Anatomic Pathology / HYALINIZING TRABECULAR ADENOMA

Smears of fine-needle aspiration (FNA) biopsy specimens of 24 histologically proven and MIB-1– positive hyalinizing trabecular adenomas stained with Papanicolaou or Diff-Quik were stripped from the original slides, transferred to charged slides, destained, and restained with MIB-1. Positive immunoreactivity with MIB-1 in a peripheral cytoplasmic pattern was present in 17 specimens, no immunoreactivity was seen in 4 specimens, and equivocal staining was seen in 3 specimens. Papanicolaou-stained smears of papillary thyroid carcinoma, medullary thyroid carcinoma, thyroid follicular neoplasm, and Hürthle cell adenoma (5 specimens each) were treated similarly. None of the control cases stained with MIB-1 had the peripheral cytoplasmic pattern. Hyalinizing trabecular adenoma can be distinguished reliably from other thyroid neoplasms by MIB-1 staining of destained cells from FNA biopsy specimens. Hyalinizing trabecular adenoma (HTA) of the thyroid gland is a rare benign neoplasm described by Carney et al in 1987.1 It is characterized by circumscription or encapsulation, trabecular growth pattern, polygonal and elongated cells, nuclear cytoplasmic inclusions and grooves, hyaline material, dilated sinusoids, laminated calcospherites, and cytoplasmic yellow bodies.1-3 Molecular genetic studies have allied the tumor with papillary thyroid carcinoma (PTC), but other findings, in particular the absence of immunostaining for cytokeratin 19 and high-molecular-weight keratin, do not support this relationship.4 For this reason, it has been suggested that the lesion be referred to as hyalinizing trabecular tumor.5-9 Of great interest is the remarkable appearance of the HTA tumor cells with MIB-1 immunostaining—heavy staining of a narrow peripheral rim of the tumor cell cytoplasm10,11—an appearance not shared with other thyroid tumors. MIB-1 is a monoclonal antibody raised against the recombinant part of the Ki-67 antigen and ordinarily is expressed in the nuclei during active parts of the cell cycle.12,13 HTA is misdiagnosed almost uniformly in fine-needle aspiration (FNA) biopsy specimens, because of the confusing similarity of its nuclear features to those of PTC and the presence of a misleading hyaline material in the tumor that mimics amyloid, and often is diagnosed as medullary thyroid carcinoma (MTC).14-23 We described the cytologic findings in specimens from 29 patients with HTA stained with the Papanicolaou and DiffQuik methods.17 Important features of the neoplasm included a bloody background; cells with ample cytoplasm arranged in cohesive aggregates, often radially oriented around hyaline material; and polygonal nuclei with abundant grooves and cytoplasmic invaginations. These and other features Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2004;122:506-510 507 507 DOI: 10.1309/5KJU9LV8LLA6CL76 507 © American Society for Clinical Pathology permitted diagnosis or suspicion of HTA in the majority of the 29 cases. In the remainder, the cytologic diagnosis was not possible, usually because the smears were poorly preserved or hypocellular, lacked hyaline material, or showed combinations of these findings. We investigated whether the unique MIB-1 cytoplasmic staining characteristic of HTA seen in tissue sections could be seen in FNA biopsy samples suggestive of the neoplasm. Unstained cytologic slides of the neoplasm were not available, so previously stained FNA biopsy samples were used from patients with HTA proven by MIB-1 staining of tissue sections. The stained cells were transferred to charged slides, destained, and restained with MIB-1. The restained cells showed the typical peripheral cytoplasmic positivity of the neoplasm in 17 (71%) of 24 cases. The results indicate that this method is practical and should prove useful in the cytodiagnosis of suspected cases of HTA. Materials and Methods This study was approved by the Mayo Foundation Institutional Review Board. We retrieved 29 specimens from HTA cases from the surgical pathology collection of Mayo Clinic, Rochester, MN, and our own collection. FNA biopsy specimens were present from each patient, in addition to tissue sections. Six biopsy samples were excluded, 2 because of insufficient cells and 4 because permission to reprocess the original slides was denied. The remaining 23 cases and an additional recent case formed the study group. H&E-stained slides of the tumor sections were reviewed, and the diagnosis of HTA was confirmed histologically in all ❚Image 1A❚. Twenty-three samples had diffuse, typical MIB-1 positivity ❚Image 1B❚; in the remaining case, the MIB-1 staining was focal (<5% of tumor cells showed staining). The original FNA biopsy cytologic diagnoses in the 24 cases had been PTC in 11 cases, “suspicious for” PTC in 8 cases, follicular neoplasm in 4 cases, and indeterminate in 1 case. The cytologic material available comprised Papanicolaou-stained (19 cases) or Diff-Quik–stained (5 cases) FNA biopsy smears. The smears featured cohesive aggregates of cells, often radially oriented around hyaline material, and found singly less frequently. Cytoplasm was abundant. Intranuclear cytoplasmic inclusions, nuclear grooves, and nuclear overlapping were very common and best seen with Papanicolaou stain. Diff-Quik–stained smears highlighted the metachromatic hyaline material, the perinucleolar clearing, and the cytoplasmic bodies. Cells from the previously stained smears were stripped from the original slides, using the “peel and stick” technique.24 This method was used instead of direct destaining and restaining of the original smear because it permitted the use of multiple stains in addition to MIB-1. The original peeled smear was cut into several pieces, and only 1 was used for MIB-1 staining. The stained slides were placed in xylene to loosen the coverslip so that it could be removed. The exposed cells were covered with organic solvent–based mounting media (Krystalon, EM Science, Gibbstown, NJ), placed in an 80°C to 90°C oven for 6 hours, permitted to cool to room temperature, and placed in 90°C distilled water for 20 to 30 seconds. The hardened mountant with the cells attached to it was undermined and separated from the slide, using a single-edged razor blade held at a 45° angle. Heated (90°C) distilled water was flooded onto charged glass slides, and the removed mountant with attached cells was placed on a new slide. The new slides then were placed in an 80°C to 90°C oven for 30 minutes. They were removed, permitted to return to room temperature, and placed in xylene to remove the mountant. The slides were rehydrated in 70% alcohol and were decolorized in 0.3% acid alcohol, washed in running tap water for 5 minutes, and rinsed in distilled water. The samples then were restained with MIB-1 antibody (clone MIB-1, DAKO, Carpinteria, CA). Immunostaining was performed at a 1:800 dilution, using avidin-biotin complex detection chemistry and 3,3'-diaminobenzidine as the chromogen. A B ❚Image 1❚ Histology of hyalinizing trabecular adenoma. A, Section showing a delicately encapsulated tumor with trabecular growth pattern, oval nuclei, and acellular hyaline material (arrows) (H&E, original magnification ×400). B, Tissue section from Image 1A (after destaining) showing peripheral cytoplasmic staining of the tumor cells. Rare diffuse nuclear staining is present (MIB-1, original magnification ×200). Casey et al / HYALINIZING TRABECULAR ADENOMA 508 Am J Clin Pathol 2004;122:506-510 508 DOI: 10.1309/5KJU9LV8LLA6CL76 © American Society for Clinical Pathology Papanicolaou-stained FNA smears of PTC, MTC, thyroid follicular neoplasm, and Hürthle cell adenoma (5 cases each) were treated similarly.