Relapse Of Acute Leukemia Research Articles

Minimal Residual Disease Status at Day +100 Post Allogeneic Hematopoietic Stem Cell Transplantation Is a Powerful Predictor for Post-Transplant Outcome In Patients with High Risk Acute Leukemia

AbstractAbstract 3550Background and Objectives Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still a major cause for the failure in treatment. It has been shown that there was a closely relationship between the level of minimal residual disease (MRD) and relapse in acute leukemia (AL) patients; However, the application of multiparameter flow cytometry (MFC) for MRD assessment in high risk patients with AL who undergoing allo-HSCT is little concerned. We retrospectively analysed the serial results of MRD of 52 high risk patients with AL to evaluates the prognostic value of MRD pre and post transplantation. Methods52 patients with a median age of 29 (13–55) years have been enrolled on this study in our hospital from January 2003 to September 2008.Diagnoses included AML (n=27) and ALL (n=25). The patients had been analyzed retrospectively the level of MRD pre-(day-30)and post-HSCT(day+30 and +100)using three color FCM with CD45/SSC gating and a comprehensive panel of monoclonal antibodies, at least one leukemia associated aberrant immunophenotype (LAIP) at diagnosis. According to the cutoff value 0.1%, two groups were defined based on the level of patient's MRD level< (low level group) or >= (high level group) 0.1%. ResultsThe median follow up were 23 (range 1–60) months. 1.MRD level declines significantly (P=0.03) post transplant. 2. There were significantly difference between low level and high level group at day -30 before transplant with 3 years event free survival(EFS) and relapse free survival (RFS)(77.4% and 88.4% vs. 22.3% and 25.7%, p=0.007and p=0.001 respectively). 3. Concerning about MRD at day +100 after transplant, outcome was significantly better among patients with low level MRD group versus high group including 3 years OS,EFS and RFS(84.2%, 79.5% and 89.5% versus 22.9%, 9.5% and 11.2%).4. The median time from high level MRD detected first time to clinical relapse was 2.5 (range from 1 to 33) months in relapsed patients. 5. The patients with cGVHD had better 3 years OS and EFS than that without cGVHD(86.3% vs 12.1%, p<0.001 and 65.3% vs.14.8%, p< 0.001 respectively). 6. Multivariate Cox regression analysis revealed that MRD on day +100 as well as chronic GVHD were independent parameters predictive for OS and EFS. ConclusionsMRD monitoring pre- and post-transplant is an important tool to predict the outcome of transplantation for patients with high risk AL. The MRD check point at day +100 should be considered crucial for subsequent therapeutic decisions after allogeneic transplantation. Disclosures:No relevant conflicts of interest to declare.

Intact Apoptosis Signaling In Pediatric ALL Xenograft Leukemia Is Associated with Favorable Patient Outcome, Prolonged NOD/SCID Engraftment and Low Expression of Anti-Apoptotic Molecules

AbstractAbstract 2722Defects in cell death signaling might be responsible for treatment failure and development of relapse in acute leukemia. In two retrospective studies we recently investigated the functional integrity of apoptosis signaling in primary patient leukemia samples and reported an importance of intact apoptosis signaling for good outcome in pediatric ALL and AML. Using a NOD/SCID/huALL mouse xenotransplant model for pediatric ALL we have also shown that rapid engraftment of primary ALL cells (short time to leukemia/TTLshort) is characteristic for early relapse of the corresponding patient.The aim of this prospective study was to analyze the impact of the functional integrity of apoptosis signaling in xenograft ALL samples on NOD/SCID engraftment and patient outcome. Furthermore, expression of apoptosis regulating molecules was investigated and correlated to NOD/SCID engraftment, response to treatment and apoptosis signaling.Primary BCP-ALL samples (N=20) obtained at diagnosis were transplanted onto NOD/SCID mice and time to leukemia (TTL) was estimated as weeks from transplant to onset of disease for each sample transplanted. Apoptosis signaling was investigated in xenograft leukemia samples analyzing two key apoptogenic events by flowcytometry, cytochrome c release and caspase-3 activation. Transcript levels of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin was analyzed by quantitative RT-PCR.Of the 20 ALL samples transplanted 6 led to rapid leukemia manifestation in the recipient animals (TTLshort) with an inferior relapse free survival of the corresponding patients in contrast to 14 patients with TTLlong phenotype (log rank P.002).Apoptosis signaling was investigated in xenograft leukemia samples by analysis of caspase-3 activation and cytochrome c release. Both events were closely correlated to each other indicating intact signaling in xenograft leukemia derived from patients stratified into non-high risk groups, patients showing good response to treatment (remission on day 15, negative MRD on day 33), and patients without relapse. In contrast, no correlation was found in xenografts from patients with poor outcome. Most importantly, intact apoptosis signaling was also found in TTLlong but not in TTLshort patients strongly indicating that mitochondrial cytochrome c release and consecutive apoptosome formation resulting in activation of downstream effector caspases such as caspase-3 is characteristic for a long engraftment phenotype and prognostic favorable ALL.The functional integrity of this apoptogenic checkpoint is subsumed by the parameter cytochrome c related activation of caspases (CRAC). Patients with positive CRAC values reflecting intact apoptosis signaling showed a significantly superior relapse free survival in contrast to patients with disturbed cytochrome c related caspase activation/negative CRAC (log rank, P<.001).Transcript expression of apoptosis regulating molecules was analyzed. A correlation of cytochrome c release and caspase-3 activation indicating proficient apoptosis signaling was exclusively observed in leukemia samples with low expression of the anti-apoptotic molecules Mcl-1, XIAP, Bcl-2 and Livin. Furthermore, patients samples with low Mcl-1 expression showed a significantly longer time to leukemia (TTL) and lower blast cells on day 8 than samples with high Mcl-1 expression.In summary, the propensity of leukemia cells to undergo apoptosis (CRAC positive) leads to prolonged engraftment upon transplant in the NOD/SCID/huALL model (TTLlong), is associated with low expression of anti-apoptotic molecules, and results in favorable treatment response and superior survival of pediatric ALL patients. Disclosures:No relevant conflicts of interest to declare.

Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells

AbstractAbstract 1695 Introduction:The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods:The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results:A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions:Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures:No relevant conflicts of interest to declare.

Treatment of Molecular and Clinical Relapse of Acute Promyelocytic Leukemia (APL) with Arsenic Trioxide: Results of the European Registry of Relapsed APL

AbstractAbstract 15Arsenic trioxide (ATO) is presently regarded as the best treatment option in relapsed PML-RARA+ acute promyelocytic leukemia (rAPL). However, given the relative rarity of relapses following standard front-line APL therapy, the optimal management of this group of patients remains to be firmly established. For this reason, and in order to ensure homogeneity in patient data collection and gain insights into the epidemiology and clinical aspects of rAPL, a European registry of rAPL was established in 2008 under the auspices of the European LeukemiaNet (ELN). Patients with a genetically confirmed (PML-RARA+) molecular or clinical relapse of APL occurring from January 2003 onwards were eligible for registration using uniform CRFs. Management of relapse was at the discretion of the treating physician, but could be based on the treatment recommendations developed by an expert panel provided on the ELN website (www.leukemia-net.org/content/). By 15 July 2010, 122 cases of rAPL had been registered by participants of 7 European countries (Spain, Italy, UK, France, Germany, Greece, Poland) representing the starting group of this ongoing initiative open for participation. In 80 pts (69 in first, 11 in 2nd and later relapse), information was available on ATO salvage therapy. At time of first diagnosis median age was 43 years (14-77); all pts had received all-trans retinoic acid (ATRA) and anthracyclines ± ara-C as first-line therapy. No patients received ATO as part of front-line therapy. According to Sanz Score, 26% were low risk, 45% intermediate and 29% high risk. 39% of pts had the bcr1/2 and 61% the bcr3 isoform of PML-RARA. Median duration of first remission was 493 days (93d to 5.7 years). The usual dosage of ATO was 0,15mg/kg/day, or regimens with the same cumulative dose were used. ATO induction therapy was combined with ATRA (45 mg/m2/day) in 40% of pts and during the consolidation cycle in 50% of pts, respectively. The median duration of ATO (± ATRA) for induction was 30 days and for consolidation 25 days. CNS relapses were treated with intrathecal application of methotrexate ± ara-C ± hydrocortisone and ATO. Among the 69 pts with first relapse, there were (Group A) 25 molecular and (Group B) 42 hematological relapses (bone marrow), in three pts combined with CNS involvement. Two pts had isolated CNS or extramedullary relapse. Main characteristics of pts at start of induction therapy in Group A vs. B were: median WBC count (x109/L) 4.4 (1.9-6.2) vs. 2.3 (0.5-102) (p=0.015); median platelet count (x109/L) 178 (40-392) vs. 87 (9-273) (p=0.0007); median Hb (g/L) 14 (10.6-15.7) vs. 12 (7.6-14.8) (p=0.0008). In Group A, none of the pts had coagulopathy compared to 8 of 25 (32%) pts with coagulopathy in Group B (p=0.004). In Group A, ATO therapy was not complicated by hyperleukocytosis and no episodes of differentiation syndrome were observed. In Group B, 9 (21%) pts had suspected or manifest differentiation syndrome (p=0.02), and additional chemotherapy was administered in 14 (33%) pts to control hyperleukocytosis. Other toxicities were rare and had no significant influence on therapy. In Group A, the rate of 2nd molecular remission after induction was 52% (13 of 25 pts) and after consolidation therapy 64%. No pt died. In Group B, 39 (88%) pts achieved 2nd CR, 2 (5%) pts were resistant and 3 (7%) pts died early from cerebral bleeding. Second molecular remission was reached in 12 of 32 (38%) pts after induction and in 62% after consolidation. Information on postconsolidation therapy was available in 45 pts of Group A and B. Of these, 14 pts underwent allogeneic, 18 pts autologous transplantation and 13 pts further therapy with ATO ± gemtuzumab ozogamicin. 11 of the 45 (24%) pts relapsed (median 2nd remission duration 10 months, 4 to 26), and 9 deaths were reported between 9 and 14 months after the end of consolidation therapy. In pts with 2nd and later relapse, despite previous exposure to ATO, further responses with this agent were observed. Conclusions:ATO is an effective therapy for all stages of relapsed APL. The results argue in favor of serial PML-RARA monitoring in front-line therapy of APL to allow early treatment of emergent relapses, avoiding hyperleukocytosis and risk of death due to bleeding and differentiation syndrome. Furthermore, the favorable toxicity profile of ATO seems to benefit the feasibility of subsequent transplantation. Disclosures:Lengfelder:Cephalon: Research Funding. Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Honoraria, Research Funding.

