Cytokeratin 7 and Cytokeratin 20

Cytokeratins (CKs) are a family of intracytoplasmic intermediate filament proteins present in almost all epithelia. Expression of each CK molecule depends on cell type and differentiation status; therefore specific CKs can be used as markers to identify particular types of epithelial tumors ( Table 17.1 ). CK7 is found in a wide variety of epithelia, including the columnar and glandular epithelium of the lung, cervix, and breast, as well as in the bile duct, collecting ducts of the kidney, urothelium, and mesothelium, but it is not in most gastrointestinal (GI) epithelia, hepatocytes, proximal and distal tubules of the kidney, and squamous epithelium. In contrast, CK20 shows relatively restricted expression and is present in GI epithelium, Merkel cells of the epidermis, and urothelium.

CK7 expression is observed in the majority of URCa, whereas CK20 expression in URCa has been reported to vary from 15% to 97% in different studies. Bassily and colleagues showed that CK20 is more frequently positive in low-grade tumors (83%) than in high-grade tumors (52%). However, Desai and associates showed higher expression in high-grade tumors; thus most cases of URCa are positive for both CK7 and CK20. This immunoprofile (CK7+/CK20+) is helpful, particularly in the differential diagnosis of metastatic neoplasm of uncertain primary, although other examples of CK7-positive, CK20-positive tumors include carcinomas of extrahepatic bile duct and gallbladder, pancreatic adenocarcinoma, endocervical adenocarcinoma, mucinous tumors of the ovary and upper GI tract, and mucinous bronchioloalveolar adenocarcinoma.

More than half of all cases of primary adenocarcinoma of the bladder are also positive for both CK7 and CK20. However, given that intestinal-type primary adenocarcinomas of bladder are likely to be CK7 negative and CK20 positive, such a panel has only a limited role in the differential diagnosis with secondary bladder involvement by adenocarcinoma of colorectal origin. The utility of the combination of CK7 and CK20 IHC can be further enhanced by the addition of tissue-specific markers such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), P501S, thyroid transcription factor 1 (TTF-1), and so on. For example, prostatic adenocarcinomas that are occasionally CK7 and CK20 positive could be distinguished from URCa by their positivity for PSA. Unequivocal strong or extensive PSA positivity should not be encountered in URCa.

Several studies have suggested a diagnostic role for CK20 expression pattern in distinguishing flat URCa in situ (CIS) from reactive urothelial atypia. In reactive nonneoplastic lesions, CK20 expression is usually restricted to surface “umbrella” cells. In contrast, the majority of urothelial dysplasia or CIS will show at least focal positive transmucosal CK20 expression in all layers of urothelium. CK20 staining in conjunction with Ki-67 proliferation index and p53 and p16 expression have also been suggested to be of value in distinguishing reactive atypia from CIS. We do not routinely resort to immunostaining in establishing the diagnosis of CIS.

Finally, a role for CK20 expression pattern as a predictor of recurrence in low-grade urothelial neoplasms has also been proposed.

Uroplakin

Uroplakins (UPs) are urothelium-specific transmembrane proteins present in terminally differentiated superficial urothelial cells; therefore expression of UPs is expected to diminish during urothelial tumorigenesis. The majority of noninvasive and up to two-thirds of advanced invasive and metastatic URCas have been shown to retain UP expression, as assessed by UPIII IHC. Interestingly, in some of these studies, loss of UPIII expression was associated with significantly worse prognosis, even in patients with advanced disease. The latter was true on multivariate analysis when established prognostic parameters, such as stage and presence of lymph node metastasis, were included. Although highly specific for urothelial differentiation, IHC expression of UPIII is only of moderate sensitivity (as low as 40%), given that UPIII messenger RNA (mRNA) can often be detected in the absence of UPIII protein. Attesting to their suggested urothelial histogenesis, benign Brenner tumors of the ovary also stain for UPIII. Interestingly, only a slim minority of malignant Brenner tumors and primary ovarian URCa (6%) stained positive for UPIII in the study by Logani and associates.

Thrombomodulin

Thrombomodulin (TM), also designated CD141 , is an endothelial cell–associated cofactor for the thrombin-mediated activator of protein C. TM expression, predominantly as membranous staining, has been found in 69% to 100% of URCa. TM expression is particularly useful in differentiating URCa from high-grade prostatic adenocarcinoma, renal cell carcinoma (RCC), and adenocarcinoma of the colon and endometrium, in which TM is rarely positive. However, it should be kept in mind that TM is also expressed by nonurothelial tumors such as vascular tumors, mesothelioma, and squamous cell carcinoma (SCC). Compared with UPIII, TM has a higher degree of sensitivity but lower specificity as a marker for URCa.

TP63

As mentioned in the section on prostate carcinoma (PCa), TP63 , a homolog of the TP53 tumor suppressor gene, encodes at least six different proteins with a wide range of biologic functions, including a role in urothelial differentiation. Immunostaining for p63 is normally present in more than 90% of urothelial nuclei. The majority of URCa retains a normal expression pattern of p63, but expression may be partially lost in higher grade invasive URCa. However, in a recent study, we found p63 to be superior to TM as a urothelial marker of differentiation in high-grade tumors. In combination with prostate lineage markers, p63 is a valuable marker in differentiating URCa from high-grade prostatic adenocarcinoma secondarily invading the bladder.

High-Molecular-Weight Cytokeratin

As already mentioned in the discussion on PCa, the monoclonal antibody 34βE12 reacts specifically with high-molecular-weight cytokeratins (HMWCKs) CK1, CK5, CK10, and CK14. In addition to its aforementioned utility in labeling the basal cell layer of prostatic glands, HMWCK is a highly sensitive marker of urothelial differentiation, matching the sensitivity of p63 and surpassing that of TM and UPIII.

HMWCK is useful in differentiating URCa, in which it is normally positive, from PCa, which is usually negative for HMWCK. However, a cautionary note is warranted, given that HMWCK labels squamous epithelia, including areas of squamous differentiation, in posttherapy recurrent PCa lesions. HMWCK positivity restricted to areas of squamous differentiation does not exclude the diagnosis of PCa.

IHC for HMWCKs has been cited to be helpful in distinguishing urothelial dysplasia, which shows only basal staining, from flat urothelial CIS, in which a transmucosal staining of urothelial layers is expected. Diffuse expression of HMWCK in low-grade papillary URCa has been reported to be a strong predictor of tumor recurrence. Finally, CK5 expression has recently been utilized as a surrogate immunostain marker for the “basal/squamous-like” subtype in a novel genomic taxonomy for bladder MIBC based on recently published TCGA analysis.

GATA3

GATA3 (GATA binding protein 3 to DNA sequence [A/T]GATA[A/G]) is a member of a zinc finger transcription factor family that plays an important role in promoting and directing cell proliferation, development, and differentiation. Several recent studies have pointed to its utility as a marker for the diagnosis of URCa.

In the most recent and largest study by Liu and colleagues, applying a commercial antibody in tissue microarray analysis (TMA) sections constructed from 1110 carcinomas and 310 normal tissues of various organs, 62 of 72 URCas (86%) were positive for GATA3. The nuclear staining is usually diffuse in more than 50% of cells. It is important to remember that in addition to URCa, 91% of ductal mammary carcinomas and 100% of lobular carcinomas also tested positive for GATA3 in the same study. Rare staining (<2% of cases) was found in endometrioid-type endometrium, whereas SCC of lung origin, prostatic carcinoma, and clear cell and papillary RCC have been shown to be negative.

Anaplastic Lymphoma Kinase

Anaplastic lymphoma receptor tyrosine kinase ( ALK ) is a cytoplasmic membrane tyrosine kinase receptor expressed in anaplastic large cell lymphoma. ALK expression has been detected in approximately two-thirds of inflammatory myofibroblastic tumors (IMTs) of the urinary tract, also termed postoperative spindle cell nodule, inflammatory pseudotumor , and pseudosarcomatous fibromyxoid tumor . The ALK gene is located on chromosome 2p23, and rearrangements of this gene through translocations with various gene partners have been identified in IMT from a number of anatomic sites, including the urinary bladder. Two recent studies by Montgomery and associates and Sukov and colleagues showed rates of 72% and 67%, respectively, of ALK gene rearrangement in bladder IMT using a split-apart fluorescence in situ hybridization (FISH) probes strategy. Sukov and colleagues saw a tighter correlation between positive expression and rearrangement in their study. Given the close morphologic and immunophenotypic similarities between IMT and malignant spindle cell urinary bladder tumors, demonstration of ALK immunostaining or gene rearrangement by FISH can prevent unnecessary radical surgery.

