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Programmed Death Ligand-1 expression in stage II colon cancer - experiences from a nationwide populationbased cohort

Programmed Death Ligand-1 expression in stage II colon cancer - experiences from a nationwide... Background: Patients suffering from high risk stage II colon cancer (CC) may benefit from adjuvant onco-therapy, but additional prognostic markers are needed for better treatment stratification. We investigated the prognostic value of Programmed Death Ligand-1 (PD-L1) in a true population-based cohort of patients with stage II CC. Methods: PD-L1 expression on tumour cells was evaluated by immunohistochemistry in 572 colon cancers. Whole sections from tumour blocks representing the deepest invasive front of the primary tumour were used for analysis. A cut-off of 5% positivity was used for dichotomizing the data. The prognostic value was investigated in Cox proportional hazard models for recurrence-free survival (RFS) and overall survival (OS). Results: Overall, 6% of the tumours were classified as high PD-L1. High PD-L1 was related to female gender (p =0.028) , high malignancy grade (< 0.001), right side localization (p < 0.001) and microsatellite instability (MSI) (p < 0.001). Thirty-one (18%) of the MSI and 4 (1%) of the microsatellite stable tumours were classified as high PD-L1, respectively. PD-L1 expression provided no prognostic value as a single marker. In patients with MSI tumours, high PD-L1 expression had no significant impact regarding OS or RFS. Conclusions: PD-L1 expression in tumour cells of stage II CC did not provide any prognostic impact, neither in the entire population-based cohort nor in the group of MSI patients. Additional investigations of the immunogenic microenvironment are needed for evaluating the prognostic information in CC. Keywords: Colon cancer stage II, Prognostic markers, Programmed death ligand-1 Background However, the currenct high risk factors are questionable Colon cancer is one of the most common cancers in [4], and there is a need for additional prognostic markers the Western world. About 1/3 have stage II disease, for better treatment stratification. and this group of patients are in general having a In recent years, the tumour microenvironment has been good prognosis with a 5-year overall survival (OS) of investigated, and the role of the interaction between cancer approximately 70–80% after surgery alone [1]. Current cells and the immune system in cancer surveillance has been international guidelines (ASMO and ESMO) do not emphasized [5]. Tumour-infiltrating lymphocytes (TILs) are recommend routine adjuvant chemotherapy in stage considered as the host’s immune response against solid II CC, but rather that this treatment be limited to tumours, and infiltration by activated CD8+ cytotoxic patients having a high risk of recurrence, based on an T-lymphocytes is correlated with better survival of colorectal individual evaluation including high-risk markers [2, 3]. cancer (CRC) patients [6]. Activated lymphocytes expressing Programmed Death Receptor-1 (PD-1) can bind specific to the ligand Programmed Death Ligand-1 (PD-L1), which is * Correspondence: ann.christina.eriksen@rsyd.dk expressed on the cell membrane in malignant epithelial Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark tumours, including colorectal adenocarcinomas [7]. This Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark immune-checkpoint is up-regulated in many tumours, and Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Eriksen et al. BMC Cancer (2019) 19:142 Page 2 of 9 the interaction of PD-L1 on tumour cells with its receptor The tissue blocks were stored and transported at room PD-1 on the activated T-cells induces a down-regulation of temperature. One tumour block representing the deepest the antigen-stimulated lymphocyte proliferation and cytokine invasive margin, was selected from each patient. Prior to production, resulting in an inhibition of the host-immune re- inclusion, all histological slides from each tumour were sponse [8]. evaluated by first a trainee and afterwards an experienced Current results of the prognostic value of PD-L1 in pathologist. CRC are controversial. Some studies only report on trends towards worse prognosis for high PD-L1 expres- Immunohistochemistry sion [9, 10], while others identify high PD-L1 to be inde- From the selected tumour blocks serial 4 μm thick tissue pendently associated with worse recurrence free survival sections were cut and mounted on FLEX IHC Micro- (RFS) [11, 12]. In contrast, other studies report no prog- scope Slides (K8020, DAKO, Glostrup, Denmark). One nostic impact [13, 14], or even a tendency of high ex- whole tumour section per patient was used for the pression of PD-L1 to be related to a better prognosis evaluation of PD-L1 expression. Staining was performed [15]. However, studies vary greatly in methods and the using a Ventana BenchMark ULTRA (Ventana Medical study populations are highly heterogeneous, including Systems, Tucson, Arizona, USA) automated immunohis- different stages of disease, and no studies have previ- tochemistry (IHC) slide staining system. Tissue sections ously investigated the expression of PD-L1 in a cohort were heated and deparaffinised in EZprep (no.950–102, exclusively of patients with stage II CC. Ventana). Pre-treatment and demasking were carried With this motivation, the aim of the present study was out using ULTRA CC1 (no. 950–224) and ULTRA CC2 to evaluate the prognostic impact of PD-L1 in a nation- (no. 950–223), and endogenous peroxidase activity was wide, population-based cohort of stage II CC. blocked by Optiview Peroxidase Inhibitor (no. 760–700 Ventana). The slides were incubated with a rabbit monoclo- Methods nal anti-PD-L1 (clone SP263A, no. 790–4905/741–4905 This study is reported in accordance with the REMARK Ventana) for 16 min. This clone was chosen based on guidelines [16]. a pilot study. For amplification Optiview HQ Universal Linker (no. 760–700, Ventana) and Optiview HRP Multimer Patient population (no. 760–700, Ventana) was each used for 8 min. The population and sources of data has previously been The primary antibody was visualized using Optiview descreibed in detail [17]. In brief all patients surgically H O and DAB (no. 760–700, Ventana), followed by 2 2 treated for stage II CC in 2002 in Denmark were identified Optiview Copper (no. 760–700, Ventana). Counterstain by a search in the nationwide registry administrated by was done using Hematoxylin II (no. 790–2208, Ventana) the Danish Colorectal Cancer Group (DCCG) (N =746). and bluing Reagent (no. 760–2037, Ventana). Finally the Exclusion criteria were as follows: missing tumour block histological slides were cover slipped with Tissue-Tek (N = 11), insufficient tissue for analyses (N = 2), incor- PERTEX (Histolab Products AB, Göteborg, Sweden). rectly staged patients (N = 25), treatment with adjuvant Evaluation of microsattellite instability (MSI) was per- chemotherapy (N = 26)/radiotherapy (N = 1) and death formed using IHC. Tumours displaying loss of one or within 90 days after the operation (N = 75). Furthermore more of the 4 mismatch repair proteins (MLH1, MSH2, patients with loco-advanced disease (N = 8) and patients MSH6 and PMS2) were considered as MSI, whereas tu- diagnosed with another malignancy prior to CC were ex- mours with intact mismatch repair proteins were consid- cluded from the study (N = 26), and the final study popu- ered as microsatellite stable (MSS). Staining of mismatch lation comprised 572 patients. repair proteins was performed using a DAKO Autostai- Histopathological data were obtained by microscopic ner Link 48 (DAKO) with monoclonal mouse antibody examination and from the national Patobank containing against MLH1 (Novocastra, Leica, Germany, clone ES05, all pathology reports in Denmark. The term “not assessed” dilution 1:100, product code NCL-L-MLH1), MSH2 was used if the pathological feature was not described. (Novocastra, Leica, clone 25D12, dilution 1:100, product Clinical data were obtained from The National Patient code NCL-L-MSH2), MSH6 (BD Transduction Laboratories, Registry. clone 44/MSH6, dilution 1:200, material number 610919), Recurrence primarily occurred within the first five and PMS2 (BD Pharmingen, clone A16–4, dilution 1:500, years of follow-up and to encompass the majority of re- material number 556415). Tissue sections were incubated currences a follow-up period of seven years was selected. for 30 min at room temperature with the primary antibodies diluted in Envision Flex antibody diluent (code S2022 Samples DAKO). The antibody signal was amplified using Envision Formalin-fixed, paraffin-embedded tissue blocks were col- Flex+ Mouse(Linker) (DAKO) for 20 min. Bound antibodies lected from the departments of pathology in Denmark. were detected using Envision FLEX/HRP (DAKO) and Eriksen et al. BMC Cancer (2019) 19:142 Page 3 of 9 visualized by Envision FLEX DAB (DAKO) and chromogene used to