CONSORT indicates Consolidated Standards of Reporting Trials; ECOG, Eastern Cooperative Oncology Group; and EGFR-TKIs, epidermal growth factor receptor–tyrosine kinase inhibitors.
A, Median progression-free survival is 9.9 (95% CI, 7.5-12.2) months for EGFR-TKIs vs 13.1 (95% CI, 9.8-16.3) months for EGFR-TKIs plus metformin (HR, 0.60; 95% CI, 0.40-0.94; P = .03). B, Median progression-free survival is 9.7 (95% CI, 5.1-14.3) months for EGFR-TKIs vs 11.8 (7.3-20.6) months for EGFR-TKIs plus metformin (HR, 0.64; 95% CI, 0.41-0.99; P = .049). C, Median objective response rate is 54.3% (38 of 70) for EGFR-TKIs vs 71.0% (49 of 69) for EGFR-TKIs plus metformin (P = .04). D, Median overall survival is 17.5 (95% CI, 11.4-23.7) months for EGFR-TKIs vs 31.7 (95% CI, 20.5-42.8) months for EGFR-TKIs plus metformin (HR, 0.50; 95% CI, 0.28-0.90; P = .02). EGFR-TKIs indicates epidermal growth factor receptor–tyrosine kinase inhibitors; HR, hazard ratio; and RECIST, Response Evaluation Criteria in Solid Tumors guideline (version 1.1).
eFigure 1. Molecular effects of metformin on lung cancer
eFigure 2. Progression-free survival (radio-oncologist 2)
eFigure 3. (A) Kaplan-Meier curves for overall survival among patients who received second-line treatment and (B) patients who did not receive a second-line of treatment
eFigure 4. Kaplan-Meier curves for (A) Progression-free survival comparison between patients with negative LKB1 expression according to the therapeutic arm. (B) Overall survival comparison between patients with negative LKB1 expression according to the received treatment. (C) Progression-free survival comparison between patients with positive LKB1 expression according to the received treatment. (D) Overall survival comparison between patients with positive LKB1 expression according to therapeutic arm
eFigure 5. Box-plots of the serum levels of Glucose (A), HbA1c (B), Interleukin-6 (C), and Insulin growth factor receptor (D) at baseline, the endo of 1st, and 2nd EGFR-TKIs cycle
eMethods 1. Supplementary laboratory methods
eMethods 2. Supplementary log file for statistical analysis
eTable 1. Concordance agreement assessment among radio-oncologists (n = 2) for the radiographic evaluation of the best response rate through RECIST v1.1
eTable 2. Univariate and multivariable analysis of the clinic-pathological factors associated with progression-free survival according to radio-oncologist 2
eTable 3. Crude and adjusted Hazard Ratios for PFS by Radio-oncologists 1 and 2
eTable 4. Adverse events
eTable 5. Details of metformin treatment
eTable 6. Further lines of treatment
eTable 7. Overall survival among patients who did not receive a second-line of therapy after intervention
eTable 8. Overall survival among patients who received a second-line of therapy after intervention
eTable 9. Univariate and LKB1-stratified analysis of the clinic-pathological factors associated with progression-free survival
eTable 10. Univariate and LKB1-stratified analysis of the clinic-pathological factors associated with overall survival
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Arrieta O, Barrón F, Padilla MS, et al. Effect of Metformin Plus Tyrosine Kinase Inhibitors Compared With Tyrosine Kinase Inhibitors Alone in Patients With Epidermal Growth Factor Receptor–Mutated Lung Adenocarcinoma: A Phase 2 Randomized Clinical Trial. JAMA Oncol. 2019;5(11):e192553. doi:10.1001/jamaoncol.2019.2553
Does the addition of metformin hydrochloride to standard epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors for EGFR-mutated lung adenocarcinoma improve progression-free survival compared with EGFR–tyrosine kinase inhibitors therapy alone?
In this phase 2 randomized clinical trial that included 139 patients, the addition of metformin to standard EGFR–tyrosine kinase inhibitors significantly improved both progression-free survival and overall survival.
In this phase 2 trial of patients with EGFR-mutated lung adenocarcinoma, the addition of metformin increased survival, warranting the design of a larger phase 3 study.
