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Immunohistochemical Pitfalls in Genitourinary Pathology

2018 Update

Cox, Roni M., MD; Magi-Galluzzi, Cristina, MD, PhD; McKenney, Jesse K., MD

doi: 10.1097/PAP.0000000000000205
Review Articles

Immunohistochemistry may be a very useful adjunct to morphologic diagnosis in many areas of surgical pathology, including genitourinary pathology. In this review, we address common diagnostic dilemmas where immunophenotypic analysis may be utilized and we highlight pitfalls specific to each scenario. For prostate, we review the diagnosis of limited prostatic adenocarcinoma and the distinction of high-grade prostatic adenocarcinoma from urothelial carcinoma. We also cover markers of urothelial lineage in the diagnosis of metastatic carcinoma of unknown primary site. In the kidney, distinction of poorly differentiated renal cell carcinoma from urothelial carcinoma and epithelioid angiomyolipoma, adjuncts to the recognition of hereditary renal neoplasia, and the diagnosis of metastatic renal cell carcinoma are discussed. Finally, for testis we address distinction of germ cell tumors from sex cord-stromal tumors, as well as the diagnosis of metastatic germ cell tumors.

Robert J. Tomsich Institute of Pathology and Laboratory Medicine, Cleveland Clinic, Cleveland, OH

The authors have no funding or conflicts of interest to disclose.

Reprints: Jesse K. McKenney, MD, Cleveland Clinic, Robert J. Tomsich Institute of Pathology and Laboratory Medicine, Anatomic Pathology, 9500 Euclid Avenue, L25, Cleveland, OH 44195 (e-mail: mckennj@ccf.org).

All figures can be viewed online in color at www.anatomicpathology.com.

Immunohistochemistry can be an invaluable tool in the practice of surgical pathology of the genitourinary tract; however, it also has the potential to cause great diagnostic confusion. Immunostains must be carefully interpreted in the context of morphologic findings with a thorough knowledge of sensitivity and specificity in the specific context where they are being utilized. Unfortunately, as antibodies are described and studied, their specificity invariably declines over time. In essence, immunophenotypic evaluation is an exercise in assessing probability based on the most commonly expected patterns of staining, so exceptions will always be encountered, and nothing is 100% sensitive and specific. We, therefore, provide an up-to-date review of the potential pitfalls that may arise in using diagnostic immunohistochemistry for several recurring diagnostic problems in genitourinary pathology, based on the published literature and our own experience of “what works” in a high volume subspecialty genitourinary practice. The specific topics addressed include: (1) the diagnosis of small atypical glands in prostate biopsy, (2) distinguishing carcinoma of prostatic origin from urothelial origin, (3) distinguishing carcinoma of renal epithelial origin from urothelial origin, (4) diagnosing renal angiomyolipoma, (5) diagnosing metastatic renal cell carcinoma (RCC), (6) diagnosing metastatic urothelial carcinoma, (7) surrogate testing for hereditary renal neoplasia, and (8) efficient use in the diagnosis of testicular neoplasms.

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PROSTATE

Evaluation of Small Glandular Lesions on Needle Core Biopsy

In most cases, prostatic adenocarcinoma is easily diagnosed histologically on needle biopsy without the assistance of immunohistochemistry, assuming the neoplastic population of glands is present in sufficient quantities for their infiltrative nature and cytologic atypia to be recognizable. When only a small focus of suspicious glands is present in a biopsy (or in carcinomas that have a morphologically subtle variant pattern), immunohistochemical staining may be a useful adjunct to assist in accurate diagnosis. Evaluation for the presence or absence of basal cells with high molecular weight cytokeratin (HMWCK), cytokeratin 5/6, or p63 may be helpful, as the absence of staining supports a diagnosis of prostatic adenocarcinoma.1–6 α-Methyl-CoA-racemase (AMACR) also has utility in the evaluation of prostate cancer, with strong circumferential luminal reactivity identified in the majority of cases (80% to 100%).7–13 Unfortunately, AMACR is less sensitive in some variant morphologies such as atrophic, foamy gland, and pseudohyperplastic prostatic adenocarcinomas.14 In morphologically unequivocal carcinoma cases, one should not require AMACR reactivity for diagnosis. Many institutions now use multiplexed antibodies on a single slide (often with dual chromogens) to preserve tissue and ensure ability to evaluate focal atypical glands that might be lost in deeper sections, including various combinations of p63, CK5/6, and/or HMWCK with AMACR.15–20 The International Society of Urologic Pathology (ISUP) has recommended that any of these basal cells markers may be utilized for evaluating small atypical glandular foci on needle biopsy, but states that they are not needed in the setting of obvious carcinoma.21 Moreover, we would emphasize that blind immunohistochemical screening of prostate biopsies is unwarranted and leads to unnecessarily increased costs as well as more interpretation problems.

