ABL Transcripts Research Articles

Hematopoietic progenitor cell collection in patients with chronic myelogenous leukemia in complete cytogenetic remission after imatinib mesylate therapy

The introduction of BCR–ABL tyrosine kinase inhibitors such as imatinib has changed the treatment of chronic myelogenous leukemia (CML). More than 75% of patients achieve complete cytogenetic remission (CCR) after treatment with imatinib, which provides an opportunity to collect minimally involved hematopoietic progenitor stem cell (HPC) products. In order to assess the feasibility of HPC collection in patients with CML, we prospectively enrolled 24 patients who achieved CCR on therapy with imatinib. Two patients could not undergo HPC collection because of coagulopathy. A CD34+ cell yield of ≥2.0 × 106/kg body weight was obtained in 16/22 (73%) patients. Patients who stopped imatinib for at least 3 weeks prior to HPC collection had significantly higher CD34+ cell yields (median: 6.52 × 106/kg body weight) when compared with patients who continued imatinib through the collection (median: 3.74 × 106/kg body weight). Mobilization with granulocyte colony-stimulating factor (G-CSF) did not increase the levels of BCR–ABL transcript. With a mean follow-up of 46 months, all patients but one were in CCR. In conclusion, a significant number of CD34+ cells can be safely collected in patients with CML who are on imatinib therapy, but CD34+ cell yields improve when imatinib is temporarily withheld.

A unique BCR–ABL1 transcript with the insertion of intronic sequence from BCR and ABL1 genes in a patient with Philadelphia-positive chronic myeloid leukemia: a case study

The BCR–ABL1 fusion gene results from a reciprocal translocation rearrangement, t(9;22)(q34;q11.2), and is a hallmark of chronic myeloid leukemia (CML). The breakpoint on chromosome 9 is mostly 5′ to ABL1 exon 2, whereas on chromosome 22, the breakpoint can occur in various regions involving the major breakpoint cluster region (M-bcr) in CML and the minor breakpoint cluster region (m-bcr) in acute lymphoblastic leukemia. Described here is a rare case of Philadelphia-positive CML with intronic splice sites. This atypical BCR–ABL1 transcript was detected along with a classic e13a2 transcript, using reverse transcription polymerase chain reaction (RT-PCR). Nucleotide sequence analysis revealed a joining of BCR intron 13 with ABL1 intron 1a. Both transcripts were detected when the patient was on hydroxyurea treatment; with imatinib mesylate therapy, the atypical transcript disappeared. To our knowledge, this is the first report of BCR–ABL1 transcript with breakpoint occurring within both BCR and ABL1 introns and fusion of intronic sequences from both BCR and ABL1 genes.

Mutations in ABL kinase domain are associated with inferior progression-free survival

Imatinib mesylate is currently the drug of choice for all phases of chronic myeloid leukemia (CML). Despite the initial high rates of hematological and cytogenetic responses, many patients develop resistance. We prospectively studied 40 patients with CML with primary cytogenetic resistance to imatinib mesylate. A semi-nested reverse transcriptase-PCR approach was used for amplification of the ABL kinase domain, which was then subjected to direct sequencing for the detection of point mutations. Expression of BCR–ABL transcripts was quantified using the real-time Taqman assay, and BCR–ABL gene amplification was detected using fluorescence in situ hybridization. Twelve different point mutations were detected in 18/40 (45%) patients. Five patients had mutations in the P-loop region and 13 had mutations in other regions of the BCR–ABL kinase domain. Progression-free survival at 2 years was inferior for patients with mutations compared to those without mutations (72% vs. 95%, p < 0.0045). The BCR–ABL fusion gene was over-expressed in five patients (5/18); the mean BCR–ABL/ABL ratio was 75.38 vs. 28.72 for imatinib responders, p < 0.001. Amplification of the BCR–ABL fusion gene was detected in 4/40 (10%) patients. Point mutation was the major mechanism of primary cytogenetic resistance to imatinib mesylate in the present study. Patients with mutations had inferior progression-free survival compared to those without mutations. Regular monitoring for the presence of point mutations in the ABL kinase region may identify such patients early, with an opportunity to intervene.

BCR-ABL1INS35 Is Not Uncommon in CML Patients and Is Related to Resistance and Sensitivity to Inhibitors in CML Treatment - Response

Santamaria et al. report the detection of the BCR-ABL1INS35 in 27% of 18 patients with CML being treated with imatinib. This is consistent with our finding of 2 of 21 (10%) at 3 months, 1 of 20 (5%) at 6 months, 4 of 25 (16%) at 9 months, and 6 of 23 (26%) at 12 months of imatinib therapy (1). However, it is important to point out that the relative level of expression of the BCR-ABL1 INS35 as compared with the wild-type fusion BCR-ABL1 transcript is very low in these patients (<20%). As we discussed in our article and based on additional data that we generated, the level of resistance is “dose-dependent”.[1][1] In that, the level of resistance to imatinib is dependent on the level of the BCR-ABL1INS35 expression as compared with the wild-type BCR-ABL1 and not with any other control, such as GUS as reported by Santamaria et al. Our data show that the longer the patients are on therapy, the higher the percentage of patients expressing low levels of BCR-ABL1INS35. For these patients, becoming overtly resistant to therapy requires either a substantial increase in the level of expression of the BCR-ABL1INS35 or the development of additional mutations in BCR-ABL1 or somewhere else in the genome. As for the detection of alternative splicing in normal ABL1 transcript, we have detected the presence of low levels of this alternatively spliced ABL1 transcript in normal tissue using a primer set that amplifies only the ABL1.[1][1] This has also been reported by others (2). We think that this low expression of the ABL1INS35 in normal tissue may provide some protection to normal tissue against the effects of imatinib. Therefore, in the quantification of the BCR-ABL1INS35, it is important to be careful and not amplify the native ABL1 from normal cells. As we have described in our article, we used a nested assay in which the fusion BCR-ABL1 transcript was first amplified, then we took the fusion transcript in the next step of the nested PCR and quantified the BCR-ABL1INS35, so we prevented the amplification of the normally expressed ABL1INS35. In our hands, using our procedure, we could not detect ABL1INS35 in normal tissue. If this procedure is not stringent enough, the ABL1INS35 transcript from the normal cells may contaminate final product of the BCR-ABL1INS35 and this may confuse results. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. [1]: #fn-1

