Squamous neoplasia of the uterine cervix: classification, biomarkers
and recommendationsJaume Ordi M.D., Ph.D.Professor of Pathology
Department of PathologyHospital Clínic. University of Barcelona
Conflict of interest/Funding
X None
Company:____________________________
□ Product royalties
□ Paid consultant
□ Research support
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
LSIL HSIL
TreatmentFollow-up
LAST/WHO 2014 classification
Grade HPV infection Significance Management
Low-grade SIL Transient - Follow-up
High-grade SIL Persistent Pre-cancer Conization
• Two categories:
Use of biomarkers in LAST/WHO 2014• Biomarkers should be used in several circumstances to adequately
classify HPV associated lesions
• p16
• Ki67
• HPV L1
• nm 23
• ISH (HPV)
• TOP2A
• MCM2
• ProEx C (TOP2A+MCM2)
Use of biomarkers in LAST• Biomarkers should be used in several circumstances to adequately
classify HPV associated lesions
• p16
(+)
(-)
LAST recommendations for p161. Differential diagnosis between pre-cancer (HSIL) and mimickers
(immature metaplasia, atrophy, reparative changes, tangential cutting)
p16 and CIN2+ Sensitivity
Klaes (Am J Surg Pathol, 2002) 100%
Hariri (Int J Gynecol Pathol, 2007) 100%
Ordi (Int J Gynecol Pathol, 2009) 99%
Benevolo (Histopathology, 2010) 96%
0.4-0.6 Moderate
Inter-observer agreement. H&E vs p16
Bergeron C, Ordi J, et al. Am J Clin Pathol 2010; 133: 395
0.6-0.8 Substantial
• Kappa 0.58 to 0.73 (CERTAIN study) (Stoler MH, et al. Am J Surg Pathol 2018; 42 1001-9)
LAST recommendations for p16
p16 and CIN2+ Sensitivity
Klaes (Am J Surg Pathol, 2002) 100%
Hariri (Int J Gynecol Pathol, 2007) 100%
Ordi (Int J Gynecol Pathol, 2009) 99%
2. Differential diagnosis between LSIL and HSIL/CIN2
a. Positive staining supports HSIL
b. Negative staining favors LSIL (or a non-HPV-associated lesion)
p16 and CIN2+ Sensitivity
Klaes (Am J Surg Pathol, 2002) 100%
Hariri (Int J Gynecol Pathol, 2007) 100%
Ordi (Int J Gynecol Pathol, 2009) 99%
Benevolo (Histopathology, 2010) 96%
Galgano (Am J Surg Pathol, 2010) 87%
Kong (Am J Surg Pathol, 2007) 82%
Wang (Clin Cancer Res, 2004) 81%
Guo (Am J Clin Pathol, 2011) 79%
LSIL HSIL
Treatment
p16 in CIN2: increasing the threshold
p16 special circumstance (LAST)• Biopsy specimens interpreted as ≤ LSIL/CIN1 at high risk for missed
high grade disease
• Pap smear with HSIL, ASC or AGC result
• HPV16
• Biopsy specimens interpreted as ≤ LSIL/CIN1 at high risk for missed high grade disease
• Pap smear with HSIL, ASC or AGC result
• HPV16
• Any identified p16 positive area must meet H&E morphological criteria for HSIL to be diagnosed as such
p16. HPV+ and negative biopsy
Ordi J, et al. Int J Gynecol Pathol 2009; 28: 90-97
Final biopsy evaluation
p16 N No lesion (n=107)
LSIL/CIN1 (n=13)
HSIL/CIN2-3 (n=13)
p value
Negative 105 103 (98%) 2 (2%) 0 (0%) <0.001
Focal 10 4 (40%) 6 (60%) 0 (0%) <0.001
“Block” staining 24 0 (0%) 5 (21%) 19 (79%) <0.001
• p16 and histological revision in 139 women with negative biopsy and simultaneous positive HPV testing
- 18 -
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
• LAST/WHO2014: HSIL is a single category (May be further qualified with the CIN terminology [CIN2, CIN3])
