Cytological Remission Research Articles

Acute Myeloblastic Leukemia in Chile. Treatment and Outcomes in Patients Admitted at the Hospital Clinico De La Pontificia Universidad Catolica De Chile Between 2010–2014

Introduction: Acute Myeloblastic Leukemia (AML) is the most frequent acute leukemia in the adults and its incidence increases with age. There are few studies about the demography and outcomes of AML patients in Chile and the only report belongs to a public hospital from 2000. We discuss the results of patients treated in our institution with AML non promyelocytic.Patients and Methods: Retrospective analysis of the epidemiologic, clinical and laboratory characteristics of diagnosis (cytology and flow cytometry) and treatment of AML non promyelocytic patients between 2010-2014. Statistical analysis of the data was performed using SPSS Statistics v21 software.Results: 63 patients were diagnosed with AML non M3, 52 males (66%), with a median age of 55.4 years (range: 16 - 89). Diagnosis laboratory tests (mean values and ranges) were: WBC 45.989/mm3 (range: 700 - 405.000); hemoglobin 9,1 g/dl (range: 5,2 - 14,1); platelets 75.548/mm3 (range: 10.000 - 454.000); peripheral blood blasts 38% (range 0 - 100); bone marrow blasts 74% (range 25 - 100%). The cytogenetic risk groups were: favorable (n=5, 8%), intermediate (n=33, 52%), adverse (n=8, 13%) and unknown (n=17, 27%). Of all the patients, 75% (n=47) received induction chemotherapy (CT) and 25% (n=16) palliative care. The mean age of the group with cytogenetic analysis was 51.2 years and only 8.6% did not receive consolidation CT. On the other hand, the group of patients with unknown cytogenetics had a mean age of 68 years and 57% did not receive consolidation CT. The mean survival of the CT group was 27.3 month (range: 0 - 53). By contrast, the mean survival in the palliative care group was 1 month (range: 0 - 6). The mean follow up in all patients was 13 months, (range: 1 - 55) and 17 months (range: 1 - 54) in the group that received CT. 87% (n=41) of patients with CT had febrile neutropenia with respiratory and intestinal focus most commonly identified. The induction mortality was 4,2% (n=2). Complete cytologic remission was achieved in 70% (n=33). The 3-year relapse free survival (RFS) and overall survival (OS) in the CT group were 25% and 31%, respectively. The multivariate survival analysis using Cox’s regression demonstrated that the variables that had significant impact in RFS and OS were: age at diagnosis (<60 years), achievement of disease remission and the use of induction and consolidation CT (high dose cytarabine versus others). In this analysis the cytogenetic risk did not have any impact in OS. The patients that only had induction CT (but not consolidation) had significantly better survival rates compared to the group in palliative care (6 months vs. 1 month, respectively, p=0.001). The mortality during the follow up of patients who had survived the induction CT was 47% (n=22), 2/3 of leukemia and 1/3 of infections.Conclusions: Our study shows that in our center, CR rates and OS rates after induction and consolidation chemotherapy are similar to those reported in international series, and are better than the data that was previously reported in our country. Low induction CT mortality, and the efficacy of CT in patients younger than 60 years old stand out in our report and validate the efficacy of intensive CT. DisclosuresNo relevant conflicts of interest to declare.

Detection of dicentric chromosome (9;20) in paediatric B-cell acute lymphoblastic leukaemia: prognostic significance.

The dicentric chromosome (9;20) (dic(9;20)) is described in 2% of childhood B-acute lymphoblastic leukaemia. Fluorescence in situ hybridization (FISH) is the most reliable method to identify dic(9;20) when compared with conventional cytogenetics. To define the prognostic importance of dic(9;20), we evaluated treatment response and patient survival. This was a retrospective study in three French university centres. Patients' clinical and laboratory characteristics and treatment response are described. Nine children with dic(9;20) have been identified since 1995. All patients had at least one poor prognostic feature either among the clinical features, the initial laboratory results or in the initial treatment response: central nervous system involvement (2/9), high median leucocyte count (≥50G/L) (8/9) and poor response to prednisone (2/9). All patients were in complete cytological remission after induction therapy but only three had a good molecular response with minimal residual disease (MRD) <10(-3). Five out of nine patients relapsed and two died, 4 and 12months after diagnosis, respectively. The event-free survival rate in this population was 44% (95% confidence interval (CI) = 0.09-0.79) and overall survival 78% (95% CI = 0.51-1.05). In this population, dic(9;20) is associated with a relatively poor prognosis. Patients showing dic(9;20), whether this cytogenetic abnormality is associated with other poor prognostic factors or not, should be identified at the outset in order to be offered a more intensive treatment protocol.

