Prognostic evaluation of clear cell renal cell carcinoma

Transcription

1 ORIGINAL ARTICLE Architectural Patterns are a Relevant Morphologic Grading System for Clear Cell Renal Cell Carcinoma Prognosis Assessment Comparisons With WHO/ISUP Grade and Integrated Staging Systems Jérôme Verine, MD, PhD,* Delphine Colin, MD, Mary Nheb, MD,* Dominique Prapotnich, MD, Guillaume Ploussard, MD, PhD,# Xavier Cathelineau, MD, François Desgrandchamps, MD, PhD, # Pierre Mongiat-Artus, MD, PhD, # and Jean-Paul Feugeas, MD, PhD** Abstract: We developed and validated an architecture-based grading for clear cell renal cell carcinoma (ccrcc) in an observational retrospective cohort study including 506 tumors (principal cohort, n = 254; validation cohort, n = 252). Study endpoints were disease-free survival (DFS) and cancer-specific survival (CSS). Relationships with outcome were analyzed using Harrell concordance index, time-dependent receiver operating characteristic curve, area under curve, and Cox regression model. An architecture-based grading was devised on positive likelihood ratio (LR+) for DFS at 50 months as follows: grade 1 (LR+ < 0.8), cystic, compact, acinar, clear cell papillary RCClike, and/or regressive patterns; grade 2 (1.2 LR+ < 5), large nest, alveolar, papillary, chromophobe/oncocytic cell-like, eosinophilic hyaline globule, and/or intratumoral inflammatory reaction patterns; grade 3 (5 LR+ < 10), rhabdoid, tumor giant cell, enlarged vascular space, and/or hereditary leiomyomatosis renal cell carcinoma (HLRCC)-like patterns; grade 4 (LR+ 10), sarcomatoid, infiltrative growth patterns, and lymphatic invasion. In the principal cohort, 3-tier (grades 1-2, 3, and 4) and 4-tier architectural scores outperformed World Health Organization/International Society of Urological Pathology, and World Health Organization/ International Society of Urological Pathology+necrosis gradings for DFS and CSS, and constituted an independent predictor for DFS (hazard ratio [HR] = 5.91; P < 6.7E-10) and CSS (HR = 4.49; P = 2.2E-03), retained in the localized (pt1-3n0m0) ccrcc subgroup (HR = 6.10; From the *Department of Pathology, Saint-Louis Hospital, AP-HP; CEA, Institute of Emerging Diseases and Innovative Therapies (imeti), Research Division in Hematology and Immunology (SRHI); University Paris Diderot, Sorbone Paris Cité, UMR E_5, Saint-Louis Hospital; University Paris Diderot, INSERM, UMR_S1165; Department of Pathology; Department of Urology, Institut Mutualiste Montsouris; #Department of Urology, Saint-Louis Hospital, AP-HP; **University Paris Diderot, INSERM, IAME, UMR_1137, Paris; and University of Franche Comté, INSERM, U1098, Besançon, France. Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Correspondence: Jérôme Verine, MD, PhD, Laboratoire de Pathologie, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, Paris 75010, France ( jerome.verine@aphp.fr). Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. P = 1.3E-07 for DFS, and HR = 20.09; P = 9.4E-05 for CSS). On comparing with integrated staging systems, architectural grade with 1 morphologic datum remained an independent predictor of CSS, as did University of California Los Angeles Integrated Staging System and SSIGN, and was associated with the highest HR (HR = 2.60; P = 9.1E-04 in all patients; HR = 4.38; P = 2.0E-05 in the localized ccrcc subgroup). Architecture-based score for ccrcc outperforms all other morphologic grading systems and constitutes an independent predictor for DFS and CSS. As the predictive values of 3-tier and 4-tier architecture-based scores were similar throughout the study, we proposed to keep the simplified version as the final score, and to define 3 risk groups as follows: low risk (grades 1 to 2), intermediate risk (grade 3), and high risk (grade 4). Key Words: clear cell renal cell carcinoma, pathology, grade, tumor architecture, prognosis (Am J Surg Pathol 2018;00: ) Prognostic evaluation of clear cell renal cell carcinoma (ccrcc) is mainly based on tumor stage and grade, and is used to guide disease treatment and monitoring. CcRCC has long been known to be a very heterogenous morphologic tumor with distinct architectural patterns that are not fully reflected by the usual grading systems. Moreover, our perception of ccrcc progression has dramatically changed with the demonstration of ccrcc intratumor genomic heterogeneity, 1 which is clearly linked to morphologic heterogeneity. Nevertheless, among the numerous RCC grading systems described in the literature, few were solely based on tumor architecture. 2 Until recently, the nuclear grading system described by Fuhrman et al 3 has been endorsed for routine clinical use despite its methodological issues related to its application, reproducibility, and validity. Recently, a new grading system was described and adopted by both the 2012 International Society of Urological Pathology (ISUP) Vancouver consensus conference, 4 andtheworldhealthorganization(who). 5 WHO/ISUP grading system integrates architectural patterns but only into Am J Surg Pathol Volume 00, Number 00,

