Ancillary Tests

Satish K. Tickoo

Debra L. Zynger

Ying-Bei Chen

RENAL IMMUNOHISTOCHEMISTRY

Unlike that for resection specimens, most pathologists have relatively limited experience with renal needle core biopsies or cytology preparations. It is therefore expected that ancillary studies, particularly immunohistochemistry (IHC), are used by most pathologists to help them arrive at specific diagnoses. At the same time, it needs to be kept in mind that often, only the better differentiated (low-grade) tumors show the typical immunophenotype, whereas poorly differentiated (high-grade) tumors are more likely to lose the staining characteristics of the specific tumor type. Aberrant or unexpected expression of antigens is also a possibility, particularly in less differentiated tumors. Although IHC studies can be very helpful in resolving difficult differential diagnostic considerations, they should not be requested at random in all cases. The choice of requested IHC markers must depend on careful light microscopic analysis and formulation of differential diagnostic possibilities. A number of IHC markers have been used in the differentiation of renal cell tumors; some of the useful ones are detailed in Table 13.1. However, in the vast majority of cases, only a limited number of antibodies among those listed in the Table 13.1 are required to arrive at proper diagnosis on biopsy material (Table 13.2).

The following is a brief description of some of the most commonly used antibodies in the differential diagnosis of renal tumors.

Carbonic Anhydrase IX

An important downstream molecule in the hypoxia-inducible factor (HIF) pathway, carbonic anhydrase IX (CA-IX) is a very useful marker in the differential diagnosis of renal tumors. Its consistent, diffuse membranous positivity in overwhelming majority of clear cell renal cell carcinomas (RCCs) (1,2,3,4,5,6,7), at both the primary (Fig. 13.1A,B, eFig. 13.1) and metastatic sites (8,9,10,11) (Fig. 13.2A,B), as well as in both the epithelial and spindle cell components of sarcomatoid clear cell RCC in a large proportion of cases (12) (Fig. 13.3A,B), makes its utility on limited material quite valuable. However, interpretation of the staining—particularly in biopsies and cytologic cell blocks—requires considerable attention to detail. Because CA-IX is also expressed in hypoxic

areas in most tumorous and nontumorous tissues (eFig. 13.2A,B), biopsies showing tumor necrosis merit interpretation with particular caution; perinecrotic areas almost always show strong membranous positivity for CA-IX (Fig. 13.4A,B). Thus, interpretation is quite problematic in biopsies with predominantly necrotic tumors in which the few viable areas often show diffuse membranous positivity. In such cases, the diagnosis of clear cell RCC cannot be rendered with certainty. Similarly, patchy staining in a biopsy— which may be a result of tumor ischemia—does not support the diagnosis of a clear cell RCC. Occasionally, CA-IX may show granular cytoplasmic staining in some tumors, mostly in those with eosinophilic cytoplasm (Fig. 13.5). Because CA-IX is a transmembrane molecule, cytoplasmic staining without membranous positivity is nonspecific and should not be regarded as a positive result. Among the renal cell tumors, CA-IX is also diffusely expressed in clear cell papillary RCC; however, the staining pattern is usually quite different (cup-shaped in clear cell papillary versus box-shaped in clear cell RCC) (13) (Fig. 13.6A,B, eFig. 13.3A,B). The other caveat is that CA-IX is often expressed in a diffuse, membranous pattern in mesotheliomas (10,11) and may also be expressed in urothelial carcinomas (14). Therefore, correlation with morphology, clinical history, and usage of other markers in the form of a panel is mandatory (Table 13.2). Most of the non-clear cell RCC that express CA-IX show positivity in a patchy manner and often will also show immunoreactivity for markers appropriate for that tumor.

