Somatic mutations detected by mini- and microsatellite DNA markers reveal clonal intratumor heterogeneity in gastrointestinal cancers.

We investigated clonal intratumor heterogeneity by comparing different areas of each tumor in 20 gastrointestinal cancers from female patients (1 esophageal cancer, 5 stomach cancers, and 14 colorectal cancers). In all 19 cases informative for X-inactivation analysis with the M27 beta and/or the phosphoglycerate kinase probes, the tumors were clonal. Separate areas from a given tumor showed identical X-inactivation patterns, providing evidence for its single-cell origin. Of 20 cancers, 11 showed p53 gene mutations (base pair insertions, point mutations, and one case of a base pair deletion) in exons 5-8. A particular p53 gene mutation was identical in all tumor areas investigated per case. The minisatellite probes detected loss of heterozygosity or new mutant alleles at 1p33, 1q21, 5q35, 17p13, or 18q21. In seven cases mutations at particular loci were restricted to one or two areas per tumor, while in another seven cases they were common to all tumor areas. Loss of heterozygosity or new alleles detected at the microsatellite loci D2S123, D3S1611, D5S107, D17S261, or D18S34 [(CA)n repeats] were common to all tumor areas in 7 of 19 cases. In another seven cases, however, microsatellite mutations at these loci were restricted to one to three areas per tumor. Tracing clonal intratumor heterogeneity would permit one to study the hierarchy of mutational events in cancers where no premalignant lesions can be harvested. Most important, our study indicates that clonal intratumor heterogeneity might lead to sampling errors in the molecular diagnosis of cancer biopsy specimens when using mini- or microsatellite markers.

Development and validation of a new reversed-phase ion pairing liquid chromatographic method with fluorescence detection for penciclovir analysis in plasma and aqueous humor.

A simple, sensitive and reliable HPLC ion-pairing method with fluorescence detection, was developed for penciclovir determination in plasma and aqueous humor, with a Zorbax SB-aq C18 (100 mmx2.1 mm) column. Plasma samples were treated by solid-phase extraction with Oasis MCX (30 mg) cartridges. Ganciclovir, an antiviral drug structurally related to penciclovir, was used as internal standard (I.S.). Aqueous humor samples were directly injected into the chromatographic system. Separation was performed by a gradient elution with a mobile phase consisting of a mixture of acetonitrile and phosphate buffer 50mM containing 5mM of sodium octanesulfonate, pH 2.0, at a flow rate of 0.3 ml/min. The method was validated and showed good performances in terms of linearity, sensitivity, precision and trueness. Quantification limit was obtained at 0.05 microg/ml for aqueous humor and at 0.1 microg/ml for plasma. Finally, the proposed analytical method was used to measure penciclovir in clinical samples for a pharmacokinetic study, after oral administration of famciclovir.

Polyclonal and monoclonal thyroid nodules coexist within human multinodular goiters.

Although somatic mutations have been identified in a subset of thyroid nodules, the pathogenesis of nodules in multinodular goiters remains unclear. Clonal analysis indicates whether a nodule arises from the polyclonal proliferation of a group of cells or forms a clone from a genetically altered cell. Individual thyroid nodules have been shown to be of polyclonal or monoclonal origin. In this study we examined the clonality of several different nodules in patients with multinodular goiters. Clonality was established using the X-chromosomal probe M27 beta, which detects a multiallelic polymorphism at the locus DXS255 in 90% of females. Twenty-five nodules from 9 multinodular goiters were analyzed; 9 nodules were polyclonal, and 16 were monoclonal. Three goiters contained only polyclonal nodules, whereas 3 contained only monoclonal nodules. Polyclonal and monoclonal nodules coexisted in 3 goiters. In 2 goiters, the monoclonal nodules were shown to derive from different progenitor cells. We conclude that polyclonal and monoclonal nodules may coexist in multinodular goiters and that monoclonal nodules can originate from different cells. The coexistence of polyclonal and monoclonal nodules suggests that different pathogenic mechanisms occur simultaneously or that monoclonal nodules emerge secondarily from a polyclonal population due to a growth advantage from a genetically altered cell.

The degree of inhibition of thyroid follicular cell proliferation by iodide is a highly individual characteristic of each cell and differs profoundly in vitro and in vivo.

