Indoleamine 2,3-dioxygenase expression predicts impaired survival of invasive cervical cancer patients treated with radical hysterectomy

Indoleamine 2,3-dioxygenase expression predicts impaired survival of invasive cervical cancer patients treated with radical hysterectomy

Gynecologic Oncology 117 (2010) 423–428 Contents lists available at ScienceDirect Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s ...

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Gynecologic Oncology 117 (2010) 423–428

Contents lists available at ScienceDirect

Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o

Indoleamine 2,3-dioxygenase expression predicts impaired survival of invasive cervical cancer patients treated with radical hysterectomy Tomoko Inaba a, Kazuhiko Ino a,⁎, Hiroaki Kajiyama a, Kiyosumi Shibata a, Eiko Yamamoto a, Shinji Kondo a, Tomokazu Umezu a, Akihiro Nawa a, Osamu Takikawa b, Fumitaka Kikkawa a a b

Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Japan

a r t i c l e

i n f o

Article history: Received 13 November 2009 Available online 28 March 2010 Keywords: Cervical cancer Immunohistochemistry Indoleamine 2,3-dioxygenase Prognostic factor Survival

a b s t r a c t Objective. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces tolerance to host immune surveillance within the tumor microenvironment. The present study aimed to investigate IDO expression and its prognostic significance in invasive cervical cancer. Methods. Immunohistochemical expression of IDO in tumor tissues and its association with clinicopathological factors and survival were analyzed in 112 stage IB–IIB cervical cancer patients treated with radical hysterectomy and pelvic lymphadenectomy. Results. IDO was diffusely expressed in tumor cells in 29 (26%) cases and focally expressed at the invasive front in 29 (26%) cases, while the other 54 (48%) cases were IDO-negative. IDO expression was positively correlated with clinical stage, lymph node metastasis, and lymph-vascular space invasion, but not with histological type. Patients with diffuse IDO expression had significantly reduced overall survival (OS) and disease-free survival (DFS) compared to patients with no IDO expression. The 5-year OS/DFS rates for the IDO-negative, focally positive, and diffusely positive groups were 92.3%/84.9%, 89.5%/75.8%, and 65.5%/ 51.7%, respectively. When we analyzed patients with stage IB disease alone (n = 67), the OS and DFS for the IDO-diffusely positive group were significantly lower than those for the IDO-negative group. In multivariate analysis, diffuse IDO expression was found to be an independent prognostic factor for impaired OS and DFS. Conclusions. Diffuse expression of IDO in the tumor obtained from Stage IB–IIB cervical cancer patients who underwent radical hysterectomy was correlated with an unfavorable clinical outcome. These findings suggest that IDO may be a novel post-operative prognostic indicator for stage IB–IIB cervical cancer. © 2010 Elsevier Inc. All rights reserved.

Introduction Cervical cancer is the second most frequent cancer in women worldwide [1] and is generally treated by surgery, radiotherapy, or both. While primary concurrent chemoradiation therapy without surgery has recently been selected not only for advanced disease, but also for early-stage locally advanced disease [2–4], most patients with FIGO stage IB through IIB disease are treated with radical hysterectomy followed by adjuvant therapy at many institutions [5–8]. Despite the generally good prognosis for stage IB–IIB cervical cancer, significant numbers of patients develop localized recurrence or distant metastases following surgery. Several clinicopathological parameters are currently used to assess the risk of relapse and death, including age, histological subtype, lymph node status, tumor size, depth of stromal invasion, parametrial invasion, and lymph⁎ Corresponding author. Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax: +81 52 744 2268. E-mail address: [email protected] (K. Ino). 0090-8258/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2010.02.028