Metformin hydrochloride is emerging as a repurposed anticancer drug. Preclinical and retrospective studies have shown that it improves outcomes across a wide variety of neoplasms, including lung cancer. Particularly, evidence is accumulating regarding the synergistic association between metformin and epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors (TKIs).
To assess the progression-free survival (PFS) in patients with advanced lung adenocarcinoma who received treatment with EGFR-TKIs plus metformin compared with those who received EGFR-TKIs alone.
Design, Setting, and Participants
Open-label, randomized, phase 2 trial conducted at the Instituto Nacional de Cancerología (INCan), Mexico City, Mexico. Eligible patients were 18 years or older, had histologically confirmed stage IIIB-IV lung adenocarcinoma with an activating EGFR mutation.
Patients were randomly allocated to receive EGFR-TKIs (erlotinib hydrochloride, afatinib dimaleate, or gefitinib at standard dosage) plus metformin hydrochloride (500 mg twice a day) or EGFR-TKIs alone. Treatment was continued until occurrence of intolerable toxic effects or withdrawal of consent.
Main Outcomes and Measures
The primary outcome was PFS in the intent-to-treat population. Secondary outcomes included objective response rate, disease control rate, overall survival (OS), and safety.
Between March 31, 2016, and December 31, 2017, a total of 139 patients (mean [SD] age, 59.4 [12.0] years; 65.5% female) were randomly assigned to receive EGFR-TKIs (n = 70) or EGFR-TKIs plus metformin (n = 69). The median PFS was significantly longer in the EGFR-TKIs plus metformin group (13.1; 95% CI, 9.8-16.3 months) compared with the EGFR-TKIs group (9.9; 95% CI, 7.5-12.2 months) (hazard ratio, 0.60; 95% CI, 0.40-0.94; P = .03). The median OS was also significantly longer for patients receiving the combination therapy (31.7; 95% CI, 20.5-42.8 vs 17.5; 95% CI, 11.4-23.7 months; P = .02).
Conclusions and Relevance
To our knowledge, this is the first study to prospectively show that the addition of metformin to standard EGFR-TKIs therapy in patients with advanced lung adenocarcinoma significantly improves PFS. These results justify the design of a phase 3, placebo-controlled study.
ClinicalTrials.gov identifier: NCT03071705
Lung cancer remains a major public health problem, with more than 1.6 million deaths every year; it is the leading cause of cancer-related deaths worldwide.1,2 In addition to the alarming incidence rate, 75% of patients with lung cancer are diagnosed in the late-stage setting, when therapeutic options are often inefficient at achieving long-term disease control.3 Nonetheless, the discovery of targetable molecular alterations has outlined a subset of patients with lung cancer who are sensitive to targeted therapy.4 Particularly, the epidermal growth factor receptor gene (EGFR [OMIM 131550]), a common genetic alteration in lung cancer,4 renders potential sensitivity to specific tyrosine kinase inhibitors (TKIs).5 The use of epidermal growth factor receptor (EGFR)-TKIs has dramatically improved patient outcomes; however, patients ultimately progress due to acquired resistance.6 Current research is focusing on therapeutic combinations that might increase long-term efficacy of EGFR-TKIs, including the addition of antiangiogenics, immune checkpoint inhibitors, and poly–adenosine diphosphate ribose polymerase (PARP) inhibitors, among others. Nevertheless, some of these combinations come with a high risk of toxic effects and in some cases have failed to show a survival advantage.7-9
Previous reports have identified that metformin hydrochloride, an oral biguanide used for treating type 2 diabetes, might be beneficial when repurposing its use as an antineoplastic agent.10-14 Metformin affects the mitochondrial respiration rate, as well as adenosine triphosphate (ATP) production. In addition, it activates adenosine monophosphate (AMP)–activated protein kinase (AMPK) by liver kinase 1 (LKB1), which decreases cellular proliferation (eFigure 1 in Supplement 1). A recent phase 2 trial15 showed a significant benefit in progression-free survival (PFS) for patients treated with carboplatin, paclitaxel, bevacizumab, and metformin compared with historical controls treated without metformin, without added toxic effects. Evidence has also been accumulating in terms of the synergistic association between metformin and EGFR-TKIs both in vitro and in retrospective studies.16-18 However, the efficacy of metformin use on the outcomes of patients with lung adenocarcinoma has not been previously validated to our knowledge in prospective trials among specific oncogene-addicted subsets. We present the results from a randomized, phase 2 trial that evaluated the effect of adding metformin to a standard treatment with EGFR-TKIs in patients with EGFR-mutated lung adenocarcinoma.