New Translocation (7:17) In Variant Acute Promyelocytic Leukemia With ATRA Resistance

BackgroundAcute promyelocytic leukemia (APL) is characterized by pathologic promyelocytes, chromosomal translocations t(15:17) (q22; q21) and clinical and cytological response to all-transretinoic acid (ATRA). A significant minority of patients with APL have variant translocations involving RARα gene with a different partner, not involving PML. These APL variant translocations may or not respond to ATRA treatment. Case reportA routine laboratory evaluation, before minor surgery, revealed leukocytosis, thrombocytopenia and blasts presence in peripheral blood (PB), in an otherwise asymptomatic 70-year-old man. He had a previous history of hypertension, dyslipidemia, ischemic heart disease, double coronary bypass and prostatectomy for non-malignant prostatic hypertrophy.Peripheral blood (PB) count: hemoglobin concentration 12g/dL, platelet count: 21x109/L and white blood cell count (WBC): 35x109/L. Peripheral blood smear: myelemia exhibiting 74% blasts (including myeloid blast and promyelocytes) with fine chromatin pattern, heterogeneous and frequently convoluted nuclei and one to three prominent nucleoli. The cytoplasm of these cells was basophilic with scanty purple granules. No Auer rods were found. The basic coagulation tests were normal(INR, APTT and fibrinogen) but D Dimer was 33.87 µg/ml. Normal biochemistry was shown, except for GGT 85 U/L and lactate dehydrogenase 2244 U/L.Bone marrow (BM) aspirate was unsuccessful (”dry tap”) but the bone trephine revealed an infiltration of 80% myeloid blasts. Cytochemistry: myeloperoxidase stain was deeply positive and chloroacetate and non-specific esterase stains were negative. Flow cytometry immunophenotype on PB was consistent with AML, likely M3 leukemia, with an atypical phenotype (MPO+, CD117+low, CD34+, CD13++, CD45+, CD15-,CD133-, CD33+, HLA-DR+low het , CD5-, CD7-, CD11b- , CD56+het, CD10-, CD8-,CD4-, CD45RA+, CD19-, CD2-, Glya-, CD71+low, CD38-, CD1a-, CD41-, CD14-).The analysis of the PML/RARα fusion gene, according to standard protocols, was negative. FISH studies showed 2 PML signals and 3 RARα copies, suggesting a possible variant rearrangement of this gene. FISH and molecular biology did not detect transcript for t(15:17), (5:17) or (11:17); so RARα was translocated with a strange partner.Suspecting of a variant APL, the patient was treated with ATRA plus Idarrubicin. After 4 weeks treatment, deep neutropenia was observed but no granulocytic maturation. That suggested us resistance to ATRA. Thus, we changed the treatment to a classical induction scheme for AML. The patient attained cytologic remission, which he mantains so far with conventional chemotherapy.Karyotype study was subsequently performed, identified an unbalanced rearrangement between 7q and 17q. Currently, we are identifying and sequencing the fusion partner of RARα. ConclusionWe report a novel case of variant acute promyelocytic leukemia with the karyotype der (7) t(7;17) (q11;q21). The morphology of PB and BM was critical for initial diagnosis and treatment decisions. Disclosures:No relevant conflicts of interest to declare.

Cytological and molecular remissions with blinatumomab treatment in second or later bone marrow relapse in pediatric acute lymphoblastic leukemia (ALL).

10007 Background: Pediatric B-precursor acute lymphoblastic leukemia (ALL) in second or later relapse is an aggressive malignancy that needs therapeutic approaches with new mechanisms of action. Blinatumomab, a bispecific T-cell engaging (BiTE) antibody, has shown a hematological remission rate of 69% in adult patients with relapsed/refractory ALL. In order to establish a recommended dose in pediatric patients, a phase I multicenter trial was initiated. Methods: The primary endpoint is to determine the maximum tolerable dose defined by ≤1 of 6 patients experiencing dose limiting toxicity (DLT) within the 1st course of treatment. Up to 6 different dose levels of blinatumomab are being evaluated. Eligible patients must be &lt;18 years old and have B-precursor ALL that is refractory or in second or later bone marrow relapse, or in any marrow relapse after allogeneic hematopoetic stem cell transplantation (HSCT). Blinatumomab is administered by continuous IV infusion over 28 days followed by a 14-day treatment-free interval (up to 5 cycles). To date, 3 dose levels have been explored (Table). Results: Seventeen patients have been treated thus far with a total of 32 cycles. One DLT (gastrointestinal hemorrhage) at dose level 2 (15 µg/m²/d) and two DLTs at dose level 3 (30 µg/m²/d; both cytokine release syndrome) with 1 death have been observed. One patient had generalized seizures on the 3rd day of the 2nd treatment cycle at the first dose level of 5 µg/m2/d, which was completely reversible. The patient successfully underwent an allogeneic HSCT after blinatumomab. Eight (47%) of the 17 patients reached a cytological complete remission in bone marrow and a molecular remission defined as MRD by PCR &lt;10-4. Conclusions: A phase I trial of blinatumomab in patients with relapsed/refractory pediatric ALL has shown hematological and molecular remissions. Dose-limiting cytokine release syndrome has been noted. Alternative dosing regimens are being explored in current cohorts to refine the recommended dose of blinatumomab in this patient population. Clinical trial information: NCT01471782. [Table: see text]

Severe hemorrhagic colitis in a patient with chronic myeloid leukemia in the blastic phase after dasatinib use

Dasatinib is a second-line tyrosine kinase inhibitor used in patients with imatinib resistant or intolerant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute leukemia. Gastrointestinal bleeding may occur in up to 7% of patients using dasatinib, although, severe dasatinib-related acute colitis had rarely been reported. Here, we present the case of a 36-year-old female who progressed to acute myeloid leukemia after fourteen months of receiving imatinib for CML in the chronic phase and was treated with a dasatinib-containing chemotherapy regimen. On day 34 of treatment, the patient developed moderate abdominal pain and bloody diarrhea with mucous. Analyses of stool specimens were negative for parasites, Clostridium difficile, and other pathogenic bacteria. The cytomegalovirus pp65 antigen was negative in her blood leukocytes. A colonoscopy revealed acute colitis, and a mucosal biopsy showed nonspecific colitis. The patient was treated with broad-spectrum antibiotics, bowel rest and hydration, and dasatinib treatment was stopped. Her bloody diarrhea improved within 72 h. After confirming cytological remission, the patient received initial course of consolidation, and dasatinib treatment was reinstated. However, hemorrhagic colitis recurred. After discontinuing dasatinib, herhemorrhagic colitis drastically improved and did not recur following the administration of nilotinib. The characteristics of our patient suggest that dasatinib treatment can lead to hemorrhagic colitis, which typically resolves after discontinuation of the drug.

