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. 2017 Apr 12;66(7):927–939. doi: 10.1007/s00262-017-1999-6

Prognostic implication of CD274 (PD-L1) protein expression in tumor-infiltrating immune cells for microsatellite unstable and stable colorectal cancer

Kyu Sang Lee 1, Yoonjin Kwak 1, Soyeon Ahn 2, Eun Shin 1, Heung-Kwon Oh 3, Duck-Woo Kim 3, Sung-Bum Kang 3, Gheeyoung Choe 1,4, Woo Ho Kim 4, Hye Seung Lee 1,4,✉
PMCID: PMC11028600  PMID: 28405764

Abstract

In this study, we investigated the clinical relevance of CD274 (PD-L1) protein expression by tumor cells and tumor-infiltrating immune cells in colorectal cancer (CRC). To this end, 186 microsatellite instability-high (MSI-H) and 153 microsatellite stable (MSS) CRCs were subjected to immunohistochemistry (IHC) analysis for the expression of CD274 and mismatch repair proteins. CD274 expression was evaluated in tumor cells at the center (TC) and periphery (TP), and immune cells at the center (IC) and periphery (IP) of CRC. IHC slides stained for CD3 and CD8 were scanned using an Aperio ScanScope for precise calculation of tumor-infiltrating T cell density. Additionally, samples were screened for the B-Raf (BRAF)-V600E mutation using a Cobas 4800 System and IHC. In total, CD274TC, CD274TP, CD274IC, and CD274IP were observed in 43 (23.1%), 47 (25.3%), 107 (57.5%), and 102 (54.8%) of the MSI-H CRCs examined, and in three (2.0%), four (2.6%), 47 (30.7%), and 56 (36.6%) of the 153 MSS CRCs tested. Meanwhile, intratumoral heterogeneity of CD274 expression in tumor cells and immune cells was detected in 24 (12.9%) and 47 (25.3%) MSI-H CRCs, respectively. Notably, in both MSI-H and MSS CRC, CD274IC and CD274IP were independently associated with improved prognosis (P < 0.05), while BRAF mutation was associated with CD274TP, poor differentiation, sporadic type, and hMLH1(−)/hMSH2(+)/hMSH6(+)/PMS2(−) in MSI-H CRC (P < 0.006). In conclusion, CD274 expression in tumor-infiltrating immune cells was an independent factor for improved prognosis in CRC patients. A deeper understanding of CD274 status may yield improved responses to future CRC immunotherapies.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-017-1999-6) contains supplementary material, which is available to authorized users.

Keywords: Colorectal cancer, CD274, PD-L1, Tumor-infiltrating immune cells, Microsatellite instability, Prognosis

Introduction

The remarkable development of immunotherapeutics for several cancers has changed the anti-cancer therapeutic paradigm. Particularly, targeting of PDCD1 (PD-1) and its ligand, CD274 (PD-L1), has demonstrated excellent anti-tumor effects in various tumors [1, 2]. The PDCD1/CD274 checkpoint is thought to comprise a key mechanism of host immune system evasion in malignancy [3], and anti-PDCD1 antibodies have been approved by the US Food and Drug Administration (FDA) for metastatic non-small-cell lung cancer patients [4]. Interestingly, CD274 expression has primarily been observed in MSI-H CRCs, and is only rarely seen in microsatellite stable (MSS) CRCs [5, 6]. Moreover, a recent study indicated that PDCD1 blockade using drugs such as pembrolizumab comprises a specific and highly effective method for treating microsatellite instability-high (MSI-H) CRC [7]. As such, the FDA has granted a priority review to evaluate the efficacy of pembrolizumab for treating MSI-H advanced CRC.

Many studies have examined the prognostic impact of CD274 status and its predictive value in various malignancies. Recent studies showed that CD274 was expressed in several malignant tumors, where it tended to be correlated with decreased survival [1, 8]. However, the prognostic value of CD274 expression remains to be determined, as other studies have detected debatable correlations [9–11], or even no correlation [5, 6, 12], between prognosis and CD274 expression in CRC patients.

Interestingly, a previous report observed an improved response to PDCD1 blockade therapy in patients exhibiting CD274 expression on tumor-infiltrating immune cells [13]. In particular, abundant tumor-infiltrating immune cells are frequently observed in MSI-H CRC [14]. Thus, further evaluation of CD274 expression by immune cells, as well as tumor cells, is needed to facilitate CRC immunotherapy.

Frequently, certain subsets of cancer patients will show decreased responses to targeted therapies due to regional heterogeneity of target molecules, and immunotherapy seems to be no exception. CD274 expression likely has more regional heterogeneity than other mutational alterations, because the PDCD1/CD274 axis is part of a dynamic immune reaction. Indeed, certain recent studies suggest that the value of CD274 immunohistochemistry (IHC) as a predictive and prognostic marker is debatable due to frequent regional heterogeneity [2, 15]. Therefore, regional heterogeneity of CD274 expression should be evaluated in detail in CRC.

This study was conducted to investigate the clinical relevance of CD274 expression by tumor cells and immune cells in CRC, focusing primarily on the MSI-H subgroup. We also analyzed intratumoral heterogeneity and precisely determined the tumor-infiltrating lymphocyte density in CRC using an Aperio image analysis system.

