Cumulative interfraction interval analysis of time and fraction size in radiotherapy.

AIMS: Overall treatment time (OTT) and fraction size influence treatment results with radiotherapy; yet, being inversely related, it is difficult to distinguish their effect independently of each other. This paper proposes a new approach for analysing the interrelationship of time and fraction size in terms of cumulative interfraction intervals (CIFI) (i.e. intervals between non-successive fractions of radiotherapy). MATERIALS AND METHODS: We analysed the influence of CIFI1-6, CIFI1-11 and OTTon tumour control by Kaplan-Meier calculation of the primary relapse-free survival (PRFS) and Mann Whitney 'U' test in 242 patients with epidermoid cancer of the buccal mucosa-gingiva-palate region treated by either 2.4 Gy/fraction (60 Gy/5 weeks) or 3.5 Gy/fraction (52.5 Gy/3 weeks). RESULTS: The results showed that (1) prolongation of CIFI1-11 significantly decreased tumour control in the 2.4 Gy but not 3.5 Gy schedule: (2) 3.5 Gy schedule was superior to 2.4 Gy schedule, particularly when CIFI1-11 was prolonged (5-year PRFS 80.3% vs 30.9%); and (3) OTT did not influence either schedule. CONCLUSIONS: Prolonging interfraction intervals (by treatment interruptions, weekend gaps, etc.) in the first 2 weeks (when accelerated repopulation attempts are maximal) affects treatment results with low fraction sizes but not high fraction sizes; OTT probably vicariously reflects the effect of prolonging the intervening CIFI.

Radiation-induced changes in oral carcinoma cells - a multiparametric evaluation.

The aim of this study was to see whether serial cytological evaluation of various cellular abnormalities in tumours from patients receiving fractionated radiotherapy can predict radio-response in oral carcinoma. Cytological assessment was carried out in scrape smears collected prior to and during the course of radiotherapy in 68 patients with squamous cell carcinoma of the oral cavity planned for radical radiotherapy with accelerated fraction schedule. Smears were evaluated for a set of 15 radiation-induced cellular abnormalities. The relationship between the cellular alterations and the cumulative radiation dose was analysed by Kruskal-Wallis one-way anova. The results showed that among the various quantifiable changes that occur in irradiated cancer cells, karyolysis, karyorrhexis, pyknosis, cytolysis, multinucleation, micronucleation and nuclear budding show significant increase depending on the dose of radiation. The radio-resistant group of patients exhibited a lesser degree of change compared with the radio-sensitive group. This suggests that radio-resistance may be due to the defective induction of cell damage and that these cytological features may have potential use as predictive markers of radio-sensitivity in oral carcinoma.

Cytotoxic activity of amooranin and its derivatives.

Amooranin, 25-hydroxy-3-oxoolean-12-en-28-oic acid, is a triterpene acid isolated from Amoora rohituka stem bark. The cytotoxic effects of amooranin and its derivatives were studied. Amooranin and its methyl ester showed greater cytotoxicity against MCF-7 and HeLa cells derived from tumour tissues with a 50% inhibitory concentration (IC(50)) of 1.8-3.4 microg/mL, compared with Chang liver cells from normal tissue with an IC(50) of 6.2-6.4 microg/mL, but amooranin exhibited no activity on HEp-2 and L-929 cells. However, its monoacetate derivative showed no inhibitory activity at 1-10 microg/mL dose levels. Of the cytotoxic isolates, the methyl ester derivative was inactive in in vivo evaluations in the Ehrlich ascites tumour cells at 50 and 100 mg/kg/day, demonstrating T/C values of 106% and 114%, respectively.

Amitotic cell divisions and tumour growth: an alternative model for cell kinetic compartments in solid tumours.

