ALBERT

Description: Features of herpesvirus infections (CMV, EBV, HHV6) and immune reconstitution after CB transplantation (CBT) has been little studied thus far. Here, we report a retrospective comparison between 15 consecutive adult CBT recipients (CBT group) and 40 patients who received allo-SCT using MUD (MUD group). The 2 groups had no significant differences in terms of gender, age, disease features, transplant characteristics (CB: meloablative conditioning 46% vs. MUD: 35%; P=NS). However, in vivo T-cell depletion was more frequently used in the MUD group (62.5% vs 6%, P=0.0002). As expected, median number of CD34+ stem cells infused was significantly lower in the CBT group (0.15 10*6/Kg vs 8.2 10*6/Kg, P

Description: Although great progress has been made in the treatment of acute lymphoblastic leukemia (ALL), relapse remains a major issue in the follow-up of these patients. Recent data about the emergence of subclones during haematological malignancies suggest that relapses could result from resistant cells initially in minority or from cells driven to resistance by previous treatments. Among the tools allowing for the characterization of leukemic cells, flow cytometry (FCM) is an essential approach. Increasingly used to evaluate minimal residual disease (MRD) based on the immunophenotypic features of the blasts at diagnosis, it can also allow to identify immunophenotypic shifts related to clonal evolution. Such an approach would be best studied by comparing follow-up samples from the same patients. In order to be thorough, this would however require that conditions as similar as possible are applied to both types of cells. This work was designed 1) to compare the immunophenotypic features of B-ALL blast cells with those of normal hematogones, 2)to assess potential immunophenotypic shifts at relapse 3)to determine the stability of markers not classically used at diagnosis during follow-up and their potential utility for MRD. FCM was performed simultaneously on thawed paired samples from 15 patients (9 children aged between 1 and 12 years old and 6 adults aged between 23 and 71 years old) with B-lineage ALL. With a three-tubes 8 colours panel comprising a backbone of CD45, CD34, CD22 and CD10, the expression of 8 markers was examined and compared to that observed on normal hematogones contained in 29 bone marrow samples from healthy donors. These 8 markers were CD7, CD19, CD20, CD24, CD38, CD58, CD81, CD123. Moreover, an additional four colours panel was used to examine the more recently described antigens CD44, CD200, CD304 and Her2Neu. The presence of leukemia associated immunophenotypes (LAIP) was defined as a difference in mean fluorescence intensity (MFI) between hematogones and blasts of at least 2 standard deviations. At diagnosis, the expression of each marker was at variance from that on hematogones yielding LAIP in all patients, with at least 4 aberrant markers (up to 11). Antigens with the most abnormal expression were CD10 (100%), CD24 (93.3%), CD81 (80%), CD38 (60%) and CD58 (53.3%). Antigens with the least aberrant expression were CD19 (20%), CD22 (20%), CD123 (20%), CD34 and CD20 (46,7%). CD44 which is at a low level on hematogones, was present for 80% of the patients at diagnosis and overexpressed in ALL with MLL rearrangement (3/15 cases). CD200 was overexpressed in 73% of the patients while CD304 was present in only 40% of the patients. A single patient was positive for Her2Neu, which remained present at relapse. All patients retained at relapse the same global immunophenotype without any change in the EGIL classification (3 B-I, 8 B-II, 4 B-III) and the difference with hematogones remained. The expression of most markers was similar at diagnosis and relapse. There was no change at all for the expression of CD38 which therefore appears as the most interesting marker for follow-up and MRD in ALL. Only one patient each showed a change in the expression of CD44, CD58 or CD123. As a whole, stable markers were CD58, CD44, CD200, CD81 and CD24 in contrast with CD19, CD22, CD123, CD304, CD24 and CD20 which changed in 27 to 67% of the patients. Four patients displayed no immunophenotypic change at relapse while 3 showed a modification of a single marker. For 5 patients, with respectively 6 and 7 LAIP, two markers were modified at relapse. Three markers changed for the patient with Her2Neu expression. Finally, only two patients (13%) showed major changes possibly associated with the emergence of a new clone. This study confirms that B-ALL blast cells differ immunophenotypically from hematogones, although the latter have been reported to possibly be their normal counterpart. These data moreover comfort the interest of using LAIP in the detection of MRD in multiparameter FCM. Finally, since molecular targets of therapeutic monoclonal antibodies do not shift sensibly, their use can also be considered at relapse. Disclosures: No relevant conflicts of interest to declare.

