Text

theses

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell cancers with overlapping laboratory, cytogenetic, molecular, and clinical features. According to the World Health Organization (WHO, 2016), there are three classic Philadelphia chromosome negative MPN (Ph- MPN) entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).

Currently, the genomic characterization of these diseases is based on conventional cytogenetics and the mutational status of three driver genes (JAK2, CALR, and MPL). Although other non-driver mutations may be present in up to 40% of patients, their clinical and prognostic relevance, at present, is not entirely clear. Cytogenetic abnormalities are absent in 90% of patients with ET, 70% of patients with PV, and 50% of patients with PMF. Moreover, up to 10% of patients do not harbor mutations in any of the three driver genes. Chromosomal microarray (CMA) abnormalities have been identified in ~80% of MPN patients but the application of this technology for prognostic clinical purposes has not been widely used. The goal of this dissertation was to gain additional insight into the genetic basis of these diseases by integrating three genomic technologies; conventional cytogenetics, next-generation sequencing (NGS), and CMA, each with its advantages and limitations. To achieve this goal, three groups of patients were investigated: (1) patients with advanced forms of Ph- MPN who required therapeutic splenectomy, (2) patients that progressed from the chronic form to more advanced forms of Ph- MPN that were associated with specific chromosomal abnormalities, and (3) cytogenetically normal patients with Ph- MPN that represent early stages of their disease.

The hypothesis in aim 1 was to determine whether the spleens of patients with advanced forms of Ph- MPN, such as myelofibrosis requiring therapeutic splenectomy, contain hematopoietic stem cells which harbor unique genomic aberrations that contribute to disease progression. The results of the integrated analysis showed a high concordance of genomic lesions between spleen and bone marrow cells suggesting a similar clonal architecture between these two tissues. In spite of the overall similarity, CMA identified additional novel genomic changes present in the spleen indicating that there are genomic differences between myelofibrosis hematopoietic cells that reside in the spleen and bone marrow. CMA results demonstrated for the first time recurrent gains within four genes: RHOC, RASA3, NCOR2, and TAF15, strongly suggesting higher genomic complexity of spleen cells. These candidate genes may play a role in the pathogenesis PMF and support the hypothesis that integrating genomic technologies is a valuable tool in the discovery of new genes that may be implicated in the genetic landscape of myelofibrosis.

Based on the above novel observations, it was postulated in aim 2 that application of this strategy may provide the molecular understanding of recurrent chromosomal abnormalities such as the gain of chromosome 1q and rearrangements of 12q that are present in patients with Ph- MPN who progress to advanced forms of the disease. Results from aim 2 showed that recurrent gain of 1q, identified in 6% (n=72) of 1,294 evaluated patients with MPN was accompanied by an increase in transcript levels of MDM4. Single cell assays demonstrated that gain of 1q was present in CD34+ cells. Further analysis also identified candidate genes among patients (n=24) with recurrent chromosome 12q rearrangements (MDM2, HMGA2, and WIF1). The combined application of both cytogenetic and CMA identified large and cryptic deletions of TP53, a known tumor suppressor gene. These observations strongly link, for the first time, recurrent chromosomal abnormalities and an increased level of MDM4 transcripts with perturbations of the TP53 pathway.

Finally, in aim 3, a set of nine genes implicated in the advanced forms of Ph- MPNs (RASA3, NCOR2, RHOC, TAF15, MDM4, MDM2, TP53, HMGA2, and WIF1), were validated to determine whether they are also present in patients who were cytogenetically normal. Integrated analysis of 184 patients with Ph- MPN yielded informative results in 96.1% indicating that it is possible to characterize, at the genomic level, the majority of MPN patients at diagnosis. Moreover, genomic changes in the nine gene panel did occur in patients who were karyotypically normal, although the frequency was higher in the cytogenetically abnormal patients. Among the cytogenetically normal patients, MDM4 was a sole CMA abnormality but when compared to the cytogenetically abnormal patients MDM4 co-occurred with six other genes. Aberrations involving TP53 occurred only in the cytogenetically abnormal patients and did not co-occur with MDM4. These findings suggest that abnormalities involving MDM4 may be an early genomic event in patients with PV and ET and may represent an alternative route to TP53 dysregulation.

The results and observations provided in this dissertation strongly suggest that the integration of these technologies provide novel insight into the genomics of patients with Ph- MPN that could not be achieved if each methodology was performed individually. The results also show that 96% of patients with Ph- MPN acquire different lesions, such as mutations, large structural and numerical aberrations, and for the first time intragenic as well as whole gene gains and losses of chromosomal regions. These lesions may be also detected in patients who are chromosomally normal and do not have driver gene mutations. The implication of this approach may be beneficial for both clinical and research purposes.

In conclusion, genomic lesions in Ph- MPNs may be present in different genes in different patients affecting the same pathway. Since one method alone cannot uncover the diversity of genomic lesions, to fully characterize these patients an integrated approach utilizing various testing modalities should be employed to fully understand the interplay of these genes in the pathogenesis of these neoplasms.

ETD_9952

Ph.D.

Includes bibliographical references

doi:10.7282/t3-pssr-wt05

ETD doctoral

The author owns the copyright to this work.