This review on the applications of immunohistochemistry (IHC) to endocrine pathology will focus on the utilization of IHC in tumors of the thyroid, parathyroids, adrenals, and paraganglia in adults. For each organ, IHC to identify tumors of that site will be discussed with an emphasis on histologic findings and clinical scenarios in which IHC may be most helpful. Potential pitfalls due to lack of specificity of stains will also be reviewed. Utilization of IHC for subtyping tumors is most relevant in thyroid where it can help identify rare papillary thyroid carcinoma (PTC) variants such as cribriform morular variant and identify tumors with focal progression to poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma. BRAF V600E IHC is another valuable tool in thyroid diagnostics. For example, in ATC it is increasingly being used to rapidly identify patients with tumors that might respond to BRAF specific inhibitors. Although the vast majority of parathyroid specimens are adenomas that do not require IHC, for cases that are worrisome for carcinoma, IHC to evaluate for parafibromin expression (which is lost in the majority of parathyroid carcinomas) may be considered. Because of the rarity of tumors of the adrenal gland and the wide range in appearance of adrenocortical carcinoma (ACC), IHC is often used to confirm a diagnosis of ACC. In addition, assessment of IGF-2 and Ki67 proliferative index (PI) by MIB-1 can be used to support a diagnosis of low-grade ACC. This review will finish with the role of IHC in pheochromocytomas and paragangliomas. This is an exciting area as there are now IHC stains to aid in the identification of tumors arising as part of inherited syndromes. Finally, it should be noted that reviewing every IHC marker studied in tumors of the thyroid, parathyroid, adrenal, and paraganglia would not be possible in one review; therefore, this review focuses on IHC markers that are diagnostic heavy lifters, have results with established or emerging prognostic/treatment implications, or highlight the emerging role of IHC as a substitute for genetic testing.
Thyroid carcinomas include tumors of follicular-cell and C-cell origin. Follicular cell–derived tumors can be divided into differentiated thyroid carcinomas, poorly differentiated thyroid carcinoma, and anaplastic thyroid carcinoma. Differentiated follicular cell–derived tumors include PTC, follicular thyroid carcinoma, and Hurthle cell carcinoma. Medullary carcinoma is derived from C cells.
Differentiated Follicular Cell–derived Tumors
Together PTC, follicular thyroid carcinoma, and Hurthle cell carcinoma comprise over 90% of thyroid carcinomas. Most cases do not require IHC; however, the first scenario in which IHC may be used for these tumors is to confirm thyroid origin in the setting of a metastasis (Fig. 1). PAX8 is a paired-box gene that plays an important role in thyroid, kidney, and Müllerian tract development.1,2 Monoclonal PAX8 antibodies are specific for PAX8, whereas the polyclonal PAX8 antibody cross reacts with other PAX family members, including PAX3, PAX5, and PAX6.3 Diffuse, strong nuclear PAX8 expression is seen in 91% of thyroid tumors, including virtually all PTCs, follicular thyroid carcinomas, and Hurthle cell carcinomas.4,5 PAX8 is also positive in 90% of renal cell carcinomas, 90% to 100% of endometrial and high-grade ovarian serous carcinomas, 80% of thymic carcinomas, and 50% to 65% of metastatic well-differentiated pancreatic neuroendocrine tumors.4–9 However, it is negative in the vast majority of other tumors including breast carcinomas, lung adenocarcinomas (a subset of squamous cell carcinomas of the lung have been reported to express PAX8), head and neck carcinomas, and the vast majority of gastrointestinal carcinomas.4,6,8 PAX8 positivity is extremely helpful in identifying thyroid as a potential site of primary; however, because it is expressed in other carcinomas, it is often used in conjunction with TTF-1 and thyroglobulin. TTF-1 is a transcription factor normally expressed in adult thyroid and lung tissue. In the thyroid, it regulates the expression of multiple thyroid-specific proteins, such as thyroglobulin and thyroperoxidase.10 Diffuse, strong nuclear TTF-1 expression is seen in virtually all PTCs, follicular thyroid carcinomas, and Hurthle cell carcinomas.4,11,12 Although the combination of PAX8 and TTF-1 expression suggests spread from a thyroid primary, some gynecologic tumors (including a subset carcinomas of the uterus, ovary, and cervix) can be positive for both stains (Fig. 1).13–15 The glycoprotein thyroglobulin is a thyroid hormone precursor that is produced by follicular cells and is the main component of colloid. Thyroglobulin is highly specific for thyroid and it is expressed in >95% of PTCs and follicular thyroid carcinomas and in >90% of Hurthle cell carcinomas.16–19 Cytoplasmic thyroglobulin staining is often variable throughout the tumor.19,20 Although thyroglobulin expression is maintained in metastatic disease, because of the patchy staining pattern of thyroglobulin, absence of staining in a small biopsy sample does not entirely exclude a thyroid primary. If a small biopsy of a metastatic tumor with morphology suggestive of PTC is positive for PAX8 and TTF-1, but negative for thyroglobulin, a BRAF V600E stain could be considered. The BRAF V600E mutation is present in roughly 70% of classical and >95% of tall cell variant of PTC, but absent in benign thyroid tumors, follicular thyroid carcinoma, Hurthle cell carcinoma and medullary thyroid carcinoma.21,22 BRAF V600E IHC shows an excellent correlation with mutation status as assessed by molecular analysis in PTC.23–26 Although other tumors (including lung adenocarcinomas) can have a BRAF V600E mutation,27 the finding of cytoplasmic BRAF V600E immunohistochemical staining, along with PAX8 and TTF-1 expression, would be highly suggestive of spread from a thyroid primary. The expression of PAX8, TTF-1, thyroglobulin, and BRAF V600E in poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma is discussed below. Finally, it is worth noting that Napsin A, an IHC marker used to identify lung adenocarcinoma, is positive in approximately 5% of PTC (with higher rates in some variants such as tall cell variant and hobnail variant).28,29
