Relapsed Acute Promyelocytic Leukemia Research Articles

Hemorrhagic cystitis in a patient receiving gemtuzumab ozogamicin for relapsed acute promyelocytic leukemia after cord blood transplantation

Dear Editor, We experienced hemorrhagic cystitis (HC) after gemtuzumab ozogamicin (GO) administration for a patient with acute promyelocytic leukemia (APL). A 52-year-old male with APL in the second relapse underwent unrelated cord blood transplantation (uCBT). The preparative conditioning regimen consisted of 12 Gy total body irradiation, 120 mg/kg cyclophosphamide, and 12 g/m 2 cytarabine. He developed HC on day 20 which promptly improved by hyperhydration. This HC was associated with adenovirus (ADV) and BK virus, both of which were confirmed in urine by polymerase chain reaction (PCR) assays. Primary engraftment was achieved on day 20, and complete donor chimerism and molecular complete remission (CR) were attained on day 28.

Hematopoietic stem cell transplantation for acute promyelocytic leukemia in second or third complete remission: a retrospective analysis in the Nagoya Blood and Marrow Transplantation Group

Acute promyelocytic leukemia (APL) is the most curable subtype of acute myeloid leukemia. Second complete remission (CR2) can be easily achieved with several therapeutic options even after relapse. However, the optimal strategy to treat APL in CR2 is still controversial. We retrospectively compared the outcome of autologous (auto) and allogeneic (allo) hematopoietic stem cell transplantation (HSCT) for patients with APL in CR2 or CR3. Fifteen patients received auto and 13 received allo HSCT between 1999 and 2004 at eight hospitals belonging to the Nagoya Blood and Marrow Transplantation Group. Four-year disease-free survival (DFS) and overall survival (OS) for autografted patients were 68.9 and 75.8%, whereas those for allografted patients were 46.2 (P = 0.350) and 46.2% (P = 0.185), respectively. Three autografted patients and one allografted patient relapsed, and one autografted patient and five allografted patients died without leukemia relapse. Among 14 autografted patients who were evaluated for MRD with molecular analysis, relapse occurred in one with positive MRD (n = 2) and two with negative MRD (n = 12). These data suggest that auto HSCT is very effective for APL in CR2 or CR3, and may be preferable to allo HSCT for a portion of patients. Prospective studies are required to define the role of auto HSCT in the treatment of relapsed APL.

Phase 1 trial and pharmacokinetic study of arsenic trioxide in children and adolescents with refractory or relapsed acute leukemia, including acute promyelocytic leukemia or lymphoma

Arsenic trioxide (ATO) induces remission in 85% of adults with refractory acute promyelocytic leukemia (APL). We conducted a phase 1 trial of ATO in children (median age 13 y, range, 2-19) with refractory leukemia. ATO was administered intravenously over 2 hours, 5 d/wk for 20 doses/cycle. Patients with APL (n=13) received 0.15 mg/kg per day, and patients with other types of leukemia received 0.15 mg/kg per day (n=2) or 0.2 mg/kg per day (n=4). Nineteen of the 24 enrolled patients were fully evaluable for toxicity. At 0.15 mg/kg per day, 2 of 15 patients experienced dose-limiting corrected QT interval (QTc) prolongation, pneumonitis, or neuropathic pain. At 0.2 mg/kg per day, 2 of 4 patients had dose-limiting QTc prolongation or pancreatitis. Non-dose-limiting toxicities included elevated serum transaminases, nausea, vomiting, abdominal pain, constipation, electrolyte imbalance, hyperglycemia, dermatitis, and headache. At 0.15 mg/kg per day, the median (range) plasma arsenic maximum concentration (Cmax) was 0.28 microM (0.11-0.37 microM) and at 0.2 mg/kg per day, Cmax was 0.40 and 0.46 microM; area under the concentration times time curve (AUC0-24) was 2.50 microM-hr (1.28-3.85 microM-hr) and 4.37 microM-hr and 4.69 microM-hr, respectively. Morphologic complete response (CR) was achieved in 85% of patients with APL; no responses were observed in non-APL patients. ATO is well-tolerated in children at the recommended dose of 0.15 mg/kg per day. The response rate in children with relapsed APL is similar to the response rate in adults. This trial was registered as #NCT00020111 at www.ClinicalTrials.gov.

Comparison of Clinical Outcomes of Relapsed APL Patients Induced with ATO and Consolidated with Either an Autologous SCT or ATO Based Treatment Regimen.

