Aims We investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis (PMF).

Methods Pyrosequencing was used to detect JAK2 ^V617F mutational status and measure allele burden, while MPL (exon 10) mutations were analysed by PCR assay and then by direct sequencing. CALR exon 9 mutations were first screened for length changes by GeneScan followed by sequencing. The allele burden of the mutated CALR gene was measured by pyrosequencing. The EEC assay was conducted using a serum-free culture system.

Results The frequencies of the three driver mutations and triple-negative status were similarly distributed between pre-PMF and overt PMF patients, except that pre-PMF patients had a higher incidence of CALR type 2/type-2 like mutations and a lower JAK2 ^V617F allele burden. EEC growth and CALR mutations conferred favourable overall survival (OS). A lower JAK2 ^V617F allele burden and grade 3 bone marrow fibrosis were associated with shorter OS and decreased leukaemia-free survival (LFS). Type 2/type 2-like CAL mutations were associated with better LFS compared with type1/type 1-like mutations. Patients with triple-negative mutation status had significantly worse OS and LFS. The allele burden of CALR mutations remained unchanged, while some JAK2 ^V617F mutations showed clonal expansion in patients during secondary acute myeloid leukaemia transformation.

Conclusions Our study showed that EEC growth, a higher JAK2 ^V617F allele burden and CALR mutations, especially type 2, were independent predictors for better outcomes in PMF. The allele burden of CALR mutations remained stable, but the allele burden of JAK2 ^V617Fmutations was variable during leukaemia transformation.