Majda Haznadar

Noninvasive Urinary Metabolomic Profiling Identifies Diagnostic and Prognostic Markers in Lung Cancer

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

Biomarkers of Coordinate Metabolic Reprogramming in Colorectal Tumors in Mice and Humans

BACKGROUND & AIMS There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis. METHODS Mass spectrometry-based metabolomic analysis of urine and tissues from wild-type C57BL/6J and Apc(Min/+) mice, as well as from mice with azoxymethane-induced tumors, was employed in tandem with gene expression analysis. Metabolic profiling was also performed on colon tumor and adjacent nontumor tissues from 39 patients. The effects of β-catenin activity on metabolic profiles were assessed in mice with colon-specific disruption of Apc. RESULTS Thirteen markers were found in urine associated with development of colorectal tumors in Apc(Min/+) mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice revealed that derangement of metabolites were a reflection of coordinate metabolic reprogramming in tumor tissue. Similar changes in urinary metabolites were observed in mice with azoxymethane-induced tumors and in mice with colon-specific activation of β-catenin. The metabolic alterations indicated by markers in urine, therefore, appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 17 metabolites associated with the same metabolic pathways identified in mice. Ten metabolites that were increased in tumor tissues, compared with nontumor tissues (proline, threonine, glutamic acid, arginine, N1-acetylspermidine, xanthine, uracil, betaine, symmetric dimethylarginine, and asymmetric-dimethylarginine), were also increased in urine from tumor-bearing mice. CONCLUSIONS Gene expression and metabolomic profiles of urine and tissue samples from mice with colorectal tumors and of colorectal tumor samples from patients revealed pathways associated with derangement of specific metabolic pathways that are indicative of early-stage tumor development. These urine and tissue markers might be used in early detection of colorectal cancer.

Urinary Metabolite Risk Biomarkers of Lung Cancer: A Prospective Cohort Study

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.

Navigating the road ahead: addressing challenges for use of metabolomics in epidemiology studies

Metabolomics platforms allow for the measurement of hundreds to thousands of unique small chemical entities, as well as offer extensive coverage of metabolic markers related to obesity, diet, smoking, and other exposures of high interest to health scientists. Nevertheless, its potential use as a tool in population-based study design has not been fully explored. As the field of metabolomics continues to mature, and in part, accelerate through the National Institutes of Health (NIH) investment of ≤65 million in the Common Funds Metabolomics Program (https://common fund.nih.gov/metabolomics/index), it is time to consider those challenges most pertinent to epidemiologic studies.

Abstract PR02: Diagnostic and prognostic utility of urinary creatine riboside for early stage non-small cell lung cancer

Lung cancer is the leading cause of cancer-related death worldwide. With the advent of low-dose computed tomography screening, it is expected that the number of lung cancers diagnosed at an early stage will rise sharply. The recommended treatment for stage I non-small cell lung cancer (NSCLC) patients is tumor resection, which may be followed by chemotherapy in patients with pathologically high-risk, margin-negative stage IB tumors. Still, up to 30% surgically treated stage I patients experience recurrence leading to death. Therefore, biomarkers that molecularly categorize stage I patients after tumor resection and stratify high-risk patients who may benefit from adjuvant chemotherapy would lead to improved clinical management. We previously conducted metabolomic profiling of urines collected from 469 NSCLC patients and 536 population controls using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS) and found that creatine riboside, a novel metabolite identified by the study, is significantly elevated in stage I and II NSCLC patients compared to controls. We also found that creatine riboside levels are 19-fold higher in tumor tissues compared to adjacent normal lung tissues (P 2 =0.87 by Spearman’s rank order test). These data indicate that creatine riboside may be a product of deregulated tumor metabolism that is detectable in urine, making urinary creatine riboside a potentially useful liquid biopsy biomarker for surveillance after surgery in early stage NCSLC patients. To further evaluate the utility of creatine riboside as a liquid biopsy biomarker, urines from 34 stage I and II NSCLC patients from Lung Cancer Biospecimen Resource Network (LCBRN), collected at the time of diagnosis and 6, 12, 18 and 24 months after surgery, were evaluated in the current study. The urinary levels of creatine riboside were quantitated using UPLC-MS/MS and were analyzed for association with cancer-specific survival and disease-free survival. As a result, in non-recurrent cases (n=23), creatine riboside levels showed a significant decreasing trend over 24 months after surgery (P=0.03). These data further support previous evidence that creatine riboside is a product of deregulated tumor metabolism that can be detected in urine. In addition, Kaplan Meier survival estimates demonstrated that high creatine riboside levels at the time of diagnosis correlated significantly with worse cancer-specific survival and disease-free survival (P=0.005 and P=0.003, respectively). In summary, urinary creatine riboside may have potential as a liquid biopsy biomarker for early stage NSCLC that aids surveillance after surgery as well as to stratify patients with worse prognosis. This abstract is also being presented as Poster A18. Citation Format: Takahiro Oike, Yasuyuki Kanke, Amelia Parker, Majda Haznadar, Kristopher W. Krausz, Elise D. Bowman, Ana I. Robles, Frank J. Gonzalez, Curtis C. Harris. Diagnostic and prognostic utility of urinary creatine riboside for early stage non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR02.