Quantitative Real-Time PCR Detection of Wilms' Tumor Gene (WT1) Transcript in Autologous Peripheral Blood Stem Cell (PBSC) Products Predict the Risk of Acute Myeloid Leukaemia (AML) Relapse After Autologous Transplantation (ASCT).

Abstract 4690 Introductionwhen allogeneic transplantation is not feasible, ASCT is an alternative option as post-remission therapy for patients (pts) with AML, as it can prolong their disease-free survival (DFS). Relapse is the main cause of AML treatment failure after initial complete response; AML relapse after ASCT is partly due to contamination with leukemic blasts of the PBSC products, although neither morphological nor genetic evidence of disease are detected in the bone marrow before leukapheresis. Thus, identification and quantification of a reliable minimal residual disease (MRD) marker in the collected PBSC could be relevant in determining the relapse risk after ASCT. The WT1 gene is overexpressed in leukemic blasts of most AML cases; several studies have shown that quantification with RT-PCR of WT1 in the bone marrow and peripheral blood of AML pts in complete remission has prognostic value. We have determined the WT1 transcript levels in autologous PBSC of AML pts autografted for complete remission (CR) consolidation; preliminary results and data interpretation are here presented. Aimto evaluate quantitative WT1 transcript levels in autologous PBSC collections and to compare results with outcome in AML patients who received an ASCT as consolidation of CR, at our Institute. Patients and Methods9 pts, period 06/2006-03/2009, median age 68 years (range 41-76). At diagnosis cytogenetic prognostic risk was intermediate for all pts. All pts were in morphological and genetic CR at the time of PBSC collection and before ASCT. Eight patients were in first and 1 in second CR. PBSC collection by leukapheresis (COBE Spectra cell separator): median count of CD34+ 9.75 ×106/kg (3.79-32). Conditioning regimen: Treosulfan 30 mg/sqm, Fludarabine 150 mg/sqm and Cytarabine 5 g/sqm. Median count of CD34+ cells infused: 5×106/kg (range 3.3-8.5×106/kg). RT-PCR quantification of WT1 transcript was performed using TaqMan technology starting from 1 μg of RNA extracted from mononucleated cells of fresh (4) or cryopreserved (5) PBSC samples. The housekeeping gene ABL was used as the control gene for these quantifications with WT1 level being normalised to 104 copies of ABL per sample. We used the Mann-Whitney-U-test to determine if median WT1 levels in the PBSC products of relapsed and not-relapsed pts was statistically different. Than we used the log-rank test to compare RFS and median WT1 value in the PBSC products. Resultsat last follow-up 4 pts relapsed and 5 were still in CR. The WT1 levels in the autologous PBSC of the 4 relapsed pts were 89.96, 193.87, 779.43 and 839.63, of the 5 CR pts were 8.40, 16.96, 36.45, 74.89 and 82.49. Overall median WT1 in the PBSC products was 82,49 copies. The median WT1 levels in the PBSC products of relapsed and not-relapsed pts were 486.65 (89.96–839.63) and 36.35 (8.40–82.49) copies, respectively; this difference was statistically significant (p<0.05). Overall, median relapse free survival (RFS) from ASCT was 534 (93-1096) days. Median RFS was 360 days for pts with a WT1 level > 82,49 copies (n=4), and has not been reached for pts with a WT1 level ' 82,49 (n=5) (p=ns). Conclusionsthese results suggest that RT-PCR quantification of the WT1 transcript in autologous PBSC could predict AML relapse in pts who receive ASCT in CR. Higher WT1 levels should reflect higher PBSC product contamination with leukemic blasts, indicating an increased risk of relapse after ASCT. These last pts could probably benefit of other strategies than ASCT. We conclude that, if our preliminary data will be confirmed in a larger number of pts, RT-PCR quantification of WT1 transcripts should be used for MRD detection and quantification in autologous PBSC, before proceeding to ASCT; according to our preliminary data the cut-off level could be about 80 WT1 copies to discriminate which pts should receive the ASCT and which should not. Disclosures:No relevant conflicts of interest to declare.

WT1 Expression Levels at Diagnosis and as a Marker of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukaemia (AML).

