Relationship between tumor thickness and GATA3 immunoexpression in lip and tongue squamous cell carcinomas

doi:10.21203/rs.3.rs-3147311/v1

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Relationship between tumor thickness and GATA3 immunoexpression in lip and tongue squamous cell carcinomas

https://doi.org/10.21203/rs.3.rs-3147311/v1

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Objectives

Our aim was to evaluate tumor thickness in cases of oral squamous cell carcinoma (SCC) and to correlate it with histological grade of malignancy and GATA3 immunoreactivity.

Materials and Methods

Sixty specimens (30 lower lip SCCs [LLSCCs] and 30 oral tongue SCCs [OTSCCs]) were scanned and digitized for the subsequent measurement of tumor thickness, histopathological examination, and quantitative analysis of GATA3 in the parenchyma and stroma of the tumors.

Results

Tumor thickness was lower in LLSCC cases compared to OTSCCs (p: 0.000). Immunohistochemical analysis of GATA3 in parenchyma, stroma and both compartments showed higher immunoreactivity in LLSCCs compared to OTSCCs (p: 0.000). We observed a negative correlation between tumor thickness and GATA3 expression in parenchyma (p: 0.014), stroma (p: 0.032), and both compartments (parenchyma and stroma) (p: 0.012).

Conclusions

Our results revealed the presence of GATA3 in all cases both in the parenchyma and in the stroma. Higher expression was more related to LLSCCs, which are known to be less aggressive tumors than OTSCCs. A greater tumor thickness was found in OTSCCs, which was correlated with lower expression of GATA3, suggesting that this protein is involved in the inhibition of proliferative, migratory, and invasive capacity.

Clinical relevance:

These findings can provide a basis for the identification of new therapeutic targets for OSCC, reinforcing the use of GATA3 as a biomarker in the neoplasms studied.

GATA3 transcription factor

Neoplasm invasion

Immunohistochemistry

Tongue neoplasms

Lip neoplasms

Head and neck cancer is the sixth most common type of malignancy in the world. The predominant type of this cancer is oral squamous cell carcinoma (OSCC), whose prevalence is increasing particularly in developing countries [1]. The pathogenesis of OSCC is varied and is related to genetic and environmental (non-genetic) factors such as smoking, alcohol, tobacco or areca nut chewing, viral infections, and a low socioeconomic status [2]. Other risk factors that have been linked to OSCC are immune status, eating habits, vitamin deficiencies, environmental pollutants [3], and exposure to ultraviolet radiation from sun exposure in cases of lip cancer [4]. In the case of OSCC, the location of the tumor is known to be directly associated with diagnosis and prognosis. Within this context, lip OSCC has the best prognosis because it is easily identified and can be treated early, while OSCC of the tongue has been associated with a poor prognosis [5].

Among other criteria, the choice of cancer treatment highly depends on the stage and grade of the tumor. Although the WHO histopathological classification of head and neck tumors often lacks significant prognostic value, clinical staging and TNM classification represent important prognostic factors according to the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) [6]. Within this context, the 8th edition of the AJCC and UICC staging manual introduced two important changes for OSCC, i.e., the inclusion of the depth of tumor invasion (DOI) in stage T and of extracapsular spread in stage N [7].

Advanced cases of OSCC are prone to lymph node metastases and recurrence. It is worth noting that in cases of metastasis outside the anterior lymph nodes, distant metastases involving other organs are usually observed. Thus, patients with advanced OSCC have a poor prognosis, with a survival rate of about 30%, and the identification of biomarkers for the diagnosis and treatment of OSCC is therefore of extreme clinical importance [8].

The GATA family of transcription factors comprises six members. GATA3 plays a key regulatory role in the initiation and progression of different cancers and its overexpression appears to be associated with a less aggressive tumor stage. This transcription factor has recently been reported to play a critical role in the regulation of the anticancer immune response in the tumor microenvironment [9].

