D. E. Thrall

Proliferation and hypoxia in human squamous cell carcinoma of the cervix: First report of combined immunohistochemical assays

PURPOSE To characterize the distribution of hypoxia and proliferation in human squamous cell carcinoma of the cervix via an immunohistochemical approach prior to initiation of therapy. METHODS AND MATERIALS Patients with primary squamous cell carcinoma of the cervix uteri received a single infusion of the 2-nitroimidazole, pimonidazole (0.5 g/m2 i.v.), and 24 h later punch biopsies of the primary tumor were taken. Tissue was formalin fixed, paraffin embedded, and sectioned for immunohistochemistry. Hypoxia was detected by monoclonal antibody binding to adducts of reductively activated pimonidazole in malignant cells. Staining for endogenous MIB-1 and PCNA was detected in tumor cells via commercially available monoclonal antibodies. Point counting was used to quantitate the fraction of tumor cells immunostained for MIB-1, PCNA, and hypoxia marker binding. RESULTS Immunostaining for pimonidazole binding was distant from blood vessels. There was no staining in necrotic regions, and only minimal nonspecific staining, mostly in keratin. In general, cells immunostaining for MIB-1 and PCNA did not immunostain for pimonidazole binding. Cells immunostaining for MIB-1 and PCNA showed no obvious geographic predilection such as proximity to vasculature. Quantitative comparison showed an inverse relationship between hypoxia marker binding and proliferation. CONCLUSIONS Immunohistochemical staining for pimonidazole binding is consistent with the presence of hypoxic cells in human tumors and may be useful for estimating tumor hypoxia prior to radiation therapy. Immunostaining for pimonidazole binding is an ideal complement to immunohistochemical assays for endogenous proliferation markers allowing for comparisons of tumor hypoxia with other physiological parameters. These parameters might be used to select patients for radiation protocols specifically designed to offset the negative impact of hypoxia and/or proliferation on therapy. The inverse relationship between pimonidazole binding and proliferation markers is a preliminary result requiring verification.

Measuring tumor hypoxia

A variety of techniques for measuring oxygen in normal and tumor tissue has been developed over the years in response to the realization that hypoxia is important in a number of pathophysiological conditions in normal tissues and in the response of tumors to radiation treatment. A review of the techniques for measuring tissue oxygenation is presented with an emphasis on clinical results. Histomorphometry, DNA strand break analysis (comet assay), oxygen microelectrodes and hypoxia markers are highlighted. Comparison among techniques is touched on and a short discussion of the possibility that hypoxia is not an independent variable in determining the outcome of radiation therapy is presented.

Serious toxicity associated with annular microwave array induction of whole-body hyperthermia in normal dogs

Using a regional annular microwave array it was possible to produce a systemic temperature of 42 degrees C in approximately 80 min with applied net power levels of approximately 150 W. Resulting temperature distributions were non-uniform. Sites within the array were above systemic temperature during heating but approximated systemic temperature during the plateau phase. Sites outside of the array were lower than systemic temperature during heating and plateau phases. Dogs allowed to recover from the procedure experienced severe toxicity consisting of lumbar muscle haemorrhage, pain and swelling, and pelvic limb paralysis. Histologically, there was severe myopathy and haemorrhage and oedema in neural tissue in the caudal lumbar spine. Acute necrosis of lymphoid tissue was observed in all dogs. Temperatures in muscle reached 43-46 degrees C and were higher than at other measured sites. Spinal canal temperatures were essentially equal to rectal temperature, approximating 42-43 degrees C during heating and plateau phases. These data suggest regionally induced whole-body hyperthermia may result in: (1) power deposition non-uniformity leading to muscle and spinal canal temperatures which exceed systemic temperature and which are sufficient to cause serious toxicity; (2) systemic temperature non-uniformity which is undesirable for systemic thermochemotherapy; and (3) possible immunological dysfunction associated with lymphoid necrosis. Extreme caution must be exercised in administering energy to localized regions of human patients with the intent of elevating systemic temperature.

Spontaneous Pet Animal Cancers

A significant amount of the in vivo work assessing cancer biology or experimental therapeutics is conducted in small laboratory animals. Indeed, the vast majority of this book is based on in vivo laboratory animal systems that have enabled significant advances to be made in the understanding of cancer. Clearly, there are many advantages associated with these models. Alternatively, however, studies of spontaneous cancer can provide information that cannot be obtained elsewhere, except for the study of cancer in humans. Spontaneous tumors in large animals (dogs and cats) can provide a bridge between novel discoveries in the laboratory and implementation of new therapies in humans in a number of novel ways. This chapter reviews some of the basic aspects of spontaneous large animal tumors and provides specific examples of results that have important implications for human cancer therapy.

