|Ahead of print
Lung cancer in the older population:Interactive effects of angiotensin converting enzyme gene polymorphism (rs 4340 ID) and tobacco addiction in risk assessment
Joyita Banerjee1, Abhishek Gupta2, Vertica Agnihotri2, Rashmita Pradhan1, Ramesh Kandel1, Ashish D Upadhyay2, Sadanand Dwivedi2, Lalit Kumar3, Sharmistha Dey2, Aparajit B Dey1
1 Department of Geriatric Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
3 Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||30-Dec-2019|
|Date of Decision||10-Jan-2020|
|Date of Acceptance||20-Jun-2020|
|Date of Web Publication||21-Jun-2021|
Aparajit B Dey,
Department of Geriatric Medicine, All India Institute of Medical Sciences, New Delhi
Department of Biophysics, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Background: rs4340ID polymorphism of angiotensin-converting enzyme (ACE) correlates with serum ACE levels in many known cancers. This study analyzed ACE rs4340 ID polymorphism in lung cancer (LC) in older patients of North India and correlated it with addiction status.
Methods: The study enrolled all subjects aged 60 years and above with 154 LC and 205 healthy controls. Genotyping was done by polymerase chain reaction (PCR) and validated by sequencing of 10% of the sample. Statistical analysis was done by SPSS Statistics 21.
Results: Genotype II was observed to have a significant 2.21-fold increased risk of LC as compared to the DD genotype and 3.43-folds enhanced risk with interaction of I allele with tobacco consumption habits as compared to D allele in LC was seen.
Conclusion: The risk of LC was higher with II genotype as compared to DD genotype. Interactive effect showed that I allele with tobacco habits may increase the risk of LC.
Keywords: Angiotensin converting enzyme, elderly, lung cancer, polymorphism, tobacco
Key Message:Genotype II of angiotensin-converting enzyme shows higher risk for lung cancer as compared to DD genotype. Interactive effect of II genotype with tobacco may also increase the risk of lung cancer in older adults.
|How to cite this URL:|
Banerjee J, Gupta A, Agnihotri V, Pradhan R, Kandel R, Upadhyay AD, Dwivedi S, Kumar L, Dey S, Dey AB. Lung cancer in the older population:Interactive effects of angiotensin converting enzyme gene polymorphism (rs 4340 ID) and tobacco addiction in risk assessment. Indian J Cancer [Epub ahead of print] [cited 2022 May 29]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=318894
| » Introduction|| |
Lung cancer (LC) is one of the most common cancers related to tobacco use. LC is the only cancer which has been implicated among 15 most burdensome disorders in the elderly. This is highly prevalent in India due to rampant tobacco use in different local forms. LC is the most prominent cancer in the geriatric population.
Angiotensin-converting enzyme (ACE) gene plays key role in the renin–angiotensin system (RAS). ACE catalyzes the conversion of inactive angiotensin I to active angiotensin II, a vasoactive and aldosterone-stimulating peptide which is involved in the control of fluid-electrolyte balance, systemic blood pressure, and also inactivates bradykinin. Angiotensin II is a potent vasoconstrictor and mediates cell growth and proliferation by stimulating various cytokines and growth factors. ACE gene is located on chromosome 17q23 and consists of 26 exons and 25 introns. The rs4340 insertion/deletion (ID) polymorphism in this gene refers to an Alu (Arthrobacter luteus) repetitive sequence of 287 base pair (bp) long. This is present in intron 16 of the ACE gene. Presence or absence of Alu repetitive sequence forms three genotypes, deletion/deletion (DD), insertion/insertion (II) homozygotes, and insertion/deletion (ID) heterozygotes. More than 160 ACE gene polymorphisms have been reported so far, most of which are single nucleotide polymorphisms (SNPs). An association of ACE rs4340 ID polymorphism with various cancers have been carried out lately in a number of studies as it has been proved that angiotensin II is a key enzyme involved in neovascularization, cell proliferation, inflammation and cell adhesion.,,,,, rs4340 ID ACE gene polymorphism has been reported to be implicated in pancreatic, breast, lung, gastric, esophageal, as well as in colorectal cancer.
