AJCP / Original Article
The LAST Guidelines in Clinical Practice
Implementing Recommendations for p16 Use
Lani K. Clinton, MD, PhD,1,2 Kyle Miyazaki,1 Asia Ayabe,1 James Davis, PhD,2
Pamela Tauchi-Nishi, MD,1,2 and David Shimizu, MD1,2
From the 1Hawaii Pathologists’ Laboratory, Queen’s Medical Center, Honolulu, and 2John A. Burns School of Medicine, University of Hawaii, Honolulu.
Key Words: p16; LAST guidelines; Cervical pathology; Gynecologic pathology
Am J Clin Pathol December 2015;144:844-849
DOI: 10.1309/AJCPUXLP7XD8OQYY
ABSTRACT
Objectives: To determine the impact of implementing p16
Lower Anogenital Squamous Terminology Standardization
Project for HPV-Associated Lesions (LAST) guidelines,
we compared p16 use and follow-up data before and after
implementation of the guidelines.
Methods: We reviewed all cervical biopsy specimens
diagnosed by two pathologists before and after
implementation of the LAST guidelines and calculated the
rate of and reason for p16 use across all biopsy specimens,
high-grade squamous intraepithelial lesion (HSIL) detection,
and follow-up.
Results: In total, 1,829 and 1,623 cervical biopsy
specimens were reviewed in periods A and B, respectively.
Overall p16 use increased from 2.8% to 6.2% (P < .001).
Recommendations 2 and 4 increased from 0.16% and 0% of
all cervical biopsy specimens in period A to 1.4% and 1.9%
in period B, respectively (P < .0001). p16+ HSIL increased
from 1.4% to 2.3% (P < .05). The positive predictive value of
p16+ HSIL increased from 48% to 76% (P < .05).
Conclusions: Implementation of the p16 LAST guidelines
resulted in a significant increase in p16 use and a significant
increase in the positive predictive value of p16+ HSIL.
844 Am J Clin Pathol 2015;144:844-849
DOI: 10.1309/AJCPUXLP7XD8OQYY
The human papillomavirus (HPV)–associated squamous lesions of the lower anogenital tract (LAT) have
long presented a challenge with respect to terminology and
diagnosis. HPV-infected squamous epithelium responds
biologically in a process that results in a transient low-grade
lesion or a more stable precancerous lesion. Interestingly, it
is nearly impossible to differentiate between intraepithelial
lesions from different LAT sites, including the cervix, anus,
or penis.1,2 The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions
(LAST Project)3 aimed to align terminology for HPV-associated squamous lesions of the LAT with current scientific
knowledge by proposing a two-tiered system—low-grade
squamous intraepithelial lesion (LSIL) and high-grade
squamous intraepithelial lesion (HSIL)—across all LAT
sites in both sexes.
The LAST Project also suggested guidelines for the use
of biomarkers, and immunohistochemical staining with p16
was selected as the preferred biomarker for use in cervical
lesions.3 Recommendation 1 is to use p16 to differentiate
between HSIL and mimics such as atrophy, immature squamous metaplasia, or tangential cuts. Recommendation 2 is
to use p16 if a diagnosis of cervical intraepithelial neoplasia
grade 2 (CIN2) is considered, which morphologically falls
between a low-grade lesion and a precancerous lesion. Recommendation 3 is to employ p16 in the case of professional
disagreement. Recommendation 4a is for the use of p16 in
cases of high-risk colposcopic referral situations where the
H&E biopsy specimen is interpreted as LSIL or lower.
The aim of this study is to determine if clinical implementation of the LAST Project recommendations for p16
use results in improved patient care. We hypothesize that
© American Society for Clinical Pathology
AJCP / Original Article
following the LAST Project recommendations for p16 will
enhance pathologists’ ability to reliably and accurately differentiate between precancerous lesions and mimics. This
will result in more appropriate treatment, decreasing the risk
of cervical cancer, as well as the morbidity associated with
unnecessary intervention.
Materials and Methods
After obtaining approval from our institutional review
board, the CoPath (Sunquest Information Systems, Tucson,
AZ) records of our institution were searched for all cervical
biopsy cases from May 1, 2011, through October 31, 2013.
