ORIGINAL PAPER
Ai-Min Liu . Shotaro Maeda . Masaru Hosone
Keiko Azuma · Hironori Katayama
Munehiro Yokoyama · Zenya Naito · Yuuichi Sugisaki Goro Asano
Use of electron microscopic evaluation for the diagnosis of adrenal cortical carcinoma in fine needle aspiration cytology: a case report and review of the literature
Received: June 18, 2001 / Accepted: August 7, 2001
Abstract Bilateral adrenal tumors were detected in a 72- year-old man who had a history of hepatic inflammatory pseudotumor. Computet tomography (CT)-guided fine needle aspiration cytology (FNAC) of the adrenal glands was performed. The cytologic findings were similar to the previous diagnosis of “inflammatory pseudotumor” in the liver. However, the origin of some aggregated large atypical cells observed in the adrenal FNAC specimens was not known. Immunocytochemically, these large atypical cells were positive for vimentin and negative for cytokeratin and chromogranin A. An electron-microscopic study showed that these large atypical cells contained mitochondria with tubulovesicular cristae and smooth endoplasmic reticulum arranged in whorled and laminated patterns, and these find- ings confirmed diagnosis of primary adrenal cortical carci- noma. The histopathological diagnosis of the resected bilateral adrenal tumor was adrenal cortical carcinoma. The patient died 7 months after surgery, with recurrence of the bilateral adrenal cortical carcinoma and extensive metastases. A diagnosis of primary adrenal cortical carci- noma with extensive metastases was finally demonstrated by autopsy. Retrospectively, the previous liver tumor was determined to be a metastatic lesion.
Key words Adrenal cortical carcinoma . Fine needle aspiration cytology · Electron microscopy · Immunocy- tochemistry
Introduction
Adrenal cortical carcinoma is a very rare but highly malig- nant tumor in both children and adults, accounting for 0.02% to 0.04% of all cancers. The tumor affects all age groups, from 6 months to 72 years, with a median age at detection of 40 years, and a mean age of 37.6 years. Its diagnosis is often difficult and delayed. Liver metastasis is often involved and the condition continues to carry a poor prognosis.14 The mean or median survival time of the pa- tients reported has ranged from 4 to 30 months.1,5
Computet tomography (CT)-guided fine needle aspira- tion cytology (FNAC) can localize a tumor accurately, and can thus help in the aspiratation of diagnostic material with minimal adverse effects; this technique is therefore being increasingly utilized in the diagnostic evaluation of superfi- cial as well as deeply situated tumors.6,7 Therefore, FNAC has become the procedure of choice for the initial diagnosis of adrenal masses.8-12
We report here a case of primary adrenal cortical carci- noma in a 72-year-old man. FNAC of adrenal masses showed aggregation of large atypical cells in an inflam- matory background, mimicking the previous diagnosis of inflammatory pseudotumor of the liver in the patient. However, FNAC confirmed the diagnosis of primary adre- nal cortical carcinoma, and electron microscopic findings of portions of the aspirated material led to a demonstration of the diagnosis.
A .- M. Liu () · Y. Sugisaki Central Institute for Electron Microscopic Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan Tel. +81-3-3822-2131 (ext. 5211); Fax +81-3-5685-5517 e-mail: Liu_Aimin/emlab@nms.ac.jp
S. Maeda · M. Hosone · K. Azuma · H. Katayama Department of Pathology, Tama-Nagayama Hospital, Nippon Medical School, Tokyo, Japan
M. Yokoyama · Z. Naito · G. Asano Department of Pathology, Nippon Medical School, Tokyo, Japan
Case report
A 72-year-old man had a hepatic inflammatory pseudo- tumor in the right lobe that was resected in October, 1998. Six months after the operation, the patient had a relapse of fever and an elevated serum C-reactive protein (CRP) level (13.8mg/dl). CT scan and magnetic resonance imag- ing (MRI) detected bilateral adrenal tumors; the left measured 4cm and the right measured 7cm in diameter
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(Fig. 1). CT-guided FNAC was performed. The cytologic findings were similar to those that led to the previous diagnosis of hepatic inflammatory pseudotumor. However, the origin of some aggregated large atypical cells in the adrenal FNAC specimens was not known. An immunocy- tochemical examination of the FNAC material suggested adrenal cortical carcinoma and, electron microscopic observations confirmed a diagnosis of primary adrenal cor- tical carcinoma. The bilateral adrenal glands were then excised surgically. The pathological diagnosis was adrenal cortical carcinoma. Postoperatively, palliative chemo- therapy with mitotane was instituted. The patient died 7 months after this surgery, with recurrence of the bilateral adrenal cortical carcinoma and extensive metastases to the liver, lungs, spleen, pancreas, kidneys, and periaortic lymph nodes. Autopsy was performed and primary adrenal cortical carcinoma with extensive metastases was demonstrated.
