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Abstract

Objective

To investigate if the administration of neoadjuvant chemotherapy (NACT) reduces pelvic lymph node metastasis by inducing tumour cell apoptosis in patients with cervical cancer.

Methods

This study enrolled patients with stage Ib₂–IIb cervical cancer who underwent surgery with (NACT group) or without (control group) prior cisplatin-based chemotherapy. Immunohistochemical staining of caspase-3 and an in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay were used to measure the levels of apoptosis in primary tumours and pelvic lymph nodes.

Results

A total of 185 patients participated in the study: 102 in the NACT group and 83 in the control group. Treatment was considered to be clinically effective in 69.6% (71/102) of the NACT group. The rate of metastasis in the NACT group (20.6%; 21/102) was significantly lower than the control group (42.2%; 35/83). The level of caspase-3 immunostaining and the rate of apoptosis in primary tumours and pelvic lymph nodes in the NACT group were significantly higher than in the control group.

Conclusions

NACT appeared to limit pelvic node metastasis by inducing tumour cell apoptosis in patients with cervical cancer.

Keywords

Cervical cancer neoadjuvant chemotherapy apoptosis nodal metastasis

Introduction

In the past two decades, neoadjuvant chemotherapy has been shown to be effective in improving short-term treatment efficacy when used prior to surgical resection in early stage, bulky cervical cancer.^1,2 Neoadjuvant chemotherapy can reduce local lesion volume, alleviate tumour burden, increase the likelihood of being able to operate, and decrease subclinical lesions within lymph vessels and other pathological risk factors.^3,4 However, there are few reports on the effects of neoadjuvant chemotherapy in controlling lymph node metastasis.⁵

This present study compared the levels of apoptosis in primary tumours and pelvic lymph nodes from patients with cervical cancer who had been treated with neoadjuvant chemotherapy prior to surgery with those of patients who underwent surgery immediately after diagnosis without neoadjuvant chemotherapy.

Patients and methods

Patient population

This retrospective study analysed the clinical and pathological data collected from consecutive patients with stage Ib₂–IIb cervical cancer who received surgical treatment in the Department of Gynaecology and Obstetrics, Xiangya Hospital of Central South University, Changsha, Hunan Province, China, between February 2002 and October 2010. Patients either underwent surgery without neoadjuvant chemotherapy (control group) or they underwent neoadjuvant chemotherapy prior to surgery (NACT group). Complete pelvic lymphadenectomy up to the common iliac nodes was performed in all patients, while sampling of para-aortic nodes was limited to patients with suspected nodal metastasis at surgical exploration.

Tissue samples were collected from all patients during surgery according to the pathological results of the pelvic lymph node examinations made during surgery. If patients showed lymph node metastasis, positive lymph nodes were selected for caspase-3 detection using immunohistochemistry (see below); otherwise, lymph nodes were selected randomly. Caspase-3 was measured as a marker of the level of apoptosis. Samples of primary tumours were also collected for caspase-3 immunohistochemistry. Lymph node samples were collected from all patients during surgery, but only a proportion of patients provided samples of primary tumours because of the difficulty associated with collecting these samples.

The study protocol was approved by the Ethics Committee of Central South University. All patients provided written informed consent to participate in the study.

Caspase-3 immunohistochemistry

All surgical tissue samples used in this immunohistochemical study had been previously fixed in 10% neutral-buffered formalin and embedded in paraffin wax. Immunohistochemical staining was performed on 4-µm-thick sections of whole tissue using the Leica BOND-MAX™ fully automated immunohistochemical system (Leica Microsystems, Shanghai, China). After the sections had been deparaffinized and rehydrated in a descending series of alcohol solutions, they were heated in a 900-W microwave oven at maximum power for 15 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval, then cooled to room temperature. Rabbit antihuman caspase-3 polyclonal antibody (SC-7148; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied at a 1:100 dilution overnight at 4℃. The sections were then washed three times in 0.1 M phosphate-buffered saline (PBS; pH 7.2) followed by incubation in horseradish peroxidase-conjugated goat antirabbit antibody (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 30 min. The sections were then washed three times in 0.1 M PBS (pH 7.2). Immunostaining was visualized using a VECTASTAIN® Universal Elite ABC Kit (PK-6200; Vector Laboratories, Burlingame, CA, USA).

