PCR-Based MRD Detection in AML with Normal Karyotype or Other intermediate Risk Aberrations by the Use of FLT3-LM, NPM1-Mutations, MLL-PTD or WT1-Overexpression.

The prognostic value of MRD detection in AML has been shown for PML-RARA, AML1-ETO, and CBFB-MYH11positive AML, a group which accounts for only 20–25% of AML. The largest group of AML is represented by cases with normal karyotype or unbalanced intermediate risk group karyotypes. In this group different molecular mutations occur, which are associated with heterogeneous clinical outcome. Thus, different MRD patterns can be anticipated. To expand the spectrum of molecular targets in intermediate risk AML, we used MLL-PTD, FLT3-LM, and NPM1 which are detectable in 45–50% of all intermediate risk group pts for MRD detection. In addition, overexpression of WT1 for MRD detection was used to even include pts. without detectable mutations. In total 996 samples (spl) of 234 patients (pts) were analysed by quantitative real time PCR. For MLL-PTD (321 spl of 78 pt), and WT1 (336 spl of 66 pts) universal assays were used. For NPM1 mutation specific assays (182 spl of 54 pts (42 x type A, 2 x type B, 7 x type D, 3 x rare not yet defined types)) and for FLT3-LM (161 spl of 18 selected pts) patient specific assays were used. All assays were RNA based. Sensitivity of the assays were between 1:100.000 to 1:1.000.000 for the FLT3-LM and NPM1, depending on pts specific assay. Sensitivity was 1:10.000 to 1:100.000 for MLL-PTD due to low background levels detectable in healthy controls. The sensitivity of WT1 was relatively low with 1:100 at most, as there was no high WT1 expressor at diagnosis in this cohort. With all markers the clinical course of the disease was clearly be reflected and all 84 relapses were detectable due to recurring high expression rates. In 17 cases, were samples 2–4 months before clinical relapse were available relapses were predictable based on increasing transcript levels. Five different follow up intervals (int) were defined:

1. up to day 21;

2. days 22–60;

3. days 61–120;

4. day 121–365,

5. later.

The log change from diagnosis to defined follow up intervals was analyzed. A rapid decline of median transcript ratios in the NPM1 group (int 1: 2 log; int 2 to 4: 4 log) was observed. Relapses in the NPM1 group occurred earliest after one year. AML with FLT3-LM similarly showed good responses with 1–2 log decreases in int 1 and 2 and 3 log in int 3 and 4. Also this group revealed the first relapses in int 5. The MLL-PTD group was characterized by slow response rates with only 0.2 log reduction in int 1 and 2 and hardly 3 log in int 3. In this group many relapses occurred in int 4 and 5. These data reflect the biological differences of these molecular subgroups: NPM1 as a favourable group, FLT3-LM as a slightly unfavourable group with good response rates but high relapse rates, and MLL-PTD as an entity with bad prognosis due to poor response rates and high relapse rates. Due to low sensitivity WT1 reflected only 0.2 log in interval 1 up to 2 log in interval 3. Seven cases were analyzed in parallel for WT1 and MLL-PTD, 6 for WT1 and FLT3-LM, and one for FLT3-LM and MLL-PTD. Although the correlation of parallel assessment was high (R=0.993) the median differences of log changes of FLT3-LM and NPM1 was one log larger than for MLL-PTD and three log larger than for WT1, depending on the initial sensitivity of the assays. In conclusion:

1. MRD detection is feasible in the karyotypically intermediate risk group.

2. it nicely reflects biological difference in this group

3. NPM1, MLL-PTD and FLT3-LM are better MRD-markers than WT1, which may only be investigated in cases without any other available marker.