ALBERT

Description: Reduced intensity allogeneic stem cell transplantation (RIC alloSCT) is a therapeutic option for poor risk relapsed chronic lymphocytic leukemia (CLL) and can lead to 50% of progression-free survivors. The aims of this study were: to assess the “molecular quality” of clinical remission after RIC alloSCT in CLL patients; to investigate whether molecular remission (MR) can correlate with a lower relapse risk; to understand the clinical role of molecular monitoring in post-transplant immunotherapy. Twenty-nine patients with a molecular marker (heavy chain gene immunoglobulin rearrangement, IgH) were monitored for minimal residual disease (MRD). All patients had a relapsed disease; 75% had an unmutated IgH; cytogenetic analysis was available in 13 patients at first relapse, and 5 of them (38%) showed a 17p deletion. Median age at transplant was 60 years (range, 44–69). Median number of previous chemotherapy was 3 (range, 1–6) and 29% of patients failed autologous transplant. Eleven patients (38%) were chemorefractory before transplant. The conditioning regimen included thiotepa-cyclophosphamide-fludarabine in HLA identical sibling transplants (n=21), and an in vivo T cell depletion in haploidentical and unrelated alloSCT (n=8). Molecular monitoring was performed by nested-PCR on bone marrow using CDR-2 and CDR-3-derived patient-specific primers. For real-time PCR a FR3-derived probe was used. Post-transplant samples were amplified including the pre-transplant sample as positive control, and ΔCT was calculated after normalization for GAPDH gene. Eight patients (28%) were PCR-negative: 4 of them (14%) have been always PCR-negative while 4 patients (14%) experienced a delayed clearance of MRD during the first year after transplant. All these patients are alive and in CR at the median follow-up of 24 months (range, 3–71). Seven patients (24%) showed a mixed pattern of PCR positivity and negativity: one patient died of secondary acute leukemia, another patient had a nodal relapse, the others are in CR at a median follow-up of 30 months (range, 6–60). Fourteen patients (48%) were always PCR-positive: 7 of them relapsed after a median time of 9 months (range, 3–12); 2 patients died of TRM in MR; 5 patients are alive and in CR after a median follow-up of 15 months (range, 3–24). Relapse risk was higher for PCR-positive patients compared to PCR-mixed/negative patients (p=0.014). Eighty percent of PCR-mixed/negative patients experienced GVHD compared to 43% of PCR-positive patients (p=0.06). In 5 PCR-positive patients real-time PCR was carried out. In 3 patients that did not relapse a decreasing tumor load was detected; these patients were affected by extensive chronic GVHD. In the other 2 patients after an initial reduction, the tumor load increased on day +300 and +270: both patients relapsed on days +420. In conclusion, in poor risk relapsed CLL clinical and molecular remission can be achieved in a sizeable fraction of patients after RIC alloSCT. Persistent PCR positivity correlates with a high incidence of relapse and requires novel treatments, while a mixed-PCR pattern can be observed without clinical relapse.

Description: Haploidentical stem cell transplantation (SCT) can be used in relapsed haematological malignancies for patients lacking a matched sibling or unrelated donor. Major barriers of this strategy are the poor immune reconstitution and the high risk of relapse. Here, we report results of a phase I–II trial evaluating early add-backs of CD8-depleted donor lymphocytes (DLIs) (from 1x10^4 up to 1x10^5 cells/kg starting at day+45 up to day +105 at monthly intervals) after a reduced intensity conditioning (RIC) regimen [thiotepa (10 mg/kg), fludarabine (120 mg/sqm), cyclophosphamide (60 mg/kg) and TBI (2 Gy)]. Ex-vivo and in-vivo TCD were carried out by CD34+ cell selection using the CliniMACS device and alemtuzumab (15mg/m2, day-2), respectively. Twenty-one patients [n= 10 NHL (n=5 CLL, n=5 high-grade NHL), n=7 HL, n=1 MM, n=1 ALL, n=2 AML] were transplanted with advanced disease: 16 (76%) failed a previous autograft and 13 (62%) had refractory disease. A median of 10.4x10^6/Kg CD34+, 1x10^4/kg CD3+, 10x10^4/kg CD19+, 0.9 x10^4/kg NK+ were infused. All patients engrafted with full donor chimerism from day +90. At a median follow-up of 12 months (range, 4–41 months), 12 of 21 pts are alive (7 CR, 2 PR and 3 PD) and 9 died [n=3 infection with GVHD (+610, +187, +253), n=6 disease]. The estimated 2-year overall survival was 49%: pts transplanted in remission had better outcome (83% versus 31%, p=0.13). The estimated 2-year cumulative incidence of TRM and relapse were 27% and 58%, respectively. CMV reactivation and hospital readmissions for opportunistic infections occurred in 76% and 57% of patients, respectively. For CD8 depletion of donor leukaphereses, a new depletion protocol using clinical grade CD8 microbeads (Miltenyi) was applied. This procedure is efficient to reduce the content of CD8 T cells by 3 logs while the median cell recovery of CD3+, CD4+, CD56+/CD3+, CD 20+ was 60%, 86%, 54%, 72%, respectively. Before DLIs, only 2 of 21 patients (10%) developed acute GVHD (no grade III–IV). A total of 36 CD8-depleted DLIs were administered to 17 pts without any acute toxicity. Following DLIs, 6 pts (35%) developed acute GVHD (grade II) and 5 (30%) chronic GVHD (n=2 limited, n=3 extensive). Overall, the incidence of acute GVHD is higher (50% vs 22%, p=0.33) in pts receving larger numbers of donor cells (10–15x10^4/kg versus 3–5x10^4/kg CD8-depleted DLIs). The median values of CD4+/uL, CD8+/uL and NK+ were 100, 280 and 680 at 4 months and 220, 200, 500 at 6 months after SCT in patients receiving CD8-depleted DLIs. Measurable TREC/ucg DNA (mean value 316; mean value donors 3740) and polyclonal T cell repertoire, evaluated by spectratyping, were observed at 9 months in patients younger than 40 years and/or without GVHD. Our results suggest that: haploidentical SCT with RIC regimen provides high engraftment rate T-cell addback allows the achievement of more than 100/uL CD4+ at 4 months after SCT in the majority of patients Survival rate is promising in patients who had transplantation in remission suggesting that this strategy should be evaluated earlier in high risk haematological diseases.

