Leukemia (2013) 27, 208–212

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ORIGINAL ARTICLE
Suppression of uninvolved immunoglobulins defined by
heavy/light chain pair suppression is a risk factor for progression
of MGUS
JA Katzmann1, R Clark1, RA Kyle2, DR Larson3, TM Therneau3, LJ Melton III4, JT Benson3, CL Colby3, A Dispenzieri2, O Landgren5,
S Kumar2, AR Bradwell6, JR Cerhan4 and SV Rajkumar2

We hypothesized that the suppression of uninvolved immunoglobulin in monoclonal gammopathy of undetermined significance
(MGUS) as detected by suppression of the isotype-specific heavy and light chain (HLC-pair suppression) increases the risk of
progression to malignancy. This approach required quantitation of individual heavy/light chains (for example, IgGl in IgGk MGUS
patients). Of 1384 MGUS patients from Southeastern Minnesota seen at the Mayo Clinic from 1960 to 1994, baseline serum samples
obtained within 30 days of diagnosis were available in 999 persons. We identified HLC-pair suppression in 27% of MGUS patient
samples compared with 11% of patients with suppression of uninvolved IgG, IgA or IgM. HLC-pair suppression was a significant
risk factor for progression (hazard ratio (HR), 2.3; 95% confidence interval (CI) 1.5–3.7; Po0.001). On multivariate analysis, HLC-pair
suppression was an independent risk factor for progression to malignancy in combination with serum M-spike size, heavy chain
isotype and free light chain ratio (HR, 1.8; 95% CI, 1.1–3.00; P ¼ 0.018). The finding that HLC-pair suppression predicts progression
in MGUS and occurs several years before malignant transformation has implications for myeloma biology.

Leukemia (2013) 27, 208–212; doi:10.1038/leu.2012.189

Keywords: MGUS; multiple myeloma; prognosis; suppression; heavy/light

INTRODUCTION HLC-pair in order to more broadly test the impact of

Monoclonal gammopathy of undetermined significance (MGUS) is immunoparesis. The purpose of this study was to determine the
a common premalignant plasma cell proliferative disorder that is a prognostic value of isotype-specific suppression of immuno-
precursor of multiple myeloma (MM).1–3 A number of prognostic globulins in a large population-based cohort of patients with
factors for progression of MGUS to MM have been identified. MGUS. Identification of additional biomarkers that predict the risk
These include the size of the M-spike, heavy chain isotype, of progression in MGUS is needed not just from a clinical stand
detection of urinary monoclonal light chain, percentage of point but also from a biological stand point to better understand
bone marrow plasma cells, isotype suppression of uninvolved the pathogenesis of myeloma.
immunoglobulins, and serum-free light chain (FLC) k/l ratio.4–7
By combining three of these variables (M-spike size, heavy chain
isotype and serum FLC ratio), we have constructed a model that MATERIALS AND METHODS
provides approximately a 10-fold difference in risk for MGUS The study population was derived from a cohort of 1384 southeastern
progression.6 By contrast, suppression of uninvolved, polyclonal Minnesota patients with MGUS who were seen at the Mayo Clinic from
immunoglobulins (immunoparesis) has not been a consistent 1 January 1960 through 31 December 1994; the characteristics of this
group have previously been described.4 Our study group consisted of 999
predictor of progression in MGUS.7,8 Immunoparesis has always of the MGUS cohort on whom cryopreserved serum samples collected
been defined as suppression of uninvolved immunoglobulins (for within 30 days of initial diagnosis were available for assay (Table 1).
example, suppression of IgM and IgA in a patient with IgG MGUS). Each sample was tested for total immunoglobulin concentrations (that
The effect on normal, polyclonal IgG could not be ascertained in a is, IgG, IgA and IgM), as well as for HLC concentration (that is, IgGk, IgGl,
patient with IgG MGUS, as standard nephelometric assays do not IgAk, IgAl, IgMk and IgMl). Total immunoglobulins were quantified by
differentiate between monoclonal and polyclonal IgG. However, immunonephelometry (BNII, Siemens Diagnostics, Marburg, Germany). HLC
we hypothesized that immunoglobulin heavy chain isotype- concentrations were quantified by immunonephelometry using Hevylite
specific suppression (for example, IgG l suppression in the case reagents provided by the manufacturer (The Binding Site, Birmingham,
UK). The IgG, IgA and IgM reference ranges are from the Mayo Clinic
of IgG k MGUS) is a marker of a more clonally advanced MGUS
routine Immunology Laboratory. The reference ranges for each HLC and
stage and will be associated with a greater risk of progression to the HLC-pair ratios were derived from 129 normal serum donors aged
MM. The novel Hevylite assay now enables us to accurately 22–77 years. There was no apparent age or sex effect on the HLC reference
measure each isotype-specific heavy and light chain (HLC) (that is, ranges. An abnormal HLC-pair ratio was defined as being outside (either
IgGk, IgGl, IgAk, IgAl, IgMk and IgMl).9 Thus, for the first time, high or low) the reference range. Isotype-specific suppression of the
we can measure isotype-specific suppression of the uninvolved uninvolved HLC (HLC-pair suppression) was defined as a concentration of

