一种新型肿瘤药敏试验方法的建立及初步应用研究
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摘要
恶性肿瘤是一类发病率和死亡率都非常高的疾病,严重危害着人类的生命和健康。科研人员和临床医生长期致力于肿瘤治疗方面的探索和实践。目前,恶性肿瘤的治疗,包括手术治疗、放疗、化疗、内分泌治疗和生物免疫治疗等,其中以前三种为主。化疗,作为一种全身性的治疗手段,因能广泛杀灭患者体内的肿瘤细胞,提高患者的生存率和生存期,而在治疗中占有重要地位。
肿瘤的发生发展,和细胞周期调节紊乱有关。细胞周期检测点是细胞周期调控的一种重要机制,检测点功能的正常发挥,可以确保周期运行中每一时相事件有序、完整的完成。当监测细胞周期运行的检测点功能丧失后,细胞周期调节发生紊乱,细胞出现无限增殖,从而引起了肿瘤的发生发展。化疗药物抗肿瘤的机制大多是干扰肿瘤的细胞周期,诱导肿瘤细胞凋亡,而这一功能的发挥则有赖于检测点功能的正常。因为不同化疗药物的具体作用机制和靶点不同,不同肿瘤检测点功能的丧失和保留程度不同,加之肿瘤细胞结构和功能上异质性的存在,所以,对大多数恶性肿瘤而言,即使对这类肿瘤患者全部应用治疗该类肿瘤疗效最好的化疗方案,往往也会有一部分病例治疗无效,最终依然会导致肿瘤的恶性进展、复发及转移。
因此,化疗方案的选择至关重要。近年来,临床肿瘤药敏试验的开展,使化疗方案的选择趋于个体化和有针对性,明显地提高了化疗的有效率,改善了患者的预后。但也不可否认,目前广泛应用的几种肿瘤药敏试验方法,普遍存在着一些缺陷,如需要较高水平的原代细胞培养技术、耗时较长、样品易污染、灵敏度较低等等。这些缺点均会对实验结果的准确性造成干扰,进而影响到化疗方案的正确选择和最终疗效,一定程度上限制了药敏试验的推广,使其陷入徘徊不前的局面。
鉴于以上一些问题,在本研究中,我们试图建立一种新型体外肿瘤药敏试验方法。因为化疗方案的选择是基于人体各种细胞群体对化疗药物的敏感性不同。人体内不同组织来源的细胞增殖能力是不同的,肿瘤组织通常较人体其它组织增殖力旺盛,此外,肿瘤组织内部不同细胞亚群之间也存在着增殖能力的巨大差异,一系列研究认为,化疗杀伤的主要是肿瘤组织中这一部分增殖活跃的细胞群体。因此,从这一点出发,首先,我们在检测化疗药物对原代肿瘤细胞的杀伤能力的同时,也对这群细胞的增殖能力加以检测,以Ki-67为标志检测细胞增殖比例。原代肿瘤细胞在体外加入化疗药物共同培养后,以Annexin-V和PI联合标记来检测细胞凋亡率。以增殖群体被药物杀伤后发生凋亡的比例来衡量药物的疗效。大量病例数据统计分析表明,整体上,细胞凋亡率和细胞增殖率波动在近似范围,这种现象也反映了化疗的实质。因此,我们提出将增殖和凋亡相结合来衡量肿瘤化疗敏感性的新理念,并初步建立了一种新型体外肿瘤药敏试验方法,即药物敏感性=细胞凋亡率/增殖细胞比例×100%。进而,为了验证我们新建立的肿瘤药敏试验方法的灵敏度和特异性,我们首先用原代胃肠道肿瘤细胞建立了裸鼠移植瘤模型,运用不同化疗方案治疗后,治疗效果与同一肿瘤原代细胞体外药敏试验的结论进行比较,结果证实我们新提出的肿瘤药敏实验方法灵敏度和特异性均高于传统的MTT肿瘤药敏试验,说明新方法能够有效预测肿瘤的药物敏感性。然后,我们选取了一部分胃肠道肿瘤病例,术后取肿瘤组织依据新型肿瘤药敏试验方法进行药物敏感性检测,且入选患者术后都进行了FOLFOX方案的化疗,并密切跟踪随访。结果表明,经新型肿瘤药敏试验检测显示肿瘤对FOLFOX方案有效的患者群体,其预后明显优于检测显示肿瘤对FOLFOX方案耐药的患者群体,这表明,我们建立的新型肿瘤药敏试验方法可以反映肿瘤的化疗敏感性,对临床肿瘤化疗具有指导意义。
我们从细胞增殖和凋亡的内在联系入手,建立了一种新型体外肿瘤药敏试验方法,并在动物模型和临床病例研究中证实了这种方法的有效性。该肿瘤药敏试验将有助于提高肿瘤的化疗效果,指导肿瘤化疗方案的选择。
Malignant tumor is a kind of disease with high incidence and mortality, which is hazardous to people's life and health. Researchers and clinicians have dedicated to scientific exploration and clinical practice in tumor treatment for a long time. Currently, cancer treatments mainly include surgery, radiotherapy, chemotherapy, endocrine therapy and immune therapy. Surgery, radiotherapy, chemotherapy are more widely used than others. Chemotherapy, as a systemic treatment which can kill tumor cells maximally in the patients'body, can increase patients'survival greatly, and is very important in cancer treatment.
