of an effective immune system. In our trial 29 patients (median age 52y) were transplanted for high-risk leukemia. Seventeen pts received TK-DLI starting at day +42, 14 pts obtained prompt immune reconstitution with CD3+ 〉100/mcl at day +86 (median) from SCT and day +21 from TK-DLI. Twelve/14 experienced CMV reactivation, 3 reactivation/patient (median), each reactivation required 14 days (median) of pre-emptive treatment with foscarnet to achieve a complete clearance; in 2/38 events, a CMV antigenemia persistent during foscarnet treatment was cleared by ganciclovir administration. In this series, the last CMV reactivation requiring pre-emptive treatment happened at day +84 from SCT and day +19 from TK-DLI. Nine/14 experienced EBV reactivation, 1 reactivation/patient (median), 6 patients required treatment with rituximab (375 mg/mq weekly) for 3 cycles (median). In 3 cases we documented EBV lymphoproliferative disease, that were completely controlled with the treatment. The proportion of lymphocytes committed to produce IFN-γ upon CMV or EBV stimulation normalized within the first 3 months post transplant. The analysis of CMV and EBV T-cell response showed a prevalent host-restriction pattern, suggesting active modulation of the T-cell repertoire by the host environment. The TCR-Vβ repertoire progressively changed from oligoclonal into polyclonal and normalized by 1 year after transplant. The progressive acquirement of a protective, host-restricted, anti-viral response, highly correlated with a decline in the occurrence of any infectious event, that were nearly abrogated by 6th month post transplant. The overall cumulative infectious mortality beyond day +100 post transplant was 12,5% in TK treated patients demonstrating the efficacy of this strategy in building a protective immune system.

Description: The management of disease relapse following an allogeneic transplant is problematic. For patients with some diseases treatment with DLI alone can be successful. The use of a second allogeneic procedure has been associated with a prohibitively high TRM, especially when using myeloablative conditioning. Here we report the results of a British Society for Blood and Marrow Transplantation (BSBMT) registry retrospective multicentre analysis of 70 patients receiving a second allogeneic transplant using reduced intensity conditioning (RIC) after disease relapse following an initial allogeneic transplant. In 41 cases the first procedure was myeloablative and in 17 this was with RIC (unknown: 12). Over half of the first grafts were T cell depleted (TCD). The donor was an HLA identical sibling (48), unrelated (17) or other family member (2). In 11 cases a different donor was used at second transplant. The median age at first transplant was 39 (range: 8–69). The underlying diseases are as follows: AML 21, MDS 13, ALL 13, Lymphoma 11, CML 7, MPD 3, MM 2. For the second transplant, a variety of RIC regimens were used. Of these, 85% were fludarabine based and less than 20% include TCD. At the time of analysis, 27 patients were alive at a median follow-up of 1.7 years from second allograft (range: 0.3–7.4 years). In the cohort overall, the predicted overall survival and TRM at 2 years post second allograft were 27% and 27% respectively. Disease type was significantly associated with outcome (OS 1yr: lymphoma 82%, AML 32%, MDS 39%, ALL 41%, p=0.049). Age and disease status did not impact on OS or TRM. The median time to relapse after the first transplant was 1 year (range: 0.1–12.2). There was a significant survival advantage to those who relapsed more than a year post first allograft (2yr post second allograft: 33% vs 21%, p=0.038). In addition, patients with later relapses had a TRM of 12% at 2 years compared to 39% in those relapsing within a year (p=0.009). The incidence of acute GvHD was higher following the second procedure (43%) than the first (28%). Although aGvHD at any time did not impact on OS in the whole cohort, in those who relapsed prior to a year the presence of acute GvHD post second allograft was significantly associated with a superior outcome (p=0.001). The median time to relapse from the second transplant was 0.73 years (range = 0.1 – 1.6). The predicted relapse risk post second transplant was 52 % at 2 years. Those under the median age (p=0.029) and with acute leukaemia (1 yr post second transplant risk: AML 57%, MDS 34%, ALL 54%, lymphoma 14%; p=0.056) were at higher risk. Remission status prior to either transplant, acute or chronic GvHD post second allograft, donor type and time to first relapse did not impact significantly on relapse risk. In conclusion, compared to second myeloablative transplants, a second allograft using RIC can be performed with a low TRM in those relapsing more than one year after a first allograft, even when using UD. These patients achieved an encouraging OS of 〉 30% at 2 years. In those relapsing within a year of first transplant the development of GvHD was strongly associated with a better outcome.

