Minimal Residual Disease Clearance Research Articles

Busulfan (Bu) in Combination with Double Nucleoside Analogues Fludarabine (Flu) and Clofarabine (Clo) Plus Vorinostat As a Novel Reduced Toxicity Conditioning Regimen for Allogeneic Hematopoietic Cell Transplantation (HCT) in Patients (pts) with Acute Leukemia

The combination of IV Bu and Flu is an effective and well-tolerated HCT conditioning regimen for patients with advanced acute leukemia. However, relapse remains the main reason for treatment failure. We combined two nucleoside analogues Flu and Clo, and showed cytotoxic synergy when combined with Bu, first in pre-clinical models, and subsequently in a randomized clinical study (Andersson BBMT 2011). We selected the optimized regimen from Andersson et al, and combined with escalating doses of the histone deacetylase inhibitor vorinostat in attempts to augment disease control without increasing toxicity. Herein, we describe our initial experience with this novel regimen for patients with advanced acute leukemia or MDS undergoing allogeneic HCT.Methods: Vorinostat (200mg to 1200mg) was escalated in cohorts of 3 patients, and administered 1 hour prior to the fixed regimen of Flu 10 mg/m2 followed by Clo 40 mg/m2 followed by Bu. The nucleoside analogs were infused over 1 hour once daily for 4 days, followed on each day by Bu, infused over 3 hours to a pharmacokinetically targeted daily area under the curve (AUC) of 5500 μMol-min ± 5%. Dilantin was given for seizure prophylaxis. GVHD prophylaxis was based on tacrolimus andmini-MTX, with the addition of rabbit anti-thymocyte globulin (4 mg/kg total dose) for unrelated donor transplants. The presence of minimal residual disease (MRD) was determined by multiparameter flow cytometry.Results: 21 patients (7 AML, 14 ALL) with median age 43 years (range 19-60) received an allogeneic matched sibling (n=10) or unrelated donor (n=11) HCT in CR1 (n=13), CR2 (n=1), primary induction failure (n=5), or relapse (n=2). Twelve patients had MRD or overt disease present at time of HCT. Ten patients had high-risk karyotype defined by t(9;22) (n=3), t(4;11) (n=2), complex (n=2), or del 5 or 7 (n=3). Median time from diagnosis to HCT was 5 months (range 3-48). Median time to ANC > 0.5 x 109/L and platelets > 20 x 109/L were 11 (range 10-15) and 12 days (5-30), respectively. Excluding grade 1 toxicities, the most commonly reported toxicities were nausea with maximum grade 2 (71%), followed by grade 2 or 3 mucositis (64%). Grade 2 or 3 reversible elevation of liver function tests was noted in 54% of pts, including 1 case of reversible VOD. No renal toxicities were noted. Two patients with prior systemic treatment for acute GVHD died of infections (disseminated adenovirus and E.coli sepsis). Ten patients achieved CR and clearance of MRD by day +30 after HCT; one patient had progressive disease and on patient died before disease restaging. Eighteen patients achieved 100% donor chimerism at 30 days following HCT, 2 patients remain with mixed chimerism by 100 day assessment following HCT, and 1 patient died before assessment. The incidence of grades II-IV acute GVHD is 43% and chronic extensive GVHD among 18 evaluable patients is 24%. With a median follow-up of 10 months among surviving patients (1-22), overall and progression-free survival at 6 months is 95% and 71%, respectively. Early results suggest excellent disease control in MRD negative patients (Figure).Conclusion: The Vorinostat-Flu-Clo-Bu combination is well-tolerated and did not add appreciable toxicity in these patients with high-risk leukemia. Longer follow-up is needed to better assess disease control. [Display omitted] DisclosuresCiurea:Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership. Patel:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties.

TCR Clonal Evolution in AML Patients in Morphologic Remission Treated with Anti-PD1 Antibody, Nivolumab

We are conducting a clinical trial titled "Randomized Phase II Study to Assess the Role of Nivolumab as Single Agent to Eliminate Minimal Residual Disease and Maintain Remission in Acute Myelogenous Leukemia (AML) Patients After Chemotherapy" (REMAIN trial) (NCT02275533).A critical barrier in developing immunotherapies is the identification of predictive biomarkers of response to therapy. T lymphocytes play critical roles in response to immunotherapies but their clonality and temporal changes in the T cell repertoire during treatment have not been well investigated. Recent advances in deep sequencing technology make it possible to characterize the T cell receptor (TCR) repertoire generated following immunotherapy.In this study, we characterized T cell repertoire in peripheral blood and/or bone marrow samples of three AML patients on the REMAIN trial before and after nivolumab treatment. Using Illumina MiSeq sequencer and total RNA from each sample, we conducted deep sequencing of TCR-α and -β chains, and calculated the diversity index (inverse Simpson’s index) in their CDR3 sequences to assess overall clonality of T cells. We obtained total CDR3 clonotypes of 420,765 ± 155,449 (average ± standard deviation) for TCR-α and 410,786 ± 115,219 for TCR-β per each sample. Interestingly, we found that certain TCR-α and -β clonotypes were drastically enriched in the bone marrow samples after nivolumab treatment. Many of these enriched TCR clonotypes were minimal or undetectable before nivolumab treatment, indicating that nivolumab might induce expansion of anti-AML T cell subclones. Particularly, nivolumab treatment led to marked reduction of TCR diversity indexes in both peripheral blood and bone marrow samples of one AML patient, who had shown a clearance of minimal residual disease as detected by WT1 qRT-PCR.Our results thus far indicate the feasibility of this type of comprehensive analysis of TCR repertoire in the context of immunotherapy for AML. Preliminary results suggest that such analysis may be utilized to predict response of immune checkpoint blockade, and could also be useful to identify high-affinity TCRs for adaptive T cell therapy approaches. DisclosuresLiu:BMS: Research Funding; Karyopharm: Research Funding. Odenike:Incyte: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Suneisis: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Geron: Research Funding; CTI/Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Spectrum: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Algeta: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Stock:ADC Therapeutics: Honoraria; Amgen: Honoraria; Gilead Sciences: Honoraria; Sigma-Tau: Honoraria, Research Funding; Royalties for a chapter in Up to Date: Patents & Royalties.

Early Stage Follicular Lymphoma. Predictive Role of Minimal Residual Disease (MRD) and Impact of MRD-Driven Treatment with Radiotherapy and Rituximab on Clinical Outcome

