Intact immunoglobulin multiple myeloma (IIMM) - theoretical considerations of sFLC and Ig measurements

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10.1. Introduction

Serum concentrations of FLCs and intact monoclonal immunoglobulins result from the balance between production and clearance rates. Production rates vary, not only between different patients but also in individual patients as their tumours progress or respond to treatment. Salmon and Smith in 1970 ^[1], showed that for IgG myeloma, average synthetic rates per myeloma cell, per minute for IgG varied from 12,500 to 85,000 molecules between different patients. However, this was constant over time which means that changes in total synthetic rates reflect changes in tumour mass for an individual patient. This is presumably also true for monoclonal FLC synthesis.

Clearance rates are more complicated. For sFLCs, there is normally rapid clearance by renal glomeruli but in renal failure, clearance is by general pinocytosis with a half-life of 2-3 days. Between these two half-lives there is a continuous range depending upon the degree of renal impairment. For IgA and IgM the half-lives appear constant at 5-6 days, with clearance by pinocytosis, while for IgG, clearance is prolonged to 21 days by saturable recycling receptors (Brambell receptors; more recently called neonatal receptors [FcRn]). These various influences on the serum concentrations of FLCs and immunoglobulins mean that both isolated and serial measurements may not reliably relate to tumour size or changing tumour size.

These variable clearance rates apply equally to monoclonal immunoglobulins and their normal counterparts. Hence the value of sFLC κ/λ ratios, that inherently compensate for varying clearance rates as renal function changes. The same argument applies to IgG monoclonal immunoglobulins; ratios of the monoclonal Igs to normal immunoglobulins (eg. IgGκ/IgGλ) numerically compensates for varying removal rates. This is discussed in detail later (see Hevylite, Chapter 32).

In addition, other factors need consideration. Large molecules such as IgM and to a lesser extent IgA and IgG are affected by changes in blood volume. In contrast, the smaller FLC molecules are 70-80% extravascular and are least affected. Furthermore, sFLC κ/λ ratios automatically compensate for volume changes.

The purpose of this chapter is to analyse the theoretical basis for the utility of sFLC measurements in patients with multiple myeloma who produce both intact monoclonal immunoglobulins and FLCs.

10.2. Half-life of serum free light chains

Figure 10.1 Calculated serum halflife curves for different immunoglobulin molecules. (A) monomeric κ; (B) dimeric λ; (C) monomeric κ with renal failure; (D) IgA and (E) IgG. (Courtesy of N Evans and M Chappell).

Figure 10.2 Diagram of FcRn structure with binding of IgG and albumin molecules. Albumin and IgG are bound non-competitively (Courtesy of J Hobbs).

Figure 10.3 Recycling of IgG molecules by FcRn receptors. (Courtesy of J Hobbs).

As indicated in Chapter 3, the dominating clearance mechanism for sFLCs is filtration through the renal glomerular fenestrations. At 25kDa, monomeric FLC molecules (usually κ) have a half-life of 2 hours while dimeric molecules (usually λ) have a halflife of 4-6 hours (Figure 10.1). Serum FLCs that are more highly polymerised have longer half-lives. The 200 to 300-fold shorter serum half-life of FLCs compared with IgG (21 days) allows a much more sensitive evaluation of changing monoclonal protein production and hence tumour size, during treatment ^[2].

The half-life of FLCs becomes prolonged to 2-3 days when there is renal failure. Under such circumstances, clearance is by general pinocytotic mechanisms that account for the removal of many serum proteins, including albumin. This is particularly relevant at high sFLC concentrations when they directly cause renal damage, thereby further increasing sFLC concentrations. The relationship between sFLCs, urine FLCs and tumour progression is shown in Figure 3.10. Fast rises in sFLC concentrations suggest accelerating tumour growth but a contributing cause is prolongation of the half-life from renal failure. sFLC κ/λ ratios correct for varying clearance rates because both FLCs are removed by the same mechanisms. Such measurements are helpful when renal function is abnormal or changing.

