Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection

Colorectal cancer (CRC) mortality remains one of the highest among all cancers, second to only lung cancer (Canadian Cancer Statistics, 2008). Despite the known benefits of early detection, screening programmes based on colonoscopy and fecal occult blood testing have been plagued with challenges such as public acceptance, cost, limited resources, accuracy and standardization. There is consensus in the field that the use of colonoscopy alone for CRC screening is not practical [1], and that a minimally-invasive serum-based test capable of accurately identifying subjects who are high risk for the development of CRC would result in a higher screening compliance than current approaches and better utilization of existing endoscopy resources [1–3]. Although there have been multiple reports of altered transcript levels [4–11], aberrantly methylated gene products [12–14] and proteomic patterns [15–18] associated with biological samples from CRC patients, few if any have advanced into clinically useful tests. This may be due to a number of reasons including technical hurdles in assay design, challenges obtaining reproducible results, costs and lengthy regulatory processes. Furthermore, most of the tests currently used or in development are based upon the detection of tumour-specific markers and have poor sensitivity for identifying subjects who are either very early stage, or are predisposed to risk but show no clinical presentation of disease.

Although causal genetic alterations for CRC have been well characterized, the number of cases due to adenomatous (APC) and hereditary nonpolyposs colorectal cancer are less than 5% of the total, with approximately 15% claimed to be attributable to inheritable family risk likely due to complex patterns of low penetrance mutations which have yet to be delineated [19]. The fact remains that approximately 80% of CRC cases are thought to arise sporadically, with diet and lifestyle as key risk factors [20, 21]. In addition, an individual's microbiome is intricately linked to their gastrointestinal physiological status and may itself be involved as a risk factor [22]. Given that metabolism is heavily influenced by both diet and lifestyle and that the microbiome contributes its own metabolic processes, it is surprising that there has been little effort aimed identifying metabolic markers as risk indicators of CRC. This may, in part, have been due to the lack of platform technologies and informatics approaches capable of comprehensively characterizing metabolites in a similar way that DNA microarrays or surface-enhanced laser desorption ionization can characterize transcripts or proteins, respectively.

Recently, however, there have been rapid advances made in mass spectrometric-based systems which can identify large numbers of metabolic components within samples in a parallel manner [23–25]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is based upon the principle that charged particles exhibit cyclotron motion in a magnetic field, where the spin frequency is proportional the mass [26]. FTICR-MS is known for its high resolving power and capability of detecting ions with mass accuracy below 1 part per million (ppm). Liquid sample extracts can be directly infused using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) without chromatographic separation [23], where ions with differing mass to charge (m/z) ratios can be simultaneously resolved using a Fourier transformation. Using informatics approaches, spectral files from multiple samples can be accurately aligned and peak intensities across the samples compared [23]. High resolution also enables the prediction of elemental composition of all ions detected in a sample, providing a solid foundation for metabolite classification and identification, as well as the ability to construct de novo metabolic networks [23, 27]. The combination of liquid extraction, flow injection, high resolution and informatics affords a unique opportunity to broadly characterize the biochemical composition of samples, with no a priori knowledge about the sample itself, to a degree which was not previously possible. This 'non-targeted' approach has the advantage of detecting novel compounds and is therefore ideally suited for biomarker-driven discovery applications. Using a MS-based discovery platform for metabolic biomarker identification also has the added advantage of straightforward translation into a quantitative method based upon triple-quadruple multiple-reaction-monitoring (TQ-MRM), similar to the clinical methods used to screen for inborn errors of metabolism [28].

Here we report on the application of this technology for characterizing the serum metabolomes of treatment-naive CRC patients and healthy asymptomatic subjects. A specific metabolic perturbation was discovered in the serum of CRC patients compared to controls in three independent and unrelated sets of samples (total n of 222). We further verify the perturbation using a tandem MS (MS/MS) approach in two additional independent case-control populations totalling 220 subjects. Implications of the findings for CRC screening are discussed.

