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Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12290
ORIGINAL ARTICLE
Interleukin-6 gene-174 G/C promoter polymorphism and its association with clinical profile of patients with multiple myeloma Baidarbhi CHAKRABORTY,1 Gaurav VISHNOI,2 Srinivas H GOWDA3 and Binita GOSWAMI4 1
Department of Biochemistry, Dr. D.Y. Patil Medical College, Navi Mumbai, Maharashtra, and 2Department of Pharmacology, Maulana Azad Medical College, 3Department of Biochemistry, AIIMS, 4Department of Biochemistry, Lady Hardinge Medical College, Delhi, New Delhi, India
Abstract Aim: In multiple myeloma (MM), the growth and survival of myeloma cells is controlled by interleukin-6 (IL-6), the plasma levels of which is controlled by a guanine/cytosine substitution occurring in position -174 of IL-6 gene promoter region. We studied the occurrence of IL-6-174 G/C polymorphism in patients of MM and correlated the presence of genotypes with serum IL-6 levels and tumor staging. Methods: One hundred three patients with MM and 117 age- and sex-matched healthy controls were staged by International Staging System. IL-6 genotypes were evaluated by polymerase chain reaction and restriction enzyme analysis. Serum levels of IL-6 were assessed by enzyme-linked immunosorbent assay. Results: Frequency of GG, GC and CC genotypes did not differ significantly between cases (GG 52%, GC 40%, CC 9%) and controls. The median serum level of IL-6 was significantly higher among the GC genotype versus other genotypes (24 ng/mL, P = 0.007) as compared with the GG versus other genotypes (12 ng/mL, P = 0.001). GC was associated more with stage 3 disease (27%) than was GG (11%) or CC (22% P = 0.001). Conclusions: At position 174 of the IL-6 promoter, patients with GC genotype had higher serum levels of IL-6 and presented with more severe disease compared with patients with GG or CC genotype. Key words: interleukin-6, lipid, polymorphism, ratio.
INTRODUCTION Multiple myeloma (MM) is a B-cell neoplasm accounting for 15% of hematological malignancies and 1% of total malignancies.1 It is characterized by clonal expansion of plasma cells and increased production and secretion of monoclonal immunoglobulins2 which lead to a
Correspondence: Dr Baidarbhi Chakraborty MD, Flat No. E-801, Army Cooperative Housing Society, Plot-6, Dara Enclave, Sector-9, Nerul, Navi Mumbai 400706, Maharashtra, India. Email: [email protected] Conflict of interest: none Accepted for publication 7 September 2014.
© 2014 Wiley Publishing Asia Pty Ltd
spectrum of clinical symptoms including osteolytic bone lesions, anemia and end organ renal disease.3 MM has a multifactorial etiology which brings into play the role of inflammation, oxidative stress, genetic mutations and various single nucleotide polymorphisms.4 Interleukin-6 (IL-6) is a proinflammtory cytokine which is a crucial player in the pathogenesis of MM. It acts as a growth factor for many myeloma cell lines and helps in plasma cell proliferation and survival via activation of STAT3 and ERK pathways.5,6 It also induces polyclonal B-cell activation leading to hypergammaglobulinemia. Higher levels of this cytokine have been essentially linked with higher staging and poorer prognosis of the disease.5,7 IL-6 has a very short plasma half-life and is rapidly cleared from the blood. Hence, its
2
circulating levels are determined by its level of expression.8 Several single nucleotide polymorphisms of this proinflammatory cytokine modulate the plasma levels and biological activity of IL-6 and thus influence the disease process and clinical outcome. IL-6-174 G/C polymorphism (rs1800795) is one of the four major polymorphisms described at the promoter region of IL-6.8 The region between -225 and -164 of the IL-6 promoter region containing the G/C polymorphism has previously been reported to demonstrate a negative regulatory effect on reporter gene expression.9 Polymorphism at -174 site is important as it is close to the site for glucocorticoid receptor binding (-201).10 Secondly, G to C change creates a binding site for NF1.11 This explains our rationale for choosing polymorphism at -174 region. There is, however, a lack of data regarding the prevalence of this polymorphism and its association with the prognosis of MM in Indian patients. Hence, we studied the occurrence of IL-6-174 G/C polymorphism and evaluated its association with the serum levels of IL-6 and staging of MM in Indian patients.
SUBJECTS AND METHODS Subjects The study was conducted in the Department of Biochemistry, Maulana Azad Medical College and Dr. D.Y. Patil Medical College. The study was a hospital-based case control study and comprised of 103 cases and 117 healthy controls. The cases were clinically diagnosed of having MM and were recruited after confirmation of disease by serum electrophoresis. The control group comprised of age- and sex-matched healthy individuals who were recruited from volunteers and healthy persons accompanying the patients in general outpatient (OPD). Patients with history of chronic inflammatory disorders, surgery or trauma in the past 3 months, anemia, renal or hepatic disorders, and history of regular immunosuppressive or analgesic therapy were excluded. Informed consent was obtained from all subjects before collection of information. The study protocol was passed by the institutional ethical committee.
Clinical examination A detailed history and clinical evaluation was carried out. Assessment of patients was done at day 1 following admission. International Staging System (ISS)12 was used for staging of MM.
© 2014 Wiley Publishing Asia Pty Ltd
B Chakraborty et al.
