Homozygosity of Mutated Calreticulin in Myeloproliferative Neoplasms By an Acquired Chromosome 19p UPD Is Associated with Deletions in the Long Arm of Chromosome 5 and SF3B1 Mutations

Background: CALR is mutated in about 70% of patients with essential thrombocythemia and primary myelofibrosis lacking mutations in JAK2 and MPL. High mutational loads (≥ 60%) are seen less frequently in CALR mutated cases than in JAK2 or MPL mutated cases. Especially for JAK2 uniparental disomy (UPD) of 9p24 is commonly detected leading to homozygosity of JAK2V617F and progressive disease. Until now, in CALR mutated patients UPD has only rarely been detected and data on these cases are scant.

Aim: (1) Detection of cases with CALR UPD in a cohort of MPN cases with high CALR mutation loads and/or progressive disease, (2) cytogenetic and molecular genetic characterization of these cases in comparison to CALR mutated patients without UPD.

Patients and Methods: Overall 50 cases with a CALR mutation were analyzed by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). Caseswere selected as follows: (1) mutation load ≥ 60% determined by gene scan analysis (n=26), (2) progressive disease (accelerated phase: n=7, blast crisis: n=5) according to cytomorphology and/or (3) displaying mutations in ASXL1, SRSF2, EZH2 and/or IDH1/2 as markers for progression (n=12). Additionally, amplicon next-generation sequencing was performed to detect mutations in ASXL1, CBL, DNMT3A, EZH2, IDH1/2, KRAS, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53 and U2AF1. Variants of unknown significance were excluded from statistical analysis.

Results: 11/50 cases (22%) with UPD were identified by array CGH. While the type 1 (c.1099_1150del52) CALR mutation was the most frequent mutation in cases without UPD (25/39, 64%), type 1 mutation was rare in CALR UPD cases (1/11, 9%; p=0.009). Thus, in cases with UPD, mainly the type 2 (c.1154_1155insTTGTC) mutation (5/11, 45%) and rare mutation types were detected (5/11, 45%). Of note, the only case with CALR UPD showing the type 1 mutation was found to harbor a TET2 mutation and UPD encompassing 4q23q28, which includes the TET2 gene. As the CALR mutation load was clearly lower compared to the TET2 mutation load (50% vs. 100%), the CALR mutation might only belong to a subclone in this case.

Cases with CALR UPD showed a higher mutation load than cases without UPD (mean: 77% vs. 48%, p<0.001) and presented more frequently with MPN in acceleration (5/11, 46% vs. 6/39, 15%, p=0.048). Almost all of the patients (4/5, 80%) in accelerated phase with UPD showed a mutation load ≥60%.The remaining case was the patient with concomitant TET2 and CALR UPD.

Regarding chromosomal aberrations, del(13q) (12/50, 24%), del(5q) (9/50, 18%), del(20q) (6/50, 12%) and gain of 1q (5/50, 10%), have been detected as recurrent abnormalities by array CGH analyses in the total cohort. Del(5q) showed a trend to be more frequent in cases with UPD (4/11, 36% vs. 5/39, 13%; p=0.093). Corresponding to the advanced disease state of the selected cohort, mutation analyses revealed a high frequency of ASXL1 mutations (44%), followed by mutations in TET2 (19%), EZH2 (13%), TP53 (13%), U2AF1 (9%), NRAS (9%) and SF3B1 (7%). Interestingly, SF3B1 mutations were exclusively detected in cases with CALR UPD (3/11, 27% vs. 0/34, 0%; p=0.012), whereas ASXL1 mutations tended to be more frequent in cases without UPD (19/37, 51% vs. 2/11, 18%, p=0.083). TP53 mutations were also detected more often in the subgroup of cases with CALR UPD, although this was not statistically significant (3/11, 27% vs. 3/35, 9%). Additionally, TP53 mutations showed a significant correlation to del(5q) (TP53 mutation in cases with del(5q): 5/9, 56% vs. TP53 mutation in cases without del(5q): 1/37, 3%, p=0.001). Comparison of the CALR mutation loads with mutation loads of accompanying mutations revealed that in most cases, the CALR mutation is found in the main clone.

Conclusions: In cases with high CALR mutation loads and/or progressive disease UPD of 19p13 is rather common (22%). These cases show a distinct pattern of chromosomal aberrations and additional molecular mutations, as they are associated with del(5q) and mutations in SF3B1 and TP53, whereas ASXL1 mutations are less frequent. Due to the limited number of cases, these results have to be verified in future analyses.