MYB-NFIB Translocation by FISH in Adenoid Cystic Carcinoma of the Head and Neck in Nigerian Patients: A Preliminary Report

Abstract

Adenoid cystic carcinoma (AdCC) is a relatively rare malignancy of head and neck sites such as the salivary glands, lacrimal gland, sinonasal region, and pharynx and may arise in other exocrine glands. The oncologic event in AdCC is the translocation between MYB proto-oncogene transcription factor (MYB) and nuclear factor I/B (NFIB) resulting in t(6;9)(q22-23;p23-24). We carried out a preliminary evaluation of MYB-NFIB translocation by fluorescence in-situ hybridization on seven archived formalin-fixed paraffin-embedded tissues of AdCC of Nigerian patients and its clinicopathologic features. Only 3 of the 7 cases were successfully hybridized, all featuring MYB-NFIB translocations with a range of 14.7–83.3% of translocated cells in 60 cells examined. The 3 translocation positive cases were located in the maxillary sinus, buccal mucosa and parotid. Their morphologic appearances were cribriform-solid (1) & cribriform (2) and classified as grades III (1) & I (2), respectively. These patients may potentially benefit from MYB-targeted anti-neoplastic therapy.

Introduction

Adenoid cystic carcinoma (AdCC) is the second most common salivary gland malignant neoplasm after mucoepidermoid carcinoma [1]. AdCC may arise in other exocrine glands of the tracheobronchial tree, lacrimal gland, breast, cervix, vulva, and prostate. AdCCs of the head and neck are characterized by protracted clinic course, extensive perineural invasion, metastasis, and local and distant recurrences [1–5]. Histologically, AdCC is a biphasic tumor of ductal and myoepithelial cells arranged in heterogeneous morphologic growth patterns: cribriform, tubular, and solid in that order of frequency. The solid type (containing > 30% of solid pattern) of AdCC is associated with increased risk of recurrence and poor prognosis [6].

The genetic hallmark and key oncologic event in AdCC is the balanced translocation between MYB proto-oncogene transcription factor (MYB) and nuclear factor I/B (NFIB) resulting in t(6;9)(q22-23;p23-24), associated with overexpression of transcriptionally active 5’ end of MYB and a subsequent increase in protein expression detected by immunohistochemistry [7–9]. MYBL1 (another member of the MYB family) is involved in the oncogenesis of AdCC in patients that do not harbor the MYB-NFIB fusion [10, 11]. MYB promotes cellular proliferation and metastasis in AdCC and may therefore be a potential therapeutic target [12]. MYB-NFIB fusion has not been reported in any other salivary gland neoplasm [7, 13, 14]. To our knowledge, no detailed molecular characterization of AdCC of the head and neck has been described in the Nigerian population. Thus, the aim of this study is to carry out a preliminary evaluation of MYB-NFIB translocation by fluorescence in-situ hybridization on seven archived formalin fixed paraffin embedded tissue of AdCC of Nigerian patients and review their clinicopathologic features.

Materials and Methods

The pathology files of the Department of Oral and Maxillofacial Surgery and Oral Pathology, Faculty of Dentistry, Obafemi Awolowo University, Ile-Ife, Nigeria and the Department of Morbid Anatomy and Forensic Medicine, Obafemi Awolowo University Teaching Hospitals Complex, were searched for the diagnosis of adenoid cystic carcinoma of the salivary gland from 2017 to 2019. Only cases with available material for molecular work-up were included in this study. The cases were reviewed for grading according to the criteria of Szanto et al.: grade I, tumors with tubular and cribriform areas but without solid components; grade II, cribriform or cribriform/tubular with less than 30% of solid areas; and grade III, tumors with a solid pattern > 30%, and the presence of perineural invasion [6]. The following clinical information was retrieved: age at diagnosis, gender and anatomic site. Fluorescence in-situ hybridization for MYB-NFIB translocation was performed on all cases retrieved at the University of Pittsburgh, Pennsylvania, as previously described [15]. The cut-off for a positive MYB-NFIB translocation was 10% of translocated cells in 60 cells evaluated. This study was exempt from review by the Institutional Review Board of A.T. Still University and Obafemi Awolowo University.

Results

Clinical Characteristics of Cohort

A total of 7 adenoid cystic carcinoma of salivary gland cases were retrieved. Patient demographics are presented in Table 1. There were 4 (57%) females and 3 (43%) males, with ages ranging from 39 to 52 years old (median, 41 years). The tumor sites were the palate in 2 cases and a case each from the maxillary sinus, submandibular gland, parotid gland, nasal cavity, and buccal mucosa.

Table 1.