In view of these considerations, the aim of the present study was to measure tumor thickness in lower lip (LLSCC) and oral tongue (OTSCC) squamous cell carcinoma and to correlate it with the histological grade of malignancy and GATA3 immunoexpression in these tumors.

Sixty specimens (30 LLSCCs and 30 OTSCCs) obtained from the Federal University of Rio Grande do Norte (UFRN) Oral Pathology Department, were randomly selected for this research. The degree of histological differentiation was classified as well differentiated (WD), moderately differentiated (MD), or poorly differentiated (PD) according to the World Health Organization (WHO) guidelines [10]. The study was approved by the Research Ethics Committee of UFRN, Natal, Brazil (Protocol No.2.283.894), and was conducted in accordance with the 1964 Helsinki declaration and its later amendments.

Measurement of tumor thickness

Tumor thickness was measured following the method proposed by Almangush et al [11]. from the surface to the deepest point of the invasive tumor. The cut-off point used to stratify LLSCC and OTSCC into low- and high-risk tumors was 4 mm. All slides were scanned with a digital slide scanner system (3DHISTECH®, Budapest, Hungary) and tumor thickness was measured using the Case Viewer 2.3.0.99276 software (3DHISTECH®, Budapest, Hungary).

Immunohistochemistry

Three-µm sections were cut from paraffin-embedded tissue blocks. The tissue sections were deparaffinized and immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were then washed in phosphate-buffered saline (PBS). The primary antibodies used were Mouse Anti-Human GATA3 Monoclonal antibody (1:50; B10; sc-137152; Santa Cruz Biotechnology Inc. Dallas, USA). After treatment with normal serum, the sections were incubated with the primary antibodies, in a moist chamber, followed by washing twice with Tween 20 solution (1%) in TRIS-HCL (pH 7.4). Then they were treated at room temperature with a HRP polymer-system, HiDef detection (Cell Marque, California, USA) for anti-GATA-3. Peroxidase activity was developed by immersing the tissue sections in diaminobenzidine (Liquid DAB + Substrate; Dako), resulting in a brown reaction product. Finally, the sections were counterstained with Harris’s hematoxylin, and coverslipped. According to the manufacturer’s specifications (Santa Cruz), GATA3 (B10) was routinely validated by Western Blot (WB) analysis of GATA-3 expression in JURKAT (A), CCRF-HSB-2 (B), and MOLT4 (C) whole cell lysates, where the antibody labeled a band of approximately 50 kDa. Fragments of normal mucosa were used as control.

Immunohistochemical analysis

All immunohistochemical slides were scanned with a digital slide scanner system (3DHISTECH®, Budapest, Hungary) and were subsequently analyzed by two previously trained examiners, who used Case Viewer 2.3.0.99276 software (3DHISTECH®, Budapest, Hungary). Immunohistochemical GATA-3 expression was quantitatively assessed by using an adaptation of a method described by Fang et al. [12] For quantification of the GATA3 cells, this assessment considered the analysis of 10 fields: 5 in parenchyma, and 5 in stroma. All fields selected were digitally photographed at 400x magnification, each field corresponded to an area of 0.0998 mm². Cells were counted with the use of ImageJ® software (National Institute of Mental Health, Bethesda, MD, USA).

Statistical analysis

The data obtained were submitted to the Kolmogorov-Smirnov test, which revealed normal data distribution for cases in which GATA3 immunostaining was performed, therefore the ANOVA test was applied. The Pearson correlation tests were applied, using the Statistical Package for the Social Sciences software (SPSS version 22.0; IBM® Company). The Study hypotheses were tested by adopting a level of significance of 5%, with p values ≤ 0.05 considered significant.