Background. In localized follicular lymphoma (FL, stage I-II), BCL2/IGH+ cells can be detected in the peripheral blood (PB) and/or bone marrow (BM) in 66.7% of cases (Pulsoni et al, BJH 2007). We hereby analyzed the prognostic impact of MRD in localized FL and explored the possibility of a MRD-guided therapeutic approach on a series of patients with a long follow-up.Methods. Between April 2000 and February 2015, 67 consecutive patients with a confirmed histologic diagnosis of stage I/II FL followed at our Center were enrolled in the study. PB and BM samples were collected at enrollment in all patients and investigated by qualitative PCR to identify the presence of a BCL2/IGH rearrangement. Paraffin-embedded lymph nodes (LN) were studied when available. Patients who proved positive at baseline were studied for MRD every 6 months. Real-Time Quantitative PCR (RQ-PCR) was retrospectively performed according to material availability. All patients were treated with involved field radiotherapy (RT) (24-30 Gy); from 2005, patients who were MRD+ after RT received rituximab (R) (375 mg/m2, 4 weekly administration). The median follow-up is 67 months (17-183); 21 patients (31%) have relapsed after a median of 37 months (17-165) from diagnosis.Results. At baseline, a clonal marker was found by qualitative PCR in 48/67 cases (72%): 36 were MBR+ (54%), 6 mcr+ (9%), 6 showed a minor BCL2 rearrangement (9%), while 19 (28%) were negative. Fifteen of the latter 19 were analyzed by RQ-PCR and 4 proved MBR+. Of the 13 available LNs, 11 showed the same molecular marker identified in the PB/BM; 2 cases, negative in the PB/BM, showed a rearrangement in the LN only. After RT, 40/42 MBR+/mcr+ patients were analyzed: 20 resulted MRD-, while 20 persisted MRD+. Regardless of the post-RT MRD status, an equal number of relapses was recorded in both groups (7 each). R treatment was administered to the 20 MRD+ patients after RT. Sixteen (80%) achieved a MRD- status after R: over time, 7/16 patients converted to MRD+ and 4 relapsed, whilst 9/16 patients (56.2%) remain persistently MRD- and none has relapsed so far.To evaluate the impact of R, we considered a series of 27 patients MRD+ after RT or who were MRD- and became MRD+ during the follow-up. Of the 19 patients who received R (1 could not be studied), 15 (79%) did not relapse, while of the 8 untreated patients (pre-2005), 6 (75%) relapsed (p=0.025). Progression-free survival (PFS) was significantly longer for R-treated patients (p=0.0412) (Fig. 1).To define the predictive role of MRD in the entire cohort regardless of post-RT treatment, we considered the 39 patients with molecular follow-up. Thirteen have relapsed: 10/13 (77%) were MRD+ in the follow-up, including the pre-relapse time point, while 3 resulted persistently MRD-. Contrariwise, of the 26/39 patients in continuous remission, 18 (69%) were persistently MRD- while 8 were MRD+ (p=0.015). PFS was significantly better for MRD- patients (p=0.0163) (Fig. 2).RQ-PCR was performed in 30 MBR+ patients: 17 (57%) showed a tumor burden ≥10-5 and 13 <10-5. Tumor burden at diagnosis predicted the MRD clearance following RT: 9/13 (69%) cases with low tumor burden resulted MRD- after RT compared to 2/17 (12%) cases with high tumor burden (p=0.0027). Contrariwise, tumor burden did not predict the occurrence of relapse.Conclusions. Early stage FL at diagnosis can have a heterogenous disease extension: 2 of our cases were truly localized, showing a molecular marker only in the LN. However, in most cases the use of combined qualitative approaches, including canonical MBR/mcr and minor rearrangements, together with RQ-PCR has allowed to identify circulating BCL2/IGH+ cells (52/67 cases: 77.6%), despite a negative BM biopsy. RT induced a MRD negativity in 50% of BCL2/IGH+ patients, but this did not impact on clinical outcome. The administration of R in MRD+ patients decreased significantly the risk of a subsequent relapse and improved PFS. Regardless of treatment, MRD positivity during the follow-up is a predictor of relapse and PFS. Tumor burden at diagnosis is associated with MRD clearance after RT. We support the use of a MRD-driven treatment with anti-CD20 monoclonal antibodies in patients with localized FL after RT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Minimal Residual Disease in Patients with Follicular Lymphoma Treated with Obinutuzumab or Rituximab As First-Line Induction Immunochemotherapy and Maintenance in the Phase 3 GALLIUM Study

Fludarabine, Cytarabine, Daunoxome Plus Dasatinib Has High Efficacy with an Acceptable Toxicity Profile As Either Consolidation or Salvage Regimen in Adult Philadelphia Positive Acute Lymphoblastic Leukemia Patients

BACKGROUND AND AIMSThe prognosis of Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) patients has improved since the introduction of tyrosine kinase inhibitors (TKI). The inclusion of TKIs in standard ALL protocols allows a great increase in complete molecular responses, but at the price of non negligible toxicities and high rates of toxic deaths. On the other and TKI monotherapy as induction treatment allows to rapidly achieve complete hematologic remission (CR) but only a minority of patients achieve a complete molecular response with high risk of relapse. On the other hand,In the last years we tested a combination of Fludarabine, Cytarabine, Daunoxome (FLAD) with or without TKIs (mainly Dasatinib) as salvage regimen in relapsed-refractory ALL, with acceptable toxicity and good efficacy. We decided to apply the same schedule in newly diagnosed Ph+ ALL as consolidation treatment after a two months TKI (Dasatinib) monotherapy induction on a minimal residual disease condition.MATERIALS AND METHODSFLAD regimen consisted with a three-days administration of Fludarabine 30 mg/sqm followed four hours later by Cytarabine 2000 mg/sqm and Daunoxome 100 mg/sqm. TKI were suspended during chemotherapy administration and were re-administrated starting from day 5. G-CSF was given to all patients from day 4 to complete hematological recovery. FLAD was administrated for up to two cycles; all patients with available donor proceeded to allogeneic bone marrow transplantation (allo-BMT) after FLAD. Minimal residual disease (MRD) was evaluated in all patients after each FLAD either by RQ-PCR for VDJ rearrangements, multicolor flow cytometry (MFC) and RQ-PCR for BCR/Abl.Ten Ph+ ALL have been treated with FLAD + TKIs from January 2008 to December 2014: six patients received FLAD as salvage regimen, two of them in post allo-BMT setting, whereas four patients were treated frontline, after hematological CR was obtained with Dasatinib + steroids induction. All frontline patients proceeded to allo-BMT after two FLAD. Median age for frontline patients was 50 years (range 29-58), median follow-up was 20 months.RESULTSAs salvage regimen, 5/6 patients achieved hematological CR after FLAD, with three patients achieving also MFC MRD negativity and clearance of VDJ and BCR/Abl transcript. All patients who did not receive subsequent BMT relapsed, whereas of the two transplanted patients one is still in CR after a follow-up of 38 months.In the frontline setting, all patients received 70 days induction of Dasatinib + Steroids and achieved CR with complete hematological recovery. BCR/Abl transcript could be detected in all patients on BM samples on day 33 and on day 70 (Fig. 1), two patientshad MFC MRD positivity both on day 33 and on day 70, whereas two patients achieved MFC MRD negativity on day 33.FLAD was very well tolerated, with negligible non hematological toxicity, with a median duration of ANC <500 and PLT <20000 of 7 and 9 days, respectively, slightly higher in the second course. Median time between the beginning of first and second course was 35 days, whereas median time from second course to allo-BM was 44 days.Two patients achieved BCR/Abl negativity after first FLAD. All patients achieved molecular complete response after the second course (Fig. 1). No patient experienced relapse, whereas one patient died in CR on day +289 after allo-BMT due to myocardial viral infection.CONCLUSIONSFLAD has a very good efficacy in adult Ph+ ALL, with an acceptable toxicity profile. Deep responses have been observed in relapsed patients, and all newly diagnosed patients who received FLAD as consolidation regimen had achieved molecular CR before allo-BMT. Achieving complete hematological response with Dasatinib + steroids allowed us to safely administer two FLAD courses. [Display omitted] DisclosuresOff Label Use: Use of liposomal daunorubicin in the treatment of ALL.

