ALBERT

Description: GVHD remains a matter of concern after RIC allo-SCT. Our knowledge of aGVHD risk factors is still primarily based on historical analyses performed in the myeloablative allo-SCT setting. Thus, modern approaches to predict the occurrence/severity of aGVHD are needed. This study aimed to identify a plasma protein signature correlating with occurrence of early aGVHD. We performed Surface-Enhanced Laser Desorption/Ionization-time (SELDI) of flight mass spectrometry profiling of plasma from 88 patients who received a RIC allo-SCT from HLA-identical siblings. Patients characteristics were: median age was 51 (range, 18–70) y. 41 patients (47%) had a myeloid malignancy, whereas 30 patients (34%) had a lymphoid malignancy. The remaining 17 patients (19%) were treated for metastatic non-hematological malignancies. The majority of patients (n=70, 80%) had an advanced disease with high risk clinical features precluding the use of standard myeloablative allo-SCT. All patients (100%) received G-CSF-mobilized PBSCs. The RIC regimen consisted of fludarabine, busulfan and ATG in 53 patients (60%) and low dose irradiation in 35 patients (40%). With a median follow-up of 400 (range, 127–829) d, 20 patients (23%) experienced early (prior to day 35 after allo-SCT) grade 2–4 acute GVHD (12 grade 2 and 8 grade 3–4). Denatured plasma samples (collected at a median of 28 days after allo-SCT) were incubated with H50 and CM10 ProteinChip arrays and subjected to SELDI analysis. Patient population was divided into a training (n=59) and a validation set (n=29). In the training set, 36 protein peaks were differentially expressed according to early aGVHD occurrence. By combining partial least squares and logistic regression methods, we built a multiprotein model that correctly predicted outcome in 96% of patients (14/14 patients with early aGVHD; specificity, 96%). The observed correct prediction rate in the validation set was 69% with a sensitivity of 67%, and a specificity of 70%. While negative predictive value of the model was only 36%, predictive positive value was estimated to 89% in the validation set. The performances of the model remained very similar after iterative (500 times) random resampling (correct prediction rate: 74%, median sensitivity: 48%, median specificity: validation set: 83%). Univariate and multivariate analyses of known risk factors (demographic features, diagnoses and transplant procedures) for occurrence of an early grade 2–4 acute GVHD did not show any statistically significant difference between the group of 20 patients who had early grade 2–4 aGVHD as compared to the remaining patients, and suggested that the multiprotein index is likely to be the only independent prognostic parameter. Major components of this multiprotein index are currently being characterized and will be presented. Obviously, larger prospective studies are still needed, but our results already suggest that proteomic analysis of plasma will prove increasingly important in the early and clinical diagnosis of aGVHD allowing improving patient outcome.

Description: In the setting of RIC for allo-SCT, long term outcomes are still poorly defined. Of note, the epidemiology of long term transplant-related infections is still sparse. This prospective report describes infectious complications occurring beyond 6 months after allo-SCT, in 159 consecutive patients who received a RIC allo-SCT from an HLA-identical sibling. Patients characteristics are as follow: median age was 50 (range, 18–68) years. 68 patients (43%) had a myeloid malignancy, whereas 66 patients (41%) had a lymphoid malignancy. The remaining 25 patients (16%) were treated for metastatic non-hematological malignancies. The majority of patients (n=126, 79%) had an advanced disease with high risk features precluding the use of myeloablative allo-SCT. 24 patients (15%) received donor bone marrow (BM), while the remaining 135 patients (85%) received PBSCs. In addition to fludarabine and busulfan, the RIC regimen included high dose ATG in 20 patients (13%) and low dose ATG in 95 (60%). 24 patients (15%) received fludarabine, busulfan and TLI, while the remaining 24 patients (15%) received fludarabine and low dose TBI. With a median follow-up of 19 (range, 6–90) months, 120 patients (75%) experienced at least one infectious episode (total number of episodes, 366) beyond the first six months after allo-SCT developing at a median of 8 (range, 6–34) months. In all, 212 infectious episodes (58%) required hospitalization (7% in the intensive care unit) for a median duration of 10 (1–91) days. 144 episodes (39%) could be documented (bacterial, n=48; viral, n=78; fungal, n=18). Microbiologically documented infections were distributed as follow: gram negative bacteria (18%), other bacteria (15%), CMV positive antigenemia (17%), HSV (19%), VZV (15%), other viruses (3%), aspergillus (6%), candida species (6%), other (1%). 76% of patients with an infection were under systemic immunosuppressive therapy for chronic GVHD at time of infection. Moreover, 85 patients (71%) experienced more than one infectious episode (median, 2; range, 1–12). In multivariate analysis, active or prior history of extensive chronic GVHD and the use of a BM graft were the strongest factors significantly associated with an increased risk of long term infections (P=0.0003; RR=2.04; 95%CI, 1.4–3.0; and P=0.005; RR=2; 95%CI, 1.2–3.2 respectively), highlighting the raising concern about the deleterious impact of severe chronic GVHD occurring after RIC allo-SCT, but also the protective effect of donor origin immunity based on graft origin and content. In this series of patients surviving at least 6 months after RIC allo-SCT, the overall long term transplant-related mortality was 11% (n=18), of whom 12 deaths were attributed to chronic GVHD and its complications including infections, and 5 deaths solely attributed to infections. In all, these results suggest that, despite reduction in early toxicity associated with the use of RIC regimens, long term debilitating chronic GVHD and its corollary of continuous immunosuppression and subsequent infections are still a matter of concern. Prospective efforts to develop optimal antimicrobial preventive strategies are needed to further improve the safety of the procedure and the overall benefits of RIC preparative regimens before allo-SCT.

