ABCC2 brush‐border expression predicts outcome in papillary renal cell carcinoma: a multi‐institutional study of 254 cases

Papillary renal cell carcinoma (PRCC) histologic subtyping is no longer recommended in the 2022 WHO classification. Currently, WHO/ISUP nucleolar grade is the only accepted prognostic histologic parameter for PRCC. ABCC2, a renal drug transporter, has been shown to significantly predict outcomes in PRCC. In this study we evaluated the prognostic significance of ABCC2 IHC staining patterns in a large, multi‐institutional PRCC cohort and assessed the association of these patterns with ABCC2 mRNA expression.


Introduction
Renal cell carcinoma (RCC), which arises from renal tubular epithelial cells is the most common kidney tumour in adults and ~15% of RCCs, are papillary renal cell carcinoma (PRCC).PRCC is the second most common type of RCC, following clear-cell RCC (CCRCC). 1,2Compared to CCRCC, PRCC displays a worse prognosis in advanced or disseminated disease. 3RCC is, however, a heterogeneous disease characterized by a spectrum of clinical, morphologic, and molecular features. 4To address this, PRCC was historically categorized into indolent type 1 and aggressive type 2, exclusively based on histomorphologic features. 5Several issues rendered this subdivision of limited value, including: the occurrence of overlapping types 1 and 2 morphology in about half of the cases, leading to high interobserver reporting variability [6][7][8] ; the inclusion of other, more recently recognized tumour entities in the type 2 PRCC; for example, MiTfamily translocation RCC and fumarate hydratase (FH) deficient RCC 4 ; and the lack of consistent evidence of prognostic significance of type 1 versus type 2 division. 9,10Consequently, the recent 2022 World Health Organization (WHO) classification eliminated the traditional histologic subtyping of PRCC. 11urrently, WHO/ISUP (International Society of Urological Pathology) nucleolar grade is accepted as the only prognostic histopathologic marker for PRCC. 124][15] In addition, assigning grade on biopsy can be inaccurate in more than 1/3 of PRCCs, particularly in cases with mixed morphology. 16herefore, exploring other molecular markers that can potentially offer a better patient stratification is warranted.
ATP-binding cassette subfamily C member 2 (ABCC2) is a transmembrane protein that plays an important role in the transport of endogenous and exogenous substances. 17In the kidney, it is expressed in the apical membranes of the proximal tubular epithelial cells. 17It has been implicated in chemotherapeutic resistance in various tumours and also plays an active role in cancer progression. 18Overexpression of ABCC2 at the transcriptomic level was reported in aggressive PRCC. 7We have previously shown that ABCC2 expression by immunohistochemistry (IHC) is associated with significantly worse disease outcomes in PRCC. 7,15However, the ABCC2 IHC assay has variable cell patterns of reactivity that include weak cytoplasmic and brush-border staining.The significance of these different ABCC2 staining patterns has not been previously studied.
In the current study we evaluated the prognostic significance of the ABCC2 IHC reactivity patterns in a large multi-institutional PRCC cohort.Recently recognized entities with papillary architecture, including translocation-associated RCC, FH-deficient RCC, and clear-cell papillary renal cell tumour were excluded after histomorphologic and immunohistochemical evaluation for TFE3, pankeratin, epithelial membrane antigen, Cathepsin K, and FH.Cases diagnosed as papillary renal neoplasm with reverse polarity were also excluded.

Materials and methods
Clinicopathologic data and follow-up information were collected.All tumours were graded using the WHO/ISUP nucleolar grading system 12 and were staged according to the American Joint Committee on Cancer (AJCC) Staging Manual, 8th edition. 19isease-free survival (DFS) was used to assess prognosis.DFS was calculated as the time interval from the date of the surgery till the last recorded follow-up or the documented adverse event (local recurrence, metastasis, or cancer-related death).

A B C C 2 I M M U N O H I S T O C H E M I S T R Y
Tissue microarrays (TMA) were constructed from the representative FFPE blocks for 211 cases, using three cores per case.Whole FFPE tissue blocks were used for the remaining 43 samples.Immunohistochemistry (IHC) was performed using a validated, rabbit monoclonal antibody against MRP2 (ABCC2) (ABCAM, Boston, MA, USA, cat.no.ab187644, clone EPR10997(2), dilution 1:100) as previously described. 15The results were then stratified into: negative, cytoplasmic, brush-border <50%, and brush-border ≥50% (Figure 1).
Initially, the IHC slides were interpreted independently by three pathologists (R.S., M.M., and V.C.), without any knowledge of the relevant clinicopathologic information.Subsequently, all the cases were reviewed jointly by all three pathologists; in cases of discrepancy, a final consensus of ABCC2 status was assigned.