Klaes et al, Am J SurgPathol 2002 26:1389
Study CIN3 CIN2
Klaes (Am J Surg Pathol 2002) 100% 100%
Ordi (Int J Gynecol Pathol 2009) 100% 99%
• Poor inter-observer reproducibility of CIN2-3 grading
• No differences in terms of biomarker abnormalities (p16)
HSIL a single category: rationale
Spontaneous regression of CIN2
Tainio K, et al. BMJ 2018; 360: K499
del Pino M, et al. ObstetGynecol 2010; 116: 1324-31
Regression of CIN1
Spontaneous regression of CIN2 • Spontaneous regression particularly frequent in women ≤30y: risk of
overtreatment of potentially non-progressive or regressive lesions
• Most investigators in the HPV field, separate HSIL/CIN2 and HSIL/CIN3
Moscicki AB, et al. ObstetGynecol 2010; 116: 1373-80
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
p16 in LSIL/CIN1
Ordi J, et al. Int J Gynecol Pathol 2009; 28:90-97
p16 immunostaining
Final diagnosis nNegative (n=182)
Focal(n=38)
Block pattern(n=106)
No lesion 161 153 (95%) 8 (5%) 0 (0%)
LSIL/CIN1 85 29 (34%) 29 (35%) 27 (32%)
HSIL/CIN2-3 80 0 (0%) 1 (1%) 79 (99%)
p16 as progression marker in LSIL
Author n Sensitivity Specificity PPV NPV
Hariri (2007) 91 96 37 35 96
del Pino (2009) 138 100 49 18 100
p16 as progression marker in LSIL
Author n Sensitivity Specificity PPV NPV
Hariri (2007) 91 96 37 35 96
del Pino (2009) 138 100 49 18 100
Cortecchia (2013) 610 59 72 18 94
Mills (2015) 524 59 54 15 91
Sagasta (2016) 507 71 56 25 90
Sagasta A, et al. Mod Pathol; 2016; 29: 51-59
- 27 -
Other biomarkers of progression• HPV16 and 18 have
significantly higher risk of progression compared with other HPV types
Rodriguez-Trujillo A, et al. Am J Clin Pathol 2018; 150:432-40
- 28 -
Other biomarkers of progression
del Pino M, et al. Int J Mol Sci; 2019; 20: E2262 Leeman A, et al. In J Cancer 2019; 144: 160-8
CADM1 MAL miR124
• Promoter methylation of tumor suppressor genes (FAM19A4, CADM1, MAL, miR124-2, HPV CpG sites) increases with the severity of the lesion
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
HPV and cervical carcinoma
de Sanjose S, et al. Lancet Oncol 2010; 11: 1048-56
n= 136
HPV-negative cervical cancer
Rodriguez-Caruchio L, et al. BJOG 2014
HPV-negative cervical cancer
Rodriguez-Caruchio L, et al. BJOG 2014
HPV-negative (n=21)
HPV-positive (n=193)
p
Age * 57.7 (15.9) 50.5 (15.3) 0.04Clinical presentation <0.01
Clinical symptoms 20 (95.2) 119 (61.7)Abnormal Pap smear 1 (4.8) 74 (38.2)
Histological type <0.01Squamous cell carcinoma 12 (57.1) 156 (80.8)Adenocarcinoma 6 (28.6) 33 (17.1)Adenosquamous carcinoma 1 (4.8) 3 (1.6)Neuroendocrine carcinoma 2 (9.5) 1 (0.5)
HPV-negative cervical cancer
Nicolas I, et al. Mod Pathol 2019; 32: 1189-96
Case Age Histological type p16 p53 FIGO1 64 SCC, non-keratinizing - nm IIIB2 57 SCC, sarcomatoid - nm IVA3 83 SCC, non-keratinizing - abn IIIB4 69 Neuroendocrine carcinoma - abn IVB5 85 SCC, non-keratinizing - nm IIIA6 51 Neuroendocrine carcinoma - nm IIIB7 49 ADC, mucinous - abn IIIB8 41 ADC, mucinous - abn IIB9 61 SCC, keratinizing - abn IIB10 53 SCC, non-keratinizing + abn IIIB11 54 ADC, mucinous + abn IIIB12 36 Adenosquamous carcinoma + nm IIB13 82 ADC, mucinous + abn IIIB14 38 SCC, non-keratinizing + abn IB115 53 SCC, non-keratinizing + abn IIIB16 32 ADC, mucinous + abn IB117 64 SCC, non-keratinizing + abn IIB18 40 SCC, non-keratinizing + abn IIIB19 66 ADC, mucinous + nm IVB20 54 SCC, non-keratinizing + abn IIB21 80 SCC, non-keratinizing + abn IVA
