Monitoring IIMM patients using sFLCs

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12.1. Introduction

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.

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.

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

Figure 12.5 Serum FLCs and IFE during the evolution of patients with MM into remission. (Courtesy of N Kroger).

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

Figure 12.7 Serum FLC concentrations in 36 patients with LCMM in complete remission or with stable disease.

Serum FLC measurements are being widely used for monitoring patients with IIMM. Monoclonal sFLCs are produced in ~95% of patients, are independent of intact monoclonal immunoglobulin production and have a short half-life (Chapter 10 and 11). Therefore, they are of use in all stages of the disease; during early treatment, assessing response rates, for indicating remission and residual disease, and during relapse. This chapter analyses numerous publications that have addressed these various issues. Many other studies support these results but not all can be discussed in detail for lack of space. Therefore, publications containing the largest series of patients or the most important results have been selected. A full list of publications is shown in Chapter 31.

12.2. Overview of sFLCs for monitoring patients with IIMM

The various attributes of sFLC for monitoring patients are best demonstrated using some patient examples. Detailed analysis of the various aspects of the sFLC responses are provided in the subsequent sections.

Figure 12.1 shows a patient with MM producing monoclonal IgGκ and monoclonal κ serum FLCs (but no urine FLCs). During the initial therapy, κ concentrations rapidly fell with a half-life of approximately 20 days, while IgGκ, quantified from SPE gels, only gradually returned to normal. The apparent half-life of IgGκ was between 100 and 200 days. (This depended, in part, upon which densitometric scanning device was used: white light scanners may 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 vunerable patients it may be of no harm to stop chemotherapy if sFLC levels have normalised in order to minimise drug side effects.

12.3. Bone marrow responses and short sFLC half-life

Table 12.1. Comparison of bone marrow plasma cell counts in MM using different tests for monoclonal proteins.

The value of sFLCs rather than IgG levels for accurately assessing 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 51 patients by Mead et al. ^[1], (Table 12.1) and others ^[2], sFLC concentrations were better than both uFLCs and SPE/sIFE for assessing disease status. Many patients with normal bone marrow biopsies had elevated monoclonal immunoglobulins by SPE, reflecting slow clearance rates of IgG. 4 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.

12.4. Speed of response using sFLCs

The fast rate of fall of sFLCs compared with IgG has been shown in many studies. It is most apparent with drugs that produce a fast tumour response. Thus, after high dose melphalan, sFLC falls are seen within days (also see 12.8 and 12.9). Dispenzieri et al. ^[3], showed that in an intensive testing study in which FLCs were tested on most days post-transplant, a 90% reduction by day 7 predicted complete response.

Bortezomib (Velcade) is a rapidly acting chemotherapeutic agent in which particularly fast tumour responses occur. Das et al. ^[4], showed that 6 of 8 patients had responses of which 3 showed 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 times. 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 that could allow relevant changes of treatment strategy. This may have 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. 12 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 clinically responding 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 serious side-effect profiles might be possible when using sFLC measurements.

The fast response of sFLCs to chemotherapy was recently 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, assessement 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.

Cavallo et al., studied 140 patients, comparing early sFLC responses with subsequent outcome. They found that normalization of sFLCs increased the probability of subsequent complete remission when assessed both at 30 days (p=0.002) and prior to 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. There was a high correlation between the two measurements for predicting outcome (p<0.002) but the PET scans normalised faster ^[10].

Mösbauer et al. ^[11], evaluated the sensitivity of sFLCs in 26 MM patients for early detection of remission. 3 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).

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

Orlowski et al. ^[12], 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 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 for 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. ^[13], and Nakorn et al. ^[14], showed similar results. Serum FLC 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 12.8 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 well described as prognostic for good survival. However, their slow clearance and the relatively poor sensitivity of IFE suggests that sFLC analysis should be a better prognostic marker, or at least 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 in the International Uniform Response Criteria for MM (Chapter 25) . Several studies, described below, have confirmed the high sensitivity and value of sFLCs for assessing complete responses.

