PML-RARA Transcript Research Articles

Auer rods and differentiation in acute promyelocytic leukemia

Dalal et al (2007) recently reported that the residual detection of Auer rods one month after treatment of an Acute Promyelocytic Leukemia (APL) patient was a sign of maturing granulocytes under differentiation therapy but not of refractory leukemia. Auer rods deriving from the crystallisation of myeloperoxidase (MPO) granules are the hallmark of Acute Myeloid Leukemia (AML), but can also be observed in the Myelodysplastic Syndromes in which they have been associated with a more aggressive disease (Seymour & Estey, 1995). Among AML, two characteristics are specific to APL (AML-M3 subtype in the French-American-British classification): 1) the presence in more than 90% of cases of the oncogenic PML-RARα fusion protein and 2) an exquisite sensitivity to the differentiation induced by All-trans retinoic acid (ATRA) (Cassinat & Chomienne, 2001). We report herein an unexpected correlation between the presence of Auer rods in APL blast cells at diagnosis and ATRA differentiation efficacy. We have been routinely assessing primary APL cells at diagnosis to evaluate their sensitivity to differentiation induction by ATRA. As previously reported (Cassinat et al, 2001), although the percentage (%) of differentiated cells (estimated by the percentage of nitroblue tetrazolium (NBT) positive cells) after 6 d of culture was very high in almost all cases, the levels were more heterogeneous after 3 d of culture (ranging between 0% and 100%) indicating different levels of differentiation sensitivity. 128 consecutive patient samples showed an inverse correlation between the percentage of cells harbouring Auer rods and the percentage of differentiated cells at day 3 (P < 0·015, Pearson test). When patients were studied as three groups according to the proportion of cells with Auer rods (Fig 1A), the median percentages of differentiated cells after 3 d of culture were significantly lower in patients with a higher level of Auer rods (P = 0·014, Kruskal-Wallis non parametric test). (A) Results of ATRA-induced differentiation (measured by NBT test after 3 d of culture) according to the percentage of Auer rod-positive cells at diagnosis. Box 1: 0 to 1% Auer rod-positive cells (n = 48 patients). Box 2: 2 to 15% Auer rod-positive cells (n = 55 patients). Box 3: at least 15% Auer rod-positive cells (n = 25 patients). (B) Distribution of the percentage of Auer rod-positive cells in patients that relapsed (n = 8) or not (n = 30), in the group of patients with a PML-RARα transcript of the Bcr1 subtype. In 62 of these patients, clinical outcome was obtained from the APL93 and APL 2000 trials registries (median follow-up, 9 and 4 years, respectively) (Fenaux et al, 1999; Ades et al, 2006). It appeared that patients with higher levels of Auer rod-positive cells tended to have a higher risk of relapse (Table I). Accordingly, the median percentage of Auer rod-positive cells was higher in the group of patients (n = 12) that relapsed compared to patients that did not relapse (n = 50) (8·5% vs. 4·5%). This difference only trended toward significance when the whole population was considered (P = 0·14; Kruskal-Wallis non parametric test), but was in accordance with our previous observation that in vitro differentiation could be correlated to patient prognosis (Cassinat et al, 2001). Interestingly, when a more homogeneous population, such as patients harbouring a Bcr1 transcript (the most frequent transcript) were analysed (n = 38) (Fig 1B), the percentage of Auer rod-positive cells in the group of patients that relapsed was significantly higher compared to the patients that did not relapse (13·5% vs. 5%, P = 0·045, Kruskal-Wallis non parametric test). These data suggest a link between the presence of Auer rods in APL cells at diagnosis and the sensitivity of these cells to the differentiation effect of ATRA and hence with the clinical outcome. Given that Auer rods are supposed to be a result of MPO crystallisation and their presence is inconstant in AML patients, one hypothesis could be that presence of Auer rods is dependent on a particular MPO genotype. Indeed, several polymorphisms have been reported in the MPO promoter or coding regions and correlation may exist with several types of cancer, such as lung cancer (Gresner et al, 2007) or leukemia (Zhang et al, 2007). We have recently shown that MPO expression during ATRA treatment differs between patients with good or poor differentiation response (Quere et al, 2007). Thus, the present study could be the basis for testing the hypothesis that the presence of Auer rods could reflect a specific genotype correlated to ATRA sensitivity.