10.3. Half-life of IgG, IgA and IgM and recycling by FcRn receptors

Under normal circumstances, serum proteins that are too large for renal filtration (>65kDa) have a half-life of 2-3 days. The removal mechanism is by pinocytosis which occurs in all nucleated cells as they obtain their essential nutrients from plasma. However, for IgG the half-life is prolonged to 20-25 days by FcRn receptors ^[3][4][5]. These proteins have a structure similar to Class I MHC molecules with a heavy chain of 3 domains and a single domain light chain comprising β₂-microglobulin (Figure 10.2). Furthermore, these are the same receptors that transport IgG from the pregnant mother to the developing foetus in the last trimester of pregnancy.

These heterodimeric FcRn molecules protect both IgG and albumin from acid digestion and recycle them back to the cell surfaces to be released in the slightly alkaline environment of the blood. The process occurs many times under normal circumstances so that the halflives of both IgG and albumin extend from 3 to 21 days (Figure 10.3). Interestingly, IgG and albumin molecules do not compete for the same receptor sites, but the exact mechanism and sites of binding are unknown.

When there are no functioning FcRn receptors as in familial hypercatabolic hypoproteinaemia (a disease associated with a genetic deficiency of β₂-microglobulin) the half-lives of IgG and albumin are only 3 days. Such patients have hypogammaglobulinaemia, not from failure of production, but simply from excess catabolism.

10.4. Tumour killing rates and half-lives of immunoglobulins

As a consequence of the long half-lives of intact immunoglobulins and particularly IgG, serum concentrations respond slowly to changing tumour production. This is evident from calculations of serum half-life curves for the different Igs and sFLCs in relation to tumour cell killing rates. Figure 10.4 shows calculated half-life curves for serum monomeric κ and serum IgG when the tumour cells are killed at different rates. The half-life of sFLCs adds only a few hours to the changing tumour production rates. Hence, tumour responses and complete remission can be rapidly identified. In contrast, the 21-day serum half-life of IgG is so long that it largely obscures differences in rates of tumour destruction. Furthermore, complete remission is identified many months later using IgG levels compared with FLCs. For the first time in MM, a short half-life marker is available that allows reliable assessments of tumour cell killing rates and earlier identification of tumour remission ^[6].

Figure 10.4 Calculated serum half-life curves for monomeric κ (A,B,C) and IgGκ (D,E,F) when the tumour cells are killed at: 50% per day (A, D); 50% in 4 days (B, E) and 50% in 2 weeks (C, F). (Courtesy of N Evans and M Chappell).

Figure 10.5 Calculated changes in κ FLCs and IgG during tumour relapse following chemotherapy. An increase in κ is seen months before an increase in IgG (arrows). Tumour kill rate of: 50% per day for κ (A) and IgG (D); 50% in 4 days for κ (B) and IgG (E); 50% in 2 weeks for κ (C) and IgG (F). 0.1% residual tumour with a doubling time of 30 days after the initial chemotherapy, has been assumed. (Courtesy of N Evans and M Chappell).

Figure 10.6 Variations in half-lives of IgG, IgA, IgM and other molecules (in days) at different concentrations. (Reproduced with permission from Trends Immunol^[5])

Figure 10.7 Changes in plasma and red cell volumes during treatment. Falls in serum immunoglobulin concentrations do not take account of these volume changes. Normal range (curved lines) and patients during treatment (This research was originally published in Blood ^[7] © the American Society of Hematology).

Production of both FLCs and intact immunoglobulins usually return when MM relapses (Chapter 12). When tumour recurrence is associated with synthesis of both types of molecule then they typically become detectable again at approximately the same time. (In practice, re-synthesis is not always synchronous so one type of immunoglobulin molecule may return before the other - see FLC breakthrough). If tumour re-growth occurs before all the monoclonal IgG has disappeared, then FLC measurements will be more sensitive for detecting tumour recurrence (Figure 10.5). This is because the rising concentrations of monoclonal IgG from tumour relapse are superimposed upon falling concentrations from the initial tumour response, thereby obscuring an early IgG increase. In contrast, sFLCs normalise early so increasing production by the relapsing tumour is not hidden by residual falling levels.