In this study, the primary objective was to analyze rs4340ACE ID polymorphism in LC in older patients (60 years and above) in a North Indian population attending a tertiary care center in northern part of India.
| » Materials and Methods|| |
Enrolment of patients and clinical assessment
LC patients and controls were enrolled from the Department of Geriatric Medicine of All India Institute of Medical Sciences (AIIMS), New Delhi. Controls were mainly healthy caregivers and spouses, aged 60 years and above who had accompanied the patients to the hospital. Institute Ethics Subcommittee approval was obtained for this study (IESC/T-107/01.03.2013) and written informed consent was recorded from the participants.
The diagnosis and staging of LC were done as per established clinical protocol. Demographic and addiction data from all subjects were collected. Subjects were grouped under the following groups for addiction:
Tobacco users: (i) current users: Those who had history of smoking at least 5 cigarettes or beedi per day and/or chewed at least 4 packets/day of tobacco were included in the “current user” group; (ii) former/never users: Those who did not take tobacco in any form or had not consumed in the last 15 years were considered in the “former/never user” category. Different papers cite different time intensity exposure of cigarette/tobacco smoking. In a study done by Markaki et al., those who responded positively to the question “Smoke daily now or ever?” were defined as ever-smokers; those who answered negative were defined as never-smokers. The National lung screening trial (NLST) considered heavy smokers as those smoking 30 pack-years, and less than 15 years quit time in an age group of 55–74 years. Taking these former studies in consideration, we have grouped our participants as current users and former/never users as described above. For the purpose of analysis, we have divided these above groups of current smokers and former/never smokers as smoking and non-smoking respectively and studied the effect of interaction of these 2 groups with ACE polymorphism in LC patients aged 60 years and above.
It will be noteworthy to say that we had tried to group subjects as never users (who had never consumed tobacco in any form in their life) and former users (who used to consume tobacco regularly but have given up since last 15 years). Since the former group (never users) was very small, the two groups were clubbed together as “Former/never users.”; It has been shown in previous studies that smoking cessation can reduce the risk of lung cancer by approximately 50% with 10 years of abstinence. Similarly for alcohol, subjects who consumed alcohol 3–4 times a week were considered under the “current” user category and those who had never taken alcohol or not taken since the last 15 years were considered in the “former/never” user category.
A brief geriatric assessment was done to assess functionality of the patients. A Barthel's activity of daily living (ADL) scale was used to assess whether the subjects could carry on daily activities at home or had functional impairment and were dependent on others for help. The nutritional status was checked by the short form of Mini Nutritional Assessment (MNA) scale and the BMI (Body Mass Index) was calculated according to World Health Organization (WHO) standards for Asian population. Geographically, the samples included in the study belong to both urban and rural North-Indian population, attending the tertiary care facilities of the hospital.
DNA extraction and genotyping
Venous blood of 200 μl was collected from each of the participants in EDTA vacutainers and was used for genomic DNA isolation. DNA was extracted, using the Medi G blood genome DNA miniprep kit according to the manufacturers' instruction.
ID polymorphism was analyzed by polymerase chain reaction (PCR). PCR was run with 50 μl of total reaction volume, which contains: 100 ng of genomic DNA, 10X Taq buffer 5 μl (New England Bio labs), 10 mM dNTP mix 0.5 μl 1.25 U (New England Bio labs), 0.5 μl (20 μM) of each primers, forward primer: CTG GAG ACC ACT CCC ATC CTT TCT and reverse primer: GAT GTG GCC ATC ACA TTC GTC AGA. PCR reaction conditions were: initial denaturation at 95°C for 2 minutes followed by 34 cycles of cycle denaturation at 95°C for 40 seconds, annealing at 56°C for 3 minutes, extension at 72°C for 40 seconds, final extension at 72°C for 10 minutes.
