p16 Cervical HISTOLOGY Histology Compendium & Staining Atlas

Transcription

1 p16 Cervical HISTOLOGY Histology Compendium & Staining Atlas

2 Chapter 1: An Introduction to p Normal Cervical Epithelium and the Cell Cycle....4 HPV Infection and Cervical Disease p16: A Biomarker for Cervical Disease Chapter 2: Interpreting p16 Staining Patterns Squamous Epithelium p16 Positive p16 Negative Endocervical Epithelium p16 Positive p16 Negative Chapter 3: Clinical Utility of p16 for High-Grade CIN Confirmation of CIN2/ Differential Diagnosis of High-Grade Dysplasia Squamous Atrophy Atypical Squamous Metaplasia Chronic Cervicitis with Reactive Atypia Chapter 4: Clinical Utility of p16 for Low-Grade CIN Chapter 5: Clinical Utility of p16 for Endocervical Neoplasia

3 contents CINtec Histology Staining Case Examples Case 1: High-grade squamous dysplasia and AIS Case 2: CIN Case 3: Inflammatory atypia versus CIN Case 4: Glandular atypia, differential diagnosis tubo-endometrial metaplasia versus AIS Case 5: Inflammatory atypia versus CIN Case 6: Atypical immature metaplasia versus CIN Case 7: Atypical immature metaplasia versus CIN2/ Case 8: Atrophic cervicitis versus high-grade dysplasia Case 9: Chronic cervicitis with reactive atypia versus CIN2/ Case 10: Reserve cell hyperplasia versus CIN Case 11: Reactive atypia versus CIN Case 12: Reactive atypia versus CIN Case 13: Disrupted fragments of cervical mucosa, tangentially sectioned, atrophy versus CIN2/ Case 14: CIN 2/3, small lesion Case 15: CIN 2/3, small lesion, disrupted mucosa Case 16: CIN1, small lesion, endocervical glandular involvement Bibliography

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5 An Introduction to p16 Chapter 1 An Introduction to p16 The histologic diagnosis of Cervical Intraepithelial Neoplasia (CIN) has historically been based only on the morphologic evaluation of disordered squamous maturation and the identification of Human Papilloma Virus (HPV)-mediated viral cytopathic effects. While the differential diagnosis of high-grade dysplasia (CIN2/3) versus normal squamous mucosa may be definitive in optimal cases, the subjective nature of the diagnostic processes and the inconsistent reproducibility in histologically subtle or morphologically equivocal cases may lead to false positive or false negative diagnoses. Published clinical research studies have shown that the accuracy of community-based pathologists for diagnosing high-grade CIN on cervical biopsy specimens can be significantly increased by the conjunctive interpretation of p16-immunostained slides along with the routine H&E-stained slide interpretation. 1 And, the agreement of community-based pathologists for categorizing lesions as highgrade CIN vs CIN 1 or Negative for dysplasia significantly improved with H&E- plus p16-stained slides. 1 An additional series of reports have suggested that the detection of the overexpression of p16 in cervical lesions may allow the progression risk of low-grade cervical dysplasia to be predicted. 3,17

6 Normal Cervical Epithelium and the Cell Cycle Extensive research over the past 20 years has provided strong evidence that persistent infections with high-risk types of human papillomaviruses (HR-HPV) may cause cervical cancer.16 Normal ectocervical mucosa includes a basal cell layer, parabasal cells, intermediate cells, and superficial cells. As squamous cells mature, they migrate toward the mucosal surface (Fig. 1-1). Superficial cells Terminally differentiated cells Largeintermediate glycogenatedcells Differentiating cells Smallintermediate cells Commitment to terminal differentiation Parabasal cells Actively replicating cells Basal cells Stem cell compartment Fig. 1-1 Normal Squamous Maturation. Proliferation is limited to the basal and parabasal cells. Maturation occurs in the intermediate zone and the superficial layer contains terminally differentiated squamous cells that eventually exfoliate. Cell replication is controlled through a complex mechanism involving many regulatory pathways. One of these is the retinoblastoma protein (prb) pathway which controls cell cycle progression and cellular proliferation. Under normal conditions, the bound complex of transcription factor E2F and prb is a critical control mechanism that prevents cells from continuously replicating their genomes and proliferating (Fig 1-2A). p16 normally serves as a negative regulator of cell cycle progression that blocks proliferation by promoting the interaction between E2F and prb (Fig 1-2B). 2

