—  SHORT COURSE #12  —

Molecular Analyses in Endocrine Pathology
Dr. George Kontogeorgos
Dr. Robert Yoshiyuki Osamura
Dr. Jennifer Hunt

Section 2 - Case Reports: Case 1

George Kontogeorgos
Department of Pathology,
G. Gennimatas General Hospital,
Athens, Hellas


Case 1
A 33-yr-old woman was referred to G. Gennimatas General Hospital of Athens with rapidly developing symptoms/signs of hyperandrogenism, secondary amenorrhea, infertility and markedly elevated serum LH and androgen. Her menarche was at the age of 15 years; after a period of regular menstruation she developed oligomenorrhea and hirsutism and the diagnosis of PCOS was made. At the age of 22 years, following a 3-month treatment with oral clomiphen citrate and intramuscular β-hCG injections, the patient became pregnant and underwent a successful pregnancy and delivery. After delivery, she again developed oligomenorrhea and despite a 4-years treatment with the same scheme, she failed to conceive again. Two years before this admission she developed severe acne, deepening of her voice, amenorrhea and worsening hirsutism. Endocrine investigations revealed high serum androgen and LH levels, but normal serum DHEAS. Ovarian ultrasonography revealed a 2.8 cm in diameter left ovarian solid tumor and slight enlargement of the right ovary. Computer tomography (CT) and magnetic resonance imaging (MRI) of the pituitary were normal. The patient underwent resection of the left ovarian tumor and a wide wedge resection of the right ovary.


Case 1 - Figure 1 - Histology of ovarian luteinized granulosa-theca cell tumor (H&E 10X).
Case 1 - Figure 2 - Typical tumor composed of sheets of ovoid or polyhedral cells with plump acidophilic cytoplasm (H&E 20X).
Case 1 - Figure 3 - With higher magnification, the cells display round nuclei with conspicuous nucleolus (H&E 40X).

Case 1 - Figure 4 - Strong and diffuse cytoplasmic immunoreactivity for -inhibin (ABC 20X).
Case 1 - Figure 5 - Strong and diffuse cytoplasmic immunoreactivity for -inhibin (ABC 20X).

By light microscopy, the ovarian tumor was composed of clusters or sheets of ovoid or polyhedral cells with plump acidophilic cytoplasm and discerning cytoplasmic borders (Fig. 1,2,3). The nuclei were small, round possessing small nucleoli. By immunohistochemistry, the tumor cells were selectively positive for α- and β-inhibin showing strong and diffuse cytoplasmic reactivity (Fig. 4,5). The adjacent parenchyma included multiple atretic cystic follicles showing luteinization of the inner theca at various stages of regression. In addition, multiple primary ovarian follicles were noted. The initially resected right ovarian wedge showed foci of nodular hyperthecosis. Based on the histological findings, the diagnosis of luteinized granulosa-theca cell tumor was made.

Postoperatively, androgen serum levels markedly decreased, but serum LH levels remained markedly elevated. As pituitary imaging excluded the presence of an LH-secreting pituitary tumor a presumptive diagnosis of severe PCOS was made and the patient was started on treatment with a long acting GnRH analogue. However, after a 3-month treatment there was no reduction of the elevated LH serum levels, whereas serum androgens rose to the pre-operative levels. Although imaging with helical CT-scan of the chest, CT and MRI of the abdomen and scintigraphy with 111In-octreotide scan were suggested in order to localize the tumor, the patient decided to proceed to a right ovariectomy; histological findings were also consistent with a typical luteinized granulosa-theca cell tumor.


Case 1 - Figure 6 - This abdominal MRI shows a tumor at the tail of the pancreas, hypointense on T1-weighted images.
Case 1 - Figure 7 - On T2-weighted images the well-demarcated tumor appeared hyperintense.
Case 1 - Figure 8 - The octreoscan shows an abdominal of [111In]octreotide uptake corresponding to a 7-cm tumor at the tail of the pancreas.

On admission, a repeated MRI scan of the pituitary showed no evidence of a pituitary lesion. Abdominal CT and MRI scans revealed normal adrenal glands and a 7 cm in diameter tumor at the tail of the pancreas (Fig. 6,7), which corresponded to a well-demarcated area of intense uptake on 111In-octreotide scanning (Fig. 8). In view of these findings a presumptive diagnosis of ectopic LH secretion from a pancreatic endocrine tumor possessing somatostatin receptors was made. The patient underwent surgical exploration and removal of the pancreatic tumor.


Case 1 - Figure 9 - Low magnification of an endocrine tumor separated by thick fibrous band from the adjacent to pancreatic parenchyma (H&E 1X).
Case 1 - Figure 10 - Typical histology of endocrine tumor consisted of highly cellular anastomosing solid nests or trabecules, separated by delicate vascularized connecting tissue (H&E 10X).
Case 1 - Figure 11 - Small round tumor cells with pseudorosette formation (H&E 20X).

Case 1 - Figure 12 - Spherical nucleus with focally conspicuous nucleolus (H&E 20X).
Case 1 - Figure 13 - Area of tumor cells with prominent nucleoli (H&E 40X).