The slides were evaluated using light microscopy by two pathologists (W.P. and Y.Z.) to improve the accuracy of the results. If there was a discrepancy in the interpretation of the same slide, the evaluation was repeated until consensus was obtained. Nonrepresentative samples or samples with only a few tumour cells (at ×100 magnification) were excluded from the data analysis. Immunoreactivity was quantified within five random high-power fields at ×100 magnification. A high-resolution pathological image analysis system (HPIAS-1000; Champion Image, Wuhan, China) was used for the quantitative analysis of the levels of caspase-3 immunostaining. The mean integral optical density (IOD) of each visual field was measured automatically using a personal computer (ThinkPad E40; Levon, Beijing, China).

Cell apoptosis detection

Apoptotic cells were detected in samples of primary tumour and pelvic lymph nodes using a commercially available terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay kit (In Situ Cell Death Detection Kit, POD; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Dark brown staining indicated positive TUNEL staining when the slides were examined under a light microscope (Olympus BX53® Research Microscope; Olympus Optical, Tokyo, Japan). Cells were manually examined in five random high-power (×100 magnification) fields (>1000 cells) and the apoptosis index (AI) was determined as follows: AI = number of positively stained cells/total number of cells counted.

Treatment options and clinical observations

Patients with stage Ib₂–IIb cervical cancer in the NACT group received cisplatin-based chemotherapy either administered as intravenous chemotherapy or as femoral arterial chemotherapy. Patients in the NACT group received either one, two or three courses of neoadjuvant chemotherapy prior to surgery according to the clinical efficacy of the chemotherapy and the aggressiveness of the tumour. The outcome of the chemotherapy was estimated at 3 weeks after the final course of chemotherapy was administered and immediately before surgery was undertaken. It was defined as complete remission, partial remission, stable disease and progressive disease.⁶ Complete and partial remission were considered to indicate that the anticancer therapy had been clinically effective (i.e. remission induced), while stable and progressive disease were considered to indicate that the anticancer therapy had been clinically ineffective. Patients with stage Ib₂–IIa cervical cancer in the control group received radical hysterectomy and pelvic lymphadenectomy after diagnosis. Pathological remission was defined as having occurred when the pathological results after surgery showed no evidence of invasive carcinomas.

Statistical analyses

Data are expressed as mean ± SD (all SDs were < 5%) of three experiments. All statistical analyses were performed using the SPSS® statistical package, version 18.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Statistical comparisons between the groups were undertaken using Student’s t-test. The association between the AI in the pelvic lymph nodes and the AI in the primary tumours was analysed in all study participants using Pearson’s correlation coefficient analysis. A P-value < 0.05 was considered statistically significant.

Results

A total of 185 patients with stage Ib₂–IIb cervical cancer participated in the study. The NACT group comprised 102 patients who received cisplatin-based chemotherapy prior to surgery; of these, 77 patients received intravenous chemotherapy and 25 patients received femoral arterial chemotherapy. In the NACT group, 68 patients were treated with one course of chemotherapy, 25 were treated with two courses of chemotherapy, and nine were treated with three courses of chemotherapy. In the control group, 83 patients received radical hysterectomy and pelvic lymphadenectomy after diagnosis. Due to the difficulties of sampling from patients during surgery, primary tumour samples were only collected from 49 patients in each group. Table 1 presents the demographic and clinical characteristics of the study participants. There were no significant differences between the four groups of patients.

Table 1.

Demographic and clinical characteristics of patients with cervical cancer who were either treated with surgery alone (control group) or with neoadjuvant chemotherapy prior to surgery (NACT group).

Characteristic	NACT group: pelvic lymph nodes, n = 102	Control group: pelvic lymph nodes, n = 83	NACT group: primary tumours, n = 49^a	Control group: primary tumours, n = 49^a
Age, years	40.3 ± 7.4	41.8 ± 8.5	39.4 ± 8.1	40.9 ± 7.9
Clinical stage
Ib₂	40	55	18	42
IIa	30	28	15	7
IIb	32	0	16	0
Pathological types
Squamous carcinoma	85	51	41	43
Adenosquamous carcinoma	7	10	2	5
Adenocarcinoma	10	22	6	1

Data presented as mean ± SD or n of patients.

All patients in each group provided samples of pelvic lymph nodes for analysis, but only 49 patients in each group provided samples of primary tumour.

