Transcript Slide 1

Free light chains and
Free light chain assays
Ali Bazargan
Haematology Registrar
•An Ig molecule is composed of four
polypeptides, 2 light and 2 heavy chains.
•There are two types of light chain
•There are five types of heavy chain: , ,  (4
subtypes), , and . The type of heavy chain
defines the class of Ig: IgM, IgD, IgG, IgE, and
IgA, respectively.
Each heavy or light chain is composed of a
relatively invariable constant part and a
variable part. However, there are also
hypervariable regions interspersed throughout
the variable part of each chain. These form
the 3 dimensional structure of the Ag binding
site.
© New Science Press Ltd. 2003
The heavy chains have 4 domains each.
The amino terminal variable domains (VH)
are at the tips of the Y. They are followed by
3 constant domains: CH1, CH2, and the
carboxy terminal CH3, at the base of the
stem.
The hinge connects CH2 and CH3 (the Fc
fragment) to the remainder of the Ab (the
Fab fragments).
The light chains consists of 2 domains each,
variable (VL) and constant (CL), each
connected by a switch.
Functions of immunoglobulins
• The variable region of the Ig molecule
interacts with antigen to effect several
phenomena, including precipitation,
agglutination, and neutralization.
• Utilized in a variety of diagnostic laboratory
tests,
- immunoelectrophoresis,
-ELISAs,
-immunohistochemistry, and agglutination
tests.
• The functions of the constant region Ig:
-activation of the complement system,
-binding to cell surface receptors on
granulocytes or macrophages to initiate
phagocytosis and/or other activities.
Immunoglobulin gene rearrangements
•
During early B-cell differentiation in the
bone marrow (BM) the variable (V),
diversity (D), and joining (J) gene
segments of the immunoglobulin (Ig)
genes are rearranged in an ordered
fashion to generate the primary Ig
repertoire.
•
Ig heavy chain gene (IGH)
rearrangement precedes Ig light chain
gene rearrangement, and DH to JH
joining precedes VH to DJH joining.
•
These rearrangements are mediated
by a tightly regulated enzymatic
machinery involving several different
proteins operating at theDNAlevel,
which are controlled by the
recombination signal sequences
(RSSs) flanking each gene segment
Immunoglobulin gene rearrangements
• The process of allelic exclusion ensures that once a
functional VDJH rearrangement has been achieved, the
other IGH allele is generally excluded from further
recombination attempts.
• Following successful IGH recombination, the Ig light chain
loci proceed to rearrange
• Initial attempts occur at the Ig kappa locus (IGK), and if a
functional IGK rearrangement is not achieved the Ig lambda
locus (IGL) undergoes recombination.
• Usually, rearrangements of the IGL locus are accompanied
by deletion of the nonfunctional IGK rearrangements
Schematic diagram of IGH gene rearrangement,
Recombination signal sequence
Schematic diagram of the ordered IGK
and IGL gene rearrangement
Free light chains
•Serum FLC levels in normal individuals
-a median serum K level of 7.3 mg/l (95% range: 3.3–19.4 mg/l)
-a median L level of 12.7 mg/l (95% range: 5.7–26.3 mg/l)
(Bradwell et al, 2001; Katzmann et al, 2002
•L and K free light chains are cleared and metabolised by the kidneys.
•K light chains have a half-life of 2–4 h
•L light chains are cleared slightly more slowly at 3–6 h
•Up to 10–30 g of FLC can be processed by the proximal tubules via the
megalin/cubulin scavenger receptor (Batuman et al, 1998)
Free light chains
•With advanced renal failure the serum half-life of serum FLC is increased to
2–3 d (R/O by pinocytosis by reticulo-endothelial cells).
•Advanced renal failure: FLC levels 20–30 times normal (Bradwell et al, 2005)
•Ability of the kidneys to clear K faster than L is lost with a slight increase in
the K/L ratio. (K/L ratio from 0.49 to 0.7) (Katzmann et al, 2002).
•Serum FLC concentrations increase with polyclonal immunoglobulin
production due to generalised B-cell activation in a broad range of
inflammatory or infective conditions (Dispenzieri et al, 2001;
Renal metabolism of FLC.
•Once the tumour FLC production exceeds the capacity of proximal tubular
reabsorption there is an exponential increase in urinary FLC
• increasing damage to the proximal tubules, capacity to remove FLC
becomes increasingly impaired.
• with end-stage renal failure there is a fall in glomerular filtration and a
failure to excrete FLC and an exponential rise in both serum FLC and a fall
in urinary FLC.
Analytical issues with serum FLC assays
• Serum free light chain immunoassays consist of
polyclonal latex-conjugated anti-free light chain
antibodies
• The binding of the antibodies to light chain can be
measured by nephelometry or turbidimetry
Analytical issues with serum FLC assays
•No international standard exists for free light chains
•batches of antisera are subject to small variation
•Significant inter-instrument variability exists as shown in
National External Quality Assessment Service (NEQAS)
•The ideal practice is to re-run a previous sample alongside any
new sample and to use local standards or reference material for
controls.
