CINtec PLUS Cytology. Interpretation training

CINtec PLUS Cytology Interpretation training

Objectives After reviewing this learning module, you will have a basic understanding of how to interpret CINtec PLUS Cytology, including: The mechanism of action for p16 in cervical disease The functional utility of CINtec PLUS Cytology Principles of interpretation Troubleshooting pitfalls and artifacts 2

Agenda Interpretation training for CINtec PLUS Cytology CINtec PLUS Cytology: Principle Interpretation of CINtec PLUS Cytology Isolated cells Cell cluster Troubleshooting Practical slide review training May be included after completing the module Microscope session (multihead and/or single microscopes) Quiz slides (single microscopes) 3

The mechanism of action for p16 in cervical disease The functional utility of CINtec PLUS Cytology Principles of interpretation Troubleshooting pitfalls and artifacts 4

p16 protein expression p16, negative cell cycle regulator Anti-proliferative effect in physiologically normal cells In cervical dysplasia, p16 cannot block cell cycle progression (proliferation), as HPV viral E7 oncoproteins inactivate the tumour suppressor prb upstream from p16 p16 over-expression in CIN Cervical intraepithelial neoplasia 5

Normal cell cycle arrest (not dividing) No mitosis Retinoblastoma protein (prb) binds to the transcription factor E2F The prb-e2f protein complex blocks transcription of genes that promote cell cycle progression and proliferation 6

Normal cell cycle arrest (dividing) Progression Arrest Feedback control mechanism maintaining cell cycle proliferation and arrest The release of E2F from prb results in cell cycle progression, mitotic replication and activation of the p16 gene. This enables p16 protein production and facilitates the re-binding of prb to E2F, leading to cell cycle arrest. 7

HPV infection The onset of HPV-mediated cervical disease occurs when HR-HPV types infect the basal cells of the epithelium The vast majority of HPV infections are transient and clear within 6-12 months 8

Transient HPV infection Progression Arrest 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. 9

Transforming HPV infection Some HR-HPV infections persist and produce levels of viral E6 and E7 oncoproteins that can mediate oncogenic transformation by disrupting the cell cycle regulatory mechanism. 10

Transforming HPV infection Fully transformed cells are characterized by unregulated cell-cycle progression, disrupted maturation and the ability to invade underlying cervical stroma, resulting in cervical cancer. 11

Transforming infection: oncogenesis In cells with transforming HPV infections, HPV viral oncoprotein E7 impairs the function of prb, disrupting its ability to bind to E2F This leads to deregulated cell proliferation, genetic instability and p16 over-expression Detectible by immunohistochemistry and immunocytochemistry staining 12

Knowledge check The normal function of p16 is to place a dividing cell into. a. proliferation b. cell arrest c. a malignant state d. None of the above Over-expression of p16 in a cervical squamous cells may indicate the following? a. normal cell division process b. cell cycle de-regulation c. The binding of E7 onco-protein to prb d. B and C 13

The mechanism of action for p16 in cervical disease The functional utility of CINtec PLUS Cytology Principles of interpretation Troubleshooting pitfalls and artifacts 16

p16 mechanism principle summary An objective biomarker for cervical disease p16 plays a key role in cell cycle regulation, and as a result, has potential applications in other cancers Cells where HPV oncoproteins have started cellular transformation processes (oncogenesis) have been shown to over-express p16 Therefore, p16 over-expression can be used as a marker for hr-hpv related cervical disease, measuring the carcinogenic activity at the cellular level, independent of hr-hpv subtype or patient age Data produced through clinical trials and KOL-lead studies demonstrates a marked increase in specificity for high-grade disease 17

Proliferation marker Ki-67 Nuclear protein can be detected in proliferating cells with Ki-67 Expression restricted to the G1-, S-, G2 and M-phase of the cell cycle Marker of cell proliferation No expression in non-dividing cells Absent in G0-phase of the cell cycle Proliferating cells in normal squamous cervical epithelium show nuclear Ki-67 staining Normal squamous epithelium 18

p16 P16 expression in normal cells Cell cycle arrest 19

p16, Ki-67 P16 expression in normal cells Ki-67 expression in normal cells Cell cycle arrest Cell cycle progression/ proliferation 20

p16, Ki-67 and co-expression CINtec PLUS Cytology P16 expression in normal cells Ki-67 expression in normal cells Simultaneous co-expression Cell cycle arrest Cell cycle progression/ proliferation Cell cycle deregulation 21

p16, Ki-67 and co-expression CINtec PLUS Cytology Simultaneous expression of p16 and Ki-67 is mutually exclusive of each other in cells with intact cell cycle control p16/ki-67 dual staining in the same cell indicates cell cycle deregulation Identification of double-immunoreactive cells in cervical cytology preparations can be an indicator for the presence of highgrade cervical dysplastic lesions p16 Ki-67 22

