Conflict of interest: The authors declare no conflict of interest.
Correspondence: Naoto Kuroda, MD, Department of Diagnostic Pathology, Kochi Red Cross Hospital, Shin-honmachi 2-13-51, Kochi City, Kochi 780-8562, Japan. Email: email@example.com
The recent classification of renal tumors has been proposed according to genetic characteristics as well as morphological difference. In this review, we summarize the immunohistochemical characteristics of each entity of renal tumors. Regarding translocation renal cell carcinoma (RCC), TFE3, TFEB and ALK protein expression is crucial in establishing the diagnosis of Xp11.2 RCC, renal carcinoma with t(6;11)(p21;q12), and renal carcinoma with ALK rearrangement, respectively. In dialysis-related RCC, neoplastic cells of acquired cystic disease-associated RCC are positive for alpha-methylacyl-CoA racemase (AMACR), but negative for cytokeratin (CK) 7, whereas clear cell papillary RCC shows the inverse pattern. The diffuse positivity for carbonic anhydrase 9 (CA9) is diagnostic for clear cell RCC. Co-expression of CK7 and CA9 is characteristic of multilocular cystic RCC. CK7 and AMACR are excellent markers for papillary RCC and mucinous tubular and spindle cell carcinoma. CD82 and epithelial-related antigen (MOC31) may be helpful in the distinction between chromophobe RCC and renal oncocytoma. WT1 and CD57 highlights the diagnosis of metanephric adenoma. The combined panel of PAX2 and PAX8 may be useful in the diagnosis of metastatic RCC.
The recent classification of renal tumors is based on the difference of clinical, morphological and molecular genetic characteristics.[1, 2] In this review, we present immunohistochemical characteristics of each tumor entity, with focus on newly recognized tumor categories, and describe the caution of interpretation of immunohistochemical results, particularly in translocations-associated renal cell carcinoma (RCC). Antibodies frequently employed in the histological diagnosis of renal tumors are summarized in Table 1. Additionally, positive immunohistochemical markers in each tumor entity are summarized in Table 2.
Table 1. Antibodies frequently employed in the diagnosis of renal tumors
Table 2. Positive markers in each tumor category
Xp11.2 translocation renal cell carcinoma (RCC)
6p21 translocation RCC
ALK, PAX2, PAX8
Acquired cystic disease-associated RCC
AMACR, RCC Ma, CD10
Clear cell papillary RCC
CK7, AMACR, CD10
Clear cell RCC
CA9, RCC Ma, CD10
Multilocular cystic RCC
CK7 (type 1 > 2), AMACR
Collecting duct carcinoma
CK7, CK19, PAX8
Mucinous tubular and spindle cell carcinoma
Translocation-Associated Renal Cell Carcinomas
Renal carcinoma associated with Xp11.2 translocations/TFE3 gene fusions (Xp11.2 RCC)
This tumor predominantly affects children and young adults. This tumor forms a microphthalmia transcription factor/transcription factor enhancer (MiTF/TFE) translocations-associated RCC family with renal carcinoma with t(6;11)(p21;q12).[3, 4] This tumor bears chromosomal translocation involving TFE3 gene located on Xp11.2 and several kinds of partners including ASPL on chromosome 17p25, PRCC on chromosome 1q21, PSF on chromosome 1p34 or CLTL on chromosome 17q23. Additionally, inv(X)(p11;q12) results in formation of a fusion gene, NoNo-TFE3. A partner gene of t(X;3)(p11;q23) is unknown. Xp11.2 RCC is histologically characterized by mixed papillary nested/alveolar growth pattern and tumor cells with clear and/or eosinophilic voluminous cytoplasm. In ASPL-TFE3 RCC, tumor cells have distinct cell border, vesicular chromatin, and prominent nucleoli. Psammoma bodies and hyaline nodules are frequently seen in the stroma. In PRCC-TFE3 RCC, tumor cells have a solid, compact architecture, slightly less voluminous cytoplasm, usually less frequent psammoma bodies and hyaline nodules, and less prominent nucleoli. Different TFE3 gene fusions constantly lead to overexpression of fusion protein, compared with native TFE3. Hence, the strong immunohistochemical nuclear labeling for TFE3 is a sensitive and specific marker for this tumor (Fig. 1). However, a subset of the usual type of RCCs, characterized by unfavorable clinical behavior and poor patient survival, may lead to the TFE3 activation through the increase of TFE3 copy numbers. Additionally, the FLCN inactivation in Birt-Hogg-Dubé syndrome may give rise to the increase of the TFE3 protein level. As the TFE3 is ubiquitously distributed in the normal human