ALBERT

Description: The curative potential of allo-SCT for malignancies derives from the progressive reconstitution of the immune system and the development of effective anti-tumor immunity, but GVHD and disease relapse remain considerable obstacles to improvement in overall outcomes. Because in recipients target antigens are persisting, donor-derived T-cell responses may be expected to lead to the accumulation of a sizable proportion of differentiated T-cells, as happens following infection with persisting pathogens. A few cross-sectional studies have pointed to the preponderance of certain memory T-cell subsets associated with chronic GVHD (cGVHD), but the subset identified differed between studies. Inasmuch as qualitative T-cell recovery takes months to years to complete and there is substantial variability in time to development of GVHD or relapse, serial analysis might be more suitable to unveil early changes in T-cell subset composition attributable to transplantation-related events. From October 2003 on, 55 pts who underwent an allo-SCT after myeloablative conditioning were monitored prospectively in terms of clinical post-graft complications, including graft rejection, infections, GVHD and relapse. Blood samples were obtained on days 30±2, 60±3, 90±5, 180±10 and 365±15 post-transplant. Naive (CD45RA+CCR7+), central memory (TCM, CD45RAnegCCR7+), effector memory (TEM, CD45RAnegCCR7neg), and terminally differentiated effector (TTD, CD45RA+CCR7neg) were enumerated within the CD4+ and CD8+ pools, and the percentage of cells coexpressing CD28 was calculated within each eight subsets. The degree of donor-derived T-cell chimerism was assessed by real time PCR (sensitivity ≤ 1%). Median follow-up was 733 d (404–1251). Dynamics of CD4+ and CD8+ naive, TCM, TEM, and TTD were similar between the pts who developed cGVHD (n=15) and those who did not and between pts who relapsed and those who did not. However, costaining to detect CD28 demonstrated contrasting differences between cGVHD and relapse. At day 30, pts who subsequently relapsed (n=17) had elevated percentages of cells keeping CD28 expression within CD8+ T-cell subsets (TCM, p=.001; TCM, p=.021; and TTD, p=.007). Conversely, pts who subsequently developed cGVHD (n=15; only one relapsed) had diminished percentages of CD28+ cells within the two CD8+CCR7+ subsets at day 30 (p=.002 and p=.034, respectively). Loss of CD28 expression is known to be a hallmark of CMV infection but multivariate analysis ruled out, however, a confounding effect of CMV. Adjusted hazard ratios were 0.10 (95% CI, 0.01-0.76; p=.026) and 5.56 (95% CI, 1.16-25.00; p=.032) with CD28neg cells 16.7% of all CD8+ TCM at day 30 for relapse and cGVHD, respectively. Furthermore, pts with relapse had more often mixed chimerism at day 30 while those with cGVHD had more often full-donor chimerism (p=.042 and p=.023, respectively). CONCLUSION: This prospective study is the first to associate an early contrasting change in CD8+CD28neg T-cells with the risk of relapse and cGVHD after a myeloablative conditioning. Determination at day 30 of the proportions of CD8+ T-cell subsets expressing CD28 and of the level of T-cell chimerism could assist in predicting risk of relapse and cGVHD and help build an algorithm for the management of immunosuppressive treatment.

