Does the addition of AMACR to CK20 help to diagnose challenging cases of urothelial carcinoma in situ?

Urothelial carcinoma in situ (CIS) is a high-risk subtype of non-invasive bladder cancer that has a substantial rate of invasion and death, with a 5-year risk of progression up to 45% [1‐3]. However, if appropriate intervention is made, risk of progression is significantly reduced with improved disease-free survival [3]. It is important to accurately identify this lesion in a timely manner so that appropriate treatment can be administered. The initial treatment is typically intravesical Bacillus Calmette-Guerin (BCG) therapy. For refractory urothelial CIS, radical cystectomy is the recommended approach [4‐6]. Thus, it is essential to distinguish between CIS and non-neoplastic tissue because underdiagnosis can lead to invasion, while overdiagnosis can lead to harmful and unnecessary treatment.

Distinguishing urothelial CIS from non-neoplastic urothelium using hematoxylin and eosin (H&E) staining alone can be difficult for multiple reasons including denudation of tissue, artifact from procedure (fragmentation, crush, and/or cautery), inadequate sample size, or therapy-related changes. Furthermore, some morphologic growth patterns of urothelial CIS can be difficult to diagnose, such as pagetoid spread or clinging carcinoma [3]. Utilization of immunohistochemistry (IHC) can be helpful in difficult-to-diagnose cases of CIS. Immunostains that have been shown to be of some benefit in diagnosis include CK20 [7‐14], CD44s [7, 13], Ki67 [7, 10‐12], and p53 [7, 8, 12‐14]. Markers such as CD44s, Ki67, and p53 are problematic due to overlapping expression profiles of CIS and reactive nonneoplastic urothelium.

AMACR has been found to be overexpressed in urothelial carcinoma, with amount of expression correlating with tumor grade [15‐18]. Like CK20, prior studies described increased expression of AMACR in urothelial CIS [19, 20]. Based on this these publications, our institution has utilized AMACR in addition to CK20 in challenging cases. However, there are currently no studies that evaluate the expression of AMACR in cases that were equivocal on H&E in which staining was performed during clinical practice. The aim of this investigation was to assess the expression of AMACR in difficult bladder cases in which testing was performed in clinical practice and compare its utility to that of CK20 for diagnostic urothelial CIS.

AMACR overexpression has been described in urothelial carcinoma in situ [19, 20]. CK20 also has increased expression in urothelial CIS [7‐14]. Thus, both AMACR and CK20 may be helpful in diagnosing challenging cases of CIS. To the best of our knowledge, there are no published studies that have examined the expression of AMACR in difficult cases as a part of clinical practice and compare these results to CK20.

In straightforward cases of urothelial CIS in which the diagnosis was unequivocal on H&E light microscopy alone, we found AMACR positivity, defined as expression in > 1/3 of the apical urothelial thickness, to have 100% sensitivity. Staining was granular, cytoplasmic, and variable in strength. The sensitivity in our study was comparable to that reported by Fellegara et al. (96%) [20]. This group defined AMACR positivity by diffuse, microgranular, cytoplasmic staining confined to the superficial 2/3rd’s of the urothelium. Aron et al. reported a lower sensitivity of AMACR for urothelial CIS, with AMACR positivity in 78% of untreated urothelial CIS and 50% of post-treatment cases [19]. Aron et al. defined positive as > 5% staining, which differed from our scoring criteria and that of Fellegara et al. We found that in cases that were equivocal on H&E and required IHC to assist in the diagnosis of urothelial CIS in clinical practice, AMACR had a sensitivity of 73% (n = 22), significantly less than that of CIS not requiring IHC for diagnosis. Additionally, AMACR can assist in recognition of CIS with pagetoid growth, in which neoplastic cells exist among non-neoplastic cells. This particular advantage of AMACR was also described previously by Fellegara et al. [20], who found AMACR to be useful in recognizing this urothelial CIS growth pattern.

The specificity of AMACR in our investigation for non-neoplastic urothelium (diagnosed as reactive urothelium) with IHC was 97%. Only 1 case of non-neoplastic, reactive urothelium had partial positive expression of regenerating cells in a patient status post multiple rounds of BCG therapy. This was similar to the specificity reported by Aron et al. (100%) and Fellegara et al. (93%) [19, 20]. However, one potential pitfall of AMACR is that it frequently is expressed in umbrella cells or surface urothelium. Almost half (45%) of non-neoplastic cases exhibited some surface expression confined to the upper 1/3rd urothelium or umbrella cells. Previous reports described similar staining of surface epithelium in non-neoplastic cases [19, 20].

CK20 had a similar pattern of expression as AMACR but was more sensitive (95% vs 73%) and less specific (80% vs 97%). The intensity of CK20 expression was consistently strong, allowing for easier interpretation compared to AMACR, which was highly variable in intensity. In addition, CK20 shares the same advantage as AMACR in that it easily demonstrated pagetoid growth, but also shared the potential pitfall of often showing surface expression in non-neoplastic urothelium. The only published study that directly compared the expression of CK20 with AMACR in urothelial CIS found CK20 to be less sensitive than AMACR (79% vs 96%) but more specific (100% vs 93%) [20]. However, there was no comparison of AMACR to CK20 in difficult cases in which IHC was performed in clinical practice prior to diagnosis, as was done in our study. Based on our results, CK20 was superior to AMACR for the indication of identifying CIS in challenging cases due to its higher sensitivity and stronger staining intensity.

In cases of classic urothelial CIS, AMACR sensitivity was 100%, and thus there was no difference in rate of positive staining based on BCG therapy history, and although cases with a history of BCG therapy did have a higher mean intensity, the difference was not significant. Fellegara et al. also did not observe a difference in AMACR expression based on treatment history [20]. However, this differed from the findings of Aron et al., in which pre-treatment samples had a sensitivity of 78%, while post-treatment sensitivity was 50% [19]. In our cohort, treatment was limited to BCG while in the prior two studies treatment was not specified for all cases.

AMACR can be used to confirm the diagnosis of urothelial CIS with high sensitivity and specificity, both in classic urothelial CIS and in challenging cases. However, it should be noted that the sensitivity of AMACR is decreased in CIS that is equivocal on H&E. Furthermore, despite similar patterns of staining, CK20 showed stronger and more consistent intensity of staining, making it easier to interpret than AMACR. Therefore, CK20 is recommended instead of AMACR for this indication.