Associated Acute Myeloid Leukemia Research Articles

The new diagnostic criteria for myelodysplasia-related acute myeloid leukemia is useful for predicting clinical outcome: comparison of the 4th and 5th World Health Organization classifications.

Mutations in myelodysplasia-related (MR) genes, rather than morphological features, have been included in the diagnostic criteria of the new 5th World Health Organization (WHO) classification for myelodysplastic syndrome (MDS)-associated acute myeloid leukemia (AML). This study compares the clinical relevance of the new criteria with those of the previous version. In a cohort of 135 patients with newly diagnosed AML, the MDS-related AML patients were classified according to the 5th and 4th edition of the WHO classification (AML, myelodysplasia-related [AML-MR5th] and AML with myelodysplasia-related changes [AML-MRC4th], respectively). The median age of the patients was 70.4years. MR gene mutations were found in 48 patients (35.6%). Sixty-one patients (46.6%) were diagnosed with AML-MRC4th, while 71 patients (53.0%) were diagnosed with AML-MR5th. Patients with AML-MR5th were significantly older with significantly lower treatment response rate, higher recurrence rate, and shorter relapse-free survival after chemotherapy, whereas AML-MRC4th patients did not show any association with the treatment outcome. Overall, the following prognostic factors for survival were identified: age over 75years, antecedent MDS or MDS/myeloproliferative neoplasm, chromosome 5 or 7 abnormalities, and KRAS and ZSZR2 mutations. The 5th WHO classification is more useful for predicting the treatment response of patients with AML-MR than the previous version. Among the MR genes, ZSZR2 mutations were found to be independent prognostic factors affecting survival.

Long-Term Follow-up on Adult Patients with Philadelphia-Chromosome Positive Acute Myeloid Leukemia Who Were Treated with Interim Imatinib Followed By Allogeneic Hematopoietic Cell Transplantation

▪Background: The incidence of Philadelphia chromosome (Ph)-positivity in adult patients with acute myeloid leukemia (AML) is very low ranging from only 1 to 2%. Ph-positive AML has been included in the high-risk group presenting lower complete remission (CR) rate and early relapse after standard chemotherapy. However, the treatment outcome in a large cohort is not well elucidated due to its low incidence and variable treatment strategies including the use of tyrosine kinase inhibitors. We attempted to unveil clinical outcomes of Ph-positive AML in association with concomitant chromosomal abnormalities.Methods: We retrospectively analyzed 29 patients with Ph-positive AML (median age: 43 years old [range; 18-80]) from 2002 to 2016. Except for the 2 patients who did not get treated at all, 3 were treated repeatedly with low-dose cytarabine and 24 were treated with 3+7 standard induction chemotherapy, followed with the application of interim imatinib 400mg until the starting day of next chemotherapy or conditioning for hematopoietic cell transplantation (HCT). Among them, 17 underwent allogeneic HCT from sibling donors (n=6), unrelated donors (n=7), and haploidentical donors (n=4).Results: In total of 29 patients, 7 (24.1%) had additional inv(16). There were 3 therapy-related AML in Ph-positive AML subgroup with inv(16), while none were observed in the subgroup without inv(16). In the Ph-positive AML subgroup with inv(16), median age was younger (31 vs. 44 years old, p= 0.083) and CR rate was higher (85.7% vs. 54.5%, p= 0.214) than the opposite. Among 27 treated patients, 20 (74.1%) patients achieved CR after standard chemotherapy with interim imatinib, and 2 achieved CR after low-dose cytarabine with interim imatinib. In the entire patients group, after a median follow-up duration of 41.5 months (range: 0.2 to 156.6), 5-year overall survival (OS) was 40.1%. However, 5-year OS of 17 patients (11 in subgroup without inv(16) and 6 in subgroup with inv(16)) treated with allogeneic HCT was 69.3%, and 6 patients among them with Ph-positive AML with inv(16) were all alive after allogeneic HCT. In contrast, all 12 patients who were not treated with allogeneic HCT expired within a median of 6.3 months [range: 0.2-18].Conclusion: This study revealed that interim imatinib combined chemotherapy produced an acceptable CR rate, and allogeneic HCT as a post-remission therapy was a feasible choice for providing a long-term disease control in adult patients with Ph-positive AML. We also identified that inv(16) was related to favorable outcomes regardless of the coexisting t(9;22) and to therapy-related AML. DisclosuresKim:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Interaction between 12p chromosomal abnormalities and Lnc-HOTAIR mediated pathway in acute myeloid leukemia.