While these stains are valuable in supporting a diagnosis of prostate cancer in difficult cases, it is necessary for the pathologist to be aware of some potential pitfalls. Some benign mimickers of prostatic adenocarcinoma may be mistaken for carcinoma even with immunohistochemistry, particularly when present in small quantities. Partial atrophy and adenosis can both display variable cytoplasmic reactivity with AMACR (Fig. 1).22,23 In addition, basal cell markers are often present in a patchy distribution within partial atrophy and may be absent if only a small focus of glands is represented (Fig. 1).24,25 High-grade prostatic intraepithelial neoplasia (HGPIN) often shows positive staining for AMACR and the basal cell layer may also have a patchy distribution, another potential pitfall.26 Caution must be taken when diagnosing a small focus of adenocarcinoma with features overlapping those of partial atrophy, adenosis, or HGPIN, with an infiltrative architecture or a sufficient sample of glands available for adequate evaluation.

FIGURE 1

FIGURE 1

Uncommon cases of prostate cancer may show “positive” staining for basal cell markers, and careful consideration of their specific pattern of reactivity is essential. The most common cause of positive staining for basal cell markers in prostate cancer is simply nonspecific or “aberrant” staining for HMWCK in the tumor cells (ie, a nonbasal pattern).27,28 On close evaluation, the staining is not of flattened abluminal basal cells, but often weak cytoplasmic staining of the neoplastic secretory cells in a patchy distribution. Uncommonly, rare scattered individual tumor cells may show nuclear p63 reactivity (0.3% of cases in one series).27 Although not as diagnostically problematic, aberrant staining may also occur in high Gleason grade prostatic adenocarcinoma, and again the staining consists of scattered positive cells in a nonbasal pattern. Nuclear p63 staining in a diffuse, nonbasal pattern has been described in a rare subset of prostatic adenocarcinomas thought to represent a unique morphologic variant (Fig. 2).29–31 These carcinomas often have a distinct morphology consisting of infiltrative cords, nests, and glands with an atrophic morphology, nuclear hyperchromasia, and prominent nucleoli. Immunohistochemistry for HMWCK or CK5/6 confirms an absence of basal cells in these unique cancers (Fig. 2).

FIGURE 2

FIGURE 2

Although extraordinarily rare cases of prostatic adenocarcinoma show retention of basal cells in a portion of the glands,32 this is the exception to the rule and should only be accepted in morphologically unequivocal cases. For practical purposes, the presence of basal cells excludes the diagnosis of invasive carcinoma.

