Development of a Digital-PCR Assay for Screening and Quantitative Monitoring of Calreticulin (CALR) Type-2 Positive Patients with Myelofibrosis Following Allogeneic Stem Cell Transplantation
Authors: Fehse Boris , Badbaran Anita , Christopeit Maximilian , Aranyossy Tim , Ayuk Francis , Wolschke Christine , Kroeger Nicolaus 
Published: October 2019

Background:

The recent discovery of the somatically acquired calreticulin mutation in about 30% of the myelofibrosis patients provided a new diagnostic marker, which can also be used as marker of minimal residual disease (MRD) following curative treatment with allogeneic stem cell transplantation (ASCT). More than 80% of CALR mutations are of two types: type-1 variants result from a 52-bp deletion and produce the protein change p.L367fs*46, and type-2 variants are caused by a 5-bp insertion and produce the protein change p.K385fs*47. Whereas Sanger as well as next-generation sequencing (NGS) readily allow detection of CALR mutations in newly diagnosed patients, their applicability for MRD detection is limited. Real-time quantitative PCR (qPCR) has therefore been suggested as a potential alternative. In order to combine the increased sensitivity of qPCR with the excellent accuracy of digital PCR (dPCR) we developed a duplex dPCR assay detecting the CALR type-2 mutation in combination with its wild-type allele.

Methods and Patients:

To address sensitivity and reliability of the novel dPCR assay we first tested it on a new, UKE-1 derived cell line harboring one copy of the CALR type-2 mutation (UKE-1CALR2), which was established by lentiviral gene transfer and single-cell sorting. We generated serial dilutions of UKE-1CALR2 in buffy-coat (BC) cells, isolated DNA and submitted it to dPCR. Using 120 ng EcoRI-restricted genomic DNA we detected up to one UKE-1CALR2 in 10,000 BC cells (0.01%) indicating excellent sensitivity. As expected the detection limit could be further increased by applying the p.K385fs*47-specific dPCR as a singleplex assay.

Results:

Using our new technique we next performed MRD analysis in CALR+ patients who underwent allogeneic stem cell transplantation and compared results with respective qPCR data. Out of 143 patients with myelofibrosis who underwent allogeneic SCT 92 were JAK2V617 positive, 4 MPL positive and 35 CALR positive. Out of these 35 patients 21 harbored the CALR type-1 and eight the CALR type-2 mutation. In seven out of those eight patients both qPCR and digital PCR could be applied for MRD monitoring after SCT. In 3/7 patient qPCR as well as dPCR were negative on day +20, +100 and +180 after transplantation, respectively. In 2 patients dPCR remained positive at days +100 and +180 days before turning negative, whereas qPCR was already negative. In one patient dPCR remained positive 6 months after SCT, whereas qPCR was negative since day +80. None of the above patients had experienced clinical relapse. In contrast, there was one patient who relapsed 28 months after transplantation. That patient was MRD-negative by qPCR until one months before relapse, whereas dPCR, which initially also became negative after SCT (day +180), converted to positivity already 1 year after transplantation and was steadily increasing until clinical relapse.

Conclusion:

Our data indicates that the new CALR type-2-mutation specific dPCR assay combines excellent accuracy with high sensitivity thus allowing the monitoring of deep molecular remission and the early detection of MRD in relapsing patients with myelofibrosis after stem cell transplantation

* BF and AB contributed equally

Disclosures

No relevant conflicts of interest to declare.