Description: Abstract 4494 1.Background Mismatches of minor Histocompatibility antigens (mHAg) have been considered as an important immunogenetic factor influencing outcomes and immune responses following allogeneic stem-cell transplantation (alloSCT) despite fully matched HLA of donor and recipient. 2. Aim Aim of this study was to assess whether mHAg incompatibilities may affect overall survival (OS), progression free survival (PFS) and GVHD incidence (acute and chronic, aGVHD and cGVHD) in patients receiving alloSCT for lymphoid malignancies. 3. Methods Sixty-four consecutive patients with B-cell lymphomas who underwent alloSCT were studied. Ten patients had chronic lymphocytic leukemia (CLL, 15.8%), 3 had follicular lymphoma (FCL, 4.7%), 1 had diffuse large B cell lymphoma (DLBCL, 1,5%), 17 had Hodgkin's lymphoma (26.5%) and 33 had multiple myeloma (51.5%). All underwent peripheral blood stem-cells allograft with non-myeloablative (13 patients, 20%) or reduced intensity (51 pts, 80%) Fludarabine-based conditioning; GVHD prophylaxis included methotrexate and oral cyclosporine +/- micomofetil fenolate in case of matched unrelated donors. Median age was 51 years (range 18-66); 35 patients were male (55%), median number of previous chemotherapies was 3 (0-7), 49 patients had a previous autologous transplant (76%). Twenty-five patients were in complete remission (CR, 39%), 30 and 9 were in partial response and progression (PR 47% and PD 14%). Karnofsky performance status (PS) was 〉80% in 50 patients (78%). Forty-four patients allografted from HLA-matched siblings (69%), 20 from matched unrelated donor (31%): all were matched at allelic level for HLA-A, -B, -Cw, -DRB1 and -DQB1 loci. Allelic mHAs typing was performed by PCR with sequence-specific primers for 14 autosomic mHAg and H-Y. Host versus Graft or Graft versus Host direction of immune responses in donor/recipient pairs was analyzed with use of the minor Histocompatibility Database of Leiden University Medical Center. OS and PFS were analyzed with Kaplan-Meier method and log-rank test. aGVHD and cGVHD were analyzed with a multivariate logistic regression including as covariates age, sex, previous lines of chemotherapy, disease, pre-transplant status, PS and mHAs mismatches; grade 〉=2 aGVHD and extensive cGVHD were considered events. 4. Results Median follow-up was 34 months (2-83). One-year OS was 85%, 2- and 3-years OS were 82% and 77%. One-year PFS was 61%, 2- and 3-years PFS were 49% and 41%. The univariate analysis which considered transplant characteristics showed that OS and PFS were significantly affected by disease status at transplant (p