All glandular lesions of the cervix were excluded from our
study.
The following data were recorded for each patient: (1)
the pathologic diagnosis, (2) recommendation category for
p16 use, and (3) interpretation of p16 immunostain. The recommendation categorization of p16 use was per the LAST
guidelines as follows and will be referred to as recommendations 1, 2, 3, and 4a throughout this article:
Recommendation 1: HSIL vs mimics
Recommendation 2: Possible CIN2
Recommendation 3: Professional disagreement
Recommendation 4a: High-risk colposcopic referral situations with H&E biopsy specimens initially LSIL or lower
For recommendation 4a, we used HSIL or atypical
squamous cells, cannot rule out HSIL (ASC-H), as the criterion for the high-risk colposcopic referral situations. We
calculated the total number of cervical biopsy specimens
for two time periods. The 12-month time period before
implementation of the LAST guidelines from May 1, 2011,
through April 30, 2012, was designated period A. We
implemented the LAST guidelines in our laboratory in May
2012, but we allowed for a 6-month transition period. The
12-month time period after implementation of the LAST
guidelines from November 1, 2012, through October 31,
2013, was designated period B. In addition, the frequency
of p16 use, category of p16 use, and the number of cases in
which the HSIL diagnosis was made with the assistance of
p16 were calculated for each time period.
For period B, the reason for the p16 immunostain was
indicated at the time ordered by the sign-out pathologist per
the LAST guidelines. For period A, the cases with p16 were
identified from CoPath using a keyword search, and the
glass slides were pulled from the slide archives and reviewed
to designate a reason for the p16 immunostain.
According to our standard histopathologic protocol for
cervical biopsy specimens and endocervical curettage, an
initial slide with at least three 4-μm–thick serial sections was
prepared from formalin-fixed, paraffin-embedded (FFPE)
blocks followed by three deeper levels (D1, D2, and D3).
© American Society for Clinical Pathology
Each level, D1 to D3, was approximately 100 μm deeper
in the FFPE block and contained at least three 4-μm–thick
serial sections. The slides were subsequently stained with
H&E. A blank serial slide was collected after D1 and was
held for potential p16 immunostain if deemed necessary
and thus ordered by the sign-out pathologist. Immunostaining for p16 (clone JC8; Santa Cruz Biotechnology, Dallas,
TX) was performed at our laboratory using the Bond III
immunostainer (Leica Microsystems, Buffalo Grove, IL).
The antibody was diluted 1:800 with 20 minutes of heatinduced epitope retrieval. Positive controls were run with
each sample. Strong and diffuse block staining with p16 was
interpreted as positive (ie, p16-positive HSIL diagnosis), and
patchy, incomplete p16 staining was interpreted as negative.
We obtained follow-up data by searching CoPath for loop
electrosurgical excision procedure (LEEP) or cervical conization specimens for the HSIL cervical biopsy cases. For those
patients with an identifiable follow-up LEEP or cone specimen, we recorded the excision diagnoses and calculated the
positive predictive values of a cervical biopsy HSIL diagnosis
before and after implementation of the LAST guidelines.
All statistical calculations were performed by using
SAS statistical software version 9.3 (SAS Institute, Cary,
NC) in collaboration with a biostatistician. A P value of less
than .05 was considered significant.
Results
Implementation of LAST Guidelines
We reviewed 1,829 and 1,623 cervical biopsy cases in
periods A and B, respectively. The rate of p16 use significantly increased from 2.79% to 6.16% in periods A to B (P
< .001) ❚Figure 1A❚. In addition, there were significant shifts
with respect to the recommendation category of p16 use.
There was no change in the use of p16 per recommendation
1, but there was a marked increase in the utilization of recommendation 2, when the pathologist suspected CIN2, from
0.16% to 1.42% of all cervical biopsy specimens (P < .0001;
Figure 1A). There was also an increase in recommendation
4a from 0% to 1.91% of all cervical biopsy specimens (P <
.0001; Figure 1A).