Materials and methods

Patients and samples

In total, 339 CRC patients who underwent surgical resection at Seoul National University Bundang Hospital were included in our study. The patient cohort was composed of two groups: 186 MSI-H CRC patients who were surgically treated between 2003 and 2012, and for comparison, 153 consecutive MSS CRC patients who underwent surgical treatment during 2005. Patients who had received pre-operative chemotherapy or radiotherapy were excluded from the cohort. Among the MSI-H CRC patients, 104 received post-operative chemotherapy, while 74 were only treated surgically; the post-operative history for the remaining eight patients was unobtainable. Meanwhile, 107 and 46 of the MSS CRC patients received post-operative chemotherapy or surgery alone, respectively. Two pathologists (Kyu Sang Lee and Hye Seung Lee) histologically reviewed each CRC case. Clinicopathological data were obtained from hospital medical and pathologic reports. Cancer stage was determined according to the American Joint Committee on Cancer (AJCC), 7th edition. Suspected Lynch syndrome (LS) patients were selected from the MSI-H CRC cohort according to the Bethesda guideline (2004) [16]. These patients did not fulfill the diagnostic criteria for LS, as the MMR mutation test was not performed. Follow-up information collected included patient outcome and the interval between the date of surgical resection and the date of death by any cause or censoring (overall survival).

The use of medical record data and patient tissue samples in this study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (reference: B-1511/322-306).

Tissue array method

A tissue microarray (TMA) was constructed using representative lesions of formalin-fixed paraffin-embedded (FFPE) CRC tissues (SuperBioChips Laboratories, Seoul, South Korea) [17]. Two TMA (2 mm in diameter) single cores were placed at the tumor center and periphery.

Microsatellite instability

Microsatellite instability (MSI) was examined by fragmentation assay analysis using an automated DNA sequencer (ABI 3731 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA) with the following five microsatellite markers, according to previously described methods: BAT-26, BAT-25, D5S346, D17S250, and D2S123 [18].

Immunohistochemistry

IHC analysis was performed using antibodies specific to CD274 (PD-L1, E1L3 N, 1:50; Cell Signaling Technology, Danvers, MA, USA), CD3 (1:100; Dako, Glostrup, Denmark), CD8 (1:100; Dako), B-Raf (BRAF) (Ventana, Tucson, AZ, USA), MLH1 (Ventana), MSH2 (Cell Marque, Rocklin, CA, USA), MSH6 (Cell Marque), and PMS2 (Ventana). Immunostaining was conducted using the Ventana Bench mark XT autostainer (Ventana) and the ultraView Universal DAB kit (Ventana), according to the manufacturer’s recommendations. Normal colonic epithelial cells were utilized as internal negative controls. Tissues were considered CD274-positive when more than 5% of neoplastic cells showed membrane staining of any intensity (Fig. 1) [5]. Meanwhile, tissues were considered BRAF positive when at least 80% of the tumor cells exhibited moderate to strong diffuse cytoplasmic staining (Fig. 1) [19]. Mild cytoplasmic staining in neoplastic cells was considered equivocal. Lastly, tissues containing neoplastic cells that exhibited nuclear staining for the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2, respectively, were considered positive.

Fig. 1.

Fig. 1

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Immunohistochemical staining of colorectal cancer patient samples. Images of tumor cells exhibiting a a lack of CD274 expression and b CD274 expression at the cell surface (×40). c CD274 expression on tumor-infiltrating immune cells (×40). d CD3 expression on tumor-infiltrating lymphocytes (×40). e Aperio image analysis using the Nuclear v9 algorithm: blue score 0; yellow score 1; orange score 2; red score 3 (×40). f B-Raf (BRAF) expression (×40). All immunohistochemistry images depict representative results

CD3 and CD8 IHC slides were scanned on an Aperio ScanScope (Aperio Technologies, Inc., Vista, CA, USA) at 20× magnification. For precise calculation, CD3+ and CD8+ tumor-infiltrating lymphocytes (TILs) were quantified with an ImageScope computerized image analysis system (Aperio Technologies) using the Nuclear v9 algorithm. A score of 2–3 indicated CD3+ and CD8+ T cells (Fig. 1). TIL density was calculated by dividing the percentage (%) of positive nuclei by the core area (mm2). We arbitrarily defined the cutoff value for TIL density as the median, and thereby divided TIL density into two groups: high and low.

BRAF mutation analyses

To identify BRAF mutations, DNA was harvested from 40 MSI-H CRC patient tissues including 26 BRAF IHC-positive and 14 equivocal tissues, using a Cobas DNA Sample Preparation Kit (Roche, Basel, Switzerland), according to the manufacturer’s instructions and as described previously [20]. Samples were then screened for the BRAF (V600E) mutation using a Cobas 4800 System (Roche).

Statistical analyses

Categorical variables were compared using the Chi square or Fisher’s exact test, as appropriate. The correlation between TIL density and CD274 expression was analyzed by determining Pearson’s correlation coefficients. The McNemar test was used to determine intratumoral CD274 expression heterogeneity. The association between survival and CD274 expression was evaluated using Kaplan–Meier curves with the log-rank test and Cox’s proportional hazards model. A threshold of P < 0.05 was considered statistically significant. To prevent inflation of type I error, data were subjected to multiple testing correction by Bonferroni adjustment [21]. The adjusting P values in Tables 1, 2, and 3 were 0.003, 0.004, and 0.006, respectively. IBM SPSS statistics version 21 (IBM, Armonk, NY, USA) was utilized for all statistical analyses.

Table 1.

