Ann Thorac Surg 2005;80:1046-1051
© 2005 The Society of Thoracic Surgeons
Original article: General thoracic
Can Surgery for Cancer Accelerate the Progression of Secondary Tumors Within Residual Minimal Disease at Both Local and Systemic Levels?
Syed S.A. Qadri, FRCSI
a
,
Jiang-Huai Wang, PhD
b
,
J.C. Coffey, AFRCSI, PhD
a
,
Mahmood Alam, FRCSI
a
,
Aonghus O’Donnell, FRCSI
a
,
Thomas Aherne, FRCSI
a
,
Henry P. Redmond, MCh, FRCSI
a
,
b
,
*
a Cork University Hospital, Wilton, Cork, Republic of Ireland
b University College Cork, Wilton, Cork, Republic of Ireland
Accepted for publication March 16, 2005.
* Address reprint requests to Professor Redmond, Department of Academic Surgery, Cork University Hospital, Wilton, Cork, Republic of Ireland (Email: redmondhp{at}shb.ie).
Presented at the Forty-first Annual Meeting of The Society of Thoracic Surgeons, Tampa, FL, Jan 24–26, 2005.
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Abstract
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BACKGROUND: Surgical removal remains the principal treatment modality in the management of lung cancer. Our aim is to characterize the effects of tumor removal on subsequent tumor recurrence at both local and systemic levels.
METHODS: C57/BL6 mice [10/group] underwent a mammary fat pad inoculation of 3LL cells [5 x 105/animal] and were divided into two groups. Group 1 served as control while mice in group 2 were further subdivided into groups 2A and 2B. After 2 weeks, all mice in 2A were killed, and primary tumors and lungs were excised. At 2 weeks, primary tumors were excised completely for all mice in group 2B. These mice were then recovered and recurrent tumor growth evaluated for a further 2 weeks. Four weeks from the onset of the study, all remaining primary tumors and lungs were excised from groups 1 and 2.
RESULTS: After 4 weeks undisturbed growth, primary tumors in group 1 reached a mean size of 2.85 ± 0.33 cm. After 2 weeks growth, primary tumors in groups 2A and 2B were comparable at 1.36 ± 0.44 m and 1.53 ± 0.29 cm, respectively. Two weeks after primary tumor excision, recurrent tumors in group 2B had reached a mean size of 2.65 ± 0.74 cm. Moreover, for several animals, recurrent tumors rapidly reached similar volumes to that of primary tumors in group 1. Primary tumors were typically encapsulated and nonadherent. In contrast, recurrent tumors were locally invasive and adherent to chest wall and wound. Interestingly, pulmonary metastatic burden was increased in group 2B relative to group 1. Histologic examination revealed increased mitosis in recurrent tumors when compared with primary tumors.
CONCLUSIONS: Tumor removal is followed by accelerated growth of locally recurrent tumors and metastases. Moreover, recurrent tumors are more locally invasive than primary tumors. These findings strongly indicate that resection may be followed by tumor progression in residual disease.
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Introduction
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Lung cancer is currently one of the leading causes of death in both men and women. Despite a number of treatment options for lung cancer including resection, the status of the treatment of lung cancer is no more encouraging and worldwide mortality rates remain unacceptably high. Approximately one million people worldwide die of this disease each year [1]. Increasing evidence demonstrates that the process of tumor removal itself stimulates accelerated regrowth. This phenomenon may account for the poor long-term survival even after apparently curative resection [2].
For localized lung cancer the expectations of treatment, while better, remain limited. For patients with potentially resectable lung cancer, 5-year survival rates are 67% for stage-IA, 57% for stage-IB, 55% for stage-IIA, 39% for stage-IIB, and 23% for stage-IIIA [3]. The Lung Cancer Study Group has reported that lung cancer recurrence rates are 75% after limited resection due to tripling of local recurrence [4]. This results in a 50% increase in mortality [4]. Studies have shown that 5-year recurrence rates are up to 39% after complete excision of stage-I lung cancer [5]. Most recurrences are systemic but local recurrence occurs as well [6].