Clonal Evolution of Multiple TET2mutations In the Transformation of Chronic Myelomonocytic Leukemia to Acute Myeloid Leukemia and Its Subsequent Relapse

AbstractAbstract 2699Transformation from chronic myelomonocytic leukemia (CMML) to acute myeloid leukemia (AML) is associated with a poor prognosis. Clinical features have been used to score those at high risk, e.g. increased marrow blast percentage. Recently, several gene mutations have been identified including TET2, KRAS, NRAS, CBL and RUNX1, of which TET2 is the most frequent (Kohlmann et al 2010, Kosmider et al 2009). Initial descriptions of TET2 mutations in myeloid malignancy suggested that they were early mutations (Delhommeau et al 2009), however subsequent studies have shown in some patients with myeloproliferative neoplasia (MPN) they can be preceded by mutations in JAK2 (Schaub et al 2010). We report a male patient who was diagnosed with CMML aged 43 years. His marrow blast count was 6% and within 2 months he had transformed to AML (FAB M4) with marrow blasts of 50%. He was treated with daunorubicin and AraC (Barts 12 schedule) and obtained a complete morphological remission (CR). However he relapsed 88 days after CR in his skin, central nervous system and marrow. He died with conservative treatment after 6 months. Using this case as a model for transforming CMML, we aimed to identify clonal changes characteristic of the three stages of his disease, chronic leukaemia, transformation to acute leukemia and subsequent relapse. We used cytogenetic analysis, single nucleotide polymorphism (SNP) arrays (Affymetrix SNP 6.0 and 10K; 10K data previously reported by Raghavan et al 2008) to identify the clonal changes. Gene mutation analysis of genomic and cDNA was performed using PCR, cloning and Sanger and amplicon deep-sequencing for the mutational burden of TET2, CBL and KRAS (454 Life Sciences, Branford, CT). At all stages of his disease, he had a normal karyotype, a NPM1 mutation, but no mutations of FLT3, JAK2, KRAS or CBL (Table). Uniparental disomy (UPD) was demonstrated on chromosome 4q at relapse but not present at diagnosis, suggesting an associated TET2 mutation that would be heterozygous at diagnosis and homozygous at relapse. The heterozygous mutation was indentified in the CMML and at transformation, in a region adjacent to the splice site of the end of exon 7. The transcript resulted in a mis-splicing event leading to a stop codon. A second truncated mutant due to a deletion in exon 6 was found expressed at low level. Deep-sequencing of genomic DNA confirmed a deletion in exon 6 consistent with this product at 2.4% (918-fold coverage) in the CMML, and increased to 5.7% (865-fold coverage) burden at transformation. At relapse, the genomic mutation was homozygous. Expression of only the mis-spliced product was observed, with no wild type TET2, and no evidence of the second truncated product by cloning or deep-sequencing (832 reads). Homozygous TET2 mutations are associated with 4qUPD in MPN and myelodysplastic syndromes (Mohamedali et al 2009, Janowska et al 2010), but this is the first description of clonal evolution of a hetero- to homozygous TET2 mutation at disease progression. The case is consistent with the hypothesis that TET2 mutations evolve early in the pathogenesis of myeloid malignancies. However, it also shows there is considerable instability of TET2 mutations, with multiple mutations at various levels, and with mitotic recombination being a late event leading to relapse.CMMLTransformed AMLRelapsed AMLCGN46,XY46,XY46,XYSNP arrayNot doneNo somatic copy number abnormalities detected4qUPD 88201900-qterTET2c4340+5G>T+10_+21del het result: Cys1298Xc4340+5G>T+10_+21del het result: Cys1298Xc4340+5G>T+10_+21del hom result: Cys1298Xc3732_3733delCT 2.4% result: Tyr1245LeufsX22c3732_3733delCT 5.7% result: Tyr1245LeufsX22AbsentNPM1Dup956_959TCTG hetDup956_959TCTG hetDup956_959TCTG het Disclosure:No relevant conflicts of interest to declare.