CINtec PLUS Cytology Immunocytochemistry procedure p16 Ki-67 23

Knowledge check Ki-67 protein can be detected in cells that are. a. Proliferating b. At cell arrest c. Dying d. A and C The co-localization of p16 and Ki-67 in a Cervical Squamous cell can indicate. a. The presence of high-grade cervical dysplastic lesions b. Cell cycle de-regulation c. The binding of E7 HPV Oncoprotein to prb d. All of the above 24

The mechanism of action for p16 in cervical disease The functional utility of CINtec PLUS Cytology Principles of interpretation Troubleshooting pitfalls and artifacts 27

Interpretation of CINtec PLUS Cytology p16/ki-67 Dual stain Locator function: Ki-67 Screen slide for p16/ki-67 Dual-stained cells Interpreter function: p16 At least one Dual-stained cell present? Yes No CINtec PLUS Positive CINtec PLUS Negative 28

Definition of a dual-stained cell The cytoplasm is stained brown and the nucleus stained red p16 signal (brown) and Ki-67 signal (red) within the same cell Any level of expression, weak or strong The red stained nucleus and the brown stained cytoplasm must be within the same plane of focus (level) Interpretation is independent from morphologic criteria One dual-stained cell indicates a positive test result 29

Single and dual-stained cells Example from the same case Single p16 Single Ki-67 Dual-stained cell 30

Only p16 stained cells 31

Only p16 stained cells 32

Only Ki-67 stained cells 33

Single dual-stained cells 34

Dual-stained cells 35

Group of dual-stained cells 36

Group with dual-stained and p16 only cell 37

Dual-stained cells, low level expression Weak brown or red staining, is positive with co-expression 38

Cluster with dual-stained cells 20x 39

Cluster with dual-stained cells 60x 40

Cluster with dual-stained cells 40x 41

CINtec PLUS Cytology staining results Negative Result No dual-stained cells present Positive Result Detection of at least one dual-stained cell Isolated cell(s) or within cell cluster 42

Recommended screening procedure CINtec PLUS Cytology Systematic overlapping fields of view Initially search for isolated dual-stained cells Easiest to evaluate If an isolated dual-stained cell has been identified, the stained nucleus and cytoplasm must be clearly allocated to the same cell Verify the cytoplasm and nucleus are in the same plane of focus (no overlying or underlying red nucleus) When evaluating cell clusters with red and brown staining, an interpretation algorithm must be followed 43

Interpretation algorithm for cell clusters Interpretation of red and brown stained cell clusters 1. Look for dual-stained cells at the edge of the cluster If yes test result is positive, dual-stained cells present If no proceed with step 2 2. Check if the p16 staining of the cluster can be defined as diffuse or focal Focal p16 staining: no further evaluation of this negative cluster Diffuse p16 staining: proceed with step 3 3. Check if the p16 staining is specific to the cytoplasm p16 staining is not specific (mucus or non-specific background staining) no further evaluation, a negative cluster Specific p16 staining: proceed with step 4 4. Are the Ki-67 staining, red nuclei over/underlying or embedded? (use the fine focus knob) Ki-67 staining, red nuclei are over/underlying no further evaluation, a negative cluster Ki-67 staining, red nuclei are embedded test result is positive dual-stained cells present 44

Interpretation algorithm for cell clusters Cell cluster checklist Separated cells at the edge of the cluster yes / no P16 staining pattern focal / diffuse Specific staining yes / no Ki-67-positive nuclei embedded in the cluster (same plane of focus) yes /no 45

Knowledge check The 2 cell group in the image indicates the following: a. A positive CINtec PLUS Cytology result b. A negative CINtec PLUS Cytology result c. Is difficult to evaluate d. None of the above True or False: p16 staining reaction is red and Ki- 67 staining reaction is brown True False 46

Cluster examples 49

Cluster with dual-stained cells 50

Cluster with dual-stained cells 51

One isolated dual-stained cell at the edge 52

Cluster with dual-stained cells 53

No dual stain in individual cells 54

Cluster with no dual-stained cells 55

The mechanism of action for p16 in cervical disease The functional utility of CINtec PLUS Cytology Principles of interpretation Troubleshooting pitfalls and artifacts 56

Non-specific background staining Detection chemistry chromogen can become trapped within cells, mucus or other cellular debris and could cause: Various intensities: low/medium/high Various distribution: few cells to all cells Homogeneous/heterogeneous 57

Non-specific brown background staining 20X Dual stained cells (+) 58

Non-specific brown background staining 40X Dual stained cells (+) 59

Non-specific brown background staining 60X 60

Specific staining Ki-67 (red) only Proliferating cells show only ki-67 signal (red) without background without specific p16 signal negative test result 61

Non-specific brown background staining Be aware to avoid false positive results Proliferating cells (Ki-67 positive) in combination with non-specific background staining could cause a false positive result 62

Interpretation with non-specific brown background Key recommendation for interpretation The brown p16 stain must be more intense than the nonspecific surrounding brown background to evaluate the cell(s) as positive for p16 & Ki-67 Do not evaluate cells with nonspecific brown background, regardless of the Ki-67 staining 63