Value of Pretransplantation Minimal Residual Disease in Acute Myeloid Leukemia and Myeloablative Hematopoietic Cell Transplantation.

AbstractAbstract 2510Relapse remains the main cause of treatment failure in patients with acute myeloid leukemia (AML) in first remission (CR1) after allogeneic hematopoietic stem cell transplantation (SCT). The detection of minimal residual disease (MRD) in AML has improved in the past years with multiparametric flow cytometry (MFC) and molecular analysis (RT-PCR). However the prognostic impact of pre-transplantation MRD and the outcome after SCT has not been well studied. The aim of this study was to evaluate pre-transplantation MRD in patients in first remission undergoing myeloablative allogeneic SCT.We retrospectively studied 35 consecutive patients receiving myeloablative SCT for AML in first cytologic remission after intensive chemotherapy with available MRD determination before transplant. MRD was studied by 4-color MFC on bone marrow aspirates, and quantitative RT-PCR (NPM1, WT1, MLL) on bone marrow and/or peripheral blood samples obtained within thirty days before transplant.Thirty-five patients consecutively transplanted in our institution between 1999 and 2012, and for which pre-transplant MRD data was available were analyzed (Table 1). Eighteen showed negative MRD pre-transplant whereas 17 showed positive values. Characteristics of patients were homogeneous between both groups, including number of chemotherapy cycles received before transplantation (Table 1).Within the MRD-negative group, 17 patients showed negative MRD by MFC (12 of them showed negative values also by PCR) and 1 patient showed negative MRD by PCR (MFC not available). Within the MRD-positive group, 9/17 (52%) patients showed MRD-positive values by MFC: in 5 cases MRD was also detected by PCR, only 1 showed negative PCR and in the remaining 3 cases, PCR was not available. On the other hand, in 8/17 (47%) patients MRD was not detected by MFC, however, PCR detected MRD in all of the cases in bone marrow (2), peripheral blood (4) or both samples (2).With a median follow-up of the whole series of 23 months, 2-years estimates of overall survival were 82% (95% CI, 97–55) and 30% (95% CI, 3–71) for MRD-negative and MRD-positive patients (p=0.045), respectively. Cumulative incidence of relapse were 21% (95% CI, 5–48) and 56% (95% CI 10–73) for MRD-negative and MRD-positive patients (p=0.11).In the MRD-negative group, cause of death was toxicity in 11% of the cases and relapse in 11%, while in the MRD-positive group, 17% of patients died due to toxicity and 23% due to relapse.Conclusions: Our data shows that the presence of MRD before allogeneic SCT in patients with AML in CR1 is associated with a significant worse OS rate compared to patients with negative MRD, as well as a tendency towards a higher risk of relapse. The detection of MRD by MFC correlates with the detection by PCR in most of the cases. However, in a significant group of patients, MRD was detected only by PCR. This could be related to differences in sensitivity between both methods. Further studies including larger series are needed to confirm these observations.Table 1Patient and Disease Characteristics by MRD statusPre-transplant MRDMRD-negativeMRD-positiveN=351817Age at transplant (median, R)36 (19-62)42 (19-68)Gender (male/female)8/1012/5Cytogenetics and molecular markersNormal/Intermediate risk77%83%FLT3+28%27%NPM1+/FLT3-6%7%FLT3-/NPM1-28%40%High-risk22%17%Secondary AML16%6%Cycles pre-transplantation (median, R)3 (2-5)3 (2-5)MRD detectionMFC only27%18%RT-PCR only5%0%Both68%82%Donor*HLA-identical sibling50%35%HLA-matched unrelated22%29%SCU-dual22%24%HLA-haploidentical related6%12%acute GVHD II-IV (n°/patients at risk)44% (8/18)24% (4/17)chronic GVHD lim/ext (n°/patients at risk)53% (8/15)57% (8/14)MRD: minimal residual disease, MFC: multiparametric flow cytometry, RT-PCR: real time PCR, GVHD: graft vs host disease, SCU-dual: single cord blood with co-infusion of selected CD34+ cells from a third party HLA-mismatched donor.*The MRD negative group includes 2 MAC SCU-dual cases with primary graft failure rescued immediatly by a second graft (1 Dual and 1 Haplo) using a RIC regimen. The MRD positive group includes 1 MAC SCU-dual case rescued immediatly by a second graft (1 Haplo). Disclosures:No relevant conflicts of interest to declare.

Safety of Clofarabine with Multiagent Chemotherapy in Childhood High Risk Relapse of Acute Lymphoblastic Leukemia (Vandevol study of the French SFCE Acute Leukemia Committee).

