Multiple myeloma pathophysiology

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Hannan Javed, M.D.[2]; Haytham Allaham, M.D. [3]; Shyam Patel [4]

Overview[edit | edit source]

Multiple myeloma, a disorder of clonal late B-cells, arises from post-germinal center plasma cells that are normally involved in production of human immunoglobulins.^[1][2][3] Although the exact pathogenesis and the stage at which myeloma cells arise from post-germinal B-cells remain unclear, a variety of factors have been implicated in pathogenesis of multiple myeloma. Of these, chromosomal abnormalities are thought to be the most important. It has been suggested that all cases of multiple myeloma pass through MGUS. Renal involvement by multiple myeloma is catergorized into three entities: light chain cast nephropathy, monoclonal immunoglobulin deposition disease, and amyloidosis. Osseous involvement by multiple myeloma is based on cytokine and cellular interactions that lead to bone breakdown. On microscopic histopathological analysis, abundant eosinophilic cytoplasm, eccentrically placed nucleus, and Russell bodies are characteristic findings of multiple myeloma.^[4]

Pathophysiology[edit | edit source]

Normal physiology and development of plasma cells[edit | edit source]

• Plasma cells are terminally differentiated B-cells which function to produce immunoglobulins. plasma cells arise from B lymphocytes through a series of events. These events take place in bone marrow and secondary lymphoid tissues.
• These events include immunoglobulin heavy-chain (IgH) VDJ gene rearrangement, migration into lymphoid tissues, somatic hypermutation (SMH) in the IgH and light-chain genes, antigen selection, switch in Ig class from IgM to IgG, IgA, IgD, or IgE, migration back to bone marrow and terminal differentiation into plasma cells.^{[5] [6][7]}

Stem cells → Pre-B cells → Immature B-cells → Mature B-cells (naïve) → Activated B-cells → Memory B-cells and Plasmablasts → Plasma cells

• During these events, B cells express variety of surface markers which may be used to denote their developmental stage such as CD19, CD20, CD27, CD38, CD10, CD138. These markers and IgH chain gene sequences are important to define the nature of myeloma cells.^[5][6][7]
• Plasma cells secrete antibodies which function in humoral immunity.^{[5][6] [7]}
• Plasma cells are typically polyclonal and can respond to a variety of antigens, which helps combat infections. Under normal circumstances, there is no monoclonality amongst the plasma cell population in a person. These plasma cells are functionally intact in their ability to contribute to humoral immunity.^[5][6][7]
• Transcription factors such as interferon regulatory factor 4 (IRF4), BCL6, B-lymphocyte-induced maturation protein 1 (BLIMP1, also known as PRDM1), paired box gene 5 (PAX5) and X box binding protein 1 (XBP1) play an important role in differentiation and survival of plasma cells.^[8][9][10]

Interferon regulatory factor 4 (IRF4) → Down-regulation of BCL6 → Up-regulation of B-lymphocyte-induced maturation protein 1 (BLIMP1) → Down-regulation of paired box gene 5 (PAX5) and Up-regulation of X box binding protein 1 (XBP1).^[11][12][13]

For more information on plasma cells, click here.

Normal physiology and development of Immunoglobulins[edit | edit source]

• Immunoglobulins (also known as Antibodies) are proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
• They are made of a few basic structural units called chains; each antibody has two large heavy chains H and two small light chains L. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess.^[14][15]
• Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.^[14][15]
• Class switch recombination (CSR), a region specific deletion recombination process, generates different immunoglobulin (Ig) isotypes by replacing one switch region with another. This process leads to enhanced functionality of immunoglobulins.^[16][17]
• Class switch recombination (CSR), just like somatic hypermutation (SMH), requires the expression of activation-induced deaminase (AID) and both are dependent on creation of double-stranded DNA breaks (DSB) in the Ig loci.^[18][19][20]

For more information on immunoglobulins, click here.

Pathogenesis[edit | edit source]

The pathogenesis of multiple myeloma is complex and probably is a result of multiple and multi-step oncogenic events such as hyperdiploidy and deregulation of cyclin D1, and interaction of myeloma cells with marrow environment. Recently it has been suggested that all cases of multiple myeloma pass through an MGUS phase. The events surrounding the progression of MGUS into multiple myeloma are not well-defined but environmental and genetic factors have been proposed to have an association. A brief description of events thought to play a role in pathogenesis of multiple myeloma is given here.

