Lecture: Dr Ming Zhou, Cleveland Clinic, Cleveland, Ohio, USA Uncommon renal tumour: Morphologic and molecular characteristics

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1 Australasian Division of the International Academy of Pathology Limited 35 th Annual Scientific Meeting June 4-6, 2010 Venue: Sydney Convention & Exhibition Centre, Darling Harbour, NSW International Society of Urological Pathology (ISUP) COMPANION MEETING Friday, 4 th June, 2010 Bayside Room 104 5:15 7:00 Convenor: Dr Hema Samaratunga, Aquesta Pathology, Milton, Qld Lecture: Dr Ming Zhou, Cleveland Clinic, Cleveland, Ohio, USA Uncommon renal tumour: Morphologic and molecular characteristics Case Presentations: 1. Dr Penelope Cohen, SA Pathology, Adelaide, SA 2. Dr Glen Baxter, Gold Coast Hospital, Qld 3. Dr Shahin Sharifi, Gold Coast Hospital, Qld 4. Dr Anthony Gill, University of Sydney and Royal North Shore Hospital, Sydney, NSW

2 Uncommon Renal Tumors: Morphologic and Molecular Characteristics Ming Zhou, MD, PhD Departments of Anatomic Pathology, Urology, Cancer Biology and Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio, USA Cleveland Clinic

3 Case 1 47 year-old male with right lower abdominal pain Abdominal CT: 2.5 cm right kidney mass Mass unchanged in size in 4 months Right partial nephrectomy

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7 Diagnosis Renal Tubulocystic Carcinoma

8 Renal Tubulocystic Carcinoma Rare form of renal tumors (Farrow et al, USCAP abstract) Low grade RCC of collecting duct origin (MacLannen et al, Urology 1997) Predominantly in men; low stage (T1N0M0) with excellent prognosis; new RCC subtype (Azoulay et al, Virchows Arch 2007; Yang et al, AJSP 2008; Amin et al, AJSP 2009)

9 Clinical and Pathologic Features of 13 Renal Tubulocystic Carcinoma (Yang et al, Am J Surg Pathol, 2008) Clinical features Mean Age 62.9 (range years) Male/female= 10/3 (3.3/1) Pathological findings Tumor size 2.8 ( ) cm 3/13 (23.1%) multi-focal 5/13 (38.5%) with concomitant papillary tumors 12/13 (92.3%) T1, 1/13 T2, 1/13 N2 Clinical outcomes F/U available in 11/ (1-90) months 10 no evidence of disease; 1 alive with disease (PRCC)

10 Yang et al, AJSP, 2008

11 Immunohistochemical Profile of Renal Tubulocystic Carcinoma Yang et al, Am J Surg Pathol, 2008 Azoulay et al, Virchows Arch, 2007 CD10 CK7 AMACR Tubulocystic Ca PRCC Collecting duct Ca CD CK HMWCK AMACR + + -

12 Tubulocystic Ca PRCC, type 1 Clear cell RCC PRCC, type 2 Normal Urothelial Ca Chromophobe RCC Oncocytoma Yang et al, Am J Surg Pathol, 2008

13 Comparative Genomic Microarray Analysis Tubulocystic Ca PRCC Chr 7 Chr 17 Yang et al, Am J Surg Pathol, 2008

14 Case No Renal Tubulocystic and Papillary RCC Are Related Lesions: Pathologic Evidence (Zhou, et al, AJSP, 2009) Solitary/multifocal Solitary Solitary Solitary Solitary Multifocal Solitary Solitary Multifocal Solitary Solitary Solitary Multifocal Solitary Multifocal Solitary Solitary Solitary Solitary Solitary Solitary Tumor size (cm) PRCC, 11 cm - PRCC, 7 cm PRCC, 1cm P Ad, 0.2 cm - - Multiple PRCC, cm PRCC, 4.5 cm Associated Papillary Lesions Within the same kidney, but separate from TCCa PRCC, 1.1 cm, 5 additional pap adenomas Admixed TCCa and PRCC TCCa 20%; PRCC 80% - TCCa 80%; PRCC 20% TCCa 70%; PRCC 30% - TCCa 99%; PRCC 1%

15 Tubulocystic Carcinoma with Admixed Papillary Renal Cell Carcinoma

16 Tubulocystic Carcinoma with Admixed Papillary Renal Cell Carcinoma

17 Tubulocystic Carcinoma with Admixed Papillary Renal Cell Carcinoma Zhou, et al, AJSP, 2009

18 Gain of Chromosomes 7 and 17 in Renal Tubulocystic Carcinoma Normal DAPI Chr 7 Chr 17 Tubulocystic ca DAPI Chr 7 Chr 17 Zhou, et al, AJSP, 2009

19 Chromosomal Changes in Renal Tubulocystic Carcinomas Chromosome Copy Number Chromosome Abnormality Diagnosis Case # Chromosome 7 Chromosome 17 Chromosome Y + Chr 7 + Chr 17 -Chr Y > 3 > 3 0 Tubulocystic carcinoma 2 3, tubulocystic , papillary , tubulocystic , tubulocystic , tubulocystic , tubulocystic , tubulocystic , papillary , tubulocystic , papillary Normal tissue Mean +3 S.D

20 Renal Tubulocystic: Summary Renal tubulocystic carcinoma is a rare morphologic variant of RCC Predominantly affects male patients Presents as low stage tumor with excellent prognosis Molecular, cytogenetic and histological evidence suggests tubulocystic and papillary RCC are closely related lesions A distinct subtype?

21 Case 2 49 year-old female with abdominal pain Abdominal CT: 1.4 cm enhancing cortical mass in lateral interpolar left kidney Left partial nephrectomy Gross pathology 1.2 cm yellow-tan cystic mass with thick capsule

22 Thick Prominent fibrous fibrotic capsule stroma

23 Branching acini Cysts Papillae

24 Cytokeratin 7 Carbonic anhydrase 9 AMACR CD10

25 Diagnosis Clear Cell Tubulopapillary Renal Cell Carcinoma

26 Similar Tumors in the Literature Renal angiomyoadenomatous tumor (Michal, Diagn Pathol, 2000; Michal, Vichows Arch 2009) 6 cases Epithelial component: CK7 +++, CD10- Leiomyomatous stroma, abortive vessels No VHL gene mutation; no 3p LOH Sporadic clear cell RCC with diffuse CK7 immunoreactivity (Mai, Pathology, 2008) 21 small tumor nodules from 10 patients Distinct tubular, cystic and papillary architecture Cytokeratin but CD10- Clear cell papillary RCC (Gobbo, AJSP, 2008) 7 renal tumors composed mainly of cells with clear cytoplasm arranged in papillary patterns in kidneys unaffected by end-stage renal disease Cytokeratin 7 +++, CD10- No chromosome 7 and loss of Y chromosome, no deletion of 3p

27 Clear Cell Tubulopapillary Renal Cell Ca: A Study of 36 Distinctive Low Grade Tumors Aydin et al (Manuscript submitted) 36 tumors from 33 patients between 1991 and 2009 Cases from represented consecutive cases identified in daily sign-out and accounted for 0.8% (23/3000) of renal tumors Clinical features Gender: Male/female= 17/16 Age: mean (range) = 60.4 (26-88) years 3 patients with bilateral tumors 1 patient with von Hippel-Lindau disease

28 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Gross Findings 82% (23/28) cystic, 18% (5/23) solid A thick fibrous capsule was often present Mean tumor size: 2.4 cm (range ) Six (17%): multi-focal disease (number of tumor nodules: 2 to 6)