Post-Remission Treatment with Chemotherapy or Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) of High-Risk (HR) Philadelphia Chromosome-Negative (Ph-neg) Adult Acute Lymphoblastic Leukemia (ALL) According to Minimal Residual Disease (MRD). Preliminary Results of the Pethema ALL-HR-11 Trial

▪Introduction: Recent studies have shown that young to middle-aged adults who receive a pediatric-inspired chemotherapy regimen for treatment of Ph-neg ALL do not appear to require an alloHSCT if they achieve good response on MRD testing after induction therapy. Patients (pts) who are not good MRD responders achieve better outcomes with alloHSCT than their counterparts who do not receive alloHSCT. However, it is not clear if this approach can be translated to adult ALL pts with HR features at baseline. The aim of the prospective ALL-HR-11 trial from the Spanish PETHEMA Group was to evaluate the response to a differentiated post-induction therapy (chemotherapy or alloHSCT) according to MRD levels (assessed by 8-color, centrally-performed flow cytometry at the end of induction-week 5- and consolidation therapy-week 17-) in HR Ph-neg adult ALL patients. Patients and methods: HR ALL included one or more of the following parameters at baseline: age 30-60 yr, WBC count >30x109/L for B-cell precursor ALL or >100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or MLL rearrangements or complex karyotype. Induction therapy included vincristine, prednisone, daunorubicin and asparaginase (E coli native or PEG according to center availability) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in pts not achieving CR or those in CR with MRD≥0.1% at the end of induction. For pts in CR and MRD<0.1% early consolidation therapy included 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and high-dose asparaginase (E coli native or PEG). These pts continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2 yr. in CR if MRD levels after consolidation were <0.01%, otherwise they were assigned to alloHSCT. Pts in CR after Induction-2 received one consolidation cycle and were assigned to alloHSCT. Results: On June 2015, 115 HR ALL pts were evaluable [mean (SD) age 38(13) yr, 67 males, 80/114 precursor B-ALL, 34/114 T-ALL, WBC count 56(96) x109/L]. Results of Induction-1: therapy-related death: 4(4%), resistance: 11 (10%), CR: 95(86%). MRD<0.1% at the end of induction was observed in 75% of CR patients. Induction-2 was administered to 33 patients (no CR: 11, CR and MRD≥0.1%: 22). No differences in the CR rate or in the rate of MRD clearance after induction were observed according to the type of asparaginase administered, although significantly increased hepatic toxicity in consolidation was observed in patients treated with PEG-asparaginase. The 2-yr DFS and OS probabilities for whole series were 51%±18% and 62%±13%. By intention-to treat after Induction-1 36 pts were assigned to alloHSCT and 68 to delayed consolidation and maintenance. The 2-yr DFS and OS probabilities were 54%±25% and 49%±20%, respectively, for pts assigned to alloHSCT, and 50%±22% and 73%±17%, respectively, for those assigned to chemotherapy (P =0.002 for OS comparison). Patients with MRD<0.1% at the end of induction and <0.01% at the end of consolidation (n=51) showed a 2-yr DFS and OS of 55%±25% and 81%±18%, respectively. Conclusions: The preliminary results of this trial, in which the post-induction therapy decision is only based on MRD results, suggest that in HR, Ph-neg adult ALL pts with adequate MRD response after induction and after consolidation the results of therapy are not hampered by avoiding alloHSCT. Supported by grants RD12/0036/0029 (RTICC, FEDER), PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain DisclosuresNo relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation for poor-risk CLL: dissecting immune-modulating strategies for disease eradication and treatment of relapse.

To elucidate factors contributing to the effectiveness of allogeneic hematopoietic stem cell transplantation (alloHCT) in high-risk CLL, immune interventions, GvHD and clinical outcome of 77 consecutive patients allografted for CLL were analyzed. Immune modulation (immunosuppression tapering, rituximab-augmented donor lymphocyte infusions) was guided by minimal residual disease (MRD) monitoring and commenced at a median of 91 (22-273) days after alloHCT, resulting in a probability of being event free and MRD-negative 1 year after transplant of 57% (84% in those encountering chronic GvHD). Patients who were event free and MRD-negative at the 12-month landmark had a 4-year PFS of 77% and largely remained durably MRD-negative if MRD clearance had occurred subsequent to immune modulation. Three-year overall survival, PFS, relapse incidence and non-relapse mortality of all 77 patients were 69, 57, 26 and 24%, respectively. Survival was not affected by EBMT risk category but by active disease at alloHCT, which could not be overcome by intensification of conditioning. Twenty-three patients who experienced relapse post alloHCT had a survival of 56% at 2 years after CLL recurrence. In conclusion, MRD-guided immune modulation after alloHCT for high-risk CLL can provide durable MRD clearance in more than half of the patients.

Long-Term Follow-up Data Support the Curative Potential of Allogeneic Hematopoietic Cell Transplantation in Patients with Chronic Lymphocytic Leukemia: A Retrospective Analysis from the Chronic Malignancies Working Party of the EBMT

Objectives: In patients with refractory chronic lymphocytic leukemia (CLL), the potential to cure is unique to allogeneic stem cell transplantation (HSCT). Clearance of minimal residual disease by suspected graft-versus-leukemia effects has been described elegantly and documented in several independent patient cohorts. Yet data from large numbers of patients which support the concept of cure by allogeneic transplantation have not been published. With the advent of new targeted therapies for patients with advanced chemo-refractory CLL, this information becomes crucial for clinical decision making and patient counselling. Therefore, we aimed at the description of long-term survival outcomes, and the estimation of excess mortality compared to the age- and sex-matched general population.Patients and Methods: Data from patients with CLL who had received a first allogeneic HSCT from an HLA-identical sibling (SIB) or alternative donor between January 2000 and December 2010, and who were registered with the EBMT database, were analyzed. Patients with Richter’s syndrome and with syngeneic donors were excluded from this analysis. Survival probabilities were calculated by means of the Kaplan-Meier estimator both in the total population, and in patients who passed the 2- and 5-year landmark without previous relapse or progression. Excess mortality of the landmark populations compared to an age-, sex- and calendar year-matched general population was estimated with a Cox regression model for relative survival using the R-package “relsurv”.Results: In total 2589 patients were included into the analysis. The median follow-up of patients alive at the end of follow-up was 4.0 years (range: 1 to 161 months). The median age at HSCT was 55 years (range: 12 to 74 years). One hundred and fifty eight patients (6.1%) were below 40 years of age at the time of transplantation. Seventy-four percent of patients were male. The remission status at the time of transplantation was reported as complete remission in 15%, partial remission in 47%, and stable disease or progressive disease in 32%. Information on the remission status was not available for 6% of the patients. Fifty-one percent of the patients had an HLA-matched sibling donor and seventy-seven percent of patients received reduced-intensity conditioning.For the whole cohort of patients, the 5- and 10-year overall survival (OS), progression-free survival (PFS), and non-relapse mortality (NRM) were 45%, 35%, 36%, and 35%, 28%, 40%, respectively. The cumulative incidence of relapse (CIR) was 21% at two years, 29% at five years, and 32% at ten years. A total of 1023 patients and 394 patients were alive without relapse or progression, and in follow-up at two and five years after HSCT. Five years after patients had passed the 2- and 5-year landmark, OS, CIR and NRM were 73%, 22%, 16%, and 83%, 11%, 10% respectively.Compared to the general population excess mortality of the 5-year landmark population in the subsequent five years was estimated to be 3% for male patients at an age of 45 years, 10% for male patients at an age of 55 years, and 24% for male patients at an age of 65 years (see Figure 1). For female patients in this 5-year landmark population, the corresponding excess mortality rates were 4%, 11%, and 27%.Patients without progression and with CR at any time from HSCT to the two and five-year landmarks had a slightly better outcome than those who had never had CR. Surprisingly, this was not a result of a lower CIR but of a lower NRM.Conclusion: Long-term follow-up data derived from the EBMT registry show a steady decline in hazard of relapse after allogeneic HSCT, yet relapse continues to be a threat. Moreover, even patients alive and disease-free after 5 years are still confronted with substantial NRM. These results show that there is room for improvement of long-term patient care.By comparing mortality of younger patients who passed the 5-year landmark with the general population, only marginal excess mortality was observed, while elderly patients still had substantial excess mortality beyond this landmark. Nevertheless, the results indicate that a significant fraction of patients can be cured by allogeneic HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Excellent Outcome Of MRD Low Risk Patients Is Sustained With Greater Than 10 Years Follow Up – Results Of UK ALL Trials 1997 -2003

