Journal of

Clinical Medicine

Article
Impact of PD-L1 Scores and Changes on Clinical
Outcome in Rectal Cancer Patients Undergoing
Neoadjuvant Chemoradiotherapy
Florian Huemer 1 , Eckhard Klieser 2 , Daniel Neureiter 2,3 , Verena Schlintl 1 ,
Gabriel Rinnerthaler 1,3 , Franck Pagès 4,5 , Amos Kirilovsky 4,5 , Carine El Sissy 4,5 ,
Wolfgang Iglseder 6 , Franz Singhartinger 7 , Tarkan Jäger 7 , Adam Dinnewitzer 7 ,
Nadja Zaborsky 1 , Markus Steiner 1 , Richard Greil 1,3 and Lukas Weiss 1,3, *
1 Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology
and Rheumatology, Oncologic Center, Salzburg Cancer Research Institute-Laboratory for Immunological
and Molecular Cancer Research (SCRI-LIMCR), Paracelsus Medical University, 5020 Salzburg, Austria;
f.huemer@salk.at (F.H.); v.schlintl@salk.at (V.S.); g.rinnerthaler@salk.at (G.R.); n.zaborsky@salk.at (N.Z.);
mark.steiner@salk.at (M.S.); r.greil@salk.at (R.G.)
2 Institute of Pathology, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria;
e.klieser@salk.at (E.K.); d.neureiter@salk.at (D.N.)
3 Cancer Cluster Salzburg, 5020 Salzburg, Austria
4 Laboratory of Integrative Cancer Immunology, INSERM UMRS1138, Immunology and Cancer Department,
Cordeliers Research Center, 75006 Paris, France; franck.pages@aphp.fr (F.P.);
amos.kirilovsky@gmail.com (A.K.); carineelsissy@hotmail.com (C.E.S.)
5 Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Université de Paris,
Faculté de santé, 75015 Paris, France
6 Department of Radiation Oncology, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria;
w.iglseder@salk.at
7 Department of Surgery, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria;
f.singhartinger@salk.at (F.S.); ta.jaeger@salk.at (T.J.); adam.dinnewitzer@ooeg.at (A.D.)
* Correspondence: lu.weiss@salk.at; Tel.: +43-57255-57892




Received: 2 July 2020; Accepted: 24 August 2020; Published: 27 August 2020


Abstract: Reports on the prognostic role of programmed death-ligand 1 (PD-L1) expression in rectal
cancer are controversial. We investigated expression patterns and changes of PD-L1 in rectal cancer
patients undergoing neoadjuvant chemoradiotherapy (CRT). Seventy-two patients diagnosed with
rectal cancer and/or treated with fluorouracil-based neoadjuvant CRT at the Department of Internal
Medicine III of the Paracelsus Medical University Salzburg (Austria) between January 2003 and
October 2012 were included. PD-L1 scoring was performed according to the tumor proportion score
(TPS), combined positive score (CPS), and immune cell score (IC). PD-L1 TPS prior to neoadjuvant
CRT had a statistically significant impact on survival (median: ≤1%: 95.4 months (95% CI: 51.8—not
reached) vs. >1%: not reached, p = 0.03, log-rank). Patients with a PD-L1 TPS ≤1% prior to and
after CRT showed an inferior survival compared to all other patients (median: 56.7 months (95% CI:
51.4—not reached) vs. not reached, p = 0.005, log-rank). In multivariate analysis, PD-L1 TPS prior
to neoadjuvant CRT (>1% vs. ≤1%, hazard ratio: 0.29 (95% CI: 0.11–0.76), p = 0.01) remained
independently associated with survival. In conclusion, low PD-L1 TPS was associated with inferior
survival in rectal cancer patients undergoing neoadjuvant CRT. A prospective validation of the
prognostic value of PD-L1 expression in rectal cancer patients within a clinical trial is necessitated.

Keywords: programmed death-ligand 1; TPS; CPS; IC; survival

J. Clin. Med. 2020, 9, 2775; doi:10.3390/jcm9092775 www.mdpi.com/journal/jcm

J. Clin. Med. 2020, 9, 2775 2 of 16

1. Introduction
The treatment of stage II-III rectal cancer usually consists of neoadjuvant capecitabine or
5-fluorouracil (5-FU)-based long-course chemoradiotherapy (CRT) followed by surgery [1,2]. Despite
the application of adjuvant chemotherapy, 32–43% of rectal cancer patients experience a disease relapse
within the first six years [3]. Immune checkpoints, such as programmed death-ligand 1 (PD-L1),
can be therapeutically targeted, which has led to dramatic clinical improvements in various tumor
entities [4–12]. However, in metastatic colorectal cancer (CRC), the clinical benefit of this therapeutic
approach is almost exclusively restricted to tumors with microsatellite instability (MSI) [13–15]
and the addition of an immune-checkpoint inhibitor to adjuvant chemotherapy in stage III colon
cancer with MSI is currently being investigated in the ATOMIC trial [16] (NCT02912559). Studies
specifically investigating the prognostic role of PD-L1 expression in rectal cancer are sparse. The applied
anti-PD-L1 monoclonal antibodies, the time point of PD-L1 testing, as well as the applied PD-L1
scores were heterogeneous in these studies and the impact of PD-L1 expression on clinical outcome
was controversial [17–21]. The aim of our retrospective single-center analysis was to evaluate PD-L1
expression patterns and time-dependent changes according to three established PD-L1 scores and the
impact on clinical outcome in a well-defined rectal cancer cohort undergoing neoadjuvant CRT.