2 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 grade 4, that is, sarcomatoid and/or rhabdoid features, as does Fuhrman grade. It also considered as grade 4 a recently identified morphologic pattern, that is, syncytial-type multinucleated giant tumor cell. 6 Nevertheless, several recent studies also defined new morphologic patterns of ccrcc associated with a favorable outcome, 7,8 or outlined borderline features between ccrcc and an indolent form of RCC, that is, clear cell papillary RCC. 9 All these elements demonstrate a renewed interest in considering tumor architecture as a potential morphologic predictor of ccrcc outcome. Therefore, we developed a new morphologic grading system, founded on tumor architectural patterns, and tested it in 2 large retrospective series of surgically treated ccrcc patients. MATERIALS AND METHODS Study Design This observational retrospective population-based study included consecutive ccrcc patients who underwent a partial or radical nephrectomy without neoadjuvant therapy at Hôpital Saint-Louis (Paris, France) between January 1, 1998, and December 31, Only patients with available follow-up information superior to 18 months or an early metastatic evolution (< 12 mo) were selected for detailed study. Confirmation of ccrcc diagnosis was performed for each case by a single experienced uropathologist (J.V.). A minimum of 1 block/cm of tumor (from gross report) was defined as inclusion requirement in the principal cohort. The study was approved by the institutional review boards of Hôpital Saint-Louis and Institut Mutualiste Montsouris. Data Collection Clinical data were collected from the electronic patient records, including age, sex, symptoms at presentation according to Patard classification, 10 Eastern Cooperative Oncology Group performance status, node status, and distant metastases. The following morphologic data were also recorded: WHO/ISUP grade, modified WHO/ISUP grade incorporating the presence or the absence of tumor necrosis into the tumor grade as previously described, 11 microscopic tumor necrosis, tumor size (from gross report), 2017 primary tumor TNM classification, SSIGN (Stage, SIze, Grade, and Necrosis), 12 UISS (University of California Los Angeles Integrated Staging System), 13 and Leibovich prognosis 14 scores. For each patient, follow-up data were based on a complete review of the medical charts. Outcomes The primary end point was disease-free survival (DFS),calculatedfromthedateofsurgerytothedateof first documentation of recurrence, or death for any cause. The secondary end point was cancer-specific survival (CSS), calculated from the date of surgery to the date of death from ccrcc. Survival of patients who were lost to follow-up was censored as of the date of last contact. Outcome data were retrieved from the database on April 30, TABLE 1. Descriptive Characteristics of 254 Patients With ccrcc Included in the Principal Cohort Clinicopathologic Features n (%) Age (median [range]) (y) 61 (30-90) Sex Male 176 (69.3) Female 78 (30.7) Mode of presentation S1 (incidental) 184 (73.9) S2 (local symptoms) 37 (14.9) S3 (systemic symptoms) 28 (11.2) ECOG performance status (93.2) 1 17 (6.8) Pathologic tumor size (median [range]) (mm) 50 (10-180) 4-tier WHO/ISUP grading 1 14 (5.5) 2 78 (30.7) 3 88 (34.7) 4 74 (29.1) 3-tier WHO/ISUP grading 1/2 92 (36.2) 3 88 (34.7) 4 74 (29.1) Coagulative tumor necrosis Absent 173 (68.1) Present 81 (31.9) WHO/ISUP grade+necrosis Groups 1/2 91 (35.8) Groups 3/4 73 (28.8) Groups 5/6 26 (10.2) Groups 7/8/9 64 (25.2) 4-tier architectural grading system 1 93 (36.6) 2 67 (26.4) 3 49 (19.3) 4 45 (17.7) 3-tier architectural grading system 1/2 160 (63.0) 3 49 (19.3) 4 45 (17.7) 2010 primary tumor classification pt1 138 (54.3) pt2 29 (11.4) pt3+pt4 87 (34.3) 2010 regional lymph node involvement pnx+pn0 242 (95.3) pn1 12 (4.7) 2010 distant metastases M0 222 (87.4) M1 32 (12.6) Follow-up period (median [range]) (mo) 48 (1-216) ECOG indicates Eastern Cooperative Oncology Group. Scoring System We approached the study in 3 stages. The purpose of the first stage was to identify the largest number of distinct architectural patterns of ccrcc present in the principal cohort, independently of the tumoral surface area occupied by each pattern. Microscopic analysis was performed using a BX51 microscope (Olympus France S.A.S, Rungis). The distinct architectural patterns were determined on routine hematoxylin and eosin stained tumor sections without the aid of immunohistochemistry by a pathologist (J.V.) blinded to clinical data. In the second stage, we 2 Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

