Programmed Death Ligand-1 Expression Is Associated
With Poorer Survival in Anal Squamous Cell Carcinoma
Ashley L. Monsrud, MD; Vaidehi Avadhani, MD; Marina B. Mosunjac, MD; Lisa Flowers, MD; Uma Krishnamurti, MD, PhD
 Context.—Upregulation of programmed death ligand-1
(PD-L1), an immunoregulatory protein, is associated with
an adverse outcome in several malignancies. Very few
studies have evaluated PD-L1 expression in invasive anal
squamous cell carcinoma (ASCC).
Objective.—To assess PD-L1 expression in patients with
ASCC and correlate it with clinicopathologic factors and
clinical outcomes.
Design.—Fifty-one cases of ASCC were immunostained
for PD-L1. PD-L1 expression by combined positive score
and tumor proportion score was correlated with age, sex,
HIV status, HIV viral load, CD4 count, stage, and
outcomes. Kaplan-Meier curves for overall survival were
plotted and compared using the log-rank test. Cox
regression analysis was performed to identify significant
prognostic factors (2-tailed P , .05 was considered
statistically significant).
A lthough anal squamous cell carcinoma (ASCC) is a rare
malignancy, making up only 0.5% of all cancer cases in
the United States, alarmingly, the annual incidence contin-
ues to rise both globally and in the United States.1 A
particularly steep rise in the incidence of ASCC is noted in a
high-risk population that includes HIV-positive and other
immunocompromised patients, men who have sex with
men, smokers, persons practicing anal receptive intercourse,
and women with multifocal and multicentric lower genital
tract squamous intraepithelial lesions.2 There is a clear
association between human papillomavirus and ASCC, with
human papillomavirus 16 being the most frequent genotype
associated with nearly 90% of cases.3 Despite morphologic
similarities, association with human papillomavirus, and
shared risk factors with cervical squamous cell carcinoma,
molecular and biological outcome predictors for survival are
Accepted for publication August 12, 2021.
Published online December 22, 2021.
From the Departments of Pathology & Laboratory Medicine
(Monsrud, Avadhani, Mosunjac, Krishnamurti) and Gynecology &
Obstetrics (Flowers), Emory University, Atlanta, Georgia. Krishna-
murti is now with the Department of Pathology at Yale School of
Medicine.
The authors have no relevant financial interest in the products or
companies described in this article.
Presented in part at the American Society of Clinical Pathology
2020 annual virtual meeting; September 9–12, 2020.
Corresponding author: Uma Krishnamurti, MD, PhD, Yale New
Haven Hospital, Ste 732B, East Pavilion, 2nd floor, 20 York St, New
Haven, CT 06510 (email: uma.krishnamurti@yale.edu).
1094 Arch Pathol Lab Med—Vol 146, September 2022
Results.—PD-L1 was positive in 24 of 51 cases (47%) by
combined positive score and in 18 of 51 (35%) by tumor
proportion score. The median cancer-specific survival and
5-year overall survival were significantly lower in PD-L1þ
patients. Age, sex, HIV status, HIV viral load, stage, and
cancer progression were not significantly different be-
tween the 2 groups. CD4 count of more than 200/lL was
significantly higher in PD-L1þ patients. PD-L1þ status
remained statistically significant for worse overall survival
on multivariate analysis.
Conclusions.—PD-L1þ status is an independent adverse
prognostic factor for overall survival in ASCC. This study
highlights the potential of PD-L1 targeted therapy in better
management of ASCC.
(Arch Pathol Lab Med. 2022;146:1094–1101; doi:
10.5858/arpa.2021-0169-OA)
still not understood and may differ from those in cervical
squamous cell carcinoma. Moreover, anal cytology has failed
as an adequate screening tool with its limited specificity and
low accuracy. There are no standardized screening and
management guidelines for anal cancer in a high-risk
population.4
Most patients with newly diagnosed ASCC were not
screened by anal Papanicolaou and present with locally
advanced disease.5 The standard chemoradiation protocol
described initially by Nigro et al6 in 1974 has not changed
much today and results in a relatively high cure rate in early
stages. However, 10% of patients present in stage IV, and
almost one-fourth of patients develop metastatic disease.6,7
Chemoradiation can cause a variety of short-term and long-
term side effects that may severely impair quality of life.
Furthermore, almost 30% of patients do not respond to
chemoradiation or cannot be treated because of contrain-
dications.8,9 Therefore, new therapeutic options have been
recently explored.
The introduction of targeted therapies and immune
checkpoint blockade has transformed cancer patients’
treatment options in the last decade.10 Programmed death
receptor-1/programmed death ligand-1 (PD-1/PD-L1) is
one of the most well-known and investigated essential
immune checkpoints.11 T-cell activation is downregulated
when PD-L1 expressed on tumor cells or antigen-present-
ing cells is bound to its inhibitory receptor PD-1 on the
surface of T cells. When this interaction is inhibited, T-cell
activation is reinitiated, and tumor manipulation of the
immune checkpoint system is significantly impaired. Tar-
PD-L1 in Anal Cancer—Monsrud et al
geting the PD-1/PD-L1 axis with specific antibodies has
achieved significant results in several malignancies, includ-
ing melanoma, non–small cell lung cancer, and head and
neck squamous cell carcinoma.11,12 However, an association
of PD-L1 expression and survival outcome has shown
controversial results in different malignancies. In cancers
such as renal cell carcinoma, non–small cell lung carcinoma,
esophageal carcinoma, gastric carcinoma, and melanoma,
PD-L1 expression is associated with a poor prognosis.12 PD-
L1 positivity in gastric carcinoma was found to be an
independent prognostic factor and associated with a
decreased survival time and higher stage.13 Conflicting data
on breast cancer and PD-L1 expression exist, showing PD-
L1 as a favorable prognostic marker in some studies and as a
negative in others.14,15
Very few studies have evaluated PD-L1 expression in anal
carcinomas. Based on 2 trials with 37 and 24 patients enrolled
in each, the US Food and Drug Administration has approved
pembrolizumab and nivolumab, and these have been added
to the National Comprehensive Cancer Network guidelines
for subsequent systemic therapy for anal carcinoma in
2018.16,17 This study aims to assess PD-L1 expression in a
relatively large cohort of patients with ASCC and correlate it
with clinicopathologic factors and clinical outcomes.
