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Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity
E\_ADIOTHEIL~PY & ONCOLOGY
ELSEVIER
Radiotherapy and Oncology 41 (1996) 139-142
Short Communication
Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity V.N. Bhattathiri”,“,
L. Bindub, P. Remanic, B. Chandralekhad, C.A. Davis”, M. Krishnan Nair” “Department of Radiotherapy, Regional Cancer Centre, Trivandrum, India 695011 “Research Fellow and Cytotechnologist, Regional Cancer Centre, Trivandrum, India 695011 ‘Department of Research, Regional Cancer Centre, Trivandrum, India 695011 ‘Department of Pathology, Regional Cancer Centre, Trivandrum, India 6.9501 I ‘Department of Radiation Physics, Regional Cancer Centre, Trivandrum, India 695011
Received 27 February 1996; revised 26 June 1996; accepted 27 July 1996
Abstract Background and purpose: The micronucleus test, generally done in cultured tumour cells irradiated in vitro, has not gamed 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 Gyf 25 fractions/S 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,.‘%@ 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. Conclusion: 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. Keywords:
Micronucleus;
Cytology; Radiosensitivity;
Oral cancer; Repopulation
1. Introduction Micronuclei are chromosomesand/ or chromosomefragments, usually acentric, which lag behind during mitosis, fail to incorporate into the daughter nucleus and remain outside the main nucleus. Presence of a micronucleus suggeststhat the cell has suffered chromosome damage. Radiation or chemicals can induce micronuclei, and micronucleated cells (MNC) are considered as dying cells [7,10,16]. Micronucleus induction by radiation is doserelated, as first reported by Evans et al. [4]. The micronucleus test was then recognised as a quick method to *Corresponding author. Tel.: +91 471 445435; fax: +91 471 447454. 0167-8140/96/$1S.O0 0 PIZ SO167-8140(96)01810-S
1996 Elsevier
Science
Ireland
Ltd. All rights
reserved
evaluate chromosomal damage by radiation [8,11]. Cell survival [5,6] assayedby the micronucleus test correlates well with colony forming assay[ 14,151.Its potential value as a predictive test of human cancer radiosensitivity was recognisedby Streffer, more than a decadeago [21]. These tests are generally done by evaluation of micronuclei either in biopsy specimenstaken before and after a course of radiotherapy [22,27], or in tumour cell cultures irradiated in vitro [ 181. These studies suggestedthat the micronucleus test can predict radiosensitivity [ 121. Yet, experiments with multiple cell lines showed that the radiosensitivity ranking given by micronucleus test was different from that given by clonogenic assay [2,24]. Cytological screeningof oral mucosalcells for micronu-
140
VM. Bhattathiri
et al. I Radiotherapy
clei is frequently done to assess genotoxicity of carcinogens and evaluate the efficacy of presumed chemopreventive agents like betacarotene and vitamin A [13,20]. By cytology, Tolbert et al. showed that the micronucleus count in normal cheek mucosa is elevated after exposure to radiation [23]. However, micronucleus test by scrape smear cytology has not been tried as a method to predict human cancer radiosensitivity. The present study was undertaken to see whether serial cytological evaluation of micronucleus induction in tumours of patients receiving fractionated radiotherapy can predict oral cancer radiosensitivity.
and Oncology
41 (1996)
139-142
analyzed in relation to cumulative dose and overall treatment time. Within-group analysis was done by KruskalWallis one-way analysis of variance and between-group by Mann-Whitney U-test. A P value CO.05 was considered as significant. The relation between dose and MNC count was also checked by calculating the Pearson’s correlation coefficient, 1. Changes in relation to time was additionally analyzed in terms of mean daily MNC production, i.e. MNC count/number of days of treatment completed.