Factors affecting the rate of return of monoclonal proteins include: residual tumour production rates, length of time of complete remission, tumour doubling times and renal function. The interplay of these factors is complex and is different for each patient (see below and Chapter 12 for clinical examples).

An additional feature of the FcRn recycling system is that it saturates, presumably because of limited supplies of FcRn molecules. This results in a continuous relationship between IgG concentrations and its serum half-life (Figure 10.6). IgG half-life is 21 days at normal concentrations because most of the molecules are protected from acid digestion. At high IgG concentrations this falls towards 3 days as there are insufficient FcRn receptors to protect all IgG molecules. Hence, a patient presenting with, for example, a monoclonal IgG of 90g/L is producing far more than 3 times the amount of IgG than a patient presenting with 30g/L of IgG. In contrast, at low IgG concentrations, when FcRn receptor protection is maximal, IgG half-life extends to many weeks. For IgA and IgM, clearance rates are not concentration dependent, although there is presumably some mechanism that prolongs their half-lives beyond 2-3 days (5 days for IgM and 6 days for IgA compared with only 3 days for IgD).

This variation in catabolic rate has several consequences for serum measurements of IgG in patients with IIMM:-

1. Concentrations do not accurately relate to tumour production rates (in addition to any variations in the efficiency of Ig production by myeloma cells).
2. Incremental increases in concentrations under-estimate tumour growth.
3. Incremental falls in concentrations under-estimate reductions in tumour size.
4. At high concentrations, the rate of IgG fall after successful tumour killing is much faster than 21 days, while for low concentrations it is slower.
5. Background IgG is low when monoclonal IgG is high because of FcRn saturation. This is manifest as suppression of polyclonal IgG ^[8]. There is additional bone marrow suppression of IgG producing plasma cells in many patients.

Hence, the apparent speed of tumour response is much greater when monoclonal IgG concentrations are very high compared with when they are low. Since high concentrations are associated with larger tumour masses, and normally a worse outcome, these patients may have a fast initial fall in IgG concentrations but then may relapse quickly. Therefore, landmark studies of patients at 6 or 12 months, where patients have responded fast initially may identify patients with worse prognosis ^[9].

These processes mean that measurements of, and changes in, monoclonal IgG concentrations bear less relationship to tumour production rates than might be hoped. Early guidelines for MM included measurements of monoclonal IgG, IgA and IgM concentrations for tumour staging but it was later realised that such measurements were unhelpful. This was probably because of these mechanisms (Chapter 25 guidelines). Sullivan and Salmon, in 1972 ^[10], used this information to assess MM growth and regression and more recently (2007), Dingli et al.^[11], studied MM after ASCT to try and improve the design of clinical MM trials.

10.5. Blood volume changes in monoclonal gammopathies

Figure 10.8 Serum IgAλ and λ FLC following highdose melphalan and PBSCT. A half-life curve has been fitted to the λ concentrations. (Courtesy of G Pratt).

Figure 10.9 Concentrations of sFLC, total IgG and monoclonal IgGκ after high-dose melphalan. (Courtesy of G Pratt).

Changes in red cell volume (haematocrit) affect measurements of serum immunoglobulins in a direct manner ^[12]. If, for example, haematocrit rises from 20 to 40% during treatment there is less blood volume available for immunoglobulin molecules so their concentrations increase (assuming no changes in immunoglobulin mass). Moreover, changes in haematocrit do not affect the serum concentrations of IgG, A and M to the same extent. Because of their differing molecular sizes, 90% of the IgM, 50% of IgG and only 20% of FLCs are located in the vascular compartment. Hence, sFLCs are least affected because of their large extravascular distribution. Furthermore, sFLC ratios compensate for any changes in the individual FLCs or immunoglobulin concentrations, making them inherantly more reliable for judging changes in tumour mass.

