Fusion Transcript Levels Research Articles

Quantification of Fusion Transcripts Reveals Slower Treatment Kinetics As Compared with Multiparameter Flow Cytometry during Induction Treatment of Acute Myeloid Leukemia in Children

The prognosis of children with acute myeloid leukemia (AML) is improving but still unsatisfactory. We and others have shown that measurable residual disease (MRD) as measured with multiparameter flow cytometry (MFC) is a very strong prognostic factor, however this technique cannot reliably identify low-risk cases. An alternative approach for MRD analysis is quantification of leukemia-specific transcripts using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). This approach has shown strong prognostic value in adult AML but its significance in childhood AML is less studied. In this retrospective study, we evaluated early treatment response with RT-qPCR and MFC in parallel to determine treatment kinetics and the relationship between results of the two methods. The study included 15 children (7 females, 8 males, median age 6 years (range 1-16)), diagnosed 2004-2011 with de novo AML with a quantifiable fusion transcript (8 with RUNX1-RUNX1T1, one with CBFB-MYH11 and 6 with KMT2A-MLLT3), all treated in the NOPHO AML-2004 trial. MRD analyses were performed on samples from day 15 from start of the first induction course and just before the first consolidation course. MRD analysis with MFC was performed in bone marrow samples by identifying leukemia-associated immunophenotypes (LAIP), and reported as percent of viable cells. Fusion transcripts were analyzed with RT-qPCR in bone marrow and blood according to the Europe against cancer program, and reported as percentage of diagnostic level in bone marrow. Results of all MRD analyses were reported to clinicians but not used for treatment decisions. All patients achieved complete remission. 9 patients relapsed (median time from diagnosis 12 months, range 8-16); 5 with RUNX1-RUNX1T1 and 4 with KMT2A-MLLT3. When we compared results of RT-qPCR in blood and bone marrow day 15 and before consolidation, there was a high correlation; 0.93, p<0.01. When 0.1% was used as cut-off for positivity, 20/22 samples were concordant (Cohens kappa test K value 0.818, P<0.001) with the two disconcordant samples positive in bone marrow but negative in blood before consolidation. Of the concordant samples, 9 were ≥0.1% and 11 were <0.1% in both bone marrow and blood. There was no significant correlation between results obtained with MFC and RT-qPCR, neither in bone marrow nor in blood. When comparing MFC and RT-qPCR results in bone marrow using the 0.1% cut-off, only 12 of 22 samples were concordant (4/10 at day 15, 8/12 before consolidation, in total 55% agreement; K value 0.141, P=0.36). Most discordant results (9/10) were ≥0.1% with RT-qPCR but negative (<0.1%) with MFC. There was a pattern of slower treatment kinetics with higher MRD results obtained with RT-qPCR than MFC. In cases with RUNX1-RUNX1T1 fusion transcript levels were 22% (median, range 12-63%) of diagnostic value day 15 and 0.03% (0-1.4%) before consolidation. In cases with KMT2A/MLLT3, the corresponding levels were 15% (0 - 40%) and 0 (0-0.038%). There was no difference in relapse frequency between children with fusion transcripts ≥0.1% or <0.1% day 15. On the other hand, two patients with RUNX1-RUNX1T1 fusion transcript level ≥0.1% but negative with MFC before consolidation both relapsed. In order to find the reason for remaining high levels of fusion transcripts day 15 when the bone marrow is usually hypoplastic, we reanalyzed flow cytometry files from four cases with remaining fusion transcript >0.1%. These analyses confirmed the absence of immature cells with LAIP and detected either more mature myeloid cells or immature cells without LAIP. We hypothesize that fusion transcripts can be present in mature cells not detected as MRD using MFC. This was supported by analyzing sorted immature and mature cells from bone marrow samples at diagnosis of AML (n=4, adults). In CD34-CD117+ cells, leukemic transcripts (RUNX1-RUNX1T1/CBFB-MYH11/mutated NPM1) were 67±10% (mean±SEM) of the level in the most immature cells, CD34+CD117+, from the same patients and in mature granulocytes (CD34-CD117-, high SSC) 88±9%. In conclusion, RT-qPCR and MFC provide different information during induction treatment. RT-qPCR has a stronger ability to detect remaining leukemic burden, but whether this burden is clinically relevant remains to be shown. The impact of fusion transcript levels on relapse risk is currently investigated in the NOPHO-DBH AML-2012 trial in which MFC is used for risk stratification. DisclosuresNo relevant conflicts of interest to declare.

Development of Sample Preparation Procedures for Testing Specimens with High White Blood Cell Counts for Xpert® BCR-ABL Ultra¥

