When Immunostains Can Get You in Trouble: Gynecologic Pathology p16: Panacea or Pandora s Box?

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1 When Immunostains Can Get You in Trouble: Gynecologic Pathology p16: Panacea or Pandora s Box? Teri A. Longacre, MD Stanford Medicine Stanford California

2 pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

3 Disclosure of Relevant Financial Relationships USCAP requires that all planners (Education Committee) in a position to influence or control the content of CME disclose any relevant financial relationship WITH COMMERCIAL INTERESTS which they or their spouse/partner have, or have had, within the past 12 months, which relates to the content of this educational activity and creates a conflict of interest. Dr. Teri A. Longacre declares she has no conflict(s) of interest to disclose. pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

4 Use of p16 in Cervical Squamous Lesions Tumor-suppressor p16 is overexpressed in cervical carcinomas p16 expression is altered by the effect of HPV on the retinoblastoma protein IHC staining for p16 has become standard practice in the evaluation of cervical lesions Although considered a surrogate marker for HPV infection in the appropriate setting, p16 does not, in general, act as a surrogate marker for HPV infection pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

5 Stimulation of cell-cycle progression by high-risk HPV Clin. Sci. (2006) 110,

6 p16 Immunohistochemistry Positive stain Diffuse (>80%) strong nuclear or nuclear and cytoplasmic reactivity block positive involving at least 1/3 basal layer: Correlates with presence of HR-HPV and diagnosis of dysplasia Grading of dysplasia MUST be based on histology J Low Genit Tract Dis. 2012;16: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

7 pi6 in Gynecologic Pathology: Panacea or Pandora s Box? Block positive p16

8 HSIL HSIL LSIL LSIL

9 p16 Immunohistochemistry Negative stain Cytoplasmic only staining Focal or patchy staining Discontinuous staining of basal layer Staining of upper layers but not basal layer J Low Genit Tract Dis. 2012;16: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

10 Cytoplasmic only interpret as negative

11 Discontinuous basal p16 interpret as negative

12 p16 Recommendations USE: HSIL vs. benign mimic Confirm diagnosis of CIN2 Disagreement about diagnosis of HSIL Negative biopsy with prior high-risk cytology: HSIL, ASC-H, AGC-NOS, ASC-US/HPV16+ pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

13 p16 Recommendations DO NOT USE: LSIL vs. benign mimic Morphology unequivocally diagnostic of: LSIL (CIN1) HSIL (CIN3) Negative for dysplasia pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

14 Squamous Morules p16

15 Neuroendocrine Carcinoma p16

16 Cervical Neuroendocrine Carcinoma <5% of all cervical cancer Most in mid 50 s Clinically aggressive Most bulky & deeply invasive with necrosis 50% high stage (FIGO III/IV) pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

17 Cervical Neuroendocrine Carcinoma Most express one or more neuroendocrine markers Often associated with AIS, HSIL, and conventional invasive cervical adenocarcinoma Both small and large cell types are p16-positive and harbor high-risk HPV (esp HPV 18) TREATED like neuroendocrine carcinoma elsewhere NOT like cervical cancer pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

18 Endometrial Neuroendocrine Carcinoma Most express one or more neuroendocrine markers Often associated with conventional endometrioid carcinoma Predominantly large cell type followed by mixed, and small cell Often PAX-8 negative May exhibit MMR deficiency (MLH1/PMS2 most common) May be p16-positive (24%) Am J Surg Pathol 2016;40: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

19 Summary: p16 in Cervical Squamous Lesions Should be strong, diffuse block-positive staining Many other lesions can have focal strong staining patterns Caution on limited sampling Do not use on LSIL lesions significance unknown Be clear about the question you are asking and be clear that you are using the right bioassay for that question pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

20 Use of p16 in Cervical Glandular Lesions Since AIS is assoc w/ high-risk HPV, AIS demonstrates diffuse, strong expression of p16 Ki-67 is also elevated & can be used as complimentary marker This can be used in confirming diagnosis of AIS in biopsy or curettage samples, but requires experience pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

