ALBERT

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: Abstract 135 From December 2000 to June 2010, 1201 children with SR-BCP-ALL (age: 1–9 years, WBC

Beschreibung: 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-

Beschreibung: Autoimmune lymphoproliferative syndrome (ALPS) is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, accumulation of double-negative TCRαβ+ CD4−CD8− T cells (DNT cells), and autoimmunity. Previously, DNT cell detection and a functional defect of T cells in a FAS-induced apoptosis test in vitro had been used for ALPS diagnosis. However, a functional defect can also be detected in mutation-positive relatives (MPRs) who remain free of any ALPS-related disease. In contrast, lymphocytes from patients carrying a somatic mutation of FAS exhibit normal sensitivity to FAS-induced apoptosis in vitro. We assessed the soluble FAS-L concentration in the plasma of ALPS patients carrying FAS mutations. Overall, we showed that determination of the FAS-L represents, together with the IL-10 concentration and the DNT cell percentage, a reliable tool for the diagnosis of ALPS.

Beschreibung: Abstract 2570 Background. Current outcome of very early relapse of ALL in children remains poor. Clofarabine, a novel nucleoside analog has proven effective as a single agent in childhood ALL second relapse, providing a CR+CRp ratio of 20 to 28 % (Jeha 2006, Kearns 2006). Clofarabine in combination with cyclophosphamide and Etoposide provided a 44% overal response rate rate in children with advanced ALL (Hijiya 2011) and further multi-drug combinations need to be investigated. The VANDA regimen that has proven effective in high risk ALL and in ALL relapse (Domenech 2008) was used as a template, Cytarabine being replaced by Clofarabine in this combination. Procedure. A phase I study combining escalating doses of Clofarabine with fixed doses of Mitoxantrone, Etoposide, Asparaginase and Dexamethasone was undertaken in children presenting very early (18 months or less after initial diagnosis) medullary or combined ALL relapse or children with second relapse or post transplant relapse. A 3+3 rolling design was used for escalating clofarabine doses with 25% increments from 20 to 40mg/m2/d. Results. Sixteen patients were enrolled, eleven in first marrow relapse, four in second relapse. One patient with an initially refractory disease was secondary excluded as he didn't fulfill the inclusion criteria. Four patients had previously been allografted. Three patients were enrolled at dose level 1 (clofarabine 20 mg/m2), and 3 patients at dose level 2 (clofarabine 26 mg/m2) without any significant dose-limiting toxicity (DLT). Six patients were enrolled at dose level 3 (clofarabine 32 mg/m2), one patient experienced a liver DLT (gamma GT elevation) exceeding 7 days. At the last planned dose level (clofarabine 40 mg/m2), two patients had a grade 3 transaminase elevation exceeding 7 days and 2 patients had a severe fungal infection. Median duration of neutropenia was 20 days (15–31). Other expected toxicities included 3 documented bacterial infections, 3 mucosal toxicities needing parenteral support, one lung aspergillosis, one transient insulin dependant hyperglycemia. There was no toxic death. Out of 15 evaluable patients, 11 (73%) achieved a complete cytological remission (CR). 4 relapses occurred at 1, 1, 2 and 3 months. 6 patients proceeded to allo-SCT with 2 second allografts. One allografted patient died from EBV-PTLD, the others are alive at 2, 13, 13, 15, and 29 months post-transplant. Conclusion. Clofarabine in combination with four major antileukemic drugs appeared feasible and effective in inducing complete remission in those high risk patients. Clofarabine MTD was 32 mg/m2/d for five days and this dose step is currently being expanded to a total of 10 patients, before a phase II study will be proposed for early and very early ALL relapses. Study registered at ClinicalTrials.gov (http://clinicaltrials.gov/ct2/show/NCT01279096). Disclosures: Off Label Use: off label use of clofarabine.

Beschreibung: Evans syndrome (ES) is a rare severe autoimmune disorder characterized by the combination of autoimmune hemolytic anemia and immune thrombocytopenia. In most cases, the underlying cause is unknown. We sought to identify genetic defects in pediatric ES (pES), based on a hypothesis of strong genetic determinism. In a national, prospective cohort of 203 patients with early-onset ES (median [range] age at last follow-up: 16.3 years ([1.2-41.0 years]) initiated in 2004, 80 nonselected consecutive individuals underwent genetic testing. The clinical data were analyzed as a function of the genetic findings. Fifty-two patients (65%) received a genetic diagnosis (the M+ group): 49 carried germline mutations and 3 carried somatic variants. Thirty-two (40%) had pathogenic mutations in 1 of 9 genes known to be involved in primary immunodeficiencies (TNFRSF6, CTLA4, STAT3, PIK3CD, CBL, ADAR1, LRBA, RAG1, and KRAS), whereas 20 patients (25%) carried probable pathogenic variants in 16 genes that had not previously been reported in the context of autoimmune disease. Lastly, no genetic abnormalities were found in the remaining 28 patients (35%, the M− group). The M+ group displayed more severe disease than the M− group, with a greater frequency of additional immunopathologic manifestations and a greater median number of lines of treatment. Six patients (all from the M+ group) died during the study. In conclusion, pES was potentially genetically determined in at least 65% of cases. Systematic, wide-ranging genetic screening should be offered in pES; the genetic findings have prognostic significance and may guide the choice of a targeted treatment.

