- Email: [email protected]
The significance of tumor-associated neutrophil density in uterine cervical cancer treated with definitive radiotherapy
Gynecologic Oncology 145 (2017) 469–475
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
The significance of tumor-associated neutrophil density in uterine cervical cancer treated with definitive radiotherapy Yuri Matsumoto, Seiji Mabuchi ⁎, Katsumi Kozasa, Hiromasa Kuroda, Tomoyuki Sasano, Eriko Yokoi, Naoko Komura, Kenjiro Sawada, Tadashi Kimura Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
H I G H L I G H T S • The CD66b-positive cells (TAN) were observed in 83.6% of cervical cancer specimens. • The increased intratumoral TAN density was significantly associated with shorter PFS. • Intratumoral TAN may be a promising therapeutic target in cervical cancer patients.
a r t i c l e
i n f o
Article history: Received 5 December 2016 Received in revised form 2 February 2017 Accepted 4 February 2017 Keywords: Tumor-associated neutrophils Cervical cancer Radiotherapy Survival
a b s t r a c t Objective. The aim of this study was to investigate the prognostic significance of tumor-associated neutrophil (TAN) density in cervical cancer patients that were treated with definitive radiotherapy. Methods. The baseline characteristics and outcome data of FIGO stages IB-IVA cervical cancer patients who were treated with definitive radiotherapy between January 1996 and December 2011 were collected. Using biopsy samples obtained at the time of the initial diagnosis, the expression levels of CD66b in the patients' cervical tumors were evaluated by immunohistochemistry. Univariate and multivariate analyses were performed to evaluate the relationships between intratumoral TAN density and various clinicopathological features as well as progression-free survival (PFS) in these patients. Results. The CD66b-positive cells (TAN) were observed in 209 (83.6%) of 250 cervical cancer specimens. The TAN density was significantly associated with shorter PFS. Multivariate analysis identified an increased number of TAN (hazard ratio [HR]: 4.95; 95% confidence interval [CI]: 2.51–10.7; p b 0.0001), FIGO stage IVB disease (HR: 2.64; 95% CI: 1.38–5.01; p = 0.01), non-squamous cell carcinoma (SCC) histology (HR: 2.50; 95% CI: 1.23–4.64; p = 0.01), larger tumors (HR: 1.58; 95% CI: 1.03–2.40; p = 0.04), and pelvic lymph node metastasis (HR: 2.24; 95% CI: 1.48–3.38; p = 0.0001) as independent prognostic factors for short PFS. Conclusion. Intratumoral TAN density is an independent prognostic factor for short PFS in cervical cancer patients treated with definitive radiotherapy. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Cervical cancer is the second most common type of cancer affecting women worldwide and displays an annual incidence of 530,000 new cases [1]. Although concurrent chemoradiotherapy (CCRT), the current standard treatment for invasive cervical cancer, is potentially curative, a significant number of patients develop recurrent disease (the risk of recurrence is 10–20% for FIGO stages IB-IIA and 50–70% in stages IIBIVA) [2–4]. Thus, in addition to developing more effective treatments, ⁎ Corresponding author. E-mail address: [email protected] (S. Mabuchi).
http://dx.doi.org/10.1016/j.ygyno.2017.02.009 0090-8258/© 2017 Elsevier Inc. All rights reserved.
identifying new prognostic factors that could be used to select patients who are likely to exhibit resistance to the standard treatment is of great importance. Systemic leukocyte alterations have recently become a focus of attention for physicians and researchers investigating cancer treatment [5]. So far, increased neutrophil [5,6] or monocyte counts [7], a reduced lymphocyte count [8], and an increased neutrophil to lymphocyte ratio (NLR) [9] or platelet to lymphocyte ratio (PLR) [10] have been reported to be associated with poor prognosis in patients with various solid cancers. Concerning uterine cervical cancer, previous studies have demonstrated that leukocytosis [11–14], neutrophilia [11–14], and lymphopenia [11] all have a negative prognostic impact. In addition, the NLR and PLR have also been identified as poor prognostic indicators
470
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
[15]. However, the mechanisms behind the aggressive nature of cancers displaying systemic leukocyte alterations remain to be elucidated. Neutrophils, which are known for their antimicrobial functions, are the most abundant circulating leukocytes (about 60%) and are the first line of defense at sites of infection or inflammation. Inflammatory neutrophils engulf bacteria and activate the immune system. In addition, tumor-associated neutrophils (TAN), a subgroup of neutrophils, function as immunosuppressive cells in the tumor microenvironment (TME). According to previous studies, TAN suppress T cell functions and contribute to multiple cancer processes, including tumor initiation, angiogenesis, invasion, and dissemination [16]. In a recent investigation of the immunological TME based on gene expression and clinical outcome data from ~18,000 human tumors across 39 malignancies, Gentles et al. demonstrated that the TAN count was a significant predictor of survival in patients with solid cancer [17]. In another study, the TAN count was shown to be an independent prognostic factor for recurrence in FIGO stage IB and IIA cervical cancer. However, all of the patients included in the latter study exhibited squamous cell carcinoma (SCC) histology, and the majority (86%) of them had been treated with radical hysterectomy [18]. Thus, the prognostic significance of TAN in terms of their effects on the radiosensitivity of cervical cancer remains unknown. In the present study, using tissue samples obtained at the initial diagnosis, we investigated the prognostic significance of intratumoral TAN (CD66b+ neutrophils) density in cervical cancer patients that were treated with definitive radiotherapy.
2. Methods 2.1. Patients and treatments Permission to proceed with the data acquisition and analysis was obtained from Osaka University Hospital's institutional review board. A list of patients who had been treated with definitive radiotherapy for International Federation of Gynecology and Obstetrics stage IB1 to IVA cervical cancer at Osaka University Hospital from November 1993 to December 2011 was generated from our institutional tumor registry, and their clinical data were retrospectively analyzed. Appropriate informed consent was obtained from each patient, and their clinical data, tissue biopsy specimens, and blood samples were examined. The patients were treated with definitive radiotherapy consisting of external beam radiotherapy followed by high-dose-rate brachytherapy with or without platinum-based concurrent chemotherapy (i.e., CCRT), as described previously [19]. The follow-up examinations performed after the initial treatment were conducted by gynecological oncologists and radiation oncologists at regular intervals in an outpatient clinic, as reported previously [20]. Recurrent disease was treated with radiotherapy, chemotherapy, or palliative care in accordance with the institutional treatment guidelines.
2.3. Immunohistochemistry Tumor biopsy samples that were obtained at the initial diagnosis were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and processed for hematoxylin and eosin or immunohistochemical staining. The slides were examined using a bright field microscope by two observers (Y.M. and E.Y.), who were blinded to the patients' clinical data, and were scored. The tumor sections were examined at high power magnification (× 400), and the numbers of immunolabeled cells in the intratumoral regions was evaluated. Areas of necrosis or artifacts were ignored. A score of 0 indicated no staining, +1 was indicative of focal staining (1–99 cells/high powered field [HPF] were stained), and + 2 was indicative of clearly positive staining (N 100 cells/HPF were stained), as described in detail elsewhere [14]. The tumors with staining scores of 0 were grouped into the TAN-Low group, whereas those with staining scores of +1 and +2 were grouped into the TAN-Med and TAN-High groups, respectively. In the current study, the significance of TAN in surrounding non-neoplastic stroma was not evaluated. 2.4. Statistical analysis Continuous data were compared between the groups using the Student's t-test, Wilcoxon rank sum test, or median test, as appropriate. Frequency counts and proportions were compared between the groups using the chi-squared test or the 2-tailed Fisher's exact test, as appropriate. Survival was defined as the time from the primary diagnosis to death or the last observation. We performed a univariate analysis by comparing the Kaplan-Meier curves for each subgroup using the logrank test. Cox proportional hazards regression analysis with stepwise variable selection was conducted to identify independent prognostic factors for survival. p-Values of b0.05 were considered statistically significant. All analyses were performed with SAS version 9.1 for Windows (SAS Institute Inc., Cary, NC). 3. Results 3.1. Patients A total of 250 consecutive patients with cervical cancer were included in this retrospective study. The patients' clinicopathological characteristics are summarized in Table 1. Their median age was 65 years (range: 26–87 years). One hundred and sixteen patients had FIGO stage IIIA-IVA disease, and 30 had stage IVB disease. Two hundred and thirty-six patients had tumors that displayed SCC histology, and 14 had tumors that exhibited non-SCC histology. Seventy-one patients had pelvic lymph node metastasis. The patients' mean pretreatment hemoglobin concentration was 12.3 g/dl (range: 7.8–14.8 mg/dl), and the mean duration of radiotherapy was 45 days (range: 30–67 days). Overall, the 5-year progression-free survival (PFS) and overall survival (OS) rates for stage IA-IIA, IIB-IVA, and IVB disease were 65.8%, 39.5%, and 5.0% and 69.8%, 49.2%, and 7.2%, respectively.