IHC can also facilitate diagnosis of uncommon PTC variants, such as cribriform morular variant of PTC and hobnail variant of PTC (Fig. 2). Cribriform morular variant of PTC accounts for <1% of thyroid tumors. Although cribriform morular variant of PTC can occur sporadically, identification of this variant is important because approximately 40% of cases are associated with familial adenomatous polyposis (FAP).30 Thus, a diagnosis of cribriform morular variant of PTC should raise the possibility of this familial tumor syndrome. In a series by Ito et al,30 the diagnosis of cribriform morular variant of PTC was the first detected manifestation of FAP in 42% of the cohort, a finding that emphasizes the importance of recognizing this tumor at the time of thyroidectomy. Cribriform morular variant of PTC is characterized histologically by its cribriform architecture, lack of associated colloid, and morules. Nuclear features of PTC are variably present. FAP-associated cribriform morular variant of PTC is often multifocal. This tumor can be challenging to recognize when morules are not evident and when a papillary architecture predominates. In such cases, the main pitfall is rendering a diagnosis of a classical PTC. In other cases, because of diffuse stromal hyalinization, the tumor may be confused with a hyalinizing trabecular tumor. IHC for β-catenin can be used to confirm the diagnosis of cribriform morular variant of PTC.31–33 FAP-associated cribriform morular variant of PTC has an underlying germline APC mutation. Sporadic cribriform morular variant of PTC can have somatic APC mutations or mutations in other genes in the Wnt signaling pathway, including CTNNB1 (the gene that encodes β-catenin).34,35 APC and CTNNB1 mutations result in nuclear and cytoplasmic accumulation of β-catenin in cribriform morular variant of PTC, in contrast to the membranous staining see in normal thyroid parenchyma. Cytoplasmic and nuclear β-catenin staining has been shown to be specific for cribriform morular variant of PTC.32
Hobnail variant comprises approximately 1% of PTCs, but is important to recognize because of its aggressive clinical course (most patients die in <5 y).36–38 The histologic features of this tumor have been well-described.36–38 They are usually large, invasive tumors with a papillary or micropapillary architecture (though areas of follicular architecture may also be present). They are characterized by nuclear pseudostratification and a loss of cellular polarity with enlarged nuclei jutting out from the apical surface. The nuclei are often more atypical than those of classical PTC; however, nuclear features of PTC are maintained. Because hobnail variant is rare and because it has features similar to those of classical PTC (ie, a papillary architecture and nuclear features of PTC), it can be mistaken for classical PTC. Conversely, because classical PTC can show degenerative changes resulting in a hobnail appearance, classical PTC can be confused with hobnail variant. IHC can be helpful in rendering a diagnosis of hobnail variant. Similar to classical PTC, hobnail variant is positive for PAX8, TTF-1, and thyroglobulin, though metastatic disease can show loss of thyroglobulin expression.37 The Ki67 PI is below 5% in 95% of differentiated thyroid carcinomas.39 Although not all reported cases of hobnail variant have a Ki67 PI above 5%, the mean Ki67 PI is 8% to 10%.37,38 Therefore, an elevated Ki67 PI supports the diagnosis of hobnail variant. Similarly, whereas p53 overexpression and TP53 mutations are essentially absent in classical PTC,21 just over half of hobnail variants harbor a TP53 mutation.40 As a result, overexpression of p53 also can be used to support a diagnosis of hobnail variant (it should be strong, diffuse positivity in contrast to the patchy, weak staining pattern seen with wild-type p53).
IHC can also be used in the evaluation of a few tumors in the differential with PTC, including noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), hyalinizing trabecular tumor, and primary secretory carcinoma of the thyroid (Fig. 2). NIFTP is an encapsulated or well-circumscribed follicular cell–derived tumor that lacks invasive or infiltrative growth, has an entirely follicular architecture, and has “papillary-like” nuclear features.41 BRAF V600E IHC can be used to help differentiate follicular-predominant classical PTC from NIFTP. Although a diagnosis of NIFTP does not require molecular analysis, if a BRAF V600E mutation is detected, a diagnosis of NIFTP should probably be avoided.42,43 This approach is advised based on the fact that the studies establishing the indolent biology of NIFTP lacked tumors with a BRAF V600E mutation.41,44–46 In addition, the definition of NIFTP is quickly evolving to exclude cases with any papillae.42,43 Many, or perhaps most, reported cases of NIFTP with a BRAF V600E mutation have a small papillary component (<1%).47 Therefore, BRAF V600E IHC could be considered when evaluating cases of potential NIFTP that have questionable papillae or other features that are more frequently seen in classical PTC such as pseudoinclusions or diffuse, pronounced nuclear features of PTC.44 Although the absence of BRAF V600E staining does not definitively establish a tumor as NIFTP, positive staining can be used to exclude the diagnosis. Finally, it is worth noting that with the introduction of NIFTP, IHC stains for CK19, HBME-1, and galectin-3 (stains that had previously been used with variable purported success in the identification of follicular variant of PTC),48,49 now have less diagnostic utility since they would not be expected to differentiate PTC from NIFTP.