3. Results 2. Materials
and methods
Forty nine previously untreated patients with squamous cell carcinoma of the oral cavity (buccal mucosa: 29, gingiva: 13, hard palate: 5, and floor of mouth: 2), treated by radical radiotherapy (60 Gy/25 fractions/5 weeks, 2.4 Gyifractionlday, 5 days/week), with or without concurrent chemotherapy were included in the study. Clearance from the institutional screening committee and informed consent from the patients were obtained. Treatment was by tele-cobalt or 4 MV linear accelerator, by lateral and anterior, or single lateral portals. The doses were prescribed to the intersection or a depth, with homogeneity of better than 90%. At the time of analysis the median follow-up period is 14 months and the minimum 9 months. Pre-treatment scrape smears were collected from the tumour of all patients. Subsequently, a minimum of three smears were collected from each patient, after delivery of 2, 5, 8 or 12 fractions. Care was taken to ensure that smears were taken only from the tumour, avoiding adjacent normal areas. (Smears were not collected after 12 fractions because radiation mucositis made it difficult to distinguish tumour from normal tissue). Scraping was done with a previously wetted wooden spatula and the material immediately spread on a clean glass slide. It was then air dried for 30 min, stained with Giemsa’s, dehydrated and mounted on DPX. The slides were evaluated by light microscopy, and the number of nuclei and micronuclei in the tumour cells scored. Well defined round or oval bodies, less than a third of the nuclear size, with staining characteristics similar to the main nucleus and distinctly separate from it were considered as micronuclei. Cells containing one, two, and more than two nuclei or micronuclei were recorded as separate events. Around 500-1500 cells were evaluated on each occasion and results expressed as MNC count/ 1000 cells. During follow-up, 57.1% (28/49) of the patients developed recurrence of the primary tumour. For purposes of analysis, they were regarded as being ‘radioresistant (Resistant group). The remaining 21 (42.9%) who had no recurrence were grouped as being radiosensitive (Sensitive group). Changes in MNC count in these groups were
In both groups many tumours had MNC even before treatment. The mean values (range) were 4.17 (O-18.5) in the resistant and 4.12 (O-9.8) in the sensitive groups; the difference was not significant. Seven tumours of the sensitive (33.3%) and 5 (17.9) of the resistant groups did not have any MNC before treatment. There was statistically significant (P
Table 1 Change in MNC Dose (Gy)
0 4.8 12.0 19.2 24.0
count in relation Mean
to dose and time
time in days
MNCllOOO
cells (variance)
Sensitive
Resistant
Sensitive
Resistant
0 2.9 7.6 12.3 18.4
0 2.5 7.3 12.6 18.0
4.1 7.5 8.5 12.2 25.2
4.2 7.8 11.3 16.6 15.0
(12.3) (29.2) (29.2) (59.3) (529.0)
(31.4) (36.0) (34.8) (255.0) (184.9)
KN. Bhattathiri et al. I Radiotherapy and Oncology 41 (1996) 139-142
8‘.Oi5o.8. 0.6s
Fig.
1. Mean
daily
MNC
induction.
4. Discussion The MNC frequency of normal oral mucosa is about 1.3-2.6/1000 cells, with higher frequencies in those habituated to tobacco, arecanut, etc. [13,20]. In the present study, both resistant and sensitive tumours had MNC even before treatment. Presenceof a micronucleusin a cell with no known exposure to any genotoxic agent reflects its inherent chromosomal instability [17]. Since mitosis is necessaryfor expression of micronuclei, the pretreatment MNC count reflects both chromosomal instability and growth rate of the tumours, though the relative importance of the two may be different for sensitive and resistant tumours. Zolzer et al. [27] reported that cervical cancers with lower pre-treatment MNC frequency had better control with radiotherapy. Streffer et al. [21] reported that rectal cancers with elevated S-phase fraction had greater MNC count and suggestedthat the S-phase/micronucleus ratio is an index of cell turnover. In the present study, though the mean pre-treatment MNC count was similar in both groups, the variance was higher in the resistant group