AbstractAbstract 2570 Background.Current outcome of very early relapse of ALL in children remains poor. Clofarabine, a novel nucleoside analog has proven effective as a single agent in childhood ALL second relapse, providing a CR+CRp ratio of 20 to 28 % (Jeha 2006, Kearns 2006). Clofarabine in combination with cyclophosphamide and Etoposide provided a 44% overal response rate rate in children with advanced ALL (Hijiya 2011) and further multi-drug combinations need to be investigated. The VANDA regimen that has proven effective in high risk ALL and in ALL relapse (Domenech 2008) was used as a template, Cytarabine being replaced by Clofarabine in this combination. Procedure.A phase I study combining escalating doses of Clofarabine with fixed doses of Mitoxantrone, Etoposide, Asparaginase and Dexamethasone was undertaken in children presenting very early (18 months or less after initial diagnosis) medullary or combined ALL relapse or children with second relapse or post transplant relapse. A 3+3 rolling design was used for escalating clofarabine doses with 25% increments from 20 to 40mg/m2/d.Dexamethasone20 mg/m2D1 to D5Clofarabine20 mg/m2 (26, 32, 40)D1 to D5Mitoxantrone8 mg/m2D3, D4Etoposide150 mg/m2D 3, 4, 5E-Coli Asparaginase10 000 UI/m2D7, 9, 11, 13 Results.Sixteen patients were enrolled, eleven in first marrow relapse, four in second relapse. One patient with an initially refractory disease was secondary excluded as he didn’t fulfill the inclusion criteria. Four patients had previously been allografted. Three patients were enrolled at dose level 1 (clofarabine 20 mg/m2), and 3 patients at dose level 2 (clofarabine 26 mg/m2) without any significant dose-limiting toxicity (DLT). Six patients were enrolled at dose level 3 (clofarabine 32 mg/m2), one patient experienced a liver DLT (gamma GT elevation) exceeding 7 days. At the last planned dose level (clofarabine 40 mg/m2), two patients had a grade 3 transaminase elevation exceeding 7 days and 2 patients had a severe fungal infection. Median duration of neutropenia was 20 days (15–31). Other expected toxicities included 3 documented bacterial infections, 3 mucosal toxicities needing parenteral support, one lung aspergillosis, one transient insulin dependant hyperglycemia. There was no toxic death. Out of 15 evaluable patients, 11 (73%) achieved a complete cytological remission (CR). 4 relapses occurred at 1, 1, 2 and 3 months. 6 patients proceeded to allo-SCT with 2 second allografts. One allografted patient died from EBV-PTLD, the others are alive at 2, 13, 13, 15, and 29 months post-transplant. Conclusion.Clofarabine in combination with four major antileukemic drugs appeared feasible and effective in inducing complete remission in those high risk patients. Clofarabine MTD was 32 mg/m2/d for five days and this dose step is currently being expanded to a total of 10 patients, before a phase II study will be proposed for early and very early ALL relapses. Study registered at ClinicalTrials.gov (http://clinicaltrials.gov/ct2/show/NCT01279096). Disclosures:Off Label Use: off label use of clofarabine.

Frequency and Prognosis of Clonal Chromosomal Abnormalities Following Stem Cell Transplantation (SCT) in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS)

Abstract 4897 Objective.To study the frequency, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML and MDS. Patients and methods.One hundred thirty patients were studied between 2000 and 2010. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2009. Results.Clonal abnormalities were observed in 36/130 patients (28%) with a median follow-up was 11 months (range 3–131). Initial diagnosis (OMS 2008): AML with maduration (8 patients), acute erythroid leukaemia (4), acute monocytic leukaemia (3), AML with multilineage dysplasia (3), AML with myelodysplasia-related changes (3), AML with inv(16)(p13q22) (2), acute monoblastic leukaemia (2), AML with minimal differentation (2), AML without maduration (1), acute myelomonocytic leukaemia (1), refractory anaemia with excess blasts (4), chronic myelomonocytic leukaemia (2) and refractory cytopenia with multilineage dysplasia (1). Treatment before SCT: idarubicin, cytarabine, etoposide and mitoxantrone (25), idarubicin, cytarabine and etoposide (6), idarubicin, cytarabine, etoposide and gemtuzumab (2), FLAG-ida (2) and azacitidine (1). SCT type: autologous (23 patients), allogeneic of reduced intensity (7), allogenic (4), umbilical cord blood (1) and syngeneic transplant (1). Conditioning regimen: TBI and cyclophosphamide (24 cases), fludarabine and busulfan (7), busulfan and cyclophosphamide (4) and thymoglobulin, thiotepa, fludarabine and busulfan (1). The median time between SCT and the appearance of clonal abnormalities was 6.5 months (range 2–51). At the time of clonal abnormality detection, 32 patients were in cytological and/or clinical relapse and 4 in cytological remission. In 15 cases the initial clone reappeared, 2 showed the initial abnormalities with an acquired abnormality and 19 presented de novo clonal abnormalities (53%). The most frequent clonal chromosomal abnormalities observed were: complex karyotype (22%), estructural abnormalities with afection of chromosomes 1, 4, 6, 7, 10, 11, 12, 16 and 17 (22%), +21 (5%) and +11 (3%). The median survival from the appearance of clonal abnormalities was 2.5 months (range 1–100). There were no differences when we compared the survival of patients with de novo clonal abnormalities with that of patients with initial abnormalities (including those with acquired abnormality). Furtheremore there were no differences when we compared the median survival of relapsed patients with normal Karyotype with that of patients with clonal chromosomal abnormalities following SCT (2.5 months [range 1–25] and 2.5 months [range 1–100] respectively). At the time of the analysis only 1 patients are alive and in complete remission (100 months). Conclusions.1. The appearance of clonal abnormalities following SCT in patients with AML and MDS is frequent and the majority of cases are detected at the time of relapse. Half of cases presented de novo clonal abnormalities and the majority were not related with prior irradiation exposition, alkylating agents neither topoisomerase II inhibitors. The prognosis of patients with acquired clonal abnormalities after SCT is poor. Disclosures:No relevant conflicts of interest to declare.

