Neuroendocrine Carcinoma of the Paranasal Sinus A Morphological and Endocrinological Study
TORU KAMEYA, MD,* YUKIO SHIMOSATO, MD,* ISAMU ADACHI, MD,+ KAORU ABE, MD,t SATOSHI EBIHARA, MD,# AND ISAMU ONO, MD#
Four rare cases of small cell carcinoma of the paranasal sinuses were studied histologically, ultrastructurally, and endocrinologically. All tumors showed features of undifferentiated carcinoma with alveolar patterns but without acinar or squamous differentiation and contained cells possessing endocrine-type small secretory granules, which were indistinguishable from non-neoplastic neuro- secretory granules. The three cases also possessed a small number of microtubules, and in one of these fine cytoplasmic filaments were observed. One case showed elevated plasma levels of cortisol and adrenocorticotropic hormone associated with adrenocortical hyperplasia and Crooke’s changes of the pituitary gland. Another case showed hypercalcemia with bone metastasis, hypercalcitonemia with a high content of calcitonin in the tumor tissue, calcitonin-positive tumor cells, and C-cell hyper- plasia of the thyroid. It was concluded that all four cases should be called neuroendocrine carcinoma, which might be related to neoplasms derived from amine precursor uptake and decarboxylation cell series rarely encountered in the paranasal sinuses.
Cancer 45:330-339, 1980.
1 N NASAL AND PARANASAL MALIGNANCIES, squamous cell carcinoma is the most common, followed by adenocarcinoma, malignant lymphoma, plasma cell tumor, malignant melanoma, and olfactory neuroblas- toma (in order of frequency).
We present 4 cases of tumors in this location in which definite histological typing was extremely dif- ficult, and undifferentiated carcinoma, atypical car- cinoid, malignant paraganglioma or poorly differen- tiated olfactory neuroblastoma were suspected. Ultra- structural, immunohistochemical and endocrinological studies threw some light on these difficult cases. A comparative analysis with other histologically similar tumors based on morphology and polypeptide hormone production led us to conclude that these four cases were related to neoplasms derived from the diffuse endocrine
cell system or neural crest system-the so-called APUD (amine precursor uptake and decarboxylation) cell series,33 whose presence in the nasal cavity and paranasal sinuses has not yet been proved.
Materials and Methods
Since the primary tumors of all 4 cases presented were degenerated due to irradiation and chemotherapy, neck node metastases were used for the study im- mediately after removal in cases 1, 2 and 3. In Case 4, autopsy material was used. Tissue for examination by light microscopy was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin, periodic acid-Schiff and Alcian blue for mucin, silver impregnation for reticulin, and Fontana-Masson and Grimelius stains for argentaffin and argyrophil cells.
In cases 2 and 4, freeze-dried sections treated with formalin vapor at 80 C for 10 minutes were observed for fluorescence of catecholamines and serotonin, along with the human adrenal medulla as a positive control specimen.12 Indirect immunofluorescent reac- tions for adrenocorticotropic hormone (ACTH) and calcitonin (CT)23 were performed on deparaffinized sections of paraformaldehyde-, formalin- or Bouin- fixed tissue in all 4 cases. All immunostained sections were observed by a fluorescence microscope equipped with a Model A-RFL (Olympus, Tokyo, Japan). The specificity control consisted of substituting normal serum or other unrelated antisera (anti-human chorionic
From the *Pathology and tEndocrinology Division, National Can- cer Center Research Institute, Tokyo and Head and Neck Division, National Cancer Center Hospital, Tokyo.
Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (201005, 201592) and for Cancer Research and Specific Diseases from the Ministry of Health and Welfare (51-1, 53-4, 53-5), Japan.
Address for reprints: Toru Kameya, Pathology Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo (104) Japan.
We thank Dr. T. Takeuchi, Biochemistry Division, National Cancer Center Research Institute, for tumor serotonin assay, Messrs. M. Tsumuraya and T. Koide, Mmes. M. Ikeda and R. Imagiire and Miss S. Miyakawa for their excellent technical assistance.