Previous studies on cytological assay of amitotic changes such as micronucleation or nuclear budding (akaryokinesis) and multinucleation (acytokinesis; Bhattathiri et al., Serial cytological assay of micronucleus induction--a new tool to predict human cancer radiosensitivity. Radiother Oncol 1996; 41: 139-142; Serial cytological assay of micronucleus induction--a new tool to predict human cancer radiosensitivity. Radiother Oncol. 1997; 41: 139-142; Radiation-induced acute immediate nuclear abnormalities in oral cancer cells. Serial cytologic evaluation. Acta Cytol 1998; 42: 1084-1090) had suggested that, in addition to predicting radiosensitivity, they may be related to proliferation characteristics of tumours. Hence the present study was undertaken to see if their pre-treatment frequency was related to the clinical growth characteristics of tumours. Smears from 121 untreated oral cancers were stained with Giemsa and the frequency, in percentage of total cells counted, of micronucleated or nuclear budded, binucleated and multinucleated cells taken as the akaryokinesis index (AKI), mitotic index (MI) and acytokinesis index (ACI), respectively were evaluated. The sum of AKI and MI was taken as the amitotic index (AMI), and the sum of AMI and MI as the cell division index (CDI). The tumours were divided to three groups according to size: <2 cm (Tsize1); 2-4 cm (Tsize2) and >4 cm (Tsize3). The tumours were divided to those with duration of <4 months and > or =4 months, this data having been collected from the patient. The differences in the frequency of the indices among the three size groups were analysed by Kruskall-Wallis one-way analysis of variance. Within each size group, the differences in frequency of the indices betwen the two duration groups was analysed by Mann-Whitney U test. Larger tumours had significantly higher CDI, the median frequency being 1.2, 2.29 and 3.28% in Tsize1, Tsize2 and Tsize3 tumours, respectively (P=0.0025). In the case of AMi, the frequency was significantly higher the median values being 1.0, 6.3 and 10.05%, respectively (P=0.0015). The difference was not significant in the case of the MI, the frequency being 0.69, 0.99 and 1.32%, respectively. AKI and ACI also showed significant increase. As regards relation to clinical growth, those tumours which reached larger size in shorter duration had higher frequency of MI. Those tumours which remained small in spite of a longer duration of growth had statistically significantly higher AKI whereas tumours which reached Tsize2 in shorter duration of growth had significantly higher ACI. The results suggests that cytological evaluation of mitoses and amitoses can be helpful in evaluating proliferation characteristics of solid tumours. Micronucleated and multinucleated cells are clonogenically dead, but not physically so, but continue to divide for a few times before dying. The significant increase in AMI with size suggests that induction of amitoses is an important mechanism of cell loss as tumours enlarge, probably induced by associated hypovascularity, and precedes necrosis. The high frequency of amitoses in untreated tumours suggest that proliferation markers such as Tpot, growth fraction, etc., as measured by techniques such as flowcytometry, thymidine and BrdUrd labelling, etc., which do not evaluate amitoses, may essentially be wrong. Based on the findings of the study, an alternative model for tumour cell kinetic compartmentalisation, which includes a non-clonogenic compartment in addition to clonogenic and quiscent compartments, is presented.

Relation of erythrocyte and iron indices to oral cancer growth.

BACKGROUND AND PURPOSE: Anaemia is known to influence prognosis of head and neck cancer patients, but how anaemia and tumour growth influences each other is not clear. The present study investigates the relation of erythrocyte and iron indices of oral cancer patients to primary tumour size (Tsize), invasiveness and lymph node involvement. MATERIALS AND METHODS: The haemoglobin (Hb), erythrocyte count (RBC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), Serum iron (SFe), transferrin iron-binding capacity (TIBC) and transferrin saturation (%Fe) were evaluated in 217 untreated patients with epidermoid cancer of the bucco-gingivo-palatine area. The association of erythrocyte and iron indices with sex, tumour size groups, invasion of adjacent structures and lymph node involvement, as well as the relation of SFe to Hb were analyzed. RESULTS: Most of the patients were anaemic in terms of Hb (63%), RBC (43%) and PCV (48.4%) but almost all had normal or higher MCH (97.3%) and MCV (93.3%) though MCHC was less than normal in 70.7%. Normal or higher SFe was seen in nearly 70% and TIBC in 45% of patients. Hb, RBC and PCV were significantly lower in women, but there was no difference between men and women in the case of MCV, MCH and MCHC. Primary tumour size showed negative association with Hb, RBC and PCV but positive association with MCH (< 2 cm: 29.7 pg; 2-4 cm: 31.4 pg; > 4 cm: 31. 7 pg; P = 0.04) and MCHC (< 2 cm: 29.9; 2-4 cm: 31.5; > 4 cm: 32.1; P = 0.006). MCV, SFe, TIBC and %Fe did not show any relation to primary tumour size. None of the indices had any relation to invasion of adjacent structures or lymph node involvement. MCH, MCHC and MCV were not different in men and women but women had significantly lower Hb, RBC and PCV. The SFe showed poor correlation with Hb. CONCLUSIONS: The negative association of Hb, RBC and PCV with tumour size is most likely due to chronic RBC destruction, probably tumour induced, with the products of haemolysis such as polyamines, glutathione, iron, etc., promoting tumour growth, and the positive association with MCH and MCHC reflects compensatory regeneration attempts by bone marrow. Lack of relation between the iron indices and tumour parameters and the poor correlation between SFe and Hb is probably due to utilization of iron by both bone marrow and tumours. Lack of difference in MCH and MCHC between men and women obviates the need of using separate cut-off values for the two sexes, unlike Hb, RBC and PCV. The study suggests that anaemia in oral cancer patients represents a tumour-host interaction and that evaluation of all erythrocyte indices should be part of research on cancer related anaemia.

