Background: Lung cancer is a major health burden causing 160,000 and 1.6 million deaths annually in the United States and worldwide, respectively.

Methods: While seeking to identify stable and reproducible biomarkers in noninvasively collected biofluids, we assessed whether previously identified metabolite urinary lung cancer biomarkers, creatine riboside (CR), N-acetylneuraminic acid (NANA), cortisol sulfate, and indeterminate metabolite 561+, were elevated in the urines of subjects prior to lung cancer diagnosis in a well-characterized prospective Southern Community Cohort Study (SCCS). Urine was examined from 178 patients and 351 nondiseased controls, confirming that one of four metabolites was associated with lung cancer risk in the overall case–control set, whereas two metabolites were associated with lung cancer risk in European-Americans.

Results: OR of lung cancer associated with elevated CR levels, and adjusted for smoking and other potential confounders, was 2.0 [95% confidence interval (CI), 1.2–3.4; P= 0.01]. In European-Americans, both CR and NANA were significantly associated with lung cancer risk (OR = 5.3; 95% CI, 1.6–17.6; P= 0.006 and OR=3.5; 95% CI, 1.5–8.4; P= 0.004, respectively). However, race itself did not significantly modify the associations. ROC analysis showed that adding CR and NANA to a model containing previously established lung cancer risk factors led to a significantly improved classifier (P= 0.01). Increasing urinary levels of CR and NANA displayed a positive association with increasing tumor size, strengthening a previously established link to altered tumor metabolism.

Conclusion and Impact: These replicated results provide evidence that identified urinary metabolite biomarkers have a potential utility as noninvasive, clinical screening tools for early diagnosis of lung cancer. Cancer Epidemiol Biomarkers Prev; 25(6); 978–86. ©2016 AACR.

Lung cancer continues to be a significant burden in the United States and worldwide (1), with still over 40 million smokers in the United States, approximately 23% of men and 19% of women ages 35 to 64, and even higher percentages among lower socioeconomic groups (2). A substantial challenge with this disease is that at present, more than 61% of patients are diagnosed in later stages III and IV, when therapeutic options are limited, and the 5-year survival is only 4%. In contrast, the 5-year survival of patients with early stage and localized lung cancer is about 50%, and is thus considerably improved compared with later stages (1). These exceptionally bleak statistics provide strong motivation to search for biomarkers that could aid currently established screening methods for early detection of lung cancer. To date, no such biomarkers are clinically utilized.

A seminal report by the National Lung Screening Trial (NLST) in 2011 showed for the first time a significant 20% reduction in lung cancer mortality from low-dose CT (LDCT) screening when compared with the chest X-ray (3). Since then, there have been a number of recommendations for screening high-risk populations of ages 55–79 years, with a 30-pack-year history of smoking (4–7), triggering coverage by Medicare and private health insurers under provisions of the Affordable Care Act (8). However, the major problem with LDCT is a high false-positive rate of 96%, which can lead to unnecessary downstream diagnostic procedures that may result in patient anxiety, higher health care spending, and increased mortality (3). Despite encouraging results of the NLST, there are concerns related to the cost benefit of LDCT for lung cancer screening, risks of radiation exposure, overdiagnosis (9), and expected epidemic rise in indeterminate pulmonary nodules (IPN) diagnoses, only some of which present a higher risk for developing lung cancer (10). The use of complementary and accurate biomarkers would be a useful strategy to ameliorate most of these issues. However, most of the available molecular factors incorporated in diagnostic algorithms that guide therapy decisions are established by using invasively collected tissue specimens (11, 12). Noninvasively collected biospecimens, such as urine and blood, are gaining a significant interest as methods for measuring robust biomarkers of risk and prognosis (13–15).

While several circulating biomarkers have been evaluated in association with lung cancer risk (16–23), a large majority are tobacco-related carcinogens (24–27), which are markers of exposure and do not present targets for potential therapeutic interventions. Other studies have evaluated demographic and smoking-related behavioral information to aid in the selection criteria for lung cancer screening, much of which is self-reported (28). Nevertheless, to increase the specificity of LDCT, the ultimate goal is to identify stable, reproducible, and noninvasively measured biomarkers. If said biomarkers are also products of deregulated tumor metabolism, they may have an ability to distinguish benign from malignant nodules. Despite intensive research and major advances in the field, there are still a number of challenges before biomarker panels can be used routinely in clinical practice, the most important of which is a necessity to validate them. The National Cancer Institute (NCI) previously created the Early Detection Research Network to promote biomarker discovery, validation, and translation into clinical practice (29). To fully assess and develop reliable biomarkers for monitoring asymptomatic patients for lung cancer, there is an urgent need to evaluate their utility in prediagnostic samples.

To that extent, we have evaluated a previously identified panel of urinary metabolite lung cancer biomarkers (30) in a well-characterized prospective Southern Community Cohort Study (SCCS): creatine riboside (CR), N-acetylneuraminic acid (NANA), cortisol sulfate, and a glucuronidated, indeterminate metabolite referred to as 561+. Previously, novel CR and NANA were found to be overexpressed in tumor when compared with the adjacent nontumor tissue, with levels positively correlated between the tissue and urine (30). Therefore, if their association with lung cancer is validated, these metabolites present promising molecular markers for clinical evaluation as a method to aid LDCT, especially considering that metabolites are more proximal to disease phenotype than other “omics” markers.