29 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Microscopic Findings 64% (23/36) multi-cystic Thick circumferential fibrous capsule in 72% (26/36); partial capsule in 28% (10/36) 61% (22/36) had prominent fibrotic stroma 3% (1/36) had diffuse smooth muscle bundles; 17% (6/36) had focal smooth muscle bundles within the stroma

30 Desmin Desmin

31 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Microscopic Findings Cysts of variable sizes Tubulo-acini Papillae Clear cell nests

32 Cysts 92% (33/36)

33 Tubules/Acini 97% (35/36) Solid tubules 97% (35/36) Branching acini 92% (33/36) Clear cell ribbon 44% (16/36)

34 Papillae: 81% (29/36)

35 Clear cell nests 72% (28/36)

36 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Microscopic Findings Morphologic patterns co-exist within the same tumor 2 tumors had only 1 pattern 17% (6/36) had 2 patterns 6% (2/36) had 3 patterns 72% (26/36) had all 4 patterns

37 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Immunohistochemical Profiles Marker % + Tumor % + Tumor cells (range) CCRCC* PRCC* CK7 100 (36/36) 95 (90-100) - + CD10 19 (7/36) 4 (1-10) + + CA9 92 (33/36) 61 (5-100) + - AMACR TFE * Literature

38 Papillae: 81% (29/36) CK7 CD10

39 Clear cell nests CK7 CA9 CD10

40 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Molecular Features Case # Chromosomal alteration -3p * +* * Low percentage gain von Hippel-Lindau gene Mutation Promoter methylation

41 Clear Cell Tubulopapillary RCC: A Study of 36 Distinct Low Grade Renal Tumors Pathological and Clinical Outcomes Pathological parameters Pathologic stage pt1a: 94% pt1b: 6% No necrosis, sarc diff or microscopic vascular invasion Fuhrman grade: Grade 1: 42% Grade 2: 58% Clinical outcomes Follow up: 60% (20/33) Follow up length: 27.4 months (range 1-85) Follow up outcomes 19/20: no evidence of disease 1/20: alive with disease (VHLD)

42 Clear Cell Tubulopapillary RCC Conclusions Distinct renal tumors with characteristic morphology Small cystic tumor with fibrotic stroma Two characteristic morphologic patterns o Branching glands o Clear cell ribbons Varying cystic, tubulo-acinar and papillary components represent morphological spectrum Immunohistochemical profile CK7: diffusely positive CD10: negative Clinical behavior Benign Low grade malignancy?

43 Case 3 56 year-old female with an incidentally discovered right kidney mass Abdominal CT 7.8 cm cystic mass Numerous septation 1-2 mm Radical nephrectomy

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46 Diagnosis Cystic Nephroma

47 Cystic Nephroma & Mixed Epithelial and Stromal Tumor (WHO blue book 2004) Cystic Nephroma Benign cystic neoplasm composed of epithelial and stromal elements Presents > age 30 Different from pediatric CN (a form of Wilms tumor) Female: male = 8: 1 Well-demarcated entirely cystic without solid area or necrosis Cysts lined with single layer of flat, low cuboidal or hobnail cells Cystic septa: paucicellular or cellular, ovarianlike stroma; may contain mature tubules

48 Cystic Nephroma & Mixed Epithelial and Stromal Tumor (WHO blue book 2004) Mixed Epithelial and Stromal Tumor Complex tumor of a mixture of stromal and epithelial elements Cystic hamartoma of renal pelvis Adult mesoblastic nephroma Female: male = 6: 1 Perimenopausal History of estrogen use Multiple cysts and solid areas Complex architecture of cysts, tubules, and stroma of variable cellularity Stromal cells + for ER, PR

49 Are CN and MEST related lesions? Female gender predilection Similar age distribution Similar morphology Similar immunohistochemical profile Molecular data lacking Turbiner et al: Am J Surg Pathol, 2007 Antic et al: Arch Pathol Lab Med, 2006 Jeveremovic et al: Annals of Diagnostic Pathology, 2006

50 Clinical Features of CN and MEST Clinical Features Median age, years (range) Female: Male Body Mass Index (range) % Obese (BMI>30) Symptomatic Menopausal status: Pre-menopausal Peri-menopausal Post-menopausal Exogenous hormones: Progesterone Tamoxifen Testosterone Personal history of cancer Breast Ovarian Cervical Pancreatic Colon Mean follow-up, years (range) CN (n=21) 55 (39 79) 19: 2 25 (20 45) 25% 16 (76%) 2 (11%) 10 (53%) 7 (37%) 3 (14%) (14%) (1.0 18) MEST (n=9) 52 (39 67) 9: 0 28 (21 42) 29% 6 (67%) 2 (22%) 4 (44%) 3 (33%) 2 (22%) (22%) (1.1 12) p value 0.3 NS NS NS NS NS NS NS No evidence of disease 21 (100%) 9 (100%) NS Lane et al, Urology, 2008

51 Stroma/cyst Ratio & Septal Thickness Zhou, et al, AJSP, 2009

52 Stroma desmin

53 Stroma

54 Cystic Epithelium

55 Complex Branching Glands (Specific for MEST)

56 Pathological Features of CN and MEST Pathologic Features CN (n=26) MEST (n=12) P value Median size, cm (range) 6.7 (1.7 13) 6.9 (2 18) 0.66 % Stroma (range) 28.3% (10-40) 52.5% (30-90) <0.001 Stroma Hypocellular Hypocellular > hypercellular Hypercellular > hypocellular Hypercellular 3/26 (11.5%) 7/26 (26.9%) 16/26 (61.5%) 0/26 (0%) 0/26 (0%) 2/12 (16.7%) 7/12 (58.3%) 3/12 (25.0%) Septal thickness, mm (range) 2.6 (1-5) 7.8 (6-12) <0.001 Complex branching glands 0 / 22 (0%) 5 / 10 (50%) <0.001 Ovarian-type stroma 10 / 26 (38.5%) 8 / 12 (66.7%) Smooth muscle metaplasia 2 / 26 (7.7%) 4 / 12 (33.3%) Prominent vessels in septa 0 / 26 (0%) 4 / 12 (33.4%) Calcification 11 / 26 (42.3%) 1 / 12 (8.3%) Zhou et al, AJSP, 2009

57 CN and MEST Have Overlapping Histological Features Number of Cases :9 2:8 3:7 4:6 5:5 6:4 7:3 8:2 9:1 Stroma/cyst Ratio CN MEST

58 Cystic nephroma MEST Urothelial Ca PRCC, type 2 PRCC, type 1 Chromophobe RCC Clear cell RCC Oncocytoma Normal

59 CN and MEST: Conclusions CN and MEST are related lesions Represent morphological continuum Rename as renal epithelial and stroma tumor (REST) (Turbiner et al, Am J Surg Pathol, April, 2007)

60 Case 4 64 year-old man with work up for prostate cancer Abdominal CT Bilateral renal masses Left: 3 masses, largest 10 cm Right: 2 masses, larger 1.2 cm Right radical nephrectomy

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66 Diagnosis: Hybrid oncocytic epithelial tumor with small focus of chromophobe RCC (5%)

67 Hybrid Oncocytic Renal Tumor Different names Hybrid oncytic tumor (HOT) Hybrid chromophobe RCC Mainly seen in Birt-Hogg-Dube and renal oncocytosis Morphologic spectrum of tumors derived from intercalated cells of collecting ducts Oncocytoma Chromophobe RCC HOT? Clinical outcomes poorly defined 5 sporadic HOT with benign clinical course (follow-up 1-14 years) (Mai et al, Pathol Res Prac, 2002) 16 Chromophobe-oncocytoma hybrid tumors with no recurrence 50 months after resection (Waldert et al, Eur Urol, 2010)