Description: Purpose: Hyper-CVAD developed by the MD Anderson group a few years ago, is one of the standard salvage regimen used for younger relapsed/refractory ALL patients. Recently, targeted therapies using monoclonal antibodies directed against such surface antigens as CD19, CD20 or CD22 have allowed to obtain complete remission (CR) in B ALL expressing these markers. We hypothesized that combining Hyper-CVAD and an anti-CD22 monoclonal antibody could improve the response of such patients. Materials and Methods: This study evaluated the Cheprall salvage regimen, where epratuzumab, a humanized therapeutic monoclonal antibody against CD22 with mainly ADCC property, was associated to Hyper-CVAD, in younger patients (18-59 years old) with relapsed/refractory CD22+ (〉30% of expression) B-ALL. Cheprall consisted of epratuzumab 360 mg/m²/d iv on days 1, 8, 15 and 22, cyclophosphamide 300 mg/m²/12h iv on days 1 to 3, vincristine 2 mg iv on days +4 and +11, doxorubicin 50 mg/m² iv on day +4 and dexamethasone 40 mg po on days 1 to 4 and 11 to 14. The main objective of the study was the overall response rate (CR + CR with incomplete platelets recovery (=50% of bone marrow (BM) blasts decrease or CR with persistent extramedulladory disease) evaluated between 4 and 6 weeks from day+1. Secondary objectives were overall (OS) and leukemia free (LFS) survivals and minimal residual disease (MRD) evaluated by flow cytometry. Results: Between January 2011 and April 2016, 31 patients from 11 French centres were enrolled in the study. A combination of epratuzumab + vincristine and dexamethasone (EVD) only was given to one patient subsequently excluded from the analysis. Among the 30 patients ultimately considered for analyses, 19 were males and the median age was 35 years (range: 21-59). The median time between diagnosis and Cheprall was 14.5 months (range: 4-130) and 13 patients had been allotransplanted. Disease status at time of Cheprall was as follows: primary refractory n=3; first relapse non treated n=13; refractory first relapse n=6, second relapse non treated n=7 and fourth relapse n=1. Median percentage of white blood cells and BM blasts were 4525/mm3(range: 90-86790) and 60% (range: 15-100), respectively. The median CD22 expression of BM blasts was 100% (range: 36-100). Four patients had extramedullary disease: breast n=2, parotid n=1, nervous central system n=1 (deviation). Cheprall was overall well tolerated including mostly pancytopenia as grade ¾ toxicities. Three patients died during aplasia (septis n=1; cerebral haemorrhage n=1, fusariosis n=1) and were not evaluable for response. The overall response rate was 50% (n=15) including 9 CR (30%), 1 CRp (3%) and 5 PR (17%). The number of CR/CRp was higher for patients in first non-treated relapse (54% vs 18%) with an age below 36 years (50% vs 14%), with 18 months (54% vs 17%). Four out of 9 evaluated CR/CRp patients (45%) were documented with negative MRD. All patients in CR/CRp and 1 patient in PR received a consolidation consisting of a second cycle of Cheprall n=5, EVD n=5 or blinatumomab n=1. At the time of analysis (July 2016), all patients have died (during aplasia n=3, progression n=23, multiple organ failure n=1), except three responders still in CR, but yet recently enrolled (2015 n=1, 2016 n=2). Six patients received allogeneic transplant after Cheprall: 4 in CR2, 1 as salvage treatment and 1 in CR3. The last patient included and who achieved CR2 should be allografted in August 2016. Median OS was 3 months (range: 0.2-34.8). Median LFS for those achieving CR/CRp was 4.5 months (range: 1-12). Conclusion: Hyper-CVAD + epratuzumab allowed to obtain 50% of response in this cohort of patients at high risk of failure with refractory/relapsed younger CD22+ B-ALL. Disease improvement was however short-lived, which could be explained either by an insufficient disease load decrease and/or by escape of the blast cells to epratuzumab. This partial efficacy in a population of poor prognosis may suggest that epratuzumab should be tested within first-line chemotherapies as it may participate to decrease MRD level, especially before transplantation. The trial was registered at http://clinicaltrials.gov/ct no.NCT01219816. This study was supported by a grant from the French National Cancer Institute (PHRC 2010). Disclosures Huguet: Pfizer, Novartis, BMS, Ariad, Jazz, Amgen: Membership on an entity's Board of Directors or advisory committees. Thomas:Pfizer: Consultancy. Goldenberg:Immunomedics: Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Wegener:Immunomedics: Employment, Honoraria.