vascular space invasion (LVSI) [5–8]. For patients belonging to highrisk groups, post-operative radiotherapy with or without chemotherapy has been performed. However, the selection of patients for adjuvant therapy and its effectiveness, especially its impact on survival, remain controversial [9]. Thus, in addition to the conventional clinicopathological parameters, the identification of molecular markers more closely related to the intrinsic biological behavior of cervical cancer and the individualization of adjuvant therapy based on more reliable prognostic indicators would be helpful for improving the survival of patients with this disease, as well as for preventing the unnecessary use of adjuvant therapy. The development of cervical cancer is a multistep process initiated by persistent infection with human papillomavirus (HPV) [10]. After high-risk HPV infection, cervical lesions progress via cervical intraepithelial neoplasia (CIN) to invasive cervical cancer, in which most tumor cells constitutively express HPV-encoded E6 and E7 oncoproteins. Thus, host immune surveillance and its suppressive status within the tumor microenvironment are closely involved not only in cervical carcinogenesis [11], but also in further tumor growth and metastasis [12–14]. The presence of an immunosuppressive state

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within tumors involving the down-regulation of human leukocyte antigen (HLA) class I, production of immunosuppressive cytokines, and a low density of CD8+ tumor-infiltrating lymphocytes (TIL) combined with the induction of Foxp3+ regulatory T cells (Treg) was shown to be associated with disease progression and impaired survival in cervical cancer patients [15–18]. In addition, recent studies have suggested that indoleamine 2,3-dioxygenase (IDO) is involved in the carcinogenesis of cervical cancer [11,19]. IDO is a tryptophan-catabolizing enzyme that induces immune tolerance by depleting tryptophan and producing toxic tryptophan catabolites, which causes growth arrest of alloreactive T cells and natural killer (NK) cells and suppression of their killer function [20– 23]. In tumor-bearing mice, IDO was expressed in tumor cells and dendritic cells, both of which induce tolerance to tumor-derived antigens and also activation of Tregs [24–27]. In human cancer, IDO expression was found to be associated with poor clinical outcome in colorectal cancer [28] and ovarian cancer [29]. Recently, we have demonstrated that IDO expression is correlated with disease progression and impaired patient survival in endometrial cancer and ovarian cancer, and that high tumoral IDO expression induces rapid tumor growth and spread together with the suppression of TIL and NK cells [30–33]. These findings prompted us to investigate the expression of IDO in cervical cancer and its prognostic significance. In the current study, we performed immunohistochemical analysis of IDO expression in cervical cancer and evaluated its correlation with clinicopathological factors and patient survival. We demonstrate here that IDO is a reliable molecular marker for predicting poor prognosis in invasive cervical cancer patients following radical hysterectomy. Materials and methods Patient selection One hundred and twelve patients with stage IB–IIB invasive cervical cancer who underwent radical hysterectomy and pelvic lymphadenectomy at Nagoya University Hospital between 1992 and 2003 were included in this study. All patients were staged according to the International Federation of Gynecology and Obstetrics (FIGO) criteria: 67 were stage IB, 10 were stage IIA, and 35 were stage IIB. Histological subtype was assigned according to the criteria of the World Health Organization (WHO) classification: 88 cases were squamous cell carcinoma (SCC), 18 were adenocarcinoma (AC), and 6 were adenosquamous carcinoma (ASC). The FIGO stage IB patients with lymph node metastasis or LVSI and all FIGO stage II patients received post-operative adjuvant therapy involving whole pelvic irradiation or chemoradiation with cisplatin plus 5-fluorouracil [34]. Patients receiving primary radiotherapy/concurrent chemoradiation therapy without surgery, or receiving any preoperative treatment were excluded from this study. Informed consent was obtained from all patients for the use of their tissue samples. This study was approved by the ethics committee of Nagoya University Hospital.

