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Abstract

BACKGROUND:

Matrix metalloproteinase-9 (MMP-9) is an important mediator of tumor initiation and progression. The MMP-9 promoter -1562C/T functional polymorphism increases gene expression and was identified as a susceptibility factor for various cancers.

OBJECTIVE:

To evaluate the influence of the MMP-9 promoter genotype on the risk of developing papillary thyroid cancer (PTC) and to correlate cancer patient genotype with the clinical and pathological phenotype.

METHODS:

We evaluated 236 patients with nodular thyroid disease pre-thyroidectomy (119 benign disease, 117 PTC). Genomic DNA was isolated from whole blood and the MMP-9 -1562C/T genotype was evaluated by PCR-RFLP analysis.

RESULTS:

Genotype frequencies were in Hardy-Weinberg equilibrium for all groups. The T allele was significantly more frequent in cancer compared to benign disease (17.5% vs 10.1%), $p=$ 0.019. Patients with the CT or CT $+$ TT genotype had an increased risk of developing PTC, specifically micropapillary thyroid carcinoma (MPTC) (CT genotype: OR $=$ 6.467, $p=$ 0.00006; CT $+$ TT: OR $=$ 6.859, $p=$ 0.00002), but not more advanced stages (CT: $p=$ 0.094; CT $+$ TT: $p=$ 0.157). The -1562C/T genotype did not significantly correlate with tumor histological subtype, invasion or TNM stage.

CONCLUSION:

The MMP-9 -1562C/T functional polymorphism may indicate susceptibility to develop thyroid cancer, specifically intrathyroidal clinically non-relevant MPTC. This suggests that although this genotype might be a predisposing factor, other genetic/epigenetic events are needed for cancer progression.

Keywords

Metalloproteinase-9 MMP-9 polymorphism cancer susceptibility thyroid cancer

1. Introduction

Matrix metalloproteinases (MMPs) are zinc- dependent endoproteinases with enzymatic activity directed against components of the basement membrane and extracellular matrix. Matrix metalloproteinase-9 (MMP-9) is involved in regulating extracellular homeostasis and innate immune responses and has low-level physiological expression, but it is overexpressed in almost all human cancers [1, 2, 3, 4].

In differentiated thyroid cancer (DTC) high MMP-9 expression is associated with invasion, lymph node metastasis and generally higher tumor stages [5, 6, 7, 8, 9, 10]; furthermore, high MMP-9 expression can be used to distinguish malignant from benign tumors [7, 8].

High peripheral blood levels of MMP-9 are also generally related to poor outcomes in cancer [4, 11, 12, 13, 14, 15, 16, 17]. In papillary thyroid cancer (PTC) we have previously shown that high pre-surgical serum MMP-9 levels may identify patients at higher risk of persistent disease who require intensive treatment [18].

We know that MMP-9 expression is primarily controlled at the level of gene transcription [2, 3]. Under physiological and pathological conditions, adhesion molecules, cytokines, growth factors and hormones act through different signaling pathways to induce or repress transcription of the MMP-9 gene [2]. However, in addition, functional polymorphisms in the promoter region of MMP-9 have also been shown to influence the level of gene expression [19] and thus might contribute to individual susceptibility for cancer initiation and progression.

A single polymorphism arising from a cytosine (C) to thymidine (T) substitution at position -1562 (rs3918242) significantly influences MMP-9 gene promoter activity, resulting in a 1.5 fold increase in gene expression in carriers of the T-allele compared to the C-allele [19]. Originally described in association with coronary atherosclerosis [19], the functional MMP-9 1562C/T polymorphism was subsequently shown to increase susceptibility to several cancers [20], to increase the risk of progression of gastric cancer [21] and the overall risk of metastasis in the Asian but not the European population [22]. In papillary thyroid cancer, the only other study published so far revealed that the presence of the T allele was associated with increased MMP-9 immunohistochemical expression and increased risk for extrathyroidal extension and higher tumor stages [23].

