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INTRODUCTION . Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD, and Transient Abnormal Myelopoiesis, TAM) occurs in 10-30% of newborns with Down syndrome (DS). Approx. 20% of infants with TL go on to develop acute myeloid leukemia of DS (ML-DS), typically within the first four years of life. Somatic, clone-specific mutations of GATA1 are found both in the blasts of TL and ML-DS, are concordant within the same individual and thought to function as initiating event in the development of ML-DS. In contrast, additional mutations of cohesin complex and related genes (e.g. RAD21, STAG2, CTCF), epigenetic regulators (e. g. EZH2) and signal transducers (e.g. within RAS, JAK signaling pathways) have been identified only in ML-DS blasts and are thought to cooperate with mutant GATA1 in the progression from TL to ML-DS. It is not known whether these cooperating mutations already mark a minor subclone of TL blasts at birth - allowing, at least in principle, a genetic risk stratification of TL - or are acquired postnatally during the first four years of life. OBJECTIVES . We tested the functional impact of impaired function of cohesin complex genes, CTCF and EZH2 on the progression of TL to ML-DS. We asked if mutations representing putative genetic progression events were already detectable at birth in a minor clone of TL blasts or were acquired postnatally (during the first four years of life). METHODS. The spectrum of GATA1 and cooperating mutations was determined by whole exome sequencing in fractions of TL and ML-DS blasts sorted from blood and bone marrow samples of five patients who had successively developed both disorders including one with a relapse of ML-DS. Corresponding normal T lymphocyte fractions of each patient at the stage of TL and ML-DS served as controls. Numbers of blasts harboring specific mutations were quantified by digital droplet PCR (BioRad, Inc.). Primary TL cells were transduced with lentivirus encoding shRNA (pLVX-shRNA, Clontech, Inc.) to suppress expression of cohesin complex genes, CTCF and EZH2 and intrafemurally injected into 8 week old NSG recipient mice. Engraftment in the bone marrow was assessed 8 weeks later by flow cytometry and GATA1 mutational analysis and compared to TL cells transduced with control vector. RESULTS. TL blasts harbored fewer mutations than those of ML-DS. GATA1 mutations were concordant in TL and ML-DS blasts in the same patient, consistent with development of ML-DS from subclone of TL. Knockdown of RAD21 expression in primary TL blasts, mimicking loss of function mutation of a cohesin complex gene, resulted in significantly increased engraftment of transduced cells in xenograft recipients compared to controls. This finding is consistent with RAD21 loss of function mutations playing the role of a progression event. Mutations of cohesin complex genes (SMC1A, STAG2, RAD21), NRAS and other putative cooperating mutations (with mutant GATA1) were not detectable in any sample of primary TL blasts by either whole exome sequencing or digital droplet PCR. The same result was obtained with control T lymphocytes sorted from TL samples. ML-DS blasts in one case were oligo-clonal with regard to cohesin complex gene mutations. Relapse in this patient arose from a minor clone as defined by cohesin complex gene mutations; mutations of NRAS, KNASL1 and SMC1A were present in ML-DS blasts but absent at relapse. CONCLUSIONS . Increased engraftment of TL cells with suppressed RAD21 expression is consistent with a model in which RAD21 loss of function mutations function as a progression event in the development of ML-DS. Absence of detectable cohesin complex gene mutations and other putative cooperating events in TL blasts suggests these mutations are acquired during the first four years of life and do not mark a minor clone of TL blasts present at birth. Genomic screening of TL blasts at birth therefore is unlikely to predict the risk for development of ML-DS. Disclosures No relevant conflicts of interest to declare.

Background: Transient leukemia (TL) occurs in 30% of newborns with Down syndrome (DS) and typically resolves spontaneously. Approximately 20% of infants with TL go on to develop acute myeloid leukemia of DS (DS-AML) within the first four years of life. The blasts of both TL and DS-AML harbor somatic mutations of GATA1 . The objective of this study was to identify additional genetic events, which associated with the progression of TL to DS-AML. Methods: Leukemic blasts of TL, DS-AML and normal T lymphocytes were sorted from blood and bone marrow samples of five patients who successively developed both disorders. In addition, blasts of one patient with subsequent relapse of DS-AML were analyzed. Mutational spectrum and gene expression and were determined by exome sequencing and RNASeq (Illumina HiSeq2000). The presence of mutations, which were identified with this approach in DS-AML blasts, was examined by droplet digital PCR in TL blasts (BioRad QX200). Results: Blasts of TL overall harbored fewer mutations than those of DS-AML. Mutations of cohesin and RAS pathway genes were identified in a subset of DS-AML but not TL. In the patient who developed a relapse, different cohesin gene mutations were detected at initial diagnosis of AML and relapse; a minor clone present at initial diagnosis of AML emerged as the predominant clone at relapse. Concordant somatic GATA1 mutations were present in both TL and DS-AML blasts derived from the same patient. In contrast, other genetic events, which were detected in DS-AML blasts by exome sequencing, were confirmed to be absent in TL (by droplet digital PCR). The majority of differentially expressed genes showed higher expression levels in blasts of TL compared to DS-AML. They included genes encoding chemokines and related to IL1 and TGFb signaling. Conclusions: The pathogenic sequence starting with TL and culminating in AML is uniquely initiated in children with DS by somatic mutation of GATA1. In contrast, the events associated with the transformation of TL to DS-AML resemble progression factors also found in non-DS AML. These progression events were not detectable even in minor subclones of TL suggesting they are acquired after the onset of TL. This research was supported by funding from the Canadian Cancer Society Research Institute and Ontario Institute for Cancer Research. Disclosures No relevant conflicts of interest to declare.