Ini J Rndmum Oncology Bml. Phys Vol. 24, PP. 675-680 Printed in the U.S.A. All rights reserved.
copyright
0X0-3016/92 $5.00 + .W 0 1992 Pergamon Press Ltd.
??Biology Original Contribution
RADIOSENSITIVITY J. RAMSAY, ANDN. MRC Unit and University
TESTING M.Sc.,
M.R.C.P.,
M. BLEEHEN,
Department
OF HUMAN F.R.C.R.,
F.R.C.P.,
of Clinical Oncology
F.R.C.R.,
MALIGNANT R. WARD, HON.
and Radiotherapeutics,
GLIOMAS
B.Sc.
F.A.C.R.
Hills Road, Cambridge,
CB2 2QR UK
Radiotherapy remains the main treatment modality for patients with malignant gliomas and is the only treatment which significantly prolongs survival. Clonogenic and tetrazolium based calorimetric assays (MTT) of early passage cultures have been performed following 2 Gy doses of x-rays in order to determine if in vitro radiosensitivity is a factor in response to treatment. Of 47 biopsies received, 39 were established in primary culture. A value of surviving fraction to 2 Gy (SF2) was obtained in 85% of growth assays and 64% of clonogenic assays. The mean SF2 value for the MIT was 0.56 which was significantly higher than the 0.42 obtained for the clonogenic assay. There was, however, reasonable qualitative agreement in assessing relative radiosensitivity/radioresistance (r = 0.7). Mean SF2 values for grade 3 tumors were 0.52 (M’IT) and 0.35 (clonogenic) as against mean SF2 values of 0.63 (MIT) and 0.47 (clonogenic assay) for grade 4 tumors. In 24 patients with adequate follow-up, no direct correlation was found between SF2 and survival, although mean SF2 values for patients surviving greater than 18 months was significantly less (p = 0.01) than patients surviving less than 18 months as determined by the M’IT assay. Malignant gliomas, Radiosensitivity, MTT assay.
has been directly to compare the use of a semiautomated tetrazolium based calorimetric assay (MTT) (17) with standard clonogenic assay for these measurements. The MTT assay has been used extensively for in vitro chemosensitivity testing (1, 4, 23) and in a more limited capacity, for radiosensitivity testing (5, 18).
INTRODUCTION There is considerable interest in measuring the intrinsic radiosensitivity of human tumors. Historically, it has long
been recognised that tumors of different histological type have varying clinical radiosensitivity. More recently it was shown that the observed clinical variation in radiosensitivity could be explained in part by differences of in vitro radiosensitivity. In particular, it was shown that these differences were best distinguished by the response to low doses of 1.5 Gy to 2 Gy (7, 8, 9, 14). Within each histological type, however, it was clear that there was considerable heterogeneity between different cultures. It is this spread of radiosensitivities within a particular tumor type which has encouraged investigators to examine if in vitro radiosensitivity measurement can be used to predict radiation response in individual patients. Mathematical modeling would suggest that this has potential for being a good predictor (22) and there have been reports of correlation with patient outcome (2,3,6). We have examined the in vitro radiosensitivity of early passage cultures established from biopsies of human malignant gliomas (anaplastic astrocytomas and glioblastoma multiforme). Values of surviving fraction to 2 Gy (SF2) have been obtained to examine for heterogeneity between cultures and to see if this heterogeneity is sufficient for predicting response to radiotherapy. An important part of the work
Reprint requests to: Dr. J. Ramsay, stitute. Brisbane Qld 4029, Australia.
Queensland
Radium
METHODS
AND
MATERIALS
Establishment of cultures Biopsies were obtained from patients undergoing craniotomy for intracranial tumor. Histology was confirmed by neuropathologists at Addenbrookes Hospital as Grade 3 or 4 malignant glioma (anaplastic astrocytomas or glioblastoma multiforme). Samples were collected in Hams F 10 culture medium supplemented with penicillin, streptomycin and amphotericin B. Disaggregation of samples was initiated within 6 hr of surgery. The specimen was cut into l-2 mm pieces and then suspended in 9 ml of media with 1 ml of collagenase to give a concentration of 200 units ml-‘. After 1-2 hr of incubation the samples were disaggregated further with pipetting and the resulting cell suspension washed in fresh media and transferred to a 25 ml flask. Depending on growth rate and size of sample a confluent monolayer took 2-6 weeks to form. For the radiation experiments cells were subcultured and used at
Accepted
In-
675
for publication
2 1 April 1992.
