Exploratory cross-sectional study to evaluate the metalloproteinases role as ‘immunoscore’ for HPV-induced cervical lesions

doi:10.21203/rs.3.rs-2231649/v1

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Exploratory cross-sectional study to evaluate the metalloproteinases role as ‘immunoscore’ for HPV-induced cervical lesions

https://doi.org/10.21203/rs.3.rs-2231649/v1

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Cervical cancer (CC) is the fourth most common type of cancer among women and is responsible for about 8% of female cancer deaths worldwide. Understanding how the tumor microenvironment behaves is essential to realize the carcinogenic process, and thus infer possible prognostic biomarkers in the CC development. One of the prognostic factors that has aroused interest in recent years is the increased expression of metalloproteinases (MMPs) in tumor tissues, which is associated with tumor growth and metastasis, and recurrence of degradation of extracellular matrix (ECM) components in tissues of different tumors. Thus, our objective is to evaluate the in situ distribution of MMPs⁺ cells (MMP-2, -7, -9), as well as TIMP-2⁺, EphA2⁺ and EfrinA1⁺ cells in intraepithelial (IE) and marginal stroma (MS) areas using the technique of immunohistochemistry and immunoscore analysis in low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) and CC. We observed in MS, a high distribution of MMPs⁺ (-2, -7 and − 9), TIMP2⁺ and EPHA2⁺ cells in the HSIL and CC group compared to the control. Regarding IE, we observed this same pattern, except in EPHA2 where there was a decrease in the positive cells distribution in CC compared to control and LSIL. Regarding the “immunoscore”, from 15 possible profiles, we found statistical differences in only 9 (MMP-2⁺/MMP-9⁺, MMP-2⁺/EphA2⁺, MMP-7⁺/MMP-9⁺, MMP- 7⁺/TIMP-2⁺, MMP-7⁺/EphA2⁺, MMP-9⁺/EphA2⁺, TIMP-2⁺/EphA2⁺, TIMP-2⁺/EphrA1⁺ and EphA2⁺/EphrA1⁺) when the groups were compared. However, only the MMP-7⁺/MMP-9⁺ profile can be used as a prognostic factor for the development of CC and the MMP-7⁺/EphA2⁺, MMP-9⁺/EphA2⁺ and TIMP-2⁺/EphA2⁺ profiles may be related to the development of precancerous lesions. A more comprehensive review of longitudinal studies should be performed to link these “immunoscores” to CC progression and validate their value as a prognostic method.

Cervical cancer

HPV

metalloproteinase

Cervical cancer (CC) is the fourth most common type of cancer among women worldwide (1) and its association with human papillomavirus (HPV) has been established by detecting its DNA in more than 95% of all CC, being the oncotypes HPV16 and HPV18 most found (2). In Brazil, CC is the third most frequent tumor in the female population, behind breast cancer, colon and rectum cancer (3). Cervical carcinogenesis is a complex process involving a large number of genetic and epigenetic factors. Among them, behavioral factors such as early onset of sexual activity, multiple partners, smoking, and the concomitant presence of other sexually transmitted infections is related to HPV outcome (4). In addition, other mechanisms such as stromal invasion and metastasis play a key role in the progression of the cervical tumor that involves the degradation and remodeling of extracellular matrix components (EMC) (5). Tumor invasion and metastasis define malignancy and are the principal causes of cancer-associated death. Tumor cells are surrounded by the ECM comprising of proteoglycanes and non-proteoglycanic matrix components (collagen, laminin, fibronectin and elastin). Degradation of the ECM allows tumor cells to detach from the primary tumor mass, invade local tissue and build new metastatic formations (6). Matrix metalloproteinases (MMPs) are involved in the degradation of the ECM and thus play an important role in tumor invasion and metastasis in cancers (6, 7). MMPs belong to a family of zinc dependent enzymes involved in the degradation of EMC and tissue remodeling (8). In physiological conditions, its activities are regulated transcriptionally, through activation of the enzyme in its inactive form (pre-MMP) and inhibition of tissue MMP inhibitors (TIMP), and, therefore, minimize matrix degradation (9). In cancer, there is a clear association between increased MMP expression (especially MMP-2 and MMP-9) and metastasis, and so, they can be used as prognostic markers for carcinogenic process (10). In CC, MMPs also can be associated with tumor progression (11) and poor survival in CC patients (12).