Study subjects

With recruitment completed in 2009, the Southern Community Cohort Study (SCCS) comprises a population of adults ages 40–79 years who reside in the southeastern United States and belong to historically underrepresented groups in major cancer research studies (e.g., African-Americans; ref. 31), with a high prevalence of behaviors associated with increased disease risk (e.g., smoking). Key strengths of this cohort are that subjects come from similar socioeconomic backgrounds (lower compared with other established cohorts), thereby diminishing this factor as a potential confounder, and an established Biospecimen Repository, containing frozen stored blood, buccal cells, and urine.

Detailed procedures for data linkage, processing and quality control have been established with the 12 state cancer registries covering the SCCS catchment area, providing the primary means of identifying incident cancer diagnoses. Although reporting lags are common, the registries provide nearly complete and unbiased ascertainment of cancers diagnosed among the participants after their entry into the SCCS. Three of the registries are SEER registries, and all 12 registries have North American Association of Central Cancer Registries gold- (N= 11) or silver-level (N= 1) certification. Cohort member deaths are identified through annual linkages with both the National Death Index (NDI) and Social Security Administration (SSA), well-established and reliable means of identifying deaths in the United States (32, 33). In addition, social security numbers (an ideal linking criterion) were collected from 97% of SCCS participants. Informed consent was obtained from all study participants. This study was approved by the Institutional Review Boards of the involved institutions.

At the commencement of the study, there were 854 incident lung cancer cases and 178 of them had available urine samples for the metabolomics analysis, thereby forming the case group for the nested case–control analysis. Corresponding individually matched controls with stored urine specimens were randomly selected in a 2:1 ratio to cases using incidence density sampling among cohort members matched by age (±2 years), sex, race, CHC recruitment site, menopausal status (women), and date of sample collection (±6 months), the latter to ensure similar specimen storage durations (Table 1). Of note, we conducted an analysis to compare the main variables used for matching or adjustment of analyses between the cases who had and those who did not have urine samples. We found no significant differences except in the race distribution (P = 0.03), with a higher number of African-American subjects in those without urine samples when compared with those with (65%, vs. 54%), and self-reported prior history of chronic obstructive pulmonary disease (COPD; P = 0.05). Therefore, we believe that the results presented herein are generalizable to the entire SCCS cohort.

Table 1.

Demographic and clinical characteristics of the nested SCCS case–control sample set

CharacteristicsCasesControlsTotalPa
N 178 351 529 0.83 
Age; mean ± SD 57.7 ± 8.6 57.3 ± 8.5 57.4 ± 8.6  
Race; n(%) 
 African-American 96 (54) 186 (53) 282 (53) 0.92 
 European-American 74 (42) 146 (42) 220 (42)  
 Other 8 (4) 12 (3) 20 (4)  
Refused  1 (0) 1 (0)  
Missing  6 (2) 6 (1)  
Gender; n(%) 
 Male 101 (57) 194 (56) 295 (56) 0.88 
 Female 77 (43) 152 (44) 229 (44)  
Smoking status; n(%) 
 Current 127 (73) 140 (42) 267 (52) <0.0001 
 Former 39 (23) 99 (29) 138 (27)  
 Never 7 (4) 97 (29) 104 (20)  
Histology; n(%) 
 Adenocarcinoma 59 (33)    
 Squamous cell carcinoma 36 (20)    
 Non-small cell carcinoma 19 (11)    
 Small cell carcinoma 29 (16)    
 Large cell carcinoma 9 (5)    
 Otherb 13 (7)    
 Unknown 13 (7)    
Stagec; n(%) 
 Stage occult 2 (1)    
 I 16 (9)    
 II 13 (7)    
 III 41 (23)    
 IV 77 (43)    
 Unknown 29 (16)    
CharacteristicsCasesControlsTotalPa
N 178 351 529 0.83 
Age; mean ± SD 57.7 ± 8.6 57.3 ± 8.5 57.4 ± 8.6  
Race; n(%) 
 African-American 96 (54) 186 (53) 282 (53) 0.92 
 European-American 74 (42) 146 (42) 220 (42)  
 Other 8 (4) 12 (3) 20 (4)  
Refused  1 (0) 1 (0)  
Missing  6 (2) 6 (1)  
Gender; n(%) 
 Male 101 (57) 194 (56) 295 (56) 0.88 
 Female 77 (43) 152 (44) 229 (44)  
Smoking status; n(%) 
 Current 127 (73) 140 (42) 267 (52) <0.0001 
 Former 39 (23) 99 (29) 138 (27)  
 Never 7 (4) 97 (29) 104 (20)  
Histology; n(%) 
 Adenocarcinoma 59 (33)    
 Squamous cell carcinoma 36 (20)    
 Non-small cell carcinoma 19 (11)    
 Small cell carcinoma 29 (16)    
 Large cell carcinoma 9 (5)    
 Otherb 13 (7)    
 Unknown 13 (7)    
Stagec; n(%) 
 Stage occult 2 (1)    
 I 16 (9)    
 II 13 (7)    
 III 41 (23)    
 IV 77 (43)    
 Unknown 29 (16)    

aTwo-sided χ2 test.

bOther includes: adenosquamous carcinoma, carcinoid tumor malignant, carcinoma, neoplasm, and neuroendocrine carcinoma.

cOnly cases identified through cancer registry contain staging information. The original staging was based on the 6th (N = 115) and 7th (N = 50) edition of the AJCC staging manual. Staging presented in the table is based on the 7th edition, wherein all cases with sufficient tumor size, metastases status, and lymph node involvement data previously staged based on the 6th edition were restaged based on the 7th edition for consistency (N total 7th edition staging = 149).