ERG is a relatively new immunohistochemical marker with conflicting studies on its potential usefulness in the evaluation of small foci of atypical glands on needle biopsy.33–36 TMPRSS2-ERG fusion gene combines the transcription factor ERG (located on chromosome 21q22.2) with TMPRSS2, an androgen-driven promoter.37 Strong nuclear overexpression of ERG by immunohistochemistry correlates with the fusion,38 and increasing numbers of diagnostic immunohistochemistry labs now stock the antibody for its alternative use as an endothelial marker. There are some notable obstacles to ERG being of daily diagnostic utility in prostate needle biopsy evaluation. It is true that ERG is rather specific for prostate carcinoma, with studies reporting negative staining in benign mimics such as partial atrophy, postatrophic hyperplasia, and adenosis.36,39,40 Despite the improved specificity of ERG compared with AMACR, it has a much lower sensitivity (40% to 50% of adenocarcinomas). Another problem is that HGPIN and intraductal carcinoma may show nuclear ERG expression, particularly the latter.36,41 In addition, in rare cases benign glands have shown positive staining;36,38 therefore, as with other markers, histologic correlation remains essential. Finally, the pattern of nuclear ERG reactivity is heterogenous in about 16% to 28% of cases, further increasing the chance that a small focus of carcinoma may show negative staining.42,43 Andrews and Humphrey questioned the cost-effectiveness of ERG immunohistochemistry, reporting that it provided useful information (beyond other stains) in only 29% of cases.33 For all of these reasons, we only rarely employ ERG immunohistochemistry in our routine diagnostic practice.

Nephrogenic adenoma is another histologic mimic of prostate cancer. Although uncommonly sampled on prostate needle biopsy, it may be seen more frequently in transurethral resections of prostate where suburothelial tissue is sampled. The tubular architecture and the nuclear features (with nucleoli in some) are easily mistaken for a small focus of prostatic adenocarcinoma if nephrogenic adenoma is not considered (Fig. 3). In our experience, the immunophenotypic findings of nephrogenic adenoma are very heterogeneous, even within the same lesion. AMACR (racemase/P504S) staining is often positive in nephrogenic adenoma,44,45 and was recently reported to be reactive in 55% of cases using a p63, HMWCK, AMACR triple antibody multiplex stain using a biotin free system.46 Although HMWCK was positive in 97% of the nephrogenic adenomas in this study, 13% of cases had only focal HMWCK staining.46 In addition, p63 showed only rare cases with positive nuclear staining (3%). Overall, only 3% of nephrogenic adenomas had an immunophenotype completely overlapping with prostate cancer based on the triple multiplex stain. Since individual cases of nephrogenic adenoma may show scattered foci with diffuse AMACR staining and no p63 or HMWCK, extreme caution is warranted. In difficult cases, a PAX8+/NKX3.1-immunoprofile strongly supports a diagnosis of nephrogenic adenoma (assuming clear cell adenocarcinoma of the urinary tract is histologically excluded).47–49 As with most morphologic pitfalls regarding benign mimics of cancer, the most important step is simply considering the possibility. Although not relevant to the differential of prostatic adenocarcinoma, it should also be noted that nephrogenic adenomas express nuclear GATA3 in up to 40% of cases.46

FIGURE 3

FIGURE 3

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Distinction of Prostate and Urothelial Carcinoma

In some cases, the distinction between high-grade prostatic adenocarcinoma and urothelial carcinoma can be very difficult by morphology alone. Histologic features typically provide useful clues in establishing the correct diagnosis. Prostatic adenocarcinoma, even when high grade, commonly maintains monomorphic round nuclei with central prominent nucleoli and often at least focal acinar formation. The nuclear pleomorphism in urothelial carcinoma is typically a more prominent feature than that of high-grade prostate cancer, and desmoplasia and variant patterns would favor urothelial lineage. A subset of reported prostatic adenocarcinomas have significant histological overlap with urothelial carcinoma,50 and adjunctive immunohistochemistry may be needed in such cases.

CK7+/CK20+ immunoprofile is often used for distinguishing carcinoma subtypes (including urothelial and prostatic primaries); however, this data may cause significant confusion because prostatic adenocarcinomas (particularly when high grade) may have unexpected phenotypes for CK7 and CK20.51,52 In fact, high-grade prostatic adenocarcinomas may show significant CK20 reactivity (reportedly from 8% to 26% of cases depending on the grade). Because of this overlapping CK7/CK20 phenotype, we generally discourage their use in this setting and would argue that much better markers are available.

