Fatty acid metabolism and colon cancer protection by dietary methyl donor restriction.

INTRODUCTION: A methyl donor depleted (MDD) diet dramatically suppresses intestinal tumor development in Apc-mutant mice, but the mechanism of this prevention is not entirely clear. OBJECTIVES: We sought to gain insight into the mechanisms of cancer suppression by the MDD diet and to identify biomarkers of cancer risk reduction. METHODS: A plasma metabolomic analysis was performed on ApcΔ14/+ mice maintained on either a methyl donor sufficient (MDS) diet or the protective MDD diet. A group of MDS animals was also pair-fed with the MDD mice to normalize caloric intake, and another group was shifted from an MDD to MDS diet to determine the durability of the metabolic changes. RESULTS: In addition to the anticipated changes in folate one-carbon metabolites, plasma metabolites related to fatty acid metabolism were generally decreased by the MDD diet, including carnitine, acylcarnitines, and fatty acids. Some fatty acid selectivity was observed; the levels of cancer-promoting arachidonic acid and 2-hydroxyglutarate were decreased by the MDD diet, whereas eicosapentaenoic acid (EPA) levels were increased. Machine-learning elastic net analysis revealed a positive association between the fatty acid-related compounds azelate and 7-hydroxycholesterol and tumor development, and a negative correlation with succinate and ß-sitosterol. CONCLUSION: Methyl donor restriction causes dramatic changes in systemic fatty acid metabolism. Regulating fatty acid metabolism through methyl donor restriction favorably effects fatty acid profiles to achieve cancer protection.

An Immunotherapeutic CD137 Agonist Releases Eomesodermin from ThPOK Repression in CD4 T Cells.

Agonists to the TNF/TNFR costimulatory receptors CD134 (OX40) and CD137 (4-1BB) elicit antitumor immunity. Dual costimulation with anti-CD134 plus anti-CD137 is particularly potent because it programs cytotoxic potential in CD8+ and CD4+ T cells. Cytotoxicity in dual-costimulated CD4 T cells depends on the T-box transcription factor eomesodermin (Eomes), which we report is induced via a mechanism that does not rely on IL-2, in contrast to CD8+ CTL, but rather depends on the CD8 T cell lineage commitment transcription factor Runx3, which supports Eomes expression in mature CD8+ CTLs. Further, Eomes and Runx3 were indispensable for dual-costimulated CD4 T cells to mediate antitumor activity in an aggressive melanoma model. Runx3 is also known to be expressed in standard CD4 Th1 cells where it fosters IFN-γ expression; however, the CD4 T cell lineage commitment factor ThPOK represses transcription of Eomes and other CD8 lineage genes, such as Cd8a Hence, CD4 T cells can differentiate into Eomes+ cytotoxic CD4+CD8+ double-positive T cells by terminating ThPOK expression. In contrast, dual-costimulated CD4 T cells express Eomes, despite the continued expression of ThPOK and the absence of CD8α, indicating that Eomes is selectively released from ThPOK repression. Finally, although Eomes was induced by CD137 agonist, but not CD134 agonist, administered individually, CD137 agonist failed to induce CD134-/- CD4 T cells to express Eomes or Runx3, indicating that both costimulatory pathways are required for cytotoxic Th1 programming, even when only CD137 is intentionally engaged with a therapeutic agonist.

A novel bioactive derivative of eicosapentaenoic acid (EPA) suppresses intestinal tumor development in ApcΔ14/+ mice.

Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of hundreds of polyps throughout the colon. Without prophylactic colectomy, most individuals with FAP develop colorectal cancer at an early age. Treatment with EPA in the free fatty acid form (EPA-FFA) has been shown to reduce polyp burden in FAP patients. Since high-purity EPA-FFA is subject to rapid oxidation, a stable form of EPA compound has been developed in the form of magnesium l-lysinate bis-eicosapentaenoate (TP-252). We assessed the chemopreventive efficacy of TP-252 on intestinal tumor formation using ApcΔ14/+ mice and compared it with EPA-FFA. TP-252 was supplemented in a modified AIN-93G diet at 1, 2 or 4% and EPA-FFA at 2.5% by weight and administered to mice for 11 weeks. We found that administration of TP-252 significantly reduced tumor number and size in the small intestine and colon in a dose-related manner and as effectively as EPA-FFA. To gain further insight into the cancer protection afforded to the colon, we performed a comprehensive lipidomic analysis of total fatty acid composition and eicosanoid metabolites. Treatment with TP-252 significantly decreased the levels of arachidonic acid (AA) and increased EPA concentrations within the colonic mucosa. Furthermore, a classification and regression tree (CART) analysis revealed that a subset of fatty acids, including EPA and docosahexaenoic acid (DHA), and their downstream metabolites, including PGE3 and 14-hydroxy-docosahexaenoic acid (HDoHE), were strongly associated with antineoplastic activity. These results indicate that TP-252 warrants further clinical development as a potential strategy for delaying colectomy in adolescent FAP patients.

Associations of dietary fat with risk of early neoplasia in the proximal colon in a population-based case-control study.

PURPOSE: Excess dietary fat consumption is strongly associated with the risk of colorectal cancer, but less is known about its role in the earliest stages of carcinogenesis, particularly within the proximal colon. In the following case-control study, we evaluated the relationship between the intake of dietary fats and the frequency of early proximal neoplasia [aberrant crypt foci (ACF) or polyps], detectable by high-definition colonoscopy with contrast dye-spray. METHODS: Average-risk screening individuals underwent a high-definition colonoscopy procedure as part of larger ongoing clinical study of precancerous lesions in the proximal colon. Dietary fat intake was assessed using the Block Brief Food Frequency Questionnaire, which estimates average dietary intake based on 70 food items. The diets of individuals with no endoscopically identifiable lesions (n = 36) were compared to those with either ACF or polyps detected in the proximal colon. RESULTS: In multivariate analysis, high dietary intake of total polyunsaturated fatty acids (PUFAs) and intake of omega-6 and omega-3 fatty acids were positively associated with neoplastic lesions in the proximal colon. When comparing ACF and polyp groups separately, a positive association was observed for both proximal polyps (OR 2.28; CI 1.16-7.09) and ACF (OR 2.86; CI 1.16-7.09) for total PUFA intake. Furthermore, the prevalence of proximal ACF was increased with higher intake of omega-6 (OR 3.54; CI 1.32-9.47) and omega-3 fatty acids (OR 2.29; CI 1.02-5.13), although there was no discernible difference in the omega-6/omega-3 ratio. CONCLUSIONS: These results suggest that dietary PUFAs may be positively associated with risk of early neoplasia in the proximal colon. This study provides further evidence that dietary PUFA composition may play an important role in altering the microenvironment within the human colon.

Neglecting human ecology: The common element of global health failures.

Attempts to control malaria, AIDS, and maternal mortality in Africa have been woefully inadequate. This has involved adopting an almost exclusively technical preventive approach in the context of AIDS even though emphasizing human behavior holds the most promise. But on the other hand, it has also involved abandoning highly effective technical measures, as in the case of malaria. This suggests that the failure, at root, is anthropological in nature. The common element, it is argued here, is the failure to place the human ecology resolutely above destructive ideologies. Sound public-health approaches have been spurned in favor of predetermined preventive approaches in the service of ideological aims rather than of man and the common good. This article examines the ideological forces that have ultimately driven global health policy, and proposes that a more humane anthropology would be beneficial. Lay Summary: The scourges of malaria, AIDS, and maternal mortality have persisted in Africa, even though sensible and available means of addressing these epidemics, when stressed, have met with success. The reluctance to consistently emphasize the soundest public-health approaches-whether technical or behavioral in nature-indicate that global health policy has to a large extent been improperly concerned with advancing ideological agendas. The challenge we face today is not primarily technical but philosophical; the healing professions would perform a service by cultivating a higher view of man and an appreciation for objective moral truths that protect him.

Osimertinib and Selpercatinib Efficacy, Safety, and Resistance in a Multicenter, Prospectively Treated Cohort of EGFR-Mutant and RET Fusion-Positive Lung Cancers.

PURPOSE: Acquired RET fusions have been reported at resistance to treatment with EGFR inhibitors in EGFR-mutant non-small cell lung cancer (NSCLC); however, a multicenter cohort of patients with EGFR-mutant lung cancers treated with osimertinib and selpercatinib for RET fusion-mediated osimertinib resistance has not previously been published. PATIENTS AND METHODS: Patients who received selpercatinib in combination with osimertinib on a prospective expanded access clinical trial (NCT03906331) and single-patient compassionate use programs across five countries were centrally analyzed. All patients had advanced EGFR-mutant NSCLC with a RET fusion detected from tissue or plasma following osimertinib therapy. Clinicopathologic and outcomes data were collected. RESULTS: Fourteen patients with EGFR-mutant and RET fusion-positive lung cancers who experienced prior progression on osimertinib received osimertinib and selpercatinib. EGFR exon 19 deletions (±T790M, 86%) and non-KIF5B fusions (CCDC6-RET 50%, NCOA4-RET 36%) predominated. Osimertinib 80 mg daily and selpercatinib 80 mg twice daily were the most commonly administered dosages. The response rate, disease control rate, and median treatment duration were 50% [95% confidence interval (CI), 25%-75%, n = 12], 83% (95% CI, 55%-95%), and 7.9 months (range, 0.8-25+), respectively. Resistance was complex, involving EGFR on-target (EGFR C797S), RET on-target (RET G810S), and off-target (EML4-ALK/STRN-ALK, KRAS G12S, BRAF V600E) mechanisms; RET fusion loss; or polyclonal mechanisms. CONCLUSIONS: For patients with EGFR-mutant NSCLC with an acquired RET fusion as a mechanism of EGFR inhibitor resistance, the addition of selpercatinib to osimertinib was feasible and safe and offered clinical benefit, supporting the prospective evaluation of this combination. See related commentary by Krebs and Popat, p. 2951.

Quantification of gadolinium in fresh skin and serum samples from patients with nephrogenic systemic fibrosis.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) is a rare, potentially fatal fibrosing disorder associated with renal insufficiency and gadolinium (Gd)-based contrast exposure. The cause remains unknown. To date, all efforts to investigate skin Gd concentrations in patients with NSF have been performed on paraffin-embedded samples, and Gd deposition has not been correlated with disease activity by a statistically significant analysis. OBJECTIVE: We sought to: (1) quantify Gd concentration in fresh tissue skin biopsy specimens; (2) quantify and compare synchronous Gd concentration of affected skin and unaffected skin in patients with NSF (n = 13) with a control group (n = 13); and (3) quantify serum Gd. METHODS: We used inductively coupled plasma mass spectrometry. RESULTS: In patients with NSF, the mean ratio of paired Gd concentrations of affected skin to unaffected skin was 23.1, ranging from 1.2 to 88.9. Mean serum Gd concentrations in patients with NSF were 4.8 ng/mL, which is more than 10 times the level in control patients. A statistically significant correlation existed between serum and affected skin Gd concentrations (r(2) = .74, P < .0001). LIMITATIONS: Because of the feasibility of this study, the main limitation was the small sample size (n = 13 affected and 13 control). CONCLUSIONS: Determination of Gd concentrations in fresh skin samples and serum using inductively coupled plasma mass spectrometry demonstrates significant differences in the amounts of Gd in involved versus nonlesional skin of patients with NSF. This supports the role of differential free Gd deposition from Gd-based contrast in the pathogenesis of NSF.

Reliable, Rapid, and Robust Speciation of Arsenic in Urine by IC-ICP-MS.

BACKGROUND: Arsenic is a naturally occurring element with varying species and levels of toxicity. Inorganic arsenic (e.g., arsenite (AsIII) and arsenate (AsV)) are toxic, while its metabolites (e.g., monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) are less toxic). Symptoms of exposure can include headaches, confusion, diarrhea, and drowsiness. As these symptoms overlap with many other conditions, arsenic exposure can often be overlooked as a cause. Arsenic toxicity may be treated with chelation and/or electrolyte replacement therapy. However, treatment is not without risks and is unnecessary for exposure to organic (nontoxic) forms of arsenic. This makes screening and differentiation of arsenic important for clinical testing. METHOD: An IC-ICP-MS method was developed using a Dionex 5000 with ion exchange chromatography for separation and iCAP Q for detection. Nontoxic species are arsenobetaine and arsenocholine, and toxic species are AsIII, DMA, MMA, and AsV. RESULTS: Precision, linearity, and specificity studies produced acceptable results. For accuracy, proficiency testing and method comparison samples were analyzed and produced acceptable results. Carryover studies demonstrated single species carryover from the diluter at levels of 500 µg/L, which can be avoided by analysis rules in the standard operating procedure. Limit of detection studies yielded a lower limit of quantitation of 1 µg/L per species. CONCLUSIONS: Here, we present a rapid and reliable method for quantifying and differentiating toxic and nontoxic forms of arsenic to allow for swift and appropriate management of patients with exposure.

Methyl Donor Deficiency Blocks Colorectal Cancer Development by Affecting Key Metabolic Pathways.

Our understanding of the role of folate one-carbon metabolism in colon carcinogenesis remains incomplete. Previous studies indicate that a methyl donor-deficient (MDD) diet lacking folic acid, choline, methionine, and vitamin B12 is associated with long-lasting changes to the intestinal epithelium and sustained tumor protection in Apc-mutant mice. However, the metabolic pathways by which the MDD diet affects these changes are unknown. Colon samples harvested from ApcΔ14/+ mice fed the MDD diet for 18 weeks were profiled using a GC-MS and LC-MS/MS metabolomics platform. Random forest and pathway analyses were used to identify altered metabolic pathways, and associated gene expression changes were analyzed by RT-PCR. Approximately 100 metabolites affected by the MDD diet were identified. As expected, metabolites within the methionine cycle, including methionine (-2.9-fold, P < 0.001) and betaine (-3.3-fold, P < 0.001), were reduced. Elevated homocysteine (110-fold, P < 0.001) was associated with increased flux through the transsulfuration pathway. Unexpectedly, levels of deoxycholic acid (-4.5-fold, P < 0.05) and several other secondary bile acids were reduced. There were also unexpected reductions in the levels of carnitine (-2.0-fold, P < 0.01) and a panel of acylcarnitines involved in fatty acid ß-oxidation. Finally, metabolites involved in redox balance, including ascorbate and hypotaurine, were found to be persistently elevated. These findings provide clues to the molecular changes underlying MDD-mediated tumor protection and identify regulatable metabolic pathways that may provide new targets for colon cancer prevention and treatment. IMPLICATIONS: Metabolomic profiling reveals molecular changes underlying MDD-induced tumor protection and may provide new targets for colorectal cancer prevention and treatment.

Clinically Meaningful Laboratory Protocols Reduce Hospital Charges Based on Institutional and ACS-NSQIP® Risk Calculators in Hepatopancreatobiliary Surgery.

Postoperative laboratory testing is an underrecognized but substantial contributor to health-care costs. We aimed to develop and validate a clinically meaningful laboratory (CML) protocol with individual risk stratification using generalizable and institution-specific predictive analytics to reduce laboratory testing and maximize cost savings for low-risk patients. An institutionally based risk model was developed for pancreaticoduodenectomy and hepatectomy, and an ACS-NSQIP®-based model was developed for distal pancreatectomy. Patients were stratified in each model to the CML by individual risk of major complications, readmission, or death. Clinical outcomes and estimated cost savings were compared with those of a historical cohort with standard of care. Over 34 months, 394 patients stratified to the CML for pancreaticoduodenectomy or hepatectomy saved an estimated $803,391 (44.4%). Over 13 months, 52 patients stratified to the CML for distal pancreatectomy saved an estimated $81,259 (30.5%). Clinical outcomes for 30-day major complications, readmission, and mortality were unchanged after implementation of either model. Predictive analytics can target low-risk patients to reduce laboratory testing and improve cost savings, regardless of whether an institutional or a generalized risk model is implemented. Broader application is important in patient-centered health care and should transition from predictive to prescriptive analytics to guide individual care in real time.

Dietary Methyl Donor Depletion Suppresses Intestinal Adenoma Development.

The role of folate one-carbon metabolism in colorectal cancer development is controversial, with nutritional intervention studies producing conflicting results. It has been reported that ApcMin/+ mice maintained on a diet deficient in the methyl donors folic acid, methionine, choline, and vitamin B12, and supplemented with homocysteine, show a greater than 95% reduction in intestinal tumor development. The present study extends these findings and shows that tumor protection afforded by dietary methyl donor deficiency (MDD) is long-lasting. After 11 weeks of MDD, tumor protection persisted for at least an additional 7 weeks of methyl donor repletion (22.2 ± 3.5 vs. 70.2 ± 4.6 tumors per mouse; P < 0.01). Sustained tumor protection was associated with a reduction in intestinal crypt length (26%, P < 0.01), crypt cell division and crypt fission, and an increase in apoptosis of both normal crypts and tumors (4.9- and 3.2-fold, respectively, P < 0.01). MDD also caused a significant reduction in the number of Dclk1-positive cells in the intestine (62%, P < 0.01), a long-lived crypt cell with cancer stem cell potential. Several undesirable effects associated with methyl donor restriction (e.g., reduced body weight gain) were shown to be transient and readily reversible following methyl donor repletion. Taken together, these results indicate that even temporary dietary methyl donor restriction in adenoma-prone mice can induce persistent changes to the intestinal epithelium and provide long-lasting tumor protection. These data also suggest that transient reductions in dietary methyl donor consumption should be considered when studying the impact of folate on colon cancer risk in humans. Cancer Prev Res; 9(10); 812-20. ©2016 AACR.

Late Amputation Following Refracture After Removal of Multiplanar Circular External Fixator for Treatment of Severe Combat-Related Tibial Fracture: A Case Report.

CASE: The multiplanar circular external fixator is commonly used in the treatment of severe combat-related tibial fractures. We present the case of a patient who sustained a refracture after removal of such a fixator. This complication contributed to failure of the limb salvage and ultimately resulted in the patient undergoing transtibial amputation. CONCLUSION: The choice to pursue limb salvage or amputation must be a shared decision between the patient and provider. This discussion must now include the possibility of refracture if limb salvage is pursued using multiplanar circular external fixation. Further study is also required to define fracture stability after the removal of a multiplanar circular external fixator.

One-Carbon Metabolism and Colorectal Cancer: Potential Mechanisms of Chemoprevention.

For many years folic acid has been evaluated for its utility as a chemopreventive agent due to its position at the center of the one-carbon metabolic network. This network is responsible for generating precursors to nucleotide synthesis as well as the one-carbon moieties used in DNA methylation reactions, two mechanisms which are frequently disrupted during carcinogenesis. While the use of folic acid for the chemoprevention of colorectal cancer is still controversial, there is evidence that folic acid intake has significant influence on these fundamental cellular mechanisms. Folic acid has a dual role with regards to nucleotide synthesis and colorectal cancer (CRC) prevention; in a healthy colon, adequate folate status is important for nucleotide metabolism homeostasis and the maintenance of DNA integrity, however in a colon harboring premalignant lesions lowered folate status may help to eliminate transformed cells. In addition, folic acid is important for the generation of the one-carbon groups used in DNA methylation reactions, and modulation of folic acid metabolism may be useful in combating the aberrant DNA methylation during carcinogenesis. Interestingly, it has been revealed that decreased folic acid intake can dampen the inflammatory response, which has recently been a popular strategy for colorectal cancer chemoprevention. In this review we discuss the molecular mechanisms influenced by folic acid intake and how they might be relevant to cancer chemoprevention in greater detail.

Heterotopic Ossification following Tissue Transfer for Combat-Casualty Complex Periarticular Injuries.

BACKGROUND: Although mechanisms underlying heterotopic ossification remain unknown, certain risk factors can influence heterotopic bone formation. The purpose of this study was to determine whether flaps used in periarticular reconstruction had any effect on heterotopic ossification formation. METHODS: A retrospective review of periarticular injuries requiring flap coverage from 2003 through 2014 was performed. Flap types, Injury Severity Scores, functional outcomes, and complications were reviewed. Radiology findings were assessed to determine heterotopic ossification rates and grades. RESULTS: Three hundred eighty-nine flaps were performed for traumatic extremity coverage over the 13-year study period. Sixty-nine of these flaps were used for periarticular coverage. The rate of periarticular heterotopic ossification was 47 percent for fasciocutaneous versus 54 percent for muscle-based flap coverage (p = 0.88). There were no significant differences in Injury Severity Score (p = 0.44) or overall heterotopic ossification formation (p = 0.97) between groups; however, the grade of heterotopic ossification within muscle-based flap cohort was significantly higher (1.70 for muscle versus 1.06 for the fasciocutaneous cohort; p = 0.002). CONCLUSIONS: Combat-related trauma is associated with high rates of heterotopic ossification, with an overall formation rate exceeding 85 percent for our patients requiring periarticular flap coverage. Although no difference in the rate of heterotopic ossification formation was found between fasciocutaneous and muscle flap coverages, a significantly increased severity of heterotopic ossification was seen in periarticular muscle-based flaps. These findings suggest that flap composition might not affect the rate of heterotopic ossification formation but may have an effect on ectopic bone formation severity.

Fecal electrolyte testing for evaluation of unexplained diarrhea: Validation of body fluid test accuracy in the absence of a reference method.

BACKGROUND: Validation of tests performed on body fluids other than blood or urine can be challenging due to the lack of a reference method to confirm accuracy. The aim of this study was to evaluate alternate assessments of accuracy that laboratories can rely on to validate body fluid tests in the absence of a reference method using the example of sodium (Na(+)), potassium (K(+)), and magnesium (Mg(2+)) testing in stool fluid. METHODS: Validations of fecal Na(+), K(+), and Mg(2+) were performed on the Roche cobas 6000 c501 (Roche Diagnostics) using residual stool specimens submitted for clinical testing. Spiked recovery, mixing studies, and serial dilutions were performed and % recovery of each analyte was calculated to assess accuracy. Results were confirmed by comparison to a reference method (ICP-OES, PerkinElmer). RESULTS: Mean recoveries for fecal electrolytes were Na(+) upon spiking=92%, mixing=104%, and dilution=105%; K(+) upon spiking=94%, mixing=96%, and dilution=100%; and Mg(2+) upon spiking=93%, mixing=98%, and dilution=100%. When autoanalyzer results were compared to reference ICP-OES results, Na(+) had a slope=0.94, intercept=4.1, and R(2)=0.99; K(+) had a slope=0.99, intercept=0.7, and R(2)=0.99; and Mg(2+) had a slope=0.91, intercept=-4.6, and R(2)=0.91. Calculated osmotic gap using both methods were highly correlated with slope=0.95, intercept=4.5, and R(2)=0.97. Acid pretreatment increased magnesium recovery from a subset of clinical specimens. CONCLUSIONS: A combination of mixing, spiking, and dilution recovery experiments are an acceptable surrogate for assessing accuracy in body fluid validations in the absence of a reference method.

Chelation of gadolinium with deferoxamine in a patient with nephrogenic systemic fibrosis.

A 65-year-old female with biopsy-confirmed nephrogenic systemic fibrosis (NSF) received a kidney transplantation. Despite good kidney function, her symptoms continued to progress. Deferoxamine was administered intramuscularly at 500 mg/day and later 1000 mg/day after 1 week with no adverse effects. Urine excretion of gadolinium increased from 6.0 µg/day to 11.6 µg/day and subsequently to 13.0 µg/day with 500 mg/day and 1000 mg/day of deferoxamine, respectively. Serum levels, however, remain unchanged from 1.7 ng/ml to 1.4 ng/ml. Although chelation therapy may have a role in the treatment of NSF, deferoxamine is too weak and a stronger chelator is needed.