In cases of LLSCC (Fig. 1A), the smallest tumor thickness was 280 µm (0.280 mm), while the largest thickness was 2,743 µm (2.743 mm). In cases of OTSCC (Fig. 1B), the smallest and largest tumor thicknesses were 2,907 µm (2.907 mm) and 6,531 µm (6.531 mm), respectively. Statistical analysis of all cases showed a significantly lower tumor thickness (p: 0.000) in cases of LLSCC (median: 764.50 µm/0.76450 mm) compared to OTSCCs (median: 4,331 µm/4.331 mm). Regarding the cut-off point for identifying low- and high-risk tumors, we found that 18 of the 30 OTSCC cases analyzed were high risk (tumor thickness > 4 mm) (Table 1), while all 30 LLSCC cases were low risk (tumor thickness < 4 mm) (Table 2).

Table 1

Histological grading of malignancy (HGM), measurement of tumor thickness in micrometers (µm) and millimeters (mm) of lower lip squamous cell carcinoma (LLSCC) cases (n: 30).
Case	HGM	Tumor thickness		Case	HGM	Tumor thickness
Case	HGM	µm	mm	Case	HGM	µm	mm
1	WD	280	0.280	16	MD	585	0.585
2	MD	294	0.294	17	MD	876	0.876
3	WD	304	0.304	18	WD	883	0.883
4	WD	711	0.711	19	WD	604	0.604
5	WD	1184	1.184	20	MD	1475	1.475
6	WD	745	0.745	21	WD	719	0.719
7	WD	853	0.853	22	MD	576	0.576
8	WD	384	0.384	23	PD	835	0.835
9	WD	921	0.921	24	WD	974	0.974
10	WD	634	0.634	25	WD	818	0.818
11	MD	779	0.779	26	WD	961	0.961
12	PD	750	0.750	27	WD	927	0.927
13	WD	490	0.490	28	WD	1637	1.637
14	WD	714	0.714	29	MD	1462	1.462
15	MD	532	0.532	30	PD	2743	2.743
WD: Well Differentiated; MD: Moderately Differentiated; PD: Poorly Differentiated.

Table 2

Histological grading of malignancy (HGM), measurement of tumor thickness in micrometers (µm) and millimeters (mm) of oral tongue squamous cell carcinoma (OTSCC) cases (n: 30).
Case	HGM	Tumor thickness		Case	HGM	Tumor thickness
Case	HGM	µm	mm	Case	HGM	µm	mm
1	WD	6125	6.125	16	MD	2925	2.925
2	MD	4319	4.319	17	WD	3859	3.859
3	PD	4905	4.905	18	MD	3996	3.996
4	WD	3944	3.944	19	MD	2907	2.907
5	PD	6327	6.327	20	MD	4014	4.014
6	MD	3501	3.501	21	MD	4574	4.574
7	WD	5169	5.169	22	WD	5516	5.516
8	WD	4629	4.629	23	MD	4343	4.343
9	WD	3102	3.102	24	MD	5515	5.515
10	MD	3802	3.802	25	WD	3289	3.289
11	MD	6531	6.531	26	WD	6429	6.429
12	MD	5978	5.978	27	PD	3602	3.602
13	MD	4311	4.311	28	PD	4594	4.594
14	WD	5044	5.044	29	MD	3676	3.676
15	WD	3770	3.770	30	MD	5635	5.635
WD: Well Differentiated; MD: Moderately Differentiated; PD: Poorly Differentiated.

Histological grading of the LLSCC cases (Table 1) revealed 19 (63%) well-differentiated cases, 8 (27%) moderately differentiated cases, and 3 (10%) poorly differentiated cases. The OTSCC cases (Table 2) were categorized as well differentiated in 37% (n: 11), moderately differentiated in 50% (n: 15), and poorly differentiated in 13% (n: 4).

