- Email: [email protected]
programmed cell death ligand 1 in the tumor microenvironments of primary gastrointestinal diffuse large B cell lymphomas
Pathology - Research and Practice 214 (2018) 507–512
Contents lists available at ScienceDirect
Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
Expression of programmed cell death 1/programmed cell death ligand 1 in the tumor microenvironments of primary gastrointestinal diffuse large B cell lymphomas
T
⁎
Yang Liua, Jing Maa, Kangjie Yub, Mingyang Lia, Fang Liua, Qingguo Yana, Zhe Wanga, , Shuangping Guoa a
State Key Laboratory of Tumor Biology, Department of Pathology, The Basic Medicine Science and Xi Jing Hospital, The Fourth Military Medical University, Xi’an, Shaan Xi Province, 710032, China Student Team 1, Class 3, The Fourth Military Medical University, Xi’an, Shaan Xi Province, 710032, China
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Programmed cell death 1 Programmed cell death ligand 1 Expression Gastrointestinal diffuse large B cell lymphoma
Background: Gastrointestinal diffuse large B cell lymphoma (GI DLBCL) is the most common gastrointestinal lymphoma. However, there has not been a comprehensive investigation into the expression patterns of programmed cell death 1 (PD-1) and programmed cell death ligand 1(PD-L1) in GI DLBCL tissues. Methods: PD-1 protein expression in tumor-infiltrating lymphocytes (TILs) was evaluated by immunohistochemical staining, and expression of PD-L1 was evaluated by using PD-L1/PAX5 immunohistochemical double staining in 92 GI DLBCL specimens. Results: The prevalence of positive PD-L1 expression (PD-L1 + ) in GI DLBCL cells and positive PD-L1 expression in non-cancer cells of the GI DLBCL microenvironment (microenvironmental PD-L1, mPD-L1) were 11.96% (11 of 92) and 41.98% (34 of 81), respectively. PD-L1 expression in GI DLBCL was significantly associated with involvement of extranodal sites ≥ 2 (P = 0.034) and mPD-L1 expression was significantly associated with ECOG performance status (score ≥ 2) (P = 0.041). PD-L1 expression and mPD-L1 expression had no prognostic significance (P > 0.05) on disease outcome. PD-1+ TILs were significantly lower in patients with extranodal site involvement (P = 0.011) and the quantity of PD–1 + TILs correlated positively with the level of PDL1 expression in non malignant microenvironment cells (P = 0.001). Patients with high levels of PD-1+ TILs had better prognosis (P = 0.0005). Conclusions: The expression patterns of PD-L1 in patients with GI DLBCL are different from patients with common DLBCL. Immunotherapies that target the PD-1/PD-L1 pathway may have therapeutic potential in GI DLBCL.
1. Introduction Diffuse large B cell lymphoma (DLBCL), one of the most common and aggressive forms of B-cell lymphomas, accounts for 40% of B-cell malignancies, and is highly heterogeneous [1]. DLBCL has been subdivided into several distinct disease entities based on morphological, biological, and clinical characteristics, although a large number of cases remain heterogeneous [2]. Despite the success of rituximab and doxorubicin treatment in improving the survival of patients with DLBCL, approximately one-third of patients will relapse or develop refractory disease [3]. It is therefore necessary to develop novel therapeutic strategies to treat DLBCL.
Gastrointestinal DLBCL (GI DLBCL) is the most common GI lymphoma [4]. The GI tract is a complex system with an immunologically specific environment where antigen stimuli can result in rapid and severe pathologies, such as occurs in Helicobacter pylori infection; on this basis, we believe that primary GI DLBCL differs from DLBCL of lymph node origin. Programmed cell death 1 (PD-1), a member of the B7 receptor family, is expressed on B cells, monocytes, activated natural killer T cells, and immature Langerhans’ cells. The functional and biochemical properties of PD-1 have been best studied in T cells and up-regulation of PD-1 is a natural consequence of T-cell activation, and is necessary for termination of the immune response [5]. The ligand for PD-1,
⁎
Corresponding authors at: Department of Pathology, Fourth Military Medical University, Chang le west Rd, Xi’an, Shaan Xi Province,710032, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Ma), [email protected] (K. Yu), [email protected] (M. Li), [email protected] (F. Liu), [email protected] (Q. Yan), [email protected] (Z. Wang), [email protected] (S. Guo). https://doi.org/10.1016/j.prp.2018.03.001 Received 17 December 2017; Received in revised form 5 February 2018; Accepted 2 March 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
for horseradish peroxidase. Tissue sections were counterstained with Harris’ hematoxylin and then mounted. For negative controls, phosphate-buffered saline was used instead of primary antibody. PD-L1+ DLBCL was defined as 30% or more of the lymphoma cells showing distinct membranous and/or cytoplasmic PD-L1 staining and PAX5 nuclear staining, regardless of the PD-L1 positivity of non malignant microenvironment cells. Among PD-L1-negative DLBCL cases, in which PD-L1+ non malignant microenvironment cells represented 20% or more of the total tissue, cellularity was defined as microenvironmental PD-L1 (mPD-L1+) DLBCL [12].
programmed cell death ligand 1 (PD-L1), is expressed on T-cells, B-cells, antigen presenting cells (APCs), monocytes, epithelial cells, and tumor cells. The PD-1/PD-L1 pathway is an inhibitory immune checkpoint that can suppress T cell immune activity, and plays a vital role in the maintenance of peripheral tolerance by protecting bystander tissues from immune-mediated damage. On the other hand, the PD-1/PD-L1 pathway inhibits the immune activity of tumor specific CD8+ T cells, allowing tumor cells to escape T-cell–mediated tumor-specific and pathogen-specific immunity, thereby promoting tumor development. Increased expression of PD-L1 has been associated with poor prognosis in several cancers, including melanoma, lung cancer, ovarian cancer, and DLBCL [6–9]. Blockade of the PD-1/PD-L1 pathway demonstrated beneficial therapeutic effects in extensively pretreated patients with relapsed or refractory DLBCL [10]. However, no studies to date have comprehensively investigated the expression patterns of PD-1 and PDL1 in GI DLBCL tissues. In this study we evaluated the expression of PD1/PD-L1 in primary gastrointestinal diffuse large B cell lymphoma, and determined the association of PD-1/PD-L1 expression with clinicopathological features in a cohort of 92 primary GI DLBCL cases.
2.4. Statistical analyses
2. Materials and methods
Statistical analysis was performed using IBM-SPSS Statistics version 20 (IBM). The association between PD-L1 expression and clinicopathological features of GI DLBCL was investigated using Pearson’s χ2 test. PD-1–positive cells were analyzed as a continuous variable. An independent T-test was used to evaluate the relationship between PD–1 + TILs and clinicopathological features. Survival analysis was performed using Kaplan–Meier analysis (the event was death). All Pvalues were two-sided; P < 0.05 was considered statistically significant.