21 p16

22 p16

23 Cervical Endometriosis p16

24 IHC Endometriosis AIS p16 Patchy Diffuse, continuous Ki-67 Variable High

25 Uterine Endometrioid Adenocarcinoma p16

26 Uterine Serous Adenocarcinoma p16

27 p16 Predicament Strong, diffuse p16 in up to 10% of uterine corpus cancers in small samples p16 can be misleading in small samples Minimal deviation adenocarcinoma & other cervical special variant carcinomas are p16-negative p16 may not be sufficiently discriminating to use alone some use Ki-67 (mib1) pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

28 False Positive p16 Benign Lobular endocervical hyperplasia Malignant HPV-negative adenocarcinoma (55% patchy/diffuse p16) Am J Surg Pathol 2011; 35:633-46; Histopathology 2010;57: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

29 Summary: p16 in Cervical Glandular Lesions Should be strong, diffuse staining Many other lesions can have focal strong staining patterns Caution on limited sampling Be clear about the question you are asking and be clear that you are using the right bioassay for that question Use only after you have developed some experience with this pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

30 Use of p16 in Vulvar Squamous Lesions Exceptions: Differentiated (simplex) VIN Verrucous carcinoma Basal cell carcinoma Paget s disease pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

31 Differentiated (Simplex) VIN 10% of vulvar intraepithelial neoplasia Post-menopausal women Associated with lichen sclerosus, not HPV-related Typically identified adjacent to well-differentiated keratinizing carcinoma No counterpart in the cervix pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

32 Differentiated (Simplex) VIN Clinical appearance: Rough area with gray-white discoloration, or Ill-defined raised white plaque pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

33 Differentiated (Simplex) VIN: Architecture Expansion of basal layer with elongated, narrow rete ridges Epithelial thickness: acanthotic to atrophic Prominent parakeratosis pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

34 pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

35 Differentiated (Simplex) VIN: Cytology Abnormal maturation Enlarged keratinocytes with abundant, markedly eosinophilic cytoplasm in mid-to-superficial layers Superficial cell enlargement with pallor Nuclear atypia of the basal cell layer Enlarged, pleomorphic nuclei or Relatively small, hyperchromatic irregular nuclei pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

36

37

38 Differentiated (Simplex) VIN: Dermal Changes Fibrosis Lymphocytic infiltrate, may be lichenoid pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

39

40

41 Are There Hybrid Types? Usual HPV-related VIN in patients with lichen sclerosus Usual HPV-related VIN and non-hpv-related (differentiated [simplex]) VIN in same patient Usual basaloid non-hpv related VIN J Clin Pathol. 2007;60; ; Am J Surg Pathol. 2009;33: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

42 Vulvar Verrucous Carcinoma Rare, special variant of SCC Slow-growing, minimal metastatic potential Typically low-risk HPV (6, 11) Histology: Exophytic, hyperkeratotic fronds Cytologically bland squamous epithelium Well-circumscribed pushing border with chronic inflammation pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

43

44

45 Anogenital Basal Cell Carcinoma No squamous intraepithelial component Stromal retraction but may not be present CK5/6-pos, p63-pos BerEp4-pos, BCL2-pos p16 may be focally strong positive limited (small) sampling problem pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

46

47

48

49 p16

50 p63

51 BerEp4

52 Anogenital Paget s Disease Squamous hyperplasia with hyperkeratosis and parakeratosis in 90% of anal extra-mammary Paget s Pseudoepitheliomatous hyperplasia Fibroepithelioma-like Papillomatous (mimics HPV) Paget cells may be unapparent Paget cells may strongly express p16 Am J Surg Pathol 2000;24: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

53

54

55

56

57

58 CK7

59 p16

60 Anogenital Paget s disease Primary (may rarely invade) CK7+/CK20-/HER2neu+ Secondary to anorectal or bladder cancer CK7+/CK20+/GATA3+ CK7+/-/CK20+/-/CDX2+ pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

