Impact of pretransplant mutation status on survival after allogeneic stem cell transplant for acute myeloid leukemia

1 Department of Pathology, UMassMemorial Medical Center, University ofMassachusetts,Worcester, Massachusetts, USA 2 Department of Hematology-Oncology, UMassMemorial Medical Center, University ofMassachusetts,Worcester, Massachusetts, USA 3 Department of Hematology, Medical Oncology, BannerMDAnderson Cancer Center Clinic, Gilbert, Arizona, USA 4 Bristol Myers Squibb Company, Cambridge, Massachusetts, USA


INTRODUCTION
Allogeneic stem cell transplant (Allo-SCT) is a powerful tool in the treatment of acute myeloid leukemia (AML) and offers the only potential chance of cure. Crucial to the success of Allo-SCT is the elimination of leukemic blasts prior to transplant. Evaluation of residual disease historically has been via morphologic review, with complete remission defined as fewer than 5% blasts [1]. Although thresholds lower than 5% are prognostically significant [2], morphologically it is usually impossible to distinguish normal regenerating bone marrow blasts from leukemic blasts.
Distinction of pathogenic from regenerating blasts has been achieved via flow cytometry and by PCR or cytogenetic studies in cases with recurring genetic abnormalities. Unfortunately flow cytometry is plagued by lack of standardization and the requirement for analytic expertise for interpretation, especially in the context of AML residual disease monitoring. AML demonstrates vast phenotypic and genotypic heterogeneity, and FISH and PCR are applicable only in those cases with detectable genetic abnormalities, limiting the utility of these modalities. AML is also temporally heterogeneous, as the dominant phenotypic and genotypic features present at diagnosis can change significantly over the course of disease. This makes residual disease testing at times a "moving target. tions across a broad array of commonly altered genes in a single assay. In the context of residual disease testing for leukemia, it allows detection of dominant and subclonal mutations both at diagnosis and in posttreatment monitoring and has the ability to detect molecular aberrations suspicious for persistent leukemia at low levels and across the gamut of subtypes.
The sensitivity of NGS for detecting residual AML and the prognostic significance of post-induction residual low-level leukemiaassociated mutations has previously been established by others. [3] Jongen-Lavrencic et al showed specifically that residual mutations in genes other than DNMT3A, TET2, and ASXL1 detectable after induction confer worse post-induction survival. [3 ,4] In the current study we endeavored to assess the significance of persistent AML-associated mutations in the pre-Allo-SCT setting since relatively few studies have examined their significance in this context. We explored the impact of mutations present at diagnosis that remained detectable within 30 days of transplant on overall survival and disease-free survival. Prior studies have suggested that mutations in genes involved in modification of the epigenome -either through DNA methylation regulation or histone modification -tend to be permissive of AML transformation but not sufficient in and of them to give rise to leukemia. [3,5] Therefore, we focused on the implications of mutations in epigenetic modifiers, specifically IDH1/2, DNMT3A, ASXL1, TET2, and EZH2 versus others (the acronym IDATE will be used to refer to these epigenetic modifier genes henceforth in this paper). We sought to explore whether the survival impact of these mutations purported to be merely "preleukemic" would be as significant as mutations in other classes of genes. We also evaluated the stability of these epigenetic modifier mutations by comparing their tendency to be eradicated through pretransplant conditioning (also known as rate of clearance) versus other pathogenic mutations.
We performed a retrospective analysis of patients who received NPM1 and FLT3 mutation status was tested in the diagnostic specimens of all patients using PCR fragment analysis. In those patients with either mutation detected at diagnosis, supplemental PCR testing for these common insertion mutations was repeated in the pretransplant specimens.
We evaluated overall survival and relapse free survival according to the presence or absence of morphologic and molecular residual disease. We categorized residual mutations according to type/affected pathway (e.g., epigenetic vs. signaling, etc.). Mutation status at diagnosis was compared to mutation status immediately before transplant.
Overall survival and relapse free survival were compared across mutation class categories with particular emphasis on epigenetic modifier mutations versus all others using the Kaplan--Meier method and the log-rank test. Overall survival was censored for patients lost to follow-up. Relapse free survival was censored for patients that were lost to follow-up or who died without diagnosed relapse. We also examined clearance rates of somatic mutations by subtype (using chi-square test with Yates correction) to determine which mutations showed the greatest propensity to persist through pretransplant conditioning (i.e., remain detectable in the window between the end of chemotherapy and the initiation of transpant).
Patient median age at diagnosis was 59 (range 27-80), M:F 1.8:1 (Table 1). Thirteen patients showed normal karyotype, two had disease defining translocations, and two had other cytogenetic abnormalities. These findings are consistent with previous studies that have suggested that epigenetic modifier mutations as a class seem to be merely pre-leukemic rather than true drivers of AML. [5,6] It is well-established that mutations in epigenetic modifiers (particularly DNMT3A, TET2, and ASXL1) are frequently present in preleukemic states such as clonal hematopoiesis of indeterminate potential [7].
Although they confer greater risk for leukemic transformation, they  In a recent paper Press et al similarly established the prognostic impact of pre-stem cell transplant MRD on posttransplant leukemia-free survival in their cohort [13]. However, none of these studies focused on the specific survival implications of non-IDATE mutations versus IDATE mutations. The current study lends further evidence for the unfavorable outcome predicted by persistent mutations prior to Allo-SCT, but it also specifically helps to confirm that the preponderance of added risk is attributable to non-epigenetic modifier (non-IDATE) mutations.

Findings in pretransplant specimen
Although our investigation produced significant results, it is limited by relatively small sample size necessitating a focus on classes of mutations rather than individual genes. Additionally there was some disparity among patient and tumor characteristics and among pretransplant conditioning regimens in our cohort. Larger studies with the statistical power to delve with greater granularity into the prognostic impact of individual mutations (with a particular focus on IDATE vs. others) and to adjust for varying patient, tumor and treatment characteristics are merited. Chen, and Jan Cerny reviewed and revised the paper.