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High-Producer Haplotypes of Tumor Necrosis Factor Alpha and Lymphotoxin Alpha Are Associated With an Increased Risk of Myeloma and Have an Improved Progression-Free Survival After Treatment

From the Department of Haematology, Leeds General Infirmary and University of Leeds; Leukaemia Research Fund Clinical Epidemiology Unit, University of Leeds, Leeds; and Department of Immunology, University of Birmingham, Birmingham, United Kingdom.

Address reprint requests to Gareth Morgan, PhD, Department of Haematology, Algernon Firth Building, University of Leeds, Great George St, Leeds, LS1 3EX United Kingdom; email garethm{at}pathology .leeds.ac.uk.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the effect of polymorphic variations in the tumor necrosis factor alpha (TNF) and lymphotoxin alpha (LT) genes on the predisposition to myeloma and the effect of these polymorphisms on response to treatment and overall survival.

PATIENTS AND METHODS: Genotype distribution was determined in 63 patients with monoclonal gammopathy of uncertain significance (MGUS) and 198 patients with myeloma and compared with that in 250 age- and sex-matched population-based controls. The effect on treatment response and survival was determined in 171 myeloma patients treated with either conventional or high-dose chemotherapy.

RESULTS: Comparison of the extended TNF/LT haplotype in the myeloma cases and controls showed a significant excess of high-producer alleles in the cases. The double heterozygotes TNF1/2 and LT10.5/5.5 were present in 35.8% of cases but in only 18% of the controls; this presence was associated with a significant increased risk of myeloma (odds ratio, 2.05; 95% confidence interval, 1.26 to 3.35). A similar odds ratio was seen in the MGUS cases, suggesting that this genotype is associated with the initiation of plasma-cell disorders rather than the progression of MGUS to myeloma. The median overall survival time of myeloma patients was 53.8 months and showed no difference with regard to TNF/LT polymorphic status. A trend toward an improved progression-free survival was apparent in cases with a high-producer haplotype, although this effect was seen only in patients receiving high-dose chemotherapy.

CONCLUSION: Individuals with polymorphisms associated with a high production of TNF/LT are at a significantly increased risk of developing MGUS and myeloma. The impact of polymorphic status on overall survival is minimal, although there is a trend toward an increased progression-free survival in the high-producer group.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
A NUMBER OF factors have consistently been identified as important in the etiology of myeloma. These include exposure to ionizing radiation, benzene, and solvents, and occupational links, such as farming.^1-4 Genetic factors—especially events such as switch recombination and affinity maturation, which occur during normal B-cell development—are known to exert an effect. Gene-environment interactions are also important in the etiology of many tumors, and chronic immune-system stimulation has been suggested as a potential etiologic factor in myeloma. In experimental studies, plasma-cell dyscrasias have been associated with protracted stimulation of the reticuloendothelial system; however, the results from case-control studies are inconsistent and provide little evidence to support this hypothesis.^3-7 Unfortunately, these studies are limited by interviewing techniques, the lack of accurate medical records, and difficulties in the quantification of recurrent and chronic infections. In contrast, inherited genetic polymorphisms at cytokine loci, acting early in the immune response, may mediate immune-system responsiveness and are readily quantifiable using polymerase chain reaction (PCR)–based techniques.

Tumor necrosis factor alpha (TNF) and lymphotoxin alpha (LT, formerly known as tumor necrosis factor beta) are two critical cytokines produced early in the inflammatory reaction process. The major sources of these cytokines are macrophages, monocytes, and T cells, although TNF can also be produced by virtually all cells on activation; in addition, LT is expressed on B lymphocytes and natural killer cells. Both cytokines share a pair of receptors (tumor necrosis factor receptors I and II), and signaling via these pathways can result in cell activation, proliferation, or apoptosis, depending on the cell lineage, metabolic state of the cell, and kinetics of ligand binding.⁸ TNF and LT are also involved in T-cell–dependent B-cell responses, T-cell proliferation and receptor expression, natural killer cell activity, and dendritic-cell maturation. Their role in B-cell development has been shown in studies of knockout mice,^9-11 in which distinct roles for both cytokines in organogenesis and spatial organization of lymphoid tissue have been demonstrated. TNF/LT knockout mice show no germinal-center formation and show defects in the regulation of isotype switching and problems with both primary and secondary immune responses. In addition to these normal functions, a number of studies have suggested an important role in the pathogenesis and maintenance of the malignant clone in myeloma. Early studies showed that TNF secretion from myeloma bone marrow cells was significantly greater than that from normal bone marrow cells.¹² TNF and LT are potent inducers of the production of interleukin-6 (IL-6), a major growth factor for myeloma cells, and may therefore indirectly stimulate plasma-cell growth.¹³ Finally, TNF also seems to play a role in the differentiation of malignant plasma cells, as monoclonal plasma cells are produced when peripheral-blood mononuclear cells from myeloma patients are exposed in vitro to TNF and IL-4.¹⁴