test for differences between groups. The multi- diluted in Envision Flex Substrate Buffer (DAKO). The sec- variable Cox-regression model was used to test for inde- tions were incubated in 0.5% CuSO in TBS buffer pH 7.6 pendent prognostic value with hazard ratio (HR) of 1.0 for 10 min to enhance the immunohistochemical staining. as reference and a 95% confidence interval (CI). A Sections were counterstained with Meyer’s hematoxylin cut-off significance level of 0.10 was pre-specified for a (Merck, Damstadt, Germany). variable to be included in the multivariable Cox regres- sion model. Scoring of PD-L1 expression Chi -statistics were used to test associations between Tumour PD-L1 expression was evaluated based on clinicopathological variables. A p-value < 0.05 was con- immunostaining of the cell membrane of the epithelial sidered significant. The statistical analyses were per- tumour cells. The immunostaining of the stromal cells formed using STATA software version 14.0 (StataCorp, were not evaluated. Tumour cells were considered Texas, USA), and all statistical tests were two-sided. positive when any cell membrane staining (partial of complete) was present. Staining intensity was not evalu- Results ated and cytoplasmatic immunoreaction was not Patient characteristics considered. Patient characteristics are summarized in Table 1. In the The percentage of positive tumour cells were scored follow-up period of seven years, 266 (46.5%) patients semi-quantitatively as 0 (no positive tumour cells), 1 died; 110 (19.2%) patients experienced disease recur- (≤1% positive), 2 (> 1 and ≤ 5% positive), 3 (> 5 and ≤ rence and 78 (13.6%) patients were diagnosed with an- 20% positive), 4 (> 20 and ≤ 50% positive) and 5 (> 50% other cancer. The median age at time of surgery was 73 positive) (Fig. 1). A subset of 50 randomly selected tu- years (range 29–95), and the mean follow-up time was mours was examined by a second pathologist in order to 6.9 years (range 3–84 months). assess inter-observer variation. For prognostic evaluation data were dichotomized, using 5% PD-L1 expression as cut-off. In absence of a standardized scoring system the PD-L1 expression cut-off was based on previously studies [9, 12, 14]. The IHC staining of PD-L1 often had a highly heteroge- neous expression both between the central part of the Statistics tumour and the invasive margin and along the invasive The inter-observer reproducibility of PD-L1 scoring was tumour front (Fig. 2). evaluated by kappa statistics. Simple and weighted kappa Results regarding PD-L1 expression are displayed in (κ) values were calculated, and agreement was described Table 2. Nearly half of the population (46%) had no PD-L1 according to Landis et al [18] as moderate, substantial, expression in tumour cells. After dichotomization, using 5% and almost perfect for κ values of 0.41–0.60, 0.61–0.80, PD-L1 expression as cut-off, 35 (6%) of the tumours were and 0.81–1, respectively. classified as high PD-L1. In the group of MSI 31 (18%) of The endpoint OS was defined as time from operation the tumours were classified as high PD-L1 and in the to death of any cause or last follow-up. RFS was defined subgroup of MSS 4 (1%) were classified as high PD-L1. High as time from operation to death of any cause or recur- PD-L1 was related to female gender (p = 0.028), high malig- rence of CC. Patients later diagnosed with another can- nancy grade (< 0.001), right side localization (p < 0.001) and cer were censored at the date of diagnosis (N = 102). MSI (p <0.001). The median age was used as cut-off to dichotomize the The inter-observer agreement for the semi-quantitative parameter age. Survival curves were generated according evaluation of PD-L1 expression was moderate with κ=0.418 to the Kaplan-Meier method and the log-rank test was and weighted κ = 0.573. The agreement improved to Fig. 1 Example of immunohistochemically staining of programmed death ligand-1. a ≤ 1% positive tumour cells, b 5% < positive tumour cells ≤20%, and c > 50% of the tumour cells are positive Eriksen et al. BMC Cancer (2019) 19:142 Page 4 of 9 Table 1 Clinico-pathological characteristics and association to Table 1 Clinico-pathological characteristics and association to PD-L1 (cut-off 5%) PD-L1 (cut-off 5%) (Continued) Number PD-L1 Number PD-L1 (N = 572) (%) Low (%) High (%) p-value (N = 572) (%) Low (%) High (%) p-value Age (years at diagnosis) Mismatch repair status Median 72 MSS 400 (70) 396 (74) 4 (11) < 0.001 Range 29–95 MSI 172 (30) 141 (26) 31 (89) Abbreviations: MSI microsatellite instability, MSS microsatellite stable, NOS not ≥ 73 267 (47) 253 (47) 14 (40) 0.414 otherwise specified < 73 305 (53) 284 (53) 21 (60) Including mucinous adenocarcinomas and signet-ring cell carcinomas P-values are obtained using chi -test. Statistically significant p-values are Gender highlighted in bold Male 283 (49) 272 (51) 11 (31) 0.028 Female 289 (51) 265 (49) 24 (69) substantial, when categorizing the data into high PD-L1 ex- T-stage pression (> 5%) or low PD-L1 expression (≤5%), κ=0.691. pT3 500 (87) 472 (88) 28 (80) 0.172 pT4 72 (13) 65 (12) 7 (20) Survival analysis Histology (WHO) The 5-year RFS for the population with low PD-L1 was Adenocarcinoma NOS 515 (90) 481 (90) 34 (97) 0.348 69.2% versus 67.7% in the group with high PD-L1 ex- Mucinous 55 (10) 54 (10) 1 (3) pression, and OS was 74.7% versus 70.5%, respectively. adenocarcinoma When considering patients with MSI tumours the 5 year Signet-ring cell 2 (0) 2 (1) 0 (0) RFS for low PD-L1 was 77.4% versus 67.5% in the group carcinoma of high PD-L1, and OS was 79.4% versus 70.7%. No Malignancy grade significant differences in survival rates were observed, Medium + low 450 (79) 436 (81) 14 (40) < 0.001 considering the entire cohort (Fig. 3). In the group of High 122 (21) 101 (19) 21 (60) patients with MSI tumours the Kaplan Meier curves were separated for RFS, but results were insignificant, p =0.256 Localisation (Fig. 4). This also accounted the group of patients with Right 273 (48) 242 (45) 31 (89) < 0.001 MSI T3 tumours (N =155), p =0.149. Left 299 (52) 295 (55) 4 (11) Outcomes from the corresponding univariable Cox Tumour perforation regression analyses are shown in Table 3. Patients with Yes 530 (93) 499 (97) 31 (94) 0.313 MSI tumours and high PD-L1 expression did not have a No 17 (3) 15 (3) 2 (6) significant worse OS or RFS, HR = 1.104 (0.604–2.016), p = 0.748 and HR = 1.429 (0.769–2.653), p = 0.258, Unknown 25 (4) respectively. Age ≥ 73 years, T4 tumour and perfor- Lymph nodes ation were significantly related to an adverse outcome Median 10 of both OS and RFS. Patients with MSI T3 tumours and Range 0–41 high PD-L1 expression did neither have a significant < 12 nodes 351 (61) 329 (61) 22 (63) 0.851 worse OS or RFS, HR = 1.531 (0.757–3.100), p = 0.236 and ≥ 12 nodes 221 (39) 208 (39) 13 (37) HR = 1.637 (0.833–3.216), p = 0.153, respectively. Multivariable Cox regression analyses were not per- Perineural invasion formed, as PD-L1 had p-value > 0.10 in the univariable Yes 26 (5) 25 (7) 1 (4) 0.635 Cox-regression analysis. No 359 (63) 337 (93) 22 (96) Not assessed 187 (32) Discussion Vascular invasion In this study, we investigated the prognostic value of Yes 43 (7) 38 (9) 5 (22) 0.054 PD-L1 expression on tumour cells in an unbiased, na- No 386 (68) 368 (91) 18 (78) tionwide and population-based cohort of patients with Not assessed 143 (25) stage II CC, treated exclusively with surgery. PD-L1 ex- pression as a single marker did not provide any signifi- cant prognostic value regarding OS or RFS, neither in the the entire cohort nor in the subgroup of patients with MSI tumours. Eriksen et al. BMC Cancer (2019) 19:142 Page 5 of 9 Fig. 2 Example of heterogeneity of the expression of programmed death ligand-1. a Overview of the heterogenetic area. Frame one is presented in higher magnification in (b) and frame two in (c). The membranous expression pattern is displayed in (d) In the entire cohort we found 6% of the tumours to and MSS tumours has previously been descriebed in have a high expression of PD-L1 on tumour cells, which studies using a different scoring system [11, 19], al- is in accordance with other studies of CRC, reporting 5% though a recent study reported no differences in PD-L1 positivity [11]. Likewise we found high PD-L1 expression positivity among MSI and MSS tumours [20]. associated to female gender, high malignancy grade, right The association between MSI and high PD-L1 expres- sided localisation, and MSI, which also has been found sion may be explained by the abundant infiltration of by Lee et al, who investigated all stages of CRC [11]. TILs in these tumours. Deficiency of the mismatch re- Regarding MSI and MSS tumour subgroups, we found pair proteins results in a number of mutations. There- 18% of the MSI tumours to have high PD-L1 expression fore MSI tumours have a high load of tumour specific and 1% of the MSS tumours to have high PD-L1 expres- neo-antigens, which can induce an immunological re- sion. The difference in PD-L1 