Introduction: Monitoring BCR-ABL fusion transcript levels in peripheral whole blood (WB) of patients on tyrosine kinase inhibitor (TKI) therapy using real-time quantitative PCR (RT-qPCR) is standard of care in the management of Chronic Myeloid Leukemia (CML). GeneXpert® BCR-ABL V2¥¥ or Xpert® BCR-ABL Ultra¥ (Ultra), a cartridge-based assay for use on the GeneXpert Instrument System, automates and standardizes the RT-qPCR process in less than 2 hours, using a lysate prepared from 4mL WB, resulting in an effective WB input volume of 600μL. There are, however, clinical situations where the total RNA isolated from high numbers of white blood cells (WBC) circulating in the patient's blood or in bone marrow (BM) can overload the Ultra cartridge and other quantitative BCR-ABL assays, requiring subsequent dilution of the sample to generate valid assay results.In this set of experiments, we sought to define the WBC input limits for Ultra that would predict cartridge overload, and thereby provide guidance regarding when to dilute the patient's sample in the presence of high WBC.Methods: Serial dilutions of WB specimens were tested in Ultra to determine the upper and lower WBC input limits corresponding to the valid ABL reference gene Ct cutoffs of 10 and 18, respectively. Sample prep procedures were developed to allow using 50μL or lower input volume from WB, BM, or the 1st sample lysate, with or without the WBC count (WBCC) information. BCR-ABL log% IS results from a subset of samples prepared and tested with various specimen input volumes in Ultra were compared in linear regression analyses with results from the Qiagen Ipsogen BCR-ABL1 Mbcr IS-MMR assay (IS-MMR).Results: The WBC input number of ~20 million cells/mL WB corresponded to the upper limit, and ~150K cells/mL WB corresponded to the lower limit, of the valid ABL Ct range for Ultra. For CML specimens with WBCCs <20 million cells/mL WB, the standard 4mL WB input yielded ABL Ct within acceptable limits. For specimens with WBCCs ≥20 million cells/mL, reaching as high as >500 million, sample prep procedures were developed to use 50μL WB, BM, or 1st lysate input volume. When 50μL was used but still yielded ABL Ct <10, a lower input volume (10μL or lower) can be used (Table 1). For situations in which the WBCC information is not available, but there is suspicion of high WBC yielding a very viscous 1st lysate that is hard to pipette, a 1st lysate treatment procedure for dilution was developed to allow for testing with 50μL or lower WB or BM input volume. These procedures were validated in Ultra by testing in CML specimens with various WB or BM input volume and showed high concordance (R2=0.98) when compared to the IS-MMR.Conclusions: In summary, sample prep procedures were developed in cases where high WBCC is known, or is suspected, or when repeat testing is needed for Invalids with ABL Ct <10, allowing the use of Ultra with various input sample volume for WB or BM in a wide variety of clinical situations.¥In vitro diagnostic medical device. May not be available in all countries. Not available in the United States.¥¥ RUO; For research use only. Not for diagnostic use. Not reviewed by any regulatory body. DisclosuresDay:Cepheid: Employment.

Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion genes resulting from the translocation t(2;5)(p23;q35) are present in almost 90% of childhood ALK-positive anaplastic large-cell lymphomas (ALCL). Detection and quantification of minimal disseminated disease (MDD) by measuring NPM-ALK fusion transcript levels in the blood provide independent prognostic parameters. Characterization of the genomic breakpoints provides insights into the pathogenesis of the translocation and allows for DNA-based minimal disease monitoring.We designed a nested multiplex PCR assay for identification and characterization of genomic NPM-ALK fusion sequences in 45 pediatric ALCL-patients, and used the sequences for quantitative MDD monitoring. Breakpoint analysis indicates the involvement of inaccurate non-homologous end joining repair mechanisms in the formation of NPM-ALK fusions. Parallel quantification of RNA and DNA levels in the cellular fraction of 45 blood samples from eight patients with NPM-ALK-positive ALCL correlated, as did cell-free circulating NPM-ALK DNA copies in the plasma fraction of 37 blood samples. With genomic NPM-ALK fusion sequence quantification, plasma samples of ALCL patients become an additional source for MRD-assessment. Parallel quantification of NPM-ALK transcripts and fusion genes in ALCL cell lines treated with the ALK kinase inhibitor crizotinib illustrates the potential value of supplementary DNA-based quantification in particular clinical settings.

Treatment Free Remission (TFR) in Chronic Myeloid Leukaemia (CML): The Tawam Hospital Experience

Introduction: CML is a chronic myeloproliferative neoplasm that is characterized by presence of Philadelphia chromosome (a translocation between chromosomes 9 and 22 leading to BCR-ABL1 fusion gene and protein). This fusion can be detected by karyotyping, FISH and polymerase chain reaction (PCR). The therapy of CML has evolved from hydroxyurea, interferon, allogeneic stem cell transplant and lately tyrosine kinase inhibitors (TKI). The target of therapy is molecular response (MR) and has also evolved from the major molecular response (MMR), defined as ≥3-log reduction in BCR/ABL1 fusion transcript using real-time quantitative PCR (RQ-PCR) to deep molecular response (DMR, ≥4-log reduction). The experience from a number of clinical trials, including STIM, TWISTER, A-STIM and KIDS, has generated interest in stopping TKIs in patients who have reached a sustained DMR. Hughes et al (Blood 2016) have published practical guidelines for selection of candidates for TFR and their subsequent management. Here we report our experience with TFR in patients with CML in DMR. Method: We searched our database for CML patients on continuous TKI therapy and monitoring. Patient not followed for more than 6 months were excluded from analysis. Molecular response was monitored in all patients at intervals defined by National Comprehensive Centre Network (NCCN) guidelines (available at www.nccn.org). Peripheral blood samples were collected in Tempus™ Blood RNA Tubes and analysed in a central Molecular Diagnostics Laboratory. Real-time quantitation of BCR-ABL1 gene transcripts was performed using the Qiagen-Ipsogen BCR-ABL1 Mbcr IS-MMR Dx® kit. The results were expressed as a percent ratio of BCR-ABL1 to ABL1 transcripts on the International Scale (IS). The assay can quantitate up to 4.96 log reductions (0.0011%IS) calculated according to the recommendations of Cross et al (Leukemia 2015). Result: We identified One hundred and seventeen CML patients on continuous TKI therapy and monitoring in our database. The median age was 36 years (range, 15-73 years). In our cohort 66/117 (56%) had achieved a DMR, while 20/117 (17%) had achieved undetectable levels of fusion transcript (≥4.5-log reduction, MR4.5). Fifteen out of the twenty (75%) with MR4.5 were candidates for TFR as they fulfilled the criteria proposed by Hughes et al (Blood 2016) of being more than 8 years on therapy with optimal response. Nine patients were counselled about TFR, monitoring and definition of progression and risk of progression. Six patients from this cohort have been initiated on TFR (Table 1). The median period of TKI therapy was 11 years. All six had undetectable levels of BCR-ABL1 transcript for more than 2 years. All were followed monthly except for the patient TW0012. Three patients have reached the six month milestone with one patient having MR 4.49 while the other 2 remain at undetectable levels. None of the patients experienced TKI withdrawal syndrome (myalgia/arthralgia) or expressed psychological anxiety for relapse. Conclusion: TFR is a very important goal especially in a young patient population. Continuous and chronic TKI therapy not only adds economic burden to the patient and healthcare system, but can also have undesirable side effects. Our limited experience outside of a clinical trial and in real world shows that willing and compliant patients and a reliable molecular laboratory will be the cornerstones of TFR as the future goal of CML. As our confidence builds with TFR, we anticipate extending it to candidates with MR4.5 when they reach 8 years or more of TKI therapy threshold. Disclosures Alam: BMS: Honoraria; Biologix: Honoraria.

