HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
CINtec PLUS and the
Pap smear:
a co-testing alternative
Rosemary Tambouret MD
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
p16/Ki67 (CINtec
PLUS) and the Pap
smear
Rosemary Tambouret MD
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
CINtec PLUS dual stain: p16 and Ki67
• p16 is anti-proliferative marker
• cell cycle regulator by inhibition of CDK4/ CDK6 leading to cell cycle arrest in epithelial cells undergoing cellular differentiation.
• In response to transforming hrHPV, levels of p16 rise; p16 over expression is considered to be a surrogate marker for transforming hrHPV infection
• Ki67 is a marker of cycling cells
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
CINtec PLUS
p16 in normal squamous cells: no staining or brown cytoplasmic staining and no red stain of Ki67as the cell is in G0 Clone E6H4
Ki67 nuclear stain if normal cell is cycling but no p16 staining is identified Clone 274-11 AC3
Dual staining for p16 and Ki67 correlates with cell cycle dysregulation and with the presence of high risk HPV in women at risk for HSIL (independent of morphology)
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Screening for cervical cancer
• Single test: Pap, hrHPV
• Single test with triage:
• Pap -> hrHPV
• hrHPV -> Pap or genotyping
• hrHPV-> CINtec PLUS or other tests
• Co-testing:
• Pap and hrHPV
• Pap and CINtec PLUS
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Studies evaluating use of dual p16/Ki67 stain
in cervical cancer screening
• Co-testing with cytology and hrHPV • Ikenberg H et al. J Natl Cancer Inst 2013;105:1550-7
• Cytology negative, hrHPV positive • Petry KU et al. Gynecol Oncol 2011; 121:505-9
• Wentzensen N et al JNCI 2015:107: (12)
• Abnormal cytology, hrHPV positive • Schmidt D et al. Cancer Cytopathol 2011;119:158-66
• Uijterwaal MH et al BJCancer 2014; 110:1579-86
• Bergeron C et al Cancer Cytopathol 2015;123:373-81
• Wentzensen N et al J Natl Cancer Inst 2015;107 (12)
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Screening for cervical cancer precursors with
p16/Ki67 dual-stained cytology: PALMS
• 25,577 women >18 years at 196 centers
• Screening with hrHPV (HC2), cytology (CS,
TP or SP), dual stain
• 21,909 all negative (86%)
• Positive tests
• 5.4% dual stain
• 5.2% cytology ASC-US+
• 10.7% HC2
Ikenberg H et al. J Natl Cancer Inst 2013;105:1550-7
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Screening for cervical cancer precursors with
p16/Ki67 dual-stained cytology: PALMS
• Colposcopy done following any positive
test except in women <30 years old with
only HPV positive
• Cervical biopsies taken as clinically
indicated
• When no biopsy taken, the patient was
considered to have no clinical disease
Ikenberg H et al. J Natl Cancer Inst 2013;105:1550-7
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
PALMS: Test results
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Sensitivity, specificity, predictive value
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
PALMS: Diagnostic performance for CIN2+
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
PALMS: Conclusions
• Because 3 tests applied to all women, can tease out “Pap-dual stain co-testing” results
• hrHPV testing is most sensitive test, suggesting may be used as primary screen
• Dual stain has higher sensitivity than Pap but nearly equivalent specificity
• Confirmed that hrHPV prevalence too high in women <= 30 years
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
PALMS: Dual stain triage for abnormal cytology Bergeron C et al Cancer Cytopathol 2015;123:373-81
• Large prospective study, >1100 women with ASCUS/LSIL
cytology
• ASCUS triage: DS sensitivity near or same as HPV, but
specificity higher for DS
• Women < 30 with ASCUS would have almost 50% less
referral to colposcopy
• LSIL triage: HPV more sensitive than DS but much less
specific;
• referral rates for DS slightly >50% and risk CIN3+ <2% in
DS neg group
• Results in LSIL group vary by cytology method
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
