Immunoproliferative disorders 28/02/23

Question 1

What can hypergammaglobinlinaemias?

Answer 1

They can be monoclonal or polyclonal.

Question 2

What are gammapathies?

Answer 2

A disorder characterised by abnormality of gamma globulins (antibodies).

Question 3

Can monoclonal gammopathies be benign or malignant?

Answer 3

They can be benign or malignant and results from a single clone of plasma cells producing high levels of a single class and type of antibody, referred to as monoclonal protein, M protein or Paraprotein.

Question 4

What are examples of monoclonal gammopathies?

Answer 4

Multiple myeloma, Waldenstrom primary macroglobulinemia, light chain disease, heavy chain disease, and monoclonal gammopathy of undetermined significance.

Question 5

What are polyclonal gammopathies?

Answer 5

Polyclonal gammopathy is a secondary disease, with increased levels of 2 or more antibodies, produced by several clones of plasma cells.

Question 6

What is lambda?

Answer 6

A free light chain which is a dimer/large polymer.

Question 7

What is Kappa?

Answer 7

A free light chain which is a monomer.

Question 8

How are excess light chains removed from the body?

Answer 8

The excess light chains enter the kidneys and because of 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.

Question 9

How much light chain is secreted normally?

Answer 9

Normally 1-10 mg of light chains daily reach distal tubule and into urine.

Question 10

How much light chain is secreted in monoclonal gammopathies?

Answer 10

In monoclonal gammopathies the capacity of the proximal tubule can be overwhelmed, so much higher levels of light chains is seen in the urine.

Question 11

What is a monoclonal gammopathy?

Answer 11

Monoclonal gammopathy is defined as a disease characterised by monoclonal immunoglobulin in the serum and/or urine.

Question 12

What causes the total plasma immunoglobulin levels?

Answer 12

Total plasma immunoglobulin results from millions of plasma clones, polyclonal antibody response.

Question 13

What are plasma cells regulated by and why does it go wrong?

Answer 13

Plasma cell production regulated by homeostasis. Chromosome abnormalities allow plasma cells to overcome growth restraints. Growth of abnormal plasma clone which secretes its antibody (monoclonal antibody, paraprotein or M protein).

Question 14

What causes the B cells to become malignant and become malignant plasma cells?

Answer 14

In a B-cell tumour every cell has an identical immunoglobulin gene rearrangement. This proves they have originated from the same cell. B cell tumours from different patients have different rearrangements and this reflects 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 slow 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 naïve B cells are found in lymph node follicles, forming follicular centre cell lymphoma, whereas plasma cell tumours (called myelomas) are 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.

Question 15

What is MGUS?

Answer 15

MGUS is the commonest subtype of monoclonal gammopathy. Patient does not have symptoms due to the monoclonal gammopathy, so they are picked up doing investigations for another condition.

-Monoclonal band is less than 30g/L.
-Less than 10% plasma cells in the bone marrow.
-The incidence of MGUS increases with age, affecting 1% in >50s, and increasing to 10% in >80s.
-It has a Higher incidence in African-Caribbean patients.
.Most patients 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.

Question 16

What is multiple myeloma?

Answer 16

Multiple myeloma demonstrates the following characteristics:
-Cancer of the bone marrow
-Malignant monoclonal proliferation of bone marrow marrow plasma cells, and accumulation of plasma cells in the bone marrow
-Lytic bone lesions
-Monoclonal immunoglobulin in serum and/or urine
-Typically present with bone pain, anaemia, infections, renal failure, and sometimes hyper viscosity syndrome

Question 17

What are the typical laboratory tests and results for MM patients?

Answer 17

Laboratory features:
-Electrophoresis of serum or urine shows a monoclonal protein in 90% of patients
-Increased serum calcium, low haemoglobin, raised mean cell volume (MCV) and increased erythrocyte sedimentation rate

Question 18

What is the incidence of MM?

Answer 18

Give incidence overall it is a relatively common malignancy in the elderly, affecting 3 per 100,000, but is very rare in those less than 40 years. MM thought to possibly result from excess production of IL-6. Rapidly progressive if untreated, patients usually die within a year. With standard chemo, life expectancy following diagnosis was 3 years, although survival is now increasing due to more aggressive treatments. Median age for the disease is 65-70 years old.

Question 19

What are the types of paraprotein secreted?

Answer 19

The type of protein produced by the clone of abnormal plasma protein cells varies.
Most produce a whole immunoglobulin molecule comprising heavy and light chain molecules.