To investigate the correlation of homeobox (HOX) transcript antisense RNA expression with clinicopathological features and the clinical prognosis of the patients with chromosome 12p abnormalities associated acute myeloid leukemia (AML). We also investigate the association of 12p chromosomal on the expression of HOTAIR, miRNA-193a, and c-kit gene as targeting genes for HOTAIR in AML. AML patients with 12p chromosomal abnormalities were recruited and compared to AML with other chromosomal abnormalities rather than 12p. The long noncoding RNA (lncRNA) "HOTAIR," miR-193a, and c-Kit genes expression were measured in bone marrow samples using Syber green based real-time polymerase chain reaction. We found a significant difference for the expression levels of HOTAIR, c-kit, and miR-193a between 12p abnormalities associated AML and those without. The survival analysis revealed that patient's with low expression levels of HOTAIR and c-kit had significantly better survival and leukemia free survival. In contrast, miR-193a was associated with better overall survival but not leukemia free survival. 12p abnormalities associated AML were associated with worse prognosis. Our results proved that HOTAIR, miR-193a, and c-kit genes are independent prognostic predictors in 12p chromosomal associated AML; therefore it may represent a novel therapeutic application in AML in the future.

Role of cluster of differentiation 39 (CD39) in the immunosuppressive status associated Acute myeloid leukemia (AML)

Role of cluster of differentiation 39 (CD39) in the immunosuppressive status associated Acute myeloid leukemia (AML)

Role of cluster of differentiation 39 (CD39) in the immunosuppressive status associated Acute myeloid leukemia (AML)

Background: CD39 is an integral membrane protein (ectoenzyeme) that phosphohydrolyzes ATP in a Ca2+- and Mg2+-dependent fashion to yield AMP, Can be viewed as immunological switch, and is expressed in spleen, thymus, lung, and placenta, and in these organs it is associated primarily with endothelial cells and immune cell populations, such as B cells, NK cells, dendritic cells, Langerhans cells, monocytes, macrophages, mesangial cells, neutrophils, and regulatory T cells. Aim of the study is to assess the expression of CD39 on CD4+ CD25high T-reg cells in patients with acute myeloid leukemia to identify its role in immunosuppressive status of AML patients. Methods: Flow cytometric analysis of T-reg cells using CD4 CD25 and CD39 in 60 AML patients and 15 apparently healthy controls with matched age and sex was done. Results: There was a significant difference between cases and controls as regards expression of CD39 on T-helper cells. A highly significant elevation of expression of CD39 has been obtained on T-reg cells in AML patients compared with Controls with increased expression of CD39 on T-reg cells than on helper T cells. Conclusion: CD39 plays a significant role in immunosuppressive status of AML patients particularly with expressing the receptor of α chain of IL-2 (CD25)

Subsequent primary malignancies and acute myelogenous leukemia transformation among myelodysplastic syndrome patients treated with or without lenalidomide.

The few studies that have examined rates of acute myeloid leukemia (AML) transformation in lenalidomide‐treated myelodysplastic syndrome (MDS) patients have been limited to deletion 5q MDS. The association between lenalidomide and subsequent primary malignancies (SPMs) in MDS patients has not been evaluated previously. We conducted a retrospective cohort study to evaluate the risk of both SPM and AML in association with lenalidomide. A cohort of MDS patients (n = 1248) treated between 2004 and 2012 at Moffitt Cancer Center were identified, and incident cases of SPM and AML transformation were ascertained. Using a nested case–control design, MDS controls were 1:1 matched to SPM (n = 41) and AML (n = 150) cases, on age and date of MDS diagnosis, gender, follow‐up time, IPSS, and del (5q). Associations between lenalidomide and (1) SPM incidence and (2) AML transformation were estimated with hazards ratios (HR) and 95% confidence intervals (CIs) in the cohort and odds ratios (OR) in the case–control analysis. SPM incidence did not differ significantly between cohort MDS patients treated with (0.7 per 100 person‐years) or without lenalidomide (1.4 per 100 person‐years) (HR = 1.04, 95% CI = 0.40–2.74), whereas a significantly reduced SPM risk was observed in the case–control sample (OR = 0.03, 95% CI = <0.01–0.63). Lenalidomide was not associated with AML transformation in the cohort analysis (HR = 0.75, 95% CI = 0.44–1.27) or in the case–control analyses (OR = 1.16, 95% CI = 0.52–2.56), after adjustment for potential confounders. Lenalidomide was not associated with increased risk of SPM or AML transformation in a large cohort of MDS patients mostly including nondeletion 5q MDS.