1
Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA; 2Division of Hematology, Department of Internal Medicine,
Mayo Clinic, Rochester, MN, USA; 3Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA; 4Division of Epidemiology,
Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA; 5Multiple Myeloma Section, National Cancer Institute, Bethesda, MD, USA and 6The Binding Site, Birmingham,
UK. Correspondence: Dr J Katzmann, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine, Mayo Clinic, 200 1st street SW, Rochester, MN 55905, USA.
E-mail: Katzmann@mayo.edu
Received 11 May 2012; accepted 2 July 2012; accepted article preview online 11 July 2012; advance online publication, 3 August 2012
 Suppression of polyclonal Ig and MGUS progression
JA Katzmann et al
209
Table 1. Characteristics of gender, age, heavy chain isotype and Table 2. Normal reference ranges for immunoglobulin isotypes,
M-spike size in the 999 MGUS patient cohort heavy/light chains and HLC-pair ratios

Characteristic Level N (%) Ligand Median 95% Reference range

Sex Female 449 (44.9) IgG 1000 mg/dl 767–1600 mg/dl

Male 550 (55.1) IgGk 645 mg/dl 434–1080 mg/dl
IgGl 327 mg/dl 177–531 mg/dl
Age (years) 0–49 59 (5.9) Gk/Gl ratio 2.0 1.3–3.7
50–59 97 (9.7)
60–69 255 (25.5) IgA 197 mg/dl 61–399 mg/dl
470 588 (58.9) IgAk 122 mg/dl 53–262 mg/dl
IgAl 92 mg/dl 38–181 mg/dl
Heavy chain isotype IgG 726 (72.7) Ak/Al ratio 1.4 0.7–2.2
IgA 117 (11.7)
IgM 156 (15.6) IgM 95 mg/dl 50–267 mg/dl
IgMk 64 mg/dl 22–143 mg/dl
M-spike (g/dl) p0.5 249 (24.9) IgMl 41 mg/dl 10–94 mg/dl
0.6–1.0 197 (19.7) Mk/Ml ratio 1.6 1.0–2.4
1.1–2.0 490 (49.0)
2.1–3.0 63 (6.3) Abbreviations: HLC, heavy and light chain; Ig, immunoglobin.

Abbreviation: MGUS, monoclonal gammopathy of undetermined

significance.