Correlation between clinicopathological factors and CD274 expression in 186 MSI-H CRC patients

Total no. of cases CD274TC CD274TP CD274IC CD274IP
Negative Positive P value Negative Positive P value Negative Positive P value Negative Positive P value
Age
 Mean 186 58.1 65.6 0.003 57.6 66.4 <0.001 59.7 59.9 0.899 59.8 59.8 0.997
Sex
 Male 86 (46.2%) 74 (51.7%) 12 (27.9%) 0.006 70 (50.4%) 16 (34.0%) 0.049 35 (44.3%) 51 (47.7%) 0.650 40 (47.6%) 46 (45.1%) 0.731
 Female 100 (53.8%) 69 (48.3%) 31 (72.1%) 69 (49.6%) 31 (66.0%) 44 (55.7%) 56 (52.3%) 44 (52.4%) 56 (54.9%)
Location
 Cecum to D-colon 154 (83.2%) 114 (80.3%) 40 (93.0% 0.050 112 (80.6% 42 (91.3%) 0.091 66 (83.5%) 88 (83.0%) 0.925 69 (82.1%) 85 (84.2%) 0.715
 Rectosimoid colon 31 (16.8%) 28 (19.7%) 3 (7.0%) 27 (19.4%) 5 (8.7%) 13 (16.5%) 18 (17.0%) 15 (17.9%) 16 (15.8%)
Hereditary vs. sporadic
 Sporadic 132 (71.0%) 96 (67.1%) 36 (83.7%) 0.036 91 (65.5%) 41 (87.2%) 0.004 55 (69.6%) 77 (72.0%) 0.728 59 (70.2%) 73 (71.6%) 0.842
 Suspected LS 54 (29.0%) 47 (32.9%) 7 (16.3%) 48 (34.5%) 6 (12.8%) 24 (30.4%) 30 (28.0%) 25 (29.8%) 29 (28.4%)
Differentiation
 Low grade 143 (76.9%) 125 (87.4%) 18 (41.9%) <0.001 123 (88.5%) 20 (42.6%) <0.001 59 (74.7%) 84 (78.5%) 0.541 64 (76.2%) 79 (77.5%) 0.839
 High grade 43 (23.1%) 18 (12.6%) 25 (58.1%) 16 (11.5%) 27 (57.4%) 20 (25.3%) 23 (21.5%) 20 (23.8%) 23 (22.5%)
Mucinous component
 Absent 117 (62.9%) 84 (58.7%) 33 (76.7%) 0.032 79 (56.8%) 38 (80.9%) 0.003 43 (54.4%) 74 (69.2%) 0.040 48 (57.1%) 69 (67.6%) 0.140
 Present 69 (37.1%) 59 (41.3%) 10 (23.3%) 60 (43.2%) 9 (19.1%) 36 (45.6%) 33 (30.8%) 36 (42.9%) 33 (32.4%)
Tumor border
 Expending 71 (38.2%) 61 (42.7%) 10 (23.3%) 0.022 54 (38.8%) 17 (36.2%) 0.744 22 (27.8%) 49 (45.8%) 0.013 24 (28.6%) 47 (46.1%) 0.014
 Infiltrating 115 (61.8%) 82 (57.3%) 33 (76.7%) 85 (61.2%) 30 (63.8%) 57 (72.2%) 58 (54.2%) 60 (71.4%) 55 (53.9%)
T stage
 1–3 161 (86.6%) 127 (88.8%) 34 (79.1%) 0.101 121 (87.1%) 40 (85.1%) 0.736 66 (83.5%) 95 (88.8%) 0.300 68 (81.0%) 93 (91.2%) 0.042
 4 25 (13.4%) 16 (11.2%) 9 (20.9%) 18 (12.9%) 7 (14.9%) 13 (16.5%) 12 (11.2%) 16 (19.0%) 9 (8.8%)
N stage
 0 165 (89.2%) 128 (90.1%) 37 (86.0%) 0.449 122 (88.4%) 43 (91.5%) 0.557 65 (83.3%) 100 (93.5%) 0.029 70 (84.3%) 95 (93.1%) 0.055
 1–2 20 (10.8%) 14 (9.9%) 6 (14.0%) 16 (11.6%) 4 (8.5%) 13 (16.7%) 7 (6.5%) 13 (15.7%) 7 (6.9%)
M stage
 0 177 (95.2%) 137 (95.8%) 40 (93.0%) 0.456 133 (95.7%) 44 (93.6%) 0.568 71 (89.9%) 106 (99.1%) 0.004 76 (90.5%) 101 (99.0%) 0.007
 1 9 (4.8%) 6 (4.2%) 3 (7.0%) 6 (4.3%) 3 (6.4%) 8 (10.1%) 1 (0.9%) 8 (9.5%) 1 (1.0%)
pTMN stage
 I–II 121 (65.4%) 98 (69.0%) 23 (53.5%) 0.061 92 (66.7%) 29 (61.7%) 0.537 44 (55.7%) 77 (72.6%) 0.017 49 (58.3%) 72 (71.3%) 0.065
 III–IV 64 (34.6%) 44 (31.0%) 20 (46.5%) 46 (33.3%) 18 (38.3%) 35 (44.3%) 29 (27.4%) 35 (41.7%) 29 (28.7%)
Venous invasion
 Absent 177 (95.2%) 136 (95.1%) 41 (95.3%) 0.948 132 (95.0%) 45 (95.7%) 0.829 74 (93.7%) 103 (96.3%) 0.416 78 (92.9%) 99 (97.1%) 0.184
 Present 9 (4.8%) 7 (4.9%) 2 (4.7%) 7 (5.0%) 2 (4.3%) 5 (6.3%) 4 (3.7%) 6 (7.1%) 3 (2.9%)
Lymphatic invasion
 Absent 124 (66.7%) 104 (72.7%) 20 (46.5%) 0.001 103 (74.1%) 21 (44.7%) <0.001 46 (58.2%) 78 (72.9%) 0.036 54 (64.3%) 70 (68.6%) 0.532
 Present 62 (33.3%) 39 (27.3%) 23 (53.5%) 36 (25.9%) 26 (55.3%) 33 (41.8%) 29 (27.1%) 30 (35.7%) 32 (31.4%)
Perineural invasion
 Absent 166 (89.2%) 126 (88.1%) 40 (93.0%) 0.362 122 (87.8%) 44 (93.6%) 0.263 68 (86.1%) 98 (91.6%) 0.230 75 (89.3%) 91 (89.2%) 0.988
 Present 20 (10.8%) 17 (11.9%) 3 (7.0%) 17 (12.2%) 3 (6.4%) 11 (13.9%) 9 (8.4%) 9 (10.7%) 11 (10.8%)
Eosinophilic infiltration
 Minor 116 (62.4%) 91 (63.6%) 25 (58.1%) 0.514 85 (61.2%) 31 (66.0%) 0.557 51 (64.6%) 65 (60.7%) 0.596 52 (61.9%) 64 (62.7%) 0.906
 Dominant 70 (37.6%) 52 (36.4%) 18 (41.9%) 54 (38.8%) 16 (34.0%) 28 (35.4%) 42 (39.3%) 32 (38.1%) 38 (37.3%)
CD8-positive TIL
 Low 93 (50.0%) 79 (55.2%) 14 (32.6%) 0.009 83 (59.7%) 10 (21.3%) <0.001 53 (67.1%) 40 (37.4%) <0.001 62 (73.8%) 31 (30.4%) <0.001
 High 93 (50.0%) 64 (44.8%) 29 (67.4%) 56 (40.3%) 37 (78.7%) 26 (32.9%) 67 (62.6%) 22 (26.2%) 71 (69.6%)
CD3-positive TIL
 Low 93 (50.0%) 80 (55.9%) 13 (30.2%) 0.003 84 (60.4%) 9 (19.1%) <0.001 52 (65.8%) 41 (38.3%) <0.001 63 (75.0%) 30 (29.4%) <0.001
 High 93 (50.0%) 63 (44.1%) 30 (69.8%) 55 (39.6%) 38 (80.9%) 27 (34.2%) 66 (61.7%) 21 (25.0%) 72 (70.6%)
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CRC colorectal cancer, D descending; high grade, poorly and undifferentiated, IC immune cells at the center, IP immune cells at the periphery; low grade, well and moderately differentiated, LS Lynch syndrome, M metastasis, MSI-H microsatellite instability-high, N lymph node, No number, T tumor, TC tumor cells at the center, TIL tumor-infiltrating lymphocyte, TP tumor cells at the periphery

