Combined Immunohistochemistry for the “Three 7” Markers (CK7, CD117, and Claudin-7) Is Useful in the Diagnosis of Chromophobe Renal Cell Carcinoma and for the Exclusion of Mimics: Diagnostic Experience from a Single Institution

Background There is a morphological overlap among renal epithelial tumors, particularly chromophobe renal cell carcinoma (CHRCC), clear cell renal cell carcinoma (CCRCC), renal oncocytoma (RO), and papillary renal cell carcinoma (PRCC). Discriminating between these tumors is important but sometimes challenging. This study is aimed at evaluating the clinical usefulness of the combined immunochemistry for the “three 7” markers (CK7, CD117, and Claudin-7) to distinguish chromophobe renal cell carcinoma from these mimics. Methods Immunochemical staining for CK7, CD117, and Claudin-7 was performed in 68 CHRCCs, 199 CCRCCs, 32 ROs, and 30 PRCCs. Fluorescence in situ hybridization (FISH) was performed in some cases to exclude CCRCC and PRCC. The sensitivity (SE) and specificity (SP) for CHRCC as well as the immunoreactivity of each marker and their combinations were statistically evaluated. Results High positive rates for CK7 (94%), CD117 (87%), Claudin-7 (94%), and their combinations (CK7+CD117, 79%; CK7+Claudin-7, 88%; CD117+Claudin-7, 82%; CK7+CD117+Claudin-7, 76%) were observed in CHRCC compared to those in CCRCC, RO, and PRCC, with increasingly higher SP when combinations of the “three 7” markers were applied (CK7, 0.80; CD117, 0.82; Claudin-7, 0.78; CK7+CD117, 0.95; CK7+Claudin-7, 0.97; CD117+Claudin-7, 0.97; CK7+CD117+Claudin-7, 1). Conclusion CK7, CD117, and Claudin-7 are frequently expressed in CHRCC with high specificity. We recommend the routine use of these 3 markers as a routine panel when making a differential diagnosis of CHRCC and excluding other mimics.


Background
Chromophobe renal cell carcinoma (CHRCC) is the third most common renal cell carcinoma (RCC, 5%) and is inferior to clear cell renal cell carcinoma (CCRCC, 70-80%) and papillary renal cell carcinoma (PRCC, 15%) [1]. CHRCC is considered to have low malignant biologic behavior with a 5-year survival rate of 78-100% [2]. The somatic genomic landscape of CHRCC reveals its distal nephron origin [3]. Histologically, CHRCC is typically arranged in a sold-sheet pattern separated by a thin, incomplete, and hyalinized vascular septa [4]. Other configurations, such as nested, tubular, trabecular, cystic, alveolar, and focal papillary areas, have also been appreciated [4]. Two distinct subtypes of CHRCC have been described, that is, a typical variant and an eosinophilic variant; the classical type features a predominance of large polygonal cells with a pale and distinct cell membrane, and the eosinophilic variant demonstrates smaller cells with fine oxyphilic granularity [2,5].
For a subset of difficult cases showing overlapping morphological and immunohistochemical features, FISH was additionally applied (CCRCC, loss of chromosome 3p; PRCC, trisomy of 7 or/and 17 or loss of the Y chromosome). The procedure has been previously described [18,19]. The probes included CEP7, CEP17, SEY (Vysis, Downers Grove, IL, USA), and CSP3+GSP 3p (LBP, Guangzhou, Guangdong, China). The signals from 100 nonoverlapping intact nuclei were counted for each lesion. Chromosome loss (or gain) was defined as the percentage of nuclei with single (or 3) signals greater than the normal tissue means for that chromosome, within 4 times the normal tissue mean for that chromosome, and within 4 times the normal standard deviation for that chromosome, as described previously [19]. Thus, for CEP7 or CEP17, the percentages of 3 or more signals of more than 10% were considered as trisomy; for CEP Y and 3p, the percentages of single signals of more than 70% and 40%, respectively, were considered as chromosome loss in this study.
2.3. Data Analysis. The sensitivity (SE) and specificity (SP) of CHRCC compared to those of the other 3 types of renal cell tumors (CCRCC, PRCC, and RO) were calculated using standard formulas. The differences in the immunoreactivity of each marker and their combinations were evaluated using the chi-squared test between CHRCC and CCRCC, PRCC (type 1 and type 2), or RO.
The SE and SP of the "three 7" markers in CHRCC and comparisons of CHRCC with CCRCC, PRCC, and RO are summarized in Table 2. The SE and SP of CK7 in CHRCC were 0.91 and 0.80, respectively. Claudin-7 showed higher SE and slightly lower SP (SP = 0:78) than those of CK7. The SP of CD117 was 0.82, while the SE (SE = 0:94) was relatively higher than that of CK7. The SP for the combination of the 3 markers was significantly enhanced, but the SE was consequently decreased. The results are shown in descending order of SP as follows: CK7+CD117+Claudin CD117 or Claudin-7 between CHRCC and type 1 PRCC and CD117 between CHRCC and RO.