Flow Cytometric Immunophenotypic Analysis of Cerebrospinal Fluid Cells In Hematological Malignancies

AbstractAbstract 5094 Background.Leptomeningeal involvement is an adverse complication of haematological malignancies, acute leukemias and lymphomas. The diagnostic gold standard to identify cerebrospinal fluid (CSF) involvement is conventional cytological analysis, technique characterized by low sensitivity and specificity. Recent papers make evidence that the efficiency of CSF involvement detection could improve by use of Flow Cytometry (FCM) immunophenotypic analysis of CSF samples. We describe the FCM method used in our hospital and we compare the results with cytological findings. Methods.CSF samples should be processed immediately, within 1 hour after collection to avoid cell's deterioration; in B Non Hodglin Lymphomas (B-NHL) we use two steps FCM approach, in four color staining (FITC/PE/PerCP/APC). The first step was two tubes: kappa/lambda/CD45/CD19 and CD5/CD20/CD45/CD3 or CD3/CD20/CD45/CD10 on the basis of a previous know immunophenotype or not. If a pathological population is identified, we complete immunophenotype characterization with a second step, using antibody panels suitable for the suspected malignancy. In case of suspected meningeal relapse of Acute Leukemia we use a monoclonal antibody combination select for Minimal Residual Disease. Results.Between 2006 and 2009 we studied by FCM analysis 53 CSF samples of 36 patients. 7 CSF of 29 B-NHL newly diagnosed resulted positive at FCM analysis (24,1%), 1 CSF of 7 Acute Leukemias shows disease relapse. The conventional cytology was positive only in 1 case of B-NHL. Conclusion.Our study confirms that immunophenotypic flow cytometric analysis of CSF cells markedly improves sensitivity to identify CSF involvement in haematological malignancies. Disclosures:No relevant conflicts of interest to declare.

THE SIGNIFICANCE OF DETECTING WT1 EXPRESSION IN CHILDHOOD ACUTE LEUKEMIAS

WT1 (Wilms' tumor gene 1) overexpression is implicated in the prognosis of acute leukemia. The purpose of this study was to investigate WT1 expression and its clinical implication in childhood acute leukemia (AL) in Chinese population. Bone marrow specimen from 200 children at different stages of acute leukemia and from 21 children without leukemia were studied. The WT1 expression at diagnostic marrow specimen in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) was higher than control group, whereas WT1 expression in AML was higher than in ALL, and WT1 expression level in relapse in ALL increased more significantly than in AML. The WT1 expression level showed positive correlation with the hypodiploidy and BCR-ABL fusion gene in acute leukemia. A rapidly decrease of WT1 expression level predicted a good response to the induction therapy and low expression of WT1 correlates with remission status. This study suggested that WT1 expression levels in acute leukemia can potentially be a marker for evaluating therapeutic efficacy, correlating with monitoring minimal residue disease, and predicting hematological relapse in children acute leukemia.

Adoptive immunotherapy, chemotherapy, and second allogeneic transplant in the treatment of post-transplant relapse of acute leukemia in children: a single center experience

Relapse of acute leukemia (AL) remains a major cause of treatment failure in allogeneic hematopoietic stem cell transplant (HSCT) in children. The prognosis of patients with post-transplant relapse...

FLANG salvage chemotherapy is an effective regimen that offers a safe bridge to transplantation for patients with relapsed or refractory acute myeloid leukemia

The efficacy of fludarabine in combination with an intermediate dose of cytosine arabinoside, mitoxantrone, and G-CSF (FLANG; fludarabine 30 mg/m(2)/day, cytosine arabinoside 1 g/m(2)/day, mitoxantrone 10 mg/m(2)/day, and G-CSF 300 μg/day for 5 days) was evaluated in patients with refractory or relapsed acute myelogenous leukemia (AML). Between January 2004 and December 2006, 27 patients with relapsed or refractory AML were enrolled in the present study. In total, 14 patients had experienced an early relapse, 10 had experienced a late relapse, and the remaining three (11%) had developed primary refractory leukemia at the time of study entry. Most patients (n = 17, 63%) had post-transplant relapse, and 10 of them relapsed after allogeneic hematopoietic stem cell transplantation (SCT). After FLANG treatment, 15 patients (56%) achieved a complete response (CR), and three patients died during reinduction chemotherapy. After achieving a CR, eight patients received SCT (seven allogeneic (sibling = 4, unrelated = 2, and haploidentical familial = 1) and one autologous SCT), one received donor lymphocyte infusion, three received consolidation chemotherapy, and the remaining three refused further therapy. Eight patients were alive during continuous CR, with an event-free survival (EFS) rate of 30% after a median follow-up of 42.1 months. The survival outcome of patients who received SCT was remarkable (EFS of 75%). Additionally, no toxicity severe enough to preclude transplantation was evident after or during FLANG. The findings of the present study suggest that FLANG salvage chemotherapy is an effective regimen and that it offers a safe bridge to SCT. Furthermore, this regimen prompts efforts to proceed to SCT as post-remission therapy for patients in greater than first CR.