The most established prostatic markers are PSA and PSAP. These markers are useful, especially PSA, as they are highly specific for prostate carcinoma, with PSA showing greater specificity than PSAP. Unfortunately, these markers show decreased staining in higher grade prostatic adenocarcinomas.51 Moreover, when positive, patchy weak or focal staining is common. The degree of weak cytoplasmic staining in these cases may prove difficult to differentiate from nonspecific background staining, further diminishing one’s confidence. Newer prostatic epithelial markers have been developed in attempts to remedy the problems with PSA and PSAP. PSMA, prostein (P501S), NKX3.1, and ERG are some recently described markers which have proven useful in confirming a prostate primary origin. PSMA has a very high sensitivity for prostatic carcinoma but is also positive in up to 17% of urothelial carcinomas.53,54 Prostein (P501S) has been reported to have higher sensitivity than PSA/PSAP in high-grade prostate cancer, and the characteristic coarse granular staining pattern is easier to differentiate from nonspecific staining; however, results have been variable.54–57 As discussed previously, ERG is specific for prostatic adenocarcinoma in the differential diagnosis with urothelial carcinoma, but suffers from low sensitivity (only 40% to 50%).58,59 NKX3.1 is both sensitive and specific for prostatic adenocarcinoma (even in high-grade carcinomas), with a clean nuclear staining pattern (Fig. 4).60–62 In fact, most large centers now consider NKX3.1 as the prostatic epithelial marker of choice, as do we.54 Outside of the differential diagnosis with urothelial carcinoma, NKX3.1 expression has also been reported in breast carcinoma and normal testis.60

FIGURE 4

FIGURE 4

HMWCK and p63 are 2 widely available immunostains that have historically been used as urothelial markers in this setting, and are positive in most urothelial carcinomas.63,64 HMWCK may show aberrant positive staining in a small proportion of high-grade prostatic adenocarcinoma, but the staining is usually only focal as opposed to the more diffuse staining common in urothelial carcinoma.27 The uroplakins are specific for urothelial carcinoma, but are less widely available and suffer from low sensitivity.65,66 Newer uroplakin antibodies, such as uroplakin II, have been reported as having improved sensitivity, but it is still lower than other available markers.65,67,68 Thombomodulin has been reported as more sensitive than the uroplakins in some studies, but is also not widely available and is less specific, showing positive staining in squamous cell carcinomas, mesotheliomas, and rarely prostatic adenocarcinoma.61,66,69 GATA3 is rapidly becoming widely available and is highly sensitive for urothelial carcinoma. To date, prostatic adenocarcinomas have been negative for GATA3, but a potential pitfall is the positive GATA3 staining in prostatic basal cells. It was recently reported that benign prostate glands with radiation atypia show GATA3 expression, a finding that we have also seen cause diagnostic confusion.70,71 Also, it must be realized that GATA3 is very specific for urothelial origin only in the differential with prostate cancer, but not in an unknown primary setting (discussed fully in the section on metastatic urothelial carcinoma).

Overall, we generally recommend an approach using multiple markers for prostatic versus urothelial carcinoma. What antibodies to use is debatable, but a panel consisting of PSA and NKX3.1 (for prostatic) and p63 and GATA3 (for urothelial) will lead to a definitive diagnosis in the vast majority of cases (Fig. 4). In those rare cases where the diagnosis remains unclear, staining with additional markers such as prostein (P501S), PSMA, HMWCK, uroplakin, or thrombomodulin might be considered; however, in such undifferentiated tumors, a definitive diagnosis may not be possible. More recent studies have proposed HOXB13 as a sensitive and specific marker of prostate epithelial origin, but there is currently less practical diagnostic experience with that antibody, compared with NKX3.1.54,72–74

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BLADDER/UROTHELIAL

Diagnosis of Metastatic Urothelial Carcinoma

In our experience, there is no single antibody with sufficient specificity to render a definitive diagnosis as urothelial lineage based on immunohistochemistry alone. Therefore, it is our strongly held opinion that the diagnosis of metastatic urothelial carcinoma always relies on morphologic, immunophenotypic, and clinical correlation. We have seen numerous cases misdiagnosed because of a misleading immunoprofile. The sensitivity and specificity of urothelial markers were reviewed in the section on “Distinction of Prostate and Urothelial Carcinoma” and will not be repeated here. We will discuss 2 specific problems: over-reliance on GATA3 and carcinomas with alternative differentiation.

