Circulating tumor DNA as a potential marker of adjuvant chemotherapy benefit following surgery for localized pancreatic cancer.

BACKGROUND: In early-stage pancreatic cancer, there are currently no biomarkers to guide selection of therapeutic options. This prospective biomarker trial evaluated the feasibility and potential clinical utility of circulating tumor DNA (ctDNA) analysis to inform adjuvant therapy decision making. MATERIALS AND METHODS: Patients considered by the multidisciplinary team to have resectable pancreatic adenocarcinoma were enrolled. Pre- and post-operative samples for ctDNA analysis were collected. PCR-based-SafeSeqS assays were used to identify mutations at codon 12, 13 and 61 of KRAS in the primary pancreatic tumor and to detect ctDNA. Results of ctDNA analysis were correlated with CA19-9, recurrence-free and overall survival (OS). Patient management was per standard of care, blinded to ctDNA data. RESULTS: Of 112 patients consented pre-operatively, 81 (72%) underwent resection. KRAS mutations were identified in 91% (38/42) of available tumor samples. Of available plasma samples (N = 42), KRAS mutated ctDNA was detected in 62% (23/37) pre-operative and 37% (13/35) post-operative cases. At a median follow-up of 38.4 months, ctDNA detection in the pre-operative setting was associated with inferior recurrence-free survival (RFS) [hazard ratio (HR) 4.1; P = 0.002)] and OS (HR 4.1; P = 0.015). Detectable ctDNA following curative intent resection was associated with inferior RFS (HR 5.4; P < 0.0001) and OS (HR 4.0; P = 0.003). Recurrence occurred in 13/13 (100%) patients with detectable ctDNA post-operatively, including in seven that received gemcitabine-based adjuvant chemotherapy. CONCLUSION: ctDNA studies in localized pancreatic cancer are challenging, with a substantial number of patients not able to undergo resection, not having sufficient tumor tissue for analysis or not completing per protocol sample collection. ctDNA analysis, pre- and/or post-surgery, is a promising prognostic marker. Studies of ctDNA guided therapy are justified, including of treatment intensification strategies for patients with detectable ctDNA post-operatively who appear at very high risk of recurrence despite gemcitabine-based adjuvant therapy.

Serous cystic neoplasm of the pancreas: a multinational study of 2622 patients under the auspices of the International Association of Pancreatology and European Pancreatic Club (European Study Group on Cystic Tumors of the Pancreas).

OBJECTIVES: Serous cystic neoplasm (SCN) is a cystic neoplasm of the pancreas whose natural history is poorly known. The purpose of the study was to attempt to describe the natural history of SCN, including the specific mortality. DESIGN: Retrospective multinational study including SCN diagnosed between 1990 and 2014. RESULTS: 2622 patients were included. Seventy-four per cent were women, and median age at diagnosis was 58 years (16-99). Patients presented with non-specific abdominal pain (27%), pancreaticobiliary symptoms (9%), diabetes mellitus (5%), other symptoms (4%) and/or were asymptomatic (61%). Fifty-two per cent of patients were operated on during the first year after diagnosis (median size: 40 mm (2-200)), 9% had resection beyond 1 year of follow-up (3 years (1-20), size at diagnosis: 25 mm (4-140)) and 39% had no surgery (3.6 years (1-23), 25.5 mm (1-200)). Surgical indications were (not exclusive) uncertain diagnosis (60%), symptoms (23%), size increase (12%), large size (6%) and adjacent organ compression (5%). In patients followed beyond 1 year (n=1271), size increased in 37% (growth rate: 4 mm/year), was stable in 57% and decreased in 6%. Three serous cystadenocarcinomas were recorded. Postoperative mortality was 0.6% (n=10), and SCN's related mortality was 0.1% (n=1). CONCLUSIONS: After a 3-year follow-up, clinical relevant symptoms occurred in a very small proportion of patients and size slowly increased in less than half. Surgical treatment should be proposed only for diagnosis remaining uncertain after complete workup, significant and related symptoms or exceptionally when exists concern with malignancy. This study supports an initial conservative management in the majority of patients with SCN. TRIAL REGISTRATION NUMBER: IRB 00006477.