Monitoring of minimal residual disease by quantitative WT1 gene expression following reduced intensity conditioning allogeneic stem cell transplantation in acute myeloid leukemia

WT1 is well-known to be a panleukemic marker that is expressed in 90% of acute myeloid leukemias (AML). Quantification of WT gene expression in bone marrow (BM) samples may be useful as a marker of minimal residual disease (MRD) during and after treatment for early prediction of relapse. We evaluated the validity of this AML-MRD marker after reduced intensity conditioning (RIC) allogeneic stem cell transplantation (SCT). The quantitative assessment of WT1 expression by real-time quantitative PCR (RQ-PCR) was measured in 25 patients (pts) with AML at diagnosis, at the time of RIC-SCT and after transplant at precise time points. All pts showed high WT1 levels at diagnosis with a mean of 4895 (SD 4462) and a median of 3679 (range 454-16,853) copies WT1/10(4) ABL. At transplant, 18/25 pts (72%) were in complete cytologic remission (CcR) and 7/25 (28%) had refractory AML. At the pre-SCT evaluation, BM samples from pts transplanted in CcR showed significantly lower WT1 expression levels compared to the samples from pts with refractory AML (p = 0.002). Median follow-up after RIC-SCT was 18 months (range 2-54). On 18 pts transplanted in CcR, those (17/18) who maintained CcR after RIC-SCT displayed a WT1 copy number persistently low during all the follow-up period. In patients who received RIC-SCT with active disease obtaining a sustained CcR after transplant (3/25), WT1 levels decreased to normal range in the first two months after RIC-SCT and remained low through the entire study period. All pts who relapsed after RIC-SCT (4/25) had a high WT1 copy number before the cytologic relapse. In 50% of these cases, an increase in WT1 expression was documented before molecular chimerism decreasing. With this experience, taking into account the limited number of pts, we confirmed a concordance between WT1 expression levels (measured by RQ-PCR at precise and sequential time points) and status of AML before and after RIC-SCT and we found a concordance between WT1 expression levels and hematopoietic chimerism status. Our data suggest that, in the RIC-SCT setting, the sequential and quantitative analysis of WT1 may be useful as a leukemia marker for monitoring MRD and as a predictor of overt AML cytologic relapse.

Liposomal cytarabine is effective and tolerable in the treatment of central nervous system relapse of acute lymphoblastic leukemia and very aggressive lymphoma

Treatment of central nervous system relapse in adult acute lymphoblastic leukemia is a challenge and outcome is poor. Liposomal cytarabine has a prolonged half-life and, given intrathecally, has produced high response rates in patients with central nervous system relapse of non-Hodgkin's lymphoma. The aim of this study was to evaluate the efficacy and tolerability of liposomal cytarabine in central nervous system relapse of acute lymphoblastic leukemia or Burkitt's lymphoma/leukemia. Liposomal cytarabine (50 mg) was given intrathecally together with systemic or intrathecal dexamethasone once every 2 weeks in a phase II European trial. The primary end-point, cytological response in the cerebrospinal fluid after one or two cycles, was evaluated at the time of next treatment. Nineteen heavily pretreated patients (median age, 53 years; range 24-76 years) were evaluable: 14 with acute lymphoblastic leukemia and 5 with Burkitt's lymphoma/leukemia). Complete cytological remission as best response after two cycles of liposomal cytarabine was confirmed in 74% of the patients: 86% of those with acute lymphoblastic leukemia and 40% of those with Burkitt's lymphoma/leukemia). Nine of the 14 patients who achieved complete remission relapsed after a median of 7 months. The median overall survival was 11 months. Adverse events were observed in 89% of the patients (57% of cycles). Grade III-IV events with potential correlation to liposomal cytarabine occurred in 32% of the patients. The most frequent adverse event was headache. One patient developed severe neurological complications with loss of vision and a conus syndrome. Overall, liposomal cytarabine showed excellent antileukemic activity. Toxicity was acceptable but appeared to increase with the number of cycles. Future evaluation in prophylaxis is of interest.

