Epigenetic liquid biopsies for minimal residual disease, what's around the corner?

Liquid biopsy assays for minimal residual disease (MRD) are used to monitor and inform oncological treatment and predict the risk of relapse in cancer patients. To-date, most MRD assay development has focused on targeting somatic mutations. However, epigenetic changes are more frequent and universal than genetic alterations in cancer and circulating tumor DNA (ctDNA) retains much of these changes. Here, we review the epigenetic signals that can be used to detect MRD, including DNA methylation alterations and fragmentation patterns that differentiate ctDNA from noncancerous circulating cell-free DNA (ccfDNA). We then summarize the current state of MRD monitoring; highlight the advantages of epigenetics over genetics-based approaches; and discuss the emerging paradigm of assaying both genetic and epigenetic targets to monitor treatment response, detect disease recurrence, and inform adjuvant therapy.

Oncomicrobial Community Profiling Identifies Clinicomolecular and Prognostic Subtypes of Colorectal Cancer.

BACKGROUND & AIMS: Dysbiosis of gut microbiota is linked to the development of colorectal cancer (CRC). However, microbiota-based stratification of CRC tissue and how this relates to clinicomolecular characteristics and prognosis remains to be clarified. METHODS: Tumor and normal mucosa from 423 patients with stage I to IV CRC were profiled by bacterial 16S rRNA gene sequencing. Tumors were characterized for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), APC, BRAF, KRAS, PIK3CA, FBXW7, SMAD4, and TP53 mutations, subsets for chromosome instability (CIN), mutation signatures, and consensus molecular subtypes (CMS). Microbial clusters were validated in an independent cohort of 293 stage II/III tumors. RESULTS: Tumors reproducibly stratified into 3 oncomicrobial community subtypes (OCSs) with distinguishing features: OCS1 (Fusobacterium/oral pathogens, proteolytic, 21%), right-sided, high-grade, MSI-high, CIMP-positive, CMS1, BRAF V600E, and FBXW7 mutated; OCS2 (Firmicutes/Bacteroidetes, saccharolytic, 44%), and OCS3 (Escherichia/Pseudescherichia/Shigella, fatty acid ß-oxidation, 35%) both left-sided and exhibiting CIN. OCS1 was associated with MSI-related mutation signatures (SBS15, SBS20, ID2, and ID7) and OCS2 and OCS3 with SBS18 related to damage by reactive oxygen species. Among stage II/III patients, OCS1 and OCS3 both had poorer overall survival compared with OCS2 for microsatellite stable tumors (multivariate hazard ratio [HR], 1.85; 95% confidence interval [CI], 1.15-2.99; P = .012; and HR, 1.52; 95% CI 1.01-2.29; P = .044, respectively) and left-sided tumors (multivariate HR, 2.66; 95% CI, 1.45-4.86; P = .002; and HR, 1.76; 95% CI, 1.03-3.02; P = .039, respectively). CONCLUSIONS: OCS classification stratified CRCs into 3 distinct subgroups with different clinicomolecular features and outcomes. Our findings provide a framework for a microbiota-based stratification of CRC to refine prognostication and to inform the development of microbiota-targeted interventions.

DNA Methylation Cancer Biomarkers: Translation to the Clinic.

Carcinogenesis is accompanied by widespread DNA methylation changes within the cell. These changes are characterized by a globally hypomethylated genome with focal hypermethylation of numerous 5'-cytosine-phosphate-guanine-3' (CpG) islands, often spanning gene promoters and first exons. Many of these epigenetic changes occur early in tumorigenesis and are highly pervasive across a tumor type. This allows DNA methylation cancer biomarkers to be suitable for early detection and also to have utility across a range of areas relevant to cancer detection and treatment. Such tests are also simple in construction, as only one or a few loci need to be targeted for good test coverage. These properties make cancer-associated DNA methylation changes very attractive for development of cancer biomarker tests with substantive clinical utility. Across the patient journey from initial detection, to treatment and then monitoring, there are several points where DNA methylation assays can inform clinical practice. Assays on surgically removed tumor tissue are useful to determine indicators of treatment resistance, prognostication of outcome, or to molecularly characterize, classify, and determine the tissue of origin of a tumor. Cancer-associated DNA methylation changes can also be detected with accuracy in the cell-free DNA present in blood, stool, urine, and other biosamples. Such tests hold great promise for the development of simple, economical, and highly specific cancer detection tests suitable for population-wide screening, with several successfully translated examples already. The ability of circulating tumor DNA liquid biopsy assays to monitor cancer in situ also allows for the ability to monitor response to therapy, to detect minimal residual disease and as an early biomarker for cancer recurrence. This review will summarize existing DNA methylation cancer biomarkers used in clinical practice across the application domains above, discuss what makes a suitable DNA methylation cancer biomarker, and identify barriers to translation. We discuss technical factors such as the analytical performance and product-market fit, factors that contribute to successful downstream investment, including geography, and how this impacts intellectual property, regulatory hurdles, and the future of the marketplace and healthcare system.

