Immunohistochemical assessment of the OVOL1 in pleomorphic adenoma and its malignant counterpart Key word OVOL1 Pleomorphic adenoma Carcinoma x pleomorphic adenoma

doi:10.21203/rs.3.rs-5307225/v1

Background

Pleomorphic adenoma (PA) is one of the few benign salivary gland tumors (SGT) that can undergo malignant change. Accordingly, a detailed study of the OVOL1 in PA and carcinoma x pleomorphic adenoma (CXPA) would be expected to contribute to the basic explanation of the mechanisms underlying the development of benign and malignant PA diagnosis for better evaluation of tumor biologic behavior.

Method

This retrospective study included 40 eligible cases (20 cases of PA and 20 cases of CXPA). The medical electronic records of patients were collected. Data regarding age, gender, tumor size, lymph node status, and TNM staging. Re-examination and revision of hematoxylin and eosin (H&E) stained slides were performed by expert pathologists. The OVOL1 immunostaining was used to differentiate between PA and CXPA.

Results

The OVOL1 level was higher significantly in PA lesions compared to CXPA.

Conclusion

OVOL1 down-expression may be contributing to the progression of PA to CXPA, which may provide a marker for malignant potentiality in PA and CXPA.

Pleomorphic Adenoma (PA) is the most common benign salivary gland tumor (SGT), representing 70% of all SGTs, and 33–41% of minor gland tumors (Nishida et al. 2023). Histologically, PA is a benign neoplasm consisting of cells exhibiting the ability to differentiate into epithelial (ductal and non-ductal) cells and mesenchymal (chondroid, myxoid, and osseous) cells [1].

Moreover, the risk of malignant transformation from PA to carcinoma x pleomorphic adenoma (CXPA (, the sixth most frequent salivary gland carcinoma, is notorious as it may lead to dilemmas in both pathological diagnosis and surgical/adjuvant radiation treatment of primary and recurrent PA [2]. The CXPA is a rare type of salivary gland malignancy, accounting for 5 to 15% of all salivary gland malignancies, and it has been estimated to develop in 6% of all PA [3]. The CXPA is an epithelial and/or myoepithelial malignancy that arises in association with a primary or recurrent PA [4].

In a recent WHO classification, recurrent PA malignant transformation rate is around 3%. According to WHO classification 2022, the carcinoma component can be of any type but the most common is salivary duct carcinoma (SDC), followed by myoepithelial carcinoma (MC), and Adenocarcinoma-not otherwise specified (Adeno-NOS) [5].

Furthermore, malignant change in PA is represented by obvious carcinomatous features characterized by cellular pleomorphism and abnormal mitotic activity adjacent to the benign cellular picture of the remainder of the benign tumor. This is typically a late phenomenon, usually only after the tumor has been present for many years or in recurrences after inadequate excision [6].

One of the more challenging dilemmas in salivary gland pathology is differentiating a benign neoplasm such as cellular PA (solid) from a low-grade carcinoma of CXPA. In addition, a major diagnostic concern is the ability to detect early malignant transformation in the absence of invasion in a PA [7].

The process of transformation from a benign to a malignant neoplasm has been reported to be controlled by a plethora of mechanisms including epithelial-mesenchymal transition (EMT). The Ovo-like transcriptional repressor 1 (OVOL1) has been considered a critical regulator of normal epithelial differentiation as well as cancer cell differentiation [8]. Moreover, OVOL 1 is one of a group of zinc finger protein family transcription factors that regulate gene expression in various differentiation processes [9].

Although the malignant transformation of benign salivary tumors has been documented in the literature [7], this process is not yet well understood in PA. Accordingly, a detailed study of the OVOL1 in PA and CXPA would be expected to contribute to the fundamental elucidation of the underlying development of PA and CXPA diagnosis for better evaluation of tumor biologic behavior.

This retrospective study included 40 eligible cases (20 cases of PA and 20 cases of CXPA) diagnosed during the period between 10 May and 15 August 2023 in the surgical pathology laboratory in our Oncology Center. Inclusion criteria were the availability of tissue blocks and complete clinical and follow-up data. Exclusion criteria included incomplete clinical data or unavailability of paraffin blocks. The medical electronic records of patients were reviewed. Data regarding age, gender, tumor size, lymph node status, TNM staging according to (AJCC) 2017, treatment, and follow-up were revised. Re-examination and revision of hematoxylin and eosin (H&E) stained slides were performed by expert pathologists.