Inverse association of vitamin D3 levels with lung cancer mediated by genetic variation

Vitamin D is an essential micronutrient required for normal physiological function and recognized for its role regulating calcium metabolism. Recent work is beginning to emerge demonstrating a role for vitamin D in chronic illnesses, such as cancer. Circulating serum levels of 25(OH)D2/3 were quantitatively measured using sensitive ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC‐MS/MS) in 406 lung cancer cases and 437 population controls, while 1,25(OH)2D2/3 levels were measured in a subset of 90 cases and 104 controls using the same method, from the NCI‐MD case–control cohort. 25(OH)D3 levels were inversely associated with lung cancer status across quartiles (Q2 vs. Q1: ORadjusted = 0.5, 95% CI = 0.3–0.8; Q3 vs. Q1: ORadjusted = 0.5, 95% CI = 0.3–0.8; Q4 vs. Q1: ORadjusted = 0.5, 95% CI = 0.2–0.9; Ptrend = 0.004). Levels of 1,25(OH)2D3 were also inversely associated with lung cancer status (Q2 vs. Q1: ORadjusted = 0.2, 95% CI = 0.03–0.7; Q3 vs. Q1: ORadjusted = 0.1, 95% CI = 0.01–0.4; Q4 vs. Q1: ORadjusted = 0.04, 95% CI = 0.01–0.3; Ptrend<0.0001). Although the observed trends were similar for the 25(OH)D2 (Ptrend = 0.08), no significant associations were seen between vitamin D2 and lung cancer status. Additionally, genotyping of 296 SNPs in the same subjects resulted in findings that 27 SNPs, predominantly in CYP24A1 and VDR genes, were significantly associated with lung cancer status, affected mRNA expression, and modulated vitamin D levels. These findings suggest a protective role for vitamin D3 in lung cancer, with similar trends but insignificant findings for D2. Vitamin D3 levels appeared to be modulated by genetic variation in CYP24A1 and VDR genes. Additional research to illuminate the mechanism(s) through which vitamin D exacerbates effects against lung carcinogenesis is warranted.

Abstract 4320: Circulating vitamin D2 and D3 levels and single nucleotide polymorphism associations with lung cancer status: A case-control study