The PRKAR1A gene is fused to RARA in a new variant acute promyelocytic leukemia

AbstractWe report the molecular and cytogenetic characterization of a novel variant of acute promyelocytic leukemia (APL). The bone marrow showed 88% hypergranular promyelocytes, and the karyotype was 47,XY,+22 [5]/46,XY[30]. Fluorescence in situ hybridization (FISH) indicated disruption and deletion of the 5′-end of the RARA gene. Treatment with all-trans retinoic acid, idarubicin, and arsenic trioxide induced cytogenetic complete remission without morphologic evidence of residual leukemia. The diagnostic marrow was negative for PML-RARA transcripts by reverse transcription–polymerase chain reaction (RT-PCR), but an atypical product was observed. Sequencing showed partial homology to the PRKAR1A gene, encoding the regulatory subunit type I-α of cyclic adenosine monophosphate–dependent protein kinase. RT-PCR using specific primers for PRKAR1A and RARA amplified 2 transcript splice variants of a PRKAR1A-RARA fusion gene, and PRKAR1A and RARA FISH probes confirmed the fusion. This novel PRKAR1A-RARA gene rearrangement is the fifth variant APL in which the RARA partner gene has been identified and the second known rearrangement of PRKAR1A in a malignant disease. This trial was registered at www.actr.org.au with the Australian Clinical Trials Registry as number 12605000070639.

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.

Detecting PML-RARα transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR

Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005. All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7,500 platform. The quantitation of PML-RARalpha transcripts was represented by the normalized quotient, that is, PML-RARalpha transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n = 23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n = 13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone. The sensitivity of RQ-PCR was 1 per 10(5) cells and 5 copies of the PML-RARalpha transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P = 0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P = 0.036). Compared with pretreatment, median reduction of the PML-RARalpha transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P = 0.024). Interestingly, we found that PML-RARalpha transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups. The RQ-PCR assay is reliable for the detection of PML-RARalpha transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.

A randomized study with or without intensified maintenance chemotherapy in patients with acute promyelocytic leukemia who have become negative for PML-RARα transcript after consolidation therapy: The Japan Adult Leukemia Study Group (JALSG) APL97 study

To examine the efficacy of intensified maintenance chemotherapy, we conducted a prospective multicenter trial in adult patients with newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Of the 302 registered, 283 patients were assessable and 267 (94%) achieved complete remission. Predicted 6-year overall survival in all assessable patients and disease-free survival in patients who achieved complete remission were 83.9% and 68.5%, respectively. A total of 175 patients negative for PML-RARalpha at the end of consolidation were randomly assigned to receive either intensified maintenance chemotherapy (n = 89) or observation (n = 86). Predicted 6-year disease-free survival was 79.8% for the observation group and 63.1% for the chemotherapy group, showing no statistically significant difference between the 2 groups (P = .20). Predicted 6-year survival of patients assigned to the observation was 98.8%, which was significantly higher than 86.2% in those allocated to the intensified maintenance (P = .014). These results indicate that the intensified maintenance chemotherapy did not improve disease-free survival, but rather conferred a significantly poorer chance of survival in acute promyelocytic leukemia patients who have become negative for the PML-RARalpha fusion transcript after 3 courses of intensive consolidation therapy.

Prognostic significance of initial clinical and molecular genetic features of acute promyelocytic leukemia (APL): Results from the North American Intergroup Trial, C9710