Rac1 GTPase Promotes Interaction of Hematopoietic Stem/Progenitor Cell with Niche and Participates in Leukemia Initiation and Maintenance in Mouse.

Interaction between hematopoietic stem/progenitor cells (HSPCs) with their niche is critical for HSPC function. The interaction also plays an important role in the multistep process of leukemogenesis. Rac1 GTPase has been found to be highly expressed and activated in leukemia patients. Here, by forced expression of constitutively active form of Rac1 (Rac1-V12) in HSPCs, we demonstrate that active Rac1 promotes interaction of HSPC with niche. We then established an active Rac1 associated acute myeloid leukemia (AML) model by expression of Rac1-V12 cooperated with AML1-ETO9a (AE9a) in mouse HSPCs. Compared with AE9a alone, Rac1-V12 cooperated with AE9a (AER) drives an AML with a short latency, demonstrating that activation of Rac1 GTPase in mice promotes AML development. The mechanism of this AML promotion is by a better homing and lodging of leukemia cells in niche, which further enhancing their colony formation, quiescence and preventing leukemia cells from apoptosis. Further study showed that an inhibitor targeting activated Rac1 can increase the efficacy of chemotherapeutic agents to leukemia cells. This study provides evidence that activation of Rac1 promotes leukemia development through enhancing leukemia cells' homing and retention in niche, and suggests that inhibition of Rac1 GTPase could be an effective way of eliminating AML cells. Stem Cells 2016;34:1730-1741.

The Novel FLT3-N676K Mutant Induces Acute Leukemia Independently of the Inv(16) Chimeric Gene CBFB-MYH11

Using high-throughput sequencing, an increased number of gene mutations has been identified in cancer. Among the up to hundreds of acquired mutations in cancer clones, only a few cooperating mutations are believed to be needed for initiation of the malignant disease. Recently, we reported a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in one patient with FLT3-ITD associated acute myeloid leukemia (AML). The FLT3-N676K mutation was more recently identified independently in up to 6% of de novo AML patients with inv(16) by other groups. As FLT3-TKD mutations are strongly associated with inv(16) in AML and particularly FLT3-N676K was found almost exclusively in AML patients with inv(16), this prompted us to investigate the transforming activity of FLT3-N676K and to test whether FLT3-N676K would cooperate with inv(16) to promote AML. First, we analyzed in vivo leukemogenesis mediated by FLT3-N676K. Retroviral expression of FLT3-N676K in myeloid 32D cells induced AML in syngeneic C3H/HeJ mice (n=11/13, latency ~8 weeks), with a transforming activity similar to FLT3-ITD (n=8/8), FLT3-TKD D835Y (n=8/9), and FLT3-ITD-N676K (n=9/9) mutations. Three out of 14 C57BL/6J mice transplanted with FLT3-N676K-transduced primary lineage negative (Lin-) bone marrow cells died of acute leukemia (latency of 68, 77, and 273 days), while none of 16 animals in the control groups including FLT3-ITD and CBFß-SMMHC developed any hematological malignancy. Secondly, co-expression of FLT3-N676K and CBFß-SMMHC did not promote acute leukemia in 3 independent experiments using C3H/HeJ and C57BL/6J mice (n=16). So far only 1 out of 11 C57BL/6J mice co-expressing FLT3-N676K and CBFß-SMMHC developed acute leukemia (AML with latency of 166 days). In comparison with FLT3-ITD, FLT3-N676K tended to result in stronger phosphorylation of FLT3, MAPK and AKT, and diseased animals carrying FLT3-N676K demonstrated much lower frequency of leukemic stem cells in the majority of analyzed cases. Importantly, leukemic cells co-expressing FLT3-N676K and CBFß-SMMHC were still highly sensitive to the FLT3 inhibitor AC220. Taken together, we show that FLT3-N676K mutant is potent to transform murine hematopoietic stem/progenitor cells in vivo independently of the inv(16) chimeric gene CBFB-MYH11. This is the first report of acute leukemia induced by an activating FLT3 mutation in C57BL/6J mice. Moreover, our data suggest that targeting FLT3-N676K mutation may be an attractive treatment option for FLT3-N676K-positive patients without concurrent ITD. Our data emphasize more careful analysis of the cooperating network of mutations identified in AML by high-throughput sequencing.This work was supported by DJCLS (grant: 13/22) and the Deutsche Forschungsgemeinschaft (grant: Li 1608/2-1). KH and ZP were supported by the China Scholarship Council (2011638024 and 201406100008). DisclosuresHeidel:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Association of IDH1 mutations in normal karyotype acute myeloid leukemia samples with higher HIF-1α and VEGF-A expression.

