Impact of primary molecular cytogenetic abnormalities and risk of progression in smoldering multiple myeloma

Abstract

We studied 351 patients with smoldering multiple myeloma (SMM) in whom the underlying primary molecular cytogenetic subtype could be determined based on cytoplasmic immunoglobulin fluorescent in situ hybridization studies. Hundred and fifty-four patients (43.9%) had trisomies, 127 (36.2%) had immunoglobulin heavy chain (IgH) translocations, 14 (4%) both trisomies and IgH translocations, 53 (15.1%) no abnormalities detected and 3 (0.9%) had monosomy13/del(13q) in the absence of any other abnormality. Among 127 patients with IgH translocations, 57 were t(11;14), 36 t(4;14), 11 musculoaponeurotic fibrosarcoma (MAF) translocations, and 23 other or unknown IgH translocation partner. Time to progression (TTP) to symptomatic multiple myeloma was significantly shorter in patients with the t(4;14) compared with patients with t(11;14), median 28 versus 55 months, respectively, P = 0.025. The median TTP was 28 months with t(4;14) (high-risk), 34 months with trisomies alone (intermediate-risk), 55 months with t(11;14), MAF translocations, other/unknown IgH translocations, monosomy13/del(13q) without other abnormalities, and those with both trisomies and IgH translocations (standard-risk), and not reached in patients with no detectable abnormalities (low-risk), P = 0.001. There was a trend to shorter TTP with deletion 17p (median TTP, 24 months). Overall survival from diagnosis of SMM was significantly inferior with t(4;14) compared with t(11;14), median 105 versus 147 months, respectively, P = 0.036.

INTRODUCTION

Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by osteolytic bone lesions, anemia, hypercalcemia and renal failure.^1,2 Despite major advances, most patients eventually relapse and die of the disease.^3–6 MM is almost always preceded by an asymptomatic premalignant period that when detected clinically is termed either monoclonal gammopathy of undetermined significance (MGUS) or smoldering MM (SMM) depending on the extent of bone marrow involvement and monoclonal (M) protein levels.^7,8 MGUS is present in ~ 3 to 4% of the population above the age of 50, and carries a risk of progression to MM or related malignancy of 1% per year, a rate that makes it difficult to study the impact of biological risk factors.^9–11 SMM represents an intermediate stage between MGUS and MM, and has a risk of progression to MM or related malignancy of ~ 10% per year making it well suited for trials of preventive therapy as well as studies of risk factors and biomarkers of disease progression.^12–14

The disease phenotypically referred to as MM at a molecular level consists of several distinct subtypes, each of which could be considered a unique disease entity.^1,2,15,16 At a high level, MM consists of three main cytogenetic subtypes: trisomies (involving the odd-numbered chromosomes), immunoglobulin heavy chain (IgH) translocations involving chromosome 14q32, and a small subset in which there is evidence of both trisomies and IgH translocations. The IgH translocated subgroup includes several distinct cytogenetic types based on the specific partner chromosome and the oncogene that is dysregulated as a result of the translocation. The most common reciprocal translocations in the IgH translocated subgroup of MM are t(11;14), t(4;14), t(14;16), t(14;20) and t(6;14).¹⁷ These abnormalities (trisomies and IgH translocations) are referred to as ‘primary’ cytogenetic abnormalities as they originate at the MGUS stage and are felt to be causally related to the onset of clonal plasma cell proliferation. Some patients have loss of chromosome 13 or deletions involving chromosome 13q, referred to as monosomy13/del(13q), and occasionally this occurs in the absence of any other primary cytogenetic abnormality. Monosomy 13/del(13q) may occur anytime during the disease course from MGUS to MM and is generally not thought to be causally related to the onset of clonal proliferation.^15,16,18 Other abnormalities such as deletions involving chromosome 17p, referred to as del(17p), are typically considered late events, indicative of MM rather than premalignancy.