Both before and after implementation of the LAST
guidelines, approximately half of the cases in recommendation 1 were p16 positive and diagnosed as HSIL,
representing 1.37% of all cases in period A and 1.29% in
period B ❚Figure 1B❚. Prior to implementation of the LAST
guidelines, approximately one-third of the cases in recommendation 2 were p16 positive and diagnosed as HSIL,
representing 0.05% of all cervical biopsy specimens in this
time period. After implementation of the LAST guidelines,
approximately one-fourth of the cases in recommendation
Am J Clin Pathol 2015;144:844-849845
DOI: 10.1309/AJCPUXLP7XD8OQYY
Clinton et al / p16 LAST Guidelines in Clinical Practice
A
B
H
SI
L
1.4%
1.4% 1.3%
0.5%
0.0%
P < .0001
P < .05
0.68%
0.37%
0.05%
al
1.0%
2.3%
vs
To
t
0.16%
Rate of p16+ HSIL
1.4%
2.8%
1.9%
M
im
Pr
C
ic
on
of
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si
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er
on
ig
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al
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C
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po
m
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en
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t
ic
R
ef
er
ra
l
0%
P < .0001
1.5%
al
2.6% 2.8%
2%
P < .0001
Period A
Period B
2.0%
H
SI
L
4%
2.5%
To
t
6%
6.2%
M
im
Pr
C
ic
on
of
es
si
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er
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ol
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R
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er
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Period A
Period B
P < .001
vs
Overall Rate of p16 Use
P < .05
8%
❚Figure 1❚ A, Overall rate of p16 use from period A to period B per Lower Anogenital Squamous Terminology Standardization
Project for HPV-Associated Lesions (LAST) recommendation category. B, Rate of p16+ high-grade squamous intraepithelial
lesion (HSIL) from period A to period B per LAST recommendation category. CIN2, cervical intraepithelial neoplasia grade 2.
2 were p16 positive and diagnosed as HSIL, a significant
increase to 0.37% of all cervical biopsy specimens (P < .05;
Figure 1B). Recommendation 4a did not exist prior to the
LAST guidelines, but approximately one-third of cases in
this category were p16 positive, representing 0.68% of all
cervical biopsy specimens. We never cited recommendation
3, professional disagreement, as a reason for p16 use. Our
practice does not employ a formal routine review of cases,
and thus the opportunity for overt professional disagreement
is minimal. In cases with intradepartmental consultation, the
reason for p16 use could be assigned to recommendation
category 1, 2, or 4a.
There was a significant increase in the total incidence
of p16-positive HSIL from 1.42% in period A to 2.34% in
period B (P < .05; Figure 1B). Representative H&E photomicrographs with corresponding positive p16 stains for
recommendations 1, 2, and 4a are shown in ❚Image 1❚.
CIN2: Biopsies
The rate of CIN2 in the p16-positive HSIL biopsy
specimens increased from 34.6% in period A to 68.4%
in period B (P < .001) ❚Figure 2❚, and the rate of CIN2 in
the non–p16-positive HSIL biopsy specimens decreased
significantly from 24.1% in period A to 4.32% in period B
(P < .0001; Figure 2). The overall rate of CIN2 decreased
significantly from 25.1% in period A to 16.5% in period B
(P < .05; Figure 2).
Follow-up
There was no significant difference in the LEEP or cone
frequency for patients with HSIL biopsy specimens between
846 Am J Clin Pathol 2015;144:844-849
DOI: 10.1309/AJCPUXLP7XD8OQYY
period A, at 70.7%, and period B, at 65.0% ❚Table 1❚. However, excision specimens that resulted from the p16-positive
HSIL biopsy specimens illustrated a significant increase in
the frequency of HSIL diagnosis, from 47.6% in period A
to 75.9% in period B (P < .05; Table 1). Nearly 50% of the
additional p16-positive HSIL diagnoses were due to the use
of p16 per recommendations 2 and 4a. The overall rate of
HSIL detection on the excision specimens trended upward
from 64.5% in period A to 70.0% in period B. Of note, 85%
of the patients who underwent excisional procedures for
HSIL were between ages 21 and 45 years, potential childbearing years.