expression between MSI sponse with recruitment and activation of T-cells [21]. One way to stimulate PD-L1 upregulation is afforded by the pro-inflammatory cytokine interferon-gamma Table 2 Programmed death ligand-1 (PD-L1) expression in (IFN-γ), which is produced by activated T-cells and colon cancer Natural Killer cells [22]. The high expression of PD-L1 in Number of PD-L1 Entire cohort MSI (N = 172) (%) MSS (N = 400) (%) MSI tumours with abundant infiltration of TILs is in ac- positive tumour (N = 572) (%) cordance with the consensus molecular subtype (CMS) cells classification. The molecular group CMS1 is characterized None 264 (46) 47 (27) 217 (54) by hypermutation, MSI and intense immune reaction [23], 0 < PD-L1 ≤ 1% 233 (41) 73 (42) 160 (40) and this immunogenic group has been documented with a 1% < PD-L1 ≤ 5% 40 (7) 21 (12) 19 (5) high PD-L1 expression [24]. < 5% PD-L1 ≤ 20% 10 (2) 8 (5) 2 (1) In the group of patients with MSI tumours, the < 20% PD-L1 ≤ 11 (2) 10 (6) 1 (0) Kaplan-Meier curves were clearly separated regarding RFS, 50% with a worse RFS related to a high PD-L1 expression, but PD-L1 > 50% 14 (2) 13 (8) 1 (0) statistical significance was not reached. This is in accord- Abbreviations: MSI Microsatellite instability, MSS sMicrosatellite stable ance with studies of tumour expression of PD-L1 in MSI Eriksen et al. BMC Cancer (2019) 19:142 Page 6 of 9 Fig. 3 Kaplan-Meier survival curves depicting overall survival (a) and recurrence-free survival (b) stratified by the expression of programmed death ligand-1 (PD-L1) in the entire cohort (N = 572). P-values were calculated by log-rank test stage I-IV CRC. Kim et al [9] reported a tendency towards PD-L1 and N status with high PD-L1 expression being a worse prognosis for tumours with high PD-L1 expression; related to higher N status. This may be part of the ex- however results were non-significant. Rosenbaum et al [10] planation for the inconsistency, as we only investigated reported no prognostic value for dichotomized data, but stage II CC. Unfortunately, Koganemaru et al did not re- the group with the highest expression of PD-L1 (≥50%) had port any data on MSI status. a markedly reduced disease-specific survival. We only Lee et al [11] documented PD-L1 expression as an found 14 patients to have PD-L1 expression ≥50%, and independent prognostic marker in patients with MSI using this cut-off in our cohort did not enhance the prog- tumours. In the present populationbased study 172 nostic impact (data not shown). Rosenbaum et al investi- patients had a MSI tumour and only 31 (18%) of these gated all stages of CRC and found PD-L1 expression tumours were classified as high PD-L1, resulting in a related to stage, which might explain the difference. small group and thus low statistical power in the Cox re- In contrast to our data, Koganemaru et al [12] re- gression analysis. This may be part of the explanation ported high PD-L1 expression being an independent for the non-significant results. prognostic marker. They used the same cut-off (5%) in Furthermore, previous studies reporting independent their evaluation of PD-L1, but they exclusively investi- prognostic impact of PD-L1, differ in investigated co- gated stage III CRC. They found an association between horts and evaluation methods. Lee et al [11]included AB Fig. 4 Kaplan-Meier survival curves depicting overall survival (a) and recurrence-free survival (b) stratified by the expression of programmed death ligand-1 (PD-L1) in the group of patients with MSI tumours (N = 172). P-values were calculated by log-rank test Eriksen et al. BMC Cancer (2019) 19:142 Page 7 of 9 Table 3 Univariable Cox regression analysis Parameter Overall survival Recurrence-free survival HR (95% CI) p-value HR (95% CI) p-value Age (years at diagnosis) <73 1 0.001 1 0.009 ≥73 2.533 (1.466–4.377) 2.194 (1.216–3.958) Gender Male 1 0.462 1 0.634 Female 0.839 (0.524–1.341) 0.881 (0.523–1.484) T-stage T3 1 0.003 1 0.002 T4 2.68 (1.409–5.128) 2.982 (1.503–5.915) Malignancy grade Medium/Low 1 0.536 1 0.636 High 1.159 (0.726–1.852) 1.134 (0.675–1.906) Localisation Right 1 0.792 1 0.150 Left 1.084 (0.594–1.981) 1.576 (0.848–2.927) Tumour perforation No 1 0.001 1 0.005 Yes 5.332 (1.924–14.776) 5.644 (1.708–18.645) Lymph nodes < 12 nodes 1 0.440 1 0.220 ≥12 nodes 1.203 (0.753–1.922) 1.386 (0.823–2.334) Perineural invasion No 1 0.630 1 0.873 Yes 1.417 (0.343–5.857) 0.850 (0.117–6.203) Vascular invasion No 1 0.878 1 0.756 Yes 0.922 (0.332–2.566) 0.830 (0.256–2.690) PD-L1 Low 1 0.748 1 0.258 High 1.104 (0.604–2.016) 1.429 (0.769–2.653) Univariable Cox regression analysis regarding overall survival and recurrence-free survival for the sub-cohort of patients with MSI tumours (N = 172). Statistically significant p-values are highlighted in bold Including mucinous adenocarcinomas and signet-ring cell carcinomas MSI microsatellite instability all stages of both colon and rectal cancers. Also their expression on tumour cells in 13% of the investigated evaluation method differed in a number of ways from tumours based on evaluation of TMAs from the central our technical approach. They used tissue microarrays and invasive tumour compartments. They included all (TMAs),asdomostother studies[9–15]. In constrast stages of CRC. In contrast we evaluated PD-L1 ex- we evaluated the PD-L1 expression in whole sections pression in a highly homogenous cohort of stage II and observed a lot of heterogeneity both between the CC patients and found a considerable intra-tumoral central part of the tumour and the invasive margin, heterogeneity. and along the invasive tumour front. The use of A standardized scoring system for PD-L1 expression TMAs may lead to selection bias, although several in CC is missing and several unvalidated methods are in studies try to avoid this by using several representa- use. In the present study, the proportion of PD-L1 posi- tive TMAs from each tumour [9, 11, 13]. Lee et al tive tumour cells was evaluated considering only mem- [14] descreibe intra-tumoral heterogeneity of PD-L1 branous staining as positive. Intensity of the staining and Eriksen et al. BMC Cancer (2019) 19:142 Page 8 of 9 cytoplasmic immunoreaction were not considered. expression in tumour cells should be seen in the context Whether to take cytoplasmic staining of PD-L1 into of the entire immune tumour microenvironment. The account differs among studies. Some investigations use expression of PD-L1 is influenced by TILs, which is a combination of membranous staining and staining related to MSI. We did not prove any prognostic value intensity [11] while others do not consider cytoplasmic of PD-L1 in patients with MSI tumours, however staining at all [10]. PD-L1 expressed on the cell surface this interaction should be taken into consideration in is essential for the interaction with the PD-1 receptor on future studies. the T-cells, indicating that only PD-L1 expressed on the membrane is of clinical importance. Furthermore, in the Conclusions evaluation of lung cancer, only membrane staining is ap- In this nationwide population-based cohort of stage II plied when evaluating the indication for immunotherapy colon cancer, we found membranous PD-L1 expression with a PD-L1 inhibitor [25]. (cut-off 5%) in tumour cells of stage II colon cancer to As mentioned above, we did not consider intensity be associated with female gender, high malignancy grade, of the staining. Intensity of IHC may be difficult to right side localisation and MSI. The expression of PD-L1 interpret. Poor reproducibility of IHC staining inten- was often highly heterogenous. No prognostic informa- sities of various proteins has been documented, while tion was detected of PD-L1 as a single biomarker in this excellent inter-observer reproducibility was found esti- cohort of stage II colon cancer. mating the fraction of positive tumour cells [26]. Abbreviations PD-L1 was not included in that study, but the same CC: Colon cancer; CRC: Colorectal cancer; DCCG: Danish Colorectal Cancer most likely applies to this protein. Furthermore, the Group; HR: Hazard ratio; IHC: Immunohistochemical; MSI: Microsatellite evaluation of staining intensity is not only influenced instability; MSS: Microsatellite stability; NOS: Not otherwise specified; OS: Overall survival; PD-1: Programmed death receptor 1; PD-L1: Programmed death ligand 1; by subjectivity. Various other factors may affect the RFS: Recurrence-free survival; TILs: Tumour infiltrating lymphocytes; TMA: Tissue staining intensity, encompassing both pre-analytical microarray and analytical factors such as fixation (time and type), storage, and IHC protocols. Also section thickness Acknowledgements We thank Birgit Roed Sørensen and Christina Braad Petersen for excellent affects staining intensity, and even modern state of technical assistance. We also thank all pathology departments in Denmark for the art microtomes produce sections with varying their support and participation in the study, which has made the population- thicknesses. based design