Tumour cell kinetics as predictors of response in canine lymphoma treated with chemotherapy alone or combined with whole body hyperthermia

Kinetic parameters including potential doubling time (Tpot), duration of S phase (Ts), labelling index (LI), and DNA index (DI) were obtained from 42 dogs with previously untreated lymphoma. Standard flow cytometric techniques using BrdUrd were employed. All dogs were treated with L-asparaginase and remission was induced in 26 dogs, which were then randomized to receive chemotherapy only (doxorubicin [DOX] alone or with lonidamine) or chemotherapy plus whole body hyperthermia (WBH). Dogs were treated every 3 weeks for up to five treatments and evaluated every 3 weeks for evidence of tumour recurrence. Within this subset of animals there was no difference in outcome based on treatment group. Median values for Tpot, Ts and LI were 3.4 days, 7.23 h and 12.49%, respectively. Dogs that had tumours with LI > or = 20% had a shorter time until recurrence than dogs with tumours characterized by LI < 20%. In dogs treated only with chemotherapy, dogs bearing tumours with longer than median Tpot and Ts values and lower than median LI had significantly longer remission duration than dogs with more rapidly proliferating tumours. Dogs treated only with chemotherapy, which had longer than median Tpot and Ts values and lower than median LI, had significantly longer remission duration than all other dogs in the study. The mechanisms in which kinetics are associated with response to chemotherapy are not clear and vary depending on tumour type and treatment regimen. More work is needed to understand factors involved in cell killing during in vivo hyperthermia.

A Clinically Proven, Prospective, Thermal Dose Descriptor Exists

In Response: We appreciate the consideration that Dr. van der Zee and her group have given to our recent hyperthermia trial in canine sarcomas published in Clinical Cancer Research ([1][1]). It is apparent that they have scrutinized our trial very carefully and we welcome this examination. However,

Inspired anaesthetic gas humidification improves thermal uniformity during canine whole body hyperthermia

Supplying warmed saturated water vapour in anaesthetic gases during whole body hyperthermia (WBH) could potentially improve thermal uniformity in the trachea and esophagus. Four normal dogs were anaesthetized for WBH at 42 degrees C. A Puritan Bennett Cascade humidifier was used to supply anaesthetic gases saturated with water vapour at an average airway temperature of either 42 degrees C or 38 degrees C. Esophageal temperature was monitored at the thoracic inlet and 5 cm cephalad. Thermal dose was estimated by calculating equivalent minutes for an isoeffect at 43 degrees C (CEM 43 degrees Tx, where Tx is the site of temperature measurement). Endotracheal mucociliary transport velocity (MCTV) was determined before and 48 h following WBH by 99mTc-MAA scintigraphy. Compared to the 38 degrees C humidified gas group, dogs receiving 42 degrees C humidified gas reached 42 degrees C faster (p = 0.02) and had CEM 43 degrees T(esophageal) values equivalent to the target CEM 43 degrees T(rectal). Endotracheal MCTV with 42 degrees C humidified gas, however, was reduced 53% from baseline 48 h following WBH (p = 0.02). With 38 degrees C humidified gas, endotracheal mucociliary transport velocity was unchanged from baseline 48 h post WBH. Tracheal histology was examined using light and electron microscopy in four additional dogs euthanatized following 90 min of 42 degrees C humidified gas combined with WBH. There was no histological evidence of tracheal or lung thermal damage with 42 degrees C humidified gas in these four dogs. However, a moderate increase in tracheal goblet cell secretory granule staining was observed. This change could imply temporary heat-induced ciliary microtubule dysfunction, rather than decreased mucus production, as the likely mechanism of reduced mucociliary transport velocity 48 h following WBH. Administration of 42 degrees C humidified anaesthetic gases with WBH improves heating rate and esophageal thermal uniformity but temporarily depresses tracheal mucociliary transport velocity.

Effect of calcitonin gene related peptide vs sodium nitroprusside to increase temperature in spontaneous canine tumours during local hyperthermia

The objectives of this study were to compare the effects of two vasodilators, sodium nitroprusside (SNP) and calcitonin gene-related peptide (CGRP) on mean arterial pressure (MAP), heart rate (HR) and temperatures in tumour and surrounding normal tissue during local hyperthermia treatment. Eleven tumour-bearing pet dogs with spontaneous soft tissue sarcomas were given SNP intravenously during local hyperthermia. The drug infusion rate was adjusted to maintain a 20% decrease in MAP. The median (95% CI) increase in the temperature distribution descriptors T90 and T50 was 0.2°C (0.0–0.4°C, p = 0.02) and 0.4°C (0.1–0.7°C, p = 0.02), respectively, in tumour. Normal subcutaneous tissue temperatures were mildly increased but remained below the threshold for thermal injury. The effects of CGRP were investigated in six tumour-bearing dogs following a protocol similar to that used for SNP. The median (interquartile (IQ) range) decrease in mean arterial pressure was 19% (15–26%) after CGRP administration and a significant increase was seen in tumour but not normal subcutaneous tissue temperatures. The median (95% CI) increase in the temperature distribution descriptors T90 and T50 was 0.5°C (0.1–1.6°C, p = 0.03) and 0.8°C (0.1–1.6°C, p = 0.13), respectively. Administration of SNP or CGRP did not result in local or systemic toxicity in tumour-bearing dogs. However, the magnitude of increase in tumour temperatures was not sufficient to improve the likelihood of increased response rates. Therefore, there is little justification for translation of this approach to human trials using conventional local hyperthermia.