TP53 Mutations, Deletions, and CN-LOH: Comparison of TP53 single Hit and Double Hit Events and Their Impact on Prognosis in Hematological Malignancies
Authors: Stengel Anna , Müller Heiko , Meggendorfer Manja , Walter Wencke , Baer Constance , Nadarajah Niroshan , Hutter Stephan , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia 
Published: November 2021

Abstract
Background: TP53 is altered in ~50% of human cancers. Alterations mainly include mutations and/or deletions, but also copy-neutral loss of heterozygosity (CN-LOH) was reported. Frequently, both TP53 alleles are altered (by mutation + deletion, mutation + CN-LOH or ≥2 mutations), leading to a "double hit" event.
Aim: Analysis of TP53 aberrations using WGS in 4646 cases with 29 different hematological malignancies, comparing (1) the frequencies of TP53 alterations, (2) occurrence of single hit vs. double hit, (3) correlation with complex karyotype and (4) impact on survival.
Methods: Whole-genome sequencing (WGS) was performed for all 4,646 patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p&lt;0.05.
Results: In the total cohort of 4,646 cases, in 582 (13%) at least one alteration (alt) involving TP53 was detected (comprising mutations (mut), deletions (del) and CN-LOH (LOH); Fig 1A,B). Cases were categorized as follows: cases with (1) 1 TP53 mut only (n=166), (2) del only (n=100), (3) LOH only (n=15), constituting the single hit events. Further, (4) cases with mut+del (might include 1 or more mut, n=211), cases with mut+LOH (≥1 mut, n=41), cases with ≥2 mut only (without del or LOH, n=49), resulting in double hit events (Fig 1B). Regarding the respective entities, high frequencies of TP53 alt were mainly detected in lymphoid malignancies (e.g. HGBL, MPAL, vHZL, MZL, MCL), whereas they were infrequent or absent in many myeloid malignancies (e.g. aCML, MPN, CMML, CML, MLN_eo; Fig 1A). For further analysis, only entities in which &gt;10 cases showed TP53 alt events were used. Comparison of single hit vs. double hit revealed that T-NHL, MM, MPN and MDS predominantly showed a single hit, whereas the double hit was frequent in MPAL, MZL, MDS/MPN-U, CLL and MCL cases (Fig 1A). However, the type of double hit differed between myeloid and lymphoid malignancies, as myeloid neoplasms showed a high frequency of cases with ≥2 mut only, whereas in many lymphoid malignancies the double hit was predominantly generated by mut+del (Fig 1A,C). All TP53-associated events (mut, del, LOH and the respective combinations) were found to be associated with a complex karyotype in the total cohort (LOH: 14% complex karyotype in cases without TP53 alt vs. 59% in cases with TP53 alt, p&lt;0.001). This association was also detected for most of the selected entities (exceptions: MZL, T-NHL). Regarding overall survival (OS), in the total cohort, all events involving TP53 impact on OS (TP53 alt: 22 months vs. 84 months, p&lt;0.001; TP53 mut: 20 vs. 82 months, p&lt;0.001; TP53 del: 20 vs. 79 months, p&lt;0.001; TP53 LOH: 20 vs. 75 months, p&lt;0.001). Moreover, although the single hit already impacts on OS, the double hit leads to an even inferior outcome (no hit vs. single hit vs. double hit: 84 vs. 39 vs. 14 months, p&lt;0.001). In the selected entities, an influence of TP53 alt on OS was detected for all malignancies except HGBL, MZL and T-NHL, for which also the presence of a double hit did not show an effect on OS. For the majority of the other entities, the double hit leads to a shorter OS than the single hit (as observed for the total cohort), with the exceptions of MCL and MPAL: in these entities, the single hit did not impact on OS, but only a double hit is associated with inferior outcome.
Conclusions: (1) Frequency of TP53 alterations and of double hit vs. single hit differs markedly between entities. (2) The kind of TP53 complexity differs between both lineages (double hit in myeloid neoplasms: often ≥2 mut only; in lymphoid malignancies: predominantly mut+del). (3) In 7% (41/582) of cases with TP53 alt, CN-LOH transforms a single hit into a double hit. (4) In the total cohort and in the majority of selected entities (except MZL and T-NHL), TP53 alt are associated with complex karyotype. (5) In the total cohort, all events involving TP53 impact on OS; cases with double hit show an inferior outcome compared to single hit. (6) Regarding OS, the selected entities can be divided into three categories: (i) no influence of TP53 alt (HGBL, MZL, T-NHL); (ii) double hit required for impact on OS (MCL, MPAL); (iii) influence of both single hit and double hit with inferior outcome of double hit (all other).
Figure 1 Figure 1.

Disclosures
Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.