Sample processing

SCCS participants were asked to provide urine samples starting in November of 2004. Participants were not required to fast before giving the samples. One-time urine specimens were collected from participants at Community Health Centers, refrigerated, and shipped overnight to Vanderbilt University (Nashville, TN) for processing and frozen storage at −80°C. Participants donated approximately 60 mL of urine which was mixed with a small amount of ascorbic acid as a preservative. Samples were deproteinated using 50% acetonitrile containing 10 μmol/L chloropropamide as internal standard. Supernatants were transferred into 96-well sample plates. All pipetting and dilutions were performed using a MICROLAB STARLET automated liquid handler (Hamilton Robotics), with microcentrifugation steps performed manually (14,000 × g for 15 minutes at 4°C).

Ultra performance liquid chromatography-mass spectrometry

Mass spectrometry (MS) was performed on a XEVO G2 ESI QTOF mass spectrometer operating in electrospray ionization (ESI) positive [monitoring CR, mass to charge ratio (m/z) = 264.1196 and retention time (RT) = 0.4 minutes, and 561+, m/z = 561.3435 and RT = 6.3] and negative modes (monitoring NANA, m/z = 308.0982, RT = 0.4 minutes, and cortisol sulfate, m/z = 441.162, RT = 5.5). Detailed methods have previously been reported (30), and are given in some detail in the Supplementary Methods.

Statistical analyses

A χ2test was performed on the dichotomized levels of CR and NANA across smoking, gender, and race characteristics, to test differences in levels across these strata. Metabolite abundances were dichotomized into high (>75th percentile) and low (≤75th percentile) groups based on the distribution of abundances in the controls from the current study population. Test for trend was performed to assess trends in metabolite levels across the tumor size categories (34).

Conditional logistic regression analyses were performed to calculate ORs and accompanying 95% confidence intervals (CIs) for lung cancer associated with high metabolite levels (normalized area under the peak of corresponding chromatographic peaks). In this hypothesis-testing analysis, type I error rates of 0.05 were applied in assessing the significance of each relationship. Covariate adjustments were made by adding terms for age at enrollment, body mass index (BMI), cigarette smoking status (current/former/never), amount smoked among smokers (pack-years), education and income level, prior history of COPD, and family history of lung cancer (yes/no/don't know). Interaction analysis to test whether race modifies observed associations was conducted using likelihood ratio test.

SCCS mortality follow-up data was used to conduct survival analyses for death due to lung cancer among cases in relation to categorical variables of dichotomized metabolite abundances using Cox regression modeling. Multivariate models were computed by adjusting for cigarette smoking status (current/former/never), amount smoked among smokers (pack-years), age at diagnosis, sex, race, stage (7th edition of the AJCC staging manual), and histology.

Subgroup analyses were performed by stratifying on race (European- vs. African-American subjects), and on reported preference for smoking menthol cigarettes (yes vs. no). Sensitivity analyses were performed by removing cases diagnosed within two years of cohort enrollment (n = 55), by removing cases and controls who had reported a previous cancer diagnosis (any tumor type; n = 60), and by excluding those individuals who did not answer the question regarding the family history of lung cancer (n = 91).

In exploratory analyses, we used ROC to assess the predictive value of identified metabolites in lung cancer diagnosis using the roccomp function. Models were built using conditional logistic regression on the continuous abundances of CR and NANA, as well as on the demographic, smoking-related and behavioral risk factors, and the combinations thereof. Difference between the predictive abilities of the model containing CR and NANA, and model without the metabolites was tested using rocreg function.

Mann–Whitney (two-sample Wilcoxon rank-sum) test was used to test differences in levels of urinary menthol glucuronide between those who reported and those who did not report a preference for smoking menthol cigarettes.

All reported P values are two-sided, and all P values less than or equal to 0.05 were considered statistically significant. All analyses were conducted in STATA (Stata Statistical Software Release 13.1).

Risk

CR, NANA, cortisol sulfate, and 561+, previously identified as diagnostic and prognostic biomarkers in a case–control study (30) were assessed for their relationship with lung cancer risk in prospectively collected urine specimens from the SCCS cohort. Of note, these specimens were collected before clinically detectable disease, and relevant demographic and clinical characteristics are given in Table 1. Out of four metabolites, only CR was associated with lung cancer in the overall case–control set, while NANA showed borderline significance; cortisol sulfate and 561+ were not associated with either risk or survival (Supplementary Tables S1 and S2). Therefore, focus of the current study is on metabolites CR and NANA.

Initially, potential correlates of CR and NANA were assessed in the control subjects alone, as the presence of the disease may introduce confounding. As illustrated in Table 2, high levels of CR and NANA (dichotomized on the basis of the 75th percentile of control abundances) are significantly higher in current- when compared with former- and never-smokers. Furthermore, high levels of NANA but not CR are more frequent in former- compared with never-smokers. However, correlation of CR and NANA levels with reported pack-years (measure of amount smoked) in former- and current smokers is not observed (Supplementary Fig. S1), indicating that perhaps the amount smoked does not influence the metabolite levels, consistent with the previous report (30). In addition, high levels of CR are significantly more elevated in males when compared with females, and in African-American when compared with European-American controls. These associations were not seen for NANA. Taking into account possible confounders, all subsequent analyses are adjusted for smoking status, race, and other potential confounding factors.

Table 2.

