Somatic Mutations and Inborn Variants in Exon 12 of ASXL1 in Different Myeloid Neoplasms

ASXL1 encodes a chromatin-binding protein that is member of the Polycomb group of proteins. Mutations in Exon 12 of ASXL1 have recently been described in a number of different myeloid malignancies. Reported frequencies in the respective entities are highly variable and may rely on in part on the difficulty to differentiate between somatic mutations and inborn polymorphisms. Especially the frequent variant G646WfsX12 of ASXL1 has been described to be most likely a polymorphism or even a technical artefact. Aim: The aim of this study was to evaluate frequencies of ASXL1 mutations in different myeloid entities, compare the mutation types and exclude polymorphisms. Methods: Exon 12 of ASXL1 was analyzed by direct Sanger sequencing. In total 1,053 patients with myeloid malignancies were analyzed. In detail, the cohort consisted of 657 patients (pts) with acute myeloid leukemia (AML), 76 pts with myelodysplastic syndrome (MDS), 21 pts with myeloproliferative neoplasias (MPN) not further classified, 14 pts with MDS/MPN overlaps, 229 pts with chronic myelomonocytic leukemia (CMML), 40 pts with chronic myeloid leukemia in blast crisis (CML-BC), and 16 pts with primary myelofibrosis (PMF). Female/male ratio was 440/613 and age ranged from 13.3–100.4 years (median, 68.0 years). In addition, we analyzed a healthy control cohort from the KORA (Cooperative Health Research in the Region of Augsburg) survey S4, which consists of 491 cases which were matched to the leukemia samples with respect to sex and age. Results: In total 273 ASXL1 variants were detected. All variants were detected with a mutation/wildtype load of 40–50%. 23 of these have been described to be rare polymorphisms (G652S: n=1, L815P: n=2, N986S: n=2, E1102D: n=15, A1312V: n=1, M1249V: n=1, S1253S: n=1) and were excluded from further calculations. Of the remaining 250 variants most were frameshifts (n=187, 74.8%) and stop mutations (n=46, 18.4%). Thus, 93.2% had disruptive character. Only 17 (6.8%) were missense mutations. The most frequent mutation was G646WfsX12 detected in 106 cases (42.4%). This variant was detected with similar frequency in all entities (AML: 49/657, 7.5%; MDS: 9/76, 11.8%; MPN: 2/21, 9.5%; MDS/MPN: 3/14, 23.4%; CMML: 37/229, 16.2%, CML-BC: 4/40, 10%, PMF: 2/16, 12.5%). G646WfsX12 has repeatedly been discussed not to be a somatic mutation but more likely a polymorphism or even a sequencing artefact due to an 8 base-pair guanine homopolymere at that site. Unfortunately, in none of these cases a healthy control tissue or a complete remission sample was available. However, we excluded a polymorphic character of G646WfsX12 in three ways: 1) a technical problem was excluded as all G646WfsX12 cases remained positive and all G646wt samples remained negative upon repeated testing. 2) In the KORA cohort of 491 samples one (0.2%) G646WfsX12 positive sample with mutation load of only 10% was identified. This incidence is significantly below that observed in myeloid malignancies and because of the low mutation load this case was interpreted as having a small premalignant clone or an early yet undetected clonal disease. 3) G646WfsX12 occurs due to three different mechanisms at the nucleic acid level: c.1934dupG (n=101), c.1927_1928insA (n=2), or c.1935dupT (n=3). Thus, we strongly believe that G646WfsX12 has to be regarded as a somatic mutation. In contrast, the so far undescribed G652S, T688M, and P722R variants were detected with a 50% load in 2, 1, and 1 KORA samples, respectively, and may be addressed as new rare germline variants. Other missense mutations observed in the patient cohort were not detected in the control cohort and were regarded as somatic mutations. Consequently, the total frequency of ASXL1 mutations in the different entities was determined as follows: AML: 100/657, 15.2%; MDS: 24/76, 31.6%; MPN: 7/21, 33.3%; MDS/MPN: 6/14, 46.8%; CMML: 94/229, 41.0%, CML-BC: 13/40, 32.5%, PMF: 6/16, 37.5%). Summary:ASXL1 mutations are frequent in all myeloid malignancies with the highest incidences in CMML, MDS/MPN overlap and CML-BC. There are no disease specific ASXL1 mutation patterns. Disrupting mutations (frameshift and nonsense) are the most common mutation types (93.2%). G646WfsX12 is the most frequent alteration and was verified here as a true somatic mutation. Missense mutations are rare and should be regarded carefully as possibly innocent bystanders or very rare polymorphisms.

Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.