Hyalinizing trabecular tumor is a benign follicular cell–derived tumor that is either encapsulated or well circumscribed, has a variable amount of hyalinized stroma, is composed of trabeculae or nests of polygonal to elongated cells with abundant eosinophilic to clear cytoplasm (sometimes with faint yellow granules, termed yellow bodies) and with nuclei with clearing, contour irregularities, grooves, and pseudoinclusions.50,51 Because the nuclear features are similar to those of PTC, hyalinizing trabecular tumor can be mistaken for PTC. However, hyalinizing trabecular tumor demonstrates membranous MIB-1 staining that is not seen with PTC or any other thyroid tumors.52,53 This staining pattern is only seen with the MIB-1 antibody to Ki67 and only when the reaction is carried out at room temperature.54
The recently described primary secretory of the carcinoma of the thyroid (harboring an ETV6-NTRK3 fusion) is histologically similar to its salivary gland counterpart and is another PTC mimic.55–57 These tumors are infiltrative and have abundant hyalinized stroma. They have a predominantly cribriform or microcystic architecture with associated colloid-like material, though papillary or micropapillary growth can be present. The tumor cells are uniform with a moderate amount of eosinophilic cytoplasm and frequent intracytoplasmic vacuoles with eosinophilic material. The nuclei are enlarged, have vesicular chromatin, and an eosinophilic central nucleolus. Nuclear features similar to those of PTC can be striking. Some cases can show high-grade features such as increased mitotic activity or necrosis.56,58 The microcystic architecture, colloid-like material, and nuclear features all can contribute to a misdiagnosis of PTC. If a diagnosis of secretory carcinoma of the thyroid is considered, IHC can be used to confirm the histologic impression. These tumors are usually positive for PAX8 (though at a decreased intensity compared to PTC); however, in contrast to PTC, secretory carcinoma lacks expression of TTF-1 and thyroglobulin, but instead is positive for mammaglobin and variably positive for S100, GATA3, and p63.55–58
IHC is not routinely used in PTC, follicular thyroid carcinoma, or Hurthle cell carcinoma for prognostic or predictive purposes. However, it is worth discussing BRAF V600E IHC in this context. As indicated previously, the BRAF V600E mutation is present in approximately 70% of classical and >95% of tall cell variant of PTC.18 In a retrospective study of 1849 patients with PTC, Xing et al59 found that the presence of a BRAF V600E mutation was significantly associated with increased cancer-related mortality. Although this association lost significance in multivariate analysis, they found a significant interaction between the presence of a BRAF V600E mutation and other conventional clinicopathologic parameters in predicting mortality risk. Moreover, they found that conventionally defined low-risk patients with tumors lacking a BRAF V600E mutation had a negligible mortality rate. In a meta-analysis that included 2470 PTC patients, presence of a BRAF V600E mutation was associated with a significantly higher recurrence rate.60 On the basis of the results of these studies and others, the American Thyroid Association concluded that BRAF V600E mutation status considered in the context of other clinicopathologic parameters incrementally improves risk stratification.61 However, because the clinical implications of this incremental difference are not clear, the American Thyroid Association does not recommend routine assessment of BRAF V600E mutation status for initial postoperative risk stratification of PTC. Nonetheless, based on the potential improvement in risk stratification, some groups choose to interrogate BRAF V600E mutation status by IHC for all cases of PTC. Finally, IHC can also be used as a screening tool for other molecular alterations in PTC. For example, IHC for ALK has been shown to be highly sensitive for an underlying ALK translocation in PTC (ALK rearrangements are found in about 2% of PTCs).62
Poorly Differentiated Thyroid Carcinoma and Anaplastic (Undifferentiated) Thyroid Carcinoma
Poorly differentiated thyroid carcinoma accounts for approximately 2% of thyroid malignancies in the United States and has a 5-year and 10-year survival rate of roughly 70% and 50%, respectively.63 Poorly differentiated thyroid carcinoma is defined as a tumor with a solid, insular, or trabecular growth pattern that lacks the nuclear features of PTC, and demonstrates the presence of at least one of the three findings: convoluted nuclei, a mitotic count ≥3 per 10 HPFs, and necrosis.64,65 IHC is not required to render a diagnosis of poorly differentiated thyroid carcinoma; however, it can be used to support the diagnosis and can be very helpful for cases in which only part of the tumor has progressed to poorly differentiated thyroid carcinoma (usually in the setting of follicular thyroid carcinoma or Hurthle cell carcinoma) (Fig. 3). Poorly differentiated thyroid carcinomas are diffusely positive for PAX8 and TTF-1, though the intensity of TTF-1 staining is often reduced compared to that of differentiated follicular cell–derived tumors.4,65 Thyroglobulin is positive in approximately 90% of poorly differentiated thyroid carcinoma and usually has a dot-like paranuclear pattern.63,65 Although this dot-like paranuclear pattern is not entirely specific for poorly differentiated thyroid carcinoma,65 it can be very helpful in highlighting areas of progression to poorly differentiated thyroid carcinoma. In a study by Asioli et al,63 the mean Ki67 PI of poorly differentiated thyroid carcinoma was 13%. Although not all poorly differentiated thyroid carcinomas have a Ki67 PI >5%, a Ki67 PI >5% can be used to support the diagnosis. The results of p53 IHC in poorly differentiated thyroid carcinoma have been variable.63,66 Given that, a recent study interrogating poorly differentiated thyroid carcinoma by next-generation sequencing found a TP53 mutation rate of only 8%,21 IHC for p53 is unlikely to contribute significantly to the diagnosis.