Another prognostic factor in the cancer development and progression involves the receptor tyrosine kinase (RTK) family expression, in particular the erythropoietin-producing human hepatocellular (Ephs) receptors, which aggregate about 14 different receptors (13). The EphA2 role and its ligand in tumor development take place in the angiogenesis induction (14). Besides, EphA2 have association with the MMPs expression in many cancers. In ovarian cancer, EphA2 is strongly associated with the MMP-9 expression in the stroma (15). This high expression may be associate with the short patient’s survival in ovarian cancer (16). EphA2 blockade is able to inhibit the tumor cells migration, both in vitro in breast cancer cells (17) as in vivo in gastric cancer (18). The Ephs family receptor interacting proteins (Ephrins) (13) membrane ligands, the EphrinA1, is the major ligand of EphA2, and was originally cloned from human umbilical vein endothelial cells as a factor induced by TNF-α (19). It has already been described that increased expression of Ephrin receptor is associated with several types of human cancers, such as breast, ovarian, prostate, pancreas and gastric (15, 20–23). In CC, both EphA2 and EphrinA1 can be associated with the early CC stage (24). Although EphA2/EphrinA1 is widely studied in other types of cancer, in CC doesn't have many studies.

Recently, new methods based on the density of lymphocyte populations have been established to predict the outcome of colon cancer (25). This method, known as ‘immunoscore’ incorporated the number, type, and distribution of immune cells in cancer tissue. Our group has been studying immunological factors (26) related to carcinogenesis and recently we demonstrated that a pattern in cervical inflammatory process was associated with an increasing CD45RA⁺/CD45RO⁺, and decreasing CCL20⁺/CCR6⁺ expressing cells in accordance with cervical intraepithelial neoplasia (CIN) severity. Taken together, these markers could be evaluated as ‘immunoscore’ predictors to CC outcome. Understanding the role of the cervical immune microenvironment in precursor lesions is crucial for impact on the progression to CC. In this study, we aimed to search for possible prognostic markers linked to the MMPs induction and their modulators, such as TIMP-2, Ephrin and EphA2, which mediate the degradation of ECM and tumor progression.

Patients

From 2015 to 2021, sixty-four patients were enrolled for this study. Women with suspicious of cervical premalignant lesion were biopsied in the colposcopy clinic at the Fernandes Figueira National Institute of Woman´s, Child´s and Adolescent´s Health at Oswaldo Cruz Foundation, Rio de Janeiro, Brazil (IFF/Fiocruz) and colposcopy clinic at the Institute of Gynecology of the Federal University of Rio de Janeiro (IG/UFRJ). In these institutes, all patients received an appropriate diagnosis and treatment when necessary to evaluate a possible cervical intraepithelial neoplasia (CIN) recurrence.

After histopathological analysis, lesions were classified in low- or high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) or CC groups by certified pathologists. The control group was composed of cervical samples obtained from uterus of hysterectomized women, without histopathological HPV-associated lesions.

All volunteers provided written informed consent for participation in this study, approved by INI and IFF/Fiocruz Institutional Ethical Review-Boards (protocols number 35589314.0.0000.5262 and 35589314.0.3001.5269), following Brazilian Ethical Committee Legislation..

Identification of MMP-2, MMP-9, MMP-7, TIMP-2, EphA2 and EphrinA1 positivecells in cervical lesion.