•Polymerisation of monoclonal free light chains and the presence
of polyclonal light chains leads to the overestimation of
monoclonal light chains.
The diagnosis and monitoring of Bence Jones
only myeloma.
•Approximately 15% of multiple myeloma are light chain only
(Bence-Jones multiple myeloma) with FLC in the urine and
serum and a lack of an intact monoclonal immunoglobulin.
•Errors and failures in collecting urine and the handling of urine are a major
problem and also make serum estimation a more practical option.
•428 patients with a monoclonal gammopathy and a monoclonal urinary protein who had serum FLC
quantification at diagnosis. Reliance on serum studies alone would have missed two patients (0.5%).
•The study concluded that discontinuation of urine studies resulted in a minimal loss of diagnostic sensitivity
(Katzmann et al, 2006).
The diagnosis and monitoring of Bence Jones
only myeloma
•UK British Committee for Standards in Haematology (BCSH)/Nordic
Myeloma Study Group stated that serum FLC assays can be used as an
alternative to quantifying urinary light chains (Smith et al, 2005) Guidelines on
the diagnosis and management of multiple myeloma 2005 BJH
•UK Medical Research Council studies following chemotherapy 32% (26/82)
of patients,in complete remission by normal urinary FLC compared with only
11% (9/82) by assessment of serum FLC (Bradwell et al, 2003).
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Serum FLC in the diagnosis and
monitoring of non-secretory myeloma
•Non-secretory myeloma accounts for 1–3% of all myelomas
•defined by the absence of monoclonal immunoglobulins in serum and urine
electrophoresis tests.
•using the serum FLC assay it appears that less than 25% are truly nonsecretory,
with 75% producing low levels of monoclonal FLC
•UK MRC, 64 patient with non-secretary MM. 19/28 sera (68%) had an identifiable
monoclonal protein with elevated tumour FLC conc. and abnormal K/L ratios and a
further four patients had abnormally low levels of either K or L (14%) and the
remaining five patients had normal FLC levels. (Drayson et al, 2001).
•Re-analysis using immunofixation electrophoresis identified monoclonal bands in six
out of 28 sera but these bands were mostly weak and diffuse.
•In non-secretory myeloma patients with elevated serum FLC, the serum FLC can act
as a sensitive tumour marker for monitoring disease, allowing early identification of
relapses and responses (Drayson et al, 2001;)
FLC assays in the diagnosis and monitoring of
intact immunoglobulin myeloma
•More than 80% of patients with multiple myeloma secrete monoclonal
intact immunoglobulins.
•In the UK Myeloma IV-VIII trials, serum FLC concentrations were
abnormal in 96% of 493 myeloma patients with monoclonal intact
immunoglobulin at presentation (Mead et al, 2004)
•Re-analysis of this data identified 89% of patients with abnormal FLC
ratios
•Serum FLC ratio is therefore normal in approximately 10% of patients
with intact immunoglobulin myeloma
•Monitoring patients serially has shown that serum FLC assays can be
used to follow the disease course in nearly all multiple myeloma
patients (Mead et al, 2004).
FLC assays in the diagnosis and monitoring of
intact immunoglobulin myeloma
•The International Myeloma Working Group defined measurable disease as
having one or more of the following: (Durie et al, 2006)
-A serum M-protein >10 g/l,
-A urinary M-protein >200 mg/24 h
-Involved FLC level >100 mg/l (provided serum FLC ratio is abnormal)
If patients have measurable disease in only one of these measurements then that
measurement will be used to monitor and assess response.
•The new international uniform response criteria define a new more stringent
complete remission (sCR) (Durie et al, 2006) which has two additional criteria
added to conventional CR, namely
-The absence of clonal cells in bone marrow (by immunohistochemistry or
immunofluorescence) and
-A normal FLC ratio.
Serum FLC in MGUS, smouldering multiple myeloma
•MGUS is associated with progression to multiple myeloma (or related
lymphoid malignancy or AL amyloidosis) at a rate of 1% per year and
this rate does not change over time (Kyle & Rajkumar, 2006).
•Previous risk factors for progression identified by the Mayo group:
-the size and type of M protein (increased risk of progression with
IgM and IgA subtypes) (Kyle et al, 2002)
-bone marrow plasma cell percentage (Cesana et al, 2002)
-the presence of circulating plasma cells (Kumar et al, 2005)
-presence of Bence- Jones proteinuria and presence of
immunoparesis (Baldini et al, 1996)
Serum FLC in MGUS, smouldering multiple myeloma
•An abnormal FLC ratio was detected in 379 (33%) of 1148 patients and the
risk of progression in patients with an abnormal FLC ratio was significantly
higher, independent of the size and type of the monoclonal paraprotein
(95% confidence interval 2.3–5.5; P < 0.001). (Rajkumar et al, 2005)
•Using a risk stratification model incorporating the three risk factors
- abnormal serum FLC ratio,
- A monoclonal protein level >15 g/l
- Non IgG MGUS),
four risk groups could be identified and the 40% of MGUS patients with no
risk factors had only a 5% risk of progression at 20 years
Prognostic and clinical correlations of serum
FLC in multiple myeloma
•High serum levels of FLC associated with a rapid reduction in response
to therapy defines an aggressive myeloma subtype with a poor prognosis
(van Rhee et al, 2007).
