Prediction of Isolated Local Recurrence After Resection of Pancreatic Ductal Adenocarcinoma: A Nationwide Study.

BACKGROUND: Distinguishing postoperative fibrosis from isolated local recurrence (ILR) after resection of pancreatic ductal adenocarcinoma (PDAC) is challenging. A prognostic model that helps to identify patients at risk of ILR can assist clinicians when evaluating patients' postoperative imaging. This nationwide study aimed to develop a clinically applicable prognostic model for ILR after PDAC resection. PATIENTS AND METHODS: An observational cohort study was performed, including all patients who underwent PDAC resection in the Netherlands (2014-2019; NCT04605237). On the basis of recurrence location (ILR, systemic, or both), multivariable cause-specific Cox-proportional hazard analysis was conducted to identify predictors for ILR and presented as hazard ratios (HRs) with 95% confidence intervals (CIs). A predictive model was developed using Akaike's Information Criterion, and bootstrapped discrimination and calibration indices were assessed. RESULTS: Among 1194/1693 patients (71%) with recurrence, 252 patients (21%) developed ILR. Independent predictors for ILR were resectability status (borderline versus resectable, HR 1.42; 95% CI 1.03-1.96; P = 0.03, and locally advanced versus resectable, HR 1.11; 95% CI 0.68-1.82; P = 0.66), tumor location (head versus body/tail, HR 1.50; 95% CI 1.00-2.25; P = 0.05), vascular resection (HR 1.86; 95% CI 1.41-2.45; P < 0.001), perineural invasion (HR 1.47; 95% CI 1.01-2.13; P = 0.02), number of positive lymph nodes (HR 1.04; 95% CI 1.01-1.08; P = 0.02), and resection margin status (R1 < 1 mm versus R0 ≥ 1 mm, HR 1.64; 95% CI 1.25-2.14; P < 0.001). Moderate performance (concordance index 0.66) with adequate calibration (slope 0.99) was achieved. CONCLUSIONS: This nationwide study identified factors predictive of ILR after PDAC resection. Our prognostic model, available through www.pancreascalculator.com , can be utilized to identify patients with a higher a priori risk of developing ILR, providing important information in patient evaluation and prognostication.

The suprachiasmatic nucleus is part of a neural feedback circuit adapting blood pressure response.

The suprachiasmatic nucleus (SCN) is typically considered our autonomous clock synchronizing behavior with physiological parameters such as blood pressure (BP), just transmitting time independent of physiology. Yet several studies show that the SCN is involved in the etiology of hypertension. Here, we demonstrate that the SCN is incorporated in a neuronal feedback circuit arising from the nucleus tractus solitarius (NTS), modulating cardiovascular reactivity. Tracer injections into the SCN of male Wistar rats revealed retrogradely filled neurons in the caudal NTS, where BP information is integrated. These NTS projections to the SCN were shown to be glutamatergic and to terminate in the ventrolateral part of the SCN where light information also enters. BP elevations not only induced increased neuronal activity as measured by c-Fos in the NTS but also in the SCN. Lesioning the caudal NTS prevented this activation. The increase of SCN neuronal activity by hypertensive stimuli suggested involvement of the SCN in counteracting BP elevations. Examining this possibility we observed that elevation of BP, induced by α1-agonist infusion, was more than twice the magnitude in SCN-lesioned animals as compared to in controls, indicating indeed an active involvement of the SCN in short-term BP regulation. We propose that the SCN receives BP information directly from the NTS enabling it to react to hemodynamic perturbations, suggesting the SCN to be part of a homeostatic circuit adapting BP response. We discuss how these findings could explain why lifestyle conditions violating signals of the biological clock may, in the long-term, result in cardiovascular disease.

A role for VGF in the hypothalamic arcuate and paraventricular nuclei in the control of energy homeostasis.

