P16 et Ki67 Biomarkers: new tool for risk management and low grade intraepithelial lesions (LGSIL): be ready for the future.
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1 P16 et Ki67 Biomarkers: new tool for risk management and low grade intraepithelial lesions (LGSIL): be ready for the future. Mark H Stoler, MD University of Virginia Health System, Charlottesville, VA, USA
2 Disclosure of Conflicts of Interest Dr. Stoler is a consultant to: Roche/Ventana, BD Diagnostics, Hologic/GenProbe, Abbott Molecular, Cepheid, InovioPharma, & Merck.
3 OBJECTIVES 1) Demonstrate the role of p16/ki-67 dual stain for LSIL and HPV positive results 2) Analyze the clinical impact of p16/ki-67 dual stain in colposcopy referral population 3) Determine the economic implications of p16/ki-67 dual stain in screening strategy
4 General Principles of Cervical Cancer Screening: Desired Information Which women are safe to keep in routine screening? Negative predictive value (NPV); rule out High sensitivity Drives the screening interval Which women are most likely to have established high-grade disease? Positive predictive value (PPV); rule in High specificity Drives the intervention protocol
5 How Can We Make Cervical Cancer Screening More Effective and Cost Efficient? Screening Patient management Higher sensitivity as compared with Pap cytology Less frequent testing based on high NPV of single screening result Provide optimal triage tool Minimize number of false-positive screening results
6 Primary Objective Optimize triage for better patient management Understand the clinical utility of p16/ki-67 dual/stained cytology for triaging screen positives
7 Boundary Problem of Cancer Screening At a given prevalence of precancer/cancer, how do we sort the population into those who need treatment and those who do not? Prevalence estimate 1% Ideal Colpo Referral: 2% How do we take:1:100 to 1:2?
8 Role of p16 p16 INK4a a CDK inhibitor Mutated in some malignancy syndromes (e.g., melanoma) hrhpv E7 mediates overexpression via transcriptional activation Paradoxically, overexpression does not prevent cell cycle progress
9 Normal Cell Cycle Progression Release of E2F from prb results in cell cycle progression, mitotic replication and lowlevel expression of p16 p16 protein facilitates rebinding of prb to E2F, leading to cell cycle arrest This feedback control mechanism is key to maintaining balance between cell cycle progression/proliferation and cell cycle arrest
10 Transforming HPV 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 overexpression detectable by immunohistochemistry staining
11 Are Biomarkers the Solution to Our Problems? p16 positive fraction 1500 adjudicated biopsies Histology p16+, % Negative 5 CIN1 39 CIN2 77 CIN3 99 Galgano MT, et al. Am J Surg Pathol. 2010;34(8):
12 Is This Not Easier? Can histological variation be minimized by use of biomarkers?
13 Utility of p16 in Cervical Cytology First-generation p16 cytology product was based on combining p16 immunoreactivity with morphological interpretation Various study results demonstrated good sensivitity and significantly improved specificity for triaging abnormal Pap cytology Still dependent on interpretation of morphological criteria, which keeps subjectivity associated with it
14 p16/ki-67 Dual-Stained Cytology (CINtec PLUS) High sensitivity and specificity in a single test A combination of p16 PLUS Ki-67 biomarkers in a single test No morphological interpretation; cells with positive staining for both proteins exhibit Brown cytoplasmic p16 stain Red nuclear Ki-67 stain