P values are calculated using χ 2 test or Fisher’s exact test

Table 2.

Correlation between clinicopathological factors and CD274 expression in 153 MSS CRC patients

Total no. of cases CD274IC P value CD274IP P value
Negative Positive Negative Positive
Age
 Mean 153 64.5 63.4 0.585 63.3 65.7 0.237
Sex
 Male 79 (51.6%) 52 (49.1%) 27 (57.4%) 0.338 48 (49.5%) 31 (55.4%) 0.484
 Female 74 (48.4%) 54 (50.9%) 20 (42.6%) 49 (50.5%) 25 (44.6%)
Location
 Cecum to D-colon 51 (33.3%) 32 (30.2%) 19 (40.4%) 0.215 35 (36.1%) 16 (28.6%) 0.342
 Rectosigmoid 102 (66.7%) 74 (69.8%) 28 (59.6%) 62 (63.9%) 40 (71.4%)
Differentiation
 Low grade 143 (93.5%) 98 (92.5%) 45 (95.7%) 0.447 90 (92.8%) 53 (94.6%) 0.654
 High grade 10 (6.5%) 8 (7.5%) 2 (4.3%) 7 (7.2%) 3 (5.4%)
Tumor border
 Expending 35 (22.9%) 26 (24.5%) 9 (19.1%) 0.465 19 (19.6%) 16 (28.6%) 0.203
 Infiltrating 118 (77.1%) 80 (75.5%) 38 (80.9%) 78 (80.4%) 40 (71.4%)
T stage
 1–3 125 (81.7%) 86 (81.1%) 39 (83.0%) 0.785 75 (77.3%) 50 (89.3%) 0.065
 4 28 (18.3%) 20 (18.9%) 8 (17.0%) 22 (22.7%) 6 (10.7%)
N stage
 0 72 (47.1%) 46 (43.4%) 26 (55.3%) 0.173 39 (40.2%) 33 (58.9%) 0.025
 1–2 81 (52.9%) 60 (56.6%) 21 (44.7%) 58 (59.8%) 23 (41.1%)
M stage
 0 135 (88.2%) 91 (85.8%) 44 (93.6%) 0.169 79 (81.4%) 56 (100.0%) 0.001
 1 18 (11.8%) 15 (14.2%) 3 (6.4%) 18 (18.6%) 0 (0.0%)
pTMN stage
 I–II 71 (46.4%) 45 (42.5%) 26 (55.3%) 0.141 37 (38.1%) 34 (60.7%) 0.007
 III–IV 82 (53.6%) 61 (57.5%) 21 (44.7%) 60 (61.9%) 22 (39.3%)
Venous invasion
 Absent 128 (83.7%) 85 (80.2%) 43 (91.5%) 0.081 78 (80.4%) 50 (89.3%) 0.153
 Present 25 (16.3%) 21 (19.8%) 4 (8.5%) 19 (19.6%) 6 (10.7%)
Lymphatic invasion
 Absent 71 (46.4%) 49 (46.2%) 22 (46.8%) 0.947 44 (45.4%) 27 (48.2%) 0.733
 Present 82 (53.6%) 57 (53.8%) 25 (53.2%) 53 (54.6%) 29 (51.8%)
Perineural invasion
 Absent 102 (66.7%) 64 (60.4%) 38 (80.9%) 0.013 59 (60.8%) 43 (76.8%) 0.044
 Present 51 (33.3%) 42 (39.6%) 9 (19.1%) 38 (39.2%) 13 (23.2%)
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CRC colorectal cancer, D descending; high grade, poorly and undifferentiated, IC immune cells at the center, IP immune cells at the periphery; low grade, well and moderately differentiated, M metastasis, MSS microsatellite stable, N lymph node, T tumor