The arcuate nucleus is the main receptive area of the brain for peripheral and central metabolic cues and its integrity is essential for the maintenance of energy homeostasis. In the arcuate nucleus, different neuronal populations process metabolic signals and transmit this information to other nuclei of the hypothalamus by means of neurotransmitters and a combination of neuropeptides whose expression is modulated by the nutritional status. Here we investigated the changes in expression and synthesis of the polypeptide VGF in the arcuate nucleus of rats, in relation to the two main categories of neurons that show colocalization with VGF: the orexigenic NPY-expressing cells and the anorexigenic POMC-expressing cells. The results show that fasting is the most important stimulus for VGF expression, and that the up-regulation of VGF mRNA is restricted to the NPY area of the arcuate nucleus. POMC neurons express VGF under all feeding conditions, but especially in ad libitum-fed and fasted-refed animals. We also show that VGF arcuate neurons project to the pre-autonomic neurons of the paraventricular nucleus of the hypothalamus, providing anatomical evidence suggesting VGF as a central modulator of the autonomic nervous system.

The NPY intergeniculate leaflet projections to the suprachiasmatic nucleus transmit metabolic conditions.

The intergeniculate leaflet (IGL) is classically known as the area of the Thalamic Lateral Geniculate Complex providing the suprachiasmatic nucleus (SCN) non-photic information. In the present study we investigated whether this information might be related to the metabolic state of the animal. The following groups of male Wistar rats were used for analysis of neuropeptide Y (NPY) and c-Fos in the IGL and SCN. (1) Fed ad libitum. (2) Fasted for 48 h. (3) Fasted for 48 h followed by refeeding for 3 h. (4) Monosodium glutamate-lesioned and 48 h fasted. (5) Electrolytic lesion in the IGL and 48 h fasted. The results were quantified by optical densitometry. Neuronal tracers were injected in two brain areas that receive metabolic information from the periphery, the arcuate nucleus (ARC) and Nucleus of the Tractus Solitarius to investigate whether there is an anatomical relationship with the IGL. Lesion studies showed the IGL, and not the ARC, as origin of most NPY projections to the SCN. Fasting induced important changes in the NPY expression in the IGL, coinciding with similar changes of NPY/glutamate decarboxylase projections of the IGL to the SCN. These changes revealed that the IGL is involved in the transmission of metabolic information to the SCN. In fasted animals IGL lesion resulted in a significant increase of c-Fos in the SCN as compared to intact fasted animals demonstrating the inhibitory influence of the IGL to the SCN in fasting conditions. When the animal after fasting was refed, an increase of c-Fos in the SCN indicated a removal of this inhibitory input. Together these observations show that in addition to increased inhibitory IGL input during fasting, the negative metabolic condition also results in increased excitatory input to the SCN via other pathways. Consequently the present observations show that at least part of the non-photic input to the SCN, arising from the IGL contains information about metabolic conditions.

Pharmaceutical preparation of oxygen-15 labelled molecular oxygen and carbon monoxide gasses in a hospital setting.

BACKGROUND: Clinical positron emission tomography (PET) requires safe and effective PET radiopharmaceuticals. Tracers used for measuring oxygen consumption and blood volume are [(15)O]O(2) and [(15)O]CO, respectively. In general, these oxygen-15 labelled tracers are produced using a cyclotron that accelerates deuterons onto a target filled with (14)N(2) containing a trace of oxygen. In recent years, cyclotrons have been developed that only are capable of accelerating protons. The purpose of this study was to validate and assess such a cyclotron for production and administration of oxygen-15 labelled gasses in an hospital setting. METHODS: An RDS111 cyclotron (Siemens-CTI, Knoxville, USA) was validated for bolus production of [(15)O]O(2) and [(15)O]CO gasses. In addition, equipment was developed to administer these tracers to patients. RESULTS: Both [(15)O]O(2) and [(15)O]CO gasses could be produced in sufficient amounts, whilst meeting European Pharmacopeia requirements. Although produced oxygen-15 gasses contained a minor level of (11)C contamination, in clinical studies it was possible to correct for this contamination by delayed blood counting. CONCLUSION: An 11 MeV proton cyclotron combined with an in-house developed gas delivery system allows for the production and administration of sufficient amounts of [(15)O]-gasses for routine clinical PET studies in an hospital setting.

Estimation of uncertainty in long term combined sewer sediment behaviour predictions, a UK case study.

There are regulatory driven requirements for UK water companies to reduce the number of properties at risk of sewer flooding. One of the potential causes of sewer flooding is the presence of persistent sediment deposits in sewers. This is a common problem in many combined sewers. Although the regulation is risk based, there is a gap in current knowledge on how risk assessment is affected by the uncertainty in sewer solids behaviour prediction. This paper describes a UK case study exploring the possibility of estimating uncertainty in sewer sediment deposit level predictions, using Monte Carlo simulations combined with a response database.