TP53

The tumor suppressor gene TP53 orchestrates the transcriptional regulation of cell-cycle control elements, and TP53 mutations represent the most common genetic alterations in human malignancies. A number of studies have revealed TP53 mutations in 40% to 60% of invasive bladder cancers and their association with a worse prognosis. The altered protein product of the mutant TP53 gene has an extended half-life, leading to its accumulation and detection by IHC techniques. Staining results may vary because of differences in specimen processing and fixation, and only modest correlation between TP53 mutations and protein overexpression has been shown. In addition, TP53 alterations in URCa have been shown to be predictive of increased sensitivity to chemotherapeutic agents that damage DNA.

As mentioned previously in the discussion of CK20 expression patterns in urothelial CIS, one potential use of TP53 immunostaining is differentiating urothelial CIS from reactive urothelial atypia. Strong, extensive p53 positivity in more than 50% of cells is encountered in CIS, whereas reactive urothelium is usually negative or demonstrates only weak, patchy TP53 nuclear staining ( Fig. 17.1A to D ).

Cytokeratin 20 (CK20) and p53 staining as an adjunct to differentiate reactive urothelial atypia (A and C) from urothelial carcinoma in situ (B and D). (A and B) CK20 immunostain. (C and D) p53 immunostain.

CDKN2A

The tumor suppressor gene CDKN2A (formerly p16 ) inhibits cyclin D-dependent protein kinases and thereby plays a vital role in the regulation of G1-S transition. In fact, CDKN2A gene (9p21) deletions and mutations are frequent in bladder cancer and appear to be more frequent in low-grade superficial tumors compared with higher grade invasive tumors. Several recent studies revealed a significant correlation between loss of p16 expression and progression in noninvasive (pTa) and superficially invasive (pT1) URCa.

Yin and colleagues recently showed increased expression of p16 protein in flat urothelial CIS compared with its uniform and weak expression in normal and reactive urothelium, suggesting a potential diagnostic role for p16 immunostaining in such settings. p16 immunohistochemistry cannot be used to distinguish secondary involvement of the bladder with uterine cervical carcinoma from URCa.

Retinoblastoma Protein

The retinoblastoma gene ( RB1 ) product was the first to be identified in human cancer. It encodes a nuclear protein, pRb, which plays a crucial role in cell-cycle progression by regulating cell-cycle arrest at the G1-S phase. Alterations in pRb may occur either as a result of RB1 gene mutations or as a result of loss of p16, which normally phosphorylates pRb. Loss of heterozygosity (LOH) of one RB1 gene allele in combination with mutation of the remaining allele is found more frequently in high-grade muscle-invasive URCa tumors. Interestingly, both overexpression and loss of pRb expression have been associated with increased risk for bladder cancer progression. It appears that alterations in pRb and p53 act in a synergistic manner to promote bladder cancer progression. Thus pRb immunostaining could be a useful prognostic marker in URCa.

Urothelial Carcinoma and Variants

The distinctive morphologic features of noninvasive papillary urothelial neoplasms—papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade and high-grade noninvasive papillary URCa—make their diagnosis easily achieved on hematoxylin and eosin (H&E) sections. Therefore the diagnostic role of IHC in URCa is practically limited to (1) distinguishing high-grade invasive URCa from tumors that secondarily involve the bladder from adjacent organs or, more rarely, from distant primary sites; (2) assigning a primary urothelial origin for a metastatic carcinoma of unknown primary; (3) potential utility in distinguishing CIS from reactive urothelial atypia; and (4) establishing the diagnosis in rare variants, such as plasmacytoid and sarcomatoid URCa.

Urinary bladder involvement by a secondary tumor, either as a metastasis or by direct extension, occurs most commonly from colorectal (33%), prostatic (12%), and cervical (11%) sites. Less common sources include breast, stomach, lung, and melanoma primaries.

Spread from a colonic or rectal primary could present a diagnostic challenge in bladder transurethral resection (TUR) samples. In fact, such secondary involvement is a more common occurrence than primary adenocarcinoma of the bladder. Differentiating a colorectal carcinoma (CRCa) spread from intestinal-type primary adenocarcinoma of the bladder cannot usually be achieved with certainty. The presence of a background of urothelial intestinal metaplasia with associated glandular dysplasia may favor a primary origin; however, the pathologist should consider the possibility of secondary colonization of bladder urothelial mucosa by a well-differentiated CRCa mimicking intestinal metaplasia/dysplasia. In general, a recommendation to clinically rule out spread from a colorectal primary by imaging techniques should be forwarded in order to avoid a potentially unjustifiable radical cystectomy procedure. Immunostains that include CDX2, β-catenin, villin, and CK7/CK20 have been shown to be helpful by some authors. However, some degree of overlap in staining patterns among primary enteric-type bladder adenocarcinomas and secondary colorectal adenocarcinomas still exists, which limits the utility of these markers on an individual case basis ( Fig. 17.2A to F ).

(A and B) Rectal adenocarcinoma secondarily involving urinary bladder. The tumor is negative for cytokeratin 7 (CK7) and is positive for CK20, CDX2, and β-catenin as shown in panels C to F, respectively.

The second most common source of secondary tumor involvement of the bladder is PCa. Even in cases in which a prior known history of PCa is given, superimposed morphologic changes, such as squamous differentiation as a result of prior hormonal or radiation treatment, lead to additional difficulty in distinguishing PCa recurrence from a second primary URCa on a transurethral resection of prostate (TURP) or needle biopsy. As mentioned in the discussion of prostate tumors, poorly differentiated PCa may have enlarged nuclei and prominent nucleoli, yet little variability is found in nuclear shape and size in PCa. High-grade URCa often reveals a more pronounced nuclear pleomorphism. URCa tends to grow in nests, even when poorly differentiated, and usually lacks the cribriform and cord-like architecture of PCa. However, in the absence of a noninvasive flat or papillary URCa component, it is difficult on limited material to distinguish high-grade PCa that involves the bladder from primary high-grade infiltrating URCa on routine H&E-stained sections. Given the crucial difference in management and prognosis, resorting to immunostains is a must if the distinction could not be made with absolute certainty on morphologic grounds.

As mentioned in the section on prostate, PSA and prostate-specific acid phosphatase (PSAP) have proved to be useful in identifying prostate lineage. However, the sensitivities of PSA and PSAP decrease in poorly differentiated PCa, and newer markers such as prostein (P501S), PSMA, proPSA (pPSA), and NKX3-1 may be of added utility. Combining the previously listed markers with urothelial lineage markers, such as TM and uroplakin, will further facilitate resolving a urothelial versus a prostatic differential ( Table 17.2 ).

It should also be kept in mind that both UPIII and TM are of only moderate sensitivity, compared with HMWCK and p63, in labeling URCa. Recent studies have documented HMWCK positivity in more than 90% of URCa. HMWCK is only rarely and focally expressed in PCa (8%), and p63 has a greater specificity for URCa, albeit lower sensitivity, compared with HMWCK (100% specificity, 83% sensitivity). Finally, our experience shows that CK7 and CK20 are of limited utility in this differential, given that they may both be positive in a subset of PCa. More recently, GATA3 has achieved better sensitivity and specificity in labeling URCa and ruling out prostate adenocarcinoma.

Among other rare sources of tumors that metastasize to the bladder, mammary carcinoma deserves a cautionary note. The possibility of a breast metastasis should be raised when epithelial infiltration is seen in the form of cords or individual plasmacytoid to signet-ring–shaped cells that involve lamina propria without associated overlying papillary urothelial proliferation or CIS. In such cases, the differential should also include a rare variant of URCa, plasmacytoid variant ( Fig. 17.3A to C ). Obtaining a proper clinical history and the use of IHC that includes estrogen and progesterone receptors, gross cystic disease fluid protein (GCDFP), UPIII, and TM will help reach a proper diagnosis. Finally, positive reactivity for CD138 in the plasmacytoid variant of URCa can lead to a misdiagnosis of plasma cell dyscrasia if a proper battery of immunostains was not utilized.

