Intact immunoglobulin MM (IIMM) - sFLCs at diagnosis

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Chapter 11	SECTION 2A - Multiple Myeloma
	Intact immunoglobulin multiple myeloma - sFLCs at diagnosis

Contents 1 2 11.1. Introduction 3 11.2. Myeloma cell diversity: production of monoclonal immunoglobulins (Igs) and FLCs 4 11.3. sFLC concentrations at disease presentation 5 11.4. Disease stage and sFLCs 6 References

In patients with intact immunoglobulin multiple myeloma (IIMM), monoclonal serum free light chains:
Are produced by clones of cells with considerable diversity. Are abnormal in 95% of patients at disease presentation. Show no correlation with intact monoclonal immunoglobulin concentrations. Are markers of disease stage. Are markers of tumour outcome. Are independent of albumin and β2-microglobulin for disease staging.

11.1. Introduction

Approximately 80% of patients with multiple myeloma (MM) produce intact immunoglobulin monoclonal proteins (Figure 7.3), of whom ~50% have excess monoclonal FLCs in urine by immunofixation electrophoresis (IFE) ^[1]. Serum protein electrophoresis (SPE) and serum IFE tests for FLCs are less frequently positive because sFLC concentrations are below the detection limits of these assays.

The first attempt to measure sFLCs in IIMM was by Sölling ^[2] in 1982. Using column chromatography, FLCs were first separated from bound light chains and then measured using antibodies against whole light chains. Using this technique monoclonal sFLCs were present in 86% of IIMM patients. Recently, using sensitive sFLC immunoassays, many authors have reported even higher prevalences of monoclonal FLCs. These studies, together with their clinical relevance will be described in this chapter.

11.2. Myeloma cell diversity: production of monoclonal immunoglobulins (Igs) and FLCs

Myeloma plasma cells have huge genetic and mophological diversity, so mixed clones occur at clinical presentation. Subsequent evolutionary selection by chemotherapy leads to escape mutants with different protein expression profiles. Predictably, myeloma cell populations with mixed monoclonal protein expression arise from this diversity, although perhaps surprisingly, this was only recently confirmed by histology. Using a double immunofluorescence staining method, Ayliffe et al. ^[3] estimated the incidences of different types of plasma cells in bone marrow biopsies (Figure 11.1). In 82% of patients, single populations of cells were present that contained either intact monoclonal immunoglobulins (with or without monoclonal FLCs [74%]) or monoclonal FLCs alone (8%). However, 18% of the samples contained a mixture of both cell populations (Figure 11.2). Progression from cells making intact monoclonal immunoglobulins to cells restricted to FLC production alone was also shown to occur in some patients during the course of their disease. Furthermore, the presence of “FLC-only” cells was associated with shortened survival. This indicates that monoclonal FLC production at a cellular level is an adverse prognostic marker both at clinical presentation and during relapse of patients with IIMM. The relationship between monoclonal FLC production and poor survival has been observed in a series of studies that are described below.

11.3. sFLC concentrations at disease presentation

Using archived samples from the UK MRC MM trials, Mead et al. ^[4] assessed sFLC concentrations at the time of presentation in a series of patients: 314 with IgG MM, 142 with IgA MM, 36 with IgD MM and 5 with IgE MM. Overall, 88% had elevated sFLCs with the following breakdown: IgG 84%, IgA 92%, IgD 94%, and all 5 of the IgE MM patients. Some of the remaining patients had normal or reduced concentrations of FLCs but their κ/λ ratios were abnormal, indicating monoclonality in association with bone marrow suppression. In total, 96% of all MM patients (including light chain [LCMM] and nonsecretory [NSMM] disease) had abnormal FLC concentrations or abnormal κ/λ ratios (Figures 11.3 to 11.6). This percentage is higher than previously reported ^[1][2], and reflects the increased sensitivity of the FLC immunoassays, in particular, the use of the suppressed alternate FLC to identify abnormal κ/λ ratios. It is also of note that there was complete concordance between the monoclonal FLC type identified by κ/λ ratios and that obtained by IFE (Figure 11.3): this provided an important specificity validation of the FLC immunoassay.