2.1. Patient samples
3. Results
All patients enrolled in this study were diagnosed with GI DLBCL at Xi Jing Hospital and Tang Du Hospital (Xi’an, China) between January 2007 and February 2017. Pathologists (S.P.G., Z.W., W.Z) re-reviewed cases and identified 92 patients presenting with GI system lesions with or without extra-GI involvement (Stomach: 52; Colon: 21; Small intestine: 19). All patients were diagnosed according to the 2008 WHO classification of tumors of hematopoietic and lymphoid tissues [1]. Clinicopathological information, including age, location, and treatment, was obtained for this patient cohort. Therapeutic information was obtained for 92 cases; eighty-three patients (90.2%) underwent surgery and received chemotherapy; one patient (1.1%) received radiation after surgery and chemotherapy. Two patients (2.2%) underwent surgery only; six patients (6.5%) received chemotherapy only. The hospital-based ethics committees approved the study and informed consent was obtained from patients.
3.1. Patient characteristics The clinicopathological features of patients with GI DLBCL in the study cohort are summarized in Table 1. Patients commonly presented with GI tract symptoms such as weight loss, fatigue, dyspepsia, abdominal distension, stomachache, intestinal obstruction, and GI bleeding. Patient age ranged from 15 to 87 years; the median age was 54 years. Thirty-three cases had B symptoms. International Prognostic Index (IPI) scores were determined for 81 cases: the IPI score was 0–1 in 37 cases and 2–5 in 44 cases. Within the cohort, 44/75 cases (58.67%) had stage I/II disease and 31/75 cases (41.33%) had stage III/IV disease, according to the tumor-node-metastasis (TNM) system [13]; 17 cases of endoscopy biopsy tissues were not staged. Colonic DLBCL more frequently showed advanced TNM stage (III–IV; P = 0.025) and higher IPI score (score = 2–5; P = 0.005) than gastric DLBCL. Small intestine DLBCL more frequently showed involvement of extranodal sites ≥ 2 (P = 0.049) and higher ECOG performance status (score ≥ 2; P = 0.005) compared with gastric DLBCL. Regarding sex, age distribution, B symptoms, Hans classification, mPD-L1 expression, and PDL1 expression there were no differences between gastric, intestinal, and colonic DLBCL. Follow-up data were available for 66 patients, with a follow-up period ranging from 1 to 78 months (median: 58 months), the number of deaths in the overall gastrointestinal DLBCL, gastric DLBCL, colic DLBCL and small intestina DLBCL was 21 (31.8%;21/66),12 (30.8%;12/39), 7 (43.8%;7/16), 2 (18.2%;2/11), respectively. Univariate survival analysis revealed that poor overall survival was more associated with higher TNM stage (P < 0.001), involvement of extranodal sites ≥ 2 (P < 0.001), non-GCB DLBCL (P = 0.010), and higher IPI risk group classification (P < 0.001).
2.2. Immunohistochemical staining Tumor tissues were fixed in 10% buffered formalin and embedded in paraffin (FFPE), and stained with hematoxylin and eosin (H&E) or immunohistochemical (IHC) stains. IHC staining was performed using the Bond-Max automated immunostainer (Vision BioSystems, Leica, Victoria, Australia), with a polymer-based detection system. The primary antibodies used were against CD3 (Clone SP7), CD10 (Clone 56C6, Ventana, Tucson, AZ, USA), H. pylori (Clone RAB-0064; Fuzhou Maixin Biotech, Fuzhou, China), BCL6 (Clone PG-B6P), CD20 (Clone L26), and MUM1 (Clone Mum1P; Dako, Glostrup, Denmark). The cases were classified into two immunophenotypic subtypes: germinal center B cell–like (GCB) and non-GCB, according to the Hans classification [11]. PD-1 (UMAB199, OriGene) was immunostained in tumor-infiltrating lymphocytes (TILs) for all cases.
3.2. Histological features
2.3. Classification of DLBCL by using PD-L1/PAX5 double staining
All cases showed diffuse infiltration, often in a sheet-like pattern, and replacement of the GI mucosa and invasion of the submucosal and muscular layers by large atypical lymphoid cells. Tumor-adjacent tissues displayed histologic features of chronic atrophic gastritis in all 52 gastric DLBCL cases; 26 cases were severe, with heavy inflammatory cell infiltration and severe atrophy of the lamina propria mucous glands, and intestinal metaplasia; the remaining cases were mild to moderate. Of 52 gastric DLBCL cases, 20 (38.46%) were positive for H. pylori; no small intestinal or colonic DLBCL cases were positive for H.
The expression of PAX5 (Clone EP156; ZSGB-BIO) and PD-L1 (Clone sp142; Roche) was performed by double IHC staining using DouMaxVision immunohistochemical double dye kits (KIT-9998; Maixin Biotech), according to the manufacturer’s instructions. For this double IHC staining, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium was used as the blue-black chromogen for alkaline phosphatase and 3-amino-9-ethylcarbazole was used as the red chromogen 508
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
Table 1 Clinicopathological features of the GI DLBCL cohort. Total
Gender Male 48 Female 44 Age (years) < 60 58 ≥60 34 TNM stage I–II 44 III–IV 31 NA 17 Extranodal sites <2 80 ≥2 12 B symptoms No 59 Yes 33 ECOG performance status <2 55 ≥2 37 IPI risk group 0–1 37 2–5 34 NA 21 Hans classification GCB 31 Non-GCB 61 mPD-L1 Negative 47 Positive 34 PD-L1 Negative 81 Positive 11
OS, P
Pathogenic site
P*
P**
P***
Stomach
Colon
Small intestine
0.904
24 28
11 10
13 6
0.796
0.114
0.349
0.268
31 21
15 6
12 7
0.427
1.000
0.738
< 0.001
31 12 9
8 12 1
5 7 7
0.025
0.055
0.607
< 0.001
43 9
18 3
19 0
0.527
0.049
0.135
0.087
33 19
13 8
13 6
1.000
0.784
0.748
0.124
37 15
12 9
6 13
0.280
0.005
0.125
< 0.001
27 12 13
5 13 3
5 9 4
0.005
0.054
0.712
0.010
17 35
5 16
9 10
0.577
0.278
0.186
0.186
29 16
7 11
11 7
0.092
1.000
0.318
0.111
45 7
18 3
18 1
0.596
0.310
0.342
Abbreviations: *P value between stomach and colon; **P value between stomach and small intestine; ***P value between colon and small intestine.
Fig. 1. Representative immunohistochemical analysis of PD-L1 and PD-1 expression in GI DLBCL (400×magnification). (A) PD-L1+ DLBCL; tumor cells were double positive for PD-L1 (red) and PAX5 (black); (B) mPD-L1+ DLBCL. Tumor cells were positive for PAX5 and negative for PD-L1; (C) low quantity of TILs; (D) high quantity of PD–1 + TILs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
509
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
significance (P > 0.05; Fig. 2A, B). In all cases, the quantity of PD-1+ TILs ranged from 0 to 300 (average of four high power fields of vision). Based on receiver operating characteristic curve (ROC) analysis(Fig. 3), we defined a prognostic cutoff value for PD–1 + TILs through Youden's indcx, and the cutoff value was 51.5. A higher quantity of PD-1+ TILs was associated with better prognosis (univariate log rank P = 0.0005; Fig. 2C). PD-1+ TILs were significantly lower in patients with involvement of extranodal sites (P = 0.011), and the quantity of PD–1 + TILs correlated positively with the level of PD-L1 expression in non malignant microenvironment cells (P = 0.001).