When the checkpoint lose of fuction, the defects of genes in the cells could not be recognized, which may lead to cell cycle disregulation and unlimited proliferation. The anticancer mechanisms of cytotoxic drugs are to interupt the tumor cell cycle and to induce tumor cell apoptosis, which depend on the normal function of checkpoints. However, as specific mechanisms and targets of anticancer drugs are different, the checkpoints remained in the tumors cells are also different, and tumor cells often show great histological and functional heterogeneity, for most kinds of malignant tumors, even the most effective chemotherapy drugs or strategies failed in some cases, leading to disease progression, relapse and metastasis. Therefore, it is important to choose appropriate chemotherapy strategy for every patient. In recent years, tumor chemosensitivity test have been conducted in clinical practice, making chemotherapy more individualized, which have increased the responds rates of the patients and improved the prognosis of the diseases. But there are still some shortcomings in the tumor chemosensitivity test methods which are widely used in our country. For example, all the methods need better techniques of primary cell culture, most are time consuming, cells may be contaminated during long time culture, and sometimes the tests show lower sensitivity, ect. All the shortcomings may interference the accuracy of the tumor chemosensitivity test, which makes the doctors not able to choose the best chemotherapy strategy, and the patient would not get the best effect after chemotherapy, all those restrict the promotion of the tumor chemosensitivity test.
To solve the problems above, in this study, we explored a new tumor chemosensitivity test in vitro. Different cell populations in human body show different chemosensitivity, which is the principle of cancer chemotherapy. Not only the tumor tissues show higher proliferation index than normal human tissues, but also different proliferative potential exists in subpopulations within cancer tissues. The aim of chemotherapy is to destroy the proliferative populations within the tumor.
First of all, because of what was mentioned above, when we examined the cytotoxic effect of the drugs to primary tumor cells from the patients, meanwhile, we also detected the proliferative potential of the same population. We suggest that the cytotoxic effect of the chemotherapeutic drugs should be measured by their ability to induce the proliferative populations to go to apoptosis. The primary tumor cells, after incubated with drugs for several hours, were stained with Annexin-V and PI, and then the apoptosis index was detected by a flow cytometer. The primary tumor cells from the same patient were also analyzed for Ki-67 expression. By the way, Ki-67 antigen was widely used as a proliferation marker. Statistical analysis of large samples showed that, the apoptosis and proliferation rates fluctuated in the same range, this phenomenon was in accordance with the theory of chemotherapy. So we suggested a new idea that the tumor chemosensitivity should be measured by the combined analysis of proliferation and apoptosis. Second, in order to validate the sensitivity and specificity of our newly established tumor chemosensitivity test in vitro, we used primary xenograft model in nude mice. After the mice being treated with different chemotherapeutic drug or their combinations, the results observed were compared with the results from in vitro tumor chemosensitivity tests. The results indicated that our newly proposed method of chemosensitivity test showed higher sensitivity and specificity than the traditional MTT test. Third, we chose some cases of patients with gastric or colorectal cancer, and they all received postoperative chemotherapy and were closely followed up. Patients were divided into drug sensitive and resistant group by our in vitro tumor chemosensitivity test. Drug sensitive group showed better prognosis than resistant group. This evidence also confirmed that our new tumor chemosensitivity test can reflect the drug chemosensitivity of primary tumor cells.
