The comet assay in clinical practice.

The comet assay is a single-cell gel electrophoresis technique that measures DNA damage in individual cells. Since radiation produces 3-4 times more DNA damage in well-oxygenated cells compared with hypoxic cells, this assay can quantify the fraction of radiation-resistant hypoxic cells found in many solid tumours. This paper summarizes our results with 73 accessible metastatic tumours irradiated with palliative intent. Hypoxic fractions ranged from 0.0 to 0.67 with a mean of 0.15; 62% of these advanced tumours showed a hypoxic fraction > 0.05. Comparisons between two sequential aspirates in 33 tumours gave a slope of 0.92 (r2 = 0.88), suggesting that a single aspirate is generally representative of the tumour. A limitation, however, is that the hypoxic fraction could not be measured in clinical samples given a conventional dose of 2 Gy.

Heterogeneity in human tumour hypoxic fraction using the comet assay.

The alkaline comet assay has previously been used to estimate the fraction of radiobiologically hypoxic cells in tumours from patients undergoing palliative radiotherapy for advanced breast and head and neck cancer. Results obtained from fine needle aspirate biopsies (FNABs) using this method indicate considerable heterogeneity in hypoxic fraction between tumours. Heterogeneity between 3 aspirates taken from the same 10 tumours immediately following single doses of 3.5 to 5 Gy is now examined. Results indicate that a single fine needle aspirate is reasonably representative for DNA damage and DNA content. However, difficulties were encountered in obtaining an adequate sample of tumour cells after the final radiation treatment. The average hypoxic fraction decreased from 14% after the first dose to 9% after the last dose, and in 3 tumours which could be evaluated after both the first and last fraction, the hypoxic fraction decreased in two but increased in the third. Rejoining of DNA strand breaks was observed between sequential aspirates indicating that pooling of samples for analysis may not be advisable using this method.

Small cell carcinoma of the cervix treated with concurrent radiotherapy, cisplatin, and etoposide.

A multimodality regimen of four cycles of cisplatin and etoposide with concurrent locoregional radiotherapy (XRT) has been, since May 1988, the standard therapy for women with small cell carcinoma of the cervix (SCCC). Prophylactic cranial irradiation was to be used in all but primary progressors. All 11 patients (median age 47; 4 with pure SCCC and 7 with mixed histology) seen by us were treated with this regimen. Only 1 patient progressed while on treatment. The 3-year overall and failure-free survivals were 28%. Four patients remain alive in first remission; the remaining 7 died (2 from toxicity, 5 from cancer). Although not statistically significant due to the small numbers, it appeared that the chance of long-term survival depended both on the amount of the cancer as indicated by the FIGO stage and size of the primary and also the performance status. The toxicity was significant with 70% experiencing severe neutropenia and 40% being admitted for control of emesis. This regimen is only appropriate for those women in whom all of the apparent tumor can be encompassed within a radiation field and who, in addition, have a performance status of 0 or 1. For the remainder it does not offer any chance of long-term survival and its toxicity renders it antipalliative.

Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers.

A new gel electrophoresis method has been used to quantify hypoxic fraction in human tumors. Radiation-induced DNA damage was measured in individual tumor cells, where the radiobiologically hypoxic cells were observed as a subpopulation showing a 3-fold reduction in DNA strand breaks. Patients receiving palliative radiotherapy for breast cancers were given a single dose of 5-10 Gy, and a fine needle aspiration biopsy was taken immediately after irradiation. Hypoxic cells were detected in seven of eight tumors. In four tumors, bivariate analyses of DNA content versus DNA damage to individual cells allowed distinction between the response of diploid normal cells and aneuploid tumor cells. These early results indicate that "comet assay" shows considerable promise for resolving the extent and significance of hypoxia in human tumors.

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.

Invasive cancer of the cervix in British Columbia: a review of the demography and screening histories of 437 cases seen from 1985-1988.

The charts of 437 patients with newly diagnosed invasive cervical cancer seen in British Columbia during 1985-1988 were reviewed to determine the age at diagnosis, stage of disease, and histology of the lesions. The patient's immigration status and country of birth were also examined. Two hundred forty-two patients (55%) were age 50 or over and 149 (34%) had preclinical stage IB disease. One hundred seventy of these patients (39%) had never had a cytologic examination before presenting with clinical symptoms, and 45 additional patients had not had cytology for 5 or more years before presenting with invasive disease; thus, a total of 215 cases (49%) fell into the category of "no cytology or cytology longer than 5 years ago." All negative smears were reviewed and in 39 patients (15%) having previous cytology, abnormal cells had been missed or undercalled. Native Indian women made up 10% of the cases of invasive carcinoma, a disproportionately large number as they represent only 2% of the British Columbian population. Of the 27 Native Indian patients in this series, 16 (59%) had never had a cytologic examination. Eighty-five (30%) of 276 women born in Canada had never had a cytologic examination, compared with 34 (71%) of 48 immigrants resident in the country for fewer than 10 years. Twenty-four patients were over the age of 60 with a history of negative Papanicolaou smears. We conclude that, if further gains are to be made in the reduction of death rates from invasive cervical carcinoma, new means must be found to encourage women to participate in Papanicolaou smear screening programs. In particular, special efforts must be made to attract specific groups, such as the Native Indian population and older recent immigrants.