All statistical analysis were performed using SPSS Statistics 21. Chi-square tests were applied for comparison of demographic characteristics between cases and controls. Frequencies of genotypes and alleles were calculated by simple calculation. nQUERY advisor software was used for sample size exploration. The Chi-square test was performed to test Hardy–Weinberg equilibrium of genotypes among the control group. The adjusted odds ratios (AORs) and 95% confidence intervals (CIs) of the association of genotype frequencies and tobacco consumption with risk were estimated using multiple logistic regression models after adjusting with confounders. For all tests, a two-sided P ≤ 0.05 was considered statistically significant.
| » Results|| |
Baseline characteristics of study group
A total of 359 subjects of age 60 years and above were enrolled in this study out of which 205 were controls and 154 LC patients. The power calculation analysis showed that the patient-control pair of 359 was sufficient to achieve 90% power for effective sample size. Men were more in number, that is, 122 (59.4%) and 117 (75.9%) in controls and LC, respectively. Tobacco and alcohol consumption were found significantly more in the LC group (P = 0.0001) as compared to the controls. A geriatric age specific assessment threw light on the functionality, nutrition status, and the BMI of patients. Functional dependence, malnutrition, weight loss, and comorbidities were much more common in cancer patients as compared to controls shown in [Table 1].
The tumor characteristics regarding histopathology and stage are depicted in [Table 2]. Out of 154 LC, 74 were squamous cell carcinoma, 64 adenocarcinoma, and 16 were small cell carcinoma. Majority of cases presented in the late stages (Stage III and IV) of the disease.
Genotyping of ACE polymorphism
The amplified product was run on 2% agarose gel and stained with ethidium bromide for visualization of bands. Two sizes of amplified products were seen: (a) 490 bp for insertion allele, (b) 190 bp for deletion allele [Figure 1]. The results of ID polymorphism were validated by sequencing of samples from each group.
|Figure 1: The pattern of ID polymorphism on 2% agarose gel. lane 1: Marker, lanes 2, 4, and 8, are heterozygous ID; lanes 3 and 4 are homozygous DD and lane 7 and 9 is homozygous II genotype|
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Association of rs4340 ID ACE polymorphisms with LC
The frequencies and association of rs4340 ID ACE genotype in LC and control are shown in [Table 3]. The risk of LC was 2.21-fold significantly (P < 0.01) higher with II genotype as compared to wild type DD genotype.
|Table 3: Distribution frequencies of rs4340 ACE ID genotype in control and LC|
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Interaction combination of rs4340 ID ACE genotype with risk factors
Interaction combinations of ACE genotype with tobacco consumption are outlined in [Table 4]. Interestingly, a significant enhanced risk was observed with interaction of I allele with tobacco consumption with an adjusted Odd's ratio 3.43 (95% confidence interval =1.08-10.8) and a P value of 0.03 as compared to I allele without tobacco consumption and D allele with and without tobacco habits in LC [Table 4].
|Table 4: Interactive effect of tobacco consumption on rs4340ID ACE polymorphism|
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| » Discussion|| |
LC is a common cancer observed in the geriatric population of India and the commonest cancer in males above 60 years of age. It is the leading cause of cancer deaths and the 5-year mortality rate is dismal. Lung carcinogenesis is a multistep process occurring due to environmental factors such as smoking and chewing tobacco, alcohol consumption, gene alterations in oncogenes, tumor suppressor genes, and viral infections.
A male predominance was seen in both cancer patients and controls in this study. Sociocultural reasons explain the gender bias in accessing health system in developing regions like India. Women's access to care might be limited by gender norms that restrict their autonomy in these regions. Moreover, LC has a strong correlation with smoking, which is commonly seen in males all over the world. This study showed significantly more tobacco and alcohol consumption habits in LC as compared to controls which strengthen the fact that these are risk factors for development of LC. Tobacco contains hundreds of carcinogenic compounds and its addiction is associated with increased risk of LC and other cancers in virtually every organ of the body. Older patients usually present with multi-morbidities and various geriatric issues. Studies have shown that older patients suffering from cancer present with more co-morbidities and geriatric issues as compared to healthy older adults., In this study, comorbidities and geriatric issues like malnutrition, loss of functionality, and reduced BMI was observed to be significantly more in LC as compared to controls.
