Molecular Triage: Partial and Extended Genotyping and More! Thomas C. Wright, Jr. MD Professor Emeritus Columbia University, New York Pathologist, Enzo Clinical Laboratories, Farmingdale, NY
Disclosures Dr. Wright is a consultant and study pathologist for Roche and BD Diagnostics and receives payment for his services. Dr. Wright is a speaker for Roche and BD Diagnostics and receives payment for his services.
Molecular Triage Partial and extended genotyping and more! Screening approaches utilizing hrhpv testing produce considerable numbers of screen positive women Does not matter whether we are discussing cotesting or HPV primary screening With cotesting, we originally used 12 month follow-up as our triage step concern about watching cancers
Original Approach to Cotesting What is the goal we are trying to achieve? Cytology Routine screening NILM / HPV- ASCUS / HPV NILM / HPV+ ASCUS / HPV+ >ASCUS 6.7% Cotesting 12 mos What if cancer? What if fail to return? HPV Testing COLPOSCOPY Wright et al. (2011) Am J Clin Pathol
HPV Genotypes in Cervical Cancer Global distribution 70% 60% 50% 40% 30% 61% Bivalent Vaccine Nanovalent Vaccine Immediate colposcopy if HPV 16/18 would identify 70% of cancers 20% 10% 0% 10% 4% 4% 6% 3% 2% 16 18 31 33 35 39 45 51 52 56 58 59 68 de Sanjose (2010) Lancet Oncology
Cotesting with Genotyping What is the goal we are trying to achieve? Cytology Routine screening NILM / HPV- ASCUS / HPV Cotesting 12 mos NILM / HPV+ 12 other 5.2% HPV Testing ASCUS / HPV+ >ASCUS 16/18/45+ COLPOSCOPY COLPOSCOPY 1.5% Wright et al. (2011) Am J Clin Pathol
Cotesting with Genotyping What is the goal we are trying to achieve? Cytology Routine screening NILM / HPV- ASCUS / HPV Cotesting 12 mos NILM / HPV+ 12 other 2.4% CIN3+ HPV Testing ASCUS / HPV+ >ASCUS 16/18/45+ COLPOSCOPY COLPOSCOPY 9.8% CIN3+ Wright et al. (2011) Am J Clin Pathol
HPV+ Women, % HPV Primary Screening Prevalence of hrhpv by age group - ATHENA 35% 30% 30.8% Abnl Pap hrhpv 16 18 25% 21.5% 20% 15% 11.6% 10% 5% 7.1% 6.0% 0% 21-24 25-29 30-39 40-49 50+ Age Group, years Wright et al. (2011) Am J Obstet Gynecol
HPV+ Women, % HPV Primary Screening Prevalence of hrhpv by age group - ATHENA 35% 30% 25% 20% 30.8% 21.5% Abnl Pap hrhpv 16 18 Overall, 10.5% of women >25 yrs will be HPV positive 15% 11.6% 10% 5% 7.1% 6.0% 0% 21-24 25-29 30-39 40-49 50+ Age Group, years Wright et al. (2011) Am J Obstet Gynecol
Primary HPV Screening - >25 yrs HPV with 16/18 Genotyping and Reflex Cytology Routine screening HPV 12 other hrhpv+ Cytology NILM Follow up in 12 months HPV Testing HPV16/18+ ASC-US COLPOSCOPY COLPOSCOPY Wright et al. (2015) Gynecol Oncol
HPV Primary Screening Women >25 yrs in ATHENA HPV n = 36,626 (89.5%) HPV Testing n = 40,901 HPV+ n = 4,275 (10.5%) Wright et al. (2015) Gynecol Oncol
HPV Primary Screening Women >25 yrs in ATHENA HPV n = 36,626 (89.5%) HPV Testing HPV+ n = 4,275 (10.5%) HPV 12 other+ n = 3,108 (7.6%) n = 40,901 HPV 16/18+ n = 1,167 (2.9%) Wright et al. (2015) Gynecol Oncol
HPV Primary Screening Women >25 yrs in ATHENA HPV n = 36,626 (89.5%) n = 2,388 (5.8%) HPV Testing HPV+ n = 4,275 (10.5%) HPV 12 other+ n = 3,108 (7.6%) NILM n = 40,901 HPV 16/18+ >ASC-US n = 1,167 (2.9%) n = 720 (1.8%)