Description: Abstract 3478 RIC regimens prior to allo-SCT are a well-established conditioning approach in elderly adult patients, or in patients with comorbidities not eligible for standard allo-SCT approach. The development of RIC allo-SCT in pediatric patients has been slower than in the adult population because children generally tolerate more intensive myeloablative regimens. However, an increasing proportion of heavily pre-treated pediatric patients might benefit from a non intensive conditioning approach prior to allo-SCT. Thus far, data regarding the efficacy of RIC approaches to treat pediatric patients is still limited, and the role of this approach in pediatric cancer has yet to be defined. The aim of this single-centre retrospective study was to assess the outcome of 19 children (Median age: 12.1 (range, 2.6–18.1) years; gender: male/female 10/9) treated with RIC allo-SCT for different hematological malignancies (n=17; ALL: 6; AML: 4; JMML: 2; NHL: 1; MDS: 1; sAML: 1; biphenotypic leukemia: 1; CML: 1), bone marrow failure (n=1) and neuroblastoma (n=1). In this series, all children were ineligible for a conventional myeloablative conditioning regimen because of severe comorbidities (n=9), a previous auto or allo-SCT (n=7) or a history of extensive chemotherapy (n=3). At time of RIC allo-SCT, most of the patients were in complete remission (n=13; 68%), 2 in partial response and 4 in stable or progressive disease. All patients received a fludarabine-based RIC regimen before allo-SCT (Flu-i.v.Bu-ATG: 8; Flu-Cy-low dose TBI: 7; other combinations: 4). The allogeneic graft was obtained from a match-related donor in 5 cases, match-unrelated donor in 6 cases, and unrelated cord blood (UCB) cells in the remaining 8 cases (42%). The median infused number of nucleated cells and CD34+ stem cells were 4.54×108 and 4.94×106 /kg for peripheral blood stem cells or bone marrow, respectively, and 0.462 and 0.155×106/kg for UCB stem cells. Two patients who received UCB failed to engraft and the median time to ANC〉500/μL was 18 days. With a median follow up of 25 (range, 12–120) months after allo-SCT, treatment related mortality incidence was 16% (n=3). The principal cause of death was relapse (n=6) which occurred at a median time of 112 (range, 29–406) days after RIC allo-SCT. Only 2 patients experienced grade 3–4 acute GVHD and one patient developed chronic GVHD. At 2 years, the Kaplan-Meier estimates of disease-free (DFS) and overall survival (OS) were 45% (95%CI, 25–67%) and 55% (95%CI, 33–75%). In all, these data suggest that favorable outcomes can be achieved with RIC allo-SCT in pediatric patients who are ineligible for standard myeloablative conditioning. Larger prospective studies focusing on decreasing relapse by decreasing tumor burden prior to RIC and by using early immunomodulating approaches after allo-SCT are needed because RIC allo-SCT may help cure many very-high-risk pediatric patients. Disclosures: Mohty: Genzyme: Consultancy, Honoraria, None, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; Janssen Cilag: Consultancy, Honoraria; Celgene: Honoraria.