Table 2 Association of PD-1 and mPD-L1 expression with clinicopathological features of GI DLBCL. Parameters
PD-1+ TILs (Mean)
P
Gender Male 85.23 0.611 Female 77 Age (years) < 60 79.65 0.792 ≥60 84.08 TNM stage I–II 88.73 0.509 III–IV 77.22 B symptoms Yes 81.03 0.966 No 81.76 ECOG performance status <2 78.98 0.728 ≥2 84.73 Extranodal sites≥2 No 89.14 0.011 Yes 29 IPI risk group 0−1 77.95 0.972 2–5 77.35 Hans classification GCB 71.52 0.388 Non-GCB 86.62 mPD-L1 Negative 62.1 0.001 Positive 114.03 PD-L1 Negative 85.98 0.083 Positive 46.82
PD-L1+ (n = 11)
PD-L1- (n = 81)
P*
P**
mPDL1+ (n = 34)
mPD-L1(n = 47)
6 5
17 17
25 22
1.000
0.824
6 5
22 12
30 17
0.379
1.000
5 2
19 13
20 16
0.384
0.809
4 7
13 21
16 31
0.608
0.815
6 5
16 18
33 14
0.474
0.041
7 4
31 3
42 5
0.034
0.549
5 2
14 15
18 17
0.251
1.000
4 7
11 23
16 31
0.544
1.000
4. Discussion In this study, we demonstrate that PD-L1 is expressed variably by tumor cells and by tumor-infiltrating lymphocytes (TILs) in GI DLBCL. We found that the prevalence of microenvironmental PD-L1 positivity (mPD-L1+) in GI DLBCL is higher than common DLBCL. We also report that elevated numbers of PD–1 + TILs significantly associates with improved prognosis. Additionally, the number of PD–1 + TILs was significantly related to PD-L1 positivity of non malignant microenvironment cells. We observed positive PD-L1 expression in tumor cells (PD-L1+ GI DLBCL) in 11.96% of GI DLBCL cases; this is consistent with earlier reports of commom DLBCL [12,14]. PD-L1 functions as an inhibitory receptor that transmits anti-apoptotic signals to cancer cells and prevents immune-mediated destruction of cancer cells and Fas-mediated killing [15]. The expression of PD-L1 on tumor cells can be mediated through various mechanisms. A recent study revealed that disruption of the PD-L1 3′-untranslated region (UTR) could serve as a genetic marker to identify cancers that actively evade anti-tumor immunity through overexpression of PD-L1 [16]. Additionally, activation of the EGFR, MAPK, and PI3K–Akt pathways, microRNAs, and elevated expression of STAT3 transcription factors can up-regulate the PD-L1 expression in various cancer types through transcriptional or post-transcriptional mechanisms [17,18]. Another notable finding of this study is that the high expression of PD-L1 was significantly associated with involvement of more than two extranodal sites. Although PD-L1 expression had no prognostic significance in GI DLBCL patients treated with R-CHOP, there is a trend that the PD-L1 positivity of GI DLBCL patients had a worse prognosis(P = 0.11), these may be due to the small sample size. In addition to tumor cells, cell surface expression of PD-L1 protein has been observed in non-cancer cells of the tumor microenvironment, including tumor-associated macrophages, dendritic cells, and stromal cells [19]. Expression of PD-L1 in non-malignant cells of DLBCL may also be regulated by other mechanisms. Inflammatory mediators can induce expression of PD-L1 on cancer cells as well as on tumor-associated macrophages, dendritic cells, and stromal cells. For example, interferon γ is a major cytokine released by T cells after antigen recognition and activation, and may be a universal inducer of PD-L1. Our study showed that the frequency of non malignant microenvironment cells expressing PD-L1 in GI DLBCL (mPD-L1+) was 41.98%, this is significantly higher than mPD-L1+ observed in common DLBCL (15.5%) [12]. We observed a significant association of mPD-L1 positivity with ECOG performance status (score ≥ 2).
Abbreviations: P, T-test to evaluate the relationship between PD–1 + TILs and clinicopathological features; P*: PD-L1+ vs. PD-L1-; P**: mPD-L1+ vs. mPDL1-.
Pylori. 3.3. Expression of PD-L1 and PD-1 in GI DLBCL Representative IHC images for PD-L1 and PD-1 are shown in Fig. 1, and the results are summarized in Table 2. Cases were divided into PDL1+ GI DLBCL and mPD-L1+ GI DLBCL based on double immunostaining for PD-L1 and PAX-5. The prevalence of PD-L1+ GI DLBCL was 11.96% (11 of 92); subtype-specific PD-L1 prevalence rates for patients with gastric DLBCL, colonic DLBCL, and small intestinal DLBCL were 7.61%, 3.26%, and 1.09%, respectively. The prevalence of mPD-L1+ GI DLBCL was 41.98% (34 of 81); subtype-specific mPD-L1 prevalence rates for patients with gastric DLBCL, colonic DLBCL, and small intestinal DLBCL were 19.75%, 13.58%, and 8.64%, respectively. PD-L1 expression was significantly associated with involvement of extranodal sites ≥ 2 (P = 0.034) and mPD-L1 expression was significantly associated with ECOG performance status (score ≥ 2; P = 0.041); however, PD-L1 expression and mPD-L1 expression had no prognostic
Fig. 2. Kaplan-Meier survival analyses with respect to the expression of (A) PD-L1, (B) mPD-L1, (C) and the quantity of PD–1 + TILs. 510
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
Fig. 3. Determine the prognostic cutoff value for PD–1 + TILs through receiver operating characteristic curve (ROC) analysis.