•In a study of 94 multiple myeloma patients a tumour FLC concentration
above the median value correlated with elevated serum creatinine, lactate
dehydrogenase, extensive marrow infiltration, light chain only myeloma
and serum FLC was an independent prognostic factor.(Median baseline
sFLCR was 3·57 in κ-MM patients, 45·09 in λ-MM) (Kyrtsonis et al,2007),
Serum FLC assays as an earlier marker of
tumour response and relapse
•A theoretical advantage of serum FLC monitoring is the short serum
half-life of FLC compared to intact immunoglobulins.
•The half-life of serum FLC is 2–6 h (monomeric K being faster than
dimeric L) compared to the serum half-lives of intact immunoglobulin
which are 20–25 d for IgG (IgG3 is 8 d), 6 d for IgA, 3 d for IgD and 2 d
for IgE.
Following chemotherapy, changes in serum FLC concentrations and
intact immunoglobulins occur in parallel but FLC levels usually fall much
more rapidly, particularly with IgG myelomas (; Mead et al, 2004; Pratt
et al, 2006).
Serum FLC assays as an earlier marker of
tumour response and relapse
•Earlier disease assessment may be of value in detecting poorly
responding patients who need alternative forms of therapy
•Conversely the most rapidly responding patients may have a
worse outcome (van Rhee et al, 2007).
•Retrospective analysis of the MRC UK trials identified a significant
proportion of longer term survivors as having a poor response to
initial treatment (Drayson et al, 2007),
Serum FLC assay in the
diagnosis and monitoring of AL amyloidosis
•A paraprotein is detectable in the serum or urine in
approximately 50% of patients with AL amyloid
•The paraprotein level is less than 20 g/l in over 70% of
patients and above 30 g/l in less that 10% of patients (Kyle &
Gertz, 1995).
•Serum FLC assays provide quantification of the monoclonal
FLC in 90–95% of patients and give an abnormal FLC ratio
in about 90% of AL amyloid patients (Lachmann et al, 2003;
Katzmann et al, 2005)
Serum FLC assay in the
diagnosis and monitoring of AL amyloidosis
•
serum FLC a marker of amyloid load (Lachmann et al, 2003;
Dispenzieri et al, 2006)
•
Correlate with risk of death and serum cardiac troponin or natiuretic
peptide type B (Dispenzieri et al, 2006;)
•
Normalisation or reduction of the FLC level following treatment
correlates with survival (Lachmann et al, 2003; Dispenzieri et al, 2006;
Palladini et al, 2006).
Serum FLC: ? an alternative to urinary screening
Two important questions:
-whether such an approach would miss any pathology
-what the cost/quality implications are for such an approach.
•
Major argument for using serum alone tests
1)physicians/patients are unreliable in providing urine samples
2)laboratories frequently do not receive a urine specimen from a patient
(Hill et al, 2006).
•
43 (out of 923) false-positive serum FLC ratios from patients referred from
both primary care and a general hospital population, many with polyclonal
immunoglobulins and reduced glomerular filtration rates.(Hill et al 2006)
•
Serum FLC cannot replace 24-h urinalysis for the assessment of
proteinuria.
Summary
• 1 Users of the serum free light chain assay need to be aware of the
requirement for internal controls and reference material.
• 2 Serum free light chain assays have been an advance in the
diagnosis and monitoring of light-chain only multiple myeloma, AL
amyloidosis, non-secretory multiple myeloma and oligo-secretory
multiple myeloma. For light chain only myeloma it should replace
urinalysis in disease monitoring.
• 3 Serum free light chains have become incorporated into the new
response criteria for both multiple myeloma and AL amyloidosis.
• 4 Serum free light chain levels at diagnosis are an independent risk
factor for progression for MGUS, smouldering (asymptomatic)
multiple myeloma and solitary plasmacytoma of bone.
Summary
5 Serum free light chains at diagnosis and their rate of fall have prognostic
value in myeloma.
6 Serum free light chains can give an earlier assessment of tumour response
than intact immunoglobulin.
7 Serum FLC is abnormal in a significant percentage of other lymphoid
malignancies, particularly WM, CLL and mantle cell lymphoma but its value,
if any, in prognosis and monitoring is not yet known.
8 As a screening test, serum FLC could replace urine analysis for Bence Jones
proteins in clinical practice. In the UK, a urine sample is received in the
laboratory from less than 50% of patients investigated for plasma cell
dyscrasias, making serum FLC a considerably safer screening test than
urinalysis. The false-positive rate of serum FLC in this setting and the cost
implications of switching from urinalysis to serum FLC need to be
established. Serum FLC cannot replace 24-h urinalysis for the assessment
of proteinuria.
Follicular Responses