Associations of smoking, gender, and race with high and low levels of (A) creatine riboside and (B) NANA in controls only

A
Creatine ribosideHigh n (%)Low n (%)χ2P
Smoking 
 Current 53 (37.9) 87 (62.1) 21.2 <0.0001 
 Former 15 (15.2) 84 (84.9)   
 Never 16 (16.5) 81 (83.5)   
Gender     
 Male 60 (30.9) 134 (69.1) 8.7 0.003 
 Female 26 (17.1) 126 (82.9)   
Race     
 European Americans 21 (14.4) 125 (85.6) 16.4 <0.0001 
 African Americans 63 (33.9) 123 (66.1)   
B 
NANA High n (%) Low n (%) χ2 P 
Smoking 
 Current 43 (30.7) 97 (69.3) 6.2 0.05 
 Former 26 (26.3) 73 (73.7)   
 Never 16 (16.5) 81 (83.5)   
Gender 
 Male 51 (26.3) 143 (73.7) 0.71 0.40 
 Female 34 (22.4) 118 (77.6)   
Race 
 European Americans 35 (24.0) 111 (76.0) 0.15 0.70 
 African Americans 48 (25.8) 138 (74.2)   
A
Creatine ribosideHigh n (%)Low n (%)χ2P
Smoking 
 Current 53 (37.9) 87 (62.1) 21.2 <0.0001 
 Former 15 (15.2) 84 (84.9)   
 Never 16 (16.5) 81 (83.5)   
Gender     
 Male 60 (30.9) 134 (69.1) 8.7 0.003 
 Female 26 (17.1) 126 (82.9)   
Race     
 European Americans 21 (14.4) 125 (85.6) 16.4 <0.0001 
 African Americans 63 (33.9) 123 (66.1)   
B 
NANA High n (%) Low n (%) χ2 P 
Smoking 
 Current 43 (30.7) 97 (69.3) 6.2 0.05 
 Former 26 (26.3) 73 (73.7)   
 Never 16 (16.5) 81 (83.5)   
Gender 
 Male 51 (26.3) 143 (73.7) 0.71 0.40 
 Female 34 (22.4) 118 (77.6)   
Race 
 European Americans 35 (24.0) 111 (76.0) 0.15 0.70 
 African Americans 48 (25.8) 138 (74.2)   

NOTE: Levels were dichotomized into high and low based on the 75th percentile of the control abundances. Bold text designates a significant P value.

Next, conditional logistic regression analysis was conducted. High levels of CR are associated with increased lung cancer risk after adjusting for age at enrollment, BMI, smoking status, pack-years, income, education level, prior history of COPD, family history of lung cancer (ORadjusted = 2.0; 95% CI = 1.2–3.4; P = 0.01), while NANA displays borderline significance (ORadjusted = 1.6; 95% CI = 1.0–2.6; P = 0.08; Table 3). Of note, we conducted a sensitivity analysis wherein we removed those subjects who did not answer the question regarding family history of lung cancer. This analysis indicates that both, CR and NANA are significantly associated with lung cancer risk in the overall cohort (overall: ORadjusted = 1.9; 95% CI, 1.0–3.6; P= 0.4 and ORadjusted= 2.0; 95% CI, 1.1–3.6; P = 0.02, respectively; Supplementary Table S3). Stratification by race suggests that these associations are stronger in European-Americans (ORadjusted = 5.3; 95% CI = 1.6–17.6 and 3.5; 95% CI = 1.5–8.4, respectively; P≤ 0.004) when compared with African-Americans (ORadjusted = 1.1; 95% CI, 0.6–2.3 and 0.9; 95% CI = 0.4–1.8, respectively; P = ns; Table 3), although the likelihood ratio tests of significance for differing ORs by race are not significant (Pinteraction = 0.25 for CR and 0.11 for NANA). Furthermore, quartile analysis indicates that CR levels are significantly associated with lung cancer risk in the highest when compared with the lowest quartile (ORadjusted = 2.0; 95% CI, 1.1–3.8; P = 0.03; Supplementary Table S4).

Table 3.

Conditional logistic regression analysis of creatine riboside and NANA in all, European-, and African-American subjects