Arsenic trioxide (ATO) is the most effective agent in inducing remission among patients with relapsed acute promyelocytic leukemia (APL). After induction of remission in relapsed patients with ATO the best consolidation regimen remains to be defined. Since January 2000, 33 patients with relapsed APL were treated at our center. The median age was 35 years (range: 6 – 57) and there were 16 (48.5%) males. The initial treatment was conventional ATRA plus chemotherapy in 22 (66.7%) and single agent ATO in 11 (33.3%). The median duration of first remission was 19.4 months (range: 2.9 – 81.2). Relapse was treated with single agent ATO in 22 (66.7%), with ATO+ATRA in 5(15.2%) and with ATO+ATRA+anthracycline in 6 (18.2%). Thirty one (94%) achieved CR while one patient died of sepsis in induction and one died from progressive disease. After remission induction a consolidation course of the same agents used in induction was administered (ATO and ATRA were administered for 28 days), 29 (88%) achieved molecular remission after this course. All patients who achieved molecular remission were offered an autologous SCT. Nine patients opted to have an autologous SCT and the remaining patients were scheduled to receive monthly cycles of ATO as a single agent (n=15) or ATO+ATRA (n=7) for 6 months. The age, sex, duration of first CR, induction and consolidation regimen used to treat relapse was comparable between the patients that underwent an autologous SCT and the remaining patients. Patients who underwent an autologous SCT were conditioned with a conventional Bu/Cy regimen and received PBSC graft with a median cell dose of 5 x 106 MNC/Kg (range: 3.54 – 9.24). There were no treatment related deaths following the autologous SCT, one patient had an isolated CNS relapse 6 months post transplant. There were no other relapses or deaths in this group. Among the remaining patients who achieved CR but did not have an autologous SCT (n=22), 14 relapsed and all except one of these patients eventually died of progressive disease at median period of 1.6 months from second relapse (range: 0 – 42.6). At a median follow up of 24 months, the 3-year Kaplan-Meier estimate of EFS and OS among those who received an autologous SCT vs. those that did not was 88.9±10.5 vs. 28.8±9.7 (log rank: P=0.019) (Figure 1) and 100±00 vs. 36.2±10.4 (log rank: P=0.009) respectively. Following remission induction with ATO based regimens in patients with relapsed APL, consolidation with an autologous SCT, in those who achieve a molecular remission, is associated with a significantly superior clinical outcome in comparison to alternative ATO/ATRA based consolidation and maintenance regimens. [Display omitted]

Evaluation of Prospective Detection of PML-RARA and RARA-PML Fusion Transcripts by Real-Time Quantitative PCR (RQ-PCR) To Direct Pre-Emptive Therapy with Arsenic Trioxide (ATO) in Acute Promyelocytic Leukemia (APL) Patients Treated in the UK MRC AML15 Trial.

A key challenge in the management of APL is to improve upon survival rates now achieved with ATRA and anthracycline-based chemotherapy. One approach involves minimal residual disease (MRD) monitoring to identify patients otherwise destined to relapse, whose outcome may be improved by pre-emptive therapy. Patients with PML-RARA+ APL (n=261) were prospectively monitored by RQ-PCR in the MRC AML15 trial, comparing a MRC treatment schedule comprising 4 courses of combination chemotherapy (ADE+ATRA,ADE,MACE,MidAC) with a less intensive PETHEMA schedule involving anthracycline monotherapy+ATRA followed by maintenance (ATRA/6MP/MTX). Paired peripheral blood (PB) and bone marrow (BM) samples were analyzed after each course, then 3-monthly for 2 years. Patients with persistent PCR positivity or molecular relapse (defined by successive PCR positive results with rising PML-RARA transcript level) received ATO and proceeded to autologous/allogeneic transplant according to molecular response; patients unfit for transplant received 3 ATO courses. 4813 samples were analyzed (median 16/pt), including 1507 paired samples. No difference in kinetics of disease response was observed between schedules (median time to molecular CR (CRm): 70d MRC v 67d PETHEMA,p=0.3). Furthermore, neither response kinetics nor MRD status during or immediately after consolidation identified those most at risk of relapse. 6/182 (3%) patients tested PCR positive after course 4. While this has been considered an indication for allogeneic transplant, serial monitoring showed progression in only 2 (given ATO pre-emptively); the other 4 remain in CRm 3–37mo later. Most patients subject to relapse tested PCR negative post-consolidation (10/12,83%), with fusion transcripts first detected at a median of 8mo (3–33mo) later. While concordance between BM and PB MRD results was high in early phases of treatment (r>.46,p<0.0001), in relapsing patients molecular conversion in BM preceded that in PB in 4/5 evaluable patients by a median of 29d, correlating with a 1.5log greater sensitivity for the former sample type. Six patients received pre-emptive ATO and remain in CR at a median follow-up of 22mo. However, half the remaining patients with recurrent disease were not treated pre-emptively; in 3 although relapse was successfully predicted by RQ-PCR, BM blasts exceeded 5% by the time ATO was commenced 23–84d following molecular conversion, 1 patient developed early CNS relapse, while in 2 patients relapse could not be predicted due to omission of scheduled MRD samples. Three of 6 patients subject to frank relapse have died, including two of hemorrhage/sepsis shortly after presentation. In 5 patients, suspected relapse of APL was excluded by PML-RARA negativity and were diagnosed as t-MDS/AML. At 3 years the overall rate of frank relapse is 3%, which compares favorably with the previous MRC AML12 trial (13% at 3 yrs) in which the same MRC treatment was used, but routine MRD monitoring was not performed, suggesting that rigorous sequential RQ-PCR monitoring of BM coupled with pre-emptive therapy provides a valid strategy to reduce rates of frank relapse in APL.

