Immunoproliferative Disorders: Monoclonal Gammopathies Flashcards
What are gammopathies?
Gammopathy is a disorder characterised by abnormality of gamma globulins
- Hypergammaglobulinaemia are monoclonal or polyclonal
- Monoclonal gammopathy can be benign or malignant and results from a single clone of plasma cells producing high levels of a single class and type on antibody, referred to as a monoclonal protein, M protein or paraprotein eg. multiple myeloma
- Polyclonal gammopathy is a secondary disease with increased levels of 2 or more antibodies produced by several clones of plasma cells.
Immunoglobulins and kidney
- Immunoglobulins are made of heavy and light chains (2 of each). They are produced in different regions of the ER and are assembled into a single functional antibody before secretion.
- But excess light chains are produced, which are secreted-k (kappa) free light chains as monomers, lambda free light chains as dimers or larger polymers
Excess light chains –> Kidney and pass through glomerulus –> Reabsorbed in proximal tubule –> Degraded into smaller peptides (REPEAT)
The excess light chains enter the kidneys and due to their low molecular weight, they pass through the glomerulus and into the proximal tubule where they are reabsorbed and degraded into smaller peptides which are then recycled.
- 1 - 10mg of light chains reach the distal tubule and into the urine daily under normal conditions
- In monoclonal gammopathies, the capacity of the proximal tubule can be overwhelmed so much that higher levels of light chains are seen in the urine.
Normal and abnormal immunoglobulin production
- Total plasma immunoglobulin results from millions of plasma cell clones in the bone marrow and lymph nodes
- The amount of plasma cells produced is regulated by homeostasis, so that it produces the required amount of antibody to deal with an infection
- However, sometimes a plasma cell clone will undergo a chromosomal rearrangement which allows it to overcome this control of cell growth, and consequently it produces millions of copies of itself
- Alongside the extensive growth replication, the cell secretes its programmed immunoglobulin isotype (class) in such large amounts it can be seen as a discrete band by electrophoresis. Because it has been produced by a single clone it can be referred to as a monoclonal immunoglobulin
- Monoclonal gammopathy is defined as a disease characterised by monoclonal immunoglobulin in the serum and/or urine
Monoclonal gammopathy
Can be divided into several types:
- Monoclonal gammopathy of undetermined significance (MGUS)
- Myeloma/multiple myeloma
- Waldenstrom primary macroglobulinaemia
- Light-chain disease
- Heavy-chain disease
B-Cells
- Each type of tumour corresponds to a normal state of B-cell development or differentiation. Tumour cells have similar properties to their normal cell equivalent
- In a B-cell tumour, every cell has an identical immunoglobulin gene rearrangement which proves they have originated from the same cell (clone)
- B-cell tumours from different patients have different rearrangements which reflects the the diversity of rearrangements found in normal B-cells of healthy people
- Tumours retain the characteristics of the cell type from which they arose, particularly when the tumour is relatively differentiated (and thus slowly growing)
- Human tumours corresponding to all stages of B-cell development have been found
- Among the characteristics retained by the tumours is their location in the lymphoid tissues: tumours derived from mature naive B-cells are found in lymph node follicles, forming follicular centre cell lymphoma, whereas plasma cell tumours (myeloma) found in the bone marrow
- B-cell tumours have been useful to learn about the immune system (because can get large quantities of the cells) the first sequences of heavy and light chains were obtained from people with multiple myeloma
Monoclonal gammopathy of undetermined significance (MGUS)
- Most common type of monoclonal gammopathy
- Patient does not have symptoms, so is picked up by doing an investigation for another condition
- Monoclonal band is less that 30g/L
- Less than 10% plasma cells in the bone marrow
- The incidence of MGUS increases with age, affecting 1% in over 50’s and increasing to 10% in over 80’s
- Higher incidence in African-Carribean patients
- Most patiets die with MGUS rather than because of it. However, 1% transform into the malignant form of MG, known as multiple myeloma
- Patients with MGUS should be monitored annually to identify patients who have progressed and then be treated
Multiple Myeloma (MM)
Characteristics
- Malignant monoclonal proliferation of bone marrow plasma cells and accumulation of plasma cells in the bone marrow
- Lytic bone lesions
- Monoclonal immunoglobulin in serum and/or urine
Clinical Features
- Bone pain