2. Experimental Section
This research project was approved by the local ethics committee of the Provincial Government of
Salzburg, Austria (415-EP/73/655-2016).

2.1. Patients
Patients with TNM stage I–IV rectal cancer diagnosed and/or treated at the tertiary cancer center
of the Paracelsus Medical University Salzburg (Austria) with neoadjuvant capecitabine/5-FU-based
CRT followed by total mesorectal excision were consecutively selected for this retrospective analysis.
Inclusion requirements were the availability of the paraffin-embedded diagnostic primary tumor
sample as well as of the primary tumor specimen obtained from definitive surgery. Decisions on the
initiation of neoadjuvant CRT and follow-up care were based on the respective National Comprehensive
Cancer Network (NCCN) and European Society of Medical Oncology (ESMO) treatment guidelines
for rectal cancer. Radiotherapy consisted of 45.0 Gy of radiation delivered to the primary tumor,
the mesorectal lymph nodes, pre-sacral lymph nodes, and internal iliac lymph nodes. The chemotherapy
backbone was either oral capecitabine or infusional 5-FU as monochemotherapy or in combination with
oxaliplatin. Surgery was performed six to eight weeks after completion of neoadjuvant CRT. Adjuvant
chemotherapy was recommended based on interdisciplinary tumor board decisions on an individual
basis. Patients were classified as low (<8), intermediate (8–16), or high (>16) risk according to the
neoadjuvant rectal (NAR) score [22], which incorporates the pathological nodal stage and primary
tumor downstaging during neoadjuvant CRT.

2.2. Immunohistochemistry
Immunohistochemical examination was performed on 4-µm formalin-fixed paraffin-embedded
(FFPE) sections. Each case was raised on adhesive glass slides and dried at 60 ◦ C for one hour.
Deparaffination, antigen retrieval, immunostaining, counter staining, dehydration, and cover slip
application as well as pre-treatment were conducted using standardized routine immunohistochemistry
(IHC) protocols. Immunohistochemical staining was performed either on a Ventana Benchmark Ultra
instrument (Ventana Medical Systems, Tucson, AZ, USA; trademark of Hoffmann-La Roche AG, Basel,
Switzerland) or on a Dako Omnis Autostainer combined with the EnVision Plus System (Dako, Vienna,
Austria). The anti-PD-L1 ready-to-use antibody (22C3, SK006, Agilent, Santa Clara, CA, USA) was
used for PD-L1 staining. Each sample was assessed by two experienced pathologists according to the
tumor proportion score (TPS: (PD-L1-stained tumor cells/total number of viable tumor cells) × 100) [23],
J. Clin. Med. 2020, 9, 2775 3 of 16

combined positive score (CPS: (PD-L1-stained tumor cells and immune cells/total number of viable
tumor cells) × 100) [24], and immune cell score (IC: (PD-L1-stained immune cells/total number of
viable tumor cells) × 100) [25] in diagnostic biopsies prior to neoadjuvant CRT as well as in specimens
obtained from definitive surgery after neoadjuvant CRT. The PD-L1 assessors were blinded to clinical
outcome. Sections were incubated with anti-CD3 (2GV6, Ventana Medical Systems, Tucson, AZ, USA;
trademark of Hoffmann-La Roche AG, Basel, Switzerland; Oro Valley, United States) and anti-CD8
(SP57 Ventana Medical Systems, Tucson, AZ, USA; trademark of Hoffmann-La Roche AG, Basel,
Switzerland; Oro Valley, United States) ready-to-use primary antibodies to assess CD3+ as well as
CD8+ T cell density (cells/mm2 ) in diagnostic biopsies prior to neoadjuvant CRT.

2.3. Microsatellite Status

MSI testing (IdyllaTM MSI Test, Biocartis, Mechelen, Belgium) was performed according to the
manufacturer’s recommendations. In short, one or more 10-µm FFPE sections containing ≥20%
neoplastic cells and an overall tissue area of >25 mm2 were used per analysis. The sections were
sandwiched in nuclease-free water wetted Whatman filter papers (grade 1, 10 mm circles, GE healthcare,
Buckinghamshire, GB) and placed inside the MSI cartridge and measured with the IdyllaTM instrument.
Seven MSI biomarkers were simultaneously analyzed (ACVR2A, BTBD7, DIDO1, MRE11, RYR3,
SEC31A, SULF2). The analysis was considered valid if ≥5 out of these 7 biomarkers showed valid marker
results. A sample was acknowledged as “microsatellite instability-high” (MSI-H) if ≥2 biomarkers were
found to be mutated. Otherwise, the sample was classified as “microsatellite stable” (MSS).

2.4. Tumor Regression Grade

The tumor regression grade (TRG) after neoadjuvant CRT was assessed by the Dworak regression
score based on the ratio of viable tumor cells to fibrosis (ranging from 0 to 4); TRG 4 defined a pathologic
complete remission (pCR) [26]. Patients achieving a TRG 3 or TRG 4 were classified as responders to
neoadjuvant CRT, and patients with a TRG 0–2 were classified as non-responders.