indicate that further studies are necessary to understand the function of PD-L1 in mPD-L1+ DLBCL. We demonstrate that increased infiltration by high PD-1+ TILs is associated with improved prognosis in GI DLBCL patients treated with R-CHOP. The number of PD-1+ TILs was significantly lower in patients with extranodal sites and the quantity of PD–1 + TILs correlated positively with the level of PD-L1 expression in non malignant microenvironment cells. These results indicate that the presence of a high number of PD-1+ TILs is a favorable prognostic factor in patients with GI DLBCL. Furthermore, these data suggest that PD-1 plays a vital role in the microenvironment of GI DLBCL, and may participate in the modulation of tumor cell behavior and influence clinical evolution of GI DLBCL. Interestingly, previous reports have shown that high numbers of PD-1–positive cells in renal cancer associates with more advanced disease and shorter survival [21]; in this context, the number of PD-1+ TILs positively correlated with tumor-specific PD-L1 expression and was a poor prognostic factor [22]. In contrast to these observations in solid tumors, the presence of a high number of PD-1+ TILs is a favorable prognostic factor in patients with DLBCL and follicular lymphoma, whereas a low number of PD-1+ TILs is associated with a higher risk of histologic transformation [23,24]; these observations areconsistent
In our study, all GI DLBCL cases showed diffuse infiltration, often in a sheet-like pattern, and replacement of the GI mucosa and invasion of the submucosal and muscular layers by large atypical lymphoid cells. A previous study reported that H. pylori infection can inhibit T cell responses and enhance immune evasion by up-regulating PD-L1 expression; our data, however, revealed no significant association between H. pylori infection and mPD-L1 positivity in gastric DLBCL(P > 0.05), perhaps due to the small number of patients in the gastric DLBCL subgroup. Nevertheless, H. pylori infection may be one of the reasons for the high prevalence of PD-L1 expression in non malignant microenvironment cells in patients with GI DLBCL. We observed a relatively high incidence of mPD-L1 positivity in T-cell/histiocyte-rich large B-cell lymphoma (55.6%) and EBV-associated DLBCL (22%); these subtypes are pathologically characterized by infiltration of numerous inflammatory cells around tumor cells, suggesting the presence of a cytokine-rich tumor microenvironment. Inflammatory cytokines may be responsible for up-regulation of PD-L1 expression on tumor infiltrating cells [12]. A previous study showed that the patients with high PD-L1 expression in TILs (mPD-L1+) exhibited better therapeutic responses and improved clinical outcomes [20]; these observations suggest that evaluating PD-L1 in immune cells may have therapeutic relevance, and 511
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
with our results. Prevailing research indicates that the role of immuneinhibitory pathways mediated by PD-1-positive lymphocytes have different pathogenetic effects in B-cell neoplasms compared to solid tumors. The favorable clinical impact of high numbers of PD-1 positive lymphocytes in follicular lymphoma may be due in part to the inhibitory effect of Treg cells on PD-1 positive T cells, because CD4 + CD25 + Treg cells directly inhibit B-cell activation in vitro and humoral immune response in vivo, which can imapact the growth of malignant B cell lymphoma cells [25,26]. It is hypothesized that the Treg cells can have an analogous inhibitory effect on PD-1 positive T cells in GI DLBCL, but additional research is needed to better understand this mechanism. In conclusion, our study demonstrates that PD-L1 is expressed variably by both tumor cells and TILs in GI DLBCL. We report that the prevalence of mPD-L1 positivity is higher in GI DLBCL than in common DLBCL. Furthermore, we show that increased infiltration by PD-1+ TILs is associated with improved prognosis in patients with DLBCL, and the quantity of PD–1 + TILs correlated positively with the level of PDL1 expression in non malignant microenvironment cells. In recent years, the introduction of drugs targeting the PD-1/PD-L1 pathway has shown promising results in the treatment of aggressive m alignancies such as melanoma, renal cancer, and lung cancer [27], and the recent clinical trials showed that PD-1 blockade with nivolumab demonstrated activity in patients with relapsed/refractory primary central nervous system lymphoma and primary testicular lymphoma [28]. Our results suggest that PD-1/PD-L1 pathwaytargeted immunotherapies may be a viable therapeutic approach in the treatment of GI DLBCL.
[4] J.M. Howell, et al., Increasing incidence rates: distribution and histological characteristics of primary gastrointestinal non-Hodgkin lymphoma in a North American population, Can. J. Gastroenterol. 26 (7) (2012) 452–456. [5] V.A. Boussiotis, Molecular and biochemical aspects of the PD-1 checkpoint pathway, N. Engl. J. Med. 375 (18) (2016) 1767–1778. [6] J. Hamanishi, et al., Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer, Proc. Natl. Acad. Sci. U. S. A. 104 (9) (2007) 3360–3365. [7] R. Hino, et al., Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma, Cancer 116 (7) (2010) 1757–1766. [8] C.Y. Mu, et al., High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation, Med. Oncol. 28 (3) (2011) 682–688. [9] D. Rossille, et al., High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial, Leukemia 28 (12) (2014) 2367–2375. [10] A.M. Lesokhin, et al., Nivolumab in patients with relapsed or refractory hematologic malignancy: preliminary results of a phase ib study, J. Clin. Oncol. 34 (23) (2016) 2698–2704. [11] C.P. Hans, et al., Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray, Blood 103 (1) (2004) 275–282. [12] J. Kiyasu, et al., Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma, Blood 126 (19) (2015) 2193–2201. [13] A. Ruskoné-Fourmestraux, et al., Paris staging system for primary gastrointestinal lymphomas, Gut 52 (6) (2003) 912–916. [14] D. Kwon, et al., Clinicopathological analysis of programmed cell death 1 and programmed cell death ligand 1 expression in the tumour microenvironments of diffuse large B cell lymphomas, Histopathology 68 (7) (2016) 1079–1089. [15] T. Azuma, et al., B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells, Blood 111 (7) (2008) 3635–3643. [16] K. Kataoka, et al., Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers, Nature 534 (7607) (2016) 402–406. [17] E.A. Akbay, et al., Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors, Cancer Discov. 3 (12) (2013) 1355–1363. [18] M. Marzec, et al., Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1: B7-H1), Proc. Natl. Acad. Sci. U. S. A. 105 (52) (2008) 20852–20857. [19] W. Zou, L. Chen, Inhibitory B7-family molecules in the tumour microenvironment, Nat. Rev. Immunol. 8 (6) (2008) 467–477. [20] M.F. Sanmamed, L. Chen, Inducible expression of B7-H1 (PD-L1) and its selective role in tumor site immune modulation, Cancer J. 20 (4) (2014) 256–261. [21] R.H. Thompson, et al., PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma, Clin. Cancer Res. 13 (6) (2007) 1757–1761. [22] J.R. Kim, et al., Tumor infiltrating PD1-positive lymphocytes and the expression of PD-L1 predict poor prognosis of soft tissue sarcomas, PLoS One 8 (12) (2013) e82870. [23] J. Carreras, et al., High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma, J. Clin. Oncol. 27 (9) (2009) 1470–1476. [24] B.E. Wahlin, et al., A unifying microenvironment model in follicular lymphoma: outcome is predicted by programmed death-1–positive, regulatory, cytotoxic, and helper T cells and macrophages, Clin. Cancer Res. 16 (2) (2010) 637–650. [25] J. Carreras, et al., High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma, J. Clin. Oncol. 27 (9) (2009) 1470–1476. [26] Z.Z. Yang, et al., Intratumoral CD4 + CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma, Blood 107 (9) (2006) 3639–3646. [27] J.R. Brahmer, et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer, N. Engl. J. Med. 366 (26) (2012) 2455–2465. [28] L. Nayak, et al., PD-1 blockade with nivolumab in relapsed/refractory primary central nervous system and testicular lymphoma, Blood 129 (23) (2017) 3071–3073.
Conflict of interest statement No conflict of interest. Ethics The study was conducted in accordance with institutional ethical regulations, and has been approved by Xi Jing and Tang Du Hospital IRB. Acknowledgements Supported by the National Natural Science Fundation of China, No 81472597. Foundation of State Key Laboratory of Tumor Biology, Xi Jing Hospital, the Fourth Military Medical University. References [1] S.H. Swerdlow, et al., WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, fourth edition, IARC Press, Lyon, France, 2008, pp. 233–237. [2] H. S, W. R, C. W, Diffuse Large B-cell Lymphoma, Not Otherwise Specified, IARC Press, Lyon, France, 2008, pp. 233–237. [3] J.W. Friedberg, Relapsed/refractory diffuse large B-cell lymphoma, Hematol. Am. Soc. Hematol. Educ. Program (2011) 498–505 2011.