Mechanical properties of soft human tissues under dynamic loading.

The dynamic response of soft human tissues in hydrostatic compression and simple shear is studied using the Kolsky bar technique. We have made modifications to the technique that allow loading of a soft tissue specimen in hydrostatic compression or simple shear. The dynamic response of human tissues (from stomach, heart, liver, and lung of cadavers) is obtained, and analyzed to provide measures of dynamic bulk modulus and shear response for each tissue type. The dynamic bulk response of these tissues is easily described by a linear fit for the bulk modulus in this pressure range, whereas the dynamic shearing response of these tissues is strongly non-linear, showing a near exponential growth of the shear stress.

Smear layer and surface state affect dentin fluoride uptake.

OBJECTIVES: Polishing generates a smear layer (SL) on in vitro dentin samples that may influence fluoride uptake. We tested two hypotheses: SL increases fluoride uptake in superficial dentin (H1) and decreases fluoride uptake in deeper layers (H2) irrespectively of the amount of fluoride administered. METHODS: Polished bovine dentin with SL present and removed by four methods (5% tannic acid, 20s [TA]; 17% EDTA, 120 s; 38% phosphoric acid, 60s [PA]; and 10s air polishing) was fluoridated with 1200 or 12000 ppm F (NaF) solution (pH 4.0). RESULTS: Scanning electron microscopy (SEM) revealed that aggressiveness of SL removal varied by method from leaving SL patches behind (TA) to collagen exposure (PA). SL increased KOH-soluble and structurally bound fluoride uptake into superficial and deeper layers compared to SL free surfaces (except PA) following 1200 ppm, but not 12000 ppm fluoridation. CONCLUSION: Presence of SL and surface conditions influence dentin fluoride uptake depending on fluoride concentration administered.

Effects of enhancement procedures on whole and re-formed beef forequarter muscles.

Beef Supraspinatus and Triceps brachii muscles were subjected to three enhancement and/or re-forming treatments: (i) injected whole @ 15%w/w with salt-phosphate solution; (ii) injected and re-formed; (iii) injected with added flavouring and re-formed. The treated muscles were compared to whole uninjected controls. All injection treatments reduced shear force values of cooked samples and in most cases these reductions were reflected in sensory panel tenderness and chewiness ratings. For example, shear values for Supraspinatus were 83N/g in control samples and 50 in whole injected samples, while corresponding sensory panel tenderness ratings were 3.6 and 5.2. Enhanced samples did not differ from controls in sliceability or in colour and binding ratings, indicating that enhancement combined with re-forming can give an acceptable roast beef product. There were no differences in drip loss and very few differences in colour L*, a* and b* values for raw samples between any of the treatments. Addition of beef stock did not result in higher flavour ratings by sensory panels. Whole injected samples scored higher for flavour than both control (p<0.01) and injected+re-formed (p<0.05) samples.

Developmental Regulation of Respiratory Activity in Pea Leaves.

The developmental pattern of mitochondrial respiratory activity in pea (Pisum sativum) leaves has been investigated in an attempt to determine changes in mitochondrial function as plant cells mature. NADH and succinate dehydrogenase and cytochrome c oxidase activities remained relatively constant during cell maturation (from d 0 to d 14). Alternative oxidase and glycine decarboxylase activity, however, were low in young leaf tissue (d 0-6) but increased substantially as the tissue matured (d 7-14) and gained photorespiratory activity. Western blot analysis of the alternative oxidase protein revealed that it was primarily in an oxidized state in young leaves (d 0-6) but switched dramatically to the reduced form of the protein as the pea cells matured (d 7-14). The switch to the reduced form of the protein correlated with an increase in alternative oxidase activity. Results are discussed in terms of the changing function of plant mitochondria during leaf development.