Serial paraffin-embedded tissue sections (3 µm) were fixed in silane-coated slides (Sigma, Missouri, EUA). To determine the cell profile, immunoperoxidase staining was used according to the REVEAL Biotin-Free Polyvalent HRP manufacturer’s instructions (Spring, CA, USA). Sections were incubated overnight at 4°C with specific antibodies against human MMP-2 (clone 4D3, dilution 1:100), MMP-7 (clone JL07, dilution 1:50), MMP-9 (clone 2C3, dilution 1:50) and TIMP-2 (clone H-140, dilution 1:50) from Santa Cruz (Oregon, USA); EphA2 (polyclonal, dilution 1:200) from B&D (New Jersey, USA) and EphrinA1 (polyclonal, dilution 1:50) from Invitrogen (Massachusetts, EUA). Positive stained cells were counted in twenty fields (400x) in the intraepithelial (IE) and in the marginal stroma (MS) areas. For this study, we defined IE as the site of the epithelium, where it presents intraepithelial lesions, and MS as the site of the stroma that borders the IE region evaluated. We limited analysis to the area directly surrounding the pathologist-identified dysplastic region and excluded areas with no dysplasia. Cell counts were performed using a grid (1 cm² divided into 10 mm²) by two different observers.

‘Immunoscore’ analysis

A ‘Immunoscore’ were built incorporating the number, type, and distribution of immune cells in all groups. Using these three factors, a score of I0 to I4 was given to the cervical lesions (Fig. 1). To perform the ‘immunoscore’ in cervical lesions, we first determined the frequency of MMP-2⁺, MMP-7⁺, MMP-9⁺, TIMP-2⁺, EphA2⁺ and EphrinA1⁺ expressing cells per mm² in all groups, in both cervical IE and MS areas. The median of all analyzed samples in IE and MS were used as a cut-of value.

Herein, we constructed a score according to the number of studied cellular marker distribution in both IE and MS, using a two-by-two ‘immunoscore’ as follows: MMP-2⁺/MMP-7⁺, MMP-2⁺/MMP-9⁺, MMP-2⁺/TIMP-2⁺, MMP-2⁺/EphA2⁺, MMP-2⁺/EphrinA1⁺, MMP-7⁺/MMP-9⁺, MMP-7⁺/TIMP2⁺, MMP-7⁺/EphA2⁺, MMP-7⁺/EphrinA1⁺, MMP-9⁺/TIMP-2⁺, MMP-9⁺/EphA2⁺, MMP-9⁺/EphrinA1⁺, TIMP-2⁺/EphA2⁺, TIMP-2⁺/EphrinA1⁺, EphA2⁺/EphrinA1⁺.

According to all possible combination between MMP-2⁺, MMP-7⁺, MMP-9⁺, TIMP-2⁺, EphA2⁺ and EphrinA1⁺ analysis in IE and MS, independently, samples were classified as low (none or one higher parameter), het (two higher parameters) and high (three and four higher parameters — Fig. 1) (25, 26). We used the χ² test in a 3×2 contingency table to calculate statistical differences between the groups using GraphPad Prism (version 5).

Statistical analysis.

After examining the distribution of means in all of analyzed groups, nonparametric Kruskal–Wallis test and post-test of Dunns for multiple comparison were performed in GraphPad Prism 5.0 software (GraphPad) for comparisons between different continuous variables according to the categories in the study. It was considered statistically significant a p value < 0.05.

A multivariate analysis was performed to evaluate the influence of clinical and environmental data in the frequencies of MMP-2⁺, MMP-7⁺, MMP-9⁺, TIMP-2⁺, EphA2⁺ and EphrinA1⁺- expressing cells, as well as, in all ‘immunoscores’ analysis. Multiple linear regression analysis was performed to determine the effect of clinical and environmental variables on the cell markers expression, and multiple logistic regression was applied to determine the influence of these variables among the ‘immunoscores’ levels. Both multiple linear and logistic regression were performed considering age (in years), tobacco use, alcohol consumption and number of abortions as independent variables. These variables were chosen based on statistical differences observed in the analysis of the clinical and environmental data considering a p value < 0.05.

Patients

Among the 64 volunteers enrolled in this study, 15 (23.4%) women were included as control group, 14 (21.9%) had diagnosis of LSIL, 15 (23.4%) of HSIL and 20 (31.3%) of CC, according to Table 1. The demographic data showed that women from control and CC groups were older than HSIL group. The number of pregnancy of CC group was higher than control and LSIL groups. No statistical differences were described related to ethnicity, alcohol and tobacco consumption among groups.