We report here on the discovery of novel hydroxylated polyunsaturated ultra long-chain fatty acids containing between 28 and 36 carbons reduced in the serum of CRC patients compared to healthy asymptomatic controls. The utility of non-targeted metabolomics using high resolution FTICR-MS coupled with flow injection technology for biomarker discovery was demonstrated by applying the technology to three independent test populations. In contrast to the 'training/test-set' approach often used by splitting a single sample set in half to validate the performance of biomarkers [36–38], which often relies on complex algorithms (see review [39]) and can result in bias [40], we carried out fully independent discovery analyses on three separate sample sets of matched cases and controls of different ethnic backgrounds collected from multiple sites around the world to ensure a high degree of robustness and minimal chance of sampling bias. Of the top 50 metabolic discriminators discovered in the Osaka set, 44 and 47 of these were also significantly changed in the GCI and Seracare sets, respectively. This remarkable inter-study agreement indicates that not only is non-targeted FTICR-MS technology a reproducible biomarker discovery engine, but that disease-related metabolomic changes can be highly conserved across geographic locations and races. The reduction of hPULCFAs in the serum of CRC patients was further validated by translation of the non-targeted FTICR-MS discovery into a simple targeted TQ-MRM method for three hPULCFAs, which was used on two further independent and ethnically diverse case-control test populations. ROC AUCs generated from the TQ-MRM method on the two validation studies were consistent with those based upon the same fatty acids detected in the three FTICR-MS discovery studies (Figures 3, 5 and 6). In total, five independent study populations collectively comprising 222 treatment-naive CRC patient samples and 220 disease-free asymptomatic controls were evaluated using two different analytical methods. Indeed, the likelihood of the reported association between the reduction of hPULCFAs and CRC being a false positive result across the five independent sets of samples is astronomically low. Meta-analysis was performed on the false positive rates using Fisher's Inverse Chi-square Method ( ; p = P-values of five independent samples, k = five different samples, C = upper tail of the chi-square distribution with 2 k degrees of freedom ( = 18.31))[41, 42]. Based upon the meta-analysis, the resulting P-values for markers 446 and 448 were more significant than the individual P-values, at 2.96 × 10^-47 and 8.11 × 10^-49, respectively. Although there were differences in the median ages between the CRC and control cohorts in two of the studies, there was no statistically significant trend between age and hPULCFA levels within the individual cohorts and we observed no significant difference between hPULCFA concentrations among the controls from the different populations (not shown). We also observed no differences between genders, and although there were slightly higher BMI levels in the control cohorts for the GCI and Seracare 1 cohorts, the BMIs were matched in the second Seracare validation population suggesting the markers are not related to BMI. A prospective analysis of disease-free subjects equally distributed across various age groups is underway specifically to address any potential age or BMI effects in more detail. Overall our results indicate with a high degree of confidence that a reduction in these metabolites is correlated with the presence of CRC.