Prognostic Factors for Outcomes of Pediatric Patients with Refractory or Relapsed Acute Leukemia Undergoing Allogeneic Progenitor Cell Transplantation

Allogeneic stem cell transplantation (SCT) is the only curative therapy for patients with refractory or relapsed acute leukemia, although the prognosis remains poor. Few reports have described outcomes of SCT in pediatric patients with refractory acute leukemia. To identify prognostic factors for these patients, we retrospectively evaluated SCT outcomes for advanced acute leukemia in 82 pediatric patients from 3 transplant units in Nagoya City between 1990 and 2008. Median age at transplantation was 8 years (range, 0.5-17 years). Transplantation was performed in the first refractory relapse for 53 patients (64.6%), in the second or subsequent relapse for 16 patients (19.5%), and during primary induction failure for 13 patients (15.9%). Only 4 patients (4.9%) underwent transplantation in the untreated first relapse, and 39 patients (47.6%) received unrelated donor progenitor cells. Of the 82 patients, 61 died (77.9%), with a median survival of 7.1 months (95% confidence interval [CI], 4.2-10.0 months). Median disease-free survival (DFS) was 4.7 months (95% CI, 2.6-6.9 months). In multivariate analysis, peripheral blood blasts, cord blood transplantation, and more than 3 courses of previous salvage chemotherapy were predictive of DFS. These results support the notion that allogeneic SCT offers only a small chance of cure for most pediatric patients with refractory or relapsed acute leukemia, and suggest that reduction of the leukemia burden and earlier optimal timing of transplantation are essential for long-term survival even in patients with refractory acute leukemia.

Hematopoietic Stem-Cell Transplantation for Acute Leukemia in Relapse or Primary Induction Failure

Patients with acute leukemia refractory to induction or reinduction chemotherapy have poor prognoses if they do not undergo hematopoietic stem-cell transplantation (HSCT). However, HSCT when a patient is not in complete remission (CR) is of uncertain benefit. We hypothesized that pretransplantation variables may define subgroups that have a better prognosis. Overall, 2,255 patients who underwent transplantation for acute leukemia in relapse or with primary induction failure after myeloablative conditioning regimen between 1995 and 2004 were reported to the Center for International Blood and Marrow Transplant Research. The median follow-up of survivors was 61 months. We performed multivariate analysis of pretransplantation variables and developed a predictive scoring system for survival. The 3-year overall survival (OS) rates were 19% for acute myeloid leukemia (AML) and 16% for acute lymphoblastic leukemia (ALL). For AML, five adverse pretransplantation variables significantly influenced survival: first CR duration less than 6 months, circulating blasts, donor other than HLA-identical sibling, Karnofsky or Lansky score less than 90, and poor-risk cytogenetics. For ALL, survival was worse with the following: first refractory or second or greater relapse, > or = 25% marrow blasts, cytomegalovirus-seropositive donor, and age of 10 years or older. Patients with AML who had a predictive score of 0 had 42% OS at 3 years, whereas OS was 6% for a score > or = 3. Patients with ALL who had a score of 0 or 1 had 46% 3-year OS but only 10% OS rate for a score > or = 3. Pretransplantation variables delineate subgroups with different outcomes. HSCT during relapse can achieve long-term survival in selected patients with acute leukemia.

Sequence of administration and methylation of SOCS3 may govern response to gemtuzumab ozogamicin in combination with conventional chemotherapy in patients with refractory or relapsed acute myelogenous leukemia (AML)

In older patients suffering from acute myelogenous leukemia (AML), aggressive chemotherapy is accompanied with high treatment-related morbidity and mortality. Gemtuzumab ozogamicin (GO), a humanized monoclonal anti-CD33 antibody, represents a well tolerated treatment option, but optimal treatment schedules are still unknown. Additionally, Suppressor of cytokine signaling 3 (SOCS3) inhibits the CD33-induced block on cytokine-induced proliferation. Consequently, a variable response of AML cells to anti-CD33-targeted therapy may be caused by modulation of SOCS3 expression. Twenty-four patients with refractory or relapsed CD33-positive AML received GO as a single agent before or after conventional chemotherapy. The methylation status of the SOCS3 CpG island was assessed by methylation-specific polymerase chain reaction. Response (RR) and overall survival (OS) were significantly higher in 16 patients receiving chemotherapy before GO (RR 81%, OS 14.8 months) compared to three patients who received GO single agent therapy (RR 33%, OS 7.2 months) or 16 with GO before chemotherapy (RR 0% OS 2.2 months, P = 0.01 for RR and P < 0.001 for OS). Methylation of the SOCS3 CpG island was found in 8/24 patients. There was a trend towards a higher RR and longer OS in patients with SOCS3 hypermethylation (RR 86%, OS 25.1 months) compared to unmethylated SOCS3 (RR 56%, OS 10.3 months, P = 0.09). Administration of GO a few days after chemotherapy seems to provide better response and survival compared to administration of GO directly before chemotherapy. The potential role of SOCS3 hypermethylation as a biomarker should be further investigated in patients undergoing GO containing therapies.