Myeloperoxidase Is Required for the Combined Apoptotic Effects of Arsenic Trioxide with Ascorbic Acid or Epigallocatechin-3-Gallate in Myeloid Leukemia Cells.

Arsenic trioxide (As2O3) is a drug used world-wide that selectively causes the death of acute promyelocytic leukemia (APL) cells by novel mechanisms and induces complete clinical remission in 90% of patients without significant toxicity. The outstanding success of As2O3 therapy in relapsed APL patients has not been replicated by an equivalent success in other types of acute myeloid leukemia (AML). We have studied As2O3 mechanism(s) of action and identified agents to use in combination to improve the use of As2O3 as a treatment for other types of AML. As2O3 produces higher levels of H2O2 and apoptosis in APL NB4 cells than in other AML cells at therapeutic concentrations of 1-2 uM. As2O3 does not induce apoptosis in HL-60 cells but is synergistic with ascorbic acid (AA) or epigallocatechin-3-gallate (EGCG). Both AA and EGCG produce H2O2 which is augmented by the addition of As2O3. Apoptosis induction by As2O3 in combination with AA or EGCG is inhibited by catalase and the antioxidant N-acetylcysteine. Myeloperoxidase (MPO), a major neutrophil enzyme, augments H2O2-induced apoptosis by converting it into more potent reactive oxygen species. HL-60, NB4, SKNO-1 and PLB985 cells which express high level of MPO, but not U937 and K562 cells without expression of MPO, are responsive to As2O3 plus AA or EGCG-induced apoptosis. HP-100 cells, a subclone of HL-60 cells without MPO expression, are resistant to both combination treatments. MPO stable transfection sensitizes K562 cells to apoptosis following treatment with As2O3 and AA or EGCG. NADPH oxidase is an enzyme complex which generates O2− and H2O2 in neutrophils. In APL NB4 cells, As2O3 induces expression of NADPH oxidase members and is thought to participate in H2O2 production required for As2O3 response. X-CGD cells, a subclone of PLB-985 with targeted disruption of the gp91phox gene (Zhen et al, PNAS 90:9832, 1993) and loss of NADPH oxidase activity but with high levels of MPO activity, are responsive to treatment with As2O3 and AA or EGCG. These results suggest that AA and EGCG enhance As2O3-induced apoptosis through a synergistic production of H2O2 which is independent NADPH oxidase activation in non-APL AML cells. MPO augments the apoptotic effect of H2O2 produced by As2O3 plus AA or EGCG and it could be used as a marker to predict the sensitivity of AML cells to both combination treatments.

PML-RARα inhibitors (ATRA, tamibaroten, arsenic troxide) for acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by generation of the PML-RARalpha fusion gene. PML-RARalpha can homodimerize with another PML-RARalpha, and the hybrid binds the histone-deacetylase recruiting co-repressor complex with higher affinity than the wild-type RARalpha. However, the co-repressor complex is releasable by pharmacological doses of all-trans retinoic acid (ATRA). More than 90% of patients with APL achieve a complete remission (CR) with differentiation therapy consisting of ATRA combined with chemotherapy. A new synthetic retinoid, tamibaroten, showed therapeutic effectiveness in patients with ATRA-resistant APL with increased expression of cellular retinoic acid binding protein (CRABP), and about 60% of patients with relapsed APL achieved a CR. Arsenic trioxide triggers the rapid degradation of PML-RARalpha through the targeting of the PML moieties of the fusion protein and showed a high CR rate in relapsed APL. The combination of ATRA, chemotherapy, and/or new agents improved the long-term survival in patients with APL.

Arsenic trioxide entered cerebrospinal fluid with the help of mannitol overwhelm the meningeal relapse of acute promyelocytic leukemia

Central nervous system (CNS) relapse of acute promyeloytic leukemia (APL) is increasingly reported after treatment with arsenic trioxide (As2O3),[1][1] the optimal therapy for this case remains unclear. As2O3 highly effective on APL marrow relapse, but its efficacy in CNS relapse is undefined.