Discussion
There are overlapping morphological features among CHRCC, PRCC, CCRCC, and RO, often leading to diagnostic challenges when encountering difficult cases. Immunohistochemical staining, compared to electron microscopy or Hale colloidal iron staining, is an easier way to facilitate the discrimination of these carcinomas [20]. Although a collection of immunostaining markers and/or morphologic features have been described, none of these molecules is pathognomonic. Similarly, various markers and their combinations have been applied to differentiate CHRCC and other mimics, but none of these proteins is absolutely specific to the diagnosis of CHRCC [6,8,11,13,21]. Notably, 60-100% of CHRCCs are positive for CK7, with a typically diffuse (90-100% of tumor cells) and strong staining pattern [21,22]. Consistently, our study showed high SE for CK7 (94%) in CHRCC but relatively lower SP (0.87) than that in CCRCC, PRCC, and RO. The proportion of negative cases may lead to diagnostic dilemmas if one relies only on CK7 and histomorphology. Only limited CCRCCs and ROs are positive for CK7, and even if positive, these tumors usually demonstrate a focal pattern with mild-to-moderate intensity. In contrast to type 2 PRCC, CK7 is frequently labeled in type 1 carcinomas, suggesting that this marker is useless for differential diagnosis (p = 0:5497). Some negative cases can show marked focal positivity for CK7 in either CCRCC or RO [23]. The scattered expression of CK7 can be observed in some cells of high-grade tumors or the lining of cystic walls in CCRCC [24]. According to our experience, CK7 may also be positive in the clear cells of CCRCCs with a superimposed tall/columnar or eosinophilic appearance, but these areas are frequently very limited (usually <10%). CD117 labels both the majority of CHRCCs and ROs, commonly with strong and diffuse staining patterns [25]. The SP of CK7 in CHRCC should be higher than that in RO, which is not consistent with the results in our study (CK7 vs. CD117, 0.87 vs. 0.88) because the proportion of RO cases was significantly less than that of other CK7-negative renal cell carcinomas (particularly, CCRCC). Therefore, CK7 is useless for differentiation from ROs intimately mimicking CHRCCs only by the expression of CD117, but this molecule can facilitate the separation of CCRCC (0.5%) from PRCC (16%). The latter two demonstrate focal and mild-tomoderate positivity, even if these carcinomas express CD117. Previous gene expression microarray analysis revealed that Claudin-7 was overexpressed in CHRCC versus oncocytoma and other tumor subtypes [26]. Analogous to the expression of CK7, Claudin-7 usually shows diffuse positivity in the majority of CHRCCs (80%-100%) [27][28][29][30]. Although a subset of CCRCCs (0-26.1%), PRCCs (28-90%), and OCs (26%-81.8%) can be reactive for Claudin-7 [26,[28][29][30][31], the positive results of Claudin-7 expression were variable, with a wide range among different research groups. The main reason for this discrepancy is likely the different criteria for the interpretation of Claudin-7 positivity. In our case series, only Claudin-7 reactivity greater than 10% was considered positive. Claudin-7 usually shows mild-to-moderate cytoplasmic reactivity and mild and discontinuous membranous reactivity in some renal cell carcinomas, all of which should not be counted as positive staining in diagnostic practice. Although a subset of CCRCCs, PRCCs, and ROs express Claudin-7, most of these carcinomas demonstrate   cytoplasmic positivity is basically useless for the diagnosis of CHRCC if considered positive staining. In addition, positive Claudin-7 staining is not uncommon in type 1 PRCC, and therefore, there is limited discrimination between CHRCCs and type 1 PRCCs.
The recently described HOCTs have been observed in distinct clinical settings, including renal oncocytosis, Hogg-Dubé syndrome, or sporadic RCC [32]. HOCTs are characteristic of a mixture of RO-like and CHRCC-like tumor cells, either as distinctly separate tumor groups adjacent to one another, intimately admixed with one another, or showing a gradual transition from one typical region to another [33]. Although HOCT, as a variant, is under the umbrella of CHRCC, increasing evidence shows different genomic features supporting its distinct nature from CHRCC [34]. Nevertheless, the distinct components of HOCT still adhere to the expression pattern of the "three 7" markers observed in their corresponding renal tumors. These different expression modes essentially contribute to the diagnosis of HOCTs.
Although there were papers applying CK7, CD117, or Claudin-7 with other markers to make differential diagnoses, the combination of these 3 markers has never been reported.
In diagnostic practice, differential diagnoses of CHRCC, PRCC, CCRCC, and RO are routinely performed before the given diagnosis of any of these 4 tumors is made. In this scenario, the specificity of immunostaining markers is a priority but the sensitivity is secondary. The diagnostic SP for CHRCC of the combination of any two of the markers CK7, CD117, and Claudin-7 was significantly enhanced compared to that of a single immunostaining marker. The application of these "three 7" markers together reached 100% SP in our study. In the past 10 years of practice, we have routinely used these 3 markers when differentiating CHRCC from other renal cell carcinomas. When histologic overlapping exists between CHRCC and other renal cell carcinomas, more than 2 positive markers are more likely to support the diagnosis of CHRCC (SP, 0.95-1). If only one or none of the 3 markers was positive, then the diagnosis of CHRCC is suggestive but uncertain, and therefore, further investigations should be carried out. We usually perform special staining colloidal iron and FISH tests (CEP7, CEP17, SEY, and CSP3+GSP 3p) to exclude the other mimics. However, morphology is a priority, when there is a paradox between the typical histological features of a given carcinoma and the immunohistochemical staining. Cases that do not benefit from "three 7" immunostaining (only 1 or no marker positive) without superimposed classical morphology and distinct molecular markers present truly difficult situations, and most of these cases are designated as unclassified renal cell carcinomas. However, additional markers need to be investigated in the future.

Conclusions
In conclusion, our study and experience demonstrated that CK7, CD117, and Claudin-7 are frequently expressed in CHRCC with high specificity. Knowing the expression features and patterns facilitates the interpretation of positive staining in the renal cell carcinomas. Application of the "three 7" markers, if necessary, with some special staining and/or molecular tests can resolve a majority of the diagnostic issues for CHRCC. We recommend that these 3 markers are used as a routine panel in the differential diagnosis of CHRCCs from other mimics.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.