(A and B) Plasmacytoid variant of urothelial carcinoma. (C) High-molecular-weight cytokeratin highlights the tumor cells.

In the workup of metastatic carcinoma of unknown primary origin, inclusion of GATA3, CK7, CK20, HMWCK, TM, and UPIII is needed to rule out a urothelial primary. Using a panel of four of the latter markers, excluding CK7, in a wide range of 112 urothelial tumors, Parker and colleagues revealed that expression pattern varied with tumor grade and stage ( Table 17.3 ). Variant morphologic subtypes showed staining similar to that of conventional URCas. In the same study, TMA showed no UPIII immunoreactivity in tissue cores of nonurothelial tumors, rendering the expression of UPIII in a tumor almost diagnostic of urothelial origin. Although coexpression of TM, HMWCK, and CK20 strongly suggests urothelial origin, none of these markers is as specific as UPIII and GATA3, given that TM is expressed in nonurothelial tumors, such as non–small cell lung carcinomas (27%) and rare lymphomas, and given that HMWCK is expressed by 43% of non–small cell lung carcinomas and mesotheliomas, among others.

Among URCa variants, sarcomatoid carcinoma deserves special attention because of its likelihood to be confused with “true” mesenchymal neoplasms, such as leiomyosarcoma, osteosarcoma, and rhabdomyosarcoma. This is especially likely when heterologous elements are displayed and noninvasive papillary or in situ urothelial components are not evident. Reactivity for one or more of the markers AE1/AE3, CAM5.2, epithelial membrane antigen (EMA), HMWCK, p63, CK7, and CK20 supports the diagnosis of sarcomatoid carcinoma, although caution should be used with positivity for CAM5.2 and p63 because they can be seen in sarcomas. Positive reactivity for actin can be encountered in sarcomatoid carcinoma and should not mislead the observer to a diagnosis of leiomyosarcoma. As discussed later, attention to differentiating sarcomatoid carcinoma from IMT is also crucial.

Urinary Bladder Adenocarcinoma

Primary adenocarcinomas of the bladder are relatively rare; therefore establishing their diagnosis requires the exclusion of secondary involvement by direct extension or metastatic spread. Bladder adenocarcinoma variants include signet-ring cell carcinomas and urachal, mucinous, and enteric adenocarcinomas. Distinguishing a PCa that extends into the bladder from a primary bladder adenocarcinoma is important and has both clinical and management implications; IHC markers of prostate lineage are of great utility in this regard. Although the specificity of newer prostate lineage markers have been tested against bladder URCa, the same cannot be said about their pattern of reactivity in bladder adenocarcinoma. In a recent IHC study to evaluate 37 adenocarcinomas of the bladder, we demonstrated that a minority of bladder adenocarcinomas are positive for prostate antigens P501S and PSMA. P501S showed moderate diffuse cytoplasmic staining in 11% of cases, including enteric-type and rare mucinous adenocarcinomas. The granular perinuclear staining pattern of P501S typically seen in prostatic adenocarcinoma was absent in all cases of bladder adenocarcinoma. In addition, PSMA showed diffuse cytoplasmic or membranous staining in 21% of bladder adenocarcinomas, including signet-ring, urachal, mucinous, and enteric-type variants. All cases were negative for PSA and PSAP; therefore immunoreactivity for P501S and PSMA should be interpreted with caution in such settings ( Fig. 17.4A to C ). The lack of granular perinuclear staining for P501S and the absence of membranous PSMA staining both favor a bladder adenocarcinoma. Membranous PSMA staining indistinguishable from that seen in PCa can be seen in less than 10% of bladder adenocarcinoma.

Primary adenocarcinoma of urinary bladder (A) showing cytoplasmic positivity for prostate markers P501S (B) and prostate-specific membrane antigen (C).

Benign Mimics of Bladder Carcinoma

We will limit our discussion to two of the benign mimic of bladder tumors: nephrogenic adenoma (NA) as a mimic of both URCa and adenocarcinoma, and IMT as a mimic of sarcomatoid carcinoma or sarcomas.

Nephrogenic Adenoma

Typically, NA displays tubulopapillary structures lined by a single layer of bland cuboidal epithelial cells with low mitotic activity. Tubular structures are frequently surrounded by a distinct ring-like basement membrane and may contain eosinophilic or mucinous secretions. The tubular lining cells frequently display hobnail nuclei. Other tubules can have a flattened lining, thus leading to a false impression of lymphatic structures. Rarely, intracytoplasmic lumina can form in single infiltrating cells that mimic signet-ring carcinoma. Finally, rare examples in which hyalinized myxoid stroma “suffocates” the compressed tubular structures, termed fibromyxoid variant of NA , can be confused with mucinous adenocarcinoma of the bladder. When typical, NA is easily recognized in TURB samples. In difficult examples, the diagnosis of NA can be supported by their unique positivity for PAX2 and PAX8. NA is negative for HMWCK and p63 in two-thirds of cases.

A word of caution is merited in this setting, regarding the fact that clear cell adenocarcinoma of bladder will share the noted immunophenotype with NA and should be recognized by the higher degree of cytologic atypia and mitotic activity found in clear cell bladder adenocarcinoma, which can be further illustrated by a high Ki-67 index compared with NA ( Fig. 17.5A to D ).

Key Diagnostic Points Nephrogenic Adenoma

• •
  

  Variable expression of high-molecular-weight cytokeratin/p63

  
  
• •
  

  Positive for PAX2 and PAX8

  
  
• •
  

  Lacks malignant cytology of clear cell carcinoma

  
  
• •
  

  Low Ki-67

(A to C) Nephrogenic adenoma of the urinary bladder. (D) Nuclear positivity for PAX8 is shown.

Inflammatory Myofibroblastic Tumor

IMT of the bladder may arise either spontaneously or as a result of a prior instrumentation of the bladder. IMTs are benign mesenchymal neoplasms composed of a proliferation of relatively monotonous myofibroblastic cells (typical tissue culture appearance) in a richly vascularized background with red blood cell extravasation and lymphoplasmacytic inflammatory infiltrate. Mitotic activity ranges from absent to brisk, and abnormal mitotic figures are not present. Although IMTs may occasionally recur (25%), only one case of malignant transformation of IMT has been reported in the genitourinary tract. As mentioned previously in the section on anaplastic lymphoma kinase, two-thirds of IMTs contain rearrangement of the ALK gene on chromosome 2p23 with different translocation partners. The latter can be demonstrated by split-apart interphase cytogenetic FISH techniques ( Fig. 17.6A and B ). The translocation leads to ALK protein overexpression on IHC ( Fig. 17.7A to C ). IMTs are frequently immunoreactive for pancytokeratin and CAM5.2, a fact worth remembering when attempting to differentiate IMT from sarcomatoid carcinoma. IMTs are also frequently positive for smooth muscle actin (SMA) and desmin, and the presence of desmin may lead to their misinterpretation as leiomyosarcoma. IMT is usually negative for CD34, S100 protein, and CD117 ( Table 17.4 ).

(A and B) Interphase fluorescence in situ hybridization analysis for ALK1 gene in inflammatory myofibroblastic tumor by using split-apart probes. The presence of a set of one green and one red signal in addition to a juxtaposed red-green (yellow overlap) set indicates a rearrangement in one of the two ALK1 alleles.

(A and B) Inflammatory myofibroblastic tumor of the urinary bladder. (C) Positive ALK1 staining is shown.

Table 17.4

Immunohistochemistry of Spindle Cell Neoplasms of the Urinary Bladder

	IMT	Sarcomatoid URCa	Leiomyosarcoma	Rhabdomyosarcoma
Keratin	+	+	S	−
EMA	−	+	−	−
Vimentin	+	+	+	+
Desmin	+	−	+	+
MSA	+	S	+	+
Alk	S	−	−	−

 

Alk , Anaplastic lymphoma kinase; EMA , epithelial membrane antigen; IMT , inflammatory myofibroblastic tumor; MSA , muscle-specific actin; URCa , urothelial carcinoma.

Reactivity: + almost always positive; S, sometimes positive; −, negative.