Anaplastic thyroid carcinoma accounts for approximately 2% of all thyroid carcinomas in the United States. There is a considerable variation in the microscopic appearance of anaplastic thyroid carcinoma. Anaplastic thyroid carcinoma can have a spindle cell, pleomorphic giant cell, epithelioid, or squamoid morphology (or a combination of these morphologies). Although anaplastic thyroid carcinoma is usually a straight-forward diagnosis based on marked pleomorphism, high mitotic rate, and associated necrosis. There are a couple of scenarios in which IHC may be required to render a diagnosis of anaplastic thyroid carcinoma. The first is in the metastatic setting. Because of the undifferentiated nature of this tumor, it morphologically cannot be recognized as being of thyroid origin. PAX8 expression is maintained in up to 80% of anaplastic thyroid carcinomas,4,67,68 making it the most sensitive IHC marker for anaplastic thyroid carcinoma (Fig. 4). Bishop et al67 reported PAX 8 expression in 76% of anaplastic thyroid carcinoma overall, with 100% positivity in squamoid anaplastic thyroid carcinoma (and with negative PAX8 staining in all head and neck squamous cell carcinomas they tested). In contrast, TTF-1 is expressed only focally in approximately 15% of anaplastic thyroid carcinoma, and thyroglobulin is essentially always negative.4,68,69 Keratin expression is also variable in anaplastic thyroid carcinoma, with tumors with a spindle cell morphology showing the lowest rate of keratin expression, often with only scattered positive cells.69 Because 25% to 45% of anaplastic thyroid carcinoma have a BRAF V600E mutation,70,71 BRAF V600E IHC may also be used to support a diagnosis of anaplastic thyroid carcinoma (Fig. 4). Although BRAF V600E staining is not specific for anaplastic thyroid carcinoma in the setting of an undifferentiated tumor, positive staining could be used along with clinical history to support the diagnosis. The second scenario in which ancillary IHC may be required to render a diagnosis of anaplastic thyroid carcinoma is when a tumor shows only focal progression to anaplastic thyroid carcinoma. In these cases, demonstrating maintained PAX8 expression with reduction/loss of TTF-1 and thyroglobulin can support dedifferentiation. In addition, demonstrating a Ki67 PI over 30% also supports the diagnosis.64 In contrast to the infrequent TP53 mutations present in poorly differentiated thyroid carcinoma, 25% to 70% of anaplastic thyroid carcinomas have been reported to harbor a TP53 mutation;70,71 thus, overexpression of p53 can also be used to support the diagnosis of anaplastic thyroid carcinoma. Finally, p63 can be used to highlight squamoid anaplastic components. Although p63 may also be expressed in squamous metaplasia associated with PTC, in contrast to squamous metaplasia, a squamoid anaplastic thyroid carcinoma component will express p63 in conjunction with an elevated Ki67 PI.
IHC may also be used in poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma to guide treatment. BRAF V600E IHC has been shown to correlate with BRAF mutation status assessed by molecular analysis in anaplastic thyroid carcinoma.72 Recent studies have reported promising results for BRAF V600E-mutant anaplastic thyroid carcinomas treated with selective BRAF inhibitors alone or in combination with a MEK inhibitor.73–75 Moreover, dabrafenib and trametinib (a BRAF and a MEK inhibitor, respectively) have recently been FDA approved for the treatment of BRAF V600E-mutant anaplastic thyroid carcinoma. Because IHC results are rapid and anaplastic thyroid carcinoma requires immediate treatment, BRAF V600E IHC is increasingly being utilized to guide treatment in anaplastic thyroid carcinoma patients. Finally, approaching 5% of poorly differentiated thyroid carcinomas and anaplastic thyroid carcinomas have been reported to harbor ALK translocations.76 IHC for ALK could be used in poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma to identify patients that might respond to the ALK inhibitor crizotinib.76
Medullary Thyroid Carcinoma
Medullary thyroid carcinoma, derived from C cells (the calcitonin-producing neuroendocrine cells of the thyroid), accounts for approximately 1% to 2% of thyroid tumors.77 It is usually a well-circumscribed tumor that has a solid architecture and round/polyhedral, plasmacytoid, or spindled cells. However, variants can exhibit a broad range in morphologies including tumors with deceptive papillary or glandular/tubular/follicular architecture. In addition, non-neoplastic thyroid follicles can be entrapped within the tumor, which can make a diagnosis of medullary thyroid carcinoma harder to render. In cases of variant morphology, or in the metastatic setting, IHC may be employed to support a diagnosis of medullary thyroid carcinoma. Approaching 90% of medullary thyroid carcinoma express TTF-1 and up to 75% express PAX8, though the staining for both markers is often weaker or more focal than that seen