Vitamin D is an essential micronutrient, required for normal physiological function and classically recognized for its role regulating calcium metabolism. Recent work is beginning to demonstrate a role of vitamin D in chronic illnesses, such as cancer, but questions addressing how depletion of this nutrient affects cancer risk remain largely unanswered. In 2011, the Institute of Medicine report highlighted the need for more research to explore the role of vitamin D in non-skeletal health outcomes. Previously, vitamin D has largely been measured using radio- and chemiluminescence immunoassays, unable to distinguish between D2 (from plant dietary sources and supplements) and D3 forms. We measured circulating serum levels of both, inactive (25(OH)D), and active (1,25(OH)2D) forms of D2 and D3 using sensitive liquid chromatography coupled to mass spectrometry in 406 lung cancer cases and 437 controls. We observed that levels of inactive D3 are associated with decreased lung cancer risk across quartiles, after adjustment for age, gender, race, smoking status, pack years, interview year and blood collection month (ORadjusted = 0.5 (95% CI = 0.2, 0.9), P Citation Format: Majda Haznadar, Kristopher W. Krausz, Ezra Margono, Elise D. Bowman, Brid M. Ryan, Frank J. Gonzalez, Curtis C. Harris. Circulating vitamin D2 and D3 levels and single nucleotide polymorphism associations with lung cancer status: A case-control study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4320.

Abstract 3447: Evaluation of NIH funding for metabolomics research in epidemiologic studies: opportunities and challenges

Metabolomics, an emerging area of interest in the field of epidemiology, has significant potential to evaluate the effects of nutritional, environmental, and pharmaceutical exposures; conduct risk assessments; predict disease development; and diagnose diseases. To understand current funding and potential gaps for population-based metabolomics research, we cross-sectionally evaluated the National Institutes of Health (NIH)-supported research grant awards related to metabolomics and epidemiology that were active on February 9, 2015. The grant portfolio was analyzed using NIH9s Query View Report (QVR) software, using relevant search terms, which included metabolome, metabonome, metabolomic(s), metabonomic(s), metabolic profile, metabolite profile, metabolic signature, glycomic(s), and lipidomic(s). Basic descriptive statistics on technology platform, approach, disease phenotype, biospecimen type, exposure, study design, and population were examined. The search terms initially identified 473 grants. After exclusion of grants with Citation Format: Krista A. Zanetti, Mukesh Verma, Scott Rogers, Majda Haznadar, Padma Maruvada, Holly L. Nicastro, Mary C. Playdon, Sharon A. Ross. Evaluation of NIH funding for metabolomics research in epidemiologic studies: opportunities and challenges. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3447.

Abstract 935: Lung cancer metabolomics identifies metabolites as robust risk biomarkers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Lung cancer is a major health burden on the general population, as it will kill 160,000 people annually in the US, making it the number one cause of cancer related deaths. The prognosis for lung cancer is still very poor, with fewer than 15% of patients surviving 5 years after primary diagnosis, indicating the enormous necessity to search for biomarkers for early lung cancer detection when amenable to treatment. Currently accepted methods for early detection of lung cancer are limited to low-dose spiral CT (LDCT) scanning in high-risk populations. However, the specificity of LDCT is low, with a false positive rate of 96%. Our goal is to identify stable and reproducible biomarkers in non-invasively collected biofluids, namely urine, that may be powerful risk assessment screening tools. To that extent, we first conducted a first of its kind effort using mass spectrometry-based untargeted metabolic profiling of urine samples from 469 lung cancer patients and 536 healthy population controls in the NCI-MD case-control study, We found four metabolites to be independent and robust classifiers of lung cancer diagnosis and prognosis: novel and previously un-described creatine riboside, N-acetylneuraminic acid (NANA), cortisol sulfate and a metabolite with a mass to charge ratio of 561 detected in electrospray ionization (ESI) positive mode. Furthermore, creatine riboside and NANA were also found to be elevated in 48 stage I lung tumors matched to adjacent non-tumor tissues, linking them directly to deregulated tumor metabolism. The next step was to assess whether these metabolites are elevated in subjects prior to lung cancer diagnosis. To that end, we analyzed the urine of 180 patients and 349 non-diseased controls from the prospective Southern Community Cohort Study (SCCS). Controls were individually matched to cases in a 2:1 ratio using incidence density sampling by age (+/- 2 years), sex, race, recruitment site, menopausal status (women), and date of sample collection (+/- 6 months). We confirmed that two of the four metabolites, creatine riboside and NANA were associated with lung cancer risk with smoking-adjusted Odds Ratios (OR) of 1.8 and 1.5, respectively (P <0.02). These metabolites, having been observed as deregulated in tumor metabolism, may become interesting targets for future studies regarding therapeutic interventions. The associations with risk were stronger among European than African Americans, and among whites were observed in individuals having been diagnosed within two years of cohort enrollment (OR = 2.7 and 2.5, respectively, for creatine riboside and NANA; P = 0.004) and two or more years after cohort enrollment (OR = 3.6 and 3.3, respectively; P = 0.02). These results provide strong preliminary data that identified metabolite biomarkers have a potential utility as clinical screening tools for early diagnosis of lung cancer, when prognosis is significantly better. Citation Format: Majda Haznadar, Qiuyin Cai, Kristopher W. Krausz, Elise D. Bowman, William J. Blot, Frank J. Gonzalez, Curtis C. Harris. Lung cancer metabolomics identifies metabolites as robust risk biomarkers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 935. doi:10.1158/1538-7445.AM2015-935