676
I. J. Radiation Oncology 0
Biology??Physics
Volume 24, Number 4, 1992
passages of 1 to 10. Cultures were maintained with Hams F 10 supplemented with 10% foetal calf serum and buffered with sodium bicarbonate and 5% COZ. Radiation assays Monolayer cultures were trypsinized in exponential growth phase and counted using a haemocytometer. Cells were irradiated at room temperature using a 250kV x-ray machine at a dose rate of 67 cGy/min. Doses used were 1 to 5 Gray. For clonogenic assays cells were plated on 9 cm plastic plates with 3 to 6 plates used for each point. After 4 weeks of incubation, the dishes were washed in saline, fixed in ethanol, and stained with 0.5% crystal violet. Colonies with greater than 50 cells were counted and survival was expressed by comparing the number of colonies in control dishes with irradiated dishes. Average plating efficiency was 3.2% with a range of 0.4 to 14%. MTT assays were carried out at the same time as the clonogenic assay. The assay involves the ability of cells to convert a soluble tetrazolium salt 3-4,5 dimethylthiazol2, 5 dipheryl tetrazolium bromide into an insoluble formazan precipitate. The purple colored crystals are then dissolved in an organic solvent and the optical density of the solution measured on a multiwell spectrophotometer. Cells were irradiated in suspension and then immediately plated out on 96 well microtiter plates at 2,000 cells well. Eight to 16 wells were used for each radiation dose. Time of analysis was determined individually for each culture. Control cells were counted once or twice weekly by trypsinizing eight wells and obtaining the mean cell count per well. When this had increased to greater than 16,000 cells well (three doublings), the analysis was performed. This point was chosen as a compromise between allowing the irradiated cells to express radiation damage and before the control cells began to plateau out (> four doublings) (Fig. I). Taking a fixed time point after radiation would not have taken into account the widely different growth rates of individual cultures. In six cultures, little growth was obtained over 3-4 weeks and these assays were aborted. The assay was performed by adding 20 ~1 of MTT (dissolved in phosphate buffered saline at a concentration of 5 mg/ml-‘) to each well and the plates incubated for 5 hr at 37°C. The medium was then aspirated carefully and 200 ~1 of DMSO added to each well. The plates were agitated on a plate shaker for 10 min and optical density measurements carried out immediately using a Titertek Multiskan plate recorder.* The surviving fraction was obtained by comparing the irradiated optical density with control optical density. Clinical information To determine if there was any relationship between in vitro radiosensitivity as measured by SF2 values and patient outcome, the clinical records of all the patients from
* Eflab Oy, Helsinki, Finland.
IO
Days
+ ,L
1Gy
U
2Gy
*
3Gy
IO
20
Days Fig. 1. The value for SF2 using the MTT assay was determined by counting control cells on Day 7, 12, 15, and 19. By Day 12 control cells had reached 19,000 cells/well at which time the MTT would be performed to give control O.D. of 0.98 and 2 Gy O.D. of 0.81 (SF2 calculated as 0.83). If analysis delayed until Day 19, then growth of control cells had slowed and SF2
would be calculated as 0.96 (Data from culture G24). whom biopsies were received were reviewed. Details of final histology, age, performance status, treatment details, and survival were recorded. Statistical analysis Significance of results was assessed using Students 2 tailed t-test. RESULTS
Assay success rate Of 47 specimens confirmed as histological Grade 3 or 4 gliomas, 39 (83%) were established in primary culture. Four specimens were contaminated and four failed to
Radiosensitivity of human malignant gliomas 0 J.
RAMSAY
677
ef a/.
grow. Radiosensitivity testing was performed using concurrent clonogenic and MTT assays. A test result was obtained in 33 MTT assays and 25 clonogenic assays. Overall success rate was thus 70% for the MTT assay and 53% for the clonogenic assay. Distribution oj’SF2 values Figure 2 shows the distribution of SF2 values obtained with the two assays. Mean value for the MTT assay was 0.56 (range 0.33-0.91). This was significantly higher (p < 0.01) than the value obtained for the clonogenic assay of 0.42 (range 0.12-0.66). Heterogeneity between the cultures gave a coefficient variation (CV) of 30% for the MTT assay and 36% for the clonogenic assay.
0.04 0.0
I
I
0.2
0.4
.
I
I
0.6
0.8
’
1.0
SF 2Gy Clonogenic
Experimental variation Repeat assays were performed on the same culture to determine the amount of experimental variation. Three to 5 repeat assays were performed on six cultures. The CV for repeat assays was 15% for MTT assay and 13% for clonogenic assay. Thus the variation between individual cultures is considerably greater than the experimental variation. It is this difference between the overall CV and the experimental CV which determines the usefulness of the assay for predicting response in individual patients. Correlation between assays Results for both assays carried out concurrently were obtained in 24 cases allowing direct comparison of results. In Figure 3, individual values of SF2 obtained for the 24 tumors are plotted. Although the MTT assays gave an average higher value for SF2 than the clonogenic assay, there was reasonable qualitative agreement (correlation coefficient = 0.7). In Table 1 the cultures have been divided into three equal groups: a radiosensitive group, an average sensitivity group and a resistant group. In only
60
Fig. 3. Values of SF2 for MTT and clonogenic assays are plotted for 24 cultures in which results were obtained for both assays carried out concurrently.