Immunohistochemistry

Immunohistochemical staining was performed on 4 µm thick, formalin-fixed tissue sections, paraffin-embedded. Epitope retrieval was achieved by subjecting the samples to a Tris-EDTA buffered solution with a pH of 9.0 (Target Retrieval Solution; Dako, Glostrup, Denmark) in a water bath at 97 ◦C. Hydrogen peroxide (Peroxidase-Blocking Reagent; Dako) was used to block endogenous peroxidase activity. Polyclonal anti-human OVOL1 (Dilution of 1: 100, Cat. ARC0038, from Medaysis, USA) was applied as the primary antibody. The antigen-antibody complexes were detected using dextran-polymer conjugated horseradish-peroxidase and 3,3-diaminobenzidine. Finally, all specimens were counterstained with hematoxylin.

For OVOL1 staining, cases were interpreted as positive immunoreaction through the brown cytoplasmic and membranous staining of neoplastic cells and negative when no immune stain was found. Positive and negative controls were run in parallel. Scoring was calculated as the product of the staining intensity score and the extent of the immunoreactivity score. The intensity was graded on a semi-quantitative scale using the staining of the positive control as a reference and scored from 0 to 3 as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The positive stained was assessed as the area percentage ranged from 0 to 100% and evaluated as follows: (1) area percentage of positively stained cells ≤ 5%, scored 0; 6–25%, scored 1; 25–50%, scored 2; 51–75%, scored 3; and > 75%, scored 4 [10] and thus the immune reactive scoring (IRS) system was defined as the low expression for scores of 0–3 and as the high expression for scores of 4–12 [11].

The ImageJ software (Schneider, Rasband et al. 2012) was used for the precise evaluation of the area percentage of OVOL1 staining and staining intensity, as previously described. This measurement relied on the color intensity of UTF-8 grayscale pixel values to calculate the mean intensity within predefined regions of interest, for assessing intensity scores for each IRS. The software systematically assessed parameters such as integrated density, mean intensity, and the fractional proportion of stained pixels within specified areas.

Statistical methods

Statistical analysis Data were fed to the computer and analyzed using IBM SPSS Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. Chi-square test was used for comparison of two or more groups. The significance of the results obtained was judged at the (0.05) level. Spearman’s rank-order correlation is used to determine the strength and direction of a linear relationship between two non-normally distributed continuous variables and ordinal variables.

Our sample consisted of 20 PAs and 20 CXPAs. Of the 20 PAs, 65% of cases were female. The mean age of the patients was 47.05±13.43, with the youngest patient being 30 years old and the oldest being 80 years old. Meanwhile, most CXPA was more common in males than females. Moreover, 50% of CXPAs were presented among the age category (30-50 years), with a mean age of 53.2±17.11. The most frequent location was in the parotid gland accounts for 65.0% of PAs and 70% of CXPA. Furthermore, the histological subtypes of malignant components of CXPA according to WHO (2022) were: six cases of epithelial-myoepithelial carcinoma EMC ex-PA, four cases of myoepithelial carcinoma (MC) ex-PA, seven cases of salivary duct carcinoma (SDC) ex-PA, and three cases of NOS ex-PA.

Histopathological examination of the PAs showed preserved lobular structure in some cases, and it may be encapsulated or showed incomplete capsulation. The PA showed morphological diversity in the form of an admixture of bi-layered ducts (epithelial) with the myoepithelial cells interspersed with the stroma. Noticeably, the duct or duct-like structures showing mucoid material were observed in (85%) of the cases sometimes demonstrating cystic pattern and cribriform pattern. The proportion of the three components (epithelial, myoepithelial, stromal cell) varied in each tumor, and one element may predominate. Therefore, it was observed that 45% of PA was of classic phenotype (with an approximately equal proportion of cellular and stromal elements), 30% of cases were cellular phenotype (epithelial cell-rich or myoepithelial cell-rich which was dominant over the stromal elements), while 25% were stromal phenotype (with a predominance of chondroid, fibro myxoid or hyalinized stroma). The cellular phenotype, particularly of the myoepithelial cells, was broad and included epithelioid, stellate, spindle, clear cells, and plasmacytoid morphology. The stroma was variable from being mucoid, myxoid, hyalinized, and chondroid. Moreover, chondromyxoid stroma was displayed in 65% of cases, while 35% of cases exhibited fibro myxoid stroma. Four cases displayed squamous metaplasia with keratin formation whereas 2 cases showed focal areas of calcification.