Acute intestinal pseudo‐obstruction after induction treatment of relapsed acute promyelocytic leukemia with arsenic trioxide

Arsenic trioxide (As(2)O(3)) is an effective agent for the treatment of relapsed acute promyelocytic leukemia (APL). We report a patient with intestinal pseudo-obstruction, which occurred while treating relapsed APL with As(2)O(3). A 6-year-old female with relapsed APL developed paralytic ileus, hyperleukocytosis, and a high fever while being treated with As(2)O(3). Although As(2)O(3) was discontinued and dexamethasone was administered, vomiting and abdominal distension worsened. An ileostomy was performed and diffuse patch-like infiltrations on the bowel surface were noted. Pathologic findings revealed APL cells involving the entire intestinal layers. This case history suggests that As(2)O(3) when used for reinduction therapy for APL may adversely affect the intestine and cause acute intestinal pseudo-obstruction.

Arsenic metabolism in multiple myeloma and astrocytoma cells

Arsenic trioxide (As2O3, Trisenox) is used to treat patients with refractory or relapsed acute promyelocytic leukemia (APL). Its ability to induce apoptosis in various malignant cell lines has made it a potential treatment agent for other malignancies and many clinical trials are currently in progress to evaluate its clinical usefulness for multiple myeloma and glioblastoma cancer. In the present study, we investigated the metabolism of As2O3 regarding its cellular biotransformation and interaction with metallothionein (MT) as a possible protective responses of cells to arsenic-induced cytotoxicity. The study was performed on two types of cell treated with As2O3: (1) human astrocytoma (glioblastoma) cell line U87MG treated with 0.6 microM arsenic for 0, 3, 12, 24, and 48 h or 12 microM arsenic for 3, 6, 12, 24, and 48 h and (2) bone marrow cells (BM) from two patients with multiple myeloma (MM) treated with 7 microM arsenic for 0, 43, and 67 h. Cotreatment with vitamin C (1 mg/mL) was tested in longer exposure of MM BM cells. Traces of methylation products (mainly monomethylarsenic acid) were detected in cell lysates of both cell types and in pellets of U87 MG cells, although we found problems with column-sample interactions in cases where methanol pretreatment of the sample was not used. Pentavalent inorganic arsenic (AsV) was identified in both cell types, and up to 80% of total As in MM bone marrow cell lysates was present as AsV. Such an occurrence (generation) of pentavalent arsenic after As2O3 treatment demonstrates the presence of biological oxidation of trivalent arsenic, which could represent an additional protective mechanism of the cell. Vitamin C decreased As cell content and increased the percentage of pentavalent inorganic arsenic (in the growth medium and cells). The presence of metallothionein (MT) and its response to arsenic treatment was checked in all U87 MG cells, in the control, and in one exposed sample of MM BM cells. During 48 h exposure to 0.6 or 12 muM arsenic MTI/II levels increased in U87 MG cells, but with variable Zn levels, increased Cu levels, and As binding observed in traces only. Involvement of the MT-III isoform was negligible. In contrast, 43 h exposure to 7 microMarsenic did not increase MT content in multiple myeloma cells, and the levels even decreased with respect to the control. To evaluate the importance of the observed processes, MTs in U87 and AsIII-AsV conversion in MM BM cells, which could represent a resistance response of cancer cells treated by As2O3, longer-term observation with different arsenic concentrations should be performed.

Treatment of acute promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide: a paradigm of synergistic molecular targeting therapy