A B C C 2 R N A I N S I T U H Y B R I D I Z A T I O N
To evaluate ABCC2 mRNA expression, 65 FFPE tumour samples were randomly selected for RNA in situ hybridization (ISH) analysis, using the RNAscope 2.5 HD assay (ACDBio, Newark, CA, USA, cat.no.322350) according to the manufacturer's protocol. 20A probe (ACDBio, cat.no.1140921-C1) targeting the ABCC2 mRNA was used to detect ABCC2 expression.HALO image analysis platform by Indica Labs (Albuquerque, NM, USA) was utilized for quantitative analysis of the ABCC2 RNA-ISH signal.ABCC2 transcript levels were assessed using the following formula: [(strong red 9 1.5 + moderate red 9 1.2 + weak red)/ strong blue] 9 100.

S T A T I S T I C A L A N A L Y S I S
Differences in mRNA expression among the ABCC2 groups and between the combined ABCC2 groups (negative/cytoplasmic versus brush-border) were calculated using a one-way analysis of variance (ANOVA) test and independent t-test, respectively.Univariate analysis using the log-rank test and multivariate analysis using the Cox proportional hazard regression model were used to compare the DFS distribution among the different groups.Statistical significance was set at a P < 0.05.Statistical analysis was performed using GraphPad Prism (GraphPad Software, v, 9.0, San Diego, CA, USA) and SPSS (IBM, v. 29.0, Armonk, NY, USA).

C L I N I C O P A T H O L O G I C C H A R A C T E R I S T I C S
The clinicopathologic characteristics of the cohort are summarized in Table 1.The cohort had a median follow-up of 60 months (range, 1-242 months).A total of 22 patients (8.7%) had adverse events (two cases had local recurrence and 20 had metastases).Six patients (2.4%) died of disease.
Univariate and multivariate survival analyses are summarized in Figures 3 and 4, and in Table 2. DFS outcomes were stratified by the ABCC2 IHC patterns   (P < 0.001; Figure 3A).Specifically, the ABCC2 brush-border ≥50% (P < 0.001) and <50% (P = 0.024) groups were associated with significantly worse DFS compared to the group with negative ABCC2 staining (Figure 3A).Additionally, the ABCC2 brush-border ≥50% group had significantly worse DFS than the ABCC2 cytoplasmic group (P = 0.002).The ABCC2 cytoplasmic group, however, did not show a significant DFS difference compared to the negative group.Combined ABCC2 with any brushborder pattern versus the ABCC2 negative/cytoplasmic group also showed a significant DFS difference (P < 0.001) (Figure 3B).Similarly, combined WHO/ ISUP grades (high versus low grade) (P = 0.007) showed a significant association with the DFS in univariate analysis (Figure 3C).
In the pT1a subgroup (n = 120), the presence of any ABCC2 brush-border reactivity had significantly worse DFS (P = 0.014) compared to the PRCC group with cytoplasmic and negative ABCC2 staining (Figure 4A).ABCC2 brush-border reactivity also stratified the DFS in the pT1 (n = 185; P = 0.013; Figure 4B) and the ≤4 cm subgroups (n = 133; P = 0.041; Figure 4C).Furthermore, in the highstage PRCC (combined stages III and IV), the ABCC2 brush-border reactivity was associated with significantly worse DFS (P = 0.014; Figure 4D).In contrast, the WHO/ISUP grade did not show any significant DFS differences in these groups (Figure S2).
In multivariate survival analysis, after adjusting for age, tumour size, grade, and stage, the ABCC2 IHC reactivity patterns remained significant predictors of DFS (P = 0.022).In particular, both ABCC2 brush-border ≥50% reactivity (P = 0.003) and brush-border <50% reactivity (P = 0.037) were associated with significantly worse DFS outcomes.Conversely, the WHO/ISUP grade failed to demonstrate significant difference in DFS in multivariate analysis.Similar to the ABCC2 brush-border pattern, high stage was associated with worse clinical outcome (P < 0.001), when other variables were accounted for (Table 2).