MATERIALS AND METHODS
Study Cohort
After institutional review board approval, we performed a search
for cases of ASCC for the period of 2010–2018 within Epic (Epic
Systems Corporation; the patient electronic medical record
database) of Grady Memorial Hospital, a major urban hospital in
Atlanta, Georgia. Pretreatment specimens were selected. In cases
with multiple blocks, the block with the best representative section
of tumor was selected. Paraffin-embedded blocks were retrieved in
58 cases. Only 2 cases had resections; the remaining cases were
pretreatment biopsies. Clinicopathologic features including age,
sex, HIV status, HIV viral load, CD4 count, and stage of cancer
were collected. HIV viral load and CD4 values selected were those
reported at the time of diagnosis of ASCC or the closest available to
that date. HIV viral load was subcategorized as negative (,1.6 log10
copies/mL), low (1.6–4 log10 copies/mL), or high (.4 log10 copies/
mL). The CD4 count was subdivided into 2 groups: less than and
more than 200/lL. Staging in these cases was clinical and based
primarily on imaging studies. One resection case had lymph nodes
resected. Fine-needle aspiration of lymph nodes confirming
metastatic carcinoma was performed on 2 cases. Outcome analysis
included disease progression, cancer-specific survival, and overall
survival. For the outcome analysis, all cases were reviewed in Epic
until March 2020 and patients’ survival was followed. Cancer-
related deaths included patients who died secondary to metastatic
disease and/or complications from treatment (chemotherapy or
radiation) such as sepsis. Deaths related to HIV status but not
directly to anal carcinoma were included in overall deaths but not
in our cancer-related deaths. We use the term disease progression in
our study to include persistent disease, regional recurrence,
appearance of positive lymph nodes for metastatic disease, and
distant metastasis. We used information from all the radiologic
imaging reports (computerized tomography, positron emission
tomography scan, and/or magnetic resonance imaging) and clinical
follow-up visit notes available to determine disease progression. All
patients documented with a greater than 60-month (5-year)
survival were still alive at the time of the most recent encounter
available in our database beyond the 60 months.
Immunohistochemistry
Fifty-eight cases were immunostained with PD-L1 mouse
monoclonal antibody (22C3, Dako, Carpinteria, California). Four-
Arch Pathol Lab Med—Vol 146, September 2022
to 5-micrometer-thick sections of formalin-fixed, paraffin-embed-
ded tissue were tested for the presence of PD-L1 using the Dako
PD-L1 IHC 22C3 pharmDx kit. The sections were deparaffinized
and rehydrated to deionized water. They were then antigen
retrieved in a low pH target retrieval solution using Dako PT link
module. All slides were loaded on an automated system (Dako
Auto Stainer Link48) and incubated with a mouse monoclonal
antibody to PD-L1 or the negative control reagent. This was
followed by a mouse linker composed of rabbit anti-mouse
antibody and then incubation with a ready-to-use visualization
reagent consisting of a goat secondary antibody and horseradish
peroxidase molecules coupled to a dextran polymer backbone. The
enzymatic conversion of the subsequently added diaminobenzidine
chromogen results in precipitation of a visible reaction product at
the site of antigen. The color of the chromogenic reaction is
modified by a chromogen enhancer. The specimens were then
counterstained with hematoxylin and coverslipped. All incubations
were performed at room temperature; between incubations,
sections were washed with Tris-buffered saline. Results were
interpreted using a light microscope. Of these, 51 cases could be
evaluated for PD-L1 expression, and 7 cases had to be excluded
because of the loss of or insufficient tumor tissue. Of the 51 cases,
49 were biopsies and 2 cases had resection. We evaluated PD-L1
expression using both combined positive score (CPS) and tumor
proportion score (TPS). Combined positive score (CPS ¼ [No. of
PD-L1 staining cells (tumor cells, mononuclear inflammatory that
are within the tumor nests and/or adjacent supporting stroma)/total
No. of viable tumor cells] 3 100) of 1% or higher was defined as
PD-L1 positive (PD-L1þ) in the Keynote-028 clinical trial16 and has
also been used in a few other studies.16,18–20 Because other studies
evaluating PD-L1 in anal cancer have used TPS, we also evaluated
TPS.21–25 Tumor proportion score is the percentage of viable tumor
cells showing partial or complete membrane staining and of any
intensity (TPS ¼ [No. of PD-L1þ tumor cells/total No. of PD-L1þ
cells þ PD-L1 negative (PD-L1) tumor cells] 3 100). Any nuclear
staining or granular cytoplasmic staining should not be interpreted
as PD-L1 positivity. Mononuclear immune cells with complete
membrane staining and tumor cells with partial or complete
membrane staining were interpreted as PD-L1þ. There can be
heterogeneity in the staining, and we determined the overall
percentage of PD-L1 positivity. Overall, average CPS of 1% or
higher and TPS 1% or higher was interpreted as PD-L1þ. Scoring
was first done by one of the authors and subsequently by another
author. At the end of the first round of scoring, overall agreement
between the 2 observers in assigning PD-L1 as negative or positive
was 94%, with 100% agreement on PD-L1 cases. Three cases with
borderline overall TPS and CPS scores between 0 and 3 were
reviewed together to decide the final score. In other PD-L1þ cases
the differences in scores were between 5 and 10 and the mean
score was recorded. PD-L1 expression was correlated with age, sex,
HIV status, HIV viral load, CD4 count, stage of cancer, disease
progression, cancer-specific survival, and overall survival.