Laboratory investigations Routine hematological and biochemical assessments including serum electrophoresis were carried out. Radiological skeletal survey was carried out to assess skeletal involvement. Single blood samples were taken from patients on the day of admission. In the control group, blood samples were taken in the OPD. Half of the 10 mL of whole blood sample was transferred to an EDTA (ethylenediaminetetraacetic acid) vial containing 1.8 mg EDTA per mL of blood. From the other half, the serum was separated by centrifuging at 1000 g for 15 min for cytokine determination. Serum was then stored at −80°C until further analysis. Serum IL-6 level was estimated using commercially available ELISA (enzyme-linked immunosorbent assay) kit supplied by Diaclone Research, France.
Genotype determination Total genomic DNA was isolated from whole blood using MB504 HiPura ATM DNA miniprep purification spin kit from HiMedia Laboratories (Mumbai, India). IL-6 promoter region was amplified using the primers 5′-TGA CTT CAG CTT TAC TCT TTG T-3′ (forward primer) and 5′-CTG ATT GGA AAC CTT ATT AAG-3′ (reverse primers) (GenxBio Health Sciences, Delhi, India) under standard conditions as described by Fernandez et al.13
Statistical analysis Statistical analysis was done with Statistical Package for the Social Science 17.0 (SPSS Inc, Chicago, IL, USA). Categorical data were summarized as frequencies and percentages. Continuous data were presented as average (minimum, maximum). X2 test with Yates correction and Fisher’s exact test were done when appropriate and the odds ratio (OR) along with 95% CI was used to compare proportions between the groups. Nonparametric (Mann–Whitney) analyses were used if variance was high. The level of significance was chosen to be P < 0.05.
RESULTS The clinical profile of the patients is depicted in Table 1. Of the 103 patients enrolled in the study, the majority (54.3%) presented in stage 2 of the disease using ISS. The most common radiological finding was skull lytic lesions (35%), followed by vertebral and humerus fractures. Immunoglobulin G-kappa light chain was the most common monoclonal antibody detected by immunofixation technique.
Asia-Pac J Clin Oncol 2014
3
IL-6 polymorphism and multiple myeloma
A total of 69 men and 34 were enrolled in the study. The mean age of study population was 56.0 ± 9.9 years and this did not significantly differ from the control group. The control group was age and sex matched, and as was expected serum values of urea, creatinine and calcium were found significantly more often in cases than in controls (P < 0.05). Most patients had anemia and a significantly low A/G ratio (Table 2). Genetic analysis for IL-6 polymorphism was done for all 103 patients and 117 controls. Table 3 shows the
Table 1
Clinical profile of the multiple myeloma patients
Characteristics International Staging System I II III Skeletal survey Skull lytic lesions Vertebral fracture Humerus fracture Rib fracture Bone marrow analysis Plasma cells M : E ratio Immunofixation pattern IgG lambda IgG kappa Miscellaneous gammopathy IgA IgM IgD Bence Jones protein in urine
Table 2
Number of patients (n %) 28 (27.1) 56 (54.3) 19 (18.4) 36 (34.9) 28 (27.1) 29 (28.1) 22 (21.3) 13–85% 3:1–4:1 57 (55.3) 26 (25.2) 20 (19.4) 10 (9.7) 8 (7.7) 2 (1.9) 51 (49.5)
distribution of the IL-6-174 G/C genotypes and its alleles among cases and controls. Genotypes were in Hardy–Weinberg equilibrium (P = 0.15). GG polymorphism was the most prevalent genotype in both cases and control groups, while CC was least common (GG 52.4% vs 58.1%, P = 0.23; GC 38.8% vs 32.4%, P = 0.19; CC 8.7% vs 9.4%, P = 0.52). Risk of MM does not increase significantly with the various genotypes or the alleles in the present study. The OR for GG genotype was 0.7(CI 0.4–1.3) which was not significantly associated with MM. We also did not find any association between the GC (OR 1.3, CI 0.7–2.2) or CC genotypes (OR 0.9, CI 0.3–2.3) and MM (Table 3). We measured the serum IL-6 levels in cases and controls and correlated the levels with ISS. The IL-6 levels in cases (mean 31 ± 38, median 15 pg/mL, range 2–190) were significantly higher (P = 0.000) as compared with controls (mean 2.30 ± 1.1, median 2 pg/ mL, range 1–7). We found a positive correlation between serum IL-6 levels and staging of the disease (r2 = 0.4, P = 0.01). We also studied the association between serum IL-6 levels with the IL-6-174 G/C polymorphism. The median value of IL-6 levels was significantly higher in the GC genotype (24 pg/mL, P = 0.007) when compared with other genotypes, whereas the GG genotype was associated (12 pg/mL, P = 0.001) with significantly lower values of IL-6 values. Though the mean value 52 ± 60 pg/mL was apparently high, we could not establish any relationship between CC genotype and IL-6 levels (18 pg/mL, P = 0.2) (Table 4). This could be attributed to the less occurrence of this genotype. The prognosis of MM was assessed by ISS. When comparing the stage of disease with genotype, we found
Demographic profiles of study group and control
Age (mean ± SD) Gender (male : female) Men (mean ± SD) Women (mean ± SD) Hb TLC A : G ratio Urea Creatinine Calcium Phosphorus
Cases (n = 103)
Controls (n = 117)
p-value (Mann–Whitney test)
56.0 ± 9.9 69:34 56.3 ± 8.9 49.0 ± 13.8 8.3 ± 2.9 12 451 ± 6233 0.7 ± 0.29 141 ± 61 5.9 ± 2.6 10.1 ± 1.74 4.8 ± 2.1
52.5 ± 9.8 83:34 54.0 ± 9.9 49.0 ± 8.8 14.2 ± 5.1 8456 ± 2673 1.2 ± 0.2 32 ± 5 1.0 ± 0.9 8.7 ± 1.1 4.1 ± 1.9
NS NS NS NS S* S* S* S* S* S* NS
*P-value < 0.05 is considered significant (S). NS, not significant.