Although several biomarkers have been described to predict risk of progression in MGUS and SMM,^19–22 there are no studies investigating risk of progression according to the underlying cytogenetic subtype of the disease. Similarly, although numerous studies have described prognostic differences in patients with MM based on the underlying cytogenetic type,^15,16,23,24 there are no data on whether the risk of progression from premalignancy to MM differs based on the primary cytogenetic abnormality. The purpose of this study was to examine the impact of the underlying cytogenetic subtype on the risk of progression to malignancy in a well-defined cohort of patients with SMM.

PATIENTS AND METHODS

Study cohort

Patients were identified by searching a computerized database and reviewing the medical records of all patients meeting the International Myeloma Working Group definition of SMM in whom the underlying molecular cytogenetic subtype could be determined based on cytoplasmic immunoglobulin fluorescent in situ hybridization (FISH) studies of bone marrow plasma cells.^25,26 Patients were seen at the Mayo Clinic from January 1991 through June 2010. All patients had ≥ 10% bone marrow plasma cells and/or serum M protein ≥ 3 g/dl, plus absence of hypercalcemia, renal insufficiency, anemia or lytic bone lesions attributable to a plasma cell disorder. Patients who had received prior chemotherapy or had an existing diagnosis of AL amyloidosis at the time of SMM diagnosis were excluded. Approval for the study was obtained from the Mayo Clinic Institutional Review Board according to federal regulations, and in accordance with the Declaration of Helsinki.

Mayo Clinic electronic medical records, including demographic data; physician notes; laboratory tests; imaging studies; and pathologic reports, such as bone marrow aspirate and biopsy, were reviewed. Relevant laboratory data including bone marrow plasma cell percentage, serum and urine M protein, free light chain (FLC) ratio, hemoglobin, calcium and creatinine were abstracted for analysis.

Molecular cytogenetic classification

All cytoplasmic immunoglobulin FISH studies were performed for clinical purposes at the Mayo Clinic, Rochester, MN, USA, as previously described.^27,28 Briefly, aspirate samples were enriched for mononuclear cells using the ACK lyse and cytospin slides were prepared. FISH analysis was performed using the following probes: 3cen (D3Z1), 7cen (D7Z1), 9cen (D9Z1), 15cen (D15Z4), 11q13 (CCND1-XT), 14q32 (IGH-XT), 13q14 (RB1), 13q34 (LAMP1), 14q32 (5′IGH,3′IGH), 17p13.1 (p53) and 17cen (D17Z1). Additional probes as needed were used to detect t(4;14), t(14;16), t(14;20) and other abnormalities based on the results of the initial screen. For the purposes of this study, presence of trisomies of one or more odd-numbered chromosomes was classified as trisomies. A patient was classified into the specific trisomies and IgH translocation categories regardless of when these abnormalities were detected in the course of the disease, including after progression to MM, as these abnormalities are considered primary and present from the initial MGUS stage. Conversely, monosomy13/del(13q) and del(17p) were considered only if they were detected when the patient was in the SMM stage, and at least 6 months or more before any progression event. Patients were initially classified into eight non-overlapping primary cytogenetic groups: trisomies, t(11;14), t(4;14), musculoaponeurotic fibrosarcoma (MAF) translocations (t(14;16) or t(14;20)), unknown/other IgH translocation partner, both trisomies and IgH translocations, monosomy13/del(13q)in the absence of any other primary cytogenetic abnormality, and normal or insufficient plasma cells. Subsequently, groups were pooled together for additional analyses. The prognostic value of monosomy13/del(13q) and del(17p) detected before disease progression was studied separately, comparing patients with and without these abnormalities, regardless of other primary cytogenetic abnormalities.

Statistical analysis

Calculations were performed using SPSS version 19.0 (IBM Corporation, Armonk, NY, USA). Two-sided Fisher exact tests were used to test for differences between categorical variables. Two-sided Wilcoxon rank-sum tests were used to compare continuous variables. Time to progression (TTP) measured from the date of SMM diagnosis until progression to MM was the primary end point. Secondary endpoints studied included TTP from SMM diagnosis until progression to MM or related disorder (AL amyloidosis and plasmacytoma), and overall survival. Kaplan–Meier analysis was performed to generate survival curves. Groups were compared with the two-tailed log-rank test. For univariate and multivariate analysis, bone marrow plasma cell percentage, serum M protein size and serum-FLC ratio were studied as continuous variables. Multivariate analysis was performed using Cox’s proportional hazards model. Median follow-up time was calculated using the reverse Kaplan–Meier method.