P values are calculated using χ 2 test or Fisher’s exact test

Table 3.

Multivariate Cox proportional hazard models for predictors of overall survival

Factors Univariate survival analysis Multivariate survival analysis
HR (95% CI) P value HR (95% CI) P value
MSI-H cohort
 CD274IC expression 0.211 (0.069–0.649) 0.007 0.241 (0.071–0.824) 0.023
 Age 1.04 (1.01–1.09) 0.019 1.05 (1.00–1.10) 0.032
 Tumor border (infiltrative vs. expending) 11.5 (1.52–86.8) 0.018 3.93 (0.478–32.4) NS (0.203)
 Perineural invasion 5.07 (1.87–13.7) 0.001 3.734 (1.17–11.9) 0.026
 T stage (1–3 vs. 4) 6.84 (2.59–18.1) <0.001 1.84 (0.572–5.91) NS (0.307)
 N stage (0 vs. 1, 2) 6.23 (2.26–17.2) <0.001 2.97 (0.974–9.05) NS (0.056)
 M stage (0 vs. 1) 11.6 (3.22–42.9) <0.001 3.03 (0.642–14.3) NS (0.161)
CD274IP expression 0.232 (0.076–0.712) 0.011 0.274 (0.081–0.921) 0.036
 Age 1.05 (1.01–1.09) 0.019 1.04 (0.999–1.09) NS (0.056)
 Tumor border (infiltrative vs. expending) 11.5 (1.52–86.8) 0.018 3.87 (0.470–31.8) NS (0.209)
 Perineural invasion 5.07 (1.87–13.7) 0.001 3.40 (1.11–10.4) 0.032
 T stage (1–3 vs. 4) 6.84 (2.59–18.1) <0.001 1.79 (0.558–5.72) NS (0.328)
 N stage (0 vs. 1, 2) 6.23 (2.26–17.2) <0.001 3.41 (1.04–11.2) 0.043
 M stage (0 vs. 1) 11.6 (3.22–42.9) <0.001 3.40 (0.731–15.8) NS (0.119)
MSS cohort
 CD274IC expression 0.355 (0.150–0.844) 0.019 0.408 (0.169–0.985) 0.046
 Lymphatic invasion 3.95 (1.89–8.25) <0.001 1.96 (0.852–4.51) NS (0.113)
 Venous invasion 4.54 (2.44–8.48) <0.001 1.33 (0.583–3.05) NS (0.495)
 Differentiation (low vs. high grade) 6.31 (2.76–14.4) <0.001 3.67 (1.48–9.13) 0.005
 T stage (1–3 vs. 4) 4.24 (2.30–7.82) <0.001 1.98 (0.943–4.14) NS (0.071)
 N stage (0 vs. 1, 2) 4.20 (2.01–8.79) <0.001 2.09 (0.919–4.74) NS (0.079)
 M stage (0 vs. 1) 6.87 (3.58–13.2) <0.001 3.06 (1.39–6.71) 0.005
CD274IP expression 0.211 (0.083–0.537) 0.001 0.290 (0.109–0.771) 0.013
 Lymphatic invasion 3.95 (1.89–8.25) <0.001 2.12 (0.921–4.69) NS (0.077)
 Venous invasion 4.54 (2.44–8.48) <0.001 1.59 (0.697–3.62) NS (0.271)
 Differentiation (low vs. high grade) 6.31 (2.76–14.4) <0.001 3.82 (1.53–9.54) 0.004
 T stage (1–3 vs. 4) 4.24 (2.30–7.82) <0.001 1.92 (0.945–3.92) NS (0.071)
 N stage (0 vs. 1, 2) 4.20 (2.01–8.79) <0.001 1.96 (0.861–4.48) NS (0.109)
 M stage (0 vs. 1) 6.87 (3.58–13.2) <0.001 2.08 (0.919–4.69) NS (0.079)
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CRC colorectal cancer; high grade, poorly and undifferentiated, HR hazard ratio, IC immune cells at the center, IP immune cells at the periphery; low grade, well and moderately differentiated, M metastasis, MSI-H microsatellite instability-high, MSS microsatellite stable, N lymph node, T tumor, TC tumor cells at the center, TP tumor cells at the periphery

