The Role of PPM1D Mutations in Lenalidomide Resistance and Progression in Patients with MDS and Deletion of Chromosome 5q
Authors: Panagiota Victoria , Meggendorfer Manja , Kubasch Anne Sophie , Gabdoulline Razif , Krönke Jan , Shahswar Rabia , Mies Anna , Kandziora Christian , Klement Piroska , Schiller Johannes , Göhring Gudrun , Haferlach Claudia , Ganster Christina , Shirneshan Katayoon , Gutermuth Annika , Thiede Christian , Germing Ulrich , Schroeder Thomas , Kobbe Guido , Klesse Sabrina , Koenecke Christian , Schlegelberger Brigitte , Kroeger Nicolaus , Haase Detlef , Döhner Konstanze , Sperr Wolfgang R. , Valent Peter , Ganser Arnold , Thol Felicitas , Haferlach Torsten , Platzbecker Uwe , Heuser Michael 
Published: October 2019

Abstract
Background: PPM1D is a serine/threonine phosphatase that inactivates p53 tumor suppressor pathway. Recently, PPM1D mutations have been described in clonal hematopoiesis and are more frequently found in therapy-related MDS than in primary MDS (15% vs. 3%). Del(5q), is the most prevalent cytogenetic abnormality in MDS. A high proportion of MDS del(5q) patients respond to lenalidomide, but almost 40% of them progress to AML. Scharenberg et al. identified recurrent mutations in a limited number of genes i.e. TP53, RUNX1, and TET2 in a longitudinal cohort of 35 MDS del(5q) patients that progressed to AML. The clinical impact and occurrence of PPM1D mutations in MDS del(5q) patients remains unknown.
Aim: To determine the clinical impact of PPM1D mutations in MDS del (5q) patients on lenalidomide resistance and AML progression.
Methods: We studied a cohort of 243 patients with MDS or AML following MDS and 5q deletion diagnosed according to the 2008 WHO classification. Patients were cytogenetically characterized by chromosome banding analysis and followed for disease progression, treatment and survival. From 22 del(5q) patients treated with lenalidomide, follow-up (FU) material was available before and after treatment. Molecular analysis for mutations in all 6 exons of PPM1D was performed by Sanger and/or a next-generation sequencing panel covering mutations in 46 genes frequently mutated in MDS, including TP53 and CSNK1A1.
Results: At the time of diagnosis 14 PPM1D mutations were detected in 13 of 243 (5.3%) MDS patients with del(5q), 12 of which were found in the previously described hotspot region of PPM1D between amino acids 427 and 542. Six patients had nonsense mutations, 3 patients had frameshift mutations (one patient with 2 frameshift mutations), and 4 patients had missense mutations. TP53 mutations were found in 34 of 243 (14%) MDS patients with del(5q). Three TP53 mutated patients, two with complex karyotype, carried an additional PPM1D mutation. Co-occurrence of PPM1D and CSNK1A1 mutations was not observed in any patient.
In total, 71 of 243 patients were treated with lenalidomide and had available information about treatment response. Eleven patients (15.5%) did not respond to lenalidomide and 17 patients (24%) progressed to AML. Nine of 71 (12.6%) patients were TP53 (n=5, 7%) or PPM1D mutated (n=4, 5.6%). For 22 of 71 patients who either achieved a complete remission (n=5), developed resistance to lenalidomide followed by MDS progression (n=7) or AML transformation (n=10), FU samples were available before and after lenalidomide treatment.
Of the 5 patients with complete remission 4 patients displayed no mutations, while 1 patient was PPM1D- and ASXL1-mutated with a variant allele frequency (VAF) of 27.6% and 12.1%, respectively, prior to lenalidomide treatment. After 76 months on lenalidomide, both mutations had disappeared. Of the 17 patients with lenalidomide resistance/AML progression, 5 patients (29.4%) carried mutations either in PPM1D (n=2) or in TP53 (n=3) prior to lenalidomide treatment, with a mean VAF of 15.3% and 13.5%, respectively. The 2 PPM1D-mutated patients progressed to AML 59.4 and 79.6 months after diagnosis. None of the 3 initially TP53-mutated patients progressed to AML. All 3 TP53-mutated patients co-expressed SF3B1 mutations.
At the time of lenalidomide resistance/AML progression, we observed 2 known and 1 novel PPM1D mutation in a patient previously wildtype for PPM1D and TP53, 3 known and 6 novel TP53 mutations in 5 patients previously wildtype for PPM1D and TP53, and 1 novel TP53 mutation in a patient who was previously found mutated in PPM1D. Thus, at the time of lenalidomide resistance or AML progression 10 of 17 patients (58.8%) were mutated for PPM1D (n=3, 18%) and/or TP53 (n=9, 53%; 2 of 9 co-expressed PPM1D mutations). At the time of lenalidomide resistance/AML progression, VAF increased from 10.2% to 23.3% for PPM1D and from 4% to 16.9% for TP53 mutations, indicating expansion of the mutated clone under the selective pressure of lenalidomide.
Conclusion: PPM1D mutations are recurrently found in MDS del(5q) patients at a frequency of 5.3% and may be coexpressed with TP53 mutations in 5q- MDS/AML cells. Frequency at resistance/AML progression was 18% for PPM1D and 53% for TP53 mutated patients, respectively. Our findings indicate an association of PPM1D mutations in addition to the previously described TP53 mutations with lenalidomide resistance and AML progression.

Disclosures
Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Krönke:Celgene: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Kobbe:Amgen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Celgene: Honoraria, Other: Travel Support, Research Funding. Koenecke:Amgen: Consultancy; Roche: Consultancy; abbvie: Consultancy; BMS: Consultancy. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Platzbecker:Celgene: Research Funding. Heuser:BergenBio: Research Funding; StemLine Therapeutics: Consultancy; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer Pharma AG: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Karyopharm: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding.