Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

Discovery, validation, and diagnostic ability of multiple protein-based biomarkers in saliva and gingival crevicular fluid to distinguish between health and periodontal diseases.

AIM: To discover and validate differential protein biomarker expression in saliva and gingival crevicular fluid (GCF) to discriminate objectively between periodontal health and plaque-induced periodontal disease states. MATERIALS AND METHODS: One-hundred and ninety participants were recruited from two centres (Birmingham and Newcastle upon Tyne, UK) comprising healthy, gingivitis, periodontitis, and edentulous donors. Samples from the Birmingham cohort were analysed by quantitative mass spectrometry proteomics for biomarker discovery. Shortlisted candidate proteins were then verified by enzyme-linked immunosorbent assay in both cohorts. Leave-one-out cross validation logistic regression analysis was used to identify the best performing biomarker panels. RESULTS: Ninety-five proteins were identified in both GCF and saliva samples, and 15 candidate proteins were selected based upon differences discovered between the donor groups. The best performing panels to distinguish between: health or gingivitis and periodontitis contained matrix metalloproteinase-9 (MMP9), S100A8, alpha-1-acid glycoprotein (A1AGP), pyruvate kinase, and age (area under the curve [AUC] 0.970); health and gingivitis contained MMP9, S100A8, A1AGP, and pyruvate kinase, but not age (AUC 0.768); and mild to moderate and advanced periodontitis contained MMP9, S100A8, A1AGP, pyruvate kinase, and age (AUC 0.789). CONCLUSIONS: Biomarker panels containing four proteins with and without age as a further parameter can distinguish between periodontal health and disease states.

Comparison of faecal protein biomarkers' diagnostic accuracy for colorectal advanced neoplasms: a systematic review and meta-analysis.

Early diagnosis of colorectal advanced neoplasms (ANs), including colorectal cancer (CRC) and advanced adenoma (AA), has a positive effect on the survival rate. As a first attempt, the aim of this meta-analysis was to compare the diagnostic accuracy of faecal protein biomarkers for the detection of colorectal neoplasms with consideration of a wide range of covariates. A systematic literature search was performed up to Jun 10, 2021 on Web of Sciences, Scopus and PubMed. The diagnostic accuracies were calculated using the bivariate/hierarchical random effect model. Biomarkers were determined to be clinically applicable (CA) if they had areas under the curve > 0.70 and positive and negative likelihood ratios > 2 and < 0.5, respectively. A total of 47,059 test results were extracted from 16 immunochemical faecal occult blood test (iFOBT), 26 pyruvate kinase-M2 (PK-M2) and 23 faecal calprotectin (FC) studies. Only iFOBT, PK-M2 and FC for CRC plus iFOBT and PK-M2 for AN were CA. iFOBT had significantly superior accuracy (P = 0.02 versus PK-M2 and P < 0.01 versus FC for CRC; P < 0.01 versus PK-M2 for AN). Regarding covariates, the lateral flow method of PK-M2 measurement increased its accuracy for CRC detection compared to the enzyme-linked immunosorbent assay (P < 0.01). iFOBT is recommended as the most accurate faecal biomarker for CRC and AN diagnosis.

Simple and Minimally Invasive SID Devices for Native Mass Spectrometry.

We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ∼6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

Development of an Automated Enzymatic Method to Quantify Pyruvate Kinase in Red Blood Cells.

BACKGROUND: Pyruvate kinase (PK) deficiency is the most common cause of nonspherocytic hemolytic anemia owing to defective glycolysis. This study developed and validated an automated method to measure PK activity in red blood cells (RBCs). METHODS: PK catalyzes the reaction of phosphoenolpyruvate with ADP to form pyruvate and ATP. The pyruvate is reduced in the presence of lactate dehydrogenase and NADH to produce lactate and NAD+. The rate of absorbance decrease at 340 nm is proportional to PK activity. PK and hemoglobin (Hb) measurements were performed on a Roche cobas c501 analyzer. After establishing a k-factor, accuracy, linearity, imprecision, sensitivity, and stability were validated and the reference interval was verified. RESULTS: The k-factor was -9477. Accuracy was evaluated by method comparison (n = 56). Linear regression yielded y = 1.0x - 0.57, and R2 of 0.93. Linearity was determined by combining a high sample with hemolyzing solution in 6 different ratios. Linear regression analysis yielded y = 1.02x - 2.68, and R2 of 1.0. The assay was linear to 87 U/dL. Precision was evaluated by testing hemolysates in 3 replicates/day for 10 days. Within-run imprecision was 1.9% and 2.5% and total imprecision was 4.0% and 5.6% at 14.0 and 8.1 U/g Hb, respectively. The limit of blank was 0.0, and the limit of detection was 1.0 U/dL. Stability was determined in 4 sample types at 3 different temperatures; the changes were all <10% when compared with t0. The current PK reference interval of 4.6 to 11.2 U/g Hb was verified. CONCLUSIONS: This automated assay for quantifying PK in RBCs has acceptable performance characteristics and is fit for intended use.

Biliary calprotectin, lactoferrin and dimeric pyruvate kinase after liver transplantation are associated with biliary damage and graft survival in a case-control study.

BACKGROUND: After liver transplantation (LT), biliary complications are associated with reduced graft survival. We tested inflammation markers for their association with biliary damage and graft loss in bile. MATERIAL AND METHODS: The study design was a retrospective case-control study. Calprotectin, lactoferrin and pyruvate kinase were measured in endoscopically retrieved bile with ELISA. RESULTS: Calprotectin and lactoferrin were significantly higher in bile of ischemic-type biliary lesions and donor duct non-anastomotic strictures than in control, bile leakage, Cytomegalovirus infection, anastomotic stricture or acute cellular rejection patients (p<0.001) independent of serum liver values at endoscopy. Calprotectin (p=0.02) was independently associated with retransplantation free survival in multivariate analysis, as was γGT (p=0.03) but not ERC radiographic classification of the bile duct or cold ischemia time. CONCLUSION: Calprotectin and lactoferrin are bile markers for biliary damage and are associated with re-transplantation free survival. They can differentiate progressive biliary damage from non-biliary liver value alterations after LT.

Faecal biomarkers do not always identify pre-cancerous lesions in patients who present in primary care with bowel symptoms.

AIM: To determine if tumour-derived M2-PK is potentially a more accurate biomarker of pre-cancerous bowel lesions in patients who present in primary care with bowel symptoms than the detection of faecal haemoglobin. METHODS: Patients requested by their general practitioners (GPs) to provide a stool sample to determine the presence of faecal haemoglobin consented for the same stool samples to be tested for the presence of M2-PK. For comparison M2-PK levels were also measured in stool samples from patients recently identified with colorectal cancer and healthy controls who self-reported no bowel problems at the time of sampling. RESULTS: M2-PK levels measured in 185 GP-derived samples were comparable to the control cohort (57 healthy controls) and notably lower than those in the 57 patients with CRC (2.6, 3.2 and 18.2U/ml-1, respectively). Sixty-seven of the GP patients were referred for colonoscopy. While 26 of these patients had a positive M2-PK, only 10 were found to have colonic lesions. Conversely, 18 of the 41 patients who had a negative M2-PK were found to have lesions that included one CRC, 13 adenomas and four other polyps. The FIT also failed to identify colonic disease in 19 of 48 patients referred for colonoscopy. There was, however, a significant association between lesions greater than 1cm and a positive FIT (p<0.02) that was not the case with M2-PK. A positive FIT identified one patient in the GP patient cohort subsequently diagnosed with colorectal cancer at follow-up colonoscopy whereas the same stool sample tested negative for M2-PK. CONCLUSIONS: Measurement of faecal M2-PK levels lacks the specificity and sensitivity (and therefore diagnostic accuracy) to identify the individuals who should be progressed for clinical follow-up. Accordingly, M2-PK is not is not a robust biomarker for identifying pre-cancerous bowel lesions in a primary care setting.

Identification of pyruvate kinase 2 as a possible crab allergen and analysis of allergenic proteins in crabs consumed in Taiwan.

In Taiwan, crab is one of the main causes for food allergy. Several proteins are recognized as crustacean allergens, and tropomyosin is known to be the major one. However, sensitization patterns of Taiwanese patients to crustacean allergens remain unclear. Therefore, we analyzed the specific-IgE binding ability of crucifix crab (Charybdis feriatus) allergens by western blot using patients' sera. In particular, we found a 56 kDa protein in crucifix crab reacted with specific-IgEs in patients' sera, and we further identified the protein as a novel crab allergen pyruvate kinase 2. Additionally, little is known about tropomyosin contents in crabs consumed in Taiwan. Thus, we also quantified the levels of tropomyosin by using enzyme-linked immunosorbent assay (ELISA) among raw and cooked crab species. Our results showed tropomyosin levels varied depending on crab species. In summary, these findings improve the understanding of crustacean allergens and contribute to the clinical diagnosis of crustacean allergies.

Examination of the temporal variation of peptide content in decomposition fluid under controlled conditions using pigs as human substitutes.

We report the preliminary observations of the peptide content of decomposition fluid produced under controlled laboratory conditions and in the absence of a soil matrix. Four domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition over a four-week trial period; physical characteristics were recorded and the peptide components of decomposition fluid was analysed using high performance liquid chromatography-time of flight mass spectrometry. Preliminary data analysis indicated that a range of peptides were consistently detected across the course of the trial period and 27 of these were common to all four cadavers; 22 originating from haemoglobin. The peptides associated with haemoglobin subunit alpha and beta displayed a breakdown pattern that remained consistent for all cadavers for the duration of the trial. Though identification of peptides during decomposition has potential for estimating the time since death, quantification of selected peptides is likely to be essential to identify time-dependent trends.

Effects of bovine enterovirus and type 1 diabetes on liver and kidney pyruvate kinase activity in an animal model

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic beta cells. In addition to genetic factors, enteroviruses have been considered the main environmental factor involved in this pathology. Therefore, the objective of this study was to evaluate the effects of streptozotocin-induced diabetes and bovine enterovirus (BEV) on liver and kidney pyruvate kinase activity in rats. Fourteen male Wistar rats were divided in three groups: control, diabetes and a third group, which was fed with water experimentally contaminated by BEV. Increased blood glucose levels were found in both diabetes and enterovirus groups, whereas there were no alterations in the lipid profile. A reduced pyruvate kinase activity was observed in the liver and kidney of animals from diabetes and enterovirus groups. Under our experimental conditions, the ingestion of water experimentally contaminated by BEV induced alterations in glycaemia, and also interfered in the pyruvate kinase activity in liver and kidney of the rats, which might be one of the possible mechanisms involved in the T1D development.

The Multifarious Functions of Pyruvate Kinase M2 in Oral Cancer Cells.

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancies worldwide. OSCC frequently leads to oral dysfunction, which worsens a patient's quality of life. Moreover, its prognosis remains poor. Unlike normal cells, tumor cells preferentially metabolize glucose by aerobic glycolysis. Pyruvate kinase (PK) catalyzes the final step in glycolysis, and the transition from PKM1 to PKM2 is observed in many cancer cells. However, little is known about PKM expression and function in OSCC. In this study, we investigated the expression of PKM in OSCC specimens and performed a functional analysis of human OSCC cells. We found that the PKM2/PKM1 ratio was higher in OSCC cells than in adjacent normal mucosal cells and in samples obtained from dysplasia patients. Furthermore, PKM2 expression was strongly correlated with OSCC tumor progression on immunohistochemistry. PKM2 expression was higher during cell growth, invasion, and apoptosis in HSC3 cells, which show a high energy flow and whose metabolism depends on aerobic glycolysis and oxidative phosphorylation. PKM2 expression was also associated with the production of reactive oxygen species (ROS) and integration of glutamine into lactate. Our results suggested that PKM2 has a variety of tumor progressive functions in OSCC cells.

Age-related levels of fecal M2-pyruvate kinase in children with cystic fibrosis and healthy children 0 to 10years old.

BACKGROUND: The pathogenesis of gut inflammation, bacterial dysbiosis and increased rates of malignancy in CF is unclear. Fecal M2-pyruvate kinase (M2-PK) is a biomarker indicative of cellular proliferation that may be raised in intestinal malignancy and inflammation. Biomarkers, including M2-PK, may be useful in assessing effects of novel therapies on the gastrointestinal tract. METHODS: M2-PK was measured in stools collected from patients with CF and HC (0-10years). Linear mixed model analysis was used. RESULTS: M2-PK levels did not significantly change in children with CF (36 patients, 77 samples) (P=0.998) or HC (45 patients, 45 samples) (P=0.21), over the age range 0-10years. Patients with CF had elevated M2-PK compared to HC (median [IQR; range]: 10.7 [5.7-28.6; 1.0-239.1] (n=77) vs. 1.0 [1.0-1.0; 1.0-50.0] (n=45) U/mL, respectively; P=0.001). CONCLUSIONS: Fecal M2-PK was elevated in children with CF compared with HC during infancy and throughout childhood suggesting abnormalities in the CF gut exist in early life.

Thiol/disulfide status regulates the activity of thiol-containing kinases related to energy homeostasis in rat kidney.

Considering that thiol-containing enzymes like kinases are critical for several metabolic pathways and energy homeostasis, we investigated the effects of cystine dimethyl ester and/or cysteamine administration on kinases crucial for energy metabolism in the kidney of Wistar rats. Animals were injected twice a day with 1.6 µmol/g body weight cystine dimethyl ester and/or 0.26 µmol/g body weight cysteamine from the 16th to the 20th postpartum day and euthanized after 12 hours. Pyruvate kinase, adenylate kinase, creatine kinase activities and thiol/disulfide ratio were determined. Cystine dimethyl ester administration reduced thiol/disulfide ratio and inhibited the kinases activities. Cysteamine administration increased the thiol/disulfide ratio and co-administration with cystine dimethyl ester prevented the inhibition of the enzymes. Regression between the thiol/disulfide ratio, and the kinases activities were significant. These results suggest that redox status may regulate energy metabolism in the rat kidney. If thiol-containing enzymes inhibition and oxidative stress occur in patients with cystinosis, it is possible that lysosomal cystine depletion may not be the only beneficial effect of cysteamine administration, but also its antioxidant and thiol-protector effect.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia.

Power of screening tests for colorectal cancer enhanced by high levels of M2-PK in addition to FOBT.

Colorectal cancer (CRC) is a multistep process that involves adenoma-carcinoma sequence. CRC can be prevented by routine screening, which can detect precancerous lesions. The aim of this study is to clarify whether faecal occult blood test (i-FOBT), tumor M2 pyruvate kinase (t-M2-PK), and endocannabinoid system molecules (cannabinoid receptors type 1-CB1, type 2-CB2, and fatty acid amide hydrolase-FAAH) might represent better diagnostic tools, alone or in combination, for an early diagnosis of CRC. An immunochemical FOB test (i-FOBT) and quantitative ELISA stool test for t-M2-PK were performed in 127 consecutive patients during a 12 month period. Endocannabinoid system molecules and t-M2-PK expression were detected by immunostaining in healthy tissues and normal mucosa surrounding adenomatous and cancerous colon lesions. i-FOBT and t-M2-PK combination leads to a better diagnostic accuracy for pre-neoplastic and neoplastic colon lesions. T-M2-PK quantification in stool samples and in biopsy samples (immunostaining) correlates with tumourigenesis stages. CB1 and CB2 are well expressed in healthy tissues, and their expression decreases in the presence of advanced stages of carcinogenesis and disappears in CRC. FAAH signal is well expressed in normal mucosa and low-risk adenoma, and increased in high-risk adenoma and carcinoma adjacent tissues. This study shows that high levels of t-M2-PK in addition to FOBT enhance the power of a CRC screening test. Endocannabinoid system molecule expression correlates with colon carcinogenesis stages. Developing future faecal tests for their quantification must be undertaken to obtain a more accurate early non-invasive diagnosis for CRC.

Accurate cut-offs for predicting endoscopic activity and mucosal healing in Crohn's disease with fecal calprotectin.

BACKGROUND: Fecal biomarkers, especially fecal calprotectin, are useful for predicting endoscopic activity in Crohn's disease; however, the cut-off point remains unclear. The aim of this paper was to analyze whether faecal calprotectin and M2 pyruvate kinase are good tools for generating highly accurate scores for the prediction of the state of endoscopic activity and mucosal healing. METHODS: The simple endoscopic score for Crohn's disease and the Crohn's disease activity index was calculated for 71 patients diagnosed with Crohn's. Fecal calprotectin and M2-PK were measured by the enzyme-linked immunosorbent assay test. RESULTS: A fecal calprotectin cut-off concentration of ≥ 170 µg/g (sensitivity 77.6%, specificity 95.5% and likelihood ratio +17.06) predicts a high probability of endoscopic activity, and a fecal calprotectin cut-off of ≤ 71 µg/g (sensitivity 95.9%, specificity 52.3% and likelihood ratio -0.08) predicts a high probability of mucosal healing. Three clinical groups were identified according to the data obtained: endoscopic activity (calprotectin ≥ 170), mucosal healing (calprotectin ≤ 71) and uncertainty (71 > calprotectin < 170), with significant differences in endoscopic values (F = 26.407, p < 0.01). Clinical activity or remission modified the probabilities of presenting endoscopic activity (100% vs 89%) or mucosal healing (75% vs 87%) in the diagnostic scores generated. M2-PK was insufficiently accurate to determine scores. CONCLUSIONS: The highly accurate scores for fecal calprotectin provide a useful tool for interpreting the probabilities of presenting endoscopic activity or mucosal healing, and are valuable in the specific clinical context.

Accuracies of fecal calprotectin, lactoferrin, M2-pyruvate kinase, neopterin and zonulin to predict the response to infliximab in ulcerative colitis.