68 Oncocytic Tumors of the Kidney: Histologic Spectrum and Clinical Behavior Aydin et al (USCAP, 2010) 461 renal oncocytic tumors from 425 patients treated with partial or radical nephrectomy between 1981 and 2004

69 Pathological Classification Oncocytoma, classic type Oncocytoma with atypical features (atypical oncocytoma) Focal or diffuse nuclear atypia Perinuclear clearing Clear cytoplasm Extension to perirenal fat Vascular invasion

70 Pathologic Classification Oncoctyic tumor, not otherwise specified (Oncocytic tumor, NOS) Hybrid oncocytic tumor (HOT) ChRCC, eosinophilic type (> 50 % eosinophilic component) ChRCC, classic type

71 Oncocytoma

72 Atypical Oncocytoma (Nuclear atypia)

73 Atypical Oncocytoma (Perinuclear clearing)

74 Atypical Oncocytoma (Clear cells)

75 Atypical Oncocytoma (Extension into fat)

76 Oncocytic Tumor, NOS

77 Hybrid Oncocytic Tumor (HOT)

78 Chromophobe RCC, eosinophilic variant

79 Chromophobe RCC, classical type

80 Clinical Features of Oncocytic Tumors Classification n (%) Age (range) M/F ratio ONCO 207 (44.9) 65.7 (32-90) 157/50 ONCO, atypical 78 (16.9) 68.5 (43-88) 60/18 Oncocytic tumor, NOS 36 (7.8) 63.2 (43-88) 19/17 HOT 27 (5.9) 65.3 (39-88) 20/7 ChRCC-eo 30 (6.5) 59.7 (30-80) 13/17 ChRCC-classic 83 (18) 55.6 (29-83) 48/35

81 Pathology of Oncocytic Tumors Classification n Mean Size (range) Multifocal n (%) Bilateral n (%) ONCO (0.5-16) 35 (16.9) 41 (19.8) ONCO, atypical (1-12.5) 28 (35.9) 26 (33.3) Oncocytic tumor 36 4 ( ) 12 (33.3) 10 (27.8) HOT (2-16) 6 (22.2) 6 (22.2) ChRCC- eo ( ) 4 (13.3) 1 (3.3) ChRCC, classic (1.3-17) 3 (3.6) 5 (6)

82 Clinical Follow-up No evidence of disease by imaging or clinically New disease confined to kidney Ipsilateral Contralateral Bilateral Metastasis / death due to renal tumor Death due to other cause

83 Clinical Follow-up OUTCOME Classification F/U available n (%) F/U length month (range) NEDimaging n (%) NEDclinical n (%) Ipsilateral disease n (%) Contralateral disease n (%) Bilateral disease n (%) Distant metastasis n (%) DOC n (%) ONCO 26 (12.6) 64.5 ( ) 18 (69.2) 3 (11.5) 1 (3.8) (15.4) ONCO, atypical 56 (71.8) 74.2 ( ) 30 (63.6) 13 (23.2) 2 (3.6) 4 (7.2) (12.5) Oncocytic tumor 18 (50) 88.3 (16-228) 10 (55.6) 4 (22.2) 1 (5.6) 0 1 (5.6) 0 2 (11.2) HOT 19 (70.4) 51.4 (0.5-99) 13 (68.4) 3 (15.8) (15.8) ChRCC, eo 21 (70) 44.8 (2-98) 16 (76.2) 1 (4.8) 1 (4.8) 1 (4.8) (4.8) ChRCC, classic 7 (8.4) 54.6 (2-141) 2 (29) 1 (14.3) (29) 2 (29)

84 Renal Oncocytic Tumors : A Morphologic Spectrum of Many Different Tumors Oncocytoma Atypical Oncocytoma Oncocytic Tumor, NOS Hybrid Oncocytic tumor Eosinophilic ChRCC ChRCC

85 Oncocytic Tumors of the Kidney: Clinical Behavior Oncocytoma Benign Atypical Oncocytoma Oncocytic Tumor, NOS Benign or indolent (no mets or death due to diseases) May develop new lesions Hybrid Oncocytic tumor Eosinophilic ChRCC ChRCC Malignant

86 Acknowledgment Hakan Aydin, M.D. Linda Sercia, B.S. Kelly Simmerman, B.S.

87 *Manuscript (include title page, abstract, references, figure legends) Click here to download Manuscript (include title page, abstract, references, figure legends): CCTPRCC7_7_10.doc Clear Cell Tubulopapillary Renal Cell Carcinoma: A Study of 36 Distinctive Low Grade Epithelial Tumors of the Kidney Hakan Aydin 1, Longwen Chen 1, Liang Cheng 3, Susan Vaziri 2, Huiying He 1, Ram Ganapathi 2, Brett Delahunt 4, Cristina Magi-Galluzzi 1, Ming Zhou 1. 1 Pathology and Laboratory Medicine Institute and 2 Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44139, 3 Department of Pathology, Indiana University Medical School, Indianapolis, IN, 4 Department of Pathology and Molecular Medicine, University of Otago, Wellington, New Zealand Key words: kidney, renal neoplasm, clear cell tubulopapillary renal cell carcinoma, low grade neoplasm, cytogenetics, clear cell renal cell carcinoma, papillary renal cell carcinoma Running Title: clear cell tubulopapillary renal cell carcinoma Corresponding Author: Ming Zhou, MD, PhD Pathology and Laboratory Medicine Institute Cleveland Clinic 9500 Euclid Ave/L25 Cleveland, OH, Phone: (216)

88 FAX : (216) zhoum@ccf.org

89 Abstract Recently several low grade renal cell tumors, distinct from those recognized by the 2004 World Health Organization classification of renal tumors, have been described. These tumors had similar clinicopathological features, being low stage tumors with cystic, tubuloacinar and/or papillary architecture. The tumor cells were low grade with variable amounts of clear cytoplasm that was positive for cytokeratin 7 (CK 7) but negative for CD10. Genetic changes characteristic of clear cell or papillary renal cell carcinoma were not seen in these tumors. We investigated the morphological, immunohistochemical and genetic features of 36 additional tumors. Immunohistochemistry was performed for CK 7, carbonic anhydrase 9 (CA 9), alpha-methylacyl-coa racemase (AMACR), CD 10, TFE- 3 and desmin. Interphase fluorescence in situ hybridization was performed with centromeric probes for chromosomes 3, 7, 17, and a sub-telomeric probe for 3p25. Sequencing of von Hippel-Lindau gene and analysis of the methylation status of the promoter region was also performed in two tumors. 36 tumors from 33 patients (mean age: 60.4 years, range: 26-88; 17 men and 16 women) were studied. Three patients had bilateral tumors and one patient had von Hippel-Lindau disease (VHLD). Follow-up was available in 60 % (20/33) of the patients for a mean of 27.4 (range 1-85) months. No patient had evidence of disease after surgery except for the patient with VHLD who was alive with stable disease in the contralateral kidney. All 36 tumors were small (mean size 2.4 cm; range cm) and low stage (pt1). The majority was cystic and had prominent fibrous capsule and stroma. The tumors comprised variable amount of cysts, papillae, tubules, acini and solid nests. The most characteristic histological features were branching tubules and acini and anastomosing clear cell ribbons with low grade nuclei. 3