Table 3. Impact of heavy chain isotype and M-spike concentration on

the incidence of abnormal HLC-pair ratios, HLC-pair suppression and
the polyclonal member of the HLC-pair below the lower limit of the isotype suppression
reference range concentration (for example, in a MGUS patient with a
monoclonal IgGk protein, suppression of IgGl below the lower limit of the MGUS M-spike Cases Abnormal HLC-pair Uninvolved
reference range for IgGl). Suppression of uninvolved immunoglobulins isotype (g/dl) (n) HLC ratio suppression immunoglobulin
was defined as either of the other two polyclonal heavy chain isotypes (%) (%) suppression (%)
being below the lower limit of the reference range (for example, in a MGUS
patient with a monoclonal IgG protein, suppression of IgA and/or IgM IgG 726 56 29 5
below the lower limits of their reference ranges). p0.5 161 24 19 4
0.6–1.0 150 57 35 7
Statistical considerations 1.1–2.0 365 65 29 5
2.1–3.0 50 90 50 10
The prognostic effect of HLC-pair suppression on the progression of MGUS
was studied in the context of other known risk factors. The primary end IgA 117 97 36 33
point was progression to MM or a related disorder. Progression end points p0.5 39 95 23 41
were examined both as a cumulative probability of progression and 0.6–1.0 19 100 47 37
cumulative incidence. The former was computed using an ordinary 1.1–2.0 55 96 44 27
Kaplan–Meier estimate10 where patients who died were censored; curves 2.1–3.0 4 100 0 25
were compared using the log-rank test. The cumulative incidence curve,
on the other hand, explicitly accounts for death from other causes (such as IgM 156 90 10 21
cardiovascular disease, cerebrovascular disease or unrelated malignancy) p0.5 49 84 2 18
as a competing risk and was estimated using the method of Gooley.12 The 0.6–1.0 28 93 14 21
effects of potential risk factors on progression rates were examined using a 1.1–2.0 70 93 13 20
Cox proportional hazards model.11 2.1–3.0 9 100 22 33
All cases 999 66 27 11
RESULTS Abbreviations: HLC, heavy and light chain; Ig, immunoglobin; MGUS,
Clinical characteristics and reference levels monoclonal gammopathy of undetermined significance.
The adult 95% reference ranges for each HLC and HLC-pair ratio
are shown on Table 2. No age or gender effects existed for any of
these assays. The HLC ranges are similar to previously published Abnormal HLC-pair ratios were common in the IgA and IgM MGUS
ranges,9 and, similar to that study, the HLC-pair ratios were patients even at low M-spike concentrations.
different for each heavy chain isotype. The median of the normal These same data were evaluated for HLC-pair suppression and
IgGk/IgGl ratio was 2.0, whereas the IgAk/IgAl ratio median was uninvolved immunoglobulin suppression (Table 3). HLC-pair
1.4 and the IgMk/IgMl ratio median was 1.6. suppression was present in 27% of the MGUS patients, but
uninvolved immunoglobulin suppression was observed in only
HLC-pair ratio and HLC-pair suppression 11% of the cases. The higher frequency of HLC-pair suppression
Table 3 shows the occurrence of abnormal HLC-pair ratios, HLC- compared with uninvolved immunoglobulin suppression was
pair suppression and uninvolved isotype suppression segregated most obvious in the IgG MGUS patients, who had 29% HLC-pair
by MGUS heavy chain type and M-protein size. An abnormal HLC- suppression compared with 5% uninvolved isotype suppression.
pair ratio was detected in 66% of MGUS patients, but the
frequency differed depending on the isotype of the M-protein. Outcomes
Patients with monoclonal IgG proteins presented with an For our initial assessment of the effect of HLC-pair suppression,
abnormal HLC-pair ratio in only 56% of cases, whereas IgA and we identified the lowest 5% HLC polyclonal concentrations in
IgM MGUS had abnormal HLC-ratios in 97% and 90% of cases, the IgG, IgA and IgM MGUS isotype groups and evaluated
respectively. As the monoclonal IgG concentration increased, the effect on progression to MM. There were 13.5 expected
the prevalence of an abnormal HLC-pair ratio also increased. progressions in this group but 22 observed progressions (1.6-fold

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 Suppression of polyclonal Ig and MGUS progression
JA Katzmann et al
210
Table 4. Univariate analysis of prognostic factors for progression of MGUS to MM