The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria.

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.

Electron Partitioning between the Cytochrome and Alternative Pathways in Plant Mitochondria.

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.

Characterization of a cytosolic triiodothyronine binding protein in atrium and ventricle of rat heart with different sensitivity toward thyroid hormone levels.

Cytosolic T3-binding protein (CTBP) has been identified in both the ventricle and atrium of adult rat hearts. Its biochemical characteristics and concentration have been determined in the two tissues as a function of thyroid hormone level. In both tissues association and dissociation constants were, respectively, k+1 = 1.3 x 10(8) M-1/min and k-1 = 0.025 min-1. Scatchard analysis of T3 equilibrium binding data revealed a single class of binding sites (Ka = 3.8 x 10(8) M-1). The maximal binding capacity (MBC) was 1400 fmol/mg protein in the ventricle and 730 fmol/mg protein in the atrium. The apparent mol wt of CTBP, determined by gel filtration, was 63.000. Among the thyroid hormone analogs tested in ventricular cytosol, D-T3 had the highest affinity, followed by L-T3, L-T4, 3,3',5-triiodothyroacetic acid, and rT3. These characteristics were very similar to those previously described for rat brain, and dog and rat liver and kidney CTBP. In hypothyroid rats MBC was only increased in the atrium (50-100%); after a single injection of T4 (2 micrograms/10 g BW 3 or 18 h before death) values returned to normal in the atrium and declined in the ventricle (-35%). During postnatal development, the highest MBC value (2000 fmol T3/mg protein) was observed in atria on day 10, i.e. when the serum T4 level was still low, and in the ventricle on day 30 (4000 fmol T3/mg protein) when the serum T4 level was at its highest. Binding affinities were similar in the two tissues at all ages studied. It was twice as high in both these tissues during the first week of development than in adulthood. These results favor a thyroid hormone down-regulation of the binding capacity of CTBP that would be more sensitive to the hormone in the atrium than in the ventricle.

Induction of type III deiodinase activity in astroglial cells by thyroid hormones.

The type III deiodinase (D-III) activity in astroglial cells is induced by multiple pathways activated by cAMP, 12-O-tetradecanoylphorbol-13-acetate (TPA), and fibroblast growth factors (FGFs). This study examines the effects of thyroid hormones on D-III activity in astroglial cells with or without induction by these factors. Addition of 10 nM T3 to the culture medium caused a slow increase in D-III activity, which reached a plateau after 48 h. This increase was concentration dependent (maximal response at 10 nM). Doses as low as 0.3 nM caused significant increases in D-III activity. The effect of T3 was reversible. A dose of 10 nM L-T3, D-T3, T4, 3,5,3'-triiodothyroacetic, or 3'-isopropyl-3,5-diiodothyronine produced 5- to 15-fold increases in D-III activity after 48 h. In contrast, 10 nM L-thyronine, 3-monoiodothyronine, 3,3'-diiodothyronine, 3,5-diiodothyronine, and rT3 were without effect. A dose of 10 nM T3 or T4 amplified the D-III activity stimulated by 0.1 microM TPA, 20 ng/ml acidic FGF, or 1 mM 8-bromo-cAMP 3- to 8-fold. Otherwise, T3 rapidly inhibited D-II activity. This inhibition was concentration dependent, with a half-maximal effect around 10 nM. In conclusion, thyroid hormones induce D-III activity and potentiate the D-III activity induced by cAMP, TPA, and FGFs in astroglial cells. These reversible effects together with inhibition of D-II activity may contribute to protect the brain against hyperthyroidism.

Sulfation after deiodination of 3,5,3'-triiodothyronine in rat cultured astrocytes.

