Vitamin B supplementation can have side effects for human health, including cancer risk. We aimed to elucidate the role of vitamin B12 in lung cancer etiology via direct measurements of pre‐diagnostic circulating vitamin B12 concentrations in a nested case–control study, complemented with a Mendelian randomization (MR) approach in an independent case–control sample. We used pre‐diagnostic biomarker data from 5183 case–control pairs nested within 20 prospective cohorts, and genetic data from 29,266 cases and 56,450 controls. Exposures included directly measured circulating vitamin B12 in pre‐diagnostic blood samples from the nested case–control study, and 8 single nucleotide polymorphisms associated with vitamin B12 concentrations in the MR study. Our main outcome of interest was increased risk for lung cancer, overall and by histological subtype, per increase in circulating vitamin B12 concentrations. We found circulating vitamin B12 to be positively associated with overall lung cancer risk in a dose response fashion (odds ratio for a doubling in B12 [OR log2B12 ] = 1.15, 95% confidence interval (95%CI) = 1.06–1.25). The MR analysis based on 8 genetic variants also indicated that genetically determined higher vitamin B12 concentrations were positively associated with overall lung cancer risk (OR per 150 pmol/L standard deviation increase in B12 [OR SD ] = 1.08, 95%CI = 1.00–1.16). Considering the consistency of these two independent and complementary analyses, these findings support the hypothesis that high vitamin B12 status increases the risk of lung cancer.

Introduction The potential role of B vitamins in relation to cancer risk has been reported previously.1-3 Two large randomized controlled trials of B vitamin supplementation in Norway identified an increased risk for overall cancer among subjects who received both vitamin B12 and B9 (folate), a result that was primarily driven by lung cancer.4 More recently the Vitamins and Lifestyle (VITAL) cohort study5 reported increased lung cancer risks among men who used high amounts of vitamin B12 and B6 supplementation. These results4, 5 argue against any chemo preventive effect of vitamin B12 in lung cancer, and instead are consistent with high concentrations of vitamin B12 increasing risk. To further elucidate the role of vitamin B12 in lung cancer etiology, we conducted two large and complementary analyses based on (i) directly measured circulating vitamin B12 concentrations in pre‐diagnostic samples from over 5000 case–control pairs, and (ii) a Mendelian randomization (MR) analysis based on genetic data on close to 30,000 cases and 60,000 controls.