body, immunohistochemical techniques such as excessive antigen retrieval, high antibody concentration, and excessive signal amplification could lead to false-positive results. Accordingly, we recommend that appropriate positive (alveolar soft part sarcoma or another Xp11.2 RCC definitely diagnosed) and negative (for example, clear cell RCC) controls for TFE3 immunohistochemistry should be prepared in the diagnosis of Xp11.2 RCC. Positivity for negative control suggests the detection of native TFE3. We have experienced that this reaction is often caused by the immunohistochemistry using an autoimmunostainer. Therefore, TFE3 immunohistochemisty should be performed carefully. Thus, pathologists should recognize some possibilities of the positivity of TFE3 protein except for Xp11.2 RCC. Cathepsin-K is frequently positive in Xp11.2 RCC cases and available in distinguishing MiTF/TFE translocation type RCCs from other adult renal neoplasms except for angiomyolipoma, including the epithelioid variant. The clinical behavior of Xp11.2 RCC cases in children and young adults is indolent, but adult Xp11.2 RCC cases behave in a more aggressive fashion, of which the underlying reason is unknown.
Renal carcinoma with t(6;11)(p21;q12) (6p21 RCC)
This tumor also occurs predominantly in children and young adults. This tumor bears the translocation between the TFEB gene located on chromosome 6p21 and the Alpha gene located on chromosome 11q12. Histologically, this tumor is characterized by the nests of cells to clear to granular cytoplasm and the two cells pattern consisting of large and small tumor cells. The small cells tend to surround basement membrane materials, forming rosette-like structures, which could be a key diagnostic clue.[6, 7] The strong nuclear labeling for TFEB is a highly sensitive and specific marker for 6p21 RCC (Fig. 2). As TFEB is ubiquitously expressed at the mRNA level, the immunohistochemical technique such as excessive antigen retrieval, use of high concentration antibody and excessive signal amplification may lead to false-positive results for the TFEB protein. This tumor is frequently positive for melanocytic markers including Melanosome-associated antigen (detected by HMB-45) and Melan-A, but does not typically express epithelial markers including pancytokeratin and epithelial membrane antigen (EMA). Cathepsin-K may also be an important marker for the accurate diagnosis of this tumor. In the differential diagnosis, the distinction from renal epithelioid angiomyolipoma (eAML) is important because the immunophenotype of this tumor resembles that of eAML, as well as its morphology. In this setting, the positivity for TFEB is crucial. The number of reported cases for this tumor is too small to determine the clinical behavior.
Renal carcinoma with ALK gene rearrangement
This tumor has been recently identified, and seems to affect relatively young adults.[8-11] This tumor is very rare in incidence and comprises 2.3% of non-clear cell RCC. Histologically, this tumor displays a cribriform pattern of tumor cells with eosinophilic cytoplasm or papillary architecture with stromal mucin deposition. Additionally, the tumor resembling renal medullary carcinoma or the tumor with mixed histology of renal medullary carcinoma, chromophobe RCC, and urothelial carcinoma (UC) have been reported in patients bearing sickle cell anemia.[8, 9] A fusion of ALK gene on chromosome 2p23 and other genes including Vinculin (VCR) gene on chromosome 10q22.2, Tropomyosin 3 (TPM3) gene on chromosome 1q23 and EML4 gene on chromosome 2p21 has been identified to date.[8-10] Immunohistochemically, this tumor is positive for ALK protein, but usual methodology may be negative because promoter activity of the partner gene is probably low. Accordingly, the intercalated antibody-enhanced polymer (iAEP) method may be useful in the immunohistochemical screening of this tumor. The increase of copy numbers of the ALK gene comprises 10% of total RCCs, but ALK gene translocation seems to be rare. In order to exclude the possibility of metastatic lung adenocarcinoma with ALK rearrangement, the immunohistochemical panel of thyroid transcription factor-1 (TTF-1), surfactant apoprotein A (SPA), PAX2 and PAX8 may be available. In this setting, Napsin A is not useful because Napsin A is expressed both in normal lung and in kidney. In order to elucidate the clinical behavior of this tumor, a large scale study is required.
