Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer Yan Fu, Jia Hu, Nan Du, Shunchang Jiao, Fang Li, Xiaosong Li, Junxun Ma, Hui Zhao & Huanrong Kang To cite this article: Yan Fu, Jia Hu, Nan Du, Shunchang Jiao, Fang Li, Xiaosong Li, Junxun Ma, Hui Zhao & Huanrong Kang (2016) Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer, Journal of Chemotherapy, 28:3, 218-224, DOI: 10.1179/1973947815Y.0000000045 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000045
Published online: 14 Jul 2016.
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Date: 12 September 2016, At: 18:24
Anticancer Original Research Paper
Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer Yan Fu1, Jia Hu1, Nan Du1, Shunchang Jiao2, Fang Li2, Xiaosong Li1, Junxun Ma1, Hui Zhao1, Huanrong Kang1 1
Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China, Cancer Center, Chinese PLA General Hospital, Beijing 100853, China
2
This retrospective analysis evaluated the mechanism of bevacizumab plus chemotherapy (BVzCT) for preventing brain metastasis derived from lung cancer. From the total of 159 patients with advanced non-small cell lung cancer (NSCLC), 110 received BVzCT and 49 received CT. After medication, both groups had 15 patients with brain metastases (14 vs 31%, Pv0.05). With BVzCT treatment, 40 patients (33.89%) survived, whereas only 11 patients (18.64%) survived with CT treatment. The outcome for the BVzCT group was significantly better than that for the CT group only for vascular endothelial growth factor (VEGF)-positive patients. A post-treatment with BVzCT was significantly reduced than with CT only for patients with high carbonic anhydrase-9 (CA9) expression. This retrospective analysis provides supportive evidence that BVzCT can significantly reduce the incidence of brain metastasis in patients with advanced NSCLC compared with CT alone. Vascular endothelial growth factor -positive patients may benefit more from BV treatment and the outcomes with BV may be related to CA9 expression. Keywords: Bevacizumab, Brain metastasis, NSCLC
Introduction The incidence of brain metastasis derived from advanced non-small cell lung cancer (NSCLC) is 23–65%1 and accounts for 40–60% of brain metastases from all malignant tumours. Without treatment, the expected survival time of patients with brain metastasis is about 1 month.2–4 Although CV plus radiotherapy can reduce the incidence of extracranial metastases in patients with advanced NSCLC, it cannot reduce the incidence of brain metastases. Tumour angiogenesis is related to various factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and others. In addition, tumour angiogenesis is related to metastatic potential. Thus, anti-angiogenic therapy has become a promising cancer treatment. Basic research has confirmed that the spread and growth of tumour cells inside the brain parenchyma depend on VEGF.5 Clinical studies have also found that VEGF protein expression is related to the
Correspondence to: Nan Du, Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China. Email: [email protected]
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ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000045
prognosis of brain metastases derived from lung cancer.6 One study attempted to inhibit this signalling molecule and found that this effectively reduced brain metastasis in animal models.7 Carbonic anhydrase-9 (CA9) is a member of the carbonic anhydrase family. It is highly expressed under hypoxic conditions and is related to tumour cell proliferation, cell adhesion and tumour progression. It was reported that there was an apparent correlation between CA9 mRNA levels and VEGF expression levels in NSCLC tissues. Moreover, the levels of CA9 expression in the tissues of early stage lung cancer are closely related to prognosis, and CA9 expression in lung tissue is highly consistent with CA9 expression in brain metastasis derived from lung cancer.8 Bevacizumab is a monoclonal antibody directed against VEGF that has been shown in many clinical trials to be effective for treating brain metastases derived from lung cancer, breast cancer, malignant melanoma and others when used in conjunction with CV.9–11 However, the mechanism by which BV prevents brain metastasis and reduces the incidence of brain metastasis has not been thoroughly investigated.
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Materials and Methods Patients and clinical information Patients who were diagnosed between January 2009 and December 2010 with stage IIIB–IV NSCLC using pathological or cytological examinations were included in our retrospective analysis. Patients with stage IIIB– IV NSCLC who initially underwent systemic treatment were included. Patients who had previously undergone systemic CV for stage IIIB–IV NSCLC that subsequently relapsed were also included. The interval between the last treatment and the present treatment was more than three months. These patients had normal liver and kidney function, normal EKG readings and no vital organ dysfunction. We excluded patients with previous or concurrent primary tumours in other sites, patients with newly developed brain metastases within six courses of BV therapy, and patients who could not receive six courses of treatment due to various reasons, such as midway conversion to other therapy. Baseline evaluations were completed within 1 week before systemic CV, which included a clinical evaluation, chest CT, vital organ evaluations, including liver and bone and enhanced brain MRI examination. After systemic treatment, lesions were evaluated once every 8 weeks for both groups. In the BVzCT group, all patients received treatment with gemcitabine (1000 mg/m2) z carboplatin (ACU55–7) z BV (5 mg/kg) for six consecutive courses. Patients with no progression based on CT examinations after this treatment received BV maintenance therapy (5 mg/kg, once every three weeks) until the patient showed progression or could not tolerate drug toxicity. In the CT group, all patients received treatment with gemcitabine (1000 mg/m2) z carboplatin (ACU55–7) for six consecutive courses. After that, these patients received gemcitabine (1000 mg/m2) maintenance therapy once every 3 weeks.
Outcome evaluations The cumulative incidence of brain metastases was the main outcome and overall survival (OS) was the secondary outcome. Time to occurrence of brain metastasis (TTBM) was defined as the period from the initial diagnosis of advanced lung cancer to an imaging diagnosis of brain metastasis. The survival time was defined as the period from the initial diagnosis of advanced lung cancer to the date of death or to the end of follow-up.