Table 1

Demographic data in cervical HPV-associated lesions and cervical cancer.
	Control (n = 15)	LSIL (n = 14)	HSIL (n = 15)	CC (n = 20)
Age years (Mean ± SD)	44.3 ± 6.6	38.4 ± 14.2	33.7 ± 8.0 ¹	46.3 ± 13.8 ²
Ethinicity n (%)
White	6 (40.0)	1 (7.1)	0 (0.0)	3 (15.0)
Afro-Brazilian	8 (53.4)	6 (42.8)	15 (100.0)	6 (30.0)
Alcohol consumption n (%)
Yes	2 (13.3)	2 (13.3)	3 (20.0)	2 (10.0)
No	13 (86.7)	5 (33.3)	12 (80.0)	18 (90.0)
Tobacoo use n (%)
Yes	3 (20.0)	1 (6.7)	11 (73.3)	5 (25.0)
No	12 (80.0)	4 (26.7)	4 (26.7)	15 (75.0)
Age at first sexual intercourse (average ± SD)	17.5 ± 1.9	17.3 ± 2.1	16.0 ± 2.1	15.1 ± 2.3
Number of pregnancies (average ± SD)	1.4 ± 1.4	1.1 ± 1.2	1.9 ± 1.3	3.5 ± 2.1 ^{3; 4;}

¹p<0.05, HSIL vs control; ²p<0.05, CC vs HSIL; ³p<0.01, CC vs controle; ⁴p<0.01, CC vs LSIL.

Expression Of Mmps And Their Regulators In Cervical Lesions

We analyzed the expression of MMP-2, -7 and − 9 in IE and MS areas from cervical lesions. Figure 2 represents the mean of MMPs expressing cells in both areas and its representative image in the immunohistochemistry.

The frequency of MMP-2 expressing cells in IE area had no significant difference between groups. However, an increased MMP-2 expression in HSIL and CC groups was observed when compared to control group in MS area (p < 0.05, for both groups, Fig. 2A). Regarding MMP-7 expression, an increase in positive cells in CC group was seen in IE area when compared to control group (p < 0.001). In MS an increased in positive cells in HSIL and CC groups were observed when compared to control group (p < 0.001 for both groups). In addition, HSIL and CC groups showed enhanced MMP-7 expression than LSIL group (p < 0.05, for both groups), as seen in Fig. 2B. MMP-9 expressing cells in IE area was increased in CC group when compared to control and HSIL groups (p < 0.001 e p < 0.01, respectively). In addition, an MMP-9 increase in LSIL group was observed when compared to control group (p < 0.05). In MS area, an increased in MMP-9 positive cells in HSIL and CC groups were observed when compared to control group (p < 0.001 for both groups) and an increase in CC group compared to LSIL group (p < 0.05 – Fig. 2C).

Regarding the TIMP-2 expression in situ, an increase in LSIL, HSIL and CC groups were seen compared to control group (p < 0.001, p < 0,01 and p < 0.001, respectively) in IE area. In MS area, CC and LSIL groups showed enhanced TIMP-2 expression than control group (p < 0.001 and p < 0.01, respectively – Fig. 3A). Interestingly, the EphA2 expression was decreased in CC group when compared to HSIL group in both cervical areas (p < 0.01 in IE and p < 0.001 in MS), or when compared to control group (p < 0.01) in MS area. On the other hand, HSIL group showed an increased expression of EphA2 expressing cells when compared to control and LSIL groups (p < 0.05 and p < 0.001, respectively), as demonstrated in Fig. 3B. No statistical differences was observed in the expression of EphrinA1 (Fig. 3C)

To evaluate the combined expression patterns of the 6 studied markers in studied areas, radar plot analyzes were used. Regarding IE, we observed a homogeneity in the expression of all markers in all groups, with the exception of MMP9, which showed a greater dispersion in CC (Fig. 4A). However, in MS, we observed different patterns in the study groups, with higher dispersions in HSIL (MMP-2 and EphA2) and in CC (MMP-2 and MMP-9) (Fig. 4B).

