Introduction
Salivary glands tumours are a histologically heterogeneous group of lesions [1]. Pleomorphic adenoma (PA) is the most common benign salivary gland tumour. It occurs slightly more often in women between 40 and 50 years of age. Most cases are recognised in parotid glands. Among risk factors, radiation exposure seems most significant [2–5]. Superficial or total parotidectomy with facial nerve preservation are the best treatment options [6]. The risk of recurrence of PA amounts to nearly 3% and may be associated with margin-positive resection and younger age. Approximately 5–15% of pleomorphic adenomas may transform to carcinoma ex-pleomorphic adenomas (Ca ex PA), an aggressive malignancy [5, 7]. The malignant component of Ca ex PA is most frequently adenocarcinoma not otherwise specified [8], followed by myoepithelial carcinoma (MECA) [9]. In the study by Zbären et al. only 21% of malignant salivary neoplasms led to clinical symptoms [10]. Therefore, the differentiation between recurrent PA and malignancy can be a huge challenge and lead to misdiagnosis. In the available literature, many cases with false diagnosis of Ca ex PA as PA were recognized [8, 11–15]. Xu et al. reported the misdiagnosis of MECA ex PA with the benign myoepithelioma [12].
Myoepithelial carcinoma occurs very infrequently. It is estimated that less than 2% of all cases are confirmed. Apparently, the number is higher because of the difficulty in proper diagnosis [12, 16]. It has been proven that no predilection occurs in sexes [16–18]. The prognosis for patients with MECA is poor and related to early local and distant metastases [12, 16, 19].
Some researchers proved that MECA de novo is characterized by worse outcomes than MECA ex PA [18, 20]. At the same time, other studies suggest that MECA ex PA is characterized by higher aggressiveness than de novo lesions, even though it is intracapsular or of minimal invasiveness [16, 21, 22]. Additionally, MECA ex PA are detected more commonly than de novo le- sions [22]. The major issue is a proper diagnosis because MECA may mimic other lesions, especially PA. This leads to frequent misdiagnoses and delays in appropriate treatment and recovery [12].
Though salivary gland cancers occur very rarely, they are characterized by considerable aggressiveness and mortality. Nowadays, we are facing a continual lack of prognostic as well as predictive markers that would enable more personalized treatment and improve the outcomes.
The aim of this study was to identify genetic rearrangements that might be responsible for dynamic MECA progression in patients with primary radical PA excision.
Material and methods
The study was conducted in accordance with national guidelines and regulations. The Bioethics Committee at the Medical University of Warsaw approved the protocol of the study (No. AKBE/175/2021). The tissue material was collected from 2 patients treated in the tertiary Otorhinolaryngology, Head and Neck Surgery Department. The material consisted of 4 formalin-fixed, paraffin-embedded blocks with primary and recurrent tumour tissues. The next-generation sequencing (NGS) was performed in both retrieved PA and MECA samples. DNA was isolated with E.Z.N.A. FFPE DNA Kit (Omega Bio-Tek), and for each sample 100 ng were converted to genomic libraries using KAPA HyperPlus Kit (Roche). Libraries were then enriched using SeqCap EZ probes (Roche), capturing 8.4 Mb and ~1500 cancer-associated genes and sequenced on Illumina HiSeq1500 instrument using 2 × 100 bp reads. Mean coverage was in the range 174–215×, and ge20 was > 91 for all samples. Raw sequencing data processing was done according to Broad Institute recommendations [23] and involved quality control of FASTQ files, adapter trimming and low-quality read removal using Trimmomatic [24], read mapping to hg19 genome using BWA-MEM [25], duplication removal, local realignment and quality recalibration using GATK and Picard, and variant calling using HaplotypeCaller and Mutect2. Downstream analysis involved identification of small-scale mutations, as well as focal and chromosomal arm-level copy number changes and was conducted as described previously [26]. Briefly, common variants were filtered out using public and internal databases, and the remaining, rare variants were classified with the aid of bioinformatics predictors, public databases, and published data. Finally, copy-number variations (CNV) were identified with CNVkit 0.9.5 [27] and copy-neutral losses-of-heterozygosity were identified using an in-house script.