Statistical Analysis
All data were analyzed using the SPSS statistical software
package (version: 26.0; IBM Corporation). The v2 and Fisher exact
probability tests were used to analyze the differences between
qualitative data. The Mann-Whitney U test was used to compare
the medians. Survival curves were plotted by the Kaplan-Meier
method and compared using the log-rank test. Univariate and
multivariate Cox regression analyses were performed to identify
significant prognostic factors. Only variables with a P value of ,.5
in the univariate analysis were taken for the multivariate analysis. A
2-tailed P value of less than .05 was considered statistically
significant.
RESULTS
PD-L1 Expression
Forty-nine of the 51 cases were pretreatment biopsies.
Only 2 cases had resections. PD-L1 was positive in 24 of 51
PD-L1 in Anal Cancer—Monsrud et al 1095
Figure 1. Programmed death ligand-1 (PD-L1) in anal invasive squamous cell carcinoma. A through D, PD-L1 immunohistochemistry. A, Negative
PD-L1, combined positive score (CPS) and tumor proportion score (TPS) ¼ 0. B, Positive PD-L1 by CPS only (overall CPS ¼ 5, overall TPS ¼ 0). C,
Positive PD-L1, a case with overall CPS and TPS 1 and , 10. D, Positive PD-L1, a case with overall CPS and TPS ¼ 50 (original magnification 320 [A
through D].

cases (47%) by CPS and in 18 of 51 (35%) by TPS. Staining
was heterogeneous in all PD-L1þ cases; invasive foci ranged
from being completely negative for PD-L1 to foci with 100%
positivity. The overall average CPS and TPS ranged from 1%
to 50%. Of the 2 resection cases, 1 was PD-L1þ. There was
no difference in staining between the deep and superficial
aspects of the tumor in this case. The majority of PD-L1þ
cases had overall low scores of less than 10 (67% [16 of 24]
of CPS and 67% [12 of 18] of TPS). Thirteen percent (3 of 24)
of CPS and 17% (3 of 18) of TPS were higher than 25 (Figure
1, A through D).
Correlation of PD-L1 Expression With Age, Sex, and HIV
Status
The distributions of age, sex, and HIV status in the study
population by CPS and TPS are given in Table 1. Age range
in our patient population was 25 to 68 years. Age ranges for
PD-L1 and PD-L1þ cases were similar: 25 to 67 years and
29 to 68 years, respectively, with no significant difference in
the median age of PD-L1 and PD-L1þ groups by CPS and
TPS. There were 40 men and 11 women, with a male to
female ratio of 3.6:1. The majority of patients were male, and
1096 Arch Pathol Lab Med—Vol 146, September 2022
the proportion was not significantly different between the
PD-L1 and PD-L1þ groups. A majority of all patients in our
study were African American (AA) (74%; 38 of 51) and HIV
positive (71%; 36 of 51). HIV-positive status was not
significantly different between the PD-L1 and PD-L1þ
groups.
Correlation of PD-L1 Expression With Stage of Disease
The distribution of stage of disease in the 2 groups is given
in Table 1. Distribution of cancer stages 1 through 4 was
similar and not significantly different between the PD-L1
and PD-L1þ groups (P ¼ .99 by CPS and P ¼ .97 by TPS).
Correlation of PD-L1 Expression With HIV Viral Load and
CD4 Counts
Distribution of HIV viral load and CD4 counts are given in
Table 1. HIV viral load for the categories of negative and low
combined versus high was not significantly different
between the PD-L1þ and PD-L1 patients. A CD4 count
of higher than 200/mL was significantly higher in PD-L1þ
patients compared with PD-L1 patients, with a greater
difference by TPS compared with CPS (83.3% versus 42.8%,
P ¼ .03 by TPS; 75% versus 41.2%, P ¼ .049 by CPS).