RESULTS

Patient characteristics

A total of 351 patients (172 women and 179 men) were studied. Patient characteristics at SMM diagnosis are shown in Table 1. The median follow-up for the cohort was 82 months from diagnosis of SMM. The median age at SMM diagnosis was 63 years (range, 26–90 years). The light chain type was κ in 60%, λ in 37% and biclonal in 3%. The heavy chain type was IgG in 73%, IgA in 16%, 9% other (biclonal, light chain only and IgM), and 1% negative or indeterminate. The serum-FLC level at baseline was available in 246 patients (70%). The FLC ratio was abnormal (reference range <0.26 or >1.65) in 84% of patients (n =206).

Table 1.

Baseline characteristics of patients with smoldering multiple myeloma

	Patients N = 351
Median age, years (range)	63 (26–90)
Female sex, N (%)	172 (49)
Serum monoclonal protein spike (n = 302), median (range) (g/dl)	2.15 (0.0–4.9)
Urine monoclonal protein spike (n = 258), N (%)
Not present	125
Detected on immunofixation only	64
Less than 0.5 g per 24 h	51
0.5 grams per 24 h or more	18
Bone marrow plasma cell percentage (n = 351), median (range)	20 (4–90)
Serum-FLC assay (n = 246), median (range)
Serum-FLC involved/uninvolved ratio, median (range)	11.6 (1.09–7905.66)
Abnormal kappa/lambda FLC ratio (< 0.26 or > 1.65) (%)	84
Abnormal involved/uninvolved FLC ratio ≥ 8 (%)	69

Abbreviation: FLC, free light chain.

Cytogenetic classification

The distribution of patients by the various primary cytogenetic categories is given in Table 2. Overall, 154 patients (43.9%) had trisomies, 127 (36.2%) had IgH translocations, 14 (4%) had both trisomies and IgH translocations, 53 (15.1%) had no abnormalities detected (36 normal and 17 insufficient) and 3 patients (0.9%) had monosomy13/del(13q) in the absence of any other abnormality. Among patients with MAF translocations (n =11), eight had translocation t(14;16) and three had t(14;20). Of the patients with both trisomies and IgH translocations (n =14), two had t(4;14), three had t(11;14), one had t(14;20), and the rest had other or unknown partner IgH translocation. Monosomy13/del(13q), with or without primary cytogenetic abnormalities, was detected in 42 patients before disease progression to MM, while del(17p) before disease progression was present in 6 patients.

Table 2.

Distribution of primary cytogenetic categories of smoldering multiple myeloma

Cytogenetic classification by fluorescent in situ hybridization	Overall (n = 351) No. of patients (%)
Trisomy(ies) without IgH translocation	154 (43.9%)
t(11;14)(q13;q32)	57 (16.2%)
t(4;14)(p16;q32)	36 (10.3%)
MAF translocations (t(14;16)(q32;q23) and t(14;20)(q32;q11))	11 (3.1%)
Other/unknown IgH translocation partner	23 (6.6%)
Both IgH translocation and trisomy (ies)	14 (4%)
Monosomy13/del(13q) in absence of IgH translocation or trisomies	3 (0.9%)
Normal or insufficient	53 (15.1%)

Abbreviation: MAF, musculoaponeurotic fibrosarcoma.

Outcomes

During the follow-up period, 219 SMM patients (62.4%) progressed to symptomatic MM. Eight additional patients developed progression to related disorders (AL amyloidosis). The median TTP to MM was 48 months (95% confidence interval, 37–59); TTP to MM or related disorder was 43 months (95% confidence interval, 35–51). On univariate analysis previously established, prognostic markers were significantly associated with risk of progression to MM, including size of the serum M spike (P =0.05), abnormal FLC ratio <0.26 or >1.65 (P =0.001), and bone marrow plasma cell percentage (P =0.001). The median overall survival was 135 months (95% confidence interval, 118–152).