To turn a disease from highly fatal to highly curable is extremely difficult, especially when the disease is a type of cancer. However, we can gain some insight into how this can be done by looking back over the 50-year history of taming acute promyelocytic leukaemia (APL). APL is the M3 type of acute myeloid leukaemia characterized by an accumulation of abnormal promyelocytes in bone marrow, a severe bleeding tendency and the presence of the chromosomal translocation t(15;17) or variants. APL was considered the most fatal type of acute leukaemia five decades ago and the treatment of APL was a nightmare for physicians. Great efforts have been made by scientists worldwide to conquer this disease. The first use of chemotherapy (CT) was unsuccessful due to lack of supportive care and cytotoxic-agent-related exacerbated coagulopathy. The first breakthrough came from the use of anthracyclines which improved the complete remission (CR) rate, though the 5-year overall survival could only be attained in a small proportion of patients. A rational and intriguing hypothesis, to induce differentiation of APL cells rather than killing them, was raised in the 1970s. Laudably, the use of all-trans retinoic acid (ATRA) in treating APL resulted in terminal differentiation of APL cells and a 90-95% CR rate of patients, turning differentiation therapy in cancer treatment from hypothesis to practice. The combination of ATRA with CT further improved the 5-year overall survival. When arsenic trioxide (ATO) was used to treat relapsed APL not only the patients but also the ancient drug were revived. ATO exerts dose-dependent dual effects on APL cells: at low concentration, ATO induces partial differentiation, while at relatively high concentration, it triggers apoptosis. Of note, both ATRA and ATO trigger catabolism of the PML-RARalpha fusion protein which is the key player in APL leukaemogenesis generated from t(15;17), targeting the RARalpha (retinoic acid receptor alpha) or promyelocytic leukaemia (PML) moieties, respectively. Hence, in treating APL both ATRA and ATO represent paradigms for molecularly targeted therapy. At molecular level, ATRA and ATO synergistically modulate multiple downstream pathways/cascades. Strikingly, a clearance of PML-RARalpha transcript in an earlier and more thorough manner, and a higher quality remission and survival in newly diagnosed APL are achieved when ATRA is combined with ATO, as compared to either monotherapy, making APL a curable disease. Thus, the story of APL can serve as a model for the development of curative approaches for disease; it suggests that molecularly synergistic targeted therapies are powerful tools in cancer, and dissection of disease pathogenesis or anatomy of the cancer genome is critical in developing molecular target-based therapies.

Minimal Residual Disease (MRD) and Risk of Relapse in Acute Promyelocytic Leukemia (APL): Insights from the North American Intergroup Phase III Trial C9710.

MRD results during and following treatment (Rx) of APL have become an important predictor of clinical outcome and are beginning to be used to guide Rx for this highly curable leukemia, but important questions remain. Some studies suggest that low/intermediate (LI)-risk patients (pts), as defined by the Sanz criteria, who achieve a negative PCR result after intensive consolidation Rx have a very low risk of relapse and may not require maintenance Rx, while those with MRD might benefit from hematopoietic stem cell transplantation. However, these data are controversial. We studied whether a single quantitative MRD test obtained immediately after completion of C9710 consolidation Rx (with all-trans retinoic acid, daunorubicin, with or without arsenic trioxide) predicted relapse and could be used to guide further Rx. Real-time quantitative RT-PCR( RQ-PCR) was used to measure PML-RARα in bone marrow (BM) and blood (B) of 43 pts treated on C9710. All 43 pts were in first complete remission after completion of consolidation Rx when MRD was measured, but all of them later relapsed during, or after completion of maintenance Rx. By Sanz criteria, 14/43 were high risk and 29/43 LI-risk at diagnosis. 29 had S-isoform, 12 had L-isoform, and 2 had V-isoform. Median time from MRD assessment to relapse was 253 days (range: 44–1231). RQ-PCR results were expressed as a normalized quotient (NQ) of PML-RARα/GAPDH or ABL control. The sensitivity of the assay was 1 in 104–5. A NQ of >10−5 was found in the prior intergroup study, INT0129, to be strongly associated with risk of relapse and was set as the threshold for considering a result “positive”. MRD with NQ >10−5 was detected in only 4 of 43 (9%) pts after completion of consolidation Rx. 8 others had detectable PML-RARα transcript but had NQs < 10−5. The remaining 31 pts had no evidence of MRD. There was excellent concordance between MRD measurements in 26 paired B and BM NQ values and a strong correlation between NQ values in B and BM using either GAPDH or ABL controls (p = 0.0001 and p <0.0001, respectively). To determine whether a more sensitive assay might identify a higher percentage of pts with MRD, we also evaluated the post-consolidation samples in 19 of these pts using a qualitative nested RT-PCR assay with a sensitivity of detection of 1 in 106. We found 13 of 19 (68%) pts had detectable MRD. In conclusion, RQ-PCR analysis of MRD using a threshold NQ of >10−5 at a single post-consolidation Rx timepoint did not predict relapse of APL in pts on C9710. Setting a lower NQ threshold for the RQ-PCR assay or using a more sensitive, but qualitative PCR assay improved the detection rate; however, neither approach identified all pts destined to relapse, nor has the prognostic value of a positive result using the more sensitive assay been determined. Based on these results, we caution against the use of a single MRD test at the end of consolidation Rx to determine further Rx. Ongoing analysis of serial samples obtained during and following completion of Rx might provide further insights into optimal timing and frequency of MRD measurements needed to identify APL pts at high risk of relapse.

Lack of Increased Gene Promoter Methylation at Relapse in Acute Promyelocytic Leukemia (APL).