Biology of myeloma cells[edit | edit source]

• Myeloma cells are malignant plasma cells which exhibit the morphology of mature plasma cells or plasmablasts. Majority of these cells seem to be mature, differentiated and quiescent, appearing to be without long-term proliferative potential.^[21][22]
• Cells with similar morphology to mature B-cells but with immunoglobulin gene sequences and idiotype similar to myeloma cells have also been found in myeloma patients, both in bone marrow and the peripheral blood.^[23][22]
• Current hypothesis is the presence of functional heterogeneity in myeloma cells with only a minor group of specialized myeloma cells exhibiting clonogenic growth potential. Although studies have shown the presence of myeloma cells sub-populations with distinct phenotypes and functionality in multiple myeloma such as presence of CD138+ and CD138− sub-populations, the phenotype of these so-called clonogenic cells is yet to be determined. ^[23][22][24]
• Myeloma cells express surface markers associated with plasma cells such as CD138, natural killer (NK) cells such as CD56/NCAM, T cells such as CD28, and sometimes the pan-B-cell marker CD20. Presence of CD19 and CD20 on sub-population of myeloma cells may suggest either early-lineage precursor for myeloma cells or possible de-differentiation of myeloma cells.^{[5][22][25][26][27]}
• Myeloma cells show complex chromosomal abnormalities and mutations. Studies have demonstrated the presence of translocations in up to 75% of the myeloma cases.^[5]

Environmental and hereditary factors[edit | edit source]

• The Key environmental and hereditary factors thought to confer a greater risk of developing multiple myeloma or play a part in pathogenesis are summarized in the table below.^{[8][28][29][30][31][32][33][34][35][36][37]}

Chromosomal aberrations[edit | edit source]

• Two major sets of chromosomal abnormalities seen in multiple myeloma are translocations with extensive IgH rearrangements and numerical aberrations, either trisomy or monosomy.^[8][5]

Translocations[edit | edit source]

• Majority of translocations in multiple myeloma take place through class switch recombination (CSR). But few may occur through D_H–J_H recombination, possibly in early stages of B-cell development such as pre-B cell stage.^[8][38]
• Majority of these translocations put oncogenes such as cyclin D1 (CCND1), CCND3, fibroblast growth factor receptor 3 (FGFR3), multiple myeloma SET domain (MMSET; also known as WHSC1), MAF and MAFB under the influence of enhancers present at Ig loci. .^{[5][8][39][40]}
• These translocations cause over-expression of these oncogenes that in turn leads to dysregulation of cell-cycle such as increased cell survival, growth, progression and DNA repair. One of the key abnormality being the increased G1/S transition during the cell-cycle.^[8][41]
• Many other translocations may occur in later phases of multiple myeloma. One important example is translocation of MYC, an oncogene that encodes transcription factors.^[8][42][43]

Hyperdiploidy[edit | edit source]

• Gain of the odd numbered chromosomes including 3, 5, 7, 9, 11, 15, 19 and 21 is the other major abnormality observed in multiple myeloma.^{[5] [8]}
• The mechanism of this gain phenomena is not well understood, one hypothesis being the gain of chromosome during single mitosis.^[8]

Mutations in myeloma[edit | edit source]

• Studies have demonstrated the presence of approximately 35 mutations per sample in multiple myeloma. These mutations cause loss of tumor suppressors and NFKB alterations.^[8]

Tumor suppressors[edit | edit source]

• These mutations result in cell-cycle dysregulation with an increase in G1/S transition. Some of these mutations are mentioned in the table below.^{[8][44][45][46]}
• The loss of tumor suppressors allows the cells to survive and grow without check points.