Relapsing acute myeloid leukemia beginning with a penile localization

We report the case of a 52-year-old man who was diagnosed for an acute myeloid leukemia (FAB classification: M1) with abnormalities in conventional caryotype showing trisomy 7 and recombination of MLL-AML1. His leukemia was discovered in front of neutropenic fever and circulating blastic cells. He was first treated with three cycles of intensive chemotherapies using high doses of cytarabine and idarubicine in association, followed by a nonmyeloablative allogeneic bone marrow-related transplantation based on busulfan and fludarabine conditioning. He relapsed 2 months later with pancytopenia and was, therefore, treated with a myeloablative bone marrow-related transplantation using melphalan 200 mg/m conditioning. He stayed in complete clinical and cytological remission for 16 months, without any signs of GVHD. He then started to complain of a painfull spot on his penis. On examination, he had first a nodular painful lesion of the dorsal side of the penis, with no contiguous lymph nodes. No peripherical or medullar blastic infiltration was seen. Axial and sagittal fused FDG PET-CT images showed an intense and isolated FDG uptake of the distal corpus cavernosa (Image 1A). A surgical excision with anatomopathological analysis confirmed the leukemic origin of the lesion showing myeloblastic cells CD34 1 (Image 1B). The margins were safe from leukemia cells. However, even before beginning a treatment, he relapsed with a bilateral infiltration of corpus cavernosa, the penile skin, and an inguinal adenopathy. Because surgery did not limit the extension of leukemia as already report, treatment consisted in an association of a systemic donor lymphocytes injection with gemtuzumab ozogamicin (at 3 mg/ṁ) and a local radiotherapy at 40 Gy on penis and 30 Gy onto the inguinal lymph node areas, seen on sagittal CT-scan (Image 1C). A complete local remission was observed, but 3 months later, a new relapse in the gums was observed. A palliative therapy was begun, but he died 2 months later from a severe dyspnoea, due to a blastic localization in oropharynx and blastic hyperleucocytosis at 50G/L. To date, only five cases of penile localization of acute leukemia have been listed: two of them were AML [1,2]. There were all FAB M4 subtype, and the penile metastasis was seen at relapse. The first patient was treated with radiotherapy exclusively and the second by surgery, but the two patients died for disseminated disease. Three others Image 1. A: Sagittal and axial FDG PET-CT, with fused images, showing intense nodular FDG uptake of the distal corpus cavernosa. B: Massive tissue infiltration by myeloblastic cells, confirmed by positive CD34 staining. C: First dosimetry planning for radiotherapy treatment. Sagittal CT-scan showing important oedema of all the penis. Radiotherapy treatment neaded two other dosimetry planning due to the good results it gave.

Quantitative assessment of WT1 gene expression after allogeneic stem cell transplantation is a useful tool for monitoring minimal residual disease in acute myeloid leukemia

WT1 overexpression is described in several oncological diseases including acute myeloid leukemia (AML). Quantification of WT1 in bone marrow samples may be useful as a marker of minimal residual disease (MRD) and may predict the relapse of AML after allogeneic hematopoietic stem cell transplant (HSCT). The quantitative expression of WT1 was measured in 38 AML patients (16 males and 22 females) at diagnosis, at the time of transplant and after the allogeneic HSCT (at precise time points). All cases showed high WT1 expression levels at diagnosis with a mean of 4189 (SD 3325) and a median of 3495 (range 454-13923) copies WT1/10(4)Abl. At transplant, 25 patients (66%) were in complete cytologic remission (CcR) and 13 (34%) had refractory or relapsed AML. Bone marrow samples from patients transplanted in CcR showed significantly lower WT1 expression levels during HSCT compared with the samples from patients with a relapsed or refractory AML (P = 0.004). After HSCT, a rapid decline in WT1 expression levels was observed in all patients who attained or maintained a condition of CcR. Six of 38 patients (13%) relapsed after HSCT and all of them had an increase in WT1 expression at/or before relapse. Five of these six patients died of leukemia and one was successfully reinduced with donor lymphocyte infusion (DLI) + chemotherapy with a rapid reduction of WT1 levels. Besides, we found a complete concordance between WT1 expression levels and other disease markers (when available). In our experience, there was a complete concordance between WT1 expression levels (measured by quantitative RT-PCR at precise time points) and status of AML before and after allogeneic HSCT. WT1 may be useful as a non-specific leukemia marker for monitoring MRD and as a predictor of AML clinical relapse. Based on these results, cases with increase of WT1 levels after HSCT and without graft vs. host disease may be candidate to discontinuation of immunosuppression and/or DLI therapy.

Complete Hematologic and Cytologic Remission in M6A Erythroleukemia Using Decitabine Hypomethylation Monotherapy