Abstract 2639▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-615 Background.-As wt1 expression is elevated in the majority (80%) of acute myeloid leukemias at diagnosis and relapse, a validated wt1 assay would be a powerful adjunctive tool for evaluation and monitoring of patients during their treatment. Monitoring of disease status with wt1 is applicable to more AML patients in a single assay than any other leukemic marker and includes patients that are cytogenetically normal and may not otherwise be amenable to molecular MRD evaluation. We aimed to analyse wt1 expression in the peripheral blood and bone marrow of patients with de novo AML, when measured at diagnosis, post-induction and post-consolidation; and to correlate these wt1 expression levels with patient characteristics and outcome. Methods-This is a prospective, longitudinal study in which the levels of wt1 expression from 99 patients with de novo AML are measured from bone marrow (59 patients) and peripheral blood (94 patients) specimens at the time of diagnosis and at various time-points along their treatment and clinical courses. The specimens were prospectively stored in the PwC Australasian Leukaemia and Lymphoma Group (ALLG) Tissue Bank. Samples analysed included: 59 bone marrow (bm-wt1) and 94 peripheral blood (pb-wt1) at diagnosis; 54 bm-wt1 and 57 pb-wt1 post-induction; and 46 bm-wt1 and 41 pb-wt1 post-consolidation. 90 of the 99 patients were treated with standard induction chemotherapy on a variety of protocols. RNA was extracted from 107 leukocytes purified from blood and bone marrow using TRIzol® (Invitrogen) extraction. Reverse transcription was performed using Superscript ® (Invitrogen) and RQ-PCR was performed using multiplexed 5' nuclease assay with Black Hole Quencher labelled probes according to published methods. Copy number of wt1 was normalised to 104 copies of ABL. Baseline patient characteristics were correlated with baseline wt1 expression levels and the impact of baseline and post-treatment wt1 expression on leukemia-free survival (LFS) was assessed. Results-99 AML patients were studied: median age 55years, 53% male. Cytogenetic prognostic groups (SWOG) were favourable – 12%, intermediate – 59% and poor – 29%. 90 patients were treated with curative intent and 83% achieved CR, 6% had refractory disease and 11% died during induction. With a median follow-up of 33 months, the median LFS was 12 months and median OS was 24 months. Median pb-wt1 levels at diagnosis were 5122 (range, 1 to 36125) and varied significantly according to: cytogenetic risk group (good risk 12745, intermediate risk 2632, poor risk 4554 (P = 0.009)) and marrow blast percentage (P = 0.02). Diagnostic bm-wt1 showed similar correlations with baseline parameters, but did not reach statistical significance. Median pb-wt1 at relapse was 3781. One of 21 relapsed patients with elevated pb-wt1 expression at diagnosis did not express wt1 at relapse. In a multivariate Cox regression analysis model including age and cytogenetic risk-group, increased expression of wt1 from peripheral blood at diagnosis is predictive of decreased LFS (P=0.04). This correlation was not seen with wt1 measured from bone marrow aspirates. The wt1 expression levels were reduced following induction chemotherapy with a median 2.6 log reduction (range, 0 to 4.2) seen in marrow aspirate and 3.2 log reduction (range, 0 to 4.3) seen in peripheral blood. There was a trend for lower post-induction peripheral blood wt1 to correlate with better LFS when measured as a continuous variable (P=0.067) or undetected vs detected (P=0.051). Lower post-consolidation pb-wt1 significantly correlated with better LFS when measured as a continuous variable (P=0.002) or undetected vs detected (P=0.02) and continued to be significant in multivariate analysis. Bone marrow aspirate wt1 levels post-induction or post-consolidation did not correlate with LFS. Conclusion-This study demonstrates a correlation between wt1 expression levels and known risk factors for early relapse. In the MRD setting, detectable wt1 levels in peripheral blood post-induction and post-consolidation predicted very poor leukaemia free survival. Analysis is ongoing to validate these findings in a uniformly treated cohort of patients enrolled on the current ALLG clinical trial. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinical and Molecular Response in Core Binding Factor Acute Myelogenous Leukemia with Fludarabine, Cytarabine, G-CSF and Gemtuzumab Ozogamicin.

Abstract 2056Poster Board II-33 Background:Core binding factor associated acute myelogenous leukemias (CBF AML) are characterized by sensitivity to high dose cytarabine (ara-C). Attempts to modulate intra-cellular ara-C tri phosphate (ara-CTP) by induction/consolidation therapy with fludarabine, cytarabine and granulocyte colony stimulating factor (GCSF) (FLAG) has resulted in improved event free survival (EFS) (median EFS not reached after median follow-up of 118 weeks) in patients with CBF AML (Borthakur et al. 2008, Cancer 113:3181). Addition of gemtuzumab ozogamicin (GO) to induction and consolidation has resulted in improved disease free survival (DFS) in patients with AML and “favorable cytogenetics” (Burnett,A. et al. ASH 2006#13). Method:In an attempt to improve upon these results we have initiated a study incorporating GO with FLAG (GO-FLAG) in induction/consolidation for treating patients with newly diagnosed CBF AML. Patients with CBF AML, untreated or who received one cytarabine based induction regimen are eligible. Induction regimen comprises of fludarabine 30 mg/m2 IV days 1- 5, cytarabine 2 g/m2 IV days 1- 5 (3.5 h after completion of that day's fludarabine infusion, Gemtuzumab ozogamicin 3 mg/m2 IV on Day 1, filgrastim (G-CSF) 5 mcg/kg body weight starting day-1 till recovery of absolute neutrophil count (ANC) to 1.0 × 109/L or above (filgrastim is started on day 2 for patients with presenting WBC count > 10 × 109/L). We report on clinical and molecular responses in patients enrolled in this study. Extracted RNA is reverse transcribed and analyzed by real-time quantitative PCR for the fusion transcript associated with CBF leukemias. Values are expressed as a percentage of fusion transcript to normalizing ABL transcripts. The sensitivity of detection is approximately 1 in 100,000 for AML1-ETO and 1 in 10,000 for CBFb-MYH11. Results:Thirty-four (Inv 16=10/t 8;21=24) patients (male=20)[median age 48 years (range, 29-73)]have been treated to date with a median follow-up of 11 months (range,1-24). Patient characteristics at presentation include median WBC 12.3 (range, 1.3-97.2) x 109/L, hemoglobin 9.1 (range, 7.8-11.9) gm/dL, platelet count 22 (range, 6-365) x 109/L. Additional cytogenetic abnormalities were present in 18 patients; +8 and –Y being most frequent. Three patients had kit mutations. Five patients were in complete remission at start (from other ara-C based induction) and except two deaths in induction all others achieved CR or CR with incomplete platelet recovery (CRp). Two patients died in CR from infectious complications. One patient has relapsed (initially resistant to “3=7” regimen). Median pre-treatment value for fusion transcript to Abl ratio by Q-PCR is 100 (range, 100-857). More than 3 log reduction in this median value has sustained post induction/consolidation in follow up evaluations extending up to 9 months at the time of this report. Grade≥3/4 induction toxicities included liver dysfunction (N=3, 9%), atrial fibrillation, pancreatitis, respiratory failure 1 patient each and 2 deaths in induction (one from pulmonary hemorrhage and other from intra-cranial bleed). Conclusion:Induction/consolidation therapy with GO-FLAG regimen in CBF AML results in excellent clinical and molecular response. Accrual and multi-variate comparison with historical regimens used to treat patients with CBF AML at MDACC are ongoing. Disclosures:Off Label Use: Use of Gemtuzumab Ozogamicin in frontline therapy of AML.