Description: Outcomes in the treatment of sclerodermatous chronic graft-versus-host disease (cGVHD) are generally disappointing. Imatinib mesylate enables selective, dual inhibition of the transforming growth factor beta (TGFβ) and platelet-derived growth factor (PDGF) pathways. Recently, the drug’s effects on fibroblasts have been reported in both in vitro and in vivo studies. Inhibiting fibroblast growth (and thus decreasing collagen production in dermal fibroblasts) is thus a logical therapeutic approach. Here, we report on our experience with 12 patients who received imatinib mesylate for refractory sclerodermatous cGVHD following allogeneic stem cell transplantation (allo-SCT). The patients’ characteristics were as follows: median age, 35 years (range: 15–59); 7 male recipients, 6 female donors; 4 cases of CML, 4 MDS, 2 ALL and 2 Hodgkin’s lymphoma. The patients had received either myelo-ablative conditioning with standard GVHD prophylaxis based on cyclosporine and short-course MTX (n=9) or nonmyelo-ablative conditioning with cyclosporine and MMF. Seven patients received a marrow graft and 5 received a peripheral blood graft. All displayed refractory, chronic, sclerodermatous GHVD with at least 3 lines (range 3–6) of prior immunosuppressive therapy. The modified Rodnan skin score was used to assess the extent of skin damage. Glivec was initiated at a dose of 400 mg/day between 16 and 119 months post-transplantation (median: 44). Despite an imatinib dose reduction and the administration of various symptomatic treatments, 4 patients (33%) had to discontinue their treatment soon after its initiation (range: 16–64 days) because of intolerance (especially muscle cramps) and were not evaluable in terms of the efficacy criterion. Other side effects reported were parenthesis, diarrhea and edema. In the remaining patients, the scleroderma symptoms improved within three months of treatment initiation. At the time of this report, all patients were alive and those who tolerated imatinib mesylate have experienced a complete or near-complete response (n=4) or partial response (n=4). All responders (except for one who discontinued the drug 157 days after initiation, due to cramps) were still on the treatment, after a median time period of 216 days (range: 80–2053). This retrospective report shows that when imatinib mesylate is well tolerated, it is effective in patients with refractory sclerodermatous cGVHD and is thus a promising candidate for the treatment of this complication. This study should provide useful background information for building prospective, multicenter studies.

Description: Although allogeneic hematopoietic stem cell transplantation is presently the only curative option for many patients with AML or MDS, relapse remains the main cause of morbidity and mortality. Strategies are therefore developed to prevent relapse. Post-transplant immune intervention with administration of prophylactic or pre-emptive donor lymphocyte infusion (DLI) and/or chemotherapy maintenance using DNA-demethylating agents such as azacitidine (aza) is being investigated. The exact mechanism of action of aza remains obscure and might be due in part to tumor antigen upregulation or other gene induction by tumor cells causing an immune response. We enrolled, in a clinical trial (ClinicalTrials.gov Identifier:NCT01541280), patients (pts) with high risk AML or MDS and candidates for allo-HSCT transplantation to receive azacitidine (aza) and DLI post-transplant as prophylactic treatment with the primary objective to reduce the relapse rate at 2 years following allo-HSCT and secondary objectives to increase disease free survival at 2 years post-transplant, increase overall survival at 2 years, investigate feasibility and safety of maintenance strategy combining chemotherapy and immunotherapy and follow the incidence and severity of acute and chronic GVHD. High risk AML was defined as AML in CR1 with unfavorable cytogenetics, AML in CR2 or greater remission, refractory AML or in relapse prior allogeneic transplantation. High risk MDS was defined as MDS with intermediate-2 group and higher risk group according to IPSS criteria. Aza was scheduled to begin between d+56 and d+112 post-transplant at the doses of 32 mg/m²/d sc for 5 days every 28 days for up to a total of 12 cycles if the pt had not acute GVHD 〉1 or severe