with differentiated follicular cell–derived tumors.4,6 Medullary thyroid carcinoma is negative for thyroglobulin, though staining must be interpreted with caution since thyroglobulin staining may be seen in non-neoplastic follicles entrapped within the tumor. Because medullary thyroid carcinoma is a neuroendocrine tumor, it stains with the neuroendocrine markers chromogranin A and synaptophysin. Chromogranin A, a major component of neuroendocrine cell secretory granules, is the most specific marker of neuroendocrine cells. Synaptophysin, present within neuroendocrine cell microvesicles, is less specific, but more sensitive for neuroendocrine tumors (especially high-grade neuroendocrine carcinomas). Consistent with their C-cell origin, virtually all medullary thyroid carcinomas express calcitonin.78,79 Although most tumors show diffuse positivity, a subset shows more focal expression. Calcitonin staining can also be used to evaluate for C-cell hyperplasia, a recognized precursor of medullary thyroid carcinoma in hereditary forms of thyroid carcinoma. Monoclonal CEA can be used to further support a diagnosis of medullary thyroid carcinoma in tumors with focal or borderline calcitonin expression since cytoplasmic expression is seen in medullary thyroid carcinoma but absent in other thyroid carcinomas.80 It is worth noting that stains for medullary thyroid carcinoma must be evaluated in the context of clinical history since each stain is not entirely specific. For example, TTF-1 stains a subset of neuroendocrine tumors of the lung (and many high-grade neuroendocrine carcinomas regardless of site of origin), PAX8 stains roughly half of well-differentiated pancreatic neuroendocrine tumors, and even calcitonin stains occasional pulmonary and pancreatic neuroendocrine tumors and rare parathyroid adenomas.7,9,81–85 Finally, solid cell nests are ultimobranchial body remnants that are incidental findings in thyroidectomy specimens. Although they are usually recognized histologically, they can be misinterpreted as a medullary microcarcinoma (or an incidental papillary microcarcinoma). If a differential of solid cell nests and medullary microcarcinoma is being considered, IHC is very helpful. Solid cell nests are positive for p40, p63, GATA3, and TTF-1, and occasionally monoclonal CEA, but negative for PAX8 and calcitonin.86
IHC may also be used to guide genetic testing in medullary thyroid carcinoma. Approximately a quarter of patients with medullary thyroid carcinoma have a germline RET mutation.77 RET mutations are also the most common somatic alteration (seen in roughly half of cases), with HRASQ61R being the second-most frequent somatic mutation.77,87 Reagh et al88 demonstrated that the mutation-specific antibody for NRASQ61R can be used to identify medullary thyroid carcinomas with an HRASQ61R mutation (because the amino acid sequences for HRAS and NRAS proteins at codon 61 are identical). Genetic evaluation is advocated for all patients with medullary thyroid carcinoma because of the significant rate of germline RET mutations. Because RET and RAS mutations are mutually exclusive in MTC, IHC for NRASQ61R could be utilized to help triage medullary thyroid carcinoma patients for genetic evaluation. Finally, IHC for ALK could potentially be used to identify rare ALK-rearranged medullary thyroid carcinomas.87 Ji et al87 recently reported a marked treatment response with the ALK inhibitor crizotinib in a case of medullary thyroid carcinoma with an EML4-ALK fusion, a finding that highlights the potential clinical impact of identification of ALK fusions.
Multiple Adenomatous Nodules and Cowden Syndrome
Cowden syndrome is an autosomal dominant disorder with an underlying germline mutation in PTEN that is characterized by the development of multiple hamartomas and an increased risk of carcinomas of the thyroid, breast, and uterus.89 Recognition of Cowden syndrome is important so that cancer screening and genetic counseling can be initiated. Pathologic findings in thyroidectomy specimens suggestive of Cowden syndrome include multiple adenomatous nodules, follicular adenomas, and nodular hyperplasia, with or without follicular thyroid carcinoma or PTC.90,91 Although this constellation of histologic findings in thyroidectomy specimens should raise the possibility of a diagnosis of Cowden syndrome, the findings are not specific. For example, radiation can produce similar findings, though there is usually more fibrosis and atrophy in the gland.92 Loss of PTEN expression by IHC in adenomatous nodules, in contrast to the nuclear and cytoplasmic staining seen in the background thyroid parenchyma and in adenomatous nodules in patients without Cowden syndrome, has been found to be both sensitive and specific for Cowden syndrome (Fig. 2).92 Interestingly, some Cowden syndrome cases show heterogeneous loss of expression in the adenomatous nodules, with some nodules showing complete loss of expression and others showing intact expression.
