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Free light chains and Free light chain assays Ali Bazargan Haematology Registrar.

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Presentation on theme: "Free light chains and Free light chain assays Ali Bazargan Haematology Registrar."— Presentation transcript:

1 Free light chains and Free light chain assays Ali Bazargan Haematology Registrar

2 © New Science Press Ltd. 2003 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.

3 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.

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6 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.

7 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

8 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

9 Schematic diagram of IGH gene rearrangement,

10 Schematic diagram of the ordered IGK and IGL gene rearrangement Recombination signal sequence

11 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)

12 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;

13 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.

14 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

15 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.

16 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).

17 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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20 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;)

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22 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).

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24 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.

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26 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)

27 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

28 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 ),

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31 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).

32 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),

33 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)

34 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).

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38 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.

39 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.

40 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.

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