MicroRNA-126 Orchestrates a Stem Cell-like Program in AML and B-ALL and Can be Exploited to Dissect Disease Heterogeneity

MicroRNA-126 reinforces hematopoietic stem cell (HSC) quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in human AML, it was required to maintain leukemic stem cell (LSC) quiescence (Lechman et al, Cancer Cell 2016), while its ectopic expression in mouse HSC induced leukemia (75% B-ALL, 25% AML) that fully regressed when switching a tetracycline-repressible miR-126 cassette off (Nucera et al, Cancer Cell 2016). RNA sequencing showed that miR-126 targeted cell cycle, apoptosis and p53 response genes, prevented differentiation and sustained oncogenic/pro-survival pathways typically associated with stem and progenitor cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We quantified miR-126 expression levels in a cohort of 45 newly diagnosed AML patients (n=38 de novo, of which 37% favorable, 34% int-1, 16% int-2, 13% adverse according to the ELN classification) presenting at the San Raffaele Hospital between 2010 and 2015 using a robust digital droplet PCR assay. In addition to core-binding factor (CBF) mutated AML, we found that the group of AML with chromosomal aneuploidy showed significantly elevated miR-126 levels, suggesting that this subgroup is characterized by high LSC frequencies and/or a specific need to suppress p53 responses, a hypothesis supported by the data obtained in our mouse model. To measure miR-126 at single cell resolution, we stably transduced primary AML blasts (n=5 diseases) with a lentiviral miR-126 reporter vector, xenotransplanted them into NSG mice and quantified miR-126 activity by FACS in the engrafted cells recovered from the mice's bone marrow. Across different genetic subgroups including CBF leukemia, cells with the highest miR-126 activity were enriched in the CD34+ or CD34+CD38- fractions, consistent with LSC. To facilitate prospective LSC isolation based on miRNA activity, we screened a series of combination miRNA reporters incorporating response elements for LSC-enriched and LSC-depleted miRNAs and have now identified an optimized construct that highlights easily sortable, distinct subpopulations that are currently undergoing functional validation. Applying this tool to AML with complex and monosomal karyotype, we are addressing whether high miR-126 expression refers to elevated LSC frequency or is a specific feature of this clinically relevant AML entity. We next applied the miR-126 reporter to n=15 primary, human B-ALL measuring miR-126 activity in the xenograft. Surprisingly, we identified well-separated blast subpopulations that differed in miR-126 activity within single diseases. Heterogeneity for miR-126 appeared to be a general feature of B-ALL as we detected 2-3 subpopulations in most of the diseases studied. We purified miR-126(high) and miR-126(low) B-ALL subpopulations from the primografts and verified up to 1log differences in miR-126 expression, while we detected equal levels of the BCR-ABL fusion transcript in all subpopulations from Philadelphia+ B-ALL confirming their neoplastic nature. When transplanting miR-126(high) and miR-126(low) subpopulations into secondary or tertiary recipients, post-sorting miR-126 levels were tightly maintained indicating that miR-126 levels were static rather than dynamically regulated in distinct B-ALL subpopulations, as expected from a subclonal architecture. RNAseq performed on miR-126(high) and miR-126(low) human B-ALL fractions evidenced a miR-126 signature reminiscent of the one obtained in the B-ALL mouse model and cord blood CD34+ cells, uncovering physiological miR-126 activity in human primary B-ALL. Taken together, these data support a broad, pathogenetically important role for miR-126 in human AML and B-ALL that goes beyond LSC and open up opportunities to better understand leukemia disease biology, dissect intratumoral heterogeneity and therapeutically target resistant and refractory disease, considering that miR-126 knockdown expands normal HSC while depleting the leukemia. DisclosuresCiceri:MolMed SpA: Consultancy.

Deeper and Early Molecular Response is a Good Predictor for Risk of Relapse in Patients with Acute Promyelocytic Leukemia

BackgroundConventional treatment of acute promyelocytic leukemia (APL) including all-trans retinoic acid (ATRA) with anthracycline had led to good patient response and survival. However, instances of relapse continue to occur, and remain the major problem with this course of treatment. Patients and MethodsWe collected the data of patients with newly diagnosed APL for the period 2004 to 2014, retrospectively. Data analysis included age, sex, initial white blood cell (WBC) count, coagulopathy and PML-RARα subtype. We also correlated PML-RARα fusion transcript level during treatment with outcome. ResultsA total of 33 patients were ultimately included in this study. Twenty-nine patients achieved complete hematologic remission(CHR) and 24 patients achieved complete molecular remission(CMR). The median overall survival duration was 92.8 months. Patient relapse and initially high WBC count were 2 factors associated with poor survival (P = 0.003; P = 0.015). Eight patients relapsed and the relapse-free survival (RFS) was 41.8 months. A reduction exceeding 4 log of PML-RARα transcript level after induction and undetectable PML-RARα transcript after treatment was associated with better RFS (P = 0.028). ConclusionsDeeper molecular response after induction therapy was associated with a reduction in relapse and could be used to predict risk of relapse in newly diagnosed patient under conventional treatment.