GATA3, although a sensitive marker of urothelial origin, has a very low specificity with expression in a large number of benign and malignant tissues and should be utilized very cautiously in a work-up for carcinoma of unknown origin. Table 1 summarizes the known spectrum of GATA3 expression.

TABLE 1

TABLE 1

Metastatic urothelial carcinomas can show divergent squamous differentiation, which may cause significant overlap with a metastatic squamous cell carcinoma from other anatomic sites (eg, penis, urethra, uterine cervix, or vagina). Although there is literature dealing with this immunohistochemical distinction, in our practical experience there is a significant degree of immunophenotypic overlap with regard to p16, GATA3, squamous lineage markers (p40, CK5/6, p63), and urothelial markers (uroplakins and thrombomodulin). We would recommend extreme caution with the use of p16 in bladder tumors, as we try to avoid its use in that setting.75 With potential metastasis (or with direct extension to the urinary tract) from a uterine cervical carcinoma, testing for HPV with in situ hybridization techniques is now practical and can be very useful in confirming an HPV associated carcinoma.76,77 Finally, urothelial carcinomas with glandular differentiation, and primary vesical adenocarcinomas, often have both enteric morphology and immunophenotype; therefore, demonstration of CDX2 or SATB2 nuclear staining is not helpful.78 Finally, though nuclear β-catenin expression is common in colorectal adenocarcinoma, reactivity patterns are not 100% specific in the distinction form primary bladder adenocarcinoma.79,80

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KIDNEY

Distinction of Urothelial Carcinoma From Renal Cell Carcinoma (Including High-grade Forms)

The differential diagnosis of high-grade carcinoma involving the renal hilum and medulla can be difficult to navigate. The most common challenge is differentiating urothelial carcinoma originating in the renal pelvis from a poorly differentiated RCC involving the renal medulla and hilum, which may include renal medullary carcinoma, HLRCC-associated RCC, and carcinomas historically classified as collecting duct carcinoma. The simplest solution is submitting additional sections of the renal pelvis in order to identify urothelial carcinoma in situ or other precursor lesions. In cases where an in situ lesion cannot be found (or in biopsies), a panel of immunohistochemical stains may prove useful, with the most common panel consisting of PAX8, p63, and GATA3. These stains are useful in many circumstances, but some important caveats must be kept in mind in interpreting the results. PAX-8 is a widely used marker which shows high sensitivity for RCC, but upper tract urothelial carcinoma may stain positive for PAX-8 in approximately 25% of cases.81–83 Most cases show focal staining, but occasional cases can be diffusely positive. GATA3 is more sensitive than the uroplakins and thombomodulin, and commonly shows more diffuse staining, which is helpful when evaluating small biopsies. In the majority of cases, GATA3 staining in renal pelvis carcinomas signifies urothelial carcinoma, but there are some variants of RCC that may be positive for GATA3. Chromophobe RCC (51% of studied cases) and renal oncocytomas (17% of cases) were positive in one study evaluating GATA3 staining.84 A recent study has also shown GATA3 staining in 76% of clear cell-papillary RCCs.85 Only rare cases of collecting duct carcinoma stained positive for GATA3, with 6% (1/18) of cases studied showing positive staining.83 A PAX8+/GATA3−/p63− immunoprofile favors RCC,86 and a PAX-8 negative lesion with positive staining for GATA3 and/or p63 favors urothelial carcinoma; however, problems arise with interpretation of other staining combinations, such as negative staining for all markers, in which the diagnosis may unfortunately remain unresolvable. The classic immunophenotypic patterns have lower sensitivity in sarcomatoid tumors, but may still prove useful in individual cases.87

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Differential Diagnosis of Renal Angiomyolipoma