15 p16/ki-67 Dual-Stain (CINtec PLUS Cytology) Identifying women with CIN lesions p16 Mitosis Dualstained G2 G1/G0 S Cell-cycle arrest Ki-67 Mitosis G2 G1/G0 S Cell-cycle progression Coexpression of p16 and Ki-67: Indicates cell-cycle deregulation Hallmark of transforming HPV infections
16 p16/ki-67 Dual-Stained Cytology Need only one or more dual-stained cell(s) on a cervical cytology slide to define a positive test result
17 Diagnostic Challenges in Optimal Triage LSIL LSIL is a relatively common cytological diagnosis in the United States, median rate is 2.5%-3.0% hrhpv testing can be used to triage LSIL, but it works poorly for younger women ASC-US 5%-10% of Pap examinations; role reversal in primary screening HPV testing works for ASC-US triage, but a more specific test could work better NILM 5% of women aged > 30 years are hrhpv+/nilm Need triage to optimize colposcopy referral rates
18 p16 and Ki-67 Dual Staining for Cytology Clinical studies to validate clinical utility in ASC-US/LSIL PALMS trial: prospective screening trial in more than 27,000 women across Europe EEMAPS: retrospective ASC-US/LSIL triage study Waldstrom et al: comparison of APTIMA mrna HPV assay with p16/ki-67 in women with LSIL Wentzensen et al: colposcopy clinic cohort study Killeen et al: prospective colposcopy clinic cohort
19 Primary, ASC-US, LSIL Marker Study:(PALMS) Multinational, multicenter, prospective diagnostic research study Performed in Italy, Germany, France, Spain and Belgium Screening population Enrollment of more than 27,000 women attending routine cervical cancer screening Pap cytology, dual stain test, and hc2 HPV test performed on all women Biopsy-confirmed CIN2 as disease gold standard Ikenberg H, et al. J Natl Cancer Inst. 2013, in press.
20 PALMS: Triage of LSIL Cytology (n = 526) p16/ki-67 dual-stained cytology vs HPV testing 1,0 0,8 85% 98% Dual stain (+) hrhpv (+) 84% 0,6 0,4 0,2 0,0 54% 52% 19% Sensitivity Specificity Referral Rate Biopsy-confirmed CIN2 cases: n = 63.
21 PALMS: Triage of ASC-US cytology (n=575) p16/ki-67 dual-stained cytology vs HPV testing 1,0 94% 100% Dual stain (+) hrhpv (+) 0,8 0,6 0,4 78% 61% 26% 42% 0,2 0,0 Sensitivity Specificity Referral Rate Biopsy-confirmed CIN2 cases: n = 18.
22 Performance of Dual Staining in LSIL Comparison of different studies % Referral Sensitivity CIN2 Study hrhpv CINtec Plus hrhpv CINtec Plus Wentzensen a 76% 59% 94% 86% Waldstrom 69% 56% 98% 89% PALMS 84% 52% 98% 85% EEMAPS 86% 53% 96% 94% a Both LSIL and hrhpv (+) ASC-US patients.
23 Performance of Triage Tests for CIN2 LSIL and hrhpv (+) ASC-US population Sensitivity 1 0,8 0,6 0,4 0,2 HPV16/18 CINtec PLUS hrhpv Sensitivity 1-Specificity 0 0 0,2 0,4 0,6 0,8 1 1 Specificity Wentzensen N, et al. Clin Cancer Res. 2012;18(5):
24 Triage of Pap /HPV+ Women Aged 30 Years Wolfsburg study Women aged 30 years and participating in Pap/HPV cotesting program LBC (ThinPrep) Pap cytology and hc2 HPV testing Women positive on either test were referred for colposcopy p16/ki-67 dual staining retrospectively performed on residual LBC vial (at baseline) in Pap /HPV+ women Petry KU, et al. Gynecol Oncol. 2011;121(3):
25 Triage of Pap /HPV+ Women Aged 30 Years Wolfsburg study Pap /HPV+ N = 425 Dual stain positive n = % 75% Dual stain negative n = % < CIN2 n = 74 31% 99% 1% CIN2 < CIN2 CIN2 n = 34 n = 314 n = 3 Petry KU, et al. Gynecol Oncol. 2011;121(3):
