Pathogenesis

The development of myeloma is a multiple-step process that requires accumulation of genome alterations in the neoplastic plasma cell clone, and changes in the BMM that facilitate the clone’s expansion.

1.5.3.1 Aberrant gene mutations

The normal counterparts of the malignant plasma cells (myeloma cells) are the terminally differentiated B cells, which are known as the plasma cells or antibody- producing B cells. The molecular basis underlying the initiation of myeloma is mainly due to the normal biological functions of the B cells. B cells acquire the ability to produce specific immunoglobulin (Ig) through multiple rounds of somatic hypermutation, rearrangement of the Ig genes, and class switch recombination. B cells

< 10 % plasma cells ≥ 10 % plasma cells, ≥ 30 g/L paraprotein

Asymptomatic! CRAB events!

MGUS! SMM! Multiple Myeloma!

Multiple Myeloma Smouldering Myeloma Early!! Myeloma! MGUS!! like! MGUS

become activated when in contact with antigens, otherwise, these B cells normally arrest in the G0/G1 phase of the cell cycle as long-lived plasma cells in the BM or as memory B-cells in the lymph nodes until the next antigen stimulation. The genetic events needed for B cell development into plasma cells require DNA double strand breaks which can result in aberrant chromosomal translocations, some of which could lead to oncogene activation and tumour suppressors mutations [250, 251].

The progression to MM requires the accumulation of aberrant gene mutations. The primary early genetic abnormalities initiated in myeloma development are defined as the immunoglobulin heavy chain translocations, which are shared by MM and MGUS. In addition to the primary early gene mutations, the secondary late-onset gene mutations promote further myeloma progression from MGUS to MM. Complex chromosomal abnormalities generated by abnormal class switch recombination can result in the activation of oncogenes such as MYC and fibroblast growth factor receptor 3 (FGFR3) [252], loss of expression or mutation in tumour suppressor p53, and the inactivation of cyclin-dependent kinase inhibitors. These abnormalities are normally found in MM but rarely in MGUS.

Certain chromosomal abnormalities such as t(4;14), t(14;16), t(14;20), chromosome 13 deletion, chromosome 1 abnormalities and 17p13 deletion are associated with poor prognosis and resistance to therapy [253-256]. The t(14;16), t(14;20) chromosome abnormalities are associated with increased oncogene c-MAF expression and indicate poor prognosis in MM but not in MGUS [257]. The high expression of MAF protein in myeloma due to t(14;16), t(14;20) translocation confers innate resistance to proteasome inhibitor (bortezomib) [258]. The t(4;14) translocation occurs frequently in MM and results in the simultaneous deregulated expression of two oncogenes, FGFR3 and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 [252]. Deletions of 17p13, the genomic locus of the tumour suppressor gene p53, have been associated with a poor patient outcome [259]. Other genetic abnormalities involve epigenetic dysregulation and copy number abnormalities. Chromosomal abnormalities are present in 20-60% of newly diagnosed MM patients and in 60-70% of patients with progressive disease, indicating their importance in the pathogenesis of the disease [242]. This feature of dynamic gene mutations and chromosomes abnormalities makes MM

patients vulnerable to disease progression and prone to disease relapse, as patients easily present innate and develop acquired drug resistance [260, 261].

Figure 1.14 The pathogenesis of multiple myeloma. The progression of MM requires the accumulation of gene mutations and chromosomal abnormalities. Primary early mutation of the late stage matured B cells initiate the MM disease. The secondary accumulation of further gene abnormalities drives further disease progression. In rare cases, MM cells can also metastasise from the bone marrow into the blood stream causing plasma cell leukaemia. Illustration modified from Morgan et al. [251].

1.5.3.2 Bone marrow microenvironment and pathogenesis

Besides genetic mutations of myeloma cells, the hallmarks of MM progression include abnormal interactions between myeloma cells and the BMM, as well as aberrant angiogenesis [262]. BM is the semi-solid, spongy, gelatinous compartmentalized tissue found in the hollow spaces within the interior of bones. It is the major haematopoietic organ and a primary lymphoid tissue. BM consists of haematopoietic tissue and the associated supporting stroma. The BM haematopoietic tissue contains haematopoietic stem cells (HSCs), which can differentiate into precursors of all types of blood cells, erythrocytes, granulocytes, monocytes, lymphocytes and platelets [263]. The mesenchymal stem cells of the stroma can differentiate into a variety of supportive cells, such as osteoblasts, osteoclasts, chondrocytes, myocytes, fibroblasts, macrophages,

Post-germinal-

center B cell MGUS Smouldering myeloma Myeloma Plasma cell leukaemia

Ini$a$on' Progression'

Germinal'center'' Bone'marrow'' Peripheral'blood''

Primary'gene$c'events:'' •'Ig'heavy'chain'transloca$ons'' •'Cytogene$c'abnormali$es' ''' ' Secondary'gene$c'events:'' •  Acquired'muta$ons'' •  Oncogenic'ac$va$on'or'muta$on' •  Epigene$c'deregula$ons'' ''' ' Bone'destruc$on' Angiogenesis'

adipocytes, and endothelial cells [264-268]. Myeloma cells can manipulate and hijack this BM microenvironment for their own survival and progression.