Hypomethylating drugs (e.g. Azacitadine, Decitabine) are becoming increasingly attractive choices for the treatment of acute myelogenous leukemia (AML), particularly in populations deemed inappropriate for standard anthracycline-based induction chemotherapy (i.e., 7+3), such as the elderly. Acute erythroleukemia (French-American- British M6) is a rare, heterogeneous disorder involving increased red cell precursors and myeloblasts, that accounts for 3–5% of de novo AML and 20–30% of secondary AML. Three subsets have been described: M6A (myeloblast-rich erythroleukemia); M6B (proerythroblast-rich erythroleukemia); and M6C (myeloblast- and proerythroblast-rich mixed variant). Little data exists on the efficacy of hypomethylation therapy in this uncommon subtype of AML. We report the rapid induction of a complete hematologic and cytologic remission in a patient with the M6A variant erythroleukemia using decitabine monotherapy. A 75 year-old white male with a history of coronary artery disease, was admitted for elective cardiac catheterization because of recurrent exertional chest pain. A pre-catheterization complete blood count (CBC) revealed pancytopenia with a white blood cell (WBC) count of 1400/mm3, hemoglobin of 6.2 g/dL (mean cell corpuscular volume of 113 fL), and a platelet count of 51,000/mm3. The differential showed neutropenia, but was otherwise normal with no circulating blasts. Blood transfusion completely relieved his chest pain, and his catheterization was uneventful. A bone marrow aspirate and biopsy demonstrated a hypocellular marrow (15–20%) with erythroid hyperplasia (70% of total cellularity) and increased blasts (10% of total cellularity, 30% of non-erythroid cells), most compatible with acute M6A erythroid/myeloid leukemia. Cytogenetic studies revealed a normal male karyotype, but a single cell with a 5q- deletion. Subsequent fluorescent in situ hybridization (FISH) analysis using an LSI DNA probe (Vysis Inc.) for detection of the EGR1 gene on chromosome 5q, failed to confirm the 5q deletion, consistent with normal cytogenetics. Because of the patient's advanced age and co-morbidities, induction with hypomethylation monotherapy was begun with intravenous decitabine (Dacogen®) 20 mg/m2 daily for five consecutive days, repeated every four weeks. Growth factor support (Neupogen® 375 mcg daily) and antibiotic prophylaxis (ciprofloxaxin, acyclovir, fluconazole) were only required after the first cycle of decitabine because neutropenia did not occur with subsequent cycles. WBC and platelet counts normalized shortly after cycle one, while the hemoglobin normalized by cycle five. An interim bone marrow aspirate/biopsy was repeated after cycle three, showing normocellularity (30%), and no evidence of residual leukemia. Other than a localized herpetic reactivation following cycle one, the patient has tolerated hypomethylation therapy extremely well. He has now completed eight cycles of therapy, and remains in complete remission. Epigenetic alterations (such as DNA methylation) may play a role in AML leukemogenesis by inducing the inhibition of tumor suppressor genes. Recent studies have also shown that high estrogen receptor-α and p15INK4B methylation levels in AML patients in clinical remission, were associated with a high risk for leukemia relapse and poor relapse-free survival. However, little data exist for epigenetic dysregulation in the etiology of human erythroleukemia. Cell culture studies using human erythroleukemia cell lines indicate that DNA methylation may have functional consequences for gene activity, including globin gene expression and cellular differentiation. The current case indicates a potential role for DNA methylation in the pathogenesis of human erythroleukemia, and argues for further investigation into this potential mechanism of leukemogenesis. Future studies will include the effect of hypomethylating compounds on the growth and differentiation of human erythroleukemia cells, and the evaluation of DNA methylation levels in bone marrow biopsy samples from M6 patients.

Isolated central nervous system relapse during cytologic and molecular hematologic remission in two patients with acute promyelocytic leukemia

Extramedullary involvement in the absence of bone marrow disease is rare in patients with acute promyelocytic leukemia (APL). We report two patients with APL who had central nervous system (CNS) relapse without evidence of cytologic and molecular disease of bone marrow after all-trans-retinoic acid (ATRA) treatment. Both of the patients were treated successfully with combination of intrathecal chemotherapy and radiotherapy with or without systemic chemotherapy. Although increasing number of cases with extramedullary involvement of APL after ATRA including therapy have been reported, further studies with a large series of patients are necessary to determine whether ATRA increases the risk of development of extramedullary involvement of disease in patients with APL.

Immunotherapy of high-risk acute leukemia with a recipient (autologous) vaccine expressing transgenic human CD40L and IL-2 after chemotherapy and allogeneic stem cell transplantation

CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. We studied the feasibility, safety, and immunologic efficacy of an IL-2- and CD40L-expressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including 7 children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to 6 subcutaneous injections of the IL-2/CD40L vaccine. None of the patients were receiving immunosuppressive drugs. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)-restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferon-gamma, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27 to 62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived antileukemia vaccines can induce immune responses reactive against leukemic blasts. This approach may be worthy of further study, particularly in patients with a high risk of relapse.

Real-time RT-PCR quantification of PRAME gene expression for monitoring minimal residual disease in acute myeloblastic leukaemia

Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. PRAME transcripts were quantified by real-time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME-positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME. The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME-positive leukaemia.

A Deletion 7q in a M0 Adult Acute Myeloid Leukemia (AML) with a Complete Cytogenetic Response to Idarubicin.

The alteration of chromosome 7 is associated with a reported unfavorable prognosis in acute myeloid leukemia. The deletion of the 7q is seen alone in approximately 1% of AML cases. This finding is associated with an AML M0, M1, and M2 FAB classification. Treatment with idarubicin is thought to be equivalent to other anthracyclines in the induction of a complete remission in acute myeloid leukemia. In previous studies younger patients have responded with improved complete remission rates, when idarubicin is used. A unique case with a 7q single aberration and an AML M0 FAB classification is reported with a complete and rapid response of less than 32 days after one cycle of idarubicin at a dose of 12 mg /m2/day for three days accompanied by a cytosine arabinoside intravenous infusion at a dose of 100 mg/m2/day for seven days. A complete cytologic and genetic remission was obtained after one cycle of induction. The patient received high dose cytosine arabinoside consolidation and will be considered for autologous stem cell transplant. The patient remains in a complete genetic and cytologic remission. In this 25 year old african american female a complete cytologic and genetic remission by FISH is obtained in a rare acute myeloid leukemia genotype with a reportedly unfavorable prognosis using idarubicin as the anthracycline in the 3+7 combination.Conclusion: Further studies are required to better define the appropriate anthracycline in acute myeloid leukemia by using age and cytogenetic markers. In this rare case presentation, idarubicin is shown to be effective in inducing a complete and rapid cytogenetic remission in a young adult patient with a chromosome 7 cytogenetic aberration and an unfavorable prognosis.