infection. The first DLI was started following 3 cycles of aza and discontinuation of immunosuppressive prophylaxis, and if the pt had no clinical signs of GVHD, uncontrolled infection or a recent history of gr〉2 acute GVHD. Two other DLI were scheduled every 8 weeks after the 5th and 7th cycle of aza. The doses of DLI 1, 2 and 3 were respectively 5x106, 1x107, 5x107 CD3+cells/kg for related donor, and 1x106, 5x106, 1x107 CD3+cells/kg for unrelated donor. Sixty-four patients were pre-included prior transplantation, 30 pts were subsequently included, 20 pts with AML and 10 pts with MDS, median age 58 y (22-70). The status at transplantation was: CR1 = 16 pts (53%), CR2 = 6 pts (20%), refractory = 5 pts (16%), upfront transplantation for MDS = 3 pts (10%). Cytogenetics was normal or intermediate for 15 pts and unfavorable for 15 pts (namely 8 pts with complex caryotype). Conditioning was myeloablative for 11 pts, reduced for 19 pts (including 2 sequential). Donors were unrelated volunteers in 18 pts (60%).The time between allografting and first aza cycle was 66 days (38-93). The median number of cycles of aza administered was 5 (1-12) with 10 pts (33%) completing the 12 cycles. Forty one DLI were injected in 17 pts: 5 pts received one DLI, 2 pts received 2 DLI, 8 pts received 3 DLI. Two additional pts received 4 and 5 DLI because of a mixed chimerism. The first DLI was given at a median of 142 d (129-221) post transplantation. Aza was well tolerated, but was discontinued in 20 pts: because of GVHD (n=11), relapse (n=5), GVHD/infection (1pt), sudden death due to heart failure (n=1), withdrawal of consent (n=2, one after 1 cycle and another after 5 cycles). Four months following transplantation, 24 (80%) demonstrated full donor chimerism (〉95%) in CD3+ cells. Nine patients developed grade 1 to 3 acute GVHD (CI 29.8±9%), 6 who did not receive DLI and 3 following DLI (grade 1 n=2, grade 2 n=3, grade 3 n=4). No grade 4 acute GVHD was observed. Nine pts developed chronic GVHD (2 limited, 7 extensive), 3 who did not receive DLI, 6 following DLI. Twenty patients are alive. With a median follow-up from the allotransplant for those alive of 36 months (range 12 - 46 months), the survival at three years is 66%. Causes of death were infection (n=1), relapse (n=8), sudden death due to heart failure (n=1). The median time to relapse was 5 months (2.5-9) and the cumulative incidence of relapse at 3 years 28.1±8.5%. These results confirm that aza is well tolerated as a prophylactic treatment to reduce the risk of post-transplantation relapse. The incidence of GVHD following aza + DLI was not overwhelming. Analysis of T cell population and immune response as well as comparison to matched-pair control are currently performed and will be presented. Disclosures Moreau: Amgen: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria.

Description: Allogeneic stem cell transplantation has become standard therapy for haematological malignancies through the positive immunologic graft-versus-leukaemia effect. Initial immune recovery relies on peripheral expansion of infused T-cells which switch to a memory-like phenotype. This study prospectively investigated whether changes in subset composition precedes late complications after myeloablative HLA-matched transplantation. Of 80 recipients, 51 experienced neither early infection nor acute graft-versus-host disease (GVHD), of whom 18 were still free of clinical complication throughout 395 – 1564 days of follow-up. Compared with this complication-free subgroup, patients who developed chronic GVHD as the only event recovered similar numbers of circulating T-cells with predominance of CD8+ T-cells lacking CC-chemokine receptor-7 and CD28 expression. Conversely, poor CD8+ T-cell recovery with diminished numbers of CD28neg CD8+ T-cells (~1/4th of that of relapse-free patients) preceded occurrence of relapse. In multivariate analysis, lower CD28neg CD8+ T-cell counts by day 60 were associated with greater risk of subsequent relapse (HR 0.33; 95% CI 0.14 - 0.76; P = 0.01). Enumeration of CD28neg CD8+ T-cells in patients without early clinical complication could assist in predicting risk of relapse and help build an algorithm for accelerating the immune recovery by reducing the immunosuppressive regimen and considering the introduction of prophylactic donor lymphocyte infusions.