This tumor affects patients receiving long-term dialysis, particularly for more than 10 years, and exclusively occurs with the background of ACD. This tumor is histologically characterized by cribriform architecture containing numerous microcysts with deeply eosinophilic or oncocytic cytoplasm, prominent nucleoli and the deposition of intratumoral calcium oxalate crystals.[12-15] Comparative genomic hybridization or FISH analyses show gain of clustering on chromosomes 3, 7, 16, 17, and sex chromosome. Immunohistochemically, tumor cells are positive for AMACR, RCC Ma, and CD10, but negative for cytokeratin (CK) 7. This immunophenotype seems to resemble that of papillary, type 2. Antimitochondrial antibody is strongly immunoreactive in the tumor cytoplasm. A close relationship to ACD-associated RCC and papillary RCC in genetic aspect of dialysis-related renal tumors has been suggested.[13, 15, 16] This tumor generally shows a favorable prognosis, but tumors with sarcomatoid change pursue a worse clinical course.
Clear cell papillary renal cell carcinoma
This tumor occurs in both end-stage kidney disease without cystic lesions and ACD. However, this tumor occurs also in kidney without underlying disease. Macroscopically, this tumor often forms cystic space. The tumor histologically consists of papillary configurations lined by cells with clear cytoplasm and low-grade nuclei.[12, 14, 17] Genetically, this tumor generally notes no characteristics of clear cell RCC or papillary RCC. Immunohistochemically, neoplastic cells are positive for CK7 and CA9, but negative for RCC Ma, CD10, alpha-methylacyl-CoA racemase (AMACR), and TFE3. These immunohistochemical phenotypes seem to be basically different from papillary RCC, type 1, or type 2. This tumor is generally indolent in its clinical behavior.
This tumor macroscopically shows well-circumscribed and spongy ‘bubble wrap’ appearance. Histologically, this tumor consists of cysts and tubules lined by a single layer of flat, hobnail, cuboidal and columnar cells with eosinophilic cytoplasm and Fuhrman Grade 3 nuclei. Most tumors are positive for cytokeratin 7, AMACR, and CD10.[18, 19]
Renal Tumor Entities in World Health Organization (WHO) Classification at 2004
Clear cell renal cell carcinoma
This tumor is the most common type and accounts for approximately 70 to 80% of all renal tumors. Histologically, neoplastic cells with clear cytoplasm proliferate with alveolar, acinar, solid, and cystic pattern. Infrequently, a tubular or pseudopapillary pattern may be seen. Cytoplasmic granularity tends to increase as the nuclear grade elevates. Approximately 60 to 80% of clear cell RCC cases show biallelic inactivation of von Hippel-Lindau (VHL) gene and the majority of clear cell RCC demonstrates loss of heterozygosity (LOH) of chromosome 3p. Immunohistochemically, neoplastic cells are positive for vimentin, EMA, CA9, RCC Ma, CD10, PAX2, and PAX8, but negative for CK7, high molecular weight CKs, E-cadherin, and kidney-specific cadherin.[20-28] Clear cell RCC more often and more consistently demonstrate high (85%) CA9 expression than any other tumor types (Fig. 3). The prognosis of this tumor depends on nuclear grade, pathological ,stage and the presence of sarcomatoid change.
Multilocular cystic renal cell carcinoma
This tumor is rare and low-grade renal neoplasm associated with a favorable prognosis. Macroscopically, this tumor is well circumscribed, has fibrous capsule and no expansile nodule. This tumor consists of thin fibrous septa lining multiple cystic spaces and clear cells.[30, 31] Molecular genetic alterations including VHL gene inactivation and LOH of chromosome 3p seem to resemble those of clear cell RCC, but its frequency in multilocular cystic RCC is lower than those of clear cell RCC. Immunohistochemically, neoplastic cells are positive for CA9, PAX2, CD10, EMA, CK7, but negative for AMACR. Immunoreactivity for cytokeratin 7 is different from clear cell RCC, but may resemble that of clear cell papillary RCC characterized by prominent cystic architecture. However, clear cell papillary RCC is generally negative or focally positive for CD10.
