Characteristic morphology and immunohistochemical patterns of clear cell papillary renal cell tumours may be observed in renal cell carcinomas, a critical pitfall in renal biopsy cytopathology

Clear cell papillary renal cell tumour (CCPRCT) was renamed from previous clear cell papillary renal cell carcinoma (CCPRCC) in the latest WHO Classification of Tumours. It is essential to differentiate RCC from CCPRCT in renal mass biopsies (RMB).


| INTRODUC TI ON
Clear cell papillary renal cell tumour (CCPRCT) has been revised from its previous designation as clear cell papillary renal cell carcinoma (CCPRCC) in the latest WHO Classification of Tumours: Urinary and Male Genital Tumours, 2022 (5th edition). 1This alteration was made because metastasis or aggressive behaviour was not reported previously.CCPRCT was initially believed to occur in the end-stage kidneys but was later found to develop in normal kidneys sporadically. 2,3CCPRCT tumours can manifest as either solid or cystic masses. 3,4All these clinical and imaging features are also seen in more aggressive types of renal cell carcinoma (RCC), such as clear cell renal cell carcinoma (CCRCC) and papillary renal cell carcinoma (PRCC), among others.CCPRCT is currently considered as a tumour with an indolent clinical course and excellent prognosis.Therefore, it is essential to distinguish aggressive RCC from CCPRCT when conducting renal mass biopsy (RMB) cytology.
Fine needle aspiration (FNA) and touch preparation of cores show that CCPRCT tumour cells are arranged in tubules, nests, and papillae, and exhibit clear/vacuolated cytoplasm and peripherally located low-grade nuclei, [3][4][5] some of which can also be seen in CCRCC or PRCC. 5

| Case selection
This study has been approved by the Institutional Review Board of Northwestern University.RMB cases with FNA and/or core needle biopsy (CNB) as well as corresponding nephrectomy cases were retrieved from the Northwestern Memorial Hospital pathology database between January 2016 and April 2023.The renal neoplasms were classified according to the 2022 WHO Classification of Urinary and Male Genital Tumours. 1,6e following variables were recorded: cytology microscopic description, IHC stains, cytologic diagnosis, surgical resection pathology diagnosis, mass size, therapy, and metastatic status.Only cases at least focally showing CCPRCT morphologic and/or IHC features were collected.4][5] The histologic features for diagnosis CCPRCT include tumour cells arranged in nested, tubular, or papillary patterns with surface-oriented small nuclei without prominent nucleoli. 1,2,5The immunostaining patterns for diagnosis of CCPTCT include cup-like CA9 and strong stain of CK7. 1-5

| Core needle aspiration (CNB) with touch preparation
Percutaneous CNB was performed under local anaesthesia and guidance of CT scan or ultrasound imaging, using 20-gauge core needle devices.The touch preparations of CNB were air-dried and stained with modified Giemsa (Diff-Quik) stain for rapid on-site evaluation (ROSE) for adequacy and immediate interpretation by boardcertified cytopathologists.

| Correlation of cytology and nephrectomy diagnoses
Diagnoses derived from nephrectomy specimens and CNBs from metastatic RCCs or renal masses (for the cases without nephrectomy) were together used as the final diagnoses.Diagnostic accuracy was calculated as the case number with definitive diagnosis divided by the total case number.The risk of malignancy (ROM) was calculated as the case number with final diagnosis of RCC divided by the total number in the diagnostic category.

| Clinical management of patients following renal mass after biopsy
The electronic medical records were reviewed to gather patient management information following RMB.

| Clinical information of patients
Fifteen cases from 14 patients displaying at least focal morphologic features or IHC patterns of CCPRCT were identified during reviewing retrieved RMB cytology cases and corresponding nephrectomy cases or signing out cytology RMB cases from January 2016 to April 2023.The patients consisted of 11 male and 3 female patients, ranging in ages from 27 to 84 years, with an average age of 59.9 ± 14.2 years.Tumour sizes ranged from 1.8 to 16.0 cm, with an average size of 6.5 ± 4.9 cm.