Various studies have shown that primary tumor excision somehow accelerates minimal residual disease resulting in rapid cancer recurrence [2, 7, 8]. It has been reported [9, 10] that primary tumor excision in various cancers results in rapid acceleration of local residual disease and distant dormant metastases. Nissen-Meyer and colleagues [11] have shown that recurrence and death rates are significantly reduced in patients who were treated with postoperative chemotherapy after mastectomy. The Medical Research Council (MRC) OEO2 study has shown that preoperative chemotherapy improves significant overall and disease-free survival compared with surgical resection alone in patients with resectable esophageal cancer [12]. The beneficial effects of perioperative chemotherapy point to the activities of factors present during the perioperative period, which modify the subsequent growth of residual disease. Perioperative chemotherapy appears to protect against these factors. Several mechanisms have been proposed by which cytoreduction may alter growth of residual neoplastic disease. These include mechanisms that disseminate neoplasia during tumor manipulation [13], facilitate neoplasia during postoperative immunosuppression [14], or alter biological properties of neoplastic cells (ie, increased mitosis and decreased apoptosis) in contrast to primary cancer tissue [15]. It has also been reported that two endogenous angiogenesis inhibitors (ie, angiostatin and endostatin), secreted by the primary neoplasm, play a role in the inhibition of metastatic growth and that resection of primary tumor removes this inhibitory effect on secondary tumor growth [16].
Herein we describe a model in which complete tumor removal results in accelerated local systemic recurrence. The aim of this study was to characterize the growth kinetics of local recurrence and to determine the biological phenomena that underpin these kinetics.
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Material and Methods
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Cell Line and Reagents
The murine Lewis Lung (3LL) carcinoma cell line was a generous gift from Dr Alan Alfieri (Albert Einstein College of Medicine, Department of Pathology, New York). Cells were grown in Dulbecco’s modified eagle medium (DMEM) culture medium supplemented with 10% fetal calf serum, penicillin (100 units/mL), streptomycin sulfate (100 µg/mL), and 2.0 mM glutamine. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere and subcultured by trypsinization with 0.05% trypsin-0.02% ethylenediaminetetraacetic when cells became confluent.
Animals and In-Vivo Experiments
Six-week to 8-week-old C57BL/6 mice were used for all the experiments. These were treated in accordance with approved institutional protocols and following the guidelines of the Department of Health. Animals were bred in a standard laboratory and allowed free access to food and water in a temperature-controlled environment with a 12-hour light and dark cycle.
General Analysis of 3LL Tumor Growth After Cytoreduction
A suspension of 0.5 x 105 3LL cells in 0.2 mL of phosphate-buffered saline (PBS) was inoculated into the mammary fat pad (MFP) of C57BL/6 mice (n = 30, 10/group) (Fig 1
). Age and weight-matched animals were divided into two groups, the first of which served as an overall control (group 1) and tumors grew undisturbed for 4 weeks. Mice in group 2 were further subdivided into groups 2A and 2B. After two weeks tumor growth, all mice in 2A were killed. Primary tumors and lungs were harvested from these. At two weeks, primary tumors grown at MFP were excised completely from all mice in group 2B. All attempts were made to ensure complete excision. As all primary tumors were encapsulated, this meant that these could easily shell out in situ. These mice were then recovered and recurrent tumor growth evaluated at the site of primary implantation for a further two weeks. Four weeks from the onset of the study all remaining primary tumors and lungs were excised from groups 1 and 2.
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Fig 1. Schematic diagram. Mice are grouped and subgrouped as above. After two weeks, mice in 2A were killed. At two weeks, primary tumors were excised completely in group 2B. Four weeks from the onset of the study all animals were killed. Tumors and lungs were excised from all animals.
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During the course of the study, tumor growth kinetics and volumes were evaluated for each group. Pulmonary metastatic burden was assessed by counting macroscopic metastatic pulmonary nodules and histologic examination. Tumor invasiveness was identified both macroscopically and microscopically and defined by the involvement of locally adjacent structures such as chest wall and pleural involvement. Invasiveness was taken as implicit in the finding of metastases. All mice were survived and completed the study.
Histopathologic Analysis
All specimens were paraffin-embedded after fixation with 4% formaldehyde in PBS. Tissue sections, 7-µm thick, were stained with hematoxylin and eosin (H&E). Apoptotic and mitotic cells were enumerated.
Statistical Analysis
Results are presented as the mean ± standard deviation. Statistical analysis was performed using analysis of variance. Statistical significance was accepted at a p value less than 0.05.