Asia-Pac J Clin Oncol 2014
© 2014 Wiley Publishing Asia Pty Ltd
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Table 3
B Chakraborty et al.
Association of IL-6-174 G/C genotype with MM
IL- 6-174 G/C polymorphism
Cases n = 103 (%)
Controls n = 117 (%)
Odds ratio
95% CI
p-value
54 (52.4) 40 (38.8) 9 (8.7)
68 (58.1) 38 (32.4) 11 (9.4)
0.7 1.3 0.9
0.4–1.3 0.7–2.2 0.3–2.3
0.23 0.19 0.52
148 (71.8) 58 (39.1)
174 (74.3) 60 (25.6)
0.88 1.15
0.5–1.3 0.7–1.7
0.5 0.5
Genotype GG GC CC Alleles G C *P-value < 0.05 is considered significant.
Table 4
Serum levels of IL-6 in different genotypes of IL-6-174 G/C polymorphism IL-6 levels (pg/mL)
Genotype present
Range
Mean ± 2 SD
Median
p-value (Mann–Whitney)
GG GC CC
2–80 4–190 10–180
17 ± 16 43 ± 47 52 ± 60
12 24 18
0.001* 0.007* 0.2
*P-value < 0.05 is considered significant.
Table 5
ISS and association of IL-6-174 G/C polymorphism ISS Stage 1 n (%)
Stage 2 n (%)
Stage 3 n (%)
24 (44.4) 4 (8.2) 3 (7.5) 25 (39.7) 2 (22.2) 26 (27.7)
24 (44.4) 32 (65.3) 26 (65) 30 (47.6) 5 (55.6) 51 (54.3)
6 (11.1) 13 (26.5) 11 (27.5) 8 (12.7) 2 (22.2) 17 (18.1)
Genotype present GG GC+CC GC GG+CC CC GG+GC
p-value 0.001* 0.001* 0.91
*P-value < 0.05 is significant.
that most patients in GG genotype had stage 1 and stage 2 diseases (44.4% each) which was significantly higher compared with other genotypes (P = 0.001). Patients with GC genotype mostly had stage 2 (65%) and stage 3 diseases (27.5%, P = 0.001). The CC genotype was associated with stage 2 disease but it did not reach the level of significance due to less number of cases. (Table 5).
DISCUSSION Recent research in the field of carcinogenesis has highlighted the pertinent link between inflammatory mediators and inflammation-related gene polymorphisms and
© 2014 Wiley Publishing Asia Pty Ltd
cancer. MM–prototype malignancy of plasma cells also follows this proinflammatory signage. The serum level of IL-6 is dictated by the polymorphisms present at the promoter site which further regulates the levels of other inflammatory molecules and maintains the smoldering inflammatory microenvironment required for the progression of the disease. It has been suggested that the NFκB and STAT3 pathways are the primary regulators of the inflammatory pathway as they modulate cytokine release and their consequent cellular and molecular repercussions. The low-grade inflammatory milieu in the tumor acts as a stimulus for these pathways leading to the setting in of a vicious cycle.14 This reiterates the importance of understanding
Asia-Pac J Clin Oncol 2014
IL-6 polymorphism and multiple myeloma
the genetic polymorphisms regulating the IL-6 gene expression. The prevalence of IL-6-174 G/C promoter polymorphism in Indian patients of MM is not known. Further, no study has reflected on the association of the genotypes with the severity of the disease or correlated the serum levels of IL-6 with the polymorphism. To the best of our knowledge, this is the first time that the distribution of IL-6-174 G/C polymorphism and its association with severity of MM in Indian patients has been studied. We studied the distribution of IL-6-174 G/C polymorphism in Indian patients of MM and found no difference in the occurrence of the genotypes between the cases and the controls. The GG genotype was most abundantly present both in cases and controls, followed by the GC and CC genotypes. Though no other Indian study evaluated the polymorphism in MM, the frequencies stated in our work are in accordance with various other studies done in diseases like stroke15 and Alzheimer’s disease16 in the same population. GG genotype was also the most prevalent in their study and CC was least frequent. Thus, it can be implied that for Indian population the presence of the polymorphism does not increase susceptibility to MM. These results echoed the finding of Zheng et al.,17 Cozen et al.,18 Duch et al.,19 Mazur et al.20 and Iakupova et al.21 who had studied the impact of the polymorphism in Spanish, American, Brazilian, Polish and Russian populations, respectively, and found no association between the polymorphism and MM. This suggests this polymorphism does not predispose individuals to MM. We further measured the serum levels of IL-6 in both cases and controls and observed that our cases had significantly higher levels of IL-6 (median 15 pg/mL) as compared with controls (median 2 pg/mL). We also established a positive correlation between the ISS of MM and serum levels of IL-6 (r2 = 0.4, P = 0.01). Our findings were in concordance with that of Nachbaur et al.22 who studied MM in Austrian population and established that IL-6 levels were significantly higher at advanced stages (II/III). This led them to propose that in myeloma patients serum IL-6 levels may reflect disease activity and tumor cell mass. Bataille et al.23 concluded in French population that serum IL-6 levels are significantly increased in patients with fulminating MM, suggesting that IL-6 is an important factor for the progression of MM. Other studies done by Pelliniemi et al.24 also proved that immunoreactive IL-6 was a prognostic factor in MM. Thus, increased IL-6 levels