Leucemia mínima residual: nuevo concepto de remisión completa

Early response to induction treatment is one of the most important prognostic factors in children with acute lymphoblastic leukemia (ALL). Cytological remission is currently achieved in 95-98 % of these patients, although a significant proportion will later relapse. More sensitive techniques are required to measure residual leukemia and establish a new definition of complete remission. To identify minimal residual disease (MRD) by immunological techniques and define its prognostic impact in children with ALL. MRD was studied by flow cytometry in 53 children diagnosed in our department between June 1999 and April 2003 and treated using the Pethema protocols. All the children achieved complete cytological remission (< 5 %) with the induction treatment and had at least one useful phenotype for follow-up: 11 % were T phenotype, one was biphenotypic and the remainder were B cell leukemias. Bone marrow samples were analyzed post-induction, post-consolidation, after 6 and 11 months of maintenance treatment, at the end of treatment, and 3 months later. The positivity threshold was set at 0.01 % and the sensitivity of the technique was 1 x 10(-4)-1 x 10(-5). A total of 199 samples were analyzed. Thirty-seven percent of the post-induction and 20 % of the post-consolidation samples analyzed were MRD-positive. Elimination was slower in patients with a T phenotype and in high-risk patients according to the traditional classification. After a median follow-up of 26 months, event free survival (EFS) in the group as a whole was 92 %. The EFS rate in the patients who were MRD-positive post-induction was 79 %. None of the patients who were MRD-negative post-induction has developed recurrence. Study of MRD is essential and should be included in all current treatment protocols for children with ALL. Its usefulness derives from the prognostic impact of the response to induction treatment. Continued sequential monitoring may predict recurrence before the onset of clinical or hematologic manifestations.

Cytogenetic Remission Status after Induction Chemotherapy for AML and High-Risk MDS Predicts Long-Term Outcome.

Cytogenetic findings at diagnosis have an important impact on prognosis in AML and MDS. Therefore, treatment is commonly stratified according to karyotype. Another important prognostic factor is remission status after induction chemotherapy. However, the diagnosis of remission, and the resulting treatment decisions, are usually not based on cytogenetic findings, but rely on peripheral blood counts and bone marrow blast cell counts. We analysed the prognostic impact of cytogenetic remission status in 115 patients with abnormal karyotype who received induction chemotherapy because of AML (n=102) or MDS (n=13). Initial karyotypes were as follows: 14x t(15;17), 9x t(8;21), 5x inv(16), 55 intermediate-risk, and 32 complex karyotypes. The following induction protocols were used: 38x TAD, 4x HAM, 25x Ida-Ara, 36x ICE, and 3x other protocols. Eleven patients received induction chemotherapy plus ATRA. 23 patients underwent allogeneic transplantation after induction therapy, either as late consolidation in cytologic remission (CR), or as standard treatment in high-risk patients. 78 patients achieved CR, 21 achieved partial remission (PR), and 16 patients were non-responders (NR).There were 26 patients who reached cytological CR but still showed an abnormal karyotype after induction. 17 of these 26 patients had been classified as standard-risk and therefore did not receive intensified consolidation. In 59 patients, a normal karyotype was found after induction therapy. 24 of 28 patients belonging to low-risk cytogenetics (t15;17, t8/21, or inv16) achieved cytogenetic complete remission (CCR). The CCR rate was much lower in patients with intermediate-risk cytogenetics (24/55) and patients with complex karyotypes (11/32). Median survival in the group achieving CCR was 40 months, as compared to 11 months in patients with persistence of abnormal karyotype after induction (p<0,00005). The difference remained highly significant when calculated only for patients with complex or intermediate-risk karyotypes (median survival 29 vs 11 months).Conclusions: Cytogenetic analysis after induction chemotherapy is useful to detect residual disease and therefore provides meaningful information in addition to cytology. Achieving cytogenetic remission after induction therapy is strongly correlated with a better long-term outcome. Patients with a persisting abnormal karyotype must be regarded as high-risk patients who should receive intensified treatment.

Immunosurveillance of childhood ALL: polymorphic interferon-γ alleles are associated with age at diagnosis and clinical risk groups

Interferon-gamma (IFN-gamma) has been implicated as an important mediator of antitumor immunity in murine model systems. To determine whether a CA-repeat associated with differential NFkappaB-binding and IFN-gamma-expression levels may influence the incidence, manifestation and early clinical treatment response of childhood acute lymphoblastic leukemia, we performed PCR-based genotyping of 393 patients with ALL and 207 healthy controls. We could not find any differences in the allele distribution comparing patients and controls. However, when we further analyzed the allele frequencies with respect to age of clinical manifestation, we found that patients with B-lineage ALL showing the IFN-gamma high-expressing genotype presented at a more advanced age compared to those patients with intermediate and low-expressing genotypes (median 6 vs 4.4 years, P=0.01). Furthermore, we found a significantly higher number of low expressors in the group of high-risk patients (HR n=32 and MR/SR n=266, P=0.025, defined by prednisone response, cytological remission and minimal residual disease (MRD)) with B-lineage ALL. Thus, we provide evidence that polymorphic IFN-gamma alleles are associated with age at clinical presentation and risk groups such as prednisone response in B-lineage ALL, suggesting distinct effects of IFN-gamma in immunosurveillance and early response to steroid therapy.