2.2. Response evaluation The response to treatment was assessed according to the Response Evaluation Criteria in Solid Tumors after the completion of radiotherapy. A complete response (CR) was defined as the disappearance of all target and non-target lesions and no new lesions being documented after 2 assessments that were at least 4 weeks apart. A partial response (PR) was defined as the detection of at least a 30% reduction in the sum of the longest dimensions of the target lesions in 2 assessments that were at least 4 weeks apart. Progressive disease (PD) was defined as a 20% increase in the sum of the longest dimensions of the target lesions or the development of new lesions. Stable disease (SD) implies that none of the above applies. Treatment failure was defined as PR, SD, or PD.
3.2. Correlations between intratumoral TAN density and clinicopathological parameters To establish the TAN density of the patients' cervical tumors, biopsy samples that were obtained at the initial diagnosis were subjected to immunohistochemical staining with a specific antibody against CD66b, as described in the Methods section. Representative photographs of intratumoral CD66b+ TAN are shown in Fig. 1A. When the intratumoral CD66b+ TAN density was scored, of a total of 250 patients, high, intermediate, and low CD66b immunoreactivity was observed in 41 (16.4%), 156 (62.4%), and 53 (21.2%) cases, respectively. We next investigated the relationships between various patient characteristics and intratumoral CD66b+ TAN density. As shown in
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
471
Table 1 Patients enrolled in the study. Clinicopathologic variables
All cases Age (years) Clinical stage
Histology Tumor size (mm) Pretreatment hemoglobin level (mg/dl) Pelvic lymph node metastasis Distant metastasis Duration of RT (days) WBC (/μl) Neu (/μl)
Number of each group
≤50 51≤ IA2-IIB IIIA-IVA IVB SCC Non SCC ≤60 61≤ ≤10.0 10.0b No Yes No Yes ≤55 56≤ b10,000 10,000≤ b7500 7500≤
250 45 205 104 116 30 236 14 192 58 30 220 179 71 220 30 235 15 219 31 217 33
CD66b IHC
p-Value
Low
Medium
High
41 (16.4%) 2 (4.4%) 39 (19.0%) 25 (24.0%) 14 (12.1%) 2 (6.7%) 39 (16.5%) 2 (14.3%) 35 (18.2%) 6 (10.3%) 1 (3.3%) 40 (18.2%) 34 (19.0%) 7 (9.9%) 39 (17.7%) 2 (6.7%) 40 (17.0%) 1 (6.7%) 41 (18.7%) 0 41 (18.9%) 0
156 (62.4%) 22 (48.9%) 134 (65.4%) 65 (62.5%) 74 (63.8%) 17 (56.7%) 147 (62.3%) 9 (64.3%) 121 (63.0%) 35 (60.3%) 16 (53.3%) 140 (63.6%) 114 (63.7%) 42 (59.2%) 139 (63.2%) 17 (56.7%) 147 (62.6%) 9 (60.0%) 148 (67.6%) 8 (25.8%) 146 (67.3%) 10 (30.3%)
53 (21.2%) 21 (46.2%) 32 (15.6%) 14 (13.5%) 28 (24.1%) 11 (36.7%) 50 (21.2%) 3 (21.4%) 36 (18.8%) 17 (29.3%) 13 (43.3%) 40 (18.2%) 31 (17.3%) 22 (31.0%) 42 (19.1%) 11 (36.7%) 48 (20.4%) 5 (33.3%) 30 (13.7%) 23 (74.2%) 30 (13.8%) 23 (69.7%)
b0.0001 0.008
0.98 0.13 0.003 0.03 0.049 0.36 b0.0001 b0.0001
Table 1, the presence of CD66b-positive TAN was significantly associated with a significantly younger age (p b 0.0001), an advanced clinical stage (p = 0.008), a lower hemoglobin level (p = 0.003), pelvic lymph node metastasis (p = 0.03), and distant metastasis (p = 0.049). Moreover, the CD66b-positive TAN density was found to be significantly associated with the pretreatment white blood cell (WBC) count and the pretreatment neutrophil count in the peripheral blood, and roughly 70% of patients who displayed pretreatment leukocytosis (N10.000 μ/l) or neutrophilia (N7500 μ/l) exhibited significantly increased numbers of CD66b-positive TAN (Table 1 and Fig. 1B).
to have a greater prognostic impact in the patients with IB-IVA disease than in those with stage IVB disease (Fig. 2). As the univariate analysis might have been affected by confounding factors, we next performed a multivariate analysis. As shown in Table 4 and Supplemental Table 1, in addition to clinical stage, tumor size and intratumoral CD66b+ TAN density were also shown to be independent prognostic factors for PFS and OS. Although histology and pelvic lymph node metastasis were found to be independent predictors of PFS, they did not provide prognostic information about OS (Supplemental Table 1).
3.3. Prognostic significance of intratumoral TAN density in cervical cancer
4. Discussion
After a median follow-up period of 60 months, 131 patients (52.4%) had developed recurrence, and 110 patients (44.0%) had died of disease progression. The recurrence rate was significantly associated with the intratumoral CD66b+ TAN density (Table 2). When the pattern of recurrence was examined according to TAN density, it was found that the patients in the TAN-High group were more likely to develop pelvic recurrence than those in the TAN-Low and TAN-Med groups (p b 0.0001). To explore the prognostic significance of intratumoral TAN density in cervical cancer patients that were treated with definitive radiotherapy, we investigated the tumor response to radiotherapy according to intratumoral TAN density (Table 3). The CR ratio of the patients in the TAN-High group was significantly lower than those of the patients in the TAN-Low and TAN-Med groups (p b 0.0001). We then conducted univariate analyses of PFS and OS. The univariate analyses showed that intratumoral CD66b + TAN density, age, clinical stage, tumor size, the pretreatment hemoglobin level, and pelvic lymph node involvement were correlated with PFS and OS (Table 4 and Supplemental Table 1). Although non-SCC histology was found to be an independent predictor of PFS, it did not provide prognostic information about OS. Statistically significant differences in PFS and OS were observed according to intratumoral CD66b+ TAN density (log-rank; p b 0.0001 and p b 0.0001, respectively; Fig. 1C). The estimated 5-year PFS rates of the patients in the TAN-Low, TAN-Med, and TAN-High groups were 73.8%, 50.7%, and 11.3%, respectively. The estimated 5year OS rates of the patients in the TAN-Low, TAN-Med, and TAN-High groups were 77.5%, 58.5%, and 17.0%, respectively. When examined according to clinical stage, the intratumoral CD66b+ TAN density seemed
To the best of our knowledge, this is the first study to identify intratumoral TAN density as an independent poor prognostic factor for survival in patients with cervical cancer that were treated with definitive radiotherapy. In the current study, an increased intratumoral TAN density was found to be significantly associated with shorter PFS. Moreover, an increased intratumoral TAN density was shown to be significantly correlated with lymph node metastasis, a lower CR rate, and a higher recurrence rate. Thus, although this study was retrospective in nature its findings strongly suggest that TAN exhibit pro-tumorigenic activity in TME. Our findings are consistent with those of a previous study that demonstrated that the TAN count is an independent predictor of recurrence in cervical cancer patients [18]. The patients included in the latter study had early stage disease and were mainly treated with surgery. In contrast, in the current study most patients had advanced stage disease and were treated with definitive radiotherapy. Collectively, these results suggest that TAN are prognostically significant in cervical cancer patients, irrespective of clinical stage or the initial treatment. The findings obtained in the current study could have important clinical implications. By subjecting biopsy samples obtained at the initial diagnosis to immunostaining for CD66b before initiating radiotherapy, it might be possible to predict which patients are at high risk of developing recurrence after definitive radiotherapy. Moreover, as 91% and 90% of the cervical cancers with increased TAN densities were found in patients who displayed pretreatment leukocytosis and neutrophilia, respectively, by performing simple and low-cost peripheral blood examinations before radiotherapy, it might be possible to identify
472
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Fig. 1. Prognostic significance of intratumoral TAN density. A, Intratumoral TAN density was evaluated based on CD66b immunoreactivity CD66b immunoreactivity was scored, as described in the Methods. Representative photographs from the TAN-Low, TAN-Med, and TAN-High groups are shown. B, (i), Correlation between the peripheral WBC count and intratumoral TAN density A positive correlation was detected between the peripheral WBC count and intratumoral TAN density (analysis of variance, p b 0.0001). (ii), Correlation between the peripheral neutrophil count and intratumoral TAN density A positive correlation was detected between the peripheral neutrophil count and intratumoral TAN density (analysis of variance, p b 0.0001). C, Kaplan-Meier estimates of survival according to intratumoral TAN density (i), PFS The patients in the TAN-High group exhibited significantly shorter PFS than those in the TAN-Med and TAN-Low groups (TAN-Low vs. TAN-Med, log-rank, p = 0.009; TAN-Low vs. TAN-High, log-rank, p b 0.0001; TAN-Med vs. TAN-High, logrank, p b 0.0001). (ii), OS The patients in the TAN-High group displayed significantly shorter OS than those in the TAN-Med and TAN-Low groups (TAN-Low vs. TAN-Med, log-rank, p = 0.02; TAN-Low vs. TAN-High, log-rank, p b 0.0001; TAN-Med vs. TAN-High, log-rank, p b 0.0001).