Case reports
Case 1 concerned an 84-year-old woman, who had 30 years history of right submandibular gland tumour. Case 2 was a 63-year-old female, who had a tumour in the deep part of the left parotid gland, progressing for 10 years. Initially, the radical surgical resection was performed in both cases and the PA was confirmed. Unfortunately, both patients after 6 and 9 months, respectively, had the regrowth of the lesion and the PA recurrences were suspected. However, after the revision surgery and resection, histopathological examination showed multifocal MECA ex PA. Histopathological re-assessments of primary lesions were performed to exclude the possibility of misdiagnosis. The re-analysis, however, did not reveal any malignancy in the primary tumour. The presence of PA cells was confirmed. A rapid progression of malignancies after PA excision encouraged us to analyse both PAs’ genetic materials and the secondary malignancy to detect genetic patterns that may be responsible for the development of multifocal myoepithelial carcinomas. Additional radiotherapy was administered in the first patient, and chemoradiotherapy in the second case. The overall survival of the first patient was 3 years. The second patient died after one year, due to disease progression. The comprehensive description of both patients’ clinical symptoms, treatment, and histopathology analysis was previously presented by Szablewska et al. [28]. A summary of patients’ data is collected in Table 1.
Table 1
Patients’ characteristics
Results
Copy number variation
Analysis of tumour samples revealed multiple CNVs on focal and chromosomal-arm levels is presented in Table 2. The patterns were different for each patient, but aberrations remained mostly stable in PA and MECA tissues. Specifically, in Patient 1, FGFR1 and CDKN2A were affected by amplification and homozygous deletion, respectively.
Table 2
Copy number alterations in patients’ samples
Somatic mutations
Among notable genetic aberrations, FGFR2 mutation was discovered in both cases. In Patient 1’s PA and MECA samples the variant allele frequency (VAF) of pathogenic p. Pro253Arg/c.758 C > G variant was nearly 100% and was related to copy-neutral duplication of chromosome 10. This mutation was accompanied by FGFR1 and IGF1R amplifications and elevated copy numbers of EGFR, MET, ERBB2, and ERBB3, suggesting dependence of cancer cells to receptor tyrosine kinase signalling. Furthermore, a variant of unknown significance in the APC gene was identified in both samples F while somatic mutations of KDM6A and ZFHX3 were associated only with PA. In Patient 2, VAF of pathogenic p.Leu550Phe/c.1648C > T variant in FGFR2 was over 45% in the samples of PA and MECA. Selected variants identified by NGS in our study are collected in Table 3.
Table 3
Selected variants identified by next-generation sequencing
Discussion
Due to the histological heterogeneity of salivary gland tumours and inconclusive data concerning prognostic factors, current research focuses on specific genetic alterations. It is believed that a better understanding of carcinogenesis in these tumours may contribute to the improvement and more individual approach to treatment.
The most commonly occurring genetic changes in benign PA are associated with the PA gene 1(PLAG1) and the high-mobility group AT-hook 2 (HMGA2) genes [29]. The fusions of PLAG1 and HMGA2 constitute diagnostic biomarkers, enabling differentiation of PA from other salivary lesions. These are also important markers to identify whether Ca ex PA developed from PA or de novo. However, translocations in these genes were described also in MECA de novo [30]. According to researchers, TGFBR3-PLAG1 fusion is unique to MECA. EWSR1-ATF1 and MSN-ALK were detected only in de novo lesions. The FGFR1-PLAG-1 was primarily considered characteristic only for MECA ex Pa [19]. However, Freiberger et al. confirmed this fusion also in PA, Ca ex PA, and MECA de novo [31]. The most commonly described genetic rearrangements in MECA are EWSR, PIK3CA, and HRAS mutations [30].
Our knowledge about genetic changes in salivary gland tumours is evolving rapidly, but the results are not conclusive. The genetic alterations that were identified unique for benign lesions have been confirmed also in malignant tissue. Therefore, there is still a need for reliable differential indicators for the improvement of the diagnosis and the optimal therapy.