indicating greater heterogeneity in proliferation and inherent chromosomal instability. Both groups of tumours showed statistically significant dose-related increase in MNC count with radiotherapy. The maximum MNC count was higher and correlation with dose better in the sensitive group than in the resistant group. Also, when compared to the pre-treatment values, the maximum was greater in the sensitive group than in the resistantgroup, being 6.1 times and 3.6 times, respectively. These findings suggest that MNC induction is related to radiosensitivity. It is now well recognised that rapid proliferation is as important as intrinsic radioresistance in contributing to tumour recurrence following radiotherapy. Flowcytometric and bromodeoxyuridine studies have shown that epidermoid cancers of the head and neck can have potential doubling times (T,,,) as low as 2 days, with less than 5
141
days in the majority [1,25], and it is the rationale of accelerated treatment regimes like CHART [3]. In the present study, the sensitive and resistant tumours had different patterns of increase in MNC count in relation to overall treatment time, this being earlier in the resistant tumours. It is logical to assumethat the higher growth rate of the resistant tumours, which contributed to their recurrence, also resulted in the earlier expressionof micronuclei by them. The initial decreasein daily MNC production in sensitive tumours (Fig. 1) suggeststhat radiation-induced cell cycle delay may be more in sensitive/slowly proliferating tumours than in resistant/rapidly proliferating tumours, and this could be another reasonfor the differing patterns of MNC increase.A similar relation between cell cycle delay, proliferation and radiosensitivity has been reported in murine tumours [26]. In the caseof micronucleus induction by chemicals, it is seenthat constant, repeatedinjury with the samedoseof a chemical increasesthe number of MNCs to a plateau [9]. This indicates that a balancehas been reachedbetween cell loss and repopulation. The same is probably true for micronucleus induction by radiation, too. The patients in our study were receiving radiation at a doseof 2.4 Gy /day, almost daily. The plateau in MNC count in resistant tumour suggeststhat due to the combined effects of low radiosensitivity and high repopulation rate, a balance has been reached between cell kill and tumour repopulation, ultimately leading to tumour recurrence. In sensitive tumours the count does not reach a plateau becausecell death exceeds repopulation. The main drawback of our study is that the betweengroup differences in MNC count lack statistical significance. This is due to influence of other prognostic factors like tumour size, gross type, addition of chemotherapy, etc., and other modes of cell death like apoptosis, and presenceof other radiation-induced morphological abnormalities like multinucleation which were not consideredin this analysis. The micronucleus test as practiced now, in cultured tumour cells, is of uncertain value as a predictive assay of radiosensitivity. One plausible reason is that this gives no information regarding the proliferation characteristics of the tumour, particularly the changes induced by radiotherapy. The importance of monitoring both radiosensitivity and proliferation is emphasized by the report of Zolzer et al. [27]. They found that combined evaluation of radiosensitivity (by micronucleus assay) and growth fraction (by S-phasedetermination) can predict radiosensitivity of cervical cancers. Serial cytological assay of micronucleus induction can potentially identify both radiosensitivity and proliferation characteristics.The technique is almost completely non-invasive and easily done in accessible primary cancerslike that of oral cavity and uterine cervix. In other sites, fine needlecytology can be tried, asreported by Shidnia et al. [19] in a study of four patients with lymph node secondaries.
142
VT. Bhattathiri
et al. / Radiotherapy
Acknowledgments
This work was supported by a grant from the Science, Technology and Environmental Committee, Government of Kerala, India.
