Juliane Grimm

Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients

Allogeneic hematopoietic stem cell transplantation is an established consolidation therapy for patients with acute myeloid leukemia. However, relapse after transplantation remains a major clinical problem resulting in poor prognosis. Thus, detection of measurable (“minimal”) residual disease to identify patients at high risk of relapse is essential. A feasible method to determine measurable residual disease may be digital droplet PCR (ddPCR) that allows absolute quantification with high sensitivity and specificity without the necessity of standard curves. Using ddPCR, we analyzed pre-transplant peripheral blood and bone marrow of 51 NPM1-mutated acute myeloid leukemia patients transplanted in complete remission or complete remission with incomplete recovery. Mutated NPM1 measurable residual disease-positive patients had higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.014). Restricting the analyses to patients receiving non-myeloablative conditioning, mutated NPM1 measurable residual disease positivity is associated with higher cumulative incidence of relapse (P < 0.001) and shorter overall survival (P = 0.006). Positive mutated NPM1 measurable residual disease status determined by ddPCR before allogeneic stem cell transplantation is associated with worse prognosis independent of other known prognostic markers—also for those receiving non-myeloablative conditioning. In the future, mutated NPM1 measurable residual disease status determined by ddPCR might guide treatment and improve patients’ outcomes.

Prognostic relevance of DNMT3A R882 mutations in AML patients undergoing non-myeloablative conditioning hematopoietic stem cell transplantation

Mutations in the epigenetic modifying gene DNA methyltransferase 3A (DNMT3A) are found in 14–35% of acute myeloid leukemia (AML) patients [1–6]. The majority of DNMT3A mutations cluster at codon R882 in exon 23 [1–6]. Most of the studies that have investigated the prognostic impact of these alterations reported inferior outcomes associated with the presence of DNMT3A mutations—particularly in codon R882 [1–4, 6–9]. However, the negative impact on outcomes may be dependent on the clinical and biological context, such as the presence of concurrent gene mutations (i.e. NPM1 mutations and/or presence of FLT3ITD) and the DNMT3A mutation type (i.e. R882 vs. nonR882) [1–6, 9]. In older AML patients, DNMT3A mutations are more prevalent [7] and have been associated with poor outcomes [1, 6, 8]. DNMT3A mutations are often found to persist during remission (possibly due to a preexisting clonal hematopoiesis) and at AML relapse, and might contribute to chemotherapy resistance [10]. This raises the issue of allogeneic hematopoietic stem cell transplantation (HSCT) as a therapeutic approach specifically for DNMT3A mutated AML. Studies that included HSCT treated patients—most of which focused on younger AML patients—observed no influence of an HSCT on the negative prognostic impact of DNMT3A mutations [3–5, 9, 11]. For example, Gaidzik et al. [5]. described a shorter relapse-free survival in younger DNMT3A R882 mutated AML patients independent of the use of HSCT. Similarly, Ahn et al. [9]. and Xu et al. [11]. reported a negative impact of DNMT3A R882 mutations after HSCT in mainly younger patients with normal karyotype (NK) AML. Since DNMT3A R882 mutations occur more frequently with age [7] and data on the influence of DNMT3A R882 mutations on outcome in older AML patients receiving HSCT are limited, we analyzed the prognostic impact of DNMT3A R882 mutations at diagnosis among mainly older (>60 years) AML patients who underwent nonmyeloablative (NMA)-HSCT. NMA-HSCT relies on an immunological donor leukocyte-mediated GvL effect and, because of the lower treatment-related toxicity, represents a feasible consolidating therapy for older AML patients and/or those with comorbidities [12, 13]. The patients studied here were treated with cytarabinebased chemotherapies followed by conditioning with fludarabine and 2 Gy TBI or 2 Gy TBI only before NMAHSCT (for further details, see Supplementary Information) at the University Hospital Leipzig between February 2003 and December 2012. We retrospectively analyzed the presence of DNMT3A R882 mutations in pre-treatment bone marrow (n= 98) or peripheral blood (n= 10) samples of 108 AML patients (for further details, see Supplementary Information). All patients gave written informed consent in accordance with the declaration of Helsinki. Pre-treatment cytogenetics and the mutation status of NPM1, CEBPA, and the presence of FLT3-ITD were determined as previously Laura K. Schmalbrock and Lynn Bonifacio contributed equally to this work.

High BAALC copy numbers in peripheral blood prior to allogeneic transplantation predict early relapse in acute myeloid leukemia patients

High BAALC expression levels at acute myeloid leukemia diagnosis have been linked to adverse outcomes. Recent data indicate that high BAALC expression levels may also be used as marker for residual disease following acute myeloid leukemia treatment. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a curative treatment for acute myeloid leukemia patients. However, disease recurrence remains a major clinical challenge and identification of high-risk patients prior to HSCT is crucial to improve outcomes. We performed absolute quantification of BAALC copy numbers in peripheral blood prior (median 7 days) to HSCT in complete remission (CR) or CR with incomplete peripheral recovery in 82 acute myeloid leukemia patients using digital droplet PCR (ddPCR) technology. An optimal cut-off of 0.14 BAALC/ABL1 copy numbers was determined and applied to define patients with high or low BAALC/ABL1 copy numbers. High pre-HSCT BAALC/ABL1 copy numbers significantly associated with higher cumulative incidence of relapse and shorter overall survival in univariable and multivariable models. Patients with high pre-HSCT BAALC/ABL1 copy numbers were more likely to experience relapse within 100 days after HSCT. Evaluation of pre-HSCT BAALC/ABL1 copy numbers in peripheral blood by ddPCR represents a feasible and rapid way to identify acute myeloid leukemia patients at high risk of early relapse after HSCT. The prognostic impact was also observed independently of other known clinical, genetic, and molecular prognosticators. In the future, prospective studies should evaluate whether acute myeloid leukemia patients with high pre-HSCT BAALC/ABL1 copy numbers benefit from additional treatment before or early intervention after HSCT.