Myelofibrosis (MF) is one of the BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs), characterised by clonal expansion of abnormal haematopoietic progenitors and frequently (>50%) associated with the presence of JAK2V617F mutations. At present, the only curative option for MF patients is allogeneic stem cell transplantation (ASCT). A highly sensitive quantification of JAK2V617F mutations can be useful to assess minimal residual disease (MRD) in ASCT. Droplet digital polymerase chain reaction (ddPCR) is a quantitative approach for the detection of rare alleles with a high level of sensitivity and specificity.
The aim of this work was to evaluate the efficacy of a ddPCR JAK2V617F mutation detection assay in monitoring the MRD level at consecutive time points in a small cohort of MF, or secondary to MF, acute myeloid leukaemia patients (AML) (N=9) who underwent ASCT.
The DNA samples were collected serially with a follow-up of 60–2,500 days from ASCT from nine patients (one primary MF, five secondary MF, two AML evolved from a previous MF, and one myelodysplastic neoplasm/myeloproliferative neoplasm). These samples were analysed for JAK2V617F mutations, both by allele-specific oligonucleotide PCR (ASO-PCR) and by a validated ddPCR mutation detection assay (Bio-Rad, Hercules, California, USA). Moreover, the haematologic chimerism was evaluated by the PowerPlex® System (Promega, Madison, Wisconsin, USA). The ddPCR technology is able to separate into a large number of partitions and the reaction is carried out individually in each one. The PCR amplification is prepared using two hydrolysis probes labelled with two different reporter fluorophores, one specific for the wild type allele and the other for the mutated allele. Following PCR, each droplet is analysed in a flow cytometer to determine the fraction of PCR-positive droplets in the original sample.
Our experiments confirmed that the JAK2V617F ddPCR mutation assay was able to detect a low mutation load (≤0.006%), demonstrating much greater sensitivity than ASO-PCR (0.5–2%). In four patients, soon after transplantation, a low level of MRD was observed by ddPCR, which progressively increased during the follow-up and anticipated a decreased level of donor chimerism and a worsening clinical situation. In two patients, who always showed a full donor chimerism and complete haematologic remission of the disease, very low levels of MRD (1–0.006%) could be detected by ddPCR in the 2 years after ASCT. With a longer follow-up, a full molecular remission was achieved, as demonstrated by ddPCR. In two other patients, we observed the achievement of molecular negativity within 90 days post-haematopoietic stem cell transplantation, also shown by ddPCR, and a full donor chimerism. These patients entered a complete haematologic remission of the disease, which still persisted after 1 and 5 years post-transplantation. Interestingly, in one patient whose post-transplant haematopoiesis showed full donor chimerism and negativity for JAK2V617F mutations by ddPCR for 2 years, a late positive signal by ddPCR (0.075%) became apparent. This low-level positivity anticipated an extra-haematologic relapse localised in the skin and bone. This subject was treated by a subsequent second ASCT from the same sibling donor. This second ASCT allowed the clinical and molecular remission to be restored, which indicated negative results since the first evaluation after transplantation (30 days after ASCT).
We can conclude that ddPCR is a sensitive and accurate method of detecting JAK2V617F mutations and a valid tool for MRD monitoring. Moreover, the ddPCR method can also be used to detect other molecular markers, such as MPL, CALR, and ASXL1, allowing the therapeutic effect to extend the monitoring of MRD status also to JAK2V617F negative patients. Nevertheless, the use of this highly sensitive PCR should be considered with caution in the clinical management of transplanted patients to avoid inappropriate use of donor leukocyte infusion and tampering of immunosuppression. To better understand the clinical significance of low mutation load, a larger cohort should be studied with ddPCR.