2.5. Statistical Analysis

For PD-L1, established dichotomized cut-off values from phase III clinical trials [5,11,12,27] as
well as quartiles were chosen to assess the impact on clinical outcome. Kaplan–Meier survival curves
together with log-rank testing were used to compare survival distributions between patient groups.
Disease-free survival (DFS) was calculated from the date of surgery of the primary tumor until the
date of relapse or date of last known follow-up for stage I–III rectal cancer patients. Patients without
recurrence at the last contact were censored. Overall survival (OS) was calculated from the date of
initial diagnosis of rectal cancer stage I–III until the date of death or date of last known follow-up.
Patients alive at the last contact were censored. Stage IV rectal cancer patients were excluded from DFS
and OS analyses. Continuous data, such as age, were summarized using medians and ranges and
compared between groups with the Mann–Whitney test. Correlations were tested using the Spearman
test. Parameters that proved statistically significant in univariate analysis (p < 0.05) were included in
multivariate analysis. p-values < 0.05 were considered to indicate statistical significance. SPSS IBM
(version 23.0, New York, USA) and R (version 3.5.1, www.R-project.org, Vienna, Austria) including
“package” survival were used for statistical analysis.

3. Results

3.1. Patient and Treatment Characteristics

This retrospective analysis was based on the data of 72 rectal cancer patients diagnosed and/or
treated at the tertiary cancer center in Salzburg, Austria, between January 2003 and October 2012.
The baseline characteristics are depicted in Table 1.
J. Clin. Med. 2020, 9, 2775 4 of 16

Table 1. Baseline characteristics.

Parameter N = 72 (%)
Age
≤65 years 44 (61)
>65 years 28 (39)
Sex
female 23 (32)
male 49 (68)
ypN stage
N- 48 (67)
N+ 24 (33)
cTNM stage
I 1 (1)
II 29 (40)
III 37 (52)
IV 5 (7)
Histologic grade
I 1 (1)
II 61 (87)
III 8 (12)
NA 2
Dworak tumor regression grade
0 3 (4)
I 16 (22)
II 25 (35)
III 20 (28)
IV 8 (11)
Microsatellite status
MSS 56 (98)
MSI 1 (2)
NA 15
CRT backbone
5-FU or capecitabine 29 (40)
5-FU + oxaliplatin or capecitabine + oxaliplatin 43 (60)
NAR score
low 11 (15)
intermediate 36 (50)
high 25 (35)
Adjuvant chemotherapy
yes 36 (52)
no 33 (48)
NA 3
CRT: chemoradiotherapy; MSI: microsatellite instability; MSS: microsatellite stability; NA: not available, NAR score:
neoadjuvant rectal score; PD-L1: programmed death-ligand 1.

3.2. PD-L1 Expression Prior to Neoadjuvant CRT and PD-L1 Changes after Completion of CRT
PD-L1 expression status prior to neoadjuvant CRT was evaluable in 70 (97%) patients. PD-L1
positivity (≥1%) according to TPS, CPS, and IC was found in 93%, 97%, and 97%, respectively. PD-L1
expression significantly decreased after completion of neoadjuvant CRT according to TPS (median:
4.0 vs. 0.0, p < 0.001), CPS (median: 18.5 vs. 3.0, p < 0.001) and IC (median: 13.0 vs. 3.0, p = 0.001)
(Figure 1A). The decline of PD-L1 expression was independent of the chemotherapy backbone
(5-FU/oxaliplatin-based doublet chemotherapy vs. 5-FU-based monotherapy, Figure 1B,C). PD-L1
expression and CD8+ T cell density prior to neoadjuvant CRT showed a statistically significant positive
J. Clin. Med. 2020, 9, 2775 5 of 16

correlation across all PD-L1 scores (TPS: r = 0.28, p = 0.03; CPS: r = 0.32, p = 0.01; IC: r = 0.28, p = 0.03).
A statistically significant positive correlation between PD-L1 expression and CD3+ T cell density was
J. Clin. Med. 2020, 9, x FOR PEER REVIEW 6 of 17
found for IC (r = 0.30, p = 0.02); this was not the case for TPS (r = 0.10, p = 0.45) or CPS (r = 0.24, p = 0.06).

Figure PD-L1TPS,
Figure1.1. PD-L1 TPS,CPS,
CPS,andand IC changes
IC changes during
during neoadjuvant
neoadjuvant CRT.TPS,
CRT. PD-L1 PD-L1
CPSTPS, CPS
and IC and IC
changes
changes during neoadjuvant CRT (A) in the entire cohort, (B) in patients with doublet chemotherapy,
during neoadjuvant CRT (A) in the entire cohort, (B) in patients with doublet chemotherapy, and (C)
and
in (C) in patients
patients with monochemotherapy.
with monochemotherapy. CPS: combined
CPS: combined positivepositive score;chemoradiotherapy;
score; CRT: CRT: chemoradiotherapy;
IC:
IC:immune
immune cell score; PD-L1: programmed death-ligand 1; TPS: tumor proportion score.score.
cell score; PD-L1: programmed death-ligand 1; TPS: tumor proportion

3.3. PD-L1 Expression Prior to Neoadjuvant CRT and Association With Surrogate Endpoints
J. Clin. Med. 2020, 9, 2775 6 of 16
J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 17