7 HISTOLOGY A Fig. 1-2, Panel A Normal regulation of cell cycle; Arrest. prb binds to the transcription factor E2F. This prb-e2f protein complex blocks the transcription of the genes that promote cell cycle progression and proliferation, as well as the gene that encodes for p16, a negative regulator of cell cycle progression. B Progression Arrest Fig. 1-2, Panel B Normal regulation of cell cycle; Progression. The release of E2F from prb results in cell cycle progression, mitotic replication, and activation of the p16 gene, enabling p16 protein production. p16 protein in turn facilitates the re-binding of prb to E2F, leading to cell cycle arrest. This feedback control mechanism is key to maintaining the balance between cell cycle progression/ proliferation and cell cycle arrest.

8 The onset of HPV mediated cervical disease occurs when HR-HPV types infect the basal cells of the epithelium (Fig 1-3). The vast majority of HPV infections are transient and typically clear within 6-12 months. When transient, an HR-HPV infection does not significantly affect the regulatory mechanisms and cells undergo normal cell cycle (Fig 1-4). Fig. 1-3 A breach in the cervical mucosa may allow viral particles to reach the basal layer where they may establish a productive infection. 4

9 HISTOLOGY Progression Arrest Fig 1-4 Although transient HPV infection may result in increased cell proliferation, these infections do not disrupt the balance between prb and E2F or the control of p16 expression. HPV Infection and Cervical Disease Some HR-HPV infections, however, persist and produce levels of viral E6 and E7 oncoproteins that can mediate oncogenic transformation through the disruption of the cell cycle regulatory mechanism (Fig 1-5). This transformation can lead to the development of high-grade cervical dysplasia (CIN 2/3), adenocarcinoma in situ (AIS), and invasive cervical carcinomas. The cellular protein p16 is a useful marker to identify high-grade CIN lesions, as it is over-expressed in cervical cells undergoing cellular transformation in response to the expression of the HR-HPV E7 oncoprotein. 16,15,9,26 (Fig 1-6).

10 A E6 Fig. 1-5, Panel A HR-HPV E6 and E7 mediate oncogenic transformation. B Fig. 1-5, Panel B Fully transformed cells are characterized by unregulated cell cycle progression, disruptedmaturation,andtheabilitytoinvadeunderlying cervical stroma. Cellular evidence of transformation includes an increase in nuclear/cytoplasmic ratios, anisonucleosis, and hyperchromasia.2 p16: A Biomarker for Cervical Disease DNA testing for HPV cannot differentiate transient infections, which make up a majority of cases, from transforming infections, which occur less frequently. Therefore, testing for HPV is of limited use in pinpointing clinically significant disease, especially in younger women where the prevalence of HPV infection can be as high as 30%. 6

11 Diffuse p16 staining; however, results from E7-mediated inactivation of the prb-e2f control mechanism, which is a central hallmark of the oncogenic process. Thus, in contrast to HPV DNA detection, the immunohistochemical detection of a diffuse pattern of p16 over-expression identifies lesions of cervical mucosa in which molecular mechanisms leading to oncogenic 28, 8, 27, 11 transformation are active. Fig. 1-6 In cells with transforming HPV infections, HPV viral oncoprotein E7 impairs the function of prb, disrupting its ability to bind to transcription factor E2F. This leads to deregulated cell proliferation, genetic instability and p16 protein over-expression whichisdetectablebyimmunohistochemistry.