Description: Purpose: Haploidentical hematopoietic stem cell transplantation (haplo-SCT) provides an option for cancer patients lacking a compatible donor. However, the delayed immune reconstitution increases incidence of opportunistic infections and disease relapse. We conducted a phase I-II prospective trial to evaluate the effect of escalating doses of CD8-depleted (Miltenyi Device) donor lymphocyte infusions (DLIs) following RIC haplo-SCT. Here, we present the data on the immune reconstitution. Patients and Methods: Twenty-eight patients (pts) with advanced lymphoproliferative diseases (n=24) or acute myeloid leukaemia (n=4) were enrolled in this study. Fifteen (54%) of 28 pts had refractory disease. All pts received a thiotepa-fludarabine based RIC regimen, with an ex vivo and in vivo T-cell depletion performed by CD34+ cell selection and alemtuzumab infusion. Fifty-four CD8-depleted DLIs were administered to 23 pts [dose level 1 (n=4): 1×104/kg on days + 45, +75, +105; dose level 2 (n=11): 5×104/kg on days + 45, +75, +105; dose level 3 (n=8): 1×104/kg on day+ 45, 5x104/kg on days +75 and +105]. Results: At a median follow-up of 29 months, 12 pts are alive (n=3 Hodgkin Lymphoma, n=6 Non-Hodgkin Lymphoma, n=1 Multiple Myeloma, n=2 Acute Myeloid Leukemia) and 16 died from any cause [n=6 for non-relapse mortality (NRM), n=10 for disease progression] with a 2-year estimates of overall survival of 39%. The 2-year cumulative incidence of NRM and relapse were 26% and 51%, respectively. Six pts (26%) developed grade II-IV acute graft-versus-host disease (GVHD) (dose level 1: no GVHD; dose level 2: 5 cases; dose level 3: 1 case). Following CD8-depleted DLIs, the major findings were:(i) a significant expansion of memory CD4+ cells and CD19+ cells (median value 107/μL and 135/μL, respectively) at 120 days after haplo-SCT; (ii) de-novo T-cell responses to cytomegalovirus (CMV) peptides performed by activation-induced CD137 expression: the median frequency of CMV-specific CD8+/CD137+ and CD4+/CD137+ cells were 2% and 0.4% at 200 days after haplo-SCT, respectively, as evaluated in a subset of pts at risk for CMV reactivation (R CMVpos/D CMVpos/neg); this frequency was associated to a clearance of CMV antigen; (iii) recovery of thymopoiesis: 10 of 20 (50%) pts showed measurable TREC at 1 year after haplo-SCT; (iv) the increase of CD19+ cells was associated to reconstitution of B lymphocyte repertoire as analysed by Immunoglobulin heavy chain (IgH) CDR3 spectratyping: median number peaks in normal donors and pts, at day 360 after haplo-SCT, were 17 (range, 14–21) versus 12 (range, 7–14), respectively. Conclusions: Our study showed that CD8-depleted DLIs are a feasible procedure with low acute GVHD when using dose level 3. Long-term disease remissions can be observed in advanced lymphoid malignancies. Escalated doses of CD8-depleted DLIs exert complex effects on immune reconstitution: (i) provide help to expansion of CD8 T-cell clones, as suggested by the occurrence of the anti-CMV reactivity; (ii) support B-cell recovery, as demonstrated by a partial recovery of CDR3 polyclonality.

Description: Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34+ cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

Description: Allogeneic stem cell transplantation (SCT) from an haploidentical family donor has been reported as a viable option in acute leukemias when matched donors are unavailable. However, the extensive T-cell depletion (TCD), required to prevent graft-versus-host disease (GVHD), is associated to delayed immune recovery and high transplant-related mortality. In an ongoing phase I–II trial for patients (pts) with advanced hematological malignancies, we combined a RIC regimen, including thiotepa (10 mg/kg), fludarabine (120 mg/ms), cyclophosphamide (60 mg/kg) and TBI (2 Gy), with pre-emptive administration of CD8-depleted DLIs (starting from 1x104 up to 1x105 cells/kg). Ex-vivo and in-vivo TCD were carried out by CD34+ cell selection using the CliniMACS device and alemtuzumab (30 mg day −2), respectively. The aim of the study was to investigate in a dose finding study the safety and the impact on immune-reconstitution of CD8-depleted DLIs. Six-teen pts with hematological malignancies (n=14 NHL/HD, n=1 ALL, n=1 AML) were included, median age was 35 years (range, 15–65), 10 (63%) were chemorefractory, and 68% and 75% had failed a previous auto or at least 3 CT lines, respectively. Pts received a median of 10.6 x106/Kg CD34+ and 1x104/kg CD3+. All pts engrafted with full donor chimerism from day +30. At a median follow-up of 9 months, 12 pts were alive and 4 died (n=1 infection, n=3 disease). The estimated OS at 2 years was 58%; 7 of 16 (44%) relapsed at median time of 100 days after SCT. CD8-depletion of 14 donor lymphocyte aphereses was performed with a new depletion protocol (Clinimacs CD8-Microbeads) that reduces the content of CD8+ cells by at least 3 logs. The median CD3+, CD4+, CD56+/CD3+, CD20+ cell recovery were 62% (range, 35–91%), 88% (63–128%), 51% (8–78%), 76% (33–128%), respectively. Before DLIs, only 1 of 16 pts (6%) developed de novo acute GVHD (grade II). A total of 22 CD8-depleted DLIs were administered to 9 of 16 pts without any engraftment problem. The first cohort of pts (n=5) received a total dose of 3–6x104/kg CD8depleted DLIs starting at day +45 divided in 3 monthly infusions: none of them developed aGVHD. Given no toxicity, we escalated doses and the second cohort (n=4) received a total dose of 10–25x104/kg CD8-depleted DLIs divided in 3 monthly infusions: 3 pts had acute GVHD (grade II). Overall, the incidence of acute GVHD was higher (75% vs 0%, P