Quantitative immunohistochemical analysis of GATA3 (Fig. 2ABCD) in the parenchyma of the tumors studied showed higher immunoreactivity (p: 0.000) in cases of LLSCC (median: 884.50) compared to OTSCCs (median: 478). The same difference (p: 0.000) was observed in the tumor stroma, with higher immunostaining in LLSCCs (median: 1.164) when compared to OTSCCs (median: 669.50). Considering both compartments (parenchyma and stroma) in the quantitative analysis, immunoexpression of GATA3 continued to be higher (p: 0.000) in LLSCCs (median: 2.054) compared to OTSCCs (median: 1.124,50) (Fig. 3ABCD).

Analysis using Pearson’s correlation coefficient (r) revealed a negative correlation between tumor thickness and GATA3 expression in parenchyma (r: -0.315; p: 0.014), stroma (r: -0.278; p: 0.032), and both compartments (parenchyma and stroma) (r: -0.321; p: 0.012).

Although many authors use DOI and tumor thickness as synonyms, the terms are not the same and a distinction must be made. The DOI is defined as the extent of invasion below the epithelial basement membrane, while tumor thickness is measured from the mucosal surface of the tumor to the deepest point of tumor invasion [13]. For the cases of the present study, we chose to use tumor thickness because of its easier measurement since we used specimens obtained from incisional biopsies. Furthermore, considering the pattern of neoplastic proliferation and invasion, it was sometimes not possible to identify the basement membrane area, impairing the measurement of DOI.

In our study, the measurement of tumor thickness showed greater depths in cases of OTSCC compared to LLSCCs, confirming the trend that more aggressive tumors such as OTSCC have a greater invasive capacity and consequently larger tumor thickness. Wetzel et al [14], comparing aggressive and non-aggressive cases of basal cell carcinoma, found a greater depth in the most aggressive tumors; however, the authors observed that less aggressive tumors exhibited deeper extension than previously documented. We did not observe this finding of Wetzel et al [14] in the present study in which all greater depths of the less aggressive cases (LLSCCs) were lower than the smallest depth detected in OTSCCs.

Regarding the possible correlation between histological grade (WHO) and tumor thickness, we found larger and smaller tumor thicknesses (depths) regardless of the histological grade of malignancy (well differentiated, moderately differentiated, or poorly differentiated); hence, such relationship was observed neither in LLSCCs nor in OTSCCs. In agreement with our findings, the current edition of the Classification of Head and Neck Tumors recognizes that “the conventional classification into well differentiated, moderately differentiated and poorly differentiated, by itself, does not correlate well with prognosis”. Many studies indicate a low or no prognostic value of the WHO classification system [7].

GATA3 is known to be involved in the development of both normal and neoplastic tissues. This transcription factor plays a critical role in the development of the nervous system, mammary glands, parathyroids, kidneys, inner ear, skin, and lymphoid cell lineage [15]. Regarding neoplasms, GATA3 positivity has been reported in paragangliomas, acute leukemias with T-cell differentiation, SCCs (mainly in the skin but also in the cervix, larynx, and lung), salivary duct carcinomas, malignant mesotheliomas, germ cell tumors, and pancreas, liver, thyroid and, very rarely, prostate cancer [16]. We detected GATA3 immunoexpression in both LLSCCs and OTSCCs, suggesting that, like in the other neoplasms cited, this protein participates in tumor development and progression.

Immunopositivity for GATA3 was detected in the nucleus and/or cytoplasm in both the parenchyma and stroma of all cases of LLSCC and OTSCC, with higher expression in LLSCCs in all regions analyzed. These findings are in line with the study by Zhang et al [9] who found that higher GATA3 immunoexpression was associated with low-grade bladder cancer. Zhang et al [17] made a similar observation in a study following up patients with hepatocellular carcinoma (HCC), in which lower GATA3 expression was associated with a poor prognosis. The authors suggested that overexpression of GATA3 decreased the proliferative, migratory and invasive capacity of HCC cell lines, while inhibition of GATA3 stimulated proliferation, migration, and invasion. Within the same line of reasoning, Medeiros Souza et al [18] found GATA3 positivity in 103 cases (98.1%) of breast carcinoma. High expression of this marker was significantly associated with low histological and nuclear grade, positive hormone receptors, and lower proliferation activity evaluated by Ki-67 expression. Based on the evidence of higher expression of GATA3 in the stromal component of LLSCCs compared to OTSCCs obtained in the present study, we suggest greater skewing towards Th2 differentiation in the tumor microenvironment of the LLSCC cases studied.