Discussion
In the era of precision medicine, identification of biomarkers that have a predictive value for targeted therapies may improve clinical outcomes. 22][23][24] ABC proteins also play an active role, beyond multidrug resistance, in tumour development and aggressiveness. 18Our previous study using in vitro and in vivo experiments on PRCC2 cell lines showed that MK571, an ABCC2 blocker, on its own showed a significant response to therapy, suggesting that ABCC2 may contribute to tumour growth. 25hus, targeting ABCC2 could potentially become an important treatment strategy for PRCC stratified by the ABCC2 expression.In silico analysis of The Cancer Genome Atlas data 26 showed that aggressive PRCCs have high expression of ABCC2 at the transcriptomic level.For example, ABCC2 expression was 79 times greater in the traditional PRCC type 2 category, compared to the PRCC type 1 cohort. 27ABCC2 is considered part of the NRF2-ARE response pathway, which is known to be implicated in PRCC type 2. 26 In this study we emphasize further the value of ABCC2 as a specific prognostic biomarker in PRCC in a large multi-institutional cohort.This study builds on our previous research with the use of a new ABCC2 antibody [clone EPR10997 (2)] that has a more specific brush-border reactivity in the proximal renal tubules. 15The presence of such pattern correlated with the ABCC2 protein product, because this protein is normally localized in the apical surface of the proximal tubular epithelial cells. 17We observed that tumours with brush-border staining had either focal or diffuse reactivity, and set a cutoff of 50% (<50% and ≥50%) reactivity that was deemed reproducible and easily applicable.Brush-border expression showed significant predictive capacity regarding disease progression, i.e.DFS outcome in both univariate and multivariate analyses.
To validate this finding, we utilized RNA-ISH to determine the mRNA expression of the selected PRCC samples.Based on the RNA-ISH assay, any ABCC2 brush-border expression was characterized by gradually higher transcript levels that correlated with the IHC reactivity, compared to the ABCC2 cytoplasmic and negative staining.This confirms the significance of the brush-border staining identified on IHC, which is associated with ABCC2 mRNA overexpression in PRCC.However, we found no significant gene expression difference between the ABCC2 negative and cytoplasmic groups.The presence of weak, granular cytoplasmic staining in PRCC cells may represent a low level of ABCC2 protein expression in the cytoplasm, likely distributed on the membranes of cell organelles. 28Such cytoplasmic expression of ABCC2, that is often weak and may potentially result in interobserver variability, was not associated with worse DFS in the evaluated PRCC cohort.Therefore, we considered the cytoplasmic ABCC2 pattern together with the group showing an absence of any ABCC2 reactivity by IHC, because of the lack of significant biological and clinical differences between these groups.
Assessment of the ABCC2 IHC patterns in PRCC tumours provided relevant prognostic information, as shown in both univariate and multivariate analyses.In univariate analysis, the PRCC group with either focal/patchy (<50%) or prominent (≥50%) ABCC2 brush-border reactivity was associated with worse DFS, compared to the PRCC group with negative and cytoplasmic reactivity.We also demonstrated the ABCC2 prognostic significance in our previous study, although we did not previously evaluate the specific pattern reactivity. 15Importantly, in the current study the ABCC2 brush-border pattern maintained its prognostic value in multivariate analysis, after adjusting for age, tumour size, grade, and stage.Thus, ABCC2 represents an independent prognostic biomarker, which is useful and easily reproducible in PRCC, as shown in the current study and in our previous study, in which we used smaller and different cohorts. 150][31] Although the prognostic significance of WHO/ISUP grade was demonstrated in univariate analysis, we found, however, that WHO/ISUP grade was not significant in predicting survival when controlling for other variables, as already reported. 13,14,31In the current practice, WHO/ISUP grade still remains a relevant histologic parameter in PRCC and we propose that ABCC2 can be further validated by using it in conjunction with the WHO/ISUP grade in independent PRCC cohorts for more accurate prognostication.
In this study the TNM (AJCC) stage was found to be a prognostic risk factor in both univariate and multivariate analyses, which is consistent with previous studies in PRCC. 10,13,30Other variables, such as age and tumour size had no independent prognostic value in multivariate analysis for the DFS in PRCC, as already reported. 10,14,30e also demonstrated the prognostic value of ABCC2 in pT1a and tumours ≤4 cm, also referred to as "small renal masses", subsets with low malignant and metastatic potential that may benefit from active surveillance. 32In this study the tumours in these subgroups that had ABCC2 brush-border reactivity also had a significantly worse DFS.This is consistent with our previously published data regarding the predictive value of ABCC2 in pT1a tumours. 15In contrast, dividing these subgroups using the WHO/ISUP grade had no significance on the survival outcomes.Therefore, ABCC2 can also be used for prognostic separation of the small renal masses to guide clinicians in opting for active surveillance in a selected patient population.In fact, ABCC2 was assessed on TMA cores (each 1.2 mm in diameter, average 3 cores per case) in most of our PRCC cohort, which substantiates the utility of assessing ABCC2 in limited core biopsy samples.
Tumour heterogeneity in the brush-border % staining was sometimes observed in both whole slides and TMA cores (Figure S3).The average score of the three TMA core samples was used for the final brushborder status; similarly, the average score of the whole-slide staining was considered for the corresponding cases.Of note, the variation of ABCC2 staining was mostly seen in cytoplasmic and brush-border <50% groups.Given the challenge of addressing tumour heterogeneity, we still have shown significant prognostic results.Having said this, further studies should be performed to properly address the tumour heterogeneity of ABCC2, which is beyond the scope of this study.
In conclusion, ABCC2 brush-border expression evaluated by IHC in PRCC correlates with higher gene expression.We showed that ABCC2 IHC is a reproducible biomarker test in predicting survival outcomes in a multi-institutional PRCC cohort and provides a prognostic stratification beyond the WHO/ ISUP grade.Importantly, ABCC2 IHC can be an effective assay in small PRCC tumours that may guide active surveillance decisions in this PRCC group.

