Renal tumours: use of immunohistochemistry & molecular pathology. Dr Lisa Browning John Radcliffe Hospital Oxford

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1 Renal tumours: use of immunohistochemistry & molecular pathology Dr Lisa Browning John Radcliffe Hospital Oxford

2 Renal tumours: the use of immunohistochemistry & molecular pathology Classification of RCC important for prognosis and management IHC & molecular pathology useful for classification WHO 2016 classification incorporates some tumours defined by molecular pathology or IHC profile Renal tumours may have overlapping morphological features, especially when high grade?future use of IHC +/- molecular pathology to aide better prognostication and prediction of response to systemic therapies

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4 Renal tumours: the use of immunohistochemistry & molecular pathology Overview of immunohistochemistry for diagnosis Including biopsies Overview of molecular pathology and renal tumours Molecular pathology and renal tumour classification Consideration of genetic testing

5 Role of immunohistochemistry in renal tumours Primary renal tumour Establish the diagnosis of renal tumour Classify the tumour Particularly valuable in renal mass biopsies Metastatic tumours Establish the tumour as being of primary renal origin Classify the renal tumour?future use of IHC to aide prediction of response to systemic therapies

6 Immunohistochemistry Clear cell RCC Papillary RCC Chromophobe RCC Oncocytoma Collecting duct RCC Urothelial carcinoma AMACR 22-67% 100% 0-29% 15-25% 12% CK7 6-20% 44-87% 64-92% 7-14% 33-83% % type I>type II diffuse focal cytoplasmic c-kit 0-3% 0-5% % % 0% 20% MOC31 4% 11% 79% 0% 20% CD10 70->90% 45% type I 0-32% 0-58% 0-40% 54% low>high grade 88% type II Vimentin 64-87% % 0-21% 0-32% 100% 4-33% RCC 47-85% 63-91% 0-4% 0-14% 40% HMWCK 13% 33% 0% 10% 67% 100% (34BE12) CA IX positive negative (avoid necrosis) negative negative negative may be positive Usually use a panel of IHC guided by tumour morphology

7 Immunohistochemistry PAX8 Expressed in all RCC subtypes (95% sensitivity) PAX8 most useful IHC marker for establishing a diagnosis of metastatic RCC Positivity is NUCLEAR PAX8 is a transcription factor Monoclonal antibody demonstrates less cross-reactivity Other PAX8 + tumours Mullerian origin Thyroid 20% urothelial ca (renal pelvis)

8 Immunohistochemistry metastatic RCC PAX8 most useful IHC marker for establishing a diagnosis of metastatic RCC CD10 and RCC marker sensitive but not specific Clear cell RCC almost always CD10 + Additional IHC which is usually negative in RCC TTF-1 CDX2 p63 PSA ER Advise correlation with clinical and radiological features

9 Immunohistochemistry classification of renal tumours

10 Immunohistochemistry clear cell tumours Differential diagnosis Clear cell RCC (majority) Clear cell papillary RCC Requires specific IHC profile for diagnosis Chromophobe RCC MiT family translocation RCC

11 Clear cell RCC Immunohistochemistry CK and vimentin positive CA IX positive (diffuse, membranous) Usually CK7 negative (or focal) C-kit negative CD10 positive (membranous) AMACR negative HMB45 negative TFE3 negative CA IX

12 Clear cell papillary RCC CK7 & HMWCK + CD10 & AMACR CAIX + (cup-like, luminal sparing) CK7 CAIX Srigley et al. Am J Surg Pathol 2013;37(10): AMACR Clear cell papillary RCC Type I papillary RCC

13 Chromophobe RCC

14 Clear Cell vs Chromophobe RCC Clear Cell RCC Chromophobe RCC CK7 - (usually or focal) CAIX + - CD117/ckit - + vimentin + - CD RCC + - +

15 Chromophobe RCC CK 7

16 MiT family translocation RCC Immunohistochemistry Usually CK and EMA negative PAX8 positive (almost 100% cases) 15% express melan A, HMB45 May express CAIX Xp11 translocation RCC TFE3 IHC positive (nuclear) Antibody fixation dependent FISH more reliable TFE3