Relation of transmembrane potential to cell survival following hyperthermia in HeLa cells.

Hyperthermia induces cell death and the usual endpoint to study this is the ability of the cells to form colonies. Hyperthermia is also known to alter membrane characteristics, especially transmembrane potential and this has been correlated with duration and degree of heating. The aim of the present study was to see the correlation between changes in membrane potential and clonogenic ability of HeLa cells after heat treatment of 41-44 degrees C. Membrane potential was measured by the fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine by flow cytometry. Cell survival was assessed by colony formation assay. The fluorescence intensity increased and cell survival decreased with an increase in temperature. The fall in survival following heat treatment closely paralleled the increase in fluorescent intensity, especially heat treatments of 60 min or more. After 2 h of heating at 44 degrees C, the surviving fraction decreased to 1% and the fluorescence intensity increased to 154.84% of the unheated controls. This study suggests that measurement of membrane potential by flow cytometry may potentially be an alternative to colony forming assay for assessing cell survival. Since the results of membrane potential measurements are available immediately, this has implications for its potential use as a predictive assay of thermosensitivity.

Correlation of lectin binding with lymph node metastasis in oral cancers.

Lymph node metastasis is an important factor that influences the treatment policy and prognosis of oral cancers. The cell membrane is known to play a role in the metastatic process. The aim of the present study was to evaluate the relationship of lectin binding frequency of oral cancer cells to their capacity to metastasize to the lymph node. Smears collected from tumours of 66 untreated patients were stained with Jackfruit lectin (JFL) conjugated with horseradish peroxidase (HRP), with diaminobenzidine dihydrochloride (DAB) as the visualant. The frequency of cells showing lectin binding was evaluated. The results showed that tumours with a high frequency of JFL binding cells had higher risk of lymph node metastasis (RR = 1.5). It was also found that a combined score integrating the percentage of lectin binding cells with known clinical parameters, like primary tumour size, local invasion and histological subgroup, had better utility than any of these individually in assessing risk of lymph node metastasis. The density of sugar residues on the cell surface may be of importance in determining the lymph node metastatic potential of oral cancers, and the present study suggests the need for further research in this area.

Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity.

BACKGROUND AND PURPOSE: The micronucleus test, generally done in cultured tumour cells irradiated in vitro, has not gained wide acceptance in predicting human cancer radiosensitivity. The purpose of this study was to see if micronucleus assay by serial scrape smear cytology can predict oral cancer radiosensitivity. MATERIALS AND METHODS: Forty nine oral cancer patients given radiotherapy (60 Gy/25 fractions/5 weeks) form the study population. Serial scrape smears were taken from their tumours before treatment and after delivery of 2, 5, 8 and 12 fractions, stained by Giemsa and the number of micronucleated cells (MNC) noted. The patients were grouped to those who developed tumour recurrence ('Resistant') and those who did not ('Sensitive'), and the pattern of micronucleus induction compared. RESULTS: Both groups of tumours had MNC even before treatment, with statistically significant dose-related increase with radiotherapy. The sensitive group had a higher mean increase in MNC count than the resistant group (6.1 times and 3.6 times the pre-treatment value, respectively) and better correlation with dose (r = 0.54 vs. 0.43). The increase in MNC count occurred earlier in the resistant group than in the sensitive, the TMNC (time for the pre-treatment value to double) being 3.3 days and 7.6 days, respectively. Also, the resistant group showed a plateauing of the MNC count which the sensitive group lacked. CONCLUSIONS: The higher MNC induction in the sensitive tumours suggests the usefulness of the assay as a test of radiosensitivity. The differing patterns of MNC increase suggest that differences in proliferation rate is an important cause of tumour failure. Serial cytological assay of micronucleus induction can identify both radiosensitivity and proliferation characteristics of tumours, and thus may turn out to be a useful test of radiocurability.