In this context we aimed to: i) evaluate the impact of the MMP-9 promoter genotype on the risk of developing papillary thyroid cancer (PTC) and ii) correlate cancer patient genotype with clinical and pathological features of tumor aggressiveness.

2. Patients and methods

2.1 Patient selection, clinical and pathology data

We evaluated the presence of the MMP-9 -1562C/T polymorphism in 236 patients with nodular thyroid disease who were referred for endocrine evaluation and thyroid surgery in the “CI Parhon” National Institute of Endocrinology between December 2012 and November 2016. The patients were enrolled consecutively, were informed about the study procedures and gave their written informed consent. The study was performed in strict agreement with the Helsinki declaration concerning ethical principles for medical research involving human subjects. The Ethics Committee of the Institute reviewed the study protocol and agreed on the research.

Based on the pathology report the patients were divided into the two study groups: benign disease (BD) ( $N=$ 119) and papillary thyroid cancer (PTC) ( $N=$ 117). Patients with other type of thyroid neoplasia (medullary thyroid carcinoma, anaplastic carcinoma and intra-thyroidal metastases) were excluded from the analysis.

In patients with papillary thyroid cancer, clinical and imaging data obtained prior to surgery, as well as pathology by haematoxylin-eosin staining were used to classify patients according to the histological subtype, presence or absence of extra-thyroidal invasion, size, pT and TNM stage according to the AJCC 7th Edition/TNM Classification System for Differentiated Thyroid Carcinoma [24] and the initial risk of recurrence group according to the 2015 American Thyroid Association risk stratification [25].

From a histological perspective, patients were divided into three groups: classic PTC (CLS) ( $N=$ 40), follicular variant (FV) ( $N=$ 53), and aggressive forms (AGR) ( $N=$ 24), including poorly differentiated, tall cell, insular, solid, sclerosing subtypes and oxyphil variant of PTC. In terms of the pathology pT stage, intrathyroidal micropapillary thyroid carcinomas (pT1a) were noted and analyzed as a subgroup, considering that these tumors are frequently very low risk, indolent in clinical behavior and might represent a distinct entity within the differentiated thyroid cancer pool [26].

2.2 Genomic DNA isolation

Blood samples were collected in all patients 1–2 days before surgery. Genomic DNA was isolated from whole blood in all patients using Wizard ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Genomic DNA Purification Kit (Promega, USA) starting from 3 mL of whole blood and was carried out using the manufacturer’s protocol. DNA concentration was measured using Infinite200 NanoQuant reader (Tecan, Switzerland).

2.3 Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis

The presence of the MMP-9 -1562C/T polymorphism was evaluated by PCR-RFLP analysis, using the following primers: 5’-GCCTGGCACATAGTAGGCCC-3’ (forward), 5’-CTTCCTAGCCAGCCGGCATC-3’ (reverse). The amplification protocol consisted of initial denaturation, 10 minutes at 95 ${}^{\circ}$ C; 35 cycles at 94 ${}^{\circ}$ C for 30 seconds, 59 ${}^{\circ}$ C for 30 seconds, 72 ${}^{\circ}$ C for 30 seconds and final elongation at 72 ${}^{\circ}$ C for 10 minutes. The amplification products were digested with 10 U of SphI restriction enzyme (New England Biolabs, USA) at 37 ${}^{\circ}$ C for 3 hours producing fragments of 247 bp and 188 bp in case of T allele, or undigested product of 435 bp in case of C allele. The fragments underwent electrophoretic migration on 2% agarose gel stained with ethidium bromide and visualized under UV light (Gel Doc EZ System, Bio-Rad Laboratories – Dubai Branch, United Arab Emirates). CC genotype showed one band at 435 bp, TT genotype presented as two bands at 247 bp and 188 bp, while CT genotype had three bands at 435 bp, 247 bp and 188 bp.