Increasing methylation of gene-regulatory DNA sequences has been associated with the progression of malignancies, and, in APL, the pathogenetic PML-RARα fusion gene has been associated with DNA methyltransferase activity, which might contribute to this process. We, therefore, hypothesized that promoter methylation levels would be increased in APL samples at disease relapse. Preliminary studies of a few CpG-enriched gene promoters, using highly quantitative pyrosequencing technology, demonstrated considerable individual-specific variation in DNA methylation. Hence, we used matched pretreatment and relapse bone marrow and/or peripheral blood samples with >85% promyeloblasts after low-density gradient enrichment from 4 APL patients. High quality DNA was prepared from fresh samples by a guanidinium-cesium chloride gradient procedure. Global analysis of CpG-enriched DNA was performed by the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay (Khulan, et al, Genome Res. 16, 1046Khulan, et al, Genome Res. 16, 2006), using a microarray representing the promoters of 24,000 human genes. The HELP assay relies upon a comparative genomic hybridization of HpaII (methylation-sensitive) versus MspI (methylation-insensitive) digested genomic DNA and is able to distinguish populations of methylated loci from hypomethylated (HO) loci. Based on the mean centered log ratio (MCLR) of HpaII/MpaI (Khulan, et al), there were negligible global changes in methylation between the 4 matched pretreatment and relapse samples. Using cut-off values to select for hypermethylated (HR) DNA (MCLR ≤0.0) and more completely HO DNA (MCLR ≥1.0) revealed some decrease in the ratio of HR:HO DNA at relapse in 3 of the 4 patients, primarily due to an increased fraction of HO DNA (see table). [Display omitted] We then assessed relapse-associated changes in methylation in 3,908 promoters in which the change from pretreatment to relapse was associated with p <0.05 (paired t-tests) across all 4 data sets. A change from HO to HR was defined as a decrease in the MCLR by ≥2-fold in the relapse group; a change from HR to HO was the opposite, i.e., an increase in MCLR by ≥2-fold. There was a change from pretreatment HO to relapse HR for 563 loci (7.5% of effective HO promoters) and from pretreatment HR to relapse HO for 949 promoters (8.6% of effective promoters). More stringently, when promoters were selected for a change in MCLR from pretreatment HO ≥1.0 to relapse HR ≤0.0 or, conversely from pretreatment HR ≤0.0 to relapse HO ≥1.0, 40 and 82 promoters, respectively, were identified with p ≤0.05. These results indicate that changes in gene promoter methylation from pretreatment HR to relapse HO are more common than the reverse, and they are contrary to our pre-study hypothesis that increased global DNA methylation might be a major contributor to molecular progression processes in APL cells leading to clinical relapse.

Real-Time Detection of PML-RARA and RARA-PML Fusion Transcripts in High Risk Acute Promyelocytic Leukemia (APL) Treated with Arsenic Trioxide (ATO): Implications for Realization of Pre-Emptive Therapy for Molecular Relapse.

A key challenge in the management of APL is to improve upon survival rates now achieved with ATRA and anthracycline-based chemotherapy. One approach involves minimal residual disease (MRD) monitoring to identify patients otherwise destined to relapse, whose outcome may be improved by pre-emptive therapy. There is also scope for improvement through reduction in induction deaths and treatment-related mortality, with recent studies suggesting that molecularly targeted therapy involving ATO can achieve long term remissions with limited toxicity. However, such strategies are critically dependent upon optimized schedules for MRD detection to rapidly identify those patients needing additional treatment. Yet, there are limited data concerning assay sensitivity, optimal sample type or kinetics of relapse, that are fundamental to delivery of MRD-directed therapy. We determined expression levels of PML-RARA and reciprocal RARA-PML transcripts in diagnostic BM samples from 102 patients using real-time quantitative PCR (RQ-PCR). Normalized PML-RARA transcript levels varied by 3-logs, equating with marked inter-patient variability in maximal achievable sensitivities for MRD detection, ranging between 1 in 102 and 1 in 105 (med. 1 in 103.8). RARA-PML fusion transcripts were expressed in 68/102 (67%). Parallel detection of both transcripts increased sensitivity for MRD detection in 35/102 (34%); yet, the detection limit was still <1 in 104 in 49% due to the low level of fusion transcript expression. Serial MRD monitoring was undertaken in patients (n=32) treated with ATO for relapsed APL in UK and Eire since Nov 02: 22 in hematologic relapse (HR) and 10 treated pre-emptively in 1st molecular relapse (MR). RQ-PCR assessment prior to MR/HR showed a median increase in PML-RARA transcripts of 0.8 log/month (range 0.2–1.9). Analysis of paired BM and blood (PB) samples revealed that molecular conversion in BM invariably preceded that in PB; including a patient testing negative in PB despite a significant level of disease in BM (6% by FISH). Parallel use of the RARA-PML assay in evaluable patients increased rates of MRD detection, in some instances providing earlier warning of impending relapse. ATO was given using the conventional schedule (0.15mg/kg/d - Regimen A) or a regimen involving loading followed by intermittent dosing (0.3mg/kg/d x 5d, then 0.25mg/kg 2x/wk - Regimen B). No difference in kinetics of molecular response was observed; however amongst patients treated in HR, hyperleucocytosis was less common with Regimen B (peak WBC 5 vs 126 x 109/l, p=0.007), indicating that this schedule may favor apoptosis over differentiation and merits evaluation in newly diagnosed APL. OS from time of relapse was 60% at 3yrs; of 10 patients treated pre-emptively, 7 are in 2nd molecular CR at median of 23mo (7–40mo), 2 died in relapse, treatment is on-going in 1. This study highlights sample type, assay sensitivity (which demonstrates inter-patient and -sample variability) and kinetics of relapse as critical factors that need to be taken into account for the successful delivery of MRD-directed therapy, which is becoming increasingly pertinent to the management of other subsets of acute leukemia.