Qualitative and Quantitative Correlation of PML-Rara Fusion Transcript from Peripheral Blood and Bone Marrow Samples By Quantitative Real-Time PCR in Patients with Acute Promyelocytic Leukemia

Introduction: Acute promyelocytic leukemia (APL) is biologically distinct subtype of acute myeloid leukemia characterized by PML-RARA fusion transcripts, and its survival has significantly improved with all-trans retinoic acid (ATRA). Monitoring PML-RARA fusion transcripts with quantitative real-time polymerase chain reaction (RT-PCR) is the standard of care for the evaluation of response in patients with acute promyelocytic leukemia. We sought to determine the correlation between peripheral blood (PB) and bone marrow (BM) samples in patients treated on our frontline studies.Methods: We correlated results from BM and PB samples obtained from patients with newly diagnosed APL treated on three consecutive prospective clinical trials of combination of arsenic trioxide (ATO) and ATRA with or without gemtuzumab ozogamycin (ID01-014; NCT01409161; and NCT00413166) at our institution. Qualitative RT-PCR was performed on reverse-transcribed RNA from PB and BM samples for the short and long isoforms of PML-RARA, and the percent ratios of PML-RARA to ABL1 transcript levels were calculated. The sensitivity of detection with RT-PCR was 1 in 100,000. For this analysis, we selected samples with the time interval of collection within 1 day. Qualitative correlation was assessed using Kendall tau rank correlation coefficient test. Spearman's correlation coefficient was computed for quantitative variables to measure the extent of the association between samples. P values were two-sided and a p value of <0.05 was considered as statistically significant.Results: From July 2002 to May 2015, 184 patients were enrolled in the clinical trials. RT-PCR for PML-RARA was performed in 2077 samples including 1261 BM samples and 816 PB samples. In total, 584 samples (292 sample pairs) from BM and PB were identified within 1-day time interval. PML-RARA levels from PB samples with RT-PCR were strongly qualitatively and quantitatively correlated with those from BM samples (r= 0.831, p<0.001; τ= 0.792, p<0.001, respectively). Minimal qualitative discrepancy was observed in 13 samples from 11 patients: 8 BM samples from 8 patients with PML-RARA/ABL1 ≤0.01% (4 short form; 4 long form), with undetectable PML-RARA from concurrent PB; and 5 PB samples from 4 patients with PML-RARA/ABL1 ≤0.01% (2 short form; 3 long form), with undetectable PML-RARA from concurrent BM samples. Of 8 BM samples, 4 BM samples were obtained after 2 cycles, 3 cycles, 3 cycles, and 4 cycles of ATRA + ATO, respectively; 4, 1 month, 9 months, 9 months, and 10 months after completion of ATO + ATRA. The discrepancy of 4 BM samples during ATRA + ATO was resolved with undetectable PML-RARA level from peripheral blood samples and bone marrow samples after 1 additional cycle of ATO + ATRA. Of 4 BM samples after completion of ATO + ATRA, previous PML-RARA showed undetectable PML-RARA level, and repeat PML-RARA showed undetectable PML-RARA level from bone marrow and peripheral blood samples without intervention. Of 5 PB samples from 4 patients, 3 PB samples from 2 patients were obtained after 3 cycles, 3 cycles, and 4 cycles of ATRA. One patient had persistent PML-RARA/ABL1 <0.01% (short form) from peripheral blood samples with undetectable PML-RARA from concurrent BM samples after 3 cycles and 4 cycles of ATO + ATRA. Two PB samples from 2 patients were obtained 1 month after completion of ATO + ATRA.Conclusions: Strong correlation between PB and BM results indicate that PML-RARA fusion transcript levels in patients with APL can be monitored using PB samples. [Display omitted] DisclosuresVerstovsek:Incyte Corporation: Research Funding. Estrov:incyte: Consultancy, Research Funding. Cortes:Novartis: Consultancy, Research Funding; Teva: Research Funding; BerGenBio AS: Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy.

Prevalence and Prognostic Significance of c-KIT Mutations in Core Binding Factor Acute Myeloid Leukemia: A Comprehensive Large-Scale Study from a Single Chinese Center