3 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc TABLE 2. Distinct Architectural Patterns Noted in the Principal Cohort (n = 254) Architectural Patterns N (%) Compact or small nests 175 (68.9) Regressive 164 (64.6) Cystic or tubulocystic with or without hemorrhagic 130 (51.2) contents, thyroid-like and/or Paneth cell-like features Large nests 128 (50.4) Rhabdoid dedifferentiation 67 (26.4) Syncytial multinucleated tumor giant cell 40 (15.7) Intratumoral inflammatory reaction 40 (15.7) Acinar 40 (15.7) HLRCC-like 37 (14.6) Enlarged vascular spaces 34 (13.4) Chromophobe/oncocytic cell-like 31 (12.2) Infiltrative growth pattern with entrapped glomeruli 29 (11.4) Eosinophilic hyaline globules 27 (10.6) Papillary or micropapillary 26 (10.2) Sarcomatoid dedifferentiation 25 (9.8) Foamy 20 (7.9) Alveolar 15 (5.9) Clear cell papillary RCC-like 14 (5.5) Lymphatic invasion 11 (4.3) HLRCC indicates hereditary leiomyomatosis renal cell carcinoma. devised a 4-tiered grading system on the basis of the ability of architectural patterns to predict distant metastatic evolution assessed by the positive likelihood ratio (LR+) at 50 months. LRs constitute one of the best ways to measure and express diagnostic accuracy. 15,16 Moreover, unlike predictive values, LRs share the feature of being independent of disease prevalence, suggesting that their meaning can be transferred beyond study. 15,16 LR+ of each architectural pattern was thus calculated according to the following formula: sensitivity/(1 specificity). LR+ illustrates how many times more likely a positive test result occurs in subjects with the disease than in those without the disease. It may range from 0 to infinity. Findings with LR+ > 1 argue for the diagnosis of interest: the higher the number, the more convincingly the finding suggests that disease. Findings whose LR+ lie between 0 and 1 argue against the diagnosis of interest: the closer the LR+ is to 0, the less likely the disease. In the third stage, only architectural patterns associated with a relevant diagnostic accuracy were considered for the constitution of the architecture-based grading system. Consequently, tumoral patterns with LR+ close to 1, that is, 0.8 < LR + < 1.2, lacking diagnostic value, were not considered for the establishment of the grading. As ccrcc commonly presented distinct patterns, the architectural pattern associated with the highest grade defined the architectural grade of each tumor independently of its surface area. Independent Validation Cohort The external validation population included 252 patients suffering from ccrcc, referred to Institut Mutualiste Montsouris (Paris, France). Partial or radical nephrectomy was performed between January 1, 2007, and December 31, Data from Institut Mutualiste Montsouris were retrieved from the database of the Urology Department on January 28, The same inclusion criteria were used for the principal cohort (Hôpital Saint-Louis) and the validation cohort. TABLE 3. Definition of the Architectural Grading According to the Positive Likelihood Ratios (LR+) for DFS at 50 Months in the Principal Cohort (n = 156) Architectural Patterns Positive Likelihood Ratios 95% CI Architectural Grades No. Cases (N [%]) No. Cases With Metastatic Evolution at 50 mo (N [%]) Clear cell papillary RCC-like pattern Grade 1 12 (7.7) 2 (16.7) Acinar pattern Grade 1 31 (19.9) 9 (29.0) Cystic or tubulocystic pattern with or without Grade 1 73 (46.8) 20 (27.4) hemorrhagic contents, thyroid-like and/or Paneth cell-like features Regressive pattern with or without Grade 1 88 (56.4) 26 (29.5) hemangioblastoma-like features Compact (or small nest) pattern Grade 1 99 (63.5) 31 (31.3) Large nest pattern Grade 2 89 (57.0) 44 (49.4) Alveolar pattern Grade 2 14 (9.0) 7 (50.0) Chromophobe/oncocytic cell-like pattern Grade 2 21 (13.5) 11 (52.4) Intratumoral inflammatory reaction Grade 2 36 (23.1) 20 (55.6) Papillary or micropapillary pattern Grade 2 24 (15.4) 15 (62.5) Eosinophilic hyaline globule pattern Grade 2 21 (13.5) 15 (71.4) Syncytial multinucleated tumor giant cell pattern Grade 3 34 (21.8) 27 (79.4) Rhabdoid dedifferentiation Grade 3 55 (35.2) 44 (80.0) Enlarged vascular space pattern Grade 3 30 (19.2) 25 (83.3) HLRCC-like pattern Grade 3 31 (19.9) 26 (83.9) Lymphatic invasion Grade 4 11 (7.0) 10 (90.9) Infiltrative growth pattern with entrapped Grade 4 27 (17.3) 25 (92.6) glomeruli Sarcomatoid dedifferentiation Infinity Grade 4 23 (14.7) 23 (100.0) HLRCC indicates hereditary leiomyomatosis renal-cell carcinoma. Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 3

4 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 Interobserver Reproducibility A total of 50 randomly selected cases were independently reviewed by 2 other pathologists to evaluate interrater agreement: a general pathologist with no specific interest or expertise in uropathology (D.C.), working in an organization model with pathologists seeing all types of histologic specimens, and a second-year anatomic pathology trainee (M.N.). In each case, both reviewers determined WHO/ISUP and architecture-based grading systems. Before performing this review, these pathologists received a short training for architecture-based and WHO/ISUP grading systems by the uropathologist (J.V.), in the form of a review of 10 representative ccrcc cases. References outlining criteria for WHO/ISUP grading 4 and distinct FIGURE 1. Patterns defining architectural grade 1, macrocystic (A) or microcystic, tubulocystic (B), compact or small nests (C), acinar with serrated gland lumens (D), regressive (E), or clear cell papillary RCC-like (F) patterns. 4 Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

5 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc pictures of all tumor patterns identified in this study were also provided. The final score for each case to be used in all subsequent statistical analyses was that determined by the uropathologist (J.V.). Statistical Analysis Statistical analyses were performed using R software (version 3.2.1; The R Foundation for Statistical Computing, Vienna, Austria) on the supercomputer facilities of the Mésocentre de calcul de Franche-Comté. Data were FIGURE 2. Patterns defining architectural grade 2, large nests (A, B), alveolar (C), papillary (D), chromophobe/oncocytic cell-like (E-F), inflammatory reaction (G), or eosinophilic hyaline globule (H) patterns. Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 5

6 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 descriptively summarized using frequencies and percentages for categorical variables, and medians and ranges for continuous variables. Differences between the 2 cohorts of patients (Hôpital Saint-Louis and Institut Mutualiste Montsouris) were assessed by Fischer exact test for categorical variables and by Student t test for continuous variables. Interobserver agreement was statistically assessed using Fleiss or Cohen generalized kappa. DFS and CSS were plotted on Kaplan-Meier FIGURE 3. Patterns defining architectural grade 3, rhabdoid dedifferentiation (A, B), syncytial-type multinucleated tumor giant cell (C), hereditary leiomyomatosis RCC-like (D), or enlarged vascular space (E-G) patterns. 6 Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