Variable Level N Events Hazard ratio (95% CI) P-value 10-Year rate in % (95% CI)

Serum M-spike o1.5 715 36 6.1 (3.8, 8.4)

X1.5 284 41 3.0 (1.9, 4.7) o0.001 16.5 (10.5, 22.0)

IgA or IgM heavy chain IgG 726 41 6.1 (3.7, 8.4)

IgA/IgM 273 36 2.7 (1.7, 4.2) o0.001 17.2 (11.1, 22.9)

FLC ratio Normal 669 30 5.5 (3.2, 7.7)

Abnormal 330 47 3.3 (2.1, 5.3) o0.001 16.6 (11.0, 21.8)

HLC-pair ratio Normal 337 10 3.9 (1.0, 6.7)

Abnormal 662 67 3.1 (1.6, 6.1) o0.001 11.5 (8.3, 14.6)

HLC-pair suppression No_Supp 727 42 6.9 (4.4, 9.2)

HLC_Supp 272 35 2.3 (1.5, 3.7) o0.001 15.3 (9.1, 21.1)

Uninvolved Ig suppression No_Supp 889 65 8.1 (5.7, 10.4)

Ig_Supp 110 12 2.1 (1.1, 3.9) 0.017 19.0 (7.1, 29.3)

999 77 9.1 (6.7, 11.4)

Abbreviations: CI, confidence interval; FLC, free light chain; HLC, heavy and light chain; Ig, immunoglobin; MGUS, monoclonal gammopathy of undetermined
significance; MM, multiple myeloma.

group, the inclusion of HLC-pair suppression further divided the

Table 5. Multivariate analysis models of prognostic factors for
progression of MGUS to MM
groups into lower and higher risk. We then developed a modified
risk stratification model using the 4 variables of M-spike
Model Prognostic factor Hazard ratio P-value concentration, FLC ratio, heavy chain isotype and HLC-pair
(95% CI) suppression is shown in Figure 1. The model has five groups (0,
1, 2, 3 or 4 adverse risk factors), and the probability of progression
HLC-pair suppression 1.8 (1.1, 3.0) 0.018 to MM increases with the number of risk factors.
Serum M-spike X1.5 gm/dl 2.3 (1.5, 3.8) o0.001
Abnormal FLC k/l ratio 2.0 (1.2, 3.4) 0.007
IgA or IgM heavy chain 2.7 (1.6, 4.6) o0.001
DISCUSSION
Abbreviations: CI, confidence interval; FLC, free light chain; HLC, heavy The term MGUS was first coined over 30 years ago.13 Studies have
and light chain; Ig, immunoglobin; MGUS, monoclonal gammopathy of shown that MGUS almost always precedes MM,2,3 and that the risk
undetermined significance; MM, multiple myeloma.
of progression of MGUS to MM or related malignancy is
approximately 1% per year.4 Moreover, there is no decline in the
risk of progression even after 25–35 years, raising the need for
lifelong follow-up by primary-care providers necessary in all
increase over expected). This excess risk was seen in IgG MGUS persons identified with MGUS. More accurate stratification of risk
(1.8-fold), IgA MGUS (1.2-fold) and IgM MGUS (1.7-fold). The effect is essential if we are to consider early intervention strategies and
of HLC-pair suppression, abnormal HLC-pair ratio, uninvolved better understand the mechanism of progression of MGUS to
immunoglobulin suppression, as well as previously identified MM. Unfortunately, distinguishing the patients at high risk of
variables of M-protein size, heavy chain isotype and FLC ratio, progression is difficult when MGUS is initially recognized, as the
were tested in univariate analysis for their significance as distinction between MM and its precursor conditions is not based
prognostic factors for progression of MGUS (Table 4). All of these on histopathology or laboratory methods but on the manifesta-
variables were significant in univariate analysis. tion of end organ damage.7 Consequently, patients are typically
The prognostic effect of the risk factors significant on univariate referred to hematologists in the face of a rising M-spike on follow-
analysis for the progression of MGUS to MM was then studied by up or when other symptoms and signs suggestive of myeloma or
multivariate analysis (Table 5). HLC-pair suppression remained related malignancy (for example, anemia, hypercalcemia, bone
significant when serum M-protein size and FLC ratio were pain, lytic bone lesions and renal failure) have developed.
analyzed in the multivariate model as continuous variables. By However, this does not allow identification of candidates at
contrast, abnormal HLC-pair ratio (hazard ratio (HR) ¼ 1.4, P ¼ 0.38) highest risk of progression for possible intervention strategies, nor
and suppression of uninvolved immunoglobins (HR ¼ 1.1, optimization of follow-up, as the efficacy and recommendations
P ¼ 0.74) were not significant for MGUS progression on multi- for monitoring vary with risk.14,15
variate analysis. When we analyzed the effect of HLC-pair Our previous studies have indicated that the size of the M-spike,
suppression separately with each of the other risk factors in this the heavy chain isotype and the secretion of excess FLC as
multivariate model, the HR for HLC-pair suppression was 2.6 in detected by an abnormal serum FLC ratio are all predictive of
combination with IgA or IgM heavy chain (Po0.001), 2.0 in progression, and they can be used to stratify risk at initial
combination with M-spike size (Po0.002), and 1.5 in combination diagnosis of MGUS.6 In our earlier studies, we showed that these
with FLC ratio (Po0.082). three variables each carry a 2–3-fold increased risk for progression.
Moreover, combining all three variables identifies low- and high-
Risk stratification model risk patients with a 10-fold difference in risk: low-risk patients have
The effect of adding HLC-pair suppression to our previous risk only a 0.1%/year risk for progression to MM compared with high-
assessment model6 is shown in Table 6. Except for the lowest risk risk patients with a 1.4%/year risk. In addition to the need for