Impact of cytogenetic abnormalities on progression

Patients with t(4;14) had a significantly higher risk of progression compared with t(11;14), median TTP to MM 28 months versus 55 months, respectively, HR, P =0.025, (Figure 1). Corresponding values for median TTP to MM or related disorder were 28 versus 50 months, respectively, P =0.04.

Figure 1.

TTP of SMM to symptomatic MM. The TTP was significantly inferior in patients with the t(4;14) (HR) compared with patients with t(11;14) (SR), median 28 versus 55 months, respectively, P = 0.025.

The risk of progression to SMM was not affected by the presence of monosomy13/del(13q) at baseline, median TTP 48 versus 55 months, respectively, P = 0.9. There were too few patients with del(17p) at baseline (n =6) to make definitive comparisons, but there was a trend to higher risk of progression with a median TTP of 24 months among patients with del(17p) compared with 50 months in patients without del(17p). All six patients with del(17p) had concurrent trisomies.

Patients were categorized into four cytogenetically distinct groups based on risk of progression (Figure 2). Patients with t(4;14) were considered high-risk (HR); trisomies alone were considered intermediate-risk; those with t(11;14), MAF translocations, other/unknown 14q32 translocations, monosomy13/del(13q) alone without other abnormalities, and those with both trisomies and IgH translocations considered standard-risk; and patients with normal FISH or insufficient plasma cells were considered low-risk. The risk of progression to MM was significantly different between the four groups, with median TTP to MM 28, 34, 55 months, and not reached, respectively, P = 0.001 (Figure 2). The corresponding values for median TTP to MM or related disorder were 28, 34, 50 and 101 months, respectively, P = 0.002. The median TTP to MM for the trisomies group and the overall P-value (P = 0.001) were unchanged when the analysis was repeated after removing the six patients with del(17p).

Figure 2.

TTP of SMM to symptomatic MM according to four risk groups defined by primary cytogenetic abnormalities. The median TTP was 28 months with the t(4;14) (HR), 34 months with trisomies alone (IR), 55 months with t(11;14), MAF translocations, other/unknown 14q32 translocations, monosomy13/del(13q) without other abnormalities, and those with both trisomies and IgH translocations (SR), and not reached in patients with no detectable abnormalities (LR), P = 0.001.

As patients with del(17p) had a median TTP to MM of 24 months, the analysis by the four risk groups was performed after amending the HR group to include del(17p). In this modified analysis, the two patients with t(4;14) who also had trisomies (one progressed after 13 months and one was progression free at 21 months) were also considered HR (Table 3). The risk of progression to MM was significantly different between the revised four groups, with median TTP to MM of 24, 34, 55 months, and not reached, respectively, P = 0.001 (Figure 3).

Table 3.

Cytogenetically defined risk-based classification of SMM

Risk	Cytogenetic class	No. of patients (%)a	Median TTP to myeloma (months)*	Median TTP to myeloma or related disorder (months)**	Median OS from SMM diagnosis (months)***	Median OS from MM diagnosis (months)****,b
High-riska	t(4;14), del(17p)	44 (12.5%)	24	24	105	60
Intermediate- risk	Trisomy (ies) without IgH translocation	148 (42.2%)	34	34	135	77
Standard-risk	t(11;14) MAF translocations, t14;16 or t(14;20) Other/unknown IgH translocation partner Both trisomies and IgH translocation except t(4;14) Monosomy13/del(13q) in absence of IgH translocation or trisomies	106 (30.2%)	55	54	147	86
Low-risk	No abnormalities (normal or insufficient)	53 (15.1%)	Not reached	101	135	112

Abbreviations: IgH, immunoglobulin heavy chain; MAF, musculoaponeurotic fibrosarcoma; MM, multiple myeloma; OS, overall survival; SMM, smoldering multiple myeloma; TTP, time to progression.