7016 Background: The impact of previously defined prognostic variables can change with advances in treatment. Thus, the significance of initial clinical and molecular genetic features of APL were explored in the context of the first randomized trial designed to evaluate the potential benefit of adding As2O3 consolidation into front-line all-trans retinoic acid (ATRA)-based therapy of APL (Powell et al, ASCO 2007). Methods: We evaluated pre-treatment white blood cell (WBC) and platelet count, age, PML-RARA transcript level and isoform type in the first 180 untreated APL patients (pts) who underwent molecular analysis on C9710 and explored their relationship to disease- free (DFS) and overall survival (S). PML-RARA transcripts were measured using real-time quantitative RT-PCR and expressed as a normalized quotient (NQ) of PML-RARA/GAPDH. Results: Using a multivariate proportional hazard model, pre-treatment PML-RARA level and WBC count were independently associated with DFS; p = 0.0073 and p = 0.05, respectively. Pre-treatment WBC count was the only feature significantly associated with S; p&lt;0.0001. With a median follow-up of 29 months, neither median DFS nor S have been reached and only 30 DFS events have been reported among these 180 pts. Pts with higher presenting WBC &gt; 10K/μl had both shorter DFS and S with hazard ratios (HR) of 2.3 and 5.5, respectively. The relationships between treatment arm and pre-treatment WBC and PML-RARA transcript level were explored by categorical analyses. For non-As2O3-treated pts, a striking difference was observed in DFS at 2.5 years between those above or below cut-off values: WBC &lt;10K, 78%; WBC &gt;10K, 50%; NQ &lt;median, 87%; NQ &gt;median, 60%. For As2O3-treated pts smaller and the reverse differences were observed: WBC &lt;10K, 94%; WBC &gt;10K, no DFS events reported; NQ &lt; median, 93%; NQ &gt;median, no DFS events reported. Conclusion: These preliminary results (based on analysis of 31% of total C9710 pts) indicate that pre-treatment PML-RARA transcript level and WBC are prognostic variables for newly diagnosed APL pts in first remission treated with standard ATRA-based chemotherapy but suggest that they may not apply in pts receiving two 25-day courses of As2O3 as first consolidation. No significant financial relationships to disclose.

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.

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.

A Randomized Study with or without Intensified Maintenance Chemotherapy in Patients with Acute Promyelocytic Leukemia Who Had Become Negative for PML-RARα Transcript after Consolidation Therapy: The Japan Adult Leukemia Study Group (JALSG) APL97 Study.

The use of all-trans retinoic acid (ATRA) has markedly improved therapeutic outcome in patients with acute promyelocytic leukemia (APL). Non-cross resistance between ATRA and chemotherapeutic drugs has contributed to not only a high complete remission (CR) rate but also a decrease in the relapse rate, leading to the significant improvement in disease-free survival (DFS) and overall survival (OS) rates. However, it is not clear whether maintenance chemotherapy actually prevents relapse in APL patients treated with ATRA and chemotherapy. If short-term therapy without maintenance shows identical DFS rates as compared to long-term therapy with maintenance, it would be beneficial for quality of life of patients as well as to medical costs. In these aspects, to determine an efficacy of maintenance/intensification chemotherapy, this study was designed to compare the DFS and OS rates in previously untreated adult patients with APL who showed absence of PML-RARα fusion transcript at the end of consolidation therapy and were randomly allocated to either maintenance therapy or observation. Of 302 registered, 283 patients were assessable and 267 (94%) achieved a CR. Predicted 6-year OS and DFS rates were 83.7% and 69.2%, respectively. The PML-RARα fusion gene was amplified using bone marrow samples at the diagnosis and after consolidation therapy by the reverse transcriptase-polymerase chain reaction analysis. The detection limit of PML-RARα fusion transcript in this assay was 10-4. Among 235 patients who completed 3 courses of consolidation chemotherapy, five (2.1%) were positive for the PML-RARα fusion transcript. Three of them subsequently relapsed and another one patient received allogeneic hematopoietic stem cell transplantation. On the other hand, 230 patients (97.9%) showed no PML-RARα transcript in the bone marrow cells at the end of consolidation. Of these, 175 patients were randomly assigned to receive moderately intensive and intermittent maintenance chemotherapy (n=89) or to observation (n=86). Predicted 6-year DFS was 63.2% for the chemotherapy group and 81.8% for the observation group, showing no statistically significant difference (p=0.102). Predicted 6-year OS in patients assigned to observation was 98.7% and was significantly higher than 85.9% in those allocated to maintenance therapy (p=0.013). These results indicate no benefit from adding maintenance chemotherapy in APL patients who are negative for PML-RARα fusion transcript after 3 courses of intensive consolidation.

Relapse-Risk Stratification in Acute Promyelocytic Leukemia Patients by PML-RARa Transcript Quantification.