26 CAN WE REFINE PRIMARY SCREENING TO IMPROVE RISK STRATIFICATION?
27 p16/ki-67 Dual-Stained Cytology vs Pap Cytology for Triaging HPV+ Women Sub-study nested into ATHENA trial Retrospective analysis using archived residual LBC specimens > 7700 women 25 years old who were referred to colposcopy in ATHENA p16/ki-67 dual-stained cytology slides prepared (from TP archived up to 4.5 years) and interpreted; slides with 1 dual-stained cell were classified as positive Dual-stained cytology and Pap cytology results compared to histologic endpoint (243 CIN3+) TP, true positives. Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
28 NET 243 Evaluable CIN3+ Women aged 25 years with valid biopsy and cobas HPV Test result in ATHENA n = 7727 cobas HPVpos n = 3467 cobas HPVneg n = 4260 Dual stainpos n = 983 Dual stain neg n = 2355 Dual stain N/A N = 129 Dual stain pos n = 159 Dual stain neg n = 3916 Dual stain N/A N = CIN2+ 76 CIN2 166 CIN3 13 ACIS 3CxCa 109 CIN2+ 48 CIN2 60 CIN3 1 ACIS 5 CIN2+ 3 CIN2 2 CIN3 13 CIN2+ 3 CIN2 8 CIN3 2 ACIS 29 CIN2+ 21 CIN2 8 CIN3 2 CIN2+ 1 CIN2 1 CIN3 725 CIN1 572 No CIN 153 CIN1 2,246 CIN1 2,050 No CIN 196 CIN1 124 CIN1 110 No CIN 14 CIN1 146 CIN1 124 No CIN 22 CIN CIN1 3,697 No CIN 190 CIN1 183 CIN1 174 No CIN 9 CIN1 Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
29 p16/ki-67 Dual-Stained Cytology vs Pap Cytology for Triaging HPV+ Women Sensitivity Specificity PPV NPV Triage of HPV(+) Women 25 Years CIN3+ p16/ki-67 Dual-Stain Pap Cytology P Value 74.9% (69.1, 79.9) 74.1% (72.5, 75.6) 18.5% (17.1, 20.0) 97.4% (96.8, 97.9) 51.9% (45.6, 58.1) 75.0% (73.5, 76.5) 14.0% (12.5, 15.7) 95.2% (94.6, 95.8) Dual-stain cytology reduces residual risk by 50% < < < Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
30 Comparison of Strategies in 25 years 3-year cumulative incidence (CIR) of CIN3+ 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) ASC-US, atypical squamous cells of undetermined significance. NILM, negative for intraepithelial lesion or malignancy. Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file.
31 Women NOT Requiring Colposcopy 3-year CIR of CIN3+ in context of current guidelines 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file
32 Women REQUIRING Colposcopy 3-year CIR of CIN3+ in context of current guidelines 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file
33 Women NOT Requiring Colposcopy Using 3-year CIR of CIN3+ of 8% as the cutoff 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file
34 Women REQUIRING Colposcopy Using 3-year CIR of CIN3+ of 8% as the cutoff 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file
35 Women at Particularly High Risk of CIN3+ 3-year CIR of CIN3+ 3-Year CIR for CIN3+ (95% CI) HPV Status Dual-Stain (+) Dual-Stain ( ) > ASC-US ASC-US NILM HPV ( ) 3.9 ( ) 24.5 ( ) 12.5 ( ) 6.4 ( ) ( ) 9.12 ( ) 47.7 ( ) 22.7 ( ) 18.2 ( ) ( ) 4.9 ( ) 27.2 ( ) 14.5 ( ) 5.9 ( ) 12 other ( ) 2.8 ( ) 13.1 ( ) 8.8 ( ) 3.7 ( ) HPV 7.3 ( ) 0.6 ( ) 4.7 ( ) 0.2 ( ) 0.7 ( ) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1): Roche, data on file