3.3. PD-L1
Tumor Expression Prior
regression wastostatistically
Neoadjuvantsignificantly
CRT and Association
associatedwithwith
Surrogate
PD-L1Endpoints
CPS (Dworak TRG 0–2:
16.0 Tumor
(median) vs. Dworak
regression was TRG 3–4: 29.0
statistically (median), passociated
significantly = 0.02) and PD-L1
with PD-L1IC CPS
(Dworak TRGTRG
(Dworak 0–2: 0–2:
11.0
(median) vs. Dworak TRG 3–4: 18.0 (median), p = 0.02) prior to neoadjuvant
16.0 (median) vs. Dworak TRG 3–4: 29.0 (median), p = 0.02) and PD-L1 IC (Dworak TRG 0–2: 11.0CRT, while this was not
the case for PD-L1 TPS (Dworak TRG 0–2: 4.0 (median) vs. Dworak TRG 3–4: 3.0
(median) vs. Dworak TRG 3–4: 18.0 (median), p = 0.02) prior to neoadjuvant CRT, while this was not (median), p = 0.24)
(Figure
the case 2).
forThe
PD-L1pCR rate
TPS did not TRG
(Dworak correlate
0–2: with PD-L1 TPS
4.0 (median) vs. (pCR:
Dworak11.0TRG
vs. non-pCR: 3.5, p = 0.35),
3–4: 3.0 (median), CPS
p = 0.24)
(pCR: 29.0 vs. non-pCR: 17.5, p = 0.62), or IC (pCR: 17.5 vs. 13.0, p = 0.71) prior to neoadjuvant
(Figure 2). The pCR rate did not correlate with PD-L1 TPS (pCR: 11.0 vs. non-pCR: 3.5, p = 0.35), CPS CRT.
PD-L129.0
(pCR: expression prior to
vs. non-pCR: neoadjuvant
17.5, CRT
p = 0.62), or IC did
(pCR:not17.5
showvs.an association
13.0, p = 0.71)with
priorthe NAR score (TPS:
to neoadjuvant CRT.r
= −0.08, p = 0.52; CPS: r = −0.13, p = 0.29; IC: r = −0.11, p = 0.37).
PD-L1 expression prior to neoadjuvant CRT did not show an association with the NAR score (TPS:
r = −0.08, p = 0.52; CPS: r = −0.13, p = 0.29; IC: r = −0.11, p = 0.37).

Figure 2. Association between PD-L1 expression prior to neoadjuvant CRT and Dworak tumor
regression grade. CPS:between
Figure 2. Association combined positive
PD-L1 score; CRT:
expression chemoradiotherapy;
prior to neoadjuvant CRT IC:and
immune
Dworakcell tumor
score;
PD-L1: programmed death-ligand 1; TPS: tumor proportion score. * Outliers above quartile 3
regression grade. CPS: combined positive score; CRT: chemoradiotherapy; IC: immune cell score; PD- (Q3) +
1.5× interquartile range.
L1: programmed * Outliers
death-ligand above
1; TPS: quartile
tumor 3 (Q3) +score.
proportion 3× interquartile range.

3.4. PD-L1 Expression and Clinical Outcome

3.4. PD-L1 Expression and Clinical Outcome
3.4.1. PD-L1 Expression Prior to Neoadjuvant CRT
3.4.1. PD-L1 Expression Prior to Neoadjuvant CRT
Established cut-off values for PD-L1 TPS, CPS, and IC prior to neoadjuvant CRT did not separate
rectalEstablished cut-off
cancer patients values
with for PD-L1
different TPS, CPS,
risk profiles forand IC prior
OS (Table to neoadjuvant
A1). CRT did
By using quartiles not
(Q) forseparate
PD-L1
TPS (Q1: ≤1% vs. Q2–Q4: >1%), a statistically significant survival advantage for rectal cancerfor
rectal cancer patients with different risk profiles for OS (Table A1). By using quartiles (Q) PD-L1
patients
TPS higher
with (Q1: ≤1% vs. Q2–Q4:
PD-L1 >1%),
TPS prior a statisticallyCRT
to neoadjuvant significant
was shown survival advantage
(median OS: not for rectalvs.
reached cancer
95.4 patients
months
with higher PD-L1 TPS prior to neoadjuvant CRT was shown (median OS: not
(95% CI: 51.8—not reached), p = 0.03, Figure 3A). Higher PD-L1 TPS (≤1% vs. >1%) was associated reached vs. 95.4
months (95% CI: 51.8—not reached), p = 0.03,
with a trend towards better DFS (p = 0.16, Figure 3B). Figure 3A). Higher PD-L1 TPS (≤1% vs. >1%) was
associated
Patientwith a trend
baseline andtowards better DFS (p according
tumor characteristics = 0.16, Figure 3B).
to the PD-L1 TPS status (≤1% vs. >1%) prior
Patient baseline and tumor characteristics
to neoadjuvant CRT are depicted in Table A2. according to the PD-L1 TPS status (≤1% vs. >1%) prior
to neoadjuvant CRT are depicted in Table A2.
J. Clin. Med. 2020, 9, x FOR PEER REVIEW 8 of 17
J. Clin. Med. 2020, 9, 2775 7 of 16