Asia-Pac J Clin Oncol 2014
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were associated with higher stages and poorer prognosis in MM. It has been hypothesized that IL-6 expression is activated by both inflammatory and tumor cells and their presence in substantial concentration is essential for both sustaining chronic inflammation, promoting progression of tumor cells and inhibition of immune-mediated tumor surveillance. It has also been suggested that inflammation is not only responsible for increased release of cytokines but also for certain epigenetic changes that have profound implications for tumorigenesis.14 In our study, we also evaluated the presence of the genotypes with downstream protein production. The GC genotype was associated with highest levels of IL-6 (24 pg/mL, P = 0.007) and GG genotypes had significantly lower values (12 pg/mL, P = 0.001). Hence, we state that in Indian patients of MM, GC genotype is associated with increased production of IL-6 while patients with GG genotype produce less IL-6. This finding definitely has a bearing on the severity of the disease. As already discussed in the preceding paragraph, higher levels of IL-6 are associated with worse prognosis in MM, so in corollary we can state that the GC genotype is associated with more severe disease and patients with GG genotype appear to have less severe disease. The consequences of CC genotype could not be explored due to lesser number subjects who could not reach statistical significance. To further explore this result, we studied the association of genotypes with ISS of MM. Our conclusions got reinforced as we found that patients of GG genotype presented with significantly mild disease, that is, stage 1 (44.4%) and stage 2 (44,4%), whereas patients with GC genotypes had higher stages of the disease (stage 2 = 65%, stage 3 = 27.5%, P = 0.001). The GG genotype thus appears to be protective. This is in concordance with Mazur et al.20 who studied the polymorphism in Polish patients. Though they did not compare genotypes with staging, they found that the GC genotype was associated with increased mortality. This implies that patients with GC genotype have severe disease. However, our results differed from a previous work by Iakupova et al.21 who conducted the study in Russian population and stated that CC genotype was associated with low progressing MM and milder clinical signs. The discordance can be explained by differences in the ethnicity, genetic predisposition, methodology and the study design. Despite every effort being taken by us to design and perform our study meticulously, it still had some limitations. The sample size was relatively small. Larger
© 2014 Wiley Publishing Asia Pty Ltd
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prospective studies are necessary to fully ascertain the role of IL-6-174 G/C polymorphism and its genotypes (GG, GC, CC) in MM. Other functional polymorphisms may also influence the expression of IL-6 gene and their combined effect must be studied to better predict the prognosis of the disease. Furthermore, we did not follow our patients and did not explore the impact of the polymorphism on long-term outcome and mortality of MM. To conclude, we found no association between the IL-6-174 G/C polymorphism and its genotypes with MM. Patients with GC genotype had increased serum levels of IL-6 and presented with more severe disease (stage 2/stage 3) whereas the GG genotype appeared to be protective.
B Chakraborty et al.
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13
14
15
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REFERENCES 1 Angtuaco EJ, Fassas AB, Walker R, Sethi R, Barlogie B. Multiple myeloma: clinical review and diagnostic imaging. Radiology 2004; 231 (1): 11–23. 2 Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med 2004; 351 (18): 1860–73. 3 Maclennan ICM, Drayson M, Dunn J. Multiple myeloma. BMJ 1994; 308: 1033–6. 4 Stellrecht CM, Gandhi V. Myeloma antioxidant status: the good, the bad and the reactive. Leuk Lymphoma 2009; 50 (5): 691–3. 5 Hallek M, Leif Bergsagel P, Anderson KC. Multiple myeloma: increasing evidence for a multistep transformation process. Blood 1998; 91: 3–21. 6 Honemann D, Chatterjee M, Savino R et al. The IL-6 receptor antagonist Sant-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells. Int J Cancer 2001; 93: 674–80. 7 Lauta VM. A review of the cytokine network in multiple myeloma: diagnostic, prognostic, and therapeutic implications. Cancer 2003; 97: 2440–52. 8 Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. J Biol Chem 2000; 275: 18138–44. 9 Ray A, Sassone-Corsi P, Sehgal PB. A multiple cytokineand second messenger-responsive element in the enhancer of the human interleukin-6 gene: similarities with c-fos gene regulation. Mol Cell Biol 1989; 9: 5537–47. 10 Ray A, LaForge KS, Sehgal PB. On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. Mol Cell Biol 1990; 10 (11): 5736– 46. 11 Rein T, Forster R, Krause A, Winnacker EL, Zorbas H. Organization of the alpha-globin promoter and possible