The use of sequential fine-needle aspiration biopsy with flow cytometry to monitor radiation induced changes in breast carcinoma.

Thirteen patients with locally advanced or recurrent breast carcinoma were monitored during radiation therapy by multiple, sequential, fine-needle aspiration biopsies (FNAB) with flow cytometry. The material was analyzed for qualitative cytomorphological evidence of radiation effect and for DNA content and cell cycle alterations. DNA ploidy was not affected by the radiation therapy, although the aneuploid tumors showed an increased frequency of cell cycle alterations. The most common change seen was an increase in S-G2M (58%). Other changes included a decrease in the proliferative/growth fraction (17%) and no significant redistribution of cells (25%). There was a relationship between the initial proliferative activity of the tumors and the type of cell cycle change which occurred. Flow cytometric analysis was a better predictor of early clinical response than was cytomorphological assessment. Sequential FNAB with flow cytometry is an effective method of monitoring the response of breast cancer to radiation therapy.

Nuclear DNA, serum sialic acid and measured depth in malignant melanoma for predicting disease recurrence and survival.

In a previous multivariate analysis of 151 malignant melanoma patients we identified measured depth of primary lesion (Breslow) and serum N-acetyl-neuraminic acid (NANA) concentration as significant independent predictors of recurrence. Our present study examines the contribution of flow cytometric DNA analysis to prediction of recurrence and survival. Fixed, paraffin-embedded specimens of primary lesions were evaluated from 63 of the previously studied patients. These were prepared for DNA analysis. Of the 28 primaries identified as aneuploid 17 later recurred, while this was true for only 9 of the 35 diploid tumors. On multivariate analysis measured depth was again the most significant predictor of recurrence (p less than 0.001). Additional independent predictors were DNA ploidy (p = 0.02) and NANA (p = 0.05). For survival the independent predictors were measured depth (p = 0.003) and NANA (p = 0.05). Measured depth, DNA ploidy and NANA can be used to construct a model predicting the recurrence risk for stage-I melanoma.

Organisation and results of the cervical cytology screening programme in British Columbia, 1955-85.

A screening programme to detect preinvasive carcinoma of the cervix was started in British Columbia in 1949. Since 1970 the number of women who have been screened at least once has been maintained at about 85% of the population at risk. More than 500,000 cervical smears are being examined each year in the central laboratory. There has been an appreciable increase in the number of cases and rates of carcinoma in situ seen since 1970, particularly in women between 20 and 30 years of age. Since the programme started over 26,000 cases of squamous carcinoma in situ have been detected and treated. The incidence of clinically invasive squamous carcinoma of the cervix has fallen by 78% during the period under review, and mortality from squamous carcinoma of the cervix has fallen by 72%. A colposcopy programme, introduced throughout British Columbia over the past 12 years, has been important in reducing the problems of managing preinvasive lesions, particularly in younger women. It is concluded that the reduction in morbidity and mortality from invasive squamous cancer of the cervix in British Columbia over the past 30 years is directly attributable to the province wide screening programme and that a large potential increase in invasive cervical cancer rates among younger women is being prevented.

Multiple gingival metastases from angiosarcoma of the breast.

Metastases to the jaws and oral soft tissue occur rarely. Such metastases may result in the presenting symptoms and signs of malignant disease or may develop during the course of a previously diagnosed malignant condition. A case of multiple gingival metastases in a patient with angiosarcoma of the breast is reported.

A comprehensive internal quality control system for a large cytology laboratory.

The British Columbia Cervical Cytology Program is operated through the Central Laboratory at the Cancer Control Agency in Vancouver and processes all of the gynecologic Papanicolaou smears collected by 3,200 physicians throughout the province of British Columbia. The laboratory receives approximately 2,400 smears per day, and the program currently processes in excess of 500,000 smears annually. This article describes the methods that have been developed for ensuring that adequate quality control is present in the screening and interpretation of half a million smears per year, both at the cytotechnologist and at the cytopathologist level. The results of the quality control program, which was considerably modified in 1985, are also presented. The modified program shows a significant improvement in the number of undercalled and overcalled cases detected in two comparable six-month periods.

Fluorescence bronchoscopy in the localization of bronchogenic carcinoma.

Fluorescence bronchoscopy using hematoporphyrin derivative as a tumor marker has been developed for localization of early bronchogenic carcinoma. Recent clinical results have been obtained with a krypton laser and a fused quartz fiber illumination system, which is clearly superior to the mercury vapor lamp system used previously. Fifteen patients have been examined, two with the laser. The potential of the technique is demonstrated by a case study of a patient with positive sputum cytology and a negative chest radiograph. The small early invasive tumor could be detected only with difficulty by subtle mucosal changes under white light bronchoscopy. Positive fluorescence was observed in the suspicious area. Malignancy was confirmed by biopsy and histologic examination upon subsequent lobectomy.