Delayed achievement of cytogenetic and molecular response is associated with increased risk of progression among patients with chronic myeloid leukemia in early chronic phase receiving high-dose or standard-dose imatinib therapy

AbstractPatients not in complete cytogenetic response (CCyR) continuously face the competing possibilities of eventually achieving a cytogenetic response versus progressing. We analyzed the probability of achieving a CCyR, major molecular response, and progression in 258 patients with chronic myeloid leukemia in early chronic phase at 3, 6, and 12 months from imatinib start. The initial imatinib dose was 800 mg/day in 208 (81%) and 400 mg/day in 50 (19%) patients. For patients not in CCyR, the probability of achieving CCyR (P = .002) or major molecular response (P = .004) significantly decreased, whereas the risk of progression increased (P = .16) at each time point. Patients with a BCR-ABL1/ABL1 ratio greater than 1% to 10% after 3 months of imatinib had a 92% probability of achieving CCyR with continued therapy, similar to the 98% for those with 1% or less, but their risk of progression (11%) was almost 3-fold that of patients with a BCR-ABL1/ABL1 transcript ratio of 1% or less (4%) and similar to that of patients with transcript levels more than 10% (13%). These results suggest that patients not in CCyR after 12 months on imatinib have a higher risk of progression. This risk is discernible as early as 3 months into imatinib therapy by molecular analysis and may provide the rationale to institute therapies that render higher rates of early response.

Does a rise in the BCR‐ABL1 transcript level identify chronic phase CML patients responding to imatinib who have a high risk of cytogenetic relapse?

BCR-ABL1 transcript numbers were monitored in 161 patients who started treatment with imatinib early after diagnosis of chronic myeloid leukaemia in chronic phase and achieved complete cytogenetic responses (CCyR). A confirmed doubling in BCR-ABL1/ABL1 transcript levels was found to be a significant factor for predicting loss of CCyR [relative risk (RR) 8.3, P < 0.0001] and progression to advanced phase (RR 0.07, P = 0.03) provided that the eventual BCR-ABL1/ABL1 transcript level exceeded 0.05%; increases that never exceeded 0.05% had no predictive value. The finding of a kinase domain mutation in a patient in CCyR, though rare, also predicted for loss of CCyR.

Mathematical Simulation of BCR-ABL Real Time Quantitative Polymerase Chain Reaction (RQ-PCR) for Chronic Myeloid Leukemia (CML) Response Monitoring Provides Insight on the Basis of International Standardization.

BACKGROUND: Cytogenetic assessment to measure CML response to Glivec or Tasigna is expressed as a ratio of Ph+ CML to total cells. Molecular response determination for CML by RQ-PCR achieves a similar goal by obtaining a ratio of transcript levels of BCR-ABL expressed exclusively in Ph+ CML cells to the BCR or ABL control gene which is expressed in both Ph+ and normal cells. However when a RQ-PCR assay result is obtained, a different RQ-PCR ratio will be obtained for the same sample using BCR vs. ABL as control gene due to the intrinsic expression level differences in the two control genes. Experiments have demonstrated empirically that international standardization of RQ-PCR data obtained by assays using these different control genes is possible, thus allowing direct comparison of data. We have developed a mathematical simulation that explains the underlying basis for international standardization and this was experimentally confirmed to show how standardization is feasible in practice as a part of the Tyrosine Kinase Optimization and Selectivity (TOPS) trial.METHODS: We have mathematically simulated a correlation between theoretical cytogenetic response (Ph+ to total cell ratio) with the theoretical molecular response (BCR-ABL to control gene transcript ratio) by using the following assumptions; 1) BCR and BCR-ABL expression occur from similar promoters and likely have similar expression levels. 2) Ph+ cells have one normal BCR resulting in 1x level of BCR transcription and normal cells have two normal copies of BCR resulting in 2x level of BCR transcription (x=BCR transcription rate from one promoter). This simulation was extended to the ABL control gene as data of the relative expression level of ABL vs. BCR was available from samples in the TOPS trial, and the ABL transcript level is similar in both Ph+ and normal cells as the ABL PCR primers amplify both ABL and BCR-ABL transcripts. To experimentally test the simulation, RQ-PCR samples from patients in TOPS were exchanged between the study's regional RQ-PCR laboratories. Each sample was assayed at 2 labs, one using BCR and the other ABL as the control gene.RESULTS: Mathematical simulation suggests that RQ-PCR ratios with BCR but not ABL as control gene has a nonlinear relationship with the Ph+ to the total cell ratio across the entire range from 0 to 100%. However at clinically relevant RQ-PCR ratio of ≤10%, irrespective of whether BCR or ABL is used as a control gene, RQ-PCR ratios are linear but with different slopes due to higher ratios obtained with ABL as control gene. Despite the different slopes, RQ-PCR ratios obtained with either control gene can be easily inter-converted via a multiplicative factor to neutralize the bias between methods as is currently recommended in international standardization. Experimental testing in the TOPS trial showed that; 1) ABL transcript copy number is significantly lower than BCR transcript copy number leading to higher RQ-PCR ratios with ABL as control gene. 2) Mathematical correction of bias between BCR and ABL as control gene allows for direct comparison of RQ-PCR ratios with an 88.2% concordance when ratios are below 10%.CONCLUSIONS: Mathematical simulation shows that for BCR and ABL as the control gene, the basis for international standardization is a consistent linear relationship that can be easily inter-converted when PCR ratios are ≤ 10%. This can be experimentally demonstrated by the high level of concordance obtained when the same sample is analyzed using different control genes. For RQ-PCR ratios > 10%, standardization is not possible using the currently applied neutralization of bias method of conversion, but may be possible by using a formula that accounts for the non-linear relationship of BCR as a control gene. Simulation studies also highlight the importance of not only obtaining precise but also accurate RQ-PCR ratios thus enabling consistent standardization.