Figure 3. Overall survival (A) and disease-free survival (B) according to PD-L1 TPS prior to neoadjuvant
CRT. CRT: chemoradiotherapy; PD-L1: programmed death-ligand 1; TPS: tumor proportion score.
FigureThe
3.tick
Overall survival
marks on (A)represent
the curves and disease-free survival (B) according to PD-L1 TPS prior to
censored patients.
neoadjuvant CRT. CRT: chemoradiotherapy; PD-L1: programmed death-ligand 1; TPS: tumor
3.4.2. PD-L1 Changes after Completion of CRT
proportion score. The tick marks on the curves represent censored patients.
In order to evaluate the impact of PD-L1 changes on clinical outcome, patients with low PD-L1
TPS (≤1%)
3.4.2. PD-L1 Changesprior to After
and after neoadjuvantofCRT
Completion CRTwere compared to all other patients. Patients with a low
PD-L1 TPS at both time points displayed a significantly inferior OS (median OS: 56.7 months (95% CI:
In51.4—not
order toreached)
evaluate vs.the
notimpact
reached,ofp PD-L1
= 0.005, changes onand
Figure 4A) clinical
a trendoutcome, patients
towards worse DFSwith low PD-L1
(p = 0.07,
TPS (≤1%) prior
Figure 4B). to and after neoadjuvant CRT were compared to all other patients. Patients with a
low PD-L1 TPS at both time points displayed a significantly inferior OS (median OS: 56.7 months
(95% CI: 51.4—not reached) vs. not reached, p = 0.005, Figure 4A) and a trend towards worse DFS (p
= 0.07, Figure 4B).
J. Clin. Med. 2020, 9, x FOR PEER REVIEW 9 of 17
J. Clin. Med. 2020, 9, 2775 8 of 16

Figure 4. Overall survival (A) and disease-free survival (B) according to PD-L1 TPS changes during
Figure 4. Overall
neoadjuvant survival
CRT. (A) and disease-free
CRT: chemoradiotherapy; survival
PD-L1: (B) according
programmed to PD-L1
death-ligand 1; TPS: TPS
tumorchanges during
proportion
neoadjuvant
score. TheCRT. CRT:onchemoradiotherapy;
tick marks PD-L1:patients.
the curves represent censored programmed death-ligand 1; TPS: tumor
proportion score. The tick marks on the curves represent censored patients.
3.5. Univariate and Multivariate Analysis for DFS and OS
Sex (male
3.5. Univariate and vs. female), age
Multivariate Analysis for≤65
(>65 vs. DFSyears),
and OSypN stage (positive vs. negative), Dworak TRG
(3–4 vs. 0–2), histologic grade (3 vs. 1–2), application of adjuvant chemotherapy (yes vs. no), and PD-L1
Sex prior
TPS (maletovs. female), age
neoadjuvant CRT(>65 vs. ≤65
(>1% years),
vs. ≤1%) ypNtested
were stagein(positive
univariatevs.analysis
negative),
andDworak TRG (3–
multivariate
4 vs.analysis
0–2), histologic grade (3 vs. 1–2), application of adjuvant chemotherapy (yes
for DFS and OS. None of these parameters was statistically significantly associated withvs. no), and PD-L1
TPS DFS
priorinto neoadjuvant
univariate CRT
analysis (>1%
(Table 2). vs.
Age≤1%)
(>65 were tested
vs. ≤65 years,inhazard
univariate analysis
ratio: 3.12 and1.25–7.82),
(95%CI: multivariate
p = 0.02)
analysis for DFS and OS.
and PD-L1 TPSNone
prioroftothese parameters
neoadjuvant was vs.
CRT (>1% statistically significantly
≤1%, hazard associated
ratio: 0.36 (95%CI: with DFS
0.14–0.91,
p = 0.03) were
in univariate analysis (Tablewith
associated 2). Age
OS in(>65 vs. ≤65analysis
univariate years, hazard ratio: independently
and remained 3.12 (95%CI: 1.25–7.82), p = 0.02)
associated with
OS in multivariate
and PD-L1 TPS prior toanalysis (age: hazard
neoadjuvant CRTratio:
(>1%4.09 (95%CI:
vs. ≤1%, 1.54–10.86),
hazard p = 0.005;
ratio: 0.36 PD-L1
(95%CI: TPS prior
0.14–0.91, p to
= 0.03)
neoadjuvant CRT: hazard ratio: 0.29 (95%CI: 0.11–0.76), p = 0.01) (Table 3).
were associated with OS in univariate analysis and remained independently associated with OS inDFS strongly correlated
with OS among
multivariate patients
analysis (age:with stageratio:
hazard I–III rectal
4.09 cancer (r: 0.91,
(95%CI: p < 0.001).p = 0.005; PD-L1 TPS prior to
1.54–10.86),
neoadjuvant CRT: hazard ratio: 0.29 (95%CI: 0.11–0.76), p = 0.01) (Table 3). DFS strongly correlated
with OS among patients with stage I–III rectal cancer (r: 0.91, p <0.001).
J. Clin. Med. 2020, 9, 2775 9 of 16

Table 2. Univariate and multivariate analysis for disease-free survival.

UVA MVA
HR HR
p-Value p-Value
(95% CI) (95% CI)
Sex
male (n = 45) 1.86 − −
0.44
female (n = 22) (0.39–8.95)
Age
>65 (n = 25) 0.56 − −
0.47
≤65 (n = 42) (0.12–2.69)
ypN stage
N+ (n = 20) 1.21 − −
0.79
N− (n = 47) (0.30–4.84)
Dworak TRG
3–4 (n = 28) 0.74 − −
0.67
0–2 (n = 39) (0.18–2.95)
Histologic grade
3 (n = 7) 0.20 − −
0.49
1 + 2 (n = 58) (0.002–17.98)
Adjuvant chemotherapy
0.88
yes (n = 35) 0.86 − −
(0.22–3.52)
no (n = 30)
PD-L1 TPS pre-CRT
0.40
>1% (n = 48) 0.18 − −
(0.11–1.50)
≤1% (n = 17)
NAR score
intermediate/high (n = 56) 27.20
0.38 − −
low (n = 11) (0.02–43,658.20)
95% CI: 95% confidence interval; CRT: chemoradiotherapy; HR: hazard ratio; MVA: multivariate analysis; NAR
score: neoadjuvant rectal score; PD-L1: programmed death-ligand 1; TPS: tumor proportion score; TRG: tumor
regression grade; UVA: univariate analysis.