-IgG 60%
-IgA 20%
-IgD 1%
-IgE very rare
-IgM is even rarer

About 10% of myeloma patients have a clone producing one of the light chains only.
in less than 1 % of myelomas the cells are so abnormal they cannot secrete the Ig (but can be seen in the cytoplasm). This is known an 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, this is known as non-producing myeloma.

Question 20

What is the Bence Jones protein?

Answer 20

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.

Question 21

What do patients suspected of having MM require?

Answer 21

All patients with suspected multiple myeloma 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 characterized by aminoaciduria, hyponatremia, hypoglycaemia associated with glucosuria, low anion gap and hyperchloremic metabolic acidosis. If multiple myeloma is strongly suspected and electrophoresis is “normal,” serum immunofixation may be more sensitive in identifying a small M protein. Only rarely is there no monoclonal proliferation (i.e., non-secretory multiple myeloma), which occurs in 1 percent of patients.

Question 22

MM summary?

Answer 22

-All secretory myelomas produce excess free light chains
-If renal function is good it can rapidly deal with large quantities of monoclonal light chains
-Free light chains may be produced in quantities that overwhelm the tubules and appear in the urine as proteinuria
-Proteinuria is defined as the presence of protein in the urine. Proteins filtered through the glomeruli should be actively reabsorbed in the tubules and so proteinuria should normally be absent or minimal
-The Light chains are monoclonal and on urine electrophoresis form discrete bands and immunofixation determines whether κ or λ
-The monoclonal light chains are known as Bence Jones and they found in the urine of patients with renal failure
-As the myeloma progresses, the renal tubules suffer increasing damage resulting in deteriorating, and this and may be the presenting symptom

Question 23

What is immune paresis?

Answer 23

Light chain myeloma often have immune paresis. Immune paresis is 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 seen in serum electrophoresis).

Question 24

What happens to the bones during MM?

Answer 24

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 (lytic lesions/osteolytic lesions) that on x-ray look like holes punched out of bone.

Multiple myeloma is not confined to a specific bone or location within a bone (often found in skull but can affect any part of the skeleton). It tends to involve the entire skeleton. When only one lesion is found it is called a plasmacytoma. Most doctors believe that plasmacytoma is simply an early, isolated form of multiple myeloma. Plasma cells release IL-6 which acts as Osteoclast activating factor, which may be responsible for the lytic lesions and the associated generalised osteoporosis.

Question 25

What is the diagnostic criteria for MM?

Answer 25

-Paraprotein in serum and/or urine
->20% abnormal plasma cells in bone marrow or monoclonality of plasma cells (>12% with one light chain type)
-Osteolytic bone lesions

Question 26

What occurs in MM due to a decreased production of normal blood cells?

Answer 26

-Anaemia which results in fatigue, lethargy, dyspnoea
-Thrombocytopenia which results in increased bleeding
-Leukopenia which results in increased infection rate

Question 27

What occurs in MM due to the bone lesions?

Answer 27

-Bone pain, fractures, vertebral collapse
-Hypercalcaemia
-Osteoporosis

Question 28

What occurs in MM due to the increased serum Ig?

Answer 28

-Serum hyperviscosity which increases plasma volume, decreases RBC, leucocytes, and platelets
-Platelet abnormalities
-Impaired immunity due to decreased polyclonal Ig

Question 29

What occurs in MM due to increased plasma proteins and hypercalcaemia?

Answer 29

-Renal failure

Question 30

What would be seen on a MM peripheral blood film?

Answer 30

-Rouleaux formation
-Sometimes, see low numbers of neoplastic plasma cells

Question 31

What would be seen on a MM FBC and WBC diff?

Answer 31

-Anaemia which would show normocytic or macrocytic RBC
-Thrombocytopenia (low platelets)
-Increased erythrocyte sedimentation rate (ESR)

Question 32

What would be seen on a MM bone marrow biopsy?

Answer 32

-Large plasma cells making up 10-90% of the bone marrow. They are typically abnormal.

Question 33

How does the serum protein electrophoresis work?

Answer 33

In serum protein electrophoresis a small quantity of serum is loaded at one end of the agarose gel. An electric current is applied to the buffer, and current moves through the gel. As the 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 finally the gamma globulins.

The movement of each molecule depends upon:
-Charge, size, and shape of the molecule, as these affect how quickly it can move through the pores
-The Ph of buffer as this affects the charge on the molecule
-Ionic strength of the buffer as higher ionic strength can result in better separation
-Amount of current and time it is applied for as a greater current and time result in greater movement of protein
-Temperature
-Electroendosmosis, movement towards cathode because of buffers movement toward cathode

After the gels have run, the current is switched off, and the gels are fixed and 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.

Question 34

How does the immunofixation electrophoresis work?