Concerning the internal link of cell proliferation and apoptosis, after serial study, we established a new method of in vitro tumor chemosensitivity test, and the method were confirmed to be effective in animal models and clinical study, which provide a new approach to improve the effect of cancer chemotherapy.
肿瘤的发生发展,和细胞周期调节紊乱有关。细胞周期检测点是细胞周期调控的一种重要机制,检测点功能的正常发挥,可以确保周期运行中每一时相事件有序、完整的完成。当监测细胞周期运行的检测点功能丧失后,细胞周期调节发生紊乱,细胞出现无限增殖,从而引起了肿瘤的发生发展。化疗药物抗肿瘤的机制大多是干扰肿瘤的细胞周期,诱导肿瘤细胞凋亡,而这一功能的发挥则有赖于检测点功能的正常。因为不同化疗药物的具体作用机制和靶点不同,不同肿瘤检测点功能的丧失和保留程度不同,加之肿瘤细胞结构和功能上异质性的存在,所以,对大多数恶性肿瘤而言,即使对这类肿瘤患者全部应用治疗该类肿瘤疗效最好的化疗方案,往往也会有一部分病例治疗无效,最终依然会导致肿瘤的恶性进展、复发及转移。
因此,化疗方案的选择至关重要。近年来,临床肿瘤药敏试验的开展,使化疗方案的选择趋于个体化和有针对性,明显地提高了化疗的有效率,改善了患者的预后。但也不可否认,目前广泛应用的几种肿瘤药敏试验方法,普遍存在着一些缺陷,如需要较高水平的原代细胞培养技术、耗时较长、样品易污染、灵敏度较低等等。这些缺点均会对实验结果的准确性造成干扰,进而影响到化疗方案的正确选择和最终疗效,一定程度上限制了药敏试验的推广,使其陷入徘徊不前的局面。
鉴于以上一些问题,在本研究中,我们试图建立一种新型体外肿瘤药敏试验方法。因为化疗方案的选择是基于人体各种细胞群体对化疗药物的敏感性不同。人体内不同组织来源的细胞增殖能力是不同的,肿瘤组织通常较人体其它组织增殖力旺盛,此外,肿瘤组织内部不同细胞亚群之间也存在着增殖能力的巨大差异,一系列研究认为,化疗杀伤的主要是肿瘤组织中这一部分增殖活跃的细胞群体。因此,从这一点出发,首先,我们在检测化疗药物对原代肿瘤细胞的杀伤能力的同时,也对这群细胞的增殖能力加以检测,以Ki-67为标志检测细胞增殖比例。原代肿瘤细胞在体外加入化疗药物共同培养后,以Annexin-V和PI联合标记来检测细胞凋亡率。以增殖群体被药物杀伤后发生凋亡的比例来衡量药物的疗效。大量病例数据统计分析表明,整体上,细胞凋亡率和细胞增殖率波动在近似范围,这种现象也反映了化疗的实质。因此,我们提出将增殖和凋亡相结合来衡量肿瘤化疗敏感性的新理念,并初步建立了一种新型体外肿瘤药敏试验方法,即药物敏感性=细胞凋亡率/增殖细胞比例×100%。进而,为了验证我们新建立的肿瘤药敏试验方法的灵敏度和特异性,我们首先用原代胃肠道肿瘤细胞建立了裸鼠移植瘤模型,运用不同化疗方案治疗后,治疗效果与同一肿瘤原代细胞体外药敏试验的结论进行比较,结果证实我们新提出的肿瘤药敏实验方法灵敏度和特异性均高于传统的MTT肿瘤药敏试验,说明新方法能够有效预测肿瘤的药物敏感性。然后,我们选取了一部分胃肠道肿瘤病例,术后取肿瘤组织依据新型肿瘤药敏试验方法进行药物敏感性检测,且入选患者术后都进行了FOLFOX方案的化疗,并密切跟踪随访。结果表明,经新型肿瘤药敏试验检测显示肿瘤对FOLFOX方案有效的患者群体,其预后明显优于检测显示肿瘤对FOLFOX方案耐药的患者群体,这表明,我们建立的新型肿瘤药敏试验方法可以反映肿瘤的化疗敏感性,对临床肿瘤化疗具有指导意义。
我们从细胞增殖和凋亡的内在联系入手,建立了一种新型体外肿瘤药敏试验方法,并在动物模型和临床病例研究中证实了这种方法的有效性。该肿瘤药敏试验将有助于提高肿瘤的化疗效果,指导肿瘤化疗方案的选择。
Malignant tumor is a kind of disease with high incidence and mortality, which is hazardous to people's life and health. Researchers and clinicians have dedicated to scientific exploration and clinical practice in tumor treatment for a long time. Currently, cancer treatments mainly include surgery, radiotherapy, chemotherapy, endocrine therapy and immune therapy. Surgery, radiotherapy, chemotherapy are more widely used than others. Chemotherapy, as a systemic treatment which can kill tumor cells maximally in the patients'body, can increase patients'survival greatly, and is very important in cancer treatment.