C
A S E S E L E C T I O N The Institutions' Research Ethics boards approved this work.A total of 254 formalin-fixed paraffin-embedded (FFPE) specimens from surgically resected PRCC cases from 2000 to 2021 was included in this study.Participating institutions included: Sunnybrook Health Sciences Centre (n = 53), Rockyview General Hospital/ University of Calgary (n = 72), McGill University Health Centre (n = 25), Unity Health Toronto/ St. Michael's Hospital (n = 25), Unity Health Toronto/ St. Joseph's Hospital (n = 36), and Ontario Tumour Bank (n = 43).The criteria for the diagnosis of PRCC were based on the WHO Classification of Tumours of the Urinary System and Male Genital Organs, 5th edition.

Figure 1 .
Figure 1.Representative PRCC haematoxylin and eosin (H&E) and the corresponding IHC patterns: (A,B) PRCC with negative ABCC2 staining (inset; proximal renal tubules used as positive internal control and the glomeruli as negative internal control).(C,D) PRCC with cytoplasmic staining.Cytoplasmic pattern was defined as the presence of weak granular cytoplasmic staining.(E,F) PRCC with brush-border <50% staining and (G,H) PRCC with brush-border ≥50% staining.Brush-border pattern was characterized by distinct staining of the apical cell membranes with or without cytoplasmic staining.

Figure 3 .
Figure 3. DFS outcomes of different PRCC groups by ABCC2 IHC patterns in comparison to the WHO/ISUP grade.(A) Kaplan-Meier curves were stratified by ABCC2 IHC staining patterns in univariate analysis.(B) When divided into combined ABCC2 IHC groups (any brushborder reactivity versus negative/cytoplasmic reactivity), the ABCC2 group with any brush-border reactivity was associated with decreased survival.(C) The combined high WHO/ISUP grade (grade 3 + 4) also showed significantly worse DFS than the combined low WHO/ISUP grade (grade 1 + 2).[Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4 .
Figure 4. ABCC2 brush-border expression was associated with significantly worse DFS outcome in different pTNM stages and tumour size groups: In pT1a (≤4 cm tumours limited to the kidney) (A), pT1 (≤7 cm tumours limited to the kidney) (B), ≤4 cm (C), and high-stage (D) subgroups, ABCC2 brush-border staining had significantly worse patient DFS outcomes in univariate analysis.[Colour figure can be viewed at wileyonlinelibrary.com]

Table 1 .
Patient demographics and tumour characteristics

Table 2 .
Univariate and multivariate survival analysis