Monitoring IIMM patients using sFLCs

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12.1. Introduction

Serum free light chain (sFLC) measurements are being widely used for monitoring patients with intact immunoglobulin multiple myeloma (IIMM). Monoclonal sFLCs are produced in ~95% of patients, are independent of intact monoclonal immunoglobulin production and have a short half-life (Chapters 10 and 11). Therefore, they are of use in all stages of the disease: during early treatment; while assessing response rates; for indicating remission and residual disease; and during relapse. This chapter analyses the key studies that have addressed these various issues. Although many other studies support the findings, to remain concise, the assessment has been restricted to those publications that contain the largest series of patients or the most important results. A complete list of publications is presented in Chapter 31.

12.2. Overview of serum free light chains for monitoring patients with intact immunoglobulin multiple myeloma

Figure 12.1 Monitoring of a MM patient using IgGκ and κ sFLCs. SPE gels are shown for each sample.

Figure 12.2 Monitoring of MM using IgGκ and κ sFLCs. SPE gels are shown for each sample. IFN = interferon. κNR : upper limit of κ FLC normal range.

The various attributes of sFLCs for monitoring patients are best demonstrated using some patient examples. Figure 12.1 shows a patient with multiple myeloma (MM) producing monoclonal IgGκ and monoclonal κ sFLCs (but no urine FLCs). During the initial therapy, κ concentrations fell rapidly with a half-life of approximately 20 days, while IgGκ, quantified using serum protein electrophoresis (SPE), only gradually returned to normal. The apparent half-life of IgGκ was between 100 and 200 days (depending, in part, upon which densitometric scanning device was used: white light scanners can under-read at high protein concentrations). The observed response comprised the tumour kill half-life plus the half-life of sFLCs or serum IgG. A similar pattern of sFLC and IgG responses were seen during a subsequent relapse and treatment period.

A further example is given in Figure 12.2, in which the IgGκ half-life was 50 days. In contrast, sFLCs were well within the normal range at the first measurement point following treatment (and presumably earlier), yet chemotherapy was continued for many weeks longer. In highly vulnerable patients it may be feasible to stop chemotherapy if sFLC levels have normalised in order to minimise drug side effects.

12.3. Bone marrow responses and short serum free light chain half-life

Figure 12.3 Accuracy of different blood tests for assessing bone marrow plasma cell volume in MM. Because of slow catabolism, IgG concentrations lag behind reductions in bone marrow plasma cell content during treatment.

The value of using sFLCs rather than IgG levels to accurately assess rates of response in IIMM is apparent from bone marrow sampling. During treatment, monoclonal plasma cell counts correlate better with changes in sFLCs (and serum β₂ microglobulin) than intact monoclonal IgG (Figure 12.3). In a study of 45 patients by Mead et al. ^[1] sFLC κ/λ ratios had the highest concordance with bone marrow plasma cell counts, and were more accurate than than both urine free light chains (uFLCs) and SPE/serum immunofixation electrophoresis (sIFE) for assessing disease status (Table 12.1). Similar findings have been reported by other researchers ^[2]. In the Mead et al. study, many patients with normal bone marrow biopsies had elevated monoclonal immunoglobulins by SPE, reflecting slow clearance rates of IgG. Four patients had abnormal bone marrow biopsies but normal sFLC levels (non-FLC producers). Interestingly, 5 patients had abnormal sFLCs but normal marrows, suggesting that the biopsies had been taken from the wrong part of the bone marrow. In a disease with patchy distribution, a serum test that measures protein production from all tumour cells is likely to be highly sensitive for residual disease in some patients.

Table 12.1. Comparison of bone marrow plasma cell counts in MM using different tests for monoclonal proteins. sFLCs were classified as abnormal if the κ/λ ratio was outside the normal range, and for uFLC if there was >40 mg/L. The bone marrow assessment was classified as abnormal if there was ≥5% plasma cells.

12.4. Speed of response using serum free light chains

A rapid decrease in the concentration of sFLCs compared with intact IgG has been shown in many studies. It is most apparent with drugs that produce a fast tumour response. Thus, after high-dose melphalan (HDM), a reduction in sFLC concentration is seen within days (also see Sections 12.8 and 12.9 below). In an intensive testing study in which FLCs were assessed on most days post-transplant, Dispenzieri et al. ^[3] showed that a 90% reduction by day 7 predicted complete response.