Table 3. Univariate and multivariate analysis for overall survival.

UVA MVA
HR HR
p-Value p-Value
(95% CI) (95% CI)
Sex
2.24
male (n = 45) 0.15 - -
(0.74–6.76)
female (n = 22)
Age
3.12 4.09
>65 (n = 25) 0.02 0.005
(1.25–7.82) (1.54–10.86)
≤65 (n = 42)
ypN stage
1.11
N+ (n = 20) 0.84 - -
(0.42–2.91)
N− (n = 47)
Dworak TRG
1.10
3–4 (n = 28) 0.84 - -
(0.44–2.75)
0–2 (n = 39)
Histologic grade
0.67
3 (n = 7) 0.44 - -
(0.25–1.84)
1 + 2 (n = 58)
Adjuvant chemotherapy
0.72
yes (n = 35) 0.49 - -
(0.28–1.83)
no (n = 30)
PD-L1 TPS pre-CRT
0.36 0.29
>1% (n = 48) 0.03 0.01
(0.14–0.91) (0.11–0.76)
≤1% (n = 17)
NAR score
1.89
intermediate/high (n = 56) 0.40 − −
(0.44–8.17)
low (n = 11)
95% CI: 95% confidence interval; CRT: chemoradiotherapy; HR: hazard ratio; MVA: multivariate analysis; NAR
score: neoadjuvant rectal score; PD-L1: programmed death-ligand 1; TPS: tumor proportion score; TRG: tumor
regression grade; UVA: univariate analysis.
J. Clin. Med. 2020, 9, 2775 10 of 16

4. Discussion
By investigating three established PD-L1 scores in this retrospective analysis, we found a
statistically significant survival benefit for rectal cancer patients with a higher PD-L1 TPS (>1%)
compared to patients with a PD-L1 TPS ≤1% prior to neoadjuvant CRT (Figure 3A). The pre-CRT PD-L1
TPS was not associated with baseline patient and tumor characteristics (Table A2). Other clinically
established cut-off values for PD-L1 expression were not associated with OS (Table A1). Our findings
of a positive prognostic value of higher PD-L1 TPS prior to neoadjuvant CRT are in line with the
publication by Chen et al. [21], while Chiang et al. [19], Hecht et al. [18], and Ogura et al. [20] did
not show a statistically significant association between PD-L1 TPS prior to neoadjuvant CRT and
OS. The prognostic value of PD-L1 TPS was not restricted to the initial diagnostic biopsy prior to
neoadjuvant CRT in our rectal cancer cohort as patients with a PD-L1 TPS ≤1% pre- and post-CRT
displayed worse OS compared to patients with PD-L1 upregulation at any time point (Figure 4A).
The latter finding suggests that PD-L1 upregulation on cancer cells either before or after CRT is sufficient
to improve OS. PD-L1 TPS [19,21] as well as PD-L1 CPS at the invasive front [18] after neoadjuvant CRT
were associated with OS in several retrospective analyses. Neoadjuvant chemoradiotherapy is able
to trigger interferon-gamma release from cancer cells and in turn to induce PD-L1 expression [19,28].
PD-L1 expression statistically significantly decreased during CRT according to each PD-L1 score in our
cohort (Figure 1A); this was independent of the chemotherapy backbone (Figure 1B,C). Hecht et al. also
reported a statistically significantly decrease of PD-L1 expression on immune cells after neoadjuvant
CRT, whereas no changes of PD-L1 expression on cancer cells were found [18]. An increase of the
proportion of PD-L1-positive/high samples (defined by an arbitrary threshold of 5%) during neoadjuvant
CRT was described by Chiang et al. (PD-L1 expression on cancer cells) [19], Ogura et al. (PD-L1 expression
on immune cells but not on cancer cells) [20], and by Chen et al. (PD-L1 expression on cancer
cells) [21]; however, changes of the PD-L1 scores were not reported. Given the known heterogeneity
of PD-L1 expression within tumors [29], we cannot exclude bias resulting from the analysis of the
initial diagnostic biopsy, and comparison with PD-L1 expression in the surgical resectate, since it only
represents a small portion of the tumor.
As an immunosuppressive checkpoint on the one hand and a favorable prognostic factor in
rectal cancer undergoing neoadjuvant CRT on the other hand, the role of PD-L1 expression seems
contradictory. A putative explanation for this observation is the development of an immunosuppressive
tumor microenvironment by PD-L1 upregulation in immunogenic tumors during the course of disease
as it has been shown for CRC with MSI [30]. In other words, PD-L1 expression via upregulation of
interferon-gamma exhibits the capability of a viable adaptive immune response. Surgical removal of
the tumor after neoadjuvant CRT may reinvigorate pre-existing antitumor immune responses and may
in turn lead to improved clinical outcome. In a meta-analysis including stage I–IV colon and rectal
cancer patients, PD-L1 TPS proved to be a negative prognostic factor for DFS and OS. However, it is
noteworthy that PD-L1 expression was statistically significantly associated with right-sidedness and
poor differentiation, which are well-known tumor characteristics with a negative prognostic value [31].
Testing PD-L1 TPS (≤1% vs. >1%) prior to neoadjuvant CRT and established risk factors in
univariate and multivariate analysis revealed an independent favorable impact of higher PD-L1 TPS
on OS (hazard ratio: 0.29, Table 3). Hecht et al. found an independent association between PD-L1 CPS
prior to as well as after neoadjuvant CRT and OS [18] and Chiang et al. described an independent
impact of PD-L1 TPS after neoadjuvant CRT on survival [19] in multivariate analyses, respectively.
Yoshino et al. reported a 30% pCR rate by incorporating five cycles of the immune-checkpoint
inhibitor nivolumab to neoadjuvant CRT. Among patients with a PD-L1 TPS ≥1% prior to neoadjuvant CRT,
the pCR rate was doubled [32]. The latter findings are hypothesis generating and suggest therapeutic
approaches to induce or increase PD-L1 expression before the application of immune-checkpoint
inhibitors. However, the addition of oxaliplatin to a 5-FU-based CRT (Figure 1B) had no impact on
PD-L1 expression changes when compared to a 5-FU-based CRT (Figure 1C). Therapeutic approaches
to induce major histocompatibility complex I expression on cancer cells by MEK inhibitors in order to
J. Clin. Med. 2020, 9, 2775 11 of 16