Materials and Methods The first analysis was based on 5364 lung cancer cases and 5364 controls that were individually matched by age, sex, cohort and smoking status. This sample was nested within 20 individual prospective cohort studies participating in the Lung Cancer Cohort Consortium (LC3), which was initially established to interrogate a potential inverse relation between circulating concentrations of B6 and B9 with lung cancer risk.6, 7 Our study involved centralized biochemical analyses on pre‐diagnostic serum/plasma samples and their individually matched controls using a microbiological assay to measure circulating concentrations of vitamin B12,8 as well as a Liquid chromatography–tandem mass spectrometry (LC–MS/MS) based assay9 to measure cotinine. After excluding participants with missing values (n = 7) or extreme values of vitamin B12 (> 850 pmol/L, n = 174), a total of 5183 case–control pairs remained for our study (Table 1). To evaluate the relation between directly measured vitamin B12 and lung cancer risk we used conditional logistic regression, additionally adjusted for educational attainment and tobacco exposure (smoking matched by design, as well as cotinine concentrations). Adjusting for body mass index and alcohol intake status did not alter our estimates, and covariates indicating those risk factors were not included in the final model. P‐value for trend was calculated with a continuous variable as base 2 logarithm of the circulating concentrations of vitamin B12. Table 1. Baseline and sample characteristics of study participants LC3 participants TRICL‐ILCCO participants No.(%) of participants in group No.(%) of participants in group Discrete variables Cases (n = 5183) Matched controls (n = 5183) Cases (n = 29,266) Controls (n = 56,450) Sex Men 2827 (54.5%) 2827 (54.5%) 18,208 (62.2%) 27,178 (48.1%) Women 2356 (45.5%) 2356 (45.5%) 11,058 (37.8%) 24,072 (51.9%) Smoking status Never 1267 (24.4%) 1267 (24.4%) 2355 (8.0%) 7504 (13.3%) Ever (Former and current) 3916 (75.5%) 3916 (75.5%) 23,223 (79.3%) 16,964 (30.1%) Former 1458 (28.1%) 1458 (28.1%) Current 2458 (47.4%) 2458 (47.4%) Education Less than high school 1746 (33.7%) 1643 (31.7%) Completed high school 735 (14.2%) 754 (14.5%) Vocational school 862 (16.6%) 886 (17.1%) Some college 651 (12.6%) 698 (13.4%) College graduate 499 (9.5%) 480 (9.2%) Graduate studies 625 (12.2%) 677 (13.1%) Unknown 65 (1.2%) 45 (1.0%) Continuous variables, median (5th–95th percentile) Age at recruitment (years) 60 (44–72) 60 (44–72) 88% higher than 55 Vitamin B12 (pmol/L) 432 (239–747) 425 (231–733) Clinical characteristics, case participants only Age at diagnosis, median (range), (years) 69.7 (53.4 81.7) Time from blood draw to diagnosis (years) 6.4 (1.0–16.0) Histology, No. (%) Large cell carcinoma 166 (3.4%) Small cell carcinoma 481 (10.1%) 2664 (9.1%) Squamous cell carcinoma 813 (17.0%) 7426 (25.4%) Adenocarcinoma 1972 (41.2%) 11,273 (38.5%) Missing/Unknown 1751 (29.3%) 7903 (27.0%) The second investigation involved an MR analysis based on extensive genome‐wide data for lung cancer risk from 29,266 lung cancer cases and 56,450 controls of European descent. This extensive genetic data is available from the Transdisciplinary Research in Cancer of the Lung (TRICL) and The International Lung Cancer Consortium (ILCCO) collaborations (Table 1).10 In the MR framework, genetic variants that are robustly associated with circulating vitamin B12 can be used as proxies and compared between cases and controls, rather than using direct measures of circulating B12 concentrations (as in LC3). The advantage of the MR methodology is that genetic variants are not affected by reverse causation of the disease and are less sensitive to confounding.11 Single nucleotide polymorphisms (SNP) for circulating vitamin B12 that were previously identified in European populations,12 including 8 independent SNPs (linkage disequilibrium R2 < 0.1), explained 5.1% of circulating B12 variance.12 The strength of this instrument was assessed by estimating an F‐statistic (306.2), which, given the size of the instrument discovery sample (N = 45,576) gave sufficient power (80%) to detect OR estimates for lung cancer overall (1.10), adenocarcinoma (1.15), squamous cell (1.17) and small cell carcinoma (1.20). The effects on lung cancer risk for predicted B12 vitamin concentrations were estimated using a likelihood‐based approach,13 and the resulting OR estimates reflect a one standard deviation increase (SD) in vitamin B12 concentrations (150.1 pmol/l) based on the discovery study12. The instrumental SNPs could show heterogeneity of the estimated effect of vitamin B12 levels on lung cancer risk due to pleiotropic effects of these SNPs from other potential lung cancer risk factors. Thus, sensitivity analyses were performed to assess potential bias (non‐balanced pleiotropic effects) on our initial risk estimates.14 Additionally, we evaluated the association between the genetic proxies of vitamin B12 concentrations and smoking behavior using summary statistics for genetic association with smoking parameters from the Tobacco and Genetics (TAG) Consortium dataset comprising 74,035 participants15 using a similar MR approach. Finally, by way of reference with the GWAS catalog (https://www.ebi.ac.uk/gwas/) we sought to identify previously reported associations between the 8 SNPs included in this analysis and other known lung cancer risk factors beyond smoking.