Papillary renal cell carcinoma
This tumor accounts for approximately 7 to 15% of all renal tumors. This tumor is histologically subdivided into two categories. In type 1 papillary RCC, tumor cells have basophilic cytoplasm and low grade nuclei. However, nuclear pseudostratification is absent. In type 2 papillary RCC, neoplastic cells possess eosinophilic cytoplasm and high grade nuclei with pseudostratification. Gain of chromosomes 7 and 17 and loss of chromosome Y are characteristic of type 1 papillary RCC. However, these chromosomal abnormalities are infrequent in type 2 papillary RCC. CK7 and AMACR is a sensitive marker for papillary RCC (Fig. 4). However, tumor cells are also immunoreactive for hepatocyte nuclear factor 1B (HNF -1B), CD10, RCC Ma, PAX2, and PAX8.[32-35] The CK7 expression is more frequent in type 1 than type 2. Both type 1 and type 2 papillary RCCs exhibits cytoplasmic immunoreactivity for AMACR in most cases. The immunoreactivity of more than 10% of cancer cells for CD10 is observed more often in type 2 than type 1. There seems to be significant immunophenotypic overlapping between papillary RCC and mucinous tubular and spindle cell carcinoma (MTSCC). The prognosis of this tumor depends on nuclear grade, stage at presentation and sarcomatoid change. However, the prognosis of type 1 is better than that of type 2.
Chromophobe renal cell carcinoma
This tumor comprises approximately 5 to 10% of total renal neoplasms. This tumor is histologically subdivided into two categories, namely typical and eosinophilic variants. In typical variant, pale cells with relatively voluminous and reticulated cytoplasm predominantly proliferate with compact or cobblestone pattern. In eosinophilic variant, eosinophilic cells chiefly proliferate with compact pattern. Cell border is distinct and wrinkled nuclei or binucleation is seen. Perinuclear halo is often seen. Multiple losses of chromosomes 1, 2, 6, 10, 13 17 and 21 is frequently observed in chromophobe RCC. Hale's colloidal iron shows diffuse cytoplasmic positivity for chromophobe RCC, but some eosinophilic variants may be negative for this stain. Tumor cells are immunoreactive for CK7, CD82 (Fig. 5), ERA(MOC31), palvalbumin, CD117, E-cadherin, and kidney-specific cadherin, but negative for vimentin, CA9, CD10, and RCC Ma.[41-45] Immunoreactivity of parvalbumin notes intense nuclear and cytoplasmic staining pattern.[41, 42] Strong cytoplasmic staining with peripheral cell accentuation for CK7 may be characteristic of chromophobe RCC, unlike renal oncocytoma (RO). Positivity for CD117 in chromophobe RCC shows distinct peripheral cytoplasmic accentuation, unlike cytoplasmic positivity in RO. Intense cytoplasmic staining with peripheral cell membrane accentuation for antimitochondrial antibody may suggest chromophobe RCC. A subset of chromophobe RCC with aggressive behavior may express CD10. In the distinction from RO, a panel of CK 7, CD82, and MOC31 is often employed.[43-46] In the differential diagnosis between chromophobe RCC and RO, the immunohistochemical expression pattern of the discriminating panel is summarized in Table 3. Ultrastructurally, neoplastic cells contain numerous cytoplasmic microvesicles and mitochondria with tubulovesicular cristae.[40, 42] The prognosis of this tumor is better than that of clear cell RCC. However, sarcomatoid change and perinephric invasion are associated with aggressive clinical behavior.
Table 3. Immunohistochemical profile of chromophobe renal cell carcinoma (RCC) and renal oncocytoma (RO)
−, negative; +, positive.
