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Practical Applications of Immunohistochemistry in the Diagnosis of Genitourinary Tumors.

Immunohistochemical techniques with a variety of markers have been applied frequently in the diagnosis of neoplasms and have become indispensable in some situations. (1,2) In this article, we review the commonly used immunohistochemical markers in the diagnosis of genitourinary tumors. This review focuses on applying these markers in a variety of commonly encountered circumstances, particularly on the selection of markers and interpretation of immunohistochemical staining results. The newly identified diagnostic markers are discussed as well. Frequently encountered pitfalls in the practice are emphasized.


Example 1: A 56-year-old woman presented with a 6.5cm right renal mass found on computed tomography scan. Right partial nephrectomy specimen demonstrated the tumor consisted of a papillary proliferation of renal epithelial cells with papillary structure and clear to slightly eosinophilic cytoplasm. The tumor cells were positive for paired box gene 8 (PAX8), transcription factor E3 (TFE3), human melanoma black 45 (HMB45), [alpha]-methylacyl coenzyme A racemase (AMACR), and CD10, and were negative for keratin (AE1/AE3/CAM5.2), epithelial membrane antigen (EMA), cytokeratin 7 (CK7), carbonic anhydrase IX (CA-IX), CD117, melanoma antigen recognized by T cells 1 (Mart-1), microphthalmia-associated transcription factor (MiTF) and Melan-A. Expressions of fumarate hydratase (FH) and iron-sulfur subunit of complex II (SDHB) were preserved. Fluorescence in situ hybridization assay demonstrated translocations involving the TFE3 gene on chromosome X. The final diagnosis was Xp11 translocation renal cell carcinoma (RCC).

Renal cell carcinoma is the seventh most common cancer in the Western world, and its incidence has been steadily increasing during the past few decades, (3,4) representing 1% to 3% of current malignant visceral neoplasms. The major applications of immunohistochemical stain in renal neoplasms include (1) to establish kidney as the tumor origin and (2) to aid in the classification of primary renal neoplasms. (5)

Markers to Establish the Diagnosis of Renal Neoplasm (Site of Origin)

PAX8 is one of the most useful markers to establish renal epithelial neoplasm and is recommended by the International Society of Urologic Pathology for this purpose. (5) PAX8 is a 415-amino-acid transcription factor that plays important roles in the development/maintenance of the kidney. It is normally expressed strongly in the renal tubules, whereas focal PAX8 expression may be seen in the renal pelvic urothelium. It is a useful marker for primary and metastatic RCC. (6,7) PAX8 is positive in almost all RCCs, including clear cell RCC, papillary RCC, chromophobe RCC, sarcomatoid RCC, microphthalmia transcription factor (MiT) family translocation RCC, and mucinous tubular and spindle cell carcinoma (MTSC). PAX8 is also known for its expression in the lower female genital tract and thyroid tumors. (8) Compared with paired box gene 2 (PAX2), another marker for renal epithelial origin, PAX8 is more sensitive. (9) Similar markers such as RCC marker antigen, CD10, and Kspcadherin have been explored, and their utility is limited because of their relatively low specificities. (5)

Markers to Aid in the Classification of Primary Renal Neoplasms

Although a majority of renal tumors are malignant, about 10% to 30% are benign. (10-12) Among the malignant tumors in the kidney, RCCs are the most common. In addition to differentiating benign tumors from malignant ones, histologic subclassification of the RCC is critical because of the implications of the subtype in the prognosis and subsequent treatment. (13,14) The major subtypes of RCC include clear cell RCC, papillary RCC, and chromophobe RCC, together representing more than 90% of RCCs. (15) Histologic subtyping of renal neoplasm is usually straightforward by routine microscopic examination; however, immunohistochemical markers can be helpful in many challenging situations, such as renal neoplasms with atypical morphology, tumors with overlapping features, or small biopsy specimens. Although frequently encountered, renal tumors with clear cells, oncocytic cytoplasm, and papillary architecture can be diagnostically challenging (16); useful immunohistochemical panels to distinguish among the differential diagnoses are discussed here.

Immunohistochemical Markers for Renal Tumors With Clear Cell Features.--Clear cells can be seen in many renal neoplasms, including clear cell RCC, clear cell papillary RCC, papillary RCC, chromophobe RCC, MiT family translocation RCC (MiTF RCC), and epithelioid angiomyolipoma (AML). Clear cell RCC is the most common neoplasm in this category. (5) When dealing with tumors with clear cell features and morphology not typical for clear cell RCC or when limited material is available, we usually perform a panel of markers in various combinations that includes CA-IX, CK7, AMACR, CD117, transcription factor E3 and transcription factor EB (TFE3/TFEB), and HMB45. The expected staining patterns are summarized in Table 1.

Tumor cells in conventional clear cell RCC often show diffuse membranous positivity with CA-IX in a box-shape pattern (Figure 1, A). (17-19) Cytokeratin 7 is usually negative or focally positive in clear cell RCC. (5) AMACR is negative or focally expressed in clear cell RCC, and CD117, TFE3/TFEB, and HMB45 are often negative.

Clear cell (tubulo)papillary RCC is a recently recognized entity occurring in patients with or without end-stage kidney disease. (20,21) Microscopically, this tumor is characterized by proliferating papillary structures within cystic spaces. The tumor cells have uniformly clear cytoplasm and lowgrade nuclei. Other commonly seen growth patterns include cuboidal tumor cells arranged in tight tubular/acinar structures, particularly in the tumor-bearing solid areas. Characteristically, the tumor nuclei are arranged in a linear fashion, away from the basement membrane, resembling piano keys. The immunohistochemical features of this tumor are quite characteristic. The tumor cells express CAIX diffusely in a cup-shape membrane distribution, but lack staining along the luminal border (Figure 1, B). (19) The characteristic cup-shape immunohistochemical labeling for CA-IX in clear cell papillary RCC differs slightly from that of clear cell RCC with box-shape pattern (Figure 1, A). Clear cell papillary RCC is often CD10 negative, diffusely positive for CK7, and negative for AMACR. (19,22) Therefore, clear cell RCC can be distinguished from clear cell papillary RCC using a panel that includes CA-IX, CD10, CK7, and AMACR. However, extra caution should be taken when dealing with limited tissue specimens. A small portion of clear cell RCCs containing overlapping features with clear cell papillary RCC demonstrate some degree of overlap in immunohistochemical phenotype as clear cell papillary RCC, according to our own experience and published reports. (23,24) Usually, the immunohistochemical staining pattern is not fully characteristic for either one in these cases.