512
Contents lists available at ScienceDirect
Pathology - Research and Practice journal homepage: www.elsevier.com/locate/prp
Expression of programmed cell death 1/programmed cell death ligand 1 in the tumor microenvironments of primary gastrointestinal diffuse large B cell lymphomas
T
⁎
Yang Liua, Jing Maa, Kangjie Yub, Mingyang Lia, Fang Liua, Qingguo Yana, Zhe Wanga, , Shuangping Guoa a
State Key Laboratory of Tumor Biology, Department of Pathology, The Basic Medicine Science and Xi Jing Hospital, The Fourth Military Medical University, Xi’an, Shaan Xi Province, 710032, China Student Team 1, Class 3, The Fourth Military Medical University, Xi’an, Shaan Xi Province, 710032, China
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Programmed cell death 1 Programmed cell death ligand 1 Expression Gastrointestinal diffuse large B cell lymphoma
Background: Gastrointestinal diffuse large B cell lymphoma (GI DLBCL) is the most common gastrointestinal lymphoma. However, there has not been a comprehensive investigation into the expression patterns of programmed cell death 1 (PD-1) and programmed cell death ligand 1(PD-L1) in GI DLBCL tissues. Methods: PD-1 protein expression in tumor-infiltrating lymphocytes (TILs) was evaluated by immunohistochemical staining, and expression of PD-L1 was evaluated by using PD-L1/PAX5 immunohistochemical double staining in 92 GI DLBCL specimens. Results: The prevalence of positive PD-L1 expression (PD-L1 + ) in GI DLBCL cells and positive PD-L1 expression in non-cancer cells of the GI DLBCL microenvironment (microenvironmental PD-L1, mPD-L1) were 11.96% (11 of 92) and 41.98% (34 of 81), respectively. PD-L1 expression in GI DLBCL was significantly associated with involvement of extranodal sites ≥ 2 (P = 0.034) and mPD-L1 expression was significantly associated with ECOG performance status (score ≥ 2) (P = 0.041). PD-L1 expression and mPD-L1 expression had no prognostic significance (P > 0.05) on disease outcome. PD-1+ TILs were significantly lower in patients with extranodal site involvement (P = 0.011) and the quantity of PD–1 + TILs correlated positively with the level of PDL1 expression in non malignant microenvironment cells (P = 0.001). Patients with high levels of PD-1+ TILs had better prognosis (P = 0.0005). Conclusions: The expression patterns of PD-L1 in patients with GI DLBCL are different from patients with common DLBCL. Immunotherapies that target the PD-1/PD-L1 pathway may have therapeutic potential in GI DLBCL.
1. Introduction Diffuse large B cell lymphoma (DLBCL), one of the most common and aggressive forms of B-cell lymphomas, accounts for 40% of B-cell malignancies, and is highly heterogeneous [1]. DLBCL has been subdivided into several distinct disease entities based on morphological, biological, and clinical characteristics, although a large number of cases remain heterogeneous [2]. Despite the success of rituximab and doxorubicin treatment in improving the survival of patients with DLBCL, approximately one-third of patients will relapse or develop refractory disease [3]. It is therefore necessary to develop novel therapeutic strategies to treat DLBCL.
Gastrointestinal DLBCL (GI DLBCL) is the most common GI lymphoma [4]. The GI tract is a complex system with an immunologically specific environment where antigen stimuli can result in rapid and severe pathologies, such as occurs in Helicobacter pylori infection; on this basis, we believe that primary GI DLBCL differs from DLBCL of lymph node origin. Programmed cell death 1 (PD-1), a member of the B7 receptor family, is expressed on B cells, monocytes, activated natural killer T cells, and immature Langerhans’ cells. The functional and biochemical properties of PD-1 have been best studied in T cells and up-regulation of PD-1 is a natural consequence of T-cell activation, and is necessary for termination of the immune response [5]. The ligand for PD-1,
⁎
Corresponding authors at: Department of Pathology, Fourth Military Medical University, Chang le west Rd, Xi’an, Shaan Xi Province,710032, China. E-mail addresses: [email protected] (Y. Liu), [email protected] (J. Ma), [email protected] (K. Yu), [email protected] (M. Li), [email protected] (F. Liu), [email protected] (Q. Yan), [email protected] (Z. Wang), [email protected] (S. Guo). https://doi.org/10.1016/j.prp.2018.03.001 Received 17 December 2017; Received in revised form 5 February 2018; Accepted 2 March 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
for horseradish peroxidase. Tissue sections were counterstained with Harris’ hematoxylin and then mounted. For negative controls, phosphate-buffered saline was used instead of primary antibody. PD-L1+ DLBCL was defined as 30% or more of the lymphoma cells showing distinct membranous and/or cytoplasmic PD-L1 staining and PAX5 nuclear staining, regardless of the PD-L1 positivity of non malignant microenvironment cells. Among PD-L1-negative DLBCL cases, in which PD-L1+ non malignant microenvironment cells represented 20% or more of the total tissue, cellularity was defined as microenvironmental PD-L1 (mPD-L1+) DLBCL [12].
programmed cell death ligand 1 (PD-L1), is expressed on T-cells, B-cells, antigen presenting cells (APCs), monocytes, epithelial cells, and tumor cells. The PD-1/PD-L1 pathway is an inhibitory immune checkpoint that can suppress T cell immune activity, and plays a vital role in the maintenance of peripheral tolerance by protecting bystander tissues from immune-mediated damage. On the other hand, the PD-1/PD-L1 pathway inhibits the immune activity of tumor specific CD8+ T cells, allowing tumor cells to escape T-cell–mediated tumor-specific and pathogen-specific immunity, thereby promoting tumor development. Increased expression of PD-L1 has been associated with poor prognosis in several cancers, including melanoma, lung cancer, ovarian cancer, and DLBCL [6–9]. Blockade of the PD-1/PD-L1 pathway demonstrated beneficial therapeutic effects in extensively pretreated patients with relapsed or refractory DLBCL [10]. However, no studies to date have comprehensively investigated the expression patterns of PD-1 and PDL1 in GI DLBCL tissues. In this study we evaluated the expression of PD1/PD-L1 in primary gastrointestinal diffuse large B cell lymphoma, and determined the association of PD-1/PD-L1 expression with clinicopathological features in a cohort of 92 primary GI DLBCL cases.
2.4. Statistical analyses
2. Materials and methods
Statistical analysis was performed using IBM-SPSS Statistics version 20 (IBM). The association between PD-L1 expression and clinicopathological features of GI DLBCL was investigated using Pearson’s χ2 test. PD-1–positive cells were analyzed as a continuous variable. An independent T-test was used to evaluate the relationship between PD–1 + TILs and clinicopathological features. Survival analysis was performed using Kaplan–Meier analysis (the event was death). All Pvalues were two-sided; P < 0.05 was considered statistically significant.