UnivariateMultivariate
MetaboliteaN (%) CasesN (%) ControlsOR (95% CI)PbN (%) CasesN (%) ControlsOR (95% CI)cPb
All 
 Low 
  Referentcr 109 (61) 262 (75) 1.00  101 (60) 237 (25) 1.00  
  Referentnana 109 (61) 262 (75) 1.00  101 (60) 231 (74) 1.00  
 High 
  Creatine riboside 69 (39) 87 (25) 2.0 (1.3–3.0) 0.001 66 (40) 77 (25) 2.0 (1.2–3.4) 0.01 
  NANA 69 (39) 87 (25) 2.0 (1.3–3.0) 0.001 66 (40) 83 (26) 1.6 (1.0–2.6) 0.08 
European-Americans 
 Low 
  Referentcr 51 (69) 123 (85) 1.00  49 (68) 116 (87) 1.00  
  Referentnana 40 (54) 109 (76) 1.00  38 (53) 99 (74) 1.00  
 High 
  Creatine riboside 23 (31) 21 (15) 2.7 (1.3–5.4) 0.005 23 (32) 18 (13) 5.3 (1.6–17.6) 0.006 
  NANA 34 (46) 35 (24) 2.8 (1.5–5.2) 0.001 34 (47) 35 (26) 3.5 (1.5–8.4) 0.004 
African-Americans 
 Low 
  Referentcr 52 (54) 122 (66) 1.00  48 (53) 113 (66) 1.00  
  Referentnana 64 (67) 137 (74) 1.00  59 (66) 124 (73) 1.00  
 High 
  Creatine riboside 44 (46) 63 (34) 1.6 (1.0–2.7) 0.06 42 (47) 58 (34) 1.1 (0.6–2.3) 0.73 
  NANA 32 (33) 48 (26) 1.4 (0.8–2.4) 0.20 31 (34) 47 (27) 0.9 (0.4–1.8) 0.69 
UnivariateMultivariate
MetaboliteaN (%) CasesN (%) ControlsOR (95% CI)PbN (%) CasesN (%) ControlsOR (95% CI)cPb
All 
 Low 
  Referentcr 109 (61) 262 (75) 1.00  101 (60) 237 (25) 1.00  
  Referentnana 109 (61) 262 (75) 1.00  101 (60) 231 (74) 1.00  
 High 
  Creatine riboside 69 (39) 87 (25) 2.0 (1.3–3.0) 0.001 66 (40) 77 (25) 2.0 (1.2–3.4) 0.01 
  NANA 69 (39) 87 (25) 2.0 (1.3–3.0) 0.001 66 (40) 83 (26) 1.6 (1.0–2.6) 0.08 
European-Americans 
 Low 
  Referentcr 51 (69) 123 (85) 1.00  49 (68) 116 (87) 1.00  
  Referentnana 40 (54) 109 (76) 1.00  38 (53) 99 (74) 1.00  
 High 
  Creatine riboside 23 (31) 21 (15) 2.7 (1.3–5.4) 0.005 23 (32) 18 (13) 5.3 (1.6–17.6) 0.006 
  NANA 34 (46) 35 (24) 2.8 (1.5–5.2) 0.001 34 (47) 35 (26) 3.5 (1.5–8.4) 0.004 
African-Americans 
 Low 
  Referentcr 52 (54) 122 (66) 1.00  48 (53) 113 (66) 1.00  
  Referentnana 64 (67) 137 (74) 1.00  59 (66) 124 (73) 1.00  
 High 
  Creatine riboside 44 (46) 63 (34) 1.6 (1.0–2.7) 0.06 42 (47) 58 (34) 1.1 (0.6–2.3) 0.73 
  NANA 32 (33) 48 (26) 1.4 (0.8–2.4) 0.20 31 (34) 47 (27) 0.9 (0.4–1.8) 0.69 

NOTE: Bold text designates significant results.

aLevels dichotomized into high and low based on the 75th percentile of population control abundances (low = referent).

bStatistically significant; P-value < 0.05.

cMultivariate conditional logistic regression adjusted for age, BMI, income, education level, prior history of COPD, family history of lung cancer, smoking status, and pack years. Individually matched controls selected in 2:1 ratio to cases using incidence density sampling matched by age (±2 years), sex, race, CHC recruitment site, menopausal status (women), and date of sample collection (±6 months).

CR and NANA were previously shown as elevated in the stage I tumor when compared with the adjacent nontumor tissue, linking them directly to altered tumor metabolism (30). Hence it is plausible that these endogenous metabolites are involved with early processes contributing to neoplastic transformation, such as inflammation. To test this hypothesis, we evaluated whether the levels of the markers are predictive of lung cancer risk two or more years before diagnosis by excluding the cases diagnosed within two years of the cohort enrollment (Table 4). While the ORs remain in the same direction for both CR and NANA, the associations are attenuated and no longer significant in the overall cohort after the adjustment for a number of putative confounders, possibly due to insufficiently powered analysis. However, the associations with high CR and NANA remain significant in European-Americans when individuals diagnosed within two years of cohort enrollment are removed (ORadjusted = 6.7; 95% CI, 1.6–27.6 and 3.8; 95% CI, 1.3–11.5, respectively; P ≤ 0.02; Table 4). Next, aware of a possibility that CR and NANA may be associated with other cancer types, sensitivity analysis was performed wherein the cases (N = 28) and controls (N = 32) who had reported a previous cancer diagnosis at the time of the enrollment were removed. The ORs associated with high CR remain significantly elevated (ORadjusted= 2.0; 95% CI, 1.1–3.6; P = 0.03), while NANA is only significant in the unadjusted model, but the effect size remains similar after the adjustment (ORadjusted= 1.4; 95% CI, 0.8–2.5; P = 0.28). Both metabolites are significantly associated with lung cancer risk in European-Americans (ORadjusted = 19.3; 95% CI, 2.6–142.7) and 5.4 (95% CI, 1.5–20.1), respectively; P ≤ 0.01; Supplementary Table S5).

Table 4.

Conditional logistic regression analysis of creatine riboside and NANA in all, European-, and African-American subjects after removal of cases diagnosed within two years (N = 55) of cohort enrollment

