Abstract
Memory T cells survive for many months and years and are critically important for host defence in humans. In tumour immunity, they have been also suggested to play a significant role in tumour progression and metastasis. However, the role of memory T cells in actual human cancer remains largely unknown. In this study, the clinical importance of tumour-infiltrating CD45RO+ memory T cells was investigated in human oesophageal squamous cell carcinoma (OSCC). CD45RO+ T cells were evaluated by immunohistochemistry in primary OSCC tumours from 105 patients. Patients were classified into two groups as CD45RO+hi or CD45RO+lo based on the number of cells stained positively for CD45RO. No significant difference was observed between CD45RO status and several clinicopathological prognostic factors. However, the postoperative overall and disease-free survival for CD45RO+hi patients was significantly better than for CD45RO+lo patients. Furthermore, there were significant correlations of CD45RO status in the primary tumour with postoperative lymph node and pulmonary recurrence, suggesting that memory T cells may control postoperative metastatic recurrence. Most importantly, CD45RO+ memory T cell status has a significant prognostic value for OSCC independently of conventional tumour–node–metastasis (TNM) classification. Our study may provide a rationale for developing a novel immunotherapy in intentional induction of memory T cells for the treatment of oesophageal cancer.
Keywords: CD45RO, memory cells, oesophagus, prognosis, surgery
Introduction
Human oesophageal cancer is one of the most intractable gastrointestinal tumours [1]–[3]. Because it is usually diagnosed at advanced stages, and rarely curable, overall survival remains poor. Surgery is standard treatment for localized and resectable oesophageal cancer [2]. However, patients often experience distant metastasis or local recurrence even after curative operation [3]. The overall 5-year postoperative survival rate in patients is generally less than 40% [2]. Therefore, some multi-disciplinary treatments to combine pre-operative chemotherapy and/or radiotherapy with surgery have been evaluated and are currently undergoing clinical evaluation [1]. Nevertheless, previous clinical trials have often failed to show survival benefit and efficacy of conventional anti-cancer treatments seems to be limited [4]. Thus, to improve patients' prognosis, the underlying mechanisms toward novel therapeutic strategies need to be clarified. During recent years, the biological behaviour of oesophageal cancer has been investigated extensively. As a result, several molecular mechanisms have been revealed, and thereby potential targets have been proposed [5]–[8]. They include genetic alteration and angiogenesis, as well as growth factors. Although tumour immunity may be also involved critically in tumour growth and metastasis in oesophageal cancer, relatively few studies have been reported [9]–[11].
CD45 is a leucocyte-common antigen and functions as a tyrosine phosphatase in leucocyte signalling. The expression of different CD45 isoforms is cell-type-specific and depends on the stage of differentiation and activation state of the cells. CD45RO is one of the most suitable single markers for human memory T cells that could accurately represent the activation status of T cells [12]. Memory T cells are generated during cell-mediated immunity responses, and survive for many months and years after the antigen is eliminated. These memory T cells are responsible for more rapid and amplified responses to second and subsequent exposures to antigens. It is well known that they play critically important roles in host defence for infection. Furthermore, they have been reported recently to be associated with prognosis in a few malignant tumours, including colorectal and gastric cancer [13]–[15]. Furthermore, CD45RO+ T cells have also been reported recently to have prognostic value in oesophageal adenocarcinoma [16]. Although these studies have suggested that memory T cells play an important role in human tumour immunity, the clinical importance of memory T cells in oesophageal squamous cell carcinoma (OSCC) has not been evaluated previously. Several previous studies have reported that tumour-infiltrating CD4+ or CD8+ T cells have a better impact on prognosis in OSCC [11],[17]–[19]. However, the significance of each T cell subset in OSCC is still controversial, as results are not always consistent between different studies.
The aim of this study was to investigate the clinical significance of CD45RO+ memory T cells infiltrating in human oesophageal squamous cell carcinoma.
Patients and methods
Patients
We examined 105 patients with oesophageal cancer who underwent surgery at Department of Surgery, Nara Medical University, between November 1995 and May 2007. The median age of the patients was 61·4 years (range 42–78 years). When distant lymph node metastasis was solitary and resectable, subtotal oesophagectomy was performed with the combined resection of distant lymph node metastasis. None of the patients received pre-operative treatment, such as radiation or chemotherapy. Postoperative histopathological analysis indicated that all tumours evaluated in this study were squamous cell carcinoma (SCC). All tumours were staged on the basis of the pathological tumour–node–metastasis (TNM) classification of the International Union Against Cancer [20]. Documented informed consent was obtained from individual patients for use of their tissue samples.