Description: Background: Gemtuzumab Ozogamicin (GO) is a humanized anti-CD33 monoclonal antibody linked to calicheamicin. It was developed to treat relapsed CD33+ AML patients, first at the dose of 9 mg/m² administered twice at day +1 and +14, than combined with chemotherapy but with reduced dose to lower toxicity. In 2008, we reported a 63% (50% of complete remission (CR) + 13% of CR with incomplete platelets recovery (CRp), platelets

Description: Reduced intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT) is increasingly used in children, especially in those who have been heavily pre-treated or who have received prior autologous or allo-SCT. Of note, use of cord blood as stem cell source is currently a standard of care in pediatric patients lacking a sibling or an unrelated donor. Currently, comparison between matched siblings (SIB), unrelated donors (URD), or umbilical cord blood (UCB) allo-SCT has not yet been reported in the RIC setting in children. Here, we compared reduced-intensity conditioning allo-SCT performed between January 1998 and December 2012 in 124 consecutive children (= 10 years at time of transplant. Regarding RIC regimen, fludarabine and iv busulfan was the most commonly used regimen for SIB and URD cases while it was fludarabine + low-dose total body irradiation (Minneapolis approach) for UCB patients. The use of in-vivo T cell depletion (ATG) was significantly less frequent in the UCB group (16.7% vs 53.1% (SIB) vs 66.1% (URD), p

Description: Introduction The FLAMSA sequential treatment with chemotherapy followed by reduced-intensity conditioning (RIC) for allogeneic stem cell transplantation (allo-SCT) has been introduced few years ago for adult refractory acute myeloid leukemia (AML) showing high activity and relatively good survivals in this particular setting (Schmid, Blood, 2006). There is no study at our knowledge reporting the results of the sequential approach in pediatric refractory AML patients. Here we report our own experience in 6 children using a debulking chemotherapy combining clofarabine and Ara-C followed by RIC before allo-SCT. Patients and Methods These preliminary results included 3 males and 3 females with a median age of 7 years (range: 4-11). All cases had received a sequential regimen before allo-SCT at the CHU of Nantes (n=5) or at the CHU of Montpellier (n=1) for primary refractory AML (n=1), refractory relapsed AML (n=3), slow responder relapsed AML (n=1) and blastic JMML (n=1). Sequential regimen consisted of 1) clofarabine 30 mg/m²/d days-13 to -9, Ara-C 1g/m²/d days-13 to-9 followed by RIC combining cyclophosphamide 60 mg/kg/d day-5, iv Busulfan 3.2 mg/Kg/d days -4 to -3 and ATG 2.5 mg/Kg/d days -3 to -2 in 5 patients and 2) clofarabine 30 mg/m²/d days-13 to -9, Ara-C 1g/m²/d days-13 to-9 followed by RIC total body irradiation 4 grays day-5, cyclophosphamide 40 mg/Kg/d days -4 to -3, and ATG 2.5 mg/Kg/d days -3 to -2 in 1 patient. One patient received a graft from a sibling donor while the five other patients received a graft from an unrelated donor (10/10 n=3; 9/10 n=2). All patients received PBSC as stem cells graft. Ciclosporine alone was used in case of related donor while ciclosporin+MMF were used in case of an unrelated donor. Results Engraftment was observed in 4 patients (67%) and 3 patients out of the 5 refractory cases achieved complete remission (CR) after transplant. The 2 patients who had an autologous reconstitution, relapsed and died rapidly. Considering the 4 patients achieving full engraftment and CR, only one relapsed at day+60 and died of relapse. The three other patients are alive in CR at +12, +35 and +51 months post-transplant. Conclusion To our knowledge, this is the first report of a sequential allo-SCT approach for refractory pediatric AML patients. Although the number of patients is limited in our cohort, the results showed here are very encouraging as half of the patients are alive in CR with full engraftment. These results have to be confirmed prospectively. Disclosures: No relevant conflicts of interest to declare.