Chromophobe RCC accounts for 5% of renal epithelial tumors and is characterized by voluminous cytoplasm with distinct cell borders. (13) Chromophobe RCC frequently comes into the differential diagnosis of a renal neoplasm with clear cells. A very helpful distinguishing panel includes CA-IX, CD117, and CK7. Chromophobe RCC does not express CAIX, and is diffusely positive for CK7 and CD117. (25) Both clear cell RCC and clear cell papillary RCC are CD117 negative. (19,22)

The 2013 International Society of Urologic Pathology Vancouver classification of renal neoplasia grouped 2 neoplasms, Xp11 translocation RCC and t(6;11) translocation RCC, together in the category of MiT family translocation RCC. (26) The t(6;11) translocation RCCs are less common compared with the Xp11 translocation RCCs, with only about 50 cases reported in the literature. The Xp11 translocation RCCs harbor TFE3 gene translocation; the t(6;11) translocation RCCs harbor a MALAT1-TFEB gene fusion. Both TFE3 and TFEB are members of the MiT family. The most distinctive histologic feature of the Xp11 translocation RCC includes presence of clear cells and papillary architecture, which requires morphologic distinction from clear cell and papillary RCC. (27) The t(6;11) TFEB RCCs usually have a biphasic appearance with peripherally arranged larger cells and centrally located smaller epithelioid cells admixed with micronodules of basement membrane material. However, the morphology of these 2 neoplasms can overlap, with one mimicking the other. The former expresses TFE3 protein (Figure 1, C), whereas the latter stains positive for TFEB. (28,29) TFE3 and TFEB proteins are the most sensitive and specific immunohistochemical markers that can distinguish the translocation RCCs from other RCCs. Unlike other RCCs, translocation RCCs frequently express melanocytic markers like HMB45 and Melan-A and often lose the expression of cytokeratin markers and EMA. (28,30,31) These unusual immunohistochemical phenotypes can be a useful hint for translocation RCC when dealing with a high-grade RCC. Dual-color breakapart fluorescence in situ hybridization for TFE3 or TFEB gene rearrangement can also confirm the diagnosis when needed. (32)

The epithelioid variant of AML consists of polygonal cells with clear, granular or densely eosinophilic cytoplasm, which can be misinterpreted as clear cell RCC. The spindle cell and pleomorphic features could lead to a misdiagnosis of sarcoma or sarcomatoid carcinoma. Despite clear and epithelioid cells, AML does not express epithelial markers and is negative for CA-IX, CD10, and AMACR. Melanocytic markers, including Melan-A, MiTF, Mart-1, and HMB45, can be diffusely or focally positive (Figure 1, D). (5) Given the variable expression of melanotic markers in AML, it is advisable to order more than one such marker when considering this diagnosis. Most AMLs are entirely negative for TFE3 and TFEB, which helps distinguish them from translocation RCCs. Angiomyolipoma is also positive for smooth muscle markers, which can distinguish it from other renal tumors in this category.

Immunohistochemical Markers for Oncocytic Renal Tumors.--Many renal tumors may exhibit some degree of cytoplasmic eosinophilia; these tumors include eosinophilic/ granular variant of clear cell RCC, papillary RCC type 2, eosinophilic variant of chromophobe RCC, renal oncocytoma, and epithelioid AML. Histologic classification of renal tumors with oncocytic cytoplasm is usually challenging because of multiple differential diagnostic considerations. A helpful panel here includes CD117, CK7, CA-IX, AMACR, and HMB45 (Table 2).

Distinguishing a renal oncocytoma from an eosinophilic variant of chromophobe RCC can sometimes be particularly challenging. (33) Yet the distinction is very important, as chromophobe RCC is a malignant neoplasm and oncocytoma is benign. Two useful markers are CK7 and CD117. Although both chromophobe RCC and oncocytoma are positive for CD117, they show different staining patterns of CK7. The majority of chromophobe RCCs diffusely express CK7 in a membrane-predominant pattern (Figure 2, A), whereas oncocytomas are typically negative or focally positive for CK7 in scattered cells (Figure 2, B). (34-38) Hale colloidal iron histochemical stain has been used for this distinction, but the interpretation can be difficult. Characteristically, it shows a diffuse and strong reticular pattern in almost 100% of chromophobe RCC, but the stain in oncocytoma is patchy and focal. (39-41)

Many clear cell RCCs, particularly higher-grade tumors, can have focal or predominant granular cells resembling chromophobe RCC. (42) The most useful markers in separating these 2 tumors are CK7, CD117, and CA-IX. (5,17,25,36) Chromophobe RCC is typically positive for CK7 and CD117 and negative for CA-IX; in contrast, clear cell RCC is often negative for CK7 and CD117, but positive for CA-IX.

The immunoprofile of papillary RCC type 2 is often variable, and typically shows positivity for AMACR and CK7. (43) Epithelioid AML often can be eosinophilic in appearance and can be mistaken for chromophobe RCC or clear cell RCC. A clue to the correct diagnosis is the presence of occasional adipocytes in close vicinity to the eosinophilic epithelioid cells in the fat-poor AML. (26) Epithelioid AML is positive for melanocytic markers such as HMB45 as well as smooth muscle markers, which can distinguish it from other renal tumors with oncocytic cell features. (5)

Immunohistochemical Markers for Papillary Renal Tumors.--Papillary architecture is not unique to papillary RCC. Many nonpapillary RCCs may show prominent papillary architecture focally, such as clear cell RCC with pseudopapillary growth, clear cell papillary RCC, MiTF RCC, and rarely collecting duct carcinoma and chromophobe RCC. Distinction of these from papillary RCC is essential, as their biologic behavior and potential therapeutic options are different. A panel including CA-IX, CK7, CD10, AMACR, TFE3/TFEB, and HMB45 can be helpful (Table 3).