with horseradish peroxidase-conjugated streptavidin and finally treated with 3,3′-diaminobenzidine tetrahydrochloride. The slides were then counterstained with Meyer's hematoxylin. As a negative control, the primary antibody was replaced with normal mouse IgG at an appropriate dilution. As a positive control, tissue sections of normal placenta were used [30]. IDO expression was classified into three categories based on the proportion of positively stained tumor cells; “negative” when b10% of tumor cells were stained, “focally positive” when 10–50% of tumor cells were stained, and “diffusely positive” when N50% of tumor cells were stained. In the IDO-focally positive cases, most IDO-positive tumor cells were distributed at the invasive front of tumors. In this study, IDO expression in non-tumor cells in the tumor stroma was not considered because their IDO immunostaining was not marked or absent. The results of immunohistochemistry were evaluated by two independent observers who did not have any knowledge of the clinical data. The concordance rate between the observers was over 95%. Statistical analysis Fisher's exact test was used to analyze the correlation of IDO expression with clinicopathological parameters. Overall survival (OS) was calculated from the date of surgery to the date of death, and disease-free survival (DFS) was calculated from the date of surgery to the date of recurrence. Survival analyses were performed according to the Kaplan–Meier method. A comparison of the survival between groups was performed with the log-rank test. Cox proportionalhazard analysis was used for univariate and multivariate analyses to explore the impact of individual variables on survival. Stat View software version 5.0 (SAS Institute Inc., Cary, NC) was used for all statistical analyses, and a p-value of b0.05 analyzed by two-tailed tests was considered significant. Results Immunohistochemical expression of IDO in cervical cancer tissues As shown in Fig. 1, IDO immunoreactivity was localized in the cytoplasm of cancer cells, while it was very faint or absent in the adjacent tumor–stromal cells. Of the 112 specimens examined, IDO was negative in 54 (48.2%) cases (Fig. 1A), focally expressed in 29 (25.9%) cases (Fig. 1B), and diffusely expressed in 29 (25.9%) cases (Fig. 1C). IDO was also markedly expressed in cervical adenocarcinoma cells (Fig. 1E and F). In contrast, IDO was not expressed in the normal cervical squamous epithelium (Fig. 1D) or endocervical glands (Fig. 1E). The correlation of IDO expression with various clinicopathological parameters is summarized in Table 1. IDO expression was positively correlated with FIGO stage (p = 0.0178), lymph node metastasis (p = 0.0439), and LVSI (p = 0.0052), but not with histological subtype (p = 0.5775), age (p = 0.6870), or adjuvant therapy (p = 0.2719).

Immunohistochemistry Correlation of IDO expression with patient survival Surgical specimens were fixed in 10% formalin, embedded in paraffin, and cut at a thickness of 4 mm. Deparaffinized sections were treated in Target Retrieval Solution (DAKO, Glostrup, Denmark) at 95 °C for 30 min. Immunohistochemical staining was performed using the avidin–biotin immunoperoxidase method. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2, and nonspecific immunoglobulin binding was blocked by incubation with 10% normal goat serum. The sections were incubated at room temperature for 2 h with anti-human IDO monoclonal antibody at a 1:200 dilution. This antibody was prepared as previously described [35]. The sections were rinsed and incubated with a biotinylated secondary antibody. After being washed, the sections were incubated

During the follow-up period, disease recurrence was observed in 31 (27.7%) of 112 patients, of which 23 (20.5%) died. The 5-year OS rates for the IDO-negative, -focally positive, and -diffusely positive groups were 92.3%, 89.5%, and 65.5%, respectively, and the 5-year DFS rates for the IDO-negative, -focally positive, and -diffusely positive groups were 84.9%, 75.8%, and 51.7%, respectively (Table 2). Patients who demonstrated diffuse IDO expression had significantly reduced OS (p = 0.0008) and DFS (p = 0.0012) rates compared with patients with no IDO expression (Fig. 2A and B). Next, we analyzed the impact of IDO expression on OS and DFS in the patients with FIGO stage IB disease (n = 67). Of 35 stage IB patients with

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Fig. 1. Representative immunohistochemical staining of indoleamine 2,3-dioxygenase (IDO) expression. A: Negative IDO expression in SCC; B: Focal IDO expression at the invasive front (arrowheads) of SCC; C: Diffuse IDO expression in SCC. D: No IDO expression in the normal cervical squamous epithelium; E: IDO was focally expressed in cervical adenocarcinoma cells (arrowheads), but absent in normal endocervical glands (arrows). F: Diffuse IDO expression in cervical adenocarcinoma; G: Negative control. Original magnification: × 200.