References [1] Bennett, M.H., Wilson, G.D., Dische, S., Saunders, MI., Martindale, C.A., Robinson, B.M., O’Halloran, A.E., Leslie, M.D. and Laing, J.H.E. Tumour proliferation assessedby combined histological and flow cytometric analysis: implications of therapy in squamous cell carcinomas of head and neck. Br. J. Cancer 65: 870-878, 1992. [2] Bush, T. and McMillan, T.J. Micronucleus formation in human tumour cells: lack of correlation with radiosensitivity. Br. J. Cancer 67: 102-106, 1993 [3] Dische, S. and Saunders, M.I. The rationale for continuous hyperfractionated radiotherapy (CHART). Jnt. J. Radiat. Oncol. Biol. Phys. 19: 1317-1320, 1990. [4] Evans, H.J., Neary, G.J. and Williamsons, ES. The relative biological effectivenessof fast neutrons and gamma rays on vicia faba roots and the effects of oxygen. Part 2. Chromosome damage: the production of micronuclei. Int. J. Radiat. Biol. 3: 216-229, 1959. [5] Fenech, M. and Morley, A.A. Measurement of micronuclei in lymphocytes. Mutat. Res. 147: 29-36, 1985. [6] Geard, C.R. and Chen, C.Y. Micronuclei and clonogenicity following low and high dose rate irradiation. Radiat. Res. 124: 556-561, 1990. [7] Grote, S.J., Joshi, G.P., Revell, S.H. and Shaw, C.A. Observations of radiation-induced chromosome fragment loss in live mammalian cells in culture and its effect on colony forming ability. Int. J. Radiat. Biol. 39: 395-408, 1981. [8] Heddle, J.A. A rapid in vivo test for chromosomal damage. Mutat. Res. 18: 187-190, 1973. [9] McGregor, J.T. Micronucleus assay protocols. Mutat. Res. 259: 123-125, 1991. [lo] Midander, J. and Revesz, L. The micronucleus (MN) in irradiated cells as a measure of survival. Br. J. Cancer 41: 204-212, 1980. [ 111 Malls, M., Streffer, C. and Zamboglu, N. Micronucleus formation in preimplanted mouse embryos cultured in vitro after irridiation with X-rays and neutrons. Int. J. Radiat. Biol. 39: 307-314, 1981. [12] Muller, W.U. Micronucleus Assay, an indicator for radiation damage and predictor for radiosensitivity in tumour therapy. In: Radiobiological Concepts in Radiotherapy, pp. 47-52. Editors: D. Bhattachargee and B.B. Singh. Narosa, Bombay, 1995. [ 131 Nair, U., Obe, G., Nair, J., Maru, G.B., Bhide, S.V., Pieper, R. and Bartsch, H. Evaluation of frequency of micronucleated oral mucosal cells as a marker for genotoxic damage in chewers of betelquid with or without tobacco. Mutat. Res. 261: 163-168. 1991.
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41 (1996)
139-142
1141 Ono, K. Prediction of radiosensitivity by micronucleus assay. Gan No Rinsho Jpn. J. Cancer Clin. 35: 1566-1571, 1989. (abstract only) u51 Prosser, J.S., Moquet, J.E., Lloyd, D.C. and Edwards, A.A. Radiation induction of micronuclei in human lymphocytes. Mutat. Res. 199: 37-45, 1988. [161 Roberts, C.J., Morgan, G.R. and Holt, PD. A critical comparison of the micronucleus yield from high and low LET irradiation of plateau phase cell populations. Mutat. Res. 160: 237-242, 1986. u71 Rudd, N.L., Hoar, D.I., Greentree, CL., Dimnik, L.S. and Hennig, U.G. Micronucleus assay in human tibroblasts: a measure of spontaneous chromosomal instability and mutagen hypersensitivity. Environ. Mol. Mutagen. 12: 3-13, 1988. WI Shibamoto, Y., Shibata, T., Miyataka, S., Oda, Y., Manabe, T., Ohshio, G., Yagi, K. and Streffer, C. Assessment of proliferation activity and radiosensitivity of human tumours using the cytokinesis block micronucleus assay.Br. J. Cancer 70: 67-71, 1994. 1191 Shidnia, H., Crabtree, W., Hornback, N., Young, I?, Hartson, M. and Shen, R.N. Micronuclei assay - a predictive variable for tumour response to treatment. Adv. Exp. Med. Biol. 267: 51-55, 1990 (abstract only). WV Stich, H.F., Rosin, M.P., Homby, A.P., Mathew, B., Sankaranarayanan, R. and Nair, M.K. Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with betacarotene and beta-carotene plus vitamin A. Int. J. Cancer 42: 195-199, 1988. WI Streffer, C., van Beuningen, D. and Molls, M. Possibilities of the micronucleus test as an assay in radiotherapy. In: Progress in Radio-Oncology, pp. 243-251. Editors: K.H. Karcher, H.D. Kogelink and G. Reinartz. Raven Press, New York, 1981. WI Streffer, C., van Beuningen, D., Gross, E., Schabronath, J., Eigler, F.W. and Rabmann, A. Predictive assays for the therapy of rectum carcinoma. Radiother. Oncol. 5: 303-310, 1986. 