Chromosomal Numerical Alteration

Chromosome 9 alterations are the earliest genetic alterations in both of the divergent pathways of BC development described previously. They are responsible for providing the necessary milieu of genetic instability that, in turn, allows for the accumulation of subsequent genetic defects. Several additional structural/numerical somatic chromosomal alterations are also a common occurrence in BC. Among these, gains of chromosomes 3q, 7p, and 17q and 9p21 deletions ( CDKN2A locus) are of special interest, given their potential diagnostic and prognostic value. A multitarget interphase FISH-based urine cytogenetic assay was developed based on the previous numerical chromosomal alterations and is now commercially available and commonly used in clinical management ( Fig. 17.10 ). Initially approved by the Food and Drug Administration (FDA) for surveillance of recurrence in previously diagnosed BC patients, the test subsequently gained approval for screening in high-risk (smoking exposure) patients with hematuria. The multicolor FISH assay appears to enhance the sensitivity of routine urine cytology analysis and can be used in combination with routine cytology as a reflex testing in cases with atypical cytology. A sensitivity range of 69% to 87% and a specificity range of 89% to 96% have been reported with the multitarget interphase FISH assay. With the exception of one study, the multitarget FISH urine assay has been shown to be more sensitive than routine cytology. An additional advantage of urine-based FISH testing could be the anticipatory positive category of patients identified by such assay. This refers to patients in whom FISH assay detects molecular alteration of BC in urine cells several months before cancer detection by cystoscopy or routine cytology. In the study by Yoder and colleagues, two-thirds of the 27% of patients categorized as “anticipatory positive” developed BC that was detected by cystoscopy up to 29 months later. Such encouraging results point to the great potential of molecular testing in early detection and allocation of vigorous, frequent follow-up cystoscopy in at-risk patients.

Interphase fluorescence in situ hybridization urine cytology analysis by using Vysis UroVysion probe sets (Abbott Molecular, Abbott Park, IL) for chromosomes 3q (red), 7p (green), and 17q (aqua) and 9p21 deletions (p16 locus: gold). Note polysomy for 17q (aqua) and deletion of 9p21 loci (absence of gold signals) in two urothelial carcinoma cells.

(Modified from Moonen PM, Merkx GF, Peelen P, et al. UroVysion compared with cytology and quantitative cytology in the surveillance of non–muscle-invasive bladder cancer. Eur Urol 2007;51:1275–1280.)

Finally, several recent studies have pointed to the potential prognostic role for multitarget FISH analysis. Maffezini and colleagues were able to demonstrate that low-risk FISH-positive patients, defined as those with 9p21 loss and chromosome (Ch) 3 abnormalities, had a higher rate of recurrence compared with FISH-negative patients. The recurrence rate was even greater in patients with a high-risk positive FISH (Ch7/Ch17 abnormality). Kawauchi and colleagues used bladder washings, and Kruger and associates used formalin-fixed paraffin-embedded (FFPE) transurethral biopsy samples and both independently found loss of 9p21 to predict recurrence but not progression in NMI-BC. Furthermore, both Savic and associates and Whitson and colleagues found urine cytology and FISH in post-BCG bladder washings to be predictive of failure of BCG therapy in patients with non-muscle-invasive disease. Such a promising prognostic role for multitarget FISH awaits prospective randomized trials before clinical integration into a practice algorithm. Clear guidelines for interpretation and test performance parameters in terms of interobserver reproducibility are also needed.

Receptor Tyrosine Kinases

Recent studies have pointed to the potential prognostic value of evaluating the expression of receptor tyrosine kinases (RTKs), such as FGFR3 , EGFR , and other ERB family members ( ERBB2 [formerly HER2/neu ] and ERBB3 ) in non-muscle-invasive and muscle-invasive bladder cancer.

FGFR3 mutations are a common occurrence in NMI-BC and can theoretically be used alone or combined with RAS and PIK3CA oncogenes as markers of early recurrence during surveillance. Both Zuiverloon and colleagues and Miyake and associates independently developed sensitive polymerase chain reaction (PCR) assays for detecting FGFR3 mutations in voided urine. A positive urine sample by the assay developed by Zuiverloon’s group was associated with concomitant or future recurrence in 81% of NMI-BC cases. An even higher positive predictive value of 90% was achieved in patients with consecutive FGFR3- positive urine samples. Similarly, Miyake and associates were able to detect FGFR3 mutations in 53% of their 45 patients, and found their assay to be superior to cytology (78% vs. 0%) in detecting post-TURB recurrence in NMI-BC harboring FGFR3 mutations in primary tumors.

Kompier and colleagues were recently able to develop a multiplex PCR assay for mutational analysis that detects the most frequent mutation hot spots of HRAS , KRAS , NRAS , FGFR3 , and PIK3CA in FFPE TURB samples. They demonstrated evidence of at least one mutation in up to 88% of low-grade NMI-BC samples. Hernandez and colleagues revealed that FGFR3 mutations were more common among low malignant potential neoplasms (LMPNs; 77%) and TaG1 and TaG2 tumors (61% and 58%) than among TaG3 tumors (34%) and T1G3 tumors (17%). On multivariable analysis, mutations were associated with increased risk of recurrence in NMI-BC.

Van Rhijn and associates previously proposed a molecular grade parameter based on a combination of FGFR3 gene mutation status and MIB-1 index as an alternative to pathologic grade in NMI-BC. Recently, the same group elegantly validated their previously proposed molecular grade parameter and compared it with the European Organization for Research and Treatment of Cancer (EORTC) NMI-BC risk calculator, a weighted score of six variables that includes 1973 WHO grade, stage, presence of CIS, multiplicity, size, and prior recurrence rate. The molecular grade was more reproducible than the pathologic grade (89% vs. 41% to 74%). FGFR3 mutations significantly correlated with favorable disease parameters, whereas increased MIB-1 was frequently seen with pT1, high grade, and high EORTC risk scores. The EORTC risk score remained significant in multivariable analyses for recurrence and progression. Importantly, molecular grade also maintained independent significance for progression and disease-specific survival, and the addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7%.

Several studies have suggested a negative prognostic role for ERBB2 amplification and/or overexpression in MI-BC. Most recently, Bolenz and colleagues found ERBB2-positive MI-BC patients to be at twice the risk for recurrence and cancer-specific mortality on multivariable analyses adjusted for pathologic stage, grade, lymphovascular invasion, lymph node metastasis, and adjuvant chemotherapy.

TP53 , Cell-Cycle Regulators, and Proliferation Index Markers

Early studies by Sarkis and associates revealed TP53 alterations to be a strong independent predictor of disease progression in BC (NMI-BC, MI-BC, and CIS), and TP53 has also been shown to be predictive of increased sensitivity to chemotherapeutic agents that lead to DNA damage. Recent studies have further supported the prognostic role of TP53 in pT1-pT2 patients following cystectomy, showing an independent role for TP53 alteration in predicting disease-free survival (DFS) and disease-specific survival (DSS).

Among other G1-S phase cell-cycle regulators, cyclins D1 and D3, p16, p21, and p27 have also been evaluated as prognosticators in NMI-BC. Lopez-Beltran and associates confirmed their initial finding of the independent prognostic role of cyclin D1 and D3 overexpression in predicting progression in pTa and pT1 tumors. Their findings, however, are in contrast to subsequent findings by Shariat and colleagues, emphasizing the need for further validation in multiinstitutional large cohorts of patients.

A synergistic prognostic role for combining TP53 evaluation with other cell-cycle control elements such as pRb, cyclin E1, p21, and p27 is emerging both in NMI-BC and MI-BC. In a study by Shariat and colleagues, NMI-BC patients with TURB who demonstrated synchronous IHC alterations in all four tested markers—p53, p21, pRb, and p27—were at significantly lower likelihood of sustaining DFS compared with patients with only three markers. The negative predictive effect was decreased with decreasing number of altered markers (3 vs. 2 vs. 1; Fig. 17.11 ). Similarly, some of the same researchers later found that combining p53, p27, and Ki-67 assessment in pT1 radical cystectomy specimens improved the prediction of DFS and DSS.

Synergistic prognostic role of immunohistochemical analysis of four markers—p53, p21, pRb, and p27—in superficial urothelial carcinoma. SE , Standard error.

Chatterjee and associates demonstrated a similar synergistic prognostic role for the assessment of immunoexpression of multiple molecular markers (p53, pRb, and p21) in patients undergoing cystectomy for MI-BC. The superiority of a multimarker approach compared with the prior single-marker approach certainly merits further assessment. Such a multimarker approach of prognostication could soon be integrated in the standard of care in BC management once additional multiinstitutional prospective trials confirm these promising findings.

