J Neurosurg 48:580-586, 1978
Variations in response of human brain tumors to B C N U in vitro PAUL L. KORNBLITH, M . D . , AND PAULA E. SZYPKO, B.S.
Neurosurgical Service, Massachusetts General Hospital, Boston, Massachusetts
v" A microtiter assay was used to study the sensitivity of 24 cultured human astrocytomas to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). The tumor cell lines tested originated from patients who were being treated with postoperative chemotherapy within the past 24 months. At a dose level comparable to the maximum in vivo therapeutic dose level, 18 of the 24 cell lines tested showed a significant response to the drug. If in vitro response can eventually be correlated with clinical response, this microtiter assay could help to form a basis for planning more specific chemotherapeutic treatment. KEY WORDS tissue culture
A
9 BCNU
N in vitro determination that could predict the response of a specific patient's malignant brain tumor cells to anti-cancer agents would be a useful tool in the rational planning of therapy for patients with such tumors. The nitrosourea deriva tives are considered to possess pharmacophysiological characteristics favorable for the treatment of malignant brain tumors, x8,21 and in carefully studied controlled evaluations show m o d e r a t e but significant value in the treatment of malignant brain tumors. L9 Since approximately 45% of the brain-tumor patients t r e a t e d with these drugs respond clinically) ,ls,2~ it would be advantageous to determine which patients would benefit most from nitrosourea therapy, especially in view of the toxic side effects of these drugs, 18 and to identify those patients who should be offered alternative or additional drugs. In the present study, an in vitro microtiter assay has been used to measure the in vitro response of patient's cultured glial cells to 1,3bis(2-chloroethyt)-l-nitrosourea (BCNU). 580
brain tumor
9 chemotherapy
9
The series of cell lines tested were found to exhibit a range of responses to the drug. For this r e a s o n we have sought to s t u d y systematically the in vitro responses of individual patients' tumors to B C N U to identify those tumors which are responsive to the agent. This is the fi~'st step in establishing a system to correlate in vitro chemotherapeutic responses with the in vivo clinical response. Materials and Methods
Tissue obtained during surgery was minced into 1 cu m m pieces, and planted in plastic flasks,* using sterile technique. Cell cultures were grown as monolayers in F-10 nutrient medium containing 12% fetal calf s e r u m . t *Falcon No. 3024 flasks manufactured by Falcon Plastics, Division of Becton, Dickinson and Co., 1950 Williams Drive, Oxnard, California. tF-10 nutrient medium available from GIBCO (Grand Island Biological Company), Grand Island, New York. J. Neurosurg. / Volume 48 / April, 1978
H u m a n b r a i n t u m o r response to B C N U in vitro When a flask of cells reached confluency, subculturing was performed as follows: the cells were washed twice with 5 ml Hanks' balanced salt solution (Ca ++ and Mg++-free), and then treated with 1.5 mi of trypsin-EDTA solution (0.05% trypsin, 0.02% E D T A (ethylenediaminetetraacetate)) to remove the cells from the surface of the culture flask. Cells were characterized by morphological, ultrastructural, biophysical, and biochemical techniques. 