Introduction CBF-acute myeloid leukemia (AML) is a heterogeneous disease that requires prognostic factors for patient differentiation. c-KIT mutations are predominantly detected in CBF-AML among AML cases, and reported frequencies vary significantly. To date, the clinical significance of c-KIT mutations in CBF-AML has been intensively studied; however, the results are controversial and a final conclusion has not been reached. Large-scale studies with subgroup analysis should be performed to elucidate the clinical effects of c-KIT mutations in CBF-AML.Methods A total of 351 patients with CBF-AML [t(8;21) (n = 253) or inv(16) (n = 98)] were included in this study. Overall, 250 patients [42 pediatric t(8;21);146 adult t(8;21); 11 pediatric inv (16) and 51 adult inv (16)] received standard treatment and underwent follow up. Induction therapy consisted of one or two cycles of combined anthracycline and cytarabine for adult patients, whereas cytarabine, idarubicin, and etoposide were used for pediatric patients. 131, 17, and 93 of the patients further received intermediate-dose cytarabine-based chemotherapy, chemotherapy with auto-hematopoietic stem cell transplantation (HSCT), and chemotherapy with allogeneic (allo)-HSCT for consolidation, respectively. c-KIT mutations in exon 8 and 17 were screened by direct sequencing. Patients who received allo-HSCT were censored at the time of transplantation for relapse and survival analysis to exclude the effects of allo-HSCT on outcome. The median follow up time was 10 months (3 to 93 months) for the 211 living patients.Results Overall, 36.5% (128/351) of the patients were found to have a c-KIT mutation. The frequencies of c-KIT mutations were similar between pediatric and adult patients with t(8;21) AML (41.7% vs. 38.5%, P > 0.05), whereas pediatric patients tended to have higher frequency of c-KIT mutations than the adult patients with inv(16) AML (53.8% vs 25.9%, P = 0.053). In adults, the frequency of c-KIT mutations was significantly higher in patients with t (8;21) than those with inv(16) (P = 0.043). In adults, the frequency of mutations in exon 17 was significantly higher in t(8;21) than inv(16) AML (P < 0.0001). In pediatric group, the frequencies of mutations in exon 17 and exon 8 were similar for t(8;21) and inv(16) AML (P > 0.05). The low PLT count (¡Ü 30∙109/L) in pediatric t(8;21) AML tended to be associated with the presence of c-KIT mutations ( P = 0.063). High white blood cell (WBC) count (>10∙109/L), WBC index, and AML1-ETO transcript levels in adult t(8;21) AML patients were significantly correlated with the presence of c-KIT mutations (P = 0.015, 0.0051, and 0.030, respectively). Moreover, low CBF¦Â-MYH11 transcript levels in adult inv(16) AML tended to be significantly related to c-KIT mutations (P = 0.059). No difference was observed in CR rates between patients with and without c-KIT mutations (P > 0.05). c-KIT mutations were significantly associated with less reduction in fusion transcript levels after the first induction therapy in adult inv(16) patients (P = 0.021). c-KIT mutations in adult t(8;21) AML patients (n = 137) significantly correlated with higher CIR rates at 2 years and low 2-year DFS and OS rates (73.2% vs. 46.2%, P = 0.0070; 26.8% vs. 49.1%, P = 0.013; 48.9% vs. 65.7%, P = 0.0055, respectively. Figure 1). In contrast, c-KIT mutations had no impact on CIR, DFS, and OS rates in pediatric t(8; 21)(n = 42), as well as in adult inv (16) (n = 51; all P > 0.05. Figure 1). Multivariate analysis demonstrated that c-KIT mutation was the only independent prognostic factor for relapse (hazard ratio (HR) = 2.47, 95% CI = 1.25 to 4.86, P = 0.009) and DFS (HR = 2.23, 95% CI = 1.17 to 4.41, P = 0.016). Moreover, c-KIT mutation and PLT count were independent prognostic factors for OS (HR = 3.68, 95% CI = 1.57 to 8.63, P = 0.003; HR = 0.37, 95% CI = 0.16 to 0.87, P = 0.023).Conclusion CBF-AML is a heterogeneous disease with regard to c-KIT mutations. c-KIT mutations have a strong adverse impact on relapse and survival in adult t(8;21) AML.Grant support Nature Science Foundation of China (81170483 and 81370639) and Beijing Municipal Science & Technology Commission£¨Z141100000214011). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ponatinib Is Well Tolerated and Active In Patients With Relapsed/Refractory Philadelphia Positive Acute Lymphoblastic Leukemia (PH+ ALL) and Advanced Phase Of Chronic Myelogenous Leukemia (CML) Harbouring T315I Mutation: The Bologna Experience

BackgroundPonatinib, a potent third generation pan BCR-ABL inhibitor, has recently shown relevant activity against native and mutant forms of BCR-ABL, including the TKI resistant T315I mutant. The aim of this compassionate protocol was to confirm and evaluate the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML.Design and MethodsPonatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F: 8/9) have been treated with Ponatinib (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL (10 p190, 4 p210) and 3 blast phases of CML (2 Myeloid and 1 Lymphoid, p210). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 754 days (range 46-2264). All the patients were resistant or intolerant to previous TKIs (median number of previous TKIs: 2; range 1-3). Standard chemotherapy was previously performed in 7/17 patients (41%). Four patients (23%) had previously received allogeneic stem cell transplantation. At the time of enrolment, median Hb, PLTs and WBC values were 10,9 g/dl (range 8.6-13.9), 139000/mmc (range 14000-325000) and 4300/mmc (range 1700-17000), respectively. In 6 out of 17 patients, additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (9 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). No mutations were detected in 4 patients. ResultsThe median treatment duration was 139 days (range 14-540+). Causes of treatment stop were: progression disease (5 patients), savage allogenic stem cell transplantation (6 patients), drug intolerance (1 patient), consisting in grade III headache.With a median follow up of 284 days (range 8-540+), a maHR was obtained in 13/17 patients (76%). After one month of treatment, a reduction of BCR-ABL fusion transcript level was observed in 9/15 patients (60%). For two patients the follow up is too short to be evaluable. The level became undetectable in 4 patients (3 presenting with T315I mutation).After treatment, T315I mutation disappeared in 6 out of the 9 patients who showed this molecular alteration at the beginning of therapy. At the time of this report, 6/17 patients are still on study (35%). Five patients died due to progression disease. As expected, the drug was well tolerated. Non-hematologic adverse events were described in 7/17 patients (grade >III skin rash in 3 patients; grade>II serum lipase increase in 2 patients; grade>II myalgia in 1 patient; grade III headache). ConclusionIn our experience, the activity of Ponatinib in advanced Ph+ leukemias, mainly in T315I mutated patients, was confirmed. No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients early on who could take advantage of this treatment. AcknowledgmentsWork supported by European LeukemiaNet, AIRC, PRIN 2010-2011, University of Bologna and BolognAIL. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Two Cases of Myeloproliferative Neoplasm with a ConcurrentJAK2V617FMutation andBCR/ABLTranslocation without Chronic Myelogenous Leukemia Phenotype Acquisition during Hydroxyurea Treatment