The tumor proliferation index measured immunohistochemically by either Ki-67 or MIB-1 has been consistently shown to be a prognosticator in bladder cancer. As mentioned earlier, tumor proliferation index (MIB-1) in NMI-BC plays a prognostic role as one of the elements of the molecular grade parameter forwarded by Van Rhijn and colleagues. The independent prognostic role of proliferation index measured by Ki-67 has also been shown. In the study by Quintero and colleagues, Ki-67 index in NMI-BC TURB was predictive of progression-free survival (PFS) and DSS.

A similar role for proliferation index assessment as a prognosticator has been established in MI-BC. Building on initial findings of significance in an organ-confined subset of MI-BC by Margulis and colleagues, a recent report of the bladder consortium multiinstitutional trial (7 institutions, 713 patients) again confirmed the role of the proliferation index, measured in cystectomy specimens. In the later study, Ki-67 improved prediction of both PFS and DSS when added to standard prediction models, supporting a role for proliferation index assessment in stratifying patients for perioperative systemic chemotherapy. This approach has certainly taken Ki-67 assessment a step closer to clinical applicability in MI-BC.

Epigenetic Alterations

Epigenetic analysis is also gaining momentum in BC as a noninvasive diagnostic tool for screening and surveillance. As a prognostic tool, epigenetic analysis has similarly shown promising potential in BC patients.

In an early study by Catto and colleagues, hypermethylation analysis at 11 C-phosphate-G (CpG) promoter islands was performed by methylation-specific PCR (MSP-PCR) in 116 bladder and 164 upper urinary tract tumors. Promoter methylation was found in 86% of all tumors, and the incidence was relatively higher in upper-tract tumors compared with BC. Methylation was associated with advanced tumor stage and higher tumor progression and mortality rates. Most important, on multivariate analysis, methylation at the RASSF1 and DAPK1 gene promoters was associated with disease progression independent of tumor stage and grade.

The same group, using quantitative MSP-PCR at 17 candidate gene promoters, found five loci to be associated with progression: RASSF1 , E-cadherin, tumor necrosis factor (TNF) SR25 , EDNRB , and APC . Multivariate analysis revealed that the overall degree of methylation was more significantly associated with subsequent progression and death than tumor stage. An epigenetic predictive model developed by using artificial intelligence techniques identified the likelihood and timing of progression, with 97% specificity and 75% sensitivity.

Among the studies to evaluate the diagnostic role of promoter hypermethylation, the study by Lin and colleagues used MSP assay in four genes— CDH1 , CDKN2A , p14 , and RASSF1 —in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. MSP detected hypermethylation in the urine of 80% of tested patients. Hypermethylation analysis of CDH1 , p14 , or RASSF1 in urine sediment DNA detected 85% of superficial and low-grade BC, 79% of high-grade BC, and 75% of invasive BC. The study highlighted the great potential of such tests in detecting NMI-BC. A similar diagnostic role was also found by Cabello and associates using a novel technology, methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLAP), to analyze 25 tumor suppressor genes (TSGs) thought to play a role in BC oncogenesis. The TSGs included PTEN , CD44 , WT1 , GSTP1 , BRCA2 , RB1 , TP53 , BRCA1 , TP73 , RARB , VHL , ESR1 , PAX5 , CDKN2A , and PAX6 . The authors found BRCA1 , WT1 , and RARB to be the most frequently methylated TSGs, and receiver operating characteristic curve analyses revealed significant diagnostic accuracies in two additional validation sets.

Finally, assessment of promoter hypermethylation offers additional insights into BC oncogenesis. Promoter hypermethylation of CpG islands and “shores” controlling microRNA (miRNA) expression is one such example.

Gene Expression and Genomic Analysis

Genomic studies have validated previously deciphered genetic pathways of bladder cancer development and unmasked additional crucial driver genetic alterations. Whereas earlier array-based gene expression studies highlighted differentially expressed genetic signatures capable of predicting recurrence and progression, recent integrated genomic and protein analysis studies have better defined clinically relevant molecular subtypes of bladder cancer. By integrating genomic data from aCGH, gene expression arrays, targeted mutation sequencing analysis, and protein analyses, Lindgren and colleagues brought into focus two main genomic molecular circuitry in URCa: the first characterized by FGFR3 alterations, overexpression of CCND1, and deletions in 9q and CDKN2A, and the second characterized by E3F3 amplifications, RB1 and PTEN deletions, gains of 5p, and overexpression of CDKN2A. Advanced tumors in both groups demonstrated TP53/MDM2 gene alterations. The Lindgern and colleagues study was the first to point to the significantly worse prognosis associated with a gene expression profile of a keratinized/squamous phenotype (CK6+). The aggressive behavior of the latter molecular subtype was further illustrated by Choi and colleagues Termed “basal-like” and characterized by p63 activation, squamous differentiation, positive CK5/6, EGFR and CD44 expression, and lack of CK20 ( Fig. 17.12 ), the subtype is sensitive to neoadjuvant chemotherapy. The study also characterized a “luminal” subtype that is typically enriched for activating FGFR3 mutations, active estrogen receptor pathway, and ERBB2 and PPARγ expression profile (all potential targets of therapy), and a third subtype, characterized by wild-type TP53 gene expression signature. The latter was strongly associated with resistance to neoadjuvant MVAC therapy. Interestingly, tumors from the luminal and “basal-like” subtypes also displayed the TP53 wild-type expression signature upon resistance to chemotherapy.

Immunohistochemical analysis of basal and luminal markers expression in bladder cancer. The basal and luminal immunoprofiles (CK5/6+, CD44+, CK20− and CK5/6−, CD44−, CK20+, respectively) are shown in basal (left) and luminal (right) tumors that were categorized as such based on gene expression profiling is shown.

(Modified with permission from Choi W, Porten S, Kim S, et al. Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy. Cancer Cell . 2014;25[2]:152–165.)

Finally, the most comprehensive characterization of the molecular landscape of bladder cancer has been recently concluded as part of The Cancer Genome Atlas project (TCGA). The integrated genomic analysis of 131 MI-BCs revealed a staggering 302 mutations, 204 segmental copy number alterations (CNA), and 22 rearrangements on average per tumor. Recurrent “driver” mutations in 32 genes were found. They include genes involved in cell-cycle regulation, chromatin regulation, kinase signaling pathways, and nine additional genes (e.g., MLL2 , ERCC2 , ELF3 , KLF5 , RXRA , and CDKN1A ) not previously shown to have recurrent mutational pattern in other tumors. Integration of mRNA and miRNA sequencing data and protein expression analysis revealed four major expression clusters. Among them are “papillary-like” clusters (cluster I), enriched for FGFR3 gene alterations and together with cluster II share expression of luminal urothelial differentiations markers (activated expression of ER, GATA3, uroplakin, and ERBB2), and cluster III “basal/squamous-like” as its counterpart in the Choi and colleagues study, characterized by CK5/6 and EGFR expression ( Fig. 17.13 ).

Integrated analysis of mRNA, miRNA and protein data pointed to four clusters of urothelial carcinoma based on expression profiles. (A) Papillary histology, FGFR3 alterations, FGFR3 expression, and reduced FGFR3 -related miRNA expression were encountered in cluster I. (B) High expression of epithelial markers and stem/progenitor cytokeratins is demonstrated in cluster III tumors that occasionally display a squamous histology. (C) Luminal breast and urothelial differentiation markers are enriched in clusters I and II. (D) ERBB2 mutation and estrogen receptor beta (ESR2) expression are enriched in both clusters I and II.

(Adapted with permission from Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature . 2014;507(7492):315–322.)

Targeted Therapy and Predictive Markers in Bladder Cancer

The TCGA and the comprehensive genomic profiling and molecular studies that preceded it uncovered a wide range of therapeutic targets that exist in the majority (>70%) of muscle invasive bladder cancer ( Fig. 17.14 ). Many of such targeted therapeutic strategies and correlate predictive markers are under investigation. The list includes PI3KCA/AKT/mTOR pathways ; RTK/MAPK pathways, including EGFR , FGFR3 , and ERBB2 ; ER pathways; immune response check point modulators ; and chromatin regulation and remodeling targets.