Bortezomib (Velcade) is a rapidly acting chemotherapeutic agent, which is associated with particularly fast tumour responses. Das et al. ^[4] reported responses in 6 of 8 patients, 3 of whom exhibited repeated falls and rises of sFLCs co-incident with treatment cycles (Chapter 10, Figure 10.10). The relapse of sFLC was very rapid, with doubling times of less than 10 days. Such rapid changes presumably correspond with the biological half-life of proteosome inhibition and recovery rather than tumour killing and regrowth. By comparison, the intact monoclonal immunoglobulins did not show the same peaks and troughs. Similar observations have been made by others using bortezomib ^[5][6].

These patterns of response can only be observed with the use of tumour markers that are rapidly cleared from the serum, together with short sampling intervals. Generally, the sFLC levels indicated disease response earlier than the immunoglobulin assays. The authors concluded that monitoring patients with sFLC provided an opportunity to follow the kinetics of tumour killing, which is otherwise obscured by the slow clearance of intact monoclonal immunoglobulins. They added that sFLC measurements rapidly indicated tumour responses, which could facilitate relevant changes of treatment strategy. This may have a major bearing on cost of treatment and utilisation of resources.

In a study by Patten et al. ^[7], the short serum half-life of FLCs was used to assess early treatment responses to Actimid (a thalidomide analogue) in relapsed, refractory MM. Twelve patients were treated while being observed for changes in monoclonal immunoglobulins (Table 12.2). A beneficial change in the sFLC κ/λ ratio of greater than 50% by day 7 or 28 predicted clinical response or stable disease. Changes of less than 25% by day 28 predicted clinical progression or stable disease. There was a relatively poor correlation between parameters but in responding patients, reductions in sFLC concentrations predicted outcome earlier than intact monoclonal immunoglobulin measurements. The study concluded that sFLC κ/λ ratios allow early risk stratification and should allow tailoring of therapy in this difficult group of patients. In addition, faster dose escalation studies for drugs with poor tolerability profiles might be possible when using sFLC measurements.

Table.12.2. sFLCs and monoclonal intact immunoglobulin concentrations in 12 patients treated with the thalidomide analogue, Actimid. ^*FLC responses at 7 or 28 days predicted relapsing or stable disease. ^**Early response seen in sFLCs. (Courtesy of S Schey).

The fast response of sFLCs to chemotherapy was studied by Hassoun et al. ^[8][9] They found that normalisation of the FLC κ/λ ratio after the first or second cycle was highly predictive of outcome. “Since the aim of therapy is to achieve a complete response, assessment of sFLCs after 1 or 2 cycles may be an important milestone in the decision-making for these patients”. Under other circumstances, sFLC measurements may indicate that a longer duration or a change of chemotherapy is appropriate.

Figure 12.4 Normalisation of sFLCs prior to PBSCT in MM is a good indication of subsquent complete response after treatment. (Courtesy of G Tricot).

Figure 12.5 sFLCs and IFE during the evolution of patients with MM into remission. (Reproduced with permission from Haematologica ^[10]).

Cavallo et al. ^[11] studied 140 patients, comparing early sFLC responses with subsequent outcome. They found that normalisation of sFLCs increased the probability of subsequent complete remission when assessed both at 30 days (p=0.002) and prior to peripheral blood stem cell transplant (PBSCT) (p=0.001) (Figure 12.4). They also observed that sFLCs were highly correlated with cytogenetic abnormalities and MM staging - the 2 most important prognostic factors for event-free survival and overall survival. They concluded that “sFLCs can be expected to have a major independent predictive power for outcome with longer follow-up”. Comparison was also made between sFLC responses and functional positron emission tomography. A strong correlation was observed between the two measurements for predicting outcome (p<0.002) but the PET scans normalised faster ^[12].