patients who are at high risk of developing recurrence. Thus, patients with increased intratumoral TAN densities, neutrophilia, or leukocytosis should be considered for closer follow-up.
Considering the high frequency of treatment failure after platinum-based chemoradiotherapy for cervical cancer, it is of utmost importance to develop effective treatments for this patient
Table 2 Pattern of recurrence. Total (n = 250)
CD66b IHC
p-Value
Low (n = 41)
Medium (n = 156)
High (n = 53)
Number of recurrence
131 (52.4%)
10 (24.4%)
74 (47.4%)
47 (88.7%)
p b 0.0001
Pattern of recurrence Pelvic Distant only Pelvic + distant
79 (31.6%) 53 (21.2%) 17 (7.2%)
7(17.1%) 3 (7.3%) 1 (2.4%)
41 (26.3%) 34 (21.8%) 8 (5.1%)
31 (58.5%) 16 (30.2%) 8 (15.1%)
p b 0.0001 p = 0.03 p = 0.02
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
473
Table 3 Response to definitive radiotherapy. CD66b immunohistochemistry
Response
CR (n = 211) Non CR (n = 39)
p-Value
Low (%) (n = 41)
Medium(%) (n = 156)
High (%) (n = 53)
39 (95.1)
138 (88.5)
34 (64.2)
2 (4.9)
18 (11.5)
19 (35.9)
p b 0.0001
CR; complete response.
population. One possible strategy is the use of intensive concurrent chemotherapy to improve the efficacy of chemoradiotherapy. Recent studies, including a meta-analysis, have shown that the concurrent administration of platinum-based doublet chemotherapy and radiotherapy is associated with improved OS and PFS compared with radiotherapy and concurrent cisplatin chemotherapy in patients with cervical cancer [2], suggesting that the concurrent administration of platinum-based doublet chemotherapy and pelvic radiotherapy might result in improved survival and locoregional control. Another possible strategy is the addition of consolidation chemotherapy after definitive CCRT. A 2008 meta-analysis of randomized controlled trials strongly suggested that the addition of consolidation chemotherapy to CCRT is beneficial [4]. Currently, the efficacy of administering platinum-based consolidation chemotherapy against cervical cancer after pelvic CCRT is being evaluated in phase III studies in both the postoperative adjuvant radiotherapy [21] and definitive radiotherapy settings [22].
Table 4 The univariate/multivariate cox-proportional hazard regression analysis of prognostic factors for progression-free-survival. Variables
CD66b IHC Low Medium High Age (years) ≤50 51≤ Clinical stage IA2-IIB IIIA-IVA eIVB Histology SCC Non SCC Tumor size (mm) e≤60 61≤ Pretreatment hemoglobin level (mg/dl) ≤10.0 10.0b Pelvic lymph node metastasis No Yes Duration of RT (months) ≤55 56≤
Univariate
Multivariate
Hazard 95%CI ratio
p-Value
1 2.32 7.66
1.26–4.79 4.01–16.2
b0.0001 1 1.81 4.95
1 0.48
0.33–0.73
1 0.81
0.53–1.29
1 2.15 7.29
1.44–3.27 4.33–12.2
b0.0001 1 1.33 2.64
0.84–2.14 1.38–5.01
1 2.31
1.17–4.10
1 2.50
1.23–4.64
1 2.51
1.74–3.59
b0.0001 1 1.58
1.03–2.40
1 0.45
0.29–0.72
1 0.97
0.60–1.60
1 3.13
1 2.20–4.43 b0.0001 2.24
1.48–3.38
1 1.48
0.73–2.68
0.0002
0.006
0.0002
0.22
Hazard 95%CI Ratio
1 0.76
p-Value
b0.0001 0.97–3.75 2.51–10.7 0.37
0.01
0.01
0.04
0.89
0.0001
0.45 0.34–1.51
A third option is the development of a novel treatment strategy that targets neutrophils themselves. It has been reported that TAN production can be induced by upregulating the expression of hematological growth factors, including transforming growth factor-β, granulocyte colony-stimulating factor, or granulocyte macrophage colony-stimulating factor [23]. Thus, these mediators, which stimulate neutrophil production, might have therapeutic effects in cervical cancer patients. The inhibition of TAN migration into tumors might also be an effective strategy. It has recently been shown that tumor-derived cysteine-x-cysteine ligands (such as CXCL2) and the cysteine-X-cysteine chemokine receptor-2 (CXCR2), which are expressed on the surfaces of neutrophils, (the CXCL2/CXCR2 axis) play an important role in TAN migration into tumors [16,23]. CXCR2 antagonists were demonstrated to have antitumor effects on TAN in a murine tumor study [24] and have been shown to be safe in humans [25]. Thus, the efficacy of novel treatments targeting the abovementioned molecules needs to be investigated in the future. The last strategy may be targeting the mechanisms of TAN generation. A recent study have shown that Human papillomavirus (HPV) positive cervical cancer cells express IL-16 and IL-17, stimulate the myeloid cell proliferation in the bone marrow, and increase the leukocyte recruitment to the tumor microenvironment [26]. Thus, HPV might be involved in the induction of TAN in cervical cancer patients. Recently, platelet-neutorphil interaction in cancer has gained attentions [27]. Roughly a decade ago, a phenomenon of platelets adhering to neutrophils was described and referred to as platelet satellitism, but the cause or significance of the finding was not further explored [28]. However, recent investigations strongly suggested that the enhanced platelet activation in cancer patients does not only contribute to thrombosis but also to tumor progression by stimulating neutrophil function [27]. Thus, we think that the association between HPV status, platelet counts and TANs density in cervical cancer should be investigated in the future. The limitations of our study have to be addressed. The first is that our study was conducted at a single institution. The retrospective nature of the current study is the second limitation. Third, as this study covers a long period, the contents of patient work-ups and radiotherapy, salvage treatment, and supportive care regimens have changed, which might have affected patient survival. To eliminate these potential biases, prospective multi-institutional investigations need to be conducted. Fourth, in the current study we employed CD66b+ as a marker to identify TAN. However, the optimal marker for TAN has not been defined. Although CD66b+ seems to be a reasonable marker for identifying TAN, further studies investigating more specific markers are needed. Finally, although the prognostic significance of TAN was demonstrated in the current study, the mechanisms by which TAN promote cervical cancer progression remain unknown. To improve patient prognosis, future mechanistic studies of this issue are required. In conclusion, an elevated CD66b+ TAN count is an independent prognostic factor for short PFS in cervical cancer patients that are treated with definitive radiotherapy. Larger studies in the prospective setting are required to validate our findings.
474
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Fig. 2. Impact of intratumoral TAN density on survival according to clinical stage. A, FIGO stage IB-IIB patients (i) PFS A higher intratumoral TAN density was found to be significantly correlated with shorter PFS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). (ii) OS A higher intratumoral TAN density was demonstrated to be significantly correlated with shorter OS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). B, FIGO stage IIIA-IVA patients (i), PFS A higher intratumoral TAN density was found to be significantly correlated with shorter PFS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). (ii), OS A higher intratumoral TAN density was demonstrated to be significantly correlated with shorter OS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). C, FIGO stage IVB patients (i) PFS The patients in the TAN-High group exhibited slightly shorter PFS than those in the TAN-Med and TAN-Low groups, but the difference was not statistically significant (log-rank, p = 0.24) (ii) OS The patients in the TAN-High group displayed slightly shorter OS than those in the TAN-Med and TAN-Low groups, but the difference was not statistically significant (log-rank, p = 0.08).