1231 Tolbert, P.E., Shy, C.M. and Allen, J.W. Micronuclei and other nuclear anomalies in buccal smears: a field test in snuff users. Am. J. Epidemiol. 134: 840-850, 1991. [241 Villa, R., Zaffaroni, N., Gomatil, D., Costa, A. and Silverstrini, C. Lack of a correlation between micronucleus formation and radiosensitivity in established and primary cultures of human tumours. Br. J. Cancer 70: 1112-1111, 1994. ~251 Wilson, G.D., McNally, N.J., Dische, S. and Bennett, M.H. Cell proliferation in human tumours measured by in-vivo labelling with bromodeoxyuridine. Br. J. Radiol. 60: 419-422, 1988. WI Wilson, G.D., Martindale, CA., Soranson, J.A., Bourhis, J., Carl, U.M. and McNally, N.J. Radiation-induced cell cycle delay measured in two mouse tumours in vivo using bromodeoxyuridine. Radiat. Res. 137: 177-185, 1994. v71 Zolzer, F., Alberti, W., Pelzer, T., Lamberti, G., Hulskarnp, F.H. and Streffer, C. Changes in S-phase fraction and micronucleus frequency as prognostic factors in radiotherapy of cervical carcinoma. Radiotber. Oncol. 36: 128-132, 1995.
ELSEVIER
Radiotherapy and Oncology 41 (1996) 139-142
Short Communication
Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity V.N. Bhattathiri”,“,
L. Bindub, P. Remanic, B. Chandralekhad, C.A. Davis”, M. Krishnan Nair” “Department of Radiotherapy, Regional Cancer Centre, Trivandrum, India 695011 “Research Fellow and Cytotechnologist, Regional Cancer Centre, Trivandrum, India 695011 ‘Department of Research, Regional Cancer Centre, Trivandrum, India 695011 ‘Department of Pathology, Regional Cancer Centre, Trivandrum, India 6.9501 I ‘Department of Radiation Physics, Regional Cancer Centre, Trivandrum, India 695011
Received 27 February 1996; revised 26 June 1996; accepted 27 July 1996
Abstract Background and purpose: The micronucleus test, generally done in cultured tumour cells irradiated in vitro, has not gamed 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 Gyf 25 fractions/S 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,.‘%@ 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. Conclusion: 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. Keywords:
Micronucleus;
Cytology; Radiosensitivity;
Oral cancer; Repopulation
1. Introduction Micronuclei are chromosomesand/ or chromosomefragments, usually acentric, which lag behind during mitosis, fail to incorporate into the daughter nucleus and remain outside the main nucleus. Presence of a micronucleus suggeststhat the cell has suffered chromosome damage. Radiation or chemicals can induce micronuclei, and micronucleated cells (MNC) are considered as dying cells [7,10,16]. Micronucleus induction by radiation is doserelated, as first reported by Evans et al. [4]. The micronucleus test was then recognised as a quick method to *Corresponding author. Tel.: +91 471 445435; fax: +91 471 447454. 0167-8140/96/$1S.O0 0 PIZ SO167-8140(96)01810-S
1996 Elsevier
Science
Ireland
Ltd. All rights
reserved
evaluate chromosomal damage by radiation [8,11]. Cell survival [5,6] assayedby the micronucleus test correlates well with colony forming assay[ 14,151.Its potential value as a predictive test of human cancer radiosensitivity was recognisedby Streffer, more than a decadeago [21]. These tests are generally done by evaluation of micronuclei either in biopsy specimenstaken before and after a course of radiotherapy [22,27], or in tumour cell cultures irradiated in vitro [ 181. These studies suggestedthat the micronucleus test can predict radiosensitivity [ 121. Yet, experiments with multiple cell lines showed that the radiosensitivity ranking given by micronucleus test was different from that given by clonogenic assay [2,24]. Cytological screeningof oral mucosalcells for micronu-
140
VM. Bhattathiri
et al. I Radiotherapy
clei is frequently done to assess genotoxicity of carcinogens and evaluate the efficacy of presumed chemopreventive agents like betacarotene and vitamin A [13,20]. By cytology, Tolbert et al. showed that the micronucleus count in normal cheek mucosa is elevated after exposure to radiation [23]. However, micronucleus test by scrape smear cytology has not been tried as a method to predict human cancer radiosensitivity. The present study was undertaken to see whether serial cytological evaluation of micronucleus induction in tumours of patients receiving fractionated radiotherapy can predict oral cancer radiosensitivity.