Targets of therapy suggested by TCGA study in bladder cancer. (A) Mutually exclusive alterations in the PI3K/AKT/mTOR pathway are shown. AKT3 overexpression is found in 10% of samples, independent of copy number (right panel) . (B) Alterations in receptor tyrosine kinases (amplification, mutation, or fusion) are found in 45% of tumors. (C) Recurrent mutations in ERBB2 and ERBB3 . The mutations shown in black are either recurrent in the TCGA data set or reported in COSMIC. (D) ERBB2 amplification and recurrent mutations in other cancers profiled by TCGA. Hetloss, Heterozygous loss.

(Adapted with permission from Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature . 2014;507[7492]:315–322.)

Among “precision” therapeutic strategies under investigation, small molecule pan-FGFR inhibitors demonstrated encouraging results in bladder cancer subsets harboring activating FGFR mutations or translocations (luminal/papillary-like subtypes). Among receptor tyrosine kinases, EGFR inhibitors may be effective in chemotherapy naïve bladder cancer with EGFR or ERBB2 overexpression. Likewise, encouraging preclinical results have been achieved with trastuzumab conjugated with a cytotoxic agent DM1 (derivative of maytansine 1; T-DM1) in HER2-positive tumors. Based on prior HER2-targeted trials in bladder cancer, evidence of tumor HER2 positivity by either immunohistochemistry or FISH could be used to guide therapy. In contrast to breast cancer, the majority of HER2 overexpression in bladder cancer are not associated with HER2 gene amplification. mTOR pathway inhibitors in combination MEK inhibitors and inhibitors of cell cycle regulators (aurora kinase, PLK1 , and cyclin-dependent kinase 4 ) are under investigation in combination with chemotherapy.

In 2014, the FDA granted MPDL3280A, a PDL1 monoclonal antibody inhibitor, a breakthrough therapy designation based on the results of a phase I trial of patients with metastatic urothelial bladder cancer, which showed up to a 43% response rate in patients with 2+/3+ PDL1 positivity by immunohistochemistry. Phase II and phase III studies (IMvigor 210 and IMvigor 211, respectively) evaluating anti-PDL1 agent atezolizumab in adjuvant therapy in patients with PD-L1-positive high-risk MI-BC (NCT02450331) are ongoing. The primary analysis of the phase II trial showed significant objective response that was highest in tumors with 2+/3+ PDL1 expression in immune cell (IC2/3: 27% [95% CI: 19 to 37], P < .0001; IC1/2/3: 18% [95% CI: 13 to 24], P = .0004).

Finally, molecular biomarkers to predict response to neoadjuvant chemotherapy (NAC) in the limited subset of cystectomy patients that achieve pathologic complete response (20% to 30%) are crucially needed. Molecular signatures revealed in the previously discussed genomic studies (basal/squamous-like, luminal/papillary-like) can help define the subsets of patients that will respond and achieve higher survival rate while sparing others (p53 wild-type signature) from therapy-related toxicity and delay of cystectomy. A clinical trial to compare the clinical efficacy of the two frontline chemotherapy regimens (gemcitabine plus cisplatin versus MVAC) and the ability of novel gene expression profiling-based algorithm (CoXEN [Co-eXpression ExtrapolatioN]) to predict complete pathologic response was launched by SWAG.

In summary, as our understanding of the complex molecular mechanisms involved in BC development has come into sharper focus, our approaches to diagnosis and management of bladder cancer continue to evolve. In the not so distant future, the current paradigm of the clinicopathologic-based prognostic approach to predicting progression in NMI-BC will be supplemented by a molecularly guided approach. Several new targeted therapy agents are under investigation in randomized trials in combination with standard chemotherapy agents, either as first-line treatment or on a maintenance basis, to prolong response in patients with advanced BC.

Renal Cell Carcinoma Antibody

RCC antibody binds to a 200-kD glycoprotein (gp200) shown to be expressed in epithelial cells lining normal renal proximal tubules and renal carcinoma cells. Several studies have established the utility of RCC in labeling clear cell and papillary variants of renal carcinoma. Avery and colleagues revealed membranous RCC reactivity in up to 85% of clear cell renal cell carcinoma (CCRCC). Almost all tested papillary renal cell carcinoma (PRCC) was also strongly positive for RCC. In contrast, chromophobe renal cell carcinoma (ChRCC) and oncocytoma were completely negative.

CD10/Acute Lymphocyte Leukemia Antigen

CD10, also known as acute lymphocyte leukemia antigen (CALLA), is expressed on the brush border of renal tubular epithelial cells. In the previously cited study by Avery and colleagues, CD10 demonstrated a similar profile to that of RCC antibody, with 94% of CCRCC and the majority of PRCC studied showing positivity for CD10. A similar profile was encountered by Bazille and associates. Variable CD10 staining has been reported in ChRCC, and negative staining to an almost 45% positive CD10 staining rate has been described. Approximately one-third of oncocytomas stain positively for CD10.

PAX2/PAX8

PAX2 and PAX8 are members of the paired box ( PAX ) gene family, which includes nine transcription factors ( PAX1 through PAX9 ) involved in the development of several organ systems by preventing terminal differentiation and maintaining a progenitor cell state while inducing cell-lineage commitment. Consequently, PAX gene expression is cell-lineage restricted; PAX8 is expressed in thyroid, and PAX8 and PAX2 are expressed in wolffian (nephric) ducts and müllerian ducts. PAX2 and PAX8 have also been detected in epithelial neoplasms arising in these areas, including renal cell and ovarian tumors.

IHC expression of PAX2 has been demonstrated in CCRCC, PRCC, and ChRCC subtypes, in addition to collecting duct carcinoma and mucinous tubular and spindle cell carcinoma (MTSC) renal tumors.

Gokden and colleagues showed that 85% of metastatic CCRCCs had nuclear immunoreactivity for PAX2. The marker, however, is not entirely specific, because one-third of CCRCC mimics—such as parathyroid carcinoma and ovarian clear cell carcinoma—also express PAX2 . Furthermore, PAX2 expression has been demonstrated in other genital tumors such as serous ovarian tumors, endometrioid carcinoma, and epididymal tumors. PAX2 protein is also a reliable marker for NA.

PAX8 is structurally and functionally related to PAX2 , and PAX8 is also expressed in normal and neoplastic tissues of renal tubular cell origin. IHC expression of PAX8 has been demonstrated in CCRCC, PRCC, and ChRCC subtypes and in collecting duct carcinoma, MTSC, and metastatic renal carcinomas ( Fig. 17.15A to D ). As with PAX2 , PAX8 expression has also been demonstrated in NA and clear cell adenocarcinoma of the lower urinary tract. We have found PAX8 to have higher sensitivity than PAX2 in identifying RCC.

PAX8 immunoexpression in primary clear cell renal cell carcinoma (CCRCC) (A), papillary RCC (B), chromophobe RCC (C), and metastatic CCRCC (D).

Epithelial Cell-Adhesion Molecule

Epithelial cell-adhesion molecule (EpCAM)—also known as KSA, KS1/4, and 17-1 antigen—is a 34- to 40-kD glycosylated transmembrane cell-surface epithelial protein of 232 amino acids. Recently, EpCAM has gained interest as a potential therapeutic target because of its wide-spectrum expression in many epithelial malignancies. EpCAM is consistently expressed in the distal nephron on normal renal epithelium; CCRCC shows minimal and infrequent EpCAM expression. Almost half of PRCC is positive for EpCAM, whereas intense and frequent expression is the rule in ChRCC and collecting duct carcinoma. A recent study by Liu and associates confirmed the utility of EpCAM in differentiating eosinophilic variant of ChRCC from oncocytoma and CCRCC. EpCAM protein was expressed diffusely (>90% of cells) in all 22 cases of ChRCC analyzed, whereas less than one-third of oncocytomas displayed positivity for EpCAM, and only in single cells or small cell clusters of distribution. Combining EpCAM with other markers such as vimentin, glutathione S-transferase alpha (GST-α), CD117, and CK7 can be of utility in resolving the differential diagnosis of ChRCC. However, EpCAM is not recommended for routine use because others have not reproduced these findings in clinical practice.

Kidney-Specific Cadherin

Cadherins are a large family of cell-to-cell adhesion molecules that act in a homotypic, homophilic manner and play an important role in the maintenance of tissue integrity. In the human kidney, several members of the cadherin family—epithelial (E) and neuronal (N) cadherin and cadherins 6, 8, and 11—are expressed in a controlled spatiotemporal pattern. Cadherin-16, also referred to as kidney-specific cadherin (Ksp-cadherin), is exclusively expressed in epithelial cells of the adult kidney. In RCCs, a complex pattern of cadherin expression is observed.