one case was a tumor tested as radiosensitive by one assay and as resistant by the other. SF2 and tumor grade Of the 33 patients on whom SF2 data was obtained, 18 had Grade 3 tumors, 13 Grade 4 tumors and 2 mixed Grade 3 and 4. Mean SF2 with the MTT assay was 0.52 for Grade 3 tumors and 0.63 for Grade 4 tumors (p = 0.06). For clonogenic assay, mean SF2 was 0.35 for Grade 3 tumors and 0.47 for Grade 4 tumors (p = 0.05). SF2 and survival Of 33 patients on whom SF2 data was obtained, 24 were evaluable to determine if there was any relationship with SF2 and treatment outcome. Patients were only included if they received radical radiotherapy (45-60 Gy) and had either died of uncontrolled glioma or survived for greater than 18 months after diagnosis. Two of the nine patients excluded were alive with follow-up less than 18 months, six had not received radiotherapy, and one had died of an unrelated cause. Individual values for SF2 and survival are plotted in Figure 4. For both assays there is no direct correlation between SF2 and length of survival. Analysis of long term survivors of greater than 18 months, shows that they had a mean SF2 of 0.44 (MTT) and 0.32 (clonogenic) comTable 1. Clonogenic
u.0
0.2
0.4
0.6
0.8
1 _I
SF2 Fig. 2. Comparison of the cumulative frequency distribution of SF2 values obtained for 33 tumors using the MTT assay and 25 tumors using the clonogenic assay.
MTT
Sensitive SF2 5 .48 Average .61 .48 Resistant SF2 z .61
Sensitive SF2 5 .39
Average .47 i SF2 > .39
Resistant SF2 2 .41
6
2
0
I
4
2
1
2
6
678
I. J. Radiation Oncology 0 Biology0 Physics
Survival
(weeks)
0 0
0.6 -
0
0
910Oo
0.6 -
0.4 -
00 0 0
0 0
8
0 0
0
0
0.2 -
o.oI 0
100 Survival
Fig. 4. Individual weeks.
200
(weeks)
values of SF2 are plotted against survival in
pared to SF2 values of 0.62 (MTT) and 0.45 (clonogenic)
for patients surviving less than 18 months. This difference was significant for the MTT assay (p = 0.0 1). All patients surviving more than 18 months had Grade 3 tumors. DISCUSSION
The overall results of treatment of high grade glioma are poor, with a median survival of around 9 months (12). Radiotherapy is the only treatment that has been consistently shown to extend survival after surgery (2,24) and a few patients will be long term survivors. Retrospective studies (25) have shown evidence of a dose response for radiotherapy and this has recently been confirmed in a random&d study (15) showing a significant increase in median survival with 60 Gy versus 45 Gy. In view of this dose response observed and the fact that most patients die of uncontrolled local disease, there is considerable interest in the use of new techniques to increase
Volume 24, Number 4, 1992
the total dose administered either by interstitial implantation ( 11) or radiosurgery (13). One factor that has been considered to be important clinically is the relative radioresistance of this group of tumors. In vitro radiosensitivity on established cell lines would tend to confirm this with values of SF2 often greater than 0.5 (10, 14, 19, 2 1, 27). In the present study the mean value for a large number of early passage cultures tested by clonogenic assay was less at 0.42 but with a wide range of 0.12 to 0.66. Suit, (20) in a review of the data, has shown that the actual values obtained will vary considerably with the assay conditions. For example, for squamous cell carcinomas SF2 values of 0.5 were obtained on established cultures compared to 0.25 for primary cultures. This difference between early passage/primary cultures and established cell lines is not clear but may be due to the selective growth of more resistant clones with increasing passage, or due to the influence of normal stromal cells on the overall radiation response in primary cultures. Repeat assays, however, on our cultures from Pl to PlO have not shown a trend towards increasing radioresistance. It should also be noted that most published data is on Grade 4 tumors rather than Grade 3 tumors. In the present studies, the mean SF2 value for Grade 4 tumors using clonogenic assay is higher at 0.47. Seven tumors analysed had SF2 values of less than 0.3 ranking them among the most radiosensitive of tumors. DNA analysis of this subgroup has shown that six are aneuploid confirming their malignant origin with the remaining diploid tumor having the loss of one sex chromosome on karyotype analysis. While three of the six on whom clinical data is available survived greater than 18 months, two of the remaining had the shortest survival recorded. It is thus likely that factors, other than radiation resistance, are important in the poor treatment results such as lack of host immunological response and tumor physiology. Of interest is that a radiosensitive tumor such as lymphoma when arising as a primary tumor in the brain is rarely controlled with radiotherapy alone. The MTT assay under our experimental conditions gave a higher mean value for SF2. It is clear, however, that the actual values obtained for a growth assay such as the MTT will vary considerably with the assay conditions (18). By using established cell lines Carmichael (5) showed that delaying analysis until control cells had undergone at least six doublings gave results comparable to clonogenie assay. In the present studies, it was not possible to maintain relatively slow-growing cells in the multiwell plates for this number of doublings and, as a compromise, analysis was performed after three doublings. This allowed 2,000 cells to be plated for each well and analysis performed when the control cell number reached approximately 16,000. For most cell lines, this gave a control optical density of 0.8-1.0 which is optimal for dose response studies. While actual values obtained for SF2 were usually higher, there was reasonable qualitative agreement between the assays. It is also worth considering whether
Radiosensitivity of human malignant gliomas 0 J. RAMSAY el al.