Results

Frequency and clinicopathological features of CD274 expression in MSI-H and MSS CRC patients

We investigated CD274 expression in CRC in four different tissue lesions and cell types: tumor cells at the center (TC) and at the periphery (TP), and immune cells at the center (IC) and at the periphery (IP). CD274-expressing immune cells consisted primarily of macrophages, plasma cells, and lymphocytes. In total, CD274TC was observed in 43 (23.1%) of the 186 MSI-H samples, while CD274TP was observed in 47 (25.3%) samples, CD274IC in 107 (57.5%) samples, and CD274IP in 102 (54.8%) samples. Table 1 summarizes the correlations detected between clinicopathological features and CD274 expression in MSI-H CRCs. CD274 expression in tumor cells at both the center and periphery tended to be associated with old age, high grade of histologic differentiation, non-mucinous type, lymphatic invasion, and CD3+ and CD8+ T cell infiltration, in accordance with the previous studies (P < 0.003) [5, 6]. Meanwhile, CD274 expression on tumor-infiltrating immune cells at the center and periphery tended to be associated with CD3+ and CD8+ TIL infiltration (P < 0.003).

In contrast to MSI-H tissues, CD274 expression on tumor cells was rarely observed in the 153 MSS CRC tissues examined; CD274TC and CD274TP were observed in only three (2.0%) and four (2.6%) cases, respectively. In contrast, CD274 expression was frequently observed on immune cells, with CD274IC and CD274IP being observed in 47 (30.7%) and 56 (36.6%) cases, respectively. The correlations detected between clinicopathological features and CD274 expression on tumor-infiltrating immune cells of MSS CRCs are summarized in Table 2. Notably, CD274IP was associated with the absence of metastasis of MSS CRC (P < 0.004).

Correlation between peripheral CD274 expression and TIL density in MSI-H CRC patients

We investigated the correlation between CD274 expression and TIL density at the tumor periphery in the 186 MSI-H CRC patients. The density (%/mm2) of CD3+ TILs was higher [median, interquartile range (IQR): 422, 196–685] than that of CD8+ TILs [median, IQR: 125, 65.6–242]. CD274TP expression exhibited a moderate positive correlated with CD3+ (ρ, 0.538; P < 0.001) and CD8+ (ρ, 0.546; P < 0.001) TILs, according to Dancey and Reidy’s categorization method (2004) [22]. Meanwhile, CD274IP expression was moderately correlated with CD3+ (ρ, 0.438; P < 0.001), but weakly correlated with CD8+ TILs (0.365; P < 0.001).

Intratumoral heterogeneity of CD274 expression in MSI-H and MSS CRC patients

All 339 CRC cases were screened for CD274 expression at the tumor center and periphery to evaluate intratumoral heterogeneity (Supplementary Table 1). Intratumoral heterogeneity of CD274 expression was not uncommon in CRC. Among the 186 MSI-H CRC cases, discordance between CD274TC and CD274TP was observed in 24 (12.9%) cases, and discordance between CD274IC and CD274IP was observed in 47 (25.3%) cases. In the MSS CRC cohort, evaluation of intratumoral heterogeneity of CD274 expression of tumor cells was meaningless due to the low incidence of positivity. However, discordance between CD274IC and CD274IP was found in 37 (24.1%) out of 153 MSS CRC cases. The difference in intratumoral heterogeneity of CD274 expression between MSI-H and MSS CRC was not significant.

Prognostic impact of CD274 expression in MSI-H and MSS CRC patients

In MSI-H CRC, CD274TC and CD274TP were not associated with survival (Supplementary fig. 1; P > 0.05). Conversely, CD274IC and CD274IP were significantly associated with improved survival (Fig. 2; P = 0.003 and P = 0.005, respectively). However, the density of CD3+ and CD8+ TILs did not correlate with patient survival (Supplementary fig. 1; P > 0.05). As mentioned above, survival analysis of CD274 expression of tumor cells in the MSS cohort was meaningless due to a low incidence of positivity. However, CD274IC and CD274IP were significantly associated with improved prognosis in the MSS cohort (Fig. 2; P = 0.014 and P < 0.001, respectively).

Fig. 2.

Fig. 2

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Kaplan–Meier survival curves illustrating the prognostic effect of CD274 expression on tumor-infiltrating immune cells in colorectal cancer (CRC). CD274 expression on tumor-infiltrating immune cells at a the tumor center and b the tumor periphery in microsatellite instability-high CRC tissues. CD274 expression on tumor-infiltrating immune cells at c the tumor center and d the tumor periphery of microsatellite stable CRC tissues

Notably, multivariate Cox proportional hazards analysis indicated that CD274 expression on tumor-infiltrating immune cells independently predicted improved prognosis in both MSI-H and MSS CRC cohorts (Table 3).