The metabolism of [125I]T3 by rat astrocytes in culture was analyzed by Sephadex LH-20 chromatography and HPLC. The conjugates isolated on LH-20 were not hydrolyzed by glucuronidase, indicating the absence of glucuroconjugates. 3,3'-Diiodothyronine (3,3'T2) sulfate (3,3'T2-S) was the main product that accumulated in the medium over the T3 concentration explored (10 pM to 10 nM). The identity of the peak eluted as 3,3'T2-S was ascertained by its hydrolysis with sulfatase and the generation of 3,3'T2 identified by HPLC. 3'-Monoiodothyronine sulfate was also found in cells treated with 1 microM retinoic acid, i.e. with high type III deiodinase activity. No T3 sulfate (T3-S) was found as a metabolite of T3. Astrocytes did not break down 1 nM [125I]T3-S added to the medium. Astrocytes pretreated for 3 days with 10 nM T3 showed increased production of 3,3'T2-S from 10 nM [125I]T3. Exogenous [125I]3,3'T2 (20 nM) was conjugated to 3,3'T2-S released into the medium. Pretreatment of astrocytes with 10 nM T3 did not alter the production of 3,3'T2-S from 3,3'T2. Thus, T3 is metabolized in astrocytes by direct 5-deiodination, followed by sulfation. Whereas T3 induces its own deiodination and type III deiodinase activity, T3 does not regulate the sulfation of its main metabolite, 3,3'T2. This demonstration of sulfation of iodothyronines in cells originating from the brain raises the question of the role of this TH metabolic pathway in the brain.

Regulation of type 3 iodothyronine deiodinase expression in cultured rat astrocytes: role of the Erk cascade.

The type 3 iodothyronine deiodinase (D3) metabolizes thyroid hormones to inactive metabolites in many tissues, including the brain. In the present studies, we have examined the mechanisms by which T3 (T3), retinoic acid, 12-O-tetradecanoyl phorbol 13-acetate (TPA), and basic fibroblast growth factor (bFGF) induce D3 expression in primary cultures of neonatal rat astrocytes. In untreated cells, D3 messenger RNA (mRNA) was essentially undetectable by Northern analysis and RT-PCR. However, all four agents induced expression of a 2.4-kb D3 transcript as well as D3 activity. Induction of D3 by TPA and bFGF was more rapid than that by T3 and retinoic acid, and T3 potentiated the stimulatory effects of TPA and bFGF. D3 induction by TPA was blocked by GF 109203X, an inhibitor of protein kinase C. In addition, the effects of TPA and bFGF were partially prevented by PD 98059, a specific inhibitor of MEK and the Erk signaling cascade. These studies demonstrate that multiple growth factors and hormones regulate D3 activity in cultured astrocytes by inducing D3 mRNA expression. In addition, the stimulatory effects of TPA and bFGF on D3 mRNA and activity appear to be mediated at least in part by activation of the MEK/Erk signaling cascade.

Cytosolic thyroxine-binding protein and brain development.

The properties of cytosolic thyroxine binding protein were studied in the cortex and cerebellum of the rat at different stages of postnatal development: (1) Polyacrylamide-gel electrophoretic analysis showed that rat-brain cortex and cerebellum contain the same cytosolic thyroxine-binding protein which is very similar to the liver-corresponding entity. No changes in the electrophoretic mobility were seen during development in the 2 brain regions. In contrast, no defined triiodothyronine-binding component could be observed by the same technique. (2) Kinetic analysis studies revealed that the equilibrium of binding is reached in approximately 10 min whatever the brain region, the concentration of cytosolic protein and the stage of development. In all these cases saturation was obtained with the same thyroxine concentration (approximately 5 x 10(-7) M). Scatchard analysis also showed that whatever the experimental conditions, brain cytosolic protein contains a single class of thyroxine-binding sites with a K A of approximately 8 x 10(7) M-1. (3) Comparison of the K A during development showed that this constant remains unchanged from day 3 after birth until day 35 in both the cortex and the cerebellum. In contrast the number of binding sites significantly decreases in the cortex (approximately 2-fold; p less than 0.001) from day 3 to 35 with an already significant decline from day 3 to 6 (p less than 0.001). In the cerebellum this decline was even more marked since almost no binding activity was left at adulthood. Comparison of cortex and cerebellum binding activities also showed that this latter region contains approximately half the binding sites (p less than 0.001) at every stage of development studied.