9'H'~7'24 We have had success in minimizing non-astrocytic cell growth by incubating glial cultures at 35 ~ C, so cultures were maintained at that temperature. A total of 24 lines were tested for sensitivity to BCNU.:~ Fifteen of the cell lines tested were derived from tissue diagnosed as either glioblastoma multiforme or astrocytoma Grade III to IV; the remaining nine lines were derived from other intracranial tumor types, including mixed glioma, lowgrade a s t r o c y t o m a , and meningioma. Twenty-one of the lines tested were from patients who were being treated postoperatively with B C N U a n d / o r C C N U (1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea) within the past 24 months; chemotherapy had been planned for the other three patients, but was decided against following surgery. When a cell line had been successfully subcultured at least twice, it was considered ready for testing. To perform the microtiter assay, the cells in a flask of the selected test cell line were washed with Hanks' balanced salt solution (Ca ++ and Mg++-free) and treated with trypsin-EDTA solution (0.05% trypsin~ 0.02% EDTA) to remove the cells from the surface of the culture flask. A cell count was done on the resulting cell suspension. The suspension was diluted with F-10 medium containing 12% fetal calf serum supplemented with 100 units/ml penicillin and 100 u g / m l streptomycin to yield approximately 100 cells/well. A six-barrel Terasaki syringew was used to deliver 0.01 ml aliquots of the cell suspension into each of 60 wells of several Falcon
Microtest I plates. II Plates were incubated at 37 ~ C in a humidified atmosphere containing 5% CO2 for 20 to 30 minutes. The plates were then examined microscopically to insure that cells had begun to adhere to the surface of the well. A B C N U stock solution was prepared as for clinical use, first diluting the drug in absolute ethanol and then in sterile distilled water. Fresh stock solution was prepared for each experiment just before use. Ten dilutions were then prepared serially from the stock solution, using F-10 medium which had been warmed to 37 ~ C as a diluent. The dilution concentrations ranged from 11 to 330 # g / m l BCNU. The B C N U dilutions were added to the plates using a H a m i l t o n m i c r o t i t e r syringe with repeating dispenser* which delivers 0.01 ml of test solution to each well. Since 0.0! ml of nutrient medium was already present in each of the wells, the ultimate dose levels to which the cells were exposed ranged from 5.5 to 165 # g / m l B C N U . One row of six replicate wells was used for each test dilution; each plate also included three separate rows of six wells each containing F-10 medium as a control. To avoid possible effects at the edges of the plates, TM the wells in Rows 1 and 10 were not used, and the test solutions were added to the plates using a diagonal scheme. After a l-hour incubation period at 37 ~ C, the plates were blotted with a sterile gauze sponge to remove the fluid above the cells including the drug dilutions. The plates were washed twice with Hanks' balanced salt solution, then flooded with medium. As there is no standard means for determining the in vitro concentration that would be equivalent to the concentration achieved in vivo, we chose the method recommended by Dickson and Suzangar, ~ which takes into account the level of the agent to which the tumor cells would initially be exposed. In our study, the in vitro concentration of B C N U was correlated with the patient dosage of 100 mg/sq m on the basis of a median body sur-
:I:BCNU: NSC-409962, 1,3-bis(2-chloroethyl)-lnitrosourea, distributed by Division of Cancer Treatment, N.C.I., N.I.H., Bethesda, Maryland, manufactured for N.C.I. by Ben Venue Laboratories, Inc., Bedford, Ohio. w syringe manufactured by Hamilton Co., Reno, Nevada.
IIFalcon No. 3034 Microtest I plates manufactured by Falcon Plastics, Division of Becton, Dickinson and Co., 1950 Williams Drive, Oxnard, California. *Hamilton microtiter syringe manufactured by Hamilton Co., Reno, Nevada.
J. Neurosurg. / Volume 48 / April, 1978
581
P. L. Kornblith and P. E. Szypko 1.0 r 0.8
~
/ X~lt
CL
.,,,,.,.,B
~ 0.6 ~ 0.4 ~ 0.2 0 0
|
50
A
100
BCNU )tg/ml
I
150
FIG. I. BCNU concentration versus cytotoxic index for two representative cell lines: Case 1 (crosses) and Case 22 (circles).
