Niklas Zojer

Chromosomal imbalances in primary and metastatic pancreatic carcinoma as detected by interphase cytogenetics : basic findings and clinical aspects

To date, cytogenetic studies on pancreatic carcinoma are rare, and little is known about the frequency of cytogenetic aberrations in primary carcinomas compared with metastatic tumour cells. We therefore evaluated the frequency of chromosomal aberrations in 12 primary pancreatic carcinomas and in effusion specimens from 25 patients with pancreatic cancer by using interphase fluorescence in situ hybridization (FISH) and a panel of four centromeric probes. Hyperdiploidy and chromosomal imbalances, predominantly affecting chromosome 8, were a constant finding in metastatic effusion cells, whereas concordant gain of chromosomes or relative loss of chromosome 18 characterized primary pancreatic carcinomas. The potential role of oncogenes located on chromosome 8 for pancreatic cancer progression was further investigated by double-hybridization studies of aneuploid effusion cells with a probe to 8q24 (MYC) and a centromeric probe to chromosome 8, which demonstrated amplification of the MYC oncogene in two of ten cases (20%). Finally, a potential application of basic findings in the clinical setting was tested by searching for micrometastatic cells in effusions from pancreatic cancer patients primarily negative by FISH. Two-colour FISH in combination with extensive screening (>10,000 nuclei) seems to be a useful tool to unequivocally identify micrometastatic cells by demonstrating hyperdiploidy and intranuclear chromosomal heterogeneity.

Interphase fluorescence in situ hybridization improves the detection of malignant cells in effusions from breast cancer patients.

In diagnostic evaluation of effusions, difficulties are encountered when atypical reactive mesothelial cells have to be differentiated from malignant cells. We tested the impact of fluorescence in situ hybridization (FISH) to identify metastatic cells in breast cancer effusions by detection of numerical chromosomal changes. Pleural and ascitic fluid samples (n=57) from 41 breast cancer patients were concomitantly evaluated by routine cytology and FISH, using centromere-specific probes representing chromosomes 7, 11, 12, 17 and 18. After setting stringent cut-off levels deduced from non-malignant control effusions (n=9), the rates of cells with true aneuploidy were determined in each effusion sample from breast cancer patients. The occurrence of aneuploid cells, as detected by FISH and indicative of malignancy, was correlated with the cytological findings. Routine cytology revealed malignancy in 60% of effusions. Using FISH, aneuploid cell populations could be observed in 94% of cytologically positive and in 48% of cytologically negative effusions, thus reverting diagnosis to malignancy. To confirm malignancy in cases with a low frequency of aneuploid cells, two-colour FISH was additionally performed and indeed showed heterogeneous chromosomal aneuploidy within single nuclei. We conclude that FISH is a valuable tool in the diagnosis of malignancy and may serve as an adjunct to routine cytological examination, as demonstrated here for breast cancer effusions.

Frequency-dependent effects of amitriptyline and maprotiline on conduction in the guinea pig His-Purkinje-system in vivo

In the Cardiac Arrhythmia Suppression Trial antiarrhythmic drug therapy with slow kinetic sodium channel blockers (class Ic antiarrhythmic drugs) was associated with excess mortality, presumably due to drug induced proarrhythmia. It has been suggested that the degree of rate-dependent conduction slowing produced by agents that have sodium channel blocking properties may be related to the proarrhythmic propensity of these agents.In the present study, rate-dependent conduction slowing by the antidepressants amitriptyline and maprotiline was investigated in anesthetized guinea pigs. After electrical ablation of the sinus node the left atrium was stimulated at cycle lengths between 200 ms and 500 ms. His bundle electrograms were registered by means of an epicardial electrode. Drugs were administered by i.v. infusion of 0.2 mg kg−1 min−1 for 30 min followed by 0.1 mg kg−1 min−1 for up to 30 min. Both drugs produced substantial rate-dependent conduction slowing within the His-Purkinje-system. The relationship between pacing rate and conduction slowing was well fitted by linear regression. The steepness of the regression line was significantly greater for amitriptyline than for maprotiline (slope factors: 9.10×10−4±7.85 × 10−5, n = 6, vs. 6.29×10−4±2.97×10−5, n=6, P<0.001), indicating that conduction slowing by amitriptyline exhibits a greater degree of rate-dependence than conduction slowing by maprotiline. On abruptly changing the driving cycle length from 500 ms to 300 ms, conduction slowing reached a new steady state with rate constants of 0.83±0.093 beat−1 (amitriptyline) and 0.14±0.05 beat−1 (maprotiline, P<0.001). Following interruption of rapid pacing at a cycle length of 250 ms, conduction slowing recovered with time constants of 332.4±52.8 ms (amitriptyline) and 1088.1 ± 143.5 ms (maprotiline, P = 0.001). Thus, amitriptyline exhibited fast kinetic properties similar to class Ib antiarrhythmic action while the slower kinetic properties of maprotiline resembled those of class Ia agents.