The pancreatic tumor admitted for histology was ovoid well-demarcated and encapsulated measuring 6.5X4.5X4.5 cm. On sectioning, it was solid, soft and yellow showing a homogeneous, partly lobular appearance. Histologic examination was consistent with the diagnosis of a well-differentiated pancreatic endocrine tumor. The tumor consisted of highly cellular solid nests or anastomosing trabecules, separated by delicate vascularized connecting tissue (Fig. 9,10,11). The tumor cells were small round with spherical nucleus and focally conspicuous nucleolus (Fig. 12,13).


Case 1 - Figure 14 - The tumor was diffusely positive for synaptophysine (ABC 2.5X).
Case 1 - Figure 15 - Higher magnification shows strong reactivity for synaptophysine (ABC 10X).
Case 1 - Figure 16 - Extensive immunoreactivity of tumor cells for -LH (ABC 10X).

Case 1 - Figure 17 - Higher magnification of -LH immunopositive tumor cells (ABC 10X).
Case 1 - Figure 18 - Extensive immunoreactivity of tumor cells for -SU (ABC 10X).
Case 1 - Figure 19 - Higher magnification of SU immunopositive tumor cells ABC X

Mitotic figures were rare. The tumor was immunoreactive for synaptophysin CGR and NSE (Fig. 14,15). The tumor cells were strongly immunoreactive for β-LH (Fig. 16,17) and α-SU (Fig. 18,19).


Case 1 - Figure 20 - Sustentacular cells positive for S-100 protein (ABC10X).
Case 1 - Figure 21 - Low Ki-67 labeling index as estimated at <1% (ABC10X).
Case 1 - Figure 22 - Electron microscopy of endocrine tumor cells with moderate development of membranous organelles, and scarce and small secretory granules mostly accumulated in the cytoplasmic processes (11,000X).

Case 1 - Figure 23 - -LH mRNA expression in tumor cells by in situ hybridization (20X).
Case 1 - Figure 24 - Expression of -SU mRNA in tumor cells by in situ hybridization (20X).

Immunostain for S-100 protein disclosed several scattered cells representing sustentacular cells (Fig. 20). The Ki-67 labeling index was estimated at <1% (Fig. 21). Immunostains were negative for insulin, glucagon, somatostatin, PP, VIP, bombesin, hCG, GH, PRL, ACTH, β-TSH and β-FSH. By electron microscopy, the tumor consisted of polyhedral or polar cells with spherical nuclei. The cytoplasm contained moderately developed Golgi apparatus and several parallel profiles of rough endoplasmic reticulum. A fair number of mitochondria and scarce secretory granules of 100 to 150 nm mostly accumulated in the cytoplasmic processes (Fig. 22). In situ hybridization showed positivity in the tumor cells with the β-LH α-SU mRNA antisense probes, whereas the sense probes produced no staining (Fig. 23,24). Experiments of dispersed cell cultures following incubation with somatostatin resulted in suppression of LH secretion by approximately 90% to 95%.

Postoperatively, serum LH levels significantly decreased, but still remained within the postmenopausal range . After 2-month administration of long-acting GnRH analog (triptorelin depot), serum LH levels decreased significantly, but failed to suppress completely, suggesting that part of the tumor was left behind. A subsequent whole body octreoscan and abdominal MRI revealed a residual tumor of 1.5 cm in the pancreatic tail. During the last 5 yr follow-up, no change in the size of the remaining tumor was noted.

Discussion
Pancreatic endocrine tumors are rare neoplasms that occur either sporadically or as part of the multiple endocrine neoplasia type 1 syndrome. According to the 2004 WHO classification they represent 1-2% of all pancreatic neoplasms; their reported incidence is estimated at 1/100,000 population per year [1].

Approx. 40% of them are nonsynsdrominc (nonfunctioning). Functioning tumors secrete a variety of peptide hormones; hypersecretion of such peptides is associated with characteristic clinical syndromes. Among them, insulinomas represent the most common, followed by gastrinomas, glucagonomas, VIPomas and somatostatinomas [1]. Pancreatic endocrine tumors may also secrete peptide hormones that originate from cells eutopically expressed in endocrine pancreatic tissue [2].

Several examples of Cushing's syndrome due to the ectopicsecretion of either ACTH or CRH and cases of acromegaly due to GHRH secretionhave been documented. Other less commonly secreted peptide hormones, such as growth hormone, parathyroid hormone-related protein, calcitonin, calcitonin gene related peptide, pro-opiomelanocortin-derived peptides, neuropeptide Yand GHrelin have also been described [3, 4, 5, 6, 7, 8, 9, 10]. Until recently only one case of documented ectopic LH production from a pancreatic endocrine tumor in a 40-year-old woman has been reported [11].

