Ultra-Deep Sequencing As a Predictive Tool for MDS Progression after Allogeneic Hematopoietic Cell Transplant

Background:

Allogeneic hematopoietic cell transplantation (alloHCT) is the only curative treatment for patients with myelodysplastic syndromes (MDS). Up to 40% of MDS patients will die within the first year post-alloHCT due to progressive disease or treatment-related mortality. Predictors of post-alloHCT progression are needed. While recent data have shown that gene mutations in pre-transplant samples, including TP53, are associated with worse outcomes post-alloHCT, it is not known whether persistence of patient-specific mutations after alloHCT are predictive of progression. In this study we identified somatic mutations in MDS patients prior to alloHCT and then used ultra-deep sequencing of day 30 and day 100 post-alloHCT bone marrows to determine whether the extent of mutation clearance was associated with outcome.

Methods:

We selected 55 MDS patients who underwent alloHCT with a mean age of 55.6 years (range 23-71) and IPSS-R scores of very low (1), low (12), intermediate (6), high (23), and very high (13). Transplants included myeloablative and non-myeloablative conditioning regimens with related and unrelated donors. Of the 55 patients, 19 relapsed post-alloHCT and 36 did not with a mean of 242 days to relapse (range 27-1015) and a mean follow-up of 883 days (range 103-2106), respectively. Initial pre-alloHCT bone marrows and paired normal skin (as a source of control DNA) were enhanced exome sequenced (EES) to yield higher coverage in known recurrently mutated AML/MDS genes. To provide high sensitivity at post-alloHCT time points, an ultra-sensitive, error-corrected sequencing approach using unique molecular identifiers (UMIs) was combined with high-coverage depths. Probes were designed to target mutations detected by EES in the pre-alloHCT samples. Initial diagnostic marrow, skin, and day 30 post-alloHCT marrow samples were sequenced in all patients and day 100 post-alloHCT marrows were available in 37 patients. Variant allele fractions (VAFs) of somatic single nucleotide variant (SNV) mutations at day 30 and day 100 post-alloHCT were detected using a custom analysis pipeline employing read-level UMI-based error correction and base-level background error rate corrections. SNV calls were correlated with post-alloHCT progression-free survival and overall survival using Kaplan-Meier and Cox proportional hazards models, respectively.

Results:

EES was performed to a mean of 230x depth (1310x for recurrently mutated genes). A mean of 40.3 (range 17-151) somatic variants per case were identified with a mean VAF of 29.6%. Of these, a mean of 23 (range 1-70) SNVs per case were targeted and analyzed. Day 30 post-alloHCT marrows were sequenced to a mean depth of 23,062x corresponding to 2,753x unique coverage; day 100 samples were sequenced to 27,936x corresponding to 3,430x unique coverage. A SNV was included in the analysis if the error-corrected pre-transplant VAF was >10% and had >100x unique coverage at day 30. Nineteen of 55 (35%) and 18 of 37 (49%) cases had no mutations detected on post-alloHCT days 30 and 100, respectively. For patients with detectable mutations, the mean VAF per patient at day 30 was 2.35% (range of detected mutations 0.001-25.81%) and 3.44% (range 0.001-21.45%) at day 100. For patients with detectable mutations, the mean maximum VAF per patient at day 30 was 7.21% (range 0.020-88.19%) and 3.44% (range 0.023-38.98%) at day 100.

The presence of at least 1 mutation with a VAF greater than 0.5% (equivalent to 1 heterozygous mutant cell in 100) at day 30 post-alloHCT was associated with a worse progression-free survival (median 3.8 vs. 21.9 months; p=0.031). Similar results were obtained using VAF cutoffs of 1% and 2.5% (p= 0.018, p=0.0002, respectively). In contrast, day 100 post-alloHCT marrows did not show a similar correlation. The effect of persistent mutations on overall survival was assessed with a Cox proportional hazards model. A higher mean VAF at day 30 post-alloHCT was associated with worse overall survival (p=0.028); for each mean VAF increase of 1% at day 30, the risk of death increased by about 10% [HR 1.10, 95% CI (1.01,1.20)].

Conclusions:

Detection of persistent mutations in the bone marrow by ultra-sensitive, error-corrected sequencing at day 30 post-alloHCT is associated with an increased risk of progression. These findings suggest that sequencing could be used as an individualized risk-assessment marker in MDS patients who have undergone alloHCT.