BackgroundSensitive measurement of minimal residual disease (MRD) at the end of induction, to a minimum limit of detection of 0.01% is known to highlight a large group of patients (>40%) with an excellent (>90%) short term EFS. The UKALL 2003 study showed treatment reduction is feasible in children and young adults with no MRD >0.01% at day 28 of therapy (Vora et al, Lancet Oncology 2013). Nevertheless, follow up in that study and other recent trials is relatively short, raising concerns about using this result to infer the safety of further therapy reduction in the future. In order to provide insight into the applicability of this result, we have studied the longer term outcome of patients with MRD <0.01% at day 28 in our non-interventional pilot studies; UK ALL 97, 97/99 and 2003. MethodsWe examined 225 patients treated on one of the 3 trials between 1997 and 2003 for whom MRD results were available. The UK Medical Research Council (MRC) protocol ALL97 (1997–1999) had a 5 year EFS of 74% and OS 83.5%, and its amended version ALL 97/99 (1999–2002) had a 5 year of EFS 80% and OS 88%. These studies both compared in a randomised fashion, the efficacy and toxicity of dexamethasone with prednisolone, and 6-thioguanine with 6-Mercaptopurine. The successor trial ALL 2003, had a 5 year EFS of 87.7% and an OS of 91.3%, using more intensive induction including dexamethasone for all patients and PEGylated asparaginase. These trials provided an opportunity to determine the impact of MRD clearance on EFS and OS, with extended follow-up. MRD status at the end of induction chemotherapy was defined as low risk if no MRD was detected by at least one marker sensitive to 0.01%. In the ALL 97 study, MRD was measured by radiolabelled allele specific oligoprobing (sensitive to 0.01%); in ALL 99 and 2003 it was measured using the Euro-MRD RQ PCR of antigen receptor genes (quantitative range 0.01%). Results100 patients (44%) were low risk by MRD at end of induction. The median range of follow-up across the trials was 7 years 4 months to 12 years 11 months (longest individual follow up 14 years 1 month). There was no significant difference in NCI risk group between MRD positive and negative patients. DiscussionOur pilot data defines a cohort of 44% of children with ALL who have a very good EFS and an excellent long term OS. Of 100 day 28 MRD low risk patients, 6 died: 1 because of toxicity at 1 month post diagnosis; and 1 from influenza 147 months post diagnosis whilst in CR; only 4 patients had relapses, at 17, 45, 74 and 125 months post diagnosis. ConclusionThe excellent outcome for childhood ALL with low risk MRD after induction chemotherapy is sustained in the intermediate to long term. This result supports the potential for further reduction in therapy in subsequent trials for these children, without risk of worsening outcome. Disclosures:No relevant conflicts of interest to declare.Table 1EFS and OS by trial for MRD low risk patientsMRD low risk end of induction (%)Median follow-up10 year EFS MRD low risk group (%)10 year OS MRD low risk group (%)MRC ALL 9739 (n=26)12y11m (range 12y2m - 14y1m)8592MRC ALL 97/9925 (n=19)10y7m (range 9y4m -11y5m)9595MRC ALL 2003 pilot66 (n=55)7y4m (range 1m - 7y11m)9395

Treatment reduction for children and young adults with low-risk acute lymphoblastic leukaemia defined by minimal residual disease (UKALL 2003): a randomised controlled trial

Minimal residual disease (MRD) is the most sensitive and specific predictor of relapse risk in children with acute lymphoblastic leukaemia (ALL) during remission. We assessed whether treatment intensity could be adjusted for children and young adults according to MRD risk stratification. Between Oct 1, 2003 and June 30, 2011, consecutive children and young adults (aged 1-25 years) with ALL from the UK and Ireland were recruited. Eligible patients were categorised into clinical standard, intermediate, and high risk groups on the basis of a combination of National Cancer Institute (NCI) criteria, cytogenetics, and early response to induction therapy, which was assessed by bone marrow blast counts taken at days 8 (NCI high-risk patients) and 15 (NCI standard-risk patients) after induction began. Clinical standard-risk and intermediate-risk patients were assessed for MRD. Those classified as MRD low risk (undetectable MRD at the end of induction [day 29] or detectable MRD at day 29 that became undetectable by week 11) were randomly assigned to receive one or two delayed intensification courses. Patients had received induction, consolidation, and interim maintenance therapy before they began delayed intensification. Delayed intensification consisted of pegylated asparaginase on day 4; vincristine, dexamethasone (alternate weeks), and doxorubicin for 3 weeks; and 4 weeks of cyclophosphamide and cytarabine. Computer randomisation was done with stratification by MRD result and balancing for sex, age, and white blood cell count at diagnosis by method of minimisation. Patients, clinicians, and data analysts were not masked to treatment allocation. The primary outcome was event-free survival (EFS), which was defined as time to relapse, secondary tumour, or death. Our aim was to rule out a 7% reduction in EFS in the group given one delayed intensification course relative to that given two delayed intensification courses. Analyses were by intention to treat. This trial is registered, number ISRCTN07355119. Of 3207 patients registered in the trial overall, 521 MRD low-risk patients were randomly assigned to receive one (n=260) or two (n=261) delayed intensification courses. Median follow-up of these patients was 57 months (IQR 42-72). We recorded no significant difference in EFS between the group given one delayed intensification (94·4% at 5 years, 95% CI 91·1-97·7) and that given two delayed intensifications (95·5%, 92·8-98·2; unadjusted odds ratio 1·00, 95% CI 0·43-2·31; two-sided p=0·99). The difference in 5-year EFS between the two groups was 1·1% (95% CI -5·6 to 2·5). 11 patients (actuarial relapse at 5 years 5·6%, 95% CI 2·3-8·9) given one delayed intensification and six (2·4%, 0·2-4·6) given two delayed intensifications relapsed (p=0·23). Three patients (1·2%, 0-2·6) given two delayed intensifications died of treatment-related causes compared with none in the group given one delayed intensification (p=0·08). We recorded no significant difference between groups for serious adverse events and grade 3 or 4 toxic effects; however, the second delayed intensification course was associated with one (<1%) treatment-related death, and 74 episodes of grade 3 or 4 toxic effects in 45 patients (17%). Treatment reduction is feasible for children and young adults with ALL who are predicted to have a low risk of relapse on the basis of rapid clearance of MRD by the end of induction therapy. Medical Research Council and Leukaemia and Lymphoma Research.