no IDO expression, only two (5.7%) exhibited recurrence and died, and of 21 IDO-focally positive stage IB patients, five (23.8%) showed recurrence and three (14.3%) died. In contrast, of 11 IDO-diffusely positive stage IB patients, six (54.5%) had recurrence and four (36.4%) died. The 5-year OS rates for the IDO-negative, -focally positive, and diffusely positive groups were 94.3%, 89.6%, and 72.7%, respectively, with a significant difference between the IDO-diffusely positive and negative groups (p = 0.0055) (Fig. 3A). Similarly, the 5-year DFS rates for the IDO-negative, -focally positive, and -diffusely positive groups were 94.3%, 81.0%, and 45.5%, respectively, with significant differences between the IDO-focally positive and -negative groups (p = 0.0464), and between the IDO-diffusely positive and -negative groups

(p = 0.0001) (Fig. 3B). These results suggest that IDO has a significant impact on disease recurrence and survival even in early-stage invasive cervical cancer patients who have undergone radical hysterectomy. Finally, we analyzed the correlation of IDO with OS and DFS in groups stratified according to histological subtype. In SCC patients (n = 88), the 5-year OS/DFS rates for the IDO-negative, -focally positive, and -diffusely positive groups were 92.4/89.9%, 90.5/79.1%, and 62.4/58.1%, respectively. There was a significant difference in OS Table 2 Univariate analyses of clinicopathological parameters in relation to survival. Characteristics No. Overall survival

Age (years) b50 59 ⪴50 53 FIGO stage IB 67 IIA/IIB 45 Histological type SCC 88 AC/ASC 24 Nodal status Negative 79 Positive 33 LVSI Absent 50 Present 62 IDO expression Negative 54 Focal 29 Diffuse 29 Adjuvant therapy No 29 Yes 83

Table 1 Correlation of indoleamine 2,3-dioxygenase (IDO) expression with clinicopathological factors in cervical cancer. Characteristics No.

IDO expression Negative (%)

All cases 112 Age (years) b50 59 ⪴50 53 FIGO stage IB 67 IIA/IIB 45 Histological type SCC 88 AC/ASC 24 Nodal status Negative 79 Positive 33 LVSI Absent 50 Present 62 Adjuvant therapy No 29 Yes 83

p-value* Focal (%)

Diffuse (%)

54

(48.2) 29

(25.9) 29

(25.9)

26 28

(44.1) 17 (52.8) 12

(28.8) 16 (22.6) 13

(27.1) (24.5) 0.6870

35 19

(52.2) 21 (42.2) 8

(31.3) 11 (17.8) 18

(16.4) (40.0) 0.0178

40 14

(45.5) 24 (58.3) 5

(27.3) 24 (20.8) 5

(27.3) (20.8) 0.5775

42 12

(53.2) 22 (36.4) 7

(27.8) 15 (21.2) 14

(19.0) (42.4) 0.0439

31 23

(62.0) 13 (37.1) 16

(26.0) 6 (25.8) 23

(12.0) (37.1) 0.0052

18 36

(62.1) 6 (43.4) 23

(20.7) 5 (27.7) 24

(17.2) (28.9) 0.2719

SCC: squamous cell carcinoma, AC: adenocarcinoma, ASC: adenosquamous carcinoma, LVSI: lymph-vascular space invasion. ⁎Fisher's exact test.

Disease-free survival

5-year survival (%) p-value

5-year survival (%) p-value

87.9 76.4

0.0474

84.7 61.7

0.0101

89.3 71.7

0.0135

82.1 61.3

0.0127

83.7 77.7

0.2476

78.3 57.1

0.0346

87.1 70.7

0.0032

79.2 59.1

0.0057

91.7 74.9

0.0026

85.9 64.2

0.0052

92.3 89.5 65.5

84.9 0.3137a 75.8 b 0.0008 51.7

96.6 77.3

0.0081

86.2 69.5

0.1952a 0.0012b

0.057

SCC: squamous cell carcinoma, AC: adenocarcinoma, ASC: adenosquamous carcinoma, LVSI: lymph-vascular space invasion. IDO-focally positive vs. IDO-negative. b IDO-diffusely positive vs. IDO-negative. Note that there was no significant difference in overall survival (p = 0.0604) or diseasefree survival (p = 0.1093) between IDO-focally positive and IDO-diffusely positive groups. a

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Fig. 2. Overall survival (A) and disease-free survival (B) curves in stage IB–IIB cervical cancer patients (n = 112).