Lymphoid aggregate outside the capsule was observed in 25% of cases. Only one case infiltrated the muscle fibers (Figure 1).

The malignant transformation from PA to CXPA appeared as dysplastic changes including pleomorphism, increased mitotic activity, abnormal mitosis, and hyperchromatic nuclei of ductal and myoepithelial cells. Moreover, it has been observed that the benign PA appeared intermixed with the carcinoma or appeared as a discrete nodule (Figure 2).

Immunohistochemical results

OVOL1 positive expression was observed among all of the studied cases of PAs and CXPAs. It was detected as brown staining in the membrane and cytoplasm of tumor cells. The nuclear reaction was negative among all studied PA and CXPA types. Furthermore, the surrounding stromal cells, neural tissue, and vascular wall showed a negative reaction to the OVOL1 antibody. The present work was focused only on the epithelial component of tumors. The positive immunoreactivity for OVOL1 was found among 100% of the studied PA and CXPA. Most of the studied PA and 15% of CXPA revealed high OVOL1 expression. There was a statistically significant difference between IRS for OVOL1 among the studied PA and CXPA, Table (1).

Table (1): The distribution of OVOL1 immunoreactivity between the studied PA and CXPA.

	PA n=20	CXPA n=20	p- Value
Low	8 (40%)	17 (85%)	0.0032*
High	12 (60%)	3 (15%)
Total	20	20

*Significant difference, (p < 0.05). PA: Pleomorphic adenoma, CXPA: Carcinoma ex polymorphic adenoma. Chi-square test.

All of the presented CXPA cases showed positive immunostaining for OVOL1 with different proportions of low and high immunoreactions based on the presented histological subtypes of these carcinomas. Noticeably, all of the studied EM ex PA, MC ex PA, and 85.7% of SDC ex PA exhibited low IRS. However, 66% of NOS ex-PA revealed a high reaction. Statistically, there was a significant difference between IRS for OVOL1 expressions among the histological subtypes of the studied CXPA (Table 2, figure 3).

Table (2): The distribution of IRS for OVOL1 among the histological subtypes of the studied CXPA

IRS	EMC n=6	MC n=4	SDC n=7	NOS n=3	Total	P- value
Low	6 (100%)	4 (100%)	6 (85.7%)	1 (33.3%)	17 (85%)	0.0450*
High	0	0	1 (14.3%)	2 (66.7%)	3 (15%)
Total	6 (100%)	4 (100%)	7 (100%)	3 (100%)	20 (100%)

Chi-Square test. *Significant difference, (p<0.05), n=number, percentage (%). IRS=immunoreactive Score.

As regards the correlation between the OVOL1 IHC staining and clinical staging of the studied CXPA cases, we found low OVOL1 reaction among all cases in stage IVA, 83 % of cases in stage III, and 75% of cases in stage II. Meanwhile, in stage I, there was an equal distribution between low and high IRS. Based on the statistical analysis, there was no statistically significant difference observed between IRS for OVOL1 among the studied CXPA regarding the clinical stages (Table 3).

Table (3): The distribution of IRS for OVOL1 among the TNM of the studied CXPA.

TNM	IRS	CXPA n=20	Total	P-value
stage I	Low	1 (50%)	2 (10%)	0.301
stage I	High	1 (50%)	2 (10%)
stage II	Low	3 (75%)	4 (20%)
stage II	High	1 (25%)	4 (20%)
stage III	Low	5 (83.3%)	6(30%)
stage III	High	1 (16.7%)	6(30%)
stages IVA	Low	8 (100%)	8 (40%)
stages IVA	High	0	8 (40%)
Total		20 (100%)	20

Chi-Square test. *Significant difference, (p<0.05), n=number, percentage (%). IRS=immunoreactive Score.

Only 5% showed recurrence in PA cases and all of them showed high immunoreactivity for OVOL1. Meanwhile, 80% of CXPA cases that had recurrence revealed low IRS. Based on the statistical analysis, there was no significant difference between recurrent and non-recurrent cases related to OVOL1 among either PA or CXPA. However, there was a statistically significant difference in IRS for OVOL1 among the studied PA and CXPA concerning tumor recurrence (Table 4).