UnivariateMultivariate
MetaboliteaN (%) CasesN (%) ControlsOR (95% CI)PbN (%) CasesN (%) ControlsOR (95% CI)cPb
All 
 Low 
  Referentcr 79 (64) 178 (74) 1.00  73 (64) 163 (74) 1.00  
  Referentnana 76 (62) 183 (76) 1.00  70 (61) 163 (74) 1.00  
 High 
  Creatine riboside 44 (36) 62 (26) 1.7 (1.0–2.7) 0.04 42 (36) 56 (26) 1.6 (0.9–3.1) 0.14 
  NANA 22 (45) 23 (24) 2.2 (1.3–3.6) 0.003 45 (39) 56 (26) 1.7 (0.9–3.1) 0.10 
European-Americans 
 Low 
  Referentcr 33 (67) 82 (85) 1.00  31 (66) 77 (87) 1.00  
  Referentnana 27 (55) 73 (76) 1.00  25 (53) 66 (74) 1.00  
 High 
  Creatine riboside 16 (33) 14 (15) 3.0 (1.3–7.3) 0.01 16 (34) 12 (13) 6.7 (1.6–27.6) 0.008 
  NANA 22 (45) 23 (24) 3.1 (1.4–7.0) 0.007 22 (47) 23 (26) 3.8 (1.3–11.5) 0.02 
African-Americans 
 Low 
  Referentcr 44 (62) 90 (65) 1.00  40 (61) 83 (65) 1.00  
  Referentnana 47 (66) 104 (75) 1.00  43 (65) 94 (74) 1.00  
 High 
  Creatine riboside 27 (38) 48 (35) 1.2 (0.6–2.1) 0.64 26 (39) 44 (35) 0.6 (0.3–1.5) 0.31 
  NANA 24 (34) 34 (25) 1.6 (0.8–3.1) 0.15 23 (35) 33 (26) 1.0 (0.4–2.5) 0.98 
UnivariateMultivariate
MetaboliteaN (%) CasesN (%) ControlsOR (95% CI)PbN (%) CasesN (%) ControlsOR (95% CI)cPb
All 
 Low 
  Referentcr 79 (64) 178 (74) 1.00  73 (64) 163 (74) 1.00  
  Referentnana 76 (62) 183 (76) 1.00  70 (61) 163 (74) 1.00  
 High 
  Creatine riboside 44 (36) 62 (26) 1.7 (1.0–2.7) 0.04 42 (36) 56 (26) 1.6 (0.9–3.1) 0.14 
  NANA 22 (45) 23 (24) 2.2 (1.3–3.6) 0.003 45 (39) 56 (26) 1.7 (0.9–3.1) 0.10 
European-Americans 
 Low 
  Referentcr 33 (67) 82 (85) 1.00  31 (66) 77 (87) 1.00  
  Referentnana 27 (55) 73 (76) 1.00  25 (53) 66 (74) 1.00  
 High 
  Creatine riboside 16 (33) 14 (15) 3.0 (1.3–7.3) 0.01 16 (34) 12 (13) 6.7 (1.6–27.6) 0.008 
  NANA 22 (45) 23 (24) 3.1 (1.4–7.0) 0.007 22 (47) 23 (26) 3.8 (1.3–11.5) 0.02 
African-Americans 
 Low 
  Referentcr 44 (62) 90 (65) 1.00  40 (61) 83 (65) 1.00  
  Referentnana 47 (66) 104 (75) 1.00  43 (65) 94 (74) 1.00  
 High 
  Creatine riboside 27 (38) 48 (35) 1.2 (0.6–2.1) 0.64 26 (39) 44 (35) 0.6 (0.3–1.5) 0.31 
  NANA 24 (34) 34 (25) 1.6 (0.8–3.1) 0.15 23 (35) 33 (26) 1.0 (0.4–2.5) 0.98 

NOTE: Bold text designates significant results.

aLevels dichotomized into high and low based on the 75th percentile of population control abundances (low = referent).

bStatistically significant; P-value < 0.05.

cMultivariate conditional logistic regression adjusted for age, BMI, income, education level, prior history of COPD, family history of lung cancer, smoking status, and pack years. Individually matched controls selected in 2:1 ratio to cases using incidence density sampling matched by age (± 2 years), sex, race, CHC recruitment site, menopausal status (women), and date of sample collection (±6 months).

Finally, to evaluate the ability of CR and NANA alone and in combination to classify lung cancer in this nested case–control set, conditional logistic regression models and ROC analysis were performed. A model containing variables previously found to be most robust in selecting high-risk individuals for downstream screening comprising smoking status, pack years, age, BMI, income and education levels, previous history of COPD, and family history of lung cancer (28), was compared with a model also comprising CR and NANA, resulting in a significant improvement indicated by the increased AUC (0.78 compared with 0.80 after the addition of the metabolites, P = 0.01; Fig. 1A). Of note, a selected cut-off point with the most robust ability to correctly classify subjects, at 50% sensitivity and 86% specificity, leads to a positive predictive value (PPV) of 63%, a negative predictive value (NPV) of 79%, and correct classification of 75% subjects. We assessed the ability of the models to classify lung cancer cases in European-Americans also, due to observed stronger associations in this racial group. We detected a significant improvement in the AUC after the addition of CR and NANA to the model containing risk factors alone (from 0.84 to 0.90, respectively; P = 0.002), with a selected cut-off point leading to a correct classification of 84% of subjects (PPV = 75%; NPV = 89%; Fig. 1B).

Figure 1.

ROC analysis of selected demographic, smoking, and behavioral characteristics associated with lung cancer risk, CR, NANA, and combinations thereof in all subjects (A), and European-Americans (EA) (B). CR, creatine riboside; NANA, N-acetylneuraminic acid; PPV, positive predictive value; NPV, negative predictive value. Risk factors model comprises: age (age at enrollment), education (achieved level of education), income (household income), smoking [smoking status (current-, former-, never-smoker)], pack-years (amount smoked indicated by pack-years), BMI (body mass index), COPD (history of chronic obstructive pulmonary disease), and family history of lung cancer.

Figure 1.

ROC analysis of selected demographic, smoking, and behavioral characteristics associated with lung cancer risk, CR, NANA, and combinations thereof in all subjects (A), and European-Americans (EA) (B). CR, creatine riboside; NANA, N-acetylneuraminic acid; PPV, positive predictive value; NPV, negative predictive value. Risk factors model comprises: age (age at enrollment), education (achieved level of education), income (household income), smoking [smoking status (current-, former-, never-smoker)], pack-years (amount smoked indicated by pack-years), BMI (body mass index), COPD (history of chronic obstructive pulmonary disease), and family history of lung cancer.