No statistically significant between-group differences (P ≥ 0.05, Student’s t-test).

The immunohistochemical analyses demonstrated that caspase-3 protein was located mainly in the cytoplasm and in part of the nucleus. The mean IOD was used to represent the intensity of the caspase-3 immunostaining observed in the tissue specimens (Table 2). The results demonstrated that the immunohistochemical levels of caspase-3 were significantly increased in the pelvic lymph nodes and primary tumours from patients in the NACT group compared with the control group (P < 0.001 for both comparisons). When the patients in the NACT group were further analysed, there were no significant differences in the intensity of the caspase-3 immunostaining in either the pelvic lymph nodes or primary tumours between patients treated with the either mode of chemotherapy (intravenous chemotherapy versus femoral arterial chemotherapy) or in patients with and without lymph node metastasis. There was no significant difference between caspase-3 staining intensity in pelvic lymph nodes and primary tumours from patients in the NACT group with or without lymph node metastasis (analysed using Student’s t-test). Representative photomicrographs of caspase-3 immunostaining in metastatic lymph nodes from patients in the NACT and control groups are shown in Figure 1.

Figure 1.

Representative photomicrographs showing caspase-3 staining intensity as determined by immunohistochemistry in metastatic lymph nodes from patients with cervical cancer who were either treated with neoadjuvant chemotherapy prior to surgery (NACT group) (A) or with surgery alone (control group) (B). Scale bar, 25 µm. The colour version of this figure is available at: http://imr.sagepub.com.

Table 2.

The integrated optical density (IOD) value for caspase-3 immunostaining intensity in samples of primary tumours and pelvic lymph nodes from patients with cervical cancer who were either treated with surgery alone (control group) or with neoadjuvant chemotherapy prior to surgery (NACT group).

Groups	Pelvic lymph nodes				Primary tumours^a
Groups	n	IOD	t	Statistical significance^b	n	IOD	t	Statistical significance^b
NACT group	102	0.192 ± 0.223	49	0.153 ± 0.028
Control group	83	0.102 ± 0.048	3.472	P < 0.001	49	0.097 ± 0.016	2.982	P < 0.001
NACT group
Intravenous chemotherapy	77	0.162 ± 0.044	31	0.158 ± 0.024
Femoral arterial chemotherapy	25	0.185 ± 0.039	0.524	NS	18	0.129 ± 0.017	0.872	NS
Lymph node metastasis in the NACT group^c
Yes	21	0.227 ± 0.142	12	0.139 ± 0.027
No	81	0.154 ± 0.038	0.792	NS	37	0.145 ± 0.071	0.358	NS

Data presented as mean ± SD.

All patients in each group provided samples of pelvic lymph nodes for analysis, but only 49 patients in each group provided samples of primary tumour.

Student’s t-test.

The present analysis only focused on the influence of neoadjuvant chemotherapy on lymph node metastasis so the control group was not analysed.

NS, no significant between-group differences (P ≥ 0.05).

The results of the TUNEL assay demonstrated that labelling was mainly located in the nucleus and the AI value provided an estimate of the apoptotic status of the tissue samples (Table 3). The results demonstrated that the AIs were significantly higher in the pelvic lymph nodes and primary tumours from patients in the NACT group compared with the control group (P < 0.05 for both comparisons). When compared with the control group, patients who developed metastases but who had received neoadjuvant chemotherapy demonstrated a higher rate of apoptotic cancer cells, and the cancer cells were surrounded by apoptotic lymphocytes (Figure 2). The AI in the lymph nodes and primary tumours of patients in the NACT group who underwent femoral arterial chemotherapy was higher than that of patients who underwent intravenous administration. The AI in the lymph nodes and primary tumours of patients in the NACT group without lymph node metastasis was higher than that of patients with metastasis. None of these differences was significant, however. The AI in the pelvic lymph nodes was positively associated with the AI in the primary tumours (r = 0.316, P = 0.01; analysed in all study participants using Pearson’s correlation coefficient analysis).

Figure 2.

Representative photomicrographs showing apoptotic cells as detected using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay in metastatic lymph nodes from patients with cervical cancer who were either treated with neoadjuvant chemotherapy prior to surgery (NACT group) (A) or with surgery alone (control group) (B). In the NACT group, the cancer cells are surrounded by apoptotic lymphocytes (black arrows). Scale bar, 25 µm. The colour version of this figure is available at: http://imr.sagepub.com.