Description: One of major hurdles to achieving good patient outcomes and survival rates in allogeneic stem cell transplantation (allo-SCT) after myeloablative conditioning is the high rate of transplant-related mortality (TRM). Much progress in supportive patient care has been accomplished over the last decade-notably the use of allelic HLA-matching (Yakoub-Agha, JCO 2006), the introduction of enteral nutrition (Seguy, Transplantation 2004), the development of wireless video-capsule endoscopy for the management of post-transplant diarrhea (Yakoub-Agha, Transplantation 2005), the availability of broad-spectrum antifungal prophylaxis, the use of busulfan IV instead of PO in the conditioning regimen, limitation of the use of ATG in graft-versus-host disease (GVHD) treatment and dose reduction when the latter drug is used in conditioning. Although all these various modifications have had a positive impact on short-term patient outcomes, their impact on long-term survival is still unclear. Hence, the main objective of the present study was to evaluate allo-CST outcomes as a function of the transplantation period. A total 445 patients have undergone post-myeloablation allo-CST in our department. The patient distribution over the time was as follows: prior to 1998 (first period): n=133; between 1998 and 2003 (second period): n=154; between 2003 and 2007 (third period): n=158. Only the first transplant for a given individual was taken into account. Kaplan-Meyer curves were plotted for 100-day survival, 180-day survival and 3-year survival for each time period. Setting aside the clear differences in supportive care methods, the three groups were well matched in terms of disease diagnosis, disease status at transplant and the main recipient and donor characteristics. It is noteworthy, however, that the median age of patients increased over time. Mean 100-day survival was 86 days (95% CI: 81–90), 93 days (95% CI: 90–96) and 96 days (95% CI: 94–98) for the first, second and third periods, respectively (p

Description: Acute graft versus host disease (aGvHD) represents the first complication after allogeneic stem cell transplantation (allo-HCT). During conditioning regimen (CR) intestinal damage induces microbial translocation, which primes macrophage reactivity and leads to donor-derived T cells alloreactivity. To date, little is reported on intestinal status and macrophage reactivity prior to CR. This study aimed to assess citrulline, an amino acid reflecting intestinal damage, and circulating monocytes-derived macrophage reactivity 30 days before CR. Blood samples from consecutive patients undergoing allo-HCT for hematological malignancies and from 10 healthy donors were analyzed. Citrulline levels were assessed by high performance liquid chromatography associated to spectrometry. Monocytes were isolated and stimulated with pathogen-associated molecular patterns as LPS, PAM, flagelline, MDP, curdlan and PBS as control. In each condition, cytokines (TNF-α, IL-1β, IL-6, IL-10) releasing was evaluated by multiplex fluorescent immunoassay. Citrulline and cytokine levels were analyzed relatively to aGvHD onset within 100 days post-transplant. Forty-seven patients underwent allo-HCT for different indications, including acute leukemia (n=26) and myelodysplastic syndrome (n=11). Twenty patients developed an aGvHD I-IV. Among the groups, differences were depicted for levels of citrulline, IL-1β, IL-6 and IL-10. Citrulline levels were lower in aGvHD group than in no-aGvHD group (p

Description: Introduction: Treatment of refractory gastro-intestinal GVHD (rGI-GVHD) is still disappointing. Indeed, combination of or escalating dose of immunosuppressive treatments not only have no beneficial effects on disease progression but also are responsible for an increased risk of infections with short-term survival rate less than 20%. GI surgery has been for long time offered to patients with Crohn disease; this approach is usually restricted to patients with GVHD-related GI occlusion. Given the similarity between the two diseases, and the predominance of GI-GVHD damages in the distal part of the small bowel, we have considered surgery in 8 patients with rGI-GVHD. We performed an end ileostomy in order to circumvent the affected portion of the intestine and limit the use of immunosuppressive treatment. Here, we report our experience on patients with rGI-GVHD who underwent GI-surgery. Methods: Eight patients with acute grade 4 GVHD according to standard criteria were retrospectively analyzed. First line treatment consisted of steroids at the dose of 2 mg/kg/day for all patients. Second-line therapy consisted of anti-IL2R monoclonal antibodies (n=6) or anti-thymocyte globulin (n=2). Six patients had received 2 lines of therapy before surgery while 1 had received 4 lines and 1 after 5 lines. The diagnosis of acute GI-GVHD was confirmed by comprehensive GI examination including upper and lower endoscopies with biopsies, wireless video-capsule endoscopy and investigation for eliminate infection. In addition, barium small follow-through was performed to evaluate the extension of the GI damages before surgery in order to guide the intervention. After surgery, oral feeding was encouraged and patients received both enteral and parenteral nutrition until GI-lesions recovery and restoration of intestinal continuity with end-to-end anastomosis. Results: Table 1 summarizes patient's characteristics and outcome. The median occurrence of aGVHD was 30 days (25-34) from transplant. Four patients are alive and GVHD free, 81, 71, 11 and 6 months after transplant. The first patient underwent an initial resection of 150cm of distal ileum with immediate intestinal continuity restoration for a progressive obstructive syndrome. The other patients had proximal ileostomy at 100 cm to 150cm after the post duodenal angle. For one patient, perendoscopic gastrostomy was also realized during surgery to simplify enteral nutrition. Two patients (#4 and #5) died of infections 6 and 13 days after surgery. They were in very bad condition at surgery. Patient #3 who improved his GVHD after surgery, died 225 days later of severe flu infection. Conclusion: Despite its limited size, this cohort of patients with severe rGI-GVHD treated with ileostomy appears promising. Whenever possible, this original and multidisciplinary approach should be considered in patients with grade IV rGI-GVHD after 2 lines of immunosuppressive treatment. A prospective study will start soon to evaluate the safety and efficacy of GI surgery in GI-GVHD after one line of therapy. Table 1: Patients and GVHD characteristics and outcome. # Age Gender Onsetof aGVHDfrom transplant(Days) Number of Prior line of immunosuppressive therapy(Lines) Time of surgery from GVHD (Days) Outcome(Time from transplant) 1 41 1 30 2 185 Alive, GVHD-free (81 months) 2 46 2 30 4 93 Alive, GVHD-free (71 months) 3 61 2 30 2 140 Dead (13months) 4 33 1 26 5 64 Dead (3 months) 5 27 2 25 2 49 Dead (3 months) 6 44 2 34 2 22 Alive, GVHD-free (11 months) 7 21 1 30 2 25 Dead (4 months) 8 33 1 35 2 120 Alive, GVHD free (6 months) Disclosures No relevant conflicts of interest to declare.