Cellular location of cytosolic triiodothyronine binding protein in primary cultures of fetal rat brain.

The evolution of a cytosolic triiodothyronine (T3) binding protein was studied in primary cultures of fetal rat brain. These cultures exhibited neuronal characteristics during the first week. T3 binding activity in cell supernatants increased during this period from 39 +/- 7 (mean +/- SD) to 159 +/- 24 fmoles T3/culture flask. A similar increase was observed in the soluble proteins. After day 8, neuronal death occurred and glial cells multiplied and differentiated. On day 11 an 86% drop in the binding activity was observed (24 +/- 7 fmoles T3/culture flask); the pool of soluble proteins remained stable. Scatchard analysis revealed two types of binding site in both 7- and 14-day cultured cell cytosols. Binding affinities were similar in both cytosols (KA1 approximately 1.5 X 10(9) M-1, KA2 approximately 1 X 10(8) M-1); in contrast, the number of sites was 4-fold smaller in 14-day cytosols. In subcultures mostly composed of glial cells, almost the same affinities were measured, but the numbers of both types of sites were 20 times smaller than in 7-day cells. These results show that in cell cultures from embryonic rat telencephalon, cytosolic T3 binding protein is mainly located in the neurons.

Peritoneal involvement in stage II colon cancer.

A pathologist (K.S.) reviewed histologic slides for peritoneal involvement by tumor cells for 118 patients with stage II colon cancer. Patients were followed up for a median of 6 years. Tumor cells were found free in the peritoneal space in 16 cases (13.6%). The presence of cancer cells free in the peritoneal space was associated with lymphovascular invasion (P = .001) and neural invasion (P < .001). The overall 5-year survival was 80% in the patient population, but was 39% and 86% for those with and without tumor cells free in the peritoneal space, respectively (P < .0001). Multivariate analysis confirmed that free tumor cells within the peritoneal space (P < .0001) and lymphovascular invasion (P = .007) were related independently to outcome. Peritoneal involvement with tumor cells free in the peritoneal space in stage II colon cancer is a powerful indicator of outcome; patients have a survival similar to that for patients with stage III disease.

Effect of mineral supplements to citric acid on enamel erosion.

The aim of this study was to evaluate the effect of mineral supplements to citric acid (1%; pH 2.21) on enamel erosion under controlled conditions in an artificial mouth. From each of 156 bovine incisors one polished enamel sample was prepared. The samples were divided among 13 experimental groups (n=12). In group 1 citric acid only was used (control). In groups 2-10 either calcium, phosphate or fluoride in various low concentrations was admixed to the citric acid. In groups 11-13 the citric acid was supplemented with a mixture of calcium, phosphate and fluoride. For demineralisation the specimens were rinsed with the respective solution for 1 min, immediately followed by a remineralisation period with artificial saliva (1 min). The specimens were cycled through this alternating procedure five times followed by rinsing for 8 h with artificial saliva. The de- and remineralisation cycle was repeated three times for each specimen interrupted by the 8 h-remineralisation periods. Before and after the experiments, the specimens were examined using microhardness testing (Knoop hardness) and laser profilometry. Hardness loss and enamel dissolution was significantly higher for the controls as compared to the remaining groups. Significantly lowest hardness loss for all groups was recorded for group 12 with admixture of calcium, phosphate and fluoride to citric acid. The significantly highest enamel loss was recorded for the controls compared to all other samples. Groups 3 and 4 revealed significantly lower and higher tissue loss compared to the remaining groups (2-13), respectively. The other groups did not differ significantly from each other. Modification of citric acid with calcium, phosphate and fluoride exerts a significant protective potential with respect to dental erosion. However, with the low concentrations applied enamel dissolution could not be completely prevented.