This prospective, open-label, randomized, phase 2 trial recruited patients at the Instituto Nacional de Cancerología (INCan), Mexico City, Mexico, between March 31, 2016, and December 31, 2017. Eligible patients were 18 years or older and had histologically confirmed stage IIIB/IV lung adenocarcinoma with an activating EGFR mutation. All patients were naive to EGFR-TKIs treatment and had an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, a life expectancy of at least 12 weeks, and measurable lesions based on the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1). Main exclusion criteria included the presence of Thr790Met mutation, history of diabetes, and the use of any hypoglycemic drug. This study was performed in accord with the Declaration of Helsinki19 and the principles of good clinical practice. All patients provided written informed consent to participate, and the study was approved by the scientific and bioethical committees of the Instituto Nacional de Cancerología (INCan) (CEI/1019/16). The trial protocol is available in Supplement 2. This study followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guidelines.
The sample size was calculated for a 2-sample comparison PFS function with the log-rank test using the method by Freedman20 to observe an effect size corresponding to a 0.47 hazard ratio (HR)16 between the therapeutic arms to prove the next hypothesis for H0: S1(t) = S2(t), where H0 indicates the null hypothesis; S1(t), the survival function for the experimental arm; and S2(t), the survival function for the control arm. In addition, study power was set at 0.80, and the 2-sided type I error (α) was set at 0.20. Therefore, the estimated sample size was 124. Finally, 14 patients (10%) were added to the whole sample size to account for losses, with an expected sample of 138.
Eligible patients were randomly assigned in a 1:1 ratio using a random-numbers table to receive treatment with EGFR-TKIs or EGFR-TKIs plus metformin. The method was simple randomization; allocation was not masked, so both the patient and health care provider were aware of the assignment.
The primary outcome was PFS in the intent-to-treat population. Secondary outcomes included objective response rate (ORR), disease control rate (DCR), overall survival (OS), and safety. For PFS, time to event was defined and calculated from the date of randomization until radiographic disease progression or treatment discontinuation due to either unacceptable toxic effects or death by any cause. For OS, time to event was defined as time from randomization until death by any cause or loss to follow-up. Observations for patients who did not experience an event were censored at patient-specific last follow-up. The radiographic response was assessed by 2 independent masked radiooncologists (M.Y.R. and a nonauthor) according to the RECIST guideline (version 1.1) by comparing the pretreatment and posttreatment images. Any in-field tumor progression or the appearance of new malignant lesions denoted progressive disease. The ORR was defined as the sum of complete and partial response, whereas DCR was defined as the sum of ORR and stable disease. In addition, an exploratory analysis was performed to evaluate the usefulness of LKB1 as a prognostic biomarker in patients with non–small cell lung cancer (NSCLC) treated with EGFR-TKIs and metformin.
Patients in the EGFR-TKIs group received treatment with erlotinib hydrochloride, afatinib dimaleate, or gefitinib at standard dosage according to physician preference. Patients in the experimental arm received treatment with EGFR-TKIs plus metformin hydrochloride (500 mg twice a day). Metformin was started concomitantly with EGFR-TKIs therapy. Treatment was continued until intolerable adverse events or withdrawal of consent. Toxic effects were assessed according to the Common Terminology Criteria for Adverse Events (version 4.0) at each visit. Patients were clinically assessed every 4 weeks. A dosage reduction for metformin hydrochloride (500 mg daily) was permitted in the case of intolerable grade 3 to 4 drug-related adverse events. Patients who progressed to EGFR-TKIs were switched to a different systemic therapy; however, metformin treatment was continued until occurrence of intolerable toxic effects. A 28-day treatment interruption to recover from toxic effects was allowed. Patients in both groups were assessed for progression every 8 weeks (±1 week) with computed tomography or magnetic resonance imaging. After disease progression, patients continued to receive metformin, in addition to further therapy recommended by their treating physician. Additional information regarding laboratory methods can be found in eMethods 1 in Supplement 1.