The World Health Organization Classification of the Renal Tumors (2016) divides papillary RCC into 2 types, namely type 1 and type 2. (130) Type 1 papillary RCCs contain papillae covered by small cells with scant cytoplasm arranged in a single layer; type 2 tumors are characterized by tumor cells of higher nuclear grade, pseudostratification of nuclei, and abundant eosinophilic cytoplasm (Figure 3, A). (44) These 2 types differ in terms of prognosis, with better prognosis associated with type 1 papillary RCC. (44) Most papillary RCCs show diffuse cytoplasmic granular staining for AMACR (Figure 3, B) and membranous and cytoplasmic staining for CK7 (Figure 3, C), (43) more frequently seen in type 1 than in type 2 tumors. (45) CD10 is often positive in papillary RCC.

Occasionally clear cell RCC may have a papillary component, either focally or in a pseudopapillary growth pattern usually associated with high-grade tumors because of cell drop-off. A practical initial panel for the differential diagnosis of clear cell RCC with pseudopapillary growth pattern and other RCCs with papillary architecture consists of CA-IX, AMACR, and CK7. CA-IX is diffusely positive (membranous, box shape) in most clear cell RCCs with pseudopapillary growth pattern, whereas CK7 is often negative and AMACR can be variably positive in clear cell RCC. (17-19)

Another group of tumors that may be potentially considered in the differential

diagnosis of papillary RCC, especially type 2 papillary RCC, is the Xp11 translocation RCCs because of their prominent papillary architecture in some cases. Useful clues to suspect the diagnosis of Xp11 translocation RCC include younger patient age at diagnosis, stromal psammomatous calcifications, and negative or weak immunoreactivity with epithelial markers such as cytokeratins and EMA. Additionally, Xp11 translocation RCCs consistently express CD10 and RCC marker, and the majority express PAX2 and PAX8. (46) Antibodies against TFE3 or TFEB proteins are highly specific for MiTF RCC. (28,43) TFE3- and TFEB-positive immunore activity supports the diagnosis of MiTF RCC and can be further confirmed by fluorescence in situ hybridization testing for TFE3 or TFEB gene rearrangement.


Example 2: A 46-year-old woman presented with bladder tumor and had a transurethrally resected urinary bladder tumor that was diagnosed as carcinoma consistent with transitional cell carcinoma at an outside hospital. The patient came to one of our hospitals for treatment and brought the slides for rereview. The lesion consisted of a spindle cell proliferation in loose edematous stroma and infiltrating muscularis propria. The spindle cells were monotonous with low cellularity and not associated with significant cytologic atypia. The tumor cells were diffusely positive for vimentin and an antibody specific for anaplastic lymphoma kinase 1 (ALK-1) fusion protein associated with ALK-1 gene translocation. The tumor cells were also focally positive for desmin, smooth muscle actin (SMA), and cytokeratin (AE1/AE3/CAM5.2), and negative for p63, GATA3, CK7, CK34[beta]E12, and myogenin. The final diagnosis was inflammatory myofibroblasts tumor (IMT).

Major applications of immunohistochemistry in the pathology diagnosis of bladder tumors include (1) to establish a urothelial origin at a metastatic site or in a bladder tumor with unusual histology; (2) to distinguish between reactive atypia and carcinoma in situ (CIS); (3) to assist the staging of bladder cancer; (4) to distinguish various spindle cell lesions of the bladder; and (5) to rule out a metastasis to the bladder, which may simulate urothelial primary.

Novel and Traditional Markers Associated With Urothelial Differentiation

Only a few markers can be used to support urothelial differentiation. The markers used in the past include CK7, CK20, high-molecular-weight cytokeratin (HMWCK, 34[beta]E12), p63, and CK5/6. Urothelial carcinoma is one of the few tumors that frequently coexpresses CK7 and CK20, with coexpression of CK7 and CK20 reported in 50% to 62% of urothelial carcinomas. (47) Other markers such as HMWCK, p63, and CK5/6 are not specific for urothelial carcinoma. GATA3, a newly identified marker, is the firstline marker that the International Society of Urologic Pathology recommends to confirm urothelial differentiation. (48) GATA3 is expressed in 80% to 90% of urothelial carcinomas with nuclear staining. (49-51) In addition to urothelium, GATA3 is frequently expressed in breast. (49,52) Recent studies have shown GATA3 expression in many other tumor types, including cutaneous basal cell carcinomas, trophoblastic and yolk sac tumor (YST), squamous cell carcinoma, mesothelioma, salivary gland tumors, pancreatic ductal carcinomas, endometrial adenocarcinomas, and paragangliomas. (49,53,54) However, the results of these studies are poorly reproducible, mainly because of the different antibodies used. In our experience, GATA3 is a very reliable marker for both urothelial and breast primaries. Placental S100, uroplakin 2, and uroplakin 3 are other emerging urothelial markers. Placental S100 (55) and uroplakin (56,57) are expressed in 78% and 77.7% to 83.0% of urothelial carcinomas, respectively. Both uroplakin 2 and uroplakin 3 are highly specific urothelial markers, but their sensitivity is slightly lower than GATA3. Smith et al (58) showed that uroplakin 2 outperforms uroplakin 3 with better sensitivity.

Distinction of Reactive Atypia From Urothelial CIS

Urothelial cell CIS, by definition, is a high-grade flat lesion composed of malignant cells with hyperchromatic nuclei, nucleomegaly, pleomorphism, and frequent mitoses (Figure 4, A). (59) Reactive urothelial atypia is often seen in association with intravesical therapy (such as bacillus Calmette-Guerin treatment), calculi, infection, inflammation, or instrumentation. Reactive atypia may show some overlapping features with CIS and can be difficult to distinguish from it, particularly when only small amounts of tissue are available. Yet this distinction is critical because of the therapeutic and prognostic implications. Carcinoma in situ is a high-grade disease and associated with high likelihood of developing muscle-invasive cancer. Thus, the choice of treatment for bacillus Calmette-Guerin-refractory CIS is radical cystectomy. Although morphology remains the gold standard, immunohistochemistry can be helpful in establishing a diagnosis in difficult cases.