Collecting duct carcinoma of Bellini (CDC)
This tumor accounts for less than 1% of all renal tumors. Histologically, various configurations of tumor cells with marked cytologic atypia such as tubular, papillary, solid or hobnail pattern are observed. The stromal desmoplasia or inflammation is prominent. Atypical collecting ducts may be seen in the interface between the tumor and surrounding renal parenchyma. It seems to be very important for the pathological diagnosis of this tumor to exclude the possibilities of other renal tumors. There has been no report of consistent genetic abnormality for CDC to date. Tumor cells are positive for vimentin, CK7, CK19, PAX2, PAX8, but negative for CK5/6, CK17, kidney-specific cadherin, CD10, RCC Ma, CD117, p63, thrombomodulin, uroplakin III, and CK20. Immunoreactivity for high molecular weight cytokeratins and Ulex europaeus lectin agglutinin is described as one of diagnostic criteria of the present WHO classification. However, in our experience, these markers seem to be not necessarily available for the diagnosis of CDC. The immunohistochemical panel of CK5/6, CK17, and vimentin or the immunohistochemical combination of PAX8 and p63 may be available in the distinction for UC.[23, 48] This tumor generally has a poor prognosis.
Mucinous tubular and spindle cell carcinoma (MTSCC)
MTSCC accounts for less than 1% of all renal tumors. Histologically, tumor cells consist of elongated tubules showing frequent fusion and spindle cells with low nuclear grade. The stroma demonstrates a significant amount of mucin. Mucin-poor variant or spindle-cell dominant variant has been also reported. MTSCC seems to have a considerable immunophenotypic overlapping to papillary RCC. Tumor cells are frequently positive for CK 7, AMACR, and EMA. The immunoreactivity for RCC Ma and CD10 is variable. HNF1B may express in some MTSCC cases. In CGH or FISH analyses, multiple genetic losses involving chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22 are seen in MTSCC. This tumor behaves in a favorable fashion, but aggressive tumors have been reported recently.
Renal oncocytoma (RO)
This tumor comprises about 3 to 5% of total renal tumors. Macroscopically, the cut surface of the tumor shows mahogany brown. Larger tumors may possess a central scar. Histologically, the tumor consists of compact or nesting growth of neoplastic cells with deeply eosinophilic or oncocytic cytoplasm. Nuclei are generally round, centrally located and have smooth margin. Chromosomal analysis shows normal in most RO cases. However, losses of chromosomes Y and 1, deletion from chromosome 14, or alteration of 11q13 including translocation may be observed in some cases. Mitochondrial DNA alteration in RO has been reported. Hale's colloidal iron stain in RO shows a negative or luminal staining pattern. In immunohistochemical analysis, neoplastic cells show positivity for CD117, E-cadherin, and S100A1, but negativity for RCC Ma and CD10. In the distinction from chromophobe RCC, the utility of S100A1 positivity in RO has been reported in some studies. Positivity for CD117 in RO is seen predominantly in the cytoplasm. The frequency of CK7 positivity in RO accounts for 0–10%, but the reactivity is usually focal. Diffuse granular cytoplasmic staining for antimitochondrial antibody may suggest the diagnosis of RO. Immunoreactivity for parvalbumin in RO is focal cytoplasmic staining pattern. Ultrastructurally, neoplastic cells contain numerous mitochondria with lamellar cristae. This tumor is benign. However, this tumor should be strictly distinguished from sporadic hybrid oncocytic/chromophobe tumor (SHOCT) or chromophobe RCC, oncocytic variant.
This tumor is rare and generally affects middle-aged women. Macroscopically, the tumor is well circumscribed, but unencapsulated. Histologically, the tumor cells with scant basophilic cytoplasm proliferate with acinar, tubular and papillary architectures. Glomeruloid structures may be also seen. Psammoma bodies are frequently seen in the stroma. The potential tumor suppressor gene located on chromosome 2p13 has been suggested. Immunohistochemically, tumor cells are positive for CD57, WT1, PAX2, and PAX8 but negative for CK7. Unlike nephroblastoma, the blastemal cells positive for CD56 are absent. Positivity for CK7 and AMACR generally distinguishes papillary RCC from metanephric adenoma. Because of the benign nature of metanephric adenoma, these differential diagnoses are very important.
Immunohistochemical Application of Needle Biopsy
Demand for needle biopsy for renal tumors is gradually increasing because of active surveillance in elderly or comorbidly ill patients, or the application of radiofrequency ablation and cryoablation. It is difficult for pathologists to accurately diagnosis all renal tumors in only routine hematoxylin and eosin stain of needle biopsy specimens. Accordingly, the immunohistochemical panel of CA9, CD117, AMACR, CK7, and CD10 may be recommended in the establishment of accurate pathological diagnosis for diagnostically difficult cases. Diffuse membranous positive for CA9 and CD10 supports the diagnosis of clear cell RCC. Diffuse reactivity for AMACR (cytoplasmic) and CK7 (membranous) suggests the diagnosis of papillary RCC. Diffuse membranous positivity for CK7 and CD117 should be considered to be chromophobe RCC. If all markers are negative, the diagnosis of RO should be considered.