Beschreibung: Introduction: CD38 is a type II transmembrane glycoprotein expressed on immature T and B lymphocytes (thymocytes and hematogones), NK cells, activated T-cells, plasma-cells, monocytes and red blood cells. This ectoenzyme is a ribonucleosyl cyclase (Cyclic ADP ribose hydrolase) involved in the regulation of calcium fluxes. CD38 is absent from quiescent lymphocytes. The lowest levels are present on erythrocytes while CD38 is brightly expressed by hematogones and plasma-cells. The latter has led to the development of daratumumab, a therapeutic monoclonal antibody used in the treatment of multiple myeloma. Because of the wide distribution of this antigen, other diseases could be considered for such a therapy. Among them, acute lymphoblastic leukemia (ALL) could represent an interesting target. We thus investigated the level of expression of CD38 in a cohort of 128 samples from B-lineage ALL (113 at diagnosis and 15 at relapse). We also compared it to that of the normal counterpart of these blasts, hematogones, because of the potential use of CD38 as a leukemia associated immunophenotype (LAIP). Moreover, in 15 paired samples of diagnosis/relapse, we examined the stability of this expression during disease evolution. Patients, Material and Methods: A total of 62 females and 66 males were included in the cohort, with a median age of 20 years old (range 4 months to 90 years). There were 57 children (below 15 yo), 13 adolescents and young adults (15-25 yo) and 58 adults. CD38 expression was investigated in 45 bone marrow (BM) samples and in 83 peripheral blood (PB) samples. The median level of blasts was 62% in BM and 48% in PB. According to EGIL classification, there were 19 B-I, 66 B-II, 38 B-III, 2 B-IV and intracytoplasmic mu chain was not investigated in 3 cases. In parallel, 26 samples of BM with morphologically evidence hematogones were used to compare the level of expression of CD38 on these cells. Immunophenotyping panels comprised CD38 antibodies conjugated to allophycocyanin and all samples were analyzed on a Canto II flow cytometer (BD biosciences, San Jose, CA). Results: CD38 was always present on hematogones. It was expressed by 122 of the 128 B-ALL samples tested (95,3%). Partial expression, between 20 and 70% of the blasts was noted for 9 patients (7%) while 113 patients (88%) had more than 70% positive blasts. For 102 patients (79,7%), the whole population was stained (100% of the blasts). Among the 15 samples obtained at relapse, CD38 was always expressed, partially only in one case. The mean fluorescence intensity (MFI), expressed using the flow cytometer arbitrary units (AI) ranged between 510 and 1396 AI (median 878 AI) in the group of patients with 20-70% CD38+ blasts (n=9) and between 317 and 26466 AI (median 4704 AI) for the 113 patients with more than 70% CD38+ blasts (n=113). The level of fluorescence was always higher on hematogones compared to blasts, with a median MFI of 14915 AI (range 3834-34501 AI). CD38 expression is also used as a LAIP, to discriminate minimal residual disease (MRD) from regenerating hematogones. In our department, a threshold of 6300 AI (median of hematogones - 2 SD) is used to define the LAIP of CD38low blasts. In the cohort reported here, 82 patients presented this LAIP (64%). Conversely, high expression of CD38 with an MFI 〉10 000 AI was present for 23 of the 128 patients. For the 15 patients with paired samples of diagnosis and relapse, 9 had low levels of CD38 (LAIP). In none of these cases modulation of CD38 was observed, this LAIP remaining stable over time. Conclusion: CD38 has been reported by several authors as one of the best LAIP marker for the detection of MRD in B-ALL, allowing a good follow-up of the patients because of its stability. Moreover, this study confirms that CD38 could be a valuable therapeutic target in most of B-ALL cases, being expressed in over 95% of the cases. Used together with chemotherapy, daratumumab could thus be useful to treat B-ALL both in first line or at relapse. Disclosures Moreau: Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau.