TABLE 13.1 List of Antibodies Used in the Diagnosis of Renal Tumors

	Clear Cell RCC	Clear Cell Papillary RCC	Papillary RCC, Type 1	Papillary RCC, Type 2	Chromophobe RCC	Renal Oncocytoma
PAX8/PAX2	+	+	+	+	+/-	+/-
CA-IX (diffuse, membranous)	+ (box-like pattern)	+ (cup-shaped pattern)	–	–	–	–
CD10	+	–	+ (often luminal)	+/-	+/-	–
CK7	–	+	+	+/-	+/-	-/+
AMACR	–	–	+	+/-	–	–
CD117	–	–	–	–	+	+
Vimentin	+/-	+	+/-	-/+	–	–
Parvalbumin	–	ND	–	–	+	+
E-cadherin	–	ND	–	–	+	+
EpCAM	-/+	ND	+/-	–	+	-/+
CD82	ND	ND	ND	ND	+	–
TFE3/TFEB	–	–	–	–	–	–
Cathepsin-K	–	–	–	–	–	–
HMB-45	–	–	–	–	–	–
Melan-A	–	–	–	–	–	–
34βE12 (HMWCK)	–	+	–	–	–	–
p63	–	–	–	–	-/+	-/+
GATA3	–	–	–	–	–	–
INI1 (BAF47)	+	+	–	+	+	+
OCT-4	–	–	–	–	–	–
CD57	ND	ND	–	ND	ND	ND
	MTSCC	Collecting Duct Carcinoma	Renal Medullary Carcinoma	Metanephric Adenoma	Translocation RCC	Urothelial Ca
PAX8/PAX2	+	+/-	+/-	+	+	-/+
CA-IX (diffuse, membranous)	–	–	–	–	-/+(focal)	-/+
CD10	+/-	–	–	+	+	–
CK7	+	-/+	-/+	-/+	–	+
AMACR	+	-/+	-/+	+/-	–	-/+
CD117	–	–	–	–	–	–
Vimentin	–	–	–	–	–
Parvalbumin	ND	-/+	ND	–	–	–
E-cadherin	ND	ND	ND	–	–	–
EpCAM	ND	ND	ND	–	–	–
CD82	ND	ND	ND	–	–	–
TFE3/TFEB	ND	–	–	–	+	–
Cathepsin-K	–	–	–	–	+	–
HMB-45	–	–	–	–	-/+	–
Melan-A	–	–	–	–	-/+	–
34βE12 (HMWCK)	–	+/-	-/+	–	–	+
p63				–	–	+
GATA3	–	–	–	–	–	+
INI1 (BAF47)	+	+	–	+	+	+
OCT-4	–	–	+	ND	ND	ND
CD57	ND	ND	ND	+	–	–
RCC, renal cell carcinoma; MTSCC, mucinous tubular and spindle cell carcinoma; Ca, carcinoma; HMWCK, high-molecular-weight cytokeratin; ND, not done.

TABLE 13.2 Most Useful, Limited Differentiating Immunohistochemical Panels for Tumors in the Kidney^a