Other large studies of MM at disease presentation have shown similar results. Orlowski et al. ^[5] studied sFLCs in 487 patients and noted that 94% had abnormal sFLC κ/λ ratios. Snozek et al. ^[6] reported that monoclonal sFLCs were present in 95% of 576 MM patients at disease presentation, a figure identical to that observed by Owen et al. ^[7] in 207 patients entered into the UK MRC trials. Dispenzieri et al. ^[8] observed abnormal sFLC κ/λ ratios in 96% of 399 patients and, most recently, Katzman et al. observed abnormal ratios in 96.8% of 467 patients with MM ^[9].

As sFLC concentrations are normal in some patients with IIMM, it is clear that serum electrophoretic tests are essential for MM diagnosis. In contrast, sFLC assays are more sensitive for the identification of FLCs in LCMM and NSMM. Therefore, when MM is suspected, the optimum laboratory practice should be to test sera by both SPE/IFE and sFLC assays.

In the study by Mead et al. ^[4], sFLC concentrations were higher in IgA than IgG patients but highest in IgD patients (similar to LCMM patients (see Figure 11.4). High levels of urine FLC (uFLC) excretion and excess of λ compared with κ are typical of IgD MM ^[10][11] . Five patients with IgE MM are included in Figure 11.4. Although the number of patients is small, the FLC results are presumably representative of the disease.

A subgroup of 69 patients with IIMM (55 IgG and 14 IgA), each with no detectable uFLC excretion (less than 40mg/L), is shown in Figure 11.6. Serum analysis showed that 95% of the patients had abnormal FLC κ/λ ratios. It is of note that none of the patients had increased concentrations of the alternate sFLC, indicating there was insignificant renal impairment. At clinical presentation there was no correlation between serum creatinine and sFLC levels (as for LCMM - Figure 8.3).

It was also apparent that sFLC measurements showed no significant correlation with serum levels of intact monoclonal immunoglobulins (by Pearson correlation coefficient r). Results for IgGκ and IgGλ are also shown in Figures 11.7 and 11.8.

IgGκ vs κFLC: r = -0.0145 (n=150). IgGλ vs λFLC: r = 0.0037 (n=116). IgAκ vs κFLC: r = 0.212 (n=68). IgAλ vs λFLC: r = -0.0330 (n=64).

This lack of correlation is an important issue because it indicates that sFLC concentrations are independent markers of the disease process in IIMM. sFLCs provide additional disease information, both at initial presentation and when monitoring patients.

11.4. Disease stage and sFLCs

It has been known for some time that in MM, high monoclonal uFLC excretion at clinical presentation is predictive of poor survival, and hence, disease stage. For example, the outcome for 351 patients with LCMM was compared with 1,512 IgG and 717 IgA patients entered into the UK MRC myeloma multicentre trials ^[13]. LCMM patients had the shortest median survival of 1.9 years (P<0.001) compared with 2.3 years for IgA patients and 2.5 years for IgG patients. It follows that sFLC measurements should be of more value than uFLCs, due to their greater clinical sensitivity and analytical accuracy.

Several recent studies have assessed the relationship between sFLCs at disease presentation and subsequent outcome. Kyrtsonis et al. ^[14] investigated the prognostic value of baseline sFLC κ/λ ratios in 94 MM patients. The median baseline ratio for κ MM (κ/λ ratio) was 3.57 and for λ MM (λ/κ ratio) was 45.1. sFLC ratios above the observed median values correlated with elevated serum creatinine and lactate dehydrogenase, extensive marrow infiltration and κ or λ light chain type of the intact monoclonal immunoglobulins. Importantly, the 5-year disease specific survival was 82% in patients with sFLC κ/λ ratios lower than the median compared with 30% for κ/λ ratios equal to or greater than the median (P<0.001) (Figure 11.9A).