Comparison of Cytogenetic Clonal Evolution Patterns following Allogeneic Hematopoietic Transplantation versus Conventional Treatment in Patients at Relapse of AML

Relapse of acute myelogenous leukemia has been associated with clonal cytogenetic evolution, but no study focused specifically on relapse after allogeneic hematopoietic stem cell transplantation (HSCT). We compared karyotypes in 160 patients at both diagnosis and relapse either after allo-HSCT (n = 26) or standard chemotherapy (n = 134) using chromosome banding analysis combined with fluorescein in situ hybridization. There were 71 females and 89 males (19.7-80.6 years). At diagnosis, aberrant karyotypes were more frequent in the HSCT than in the chemotherapy cohort (16 of 26; 61.5% versus 63 of 134; 47.0%). This was most obvious in patients with unfavorable cytogenetics (8 of 26; 30.8% versus 19 of 134; 14.2%; P = .032). Differences in the karyotypes between diagnosis and relapse were more frequent in the allo-cohort (14 of 26; 53.8% versus 49 of 134; 36.6%) than in the conventional cohort (n.s.), mainly because of newly emerging cytogenetic alterations. Appearance of ≥ 3 new clonal alterations was more frequent in the allo-cohort (6 of 12; 50.0% with clonal evolution versus 5 of 41; 12.2%, P = .005). The mean number of cytogenetic alterations per patient was increasing from 2.0 at diagnosis to 4.0 at relapse in the allo-cohort, in the conventionally treated patients from 0.9 to 1.3 (both P < .001). Thus, higher frequencies of clonal evolution and increasing cytogenetic complexity were observed in the stem cell recipients probably related to the more unfavorable cytogenetic profiles already depicted at diagnosis.

Comprehensive flow cytometry phenotype in acute leukemia at diagnosis and at relapse

Multiparameter flow cytometry (MFC) plays a vital role in the detection of minimal residual disease (MRD) and diagnosis of relapse in acute leukemia. However, application of a limited panel of antibodies in MFC leads to high rates of false-negative and false-positive results. Thirteen patients with acute lymphoblastic leukemia (ALL) and 12 patients with acute myeloid leukemia (AML) were immunophenotyped by MFC at diagnosis and at relapse using a comprehensive panel of monoclonal antibodies (McAbs) to 27 antigens and CD45/SSC gating. In 23 of 25 patients (92.3%), changes in at least one of progenitor-associated, myeloid and lymphoid antigens between diagnosis and relapse were observed. Antigen changes were observed in 92 of 239 antigens (38.5%) expressed in 25 patients, in 49 of 117 antigens (41.9%) expressed in 13 ALL patients, and in 43 of 122 antigens (35.2%) expressed in 12 AML patients. Phenotypic changes were characterized by the expression of cross-lineage antigens. The intralineage change was observed in the majority of patients. However, myeloid lineage shift was identified by MFC in two patients with T-ALL. Multiple panels of three or more McAbs are likely to be required in the monitoring of MRD and diagnosis of relapse in acute leukemia by MFC.

External Auditory Canal and Middle Ear Relapse of Acute Promyelocytic Leukemia Treated With Arsenic Trioxide

Extramedullary involvement occurs infrequently in acute promyelocytic leukemia and is said to be more common after treatment with all-trans retinoic acid. We describe a 9-year-old girl who had an isolated external auditory canal and middle ear relapse after treatment with all-trans retinoic acid and chemotherapy. A patient with cytogenetically and molecularly confirmed acute promyelocytic leukemia developed isolated extramedullary relapse in the auditory canal and middle ear 4 years and 9 months after initial diagnosis, while in hematologic and molecular remission, successfully treated with arsenic trioxide alone.

Molecular or cytogenetic monitoring and preemptive therapy for central nervous system relapse of acute promyelocytic leukemia

We read with great interest the article by Montesinos et al .[1][1] concerning central nervous system (CNS) relapse of acute promyelocytic leukemia (APL). They reported a low incidence of CNS involvement at first relapse in APL patients following therapy without CNS prophylaxis.[1][1] The optimal

Rapid and reliable confirmation of acute promyelocytic leukemia by immunofluorescence staining with an antipromyelocytic leukemia antibody

The authors evaluated the utility of immunofluorescence staining with an antipromyelocytic leukemia (anti-PML) antibody for patients with a suspected diagnosis of new or relapsed acute promyelocytic leukemia (APL) and correlated the findings with the results of other established diagnostic modalities. Bone marrow (BM) and/or peripheral blood (PB) smears from 349 patients in whom the diagnosis of APL was considered were assessed with the anti-PML antibody using immunofluorescence. The study group included 199 patients with confirmed APL and 150 with other conditions. The results of conventional cytogenetics, reverse transcription polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) performed on these patients were correlated with the PML results. Among patients with confirmed APL, anti-PML antibody was positive in 182 of 184 BM and 32 of 33 PB smears. Conventional cytogenetics demonstrated t(15;17)(q22;q12) in 166 of 182 (91%) patients; 10 had a normal karyotype, 4 had insufficient mitoses to grow in culture, 1 was inconclusive, and 1 was 48, XX, +8, +8. Anti-PML staining was positive in 9 of 10 with a normal karyotype and in all 4 cases with insufficient mitoses. RT-PCR and FISH were positive for PML-retinoic acid receptor-alpha in 169 of 172 (98%) and 90 of 94 (96%) cases, respectively. Among the patients without APL, 148 of 150 (98.6%) were negative with anti-PML antibody. The sensitivity and specificity of the test were 98.9% and 98.7%, respectively. PML immunofluorescence staining is a rapid (<4 hours turnaround time) and reliable frontline diagnostic approach that can facilitate initiation of targeted therapy, particularly in clinical settings where cytogenetic and molecular testing are not readily available.

