PMC5283962_01
The clinicopathological appearance of the adrenal composite pheochromocytoma. Computed tomography revealed a right adrenal tumor which had a heterogeneous internal structure on a plain image (A) and was inhomogeneously enhanced with contrast medium (B). The tumor exhibited high signal intensities on T2-weighted magnetic resonance imaging (C). On gadolinium-enhanced T1-weighted imaging, the tumor also presented as an irregular-shaped low-signal-intensity area partially surrounded by strongly enhanced outer regions (D). On gross appearance, the cut surface of the resected tumor consisted of two different components: a brown component in the central region and a whitish component at the periphery (E), consistent with radiological images (A-D). Similarly, the two components were readily discernable on the tumor sections prepared for microscopic observation using Hematoxylin and Eosin staining (F). Of note, panel F was synthesized intentionally by combining two photos of different pathological preparations with reference to the tumor morphology in panels A-E.
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Gadolinium-enhanced T1-weighted imaging of the abdominal MRI demonstrated the presence of an irregularly shaped low-signal-intensity region within the tumor, partially surrounded by well-enhanced regions with clear demarcation (Fig. 1). Distinct solid and white regions dominated the periphery outside the brown-colored area in the central tumor (Fig. 1). The morphology of the cut section was consistent with the T2-weighted and gadolinium-enhanced T1-weighted MRI findings observed before surgical resection (Fig. 1). The PC component was located within the dark, inhomogeneous region of the central tumor, while the GNBL component existed outside of the PC region at the periphery of the tumor (Fig. 1D and E).
Pathological microphotographs of the right adrenal composite pheochromocytoma with ganglioneuroblastoma. Different sections at the same position of the tumor were stained with Hematoxylin and Eosin staining (A) and for chromogranin A (B) and S-100 protein (C). The upper and lower halves of panels A to C represent the ganglioneuroblastoma and pheochromocytoma components of the tumor, respectively. In panel D, the GNBL component comprised a mixture of ganglion-like cells in a neurofibrillary background with no primitive neuroblastic foci, presenting with a characteristic morphology of GNBL-intermixed according to the international neuroblastoma pathology classification (original magnification 100x for A, B, and C, 200x for D).
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Microscopically, the tumor consisted of two different components: PC and GNBL (Fig. 2A-C). The PC component demonstrated histological features typical of PC, consisting of polygonal tumor cells with granular and basophilic cytoplasm, round-to-oval nuclei, and a single prominent nucleolus, distributed in well-separated nests with thin stroma and rich vascularity (Zellballen pattern) (Figure 2A, 3A and the inset). Our immunohistochemical analyses demonstrated that the PC component stained diffusely positive for chromogranin A (Fig. 2B) and synaptophysin (Supplementary file 1) in the tumor cells and stained positive for S-100 protein (Figure 2C, 3D) in the surrounding sustentacular cells. In contrast, the GNBL component comprised a mixture of variably differentiated neuroblastic cells and ganglion-like cells in a neurofibrillary background, with no primitive neuroblastic foci (Fig. 2A and D). The GNBL component stained positively for anti-S-100 protein (Fig. 2C), neurofilament, and synaptophysin antibodies (Supplementary file 1), presenting with a characteristic morphology of GNBL-intermixed, according to the international neuroblastoma pathology classification.
The microscopic features of the pheochromocytoma (PC) component of the right adrenal composite PC. Focal hemorrhaging was observed in the PC component, but no confluent necrosis or mitosis was observed on Hematoxylin and Eosin (H&E) staining (A). The PC component comprised two types of tumor cells: large polygonal eosinophilic cells with pleomorphic nuclei and occasionally prominent nucleoli and coarse chromaffin (left half of inset A); and small, round chromaffin cells (right half of inset A). Both types of cells were distributed in the form of variably sized nests, called Zellballen structures (A). There were also sheet-like areas and spindle cell arrangements on H&E staining (B). Although high cellularity, cellular monotony, and tumor cell spindling were noted, neither mitosis nor necrosis was evident (A, B). There was focal vascular invasion in the PC component on Elastica-Masson's staining (C). The sustentacular cells, stained positively for S-100, partially disappeared in the PC region where the Zellballen structures were poorly aligned (D). In (D), the arrowheads indicate the sparse localization of the sustentacular cells around the disordered Zellballen nests. The PC cells were positive for the Ki-67 antigen at a maximum of 5.1% (E and the inset) but did not show any expression of vasoactive intestinal peptide (F) (original magnification 4x for A, 200x for B, 400x for C, and 100x for D-F; the insets of A and E were originally magnified at 400x).