Answer 34

The isotype (class) of the Ab produced by the tumour is determine by immunofixation electrophoresis. This involves serum protein electrophoresis (SPE) being undertaken, however 6 replicates of each serum undergo (SPE). Then antiserum to IgG, IgA, IgM, kappa light chains, lambda light chains are incubated with the gel, such that have a different antiserum added to each lane. The 6th lane is a control for total serum protein. There is a reaction of the antibody with the Ig in the patients sera which forms an immunoprecipitant reaction by forming a lattice which fixes the antibody to the immunoglobulin in the patients serum-Ig complexes in the gel. The gels are then washed in saline and this removes any proteins that have not precipitated (i.e., formed a lattice by binding to Antiserum to Ab). Gels are stained to show precipitated proteins.

Question 35

What would be seen on a MM flow cytometry?

Answer 35

-CD19: present on B cells, but lost when mature to plasma cells
-CD38: present on plasma cells
-CD45: present on all leucocytes
-CD56: found in 70-80% of cases and is prognostic marker (shows a poor prognosis)

Question 36

What are the biochemistry markers of MM?

Answer 36

-Urea
-Creatinine
-Uric acid
-Calcium

Question 37

What is the treatment for MM?

Answer 37

Patients were given a standard treatment of melphalan and prednisone (this had poor complete remission rates (5%). However, with newer high dose treatments followed by autologous stem cell transplantation (from blood or marrow), there has been a large increase in the complete remission rate to 40% and overall survival increased to beyond 5 years. There are lots of exciting treatments that are being actively researched or currently being trialled which you could incorporate into your case study. MM patients are given high dose chemo with stem cells harvested from either their BM or blood. Radiation therapy is given to reduce tumour cell growth in areas with bone pain. Plasmapheresis is done to remove Ig and may reduce the rate of renal failure in these patients.

Question 38

What are some medication treatments approved by NICE?

Answer 38

-2007: bortezomib monotherapy (Velcade) for treating relapsed multiple myeloma in adults.
-2011: bortezomib (Velcade) and thalidomide (Thalidomide Celgene) for treating multiple myeloma in adults.
-2016: panobinostat (Farydak) for treating multiple myeloma after at least 2 previous treatments.
-2019: daratumumab (Darzalex) with bortezomib and dexamethasone for previously treated multiple myeloma in adults.

Question 39

What is daratumumab?

Answer 39

Daratumumab, an anti-CD38 therapy, a cell-surface protein, resulting in tumour cell death by immune-mediated actions and apoptosis. FDA approval in 2015 and NICE approval in March 2018 (Daratumumab monotherapy for treating relapsed and refractory multiple myeloma).

Question 40

What is elotuzumab?

Answer 40

Elotuzumab is a monoclonal antibody that targets the signalling lymphocytic activation molecule family member 7 (SLAMF7) protein, thereby activating natural killer cells and mediating myeloma cell death.

Question 41

What are the supportive medication given for MM?

Answer 41

-Red cell transfusion
-Antibiotics
-Bisphosphonates

Question 42

What is Waldenstrom Primary Macroglobulinemia?

Answer 42

B cell disorder characterized by lymphoplasmacytic cell infiltration of the bone marrow and an IgM monoclonal gammopathy. Unknown cause but ~20% of patients have familial predisposition to the disease and other B cell malignancies. Significantly increased risk after exposure to Hep B and HIV. This is a malignant off shoot of B cell development, (before the myelomas), and patients have typical B cell neoplastic chromosome abnormalities (t(8;14), trisomy 12.

Question 43

What is light chains disease?

Answer 43

Accounts for 10-15% of monoclonal gammopathies. Occurs as often as Waldenstroms primary macroglobulinaemia. Only κ or λ monoclonal light chains or Bence Jones proteins are produced. Diagnostic workup is similar to other lymphoproliferative disorder.

Question 44

What is heavy chains disease?

Answer 44

Characterized by monoclonal proteins composed of the heavy chain part of the immunoglobulin molecule. Franklin disease is name for γ heavy chain disease
α heavy chain disease is the commonest type of heavy chain gammopathy, and often seen in men of Mediterranean descent. Heavy chain disease is rare.

Question 45

What type of B-cell tumor that arises from a germinal center B cell and does not express antigen receptor?

Answer 45

Hodgkin’s lymphoma arises from transformation of B cells in the germinal center. They do not express immunoglobulin, and the tumor cells are often cells of an unusual dendritic morphology (Reed-Sternberg cells).

Question 46

Which tumour has a B-cell line that has a single receptor specificity and resembles a naive B cell?

Answer 46

Mantle cell lymphoma.