For parathyroid, IHC is probably used most frequently to identify intrathyroidal parathyroid tissue or parathyroid tissue in small biopsy specimens. In both situations parathyroid tissue can be confused with thyroid parenchyma because of a lack of an adipocytic component or an acinar architecture with intraluminal secretory material that mimics colloid. Because parathyroids are neuroendocrine tissues, they stain for chromogranin A and synaptophysin. In addition, parathyroid hormone (PTH) can be used, though its expression can be variable in intensity in parathyroid neoplasms and is often negative in parathyroid cysts.93,94 PTH is highly specific for parathyroid, though ectopic PTH expression can rarely be expressed in other tumors such as small cell carcinoma of the ovary.95 Parathyroid is negative for TTF-1 and thyroglobulin, but frequently positive for PAX8.91 In addition, it is positive for the transcription factor GATA3.96,97 Betts et al96 evaluated a range of parathyroid lesions, and found that all showed nuclear GATA3 staining. Finally, glial cells missing 2 (GCM2) is another transcription factor integral to parathyroid development that has been shown to be highly sensitive and specific for parathyroid tissue.93
A diagnosis of parathyroid carcinoma is based on unequivocal lymphovascular or perineural invasion, invasion into adjacent structures, or metastatic disease. Although parathyroid carcinomas are often clinically suspected because of more pronounced manifestations of hyperparathyroidism or because gross invasion is evident at the time of surgical resection, a significant number of cases are diagnostic challenges, both clinically and histologically. Germline inactivating mutations in the tumor suppressor gene CDC73 (previously termed HRPT2), the gene that encodes parafibromin, were identified as the cause of hyperparathyroidism-jaw tumor syndrome, an autosomal dominant disease associated with hyperparathyroidism, renal lesions, fibroosseus jaw tumors, and an increased risk of developing parathyroid carcinoma.98 Subsequently, it was shown that biallelic CDC73 mutations/inactivation are present in roughly 75% of sporadic parathyroid carcinomas and absent in virtually all adenomas (with the exception of cystic adenomas with a somatic mutation rate of 4% and parathyroid adenomas in patients with hyperparathyroidism-jaw tumor syndrome).99–101 Although the majority of CDC73 mutations in clinically sporadic parathyroid carcinomas are somatic, approximately 20% are germline, indicating that all patients with parathyroid carcinoma should undergo genetic evaluation.99,101 On the basis of the above findings, Tan et al102 generated a monoclonal parafibromin antibody and found that loss of parafibromin nuclear staining was highly sensitive and specific for parathyroid carcinoma (though loss of expression is also seen in parathyroid adenomas in patients with hyperparathyroidism-jaw tumor syndrome) (Fig. 5). Other groups have confirmed these findings.103–107 Gill et al105 found complete absence of nuclear staining in 73% sporadic parathyroid carcinomas, with focal weak staining in 18%. Only 1 parathyroid carcinoma in their study exhibited diffuse strong nuclear expression of parafibromin. In contrast, 98% of the non–HPT-JT-related benign parathyroids showed diffuse, strong nuclear positivity and 2% showing weak positive staining. On the basis of their findings they concluded that, in the correct clinical and pathologic context, complete absence of nuclear staining for parafibromin is essentially diagnostic of parathyroid carcinoma or a hyperparathyroidism-jaw tumor syndrome-related adenoma. Loss of parafibromin staining has not only been shown to be useful in detecting parathyroid carcinomas, there are some data to suggest that it may be useful in determining the malignant potential of atypical parathyroid adenomas.106 Atypical parathyroid adenomas lack definitive WHO criteria of malignancy, but have concerning histologic features, such as increased mitotic activity. Kruijff et al106 reported that none of the parafibromin-intact atypical adenomas in their cohort recurred; whereas the recurrence rate was 10% in atypical adenomas with loss of parafibromin expression. Gill et al108 have recently described distinctive morphologic features of parafibromin-deficient tumors. They found that parafibromin-deficient tumors (defined as tumors with complete loss of expression of nuclear parafibromin in all neoplastic cells with an internal positive control required) had extensive sheet-like growth, a prominent arborizing vasculature, and frequently, a thick capsule and microcystic change. In addition, the tumors were composed of cells with eosinophilic cytoplasm, enlarged nuclei with distinctive coarse chromatin, and perinuclear cytoplasmic clearing (Fig. 5). These finding suggest that morphologic findings could be used in conjunction with IHC results. Finally, it should be stated that although parafibromin IHC has promising clinical utility, it can be difficult to use clinically because of technical staining issues and because of differences in interpretation of staining. For example, Gill et al108 indicated that over a 12-year period of use, to achieve positive staining in internal control tissue, various dilutions, detection systems, and antigen retrieval protocols were required, with several attempts at different titers often required to achieve an interpretable result.
Ki67 PI has been shown to be significantly higher in parathyroid carcinomas versus adenomas. Lloyd et al109 showed that the mean Ki67 PI was 7.1 for carcinomas versus 2.4 for adenomas, with no patient with benign disease showing a Ki67 PI over 5.3%. Other groups have shown that adenomas rarely have a Ki67 PI >5% and that many carcinomas have a KI67 PI of >5%. However, the reported percentage of carcinomas with a Ki67 PI >5% varies from roughly 20% to 90%.107,110,111 Therefore, although a Ki67 PI >5% would help support a diagnosis of malignancy, a low Ki67 PI does not rule out the diagnosis (Fig. 5). Additional markers that have been studied in parathyroid tumors include galectin, PGP9.5, Rb, Bcl2, p27, hTERT, mdm2, and APC, among others. Although a discussion of these markers is beyond the scope of this section, their use has recently been thoroughly reviewed.112
The main tumors of the adrenal gland are adrenocortical adenomas, ACCs, and pheochromocytomas. Pheochromocytomas and paragangliomas are tumors of neural crest origin. By convention, pheochromocytomas refer to tumors arising from the adrenal medulla and paragangliomas arise from extra-adrenal chromaffin and nonchromaffin cells of the autonomic nervous system. Because pheochromocytomas and paragangliomas have virtually indistinguishable IHC profiles for stains used routinely in clinical practice, they will be discussed together.