Description: Few data are currently available regarding platelets trasfusion needs and the kinetics and predicitive factors for platelets recovery after RIC allo-SCT. In this study, we analyzed the profile of platelets recovery and transfusion needs in the first 100 days after sibling PBSC RIC in a single institution series of 166 consecutive transplantations. Patients and graft characteristics were: age 49 y. (range: 18–70), diagnoses: 66 myeloid malignancies (40%), 64 lymphoid malignancies (39%), and 36 metastatic solid tumors (21%). 112 pts (67%) received an ATG-based RIC, while 54 pts (33%) received a low dose irradiation-based RIC. 75 pts (45%) developed grade 2–4 acute GVHD. Platelets recovery (〉20 G/L) was observed at a median of 9 days (range: 0–99). The kinetics profile of platelets recovery is shown in the figure below. In the whole study population, the nadir was observed around day +7 after allo-SCT, and a plateau was reached about day +35. Filtered and irradiated donor apheresis platelets were used and patients needed a median of 1 unit (range: 0–53). In this series, 83 pts (50%) did not require any platelets transfusion during the follow-up period (median follow-up: 442 days) and 83 patients (50%) received at least one transfusion of platelets (54 were not transfused beyond day +100 after allo-SCT). Platelets count prior to RIC allo-SCT (median count 144 G/L; HR 0.44 (0.28–0.7) p=0.002), conditioning regimen (use of ATG; HR 1.86 (1.08–3.2) p=0.025) and the occurrence of acute (HR 1.54 (1.17–2.01); p=0.001) and severe GVHD (HR 2.36 (1.38–3.05) p=0.0006; 82% of patients with grade 3–4 acute GVHD were transfused) were the parameters significantly associated with platelets transfusion needs in multivariate analysis. In this cohort, 145 pts could be assessed for platelets recovery at day +100: among them, 99 (68%) had a platelet count 〉99 G/L. Univariate analysis found a significant impact of acute GVHD (p=0.0001) and platelet count prior to conditioning (p=0.012) but only acute GVHD (HR 5.52 (2.48–12.25); p=0.001) was associated with a delayed platelet recovery in a multivariate model. No impacts of pathology, GVHD prophylaxis regimen or CD34+ cell dose were demonstrated. Overall, these observations show a significantly lower rate of platelets transfusions and a quicker kinetic of platelets recovery after RIC allo-SCT and point out the effect of acute GVHD. In addition, considering the low level of myeloablation observed, RIC could be an appropriated field of investigation for the testing of megakaryocytic stimulating agents, towards further improving the safety and outcome of RIC allo-SCT. Platelets recovery after PBSC RIC allo-SCT Platelets recovery after PBSC RIC allo-SCT