Influence of plasma GSH level on acute radiation mucositis of the oral cavity.

PURPOSE: To see how pretreatment plasma GSH level influences the severity of acute radiation mucositis of the oral cavity during therapeutic irradiation in patients with oral cancer. METHODS AND MATERIALS: Thirteen patients with squamous cell carcinoma of the oral cavity form the subject material. Radical radiotherapy (60 Gy in 25 fractions over 5 weeks) was given using telecobalt. Pretreatment plasma GSH level was measured by Beutler's method. The normal tissue reaction during radiotherapy was monitored and graded. RESULTS: The GSH levels ranged from 10.6-90.5 microM/L (mean 30.6 microM/L). Those who had higher GSH levels developed less severe mucositis. The mean GSH levels in the groups with different severity of reactions were: Grade 2 (four patients) = 50.7 microM/L; Grade 3 (five patients) = 26.1 microM/L; Grade 4 (two patients) = 20.4 microM/L and Grade 5 (two patients) = 26.1 microM/L; Grade 4 (two patients) = 20.4 microM/L and Grade 5 (two patients) = 13.6 microM/L. CONCLUSION: Plasma GSH estimation has the potential to predict individual sensitivity to acute radiation mucositis and may particularly be useful in hyperfractionated regimes. The study also affirms the radioprotective role of GSH and suggests that this effect is either due to protection against membrane lipid peroxidation (since GSH does not enter the cell freely) or DNA damage (fractionated radiotherapy may permit freer entry of GSH into cell).

Lectin cytochemistry in the exfoliative cytology of uterine cervix.

A lectin was isolated from the seeds of jack fruit (Artocarpus integrifolia) and purified using a column of immobilized N-acetyl-D-galactosamine. This jack fruit lectin (JFL) was then conjugated to horse-radish peroxidase (HRP) type VI and used to study the cell surface carbohydrate profile of the cytological smears of the uterine cervix using diaminobenzidine as substrate. Cervical smears from 15 healthy individuals and 65 patients with dysplasia, carcinoma in situ and carcinoma of uterine cervix were used for the study. Normal cells showed weak binding in the membrane as well as cytoplasm, whereas carcinomatous cells showed strong binding towards JFL. Carcinoma in situ cells showed a binding pattern similar to that of carcinoma. Dysplastic cells showed difference in binding in mild, moderate and severe dysplasia. The intensity of binding increased with the severity of the dysplasia. The nature and intensity of binding of jack fruit lectin with cancer tissues suggest that this lectin may be of use as a diagnostic aid in exfoliative cytology.

Sister chromatid exchange frequencies in carcinoma of the human oral cavity: effect of treatment.

Spontaneous and mitomycin-C-induced sister chromatid exchange frequencies were studied in the lymphocytes of 25 oral cancer patients. For these patients, the mean spontaneous SCE value was 8.31 (+/- SD), which was significantly higher than the value of 6.60 (+/- SD) obtained for the controls (p < 0.001). But there was no significant difference between the Mitomycin C induced mean SCE values of oral cancer patients and controls. Seven of these 25 oral cancer patients were selected for second sampling after surgical removal of the tumor. Six of these seven patients showed a decrease in mean SCE/cell value following surgery, which was almost equal to the SCE values obtained for the controls. This indicates that the increased spontaneous SCE rates in oral cancer patients might be due to the metabolic stress imposed by the tumor on the host body or by some clastogenic product of the malignant cells. Surgical removal of the tumour might have resulted in normalization of the lymphocytic SCE rates in postoperatively studied patients.

Periodontal space: a major route to bone involvement in oral cancer.

Oral cancers are presumed to involve mandible or maxilla by direct invasion and destruction of periosteum and underlying bone. In countries where there is a high incidence of oral cancers, most patients have poor dental hygiene and chronic periodontitis. We hypothesize that in such situations spread through the periodontal space is the major route to bone. We suggest that micro and macroscopic examination of the root surface can identify this and that this has to be done for proper 'T' grouping of tumours.