Prolongation of the QT interval by dofetilide modulates rate-dependent effects of mexiletine on intraventricular conduction

Prolongation of action potential duration during treatment with agents that possess class I antiarrhythmic activity may result in a clinically relevant increase in Na+ channel block. In order to test this hypothesis in vivo, the effect of QT prolongation on intraventricular conduction was assessed during administration of mexiletine. Epicardial His bundle recordings were made in anesthetized guinea pigs. After abolition of spontaneous sinus node activity by application of high-frequency current to the sinus node area, the hearts were paced via the left atrium. Administration of the class III antiarrhythmic agent dofetilide (10 micrograms/kg i.v.; n = 6) significantly prolonged QT intervals without a significant effect on HV intervals. Infusion of mexiletine (bolus 2 mg/kg + 0.18 mg/kg per min i.v.; n = 6) produced significant increases in HV intervals at cycle lengths of 200 and 300 ms. Subsequent addition of dofetilide (20 micrograms/kg i.v.) to mexiletine induced similar increases in QT intervals as single treatment with 10 micrograms/kg dofetilide and significantly enhanced the rate-dependent conduction slowing. Upon abruptly decreasing the pacing cycle length from 500 ms to 300 ms, conduction slowing developed with a rate constant of 1.0 +/- 0.2 beat-1 after mexiletine and with a rate constant of 1.1 +/- 0.2 beat-1 after subsequent addition of dofetilide (P = n.s.). After rapid stimulation at a cycle length of 250 ms the conduction slowing produced by mexiletine recovered with a time constant of 174 +/- 24 ms. No further change of this recovery time constant was observed after subsequent addition of dofetilide to mexiletine (160 +/- 19 ms, P = n.s.). Thus action potential duration, as reflected by the QT interval, is an important modulator of the magnitude Na+ channel block in vivo. The kinetic parameters of Na+ channel block produced by mexiletine, however, remain unchanged by prolongation of action potential duration after addition of dofetilide.

Effect of ajmaline on sustained ventricular tachycardia induced by programmed electrical stimulation in conscious dogs after myocardial infarction

SummaryAs yet the antiarrhythmic efficacy of ajmaline with regard to suppressing the induction of sustained ventricular tachycardia after myocardial infarction has not been determined. Therefore, programmed electrical stimulation was performed in 8 conscious, chronically instrumented mongrel dogs 8–20 days after a 4-hour occlusion of the left anterior descending coronary artery. At baseline all animals responded with sustained ventricular tachycardia. Thereafter, ajmaline was administered at two consecutive ix. doses: a bolus of 0.7 mg kg−1 followed by infusion of 2 mg kg−1 h−1 and infusion of 4 mg kg−1 h−1. The induction of sustained ventricular tachycardia was prevented in 2/8 animals by 2 mg kg−1 h−1 ajmaline and in 1/8 animals by 4 mg kg−1 h−1 ajmaline. During sinus rhythm only 4 mg kg−1 h−1 ajmaline significantly increased QRS-duration and intraventricular activation times, but during rapid right ventricular pacing (cycle length = 330 ms) both doses of ajmaline increased QRS duration and intraventricular conduction times. 4 mg kg−1 h−1 ajmaline also increased the cycle length of induced sustained ventricular tachycardia. In 3 animals induction of sustained ventricular tachycardia during infusion of 4 mg kg−1 h−1 ajmaline was achieved by introduction of less extrastimuli than at baseline. Furthermore the coupling intervals of extrastimuli that induced sustained ventricular tachycardia were substantially prolonged by this dose. Inhomogeneity of conduction between left ventricular normal zone and left ventricular infarct zone was significantly increased by 4 mg kg−1 h−1 ajmaline during rapid right ventricular pacing, but not during sinus rhythm.We conclude that ajmaline has only limited efficacy with regard to the prevention of induction of sustained ventricular tachycardia after myocardial infarction. Drug-induced increases in tachycardia cycle lengths, as observed under the higher dose of ajmaline, were associated with a rate-dependent rise in cardiac electrical instability.

Kinetics of rate-dependent slowing of intraventricular conduction by the class Ib antiarrhythmic agent tocainide in vivo

1 The effects of the class I antiarrhythmic agent, tocainide, on intraventricular conduction were assessed in guinea‐pigs, anaesthetized with pentobarbitone sodium 60 mg kg−1, i.p. 2 After electrical ablation of the sinus node, heart rate was controlled by atrial pacing. His bundle electrograms were recorded by means of an epicardial bipolar electrode. 3 During continuous stimulation, comparison of HV intervals measured at a cycle length of 475 ms, with HV intervals measured at a cycle length of 250 ms yielded the following results: 25.26 ± 0.64 ms versus 25.02 ± 0.70 ms (NS), at baseline, 26.65 ± 0.80 ms versus 29.881 1.13 ms (P < 0.001) after i.v. administration of 30 mg kg−1 tocainide, and 28.04 ± 0.64 ms versus 36.24 ± 1.31 ms (P < 0.001), after addition of 20 mg kg−1 tocainide. Thus, tocainide caused HV intervals to increase in a strictly rate‐dependent fashion. 4 In order to characterize the rate‐dependent class I activity of tocainide in terms of its binding kinetics to sodium channels, fractional sodium channel block was estimated from drug induced reductions of intraventricular conduction velocity (Δθ). On abruptly changing the drive cycle length from 500 ms to 250 ms, Δθ reached a new steady state with rate constants of 1.23 ± 0.09 beat−1 and 1.28 ± 0.09 beat−1, after administration of 30 mg kg−1 and addition of 20 mg kg−1 tocainide, respectively. At a basic drive cycle length of 250 ms Δθ recovered with time constants of 250.29 ± 23.32 ms and 183.04 ± 8.03 ms after administration of 30 mg kg−1 and addition of 20 mg kg−1 tocainide, respectively. 5 The experimentally determined kinetic parameters were implemented into a mathematical model that assumes drug binding to sodium channels in terms of a periodical two‐state process. Rate‐dependent reductions in conduction velocity during continuous stimulation after administration of tocainide were closely approximated by steady state reductions in sodium channel availability as calculated on the basis of the aforementioned model. 6 In agreement with previously published in vitro studies, our data, obtained in vivo, confirm the classification of tocainide as a class I antiarrhythmic agent with fast onset and offset kinetics. The kinetic parameters obtained in vivo can be used in order to predict steady state reductions in conduction velocity at a wide range of frequencies.