the results obtained from growth assays should always equate with clonogenic assay, as essentially they are measuring different things; the MTT assay is measuring changes in total cell number, while the clonogenic assay is measuring the survival of clonogens which may comprise less than 1% of the total cell number. Advantages of the MTT assay in the present study are the increased proportion of successful tests (85% vs 64%) and the shorter time taken to obtain a result. If an assay is to be useful in predicting response in individual patients, then the proportion of successful tests is critical. The semiautomated nature of the analysis also lends itself more to routine testing than the clonogenic assay. To date, a common finding is the degree of heterogeneity between tumors of the same histological type. For two large series in head and neck cancer and carcinoma of the cervix, the coefficient of variation was around 40% (3,6). In both cases this was considerably greater than the variations seen for repeat assays and variation obtained from separate biopsies from a single tumor. Similar results have been obtained in the present study, although the degree of heterogeneity was slightly less at 36%. With this degree of heterogeneity there is potential in employing
619
such measurements as predictors of outcome of radiotherapy. For a group of 24 patients we have shown that long survival (18 months) is related to a lower SF2 value and this reached statistical significance for the MTT assay. One can speculate that the MTT assay might be more likely to predict response in this group of tumors as response to radiotherapy is measured more in terms of growth delay than eradication of all clonogens. It is likely that biological measurements such as SF2 will not be clinically useful unless other clinical factors are taken into account. For example, for gliomas a prognostic index has been developed using four clinical factors which allows one to identify a group with a 2-year survival of -35% against another group with a 2-year survival of 0% (16). In summary, we have shown that the MTT assay can be used under well defined experimental conditions to give qualitatively similar results to the clonogenic assay for radiosensitivity testing. For gliomas, a wide range of SF2 values have been obtained and for the MTT assay there was a trend towards low SF2 and increased survival. However, in view of considerable overlap, it is unlikely that SF2 would be useful in the clinical management of these patients.
REFERENCES 1. Alley, M. C.; Scudero, D. A.; Morris, A.; Hursey, M. L.; Czerwnski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J.; Shoemaker, R. H.; Boyd, M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48:589-601;1988. 2. Anderson, A. P. Post operative irradiation ofglioblastomas. Results in a randomised series. Acta Radio]. 17:475484;1978. 3. Brock, W.; Baker, F.; Wike, J.; Sivon, S.; Peters, L. Cellular radiosensitivity of primary head and neck squamous cell carcinomas and local tumor control. Int. J. Radiat. Oncol. Biol. Phys. 18: 1283- 1286;1990. 4. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated calorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47:936-942; 1987. 5. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated calorimetric assay: Assessment of radiosensitivity. Cancer Res. 47:943-946;1987. 6. Davidson, S. E.; West, M. L.; Roberts, S. A.; Hendry, J. H.; Hunter, R. D. Radiosensitivity testing of primary cervical carcinoma: Evaluation of intra- and inter-tumour heterogeneity. Radioth. Oncol. 18:349-356; 1990. 7. Deacon, J.; Peckham, M. J.; Steel, G. The radioresponsiveness of human tumours and the initial slope of the cell survival curve. Radioth. Oncol. 2:317-323;1984. 8. Fertil, B.; Malaise, E. P. Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors: Analysis of 101 published survival curves. Int. J. Radiat. Oncol. Biol. Phys. 11:1699-1707;1985. 9. Fertil, B.; Malaise, E. P. Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 7:62 l-629; 198 1. 10. Gerwek, L. E.; Komblith, P. L.; Burlett, P.; Wang, J.; Sweig-
Il.
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20.
