YAP1 Rearrangement in Parotid Sclerosing Mucoepidermoid Carcinoma

ABSTRACT

Objective

This study investigates to investigate Yes-associated protein 1 (YAP1) rearrangement status in a six-case of sclerosing mucoepidermoid carcinoma (SMEC) series with reference to 20 conventional mucoepidermoid carcinomas.

Method

A retrospective analysis was conducted on a cohort of patients diagnosed with SMEC from 2018 to 2022. The presence of YAP1 rearrangement was determined using fluorescence in situ hybridization (FISH) analysis targeting the YAP1 gene located at 11q22.1-q22.2. Clinical data, including patient outcomes, were collected and analyzed. Next-generation sequencing (NGS) was performed to identify the fusion partner involved in the YAP1 rearrangement.

Results

A total of six cases of SMEC were included in the study, of YAP1::MAML2 fusion was detected and five cases showed YAP1 rearrangement on FISH testing.

Conclusion

YAP1 alteration may explain the indolent nature of SMECs, especially in cases with low cellularity. Further studies incorporating NGS analysis are warranted to unravel the fusion partner associated with YAP1 rearrangement, providing a deeper understanding of the molecular mechanisms underlying the positive outcomes observed in these patients.

KEYWORDS:

• YAP1
• sclerosing mucoepidermoid carcinoma
• FISH
• salivary gland cancers

Introduction

YAP1 (Yes-associated protein 1) is a transcriptional co-activator that plays a crucial role in the Hippo signaling pathway, which regulates organ size, cell proliferation, and apoptosis.^1,2 The loss of YAP1 expression due to YAP1 rearrangement or transcriptional repression is observed in eccrine poroma,³ Merkel cell carcinoma,⁴ and porocarcinoma.^1,4,5 YAP1 rearrangement refers to structural alterations in the YAP1 gene resulting in loss of its normal function. Transcriptional repression, on the other hand, involves the downregulation of YAP1 expression through regulatory epigenetic modifications or altered signaling pathways. In gastric carcinoma, YAP1 overexpression has been observed, and seems to be correlated with disease progression, lymph node metastasis, and poor prognosis^6; suggesting that YAP1 overexpression may predict lymph node metastasis, act as a prognostic factor in gastric carcinoma, with conflicting therapeutic implications.^7–9 The exact mechanisms underlying YAP1 overexpression in gastric carcinoma are not fully understood. However, dysregulation of the Hippo pathway, which normally restricts YAP1 activity, may contribute to YAP1 overexpression,¹⁰ indicating that YAP1 rearrangements may not always correlate with aggressive tumor behavior.

Sclerosing mucoepidermoid carcinoma (SMEC) is a rare subtype of mucoepidermoid carcinoma presenting at a younger age compared to classic mucoepidermoid carcinoma¹¹. SMEC histopathologically exhibits central sclerosis, infiltration of plasma cells, eosinophils and/or lymphocytes at the tumor periphery, and tendency to show heterogeneity.¹¹ We investigate the association between YAP1 rearrangement and prognosis in six patients with SMEC.

Materials and Methods

Paraffin blocks corresponding to 20 conventional MEC tissues were retrieved from the laboratory archives, from 2019 to 2022, after securing the ethical approval from the MU. These blocks contained tissue samples that had been preserved in paraffin wax for long-term storage. The selected paraffin blocks were visually inspected, and the desired areas of the MEC tissues were identified. Six cases of SMEC paraffin blocks were also retrieved and checked before all sectioned with a microtome set at 4 microns. Conventional MECs are well-established and widely studied, making them an ideal reference point. The prepared slides were stained by Hematoxylin and eosin (H&E) for confirmation of diagnosis. The grading of the included cases according to the AFIP and Brandwein scoring sytems was conducted using our automatic grading tool (Supplementary Mateial).