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Conflicts of interest statement The authors declare that they have no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2017.02.009. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108. [2] F. Petrelli, A. De Stefani, F. Raspagliesi, D. Lorusso, S. Barni, Radiotherapy with concurrent cisplatin-based doublet or weekly cisplatin for cervical cancer: a systematic review and meta-analysis, Gynecol. Oncol. 134 (2014) 166–171. [3] Chemoradiotherapy for Cervical Cancer Meta-analysisCollaboration (CCCMAC), Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer: individual patient data meta-analysis, Cochrane Database Syst. Rev. (2010), CD008285. . [4] Chemoradiotherapy for Cervical Cancer Meta-AnalysisCollaboration, Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer: a systematic review and meta-analysis of individual patient data from 18 randomized trials, J. Clin. Oncol. 26 (2008) 5802–5812. [5] S. Chakraborty, B. Keenportz, S. Woodward, J. Anderson, D. Colan, Paraneoplastic leukemoid reaction in solid tumors, Am. J. Clin. Oncol. 38 (2015) 326–330. [6] R. Takahashi, S. Mabuchi, M. Kawano, T. Sasano, Y. Matsumoto, H. Kuroda, T. Hisamatsu, K. Kozasa, K. Sawada, T. Hamasaki, T. Kimura, Prognostic significance of systemic neutrophil and leukocyte alterations in surgically treated endometrial cancer patients: a monoinstitutional study, Gynecol. Oncol. 137 (2015) 112–118. [7] K. Matsuo, M.S. Hom, A. Moeini, H. Machida, N. Takeshima, L.D. Roman, A.K. Sood, Significance of monocyte counts on tumor characteristics and survival outcome of women with endometrial cancer, Gynecol. Oncol. 138 (2015) 332–338. [8] K. Milne, C. Alexander, J.R. Webb, W. Sun, K. Dillon, S.E. Kalloger, C.B. Gilks, B. Clarke, M. Köbel, B.H. Nelson, Absolute lymphocyte count is associated with survival in ovarian cancer independent of tumor-infiltrating lymphocytes, J. Transl. Med. 10 (2012) 33. [9] A.J. Templeton, M.G. McNamara, B. Šeruga, F.E. Vera-Badillo, P. Aneja, A. Ocaña, R. Leibowitz-Amit, G. Sonpavde, J.J. Knox, B. Tran, I.F. Tannock, E. Amir, Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis, J. Natl. Cancer Inst. 106 (2014). [10] A.J. Templeton, O. Ace, M.G. McNamara, M. Al-Mubarak, F.E. Vera-Badillo, T. Hermanns, B. Seruga, A. Ocaña, I.F. Tannock, E. Amir, Prognostic role of platelet to lymphocyte ratio in solid tumors: a systematic review and meta-analysis, Cancer Epidemiol. Biomark. Prev. 23 (2014) 1204–1212. [11] B.M. Tavares-Murta, M.A. Mendonça, N.L. Duarte, J.A. da Silva, T.S. Mutão, C.B. Garcia, E.F. Murta, Systemic leukocyte alterations are associated with invasive uterinecervical cancer, Int. J. Gynecol. Cancer 20 (2010) 1154–1159. [12] S. Mabuchi, Y. Matsumoto, F. Isohashi, Y. Yoshioka, H. Ohashi, E. Morii, T. Hamasaki, K. Aozasa, D.G. Mutch, T. Kimura, Pretreatment leukocytosis is an indicator of poor prognosis in patients with cervical cancer, Gynecol. Oncol. 122 (2011) 25–32.
475
[13] S. Mabuchi, Y. Matsumoto, T. Hamasaki, M. Kawano, T. Hisamatsu, D.G. Mutch, T. Kimura, Elevated white blood cell count at the time of recurrence diagnosis is an indicator of short survival in patients with recurrent cervical cancer, Int. J. Gynecol. Cancer 22 (2012) 1545–1551. [14] S. Mabuchi, Y. Matsumoto, M. Kawano, K. Minami, Y. Seo, T. Sasano, R. Takahashi, H. Kuroda, T. Hisamatsu, A. Kakigano, M. Hayashi, K. Sawada, T. Hamasaki, E. Morii, H. Kurachi, N. Matsuura, T. Kimura, Uterine cervical cancer displaying tumor-related leukocytosis: a distinct clinical entity with radioresistant feature, J. Natl. Cancer Inst. 106 (2014). [15] C. Onal, O.C. Guler, B.A. Yildirim, Prognostic use of pretreatment hematologic parameters in patients receiving definitive chemoradiotherapy for cervical cancer, Int. J. Gynecol. Cancer 26 (2016) 1169–1175. [16] K. Moses, S. Brandau, Human neutrophils: their role in cancer and relation to myeloid-derived suppressor cells, Semin. Immunol. 28 (2016) 187–196. [17] A.J. Gentles, A.M. Newman, C.L. Liu, S.V. Bratman, W. Feng, D. Kim, V.S. Nair, Y. Xu, A. Khuong, C.D. Hoang, M. Diehn, R.B. West, S.K. Plevritis, A.A. Alizadeh, The prognostic landscape of genes and infiltrating immune cells across human cancers, Nat. Med. 21 (2015) 938–945. [18] A. Carus, M. Ladekarl, H. Hager, B.S. Nedergaard, F. Donskov, Tumour-associated CD66b + neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer, Br. J. Cancer 108 (2013) 2116–2122. [19] S. Mabuchi, H. Ugaki, F. Isohashi, Y. Yoshioka, K. Temma, N. Yada-Hashimoto, T. Takeda, T. Yamamoto, K. Yoshino, R. Nakajima, C. Kuragaki, K. Morishige, T. Enomoto, T. Inoue, T. Kimura, Concurrent weekly nedaplatin, external beam radiotherapy and high-dose-rate brachytherapy in patients with FIGO stage IIIb cervical cancer: a comparison with a cohort treated by radiotherapy alone, Gynecol. Obstet. Investig. 69 (2010) 224–232. [20] S. Mabuchi, F. Isohashi, S. Maruoka, T. Hisamatsu, T. Takiuchi, Y. Yoshioka, T. Kimura, Post-treatment follow-up procedures in cervical cancer patients previously treated with radiotherapy, Arch. Gynecol. Obstet. 286 (2012) 179–185. [21] NCT01755845. Clinical Trials.gov. A Service of the U.S. National Institute of Health. (http://www.clinicaltrials.gov (Accessed December 1, 2016)). [22] NCT01414608. Clinical Trials.gov. A Service of the U.S. National Institute of Health. (http://www.clinicaltrials.gov (Accessed December 1, 2016)). [23] Y. De Vlaeminck, A. González-Rascón, C. Goyvaerts, K. Breckpot, Cancer-associated myeloid regulatory cells, Front. Immunol. 7 (2016) 113. [24] T. Jamieson, M. Clarke, C.W. Steele, M.S. Samuel, J. Neumann, A. Jung, D. Huels, M.F. Olson, S. Das, R.J. Nibbs, O.J. Sansom, Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis, J. Clin. Invest. 122 (2012) 3127–3144. [25] S. Jurcevic, C. Humfrey, M. Uddin, S. Warrington, B. Larsson, C. Keen, The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions, Br. J. Clin. Pharmacol. 80 (2015) 1324–1336. [26] S.C. Stone, R.A. Rossetti, A.M. Lima, A.P. Lepique, HPV associated tumor cells control tumor microenvironment and leukocytosis in experimental models, Immun. Inflamm. Dis. 2 (2014) 63–75. [27] A.K. Olsson, J. Cedervall, NETosis in cancer - platelet-neutrophil crosstalk promotes tumor-associated pathology, Front. Immunol. 7 (2016) 373, http://dx.doi.org/10. 3389/fimmu.2016.00373. [28] C.R. Kjeldsberg, Letter: platelet satellitism, N. Engl. J. Med. 290 (1974) 165, http://dx. doi.org/10.1056/NEJM197401172900318.