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13 Chapter 2 Interpreting p16 Staining Patterns Interpreting p16 Staining Patterns

14 Squamous Epithelium p16 is a nuclear protein under normal physiological conditions, however, typically both nuclear and a cytoplasmic expression are observed when p16 is over-expressed in cervical neoplasia. 9, 24, 19, 10 In few cases, nuclear expression may be faint or undetectable, but nuclear staining is not required to interpret the p16 staining. Therefore, both nuclear and/or cytoplasmic immunostaining should be taken into account when assessing the p16 staining pattern. As with any immunohistochemical procedure, it is important to standardize the interpretation criteria for p16 by designating specific immunostaining patterns. The p16 rating system not only reflects the molecular pathogenesis of cervical dysplasia, but has also been validated by correlation with the progression of CIN in numerous studies. 1,28,7,18, 24 Interpretation of p16 staining results must take into consideration the fact that p16 at the individual cell level may be expressed in highgrade dysplastic cervical lesions and cervical cancers as well as in some conditions not associated with cervical dysplasia. Furthermore, it is important to note that p16 test results should be used only to provide additional information that must be interpreted in conjunction with the morphological classification of the lesion. Specimens stained with the CINtec Histology Kit are evaluated according to a binary rating system composed of the ratings positive and negative. The rating of a slide as positive or negative considering both nuclear and/or cytoplasmic staining is assigned as follows: 10

15 HISTOLOGY p16 Positive: A p16 test result is positive if the staining pattern is Diffuse, showing a continuous staining of cells of the basal and parabasal cell layers of the squamous cervical epithelium, with or without staining of cells of superficial cell layers. Fig 2-1 Diffuse p16 staining pattern. A B Panel A Magnification 20X Panel B Magnification 40X A diffuse staining pattern is sometimes also referred to as a confluent staining, band staining, or block staining, where typically the staining intensity of the cells within the lesion is homogeneous. Virtually all CIN3 lesions are positive for p16 28,7 and a positive test result provides high positive predictive value for high-grade CIN. 20 Although high-grade lesions typically show diffuse staining, 40-60% of CIN1 lesions can also show diffuse staining, which may indicate the onset of transforming HPV infections. 4,22,7 Therefore, the assessment of the extent of the diffuse staining within the squamous cervical epithelium itself should not be used as an independent criterion for the interpretation of cervical lesions.

16 p16 Negative: A test result is rated negative if a p16 stained slide shows either a negative staining reaction in the squamous epithelium ( negative staining pattern Fig 2-2A), or a staining of isolated cells or small cell clusters; i.e., a noncontinuous staining, particularly not of the basal and parabasal cells ( focal staining pattern Fig 2-2B). 1, 8 Fig. 2-2 Negative p16 results. A Panel A No p16 staining; magnification 20X B Panel B Focal p16 staining pattern; magnification 20X 12

17 HISTOLOGY A focal staining pattern is sometimes also referred to as patchy staining, scattered staining, or checkerboard staining. The staining intensity of the cells of the same morphological grade within the respective lesion may show substantial variability. Focal p16 staining is occasionally seen in squamous metaplasia, atypical immature metaplasia, reactive atypia, and is frequently detected in CIN1. Neg Negative: Focal p16 S Columnar cells Negative: Focal p16 Staining Columnar cells Normal Negative: Focal p16 Staining Squamous MetaplasiaColumnar cells Normal CIN1 HPV infected HPV infected p16 over-expre Negative: Focal p16 Staining Columnar cells Columnar cells Normal Normal Normal p16over-expression HPV infected HPV infected p16 over-expression Fig. 2-3 Negative p16 results. Squamous metaplasia, CIN1 (focal staining pattern) HPV infected p16 over-expression p16 over-expression

18 Endocervical Epithelium When interpreting glandular cervical epithelium, an analogous rating system may be used for the evaluation of p16 staining patterns. p16 Positive: As in squamous tissue, a diffuse staining pattern in endocervical tissue is rated as positive. A diffuse pattern requires all atypical cells in the gland to stain for p16. Diffuse p16 staining in the glands is typically observed in adenocarcinoma in situ (AIS). (Fig. 2-4, 2-5) as well as in invasive adenocarcinomas and should 8, 23 be rated as a positive test result. Fig. 2-4 Diffuse p16 staining as seen in AIS Fig. 2-5 Positive p16 test result, AIS. Note diffuse staining in atypical glandular epithelial cells and absence of staining in adjacent benign glandular mucosa (arrow) Magnification 10X 14