NFKB alterations[edit | edit source]

• NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex which regulates DNA transcription, production of cytokine and cell survival. NF-κB plays a key role in generating cellular responses to stimuli such as stress, free radicals, heavy metals, ultraviolet radiations, and foreign antigens. NF-κB is also crucial in response of the immune system towards infections.^[47][48][23]
• Deregulated NF-κB system leads to increased growth factors and cytokines such as IL-6 which promote growth and survival in myeloma cells.^[23]
• Mutations in multiple genes can cause activation of NF-κB system which in turn plays a role in pathogenesis of multiple myeloma. Some of these mutations, that have been documented in genes regulating NF-κB pathway, are mentioned below.^[49][50]

Epigenetic changes in multiple myeloma[edit | edit source]

• Epigenetic events such as methylation of DNA and histones play an important role in pathogenesis of multiple myeloma.
• Of these events, the most notable is the global hypomethylation and gene-specific hypermethylation that takes place during progression of MGUS to myeloma.^[8]
• A subset of patients with t(4;14) translocation show prominent DNA methylation changes that lead to MMSET overexpression.^[8][39]
• Other dysregulations related to epigenetics in multiple myeloma may effect KDM6A (UTX), a histone demethylase, mixed lineage leukemia (MLL) protein, lysine demethylase 6B (KDM6B) and homeobox A9 (HOXA9).^[8][51]

Bone marrow microenvironment and multiple myeloma[edit | edit source]

• Bone marrow microenvironment comprises of bone marrow stromal cells (BMSCs), extracellular matrix (ECM) proteins such as collagen, fibronectin and laminin, and the extracellular fluid containing cytokines and growth factors. This microenvironment is conducive to normal hematopoiesis but this also helps myeloma cells to have increased replication activity and anti-apoptotic resistance.^[5][8][52]
• One of the key processes is the interaction and adherence of myeloma cells with bone marrow stromal cells (BMSCs) and extracellular matrix proteins through cellular adhesion molecules (CAMs) such as CD44 (H-CAM), CD56 (N-CAM), members of the CD49 integrin family (including very-late antigen VLA-4 and VLA-5), lymphocyte function-associated antigen-1, syndecan-1, and selectin.^[5][8][53]
• This interaction leads to activation of p42/44 mitogen-activated protein kinase, and nuclear factor κB (NF-κB) which cause increased adhesion molecules, both on myeloma cells and bone marrow stromal cells. These events ultimately lead to increased production of cytokines. Of these cytokines IL-6, TNFα, BAFF, IGF and HGF are of particular importance.^{[5][8][54][55][56]}
• These events cause localization of tumor cells in bone marrow, increased proliferation and survival, and resistance to apoptosis and chemotherapy. Bone marrow niche thus plays a crucial role in pathogenesis of multiple myeloma.^{[5][8][54][55][56]}
• Decreased cellular adhesion molecules such as CD56 and chemokine receptors such as CXCR4 on myeloma cells lead to decreased adhesion to bone marrow stromal cells and immune evasion, allowing the myeloma cells to spread outside the bone marrow.^[57][58]

Cytokines in multiple myeloma pathogenesis[edit | edit source]

• A variety of cytokines have been implicated in pathogenesis of multiple myeloma. Some of the relevant cytokines, their mechanisms and effects on tumor cells have been summarized in the table below.^{[5][59][60][60][61][62][63][64][65][66][67][68]}

Pathophysiology of renal involvement[edit | edit source]

Abnormal antibody fragments are produced in multiple myeloma and are deposited in various organs, such as the kidneys. There are three major forms of renal damage in patients with multiple myeloma.

• Cast nephropathy: End-organ damage to the kidneys is typically due to light chain cast nephropathy. The pathophysiology of this type of renal involvement is based on light chain deposition in the renal tubules, which results in obstruction. Free light chains are readily filtered at the glomerulus and are reabsorbed in the proximal tubule of the nephron. This reabsorption occurs via the megalin-cubulin transport system.^[69] In patients with multiple myeloma, there is excess production of free light chains, and the ability of the nephron to resorb light chains in the proximal tubule cannot meet the demands of the freely filtered light chains. This results in excess secretion of free light chains in the urine (known as Bence-Jones protein). Eosinophilic proteinaceous casts and crystalline structures can be seen. Cast formation occurs in the tubules due to excess abundance of free light chains that interact with Tamm-Horsfall proteins in the thick ascending loop of Henle.^[69] Tubular obstruction ensues, triggering local inflammation which results in interstitial nephritis and fibrosis.^[69] The onset of cast nephropathy can be very quick, requiring prompt treatment. Risk factors for development of cast nephropathy include monoclonal immunoglobulin secretion of >10 g/day, sepsis, and volume depletion.^[70] Patients can also develop Fanconi syndrome, resulting in dysfunctional reabsorption ability by the proximal tubule, and type II renal tubular acidosis.

