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

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10.1. Introduction

Serum concentrations of free light chains (FLCs) and intact monoclonal immunoglobulins (Igs) reflect the balance between their 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 synthesis rates in different patients ranged from 12,500 to 85,000 molecules of IgG per minute per myeloma cell. However, this was constant over time which means that changes in total synthesis rates reflect changes in tumour mass for an individual patient. This is presumably also true for monoclonal FLC synthesis.

Clearance rates are more complicated. Under normal circumstances, sFLCs are cleared rapidly via renal glomeruli, but in renal failure clearance is by general pinocytosis resulting in half-lives of between 2 and 3 days, depending upon the degree of renal impairment. For IgA and IgM molecules, half-lives appear to be constant at around 5 to 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 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 rationale applies to IgG monoclonal immunoglobulins, with ratios of monoclonal Igs to normal immunoglobulins (eg. IgGκ/IgGλ) numerically compensating for varying removal rates. This is discussed in detail in Chapter 32.

In addition, other factors need to be considered. 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, which are 70-80% extravascular, are affected less. 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 multiple myeloma (MM) patients who produce both intact monoclonal immunoglobulins and FLCs.

10.2. Half-life of sFLCs

Figure 10.1 Calculated serum half-life 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 showing 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 predominant 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 half-life of between 4 and 6 hours (Figure 10.1). sFLCs that are more highly polymerised have longer half-lives. The 200- to 300-fold shorter serum half-lives 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].

As noted earlier, the half-lives of sFLCs become prolonged to 2-3 days when there is renal failure. This is particularly relevant at high sFLC concentrations as they directly cause renal damage, further increasing sFLC concentrations. The relationship between sFLCs, urine FLCs and tumour progression is shown in Figure 3.8. Accelerating rises in sFLC concentrations suggest accelerating prolongation of the half-life as a result of renal failure rather than rapid tumour growth. sFLC κ/λ ratios correct for varying clearance rates because both types of FLC 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 between 2 and 3 days and are removed by pinocytosis. This process occurs in all nucleated cells as they obtain their essential nutrients from plasma. However, for IgG (and albumin) 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). They are the same receptors that transport IgG from the pregnant mother to the developing foetus in the last trimester of pregnancy.

The heterodimeric FcRn molecules protect both IgG and albumin from acid digestion and recycle them back to the cell surfaces to be released into the slightly alkaline environment of the blood. This process occurs many times under normal circumstances, resulting in the half-lives of both IgG and albumin extending from 3 to 21 days (Figure 10.3). Interestingly, IgG and albumin molecules do not compete for the same sites on the receptor although the exact mechanism and sites of binding are unknown.

When there are no functioning FcRn receptors, as in patients with 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 excessive catabolism. FcRn receptors are functional in most nucleated cells including renal podocytes, and presumably affect urine IgG and uFLC concentrations (Chapter 24) ^{[6][7][8][9][10][11]}.

10.4. Tumour killing rates and half-lives of immunoglobulins

Due to the long serum half-lives of intact immunoglobulins, in particular IgG, their respective serum concentrations are slow to reflect changes in 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 such that 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 can only be identified many months later using IgG levels rather than FLCs. For the first time in MM, a short half-life marker is available that allows reliable assessment of tumour cell killing rates and earlier identification of tumour remission ^[12].

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 ^[13] © the American Society of Hematology).

Production of both FLCs and intact immunoglobulins usually recommence when MM relapses (Chapter 12). When tumour recurrence is associated with synthesis of both types of molecule, 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 reappear before the other - see FLC breakthrough – Chapter 12.7). 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 masked by residual falling levels.

Many factors affect the rate of return of monoclonal proteins, including 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 can reach saturation, 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). The 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, the 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 the following consequences for serum measurements of IgG in patients with intact immunoglobulin multiple myeloma (IIMM):

1. Concentrations do not accurately reflect tumour production rates (in addition to any variations in the efficiency of Ig production by myeloma cells).
2. Incremental increases in concentrations underestimate tumour growth.
3. Incremental falls in concentrations underestimate reductions in tumour size.
4. At high concentrations the rate of IgG fall after successful tumour killing is much faster, as the IgG half life is < 21 days, however, at lower concentrations where the half life is 21 days this fall is much slower.
5. Background IgG is low when monoclonal IgG is high because of FcRn saturation. This manifests as suppression of polyclonal IgG ^[14]. Additional bone marrow suppression of IgG-producing plasma cells occurs 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 ^[15].

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 the mechanisms described above. Sullivan and Salmon in 1972 ^[16] used this information to assess MM growth and regression, and more recently (2007), Dingli et al. ^[17] studied MM after autologous stem-cell transplant (ASCT) to try to improve the design of clinical MM trials.

10.5. Blood volume changes in monoclonal gammopathies

Changes in red cell volume (haematocrit) affect measurements of serum immunoglobulins in a direct manner ^[13]. 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, IgA and IgM to the same extent. Because of their differing molecular sizes, 90% of 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 inherently more reliable for judging changes in tumour mass.

Plasma volume changes also occur in monoclonal gammopathies ^[13]. 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 of the factors described above - 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, whereas sFLCs and sFLC ratios are helpful (Chapter 11). For these reasons, when sFLC ratios (and Hevylite Igκ/Igλ ratios - see Chapter 32) are abnormal 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 10.8 Serum IgAλ and λ FLC following high-dose 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).

Figure 10.10 Changes in sFLC concentrations during 6 cycles of Bortezomib (V) 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 (vincristine, adriamycin and dexamethasone) followed by high-dose melphalan (200mg/m²) on day zero, prior to peripheral blood stem cell transplant (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 contrast, 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 ^[18] and so may melphalan.

In a study of 17 patients with MM, tumour-produced sFLCs decreased 210-fold (range 2.1-1,678) whereas intact immunoglobulin reduction was only 14-fold (range 1.5-88) ^[12]. The greater range of sFLC concentrations and the speed of reduction indicate sFLC measurement 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 too low to allow reliable quantitation by serum protein electrophoresis (SPE).

Sometimes, short term changes in monoclonal production are undetectable using IgG measurements, but clearly apparent using sFLCs. Das et al. ^[19] studied sFLC concentration kinetics in patients receiving bortezomib (Velcade). Six of 8 patients showed a response to treatment by EBMT/Bladé criteria (>25% fall in paraprotein ^[20]). In 3 of these the tumour sFLC fell and then rapidly recovered 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 evident, 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 to 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 facilitate relevant changes of treatment strategy. This may have a major bearing on the costs of treatment and utilisation of resources. Similar observations have been made by others using bortezomib ^[21][22].

References