7 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc FIGURE 4. Patterns defining architectural grade 4, sarcomatoid dedifferentiation (A), lymphatic invasion (B), or infiltrative growth pattern with intratumoral entrapped glomeruli (C, D). curves according to architectural score. Time-dependent receiver operating characteristic (ROC) curve and area under curve (AUC) were computed with time ROC R package. An AUC of 0.5 to 0.7 indicated a low accuracy, an AUC of 0.7 to 0.9 indicated moderate accuracy, and > 0.9 indicated greater accuracy. 17 The Cox model was applied to analyze factors associated with DFS and CSS, using the survival R package. Concordance was extracted from coxph objects produced by this analysis. Significant levels of P-values of univariate analyses were corrected for multiple comparisons using the Bonferroni correction (0.05 was divided by the number of comparisons to provide the new significance threshold). In multivariate analyses, we used stepwise model selection by Akaike information criterion to determine the best subsets of predictors. RESULTS Baseline Characteristics of the Study Population Two hundred fifty-four patients were finally included in the principal cohort. Table 1 summarizes clinical and morphologic data in the overall population. Mean age at ccrcc diagnosis was 61 years (range, 30 to 90 y). One hundred seventy-six patients (69.3%) were men. The mode of presentation was incidental in 184 patients (79.9%), local in 37 (14.9%), and systemic in 28 (11.2%). Median follow-up was 48 months (range, 1 to 216 mo). Among 67 patients with metastatic spreading (26.4%), 32 were metastatic at presentation, and 35 developed a metastatic disease after a median follow-up period of 50 months (range, 4 to 160 mo). A total of 44 patients (17.3%) died during the follow-up period. Thirty-five patients died from advanced ccrcc (13.8%), with a median follow-up period of 14 months (range, 1 to 113 mo). Pathologic Analysis We identified a large number of distinct architectural patterns of ccrcc (see Table 2), the great majority of which was already described in the literature. These included common architectural patterns such as macrocystic or microcystic with or without hemorrhagic contents and/or thyroid-like and/or Paneth cell-like features, 18 alveolar, acinar with or without serrated lumens, papillary or micropapillary (small papillary tufts without a fibrovascular core), compact Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 7

8 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 (small nests), regressive 7,19 with or without hemangioblastomalike features, 8,20 and large nest patterns. We equally defined the enlarged vascular space pattern as tumoral islets lined by endothelial cells and surrounded by large capillary cavities. Moreover, we considered some morphologic patterns more characteristic of other tumors or tissues, that is, clear cell papillary RCC-like, 9,21,22 chromophobe/oncocytic cell-like, and HLRCC-like (large eosinophilic nucleolus surrounded by a clear halo). 23,24 In addition, some particular tumor cell appearances were recorded, that is, rhabdoid or sarcomatoid dedifferentiation, syncytial-type multinucleated tumor giant cell, foamy (tumor cells with a vacuolated and not optically empty cytoplasm), and intracytoplasmic eosinophilic hyaline globules. 18 Finally, we collected some additional findings, that is, intratumoral inflammatory reaction, lymphatic (and not major vein) invasion, and infiltrative growth pattern with intratumoral entrapped normal renal parenchyma, 25 including normal or sclerosed glomeruli. It is important to note that all ccrcc cases included in this cohort presented at least one of the following patterns: macrocystic or microcystic, compact (small nests), large nests, and regressive patterns. These tumor areas, characteristic of the ccrcc subtype, were always constituted of clear cells positive for carbonic anhydrase-ix and CD10 immunostaining. These data were considered as a prerequisite for inclusion in this study. Definition of Pattern-based Grading System To establish this new grading in a rigorous way, we determined the capacity of distinct architectural patterns to predict metastatic evolution at 50 months. One hundred fifty-six of the 254 suitable patients included in the principal study were included in this preliminary study. Among them, 32 (20.5%) were metastatic at presentation, and 30 (19.2%) became metastatic during the 50-month follow-up period. We distributed the distinct architectural patterns in 4 grades according to LR+ values (see Table 3): grade 1, no metastatic potential (LR+ 0.8), macrocystic, microcystic, or tubulocystic, compact, acinar, clear cell papillary RCC-like and regressive patterns; grade 2, low metastatic potential (1.2 LR+ <5), large nest, alveolar, papillary or micropapillary, chromophobe/oncocytic cell-like, intracytoplasmic eosinophilic hyaline globule, and intratumoral inflammatory reaction patterns; grade 3, intermediate metastatic potential (5 LR+ <10), rhabdoid dedifferentiation, multinucleate giant cells, enlarged vascular space, and HLRCC-like patterns; grade 4, high metastatic potential (LR+ 10), sarcomatoid dedifferentiation, infiltrative growth pattern with intratumoral entrapped glomeruli, and lymphatic invasion. Foamy pattern with an LR+ of 1.18 did not reach a diagnostic value and was, therefore, not included TABLE 4. Univariate and Multivariate Analysis for DFS and CSS in the Principal Cohort (n = 254) DFS CSS Factor HR 95% CI P c Index HR 95% CI P HR 95% CI P c Index HR 95% CI P Age E E Sex E E E Mode of < 1.0E E E E-01 presentation ECOG E E E E-01 Pathologic tumor E E E E-01 size 2010 primary E E E E-01 tumor classification 2010 regional lymph node involvement E E E E distant ND ND ND ND < 1.0E E-03 metastasis 4-tier WHO/ISUP E E E E-01 grading 3-tier WHO/ISUP E * E * grading Coagulative tumor necrosis < 1.0E * E * WHO/ISUP grade < 1.0E E E E-01 +necrosis 4-tier architectural grading system < 1.0E * E * 3-tier Architectural grading system < 1.0E E E E-03 c index indicates Harrell concordance index; ECOG, Eastern Cooperative Oncology Group; ND, not determined; PHR, proportional hazards regression. *Collinear variables not included in multivariate analysis. 8 Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