36 Performance of Different Triage Algorithms For Detection of CIN3+ in HPV+ Women (n= 243) Triage all HPV+ with DS Estimate (95%CI) Triage all HPV+ with Pap Cytology Estimate (95% CI) HPV16/18 to Colposcopy and Triage 12 other hrhpv+ with DS Estimate (95% CI) HPV16/18 to Colposcopy and Triage 12 other hrhpv+ with Pap Cytology Estimate (95% CI) Sensitivity 79.9% (69.0, 80.2) 51.9% (45.4, 58.3) 86.8% (81.9, 90.8) 78.2% (72.5, 83.2) [182/243] [126/243] [211/243] [190/243] Specificity 74.1% (72.5, 75.7) 75.0% (73.5, 76.5) 57.4 (55.7, 59.2) 57.6% (55.9, 59.4) [2294/3095] [2321/3095] [1777/3095] [1784/3095] PPV 18.5% (16.1, 21.1) 14.0% (11.8, 16.5) 13.8%, (12.1, 15.6) 12.7% (11.0, 14.4) NPV 97.4% (96.7, 98.0) 95.2% (94.3, 96.0) 98.2% (97.5, 98.8) 97.1% (96.2, 97.8) DLR (2.634, 3.179) (1.812, 2.377) (1.913, 2.173) (1.707, 1.996) DLR ( ) (0.562, 0.732) (0.166, 0.317) (0.298, 0.481) Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
37 Diagnostic Performance for the Detection of CIN3+ Receiver operating characteristic graph for triaging HPV-positive women ages 25 years: (i) by referring HPV16/18-positive women to colposcopy and triaging 12 other hrhpv-positive women with Pap cytology (HPV16/18 & Pap) (ii) by referring HPV16/18-positive women to colposcopy and triaging 12 other HR-HPV-positive women with dual-stained cytology (HPV16/18&DS) (iii) with cytology (Pap) (iv) with dual-stained cytology (DS) Sensitivity DS Pap HPV16/18 & DS HPV16/18 & Pap Specificity Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
38 Performance Characteristics of Different Triage Algorithms: Detection of CIN3+ vs Colposcopies Needed (n = 243) Triage Algorithm Number of CIN3+ Detected Number of Colposcopies Number of Colposcopies per CIN3+ Detected Triage all HPV(+) with dual stain 182/ Triage all HPV(+) with Pap 126/ HPV16/18 to colpo and triage 12 other hr-hpv(+) with DS HPV 16/18 to colpo and triage 12 other hr-hpv(+) with Pap 211/ / CIN3+, cervical intraepithelial neoplasia grade 3 or worse; hrhpv, high-risk human papillomavirus. Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
39 Triage of HPV Positive Women Use of p16/ki-67 dual-stained cytology for risk stratification hrhpv HPV HPV16/18+ Routine screening 12 other HPV+ Dual-stain Dual-stain ALL DS DS+ Follow up in 12 months COLPOSCOPY COLPOSCOPY Wright TC Jr, et al. Gynecol Oncol. 2017;144(1):51-56.
40 Management Based on Risk Stratification Risk of CIN3+ in ATHENA Possible Management Risk that CIN3+ is present 0.3% 0.8% 2.9% 8.3% 50% a HPV / NILM 80% a NILM ASC-US HPV+ / ASC-US ASC-H or AGC HSIL HPV-POS/DS-NEG HPV-POS/DS-POS HPV16 DS+ Wright TC, et al. Gynecol Oncol. 2015;136(2): ; Stoler MH, et al. Am J Clin Pathol. 2011;135(3): a Saslow et al. American Society for Colposcopy and Cervical Pathology Guidelines. JLGTD. 2012; 16, (3); Katki et al. Lancet Oncology. 2011; 12(7), ; Other Sources
41 p16/ki-67 Dual Staining of Cytology Data to support use in several settings As a triage method for women with ASC-US especially younger women or HPV 16/18 As a triage method for women with LSIL As a triage method for women with NILM cytology who are HPV+; likely in conjunction with genotyping for HPV 16/18 As a potential triage for HPV+ women when HPV testing is the primary cervical cancer screening
42 Conclusions Clinically validated HPV tests are specifically designed to balance sensitivity with specificity The performance of HPV testing in primary screening depends on the screening algorithm (i.e. how HPVpositive women are triaged) Data support p16/ki-67 dual-stained cytology as a better triage approach
43 Thank you!
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