- Anaemia
- Renal failure
- Infections (defects in humoral, not cell immunity)
- Hyperviscosity syndrome
- Proteinuria in 50% of patients excreting abnormal amounts of Bence-Jones proteins (light chains)
Laboratory Features
- Electrophoresis of serum/urine shows a monoclonal protein in 90% of patients, increased serum calcium, low haemoglobin, raised mean cell volume (MCV) and increased erythrocyte sedimentation rate
* Incidence is 5 - 7 per 100,000, very rare in those less than 40 years old
* Median presentation age is 65 - 70 years
* MM is thought to possibly result from excess production of IL-6
* Paraprotein or M protein - antibody produced in MM. Paraprotein is the type of antibody that is characteristic of and produced by the malignant cells in MM. Becasue this paraprotein is produced by a clone of cells, it has a single light chain type, kappa or lambda
* Rapidly progressive if untreated patients die within a year. With standard chemotherapy,life expectancy following a diagnosis was 3 years, although survival is now increasing due to more aggressive treatments
MM: Ig
The type of protein produced by the clone of abnormal plasma protein cell varies:
- IgG (60%)
- IgA (>20%)
- IgD (1%)
- IgE (very rare)
- IgM (extremely rare)
- Kappa / Lambda light chains (10%)
* Less than 1% of myelomas are so abnormal they cannot secrete the Ig, but can be seen in the cytoplasm which is known as non-secretory myeloma. On bone marrow examination, there are large numbers of plasma cells, greater than 10% of nucleated bone marrow cells and their cells are packed with one immunoglobulin isotype
* More rarely, the cells cannot produce Ig/Ig fragments which is known as non-producing myeloma
* The antibody is monoclonal which means there is one type of ab produced due to the proliferation of one particular plasma cell
* The monoclonal light chain may be synthesised in excess and as it has a low molecular weight, it is excreted by the kidney. This is known as Bence-Jones protein
* This monoclonal antibody is shown by doing serum protein electrophoresis followed by densitometry and is evident as a tall peak
* All patients with suspected MM require a 24 hour urinalysis by protein electrophoresis to determine the presence of Bence Jones proteinuria and kappa or lambda light chains
* In patients with renal involvement, Fanconi syndrome may be the presenting manifestation. This syndrome is characterised by amino aciduria, hyponatremia, hypoglycaemia associated with glucosuria, low anion gap and hyperchloremic metabolic acidosis
* Serum protein electrophoresis will identify an M protein as a narrow peak/spike in the Y, beta or x2 regions of the densitometer tracing. If MM is strongly suspected and electrophoresis is ‘normal’, serum immunofixation may be more sensitive in identifying a small protein. Only rarely is there no monoclonal proliferation (ie, non-secretory MM) which occurs in 1% of patients.
Light chain myeloma
- Light chain myeloma often have immune paresis
- Immune paresisis suppression of normal immunoglobulin production by a malignant plasma clone
- Low immunoglobulin levels may be the only sign of a small serum free light chain or an IgD monoclone (reason for doing immunofixation on samples with low immunoglobulin even if no abnormal electrophoretic band is seen in serum electrophoresis)
Multiple Myeloma: Bone
- Bone damage is due to the breakdown of the homeostatic control of bone remodelling by osteoclasts and osteoblasts. Osteoclasts dissolve bone and in the myeloma patient their activity is upregulated where the myeloma cells are found, as there is an imbalance in the regulatory system. Plasma cells often proliferate in bone, forming areas which are replaced by a circular clone of plasma cells. This results in bone lesions which look like holes punched out of the bone
- MM is not confined to a specific bone or location within a bone (often found in a skull but can effect any part of the skeleton). It tends to invove the entire skeleton. When only one lesion is found it is called a plasmacytoma which doctors believe is an early, isolated form of MM
- Plasma cells release IL-6 which act as an osteoclast activating factor which may be responsible for the lytic lesions and the associated general osteoporosis
What is an osteoclast?
A large, multi-nucleated cell formed from differentiated macrophages responsible for the breakdown of a bone
MM: Diagnostic Criteria
Two of the following criteria:
1. Paraprotein in serum and/or urine
2. >20% abnormal plasma cells in bone marrow or monoclonality of plasma cells (>12% with one light chain type)
3. Osteolytic bone lesions
Clinical Findings
- The symptoms of myeloma seem to be quite harmless and are most often due to the organ system most affected by the disease. The patient may present with renal failure, anaemia, or severe bone pain.