17 Multilocular cystic renal neoplasm of low malignant potential

18 Multilocular cystic renal neoplasm of low malignant potential Previously multilocular cystic RCC Tumour composed of multiple cysts Septae of cysts contain groups of clear cells No expansile growth/nodules of clear cells IHC useful to confirm presence of clear cells in septae

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20

21 EMA

22 EMA

23 Immunohistochemistry eosinophilic tumours Differential diagnosis Clear cell RCC Chromophobe RCC (eosinophilic variant) Oncocytoma Papillary RCC (especially Eble type 2) Epithelioid AML

24 Clear cell RCC

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26

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28 PanCK vimentin Ca IX

29 Ca IX

30 Ca IX

31 Oncocytoma

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33 CK 7 Internal control tubular epithelium

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35 CD34

36 Oncocytoma vs chromophobe RCC Oncocytoma Chromophobe RCC (eosinophilic variant) CK 7 - or cytoplasmic patchy Membrane strong diffuse + MOC RCC - + (50%) Hale s colloidal iron - +

37 Papillary renal cell carcinoma Eble type 1 and 2 type 2 usually eosinophilic Immunohistochemistry CK7 positive (may be negative in high grade or type 2 tumours) AMACR positive (cytoplasmic) CAIX negative (beware necrosis) TFE3 negative IHC not useful if DD is mucinous tubular and spindle cell carcinoma

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39

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41 PAX8

42 CK7

43 AMACR

44 CD10

45 Immunohistochemistry papillary tumours Differential diagnosis Papillary RCC (majority) Clear cell papillary RCC MiT family translocation RCC Clear cell RCC may occasionally have focal papillary component (rare)

46 Papillary RCC vs metanephric adenoma Type 1 PRCC can be variably solid DD may then include metanephric adenoma Benign vs malignant tumour Distinction usually possible with IHC If IHC not characteristic guidance suggests FISH for trisomy ch 7/17 Wilms tumour is a DD but very rare outside paediatric population

47 Papillary RCC vs metanephric adenoma Metanephric Adenoma Solid variant papillary RCC Epithelial predominant Wilms tumour CK CD AMACR WT EMA - + FISH Chr 7, 17 gains (trisomy)

48 RED = chromosome 1 (control) AQUA = chromosome 7 GREEN = chromosome 17 NORMAL = 2 RED signals 2 AQUA signals 2 GREEN signals Images courtesy of Melody Tabiner

49 Collecting Duct Carcinoma Rare Derived from cells of the collecting duct of Bellini ( Bellini cell Ca ) Solid, tubular or papillary growth & stromal reaction Aggressive tumours with poor prognosis Medullary Ca looks similar but occurs in young patients with Sickle Cell disease Characteristically 34betaE12 positive, whereas other variants of RCC are (usually) negative Exclude urothelial carcinoma

50 Collecting Duct Carcinoma Exclude urothelial carcinoma There is an overlap in IHC with urothelial carcinoma (UC) In CDC PAX8 = almost 100%, in UC = 17-20% In UC p63 = almost 100%, in CDC = 14%

51 Collecting Duct Carcinoma ISUP 2013 recommendations Diagnosis requires the following features; at least some of the lesion involves the medullary region there is a predominant formation of tubules a desmoplastic stromal reaction should be present cytologic features are high grade growth pattern is infiltrative there is an absence of other typical RCC subtypes or urothelial carcinoma when a tumour has >95% morphology of CDC but focal urothelial carcinoma is present, it is designated as urothelial carcinoma with prominent glandular differentiation in the setting of undifferentiated carcinoma, if any of the tumour meets criteria for CDC, it is designated as poorly differentiated CDC CDC are high grade, and therefore should not be assigned a grade

52 Immunohistochemistry - biopsies

53 Immunohistochemistry - biopsies Increasing number of renal tumour biopsies undertaken Confirm presence of renal tumour Guide management Surgical excision vs surveillance Tissue diagnosis in patients with metastatic RCC Interpretation of morphology aided by immunohistochemistry Neither reviewed in isolation Challenging area of renal tumour pathology