(p = 0.0018) and DFS (p = 0.0159) between the IDO-diffusely positive and -negative groups. In contrast, there was no significant difference in OS or DFS in AC (n = 18) or ASC (n = 6) patients, although the numbers of patients in these groups were very small.

Univariate and multivariate analyses of prognostic variables in cervical cancer patients Univariate analyses demonstrated that age (p = 0.0474), FIGO stage (p = 0.0135), lymph node metastasis (p = 0.0032), LVSI (p = 0.0026), and IDO expression (p = 0.0008, diffusely positive vs. negative), but not histological subtype (p = 0.2476), were significant prognostic factors for OS, while these six variables were significant prognostic factors for DFS (Table 2). In this study, the patients who had undergone post-operative adjuvant therapy such as radiotherapy or chemoradiation showed a significantly reduced OS rate (p = 0.0081) as compared to those without adjuvant therapy (Table 2). This may be because the application of adjuvant therapy was limited to high-risk patients who were stage IB with positive lymph node metastasis or LVSI and stage II. Multivariate analyses demonstrated that only diffuse IDO expression (hazard ratio (HR) = 3.103, 95% confidence interval (CI) = 1.115–8.637, p = 0.0302) was an independent prognostic factor with respect to OS (Table 3). In contrast, both diffuse IDO expression (HR = 2.651, 95% CI = 1.087–6.470, p = 0.0321) and age (HR = 2.723, 95% CI = 1.246–5.950, p = 0.0120) were found to be independent prognostic factors with respect to DFS (Table 3).

Discussion In the present study, IDO was expressed in 52% of cases including SCC, AC, and ASC, which was consistent with the results shown by our prior studies in endometrial and ovarian cancers [30,33]. We showed that IDO expression was correlated with FIGO stage, positive lymph node metastasis, and LVSI, but not with histological subtype. These results indicate that tumoral IDO is involved in the progression of cervical cancer in both SCC and AC. The treatment options for cervical cancer differ depending on the clinical stage at the initial diagnosis and other factors, such as patient age, performance status, and the necessity of preserving fertility [2,3]. Radiotherapy, concurrent chemoradiotherapy, and various types of surgery including hysterectomy, cervical conization, and trachelectomy can be applied to the treatment of this tumor, and the choice of the initial therapy may affect the clinical outcome. To exclude the initial treatment-based bias and strictly assess the impact of IDO on survival, we focused on FIGO stage IB–IIB patients who underwent radical hysterectomy and pelvic lymphadenectomy. It should be noted that patients with FIGO stage IA, III, or IV disease were excluded from this study, because none of these patients had undergone radical hysterectomy in our institution. Our data clearly demonstrated that patients with diffuse tumoral IDO expression had a poor clinical outcome. Indeed, of the 54 patients with no IDO expression, only 6 patients died (5-year OS = 92.3%), while 12 of the 29 with diffuse IDO expression died (5-year OS = 65.5%). More importantly, our analysis of FIGO stage IB disease showed that only 2 of 35 patients with no IDO expression developed

Fig. 3. Overall survival (A) and disease-free survival (B) curves in stage IB cervical cancer patients (n = 67).