Description: The efficacy of induction chemotherapy in childhood acute lymphoblastic leukemia (ALL) is usually evaluated on day 35. However, at this stage, many patients have already begun to recover and present with a regenerative bone marrow (BM) where hematogones may make the identification of residual blast cells problematic both in morphology and in flow cytometry (FCM). In the FRALLE (French Acute Lymphoblastic Leukemia) trials, evaluation is proposed on days 8, 21 and 35. Here we evaluated whether FCM performed on day 21 (D21), when hematogones are still absent, would prove informative. The cohort reported here was constituted of 45 children aged between 1 and 20 years old (median 6) treated for ALL according to the FRALLE recommendations since 2006. There were 81% B-ALL, 17% T-ALL and 2% of mixed phenotype acute leukemia (MPAL, T/My). At diagnosis, the mean percentage of BM blasts was 50%. Classification according to the European Group for Immunophenotyping of Leukemia (EGIL) was 3 B-I, 21 B-II, 11 B-III, 2 B-IV and 1 T-I, 2 T-II and 4 T-III. Extensive immunophenotyping at diagnosis identified a median of 3 leukemia associated immunophenotypes (LAIP, range 1-5), defined as discriminant from hematogones. Corticosensitivity was defined on a complete blood count (CBC) as less than 1 G/L of blast cells on day 8. Chemosensitivity was assessed on a bone marrow aspiration at day 21, both morphologically (〈 5% blasts) and in FCM (MRD0). Molecular biology (according to Biomed2) was performed on BM samples collected on days 35 (MRD1) and 70 (MRD2). Follow-up median time was 59 months (3-276). Corticosensitivity was observed for 39/43 patients (one had received corticosteroids for a tonsillitis before being referred and diagnosed with ALL and another one had less than 1 G/L of blasts at diagnosis). Five/44 patients were identified as chemoresistant by morphology on D21 (one aplastic sample). Enough cells were available for minimal residual disease (MRD) by FCM in 43 patients, on bone marrow collected on EDTA. As a mean, 586 328 total nucleated cells were acquired in FCM (range 9 616 - 1 751 000) thereby providing good sensitivity. Multiparameter FCM in 6 to 8 colors was performed on a single tube, customized according to each patient’s LAIP. Five MRD thresholds were defined as follows : level 1, 〉10-2 detected blasts; level 2, 10-3-

Description: Blinatumomab is a monoclonal bispecific antibody, combining two binding sites: a CD3 site for T cells and a CD19site for the target B cells. Blinatumomab has demonstrated efficacy and safety in adult relapsed/refractory B-precursor ALL (R/R ALL) populations, but also in the pediatric R/R ALL population as shown by interim results from the Phase 1b/2 study MT103-205 (Gore L et al, ASH 2014). Patients and methods: this retrospective study aims to evaluate safety and efficacy of Blinatumomab in 17 pediatric pts with B-precursor ALL treated in 4 french hematological centers between April 2013 and December 2015 within a compassionate use frame. These 17 pts represent the whole population of French children treated with Blinatumomab outside a clinical trial but under a Temporary Authorization for Use (ATU) given by the French Regulatory Agency (ANSM) within this period. Median age was 6.6 years (8 months - 16.6 years). Results: 1)7 pts (41%) received Blinatumomab at a dose of 15 mcg/m2/day for 28 days/cycle in first or second complete remission for minimal residual disease (MRD) persistence. Six of those 7 pts had a molecular remission (MRD

Description: We studied the impact of a set of immune cells contained within granulocyte colony-stimulating factor–mobilized peripheral blood stem cell grafts (naïve and memory T-cell subsets, B cells, regulatory T cells, invariant natural killer T cells [iNKTs], NK cells, and dendritic cell subsets) in patients (n = 80) undergoing allogeneic stem cell transplantation (SCT), using the composite end point of graft-versus-host disease (GVHD)-free and progression-free survival (GPFS) as the primary end point. We observed that GPFS incidences in patients receiving iNKT doses above and below the median were 49% vs 22%, respectively (P = .007). In multivariate analysis, the iNKT dose was the only parameter with a significant impact on GPFS (hazard ratio = 0.48; 95% confidence interval, 0.27-0.85; P = .01). The incidences of severe grade III to IV acute GVHD and National Institutes of Health grade 2 to 3 chronic GVHD (12% and 16%, respectively) were low and associated with the use of antithymocyte globulin in 91% of patients. No difference in GVHD incidence was reported according to the iNKT dose. In conclusion, a higher dose of iNKTs within the graft is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content to improve allo-SCT outcome.