Detection of the BRAF-V600E mutation in MSI-H CRC patients

We investigated BRAF IHC status in 186 MSI-H CRC cases. To validate BRAF-V600E IHC, 26 IHC-positive and 14 IHC-equivocal cases were subjected to BRAF (V600E) mutation analysis using the Cobas 4800 System. Each of the 26 BRAF IHC-positive cases, but none of the IHC-equivocal cases, contained the V600E mutation. Notably, BRAF mutation was associated with CD274TP, distinguishing expression pattern of MMR proteins [hMLH1(−)/hMSH2(+)/hMSH6(+)/PMS2(−)], sporadic type, and high grade of histologic differentiation (P < 0.006), in agreement with previous studies (Table 4) [23, 24]. While BRAF mutation did not predict overall survival in MSI-H CRC patients (P = 0.987; Supplementary Figure 1), this mutation was significantly associated with worse prognosis in a subgroup of patients who had received post-operative chemotherapy (P = 0.041; Supplementary Figure 1). As such, BRAF mutation seems to be associated with worse prognoses only at the later stages of MSI-H CRC, which is an indication for post-operative chemotherapy.

Table 4.

The correlation between clinicopathological factors and BRAF mutation in 186 MSI-H CRC patients

Total no. of cases BRAF mutation
Negative Positive P value
CD274TC
 Negative 143 128 (80.0%) 15 (57.7%) 0.012
 Positive 43 32 (20.0%) 11 (42.3%)
CD274TP
 Negative 139 126 (78.8%) 13 (50.0%) 0.002
 Positive 47 34 (21.3%) 13 (50.0%)
CD274IC
 Negative 79 69 (43.1%) 10 (38.5%) 0.655
 Positive 107 91 (56.9%) 16 (61.5%)
CD274IP
 Negative 84 75 (46.9%) 9 (34.6%) 0.244
 Positive 102 85 (53.1%) 17 (65.4%)
CD8-positive TIL
 Low 93 84 (52.5%) 9 (34.6%) 0.091
 High 93 76 (47.5%) 17 (65.4%)
CD3-positive TIL
 Low 93 84 (52.5%) 9 (34.6%) 0.091
 High 93 76 (47.5%) 17 (65.4%)
MMR protein (hMLH1/hMSH2/hMSH6/PMS2)
 (+/+/+/+) 9 9 (5.6%) 0 (0.0%) 0.001
 (−/+/+/−) 113 87 (54.4%) 26 (100.0%)
 (+/−/−/+) 41 41 (25.6%) 0 (0.0%)
 (+/+/−/+) 8 8 (5.0%) 0 (0.0%)
 (+/+/+/−) 15 15 (9.4%) 0 (0.0%)
Hereditary vs. sporadic
 Sporadic 132 106 (66.3%) 26 (100.0%) <0.001
 Suspicious LS 54 54 (33.8%) 0 (0.0%)
Differentiation
 Low grade 143 129 (80.6%) 14 (53.8%) 0.003
 High grade 43 31 (19.4%) 12 (46.2%)
Open in a new tab

CRC colorectal cancer; high grade, poorly and undifferentiated, IC immune cells at the center, IP immune cells at the periphery; low grade, well and moderately differentiated, LS Lynch syndrome, MMR mismatch repair, MSI-H microsatellite instability-high, No number, TC tumor cells at the center, TP tumor cells at the periphery

P values are calculated using χ 2 test or Fisher’s exact test

Discussion

The high density of TILs that is commonly observed in MSI-H CRCs [14] suggests that these cancers vigorously induce a host immune reaction, which could be due to the higher mutational burden of these tumors; next-generation sequencing (NGS) studies have reported that MSI-H CRCs contain 10–50 times more mutations than MSS CRCs [25]. These molecular alterations produce abnormal neoantigens, which have the potential to result in increased numbers of TILs. Giannakis et al. showed that increased neoantigen load was positively correlated with TILs density and improved prognosis in CRC [26]. Actually, these data indicate that neoantigen load is more significant than MSI in clinical implication. Moreover, a recent study demonstrated that MSI-H CRCs show stronger expression of immune-regulation gene clusters than MSS CRCs [27, 28]. These clusters predominantly consist of gene-related T-helper 1 (Th1) and immune checkpoint receptors including PDCD1, CD274, CTLA-4, and LAG-3 [28, 29]. Notably, elevated expression of immune checkpoint molecules can create an immune-suppressive microenvironment [30], which could yield improved responses to immune checkpoint blockade.

The relationship between CD274 expression on tumor cells and prognosis in CRC is highly variable and controversial. While Droeser et al. demonstrated that CD274 expression was associated with better prognosis in MMR-proficient CRC [9], another study reported that the occurrence of CD274 expression on tumor cells with PDCD1 expression on TILs resulted in a worse CRC prognosis [11]. In the current study, CD274 expression on tumor cells was associated with a tendency toward a favorable prognostic value; however, this result had limited significance (Supplementary fig. 1) [6].