Accepted for publication February 19, 1979.
| Case no. | Age | Sex | Location of the tumor | Clinical course | Size (nm) of secretory granules (mean ± standard error) | Endocrinologic data |
|---|---|---|---|---|---|---|
| 1 | 50 | male | Right maxillary and ethmoid sinus | 5 recurrences of the primary focus, and neck node metastatis. Death after 10 year clinical course. | 100-240 (157 ± 25.1) | High plasma cortisol, ACTH and B-MSH. Obesity and adrenal cortical hyperplasia. |
| 2 | 56 | male | Right ethmoid and maxillary sinus and nasal cavity | 4 recurrences of the primary focus, and neck node metastasis. Death after 6 year clinical course. | 80-210 (136 ± 17.5) | High content of calcitonin in tumor. |
| 3 | 33 | female | Right ethmoid and frontal sinus | Death due to postoperative hemorrhage and respiratory distress, 50 days after neck dissection for metastasis. | 120-250 | - |
| 4 | 70 | male | Right maxillary sinus | Death, with hypercalcemic symptoms, after 6 month clinical course. | 270-360 (331 ± 38.8) | High serum calcium and in- creased urinary excretion of calcium. High serum and tumor calcitonin and presence of calcitonin- containing cells in tumor. C-cell hyperplasia of the thyroid. |
gonadotropin, and anti-glucagon) for a specific anti- serum.
For electron microscopy, tissues were fixed in phos- phate buffered 5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated by graded alcohols and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate. In Case 4, materials for electron microscopy were obtained from formalin-fixed tissue, washed in tap water overnight, refixed briefly in glutaraldehyde and then processed as described above.
Plasma levels and tumor contents of ACTH, ß- melanocyte stimulating hormone (3-MSH), CT and parathyroid hormone (PTH) were determined by their respective radioimmunoassays.1,35 Tumor ACTH, ß- MSH and CT were extracted by Payne’s method modified as reported previously.1 PTH was extracted using the method reported by O’Riordan et al.30 The extracts were assayed with three or more dilu- tions to obtain a dose-response curve, and when sig- nificant displacement was observed in only one dilu- tion, the sample was considered undetectable. In case 4, which showed hypercalcemia and hypercalcitonemia, the right lateral lobe of the thyroid was sectioned in 12 slices at a thickness of 2-4 mm in a plane per- pendicular to the longitudinal axis, according to Wolfe et al.39 Alternate slices of tissue were then taken at each level for determination of CT content and im- munofluorescent localization of C-cells using the anti- CT serum used for radioimmunoassay.
Urinary and serum serotonin and 5-hydroxyindole- acetic acid (5-HIAA) were measured by the methods of Davis9 and Udenfriend.37
For tumor serotonin, tissue was homogenized with 0.4 M perchloric acid and centrifuged. Serotonin was extracted from the supernatant with n-butanol and assayed in a spectrofluorometer.37
Results
Case 1: A 50-year-old male patient, with an initial symptom of nasal bleeding, had a tumor invading the right maxillary and ethmoid sinuses. Partial and total maxillectomies and radiotherapies were performed for the primary and recurrent tumors, followed by radical right neck dissection. The patient died after a ten year clinical course (Table 1). Tentative diagnosis made from several biopsy specimens was atypical carcinoid or malignant paraganglioma. One month before the pa- tient’s death, high plasma cortisol (31.4 µg/ml), ACTH (2300 pg/ml), and ß-MSH (400 pg/ml) had been noted, which suggested the ectopic ACTH-MSH pro- duction from the tumor, although ACTH and 8-MSH contents in the tumor tissue could not be measured. Urinary vanyl mandelic acid was negative, and serotonin was not elevated.
In well-preserved neck node metastasis, tumor cells were arranged in solid nests with thick or thin fibrovascular stroma, forming alveolar structures. They possessed round or oval and hyperchromatic
nuclei with stippled chromatin. Nucleoli were not prominent, mitoses were rare, and occasional pyknotic nuclei were observed. The amount of cytoplasm of tumor cells was small to moderate and finely granular with ill-defined cell borders (Fig. 1). Ribbon or festoon patterns and peripheral palisading as seen in certain types of carcinoid tumors were not present. Argyrophil and argentaffin cells were not found. Tubular structures or epithelial mucin were not observed.
By electron microscopy, membrane-bound, electron- dense cored granules measuring 100 to 240 nm (mean: 157 + 25.1 nm) in diameter were present in almost all tumor cells but in variable numbers. They were en- countered in the frequently observed cytoplasmic processes which formed complex cellular interdigita- tions. A few microtubules of about 20 nm in width were also present. Features seen in glandular cells were not found. Small desmosomes and bundles of tonofilaments could be found, although they were rare. Golgi complexes and free polyribosomes were well developed, but rough endoplasmic membranes were not observed (Fig. 2).