face area of 1.65 sq m and a blood volume of 5 liters. Since such a patient would be treated with 165 mg B C N U , the following calculation can be made: 165 mg BCNU 5000 ml plasma • 10s pg/ml = 33 pg BCNU/ml plasma. Therefore, 33 p g / m l B C N U represents the maximum concentration attained in the blood, assuming that the total dose was uniformly distributed in the plasma only. As the concentration of B C N U in the plasma has been found to be about equal to that in tumor tissue, 1,2~ the in vitro dose level of 33 #g B C N U / m l medium was determined to be the closest estimate of the maximum dose level to which t u m o r tissue would be exposed in vivo. After approximately 20 hours of incubation, plates were rinsed three times with Hanks' balanced salt solution, fixed with methanol for 10 minutes, and stained with Giemsa. The intact cells remaining attached to the bottom of each well were counted by projecting the light microscopic image of the well onto a semi-opaque screen. The screen was m a r k e d at the position of each intact cell and the marks were automatically tabulated. All cell counting was done by observers who were unaware of the test solutions used on the plates. The cytotoxic index (CI) of the test dilutions was calculated by the following formula: CI = 1 -
582
average number of cells in test wells average number of cells in control wells"
For each dilution tested, the mean and the variance for the cell counts in each set of wells containing the test dilution and in the wells containing F-10 medium as a control were calculated; from these values, a t value was calculated which was used to determine the statistical significance, at the 99% level of confidence, of any observed differences in cell counts between test dilutions and the control. After several passages, many of o u r cell lines are frozen in nutrient medium with 10% glycerol and stored at - 8 0 ~ C. Frozen-cell suspensions may be thawed for testing at a later date. Such a procedure may be advantageous in the case of patients with recurrent tumors. To test whether this procedure would affect the in vitro response to B C N U , a Grade III astrocytoma cell line from Case 4 was tested approximately 14 weeks after surgical resection of the tumor. The line was then frozen in suspension, thawed nearly 1 year later, and retested for sensitivity to BCNU. The CI's for the individual dosages both before and after freezing were calculated and compared. Results The results of the microtiter tests are summarized in Table 1. The series of cell lines tested exhibited a range of responses. Treatment of cells with the highest dosage of BCNU, 165 p g / m l , resulted in cell numbers which for all lines were significantly different from controls (p < 0.01). For all cell lines, the number of cells remaining in the test wells was les~ than or equal to 30% of the numbers of cells in control wells. At the lowest B C N U dose level tested, 5.5 p g / m l , the drug had no significant effect on any of the cell lines tested. Cell numbers for this dose level were not significantly different from controls, even when the p value was raised from 0.01 to 0.05. At the intermediate dose levels, however, variations in response could be observed. These differences are especially apparent at the dose level calculated to approximate the maximum therapeutic level, 33 pg/ml, as well as at dose levels of 22 p g / m l and 16.5/~g/ml (Fig. 1). When a dosage of 33 # g / m l B C N U was administered to the cells, the cytotoxic index ranged from 0.80 for the cell line for Case l, derived from a Grade II astrocytoma, to 0.04 for the cell line for Case 24, derived from a Grade III to IV astrocytoma. Eighteen of the cell lines tested (75%) with 33 J. Neurosurg. / Volume 48 / April. 1978
H u m a n brain t u m o r r e s p o n s e to B C N U in vitro TABLE 1
Summary of in vitro cytotoxieity data Case No. 1 2 3 4t 4 5
Pathological Diagnosis
NO.
of Cytotoxic Index for Dosages of BCNU (t~g/ml) Pas- 165.0 66.0 41.5 33.0w 22.0 16.5 13.0 11.0 8.5 sages