Sang Hyuk Park, M.D., Hyun-Sook Chi, M.D., Young-Uk Cho, M.D., Seongsoo Jang, M.D., Chan-Jeoung Park, M.D., Dae-Young Kim, M.D., Je-Hwan Lee, M.D., and Kyoo-Hyung Lee, M.D.2. Ann Lab Med 2013;33:229-32. https://doi.org/10.3343/alm.2013.33.3.229

Very Late Relapse of a Philadelphia Positive Acute Megakaryocytic Leukemia Post Allogenic Bone Marrow Transplant

Abstract 4518A 33 year old male initially diagnosed with myelodysplastic syndrome presented with acute leukemia, his bone marrow demonstrating sheets of abnormal megakaryocytes. Cytogenetics revealed two cell lines, 44X –Y, –C and 46XY, both positive for the Philadelphia chromosome mutation. He underwent busulphan and cyclophosphamide conditioning and allogeneic bone marrow transplant in the absence of a course of induction chemotherapy. One year later he was free of leukemia. Ten years after the transplant he was discharged from hematology follow-up, declared cured.Twenty-two years later, the patient re-presented with significant anemia and circulating blasts. One year previous he had normal blood counts. Bone marrow biopsy revealed acute megakaroblastic leukemia (M7), with cytogenetics demonstrating Philadelphia chromosome positivity and two identified cell lines, one with deletion of the Y chromosome. The patient underwent induction chemotherapy with idarubicin and cytarabine in addition to imatinib. His recovery marrow revealed no residual leukemia, including normal cytogenetics. He remained BCR-ABL fusion gene transcript positive, and was maintained on single agent imatinib. Four hundred and forty one days after induction chemotherapy he received a second sibling matched allogeneic stem cell bone marrow transplant. One year later he remains free of leukemia with no detectable BCR-ABL fusion gene transcript.Acute megakaryoblastic leukemia is a rare entity representing <1% of AML seen in adults. Prognosis is poor due to a high rate of relapse with disease free survival of 17% at 5 years. Even with allogeneic stem cell transplant, overall survival at 3 years is 43% with a relapse rate of 64%. With definitive diagnostic criteria for megakaryoblastic leukemia only being first described in 1985, at the time of our patient's first transplant he was one of the first M7-AML to receive a transplant.How did this patient, with such an aggressive subtype of leukemia, manage to stay in remission for over 20 years? Looking at all types of AML relapse, very late relapse, defined as occurring >5 years from the day that complete remission, occurs in 1–3% of cases. To our knowledge, this is the first reported case of very late relapse in an M7 leukemia. Fortunately, the rate of achieving a second complete remission state is higher in those patients who have a very late relapse with a second remission attained in up to 87% of cases. This may be influenced by the aggressiveness of the initial consolidation regime; those having more intense initial chemotherapy may have subsequently developed more resistant disease rendering them less sensitive to the second chemotherapy regimen. The fact that our patient never received induction chemotherapy when he initially presented may have contributed to the success of attaining remission when he was exposed to the standard 3+7 model used today.The pathogenesis for very late relapses remains a topic of debate. One hypothesis suggests that cells with malignant potential retain viability after chemotherapy treatments awaiting re-activation into a malignant state via either intrinsic errors in genetic division or an external stressor such as a viral infection or acquired immunodeficiency state. In this circumstance, the immune system is able to keep the tumor burden below the limits of minimal residual disease detection. It is also possible that residual very slow growing malignant cells remain after chemotherapy that are resistant to anti-tumor treatments. A third possibility is the presumed relapse is actually a de novo tumor with characteristics similar to its predecessor. Other late recurrences of AML have demonstrated both identical and different cytogenetic and molecular profiles to the original disease, which substantiates the premise for multiple mechanisms.In this case, one could also question whether the second transplant was necessary. He was maintained with a 2–3 log reduction in BCR/ABL fusion transcript level, but no morphologic evidence of leukemia, on only imatinib for over one year. The rationale for pursuing transplantation was the presumed aggressiveness of an M7 leukemia; it is unknown how long he could have been maintained on only a tyrosine kinase inhibitor. A second generation tyrosine kinase inhibitor would have been an option if the major molecular response was not maintained, and remains an option if our patient is unfortunate enough to have a second relapse. Disclosures:No relevant conflicts of interest to declare.

Results of Treatment with Tyrosine Kinase Inhibitors(TKIs) in Combination with Intensive Chemotherapy in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia(Ph+ALL)