| Cytology biopsy results of renal masses
The number of passes for obtaining adequate diagnostic material ranged from 2 to 9, with an average of 4.2 ± 1.9 passes.
One of 12 (8%) CCRCC cases was diffusely positive for CA9 (cuplike) and 7 (58%) focally.CK7 positivity was observed diffusely in 4/12 (33%) CCRCC cases and focally in 6/12 (50%).The presence of high-grade nuclei with conspicuous to prominent nucleoli and marked nuclear pleomorphism in three (25%) CCRCC cases excluded CCPRCT based on the current diagnostic criteria of CCRCT. 1 In this study, 2/12 (17%) CCRCC cases were diagnosed as CCPRCT, and 2/12 (17%) CCRCC cases included CCPRCT as a differential diagnosis on RMB.The case 13 showed CCRCC morphology and IHC patterns in RMB; however, nephrectomy specimens displayed a majority area showing characteristic CCPRCT morphology and IHC patterns for CA9 and CK7 and a minority area showing typical CCRCC morphology and IHC patterns.A prior publication documented renal neoplasms with overlapping features of CCRCC and CCPRCC (CCPRCT). 7A case report also highlighted a sarcomatoid renal cell carcinoma with clear cell papillary-like primary tumour and lymph node metastasis. 8CCRCC rarely exhibits papillary and threedimensional clusters, a helpful feature to distinguish CCPRCT from CCRCC. 5 Besides immunostains for CA9 and CK7, immunostaining for GATA3 9 CCPRCT tumour cells characteristically exhibit diffuse positivity for CA9 with a distinctive cup-like pattern, while CCRCC and PRCC tumour cells show positivity for CA9 with a box-like pattern.Furthermore, CCPRCTs and PRCCs are diffusely positive for CK7, while CCRCCs are mostly negative, with rare cases showing diffuse positivity.These immunohistochemical (IHC) stains help distinguish indolent CCPRCT from more aggressive CCRCC or PRCC.Given the clinical presentation, imaging, gross features, and histomorphologic and cytomorphologic morphology of CCPRCT and RCC with clear cytoplasm overlap, their relationship warrants further investigation.This study aimed to investigate whether RCC can coexist with morphologic and IHC features resembling CCPRCT.
nucleoli were observed in one (13%) case, and conspicuous nucleoli were seen in four (27%) cases.IHC staining for CA9 demonstrated a diffuse cup-like pattern in three (20%) cases, a focal cup-like pattern in seven (47%) cases, a diffuse box-like pattern in four (27%) cases, and negativity in one (7%) case.IHC staining for CK7 showed diffuse positivity in five (33%) cases, focal positivity in six (40%) cases, and negativity in four (27%) cases.IHC staining for TFE3 was performed on four cases, and one (7%) case was diffusely positive.A molecular test performed on the case 6 identified a TSC mutation, which case showed focal CCPRCT morphology with focal high-grade nuclei (WHO/IUSP grade 3) and diffuse CCRPCT IHC patterns for CA9 and CK7.Tempus NGS DNAseq xT (648 gene panel) on case 7 demonstrated SETD2 p.G1681 frameshift -loss of function, and RNA-seq demonstrated NRG1 overexpression, but no TFE3 translocation.
received systemic treatment (chemotherapy, radiation, and/or targeted therapy) for stage 4 RCC.The patient with stage 4 RCC with TFE3 expression received systematic treatment and deceased in 3.4 years.The patient with stage 4 RCC with TSC mutation received systemic treatment and deceased in 2 years.Additionally, one (7%) patient with a small renal mass (1.6 cm) and low-grade (G2) CCRCC on RMB underwent ablation.4| DISCUSS IONThis study demonstrated that the distinctive morphology and/or immunohistochemical patterns characteristic of CCPRCT may be observed in other RCCs, including those with TSC mutation and RCC with TFE3 expression.CCPRCT is characterized by clear tumour cells arranged in papillary, nested, tubular, and cystic architectures, featuring surfaceoriented low-grade nuclei and diffuse positivity for CA9 (cup-like) and CK7.It is difficult to interpret cup-like CA9 pattern in nonluminal areas, which might show a box-like pattern depending on the growth pattern and tangential cross-sectioning, a pitfall in interpreting CA9 in RMB.On the other hand, CCRCC exhibits clear tumour cells arranged in nested, sheet, and cystic architectures and diffuse positivity for CA9 (box-like), while mostly being negative for CK7.
and molecular tests for identification of recurrent genomic mutations linked to CCRCC have proven helpful in distinguishing CCRCC displaying CCPRCT features from CCPRCT.Therefore, because presence of CCPRCT morphology and IHC patterns for CA9 and CK7 may be observed in CCRCC as a mixed tumour, diagnosis of CCPRCT on RMB should be careful to avoid misinterpreting CCRCC as CCPRCT and undertreatment.TFE3-rRCC can present with papillae, tubules, and nests 10 and exhibit abundant clear to eosinophilic cytoplasm. 11TFE3-rRCC is typically positive for TFE3, vimentin, and AMACR, with subsets being positive for cathepsin and Melan A. Tumour cells are either negative or focally positive for CA9, and negative for CK7.In this study, two RMB cases from a stage 4 patient showing diffuse CCPRCT morphology, but featuring focal conspicuous nucleoli were focal CA9 immunoreactivity (cup-like), negative for CK7, and diffusely positive for TFE3.The tumour metastasized to a few organs confirmed by biopsies.The IHC patterns and metastatic

TA B L E 1
Diagnosis of cytologic biopsy and final diagnosis.F I G U R E 1 Characteristic morphologic or immunohistochemical features of CCPRCT may be observed in RCC, high magnification.(A to E) Case 4, CCRCC: Histology of cores shows focal CCPRCT morphology (A) and focal CCRCC morphology with conspicuous nucleoli (B).The tumour cells are positive for vimentin (C) and CA9 (box-like) (D), and negative for CK7 (E).(F to J) Case 6, RCC NOS with TSC mutation: Touch preparation cytology displays nests of tumour cells with vacuolated cytoplasm and eccentric nuclei containing conspicuous nucleoli (F, G).The histology of cores shows CCPRCT morphology (H).Tumour cells are diffusely positive for CA9 (cup-like) (I) and CK7 (J).(K to O) Case 7, RCC-TFE3 expression: Touch preparation cytology shows that tumour cells are arranged in papillae with granular and vacuolated cytoplasm and surface-oriented nuclei focally containing conspicuous nucleoli (K).Histology of cores exhibits CCPRCT morphology (L).Tumour cells are diffusely positive for TFE3 (M), focally positive for CA9 (cup-like) (N), and negative for CK7 (O).

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status contradicted the indolent CCPRCT, 1 although these cases displayed morphology similar to that of CCPRCT.IHC on the second RMB showed the tumour cells were diffusely positive for TFE3, which plus aggressive clinical behaviour (stage 4) raises the possibility of TFE3-rRCC.The patient received systemic treatment.Eleven months later, molecular tests were performed on the second RMB.Tempus NGS DNA-seq xT (648 gene panel) on case 7 demonstrated SETD2 p.G1681 frameshift -loss of function, and