Delayed Processing of Bone Marrow Samples Reveals a Prognostic Pattern of NME mRNA Expression in Cytogenetically Normal AML

Abstract

4904 Background: Prognostic molecular markers are expected to be of increasing value in stratifying treatment of patients with AML lacking cytogenetic abnormalities. While specific DNA-level mutations such as those in the FLT-3 or NPM1 genes clearly identify informative genotypes, phenotypic characterization of gene expression should be a more direct indicator of cell function. However, of the prognostic mRNA levels identified to date, such as BAALC, ERG or NME1/2, none appear to be superior to DNA mutation as indicators of prognosis. Because of the lability of mRNA expression, it is common practice to quantify levels only in freshly processed or direct-lysed samples in order to provide a snapshot of gene expression as close as possible to that of fresh tissue. However, characteristics of leukemic cells relevant to prognosis might become more apparent under challenging conditions, such as the stress environment developing in a bone marrow sample during transport or storage. The NME1 and -2 mRNAs have been reported to be prognostic indicators in AML and are multifunctional proteins coordinating metabolism, signalling and gene expression. To see how storage related stress affects the prognostic power of NME mRNA, we have assessed their prognostic relevance in CN-AML bone marrow samples which had originally been processed either directly, or following ≥ 2 days of transport from collaborating centers. Methods: A total of 78 archived CN-AML BMMNC samples were available for this analysis. All samples were taken at presentation from patients under 60ys treated within the AML 96 (OSHO #033) or AML 2002 (OSHO #061) protocols of the East German Study Group (OSHO). Of the 78 samples, 25 (32%) originated locally and had been freshly processed to cryopreserved MNC, while the remaining 53 (68%) had been submitted as bone marrow from other centers with an associated delay of 2–3 days. NME1 and NME2 mRNA levels were determined by triplicate qRT-PCR determinations normalized to RPLP0. The mean NME1 and NME2 expression values from each patient were expressed relative to the corresponding mean NME expression of 17 healthy donor BM samples (control group).To investigate directly the effects of delayed processing on NME mRNA expression, aliquots of 11 fresh CN-AML bone marrow samples were removed for analysis either immediately or following storage at room temperature for ≥ 2 days. Results: Both NME1 and -2 mRNA were increased relative to controls in 84% (21/25) of fresh samples with no prognostic relevance of expression above or below the median. Comparable results were obtained from a cohort of 59 freshly processed CN-AML samples from a separate study. In contrast, the transported samples yielded a heterogeneous pattern of NME mRNA expression above and below control levels, with a significant correlation between NME2 mRNA and event-free survival (p=0.009) independently of FLT-3ITD status. Direct analysis of aliquoted AML bone marrow samples confirmed that a delay in processing of ≥ 2 days resulted in a universal decrease in NME1 mRNA with variable changes in NME2 mRNA. Conclusion: NME1/2 mRNA levels are not indicative of prognosis in freshly processed bone marrow from CN-AML patients <60ys. However, delayed processing is associated with the development of an NME2 expression pattern with high prognostic relevance, maintenance of high NME2 in samples with reduced NME1 being a strong indicator of increased event-free survival. These results suggest that subjecting leukemic cells to defined challenges ex-vivo may reveal phenotypic properties of high relevance to treatment optimization. Disclosures: No relevant conflicts of interest to declare.