HPV-negative cervical cancer
HPV-negative (n=21)
HPV-positive (n=193)
p
Tumor size* 54.7 (22.4) 36.8 (21.2) <0.01FIGO staging <0.01
Early (IA-IB1) 2 (9.5) 83 (43.0)Advanced (IB2-IV) 19 (90.5) 110 (57.0)
Lymph node metastasis 14 (66.7) 69 (35.8) <0.01
Nicolas I, et al. Mod Pathol 2019; 32: 1189-96
HPV-negative cervical cancer
HPV-negative CC: survival
Nicolas I, et al. Mod Pathol 2019; 32: 1189-96
p=0.01 p=0.01
HPV-negative squamous CC: survival
Nicolas I, et al. Mod Pathol 2019; 32: 1189-96
HPVHPV
p=0.04p=0.01
HPV-negative CC: overall survival
Univariable Cox model Multivariable Cox model
HR 95% CI p HR CI 95% p
Non SCC histology 0.7 0.3-1.5 0.35 - - -
Negative HPV status 2.5 1.2-5.1 0.01 0.9 0.4-2.3 0.44
p16 negative 3.5 1.6-7.4 <0.01 1.9 0.8-5.1 0.14
Advanced FIGO stage 43.7 6.0-316.5 <0.01 23.8 3.1-177.6 <0.01
Lymph node metastasis 7.1 3.7-13.7 <0.01 3.2 1.7-6.0 <0.01
Nicolas I, et al. Mod Pathol 2019; 32: 1189-96
Disease-free survival HPV-negative CC
Li P et al. Oncotarget 2017; 8: 66352-9
Overall survival HPV-negative CC
Li P et al. Oncotarget 2017; 8: 66352-9
Outline1. Squamous intraepithelial lesions (SIL) of the uterine cervix:
a) LAST /WHO 2014 classification and use of biomarkers (p16)
b) Should HSIL/CIN2 and HSIL/CIN3 be considered a single disease?
c) p16 and progression risk of LSIL/CIN1. Other biomarkers of progression
2. Squamous cell carcinoma of the uterine cervix:
a) HPV negative squamous cell carcinomas: do they exist?
b) Value of HPV genotyping, p16 staining. Prognostic implications
HPV genotypes in cervical cancer
Nicolas I, et al. Mod Pathol 2019; in press
HPV16 and/or 18 (n=156)
Other HPV types(n=38)
p
Age * 49 (15) 57 (17) <0.01
Histological type 0.01
Squamous cell carcinoma 119 (76) 38 (100)
Adenocarcinoma 33 (21) - -
Adenosquamous carcinoma 3 (2) - -
Neuroendocrine carcinoma 1 (1) - -
FIGO 2009 staging 0.22
Early (IA-IB1) 62 (40) 11 (29)
Advanced (IB2-IV) 94 (60) 27 (71)
Relapse 31 (20) 11 (29) 0.22
Lymph node metastasis 55 (35) 15 (40) 0.62
HPV genotypes and survival
Nicolas I, et al. Mod Pathol 2019; in press
p16 p16 p16
Nicolas I, et al. Mod Pathol 2019; in press
p16 negativity in HPV + CC: survival
“Take home” messages (SIL)• p16 is a useful tool in the differential diagnosis between HSIL/CIN2+
and the mimics of pre-cancer and may help in identifying occult HSIL lesions in women at high risk for missed high grade disease
• CIN2 and CIN3 have probably a different behavior. The separation between HSIL/CIN2 and HSIL/CIN3 should be recommended
• p16 has little (or no) value as a marker of progression of LSIL/CIN1. Other biomarkers are promising, but have a limited utility in biopsy specimens
“Take home” messages (SCC)• A low percentage of SCCs arise via HPV-independent pathway. Many
of them are of conventional non-keratinizing type. These tumors may have impaired prognosis
• A negative p16 staining in patients with HPV-positive tumors may be a prognostic marker associated with a poor overall survival