The detection of PML-RARa by real-time PCR (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) patients; however, the clinical value of using a RQ-PCR approach remains to be determined. Our aim was to analyze the correlation between relapse risk and MRD levels assessed by RQ-PCR at different phases of treatment, and to compare these data with conventional qualitative RT-PCR. Follow-up samples from 145 APL patients treated with the PETHEMA protocols were analyzed by RQ-PCR protocol from Europe Against Cancer program and by RT-PCR method from European BIOMED-1 Concerted Action. After induction therapy, no association was found between the presence of a RQ-PCR positive result and relapse. After the third consolidation course, three cases remained positive by RQ-PCR assays and two of them (67%) relapsed. By contrast, only 16 out of 119 (13%) patients with negative RQ-PCR relapsed. During maintenance therapy and out-of treatment, a significant correlation between a RQ-PCR positive result and a poor relapse-free survival was observed (p < 0.0001). All patients with > 10 PML-RARa normalized number copies (NCN) at maintenance or out-of therapy relapsed (n=19), while all patients with < 1 NCN within out-of treatment group remained in hematological remission (n=75). 8 patients with < 1 NCN during maintenance therapy finally relapsed, but all of them appeared beyond the end of treatment. In addition, in the intermediate group of patients (NCN 1–10) (n=18) relapse probability of 40% at 5 years was observed. All hematological relapses were predicted if a follow-up sample had been obtained within the previous 4 months. Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients.

PML-RARα isoform at diagnosis is associated with disease-free survival (DFS) in patients enrolled in the intergroup trial (C-9710) for treatment of acute promyelocytic leukemia (APL)

6504 Background: Refinement of prognostic groups using molecular genetic features of the disease may facilitate the design of future risk-adapted trials for APL. Previous analyses of the prognostic value of PML-RARα transcript levels and isoform type have generated conflicting results. We sought to determine the clinical significance of measuring PML-RARα transcript level at the time of diagnosis using real-time quantitative RT-PCR (RQ-PCR) of the long (L) and short (S) isoforms of the fusion gene. Methods: PML-RARα transcript levels were measured in pretreatment bone marrow (BM) and/or paired blood (B) samples of 139 patients (pts) with newly diagnosed APL registered to C-9710, the intergroup phase III randomized study of concurrent tretinoin and chemotherapy with or without arsenic trioxide as initial consolidation therapy. Transcript levels were expressed as the normalized quotient (NQ) of PML-RARα/ABL or PML-RARα/GAPDH. Correlations between pre-treatment NQ values and other pre-treatment characteristics including age, presenting WBC and platelet count, and L or S isoform were explored. Results: Pre-treatment NQ values using ABL vs. GAPDH showed significant correlation (p &lt; 0.0001). Pre-treatment transcript levels in B and BM for 57 pts with S-form were significantly higher than 82 L-form patients (p = 0.02) when NQ was determined using GAPDH as the control gene (but not with ABL). With a median follow-up of 24.3 months for all patients, there have been 19 events. Median DFS for either isoform has not been reached, however, DFS was significantly shorter for patients with S-isoform compared to those with L-isoform (p = 0.017) with an estimated hazard ratio of 3.28. Using a multivariate proportional hazard model, no other significant relationships were found between DFS and NQ level at diagnosis, age, presenting WBC or platelet count. Conclusions: The S-isoform of the PML-RARa fusion gene was associated with shorter DFS in a subset of patients entered onto C-9710. If confirmed in a larger cohort of C-9710 pts, presence of the S-isoform should be considered as a high-risk feature in the design of future risk-adapted treatment strategies for APL. No significant financial relationships to disclose.

Evaluation of Molecular Response by Real-Time RT-PCR in Acute Promyelocytic Leukemia (APL): A Direct Comparison with Regular RT-PCR.