2.1. Patient samples
3. Results
All patients enrolled in this study were diagnosed with GI DLBCL at Xi Jing Hospital and Tang Du Hospital (Xi’an, China) between January 2007 and February 2017. Pathologists (S.P.G., Z.W., W.Z) re-reviewed cases and identified 92 patients presenting with GI system lesions with or without extra-GI involvement (Stomach: 52; Colon: 21; Small intestine: 19). All patients were diagnosed according to the 2008 WHO classification of tumors of hematopoietic and lymphoid tissues [1]. Clinicopathological information, including age, location, and treatment, was obtained for this patient cohort. Therapeutic information was obtained for 92 cases; eighty-three patients (90.2%) underwent surgery and received chemotherapy; one patient (1.1%) received radiation after surgery and chemotherapy. Two patients (2.2%) underwent surgery only; six patients (6.5%) received chemotherapy only. The hospital-based ethics committees approved the study and informed consent was obtained from patients.
3.1. Patient characteristics The clinicopathological features of patients with GI DLBCL in the study cohort are summarized in Table 1. Patients commonly presented with GI tract symptoms such as weight loss, fatigue, dyspepsia, abdominal distension, stomachache, intestinal obstruction, and GI bleeding. Patient age ranged from 15 to 87 years; the median age was 54 years. Thirty-three cases had B symptoms. International Prognostic Index (IPI) scores were determined for 81 cases: the IPI score was 0–1 in 37 cases and 2–5 in 44 cases. Within the cohort, 44/75 cases (58.67%) had stage I/II disease and 31/75 cases (41.33%) had stage III/IV disease, according to the tumor-node-metastasis (TNM) system [13]; 17 cases of endoscopy biopsy tissues were not staged. Colonic DLBCL more frequently showed advanced TNM stage (III–IV; P = 0.025) and higher IPI score (score = 2–5; P = 0.005) than gastric DLBCL. Small intestine DLBCL more frequently showed involvement of extranodal sites ≥ 2 (P = 0.049) and higher ECOG performance status (score ≥ 2; P = 0.005) compared with gastric DLBCL. Regarding sex, age distribution, B symptoms, Hans classification, mPD-L1 expression, and PDL1 expression there were no differences between gastric, intestinal, and colonic DLBCL. Follow-up data were available for 66 patients, with a follow-up period ranging from 1 to 78 months (median: 58 months), the number of deaths in the overall gastrointestinal DLBCL, gastric DLBCL, colic DLBCL and small intestina DLBCL was 21 (31.8%;21/66),12 (30.8%;12/39), 7 (43.8%;7/16), 2 (18.2%;2/11), respectively. Univariate survival analysis revealed that poor overall survival was more associated with higher TNM stage (P < 0.001), involvement of extranodal sites ≥ 2 (P < 0.001), non-GCB DLBCL (P = 0.010), and higher IPI risk group classification (P < 0.001).
2.2. Immunohistochemical staining Tumor tissues were fixed in 10% buffered formalin and embedded in paraffin (FFPE), and stained with hematoxylin and eosin (H&E) or immunohistochemical (IHC) stains. IHC staining was performed using the Bond-Max automated immunostainer (Vision BioSystems, Leica, Victoria, Australia), with a polymer-based detection system. The primary antibodies used were against CD3 (Clone SP7), CD10 (Clone 56C6, Ventana, Tucson, AZ, USA), H. pylori (Clone RAB-0064; Fuzhou Maixin Biotech, Fuzhou, China), BCL6 (Clone PG-B6P), CD20 (Clone L26), and MUM1 (Clone Mum1P; Dako, Glostrup, Denmark). The cases were classified into two immunophenotypic subtypes: germinal center B cell–like (GCB) and non-GCB, according to the Hans classification [11]. PD-1 (UMAB199, OriGene) was immunostained in tumor-infiltrating lymphocytes (TILs) for all cases.
3.2. Histological features
2.3. Classification of DLBCL by using PD-L1/PAX5 double staining
All cases showed diffuse infiltration, often in a sheet-like pattern, and replacement of the GI mucosa and invasion of the submucosal and muscular layers by large atypical lymphoid cells. Tumor-adjacent tissues displayed histologic features of chronic atrophic gastritis in all 52 gastric DLBCL cases; 26 cases were severe, with heavy inflammatory cell infiltration and severe atrophy of the lamina propria mucous glands, and intestinal metaplasia; the remaining cases were mild to moderate. Of 52 gastric DLBCL cases, 20 (38.46%) were positive for H. pylori; no small intestinal or colonic DLBCL cases were positive for H.
The expression of PAX5 (Clone EP156; ZSGB-BIO) and PD-L1 (Clone sp142; Roche) was performed by double IHC staining using DouMaxVision immunohistochemical double dye kits (KIT-9998; Maixin Biotech), according to the manufacturer’s instructions. For this double IHC staining, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium was used as the blue-black chromogen for alkaline phosphatase and 3-amino-9-ethylcarbazole was used as the red chromogen 508
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
Table 1 Clinicopathological features of the GI DLBCL cohort. Total
Gender Male 48 Female 44 Age (years) < 60 58 ≥60 34 TNM stage I–II 44 III–IV 31 NA 17 Extranodal sites <2 80 ≥2 12 B symptoms No 59 Yes 33 ECOG performance status <2 55 ≥2 37 IPI risk group 0–1 37 2–5 34 NA 21 Hans classification GCB 31 Non-GCB 61 mPD-L1 Negative 47 Positive 34 PD-L1 Negative 81 Positive 11
OS, P
Pathogenic site
P*
P**
P***
Stomach
Colon
Small intestine
0.904
24 28
11 10
13 6
0.796
0.114
0.349
0.268
31 21
15 6
12 7
0.427
1.000
0.738
< 0.001
31 12 9
8 12 1
5 7 7
0.025
0.055
0.607
< 0.001
43 9
18 3
19 0
0.527
0.049
0.135
0.087
33 19
13 8
13 6
1.000
0.784
0.748
0.124
37 15
12 9
6 13
0.280
0.005
0.125
< 0.001
27 12 13
5 13 3
5 9 4
0.005
0.054
0.712
0.010
17 35
5 16
9 10
0.577
0.278
0.186
0.186
29 16
7 11
11 7
0.092
1.000
0.318
0.111
45 7
18 3
18 1
0.596
0.310
0.342
Abbreviations: *P value between stomach and colon; **P value between stomach and small intestine; ***P value between colon and small intestine.
Fig. 1. Representative immunohistochemical analysis of PD-L1 and PD-1 expression in GI DLBCL (400×magnification). (A) PD-L1+ DLBCL; tumor cells were double positive for PD-L1 (red) and PAX5 (black); (B) mPD-L1+ DLBCL. Tumor cells were positive for PAX5 and negative for PD-L1; (C) low quantity of TILs; (D) high quantity of PD–1 + TILs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
509
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
significance (P > 0.05; Fig. 2A, B). In all cases, the quantity of PD-1+ TILs ranged from 0 to 300 (average of four high power fields of vision). Based on receiver operating characteristic curve (ROC) analysis(Fig. 3), we defined a prognostic cutoff value for PD–1 + TILs through Youden's indcx, and the cutoff value was 51.5. A higher quantity of PD-1+ TILs was associated with better prognosis (univariate log rank P = 0.0005; Fig. 2C). PD-1+ TILs were significantly lower in patients with involvement of extranodal sites (P = 0.011), and the quantity of PD–1 + TILs correlated positively with the level of PD-L1 expression in non malignant microenvironment cells (P = 0.001).
