Introduction

Branchiomas are rare benign lower neck tumors with an adult male predominance [1]. In the past, the tumor received several names trying to reflect its most likely histogenetic origin, in particular thymic anlage tumor, branchial anlage mixed tumor, or ectopic hamartomatous thymoma. However, it became evident that none of these names really describes the true nature of this lesion. In order to avoid taxonomic confusion, a descriptive term reflecting the entodermal and mesodermal origin of the tumor was introduced as a common basis for tumors arising from or recapitulating the branchial apparatus. The English literature contains 87 cases of this entity with four cases showing a concurrent malignant histology [2,3,4,5,6,7].

Branchioma (biphenotypic branchioma) is composed of an admixture of adipocytic tissue, spindled cells, and epithelial nests, the latter potentially arranged in different architectural [8]. Epithelial and spindle cells are immunoreactive for pancytokeratins, p63 and p40, but only spindle cells are positive for smooth muscle actin and CD34 [9, 10]. The last components represent delicate spindly cells, usually irregularly interspersed between the previous components, which are negative for cytokeratins and positive for CD34. The frequent expression of AR by the tumor cells has been linked to the male predominance of branchiomas [11]. In a recent case report, we published a case of branchioma with neuroendocrine-like tumor morphology showing immunohistochemical loss of Rb1 expression, while FISH showed no alteration in the Rb1 gene [8].

This study specifically aimed to establish the presence of Rb1 alterations in a series of branchiomas, especially set within the context of the differential with spindle cell lipoma and spindle cell predominant trichodiscoma (https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_oncology_500.xlsx.) [12,13,14].

Materials and methods

Histology and immunohistochemistry

For conventional microscopy, tissues were fixed in formalin, routinely processed, embedded in paraffin (FFPE), cut, and stained with hematoxylin and eosin.

For immunohistochemistry, 4-μm-thick sections were cut from paraffin blocks and mounted on positively charged slides (TOMO, Matsunami Glass IND, Osaka, Japan). Sections were processed on a BenchMark ULTRA (Ventana Medical Systems, Tucson, AZ), deparaffinized, and subjected to heat-induced epitope retrieval by immersion in a CC1 solution (pH 8.6) at 95 °C. The primary antibodies used in this study are summarized in Table 1. Visualization was performed using the ultraView Universal DAB Detection Kit (Roche, Tucson, AZ) and ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche, Tucson, AZ). The slides were counterstained with Mayer’s hematoxylin. Appropriate positive controls were employed.

Table 1 Antibodies used for the immunohistochemical study
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Molecular genetic study

Archer FusionPlex assay

The in-house customized version of Archer FusionPlex Sarcoma kit was used to construct a cDNA library for detecting fusion transcripts and point mutations in 88 and 14 genes, respectively. The complete list of genes and mutations covered by this assay has been reported previously [12]. All steps were performed according to the manufacturer’s instructions, and the library was sequenced on an Illumina platform as described previously [13].

Illumina TruSight Oncology 500 assay

The cases were analyzed using the commercially available TruSight Oncology 500 assay from Illumina. This panel can analyze both DNA and RNA. The DNA analysis interrogates 523 genes for single nucleotide variants and indels, and the RNA analysis interrogates 55 genes. The complete list of genes can be found on manufacturer’s website (https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_oncology_500.xlsx).

Briefly, DNA libraries were prepared using the TruSight Oncology 500 Kit (Illumina) according to the manufacturer’s protocol, except for DNA enzymatic fragmentation which was done using KAPA FragKit (KAPA Biosystems, Washington, MA). Sequencing was performed on the NextSeq 550 sequencer (Illumina) following manufacturer’s recommendations. Data analysis (DNA variant filtering and annotation) was performed using the Omnomics NGS analysis software (Euformatics, Finland). Custom variant filter was set up including only non-synonymous variants with coding consequences, read depth greater than 50, benign variants according to the ClinVar database were also excluded [14]. The remaining subset of variants was checked visually, and suspected artefactual variants were excluded.