VEGF expression Immunohistochemistry (IHC) was conducted as previously described. Monoclonal anti-VEGF antibody was used to incubate with tissue sections (Abcam, Cambridge, MA, UK; Catalog no. ab52917 dilution 1:100) at 4uuC overnight. After washing with PBS,
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the sections were incubated with secondary antibody, then diaminobenzidine (Dako, Glostrup, Denmark) was used for visual detection in the Envision System. A negative control was set by replacing the primary antibody with a normal murine or rabbit IgG at the same dilutions. Immunohistochemistry scoring was performed using a method described as follows: the percentage of positive cells was divided into five Grades (percentage scores): j10% (0), 11–25% (1), 26–50% (2), 51–75% (3) and w75% (4). The intensity of staining was divided into four Grades (intensity scores): no staining (0), light brown (1), brown (2) and dark brown (3). Staining positivity was determined by the formula: overall scores5percentage scorezintensity score. The total score ranged from 0 to 7, with low expression (0–2) and high expression (3–7).
Serum CA9 measurements We used an ELISA to measure serum CA9 concentrations before treatment and six weeks after treatment in both groups. Peripheral blood (5 ml) was collected in an anticoagulant tube. Within 30 minutes, a sample was spun in a centrifuge at 3000 rpm for 10 minutes at 4uuC to isolate serum. Then the soluble CA9 concentration was determined according to kit instructions (Systems Inc, Minneapolis, USA) using a 100 ml test sample
Statistical analysis STATA 10.0 software was used for statistical analyses. A t-test was used to compare mean patient age between the two groups. A chi-squared test or Fisher’s test was used compare other baseline variables. Kaplan–Meier curves were generated fort survival analysis and these curves were compared using a log-rank test. A cumulative incidence curve was generated to evaluate the cumulative risk of brain metastases. COX regression models were used for univariate and multivariate analyses to evaluate factors that influenced the occurrence of brain metastasis. All statistical tests were two tailed. A P-value of v0.05 was considered significant.
Results Baseline patient characteristics Total of 159 patients were included in this retrospective study (110 patients in the BVzCT group and 49 patients in the CT group). Their ages ranged from 37 to 77 years. The baseline characteristics of these patients are shown in Table 1. There were no significant differences between these two groups for age, at disease onset, gender, smoking history, family history, pre-treatment PS scores, pathological types, TNM stages, previous medical history or VEGF and CA9 expressions.
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Table 1 Baseline patient characteristics Bevacizumab plus chemotherapy CT (BVzCT) alone (N5110) (N549) Age, years Median (range) Gender, n (%) Male Female Smoking, history, n (%) Never-smoker Smoker Family history, Yes No ECOG PS, n (%) 0 1 2 Histology, n (%) Adenocarcinoma Large cell carcinoma TNM stage IIIB IV Prior therapy Yes No VEGF status z { Unknown CA9 status w100 pg/ml v100 pg/ml
P
63 (39–77)
62 (37–76)
0.178ª
59 (53.64) 51 (46.36)
26 (53.06) 23 (46.94)
0.946
51 (46.36) 59 (53.64)
18 (36.73) 31 (63.27)
0.258
4 (3.64) 106 (96.36)
2 (4.08) 47 (95.92)
1.000*
50 (45.45) 58 (52.73) 2 (1.82)
20 (40.82) 28 (57.14) 1 (2.04)
0.824*
103 (93.64) 7 (6.36)
46 (93.88) 3 (6.12)
1.000*
17 (15.45) 93 (84.55)
6 (12.24) 43 (87.76)
0.595
25 (22.73) 85 (77.27)
11 (22.45) 38 (77.55)
0.969
56(50.91) 52(47.27) 2(1.82)
25(51.02) 24(48.98)
0.923
29(26.3) 81(73.6)
13(26.5) 36(73.4)
0.982
Outcome evaluations for BVþ CV The median follow-up period was 20.7 months (95% CI: 18.5–22.9 months). Up to 31 December 2012, both BVzCT and the CT groups had 15 patients with brain metastases (14 vs 31%, Pv0.05). In the BVzCT group, the 6-, 12- and 24-month cumulative risks of brain metastases were 1.0, 7.5 and 14.0%, respectively, compared with 6.7, 18.8 and 31% in the CT group. As shown in Figure 1, there were significant differences between these two groups in their cumulative incidence curves for brain metastases (Pv0.01).
Figure 1 Cumulative incidence of brain metastasis in all eligible patients.
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Relationship between VEGF expression status and BV efficacy We did an analysis for the impact of VEGF expression on BV efficacy. For VEGF-positive patients, compared to CT alone, BVzCT reduced the risk of brain metastasis (P50.003; Figure 2) and prolonged overall patient survival (P50.007).
Association between CA9 expression status and BV efficacy
ª : t-test. * : Fisher’s exact.
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In addition, at the end of follow-up, 40 patients (36%) had survived in the BVzCT group with a median OS of 19.9 months (95% CI: 16.273–23.587 months). In the CT group, 11 patients (22%) had survived with a median OS of 15.7 months (95% CI: 13.742– 17.658 months). There was a significant difference in the OS curves for these two groups (P50.001).
