Longitudinal Sequencing to Investigate Clonal Evolution in Myeloid Neoplasms
Authors: Shukla Asmita , Padmanabhan Dakshin , Kim Paul , Aguilar Jeff , Nanja Reddy Sushmitha , Dhillon Vikram , Carr David , Yang Jay , Boerner Julie , Balasubramanian Suresh Kumar 
Published: December 2024

Introduction:
Next-generation sequencing has revolutionized the risk stratification, prognosis, and treatment landscape of myeloid neoplasms (MN). Large-scale sequencing studies have shed light on the mutational architectures in different MN. However, there is limited information on the clonal trajectory of MN due to the lack of longitudinal gene sequencing of serial samples in published literature. Such serial monitoring of myeloid disease-related gene sequencing can capture the temporal heterogeneity with respect to treatment impact and associated mutations that influence treatment outcomes. By analyzing genetic profiles at diagnosis, during treatment, and at relapse, one can detect molecular changes linked to disease progression and relapse. The emergence of new mutations or expansion of subclones with existing mutations may provide clues to therapeutic resistance and prognosis.
Methods:
We included all patients diagnosed with myeloid neoplasms treated between 2016-2023 at Karmanos Cancer Institute. Targeted next-generation sequencing was performed (Illumina's MiSeqDx) using a 54-gene Illumina TruSight Myeloid Panel. Baseline demographics and clinical characteristics were noted. Chi-square test was used to study various parameters as described in the results. Kaplan-Meier curves were used for survival analyses.
Results:
We analyzed 851 patients with MN at Karmanos Cancer Institute; 444 with serial samples. Among these patients, 174 (39%) had primary AML (pAML), 144 (32%) had secondary AML (sAML), 58 (13%) had MDS, 18 (4%) had MDS/MPN and 50 (11%) had MPN. Median age for primary AML was 56 ys (19-82), secondary AML was 64 ys (31-86) and MDS was 66 ys (23-90). 90 (51%), 85 (59%), and 36 (62%) were male patients with primary AML, secondary AML, and MDS, respectively. 56/140 (40%), 24/43 (55%), and 7/15 (47%) patients had abnormal cytogenetics in pAML, sAML, and MDS, respectively. 124 (70%), 103 (72%), 37 (64%) had transplant in pAML, sAML, and MDS, respectively. 74 (42%) of pAML patients, 32 (22%) of sAML, and 10 (17%) of MDS patients had relapsed in their clinical course.
At diagnosis, primary AML 31/104 had FLT3 (30%), 31/104 had DNMT3A (30%), and 14/104 TET2 (14%) as the most common co-mutations. 7 patients (22%) with FLT3 and 3 (10%) with DNMT3A mutations who had persistence of mutations post-induction treatment eventually relapsed and were still positive post-transplant as well. Of 88 pts at diagnosis, 31 (35%) TP53, 19 (21%) DNMT3A, 18 (20%) TET2 were the most common co-occurring mutations in patients with sAML. Among 13 sAML patients post induction treatment, 7 (54%) TP53 and 3 (23%) DNMT3A persisted that were also present at clinical relapse. In 24 MDS patients, at diagnosis 9 (37%) DNMT3A, 7 (29%) TP53, 5 (21%) SF3B1, and 5 (21%) RUNX1. Among post-induction treatment specimens, there was persistence of 2 RUNX1 (22%) and 1 SF3B1 (11%), which eventually had clinical relapse. At relapse, 5 MDS patients gained 3 (60%) TP53 and 2 (40%) DNMT3A. The median OS in the pAML cohort was 18 months. After analyzing the cohort based on good and poor survival data, NPM1 and SRSF2 were found to be most frequent in the good OS group (43% and 37%, respectively). DNMT3A and STAG2 were the most frequent mutations noted in the poor OS group (39% and 21% respectively).
Conclusion
Existing risk stratification schema is largely applicable for newly diagnosed MN patients especially in AML. Longitudinal sequencing studies will help establish molecular risk stratification systems that would classify MN in various relapse settings based on the evolving clonal signatures. Ongoing work would further dissect the exact impact of resistant/relapse signature and a weighted impact on survival and outcomes. Ongoing analysis in other disease groups will be presented at the ASH conference.