Characterization of a single LSO crystal layer high resolution research tomograph.

The purpose of this study was to determine the performance of a single lutetium oxy-orthosilicate (LSO) crystal layer High Resolution Research Tomograph (HRRT) positron emission tomography (PET) scanner. The HRRT is a high resolution PET scanner designed for human brain and small animal imaging. The scanner consists of eight panel detectors, which have one layer of 2.1 x 2.1 x 7.5 mm thick LSO crystals. Several phantom studies were performed to determine scanner characteristics, such as resolution, scatter fraction, count rate and noise equivalent count rates (NECR). NECR curves were measured according to both NEMA NU2-1994 and NU2-2001 for three different energy windows, i.e. lower level discriminators (lld) of 350, 400 and 450 keV and an upper level discriminator (uld) of 650 keV. Accuracy of scatter and single photon attenuation corrections was evaluated according to NU2-1994. Data were acquired using a ring difference of 67 and a span of 9. Reconstructions were performed using FORE + 2D FBP or OSEM. Transaxial resolution varied from 2.7 to 2.9 mm FWHM between I and 10 cm off centre locations, and axial resolution varied from 3.2 to 4.4 mm FWHM. Scatter fractions (NU2-1994) equalled 0.31, 0.42 and 0.54 for lld of 450, 400 and 350 keV, respectively. NECR data were highest for an lid of 400 keV and showed a maximum of 46 kcps at 38 kBq cm(-3). Lower NECR values were observed according to NU2-2001, but were still optimal for an lld of 400 keV. After scatter and attenuation corrections, pixel values within water, air and teflon inserts of the NU2-1994 phantom were 14, 4 and 35% of the background activity, respectively. The single layer LSO HRRT scanner shows excellent spatial resolution, making it suitable for small animal studies. The low count rate performance, due to the small amount of LSO, prohibits studies of the human brain, but is sufficient for studies in small laboratory animals.

Use of an in-field-of-view shield to improve count rate performance of the single crystal layer high-resolution research tomograph PET scanner for small animal brain scans.

The count rate performance of the single LSO crystal layer high-resolution research tomograph (HRRT-S) PET scanner is limited by the processing speed of its electronics. Therefore, the feasibility of using an in-field-of-view (in-FOV) shield to improve the noise equivalent count rates (NECR) for small animal brain studies was investigated. The in-FOV shield consists of a lead tube of 12 cm length, 6 cm inner diameter and 9 mm wall thickness. It is large enough to shield the activity in the body of a rat or mouse. First, the effect of this shield on NECR was studied. Secondly, a number of experiments were performed to assess the effects of the shield on the accuracy of transmission scan data and, next, on reconstructed activity distribution in the brain. For activities below 150 MBq NECR improved only by 5-10%. For higher activities NECR maxima of 1.2E4 cps at 200 MBq and 2.2E4 cps at 370 MBq were found without and with shield, respectively. Listmode data taken without shield, however, were corrupted for activities above 75 MBq due to data overrun problems (time tag losses) of the electronics. When the shield was used data overrun was avoided up to activities of 150 MBq. For the unshielded part of the phantom, transmission scan data were the same with and without shield. The estimated scatter contribution was approximately 8.5% without and 5.5% with shield. Reconstructed emission data showed a difference up to 5% in the unshielded part of the phantom at 5 mm or more from the edge of the shielding. Of this 5% about 3% results from the difference in the uncorrected scatter contribution. In conclusion, an in-FOV shield can be used successfully in an HRRT PET scanner to improve NECR and accuracy of small animal brain studies. The latter is especially important when high activities are required for tracers with low brain uptake or when multiple animals are scanned simultaneously.

Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction.