3
According to a related study,8 a pre-treatment serum CA9 level of w100 pg/ml was defined as high CA9 expression and a pre-treatment serum CA9 level of v100 pg/ml was defined as low CA9 expression. In the BVzCT group, high CA9 expression was found in 29 patients (26.4%) and low CA9 expression was found in 81 patients (73.6%). In the CT group, high CA9 expression was found in 13 patients (26.5%) and low CA9 expression was found in 36 patients (73.5%). In the BVzCT group, there was no significant difference between patients with high CA9 expression and patients with low CA9 expression in terms of a decreased risk for brain metastasis (P50.252; Figure 3). In the CT group, there was also no significant difference between patients with high CA9 expression and patients with low CA9 expression for a decreased risk for brain metastasis (P50.517). The mean pre- and post-treatment concentrations of serum CA9 were 115.84 and 74.21 pg/ml, respectively, in the BVzCT group (Pv0.05). The mean pre- and posttreatment concentrations of serum CA9 were 154.88
Figure 2 Cumulative incidence of brain metastasis in patients with positive expression of vascular endothelial growth factor (VEGF).
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By univariate analysis, there were associations between the risk of brain metastasis and the treatment method and medical history. Previous treatment (HR50.235, 95% CI: 0.056–0.988, P50.048) and BVzCT (HR50.263, 95% CI: 0.128–0.541, Pv0.001) reduced the risk of brain metastases in patients with advanced lung cancer. However, by multivariate analysis, only the use of BV was an independent factor for determining the risk of brain metastases (HR50.366, 95% CI: 0.161–0.829, P50.016). Figure 3 Cumulative incidence of brain metastasis in patients with negative expression of vascular endothelial growth factor (VEGF).
and 141.02 pg/ml, respectively, in the CT group (Pw0.05). The risk of brain metastasis was significantly reduced in patients with high CA9 expression in the BVzCT group compared to that in the CT group (P50.021; Fig. 4). The mean pre- and post-treatment concentrations for patients with low CA9 expression in the BVzCT group were 51.83 and 41.18 pg/ml, respectively (Pw0.05), and the mean pre- and posttreatment concentrations for patients with low CA expression were 55.87 and 49.10 pg/ml in the CT group (Pw0.05). There was no significant difference in the risk of brain metastasis when comparing patients with low CA9 expression in the BVzCT group to those in the CT group (P50.108).
Factors related to the risk of brain metastasis Possible factors related to a risk of brain metastasis were analysed using COX regression models. Factors included were the treatment group, age, gender, smoking history, pre-treatment PS score, pathological classification, TNM staging, medical history and VEGF expression status. Because only a few patients had positive family histories, family history was not included. These results are shown in Table 2.
Figure 4 Cumulative incidences of brain metastases of the patients with high Carbonic anhydrase-9 (CA9) expression in two groups (P50.021).
Discussion Brain metastasis is a common complication with advanced NSCLC. It causes severe damage to neurocognitive functions and affects patients’ quality of life and prognosis. Current treatment methods include whole brain radiotherapy (WBRT), stereotactic radiosurgery (SRS), surgery and CV. However, the outcomes with these treatments are not satisfactory. The incidence of brain metastasis within 2 years after simple CV is 32%. With the development of targetted drug therapy, the survival of patients with advanced lung cancer has improved greatly. Thus, it needs to be determined whether the application of targetted drug therapy can open new avenues for treating brain metastasis in patients with advanced lung cancer. Heon et al. demonstrated that gefitinib or erlotinib could significantly reduce the risk of tumours invading the central nervous system for patients with advanced NSCLC accompanied by a somatic mutation in EGFR.12,13 Angiogenesis is very important for tumour occurrence and development. Vascular endothelial growth factor is a key factor that mediates tumour angiogenesis. It has been shown that vascular permeability and interstitial fluid pressure are significantly increased in tumour tissues and that peripheral cell coverage is reduced, which results in tumour hypoxia and upregulated VEGF expression.14 Thus, anti-angiogenic therapy has become a new target for anti-cancer therapy. Bevacizumab can directly act on VEGF to inhibit tumour angiogenesis, as it promotes the degeneration of tumour vessels, normalizes existing tumour vessels and removes immature vasculature to inhibit the growth of tumour cells and promote their death. Clinical trials have also shown that BV was effective for treating tumours, including those of colorectal cancer, lung cancer, glioma and others.15–19 For the treatment of lung cancer, BV was the first anti-VEGF drug that improved the outcomes of standard double-agent CV for patients with advanced lung cancers. In 2006, BV was approved by the FDA for use as a first-line treatment together
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Table 2 COX regression univariate and multivariate analyses for brain metastasis incidence Univariate analysis Factor Treatment (BVzCT vs CT alone) Age Gender (female vs male) Smoking history (yes vs no) ECOG PS Histology (large cell carcinoma vs adenocarcinoma) TNM stage Prior therapy (yes vs no) VEGF status (z vs {) CA9 status (high vs low)
HR
95% CI
0.263 0.971 0.881 1.630 0.703 1.107 0.808 0.235 1.981 0.789
0.128–0.541 0.918–1.028 0.428–1.815 0.763–3.484 0.356–1.386 0.264–4.651 0.329–1.982 0.056–0.988 0.938–4.185 0.338–1.842
