Branchioma with a nested/organoid morphology: molecular profiling of a distinctive potentially misleading variant and reappraisal of potential relationship to CD34-positive/Rb1-deficient tumors of the neck

The tissue specimen was fixed in formalin, embedded in paraffin, and processed routinely for conventional microscopy using hematoxylin and eosin staining.

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. Antigen 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.

A 78-year-old man without a previous medical history of malignancy presented with a mass in the left supraclavicular area which was surgically excised. The case was sent as a consultation to our department with a diagnosis of metastatic neuroendocrine tumor of unknown origin. Computed tomography of the neck showed a homogenous mass within the left supraclavicular area near the upper margin of the clavicle (Fig. 1A). Grossly, the tumor presented as an oval, well-circumscribed nodule surrounded by a thin capsule measuring 6 × 5 × 5 cm. Cut sections revealed a solid homogenous mass, white to yellowish in color with small cystic areas, but no hemorrhage or necrosis (Fig. 1B). The patient is alive without evidence of recurrence or metastasis 8 months post-surgery.

Histologically, the tumor was well-circumscribed (Fig. 2A), and displayed two components: the first component, which corresponded to classical triphasic pattern of branchioma, showed spindle cells (20% of the tumor), and adipose tissue (5%) entrapping scattered squamoid and cystic epithelial aggregates. This classical component merged with a predominant cellular epithelial component with nested/organoid (neuroendocrine tumor-like) features comprising 75% of the whole mass (Fig. 2B). The spindle cell proliferation was arranged in a storiform, vague fascicular, or haphazard architecture and was localized rather at the periphery of the lesion (Fig. 2A, B). The cells had oval nuclei with light eosinophilic plump cytoplasm, and in some areas, they formed solid plump nests with interspersed fatty tissue (Fig. 2C). Tumor cells were admixed with scant lymphocytes which focally formed lymphoid follicles (not shown). The epithelial component grew either in a cystic formation layered by flattened bilayered epithelial cells (less than 5%) or solid/neuroendocrine tumor-like (more than 95%) architecture (Fig. 2D). The solid areas were composed of middle-sized monomorphic epithelioid cells with regular round to oval nuclei with “salt and pepper” chromatin, distinct nucleoli, and scant pink cytoplasm. These nested/organoid (neuroendocrine-like) areas showed sheets, nests, trabeculae, pseudorosettes/pseudoglandular or microglandular, and interanastomosing patterns (Fig. 2D–G). The anastomosing structures were surrounded by artificially created clefts from the surrounding loosely cellular and mildly vascularized stroma (Fig. 2E, F). Some parts of the tumor were more haphazard with small ducts composed of monomorphic cells with pink cytoplasm, rounded nuclei, and luminal formations sometimes containing dense eosinophilic homogenous material (Fig. 2G). The last component was adipose tissue which was haphazardly dispersed and admixed with the other two components (Fig. 2B, C).

Immunohistochemically, both spindled and epithelial components were strongly positive for AE1-3 and OSCAR (Fig. 3A). CD34 and SMA were biphasic and highlighted the spindle cell component (Fig. 3B, C). The p63 was expressed focally in spindle cells and stained the epithelial component (Fig. 3D). The nested/organoid (neuroendocrine-like) morphology mirrored the epithelial component immunophenotype. S100 protein was positive in fatty tissue only (not shown). The androgen receptor (AR) was expressed in 40% of tumor cells, predominantly in spindle and solid epithelial components (Fig. 3E). Neuroendocrine markers synaptophysin, chromogranin, INSM1, and CD56 were negative. MSH6 (stained due to the detected molecular alteration, see molecular findings below) was retained in tumor cells. Proliferative activity was low (the MIB1 index in hot-spots reached up to 5%). All components (epithelial, spindle cells, and adipocytes) showed loss of RB1 expression with positive internal control in lymphatic cells and/or endothelium Fig. 3F.

The tumor was tested for RB1 gene aneuploidy by FISH and underwent molecular genetic testing by TrueSight Illumina Oncology 500 NGS panel. Five pathogenic/likely pathogenic mutations were detected by Illumina TS500, including MSH6 c.3261dup p.(Phe1088LeufsTer5), MSH6 c.3202C>T p.(Arg1068Ter), PTEN c.385G>A p.(Gly129Arg), PTEN c.697C>T p.(Arg233Ter), and KRAS c.437C>T p.(Ala146Val), while no aberration of RB1 gene was found. From one hundred randomly selected nonoverlapping nuclei, 32 nuclei showed monoallelic RB1 gene loss which was below the cut-off of >45% of the counted cells defined for RB1 deletion in this study.