Episialin (MUC1) is a mucin-like glycoprotein abundantly expressed on most carcinoma cells. As a result of its extended and rigid structure, it reduces intercellular adhesion. We investigated whether this antiadhesion function allows tumor cells expressing high levels of episialin to escape from immune recognition. To test this hypothesis, we transfected episialin-negative (episialin-) melanoma cells (A375) with the MUC1 cDNA-encoding episialin. The results demonstrated that episialin-positive (episialin+) melanoma cells were significantly less susceptible to lysis than episialin- melanoma cells by both alloantigen or rIL-2-stimulated cytotoxic effector cells. In addition, cold target inhibition experiments with episialin+ and episialin- cells clearly demonstrated preferential lysis of episialin- cells. Furthermore, antibody blocking studies showed that lysis of episialin+, but not of episialin-, melanoma cells was predominantly dependent on the leukocyte function-associated Ag-1/intracellular adhesion molecule adhesion route, suggesting that episialin+ target cells adhere less efficiently to effector cells than episialin- target cells. This notion was supported by the observation that conjugate formation of the effector cells with episialin+ target cells was significantly impaired. From these results we conclude that over-expression of episialin as found on many tumor cells may indeed affect efficient lysis by cytotoxic lymphocytes and thus may contribute to escape from immune surveillance.

Suppression of cellular aggregation by high levels of episialin.

Episialin is a mucin-like molecule located at the apical surface of most glandular epithelial cells. It is present at increased levels in carcinomas, where the molecule is often distributed over the entire cell surface. We have simulated this overproduction of episialin by transfecting a normal mammary epithelial cell line and a melanoma cell line with full-length complementary DNA encoding episialin. Transfectants of both cell lines containing episialin at levels similar to that of carcinoma cell lines do not aggregate as efficiently as their control cells, which do not express exogenous episialin. In mixing experiments, episialin transfectants are excluded from aggregates formed by these control cells, indicating that high levels of episialin on one of the interacting cells is sufficient to inhibit aggregation. The effect of episialin overexpression on aggregation is probably not only due to the negative charge of its numerous sialic acid residues, since neuraminidase treatment only partially restored the aggregation capacity of the transfectants. We propose that episialin, as a result of its large, extended, and rigid structure, can mask most cell surface molecules in its immediate surroundings and that a high density of episialin can severely disturb the interaction of cell surface proteins with macromolecules on adjacent cell membranes.

Cell-associated episialin is a complex containing two proteins derived from a common precursor.

cDNA for the epithelial sialomucin episialin encodes a transmembrane molecule with a large extracellular domain, which mainly consists of repeats of 20 amino acids. Here we confirm the existence of a previously proposed proteolytic cleavage of episialin that occurs in the endoplasmic reticulum (Hilkens, J., and Buijs, F. (1988) J. Biol. Chem. 263, 4215-4222) and show that a similar cleavage takes place in in vitro translation systems. Using in vitro translation of truncated mRNAs, we map the cleavage site to a region located between 71 and 53 amino acids upstream of the transmembrane domain. Analysis of a mutant, in which this region has been deleted, indicates that the cleavage sites used in vitro and in vivo are identical or in close proximity. Both cleavage products remain associated although they are not linked through disulfide bonds. Therefore, the subunit derived from the N terminus, which represents the actual mucin-like domain, remains indirectly anchored to the cell membrane as a result of its interaction with the C-terminal subunit.

Complexity of MAM-6, an epithelial sialomucin associated with carcinomas.

The complexity of epithelial sialomucins was investigated by immunoprecipitation and membrane immunofluorescence, using monoclonal antibodies (MAbs) against MAM-6 and other sialomucins. MAbs against MAM-6 immunoprecipitated from a variety of sources either one or two sialylated glycoproteins with apparent molecular weights of over 400,000 under reducing as well as nonreducing conditions. The electrophoretic mobility of each MAM-6 glycoprotein as isolated from serum, milk, and cell lines of different individuals showed considerable variation. The differences in molecular weight of the MAM-6 glycoproteins were also reflected at the level of MAM-6 precursors which are less heavily glycosylated. Therefore, large differences in apparent molecular weight (150,000 and over) are most likely due to a variable protein backbone. We used this molecular polymorphism to prove that 11 MAbs against different sialomucins, obtained from various investigators, precipitated sialomucins generated from common precursor molecules. The pattern of reactivity of the MAbs with carcinoma cell lines was complex. All but the two MAbs, directed against putative carbohydrate epitopes, immunoprecipitated the precursor molecule from each cell line. However, some of them were unable to immunoprecipitate the mature form of MAM-6 from these cell lines. These results indicate that those epitopes are masked, probably due to cell line- or possibly cell type-dependent variations in glycosylation of the epithelial sialomucin. Even within a single cell line mature molecules with different epitopes were observed. The differential reactivity of the MAbs was confirmed by membrane immunofluorescence. These results show that MAM-6 belongs to a family of epithelial sialomucins with a polymorphic protein backbone and extensive variation in glycosylation.