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
The significance of tumor-associated neutrophil density in uterine cervical cancer treated with definitive radiotherapy Yuri Matsumoto, Seiji Mabuchi ⁎, Katsumi Kozasa, Hiromasa Kuroda, Tomoyuki Sasano, Eriko Yokoi, Naoko Komura, Kenjiro Sawada, Tadashi Kimura Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
H I G H L I G H T S • The CD66b-positive cells (TAN) were observed in 83.6% of cervical cancer specimens. • The increased intratumoral TAN density was significantly associated with shorter PFS. • Intratumoral TAN may be a promising therapeutic target in cervical cancer patients.
a r t i c l e
i n f o
Article history: Received 5 December 2016 Received in revised form 2 February 2017 Accepted 4 February 2017 Keywords: Tumor-associated neutrophils Cervical cancer Radiotherapy Survival
a b s t r a c t Objective. The aim of this study was to investigate the prognostic significance of tumor-associated neutrophil (TAN) density in cervical cancer patients that were treated with definitive radiotherapy. Methods. The baseline characteristics and outcome data of FIGO stages IB-IVA cervical cancer patients who were treated with definitive radiotherapy between January 1996 and December 2011 were collected. Using biopsy samples obtained at the time of the initial diagnosis, the expression levels of CD66b in the patients' cervical tumors were evaluated by immunohistochemistry. Univariate and multivariate analyses were performed to evaluate the relationships between intratumoral TAN density and various clinicopathological features as well as progression-free survival (PFS) in these patients. Results. The CD66b-positive cells (TAN) were observed in 209 (83.6%) of 250 cervical cancer specimens. The TAN density was significantly associated with shorter PFS. Multivariate analysis identified an increased number of TAN (hazard ratio [HR]: 4.95; 95% confidence interval [CI]: 2.51–10.7; p b 0.0001), FIGO stage IVB disease (HR: 2.64; 95% CI: 1.38–5.01; p = 0.01), non-squamous cell carcinoma (SCC) histology (HR: 2.50; 95% CI: 1.23–4.64; p = 0.01), larger tumors (HR: 1.58; 95% CI: 1.03–2.40; p = 0.04), and pelvic lymph node metastasis (HR: 2.24; 95% CI: 1.48–3.38; p = 0.0001) as independent prognostic factors for short PFS. Conclusion. Intratumoral TAN density is an independent prognostic factor for short PFS in cervical cancer patients treated with definitive radiotherapy. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Cervical cancer is the second most common type of cancer affecting women worldwide and displays an annual incidence of 530,000 new cases [1]. Although concurrent chemoradiotherapy (CCRT), the current standard treatment for invasive cervical cancer, is potentially curative, a significant number of patients develop recurrent disease (the risk of recurrence is 10–20% for FIGO stages IB-IIA and 50–70% in stages IIBIVA) [2–4]. Thus, in addition to developing more effective treatments, ⁎ Corresponding author. E-mail address: [email protected] (S. Mabuchi).
http://dx.doi.org/10.1016/j.ygyno.2017.02.009 0090-8258/© 2017 Elsevier Inc. All rights reserved.
identifying new prognostic factors that could be used to select patients who are likely to exhibit resistance to the standard treatment is of great importance. Systemic leukocyte alterations have recently become a focus of attention for physicians and researchers investigating cancer treatment [5]. So far, increased neutrophil [5,6] or monocyte counts [7], a reduced lymphocyte count [8], and an increased neutrophil to lymphocyte ratio (NLR) [9] or platelet to lymphocyte ratio (PLR) [10] have been reported to be associated with poor prognosis in patients with various solid cancers. Concerning uterine cervical cancer, previous studies have demonstrated that leukocytosis [11–14], neutrophilia [11–14], and lymphopenia [11] all have a negative prognostic impact. In addition, the NLR and PLR have also been identified as poor prognostic indicators
470
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
[15]. However, the mechanisms behind the aggressive nature of cancers displaying systemic leukocyte alterations remain to be elucidated. Neutrophils, which are known for their antimicrobial functions, are the most abundant circulating leukocytes (about 60%) and are the first line of defense at sites of infection or inflammation. Inflammatory neutrophils engulf bacteria and activate the immune system. In addition, tumor-associated neutrophils (TAN), a subgroup of neutrophils, function as immunosuppressive cells in the tumor microenvironment (TME). According to previous studies, TAN suppress T cell functions and contribute to multiple cancer processes, including tumor initiation, angiogenesis, invasion, and dissemination [16]. In a recent investigation of the immunological TME based on gene expression and clinical outcome data from ~18,000 human tumors across 39 malignancies, Gentles et al. demonstrated that the TAN count was a significant predictor of survival in patients with solid cancer [17]. In another study, the TAN count was shown to be an independent prognostic factor for recurrence in FIGO stage IB and IIA cervical cancer. However, all of the patients included in the latter study exhibited squamous cell carcinoma (SCC) histology, and the majority (86%) of them had been treated with radical hysterectomy [18]. Thus, the prognostic significance of TAN in terms of their effects on the radiosensitivity of cervical cancer remains unknown. In the present study, using tissue samples obtained at the initial diagnosis, we investigated the prognostic significance of intratumoral TAN (CD66b+ neutrophils) density in cervical cancer patients that were treated with definitive radiotherapy.
2. Methods 2.1. Patients and treatments Permission to proceed with the data acquisition and analysis was obtained from Osaka University Hospital's institutional review board. A list of patients who had been treated with definitive radiotherapy for International Federation of Gynecology and Obstetrics stage IB1 to IVA cervical cancer at Osaka University Hospital from November 1993 to December 2011 was generated from our institutional tumor registry, and their clinical data were retrospectively analyzed. Appropriate informed consent was obtained from each patient, and their clinical data, tissue biopsy specimens, and blood samples were examined. The patients were treated with definitive radiotherapy consisting of external beam radiotherapy followed by high-dose-rate brachytherapy with or without platinum-based concurrent chemotherapy (i.e., CCRT), as described previously [19]. The follow-up examinations performed after the initial treatment were conducted by gynecological oncologists and radiation oncologists at regular intervals in an outpatient clinic, as reported previously [20]. Recurrent disease was treated with radiotherapy, chemotherapy, or palliative care in accordance with the institutional treatment guidelines.
2.3. Immunohistochemistry Tumor biopsy samples that were obtained at the initial diagnosis were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and processed for hematoxylin and eosin or immunohistochemical staining. The slides were examined using a bright field microscope by two observers (Y.M. and E.Y.), who were blinded to the patients' clinical data, and were scored. The tumor sections were examined at high power magnification (× 400), and the numbers of immunolabeled cells in the intratumoral regions was evaluated. Areas of necrosis or artifacts were ignored. A score of 0 indicated no staining, +1 was indicative of focal staining (1–99 cells/high powered field [HPF] were stained), and + 2 was indicative of clearly positive staining (N 100 cells/HPF were stained), as described in detail elsewhere [14]. The tumors with staining scores of 0 were grouped into the TAN-Low group, whereas those with staining scores of +1 and +2 were grouped into the TAN-Med and TAN-High groups, respectively. In the current study, the significance of TAN in surrounding non-neoplastic stroma was not evaluated. 2.4. Statistical analysis Continuous data were compared between the groups using the Student's t-test, Wilcoxon rank sum test, or median test, as appropriate. Frequency counts and proportions were compared between the groups using the chi-squared test or the 2-tailed Fisher's exact test, as appropriate. Survival was defined as the time from the primary diagnosis to death or the last observation. We performed a univariate analysis by comparing the Kaplan-Meier curves for each subgroup using the logrank test. Cox proportional hazards regression analysis with stepwise variable selection was conducted to identify independent prognostic factors for survival. p-Values of b0.05 were considered statistically significant. All analyses were performed with SAS version 9.1 for Windows (SAS Institute Inc., Cary, NC). 3. Results 3.1. Patients A total of 250 consecutive patients with cervical cancer were included in this retrospective study. The patients' clinicopathological characteristics are summarized in Table 1. Their median age was 65 years (range: 26–87 years). One hundred and sixteen patients had FIGO stage IIIA-IVA disease, and 30 had stage IVB disease. Two hundred and thirty-six patients had tumors that displayed SCC histology, and 14 had tumors that exhibited non-SCC histology. Seventy-one patients had pelvic lymph node metastasis. The patients' mean pretreatment hemoglobin concentration was 12.3 g/dl (range: 7.8–14.8 mg/dl), and the mean duration of radiotherapy was 45 days (range: 30–67 days). Overall, the 5-year progression-free survival (PFS) and overall survival (OS) rates for stage IA-IIA, IIB-IVA, and IVB disease were 65.8%, 39.5%, and 5.0% and 69.8%, 49.2%, and 7.2%, respectively.