The FTICR-MS provided resolution sufficient for confident molecular formula predictions based upon accurate mass in conjunction with extraction, ionization, and statistical correlative information. Although multiple elemental compositions were theoretically assignable to given biomarker masses, only formulas having 28 to 32 carbons, and four to six oxygen were consistently assignable to common masses detected in two or three of the discovery sets. Given a high degree of statistical interaction between the sample-to-sample expression profiles of the hPULCFAs (that is, a high degree of correlation between the relative intensities of the markers across subjects) we suspected they were all part of the same metabolic system and should therefore show related compositions. Detection in negative ionization mode also reduced the likelihood that nitrogen was present in any of the compositions. This information in conjunction with tandem mass spectrometry showing prominent losses of water and carbon dioxide enabled the determination of molecular formulas as shown in Table 3 and Additional File 2. A number of candidate classes of molecules theoretically fitting the molecular formula class were easily excluded using tandem MS. For example, we observed no fragments indicative of condensed ring systems such as those in steroids or vitamin D, and no fragments indicative of chroman ring systems such as those observed in the vitamin E tocopherols. Several other classes of molecules including vitamin K and retinol, and bile acids such as cholic acid and 3β,7α-dihydroxy-5-cholestenoic acid also did not show comparable fragmentation patterns. However, the similarity in fragmentation pattern, particularly in the relative abundances of daughter ions resulting from losses of CO₂ and H₂O, and chain cut ions from the hPULCFAs to known hydroxy fatty acid standards as well as other fatty acids reported in the literature such as the resolvins and protectins (discussed below), allowed for the identification of the metabolites as hydroxylated polyunsaturated ultra long-chain fatty acids. Examination of the MS/MS data for the C28 series (masses 446, 448, 450, 464, 466 and 448) revealed a consistent 113 Da daughter ion, which we conjecture to represent the carboxy-terminus chain fragment -CH₂-CH=CH-CH₂-CH₂-COOH. In addition, a consistent loss of 54 (-CH=CH-CH₂-CH₂-) from the [M-(CO₂ +H₂O)] daughter ion was observed for the 446, 448, 464 and 466, but not the 450 and 468 molecules, suggesting that (1) the 450 and 468 may have a saturated carboxy terminal region and (2), that there are likely no hydroxyl moieties within this region of the molecule. MS/MS data of all the C28 and other markers also did not show the diagnostic fragment obtained with a 1,2-diol motif as observed for 1 (base peak is chain cut ion at m/z 115) and NMR on fractions enriched via flash-column chromatography showed lower than expected integration values obtained for the ¹H NMR signals at δ 2.78 (methylene interruptions between double bond carbons) and at δ 5.12 - 5.90 (hydrogen atoms on double bond carbons). Cumulatively these results suggested that the hydroxyl groups in the molecules are likely bonded to the carbon atoms between the sp² carbons at least seven carbons from the carboxy end. Confirmation of the exact positions of the hydroxyl groups and precise locations of unsaturations in individual hPULCFAs using preparatory HPLC and chemical synthesis is in progress and will be reported in subsequent publications.

Interestingly, the metabolite markers reported in this study represent a human-specific metabolic system. We analysed serum samples from multiple species, including rat, mouse and bovine, as well as multiple different sample sources including numerous cell lines, conditioned media, tumour and normal colonic tissue from patients in the GCI discovery set, and brain, liver, adipose and other tissues from various species, all of which failed to show any detectable levels of these hPULCFAs (results not shown). We also could not detect these molecules in various plant tissues or grains, including policosanol extracts which are rich in saturated C28 and longer-chain fatty acids [43, 44]. This suggests that the molecules may originate from human-specific metabolic processes, such as specific p450-mediated and/or microbiotic processes. The lack of detection in tumour or normal colonic tissue suggests that the metabolites are not 'tumour-derived markers' and, combined with the high rate of association in stage I cancer, it is not likely that the reduction is the result of tumour burden. Analysis of post-surgery samples is currently in progress to address this question. However, the further reduction of levels observed in some late stage Japanese cases (Figure 6) could be explained if lower levels of the hPULCFAs were indeed indicative of progression rate in this group. It is also important to note that in all control groups reported in this paper, subjects were not colonoscopy-confirmed to be free of tumours or advanced neoplasia. Based upon colonoscopy results by Collins et al in average-risk subjects, up to 10% of an asymptomatic population can be positive for advanced neoplasia [45]. Therefore, the ability of these metabolites to discriminate between subjects at risk and not at risk for CRC is likely under-estimated in our results. Studies are currently in progress to evaluate endoscopy-confirmed controls, to assess the effect of treatment on the markers, and to investigate any possible association with various grades of colon pathologies and non-malignant GI disorders as well as other cancers.