A potentially useful immunohistochemical panel includes CK20, p53, and CD44. (48) The typical staining patterns are outlined in Table 4. Cytokeratin 20 is one of the most useful markers. In normal and reactive urothelium, CK20 expression is seen only in umbrella cells. Full-thickness CK20 immunostaining is observed (Figure 4, B) in 75% to 94% of CIS cases.60-63 Staining patterns of p53 varied from negative to patchy staining limited in the basal cells in the normal and reactive urothelium, and showed variably full-thickness stain (Figure 4, C) in 57% to 90% of CIS. (60-63) CD44 is a surface transmembrane glycoprotein involved in cell-surface binding to hyaluronate. In normal urothelium, CD44 stains basal cells only. Reactive urothelial atypia may show a variable staining pattern, from focal basal-layer stain to diffuse full-thickness staining. However, CD44 expression is lost in most CIS. (60,64) The use of AMACR, human epidermal growth factor receptor 2, and CK5/6 in distinguishing reactive atypia from CIS has been reported, but needs further validation study. (62,63,65)

Distinction Among Spindle Cell Lesions of the Bladder

Spindle cell tumors in the bladder include mainly sarcomatoid urothelial carcinoma, IMT/pseudosarcomatous myofibroblastic proliferation, leiomyosarcoma, and rhabdomyosarcoma. (48) The distinction among these tumors can be challenging as all these lesions can have mass formation, infiltrative growth pattern, marked cellularity, mitotic activity, and necrosis. (66-69) When a predominantly spindle cell lesion with limited tissue availability is encountered in the bladder, an immunohistochemical panel composed of ALK-1, SMA, desmin, cytokeratin (AE1/AE3), p63, and HMWCK or CK5/6 is potentially helpful (Table 5). (67-70-79)

Inflammatory myofibroblastic tumor (Figure 5, A) is characterized by strong SMA positivity and frequent expression of broad-spectrum cytokeratins (for example AE1/AE3). Detection of ALK-1 protein or ALK-1 gene rearrangements helps distinguish IMT from other spindle cell tumors in the bladder. About 80% of IMTs are positive for ALK-1 by immunostain (Figure 5, B). (71) Therefore, a combination of cytokeratin, SMA, and ALK-1 positivity favors IMT. Inflammatory myofibroblastic tumor is negative for p63, HMWCK, and CK5/6 by immunostain.

Expression of several cytokeratin markers, especially HMWCK and CK5/6, together with p63-positive immuno stain favors sarcomatoid urothelial carcinoma. A recent study showed that HMWCK and CK5/6 were focally positive in 25% and 27% of sarcomatoid urothelial carcinomas of bladder, respectively, but negative in IMTs or bladder sarcomas. (70) Positive p63 staining has been reported in 36% to 50% of bladder sarcomatoid carcinomas. Actin and desmin are typically negative or weakly positive in sarcomatoid carcinoma. Leiomyosarcoma is diffusely positive for actin and/or desmin, negative to focal or weakly positive for cytokeratin (AE1/AE3), and negative for HMWCK and p63. Rhabdomyosarcoma is usually a more straightforward diagnosis because of the classic morphology and typically younger age of patients. Immunophenotypically, rhabdomyosarcoma can rarely express ALK-1, and the diagnosis can be confirmed by myogenin or MyoD1 nuclear expression.

Roles of Immunohistochemical Markers in Staging of Bladder Carcinoma

Tumor stage is the most important prognostic factor of urothelial carcinoma of the bladder. Management of bladder tumors depends heavily on the depth of tumor invasion, which the staging system is based upon. (80) However, the interpretation of the depth of tumor invasion within pT1 or pT2 stage is often difficult. Immunohistochemistry may be a useful tool in establishing the correct tumor stage. (81) Immunostain of cytokeratin (AE1/AE3, CK7, CK8, and CK18) is helpful in identifying infiltrating tumor cells when there are only a few tumor cells, especially in patients who have undergone neoadjuvant therapy. It is especially useful to identify tumor cells in fibrous scars or cauterized tissue in patients who have undergone transurethral resections previously. (82) In the latter situation, extra caution should be taken not to mistake keratin-positive myofibroblasts as keratin-positive tumor cells. Comparing the keratin immunostain with a simultaneously performed SMA stain may avoid the misdiagnosis. (82) Immunostain can be helpful in outlining muscle bundles and facilitating tumor staging (pT2 versus pT1). Desmin may highlight smooth muscle fibers not readily appreciated on hematoxylin-eosin sections, especially when desmoplastic stromal response is exuberant. (83,84) However, desmin immunostain cannot distinguish hyperplastic muscularis mucosae from muscularis propria, a potentially challenging distinction in transurethral resection specimens. (85) Smoothelin, a contractile protein expressed in fully differentiated muscle cells, is reported to be negative or weakly positive in smooth muscle bundles in lamina propria (discontinuous muscularis mucosae) but positive in the muscularis propria, including muscularis propria exhibiting cautery artifact. (83,86) However, it has not been widely used in practice because of its limitations. (87,88)


Example 3: A 63-year-old man was diagnosed with Gleason 3 + 3 adenocarcinoma, involving 5% of one core, 5 years earlier. He was subsequently treated with intensity-modulated radiotherapy. Recently, transurethral resection of the prostate for obstructive voiding symptoms demonstrated a small cell tumor. The tumor cells were diffusely positive for thyroid transcription factor 1 (TTF-1), CD56, synaptophysin, and chromogranin A; focally positive for keratins (AE1/AE3/ CAM5.2), prostate-specific antigen (PSA), and prostatespecific acid phosphatase (PSAP); and negative for p63, GATA3, and CK34pE12. The final diagnosis was small cell carcinoma (SCC) of the prostate, extensively involving all tissue chips.

The major applications of immunohistochemistry in pathology diagnosis of prostate tumors include (1) to establish a prostate primary, (2) to assist the diagnosis of prostate malignancy by markers targeting basal cells or secretory cells, and (3) to differentiate high-grade prostatic adenocarcinoma from urothelial carcinoma or other highgrade malignancies.