with platinum-based CV drugs for NSCLC patients.20 However, patients with definite brain metastases were excluded from various clinical trials on targetted anti-angiogenic drugs because these drugs were believed to increase the risk of bleeding in the central nervous system. Yet, numerous clinical trials have shown that BV did not increase the risk of cerebral haemorrhage in patients with brain metastases.21–24 Along with the wide application of BV in clinical practice, more and more studies have observed obvious outcomes with BV for treating brain metastasis.11,25 Brain metastasis is a complex multi-stage pathological process. There are significant differences between brain metastasis and metastases to other organs in terms of pathology and clinical prognosis. However, its underlying mechanism has not yet been determined. There have been many theoretical hypotheses proposed, such as the tumour microenvironment theory, the blood–brain barrier theory, the metastatic cancer stem cell theory and others. Experiments using animal models also showed that the incidence of brain metastasis could be effectively reduced by inhibiting this molecular signalling pathway.7 Detachment of tumour cells into the blood circulation is also a prerequisite for tumour metastasis.26 As a VEGF inhibitor, BV can normalize tumour blood vessels and subsequently reduce the infiltration of tumour cells into blood vessels, which may decrease the incidence of tumour metastasis. Carmeliet et al. demonstrated this using a mouse model in an experiment.27 Thus, we hypothesized that BV was not only effective for treating brain metastasis but could also prevent the occurrence of brain metastasis. Our works show that BVzCV significantly reduced the incidence of brain metastasis compared to CV alone. Moreover, there were significant differences between these two groups in their cumulative risk curves and total survival of patients with brain metastases. The results of multivariate analysis also
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v0.001 0.319 0.731 0.207 0.308 0.889 0.641 0.048 0.073 0.584
HR
95% CI
P
0.366 0.989 0.556 2.484 0.308 0.873 0.732 0.269 1.700 0.795
0.161–0.829 0.939–1.042 0.163–1.902 0.761–8.112 0.092–1.034 0.174–4.387 0.288–1.863 0.063–1.160 0.758–3.812 0.300–2.106
0.016 0.669 0.350 0.132 0.057 0.869 0.513 0.078 0.198 0.645
showed that BV was an independent factor for determining the risk of brain metastasis. This suggests that BV can prevent the occurrence of brain metastasis in patients with advanced lung cancer and prolong their total survival. Because VEGF plays an important role in the occurrence of brain metastasis, and the pharmacological basis of BV is to inhibit the effects of VEGF, is there a correlation between the effects of BV on preventing brain metastasis and VEGF expression? Our results showed that the combination of BV and CV significantly reduced the risk of brain metastasis and prolonged patient total survival compared to CV only for patients who had positive VEGF expression. However, this combined therapy provided no obvious advantage for the risk of brain metastasis or total survival for patients who had negative VEGF expression. This suggests that BV should be applied selectively to VEGF-positive patients. Carbonic anhydrase-9 is a member of the carbonic anhydrase family. Its expression significantly increases under hypoxic conditions and is related to tumour cell proliferation, cell adhesion and tumour progression. Carbonic anhydrase-9 expression is significantly enhanced in many tumours, including renal cell carcinoma, cervical cancer and lung cancer, and its expression is closely related to tumour metastasis.11 Van Baardwijk et al.28 performed PET examinations for 102 NSCLC patients and analysed the expression of HIF-1alpha (hypoxia inducible factor1alpha), Ki67 and CA9 in post-operative specimens. They found positive correlations between the uptake rate of FDG and the expressions of HIF-1alpha. Kim et al.29 conducted a comparative study on the effects of two endogenous hypoxia factors, HLF-1 and CA9, on prognosis compared their relationships to VEGF and MMP-9 expressions. They found a significant correlation between VEGF and MMP-9 expression. HLF-1 and CA9 expressions were higher in areas of moderate to severe necrosis
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compared to those in areas of mild necrosis, which suggested that they were correlated with hypoxia. Carbonic anhydrase-9 mRNA expression in NSCLC tissues was correlated with VEGF mRNA. Swinnson et al.30 studied CA9 expression in the cancer tissues of 175 NSCLC patients using IHC and found that patient prognosis was poor for those with perinuclear CA9 expression. It was also reported that there was a high degree of consistency between CA9 expression in lung cancer tissue and brain metastasis,8 in that patients with high CA9 expression in their lung cancer tissues also had relatively high CA9 expression in their brain metastasis lesions. Our results showed that the CA9 concentrations in patients who had pre-treatment serum CA9 concentrations of w100 pg/ml decreased significantly after BV treatment, and that the risk of brain metastasis was also reduced. This suggests that BV efficacy may be related to CA9 expression. There was a tendency for reduced risk of brain metastasis in those patients with pre-treatment serum CA9 concentrations of v100 pg/ml. However, the difference was not statistically significant, which may have been related to our small sample size. On the other hand, there were some limitations in this retrospective study. Many factors such as healthy conditions of patients, their economic conditions and their trusts for doctors could affect their choice for therapeutic strategies. Furthermore, in this study, the baseline variation between two groups may reduce credibility of the result. Moreover, as patient numbers involved in this study is relatively smaller, a large-scale investigation is needed to further validate the conclusion. In summary, the results of the current study suggest that BVzCV can effectively decrease the risk of brain metastasis for patients with advanced NSCLC. Vascular endothelial growth factor-positive patients may benefit more from BV treatment, and the outcomes with BV may be related to CA9 expression.
Acknowledgements Thank you for all the authors.
Disclaimer Statements Contributors Design of study: Nan Du, Shunchang Jiao. Data collection: Yan Fu, Xinrong Kang, Hui Zhao, Junxun Ma, Xiaosong Li, and Fang Li. Sample test: Yan Fu. Data analysis: Yan Fu, Jia Hu.Writing: Yan Fu. Proof: Yan Fu, Nan Du. Funding The study was financially supported by Municipal Commission of Sicience and Technology [ grant number 2131107002213040]. Conflicts of interest There are no conflicts of interest in the paper.
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Ethics approval All of the tissue specimens used in this study were obtained with patient informed consent. The Ethics Committee of The First Affiliated Hospital of Chinese PLA General Hospital approved the use of these specimens.