Interestingly, CD274 positivity was detected in >50% of tumor-infiltrating immune cells in MSI-H CRCs and >30% of those in MSS CRCs. CD274 expression on tumor and immune cells is thought to have distinct implications. However, a few other studies have also examined the clinicopathological implications of CD274 expression on immune cells. CD274 expression on immune cells shows a tendency towards decreased survival in gastric and uterine cervix adenocarcinomas [31, 32]. On the contrary, CD274 expression on immune cells was associated with improved survival in advanced urothelial carcinoma and spinal chordoma [33, 34]. Notably, our data indicated that CD274 expression on tumor-infiltrating immune cells was an independent factor for improved prognosis in both MSI-H and MSS CRC. Interestingly, in contrast to our data, Wang et al. showed that CD274 positivity on immune cells signifies worse prognoses in consecutive CRC [10]. However, a recent study supported our results that CD274 expression on immune cells results in improved survival of patients with stage IIIb CRC [35]. Similarly, another study also demonstrated that CD274 expression on immune cells showed a tendency toward improved survival in MSI-H CRCs, but this result was not statistically significant [6]. Moreover, this latter study suggested that favorable prognoses resulting from CD274 expression on immune cells was considered an effect of a high density of tumor-infiltrating T cells [6]. To rule out the causes of the favorable prognosis suggested by other groups, we precisely counted and analyzed tumor-infiltrating CD3+ and CD8+ T cells using an Aperio image analysis system, instead of the traditional method of counting by eye. Despite these elaborative analyses, the presence of CD3+ and CD8+ TILs was not significantly associated with prognosis for any of the cutoff values tested. These results support the conclusion that the prognostic value of the CD274 expression on immune cells was not a reflection of high TIL density.

CD274 has been shown to be expressed on macrophages, dendritic cells, T and B lymphocytes, and to protect tissues from excessive immune reaction [36]. While the mechanism by which expression of CD274 on TILs leads to improved patient prognoses remains unclear, there are several possible hypotheses. First, it is conceivable that active anti-tumor immune reactions could enable the positive selection of tumor cells exhibiting mutations in genes encoding human leukocyte antigen (HLA) and/or antigen-processing machinery (APM) [26]. In this case, expression of CD274 on TILs could relieve the anti-tumor immune reaction, leading to reduced numbers of such mutant tumor cells and thereby to better patient outcomes. Alternatively, Di Caro et al. suggested that chemotherapeutic effects on tumor cells seem to be enhanced under immune-escape conditions [37]. CD274 expression on TILs could, therefore, have enhanced the chemotherapeutic responsiveness of tumor cells by promoting an immune-escape condition in CRC. Another contributing factor for favorable prognosis is interferon (IFN)-r-producing tumor-infiltrating T cells. Droeser et al. demonstrated that CD274 mRNA expression was significantly correlated with IFN-γ gene expression in CRC specimens [9], and that IFN-γ might play a role in the tumor surveillance and cytotoxic anti-tumor function. However, further studies are necessary to clarify this possibility.

Recently, several studies detected regional heterogeneity of CD274 expression in various cancers, including lung cancer, melanoma, and renal cell carcinoma [15, 38, 39]. These data suggest that we should be careful in evaluating CD274 expression in routine small biopsies due to the potential for false-negative results. To the best of our knowledge, our study is the first to evaluate intratumoral heterogeneity (central and peripheral portions of the primary tumor) in CD274 expression in CRCs; our data indicate that such heterogeneity is common in resected CRC specimens (Supplementary Table 1), which are more reliable than biopsy specimens. A possible reason for this regional discordance is that various immune reactions might further affect the peripheral portion of tumors than the central portion. Additionally, hypoxic conditions in the central portion might induce intratumoral heterogeneity of CD274 expression. Several studies suggested that hypoxia can promote the expression of CD274 on tumor cells and TILs via hypoxia-inducible factor-1α (HIF-1α) up-regulation [40, 41].

Recent molecular studies indicated that CRC is a heterogeneous disease, arising from several genetic pathways. These pathways are crucial in determining patient prognosis and treatment [42, 43]. In non-small cell lung cancer, recent studies indicated that the genomic landscape determined patient response to PDCD1 blockade therapy [44], and that CD274 expression was associated with EGFR mutations [45]. Our study and others demonstrated that mutation of BRAF is significantly associated with CD274 expression in CRC tumor cells, but not in immune cells [5, 6]. Another recent study suggested that CD274 positivity is associated with the serrated pathway and stem cell features in CRC [24]. Thus, the diverse genetic alterations that correlate with CD274 expression in CRC should be further investigated.

To conclude, our study comprehensively evaluated CD274 expression in MSI-H CRCs, as well as in MSS CRCs. Notably, we found that CD274 expression on tumor-infiltrating immune cells was an independent predictive factor for improved prognosis in both MSI-H and MSS CRCs. Our findings indicate that the CD274 status may be helpful in predicting CRC patient outcomes. Moreover, our results indicate that discordance of CD274 expression between the central and peripheral portions of CRC tumors is not uncommon. Thus, evaluation of various tumor portions is recommended to enhance the validity of CD274 expression results. Further investigation of the mechanism underlying CD274 expression in immune cells as well as the predictive and prognostic role of this protein in CRC is needed.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 180 kb) (181KB, pdf)

Abbreviations

AJCC

American Joint Committee on Cancer

C

Center

CI

Confidence interval

CRC

Colorectal cancer

FFPE

Formalin-fixed and paraffin-embedded

HR

Hazard ratio

IC

Immune cells at the center

IHC

Immunohistochemistry

IP

Immune cells at the periphery

LS

Lynch syndrome

MSI-H

Microsatellite instability-high

MSS

Microsatellite stable

P

Periphery

PCR

Polymerase chain reaction

PD-1

Programmed cell death 1

PD-L1

Programmed cell death ligand 1

TC

Tumor cells at the center

TP

Tumor cells at the periphery

Compliance with ethical standards

Funding

This research was supported by a Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Republic of Korea (NRF-2016R1D1A1B03931744).

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

For this type of study formal consent is not required.

Conflict of interest

The authors declare that they have no conflict of interest.

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