Table 3.

The apoptosis index in samples of primary tumours and pelvic lymph nodes from patients with cervical cancer who were either treated with surgery alone (control group) or with neoadjuvant chemotherapy prior to surgery (NACT group).

Groups	Pelvic lymph nodes				Primary tumours^a
Groups	n	Apoptosis index	t	Statistical significance^b	n	Apoptosis index	t	Statistical significance^b
NACT group	102	0.167 ± 0.061	49	0.159 ± 0.072
Control group	83	0.092 ± 0.031	2.647	P = 0.018	49	0.102 ± 0.053	3.504	P < 0.001
NACT group
Intravenous chemotherapy	77	0.173 ± 0.052	31	0.164 ± 0.091
Femoral arterial chemotherapy	25	0.214 ± 0.085	1.701	NS	18	0.167 ± 0.085	0.284	NS
Lymph node metastasis in the NACT group^c
Yes	21	0.145 ± 0.036	12	0.109 ± 0.034
No	81	0.172 ± 0.052	1.534	NS	37	0.142 ± 0.069	1.558	NS

Data presented as mean ± SD.

All patients in each group provided samples of pelvic lymph nodes for analysis, but only 49 patients in each group provided samples of primary tumour.

Student’s t-test.

The present analysis only focused on the influence of neoadjuvant chemotherapy on lymph node metastasis so the control group was not analysed.

NS, no significant between-group differences (P ≥ 0.05).

In the NACT group, the tumour volume was reduced in 76 patients following neoadjuvant chemotherapy. Of these patients, the treatment in 71 was categorized as being clinically effective, giving an overall remission rate of 69.6% (71/102). A total of 26 patients in the NACT group had stable disease and there were no patients with progressive disease. All patients in the NACT group showed vaginal bleeding before chemotherapy was initiated, which was relieved after chemotherapy; all patients were deemed eligible for surgery after the assessment of the efficacy of the neoadjuvant chemotherapy. The pathological results suggested that 18 out of 102 patients in the NACT group showed pathological remission after surgery and there was no evidence of invasive carcinomas. In these 18 patients, nine were stage IIb, seven were stage Ib₂, and two were stage IIa at diagnosis. No evidence of lymph node metastasis was identified in these 18 patients in the NACT group who showed pathological remission. A total of 21 patients in the NACT group had evidence of lymph node metastasis during surgery; of these patients, the treatment in 12 was categorized as being clinically effective, while nine patients were categorized as having received clinically ineffective treatment. The rate of metastasis identified during surgery in the NACT group was 20.6% (21/102), which was significantly lower than that of the control group (42.2%; 35/83) (P < 0.05).

Discussion

Lymph node metastasis is the major pathway by which primary cervical tumours metastasise, and it is an important factor affecting prognosis.^7–9 Squamous cell carcinomas, which account for the majority of cervical carcinomas, are sensitive to radiotherapy.¹⁰ Since clinical and experimental data suggest that it is difficult for chemotherapeutic drugs to reach retroperitoneal lymph nodes,^11,12 regional radiotherapy is the standard treatment option for lymph node metastasis. Randomized controlled trials have shown that pelvic lymph node metastasis in patients with locally advanced cervical cancer was significantly reduced after neoadjuvant chemotherapy.^13,14 Further research has shown that although the rate of lymph node metastasis in neoadjuvant + surgery and surgery-only groups showed no significant difference, the rate in those patients who had clinically effective chemotherapy was significantly lower than in those patients who had clinically ineffective chemotherapy.¹⁵ In the present study, the rate of metastasis to the pelvic lymph nodes in the NACT group (20.6%) was significantly lower than that of the control group (42.2%) (P < 0.05). According to the current findings, 18 out of 102 patients in the NACT group showed pathological remission after surgery and there was no evidence of lymph node metastasis or lymph vessel involvement, which suggests that the neoadjuvant chemotherapy was able to limit pelvic lymph node metastasis. In the 21 patients that developed lymph node metastasis in the NACT group, 12 were categorized as having received clinically effective treatment for the primary tumour, whereas nine had received clinically ineffective treatment. Whether the treatment failure in these nine patients can be attributed to an insufficient chemotherapeutic dose or to a lack of sensitivity of the tumour cells to the chemotherapy remains to be determined. However, the results of this present study suggest that neoadjuvant chemotherapy was advantageous in the short-term, and this helped the oncologists when selecting the most appropriate chemotherapy drugs and treatment options to use after surgery.