Correlation Between Mmps And Its Modulators

We evaluated whether there was a correlation between MMPs and its modulators expressing cells distributions in both IE and MS areas in HPV induced lesions. First, we analyzed all groups showing cervical lesions. We found just a weak positive correlation between TIMP-2- and EphrinA1-expressing cells (r = 0.35, p = 0.02 – Supplemental Fig. 1).

Second, each group was analyzed individually. In CC group, no correlation was found. When evaluating LSIL group, we observed a moderate positive correlation between MMP-2⁺ and MMP-7⁺ (r = 0.70, p = 0.02 – Fig. 5A), TIMP-2 + and MMP-9+ (r = 0.66, p = 0.02 – Fig. 5B) and TIMP-2 + and EphA2+ (r = 0.70, p = 0.01 Fig. 5C) cells on IE area.

In HSIL group, we found in MS area a moderate and strong negative correlation between TIMP-2 + and EphA2+ (r= -0.70, p = 0.01 – Fig. 6A) and between EphA2 + and EphrinA1 + cells (r= -0.89, p = 0.0002 – Fig. 6B), respectively. Also in MS area, a strong positive correlation between TIMP-2 + and EphrinA1+ (r = 0.80, p = 0.002 Fig. 6C) was seen.

‘Immunoscore’ analysis

To assess the dynamic cells distribution that express MMPs and its modulators in cervical lesions microenvironment, we analyzed the “immunoscore” profile in LSIL, HSIL and CC groups, as explained above. Among the 6 markers analyzed, we evaluated all possible combinations, totaling 15 profiles. We found statistical difference in 9 profiles: MMP-2+/MMP-9+, MMP-7+/MMP-9+, MMP-2+/EphA2+, MMP-7+/TIMP-2+, MMP-7+/EphA2+, MMP-9+/EphA2+, TIMP-2+/EphA2+, TIMP-2+/EphrinA1 + e EphA2+/EphrinA1+ (Fig. 7).

According with MMP-2+/MMP-9+ ‘immunoscore’ profile, an enhanced number of patients having a high ‘immunoscore’ in the CC group (52.6%) compared to LSIL (16.7%) (x²=5.70; p = 0.03 – Fig. 7A) was observed. However, no difference was seen compared to HSIL comparing to CC.

About the MMP-2+/EphA2 + profile, we observed an increase of low ‘immunoscore’ in CC group compared to HSIL group (x²= 10.81; p = 0.003 – Fig. 7B).

A gradative enhancement of MMP-7+/MMP-9 + high profile was seen through CIN progression. An increased high ‘immunoscore’ in CC group compared to LSIL (x²= 13.04; p = 0.0009 – Fig. 7C) and HSIL (x²= 5.86; p = 0.045) groups was reported

In the MMP-7+/TIMP-2 + profile, an increase of high ‘immunoscore’ in CC group were observed when compared to HSIL (x²= 8.03; p = 0.012 – Fig. 7D) group, indicating an association with cervical cancer severity.

When we analyzed the MMP-7+/EphA2 + profile, an increase of high ‘immunoscore’ in HSIL group when compared to LSIL group (x²= 6.97; p = 0.024 – Fig. 7E) was seen in our study, indicating an increase association with lesion severity. However, we found a decrease of high ‘immunoscore’ in CC group compared to HSIL group (x²= 8.15, p = 0.012).

In the MMP-9+/EphA2 + profile, we observed an increase of high ‘immunoscore’ in HSIL group compared to LSIL group (x²= 9.33; p = 0,006 – Fig. 7F), indicating an increase association with lesion severity. However, a decrease high ‘immunoscore’ in CC group was found when compared to LSIL group (x²= 5.66; p = 0.03).

In the TIMP-2+/EphA2 + profile, we noticed the same pattern that MMP-9+/EphA2 + profile, in which the CC group had a lower number of patients with high ‘immunoscore’ compared to HSIL group (x²= 7.59; p = 0,018 – Fig. 7G).