Description: Invasive aspergillosis (IA) remains a major concern among patients with hematological malignancies who receive cytotoxic chemotherapy or allogeneic stem cell transplantation (allo-SCT). With a view toward better understanding the differences in terms of clinical significance and outcomes of IA between allo-SCT recipients and patients treated for leukemia, we report a single-center study of 739 unselected consecutive patients treated in our unit between August 2000 and February 2004. IA episodes were classified according to the 2008 EORTC/MSG criteria. Patients were hospitalized either for allo-SCT (n=135), or for the treatment of acute leukemia (n=378), chronic lymphocytic leukemia (n=116), or myelodysplasic syndrome (n=105). Probable or proven IA were observed in 29 patients, among whom 7 were undergoing allo-SCT (5.2%), 20 were treated for acute leukemia (5.3%), 1 for chronic lymphocytic leukemia (0.8%), and 1 for myelodysplastic syndrome (0.95%). Most of the clinical signs were mild and nonspecific, especially in allo-SCT recipients. Chronologically, thoracic computerized tomography (CT) showed the first signs of IA in 38% of cases, and was positive the same day as at least one biological test (bronchoalveolar lavage [BAL] and serum galactomannan antigen) in 41% of cases. BAL was contributive to the diagnosis in 66% of the 29 cases. Comparison of patients undergoing allo-SCT to the others revealed significant differences. In allo-SCT recipients, the onset of IA occurred later than in the other patients, after the neutropenic period. The median time between the last hematologic treatment and the diagnosis of IA were 231 days (range, 68-341) in allo-SCT recipients and 17 days (6-57) in the other patients (p

Description: Abstract 2378 Allogeneic stem cell transplantation (allo-SCT) offers potential curative treatment of for a wide range of otherwise fatal hematological diseases. However, only one third of patients have an HLA-identical sibling donor. We have previously reported that in patients with standard risk malignancy, transplantation from unrelated HLA-allellically matched donors (10/10) led to outcomes similar to those from HLA-identical sibling donors (Yakoub-Agha et al, JCO 2006). Indeed, with the increase in the number of single-child families, stem cell grafts from unrelated donors are being increasingly used and more than 30% of patients eligible for allo-CST, are still lacking a well-matched donor. In the attempt to investigate the impact of unrelated one-antigen HLA-mismatched graft, we report a single center retrospective study on 209 patients who underwent allo-CST from identical HLA-sibling donor (n=123), unrelated HLA-matched donor (10/10) (n=73) and unrelated one-antigen-HLA-mismatched donor (9/10) (n=13) over the last 5 years. In order to homogenize our cohort, patients with CML, aplastic anemia or lymphoproliferative disorder were excluded from the study. Therefore, underlying diseases were AML (n=104), ALL (n=54), myelodysplastic syndrome (n=30), and myeloproliferative syndrome (n=21). Of the 117 (56%) males patients, 49 (23%) received graft from female donor (classical sex-mismatch). Medians age of recipients and donors at transplantation were 45.2 years (4.4-65.5) and 40.8 years (2.0-67.5), respectively. Patients received conditioning regimens using either myeloablative (n=149) including 81 who received High-dose TBI (12Gy) or nonmyeloablative (n=60) including 48 who received low-dose TBI (2Gy). Antithymoglobulin was given to 25 pts. Bone marrow was the main source of stem cells (n=150; 72%). Results: with the median of follow-up of 37.9 months, 78 patients died including 25 from TRM. Relapse was recorded in 70 patients. Seventy-two patients experienced acute GVHD (aGVHD) including 47 with II-IV grades and 30 with III-IV grades. In multivariate