PD-L1 in Anal Cancer—Monsrud et al
 Table 1. Correlation of Programmed Death Ligand-1 (PD-L1) Status With Clinical Characteristics
PD-L1 Negative PD-L1 Positive P Value
Patient Characteristics (N ¼ 51) CPS (n ¼ 27; 53%) TPS (n ¼ 33; 65%) CPS (n ¼ 24; 47%) TPS (n ¼ 18; 35%) CPS TPS
Age, y
Median 49 48 50.5 51.5 .77 .55
Range 25–67 29–68
Sex, No. (%) .31 .48
Male 23/27 (85.2) 27/33 (81.8) 17/24 (70.8) 13/18 (72.2)
Female 4/27 (14.8) 6/33 (18.2) 7/24 (29.2) 5/18 (27.8)
HIV-positive, No. (%) 20/27 (74.1) 24/33 (72.7) 16/23 (69.6) 12/17 (70.6) .72 ..99
Stage, No. (%) .99 .97
I 3/26 (11.5) 4/32 (12.5) 3/23 (13.0) 2/17 (11.8)
II 9/26 (34.6) 9/32 (28.1) 7/23 (30.4) 6/17 (35.3)
III 10/26 (38.5) 13/32 (40.6) 9/23 (39.1) 6/17 (35.3)
IV 4/26 (15.4) 6/32 (18.8) 4/23 (17.5) 3/17 (17.6)
CD4 count .200, No. (%) 7/17 (41.2) 9/21 (42.8) 12/16 (75.0) 10/12 (83.3) .049 .03
HIV viral load, No. (%) .10 ..99
Negative and low (,4 log10 copies/mL) 14/14 (100) 15/18 (83.3) 11/15 (73.3) 10/11 (90.9)
High (.4 log10 copies/mL) 0/14 (0) 3/18 (16.7) 4/15 (26.7) 1/11 (9.1)
Abbreviations: CPS, combined positive score; TPS, tumor proportion score.

PD-L1 Expression and Outcomes
Eighty-six percent (44 of 51) of all patients underwent
radiation therapy (RT). The majority (80%; 41 of 51) of all
patients also received chemotherapy. Clinical outcomes are
summarized in Table 2. Only 2 patients were lost to follow-
up for unknown reasons, giving us an excellent retention
rate of 96%. Fifty percent (25 of 51) of all patients had
disease progression, with 1 patient of unknown status
because of loss to follow-up. Disease progression was not
significantly different between the PD-L1 and PD-L1þ
groups. By CPS, 47.8% (11 of 23) of PD-L1þ patients and
37% (10 of 27) of PD-L1 patients had disease progression,
P ¼ .57. By TPS, 42.4% (14 of 33) of PD-L1 patients and
41.2% (7 of 17) of PD-L1þ patients had disease progression,
P . .99. Progression-free survival in months was also not
significantly different between PD-L1 and PD-L1þ patients
(23 versus 22 months, P ¼ .89 by CPS; 24 versus 18 months,
P ¼ .53 by TPS).
Overall, 41.2% (21 of 51) of patients died, with 19 deaths
(90% of all deaths) secondary to the cancer diagnosis. Two
deaths, 1 in the PD-L1 and 1 in the PD-L1þ group, were
due to disseminated tuberculosis and cardiac disease,
respectively. The median cancer-specific survival was
significantly lower in PD-L1þ patients compared with PD-
L1 patients (24 versus 48 months, P ¼ .02 by CPS; 22
versus 48 months, P ¼ .01 by TPS). The ranges for disease-
specific survival for PD-L1þ and PD-L1 patients were 3–60
and 6–60 months, respectively. The Kaplan-Meier curve for
5-year overall survival was significantly lower in PD-L1þ
patients compared with PD-L1 patients (42% versus 72%,
P ¼ .03 by CPS; 41% versus 64%, P ¼ .02 by TPS) (Figures 2
and 3).
On univariate analysis, only stage and PD-L1þ status by
both CPS and TPS were significant prognostic factors for
overall survival (Table 3). On multivariate analysis, PD-L1þ
statuses by both CPS and TPS were independently
significant for worse overall survival with hazard ratio ¼
2.85 (95% CI, 1.06–7.67; P ¼ .04) by CPS and hazard ratio ¼
3.4 (95% CI, 1.18–9.76; P ¼ .02) by TPS (Table 4).
In summary, factors that were not significantly different
between the PD-L1þ and PD-L1 groups were age, sex, HIV
status, HIV viral load, stage, number of patients with cancer
progression, and progression-free survival. The median
cancer-specific survival and 5-year overall survival were
significantly lower in PD-L1þ patients compared with PD-
L1 patients. PD-L1 positivity was an independent prog-
nostic factor for worse overall survival.

Table 2. Correlation of Programmed Death Ligand-1 (PD-L1) Status With Outcomes

PD-L1 Negative PD-L1 Positive P Value
Patient Outcomes (N ¼ 51) CPS (n ¼ 27; 53%) TPS (n ¼ 33; 65%) CPS (n ¼ 24; 47%) TPS (n ¼ 18; 35%) CPS TPS
No. (%) with cancer progression 10/27 (37.0) 14/33 (42.4) 11/23 (47.8) 7/17 (41.2) .57 . .99
PFS, mo .89 .53
Median 23 24 22 18
Range 0–60 0–60
CSS, mo .02 .01
Median 48 48 24 22
Range 6–60 3–60
Abbreviations: CPS, combined positive score; CSS, cancer-specific survival; PFS, progression-free survival; TPS, tumor proportion score.
Arch Pathol Lab Med—Vol 146, September 2022 PD-L1 in Anal Cancer—Monsrud et al 1097
Figure 2. Kaplan-Meier plot of overall survival in groups with and without programmed death ligand-1 (PD-L1) expression by combined positive
score.

Figure 3. Kaplan-Meier plot of overall survival in groups with and without programmed death ligand-1 (PD-L1) expression by tumor proportion
score.