PURPOSE: In this study, we systemically compare the molecular response by regular reverse transcript polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR in newly diagnosed patients with acute promyelocyte leukemia (APL).PATIENTS AND METHODS: A total of 31 newly diagnosed patients with APL who entered remission by induction therapy with ATRA plus As2O3 were included in this study with median follow-up of 38 months (27–46 months). Serial bone marrow samples were obtained before and after the induction, consolidation chemotherapy and every 3 to 6 months during maintenance therapy. Regular RT-PCR and real-time RT-PCR were carried out to evaluate the molecular response. The PML-RARa fusion transcript level was calculated in two ways: normalized PML-RARa level as DoseN and log-reduction compared to the pre-treatment level.RESULTS: A total of 29 patients remained disease free for a median of 27 to 46 months. Comparing two PCR assays, the sensitivity limit for real-time RT-PCR was 5.7 log DoseN reduction while the limit for regular RT-PCR was about 3 log. Furthermore, we established criteria for evaluation of molecular response by real-time RT-PCR: ≥3.0 log reduction of PML-RARa transcript level as minor molecular response; ≥5.0 log reduction as major molecular response and undetectable level of PML-RARa transcript as complete molecular responses. Minor molecular response by real-time RT-PCR was achieved in 22.6% and 96.8% of patients after induction and consolidation chemotherapy, which was equivalent to molecular response by regular RT-PCR (35.5% and 96.8% respectively). The major molecular response rate was 12.9% and 90.3% after induction and consolidation whereas the complete response was at 3.23% and 77.4% respectively. All patients achieved major and complete molecular remission during and after maintenance therapy. Loss of either major or complete molecular response occurred in two patients while both patients experienced subsequent loss of minor molecular response, which coincided with CNS leukemia and bone marrow relapse.CONCLUSION: Real-time RT-PCR was more sensitive than regular RT-PCR and the evaluation criteria for real-time RT-PCR was established and proved to be clinically relevant.

Frequency and Dynamics of Bcr1/Bcr3 Isoforms of PML/RARA Fusion Gene - Results of Russian APL Multicenter Trial.

Detection and monitoring of minimal residual disease (MRD) in APL patients has become a routine practice and is essentially important for tailoring the therapeutical intervention. Two major isoforms of PML-RARa fusion gene (bcr1/2 and bcr3) are considered to determine diverse outcome with bcr3 to be less favorable. It was also shown by different study groups that among different ethnic populations the ratio of bcr1/2 and bcr3 varies. The prevalence of bcr1/2 isoform over bcr3 was reported in Latinos and Chinese population: 84% vs 16% and 73% vs 27%, respectively. Less evident but nevertheless convincing tendency was registered in USA (US Intergroup – 63% vs 37%; Memorial Sloan Kettering CRC – 66% vs 34%), Great Britain (MRC – 61% vs 39%), Italy (GIMEMA – 60% vs 40%), Spain (PETHEMA – 56% vs 44%) (Santillana S. et al Joint International Congress on APL, Rome, 2001, abstr P2.14; Dan Douer et al BJH, 2003, p.563–570). The analysis of PML-RARa transcript in 66 APL patients included in the Russian Multicenter trial revealed the inverse (statistically significant from mentioned above) ratio with 37 patients (56%) bearing bcr3 isoform and 29 (44%) – bcr1/2. There is no exact explanation for these findings but it may be due to low numbers of cases and perhaps to certain environmental and/or ethnic reasons. The treatment results in these 66 APL patients are very similar to usually reported with 7+3+ATRA protocols – 91% CR rate with 75% molecular CR after the first and 96% - after the second course. The OS and DFS do not differ according to the isoform and constitute 77% and 80% at 3 years, respectively. It is worth to stress that close monitoring of MRD (3–15 probes per patient) revealed different time adjusted dynamics in the molecular relapse incidence for bcr1/2 and bcr3 isoform. It was equal at each month during the whole period of follow-up (36 mo from treatment start) for bcr1/2 patients, and clustered in two periods (3–12 mo) and (20–36 mo) for bcr3 patients with 8 months of absence of positive signal. So we may suppose that each isoform of PML-RARa fusion gene determines its distinctive course of MRD while standard APL treatment. This data once again shows discrepancies in the biological features of APL with bcr1/2 and bcr3 isoforms.

ATRA Resolves the Differentiation Block in t(15;17) Myeloid Leukemia by Restoring PU.1 Expression.