Close modal

We have previously shown that a preference for smoking menthol cigarettes was no more harmful than smoking non-menthol cigarettes, as assessed in a larger nested case–control set in the SCCS (35). Considering that urinary menthol glucuronide may be a biomarker for smoking menthol cigarettes, the association between the levels of menthol glucuronide and lung cancer risk was investigated. Menthol glucuronide was dichotomized into high and low categories based on the 75th percentile of the control abundances. No significant association between urinary menthol glucuronide and lung cancer risk were observed in either all subjects, or across the racial strata, after adjustment for amount smoked (Supplementary Table S6). In concordance with our previous report (35), we also did not observe a significant association between a preference for smoking menthol cigarettes and lung cancer risk in this smaller nested case–control set (Supplementary Table S7). Of note, a strong association between the reported preference for smoking menthol cigarettes and urinary menthol glucuronide levels was observed [fold change (FC) = 1.9; P < 0.0001 in current- and former-smokers (Supplementary Fig. S2A), and FC = 2.5; P < 0.0001 in current-smokers only (Supplementary Fig. S2B)], with similar associations observed in European- and African-Americans.

Survival

On the basis of the previous data from the NCI-MD case–control study that showed high levels of CR and NANA to be associated with worse survival (30), the same analysis was performed in this study. Cox regression analysis adjusted for age at diagnosis, smoking status, pack-years, stage (the 7th edition of the AJCC staging manual) and histology showed no significant associations (Supplementary Table S8; Supplementary Fig. S3). This finding suggests that CR and NANA levels prior to clinically detectable disease are not associated with survival.

No significant associations between either urinary menthol glucuronide (Supplementary Table S9), nor reported preference for smoking menthol cigarettes (Supplementary Table S10) and lung cancer survival were found in this study.

Levels across tumor sizes

Considering previous evidence that CR and NANA are tumor-specific metabolites linked to altered tumor metabolism (30), it is plausible that their levels may increase with increasing tumor size. For a subset of cases with exact tumor size available (N = 108), the prevalence of high CR rose with increasing tumor size (Ptrend = 0.03, Fig. 2A). While the results are not significant for NANA, a similar trend was observed across the tumor size strata (Fig. 2B).

Figure 2.

Mean levels of creatine riboside (A) and NANA (B) in SCCS lung cancer cases across tumor size strata.

Figure 2.

Mean levels of creatine riboside (A) and NANA (B) in SCCS lung cancer cases across tumor size strata.

Close modal

Currently, there are no biomarkers that can successfully complement available screening modalities for detecting early-stage lung cancer. LDCT has been proven to reduce lung cancer mortality by 20%, as compared with chest X-ray (3). While no lesion goes undetected by LDCT, most are nonmalignant, leading to an astonishingly high false discovery rate of 96%. A panel comprising four metabolites detected in the urine was found to be most significantly associated with both lung cancer status and survival, and reproducible and stable in stored urine over a long period of time (30). The goal of the current study was to investigate whether the four metabolites, creatine riboside (CR), N-acetylneuraminic acid (NANA), cortisol sulfate, and indeterminate compound designated as 561+, were predictive of lung cancer risk in prediagnostic urine specimens, one of the crucial steps for validating promising biomarkers that may have clinical utility (29). Of these four, only CR was predictive of lung cancer risk before clinically detectable disease, with NANA displaying borderline significance. In our earlier work, we did not detect elevated levels of cortisol sulfate and 561+ in tumor tissue, which also tended to lower the importance of the associations in urine. CR and NANA, however, were significantly elevated in tumor when compared with matched, adjacent nontumor lung tissue, strengthening their importance as markers of lung cancer risk. NANA has previously been characterized and is the most common form of sialic acid that plays several roles in biology, and is thought to protect cancer cells from immunosurveilance (36). Creatine riboside is a novel metabolite previously reported for the first time by our group (30). While the characterization of this metabolite is of special importance for future studies, we speculate that CR may be a product of both, high creatine within the tumor, and high phosphate flux, as a result of a pronounced energy requirement of fast dividing tumor when compared with normal cells (37). Future studies should elucidate what role, if any, CR may play in neoplastic transformation.

While we were unable to conduct an adjusted conditional logistic analysis between CR and NANA and early stage I-II lung cancer due to a small number of patients in this group, univariate analysis suggest that these metabolites may be robust in predicting early stage lung cancer (data not shown), consistent with our previous report. The utility of these metabolites in aiding early lung cancer diagnosis remains to be established by investigating their usefulness in distinguishing benign from malignant nodules detected by LDCT.

Although the levels of the two metabolites were higher among current than nonsmokers (and levels of NANA but not CR were higher among former than never smokers), the associations between CR and lung cancer risk held after adjusting for cigarette smoking status and amount smoked. In both this and in the previous report, we did not observe any correlations between the metabolites and the amount smoked (cigarettes per day and pack years, respectively). Furthermore, a previous study showed that amino acid and lipid pathways are most significantly affected by the exposure to tobacco-smoke and reversible after smoking cessation; CR and NANA, however, are not members of these pathways (38). Thus, the levels of these metabolites may not be affected by immediate exposure to tobacco smoke, and they are not likely markers of exposure. While NANA displayed borderline significance after adjustment in the overall cohort, the levels of both metabolites were significantly associated with lung cancer risk in European-Americans. Although race did not seem to significantly modify the effect of the associations between metabolites and lung cancer risk, the associations were stronger in European- than African-Americans. The lack of observed formal interaction may be a result of decreased power, or factors associated with race (such as lifestyle), which could not be accounted for in this study. In our previous case–control study, racial differences did not attenuate associations with lung cancer status, as disease presence may override any other contributing factors, such as genetic backgrounds. Future investigation may elucidate whether or to what extent racial differences in the association with lung cancer risk may exist. It is possible that genetic variants may contribute to racial differences, as they play a role in the metabolism of both exogenous (25) and endogenous metabolites (39, 40). In addition, lifestyle and external exposures may exacerbate the observed differences, factors that could not be controlled in this study.