3. Statistical analysis

Statistical analysis was performed using Medcalc software version 14.8.1. Chi square test was used to test for deviation of the genotype distribution from the Hardy Weinberg equilibrium. In order to compare genotype and allele frequencies between patients with PTC and BD we calculated odds ratio and the corresponding confidence intervals. Chi square test was used to check for associations between genotypes and clinical and pathological phenotypes of PTC patients. $P$ less than 0.05 was considered significant.

Table 1
MMP-9 promoter genotype distribution and allele frequencies in patients with papillary thyroid carcinoma, micropapillary thyroid carcinoma and benign disease

	Genotype	Cancer $n$ (%)	BD $n$ (%)	OR	95% CI	$P$
PTC (all patients)	CC	78 (66.7%)	97 (81.5%)	1	Reference
	CT	37 (31.6%)	20 (16.8%)	2.301	1.237–4.278	0.0076
	TT	2 (1.7%)	2 (1.68%)	1.244	0.171–9.030	0.829
	CT $+$ TT	39 (33%)	22 (18.5%)	2.205	1.208–4.024	0.0092
	C	82.5%	89.9%	1	Reference
	T	17.5%	10.1%	1.894	1.104–3.251	0.019
MPTC ( $<$ 1 cm, intrathyroidal)	CC	9 (39.1%)	97 (81.5%)	1	Reference
	CT	12 (52.2%)	20 (16.8%)	6.467	2.405–17.39	0.00006
	TT	2 (8.7%)	2 (1.68%)	10.778	1.352–85.88	0.00660
	CT $+$ TT	14 (60.9%)	22 (18.5%)	6.859	2.635–17.85	0.00002
	C	65.2%	89.9%	1	Reference
	T	34.8%	10.1%	4.756	2.271–9.958	0.00001

PTC – papillary thyroid carcinoma; MPTC – micropapillary thyroid carcinoma ( $<$ 1 cm, limited to the thyroid, no invasion); BD – benign disease; OR – odds ratio; 95% CI – 95% confidence interval. Chi square test, $p<$ 0.05 considered significant.

4. Results

4.1 MMP-9 promoter genotype distribution and allele frequencies in PTC patients and controls

The MMP-9 promoter genotype distribution and allele frequencies in patients with papillary thyroid carcinoma, micropapillary thyroid carcinoma and benign disease are summarized in Table 1. Genotype frequencies were in Hardy-Weinberg equilibrium for benign disease patients and PTC patients, as well as the subgroups of patients being evaluated. The T allele was significantly more frequent in cancer patients compared to those with benign nodules (17.5% vs 10.1%), $\chi^{2}=$ 5.5, $p=$ 0.019, as was the CT genotype (31.6% vs 16.8%), $\chi^{2}=$ 7.12, $p=$ 0.0076. Only 4 patients presented with the rare TT genotype, 2 of them with micropapillary thyroid carcinoma, the other 2 with benign disease.

Using the CC genotype as reference, a significant association was found between the presence of the CT genotype alone (OR $=$ 2.301, $p=$ 0.0076), or in combination with the TT genotype (CT $+$ TT: OR $=$ 2.205, $p=$ 0.0092) and the risk of developing papillary thyroid cancer.

Patients with the CT genotype, or CT $+$ TT genotype have an even higher relative risk of developing micropapillary thyroid carcinoma, representing very early stages of PTC (size smaller than 1 cm, intrathyroidal) – for CT genotype: OR $=$ 6.467, $p=$ 0.00006; CT $+$ TT: OR $=$ 6.859, $p=$ 0.00002 (Table 1). Interestingly, presence of the T allele is not associated with an increased relative risk for more advanced PTC stages (CT: $p=$ 0.094; CT $+$ TT: $p=$ 0.157).

4.2 Association of MMP-9 promoter genotype with clinical and pathological features of PTC

The effect of the MMP-9 promoter genotype on clinical and pathological features of papillary thyroid carcinoma are presented in Table 2.