19 HISTOLOGY Negative: Both no p16 reactivity (Fig 2-6) or a focal staining pattern (Fig 2-7) are rated as negative. Focal p16 staining can be observed occasionally in normal glandular epithelium where individual cells or small groups of neighboring glandular cells may show p16 immuno-reactivity. 8 Fig. 2-6 Negative p16 result. No staining. Fig. 2-7 Negative p16 result. Focal staining pattern as can be seen in tubal metaplasia and tubo-endometrial metaplasia Fig. 2-8: Although benign mucinous glandular cells don t typically stain for p16, staining may be detected in scattered, non-mucinous/secretory cells (with or without cilia) as well as in areas of tubo-endometrial metaplasia producing a focal 8, 23, 12, 13 staining pattern. Magnification 40X

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21 Chapter 3 Clinical Utility of p16 for High-Grade CIN Clinical Utility of p16 for High-Grade CIN

22 Confirmation of CIN2/3 The primary role of the conjunctive use of p16 immunohistochemical staining is to confirm or rule out highgrade dysplasia (CIN2/3) in lesions with equivocal morphology. p16 has been found to be strongly over-expressed in over 99% of cases of high-grade cervical dysplasia. 1,4,15 In addition, a recent study has reported a high level of inter-observer reproducibility in the scoring of p16 test results. 1 A B Fig. 3-1 CIN3. Note dysplastic cervical mucosa stains diffusely for p16 indicating a positive p16 test result. The diagnosis of CIN3 is confirmed. Magnification 2.5X Panel A H&E; Panel B p16 18

23 HISTOLOGY A B Fig. 3-2 CIN3 at squamo-columnar junction. Note diffuse p16 staining throughout all layers of dysplastic cervical mucosa and absence of staining in adjacent benign glandular cells. The p16 test result is rated positive and the diagnosis of CIN3 is confirmed. Magnification 40X Panel A H&E; Panel B p16 A B Fig. 3-3 CIN2. Note the continuous p16 staining of the basal and parabasal cell layers of the dysplastic cervical mucosa. The p16 staining pattern is diffuse and rated as a positive p16 test result. Magnification 20X Panel A H&E; Panel B p16

24 Differential Diagnosis of High-Grade Dysplasia Some benign conditions, including squamous atrophy and atypical squamous metaplasia, can mimic high-grade dysplasia and represent diagnostic challenges. p16 staining in these cases can provide important data to support or exclude the final diagnosis of high-grade dysplasia, as highlighted in the 4, 8, 29 following examples. 1.Squamous Atrophy: CIN2/3 and squamous atrophy are both characterized by an arrest of normal squamous differentiation with nuclear crowding and high nuclear to cytoplasmic ratios. Whereas CIN2/3 is a precursor of cervical cancer, squamous atrophy has no premalignant potential and shows only limited focal staining or is completely negative for p16 staining. Thus, p16 staining, especially in biopsies from postmenopausal women can provide important information that can help solve the differential diagnosis of CIN2/3 versus squamous atrophy. 11 A B Fig. 3-4 Squamous atrophy versus high-grade dysplasia. Note nuclear crowding and disordered maturation, however p16 shows no immuno-reactivity. The result of the p16 test is negative helping to rule out high-grade dysplasia. Magnification 20X Panel A H&E; Panel B p16 20

25 HISTOLOGY 2. Atypical Squamous Metaplasia Atypical squamous metaplasia (ASM) encompasses the replacement of benign glandular cells with squamous metaplastic cells that may mimic high-grade disease. p16 is a useful tool to aid in differential diagnosis, a diffuse stain indicating the presence of transforming HPV infection supports the diagnosis of CIN2/3 (Fig 3-5), 29 whereas a negative p16 result favors the diagnosis of ASM (Fig 3-6). It is important to note that both focal staining and the absence of staining should be rated as a negative p16 test result. A B Fig. 3-5 ASM versus CIN3. The mucosal surface appears attenuated and could be interpreted based on H&E staining to reflect either ASM or a high-grade squamous lesion. Diffuse p16 staining is rated as a positive p16 test result and supports the diagnosis of CIN3. Magnification 40X Panel A H&E; Panel B p16