Continuous variables were summarized as arithmetic means with standard deviations or medians and ranges according to the data distribution assessed with the Kolmogorov-Smirnov test. For descriptive purposes, categorical variables were summarized as frequencies and percentages. Cross-sectional inferential comparisons were made using the Mann-Whitney test. Paired comparisons were performed using the Wilcoxon rank sum test. The χ2 test or the Fisher exact test was used for assessing statistical significance of categorical variables. Both PFS and OS were analyzed with the Kaplan-Meier method. Comparisons among the subgroups were analyzed using the log-rank test. Statistically significant (P < .05) and clinically relevant variables were included for adjustment in the multivariable Cox proportional hazards regression model, and HRs were calculated with their corresponding 95% CIs as a measure of association. Statistical significance was set at P < .05 using a 2-tailed test. SPSS software (version 20; SPSS Inc) was used for statistical analysis, and Stata (release 13; StataCorp LP) was used for plotting. This study was registered at ClinicalTrials.gov (NCT03071705) (an error occurred when uploading the information to ClinicalTrials.gov, which incorrectly lists OS as the primary end point). Further information regarding the statistical analysis is available in eMethods 2 in Supplement 1.
A total of 164 patients were assessed for eligibility. Among them, 139 patients (mean [SD] age, 59.4 [12.0] years; 65.5% female) met inclusion criteria and were enrolled. Participants were randomly assigned to receive EGFR-TKIs (n = 70) or EGFR-TKIs plus metformin (n = 69) (Figure 1). Baseline characteristics were equally distributed between the treatment groups. There were no patients with recurrent disease who met eligibility criteria during the recruitment period; therefore, the study population includes only patients with recently diagnosed lung adenocarcinoma (Table 1). The median follow-up for the entire population was 16.9 (95% CI, 7.0-24.9) months. The median PFS was 10.9 (95% CI, 9.4-12.5) months. In the univariate analysis, the addition of metformin to EGFR-TKIs treatment was associated with a better PFS (13.1; 95% CI, 9.8-16.3 vs 9.9; 95% CI, 7.5-12.2 months; P = .01). Patients allocated to receive EGFR-TKIs plus metformin had 40% less risk of progression to EGFR-TKIs (HR, 0.60; 95% CI, 0.40-0.94; P = .03) (Table 2 and Figure 2A and B). In both the univariate and multivariable analyses, the presence of liver metastases was the only clinical factor associated with a worse PFS (HR, 2.71; 95% CI, 1.20-6.30; P = .02) (Table 2).
Patients allocated to receive EGFR-TKIs plus metformin were more likely to respond to EGFR-TKIs therapy (71.0% [49 of 69] vs 54.3% [38 of 70]; P = .04). Grade 3 or 4 adverse events in both groups included diarrhea, rash, nausea, and mucositis; however, the frequency was similar across both arms. Indeed, the addition of metformin to EGFR-TKIs was associated with a lower risk of not responding to EGFR-TKIs (ORR, 0.48; 95% CI, 0.24-0.97; P = .04) (Table 1 and Figure 2C). An additional independent validation assessment of the radiographic outcomes was performed by a second masked radiooncologist, with a concordance agreement percentage of 91.7% and a κ value of 0.784 (eFigure 2 and eTables 1, 2, and 3 and in Supplement 1).
The median OS was 21.5 (95% CI, 7.9-25.1) months. Patients in the experimental arm of the trial had a statistically significant longer median OS of 31.7 (95% CI, 20.5-42.8) months compared with those in the control arm, who had a median OS of 17.5 (95% CI, 11.4-23.7) months (P = .02). In the univariate analysis, the addition of metformin to EGFR-TKIs was the only statistically significant factor associated with a better OS (Figure 2D). Disease progression to EGFR-TKIs was associated with a worse OS (5.5; 95% CI, 0.0-19.3 vs 22.5; 95% CI, 17.6-23.4 months; P = .02). In the multivariable analysis, the addition of metformin to EGFR-TKIs was the only factor independently associated with a better OS, decreasing the hazard of death by 48% (HR, 0.52; 95% CI, 0.30-0.90; P = .04) (Table 3). After progression, patients in our study were mostly treated with standard platinum-based chemotherapy. Patients in the experimental group who did not receive second-line therapy had lower hazards of death (HR, 0.32; 95% CI, 0.14-0.70; P = .005).