61 Anogenital Paget s & HSIL Rare, but may coexist Paget cells are HPV-negative HSIL is HPV-positive pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

62 Summary: Use of p16 in Vulvar Lesions LAST recommednations VIN lesions may change over time (dvin vs HPV-VIN) Consider panel to diagnose unusual vulvar lesions (CK7, p16, S100, BerEp4) pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

63 Use of p16 in Metastatic Lesions Exceptions: Transitional cell carcinoma Metastatic neuroendocrine carcinoma pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

64 p16 in Bladder Neoplasms Urothelial carcinoma with squamous differentiation (33%) Primary bladder squamous cell carcinoma (31%) Primary bladder adenocarcinoma (67%)* *Both p16 and p53 in 33% Mod Pathol 2012;25: ; PLoS One 2014; 9:e95724 pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

65 p16 in Bladder Neoplasms Use p16 in conjunction with HPV ISH in the evaluation of cervical cancer vs urinary tract cancer Not all metastatic lower genital tract squamous cancer express p16 in strong diffuse pattern pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

66 Summary: Use of p16 in Metastatic Lesions In general, cannot be relied on for determining primary site Requires careful clinicopathologic correlation Consider HPV testing (in situ hybridization) pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

67 Use of p16 in Endometrial Glandular Lesions p16 (% cells) p53 (% cells) HPV Endocervical adenocarcinoma Diffuse, mod-strong (90-100%)??? Serous adenocarcinoma Diffuse, mod-strong (90-100%) Strong, diffuse (>90%) or null 45/49 Endometrioid adenocarcinoma Variable, weak-strong (10-90%)??? Am J Surg Pathol 2009;33: ; Int J Gynecol Pathol 2007;26: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

68

69

70 p16

71 Papillary Syncytial Change (Metaplasia) Papillary syncytial change associated with stromal breakdown, atrophy, karyorrhectic debris Papillary syncytial metaplasia occurs over surface of endometrium, may be extensive, often mixed epithelial types May overlie atrophy, hyperplasia or carcinoma pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

72 Papillary Syncytial Change (Metaplasia) Decreased expression of ER Increased expression of p53 (although still wild-type staining) and p16, the latter marker typically being diffusely positive Low MIB1 proliferation index In problematic cases, IHC may result in a misdiagnosis Int J Gynecol Pathol 2012;31: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

73 Papillary Syncytial Change (Metaplasia) p53

74 Use of p16 in Uterine Mesenchymal Lesions p16 is overexpressed in uterine leiomyosarcomas by gene expression studies, but p16, p53, Ki-67, and other cell cycle regulatory markers are often not helpful in the distinction between leiomyomas with unusual features (mostly leiomyomas with bizarre nuclei) and leiomyosarcoma, as overlap in their expression is common. Am J Surg Pathol 2013;37: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

75 Leiomyoma with Bizarre Nuclei vs LMS LBN LMS Mitotic index (MF/10HPF) 10 >10 Tumor cell necrosis Absent Present Ki-67 (MIB-1) Low High p16 Low High p53 Negative Positive Am J Surg Pathol 2013;37: pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

76 Leiomyoma with Bizarre Nuclei p16

77 Summary p16 has transformed the diagnosis and treatment of lower genital tract squamous & glandular lesions p16 is only a surrogate and so there are many caveats & limitations to its utility HPV in situ or PCR may be required to establish definitive diagnosis in difficult cases Panels should always be utilized in the appropriate clinical context pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

78 Important Information Regarding CME/SAMs The Online CME/Evaluations/SAMs claim process will only be available on the USCAP website until September 30, No claims can be processed after that date! After September 30, 2017 you will NOT be able to obtain any CME or SAMs credits for attending this meeting. pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

79 pi6 in Gynecologic Pathology: Panacea or Pandora s Box?

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