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 Suppression of polyclonal Ig and MGUS progression
JA Katzmann et al
211
Table 6. Effect of HLC-pair suppression on risk stratification for progression of monoclonal gammopathy of undetermined significance

Risk group Number of Hazard ratio Absolute risk of progression at Absolute risk of progression at 20 years
patients (95% CI) 20 years in % (95% CI) accounting for death as a competing risk (%)

Low-risk (M-protein o1.5 gm/dl, IgG subtype and normal rFLC)

No HLC-pair suppression 325 1 7.8 (0.0, 17.0) 3.0
HLC-pair suppression 73 0.7 (0.1, 5.8) 0.0 (0.0, 0.0) 0.0

Low-intermediate-risk (1 of 3 adverse risk factors)

No HLC-pair suppression 265 1 21.9 (2.3, 37.6) 9.2
HLC-pair suppression 95 2.0 (0.9, 4.2) 24.9 (9.0, 38.0) 13.4

High-intermediate-risk (2 of 3 adverse risk factors)

No HLC-pair suppression 104 1 35.4 (3.2, 56.9) 16.1
HLC-pair suppression 92 1.7 (0.8, 3.6) 35.3 (15.2, 50.6) 21.5

High-risk (3 of 3 adverse risk factors)

No HLC-pair suppression 33 1 44.0 (0.0, 69.8) 23.4
HLC-pair suppression 12 1.9 (0.6, 5.8) NAa NAa
Abbreviations: CI, confidence interval; FLC, free light chain; HLC, heavy and light chain; Ig, immunoglobin; MM, multiple myeloma. The impact of HLC-pair
suppression is tested in low-risk, low-intermediate-risk, high-intermediate-risk and high-risk MGUS risk groups. aNA ¼ 20-year risk not available due to too few
observed events.