Adrenocortical adenomas account for the majority of adrenal neoplasms. These tumors are generally not diagnostic dilemmas and do not require IHC. In contrast, because of the range in morphology of ACC, the fact that the adrenal is a frequent site of metastatic disease, and the fact that pheochromocytomas and ACCs can sometimes be difficult to differentiate, IHC is frequently used to confirm a diagnosis of ACC both within the adrenal and in the metastatic setting.113 ACCs are positive for SF-1 (steroidogenic factor-1), inhibin, synaptophysin, Melan-A, and calretinin and negative or weakly positive for keratins (Cam5.2 shows the most expression).9,114,115 SF-1, a transcription factor expressed in steroidogenic organs like the adrenal glands, testes, and ovaries, plays a key role in the regulation of steroid biosynthesis.116 SF-1 is an excellent diagnostic marker for ACC (Fig. 6). In a large study by Sbiera et al116 studying 167 ACC and 73 malignancies from nonsteroidogenic organs (including carcinomas of the kidney, lung, breast, colon, pancreas, liver, prostate, endometrium, and ovary and pheochromocytomas, melanomas, lymphomas, and a germ cell tumor), they reported a specificity of 100% and a sensitivity of 98% for ACC. Sangoi et al117 reported a slightly lower sensitivity (86%) for SF-1 in adrenocortical tumors. If a pathologist does not have access to SF1, then a panel of stains including inhibin, synaptophysin, Melan-A, and calretinin can be used. A panel is advised both because of decreased specificity and decreased sensitivity of these stains compared to SF-1, with reported sensitivities ranging from approximately 60% to 95%.114,115,117 SF-1, inhibin, synaptophysin, Melan-A, and calretinin have been reported to be positive in myxoid ACC (a rare ACC variant);118 however, the sarcomatoid variant of ACC has been reported to be negative for these stains (or demonstrate staining in only the nonsarcomatoid component).119 Chromogranin A is often used to differentiate pheochromocytoma from ACC (both ACCs and pheochromocytomas are positive for synaptophysin). Though the vast majority of ACCs are negative for chromogranin, rare cases of ACC may show focal expression.114 This finding highlights why panels should be utilized and why morphology and clinical history need to be considered when interpreting IHC results. Finally, PAX8 is very helpful in distinguishing ACC from renal cell carcinoma as PAX8 is virtually always negative in ACCs, but is positive in approximately 85% of metastatic renal cell carcinomas.114,117,120
IHC has also been reported to aid in the assessment of malignant potential of adrenocortical tumors and to have prognostic value in the setting of ACC. Although most ACCs are overtly malignant, IHC may be helpful in the setting of low-grade ACCs that are histologically more challenging to diagnose. The 2 most established IHC stains used to aid in the assessment of malignancy in adrenocortical tumors are IGF-2 and Ki67 PI assessed by MIB-1 (Fig. 6). Perturbation of the IGF2 (insulin-like growth factor -2) locus is one of the most consistent genetic changes seen in ACC, with approaching 90% of ACCs harboring alterations resulting in molecular overexpression of IGF-2.121,122 In 2005, Schmitt et al123 reported that perinuclear dot-like IGF-2 staining was virtually confined to ACCs. Although some groups reported IGF-2 staining in a subset of adrenocortical adenomas,124,125 2 subsequent studies have confirmed the diagnostic utility of IGF-2, emphasizing the importance of perinuclear dot-like/juxtanuclear Golgi-pattern of staining, which both groups reported in roughly 80% of ACCs and in none of the adrenocortical adenomas tested126,127 Significantly, IGF-2 positivity occurs in both low and high-grade ACCs indicating that IGF-2 can help identify low-grade ACCs. Ki67 is the second important IHC marker used for diagnostic purposes in adrenocortical tumors. Several groups have reported a higher Ki67 PI in ACCs than in adrenocortical adenomas.114,126,128–130 Schmitt et al123 found that 88% of ACCs had a Ki67 PI >5% whereas 95% of adenomas had a Ki67 PI <5%. However, it is important to recognize that not all low-grade ACCs have a Ki67 PI >5%.126 IGF-2 and Ki67 can be used together to evaluate potential low-grade ACCs. Soon et al127 evaluated a cohort of 41 adrenocortical adenomas and 23 ACCs and found that although the Ki67 PI was high (>5%) in only 70% of ACCs, positive staining for IGF2 and/or a high Ki67 PI identified 22 of 23 ACCs (96% sensitivity) but no adrenocortical adenomas (100% specificity). The Ki67 PI is also prognostic in ACCs.129,131,132 Beuschlein et al131 found that Ki67 proliferative indices of <10%, 10% to 19%, and ≥20% provided highly significant differences for both recurrence-free survival and overall survival. Moreover, the prognostic value of the Ki67 PI was maintained in multivariate analysis. As a result, Ki67 PI may inform treatment decisions for patients with ACC.133 It is important to note that although Ki67 PI has diagnostic and prognostic utility in adrenocortical tumors, there are problems with interobserver reproducibility.134 To improve the accuracy of assessment of Ki67 PI, it should not be estimated, rather it should be determined either by manual counting (dividing number of positive cells by the number of total cells in a printed image) or by automated image analysis. Although IGF-2 and Ki67 PI are the most widely used stains in the clinical evaluation of adrenocortical tumors, it is likely that list will expand. For example, IHC for mismatch repair proteins may become part of routine clinical practice since roughly 3% of ACCs have been reported to be associated with Lynch syndrome (a prevalence similar to that seen with endometrial and colorectal carcinoma).122,135
Pheochromocytomas and Paragangliomas
Pheochromocytomas and paragangliomas are positive for synaptophysin and chromogranin A. Keratins are almost always negative (with the exception of cauda equina paragangliomas).136,137 S100 highlight sustentacular cells (non-neoplastic support cells) of pheochromocytomas and paragangliomas, though it should be noted that sustentacular cells are not specific to pheochromocytomas and paragangliomas, but can also be seen in other well-differentiated neuroendocrine tumors.3 In a subset of cases, chromaffin cells may also show S100 positivity, though the staining is weaker than that seen in the sustentacular cells. Most pheochromocytomas do not require IHC to confirm the diagnosis; however, paragangliomas are often more diagnostically challenging and warrant use of IHC (with the main differential being a metastatic well-differentiated neuroendocrine tumor) (Fig. 7). In addition to keratins, GATA3 and PHOX2B IHC can be used to differentiate paragangliomas/pheochromocytoma and well-differentiated neuroendocrine tumors. Miettinen et al138 reported GATA3 positivity in 92% of pheochromocytomas and 82% of paragangliomas, with no staining seen in small cell carcinoma and well-differentiated neuroendocrine tumors of the lung, small intestine, and pancreas. PHOX2B is a transcription factor expressed in tissues of neural crest origin.139 Recently, Lee et al140 reported PHOX2B expression in 32% of pheochromocytomas and 47% of paragangliomas (with a higher rate of staining seen when whole slides were tested instead of tissue microarrays). Moreover, although they found PHOX2B expression in 7% of poorly differentiated neuroendocrine carcinomas, all 250 well-differentiated neuroendocrine tumors in their cohort were negative for PHOX2B.