Sirohi et al. ^[15] studied sera for sFLCs from 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% 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. ^[16], measured sFLCs in 36 patients with LCMM who were clinically in complete remission or had stable disease (Figure 12.7). 17 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 ^[16], 17 had abnormal sFLC κ/λ ratios (Figure 12.8). Survival data was available for 44 of the 97 patients in these 2 groups and showed that abnormal FLC ratios were highly significant for 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 IXth trial, by Owen et al. ^[17], showed that normal sFLC results at the end of induction therapy (prior to high dose melphalan) predicted attainment of an IFE negative complete response. Thus, 5% of patients had normal sFLCs at presentation and this increased to 46% at the end of induction therapy and 79%, 100 days after high-dose melphalan. 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. ^[18], 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 FLCs during disease relapse

Figure 12.10 Serum FLCs and IFE during relapse of patients with MM. (Reproduced with permission from Haematologica^[11]).

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 (Figure 11.5), this is likely to be a common occurrence with frequent serum sampling.

Möesbauer et al. ^[11], evaluated the sensitivity of sFLCs in 9 MM patients in complete remission by IFE and sFLCs for early detection of relapse. 8 of the 9 patients showed relapse by sFLC ratios earlier than IFE positivity by a median of 98 days (range 35-238 days) (Figure 12.10). Earlier studies ^[19][20], with lower FLC sensitivity for relapse (10-15%) may be explained by less frequent serum sampling (typically 3 monthly) ^[20].

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).

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 base-line 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. Free light chain 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. Free light chain ‘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 patients producing intact monoclonal immunoglobulins relapsed with only Bence Jones proteinuria in 5% and with a relative increase in 35%. 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.

Kuhnemund et al. ^[21], recently reported 2 patients with IgA MM who relapsed with monoclonal FLCs only (Figure 12.13). Dawson et al. ^[22], 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.

In order to better establish the incidence of this phenomenon we reviewed 30 IgG and 36 IgA patients randomly selected from the UK, MRC VIIth myeloma trial ^[23]. 4 of the patients (6%) had FLC escape only (1 IgG and 3 IgA). 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 and more 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 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. Second, 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. sFLCs following high dose melphalan and bone marrow transplant

Figure 12.14 Changing immunoglobulin concentrations in a MM patient with IgGκ and FLC κ after highdose melphalan treatment (day 0) and PBSCT. (Courtesy of G Pratt).

Figure 12.15 Changes in FLCs and creatinine while monitoring a patient with MM after highdose melphalan and PBSCT.(Courtesy of G Pratt).

Figure 12.16 Concentrations of sFLC after high dose melphalan identifying a poor treatment response. (Courtesy of G Pratt).

Figure 12.17 Long term evaluation of sFLC concentrations in a patient following high dose melphalan and PBSCT.

Pratt et al. ^[24] 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 transplant, in all patients with associated intact monoclonal immunoglobulins, the tumour-produced FLC concentrations fell within 48 hours (median half-life 4.3 days) and 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/chemosensitivity (Figures 12.14 to 12.16). Figure 12.16 shows a patient treated with chemotherapy in whom there was an incomplete tumour response. This was evident earlier from the failure of λ concentrations to normalise quickly.

Figures 12.14 and 12.15 illustrate bone marrow engraftment with functioning plasma cells after high-dose melphalan and PBSCT. In Figure 12.14, 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, there were 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 12.14, 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 12.15 illustrates similar features to Figure 12.14 but there is the added complication of renal impairment from gastrointestinal bleeding at day 14. This markedly affected the concentrations of sFLCs. Initially, after high-dose melphalan, 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 12.14 and 12.15). Since early lymphocyte recovery is associated with a more favourable outcome, the time to FLC normalisation may be a useful prognostic marker.

Overall, FLC measurements provided a sensitive monitor of changes in the numbers of tumour cells after PBSCT and pre-dated 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 were reported by others ^[13].

12.9. Long term variations in sFLC 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. 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 ^[25].

An example of 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. 10-12 hours after the first dose of dexamethasone (apoptosis time), sFLC fell 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 one-by-one, may allow active components to be identified.

12.11. Utility of sFLCs in IIMM-comments on utility

References