26 A B Fig. 3-6 Atypical squamous metaplasia (ASM) versus high-grade dysplasia. Focal nuclear crowding and disrupted maturation but with minimal nuclear changes is suggestive of ASM versus CIN2/3. Focal staining for p16, indicating a negative p16 test, helps to exclude the diagnosis of CIN2/3 and supports the final diagnosis of ASM. Magnification 40X Panel A H&E; Panel B p16 3. Chronic Cervicitis with Reactive Atypia Acute and/or chronic cervicitis may lead to reactive atypia and may also obscure underlying high-grade dysplasia. In these cases, p16 immunostaining may help to confirm or rule out the presence of underlying CIN2/3 lesions that are not readily apparent on H&E stained sections. 22

27 HISTOLOGY A B Fig. 3-7 Chronic cervicitis with reactive atypia versus CIN2/3. Intense chronic inflammation of the cervical stroma with reactive atypia of the overlying squamous epithelium, suggestive but not diagnostic for high-grade dysplasia. p16 shows no immuno-reactivity and is rated a negative test result, helping to rule out CIN2/3. Magnification 10X Panel A H&E; Panel B p16 A B Fig. 3-9 Atypical squamous metaplasia versus CIN2/3 in a background of severe chronic inflammation. Note diffuse staining for p16 in lesional epithelium. The p16 test result is positive and consistent with the final conclusion that this is a case of high-grade squamous dysplasia (CIN2/3). Magnification 20X Panel A H&E; Panel B p16

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29 Chapter 4 Clinical Utility of p16 for Low-Grade CIN Clinical Utility of p16 for Low-Grade CIN

30 The primary role of the conjunctive use of p16 immunohistochemical staining in CIN1 is to provide evidence to help rule out high-grade disease with a negative test result. Tissue biopsies that are classified as CIN1 include those that represent productive HPV infections and in some cases, those that have similar morphologic characteristics but are HPV negative. Cases that have definitive features of HPV infection, including unequivocal koilocytotic atypia, may show positive or negative p16 test results. p16 negative test results are commonly seen in cases that have some degree of disordered maturation but lack unequivocal morphologic evidence of HPVassociated cytopathic effect (Fig 4-1), as well as in some cases that are morphologically definitive for HPV cytopathic effect. Nevertheless, the majority of cases that have unequivocal koilocytotic atypia show either focal staining (Fig 4-2) or diffuse staining (Fig 4-3) for p A B Fig. 4-1 CIN1 versus tangential sectioning. Crowding of nuclei in the lower third that couldhavebeeninterpretedasevidenceofcin1versustangentialsectioningofnormal mucosa. p16 shows no immuno-reactivity and the test result is rated as negative. Magnification 20X Panel A: H&E; Panel B: p16 26

31 HISTOLOGY A B Fig. 4-2 This case was classified as CIN1 based on the presence of cells with perinuclear clearing (koilocytosis). p16 staining is focal, p16 test result is rated negative. In retrospect, however, the areas of perinuclear clearing may have resulted from glycogen accumulation rather than HPV infection, which is a commonly encountered pitfall in the diagnosis of CIN1. Magnification 20X Panel A: H&E; Panel B: p16 A B Fig. 4-3 Koilocytotic atypia and dyskeratosis with crowding of nuclei in the lower third, consistent with CIN1. p16 staining is diffuse and this cases is rated as a positive test result. Magnification 40X Panel A: H&E; Panel B: p16 Several published reports indicate that CIN1 lesions that show no p16 reactivity or stain in a focal pattern may persist or regress but those that show a diffuse p16 staining pattern are at an increased risk for progression to CIN2/3 over a 2-3 year period. 3,17,5,20 More clinical research is required to validate these interesting but still preliminary findings.