90 All tumors were strongly positive for CK 7 and variably positive for CA 9, but largely negative for CD 10, and negative for AMACR and TFE-3. All but one tumor had no gains of chromosomes 7 and 17 and deletion of 3p. Only one tumor had low copy number gains of chromosomes 7 and 17. VHL gene mutation and promoter methylation were negative in two tumors analyzed. We show that these tumors, which we term as clear cell tubulopapillary renal cell carcinoma, constitute a unique subtype in the spectrum of renal epithelial neoplasia based on their characteristic morphologic and immunohistochemical features. 4

91 Introduction Recently several examples of low grade renal cell tumors, distinct from those recognized by the 2004 World Health Organization classification of renal tumors, have been described. In 2000 and 2009 Michal et al. described, in two reports, six cases of benign or indolent renal angiomyoadenomatous tumor. These were biphasic tumors with a characteristic epithelial component that exhibited immunopositivity for cytokeratin 7 (CK 7), but not for CD10, and an angioleiomyomatous stroma that was HMB45 negative. No mutations in the von Hippel-Lindau (VHL) gene, nor loss of heterozygocity involving chromosome 3p, was found in these tumors. 23,24 Tickoo et al., in their study of the epithelial neoplasms in the end-stage renal diseases, described 15 clear-cell papillary renal cell carcinoma of the end -stage kidneys, which were predominantly cystic tumors and showed prominent papillary architecture with purely clear-cell cytology. 27 Gobbo et al. later reported seven clear cell papillary renal cell carcinomas composed mainly of cells with clear cytoplasm arranged in papillary patterns in kidneys unaffected by end-stage renal disease. 10 All tumors showed strong positive staining for CK 7, but were negative for CD10. None had gains of chromosome 7 or loss of Y chromosome, typical of papillary renal cell carcinoma (PRCC), and none had deletion of 3p, a finding seen in clear cell renal cell carcinoma (CCRCC). Mai et al. described 21 small tumors from ten patients that had a distinct tubular, cystic and papillary architecture. These showed diffuse CK 7 reactivity, but were negative for CD10 and for this reason the designation sporadic clear cell renal cell carcinoma with diffuse cytokeratin 7 immunoreactivity was applied. 21 5

92 Although bearing different names, the renal tumors reported in these five studies had similar clinicopathological features, being low stage tumors with cystic, tubuloacinar and/or papillary architecture. The tumor cells were low grade with variable amounts of clear cytoplasm that was positive for CK 7 but negative for CD10. Genetic changes characteristic of CCRCC or PRCC were not seen in these tumors. In this study we have investigated the morphological, immunohistochemical and genetic features of 36 additional renal tumors that are similar to those described in the earlier studies, and which appear to constitute a novel form of low grade renal malignancy. We propose the term clear cell tubulopapillary renal cell carcinoma (CCTP-RCC) for these tumors to emphasize their dominant morphological features. This new terminology would unify this novel renal tumor that was reported under several different names in the literature and clarify confusions resulting from the polynomial nature of this tumor. Material and Methods Pathology review Tumors accessioned between 1991 and 2009 were identified from the senior authors surgical pathology files. Demographic and clinical information was collected according to protocols approved by the authors institutional review boards. The gross appearance of the tumors was retrieved from the pathology database. All the cases were reviewed in order to confirm the microscopic features. Immunohistochemistry One representative block from each case was cut at 5 thickness and stained for; CK 7 (1:40 dilution, Dako, Carpinteria, CA), racemase (AMACR) (1:100 dilution, Zeta, 6

93 Sierra Madre, CA), CD10 (1:5 dilution, Novacastra, Newcastle upon Tyne, UK), carbonic anhydrase 9 (CA9) (1:5000 dilution, Novus Biologicals, Littleton, CO), TFE3 (1:5 dilution, Abcam, Cambridge, MA) and desmin (1:10 dilution, Dako, Carpinteria, CA). Briefly, antigen retrieval was undertaken according to the specifications of the manufacturers of the primary antibodies. The slides were then incubated sequentially with primary antibody, biotinylated secondary antibody, avidin-peroxidase complex (Ventana, Tucson, AZ) and diaminobenzidine. The staining protocol for TFE3 was modified from Argani et al s method. 2 Immunostaining was performed using a Ventana Benchmark automatic stainer (Ventana, Tucson, AZ). Fluorescence in situ hybridization (FISH) For each case, a representative section was selected and a further section was cut from the paraffin embedded block at 4 thickness. The section was baked at 65 +/- 5 C overnight or for a minimum of 5 hours, de-paraffinized in xylene, washed in absolute alcohol and air dried. Pepsin was used to digest the tissue at 37 o C for 40 minutes. FISH was performed with a UroVysion bladder cancer recurrence kit that contained the centromeric probes for chromosome 7 (CEP7, spectrum green) and chromosome 17 (CEP17, spectrum aqua) (Abbott Molecular/Vysis Cat. No ). Probes for chromosome 3 (CEP3, Spectrum orange) and subtelomeric probe for 3p25 (3pTel25, Spectrum green) were also used. The slide was cover-slipped and sealed with rubber cement. Co-denaturation was performed at a melt temperature of 73 C for 5 minutes by placing the slide in Hybrite. Hybridization was carried out in a 37 C humidified C incubator overnight (12-18 hours). The coverslip was then removed and the slides were washed in 1 X SSC/0.3% NP-40 followed by wash in 2 X SSC/0.1% NP-40 at room 7

94 temperature. Slides were then air dried completely in darkness µl Vectashield with DAPI counterstain was applied to the target area of the slide. We also performed FISH assay using the same probes (CEP7, 17, 3 and 3pTel25) on sections of CCRCC and PRCC as positive controls. FISH analysis The FISH data were analyzed using methods described previously. 10,33 The corresponding H&E stained slides were reviewed before FISH study and areas of tumor, as well as normal renal tubules, were marked. For each case, 100 nuclei from normal and tumor areas were examined for signals from probes under fluorescence microscopy at 1000x magnification. Normal renal tubules were used as a control. Definitions of chromosomal gain and loss of chromosomes 7, 17 and Y were based on the Gaussian model and related to the non-neoplastic controls. Any tumor with a signal score beyond the cutoff value of Mean + 3 SD of control tissue was considered to have a gain or loss of the specific chromosome. The statistical method to analyze 3p deletion was based on previous studies. 9,10 Analysis of VHL gene sequence and promoter methylation status For VHL gene sequence analysis, PCR-based amplification of each of the 3 exons was performed as previously described, 14 using the following VHL primers sets; exon1 forward, 5 -TGGAGGATCCTTCTGCGCAC-3 ; exon 1 reverse; 5 - GGCTTCAGACCGTGCTATCG-3 ; exon 2 forward, 5- GGCTCTTTAACAACCTTTGC-3 ; exon 2 reverse, 5 - TTGGATACCGTGCCTGACATC-3 ; exon 3 forward, 5 - ACAGGTAGTTGTTGGCAAAGCC-3 ; exon 3 reverse, 5-8

95 GAAGGAACCAGTCCTGTATC-3. The amplicons were sequenced using an ABI377 automated sequencer (Applied Biosystems, Foster City, CA). Sequences derived from the amplified samples were compared to the wild-type VHL sequence (GeneBank Accession No. AF010238) using LaserGene software (DNAStar, Perkin Elmer, Foster City, CA) to identify and characterize mutations. The methylation status of the VHL gene promoter was determined using VHL methylation-specific PCR primers after DNA bisulfite modification. Genomic DNA was modified using the CpGenomeTM DNA modification kit according to the manufacturer s protocol (Chemicon International, Temecula, CA). The product then underwent PCRbased amplification using methylation-specific primers. The methylation status was determined by gel electrophoresis of the PCR products as previously described. 13 Results Thirty-six tumors from thirty-three patients were identified from the authors surgical pathology files that had been accessioned between 1991 and Cases from represented consecutive cases identified in daily sign-out and the tumors identified represented 0.8% (23/3000) of all adult renal tumors diagnosed during this period. Thirty-six tumors from thirty-three patients were identified from the authors surgical pathology files that had been accessioned between 1991 and Cases from represented consecutive cases identified in daily sign-out and the tumors identified represented 0.8% (23/3000) of all adult renal tumors diagnosed during this period. 9