The differential diagnosis of renal angiomyolipoma (AML) varies depending on the histologic features of the individual case being evaluated. AMLs are part of the PEComa tumor family and may show components with myoid appearing spindled cells, epithelioid cells (with or without pronounced cytologic atypia), and intracytoplasmic lipid mimicking adipose tissue.88

Lipid-rich angiomyolipomas can extend into the adjacent perinephric tissue and may closely mimic a well-differentiated liposarcoma arising from the retroperitoneum. MDM2 immunohistochemical stains that are used for the diagnosis of well-differentiated/dedifferentiated liposarcoma may be problematic in this specific scenario, as 23% of lipid-rich AMLs showed nuclear MDM2 expression in a recent study.89 Although the MDM2 staining in AMLs is often more focal, the interpretation can be very difficult and somewhat subjective, particularly in limited needle biopsy specimens. AMLs lack MDM2 amplification by FISH (in contrast to well-differentiated liposarcoma), so FISH amplification is the preferred test in diagnostically challenging cases.89

Epithelioid angiomyolipomas, which may be composed predominantly of cells with abundant eosinophilic cytoplasm and marked nuclear atypia, must be differentiated from RCC. PAX-8 is the most useful stain in this setting, as AMLs are negative (in contrast to most RCCs).

Another pitfall in the immunophenotypic analysis of AML is simply the expectation for expression of melanocytic markers. No matter which antibody is utilized (HMB45, Melan-A, etc), cytoplasmic reactivity is often weak and focal. This problem is further amplified in lipid-rich AMLs because the cytoplasm is displaced, and reactivity may be very subtle at the edge of the cell. In such cases, the perivascular neoplastic cells are often more strongly reactive. Cathepsin-K, although not as widely available as HMB45 and Melan-A, is positive in the vast majority of renal AML and often in a more diffuse pattern (Fig. 5).90 It should be noted that Cathepsin-K may also be positive in MiT family translocation carcinomas, which may also have reactivity to other melanocytic markers.91

FIGURE 5

FIGURE 5

Finally, AMLs with a myoid appearance may mimic a true smooth muscle tumor (ie, renal leiomyoma). One should be very suspect of a leiomyoma diagnosis in the kidney as most actually represent AML. Very rare bona fide renal leiomyomas exist, but they occur almost exclusively in women and show strong desmin expression with no staining for Cathepsin-K.92

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Diagnosis of Metastatic Renal Cell Carcinoma

For practical purposes, small occult radiographically undetectable RCCs do not present with metastases. Therefore, it is unwise to default to immunohistochemical markers of renal origin outside the context of known renal imaging studies. One caveat may be the setting of adult polycystic kidney disease, in which imaging may occasionally be difficult.

PAX8 is currently the marker of choice in confirming metastatic RCC. It is recommended by the ISUP and has largely supplanted other purported renal markers such as PAX-2, CD10, Ksp-cadherin, and RCC marker antigen (RCCma) given its superior test characteristics.93–95 PAX8 has higher sensitivity and more intense staining than PAX-2, including in cases of metastatic RCC.96 The overall sensitivity of PAX8 for RCC is high (approximately 95%), but some subtypes are more likely to be negative. Clear cell RCC and papillary RCC stain positive for PAX8 in 95% of cases, whereas chromophobe RCC has a lower proportion of positivity that is approximately 85%.96 An absence of PAX8 staining in a metastatic carcinoma with the appearance of RCC should not absolutely exclude the diagnosis, especially in cases with clinical history of renal carcinoma or a renal mass.

PAX8 can show positive staining in a vast array of tumors from other primary sites (depending on whether the monoclonal or polyclonal antibody is used), including the gynecologic tract carcinomas, thyroid gland carcinomas, urothelial carcinomas, pancreatic neuroendocrine tumors, mesothelium, thymus, Merkel cell carcinoma, and medulloblastomas.82,97–107 Background B lymphocytes can be positive for PAX8, and B cell lymphoma may show PAX8 staining when using polyclonal antibodies.108 PAX8 is an invaluable stain for supporting a renal primary, but clinical history and tumor morphology must be kept in mind and other PAX8 positive neoplasms should be excluded (Table 2).