Differential Diagnosis	Important Positive Markers	Important Negative Markers
Clear cell cytology group:
Clear cell RCC	CA-IX (box-shaped), CD10, vimentin	CD117
Chromophobe RCC	CD117, CK7, E-cadherin	CA-IX, vimentin
Clear cell papillary RCC	CA-IX (cup-shaped), CK7, HMWCK	CD10, AMACR
Translocation RCC	TFE3/TFEB, Cathepsin-K, HMB-45 (+/-), CD10	CK, EMA
Papillary RCC	AMACR, CK7 (in type 1)	CA-IX (present only focally)
Epithelioid angiomyolipoma	HMB-45, Melan-A (A103), SMA (+/-)	CK, CA-IX
Adrenal cortical neoplasm	Inhibin, Melan-A (A103), synaptophysin	CK, EMA, CA-IX, chromogranin
Eosinophilic cell cytology group:
Clear cell RCC	CA-IX, CD10, vimentin	CD117
Chromophobe RCC	CD117, CK7, CD82, E-cadherin	CA-IX, vimentin
Renal oncocytoma	CD117, CK7-/rare cells	CD82
RCC, unclassified, oncocytic	CD117, CK7 (+/-) [histology prevails]	[histology prevails]
Papillary RCC, type 2	CK7 (may be focal), AMACR	CD117, CA-IX
SDHB-RCC	Variable for usual markers	SDHB
Oncocytic angiomyolipoma	HMB-45, Melan-A (A103), SMA (+/-)	CK, CA-IX
Adrenal cortical neoplasm	Inhibin, Melan-A (A103), synaptophysin	CK, EMA, CA-IX, chromogranin
Papillary/tubulopapillary architecture group:
Papillary RCC, type 1	CK7, AMACR, CD10 (often luminal)	CA-IX (occasionally focal positive)
Papillary RCC, type 2	CK7 (+/-), AMACR, CD10 (often luminal)	CA-IX
Collecting duct carcinoma	CK7 (+/-), AMACR (+/-), HMWCK (+/-), Ulex europaeus, BAF47 (INI1)	CA-IX, CD10
Renal medullary carcinoma	Variable for CK7, AMACR	BAF47 (INI1)
HLRCC-associated carcinoma	CK7 (-/+), AMACR (+/-), 2SC, BAF47	Fumarate hydratase, others variable
Translocation RCC	TFE3/TFEB, cathepsin K, HMB-45 (+/-), CD10	CK, EMA
RCC, unclassified	Variable for usual markers, BAF47 (INI1)	Variable for usual markers, TFE3/TFEB
Clear cell papillary RCC	CA-IX (cup-shaped), CK7, HMWCK	CD10, AMACR
Metanephric adenoma	CD57, WT1	CK7 (-/+), AMACR (-/+)
Mucinous tubular and spindle cell	CK7, AMACR	CD10 (occasionally very focal positive)
Poorly differentiated histology group:
Clear cell RCC	PAX8/2, CA-IX, CD10	TTF-1, CK7, CK20, chromogranin, synaptophysin, inhibin, HMB-45, Melan-A
Epithelioid AML	HMB-45, Melan-A (A103), SMA (+/-), cathepsin K	PAX8/2, CK, EMA
Metastatic tumors	Depending on the primary source	PAX8/2 (unless GYN tract, thyroid, or occasionally mesothelial), CA-IX (unless mesothelial)
Adrenal cortical carcinoma	Inhibin, Melan-A, synaptophysin	EMA, CK, PAX8/2, CA-IX
Urothelial carcinoma	GATA3, p63, HMWCK (34βE12), CK7, CK20	PAX8/2 (occasionally positive)
Spindle cell group:
Sarcomatoid RCC	CA-IX (in sarcomatoid clear cell RCC), EMA/CK (at the most focal in spindle cell areas), PAX8/2 (+/-)	NA
Mucinous tubular and spindle cell	CK7, AMACR	CD10 (occasionally very focal+)
Sarcoma	Markers according to the subtype	PAX8/2, CK/EMA (except synovial sarcoma, leiomyosarcoma, and epithelioid angiosarcoma)
aAberrant, unexpected positivity for individual markers may be present in rare cases. RCC, renal cell carcinoma; HMWCK, high-molecular-weight cytokeratin; EMA, epithelial membrane antigen; SDHB, succinate dehydrogenase B; HLRCC, hereditary leiomyomatosis and renal cell carcinoma; AML, angiomyolipoma; NA, not applicable.

FIGURE 13.1 A: Primary clear cell RCC (hematoxylin and eosin). B: Diffuse, membranous (box-like) positivity for CA-IX.

PAX8 and PAX2

PAX8 and PAX2 are members of paired box family transcription factors that are important in the fetal development of nervous system, kidney, thyroid, eye, and female genital tract. They show nuclear expression in the vast majority of renal cortical tumors at both the primary and metastatic
sites (Fig. 13.7A,B, eFigs. 13.4A,B) (15,16,17,18,19,20,21,22,23,24). Therefore, these are very useful markers for establishing renal cell origin in the proper clinical context. Overall, PAX8 appears to be a more sensitive marker than PAX2 (23), but in rare instances, the reverse may be true (22). Although of great utility for determining renal origin of a tumor, both the markers may also be expressed in the tumors of female genital tract (18,22), clear cell
adenocarcinoma of the bladder (25,26), peritoneal mesotheliomas (27), urothelial carcinomas—particularly of the upper tract (24), and thymic tumors (28). PAX8 is also uniformly positive in the tumors of thyroid. At the same time, staining for PAX8 and PAX2 may be absent in some tumors of renal cell origin, particularly high-grade tumors such as collecting duct carcinoma, sarcomatoid RCCs, and tumors with rhabdoid features;
chromophobe RCCs and renal oncocytomas may also show negative staining (22). Thus, positive staining in isolation cannot be regarded as definitive for renal cell origin, nor does the lack of staining exclude a renal origin. Proper interpretation requires analysis in association with other appropriate stains (Table 13.2).