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33 Chapter 5 Clinical Utility of p16 for Endocervical Neoplasia Clinical Utility of p16 for Endocervical Neoplasia

34 It has been reported that normal endocervical glands are usually p16 negative (Fig 5-1) with an occasional case exhibiting focal weak staining that is also rated as a negative test result (Fig 5-5, 5-6). In contrast, most preneoplastic and neoplastic endocervical glandular lesions, as a result of their association with high-risk HPV, exhibit diffuse p16 positivity (Fig 5-2,5-3) 8, 23 Although benign mucinous columnar cells, including those in areas of microglandular hyperplasia (Fig 5-4), are consistently negative for p16, benign non-mucinous/secretory cells, tubal metaplasia, tubo-endometrial metaplasia, cervical endometriosis, and endometrial glandular cells, including lower uterine segment mucosal cells sometimes show extensive p16 immuno-reactivity (Fig 5-5, 5-6). A B Fig. 5-1 Benign endocervical mucinous glandular epithelium, negative for p16. Magnification 40X Panel A: H&E; Panel B: p16 It is very important to recognize that staining in benign glandular cells is a normal manifestation and does not denote premalignant potential. Thus, p16 staining results should be interpreted in correlation with tissue cellular morphology to avoid potential misclassification of benign cellular changes as glandular neoplasia. 30

35 HISTOLOGY A B Fig. 5-2 AIS. Note intense nuclear and cytoplasmic staining for p16 in all neoplastic cells and the absenceofstaininginbenignglandularcells (arrow).thep16stainingpatternisdiffuseindicating a positive p16 test result and confirming AIS. Magnification 40X Panel A: H&E; Panel B: p16 A B Fig. 5-3 Invasiveadenocarcinoma.Intensenuclearandcytoplasmicstainingforp16isretained in areas of invasion. The p16 staining pattern is diffuse indicating a positive p16 test result and confirming Invasive adenocarcinoma. Magnification 10X Panel A: H&E; Panel B: p16

36 A B Fig. 5-4 Microglandular hyperplasia, p16 shows no immuno-reactivity and the test result is rated negative. Magnification 40X Panel A: H&E; Panel B: p16 A B Fig. 5-5 Normal endocervical glands with mucinous and non-mucinous/secretory columnar cells.notefocalstaining,themucinouscellsarep16negativebutthenon-mucinous/secretory cells show strong staining for p16. The p16 test result is rated negative. Magnification 40X Panel A: H&E; Panel B: p16 32

37 HISTOLOGY A B Fig. 5-6 Tubal metaplasia with focal staining for p16. Normal endocervical glands with mucinous and non-mucinous/secretory columnar cells. Note that the mucinous cells are p16 negative but that the non-mucinous/ secretory cells show strong staining for p16. The p16 test result is rated negative. Magnification 20X Panel A: H&E; Panel B: p16

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39 CINtec Histology Staining Case Examples Histology Staining Case Examples

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41 HISTOLOGY CASE 1: High-grade squamous dysplasia and AIS Diffuse p16 staining pattern in both squamous and glandular components are interpreted as a positive p16 result. Final diagnosis: CIN3 with endocervical glandular involvement and AIS Magnification 20X

42 A B 38

43 HISTOLOGY CASE 2: CIN 1 Note disordered maturation in the lower third of the squamous mucosa with morphologic evidence of a productive HPV infection, including koilocytosis and hyperkeratosis. p16 shows diffuse staining throughout the full thickness of the lesion and the test result is rated positive. Magnification 40X Panel A H&E Panel B p16

44 A B 40

45 HISTOLOGY CASE 3: Inflammatory atypia versus CIN1 Histologic features include nuclear enlargement and disordered maturation but without koilocytosis. Note underlying acute inflammatory cells that extend into the overlying squamous mucosa. p16 staining shows a focal pattern and the test is rated negative. Magnification 40X Panel A H&E Panel B p16