Although fatty acids of this length containing hydroxyl groups have never been reported as far as we are aware, they appear to resemble a class of hydroxylated very long-chain fatty acids knows as the resolvins and protectins that originate from the n3 essential fatty acids EPA and DHA, respectively, which are critical in promoting the resolution of acute inflammation. The inability to sufficiently 'resolve' acute inflammation is the leading theory behind the establishment of chronic inflammatory states which underlie multiple conditions including cancer [46] and Alzheimer's Disease [47]. Of particular relevance is the effect of pro-resolution long-chain hydroxy fatty acid mediators on intestinal inflammatory conditions such as irritable bowl disease (IBD), Crohn's Disease, Colitis and colon cancer. Both Resolvin E1 (RvE1) and Lipoxin A4 (LXA4) have been implicated with protective effects against colonic inflammation. RvE1 was shown to protect against the development of 2,4,6-trinitrobenze sulphonic acid-induced colitis in mice, accompanied by a block in leukocyte infiltration, decreased proinflammatory gene expression, induced nitric oxide synthase, with improvements in survival rates and sustained body weight [48]. Similarly, LXA4 analogues have been shown to attenuate chemokine secretion in human colon ex vivo [49], and attenuated 50% of genes, particularly those regulated by NFκB induced in response to pathogenically induced gastroenteritis [50]. In vivo, LXA4 analogues reduced intestinal inflammation in DSS-induced inflammatory colitis, resulting in significantly reduced weight loss, haematochezia and mortality [50]. Structurally, resolvins and protectins (as well the n6 lipoxins) comprise mono-, di- and tri-hydroxylated products of the parent VLCFAs, catalyzed by various lipoxygenases, cyclooxygenases and p450 enzymes [51–55]. The possibility that the hPULCFAs reported here represent elongation products of these molecules cannot be excluded. Future studies will be required to address the origin, as well as the biological role, if any, that these molecules may play in defending the body against CRC development.

Although we report results from multiple case-control cohorts each having a limited sample size, the average AUC across all the samples reported here was 0.91 ± 0.04, which translates into approximately 75% sensitivity at 90% specificity with little to no disease-stage bias. The real-world screening performance is currently being evaluated through two large ethically approved prospective clinical trials, one in collaboration with the Saskatchewan Cancer Agency and the Saskatchewan Provincial Government (PDI-CT-1; n = 5000), and the other with the University of Calgary (PDI-CT-3 n = 1500). Clinically relevant questions are being addressed, including correlation between hPULCFAs and CRC in a prospective hospital screening environment, correlation with other non-malignant gastrointestinal disorders (such as IBD, Crohn's and colitis), whether there is any correlation with various stages of neoplasia or polyps and family history and whether subjects with low hPULCFA levels show higher incidence rates of CRC than subjects with 'normal' levels over time.

In summary, we have identified a consistent reduction of novel circulating hPULCFAs in CRC patients which could have considerable implications for CRC diagnosis and screening and possibly prevention and treatment. Adherence to currently recommended screening modalities, namely faecal occult blood testing and colonoscopy, is poor due to a number of factors including public acceptance, risk, cost and available resources. The use of a serum-based test to screen the population for subjects who are high risk would focus endoscopy resources on subjects who need it the most, resulting in a higher detection rate, particularly in early stages of the disease. Given the positive prognosis of early-stage therapeutic intervention, it is tempting to speculate that hPULCFA-based screening could one day result in decreased CRC mortality.

We have shown that comprehensive non-targeted metabolomics technology based upon high-resolution FTICR mass spectrometry represents a powerful and robust approach for small-molecule biomarker-driven discovery. Accurate mass measurements combined with conventional MS/MS resulted in the rapid identification of key structural characteristics of the novel metabolites discovered and the assignment of putative chemical structures. The subsequent translation of these metabolite biomarker discoveries into an efficient and clinically viable high-throughput semi-quantitative triple-quadrupole platform represents a significant advancement in the clinical implementation of biomarker discoveries. The reduction of systemic hydroxylated ultra-long chain fatty acids in CRC patients raises intriguing biological and aetiological questions given the large numbers of sporadic CRC cases and the heavy influence of lifestyle and diet on risk. Further research is ongoing regarding the potential role(s) these novel molecules play in CRC progression and whether they have any association with previously established risk factors.