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ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer Yan Fu, Jia Hu, Nan Du, Shunchang Jiao, Fang Li, Xiaosong Li, Junxun Ma, Hui Zhao & Huanrong Kang To cite this article: Yan Fu, Jia Hu, Nan Du, Shunchang Jiao, Fang Li, Xiaosong Li, Junxun Ma, Hui Zhao & Huanrong Kang (2016) Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer, Journal of Chemotherapy, 28:3, 218-224, DOI: 10.1179/1973947815Y.0000000045 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000045
Published online: 14 Jul 2016.
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Date: 12 September 2016, At: 18:24
Anticancer Original Research Paper
Bevacizumab plus chemotherapy versus chemotherapy alone for preventing brain metastasis derived from advanced lung cancer Yan Fu1, Jia Hu1, Nan Du1, Shunchang Jiao2, Fang Li2, Xiaosong Li1, Junxun Ma1, Hui Zhao1, Huanrong Kang1 1
Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China, Cancer Center, Chinese PLA General Hospital, Beijing 100853, China
2
This retrospective analysis evaluated the mechanism of bevacizumab plus chemotherapy (BVzCT) for preventing brain metastasis derived from lung cancer. From the total of 159 patients with advanced non-small cell lung cancer (NSCLC), 110 received BVzCT and 49 received CT. After medication, both groups had 15 patients with brain metastases (14 vs 31%, Pv0.05). With BVzCT treatment, 40 patients (33.89%) survived, whereas only 11 patients (18.64%) survived with CT treatment. The outcome for the BVzCT group was significantly better than that for the CT group only for vascular endothelial growth factor (VEGF)-positive patients. A post-treatment with BVzCT was significantly reduced than with CT only for patients with high carbonic anhydrase-9 (CA9) expression. This retrospective analysis provides supportive evidence that BVzCT can significantly reduce the incidence of brain metastasis in patients with advanced NSCLC compared with CT alone. Vascular endothelial growth factor -positive patients may benefit more from BV treatment and the outcomes with BV may be related to CA9 expression. Keywords: Bevacizumab, Brain metastasis, NSCLC
Introduction The incidence of brain metastasis derived from advanced non-small cell lung cancer (NSCLC) is 23–65%1 and accounts for 40–60% of brain metastases from all malignant tumours. Without treatment, the expected survival time of patients with brain metastasis is about 1 month.2–4 Although CV plus radiotherapy can reduce the incidence of extracranial metastases in patients with advanced NSCLC, it cannot reduce the incidence of brain metastases. Tumour angiogenesis is related to various factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and others. In addition, tumour angiogenesis is related to metastatic potential. Thus, anti-angiogenic therapy has become a promising cancer treatment. Basic research has confirmed that the spread and growth of tumour cells inside the brain parenchyma depend on VEGF.5 Clinical studies have also found that VEGF protein expression is related to the
Correspondence to: Nan Du, Department of Oncology, The First Affiliated Hospital of Chinese PLA General Hospital, Beijing 100048, China. Email: [email protected]
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ß 2015 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000045
prognosis of brain metastases derived from lung cancer.6 One study attempted to inhibit this signalling molecule and found that this effectively reduced brain metastasis in animal models.7 Carbonic anhydrase-9 (CA9) is a member of the carbonic anhydrase family. It is highly expressed under hypoxic conditions and is related to tumour cell proliferation, cell adhesion and tumour progression. It was reported that there was an apparent correlation between CA9 mRNA levels and VEGF expression levels in NSCLC tissues. Moreover, the levels of CA9 expression in the tissues of early stage lung cancer are closely related to prognosis, and CA9 expression in lung tissue is highly consistent with CA9 expression in brain metastasis derived from lung cancer.8 Bevacizumab is a monoclonal antibody directed against VEGF that has been shown in many clinical trials to be effective for treating brain metastases derived from lung cancer, breast cancer, malignant melanoma and others when used in conjunction with CV.9–11 However, the mechanism by which BV prevents brain metastasis and reduces the incidence of brain metastasis has not been thoroughly investigated.
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Materials and Methods Patients and clinical information Patients who were diagnosed between January 2009 and December 2010 with stage IIIB–IV NSCLC using pathological or cytological examinations were included in our retrospective analysis. Patients with stage IIIB– IV NSCLC who initially underwent systemic treatment were included. Patients who had previously undergone systemic CV for stage IIIB–IV NSCLC that subsequently relapsed were also included. The interval between the last treatment and the present treatment was more than three months. These patients had normal liver and kidney function, normal EKG readings and no vital organ dysfunction. We excluded patients with previous or concurrent primary tumours in other sites, patients with newly developed brain metastases within six courses of BV therapy, and patients who could not receive six courses of treatment due to various reasons, such as midway conversion to other therapy. Baseline evaluations were completed within 1 week before systemic CV, which included a clinical evaluation, chest CT, vital organ evaluations, including liver and bone and enhanced brain MRI examination. After systemic treatment, lesions were evaluated once every 8 weeks for both groups. In the BVzCT group, all patients received treatment with gemcitabine (1000 mg/m2) z carboplatin (ACU55–7) z BV (5 mg/kg) for six consecutive courses. Patients with no progression based on CT examinations after this treatment received BV maintenance therapy (5 mg/kg, once every three weeks) until the patient showed progression or could not tolerate drug toxicity. In the CT group, all patients received treatment with gemcitabine (1000 mg/m2) z carboplatin (ACU55–7) for six consecutive courses. After that, these patients received gemcitabine (1000 mg/m2) maintenance therapy once every 3 weeks.