In current myeloma practice, patients are categorised using the International Staging System (ISS) based upon serum albumin and β₂-microglobulin measurements alone. Kyrtsonis et al. ^[14] assessed these parameters alongside sFLC analysis and showed that κ/λ ratios were an additional independent prognostic factor. Combination of the ISS with sFLC ratios in newly diagnosed MM patients for time to progression and survival (Table 11.1) showed significantly worse survival with abnormal sFLC ratios (P<0.0001)(Figure 11.9B).

In a similar study of 576 patients at the Mayo Clinic, abnormal sFLC ratios at presentation were again important independent markers of outcome. Snozek et al. ^[6] showed that sFLC ratios assessed alongside the ISS in patients with 0, 1, 2 or 3 risk factors (sFLC κ/λ ratios <0.03 or >32, β₂-microglobulin >3.5g/L and albumin >35g/L) had median overall survival times of 51, 39, 30 and 22 months, respectively (P<0.001). Because these data provided additional outcome information, it was suggested that sFLC ratios should be incorporated into the ISS to provide a new risk stratification model.

van Rhee et al. ^[12] studied the relationship between sFLCs and outcome in 301 patients undergoing intensive treatment (Figure 11.10). They observed that baseline top-tercile sFLC levels >750 mg/L were associated with inferior overall (p=0.005) and event-free survival (p=0.007). Baseline concentrations of other serum or urine immunoglobulins (excluding β₂-microglobulin and albumin) did not identify prognostic subgroups. In an associated investigation, Cavallo et al. ^[15] indicated that sFLC concentrations and abnormal κ/λ ratios were both highly correlated with cytogenetic abnormalities and MM staging.

Patient Subgroup	Pts (%)	3-yr DSS (%)	5-yr DSS (%)
Low sFLCR and ISS <3	61 (29)	95	90
Either High sFLCR or ISS=3	96 (46)	82	56
High sFLCR and ISS=3	50 (24)	37	24

Table 11.1 Disease specific survival (DSS) in 207 newly diagnosed patients with MM according to the combined sFLC κ/λ ratios and the ISS comprising serum albumin and β₂-microglobulin. (Courtesy of M-C Kyrtsonis).

While results from these different studies show that both sFLC concentrations and sFLC ratios are prognostic, it is unclear which is preferable. Should it be sFLC κ/λ ratios, absolute values or subtracted FLC concentrations (involved minus uninvolved)? This question was recently addressed by Dispenzieri et al. ^[8], when analysing the outcome of 399 MM patients at clinical presentation. Patients were divided into terciles, and regardless of the baseline variable used (i.e. FLC ratios, involved FLCs or subtracted FLCs), patients with the lowest tercile of FLC had the best outcomes when compared with the two higher terciles. The outcomes for the two higher terciles were nearly identical (Figure 11.11). The respective median overall survivals were 49.4, 41.7 and 42.1 months, and the median progression-free survivals were 34.9, 28.7 and 29.5 months.

Since high sFLCs frequently cause renal damage because of their inherent toxicity, reduced survival might be related to renal-associated mortality. In a study of sFLC concentrations in patients presenting with renal failure, patients with the highest concentrations were more likely to have cast nephropathy and hence a higher mortality rate (Chapter 13). However, such patients are not normally included in routine clinical trials so it is unclear what role renal impairment has in determining outcome in the above studies.

Test Questions
Do MM plasma cells produce more than one type of monoclonal protein? How often are sFLCs abnormal in IgA MM? Are sFLCs independent of the ISS as risk factors in MM? Why are high sFLCs an adverse risk factor?

Answers
Yes, there is considerable diversity in bone marrow myeloma cells (Section 11.2) . In 98% of patients at clinical presentation (Section 11.3). Yes, they are independent of serum albumin and β2-microglobulin. (Section 11.4). Because their expression is increased as tumours become genetically more diverse, more aggressive (Section 11.4) and patients develop renal impairment (Chapter 13).

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