1098 Arch Pathol Lab Med—Vol 146, September 2022 PD-L1 in Anal Cancer—Monsrud et al
 Table 3. Univariate Analysis of Clinical
Characteristics and Programmed Death Ligand-1
(PD-L1) Status for Overall Survival
Variable P Value HR
Age (,45/45) .48 1.42
Sex (male/female) .98 0.98
HIV status (þ/) .30 1.65
Stage (1 þ 2/3 þ 4) .02 3.34
Viral load (negative þ low/high) .67 1.39
CD4 count (,200/.200) .79 0.85
PD-L1 þ/ by CPS .04 2.72
PD-L1 þ/ by TPS .03 2.72
Abbreviations: CPS, combined positive score; HR, hazard ratio; TPS,
tumor proportion score.
DISCUSSION
PD-L1 expression on tumor cells has been found in
various malignancies, including head and neck, gastric,
colorectal, breast, renal cell, non–small cell lung cancers,
and melanomas.12,26–30 Its expression by tumor cells has
been associated with both favorable and unfavorable
prognosis. Thus far, only a few studies have investigated
PD-L1 expression in ASCC, with controversial results.
In this study, we investigated the correlation of PD-L1
expression in ASCC with clinical outcomes, clinical charac-
teristics, and laboratory data in a unique patient population
of predominantly male, HIV-positive AAs. The 5-year
mortality rate in our study was about 41% compared with
31% for anal cancer based on SEER data from 2011 through
2017. However, 46% of cases in the SEER data were in
stages 3 and 4 combined, whereas 55% of our patients were
in stages 3 and 4 combined.1 Seventy-six percent of our
study cohort were AAs receiving care in an urban public
hospital. Racial disparity in stage distribution and survival is
multifactorial and is reported to be due to socioeconomic
factors, insurance status, comorbidities, and tumor charac-
teristics.31 Studies by Iseas et al,19 Wessely et al,22 Balermpas
et al,32 and Chamseddin et al33 showed better survival rates
from ASCC in PD-L1þ patients. Mitra et al24 did not find
PD-L1 to be prognostic but found a different biomarker,
indoleamine 2,3 dioxygenase 1 (IDO1), to be associated
with poor outcome. Zhao et al18 and Govindarajan et al21
were the first to suggest that PD-L1 positivity was
associated with worse recurrence and mortality rates in
ASCC. Steiniche et al20 also found PD-L1 positivity to be
associated with shorter overall survival. In our patient
population, the median cancer-specific survival and 5-year
survival were significantly lower in PD-L1þ patients than in
PD-L1 patients. Even though these findings are in
concordance with those of Zhao et al18 and Govindarajan
et al,21 there are significant differences in our patient
population demographics and HIV status compared with
the population sample of those 2 studies. Although in both
of those studies there was a large proportion of female
95% CI
patients (49% and 85%) with only 15% of AA and no HIV or
0.54–3.76 immunocompromised patients included in the study, our
0.34–2.97 study population was composed mostly of male, AA, and
0.64–4.27 HIV-positive patients (78%, 74%, and 71%, respectively).
1.19–9.34 The other studies that showed improved survival in PD-L1þ
0.3–6.45 ASCC also examined a distinctly different patient popula-
0.26–2.8 tion with a predominance of female, White, and non-
1.06–6.97 immunocompromised patients.19,22,32,33 The PD-L1 positivity
1.09–6.80 rate in several studies was wide, ranging from 22% to
82.5%, with Koncar et al23 reporting 22% PD-L1 positivity
using TPS and Steiniche et al20 reporting 82.5% PD-L1
positivity using CPS. Our PD-L1 positivity is within this
range. Although differences in patient population could be a
potential explanation for variation in PD-L1 positivity, the
method used for establishing PD-L1 positivity is also
different in different studies. Some studies used CPS18,19;
some used TPS21,22,25; some used greater than or equal to
1%,19,20,22 greater than 1%,24,33 or greater than 5% as a cutoff
value18; and others used ‘‘any positivity’’ as evidence of PD-
L1 expression.21 In our study the criterion of an overall
average TPS of greater than or equal to 1% of tumor cells
with partial or complete membrane staining as PD-L1þ
resulted in lower PD-L1 positivity than using CPS of greater
than or equal to 1%. Also, several different commercially
available PD-L1 antibodies are available. Each uses a
different staining protocol, threshold, and scoring algorithm
(Table 5); therefore, there is a pressing need to standardize
PD-L1 scoring in ASCC. The association of HIV status with
local antitumor immune response has been poorly under-
stood. Yanik et al34 observed no difference in PD-L1
expression of ASCC between HIV-positive and HIV-
negative patients, with 49% of total tumors demonstrating
tumor cell PD-L1 expression. Their findings are in
concordance with our results in which HIV-positive status
was similar in PD-L1 and PD-L1þ groups (73% and 71%, P
. .99). In addition, our study showed that a CD4 count of
higher than 200/lL was associated with higher tumoral
expression of PD-L1. Yanik et al34 showed no association of
peripheral CD4 count with expression of immune check-
point molecules, including PD-1, PD-L1, and LAG-3. CD4
count correlated positively with PD-L1 CPS higher than 1%
and negatively with TPS higher than 25% in HIV-positive
patients with cervical squamous cell carcinoma.35 Such
discrepancies may be attributed to a relatively small number
of cases tested and different methodology in evaluating PD-
L1 and different tumor microenvironments of cervical and
anal SCC. Besides, it has been postulated that a decrease in
CD4 count may result in decreased PD-L1 expression on

Table 4. Multivariate Cox Regression Analysis of Clinical Characteristics and Programmed Death Ligand-1 (PD-L1)
Status for Overall Survival
P Value HR 95% CI
Variable CPS TPS CPS TPS CPS TPS
Age (,45/45) .38 .80 1.62 0.16 0.55–4.75 0.38–3.52
HIV status .43 .41 0.67 0.64 0.24–1.84 0.23–1.85
Stage (1 þ 2/3 þ 4) .01 .01 4.28 4.67 1.43–12.84 1.56–14.01
PD-L1 þ/ .04 .02 2.85 3.40 1.06–7.67 1.18–9.76
Abbreviations: CPS, combined positive score; HR, hazard ratio; TPS, tumor proportion score.