9 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc in the grading system. This new morphologic grading system devised on the metastatic potential at 50 months from distinct architectural patterns is presented in Figures 1 4. To determine the best architectural scoring, we studied in parallel 3-tier (grades 1-2, 3, and 4) and 4-tier grading systems. For multivariate analyses and ROC curves, only the most efficient architectural grading in the univariate analysis was systematically retained. In the case of superimposable values, the 3-tier scoring was always preferred to simplify the assessment of this new grading system. Prognostic Impact of the Architecture-based Grading System On univariate analysis, both the 3-tier and 4-tier architecture-based scores outperformed all other morphologic FIGURE 5. Predicted survival and ROC curves in the principal cohort. Kaplan-Meier estimates of DFS and CSS according to the 3-tier architectural score in all patients (A, E) and in the localized (pt1-3n0m0) ccrcc subgroup (C, G). AUC for the distinct morphologic scores in predicting 5-year DFS and CSS in all patients (B, F) and in the localized ccrcc subgroup (D, H). Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 9

10 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 grading systems, that is, 3-tier, 4-tier WHO/ISUP gradings, and WHO/ISUP+necrosis for DFS (3-tier, c index = 0.91, P < 10E-16; 4-tier, c index = 0.92, P <10E-16) and CSS (3-tier, c index = 0.90, P = 1.5E-12; 4-tier, c index = 0.90, P = 1.4E-10) (see Table 4). Figures 5A, B, E, and F displays the Kaplan- Meier curves for DFS (Log-rank test, p < 1E-16) and CSS (Log-rank test, p < 1E-16) according to the 3-tier architectural score and the comparing ROC curves between the distinct morphologic gradings in predicting 5-year DFS and CSS. ROC curve analysis showed a greater accuracy of the 3-tier architectural score in predicting 5-year DFS (AUC, 0.94), significantly better than that observed using WHO/ISUP grade (AUC, 0.87; P = 4.5E-03) and WHO/ISUP+necrosis grade (AUC, 0.87; P = 9.6E-03). ROC analysis also provided thehighestaucwithregardtopredictingthe5-yearcss for the architectural score (3-tier, AUC, 0.91), compared with 0.86 for the WHO/ISUP grade (P = 4.8E-02) and 0.87 for the WHO/ISUP+necrosis grade (P = 8.1E-02). On multivariate analysis, gathering all noncollinear clinical and pathologic data statistically significant in univariable setting, 3-tier architectural score constituted the only independent predictor parameter of DFS (hazard ratio [HR], 5.91; 95% confidence interval [CI], ; P = 6.7E-10). Furthermore, the 3-tier architectural score was also found to be an independent indicator of CSS (HR, 4.49; 95% CI, ; P = 2.2E-03), as did distant metastases at presentation (HR, 4.89; 95% CI, ; P = 1.2E-03). We also performed the same analysis in the localized (pt1-3n0m0) ccrcc subgroup (see Table 5). On univariate analysis, the architectural score maintained its ability to predict DFS (3-tier, c index = 0.88; P = 1.1E-16; 4-tier, c index = 0.89; P = 1.1E-13) and CSS (3-tier, c index = 0.93; P = 1.2E-06; 4-tier, c index = 0.93; P = 7.5E-06). Figures 5C, D, G, and H display the Kaplan-Meier curves for DFS (Log-rank test, P < 1E-16) and CSS (Log-rank test, P < 1E- 16) according to the 3-tier architectural score and the comparison of ROC curves in predicting 5-year DFS and CSS. The architectural score described the highest AUC in predicting 5-year DFS (AUC, 0.90) versus 0.84 for the WHO/ISUP grade (P = 4.2E-02) and 0.83 for the WHO/ ISUP+necrosis grade (P = 5.5E-02). The architectural score also showed the highest AUC in predicting 5-year CSS, that is, 0.90 versus 0.85 and 0.86 for the WHO/ISUP (P = 0.24) and WHO/ISUP+necrosis (P = 0.39) grades, respectively. In TABLE 5. Univariate and Multivariate Analysis for DFS and CSS in Localized (pt1-3n0m0) ccrccs of the Principal Cohort (n = 217) DFS CSS Factor HR 95% CI P c Index HR 95% CI P HR 95% CI P c Index HR 95% CI P Age E E Sex E E Mode of E E E E-01 presentation ECOG E E E-03 Pathologic E E E E-01 tumor size 2010 primary E E E tumor classification 4-tier WHO/ E E E E-01 ISUP grading 3-tier WHO/ E * E * ISUP grading Coagulative E * E * tumor necrosis WHO/ISUP E E E E-01 grade + necrosis 4-tier E * E * architectural grading system 3-tier architectural grading system E E E E-05 c index indicates Harrell concordance index; ECOG, Eastern Cooperative Oncology Group; PHR, proportional hazards regression. *Collinear variables not included in multivariate analysis Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

11 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc a multivariable setting, 3-tier architectural score constituted the unique predictor of DFS (HR, 6.10; 95% CI, ; P = 1.3E-07). It also constituted an independent predictor of CSS (HR, 20.09; 95% CI, ; P = 9.4E-05) as did Eastern Cooperative Oncology Group (HR, 15.36; 95% CI, ; P = 9.2E-03). FIGURE 6. Predicted survival curves for DFS and CSS according to the WHO/ISUP grades for all patients in the principal cohort. Patients were stratified according to the architectural score. A, Kaplan-Meier analysis of DFS in WHO/ISUP grades 1 to 2 subgroup. B, Kaplan-Meier analysis of DFS in WHO/ISUP grade 3 subgroup. C, Kaplan-Meier analysis of DFS in WHO/ISUP grade 4 subgroup. D, Kaplan-Meier analysis of CSS in WHO/ISUP grades 1 to 2 subgroup. E, Kaplan-Meier analysis of CSS in WHO/ISUP grade 3 subgroup. F, Kaplan-Meier analysis of CSS in WHO/ISUP grade 4 subgroup. Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 11