Detection of Rb1 deletion by FISH

For the detection of Rb1 loss, the probe ZytoLight® SPEC Rb1/13q12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany) was used. The fluorescence in situ hybridization (FISH) procedure was performed as described previously [15].

FISH interpretation

One hundred randomly selected nonoverlapping tumor cell nuclei were evaluated in all analyzed samples. Rb1 gene loss was recorded as the number of cells with loss divided by the total number of cells counted. The test was interpreted as positive if > 45% of the counted nuclei had gene loss (mean + 3 standard deviations in normal non-neoplastic control tissues).

Results

Demographic and clinical features

The clinicopathological data of the 23 branchioma cases are summarized in Table 2. In addition, cases of carcinoma ex branchioma are listed in a separate Table 3. There were 21 males and two females aged between 31 and 80 years, with both median and mean age of 52 years. The tumors were localized in the supraclavicular area (n = 11), suprasternal area (n = 8), chest wall (n = 2), neck (n = 1), and in one case at the junction of the posterior axillary region and the back. The median tumor size was 35 mm (range 10–80 mm). Fourteen patients complained of slowly enlarging mass lasting from 2 months to 30 years (median 15 months). None of the cases showed signs of recurrance or metastasis. All cases were treated by simple excision without adjuvant therapy including cases with carcinoma.

Table 2 Clinical data and follow-up
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Table 3 Clinicopathological and histological data of all carcinomas ex branchioma reported in the English literature (three cases included in the recent study)
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Fifteen patients were alive without evidence of disease with a median 16 months of follow-up (mean 25.8 months, range 0–72 months), and one patient died of unknown reasons 143 months after surgery. Follow-up data were not available in seven cases.

Histological features

Key histological and immunohistochemical features of the branchioma are summarized in Supplementary file 1. Twenty tumors were well-circumscribed classic branchiomas consisting of an admixture of spindle cells, epithelial cells, and adipose tissue with interspersed bland spindle cells. The epithelial component showed either cystic structures layered by biphasic flattened epithelium or solid nests (Fig. 1A, B). Plump spindle cells were arranged in a haphazard, storiform, or fascicular fashion (Fig. 1C, D).

Fig. 1
figure 1

A classic branchioma composed of a combination of epithelial component, cystic structures, and solid epithelial nests with intermingled fatty tissue (A) and spindle cells (B). Plump epithelioid to spindle cells are arranged in haphazard (C), storiform, or fascicular fashion, resembling monophasic synovial sarcoma (D)

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In case 1, a neuroendocrine tumor-like morphology was observed (previously reported [8]), but the tumor did not express any neuroendocrine markers investigated (synaptophysin, chromogranin, and INSM1) (Fig. 2A). Another previously described case (case 5) showed extensive myoid differentiation, mainly in the myxoid areas. The spindle cells were plump and highly eosinophilic without cross striation. They were immunoreactive for smooth muscle actin, but non-reactive for rhabdomyoblastic markers including desmin, myogenin, and MyoD1 (Fig. 2B) [3]. In case 6, multinucleated giant cells were scattered throughout the tumor together with epithelial formations resembling squamous pearls mimicking the sarcomatoid subtype of squamous cell carcinoma (SCC) (Fig. 2C) [3]. In case 7, clear cells were predominant, the epithelial cells were arranged in clear cell cords reminiscent of the structure of the parathyroid gland, but the tumor was negative for parathyroid hormone (Fig. 2D) [16]. Case 8 was composed of spindle cells and epithelioid cells with the appearance of syringomatoid ducts with atypia. A granular cell component was also present (Fig. 2E).