Blood-based protein biomarker panel for the detection of colorectal cancer.

BACKGROUND: The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test. PRINCIPAL FINDINGS: In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2). CONCLUSIONS: Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

Claudin-1 Expression Is Elevated in Colorectal Cancer Precursor Lesions Harboring the BRAF V600E Mutation.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/P) are now recognised precursors of colorectal cancer (CRC) including cancers harbouring somatic BRAF (V600E) mutations. While the morphological diagnostic criteria of SSA/P have been established, distinguishing between small/early SSA/P and microvesicular hyperplastic polyps (MVHP) is challenging and may not be possible in routine practice. METHODS: Gene expression profiling of MVHP (n=5, all BRAF V600E wild-type) and SSA/P (n=5, all BRAF V600E mutant) samples was performed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical analysis was performed to verify the expression of claudin 1 (CLDN1) in MVHP and SSA/P. RESULTS: Gene expression profiling studies conducted between MVHP and SSA/P identified CLDN1 as the most statistically significant differentially expressed gene (p<0.05). Validation with qRT-PCR confirmed an up-regulation of CLDN1 in BRAF V600E mutant polyps regardless of polyp type (p<0.0005). Immunohistochemical analysis of CLDN1 expression in BRAF V600E mutant SSA/Ps (n=53) and MVHPs (n=111) and BRAF wild-type MVHPs (n=58), demonstrated a strong correlation between CLDN1 expression and the BRAF V600E mutation in both SSA/P and MVHP samples when compared to wild-type polyps (p<0.0001). CONCLUSION: This study demonstrates an up regulation of CLDN1 protein in serrated colorectal polyps including MVHP harbouring the BRAF V600E mutation. Our results demonstrated an apparent heterogeneity on the molecular level within the MVHP group and suggest that MVHP with somatic BRAF V600E mutation and up-regulated expression of CLDN1 are closely related to SSA/P and may in fact represent a continuous spectrum of the same neoplastic process within the serrated pathway of colorectal carcinogenesis.

Colorectal cancer biomarkers: to be or not to be? Cautionary tales from a road well travelled.

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.

Performance of serum lipocalin 2 as a diagnostic marker for colorectal cancer.

BACKGROUND: Lipocalin 2 has been implicated in colorectal tumorigenesis but its usefulness as a diagnostic marker for the disease has previously never been determined. METHODS: We have used ELISA immunoassay to measure the level of serum lipocalin 2 in a cohort consisting of colorectal cancer patients (n=196) and age/gender matched controls (n=99). RESULTS: The median concentration of lipocalin 2 was found to be significantly higher (p< 0.0001) in the patient group (105.9 ng/mL, range 10.8-444.7 ng/mL) when compared to the control subjects (86.4 ng/mL, range 17.1-190.0 ng/mL). Additionally, no significant difference was observed between disease stage (Dukes' or T stage) in the patient cohort. Receiver operating characteristic analysis was performed to determine its performance as a diagnostic marker. The area under the curve was found to be 0.641 (95% confidence interval 0.576-0.706). Furthermore, the sensitivity of lipocalin 2 was found to be 24% at 90% specificity. CONCLUSIONS: Our study indicates that lipocalin 2 is not a suitable serum biomarker for the diagnosis of CRC.

Colorectal carcinogenesis: a cellular response to sustained risk environment.

The current models for colorectal cancer (CRC) are essentially linear in nature with a sequential progression from adenoma through to carcinoma. However, these views of CRC development do not explain the full body of published knowledge and tend to discount environmental influences. This paper proposes that CRC is a cellular response to prolonged exposure to cytotoxic agents (e.g., free ammonia) as key events within a sustained high-risk colonic luminal environment. This environment is low in substrate for the colonocytes (short chain fatty acids, SCFA) and consequently of higher pH with higher levels of free ammonia and decreased mucosal oxygen supply as a result of lower visceral blood flow. All of these lead to greater and prolonged exposure of the colonic epithelium to a cytotoxic agent with diminished aerobic energy availability. Normal colonocytes faced with this unfavourable environment can transform into CRC cells for survival through epigenetic reprogramming to express genes which increase mobility to allow migration and proliferation. Recent data with high protein diets confirm that genetic damage can be increased, consistent with greater CRC risk. However, this damage can be reversed by increasing SCFA supply by feeding fermentable fibre as resistant starch or arabinoxylan. High protein, low carbohydrate diets have been shown to alter the colonic environment with lower butyrate levels and apparently greater mucosal exposure to ammonia, consistent with our hypothesis. Evidence is drawn from in vivo and in vitro genomic and biochemical studies to frame experiments to test this proposition.