Efficient Duplex Real-Time Quantitative RT-PCR of BCR-ABL and ABL Gene Transcripts for Monitoring of Minimal Residual Disease

BCL-ABL fusion gene can be found in 100% chronic myelogenous leukemia (CML) and about 25% adult acute lympholid leukemia where its expression level is a crucial parameter for monitoring of minimal residual disease (MRD). Depending on the breakpoint in BCR, exon 2 of ABL (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of BCR resulting in chimeric proteins of p190, p210 and p230, respectively. The c-ABL gene is one of the best controls for MRD detection by real-time quantitative RT-PCR (RQ-RT-PCR). In most studies published before, PCR probes were labeled by single fluorescence such as FAM, and BCR-ABL and ABL transcripts were detected in separate PCR reactions.Purpose: To design and evaluate a duplex, real-time quantitative RT-PCR (RQ-RT-PCR) system labeled with double fluorescence Taqman probes to simultaneously measure both BCR-ABL and ABL transcripts.Methods:Positive controls are plasmids containing full-length target sequence of BCR-ABLP210 and ABL. 70 cases of CML bone marrow samples collected in Nanfang Hospital from Jane 2005 to July 2008 were examined. Patients were untreated ones and those treated with STI571 or allo-hematological stem cell transplantation. RQ-RT-PCR value=copies of BCR-ABL/copies of ABL. The results are compared with fluorescence chromosomal in situ hybridization (FISH) for BCR-ABL.Probes and primers recommended by Europe Anti-Cancer group in 2003 were used as gold standard control; probe and primers for bcr-ablP210 gene are ENF541, ENP501 and ENR561, respectively; probe and primers for abl gene are ENPr1043, ENF1003 and ENR1063, respectively. Both Taqman probes are FAM labed.For duplex RQ-RT-PCR, HEX labeled probe is used for the ABL gene, along with corresponding primers, ENP541 labeled with FAM fluorescence and ENF501 and ENR561 were also used for BCR-ABLP210.The experiments were carried out in the same tube on a Biorad Opticon2 RQ-PCR unit. The end concentration is 0.3μmoL/L for primers and 0. 2 μmoL/L for probe. The PCR reaction was carried out in 25μL. PCR condition is at 50°C×2min+95°C×10min, then followed 95°C×15s+60°C×1min for 50cycle.Result: Testing using serial dilutions of plasmid positive control suggested that HEX-FAM duplex is readily amplified with the FAM Taqman probes of BCR-ABLP210, and the HEX-ABL results is same with those with FAM-ABL (recommeded by Europe Anti-Cancer group). Coefficiency of variation among different experiments is less than 5%. The 70 CML cases can be divide into 3 groups based on FISH value: ≥10% (n=32), 0.5% 10% (n=27) and negative (n=11). The corresponding RQ-PCR ratio of BCR-ABL/ABL are 0.590±0.264, 0.044±0.041 and (9.46±6.99)×10|4, respectively, P<0.01 between each group.Conclusion: We have established an efficient duplex RQ-RT-PCR method with FAM and HEX Taqman probes. This approach enables acquisition of more information from each test and hence reduces the amount of sample needed for each test. We believe this method will be useful to MRD monitoring in CML and BCL-ABL + B-ALL.

Leukemia Relapse after Allogeneic Transplants in AML: Predictive Role of WT1 Expression and Protective Effect of DLI.

Background: relapse remains a significant problem in patients with acute leukemia undergoing an allogeneic hemopoietic transplant (HSCT) and a predictive molecular marker of disease would be useful.Aim of the study. Assess the role of WT1 expression in AML patients undergoing an allogeneic HSCT, topredict relapse anddrive immune interventionMethods. We studied 81 patients with AML who received an allogeneic HSCT between 30/1/04 and 11/2/08 in our Unit: 52 patients were in first remission, and 29 patients had more advanced disease. Median age 45 (16–66), prepared with CY-TBI (n=52) or RIC regimen (n=29). WT1 mRNA levels of expression were normalized with respect to the number of ABL transcripts and expressed as WT1 copy numbers every 104 copies of ABL. Patients bone marrow cells were monitored for WT1 expression, and cytology, pretransplant, at day +30 and every month untill day +180, then at scheduled follow up. Patients were considered WT1 positive, when WT1 copy numbers were greater than 180. Escalating doses of donor lymphocyte infusions (DLI) were given, when possible, in WT1+ patients.WT1 pre-transplant. WT1 pre-HSCT was available in 63 patients, 39 were WT1- and 24 WT1+: their relapse risk was 10% vs 62% (p=0.00002) and their crude survival 79% vs 29% (p=0.0001). When CR1 patients were selected, relapse was 10% vs 50% for WT1- vs WT1+ patients (p=0.02), and survival 81% vs 50% (p=0.9).WT1 post-transplant. Fifty patients were always WT1- after HSCT, and 31 were found WT1+ at a median interval from HSCT of 80 days (range 24–696): relapse was 8% vs 68% for WT1- vs WT1+ patients (p=<0.0001), and survival 78% vs 26% (p=0.0001).Cox analysis: in multivariate analysis on relapse WT1 pre-transplant (p<0.0001) and post-transplant (p<0.0001) were independent predictors, whereas disease phase (CR 1/>1) was not (p=0.39). Survival was predicted by all 3 variables, WT1+ pre (p=0.001), WT1+ post (p=0.03) and phase of disease (p=0.002).Immune intervention. Of the 31 WT1+ patients, 13 had BM blasts on cytology (6– 80%) at the time of first WT1 positivity, and were treated with chemotherapy. 18 patients were WT1+ and had marrow in CR on cytology: 9 of these received DLI and 9 did not; hematologic relapses were seen in 2/9 and 7/9 respectively (p=0.02) and crude survival is 6/9 vs 2/9 (p=0.07). The median interval between WT1+ and relapse was 96 days (39– 246).Conclusions. This study suggests that WT1 positivity before and after HSCT are independent predictors of relapse and survival in patients with AML. WT1 expression predicts relapse better than disease phase. The interval between WT1 positivity and relapse is approximately 3 months: the use of DLI in this time interval appears to protect against hematologic relapse.