Fig. 2
figure 2

Case 1 was composed of sheets and cords of neuroendocrine-like epithelial cells with peripheral clefting, with intermingled spindle cells (A). Case 5 showed prominent myoid spindle cells without cross striations surrounded by myxoid stroma (B). Case 6 showed prominent squamoid cords nestled in a spindle cell background (C). Case 7 demonstrated clear cell nests or cords reminiscent of parathyroid gland (D). Case 8 had syringomatoid ducts and tumor cells with abundant granular cytoplasm (E)

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In three cases (3/23, 13%), a carcinoma developed in the background of branchioma (Table 3). All cases were previously reported [2,3,4,5,6]. In case 4, the tumor consisted of all three components typical of branchioma, but the epithelial component was arranged in solid sheets formed by squamous cells, cords, or glands with back-to-back glands mimicking ductal breast carcinoma. The tumor cells had granular cytoplasm and mitoses were unevenly dispersed [2, 3] (Fig. 3A, B). Case 13 was composed of different epithelial structures, one with classic solid to cystic structures with squamous lining resembling syringomatoid ducts and the second with adenomatoid cribriform pattern and Roman bridging resembling intraductal carcinoma (IC) of the salivary gland (Fig. 3C, D). A scanned whole slide image of the case is available at https://pathpresenter.net/public/display?token=66640ea4. The tumor cells showed granular cytoplasm and frequent mitoses, including atypical ones [2,3,4]. Case 19 showed a developmental continuum of typical and atypical ductal hyperplasia evolving into IC (Fig. 3E, F) [5, 6]. A scanned whole slide image of the case is available at https://pathpresenter.net/public/display?token=c7cc23dd.

Fig. 3
figure 3

Case 4 showed an epithelial adenomatoid component arranged in solid sheets, along with cords or glands with a back-to-back appearance similar to ductal breast carcinoma (A). Tumor cells had granular cytoplasm; mitoses were unevenly dispersed (B). Case 13 had two different epithelial components: one with classic solid to cystic structures with squamous lining resembling syringomatoid ducts and squamoid nests with multiple mitoses (C). The second component grew in an adenomatoid cribriform pattern with Roman bridging resembling intraductal carcinoma of breast and salivary gland (D). Case 19 demonstrated an epithelial proliferation with cystic changes lined by squamous epithelium (lower right) with a direct transition to solid IC (upper left) (E). The IC part was a continuum of typical and atypical ductal hyperplasia growing directly into IC with Roman bridges, and papillary or solid growth (F)

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Immunohistochemistry

Immunohistochemistry was performed in 18 cases (18/23; 78%) in which tissue blocks were available (all cases without material were classic branchiomas without malignant transformation) and the results are summarized in Table 4.

Table 4 Immunohistochemical results of 20 cases of branchiomas and three cases of carcinomas ex branchioma
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All 15 cases of typical branchioma showed a classical IHC profile. Both the spindle and epithelial components were AE1/3 (Fig. 4A) and p63 (Fig. 4B) positive. Spindle cells additionally expressed CD34 (Fig. 4C) and SMA. AR was positive in 12/15 tested cases with an average of 60% positive tumor nuclei (range 40–85%) (Fig. 4D). Proliferative activity was low with an average Ki-67 (MIB1) proliferation index of 3% (range 0 to 5%). Rb1 immunostaining was performed in 15 cases, of which six cases were completely negative, seven cases showed < 5% nuclear reactivity, and two cases were positive (maximum of 25% positive tumor cell nuclei) (Fig. 4E).

Fig. 4
figure 4

Keratin cocktail AE1/3 (A) and p63 (B) were positive in both the epithelial and spindled cells. CD34 was positive in the spindle cells only (C). Androgen receptor was positive in most cases (D). Loss of tumor cell Rb1 expression (endothelial cells are a positive internal control) (E)

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Three cases of carcinoma ex branchioma were located in the background of branchioma with a typical IHC profile. The carcinoma component showed strong expression of AE1/3 (3/3) (Fig. 5A), while CD34, SMA, SOX10, and S100 protein were negative in the carcinoma component (Fig. 5B, C). The myoepithelial layer around atypical luminal cells of IC was preserved and positive for p63 and SMA, while the latter two antibodies were also positive in the spindle cells of branchioma (Fig. 5D). Cases 4 and 19 showed strong expression of AR in 60% and 100% of the tumor cells, respectively, mainly in the IC part (Fig. 5E). Case 13 was negative for AR. Rb1 was lost in all carcinoma cases (Fig. 5F). Proliferative activity was low, with a Ki-67 (MIB1) proliferation index was 1% in cases 4 and 13, reaching up to 15% in case 19.