Plasma volume changes also occur in monoclonal gammopathies ^[12]. Immunoglobulin molecules are osmotically active so that high serum concentrations lead to increases in plasma volume. This relates to the relative amounts in serum compared with the extravascular compartment, so again, molecular size is relevant. As the mass of monoclonal immunoglobulins fall during treatment, there is a reduction in the plasma volume (Figure 10.7) and vice-versa. Thus, changes in serum measurements underrepresent changes in tumour production. Again, sFLC ratios are not affected by changes in plasma volume.

All these various factors:- long half-life of serum IgG, saturable receptors and changes in haematocrit and blood volume, may explain why intact Ig measurements at diagnosis are not helpful as prognostic markers while sFLCs and sFLC ratios are prognostic (Chapter 11). For these reasons, when sFLCs ratios are abnormal (and Hevylite Igκ/Igλ ratios - see Chapter 32) in patients with IIMM they are usually better than IgG or IgA concentrations for predicting and monitoring responses to chemotherapy.

10.6. Changes in sFLCs and immunoglobulins during treatment

Figure 10.10 Changes in sFLC concentrations during 6 cycles of Bortezomib showing rapid responses to treatment and subsequent relapses. (Courtesy of GP Mead).

Examples of the effects of the different half-lives of the various immunoglobulins during patient treatment are shown below. Other examples can be found in Chapters 11 and 12.

Figures 10.8 and 10.9 compare sFLCs and intact immunoglobulin levels in patients treated with three courses of VAD followed by high dose melphalan (200mg/m²) on day zero, prior to PBSCT. In Figure 10.8, serum λ levels fell with a half-life of 1 day while the monoclonal IgAλ concentrations showed only a slight fall over a week. In Figure 10.9 the half-life of tumour destruction was approximately 3 days as judged by sFLC changes. In contast, IgG levels fell with a half life of 10 days. This is faster than the 21 days normally expected, not because the concentrations are very high, but probably because FcRn receptor synthesis has been impaired by the chemotherapy. Dexamethasone affects FcRn synthesis ^[13] and so may melphalan.

In a study of 17 patients with MM, the tumour-produced sFLCs fell 210-fold (range 2.1-1,678) whereas the intact immunoglobulin reduction was only 14-fold (range 1.5-88) ^[6]. The greater range of sFLC concentrations and the speed of fall indicates it is more sensitive to small changes in tumour size than intact immunoglobulin levels. This is particularly useful when the concentrations of intact monoclonal immunoglobulins are sufficiently low that quantitation by SPE is unreliable.

Sometimes, short term changes in monoclonal production are invisible using IgG measurements but clearly apparent using sFLCs. Das et al.^[14], studied sFLC concentration kinetics in patients receiving Bortezomib (Velcade). Six of 8 patients showed a response to treatment by EBMT/Blade criteria (>25% fall in paraprotein). In 3 of these patients the tumour sFLC showed a fall and rapid recovery within 10-20 days of a treatment cycle. These patients were monitored over multiple treatment cycles and showed repeated falls and rises in sFLC co-incident with treatment (Figure 10.10). When seen, the relapse of sFLC was rapid with a doubling time of less than 10 days. This may correspond to the biological half-life of proteosome inhibition and recovery rather than tumour killing and regrowth. By comparison, intact immunoglobulin monoclonal proteins did not show the same peaks and troughs. Generally the sFLC levels indicated disease response earlier than the immunoglobulin measurements. They concluded that monitoring patients with sFLCs provided an opportunity to follow the kinetics of tumour kill, which is obscured by the slow clearance of intact monoclonal immunoglobulins. They added that sFLC measurements indicated early tumour responses that could allow relevant changes of treatment strategy. This may have major bearing on the costs of treatment and utilisation of resources. Similar observations have been made by others using Bortezomib ^[15][16].

References