possible. Also, we appreciate the help and advice supported by OPEN, Odense Patient data Explorative Network, Odense University Hospital, The lack of a standardized IHC method challenges Odense, Denmark. Finally, we are grateful to Roche Diagnostics for the discount the assessment of PD-L1 expression, and moreover, provided, regarding antibody expenses. different trials use different antibodies and assays. In melanoma, diverse assays have been found to variate Funding The study was conducted and affiliated under Danish Colorectal Cancer in staining sensitivity of tumour cells [27]. For future Group and Danish Colorectal Center South, Vejle Hospital, Part of Lillebaelt studies, standardized techniques for evaluating PD-L1 Hospital, Denmark. Financial support was granted by The Research Council in CC are required regarding antibodies, assays, inter- of Lillebaelt Hospital and the Danish Cancer Research Foundation. The commercial company Roche Diagnostics supported the study providing pretation, and threshold cut-off in scoring the immu- antibodies at a discount. The funding organisations did not play any role in nostain. Also, focus should be directed on how to the study design, data collection, analysis or interpretation of the data, handle the marked heterogeneity, as this might hinder decision to publish, or preparation of the manuscript. reproducibility of IHC scoring systems. Availability of data and materials The present study is limited by the retrospective de- The datasets used and/or analysed during the current study are available sign, as we had no influence on the pre-analytical phase from the corresponding author on reasonable request. of the IHC. However, we used a validated antibody on a fully automatic platform, and only considered membran- Authors’ contributions Conceived and designed the experiments: ACE, FBS, SKF, JL and TFH. ous staining according to the manufactures recommen- Performed the experiments: ACE. Analyzed the data: ACE, RD and TFH. Wrote dation. The PD-L1 antibody used in this study stained the paper: ACE, FBS, HH, JL and TFH. All authors read and approved the final both the malignant epithelial cells and immune cells in manuscript. the stroma, which made it difficult to discriminate these Ethics approval and consent to participate cellular populations when the density of immune cells in The Regional Committees on Health Research Ethics for Southern Denmark the tumour stroma interface was high. approved the study and exempted the study from the rule of informed The prognostic value of PD-L1 expression on tumour consent in their approvel (S-20140119). The study was also approved by the Danish Data Protection Agency (14/26345). All patients were screened in the cells is controversial. We investigated the expression of Danish Registry of Tissue Utilization before enrollment in the study. PD-L1 exclusively in stage II CC in a well-defined and unbiased population, and did not find any prognostic Consent for publication value of PD-L1 as a single biomarker. The PD-L1 Not applicable. Eriksen et al. BMC Cancer (2019) 19:142 Page 9 of 9 Competing interests 17. Eriksen AC, Sørensen FB, Lindebjerg J, Hager H, dePont Christensen R, The authors declare that they have no competing interests. Kjær-Frifeldt S, et al. The prognostic value of tumour infiltrating lymphocytes in stage II colon cancer. A nationwide population-based study. Transl Oncol. 2018;11(4):979–87. Publisher’sNote 18. Landis JR, Koch GG. The measurement of observer agreement for Springer Nature remains neutral with regard to jurisdictional claims in categorical data. Biometrics. 1977;33(1):159–74. published maps and institutional affiliations. 19. Korehisa S, Oki E, Iimori M, Nakaji Y, Shimokawa M, Saeki H, et al. Clinical significance of programmed cell death-ligand 1 expression and the Author details immune microenvironment at the invasive front of colorectal cancers with Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark. high microsatellite instability. Int J Cancer. 2018;142(4):822–32. Institute of Regional Health Research, University of Southern Denmark, 20. Zhang Y, Sun Z, Mao X, Wu H, Luo F, Wu X, et al. Impact of mismatch-repair Odense, Denmark. Department of Clinical Medicine, University Institute of deficiency on the colorectal cancer immune microenvironment. Oncotarget. Pathology, Aarhus University Hospital, and , University of Aarhus, Aarhus, 2017;8(49):85526–36. Denmark. Danish Colorectal Cancer Group (DCCG), Aarhus N, Denmark. 21. Boissiere-Michot F, Lazennec G, Frugier H, Jarlier M, Roca L, Duffour J, et al. Research Unit of General Practice, University of Southern Denmark, Odense, Characterization of an adaptive immune response in microsatellite-instable Denmark. Department of Pathology, Lillebaelt Hospital, Beriderbakken 4, colorectal cancer. Oncoimmunology. 2014;3:e29256. DK-7100 Vejle, Denmark. 22. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015;27(4):450–61. Received: 19 April 2018 Accepted: 1 February 2019 23. Guinney J, Dienstmann R, Wang X, de Reynies A, Schlicker A, Soneson C, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21(11):1350–6. References 24. Reilley M, Blando JM, Katkhuda R, Menter D, Sharma P, Allison JP, et al. 1. Morris M, Platell C, McCaul K, Millward M, van Hazel G, Bayliss E, et al. Immunologic profiling of consensus molecular subtype (CMS) stratified Survival rates for stage II colon cancer patients treated with or without colorectal cancer (CRC) primary and liver metastectomy specimens: chemotherapy in a population-based setting. Int J Color Dis. 2007;22(8): implications for immune targeting of proficient mismatch repair CRC. 887–95. J Clin Oncol. 2016;34(15):3520. 2. Labianca R, Nordlinger B, Beretta GD, Mosconi S, Mandala M, Cervantes A, 25. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. et al. Early colon cancer: ESMO clinical practice guidelines for diagnosis, Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. treatment and follow-up. Ann Oncol. 2013;24(6):64–72. 2015;372(21):2018–28. 3. Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, 26. Zlobec I, Terracciano L, Jass JR, Lugli A. Value of staining intensity in the et al. American Society of Clinical Oncology recommendations on adjuvant interpretation of immunohistochemistry for tumor markers in colorectal chemotherapy for stage II colon cancer. J Clin Oncol. 2004;22(16):3408–19. cancer. Virchows Arch. 2007;451(4):763–9. 4. O'Connor ES, Greenblatt DY, LoConte NK, Gangnon RE, Liou JI, Heise CP, 27. Sunshine JC, Nguyen PL, Kaunitz GJ, Cottrell TR, Berry S, Esandrio J, et al. et al. Adjuvant chemotherapy for stage II colon cancer with poor prognostic PD-L1 expression in melanoma: a quantitative Immunohistochemical features. J Clin Oncol. 2011;29(25):3381–8. antibody comparison. Clin Cancer Res. 2017;23(16):4938–44. 5. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39(1):1–10. 6. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313(5795):1960–4. 7. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8(8):793–800. 8. Keir ME, Francisco LM, Sharpe AH. PD-1 and its ligands in T-cell immunity. Curr Opin Immunol. 2007;19(3):309–14. 9. Kim JH, Park HE, Cho NY, Lee HS, Kang GH. Characterisation of PD-L1- positive subsets of microsatellite-unstable colorectal cancers. Br J Cancer. 2016;115(4):490–6. 10. Rosenbaum MW, Bledsoe JR, Morales-Oyarvide V, Huynh TG, Mino- Kenudson M. PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes. Mod Pathol. 2016;29(9):1104–12. 11. Lee LH, Cavalcanti MS, Segal NH, Hechtman JF, Weiser MR, Smith JJ, et al. Patterns and prognostic relevance of PD-1 and PD-L1 expression in colorectal carcinoma. Mod Pathol. 2016;29(11):1433–42. 12. Koganemaru S, Inoshita N, Miura Y, Miyama Y, Fukui Y, Ozaki Y, et al. Prognostic value of programmed death-ligand 1 expression in patients with stage III colorectal cancer. Cancer Sci. 2017;108(5):853–8. 13. Masugi Y, Nishihara R, Yang J, Mima K, da Silva A, Shi Y, et al. Tumour CD274 (PD-L1) expression and T cells in colorectal cancer. Gut. 2017;66(8): 1463–73. 14. Lee KS, Kwak Y, Ahn S, Shin E, Oh HK, Kim DW, et al. Prognostic implication of CD274 (PD-L1) protein expression in tumor-infiltrating immune cells for microsatellite unstable and stable colorectal cancer. Cancer immunology. Immunotheraphy. 2017;66(7):927–39. 15. Li Y, Liang L, Dai W, Cai G, Xu Y, Li X, et al. Prognostic impact of programed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor infiltrating lymphocytes in colorectal cancer. Mol Cancer. 2016;15(1):55. 16. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. REporting recommendations for tumour MARKer prognostic studies (REMARK). Br J Cancer. 2005;93(4):387–91. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Programmed Death Ligand-1 expression in stage II colon cancer - experiences from a nationwide populationbased cohort

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Springer Journals
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Copyright © 2019 by The Author(s).