The TNF and LT genes are located on chromosome 6p within the class III region of the major histocompatibility complex locus. Four polymorphic sites within the promoter region of the TNF gene and one polymorphic site within the first intron of the LT gene have been described; two of these five sites seem to have functional significance in vivo. A single base substitution at position -308 of the TNF gene results in two allelic forms of TNF, in which the presence of guanine defines the common variant TNF1 and the presence of adenine defines the less common variant TNF2.¹⁵ The polymorphism within the LT gene also results from a single base substitution in which guanine is replaced by adenine at position +252 of the first intron.¹⁶ The two forms are referred to as LT10.5 and LT5.5, respectively. It has been shown previously that these two polymorphisms are in linkage disequilibrium and that both high-producer alleles are associated with the extended HLA haplotype A1-B8-Dr3-DQ2.¹⁷

Functional studies of the -308 TNF polymorphism in B-cell lines have shown both higher constitutional and inducible TNF expression, reflecting the importance of both the activator protein-2 binding motif and the polymorphism in the promotor region of TNF.¹⁸ Similar studies have been performed on the LT +252 polymorphism, showing higher levels of LT in phytohemagglutinin-stimulated peripheral-blood mononuclear cells as a result of increased transcription.¹⁹ Further evidence for their functional significance may be drawn from clinical studies in autoimmune and infectious diseases. Individuals who carry the high-producer alleles have a severe and rapid disease course, compared with those cases with low-producer alleles.^20,21 More direct evidence was provided in a study of non-Hodgkin’s lymphoma (NHL) patients, which found that the TNF2 allele was associated with higher plasma levels of TNF, as was the presence of the LT5.5 allele.²² Interestingly, on the basis of the presence of LT5.5 alleles, no difference in plasma level was found for LT. The strong association between the TNF2 and LT5.5 alleles and data that demonstrate that both polymorphisms contribute to an excess of TNF suggests that these mutations should be analyzed as haplotypes rather than in isolation.

Given the central role of TNF and LT in the development of the immune system, we hypothesized that polymorphisms at these loci may alter the risk of developing myeloma and may also affect response to treatment and overall survival. To approach the question of whether high-producer haplotypes predispose to the development of myeloma, we compared the segregation of polymorphic variants at these loci in a large series of cases with monoclonal gammopathy of uncertain significance (MGUS), a series of cases with myeloma, and a series of population-based controls. To address the effect of these polymorphisms on response to treatment and survival, we analyzed the outcome of the myeloma cases after both conventional and high-dose chemotherapy.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study Group
Material was analyzed from 63 patients with MGUS and 198 patients with multiple myeloma who either presented to the General Infirmary at Leeds or were referred to the regional diagnostic laboratory. Because of the possible racial variations in genotypic frequency, only whites were included in the analysis. Myeloma patients were treated with either cyclophosphamide, vincristine, doxorubicin, and methylprednisolone (days 1 through 4, doxorubicin 9 mg/m² and vincristine 0.4 mg; days 1 through 5, methylprednisolone 1 g/m²; and days 1, 8, and 15, cyclophosphamide 500 mg) to maximum response, followed by melphalan 200 mg/m² with peripheral-blood stem-cell rescue, or with doxorubicin, carmustine, cyclophosphamide, and melphalan (day 1, doxorubicin 30 mg/m² and carmustine 30 mg/m²; days 22 through 25, cyclophosphamide 100 mg/m² and melphalan 6 mg/m²) to plateau. After undergoing autografting treatments or after achieving plateau, patients were maintained on interferon alfa-2a (Roferon-A; Hoffman-La Roche Inc, Nutley, NJ) 3 MU for 3 days each week. Presentation prognostic factors, including beta-2 microglobulin (ß2M), creatinine, and hemoglobin levels, immunoglobulin isotype, and response and survival data were available for 171 of these cases; details are summarized in Table 1. Of these 171 cases, 79 were treated with the conventional doxorubicin, carmustine, cyclophosphamide, and melphalan therapy, and 92 received the intensive cyclophosphamide, vincristine, doxorubicin, and methylprednisolone + high-dose melphalan regime. The mean age of the cohort was 55.8 years (range, 35.1 to 65.0 years). The immunoglobulin (Ig) isotype distribution was as follows: IgG, 60.2%; IgA, 22.2%; IgD, 2.9%; light-chain, 12.9%; and non-secretory, 1.8%. The median follow-up period for the entire cohort was 24.0 months (range, 0.4 to 69.5 months); the median follow-up for the conventional group was 22.2 months, and that for the intensive group was 26.0 months. The two groups were comparable for determinations of ß2M, hemoglobin, creatinine, stage, and sex.