12 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 Contribution of Architectural Score in the Prognostic Evaluation of ccrcc To better determine the relative contribution of the architectural and WHO/ISUP scores in ccrcc prognostic determination, we performed, in the principal cohort, an additional analysis by stratifying WHO/ISUP grades with different 3-tier architectural grades for DFS and CSS. These results are described in Figure 6. There is no gain for grades 1 and 2. However, the incrementation of architectural grading in both WHO/ISUP grade 3 and WHO/ISUP grade 4 subgroups of ccrcc patients improved outcome prediction for these 2 patient subgroups, that is, in the WHO/ISUP grade 3 subgroup, Log-rank test, P = 1.5E-09 for DFS and P = 5.9E-06 for CSS, and in WHO/ISUP grade 4 subgroup, Log-rank test, P = 2.9E-11 for DFS and P = 7.0E-05 for CSS. For WHO/ISUP grade 3, this gain was mainly linked to a relative downgrading of numerous WHO/ISUP grade 3 tumors with favorable outcome in architectural grade 1 or 2. For WHO/ISUP grade 4, the higher predictive value was essentially attributable to the rhabdoid and giant cell patterns that were regarded as grade 3 in architectural score. On the contrary, the Kaplan-Meier curves according to architectural score, stratifying by WHO/ISUP grading or tumor necrosis, did not improve the predictive value of this scoring system. Actually, with these 2 additional parameters, a minimal gain was obtained only for DFS in architectural grade 3 but failed to reach statistical significance, that is, P = for WHO/ISUP and P = for necrosis (data not shown). External Validation The external validation cohort was composed of 252 patients whose baseline characteristics are compared with those of the principal cohort in Table 6. Among 56 patients with metastatic spreading (22.2%), 17 were metastatic at presentation and 39 developed a metastatic disease after a median follow-up period of 16 months (range, 1 to 122 mo). A total of 28 patients died during the follow-up period (11.1%). Twenty-three patients died from advanced ccrcc (9.1%), with a median follow-up period of 35 months (range, 15 to 90 mo). The univariate and multivariate analyses confirmed the architectural score as the best morphologic grading system and its independent association with DFS and CSS (Table 7). This ability to predict DFS and CSS was also maintained in localized forms of ccrcc (Table 8). Architecture-based Grading and Integrated Staging Systems To compare the predictive value of our architecturebased grading to integrated staging systems, that is, SSIGN and UISS for CSS, and Leibovich score for DFS, we pooled data of patients from the principal and the validation cohorts. These distinct univariate and multivariate analyses are displayed in Table 9. In all patients and the localized ccrcc subgroup, architectural and SSIGN scores showed very close c index, that is, SSIGN TABLE 6. Comparison of Clinical and Morphologic Characteristics of ccrcc Patients Included in the Principal and Validation Cohorts Clinicopathologic Features Principal Cohort (n = 254) Validation Cohort (n = 252) P Age (median [range]) (y) 61 (30-90) 63 (35-87) 1.1E-2 Sex (n [%]) 1 Male 176 (69.3) 175 (69.4) Female 78 (30.7) 77 (30.6) Mode of presentation (n [%]) S1 (incidental) 184 (73.9) 199 (79.0) 4.0E-2 S2 (local symptoms) 37 (14.9) 40 (15.9) S3 (systemic symptoms) 28 (11.2) 13 (5.1) ECOG performance status (n [%]) (93.2) 196 (80.7) 1.0E (6.8) 47 (19.3) Pathologic tumor size 50 (10-180) 48 (12-160) 2.5E-1 (median [range]) (mm) 4-tier WHO/ISUP grading (n [%]) 1 14 (5.5) 5 (2.0) 1.0E (30.7) 118 (46.8) 3 88 (34.7) 90 (35.7) 4 74 (29.1) 39 (15.5) 3-tier WHO/ISUP grading (n [%]) 1/2 92 (36.2) 123 (48.8) 4.5E (34.7) 90 (35.7) 4 74 (29.1) 39 (15.5) Coagulative tumor necrosis (n [%]) Absent 173 (68.1) 196 (77.8) 1.6E-2 Present 81 (31.9) 56 (22.2) WHO/ISUP grade+necrosis (n [%]) Groups 1/2 91 (35.8) 118 (46.8) 2.4E-03 Groups 3/4 73 (28.8) 76 (30.2) Groups 5/6 26 (10.2) 26 (10.3) Groups 7/8/9 64 (25.2) 32 (12.7) 4-tier architectural grading system (n [%]) 1 93 (36.6) 86 (34.1) 6.7E (26.4) 93 (36.9) 3 49 (19.3) 37 (14.7) 4 45 (17.7) 36 (14.3) 3-tier architectural grading system (n [%]) 1/2 160 (63.0) 179 (71.0) 1.5E (19.3) 37 (14.7) 4 45 (17.7) 36 (14.3) 2010 primary tumor classification (n [%]) pt1 138 (54.3) 148 (58.7) 6.2E-1 pt2 29 (11.4) 26 (10.3) pt3+pt4 87 (34.3) 78 (31.0) 2010 regional lymph node involvement (n [%]) pnx+pn0 242 (95.3) 246 (97.6) 2.3E-1 pn1 12 (4.7) 6 (2.4) 2010 distant metastasis (n [%]) M0 222 (87.4) 235 (93.3) 6.5E-2 M1 32 (12.6) 17 (6.7) Follow-up period (median [range]) (mo) 48 (1-216) 52 (1-156) 3.4E-1 ECOG indicates Eastern Cooperative Oncology Group Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