Comparison of Performance HPV vs Pap Performance for CIN3+ in large studies Sensitivity* Specificity* Study HPV Pap HPV Pap Mayrand et al. 1 95 57 81 61 ATHENA study 2 92 53 60 73 Onclarity study 3 93 59 50 65 1 Mayrand et al. (2006) NEJM 2 Castle et al. (2006) Lancet Oncol 3 Wright et al. (2017) Abst at IFCCP *Crude estimates
Cumulative Detection CIN3+ Predictive Value of HPV Genotype NCI Kaiser, Portland, Oregon study 16% 12% 8% 20,817 women followed up to 15 yrs HPV 16 HPV 18 HPV 31 Other hrhpv HPV neg 4% 0% Schiffman et al. JNCI, 2011 0-5 5-10 10-15 Years of Follow-up
Percent Developing CIN 3+ 12 Yr Risk CIN 3: Women with NILM Danish Pathology Data Bank follow-up study 30% 25% 8,656 women 20-29 yrs followed up to 13.5 yrs 20% 15% 10% 5% 16 18 33 31 Other HC neg 0% 0 1 2 3 4 5 6 7 8 9 10 11 12 Years of Follow-up Kjaer et al. JNCI, 2010:102; 1478
HPV Genotypes in Cervical Cancer Global distribution 70% 60% 50% 61% Bivalent Vaccine Nanovalent Vaccine 40% 30% 7 hrhpv genotypes in the nanovalent vaccine identify 90% of cancers 20% 10% 0% 10% 4% 4% 6% 3% 2% 16 18 31 33 35 39 45 51 52 56 58 59 68 de Sanjose (2010) Lancet Oncology
HPV Genotyping Assays Each assay is designed differently Hybrid Capture 16 18 31 33 45 52 58 35 39 51 56 59 68 cobas 16 18 31 33 45 52 58 35 39 51 56 59 66 68 Aptima 16 18 45 31 33 52 58 35 39 51 56 59 66 68 Xpert HPV 16 18 45 31 33 35 52 58 51 59 39 56 66 68 Onclarity 16 18 45 31 33 58 35 39 68 51 52 56 59 66
Primary HPV Screening - >25 yrs HPV with extended genotyping Routine screening HPV HPV 35, 39, 51, 56, 59, 66, 68 + Follow up in 12 months HPV Testing HPV 16, 18, 31, 33,?(45, 52, 58) + COLPOSCOPY
p16 / Ki-67 Dual-staining of Cytology Identifying women with CIN lesions p16 Mitosis Dualstained G2 G1/G0 S Cell-cycle arrest G2 Mitosis G1/G0 Coexpression of p16 and Ki-67: Indicates cell-cycle deregulation Ki-67 S Cell-cycle progression Hallmark of transforming HPV infections
Dual-stained Cytology Liquid-based cytology specimen
P16 / Ki67 Dual Stain as Triage Evaluation for primary screening in ATHENA Archived, residual LBC specimens from women who had colposcopy in ATHENA were immunostained for p16 / Ki-67 Cytology slides were screened by a cytotechnician and then reviewed by an experienced reader Slides with one or more dual-stained cells were classified as positive Wright et al. (2017) Gynecol Oncol
Comparison of strategies in >25 years 3 year cumulative incidence (CIR) of CIN 3+ 3 yr CIR for CIN 3+ (95% CI) HPV Status Dual-stain (+) Dual-stain (-) >ASCUS ASCUS NILM HPV (+) 24.6 (21.8-27.7) 3.9 (2.4-4.2) 24.5 (20.7-28.6) 12.5 (9.2-16.7) 6.4 (5.4-7.5) 16 (+) 46.1 (39.7-52.6) 9.12 (6.4-12.8) 47.7 (39.0-56.4) 22.7 (14.4-34.0) 18.2 (14.5-22.5) 18 (+) 25.0 (16.3-36.4) 4.9 (2.4-9.6) 27.2 (16.1-42.0) 14.5 (5.5-33.1) 5.9 (3.2-10.8) 12 other (+) 13.8 (11.1-17.0) 2.8 (2.1-3.7) 13.1 (9.6-17.4) 8.8 (5.6-13.4) 3.7 (2.9-4.8) HPV (-) Roche data on file, 2011. 7.3 19.6-24.8) 0.6 (0.4-0.9) 4.7 (2.7-8.1) 0.2 (0.02-1.12) 0.7 (0.4-1.1)