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Table 3 Multivariate analyses of survival in cervical cancer patients. Variables

Categories

Overall survival HR

Age (years) FIGO stage IDO expression

Nodal status LVSI

b 50 ≧50 IB IIA/IIB Negative Focal Diffuse Negative Positive Absent Present

1 2.197 1 1.268 1 1.953 3.103 1 1.542 1 2.678

p-value 95% CI

Disease-free survival HR

0.901–5.357

0.0833

0.455–3.537

0.6497

0.580–6.573 1.115–8.637

0.2795 0.0302

0.560–4.244

0.4017

0.790–9.078

0.1137

1 2.723 1 1.206 1 2.022 2.651 1 1.463 1 2.027

p-value

95% CI 1.246–5.950

0.0120

0.508–2.864

0.6713

0.766–5.339 1.087–6.470

0.1553 0.0321

0.610–3.510

0.3936

0.763–5.382

0.1567

HR: hazard ratio, CI: confidence interval, LVSI: lymph-vascular space invasion.

recurrence, resulting in a 94.3% 5-year DFS rate; whereas, 6 of 11 patients with diffuse IDO expression had recurrence (5-year DFS = 45.5%). These results suggest that patients with diffuse tumoral IDO expression are very likely to develop recurrence and have a poor prognosis, even in cases with early-stage disease at the time of surgery. Furthermore, our multivariate analyses demonstrated that diffuse IDO expression was the only independent prognostic factor for OS, suggesting that IDO is a more reliable prognostic parameter for FIGO IB–IIB surgically treated cervical cancer than the currently used clinicopathological factors [5–8]. It was noted that some cases in our study were diagnosed and treated before the introduction of the FIGO 1994 staging system; thus, we could not analyze the correlation of IDO with the prognosis of stage IB subgroups stratified according to tumor size (IB1 and IB2). As 70–80% of surgically treated stage IB–IIB cervical cancer cases including locally advanced (bulky) disease are curable [34,36], it would be of substantial benefit to define the minority of patients who are likely to develop recurrence following surgery, and to give aggressive adjuvant therapy to these patients alone. Our findings suggest that IDO is a useful post-operative indicator of the prognosis of this disease. The mechanism by which IDO contributes to cervical cancer progression remains to be determined. Tumor escape from host immune surveillance and the acquisition of tolerance are essential to cancer growth and progression [37]. Recent studies showed that IDO inhibits the growth of tumor antigen-specific cytotoxic T cells and activates Tregs by depleting the tumor microenvironment of tryptophan and/or by generating toxic tryptophan catabolites such as kynurenine [23–26]. In human cancer, we and others have shown that IDO suppresses the infiltration of CD3+ and/or CD8+ TIL into tumors in endometrial cancer [31], ovarian cancer [33], and colon cancer [28]. As an immunosuppressive state within tumors was shown to be associated with disease progression and impaired survival in cervical cancer [15–18], the correlation of IDO expression with a poor clinical outcome might have been due to IDO-induced immunosuppression within the tumor microenvironment. Recently, Nakamura et al. [19] reported that IDO was focally expressed in CIN 2/ 3 and that its expression was increased in microinvasive cancer, but absent in the normal cervical epithelium and CIN 1, suggesting the involvement of IDO in the progression of cervical neoplasia to microinvasive cancer. They also showed that IDO was expressed at the invasive front alone in all 17 invasive SCC cases examined. In contrast, our results showed that half of IDO-positive patients (29/58) focally expressed IDO at the invasive front, while the other half diffusely expressed IDO. Furthermore, the IDO-diffusely positive patients had worse OS and DFS than the IDO-focally positive patients. It is speculated that local immune tolerance by some IDO-expressing tumor cells is initiated at the tumor–stromal interface or the tumor invasive front, and expands to the entire tumors by diffuse IDO expression. Taken together, IDO may be involved not only in cervical

carcinogenesis but also in further tumor growth and metastasis of invasive cervical cancers. Further studies are needed to clarify the functional role of IDO in the progression of this tumor. In summary, we demonstrated that IDO expression was correlated with disease progression and a poor clinical outcome in surgically treated FIGO IB–IIB invasive cervical cancer. Furthermore, the diffuse tumoral IDO expression was an independent prognostic factor for OS and DFS. Thus IDO may be a novel post-operative prognostic indicator and useful for future IDO-targeted therapy against cervical cancer. Conflict of interest statement The authors declare that they have no conflicts of interest.

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