Identification of potential pathways involved in induction of apoptosis by butyrate and 4-benzoylbutyrate in HT29 colorectal cancer cells.

Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.

A review of the potential mechanisms for the lowering of colorectal oncogenesis by butyrate.

Colorectal cancer (CRC) is a leading cause of preventable cancer deaths worldwide, with dietary factors being recognised as key risk modifiers. Foods containing dietary fibre are protective to a degree that the World Cancer Research Fund classifies the evidence supporting their consumption as 'convincing'. The mechanisms by which fibre components protect against CRC remain poorly understood, especially their interactions with the gut microbiome. Fibre is a composite of indigestible plant polysaccharides and it is emerging that fermentable fibres, including resistant starch (RS), are particularly important. RS fermentation induces SCFA production, in particular, relatively high butyrate levels, and in vitro studies have shown that this acid has strong anti-tumorigenic properties. Butyrate inhibits proliferation and induces apoptosis of CRC cell lines at physiological concentrations. These effects are attributed to butyrate's ability to alter gene transcription by inhibiting histone deacetylase activity. However, the more recent discovery of G-protein coupled receptors that bind butyrate and other SCFA and data obtained from proteomic and genomic experiments suggest that alternative pathways are involved. Here, we review the mechanisms involved in butyrate-induced apoptosis in CRC cells and, additionally, the potential role this SCFA may play in mediating key processes in tumorigenesis including genomic instability, inflammation and cell energy metabolism. This discussion may help to inform the development of strategies to lower CRC risk at the individual and population levels.

Nutrigenetics and nutrigenomics: viewpoints on the current status and applications in nutrition research and practice.

Nutrigenetics and nutrigenomics hold much promise for providing better nutritional advice to the public generally, genetic subgroups and individuals. Because nutrigenetics and nutrigenomics require a deep understanding of nutrition, genetics and biochemistry and ever new 'omic' technologies, it is often difficult, even for educated professionals, to appreciate their relevance to the practice of preventive approaches for optimising health, delaying onset of disease and diminishing its severity. This review discusses (i) the basic concepts, technical terms and technology involved in nutrigenetics and nutrigenomics; (ii) how this emerging knowledge can be applied to optimise health, prevent and treat diseases; (iii) how to read, understand and interpret nutrigenetic and nutrigenomic research results, and (iv) how this knowledge may potentially transform nutrition and dietetic practice, and the implications of such a transformation. This is in effect an up-to-date overview of the various aspects of nutrigenetics and nutrigenomics relevant to health practitioners who are seeking a better understanding of this new frontier in nutrition research and its potential application to dietetic practice.

A combined free-flow electrophoresis and DIGE approach to identify proteins regulated by butyrate in HT29 cells.

Many biologically active agents exert a pleiotropic response in cells and tissues. This presents challenges in descriptive and comparative analysis of the proteome in response to these agents. Although free-flow electrophoresis has been applied in a number of proteomic studies as a protein separation technique, the combination of free-flow electrophoresis and DIGE has not yet been investigated for comparative proteomic analysis. In this study, we have compared the effects of butyrate on HT29 colorectal cancer cells with a particular focus on apoptosis and describe the utility of a novel approach combining free-flow electrophoresis with DIGE to identify differentially expressed proteins. We verify the results obtained by the combined free-flow electrophoresis and DIGE approach with Western blot analysis of selected proteins. We also report for the first time the regulation of a number of proteins by butyrate in HT29 colorectal cells including peptidyl-prolyl cis-trans isomerase A (cyclophilin A) and profilin-1.

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response.

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.

Proteomic analysis of butyrate effects and loss of butyrate sensitivity in HT29 colorectal cancer cells.

Butyrate, a fermentation product of the large bowel microflora, is potentially protective against the development of colorectal cancer. In vitro, butyrate has been shown to induce apoptosis and inhibit proliferation in numerous cancer cell lines, including colorectal cancer. Although these tumor suppressing properties of butyrate are well-documented in experimental systems, the mechanisms underlying the induction of these effects are not fully understood. Understanding these mechanisms in cancer cells, as well as the pathways involved in a cell's ability to overcome them and progress toward malignancy, is vital to determine therapeutic approaches for disease management. We have developed a colorectal cancer cell line (HT29-BR) that is less responsive to the apoptotic effects of butyrate through sustained exposure of HT29 cells to 5 mM butyrate and have used proteomics to investigate the mechanisms involved in the development of butyrate insensitivity. Proteomic analysis identified a number of cellular processes in HT29 and HT29-BR cells influenced by butyrate including remodeling of the actin cytoskeleton, inhibition of protein biosynthesis and dysregulation of the cell stress response. We describe novel roles for butyrate in the induction of its tumor suppressing effects and outline potential cellular pathways involved in the development of butyrate insensitivity in the HT29-BR cell population.