PALMS: Dual stain triage for abnormal cytology Bergeron C et al Cancer Cytopathol 2015;123:373-81
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Triage cytology negative hrHPV positive
results with dual stain Petry KU et al. Gynecol Oncol 2011;121:505-9
• Prospective Pap (TP)/HPV (HC2) co-testing
trial 2007-8 of women >= 30 years
• In subgroup of 425 women negative TP/pos HC2, the dual stain was used
• Usual follow up for this group is by HPV and/or Pap in 6-12 months
• 147 colposcopy results (1/3 of cohort), biopsy in 132, 64 no follow-up visit
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Triage cytology negative hrHPV positive
results with dual stain Petry KU et al. Gynecol Oncol 2011;121:505-9
• 108 samples dual stain positive with 34 CIN2+
• 317 samples dual stain negative with 3 CIN2+
• Sensitivity of dual stain for CIN2+ 91.9%
• Specificity of dual stain for CIN2+ 82.1%
• Conclusion: Use of dual stain to triage Pap negative/HPV positive women will identify >90% of underlying CIN2+
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
p16/Ki67 dual stain cytology for detection of
precancer in HPV-positive women Wentzensen N et al JNCI 2015;107 (12)
• Prospective trial of HPV/cytology co-testing begun 2003 in women >=30 yrs
• Evaluated performance of dual stain in all HPV positive women and in a cohort of HPV positive/cytology negative women
• Samples collected over 3 months in 2012, dual stain on HPV pos samples from 1509 women
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
p16/Ki67 dual stain cytology for detection of precancer in
HPV-positive women Wentzensen N et al JNCI 2015;107 (12)
• ASCUS 383 (25.4%)
• LSIL 319 (21.1%)
• ASC-H 36 ( 2.4%)
• HSIL 49 ( 3.3%)
DS positivity increased with lesion severity
All women with cancer were DS positive
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Performance of DS compared to ASCUS+
Wentzensen N et al JNCI 2015;107 (12)
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Test performance of dual p16/Ki67 stain in HPV-
positive/cytology-negative women
Wentzensen N et al JNCI 2015;107 (12)
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Risk of cervical precancer for DS results
in relation to clinical mgt thresholds
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
Risk of cervical precancer for DS results
in relation to clinical mgt thresholds
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
p16/Ki67 dual stain cytology for detection of precancer in HPV-positive women: Conclusions
• DS had lower positivity and higher
sensitivity and specificity than cytology at
ASCUS threshold despite better than most
cytology screening performance at KPNC
• Using established risk thresholds to
HPV+/cyto-
• DS+ surpassed the colposcopy risk level
• DS- was below risk level for one year f/u
Wentzensen N et al JNCI 2015;107 (12)
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
European Equivocal of Mildly Abnormal Pap
Cytology Study (EEMAPS) and dual stain Schmidt D et al Cancer Cytopathol 2011;119:158-66
• Dual stain on TP from prior retrospective study using p16
• ASCUS and LSIL 5 European cytology labs
• HPV testing by HC2
• Biopsy within 6 months into 2 categories
• Disease = CIN2+
• No disease = CIN1 or negative for dysplasia
• Cases enriched for CIN2+
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
EEMAPS and dual stain results ASCUS
DS identifies about same proportion of HSIL as HPV testing
but reduces number of women referred to colposcopy, most evident
in group of women aged 18-29
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
EEMAPS LSIL Pap and dual stain results
DS in LSIL has about the same sensitivity for CIN2+ but with
a much higher specificity, DS could be used to triage LSIL
HARVARD MEDICAL SCHOOL
MASSACHUSETTS GENERAL PHYSICIANS ORGANIZATION
p16/Ki67 dual stain in screening for
cervical cancer
• Mounting evidence for a role in
• Triage of ASCUS and LSIL
• Triage of co-testing results: cytology
negative/hrHPV positive
• Triage of primary hrHPV test positive women
• Future developments
• Cost analysis
• Automation