Arch Pathol Lab Med—Vol 146, September 2022 PD-L1 in Anal Cancer—Monsrud et al 1099
 Table 5. Published Studies of Programmed Death Ligand-1 (PD-L1) Expression in Anal Squamous Cell Carcinoma:
Comparison of Demographic Characteristics, Prognostic Value, PD-L1 Antibody Clones, and Scoring Methods
Survival of
Patients, M:F HIV, AA, PD-L1, PD-L1þ PD-L1 Score;
Source, y No. Ratio % % % Cases PD-L1 Clone CPS or TPS
Zhao et al,18 2018 26 1:5.5 ... ... 46 Worse Cell Signaling .5%; TPS
Technology
13684
Iseas et al,19 2021 79 1:2.8 14 ... 57 Better Ventana SP263 1%; CPS
Steiniche et al,20 2020 40 1:1.8 ... ... 82.5 Worse Dako 22C3 1%; CPS
Govindarajan et al,21 2018 41 1.1:1 ... 15 56 Worse ABCAM 153991 Any membranous or
cytoplasmic staining
of tumor cells; TPS
Wessely et al,22 2020 54 1:1.3 13 ... 61.1 Better Ventana SP263 .1%; TPS
Mitra et al,24 2019 63 1:1.4 22 ... 41 NS Cell Signaling .1%; TPS
Technology
E1L3N
Chamseddin et al,33 2019 16 1:1.6 ... 14 62 Better ... .1%; CPS
Our study 51 3.6:1 71 76 47 (CPS), 35 (TPS) Worse Dako 22C3 1%; CPS and TPS
Abbreviations: AA, African American; CPS, combined positive score; NS, no significant difference; TPS, tumor proportion score.

tumor cells and antigen-presenting cells, secondary to
decreased interleukins (IL-2) and interferon-c in the
microenvironment. In HIV-infected patients, even after
prolonged antiretroviral therapy, fewer CD4-positive cells
may prevent IL-2 and IFN-c secretion, resulting in
decreased PD-L1 expression in antigen-presenting cells
and tumor cells. On the other hand, when CD4 count
increases, the immune system is enhanced and produces
more cytokines, increasing PD-1/PD-L1 expression on
immune cells and tumor cells.35 More studies are needed
to better understand the relationship of CD4-positive cells
and PD-L1 expression in HIV-positive patients and the
effect of antiretroviral therapies.
Furthermore, an immunosuppressive subset of CD4 T
cells (Tregs), characterized by the expression of a master
regulatory transcription factor, forkhead box P3 protein
(FOXP3), correlates with poor prognosis and low survival
rates in some cancers and is favorable in others.36 There is
accumulating evidence suggesting that an increased number
of Tregs is associated with resistance against cancer
immunotherapy.37 PD-L1 expression on tumor cells has
been found to have a direct association with the presence of
FOXP3þ Treg density at the tumor site, which could indicate
a coordinated immune suppressing action and worse
prognosis.36 In our study, we did not investigate the density
of tumor-infiltrating T cells and their subsets. Further
studies are needed to elucidate FOXP3-positive Tregs’
prognostic significance as a tool for overcoming Treg-
mediated tumor resistance and maximizing the therapeutic
efficacy and immune response in patients with ASCC.
Many immune-suppressive factors such as PD-1 and PD-
L1 are stimulated or enhanced by radiation in the absence of
immunotherapy. Therefore, it has been demonstrated that
the combination of radiotherapy and PD-1/PD-L1 check-
point blockade synergistically enhances antitumor immune
activity and increases the treatment efficacy of either therapy
alone.38
Scientists have not reached a consensus concerning the
effect of RT on Tregs. The numerical and functional
abnormality of Tregs may be influenced by radiation dose,
the form of radiation, or tumor type.39,40 A previous study of
HIV-positive patients with cervical squamous cell carcinoma
showed that PD-L1þ tumor cells are more radiosensitive and
that those patients show better response with RT.35 High
CD8:FOXP3 ratio combined with negative PD-L1 expres-
sion was an independent and significant favorable predictive
factor for local control. It is reported that it might be a useful
biomarker of radiosensitivity in patients with laryngeal SCC
receiving definitive RT.39 In our study, most patients
received RT, and the subset of PD-L1þ patients could
benefit from a combination of radiotherapy and anti–PD-L1
treatment. Further studies on CD8/FOXP3 expression on
ASCC are needed to explore future therapeutic venues.