6530 Background: Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes have been reported in normal karyotype (NK) acute myeloid leukemia in association with poor prognosis. Glioma-derived IDH1 mutations have been shown to dominantly inhibit IDH1 catalytic activity, resulting in increased conversion of 2-hydroglutarate (2-HG) from α-ketoglutarate and higher HIF-1α levels. We have previously shown that increased HIF-1α expression in NK-AML samples predicted for poor clinical outcome (Deeb et al Leukemia Research Dec 2010 Epub). Here we examined the IDH1 and IDH2 mutational status of 81 adult NK-AML marrow samples previously characterized for expression of HIF-1α and its downstream target VEGF-A. Methods: Genomic DNA was extracted from marrow mononuclear cells from diagnostic AML samples. High-throughput DNA sequence analysis (PyroMark Q24 Pyrosequencer, Qiagen) performed with custom-designed primers for IDH1 R132 and IDH2 R140 and R172 mutations was used. HIF-1α and VEGF-A expression in AML cells was determined by immunohistochemistry (see Deeb et al, Leukemia Research online 2010) and RQ-PCR analysis. Results: Among these 81 NK-AML samples, 6 (7.4%) contained IDH1-mutations, most in R132H (n=4). Eleven samples (13.6%) contained IDH2-mutations, all in R140Q. IDH1-mutated AML samples expressed higher mean ± SEM levels of HIF-1α (30±18% vs. 10±2.7%) and VEGF-A (62±14 vs. 28±3.5%) protein expression and higher HIF-1α gene (6.9±3.5 vs. 4.0±0.5 fold) expression than IDH1 wildtype samples. In contrast, IDH2-mutated AML samples expressed no detectable HIF-1α protein and lower VEGF-A protein and HIF-1α gene levels than IDH2 wildtype samples. No differences in age, survival, presenting white blood cell count, or marrow blasts was noted based on IDH1/2 status. Conclusions: Our data suggest a potential association between IDH1 mutations and higher HIF-1α and VEGF-A protein expression in AML cells as compared with IDH1 wild-type cells. These results imply that HIF-1α mediated effects may play a role in the leukemogenic effects of IDH1 (but not IDH2) mutations. How HIF-1α expression impacts on metabolic or oxidative stress responses in IDH1-mutated AML warrants further exploration.

Human Immunodeficiency Virus (HIV)-Associated Acute Myeloid Leukemia (AML) in the Era of Highly Active Antiretroviral Therapy (HAART): High Complete Remission Rate, but Poor Tolerance of Consolidation Therapy and Short Disease-Free Survival.

