The classical myeloproliferative disorders (MPD), comprising essential thrombocythaemia (ET), polycythaemia vera (PV) and idiopathic myelofibrosis (IMF), are clonal premalignant haematopoietic neoplasms associated with activating mutations in signalling pathway molecules and a variable tendency to develop acute myeloid leukaemia (AML). This thesis examined genotype-phenotype associations of JAK2 and MPL mutations, the presence of clonal diversity in the MPD and the genetic events associated with progressive disease. Mutations in MPL were identified in 4% of ET and 7% of IMF but not in PV. Three different acquired MPL mutations were identified, one of which had been reported as an inherited allele. Although MPL mutations did not delineate a distinct clinical or histopathological subtype of ET, molecular testing provides an important new tool in the diagnostic armamentarium. Clones homozygous for the JAK2 V617F mutation were identified in female but not male patients with ET, suggesting that gender differences may be important in the determination of disease phenotype. In patients with two acquired genetic alterations, a signalling pathway mutation and a cytogenetic abnormality were usually present within the same clone. By contrast, coexistence of two signalling pathway mutations indicated the presence of biclonal disease that in two patients had arisen independently and not from a shared founder clone. RAS mutations were identified as potential cooperating events in patients with JAK2 or MPL mutant IMF. In patients developing AML following a JAK2 V617F-positive MPD, those with V617F-positive leukaemia had progressed via an accelerated phase of disease and harboured acquired alterations of RUNX1 or EVI1. V617F-negative leukaemias tended to follow directly from ET or PV, and loss of the JAK2 mutation by reversion to wild-type due to mitotic recombination, gene deletion or gene conversion was excluded. The thesis concludes with a discussion of how clonal heterogeneity can be integrated into current models of MPD disease pathogenesis.

Thesis (M.A.)--Boston University

A 59-year-old man with lung cancer, peripheral blood leukocytosis and thrombocytosis without peripheral lymphadenopathy and hepatosplenomegaly was found to have pancreatic extramedullary hematopoiesis (EMH) in association with an 'atypical' myeloproliferative disorder. Studies for the Philadelphia chromosome and bcr-abl fusion product were negative. This is the first documented case in the literature of isolated EMH in the pancreas.

The formation of blood, hematopoiesis, is a dynamic process originating from a small number of hematopoietic stem cells (HSCs). To sustain hematopoiesis throughout life HSCs have the unique capacity to differentiate into all mature hematopoietic lineages as well as generating more HSCs by a mechanism referred to as self-renewal. However, the regulation of these processes is largely unknown. During embryonic development HSCs expand in the fetal liver, indicating that this environment supports HSC self-renewal. The LIM-homeobox gene Lhx2 is expressed in the fetal liver during this period and Lhx2 null mutant mice die in utero due to severe anemia caused by an environmental defect in the fetal liver. Embryonic stem cells differentiate in vitro, forming embryoid bodies (EBs) containing various tissues including hematopoietic progenitor cells. Introduction of Lhx2 into this system by retroviral transfer led to the generation of cytokine dependent HSC-like cell lines that were multipotent and expressed surface markers similar to embryonic HSCs. However, the specificity and efficiency of this event could not be elucidated. To further evaluate the function of Lhx2 expression during hematopoietic development, Lhx2 was introduced into an ES cell system where expression could be efficiently turned on. This approach revealed that Lhx2 induce self-renewal of distinct multipotent hematopoietic progenitor/stem cells present in the EB, with the ability to form HSC-like cell lines. The Lhx2 induced self-renewal is growth factor specific since stem cell factor and interleukin-6 are necessary and sufficient for this process. However, Lhx2 expression blocked erythroid differentiation and interfered with early ES cell commitment, indicating that the effect of Lhx2 is cell type specific. Since HSCs of early embryonic origin are inefficient in engrafting adult recipients upon transplantation, we wanted to address whether we could generate cell lines retaining this capacity by expression of Lhx2 in hematopoietic cells from adult bone marrow. This led to the generation of clonal and cytokine dependent HSC-like cell lines capable of generating erythroid, myeloid and lymphoid cells upon transplantation into lethally irradiated recipients. When transplanted into stem cell-deficient mice, they contributed to circulating erythrocytes for at least 18 months, revealing a remarkable potential for self-renewal and differentiation in vivo. However, expression of Lhx2 was maintained in vivo and most engrafted mice developed a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia. Thus, elucidation of the mechanism for Lhx2 function in HSC-like cell lines would give insights into both normal and pathological regulation of HSCs. Down-regulation of Lhx2 expression in HSC-like cell lines with inducible Lhx2 expression led to rapid loss of stem cell characteristics and differentiation into various hematopoietic cell types. Thus, global gene expression analysis comparing Lhx2+ HSC-like cell lines to their Lhx2- progeny would give insights into the molecular basis for Lhx2 function in stem cells. A number of differentially expressed genes overlapped with previously reported HSC enriched genes, further emphasizing the resemblance between HSCs and the HSC-like cell lines also at the molecular level. Moreover, a number of genes were identified with functions or expression patterns related to Lhx2 in other organs. Collectively, these data suggest that these HSC-like cell lines represent a relevant model system for normal HSCs on the molecular and the functional level as well as for evaluating Lhx2 function in the development of various tissues in the embryo as well as in disease.