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18
19
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role of nuclear factor I in an alpha-globin-inducible and a noninducible cell line. J Biol Chem 1995; 270: 19643–50. Greipp PR, San Miguel J, Durie BGM et al. International Staging System for multiple myeloma. J Clin Oncol 2005; 23: 3412–20. Fernandez-real JM, Broch M, Vendrell J et al. Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab 2000; 85: 1334–9. Gonda TA, Tu S, Wang TC. Chronic inflammation, the tumor microenvironment and carcinogenesis. Cell Cycle 2009; 9: 2005–13. Banerjee I, Gupta V, Ahmed T et al. Inflammatory system gene polymorphism and the risk of stroke: a case–control study in an Indian population. Brain Res Bull 2008; 75: 158–65. Mansoori N, Tripathi M, Alam R et al. IL-6–174 G/C and ApoE gene polymorphisms in Alzheimer’s and vascular dementia patients attending the cognitive disorder clinic of the all India Institute of Medical Sciences, New Delhi. Dement Geriatr Cogn Disord 2010; 30: 461–8. Zheng C, Huang D, Bergenbrant S et al. Interleukin 6, tumour necrosis factor [a], interleukin 1 [h] and interleukin 1 receptor antagonist promoter or coding gene polymorphisms in multiple myeloma. Br J Haematol 2000; 109: 39–45. Cozen W, Gebregziabher M, Conti DV et al. Interleukin6-related genotypes, body mass index, and risk of multiple myeloma and plasmacytoma. Cancer Epidemiol Biomarkers Prev 2006; 15: 2285–91. Duch CR, Figueiredo MS, Ribas C, Almeida MS, Colleoni GW, Bordin JO. Analysis of polymorphism at site −174 G/C of interleukin-6 promoter region in multiple myeloma. Braz J Med Biol Res 2005; 40: 265–7. Mazur G, Bogunia-Kubik K, Wrobel T et al. IL-6 and IL-10 promoter gene polymorphisms do not associate with the susceptibility for multiple myeloma. Immunol Lett 2005; 96: 241–6. Iakupova E, Grinchuk O, Kalimullina D et al. Molecular genetic analysis of the interleukin 6 and tumor necrosis factor a gene polymorphisms in multiple myeloma. Mol Biol (Mosk) 2003; 37: 420–4. Nachbaur D, Herold M, Maneschg A, Huber H. Serum levels of interleukin-6 in multiple myeloma and other hematological disorders: correlation with disease activity and other prognostic parameters. Ann Hematol 1991; 62: 54–8. Bataille R, Jourdan M, Zhang XG, Klein B. Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias. J Clin Invest 1989; 84: 2008–11. Pelliniemi T, Irjala K, Mattila K et al. Immunoreactive interleukin-6 and acute phase proteins as prognostic factors in multiple myeloma. Finnish Leukemia Group. Blood 1995; 85: 765–71.
Asia-Pac J Clin Oncol 2014
Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12290
ORIGINAL ARTICLE
Interleukin-6 gene-174 G/C promoter polymorphism and its association with clinical profile of patients with multiple myeloma Baidarbhi CHAKRABORTY,1 Gaurav VISHNOI,2 Srinivas H GOWDA3 and Binita GOSWAMI4 1
Department of Biochemistry, Dr. D.Y. Patil Medical College, Navi Mumbai, Maharashtra, and 2Department of Pharmacology, Maulana Azad Medical College, 3Department of Biochemistry, AIIMS, 4Department of Biochemistry, Lady Hardinge Medical College, Delhi, New Delhi, India
Abstract Aim: In multiple myeloma (MM), the growth and survival of myeloma cells is controlled by interleukin-6 (IL-6), the plasma levels of which is controlled by a guanine/cytosine substitution occurring in position -174 of IL-6 gene promoter region. We studied the occurrence of IL-6-174 G/C polymorphism in patients of MM and correlated the presence of genotypes with serum IL-6 levels and tumor staging. Methods: One hundred three patients with MM and 117 age- and sex-matched healthy controls were staged by International Staging System. IL-6 genotypes were evaluated by polymerase chain reaction and restriction enzyme analysis. Serum levels of IL-6 were assessed by enzyme-linked immunosorbent assay. Results: Frequency of GG, GC and CC genotypes did not differ significantly between cases (GG 52%, GC 40%, CC 9%) and controls. The median serum level of IL-6 was significantly higher among the GC genotype versus other genotypes (24 ng/mL, P = 0.007) as compared with the GG versus other genotypes (12 ng/mL, P = 0.001). GC was associated more with stage 3 disease (27%) than was GG (11%) or CC (22% P = 0.001). Conclusions: At position 174 of the IL-6 promoter, patients with GC genotype had higher serum levels of IL-6 and presented with more severe disease compared with patients with GG or CC genotype. Key words: interleukin-6, lipid, polymorphism, ratio.
INTRODUCTION Multiple myeloma (MM) is a B-cell neoplasm accounting for 15% of hematological malignancies and 1% of total malignancies.1 It is characterized by clonal expansion of plasma cells and increased production and secretion of monoclonal immunoglobulins2 which lead to a
Correspondence: Dr Baidarbhi Chakraborty MD, Flat No. E-801, Army Cooperative Housing Society, Plot-6, Dara Enclave, Sector-9, Nerul, Navi Mumbai 400706, Maharashtra, India. Email: [email protected] Conflict of interest: none Accepted for publication 7 September 2014.