Description: Despite continuous advances in the treatment of metastatic breast cancer, some patients have very poor outcome. Over the last period, we have investigated allogeneic immunotherapy as a possibility for tumor control. To date, we treated 31 pts with Allo SCT for advanced metastatic Breast Cancer (mBrC) in 2 successive clinical trials approved by Institut Paoli Calmette review Board, Marseille “comite de protection des personnes de Marseille” (local ethical committee) and the AFFSSAPS cellular therapies committee (national agency). The protocols were supported by special grants from the “Association pour la recherche sur le cancer (ARC)” (ARECA pole) and from the ministry of health (PHRC). All patients and donors gave informed consents. All pts (age: 43 (27–57)) underwent ASCT after the same reduced intensity conditioning (RIC) (Fludarabin (150mg/m2), Busulfan (8mg/kg) and Thymoglobulin (2,5mg/kg) or TLI (1 Cgy)) from a HLA-identical sibling (BM: 13%; PBSC: 87%) followed by CSA. All patients presented advanced disease as they have failed at least one conventional line of treatment. Indeed, prior to ASCT a median of 3 lines of treatment (1–7) have been administered over a period of 57 months (6–143). In addition, 15 (48%) pts underwent autologous SCT at a median time of 15 months (1–99) prior to ASCT. All pts were measurable and had a median of 2 metastatic sites (1–4) (liver:72%, bone:50%, lung:22% and brain:11%). We estimated disease status at transplant according to RECIST criteria: 17 (55%), 10 (32%) and 4 (13%) pts had progressive (PD), stable disease (SD) and partial response (PR) respectively. All patients engrafted. The cumulative incidences of grade 2–4 aGVHD and cGVHD were respectively 42% (25–59) and 62% (45–79) respectively. None of the 31 pts died from TRM. Seven patients achieved an objective response (CR=1; PR=6) at a median of 60 days (30–150) for a 24 % (9–39) OR cumulative incidence. Eventually all pts but 3 progressed at a median of 310 days (120–560) post transplant. Four pts are alive at median of 23 months (21–30) post transplant for a 2-year overall survival (OS) probability of 29% (16–47)). Results are dramatically different in regards to disease status at time of transplant. While outcome was uniformly poor for pts with PD (OR=0; 2 year OS probability: 6% (1–27)), patients with SD achieved a 40% (0–80) OR rate (p=.02) for a 50% (19–80) OS probability at 2 years (p=.001). Of the 4 patients in response at time of transplant, 3 increased their response (PR=2; CR=1) and 3 were alive after 2 years. This study shows that RIC-ASCT can be safely performed in BrC pts without high transplant related mortality. Data concerning quality of life (pre and post transplant) are presently under analysis. However we show evidences that pts with highly progressive disease do not benefit from this approach. In contrast, a high rate of response associated with prolonged survival can be achieved in patients with slowly progressive disease. These encouraging results for this advanced population compare favorably with non transplant literature. However most of the patients presenting objective response eventually progress. It seems that allogeneic immunotherapy could hardly cure patients with advanced mBrC. As routinely performed for acute myeloïd leukemia, this could perhaps be achieved only in pts in the initial disease phase when treatment disease resistance has not occurred. Target population would need careful selection on individual prognosis factors indicating their poor short term outcome: this represents the ultimate goal for future investigations. In this perspective, we designed a new program for the “triple negative” patients (Her2, estrogen and progesterone receptors negatives).

Description: Dendritic cells (DCs) are crucial in the induction of immune responses. Among DC subsets, the reconstitution of the natural type I-interferon-producing plasmacytoid DCs (PDC) has been proposed to play a major role in establishing immune competence, given the capacity of PDCs to efficiently expand either specific cytotoxic T lymphocytes, or to promote regulatory T cells, contributing to an impaired immune response. Therefore, we investigated the impact of circulating PDCs measured at the third month after RIC-allo-SCT, in 54 patients with hematological and non-hematological malignancies who received a RIC-allo-SCT from an HLA-identical sibling, in order to determine whether this could provide a convenient indicator for long term outcome. The median absolute count of PDCs measured at 3 months after RIC-allo-SCT was 0.725/μL (range, 0–23.2). In a multiple logistic regression analysis including all relevant parameters (demographic and graft characteristics, RIC regimens, CMV infections, and acute GVHD), only the absence of clinically significant grade II-IV acute GVHD was associated with an improved PDC recovery at 3 months (P=0.003; OR=6.4; 95%CI, 1.9–22). Being the major type I IFN-secreting cells, we also investigated whether PDCs recovered after allo-SCT are functional in response to viral stimulation. Patients experiencing grade 0-I acute GVHD could secrete significantly higher amounts of IFN-alpha as compared to patients with grade II–IV aGVHD (mean, 91 pg/ml vs. 0 pg/ml respectively; P=0.002), likely highlighting the deleterious impact of corticosteroids therapy on PDC function. The CD34+ stem cell dose and other lymphoid subsets infused with the allograft did not affect PDC recovery. Though PDC count could not predict death from progression or relapse, patients with a “high” PDC recovery profile had an improved overall survival (OS; P=0.03), in contrast to patients with a “low” PDC recovery profile who had an increased incidence of late transplant-related mortality (GVHD, infections) (P=0.03). In addition, the overall incidence of late infections (viral, fungal and bacterial) was significantly higher in the “low” PDC recovery group as compared to the “high” PDC recovery group (59% vs. 19%; P=0.002), illustrating the importance of PDCs in anti-infectious immune responses. In a multivariate analysis, only a “high” PDC count was significantly predictive of a decreased risk of death (P=0.04; RR=0.34; 95%CI, 0.12–0.96). The role and impact of rare immune effector cells would tend to be more evident in truly RIC and less toxic regimens. In this study, we could show that monitoring of PDCs may be useful for patients’ management (closer surveillance, infection prophylaxis…), and may have a significant impact on the probability of a favorable outcome in the context of RIC-allo-SCT.