When the checkpoint lose of fuction, the defects of genes in the cells could not be recognized, which may lead to cell cycle disregulation and unlimited proliferation. The anticancer mechanisms of cytotoxic drugs are to interupt the tumor cell cycle and to induce tumor cell apoptosis, which depend on the normal function of checkpoints. However, as specific mechanisms and targets of anticancer drugs are different, the checkpoints remained in the tumors cells are also different, and tumor cells often show great histological and functional heterogeneity, for most kinds of malignant tumors, even the most effective chemotherapy drugs or strategies failed in some cases, leading to disease progression, relapse and metastasis. Therefore, it is important to choose appropriate chemotherapy strategy for every patient. In recent years, tumor chemosensitivity test have been conducted in clinical practice, making chemotherapy more individualized, which have increased the responds rates of the patients and improved the prognosis of the diseases. But there are still some shortcomings in the tumor chemosensitivity test methods which are widely used in our country. For example, all the methods need better techniques of primary cell culture, most are time consuming, cells may be contaminated during long time culture, and sometimes the tests show lower sensitivity, ect. All the shortcomings may interference the accuracy of the tumor chemosensitivity test, which makes the doctors not able to choose the best chemotherapy strategy, and the patient would not get the best effect after chemotherapy, all those restrict the promotion of the tumor chemosensitivity test.
To solve the problems above, in this study, we explored a new tumor chemosensitivity test in vitro. Different cell populations in human body show different chemosensitivity, which is the principle of cancer chemotherapy. Not only the tumor tissues show higher proliferation index than normal human tissues, but also different proliferative potential exists in subpopulations within cancer tissues. The aim of chemotherapy is to destroy the proliferative populations within the tumor.
First of all, because of what was mentioned above, when we examined the cytotoxic effect of the drugs to primary tumor cells from the patients, meanwhile, we also detected the proliferative potential of the same population. We suggest that the cytotoxic effect of the chemotherapeutic drugs should be measured by their ability to induce the proliferative populations to go to apoptosis. The primary tumor cells, after incubated with drugs for several hours, were stained with Annexin-V and PI, and then the apoptosis index was detected by a flow cytometer. The primary tumor cells from the same patient were also analyzed for Ki-67 expression. By the way, Ki-67 antigen was widely used as a proliferation marker. Statistical analysis of large samples showed that, the apoptosis and proliferation rates fluctuated in the same range, this phenomenon was in accordance with the theory of chemotherapy. So we suggested a new idea that the tumor chemosensitivity should be measured by the combined analysis of proliferation and apoptosis. Second, in order to validate the sensitivity and specificity of our newly established tumor chemosensitivity test in vitro, we used primary xenograft model in nude mice. After the mice being treated with different chemotherapeutic drug or their combinations, the results observed were compared with the results from in vitro tumor chemosensitivity tests. The results indicated that our newly proposed method of chemosensitivity test showed higher sensitivity and specificity than the traditional MTT test. Third, we chose some cases of patients with gastric or colorectal cancer, and they all received postoperative chemotherapy and were closely followed up. Patients were divided into drug sensitive and resistant group by our in vitro tumor chemosensitivity test. Drug sensitive group showed better prognosis than resistant group. This evidence also confirmed that our new tumor chemosensitivity test can reflect the drug chemosensitivity of primary tumor cells.
