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1627 A Greater Mutational Complexity May Contribute to the Differential Prognostic Impact of Type 1/Type 1-like Versus Type 2/Type2-like Calreticulin Mutations in Primary Myelofibrosis

Myeloproliferative Syndromes: Basic Science
Program: Oral and Poster Abstracts
Session: 635. Myeloproliferative Syndromes: Basic Science: Poster I
Saturday, December 5, 2015, 5:30 PM-7:30 PM
Hall A, Level 2 (Orange County Convention Center)

Paola Guglielmelli, MD, PhD1,2*, Giada Rotunno, MSc1,2*, Tiziana Fanelli, MSc2*, Annalisa Pacilli, PhD1,2*, Laura Calabresi, BiSci1,2*, Alessandro Pancrazzi, BS1,2* and Alessandro M. Vannucchi1,2

1Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
2CRIMM-Centro Ricerca e Innovazione delle Malattie Mieloproliferative, Azienda Ospedaliera-Universitaria Careggi, Florence, Italy

BACKGROUND. Calreticulin (CALR) mutations (mut) are associated with a IPSS/DIPSSplus and mutated ASXL1-independent favorable outcome in patients (pts) with primary myelofibrosis (PMF) compared to those harboring JAK2V617F and MPLW515 mut and those lacking driver mutations (“triple negative”, TN). Spliceosome mut were reported to be less represented in CALR-mut pts compared with other genotypes (Leukemia 2014;28:1472), but no other molecular or cytogenetic correlate is known. It is also debated whether there are prognostic differences between type 1/type 1 like (Ty1; a 52 bp deletion; p.L367fs*46) and type 2/type 2 like (Ty2; a 5 bp TTGTC insertion; p.K385fs*47) CALR mut. Tefferi et al (Blood 2014;124:2465) reported that only Ty1 was prognostically favorable, while either the opposite or no effect was found in other studies (Leukemia 2015; 29:249).

AIMS and METHODS.  To evaluate the prognostic relevance of different types of CALR mut in a series of 396 PMF pts (2008 WHO criteria) seen in our center. CALR mut were analyzed by high-resolution capillary electrophoresis and confirmed by Sanger sequencing. Deep sequencing based on Ion Torrent PGM platform was used to analyze mutations in 17 genes previously shown to recur at discrete frequencies in chronic phase and leukemic transformation (LT) of PMF. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test.

RESULTS.      251 pts (63.4%) harbored JAK2V617F mut, 21 (5.3%) MPLW515 mut, 74 (18.7%) CALR mut, of which 53 (71.6%) Ty1 and 21 (28.4%) Ty2; 50 pts (12.6%) were TN. There was no statistically significant difference between Ty1 and Ty2 for common hematologic and clinical variables. Conversely, both Ty1 and Ty2 differed from JAK2V617F mut counterpart for younger age, lower leukocyte and higher platelet counts; males were more represented among Ty1 pts than other genotypes. Among CALR Ty1, more pts were in lower (low+Intermediate-1) IPSS risk categories (80%) compared to Ty2 (62%; P =0.04). With a median follow-up of 6.4 and 5.4 yrs for CALR mut Ty1 and Ty2 pts, respectively, there were less pts who died among Ty1 (17%) compared to Ty2 (47.6%) (P= 0.007). Median survival of CALR Ty1 was 26.4 yr (range, 15.5-37.3) vs 7.4 yr (4.6-10.2) for CALR Ty2, 7.2 yr (5.7-8.6) for JAK2V617F and 2.0 (1.6-2.4) for TN (P<.0001). The corresponding hazard ratio, taking CALR Ty1 pts as the reference, was 4.9 (95% CI, 1.8-12.9), 6.0 (95% CI, 2.7-13.4) and 20.6 (95% CI, 8.9-48.4) for CALR Ty2, JAK2V617F and TN pts, respectively. In a multivariable Cox proportional hazard regression model, CALR Ty2 (HR 4.4, 95% CI 1.6-11.7), JAK2V617F (HR 3.8, 95% CI 1.7-8.7) and TN (HR 9.2, 95%CI 3.8-22.2) all retained IPSS-independent prognostic impact on survival.

Since some subclonal mutations are prognostically relevant in PMF (Leukemia 2013; 27:1861), we compared the mutational profile of 17 such genes, functionally clustered as epigenetic (DNMT3A, EZH2 TET2, IDH2, IDH1, ASXL1), of the spliceosome (SF3B1, SH2B3 U2AF1, SRSF2) or associated with LT (cKIT, RUNX1, NRAS, KRAS, IKZF1, TP53 CBL) in 44 and 20 CALR mut Ty1 and Ty2 pts, respectively. There was a greater proportion of pts harboring at least one mutated gene among CALR Ty2 than Ty1 (70% vs 56.8%; P= 0.02). Also, the proportion of pts with >2 mut was significantly greater in CALR mut Ty2 compared to Ty1 (40% vs 18%; P= 0.01); in particular, 15% of CALR Ty2 mut pts had >3 mut compared to 2.2% in Ty1 (P=0.04). Frequency of individual mutations in Ty2 vs Ty1 was: epigenetic, 50% vs 50%; spliceasome 30% vs 4.5% (P= 0.009); LT 30% vs 13.6% (P=0.06). Of note, SF3B1 mutations were 10-fold more represented in CALR mut Ty2 (25%) compared to Ty1 (2.2%; P< 0.01). TP53 mutations were found in 10% of Ty2 and 0 in Ty 1.

CONCLUSIONS.      These findings support previous report that the prognostic advantage of CALR mutation in PMF regards only pts harboring type 1/type 1-like abnormalities. A greater mutational complexity involving subclonal mutations, particularly in genes of the spliceosome with an unusually high incidence of SF3B1 mutations, as well as increased number of mutated genes, may contribute to the dismal outcome of CALR mut Ty2 pts.

Disclosures: Vannucchi: Novartis Pharmaceuticals Corporation: Membership on an entity’s Board of Directors or advisory committees , Research Funding , Speakers Bureau ; Baxalta: Membership on an entity’s Board of Directors or advisory committees ; Shire: Speakers Bureau .

*signifies non-member of ASH