Fluorescence in situ Hybridization

In the fluorescence in situ hybridization (FISH) analysis, we employed 4-μm-thick formalin-fixed, paraffin-embedded tissue sections. To specifically target the YAP1 and MAML2 rearrangements at their respective chromosomal regions, we utilized dual color break-apart probes from Empire genomics and ZytoVision, GmbH, Germany, respectively. This allowed us to visualize and identify the presence of YAP1 and MAML2 gene rearrangements. The FISH analysis was performed at a high magnification of 1000×, and 50 nuclei were examined in each tested sample using the YAP1 and MAML2 break-apart probes [YAP1-20-OR, Fluorochromes: 5-TAMARA, Empire genomics; MAML2 Z-2014-200: ZytoLight SPEC Dual Color Break Apart Probe]. Negative signals were characterized by orange-yellow-green signals, while positive signals were determined by the separation of orange and green signals. To establish the cutoff value for positive signals, we considered more than 10% of nuclei displaying the break-apart split signals, which was determined based on the mean + 3 standard deviations in normal non-neoplastic control tissues. Two technicians, blinded to the study’s purpose, assessed the FISH signals. The DNA of interest was amplified using primers designed to flank the YAP1 gene region. Amplified DNA fragments were labeled with fluorescent molecules to allow visualization of the probes with a fluorescence microscope for FISH analysis. Different probes were labeled using different fluorochromes. The labeled DNA fragments were purified to remove unincorporated nucleotides and PCR artifacts so that only labeled probe fragments were present in the final probe mix. The purified probe was heat denatured to convert double-stranded DNA to single-stranded DNA, allowing it to hybridize with the target DNA sequence in the FISH method. Denatured fluorescently labeled probes were then applied to tissue sections on slides. The slides were then incubated in a hybridization chamber under specific conditions to allow the probes to hybridize to the YAP1 gene region. Slides were evaluated under a fluorescence microscope. We visualized the FISH signals using a ZEISS Axioscope 5 Smart Laboratory microscope (Zeiss Axioplan, Oberkochen, Germany) equipped with a triple-pass filter for DAPI/FITC/BAO. The acquired images were processed using Isis software (Metasystem, Newton, MA) and automatically integrated to generate comprehensive imaging of the detected signals against a black background.

Next Generation Sequencing (NGS)

To isolate nucleic acids for our analysis, we used tissue blocks that contained the harvested cells of interest. These tissue blocks provided a source of cellular material for further experimentation. The cells within the tissue blocks were effectively lysed by subjecting them to disruption, which allowed us to access the nucleic acids present within the cells. For RNA isolation, we employed the TRIzol/phenol-chloroform method, a widely used technique for extracting RNA from biological samples. This method ensures the isolation of high-quality RNA by effectively separating it from other cellular components. Following the extraction, we assessed the concentration and purity of the RNA using a spectrophotometer. We followed the manufacturer’s instructions to ensure accurate and reproducible cDNA synthesis.During the cDNA synthesis process, we used 500 ng of RNA as input. This amount was selected based on established protocols and guidelines for cDNA synthesis. By starting with a standardized input of RNA, we aimed to obtain consistent and reliable cDNA samples for downstream analysis.

To identify the fusion gene, we initially conducted RNA-seq using the Illumina TruSight RNA Pan-cancer panel. This panel allowed us to target genes relevant to MEC, acute lymphoblastic leukemia, and other known driver mutations, including the YAP1::MAML2 fusion was detected on NGS in two cases, using the Illumina NovaSeq 6000 platform (Illumina Inc, San Diego, CA, USA). To analyze the obtained data, we utilized the tools provided by BaseSpace Sequence Hub (Illumina).

Results

We present six cases of SMEC, characterized by the presence of central sclerohyalinization in addition to the typical mucous, intermediate and epidermoid cells seen in MEC. The cases were low to intermediate grade and showed different stages. None of the cases developed metastasis at diagnosis or during follow-up, despite moderate grade and/or stage III and/or recurrence in some cases. All tumors showed YAP1 rearrangement, except Case #6 (Table 1). Histologically, sclerosing mucoepidermoid carcinoma (SMEC) is characterized by prominent mucin-producing glandular elements and solid areas composed of squamoid cells. The neoplastic components can vary cellularity and architecture, with some cases showing highly sclerosing and inconspicuously cellular features (Figure 1), while others exhibit slightly sclerosing and cellular features(Figure 2). Additionally, all cases of SMEC demonstrate peripheral tumor-associated lymphocytic proliferations and some degree of intratumoral inflammatory infiltrates. The intermediate cells were not as common as mucocytes and squamoid cells within the neoplastic cells (Figure 3).

Figure 1. (a) Histological micrograph for SMEC with low neoplastic cellularity and extensive hyalinosclerosis (H&E, ×4); (b) high power imaging of the diagnostic areas showing mucoepidermoid carcinoma islands (H&E, ×40).

Figure 2. Histological micrograph for SMEC with high cellularity (case #1) (H&E, ×4).

Figure 3. (a) Histological micrograph for SMEC with low neoplastic cellularity showing hyalinosclerosis, peripheral tumor-associated lymphoid proliferation and moderate mucin production (H&E, ×10); (b) high power imaging of the diagnostic areas showing mucoepidermoid carcinoma islands (H&E, ×40).

Table 1. Characteristics of the patients.