Materials and methods
In this patient, FNAC of the adrenal tumor was performed using an 18-gauge needle under CT guidance. Direct smears were immediately spray-fixed in 95% alcohol for Papanico- laou stain and air-dried for May-Grunwald-Giemsa (MGG) stain.
A portion of the aspirated material was then suspended in 0.8% normal saline (NACL), and prepared in a cyto- centrifuge to make a cell block that was formalin-fixed and paraffin-embedded. The cell block sections were stained with hematoxylin and eosin (H&E).
The immunohistochemical examination was performed on the cell block sections, using the avidin-biotin- peroxidase complex (ABC) method. The primary antibod- ies used included cytokeratin (M0821, pankeratin; Dako, Tokyo, Japan), vimentin (M0725; Dako), S-100 protein (Z0311; Dako), chromogranin A (A0430; Dako), and leukocyte common antigen (LCA) (M0701; Dako).
For electron microscopic evaluation, another portion of the aspirated material was fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a model H 7100 electron microscope (Hitachi, Tokyo, Japan).
Results
Cytologic findings
Papanicolaou-stained and May-Grunwald-Giemsa-stained smears showed large atypical cells in a background of in-
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flammatory cells, that were predominantly lymphocytes and plasma cells (Fig. 2A,B). There was coarse clumping of nuclear chromatin and a centrally located obvious nucleolus in some of the large atypical cells.
The cell block sections with H&E staining showed a granulomatous lesion, consisting of marked inflammatory cell infiltration with some aggregated large atypical cells (Fig. 2C), that was cytohistologically similar to the previous hepatic inflammatory pseudotumor. These large atypical cells had ample eosinophilic cytoplasm and obvious nucleoli. Mitotic figures were seen without difficulty.
Immunocytochemical findings
Immunocytochemical examination of the cell clock sections showed that the large atypical cells were positive for vimentin (Fig. 2D), and negative for cytokeratin, S-100 pro- tein, chromogranin A, and LCA.
Electron microscopic findings
Ultrastructurally, at low magnification, we observed a granulomatous lesion that consisted of various cell compo- nents, including numerous lymphocytes and plasma cells (Fig. 3). In the granulomatous lesion, the large atypical cells
contained irregularly shaped nuclei, plentiful smooth endo- plasmic reticulum, and darkly stained mitochondria, and were partly outlined by basal lamina (Fig. 4A,B). At high magnification, the large atypical cells showed prominent nucleoli, and contained whorled and laminated structures, consisting of smooth endoplasmic reticulum, that were inti- mately mingled with mitochondria in the cytoplasm (Fig. 5B). The whorled and laminated structures suggested one of the characteristics of adrenal cortical epithelial cells. The darkly stained mitochondria contained tubulovesicular cristae (Fig. 5A), which suggested another characteristic of the adrenal cortical epithelial cells.
Histopathologic findings
In surgically excised specimens of the bilateral adrenal glands, the areas with obvious inflammation showed a granulomatous lesion that was similar to the previous liver inflammatory pseudotumor (Fig. 6A). However, large atypical cells with neoplastic proliferation were defined in the specimens. The tumor cells were in a sheet-like arrange- ment and had ample eosinophilic granular cytoplasm with vesicular nuclei and prominent nucleoli (Fig. 6B). Mitotic figures were seen without difficulty.
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Autopsy findings
Autopsy findings indicated that the adrenal cortical carci- noma had recurred in the bilateral adrenal glands and had extensively metastasized to the liver, lungs, spleen, pan- creas, kidneys, and periaortic lymph nodes. The final diag- nosis was primary adrenal cortical carcinoma with extensive metastases.
