Improved FLT3 internal tandem duplication PCR assay predicts outcome after allogeneic transplant for acute myeloid leukemia

Abstract

Patients with acute myeloid leukemia (AML) who harbor internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene carry a poor prognosis. Although allogeneic transplantation may improve outcomes, relapse occurs frequently. The FLT3/ITD mutation has been deemed an unsuitable minimal residual disease (MRD) marker because it is unstable and because the standard assay for the mutation is relatively insensitive. The FLT3 mutation is undetectable by PCR at pre- or post-transplant time points in many FLT3/ITD AML patients who subsequently relapse after transplant. We report the application of a new technique, tandem duplication PCR (TD-PCR), for detecting MRD in FLT3/ITD AML patients. Between October 2004 and January 2012, 54 FLT3/ITD AML patients in remission underwent transplantation at our institution. Of 37 patients with available day 60 marrow samples, 28 (76%) were assessable for MRD detection. In seven of 28 patients (25%), the FLT3/ITD mutation was detectable by TD-PCR but not by standard PCR on day 60. Six of 7 patients (86%) with MRD by TD-PCR have relapsed to date compared with only 2 of 21 patients (10%) who were negative for MRD (P = .0003). The ability to detect MRD by this sensitive technique may provide an opportunity for early clinical intervention.

Keywords: Acute myeloid leukemia (AML); Bone marrow transplant; FLT3; Internal tandem duplication (ITD); Minimal residual disease (MRD); Tandem duplication PCR (TD-PCR).

Figure 1

MRD results and outcomes for 28 evaluable patients.

Figure 2

A) Relapse Free Survival in 54 FLT3/ITD AML Patients Undergoing Transplant at a Single Institution. B) Overall Survival in 54 FLT3/ITD AML Patients Undergoing Transplant at a Single Institution.

Figure 2

A) Relapse Free Survival in 54 FLT3/ITD AML Patients Undergoing Transplant at a Single Institution. B) Overall Survival in 54 FLT3/ITD AML Patients Undergoing Transplant at a Single Institution.

Figure 3

A) Relapse Free Survival by Day 60 TD-PCR Status. B) Overall Survival by Day 60 TD-PCR Status.

Figure 3

A) Relapse Free Survival by Day 60 TD-PCR Status. B) Overall Survival by Day 60 TD-PCR Status.

Figure 4. TD-PCR detected MRD at 2 months post-transplant and predicted relapse

In the case of patient 2, standard PCR showed relapse of the original ITD mutant migrating at 420 bases at 13 months post-transplant (C), but not at 2 months (A) or 12 months (B) post-transplant. TD-PCR using primer pairs TD3 and TD7, however, detected the ITD mutant at 2 (D and G), 12 (E and H) and 13 months (F and I) post-transplant. TD-PCR products from the relapse specimen at 13 months were diluted 30 fold before electrophoresis. The red vertical lines indicate off-scale peaks. Arrows indicate amplicons from the same ITD mutant (with sizes in nucleotides provided in parentheses). RFU, relative fluorescence units.