• Monoclonal immunoglobulin deposition disease (MIDD): In this subtype of renal involvement by multiple myeloma, the initial pathophysiological process is filtration of monoclonal immunoglobulins and subsequent deposition of immunoglobulins along the tubular or glomerular basement membrane.^[70] Deposits of immunoglobulin can have a similar appearance as Kimmelstein-Wilson lesions (seen in diabetes). The immunoglobulins can appear fibroblast-like.

• Light chain amyloidosis: The pathophysiology of renal involvement by light chain amyloidosis begins with beta-pleated sheet formation in the tubules or glomeruli. Beta-pleated sheets form as a result of electrostatic interactions between heparan sulfate proteoglycan and amyloid proteins. Amyloid fibrils usually consist of immunoglobulin light chains (usually lambda light chain) and deposit in the basement membrane. The size of the fibrils vary from 7 to 10 nanometers. A diagnosis of this type of renal involvement is made by the visualization of apple green birefringence upon Congo red staining of the renal specimen.^[70] It is frequently associated with nephrotic range proteinuria, in which greater than 3 grams of protein is excreted daily.

Pathophysiology of osseous involvement[edit | edit source]

Bone disease characterized by progressive osteolytic bone lesions leading to bone resorption is hallmark of multiple myeloma. Abnormal bone remodeling is thought to be the cause. It has been reported that up to 80% of the patients have characteristic osteolytic bone lesions at presentation and 60% of the patients with multiple myeloma will develop at least one pathological fracture at some stage.The pathophysiology of bony involvement in multiple myeloma is complex and is briefly described here..^[71][72][73]

Increased osteoclastic activity[edit | edit source]

Osteoclasts are large, multinucleated cells of monocyte–macrophage lineage and play a crucial role in bone remodeling. Myeloma cells, in addition to their direct interaction with other cells, produce and release a number of factors which promote osteoclast differentiation and activation. Some of these factors and interactions have been described below in the tables.^[71][74][75]

.

Decreased osteoblast activity[edit | edit source]

Inhibition of osteoblasts leading to decreased bone formation is now considered to be a critical event in bone disease in multiple myeloma. This inhibition leads to bone loss as well as inability to repair the osteoytic lesions caused by increased osteoclastic activity. Several pathways and factors have been associated with suppressed osteoblastic activity. Some of them are mentioned below in the table.^[71][74][75]

Renal involvement in multiple myeloma[edit | edit source]

Renal involvement is common in multiple myeloma as up to 50% of the patients go on to develop kidney disease at some point during the disease course. Half of these patients recover kidney function while the rest of them develop chronic kidney dysfunction. The causes of renal involvement in multiple myeloma are multiple and diverse. They can broadly be classified into Ig dependent and Ig independent mechanisms. Some of them have been discussed below in the table and then described briefly.^[76][77][70]

Cast nephropathy (myeloma kidney)[edit | edit source]

• Normally circulating monoclonal free light chains are relatively freely filtered through the glomerulus and a receptor-mediated process leads to their endocytosis in proximal tubule cells. They bind to the tandem scavenger receptor system cubilin/megalin, followed by endocytosis through the clathrin-dependent endosomal/lysosomal pathway. They are then degraded within lysosomes.
• In multiple myeloma, myeloma cells produce excess of monoclonal free light chains that overwhelms the capacity of the proximal tubule cells to absorb and catabolize them. This leads to large amounts of monoclonal free light chains reaching distal renal tubules.
• In distal renal tubules, they interact with Tamm-Horsfall protein (uromodulin), a glycoprotein produced in the medullary thick ascending limb of the loop of Henle in kidneys. This interaction leads to formation of myeloma casts.
• These casts block the glomerular flow, compromising renal function and may lead to proximal tubular atrophy. This may also contribute to interstitial fibrosis.
• This blockage leads to increased luminal pressure, resulting in decreased blood flow and causing further renal injury.
• The excessive amounts of monoclonal free light chains being absorbed in proximal renal tubules leads to induction of apoptosis and DNA damage in proximal renal tubules.
• Free light chains also leads to increased pro-inflammatory cytokines such as IL-6, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor α (TNFα) through transcription factors nuclear factor kappa B (NFκB) and mitogen-activated protein kinases.
• All these events leads to kidney injury and inability of renal cells to repair the injury. On histopathological studies, chronic tubulointerstitial nephropathy with marked tubular atrophy, laminated casts in the tubules, and interstitial fibrosis may be observed.

