Assessment of<i>CALR</i>mutations in myelofibrosis patients, post-allogeneic stem cell transplantation

Abstract

The recent discovery of insertion and/or deletion mutations within the CALR gene in a significant proportion of JAK2- and MPL-unmutated essential thrombocythaemia (ET) and primary myelofibrosis (PMF) patients compels the incorporation of CALR exon 9 mutational analysis into the molecular diagnostic algorithm for these myeloproliferative neoplasms (MPN) (Klampfl et al, 2013; Nangalia et al, 2013; Tefferi & Pardanani, 2014). Identification and monitoring of the common MPN-associated mutations of JAK2 and MPL have been shown to be of prognostic value and enhance assessment of treatment effectiveness in those MPN patients undergoing allogeneic stem cell transplantation (ASCT) (Alchalby et al, 2010a,b). Despite the introduction of JAK inhibitors for the treatment of PMF, ASCT remains the only potentially curative option. Improvements in candidate patient selection and stratification, timing of transplantation and conditioning regimens have significantly reduced the transplant-related morbidity and increased the overall survival for patients undergoing this procedure (McLornan et al, 2012). Post-ASCT monitoring utilizing additional patient-specific markers could provide a more beneficial, individualized approach. As CALR mutations are likely initiating events in MPN pathogenesis, the scenario arises to assess these mutations as potential markers of residual disease in myelofibrosis patients, post-ASCT. Screening for evidence of CALR exon 9 mutations by DNA fragment analysis (Klampfl et al, 2013) of pre-ASCT samples from 15 JAK2 V617F-negative MPN patients identified four CALR mutation-positive patients, all of whom received reduced intensity conditioning (Table 1). CALR mutant allele burden was calculated using area under the peak (mutated/mutated + wild type) x100 and compared to known donor chimerism status performed according to guidelines (Lion et al, 2012). The CALR assay allowed the reproducible detection of approximately 1% mutant allele burden. All four donors were wild type CALR exon 9. A further 14 informative follow up samples evaluated for donor chimerism status were available for retrospective mutant CALR allele burden estimation. Patients 1–3 achieved full engraftment as assessed by donor chimerism (≥99%) at 5, 8 and 14 weeks respectively with very low levels of mutant CALR alleles (<1%) detectable in patients 2 and 3 at these time points (Table 1). Patient 4 failed to achieve full donor chimerism, reflected in levels of mutant CALR alleles of >1% (Fig 1). Loss of engraftment, indicated by falling donor chimerism and mirrored by a significant increase in mutant CALR allele burden, prompted donor lymphocyte infusion (DLI) at week 22 post-ASCT with subsequent attainment of 99% donor chimerism and undetectable mutant CALR alleles. The utility of MPN-specific mutations to monitor disease kinetics post-ASCT has been demonstrated for those patients harbouring a JAK2 V617F mutation. Assessment of the JAK2 V617F allele burden at 1 month post-ASCT can be highly predictive of outcome and risk of relapse (Lange et al, 2013) whereas serial monitoring of JAK2 V617F levels has been used to trigger pre-emptive intervention with DLI (Kröger et al, 2009). Initial data suggests that the CALR mutation status is a valuable prognostic marker in myelofibrosis patients who undergo ASCT, with CALR mutation-positive patients having a superior overall survival and a lower rate of non-relapse mortality (Panagiota et al, 2014). The presence of CALR mutations in the majority of JAK2 V617F- and MPL exon 10-negative MPN patients therefore allows these mutations to serve as disease-specific markers of residual disease in conjunction with donor chimerism analysis. It is envisaged that development of CALR mutation-specific quantitative polymerase chain reaction assays, similar to those established for the JAK2 V617F, will further improve the limit of mutation detection. By demonstrating both persistence and eradication of CALR mutations that correlate with the clinical course, this brief “proof of principle” study provides initial validation of serial monitoring of CALR mutations as a useful adjunct in the assessment of transplant efficacy in myelofibrosis patients. K.H., S.E.L. and K.M. performed laboratory studies. M.F.M. and E.C. provided patient care and clinical information. All authors contributed to manuscript preparation and gave final approval. All authors disclose no conflicts of interest.