About the Benefits of Immunotherapy for High-Risk Neuroblastoma

porting the Memorial Sloan-Kettering Cancer Center's institutional experience using immunotherapy for the treatment of high-risk neuroblastoma. There is an unmet need to develop novel ther- apies for children with neuroblastoma, and the longstanding efforts of the Memorial Sloan-Kettering Cancer Center team in developing immunotherapy approaches over the last three de- cades should be commended. The article 1 presents interesting data about the changing pattern of relapse after immunotherapy as well as about novel biomarkers, such as the clearance of minimal residual disease before and after immunotherapy using an mRNA marker panel andtheuseof FCGR2Apolymorphisms,humanleukocyteantigen, and killer immunoglobulin-like receptor genotyping. FivelevelsofevidencehavebeendescribedbytheOxfordCentre

How I treat relapsed childhood acute lymphoblastic leukemia

AbstractThe most common cause of treatment failure in childhood acute lymphoblastic leukemia (ALL) remains relapse, occurring in ∼ 15%-20% of patients. Survival of relapsed patients can be predicted by site of relapse, length of first complete remission, and immunophenotype of relapsed ALL. BM and early relapse (< 30 months from diagnosis), as well as T-ALL, are associated with worse prognosis than isolated extramedullary or late relapse (> 30 months from diagnosis). In addition, persistence of minimal residual disease (MRD) at the end of induction or consolidation therapy predicts poor outcome because children with detectable MRD are more likely to relapse than those in molecular remission, even after allogeneic hematopoietic stem cell transplantation. We offer hematopoietic stem cell transplantation to any child with high-risk features because these patients are virtually incurable with chemotherapy alone. By contrast, we treat children with first late BM relapse of B-cell precursor ALL and good clearance of MRD with a chemotherapy approach. We use both systemic and local treatment for extramedullary relapse, mainly represented by radiotherapy and, in case of testicular involvement, by orchiectomy. Innovative approaches, including new agents or strategies of immunotherapy, are under investigation in trials enrolling patients with resistant or more advanced disease.

Minimal Residual Disease At 3 Months, Combined to the Presence of IKZF1 Deletion in B-Lineage or Absence of NOTCH1 pathway Mutation in T-Lineage, Recapitulates the Disease Risk Assessment in Adults with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia - A GRAALL Study

Abstract 572 Aim.In recent series of adults with acute lymphoblastic leukemia (ALL), the GRAALL (ASH 2009, abstract 577) and other cooperative groups have confirmed the strong prognostic value of Ig/TCR minimal residual disease (MRD) on patient outcome. Despite this, age, WBC, CNS involvement, recurrent chromosomal translocations, and early response to steroids and chemotherapy remain frequently used to tailor post-remission therapy and envision allogeneic stem cell transplantation (SCT) in most adult ALL trials. We updated our MRD study, now with 262 patients who all achieved complete remission (CR) after the first induction and were assessed for MRD after induction (MRD1, at 6 weeks) and consolidation (MRD2, at 12 weeks). One hundred and fifty-eight patients had Philadelphia chromosome (Ph)-negative B-cell precursor ALL (BCP-ALL), while 104 had T-cell ALL (T-ALL). Since 107 of the BCP-ALL (68%) were studied for IKZF1 deletion and 90 of the T-ALL patients (87%) for NOTCH1/FBXW7 mutations, we were able to reassess the MRD significance according to these newly described oncogenic markers. These two covariates (i.e. MRD and IKZF1/NOTCH1/FBXW7 genetics) allowed us to redefine a much simpler yet more powerful stratification of disease risk in both BCP- ALL and T-ALL subsets. Methods.All 262 patients studied (median age, 31.5 years) were treated in the GRAALL-2003 and GRAALL-2005 trials. Although they were younger and had more frequently circulating blasts, other characteristics and outcome did not differ from patients treated in the same trials but not assessed for MRD. Ig/TCR MRD levels were determined according to Euro-MRD guidelines (Leukemia 2007;21:604). IKZF1 deletions were assessed by multiplex multi-fluorescent PCR. NOTCH1/FBXW7 mutations were assessed as previously described (Blood 2009;113:3918). Multivariate backward stepwise selection Cox models were used for the cumulative incidence of relapse (CIR), disease-free (DFS) and overall survival (OS) endpoints, after censoring transplanted patients at SCT. Models were always adjusted on age (35-year cutoff), WBC (30 and 100 G/L cutoff for BCP- and T-ALL, respectively), CNS involvement, and trial. Additional BCP-specific covariates included CD20 expression, t(4;11) and t(1;19) translocations, and IKZF1 deletion. Additional T-specific covariates included cortical immunophenotype according to the EGIL classification, TLX1 overexpression, and NOTCH1/FBXW7 mutation. Finally, allogeneic SCT was re-evaluated in the newly defined risk subsets, as a time-dependent covariate. Results.An initial multivariate analysis revealed that among blood response after 1 week of steroid, bone marrow response after 2 weeks of therapy, and molecular response at both MRD1 and MRD2 time-points, the MRD2 level was the main and sole independent predictor of relapse (P=0.003). In BCP-ALL patients, persistent MRD2 and IKZF1 deletion were the only two independent factors identified, the presence of at least one factor defining 51% high-risk patients with 52% versus 15% CIR (HR, 3.8; P= 0.008), 41% versus 81% DFS (HR, 3.6; P= 0.005), and 54% versus 80% OS (HR, 3.9; P= 0.015) at 4 years. Allogeneic SCT in first CR significantly decreased relapse incidence and prolonged DFS in these new high-risk BCP-ALL patients (HR, 0.23 and 0.40; P= 0.016 and 0.05, respectively). In T-ALL patients, persistent MRD2 and lack of NOTCH1/FBXW7 mutation were the only two independent factors identified, the presence of at least one factor defining 49% high-risk patients with 64% versus 12% CIR (HR, 6.4; P= 0.002), 36% versus 88% DFS (HR, 6.4; P= 0.002), and 41% versus 95% OS (HR, 7.3; P= 0.015) at 4 years. SCT had no significant effect on relapse incidence and DFS in these new high-risk T-ALL patients. Conclusion.In adult patients with Ph-negative ALL treated with the pediatric-inspired GRAALL regimen, IKZF1 deletion in BCP-ALL, NOTCH1/FBXW7 mutation in T-ALL, and MRD at 3 months in both subsets replace all classical risk factors, leading to a new simplified prognostic scoring system based only on IKZF1 and NOTCH1/FBXW7 genetics and MRD clearance. This new risk score identifies approximately half of the patients as good-risk, with a relapse incidence as low as 10–15%. It will be validated and used prospectively in the next generation of GRAALL trials, to stratify both new drug evaluation and SCT in first CR. Disclosures:No relevant conflicts of interest to declare.