and Oncology
41 (1996)
139-142
analyzed in relation to cumulative dose and overall treatment time. Within-group analysis was done by KruskalWallis one-way analysis of variance and between-group by Mann-Whitney U-test. A P value CO.05 was considered as significant. The relation between dose and MNC count was also checked by calculating the Pearson’s correlation coefficient, 1. Changes in relation to time was additionally analyzed in terms of mean daily MNC production, i.e. MNC count/number of days of treatment completed.
3. Results 2. Materials
and methods
Forty nine previously untreated patients with squamous cell carcinoma of the oral cavity (buccal mucosa: 29, gingiva: 13, hard palate: 5, and floor of mouth: 2), treated by radical radiotherapy (60 Gy/25 fractions/5 weeks, 2.4 Gyifractionlday, 5 days/week), with or without concurrent chemotherapy were included in the study. Clearance from the institutional screening committee and informed consent from the patients were obtained. Treatment was by tele-cobalt or 4 MV linear accelerator, by lateral and anterior, or single lateral portals. The doses were prescribed to the intersection or a depth, with homogeneity of better than 90%. At the time of analysis the median follow-up period is 14 months and the minimum 9 months. Pre-treatment scrape smears were collected from the tumour of all patients. Subsequently, a minimum of three smears were collected from each patient, after delivery of 2, 5, 8 or 12 fractions. Care was taken to ensure that smears were taken only from the tumour, avoiding adjacent normal areas. (Smears were not collected after 12 fractions because radiation mucositis made it difficult to distinguish tumour from normal tissue). Scraping was done with a previously wetted wooden spatula and the material immediately spread on a clean glass slide. It was then air dried for 30 min, stained with Giemsa’s, dehydrated and mounted on DPX. The slides were evaluated by light microscopy, and the number of nuclei and micronuclei in the tumour cells scored. Well defined round or oval bodies, less than a third of the nuclear size, with staining characteristics similar to the main nucleus and distinctly separate from it were considered as micronuclei. Cells containing one, two, and more than two nuclei or micronuclei were recorded as separate events. Around 500-1500 cells were evaluated on each occasion and results expressed as MNC count/ 1000 cells. During follow-up, 57.1% (28/49) of the patients developed recurrence of the primary tumour. For purposes of analysis, they were regarded as being ‘radioresistant (Resistant group). The remaining 21 (42.9%) who had no recurrence were grouped as being radiosensitive (Sensitive group). Changes in MNC count in these groups were
In both groups many tumours had MNC even before treatment. The mean values (range) were 4.17 (O-18.5) in the resistant and 4.12 (O-9.8) in the sensitive groups; the difference was not significant. Seven tumours of the sensitive (33.3%) and 5 (17.9) of the resistant groups did not have any MNC before treatment. There was statistically significant (P
Table 1 Change in MNC Dose (Gy)
0 4.8 12.0 19.2 24.0
count in relation Mean
to dose and time
time in days
MNCllOOO
cells (variance)
Sensitive
Resistant
Sensitive
Resistant
0 2.9 7.6 12.3 18.4
0 2.5 7.3 12.6 18.0
4.1 7.5 8.5 12.2 25.2
4.2 7.8 11.3 16.6 15.0
(12.3) (29.2) (29.2) (59.3) (529.0)
(31.4) (36.0) (34.8) (255.0) (184.9)
KN. Bhattathiri et al. I Radiotherapy and Oncology 41 (1996) 139-142
8‘.Oi5o.8. 0.6s
Fig.
1. Mean
daily
MNC
induction.
