Highly Sensitive Quenching Probe (QProbe) Method Is Useful to Detect c-Kit Mutation and to Predict Relapse of t(8;21)(q22;q22) Acute Myeloid Leukemia.

t(8;21)(q22;q22) acute myeloid leukemia (t(8;21) AML) show a high rate of complete remission (CR) and prolonged CR duration, especially following consolidation chemotherapy with high-dose cytarabine (HD-ARAC), and are thought to have a better prognosis than other AML patients. Nevertheless, only approximately 50% of patients were alive at 5 years. This suggests that some patients have more aggressive leukemic phenotypes and indicates the need for predictive molecular marker of relapse and treatment optimization with novel and/or more aggressive therapies such as stem cell transplantation.Recently, several groups reported that c-kit mutation (MutKIT) defined an unfavorable subgroup in t(8;21) AML. However MutKIT has been reported approximately 20% at diagnostic samples of core binding factor leukemia. It is possible that MutKIT are more high frequency at relapse sample of t(8;21) AML. In the present study, we analyzed samples collected at diagnosis and relapse to investigate the role of MutKIT s and Flt3 internal tandem duplication (ITD) in t(8;21) AML.

We analyzed MutKIT and Flt3 ITD among 32 t(8;21) AML patients diagnosed between 1991 to 2009 at the Nippon Medical School. All of them were succeeded achieving CR, but 18 patients (56.3%) relapsed, became refractory chemotherapy, and poor prognosis. Exon 8 and 17 of MutKIT were analyzed by direct sequence and QProbe-system (ARKRAY, Inc. Kyoto, Japan). QProbe-system was high sensitivity mutation screening method, detected approximately 1% MutKIT using mutation specific guanine quenching probe (Leukemia Res 32 (2008) 1462–1467). FLT3 ITD was analyzed by PCR amplification.

Using direct sequence, MutKITs were found in 5 (18.5%) of the 27 patients at diagnosis (D816V: 3 patients AN822K: 2 patients), and 7 (46.6%) of 15 patients at relapse. Interestingly 3 patients were detected MutKIT at only relapse (D816V: 2 patients AN822K: 1 patient). All mutations found in exon 17 clustered within the A-loop. Next we analyzed to detected very slight amount of mutation by QProbe-system. N822K were newly found of the 3 patients at diagnosis in spite of negative by direct sequence Finally MutKIT were found 8 (29.6%) at diagnosis. Flt3 ITD were found in 2 (9.5%) of the 21 patents at diagnosis, and none of 15 patients at relapse. 2 patients with Flt3 ITD were not accompanied MutKIT. All of N822K newly found by QProbe-system and Flt3 ITD positive patients were relapsed but turn to mutation negative. To evaluate the importance of MutKIT as a predicted relapse, we analyzed the cumulative incidence of relapse (CIR) and relapse free survival (RFS) of these patients with Kaplan-Meier method. The CIR was higher for patients with MutKIT (p=0.040, Wild type c-kit (WtKIT): 45.8% vs MutKIT: 87.5%). In direct sequencing method, The RFS tend to be shorter for patient with MutKIT, but no significant differences (p=0.260; WtKIT: 30 months vs MutKIT: 11 months).On the other hand, in QProbe-system, the median RFS was shorter for patients with MutKIT (p=0.033; WtKIT: 64 months vs MutKIT: 11 months). In addition of FLT3-ITD analysis, median RFS was shorter for patients with MutKIT or FLT3 ITD (p=0.005; Wild type of both genes: 64 months vs MutKIT or FLT3 ITD: 10 months). Additionally, we compared overall survival (OS) of wild type of both genes with MutKIT or FLT3 ITD. The OS tend to be shorter for patient with MutKIT or FLT3 ITD, but no significant differences.

In this study, we confirmed the prognostic significance of MutKIT in patients with t(8;21) AML when we use the highly sensitive quenching probe method (QProbe-system). We showed that QProbe-system is quite useful in predicting the prognosis of patients with t(8;21) AML and in determining its therapeutic strategy including tyrosine kinase inhibitors and/or up-front stem cell transplantation. We also revealed the important role of MutKIT (46.7%) at relapse, but several questions were still remained. It is unknown for some patients how to gain MutKIT at only relapse and why mutations detected at diagnosis were disappeared at relapse. We need further study to clarify the function and commitment of MutKIT in relapse of t(8;21) AML.

No relevant conflicts of interest to declare.