Type of TP53 Mutations Affects Subclonal Configuration and Selection Pressure for Acquisition of Additional Hits in Contralateral Alleles
Authors: Gurnari Carmelo , Adema Vera , Awada Hassan , Pagliuca Simona , Kerr Cassandra M , Kongkiatkamon Sunisa , Laframboise Thomas , Visconte Valeria , Voso Maria Teresa , Sekeres Mikkael A. , Carraway Hetty E. , Haferlach Torsten , Maciejewski Jaroslaw P. 
Published: February 2021

Somatic TP53 mutations are found in 10% of adult patients with MDS and de novo AML and in up to 20% of patients with therapy-related myeloid neoplasms. TP53 status is associated with complex karyotype (CK), aberrations of chromosome 5 and poor survival. Moreover, mutated TP53 (TP53MT) may be an indication for hematopoietic cell transplantation, but also predictive of relapse following the procedure, making this particular category of myeloid neoplasms (MN) a conundrum of clinical management. Unlike other tumor suppressor genes, missense (ms) mutations within the DNA-binding domain (DBD) are the most common genetic alterations in TP53 gene representing up to 80% of somatic hits, with involvement of canonical hotspots (R175H, Y220C, M237I, R248Q, R273H, R282W) in around 30% of cases. A loss-of-function (LOF) dominant-negative effect (DN) may explain the ability of TP53MT to interfere with wild type (WT) functions. Moreover, TP53 germ line (GL) mutations are responsible for Li-Fraumeni syndrome, and GL contamination may also exist in adult MN. Here we comprehensively characterize TP53MT MNs to better dissect the role of specific mutational configurations and identify the selective forces affecting outcomes in this poor prognostic MN category.
A total of 764 TP53 mutations were found in 632 MN patients. Ms mutations were the most common (75%) followed by frameshift (11%), splice site (7%), nonsense (5%) and insertion/deletions (2%), with 20% of patients harboring more than 1 lesion. Topographical annotation revealed that ms mutations typically (98%) occurred within the DBD (residues 102-292) and only 2% occurred outside this region (vs. 28% in case of truncating mutations, p&lt;.0001). Overall, 22% of patients displayed a mutation in the canonical hotspot regions. Among TP53MT, 36 cases (6%) were of possible GL origin. Focusing on the somatic lesions, a male preponderance (1.42 vs. 1.1 M:F, p=.0069) and a younger age at presentation (median 68.9 vs 71, p&lt;.00001) were found in WT vs. mutant cases, which were also less enriched in coincident de novo leukemia-driver genes mutations (e.g. NPM1, FLT3). When compared to WT MN, TP53MT cases were more likely to have CK (8% vs. 70%, p&lt;.00001), del(5q) (4% vs. 40%, p&lt;.00001), del(7q)/-7 (6% vs. 18%, p&lt;.00001) and trisomy 8 (8% vs. 49%, p&lt;.00001). Of note, deletion of the TP53 locus was found in 27% of mutated cases vs only 1% of WT counterparts (p&lt;.00001). Conversely, 77% of all MN cases with CK had either TP53 mutations (61%), del(17p) (3%) or both (36%). When classifying patients according to TP53 genomic context (30% single vs. 70% double hit, defined as a presence of biallelic, hemizigous or UPD configuration) progressive inactivation had an adverse impact on survival (p&lt;.0001).
We then hypothesized that truncating (frameshift/nonsense/deletion) alterations require additional hits as the presence of one allele may be partially protective. Consequently, the VAF for these lesions may be a result of a UPD in a smaller fraction of cells; biallelic truncation hits thus may be truly biallelic rather than clonally mosaic, which can be demonstrated only by single cell DNA sequencing. In contrast, DN mutations in canonical hotspots decrease the function beyond 50% of the activity, with further inactivation would having less of an effect, thus exerting less selection pressure for acquisition of additional lesions. Indeed, second truncating hits (including UPD and del(17p)) were common (30%), while none of the dominant ms hits had a double-mutant hotspot configuration (vs. 14% of non-canonical ms double mutant), and these canonical dominant hits were less likely to be paired with del(17p) or truncating mutations (8%). Only 25% of CK had a WT configuration of TP53, consistent with our theory that dominant ms hits were more likely to be present without del(17p). It is possible that the inability to assert clear survival differences according to the number or types of TP53 lesions may be due to an inability to resolve the intraclonal configuration of mutations using VAF calculations. We also conclude that non-canonical ms mutations (many of them classified as VUS) may have a variable impact, with functional consequences ranging from those that are less severe than truncations to various degrees of negative dominance. Analyses of the impact of ms mutations on TP53 tetramers (which may contain various doses of mutant vs WT monomer), will shed further light on the biology of TP53MT MN.

Disclosures
Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Takeda/Millenium: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; ASTEX: Other: Independent Advisory Committe (IRC); BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC). Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.