Biosynthesis of MAM-6, an epithelial sialomucin. Evidence for involvement of a rare proteolytic cleavage step in the endoplasmic reticulum.

MAM-6 is a major epithelial sialomucin which is abundantly present at the apical surface of ductal and alveolar cells of normal tissues and on many different carcinoma cells. MAM-6, as defined by monoclonal antibodies, consists of one or two sialylated glycoproteins with apparent molecular masses of over 400 kDa under reducing as well as nonreducing conditions. The mobility and number of glycoproteins immunoprecipitated vary depending on the cell line of origin. We have employed immunoprecipitation techniques to study the biosynthesis and glycosylation of this mucin. The biosynthesis of MAM-6 was studied in the ZR-75-1 breast cancer cell line. Two glycoproteins with apparent molecular masses of approximately 450 and 650 kDa, representing the mature form, were immunoprecipitated. By pulse-chase analysis, we show that the biosynthesis of the 450-kDa glycoprotein proceeded through intermediates of 220, 200, and 500 kDa which became perceptible after 1, 4, and 30 min, respectively. The biosynthesis of the 650-kDa glycoprotein followed a similar course through 380, 350, and 700-kDa intermediates. The processing of the 220- and 350-kDa precursors involves a rare proteolytic cleavage step which occurs in the endoplasmic reticulum. The late precursors of 500/700 kDa, observed after 30 min chase, and the mature glycoproteins were generated by extensive O-linked glycosylation. The formation of the 500/700-kDa precursors was not affected by monensin. However, the final step of maturation, sialylation of the 500/700-kDa precursors, could be inhibited by monensin. The extensive O-linked glycosylation causes the apparent high molecular weight as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data on the biosynthesis show that the early MAM-6 precursors contain N-linked glycans, suggesting the presence of N-linked glycans on the mature MAM-6 molecule. Although the copresence of N- and O-linked glycans has been found on many molecules, no N-linked glycans have been reported on mucus glycoproteins previously.

Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors.

Mouse monoclonal antibodies have been raised against human milk-fat globule membranes (HMFGM) to obtain reagents for mammary tumor diagnosis. A panel of 17 anti-HMFGM antibodies was selected for further investigation. Antibody-blocking studies indicated that with these antibodies at least nine different non-overlapping epitopes could be distinguished on six different molecules, MAM-1 to MAM-6. Electron microscopic studies of the cellular localization of the antigens detected by some of these antibodies revealed that they were present on the cell membrane mainly, on the microvilli, lining intercellular and intracytoplasmic lumina. The reactivity of the antibodies was studied on normal and tumor tissues and on in vitro cell lines. All antibodies reacted with the resting mammary gland while eight antibodies also bound to breast tumors. None of the antibodies was specific for the mammary gland or its tumors only, but most antibodies also reacted with other epithelial cells, especially of secretory tissues. When tested on a variety of cell lines a distribution reflecting the tissue distribution could be demonstrated. One of the antibodies reacted with nearly all carcinomas and their metastases and did not react with lymphomas, sarcomas, neuroblastomas, melanomas or nervous system tumors. The specificity of the antibodies, tested individually, was not sufficient for further differential diagnosis of the carcinomas, but when some of these antibodies were used in a panel they contribute to an important improvement of the diagnosis.

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16.

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.

Monoclonal antibodies against human acid alpha-glucosidase.

Acid alpha-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297-304). After fusion of spleen cells with myeloma cells, about 10% of the hybrid clones obtained produced antibodies against acid alpha-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid alpha-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major protein bands (mol. wt. 76000 and 70000,) a minor band of mol. wt. 9600 and several minor bands with a mol. wt. of 67000 or lower are seen. Since all these components react with the monoclonal antibodies, they must have at least one antigenic determinant in common.