Our study involved a patient population that was
predominantly male, AA, and HIV positive. The incidence
of anal cancer in HIV-infected adults is more than 30-fold
greater than in the general population, and our patient
cohort reflects this. Although this may be viewed as a
limitation, the results show that even in this unique cohort,
PD-L1 expression is associated with worse outcomes.41
In summary, we have described PD-L1 expression in a
unique population of mostly male, AA, and HIV-positive
patients with ASCC. We found that the median cancer-
specific survival and 5-year overall survival were signifi-
cantly lower in PD-L1þ patients than PD-L1 patients with
ASCC. PD-L1 expression is an independent prognostic
factor for worse overall survival and does not correlate with
age, sex, tumor stage, HIV status, HIV viral load, and
progression-free survival. HIV-positive patients with higher
CD4 count were more likely to be PD-L1þ.
As we have learned the lesson from efforts to standardize
tissue preparation, staining, and scoring of HER2, a
prognostic and therapeutic marker in breast carcinoma, we
should also strive to develop strict standardization and
accurate staining and scoring of PD-L1 in ASCC. More
studies are needed to explore the combination of RT, PD-L1
expression, and other potential targeted therapies and
immune checkpoint blockade as new venues for the
treatment of ASCC.
References
1. National Institutes of Health. Cancer stat facts: anal cancer. https://seer.
cancer.gov/statfacts/html/anus.html. Accessed June 6, 2021.
2. Deshmukh AA, Suk R, Shiels MS, et al. Recent trends in squamous cell
carcinoma of the anus incidence and mortality in the United States, 2001–2015. J
Natl Cancer Inst. 2020;112(8):829–838.

1100 Arch Pathol Lab Med—Vol 146, September 2022 PD-L1 in Anal Cancer—Monsrud et al
 3. Zou H, Fairley CK, Hocking JS, Garland SM, Grulich AE, Chen MY. The
prevalence of anal human papillomavirus among young HIV negative men who
have sex with men. BMC Infect Dis. 2012;12:341.
4. Johnson GE, Nguyen ML, Krishnamurti U, et al. Cytology as a screening tool
for anal squamous intraepithelial lesion for HIV positive men: 10-year experience
in an inner city hospital. J Am Soc Cytopathol. 2015;5(3):145–153.
5. Fish R, Sanders C, Ryan N, Van der Veer S, Renehan AG, Williamson PR.
Systematic review of outcome measures following chemoradiotherapy for the
treatment of anal cancer (CORMAC). Colorectal Dis. 2018;20(5):371–382.
6. Nigro ND, Vaitkevicius VK, Considine B Jr. Combined therapy for cancer of
the anal canal: a preliminary report. Dis Colon Rectum. 1974;17(3):354–356.
7. Martin D, Balermpas P, Winkelmann R, Rodel F, Rodel C, Fokas E. Anal
squamous cell carcinoma—state of the art management and future perspectives.
Cancer Treat Rev. 2018;65:11–21.
8. Osborne MC, Maykel J, Johnson EK, Steele SR. Anal squamous cell
carcinoma: an evolution in disease and management. World J Gastroenterol.
2014;20(36):13052–13059.
9. Morton M, Melnitchouk N, Bleday R. Squamous cell carcinoma of the anal
canal. Curr Probl Cancer. 2018;42(5):486–492.
10. Vaddepally RK, Kharel P, Pandey R, Garje R, Chandra AB. Review of
indications of FDA-approved immune checkpoint inhibitors per NCCN guide-
lines with the level of evidence. Cancers (Basel). 2020;12(3):738.
11. Chen S, Crabill GA, Pritchard TS, et al. Mechanisms regulating PD-L1
expression on tumor and immune cells. J Immunother Cancer. 2019;7(1):305.
12. Guan J, Lim KS, Mekhail T, Chang CC. Programmed death ligand-1 (PD-L1)
expression in the programmed death receptor-1 (PD-1)/PD-L1 blockade: a key
player against various cancers. Arch Pathol Lab Med. 2017;141(6):851–861.
13. Yamashita S, Abe H, Kunita A, Yamashita H, Seto Y, Ushiku T. Programmed
cell death protein 1/programmed death ligand 1 but not HER2 is a potential
therapeutic target in gastric neuroendocrine carcinoma. Histopathology. 2021;
78(3):381–391.
14. Guo H, Ding Q, Gong Y, et al. Comparison of three scoring methods using
the FDA-approved 22C3 immunohistochemistry assay to evaluate PD-L1
expression in breast cancer and their association with clinicopathologic factors.
Breast Cancer Res. 2020;22(1):69.
15. Zhao T, Li C, Wu Y, Li B, Zhang B. Prognostic value of PD-L1 expression in
tumor infiltrating immune cells in cancers: a meta-analysis. PLoS One. 2017;
12(4):e0176822.
16. Ott PA, Piha-Paul SA, Munster P, et al. Safety and antitumor activity of the
anti-PD-1 antibody pembrolizumab in patients with recurrent carcinoma of the
anal canal. Ann Oncol. 2017;28(5):1036–1041.
17. Morris VK, Salem ME, Nimeiri H, et al. Nivolumab for previously treated
unresectable metastatic anal cancer (NCI9673): a multicentre, single-arm, phase
2 study. Lancet Oncol. 2017;18(4):446–453.
18. Zhao YJ, Sun WP, Peng JH, et al. Programmed death-ligand 1 expression
correlates with diminished CD8þ T cell infiltration and predicts poor prognosis in
anal squamous cell carcinoma patients. Cancer Manag Res. 2018;10:1–11.