Concerning the internal link of cell proliferation and apoptosis, after serial study, we established a new method of in vitro tumor chemosensitivity test, and the method were confirmed to be effective in animal models and clinical study, which provide a new approach to improve the effect of cancer chemotherapy.
引文
1. Kangas L, Gronroos M, Nieminen AL. Bioluminescence of cellular ATP:a new method for evaluating cytotoxic agents in vitro. Med Biol.1984; 62(6):338-43.
2. Maehara Y, Anai H, Masuda H, et al. In vitro chemosensitivity testing evaluated by intracellular ATP level:ATP assay Gan To Kagaku Ryoho.1986 Jul; 13(7):2342-5.
3. S. P. C. Cole Rapid chemosensitivity testing of human lung tumor cells using the MTT assay Cancer Chemother Pharmacol (1986) 17:259-263
4. James Carmichael, William G. DeGraff, Adi F. Gazdar, et al. Mitchell Evaluation of a Tetrazolium-based Semiautomated Colorimetrie Assay:Assessment of Chemosensitivity Testingl CANCER RESEARCH 1987,47,936-942.
5. Jae-Gahb Park, Barnett S. Kramer, Seth M. Steinberg, et al. Chemosensitivity Testing of Human Colorectal Carcinoma Cell Lines Using a Tetrazolium-based Colorimetrie Assay CANCER RESEARCH 1987,47,5875-5879.
6. Abiko T, Kawamura M, Izumi Y, et al. Prediction of anti-tumour effect of thermochemotherapy with in vitro thermochemosensitivity testing for non-small cell lung cancer.. Int J Hyperthermia.2007 May; 23(3):267-75.
7. Cho YB, Lee WY, Song SY, et al. In vitro chemosensitivity based on depth of invasion in advanced colorectal cancer using ATP-based chemotherapy response assay (ATP-CRA). Eur J Surg Oncol.2009 Sep; 35(9):951-6.
8. Mi Z, Holmes FA, Hellerstedt B,et al. Feasibility assessment of a chemoresponse assay to predict pathologic response in neoadjuvant chemotherapy for breast cancer patients. Anticancer Res.2008 May-Jun;28(3B):1733-40.
9.Auman JT, McLeod HL. Cancer pharmacogenomics:DNA genotyping and gene expression profiling to identify molecular determinants of chemosensitivity. Drug Metab Rev.2008;40(2):303-15.
10. Nakamura R, Saikawa Y, Kubota T, et al. Role of the MTT chemosensitivity test in the prognosis of gastric cancer patients after postoperative adjuvant chemotherapy. Anticancer Res.2006 Mar-Apr; 26(2B):1433-7
11. Koshiyama M, Kinezaki M, Uchida T, et al. Chemosensitivity testing of paclitaxel versus docetaxel in human gynecological carcinomas:a comparison with carboplatin. Anticancer Res.2006Sep-Oct; 26(5B):3655-9.
12. Yongdong Feng, Jianhong Wu, Daxin Xie, et al. Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells. Oncol Rep.2007 Jun; 17(6):1437-44.
13. Huang H., Regan KM., Lou Z., et al. CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage. Science,2006,314:294-7.
14. Wu J., Feng Y., Xie D., et al. Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell Mol Life Sci.2006, 63(21):2538-45,
15. Feng Y, Wu J, Gong J. Caspase-3 and CDK1 activated by X-ray trigger apoptosis through the phosphorylation of bcl-2 in G1 MOLT-4 cells. Cytometry,59(A):93,2004 (ISAC XXII Congress, oral presentation, France,2004)
1.Kangas L, Gronroos M, Nieminen AL. Bioluminescence of cellular ATP:a new method for evaluating cytotoxic agents in vitro. Med Biol.1984; 62(6):338-43.