Mösbauer et al. ^[10] evaluated the sensitivity of sFLCs in 26 MM patients for early detection of remission. Three patients converted to IFE negativity for monoclonal immunoglobulins during follow-up. However, the corresponding sFLC concentrations became normal at a median of 128 days earlier (range 110-144 days). They concluded that sFLCs detected remission earlier than IFE (Figure 12.5).

Orlowski et al. ^[13] studied the largest cohort of patients for sFLC response rates. In 487 patients (with at least one previous relapse), they assessed the long-term impact of sFLC ratio normalisation after each 21-day cycle of bortezomib and doxorubicin. At baseline, 6% had normal ratios, but this increased to 12% after the first cycle, 17% after the second cycle and eventually to 23%. Importantly, time to progression after cycle 1 was 345 days if the ratio normalised, versus 225 days if the ratio remained abnormal (p<0.0005). After cycle 2, time to progression fell to 325 days in the normalised group versus 224 days in the abnormal group (p<0.001). Similarly, early sFLC ratio normalisation corresponded with earlier complete responses (p<0.001) and partial responses (p<0.0001) after each cycle compared with residual abnormal sFLCs. This large study confirms the beneficial impact of sFLCs as a response marker that tracks the tumour killing effectiveness of chemotherapy.

Dispenzieri et al. ^[14] and Nakorn et al. ^[15] showed similar results. sFLC responses after 2 months of therapy were superior to measurements of intact monoclonal immunoglobulins for predicting overall responses.

12.5. Complete response and residual disease

Figure 12.6 sFLC concentrations in normal individuals and 107 patients with MM in complete remission, both clinically and by serum and urine IFE.

Figure 12.7 sFLC concentrations in 36 patients with LCMM in complete remission or with stable disease. CR: complete response.

Figure 12.8 sFLC concentrations in 61 patients with IIMM whilst in complete remission, both clinically and by serum and urine IFE. CR: complete response.

Figure 12.9 A patient treated with thalidomide to normalise λ sFLCs after the IgG monoclonal protein had disappeared. (Courtesy of MR Nowrousian).

Absence of intact monoclonal immunoglobulins by IFE after chemotherapy is a recognised prognostic indicator for survival. However, the slow clearance of intact immunoglobulins coupled with the relatively poor sensitivity of IFE suggest that sFLC analysis would provide a better prognostic marker, or at least be additive to IFE. This is supported by the above observations that early normalisation of sFLCs is indicative of good responses. Hence, some patients in complete remission by IFE may be reclassified as having residual disease by the more sensitive sFLC tests. This has led to the classification of stringent complete response (sCR) in the International Uniform Response Criteria for MM and a recommendation by the International Myeloma Working Group (IMWG) that sFLCs be measured in all patients who have achieved a CR in order to determine whether they have attained a sCR (Chapter 25). Several studies, described below, have confirmed the high sensitivity and value of sFLCs for assessing complete responses.

Sirohi et al. ^[16] measured sFLCs in the sera of 107 MM patients who were in complete remission as assessed by electrophoretic tests. Many of these patients had abnormal concentrations of sFLCs (Figure 12.6). Patients with ratios outside the 95% limit of the normal range had a 1.3-fold increased risk of progression (p<0.05) and patients with results outside the 100% range had a 2.7-fold increased risk (p<0.01) (Fisher’s exact test). Patients with both FLCs elevated (but normal κ/λ ratios) were considered to have impaired renal function (Chapter 20) or disordered immune reconstitution and showed no increased risk of relapse. Thus, even though intact monoclonal immunoglobulins were undetectable by IFE in all patients, sFLCs were frequently abnormal and predicted worse outcome.

Reid et al. ^[17] measured sFLCs in 36 patients with light chain multiple myeloma (LCMM) who were clinically in complete remission or had stable disease (Figure 12.7). Seventeen of the patients had no detectable FLCs in either serum or urine by IFE (Sebia system). However, 11 of these patients had abnormal κ/λ ratios with increased FLC concentrations. In contrast, all 19 patients with abnormal IFE results had abnormal sFLC concentrations. In a further data set of 61 patients with IIMM in complete remission by conventional criteria ^[17], 17 had abnormal sFLC κ/λ ratios (Figure 12.8). Survival data were available for 44 of the 97 patients in these 2 groups and showed that abnormal FLC ratios were significantly associated with reduced overall survival (P<0.007).