Interactive feature finding in liquid chromatography mass spectrometry data.

We propose a method for finding features in liquid chromatography mass spectrometry data that is based on the isotopic pattern of peaks. Our interactive approach to feature finding is carried out across many samples simultaneously and aligns features concurrently. Our scale-independent approach prioritises potential features and is easily adaptable to look for features of a particular mass and charge, paired features in isotopically labeled samples, or differentially expressed features. We demonstrate this by identifying features from normal human adult plasma. We highlight properties of plasma data that illustrate the need to visually check the quality of features found prior to further statistical analysis.

Characterization of the in vivo forms of lacrimal-specific proline-rich proteins in human tear fluid.

The tear film is complex and is rich in both peptides and proteins. Physiological factors have been shown to alter the balance of the protein components in the tear film, however, little is known of the precise stimuli that initiate these changes, or their nature and extent. Attention has been directed at the role of tear proteins in the protection of the external ocular surface, and their potential role in the pathogenesis of inflammatory and autoimmune diseases, but few lacrimal-specific proteins have been identified and demonstrated to offer a protective function at the ocular surface. The biological importance of proline-rich proteins is uncertain, although there is some evidence to indicate a potential antimicrobial function for these proteins in saliva. Despite the detection of mRNA for proline-rich proteins in lacrimal gland, the translated protein product has not been detected in tear fluid. In this study we investigate the presence of proline-rich proteins in the tear film. Human reflex tear fluid was examined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry directly, and following size exclusion high performance liquid chromatography. This revealed significant levels of a truncated form of lacrimal proline-rich protein, and a series of peptides derived the C-terminus of this protein. None of these had previously been identified in tear. Our study highlights the dangers inherent in proteomic strategies that assign an identity to a protein based on limited coverage of tryptic peptides.

A simple and inexpensive approach to interfacing high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.

The ability to obtain the accurate mass of a protein in a complex sample mixture aids in determining its correct in vivo form. This is important when identifying post-translationally modified proteins, protein variants or isoforms. The central technique used to separate proteins, 2-dimensional gel electrophoresis offers excellent separation capabilities but does not provide adequate mass accuracy. In this study, an alternative method, liquid chromatography (LC) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS (LC-MALDI) is described. LC-MALDI-MS was used to separate and determine the mass of proteins and peptides in a complex biological sample (i.e., human pituitary gland homogenate). Peptides and proteins were first separated by capillary chromatography and the eluent mixed post-column with sinapinic acid matrix. The flow was then deposited directly onto a standard MALDI target via a capillary nebulizer. In addition to offering high mass accuracy, this method can be applied to peptide and protein quantification.

A comprehensive characterization of the peptide and protein constituents of human seminal fluid.

BACKGROUND: Knowledge of the peptide and protein components of seminal fluid and their role in prostate diseases including benign prostatic hyperplasia and prostate carcinoma is scant. We have undertaken a proteomic analysis of semen as a forerunner to identifying sensitive and specific diagnostic markers of prostatic diseases; to aid in improved therapeutic intervention; and, to enhance our understanding of prostate health and disease. METHODS: Peptide and protein components of pooled human seminal fluid (n = 5) were separated by gel electrophoresis (1D and 2D) and identified by either matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) or capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Analysis by two-dimensional electrophoresis (2DE) established that there were multiple post-translational variants of the majority of the proteins. Hormones, growth factors and bioactive peptides were detected and identified. CONCLUSIONS: We have identified over 100 protein and peptide components of normal human seminal fluid.

Practical quantitative biomedical applications of MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to obtain fast and accurate determinations of molecular mass, but quantitative determinations are generally made by other techniques. In this study we illustrate the practical utility of automated MALDI-TOFMS as a tool for quantifying a diverse array of biomolecules covering an extensive molecular weight range, and present in biological extracts and fluids. Growth hormone was measured in rat pituitary tissue; insulin in human pancreatic tissue; homovanillic acid in human urine; and LVV-hemorphin-7, epinephrine and norepinephrine in human adrenal and pheochromocytoma tissues. Internal standards including compounds of similar molecular weight, structural analogs or isotopomers were incorporated into each analysis. We report on the current practical limitations of quantitative MALDI-TOFMS and highlight some of the potential benefits of this technique as a quantitative tool.