Bendamustine and Rituximab for the Treatment of Multiply Relapsed Hairy Cell Leukemia,

Abstract 3909 Background:Hairy cell leukemia (HCL) is highly sensitive to purine analogs cladribine (CdA) and pentostatin (DCF), but patients who relapse have decreasing remission rates with each course and can eventually become purine analog-refractory. Bendamustine and rituximab (BR) have been reported as effective with acceptable toxicity in several B-cell malignancies, particularly B-cell lymphomas and chronic lymphocytic leukemia. The structure of bendamustine contains an alkylating group and also part of cladribine, suggesting it might be useful in HCL. In one case report, bendamustine achieved a transient partial response in a patient with relapsed/refractory HCL, but its activity in other patients with this disease is not reported. The combination of DCF and rituximab (DCFR) is reported to achieve high complete remission (CR) rates in HCL in retrospective series, but prospective phase II trials of this combination have not been reported. Methods:To determine the activity of BR relative to DCFR in HCL, a randomized trial was undertaken in multiply relapsed HCL comparing the 2 regimens in which each arm could constitute a prospective phase II trial, with 2-way crossover for lack of response to or relapse from the originally assigned regimen. The primary endpoint is an overall response rate of 65% for each arm and the 2 arms will be compared with respect to response and other secondary endpoints including toxicity, response duration, and eradication of minimal residual disease (MRD). Patients received 6 cycles at 4-week intervals of rituximab 375 mg/m2 days 1 and 15 with either pentostatin at 4 mg/m2 days 1 and 15, or bendamustine days 1 and 2. To test the tolerability of bendamustine prior to randomizing 56 patients between the 2 arms, 12 non-randomized patients received BR using 70 (n=6) or 90 (n=6) mg/m2/dose of bendamustine. Doses of all agents could be delayed but not reduced. Results:A total of 20 patients are so far enrolled and the 12 patients receiving the 2 dose levels of BR are evaluable for response and toxicity. Patients had 1–6 (median 3) prior courses of purine analog and 8 (67%) had prior rituximab. All toxicity was reversible and only 1 patient at 90 mg/m2 required >2-week delay due to prolonged neutropenia and thrombocytopenia. However, this delay was only between cycles 1 and 2 and not between subsequent cycles after responding to BR. Of the 36 cycles of BR administered to each group of 6 patients on the 2 dose levels of bendamustine, 90 vs 70 mg/m2/dose, common grade 3–4 toxicities included lymphopenia (28 vs 22%), leukopenia (19 vs 17%), and thrombocytopenia (14 vs 17%). Febrile neutropenia requiring hospitalization occurred just once in 3 patients at 90 vs 0 patients at 70 mg/m2/dose. Major response was achieved in 10 (83%) of 12 patients. CR was achieved in 3 (50%) of 6 patients at each dose level, while 2 (33%) at 70 and 3 (50%) at 90 mg/m2 achieved clearance of MRD at all sites including bone marrow aspirate by flow cytometry. No patient in CR has relapsed after 8–14 (median 11) months of follow-up. Of 4 patients evaluable by clone-specific real-time PCR, previously reported sensitive to 1 HCL cell in 106 normal, 3 patients at 90 mg/m2/dose were negative. Conclusions:BR can achieve responses including CRs in multiply relapsed HCL, and its safety profile permits comparison of BR with DCFR using the more common dose level of bendamustine, 90 mg/m2 days 1 and 2. Additional patients and follow-up will be required to access durability of response and long-term eradication of MRD, and to compare BR with DCFR (Supported in part by NCI, intramural research program, NIH, Genentech, Inc, and Cephalon, Inc). Disclosures:Kreitman:Cephalon: Research Funding; Genentech: Research Funding. Off Label Use: Use of bendamustine and rituximab for HCL. Arons:Genentech: Research Funding.

Identification of Post-Induction Minimal Residual Disease by Multidimensional Flow Cytometry Identifies Patients with AML at High Risk of Relapse and Poor Outcome- a Report From the Children's Oncology Group

AbstractAbstract 1702Multidimensional flow cytometry (MDF) is used to identify risk in childhood ALL; however, its utility in AML has been limited. We used 4-color MDF with standard (rather than patient-specific) panels to evaluate diagnostic and postinduction bone marrow specimens from patients treated on Children's Oncology Group (COG) study AAML03P1 for evidence of minimal residual disease (MRD). A total of 254 patients submitted marrow specimens for MRD assessment at the end of induction I, the end of induction II, and the end of therapy. Of the 222 patients with evaluable specimens at the end of induction I, 191 (86%) were in morphologic remission, and 27 (12.2%) had persistence of morphologic disease, 3 had persistent CNS disease and 1 was not evaluable for response. Of those with morphologic disease, 15 were in partial remission (PR, 5%-20% blasts), and 12 had refractory disease (RD, >20% blasts). MDF of specimens showing morphologic disease revealed that 7 (26%) did not have evidence of disease; thus, MDF identified patients with reported morphologic disease who did not have immunophenotypic evidence of disease. Overall, in 222 patients with evaluable marrow at the end of induction I, 69 (31%) had evidence of various levels of MRD by MDF (% blast range, 0.02%-43%, median, 1.5%). For the 208 patients with known cytogenetic data, the presence of MRD was evaluated in the following cytogenetic subgroups: favorable risk, defined as t(8;21) or inv(16) (the Core Binding Factor leukemias); unfavorable risk, defined as –5/del(5q) or –7; and intermediate risk, defined as all other cytogenetic subtypes. MRD prevalence at the end of Induction I in patients with favorable, intermediate-risk, or high-risk cytogenetics was 13%, 36%, and 67%, respectively. Prevalence of MRD at the end of induction I was 50% (10/20) in patients with FLT3/ITD, 40% (4/10) in patients with CEBPA mutations, and 0% (0/5) in patients with NPM1 mutations. Of the 222 patients with evaluable specimens at the end of induction I, 191 had morphologic response to the initial chemotherapy. Of those, 57 (28%) had evidence of disease by MDF. Cumulative relapse risk (RR) and disease-free survival (DFS) was assessed in those with or without MRD. Those with MRD at the end of induction I had a RR at 3 years from the end of induction of 60% vs. 29% for those without MRD (p <0.001). DFS was 32% for those with MRD and 65% for those without MRD (p<0.001). In a multivariate analysis, which included cytogenetic and molecular risk factors, the presence of MRD was highly associated with outcome and was an independent predictor of relapse (p<0.001) and worse survival (p<0.001). We further evaluated the significance of clearance of residual disease. Of the 91 patients evaluated for MRD at the end of therapy, 7 (8%) were MRD-positive (6 of whom relapsed). Of the 84 patients who were MRD-negative, 22 (26%) had previously documented MRD. For those with a history of MRD, RR from the end of therapy was 64%, and for those without previous MRD, it was 25% (p<0.001). Therefore, despite clearance of MRD, patients with previous MRD had a high RR. Given the high correlation of MRD with RR, MDF assessment of post-induction response should be incorporated into AML clinical trials for risk identification and assignment to the appropriate risk-based therapy. Disclosures:No relevant conflicts of interest to declare.

R-CHOP/R-DHAP Compared to R-CHOP Induction Followed by High Dose Therapy with Autologous Stem Cell Transplantation Induces Higher Rates of Molecular Remission In MCL: Results of the MCL Younger Intergroup Trial of the European MCL Network