Analysis of T Cell Cloanlity of Ph+ Acute Lymphoblastic Leukemia with Chronic Gvhd in Continuous Remission after Allogeneic Hematopoietic Stem Cell Transplantation

Chronic graft-versus-host disease (cGVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, recent study suggested that the presence of cGVHD might reduce the risk of relapse in Ph+ acute lymphoblastic leukemia (ALL) after allo-HSCT. GVHD is mediated by proliferation of special T-cell clones, we analyzed the T cell receptor (TCR) Vβ repertoire and cloanlity in Ph+ ALL patients undergone cGVHD after allo-HSCT in order to find the special T-cell clones associated with continuous remission.Allogeneic transplantations were performed in the first complete remission (CR) (6 ALLP190+ patients). The numbers of BCR-ABL copies in peripheral blood samples were assessed with the real-time quantitative RT-PCR (RQ-PCR) test at diagnosis, and every month after HSCT. The quantity of BCR-ABL transcript was normalized for normal ABL expression and the result was expressed as the ratio of BCR-ABL copies to ABL copies. With monitoring BCR-ABL copies of 6 BCR-ABLP190+ ALL patients after HSCT (the mean ratio of BCR-ABLP190/ABL at diagnose was 0.41 ± 0.39%), we found that 4 patients with extensive cutancous cGVHD had no evidence of leukemia recurrence, and the BCRABL transcripts of patients with cGVHD were remaining 0 copy, while 2 patients without cGVHD relapsed (the mean ratio of BCR-ABLP190/ABL was 7.56 ± 2.49%) and even dead within six months of HSCT.The expression and cloanlity analysis of TCR Vβ repertoire was detected in peripheral blood samples from patients with cGVHD by RT-PCR and genescan technique. TCR Vβ repertoires with polyclonal pattern were identified in normal controls and donor groups. However, the skew expression pattern of TCR Vβ repertoire could be detected in patients with cGVHD even more than 1 year after allo-HSCT. Among the 24 Vβ subfamilies, there were only 11~17 Vβ subfamilies expressed in cGVHD patients. Oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 1, 2, 3, 7, 8, 12, 14, 16~20, 22 and 23 subfamilies in 4 ALL patients (BCR-ABLP190 remission) with cGVHD. As a contrast, oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 2, 7, 8, 14, 17, 20, and 22 subfamilies in 2 chronic myeloid leukemia patients (BCR-ABLP210 remission) with cGVHD.In conclusion, the predominant usage and clonal expansion of TCR Vβ subfamily T cells could be found in Ph+ ALL patients with cGVHD and these groups of specific T-cell clones (Vβ 1, 3, 12, 16, 18, 19 and 23) were potentially associated with cGVHD in ALL and might be responsible for maintaining CR in Ph+ ALL patients after allo-HSCT. Research Funding.

Molecular Response in Core Binding Factor Acute Myelogenous Leukemia with Fludarbine, Cytarabine, G-CSF and Gemtuzumab Ozogamicin.

Background: Core binding factor associated acute myelogenous leukemias (CBF AML) are characterized by unique fusion transcripts that are amenable to molecular monitoring. Multiple log reduction in the fusion transcript level has been reported to be associated with lower relapse rates in CBF AML (Liu-Yin JA et al. ASH 2007# 543). Addition of gemtuzumab ozogamicin (GO) to induction and consolidation has resulted in improved disease free survival (DFS) in patients with AML and “standardrisk” or “favorable cytogenetics” (Burnett,A. et al. ASH 2006#13). Historical data from M.D. Anderson Cancer Center indicates that induction/consolidation therapy with fludarabine, cytarabine and granulocyte colony stimulating factor (GCSF) (FLAG) results in improved event free survival (EFS) (median EFS not reached after median followup of 118 weeks) in patients with CBF AML (Borthakur et al. 2008, Cancer in press).Method: In an attempt to improve upon these results we have initiated a study incorporating GO with FLAG (GO-FLAG) in induction/consolidation for treating patients with newly diagnosed CBF AML. We report on molecular responses in patients enrolled in this study. Extracted RNA is reverse transcribed and analyzed by real-time quantitative PCR for the fusion transcript associated with CBF leukemias. Values are expressed as a percentage of fusion transcript to normalizing ABL transcripts. The sensitivity of detection is approximately 1 in 100,000 for AML1-ETO and 1 in 10,000 for CBFb-MYH11.Results: Seventeen (Inv 16=6/t 8;21=11) patients (male=15)[median age 49 years (range, 29–73)]have been treated to date with a median follow-up of 10 months (range,1–16). Patient characteristics at presentation include median WBC 10.4 (range, 1.5–41.5) x 106/ml, hemoglobin 9.3 (range, 7.8–11.9) gm/dL, platelet count 22 (range, 6–365) x 109/ml. Additional cytogenetic abnormalities were present in 10 patients; +8 and –Y being most frequent. Two patients had kit mutations. All patients achieved complete remission (CR) except one (partial remission). Two patients died in CR from infectious complications. There has been no relapse. Median pre-treatment value for fusion transcript to Abl ratio by Q-PCR is 100 (range, 100–857). After induction the median Q-OCR value dropped to 0.04 (range, 0–9.17) and after three cycles of therapy the median value is 0.01 (range, 0–0.12).Conclusion: Induction/consolidation therapy with GO-FLAG regimen in CBF AML results in excellent molecular response. Further patient accrual and follow-up will generate information about impact on overall and disease/event free survival.