More results from the UK have recently been reported and further support the utility of sFLC studies for defining response rates. A study of 207 patients in the MRC Myeloma IX^th trial, by Owen et al. ^[18] showed that normal sFLC results at the end of induction therapy (prior to HDM) predicted attainment of an IFE negative complete response. Thus, 5% of patients had normal sFLCs at presentation and this had increased to 46% by the end of induction therapy and to 79%, 100 days after HDM. The sFLCs after induction therapy predicted IFE normality at day 100 (i.e, with normal sFLCs, 70% became IFE negative whereas with abnormal sFLCs, only 30% became IFE negative). Lebovic et al. ^[19] showed similar findings in a smaller study. Both studies considered the sFLC measurements to be a useful tool for defining response rates.

These results establish the use of sFLCs for assessing residual tumour but the benefit of extra treatment for patients needs to be determined in a trial setting. A few patients with residual disease, as identified by abnormalities of sFLCs alone, have been given additional high-dose therapy and PBSCT. An example of a patient treated only on the basis of residual sFLCs is shown in Figure 12.9. The patient was normal by SPE and UPE but had high λ sFLC concentrations. A successful response of sFLCs to thalidomide was observed over a six-month period.

12.6. Serum free light chains during disease relapse

Figure 12.10 sFLCs and IFE during relapse of patients with MM. (Reproduced with permission from Haematologica ^[10]).

Figure 12.11 Early tumour recurrence identified from rising λ sFLC levels while IgGλ continued to fall. Tumour relapse occurred before IgGλ had stabilised.

Relapse with monoclonal FLCs only may occur for the following reasons:

1. Since sFLC assays are intrinsically more sensitive than IFE, they will be abnormal earlier if the tumour relapses simultaneously with monoclonal FLCs and intact immunoglobulins. Since FLCs are produced by 95% of patients with IIMM (Section 11.3), this is likely to be a common occurrence with frequent serum sampling.

Mösbauer et al. ^[10] evaluated the sensitivity of sFLCs in 9 MM patients in complete remission as assessed by IFE and sFLCs for early detection of relapse. Eight of the 9 patients were identified as having relapsed earlier when sFLC ratios were used rather than IFE (median, 98 days; range 35-238 days) (Figure 12.10). Earlier studies ^[20][21] with lower FLC sensitivity for relapse (10-15%) may be explained by the use of less frequent serum sampling (typically 3 monthly).

2. When monoclonal immunoglobulin production by the tumour changes to FLCs only or a relative increase in FLCs. FLC escape/breakthrough (perhaps more appropriately called sero-conversion) may occur in 10-15% of patients with modern intensive treatment (see Section 12.7 below).

3. When relapse occurs within a few months of successful chemotherapy and intact monoclonal immunoglobulins are still abnormal by IFE. In this situation, the falling concentrations of IgG or IgA hide the early increases from the tumour relapse. In contrast, sFLCs have already normalised because of their short half-life, so the relapse occurs from lower baseline levels (Figure 12.11 and Chapter 10).

12.7. Free light chain escape (breakthrough)

Figure 12.12 Serum λ FLCs and IgAλ in a patient showing FLC escape. FLC escape was apparent as a conversion from IgA production to FLC only. (Courtesy of E Liakopoulou).

Figure 12.13 Serum λ FLCs and IgAλ in a patient during relapse. FLC escape was apparent as the IgA clone disappeared (Courtesy of M Engelhardt).

The first observation of FLC breakthrough was in 1971 by JR Hobbs when monitoring patients in the first UK myeloma trial. He noted that 5% of patients producing intact monoclonal immunoglobulins relapsed with only Bence Jones proteinuria, whilst the relative proportion of monoclonal FLCs increased in a further 35% of patients at relapse. While this has since been generally accepted as a not infrequent phenomenon, it has not been reliably documented. A typical example is shown in Figure 12.12.

Kühnemund et al. ^[22] reported 2 patients with IgA MM who relapsed with monoclonal FLCs only (Figure 12.13). Dawson et al.^[23] reported 3 patients with FLC escape, 2 of which were IgA. They noted fulminant relapses with extra-medullary features and suggested that this may be a more frequent occurrence with modern and more aggressive chemotherapy. A further example is described in Chapter 8, Clinical case history No 2.