13 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc TABLE 7. Univariate and Multivariate Analysis for DFS and CSS in the Validation Cohort (n = 252) DFS CSS Factor HR 95% CI P c Index HR 95% CI P HR 95% CI P c Index HR 95% CI P Age E E E-03 Sex E E Mode of presentation E E E E-03 ECOG E E * Pathologic tumor E E E E-02 size 2010 primary tumor classification E E E E regional lymph node involvement E E E E distant metastasis ND ND E E-04 4-tier WHO/ E E E E-01 ISUP grading 3-tier WHO/ E * E * ISUP grading Coagulative E * E * tumor necrosis WHO/ISUP E E E E-01 grade + necrosis 4-tier < 1.0E * E * architectural grading system 3-tier architectural grading system < 1.0E E E E-02 c index indicates Harrell concordance index; ECOG indicates Eastern Cooperative Oncology Group; ND, not determined; PHR, proportional hazards regression. *Collinear variables not included in multivariate analysis. c index being slightly better than the architectural score for all patients, and the reverse situation was noted in the localized ccrcc subgroup [c index = 0.92 vs (3-tier and 4-tier), and 0.87 vs (3-tier) and 0.89 (4-tier), respectively], and their AUCs in predicting 5-year CSS were not statistically different (AUCs: 0.92 vs. 0.91, P = 0.45 and AUCs: 0.86 vs. 0.89, P = 0.64, respectively; Fig. 7). In contrast, the UISS score described the lowest values (c index = 0.87 and 0.71; AUCs, 0.79 and 0.56; P = 3.5E-03 and P = 2.8E-11, compared with architectural score, respectively). On multivariate analysis, the 3-tier architectural grade remained an independent predictor of CSS in all patients and the localized ccrcc subgroup, as did UISS and SSIGN scores, and was associated with the highest HR (HR, 2.60; 95% CI, ; P = 9.1E-04 in all patients; HR, 4.38; 95% CI, ; P = 2.0E-05 in the localized ccrcc subgroup). As the Leibovich prognosis score was demonstrated to be a relevant predictor of metastatic evolution in localized ccrccs, 14,26 we also compared the architectural grade to Leibovich score for the DFS in localized ccrcc subgroup. The c index of the architectural score was of 0.87 (3-tier) and 0.88 (4-tier) versus 0.82 for the Leibovich score. AUC was 0.87 for the 3-tier architectural score in predicting 5-year DFS versus 0.81 for the Leibovich score (P = 1.6E-02). On multivariate analysis, architectural grade was a better predictor of metastatic evolution than the Leibovich score (HR, 4.27; 95% CI, ; P = 1.1E-16 vs. HR, 2.28; 95% CI, ; P = 7.2E-05). Consequently, in the current study, the architectural and SSIGN scores were very comparable in predicting CSS in all patients and the localized ccrcc subgroup, the UISS being less efficient. The architectural score was also more accurate than the Leibovich prognosis score to predict progression in patients with a localized form of ccrcc. Evaluation of the Interobserver Reproducibility of the Architectural Scoring System The overall concordance for 4-tier architectural grading was strong (k = 0.78; 95% CI, ) and substantially greater than that of 4-tier WHO/ISUP grading (k = 0.65; 95% CI, ) (Table 10). This agreement was even higher between senior pathologists (k = 0.93, % Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 13

14 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 TABLE 8. Univariate and Multivariate Analysis for DFS and CSS in Localized (pt1-3n0m0) ccrccs of the Validation Cohort (n = 229) DFS CSS Factor HR 95% CI P c Index HR 95% CI P HR 95% CI P c Index HR 95% CI P Age E E E-02 Sex E E Mode of E E E E-01 presentation ECOG E E Pathologic tumor E E E E-02 size 2010 primary E E E E-01 tumor classification 4-tier WHO/ E E E E-01 ISUP grading 3-tier WHO/ E * E * ISUP grading Coagulative E * E * tumor necrosis WHO/ISUP E E E E-01 grade +necrosis 4-tier E * E * architectural grading system 3-tier architectural grading system E E E E-02 c index indicates Harrell concordance index; ECOG indicates Eastern Cooperative Oncology Group; PHR, proportional hazards regression. *Collinear variables not included in multivariate analysis. TABLE 9. Univariate and Multivariate Analysis of Integrated Staging and Architectural Grading Systems for DFS and CSS in All (n = 506) and/or Localized (n = 448) ccrcc Patients of the 2 Cohorts Localized ccrccs Patients (n = 448) Factor HR 95% CI P c Index HR 95% CI P Leibovich score < 1.0E E-05 4-tier architectural < 1.0E * grading system 3-tier architectural grading system < 1.0E E-16 All ccrcc Patients (n = 506) Localized ccrccs Patients (n = 448) Factor HR 95% CI P c Index HR 95% CI P HR 95% CI P c Index HR 95% CI P SSIGN < 1.0E E E E-02 UISS < 1.0E E E E-02 4-tier architectural grading system < 1.0E * E * 3-tier architectural grading system < 1.0E E E E-05 c index indicates Harrell concordance index; PHR, proportional hazards regression. *Collinear variable not included in multivariate analysis Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