The transcription factor PU.1 is crucial for hematopoiesis. pu.1 knockout mice lack mature neutrophils, macrophages, B- and T-cells. PU.1 regulates important taget genes, like the M-CSF-, the G-CSF-, and the GM-CSF receptor promoter. Heterozygous mutations of the PU.1 gene have been found in some patients with AML, but not in AML with the t(15;17) encoding the PML-RARA fusion protein. These facts let us to hypothesize that decreased expression of PU.1 might contribute to the block in myeloid differentiation in t(15;17) AML. We therefore screened 141 AML patients for PU.1 mRNA expression using real-time PCR technology. We detected that t(15;17) AML patients (n=18) had significantly less PU.1 mRNA than AML-M2 patients (n=35), and 16fold less PU.1 mRNA than mature neutrophils. Moreover, the amount of PML-RARA transcripts in APL patients was inversely correlated to PU.1 mRNA levels. Conditional expression of PML-RARA in U937 cells suppressed PU.1 mRNA and PU.1 protein. HT93A cells derived from a t(15;17) AML-M3 patient nearly lack PU.1 mRNA and protein expression, and binding activity to the PU.1 site in the M-CSF-Receptor promoter is suppressed. Treatment of HT93A cells with all-trans retinoic acid (ATRA) powerfully restores PU.1 mRNA and protein and induces neutrophilic differentiation. We identified a region of extending −122 bp upstream of the translation start site in the PU.1 promoter mediates ATRA-induced activation of the PU.1 promoter in HT93A cells. Finally, conditional expression of PU.1 in HT93A cells is sufficient to trigger neutrophilic differentiation. This is the first report demonstrating that PU.1 is a critical target of the PML-RARA protein and an early ATRA-responsive gene.

Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression.

Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia. In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy. In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse. PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI). The antibody binding capacity (ABC) for each MDR protein was also calculated. At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP. PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively). By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively). No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered. Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL. The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy.

Expression of CD2 in Acute Promyelocytic Leukemia Correlates With Short Form of PML-RARα Transcripts and Poorer Prognosis

We studied the immunophenotype of 100 cases of acute promyelocytic leukemia (APL) with cytogenetic evidence of t(15;17)(q22;q21), 72 hypergranular (M3) and 28 microgranular (M3v), and correlated the results with molecular and clinical features. Most neoplasms (75/100 [75%]) had a typical immunophenotype: CD13+CD33+CD34-HLA-DR-. CD64, CD2, CD34, and HLA-DR were expressed in 27% (24/88), 23% (22/94), 21% (21/100), and 9% (9/98), respectively. CD34 expression was restricted to M3v; HLA-DR and CD2 were expressed more often in M3v than in M3 (P < .001). PML-RARalpha fusion transcripts were detected by reverse transcriptase-polymerase chain reaction in all 70 patients assessed. The short form of PML-RARalpha transcripts was found more frequently in M3v (P < .002) and CD2+ APL (P < .0001) than in M3 and CD2- APL, respectively. The median follow-up was 128 weeks. CD2+ APL was associated significantly with leukocytosis (P = .004), shorter complete remission duration (P = .03), and a trend toward shorter overall survival (P = .07) than CD2- APL. Overall survival for M3v vs M3 (P = .68) and short vs long transcripts (P = .21) was not significantly different. Immunophenotyping is useful for predicting the biologic and clinical behavior of APL.

New score predicting for prognosis in PML-RARA+, AML1-ETO+, or CBFBMYH11+ acute myeloid leukemia based on quantification of fusion transcripts

AbstractTo evaluate the prognostic significance of quantitative PML-RARA, AML1-ETO, and CBFB-MYH11 fusion transcript expression, real-time polymerase chain reaction was used to analyze bone marrow samples of 349 such patients at diagnosis and 522 samples of 142 patients also during therapy (total analyses, n = 859; median number of follow-up samples, 4/patient; median duration of assessment, 12 months). Lower expression levels at diagnosis correlated with better overall and event-free survival in all 3 leukemia subtypes. By combining the median expression ratio after consolidation therapy and the 75th percentile of the expression ratio at diagnosis, a new score was established that separates a group with 100% EFS from a significantly worse group (P < .0001) in each of the 3 acute myeloid leukemia subgroups. Eight patients showed increasing levels of expression during follow-up and all had relapse. In conclusion, patients at high risk for treatment failure can be identified by high levels of fusion gene expression at diagnosis or less than 3 logs of tumor reduction during the first 3 to 4 months of therapy. By combining the transcription ratios at these 2 checkpoints, a new powerful prognostic score has been established.