2.2. Response evaluation The response to treatment was assessed according to the Response Evaluation Criteria in Solid Tumors after the completion of radiotherapy. A complete response (CR) was defined as the disappearance of all target and non-target lesions and no new lesions being documented after 2 assessments that were at least 4 weeks apart. A partial response (PR) was defined as the detection of at least a 30% reduction in the sum of the longest dimensions of the target lesions in 2 assessments that were at least 4 weeks apart. Progressive disease (PD) was defined as a 20% increase in the sum of the longest dimensions of the target lesions or the development of new lesions. Stable disease (SD) implies that none of the above applies. Treatment failure was defined as PR, SD, or PD.
3.2. Correlations between intratumoral TAN density and clinicopathological parameters To establish the TAN density of the patients' cervical tumors, biopsy samples that were obtained at the initial diagnosis were subjected to immunohistochemical staining with a specific antibody against CD66b, as described in the Methods section. Representative photographs of intratumoral CD66b+ TAN are shown in Fig. 1A. When the intratumoral CD66b+ TAN density was scored, of a total of 250 patients, high, intermediate, and low CD66b immunoreactivity was observed in 41 (16.4%), 156 (62.4%), and 53 (21.2%) cases, respectively. We next investigated the relationships between various patient characteristics and intratumoral CD66b+ TAN density. As shown in
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
471
Table 1 Patients enrolled in the study. Clinicopathologic variables
All cases Age (years) Clinical stage
Histology Tumor size (mm) Pretreatment hemoglobin level (mg/dl) Pelvic lymph node metastasis Distant metastasis Duration of RT (days) WBC (/μl) Neu (/μl)
Number of each group
≤50 51≤ IA2-IIB IIIA-IVA IVB SCC Non SCC ≤60 61≤ ≤10.0 10.0b No Yes No Yes ≤55 56≤ b10,000 10,000≤ b7500 7500≤
250 45 205 104 116 30 236 14 192 58 30 220 179 71 220 30 235 15 219 31 217 33
CD66b IHC
p-Value
Low
Medium
High
41 (16.4%) 2 (4.4%) 39 (19.0%) 25 (24.0%) 14 (12.1%) 2 (6.7%) 39 (16.5%) 2 (14.3%) 35 (18.2%) 6 (10.3%) 1 (3.3%) 40 (18.2%) 34 (19.0%) 7 (9.9%) 39 (17.7%) 2 (6.7%) 40 (17.0%) 1 (6.7%) 41 (18.7%) 0 41 (18.9%) 0
156 (62.4%) 22 (48.9%) 134 (65.4%) 65 (62.5%) 74 (63.8%) 17 (56.7%) 147 (62.3%) 9 (64.3%) 121 (63.0%) 35 (60.3%) 16 (53.3%) 140 (63.6%) 114 (63.7%) 42 (59.2%) 139 (63.2%) 17 (56.7%) 147 (62.6%) 9 (60.0%) 148 (67.6%) 8 (25.8%) 146 (67.3%) 10 (30.3%)
53 (21.2%) 21 (46.2%) 32 (15.6%) 14 (13.5%) 28 (24.1%) 11 (36.7%) 50 (21.2%) 3 (21.4%) 36 (18.8%) 17 (29.3%) 13 (43.3%) 40 (18.2%) 31 (17.3%) 22 (31.0%) 42 (19.1%) 11 (36.7%) 48 (20.4%) 5 (33.3%) 30 (13.7%) 23 (74.2%) 30 (13.8%) 23 (69.7%)
b0.0001 0.008
0.98 0.13 0.003 0.03 0.049 0.36 b0.0001 b0.0001
Table 1, the presence of CD66b-positive TAN was significantly associated with a significantly younger age (p b 0.0001), an advanced clinical stage (p = 0.008), a lower hemoglobin level (p = 0.003), pelvic lymph node metastasis (p = 0.03), and distant metastasis (p = 0.049). Moreover, the CD66b-positive TAN density was found to be significantly associated with the pretreatment white blood cell (WBC) count and the pretreatment neutrophil count in the peripheral blood, and roughly 70% of patients who displayed pretreatment leukocytosis (N10.000 μ/l) or neutrophilia (N7500 μ/l) exhibited significantly increased numbers of CD66b-positive TAN (Table 1 and Fig. 1B).
to have a greater prognostic impact in the patients with IB-IVA disease than in those with stage IVB disease (Fig. 2). As the univariate analysis might have been affected by confounding factors, we next performed a multivariate analysis. As shown in Table 4 and Supplemental Table 1, in addition to clinical stage, tumor size and intratumoral CD66b+ TAN density were also shown to be independent prognostic factors for PFS and OS. Although histology and pelvic lymph node metastasis were found to be independent predictors of PFS, they did not provide prognostic information about OS (Supplemental Table 1).
3.3. Prognostic significance of intratumoral TAN density in cervical cancer
4. Discussion
After a median follow-up period of 60 months, 131 patients (52.4%) had developed recurrence, and 110 patients (44.0%) had died of disease progression. The recurrence rate was significantly associated with the intratumoral CD66b+ TAN density (Table 2). When the pattern of recurrence was examined according to TAN density, it was found that the patients in the TAN-High group were more likely to develop pelvic recurrence than those in the TAN-Low and TAN-Med groups (p b 0.0001). To explore the prognostic significance of intratumoral TAN density in cervical cancer patients that were treated with definitive radiotherapy, we investigated the tumor response to radiotherapy according to intratumoral TAN density (Table 3). The CR ratio of the patients in the TAN-High group was significantly lower than those of the patients in the TAN-Low and TAN-Med groups (p b 0.0001). We then conducted univariate analyses of PFS and OS. The univariate analyses showed that intratumoral CD66b + TAN density, age, clinical stage, tumor size, the pretreatment hemoglobin level, and pelvic lymph node involvement were correlated with PFS and OS (Table 4 and Supplemental Table 1). Although non-SCC histology was found to be an independent predictor of PFS, it did not provide prognostic information about OS. Statistically significant differences in PFS and OS were observed according to intratumoral CD66b+ TAN density (log-rank; p b 0.0001 and p b 0.0001, respectively; Fig. 1C). The estimated 5-year PFS rates of the patients in the TAN-Low, TAN-Med, and TAN-High groups were 73.8%, 50.7%, and 11.3%, respectively. The estimated 5year OS rates of the patients in the TAN-Low, TAN-Med, and TAN-High groups were 77.5%, 58.5%, and 17.0%, respectively. When examined according to clinical stage, the intratumoral CD66b+ TAN density seemed
To the best of our knowledge, this is the first study to identify intratumoral TAN density as an independent poor prognostic factor for survival in patients with cervical cancer that were treated with definitive radiotherapy. In the current study, an increased intratumoral TAN density was found to be significantly associated with shorter PFS. Moreover, an increased intratumoral TAN density was shown to be significantly correlated with lymph node metastasis, a lower CR rate, and a higher recurrence rate. Thus, although this study was retrospective in nature its findings strongly suggest that TAN exhibit pro-tumorigenic activity in TME. Our findings are consistent with those of a previous study that demonstrated that the TAN count is an independent predictor of recurrence in cervical cancer patients [18]. The patients included in the latter study had early stage disease and were mainly treated with surgery. In contrast, in the current study most patients had advanced stage disease and were treated with definitive radiotherapy. Collectively, these results suggest that TAN are prognostically significant in cervical cancer patients, irrespective of clinical stage or the initial treatment. The findings obtained in the current study could have important clinical implications. By subjecting biopsy samples obtained at the initial diagnosis to immunostaining for CD66b before initiating radiotherapy, it might be possible to predict which patients are at high risk of developing recurrence after definitive radiotherapy. Moreover, as 91% and 90% of the cervical cancers with increased TAN densities were found in patients who displayed pretreatment leukocytosis and neutrophilia, respectively, by performing simple and low-cost peripheral blood examinations before radiotherapy, it might be possible to identify
472
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Fig. 1. Prognostic significance of intratumoral TAN density. A, Intratumoral TAN density was evaluated based on CD66b immunoreactivity CD66b immunoreactivity was scored, as described in the Methods. Representative photographs from the TAN-Low, TAN-Med, and TAN-High groups are shown. B, (i), Correlation between the peripheral WBC count and intratumoral TAN density A positive correlation was detected between the peripheral WBC count and intratumoral TAN density (analysis of variance, p b 0.0001). (ii), Correlation between the peripheral neutrophil count and intratumoral TAN density A positive correlation was detected between the peripheral neutrophil count and intratumoral TAN density (analysis of variance, p b 0.0001). C, Kaplan-Meier estimates of survival according to intratumoral TAN density (i), PFS The patients in the TAN-High group exhibited significantly shorter PFS than those in the TAN-Med and TAN-Low groups (TAN-Low vs. TAN-Med, log-rank, p = 0.009; TAN-Low vs. TAN-High, log-rank, p b 0.0001; TAN-Med vs. TAN-High, logrank, p b 0.0001). (ii), OS The patients in the TAN-High group displayed significantly shorter OS than those in the TAN-Med and TAN-Low groups (TAN-Low vs. TAN-Med, log-rank, p = 0.02; TAN-Low vs. TAN-High, log-rank, p b 0.0001; TAN-Med vs. TAN-High, log-rank, p b 0.0001).