In the available literature, there are not many studies about genetic sporadic mutations in salivary gland tumours, especially in PA and MECA. Cormier et al. described the history of a patient, in whom metastatic MECA ex PA developed in a short period after superficial parotidectomy performed due to PA. The re-histopathological examination showed MECA misdiagnosed as PA. The genetic analysis confirmed TERT promoter mutation [11]. Currently, the meaning of this finding remains unknown. The instance proves the ongoing difficulty in differentiation in salivary gland tumours. In line with our research, Dalin et al. discovered FGFR2 mutation in a patient who developed MECA ex PA. Additionally, they also identified this alteration in the case of MECA de novo. Both tumours (MECA ex PA and MECA de novo) were associated with local recurrence and poor patients’ outcomes [19]. These findings suggest a potential association of the FGFR2 mutation with tumour development and progression. Fibroblast growth factors (FGFs) through their receptors (FGFRs) regulate proliferation, migration, differentiation, and survival in normal cells. In cancer progression, FGFs are involved in invasion and angiogenesis [32–35]. The family of FGFR is engaged in the development of a wide range of cancers, unfortunately in most cases related with poor prognosis [36, 37]. Currently, FGFR2 inhibitors are applied in the therapy in advanced cancer stages, or to patients with contraindications to surgery, and when standard systemic regiments have failed [38, 39]. Erdafitinib and Pemigatinib have been registered for urothelial cancer and cholangiocarcinoma, respectively [37, 40].
Our results are consistent with the findings of Dalin et al. and indicate that the FGFR2 mutation may be related to MECA ex PA salivary gland development and progression. These data highlight the importance of further analysis of other cases to confirm the accuracy and propose optional treatment to improve patients’ outcomes.
Other genetic aberrations of interest and with the potential for further exploration were identified in a single sample of MECA ex PA. The PIK3C2A gene and encoded proteins play a major role in the autophagy process [41]. The CDKN2A gene is located on chromosome 9p21 and encodes p14 and p16 suppressor genes, involved in the activation of p53 and Rb. Both proteins are engaged in regulation of the cell’s cycle. In human cancers with high frequency of genetic and epigenetic alterations in the CDKN2A gene, the strategies of modulation of the alteration for prevention or therapy are promising. Another identified suppressor gene, APC2, is involved in WNT-β catenin pathways and therefore in cell adhesion. Mutations of APC gene are mostly associated with colorectal cancer and familial adenomatous polyposis but occur also in other types of cancers [42]. The encoded protein prevents the uncontrolled growth of cells and controls the epithelial-mesenchymal transition.
The current direction of the research promotes the role of gene copy alterations to be responsible for malignant transformation of PA [19]. Additionally, the changes are usually associated with poor prognosis of MECA ex PA and development of metastasis. Consistently, we also detected 5p, as well as 8p and 8q amplification, not only in MECA, but also in primary PA tissue in our patients. In our study, we also found deletion of 3p22.1-p13 in both PA and MECA, but in a single patient. On this locus CTNNB1 gene is encoded, crucial for synthesis of b-catenin [43, 44]. The protein is activated in WNT pathways, involved in the regulation of cell migration, polarity, differentiation, and function. Molecular abnormalities in CTNNB1 have so far been confirmed in different types of cancers, such as colon, hepatocellular, and breast cancer [45]. In salivary gland tumour, the loss of 3p22.2–p14.3 was described by Mariano et al. in a patient who suffered from metastatic PA [46]. The most intriguing, however, are the findings by Persson et al., who suggested contribution of deletions of 5q23.2-q31.2, gains of 8q12.1 (PLAG1), and amplification of 17 chromosome, which encodes the ERBB2 gene to malignant transformation of PA to carcinoma [47]. Most of these genetic alterations were also detected in samples from our patients.
Identification of specific molecular patterns in salivary gland lesions can pose considerable diagnostic and treatment improvement. Genetic rearrangement appears to be very useful for proper diagnosis. Further studies are needed to reveal genetic patterns in the development and progression of salivary gland tumours.
Our research included the material from only 2 patients and therefore does not allow us to draw strong conclusions. However, the development of MECA in a short time after radical PA excision is quite extraordinary and may be related to some biological conditions. It is possible that mutations in FGFR2 could accelerate the tumour transformation and progression. We believe that our results may contribute to the most accurate detection of genetic alterations in salivary gland tumours and improvement of the diagnosis and treatment in the future. Additionally, identification of specific mutations in benign salivary gland lesions predisposing to malignant transformation will improve patients’ oncological supervision and prognosis.
Conclusions
Aberrations of the FGFR2 gene, identified in primary PA and MECA ex PA samples of both our patients, may be responsible for the malignant transformation and disease progression. Further studies are encouraged to confirm the relevance of these findings. The therapy option with FGFR2 inhibitors may be considered in advanced or metastatic MECA ex PA with confirmed FGFR2 mutation.
Next-generation sequencing analysis contributes to improving knowledge on the development and progression of salivary gland tumours. Identification of reliable markers for diagnosis, prognosis, and individual treatment is urgently needed in salivary gland tumours to improve outcomes.