4. Discussion The MNC frequency of normal oral mucosa is about 1.3-2.6/1000 cells, with higher frequencies in those habituated to tobacco, arecanut, etc. [13,20]. In the present study, both resistant and sensitive tumours had MNC even before treatment. Presenceof a micronucleusin a cell with no known exposure to any genotoxic agent reflects its inherent chromosomal instability [17]. Since mitosis is necessaryfor expression of micronuclei, the pretreatment MNC count reflects both chromosomal instability and growth rate of the tumours, though the relative importance of the two may be different for sensitive and resistant tumours. Zolzer et al. [27] reported that cervical cancers with lower pre-treatment MNC frequency had better control with radiotherapy. Streffer et al. [21] reported that rectal cancers with elevated S-phase fraction had greater MNC count and suggestedthat the S-phase/micronucleus ratio is an index of cell turnover. In the present study, though the mean pre-treatment MNC count was similar in both groups, the variance was higher in the resistant group indicating greater heterogeneity in proliferation and inherent chromosomal instability. Both groups of tumours showed statistically significant dose-related increase in MNC count with radiotherapy. The maximum MNC count was higher and correlation with dose better in the sensitive group than in the resistant group. Also, when compared to the pre-treatment values, the maximum was greater in the sensitive group than in the resistantgroup, being 6.1 times and 3.6 times, respectively. These findings suggest that MNC induction is related to radiosensitivity. It is now well recognised that rapid proliferation is as important as intrinsic radioresistance in contributing to tumour recurrence following radiotherapy. Flowcytometric and bromodeoxyuridine studies have shown that epidermoid cancers of the head and neck can have potential doubling times (T,,,) as low as 2 days, with less than 5
141
days in the majority [1,25], and it is the rationale of accelerated treatment regimes like CHART [3]. In the present study, the sensitive and resistant tumours had different patterns of increase in MNC count in relation to overall treatment time, this being earlier in the resistant tumours. It is logical to assumethat the higher growth rate of the resistant tumours, which contributed to their recurrence, also resulted in the earlier expressionof micronuclei by them. The initial decreasein daily MNC production in sensitive tumours (Fig. 1) suggeststhat radiation-induced cell cycle delay may be more in sensitive/slowly proliferating tumours than in resistant/rapidly proliferating tumours, and this could be another reasonfor the differing patterns of MNC increase.A similar relation between cell cycle delay, proliferation and radiosensitivity has been reported in murine tumours [26]. In the caseof micronucleus induction by chemicals, it is seenthat constant, repeatedinjury with the samedoseof a chemical increasesthe number of MNCs to a plateau [9]. This indicates that a balancehas been reachedbetween cell loss and repopulation. The same is probably true for micronucleus induction by radiation, too. The patients in our study were receiving radiation at a doseof 2.4 Gy /day, almost daily. The plateau in MNC count in resistant tumour suggeststhat due to the combined effects of low radiosensitivity and high repopulation rate, a balance has been reached between cell kill and tumour repopulation, ultimately leading to tumour recurrence. In sensitive tumours the count does not reach a plateau becausecell death exceeds repopulation. The main drawback of our study is that the betweengroup differences in MNC count lack statistical significance. This is due to influence of other prognostic factors like tumour size, gross type, addition of chemotherapy, etc., and other modes of cell death like apoptosis, and presenceof other radiation-induced morphological abnormalities like multinucleation which were not consideredin this analysis. The micronucleus test as practiced now, in cultured tumour cells, is of uncertain value as a predictive assay of radiosensitivity. One plausible reason is that this gives no information regarding the proliferation characteristics of the tumour, particularly the changes induced by radiotherapy. The importance of monitoring both radiosensitivity and proliferation is emphasized by the report of Zolzer et al. [27]. They found that combined evaluation of radiosensitivity (by micronucleus assay) and growth fraction (by S-phasedetermination) can predict radiosensitivity of cervical cancers. Serial cytological assay of micronucleus induction can potentially identify both radiosensitivity and proliferation characteristics.The technique is almost completely non-invasive and easily done in accessible primary cancerslike that of oral cavity and uterine cervix. In other sites, fine needlecytology can be tried, asreported by Shidnia et al. [19] in a study of four patients with lymph node secondaries.
142
VT. Bhattathiri
et al. / Radiotherapy
Acknowledgments
This work was supported by a grant from the Science, Technology and Environmental Committee, Government of Kerala, India.
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