Non-specific brown background Rule To avoid false-positive dual-stained cell evaluation follow this rule: A true dual-stained cell can only be called when the intensity of the brown (p16) in the cytoplasm of the cell is more intense than the surrounding brown background intensity in other cells (reference cells) 64

Dual-stained cell with non-specific background Example #1 Positive cell for CINtec PLUS Cytology: The brown p16 stain in the cytoplasm of this cell with a red Ki-67 stained nucleus is more intense than the non-specific brown surrounding background Non-specific brown background staining in reference cell 65

Dual-stained cell with non-specific background Example #2 Positive cell for CINtec PLUS Cytology: The brown p16 stain in the cytoplasm of this cell with a red Ki-67 stained nucleus is more intense than the non-specific brown surrounding background Non-specific brown background staining 66

Dual-stained cells, low level expression Example #3 Weak brown or red staining is positive with coexpression Must always compare the p16 (brown) staining to your reference cells Both negative or cells with non-specific brown background staining 67

Non-specific red background staining Red speckling /particles Non-specific red background staining will typically not interfere with interpretation If red speckling is present and is directly over or under the nucleus of a cell with p16 (brown) staining cytoplasm, use the fine focus to determine if it is true nuclear staining, or a red particle is in a different focal plane (not nuclear staining) to avoid false positive results 68

Knowledge check Non-specific background staining can be caused by detection particles (chromagen) becoming trapped within: a. Cells b. Mucus c. Cellular debris d. All of the above True or False: To successfully evaluate a dual-stained positive cell or cluster of cells, the brown background must be less than the p16 signal in the dual-stained cell or cluster? a. True b. False 69

Brown mucus background staining Artifact 72

Red bleeding into cytoplasm Artifact 73

Neutrophils staining red Artifact 74

Cornflaking Drying artifact Air drying that occurs before applying aqueous mounting media 75

Fading of the fast red stain Artifact from improper post processing procedure (mounting/coverslipping) Any contact with alcohol leads to Fading of Fast Red chromogen Aqueous mounting procedure to prevent fading The use Roche Hematoxylin, do not use alcoholic Hematoxylin Exposed to ETOH one day later: Cause: contact with alcohol during mounting/coverslipping procedure or counterstaining 76

Aqueous mounting procedure Cracking Artifact Cracks: A result of incomplete drying of aqueous mounting media 77

CINtec PLUS Cytology Virtual quiz Knowledge check 78

Endocervical glandular cells 1. Isolated cell Cluster 2. Dual stained cells at the edge of the cluster 3. p16 staining pattern focal diffuse 4. Specific p16 staining yes no 5. Ki-67-positive nuclei are embedded in the cluster (in same plane) yes no 6. Result with CINtec PLUS positive negative 79

Endometrial glandular cells 1. Isolated cell Cluster 2. Dual stained cells at the edge of the cluster 3. p16 staining pattern focal diffuse 4. Specific p16 staining yes no 5. Ki-67-positive nuclei are embedded in the cluster (in same plane) yes no 6. Result with CINtec PLUS positive negative 81

Tissue repair 1. Isolated cell Cluster 2. Dual stained cells at the edge of the cluster 3. p16 staining pattern focal diffuse 4. Specific p16 staining yes no 5. Ki-67-positive nuclei are embedded in the cluster (in same plane) yes no 6. Result with CINtec PLUS positive negative 83

Immature metaplastic cells 1. Isolated cell Cluster 2. Result with CINtec PLUS positive negative 85

Immature metaplastic cells 1. Isolated cell Cluster 2. Result with CINtec PLUS positive negative 86

Maturing metaplastic cells 1. Isolated cell Cluster 2. Dual stained cells at the edge of the cluster 3. p16 staining pattern focal diffuse 4. Specific p16 staining yes no 5. Ki-67-positive nuclei are embedded in the cluster (in same plane) yes no 6. Result with CINtec PLUS positive negative 87

LSIL 1. Result with CINtec PLUS positive negative 89

LSIL 1. Result with CINtec PLUS positive negative 90

1. Isolated cell Cluster 2. p16 signal (brown) and Ki-67 signal (red) within the same cell yes no 3. Result with CINtec PLUS positive negative 91

1. Isolated cell Cluster 2. p16 signal (brown) and Ki-67 signal (red) within the same cell yes no 3. Result with CINtec PLUS positive negative 92

1. Isolated cell Cluster 2. p16 signal (brown) and Ki-67 signal (red) within the same cell yes no 3. The Cytoplasm is stained brown and the Nucleus appears red yes no 4. Result with CINtec PLUS positive negative 93

1. Isolated cell Cluster 2. Dual stained cells at the edge of the cluster 3. p16 staining pattern focal diffuse 4. Specific p16 staining yes no 5. Ki-67-positive nuclei are embedded in the cluster (in same plane) yes no 6. Result with CINtec PLUS positive negative 95

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