Prostate Tissue-Specific Markers

Prostate-specific antigen and PSAP are widely used markers to establish prostatic origin in metastatic carcinomas. The sensitivity and specificity for both markers are high. Studies have shown that benign prostate tissue and low-grade prostate carcinoma expressed more PSA and PSAP than high-grade prostate carcinoma. (89) Prostein (P501S) is another prostate-specific marker. Its expression is seen in normal prostate tissue and prostate cancers but not in nonprostatic tissue or malignancies. (90) Comparative study showed that P501S had a similar expression pattern to PSA in benign prostate tissue, prostatic adenocarcinoma, and metastatic prostate carcinoma; therefore, P501S could serve as an alternative prostate-specific marker when PSA immunostain is negative or equivocal. (90) NKX3.1 is a homeobox protein that showed specific nuclear staining in prostate, testis, and breast. Studies showed that NKX3.1 has similar sensitivity in labeling prostate carcinoma to PSA and P501S and similar sensitivity in identifying metastatic prostate adenocarcinomas to PSA and PSAP. The characteristic nuclear staining pattern, along with its high sensitivity and specificity for prostate carcinoma, makes NKX3.1 a good second-line marker to establish prostate origin. (91) Prostate-specific membrane antigen (PSMA) is a folate hydrolase expressed by most prostate carcinomas and their metastases. Although expression of PSMA in highgrade prostate carcinoma is higher than that of PSA, the fact that PSMA is expressed in various other tumor types, including RCC, gastrointestinal neoplasms, and urothelial carcinomas, limits its utility as a prostate tissue-specific marker. (92)

Markers to Assist in the Diagnosis of Prostatic Malignancy

Markers Targeting Basal Cells.--Absence of basal cells is a key diagnostic criterion for prostate malignancy (Figure 6, A). High-molecular-weight cytokeratin or CK5/6 and p63 are frequently used basal cell markers. Antibodies against HMWCK and CK5/6 stain the cytoplasm and anti-p63 stains the nuclei of basal cells. Although the absence of basal cell markers in a lesion histologically suspicious for cancer usually supports the diagnosis of prostatic carcinoma, (93) a lack of basal cells was reported in 5% to 23% of benign prostatic glands. Therefore, absence of basal cell markers alone is not completely diagnostic of a prostatic carcinoma, and has to be interpreted in an appropriate hematoxylineosin morphology context, especially when the lesion is small. Furthermore, rare cases of prostatic carcinomas aberrantly expressing p63 have been reported. (94) Despite the positive immunostain of p63, HMWCK is reportedly negative in such cases. The false-positive immunostain of basal cell markers, especially HMWCK, in basal cell or nonbasal cell distribution is occasionally seen in prostate carcinoma based on our own experience. Conundrums like these emphasize the importance of using markers targeting basal cells in combination with a prostate carcinoma marker to increase the diagnostic accuracy.

Markers Overexpressed in Prostatic Adenocarcinoma Cells.--AMACR, encoded by the gene P504S, is a mitochondrial and peroxisomal enzyme involved in the b oxidation of branched-chain fatty acids and bile acid biosynthesis. Overexpression of AMACR is found in 83% to 100% of prostatic carcinomas regardless of Gleason score; therefore, AMACR is a great cancer marker. (95-98) However, AMACR itself is not a specific marker for prostate carcinoma, because it may be expressed in high-grade prostatic intraepithelial neoplasia and some benign mimickers of prostate carcinoma, including partial atrophy, atypical adenomatous hyperplasia, and nephrogenic adenoma. Concurrent use of AMACR along with a negative basal cell marker can increase the diagnostic accuracy of prostate cancer significantly. An antibody cocktail composed of AMACR, HMWCK, and p63 has gained popularity in pathology practice with performance superior to either marker separately (Figure 6, B) and is routinely used in many institutions. (99,100)

Fusion of the transmembrane protease serine 2 gene and ETS-related gene (TMPRSS2-ERG) is the most common molecular subtype of erythroblast transformation-specific (ETS) family gene fusions, occurring in approximately 50% of prostate carcinomas and about 20% of high-grade prostatic intraepithelial neoplasias. ERG gene fusions result in the overexpression of a truncated ERG protein product. The current commercially available anti-ERG antibody detecting overexpressed ERG is able to serve as a surrogate marker for ERG gene rearrangement. (101) Validation of anti-ERG antibody showed positive ERG stain in only about 40% of prostate carcinoma cases and rare ERG expression in benign prostate glands. (102,103) Because of its low sensitivity, ERG is sometimes used as a second-line tool given its high specificity.

Differential Diagnosis Between Poorly Differentiated Prostate Adenocarcinoma and Urothelial Carcinoma

Distinguishing poorly differentiated prostate adenocarcinoma from urothelial carcinoma is a frequently encountered dilemma and the final diagnosis has important clinical and therapeutic ramifications. Certain immunohistochemical stains can be very helpful in difficult cases (Table 6). We usually start the workup with PSA, HMWCK, and p63. If the tumor is positive for PSA with intense staining and negative for HMWCK and p63, the findings are supportive of a prostate carcinoma; if the tumor is negative for PSA, but diffusely and strongly positive for p63 and HMWCK, urothelial carcinoma (Figure 7, A through C) is favored. Additional immunostain workup with P501S, NKX3.1, and GATA3 can be used if the PSA, p63, and HMWCK immunostains are equivocal. (104) Positive immunostains of P501S and NKX3.1 together with a negative stain of GATA3 support prostate carcinoma, and diagnosis of urothelial carcinoma would be supported by GATA3 (Figure 7, D) positivity and negative stain of P501S and NKX3.1. (49)

Distinguishing Primary SCC of Prostate From High-Grade Prostate Carcinoma and Metastatic SCC

Small cell carcinoma of the prostate is rare, accounting for less than 0.5% to 1% of prostate malignancy. (105) The diagnosis of SCC can be reached by morphology alone in classical cases (similar to SCC in the lung). Occasionally, SCC is difficult to differentiate from high-grade prostate carcinoma when the morphology alone is not diagnostic, and this differentiation has important therapeutic and prognostic impacts. Small cell carcinoma in the prostate is usually positive for commonly used neuroendocrine markers, including chromogranin A, CD56, synaptophysin, and neuron-specific enolase. (106) The presence of at least one such marker is found in about 90% of prostatic SCCs. (106,107) Prostate-specific antigen and other prostatic markers, such as P501S, are positive in about 17% to 25% of SCCs, yet usually focally. (106,107) Diffuse and strong positive stain of PSA/PSAP favors high-grade prostate carcinoma over SCC. About 24% to 35% of SCCs in prostate are positive for p63 and HMWCK, markers that are typically negative in prostatic carcinoma. (107)

Many markers have been explored in distinguishing primary prostatic SCC and metastatic SCC from other organ sites, especially from bladder or lung. Studies have shown that TTF-1 is expressed in more than 50% of SCCs in the prostate, limiting its utility for this purpose. (106-108) TMPRSS2-ERG gene fusion was identified in about 50% of prostatic SCCs. Therefore, fluorescence in situ hybridization test for TMPRSS2-ERG gene fusion is a potentially useful test to distinguish prostatic SCCs from metastatic carcinomas from other organ sites.109-111 However, this test is not routinely performed or available at many institutions.