Immunohistochemistry
Immunohistochemical staining for CD45RO was performed with a Dako Envision™ kit (Dako Cytomation, Tokyo, Japan). Formalin-fixed, paraffin-embedded tissues were cut into 5-µm sections, deparaffinized and rehydrated in a graded series of ethanols. Antigen retrieval was performed by heating tissue sections using a target retrieval solution, pH 9·0 (Dako). Then, the samples were incubated for 5 min in peroxidase blocking solution (Dako) to inhibit endogenous peroxidase. The sections were then incubated overnight at 4°C with anti-human CD45RO (UHL1, monoclonal mouse; Dako). A subsequent reaction was carried out using second antibodies (Dako) at 37°C for 30 min. The sections were then washed three times with phosphate-buffered saline (PBS) and the colour was displayed subsequently with diaminobenzidine (DAB) (Dako) for approximately 5 min. Sections were counterstained with haematoxylin, dehydrated in ethanol, cleared in xylene and coverslipped. We selected five areas with the most abundant positively stained cells in each tissue under ×400 magnification. We then counted these cells and calculated the mean number of each sample.
Statistical analysis
Comparisons among the clinical and pathological features were evaluated using χ2 and Fisher's exact tests. Statistical significance between two groups of parametric date was evaluated using an unpaired Student's t-test. Survival curves were estimated using the Kaplan–Meier method, and the significant differences between survival curves were determined using the log-rank test. Multivariate comparisons of survival distributions were made using Cox's proportional hazards models. In all tests, statistical significance was set at 5%.
Results
Expression of CD45RO+ tumour-infiltrating T lymphocyte in oesophageal squamous cell carcinoma tissue
We analysed 105 patients retrospectively with OSCC without any pre-operative anti-cancer therapy. Using immunohistochemistry, CD45RO+ T cells infiltrating into OSCC tissue were evaluated (Fig. 1). CD45RO+ T cells within tumours or at the invasive margin were counted as described previously [13]. The mean number of CD45RO+ cells was 113. We defined this number as the cut-off value and classified all cases into high- or low-density groups for CD45RO, i.e. CD45RO+hi and CD45RO+lo.
Fig. 1.
Immunohistochemical staining of human oesophageal squamous cell carcinoma tissue for CDRO+ T cell. Original magnification (a) ×100; (b) ×400.
Correlation between CD45RO+ tumour-infiltrating T lymphocytes and clinicopathological characteristics
We first evaluated the association of CD45RO expression with various prognostic factors (Table 1). There was no significant correlation between CD45RO status and clinicopathological factors, including age at surgery, gender, tumour status, nodal status, metastatic status or pathological stage. Furthermore, CD45RO status also correlated with neither lymphatic nor venous invasion.
Table 1.
Relationship between clinicopathologic variables and CD45RO status in oesophageal squamous cell carcinoma.
| CD45RO expression | |||
|---|---|---|---|
| High n = 54 | Low n = 51 | P-value | |
| Age; mean ± s.d. | 61·59 ± 7·08 | 61·26 ± 8·00 | 0·819 |
| Gender male/female | 43/11 | 42/9 | 0·724 |
| TNM stage | 0·878 | ||
| I | 9 | 9 | |
| IIA | 10 | 10 | |
| IIB | 8 | 5 | |
| III | 19 | 20 | |
| IV | 8 | 7 | |
| Tumour status | 0·672 | ||
| T1 | 13 | 12 | |
| T2 | 12 | 7 | |
| T3 | 27 | 29 | |
| T4 | 2 | 3 | |
| Lymph node metastasis | 0·528 | ||
| Positive | 19 | 21 | |
| Negative | 35 | 30 | |
| Lymphatic invasion | 0·204 | ||
| Positive | 42 | 34 | |
| Negative | 12 | 17 | |
| Venous invasion | 0·674 | ||
| Positive | 18 | 19 | |
| Negative | 36 | 32 | |
TNM: tumour–node–metastasis; s.d.: standard deviation.
Association between CD45RO+ tumour-infiltrating T lymphocytes and postoperative prognosis
There was a significant correlation of CD45RO T cell status with postoperative overall survival and disease-free survival (P = 0·006 and P = 0·026, respectively, Fig. 2). The 1-, 3- and 5-year overall survival for CD45RO+hi patients was 86·8, 60·3, and 45·1%, respectively, and for CD45RO+lo patients was 62·7, 32·8, and 24·3%, respectively.