The clinical, morphologic and molecular features of adenoid cystic carcinoma

Clinical features				Morphologic			Molecular
Case	Age	Gender	Location	Subtype	PNI	Grade	MYB-NFIB FISH (% of cells translocated in 60 cells evaluated)
1	39	M	Maxillary sinus	Cribriform & Solid	No	III	Positive (83.3%)
2	39	F	Buccal mucosa	Cribriform	Yes	I	Positive (14.7%)
3	52	F	Parotid	Cribriform	No	I	Positive (56.7%)
4	40	F	Palate	Cribriform & Solid	No	III	Hybridization failed x2
5	41	M	Submandibular gland	Cribriform & tubular	Yes	I	Hybridization failed x2
6	50	M	Nasal	Cribriform & Solid	Yes	II	Hybridization failed x2
7	50	F	Palate	Cribriform & tubular	Yes	I	Hybridization failed x2

M male, F female, PNI perineural invasion

Morphologic and Molecular Characteristics

All seven cases exhibited some areas of a cribriform pattern. Tumors with any solid pattern were assigned a higher grade. As such, four cases (Cases 2, 3, 5 and 7) were categorized as grade I for their cribriform or cribriform/tubular morphology with no solid pattern (Fig. 1b, c and e, g). Three cases (Cases 1, 4 and 6) were associated with a solid morphology. Case 6 had < 30% of solid morphology, and was assigned Grade II (Fig. 1f). The two other cases (Cases 1 and 4) exhibited > 30% of solid pattern and were categorized as grade III (Fig. 1a and d). (Table 1). Perineural invasion was observed in 4 of the 7 cases, all Grades I or II (Table 1).

Fig. 1.

Histopathology of Cases 1–7. 39-year-old male, MYB-NFIB fusion positive adenoid cystic carcinoma of the maxillary sinus showing cribriform and solid morphology (a), 39-year-old female, MYB-NFIB fusion positive adenoid cystic carcinoma of the buccal mucosa showing cribriform morphology (b), 52-year-old female, MYB-NFIB fusion positive adenoid cystic carcinoma of the parotid gland showing cribriform morphology (c), 40-year-old female, adenoid cystic carcinoma of the palate showing cribriform and solid morphology (d), 41-year-old male, adenoid cystic carcinoma of the submandibular gland showing cribriform and tubular morphology (e), 50-year-old male, adenoid cystic carcinoma of the nasal region showing cribriform and solid morphology (f), 50-year-old female, adenoid cystic carcinoma of the palate showing cribriform and tubular morphology (g)

Of these 7 cases, FISH for MYB-NFIB translocations were interpretable in only 3 cases (Cases 1–3) (Fig. 1a, b and c); and MYB-NFIB translocations were detected in all 3 cases (100%) with a range of 14.7–83.3% of translocated cells in 60 cells examined (Fig. 2) (Table 1). The remaining 4 cases failed to hybridize on two attempts.

Fig. 2.

Fluorescence in-situ hybridization for MYB-NFIB in adenoid cystic carcinomas. Case 1 (a), Case 3 (b). Orange signal represents MYB/6q22-24 and green signal represents NFIB/9p24.1, overlap of the NFIB and MYB probes is indicative of a fusion-positivity

Case 1

A 39-year old male patient presented in November 2018 with a history of a maxillary mass of 18 months duration. An ulcerated palatal mass associated with loss of vision on the right eye and right nasal obstruction was observed 6 months prior to presentation. Imaging revealed an osteolytic mass in right maxillary antrum, nasal cavity and periorbital structure. An incisional biopsy was performed. The last follow up was 13 months after presentation, alive with disease.

Case 2

A 39-year old female patient presented in May 2019 with a 1-year history of a left cheek mass reported to have progressively increased in size. Clinically, it was a firm buccal mucosa swelling with cauliflower-like pattern located in the posterior retromolar region and measured 3 cm in the widest diameter. Surgical excision was performed. The last follow up was 10 months after presentation with no evidence of disease.

Case 3

A 52-year old female patient presented in July 2019 with a 12-year history of a recurrent mid-facial mass of 10 cm in size associated with superolateral displacement of the globe. Surgery had been performed 12 years prior. The mass was reported to have progressively increased in size for the past 7 years prior to presentation. Imaging revealed an osteolytic mass in the maxilla with skull base involvement. An incisional biopsy was performed. The last follow up was a month after presentation, alive with disease.