BACKGROUND: Fecal markers might predict the response to anti-TNFα in ulcerative colitis (UC). AIMS: To compare the performance of fecal calprotectin (fCal), lactoferrin (fLact), M2-PK (fM2-PK), neopterin (fNeo), and zonulin (fZon) to predict the response to therapy in active UC patients. METHODS: Disease activity from 31 consecutive patients with an active UC, treated with infliximab (IFX) was assessed by the Mayo score at baseline and at week 14 and by the partial Mayo score at W52 and stool samples collected for fecal marker measurements at W0, W2, and W14. RESULTS: At W14, 19 patients (61%) were responders to IFX induction. The median levels of fCal, fLact and fM2-PK drop dramatically from baseline to W14 in clinical responders. At W2, fM2-PK, fLact and fCal levels predicted accurately the response to IFX induction. At W14, fLact, fCal, and fM2-PK were individually reliable markers to predict sustained response at W52. The performances of fNeo and fZon were weaker in this setting. CONCLUSIONS: The performance of fM2-PK at W2 to predict response to induction therapy with IFX was superior to that of fLact and fCal, whereas monitoring fLact was the best tool to predict adequately the course of the disease at one year under maintenance IFX in UC.

Diagnostic Accuracy of Five Different Fecal Markers for the Detection of Precancerous and Cancerous Lesions of the Colorectum.

Background. Colorectal cancer (CRC) is the second deadliest malignancy worldwide. This study aimed to compare the diagnostic accuracy of different fecal markers in the detection of colorectal adenomas and cancer. Methods. Stool samples of patients referred to colonoscopy were collected for the analysis of tumor M2 pyruvate kinase (M2PK), human hemoglobin (Hb), hemoglobin/haptoglobin (Hb/Hp) complex, fecal calprotectin (FC), and matrix metalloproteinase-9 (MMP-9). Results. Sensitivity and specificity of M2PK for adenomas sized > 1 cm were 60% and 67.5% and for CRC were 94.7% and 67.5%. Sensitivity and specificity of iFOBT for adenomas sized ≥ 1 cm were 80% and 72.5% and for CRC were 94.7% and 72.5%. Sensitivity and specificity of Hb/Hp complex for adenomas sized ≥ 1 cm were 80% and 52.9% and for CRC were 100% and 52.9%. Sensitivity of FC and MMP-9 for CRC was 77.8% and 72.2%. Combined use of M2PK, iFOBT, and FC resulted in a sensitivity and specificity of 95% and 47.5% for the detection of adenomas sized ≥ 1 cm. Discussion. In CRC, sensitivity of M2PK, iFOBT, and Hb/Hp complex proved to be high. Combined use of M2PK, iFOBT, and FC may be valuable in the detection of large adenomas.

Correlation between Faecal Tumour M2 Pyruvate Kinase and Colonoscopy for the Detection of Adenomatous Neoplasia in a Secondary Care Cohort.

BACKGROUND AND AIMS: Colorectal cancer screening programmes that target detection and excision of adenomatous colonic polyps have been shown to reduce colorectal cancer related mortality. Many screening programmes include an initial faecal occult blood test (FOBt) prior to colonoscopy. To refine the selection of patients for colonoscopy other faecal-based diagnostic tools have been proposed, including tumour M2-pyruvate kinase (tM2-PK). To determine whether tM2-PK quantification may have a role in diverse settings we have assessed the assay in a cohort of patients derived from both the England bowel cancer screening programme (BCSP) and symptomatic individuals presenting to secondary care. METHOD: Patients undergoing colonoscopy provided faecal samples prior to bowel preparation. Faecal tM2-PK concentrations were measured by ELISA. Sensitivity, specificity, positive predictive value, negative predictive value and ROC analyses were calculated. RESULTS: Ninety-six patients returned faecal samples: 50 of these with adenomas and 7 with cancer. Median age was 68. Median faecal tM2-PK concentration was 3.8 U/mL for individuals without neoplastic findings at colonoscopy, 7.7 U/mL in those with adenomas and 24.4 U/mL in subjects with colorectal cancer (both, p=0.01). ROC analysis demonstrated an AUROC of 0.66 (sensitivity 72.4%, specificity 48.7%, positive predictive value 67.7%, negative predictive value 36.7%). Amongst BCSP patients with a prior positive FOBt faecal tM2-PK was more abundant (median 6.4 U/mL, p=0.03) and its diagnostic accuracy was greater (AUROC 0.82). CONCLUSION: Our findings confirm that faecal tM2-PK ELISA may have utility as an adjunct to FOBt in a screening context, but do not support its use in symptomatic patients.