54 Immunohistochemistry - biopsies Morphological assessment guides selection of IHC Careful selection of antibodies Tissue may be limited Eosinophilic tumours DD includes clear cell RCC, eosinophilic chromophobe RCC, type 2 papillary RCC, oncocytoma Oncocytic tumours Oncocytoma vs chromophobe RCC (eosinophilic) vs RCC (other)?hoct

55 Immunohistochemistry - biopsies Morphological assessment guides selection of IHC Careful selection of antibodies Tissue may be limited Eosinophilic tumours DD includes clear cell RCC, eosinophilic chromophobe RCC, type 2 papillary RCC, oncocytoma Oncocytic tumours Oncocytoma vs chromophobe RCC (eosinophilic) vs RCC (other)?hoct

56 Oncocytoma vs chromophobe RCC Oncocytoma Chromophobe RCC (eosinophilic variant) CK 7 - or cytoplasmic patchy Membrane strong diffuse + MOC RCC - + (50%) Hale s colloidal iron - +

57 Immunohistochemistry - biopsies Recent study of consecutive biopsies of oncocytic renal tumours Morphology and IHC

58 Immunohistochemistry - biopsies In their series, 16% of lesions were oncocytic tumours With morphology and limited IHC (CK7, ckit, S100A1) 78% could be classified as favour oncocytoma or favour RCC Only tumours with classic morphology and IHC were diagnosed as favour oncocytoma Otherwise diagnosis - cannot exclude RCC Propose that it is possible to classify the tumours as Favour oncocytoma Favour RCC Cannot exclude RCC

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60 Oncocytoma

61 Oncocytoma CK 7

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63 CK 7

64 C-Kit MOC31 Eosinophilic chromophobe RCC

65 Renal biopsy tract

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67 Eosinophilic chromophobe RCC

68 Oncocytoma vs chromophobe RCC Oncocytoma Chromophobe RCC (eosinophilic variant) CK 7 - or cytoplasmic patchy Membrane strong diffuse + MOC RCC - + (50%) Hale s colloidal iron - + CK7 +/- MOC31 oncocytoma vs chrcc Ckit (CD117) oncocytoma or chrcc vs other RCC Vimentin - oncocytoma or chrcc vs other RCC

69 Renal tumour biopsies postbiopsy features to be aware of

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71 Gelfoam in biopsy tract

72 Gelfoam in biopsy tract

73 Renal biopsy tract seeding

74

75

76 panck

77 Another example

78 CK7 PAX8

79 Grading tumours on biopsy

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81

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83 Molecular Pathology and Renal Tumours

84 How does Molecular Pathology apply to Renal Tumours? Molecular Pathology Tests to aide diagnosis and classification demonstration of specific gene mutation or re-arrangement. Correct classification of subtype of renal tumours Is of prognostic importance May influence management (including follow-up) May identify risk of further disease in that patient or their family?referral to clinical genetics e.g. VHL, HL-RCC, Birt-Hogg- Dube

85 How does Molecular Pathology apply to Renal Tumours? What is not so well-established in renal tumours is the use of molecular testing to predict response of the tumour to treatment

86 Molecular pathology & WHO 2016 renal tumour classification New epithelial tumors Tubulocystic renal cell carcinoma Acquired cystic disease associated renal cell carcinoma Clear cell (tubulo) papillary renal cell carcinoma MiT family translocation renal cell carcinomas Hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinoma Succinate dehydrogenase-deficient renal cell carcinoma Emerging/provisional entities Thyroid-like follicular renal cell carcinoma ALK-translocation renal cell carcinoma

87 Molecular Tests in the Classification of Renal Tumours - Value in Difficult Differential Diagnoses

88 Areas of Diagnostic Difficulty - 1 Chromophobe RCC vs Oncocytoma

89 Chromophobe RCC vs Oncocytoma Morphology IHC ChRCC: diffuse CK7+, MOC31+ Oncocytoma: usually focal CK7+, MOC31 usually FISH ChRCC (eosinophilic and classical): High frequency of losses of ch 1,2,6,10,17 Oncocytoma Occasional loss of ch 1 (10% tumours) Brunelli et al (Mod Pathol. 2005:18;161-69)