Autopsy showed the recurrent tumor destroying the ethmoid and sphenoid sinuses and invading the skull base and right frontal lobe of the brain. Distant metastasis was found only in the left femur. Slight obesity, left ventricular hypertrophy of the heart (480 g), marked diffuse cortical hyperplasia of bilateral adrenals (16 g on the left and 15 g on the right), and Crooke’s change of the pituitary gland were present. The direct cause of death was panperitonitis due to the perforation of a duodenal ulcer.
Case 2: The patient, aged 56, male, showed nasal bleeding and exophthalmos. The tumor filled the right ethmoid and maxillary sinuses and nasal cavity. In spite
of subtotal maxillectomy and two courses of radio- therapy, the tumor recurred four times. Neck node metastases were removed by radical neck dissection. The patient died 6 years after initial symptoms (Table 1). The function of the adrenal cortex appeared normal, because the diurnal rhythm of plasma cortisol was present, and the suppression after 1 mg overnight dexamethasone administration was normal.
The tumor had microscopic features similar to those described in case 1, but the nuclei were less pleo- morphic and slightly larger, with occasional prominent nucleoli. Mitotic figures were rarely observed (Fig. 3). No argyrophil or argentaffin granules were found. Freeze-dried sections exposed to formalin vapor re- vealed no fluorescence for catecholamines and sero- tonin. Ultrastructural findings were similar to those of case 1, but secretory granules were more numerous, lipid vacuoles were often seen in cells adjacent to granulated cells (Fig. 4). Secretory granules meas- ured 80-210 nm (mean 136 ± 17.5 nm) in diameter (Fig. 5). Small empty vesicles similar to synaptic vesicles in size were occasionally encountered in the cytoplasmic processes.
An autopsy revealed that the tumor had invaded the ethmoid and sphenoid sinuses and skull base, imping- ing upon the frontal lobe of the brain. No remarkable
changes were found in the endocrine and cardio- vascular systems. Testes and adrenal cortices were atrophic. No ACTH was detectable in a metastatic tumor. However, the tumor contained 6.0 ng of calcitonin per wet gram of tissue, indicating the pro- duction of the hormone from the tumor.
Case 3: The patient, aged 33, female, showed right exophthalmos with visual disturbance, and a
growth invaded the right ethmoid and frontal sinuses. Partial maxillectomy with right neck node dissection was performed (Table 1). The patient died postopera- tively because of a hemorrhage from the rupture of a carotid artery. Autopsy was not permitted. No endo- crinological studies were done.
The tumor of this case also presented solid alveolar structures supported by fibrovascular stroma. Nuclei were most pleomorphic of the 4 cases. The nucleo- cytoplasmic ratio was high, and hyperchromasia was marked with large aggregates of chromatin. Multi- nucleated cells with infoldings of nuclear membranes were constant findings. Mitotic figures were numerous. The cytoplasm was eosinophilic and finely granular, vacuolar or fibrillar, as occasionally encountered in neuroblastoma. Both silver stainings were negative. Histological diagnosis of undifferentiated small cell carcinoma or poorly differentiated olfactory neuroblas-
toma was made (Fig. 6). By electron microscopy, the nucleus showed irregular indentations with cytoplasmic extensions into the nucleoplasm, a single prominent nucleolus, and large aggregates of peripheral chromatin. Dense-cored cytoplasmic granules were scarce in num- ber and measured 120-250 nm in diameter. What was most characteristic was the presence of widely dis- tributed fine filaments in the cytoplasm running in various directions. Microtubules running in parallel to the long axis of the cell were also found in a very few cells. Golgi complexes were well developed, but rough endoplasmic membranes in stacks and vesicular form were scattered (Fig. 7).
Case 4: The patient, aged 70, male, had an initial symptom of the left nasal bleeding, which led to the discovery of a growth in the right maxillary sinus at an advanced stage. Four thousand rads and Mitomycin-C by arterial infusion were given to the maxilla. The
tumor disappeared, but two months later metastases were noted in the cervical and thoracic vertebrae, for which systemic administration of Bleomycin and Mito- mycin C was begun. High levels of serum calcium (14.0 mg/dl) with increased urinary excretion of calcium (356-560 mg/day) were noted for about 20 days before
death. Porcine calcitonin was given without effect (Table 1).