^*P = 0.001.

^**P = 0.002.

^***P = 0.12 (global); P = 0.02 (high-risk versus standard-risk).

^****P = 0.04.

^aModified to include del(17p) and t(4;14) with concurrent trisomies.

^bN = 219 patients.

Figure 3.

TTP of SMM to symptomatic MM according to modified cytogenetically defined risk-based classification. The median TTP was 24 months with the t(4;14)/del(17p) (HR), 34 months with trisomies alone (IR), 55 months with t(11;14), MAF translocations, other/unknown 14q32 translocations, monosomy13/del(13q) without other abnormalities, and those with both trisomies and IgH translocations except t(4;14) (SR), and not reached in patients with no detectable abnormalities (LR), P = 0.002.

On multivariate analysis, the increased risk of progression associated with t(4;14) remained significant (P = 0.01) in a model that included bone marrow plasma cell percentage (P = 0.001), but was not independent of the serum-FLC ratio (P-value 0.44 and 0.021, respectively). Similarly, the four-group risk model retained significance (P<0.001) in a model that included bone marrow plasma cell percentage (P = 0.001); but was not independent of the serum-FLC ratio. In a subset of 68 patients with serum M protein ≥ 3 g/dl and bone marrow plasma cell percentage ≥ 10%, the TTP to MM was 28, 21 and 55 months in the revised HR, IR and SR groups, respectively, P=0.03; there were only two patients with LR in this subset, and thus they were excluded from the survival comparisons.

Impact of cytogenetic abnormalities on overall survival in SMM

Median survival of the cohort from time of SMM diagnosis was 135 months; median survival from MM diagnosis was 77 months. The t(4;14) SMM was associated with significantly inferior overall survival measured from diagnosis of SMM compared with patients with t(11;14) SMM, 105 versus 147 months, respectively, P = 0.036 (Figure 4). When the analysis was restricted to patients with eventual progression to MM, median survival was 89 versus 141 months, respectively, P = 0.06. Overall survival measured from diagnosis of SMM appeared similar among the other cytogenetic groups of SMM; thus median survival for the four groups classified according to risk of progression was 105, 135, 141 and 135 months in the high, intermediate, standard and low-risk groups, respectively, P = 0.25. However, survival times measured from time of MM diagnosis was as expected significantly different across the four groups with median times of 51, 77, 86 and 112 months, respectively, P = 0.04 (Figure 5). The outcomes by the revised risk groups in which del(17p) patients and patients with t(4;14) and trisomies are considered HR is shown in Table 3.

Figure 4.

Overall survival from time of diagnosis of SMM. Survival was significantly inferior in patients with the t(4;14) (HR) compared with patients with t(11;14) (SR), median 105 versus 147 months, respectively, P = 0.036.

Figure 5.

Overall survival from diagnosis of symptomatic MM according to the four cytogenetically defined risk groups. Survival was 51 months with t(4;14) (HR), 77 months with trisomies alone (IR), 86 months with t(11;14), MAF translocations, other/unknown 14q32 translocations, monosomy13/del(13q) without other abnormalities, and those with both trisomies and IgH translocations (SR), and 112 months in patients with no detectable abnormalities (LR), P = 0.04.

DISCUSSION

In this study, we show for the first time the significant impact of primary cytogenetic abnormalities on progression to MM. Several studies have shown that primary cytogenetic abnormalities have a significant impact on prognosis in patients with clonal plasma cell disorders once progression to malignancy occurs.^15,16 However, despite the fact that these abnormalities are present at the MGUS and SMM stage,^27,29 the impact of the various cytogenetic subtypes on risk of malignant transformation is not known. Studies in this regard have been limited by the fact that sensitive cytogenetic testing for accurate classification has only recently been available and sufficient follow-up is required to capture risk of progression.