The frequency and severity of adverse events were evaluated among all participants. Overall, adverse events were similar in patients treated with EGFR-TKIs plus metformin and patients treated with EGFR-TKIs. Details regarding adverse events are listed in eTable 4 in Supplement 1. eTable 5 in Supplement 1 summarizes metformin dosage reductions, discontinuations, and continuations after progression. A total of 61 patients received second-line therapy (31 in the EGFR-TKIs group and 30 in the EGFR-TKIs plus metformin group). Only 17 patients received a third line of treatment (eTable 6 in Supplement 1). An additional stratified analysis was performed to assess the effect of second-line therapy on OS (eFigure 3, eTable 7, and eTable 8 in Supplement 1).
As a post hoc analysis, LKB1 expression on tissue biopsy specimens, as well as serological levels of glucose, glycated hemoglobin, interleukin 6 (IL-6), and insulinlike growth factor (IGF), was assessed in a subset of patients (n = 24) enrolled in the trial. The overall frequency of LKB1 loss of expression was 33.3%. No characteristic was associated with PFS in the stratified univariate analysis (eTable 9 in Supplement 1). When assessing OS stratified on LKB1 expression, LKB1-positive patients seemed to have a better OS when metformin was added to EGFR-TKIs therapy (not reached vs 6.7 months; P = .002). (eFigure 4 and eTable 10 in Supplement 1). The findings regarding glycated hemoglobin, IL-6, and IGF levels are shown in eFigure 5 in Supplement 1.
To the best of our knowledge, this is the first report that prospectively evaluates the use of metformin combined with EGFR-TKIs in patients with advanced NSCLC harboring EGFR mutations. Our results show that patients who receive treatment with EGFR-TKIs combined with metformin have a significantly improved PFS and ORR and a significantly longer OS compared with those who received treatment with EGFR-TKIs alone. Multivariable analysis showed that treatment with metformin is independently associated with longer PFS and OS.
Since 2005, evidence regarding the antineoplastic effectiveness of metformin has been accumulating,21,22 with the results showing that metformin use reduced the risk of pancreatic, prostate, and lung cancer,22 even when adjusting for age, smoking status, and glycated hemoglobin levels. Recently, the beneficial effectiveness of metformin in survival outcomes for patients with lung cancer has been documented in several retrospective trials, including a study16 of metformin use in patients receiving EGFR-TKIs treatment. The survival outcomes we report in our study are in concordance with the results reported in that study, with a similar ORR (71.0% vs 70.5%) and OS (31.7 vs 32.0 months) in patients treated with EGFR-TKIs plus metformin. This evidence suggests that the use of metformin as concomitant treatment of lung adenocarcinoma can be a valuable addition to improve clinical outcomes.14 Findings from in vitro studies10,23 have suggested a synergistic association between metformin and EGFR-TKIs in EGFR-mutated lung cancer cell lines, and the combination of metformin plus erlotinib or gefitinib has been shown to resensitize cell lines with EGFR-TKIs resistance. Although the exact mechanism of action remains unknown, it has been reported that the most potent antineoplastic effectiveness of metformin is by activating the LKB1-AMPK signaling pathway, which leads to an increased ratio of AMP to ATP and to AMPK activation, thus suppressing the mammalian target of rapamycin (mTOR) signaling pathway and leading to inhibition of tumor cell proliferation.24,25
LKB1 (OMIM 602216) mutations result in the loss of AMPK functions and have been associated with several types of cancer, including lung cancer.26,27 Deletions of LKB1 are the third most common type of mutation-associated NSCLC expression, ranging from 10% to 50%,28 and LKB1 loss is associated with a more aggressive phenotype in NSCLC.29,30
There are several strengths of this study, including the selection of a homogeneous population of patients with EGFR mutations. In addition, the study design reaffirms previous reports regarding the benefit of the addition of metformin to the EGFR-TKIs, suggesting the potential use of this drug in combination with osimertinib mesylate for first-line treatment, as supported by a recent preclinical study.31
