A 63-year-old Saudi female, who was known to have hypertension and hyperlipidaemia, complained of fatigue, bone and muscle pain of several years of duration. She also had a history of recurrent nephrolithiasis. Her symptoms aggravated recently with several attacks of abdominal pain, nausea and vomiting. Neck examination revealed a left lower cervical mass, consistent with thyroid enlargement. Ultrasonography showed an enlarged left lobe with multiple hypoechoid and isoechoid nodules. One of them in the anterior-inferior aspect of the left lobe displayed a rim of calcification. Thyroid function test were within the normal range. Parathyroid imaging scan using 99m Tc-SestaMIBI-99c-Pertechnetate was consistent with scintigraphic features of a parathyroid adenoma (Figure ) in the region of left lower parathyroid gland. X-ray showed a "salt and pepper" skull and diffuse osteopenia. The fine needle aspiration (FNA) of the cervical mass was performed to evaluate left thyroid pathology and showed findings consistent with parathyroid neoplasm.
Figure 1 Parathyroid carcinoma occupying nearly the whole thyroid gland (Th). Thick sharply outlined bands of acellular collagenous tissue completely divide the tumor into irregular lobules. The tumor cells are arranged in diffuse masses, solid sheets, closely (more ...)
The patient was admitted and loaded with intravenous saline followed by Furosemide (10 mg) the day before surgery, which was able to bring the serum calcium to 3.59 on the day of surgery. The patient was taken to the operating room where a video-assisted exploration of the left neck was attempted. The surgeon's plan was to remove the diseased parathyroid gland and to take out the enlarged left hemithyroid, followed by frozen section evaluation. Total thyroidectomy is only performed in our clinic, if frozen sections yield evidence of a differentiated thyroid carcinoma greater than one centimeter in diameter. A normal lower left parathyroid gland was identified in the left thymic horn and preserved. The left thyroid lobe appeared enlarged and showed only one distinct nodule near the upper pole, which was sent for frozen section diagnosis. It turned out to be benign thyroid tissue. The surgeon decided then to remove the left thyroid lobe after full neck exploration including the superior mediastinum that revealed four benign central neck lymph nodes.
The pathology report described a thyroid lobe with a large tumor mass, measuring 6 × 5 cm and weighing 8.5 g. It appeared poorly circumscribed and of hard consistence. The mass showed a gray-whitish color with occasional foci of necrosis. A firm fibrous capsule surrounded it. The tumor occupied nearly the whole thyroid gland. Histology showed that thick, sharply outlined bands of collagenous tissue extended from the thickened capsule into the interior and completely divided the tumor into irregular lobules. The tumor cells were arranged in diffuse masses, solid sheets or closely packed nests of cells (Figure ). The tumor cells mostly showed round to ovoid, markedly enlarged nuclei with prominent nucleoli and clearly demarcated cytoplasm. Parts of the tumor consisted of cells with abundant, clear to eosinophillic cytoplasm, nuclear pleomorphism, marked cellular atypia. Occasionally macronucleoli and multinucleated giant cells were found. Mitoses were difficult to detect (2 per 10 high power fields). Scattered foci of necrosis and hemorrhage occurred at the periphery of the tumor. Capsular and vascular invasion with tumor cells was observed. Extra-capsular infiltration into the adjacent soft tissue and skeletal structures was also found. The tumor obviously had destroyed most of thyroid gland, leaving only a thin rim of normal thyroid tissue at the periphery (Figure ). None of the four lymph nodes draining the suspicious left parathyroid gland showed tumor involvement.
99m Tc SestaMIBI "A" nodular left lobe with sizeable lower pole. "B" prominent focal area in the lower pole of the left lobe of the thyroid. "C" substraction image show the prominent hot area (arrow) to be located in the hot area "A".
Clarification of the molecular pathogenesis of parathyroid carcinoma can aid in diagnostically difficult cases and may provide important clues for a more effective therapy. Therefore, tumor samples (one with a histologically verified parathyroid carcinoma and four additional specimen with histologically diagnosed parathyroid adenoma) were studied using in situ hybridization and immunocytochemistry with monoclonal antibodies. Formalin-fixed (3.7%), paraffin-embedded samples were used to localize mRNA transcripts for Cyclin D1. In situ hybridisation was performed as previously described. The sequences of the oligonucleotides were designed using the program Primer3 http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
. The sequences of the antisense oligonucleotides for Cyclin D1 was as follows: 5'gtt cct cgc aga cct cca gca tcc ag3' Acc.Nr: NM_053056 (NCBI). Immunohistochemical staining was performed with monoclonal anti-Cyclin D1 anti-body (Novocastra/Vector, Burlingame, CA, USA), at dilution of 1:100, using a standard avidin/biotin complex (ABC) method. Breast cancer cases known to express Cyclin D1 served as positive control for Cyclin D1. For negative control, non-immune serum was substituted for primary antibody.
Using in situ hybridization we found a weak to distinct signal for CD1 in the cytoplasm of glandular cells of the parathyroid adenoma tissue (Figure ). No staining was seen in the fibrous stroma that occasional formed broad bands in the neoplasm. Parathyroid carcinoma cells showed a variable staining with the probe used: Cells strongly positive from CD1 mRNA varied with only weakly stained tumor cells. (Figure ). The giant cells always displayed a strong signal for CD1-mRNA. All 4 adenoma samples showed a weak to distinct immunostaining for the CD1 protein in their cytoplasm. In about 20% of the cells, also a nuclear staining occurred. The corresponding stroma in the adenomas showed no immunoreactivity for CD1. Also with immunohistochemistry, parathyroid carcinoma cells showed a variable staining for CD1 and carcinoma cells strongly positive from CD1 mRNA varied with only weakly stained tumor cells (Figure ). Therefore, the most distinct feature distinguishing parathyroid adenoma from parathyroid carcinoma cells is the occurrence of strongly CD1 positive cells in the carcinoma areas.
In situ hybridization of Cyclin D1 in a parathyroid adenoma. Cytoplasm and nuclei contain a slight to distinct signal for CD1 mRNA. Bar: 100 μm.
An interesting pattern of CD1-mRNA distribution was seen in the carcinomatous areas, where strongly staining tumor cells varied with nearly CD1-negative ones. The inset shows giant cells distinctly positive for CD1-mRNA. Bar: 100 μm.
Figure 5 Immunohistochemical localization of CD1 in parathyroid adenoma (A) and parathyroid carcinoma (Ca). Whereas the adenoma cells generally showed a weak to moderate staining, parathyroid carcinoma cells displayed a variable immunostaining for CD1: carcinoma (more ...)
At the postoperative patient visit, a week after surgery, the serum calcium has dropped to 2.38 mg/dl (2.10–2.55), the parathormone (PTH) level has returned to normal values, however alkaline phosphatase remained high (628 U/L). CT scan of the brain and lungs revealed no evidence of metastasis. At follow-up three months after surgery, the patient appeared disease free with normal levels of PTH and calcaemia and improvement of the bone disease symptoms. The patient is now, two years from surgery, disease free and her serum calcium and parathormone level remained in the normal range.