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The PC component demonstrated histological features typical of PC, consisting of polygonal tumor cells with granular and basophilic cytoplasm, round-to-oval nuclei, and a single prominent nucleolus, distributed in well-separated nests with thin stroma and rich vascularity (Zellballen pattern) (Figure 2A, 3A and the inset). Our immunohistochemical analyses demonstrated that the PC component stained diffusely positive for chromogranin A (Fig. 2B) and synaptophysin (Supplementary file 1) in the tumor cells and stained positive for S-100 protein (Figure 2C, 3D) in the surrounding sustentacular cells. The tumor cells proliferated in diffuse patterns and invaded neighboring blood vessels (Fig. 3C). Disappearance of the sustentacular cells was partially observed in the PC region (Fig. 3D). The Ki-67 proliferative index was noted to be at 5.1% in the highest of the PC regions (Fig. 3E and the inset). High cellularity, cellular monotony, and tumor cell spindling were also observed (Fig. 3B), although mitotic figures and necrosis were absent (Fig. 3A). In the present case, the PC component was found not to express vasoactive intestinal peptide (Fig. 3F) but was, however, positively immunostained for antibodies against succinate dehydrogenase subunit B (SDHB), suggesting a sporadic nature with no mutations in the SDHB gene (Fig. 4).
The immunohistochemical findings for thyrosine hydroxylase (TH), extracellular signal-regulated kinase 5 (ERK5), and ankyrin repeat domain 1 (ankrd1) protein in the pheochromocytoma (PC) and ganglioneuroblastoma (GNBL) components in the composite adrenal tumor. Both the PC and GNBL components diffusely expressed TH (A and B, respectively), but parts of the tumor cells had positive staining for anti-ERK5 (C and D, respectively) and ankrd1 (E and F, respectively) antibodies in both components (original magnification 200x).
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Fluorescence in-situ hybridization image of ganglioneuroblastoma cells displaying gain of N-myc gene copies. The fluorescence in-situ hybridization signals were scored in 200 non-overlapping tumor nuclei. Red and green fluorescence signals indicated N-myc and centrosomal protein 2 (CEP2) gene copies, respectively. Tumor nuclei were counterstained with 4',6-diamidino-2-phenylindole. Normal ploidy patterns (2 N-myc and 2 CEP2 signals) were detected in 169 tumor cells (84.5%). In 5 cells (2.5%), the number of N-myc signals (arrows) was 1 copy greater than the CEP2 probe signals (3 N-myc and 2 CEP2 signals, arrow heads). A further 5 cells (2.5%) displayed 0 to 1 N-myc and 2 CEP2 signals, and 21 cells (10.5%) exhibited a polyploidy pattern (3 to 5 N-myc and 3 to 5 CEP2 signals) in the nuclei (original magnification 1,000x).
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In the present case (Fig. 6), diploidy patterns [2 N-myc and 2 centrosomal protein 2 (CEP2) signals] were identified in 169 tumor cells (84.5%). N-myc and 3 to 5 CEP2 signals) in the nuclei. Copy number gain of the N-myc gene, 1 copy greater than the CEP2 probe signals (3 N-myc and 2 CEP2 signals), was detected in 5 GNBL cells (2.5%) (Fig. 6, arrows and arrow heads). None of the tumor cells demonstrated N-myc amplification (at least 10 copies greater than the CEP2 gene).