Description: 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 andP = .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)

Description: The use of GSCF-mobilised Peripheral Blood Stem Cells (PBSC) for unrelated donor (UD) transplantation has increased dramatically since 2000. The association of PBSC with more rapid engraftment and with an increase in chronic Graft versus Host Disease (GvHD), compared to bone marrow (BM) has been reported in a number of studies. More recently the use of PBSC has been associated with an increase in transplant related mortality (TRM) and decrease in survival (OS) in T-cell replete transplants. We sought to analyse the impact of PBSC compared to BM in a cohort of UD transplant recipients, where T-cell depleting agents (in-vivo campath in 〉90%) were included in the transplant conditioning. The study included 145 patients transplanted between January 2000 and March 2006: CML- 35 in 1CP; acute leukaemia (AML in 61, ALL in 49)-110 in CR1 or 2. All had myeloablative conditioning regimens and received grafts with 9–10/10 matched HLA alleles. 86 patients received BM and 59 PBSC. There were no associations between the stem cell source and any transplant variable (including disease and stage). There was a trend to an increased use of PBSC in patients with a single antigen mismatch (p=0.052). All evaluable patients achieved neutrophil engraftment, with a significantly faster time to engraft in recipients of PBSC compared to BM (16 vs 20 days; p=0.0003). The incidence of acute GvHD was 46% (grade I in 50%, II in 41%, III in 8%, IV in 2%). This was significantly higher in recipients of PBSC (60%) compared to BM (36%; p=0.006), however there was no increase in either II/IV (p=0.69) or III/IV (p=0.18) disease in PBSC recipients. In univariate analysis, the presence of a single HLA mismatch (p=0.026) was the only other variable to be associated with an increase in acute GvHD. In a logistic regression model including both these variables, the use of PBSC remained significantly associated with an increase in aGvHD (OR=2.3; 95% CI 1.1,4.7;p=0.020). The TRM was 14%, 27% and 39% at 100 days, 1 and 5 years respectively. At none of these time points was the stem cell source associated with a significant difference in TRM. The 5-year incidence of chronic GvHD was 58% (BM 55%, PBSC 60%; NS), extensive disease in one third, and of relapse was 61% (BM 60%, PBSC 62%; NS). The 5-years OS was 41% with a median follow-up of 3.4 years (0.5–7.1). This was 44% using PBSC and 40% using BM (NS). In conclusion, although we observed an increase in acute GVHD with PBSC this was only of grade 1 disease. We found no association between the use of PBSC and an increased risk of chronic GVHD or of a worse transplant outcome, when compared to BM, in recipients of T-cell depleted myeloablative transplants for leukaemia.

Description: 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 andP = .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)

Description: The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC 〉1.0x109/l and 13 d (11–24) for plt 〉50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ 〉100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.

Description: The FIP1L1-PDGFRA fusion gene generated by a cryptic interstitial deletion at 4q12 is a recurrent molecular lesion in idiopathic hypereosinophilic syndrome (HES), that forms a basis for diagnosis of chronic eosinophilic leukemia (CEL). This disease entity is particularly important to recognize, since presence of the FIP1L1-PDGFRA fusion predicts a favorable response to molecularly targeted therapy in the form of imatinib, with clinical remissions being achieved with lower doses than are required in BCR-ABL+ chronic myeloid leukemia (CML). In order to improve our understanding of the biology of CEL and to provide a tool to improve the management of patients with this disorder we have developed real-time quantitative reverse transcriptase PCR (RQ-PCR) assays for the FIP1L1-PDGFRA fusion. Taking into account the marked heterogeneity in upstream breakpoints within the FIP1L1 gene, RQ-PCR assays were designed for cases with FIP1L1 breakpoints leading to fusion of exon 9, 10, 11, 12 or 13 to PDGFRA exon 12. FIP1L1-PDGFRA expression was compared to that of validated Europe Against Cancer endogenous control genes - β2microglobulin (B2M) and ABL. Serial dilution of the FIP1L1-PDGFRA+ EOL1 cell-line in fusion gene negative filler cells (HL60) revealed an assay sensitivity of 1 in 105. While identification of the FIP1L1-PDGFRA fusion using conventional RT-PCR approaches can be problematic, RQ-PCR analysis undertaken in diagnostic samples from 31 patients with FIP1L1-PDGFRA+ CEL (median age 53, 31–64 years) revealed that, in the majority, the fusion transcript was expressed at relatively high level (median deltaCt FIP1L1-PDGFRA - B2M, 12.2; median deltaCt FIP1L1-PDGFRA - ABL, 2.3). The FIP1L1-PDGFRA fusion was expressed at comparable level in blood and marrow at diagnosis of CEL. Serial monitoring was undertaken in 17 patients following initiation of imatinib 100mg/d. Overall, 8/8 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcript level within the first year of therapy. In follow-up samples affording a sensitivity of at least 1 in 1000, PCR negativity by quantitative and conventional nested RT-PCR was documented in 8/17 patients following a median of 21 weeks of imatinib (4–64 weeks); in two cases profound PCR negativity (i.e. at a sensitivity level of at least 1 in 105) was documented following 13 weeks and 2 years of imatinib, respectively. Overall, these data demonstrate that CEL with the FIP1L1-PDGFRA fusion is uniquely sensitive to imatinib therapy. This contrasts with BCR-ABL+ CML, in which molecular remission is generally not achieved - a phenomenon that has been postulated to reflect the persistence of a primitive quiescent stem cell population that is resistant to this agent. Understanding the biological basis for the differences in molecular response to imatinib in CML and CEL, may yield further improvements in molecularly-targeted therapeutic approaches.