Women NOT Requiring Colposcopy 3-yr CIR of CIN 3+ in context of current guidelines 3 yr CIR for CIN 3+ (95% CI) HPV Status Dual-stain (+) Dual-stain (-) >ASCUS ASCUS NILM HPV (+) 24.6 (21.8-27.7) 3.9 (2.4-4.2) 24.5 (20.7-28.6) 12.5 (9.2-16.7) 6.4 (5.4-7.5) 16 (+) 46.1 (39.7-52.6) 9.12 (6.4-12.8) 47.7 (39.0-56.4) 22.7 (14.4-34.0) 18.2 (14.5-22.5) 18 (+) 25.0 (16.3-36.4) 4.9 (2.4-9.6) 27.2 (16.1-42.0) 14.5 (5.5-33.1) 5.9 (3.2-10.8) 12 other (+) 13.8 (11.1-17.0) 2.8 (2.1-3.7) 13.1 (9.6-17.4) 8.8 (5.6-13.4) 3.7 (2.9-4.8) HPV (-) Roche data on file, 2011. 7.3 19.6-24.8) 0.6 (0.4-0.9) 4.7 (2.7-8.1) 0.2 (0.02-1.12) 0.7 (0.4-1.1)
Women REQUIRING Colposcopy 3-yr CIR of CIN 3+ in context of current guidelines 3 yr CIR for CIN 3+ (95% CI) HPV Status Dual-stain (+) Dual-stain (-) >ASCUS ASCUS NILM HPV (+) 24.6 (21.8-27.7) 3.9 (2.4-4.2) 24.5 (20.7-28.6) 12.5 (9.2-16.7) 6.4 (5.4-7.5) 16 (+) 46.1 (39.7-52.6) 9.12 (6.4-12.8) 47.7 (39.0-56.4) 22.7 (14.4-34.0) 18.2 (14.5-22.5) 18 (+) 25.0 (16.3-36.4) 4.9 (2.4-9.6) 27.2 (16.1-42.0) 14.5 (5.5-33.1) 5.9 (3.2-10.8) 12 other (+) 13.8 (11.1-17.0) 2.8 (2.1-3.7) 13.1 (9.6-17.4) 8.8 (5.6-13.4) 3.7 (2.9-4.8) HPV (-) Roche data on file, 2011. 7.3 19.6-24.8) 0.6 (0.4-0.9) 4.7 (2.7-8.1) 0.2 (0.02-1.12) 0.7 (0.4-1.1)
Women REQUIRING Colposcopy Using 3-yr CIR of CIN 3+ of 8% as the cut-off 3 yr CIR for CIN 3+ (95% CI) HPV Status Dual-stain (+) Dual-stain (-) >ASCUS ASCUS NILM HPV (+) 24.6 (21.8-27.7) 3.9 (2.4-4.2) 24.5 (20.7-28.6) 12.5 (9.2-16.7) 6.4 (5.4-7.5) 16 (+) 46.1 (39.7-52.6) 9.12 (6.4-12.8) 47.7 (39.0-56.4) 22.7 (14.4-34.0) 18.2 (14.5-22.5) 18 (+) 25.0 (16.3-36.4) 4.9 (2.4-9.6) 27.2 (16.1-42.0) 14.5 (5.5-33.1) 5.9 (3.2-10.8) 12 other (+) 13.8 (11.1-17.0) 2.8 (2.1-3.7) 13.1 (9.6-17.4) 8.8 (5.6-13.4) 3.7 (2.9-4.8) HPV (-) Roche data on file, 2011. 7.3 19.6-24.8) 0.6 (0.4-0.9) 4.7 (2.7-8.1) 0.2 (0.02-1.12) 0.7 (0.4-1.1)
Women NOT Requiring Colposcopy Using 3-yr CIR of CIN 3+ of 8% as the cut-off 3 yr CIR for CIN 3+ (95% CI) HPV Status Dual-stain (+) Dual-stain (-) >ASCUS ASCUS NILM HPV (+) 24.6 (21.8-27.7) 3.9 (2.4-4.2) 24.5 (20.7-28.6) 12.5 (9.2-16.7) 6.4 (5.4-7.5) 16 (+) 46.1 (39.7-52.6) 9.12 (6.4-12.8) 47.7 (39.0-56.4) 22.7 (14.4-34.0) 18.2 (14.5-22.5) 18 (+) 25.0 (16.3-36.4) 4.9 (2.4-9.6) 27.2 (16.1-42.0) 14.5 (5.5-33.1) 5.9 (3.2-10.8) 12 other (+) 13.8 (11.1-17.0) 2.8 (2.1-3.7) 13.1 (9.6-17.4) 8.8 (5.6-13.4) 3.7 (2.9-4.8) HPV (-) Roche data on file, 2011. 7.3 19.6-24.8) 0.6 (0.4-0.9) 4.7 (2.7-8.1) 0.2 (0.02-1.12) 0.7 (0.4-1.1)
Sensitivity Triage of HPV Positive Women Use of dual-staining for risk stratification 1.00 HPV16/18&DS 0.75 DS HPV16/18&Pap 0.50 Pap 0.25 0.00 0.25 0.50 0.75 1-Specificity Wright et al. (2017) Gynecol Oncol
Triage of HPV Positive Women Use of dual-staining for risk stratification Routine screening HPV Testing HPV 12 other HPV+ Dual-stain HPV16/18+ Dual-stain ALL DS- DS+ Follow up in 12 months COLPOSCOPY COLPOSCOPY
Molecular Triage - Conclusions Partial and extended genotyping and more! Genotyping for HPV 16/18 currently plays an important role in managing HPV positive women Genotyping for HPV 16/18, 31, 33, 45 and possibly 52, 58 may be important in the future Other approaches such p16 Ki67 dual staining may also become important in the future