Genetic abnormalities can alter the expression of adhesion molecules such as vascular- cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule-1 (ICAM- 1), and integrin alpha 4 (VLA-4) on myeloma cell surfaces. The adhesion of myeloma cells to hematopoietic and stromal cells induces the production of cytokines and growth factors that are produced and secreted by cells in the BM microenvironment, such as interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), insulin like growth factor 1, tumour necrosis factor, transforming growth factor β1, and interleukin-10, which stimulates myeloma cells growth, survival, migration, and angiogenesis [269- 271]. The adhesion of myeloma cells to extracellular matrix proteins (e.g. collagen, fibronectin, laminin, and vitronectin) triggers the upregulation of cell-cycle regulatory proteins and anti-apoptotic proteins [262].

The approximate levels of oxygen found in normal tissues are lower than the 20% oxygen level in the air (normoxia), which averages at about 5% oxygen (ranging from 3-7.4%). Most untreated cancers are hypoxic with median oxygen levels around 2% (approximately ranging from 0.3-4.2%) [272]. The normal BM is hypoxic, but a study has shown that the myeloma-infiltrated BM has decreased hypoxia indicating that myeloma-associated angiogenesis is functional [273]. Despite high vascular density in MM, the absolute BM oxygen level is still low, ranging from 1.5-4.2% oxygen with 2.7% on average [274]. Using pimonidazole hydrochloride as a nontoxic hypoxia marker, the BM HSCs niche is hypoxic (1.3% O2) and the pimonidazole hydrochloride staining pattern correlates with that of the HIF-1α protein [275]. These hypoxic niches inside the BM harbour quiescent HSCs and are critical for HSCs self-renewal, maintenance and function [268]. The disruption of the niches can affect the normal differentiation process and could potentially lead to myeloma malignancy [268].

The hypoxic BM environment offers a favourable environment for the development and survival of haematological malignancies such as MM. Under BM hypoxic conditions, the glycolytic phenotype can be enhanced via HIF-dependent transcription activities thus providing critical advantages for cell survival under hypoxia [27, 34, 48, 276].

in MGUS cells from BM biopsy samples, indicating that up-regulated HIF-2α

expression may be correlated with the malignant status of MM cells [277]. The up- regulated expression of HIFs in MM can promote angiogenesis and thus provide a beneficial microenvironment for MM cells evolving. Strong HIFs expression correlated with high vascular density and VEGF up-regulation in BM samples from 106 MM patients. High vascular density in 37 MM patients showed a significantly worse prognosis [278]. Reviewed by Irigoyen et al., hypoxia and HIF-mediated signalling pathways promote haematological tumour progression and relapse; and the inhibition of HIF can be explored as a treatment approach [279].

In solid tumours, cancer cells in regions with restricted supply of nutrients and oxygen would rely on different metabolic pathways such as glycolysis to support their growth [25, 280]. Similar to that of the solid tumour, different vasculature perfusions and complicated structure in the BM result in the malignant cells having only varied access to nutrients and oxygen, and therefor the malignant cells in the BM also presents with metabolic heterogeneity to ensure survival. A metabolomics study has indeed shown that biopsy specimens of osteolytic lesions display heterogeneous metabolism profiles from pathological fracture sites caused by MM [281].

To summarize, MM cells originate from and reside inside the BM before developing into circulating MM cells. The hypoxic BM microenvironment and the different types of supportive cells that coexist inside the niches provide ideal growth conditions to harbour MM cells, promote their growth and metastasis, and to assist resistance to chemotherapies by a complex network of cytokines, chemokines, adhesion molecules, proteolytic enzymes and other components of the extracellular matrix [262, 265, 266, 282]. The BM microenvironment is heterogeneous in structure, physiology, and metabolism. This complicated environment offers advantages for myeloma cell progression and survival. Treatment of MM should take this complicated microenvironment into consideration. Some novel agents used in the MM treatment can target the interaction between myeloma cells and stroma, such as the proteasome inhibitor and immunomodulatory drugs. There is metabolism context in the pathogenesis of MM in the BM microenvironment. Manipulating the metabolic phenotypes should be investigated as a possible approach to myeloma therapy.