Beschreibung: Background Cranial radiotherapy (CRT) is associated with early and late side effects. Intrathecal (IT) and systemic chemotherapy could successfully replace CRT in most protocols for standard risk ALL. However, in medium and high risk ALL patients (pts) its omission is still debatable. Aim We investigated the long-term outcome, the occurrence of second malignant neoplasms (SMN) and the incidence of late toxicities in pts randomized for receiving or not CRT in the EORTC 58832 study. Methods From 1983 to 1989, ALL children under 18 years (yrs) were included in EORTC Children Leukemia Group BFM-oriented studies, either 58831, for standard risk pts (Riehm-Langerman Risk Factor (RF) 〈 1.2), or 58832, for medium risk (RF 1.2-1.69) and high risk pts (RF ≥1.7). Pts with central nervous system (CNS) involvement at diagnosis were ineligible. The present report focusses on pts included in the 58832 trial (randomized for receiving or not prophylactic CRT). Prophylactic CNS therapy consisted of 4 high-dose methotrexate (HD-MTX) injections (2500 mg/m2) during consolidation and 7 IT MTX injections scheduled during the treatment period. Pts still in complete remission (CR) after the end of late intensification were randomized for receiving prophylactic CRT (standard arm) or not (experimental arm) before the start of continuation therapy. Dose of CRT was age dependent: 24 Gy (〉 2 yrs), 20 Gy (1-2 yrs) and 16 Gy (〈 1 yr). Endpoints were: disease-free survival (DFS) (event: relapse, death in CR), incidence of SMN, event-free survival (EFS) (event: relapse, death in CR, SMN), incidence of late toxicities, and overall survival (OS) from randomization. Results 788 pts were included in the 58831/58832 study. Among them, 189 were randomized in the 58832 study to receive CRT (n=93) or No CRT (n=96). A total of 6 pts did not meet eligibility criteria, 2 had an early relapse, 3 had an early protocol violation and 2 refused allocated treatment. Finally, 176 randomized pts were included in the analyses: 84 in the CRT group and 92 in the No CRT group. The median follow-up was 20 yrs (range 4-32 yrs). Omission of CRT did not increase the 25-yr incidence of isolated CNS relapse, any CNS relapse or non-CNS relapse (4.8 vs 6.5; 11.9 vs 8.7 and 16.7 vs 21.8 in the CRT vs No CRT arms, respectively). No relapses occurred after 10 yrs. The 25-yr DFS rates were similar in both arms: 70.2% with CRT and 67.4% without CRT; No CRT vs CRT hazard ratio (HR)=1.08, 95% CI (0.63, 1.83). CRT was associated with an increase of the 25-yr SMN incidence: 13.2% with CRT and 3.9% without CRT. In the CRT arm, 9 pts (10.7%) developed SMN: 2 acute myeloid leukemias (AML), 1 non-Hodgkin lymphoma, 1 thyroid carcinoma, 4 meningiomas and 1 malignant histiocytosis. One SMN (meningioma) occurred after a CNS combined relapse. Three pts developed second SMN (meningiomas): 1 after an AML and 2 after a first meningioma. In the No CRT arm, 3 pts (3.3%) had SMN: 1 pleomorphic xanthoastrocytoma, 1 melanoma and 1 adenocarcinoma of the ileum. One SMN occurred after a bone marrow (BM) relapse. The 25-yr EFS rates were similar in both arms: 60.3% with CRT and 63.2% without CRT, HR=0.90, 95% CI (0.55, 1.46). CRT was also associated with an increase of late CNS and endocrine toxicities. Five pts (19.2% of the pts with available data) developed leukoencephalopathy in the CRT arm, versus 2 pts (8.7%) in the No CRT arm. Noteworthy, 1 of those 2 pts received CRT for a BM relapse, while the other received total body irradiation for a CNS relapse. Stroke was observed in 2 pts (7.7%) who received CRT. In contrast, there was no clear increase of the incidence of cognitive disturbance after CRT: 33.3% in the CRT arm vs 25.0% in the No CRT arm. Regarding endocrine toxicities, GH deficiency, hypothyroidism and precocious puberty were more frequent in the CRT arm: 53.1% vs 29.6%, 27.8% vs 0% and 29.4% vs 0%, respectively. Finally, the 25-yr OS rates were similar in both arms: 78.5% with CRT and 78.1% without CRT, HR=1.00, 95% CI (0.53, 1.88). Conclusion In medium and high risk pts without CNS involvement at diagnosis and treated with HD-MTX in the EORTC trial 58832 (1983-1989), omission of CRT did not increase the risk of CNS or non-CNS relapse. On long-term evaluation, CRT was associated with a higher incidence of SMN, late CNS and endocrine toxicities. These long-term results indicate that prophylactic CRT can be safely omitted in childhood medium and high risk ALL pts receiving IT and systemic chemotherapy (including HD-MTX) as CNS prophylaxis. Table Table. Disclosures No relevant conflicts of interest to declare.