AbstractAbstract 965 Background and Methods:In the MCL younger trial of the European MCL Network, patients up to 65 years with stage II-IV mantle cell lymphoma were randomized to either a standard arm with 6 cycles of 3-weekly R-CHOP, stem cell mobilization with DexaBEAM and myeloablative treatment with 12 Gy TBI, 2×60mg/kg cyclophosphamide and autologous stem cell transplantation (ASCT) (Arm A), or an experimental treatment arm including 6 cycles of alternating R-CHOP/R-DHAP followed by a high dose Ara-C containing myeloablative therapy with 10 Gy TBI, 4×1.5 g/m2 Ara-C, 140mg/m2 melphalan and ASCT (Arm B). In addition to clinical response assessment, minimal residual disease (MRD) was prospectively monitored on the molecular level by real time quantitative (RQ-) PCR in both arms. MRD samples were collected at diagnosis, at midterm (after 4 induction cycles), after induction (6 cycles), and in 3-monthly intervals after ASCT until clinical progression. MRD results were evaluated according to ESG criteria (van der Velden, Leukemia 2007) and compared to clinical response and outcome. RQ-PCR was designed to reach a sensitivity of 10E-5, MRD negativity (MRD-) was defined as a negative RQ-PCR result with a technical assay sensitivity of at least 10E-04. Molecular response (MR) was defined as MRD- in peripheral blood (PB) and/or bone marrow (BM) at any sampling time point. Results:Until May 2010, 307/422 patients randomized in Germany or France had MRD samples available and a molecular marker for RQ-PCR (158 patients of the R-CHOP arm and 149 of the R-CHOP/R-DHAP arm). Overall 2374 samples, 1639 PB and 735 BM were evaluated for MRD. Clinical parameters as stage, LDH elevation, bone marrow infiltration and MIPI were distributed equally in both groups. Based on 173/307 (56%) patients with available midterm samples, MRD clearance was significantly higher in the experimental arm with 36/84 (43%) MRD- patients compared to only 12/89 (13%) in the standard arm; p<0.0001). This difference was confirmed in both, PB (50/80 (63%) vs. 18/84 (21%); p<0.0001) and BM (31/71 (44%) vs. 12/75 (16%); p=0.0003).After completion of induction 90% of R-CHOP and 94% of R-CHOP/R-DHAP patients achieved a clinical response with a CR rate of 26% and 39%, respectively. The rate patients achieving a MR was significantly higher in the R-CHOP/R-DHAP arm (60/82 (73%) vs. 29/92 (32%) after R-CHOP; p<0.0001). BM clearance from lymphoma cells was inferior after R-CHOP only (MRD- 17/71 (24%) compared to R-CHOP/R-DHAP (MRD- 39/57 (68%)). Accordingly, MRD- in the PB was achieved after R-CHOP in only 40/86 patients (47%) compared to 63/79 (80%) in the R-CHOP/R-DHAP arm. Achievement of BM MR after induction was associated with a significantly improved remission duration in the pooled treatment arms (89% vs. 74% at 24 months, p=0.0017).High-dose consolidation followed by ASCT demonstrated a high impact on tumor reduction in the pooled treatment arms and increased the BM MR rate from 50% to 75% (p = 0.0001, paired samples). This improvement was more prominent in the R-CHOP arm (29% to 65%; p = 0.0023) than in the experimental arm (76% to 88%; p=0.18). Remarkably, sustained PB MR during the first year after ASCT was predictive for outcome in both treatment arms (pooled cohort n = 135, 94% vs. 63% ongoing remissions at 2 years, p=0.0001) R-CHOP: 92% vs. 67%, p=0.0224 and R-CHOP/R-DHAP : 96% vs. 53%, p=0.0016). Conclusion:RQ-PCR is an excellent tool to quantitatively determine the impact of different treatment modalities on tumor clearance and revealed the superiority of the high dose Ara-C containing treatment arm early during treatment. Furthermore it underlines the importance of ASCT consolidation in MCL.Thus, MRD seems to be prognostically more relevant than clinical and morphological complete response assessment and was confirmed as a reliable early predictor of clinical outcome in MCL. Disclosures:Ribrag:LFB: Honoraria, Research Funding; servier: Research Funding; celgene: Research Funding; pfizer: Honoraria; novartis: Honoraria.

UKALL 2003, A Randomised Trial Investigating Treatment Reduction for Children and Young Adults with Minimal Residual Disease Defined Low Risk Acute Lymphoblastic Leukaemia

AbstractAbstract 496UKALL 2003 is an ongoing randomised controlled trial investigating treatment modification according to Minimal Residual Disease (MRD) status at the end of induction and week 11 in children and young adults (up to age 25 years) with ALL. Whilst the randomisation testing the benefit of intensification for an MRD defined high risk group remains open, a treatment reduction randomisation for MRD low risk patients closed in August 2009 after recruiting the target number of patients. Over 2600 patients have so far been enrolled into the trial and we have observed a significant improvement in 5 year event-free (EFS) compared with its predecessor, ALL 97/99, (EFS 87 vs 80%, p<0.0005), most probably related to use of dexamethasone and pegylated asparaginase in all patients. Here we report the results of the MRD low risk randomisation.Patients in the trial are initially stratified by NCI risk, blast karyotype, and morphological marrow response at day 8/15 of induction for allocation to one of 3 treatment regimens, escalating in intensity (A, B, C). NCI standard risk (SR) patients receive a 3 drug induction (Regimen A) whilst high risk (HR) patients receive 4 drugs including daunorubicin (Regimen B). Patients with a slow early response at day 8 (NCI HR) or day 15 (NCI SR) of induction and patients with poor risk cytogenetics are transferred to Regimen C. Patients without poor risk cytogenetics and who have a rapid early response are eligible for the MRD randomisations. MRD was measured using Real Time Quantitative PCR with a sensitivity of 10−4.Five hundred and twenty-one patients who were either MRD negative at day 29 of induction or positive < 10−4 at day 29 but negative by week 11 entered a randomisation comparing two courses of delayed intensification (standard arm, n = 261) with one (treatment reduction arm, n = 260). Of these patients, 342 (65%) were NCI SR and 179 (35%) NCI HR. With follow-up to October 2009 (median 2 years 9 months), there is no difference in 5 year EFS for patients in the standard (94%) or reduced treatment arms (96%). As shown in table 1, the number of patients with relapse is similar in both arms but there was a non-significant excess of deaths in remission, serious adverse events and grade 3/4 toxicity in recipients of 2 DIs. 7/10 relapses have involved the CNS. The 3 CR deaths in the 2 DI arm were due to gram negative sepsis (DI1), pulmonary haemorrhage (DI2) and RSV pneumonitis (Maintenance, Down). There have been no relapses or CR deaths among 23 MRD low risk patients in the 16 – 25 age group.Table 11DI2DIStatistics for 2DIObs/NObs/NOdds ratio(95% CI)2pAny event6/2607/2611.19 (0.40–3.53)0.76Relapse6/2604/2610.69 (0.20–2.38)0.57Remission death0/2603/2617.40 (0.77–71.14)0.08Grade 3/4 toxicity177 (68%)191 (71%)0.2SAEs64 (25%)77 (29.5%)0.2This randomised study provides evidence that treatment can be reduced for around 40% of children and young adults who show rapid clearance of MRD by the end of induction. These patients have an excellent outcome with < 5% risk of relapse on a treatment protocol that does not include cranial radiotherapy or high dose methotrexate and gives minimal exposure to anthracyclines and alkylating agents. Future trials should test the feasibility of further reductions in treatment for this group of patients. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease (MRD) Based Preemptive 5–Azacytidine Treatment Can Prevent or Delay Imminent Relapse In Patients with High-Risk MDS or AML After Allogenic HSCT – Results of the “RELAZA” Trial