Fig. 5
figure 5

Pancytokeratins were strongly immunoreactive in both carcinoma (left) and branchioma (right) components (A). CD34 decorated vascular structures in between carcinoma nests (left) and branchioma part (right) (B). An intact myoepithelial layer around atypical luminal cells was highlighted by SMA (C) and p63 (D). Androgen receptor was seen in 100% of cells of IC, while branchioma in the background was negative or showed only patchy and weak nuclear positivity (E). Rb1 immunoexpression was present only in scattered IC cells (< 5%) while the branchioma (right) was negative (lymphocytes are a positive internal control) (F)

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Molecular testing

The results of targeted NGS and FISH are summarized in Table 5. Eight cases (8/23) had sufficient tissue and/or sufficient DNA quality for testing by NGS and/or FISH.

Table 5 Molecular genetic with immunohistochemical correlation to Rb1 of selected cases
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NGS testing was successful in six cases of classic branchiomas. In case 1, five pathogenic mutations were found, namely two different MSH6 mutations, two different PTEN mutations, and KRAS mutation. In addition, two probably germline variants of unknown significance (VUS) in ARID1A and PDGFRA were detected. Case 2 showed only VUS including BMPR1A and TET2 gene mutations. Case 14 showed a pathogenic BRCA1 mutation. In case 20, a pathogenic mutation of FANCG gene and a VUS (probably germline mutation) of NF1 gene were detected. Case 21 showed three VUS: PHOX2B, XRCC2, and PLCG2, the last two suspicious for germline origin. Finally, in case 22, NGS detected NF2 and NF1 gene mutations and two VUS including NF1 and PTCH1 genes. Tumor mutation burden was low.

In one case of carcinoma ex branchioma (case 19), we performed a microdissection of the IC component and branchioma component and we identified four pathogenic mutations in IC including HRAS, PIK3CA, CHD2, and SLIT2, the latter was suspicious for germline mutation. In addition, six suspicious germline VUS were found, including KMT2C, TET2, PIK3C2B, ABL2, FLT4, and PPARG mutations. In the branchioma part, we found only the identical SLIT2 mutation, and almost the same spectrum of VUS as in IC, while all other pathogenic mutations detected in the IC component were absent in the classical branchioma component.

In two cases (cases 14 and 18, both pure branchiomas), heterozygous deletion of Rb1 was detected by FISH with loss in 92 and 88 nuclei of 100 counted, respectively.

Discussion

Branchiomas are very rare lower neck tumors with only four malignant cases described in the literature [2, 4,5,6, 17]. The molecular genetic background of branchiomas is still poorly understood, with only few manuscripts addressing this issue [6,7,8, 18]. In a letter to the editor, four branchiomas were evaluated using PLAG1 FISH to rule out a possible relationship to pleomorphic adenomas [18]. No case from their study was positive for PLAG1 rearrangements. In another molecular study, two cases of classic branchioma and one case of carcinoma ex branchioma (the latter case is included in the current study as case 19) were evaluated [6], with a pathogenic HRAS c.181C > A (p.Gln61Lys) mutation in the carcinoma component within the current study, two additional pathogenic mutations were identified: PIK3CA c.1624G > A p.(Glu542Lys) and CHD2 c.4173dup p.(Gln1392ThrfsTer17). This case histologically, immunohistochemically, and genetically resembled salivary gland IC of apocrine subtype, in which the same HRAS and PIK3CA mutations have been reported [19, 20]. The final case report described adenocarcinoma arising from branchioma with KRAS and TP53 gene mutations detected [7]. The adenocarcinoma histologically consisted of low-grade and high-grade components, and the authors suggested the potential responsibility of KRAS mutation for the development of adenocarcinoma and TP53 alteration for the transition from low-grade to high-grade histology. The branchioma component did not contain any of the above-mentioned mutations [7].