Arsenic-induced APL differentiation in cerebrospinal fluid

Although new approaches have dramatically improved, the treatment of acute promyelocytic leukemia (APL) involvement of the central nervous system (CNS) confers a bad prognosis in the disease. Here, we report a patient who was diagnosed with relapsed APL preferentially involving the CNS. Treatment with arsenic trioxide led to impressive morphological changes in CNS cellularity consistent with the induction of a differentiation syndrome. Since arsenic trioxide could be identified in the CNS, we provide evidence that the drug can cross the blood-brain barrier and can be used for treatment of extramedullary APL.

Combination therapy with arsenic trioxide (ATO), all-trans retinoic acid (ATRA), and gemtuzumab ozogamycin (GO) in relapsed acute promyelocytic leukemia (APL)

6558 Background: Relapse occurs in 20 to 30% of patients with APL treated with ATRA. ATO is an effective agent for salvage of APL relapse, and GO has shown activity in APL due to the high expression of CD33. Methods: We investigated the efficacy of a combination regimen of ATO, ATRA and mylotarg in 8 patients with APL in 1st relapse. All patients had been previously treated with regimens that included ATRA and chemotherapy. Patients received ATO 0.15 mg/kg intravenously until marrow complete remission (CR). Once in marrow CR patient was observed until CR. Once CR was achieved or 60 days from the start of induction therapy, consolidation therapy was initiated with 1) ATO 0.15 mg/kg intravenously for 5 days per week for 4 weeks, repeated every 8 weeks for total of 5 courses, 2) ATRA 45mg/m2 orally twice daily 10 days per month for 10 months, and 3) GO 9 mg/m2 intravenously monthly for 10 months. Patients then received maintenance with 1) idarubicin 8mg/m2daily on day 1 and 2 every month for 3 months, 2) ATRA 45mg/m2orally daily 10 days per month for 14 months, 3) 6-MP 50 mg orally 3 times per day for 11 months and methotrexate 15mg/m2 orally once a week for 11 months. Results: The median age is 51years (range, 18–68 years), median WBC count 1.9 ×109/L (range 0.9–7.7 ×109/L), platelets 71 ×109/L (range, 6–164 ×109/L). The median duration of 1st CR was 7 months (range, 3 to 17 months). Seven patients achieved CR with median time to remission of 8 weeks (range 3–15 weeks). One patient achieved marrow CR after 3 weeks of therapy but is too early to evaluate for CR. All 7 are still in CR after a median follow up of 36+ months (range, 1+ to 51+ months). All seven patients achieved molecular remission. All patients had myelosuppression but there was no grade 3–4 extramedullary toxicity. Two patients died, one died secondary to complication related to metastatic lung adenocarcinoma, and the other patient died of sepsis. Conclusions: We concluded that the combination of ATO, ATRA and mylotarg is effective and may achieve durable remissions in many patients with APL in first relapse. It should be considered as an effective alternative to allogeneic or autologous transplant. No significant financial relationships to disclose.