The genotype distribution of cases (LC) and control population were performed by Hardy Weinberg equilibrium (HWE) (P > 0.05). Many studies have been done on rs4340 ID ACE polymorphism in LC so far. This study showed a statistical significance in the II genotype of rs4340 ID polymorphism of ACE with a 2.21-fold higher risk as compared to DD genotype. Interactive corelations with tobacco were found to be positive for I alleles which showed higher risk as compared to D allele without tobacco. Similar results were observed in polymorphism studies done in the Croatian and Turkish population with LC., Phukan et al. showed significant association of II genotype with tobacco smoking and chewing in LC cases on a North Eastern population of India which was consistent with our results. India presents enormous social, linguistic, and cultural diversity due to a prolonged history of migration of different races from other parts of the world. The ACE gene polymorphism shows variations with ethnicity and in this study, we attempted to find this polymorphism in a north Indian population. However, in a study done in South Indian population, heterozygous “I/D” genotype of ACE gene was found to be of high-risk for non-small cell lung cancer (NSCLC). Meta-analyses of ACE polymorphism studies in LC done by Wang et al. and Zhang et al. on different cancers like lung, oral, breast, prostate, colorectal, etc. did not find any association with rs4340 ACE ID polymorphism.
Most studies have shown a link of increased expression of ACE with cancer development by facilitating angiogenesis through higher production of angiotensin II. Angiotensin II induces vascular endothelial growth factor, which plays a pivotal role in tumor angiogenesis and correlates with aggressive behavior and poor prognosis. ACE is also known to inactivate the vasodilator bradykinin which has a role in mitogenic activity.
The present study showed significant association of rs4340 ACE ID polymorphism with increased risk of LC. As the study was done in aging population, the analysis was done considering multiple comorbidities. Literature shows evidence that these diseases or comorbidities also have individual effect on ACE gene polymorphism. ACE I/D polymorphism has been reported to be associated with essential hypertension, myocardial infarction, coronary heart disease, diabetes mellitus, and its complications in different studies.,,,, Previous studies show that the renin–angiotensin system is not only essential in cardiovascular hemodynamics but plays an important role in the development of cardiovascular diseases (CVD). ACE gene I/D polymorphism has been proposed as a genetic marker of risk of coronary heart disease. The present study takes into consideration older adults who are expected to have comorbid diseases as mentioned above. The effect of aging together with multiple comorbidities, environmental, and genetic factors also might have an effect on the polymorphism. Our sole aim in this study is to establish an interactive corelation between tobacco smoking and ACE I/D polymorphism in LC. Therefore, we have tried to show these results in our analysis by adjusting confounders like age, gender, comorbid conditions, alcohol consumption, functional dependency, and nutrition.
In this study, the unadjusted odd's ratio of the risk of lung cancers in this population with II genotype was 2.21. On adjustment with co-founders like age, gender, nutrition, BMI, comorbidity, and functionality, the risk of these cancers with II genotype was found to be 2.05-fold higher than controls [Table 3].
It can be concluded from this study that the risk of LC is higher with II genotype as compared to DD genotype. It was also observed that I allele with tobacco habits had a higher risk as compared to D allele [Table 4]. It is thus proposed that further studies with larger samples, stratified for age and different diseases and risk factors should be conducted which may throw more light on ACE polymorphism in older patients with LC.
Authors acknowledge Indian Council of Medical Research (ICMR) and National Program for Health Care of the Elderly (NPHCE) for providing fellowship for the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| » References|| |
Prince MJ, Wu F, Guo Y, Robledo LM, O'Donnell M, Sullivan R, et al
. The burden of disease in older people and implications for health policy and practice. Lancet 2015;385:549-62.
Carluccio M, Soccio M, De Caterina R. Aspects of gene polymorphisms in cardiovascular disease: The renin-angiotensin system. Eur J Clin Invest 2001;31:476-88.
Nacak M, Nacak I, Sanli M, Ozkur M, Pektaş M, Aynacioğlu AS. Association of angiotensin converting enzyme gene insertion/deletion polymorphism with lung cancer in Turkey. Cancer Genet Cytogenet 2010;198:22-6.