96 Clinical features and outcomes There were 17 men and 16 women and the mean age was 60 years (range years). Three patients had bilateral tumors. One patient, a 26-year old female, had a history of von Hippel-Lindau disease (VHLD). None had a history of tuberous sclerosis. The surgical treatment was radical/total nephrectomy for ten tumors and partial nephrectomy for 26 tumors. Follow-up was available for 60% (20/33) of patients, with a mean follow-up interval of 27.4 months (range 1-85 months). Nineteen patients had no evidence of disease during this follow-up period. The patient with VHLD was alive with stable disease in the contralateral kidney 15 months after partial nephrectomy. Pathological findings Gross findings Of the 28 tumors whose gross features were described, 23 (82%) were cystic, and 5 (18%) were solid. The mean tumor size was 2.4 cm (range cm). Six (17%) tumors were multifocal, with the number of grossly identifiable tumor nodules ranging from 2 to 6. Microscopic findings Sixty-four percent of tumors (23/36) were multi-cystic. A thick circumferential capsule was present in 72% (26/36) (Fig. 1A), and a partial capsule was present in the remaining 28% (10/36) of tumors. There was a prominent fibrotic stroma with embedded neoplastic cells in 61% of tumors (22/36) (Fig. 1B). Immunostaining for desmin showed 4 tumors (11%) to have aggregates of circumferential smooth muscle fibers (Fig. 1C). Focal smooth muscle fibers were seen in 25 (69%) tumors, while in 7 (19%) tumors no smooth muscle fibers were seen in the tumor capsule. One tumor had fascicles of smooth muscle 10

97 diffusely scattered throughout (Fig. 1D), while a further six (17%) had smooth muscle fibers present focally within the stroma. The tumors demonstrated several morphological patterns and these were found to frequently co-exist within the same tumor. In 92% (33/36) of tumors, cysts of different sizes were present and often contained serosanguineous fluid or colloid-like secretion (Fig. 2A). Short papillae arose from the cyst wall in some cases (Fig. 2B) and both the cyst wall and papillae were lined by a single layer of cuboidal cells, with scant eosinophilic cytoplasm or a moderate amount of clear cytoplasm (Fig. 2B). In 97% (35/36) of tumors tubules and acini of variable sizes and shape were present. These were lined with a single layer of cells that had scant eosinophilic cytoplasm or moderate amount of clear cytoplasm. Some tubules and acini were small with ill-formed lumens and were compressed to impart a solid appearance (Fig. 3A). More frequently tubules and acini were well-formed with intraluminal proteinaceous secretion (Fig. 3B). Occasionally the tubules and acini had branching morphology and focally formed small papillae (Fig. 3C) or formed long interconnecting ribbons that were lined with clear cells (Fig. 3D). The nuclei were often not basally oriented; rather they were oriented away from the basement membrane and towards the apical surface of the tubules, acini and papillae (Figure 3E). Branching acini (Figure 3C) and clear cell ribbons (Figure 3D) were present in 92% (33/36) and 44% (16/36) of the tumors respectively, while 94% (34/36) tumors had either feature and 42% (15/36) had both features. Papillae were present in 81% (29/36) of the tumors. One tumor was entirely papillary and consisted of thick papillae in one area (Fig. 4A) and arborizing, thin and delicate papillae in other areas (Fig. 4B). The papillae were lined with single layer of 11

98 cells with small amount of eosinophilic or clear cytoplasm (Fig. 4C). In the majority of cases, papillae were a minor component of the tumor that arose within cysts (Fig. 2B). No foamy histiocytes, psammomatous calcification or hemosiderin was present in the papillary areas. Clear cell nests that were more than 2 cells thick and without well-formed lumens similar to the cells seen in CCRCC (Fig. 5A), were present in 58% (21/36) tumors. These usually formed a minor component, constituting an average of 8% (range 0-50%) of tumor tissue. The distribution of the architectural patterns within the tumors is shown in Table 1. Two tumors contained only one pattern, one with only papillae and the other with tubules. A further 11% (4/36) had two patterns, 39% (14/36) had three patterns, and 62% (16/36) had all four patterns. Three patients had a concomitant renal cell carcinoma of a different histological type within the same nephrectomy specimen. One patient had a CCRCC with leiomyomatous stroma, while a second patient had a CCRCC. The patient with VHLD had 3 tumors in her right kidney, one of which was CCRCC (Fig. 6A-D). Pathologic stage and grade The pathologic stage was pt1a in 94% (34/36) and pt1b in 6% (2/36) tumors. Forty-two percent (15/36) of tumors were Fuhrman nuclear grade 1, while 58% (21/36) were nuclear grade 2. No coagulative necrosis, sarcomatoid de-differentiation nor microscopic vascular invasion was present. Changes of end-stage kidney, including glomerulosclerosis and tubular atrophy, were present in 9% (3/33) of kidneys. Immunophenotype 12

99 CK 7 expression was positive in all 36 tumors, with cysts, tubules/acini and papillae all being strongly and diffusely positive (Figs. 4D, 5B and 6C). Clear cell nests were also positive for CK7 in all of the 21 tumors in which clear cell nests were identified. However, the staining intensity in clear cell nests was weaker than that of the other epithelial components in 43% (9/21) tumors (Table 2 and Fig. 5C) and in two tumors only 60% and 95% of cells in clear cell nests were positive for CK7. CA9 was positive in 94% (34/36) of tumors (Fig. 5C), with a mean of 51% (range 1-100%) positive tumor cells. CA9 was positive in clear cell nests of only 38% (8/21) of the tumors (Fig. 5D). Most tumors were negative for CD10 (Figs. 4E and 5D), being positive in only 17% (6/36) of tumors examined. Where positive, CD10 expression was limited to 1-10% of tumor cells. CD10 was positive in clear cell nests in only one of the 21 tumors and in these cells there was 100% positivity. AMACR and TFE3 were negative in all the tumors. Molecular characteristics. Eleven cases selected for molecular studies were representative cases that did not significantly differ from those that were not examined in their clinical, pathological and immunohistochemical characteristics (Tables 1-3). FISH was used to enumerate the copy numbers of chromosomes 7, 17 and 3p in 10 tumors. Of these tumors only one had low copy number gain of chromosomes 7 and 17 (Table 1, tumor No. 12). This tumor showed chromosome 7 gain in 12%, and chromosome 17 gain in 6% of tumor cells and the mean + 3S.D. for chromosome 7 and 17 gain in non-neoplastic tissue was 7.5% and 3.2%, respectively. This tumor was morphologically similar to other tumors included in this study. As shown in Tables 1 and 2, this tumor comprised 75% cysts, 5% tubuloacini and 13

100 % papillae and was positive for cytokeratin 7 and CA9 but negative for CD10. For PRCC controls, the chromosomes 7 and 17 gain was 82% and 69%, respectively, in 20 tumors. Chromosome 3p deletion was not detected in any tumor. The VHL gene was sequenced and the methylation status of the VHL promoter region was analyzed by the methylation-specific PCR in two tumors. No mutation or promoter methylation was detected (Table 1). 14