Results Directly measured circulating vitamin B12 was positively associated with overall lung cancer risk in the LC3 consortium (OR for a doubling in vitamin B12 [OR log2B12 ] = 1.15, 95% confidence interval [95%CI] = 1.06–1.25, Fig. 1). Positive associations were seen for adenocarcinoma (OR log2B12 [95%CI] = 1.14 [1.00–1.30]) and small‐cell carcinoma (OR log2B12 [95%CI] = 1.20 [0.91–1.59]), but no association was seen for squamous cell carcinoma (OR log2B12 [95%CI] = 1.00 [0.81–1.23]). Subsequent analyses indicated a positive dose–response relation between directly measured circulating vitamin B12 and lung cancer risk (eTable1 in Supporting Information) that was consistently seen among all women, former and current smokers, participants with time from blood draw <72 months and > 120 months (eFig. 1 in Supporting Information), and European/Australian and Asian cohorts (eTable 1 in Supporting Information). Figure 1 Open in figure viewer PowerPoint Forest plot showing the relationship between circulating vitamin B12 and lung cancer risk from the LC3 and a Mendelian randomization analysis. Footnote: *LC3 odds ratios (OR) indicate relative risks of a doubling in circulating concentrations (base 2 logarithm transformed) adjusted for cotinine and education when relevant (95%CI: 95% confidence intervals). † Mendelian randomization ORs indicate the odds for a one standard deviation (SD) increase in circulating concentrations (approximately 150 pmol/L). ‡ P heterogeneity indicates results of chi‐square test assessing the null hypothesis of ORs being the identical. The MR analysis for circulating vitamin B12 based on 8 genetic variants was consistent with the LC3 results, showing that a one SD genetically predicted higher vitamin B12 concentration was associated with an increase in overall lung cancer risk (OR SD [95%CI] = 1.08 [1.00–1.16]). Similar to the LC3 analysis, the MR analysis stratified by histology suggested stronger associations for adenocarcinoma (OR SD [95%CI] = 1.23 [1.11–1.37]) and small‐cell carcinoma (OR SD [95%CI] = 1.17 [0.96–1.41]), but not for squamous cell carcinoma (OR SD [95%CI] = 0.97 [0.86–1.10]; p value for heterogeneity = 0.01, Fig. 1). The MR‐Egger test did not indicate bias in the risk estimates due to pleiotropy for lung overall (P value for MR‐Egger intercept [p Int ] = 0.17), nor for any histological subtype (p Int > 0.11). Furthermore, genetically predicted higher vitamin B12 concentrations were not associated with smoking parameters (OR SD being a smoker [95%CI] = 1.00 [0.91–1.11]; number of extra cigarettes smoked per day [95%CI] = −0.13 [−0.82:0.57]), indicating that our MR results on lung cancer risk were not explained by smoking as a confounder. Finally, the GWAS catalog did not list any other lung cancer risk factor in association with the 8 SNPs used for the current MR analysis. More specifically, the rs1801222 and rs602662 SNPs were associated with homocysteine levels in the one‐carbon metabolism pathway, and pediatric autoimmune diseases, respectively.

Discussion In summary, we performed two complementary and independent analyses to evaluate if elevated concentrations of vitamin B12 increased lung cancer risk.5 Circulating concentrations of vitamin B12, based on pre‐diagnostic blood samples from the LC3 consortium on over 5000 case–control pairs, were positively associated with lung cancer risk, and in contrast to the VITAL study, this association was consistently seen across sexes, former and current smokers, time from blood draw, and geographic region (eFig. 1, Supporting Information). Confirming these results, the MR analysis based on genetic data indicated that higher concentrations of vitamin B12 increased the risk of lung cancer, especially for adenocarcinoma and small‐cell carcinoma, with no association seen for squamous cell carcinoma. Generalizability of our results to populations not represented in the data used for the current analyses should be made with caution.

Conclusions Considering the consistency of these two independent and complementary analyses, as well as previously published studies,4, 5 these findings support the hypothesis that higher circulating vitamin B12 concentrations increase the risk of lung cancer.

Acknowledgements The Lung Cancer Cohort Consortium (LC3) was supported by National Institutes of Health/National Cancer Institute grant No. 1U01CA155340. The Transdisciplinary Research for Cancer in Lung (TRICL) of the International Lung Cancer Consortium (ILCCO) was supported by (U19‐CA148127 and CA148127S1). The ILCCO data harmonization is supported by Cancer Care Ontario Research Chair of Population Studies to R.H. and Lunenfeld‐Tanenbaum Research Institute, Sinai Health System. The TRICL‐ILCCO OncoArray was supported by in‐kind genotyping by the Centre for Inherited Disease Research (26820120008i‐0‐26800068‐1). The work of TLL was undertaken during a postdoctoral placement at the International Agency for Research on Cancer, within the framework of an agreement between the Research Council of Norway and the Norwegian University of Science and Technology. The funding organizations had no role in design and conduct of the study; collection, management,analysis and interpretation of the data; preparation, review, or approval of the study. The authors would like to thank all cohort participants and staff, including but not limited to, the participants and staff of the Health Professionals Follow‐up Study and Nurses' Health Study for their valuable contribution as well as the after state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY.

Supporting Information Filename Description ijc32033-sup-0001-Tables.docxWord 2007 document , 22.9 KB Supplementary tables: eTable 1 ‐ Odds ratios of lung cancer for circulating concentrations of vitamin B12. eTable 2 – β coefficients of vitamin B12 genetic instruments (SNPS) with vitamin B12 concentrations and lung cancer status overall, by histological subtypes, and smoking status. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.