Table 2 Association of PD-1 and mPD-L1 expression with clinicopathological features of GI DLBCL. Parameters
PD-1+ TILs (Mean)
P
Gender Male 85.23 0.611 Female 77 Age (years) < 60 79.65 0.792 ≥60 84.08 TNM stage I–II 88.73 0.509 III–IV 77.22 B symptoms Yes 81.03 0.966 No 81.76 ECOG performance status <2 78.98 0.728 ≥2 84.73 Extranodal sites≥2 No 89.14 0.011 Yes 29 IPI risk group 0−1 77.95 0.972 2–5 77.35 Hans classification GCB 71.52 0.388 Non-GCB 86.62 mPD-L1 Negative 62.1 0.001 Positive 114.03 PD-L1 Negative 85.98 0.083 Positive 46.82
PD-L1+ (n = 11)
PD-L1- (n = 81)
P*
P**
mPDL1+ (n = 34)
mPD-L1(n = 47)
6 5
17 17
25 22
1.000
0.824
6 5
22 12
30 17
0.379
1.000
5 2
19 13
20 16
0.384
0.809
4 7
13 21
16 31
0.608
0.815
6 5
16 18
33 14
0.474
0.041
7 4
31 3
42 5
0.034
0.549
5 2
14 15
18 17
0.251
1.000
4 7
11 23
16 31
0.544
1.000
4. Discussion In this study, we demonstrate that PD-L1 is expressed variably by tumor cells and by tumor-infiltrating lymphocytes (TILs) in GI DLBCL. We found that the prevalence of microenvironmental PD-L1 positivity (mPD-L1+) in GI DLBCL is higher than common DLBCL. We also report that elevated numbers of PD–1 + TILs significantly associates with improved prognosis. Additionally, the number of PD–1 + TILs was significantly related to PD-L1 positivity of non malignant microenvironment cells. We observed positive PD-L1 expression in tumor cells (PD-L1+ GI DLBCL) in 11.96% of GI DLBCL cases; this is consistent with earlier reports of commom DLBCL [12,14]. PD-L1 functions as an inhibitory receptor that transmits anti-apoptotic signals to cancer cells and prevents immune-mediated destruction of cancer cells and Fas-mediated killing [15]. The expression of PD-L1 on tumor cells can be mediated through various mechanisms. A recent study revealed that disruption of the PD-L1 3′-untranslated region (UTR) could serve as a genetic marker to identify cancers that actively evade anti-tumor immunity through overexpression of PD-L1 [16]. Additionally, activation of the EGFR, MAPK, and PI3K–Akt pathways, microRNAs, and elevated expression of STAT3 transcription factors can up-regulate the PD-L1 expression in various cancer types through transcriptional or post-transcriptional mechanisms [17,18]. Another notable finding of this study is that the high expression of PD-L1 was significantly associated with involvement of more than two extranodal sites. Although PD-L1 expression had no prognostic significance in GI DLBCL patients treated with R-CHOP, there is a trend that the PD-L1 positivity of GI DLBCL patients had a worse prognosis(P = 0.11), these may be due to the small sample size. In addition to tumor cells, cell surface expression of PD-L1 protein has been observed in non-cancer cells of the tumor microenvironment, including tumor-associated macrophages, dendritic cells, and stromal cells [19]. Expression of PD-L1 in non-malignant cells of DLBCL may also be regulated by other mechanisms. Inflammatory mediators can induce expression of PD-L1 on cancer cells as well as on tumor-associated macrophages, dendritic cells, and stromal cells. For example, interferon γ is a major cytokine released by T cells after antigen recognition and activation, and may be a universal inducer of PD-L1. Our study showed that the frequency of non malignant microenvironment cells expressing PD-L1 in GI DLBCL (mPD-L1+) was 41.98%, this is significantly higher than mPD-L1+ observed in common DLBCL (15.5%) [12]. We observed a significant association of mPD-L1 positivity with ECOG performance status (score ≥ 2).
Abbreviations: P, T-test to evaluate the relationship between PD–1 + TILs and clinicopathological features; P*: PD-L1+ vs. PD-L1-; P**: mPD-L1+ vs. mPDL1-.
Pylori. 3.3. Expression of PD-L1 and PD-1 in GI DLBCL Representative IHC images for PD-L1 and PD-1 are shown in Fig. 1, and the results are summarized in Table 2. Cases were divided into PDL1+ GI DLBCL and mPD-L1+ GI DLBCL based on double immunostaining for PD-L1 and PAX-5. The prevalence of PD-L1+ GI DLBCL was 11.96% (11 of 92); subtype-specific PD-L1 prevalence rates for patients with gastric DLBCL, colonic DLBCL, and small intestinal DLBCL were 7.61%, 3.26%, and 1.09%, respectively. The prevalence of mPD-L1+ GI DLBCL was 41.98% (34 of 81); subtype-specific mPD-L1 prevalence rates for patients with gastric DLBCL, colonic DLBCL, and small intestinal DLBCL were 19.75%, 13.58%, and 8.64%, respectively. PD-L1 expression was significantly associated with involvement of extranodal sites ≥ 2 (P = 0.034) and mPD-L1 expression was significantly associated with ECOG performance status (score ≥ 2; P = 0.041); however, PD-L1 expression and mPD-L1 expression had no prognostic
Fig. 2. Kaplan-Meier survival analyses with respect to the expression of (A) PD-L1, (B) mPD-L1, (C) and the quantity of PD–1 + TILs. 510
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
Fig. 3. Determine the prognostic cutoff value for PD–1 + TILs through receiver operating characteristic curve (ROC) analysis.
indicate that further studies are necessary to understand the function of PD-L1 in mPD-L1+ DLBCL. We demonstrate that increased infiltration by high PD-1+ TILs is associated with improved prognosis in GI DLBCL patients treated with R-CHOP. The number of PD-1+ TILs was significantly lower in patients with extranodal sites and the quantity of PD–1 + TILs correlated positively with the level of PD-L1 expression in non malignant microenvironment cells. These results indicate that the presence of a high number of PD-1+ TILs is a favorable prognostic factor in patients with GI DLBCL. Furthermore, these data suggest that PD-1 plays a vital role in the microenvironment of GI DLBCL, and may participate in the modulation of tumor cell behavior and influence clinical evolution of GI DLBCL. Interestingly, previous reports have shown that high numbers of PD-1–positive cells in renal cancer associates with more advanced disease and shorter survival [21]; in this context, the number of PD-1+ TILs positively correlated with tumor-specific PD-L1 expression and was a poor prognostic factor [22]. In contrast to these observations in solid tumors, the presence of a high number of PD-1+ TILs is a favorable prognostic factor in patients with DLBCL and follicular lymphoma, whereas a low number of PD-1+ TILs is associated with a higher risk of histologic transformation [23,24]; these observations areconsistent
In our study, all GI DLBCL cases showed diffuse infiltration, often in a sheet-like pattern, and replacement of the GI mucosa and invasion of the submucosal and muscular layers by large atypical lymphoid cells. A previous study reported that H. pylori infection can inhibit T cell responses and enhance immune evasion by up-regulating PD-L1 expression; our data, however, revealed no significant association between H. pylori infection and mPD-L1 positivity in gastric DLBCL(P > 0.05), perhaps due to the small number of patients in the gastric DLBCL subgroup. Nevertheless, H. pylori infection may be one of the reasons for the high prevalence of PD-L1 expression in non malignant microenvironment cells in patients with GI DLBCL. We observed a relatively high incidence of mPD-L1 positivity in T-cell/histiocyte-rich large B-cell lymphoma (55.6%) and EBV-associated DLBCL (22%); these subtypes are pathologically characterized by infiltration of numerous inflammatory cells around tumor cells, suggesting the presence of a cytokine-rich tumor microenvironment. Inflammatory cytokines may be responsible for up-regulation of PD-L1 expression on tumor infiltrating cells [12]. A previous study showed that the patients with high PD-L1 expression in TILs (mPD-L1+) exhibited better therapeutic responses and improved clinical outcomes [20]; these observations suggest that evaluating PD-L1 in immune cells may have therapeutic relevance, and 511