Discussion
Implementation of the p16 LAST guidelines in our
practice resulted in a significant increase in p16 use, more
than doubling the rate from 2.8% to 6.2%. The LAST
guidelines predicted a p16 use rate of approximately 20%.3
Although our rate of p16 use was much lower than that predicted by the LAST project, we detected significantly more
p16-positive HSIL biopsy specimens when we implemented
the LAST p16 guidelines. Unifying both the terminology of
cervical dysplasia and p16 use in cervical biopsy specimens
may also help decrease discrepant cytohistologic results.
Intrainstitutional variability in p16 use can be quite high,
ranging from 0% to 21% at one institution, and using p16
in more than 10% of cervical biopsy specimens resulted in
improved cytohistologic correlation rates and lower variability in the frequencies of histologic diagnoses.4 Moreover,
© American Society for Clinical Pathology
AJCP / Original Article
❚Image 1❚ Representative photomicrographs from the recommendation categories: high-grade squamous intraepithelial lesion
(HSIL) vs mimic (recommendation 1) (H&E and p16 immunostain, ×200); consider cervical intraepithelial neoplasia grade 2
(CIN2; recommendation 2) (H&E and p16 immunostain, ×200); and high-risk colposcopic referral (recommendation 4a) (H&E and
p16 immunostain, ×100).
Percentage of CIN2
80%
70%
P < .001
Period A
Period B
68.4%
60%
50%
40%
P < .0001
34.6%
30%
24.1%
20%
16.5%
10%
0%
P < .05
25.1%
4.32%
p16+
HSIL
Non-p16+
HSIL
Overall
CIN2
❚Figure 2❚ Cervical intraepithelial neoplasia grade 2 (CIN2)
frequency on cervical biopsy specimens. HSIL, high-grade
squamous intraepithelial lesion.
p16 has been shown to reduce interobserver variability,
particularly for CIN2+ compared with H&E alone.5 As the
cost of medicine becomes an increasingly important topic,
detecting more p16-positive HSILs on cervical biopsy
specimens with only a modest increase in p16 use may be
advantageous.
Our increase in p16 use was primarily due to increased
use for recommendations 2 (suspect CIN2) and 4a (high-risk
Papanicolaou [Pap]). Prior to the LAST guidelines, cases
with high-risk Pap smears and cervical biopsy specimens
© American Society for Clinical Pathology
that were LSIL or lower did not undergo further investigation. The addition of p16 use in this recommendation category is designed to capture potential false-negative biopsy
specimens for patients with a previous high-risk Pap. We
performed p16 immunohistochemistry on the endocervical
curettings (ECCs) and cervical biopsy specimens for these
cases; approximately 30% of these cases would have been
underdiagnosed as LSIL or lower without implementation
of the LAST guidelines. In other words, when patients had a
high-risk Pap with a cervical biopsy specimen diagnosed as
LSIL or lower, p16 staining often demonstrated small fragments of tissue with HSIL. The positive p16 staining in these
cases was typically in the ECC, and the lesion was extremely
difficult to appreciate on H&E alone. This group of patients
harbored occult HSIL detectable only by p16 stain. The
photomicrographs in Image 1 illustrate how minute, inconspicuous fragments of tissue in an ECC are easily missed on
H&E and strongly highlighted with p16.