Abstract 1503(Objective) The treatment outcome of patients with Ph+ ALL has significantly improved since the advent of TKIs such as imatinib and dasatinib. However, the long-term survival of these patients is not necessarily high when bone marrow transplantation (BMT) is not performed. The present study was aimed at developing an effective combination of a TKI and chemotherapy for long-term survival of patients with Ph+ ALL but ineligible for BMT. (Subjects and Methods) Forty-three consecutive patients (18 men, 25 women) with previously untreated Ph+ ALL who visited our hospital between June 2001 and February 2011 were treated. The age range was 13 to 84 years (median: 57), and 21 were 60 years or older. Measurement of BCR-ABL fusion transcript levels was performed immediately in these patients, and imatinib (IM) was started at 600 mg/body/day (given daily as a rule) as soon as the patient tested positive. A remission induction regimen (chemotherapy) used commonly in acute myeloblastic leukemia (AML) patients at our hospital was combined with TKIs in the first 14 patients. Another remission induction regimen often used in ALL patients at our hospital was then combined with TKIs in the subsequent 29 patients. The AML remission induction regimen used idarubicin (IDR) at 12 mg/m2 for 4 days, behenoyl-ara-C (BH-AC) at 350 mg/m2 for 10 days, and prednisolone (PSL) at 40 mg/body (P.O.) for 10 days. PSL was initially given at 100 mg/body (P.O.) and then gradually reduced in the ALL remission induction regimen. Vincristine (VCR) at 1.3 mg/m2 (Days 1, 8, 15, and 22), daunorubicin at 60 mg/m2 (Days 1, 2, and 3), cyclophosphamide at 1200 mg/m2 (Day 2), and L-asparaginase at 3000 μ/m2 (Days 11, 13, 16, 18, and 20) were administered. The remission induction therapies were given in a sterile room. Macrophage colony-stimulating factor was given for 7 days from Day 11 and granulocyte colony-simulating factor from Day 19. A post-remission therapy commonly administered to AML patients at our hospital was given after remission for 1 year (ASH, 2009, abstract, no. 1052). High-dose cytarabine (at 2 g/m2 BID for 5 days but at 1 g/m2 BID in patients aged 60 years or older) (HD-AC) and mitoxantrone (MIT) at 7 mg/m2 × 3 were initially administered after remission. Maintenance/consolidation therapy was given by alternating BH-AC at 350 mg/m2 × 4 + aclarubicin at 20 mg/body × 6 and BH-AC at 350 mg/m2 × 4 + IDR at 10 mg/m2 × 1 + VCR at 1.3 mg/m2 (each for 35 days). IM was given daily at 600 mg/day simultaneously with the post-remission therapy. Dasatinib was used in the post-remission therapy of 8 patients at 140 mg/day. All of the TKIs were given to patients for 3 years after completion of chemotherapy. (Results) Complete remission (CR) was achieved in 41/43 (95%). CR was achieved in 13 out of 14 (93%) patients treated with the AML regimen and 28 out of 29 (97%) treated with the ALL regimen. DIC occurred in 31/41 (76%) during remission induction therapy. The follow-ups of patients achieving CR lasted for 5 to 128 months (median: 24 months). Ten patients (aged 25 to 54 years [median: 37]) underwent BMT at first CR. Thirty-one patients (aged 13 to 84 years [median: 63]) were assigned to the chemotherapy group. The 10-year overall survival (OS) calculated by the Kaplan-Meier method was 57% for 41 patients who achieved CR, 66% for 20 patients younger than 60 years, and 49% for 21 patients aged 60 years or older. The OS was 80% for 10 patients who underwent BMT at first CR and 47% for 31 patients in the chemotherapy group. The 50% OS of the chemotherapy group was 36 months. The 10-year event-free survival (EFS) was 50% for patients who achieved CR, 80% for patients who underwent BMT at first CR, and 43% for the chemotherapy group. The 50% EFS of the chemotherapy group was 33 months. Fifteen patients have relapsed, and a new chromosomal aberration was observed at relapse in 11/13 (85%). (Discussion) The results of the present report indicate that BMT is the first choice for post-remission therapy of Ph+ ALL. Fifty percent of patients aged 60 years or older, who account for about half of the patients with Ph+ ALL, have achieved long-term survival with the prolonged TKI + chemotherapy used in the present report. Relapse was also rare among patients who received the combination and maintained CR for 3 years or longer. A combination of an appropriate TKI and a chemotherapy regimen as well as careful monitoring for complications is likely to further extend the survival of patients with Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.

Chromosome 20q Deletion

del(20q) can be observed in hematologic neoplasms, including chronic myelogenous leukemia (CML), and has been reported in patients undergoing blast transformation. We describe 10 patients with CML in hematologic and cytogenetic remission with del(20q) detected by conventional cytogenetics. There were 6 men and 4 women with a median age of 56 years. All patients initially had BCR-ABL1 and t(9;22) (q34;q11.2) and achieved morphologic and cytogenetic remission after therapy. del(20q) was identified before (2/10 [20%]), at the time of (3/10 [30%]), or after (5/10 [50%]) cytogenetic remission and was not associated with morphologic evidence of dysplasia. At last follow-up, no patients had a myelodysplastic syndrome (MDS). Leukocyte and platelet counts were normal; 4 of 10 patients had mild anemia. Nine patients have remained in morphologic and cytogenetic remission with stable del(20q). BCR-ABL1 fusion transcript levels were absent or low (median, 0.01%). Recently, in 1 patient, recurrent CML developed and del(20q) was lost. We conclude that del(20q) in the setting of CML in remission is not predictive of MDS or blast transformation.

Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia.

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 104−105. Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

An asymptomatic 61‐year‐old man with BCR‐ABL‐positive bone marrow following autologous transplantation for multiple myeloma

An asymptomatic 61‐year‐old man with <i>BCR‐ABL</i>‐positive bone marrow following autologous transplantation for multiple myeloma

BCR-ABL monitoring in imatinib-treated patients with chronic myelogenous leukemia.

6575 Background: Current NCCN guidelines recommend baseline and q 3 mo BCR-ABL fusion transcript levels in imatinib (IM)-treated chronic myelogenous leukemia (CML) responding to treatment (tx) to m...