Abstract 4126HIV-infected patients have a significantly longer life expectancy in the era of HAART, but also have an increasing incidence of malignancies. A 2.5-fold increase in leukemia (Patel et. Al., Annals of Int Med. 2008, 148:728-736) has been documented in HIV-infected individuals, including a two-fold increase in AML incidence, with a predominance of M2 and M4 subtypes (Aboulafia et. al., AIDS 2002, 16:865-876 and Sutton et. al., Br J Hematol 2001,112:900-908). To further characterize the biology and outcome of AML in HIV-infected patients in the HAART era, we reviewed the experience with these patients at the University of Maryland Greenebaum Cancer Center and the Johns Hopkins Kimmel Cancer Center. All patients were from greater Baltimore, a city with 37.7 new HIV cases per 100,000 population, the second highest among U.S. cities (Baltimore City HIV/AIDS Statistics Fact Sheet, April 18, 2008. Department of Health and Mental Hygiene, State of Maryland). After IRB approval was obtained, cases were identified by review of all AML patients seen at both institutions between 2002 and 2009. Data regarding patient age, gender, white blood cell count, AML subtype, karyotype, FLT3 mutation analysis, HIV treatment, CD4 count, HIV viral load, AML treatment and treatment outcome were collected. We report 8 HIV-positive AML patients diagnosed and treated in Baltimore, Maryland in the HAART era. Six patients had M2 or M4 subtype (four M2, two M4, one M1 and one M5). One M2 patient had t(8:21) and one M4 patient had inv(16); other abnormalities included del(7p) in one M4 patient, a complex karyotype in two M2 patients, one of whom had treatment-related AML. Three patients had a normal karyotype. FLT3 was wild-type in six patients, and was not studied in two. All but one patient had CD4 counts below 200 cells/μL. HAART was incorporated into the treatment of seven of our eight patients. Multiple HAART regimens were used, and treatment was variable. One patient received HAART during induction, but not consolidation. The other seven patients started or resumed HAART at some point during consolidation. AML was not treated in one patient who had rapidly progressive multiorgan failure at AML presentation. Seven patients were treated with standard induction chemotherapy; six achieved complete remission (CR), while one had refractrory disease. All six patients received consolidation with high-dose cytarabine. Complications during consolidation therapy included prolonged neutropenia in three patients, subdural hematoma secondary to prolonged, severe thrombocytopenia in one patient, and cerebellar toxicity in one patient. Disease-free survival (DFS) ranged from 15 weeks to 20 months, with a median of 10 months. One patient developed diffuse large B-cell lymphoma following completion of consolidation therapy. CD4 count, viral load and treatment of HIV did not correlate with ability to tolerate consolidation, nor with DFS. We conclude that HIV-infected AML patients achieve CR, but have poor tolerance of consolidation therapy and short DFS. Based on our data and the literature to date, CBF abnormalities may occur at an increased rate in these patients, but are not associated with favorable outcomes. Further investigation is needed to substantiate a relationship between HIV and CBF mutations, and to develop treatment strategies for AML therapy, HAART and supportive care to improve outcomes. Disclosures:No relevant conflicts of interest to declare.

A Mouse Model of Evi1-Related Leukemia.