90 Areas of Diagnostic Difficulty - 2 High grade RCC E.g. Type 2 papillary RCC vs Clear cell RCC High grade CCRCC is often eosinophilic and occasionally have papillary areas Type 2 papillary RCC may have focal clear cell areas IHC may not be useful IHC profile overlaps Type 2 PRCC may be CK7 negative Cytogenetics may be useful Clear cell RCC associated with loss of genetic material from ch 3 and mutations in VHL Papillary RCC commonly associated with trisomy of ch 7, 17 and loss of the Y chromosome

91 Areas of Diagnostic Difficulty - 3 Tumours with unusual morphology E.g. MiT family translocation RCC Xp11 translocation RCC shows fusion of TFE3 with one of at least 8 genes (usually cannot demonstrate fusion partner) The t(6;11) translocation fuses TFEB with one of 2 (currently known) fusion partners

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93 TFE3 Breakapart probe Normal 2RG Abnormal 1RG 1G 1R Images courtesy of Melody Tabiner

94 Areas of Diagnostic Difficulty - 3 Tumours with unusual morphology E.g. MiT family translocation RCC Xp11 translocation RCC shows fusion of TFE3 with one of at least 8 genes (usually cannot demonstrate fusion partner) The t(6;11) translocation fuses TFEB with one of 2 (currently known) fusion partners E.g. SDH-deficient RCC germline testing E.g. HLRCC germline testing

95 Areas of Diagnostic Difficulty - 4 Papillary RCC (type 1) vs other Papillary RCC commonly associated with trisomy/tetrasomy of ch 7,17 and loss ch Y

96 RED = chromosome 1 (control) AQUA = chromosome 7 GREEN = chromosome 17 NORMAL = 2 RED signals 2 AQUA signals 2 GREEN signals Images courtesy of Melody Tabiner

97 Areas of Diagnostic Difficulty - 4 Papillary RCC (type 1) vs Papillary RCC commonly associated with trisomy/tetrasomy of ch 7,17 and loss ch Y Mucinous tubular spindle cell carcinoma Usually lack trisomies of ch 7 and 17 and loss of ch Y Metanephric adenoma Usually IHC allows correct distinction Metanephric adenoma usually lacks trisomy ch 7 Clear cell papillary RCC Lack trisomies of ch 7 and 17 Lack mutations in VHL

98 Renal tumours associated with hereditary syndromes

99 Renal cancer in von Hippel-Lindau disease and related syndromes Baush et al. Nat Rev Nephrol 2013;9:529-38

100 Renal tumours associated with hereditary syndromes Important to consider hereditary RCCs Implications for the patient Management of tumours, e.g. partial vs radical nephrectomy Screening? Implications for their family Guidance on referral for genetic counselling?

101 Renal tumours associated with hereditary syndromes Guidance on referral for genetic counselling? Retrospective review of patients referred to genetics services with renal cancer Paper provides some suggested guidance on referral for genetic counselling

102 Available Tests Oxford Medical Genetics Laboratory Cytogenetics Available probes: Chromophobe RCC (monosomy ch 1, 2, 6, 10, 17) Papillary RCC (e.g probes for ch 7 and 17) TFE3 breakapart (Xp11 rearrangement) VHL Requirements: 4-6 slides cut at 3-5 µm Aim for report within two weeks Molecular Cancer Tests Constitutional studies (list below not exhaustive): Von Hippel Lindau Syndrome (VHL) Hereditary papillary RCC (MET) Familial paraganglioma/phaeochromocytoma syndromes (9 gene panel: SDHB, SDHC, SDHD, SDHA, RET, VHL, TMEM127, MAX, SDHAF2) Generally DNA from whole blood

103 Take home messages Renal tumour classification is of prognostic significance Majority of renal tumours can be classified on morphology Immunohistochemistry is useful in difficult differential diagnoses and is a prerequisite for some (rare) tumours Immunohistochemistry is valuable especially in biopsy material But always review morphology and IHC together Molecular pathological tests are useful in some circumstances Of particular importance if considering hereditary renal tumour syndrome

104 Questions?

105 Thank you! Acknowledgements to; Professor Ian Roberts and Dr Clare Verrill Helene Euston-Mellor Melody Tabiner and Alison Parsons

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