Description: RIC regimens for allo-CST are being explored with good results concerning feasibility and engraftment. However, little is known about the immune recovery pattern in these patients, especially the different CD4 and CD8 lymphoid T cell subsets. Here, we assessed at different time points after allo-SCT, the kinetic of recovery of naïve (CD45RA+/CD27+), central memory (CD45RA−/CD27+), and terminally differentiated (CD45RA+/CD27−) CD4+ and CD8+ T lymphocytes in 64 patients from a single center, receiving HLA-identical RIC allo-SCT. Patients and graft characteristics are: age 48 y (27–63), diagnoses: 22 myeloid malignancies (34%), 22 lymphoid malignancies (34%) and 20 metastatic solid tumors (31%). 51 pts (80%) were considered as high risk. 49 pts (77%) received a fludarabine, busulfan and ATG-based RIC, while 15 pts (23%) received a low dose irradiation-based RIC. 91% of patients received a PBSC graft, with 42 (66%) receiving CSA alone for GVHD prophylaxis and 22 (34%) receiving CSA and MMF. 24 pts (38%) developed grade 2–4 acute GVHD at a median of 49 d (26–85). In this series, in contrast to CD4+ T cell subsets, CD8+ T cell subsets had a progressive and sustained recovery in the first 3 months after allo-SCT, with acquisition of functional markers such as 2B4 and perforin. Among the different subsets analyzed, the recovery of naïve CD4+ T cells, and central memory CD4+ and CD8+ T cells, measured at day 28 after allo-SCT and before onset of grade 2–4 acute GVHD, showed a significant correlation with the risk of grade 2–4 acute GVHD (P=0.001; P=0.002 and P=0.05 respectively). Patients developing grade 2–4 acute GVHD recovered a median of 47 naïve CD4+ T cells/μL prior to onset of GVHD as compared to 11 cells/μL in patients with grade 0–1 acute GVHD (Figure). Naïve CD4+ T cell levels significantly decreased after appropriate acute GVHD treatment. In a Cox multivariate analysis taking into account all relevant risk factors for acute GVHD, early recovery of naïve CD4+/CD45RA+/CD27+ T cells in the first month after allo-SCT was the strongest parameter significantly predictive of grade 2–4 acute GVHD development (P=0.006; RR=4.0; 95%CI, 1.5–11.0). Interestingly, there was a significant correlation between the total number of CD4+ T cells infused with the allogeneic graft and the early recovery of naïve CD4+ T cells (P=0.001), suggesting that graft manipulation might represent an attractive tool towards harnessing alloreactivity after RIC-allo-SCT. Figure Figure

Description: Reduced-intensity conditioning (RIC) regimens are increasingly used for allogeneic stem cell transplantation (allo-SCT). RIC has been shown to allow engraftment with minimal early transplantation-related mortality (TRM). However, in the context of RIC, predictive factors for acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively) and their effect on outcome remain unknown. In this report, we analyzed the outcome of 101 high-risk patients (70 hematologic and 31 nonhematologic malignancies) who received an HLA-identical sibling allo-SCT after RIC, including fludarabine, busulfan, and antithymocyte globulin (ATG). The cumulative incidence of grade II-IV aGVHD was 36% (95% confidence interval [CI], 27%-45%), whereas the cumulative incidence of cGVHD at 2 years was 43% (95% CI, 33%-53%). In multivariate analysis, the incidence of aGVHD was significantly associated with the ATG dose infused during conditioning (P = .0005), whereas peripheral blood as stem cell source was the only predictive factor for the development of cGVHD (P = .0007). The 1-year cumulative incidences of disease progression or relapse in patients with (n = 69) and without (n = 31) GVHD (whatever its form or grade) were 30% (95% CI, 19%-41%) and 55% (95% CI, 37%-72%), respectively (P = .02), suggesting that a potent graft-versus-tumor (GVT) effect can be achieved in high-risk patients following RIC. Moreover, the GVT effect was closely associated with GVHD without an increased risk of TRM (cumulative incidence of TRM, 18% [95% CI, 10%-25%]). Collectively, these results provide a framework for the refinement of RIC approaches designed to enhance the GVT effect with an acceptable risk of GVHD.