Histologically, the tumor showed solid nests with intercalated, thin fibrous stroma, and in some nests peripheral palisading of tumor cells was noted. In areas, neoplastic cells were arranged in trabeculae. The
nuclei were round and small or medium in size, mitotic figures were numerous, and nucleolei were often prominent. Occasional multinucleation was found, and cytoplasm was abundant and granular. Features of squamous differentiation were lacking. Neither mucin in the cytoplasm nor acinar structure was detected
(Fig. 8). The histological diagnosis was undifferentiated carcinoma. Very few cells contained electron-dense, endocrine-type secretory granules, measuring 270- 360 nm (mean: 331 + 38.8 nm) in diameter. Other organelles were not informative, because of poor preservation of cells (Fig. 9).
The autopsy showed the left maxillary sinus lined by necrotic non-neoplastic tissue; the left upper gum and palate were destroyed. No local recurrence was seen. However, extensive metastases were found in the lungs, liver, left kidney, adrenals, cervical, thoracic and lumbar vertebrae, and pelvic bones, where osteoclasts were inconspicuous. There were calcium deposits in the renal tubules. The thyroid and parathyroid glands were histologically unremarkable. The cause of death was bronchopneumonia.
Calcitonin of 50 ng per wet gram of tissue was detected in liver metastasis in association with marked hypercalcitonemia (4600 pg/mg) that was comparable to that encountered in medullary carcinoma of the thyroid23 and in rare carcinoid cases,2 PTH was un- detectable in the tumor. In this case, tumor cells, singly or in a few clusters, revealed specific apple- green immunofluorescence for calcitonin (Fig. 10). Plasma ACTH, 8-MSH, and PTH were within normal limits. As in case 2, no specific fluorescence for
catecholamine and serotonin was seen on freeze- dried, formalin-vaporized sections.
In six tissue slices of the right lateral lobe of the thyroid, the calcitonin content was highest in the middle two slices (Fig. 11), which correlated ap- proximately with the relative numbers of C-cells (calcitonin-containing cells) revealed in per unit area of the thyroid by immunofluorescence. The calcitonin contents of the slices were two to ten times higher than those of four other non-neoplastic thyroids randomly selected at autopsy.1 The calcitonin contents were comparable to those in the tumor tissue of some cases of medullary carcinoma of the thyroid.23
Discussion
Of 313 nasal and paranasal cancers treated at National Cancer Center Hospital between 1962 and 1974, 82% were squamous cell carcinoma, 8% adeno- carcinoma, 5% malignant lymphoma and 5% other malignancies such as undifferentiated carcinoma, plasma cell tumor, malignant melanoma and olfactory neuroblastoma.26 Four cases presented in this paper were included among “others” and were distinctly dif- ferent from poorly differentiated squamous cell car- cinoma or adenocarcinoma.
Histological features of these tumors have led us to make tentative diagnoses of atypical carcinoid or malignant paraganglioma in cases 1 and 2, of undiffer- entiated small cell carcinoma or poorly differentiated olfactory neuroblastoma in case 3, and of undiffer- entiated carcinoma or malignant carcinoid in case 4. While sections stained by Grimelius and Fontana- Masson and freeze-dried, formalin-vaporized sections were not informative, ultrastructural analysis revealed the presence of endocrine-type secretion granules in all 4 cases. Secretory granules were concentrated in cytoplasmic processes, and occasional microtubules observed in cases 1, 2, and 3 were common features of 1) neuroblastoma,28,31 2) paraganglioma,18 and 3) carcinoid tumor.3,21,38 Carcinoma of the ectopic pituitary gland was also suspected in cases 1 and 2, but the presence of an ectopic pituitary gland in the paranasal sinuses has not yet been confirmed. Non-fibrillary cytoplasm, solid alveoli, and the absence of neurites in cases 1, 2, and 4 were not the features of typical neuroblastoma. As the distribution of paraganglion cells in the human fetus and newborn infant is con- siderably more extensive than in the adult,16 the occurrence of paragangliomas in these locations, where no paraganglion has been described in the adult, cannot be completely denied. The incidence of malignant carotid body tumors is very low,13 and 6.4% in a review of 500 cases of carotid body tumor.34
However, incidence may depend upon the length of follow-up.25 Although a few functioning paragangliomas secreting catecholamines were reported,7,17.19 there were very few cases in which catecholamine-secreting cells were demonstrated by formalin-induced fluores- cence.5.19 Our failure to detect catecholamines in 2 cases may be due to the absence of functioning cells.