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine, general; Medicine/Public Health, general
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1471-2407
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10.1186/s12885-019-5345-6
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Abstract

Background: Patients suffering from high risk stage II colon cancer (CC) may benefit from adjuvant onco-therapy, but additional prognostic markers are needed for better treatment stratification. We investigated the prognostic value of Programmed Death Ligand-1 (PD-L1) in a true population-based cohort of patients with stage II CC. Methods: PD-L1 expression on tumour cells was evaluated by immunohistochemistry in 572 colon cancers. Whole sections from tumour blocks representing the deepest invasive front of the primary tumour were used for analysis. A cut-off of 5% positivity was used for dichotomizing the data. The prognostic value was investigated in Cox proportional hazard models for recurrence-free survival (RFS) and overall survival (OS). Results: Overall, 6% of the tumours were classified as high PD-L1. High PD-L1 was related to female gender (p =0.028) , high malignancy grade (< 0.001), right side localization (p < 0.001) and microsatellite instability (MSI) (p < 0.001). Thirty-one (18%) of the MSI and 4 (1%) of the microsatellite stable tumours were classified as high PD-L1, respectively. PD-L1 expression provided no prognostic value as a single marker. In patients with MSI tumours, high PD-L1 expression had no significant impact regarding OS or RFS. Conclusions: PD-L1 expression in tumour cells of stage II CC did not provide any prognostic impact, neither in the entire population-based cohort nor in the group of MSI patients. Additional investigations of the immunogenic microenvironment are needed for evaluating the prognostic information in CC. Keywords: Colon cancer stage II, Prognostic markers, Programmed death ligand-1 Background However, the currenct high risk factors are questionable Colon cancer is one of the most common cancers in [4], and there is a need for additional prognostic markers the Western world. About 1/3 have stage II disease, for better treatment stratification. and this group of patients are in general having a In recent years, the tumour microenvironment has been good prognosis with a 5-year overall survival (OS) of investigated, and the role of the interaction between cancer approximately 70–80% after surgery alone [1]. Current cells and the immune system in cancer surveillance has been international guidelines (ASMO and ESMO) do not emphasized [5]. Tumour-infiltrating lymphocytes (TILs) are recommend routine adjuvant chemotherapy in stage considered as the host’s immune response against solid II CC, but rather that this treatment be limited to tumours, and infiltration by activated CD8+ cytotoxic patients having a high risk of recurrence, based on an T-lymphocytes is correlated with better survival of colorectal individual evaluation including high-risk markers [2, 3]. cancer (CRC) patients [6]. Activated lymphocytes expressing Programmed Death Receptor-1 (PD-1) can bind specific to the ligand Programmed Death Ligand-1 (PD-L1), which is * Correspondence: ann.christina.eriksen@rsyd.dk expressed on the cell membrane in malignant epithelial Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark tumours, including colorectal adenocarcinomas [7]. This Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark immune-checkpoint is up-regulated in many tumours, and Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Eriksen et al. BMC Cancer (2019) 19:142 Page 2 of 9 the interaction of PD-L1 on tumour cells with its receptor The tissue blocks were stored and transported at room PD-1 on the activated T-cells induces a down-regulation of temperature. One tumour block representing the deepest the antigen-stimulated lymphocyte proliferation and cytokine invasive margin, was selected from each patient. Prior to production, resulting in an inhibition of the host-immune re- inclusion, all histological slides from each tumour were sponse [8]. evaluated by first a trainee and afterwards an experienced Current results of the prognostic value of PD-L1 in pathologist. CRC are controversial. Some studies only report on trends towards worse prognosis for high PD-L1 expres- Immunohistochemistry sion [9, 10], while others identify high PD-L1 to be inde- From the selected tumour blocks serial 4 μm thick tissue pendently associated with worse recurrence free survival sections were cut and mounted on FLEX IHC Micro- (RFS) [11, 12]. In contrast, other studies report no prog- scope Slides (K8020, DAKO, Glostrup, Denmark). One nostic impact [13, 14], or even a tendency of high ex- whole tumour section per patient was used for the pression of PD-L1 to be related to a better prognosis evaluation of PD-L1 expression. Staining was performed [15]. However, studies vary greatly in methods and the using a Ventana BenchMark ULTRA (Ventana Medical study populations are highly heterogeneous, including Systems, Tucson, Arizona, USA) automated immunohis- different stages of disease, and no studies have previ- tochemistry (IHC) slide staining system. Tissue sections ously investigated the expression of PD-L1 in a cohort were heated and deparaffinised in EZprep (no.950–102, exclusively of patients with stage II CC. Ventana). Pre-treatment and demasking were carried With this motivation, the aim of the present study was out using ULTRA CC1 (no. 950–224) and ULTRA CC2 to evaluate the prognostic impact of PD-L1 in a nation- (no. 950–223), and endogenous peroxidase activity was wide, population-based cohort of stage II CC. blocked by Optiview Peroxidase Inhibitor (no. 760–700 Ventana). The slides were incubated with a rabbit monoclo- Methods nal anti-PD-L1 (clone SP263A, no. 790–4905/741–4905 This study is reported in accordance with the REMARK Ventana) for 16 min. This clone was chosen based on guidelines [16]. a pilot study. For amplification Optiview HQ Universal Linker (no. 760–700, Ventana) and Optiview HRP Multimer Patient population (no. 760–700, Ventana) was each used for 8 min. The population and sources of data has previously been The primary antibody was visualized using Optiview descreibed in detail [17]. In brief all patients surgically H O and DAB (no. 760–700, Ventana), followed by 2 2 treated for stage II CC in 2002 in Denmark were identified Optiview Copper (no. 760–700, Ventana). Counterstain by a search in the nationwide registry administrated by was done using Hematoxylin II (no. 790–2208, Ventana) the Danish Colorectal Cancer Group (DCCG) (N =746). and bluing Reagent (no. 760–2037, Ventana). Finally the Exclusion criteria were as follows: missing tumour block histological slides were cover slipped with Tissue-Tek (N = 11), insufficient tissue for analyses (N = 2), incor- PERTEX (Histolab Products AB, Göteborg, Sweden). rectly staged patients (N = 25), treatment with adjuvant Evaluation of microsattellite instability (MSI) was per- chemotherapy (N = 26)/radiotherapy (N = 1) and death formed using IHC. Tumours displaying loss of one or within 90 days after the operation (N = 75). Furthermore more of the 4 mismatch repair proteins (MLH1, MSH2, patients with loco-advanced disease (N = 8) and patients MSH6 and PMS2) were considered as MSI, whereas tu- diagnosed with another malignancy prior to CC were ex- mours with intact mismatch repair proteins were consid- cluded from the study (N = 26), and the final study popu- ered as microsatellite stable (MSS). Staining of mismatch lation comprised 572 patients. repair proteins was performed using a DAKO Autostai- Histopathological data were obtained by microscopic ner Link 48 (DAKO) with monoclonal mouse antibody examination and from the national Patobank containing against MLH1 (Novocastra, Leica, Germany, clone ES05, all pathology reports in Denmark. The term “not assessed” dilution 1:100, product code NCL-L-MLH1), MSH2 was used if the pathological feature was not described. (Novocastra, Leica, clone 25D12, dilution 1:100, product Clinical data were obtained from The National Patient code NCL-L-MSH2), MSH6 (BD Transduction Laboratories, Registry. clone 44/MSH6, dilution 1:200, material number 610919), Recurrence primarily occurred within the first five and PMS2 (BD Pharmingen, clone A16–4, dilution 1:500, years of follow-up and to encompass the majority of re- material number 556415). Tissue sections were incubated currences a follow-up period of seven years was selected. for 30 min at room temperature with the primary antibodies diluted in Envision Flex antibody diluent (code S2022 Samples DAKO). The antibody signal was amplified using Envision Formalin-fixed, paraffin-embedded tissue blocks were col- Flex+ Mouse(Linker) (DAKO) for 20 min. Bound antibodies lected from the departments of pathology in Denmark. were detected using Envision FLEX/HRP (DAKO) and Eriksen et al. BMC Cancer (2019) 19:142 Page 3 of 9 