19. Iseas S, Golubicki M, Robbio J, et al. A clinical and molecular portrait of
non-metastatic anal squamous cell carcinoma. Transl Oncol. 2021;14(6):101084.
20. Steiniche T, Ladekarl M, Georgsen JB, et al. Association of programmed
death ligand 1 expression with prognosis among patients with ten uncommon
advanced cancers. Future Sci OA. 2020;6(8):FSO616.
21. Govindarajan R, Gujja S, Siegel ER, et al. Programmed Cell Death-Ligand 1
(PD-L1) Expression in Anal Cancer. Am J Clin Oncol. 2018;41(7):638–642.
22. Wessely A, Heppt MV, Kammerbauer C, et al. Evaluation of PD-L1
expression and HPV genotyping in anal squamous cell carcinoma. Cancers
(Basel). 2020;12(9):2516.
Arch Pathol Lab Med—Vol 146, September 2022
23. Koncar RF, Feldman R, Bahassi EM, Hashemi Sadraei N. Comparative
molecular profiling of HPV-induced squamous cell carcinomas. Cancer Med.
2017;6(7):1673–1685.
24. Mitra D, Horick NK, Brackett DG, et al. High IDO1 expression is associated
with poor outcome in patients with anal cancer treated with definitive
chemoradiotherapy. Oncologist. 2019;24(6):e275–e283.
25. Salem ME, Puccini A, Grothey A, et al. Landscape of tumor mutation load,
mismatch repair deficiency, and PD-L1 expression in a large patient cohort of
gastrointestinal cancers. Mol Cancer Res. 2018;16(5):805–812.
26. Baptista MZ, Sarian LO, Derchain SF, Pinto GA, Vassallo J. Prognostic
significance of PD-L1 and PD-L2 in breast cancer. Hum Pathol. 2016;47(1):78–
84.
27. Chen K, Cheng G, Zhang F, et al. Prognostic significance of programmed
death-1 and programmed death-ligand 1 expression in patients with esophageal
squamous cell carcinoma. Oncotarget. 2016;7(21):30772–30780.
28. Iacovelli R, Nole F, Verri E, et al. Prognostic role of PD-L1 expression in
renal cell carcinoma: a systematic review and meta-analysis. Target Oncol. 2016;
11(2):143–148.
29. Massi D, Brusa D, Merelli B, et al. PD-L1 marks a subset of melanomas with
a shorter overall survival and distinct genetic and morphological characteristics.
Ann Oncol. 2014;25(12):2433–2442.
30. Wang A, Wang HY, Liu Y, et al. The prognostic value of PD-L1 expression
for non-small cell lung cancer patients: a meta-analysis. Eur J Surg Oncol. 2015;
41(4):450–456.
31. Sineshaw HM, Ng K, Flanders WD, Brawley OW, Jemal A. Factors that
contribute to differences in survival of black vs white patients with colorectal
cancer. Gastroenterology. 2018;154(4):906–915.e7.
32. Balermpas P, Martin D, Wieland U, et al. Human papilloma virus load and
PD-1/PD-L1, CD8(þ) and FOXP3 in anal cancer patients treated with chemo-
radiotherapy: rationale for immunotherapy. Oncoimmunology. 2017;6(3):
e1288331.
33. Chamseddin BH, Lee EE, Kim J, et al. Assessment of circularized E7 RNA,
GLUT1, and PD-L1 in anal squamous cell carcinoma. Oncotarget. 2019;10(57):
5958–5969.
34. Yanik EL, Kaunitz GJ, Cottrell TR, et al. Association of HIV status with local
immune response to anal squamous cell carcinoma: implications for immuno-
therapy. JAMA Oncol. 2017;3(7):974–978.
35. Loharamtaweethong K, Puripat N, Praditphol N, Thammasiri J, Tangitgamol
S. PD-L1 protein expression and copy number gains in HIV-positive locally
advanced cervical cancer. Ther Adv Med Oncol. 2020;12:1758835920963001.
36. Shang B, Liu Y, Jiang SJ, Liu Y. Prognostic value of tumor-infiltrating FoxP3þ
regulatory T cells in cancers: a systematic review and meta-analysis. Sci Rep.
2015;5:15179.
37. Zuazo M, Arasanz H, Bocanegra A, et al. Systemic CD4 immunity as a key
contributor to PD-L1/PD-1 blockade immunotherapy efficacy. Front Immunol.
2020;11:586907.
38. Huang Y, Huang Q, Zhao J, et al. The impacts of different types of radiation
on the CRT and PDL1 expression in tumor cells under normoxia and hypoxia.
Front Oncol. 2020;10:1610.
39. Ono T, Azuma K, Kawahara A, et al. Predictive value of CD8/FOXP3 ratio
combined with PD-L1 expression for radiosensitivity in patients with squamous
cell carcinoma of the larynx receiving definitive radiation therapy. Head Neck.
2020;42(12):3518–3530.
40. Saleh R, Elkord E. FoxP3(þ) T regulatory cells in cancer: prognostic
biomarkers and therapeutic targets. Cancer Lett. 2020;490:174–185.
41. Tong WW, Hillman RJ, Kelleher AD, Grulich AE, Carr A. Anal
intraepithelial neoplasia and squamous cell carcinoma in HIV-infected adults.
HIV Med. 2014;15(2):65–76.
PD-L1 in Anal Cancer—Monsrud et al 1101