32
470
170
12
12
87
Carcinoid tumor is believed to arise from Kultschit- zky cells, which are diffusely distributed in bronchi, in the gastrointestinal tract, and in some other organs such as the bile10 and pancreatic ducts,11 the urinary bladder,14 and the uterine cervix.15.36 However, the presence of such cells and the occurrence of carcinoid tumors in the nasal cavity and paranasal sinuses have rarely been described,40 and the tumor in this location has not been intensively investigated. In preliminary examination of the adult nasal and paranasal mucosa in 20 surgical cases, we could not find any argyrophil or argentaffin cells. Examination of the mucosa of fetal and newborn sinuses will throw some light on this problem.
Membrane-bound, electron-dense cored granules measuring 100 to 300 nm in diameter are considered to contain polypeptides and/or biogenic amines. The secretory granules cannot be differentiated from normal endocrine-type small granules of polypeptide secretion or from endocrine-type granules in neuro- blastoma,28,31 medullary carcinoma of the thyroid,23 small cell carcinoma of the lung,6,17,20,22 some types of islet cell8 and carcinoid tumors.3.4 Although his- tological features and the secretory granules observed are very similar to those of some carcinoid tumors, none of the following features was observed in four tumors reported here: acinar differentiation, mucus production, the more polarized nuclear arrange- ment at the periphery of cell nests or typical desmo- somes associated with bundles of tonofilaments. In studies of small cell carcinoma of the lung, many in- vestigators6.20.22 showed that the tumor cells possessed endocrine-type secretory granules and a small number of microtubules, leading them to interpret the cells to be of neuroendocrine type. Atypical bronchial car- cinoid suggested by Arrigoni et al.4 may also be in- cluded in this category. Tumors of cases 1 and 2 pos- sessed certain features of carcinoid, anaplastic small cell carcinoma of the lung, poorly differentiated para- ganglioma, or neuroblastoma, but yet appear to lack conclusive evidence to establish definitive diagnoses. The tumor of case 3 seems to have more character- istics of a neurogenic tumor. In case 4, anaplastic carcinoma was the diagnosis made on a small biopsy specimen. However, after detection of calcitonin in tumor tissue of the autopsy material, the possibility of an atypical carcinoid tumor was considered, al- though argyrophil cells were absent, and endocrine secretory granules were infrequently observed. There- fore, we feel that it would be better at present to lump these four tumors under the broad category of neu- roendocrine carcinoma.
Endocrinological data with high plasma ACTH and ß-MSH in case 1 indicate ectopic ACTH-MSH pro-
duction by the tumor. Marked adrenocortical hyper- plasia and Crooke’s change of the pituitary gland also support this possibility. Negative immunostaining for ACTH may not indicate its absence in the tumor, be- cause many malignancies, in which definite ACTH pro- duction was confirmed by radioimmunoassay of tumor tissue, did not show positive immunostaining. 22.23,24
In case 4, a high plasma CT level, an appreciable amount of CT in the metastatic tumor and the presence of CT-containing cells verified by immunohistochem- istry suggested that the tumor might have produced calcitonin and belonged to the APUD (amine pre- cursor uptake and decarboxylation) series.27.33 We1 de- scribed a tumor group that probably derived from APUD cells28 including medullary carcinoma of the thyroid, carcinoid, small cell carcinoma of the lung, islet cell tumor, and pheochromocytoma and that contained appreciable amounts of CT in 30 of 31 tumors (97%) in contrast to only 20 of 54 (34%) in the non-APUD tumor group.
In case 4, the coexistence of hypercalcemia and high levels of plasma and tumor CT deserves explana- tion. Hypercalcemia occurs frequently in a wide variety of malignant tumors. On occasion, tumors are known to produce PTH and PTH-like substances,29 but PTH has not been detected in case 4. Although plasma CT levels were high in about 30% of hypercalcemic pa- tients with malignant neoplasms (unpublished data), a high plasma level such as 4600 pg/ml secondary to hypercalcemic states has never been reported. A high CT content and CT-positive cells in the tumor could explain the elevated plasma level of CT, which indi- cated production and secretion by tumor cells. C-cell hyperplasia in the thyroid in this case may be second- ary to hypercalcemia caused by bone metastases. The inherent mechanism of C-cell hyperplasia in the non- neoplastic thyroid of many pathologic conditions re- mains to be elucidated.
All four tumors presented herein should be called neuroendocrine cell carcinoma of the paranasal sinus, based on histological, ultrastructural, histochemical and endocrinological data.
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