patients who are at high risk of developing recurrence. Thus, patients with increased intratumoral TAN densities, neutrophilia, or leukocytosis should be considered for closer follow-up.
Considering the high frequency of treatment failure after platinum-based chemoradiotherapy for cervical cancer, it is of utmost importance to develop effective treatments for this patient
Table 2 Pattern of recurrence. Total (n = 250)
CD66b IHC
p-Value
Low (n = 41)
Medium (n = 156)
High (n = 53)
Number of recurrence
131 (52.4%)
10 (24.4%)
74 (47.4%)
47 (88.7%)
p b 0.0001
Pattern of recurrence Pelvic Distant only Pelvic + distant
79 (31.6%) 53 (21.2%) 17 (7.2%)
7(17.1%) 3 (7.3%) 1 (2.4%)
41 (26.3%) 34 (21.8%) 8 (5.1%)
31 (58.5%) 16 (30.2%) 8 (15.1%)
p b 0.0001 p = 0.03 p = 0.02
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
473
Table 3 Response to definitive radiotherapy. CD66b immunohistochemistry
Response
CR (n = 211) Non CR (n = 39)
p-Value
Low (%) (n = 41)
Medium(%) (n = 156)
High (%) (n = 53)
39 (95.1)
138 (88.5)
34 (64.2)
2 (4.9)
18 (11.5)
19 (35.9)
p b 0.0001
CR; complete response.
population. One possible strategy is the use of intensive concurrent chemotherapy to improve the efficacy of chemoradiotherapy. Recent studies, including a meta-analysis, have shown that the concurrent administration of platinum-based doublet chemotherapy and radiotherapy is associated with improved OS and PFS compared with radiotherapy and concurrent cisplatin chemotherapy in patients with cervical cancer [2], suggesting that the concurrent administration of platinum-based doublet chemotherapy and pelvic radiotherapy might result in improved survival and locoregional control. Another possible strategy is the addition of consolidation chemotherapy after definitive CCRT. A 2008 meta-analysis of randomized controlled trials strongly suggested that the addition of consolidation chemotherapy to CCRT is beneficial [4]. Currently, the efficacy of administering platinum-based consolidation chemotherapy against cervical cancer after pelvic CCRT is being evaluated in phase III studies in both the postoperative adjuvant radiotherapy [21] and definitive radiotherapy settings [22].
Table 4 The univariate/multivariate cox-proportional hazard regression analysis of prognostic factors for progression-free-survival. Variables
CD66b IHC Low Medium High Age (years) ≤50 51≤ Clinical stage IA2-IIB IIIA-IVA eIVB Histology SCC Non SCC Tumor size (mm) e≤60 61≤ Pretreatment hemoglobin level (mg/dl) ≤10.0 10.0b Pelvic lymph node metastasis No Yes Duration of RT (months) ≤55 56≤
Univariate
Multivariate
Hazard 95%CI ratio
p-Value
1 2.32 7.66
1.26–4.79 4.01–16.2
b0.0001 1 1.81 4.95
1 0.48
0.33–0.73
1 0.81
0.53–1.29
1 2.15 7.29
1.44–3.27 4.33–12.2
b0.0001 1 1.33 2.64
0.84–2.14 1.38–5.01
1 2.31
1.17–4.10
1 2.50
1.23–4.64
1 2.51
1.74–3.59
b0.0001 1 1.58
1.03–2.40
1 0.45
0.29–0.72
1 0.97
0.60–1.60
1 3.13
1 2.20–4.43 b0.0001 2.24
1.48–3.38
1 1.48
0.73–2.68
0.0002
0.006
0.0002
0.22
Hazard 95%CI Ratio
1 0.76
p-Value
b0.0001 0.97–3.75 2.51–10.7 0.37
0.01
0.01
0.04
0.89
0.0001
0.45 0.34–1.51
A third option is the development of a novel treatment strategy that targets neutrophils themselves. It has been reported that TAN production can be induced by upregulating the expression of hematological growth factors, including transforming growth factor-β, granulocyte colony-stimulating factor, or granulocyte macrophage colony-stimulating factor [23]. Thus, these mediators, which stimulate neutrophil production, might have therapeutic effects in cervical cancer patients. The inhibition of TAN migration into tumors might also be an effective strategy. It has recently been shown that tumor-derived cysteine-x-cysteine ligands (such as CXCL2) and the cysteine-X-cysteine chemokine receptor-2 (CXCR2), which are expressed on the surfaces of neutrophils, (the CXCL2/CXCR2 axis) play an important role in TAN migration into tumors [16,23]. CXCR2 antagonists were demonstrated to have antitumor effects on TAN in a murine tumor study [24] and have been shown to be safe in humans [25]. Thus, the efficacy of novel treatments targeting the abovementioned molecules needs to be investigated in the future. The last strategy may be targeting the mechanisms of TAN generation. A recent study have shown that Human papillomavirus (HPV) positive cervical cancer cells express IL-16 and IL-17, stimulate the myeloid cell proliferation in the bone marrow, and increase the leukocyte recruitment to the tumor microenvironment [26]. Thus, HPV might be involved in the induction of TAN in cervical cancer patients. Recently, platelet-neutorphil interaction in cancer has gained attentions [27]. Roughly a decade ago, a phenomenon of platelets adhering to neutrophils was described and referred to as platelet satellitism, but the cause or significance of the finding was not further explored [28]. However, recent investigations strongly suggested that the enhanced platelet activation in cancer patients does not only contribute to thrombosis but also to tumor progression by stimulating neutrophil function [27]. Thus, we think that the association between HPV status, platelet counts and TANs density in cervical cancer should be investigated in the future. The limitations of our study have to be addressed. The first is that our study was conducted at a single institution. The retrospective nature of the current study is the second limitation. Third, as this study covers a long period, the contents of patient work-ups and radiotherapy, salvage treatment, and supportive care regimens have changed, which might have affected patient survival. To eliminate these potential biases, prospective multi-institutional investigations need to be conducted. Fourth, in the current study we employed CD66b+ as a marker to identify TAN. However, the optimal marker for TAN has not been defined. Although CD66b+ seems to be a reasonable marker for identifying TAN, further studies investigating more specific markers are needed. Finally, although the prognostic significance of TAN was demonstrated in the current study, the mechanisms by which TAN promote cervical cancer progression remain unknown. To improve patient prognosis, future mechanistic studies of this issue are required. In conclusion, an elevated CD66b+ TAN count is an independent prognostic factor for short PFS in cervical cancer patients that are treated with definitive radiotherapy. Larger studies in the prospective setting are required to validate our findings.
474
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Fig. 2. Impact of intratumoral TAN density on survival according to clinical stage. A, FIGO stage IB-IIB patients (i) PFS A higher intratumoral TAN density was found to be significantly correlated with shorter PFS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). (ii) OS A higher intratumoral TAN density was demonstrated to be significantly correlated with shorter OS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). B, FIGO stage IIIA-IVA patients (i), PFS A higher intratumoral TAN density was found to be significantly correlated with shorter PFS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). (ii), OS A higher intratumoral TAN density was demonstrated to be significantly correlated with shorter OS (TAN-High vs. TAN-Med or TAN-Low, log-rank, p b 0.0001). C, FIGO stage IVB patients (i) PFS The patients in the TAN-High group exhibited slightly shorter PFS than those in the TAN-Med and TAN-Low groups, but the difference was not statistically significant (log-rank, p = 0.24) (ii) OS The patients in the TAN-High group displayed slightly shorter OS than those in the TAN-Med and TAN-Low groups, but the difference was not statistically significant (log-rank, p = 0.08).