ert. S. Radiation sensitivity of cultured human glioblastoma cells. Radiology. 125:23 1-234;1977. Gutin, P. H.; Philips, T. L.; Wara, W. M.; Leibel, S. A.; Hosobuchi, Y.; Levin, V. A.: Weaver, K. A.; Lamb, S. Brachytherapy of recurrent malignant brain tumours with removable high activity iodine- 125 sources. J. Neurosurg. 60:6 l-68; 1984. Kelly, K. A.; Kirkwood, J. M.; Kapp. D. S. Glioblastoma multiforme: pathology, natural history and treatment. Cancer Treat. Rev. 11: l-26; 1984. Lunsford, L. D.; Flickinger, J.; Coffey, R. J. Stereotactic gamma knife radiosurgery. Initial North American experience in 207 patients. Arch. Neurol. 47: 169- 175; 1990. Malaise, E. P.; Fertil, B.; Chavandra, N.; Guichard, M. Distribution of radiation sensitivities for human tumor cells of specific histological types. Comparison of in vitro to in vivo data. Int. J. Radiat. Oncol. Biol. Phys. 12:617-624;1986. Medical Research Council Brain Tumour Working Party. A medical research council trial of two radiotherapy doses in the treatment of grades 3 and 4 astrocytoma. Br. J. Cancer (In press) 1992. Medical Research Council Brain Tumour Working Party. Prognostic factors for high-grade glioma: Development of a prognostic index. J. Neuro-Oncol. 9:47-55; 1990. Mosmann, T. Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65:55-63;1983. Price, P.; McMillan, T. J. Use of the tetrazolium assay in measuring the response of human tumor cells to ionizing radiation. Cancer Res. 50: 1392- 1396; 1990. Schultz, C. J.; Geard, C. R. Radioresponse of human astrocytic tumors across grade as a function of acute and chronic irradiation. Int. J. Radiat. Oncol. Biol. Phys. 19:13971403;1990. Suit, H. D.; Baumann, S.; Skates, S.; Convery, K. Clinical
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23.
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interest in determinations of cellular radiation sensitivity. Int. J. Radiat. Biol. 56:725-737;1989. Suit, H. D.; Zietman, A.; Tomkinson, K.; Ramsay, J.; Gerweek, L.; Sedlacek, R. Radiation response of xenografts of a human squamous cell carcinoma and a glioblastoma multiforme: A progress report. Int. J. Radiat. Oncol. Biol. Phys. 18:365-373;1990. Tucker, S. L.; Thomas, H. D. The effect ofpatient to patient variability on the accuracy of predictive assays of tumor response to radiotherapy. A theorectical evaluation. Int. J. Radiat. Oncol. Biol. Phys. 17: 146- 159; 1989. Twentyman, P. R.; Fox, N. E.; Rees, J. K. H. Chemosensitivity testing of fresh leukaemia cells using the MTT colorimetric assay. Br. J. Haematol. 7 1:19-24; 1989. Walker, M. D.; Alexander, E.; Hunt, W. E.; Maclarty,
Volume 24, Number 4, 1992 C. S.; Maholey, M. S.; Mealey, J.; Norrell, H. A.; Owens, G.; Ransohoff, J.; Wilson, C. B.; Gehan, E. A.; Strike, T. A. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. J. Neurosurg. 49:333-343; 1978. 25. Walker, M. D.; Strike, T. A.; Sheline, G. E. An analysis of dose effect relationship in the radiotherapy of malignant glioma. Int. J. Radiat. Oncol., Biol. Phys. 5: 1725- 173 1;1979. 26. Weichselbaum, R. R.; Beckett, M. A.; Schwartz, J. L.; Dritschilo, A. Radioresistant tumor cells are present in head and neck carcinomas that recur after radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 15:575-579;1988. 27. Yang, X.; Darling, J. L.; McMillan, T. J.; Peacock, J. H.; Steel, G. G. Radiosensitivity, recovery and dose-rate effect in three human glioma cell lines. Radioth. Oncol. 19:4956;1990.
copyright
0X0-3016/92 $5.00 + .W 0 1992 Pergamon Press Ltd.
??Biology Original Contribution
RADIOSENSITIVITY J. RAMSAY, ANDN. MRC Unit and University
TESTING M.Sc.,
M.R.C.P.,
M. BLEEHEN,
Department
OF HUMAN F.R.C.R.,
F.R.C.P.,
of Clinical Oncology
F.R.C.R.,
MALIGNANT R. WARD, HON.
and Radiotherapeutics,
GLIOMAS
B.Sc.
F.A.C.R.
Hills Road, Cambridge,
CB2 2QR UK
Radiotherapy remains the main treatment modality for patients with malignant gliomas and is the only treatment which significantly prolongs survival. Clonogenic and tetrazolium based calorimetric assays (MTT) of early passage cultures have been performed following 2 Gy doses of x-rays in order to determine if in vitro radiosensitivity is a factor in response to treatment. Of 47 biopsies received, 39 were established in primary culture. A value of surviving fraction to 2 Gy (SF2) was obtained in 85% of growth assays and 64% of clonogenic assays. The mean SF2 value for the MIT was 0.56 which was significantly higher than the 0.42 obtained for the clonogenic assay. There was, however, reasonable qualitative agreement in assessing relative radiosensitivity/radioresistance (r = 0.7). Mean SF2 values for grade 3 tumors were 0.52 (M’IT) and 0.35 (clonogenic) as against mean SF2 values of 0.63 (MIT) and 0.47 (clonogenic assay) for grade 4 tumors. In 24 patients with adequate follow-up, no direct correlation was found between SF2 and survival, although mean SF2 values for patients surviving greater than 18 months was significantly less (p = 0.01) than patients surviving less than 18 months as determined by the M’IT assay. Malignant gliomas, Radiosensitivity, MTT assay.
has been directly to compare the use of a semiautomated tetrazolium based calorimetric assay (MTT) (17) with standard clonogenic assay for these measurements. The MTT assay has been used extensively for in vitro chemosensitivity testing (1, 4, 23) and in a more limited capacity, for radiosensitivity testing (5, 18).
