Mutations in CALR Are Associated with Del(13q) and Are Mutually Exclusive of Monosomy 7 in BCR-ABL1-Negative MPN
Authors: Jeromin Sabine , Haferlach Claudia , Meggendorfer Manja , Kern Wolfgang , Haferlach Torsten , Schnittger Susanne 
Published: October 2019

Abstract
Introduction: Mutations (mut) in the Calreticulin gene (CALR) were recently described in BCR-ABL1-negative myeloproliferative neoplasms (MPN). They occur frequently in essential thrombocythemia (ET; 15-30%) and primary myelofibrosis (PMF; 25-35%), but not in cases with polycythemia vera (PV). Other well-known mutations in ET and PMF are JAK2V617F (50-60%) and MPLW515 (5-10%). These three mutations are nearly mutually exclusive of each other. Furthermore, also chromosomal aberrations are frequently detected in PMF (40%), whereas they are rare in ET (3%). However, no association of CALRmut with any cytogenetic aberration has been reported yet.
Aims: Investigate CALRmut and JAK2V617F in cytogenetic subgroups of BCR-ABL1-negative MPN.
Patients: We studied 220 patients with cytomorphological confirmed BCR-ABL1-negative MPN excluding PV and with following chromosomal aberrations: del(20q) (n=64), trisomy 8 (+8; n=57), +1q (n=30), del(5q) (n=25), del(13q) (n=23), monosomy 7/del(7q) (-7/del(7q); n=21). Of these 220 cases, 25 and 12 patients were in accelerated and blastic phase, respectively. The cohort comprised 85 females (38.6%) and 135 males (61.4%). Median hemoglobin (Hb) level was 12.3 g/dl (range: 6.0 - 17.8 g/dl, n=167), platelet count 277,500x109/L (range: 16,000 –1,877,000x109/L; n=170) and white blood cell (WBC) count 16,000 x109/L (range: 1,600 -305,000 x109/L, n=181).
Methods: Chromosome banding analysis was performed using standard G-banding. Screening for CALRmut was done by fragment analysis and subsequent Sanger sequencing of positive cases. JAK2V617F and MPLW515 were analyzed by melting curve analysis. MPLW515 was only analyzed in CALR/JAK2V617F-negative patients.
Results: All 220 patients were screened for CALRmut and JAK2V617F. The frequency of CALRmut was 16.8% (37/220) and of JAK2V617F 58.2% (128/220). Mutations in these two genes were mutually exclusive (p<0.001). MPLW515 occurred in 3/55 (5.5%) of CALR/JAK2V617F-negative cases. CALR mutations presented as type 1 (p.Leu367Thrfs*46) in 56.8% (21/37) and as type 2 (p.Lys385Asnfs*47) in 27.0% (10/37) according to the nomenclature of Klampfl et al. (NEJM, 2013). The remaining 6 cases represented different mutation types all resulting in the same C-terminus of the mutated CALR protein.
Analysis of gene mutations and cytogenetic aberrations showed that CALRmut associated significantly with del(13q) (with vs. without: 10/23, 43.5% vs. 27/197, 13.7%, p=0.001), whereas they were rare in +8 patients (2/57, 3.5% vs. 35/163, 21.5%, p=0.001; Figure 1). Additionally, no CALRmut was detected in patients with -7/del(7q) (0/21, 0% vs. 37/199, 18.6%, p=0.029). For JAK2V617F an association with del(20q) was detected (44/64, 68.8% vs. 84/156, 53.8%, p=0.050). Exclusion of MPN in accelerated or blastic phase from analyses resulted in the same associations between distinct cytogenetic abnormalities and CALRmut. Only the negative correlation to chromosome 7 aberrations lost its significance, probably due to low case numbers (0/9, 0% vs. 28/174, 16.1%, n.s.). For JAK2V617F the association with del(20q) was still present, even though the statistical significance was lost (37/55, 67.3% vs. 77/132, 58.3%, n.s.). Furthermore, we analyzed the distribution between type 1 and type 2 CALR mutations (n=31) in cytogenetic subgroups. Type 1 mutations were more frequent in cases with del(13q) (9/9, 100.0% vs. 12/22, 54.5%, p=0.030), whereas the frequency of type 2 mutations was higher in del(20q) (6/10, 60.0% vs. 4/21, 19.0%, p=0.040).
Analysis of clinical data showed that CALRmut vs. wild-type patients had lower Hb levels (mean: 10.9 vs. 12.1 g/dl, p=0.019) and JAK2V617F cases had lower WBC counts vs. JAK2V617F-negative patients (19,308 vs. 30,786 x109/L, p=0.041). Additionally, Hb levels were higher in JAK2V617F patients compared to cases with CALRmut (12.2 vs. 10.9 g/dl, p=0.017).
Conclusions: The highest CALR mutation frequency was observed in del(13q) cases (43.5%) and nearly all of them were type 1 mutations (90.0%). In contrast, CALRmut were rare in the cytogenetic subgroups with +8 and -7/del(7q). The highest JAK2V617F frequency was detected in patients with del(20q) (68.8%). Thus, in PMF and ET specific patterns are detectable based on cytogenetic and molecular data.
Figure 1: Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns. Figure 1:. Distribution of gene mutations in cytogenetic subgroups. The percentage of each mutation is depicted in the columns.

Disclosures
Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.