Allogeneic Hematopoietic Cell Transplantation Can Cure Some Patients with Acute Leukemia in Relapse or Primary Induction Failure: A CIBMTR Study.

Abstract 528Acute leukemia refractory to chemotherapy is uniformly fatal without hematopoietic cell transplantation (HCT). However, the benefit of transplantation for patients not in complete remission (CR) is controversial. PATIENTS AND METHODS:2,255 patients transplanted (at 252 centers in 38 countries) with an allogeneic donor after myeloablative conditioning regimen between 1995 and 2004 for acute leukemia in relapse or with primary induction failure were reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). 1,673 had acute myeloid leukemia (AML) and 582 acute lymphoblastic leukemia (ALL). Median age was 38 and 29 years for AML and ALL patients, respectively. Presence of circulating blasts, >25% marrow blasts, Karnofsky score < 90 %, or transplant in first relapse was observed in nearly half of the patients (see Table).TableAMLALLCharacteristics at HCTn=1673582> 25 % marrow blasts47%43%Circulating blasts57%38%Karnofsky < 90%48%47%Duration of CR1 < 6 months31%–1st refractory or ≥ 2nd relapse–62%Poor risk cytogenetics16%26%Outcomes (95% CI) 3-yrs OS19% (17-21)16% (13-20)aGVHD 3-423% (21-26)27% (24-31)cGVHD27% (25-30)27% (23-32) RESULTS:100 day mortality was 39% in AML and 41% in ALL. The median follow-up of survivors was 5 years. Overall survival (OS) was 19% (95% confidence interval (CI) 17-21) for AML and 16% (13-20) for ALL patients. For AML patients, five adverse pre-transplant variables significantly impacted OS : first CR duration < 6 months, blasts in the blood, donor other than HLA-identical sibling or partially matched unrelated, Karnofsky score < 90%, poor-risk cytogenetics. 106 AML patients had none of these high risk criteria, with a 3 years OS of 44% (35-54). For ALL patients, survival was worse with either: 1st refractory or ' 2nd relapse, ≥ 25% marrow blasts, CMV seropositive recipient, age ≥10 years. 8 ALL patients had no high risk criteria, with a 3 years OS of 85% (53-100). Grade 3-4 acute graft-vs-host disease (GVHD) occurred in 23% of AML and 27% of ALL patients. Chronic GVHD developed in 27%. Leukemia was the cause in 42 % of AML and 37% of ALL patients' deaths. CONCLUSIONS:1. HCT can cure some patients with acute leukemia in relapse or with PIF, particularly those lacking defined high risk features. 2. These pre-transplant variables should be considered when deciding whether or not to offer HCT to such patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thiopurine S - Methyltransferase Gene (TMPT) Polymorphisms in Population of Polish Children Treated for Acute Leukemia.

Abstract 4100 Introduction:Among drug-modifying elements age, sex, coexistent diseases, interactions with other drugs, as well as genetically determined altered drug response are included. Analysis of selected gene polymorphisms may lead to an increase treatment safety and efficacy in developing individual therapies. The objectives of the study: 1. Determination of the frequency of the allele variants: TPMT*2, *3A and *3C in population of Polish children treated for acute leukemia. 2. Analysis of the influence of TPMT genetic polymorphism on the occurrence of therapeutic and/or adverse effects such as hematological disorders, hepatotoxicity and nephrotoxicity after thiopurine administration. 3. Determination of the significance of pharmacogenetic profile for dosage optimizing of 6-mercaptopurine and 6-tioguanine. 4. Determination of the leukemia relapse risk in relation to TPMT polymorphism. Patients and Methods:The study group consisted of 210 patients (121 boys, 89 girls) treated for acute leukemia (167 ALL, 43 AML), with median age 7 (range 1-18) yrs, at following time points: on diagnosis, during intensive chemotherapy and after therapy cessation. Adverse treatment effects during the entire observation period including 6 particular stages was classified according to WHO toxicity scale in each child. Either bone marrow or peripheral blood samples were used for the analyses. DHPLC and RFLP was used for genotype determination for TPMT on selected polymorphisms: TPMT*2,*3A,*3C. DNA fragment amplification was used with relevant starters, followed by HpyF10VI (MwoI) and XmiI (AccI) restricting enzymatic digestion. RFLP marking results were validated by DHPLC. Results:1. For TPMT IM phenotype (“medium” metabolizer) was detected in 7.65% patients (n=16) and PM phenotype (“slow” metabolizer) in 0.47% cases (n=1), the most frequent polymorphic variant being TPMT*3A (72%). 2. Analysis of changes in selected morphology indicated that in TPMT *2, *3A or *3C polymorphism carriers' group there is a significant decrease in white blood count (p=0.0025), platelet count (p=0.0014) and neutrophil count (p=0.019) in comparison with individuals with the wild variant (TPMT*1/*1). In patients with TPMT allelic variants, longer periods of thrombocytopenia in relation to other hematologic parameters were observed (re-induction period: p=0.041, maintenance therapy: p=0.03). In patients with IM phenotype, significantly lower haemoglobin levels were observed only in the end stage of chemotherapy (p=0.032). In patients with ALL and IM the observed risk of leukopenia, (p=0.006), neutropenia (p=0.015) and thrombocytopenia (p=0.011) as well as hepatotoxicity: GOT (p=0.009), LDH (p=0.0014) during the entire treatment period and particular study points (WBC: re-induction p=0.004; PLT: re-induction p=0.045 and maintenance therapy p=0.043) was significantly more frequent. 4. In TPMT polymorphic variant carriers, the relapse of primary disease was statistically less frequent (p=0.0341F). 5. Death risk was not relevant to TPMT genotype. Conclusions:1. Frequency of detected TPMT single nucleotide polymorphism in children treated for acute leukemia is consistent with the published data for European population. 2. Thiopurine administration is related to the increase in hematological toxicity (leukopenia, neutropenia and thrombocytopenia) in TPMT heterozygotes. 3. Treatment with mercaptopurine or thioguanine should be regarded as an indication for genotyping. 4. TPMT single nucleotide mutation occurrence in children is related to lower risk of relapse in acute leukemia and might be included in the risk group stratification. Disclosures:No relevant conflicts of interest to declare.