46 A B 42

47 HISTOLOGY CASE 4: Glandular atypia Glandular atypia, differential diagnosis tubo-endometrial metaplasia versus AIS. The focal p16 staining pattern is rated as a negative test result and is consistent with tubo-endometrial metaplasia. Magnification 20X Panel A H&E Panel B p16

48 A C CASE 5: Inflammatory atypia versus CIN The mucosa shows dense acute and chronic inflammation with disordered maturation and nuclear crowding of the squamous epithelial cells. The differential diagnosis includes inflammatory atypia versus low-grade or high-grade CIN. p16 staining is diffuse and rated as a positive test result, supporting the final diagnosis of CIN 2/3. Note diffuse staining involving disrupted nests of squamous cells. Magnification 20X Panel A H&E Panel C p16 Magnification 40X Panel B H&E Panel D p16 44

49 HISTOLOGY B D

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51 HISTOLOGY CASE 6: Atypical immature metaplasia versus CIN2 The lesion shows nuclear crowding without enlargement or mitotic activity that was considered suggestive but not diagnostic of CIN2. p16 shows no immuno-reactivity and the test result is rated as negative, confirming that this is not high-grade dysplasia. Magnification 20X Panel A H&E Panel B p16

52 A B 48

53 HISTOLOGY CASE 7: Atypical immature metaplasia versus CIN2/3 Note squamous cells with nuclear crowding and abnormal mitotic activity and overlying mucinous columnar cells. The squamous cells are diffusely immunoreactive for p16, resulting in a positive test rating which confirms the impression of a high-grade (CIN2/3) lesion. Magnification 40X Panel A H&E Panel B p16

54 A B 50

55 HISTOLOGY CASE 8: Atrophic cervicitis versus high-grade dysplasia Immature basal-like cells are present through all layers of the squamous mucosa. The loss of maturation raises a differential diagnosis that includes high-grade dysplasia. The p16 staining pattern is focal and rated as a negative result. Focal staining is located primarily in superficial epithelial cells and in scattered inflammatory cells within the cervical stroma, helping to rule out highgrade dysplasia. Magnification 20X Panel A H&E Panel B p16

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57 HISTOLOGY CASE 9: Chronic cervicitis with reactive atypia versus CIN2/3 Intense chronic inflammation in the cervical stroma and associated reactive atypia of the overlying squamous epithelium largely obscured the presence of concurrent highgrade dysplasia that, however, is clearly detected by diffuse p16 staining. Magnification 2.5X Panel A H&E Panel B p16

58 A C CASE 10: Reserve cell hyperplasia versus CIN The mucosa is composed predominantly of basal/parabasal-like cells with nuclear crowding. p16 shows no immuno-reactivity, helping to exclude the diagnosis of CIN2/3. Magnification 20X Panel A H&E Panel C p16 Magnification 40X Panel B H&E Panel D p16 54

59 HISTOLOGY B D

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61 HISTOLOGY CASE 11: Reactive atypia versus CIN This section is cut too thick and shows some fracture artifact. Some cells show perinuclear clearing that raised suspicion for koilocytosis. The lack of p16 immunoreactivity confirms a negative p16 test result and helps to exclude the diagnosis of CIN2/3. Magnification 40X Panel A H&E Panel B p16

62 A B 58

63 HISTOLOGY CASE 12: Reactive atypia versus CIN3 The lesion shows moderate nuclear crowding, extending into the upper layers of the squamous mucosa. Diffuse p16 staining is graded as a positive result and confirms the impression of CIN3. Magnification 40X Panel A H&E Panel B p16

64 A B 60

65 HISTOLOGY CASE 13: Disrupted fragments of cervical mucosa, tangentially sectioned, atrophy versus CIN2/3 Note diffuse p16 staining, rated as positive p16 test result. Final diagnosis: CIN2/3. Magnification 10X Panel A H&E Panel B p16