Pathology - Research and Practice 214 (2018) 507–512
Y. Liu et al.
with our results. Prevailing research indicates that the role of immuneinhibitory pathways mediated by PD-1-positive lymphocytes have different pathogenetic effects in B-cell neoplasms compared to solid tumors. The favorable clinical impact of high numbers of PD-1 positive lymphocytes in follicular lymphoma may be due in part to the inhibitory effect of Treg cells on PD-1 positive T cells, because CD4 + CD25 + Treg cells directly inhibit B-cell activation in vitro and humoral immune response in vivo, which can imapact the growth of malignant B cell lymphoma cells [25,26]. It is hypothesized that the Treg cells can have an analogous inhibitory effect on PD-1 positive T cells in GI DLBCL, but additional research is needed to better understand this mechanism. In conclusion, our study demonstrates that PD-L1 is expressed variably by both tumor cells and TILs in GI DLBCL. We report that the prevalence of mPD-L1 positivity is higher in GI DLBCL than in common DLBCL. Furthermore, we show that increased infiltration by PD-1+ TILs is associated with improved prognosis in patients with DLBCL, and the quantity of PD–1 + TILs correlated positively with the level of PDL1 expression in non malignant microenvironment cells. In recent years, the introduction of drugs targeting the PD-1/PD-L1 pathway has shown promising results in the treatment of aggressive m alignancies such as melanoma, renal cancer, and lung cancer [27], and the recent clinical trials showed that PD-1 blockade with nivolumab demonstrated activity in patients with relapsed/refractory primary central nervous system lymphoma and primary testicular lymphoma [28]. Our results suggest that PD-1/PD-L1 pathwaytargeted immunotherapies may be a viable therapeutic approach in the treatment of GI DLBCL.
[4] J.M. Howell, et al., Increasing incidence rates: distribution and histological characteristics of primary gastrointestinal non-Hodgkin lymphoma in a North American population, Can. J. Gastroenterol. 26 (7) (2012) 452–456. [5] V.A. Boussiotis, Molecular and biochemical aspects of the PD-1 checkpoint pathway, N. Engl. J. Med. 375 (18) (2016) 1767–1778. [6] J. Hamanishi, et al., Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer, Proc. Natl. Acad. Sci. U. S. A. 104 (9) (2007) 3360–3365. [7] R. Hino, et al., Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma, Cancer 116 (7) (2010) 1757–1766. [8] C.Y. Mu, et al., High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation, Med. Oncol. 28 (3) (2011) 682–688. [9] D. Rossille, et al., High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial, Leukemia 28 (12) (2014) 2367–2375. [10] A.M. Lesokhin, et al., Nivolumab in patients with relapsed or refractory hematologic malignancy: preliminary results of a phase ib study, J. Clin. Oncol. 34 (23) (2016) 2698–2704. [11] C.P. Hans, et al., Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray, Blood 103 (1) (2004) 275–282. [12] J. Kiyasu, et al., Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma, Blood 126 (19) (2015) 2193–2201. [13] A. Ruskoné-Fourmestraux, et al., Paris staging system for primary gastrointestinal lymphomas, Gut 52 (6) (2003) 912–916. [14] D. Kwon, et al., Clinicopathological analysis of programmed cell death 1 and programmed cell death ligand 1 expression in the tumour microenvironments of diffuse large B cell lymphomas, Histopathology 68 (7) (2016) 1079–1089. [15] T. Azuma, et al., B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells, Blood 111 (7) (2008) 3635–3643. [16] K. Kataoka, et al., Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers, Nature 534 (7607) (2016) 402–406. [17] E.A. Akbay, et al., Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors, Cancer Discov. 3 (12) (2013) 1355–1363. [18] M. Marzec, et al., Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1: B7-H1), Proc. Natl. Acad. Sci. U. S. A. 105 (52) (2008) 20852–20857. [19] W. Zou, L. Chen, Inhibitory B7-family molecules in the tumour microenvironment, Nat. Rev. Immunol. 8 (6) (2008) 467–477. [20] M.F. Sanmamed, L. Chen, Inducible expression of B7-H1 (PD-L1) and its selective role in tumor site immune modulation, Cancer J. 20 (4) (2014) 256–261. [21] R.H. Thompson, et al., PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma, Clin. Cancer Res. 13 (6) (2007) 1757–1761. [22] J.R. Kim, et al., Tumor infiltrating PD1-positive lymphocytes and the expression of PD-L1 predict poor prognosis of soft tissue sarcomas, PLoS One 8 (12) (2013) e82870. [23] J. Carreras, et al., High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma, J. Clin. Oncol. 27 (9) (2009) 1470–1476. [24] B.E. Wahlin, et al., A unifying microenvironment model in follicular lymphoma: outcome is predicted by programmed death-1–positive, regulatory, cytotoxic, and helper T cells and macrophages, Clin. Cancer Res. 16 (2) (2010) 637–650. [25] J. Carreras, et al., High numbers of tumor-infiltrating programmed cell death 1positive regulatory lymphocytes are associated with improved overall survival in follicular lymphoma, J. Clin. Oncol. 27 (9) (2009) 1470–1476. [26] Z.Z. Yang, et al., Intratumoral CD4 + CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma, Blood 107 (9) (2006) 3639–3646. [27] J.R. Brahmer, et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer, N. Engl. J. Med. 366 (26) (2012) 2455–2465. [28] L. Nayak, et al., PD-1 blockade with nivolumab in relapsed/refractory primary central nervous system and testicular lymphoma, Blood 129 (23) (2017) 3071–3073.
Conflict of interest statement No conflict of interest. Ethics The study was conducted in accordance with institutional ethical regulations, and has been approved by Xi Jing and Tang Du Hospital IRB. Acknowledgements Supported by the National Natural Science Fundation of China, No 81472597. Foundation of State Key Laboratory of Tumor Biology, Xi Jing Hospital, the Fourth Military Medical University. References [1] S.H. Swerdlow, et al., WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, fourth edition, IARC Press, Lyon, France, 2008, pp. 233–237. [2] H. S, W. R, C. W, Diffuse Large B-cell Lymphoma, Not Otherwise Specified, IARC Press, Lyon, France, 2008, pp. 233–237. [3] J.W. Friedberg, Relapsed/refractory diffuse large B-cell lymphoma, Hematol. Am. Soc. Hematol. Educ. Program (2011) 498–505 2011.
512