Case	Age	Sex	Size (cm.)	Site	Diagnosis	Stage	MAML2	Recurrence	YAP1	NGS
1	14	F	3 × 2 × 1.5	Parotid	Low-grade SMEC	III	Rearranged	No	Rearranged	YAP1: MAML2
2	22	F	2.5 × 2 × 1	Parotid	Low-grade SMEC	II	Rearranged	No	Rearranged	Not analyzable
3	13	M	4.2 × 2.5 × 2	Parotid	Intermediate-grade SMEC	II	Rearranged	Yes	Rearranged	Not analyzable
4	24	F	5 × 3 × 2	Parotid	Low-grade SMEC	III	Rearranged	No	Rearranged	YAP1: MAML2
5	25	F	3.2 × 2.5 × 1.5	Parotid	Low-grade SMEC	II	Rearranged	No	Rearranged	Not analyzable
6	16	M	2 × 2 × 1	Parotid	Low-grade SMEC	II	Rearranged	No	Intact	Not analyzable

Within the fibrous stroma, an infiltration of inflammatory cells, including lymphocytes and plasma cells was present. The fibrous stroma remarkably imparted a dense, hyalinized appearance to the tumor. Figure 4 shows an example of YAP1 alteration in SMEC.

Figure 4. YAP1-rearrangement in SMEC (YAP1 break-apart probe, ×63).

Overall, the histology of sclerosing MEC is characterized by a combination of glandular structures, squamous cells, and a prominent fibrous hyalinizing stroma, distinguishing sclerosing MEC from other patterns of mucoepidermoid carcinoma and contributes to its unique histological appearance.

Our cases showed two distinct morphological subtypes. The first subtype showed extensive sclerosis or fibrotic tissue within the tumor. Additionally, remarkable lymphoid infiltrates were observed peripherally, along with the presence of inflammatory cells infiltrating the tumor. The production of mucin within the tumor cells and mucocytes was minimal. Notably, a significant number of squamous epithelial cells were observed in this subtype, while clear cells were absent. The second subtype was characterized as cellularly rich with minimal fibrotic tissue or sclerosis observed within the tumor. There were also minimal lymphoid infiltrates around the tumor. Conspicuously, this subtype exhibits the presence of eosinophils and IgG4 plasma cells infiltrating the tumor. Abundant mucin production was detected within the tumor cells, and there was an abundance of clear cells arranged in fascicles within the tumor. These morphological subtypes are not related to YAP1 genetic alterations and could be induced by other signaling pathways, microenvironmental factors, and immune response.

YAP1::MAML2 fusion was detected on NGS in two sclerosing MECs. Nevertheless, in a examining a reference group of 20 cases of conventional MEC, the YAP1::MAML2 fusion was not detected.

Discussion

YAP1 has diverse functions and interacts with multiple signaling pathways, which can have different effects depending on the specific cellular and molecular context of the tumor. Therefore, the impact of YAP1 rearrangements on tumoral aggression may be tumor type-specific and context-dependent^12–15. Activation of YAP1 has been associated with increased expression of EGFR (epidermal growth factor receptor) and SNAI2 (Snail family transcriptional repressor 2). EGFR is a receptor tyrosine kinase that promotes cell proliferation, survival, and migration, and its gain of function leads to increased signaling through this pathway.¹⁶ SNAI2 is a transcription factor known to promote epithelial-mesenchymal transition (EMT), a process associated with tumor invasiveness and metastasis. YAP1 activation in SCCs is often accompanied by the loss of tumor-suppressor genes. CSMD1, CDKN2A, NOTCH1, and SMAD4 are known tumor-suppressor genes involved in the regulation of cell cycle control, differentiation, and signaling pathways.¹⁶

The NOTCH pathway is a highly conserved signaling pathway that plays a critical role in cell fate determination, differentiation, and tissue development. In MEC, a recurring genetic alteration involves the rearrangement of the MAML2 gene, resulting from the gene rearrangement can interact with the NOTCH intracellular domain and other transcriptional co-activators to potentiate NOTCH signaling. This enhanced NOTCH signaling can contribute to the oncogenic properties of MEC, including increased cell proliferation, survival, and invasiveness. Interestingly, studies have suggested crosstalk between the Hippo and NOTCH pathways. Additionally, YAP activity can be regulated by NOTCH signaling through various mechanisms, including the direct regulation and modulation of YAP phosphorylation. Therefore, in cases where MAML2 is rearranged and the NOTCH pathway is dysregulated, the hyperactivation of YAP in the Hippo pathway may intersect with the dysregulated NOTCH pathway, potentially leading to an additive or synergistic effect on cell proliferation, survival, and other oncogenic processes in mucoepidermoid carcinoma. The precise molecular mechanisms underlying the interplay between the Hippo and NOTCH pathways in MEC require further investigation. However, the existing evidence suggests a potential relationship and highlights the complex nature of cancer signaling networks^16,17.