improve immune-checkpoint blockade efficacy have not resulted in better clinical outcome in metastatic
CRC [33]. Antiangiogenic therapy in combination with immune-checkpoint blockade promotes the
formation of high endothelial venules, thereby increasing tumor-infiltrating lymphocytes in tumor
mouse models [34]. In the ongoing “TARZAN“ trial (NCT04017455), the latter therapeutic approach
is being investigated by combining nivolumab with bevacizumab during neoadjuvant therapy of
localized rectal cancer.
Our retrospective analysis has several limitations. Surrogate end points, such as TRG as well
as the NAR score, did not impact OS in our rectal cancer cohort, which was likely attributable to
the limited sample size. However, the strong correlation between DFS and OS (r: 0.91) corroborates
that disease relapse was the predominant cause of death. The addition of oxaliplatin to 5-FU-based
neoadjuvant CRT increases pCR rates [35,36] but comes at the cost of a higher frequency of grade 3
and 4 adverse events [37]. More than half of our patients received a 5-FU-based neoadjuvant CRT in
combination with oxaliplatin, a therapy combination that is not recommended by the current ESMO [2]
or NCCN [1] rectal cancer guidelines. Decisions on an individual patient basis to initiate neoadjuvant
CRT with 5-FU and oxaliplatin were based on improved pCR rates and/or DFS rates reported in the
CAO/ARO/AIO-04 trial [35,38] and FOWARC trial [36]. However, CRT with 5-FU monotherapy is the
standard neoadjuvant therapy protocol at our institution. The inclusion period of rectal cancer patients
was 10 years (2003–2012) in this retrospective analysis; however, this time interval was comparable to
other reports [18–21]. The localization of CD3+ and CD8+ T cells (intratumoral vs. invasive margin)
was not taken into account. However, the standardized and validated “immunoscore” in its original
version is neither applicable to localized rectal cancer prior to neoadjuvant CRT, nor after CRT due to
post-radiogenic alterations. A recently published modified version predicting response to neoadjuvant
CRT in localized rectal cancer, the “biopsy-adapted immunoscore”, is solely based on CD3+ and CD8+
T cell density in the tumor region [39].
In conclusion, PD-L1 TPS prior to neoadjuvant CRT as well as PD-L1 TPS changes showed a
statistically significant impact on survival in rectal cancer patients undergoing neoadjuvant CRT,
and this was not the case for PD-L1 CPS or PD-L1 IC. PD-L1 TPS prior to neoadjuvant CRT proved as an
independent prognostic parameter for OS in multivariate analysis. A clinically relevant OS disadvantage
was shown for patients with PD-L1 TPS ≤1% in the initial diagnostic tumor biopsy and in the surgical
resectate after neoadjuvant CRT. Considering the published encouraging pCR rates by CRT combined
with nivolumab in PD-L1 TPS-positive rectal cancer patients, a sequential therapeutic approach with
CRT aiming at upregulating PD-L1 followed by the application of immune-checkpoint blockade
might be interesting for initially PD-L1 TPS-negative tumors. The identification of PD-L1-inducing or
-upregulating agents for the combination with immune-checkpoint inhibitors in neoadjuvant treatment
concepts of rectal cancer will be of utmost importance in future clinical trials. However, a prospective
validation of the prognostic value of PD-L1 expression in rectal cancer patients undergoing neoadjuvant
CRT is necessitated.