Y. Matsumoto et al. / Gynecologic Oncology 145 (2017) 469–475
Conflicts of interest statement The authors declare that they have no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2017.02.009. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108. [2] F. Petrelli, A. De Stefani, F. Raspagliesi, D. Lorusso, S. Barni, Radiotherapy with concurrent cisplatin-based doublet or weekly cisplatin for cervical cancer: a systematic review and meta-analysis, Gynecol. Oncol. 134 (2014) 166–171. [3] Chemoradiotherapy for Cervical Cancer Meta-analysisCollaboration (CCCMAC), Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer: individual patient data meta-analysis, Cochrane Database Syst. Rev. (2010), CD008285. . [4] Chemoradiotherapy for Cervical Cancer Meta-AnalysisCollaboration, Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer: a systematic review and meta-analysis of individual patient data from 18 randomized trials, J. Clin. Oncol. 26 (2008) 5802–5812. [5] S. Chakraborty, B. Keenportz, S. Woodward, J. Anderson, D. Colan, Paraneoplastic leukemoid reaction in solid tumors, Am. J. Clin. Oncol. 38 (2015) 326–330. [6] R. Takahashi, S. Mabuchi, M. Kawano, T. Sasano, Y. Matsumoto, H. Kuroda, T. Hisamatsu, K. Kozasa, K. Sawada, T. Hamasaki, T. Kimura, Prognostic significance of systemic neutrophil and leukocyte alterations in surgically treated endometrial cancer patients: a monoinstitutional study, Gynecol. Oncol. 137 (2015) 112–118. [7] K. Matsuo, M.S. Hom, A. Moeini, H. Machida, N. Takeshima, L.D. Roman, A.K. Sood, Significance of monocyte counts on tumor characteristics and survival outcome of women with endometrial cancer, Gynecol. Oncol. 138 (2015) 332–338. [8] K. Milne, C. Alexander, J.R. Webb, W. Sun, K. Dillon, S.E. Kalloger, C.B. Gilks, B. Clarke, M. Köbel, B.H. Nelson, Absolute lymphocyte count is associated with survival in ovarian cancer independent of tumor-infiltrating lymphocytes, J. Transl. Med. 10 (2012) 33. [9] A.J. Templeton, M.G. McNamara, B. Šeruga, F.E. Vera-Badillo, P. Aneja, A. Ocaña, R. Leibowitz-Amit, G. Sonpavde, J.J. Knox, B. Tran, I.F. Tannock, E. Amir, Prognostic role of neutrophil-to-lymphocyte ratio in solid tumors: a systematic review and meta-analysis, J. Natl. Cancer Inst. 106 (2014). [10] A.J. Templeton, O. Ace, M.G. McNamara, M. Al-Mubarak, F.E. Vera-Badillo, T. Hermanns, B. Seruga, A. Ocaña, I.F. Tannock, E. Amir, Prognostic role of platelet to lymphocyte ratio in solid tumors: a systematic review and meta-analysis, Cancer Epidemiol. Biomark. Prev. 23 (2014) 1204–1212. [11] B.M. Tavares-Murta, M.A. Mendonça, N.L. Duarte, J.A. da Silva, T.S. Mutão, C.B. Garcia, E.F. Murta, Systemic leukocyte alterations are associated with invasive uterinecervical cancer, Int. J. Gynecol. Cancer 20 (2010) 1154–1159. [12] S. Mabuchi, Y. Matsumoto, F. Isohashi, Y. Yoshioka, H. Ohashi, E. Morii, T. Hamasaki, K. Aozasa, D.G. Mutch, T. Kimura, Pretreatment leukocytosis is an indicator of poor prognosis in patients with cervical cancer, Gynecol. Oncol. 122 (2011) 25–32.
475
[13] S. Mabuchi, Y. Matsumoto, T. Hamasaki, M. Kawano, T. Hisamatsu, D.G. Mutch, T. Kimura, Elevated white blood cell count at the time of recurrence diagnosis is an indicator of short survival in patients with recurrent cervical cancer, Int. J. Gynecol. Cancer 22 (2012) 1545–1551. [14] S. Mabuchi, Y. Matsumoto, M. Kawano, K. Minami, Y. Seo, T. Sasano, R. Takahashi, H. Kuroda, T. Hisamatsu, A. Kakigano, M. Hayashi, K. Sawada, T. Hamasaki, E. Morii, H. Kurachi, N. Matsuura, T. Kimura, Uterine cervical cancer displaying tumor-related leukocytosis: a distinct clinical entity with radioresistant feature, J. Natl. Cancer Inst. 106 (2014). [15] C. Onal, O.C. Guler, B.A. Yildirim, Prognostic use of pretreatment hematologic parameters in patients receiving definitive chemoradiotherapy for cervical cancer, Int. J. Gynecol. Cancer 26 (2016) 1169–1175. [16] K. Moses, S. Brandau, Human neutrophils: their role in cancer and relation to myeloid-derived suppressor cells, Semin. Immunol. 28 (2016) 187–196. [17] A.J. Gentles, A.M. Newman, C.L. Liu, S.V. Bratman, W. Feng, D. Kim, V.S. Nair, Y. Xu, A. Khuong, C.D. Hoang, M. Diehn, R.B. West, S.K. Plevritis, A.A. Alizadeh, The prognostic landscape of genes and infiltrating immune cells across human cancers, Nat. Med. 21 (2015) 938–945. [18] A. Carus, M. Ladekarl, H. Hager, B.S. Nedergaard, F. Donskov, Tumour-associated CD66b + neutrophil count is an independent prognostic factor for recurrence in localised cervical cancer, Br. J. Cancer 108 (2013) 2116–2122. [19] S. Mabuchi, H. Ugaki, F. Isohashi, Y. Yoshioka, K. Temma, N. Yada-Hashimoto, T. Takeda, T. Yamamoto, K. Yoshino, R. Nakajima, C. Kuragaki, K. Morishige, T. Enomoto, T. Inoue, T. Kimura, Concurrent weekly nedaplatin, external beam radiotherapy and high-dose-rate brachytherapy in patients with FIGO stage IIIb cervical cancer: a comparison with a cohort treated by radiotherapy alone, Gynecol. Obstet. Investig. 69 (2010) 224–232. [20] S. Mabuchi, F. Isohashi, S. Maruoka, T. Hisamatsu, T. Takiuchi, Y. Yoshioka, T. Kimura, Post-treatment follow-up procedures in cervical cancer patients previously treated with radiotherapy, Arch. Gynecol. Obstet. 286 (2012) 179–185. [21] NCT01755845. Clinical Trials.gov. A Service of the U.S. National Institute of Health. (http://www.clinicaltrials.gov (Accessed December 1, 2016)). [22] NCT01414608. Clinical Trials.gov. A Service of the U.S. National Institute of Health. (http://www.clinicaltrials.gov (Accessed December 1, 2016)). [23] Y. De Vlaeminck, A. González-Rascón, C. Goyvaerts, K. Breckpot, Cancer-associated myeloid regulatory cells, Front. Immunol. 7 (2016) 113. [24] T. Jamieson, M. Clarke, C.W. Steele, M.S. Samuel, J. Neumann, A. Jung, D. Huels, M.F. Olson, S. Das, R.J. Nibbs, O.J. Sansom, Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis, J. Clin. Invest. 122 (2012) 3127–3144. [25] S. Jurcevic, C. Humfrey, M. Uddin, S. Warrington, B. Larsson, C. Keen, The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions, Br. J. Clin. Pharmacol. 80 (2015) 1324–1336. [26] S.C. Stone, R.A. Rossetti, A.M. Lima, A.P. Lepique, HPV associated tumor cells control tumor microenvironment and leukocytosis in experimental models, Immun. Inflamm. Dis. 2 (2014) 63–75. [27] A.K. Olsson, J. Cedervall, NETosis in cancer - platelet-neutrophil crosstalk promotes tumor-associated pathology, Front. Immunol. 7 (2016) 373, http://dx.doi.org/10. 3389/fimmu.2016.00373. [28] C.R. Kjeldsberg, Letter: platelet satellitism, N. Engl. J. Med. 290 (1974) 165, http://dx. doi.org/10.1056/NEJM197401172900318.