visualized by Envision FLEX DAB (DAKO) and chromogene used to test for differences between groups. The multi- diluted in Envision Flex Substrate Buffer (DAKO). The sec- variable Cox-regression model was used to test for inde- tions were incubated in 0.5% CuSO in TBS buffer pH 7.6 pendent prognostic value with hazard ratio (HR) of 1.0 for 10 min to enhance the immunohistochemical staining. as reference and a 95% confidence interval (CI). A Sections were counterstained with Meyer’s hematoxylin cut-off significance level of 0.10 was pre-specified for a (Merck, Damstadt, Germany). variable to be included in the multivariable Cox regres- sion model. Scoring of PD-L1 expression Chi -statistics were used to test associations between Tumour PD-L1 expression was evaluated based on clinicopathological variables. A p-value < 0.05 was con- immunostaining of the cell membrane of the epithelial sidered significant. The statistical analyses were per- tumour cells. The immunostaining of the stromal cells formed using STATA software version 14.0 (StataCorp, were not evaluated. Tumour cells were considered Texas, USA), and all statistical tests were two-sided. positive when any cell membrane staining (partial of complete) was present. Staining intensity was not evalu- Results ated and cytoplasmatic immunoreaction was not Patient characteristics considered. Patient characteristics are summarized in Table 1. In the The percentage of positive tumour cells were scored follow-up period of seven years, 266 (46.5%) patients semi-quantitatively as 0 (no positive tumour cells), 1 died; 110 (19.2%) patients experienced disease recur- (≤1% positive), 2 (> 1 and ≤ 5% positive), 3 (> 5 and ≤ rence and 78 (13.6%) patients were diagnosed with an- 20% positive), 4 (> 20 and ≤ 50% positive) and 5 (> 50% other cancer. The median age at time of surgery was 73 positive) (Fig. 1). A subset of 50 randomly selected tu- years (range 29–95), and the mean follow-up time was mours was examined by a second pathologist in order to 6.9 years (range 3–84 months). assess inter-observer variation. For prognostic evaluation data were dichotomized, using 5% PD-L1 expression as cut-off. In absence of a standardized scoring system the PD-L1 expression cut-off was based on previously studies [9, 12, 14]. The IHC staining of PD-L1 often had a highly heteroge- neous expression both between the central part of the Statistics tumour and the invasive margin and along the invasive The inter-observer reproducibility of PD-L1 scoring was tumour front (Fig. 2). evaluated by kappa statistics. Simple and weighted kappa Results regarding PD-L1 expression are displayed in (κ) values were calculated, and agreement was described Table 2. Nearly half of the population (46%) had no PD-L1 according to Landis et al [18] as moderate, substantial, expression in tumour cells. After dichotomization, using 5% and almost perfect for κ values of 0.41–0.60, 0.61–0.80, PD-L1 expression as cut-off, 35 (6%) of the tumours were and 0.81–1, respectively. classified as high PD-L1. In the group of MSI 31 (18%) of The endpoint OS was defined as time from operation the tumours were classified as high PD-L1 and in the to death of any cause or last follow-up. RFS was defined subgroup of MSS 4 (1%) were classified as high PD-L1. High as time from operation to death of any cause or recur- PD-L1 was related to female gender (p = 0.028), high malig- rence of CC. Patients later diagnosed with another can- nancy grade (< 0.001), right side localization (p < 0.001) and cer were censored at the date of diagnosis (N = 102). MSI (p <0.001). The median age was used as cut-off to dichotomize the The inter-observer agreement for the semi-quantitative parameter age. Survival curves were generated according evaluation of PD-L1 expression was moderate with κ=0.418 to the Kaplan-Meier method and the log-rank test was and weighted κ = 0.573. The agreement improved to Fig. 1 Example of immunohistochemically staining of programmed death ligand-1. a ≤ 1% positive tumour cells, b 5% < positive tumour cells ≤20%, and c > 50% of the tumour cells are positive Eriksen et al. BMC Cancer (2019) 19:142 Page 4 of 9 Table 1 Clinico-pathological characteristics and association to Table 1 Clinico-pathological characteristics and association to PD-L1 (cut-off 5%) PD-L1 (cut-off 5%) (Continued) Number PD-L1 Number PD-L1 (N = 572) (%) Low (%) High (%) p-value (N = 572) (%) Low (%) High (%) p-value Age (years at diagnosis) Mismatch repair status Median 72 MSS 400 (70) 396 (74) 4 (11) < 0.001 Range 29–95 MSI 172 (30) 141 (26) 31 (89) Abbreviations: MSI microsatellite instability, MSS microsatellite stable, NOS not ≥ 73 267 (47) 253 (47) 14 (40) 0.414 otherwise specified < 73 305 (53) 284 (53) 21 (60) Including mucinous adenocarcinomas and signet-ring cell carcinomas P-values are obtained using chi -test. Statistically significant p-values are Gender highlighted in bold Male 283 (49) 272 (51) 11 (31) 0.028 Female 289 (51) 265 (49) 24 (69) substantial, when categorizing the data into high PD-L1 ex- T-stage pression (> 5%) or low PD-L1 expression (≤5%), κ=0.691. pT3 500 (87) 472 (88) 28 (80) 0.172 pT4 72 (13) 65 (12) 7 (20) Survival analysis Histology (WHO) The 5-year RFS for the population with low PD-L1 was Adenocarcinoma NOS 515 (90) 481 (90) 34 (97) 0.348 69.2% versus 67.7% in the group with high PD-L1 ex- Mucinous 55 (10) 54 (10) 1 (3) pression, and OS was 74.7% versus 70.5%, respectively. adenocarcinoma When considering patients with MSI tumours the 5 year Signet-ring cell 2 (0) 2 (1) 0 (0) RFS for low PD-L1 was 77.4% versus 67.5% in the group carcinoma of high PD-L1, and OS was 79.4% versus 70.7%. No Malignancy grade significant differences in survival rates were observed, Medium + low 450 (79) 436 (81) 14 (40) < 0.001 considering the entire cohort (Fig. 3). In the group of High 122 (21) 101 (19) 21 (60) patients with MSI tumours the Kaplan Meier curves were separated for RFS, but results were insignificant, p =0.256 Localisation (Fig. 4). This also accounted the group of patients with Right 273 (48) 242 (45) 31 (89) < 0.001 MSI T3 tumours (N =155), p =0.149. Left 299 (52) 295 (55) 4 (11) Outcomes from the corresponding univariable Cox Tumour perforation regression analyses are shown in Table 3. Patients with Yes 530 (93) 499 (97) 31 (94) 0.313 MSI tumours and high PD-L1 expression did not have a No 17 (3) 15 (3) 2 (6) significant worse OS or RFS, HR = 1.104 (0.604–2.016), p = 0.748 and HR = 1.429 (0.769–2.653), p = 0.258, Unknown 25 (4) respectively. Age ≥ 73 years, T4 tumour and perfor- Lymph nodes ation were significantly related to an adverse outcome Median 10 of both OS and RFS. Patients with MSI T3 tumours and Range 0–41 high PD-L1 expression did neither have a significant < 12 nodes 351 (61) 329 (61) 22 (63) 0.851 worse OS or RFS, HR = 1.531 (0.757–3.100), p = 0.236 and ≥ 12 nodes 221 (39) 208 (39) 13 (37) HR = 1.637 (0.833–3.216), p = 0.153, respectively. Multivariable Cox regression analyses were not per- Perineural invasion formed, as PD-L1 had p-value > 0.10 in the univariable Yes 26 (5) 25 (7) 1 (4) 0.635 Cox-regression analysis. No 359 (63) 337 (93) 22 (96) Not assessed 187 (32) Discussion Vascular invasion In this study, we investigated the prognostic value of Yes 43 (7) 38 (9) 5 (22) 0.054 PD-L1 expression on tumour cells in an unbiased, na- No 386 (68) 368 (91) 18 (78) tionwide and population-based cohort of patients with Not assessed 143 (25) stage II CC, treated exclusively with surgery. PD-L1 ex- pression as a single marker did not provide any signifi- cant prognostic value regarding OS or RFS, neither in the the entire cohort nor in the subgroup of patients with MSI tumours. Eriksen et al. BMC Cancer (2019) 19:142 Page 5 of 9 Fig. 2 Example of heterogeneity of the expression of programmed death ligand-1. a Overview of the heterogenetic area. Frame one is presented in higher magnification in (b) and frame two in (c). The membranous expression pattern is displayed in (d) In the entire cohort we found 6% of the tumours to and MSS tumours has previously been descriebed in have a high expression of PD-L1 on tumour cells, which studies using a different scoring system [11, 19], al- is in accordance with other studies of CRC, reporting 5% though a recent study reported no differences in PD-L1 positivity [11]. Likewise we found high PD-L1 expression positivity among MSI and MSS tumours [20]. associated to female gender, high malignancy grade, right The association between MSI and high PD-L1 expres- sided localisation, and MSI, which also has been found sion may be explained by the abundant infiltration of by Lee et al, who investigated all stages of CRC [11]. TILs in these tumours. Deficiency of the mismatch re- Regarding MSI and MSS tumour subgroups, we found pair proteins results in a number of mutations. There- 18% of the MSI tumours to have high PD-L1 expression fore MSI tumours have a high load of tumour specific and 1% of the MSS tumours to have high PD-L1 