Fig. 2.
Postoperative survival in CD45RO+hi patients was significantly better than that in CD45RO+lo patients. (a) Overall survival (P = 0·006); (b) disease-free survival (P = 0·026).
Impact of CD45RO T cells on postoperative recurrence in oesophageal squamous cell carcinoma
At the time of analysis, 59 patients had postoperative recurrence. Recurrence was found in 26 lymph nodes, 22 in lung, 14 in liver and 10 in bone. We then evaluated the impact of CD45RO+ T cells infiltrating into the primary OSCC tissue on postoperative recurrence. Interestingly, there was a significant relationship between CD45RO status and recurrence (Table 2). Furthermore, CD45RO status in the primary tumour was found to have a significant impact on postoperative lymph node and pulmonary recurrence (Table 2). The difference in hepatic and bone recurrence did not reach statistical significance.
Table 2.
Impact of CD45RO status on postoperative recurrence.
| CD45RO | CD45RO | P-value | |
|---|---|---|---|
| High n = 54 | Low n = 51 | ||
| All recurrence | |||
| Presence | 25 | 34 | 0·036 |
| Absence | 29 | 17 | |
| Lymph node recurrence | |||
| Presence | 9 | 17 | 0·048 |
| Absence | 45 | 34 | |
| Pulmonary recurrence | |||
| Presence | 7 | 15 | 0·039 |
| Absence | 47 | 36 |
Prognostic value of CD45RO+ memory T cells in oesophageal squamous cell carcinoma
Finally, to determine the prognostic value of tumour-infiltrating CD45RO+ memory T cells in OSCC, we performed a multivariate analysis using a Cox regression model (Table 3). As a result, CD45RO status was defined to be a significant independent prognostic factor (P = 0·0045). Although tumour status and nodal status were also found to have significant prognostic value, metastatic status did not reach statistical significance.
Table 3.
Multivariate survival analysis in oesophageal squamous cell carcinoma.
| Variables | HR | 95% CI | P-value |
|---|---|---|---|
| CD45RO+ T cell status | 0·0045 | ||
| Hi | 1·000 | – | |
| Lo | 2·309 | 1·295–4·115 | |
| Primary tumour status | 0·0006 | ||
| T1 | 1·000 | 0·260–3·909 | |
| T2 | 1·403 | 0·351–5·558 | |
| T3 | 3·909 | 1·325–11·571 | |
| Lymph node metastasis | 0·0145 | ||
| N0 | 1·000 | – | |
| N1 | 2·551 | 1·203–5·405 | |
| Distant metastasis | 0·1560 | ||
| M0 | 1·000 | – | |
| M1 | 1·695 | 0·818–3·509 |
CI: confidence interval.
Discussion
Tumour cells have tumour-specific antigens that are capable of inducing tumour-specific cytotoxic T lymphocytes, thereby eliciting a proper host-immune response [21]–[23]. Previous studies have proved that tumour-infiltrating T lymphocytes, including CD8 and CD4 T cells, are considered to be a manifestation of the host-immune response in OSCC [11],[17]–[19]. However, the clinical significance of each T cell subset in OSCC is still controversial. Recently, the presence of CD45RO+ memory T cells in the tumour have been reported to be an independent prognostic factor in several human malignancies, including colorectal and gastric cancer [14],[15],[24]. CD45RO is the most suitable single marker for memory T cell population in humans. Therefore, these cells include both CD4+ and CD8+ lymphocytes that have been exposed to antigen. Rauser et al. have reported recently that CD45RO+ T cells were also an independent prognostic factor in oesophageal adenocarcinoma [16]. Although oesophageal adenocarcinoma (Barrett's cancer) has increased rapidly, especially in western countries, the major histological type of oesophageal cancer is still SCC in Asian countries, including Japan. In addition, there are significant differences between Barrett's cancer and OSCC. To the best of our knowledge, no studies have previously addressed the role of CD45RO+ T cells in OSCC. The most important finding in this study was that CD45RO+ memory T cell status correlated significantly with postoperative survival with OSCC. Despite different histological phenotypes, the data are similar between previous reports on adenocarcinoma and this study on SCC [16]. Thus, the clinical importance of memory T cells may be universal in oesophageal malignancy regardless of histopathological types.