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34− cell population with intrinsic resistance to imatinib

AbstractWe show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that approximately one-third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells and cell cycling, and increased clonogenic activity and expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in 2 immunodeficient mouse strains similar to Lin-CD34+ cells, whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell-cycle arrest genes and genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated compared with normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34− cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity, and clonogenic efficiency of CML Lin−CD34− cells in vitro. Moreover, leukemic CD34− cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Slow relapse in acute myeloid leukemia with inv(16) or t(16;16)

Acute myeloid leukemia (AML) with inv(16)(p13q22) or t(16;16)(p13;q22), resulting in CBFβ-MYH11 fusion transcript detectable by RT-PCR and RQ-PCR, is associated with an overall good prognosis, relapses however still occur in 30–35 % of patients, and with higher frequency in older patients.1,2 Molecular relapse generally precedes hematologic relapse in AML with balanced translocations such as t(15;17) or t(8;21), but generally by only a few weeks or a few months.3–9 Fewer data are available in AML with inv(16)/t(16;16). In 4 of the 5 relapses we observed in AML with inv(16)/t(16;16), the interval between molecular and hematologic relapse was prolonged. Between 2005 and 2009, 9 AML patients with inv(16) or t(16;16), with a median age of 60 years (range, 21–75) who had reached CR using French co-operative AML trials (anthracycline–cytarabine induction chemotherapy followed by consolidation chemotherapy with high-dose cytarabine, or intermediate dose cytarabine in elderly patients) at our center were prospectively monitored for minimal residual disease (MRD) based on CBFβ-MYH11 fusion transcript levels in bone marrow samples. RQ-PCR was performed on bone marrow cells according to the Europe Against Cancer (EAC) Program recommendations for CBFβ-MYH11 fusion transcripts (type A, D or E), using Taqman® technology, on an ABI PRISM 7000 (Applied Biosystems).10 Quantification of CBFb-MYH11 fusion transcripts was normalized to the housekeeping ABL gene. Results were expressed by the ratio CBFβ-MYH11 copy number/ABL copy number x 100 (%).10 Median follow-up after CR achievement was 18 months (range, 3–33) and median number of MRD analyses per patient was 6 (range 1–9). Molecular relapse was defined as a 10-fold or greater increase in CBFβ-MYH11 transcript level compared to the lowest level achieved. Five of the 9 patients relapsed, after 11–23 months, in the bone marrow (no extramedullary relapse was seen). In one of them, the interval between molecular relapse and hematologic relapse was short (one month). The 4 other hematologic relapses occurred slowly, and were preceded in all cases by molecular relapse detected in bone marrow samples, by ten (patient n. 1), six (patient n. 2), seven (patient n. 3) and eight (patient n. 4) months, respectively. Baseline characteristics of those 4 patients are shown in Table 1. Patients ns. 2 and 3 had c-KIT mutation in exon 8 and c-KIT D816V mutation, respectively (versus none of the patients who did not relapse) and patient n. 3 had N-RAS mutation, while no patient had FLT3-ITD or FLT3-835/I836 mutation. All 4 patients had achieved at least a 3-log reduction of the fusion transcript level, after induction therapy in 3 of them, and after the first consolidation course in patient n. 1 (Figure 1). During the period of isolated molecular relapse, blood counts and marrow aspirates remained normal in patients ns. 3 and 4, while cytopenias reappeared in patient n. 1 and abnormal marrow eosinophils in patient n. 2. A second CR was achieved in the 4 patients with chemotherapy, combined to gemtuzumab in 3 cases. Three of them were subsequently allografted, and all 4 patients were alive 2–11 months after hematologic relapse. In acute promyelocytic leukemia (APL), the median interval between molecular and hematologic relapse was 3–4 months in published litterature3–5 while in AML with t(8;21) it was generally less than six months6–8 and at a median of three months in our experience.9 Table 1. Main characteristics of the 4 relapsing patients analyzed. Figure 1. Minimal residual disease (MRD) sequentially measured by RQ-PCR in bone marrow samples in the 4 relapsing patients. MRD was expressed in CBFB-MYH11 copies per /100 ABL gene copies. Dots on curves represent MRD examinations. Full arrows indicate molecular ... In AML with inv(16)/t(16;16), few studies are available: Schnittger et al. reported 6 relapses of CBFβ-MYH11 AML with an interval between molecular and hematologic relapse ranging from 1–5 months,8 similar to what they observed in AML with PML-RAR and AML1-ETO. By contrast, Stentoft et al. reported, in 4 relapsing inv(16)/t(16;16) AML, an interval between molecular and hematologic relapse of approximately one year, with a slow molecular progression rate of about 1-log per 100 days.8 In our patients ns. 2, 3 and 4, the increase in fusion transcript levels had comparable kinetics, while in our patient n. 1, it was even slightly slower, with a molecular progression rate of about 1-log per 130 days. Thus, AML with inv(16)/t(16;16) AML may frequently relapse more slowly than other types of AML with balanced translocations. This interval between molecular and hematologic relapse may justify frequent MRD monitoring in those patients, and therapeutic intervention before overt relapse.

BCR-ABL fusion transcript types and levels and their interaction with secondary genetic changes in determining the phenotype of Philadelphia chromosome–positive leukemias

AbstractIt remains unresolved how different BCR-ABL transcripts differentially drive lymphoid and myeloid proliferation in Philadelphia chromosome–positive (Ph+) leukemias. We compared BCR-ABL transcript type and level with kinase domain (KD) mutation status, genotype, and phenotype in 1855 Ph+ leukemias. Compared with e1a2/p190 BCR-ABL cases, de novo e13-e14a2/p210 Ph+ lymphoid leukemia more frequently showed CML-type background, had higher blast-normalized BCR-ABL transcript levels, and more frequent persistent BCR-ABL transcript in the absence of detectable lymphoblasts. Secondary lymphoid blast transformation of CML was exclusively due to e13/e14a2/p210 BCR-ABL but was associated, at a much higher level than p210 myeloid transformation, with acquisition of new KD mutations and/or Ph genomic amplification. In contrast, myeloid blast transformation was more frequently accompanied by new acquisition of acute myeloid leukemia-type chromosomal aberrations, particularly involving the EVI1 and RUNX1 loci. Therefore, higher kinase activity by mutation, transcriptional up-regulation or gene amplification appears required for lymphoid transformation by p210 BCR-ABL.

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.