FIGURE 13.2 A: Clear cell RCC metastatic to liver (hematoxylin and eosin). B: CA-IX immunoreactivity in the metastatic tumor.

FIGURE 13.3 A: Spindle cell component of a sarcomatoid clear cell RCC. B: Strong, diffuse positivity for CA-IX in the sarcomatoid component of the tumor.

FIGURE 13.4 A: Needle core biopsy showing a predominantly necrotic tumor. B: Even diffuse immunoreactivity for CA-IX in the small viable perinecrotic tumor foci cannot be considered diagnostic for clear cell RCC.

FIGURE 13.5 Granular cytoplasmic immunoreactivity without membrane accentuation should not be regarded as a positive result for CA-IX.

FIGURE 13.6 A: In clear cell papillary RCC, CA-IX characteristically does not stain the luminal aspect of the cells (cup-shaped pattern). B: CA-IX immunoreactivity in clear cell RCC highlights all cell membranes (box-like pattern).

FIGURE 13.6 A: (Continued)

CD10

CD10, also known as neprilysin and common acute lymphoblastic antigen (CALLA), is a 94 kDa zinc-dependent cell membrane metalloprotein, which is widely distributed in the normal tissues, including kidney, liver, intestines, placenta, choroid plexus, brain, gonads, adrenal cortex,
and leucocytes (29). It has been widely used as a marker for clear cell RCC in its differentiation from other renal tumors in both the primary and metastatic settings (30,31,32,33,34,35,36,37,38,39,40). In the appropriate clinicopathologic setting, diffuse membranous positivity is supportive of clear cell subtype among all the RCCs (Fig. 13.8A). However, diffuse cytoplasmic staining can be seen in a number of renal and nonrenal tumors (36) and is not helpful in this distinction. At the same time, membranous reactivity is also not specific for clear cell RCC and may be present in papillary RCC (Fig. 13.8B), colorectal, pancreatic, prostatic, and some vascular tumors (30,36,41). Both benign hepatic parenchyma and hepatocellular carcinoma show canalicular pattern of staining with CD10 (42). This may be mistaken for membranous staining, particularly in the limited material of needle core biopsies or cytologic preparations. Of import, clear cell papillary RCC is often negative for CD10, in spite of being positive for some other markers of clear cell RCC (13,43,44) (Tables 13.1 and 13.2). Thus in spite of its proven utility in clear cell RCC, given its nonspecificity, CD10 should not be used in isolation for the diagnosis of clear cell RCC and should only be used as a part of immunohistochemical panel (Table 13.2).

FIGURE 13.7 A: PAX8 showing diffuse nuclear positivity in a primary RCC. B: The immunoreactivity for PAX8 is retained at metastatic sites (clear cell RCC metastatic to the liver).

FIGURE 13.7 A: (Continued)

Cytokeratin 7

Cytokeratin 7 (CK7) is a type II keratin normally present in simple, nonkeratinizing epithelium. The antibody is commonly used in surgical pathology and cytology in the differential diagnosis of tumors from a variety of organs. It is also a very useful antibody in the differential diagnosis of renal tumors (6,42,45). CK7 is diffusely positive in type 1 papillary

RCC (Fig. 13.9A), classical chromophobe RCC (Fig. 13.9B), clear cellpapillary RCC (Fig. 13.9C), mucinous tubular and spindle cell carcinoma (Fig. 13.9D), and urothelial carcinoma (6,13,42,44,45,46,47). However, papillary RCC type 2 and eosinophilic chromophobe show very variable results and may be negative or only focally positive for CK7 (48) (Fig. 13.10A,B).

Clear cell RCC, with some rare exceptions, is usually completely negative for CK7, although cystic clear cell RCC often shows patchy positivity around the cysts. CK7 cannot be used in the distinction of clear cell papillary RCC from multilocular cystic RCC, as the latter also shows diffuse immunoreactivity for CK7 (Fig. 13.11A,B) (49). Renal oncocytoma is usually
negative or shows positivity in only rare dispersed cells. Metanephric adenoma is also usually negative for CK7 unlike type 1 papillary RCC, a close differential diagnostic consideration, particularly on limited biopsy material (50). In other renal tumors, results with CK7 are variable and inconsistent. Overall, CK7 remains a useful antibody in the differentiation of a number of renal tumors. However, one cannot completely depend on the results with this antibody alone in the distinction of renal oncocytoma from eosinophilic chromophobe RCC, or papillary type 2 RCC from other high-grade RCCs with prominent papillary architecture, particularly on biopsy specimens.