expres- neo-antigens, which can induce an immunological re- sion. The difference in PD-L1 expression between MSI sponse with recruitment and activation of T-cells [21]. One way to stimulate PD-L1 upregulation is afforded by the pro-inflammatory cytokine interferon-gamma Table 2 Programmed death ligand-1 (PD-L1) expression in (IFN-γ), which is produced by activated T-cells and colon cancer Natural Killer cells [22]. The high expression of PD-L1 in Number of PD-L1 Entire cohort MSI (N = 172) (%) MSS (N = 400) (%) MSI tumours with abundant infiltration of TILs is in ac- positive tumour (N = 572) (%) cordance with the consensus molecular subtype (CMS) cells classification. The molecular group CMS1 is characterized None 264 (46) 47 (27) 217 (54) by hypermutation, MSI and intense immune reaction [23], 0 < PD-L1 ≤ 1% 233 (41) 73 (42) 160 (40) and this immunogenic group has been documented with a 1% < PD-L1 ≤ 5% 40 (7) 21 (12) 19 (5) high PD-L1 expression [24]. < 5% PD-L1 ≤ 20% 10 (2) 8 (5) 2 (1) In the group of patients with MSI tumours, the < 20% PD-L1 ≤ 11 (2) 10 (6) 1 (0) Kaplan-Meier curves were clearly separated regarding RFS, 50% with a worse RFS related to a high PD-L1 expression, but PD-L1 > 50% 14 (2) 13 (8) 1 (0) statistical significance was not reached. This is in accord- Abbreviations: MSI Microsatellite instability, MSS sMicrosatellite stable ance with studies of tumour expression of PD-L1 in MSI Eriksen et al. BMC Cancer (2019) 19:142 Page 6 of 9 Fig. 3 Kaplan-Meier survival curves depicting overall survival (a) and recurrence-free survival (b) stratified by the expression of programmed death ligand-1 (PD-L1) in the entire cohort (N = 572). P-values were calculated by log-rank test stage I-IV CRC. Kim et al [9] reported a tendency towards PD-L1 and N status with high PD-L1 expression being a worse prognosis for tumours with high PD-L1 expression; related to higher N status. This may be part of the ex- however results were non-significant. Rosenbaum et al [10] planation for the inconsistency, as we only investigated reported no prognostic value for dichotomized data, but stage II CC. Unfortunately, Koganemaru et al did not re- the group with the highest expression of PD-L1 (≥50%) had port any data on MSI status. a markedly reduced disease-specific survival. We only Lee et al [11] documented PD-L1 expression as an found 14 patients to have PD-L1 expression ≥50%, and independent prognostic marker in patients with MSI using this cut-off in our cohort did not enhance the prog- tumours. In the present populationbased study 172 nostic impact (data not shown). Rosenbaum et al investi- patients had a MSI tumour and only 31 (18%) of these gated all stages of CRC and found PD-L1 expression tumours were classified as high PD-L1, resulting in a related to stage, which might explain the difference. small group and thus low statistical power in the Cox re- In contrast to our data, Koganemaru et al [12] re- gression analysis. This may be part of the explanation ported high PD-L1 expression being an independent for the non-significant results. prognostic marker. They used the same cut-off (5%) in Furthermore, previous studies reporting independent their evaluation of PD-L1, but they exclusively investi- prognostic impact of PD-L1, differ in investigated co- gated stage III CRC. They found an association between horts and evaluation methods. Lee et al [11]included AB Fig. 4 Kaplan-Meier survival curves depicting overall survival (a) and recurrence-free survival (b) stratified by the expression of programmed death ligand-1 (PD-L1) in the group of patients with MSI tumours (N = 172). P-values were calculated by log-rank test Eriksen et al. BMC Cancer (2019) 19:142 Page 7 of 9 Table 3 Univariable Cox regression analysis Parameter Overall survival Recurrence-free survival HR (95% CI) p-value HR (95% CI) p-value Age (years at diagnosis) <73 1 0.001 1 0.009 ≥73 2.533 (1.466–4.377) 2.194 (1.216–3.958) Gender Male 1 0.462 1 0.634 Female 0.839 (0.524–1.341) 0.881 (0.523–1.484) T-stage T3 1 0.003 1 0.002 T4 2.68 (1.409–5.128) 2.982 (1.503–5.915) Malignancy grade Medium/Low 1 0.536 1 0.636 High 1.159 (0.726–1.852) 1.134 (0.675–1.906) Localisation Right 1 0.792 1 0.150 Left 1.084 (0.594–1.981) 1.576 (0.848–2.927) Tumour perforation No 1 0.001 1 0.005 Yes 5.332 (1.924–14.776) 5.644 (1.708–18.645) Lymph nodes < 12 nodes 1 0.440 1 0.220 ≥12 nodes 1.203 (0.753–1.922) 1.386 (0.823–2.334) Perineural invasion No 1 0.630 1 0.873 Yes 1.417 (0.343–5.857) 0.850 (0.117–6.203) Vascular invasion No 1 0.878 1 0.756 Yes 0.922 (0.332–2.566) 0.830 (0.256–2.690) PD-L1 Low 1 0.748 1 0.258 High 1.104 (0.604–2.016) 1.429 (0.769–2.653) Univariable Cox regression analysis regarding overall survival and recurrence-free survival for the sub-cohort of patients with MSI tumours (N = 172). Statistically significant p-values are highlighted in bold Including mucinous adenocarcinomas and signet-ring cell carcinomas MSI microsatellite instability all stages of both colon and rectal cancers. Also their expression on tumour cells in 13% of the investigated evaluation method differed in a number of ways from tumours based on evaluation of TMAs from the central our technical approach. They used tissue microarrays and invasive tumour compartments. They included all (TMAs),asdomostother studies[9–15]. In constrast stages of CRC. In contrast we evaluated PD-L1 ex- we evaluated the PD-L1 expression in whole sections pression in a highly homogenous cohort of stage II and observed a lot of heterogeneity both between the CC patients and found a considerable intra-tumoral central part of the tumour and the invasive margin, heterogeneity. and along the invasive tumour front. The use of A standardized scoring system for PD-L1 expression TMAs may lead to selection bias, although several in CC is missing and several unvalidated methods are in studies try to avoid this by using several representa- use. In the present study, the proportion of PD-L1 posi- tive TMAs from each tumour [9, 11, 13]. Lee et al tive tumour cells was evaluated considering only mem- [14] descreibe intra-tumoral heterogeneity of PD-L1 branous staining as positive. Intensity of the staining and Eriksen et al. BMC Cancer (2019) 19:142 Page 8 of 9 cytoplasmic immunoreaction were not considered. expression in tumour cells should be seen in the context Whether to take cytoplasmic staining of PD-L1 into of the entire immune tumour microenvironment. The account differs among studies. Some investigations use expression of PD-L1 is influenced by TILs, which is a combination of membranous staining and staining related to MSI. We did not prove any prognostic value intensity [11] while others do not consider cytoplasmic of PD-L1 in patients with MSI tumours, however staining at all [10]. PD-L1 expressed on the cell surface this interaction should be taken into consideration in is essential for the interaction with the PD-1 receptor on future studies. the T-cells, indicating that only PD-L1 expressed on the membrane is of clinical importance. Furthermore, in the Conclusions evaluation of lung cancer, only membrane staining is ap- In this nationwide population-based cohort of stage II plied when evaluating the indication for immunotherapy colon cancer, we found membranous PD-L1 expression with a PD-L1 inhibitor [25]. (cut-off 5%) in tumour cells of stage II colon cancer to As mentioned above, we did not consider intensity be associated with female gender, high malignancy grade, of the staining. Intensity of IHC may be difficult to right side localisation and MSI. The expression of PD-L1 interpret. Poor reproducibility of IHC staining inten- was often highly heterogenous. No prognostic informa- sities of various proteins has been documented, while tion was detected of PD-L1 as a single biomarker in this excellent inter-observer reproducibility was found esti- cohort of stage II colon cancer. mating the fraction of positive tumour cells [26]. Abbreviations PD-L1 was not included in that study, but the same CC: Colon cancer; CRC: Colorectal cancer; DCCG: Danish Colorectal Cancer most likely applies to this protein. Furthermore, the Group; HR: Hazard ratio; IHC: Immunohistochemical; MSI: Microsatellite evaluation of staining intensity is not only influenced instability; MSS: Microsatellite stability; NOS: Not otherwise specified; OS: Overall survival; PD-1: Programmed death receptor 1; PD-L1: Programmed death ligand 1; by subjectivity. Various other factors may affect the RFS: Recurrence-free survival; TILs: Tumour infiltrating lymphocytes; TMA: Tissue staining intensity, encompassing both pre-analytical microarray and analytical factors such as fixation (time and type), storage, and IHC protocols. Also section thickness Acknowledgements We thank Birgit Roed Sørensen and Christina Braad Petersen for excellent affects staining intensity, and even modern state of technical assistance. We also thank all pathology departments in Denmark for the art microtomes produce sections