Outcome evaluations The cumulative incidence of brain metastases was the main outcome and overall survival (OS) was the secondary outcome. Time to occurrence of brain metastasis (TTBM) was defined as the period from the initial diagnosis of advanced lung cancer to an imaging diagnosis of brain metastasis. The survival time was defined as the period from the initial diagnosis of advanced lung cancer to the date of death or to the end of follow-up.
VEGF expression Immunohistochemistry (IHC) was conducted as previously described. Monoclonal anti-VEGF antibody was used to incubate with tissue sections (Abcam, Cambridge, MA, UK; Catalog no. ab52917 dilution 1:100) at 4uuC overnight. After washing with PBS,
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the sections were incubated with secondary antibody, then diaminobenzidine (Dako, Glostrup, Denmark) was used for visual detection in the Envision System. A negative control was set by replacing the primary antibody with a normal murine or rabbit IgG at the same dilutions. Immunohistochemistry scoring was performed using a method described as follows: the percentage of positive cells was divided into five Grades (percentage scores): j10% (0), 11–25% (1), 26–50% (2), 51–75% (3) and w75% (4). The intensity of staining was divided into four Grades (intensity scores): no staining (0), light brown (1), brown (2) and dark brown (3). Staining positivity was determined by the formula: overall scores5percentage scorezintensity score. The total score ranged from 0 to 7, with low expression (0–2) and high expression (3–7).
Serum CA9 measurements We used an ELISA to measure serum CA9 concentrations before treatment and six weeks after treatment in both groups. Peripheral blood (5 ml) was collected in an anticoagulant tube. Within 30 minutes, a sample was spun in a centrifuge at 3000 rpm for 10 minutes at 4uuC to isolate serum. Then the soluble CA9 concentration was determined according to kit instructions (Systems Inc, Minneapolis, USA) using a 100 ml test sample
Statistical analysis STATA 10.0 software was used for statistical analyses. A t-test was used to compare mean patient age between the two groups. A chi-squared test or Fisher’s test was used compare other baseline variables. Kaplan–Meier curves were generated fort survival analysis and these curves were compared using a log-rank test. A cumulative incidence curve was generated to evaluate the cumulative risk of brain metastases. COX regression models were used for univariate and multivariate analyses to evaluate factors that influenced the occurrence of brain metastasis. All statistical tests were two tailed. A P-value of v0.05 was considered significant.
Results Baseline patient characteristics Total of 159 patients were included in this retrospective study (110 patients in the BVzCT group and 49 patients in the CT group). Their ages ranged from 37 to 77 years. The baseline characteristics of these patients are shown in Table 1. There were no significant differences between these two groups for age, at disease onset, gender, smoking history, family history, pre-treatment PS scores, pathological types, TNM stages, previous medical history or VEGF and CA9 expressions.
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Table 1 Baseline patient characteristics Bevacizumab plus chemotherapy CT (BVzCT) alone (N5110) (N549) Age, years Median (range) Gender, n (%) Male Female Smoking, history, n (%) Never-smoker Smoker Family history, Yes No ECOG PS, n (%) 0 1 2 Histology, n (%) Adenocarcinoma Large cell carcinoma TNM stage IIIB IV Prior therapy Yes No VEGF status z { Unknown CA9 status w100 pg/ml v100 pg/ml
P
63 (39–77)
62 (37–76)
0.178ª
59 (53.64) 51 (46.36)
26 (53.06) 23 (46.94)
0.946
51 (46.36) 59 (53.64)
18 (36.73) 31 (63.27)
0.258
4 (3.64) 106 (96.36)
2 (4.08) 47 (95.92)
1.000*
50 (45.45) 58 (52.73) 2 (1.82)
20 (40.82) 28 (57.14) 1 (2.04)
0.824*
103 (93.64) 7 (6.36)
46 (93.88) 3 (6.12)
1.000*
17 (15.45) 93 (84.55)
6 (12.24) 43 (87.76)
0.595
25 (22.73) 85 (77.27)
11 (22.45) 38 (77.55)
0.969
56(50.91) 52(47.27) 2(1.82)
25(51.02) 24(48.98)
0.923
29(26.3) 81(73.6)
13(26.5) 36(73.4)
0.982
Outcome evaluations for BVþ CV The median follow-up period was 20.7 months (95% CI: 18.5–22.9 months). Up to 31 December 2012, both BVzCT and the CT groups had 15 patients with brain metastases (14 vs 31%, Pv0.05). In the BVzCT group, the 6-, 12- and 24-month cumulative risks of brain metastases were 1.0, 7.5 and 14.0%, respectively, compared with 6.7, 18.8 and 31% in the CT group. As shown in Figure 1, there were significant differences between these two groups in their cumulative incidence curves for brain metastases (Pv0.01).
Figure 1 Cumulative incidence of brain metastasis in all eligible patients.
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Relationship between VEGF expression status and BV efficacy We did an analysis for the impact of VEGF expression on BV efficacy. For VEGF-positive patients, compared to CT alone, BVzCT reduced the risk of brain metastasis (P50.003; Figure 2) and prolonged overall patient survival (P50.007).
Association between CA9 expression status and BV efficacy
ª : t-test. * : Fisher’s exact.
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In addition, at the end of follow-up, 40 patients (36%) had survived in the BVzCT group with a median OS of 19.9 months (95% CI: 16.273–23.587 months). In the CT group, 11 patients (22%) had survived with a median OS of 15.7 months (95% CI: 13.742– 17.658 months). There was a significant difference in the OS curves for these two groups (P50.001).