Abstract 1901: Untargeted metabolomic profiling identifies diagnostic and prognostic biomarkers of lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Lung cancer remains the most common cause of cancer deaths world-wide. Despite the intensive research over many years, the prognosis of this deadly disease is still very poor, with fewer than 15% of the patients surviving 5 years after primary diagnosis. While there are several methodologies described and proposed for early detection of lung cancer (spiral CT, circulating pro-inflammatory cytokines IL6, IL8 and CRP), the specificity and robustness remains to be achieved. What we readily know is that cancer cells have a distinguishable metabolic fingerprint compared to normal cells. Metabolomics holds promise to be able to detect and capture subtle shifts in multiple metabolic paths and cellular modifiers that will enable identification of critical components of cancer risk and tumor behavior. We conducted a first of its kind effort using mass spectrometry-based untargeted metabolic profiling of urine samples obtained from 469 lung cancer patients and 536 healthy population controls. We identified four robust biomarkers, high levels of which are associated with lung cancer diagnosis and poorer survival. After the adjustment for potential confounding factors, all four biomarkers were significantly associated with lung cancer diagnosis (FDR-adjusted p-values <0.05, ORs ranging from 1.9 to 5.1), whereas one of four was associated with diagnosis in early I and II stages (OR =3.3, p-value =0.002). Furthermore, all four biomarkers are associated with prognosis (HRs ranging from 1.49 to 1.97, after adjustment for potential confounders, p-values <0.02), whereas two were associated with survival in stages I and II (HRs of 1.83 and 9.33, p-values 0.03 and 0.0006 respectively). A combination of the four biomarkers resulted in stronger associations, suggesting that they may be independent of one another. Significantly higher levels of these biomarkers were confirmed in an independent sample set from the same cohort, confirming our findings and eliminating storage time as a potential confounder. A targeted quantitation was carried out in a representative subset of 198 samples, further validating previous findings from the untargeted screen. Furthermore, intraclass correlation analysis revealed high repeatability of two independent measurements over a year apart (ICCs between 0.82 and 0.99). Lastly, the metabolome of 62 tumor and 62 adjacent normal tissues was profiled (stage I adeno- and squamous cell- carcinomas), linking two urinary biomarkers directly to the tumor metabolism (FCs of 1.7 and 19.0; p-values 0.03 and <0.00001, respectively). In addition to their potential to further identify those high risk groups who would most benefit from an invasive screen, thereby minimizing the false positive rate, these markers may also illuminate novel lung carcinogenesis pathways, as well as potential therapeutic targets. Mechanistic studies elucidating effected pathways are ongoing. Citation Format: Majda Haznadar, Ewy Mathe, Andrew D. Patterson, Soumen K. Manna, Kristopher W. Krausz, Elise D. Bowman, Jeffrey R. Idle, Dickran G. Kazandjian, Frank J. Gonzalez, Curtis C. Harris. Untargeted metabolomic profiling identifies diagnostic and prognostic biomarkers of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1901. doi:10.1158/1538-7445.AM2013-1901