Arsenic trioxide as primary treatment in acute promyelocytic leukemia

16533 Acute Promyelocytic Leukemia (APL) is a separate, distinct and biologically unique subset of Acute Myelogenous Leukemia (AML). The affordability of ATRA is detrimental for its use especially in the developing world. The use of Arsenic trioxide (As2O3) has radically changed the treatment, initially for relapsed APL and now is being used upfront as first line therapy in APL some studies. We accrued 11 cases (6 males, 5 females) of APL for the past 1 year. 2 patients of these had been already treated with ATRA + chemotherapy (Daunorubicin and Cytosine Arabanoside). These relapsed patients were switched over to Arsenic trioxide. One of these relapsed patients has completed the treatment and is in CR for the past 6 months after completing treatment. The other patient has recently completed the treatment and is in remission. 9 of our patients are newly diagnosed and could not afford ATRA and chemotherapy. All these patients were explained the treatment protocol and after obtaining a formal consent, patients were put on Arsenic trioxide. The protocol consists of Induction therapy with Arsenic trioxide given in a dose of 0.15 mg/kg/day till CR is achieved or to a maximum of 60 doses, followed by Consolidation treatment with Arsenic trioxide in the similar dose schedule but only for 28 days, and finally Maintenance therapy with Arsenic trioxide every 10 days a month for a total of 6 months with an interval of 4–6 weeks in between the cycles. Of the remaining patients (n = 7), 2 patients did not respond to Arsenic trioxide after 60 doses. The remaining patients (n = 5) are in different phases of treatment (all having completed Induction therapy) and are presently in CR (Mean duration to avhieve CR-57 days, documented by bone marrow examination). There were no major morbidities and none of our patients exhibited APL syndrome. The lower cost of treatment with Arsenic trioxide (Approx. Rs. 50–60,000) compared to ATRA + chemotherapy (Approx. Rs. 90,000–1,10,000), the magnitude of response and the least encountered adverse effects seem awesome in this treatment especially in the developing countries. No significant financial relationships to disclose.

Recurrent Extramedullary Relapse of Acute Promyelocytic Leukemia after Allogeneic Stem Cell Transplantation: Successful Treatment by Arsenic Trioxide in Combination with Local Radiotherapy

Isolated extramedullary relapse is rare in patients with acute promyelocytic leukemia (APL) after allogeneic stem cell transplantation (SCT), and an optimal therapy for it has not been established. We describe a patient with APL who developed serially occurring extramedullary disease (EMD) after SCT. We confirmed that EMD had arisen from the recipient's APL blasts by detecting t(15;17) and PML/RARalpha from the tumor cell suspension. The patient displayed EMD 4 times at different sites. Administration of all-trans retinoic acid with local radiotherapy and with chemotherapy for the first to third EMDs resulted in regression of the tumors. However, these regimens did not prevent the subsequent occurrence of new EMD. For the fourth EMD, intravenous administration of arsenic trioxide followed by local radiotherapy resulted in the disappearance of EMD, and no further EMD has developed to date. In the present case, the bone marrow was in morphologic and molecular remission during the course of recurrent EMD. The accumulation of detailed cases is needed to elucidate the pathogenesis, predisposing factors, and optimal therapy for EMD in APL after SCT.

Reduced intensity conditioning allogeneic bone marrow transplantation following central nervous system (CNS) relapse of acute promyelocytic leukemia: Evidence for a graft‐versus‐leukemia effect in the CNS

Reduced intensity conditioning allogeneic bone marrow transplantation following central nervous system (CNS) relapse of acute promyelocytic leukemia: Evidence for a graft‐versus‐leukemia effect in the CNS

Arsenite pretreatment enhances the cytotoxicity of mitomycin C in human cancer cell lines via increased NAD(P)H quinone oxidoreductase 1 expression

Arsenic is an effective therapeutic agent for the treatment of patients with refractory or relapsed acute promyelocytic leukemia. The use of arsenic for treating solid tumors, particularly in combination with other chemotherapeutic agents, has been extensively studied. Here, we report that arsenite-resistant human lung cancer CL3R15 cells constitutively overexpress NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme responsible for activation of mitomycin C (MMC), and are more susceptible to MMC cytotoxicity than parental CL3 cells. The effects of arsenite pretreatment on NQO1 induction were examined in CL3, H1299, H460, and MC-T2 cells. Arsenite pretreatment significantly enhanced the expression of NQO1 and susceptibility to MMC in CL3, H1299, and MC-T2 cells, but not in H460 cells that express high endogenous levels of NQO1. Alternatively, arsenic pretreatment reduced adriamycin sensitivity of CL3 cells. Arsenite-mediated MMC susceptibility was abrogated by dicumarol (DIC), an NQO1 inhibitor, indicating that NQO1 is one of the key regulators of arsenite-mediated MMC susceptibility. Various cancer cell lines showed different basal levels of NQO1 activity and a different capacity for NQO1 induction in response to arsenite treatment. However, overall, there was a positive correlation between induced NQO1 activity and MMC susceptibility in cells pretreated with various doses of arsenite. These results suggest that arsenite may increase NQO1 activity and thus enhance the antineoplastic activity of MMC. In addition, our results also showed that inhibition of NQO1 activity by DIC reversed the arsenite resistance of CL3R15 cells.