Some studies have found no significant correlation between YAP1 rearrangements and clinical outcomes. As mentioned before, the functional consequences of YAP1 rearrangements depends on the genetic and cellular context of tumor, making it challenging to generalize their impact in different tumor types.^17,18

The absence of metastasis and low recurrence rate observed in our cases suggests the favorable influence of YAP1 activation on the behavior of SMEC. Although the specific mechanisms by which YAP1 exerts its effects in SMEC are not fully understood, its involvement in maintaining tissue homeostasis and regulating key signaling pathways may contribute to the observed positive outcomes.

It seems that fusion partners of YAP1 can play a role in the aggressive nature of the neoplasm.¹⁷ YAP1 fusions create a chimeric protein that may exhibit altered functions in comparison to the normal YAP1 protein, affecting its interaction with other cellular proteins, altering their functions.^19–21 These interactions can impact downstream signaling pathways and alter rates of tumor progression and aggressiveness. According to a meta-analysis, patients with carcinoma may statistically have low overall survival time and disease-free survival time due to the strong YAP1 expression. YAP1 could serve as a future therapeutic target for these cancers.²²

However, the low-grade sclerosing mucoepidermoid carcinoma observed in young individuals typically exhibits an indolent behavior. The validity of this finding faces a challenge due to the inconsistent use of grading systems²³.

Because the reported SMEC cases in the parotid showed an indolent behavior, consistent with our findings, it is proposed that the clinically aggressive behavior that characterizes a set of YAP1-rearranged neoplasms might be mitigated by crosstalk or associated oncogenic pathways in SMEC. While rare, the coexistence of multiple driver gene fusions has been reported in other tumor types, such as secretory carcinoma of salivary glands, demonstrating a dual fusion in of ETV6::NTRK3 and MYB::SMR3B. In this regard, we confirm that YAP1::MAML2 fusion is not confined to squamous porocarcinoma.

Conclusions

The presence of YAP1 rearrangements can cause varying degrees of YAP1 activation and downstream effects in different neoplasms, leading to various effects on tumor behavior, and response to treatment, making it challenging to draw definitive conclusions about the impact of YAP1 rearrangements in various tumors. We detected YAP1 alternations in five SMEC cases while the sixth case did not show YAP1 rearrangement. We report the first YAP1::MAML2 fusion in SMEC. This fusion was previously reported salivary squamous porocarcinoma of salivary glands. Further research is warranted to elucidate the underlying molecular mechanisms of YAP1 effect on the clinical behavior of SMEC. Understanding the role of YAP1 in MEC may open up new avenues for personalized treatment approaches and improved patient management. Further investigation of similar extra-salivary morphologies, such as thyroid SMECE, is needed. The findings of this study heighten the necessity of integrating molecular alterations as a fundamental criterion for grading MECs. This advancement aims to enable a more precise, comprehensive, and clinically relevant approach to stratifying MECs. This evolution in grading methodology is essential, especially considering that most studies on MEC published before 2014 often featured conflicting grading systems and misclassified non-MEC cases as high-grade MEC.

Author Contributions

All contributed equally. For more information on using the automatic grader, please conctact the corresponding author.

Informed Consent Statement

All cases were anonymous.

Institutional Review Board Approval

Obtained.

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19424396.2023.2283225

Additional information

Funding

This research received no external funding

Notes on contributors

Sherif Y. El-Nagdy

Sherif Y. El-Nagdy is a Professor of Oral Pathology at Mansoura University, Egypt, and Horus University in Egypt. He specializes in diagnosing salivary gland carcinomas and odontogenic lesions, with a specific focus on advancing understanding regarding oncogenetic changes of odontogenic lesions in non-odontogenic topographies.

Pushparaja Shetty

Pushparaja Shetty is a Professor in the Department of Oral Pathology and Microbiology at Nitte University, holding both an MDS and a PhD. His research interests center around exploring atypical morphological presentations of oral diseases, aiming to improve the recognition and investigation of rare lesions.

Bacem Abdullah

Basem Abdullah is an Assistant Lecturer in Oral Pathology while pursuing a PhD in the same domain. His research primarily delves into molecular pathology and bioinformatics, aiming to integrate advanced technological approaches into proposing diagnostic algorithms and offering biomedical software applications.

Shahram Sabeti

Shahram Sabeti is a Board-certified Clinical Anatomical Pathologist and an Associate Professor of Pathology at Shahid Beheshti University of Medical Sciences in Iran. His research interests encompass head and neck anatomic pathology and molecular pathology, with a focus on advancing diagnostic techniques and genetic understanding in these areas.