TABLE 2

TABLE 2

As mentioned, an often unexpected pitfall is the expression of PAX8 in a subset of upper tract urothelial carcinomas, including metastases.82,83 One must consider the possibility of urothelial primary morphologically if evaluating a metastatic carcinoma in the presence of a known renal or ureteral mass (Fig. 6). Nuclear pleomorphism, stromal desmoplasia, or unusual architectural patterns (eg, micropapillary) are possible clues to urothelial origin. The coexpression of p63, HMWCK, and/or GATA3 would add support for urothelial lineage in this setting.

FIGURE 6

FIGURE 6

Pathologists should also be aware of some specific histologic mimics of metastatic clear cell RCC that, if considered, may be effectively resolved by immunohistochemistry. In bone, chordoma or notochord remnants can closely mimic metastatic RCC, but notochord-derived tissues express nuclear brachyury and lack PAX8 staining.109 Hemangioblastoma may also mimic metastatic clear cell RCC, but they express inhibin, D2-40, and GLUT-1, generally without PAX8 reactivity.110

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Surrogate Immunohistochemical Testing for Hereditary Renal Neoplasia

As more RCC subtypes are described, novel associations with hereditary neoplasia syndromes are also being recognized. In some instances, immunohistochemistry may play a role in their recognition and work-up.

Succinate dehydrogenase (SDH) deficient RCC is a recently described entity that is almost always associated with germline mutations in a succinate dehydrogenase (SDH) gene, most commonly SDHB.111–113 Although these tumors have novel histologic features, showing a low grade oncocytic appearance with distinct intracytoplasmic vacuoles, they do overlap with other subtypes. An SDHB antibody has shown great utility in diagnosing these tumors (Fig. 7).112–116 Fortunately, the antibody recognizes an epitope shared by other SDH gene products (ie, SDHC, SDHA, etc); therefore, it can be utilized as a general screen for SDH mutations. The one pitfall in the interpretation of SDHB immunohistochemistry is with renal tumors showing pale or clear cytoplasm (eg, clear cell RCC).117 Because such tumors likely have sparse mitochondria, SDHB staining may be very weak or even absent when compared with internal controls, which may lead to a false interpretation of SDH loss.

FIGURE 7

FIGURE 7

Hereditary Leiomyomatosis and Renal Cell Carcinoma syndrome (HLRCC) is caused by a germline mutation in fumarate hydratase.118 These patients most commonly present with cutaneous or early onset uterine leiomyomas, but may also harbor aggressive RCCs. The RCCs associated with fumarate hydratase mutation are morphologically heterogenous, and historically have been classified most commonly as type 2 papillary RCC, collecting duct RCC, “dedifferentiated” tubulocystic RCC, or unclassified RCC.119,120 In younger patients, they may have overlap with SDH-deficient RCC or oncocytoma.121,122 Although not always prominent, these FH deficient RCCs commonly show neoplastic cells with prominent macronucleoli and paranucleolar clearing.119,123 An FH immunostain is now available that may serve as a surrogate for mutation when cytoplasmic staining is lost in the neoplastic cell population.124,125 The problem with FH antibody screening is that not all germline mutations are associated with loss of cystoplastic FH staining, with reported sensitivity of approximately 87%.126 Moreover, a recent molecular characterization study has documented rare nonsyndromic RCCs with somatic FH mutations; therefore, specificity for the germline mutation is not 100%.127 Several studies have also validated 2SC immunohistochemistry as an ancillary tool for improving detection of these RCCs (with increased cytoplasmic and nuclear staining), and have reported it to be a useful adjunct in conjunction with the FH stain.119,126,128 However, at present, the 2SC antibody is not commercially available. Although we do use the FH immunostain in diagnostic practice, it should not replace standard genetic counseling in morphologically suspicious cases.

