TP53 Mutations Detected By Next-Generation Deep-Sequencing In Patients With Myelodysplastic Syndrome and Isolated Deletion (5q): Results From a German Multicenter Trial
Authors: Mossner Maximilian , Jann Johann-Christoph , Lauinger-Lörsch Evi , Nowak Daniel , Platzbecker Uwe , Giagounidis Aristoteles , Götze Katharina S. , Letsch Anne , Haase Detlef , Shirneshan Katayoon , Schlenk Richard F. , Kohlmann Alexander , Haferlach Torsten , Bug Gesine , Lübbert Michael , Ganser Arnold , Obländer Julia , Fey Stephanie , Hofmann Wolf-Karsten , Germing Ulrich , Nolte Florian 
Published: October 2019

Abstract

Background
Beside cytogenetic aberrations, additional gene mutations are powerful predictors of outcome in myeloid diseases. Moreover, myelodysplastic syndromes with isolated deletion (5q) (MDS del(5q)) have been regarded as one of the most favorable entities among MDS. However, a substantial proportion of MDS del(5q) patients experience transformation into AML soon after diagnosis (Germing et al. Leukemia. 2012;26:1286-1292). Mutations of TP53 gene have early been recognized as an unfavorable prognostic biomarker in MDS in general and recent data suggest a role of TP53 mutations in the transformation of MDS del(5q) into AML. Lenalidomid (Len) is now approved in the US as well as in Europe for the treatment of MDS del(5q) it is of particular interest whether Lenalidomide can alter the course of pretreatment TP53 mutated MDS del(5q).

Methods
The Le-Mon-5 trial investigated the safety and efficacy of Len in patients with MDS and isolated deletion (5q). All patients gave their written informed consent to the clinical trial and to additional molecular genetic analyses. Bone marrow aspirates were performed at screening prior treatment initiation and during follow-up every 6 months. Only freshly extracted, high-quality DNA from ficollized mononuclear cells was used for next-generation deep-sequencing analysis. For generation of PCR amplicon libraries TP53 oligonucleotide primer plate assays were used and technically validated within the IRON-II (Interlaboratory Robustness Of Next generation sequencing) research study network. Amplicon deep-sequencing of TP53 (exons 4-11) was performed on a Roche 454 GS Junior system. Mean coverage of sequenced exons was about 800-fold allowing an approximate detection sensitivity of 2% mutational burden.

Results
Central cytological, histological and cytogenetic review was performed in all patients establishing the diagnosis of MDS with isolated deletion (5q). A total of 68 patients (male: n=9) were analyzed with a median age of 71 years (range 41-88 years). TP53 mutations prior to treatment initiation with Len were found in 7 patients (10%). Mean mutation frequency was 38%. Notably, we did not find mutation frequencies lower than 15%. Of 4 evaluable patients, three patients became transfusion independent within 4 months of Len treatment. Of 2 patients we had follow-up samples available. Both patients showed no difference with regard to the mutation frequency after a follow-up of 4 and 17 months on Len treatment (27% and 51%, respectively). Noteworthy, one the two patients achieved a complete cytogenetic remission despite maintaining his TP53 mutation frequency.

Conclusion
Using freshly extracted DNA we achieved high-quality NGS results with a high mean coverage of the relevant coding region of TP53. However, prevalence of TP53 mutations in our patient cohort was lower as compared to previously published data and we did not find low-level allele burdens as published by other groups, which might be due to the different sample sources used. Transfusion independence as well as cytogenetic remissions can be achieved in patients with TP53 mutations who are treated with Lenalidomide.

Disclosures:
Platzbecker: Celgene: Honoraria. Giagounidis:Celgene: Consultancy, Honoraria. Götze:Celgene Corp.: Honoraria. Haase:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Hofmann:Celgene: Research Funding. Germing:Celgene: Honoraria, Research Funding. Nolte:Celgene: Honoraria, Research Funding.