In CCRCC, Thedieck and colleagues revealed loss of Ksp-cadherin, which was subsequently proposed to differentiate ChRCC from oncocytoma. However, additional studies failed to reveal any difference in Ksp-cadherin immunoreactivity between these two tumor types, and one study showed Ksp-cadherin at the mRNA and protein levels in approximately 80% of ChRCC and oncocytoma.

Carbonic Anhydrase IX

Carbonic anhydrase IX (CAIX) is an enzyme involved in maintaining intracellular and extracellular pH. In addition, CAIX plays a regulatory role in cell proliferation, oncogenesis, and tumor progression. CAIX expression is von Hippel–Lindau (vHL)/HIF pathway dependent. In normal renal epithelium, expression of CAIX is suppressed by wild-type vHL protein. Given loss of vHL gene function in the majority of CCRCC tumors, CAIX antigen overexpression ensues. Most IHC studies have used clone M75 as the primary CAIX antibody to show diffuse overexpression in CCRCC. CAIX expression has also been demonstrated in almost half of PRCC in a recent study by Gupta and colleagues, whereas other studies, including ours, revealed only rare PRCC staining ( Fig. 17.16A to C ). Prognostically, low CAIX expression reportedly indicates poor survival and low response to interleukin therapy in CRCC. A new commercially available antibody, clone NB100-417, was recently shown by Al-Ahmadie and associates to have a comparable expression profile. Although CAIX is of some utility in establishing a CCRCC origin of a metastatic carcinoma, it is not entirely specific, because CAIX staining has been demonstrated in normal gastric mucosa and in biliary ductules.

(A) Carbonic anhydrase IX immunoexpression in clear cell renal cell carcinoma (CCRCC). Negative staining is encountered in papillary (B) and chromophobe RCC (C).

Glutathione S-Transferase Alpha

Glutathione S-transferase alpha (GST-α) protects cells by catalyzing the detoxification of xenobiotics and carcinogens. GST-α was recently found to be of diagnostic value in renal tumors. Based on cDNA microarray findings, IHC studies have so far shown GST-α to be highly expressed in CCRCC (90%), but not in ChRCC or oncocytomas, and only occasionally in PRCC.

Renal Oncocytoma

Oncocytomas are benign renal neoplasms typically characterized grossly by a mahogany-brown color. Microscopically, the tumor is composed of islands of “oncocytic” cells with coarsely granular eosinophilic cytoplasm set in a typical edematous, myxoid, focally hyalinized stroma. Oncocytomas display round, vesicular, centrally located nuclei with conspicuous nucleoli and occasional marked nuclear polymorphism. A range of architectural features is encountered that includes nested, tubular, acinar, cystic, and solid patterns. Variable rates of mitotic activity can also be encountered. Areas of necrosis are not seen, with the exception of commonly observed central degenerative ischemic change. The diagnosis of oncocytoma should be called into question if an “oncocytic” tumor reveals papillary architecture or areas of bona fide optically clear cytoplasm. An oncoblastic pattern is well recognized in some oncocytomas, in which the tumor cells become smaller in size with increased nuclear/cytoplasmic ratio; the latter does not carry any significant prognostic connotation. A cautionary note is warranted when making a diagnosis of oncocytoma on limited material, because other types of renal neoplasms—such as PRCC, ChRCC, and CCRCC—can display focal “oncocytic” areas that share the granular cytoplasmic appearance of oncocytoma.

Immunohistochemically, oncocytomas are positive for pancytokeratin AE1/AE3, and low-molecular-weight cytokeratin (LMWCK) CAM5.2 and are negative for vimentin. In more than two-thirds of oncocytomas, c-Kit (CD117) expression is encountered, a feature also shared by ChRCC. Similarly, Ksp-cadherin is expressed in the majority of both ChRCC and oncocytoma tumors. Unlike more diffuse CK7 staining seen in chromophobe RCC, oncocytomas are typically negative or patchy for CK7, which can help in their differentiation. Oncocytoma is usually negative for RCC and only occasionally expresses antibody CD10. Commonly encountered cytogenetic findings in oncocytomas include loss of chromosome Y and chromosome 1.

Metanephric Adenoma

Metanephric adenoma of the kidney is a unique form of renal adenoma characterized by a proliferation of tubular and micropapillary to glomeruloid structures lined by bland cuboidal epithelial cells. The relatively high nuclear/cytoplasmic ratio of metanephric adenoma cells and their slightly amphophilic cytoplasmic coloration impart a typical “blue” low-power appearance to the neoplastic nodule. The latter is in contrast to the lighter eosinophilic appearance of its main differential diagnosis, the solid variant of PRCC. Like PRCC, metanephric adenoma can feature foamy histiocytes in papillary cores and occasional psammomatous calcifications. Helpful IHC features of the tumor are its positivity for Wilms tumor 1 (WT1) and negative staining for epithelial membrane antigen (EMA) and CK7, a profile that contrasts with that of solid PRCC (WT1−, EMA+, CK7+; Fig. 17.17A to D ).

Metanephric adenoma (A and B) showing positive nuclear staining for Wilms tumor 1 (C), with lack of cytokeratin 7 expression (D).

Clear Cell Renal Cell Carcinoma

CCRCC is the most common type of RCC ² and accounts for 70% to 80%. Microscopically, CCRCC is composed of cuboidal cells with typical optically cleared cytoplasm arranged in nests, tubules, and acini. CCRCC are richly vascularized tumors with areas of hemorrhage that impart a “bleeding acini” characteristic morphology. “Granular” RCC with eosinophilic granular cytoplasm is no longer considered a unique variant and is now included in the CCRCC variant. As discussed later, at the genetic level, similar to their familial counterpart, almost two-thirds of sporadic CCRCC demonstrate partial or complete chromosome 3 loss or mutation on the short arm of chromosome 3p, resulting in the loss of the VHL tumor suppressor gene, located at 3p25-26. The Key Diagnostic Points box at the end of this section summarizes the IHC profile of CCRCC.

CCRCC is usually positive for LMWCKs, such as CAM5.2, and CKs AE1/AE3 but is negative for CK7 and CK20. It is positive for EMA and vimentin. CCRCC’s negative reactivity for HMWCKs such as cytokeratin CK5/6 and 34βE12 (keratin 903) and for p63 and GATA3 is a useful feature in the differential against upper urinary tract URCa, which is regularly HMWCK and p63 positive.

It must be remembered that the range of CCRCC reactivity with many of the previous markers varies among studies, making it necessary to exercise a judicial interpretation of any IHC panel when classifying a renal tumor. In the most recent study from the MSKCC group to address the utility of IHC in needle biopsies of renal masses (taken ex vivo for the study), the extent and pattern of immunoexpression were highly useful in the diagnoses: diffuse, membranous CAIX expression was noted in CCRCC, diffuse positivity for α-methylacyl–Coenzyme-A racemase (AMACR) in PRCC, distinct peripheral cytoplasmic accentuation for CD117 in ChRCC, widespread and intense positivity for CK7 in ChRCC and PRCC, diffuse membranous reactivity in CCRCC, and patchy/luminal staining in PRCC for CD10. In conclusion, utilizing immunostains improves classification of renal tumors on needle biopsy, which may be of particular help for pathologists with limited experience. Both extent and pattern must be considered for a definitive diagnosis, and IHC results should always be integrated with the overall morphologic features of a given renal neoplasm.