101 Discussion We reported 36 renal epithelial tumors with distinctive pathological features that we term clear cell tubulopapillary renal cell carcinoma (CCTP-RCC). We believe CCTP-RCC to represent a unique subtype of renal parenchymal neoplasia that is distinct from other renal tumors. Microscopically, the presence of branching tubules/acini and/or complex clear cell ribbons in the cysts with fibrotic stroma is the key to the identification of these tumors, while the constant finding of positive CK 7 and negative CD10 immunoexpression is also an important diagnostic feature. Tumors with similar morphological and immunohistochemical features have been reported. Michal et al. described six cases of renal angiomyoadenomatous tumor which appear to be identical to CCTP-RCC reported in this study 23,24 In their studies the authors describe abortive vascular structures in the tumors, although no detailed description or illustration was provided. Contrary to this we did not observe any significant vascular component to our tumors. In addition, in Michel s report, emphasis was placed on the leiomyomatous stroma present within the tumors. 24 They used several antibodies, including caldesmon, calponin, vimentin, and smooth muscle actin, to highlight the leiomyomatous component. Although caldesmon is more specific for smooth muscle, smooth muscle actin also stains myofibroblasts within the stroma. Desmin, on the other hand, a marker more sensitive than caldesmon, only showed patchy and weak staining in their cases. We used the immunostain for desmin and found that diffuse smooth muscle fascicles within the tumor in only minority of our cases. Therefore, leiomyomatous stroma is not a conspicuous component of majority of CCTP-RCC. 15

102 Recently Tickoo et al. described 15 clear-cell papillary renal cell carcinoma of the end-stage kidneys, which were predominantly cystic tumors and showed prominent papillary architecture with purely clear-cell cytology. 27 Seven cases of clear cell papillary renal cell carcinomas were later reported by Gobbo et al., majority of which occurred in kidneys unaffected by end-stage renal diseases. 10 These were low grade and low stage renal tumors, composed mainly of cells with clear cytoplasm arranged in papillary patterns. All tumors showed strong positive staining for CK7, but were negative for CD10. None had gains of chromosome 7 or 17, loss of Y chromosome or deletion of 3p. While the papillary structures lined with a single layer of clear cells were identical to the papillary patterns seen in our tumors it is uncertain as to whether the papillary component was the only component of their tumors. Our tumors showed a spectrum of morphology and papillary structures, cysts, glands/acini and clear cell nests were frequently present within the same tumor. In only one tumor did we find an entirely papillary architecture. Mai et al. studied 21 small tumor nodules that contained clear tumor cells that were diffusely positive for CK7. 21 Architecturally, the CK7-positive tumor cells consisted of cysts and solid cell nests with tubuloacinar or papillary formations. Although the immunohistochemical findings from this series were limited, we believe that these tumors were clear cell tubulopapillary renal cell carcinomas. The current study contributed to our understanding of this unique renal tumor in three aspects. First, it expanded the morphological spectrum of renal clear cell tubulopapillary renal cell carcinomas and documented the characteristic morphological features of this tumor. Although this tumor can have a plethora of morphological patterns, 16

103 including cysts, tubules, acini, papillae and clear cell nests, branching tubules and anastomosing clear cell ribbons, however, are most characteristic. Second, it provides the molecular and genetic data on 11 additional tumors and establishes that these tumors do not have the genetic changes that are characteristic of CCRCC and PRCC. Third, we proposed a unifying term, clear cell tubulopapillary renal cell carcinoma, based on their dominant morphological features, for these tumors that shared similar morphological and immunohistochemical features but bore different names due to minor morphological variations. The clinical, pathological, immunohistochemical and molecular characteristics of 82 clear cell tubulopapillary renal cell carcinomas reported in the literature and this report are summarized in Table 4 and provide a foundation for future studies of this unique renal tumor. The overwhelming majority (80 of 82) of the clear cell tubulopapillary renal cell carcinomas described to date, including 36 cases in the present study, and 46 previously reported cases for which data were available, has been categorized as pathologic stage category pt1, and remaining two tumors were pt2. 10,21,23,24,27 All have exhibited low grade nuclear features (Fuhrman grade 1 or 2). Evidence of biological aggressiveness, such as perirenal or renal sinus invasion, vascular invasion, tumor necrosis and sarcomatoid de-differentiation, has not been observed in these tumors. Follow-up of 40 patients, including 19 in this study, showed no evidence of disease after a mean follow-up period of 28 months. The one patient with tumor recurrence in our series had VHLD and was diagnosed with stable disease in the contralateral kidney, 15 months after initial partial nephrectomy. These observations suggest that clear cell tubulopapillary renal cell 17

104 carcinomas may be biologically indolent tumors; however, further follow-up studies are needed to better define their clinical behavior. Patients may harbor multifocal renal tumors that include CCTP-RCC and other types of RCC. One patient in this study had a CCTP-RCC and a concomitant CCRCC with leiomyomatous stroma. Two other patients had CCTP-RCC with co-existing CCRCC. In those patients, clinical outcome is likely to be driven by the behavior of other renal tumors rather than that of CCTP-RCC. Several other renal tumors, including conventional CCRCC, CCRCC with leiomyomatous stroma, multilocular cystic renal cell carcinoma (MLCRCC), PRCC, and mixed epithelial and stroma tumor (MEST), have morphological features that resemble CCTP-RCC to some extent. CCRCC and PRCC are most likely to be mistaken for CCTP-RCC. Unlike PRCC these tumors have a clear cytoplasm and tumor necrosis, aggregates of foamy macrophages, psammomatous calcification and hemosiderin deposition are not observed. The presence of the clear cells lining the cysts, tubules and papillae may also raise a suspicion for CCRCC. However, these tumors lack the delicate sinusoidal vascular network typical of CCRCC and the branched tubules and complex clear cell ribbons of clear cell tubulopapillary renal cell carcinoma are not found in CCRCC. The immunophenotype of this tumor is distinct from CCRCC and PRCC. CCRCC is positive for CA9 and CD10 4,29,32 but negative for CK7 and AMACR, 16,28 while PRCC is positive for CK7, CD10 and AMACR, 8,28,32 but is usually negative for CA9. 22 All of our cases of CCTP-RCC showed strong and diffuse staining for CK7, variable staining for CA9, and negative staining for AMACR. In addition, the CD10 immunoexpression 18

105 was negative in over 80% of cases and was only focally positive in the remaining cases. Furthermore, staining for TFE3 protein, a distinctive and diagnostic feature of Xp11.2 translocation RCC, 1,3 was not identified in any of our tumors. The genetic findings in our tumors also differ from those of CCRCC and PRCC. CCRCC shows a highly specific deletion of chromosome 3p, 6 and. mutations in VHL gene and methylation of the gene promoter have been detected in the vast majority of sporadic CCRCC. 7,15 Most of the CCRCC that we used as controls showed deletion of the subtelomeric locus 3p25, whereas in our control cases of PRCC, deletion of 3p was not observed. Gains of chromosomes 7 and 17, on the other hand, are commonly identified in PRCC 5 and our PRCC controls all demonstrated these gains. In contrast to this, 3p deletion was not observed in any of our CCTP-RCCs. Two tumors whose entire VHL gene was sequenced and the promoter region was analyzed with methylationspecific PCR had no such alterations. Furthermore, gains of chromosomes 7 and 17 were not present in nine of our ten tumors tested, while the remaining tumor showed low copy number gains of chromosomes 7 and 17. CCTP-RCC should also be distinguished from rare cases of renal cell carcinomas with papillary architecture and variable proportion of cells with clear cytoplasm. 11,20 These cases represent conventional CCRCC with secondary papillary formation, PRCC with clear cell changes, composite CCRCC and PRCC or unclassified RCC with both clear cell and papillary components. Although superficially resembling CCTP-RCC, they are easily distinguishable from the former as they often exhibit pathological features of aggressive RCC, including Fuhrman grade 3 and 4 nuclei, coagulative necrosis, vascular invasion, lymph node metastasis and sarcomatoid differentiation, 20 none of which is seen 19