About the TIMP-2+/EphrinA1 + profile, we found that a gradative lost of increase of high ‘immunoscore’ in HSIL and CC groups. We found a statistical difference between HSIL and LSIL groups (x²= 6.11; p = 0.03 – Fig. 7H), indicating a lost in the cell expressing these MMP modulators.

Also when we analyzed the EphA2+/EphrinA1 + profile, an increase of low ‘immunoscore’ according the lesion severity was observed. The CC group showed an increase of low ‘immunoscore’ compared to LSIL (x²= 5.51; p = 0.03) and HSIL (x²= 11.25; p = 0.0024 – Fig. 7I) groups.

CC is the cause of about 8% of all female cancer deaths per year and about 84% of these deaths occur in low-income and developing countries. (1, 27). HPV is present in 95% of CC cases and is able to influence the tumor microenvironment, favoring the carcinogenesis process. In addition to viral infection, this process depends on other factors, including MMPs, which are enzymes that degrade the ECM and, thus, facilitate tissue invasion of tumor cells. In this study, the objective was to identify MMP-2, -7 and − 9 expression and their modulators (TIMP-2, EphA2 and EphrinA1) in different grades squamous cervical lesions to identify potential prognostic factorCC is the cause of about 8% of all female cancer deaths per year and about 84% of these deaths occur in low-income and developing countries. (1, 36). HPV is present in 95% of CC cases and is able to influence the tumor microenvironment, favoring the carcinogenesis process. In addition to viral infection, this process depends on other factors, including MMPs, which are enzymes that degrade the ECM and, thus, facilitate tissue invasion of tumor cells. In this study, we aimed to characterize the expression of MMP-2, -7 and − 9 and their modulators (TIMP-2, EphA2 and EphrinA1) in cervical lesions and to identify possible biomarker to lesion progression and carcinogenesis.

The development of CC is also influenced by behavioral factors, such as previous infections with other STIs (sexually transmitted infections) and number of sexual partners, as well as persistent HPV infection. It’s known that precancerous lesions can occur between 25 and 35 years of age and CC can occur at around 35 to 55 years of age, taking about 20 years of development if we consider that woman has been infected at the beginning of her sexual life (4, 37). In this way, our results follow what is described in literature, patients in CC group being about 12 years older than HSIL group.

MMPs are related with carcinogenesis process. In this way, an increase in MMP-2 + cells distribution in MS in HSIL and CC groups when compared to control group was seen in our study, suggesting that MMP-2 can play a role in invasion of tumor cells into stroma, thus aiding cervical carcinogenesis. Our results corroborated with, Gaiotto et al (2004) results where MMP-2 expression was elevated in HSIL and CC patients. In addition to in vivo studies, an increase in MMP-2 expression in HPV-infected cells had also been reported in in vitro studies (29). The MMP-2 expression in tissue cells lysates of women with CC was higher than in the control group and was associated with tumor histological grade (30). Besides that, MMP-2 was considered a recurrence predictor for CC by Ahmed et al (2004). The MMP-2 expression also can be associated with the early onset of sexual activity and low parity (31).

MMP-7 is also important for carcinogenesis process and metastasis. One study quantified the MMP-7 expression in tissue and serum from patients with CC and, as a result, was observed a significative increase in CC patients compared to control individuals (32). In addition, its expression was correlated with pathological grade, clinical stage and lymphatic metastasis. In our study, we found an increase in MMP-7 + cells distribution in HSIL and CC groups compared to control group. Similarly, we found an increase in HSIL group compared to LSIL group, suggesting MMP-7 as a possible biomarker for lesion progression. Corroborating our results, Guo et al (2018) reported a positive correlation of MMP-7 and MMP-9 with invasive potential in patients with early CC, associated with lymph node metastases. In addition, high expression of MMP-7 was associated with poor overall survival (OS) in patients with colon, ovarian and cervical cancer (12, 33, 34).