Carcinogenesis is associated with the failure of cells to undergo apoptosis and various antitumour therapies, including chemotherapy, aim to activate the signal transduction pathways involved in triggering apoptosis.^16,17 It does not matter whether the pathways are initiated by endogenous or exogenous signals, the final process of apoptosis is a proteolytic cascade activated by caspase.^18–20 Caspase-3 is the most important executor during cell apoptosis, and its expression level directly reflects the levels of apoptosis.^21,22 The TUNEL assay was used in this present study because it directly identifies the biochemical and morphological features of apoptosis. The results in this present study suggest that increased cell apoptosis occurred in the primary tumours and the pelvic lymph nodes after neoadjuvant chemotherapy. The levels of caspase-3 immunostaining and the rates of apoptosis in the primary tumours and the pelvic lymph nodes of the NACT group were significantly higher than in the control group. These current findings suggest that the neoadjuvant chemotherapy suppressed pelvic lymph node metastasis via the upregulation of caspase-3 and the induction of tumour cell apoptosis.

Although pelvic lymph node metastases were detected in 21 patients who received neoadjuvant chemotherapy, apoptotic tumour cells could be clearly seen in the metastatic lymph nodes (Figures 1A and 2A). The rate of apoptosis in the pelvic lymph nodes was positively associated with that in the primary tumours in all study participants. These present results indicate that neoadjuvant chemotherapy may suppress lymph node metastasis by inducing tumour cell apoptosis within the lymph nodes, and its efficacy might be associated with the responsiveness of the tumour cells to chemotherapy. Whether the occurrence of lymph node metastasis after neoadjuvant chemotherapy was due to the insensitivity of the tumour cells to the drugs or to an insufficient chemotherapeutic dose requires further investigation. In the present study, the caspase-3 immunostaining levels were significantly higher in the NACT group compared with the control group. These present findings might be explained if malignant tumour cells are able to activate and upregulate essential enzymes, such as caspase-3 and caspase-8. Mechanisms that are involved in apoptosis during metastasis might also, in turn, upregulate apoptosis-inhibiting proteins to suppress caspase activity, which would then block the apoptotic cascade and allow tumour cells to escape apoptosis.²³ This means that a high caspase-3 level might not be indicative of the overall apoptotic status of the cell, because it might fail to directly lead to an increase in the actual rate of tumour cell apoptosis if there are also high levels of apoptosis-inhibiting proteins present. Therefore, a combination of objective indicators, including apoptosis rate obtained from the TUNEL assay and electron microscopy observations of apoptotic bodies within tumour samples, are needed to confirm tumour cell apoptosis.

The results of this present study demonstrated that there were no significant differences in the intensity of the caspase-3 immunostaining or the rate of apoptosis in either the pelvic lymph nodes or primary tumours between patients treated with the either mode of chemotherapy (intravenous chemotherapy versus femoral arterial chemotherapy) or in patients with and without lymph node metastasis. The current study has several limitations, however. The number of subjects was relatively small and the number of control subjects was even smaller. The participants were Chinese women, so the findings may not be applicable to other population groups. Further studies should be done, including collection of more follow-up data, to verify the results.

In conclusion, this present study investigated the possible mechanisms of neoadjuvant chemotherapy in controlling pelvic lymph node metastases in patients with cervical cancer. The results demonstrated that both femoral arterial and intravenous neoadjuvant chemotherapy could suppress lymph node metastases by regulating the rate of cell apoptosis. However, further research is required to determine the most effective route of administration, which are the best drug regimens to use to induce tumour cell apoptosis, the timing of surgery in relation to the chemotherapy administration and the long-term effects of neoadjuvant chemotherapy on 5-year survival.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

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Detection of cell apoptosis in pelvic lymph nodes of patients with cervical cancer after neoadjuvant chemotherapy

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Patients and methods

Patient population

Caspase-3 immunohistochemistry

Cell apoptosis detection

Treatment options and clinical observations

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interest

Funding

References