Pharmacological concentrations of iodide (> 1 x 10(-6) mol/l) are known to inhibit thyroid follicular cell growth in vitro. However, the inhibitory effect varies widely, depending on experimental conditions, and usually does not exceed 50%. We demonstrate that iodide (10(-4) mol/l) inhibits the growth of FRTL-5 cells in different passages by 11-67%. When five subclones of FRTL-5 cells were compared to the wild type, iodide-induced growth inhibition varied between 25% and 46%. The individual degree of inhibition of each clone was reproducible in two subsequent passages, suggesting that it is a stable constitutive trait. When FRTL-5 cells were grown first in three-dimensional clusters and then transplanted onto nude mice with high endogenous thyrotropin secretion, iodide at a serum concentration of less than 5.7 x 10(-7) mol/l nearly completely blocked cell replication in the transplants but not in the mice's own thyroid. Five cell lines, prepared from autonomously growing hyperthyroid feline multinodular goiters, were nearly completely resistant to the growth-inhibitory effect of iodide. These observations suggest that the sensitivity towards the growth-inhibiting effect of iodide is a highly variable, stable trait of each thyrocyte, even in cloned cell populations. Some FRTL-5 cells and, even more so, cells prepared from autonomously growing nodular feline goiters are resistant constitutively to the growth-inhibiting effect of iodide.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prevention of infection at the operative site: irrigation with iodine derivatives, or NaCl. A prospective and randomized study in general surgery]. / Prévention de l'infection de la plaie opératoire: lavage par dérivé iodé, ou NaCl. Etude prospective et randomisée en chirurgie générale.

A prospective randomized study was undertaken among 540 patients submitted to a surgical operation. The operative site and the wall before skin closure have been washed either with saline or with Betadine-R solution. Bacteriological samples were taken before irrigation. The contamination rate reached 60% in visceral surgery, 30% in bone surgery. Postoperative wound sepsis nearly reached 6%. There was no difference between the NaCl and Betadine groups.

[Prospective study of administering a single-dose prophylactic antibiotic in primarily indicated abdominal cesarean section]. / Prospektive Studie zur einzeitigen Antibiotikaprophylaxe bei primär indizierter abdominaler Schnittentbindung.

A prospective study was carried out with the participation of 60 pregnant women to elucidate the question if a unilateral antibiotic-prophylaxis is also indicated for a primary caesarean section. With the help of a random-list two collectives were formed with 30 pregnant women each. After delivery of the child one collective was given 4 g Mezlocillin (Baypen) i.v., the other collective did not receive an antibiotic. We analyzed the postoperative period of storage, postoperative fever, postoperative quality of healing, endometritis, urinary tract infection and the necessity of a postoperative antibiotic therapy. In addition we investigated swabs of the ear of the newborn, swabs from the cervix of the mother and blood culture of umbilical vein. Significantly less postoperative disorder of healing could be observed in the prophylaxis collective. All other investigated parameters did not show any significant differences in both collectives. Also there were no significant differences of infections in both collectives, therefore there is no indication for an antibiotic prophylaxis at a primary caesarean section.

[The effect of food intake on the gastric emptying of gastric juice-resistant tablets and capsules]. / Einfluss der Nahrungsaufnahme auf die Magenentleerung magensaftresistenter Tabletten und Kapseln.

To test the effect of food intake on gastric emptying of gastric juice-resistant drugs, emptying time of a 11 x 6 mm tablet and a 20 x 7 mm capsule was measured by means of a metal detector in 10 healthy persons (5 men and 5 women; mean age 25 [18-30] years) after fasting and after eating three main and three in-between meals. After fasting the tablets left the stomach after 78 +/- 18 min, the capsules after 60 +/- 16 min, while meal intake delayed emptying by a factor of 10 to 12 +/- 1.3 hours and 10 +/- 1.8 hours, respectively. The slightly shorter emptying time of capsules was statistically not significant. The results indicate that gastric juice-resistant tablets taken during day-time may, if several meals are eaten, accumulate in the stomach and then be emptied together at night. It is recommended that such drugs be taken in the fasting state in the morning and between meals, while avoiding in-between meals.

[Daily variations in pregnancy hormones]. / Tagesschwankungen von Schwangerschaftshormonen.

In a prospective study estriol, estradiol, progesterone, human placental lactogen (HPL) prolactin and SP1 (a placental glycoprotein specific for pregnancy), were performed in the second half of uncomplicated pregnancies in order to answer the question if there exist a circadian rhythm of these hormones in serum. All serum concentrations were measured by radioimmunoassay at 8.00, 10.00, 16.00, 20.00, 24.00 and 4.00 o'clock. 58 pregnant women with uncomplicated pregnancies show no circadian rhythm of serum levels from estriol, progesterone, HPL and SP1, while serum levels of estradiol and prolactin have a significant circadian rhythm. The estradiol serum levels are significant higher at 8.00 and 12.00 o'clock, significant less at 24.00 and 4.00 o'clock. The prolactin serum levels are significant less at 16.00 and 20.00, at 4.00 and 8.00 significant higher. In 20 pregnancies complicated by preeclampsia and 12 pregnancies complicated by intrauterine growth retardation serum levels of all hormones were measured at 8.00, 10.00, 16.00, 20.00, 24.00 and 4.00 o'clock. The serum concentrations in complicated pregnancies show no difference to the serum levels in uncomplicated pregnancies.

alpha-MSH receptor autoradiography on mouse and human melanoma tissue sections and biopsies.