To better establish the incidence of this phenomenon, we reviewed 30 IgG and 36 IgA MM patients recruited to the UK, MRC VII^th myeloma trial ^[24][25]. Four of the patients (6%) had FLC escape only (1 IgG and 3 IgA), and a further 7 (3 IgG and 4 IgA) had a relative increase in FLCs during relapse compared with intact monoclonal immunoglobulin production (partial FLC escape). In total, 17% of patients (11/68) showed FLC escape; this was more prevalent in the IgA group (7/11). Although the patient numbers were small, there was no bias to the intensive treatment arm of the trial. It was noted that urine tests did not reliably detect FLCs in 7 of the patients. This suggests that the incidence of FLC escape is higher than suggested by evidence derived from urine studies.

The importance of this phenomenon is two-fold. First, the serological diagnosis of disease relapse cannot be relied upon by measuring intact monoclonal immunoglobulins alone. Secondly, high concentrations of monoclonal sFLCs cause renal failure. Continuous observations of sFLC during patient monitoring will provide early indications of renal impairment and any risk of renal failure. Chemotherapy can then be used to specifically reduce the load of toxic FLCs on the kidneys and lessen the incidence of renal failure in relapsing patients (Chapter 13).

12.8. Serum free light chains following high-dose melphalan and bone marrow transplant

Figure 12.14 Concentrations of sFLC after HDM identifying a poor treatment response. (Courtesy of G Pratt).

Figure 12.15 Changing immunoglobulin concentrations in a MM patient with IgGκ and κ FLC after HDM treatment (day 0) and PBSCT. (Courtesy of G Pratt).

Figure 12.16 Changes in FLCs and creatinine while monitoring a patient with MM after HDM and PBSCT. (Courtesy of G Pratt).

Figure 12.17 Long-term evaluation of sFLC concentrations in a patient following HDM and PBSCT.

Pratt et al. ^[26] analysed the detailed changes in sFLC concentrations after PBSCT in 19 patients. Before transplant (but after induction chemotherapy), 11 of the patients had elevated levels of the tumour-produced FLC with abnormal κ/λ ratios. After transplantation, in all patients with associated intact monoclonal immunoglobulins, the tumour-produced FLC concentrations fell within 48 hours (median half-life, 4.3 days) at a rate faster than the monoclonal paraprotein (median half-life, 14 days). The rate of fall and range of reduction of FLCs varied between individual patients indicating different tumour killing rates/chemosensitivities (Figures 12.14 to 12.16). Figure 12.14 shows a patient in whom there was an incomplete tumour response following chemotherapy; this was evident earlier from the failure of λ concentrations to normalise quickly.

Figures 12.15 and 12.16 illustrate bone marrow engraftment with functioning plasma cells after HDM and PBSCT. In Figure 12.15, concentrations of both FLCs fell until day 10 and then increased rapidly. Three days later, total IgG began to rise. This indicated bone marrow engraftment and occurred a few days after the return of platelet and neutrophil production. This appears to be the normal pattern of FLC response as engraftment takes place.

Minimum levels of FLCs during treatment were 3-5mg/L. This indicated that some FLC production remained, presumably associated with long-lived plasma cells that were resistant to the drugs. Since κ/λ ratios were normal, the producing cells were probably polyclonal rather than monoclonal tumour cells. For the patient in Figure 12.15, the early stages of engraftment showed greater numbers of κ plasma cells being generated. This produced a spike in the κ/λ ratio around day 12. Total IgG concentrations fell more slowly after treatment and increased later than FLCs during engraftment. By day 50, FLCs and immunoglobulins were within their normal concentration ranges.

Figure 12.16 illustrates similar features to Figure 12.15, but with the added complication of renal impairment from gastrointestinal bleeding at day 14. This markedly affected the concentrations of sFLCs. Initially, after HDM, serum λ half-life was 3 days, compared with 20 days for IgG. FLC concentrations increased from day 15 and exceeded normal levels by day 21. They returned to normal, alongside serum creatinine levels on day 24 when normal renal function was restored.