Immunohistochemistry of Some Morphological Settings
Renal tumors with clear cytoplasm
In this setting, the differential diagnosis of clear cell RCC, chromophobe RCC (typical variant), Xp11.2 RCC, 6p21 RCC, and clear cell papillary RCC. TFE3 and TFEB is useful for the identification of Xp11.2 and 6p21 RCCs. These tumors often express Cathepsin-K. In the distinction of clear cell RCC from chromophobe RCC, the panel of CD10, RCC Ma, CK7, parvalbumin and kidney-specific cadherin is available. Clear cell RCC expresses CD10 and RCC Ma, whereas chromophobe RCC expresses CK7, parvalbumin and kidney-specific cadherin. In the distinction of clear cell RCC from clear cell papillary RCC, the panel of CK7, CD10 and RCC Ma is useful. Clear cell RCC express CD10 and RCC Ma, whereas clear cell papillary RCC expresses CK7.[22-24]
Renal tumors with eosinophilic or granular cytoplasm
In this setting, the differential diagnosis of clear cell RCC (granular cell variant), chromophobe RCC (eosinophilic variant), papillary RCC (type 2), RO, 6p21 RCC and eAML are crucial. eAML co-expresses alpha smooth muscle actin and melanoma markers. 6p21 RCC is immunoreactive for TFEB. The panel of CK7, CD82, and MOC31 may assist the distinction between chromophobe RCC and RO. Namely, chromophobe RCC is positive for CK7 and CD82, but RO is negative or focally positive for CK7. Additionally, RO is often immunoreactive for S100A1. Papillary RCC express AMACR and may express CK7.[22-24]
High grade infiltrating carcinoma
In this setting, the differential diagnosis includes CDC, UC and other high grade RCCs. UC expresses CK7, CK20, p63, and thrombomodulin and CK5/6, whereas CDC expresses vimentin, PAX2, and PAX8. RCCs including clear cell RCC and papillary RCC expresses CD10, RCC Ma, and vimentin.[22-24]
Renal tumors with spindle cell morphology
In this setting, the differential diagnosis such as sarcomatoid carcinoma, sarcoma, MTSCC, and malignant lymphoma, particularly in diffuse large B-cell lymphoma, are important. In the distinction from sarcomatoid carcinoma and sarcoma including clear cell sarcoma, primitive neuroectodermal tumor, liposarcoma, leiomyosarcoma, angiosarcoma, osteosarcoma, synovial sarcoma, or undifferentiated sarcoma, the identification of epithelial component by thorough tissue sampling is most important. However, diffuse expression of epithelial markers supports the diagnosis of sarcomatoid carcinoma. However, leiomyosarcoma and synovial sarcoma can express epithelial markers. MTSCC expresses CK7, CK19, and AMACR and generally possesses low grade nuclei.
Diagnosis for Metastatic RCC
In this setting, the immunohistochemical panel composed of CD10, RCC Ma, PAX2, and PAX8 seems be available. However, the positivity for CD10 and RCC Ma seems to decrease in metastatic lesions, compared with the primary site. Accordingly, negative results for CD10 and RCC Ma in metastatic sites cannot exclude the possibility of metastatic RCC. Both PAX2 and PAX8 are diagnostically useful markers for both primary and metastatic RCC, irrespective histologic subtype. PAX8 seems to be more sensitive than PAX2 both in primary and in metastatic setting.
Even after the publication of the World Health Organization classification in 2004, some tumor entities including tubulocystic carcinoma or thyroid-like follicular carcinoma have been identified.[18, 19, 55-58] Accordingly, a large scale study of newly identified tumor entities will be expected in the near future. Furthermore, the appearance of new available immunohistochemical markers for molecular targeting therapy such as ALK protein will be expected in the near future.
We appreciate the kind direction for Dr Hideaki Enzan, Chikamori Hospital, Kochi, Japan and Dr Yuji Ohtsuki, Matsuyama-shimin Hospital, Ehime, Japan.