analyses, donor type (unrelated regardless the degree of HLA-matching vs related) and conditioning (nonmyeloablative vs myeloablative) were the most important risk factors negatively influencing the overall survival [p=.002; HR=2.038 and p=.016; HR=1.81, respectively) and event-free survival (p=.005; HR=1.783 and p=.015; HR=1.728, respectively). As expected, the only factor that influenced the risk of relapse was the conditioning type (nonmyeloablative vs myeloablative) (p=.048; HR=1.699) while donor type was found to influence TRM (p=.030; HR=2.428). Graft from unrelated one-antigen HLA-mismatched donor (9/10) was the foremost risk factor for acute grade II-IV GVHD (p=.019; HR=2.663; [95%CI: 1.178–6.019]). In conclusion, except for acute II-IV GHVD, allo-CST from unrelated one-antigen HLA-mismatched donor (9/10), seemed to led to outcomes similar to those from HLA-identical unrelated donor (10/10) and may be considered as an alternative option for patients without a full-matched donor. Prospective studies are warranted, however, to confirm our data in larger cohort of patients. Disclosures: No relevant conflicts of interest to declare.

Description: In view of the high observed frequency of bone events following allogeneic haematopoietic stem cell transplantation (allo-SCT), the aim of this prospective study was to evaluate pre-transplant bone status in allo-SCT patients. In the month before transplantation, bone-loss risk factors were documented for 27 patients. We measured the levels of bone remodelling markers (BRMs: plasma osteocalcin and bone & total alkaline phosphatases for bone formation; CTX and telopeptides for bone resorption), together with plasma creatinine, intact PTH, TSH, testosterone, LH, FSH, 25 OH vitamin D, serum calcium & phosphorus and calciuria. In addition, bone mineral density (BMD) at the lumbar spine, femoral neck and hips was measured using double-energy X-ray absorptiometry (DEXA). Spine X-ray measurements were also made. Bisphosphonate, oral calcium and vitamin D or testosterone were administered according to the results of the evaluation.Between June 2006 and March 2007, 13 males and 14 females underwent allo-CST for haematological malignancies. The median age at transplantation was 44 years (range: 22–60). Eighteen had received prior corticosteroid therapy, 10 were smokers and 2 had a history of alcohol abuse. All had received prior chemotherapy, including 2 patients having already undergone autologous SCT. Ten of the 14 women were post-menopausal but none was on hormone replacement therapy. The median BMI was 24 kg/m2 (19–35). The daily calcium intake was low, with a median value of 950 mg/day (467–1852). While serum calcium, phosphorus and creatinine levels were within the corresponding normal ranges for all patients, 15 individuals displayed vitamin D deficiency and 6 had calciuria 〈 100 mg/day. Two patients suffered from hyperparathyroidism. In terms of BRMs, the patients respectively displayed a normal profile (n=9), high bone resorption activity (n=12) or high bone formation/resorption activity (n=5). One patient had hyperthyroidism and another presented hypotestosteronaemia. Bone density results were normal in 16 patients and abnormal in 11 (41%), including 8 with osteopaenia and 3 with osteoporosis. Vertebral fractures were observed in 4 patients. Overall, 18 patients (67%) were considered as having a pathological bone status and required treatment with bisphosphonate alone (n=5), vitamin D supplementation alone (n=11) or both (n=2).This study revealed that a large proportion of allo-CST patients have pre-existing abnormal bone status and thus demonstrates the importance of pre-transplant bone status evaluation in allo-CST candidates. The implementation of appropriate bone-related treatments may reduce the frequency of post-transplant bone events.