Based on their histology, pancreatic endocrine tumors are divided into three categories: Well-differentiated endocrine tumors, and well-differentiated and poorly differentiated endocrine carcinomas. Well-differentiated tumors are confined to the pancreas with no vascular or perineural invasion and have a rather "benign" behavior. They are composed of relatively uniform cells with finely granular cytoplasm and spherical nuclei, often with conspicuous nucleoli. They form solid trabecular or glandular nests infrequently with pseudorosette arrangement. Typically the Ki-67 (clone MIB-1) index is <2%. The tumor size is crucial for biological behavior. Tumors measuring >2cm or showing Ki-67 >2% or showing vascular or perineural invasion, are expected to have uncertain behavior, even confined to the parenchyma. In general, a tumor size >2cm indicates increased risk for malignancy, whereas tumors >3 cm, as a rule, are malignant. Nonsyndromic tumors are often larger that 2 cm in diameter. Mixed endocrine-exocrine tumors are uncommon; their biological behavior depends on their exocrine cell component [1].

Ovarian stromal hyperthecosis is characterized by varying degrees of luteinized stromal cell proliferation after sustain gonadotropin stimulation. Luteinized granulosa-theca cell tumors of the ovaries are rare steroid producing lesions, under 3 cm in size, occurring mostly unilaterally in postmenopausal women. Secretion of bioactive hCG has been shown to be a strong stimulus of ovarian or testicular steroidogenesis. Similarly to hCG secreting trophoblastic tumors, pregnancy can induce extensive hyperthecosis and luteinization of the ovarian stroma and rarely it may result to granulosa-theca cell tumor, also known as pregnancy thecoma or luteoma. The present case offers a unique opportunity to investigate the effect of long-standing elevated ectopically produced bioactive LH, on ovarian morphology and function in a woman of the reproductive age [12, 13, 14, 15].

In summary, we have reported a rare case of a pancreatic endocrine tumor secreting bioactive LH leading to luteinized granulosa-theca cell tumors of both ovaries.

References
  1. Heitz PU, Dayal Y, Komminoth P, Bordi C, Perren A, Lechago J, Klimstra DS, Centeno BA, Klöppel G, in: DeLellis RA, Lloyd RV, Heitz PU, Eng C. (Eds.). WHO classification of tumours. Pathology and genetics. Tumours of endocrine organs (pp. 10-13). Lyon, France: IARC Press

  2. Rindi G, Capella C, Solcia E: Cell biology, clinicopathological profile and classification of gastroenteropancreatic endocrine tumors. J Mol Med 76: 413-420, 1998.

  3. Doga M, Bonadonna S, Burratin A, Giustina A: Ectopic secretion of growth hormone-releasing hormone (GHRH) in neuroendocrine tumors: relevant clinical aspects. Ann Oncol 12 Suppl 2:S89-94, 2001.

  4. Becker M, Aron DC: Ectopic ACTH syndrome and CRH-mediated Cushing' syndrome. Endocrinol Metab Clin North Am 23:585-606, 1994.

  5. Bouvet M, Nardin SR, Burton DW, Lee NC, Yang M, Wang X, Baranov E, Behling C, Moossa AR, Hpffman RM, Deftos LJ: Parathyroid hormone-related protein as a novel tumor marker in pancreatic adenocarcinoma. Pancreas 24:284-290, 2002.

  6. MacLeod MK, Vinic AL: Calcitonin immunoreactivity and hypercalcitoninemia in two patients with sporadic, nonfamilial, gastroenteropancreatic neuro-endocrine tumors. Surgery 111:484-488, 1992.

  7. Machens A, Haedecke J, Hinze R, Thomusch O, Schneyer U, Dralle H: Hypercalcitoninemia in a sporadic asymptomatic neuroendocrine tumor of the pancreatic tail. Dig Surg 17:522-524, 2000.

  8. Takami H, Shikata J, Kakudo K, Ito K: Calcitonin gene-related peptide in patients with endocrine tumors. J Surg Oncol 43:28-32, 1990.

  9. Waeber G, Hurlimann J, Nicod P, Grouzmann E: Immunolocalization of neuropeptide Y in human pancreatic endocrine tumors. Peptides 16:921-926, 1995.

  10. Corbetta S, Peracchi M, Cappiello V, Lania A, Lauri E, Beck-Peccoz P, Spada A: Circulating ghrelin levels in patients with pancreatic and gastrointestinal neuroendocrine tumors: identification of one pancreatic ghrelinoma. J Clin Endocrinol Metab 88:3117-3120, 2003.

  11. Hirshberg B, Conn PM, Uwaifo GI, Blauer KL, Clark BD, Nieman LK Ectopic luteinizing hormone secretion and anovulation. N Engl J Med 348:312-317, 2003.

  12. Nagamani M, Kaspar HG, Van Dinh T, Hannigan EV, Smith E: Hyperthecosis of the ovaries in a woman with a placental site trophoblastic tumor. Obstet Gynecol 76:931-935, 1990.

  13. Braithwaite SS, Erkman-Balis B, Avila TD: Postmenopausal virilization due to ovarian stromal hyperthecosis. J Clin Endocrinol Metab 46:295-300, 1978.

  14. Nagamani M, Lingold JC, Gomez LG, Garza JR: Clinical and hormonal studies in hyperthecosis of the ovaries. Fertil Steril 36:326-32, 1981.

  15. Young RH and Scally R: Ovarian sex cord-stromal tumors. Recent progress. Int J Gynecol Pathol 1:101-104, 1982.