Low Meningioma 1 (MN1) gene expression to predict outcome in cytogenetically normal acute myeloid leukemia (CN-AML): A Cancer and Leukemia Group B (CALGB) study

7011 Background: Expression of the MN1 gene, which encodes a transcription co-activator implicated in myeloid leukemogenesis, has been shown to predict outcome in CN-AML patients (pts) in 1 study (Heuser et al. Blood 2006;108:3898). Here we present the first validation of the prognostic impact of MN1 expression in CN-AML in the context of not only mutated (mut) FLT3 and NPM1 and MLL-partial tandem duplication but also other established prognostic markers (i.e., CEBPAmut, WT1mut, ERG and BAALC expression levels). Methods: MN1 expression was measured by quantitative real-time RT-PCR in pretreatment bone marrow samples from 119 de novo CN-AML adult pts <60 years with material available for MN1 expression analyses who were treated on CALGB protocols 9621 and 19808. MN1 copy numbers were measured and normalized to ABL (internal control) copy numbers. Pts were dichotomized at the MN1 median expression and analyzed for outcome. Results: At diagnosis, low MN1 expression was associated with NPM1mut (P<0.0001) and the molecular low risk group (FLT3-internal tandem duplication (ITD)negative and NPM1mut; P<0.001). Complete remission (CR) rate was higher in low MN1 pts (93% v 73%); low MN1 pts were 6 times more likely to achieve a CR than high MN1 pts (P=0.005), once adjusting for hemoglobin (P=0.01) (Table). With a median follow-up of 4.6 years, low MN1 pts also had longer disease-free survival (DFS) (P<0.005; 3-year DFS: 60% v 32%) and overall survival (OS) (P<0.001; 3-year OS: 68% v 40%) than high MN1 pts. In multivariable analyses, MN1 expression independently predicted for DFS (P=0.01) and OS (P=0.03). For low MN1 pts, the risk for an event was reduced by approximately 50% compared with high MN1 pts (Table). Conclusions: MN1 expression predicts outcome independent of other established molecular markers in CN-AML pts. Particularly, low MN1 expression defines a group of CN-AML pts with favorable clinical outcome. Multivariable analyses for achievement of CR, DFS and OS Variables in Final Models CR DFS OS OR * P HR ** P HR ** P MN1 expression, low v high 6.4 0.005 0.46 0.01 0.54 0.03 WT1, mutated v wild-type – – 5.6 <0.0001 7.0 <0.0001 FLT3-ITD/NPM1 status, high v low risk – – – – 3.0 0.005 WBC, each 50 unit increase – – 2.1 0.002 1.7 <0.0001 Hemoglobin 1.7 0.01 – – – – * OR: odds ratio; ** HR: hazard ratio. Variables considered in the models were those significant at α=0.20 in univariable analyses as follows: for CR, MN1 expression, FLT3-ITD/NPM1 status (molecular high risk is FLT3-ITDpositive and/or NPM1wt; molecular low risk is FLT3-ITDnegative and NPM1mut), WBC, hemoglobin, age, race; for DFS, MN1 expression, FLT3-ITD/NPM1 status, ERG expression, WT1 and CEBPA mutations, WBC, race; for OS, MN1 expression, FLT3-ITD/NPM1 status, ERG and BAALC expression, WT1 mutations, WBC, age, race, hemoglobin, % blood blasts, extramedullary involvement. No significant financial relationships to disclose.

Codependents in the Stress Response

Exposure to stresses, such as heat shock or toxins, causes protein processing dysfunction in the endoplasmic reticulum (ER). If proper processing cannot be restored, the cell responds by initiating apoptosis. Qi and Mochly-Rosen used a combination of cell culture and in vivo experiments to investigate the role of protein kinase C δ (PKCδ) and the Abl tyrosine kinase in relaying the apoptotic signal from ER to mitochondria, where apoptosis is initiated. PKCδ and Abl, proapoptotic proteins with stress-dependent subcellular localization dynamics, are known to interact under conditions of stress. The authors coimmunoprecipitated Abl and PKCδ from ER cell fractions of cells treated with the toxin tunicamycin to induce ER stress and found that PKCδ was not activated in stressed cells when Abl transcripts were depleted by RNA interference. Expression and catalytic activity of both PKCδ and Abl were required to induce apoptosis, as measured by c-Jun N-terminal kinase (JNK) signaling activation and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The subcellular dynamics and functional interaction of PKCδ and Abl were also supported by an in vivo rat stroke model. The authors propose a model in which ER stress induces the translocation of PKCδ to the ER, where it is activated by Abl. The PKCδ-Abl complex transduces the apoptotic signal to mitochondria by activating the JNK cascade. Whether JNK activation occurs in the ER, cytoplasm, or mitochondria is not known, nor is it known whether PKCδ and Abl interact directly or whether they are part of a larger complex. X. Qi, D. Mochly-Rosen, The PKCδ-Abl complex communicates ER stress to the mitochondria – an essential step in subsequent apoptosis. J. Cell Sci. 121 , 804-813 (2008). [Abstract] [Full Text]

Quantitative Molecular Expression of the Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia Cells as a Possible Marker for Minimal Residual Disease Detection.