Papillary Renal Cell Carcinoma

PRCC is the second most common subtype of RCC. Microscopically, this subtype contains characteristic complex papillary formations often accompanied by foamy macrophages that infiltrate the fibrovascular cores. Two subtypes of PRCC are recognized: type 1, in which the papillae are lined by a single layer of cells with scant pale cytoplasm, and type 2, in which the papillae are lined by pseudostratified cuboidal to columnar epithelial cells with abundant eosinophilic cytoplasm and prominent eosinophilic nucleoli. Type 1 tumors are usually of lower ISUP nucleolar grade and are associated with a more favorable prognosis than type 2 tumors. A solid variant of PRCC is well recognized, whereas distinct papillary structures are not easily discernible. Glomeruloid structures and overall cytologic features, together with the presence of typical host-infiltrating histiocytes, can point to the diagnosis. As mentioned previously, the solid variant of PRCC suggests the differential diagnosis of metanephric adenoma. Commonly encountered cytogenetic alterations in RCC include trisomy of chromosomes 7, 17, 3q, 8, 16, and 20, and loss of Y chromosome. A subset of sporadic PRCC (12%) exhibits c-met oncogene mutations similar to their familial counterparts in hereditary PRCC syndrome (HPRCC). Immunohistochemically, the majority of PRCCs are positive for AE1/AE3, vimentin, RCC, and AMACR. Differential EMA immunostaining was found to be useful in differentiating type 1 and type 2 tumors; polarized expression is seen in type 1, but only rare expression is seen in type 2.

Chromophobe Renal Cell Carcinoma

ChRCC is composed of characteristic large polygonal cells with clear to lightly eosinophilic reticulated cytoplasm and distinct “plantlike” cell membranes. Typical perinuclear halos are a unique feature in this type of RCC. Another helpful diagnostic feature is their diffuse cytoplasmic staining with Hale iron stain. Cytogenetically, ChRCCs are hypodiploid tumors because of commonly present loss of chromosomes 1, 2, 6, 10, 13, 17, and 21, as shown by FISH and comparative genomic hybridization (CGH) analysis.

Ultrastructural and IHC features of ChRCC point to differentiation toward the intercalated cells of renal collecting ducts.

Collecting Duct Carcinoma

Collecting duct carcinoma (CDC) of the kidney is a rare but aggressive type of RCC with presumed origin from Bellini collecting ducts.

CDC is typically centered in the medulla of the kidney and extends into the cortex. Histologic patterns include tubulopapillary, tubular, solid, and sarcomatoid types. Prominent stromal desmoplasia, angiolymphatic invasion, and host inflammatory response are commonly found. Associated “dysplastic” changes in entrapped nonneoplastic collecting ducts and the presence of intracytoplasmic or luminal mucin secretions in neoplastic glands are also helpful diagnostic features of this type of RCC. Cytogenetic features include loss of chromosomes 8p and 13. Monosomy of chromosomes 1, 6, 14, 15, and 22 are also observed.

The CDC immunoprofile is that of positive reactivity with pancytokeratins AE1/AE3 and CAM5.2 and HMWCKs CK19 and 34βE12. CDCs are positive for EMA, vimentin, and the lectin Ulex europaeus agglutinin I (UEA-1). The diagnosis of CDC carries a poor prognosis, and a majority of patients die of metastatic disease within 2 years of presentation. We found the combination of GATA3 and PAX8 positivity and TP63 negativity in CDC to be helpful in distinguishing them from URCa of the renal pelvis ( Fig. 17.18A to D ).

Renal collecting duct carcinoma (A and B) showing positive nuclear staining for PAX8 (C) with lack of p63 expression (D).

Mucinous Tubular and Spindle Cell Carcinoma

MTSC is one of the latest types of RCC to be recognized as a distinct variant. Before characterization of MTSC, such tumors were most likely classified as either a sarcomatoid variant of RCC or “low-grade CDC.”

MTSC is composed of uniform cuboidal to spindle cells with eosinophilic, focally vacuolated cytoplasm and relatively bland ovoid nuclei. Tumor cells generally form interconnecting tubules with smaller areas of solid growth. The myxoid stroma is a distinguishing feature, and mucoid material deposits at times appear as secretions within tubular or intercellular spaces.

Cytogenetically, MTSC shows multiple chromosomal losses (1, −4, −6, −8, −9, −13, −14, −15, −22). Their overall immunoprofile and ultrastructural features have suggested differentiation toward the loop of Henle, distal convoluted tubule, or collecting ducts.

MTSCs are low-grade tumors in terms of their biologic behavior with occasional recurrence on record but no known distant metastases or deaths reported. Their relatively good prognosis highlights the importance of distinguishing these “spindle cell” variants of RCC from the aggressive, lethal, sarcomatoid phenotype.

MTSC is typically positive for vimentin, CK7, AMACR, and EMA but is negative for HMWCK, CD10, and RCC. The presence of some morphologic and IHC similarities between MTSC and the solid variant of PRCC have led some to suggest a histogenetic relationship between the two subtypes; however, their distinct cytogenetic and gene-expression profile argues against such a relationship.

Angiomyolipoma

Angiomyolipoma is a member of the group of tumors that contain perivascular epithelioid cells (PECs), referred to as PEComas . Oncogenesis in PEComas is related to the genetic alterations of the tuberous sclerosis complex (TSC). Tuberous sclerosis is an autosomal-dominant genetic disease that results from losses of the TSC1 (9q34) or TSC2 (16p13.3) genes involved in the regulation of the Rheb/MTOR/p70S6K pathway. PEComas of the kidney include “classic” angiomyolipoma (AML) and its recognized cystic, epithelioid, and oncocytoma-like variants.

Classic AML represents the most common mesenchymal tumors of the kidney, which are composed of variable proportions of adipose cells and spindle and epithelioid smooth muscle cells admixed with, and at times appearing to emanate from, abnormal thick-walled blood vessels ( Fig. 17.19A to C ). In patients with TSC, multiple bilateral renal AMLs are found during the third and fourth decades of life. Sporadic AMLs are larger solitary lesions that occur in older patients.

(A and B) Renal angiomyolipoma. (C) Focal positive staining for human melanoma black 45 (HMB-45) is shown.

The epithelioid variant of AML is composed of polygonal epithelioid cells arranged in sheets and usually lacks a fat tissue component. Epithelioid AML is reactive with human melanoma black 45 (HMB-45), melan-A, tyrosinase, microphthalmia transcription factor (MITF), and smooth muscle markers (α-SMA), but is negative for epithelial markers, including CKs and EMA. Tumor cells are clear to eosinophilic, with at times considerable nuclear atypia and associated necrosis. Epithelioid AML has been associated with recurrence and metastasis; however, it has not been possible to predict its malignant behavior based on morphologic criteria.

Oncocytoma-like angiomyolipomas have distinct granular eosinophilic cytoplasm, which brings renal oncocytoma into the differential diagnosis.

The differential diagnosis of AML also includes sarcomatoid RCC. This is especially the case with epithelioid AML with significant cytologic atypia. Expression of melanocytic markers and lack of epithelial markers in AML will help differentiate it from sarcomatoid RCC. Rarely, Xp11 translocation carcinoma can enter into the differential diagnosis of epithelioid AML. In this regard, expression of some melanocytic markers (HMB-45, melan-A) in rare Xp11 translocation carcinoma should be kept in mind. Positive transcription factor E3 (TFE3) reaction is unique to Xp11 translocation carcinomas.

Novel Renal Epithelial Tumors in WHO 2016 Classification

Several new renal tumor entities have been formally accepted by the WHO 2016 working group on renal tumors. These include hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome–associated RCC, succinate dehydrogenase (SDH)–deficient renal cell carcinoma, tubulocystic RCC, and acquired cystic RCC ( Box 17.1 ).

Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) Syndrome–Associated RCC

These are rare tumors occurring in the setting of extrarenal (uterine and skin) leiomyomatosis and are frequently but not exclusively of familial inheritance and characterized by the presence of a germline fumarate hydratase (FH) mutations. They typically display a papillary architecture with eosinophilic cells containing prominent “cherry-like” nucleoli with perinucleolar halo ( Fig. 17.20 ). Unlike other familial RCC, the prognosis is poor and patients present with advanced disease.

Hereditary leiomyomatosis and renal cell carcinoma showing papillary architecture with eosinophilic cells (A) containing prominent “cherry-like” nucleoli surrounded by a clear halo (B).

Succinate Dehydrogenase–Deficient Renal Cell Carcinoma

SDH-deficient RCC is a rare renal tumor that is usually of familial inheritance and associated with germline mutation in one of SDH subunit genes (most frequently SDH-B). Except for those rare tumors with sarcomatoid differentiation, marked atypia, and necrosis, SDH-deficient RCC pursue a favorable biologic behavior. Loss of expression of SDHB is a useful diagnostic marker. The tumors are composed of sheets and nests of relatively bland cells with distinctive cytoplasmic vacuoles with flocculent inclusions ( Fig. 17.21 ).

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