IHC can also be used to help identify pheochromocytomas and paragangliomas arising in the setting of a hereditary syndrome (Fig. 7). We now know that over a third of pheochromocytomas and paragangliomas occur as part of a hereditary syndrome.141,142 Although VHL and RET may be the most widely known genes associated the development of these tumors, the most prevalent germline mutations are in the succinate dehydrogenase (SDH) genes.143 SDH is an enzyme complex localized to the inner mitochondrial membrane that plays a role in cellular metabolism.144 It is a heterotetrameric complex composed of 4 protein subunits (SDHA, SDHB, SDHC, and SDHD).145 SDH gene mutations occur in roughly 15% of pheochromocytomas and paragangliomas overall, with the rate substantially higher in paragangliomas than pheochromocytomas (over 30% vs. 4% to 5%, respectively).146–150 The frequency of the specific SDH gene mutated depends on the site of the tumor. Sympathetic paragangliomas (predominantly located in the thorax, abdomen, and pelvis) have a high rate of SDHB mutations; whereas SDHD mutations predominate in parasympathetic paragangliomas (located in the head and neck).147,150 There are phenotypic differences depending on the specific SDH gene mutation. SDHB mutations are associated with malignant behavior, whereas SDHD mutations are linked with higher rates of multifocality.146–148,150 Thus, identification of SDH mutations is important not just for genetic counseling purposes, but also for prognostication. Although clinical factors, such as age at diagnosis and personal or family history of tumors, may aid in the identification of a hereditary syndrome, a subset of tumors that appear clinically sporadic harbor germline mutations.142 IHC for SDHB can be used to detect tumors with a germline SDH mutation (SDH mutations in pheochromocytomas and paragangliomas are essentially always germline). SDHB is ubiquitously expressed (including in normal tissues of patients harboring a germline mutation in an SDH gene). Loss of heterozygosity (LOH) in a tumor along with a germline inactivating mutation of an SDH gene results in destabilization of the SDH protein complex. As a result, SDHB expression will be lost in tumors harboring an SDHA, SDHB, SDHC, or SDHD mutation. In a study of 220 pheochromocytomas and paragangliomas, van Nedeerveen demonstrated that SDHB IHC had a sensitivity of 100% regardless of which SDH gene was mutated and a specificity of 85% (though it is possible that some tumors that were negative by IHC harbored molecular alterations that escaped detection by the mutation analysis).151 If SDHB expression is lost, the next step is to perform IHC for SDHA. Tumors with SDHB, SDHC, or SDHD germline mutations will show loss of staining for SDHB but intact staining for SDHA.152 In contrast, tumors with SDHA germline mutations will show loss of staining for both SDHB and SDHA.152,153 Because IHC cannot distinguish tumors with SDHB, SDHC, or SDHD germline mutations, mutational analysis needs to be performed to determine which gene is mutated. Although SDHB IHC is a powerful tool to detect underlying SDH gene mutations, there are some caveats regarding its interpretation.149,152 The stain can only be interpreted if there is an internal positive control (endothelial cells, sustentacular cells, or adjacent non-neoplastic tissue). In addition, the staining should be granular cytoplasmic staining, reflecting the mitochondrial localization of the complex. Blushy cytoplasmic staining is not indicative of intact expression. In fact, Gill et al149 reported that blushy cytoplasmic staining was seen in some cases with an SDHD mutation. They also reported that rare tumors with VHL mutations may show decreased SDHB staining. If a pathologist is unsure of the staining result, we would advise reporting the stain as equivocal so that mutation analysis can be performed, and a potential germline mutation is not missed. Finally, rare paragangliomas and pheochromocytomas have been found to harbor germline FH and MAX mutations, and mutation status for these genes can be interrogated by IHC.154–156
In conclusion, IHC plays a critical role in the evaluation of endocrine tumors. Although most IHC in endocrine pathology is performed for diagnostic purposes, it also aids in tumor prognostication and can be used to identify molecular alterations that may prompt genetic counseling or help guide treatment. We look forward to the study of additional markers that expand this field and further inform clinical management of patients with endocrine tumors.
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