Example 4: A 45-year-old man presented with a 6.5-cm, well-circumscribed, solid mass in the right testis by ultrasound. Right radical inguinal orchiectomy specimen demonstrated mixed germ cell tumors (GCTs) (embryonal carcinoma 80%; seminoma 15%; teratoma 3%; YST 2%).

The tumor cells were positive for octamer-binding transcription factor 4 (OCT4), glypican 3 (GPC3), [alpha]-fetoprotein (AFP), and CD30.

Testicular neoplasms are relatively uncommon compared with those of other genitourinary organs, accounting for approximately 1% of human malignancies. However, they are the most common malignancy in young adults and children. The microscopic features of these neoplasms remain the gold standard for the diagnosis, and immunohistochemistry may play a significant role when the morphology is ambiguous. About less than 5% of overall testicular neoplasms require immunostaining for diagnosis. (112) However, use of immunomarkers can be very helpful in assessment of the percentage of different tumor components in a mixed GCT (Figure 8, A).

Testicular tumors can be broadly classified into GCT, sex cord stromal tumors, mixed germ cell-sex cord stromal tumors, and miscellaneous tumors. Seminomas, the most common testicular GCTs, are sensitive to chemotherapy and radiation, whereas nonseminomatous GCTs respond to chemotherapy only. In contrast, most sex cord stromal tumors in the testis are benign. The rare malignant sex cord stromal tumors are resistant to both chemotherapy and radiation therapies and are generally treated with orchiectomy and retroperitoneal lymph node dissection. The most common and significant challenges in testicular pathology include the differential diagnoses between seminoma and nonseminomatous GCTs and distinction between GCTs and non-GCTs, particularly the sex cord stromal tumors. Immunohistochemistry can be very helpful in some cases.

Testis GCT Markers

The great majority (approximately 95%) of primary testicular tumors, including seminoma, embryonal carcinoma, YST, choriocarcinoma, and others, are of germ cell origin, and accurate subtyping is important for proper management and prognosis. (113) To date there are many markers that can be applied in the diagnosis of testis GCTs; these are summarized in Table 7.

OCT4 (also known as OCT3/4 or POU5F1) is a nuclear protein that is essential for the maintenance of pluripotency in embryonic stem cells. Based on OCT4 immunoreactivity, the GCTs can be divided into 2 groups (Table 7): OCT4positive GCTs, including seminoma and embryonal carcinoma, and the OCT4-negative group, which includes YST (Figure 8, B), spermatocytic tumor, and choriocarcinoma. OCT4 is positive in nearly 100% of seminoma and embryonal carcinomas and is uniformly positive in germ cell neoplasia in situ. (114,115) An immunostain panel including AE1/AE3 cytokeratin, CD30, and CD117 can be used to separate seminoma from embryonal carcinoma. (116-118) The AE1/AE3 and CD30 stains are diffusely positive in embryonal carcinoma and negative (or focally positive) in seminoma, whereas CD117 is diffusely positive in seminoma and negative (or focally positive) in embryonal carcinoma.

Because of its complex and unusual growth patterns, YST component tends to be confused with other GCT components such as embryonal carcinoma, immature teratoma, and even seminoma. (119) So far AFP and GPC3 are the only characteristic immunohistochemical markers for YST (Figure 8, C). (119) The differential diagnosis of YST from spermatocytic tumor could be further confirmed by immunoreactivity of GPC3 and AFP in YST and negative stain in spermatocytic tumor. Immunoreactivity of [beta]-human chorionic gonadotropin supports a diagnosis of choriocarcinoma. (120)

The Differential Diagnosis of GCT From Sex Cord Stromal Tumor

Although hematoxylin-eosin morphology is the key to distinguish most sex cord stromal tumors from GCTs, occasional cases may require ancillary tests because of the overlapping morphologic features. These features include diffuse pattern of malignant Sertoli cell tumors with a clear cytoplasm, prominent nucleoli, and lymphocytic inflammation (mimicking seminoma) (121); seminomas with marked tubule formation (mimicking sex cord stromal tumors) (122); Leydig cell tumors with prominent cysts (mimicking YST); and sex cord stromal tumors with entrapped germ cells (mimicking true mixed germ cell-sex cord stromal tumors). (123) In circumstances like this, immunohistochemical stains may be very helpful tools (Table 8). Spalt-like transcription factor 4 (SALL4), [alpha]-inhibin, and calretinin are the commonly used immunohistochemical markers in this category. (112)

The transcription factor SALL4 has been extensively studied as a marker in the diagnostic distinction between sex cord stromal tumors and GCTs. It is a nuclear marker for all testicular GCTs except spermatocytic tumor. (124) The sex cord stromal tumors are negative for SALL4. (124) Currently, [alpha]-inhibin and calretinin are considered the best markers for sex cord stromal tumors of the testis. (125) Most Leydig cell tumors, but only a minority of Sertoli cell tumors, are positive for [alpha]-inhibin and calretinin. (126) The GCTs are negative for [alpha]-inhibin and calretinin. Sangoi et al (127) showed that steroidogenic factor 1 is a more sensitive and specific marker for sex cord stromal testicular tumors than [alpha]-inhibin or calretinin. When SALL4 is not available, a panel of OCT4, GPC3, [alpha]-inhibin, and calretinin can be used for the confirmation of sex cord stromal tumors, which are negative for OCT4 and GPC3. (112)

The Differential Diagnosis of GCT and Large Cell Lymphomas

Primary testicular lymphoma accounts for 1% to 2% of non-Hodgkin lymphomas and 1% to 9% of all testis tumors. (128) Diffuse large B cell lymphoma is by far the most common type of lymphoma in the testis. Distinguishing GCTs from large cell lymphomas can be assisted by an efficient initial marker panel consisting of SALL4, CD45, CD20, and CD3. (112) An important caveat concerning the algorithm is the occasional reactivity for SALL4 in myeloid leukemias, lymphoblastic lymphomas, and anaplastic large cell lymphomas. (129) In a circumstance like this, OCT4, GPC3, and AE1/AE3 can be used to replace SALL4. (112)

In summary, with the availability of an increasing number of more sensitive and specific markers, immunohistochemistry is becoming more important for the precise histologic diagnosis of genitourinary tumors. The appropriate use of immunohistochemistry can increase the diagnostic accuracy and decrease the number of uncertain diagnoses, and immunohistochemistry is almost indispensable in certain complicated situations. However, it is important to stress that the selection of an appropriate panel should be targeted at a group of well-constructed differential diagnoses based on careful microscopic examination and clinicopathologic correlation. Furthermore, we should keep in mind that every marker has its potential advantages and limitations and may be better used as part of a panel. Therefore, caution should be taken to maximize its diagnostic use and avoid possible pitfalls.