AbstractAbstract 679Besides GVHD, relapse is one of the major challenges in the care of patients with MDS or AML undergoing allogeneic hematopoietic stem cell transplantation (HSCT). However, relapse can be predicted in case of CD34-expression on the leukemic clone by a sensitive chimerism analysis in sorted CD34+ peripheral blood cells. If CD34+ donor cells drop below 80%, relapse is inevitable within 6–8 weeks in the absence of interventions like immediate cessation of immunosuppressive drugs or the administration of DLI. However, both approaches often result in clinically significant GVHD.We report results of an ongoing phase II clinical trial evaluating the efficacy of 5-azacitidine (5-aza) to treat MRD and thus prevent hematological relapse in patients with CD34+ AML or MDS and a decreasing CD34-donor chimerism after allogenic HSCT. Therefore, a total of 60 patients with CD34+ MDS (n=5) or AML (n=55) were prospectively screened after HSCT for a decreasing chimerism of donor CD34+ cells in the peripheral blood. In case of MRD and consequently imminent relapse, defined as a drop of donor CD34+ cells below 80%, 5-aza was given at a dose of 75mg/m2/d s.c. day 1–7. A total of 4 cycles every 28 days were allowed. Patients showing either insufficient CD34-chimerism response, i.e. an increase but below 80%, or again decreased below the cut-off were eligible for a second treatment phase.A median of 158 days after HSCT, 20 (CR1: n=3, CR2: n=4, PR1: n=3, PR2: n=1, PD: n=3 prior to HSCT) out of 59 patients screened entered the treatment phase of the study with a median of 21% (range 0–79%) CD34+ donor cells in the peripheral blood (PB). However, a complete overall PB donor chimerism and less than 5% marrow blasts were documented in all patients before 5-aza treatment.Currently, 19 patients are evaluable for response one month after the 4th cycle of 5-aza. As a result, 10 out of 19 patients (53%) showed a complete clearance of MRD defined as an increase of CD34-donor chimerism > 80%. In 2 patients (10%) an increase in CD34-donor cells but to less than 80% was observed. Additionally, 3 patients (16%) demonstrated no increase in CD34-donor chimerism but did not develop hematological relapse while on therapy. Four patients (21%) relapsed during therapy (between cycle 2 and 4). Out of the 10 complete responders, there were 3 patients (30%) again showing an increase in MRD after a median of 139 days after the end of 5-aza treatment with all again responding to repeated 5-aza treatment.Reversible neutropenia grade 3/4 occurred in 76% of the patients whereas thrombocytopenia grade 3/4 was observed in 65%. There were 7 patients who showed clinical signs of graft versus host disease (GvHD) under therapy with 5-aza, which in 6 of them already existed before the initiation of 5-aza treatment.Preemptive treatment with 5-aza seems to be a potent strategy to prevent or delay hematological relapse of patients with MDS or AML and MRD after allogeneic HSCT. Disclosures:Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Monitoring minimal residual disease with flow cytometry, antigen-receptor gene rearrangements and fusion transcript quantification in Philadelphia-positive childhood acute lymphoblastic leukemia

In this study, we followed minimal residual disease (MRD) in eight children with Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) using (i) flow cytometry (FCM), (ii) real-time quantitative PCR of IG/TCR gene rearrangements and (iii) RT-PCR detecting fusion gene transcripts. In six of the eight cases the kinetics of MRD clearance was comparable. One of the two discordant cases could be explained by presence of an alternative fusion transcript. The other discordant case showed high BCR-ABL1 RNA level while the other methods did not detect any MRD. In our limited material quantitative RT-PCR of fusion gene transcripts seemed particularly useful to measure MRD in Ph+ ALL. However, BCR-ABL1 expression may not reflect the percentage of leukemic cells as FCM and IG/TCR rearrangement quantification do, and these methods are thus complementary.

In Vivo Purging Followed by AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT) in PATIENTS with MULTIPLE MYELOMA (MM): Preliminary RESULTS of a Pilot STUDY

In the past decade, thalidomide, bortezomib, and lenalidomide have emerged as highly active agents in the treatment of MM. However, the optimal schedule and association of these drugs is still under investigation. Thalidomide has shown to be highly thrombogenic in induction therapy, especially in patients with high tumor burden, while Bortezomib (BOR) is not; moreover BOR does not affect peripheral blood stem cell (PBSC) collection and it has also been used in combination with Melphalan (MEL) before ASCT. Both in vitro laboratory data and preliminary phase II trials suggest synergistic effects when BOR is combined with Cyclophosphamide (CY); this drug is effective in treatment of MM and is not stem cell toxic. We started a pilot study in high risk MM patients, fit for ASCT, combining BOR, CY and dexamethasone (DEX) as induction and mobilizing therapy (CY-BOR), followed by supplemented BOR-ASCT, to determine: 1-feasibility and Response Rate (RR): percentage of complete remission (CR) and near CR (nCR) at day + 90 after ASCT; 2- clearance of Minimal Residual Disease (MRD) both in PBSC harvest and in bone marrow at day +90 after SCT; the percentage of plasma cells (PC) in PBSC harvested has been assessed by flow cytometry (FC), by using CD38, CD45, CD56, CD138, CD19, kappa and lambda staining. Patients receive three 3-weeks treatment cycles with BOR 1.3 milligrams/squared-meter/dose on days 1,4,8,11, in combination with DEX 40 milligrams/day i.v. on days 1,4,8,11 and CY i.v. 300 milligrams/squared-meter/dose on days 1,8,15. After the third course, patients achieving at least PR, undergo PBSC mobilization with BOR 1.3 milligrams/squared-meter/dose on days 1,4,8,11 in combination with DEX 40 milligrams/day i.v. on days 1,4,8,11 and CY i.v. 3000 milligrams/squared-meter/dose on day 8; Granulocyte-Colony Stimulating Factor (5 micrograms/kilogram) is given starting on day 9. Patients who successfully mobilize an adequate PBSC amount are planned to receive conditioning regimen, combining high-dose MEL(on day -1) with BOR (1.3 milligrams/squared-meter/dose on days -8 and -4). At present 10 patients (6 female and 4 male; median age: 71) have been enrolled and 8 are evaluable for response before PBSC harvest; 4 patients have been mobilized: in all of them we were able to collect a sufficient PBSC amount (median 11.1; range 3–11.2 CD34+ cells/kilograms) and to perform ASCT. Conditioning was well tolerated and was followed by quick engraftment: the median time to neutrophils>500/mcl was 9 days and 12 days for unsupported platelets count>20,000/mcl, without major extrahematological toxicity. With a minimum follow-up of 90 days after ASCT, 3 evaluable patients are alive, two in VGPR and one in CR; with a median follow up of 108 days (range 95–201) all 10 patients are alive and 100% of the evaluable patients achieved at least PR after only 2–3 courses of CY-BOR (table 1). The hematological toxicity was negligible and we did not observe neither thromboembolic events nor grade 3–4 neurotoxicity. Only one patient experienced a transient increase of liver enzymes during the first two courses of therapy. The study of MRD by flow cytometry in PBSC harvest of two evaluable patients shows complete clearance of plasmacells; in the others the MRD study is still ongoing and complete data will be further presented. This preliminary experience shows that this schedule is well tolerated and very effective also in elderly patients, allowing to collect a clonal plasmacells free harvest. Whether this will translate in an “in vivo” MRD clearance after ASCT, it should be confirmed by a longer follow up and by a PCR monitoring with patients specific probes (which is still ongoing).Pts UPNAgeSexStageISSPrevious lines of treatmentResponse before PBSC mobilizationHarvest: CD34+ cell amountSCTStatus at last Follow-up159FIII AIII1nCR3,99×106/kgYesA&W (CR)268MIII BIII2nCR11×106/kgYesA&W (nCR)373FIII AIII0VGPR11,2×106/kgYesA&W (VGPR)472FIII AII1VGPR3×106/kgYesA&W (VGPR)569MII AI0PRNENEA&W677FIII AII1PRNENEA&W752MIII AIII0PRNENEA&W874FIII BII2VGPR.NENEA&W964FII BII3N.E.NENEA&W1073MIII AII0N.E.NENEA&WTable 1: Baseline characteristics of patients and outcome Legenda: ISS: International Staging System; NE: not evaluable; A&W: alive and well; VGPR: very good partial remission