The prognostic advantage of calreticulin mutations in myelofibrosis might be confined to type 1 or type 1-like CALR variants

To the editor:

Approximately 90% of patients with primary myelofibrosis (PMF) harbor JAK2 (58%), CALR (25%), or MPL (7%) mutations.^1-3 CALR-mutated PMF patients are younger than their JAK2-mutated counterparts, and they display higher platelet count, lower leukocyte count, higher hemoglobin level, lower incidence of spliceosome mutations, and longer survival.³  More than 80% of CALR mutations constitute 1 of 2 variants: type 1, a 52-bp deletion (p.L367fs*46), or type 2, a 5-bp TTGTC insertion (p.K385fs*47).¹  Type 2, as opposed to type 1 CALR variant has been associated with higher platelet count in essential thrombocythemia⁴  and shorter survival in PMF.⁵  Furthermore, data are emerging that suggest functionally relevant structural differences between type 1 and type 2 CALR variants, including a higher α-helix content of the mutant C terminus in type 2, which is similar to what is seen with wild-type CALR, compared with type 1, which is significantly lower.⁶ 

In the current study, we used statistical models⁷  to calculate helix propensity for 31 unique amino acid sequences that were altered by CALR mutations, and we used the results to subclassify non-type 1/2 CALR mutations into “type 1-like” and “type 2-like” variants. Calculation of helix propensity was performed using AGADIR,⁷  which is a statistical approximation algorithm. Survival was calculated from the date of referral to date of death or last contact. Blast transformation (BT) replaced death as the uncensored variable to estimate leukemia-free survival. Cox proportional hazard regression model was used for multivariable analysis.

A total of 532 PMF patients were screened for JAK2, CALR, and MPL mutations; the respective mutational frequencies were 58%, 24.6%, and 7.3%. Among the 131 CALR-mutated cases, 98 (74.8%) harbored type 1, 15 (11.5%) type 2, and 18 (13.7%) other variants. Based on predicted helix propensity scale (supplemental Table 1, available on the Blood Web site), the “other” CALR mutations were subclassified as type 1-like (n = 12) or type 2-like (n = 6). The AGADIR-derived predicted helix propensity scale was 29.69 for wild-type CALR and 8.6 or 34.17 for type 1 and type 2 mutant CALR, respectively; accordingly, CALR variants with values close to or above the value for wild-type CALR were classified as type 2-like (range 26.47-36.12), and those with values close to or below the value for type 1 were classified as type 1-like (range 2.11-17.3).

A comparison of type 1/type 1-like (n = 110) and type 2/type 2-like (n = 21) CALR mutations showed the latter to be associated with higher dynamic international prognostic scoring system (DIPSS)-plus⁸  score (P = .01), EZH2 mutations (P = .0005), leukocyte count >25 × 10⁹/L (P = .0003), higher circulating blast percentage (P = .02), and palpable spleen size >10 cm (P = .008) (supplemental Table 2). Comparison of type 1/type 1-like CALR and JAK2 mutations (n = 309) showed the former to be associated with younger age, higher platelet count, higher hemoglobin level, lower leukocyte count, and lower DIPSS-plus score (P < .01 for all comparisons; supplemental Table 2). None of these associations was evident during comparison of type 2/type 2-like CALR with JAK2 mutations.

Median follow-up for the core group of 532 PMF patients was 3.4 years; during this period, 327 (61.5%) deaths and 54 BTs (10.2%) were recorded. Survival was similar between patients with type 1 and type 1-like (P = .8) and between type 2 and type 2-like (P = .63) CALR mutations; in contrast, survival was significantly shorter in patients with type 2 (HR 2.4, 95% CI 1.2-4.8) and type 2-like (HR 3.2, 95% CI 1.0-10.6), when compared with those with type 1 CALR mutations. Similarly, survival was shorter in patients with type 2/type 2-like vs type 1/type 1-like CALR mutations (P = .003; HR 2.5, 95% CI 1.4-4.5) (Figure 1), and the difference remained significant when analysis was adjusted for age (P = .047), ASXL1 (P = .003), or EZH2 (P = .001) mutations. Compared with JAK2-mutated cases (n = 309), survival was longer in patients with type 1/type 1-like (HR 0.4, 95% CI 0.3-0.5) but not in those with type 2/type 2-like (HR 0.9, 95% CI 0.5-1.6) CALR mutations (Figure 1); the difference in survival between JAK2 and type 1/type 1-like CALR-mutated cases remained significant (P < .01) when analysis was adjusted for age, ASXL1 or EZH2 mutations, or DIPSS-plus score.

Additional studies are needed to confirm or further refine our operational classification of CALR mutations into 2 prognostically distinct subgroups. In the meantime, our observations highlight the importance of clarifying the specific types of CALR mutations when discussing prognostic implication, especially with patients.