2. Maehara Y, Anai H, Masuda H, Miyamoto K, Fukuchi K, Tamada R, Sugimachi K.In vitro chemosensitivity testing evaluated by intracellular ATP level:ATP assay Gan To Kagaku Ryoho.1986 Jul; 13(7):2342-5
3. S. P. C. Cole Rapid chemosensitivity testing of human lung tumor cells using the MTT assay Cancer Chemother Pharmacol (1986) 17:259-263
4. James Carmichael,2 William G. DeGraff, Adi F. Gazdar, John D. Minna, and James B. Mitchell Evaluation of a Tetrazolium-based Semiautomated Colorimetrie Assay: Assessment of Chemosensitivity Testing1 CANCER RESEARCH 47,936-942, February 15,1987
5. Jae-Gahb Park,2 Barnett S. Kramer, Seth M. Steinberg, James Carmichael,3Jerry M. Collins,John D. Minna, and Adi F. Gazdar Chemosensitivity Testing of Human Colorectal Carcinoma Cell Lines Using a Tetrazolium-based Colorimetrie Assay CANCER RESEARCH 47,5875-5879, November 15,1987
6. Abiko T, Kawamura M, Izumi Y, Oyama T, Saito Y, Kobayashi KPrediction of anti-tumour effect of thermochemotherapy with in vitro thermochemosensitivity testing for non-small cell lung cancer.. Int J Hyperthermia.2007 May; 23(3):267-75.
7. Cho YB, Lee WY, Song SY, Choi SH, Shin HJ, Ahn KD, Lee JM, Kim HC, Yun SH, Chun HK.In vitro chemosensitivity based on depth of invasion in advanced colorectal cancer using ATP-based chemotherapy response assay (ATP-CRA). Eur J Surg Oncol. 2009 Sep;35(9):951-6. Epub2009Jan31
8. Mi Z, Holmes FA, Hellerstedt B, Pippen J, Collea R, Backner A, Bush JE, Gallion HH, Wells A, O'Shaughnessy Feasibility assessment of a chemoresponse assay to predict pathologic response in neoadjuvant chemotherapy for breast cancer patients.Anticancer Res.2008 May-Jun;28(3B):1733-40
9. Auman JT, McLeod HL. Cancer pharmacogenomics:DNA genotyping and gene expression profiling to identify molecular determinants of chemosensitivity. Drug Metab Rev.2008;40(2):303-15.
10. Nakamura R, Saikawa Y, Kubota T, Kumagai A, Kiyota T, Ohashi M, Yoshida M, Otani Y, Kumai K, Kitajima M.Role of the MTT chemosensitivity test in the prognosis of gastric cancer patients after postoperative adjuvant chemotherapy. Anticancer Res. 2006 Mar-Apr; 26(2B):1433-7
11. Koshiyama M, Kinezaki M, Uchida T, Sumitomo M. Chemosensitivity testing of paclitaxel versus docetaxel in human gynecological carcinomas:a comparison with carboplatin. Anticancer Res.2006Sep-Oct; 26(5B):3655-9.
12. Yongdong Feng, Jianhong Wu, Daxin Xie, Jichao Qin, Deding Tao, Junbo Hu, and Jianping Gong*. Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells. Oncol Rep.2007 Jun; 17(6):1437-44.
1.Clarke. R. Human breast cancer cell line xenografts as models of brest cancer.the immunobiologies of recipient mice and the characteristics of several tumorigenic lines. Breast Cancer Res Treat.1996,39,69-86.
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3. S. P. C. Cole Rapid chemosensitivity testing of human lung tumor cells using the MTT assay Cancer Chemother Pharmacol (1986) 17:259-263
4. James Carmichael,2 William G. DeGraff, Adi F. Gazdar, John D. Minna, and James B. Mitchell Evaluation of a Tetrazolium-based Semiautomated Colorimetrie Assay: Assessment of Chemosensitivity Testing1 CANCER RESEARCH 47,936-942, February 15,1987
5. Jae-Gahb Park,2 Barnett S. Kramer, Seth M. Steinberg, James Carmichael,3Jerry M. Collins,John D. Minna, and Adi F. Gazdar Chemosensitivity Testing of Human Colorectal Carcinoma Cell Lines Using a Tetrazolium-based Colorimetrie Assay CANCER RESEARCH 47,5875-5879, November 15,1987
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