Judith W.J. Bergs

Hyperthermia, cisplatin and radiation trimodality treatment: a promising cancer treatment? A review from preclinical studies to clinical application.

This review discusses available clinical and experimental data and the underlying mechanisms involved in trimodality treatment consisting of hyperthermia, cisplatin and radiotherapy. The results of phase I/II clinical trials show that trimodality treatment is effective and feasible in various cancer types and sites with tolerable toxicity. Based on these results, phase III trials have been launched to investigate whether significant differences in treatment outcome exist between trimodality and standard treatment. In view of the clinical interest, it is surprising to find so few preclinical studies on trimodality treatment. Although little information is available on the doses of the modalities and the treatment sequence resulting in the largest degree of synergistic interaction, the results from in vivo and in vitro preclinical studies support the use of trimodality treatment for cancer patients. Animal studies show an improvement in treatment outcome after trimodality treatment compared with mono- and bimodality treatment. Studies in different human tumour cell lines show that a synergistic interaction can be obtained between hyperthermia, cisplatin and radiation and that this interaction is more likely to occur in cell lines which are more sensitive to cisplatin.

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH). It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.

PCC and COBRA-FISH a new tool to characterize primary cervical carcinomas: to assess hall-marks and stage specificity

A newly developed assay based on chemically induced premature chromosome condensation (PCC) and multi-color combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) techniques have been implemented in order to investigate for the first time for recurrent cytogenetic aberrations in primary cervical carcinoma (derived directly from biopsies) at different stages of progression. The cytogenetic profiles of 17 biopsies derived from 14 and 3 cervical cancer patients with squamous-cell carcinomas (Sq) and with adenocarcinomas (Ad), respectively, were assessed. Frequencies of both structural as well as numerical aberrations were found to be higher in Sq than in Ad. The analysis revealed that even in early tumors stages (IB1) have a higher frequency of chromosome-losses and -gains as well as chromosomal alterations as compared to normal cells. A positive trend was found between stage advancement of cervical tumors and the frequency of numerical and structural aberrations. No specific and common chromosomal abnormality (e.g. distinct clones of translocation) was found among cervical carcinoma at the different stages (IB1, IIA and IIB). However, a distinct difference was found between stage IIIB and lower tumor stages, as all analyzed IIIB samples revealed a near tetraploid karyotype. Furthermore, all studied metaphases were aberrant and had a high frequency of translocations. PCC-COBRA-FISH characterization of a common type of an established culture from cervical carcinoma CSCC-1 revealed a triploidy/tetraploidy karyotype with several structural aberrations. In general, no similarity was found between this model and early stages of primary tumors. The newly established assay has a novel potential and can reveal the original status of primary tumors at different stages.

Radiosensitization with Hyperthermia and Chemotherapeutic Agents: Effects on Linear-Quadratic Parameters of Radiation Cell Survival Curves

Radiosensitization effects of hyperthermia and chemotherapeutic agents as currently exploited in the clinic are discussed with respect to the linear quadratic parameters of dosesurvival curve presentations. Studies of different human tumour cell lines show that a synergistic interaction can be obtained between hyperthermia, chemotherapy and radiation and that this interaction is more likely to occur in cell lines which are relatively sensitive to chemotherapy. The influence of modifying agents on radiation dose survival curves can adequately be analysed with the use of the linear-quadratic model: S(D)/S(O)= exp(┙D+┚D2). The linear parameter, ┙, represents lethal damage from single particle events and describes the low dose area while the quadratic parameter, ┚, indicating sub lethal damage (SLD) dominates the effectiveness in the high dose region (Barendsen, 1990, 1994, 1997). The linear-quadratic model is based on well accepted biophysical concepts, involving the assumption that lethal damage can be induced by single-particle tracks and by interaction of damage from multiple particles. It has been found to describe the low-dose region of the survival curves up to 6 Gy rather accurately. Furthermore the LQ-model has been shown to describe adequately dose fractionation effects for normal tissue tolerance and for experimental tumours. The LQ-model has also the advantage that it requires only two parameters to describe radiation dose-survival curves. It allows the separate analysis of changes in effectiveness in the low dose range, mainly determined by the linear term and in the high dose range determined mainly by the quadratic term (Barendsen, 1982; Joiner &

Transient inhibition of Calyculin A induced premature chromosome condensation by hyperthermia

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41°C to 43°C and after recovery for up to 24 h after treatment with 43°C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures ≥42.5°C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43°C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43°C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43°C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5°C.