MSH receptors and their binding characteristics of [125I]-labelled derivatives of alpha-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of alpha-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(125I)Tyr2]-alpha-MSH or [(125I)Tyr2,Nle4,D-Phe7]-alpha-MSH and specific alpha-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled alpha-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: KD = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of alpha-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.

Some fundamental aspects of morphometry in clinical pathology, demonstrated on a simple, multipurpose analysis system.

The aim of this study was (1) to investigate the value of morphometry, (2) to fix a set of parameters suitable for analyzing diagnostic problems, and (3) to create a general strategy for data storage and for user-friendly data management. The intrinsic value of morphometry lies in the fact that in contrast to other morphologic methods, it permits the presentation of findings in the form of numbers. The following set of morphometric parameters, in the broad sense of the term morphometry, is standard in our laboratory: planimetric parameters (shape descriptors), parameters of the gray value histogram (descriptors of the general gray value distribution), texture parameters (descriptors of the correlation between various image segments), invariant moments (descriptors of the size and localization of textural image segments) and densitometric parameters. The introduction of morphometric procedures into the daily routine is facilitated if data registration and evaluation are performed separately. Original data generated by direct measurement are primary or raw data, which are stored as such. In a separate, second step these raw data are used to compare more or less complex morphometric parameters, which are called "secondary data". A system designed for separate data registration and evaluation can easily be adapted to new methodologic developments. For instance, primary data on objects (gray values, coordinates of the contour) measured one time in the past can be reused at any other time for computing new features from these data. This procedure is comparable to the possibilities in immunohistochemical staining: new immunohistochemical stains can be applied to newly prepared sections of old tissue blocks.

Radioreceptor assay for alpha-MSH using mouse B16 melanoma cells+.

A radioreceptor assay for alpha-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-alpha-MSH tracer. The assay was used (1) to study the binding characteristics of alpha-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of alpha-MSH to B16 cells reached a stable plateau after 3 h at 15 degrees C. At 25 degrees or 37 degrees C, the binding was transient and at 0-1 degree C, the association was very slow. The hormone-receptor complex was relatively stable between 0 degrees and 15 degrees C whereas a 50% dissociation was reached after 90 min at 25 degrees C and after 35 min at 37 degrees C. The mean KD for alpha-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anterior lobe, alpha-MSH values are overestimated because of the large amount of ACTH present.

Growth regulation of cancer metastases by their host organ.

We analyzed mechanisms responsible for organ-specific metastasis by using two melanoma sublines derived from the same mouse tumor, of which one colonizes the lungs (F10) and the other colonizes the liver (L8) after intravenous injection. Both lines were obtained by selective growth in lung or liver after injection of tumor cells into a tail vein or portal vein. Contrary to common concepts, the cells of the liver-colonizing melanoma line do not accumulate preferentially in the liver after intravenous administration in vivo. However, the selective survival and proliferation of these melanoma cells in the target organ (liver) may be explained by the unexpected observation that they can be specifically stimulated to proliferate in the presence of hepatocytes, whereas the cells of the lung-colonizing line cannot. Growth promotion under coculture conditions in vitro was monitored both by thymidine incorporation into DNA and by increase in cell numbers. The proliferative stimulus is not mediated by an easily diffusible factor but rather depends upon direct contact between liver cells and those tumor cells that metastasize to that particular organ.

[Effect of various cytologic preparation techniques on form, texture and densitometric nuclear parameters of normal and dysplastic bladder urothelial cells]. / Einfluss verschiedener zytologischer Präparations-Methoden auf Form, Textur und densitometrische Kern-Parameter normaler und dysplastischer Zellen in Blasenspülflüssigkeiten.

An important prerequisite for densitometric and morphometric measurements on cytological specimens is the standardized cell preparation. Optimal results can only be achieved when the analyses are carried out on a regular monolayer of cell nuclei. Comparing the conventional cytological smear with a new centrifugation technique we demonstrate statistically significant differences of the two methods in texture, size and parameters describing the DNA content of normal and dysplastic urothelial cells.