Discussion

Several salivary gland neoplasms have been associated with recurrent chromosomal translocations, including pleomorphic adenoma (PLAG1, HMGA2) [16, 17], mucoepidermoid carcinoma (CRTC1/CRTC3-MAML2) [18], polymorphous adenocarcinoma/cribriform adenocarcinoma (PRKD1) [19], hyalinizing clear cell carcinoma (EWSR1-ATF1) [20], intraductal carcinoma of salivary glands (NCOA4/TRIM27-RET) [21], secretory carcinoma of salivary glands (ETV6) [22], and microsecretory adenocarcinoma (MEF2C-SS18) [23]. In 1994, Nordkvist et al. first identified the t(6;9)(q22-23;p23-24) in adenoid cystic carcinoma (AdCC) [24]. Later in 2009, the group reported that the translocation leads to the fusion of MYB and NFIB [7].

A recent systematic review and meta-analysis evaluating MYB-NFIB in head and neck AdCC from 1986 to 2018 identified the prevalence of MYB-NFIB in head and neck AdCC by all molecular techniques range from 16 to 100% [25]. This review included 23 studies from 9 countries that utilized FISH molecular analysis for evaluating MYB-NFIB in head and neck AdCC, with cases ranging from 4 to 102 from each study (making a total of 741 cases). According to the review, the prevalence of MYB-NFIB by FISH in head and neck AdCC ranged from 28 to 100% [25]. In our study, all 3 interpretable cases were positive for MYB-NFIB.

The use of FISH and reverse transcriptase-PCR (RT-PCR) are the main analytical techniques employed to detect MYB-NFIB fusion in AdCC [25]. FISH assays are based on DNA probes that are specific to a particular DNA and can predict the potential for fusion transcript; however, they do not provide information regarding specific fusion breakpoints. FISH can be carried out on formalin-fixed paraffin-embedded tissue (FFPET). RT-PCR are RNA-based assays that can detect gene fusions and also provide information regarding the fusion variants; however, this technique does not lend itself to FFPET [26]. In this retrospective study, MYB-NFIB fusion was evaluated by FISH as all cases in this study were archival FFPET. In our study, only 3 of the 7 cases analyzed by FISH for MYB-NFIB translocation were interpretable, showing the importance of proper fixation and storage of FFPET for molecular analysis by FISH. Degradation of DNA leads to failure of the FISH test.

Recent studies have shown that MYB gene product promotes cellular proliferation and metastasis in salivary AdCC and may therefore be a rational therapeutic target in the management of this tumor [12]. Other studies have also shown that AdCC with MYB translocation shows a trend towards higher local relapse rates, and tumors that lack this translocation may be associated with a better prognosis [14, 27]. MYB-NFIB fusion has not been reported in non-AdCC of the head and neck tumors [7, 13, 14], and this fusion leads to an overexpression of the MYB protein, which can be detected by immunohistochemistry [7]. However, MYB immunohistochemical (IHC) stain have been shown to be discordant with the gold standard FISH test [13, 28]. There are reports of MYB IHC expression in AdCC that are negative for MYB-NFIB fusion, suggesting that additional mechanisms maybe involved in the expression of MYB and lack of expression of MYB IHC in MYB-NFIB fusion-positive AdCC [13, 28]. Also MYB IHC has been shown to be expressed in other non-AdCC salivary gland tumors such as pleomorphic adenoma, basal cell adenoma, myoepithelioma, polymorphous adenocarcinoma, myoepithelial carcinoma, mucoepidermoid carcinoma, salivary duct carcinomas and in other non-salivary gland tumors such as lymphoma, melanoma, head and neck squamous cell carcinoma, etc [14, 29]. As such MYB IHC expression should be used with caution when making a diagnosis of AdCC. Moreover, MYB translocation may be helpful in making a diagnosis of AdCC of the head and neck.

In conclusion, we report the first series of MYB-NFIB fusion positive AdCC by FISH in Nigerian patients. Before now studies on MYB-NFIB fusion positive AdCC were from Western countries. MYB-NFIB translocations were identified in all successfully hybridized AdCC. The mainstay of management still remains surgical resection and/ or radiotherapy. Patients from this population stand to benefit from potentially MYB-targeted anti-neoplastic therapy such as RNAi and miRNA targeting MYB, Dovitinib (an FGFR inhibitor, FGF2 ligand is a downstream target of MYB), Vorinostat, and Chidamide (a HDAC inhibitor that drives the down-regulation of MYB).

Funding

All authors declare that there are no financial conflicts associated with this study and that the funding source has no role in conceiving and performing the study. No funding to declare.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no potential conflicts of interest.

Ethical Approval

This study was exempt from review by the Institutional Review Board of A.T. Still University and Obafemi Awolowo University as no human or animal subject were utilized in this study. Formal written informed consent was not required as this study did not utilize human subjects.