Treatment response and follow-up in the patients who received treatment is reported using previously described Medical Research Council response criteria.²⁴ Importantly, a complete response was defined in both groups as having occurred when no detectable paraprotein in serum or urine, as determined by immunofixation analysis on two occasions, was found, and a partial response was defined as a greater than 50% reduction in the level of serum monoclonal paraprotein.²⁵ Plateau was defined as a stable phase in which patients had minimal or no symptoms attributable to myeloma, with a stable paraprotein measurement as determined in assessments 3 months apart.

Genotype Determination
Genomic DNA was extracted from peripheral-blood or bone marrow samples by proteinase K/phenol chloroform extraction.²⁶ TNF and LT products were amplified using previously published primer sequences,^15,22 except that reactions were carried out as a multiplex using the following reaction conditions: 15 pM primers (LT and TNF primer pairs), 1 mmol/L MgCl₂, 100 µmol/L diethylnitrophenyl thiophosphate, 1x reaction buffer, and 1.25 units of platinum Taq polymerase (Gibco, Grand Island, NY), in a reaction volume of 25 µL. Products were amplified using 34 cycles of 66°C for 1 minute, 72°C for 1 minute, and 94°C for 1 minute. The GA transition at nucleotide -308 of the TNF gene results in the elimination of an NcoI restriction site. The PCR amplifies a 107–base pair (bp) fragment of the TNF gene and the wild-type allele (T1) digests to produce fragments of 87 bp and 20 bp, whereas the homozygous mutant (T2) does not digest. Heterozygote mutants (T1/T2) present as a mixture of all three bands. The AG transition at nucleotide +252 of the LT gene results in the formation of an NcoI recognition site. The PCR amplifies a 368-bp fragment of the LT gene, which the wild-type (LT10.5 Kb) product does not digest, whereas the homozygous mutant (LT5.5 Kb) digests to produce fragments of 235 bp and 133 bp. The heterozygote LT5.5/10.5 fragment presents as a combination of all three bands. Fifteen microliters of each product was then restricted using 1 µL of NcoI overnight at 37°C. The digestion products were electrophoresed on 8% polyacrylamide gels, and the products were then visualized by ethidium bromide staining. Coamplified products and digestion products were examined to ensure that no interference with genotyping had occurred. A representative gel image is shown in Fig 1.

View larger version (99K): [in this window] [in a new window]   Fig 1. A polyacrylamide gel image showing the different restriction patterns obtained after the digestion of PCR products with NcoI (TNF/LT). Lanes: 1, ladder; 2, WT/HET; 3, WT/WT; 4, HET/HET; 5, HOM/HOM; 6, WT/WT; 7, HET/HET; 8, WT/WT; 9, WT/HET; 10, WT/WT; 11, HOM/HOM; 12, WT/HOM; 13, WT/HET.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Disease Susceptibility
Myeloma. The allele frequencies in the control population for the genes studied were as follows: TNF1, 0.79; TNF2, 0.21; LT10.5, 0.65; and LT5.5, 0.35. These do not differ significantly from frequencies determined in previous studies in whites, supporting the notion that these controls were a representative group for comparison. The distribution of genotypes of both myeloma cases and controls at these two loci is listed in Table 2. As in other studies, we were able to show significant linkage between the presence of TNF1/LT5.5 and TNF2/LT5.5 (P < .0001, by ² test), supporting the notion that polymorphisms of TNF and LT should be analyzed as haplotypes. A comparison of the distribution of alleles between the myeloma cases and controls demonstrated a significant excess of the TNF2 and LT5.5 alleles in the myeloma cases, compared with the control group (OR, 3.24; 95% CI, 1.11 to 9.41; P = .0001). The distribution of haplotypes for the case and control groups is listed in Table 3 and the apparent risks of developing myeloma associated with these different distributions in Table 4. The main difference between the two groups is the excess of TNF1/2 LT10.5/5.5 haplotype in the myeloma case group (n = 71; 35.9%) and the controls (n = 45; 18.0%; OR, 2.05; 95% CI, 1.26 to 3.35). Given the increased incidence of myeloma in males, compared with females, we also looked at differences in sex distribution of TNF and LT genotype and haplotype; no sex difference was seen (TNF genotype, P = .91; LT genotype, P = .75; haplotype distribution, P = .9).