© 2014 Wiley Publishing Asia Pty Ltd
spectrum of clinical symptoms including osteolytic bone lesions, anemia and end organ renal disease.3 MM has a multifactorial etiology which brings into play the role of inflammation, oxidative stress, genetic mutations and various single nucleotide polymorphisms.4 Interleukin-6 (IL-6) is a proinflammtory cytokine which is a crucial player in the pathogenesis of MM. It acts as a growth factor for many myeloma cell lines and helps in plasma cell proliferation and survival via activation of STAT3 and ERK pathways.5,6 It also induces polyclonal B-cell activation leading to hypergammaglobulinemia. Higher levels of this cytokine have been essentially linked with higher staging and poorer prognosis of the disease.5,7 IL-6 has a very short plasma half-life and is rapidly cleared from the blood. Hence, its
2
circulating levels are determined by its level of expression.8 Several single nucleotide polymorphisms of this proinflammatory cytokine modulate the plasma levels and biological activity of IL-6 and thus influence the disease process and clinical outcome. IL-6-174 G/C polymorphism (rs1800795) is one of the four major polymorphisms described at the promoter region of IL-6.8 The region between -225 and -164 of the IL-6 promoter region containing the G/C polymorphism has previously been reported to demonstrate a negative regulatory effect on reporter gene expression.9 Polymorphism at -174 site is important as it is close to the site for glucocorticoid receptor binding (-201).10 Secondly, G to C change creates a binding site for NF1.11 This explains our rationale for choosing polymorphism at -174 region. There is, however, a lack of data regarding the prevalence of this polymorphism and its association with the prognosis of MM in Indian patients. Hence, we studied the occurrence of IL-6-174 G/C polymorphism and evaluated its association with the serum levels of IL-6 and staging of MM in Indian patients.
SUBJECTS AND METHODS Subjects The study was conducted in the Department of Biochemistry, Maulana Azad Medical College and Dr. D.Y. Patil Medical College. The study was a hospital-based case control study and comprised of 103 cases and 117 healthy controls. The cases were clinically diagnosed of having MM and were recruited after confirmation of disease by serum electrophoresis. The control group comprised of age- and sex-matched healthy individuals who were recruited from volunteers and healthy persons accompanying the patients in general outpatient (OPD). Patients with history of chronic inflammatory disorders, surgery or trauma in the past 3 months, anemia, renal or hepatic disorders, and history of regular immunosuppressive or analgesic therapy were excluded. Informed consent was obtained from all subjects before collection of information. The study protocol was passed by the institutional ethical committee.
Clinical examination A detailed history and clinical evaluation was carried out. Assessment of patients was done at day 1 following admission. International Staging System (ISS)12 was used for staging of MM.
© 2014 Wiley Publishing Asia Pty Ltd
B Chakraborty et al.
Laboratory investigations Routine hematological and biochemical assessments including serum electrophoresis were carried out. Radiological skeletal survey was carried out to assess skeletal involvement. Single blood samples were taken from patients on the day of admission. In the control group, blood samples were taken in the OPD. Half of the 10 mL of whole blood sample was transferred to an EDTA (ethylenediaminetetraacetic acid) vial containing 1.8 mg EDTA per mL of blood. From the other half, the serum was separated by centrifuging at 1000 g for 15 min for cytokine determination. Serum was then stored at −80°C until further analysis. Serum IL-6 level was estimated using commercially available ELISA (enzyme-linked immunosorbent assay) kit supplied by Diaclone Research, France.
Genotype determination Total genomic DNA was isolated from whole blood using MB504 HiPura ATM DNA miniprep purification spin kit from HiMedia Laboratories (Mumbai, India). IL-6 promoter region was amplified using the primers 5′-TGA CTT CAG CTT TAC TCT TTG T-3′ (forward primer) and 5′-CTG ATT GGA AAC CTT ATT AAG-3′ (reverse primers) (GenxBio Health Sciences, Delhi, India) under standard conditions as described by Fernandez et al.13
Statistical analysis Statistical analysis was done with Statistical Package for the Social Science 17.0 (SPSS Inc, Chicago, IL, USA). Categorical data were summarized as frequencies and percentages. Continuous data were presented as average (minimum, maximum). X2 test with Yates correction and Fisher’s exact test were done when appropriate and the odds ratio (OR) along with 95% CI was used to compare proportions between the groups. Nonparametric (Mann–Whitney) analyses were used if variance was high. The level of significance was chosen to be P < 0.05.
RESULTS The clinical profile of the patients is depicted in Table 1. Of the 103 patients enrolled in the study, the majority (54.3%) presented in stage 2 of the disease using ISS. The most common radiological finding was skull lytic lesions (35%), followed by vertebral and humerus fractures. Immunoglobulin G-kappa light chain was the most common monoclonal antibody detected by immunofixation technique.