Figure 1. Risk of progression of MGUS to MM using a risk stratification model that incorporates HLC-pair suppression, FLC k/l ratio, heavy
chain isotype and size of the serum monoclonal protein. The top curve illustrates risk of progression in patients with all four risk factors,
namely HLC-pair suppression, abnormal serum FLC k/l ratio, serum M-spike X1.5 gm/dl and non-IgG MGUS; the second gives the risk of
progression in patients with any three of these risk factors; the third curve illustrates the risk of progression with two of these risk factors; the
forth curve illustrates the risk of progression with one of these risk factors; and the bottom curve is the risk of progression for patients with
none of the risk factors.

refining prognosis for optimal follow-up and preventive strategies, to the relative insensitivity of total uninvolved immunoglobulin
identification of additional risk factors may also provide important suppression for identifying this phenomenon. The availability of
new insight into the biology and mechanisms of progression of reagents to separately quantitate the k- and l-containing proteins
MGUS to MM. of each immunoglobulin isotype now provides a unique tool to
As noted above, the progression of MGUS to MM is defined by study polyclonal isotype-specific immunoglobulin suppression
the manifestation of clinical symptoms due to the clone of in patients with MGUS. In this paper, we demonstrate the
monoclonal plasma cells. Suppression of uninvolved immunoglo- independent prognostic value of HLC-pair suppression in MGUS.
bulins is seen in many patients with MM, but it is not part of We find that the suppression of the isotype-specific HLC (for
current diagnostic criteria, and there have been conflicting reports example, suppression of the IgGl level below the reference
about the prognostic value of uninvolved immunoglobulin range in IgGk MGUS) is associated with a 2-fold excess risk of
concentrations in MGUS.4,5 We hypothesized that suppression of progression to MM after adjusting for other known risk factors,
non-clonal plasma cells is a high-risk marker for progression, and namely the size and type of the serum M-protein and the FLC
that the inconsistent prognostic results seen previously were due ratio. When added to the risk groups defined by M-spike size,

& 2013 Macmillan Publishers Limited Leukemia (2013) 208 – 212

 Suppression of polyclonal Ig and MGUS progression
JA Katzmann et al
212
heavy chain isotype and FLC ratio, the HLC-pair suppression made CONFLICT OF INTEREST
added prognostic value to each of the subgroups with the Dr Bradwell is the founder of the Binding Site, manufacturer of the Hevylite assay
exception of the lowest risk group. and has a financial investment in the company. Dr Kyle has received honoraria from
When HLC-pair suppression was tested by multivariate analysis The Binding Site for educational lectures. None of the other authors have conflicts of
in combination with only M-spike size or only FLC ratio, we noted interest pertinent to this manuscript.
a slight reduction in risk due to HLC-pair suppression. As IgG half-
life can be reduced by saturation of the IgG Fc receptors, it is
possible that some of this reduction in risk is simply due to larger ACKNOWLEDGEMENTS
M-spikes resulting in shorter half-life for the uninvolved IgG HLC.16 This study was supported in part by the National Cancer Institute (CA107476, CA100707,
Similarly, the FLC ratio may already contain some information due CA83724), National Institutes of Health, US Public Health Service, Bethesda, MD
to immune suppression. However, these interactions did not and in part by the Jabbs Foundation, Birmingham, UK and the Henry J Predolin
Foundation, USA.
abrogate the overall prognostic value of the HLC-pair suppression.
On multivariate analysis, the HLC-pair suppression contributed
unique prognostic information, independent of the serum M-spike
AUTHOR CONTRIBUTIONS
size, heavy chain type and FLC ratio.
This study was possible because we had retained serum JAK, SVR, RAK, ARB and AD designed the research. RC generated the data. DRL,
samples from a large cohort of MGUS patients, collected at the TMT, JTB and CLC analyzed the data. JAK, SVR, LJM, OL, JRC, AD, SK and RAK
time of first diagnosis, which could subsequently be tested for HLC wrote and edited the manuscript. All authors reviewed and approved the final
concentrations using the new Hevylite assay. There are a number manuscript.
of important biologic and clinical implications of our findings.
First, we show that isotype-specific suppression of polyclonal HLC
is a risk factor for progression of MGUS to MM. Second, we find REFERENCES
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