astrocytoma Gr. II 5 astroeytoma Gr. III-IV 4 recurrent meningioma 5 astrocytoma Gr. Ill 6 astrocytoma Gr. III 11 mixed astrocytoma & oligodendroglioma Gr III-IV 7 6 glioblastoma 10 7 astrocytoma Gr. II 4 8 astrocytoma Gr. Ill-IV 3 9 astrocytoma Gr. III 2 10~ glioblastoma 152 11 astrocytoma Gr. III-IV 4 12 glioblastoma 5 13 astrocytoma Gr. III-IV 3 14:~ glioblastoma 5 15 meningioma metastatic to lung 6 16 astrocytoma Gr. II 5 17 astrocytoma Gr. Ill-IV 3 18:1: astrocytoma Gr. I 3 19 astrocytoma Gr. III-IV 7 20 mixed glioma 4 21 glioblastoma 4 22 astrocytoma Gr. III-IV 3 23 glioblastoma 40 24 astrocytoma Gr. Ill-IV 13
.99* 1.00" .98* 1.00" --
.96* .95* .87* 1.00" --
.94* .92* .82* .75* --
.80* .77* .69* .67* .73*
.59" .60* .66" .42* .57*
.38* .44* .49* .42* .37*
.32* .40* .37 .35* --
.31" .24 .30 .30*
.20 .22 .31 .18
.97* .94* 1.00" .99* .97* 1.00" 1.00" .82* .98* 1.00"
.98* .93* .99* .81" .89* .98* 1.00" .61" .99* .93*
.91" .75* .74* .68* .73* .77* -.69* .82* .74*
.76* .62* .58* .52* .51" .58* .52* .29* .42* .40*
.36* .39* .21 .29 .24 .15 .48* .18" .08* .16"
.21" .34* .32* .31" .27* .03 .24* .20* .03 .00
.13" .21" .10 .13 .26* .06 -.24* .05 .01
.15" .13" .14 .18 .05 .03 .42* .16 .06 .11
.07 .17" .04 .08 .17 .01 .46* .10 .01 .15
.98* .98* 1.00" .70* .88* .82* 1.00" .91" .99* .89*
.42* .63* .61" .47* ,41" .42* .52* .27 .44* .24*
.43* .51" .38* .28* .29* .20 .18" .20 .10 .11
.31" .26* .24* .15 .24* ,16 .23* .11 .22 .12 .24 .16 .09 .06 .05 --.03 .07 .05 .04 .04
.13 .17 .19 .14 .15 .16 .22 .18 .22* .10 .06 --.04 .09 .10 .14 .05 .09 .06 .07 .08
5.5
EC 50[I (~g/ml)
.10 .13 .12 .02 .02
19.5 18.5 16.5 25.5 --
.01 .04 --.02 .03 .23 .01 --.01 --.03 -.04
26.0 27.5 28.5 32.0 33.0 31.0 28.0 54.5 34.5 35.5
.22* .18" .02 .14 .01 .03 .05 .14 .13 .06 .11 .03 .18 .10 .08 .27 .07 .12 .06 .02 --.01 .05 - . 0 3 --.02 .15 .09 .04 .05 .00 --.02
78.5 41.5 54.5 67.0 85.0 86.0 64.5 101.5 75.0 105.5
*Significantly different from control (p < 0.01). tFrozen and thawed. ~;Not treated clinically. w therapeutic level. IJConcentration of BCNU effective in reducing cell number to 50%.
u g / m l showed a significant ( p < 0 . 0 1 ) response to B C N U ( C I r a n g e 0.80 to 0.23); the r e m a i n i n g six lines showed no significant response. A t a dose level o f 22 t s g / m l B C N U , 11 lines r e s p o n d e d s i g n i f i c a n t l y , whereas 13 lines did not. A t a dose level o f 16.5 u g / m l , a level half that c o r r e s p o n d i n g to the m a x i m u m t h e r a p e u t i c level, 12 of the 24 lines responded significantly to the drug. A t d e c r e a s i n g dose levels, fewer cell lines were s h o w n to r e s p o n d significantly to B C N U t r e a t m e n t . T h e r e was n o t significant difference b e t w e e n the n u m b e r of cells in c o n t r o l wells a n d the n u m b e r of cells r e m a i n i n g in wells which had been treated with c o m p a r a b l e dilutions of ethanolic diluent. The c o n c e n t r a t i o n of B C N U which was effective in r e d u c i n g the
J. Neurosurg. / Volume 48 / April, 1978
cell n u m b e r 50% ( E C 50) was c a l c u l a t e d , a n d found to r a n g e from 16.5 u g / m l to 105.5 # g / m l ( T a b l e 1). T h e results for the cell line for C a s e 4, a G r a d e I I I a s t r o c y t o m a cell line which was tested both b e f o r e a n d after freezing, are included in T a b l e 1. A l t h o u g h only four d o s a g e s were tested b e f o r e freezing, the c y t o t o x i c indices before freezing c o r r e s p o n d closely to those o b t a i n e d after the freezing a n d t h a w i n g procedure. Discussion
The use o f in vitro d a t a to help g u i d e clinical t h e r a p y has been a n i m p o r t a n t goal o f c h e m o t h e r a p e u t i c research. Yet, a l t h o u g h 583
P. L. Kornblith and P. E. Szypko considerable in vitro testing of chemotherapeutic agents has been done using animal cell cultures, 8,15,aSa2 relatively little testing has been done using human cell cultures of solid tumors. The factors which have limited such studies have been 1) the difficulty of establishing lines from virtually all cultured tumors, 2) the difficulties in cellular characterization, 3) the slow rate of growth even in successful cases, thus making observations too late in the clinical course to be of any real value, and 4) the problems inherent in the extrapolation of in vitro data to the clinical situation. Previous studies of in vitro and in vivo effects of chemotherapeutic agents have yielded varying