Table 2
Clinical and pathological features and MMP-9 promoter genotype in patients with papillary thyroid carcinoma

	Genotype
	CC	CT $+$ TT	$p$
Gender
Female	58	30	0.940 ${}^{}$*
Male	20	9
Age
$<$ 45 years	31	16	0.947 ${}^{}$*
$\geqslant$ 45 years	47	23
Histology
CLS	23	17	0.203 ${}^{}$*
FV	36	17
AGR	19	5
Multifocality
MFoc	30	18	0.587 ${}^{}$*
Ufoc	47	21
ETE
No	44	25	0.602 ${}^{}$*
Yes	33	14
Tumor size
$<$ 1 cm	13	16	0.015 ${}^{}$*
1–2 cm	16	10
2–4 cm	31	9
$>$ 4 cm	14	3
MPTC (pT1a)	9	14	0.004 ${}^{}$*
PTC	69	25
TNM stage
1 $+$ 2	53	30	0.487 ${}^{}$*
3 $+$ 4	24	9
ATA risk
High	9	3	0.777 ${}^{}$*
Intermediate	25	12
Low	44	24

PTC – papillary thyroid carcinoma; MPTC – micropapillary thyroid carcinoma; CLS – classic PTC; FV – follicular variant PTC; AGR – aggressive histology; MFoc – multifocal; UFoc – unifocal; ETE – extrathyroidal extension. ${}^{*}$ Chi square test; $p<$ 0.05 considered significant.

Statistical analysis showed that genotype distribution was similar in the gender ( $\chi^{2}=$ 0.006, $p=$ 0.940) and age groups ( $\chi^{2}=$ 0.004, $p=$ 0.947) investigated. There was similarly no association between patient MMP-9 genotype and histological subtypes ( $\chi^{2}=$ 3.192, $p=$ 0.203), presence or absence of multifocality ( $\chi^{2}=$ 0.295, $p=$ 0.587) or invasion ( $\chi^{2}=$ 0.272, $p=$ 0.602), TNM stage ( $\chi^{2}=$ 0.483, $p=$ 0.487) and ATA risk groups ( $\chi^{2}=$ 0.506, $p=$ 0.777), suggesting that the presence of the T allele is not correlated with features of aggressiveness of papillary thyroid cancer (invasive behavior, adverse histological subtypes or advanced tumors).

Interestingly, the CT $+$ TT genotype was significantly more frequent in tumors smaller than 1 cm compared to larger tumors ( $\chi^{2}=$ 10.417, $p=$ 0.015) and particularly more frequent in small intrathyroidal tumors, i.e. micropapillary thyroid carcinoma with no extrathyroidal extension, compared to more advanced tumors ( $\chi^{2}=$ 8.287, $p=$ 0.004). This seems to suggest that the presence of the T allele is correlated with the process of tumor initiation in thyroid cancer, but not with progression.

5. Discussion

Differentiated thyroid cancer is a multi-factorial, multi-step disease, which is initiated, and then progresses based on the complex interplay of various genetic, epigenetic and environmental factors [27, 28]. The initial step in tumorigenesis is generally an activating mutation in an effector of the MAPK pathway (“BRAF-like” or “RAS-like” mutations) [29] which leads to increased constitutive activation, and thus influences cell differentiation and proliferation [30]. The accumulation of additional genetic alterations (TERT promoter, TP53) leads to a more undifferentiated phenotype and more aggressive cancers [31].

This process of tumorigenesis developing in the thyroid tissue is influenced by environmental factors (ionising radiation, dietary iodine), but also by the personal genetic background [28]. In this context, identifying potential susceptibility genes or genetic variants might help explain interpersonal differences in the risk of developing differentiated thyroid cancer.

Numerous studies have shown that MMP-9 takes part in multiple dysregulated signaling pathways in cancer that lead to both tumor cell initiation and also to invasion and metastasis [3]. The functional MMP-9 C/T -1562 polymorphism was shown to significantly influence MMP-9 gene promoter activity, increasing gene expression [19]. So in this context, our initial hypothesis was that patients carrying the “T” allele at position 1562: i) would have an increased risk to develop papillary thyroid cancer and ii) would have a higher risk of more advanced tumor stages.