In vivo experiments using animal models for the analysis of colorectal cancer (CRC) cells showed that downregulation of GATA3 expression increased the growth rate and volume of transplanted tumors, whereas overexpression of GATA3 had no significant effect on tumor growth. Furthermore, GATA3 was found to inhibit the viability of CRC cells, to promote apoptosis, and to reduce CRC cell resistance to oxaliplatin through miR-29b regulation [19].

In neoplasms, GATA3 can exert positive and negative functions, as demonstrated in a study on high-grade serous ovarian carcinoma (HGSOC) in which GATA3 acted as a tumor suppressor. Moreover, overexpression of GATA3 significantly decreased the tumor suppressor protein LSD1 in endometrial cancer. In contrast, overexpression of GATA3 in HGSOC did not alter the expression of LSD1[20].

In another study, Chi et al [21] detected GATA3 immunopositivity in 28% of the esophageal SCC cases studied; the survival rate was lower in these cases compared to patients who did not exhibit immunoreactivity. The authors concluded that GATA3 positivity was associated with a poor prognosis. Unfortunately, one limitation of our study is the lack of follow-up data and survival rates of the patients because, at our service, we only perform the incisional biopsy procedure and histopathological diagnosis; if a malignant neoplasm is diagnosed, the patient is referred to the specialized cancer service and we do not have access to these important data.

Our results revealed the presence of GATA3 in all cases of LLSCCs and OTSCCs both in the parenchyma and in the stroma. Higher expression of GATA3 was more related to LLSCCs, which are known to be less aggressive tumors than OTSCCs. A greater tumor thickness was observed in OTSCCs, which was correlated with lower expression of GATA3, suggesting that this protein is involved in the inhibition of proliferative, migratory, and invasive capacity. These findings can provide a basis for the identification of new therapeutic targets for OSCC, reinforcing the use of GATA3 as a biomarker in the neoplasms studied.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This study was funded by National Council for Scientific and Technological Development (CNPq) (Universal CNPq nº 424589/2016-8) - Pedro Paulo de Andrade Santos.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

FUNDING

This study was funded by National Council for Scientific and Technological Development (CNPq) (Universal Notice nº 424589/2016-8) - Pedro Paulo de Andrade Santos.

ETHICAL APPROVAL

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Research Ethics Committee of UFRN, Natal, Brazil (Protocol No. 2.283.894).

INFORMED CONSENT

Not required.

AUTHOR CONTRIBUTIONS

P.P.A.S, A.G.B.V and M.C.M.C performed the experiments, C.R.M, A.G.B.V, P.P.A.S and M.C.M.C analysed the data, C.R.M and A.G.B.V prepared the figures, and P.P.A.S and C.R.M wrote the main manuscript text. M.C.M.C and A.G.B.V the experiments, P.P.A.S supervised the project, and L.B.S and H.C.G reviewed the manuscript text. P.P.A.S, L.B.S and H.C.G supervised the project, offered senior supervision, and P.P.A.S reviewed the main manuscript text.

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No competing interests reported.

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Relationship between tumor thickness and GATA3 immunoexpression in lip and tongue squamous cell carcinomas

Status:

Version 1

Abstract

Objectives

Materials and Methods

Results

Conclusions

Clinical relevance:

Figures

INTRODUCTION

MATERIALS AND METHODS

Measurement of tumor thickness

Immunohistochemistry

Immunohistochemical analysis

Statistical analysis

RESULTS

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Status:

Version 1