Abstract 1958Poster Board I-981 Background:Evi1 gene is located on chromosome 3q26 and aberrantly expressed in acute myeloid leukemia (AML) patients with or without 3q26 abnormalities, and inappropriate expression of Evi1 associates with poor prognosis. Evi-1 is originally identified in a common integration site of murine leukemia retrovirus and enhanced expression of Evi1 by retrovirus integration is thought to be responsible for leukemogenesis in mouse models. However, retroviral expression of marker genes such as GFP has not induced leukemia even if some clones possessing integration at Evi1 site have been identified. These data indicate that Evi1 requires cooperative factors to induce progressive leukemia, whereas overexpression of Evi1 is enough to lead to clonal expansion of hematopoietic cells. Therefore, identifying genes collaborating with Evi1 is one of the key issues of understanding Evi1-related leukemogenesis. Recently, we demonstrated that a point mutation of the transcription factor AML1 (AML1-D171N) can induce myelodysplastic syndrome (MDS) that progresses to AML in association with overexpression of Evi1 through a mouse bone marrow transplantation model. In that work, we analyzed mice transplanted with BM cells transduced Evi1 alone as control and surprisingly confirmed that all of the mice developed leukemia within 6-11 months after the transplant. In this report, we will describe interesting findings in the novel mouse model of Evi1-induced leukemia. Result: C57BL6/Ly-5.1 murine BM cells infected with retroviruses harboring Evi1 were transplanted into irradiated syngeneic Ly-5.2 mice. The mice looked fine until 5 months, but GFP-positive-Evi1 expressing cells were gradually increased in the peripheral blood (PB), and then the mice died at 6-11 months after the transplantation. The mice showed dysplastic features in myeloid and erythroid cells, increase of blasts in the PB and the BM, hepatosplenomegaly, slight anemia, and some of the mice showed severe leukocytosis. The mice were thought to die of multiple organ failure due to invasion of leukemic cells not due to anemia. The phenotype is different from that of the mouse BMT model expressing Evi1 by retrovirus reported by another group, in which the mice died about 10 months with severe peripheral cytopenia and finally the disease did not progress to AML. Therefore, we considered that Evi1 might have collaborated with unknown genes near retrovirus integration sites in our case and analyzed integration sites by the bubble PCR method. Interestingly, frequent integration at 3' side of C/EBPb gene was found in six mice out of eight mice transplanted with Evi1-transduced BM cells. The integrations were located at 62.5-86.7kb downstream of C/EBPb gene. Next, we examined the expression level of C/EBPb, Tmem189, and Ptpn1, all of which are located near the integration site, and confirmed that C/EBPb showed elevated expression although neither Tmem189 nor Ptpn1 did. We also identified Bcas1, Rps6ka1, and Rapgef4 genes at the retroviral integration site in the other two mice without integration near C/EBPb. Discussion: C/EBPb, also known as NF-IL6, is a transcription factor that specifically binds to an IL1-responsive element in the IL-6 gene and has a role in regulation not only for the IL-6 gene but also for several cytokine genes such as TNF, IL-8, and G-CSF. The hematopoietic progenitor cells of C/EBPb-deficient mice have been reported to respond imperfectly to GM-CSF and G-CSF. Furthermore, C/EBPb is a downstream target of the Ras-Raf pathway. The locus of C/EBPb gene has been reported as a common integration site in the Retrovirus Tagged Cancer Gene Database (RTCGD), which is a database of retroviral insertional mutagenesis in mouse tumors. AKxD mice, Cdkn2a-KO mice, NUP98/HOXD13 transgenic mice, and MYC/Runx2 transgenic mice were reported to develop myeloid or lymphoid leukemia by retroviral insertion into 3' side of C/EBPb gene. In this study, we identified frequent integration at 3' side of C/EBPb gene in Evi1-transduced leukemic cells, whereas we have not identified this locus in AML1-mutants-transduced leukemic cells. Based on these findings and our results, C/EBPb is supposed to be a candidate gene to collaborate with Evi1 in leukemogenesis. Conclusion: We identified involvement of C/EBPb in Evi1-induced leukemogenesis. The novel mouse model that we generated in this study could help understanding the molecular basis of Evi1-related leukemia. Disclosures:No relevant conflicts of interest to declare.

Spontaneous remission in adult acute myeloid leukemia in association with systemic bacterial infection-case report and review of the literature.

Spontaneous remission of acute myeloid leukemia in the adult is a rare event. We report on a 31-year-old male patient suffering from acute myeloid leukemia (AML) M5a according to the French-American-British (FAB) classification with biphenotypic features in flow cytometric examination and severe bacterial infection with group G streptococci at the time of diagnosis. Because of sepsis and stable clinical conditions, chemotherapy was delayed and antibiotics were administered intravenously. Within 6 weeks a spontaneous remission of AML occurred. Remission lasted for about 2 months. At the time of relapse, a change in phenotype of the leukemic blasts with a loss of B-lymphoid markers could be demonstrated by flow cytometry. The patient was treated with an induction therapy according to the multicentric German AMLCG 2000 schedule. To our knowledge, this is the first report of a spontaneous remission in an AML FAB M5a associated with coexpression of myeloid- and lymphoid-associated antigens on the leukemic blasts. Possible mechanisms of this phenomenon are discussed with a review of the literature.

Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay.

The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.