106 in CCTP-RCC. In addition, none of these tumors were CK7 positive and AMACR negative 11,20, while all CCTP-RCC were CK7 positive and AMACR negative. Furthermore, except for one case, 11 these tumors had cytogenetic changes characteristic of either CCRCC, PRCC or both. 11,20 CCRCC with leiomyomatous stroma was recently described as tumors composed of nests of clear cells similar to those seen in CCRCC. 17,25 The neoplastic epithelial cells of these tumors are diffusely positive for CK7 and are also positive for CD10. Additionally, genetic studies of these have shown changes typical of CCRCC 25, and as such, the morphologic, immunohistochemical and genetic features of CCRCC with leiomyomatous stroma appears to differ significantly from those of CCTP-RCC. Multilocular cystic renal cell carcinoma (MLCRCC) is a variant of CCRCC characterized by multilocular cystic pattern where the cysts are lined by clear ells with low nuclear grade. 12,26. The majority of our CCTP-RCC was cystic and some had multilocular cystic component and were lined with clear cells. It is reasonable to consider and rule out MLCRCC. However, CCTP-RCC also has other components such as nests of tubules, acini and papillae that are not seen in MLCRCC and does not harbor chromosome 3p alterations as MLCRCC does. 12. Since CCTP-RCC also has a prominent fibrous stroma in addition to the epithelial components, distinction from mixed epithelial and stroma tumor (MEST) is also warranted. MEST most commonly affects young and middle-aged patients with a striking female predominance 18,31. Clinically our patients were older with no gender predilection. Ovarian-type stroma is seen in majority of MESTs 31 but is not seen in any CCTP-RCC. 20

107 Recognition of CCTP-RCC as a distinctive form of renal neoplasia may have important implications for the management of small renal tumors. Small tumors in poor surgical candidates and benign or low grade renal tumors diagnosed on renal biopsy are increasingly being managed conservatively by active surveillance 19. CCTP-RCC often contains clear cells that resemble CCRCC and may be mistaken for the latter in small needle biopsies. As a consequence CCTP-RCC should be included in the differential diagnosis of small renal tumors with clear cells and prudent use of immunostains for CK7, CD10, AMACR and CA9 should lead to a correct diagnosis. An intriguing question arises as to whether CCTP-RCC represents a precursor lesion to CCRCC, as it often contains clear cells in the form of complex clear cell ribbons and solid clear cell nests. One of the patients in our study had VHLD and had CCTP- RCC in addition to CCRCC. This may be construed to suggest that CCTP-RCC is the precursor lesion to CCRCC. However, our study has shown that CCTP-RCC and CCRCC do not share immunohistochemical and molecular features. These findings do not lend support for a precursor role of CCTP-RCC and the occurrence of this tumor in the patient with VHLD may reflect the fact that clear cell tubulopapillary renal cell carcinoma often occurs in renal cysts, which are dominant manifestation of VHLD. 30 In summary, we report a series of low grade and stage clear cell tubulopapillary renal cell carcinomas. They constitute a unique subtype in the spectrum of renal epithelial neoplasia based on their morphologic and immunohistochemical features that are distinct from other renal epithelial tumors recognized in the latest WHO classification. Their recognition is critical for diagnosis, prognosis and clinical management. 21

108 References 1. Argani P, Ladanyi M: Translocation carcinomas of the kidney. Clin Lab Med 2005, 25: Argani P, Lal P, Hutchinson B, et al.: Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay. Am J Surg Pathol 2003, 27: Argani P, Olgac S, Tickoo SK, et al.: Xp11 translocation renal cell carcinoma in adults: expanded clinical, pathologic, and genetic spectrum. Am J Surg Pathol 2007, 31: Avery AK, Beckstead J, Renshaw AA, et al.: Use of antibodies to RCC and CD10 in the differential diagnosis of renal neoplasms. Am J Surg Pathol 2000, 24: Brunelli M, Eble JN, Zhang S, et al.: Gains of chromosomes 7, 17, 12, 16, and 20 and loss of Y occur early in the evolution of papillary renal cell neoplasia: a fluorescent in situ hybridization study. Mod Pathol 2003, 16: Cheng L, Zhang S, MacLennan GT, et al.: Molecular and cytogenetic insights into the pathogenesis, classification, differential diagnosis, and prognosis of renal epithelial neoplasms. Hum Pathol 2009, 40: Cowey CL, Rathmell WK: VHL gene mutations in renal cell carcinoma: role as a biomarker of disease outcome and drug efficacy. Curr Oncol Rep 2009, 11: Delahunt B, Eble JN: Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol 1997, 10:

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113 Figure Legends Figure 1. Clear cell tubulopapillary renal cell carcinoma with a thick circumferential capsule (A). Some tumors had tumor cells embedded in a prominent fibrotic stroma that may show myxoid areas (B). Immunostains for desmin highlighted circumferential smooth muscle fibers in the tumor capsule (C) and within the stroma (D) in some tumors. Figure 2. Clear cell tubulopapillary renal cell carcinoma with cysts containing serosanguineous fluid or colloid-like secretion (A). Short blunt papillae arose from the cyst wall in some cases (2B). The cystic wall and papillae were lined with single layer of cuboidal cells with scant eosinophilic cytoplasm or moderate amount of clear cytoplasm. Figure 3. Tubules and acini in clear cell tubulopapillary renal cell carcinoma. Some tubules and acini were small with compressed lumens to impart a solid appearance (A). Some were well-formed and had pink intraluminal secretions (B). Branching pattern with papillary formations (C). In some cases, the tubules and acini formed complex interconnecting ribbons that were lined with clear cells (D). These tubules, acini and ribbons were lined with single layer of cells that had scant eosinophilic cytoplasm or moderate amount of clear cytoplasm. The nuclei were oriented away from the basement membrane and towards the apical surface of tubules (E). Figure 4. Clear cell tubulopapillary renal cell carcinoma composed exclusively of papillary architecture. Papillary structures with thick fibrotic stalk were seen in one area (A). Thin and arborizing papillae were seen in other areas of the same tumor (B). The 27

114 papillae were lined with single layer of cells with small amount of eosinophilic or clear cytoplasm (C). Immunohistochemical stains show that tumor cells are strongly and diffusely positive for CK7 (D) and negative for CD10 (E). Figure 5. Clear cell nests in clear cell tubulopapillary renal cell carcinoma. Tumor cells with clear cytoplasm formed nests that were more than 2-cell thick and did not have wellformed lumens (A). The adjacent branching glands and tubules were lined with single layer of clear cells. A stain for CD31, a marker for vascular endothelial cells, highlighted the delicate capillary network that separate clear cells into nests (B). A stain for CK7 stained tubules, glands and clear cell nests. However, the staining intensity in clear cell nests was weaker than that in tubules and glands (C). While tubules and glands were positive for CA 9, the clear cell nests were negative (D). CD10 was negative in the tumor cells (E). Figure 6. Concomitant clear cell tubulopapillary renal cell carcinoma and conventional clear cell renal cell carcinoma in a patient with von Hippel-Lindau disease. This patient had 3 tumor nodules in her right kidney. Two were clear cell tubulopapillary renal cell carcinoma (A) and the 3 rd tumor was a clear cell renal cell carcinoma (B). CK7 was positive in clear cell tubulopapillary renal cell carcinoma (C) and negative in the clear cell renal cell carcinoma (D). 28