[Drug-induced hepatic involvement, something to consider!]. / Atteintes hépatiques médicamenteuses, il faut y penser!

A retrospective study over the last 5 years on drug-induced liver injury was performed in the Internal Medicine Department of the University Hospital of Geneva. According to WHO and Council for International Organizations of Medical Sciences (CIOMS) criteria for drug-induced liver adverse events, 142 cases were identified, with 255 drugs fulfilling causality criteria of certain, probable or possible. 63 patients (44%) suffered severe consequences of liver injury, including 9 deaths. Drugs reported belonged to the therapeutic classes of antibiotics (43%), cardiovascular (20%), analgesics (10%), gastrointestinal (9%), psychotropic (7%), and others (11%). Despite bias inherent to the retrospective methodology, only 10% of the side effects were spontaneously reported to the pharmaco-vigilance board. This is in accordance with the known reporting rate. It can be concluded that spontaneous reports are to be regarded only as signals.

Contribution of cytochrome P-4502D6 phenotype to the neuromodulatory effects of dextromethorphan.

Dextromethorphan (DEM)-mediated N-methyl-D-aspartate receptor blockade may result from an action of unchanged DEM or its active metabolite, dextrorphan (DOR). In humans, DEM is metabolized into DOR by the polymorphic enzyme CYP2D6. We therefore investigated the impact of quinidine (Qd), a selective inhibitor of CYP2D6, on DEM disposition and the contribution of CYP2D6 phenotype on DEM antinociceptive and neuromodulatory effects. Using a randomized, double-blind, crossover, placebo-controlled design, healthy volunteers (n = 7) received Qd (50 mg Qd sulfate orally) or a placebo and, 12 h later, either DEM (50 mg DEM hydrobromide orally) or a placebo. DEM and DOR pharmacodynamics were assessed for their antinociceptive and neuromodulatory effects. Antinociceptive effects were assessed over 4 h by subjective pain threshold and RIII nociceptive reflex (RIII) monitoring. Neuromodulatory effects were studied using the primary and secondary hyperalgesia induced by the topical application of capsaicin. Two of seven subjects were genotypic CYP2D6 PM. Pretreatment of EM by Qd suppressed DOR formation and increased the plasma level of DEM to the levels of poor metabolizers. In poor metabolizers, DEM induced a significant increase in objective (+45%) and subjective (+35%) pain thresholds. In extensive metabolizers, only a slight and short-lasting increase in the subjective threshold was observed, whereas no effect was seen on the objective threshold. DEM modulates secondary hyperalgesia compared with DOR. The CYP2D6 phenotype affects the disposition of DEM and the production of the active metabolite DOR. The impact of the CYP2D6 phenotype is of major importance for the spinal antinociceptive and neuromodulatory effects of DEM.

[Factors associated with refractory cancer pain]. / Facteurs associés aux douleurs cancéreuses réfractaires.

Cancer pain can be effectively controlled in most patients by classical pharmacological treatment. We retrospectively studied the characteristics and factors associated with non responsive pain. Between 1989 and 1996, 1767 patients were referred to our pain center; 831 (47%) had cancer pain and from 787 evaluable cases 118 (15%) experienced non-controlled pain whereas good pain control was achieved within a few days in 669 (85%) patients. Gender, age, cancer type, metastasis, initial pain intensity, nociceptive or neuropathic components and administration of adjuvant therapies were similar in both groups. On the other hand, diffuse pain, abdominal pain, terminal care, near death and doses of strong opioids were significantly different. Factors associated with therapeutic failure were conflicts, life and complications and breakthrough pain. In the presence of refractory cancer pain the factors predictive of therapeutic failure should be identified in order to optimize individual pain treatment.

[Retrospective study of drug-induced agranulocytosis in hospitalized patients in Geneva and comparison with cases reported to IOCM]. / Etude rétrospective des cas d'agranulocytose médicamenteuse hospitalisés à Genève et comparaison avec les cas notifiés à l'OICM.

In a retrospective study, 19 cases classified as idiosyncratic drug-induced agranulocytosis were found among 162 files of patients hospitalized in internal medicine clinics of the university hospital where this diagnosis had been coded. This would give an estimated incidence of 2.6 cases per million inhabitants per year for the Geneva area. In most cases several drugs were implicated in causation of the episodes. Suspected drugs were those commonly reported in the literature, but also some drugs which might already have been taken to treat infectious complications of agranulocytosis. A comparison of the Geneva cases with those notified to the Swiss Intercantonal Office for the Control of Medicines reveals a similar profile of involved drugs.