FLC recovery occurred either after or around the time of neutrophil engraftment in all the patients (Figures 12.15 and 12.16). Since early lymphocyte recovery is associated with a more favourable outcome, the time to FLC normalisation may be a useful prognostic marker.

Overall, these studies show that FLC measurements provide a sensitive monitor of changes in the numbers of tumour cells following PBSCT and changes in FLC concentrations pre-date the intact immunoglobulin response. Further follow-up is required to ascertain whether differences in the kinetics of the FLC responses have any prognostic value. Similar findings have been reported by others ^[14].

12.9. Long-term variations in serum free light chain levels after high-dose therapy

Figure 12.18 Changes in sFLC levels and κ/λ ratios during long-term tumour monitoring. IFN: interferon.

Figure 12.19 Changes in κ sFLC concentrations in a patient with LCMM at initial treatment. The effect of dexamethasone is clearly detectable within 24 hours. Dex = dexamethasone. (Courtesy of G Galvin)

When bone marrow myeloma cells have been eradicated with intensive therapy, normal haematopoesis may not return for many months. This may show as minor fluctuations of sFLC concentrations around the normal range. Caution should therefore be taken when interpreting the data. Figures 12.17 and 12.18 show patients in whom there was a gradual change in κ/λ ratios over many months. It is unknown whether this is indicative of adverse outcome. However, when assessing residual disease or complete remission the relevance of minor fluctuations in sFLC levels needs to be considered carefully (see MM response criteria guidelines in Chapter 25).

12.10. Biological variation of monoclonal serum free light chain measurements

The long-term biological variation of monoclonal immunoglobulins has recently been studied in 158 patients with clinically stable monoclonal gammopathy ^[27]. The patients received no treatment during the study interval, had no change in clinical diagnosis, and had a <5 g/L change in serum monoclonal immunoglobulin quantification over the observation period. Protein electrophoresis was used to quantify monoclonal immunoglobulins in serum and urine, and immunonephelometry (Freelite™) was used to quantify sFLCs in at least 3 serial samples obtained up to 5 years apart.

Of the 158 patients studied, 52 patients had measurable monoclonal sFLCs (defined as involved FLC [iFLC] ≥100 mg/L in the presence of an abnormal ratio) ^[27]. The total coefficient of variation (CV) for iFLC measurements was 28.4%, which was almost entirely attributable to biological variation (CV = 27.8%) and, to a lesser extent, due to inter-assay analytic variation (CV = 5.8%). In comparison with monoclonal immunoglobulin quantification by protein electrophoresis, the variation in iFLC was more comparable to that found for urine (Total CV = 35.8%) than serum (Total CV = 8.1%). This is likely to reflect the short serum half-life of FLCs compared with intact immunoglobulins, allowing sFLC concentrations to respond rapidly to changes in plasma cell synthesis or clearance (see Chapter 10). The authors proposed that the definitions of minimal and partial response using sFLC measurements should be modified to be in line with those of urine monoclonal protein measurements (see Section 25.7).

12.11. Drug selection using rapid responses in serum free light chains

The short half-life of sFLCs may be useful for identifying effective drugs, and equally, ineffective drugs, particularly during the later stages of the disease. With each relapse, and as the disease becomes more refractory to treatment, patients are given more drugs, with many toxic side effects. The selection of a drug or drug combinations and the necessary doses can be based upon short-term responses in sFLC concentrations. When using IgG levels for such purposes it may be several weeks before it is appreciated which drug is effective ^[28].

An example of a rapid response to drugs, identified using FLCs, is shown in Figure 12.19. The patient was diagnosed with LCMM and given dexamethasone 24-hours before vincristine and adriamycin and monitored frequently for sFLCs. At 10-12 hours after the first dose of dexamethasone (apoptosis time), sFLC concentrations had fallen by 40%. Further falls followed subsequent doses. In contrast, there was no clear evidence of vincristine or adriamycin responses. These observations suggest that serial monitoring of FLCs, whilst introducing drugs sequentially, may allow active components to be identified.

12.12. Utility of serum free light chains in intact immunoglobulin multiple myeloma - comments on utility

References