Asia-Pac J Clin Oncol 2014
3
IL-6 polymorphism and multiple myeloma
A total of 69 men and 34 were enrolled in the study. The mean age of study population was 56.0 ± 9.9 years and this did not significantly differ from the control group. The control group was age and sex matched, and as was expected serum values of urea, creatinine and calcium were found significantly more often in cases than in controls (P < 0.05). Most patients had anemia and a significantly low A/G ratio (Table 2). Genetic analysis for IL-6 polymorphism was done for all 103 patients and 117 controls. Table 3 shows the
Table 1
Clinical profile of the multiple myeloma patients
Characteristics International Staging System I II III Skeletal survey Skull lytic lesions Vertebral fracture Humerus fracture Rib fracture Bone marrow analysis Plasma cells M : E ratio Immunofixation pattern IgG lambda IgG kappa Miscellaneous gammopathy IgA IgM IgD Bence Jones protein in urine
Table 2
Number of patients (n %) 28 (27.1) 56 (54.3) 19 (18.4) 36 (34.9) 28 (27.1) 29 (28.1) 22 (21.3) 13–85% 3:1–4:1 57 (55.3) 26 (25.2) 20 (19.4) 10 (9.7) 8 (7.7) 2 (1.9) 51 (49.5)
distribution of the IL-6-174 G/C genotypes and its alleles among cases and controls. Genotypes were in Hardy–Weinberg equilibrium (P = 0.15). GG polymorphism was the most prevalent genotype in both cases and control groups, while CC was least common (GG 52.4% vs 58.1%, P = 0.23; GC 38.8% vs 32.4%, P = 0.19; CC 8.7% vs 9.4%, P = 0.52). Risk of MM does not increase significantly with the various genotypes or the alleles in the present study. The OR for GG genotype was 0.7(CI 0.4–1.3) which was not significantly associated with MM. We also did not find any association between the GC (OR 1.3, CI 0.7–2.2) or CC genotypes (OR 0.9, CI 0.3–2.3) and MM (Table 3). We measured the serum IL-6 levels in cases and controls and correlated the levels with ISS. The IL-6 levels in cases (mean 31 ± 38, median 15 pg/mL, range 2–190) were significantly higher (P = 0.000) as compared with controls (mean 2.30 ± 1.1, median 2 pg/ mL, range 1–7). We found a positive correlation between serum IL-6 levels and staging of the disease (r2 = 0.4, P = 0.01). We also studied the association between serum IL-6 levels with the IL-6-174 G/C polymorphism. The median value of IL-6 levels was significantly higher in the GC genotype (24 pg/mL, P = 0.007) when compared with other genotypes, whereas the GG genotype was associated (12 pg/mL, P = 0.001) with significantly lower values of IL-6 values. Though the mean value 52 ± 60 pg/mL was apparently high, we could not establish any relationship between CC genotype and IL-6 levels (18 pg/mL, P = 0.2) (Table 4). This could be attributed to the less occurrence of this genotype. The prognosis of MM was assessed by ISS. When comparing the stage of disease with genotype, we found
Demographic profiles of study group and control
Age (mean ± SD) Gender (male : female) Men (mean ± SD) Women (mean ± SD) Hb TLC A : G ratio Urea Creatinine Calcium Phosphorus
Cases (n = 103)
Controls (n = 117)
p-value (Mann–Whitney test)
56.0 ± 9.9 69:34 56.3 ± 8.9 49.0 ± 13.8 8.3 ± 2.9 12 451 ± 6233 0.7 ± 0.29 141 ± 61 5.9 ± 2.6 10.1 ± 1.74 4.8 ± 2.1
52.5 ± 9.8 83:34 54.0 ± 9.9 49.0 ± 8.8 14.2 ± 5.1 8456 ± 2673 1.2 ± 0.2 32 ± 5 1.0 ± 0.9 8.7 ± 1.1 4.1 ± 1.9
NS NS NS NS S* S* S* S* S* S* NS
*P-value < 0.05 is considered significant (S). NS, not significant.
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Table 3
B Chakraborty et al.
Association of IL-6-174 G/C genotype with MM
IL- 6-174 G/C polymorphism
Cases n = 103 (%)
Controls n = 117 (%)
Odds ratio
95% CI
p-value
54 (52.4) 40 (38.8) 9 (8.7)
68 (58.1) 38 (32.4) 11 (9.4)
0.7 1.3 0.9
0.4–1.3 0.7–2.2 0.3–2.3
0.23 0.19 0.52
148 (71.8) 58 (39.1)
174 (74.3) 60 (25.6)
0.88 1.15
0.5–1.3 0.7–1.7
0.5 0.5
Genotype GG GC CC Alleles G C *P-value < 0.05 is considered significant.
Table 4
Serum levels of IL-6 in different genotypes of IL-6-174 G/C polymorphism IL-6 levels (pg/mL)
Genotype present
Range
Mean ± 2 SD
Median
p-value (Mann–Whitney)
GG GC CC
2–80 4–190 10–180
17 ± 16 43 ± 47 52 ± 60
12 24 18
0.001* 0.007* 0.2
*P-value < 0.05 is considered significant.
Table 5
ISS and association of IL-6-174 G/C polymorphism ISS Stage 1 n (%)
Stage 2 n (%)
Stage 3 n (%)
24 (44.4) 4 (8.2) 3 (7.5) 25 (39.7) 2 (22.2) 26 (27.7)
24 (44.4) 32 (65.3) 26 (65) 30 (47.6) 5 (55.6) 51 (54.3)
6 (11.1) 13 (26.5) 11 (27.5) 8 (12.7) 2 (22.2) 17 (18.1)
Genotype present GG GC+CC GC GG+CC CC GG+GC
p-value 0.001* 0.001* 0.91
*P-value < 0.05 is significant.
that most patients in GG genotype had stage 1 and stage 2 diseases (44.4% each) which was significantly higher compared with other genotypes (P = 0.001). Patients with GC genotype mostly had stage 2 (65%) and stage 3 diseases (27.5%, P = 0.001). The CC genotype was associated with stage 2 disease but it did not reach the level of significance due to less number of cases. (Table 5).