Our results indeed show that the “T” allele was significantly more frequent in cancer patients compared to those with benign nodules (17.5% vs 10.1%, $p=$ 0.019). Considering the CC genotype as reference, we found that the presence of the CT genotype, alone or in combination with the TT genotype, significantly increases the risk to develop papillary thyroid cancer (for CT: OR $=$ 2.301, $p=$ 0.0076; CT $+$ TT: OR $=$ 2.205, $p=$ 0.0092).

However, we identified no association between patient MMP-9 promoter genotype and the cancer histological subtype, presence of multifocality and invasion, TNM stage and ATA risk groups, which suggests that the presence of the “T” allele is not correlated with features of aggressiveness of papillary thyroid cancer. Interestingly, the CT $+$ TT genotype was significantly more frequent in patients with micropapillary thyroid carcinoma (i.e. tumors smaller than 1 cm with no extrathyroidal extension), compared to more advanced tumors ( $p=$ 0.004), which seems to suggest that the presence of the T allele is correlated with the process of tumor initiation in thyroid cancer, but not progression.

Indeed, compared to controls, patients with the “T allele” (CT or CT $+$ TT genotypes) have a significantly higher relative risk of developing intrathyroidal micropapillary thyroid carcinoma, representing very early stages of PTC (OR $=$ 6.859, $p=$ 0.00002), but not more advanced PTC stages ( $p=$ 0.157). This is an interesting observation as numerous groups have shown that these tumors are frequently very low risk, indolent in clinical behavior and might be better managed in an active surveillance setting [25, 26, 32]. Since only a small part of microcarcinomas ultimately progress toward clinically relevant cancers [32], it is likely that the indolent micropapillary thyroid carcinomas might in fact represent a distinct entity within the larger differentiated thyroid cancer group. So far, no useful genomic or transcriptomic characteristics have been identified to predict future progression of a microcarcinoma [33], but the identification of a potential susceptibility gene leading to an increased risk of early but not more advanced stages of PTC is intriguing.

Similar results have been reported for several other neoplasias: presence of the “T” allele at position 1562 in the MMP-9 gene promoter is associated with an increased risk for developing oral squamous cell carcinoma only in the early stages but not in advanced ones [20]. In contrast, no association was identified between the MMP-9 -1562C/T functional polymorphism and the risk of occurrence of nasopharyngeal carcinoma [34] or esophageal squamous cell carcinoma [35] and in patients with non-small cell lung cancer it is the presence of the -1562CC genotype that was found to be more frequent than in controls, suggesting that the T allele might in fact be a protective factor [36, 37]. Similarly, this genetic variant does not appear to influence breast cancer risk [38].

A meta-analysis including 23 studies and more than 11500 subjects with a variety of neoplasias concluded that the MMP-9 -1562C/T functional polymorphism was not associated with an increased cancer susceptibility in either the whole group analysis or in individual cancer type groups [39]. However, thyroid cancer was not included in the analysis and because of the relatively small number of studies available for each cancer type the meta-analysis lacked enough statistical power for individual cancer analysis [39]. Another meta-analysis looking at the risk of metastasis this time (again, no thyroid cancer studies included), suggested that the CT and TT genotypes were associated with an increased risk of cancer metastasis in general in Asian populations only, but no differences were identified when looking at individual cancer subtypes [40].

In the only other study ever published concerning thyroid cancer [23], PTC patients and controls did not differ significantly in genotype frequencies, but PTC patients who carried the T allele had a higher risk of developing extrathyroidal extension and more advanced stages [23]. Contrary to our results, this suggests that while the MMP-9 promoter genotype does not influence the predisposition to develop thyroid cancer, it affects the course of the disease by modulating MMP-9 expression [23].

There are several possible causes for these apparent discrepancies.