Prior to the LAST guidelines, diagnoses of CIN2 were
made based on evaluation of H&E sections alone. CIN2 is
a biologically equivocal lesion with morphologic features
intermediate between both low- and high-grade lesions.6,7
It is a poorly reproducible diagnosis with uncertain biologic implications7; thus, decreasing the frequency of this
Am J Clin Pathol 2015;144:844-849847
DOI: 10.1309/AJCPUXLP7XD8OQYY
Clinton et al / p16 LAST Guidelines in Clinical Practice
❚Table 1❚
LEEP/Cone Excisions From Patients With p16+ HSIL Cervical Biopsy Specimens
LEEP/Cone Diagnosis, No./Total No. (%)
HSIL
Rate of LEEP/Cone
Biopsy Diagnosis
A
B
P Value
A
B
P Value
p16+ HSIL
Recommendation category
1. HSIL vs mimic
2. Consider CIN2
3. Professional disagreement
4. High-risk colposcopic referral
Total
Non-p16+ HSIL
Total HSIL
10/20 (50.0)
0/1 (0)
0
0
10/21 (47.6)
110/165 (66.7)
120/186 (64.5)
10/13 (76.9)
6/8 (75.0)
0
6/8 (75.0)
22/29 (75.9)
69/101 (68.3)
91/130 (70.0)
NS
NA
NA
NA
<.05
NS
NS
20/25 (80.0)
1/1 (100.0)
0
0
21/26 (80.8)
165/237 (69.6)
186/263 (70.7)
13/15 (86.7)
8/12 (66.7)
0
8/11 (72.7)
29/38 (76.3)
101/162 (62.3)
130/200 (65.0)
NS
NS
NA
NA
NS
NS
NS
A, period A; B, period B; CIN2, cervical intraepithelial neoplasia grade 2; HSIL, high-grade squamous intraepithelial lesion; LEEP, loop electrosurgical excision procedure; NA,
not applicable; NS, not significant.
diagnosis as shown in our study may be advantageous. The
LAST guidelines recommend using p16 in all cases where
CIN2 is considered, to differentiate HSIL from LSIL. In our
practice, applying p16 in this manner resulted in an increased
frequency of p16-positive HSIL diagnoses. With an increase
in recommendation 2, one might expect a higher percentage
of CIN2 diagnoses on cervical biopsy specimens. However,
when we evaluated the breakdown of HSIL (CIN2 vs CIN3),
there was only an increase in the p16 cases, with an overall
decrease in the rate of CIN2 from 25.1% to 16.5%.
One of the most interesting findings of this study was
the increase in the positive predictive value of a p16-positive
cervical biopsy HSIL diagnosis after implementation of the
LAST guidelines, with a significantly higher percentage of
the patients in period B harboring HSIL in their excision
specimens. Indeed, nearly half of the HSIL diagnoses on
the excision specimens in period B were due to additional
p16-positive HSIL biopsy diagnoses in recommendation
categories 2 and 4a, cases that may have been missed prior
to implementation of the LAST guidelines. Furthermore, we
found a trend toward improved overall detection of HSIL
on excisions. Treatment options for high-grade cervical dysplasia such as LEEP and conization should be used appropriately, since they are not without short- and long-term
complications. Short-term morbidities include infection,
bleeding, and pain, and long-term potential complications
include preterm labor, preterm premature rupture of membranes, and cervical stenosis.8,9 These complications are of
particular importance since 85% of our patients with HSIL
who underwent LEEP/cone were of childbearing age. Our
findings illustrate the positive clinical impact of following
the p16 LAST guidelines and how the significant increase
in p16-positive HSILs (without an increase in total HSIL
diagnoses) is of great clinical importance.
In summary, we found a significant increase in overall
p16 use with the addition of recommendations 2 and 4a.
Despite higher use of recommendation 2, our overall rate
848 Am J Clin Pathol 2015;144:844-849
DOI: 10.1309/AJCPUXLP7XD8OQYY
of CIN2 diagnosis actually decreased. With overall p16 use
only one-fourth of what was predicted by the LAST project,
we showed significantly improved detection of p16-positive
HSIL. Therefore, there was a trend toward improved HSIL
detection on cervical excisions without excessive use of p16
and without overtreatment. As pathologists become more
familiar with and begin to implement the LAST guidelines,
more frequent use of p16 may further improve HSIL detection, leading to more appropriate follow-up treatment.
Corresponding author: Lani K. Clinton, MD, PhD, University of
Hawaii Residency Program, 651 Halo St #411 E, Honolulu, HI
96813; [email protected].
Biostatistical support was partially supported by grants from
the National Institute on Minority Health and Health Disparities
(U54MD007584 and G12MD007601) from the National Institutes
of Health. The views expressed in this article do not necessarily
represent those of the Queen’s Medical Center.
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Am J Clin Pathol 2015;144:844-849849
DOI: 10.1309/AJCPUXLP7XD8OQYY