Deshayes F, Nahmias C. Angiotensin receptors: A new role in cancer? Trends Endocrinol Metab 2005;16:293-9.
van der Knaap R, Siemes C, Coebergh JW, van Duijn CM, Hofman A, Stricker BH. Renin-angiotensin system inhibitors, angiotensin I-converting enzyme gene insertion/deletion polymorphism, and cancer: The Rotterdam Study. Cancer 2008;112:748-57.
Bauvois B. Transmembrane proteases in cell growth and invasion: New contributors to angiogenesis? Oncogene 2004;23:317-29.
Yoshiji H, Kuriyama S, Fukui H. Angiotensin-I-converting enzyme inhibitors may be an alternative anti-angiogenic strategy in the treatment of liver fibrosis and hepatocellular carcinoma. Tumour Biol 2003;23:348-56.
Fujita M, Hayashi I, Yamashina S, Itoman M, Majima M. Blockade of angiotensin AT1a receptor signaling reduces tumor growth, angiogenesis, and metastasis. Biochem Biophys Res Commun 2002;294:441-7.
Fendrich V, Chen NM, Neef M, Waldmann J, Buchholz M, Feldmann G, et al
. The angiotensin-I-converting enzyme inhibitor enalapril and aspirin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer. Gut 2010;59:630-7.
Puddefoot JR, Udeozo UK, Barker S, Vinson GP. The role of angiotensin II in the regulation of breast cancer cell adhesion and invasion. Endocr Relat Cancer 2006;13:895-903.
Röcken C, Lendeckel U, Dierkes J, Westphal S, Carl-McGrath S, Peters B, et al
. The number of lymph node metastases in gastric cancer correlates with the angiotensin I-converting enzyme gene insertion/deletion polymorphism. Clin Cancer Res 2005;11:2526-30.
Lang L. ACE inhibitors may reduce esophageal cancer incidence. Gastroenterology 2006;131:343-4.
Röcken C, Neumann K, Carl-McGrath S, Lage H, Ebert MP, Dierkes J, et al
. The gene polymorphism of the angiotensin I-converting enzyme correlates with tumor size and patient survival in colorectal cancer patients. Neoplasia 2007;9:716-22.
Agnihotri V, Gupta A, Kumar R, , Upadhyay AD, Dwivedi S, Kumar L, et al
. Promising link of HLA-G polymorphism, tobacco consumption and risk of Head and Neck Squamous Cell Carcinoma (HNSCC) in North Indian population. Hum Immunol 2017;78:172-8.
Markaki M, Tsamardinos I, Langhammer A, Lagani V, Hveem K, Røe OD. A validated clinical risk prediction model for lung cancer in smokers of all ages and exposure types: A HUNT Study. EBioMedicine 2018;31:36-46.
National Lung Screening Trial Research Team; Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, Fagerstrom RM, et al
. Reduced lung-cancer mortality with low-dose computed tomographic screening. N Engl J Med 2011;365:395-409.
Carson KV, Jurisevic MA, Smith BJ. Is cancer risk still reduced if you give up smoking in later life? Lung Cancer Manag 2013;2:357-68.
Mahoney FI, Barthel DW. Functional evaluation: The Barthel Index. Md State Med J 1965;14:61-5.
Rubenstein LZ, Harker JO, Salvà A, Guigoz Y, Vellas B. Screening for undernutrition in geriatric practice: Developing the short-form mini-nutritional assessment (MNA-SF). J Gerontol A Biol Sci Med Sci 2001;56:M366-72.
Misra A, Chowbey P, Makkar BM, Vikram NK, Wasir JS, Chadha D, et al
. Consensus statement for diagnosis of obesity, abdominal obesity and the metabolic syndrome for Asian Indians and recommendations for physical activity, medical and surgical management. J Assoc Physicians India 2009;57:163-70.
Vairaktaris E, Yapijakis C, Tsigris C, Vassiliou S, Derka S, Nkenke E, et al
. Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with increased risk for oral cancer. Acta Oncol 2007;46:1097-102.
Yeole BB, Kurkure AP, Koyande SS. Geriatric cancers in India: An epidemiological and demographic overview. Asian Pac J Cancer Prev 2008;9:271-4.