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Results
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Tumor Excision is Associated With Progression in Minimal Residual Neoplastic Disease
After four weeks of undisturbed growth, primary tumors in group 1 reached a mean size of 2.85 ± 0.33 cm. The growth of primary tumors in groups 2A and 2B were comparable at 1.36 ± 0.44 cm and 1.53 ± 0.29 cm at 2 weeks, respectively. Two weeks after primary tumor excision in group 2B, recurrent tumors had reached a mean size of 2.65 ± 0.74 cm (Fig 2). This was significantly greater than primary tumor growth at 2 weeks (p = 0.001) and similar to that seen in undisturbed primary tumors at 4 weeks; ie, 2.85 ± 0.33 cm (p = 0.003). Recurrent tumors in group 2 were also significantly greater in volume than corresponding primary tumors at 5.84 ± 2.92 cm-3 versus 1.61 ± 0.62 cm-3, respectively (p = 0.002). When primary tumor growth at 2 weeks in group 2A was compared with recurrent tumor growth at 2 weeks in group 2B, there was a significant increase in mean tumor volume in the recurrent tumors (1.61 ± 0.62 cm-3vs 5.84 ± 2.92 cm-3 [p = 0.003]) and it was similar to that seen in undisturbed primary tumors at 4 weeks (7.1 ± 1.72 cm-3vs 5.84 ± 2.92 cm-3 [p = 0.003]. Similarly, recurrent tumor weights in group 2B were comparable to undisturbed primary tumors at 4 weeks in group 1 (3.48 ± 2.08 gm vs 4.99 ± 0.89 gm [p = 0.048]) (Table 1).
Tumor Cytoreduction Increases the Invasiveness of Recurrent Tumor Both Locally and Systemically
Primary tumors were typically encapsulated and nonadherent. In contrast, recurrent tumors were both locally invasive and adherent to the chest wall, underlying musculature and wound. While primary tumors were soft and friable, recurrent tumors were firm and fleshy in consistency. Interestingly, pulmonary metastases were not evident when animals in group 2A were sacrificed (ie, after 2 weeks primary tumor growth). Furthermore, only one animal in group 1 had evidence of pulmonary metastatic tumor growth after 4 weeks undisturbed primary growth. In contrast, 7 of 10 mice in group 2B had developed multiple, mostly bilateral, pulmonary metastatic nodules, evident at sacrifice (Table 2
).
Histologic examination revealed that the primary tumors were encapsulated (Fig 3A) and well differentiated, while recurrent tumors were nonencapsulated (Fig 3B), poor to moderately differentiated, and invasive into adjacent muscle (Fig 3C). Recurrent tumors were also highly cellular with increased mitosis and decreased apoptosis, relative to that seen in primary tumors (p = 0.0003) (Figs 4 and 5).
The apoptotic index and apoptosis-mitosis ratio were significantly reduced in recurrent tumors (0.68 ± 0.2 vs 0.97 ± 0.2 [p = 0.04]).
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Fig 3. Invasiveness of recurrent tumors. Photomicrograph demonstrating (A) capsulated primary tumor (magnification x 10), (B) recurrent tumor without capsule (magnification x 10), and (C) invasion of recurrent tumor cells into adjacent muscles (magnification x 10).
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Fig 4. Recurrent tumors demonstrated increased mitosis (mean numbers of mitotic cells per 10 high power fields) when compared with the corresponding primary tumors; *p less than 0.05.
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Comment
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Emerging evidence indicates that tumor cytoreduction adversely alters subsequent recurrent tumor growth. Recently we developed a novel murine model that demonstrates that cytoreduction is followed by accelerated local recurrence and that this is associated with changes in apoptosis [17]. This model confirms the previously held suspicion that recurrent tumors develop at an accelerated rate. Moreover it also confirms that similar potentiating effects occur locally as do systemically after primary tumor removal. In the present study, we demonstrated that tumor recurrence after complete excision also develops at a markedly accelerated rate. Hence, cytoreductive surgery is not a prerequisite for this phenomenon. This is important as it indicates that surgery creates an extracellular milieu that promotes the resurgent growth of minimal residual disease [2].
Little is available in the literature regarding the effects of excisional surgery on lung cancer recurrence and, in particular, on surgically induced accelerated tumor growth at local and systemic levels. We found that after complete tumor removal, recurrent tumors reached dimensions in 2 weeks that were similar to those of primary tumors that have been left to grow uninterrupted for 4 weeks. These were approximately double in size relative to their corresponding primary tumors. This phenomenon was associated with increased pulmonary metastatic burden. Furthermore, recurrence exhibited enhanced local invasion (Fig 3). As most animals in nonoperated control groups had little if any evidence of pulmonary metastases, these findings indicate that tumor removal accelerates spontaneous, and not just metastatic, growth. These findings are further supported by the observation that local recurrence exhibited enhanced invasiveness. Hence, it appears that tumor removal promotes the metastatic phenotype. While the mechanisms that underpin this are not known, it is likely that they relate to an increase in exogenous factors (ie, lipopolysaccharides) that are known to be both prometastatic and proangiogenic, as well as factors associated in the wound healing.