To address other causes that may contribute to health disparities in lung cancer, a preference for smoking menthol cigarettes, which is much higher among African- than European-Americans (41–43) was investigated. We have previously reported that smoking menthol cigarettes, preferred by African-Americans, does not lead to an increased risk for developing lung cancer in the SCCS (35). In the current study, we took an additional step and measured urinary menthol glucuronide as a biomarker of exposure to menthol from tobacco smoke. Consistent with the previous report, no significant associations were observed in relationship with lung cancer risk. We are the first group to our knowledge to report this observation.

Metabolic markers have gained increased interest recently, as they are recognized as proximal markers to the disease phenotype in comparison to other “omics” markers (44–46). The footprint that products of altered tumor metabolism may leave in noninvasively collected biospecimens serves as a great opportunity for biomarker discovery. This “liquid biopsy” practice would be a high-throughput, noninvasive and inexpensive approach to allow not only cancer detection, but also patient monitoring during treatment, and would provide an ideal therapeutic strategy for precision medicine (47). There is a great necessity to identify and validate a complementary tool to the solid biopsy, which may also allow for delivery of targeted therapy. Using very precise mass spectrometry for measuring lung cancer risk markers, such as CR and NANA, presents a feasible opportunity to develop reproducible, inexpensive and high-throughput diagnostic tools to refine algorithms for early detection of lung cancer.

Caveats of the study include a relatively limited sample size, as well as the inability to control for the exogenous effects on metabolism. We adjusted for the effects of smoking, although residual confounding cannot be ruled out, but other unmeasured factors may have contributed to the differences in CR and NANA levels between those with and without lung cancer. A major strength of the study is a well-matched nested case–control sample set with its close matching on demographic variables and in methods in and timing of collection of urine samples, as well as similar socioeconomic backgrounds of the subjects enrolled in the cohort.

Overall, the results of this study suggest that CR and NANA are associated with lung cancer risk in prospective samples, more strongly in European-Americans, and therefore may have clinical utility for disease screening or diagnosis. Considering an expected forthcoming rise in the detection of indeterminate pulmonary nodules as a result of LDCT screening, it is crucial that noninvasive biomarkers be developed, with ability to distinguish malignant from indolent nodules. Future biomarker studies in LDCT screening trials are needed to determine the clinical utility of these and other biomarkers of NSCLC.

No potential conflicts of interest were disclosed.

Data on SCCS cancer cases from Mississippi were collected by the Mississippi Cancer Registry which participates in the National Program of Cancer Registries (NPCR) of the Centers for Disease Control and Prevention (CDC). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the CDC or the Mississippi Cancer Registry.

Conception and design: M. Haznadar, K.W. Krausz, C.C. Harris

Development of methodology: M. Haznadar, K.W. Krausz, C.C. Harris

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Haznadar, Q. Cai, K.W. Krausz, E.D. Bowman, E. Margono, R. Noro, M.D. Steinwandel, F.J. Gonzalez, W.J. Blot

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Haznadar, Q. Cai, M.D. Thompson, E.A. Mathé, C.C. Harris

Writing, review, and/or revision of the manuscript: M. Haznadar, Q. Cai, E.D. Bowman, E. Margono, M.D. Thompson, E.A. Mathé, H.M. Munro, F.J. Gonzalez, W.J. Blot, C.C. Harris

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Haznadar, E.D. Bowman, R. Noro

Study supervision: M. Haznadar, K.W. Krausz, C.C. Harris

The authors thank Stefan Ambs, Xin Wang, Perwez Hussain, Anu Budhu, and Brid Ryan for valuable feedback, discussion, and constructive comments; Karen Yarrick and Lisa Spillare for administrative and technical help; Jennifer Sonderman, project coordinator, at the SCCS; and Regina Courtney and Jie Wu for urine sample preparation. Data on SCCS cancer cases used in this publication were provided by the Alabama Statewide Cancer Registry; Kentucky Cancer Registry, Lexington, KY; Tennessee Department of Health, Office of Cancer Surveillance; Florida Cancer Data System; North Carolina Central Cancer Registry, North Carolina Division of Public Health; Georgia Comprehensive Cancer Registry; Louisiana Tumor Registry; Mississippi Cancer Registry; South Carolina Central Cancer Registry; Virginia Department of Health, Virginia Cancer Registry; Arkansas Department of Health, Cancer Registry, Little Rock, AR. The Arkansas Central Cancer Registry is fully funded by a grant from National Program of Cancer Registries, CDC.

The work was supported by funding from the Center for Cancer Research Intramural Research Program, National Cancer Institute, NIH, and from NIH grant R01 CA092447 (to W.J. Blot). SCCS sample preparation was conducted at the Survey and Biospecimen Shared Resource that is supported in part by the Vanderbilt-Ingram Cancer Center (P30 CA068485 to Q. Cai).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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