Quantification of Minimal Residual Disease (MRD) in MLL-PTD Positive AML Can Be Used for Prognostication in AML with Normal Karyotype.
Authors: Weisser Martin , Schoch Claudia , Kern Wolfgang , Hiddemann Wolfgang , Haferlach Torsten , Schnittger Susanne 
Published: October 2019

Abstract
Partial tandem duplications of the MLL gene (MLL-PTD) occur with the same frequency of approximately 6% as PML-RARA, AML1-ETO, and CBFB-MYH11 that have previously been shown to be relevant markers for detection of minimal residual disease (MRD). MLL-PTD has a frequency of 11% in the normal karyotype and here it is associated with an unfavourable prognosis. In the normal karyotype AML PCR-based MRD detection was not applicable so far, and thus MLL-PTD may be a promising MRD marker for this large intermediate AML subtype. So far, the clinical significance of the expression level of MLL-PTD at diagnosis or MRD during the course of therapy have not been investigated. Using real time RT-PCR MLL-PTD expression levels were quantified relative to the control gene ABL. Quantitative data were available from 143 patients at diagnosis. The range of the MLL-PTD/ABL ratios at diagnosis was 9.6–1255 (median: 149). The levels of MLL-PTD expression in 50 healthy control samples were between 0 and 0.08 (median 0.02). In total 47 of these patients with a median follow up sample number of 4 per patient (range 2–17) were evaluated for MRD during and after therapy. Twenty-one patients were evaluable for MRD analysis after 2 months after start of therapy. Thirty-one patients were evaluable for MRD analysis after 4 and 6 months after start of therapy. A 2-log reduction of MLL-PTD expression after 2 months, 4 months and 6 months was a significant prognostic factor for overall survival (OS: p=0.0179, p=0.035, p=0.048, respectively) as well as for event free survival (EFS: p=0.0047, p=0.037, p=0.020, respectively). There is no evidence that a 3-log reduction after 2 and 4 months improves OS significantly (p=0.62 and 0.24 respectively). This was mainly due to the low number of patients that achieved a 3 log decrease (6/21 and 10/31 respectively). In contrast, at 6 months a 3-log decrease had a significant impact on OS and EFS (p=0.049 and p=0.030, respectively). Neither the expression levels at diagnosis nor fusion type (involved MLL exons) showed prognostic significance. However, age above 60 years and leukocytosis above 50 G/l were associated with a worse prognosis (p=0.028 and p=0.0035, respectively). Overall, the MLL-PTD expression levels correlated well to the course of the disease and a molecular relapse was detected before clinical manifestation in 2 patients based on increasing expression ratios. Another 15 cases were assessed at clinical relapse when expression levels were again in the range of the diagnostic sample. Here the median interval between the last follow up and relapse was too long (9.2 months) for early detection of relapse. Thus, more frequent follow up analyses at least every three months should improve the early detection rate. In conclusion, this analysis confirms the significance of MRD levels as a prognostic factor in this AML subtype. 1) MLL-PTD can be applied in 11% of intermediate risk group AML. 2) Patients with an apriori high risk for relapse can be identified on the basis of a 2 log reduction. 3) Relapses can be early detected based on increasing expression ratios.