INTRODUCTION There is considerable interest in measuring the intrinsic radiosensitivity of human tumors. Historically, it has long
been recognised that tumors of different histological type have varying clinical radiosensitivity. More recently it was shown that the observed clinical variation in radiosensitivity could be explained in part by differences of in vitro radiosensitivity. In particular, it was shown that these differences were best distinguished by the response to low doses of 1.5 Gy to 2 Gy (7, 8, 9, 14). Within each histological type, however, it was clear that there was considerable heterogeneity between different cultures. It is this spread of radiosensitivities within a particular tumor type which has encouraged investigators to examine if in vitro radiosensitivity measurement can be used to predict radiation response in individual patients. Mathematical modeling would suggest that this has potential for being a good predictor (22) and there have been reports of correlation with patient outcome (2,3,6). We have examined the in vitro radiosensitivity of early passage cultures established from biopsies of human malignant gliomas (anaplastic astrocytomas and glioblastoma multiforme). Values of surviving fraction to 2 Gy (SF2) have been obtained to examine for heterogeneity between cultures and to see if this heterogeneity is sufficient for predicting response to radiotherapy. An important part of the work
Reprint requests to: Dr. J. Ramsay, stitute. Brisbane Qld 4029, Australia.
Queensland
Radium
METHODS
AND
MATERIALS
Establishment of cultures Biopsies were obtained from patients undergoing craniotomy for intracranial tumor. Histology was confirmed by neuropathologists at Addenbrookes Hospital as Grade 3 or 4 malignant glioma (anaplastic astrocytomas or glioblastoma multiforme). Samples were collected in Hams F 10 culture medium supplemented with penicillin, streptomycin and amphotericin B. Disaggregation of samples was initiated within 6 hr of surgery. The specimen was cut into l-2 mm pieces and then suspended in 9 ml of media with 1 ml of collagenase to give a concentration of 200 units ml-‘. After 1-2 hr of incubation the samples were disaggregated further with pipetting and the resulting cell suspension washed in fresh media and transferred to a 25 ml flask. Depending on growth rate and size of sample a confluent monolayer took 2-6 weeks to form. For the radiation experiments cells were subcultured and used at
Accepted
In-
675
for publication
2 1 April 1992.
676
I. J. Radiation Oncology 0
Biology??Physics
Volume 24, Number 4, 1992
passages of 1 to 10. Cultures were maintained with Hams F 10 supplemented with 10% foetal calf serum and buffered with sodium bicarbonate and 5% COZ. Radiation assays Monolayer cultures were trypsinized in exponential growth phase and counted using a haemocytometer. Cells were irradiated at room temperature using a 250kV x-ray machine at a dose rate of 67 cGy/min. Doses used were 1 to 5 Gray. For clonogenic assays cells were plated on 9 cm plastic plates with 3 to 6 plates used for each point. After 4 weeks of incubation, the dishes were washed in saline, fixed in ethanol, and stained with 0.5% crystal violet. Colonies with greater than 50 cells were counted and survival was expressed by comparing the number of colonies in control dishes with irradiated dishes. Average plating efficiency was 3.2% with a range of 0.4 to 14%. MTT assays were carried out at the same time as the clonogenic assay. The assay involves the ability of cells to convert a soluble tetrazolium salt 3-4,5 dimethylthiazol2, 5 dipheryl tetrazolium bromide into an insoluble formazan precipitate. The purple colored crystals are then dissolved in an organic solvent and the optical density of the solution measured on a multiwell spectrophotometer. Cells were irradiated in suspension and then immediately plated out on 96 well microtiter plates at 2,000 cells well. Eight to 16 wells were used for each radiation dose. Time of analysis was determined individually for each culture. Control cells were counted once or twice weekly by trypsinizing eight wells and obtaining the mean cell count per well. When this had increased to greater than 16,000 cells well (three doublings), the analysis was performed. This point was chosen as a compromise between allowing the irradiated cells to express radiation damage and before the control cells began to plateau out (> four doublings) (Fig. I). Taking a fixed time point after radiation would not have taken into account the widely different growth rates of individual cultures. In six cultures, little growth was obtained over 3-4 weeks and these assays were aborted. The assay was performed by adding 20 ~1 of MTT (dissolved in phosphate buffered saline at a concentration of 5 mg/ml-‘) to each well and the plates incubated for 5 hr at 37°C. The medium was then aspirated carefully and 200 ~1 of DMSO added to each well. The plates were agitated on a plate shaker for 10 min and optical density measurements carried out immediately using a Titertek Multiskan plate recorder.* The surviving fraction was obtained by comparing the irradiated optical density with control optical density. Clinical information To determine if there was any relationship between in vitro radiosensitivity as measured by SF2 values and patient outcome, the clinical records of all the patients from
* Eflab Oy, Helsinki, Finland.