Note: Illustration(s) are not available due to copyright restrictions


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Xiuli Xiao, MD; Rong Hu, MD, PhD; Fang-Ming Deng, MD, PhD; Steven S. Shen, MD, PhD; Ximing J. Yang, MD, PhD; Chin-Lee Wu, MD, PhD

Accepted for publication November 11, 2016.

Published as an Early Online Release June 13, 2017.

From the Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (Drs Xiao, Hu, and Wu); the Department of Pathology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China (Dr Xiao); the Department of Pathology, New York University School of Medicine, New York, New York (Dr Deng); the Department of Pathology, Houston Methodist Hospital and Weill Medical College of Cornell University, Houston, Texas (DrShen); and the Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois (Dr Yang). Drs Xiuli Xiao and Rong Hu contributed equally to this article.

The authors have no relevant financial interest in the products or companies described in this article.

Presented at the First Chinese American Pathologists Association (CAPA) Diagnostic Pathology Course: Best Practices in Immunohisto-chemistry in Surgical Pathology and Cytopathology; August 22-24, 2015; Flushing, New York.

Reprints: Chin-Lee Wu, MD, PhD, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St, Boston, MA 02114 (email:

Caption: Figure 1. Useful immunohistochemical markers in the differential diagnoses of renal tumors with clear cells. A, Clear cell renal cell carcinoma (RCC) with typical alveolar arrangement of cells. Inset shows diffuse membranous immunoreactivity for CA-IX in a box-shape pattern. B, Clear cell papillary RCC with papillary structures lined by cuboidal clear cells with piano-key nuclear arrangement. Inset shows cup-shape membranous immunoreactivity for CA-IX. C, MiT family translocation renal cell carcinoma (MiTF RCC) tumor cells with clear and eosinophilic cytoplasm. Inset demonstrates nuclear immunoreactivity for TFE3. D, Epithelioid angiomyolipoma with clear, granular or densely eosinophilic cytoplasm. Inset demonstrates diffuse cytoplasmic immunoreactivity for HMB45 (hematoxylin-eosin, original magnifications 3200 [A, B, and D] and X400 [C]; immunohistochemistry, original magnifications X200 [insets, A and D] and X400 [insets, B and C]).

Caption: Figure 2. Cytokeratin 7 (CK7) immunoreactivity assists in differentiating oncocytoma from chromophobe renal cell carcinoma (RCC). A, Eosinophilic chromophobe RCC is diffusely positive for CK7 (inset). B, Oncocytoma is focally positive for CK7 (inset) in scattered tumor cells (hematoxylin-eosin, original magnifications X400 [A] and 3200 [B]; immunohistochemistry, original magnification X400 [insets, A and B]).

Caption: Figure 3. Classic immunohistochemical profile for papillary renal cell carcinoma (RCC). Type 2 papillary RCC with eosinophilic cytoplasm (A) shows granular cytoplasmic stain for [alpha]-methylacyl coenzyme A racemase (B) and diffuse membranous and cytoplasmic positivity for CK7 (C) (hematoxylin-eosin, original magnification X200 [A]; immunohistochemistry, original magnification X200 [B and C]).

Caption: Figure 4. Use of immunohistochemical stain in diagnosis of urothelial carcinoma in situ (CIS). Urothelial CIS (A) shows full-thickness CK20 (B) and p53 (C) overexpression (hematoxylin-eosin, original magnification X200 [A]; immunohistochemistry, original magnification X400 [B and C]).

Caption: Figure 5. ALK-1 immunoreactivity supports the diagnosis of bladder inflammatory myofibroblastic tumor (IMT). A, A spindle cell bladder lesion with extravasated red blood cells and inflammatory cells. B, Granular cytoplasmic ALK-1 immunoreactivity supports the diagnosis of IMT (hematoxylineosin, original magnification X400 [A]; immunohistochemistry, original magnification X200 [B]).

Caption: Figure 6. Triple immunostain in the diagnosis of prostate adenocarcinoma. A small focus of prostatic adenocarcinoma is showing in the lower part of the core (A) and highlighted by overexpression of [alpha]-methylacyl coenzyme A racemase (red chromogen) and lack of basal cells demonstrated by negative immunostain of p63 and high-molecular-weight cytokeratin (HMWCK) (B). Adjacent benign glands (B) show basal cell stain for p63 (nuclear) and HMWCK (cytoplasmic) (hematoxylin-eosin, original magnification X100 [A]; immunohistochemistry, original magnification X100 [B]).

Caption: Figure 7. Use of immunohistochemistry in distinction of urothelial carcinoma from prostate adenocarcinoma. A transurethral resected bladder tumor resembling prostatic adenocarcinoma morphologically. Tumor cells with vesicular nuclei and prominent nucleoli (A). The tumor cells are positive for p63 (B), high-molecular-weight cytokeratin (C), and GAT A3 (D), supporting urothelial cell carcinoma (hematoxylin-eosin, original magnification X200 [A]; immunohistochemistry, original magnification X200 [B through D]) .

