Unravelling clinical heterogeneity in Philadelphia positive Acute Lymphoblastic Leukaemia (Ph+ALL)

Muir, Amy Lianne (2024) Unravelling clinical heterogeneity in Philadelphia positive Acute Lymphoblastic Leukaemia (Ph+ALL). PhD thesis, University of Glasgow.

Abstract

The understanding of the haematopoietic development process has increased exponentially over the past few decades through the advancement in technology in the field of haematology and immunology. By doing so, the classical two-er model of haematopoiesis has been enhanced to acknowledge the numerous developmental stages, cell subtypes, transcriptional alterations, transcript on factors and surface marker expression required for early lineage development. The result is a repertoire of immune cells with wide-ranging functions allowing for the rapid response to an gens and maintenance of long-term immunological memory. Hence, immune malfunction can have deleterious effects, as evidenced in leukaemia.

Development from early stem and progenitor cells to lineage-fixed effector cells has been studied in the context of leukaemia and has resulted in a deeper understanding of the biology of the disease and an improved outcome for pa ents. In both Philadelphia positive acute lymphoblastic leukaemia (Ph+ALL) and chronic myeloid leukaemia (CML), the disease driver is the constitutive activation of kinase BCR::ABL1. This kinase results from a translocation fusion event whereby the long arms of chromosomes 9 and 22 break and concurrently fuse together, producing the truncated Philadelphia chromosome. This event brings together the ABL gene on chromosome 9 and the BCR gene on chromosome 22 to form the proto-oncogene BCR::ABL1 which is found in the Philadelphia chromosome. This gene encodes for a protein of the same name which functions as the aforementioned constitutively active tyrosine kinase. BCR::ABL1 interacts with cell cycle and apoptotic pathways producing cells which rapidly and uncontrollably proliferate and which do not respond to pro-apoptotic signals. In CML, this fusion event occurs at the apex of blood cel development in haematopoietic stem cells (HSCs), resulting in the potential for BCR::ABL1 activity to affect cells in both lymphoid and myeloid lineages. In Ph+ALL however, the cell of leukaemic origin is under ques on. The orthodoxy is that the t(9;22) fusion event occurs in a lymphoid progenitor as BCR::ABL1 activity is usually exclusively observed in the lymphoid lineage by the overproduction of lymphoblasts. However, recent observa ons by Hovorkova et al (2017) has prompted further discussion into the cell of BCR::ABL1 origin in Ph+ALL.

A subgroup of paediatric Ph+ALL pa ents were observed by Hovorkova et al (2017) as having disease features similar to CML and a poor response to standard Ph+ALL therapeutic strategies. These patients were detected during investigations into minimal residual disease (MRD) monitoring where rearrangement of Ig/TCR genes (a hallmark of the la er phases of B cell development) were compared to BCR::ABL1 gene copy number. What was observed was a subgroup of patients who remained BCR::ABL1 posi ve despite being Ig/TCR negative, in essence displaying an eradication of Ig/TCR positive lymphoid cells but with a maintenance of a significant number of BCR::ABL1 positive cells. This contrasts with the current understanding of Ph+ALL where lymphoid-directed therapy would eradicate both blast cells and the leukaemic cell of origin (lymphoid progenitor) displaying concurrent negativity in both MRD methods. These patients also displayed multilineage disease involvement with large populations of myeloid cells at diagnosis, atypical in Ph+ALL where haematopoiesis is skewed toward production of lymphoid cells. The resultant theory is that this subgroup results from the BCR::ABL1 fusion event occurring prior to lineage commitment, either in a multipotent progenitor (MPP) or an HSC, just as in CML. Hence, this Ph+ALL subtype was designated as ‘CML-like Ph+ALL’ by Hovorkova et al (2017).

CML exemplifies the importance of accurately targeting and eradicating BCR::ABL1+ HSCs for the prevention of disease progression or relapse. The treatment of CML includes the direct targe ng of BCR::ABL1 activity through a class of drugs called tyrosine kinase inhibitors (TKI) in addition to chemotherapy however, Ph+ HSCs in CML (leukaemic stem cells (LSC)) are able to evade eradication by drugs through a quiescent state with little proliferative activity. Therefore, haematopoietic stem cell transplantation (HSCT) may be utilised to fully eradicate all immune cells and replace them with healthy donor cells, thus preventing disease progression or relapse. This multi-agent approach and TKI development has allowed CML survival rates to increase from almost certain death to survival of the vast majority of patients. The findings from Hovorkova et al (2017) display that the standard Ph+ALL treatment is inadequate for eradication of BCR::ABL1+ cells in pa ents with CML-like Ph+ALL, hence patients with this subtype risk relapse or disease progression similar to CML.

To determine the leukaemic origin of CML-like Ph+ALL, we utlised paediatric Ph+ALL bone marrow samples harvested at diagnosis and post-induction therapy (PI) and CML samples in the acute lymphoid blast crisis phase (CML-LBC). The current definition of CML-like Ph+ALL is the discordance between BCR::ABL1 copy number and Ig/TCR gene rearrangement MRD methods, however such information was not available for all samples used in this project and therefore, CML-like Ph+ALL samples could not be identified from the outset as specified by Hovorkova et al (2017). However, examination of Ph+ALL patient samples by flow cytometry displayed a subset of samples with large myeloid populations at diagnosis, concordant with findings from Hovorkova et al (2017). Additionally, this subset of patients displayed large numbers of HSCs and few lymphoid progenitors at diagnosis, atypical to what would be expected in a standard Ph+ALL sample. Sorted progenitor cells (HSCs and MPPs) with BCR::ABL1 fusion measured by FISH (fluorescence in situ hybridisa on) were detected in the majority of samples investigated across the Ph+ALL cohort, suggesting that BCR::ABL1 fusion in early stem and progenitor cells alone may be inadequate for establishment of the CML-like Ph+ALL subtype and perhaps, downstream transcriptional modifications determine multilineage involvement. RNAseq was used to investigate gene expression in the Ph+ALL and CML samples with findings displaying a subgroup of Ph+ALL samples which clustered distal to the rest of the cohort. Identification of differen ally expressed genes (DEGs) in these samples showed an upregulation of genes involved in myeloid leukaemia and LSC activity. All samples in this cluster had detectable Ph+ HSC/MPPs and a higher incidence of disease progression, relapse or death compared to the rest of the cohort, disease outcomes that support the findings from Hovorkova et al (2017) which display poor response to standard Ph+ALL treatment in the CML-like Ph+ALL subtype.

Our findings postulate that CML-like Ph+ALL can be characterised by atypical haematopoietic dynamics, t(9;22) occurrence in HSC/MPPs, expression of CML-associated genes and poor response to standard Ph+ALL treatment.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR180 Immunology; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Horne, Dr. Gillian A., Copland, Professor Mhairi and Halsey, Professor Chris
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84757
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Dec 2024 14:23
• Last Modified: 13 Dec 2024 12:13
• Thesis DOI: 10.5525/gla.thesis.84757
• URI: https://theses.gla.ac.uk/id/eprint/84757