The expression of the catalytic enzyme of tryptophan, indoleamine 2,3 dioxygenase (IDO) has been identified as a T-cell inhibitor effector pathway in different normal and neoplastic cells. We have recently shown that normal bone marrow (BM) cells, including hematopoietic CD34+ cells, express IDO mRNA only upon IFN-γ stimulation, whereas in a subset of human acute myeloid leukemia cells (AML) IDO is constitutively expressed both at molecular and protein level and induces immunological escape by promoting the generation of T-reg cells. To investigate whether the IDO transcript can be used as a marker for minimal residual disease (MRD) detection, we used a sensitive real-time reverse transcription-polymerase reaction assay (qRT-PCR) to quantify IDO mRNA levels in peripheral blood (PB) and BM samples of newly diagnosed AML patients. The level of IDO transcript was evaluated as IDO copy number/104 ABL copies. As control samples, we used normal PB and BM mononuclear cells (MNCs). Our data showed that normal BM and PB cells expressed minimal amount of IDO mRNA (range 100–1000). Among AML samples, we identified three subsets of patients according to IDO mRNA expression: 1) 33/74 (44.6%) IDO negative (i.e. range < 100), 28/74 (37.84%) IDO low (range 100–1000) and 13/74 (17.6%) IDO high (i.e. range > 1000). BM and PB AML blasts gave similar results. Assessment of protein expression and enzymatic activity was in accordance with molecular results. Some patients were evaluated for IDO mRNA expression before and after induction chemotherapy and the IDO levels were found to correlate with the reduction of BM blasts. Taken together, our qRT-PCR data demonstrate that1.normal PB and BM cells are negative for IDO mRNA expression, which, in turn, is significantly up-regulated in a subset of AML patients (IDO high) and2.IDO mRNA expression correlates with tumor burden,thus suggesting its possible role in the detection of MRD in IDO high patients.

Prognostic Value of Minimal Residual Disease by Real-Time Quantitative PCR in AML with CBFB-MYH11 Rearrangement: The French Experience.

Background. Despite the favorable prognosis of AML patients with inv(16)/t(16;16) leading to CBFB-MYH11 rearrangement, relapses still occur in about 40% of the cases. With the possible exception of receptor tyrosine kinase mutations, no pretreatment-factor can strongly predict risk of relapse.Methods. To explore minimal residual disease (MRD) prognostic impact, we prospectively monitored CBFB-MYH11 rearrangement by real-time quantitative PCR (RQ-PCR) in patients with inv(16)/t(16;16) treated in French cooperative trials; most patients were treated in Acute Leukemia French Association (ALFA) trials and Leucémies Aiguës Myéloblastiques de l'Enfant (LAME) Cooperative Groups trials. RQ-PCR was performed in three molecular French labs (CHU of Lille, Necker Hospital and St-Louis Hospital) according to the EAC procedure and results are expressed as CBFB-MYH11/100 ABL copies(%).Results. 61 AML patients with inv(16)/t(16;16) who had reached CR with one of the anthracyclin-aracytin regimens were analyzed. Median age was 37.5 years (range 2.1–76.5) and median baseline WBC 46 G/L (range 1.8–246). With a median follow-up of 23.3 months, median overall DFS was 23 months and median OS not reached (93.5% of patients alive). No initial clinical or hematological characteristic including age, gender and initial WBC was predictive of relapse in adults, but children had poorer DFS than adults (17.6 months vs not reached, p=0.03). Regarding chromosomal abnormalities in addition to inv(16)/t(16;16), trisomy 22 was the most frequent but no significant prognostic impact was observed. Of the 61 patients, MRD monitoring could be performed sequentially in 45. Pretreatment CBFB-MYH11 transcript level had no impact on DFS. However, after induction therapy, transcript levels >0.5% were significant predictors of poorer DFS (median 16.4 months vs not reached if <0.5%, p=0.002). Likewise, after first consolidation therapy course, transcript level >0.001% was also predictive of poorer DFS (median 22 months when >0.001% vs not reached when <0.001%; p=0.03). 34 paired analyses of bone marrow (BM) and peripheral blood (PB) samples revealed only moderate correlation (R2=0.86); especially for low MRD levels, CBFB-MYH11 transcript expression being generally higher (up to 78-fold) in BM samples than in PB samples.Conclusion. MRD monitoring by RQ-PCR appears to be a major prognostic factor of DFS in AML with CBFB-MYH11 rearrangement and could be a powerful tool to early identify good and bad responders which could have an impact on therapeutic decisions for consolidation therapy. [Display omitted]

A multiplex PCR for improved detection of typical and atypical BCR– ABL fusion transcripts

RT-PCR is the method of choice for detecting BCR– ABL in CML and ALL. The three predominant mRNA transcripts found are e1a2 (in ALL), e13a2, and e14a2 (in CML and ALL). However, a number of “atypical” BCR– ABL transcripts (e1a3, e13a3, e14a3, e19a2, e6a2, e8a2, etc.) resulting from chromosomal breakpoints outside ABL intron 1 or BCR intron 1, 13 or 14, respectively, have been reported. These atypical transcripts may escape detection when using methods that are optimized to detect just the typical ones. We present here a novel, fast, and reliable multiplex PCR for improved detection of typical and atypical BCR– ABL transcripts.

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.