with varying their support and participation in the study, which has made the population- thicknesses. based design possible. Also, we appreciate the help and advice supported by OPEN, Odense Patient data Explorative Network, Odense University Hospital, The lack of a standardized IHC method challenges Odense, Denmark. Finally, we are grateful to Roche Diagnostics for the discount the assessment of PD-L1 expression, and moreover, provided, regarding antibody expenses. different trials use different antibodies and assays. In melanoma, diverse assays have been found to variate Funding The study was conducted and affiliated under Danish Colorectal Cancer in staining sensitivity of tumour cells [27]. For future Group and Danish Colorectal Center South, Vejle Hospital, Part of Lillebaelt studies, standardized techniques for evaluating PD-L1 Hospital, Denmark. Financial support was granted by The Research Council in CC are required regarding antibodies, assays, inter- of Lillebaelt Hospital and the Danish Cancer Research Foundation. The commercial company Roche Diagnostics supported the study providing pretation, and threshold cut-off in scoring the immu- antibodies at a discount. The funding organisations did not play any role in nostain. Also, focus should be directed on how to the study design, data collection, analysis or interpretation of the data, handle the marked heterogeneity, as this might hinder decision to publish, or preparation of the manuscript. reproducibility of IHC scoring systems. Availability of data and materials The present study is limited by the retrospective de- The datasets used and/or analysed during the current study are available sign, as we had no influence on the pre-analytical phase from the corresponding author on reasonable request. of the IHC. However, we used a validated antibody on a fully automatic platform, and only considered membran- Authors’ contributions Conceived and designed the experiments: ACE, FBS, SKF, JL and TFH. ous staining according to the manufactures recommen- Performed the experiments: ACE. Analyzed the data: ACE, RD and TFH. Wrote dation. The PD-L1 antibody used in this study stained the paper: ACE, FBS, HH, JL and TFH. All authors read and approved the final both the malignant epithelial cells and immune cells in manuscript. the stroma, which made it difficult to discriminate these Ethics approval and consent to participate cellular populations when the density of immune cells in The Regional Committees on Health Research Ethics for Southern Denmark the tumour stroma interface was high. approved the study and exempted the study from the rule of informed The prognostic value of PD-L1 expression on tumour consent in their approvel (S-20140119). The study was also approved by the Danish Data Protection Agency (14/26345). All patients were screened in the cells is controversial. We investigated the expression of Danish Registry of Tissue Utilization before enrollment in the study. PD-L1 exclusively in stage II CC in a well-defined and unbiased population, and did not find any prognostic Consent for publication value of PD-L1 as a single biomarker. The PD-L1 Not applicable. Eriksen et al. BMC Cancer (2019) 19:142 Page 9 of 9 Competing interests 17. Eriksen AC, Sørensen FB, Lindebjerg J, Hager H, dePont Christensen R, The authors declare that they have no competing interests. Kjær-Frifeldt S, et al. The prognostic value of tumour infiltrating lymphocytes in stage II colon cancer. A nationwide population-based study. Transl Oncol. 2018;11(4):979–87. Publisher’sNote 18. Landis JR, Koch GG. The measurement of observer agreement for Springer Nature remains neutral with regard to jurisdictional claims in categorical data. Biometrics. 1977;33(1):159–74. published maps and institutional affiliations. 19. Korehisa S, Oki E, Iimori M, Nakaji Y, Shimokawa M, Saeki H, et al. Clinical significance of programmed cell death-ligand 1 expression and the Author details immune microenvironment at the invasive front of colorectal cancers with Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark. high microsatellite instability. Int J Cancer. 2018;142(4):822–32. Institute of Regional Health Research, University of Southern Denmark, 20. Zhang Y, Sun Z, Mao X, Wu H, Luo F, Wu X, et al. Impact of mismatch-repair Odense, Denmark. Department of Clinical Medicine, University Institute of deficiency on the colorectal cancer immune microenvironment. Oncotarget. Pathology, Aarhus University Hospital, and , University of Aarhus, Aarhus, 2017;8(49):85526–36. Denmark. Danish Colorectal Cancer Group (DCCG), Aarhus N, Denmark. 21. Boissiere-Michot F, Lazennec G, Frugier H, Jarlier M, Roca L, Duffour J, et al. Research Unit of General Practice, University of Southern Denmark, Odense, Characterization of an adaptive immune response in microsatellite-instable Denmark. Department of Pathology, Lillebaelt Hospital, Beriderbakken 4, colorectal cancer. Oncoimmunology. 2014;3:e29256. DK-7100 Vejle, Denmark. 22. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015;27(4):450–61. Received: 19 April 2018 Accepted: 1 February 2019 23. Guinney J, Dienstmann R, Wang X, de Reynies A, Schlicker A, Soneson C, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21(11):1350–6. References 24. Reilley M, Blando JM, Katkhuda R, Menter D, Sharma P, Allison JP, et al. 1. Morris M, Platell C, McCaul K, Millward M, van Hazel G, Bayliss E, et al. Immunologic profiling of consensus molecular subtype (CMS) stratified Survival rates for stage II colon cancer patients treated with or without colorectal cancer (CRC) primary and liver metastectomy specimens: chemotherapy in a population-based setting. Int J Color Dis. 2007;22(8): implications for immune targeting of proficient mismatch repair CRC. 887–95. J Clin Oncol. 2016;34(15):3520. 2. Labianca R, Nordlinger B, Beretta GD, Mosconi S, Mandala M, Cervantes A, 25. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. et al. Early colon cancer: ESMO clinical practice guidelines for diagnosis, Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. treatment and follow-up. Ann Oncol. 2013;24(6):64–72. 2015;372(21):2018–28. 3. Benson AB 3rd, Schrag D, Somerfield MR, Cohen AM, Figueredo AT, Flynn PJ, 26. Zlobec I, Terracciano L, Jass JR, Lugli A. Value of staining intensity in the et al. American Society of Clinical Oncology recommendations on adjuvant interpretation of immunohistochemistry for tumor markers in colorectal chemotherapy for stage II colon cancer. J Clin Oncol. 2004;22(16):3408–19. cancer. Virchows Arch. 2007;451(4):763–9. 4. O'Connor ES, Greenblatt DY, LoConte NK, Gangnon RE, Liou JI, Heise CP, 27. Sunshine JC, Nguyen PL, Kaunitz GJ, Cottrell TR, Berry S, Esandrio J, et al. et al. Adjuvant chemotherapy for stage II colon cancer with poor prognostic PD-L1 expression in melanoma: a quantitative Immunohistochemical features. J Clin Oncol. 2011;29(25):3381–8. antibody comparison. Clin Cancer Res. 2017;23(16):4938–44. 5. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39(1):1–10. 6. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006;313(5795):1960–4. 7. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8(8):793–800. 8. Keir ME, Francisco LM, Sharpe AH. PD-1 and its ligands in T-cell immunity. Curr Opin Immunol. 2007;19(3):309–14. 9. Kim JH, Park HE, Cho NY, Lee HS, Kang GH. Characterisation of PD-L1- positive subsets of microsatellite-unstable colorectal cancers. Br J Cancer. 2016;115(4):490–6. 10. Rosenbaum MW, Bledsoe JR, Morales-Oyarvide V, Huynh TG, Mino- Kenudson M. PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes. Mod Pathol. 2016;29(9):1104–12. 11. Lee LH, Cavalcanti MS, Segal NH, Hechtman JF, Weiser MR, Smith JJ, et al. Patterns and prognostic relevance of PD-1 and PD-L1 expression in colorectal carcinoma. Mod Pathol. 2016;29(11):1433–42. 12. Koganemaru S, Inoshita N, Miura Y, Miyama Y, Fukui Y, Ozaki Y, et al. Prognostic value of programmed death-ligand 1 expression in patients with stage III colorectal cancer. Cancer Sci. 2017;108(5):853–8. 13. Masugi Y, Nishihara R, Yang J, Mima K, da Silva A, Shi Y, et al. Tumour CD274 (PD-L1) expression and T cells in colorectal cancer. Gut. 2017;66(8): 1463–73. 14. Lee KS, Kwak Y, Ahn S, Shin E, Oh HK, Kim DW, et al. Prognostic implication of CD274 (PD-L1) protein expression in tumor-infiltrating immune cells for microsatellite unstable and stable colorectal cancer. Cancer immunology. Immunotheraphy. 2017;66(7):927–39. 15. Li Y, Liang L, Dai W, Cai G, Xu Y, Li X, et al. Prognostic impact of programed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor infiltrating lymphocytes in colorectal cancer. Mol Cancer. 2016;15(1):55. 16. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. REporting recommendations for tumour MARKer prognostic studies (REMARK). Br J Cancer. 2005;93(4):387–91.

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BMC Cancer – Springer Journals

Published: Feb 12, 2019

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