View this table: [in this window] [in a new window]   Table 2. Allele Frequency and Genotype Distribution for TNF and LT Polymorphisms

View this table: [in this window] [in a new window]   Table 3. Distribution of Haplotypes of TNF and LT Within the Myeloma Cases, Compared With Controls

Presenting Features
The effects of TNF/LT polymorphic status on the presenting features in the myeloma cases were also analyzed. There were no statistical differences seen in the distribution of haplotypes dependent on ß2M, hemoglobin, or creatinine level (data not shown).

Treatment Outcome
The overall treatment responses, including the complete response rates, were also examined. Although more patients (50% v 10%) in the intensively treated group achieved a complete response, there was no effect of TNF/LT polymorphic status on the time to achieve a complete response or the actual complete-response rate. There was also no effect of polymorphic status on the achievement of plateau in the conventional treatment group.

The median overall survival time in this study of 53.8 months (95% CI, 43.3 to 64.3 months) and progression-free survival time of 28.5 months (95% CI, 23.2 to 33.8 months) are excellent. Progression-free survival and overall survival graphs are shown in Fig 2A and 2B. The effect of TNF/LT polymorphic status on survival was analyzed on the basis of a comparison of the HET/HET group (n = 64), compared with the WT/WT group (n = 58), as suggested by the case-control comparison. No significant difference in overall survival was noted, although the median survival for the high-producer haplotypes had not been reached (Table 5). When progression-free survival time was analyzed, a difference in outcome was noted, depending on the TNF/LT haplotype. In the HET/HET group, there was a trend toward an improved progression-free survival time—a median of 30.6 months (95% CI, 23.1 to 38.0 months), compared with 23.7 months in the WT/WT group (95% CI, 14.9 to 32.4 months)—but this did not reach statistical significance (P = .56). To understand the mechanism of this effect, we determined whether it was present in both treatment groups. The median progression-free survival time in the conventional treatment group was 23.6 months (95% CI, 14.95 to 32.39 months), and that in the intensive arm was 30.6 months (95% CI, 23.1 to 38.0 months; P = .56). No difference between high- and low-producer haplotypes was seen in the conventional-treatment group; however, in the intensive-treatment arm, a trend toward an improved survival was seen (WT/WT median progression-free survival time, 30.4 months [95% CI, 16.7 to 44 months] v HET/HET median progression-free survival time, 49.3 months [95% CI, 19.7 to 78.9 months]), although this again did not reach statistical significance (P = .55; Table 5).

View larger version (14K): [in this window] [in a new window]   Fig 2. Cumulative progression-free survival and overall survival times for a group of 122 cases of myeloma. When analyzed by haplotype (HET/HET v WT/WT), a trend toward an improved progression-free survival time in the HET/HET group is seen (A). There is no statistical difference in overall survival time (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
This is the first study of its kind to address the potential for high-producer alleles at key cytokine genes to impact the likelihood of developing myeloma or affecting survival after treatment. The case series is large and composed of individuals of a homogeneous white population. The control group, with which the cases have been compared, is of a similar origin and is population-based. These features and comparisons with previous studies that have found similar allele frequencies within their control populations suggest that the effects observed are real. The main finding of this study is the increased frequency of double heterozygotes (TNF1/2 LT10.5/5.5) in the myeloma cases, compared with the controls, with an associated two-fold increased risk of developing myeloma. The confidence intervals suggest that there is both a statistically and clinically significant increased risk of developing myeloma for individuals who carry these alleles. Unfortunately, it is not possible to determine the genomic organization of the haplotypes from the data generated. We were able to demonstrate an association of the high-producer alleles at both loci, and therefore, by implication, there is likely to be an excess of TNF2/LT5.5 haplotypes in this group. To provide further support for the hypothesis that high-producer alleles are associated with an increased risk of myeloma, it would be desirable to see a dose effect with the TNF2/LT5.5 homozygotes at significantly greater risk. This was not seen, and the OR associated with this genotype approached unity. However, drawing conclusions on the basis of this group, which constitutes only 1.6% of the controls, is unsound. The effect of having three mutated alleles is more difficult to understand; the TNF2/2 LT10.5/5.5 genotype is seldom seen, and its effect cannot be interpreted. The TNF1/2 LT5.5/5.5 genotype was, however, seen in 7.6% of the control group, but none was seen in the case group, suggesting that this combination may be protective. In the absence of a coherent biologic rationale for this effect, and with the highly significant association of high-producer alleles with the myeloma case group, we believe that the effect is the result of random variation consistent with the small number of observations of this genotype.