Table (4): The distribution of IRS for OVOL1 among the tumor recurrence of the studied PA and CXPA

Tumor recurrence	IRS	PA n=20	Total	CXPA n=20	Total	P-value of PA &CXPA
Recurrent	Low	0	1(5%)	4 (80%)	4 (20%)	0.02*
	High	1 (20%)		0
Non-recurrent	Low	8 (22.8%)	19(95%)	13(37.2%)	16(80%)	0.01*
	High	11(31.6%)		3 (8.6%)
Total		20 (100%)		20 (100%)

Chi-Square test. *Significant difference, (p<0.05), n=number, percentage (%). IRS=immunoreactive Score.

Given the high prevalence of PAs and the risk of CXPA developing over time, a significant amount of attention in better describing the histological, immunophenotypic, and molecular phenotypes has become paramount for providing better treatment plans and prognostic estimations for patients.

Among the 20 cases of PAs, 45% of them were categorized as classic PAs. While 30% of cases demonstrated a greater abundance of epithelial components, which in this study represent cellular PAs. A comparable observation was also reported by another study that found classic phenotype to be the most common [12]. Other research has demonstrated that the cellular-rich phenotype was the most prevalent form of PAs, followed by the classic and stromal phenotype [13]. Although it had no prognostic value, the classification highlighted the morphological spectrum of this tumor.

The epithelial and myoepithelial cells that formed PAs were organized in a variety of patterns, and the morphology of the tumors was highly diverse. In the present study, the most common cells in the parenchymal component consisted of bi-layered ductal and myoepithelial cells. This was compatible with the study findings [14]. The molecular characteristics of PA and normal parotid gland epithelial cells showed a substantial difference in the distribution of epithelial cells from one to the other. This finding indicated that PA might have a unified cellular landscape despite the variable morphological architectures [15].

The cellular phenotype, particularly of the myoepithelial cells, was broad and included epithelioid, stellate, spindle, clear cells, and plasmacytoid morphology; this was in agreement with S. Irani, Dehghan, & Kalvandi, 2023. This was not in concordance with others [12, 13, 14]. The pathological structure of PA varied in different samples of H&E staining, which also displayed similar cell distribution in different PA samples when performing gene expression [15].

Diverse stromal types were frequently observed in PAs. The present study found that myxoid, chondromyxoid, and hyaline were the most observed types of mesenchymal-like components, which aligns with findings from other studies [13, 16]. This was contradicted by the study which reported that hyaline stroma was the most common, followed by fibrous stroma and myxomatous stroma. It was conjectured that fibrosis and hyalinization may occur because of inflammation caused by constant mechanical stimulation of the oral cavity [12]. Meanwhile, another study found myxoid to be the most common variant, followed by hyalinized stroma and chondroid. This finding is like a hypothesis that myxoid variants may express more acidic mucins and, due to a lack of differentiation, have a higher recurrence rate and poorer prognosis [14].

Squamous metaplasia was found in 20% of PAs and could manifest as diffuse sheets of epidermoid tissue. Occasionally, it was in the form of keratin pearls or cystic spaces that were filled with keratin. Similarly, 10% and 40% respectively of PAs have been reported to have squamous metaplasia by other authors [13, 14]. It is important to note that extensive squamous metaplasia does not necessarily indicate malignant transformation unless accompanied by capsular invasion, hemorrhage, and necrosis [14].

The histologic subtypes of the CXPA were identified by the histologic features depending on WHO (2022). The histological subtypes were: six 30% cases of EMC ex-PA, four 20% cases of MC ex-PA, seven 35% cases of SDC ex-PA, and three 15% cases of NOS ex-PA that were nearly compatible with previous studies of [17, 18].

Most cases arising from a pre-existing PA are like other studies [19, 20]. All cases of EMC ex-PA demonstrated infiltrating epithelial and myoepithelial cells growing in nests and trabeculae which were like other literature [18]. Moreover, SDC demonstrated nests of pleomorphic epithelial cells with central necrosis that were comparable with another study [18]. The inherent pleomorphism of many tumors contributes to the concept of CXPA being a somewhat controversial issue. A progressive transition to genetic marker identification may allow this situation to evolve [20].

All of the studied PA and CXPA showed immunopositivity to OVOL1 where more than two-thirds demonstrated a low IRS. This was consistent with other studies that found the OVOL1 transcription factor might play a dysregulation role in several types of cancer [8, 21, 22, 23].

Interestingly, two-thirds of the studied PA revealed high IRS for OVOL1 in the form of diffuse cytoplasmic/membranous immunopositivity. However, the majority of the studied CXPA showed low IRS for OVOL1. We demonstrated that OVOL1 was a unique diagnostic marker that can be used to differentiate PA from its malignant counterpart CXPA.