Our study shows that patients with t(4;14) have a significantly higher risk of progression to MM compared with the t(11;14) and other cytogenetic categories. This subgroup also had shortened overall survival from SMM diagnosis as well as after progression to MM. Patients with del(17p) at diagnosis of SMM may have a high risk of progression, but there were few cases to make any definitive conclusions. Trisomies that are associated with a favorable prognosis from time of diagnosis of MM were associated with a risk of progression that is intermediate between t(4;14) and t(11;14). This intermediate risk persisted in analysis that excluded six patients with del(17p) all of whom had trisomies. However, in the subset of patients with high M protein levels (≥ 3 g/dl), the TTP to MM with trisomies was similar to HR patients.

Patients with SMM can be broadly classified based on four cytogenetically defined groups as shown in Figure 2. Patients with no detectable abnormality (normal results or low tumor burden reflected as insufficient plasma cells for FISH analysis) had the best outcome. It is felt that all patients with clonal plasma cell disorders have underlying cytogenetic abnormalities provided more sensitive detection methods are used.¹⁶ However, the improved outcome in these patients was not fully explained by lower levels of bone marrow plasma cell involvement alone, and thus more study is needed to determine the cytogenetic nature of this subgroup of patients. Although there were limited numbers of patients with t(14;16), t(14;20) and other/unknown 14q32 translocations, these patients had a risk of progression to MM resembling t(11;14). Similarly, as a group, patients with both trisomies and IgH translocations also resembled patients with t(11;14); however, there were only two patients with t(4;14) who also had trisomies to determine whether patients with t(4;14) with concurrent trisomies have a lower risk of progression than patients without trisomies. In a revised model (Figure 3 and Table 3), patients with del(17p) and patients with t(4;14) with concurrent trisomies were also considered HR. This led to an improvement in assessment of risk, and is the model that we propose for clinical prognostic purposes.

Monosomy13/del(13q) had no impact on risk of progression. Initially it was thought that monosomy13/del(13q) was a significant adverse prognostic marker in MM, but later studies showed that even in MM the prognostic effect was seen only in karyotypic studies where the abnormality probably functions as a surrogate marker for hypodiploidy, IgH translocations or proliferation rather than being a true driver of risk.^28,30–32 However, the frequent occurrence of this abnormality in early stages of plasma cell dyscrasias despite lack of effect on outcome needs further study.

We found that the adverse impact of t(4;14) was not related to bone marrow plasma cell involvement, as both factors remained independently significant on multivariate analysis. However, the adverse effect of t(4;14) was not independent of an abnormal FLC ratio. We have previously shown that an abnormal FLC ratio is a significant risk factor for progression in SMM.^22,33 We have also shown that patients with t(4;14) MM often have markedly high FLC ratios.³⁴ The mechanism by which a high FLC ratio is associated with higher risk of progression is not clear, and is only partly related to renal failure from cast nephropathy. The result of this study suggests that part of the prognostic effect of the FLC ratio (as a predictor of risk of progression in SMM) may in part derive from the fact that it serves as a surrogate biomarker for HR t(4;14) subtype SMM.

Except in rare instances, treatment is not recommended for SMM outside the setting of a clinical trial.^14,35 The recommended clinical monitoring for SMM is serial follow-up every 3–4 months with the initiation of treatment upon symptomatic progression.³⁶ As patients with t(4;14) myeloma appear to spend a shorter duration of time in the premalignant stage, and have a worse outcome following diagnosis of MM, the type of follow-up and indications for therapy in this subgroup may need to be adjusted.

Our study provides clear evidence that clinically defined MM consists of several cytogenetically distinct diseases, with variations not just in clinical presentation and prognosis, but also the risk of progression from premalignancy to malignant disease. Patients with t(4;14) translocation have the highest risk of progression followed by patients with trisomies. Patients with del(17p) are also likely in the HR group. So far, therapy has not been recommended for SMM patients,³⁷ and trials with thalidomide,^38,39 bisphosphonates,^40,41 anakinra (IL-1 receptor antagonist)⁴² and curcumin⁴³ have not demonstrated a clear benefit of early intervention. However, as evidence favoring early intervention accumulates with newer approaches,⁴⁴ data on the effect of therapy according to the underlying molecular classification of the disease will be of critical importance.