Hypercalcemia[edit | edit source]

• Hypercalcemia in multiple myeloma primarily results from increased osteoclastic bone resorption. It is the second most common cause of renal injury in myeloma patients.
• Hypercalcemia leads to intra-tubular calcium deposition and vasoconstriction in renal vasculature. The resultant decrease in GFR not only causes renal injury by itself but also favors cast formation.
• By impairing renal concentrating ability through resistance to anti-diuretic hormone, it may also cause nephrogenic diabetes insipidus. Polyuria may develop, leading to hypovolemia. Net effect is decreased GFR, concentrated urine, reduced urine flow, and pre-renal azotemia.

Light-chain glomerulopathy[edit | edit source]

• Deposition of immunoglobulins either in the form of amyloid or non-amyloid, typically resulting in non-selective proteinuria.
• Amyloid deposits are fibrillar structures, consist of variable region fragments of the light chain, and are seen primarily within the glomeruli.
• Some patients may have predominant vascular amyloid deposits rather than glomerular deposits and present with renal failure instead of nephrotic syndrome.

Light chain deposition disease (LCDD)[edit | edit source]

• Non-fibrillar, granular deposits are topically observed in mesangial area along with thickening of basement membrane which resembles type II membranoproliferative glomerulonephritis or diabetic glomerulopathy. The light chain deposits may also be observed in renal vasculature in addition to mesangial area.
• Typical clinical picture is that of nephrotic syndrome but renal impairment is more severe with almost all patients developing renal failure.

Immune dysfunction in multiple myeloma[edit | edit source]

• Immunodeficiency is an important characteristic of multiple myeloma and is thought to associated with increased risk for infections, and affects disease progression and response to treatment.^[5][78]
• The typical clinical picture is that of global immunosuppression. The interaction between myeloma cells and bone marrow stromal cells and cytokines produced as a result of this interaction are thought to play a key role.^{[5][78][79][80]}
• Following abnormalities have been demonstrated in myeloma patients:^[79][81][80]
  • Decreased numbers of natural killer cells, B-cells, and memory T cells
  • Decreased non-myeloma immunoglobulins
  • Decreased expression of cell-surface markers on CD4+ and CD8+ T cells such as CD28 and CD152. These markers are associated with cell-signaling.
  • Abnormal signal transduction in T-cells
  • Abnormalities in cytokine production
  • Abnormalities in antibodies response and production
  • Aberrations in B-cells maturation
  • Defects in natural killer cells and antigen presenting cells

Gross Pathology[edit | edit source]

• 

  Vertebrae in multiple myeloma
  (Image courtesy of Melih Aktan M.D.)

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  Calvarium in multiple myeloma.
  (Image courtesy of Melih Aktan M.D.)

Microscopic Pathology[edit | edit source]

On microscopic histopathological analysis, multiple myeloma is characterized by the following:^[4]

• Abundant eosinophilic cytoplasm
• Eccentrically placed nucleus
• Clock face morphology of the nucleus due to chromatin clumps around the edges
• Russell bodies which are eosinophilic, large (10-15 micrometres), homogenous immunoglobulin-containing inclusions
• Dutcher bodies which are PAS stain +ve intranuclear crystalline rods

• Shown below is a series of microscopic images seen in multiple myeloma:

• 

  Bone marrow aspiration in multiple myeloma.
  (Image courtesy of Melih Aktan M.D.)

• 

  Bone marrow biopsy in multiple myeloma.
  (Image courtesy of Melih Aktan M.D.)

• 

  Bone marrow in multiple myeloma.
  (Image courtesy of Melih Aktan M.D.)

• 

  Bone marrow in multiple myeloma.
  (Image courtesy of Melih Aktan M.D.)

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  Multiple myeloma slide with intermediate magnification^[4]

• 

  Multiple myeloma slide with high magnification^[4]

• 

  Multiple myeloma slide with russell bodies^[4]

References[edit | edit source]