MGUS is a premyelomatous condition, and the data from 241 patients with MGUS who presented to the Mayo Clinic suggest that 26% of cases will progress over a median period of 10 years to develop myeloma, although this progression may take up to 29 years.²⁷ In the study presented here, we found an increased risk of developing MGUS in the cases with high-producer alleles. On the basis of the magnitude of this risk in the MGUS group, compared with the myeloma group, it seems more likely that this genotype predisposes to the development of a plasma-cell neoplasm, rather than to the progression of MGUS to myeloma.

Myeloma is twice as common in blacks, compared with whites, and two previous medical records–based case-control studies searched for risk factors that could account for such differences.^28,29 Although a family history of cancer was significant, no other factors, including infections, were found to account for the increased risk in blacks. Given that high-producer alleles of TNF and LT increase the risk of developing myeloma, it would be interesting to know the haplotype distribution within a black population. In the literature, the only study that specifically investigated a black population examined the distribution of TNF polymorphisms in Gambian children with respect to malaria susceptibility.²⁰ The distribution of genotypes within the Gambian control group was similar to the distribution in our white control group. No studies have examined the distribution of the LT polymorphisms in a black population. Our hospital serves a predominantly white population, and to avoid any introduction of bias as a result of racial variations in genotype distribution, we only analyzed white cases and controls. The small number of cases from other racial groups were excluded, because there were insufficient numbers to allow an analysis of each group independently.

The results of this study can be compared with the results of previous studies in B-cell lymphoproliferative disease. The largest of these studied a group of 273 patients with lymphoma, in which the two largest pathologically distinct subgroups were diffuse large B-cell (n = 126) and follicular lymphoma (n = 96).²² In contrast to the current study, no differences were seen between the distribution of alleles in the lymphoma cases and 96 unrelated controls. The analysis in the NHL study was developed further, with a comparison of the distribution of alleles within each pathologic subgroup. Fewer high-producer alleles were seen in the group of follicular lymphomas, suggesting a protective effect, although the differences did not reach statistical significance. To compare the results from the study presented here with those from the NHL study, we looked at the distribution of high- and low-producer haplotypes as defined in that study. The distribution of haplotypes in our control group corresponds to those published by Warzocha et al²² (high-producer haplotypes, 32%, and low-producer haplotypes, 68%, v high-producer haplotypes, 31%, and low-producer haplotypes, 69%, respectively), which provides support for the significance of the results in the myeloma cases seen in the study presented here, where there is an increase in the number of high-producer alleles (high-producer alleles, 44%, and low-producer alleles, 56%).

Previous studies in cases of NHL have associated high plasma levels of TNF and LT with anemia, cachexia, poor performance status (World Health Organization), and a poor disease outcome.³⁰ These cytokines are acute-phase proteins, therefore making the individual direct effects of high production of each cytokine difficult to interpret. A number of studies have investigated the effect of high-producer polymorphisms at these loci on survival. The largest of these studies found that high-producer haplotypes were associated with a poor outcome in a group of large-cell lymphomas, but this effect was absent in a group of follicular lymphomas.²² Two additional but small studies in chronic lymphocytic and hairy cell leukemia also could not demonstrate an effect on survival in this indolent group of tumors.^31,32 To date, the study presented here is the only study that has looked at the effect on outcome in myeloma.

In this study, the effects of TNF/LT polymorphic status on overall survival were minimal and disappeared on multivariate analysis. A trend toward an improved progression-free survival time was seen in the high-producer group; however, this was only seen in those patients who received intensive treatment. This finding contrasts what we would have predicted from previous studies, in which high levels of circulating cytokines correlated with a poor clinical outcome. In conclusion, these polymorphisms demonstrate strong effects on the likelihood of developing myeloma; unfortunately, they show little impact on survival time, resulting in a limited role clinically for their use as prognostic factors.

	ACKNOWLEDGMENTS

 
Supported by the Leukaemia Research Fund, London, and Yorkshire Cancer Research, Yorkshire, England.

	REFERENCES

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Submitted November 8, 1999; accepted April 10, 2000.

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