The acceleration in local tumor growth that followed complete tumor removal was associated with significantly reduced levels of apoptosis. A similar pattern was observed at the systemic level. These findings support the previous suggestion that tumor removal alters the neoplastic properties of residual disease at both local and systemic levels [2]
To conclude, accelerated tumor recurrent growth occurs even after complete tumor excision. This is associated with parallel increases (ie, spontaneous metastatic tumor growth) and probably reflects a more metastatic phenotype. These findings support the observation that tumor removal incurs an oncologic cost that will necessitate further investigation, and the development of targeted therapies during the perioperative period.
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Discussion
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DR M. BLAIR MARSHALL (Washington, DC): I enjoyed your talk and it generates a few questions. As you may or may not be aware, one of the first discovered inhibitors of angiogenesis, angiostatin, was actually derived from the cell line that you are using, Lewis lung carcinoma. We know that Lewis lung carcinoma has been demonstrated to produce angiostatin and that the primary tumor suppresses the growth of its metastases. If you remove the primary tumor, the lung metastases undergo exponential growth. How do you separate out the effect of surgery as a stimulus for growth versus the effect of removing the angiogenesis inhibitor?
DR QADRI: Thanks. I am aware of that. But this is not the only factor. There are multiple factors which help the secondary tumors to grow. The primary tumor secretes two anti-angiogenic hormones i.e. angiostatin and endostatin. However, manipulation of the tumor during surgery releases tumor cells in the circulation as micromets, and post-op immunocompromised status of patients facilitate these micromets to grow rapidly. Also recurrent tumors show increased mitosis and reduced apoptosis. As you mentioned, the removal of primary tumors diminish the negative feedback control system and the secondary tumors start growing. However, it does not occur only in the Lewis lung cancer cells. We have found similar effects in other human cancer cells as well.
DR MARSHALL: Have you examined this effect in other cell lines?
DR QADRI: Yes, we did. One of my colleagues has done the same experiment on other cell lines and has found same results.
DR MARSHALL: Thank you.
DR JACK A. ROTH (Houston, TX): The key issue with this study is what is the mechanism and why are these tumors recur more frequently. For example, looking at angiogenic factors that might be secreted by the tumor or a molecular analysis comparing the metastasis versus the recurrent tumor versus the primary would be very useful. Have you initiated any studies like this to look at patterns of gene expression to see if there were any factors that were being upregulated or downregulated by the manipulations?
DR QADRI: You’re right. These are not the only mechanisms that promote the secondary tumor growth. I agree with you that there are other factors. We have to consider all of these aspects as well. We have just started to look all these effects and have further plans to consider other studies as well.
DR DAVID W. JOHNSTONE (Lebanon, NH): Your conclusion that we should be applying adjuvant and neoadjuvant therapy on the basis of these findings is belied by the fact that we haven’t seen a huge difference in the patterns of recurrence in the adjuvant or neoadjuvant trials in lung cancer in humans.
DR QADRI: You’re right. In fact, we continued our study to see the effect of COX-2 inhibitors on recurrent tumors. We treated with the COX-2 inhibitor after excision of the primary tumor and found a significant reduction in the recurrent tumor growth. But you are right; we need further adjuvant and neoadjuvant trials to look at all of these effects in lung cancer in humans.
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Fig 5. Tumor excision alters the biological behavior of recurrent tumors. (A) Increased mitosis (p = 0.0003) and decreased apoptosis in recurrent tumors. (B) Primary tumor with reduced mitosis and few apoptotic cells as assessed using hematoxylin & eosin histology, revealing a decreased apoptosis-mitosis ratio in recurrent tumors (0.68 ± 0.2 versus 0.97 ± 0.2 – p = 0.04).
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Acknowledgments
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We thank Qiang Di Wu and Siobhan Blankson, and the staff of the biological services unit, University College Cork, for their technical support.
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Abstract
Introduction
= group 1; 
= group 2.