The neuroendocrine-like branchioma (case 1) demonstrated five pathogenic mutations including MSH6 mutations, two PTEN mutations, and one KRAS alteration [8]. Despite the presence of MSH6 genetic alteration, MSH6 IHC was retained in tumor cells, which can be explained by unaltered antibody epitope for MSH6 in tumor cells with its positive nuclear expression. This study presents three additional cases of branchioma with identified pathogenic gene mutations, including case 14 with BRCA1 c.160C > T p.(Gln54Ter), case 20 with FANCG c.1158del p.(Ser387ProfsTer16), and case 22 with NF2 c.1396C > T p.(Arg466Ter) and NF1 c.1765C > T p.(Gln589Ter). Despite the histological distinctness of branchiomas, the spectrum of altered genetic pathways is molecularly quite heterogeneous.

Two cases from our cohort showed Rb1 gene heterozygous deletion by FISH. This finding, together with the neck location, loss of Rb1 immunoexpression, reactivity with CD34, and a spindle cell morphologic component, places branchiomas in a differential diagnosis with spindle cell predominant trichodiscoma and spindle cell lipoma [21,22,23,24,25]. An increasing number of tumor entities, originally thought to be completely indolent with no carcinogenic potential, are now being reclassified based on the finding of cancer-causing genetic mutations in a subset of them. The above discussed findings suggest that branchiomas might also represent such a precursor lesion, as carcinoma may occasionally develop within them. In this regard, they seem somewhat similar to IC ex sclerosing polycystic adenoma of salivary glands, which shares a similar molecular genetic features with alterations in the PI3K-AKT pathway as seen in one of the carcinoma ex branchioma cases [26,27,28].

When evaluating a lateral neck mass suspicious for branchioma, a spectrum of spindle cell neoplasms with cytokeratin immunoreactivity and potentially malignant behavior should be excluded. First, metastatic poorly differentiated SCC is at the forefront of the differential diagnosis. Poorly differentiated SCCs may develop a spindle cell phenotype, are often at least focally positive for pancytokeratin, p63/p40, and CK5/6, and are usually highly mitotically active. Furthermore, metastatic SCC from the oropharyngeal region may be p16 positive as a part of HPV-associated malignancies. However, in a subset of oropharyngeal SCC, Rb1 IHC loss has been reported and associated with better disease-free survival [29]. These tumors had retained p16 immunoexpression despite HPV negativity by RNA in situ hybridization. Synovial sarcoma (SS), biphasic or monophasic, is a relatively common mimic of branchioma, expressing pancytokeratin and showing both epithelial and spindle cell morphologies. SMA is positive in almost half of SS and CD34 may be present in a small subset of monophasic SS [30]. The panel of IHC markers used for further evaluation should include SS18-SSX/SS18 antibodies [31], which were not reported in branchiomas.

Another mimic is solitary fibrous tumor (SFT) which may display a variable fatty component (lipomatous subtype) and hence mimic branchioma [32]. SFT is also positive for CD34, but negative for various cytokeratin with a strong nuclear STAT6 expression by IHC and usually retained RB1 expression. Alternatively, NAB2::STAT6 fusion can be demonstrated by molecular methods [33]. Recently, two groups have described mesenchymal-epithelial transdifferentation of SFT with well-developed epithelial cysts in the background of spindle cell proliferation and strong pancytokeratin immunoexpression [34, 35]. Thus, the demonstration of STAT6 alterations represents the most useful approach in diagnosing these tumors.

Herein, we performed the largest molecular genetic study of 23 cases of branchioma, with three cases of carcinoma arising within branchioma. These three cases had cribrifrom to solid morphology with histological resemblance to salivary IC of apocrine subtype. Five cases in our cohort showed molecular genetic alterations in different molecular genetic pathways. Eighteen cases showed loss of Rb1, and in two of these, there was a heterozygous deletion of Rb1 gene. These findings broaden the spectrum of lateral neck lesions with Rb1 IHC loss and 13q/Rb1 family of tumors.