IO
Days
+ ,L
1Gy
U
2Gy
*
3Gy
IO
20
Days Fig. 1. The value for SF2 using the MTT assay was determined by counting control cells on Day 7, 12, 15, and 19. By Day 12 control cells had reached 19,000 cells/well at which time the MTT would be performed to give control O.D. of 0.98 and 2 Gy O.D. of 0.81 (SF2 calculated as 0.83). If analysis delayed until Day 19, then growth of control cells had slowed and SF2
would be calculated as 0.96 (Data from culture G24). whom biopsies were received were reviewed. Details of final histology, age, performance status, treatment details, and survival were recorded. Statistical analysis Significance of results was assessed using Students 2 tailed t-test. RESULTS
Assay success rate Of 47 specimens confirmed as histological Grade 3 or 4 gliomas, 39 (83%) were established in primary culture. Four specimens were contaminated and four failed to
Radiosensitivity of human malignant gliomas 0 J.
RAMSAY
677
ef a/.
grow. Radiosensitivity testing was performed using concurrent clonogenic and MTT assays. A test result was obtained in 33 MTT assays and 25 clonogenic assays. Overall success rate was thus 70% for the MTT assay and 53% for the clonogenic assay. Distribution oj’SF2 values Figure 2 shows the distribution of SF2 values obtained with the two assays. Mean value for the MTT assay was 0.56 (range 0.33-0.91). This was significantly higher (p < 0.01) than the value obtained for the clonogenic assay of 0.42 (range 0.12-0.66). Heterogeneity between the cultures gave a coefficient variation (CV) of 30% for the MTT assay and 36% for the clonogenic assay.
0.04 0.0
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Experimental variation Repeat assays were performed on the same culture to determine the amount of experimental variation. Three to 5 repeat assays were performed on six cultures. The CV for repeat assays was 15% for MTT assay and 13% for clonogenic assay. Thus the variation between individual cultures is considerably greater than the experimental variation. It is this difference between the overall CV and the experimental CV which determines the usefulness of the assay for predicting response in individual patients. Correlation between assays Results for both assays carried out concurrently were obtained in 24 cases allowing direct comparison of results. In Figure 3, individual values of SF2 obtained for the 24 tumors are plotted. Although the MTT assays gave an average higher value for SF2 than the clonogenic assay, there was reasonable qualitative agreement (correlation coefficient = 0.7). In Table 1 the cultures have been divided into three equal groups: a radiosensitive group, an average sensitivity group and a resistant group. In only
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Fig. 3. Values of SF2 for MTT and clonogenic assays are plotted for 24 cultures in which results were obtained for both assays carried out concurrently.
one case was a tumor tested as radiosensitive by one assay and as resistant by the other. SF2 and tumor grade Of the 33 patients on whom SF2 data was obtained, 18 had Grade 3 tumors, 13 Grade 4 tumors and 2 mixed Grade 3 and 4. Mean SF2 with the MTT assay was 0.52 for Grade 3 tumors and 0.63 for Grade 4 tumors (p = 0.06). For clonogenic assay, mean SF2 was 0.35 for Grade 3 tumors and 0.47 for Grade 4 tumors (p = 0.05). SF2 and survival Of 33 patients on whom SF2 data was obtained, 24 were evaluable to determine if there was any relationship with SF2 and treatment outcome. Patients were only included if they received radical radiotherapy (45-60 Gy) and had either died of uncontrolled glioma or survived for greater than 18 months after diagnosis. Two of the nine patients excluded were alive with follow-up less than 18 months, six had not received radiotherapy, and one had died of an unrelated cause. Individual values for SF2 and survival are plotted in Figure 4. For both assays there is no direct correlation between SF2 and length of survival. Analysis of long term survivors of greater than 18 months, shows that they had a mean SF2 of 0.44 (MTT) and 0.32 (clonogenic) comTable 1. Clonogenic
u.0
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SF2 Fig. 2. Comparison of the cumulative frequency distribution of SF2 values obtained for 33 tumors using the MTT assay and 25 tumors using the clonogenic assay.
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Sensitive SF2 5 .48 Average .61 .48 Resistant SF2 z .61
Sensitive SF2 5 .39
Average .47 i SF2 > .39
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Survival
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Fig. 4. Individual weeks.