FIGURE 13.8 A: Diffuse membranous positivity for CD10 in a clear cell RCC. B: Papillary RCC showing the characteristic, predominantly luminal staining.

FIGURE 13.9 CK7 showing diffuse immunoreactivity in (A) type 1 papillary RCC, (B) classical chromophobe RCC, (C) clear cell papillary RCC, (D) mucinous tubular and spindle cell carcinoma, and (E) urothelial carcinoma.

FIGURE 13.9 (Continued)

FIGURE 13.10 Only focal positivity for CK7 in (A) eosinophilic chromophobe and (B) type 2 papillary RCC.

FIGURE 13.10 (Continued)

CD117 (c-Kit)

CD117 is a receptor tyrosine kinase type III, which binds to stem cell factor known as steel factor or c-Kit ligand. Its main utility in the differential diagnosis is in the separation of chromophobe RCC from eosinophilic clear cell RCC (3,42,51). Chromophobe RCC usually shows diffuse cytoplasmic immunoreactivity with peripheral accentuation with CD117 (Fig. 13.12A), whereas clear cell RCC is negative. Renal oncocytoma is also often immunoreactive for CD117; usually, the staining pattern is granular and cytoplasmic (Fig. 13.12B) (3,42). In our experience, CD117 also shows diffuse immunoreactivity in the oncocytic tumors that are not classifiable as either renal oncocytoma or eosinophilic chromophobe RCC and are often considered as oncocytic unclassified tumors.

FIGURE 13.11 CK7. A: Focal CK7 positivity in clear cell RCC in and around cystic areas. B: Like clear cell papillary RCC, multilocular cystic neoplasm of low malignant potential/multilocular cystic RCC also shows diffuse immunoreactivity with CK7. Therefore, CK7 cannot discriminate between these morphologically closely related tumors.

FIGURE 13.12 CD117 (c-Kit) showing diffuse positivity for (A) chromophobe RCC with accentuated peripheral staining and (B) renal oncocytoma with diffuse cytoplasmic staining.

Renal Cell Carcinoma Marker

RCC marker (RCC Ma), normally expressed in the proximal renal tubules, has been in use for many years as a marker for renal origin of tumors as well as in the differential diagnosis among the renal tumors. However, its sensitivity among renal tumors is quite low (27% to 90%). At the same time,
a number of nonrenal tumors, including hepatocellular carcinoma, lung adenocarcinoma, breast lobular carcinoma, ovarian clear cell carcinoma, testicular yolk sac tumor, and parathyroid carcinoma, among others, stain positive with the antibody (52). It is also expressed in greater than 40% of nonrenal tumors with papillary architecture (53). Given the availability of more sensitive and specific markers now, RCC Ma appears to have only limited utility in the diagnosis and differential diagnosis of renal tumors.

Others

A number of other markers, some as tabulated (Table 13.1), have been regarded as helpful in the differential diagnosis of renal tumors. Thus, AMACR characteristically shows diffuse, granular cytoplasmic positivity in type 1 papillary RCC (Fig. 13.13A, eFig. 13.5A,B); type 2 papillary also usually stains positive (Fig. 13.13B). Clear cell papillary RCC is typically negative, but many other tumors often show immunoreactivity with AMACR (Table 13.1). Some other markers which are particularly useful in the differential diagnosis include strong and diffuse nuclear immunoreactivity for TFE3/TFEB among translocation-associated RCC (Fig. 13.14A,B), loss of nuclear reactivity for BAF47 (INI1) in renal medullary carcinoma (Fig. 13.15A,B), and CD57 positivity in metanephric adenoma (Fig. 13.16A,B).

A general observation with IHC has been that new markers are very often initially reported as specific for a tumor type, but that “specificity” is frequently lost after greater experience with the antibody based on larger numbers and greater diversity of the cases. Finding markers that distinguish between renal oncocytoma and the eosinophilic variant of chromophobe RCC and other close mimickers has been particularly polemic in this regard. A large number of antibodies have been proposed to make this distinction but, in general, have not withstood the test of time as discriminatory markers (33,45

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