Caption: Figure 8. Use of immunohistochemistry in diagnosis of germ cell tumor (GCT). A, A mixed GCT with embryonal carcinoma and yolk sac tumor (YST) components. B, Immunostain of OCT4 is positive in the embryonal carcinoma, negative in the YST. C, Immunostain of GPC3 is positive in the YST and negative in the embryonal carcinoma (hematoxylin-eosin, original magnification X200 [A]; immunohistochemistry, original magnification X200 [B and C]) .
Table 1. Immunohistochemical Markers for Renal Tumors With Clear

Marker       Clear Cell      Clear Cell     Chromophobe
                 RCC        Papillary RCC       RCC

CA-IX       + (box shape)   + (cup shape)        -
CK7               -               +              +
AMACR            -/+              -              -
CD117             -               -              +
TFE3/TFEB         -               -              -
HMB45             -               -              -

Marker      MiTF    AML

CA-IX         -      -
CK7           -      -
AMACR        -/+     -
CD117         -      -
TFE3/TFEB     +      -
HMB45        +/-     +

Abbreviations: AMACR, a/methylacyl coenzyme A racemase; AML,
angiomyolipoma; CA/IX, carbonic anhydrase IX; CK7, cytokeratin
7; HMB45, human melanoma black 45; MiTF RCC, MiT family
translocation renal cell carcinoma; RCC, renal cell
carcinoma; TFE3/TFEB, transcription factor E3 and transcription
factor EB;+, positive;+/-, majority positive; -/+, majority
negative;-, negative.

Table 2. Immunohistochemical Markers for Renal Tumors With
Oncocytic Cytoplasm

Marker    Clear     Papillary    Chromophobe
         Cell RCC   RCC Type 2       RCC

CD117       -           -             +
CK7         -          +/-            +
CA-IX       +           -             -
AMACR      -/+          +             -
HMB45       -           -             -

Marker   Oncocytoma   Epithelioid

CD117        +             -
CK7          -             -
CA-IX        -             -
AMACR        -             -
HMB45        -             +

Abbreviations: AMACR, [alpha]-methylacyl coenzyme A racemase; AML,
angiomyolipoma; CA-IX, carbonic anhydrase IX; CK7, cytokeratin
7; HMB45, human melanoma black 45; RCC, renal cell carcinoma; +,
positive; +/-, majority positive; -/+, majority negative; -,

Table 3. Immunohistochemical Markers for Renal Tumors With
Significant Papillary Components

Marker      Papillary    Papillary    Clear Cell RCC With
            RCC Type 1   RCC Type 2     Pseudopapillary

CA-IX           -            -           + (Box shape)
CK7             +           +/-                -
CD10            +            +                 +
AMACR           +            +                -/+
TFE3/TFEB       -            -                 -
HMB45           -            -                 -

Marker       Clear Cell     MiTF
            Papillary RCC    RCC

CA-IX       + (Cup shape)     -
CK7               +           -
CD10              -           +
AMACR             -          -/+
TFE3/TFEB         -           +
HMB45             -          +/-

Abbreviations: AMACR, alpha/methylacyl coenzyme A racemase; CA/
IX, carbonic anhydrase IX; CD10, CD10 protein; CK7, cytokeratin
7; HMB45, human melanoma black 45; MiTF RCC, MiT family
translocation renal cell carcinoma; RCC, renal cell carcinoma;
TFE3/TFEB, transcription factor E3 and transcription factor EB;
+, positive; +/-, majority positive; -/+, majority negative; -,

Table 4. Commonly Used Markers in Distinction of Reactive Atypia
From Urothelial Carcinoma In Situ (CIS)

Marker       Normal           Reactive Atypia             CIS

CK20     Only umbrella     Only umbrella            Full-thickness
          cells positive     cells positive           overexpression
p53      Absent            Absent                   Strong positive
CD44     Basal cell        Increased reactivity     Absent
           positive          in all cell layers

Abbreviation: CK20, cytokeratin 20.

Table 5. Immunohistochemical Panels for Spindle Cell Lesions in
the Bladder

                   ALK-1   SMA   Desmin   Cytokeratin

IMT                 +/-     +     +/-          +
Sarcomatoid          -     -/+     -           +
Leiomyosarcoma       -      +      +           -
Rhabdomyosarcoma    -/+     +      +           -

                   p63   HMWCK or

IMT                 -        -
Sarcomatoid        +/-      +/-
Leiomyosarcoma      -        -
Rhabdomyosarcoma    -        -

Abbreviations: ALK-1, anaplastic lymphoma kinase 1; CK5-6,
cytokeratin 5 and 6; cytokeratin (AE1-AE3), pan-cytokeratin;
HMWCK, high-molecular-weight cytokeratin; IMT, inflammatory
myofibroblastic tumor; SMA, smooth muscle actin; +, positive; +/-,
majority positive; -/+, majority negative; -, negative.

Table 6. Immunohistochemical Markers Helpful in Distinguishing
Poorly Differentiated Prostatic Adenocarcinoma
From Urothelial Carcinoma

                   PSA   P501S   NKX3.1   GATA3   HMWCK   p63

Prostatic           +      +       +        -       -      -
Urothelial          -      -       -        +       +      +

Abbreviations: HMWCK, high-molecular-weight cytokeratin; P501S,
prostein; PSA, prostate-specific antigen;+, positive;-, negative.

Table 7. Testicular Germ Cell Tumor Markers

Marker       Seminoma   Embryonal Carcinoma      Yolk
                                              Sac Tumor

OCT4           +                +                 -
CD117          +                -                -/+
GPC3           -                -                 +
CD30           -                +                 -
[beta]-HCG     -                -                 -
AFP            -                -                +/-

Marker       Spermatocytic   Choriocarcinoma

OCT4              -                 -
CD117            -/+                -
GPC3              -                +/-
CD30              -                 -
[beta]-HCG        -                 +
AFP               -                 -

Abbreviations: AFP, [alpha]-fetoprotein; [beta]-hCG,
[beta]--human chorioni gonadotropin; GPC3, glypican 3; OCT4,
octamer-binding transcription factor 4;+, positive;+/-,
majority positive; -/+, majority negative; -, negative.

Table 8. Immunohistochemical Markers Useful to
Distinguish Germ Cell Tumors From Sex Cord Stromal

Marker             Germ Cell      Sex Cord
                     Tumors     Stromal Tumors

SALL4                  +              -
OCT4                   +              -
GPC3                   +              -
[alpha]-Inhibin        -            + or -
  and calretinin

Abbreviations: SALL4, spalt-like transcription factor 4; OCT4,
octamer-binding transcription factor 4; GPC3, glypican 3;+,
positive;-, negative.
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Author:Xiao, Xiuli; Hu, Rong; Deng, Fang-Ming; Shen, Steven S.; Yang, Ximing J.; Wu, Chin-Lee
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Sep 1, 2017
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