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pared to SF2 values of 0.62 (MTT) and 0.45 (clonogenic)
for patients surviving less than 18 months. This difference was significant for the MTT assay (p = 0.0 1). All patients surviving more than 18 months had Grade 3 tumors. DISCUSSION
The overall results of treatment of high grade glioma are poor, with a median survival of around 9 months (12). Radiotherapy is the only treatment that has been consistently shown to extend survival after surgery (2,24) and a few patients will be long term survivors. Retrospective studies (25) have shown evidence of a dose response for radiotherapy and this has recently been confirmed in a random&d study (15) showing a significant increase in median survival with 60 Gy versus 45 Gy. In view of this dose response observed and the fact that most patients die of uncontrolled local disease, there is considerable interest in the use of new techniques to increase
Volume 24, Number 4, 1992
the total dose administered either by interstitial implantation ( 11) or radiosurgery (13). One factor that has been considered to be important clinically is the relative radioresistance of this group of tumors. In vitro radiosensitivity on established cell lines would tend to confirm this with values of SF2 often greater than 0.5 (10, 14, 19, 2 1, 27). In the present study the mean value for a large number of early passage cultures tested by clonogenic assay was less at 0.42 but with a wide range of 0.12 to 0.66. Suit, (20) in a review of the data, has shown that the actual values obtained will vary considerably with the assay conditions. For example, for squamous cell carcinomas SF2 values of 0.5 were obtained on established cultures compared to 0.25 for primary cultures. This difference between early passage/primary cultures and established cell lines is not clear but may be due to the selective growth of more resistant clones with increasing passage, or due to the influence of normal stromal cells on the overall radiation response in primary cultures. Repeat assays, however, on our cultures from Pl to PlO have not shown a trend towards increasing radioresistance. It should also be noted that most published data is on Grade 4 tumors rather than Grade 3 tumors. In the present studies, the mean SF2 value for Grade 4 tumors using clonogenic assay is higher at 0.47. Seven tumors analysed had SF2 values of less than 0.3 ranking them among the most radiosensitive of tumors. DNA analysis of this subgroup has shown that six are aneuploid confirming their malignant origin with the remaining diploid tumor having the loss of one sex chromosome on karyotype analysis. While three of the six on whom clinical data is available survived greater than 18 months, two of the remaining had the shortest survival recorded. It is thus likely that factors, other than radiation resistance, are important in the poor treatment results such as lack of host immunological response and tumor physiology. Of interest is that a radiosensitive tumor such as lymphoma when arising as a primary tumor in the brain is rarely controlled with radiotherapy alone. The MTT assay under our experimental conditions gave a higher mean value for SF2. It is clear, however, that the actual values obtained for a growth assay such as the MTT will vary considerably with the assay conditions (18). By using established cell lines Carmichael (5) showed that delaying analysis until control cells had undergone at least six doublings gave results comparable to clonogenie assay. In the present studies, it was not possible to maintain relatively slow-growing cells in the multiwell plates for this number of doublings and, as a compromise, analysis was performed after three doublings. This allowed 2,000 cells to be plated for each well and analysis performed when the control cell number reached approximately 16,000. For most cell lines, this gave a control optical density of 0.8-1.0 which is optimal for dose response studies. While actual values obtained for SF2 were usually higher, there was reasonable qualitative agreement between the assays. It is also worth considering whether
Radiosensitivity of human malignant gliomas 0 J. RAMSAY el al.
the results obtained from growth assays should always equate with clonogenic assay, as essentially they are measuring different things; the MTT assay is measuring changes in total cell number, while the clonogenic assay is measuring the survival of clonogens which may comprise less than 1% of the total cell number. Advantages of the MTT assay in the present study are the increased proportion of successful tests (85% vs 64%) and the shorter time taken to obtain a result. If an assay is to be useful in predicting response in individual patients, then the proportion of successful tests is critical. The semiautomated nature of the analysis also lends itself more to routine testing than the clonogenic assay. To date, a common finding is the degree of heterogeneity between tumors of the same histological type. For two large series in head and neck cancer and carcinoma of the cervix, the coefficient of variation was around 40% (3,6). In both cases this was considerably greater than the variations seen for repeat assays and variation obtained from separate biopsies from a single tumor. Similar results have been obtained in the present study, although the degree of heterogeneity was slightly less at 36%. With this degree of heterogeneity there is potential in employing
619
such measurements as predictors of outcome of radiotherapy. For a group of 24 patients we have shown that long survival (18 months) is related to a lower SF2 value and this reached statistical significance for the MTT assay. One can speculate that the MTT assay might be more likely to predict response in this group of tumors as response to radiotherapy is measured more in terms of growth delay than eradication of all clonogens. It is likely that biological measurements such as SF2 will not be clinically useful unless other clinical factors are taken into account. For example, for gliomas a prognostic index has been developed using four clinical factors which allows one to identify a group with a 2-year survival of -35% against another group with a 2-year survival of 0% (16). In summary, we have shown that the MTT assay can be used under well defined experimental conditions to give qualitatively similar results to the clonogenic assay for radiosensitivity testing. For gliomas, a wide range of SF2 values have been obtained and for the MTT assay there was a trend towards low SF2 and increased survival. However, in view of considerable overlap, it is unlikely that SF2 would be useful in the clinical management of these patients.
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