Discussion
Only a few cases of adrenal cortical carcinoma diagnosed by FNAC have been reported in the English-language litera- ture.10-12 In Japan, the FNAC findings of adrenal cortical carcinoma have not been reported. However, the use of FNAC in the diagnostic evaluation of adrenal mass lesions has been demonstrated, and the cytologic features of vari- ous conditions have been characterized.
Levin,13 in 1981, first reported a case of adrenal cortical carcinoma diagnosed by preoperative percutaneous FNAC. The cytologic findings were described and correlated with the histologic findings of the surgically excised specimen. Papanicolaou staining showed individual tumor cells with specific malignant nuclear characteristics, such as coarse
clumping of the nuclear chromatin, clearing of the nucleo- plasm, and irregular thickening of the nuclear membrane. Some cells contained macronucleoli, and multinucleated cells revealed variation in the nuclei. Histologic sections of the tumor revealed nests and sheets of cells of widely vary- ing size with abundant eosinophilic cytoplasm. The nuclei showed wide variability with bizarre shapes and prominent nucleoli. Frequent mitotic figures were present in the less differentiated areas.
Since then, several other studies4-12,14,15 have demon- strated that FNAC of adrenal masses has become a useful diagnostic procedure for the evaluation of benign and ma- lignant adrenal masses, and for the differential diagnosis of adrenal cortical carcinoma and other primary or metastatic malignancies.4,6,8,10,11,12,14,15 Katz et al.12 described cytologic, histologic, and electron microscopic findings in 22 patients with adrenal masses; with the masses in 4 patients being primary adrenal cortical carcinoma. Wadih et al.10 and Nance et al.11 reported an adrenal cortical carcinoma with a predominant spindle-cell pattern from a review of 48 adre- nal FNAC, combined with ancillary immunocytochemistry and electron microscopy studies. Sharma et al.15 described the cytologic features in seven cases of adrenal cortical carcinoma, correlated them with histology, and compared the cytologic features with those of very similar renal cell carcinoma on FNAC.
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The cytologic features of adrenal cortical carcinoma in various conditions have been characterized. Papanico- laou smear and cell block sections showed extreme cellular- ity, with the cells mostly isolated, in loosely cohesive groups, or in syncytial tissue fragments. The tumor cells were uniform to pleomorphic; the nuclear/cytoplasmic (N/C) ratio was mildly increased in the well differen- tiated type, and was markedly increased in the poorly differentiated type. The nucleus was variable in size and shape, had a central or eccentric location, showed very coarse granular to chunky chromatin, and prominent nucleoli. The cytoplasm was variable, moderate to abun- dant, with clear, vacuolated, granular-to-dense patterns.9-15 Immunocytochemical stains may be useful in supporting the diagnosis of adrenal cortical carcinoma. Cytokeratins were usually negative in this tumor, in contrast to their pattern of staining in the normal adrenal cortex. Vimentin was posi- tive in the majority of adrenal cortical carcinomas, whereas normal adrenal cortex was negative, and adenomas showed
variable results.5,11,16-18 The ultrastructural studies were re- markable in terms of showing the presence of numerous cytoplasmic mitochondria and smooth endoplasmic re- ticulum. The mitochondria contained tubulovesicular or shelf-like cristae, and the smooth endoplasmic reticulum was arranged in a prominent laminated and whorled pattern. 10-12,17,19,20-22
When adrenal cortical carcinoma is present in metastatic locations, it can readily be confused with other carcinomas on light microscopy. Renal cell carcinoma, liver cell carci- noma, and melanoma head the differential diagnosis.16-18 Silva et al.19 reported that they had encountered situations in which a diagnosis of metastatic adrenal cortical carci- noma had not been considered by light microscopy and was established only from the ultrastructural findings.