Similar to MMP-2 and MMP-7, we found a distribution pattern of MMP-9 + cells, which is important for tumor invasion and angiogenesis. Our results showed an increase in MMP-9 + cells dispersion in HSIL and CC groups compared to control group, mainly in MS area, reinforcing the idea that together with MMP-2 and MMP-7, the adjacent tissue is preparing for tumor invasion. A study conducted by Westin et al (2015) demonstrated an increase of MMP-9 expression in stromal cells in all of groups (CIN I, CIN II and CIN III) compared to control group, suggesting that MMP-9 is related to interaction between the tumor and stromal cells. Corroborating with our results, Matheus et al (2014) reported an increase in MMP-9 + cells distribution in normal cells adjacent to dyskaryotic cells in cervical smear samples as lesion progressed, suggesting an induction of MMP-9 production by neighboring the tumor cells and the importance of cell-cell interaction during carcinogenesis. MMP-9 expression for tumor progression can also be seen in other cancers such as breast (37), renal cell (38) and pancreatic ductal adenocarcinoma (PDAC) (39). Mice with PDAC and MMP-9 expression deficiency had significantly larger and more invasive tumors (39). Thus, we can observe the important role of MMPs for tumor development and invasion.

Overall, the frequency of TIMP-2 expressing cells, an inhibitor of MMP-2 expression, found in our study was much lower than the frequency of MMP-2 expressing cells expression. Co-expression of E6 and E7 oncoproteins has been reported in studies to decrease RECK and TIMP-2 levels in organotypic and monolayer cultures (40). Despite this, we found an increase in TIMP-2 expression in all groups when compared to control group, mainly in MS area (with exception of HSIL group in MS), suggesting that the TIMP-2 presence can be related to precancerous lesions development and can be associated with the tumor invasion process, because the higher number of expressing cells was in the MS area.

About CC patients, those with a high MMP-2 and TIMP-2 expression in stroma had a significantly lower survival (41). Sidorkiewicz et al (2019) demonstrated an increase in TIMP-2 expression in situ in epithelial cells of CIN III and CC when compared to stromal cells. In another study, a higher TIMP-2 positivity in tissue samples of CC and precancerous lesions when compared to healthy tissue, suggesting an important role in tumor invasion and metastasis (43).

EphA2 is frequently detected at low levels in various normal epithelial cells, and its overexpression has been investigated in solid tumours. In CC, Huang et al (2021) reported that the level of EphA2 expression is positively correlated with CC tumour proliferation, invasion, patient overall survival and therapeutic resistance of CC to epirubicin. High levels of EphA2 together with moderate to high level of EphrinA-1 protein expressions in squamous cervical carcinoma were predictive for a shorter overall survival and these proteins may be valuable prognostic markers (45). Our results, however, were contradictory to the literature. We found an increase of EphA2 expression only in HSIL group, but CC group showed a decrease compared to HSIL and control groups. Holm et al (2008) reported a strong staining for EphA2 and EphrinA1 in patients with early-stage of CC, which may play an important role in development of early-stage of CC.

Our study has a some limitations, the low number of cases and the lack of CC clinical stage data. One possibility is that EphA2 has decreased in CC group because this receptor is related to early cases of CC as described by Holm and collaborators (30).

Like EphA2, EphrinA1 was associated with poor disease-free survival (24). EphA2/EphrinA1 complex was associated with lymph nodes metastases, higher histological grade and clinical stage in patients with esophageal squamous cells carcinoma (46). Despite this, no association was found between EphrinA1 expression and cervical lesions severity.

The “immunoscore” was suggested as a more accurate way of providing a clinical prognosis for cancer, taking into account other factors present in tumor microenvironment and not just the characteristics of the tumor (29). In this way we can assess the prognosis role of MMPs and their modulators in CC development.