However, the findings in this study should also be interpreted in light of its limitations, including the lack of randomization stratification according to smoking status, the EGFR mutation profile, or the EGFR-TKIs. Also, the fact that OS data might appear to subperform compared with recently published trials of first-line EGFR-TKIs (eg, the FLAURA study)32 could be explained by the high incidence of central nervous system metastases at the beginning of our trial or the tumor mutation burden in the patients studied. In the FLAURA study, 18-month survival was 71% (95% CI, 65%-76%) in the standard EGFR-TKIs group, which included patients treated with first-generation and second-generation EGFR-TKIs. However, 43% of these patients crossed over to the osimertinib arm of the trial as second-line treatment after their first progression, which might have influenced OS data. After progression, patients in our study were mostly treated with standard platinum-based chemotherapy (eTable 6 in Supplement 1). Patients in the experimental group who did not receive second-line therapy had lower hazards of death (HR, 0.32; 95% CI, 0.14-0.70; P = .005). Another important limitation is the fact that our trial was not designed as a double-blind study; therefore, several biases might be present. The presence of immortal bias must be acknowledged because patients had received previous non-TKIs treatments before the study. Therefore, despite the use of adjustment methods for survival analyses using the Cox proportional hazards regression model, future considerations of this drawback must be taken into account when designing additional phase 3 trials. It is of utmost importance that future phase 3 trials be designed as double-blind studies; they should include a stratified randomization with treatment-naive nondiabetic patients and with a mixed design that allows the simultaneous assessment of potential biomarkers to avoid these relevant limitations.
The addition of metformin to a standard EGFR-TKIs treatment in patients with EGFR-mutated lung adenocarcinoma significantly prolongs PFS and OS, without significantly increasing adverse events. The results from this phase 2 study warrant the design of a larger, phase 3, placebo-controlled study to draw more robust conclusions.
Accepted for Publication: May 8, 2019.
Corresponding Author: Oscar Arrieta, MD, MSc, Thoracic Oncology Unit, Instituto Nacional de Cancerología (INCan), Ave San Fernando, Sección XVI, Tlalpan Building 22, Mexico City 14080, Mexico (email@example.com).
Published Online: September 5, 2019. doi:10.1001/jamaoncol.2019.2553
Author Contributions: Drs Arrieta and Ramírez-Tirado had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Concept and design: Arrieta, Cardona.
Acquisition, analysis, or interpretation of data: All authors.
Drafting of the manuscript: Arrieta, Barrón, Ramírez-Tirado, Arguelles Jiménez, Vergara, Zatarain Barrón, Hernández-Pedro, Cardona, Yamamoto Ramos.
Critical revision of the manuscript for important intellectual content: Arrieta, Barrón, Salinas Padilla, Avilés-Salas, Ramírez-Tirado, Vergara, Zatarain Barrón, Cardona, Cruz-Rico, Barrios Bernal, Rosell.
Statistical analysis: Arrieta, Salinas Padilla, Ramírez-Tirado, Cardona.
Obtained funding: Arrieta, Salinas Padilla, Barrios Bernal, Yamamoto Ramos.
Administrative, technical, or material support: Arrieta, Salinas Padilla, Avilés-Salas, Vergara, Zatarain Barrón, Hernández-Pedro, Cruz-Rico, Yamamoto Ramos.
Supervision: Arrieta, Barrón, Salinas Padilla, Ramírez-Tirado, Arguelles Jiménez, Vergara.
Conflict of Interest Disclosures: Dr Arrieta reported receiving grants or personal fees from the National Council for Science and Technology in Mexico (CONACyT), Pfizer, AstraZeneca, Boehringer Ingelheim, Lilly, Merck, Bristol-Myers Squibb, and Roche. Dr Vergara reported receiving grants from CONACyT. Dr Zatarain-Barrón reported receiving grants from CONACyT. Dr Cardona reported receiving grants or personal fees from Roche, Boehringer Ingelheim, AstraZeneca, Pfizer, Celldex, Bristol-Myers Squibb, Merck Sharp & Dohme, and AbbVie and reported being cofounder of the Foundation for Clinical and Applied Cancer Research (FICMAC), Bogotá, Colombia. No other disclosures were reported.
Funding/Support: This study was funded by grant 2016-01-3160 from the National Council for Science and Technology in Mexico (CONACyT).
Role of the Funder/Sponsor: The funding source had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Data Sharing Statement: See Supplement 3.