Identification of two atypical PML–RARα transcripts in two patients with acute promyelocytic leukemia

We identified two patients with atypical PML–RARα rearrangements, 53 and 13 base pairs longer than the typical bcr1 transcript. Sequence analysis revealed a new PML breakpoint at the end of exon 7a in patient 1, and a PML exon 6 breakpoint in patient 2, with an insertion of 35 nucleotides of RARα intron 2. Patient 1 did not express RARα–PML and patient 2 showed the RARα–PML transcript, which corresponded to the typical bcr1. These results emphasize on the relevance of the correct identification of atypical PML–RARα rearrangements because of the potential implications in leukemogenesis, in the response to treatment, and for the correct monitoring of minimal residual disease.

Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RT-PCR method.

Qualitative RT-PCR methods used for monitoring minimal residual disease (MRD) in APL patients fail to predict relapse in up to 25% of patients in remission. We report here the development and evaluation of a highly sensitive (10(-5) and 10(-6) with one round and two rounds of PCR, respectively) competitive RT-PCR method to quantitate the PML-RARalpha fusion transcripts. PML-RARalpha transcript's levels were normalised to 10(5) copies of ABL transcript. Serial BM and PB samples from 16 patients with APL and t(15;17) were examined. Presentation samples from three patients (three BM, one PB) showed levels in the range of 0.7 x 10(6)-3.5 x 10(6) and 1.2 x 10(5) molecules in BM and PB samples respectively. Serial quantitation of MRD in both BM and PB samples showed significantly lower levels of PML-RARalpha transcripts in remission, although the majority of samples remain positive for the PML-RARalpha transcripts even those in long-term remission (up to 94 months). Levels of PML-RARalpha in remission samples were up to 2 x 10(2) and up to 5.2 x 10(1) molecules in BM and PB respectively. BM and PB samples taken from two patients 2-4 months before relapse showed significantly higher levels of PML-RARalpha transcripts (1.2 x 10(4) molecules in BM; 3.5 x 102, 1.2 x 10(2) and 1.2 x 10(3) in PB). The same samples, when tested with a standard qualitative RT-PCR for the amplification of PML-RARalpha (with a sensitivity of 10(-4)) produced negative results. This indicates that the qualitative methods would not have predicted relapse in these patients. Our data show that quantitating PML-RARalpha transcripts with a sensitive method may provide a superior approach for monitoring MRD in APL and identifying patients at high risk of relapse.

PML-RARA fusion transcripts in irradiated and normal hematopoietic cells.

It is believed that two important factors in the genesis of reciprocal chromosomal translocations in malignant cells are the physical proximity of the involved regions and local structural features of the chromatin fiber that make them more susceptible to breakage and rearrangement. In this work we sought to investigate whether PML-RARA fusion transcripts, characteristic of acute promyelocytic leukemia (APL), could be induced by a clastogenic agent in cells known to have, a priori, a favorable spatial distribution of these genes. A lymphoid-cell line, lacking the t(15;17) but having the PML and RARA genes in close proximity in specific phases of the cell cycle, was irradiated with 10 Gy of (60)Co, and the incidence of PML-RARA transcripts was analyzed by a highly sensitive PCR assay. Despite gene proximity, typical PML-RARA transcripts were only rarely detected in irradiated cells. The same phenomenon was observed at similar frequency in control non-irradiated cells. These findings made us investigate whether such transcripts could also be detected in peripheral blood cells from normal individuals. PML-RARA transcripts were observed at low frequencies in isolated lymphoid and granulocytic cell populations, with similar incidence in both cell types. The data thus indicate that the PML and RARA genes are not particularly susceptible to the clastogenic effects of gamma-irradiation, and that, similar to what has been reported for other chromosomal translocations, transcriptionally active PML-RARA rearrangements can be generated in normal hematopoietic cells of different lineages without apparent oncogenic consequences.