15 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc FIGURE 7. Predicted survival and ROC curves in the 2 (principal and validation) cohorts. Kaplan-Meier estimates of CSS according to the architectural score in all patients (A), and in the localized (pt1-3n0m0) ccrcc subgroup (C). AUC for the architectural score and the SSIGN and UISS model groups in predicting 5-year CSS in all patients (B), and in the localized ccrcc subgroup (D). E, Kaplan-Meier estimates of DFS according to the architectural score in the localized ccrcc subgroup. F, AUC for the architectural score and the Leibovich model groups in predicting 5-year DFS in the localized ccrcc subgroup. agreement 92% vs. k = 0.80, % agreement 82% for WHO/ ISUP grading, respectively). A perfect agreement (3/3 reviewers) was observed in 38 (76%) cases for architectural grading, and an agreement by 2/3 reviewers was seen in the remaining 12 (24%) cases. For the WHO/ISUP grading, a perfect agreement (3/3 reviewers) was observed in 33 (66%) Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 15

16 Verine et al Am J Surg Pathol Volume 00, Number 00, 2018 TABLE 10. Interobserver Agreement in 3-Tier and 4-Tier Architectural and WHO/ISUP Gradings Between 3 Categories of Pathologists 3-Tier Architectural Grading 3-Tier WHO/ISUP 2016 Grading General Pathologist Trainee General Pathologist Trainee % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) Uropathologist (0.90-1) ( ) ( ) ( ) General pathologist ( ) ( ) Weighted Cohen kappa* Overall interobserver agreement was of 0.82 (95% CI, ) for the architectural score and 0.71 (95% CI, ) for the WHO/ISUP grading (weighted Fleiss kappa) 4-Tier Architectural Grading 4-Tier WHO/ISUP 2016 Grading General Pathologist Trainee General Pathologist Trainee % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) % Agreement Kappa* (95% CI) Uropathologist (0.87-1) ( ) ( ) ( ) General pathologist ( ) ( ) Overall interobserver agreement was of 0.78 (95% CI, ) for the architectural score and 0.65 (95% CI, ) for the WHO/ISUP grading (weighted Fleiss kappa). *Weighted Cohen kappa. cases, a partial agreement (2/3 reviewers) in 16 (32%) cases, and no agreement in 1 (2%) case. The discordances noticed for the architectural grading were mainly caused by the lack of identification of the infiltrative growth pattern and by the difficulty to differentiate the compact and large nest patterns responsible for 5 (42%) and 3 (25%) of the 12 discordant cases, respectively. The interobserver reproducibility of the architectural score was even higher with the 3-tier grading system (overall concordance, k = 0.82; 95% CI, ) and always better than the 3-tier WHO/ISUP 2016 grading (overall concordance, k = 0.71; 95% CI ). Proposal for a New Morphologic Grading System All the results presented in this study demonstrate the relevance of this approach in the morphologic assessment of ccrcc prognosis. Moreover, because the predictive value of the 2 architecture-based scores developed herein (3-tier and 4-tier) was similar throughout this study, we propose to keep only the simplified version of our architecture-based grading system as the final score and define the 3 risk groups as follows: low-risk group (grades 1 to 2), intermediate-risk group (grade 3), and high-risk group (grade 4). Finally, to facilitate both the determination and the diffusion among pathologists of this new 3-tier morphologic prognostic grading, a decisional tree is presented in Figure 8. DISCUSSION We developed herein a new grading system for ccrcc on the basis of tumor architecture. Our architecture-based score outperforms all other morphologic grading systems and shows an independent prognostic value for DFS and CSS. These results are confirmed in a contemporary external validation cohort. Moreover, with only 1 morphologic datum, this new scoring system remains a predictor of DFS and CSS when multivariate analysis was performed with 3 established integrated staging systems, that is, Leibovich prognosis score for DFS, and SSIGN and UISS for CSS. Our score even outperforms 2 of them (UISS and Leibovich scores), particularly in localized ccrcc forms. This study also shows that the prognosis associated with rhabdoid and sarcomatoid features is clearly distinct (LR+ = 6.06 vs. infinity, respectively), supporting their classification in 2 different grades, that is, grade 3 for rhabdoid dedifferentiation pattern and grade 4 for sarcomatoid dedifferentiation pattern as proposed in our scoring. Moreover, additional Kaplan-Meier analysis of DFS and CSS in WHO/ISUP subgroups stratified according to architectural grades confirmed this assessment, as architectural score allowed to enhance the predictive value of WHO/ISUP grade 4 by reclassifying the rhabdoid pattern as grade 3. These data are in contradiction with the WHO/ ISUP grading system, which considers these 2 patterns as grade 4, but is in agreement with several other studies that have also described a distinct prognostic value between these 2 patterns. 27,28 The greater predictive value of our architecturebased score system also resulted in a relative downgrading of numerous WHO/ISUP grade 3 tumors with favorable outcome to grade 1 or 2 in the 4-tier architectural score. Indeed, 18.9% of patients with a WHO/ISUP grade 3 tumor developed a local recurrence and/or a metastatic evolution, and 5.7% died from ccrcc versus 46.9% and 20.4% for 4-tier architectural grade 3 tumors, respectively. This study also confirms 2 previous studies 11,29 by showing that the incorporation of tumor necrosis in 16 Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved.

17 Am J Surg Pathol Volume 00, Number 00, 2018 Architectural Pattern-based Grading for ccrcc FIGURE 8. Decision tree of the definitive architecture-based grading system for ccrcc. WHO/ISUP grading system enhances the predictive value of this morphologic grading system in predicting DFS and CSS. Conversely, the incorporation of tumor necrosis in our score did not improve significantly its predictive value for DFS and CSS. Tumor architecture has been sometimes studied among numerous morphologic parameters to determine the best predictor for RCC outcome. These studies usually showed no or weak predictive value for this parameter. 30 However, they were frequently performed on distinct RCC subtypes. When ccrcc was specifically analyzed, only a limited number of architectural patterns were identified. 30 Unlike nucleolar prominence associated with a low interobserver reproducibility, 30,31 tumor architecture is more Copyright 2018 Wolters Kluwer Health, Inc. All rights reserved. 17