Acquisition of TP53 Mutations in Chronic Lymphocytic Leukemia (CLL) Patients During the Course of Disease Is Associated with an Unmutated IGHV Status and Mutations in XPO1 and SF3B1

In chronic lymphocytic leukemia (CLL), an association between TP53 mutations (TP53mut) and a poor prognosis has been reported. Two hypotheses are discussed regarding the occurrence of TP53mut during the course of disease: 1. TP53mut are already present at first diagnosis in a small subclone, which selectively grow despite treatment, and 2. TP53mut occur de novo during disease progression.

Investigation of the acquisition of TP53mut in previously TP53wt CLL cases during the course of disease.

The discovery cohort included 20 cases, which were selected based on negativity for TP53mut at first diagnosis and acquisition of TP53mut during follow-up (7 female, 13 male patients; median age: 60.7 yrs, range: 41.3–76.6 yrs). 16/20 patients were treated according to various regimens (including fludarabine and rituximab). TP53 analyses of the discovery cohort were performed by DHPLC and subsequent direct Sanger sequencing and, additionally, by 454 deep-sequencing analyses (454 Life Sciences, Branford, CT) enabling a higher sensitivity. Further, a comprehensive molecular characterization included in addition to TP53 the following markers: IGHV mutation status, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88. All markers had been analyzed both at initial diagnosis and at the first follow-up time point that revealed an acquired TP53mut. A second cohort (n=326) was used for comparison of the mutation frequencies of IGHV, NOTCH1, FBXW7, SF3B1, XPO1, and MYD88.

Next-generation sequencing confirmed 18/20 cases as wild-type at first diagnosis (median coverage: 733-fold; range: 378-1, 369; sensitivity: <2.0%). However, in 2/20 cases deep-sequencing revealed a low-level clone of TP53 aberration (4.0% and 7.0%) that had not been detected by DHPLC and Sanger sequencing. Both patients with a detectable TP53mut at first diagnosis were excluded from further analyses.

New TP53mut occurred at a median of 39.0 months after first diagnosis (range: 13.7–68.4 months). The patients harbored between 1 and 4 TP53mut (mean: 1.3) with a median mutation load of 18.0% (range: 2.0–93.0%). Of note, 15/18 patients had received therapy prior to the acquisition of the TP53mut. In addition, 13/17 (76.5%; n=1 no data available) cases showed a concomitant TP53 deletion detected by fluorescence in situ hybridization. Only 1/10 of these cases showed a TP53 deletion already at first diagnosis (no data available: n=3).

Mutation frequencies of the NOTCH1 PEST domain, MYD88, FBXW7, XPO1, SF3B1 and the IGHV mutation status were compared between the discovery cohort and an independent cohort of 326 newly diagnosed CLL cases. Mutation frequencies in NOTCH1 PEST domain (2/18, 11.1% vs 45/326, 13.8%), MYD88 (1/18, 5.6% vs 6/326, 1.8%), and FBXW7 (0/18, 0% vs 8/326, 2.5%) were comparable between both cohorts, while mutations in XPO1 (4/18; 22.2% vs 14/326; 4.3%, P=0.010) and in SF3B1 (4/18; 22.2% vs 27/326 8.3%, P=0.067) were observed at significantly higher frequencies in the discovery cohort. Also an unmutated IGHV status was significantly more frequent in the discovery cohort compared to the unselected comparison cohort (16/18, 88.9% vs 196/326, 60.1%; P=0.01). Of note, in one case with a MYD88 mut and another case with a SF3B1 mut, these alterations were detected at first diagnosis, but disappeared during course of disease. In contrast, an XPO1 mutation was acquired during the course of disease in parallel to the TP53mut. In the remaining cases with SF3B1 (n=3), XPO1 (n=3), and NOTCH1 (n=2), mutations were observed both at first diagnosis and follow-up time point that revealed an acquired TP53mut.

1. A small fraction of CLL patients (2/20) harbored a subclone with TP53 mutations at first diagnosis which escaped detection by conventional laboratory assays (DHPLC and Sanger sequencing) and were only identified retrospectively by using a more sensitive next-generation sequencing assay. These subclones increased in size during the course of disease. 2. A subset of CLL patients acquired TP53mut during course of their disease. Thus, repeated TP53 mutation analyses are necessary for optimal treatment decisions. 3. An unmutated IGHV status and mutations in XPO1 and SF3B1 were more frequent in CLL patients who acquired TP53 mutations during the course of disease, suggesting that these are potential risk factors for the acquisition of TP53 mutations during disease progression.

Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Boeck:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.