In this study, there were no significant correlations between CD45RO+ status and various clinicopathological factors. Our results were consistent with a recent report on adenocarcinoma [16]. The data suggested that intratumour memory T cells might not influence tumour proliferation, invasiveness and metastasis directly in oesophageal cancer. In addition, there were significant correlations of CD45RO status in the primary tumour with postoperative lymph node and pulmonary recurrence. The data also suggest that memory T cells generated in the primary tumour may have the ability to control micrometastatic cancer cells in lymph nodes or distant organs in the postoperative period. However, there were no significant correlations of CD45RO status with hepatic and bone recurrence. This may be due possibly to the relatively small number of patients, because hepatic and bone recurrence was relatively rare compared to lymph node and pulmonary recurrence in this study. Taken together, memory T cells in primary tumours may affect the development of both haematogenous and lymphatic metastasis in OSCC. In order to improve the prognosis of patients with oesophageal cancer, it is essential to prevent postoperative recurrence. Therefore, our findings on the clinical importance of memory T cells may shed light on unknown mechanisms in oesophageal cancer.
It is unclear whether or not CD45RO+ T cells examined in this clinical study are tumour antigen-specific. However, similar to previous studies, we assume that CD45RO status has a strong prognostic value because of the specificity of such cells [13]–[16]. Further fundamental studies are clearly required to prove this supposition and reveal how tumour-specific memory T cells could be generated and maintained in humans. There may be complex mechanisms, including tumour antigen density, tumour environments, cytokine generation, cellular apoptosis and other regulatory functions on tumour-infiltrating lymphocytes (TILs). We have reported previously that the inhibitory interaction of programmed cell death ligand (PD-L) on tumours with TILs had a significant role in OSCC [9]. Such T cell-negative pathways, including PD-L/PD-1, B7/cytotoxic T lymphocyte antigen (CTLA)-4 and B7-homologue 3 (H3), may play roles in the control of memory T cells [25]–[27]. In addition, several immunoregulatory pathways, including Fas/FasL, have been reported to be involved in oesophageal cancer [28],[29]. Furthermore, angiogenesis and inhibitory cytokines including transforming growth factor (TGF)-β and interleukin (IL)-10 may also play critical roles in regulating memory T cells [30]. Conversely, memory T cells have been reported to be regulated by several factors, including IL-7 and IL-15 [31]. These mechanisms may not always be mutually exclusive. Further studies on memory T cells in correlation with the above potential mechanisms are warranted to develop novel therapeutic strategy.
Immunotherapy has long been expected to become a powerful anti-cancer treatment that can be tumour-specific and less toxic [32]. It includes cancer vaccine, adoptive cell therapy and monoclonal antibody-based treatment. Based on our data with other previous reports, it may be critical that any anti-cancer treatments should not prevent tumour-specific memory T cells in cancer patients [14]–[16],[24]. Furthermore, if intentional induction or maintenance of memory T cells becomes possible, such new strategy can bring about a breakthrough in cancer treatment, especially for intractable malignancies such as oesophageal cancer. However, to date, few studies have evaluated dynamic of memory T cells before and after any specific anti-cancer therapy. In animal models, a number of studies have shown the successful induction of TIL into tumours by blocking T cell inhibitory pathways [27],[33],[34]. More importantly, a recent large-scale randomized clinical trial demonstrated that immunotherapy using anti-human CTLA-4 monoclonal antibody improved overall survival in metastatic melanoma [35]. Taken together, one of candidates for the intentional induction therapy of memory T cells may be targeting T cell-negative pathways. Other treatments, including cytokine therapy and cancer vaccine, should also be evaluated from the viewpoint of memory T cells.
In conclusion, we have shown for the first time that CD45RO+ memory T cell status has a significant prognostic value for oesophageal squamous cell carcinoma independently of conventional TNM classification. Furthermore, our study has also suggested that memory T cells may play a critical role in regulating tumour metastasis and postoperative recurrence. Because metastatic recurrence is the most frequent cause of cancer-related death, our data may provide a rationale for developing a novel immunotherapy in the intentional induction of memory T cells in the treatment of oesophageal cancer.
Acknowledgments
This work was supported by the following grants: grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, no. 19591491, 21591648; research grant from Foundation for Promotion of Cancer Research in Japan; research grant from Daiwa Securities Health Foundation; research grant from the Japanese Society of Gastroenterology; and research grant from Nakayama Cancer Research Institute, Research Grant from Takeda Science Foundation (M. Sho).
Disclosure
None of the authors has any financial conflicts to disclose.
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