Author Contributions: Conceptualization: F.H., E.K., D.N., R.G., L.W.; methodology: F.H., E.K., D.N., R.G., L.W.;
software: F.H., L.W., G.R.; validation: F.H., L.W., G.R.; formal analysis: F.H., L.W.; investigation: F.H., E.K., D.N.,
V.S., G.R., F.P., A.K., C.E.S., W.I., F.S., T.J., A.D., N.Z., M.S., R.G., L.W.; resources: D.N., F.P., R.G.; data curation:
F.H., L.W.; writing—original draft preparation: F.H., L.W.; writing—review and editing: F.H., E.K., D.N., V.S.,
G.R., F.P., A.K., C.E.S., W.I., F.S., T.J., A.D., N.Z., M.S., R.G., L.W.; visualization: F.H., L.W.; supervision: L.W., R.G.;
project administration: L.W.; funding acquisition: L.W., F.H. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by a “RISE” research grant from the Paracelsus Medical University Salzburg
(RISE R15/05/075-HUE) and by a grant from the Oesterreichische Krebshilfe. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the
decision to publish the results.
J. Clin. Med. 2020, 9, 2775 12 of 16

Conflicts of Interest: Florian Huemer reports travel support from Roche, Merck, BMS, Servier and Pfizer and
an advisory role for Servier and Lilly; Eckard Klieser has nothing to disclose; Daniel Neureiter has nothing to
disclose; Verena Schlintl has nothing to disclose; Gabriel Rinnerthaler reports an advisory role for Pierre Fabre,
Roche, Novartis, Pfizer, Eli Lilly, travel support from Roche, Pfizer, BMS and Amgen, Speaker’s Bureau for Roche,
Pfizer, Eli Lilly, AstraZeneca and BMS; Franck Pagès reports grants from HalioDx, participation to scientific
advisory boards and meetings for BMS, Roche, Janssen, Merck and Gilead. Amos Kirilovsky has nothing to
disclose. Carine El Sissy has nothing to disclose. Wolfgang Iglseder has nothing to disclose. Franz Singhartinger
has nothing to disclose. Tarkan Jäger has nothing to disclose. Adam Dinnewitzer reports grants from the
Paracelsus Medical University Salzburg and from the Oesterreichische Krebshilfe. Nadja Zaborsky has nothing
to disclose. Markus Steiner has nothing to disclose. Richard Greil reports honoraria, travel accommodations,
consulting/advisory role and research funding from Celgene, Roche, Merck, Takeda, AstraZeneca, Novartis,
Amgen, BMS, MSD, Sandoz, Abbvie, Gilead, Daiichi Sankyo and Janssen. Lukas Weiss reports travel support
from IPSEN, Merck, AstraZeneca, Boehringer Ingelheim, Pharmamar and Bayer, an advisory role for Merck, Lilly,
Novocure and Nordic Pharma, honoraria from Merck, Roche, BMS, Lilly and Novocure.

Appendix A

Table A1. DFS and OS according to established PD-L1 cut-off values.

DFS OS
PD-L1 Expression Median p-Value Median p-Value
Prior to CRT (n) (months) (log-rank) (months) (log-rank)
TPS
<1% (4) 24.5 56.7
0.04 0.21
≥1% (61) NR NR
≤5% (39) NR NR
0.83 0.16
>5% (26) NR NR
≤10% (48) NR NR
0.27 0.11
>10% (17) NR NR
≤20% (56) NR NR
0.20 0.27
>20% (9) NR NR
≤50% (61) NR NR
0.41 0.23
>50% (4) NR NR
CPS
<1 (2) 24.5 44.7
0.17 0.32
≥1 (63) NR NR
≤10 (15) NR NR
0.34 0.58
>10 (50) NR NR
≤20 (34) NR 134.2
0.85 0.05
>20 (31) NR NR
≤50 (52) NR NR
0.97 0.24
>50 (13) NR NR
IC
<1% (2) 24.5 44.7
0.17 0.32
≥1% (63) NR NR
≤5% (11) NR 132.9
0.60 0.47
>5% (54) NR NR
≤10% (23) NR NR
0.87 0.66
>10% (42) NR NR
≤20% (46) NR NR
0.64 0.18
>20% (19) NR NR
≤50% (59) NR NR
0.88 0.43
>50% (6) NR NR
CPS: combined positive score; CRT: chemoradiotherapy; DFS: disease-free survival; IC: immune cell score; NR: not
reached; OS: overall survival; PD-L1: programmed death-ligand 1; TPS: tumor proportion score.
J. Clin. Med. 2020, 9, 2775 13 of 16

Table A2. Baseline patient and tumor characteristics according to PD-L1 TPS status prior to
neoadjuvant CRT.

PD-L1 TPS ≤1% PD-L1 TPS >1%

Parameter p-Value
N = 17 (%) N = 48 (%)
Age
≤65 years 11 (65) 29 (60) 0.755
>65 years 6 (35) 19 (40)
Sex
female 4 (23) 17 (35) 0.368
male 13 (77) 31 (65)
cTNM stage
I 0 (0) 1 (2)
0.559
II 9 (53) 19 (40)
III 8 (47) 28 (58)
cT stage
T2 1 (6) 1 (2)
0.662
T3 14 (82) 43 (90)
T4 2 (12) 4 (8)
cN stage
N- 9 (53) 20 (42) 0.422
N+ 8 (47) 28 (58)
Histologic grade
I 1 (6) 0 (0)
II 14 (82) 42 (91) 0.230
III 2 (12) 4 (9)
NA 0 2
Lymphovascular
invasion
no 11 (85) 30 (79) 0.657
yes 2 (15) 8 (21)
NA 4 10
Venous invasion
no 10 (83) 38 (97)
0.069
yes 2 (17) 1 (3)
NA 5 9
PD-L1: programmed death-ligand 1; TPS: tumor proportion score.

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