Mutational Profile and Dynamics of PPM1D-Mutant Clones in the Spectrum of Myeloid Disorders

Introduction
Mutations in PPM1D, a regulator of DNA damage response, are enriched in patients with clonal hematopoiesis (CH) exposed to cytotoxic treatment or with therapy-related myeloid neoplasm. However, their role in leukemic transformation is unclear. Here, we describe a large cohort of patients with PPM1D mutations from CH to acute myeloid leukemia (AML) to understand the molecular landscape of mutant PPM1D related disease and the longitudinal dynamics of CH under treatment.

Methods
We included, in a non-sequential cohort, 96 PPM1D mutated patients treated at Gustave Roussy with CH, clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) or AML. A 77 gene panel NGS analysis was performed. An additional 16 PPM1D mutated AML patients from ALFA trials (1200, 0701,0702) were included. 10 patients with ovarian cancer from this cohort had sequential blood samples (OvBIOMark trial) available prior to hematologic evaluation, from the diagnosis or first relapse of their cancer through the course of therapy. Those samples were analyzed with a UMI based 18 gene panel (HaloplexHS, Agilent). Overall survival (OS) analyses were performed with the Kaplan-Meier method from the time of diagnosis until death from any cause.

Results
Among the 112 patients with PPM1D mutations, 23 (21%) had CH, 36 (32%) CCUS, 19 (17%) MDS, 25 (22%) AML (including 4 relapses), and 9 (8%) MPN. Median age was 65 [range, 21-88] years with 67% of females. Only 18% of the patients had no previous cancer history. Most frequent primary cancers were gynecological cancer (27%), lymphoid malignancies (22%), and breast cancer (17%). Median time between primary cancer diagnosis and hematologic assessment was 5.3 [range, 1.2-8.5] years.

Eighty six percent of patients had one PPM1D mutation, 9% had 2, and 5% had 3 or more (restricted to CH and CCUS). Median variant allele frequency (VAF) was 3% [0.2-38], 3% [1-31], 2.4% [1-41], 23% [1-50], and 4% [1-42] in CH, CCUS, MDS, AML and MPN, respectively. Among CH/CCUS patients, PPM1D was the sole detected somatic mutation in 39% (23/59), compared to 9% (5/53) in MDS/AML/MPN patients (odds ratio=6, p=0.0004); 3/5 of the latter had complex karyotype. The most frequently co-mutated genes were DNMT3A (29%) and TP53 (25%), uniformly across all conditions (Fig 1A). MDS-related gene mutations, RUNX1 (7%), ASXL1 (4%), SF3B1 (4%), SRSF2 (4%), U2AF1 (4%), were specific to AML/MDS/MPN. Among 28 patients with both PPM1D and TP53 mutations, PPM1D mutations were dominant or co-dominant in 64% (18/28), and secondary in 36% (10/28). IPSS-M risk of evaluable MDS was high/very high in 54% (7/13) of patients. ELN 2022 classification of AML was adverse in 68% (17/25) of patients. AML treatment options included best supportive care for 8% of patients (2), 5-Azacytidine for 32% (8), intensive chemotherapy for 60% (15).

With a median follow up of 2.4 years, the median OS was 4.6 (CI95%; 4.1-NA), 1.31 (CI95%; 0.53-NA), 1.27 (0.43-NA), 0.66 (CI95%; 0.42-1.26) and 20.4 (CI95%; 20.4-NA) years for CH, CCUS, MDS, AML and MPN, respectively. TP53 mutation status did not stratify OS. Four ovarian cancer patients with CH/CCUS transformed to MDS/AML with a median of 5.3 [1-12] years. At transformation, 3/4 had a stable PPM1D mutation, 1/4 an increase in PPM1D VAF (2 to 28%), and 3/4 had TP53 mutations before and at transformation.

We analyzed 67 timepoints from 10 ovarian cancer patients during therapy (median 7 per patient). 10/10 received alkylating agents and 5/10 PARPi. The median number of mutations per patient was 4 and 6 at baseline and last follow-up, respectively. PPM1D-mutated clone size increased from 0.4% [0.1-3] at baseline to 7% [2-30] at last follow-up. Beyond clonal expansion, PPM1D VAF dynamics showed non-linear changes related to alkylating agents exposure: 2/10 patients had a continuous expansion, 2/10 had expansion then contraction (GR-1/3, Fig 1B), and 6/10 had expansion then stabilization (GR-2, Fig 1B).

Conclusion
We described a large cohort of PPM1D mutated patients from CH to AML. Their prognosis was poor independently of TP53 mutation at AML/MDS stage. PPM1D mutations were frequently part of the dominant clone. To confirm the clonal architecture, single cell sequencing analysis in 8 AML/MDS patients are ongoing. Trajectory of PPM1D mutations in ovarian cancer patients revealed a non-linear alkylating agent dependency which warrants further investigation.