3
According to a related study,8 a pre-treatment serum CA9 level of w100 pg/ml was defined as high CA9 expression and a pre-treatment serum CA9 level of v100 pg/ml was defined as low CA9 expression. In the BVzCT group, high CA9 expression was found in 29 patients (26.4%) and low CA9 expression was found in 81 patients (73.6%). In the CT group, high CA9 expression was found in 13 patients (26.5%) and low CA9 expression was found in 36 patients (73.5%). In the BVzCT group, there was no significant difference between patients with high CA9 expression and patients with low CA9 expression in terms of a decreased risk for brain metastasis (P50.252; Figure 3). In the CT group, there was also no significant difference between patients with high CA9 expression and patients with low CA9 expression for a decreased risk for brain metastasis (P50.517). The mean pre- and post-treatment concentrations of serum CA9 were 115.84 and 74.21 pg/ml, respectively, in the BVzCT group (Pv0.05). The mean pre- and posttreatment concentrations of serum CA9 were 154.88
Figure 2 Cumulative incidence of brain metastasis in patients with positive expression of vascular endothelial growth factor (VEGF).
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By univariate analysis, there were associations between the risk of brain metastasis and the treatment method and medical history. Previous treatment (HR50.235, 95% CI: 0.056–0.988, P50.048) and BVzCT (HR50.263, 95% CI: 0.128–0.541, Pv0.001) reduced the risk of brain metastases in patients with advanced lung cancer. However, by multivariate analysis, only the use of BV was an independent factor for determining the risk of brain metastases (HR50.366, 95% CI: 0.161–0.829, P50.016). Figure 3 Cumulative incidence of brain metastasis in patients with negative expression of vascular endothelial growth factor (VEGF).
and 141.02 pg/ml, respectively, in the CT group (Pw0.05). The risk of brain metastasis was significantly reduced in patients with high CA9 expression in the BVzCT group compared to that in the CT group (P50.021; Fig. 4). The mean pre- and post-treatment concentrations for patients with low CA9 expression in the BVzCT group were 51.83 and 41.18 pg/ml, respectively (Pw0.05), and the mean pre- and posttreatment concentrations for patients with low CA expression were 55.87 and 49.10 pg/ml in the CT group (Pw0.05). There was no significant difference in the risk of brain metastasis when comparing patients with low CA9 expression in the BVzCT group to those in the CT group (P50.108).
Factors related to the risk of brain metastasis Possible factors related to a risk of brain metastasis were analysed using COX regression models. Factors included were the treatment group, age, gender, smoking history, pre-treatment PS score, pathological classification, TNM staging, medical history and VEGF expression status. Because only a few patients had positive family histories, family history was not included. These results are shown in Table 2.
Figure 4 Cumulative incidences of brain metastases of the patients with high Carbonic anhydrase-9 (CA9) expression in two groups (P50.021).
Discussion Brain metastasis is a common complication with advanced NSCLC. It causes severe damage to neurocognitive functions and affects patients’ quality of life and prognosis. Current treatment methods include whole brain radiotherapy (WBRT), stereotactic radiosurgery (SRS), surgery and CV. However, the outcomes with these treatments are not satisfactory. The incidence of brain metastasis within 2 years after simple CV is 32%. With the development of targetted drug therapy, the survival of patients with advanced lung cancer has improved greatly. Thus, it needs to be determined whether the application of targetted drug therapy can open new avenues for treating brain metastasis in patients with advanced lung cancer. Heon et al. demonstrated that gefitinib or erlotinib could significantly reduce the risk of tumours invading the central nervous system for patients with advanced NSCLC accompanied by a somatic mutation in EGFR.12,13 Angiogenesis is very important for tumour occurrence and development. Vascular endothelial growth factor is a key factor that mediates tumour angiogenesis. It has been shown that vascular permeability and interstitial fluid pressure are significantly increased in tumour tissues and that peripheral cell coverage is reduced, which results in tumour hypoxia and upregulated VEGF expression.14 Thus, anti-angiogenic therapy has become a new target for anti-cancer therapy. Bevacizumab can directly act on VEGF to inhibit tumour angiogenesis, as it promotes the degeneration of tumour vessels, normalizes existing tumour vessels and removes immature vasculature to inhibit the growth of tumour cells and promote their death. Clinical trials have also shown that BV was effective for treating tumours, including those of colorectal cancer, lung cancer, glioma and others.15–19 For the treatment of lung cancer, BV was the first anti-VEGF drug that improved the outcomes of standard double-agent CV for patients with advanced lung cancers. In 2006, BV was approved by the FDA for use as a first-line treatment together
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Table 2 COX regression univariate and multivariate analyses for brain metastasis incidence Univariate analysis Factor Treatment (BVzCT vs CT alone) Age Gender (female vs male) Smoking history (yes vs no) ECOG PS Histology (large cell carcinoma vs adenocarcinoma) TNM stage Prior therapy (yes vs no) VEGF status (z vs {) CA9 status (high vs low)
HR
95% CI
0.263 0.971 0.881 1.630 0.703 1.107 0.808 0.235 1.981 0.789
0.128–0.541 0.918–1.028 0.428–1.815 0.763–3.484 0.356–1.386 0.264–4.651 0.329–1.982 0.056–0.988 0.938–4.185 0.338–1.842
with platinum-based CV drugs for NSCLC patients.20 However, patients with definite brain metastases were excluded from various clinical trials on targetted anti-angiogenic drugs because these drugs were believed to increase the risk of bleeding in the central nervous system. Yet, numerous clinical trials have shown that BV did not increase the risk of cerebral haemorrhage in patients with brain metastases.21–24 Along with the wide application of BV in clinical practice, more and more studies have observed obvious outcomes with BV for treating brain metastasis.11,25 Brain metastasis is a complex multi-stage pathological process. There are significant differences between brain metastasis and metastases to other organs in terms of pathology and clinical prognosis. However, its underlying mechanism has not yet been determined. There have been many theoretical hypotheses proposed, such as the tumour microenvironment theory, the blood–brain barrier theory, the metastatic cancer stem cell theory and others. Experiments using animal models also showed that the incidence of brain metastasis could be effectively reduced by inhibiting this molecular signalling pathway.7 Detachment of tumour cells into the blood circulation is also a prerequisite for tumour metastasis.26 As a VEGF inhibitor, BV can normalize tumour blood vessels and subsequently reduce the infiltration of tumour cells into blood vessels, which may decrease the incidence of tumour metastasis. Carmeliet et al. demonstrated this using a mouse model in an experiment.27 Thus, we hypothesized that BV was not only effective for treating brain metastasis but could also prevent the occurrence of brain metastasis. Our works show that BVzCV significantly reduced the incidence of brain metastasis compared to CV alone. Moreover, there were significant differences between these two groups in their cumulative risk curves and total survival of patients with brain metastases. The results of multivariate analysis also
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v0.001 0.319 0.731 0.207 0.308 0.889 0.641 0.048 0.073 0.584
HR
95% CI
P
0.366 0.989 0.556 2.484 0.308 0.873 0.732 0.269 1.700 0.795
0.161–0.829 0.939–1.042 0.163–1.902 0.761–8.112 0.092–1.034 0.174–4.387 0.288–1.863 0.063–1.160 0.758–3.812 0.300–2.106
0.016 0.669 0.350 0.132 0.057 0.869 0.513 0.078 0.198 0.645
showed that BV was an independent factor for determining the risk of brain metastasis. This suggests that BV can prevent the occurrence of brain metastasis in patients with advanced lung cancer and prolong their total survival. Because VEGF plays an important role in the occurrence of brain metastasis, and the pharmacological basis of BV is to inhibit the effects of VEGF, is there a correlation between the effects of BV on preventing brain metastasis and VEGF expression? Our results showed that the combination of BV and CV significantly reduced the risk of brain metastasis and prolonged patient total survival compared to CV only for patients who had positive VEGF expression. However, this combined therapy provided no obvious advantage for the risk of brain metastasis or total survival for patients who had negative VEGF expression. This suggests that BV should be applied selectively to VEGF-positive patients. Carbonic anhydrase-9 is a member of the carbonic anhydrase family. Its expression significantly increases under hypoxic conditions and is related to tumour cell proliferation, cell adhesion and tumour progression. Carbonic anhydrase-9 expression is significantly enhanced in many tumours, including renal cell carcinoma, cervical cancer and lung cancer, and its expression is closely related to tumour metastasis.11 Van Baardwijk et al.28 performed PET examinations for 102 NSCLC patients and analysed the expression of HIF-1alpha (hypoxia inducible factor1alpha), Ki67 and CA9 in post-operative specimens. They found positive correlations between the uptake rate of FDG and the expressions of HIF-1alpha. Kim et al.29 conducted a comparative study on the effects of two endogenous hypoxia factors, HLF-1 and CA9, on prognosis compared their relationships to VEGF and MMP-9 expressions. They found a significant correlation between VEGF and MMP-9 expression. HLF-1 and CA9 expressions were higher in areas of moderate to severe necrosis
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compared to those in areas of mild necrosis, which suggested that they were correlated with hypoxia. Carbonic anhydrase-9 mRNA expression in NSCLC tissues was correlated with VEGF mRNA. Swinnson et al.30 studied CA9 expression in the cancer tissues of 175 NSCLC patients using IHC and found that patient prognosis was poor for those with perinuclear CA9 expression. It was also reported that there was a high degree of consistency between CA9 expression in lung cancer tissue and brain metastasis,8 in that patients with high CA9 expression in their lung cancer tissues also had relatively high CA9 expression in their brain metastasis lesions. Our results showed that the CA9 concentrations in patients who had pre-treatment serum CA9 concentrations of w100 pg/ml decreased significantly after BV treatment, and that the risk of brain metastasis was also reduced. This suggests that BV efficacy may be related to CA9 expression. There was a tendency for reduced risk of brain metastasis in those patients with pre-treatment serum CA9 concentrations of v100 pg/ml. However, the difference was not statistically significant, which may have been related to our small sample size. On the other hand, there were some limitations in this retrospective study. Many factors such as healthy conditions of patients, their economic conditions and their trusts for doctors could affect their choice for therapeutic strategies. Furthermore, in this study, the baseline variation between two groups may reduce credibility of the result. Moreover, as patient numbers involved in this study is relatively smaller, a large-scale investigation is needed to further validate the conclusion. In summary, the results of the current study suggest that BVzCV can effectively decrease the risk of brain metastasis for patients with advanced NSCLC. Vascular endothelial growth factor-positive patients may benefit more from BV treatment, and the outcomes with BV may be related to CA9 expression.
Acknowledgements Thank you for all the authors.
Disclaimer Statements Contributors Design of study: Nan Du, Shunchang Jiao. Data collection: Yan Fu, Xinrong Kang, Hui Zhao, Junxun Ma, Xiaosong Li, and Fang Li. Sample test: Yan Fu. Data analysis: Yan Fu, Jia Hu.Writing: Yan Fu. Proof: Yan Fu, Nan Du. Funding The study was financially supported by Municipal Commission of Sicience and Technology [ grant number 2131107002213040]. Conflicts of interest There are no conflicts of interest in the paper.
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Ethics approval All of the tissue specimens used in this study were obtained with patient informed consent. The Ethics Committee of The First Affiliated Hospital of Chinese PLA General Hospital approved the use of these specimens.
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