Tuberous sclerosis complex (TSC), which is caused by germline mutations in TSC1 or TSC2, is commonly associated with renal angiomyolipomas, but RCC may rarely be encountered.129 Specific morphologic patterns have been described in the TSC associated renal tumors that may aid in recognition, including eosinophilic/solid/cystic (ESC) RCC, RCC with angioleiomyomatous stroma, and a spectrum of oncocytic neoplasms.130,131 Although hamartin and tuberin stains are commercially available (recognizing the protein products of the TSC genes), it is our experience that they do not correlate well with mutation status.132 Therefore, careful scrutiny of the background kidney for AML tumorlets often provides more information than immunohistochemistry.

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TESTIS

For the majority of testicular tumors, H&E morphology is sufficient for diagnosis; however, in some instances adjunctive immunohistochemistry may be needed (eg, seminoma vs. the solid pattern of yolk sac tumor). In general, immunohistochemistry tends to be overutilized for subtyping germ cell tumors or calculating their percentages. Since the immunophenotype of individual germ cell tumor subtypes has been reviewed in great detail, it is not repeated here.133–135 We will focus on distinction from sex cord stromal tumors and diagnosing metastases.

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Distinguishing Germ Cell Neoplasm Versus Sex Cord Stromal Tumor

Testicular neoplasms fall into 2 major categories: germ cell tumors and sex cord-stromal tumors (surface epithelial tumors of the testis are vanishingly rare). In cases with morphologic overlap, immunostains for germ cell markers (OCT4 and SALL4) and sex cord stromal markers (inhibin, calretinin, and/or SF-1) can help determine the correct lineage. Although it has been studied more extensively in the ovary,136,137 it is our experience that SF-1 is an excellent sex cord-stromal marker. Its nuclear pattern of reactivity is easier to read, and sensitivity and specificity are very high.138,139

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Diagnosis of Metastatic Germ Cell Tumor

Immunohistochemical markers for germ cell lineage are more commonly used in the setting of a metastatic tumor, where they may be used in an unknown primary site of origin work-up. The 2 most popular stains used in this setting are SALL4 and OCT4. SALL4 is a broad spectrum germ cell marker and is positive in seminoma, embryonal carcinoma, and yolk sac tumor. In teratomas, SALL4 expression is often limited to more primitive type gland elements. OCT4, which stains seminoma and embryonal carcinoma, is also a common stain to help determine a germ cell primary. Both of these stains are useful when used in conjunction with the histologic features, but there are some important potential pitfalls. OCT4 shows excellent sensitivity for seminoma and embryonal carcinoma and has been shown to be superior marker of embryonal carcinoma in the post-chemotherapy setting,140 where CD30 staining may be lost. OCT4 has recently been described in renal medullary carcinomas, and although rare, this aggressive carcinoma should not be misdiagnosed as germ cell tumor.141 Some hematopoietic neoplasms also stain with OCT4, including some B-cell lymphomas and acute myeloid leukemias.142 SALL4 may show positive staining in various nongerm cell neoplasms, including hepatocellular carcinoma, nonsmall cell lung carcinoma, serous carcinomas of the gynecologic tract, gastric adenocarcinomas, lymphomas, malignant rhabdoid tumor, urothelial carcinomas, and nephroblastomas.142–147 Primitive type carcinomas, such as gastrointestinal adenocarcinomas with fetal gut differentiation, may also be SALL4 positive.148 Table 3 summarizes tumors with SALL4 expression.

TABLE 3

TABLE 3

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CONCLUSIONS

In conclusion, immunohistochemistry may be a very useful adjunct for the practice of genitourinary pathology. In each of these specific differential diagnostic settings, the pathologist must know the sensitivity and specificity of the markers being utilized with regard to the specific diagnostic question being asked. In addition, one must be aware of the diagnostic pitfalls and histologic mimickers unique to each scenario. Using this approach, immunohistochemistry may be used to its full potential to provide adjunctive help in managing cases and arriving at an appropriate diagnosis.

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Keywords:

NKX3.1; SF-1; SALL4; OCT4; AMACR; FH; SDHB; GATA3

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