The present case proved difficult in regard to the initial cytologic diagnosis. In the adrenal FNAC specimen, the infiltration of inflammatory cells was extremely marked, and the cytohistologic findings were similar to those in the
Fig. 5A,B. Electron micrographs of large atypical cells. A The large atypical cell with prominent nucleolus contains dark stained mitochon- dria, plentiful smooth endoplasmic reticulum, and basal lamina. Inset, High magnification of the mitochondria containing tubulovesicular
cristae. Mi, mitochondria; BM, basal lamina. Bar, 1.4 um; inset, 0.3 um. B Whorled and laminated structures (asterisks) are shown in the cyto- plasm. Inset, High magnification of the structure. Mi, Mitochondria; BM, basal lamina. Bar, 1.2 um; inset, 0.8 um
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previous inflammatory pseudotumor of the liver. In the dif- ferential diagnosis, it was considered whether the adrenal tumor was primary or metastatic, and whether it was related to the previous liver inflammatory pseudotumor. Therefore, it was important to know the origin of the large atypi- cal cells that were observed in the Papanicolaou smear and H&E-stained cell block section. Ancillary studies, including immunocytochemistry and electron microscopy, were performed, and the results were useful in establish- ing the diagnosis of primary adrenal cortical carcinoma. Immunocytochemically, the large atypical cells were positive for vimentin, and negative for cytokeratin, S-100 protein, chromogranin A, and LCA. These findings ruled out the possibility of metastatic liver carcinoma, renal cell carcinoma, and pheochromocytoma, and supported the diagnosis of adrenal cortical carcinoma. Ultrastructurally, the large atypical cells showed mitochondria that contained tubulovesicular cristae, as well as smooth endoplasmic reticulum in a laminated and whorled pattern. These characteristics confirmed the diagnosis of adrenal cortical carcinoma.
Inflammatory pseudotumor of the liver is a rare entity and a diagnostic challenge, and it is commonly misdiag- nosed as a malignant tumor.23 Liver inflammatory
pseudotumor, mimicking a malignancy, has, in the past, been misdiagnosed as hepatocellular carcinoma, sarcoma, and metastatic carcinoma.24-26 However, adrenal cortical carcinoma mimicking an inflammatory pseudotumor has not been reported previously. Increased serum levels of CRP have been correlated with elevated levels of pro- inflammatory cytokines, such as interleukin 6 (IL-6), and such increased levels have been reported as part of a systemic inflammatory response syndrome in patients with lung, colorectal, ovarian, or oral cancer.27-30 The present patient had an increased serum level of CRP, and histo- pathologically, specimens showed marked inflammation, probably related to elevated levels of IL-6 and other cytokines, although they had not been examined. Surgically excised and autopsy specimens of the bilateral adrenal glands in our patient had indicated a final diagnosis of pri- mary adrenal cortical carcinoma, and, retrospectively, the previous liver tumor was determined to be metastatic. In this study, adrenal FNAC, combined with ancillary meth- ods, in particular the ultrastructural examination, was help- ful in indicating and in confirming the diagnosis, but the pathologist must be aware that a considerable range of fine structures may be observed in cells of adrenal cortical carci- noma. The combined clinical, cytologic, and ultrastructural presentation of adrenal cortical carcinoma is very useful for the diagnosis.
With the relatively recent advent of CT and nuclear MRI techniques, adrenal cortical neoplasms are being discovered early and are often smaller than when discovered in the past, increasing the need for more accurate diagnosis and for pathologic indicators of prognosis. In a review, Medeiros and Weiss5 proposed several pathologic indica- tors of prognosis for adrenal cortical carcinoma. These indicators included mitotic rate, stage, surgical resectability, nuclear grade, and tumor size. Of these, mitotic rate ap- peared to be the best indicator. Adrenal cortical carcinoma with a high mitotic rate behaved most aggressively. These pathologic indicators are also significant for cytologic evaluation.
In conclusion, the findings in the present patient indicate that adrenal FNAC is an accurate procedure for the diagno- sis of adrenal cortical carcinoma, and ancillary studies, in particular ultrastructural evaluation, can help to correct a previous diagnosis. The cytohistological features of adrenal cortical carcinoma diagnosed on FNAC were reviewed. Preoperative evaluation, achieved by biochemical and ra- diological methods, and an initial diagnosis by FNAC, are most important for determining the stage of the disease and for enhancing the survival of these patients with adrenal cortical carcinoma.
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