115 Tables1-4 Tumor # Table 1. Morphological Characteristics of 36 Clear Cell Tubulopapillary Renal Cell Carcinomas Morphologic pattern (% of tumor mass) Cyst Tubulo-acini Papillae Clear cell nest

116

117 Table 2: Immunohistochemical Profiles of Clear Cell Tubulopapillary Renal Cell Carcinomas Tumor Immunohistochemical Profile (% positive cells) CK7 CA9 CD10 Overall Cyst Tubuloacini Papillae Clear cell Overall Cyst Tubuloacini Papillae Clear cell Overall Cyst Tubuloacini nest nest * * N.A N.A N.A * * N.A. N.A. 100 N.A. 10 N.A. N.A. 10 N.A. 0 N.A. N.A. 0 N.A N.A N.A N.A N.A N.A N.A N.A N.A N.A * N.A. N.A N.A. N.A N.A. N.A N.A. 100 N.A. N.A. 100 N.A. 100 N.A. N.A. 0 0 N.A. N.A N.A N.A N.A. N.A N.A. N.A N.A. N.A N.A N.A N.A. N.A * N.A. 60* N.A N.A N.A Papillae Clear cell nest

118 N.A N.A N.A. 100 N.A N.A. 100 N.A N.A. N.A. N.A. 100* 0 N.A. 0 N.A N.A N.A N.A N.A N.A * *The staining intensity in clear cell nests was weaker than in other components. N.A. - the morphological component not present in the tumor. Tumor # Table 3. Molecular Characteristics of 11 Clear Cell Tubulopapillary Renal Cell Carcinomas Chromosomal alteration VHL gene Cyst -3p Mutation Promoter methylation Table 4: Clinical, immunohistochemical and molecular characteristics of clear cell tubulopapillary renal cell carcinomas reported in the literature Case Gender Age Stage ESRD Follow-up Immunohistochemistry Chromosomal alteration VHL gene Reference No^*. Duration (months) Outcome #* CK7 CD10 CA9 AMACR TFE3 7 gain 17 gain 3p loss Mutation Promoter methylation 1 M 58 1a - 9 NED + * + N.A. N.A. N.A. N.A. - - N.A. 24

119 2 M 49 1b - 12 NED + * + N.A. N.A. N.A. N.A. - - N.A. 3 M NED + * + N.A. N.A. N.A. N.A. - - N.A. 4 F 73 1a - 29 DOOC + * + N.A. N.A. N.A. N.A. - - N.A. 5 M 50 1a - N.A. N.A. + * + N.A. N.A. N.A. N.A. N.A. N.A. N.A. 6 M NED N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A M 50 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A F 35 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 9 M 50 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 10 F 56 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 11 F 57 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 12 M 78 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 13 M 60 1a + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 14 F 50 1b + N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 15 F N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 16 M N.A. N.A. # N.A. N.A. ^ N.A. N.A. N.A. N.A. N.A. N.A. 17 F 64 1a - 24 NED N.A. N.A M 63 1a - 26 NED N.A. N.A. 19 M 64 1b - 1 NED N.A. N.A. 20 M 55 1b - 48 NED + + ( N.A. N.A. 25%) 21a F 53 1a + 22 NED N.A. N.A. 21b 1a N.A. N.A. 21c 1a N.A. N.A. 24 M 60 1a - ** No Met + + N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 21 (<5%) 25 F 72 1a - ** No Met + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 26a M 56 1a - ** No Met + - N.A. + (5- N.A. N.A. N.A. N.A. N.A. N.A. 30%) 26b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 27a M 73 1a - ** No Met + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 27b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 28a F 65 1a - ** No Met + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 28b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 29a M 29 1a - ** No Met + + N.A. - N.A. N.A. N.A. N.A. N.A. N.A. (<5%) 29b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 29c 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 29d 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A.

120 30a M 56 1a - ** No Met + + N.A. - N.A. N.A. N.A. N.A. N.A. N.A. (<5%) 30b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 31a M 53 1a - ** No Met + + (5- N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 30%) 31b 1a + - N.A. + (<5%) N.A. N.A. N.A. N.A. N.A. N.A. 31c 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 31d 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 32 M 65 1a - ** No Met + + N.A. - N.A. N.A. N.A. N.A. N.A. N.A. (<5%) 33a M 57 1a - ** No Met + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 33b 1a + - N.A. - N.A. N.A. N.A. N.A. N.A. N.A. 34 M 62 1a N.A. N.A. N.A. N.A. N.A. Current study 35 F 67 1a - 12 NED + + (5%) N.A. N.A. N.A. N.A. N.A. 36 F 38 1a - 48 NED N.A. N.A. N.A. N.A. N.A. 37 M 54 1a - 85 NED N.A. N.A. N.A M 57 1a - 77 NED N.A. N.A. N.A. N.A. N.A. 39 F 44 1a - 81 NED N.A. N.A. N.A. N.A. N.A. - (10%) (1%) 40 M 56 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. 41 M 51 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. 42 M 61 1a - N.A. N.A F 67 1b - N.A. N.A N.A. N.A. 44 F 72 1a - 41 NED N.A. N.A. 45a F 61 1a - N.A. N.A N.A. N.A. 45b 1a F 57 1a - 30 NED N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. (5%) 47 F 60 1a + 33 NED N.A. N.A. 48a M 78 1b - 1 NED N.A. N.A. 48b 1a N.A. N.A. N.A. N.A. N.A. 49 M 59 1a - 21 NED N.A. N.A. 50 F 68 1a - 14 NED N.A. N.A. 51 M 79 1a - 23 NED N.A. N.A. N.A. N.A. N.A.

121 52 M 60 1a - N.A. N.A N.A. N.A. 53 M 59 1a - 22 NED N.A. N.A. 54 M 41 1a - 5 NED N.A. N.A. N.A. N.A. N.A. 55 F 72 1b - N.A. N.A N.A. N.A. N.A. N.A. N.A. 56 F 47 1a - 10 NED N.A. N.A. N.A. N.A. N.A. 57 F 26 1a - 15 AWD N.A. N.A. N.A. N.A. N.A. 58 M 53 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. 59 M 71 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. (5%) (5%) 60 M 78 1b NED N.A. N.A. N.A. N.A. N.A. (10%) 61 M 52 1a + N.A. N.A N.A. N.A. N.A. N.A. N.A. 62 M 68 1a - 3 NED N.A. N.A. N.A. N.A. N.A. 63a F 49 1a - 1 NED N.A. N.A. N.A. N.A. N.A. 63b N.A. N.A. N.A. N.A. N.A. 64 F 61 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. (1%) 65 F 79 1a - 83 NED F 88 1a - N.A. N.A N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. (5%) * reported as focally positive in 1, and negative in 4 of 5 cases. # 4 of 4 cases were positive for CK7 ^ 4 of 4 cases were negative for AMACR ** Follow-up duration months #* Follow-up outcomes: NED - no evidence of disease; AWD alive with disease; DOOC dead of other cause; No Met no metastasis ^* same number with different suffixes denotes same patient with multiple or bilateral tumors Abbreviations: M: male; F: female; ESRD: end stage renal disease; N.A.: not available or not performed; +: positive; -: negative; VHL: von Hippel Lindau

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