66 A C CASE 14: CIN 2/3, small lesion Note the complex architectural pattern, including crowded benign endocervical glands, and minute foci of metaplastic squamous cells, in a background of acute and chronic cervicitis. p16 positive results (diffuse staining pattern) serve to highlight nests of high-grade CIN. Magnification 4X Panel A H&E Panel C p16 Magnification 40X Panel B H&E Panel D p16 62

67 HISTOLOGY B D

68 A C CASE 15: CIN 2/3, small lesion, disrupted mucosa This case shows predominantly benign glandular mucosa with few squamous cells. p16 positive test results (diffuse pattern), aided in the detection of a minute focus of CIN2/3. Magnification 10X Panel A H&E Panel C p16 Magnification 40X Panel B H&E Panel D p16 64

69 HISTOLOGY B D

70 A C CASE 16: CIN1, small lesion, endocervical glandular involvement Note minute focus of squamous metaplasia adjacent to benign endocervical glands. Diffuse p16 positive test result (arrow), highlights endocervical gland involvement by CIN1. Magnification 20X Panel A H&E Panel C p16 Magnification 40X Panel B H&E Panel D p16 66

71 HISTOLOGY B D

72 1..Bergeron C, Ordi J, Schmidt D, et al. Conjunctive p16 INK4a Testing Significantly Increases Accuracy in Diagnosing High-Grade Cervical Intraepithelial Neoplasia. Am J Clin Pathol. 2010;133: Cameron RI, Maxwell P, Jenkins D, et al. Immunohistochemical staining with MIB1, bcl2 and p16 assists in the distinction of cervical glandular intraepithelial neoplasiafromtubo-endometrialmetaplasia,endometriosisandmicroglandular hyperplasia. Histopathology 2002;41: Cuschieri K, Wentzensen N. Human papillomavirus mrna and p16 detection as biomarkers for the improved diagnosis of cervical neoplasia. Cancer Epidemiol Biomarkers Prev. 2008;17: Dallenbach-Hellweg G, Trunk MJ, von Knebel Doeberitz Traditional and new molecular methods for early detection of cervical cancer. M.Arkh Patol. 2004;66: del Pino M, Garcia S, Fusté V, et al. Value of p16 INK4a as a marker of progression/ regression in cervical intraepithelial neoplasia grade 1. Am J Obstet Gynecol. 2009; 201:488.e Dijkstra MG, Heideman DA, de Roy SC, et al. p16 INK4a immunostaining as an alternative to histology review for reliable grading of cervical intraepithelial lesions. J Clin Pathol. 2010;63: Evangelou K, Bramis J, Peros I, et al. Electron microscopy evidence that cytoplasmic localization of the p16 INK4a nuclear cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas. Biotech Histochem. 2004;79: Galgano M, Castle P, Atkins K, et al. Using Biomarkers as Objective Standards in the Diagnosis of Cervical Biopsies. Am J Surg Pathol 2010;34: Ghiorzo P, Villaggio B, Sementa AR, et al. Expression and localizationofmutantp16proteinsinmelanocyticlesions from familial melanoma patients. Hum Pathol. 2004;35:25-33.

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75 bibliography 28. Wang SS, Trunk M, Schiffman M et al. Validation of p16 INK4a as a marker of oncogenic human papillomavirus infection in cervical biopsiesfromapopulation-basedcohortincostarica.cancerepidemiol Biomarkers Prev. 2004;13: Wentzensen N, von Knebel Doeberitz M. Biomarkers in cervical cancer screening. Dis Markers 2007;23: Roche greatly acknowledges the work of the authors and contributors of this Compendium & Staining Atlas: Kenneth R. Shroyer MD, PhD, Stony Brook University Medical Center Mamatha Chivukula MD, Magee Women s Hospital of University of Pittsburgh Medical Center Brigitte Ronnett MD, Johns Hopkins University School of Medicine Terry Morgan MD, PhD, Oregon State Health and Science University

76 For further information please visit us at CINtecP16.com This Compendium & Staining Atlas may not be distributed in or to the United States of America. Roche Diagnostics International Ltd. CH-6343 Rotkreuz Switzerland Roche Diagnostics International Ltd. G A G A EN MP ITREV2