DISCUSSION Recent research in the field of carcinogenesis has highlighted the pertinent link between inflammatory mediators and inflammation-related gene polymorphisms and
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cancer. MM–prototype malignancy of plasma cells also follows this proinflammatory signage. The serum level of IL-6 is dictated by the polymorphisms present at the promoter site which further regulates the levels of other inflammatory molecules and maintains the smoldering inflammatory microenvironment required for the progression of the disease. It has been suggested that the NFκB and STAT3 pathways are the primary regulators of the inflammatory pathway as they modulate cytokine release and their consequent cellular and molecular repercussions. The low-grade inflammatory milieu in the tumor acts as a stimulus for these pathways leading to the setting in of a vicious cycle.14 This reiterates the importance of understanding
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IL-6 polymorphism and multiple myeloma
the genetic polymorphisms regulating the IL-6 gene expression. The prevalence of IL-6-174 G/C promoter polymorphism in Indian patients of MM is not known. Further, no study has reflected on the association of the genotypes with the severity of the disease or correlated the serum levels of IL-6 with the polymorphism. To the best of our knowledge, this is the first time that the distribution of IL-6-174 G/C polymorphism and its association with severity of MM in Indian patients has been studied. We studied the distribution of IL-6-174 G/C polymorphism in Indian patients of MM and found no difference in the occurrence of the genotypes between the cases and the controls. The GG genotype was most abundantly present both in cases and controls, followed by the GC and CC genotypes. Though no other Indian study evaluated the polymorphism in MM, the frequencies stated in our work are in accordance with various other studies done in diseases like stroke15 and Alzheimer’s disease16 in the same population. GG genotype was also the most prevalent in their study and CC was least frequent. Thus, it can be implied that for Indian population the presence of the polymorphism does not increase susceptibility to MM. These results echoed the finding of Zheng et al.,17 Cozen et al.,18 Duch et al.,19 Mazur et al.20 and Iakupova et al.21 who had studied the impact of the polymorphism in Spanish, American, Brazilian, Polish and Russian populations, respectively, and found no association between the polymorphism and MM. This suggests this polymorphism does not predispose individuals to MM. We further measured the serum levels of IL-6 in both cases and controls and observed that our cases had significantly higher levels of IL-6 (median 15 pg/mL) as compared with controls (median 2 pg/mL). We also established a positive correlation between the ISS of MM and serum levels of IL-6 (r2 = 0.4, P = 0.01). Our findings were in concordance with that of Nachbaur et al.22 who studied MM in Austrian population and established that IL-6 levels were significantly higher at advanced stages (II/III). This led them to propose that in myeloma patients serum IL-6 levels may reflect disease activity and tumor cell mass. Bataille et al.23 concluded in French population that serum IL-6 levels are significantly increased in patients with fulminating MM, suggesting that IL-6 is an important factor for the progression of MM. Other studies done by Pelliniemi et al.24 also proved that immunoreactive IL-6 was a prognostic factor in MM. Thus, increased IL-6 levels
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were associated with higher stages and poorer prognosis in MM. It has been hypothesized that IL-6 expression is activated by both inflammatory and tumor cells and their presence in substantial concentration is essential for both sustaining chronic inflammation, promoting progression of tumor cells and inhibition of immune-mediated tumor surveillance. It has also been suggested that inflammation is not only responsible for increased release of cytokines but also for certain epigenetic changes that have profound implications for tumorigenesis.14 In our study, we also evaluated the presence of the genotypes with downstream protein production. The GC genotype was associated with highest levels of IL-6 (24 pg/mL, P = 0.007) and GG genotypes had significantly lower values (12 pg/mL, P = 0.001). Hence, we state that in Indian patients of MM, GC genotype is associated with increased production of IL-6 while patients with GG genotype produce less IL-6. This finding definitely has a bearing on the severity of the disease. As already discussed in the preceding paragraph, higher levels of IL-6 are associated with worse prognosis in MM, so in corollary we can state that the GC genotype is associated with more severe disease and patients with GG genotype appear to have less severe disease. The consequences of CC genotype could not be explored due to lesser number subjects who could not reach statistical significance. To further explore this result, we studied the association of genotypes with ISS of MM. Our conclusions got reinforced as we found that patients of GG genotype presented with significantly mild disease, that is, stage 1 (44.4%) and stage 2 (44,4%), whereas patients with GC genotypes had higher stages of the disease (stage 2 = 65%, stage 3 = 27.5%, P = 0.001). The GG genotype thus appears to be protective. This is in concordance with Mazur et al.20 who studied the polymorphism in Polish patients. Though they did not compare genotypes with staging, they found that the GC genotype was associated with increased mortality. This implies that patients with GC genotype have severe disease. However, our results differed from a previous work by Iakupova et al.21 who conducted the study in Russian population and stated that CC genotype was associated with low progressing MM and milder clinical signs. The discordance can be explained by differences in the ethnicity, genetic predisposition, methodology and the study design. Despite every effort being taken by us to design and perform our study meticulously, it still had some limitations. The sample size was relatively small. Larger
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prospective studies are necessary to fully ascertain the role of IL-6-174 G/C polymorphism and its genotypes (GG, GC, CC) in MM. Other functional polymorphisms may also influence the expression of IL-6 gene and their combined effect must be studied to better predict the prognosis of the disease. Furthermore, we did not follow our patients and did not explore the impact of the polymorphism on long-term outcome and mortality of MM. To conclude, we found no association between the IL-6-174 G/C polymorphism and its genotypes with MM. Patients with GC genotype had increased serum levels of IL-6 and presented with more severe disease (stage 2/stage 3) whereas the GG genotype appeared to be protective.
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13
14
15
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