This finding agreed with a previous study of in situ epidermal malignancy (Bowen’s disease). They concluded that OVOL1 was upregulated in Bowen’s disease but markedly downregulated in squamous cell carcinoma. Meanwhile, they suggested that the OVOL1 axis is a key modulator of expression in the shift from in situ epidermal malignancy (Bowen’s disease) to invasive squamous cell carcinoma [24].

Furthermore, other studies have shown that OVOL1 has high levels in benign and premalignant lesions as conjunctival intraepithelial neoplasia [10]. This was also compatible with the presented TNM, non-encapsulation, recurrence, lymph vascular invasion, and perineural invasion of these current tumors. In another study, OVOL1 was found to be a possible diagnostic aid to differentiate normal ocular conjunctiva from squamous cell carcinomas. Furthermore, they found that low OVOL1 scores may be associated with disease progression, indicating that OVOL1 may be a useful marker in distinguishing pre-invasive from invasive squamous neoplasms [10].

Several studies have reported that OVOL1 functions as a transcription factor to regulate gene expression during various differentiation processes and induce MET (mesenchymal-epithelial transition) in several types of cancer (breast, lung, and skin) [8, 21, 22, 23].

The results of Li strongly suggest that OVOL1 potentiates cell differentiation, contributing to the prevention of the EMT process. Loss of OVOL1 expression has been characterized by MET. This process would provide the necessary plasticity for the malignant cell to achieve stromal invasion [10, 25]. This strongly suggests that OVOL1 potentiates cell differentiation, contributing to the prevention of the EMT process [24].

In our study, there was a high statistically significant difference between IRS for OVOL1 in CXPA cases and among the studied PA and CXPA. Our result was also supported by (Ito, Tsuji et al. 2017) who observed that OVOL1 was significantly downregulated in cutaneous squamous cell carcinoma [24]. In contrast, the OVOL1 expression in breast cancer tissues was significantly higher than that in normal breast tissues [21].

These findings demonstrated that EMT (OVOL1) might play a role in the progression of PA into CXPA.

Furthermore, our study showed that OVOL1 has high levels in benign and low in malignant lesions. This was also compatible with the presented clinical staging, and recurrence of these current tumors. This finding was supported by another study in oral SCC neoplasm [26] as well as in breast cancer [21] in hepatocellular neoplasms [8], and in Renal cell carcinoma [22].

In this study, we found that analysis of OVOL1 staining has the potential to distinguish between benign PA and malignant CXPA. Low OVOL1 scores may be associated with tumor progression, indicating that OVOL1 may be a useful marker in distinguishing benign PA from malignant CXPA. These features may be useful in future assessments, planning, or progressing immune-related therapies by using OVOL1 in PA & CXPA Further studies are needed to clarify its role in the identification of risk group patients with susceptible malignant transformation.

Adeno-NOS: Adenocarcinoma-not otherwise specified.

CXPA: Carcinoma ex pleomorphic adenoma

EMT: Epithelial-mesenchymal transition

IRS: Immune reactive scoring

IHC: Immunohistochemical expression

MET: Mesenchymal-epithelial transition

MC: Myoepithelial carcinoma

OVOL1: Ovo-like transcriptional repressor 1

PA: Pleomorphic Adenoma

SDC: Salivary duct carcinoma

SGT: salivary gland tumor

Availability of data and materials

The current study's used and/or analyzed datasets are available from the corresponding author on reasonable request.

Funding

This study is self-funded, as There was no grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical approval and consent to participate.

The study was carried out according to the ethical guidelines set by the Research Ethics Committee of the Faculty of Dentistry, Mansoura University, Egypt, under approval number A0203023OP on April 15, 2023.

Competing interests

The authors have no conflicts of interest to declare.

Author Contribution

A -Aasha wrote the manuscriptb- dr. Nadia reviewed the manuscriptc- dr. Marwa reviewed the manuscriptd- dr. Ghada prepared figures c- dr. osama reviewed the manuscript

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No competing interests reported.

Immunohistochemical assessment of the OVOL1 in pleomorphic adenoma and its malignant counterpart Key word OVOL1 Pleomorphic adenoma Carcinoma x pleomorphic adenoma

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Abstract

Figures

1 Background

2 Methods

3 Results

4 Discussion

5 Conclusion

Abbreviations

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Author Contribution

References

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Status:

Version 1