The Influence of CYP2C9 and CYP2C19 Gene Polymorphism On Metabolism of Drugs Used in Therapy of Acute Leukemia in Children.

Abstract 1715Poster Board I-741 IntroductionGene polymorphism coding for drug-metabolizing enzymes may cause individual differences in effectiveness and toxicity of many medications, including cytostatics. Although intensified treatment resulted recently in better prognosis of leukemia, many adverse effects are still observed during therapy resulting from nonspecific activity and narrow therapeutic index of anti-cancer drugs. The objectives of the study: 1. Determining the frequency of selected allele variants: CYP2C9, CYP2C19 in population of Polish children treated for acute leukemia. 2. Analysis of the role of CYP2C9 and CYP2C19 in cyclophosphamide metabolism. 3. Determininig the influence of carrying selected CYP2C9 and C19 polymorphism on cyclophosphamide metabolism in form of increased adverse reaction risk (according to WHO score), relapse and other infringements in treatment protocols and procedures following the use of standard doses of alkylating agents. Patients: 184 patients (106 boys, 78 girls) were examined, age between 1 and 18 (median age 7 years, average age 7.64 years, SD=5.3) treated for acute leukemia (ALL:167, AML:17) at various time points: on diagnosis, during cytostatic treatment and after therapy cessation. All patients were treated with current uniform treatment protocols and procedures. Adverse reactions in each child was recorded according to WHO toxicity scale, during the entire length of observation period and in particular stages (6 periods). MethodsEither bone marrow or peripheral blood samples were used for the analyses. DNA isolation procedure was carried out with the use of standard tests. CYP2C9*2,*3 and CYP2C19*2 allelic variant determination was carried out with real-time PCR with double-marked probe hydrolysis with the use of Pre-Developed TaqMan Assay Reagents for Allelic Discrimination sets. PCR products fluorescence was carried out on 7900HT Fast Real-Time PCR device. Results1. For CYP2C9, IM phenotype was present in 22.3% patients (n=41) and PM phenotype in 7.6% cases (n=14), the most frequently detected polymorphic variant being CYP2C9*2 (75.3%). In case of CYP2C19, IM phenotype occurred in 22.3% patients (n=41) and PM phenotype in 7.1% (n=13). 2. Toxicity risk analysis in patients with acute leukemia did not reveal statistically significant differences depending on CYP2C9 genotype. In patients with CYP2C19*2 allelic variant, statistically lower level of alkaline phosphatase (p=0.013) was observed during the entire treatment and in particular stages (I: p=0.0096; II: p=0.022; III: p=0.038; IV:p=0.0024; V:p=0.044). Lower alkaline phosphatase levels were observed only in patients with ALL (p=0.026). Lower transaminase levels after completion of intensive treatment phase were observed in patients with CYP2C19 mutations (GOT, p=0.044). In patients with CYP2C19 and CYP2C9 polymorphic variants no dependence of risk of adverse reactions with correlation to cyclophosphamide and its dose was observed. 3. In patients with CYP2C9 and CYP2C19 polymorphism no significant relation was discovered. 4. Death risk was not relevant to CYP2C9 or CYP2C19 genotype. Conclusions 1. Occurrence of slow metaboliser phenotype for CYP2C9 and CYP2C19 is more frequent in patients with acute leukemia than in the untreated population. 2. CYP2C9 and CYP2C19 cytochromes are involved in cyclophosphamide metabolism but do not play a major role in drug metabolism. 3. CYP2C9 and CYP2C19 single nucleotide polymorphism is correlated neither with an increased risk of adverse reactions or death during therapy, nor with a relapse of acute leukemia in children. DisclosuresNo relevant conflicts of interest to declare.