Regarding MMPs, there are no studies that address this method in cervical carcinogenesis. In cases of salivary gland cancer, MMP-9 was found in the cytoplasm of tumor cells, and a high ‘immunoscore’ was associated with a more advanced stage of the tumor (47). The researchers grouped the immunoscore according to the intensity of staining of MMPs at two different sites, which is another way to assess the immunoscore. Furthermore, MMP-9 was found in pseudocysts and this expression was associated with better survival and it has been suggested that MMP-9 secreted in pseudocysts doesn’t play an active role in the tumor microenvironment (47). The expression of MMP-7 in the cytoplasm of tumors has also been reported and high MMP-7 tumoral expression was associated with a better survival for the patients (47). Nagel et al (2004) demonstrated that MMP-2 and MMP-9 were intensely found in inflammatory cells and epidermoid cells, with high MMP-2 ‘immunoscore’ associated with malignant tumors of salivary gland cancer, and moderate expression of TIMP-2 has been reported in neoplastic cells. The increase in MMP-2 expression was associated with the invasive properties and malignant potential of the tumor (48).

Our group, in a previous study, used the “immunoscore” to assess the prognostic role of CD45RA+/CD45RO + and CCL20+/CCR6 + profiles in CIN I, CIN II, CIN III and CC patients. We found that women with high-grade lesions had a pattern of high CD45RA+/CD45RO+ ‘immunoscore’ and low CCL20+/CCR6+ ‘immunoscore’, suggesting that patients with these profiles would progress to CC (26).

In present study, we found some profiles that can be considered good ‘immunoscore’ markers and need to be studied more extensively, such as MMP-7+/MMP-9+, MMP-7+/TIMP-2+, MMP-7+/EphA2+, MMP-9+/EphA2 + and TIMP-2+/EphA2+, since there are no studies using MMPs ‘immunoscore’ in CC. The MMP-7+/MMP-9 + profile could be a potential a prognostic factor for CC development while the MMP-7+/EphA2+, MMP-9+/EphA2+, and TIMP-2+/EphA2 + could be a potential marker of precancerous lesions development. MMP-7+/TIMP-2 + profile can be related to the carcinogenesis. However, these profiles need more studies.

Cohort studies to evaluate women with cervical lesions and the 'immunoscore' profiles described in this study, are necessary to validate them as prognostic factors for precancerous lesions development and CC progression.

MMPs (-2, -7, and − 9) can be associated with the severity of cervical lesions, favoring ECM degradation and tumor invasion. The TIMP-2 and EphA2 expression can be associated only with precancerous lesions progression. The MMP-7/MMP-9 ‘immunoscore’ profile can be a potential/candidate prognostic factor for CC progression.and the MMP-7/EphA2, MMP-9/EphA2, and TIMP-2/EphA2 ‘immunoscore’ profiles can be related only to precancerous lesions development. However, more studies are necessary to validate these profiles.

Competing interests: The authors declare that they have no competing interests.

Ethics approval and consent to participate: All volunteers provided written informed consent for participation in this study, approved by INI and IFF/Fiocruz Institutional Ethical Review-Boards (protocols number 35589314.0.0000.5262 and 35589314.0.3001.5269), following Brazilian Ethical Committee Legislation. The details of the study were explained to the patients and informed consent forms were obtained from all participants. All procedures performed in the studies were in accordance with the Helsinki declaration.

Consent for publication: Not applicable.

Availability of data and materials: All data generated or analysed during this study are included in this published article [and its supplementary information files].

Funding: This work received financial support by FAPERJ and INI/Fiocruz. All patients’ clinical attendance was supported by UFRJ and IFF/Fiocruz.

Authors’ contributions: ATGF and MGBA designed the study. FBR and YLF were responsible for women enrollment and diagnostic and therapeutic procedures. EAP and CVA performed the histopathological diagnosis of cervical lesions. GSL and NPR performed the experiments. ATGF, GSL, EHR and MGBA analyzed and interpreted the data. GSL and ATGF wrote the main manuscript text. GSL prepared all figures. All authors read and approved the final manuscript.

Acknowledgements: The authors would like to thank all volunteers.

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No competing interests reported.

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Exploratory cross-sectional study to evaluate the metalloproteinases role as ‘immunoscore’ for HPV-induced cervical lesions

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Introduction

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Expression Of Mmps And Their Regulators In Cervical Lesions

Correlation Between Mmps And Its Modulators

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