Valavanidis A, Vlachogianni T, Fiotakis K, Loridas S. Pulmonary oxidative stress, inflammation and cancer: Respirable particulate matter, fibrous dusts and ozone as major causes of lung carcinogenesis through reactive oxygen species mechanisms. Int J Environ Res Public Health 2013;10:3886-907.
Sen G, Ostlin P. Gender inequity in health: Why it exists and how we can change it. Glob Public Health 2008;3:1-12.
Secretan B, Straif K, Baan R, Grosse Y, El Ghissassi F, Bouvard V, et al
. A review of human carcinogens--Part E: Tobacco, areca nut, alcohol, coal smoke, and salted fish. Lancet Oncol 2009;10:1033-4.
Extermann M. Interaction between comorbidity and cancer. Cancer Control 2007;14:13-22.
Mohile SG, Magnuson A. Comprehensive geriatric assessment in oncology. Interdiscip Top Gerontol 2013;38:85-103.
Dević Pavlić S, Ristić S, Flego V, Kapović M, Radojčić Badovinac A. Angiotensin-converting enzyme insertion/deletion gene polymorphism in lung cancer patients. Genet Test Mol Biomarkers 2012;16:722-5.
Phukan RK, Borah PK, Saikia BJ, Das M, Sekhon GS, Mahanta J. Interaction of tobacco smoking and chewing with Angiotensin converting enzyme (insertion/deletion) gene polymorphisms and risk of lung cancer in a high risk area from northeast India. Asian Pac J Cancer Prev 2014;15:10691-5.
Xing J, Watkins WS, Hu Y, Huff CD, Sabo A, Muzny DM, et al
. Genetic diversity in India and the inference of Eurasian population expansion. Genome Biol 2010;11:R113.
Peddireddy V, Badabagni SP, Gundimeda SD, Mundluru HP. Association of eNOS and ACE gene polymorphisms and plasma nitric oxide with risk of non-small cell lung cancer in South India. Clin Respir J 2018;12:207-17.
Wang N, Yang D, Ji B, Li J. Angiotensin-converting enzyme insertion/deletion gene polymorphism and lung cancer risk: A meta-analysis. J Renin Angiotensin Aldosterone Syst 2015;16:189-94.
Zhang Y, He J, Deng Y, Zhang J, Li X, Xiang Z, et al
. The insertion/deletion (I/D) polymorphism in the Angiotensin-converting enzyme gene and cancer risk: A meta-analysis. BMC Med Genet 2011;12:159.
Tamarat R, Silvestre JS, Durie M, Levy BI. Angiotensin II angiogenic effect in vivo
involves vascular endothelial growth factor-and inflammation-related pathways. Lab Invest 2002;82:747-56.
Kramarenko II, Morinelli TA, Bunni MA, Raymond JR Sr, Garnovskaya MN. The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines. Cancer Manag Res 2012;4:195-205.
Mastana S, Nunn J. Angiotensin-converting enzyme deletion polymorphism is associated with hypertension in a Sikh population. Hum Hered 1997;47:250-3.
Cambien R, Poirier O, Lecerf L. Deletion polymorphism in the gene for angiotensin converting enzyme is a potent risk factor for myocardial infarction. Nature 1992;359:641-4.
Lindpaintner K, Pfeffer MA, Kreutz R. A prospective evaluation of angiotensin converting gene polymorphism and the risk factor of ischemic heart disease. N Engl J Med 1995;332:706-11.
Viswanathan V, Zhu Y, Bala K, Dunn S, Snehalatha C, Ramachandran A, et al
. Association between ACE gene polymorphism and diabetic Nephropathy in south Indian patients. JOP 2001;2:83-7.
Lee YJ, Tsai JCR. ACE gene insertion/deletion polymorphism associated with 1998 World Health Organization definition of metabolic syndrome in Chinese type 2 diabetic patients. Diabetes Care 2002;25:1002-8.
Dhar S, Ray S, Dutta A, Sengupta B, Chakrabarti S. Polymorphism of ACE gene as the genetic predisposition of coronary artery disease in Eastern India. Indian Heart J 2012;64:576-81.
[Table 1], [Table 2], [Table 3], [Table 4]