First, both the Serbian study [23] and ours enrolled a relatively small number of patients, probably not enough to assess cancer susceptibility reliably at a population level, particularly bearing in mind that there are numerous other factors likely to influence the risk of developing cancer, both environmental factors and factors related to the genetic background of the subjects, including ethnicity.

Second, we know from numerous studies that MMP-9 is involved in the development of neoplasia in both the early (cancer initiation) and late stages (metastasis), as part of a complex interplay of growth factors, cytokines and adhesion molecules [3]. It is thus likely that a genetic predisposition might manifest in terms of both the susceptibility to develop cancer and the risk of more aggressive/advanced cancer stages, and we just do not yet have studies large enough to assess this, after adjusting for the other possible confounding factors.

Third, as opposed to the study by Roncevic et al. [23], our control group consisted of patients with benign thyroid disease and not healthy volunteers, which might be a source of error, due to possible genetic heterogeneity. Benign thyroid nodules are very frequent in the Romanian population, and their incidence increases with age, due in large part to iodine deficiency, so recruiting an age matched healthy control group would be rather difficult. On the other hand, from a practical diagnostic standpoint, it is more useful to be able to identify patients who are at risk to harbor thyroid neoplasia out of the population with thyroid nodules and not necessarily those with normal thyroid morphology.

Looking at the inconsistencies between studies dealing with various neoplasias, it seems likely that the MMP-9 polymorphism might affect different cancer types in different ways, due to variations in MMP-9 regulation mechanisms in the particular microenvironment of different tissues [2, 3]. Furthermore, there is also a wide variability in the studies published so far in terms of the populations investigated, with the patients enrolled belonging to different ethnicities and being exposed to different environmental factors [20, 22, 34, 35, 36, 37, 38, 39]. These are all confounding factors, which would require larger studies in order to be adequately assessed.

6. Conclusion

Differentiated thyroid cancer is a complex entity arising from a specific combination of genetic, epigenetic and environmental factors. On this interplay of causative events, it seems likely that susceptibility genes, such as the MMP-9 promoter polymorphism, play an important role in either cancer initiation, or cancer progression, or both. We have demonstrated that the presence of the T allele at position 1562 of the MMP-9 promoter significantly increases the risk to develop papillary thyroid cancer. In our analysis, this association was restricted to early micropapillary thyroid carcinoma and not more advanced tumors, suggesting that the CT $+$ TT genotype is important for tumor initiation rather than cancer progression, however larger population studies are needed to confirm this observation.

Footnotes

Acknowledgments

This study was supported by UEFISCDI grant PN-II-PT-PCCA-2011-3.2 no.135/2012.

Conflict of interest

No competing financial interests exist.

Author contributions

Conception: RD, SS, DM, AC, CB.

Interpretation or analysis of data: RD, SS.

Preparation of the manuscript: RD.

Revision for important intellectual content: RD, SS, DM, AC, CB.

Supervision: CB.

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Matrix metalloproteinase-9 (MMP-9) promoter -1562C/T functional polymorphism is associated with an increased risk to develop micropapillary thyroid carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Patients and methods

2.1 Patient selection, clinical and pathology data

2.2 Genomic DNA isolation

2.3 Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis

3. Statistical analysis

Table 1 MMP-9 promoter genotype distribution and allele frequencies in patients with papillary thyroid carcinoma, micropapillary thyroid carcinoma and benign disease

4.1 MMP-9 promoter genotype distribution and allele frequencies in PTC patients and controls

4.2 Association of MMP-9 promoter genotype with clinical and pathological features of PTC

Table 2 Clinical and pathological features and MMP-9 promoter genotype in patients with papillary thyroid carcinoma

6. Conclusion

Footnotes

Acknowledgments

Conflict of interest

Author contributions

References

Table 1
MMP-9 promoter genotype distribution and allele frequencies in patients with papillary thyroid carcinoma, micropapillary thyroid carcinoma and benign disease

Table 2
Clinical and pathological features and MMP-9 promoter genotype in patients with papillary thyroid carcinoma