- More than 30% of the bone marrow cells can be replaced with these malignant plasma cells, and this causes a decrease in the production of normal cells, which causes the following to develop in these patients:
* Due to decreased production of blood cells due to infiltration of bone marrow:
- Anaemia
- Thrombocytopenia (decreased number of platelets) - bleeding
- Leukopenia (decreased white blood cells) - increased infection rate
* Due to bone lesions
- Bone pain, fractures
- Hypercalcaemia
- Osteoporosis
* Dues to increased serum Ig:
- The high concentration of Ig in the serum hyperviscosity (increased thickness of the serum from increaser protein concentration). To counteract this, there is an increase in plasma volume to try and reduce the serum viscosity. The increase in plasma volume results in a decrease in the number of red blood cells, leukocytes and platelets, making the anaemia worse, as well as the leukopenia, thrombocytopenia caused by the bone marrow damage. Increased serum viscosity can also lead to platelet abnormalities. Individuals can also have poor perfusion (presumably due to viscosity of blood). Individuals have impaired immunity due to low white blood cell count, the second reason is because they have large quantitites of a monoclonal antibody due to large numbers of plasma cells, and this reduces the concentation of normal polyclonal Ig.
Serum hyperviscostiy –> Increase in plasma volume –> Decrease in RBC’s, leukocytes, platelets
Due to increased plasma protein and hypercalcaemia: The high concentration of plasma protein (due to Ig levels) high levels of calcium causes kidney damage resulting in renal failure (due to deposition of paraprotein in the kidney). Patients may also present with renal failure. Usually, only proteins of small molecular size are filtered by the normal renal glomerulus, so light chains can be excreted, but whole immunogloblulin molecules cannot. In patients who have significant glomerular damage, leakage of all serum proteins into the urine may occur, including the serum paraprotein. When there is renal failure, excretion of Bence-Jones protein may be reduced, then it may be detected in the blood
MM: Laboratory Investigations
- Complete blood count and differential
- Anaemia - normocytic and macrocytic
- Thrombocytopenia
- Increased erythrocyte sedimentation rate (FSR)
- Peripheral blood film
- Rouleaux formation which is caused by the increased concentration of paraprotein)
- Sometimes there is low numbers of neoplastic cells
- Bone marrow biopsy
- 10 - 90% of plasma cells are usually abnormal
- MM results from uncontrolled proliferation of plasma cells
- These malignant plasma cells account for more than 15% of of the bone marrow cells
- Replacement of normal bone marrow cells with the plasma cells causes a decrease in the production of normal cells and accounts for the symptoms seen. Plasma cells are involved in the production of Ab, so patients with MM have very high serum Ig levels
- Very high serum Ig levels
- Due to large number of plasma cells, patients with MM have veryhigh levels of Ig in their serum but have a decrease in normal immunoglobulin production
- Ig concentrations are usually determined by:
- turbidimetry (a method for determining the concentration of a substance in a solution by the degree of cloudiness or turbidity it causes or by the degree of clarification it induces in a turbid solution)
- nephelometry (by measuring the intensity of scattered light often at right angles to the incident beam)
- IgE is often measured by ELISA
- The larger the abnormal plasma clone, the greater the immunoglobulin concentration. Therefore, it is useful to measure antibody levels to measure response to treatment
UK guidelines
- IgA and IgG isotype monoclones >30g/L
- ## IgD monoclones <10g/L
Serum protein electrophoresis
- In serum protein electrophoresis, a small quantity of serum is loaded at one end of the agrose gel. An electrical current is applied to the buffer and current moves through the gel, it brings negatively charged proteins with it.
- Albumin moves the quickest towards the anode, then alpha-1 globulins, then alpha-2 globulins, beta globulins and then gamma globulins. The movement of each molecule depends on:
1. Charge, size and shape of the molecule affects how quickly these can go through the pores
2. pH of buffer affects the charge of the molecule
3. Ionic strength of the buffer: higher ionic strength can result in better separation
4. Amount of current and time it is applied for, greater current for longer can result in more movement
5. Temperature
6. Electroendosmosis is the movement towards the cathode because of buffers movement toward cathode
After the gels have run, the current is switched off, and the gels are fixed (this keeps the separate proteins in place and prevents diffusion) and the gel is stained to make the proteins visible. The gels are then analysed by densitometry, which involves passing light through the gel. The amount of light that is absorbed by the protein bands appears as peaks on the trace. - The protein albumin is present in the serum in high amounts so appears as a darkly stained tight band on the gel as all the molecules have the same charge
- Gamma globulin is composed of many different Ig molecules with slightly different charges, so it appears in normal serum as a lighter wide band with broader range ofelectrophoretic mobilities and the densitometry tracing is a shorter, broader peak
- The serum protein electrophoresis patterns produced by tumour cells in MM and macroglobulinaemia show a sharp peak in comparison to the broad peak of a normal individual (showing a polyclonal gammopathy caused by infection - lots of different Abs present) The different tumour types show subtle differences in the serum protein electrophoresis, generally in the mobility of the Ig peak