results. In early studies, Hirschberg, et al., e reported inhibition in tissue culture, but not in vivo, with the use of quinacrine and related acridines on human glioblastoma multiforme and mouse glioma 26. The discrepancy was believed to be due to quantitative differences in drug concentrations in the two situations. In our study, we have avoided this problem by including in the dilution series a concentration of BCNU that corresponds to the comparable therapeutic dose level. Eagle and Foley4 studied the effects of 13 compounds with known carcinolytic activity in either experimental animals or man and two carcinolytically inactive compounds on six non-glial human cell lines. All 13 carcinolytic compounds were found to be cytotoxic in vitro, while the two inactive compounds had no effect in vitro, even at the highest dosages administered. This study provided one of the first pieces of evidence that a correlation may exist between the activity of a chemotherapeutic agent in vivo and in vitro.
Wright, et al., 25 studied the response of non-glial human cell lines to the particular chemotherapeutic agent being administered clinically. In vitro and clinical response were graded somewhat subjectively, and a positive correlation between the responses was found in 26 out of 40 cases. Holmes and Littld have used an in vitro microcytotoxicity test similar to the technique used in our laboratory, to test various chemotherapeutic agents against non-glial human cell culture lines. Clinical response was evaluated by measuring the size of the tumor. When in vitro results were compared 584
with clinical response, a positive correlation was found in 11 out of 12 cases. Drewinko, et al., s have tested BCNU against human lymphoma cells in order to determine cell survival rates for the drug. For the cells treated with BCNU in this study, a broad-shouldered, threshold-type survival curve was obtained. Mealey, et al., 1~ have used an in vitro microtest to study the effects of BCNU and radiation, both separately and in combination, on five cultured human glioblastoma cell lines. These studies demonstrate the additive effects of BCNU and radiation, and suggest synergistic effects between the two modalities. O f the factors stated previously which have limited the study of in vitro and in vivo correlations, the first three problems now appear to be solved. We have had success in establishing monolayer cultures in over 95% of the glial tumor specimens we receive. These cultures can be accurately characterized as to their cellular origin by a panel of morphological, ultrastructural, biophysical, and biochemical parameters. 9,xx,xT,~x,24 Since cell lines are usually subcultured 4 weeks after planting, various types of tests are possible as early as 6 to 8 weeks following surgery. In this series of experiments, 18 of the 24 cell lines studied by an in vitro microcytotoxicity test were found to have a significant response to the maximum therapeutic dose level, 33 ~g/ml, of the chemotherapeutic agent BCNU. This represented 75% of the overall test population. At half the maximum therapeutic dose level, 16.5 ug/ml, 50% of the test population responded significantly, a percentage closer to the 45% of patients reported to respond clinically in the past? ,18a~ Levin, et al., ~~ recently reported BCNU levels of-~ 1 # g / m l plasma in patients during the period 2 to 24 hours after drug treatment. In our test, however, dose levels of 5.5 #g/ml had no significant effect on the cells. As the in vivo dose level was measured by Levin, et aL, 2 hours after treatment, this level is probably lower than the maximum level achieved immediately after administration of the drug, since B C N U is known to have an in vivo half life of approximately 15 minutes? The possibility also exists, as has been suggested by Drewinko, et al., 3 that the nutrient medium exerts a partial inactivating effect when low concentrations of B C N U are tested. Our calculation of the maximum in J. Neurosurg. / Volume 48 / April, 1978
H u m a n b r a i n t u m o r r e s p o n s e to B C N U in vitro vivo B C N U level, however, is probably slightly high, as it does not take into account the fact that the drug may become diffuse throughout the tissue upon injection. The EC 50 ranged from 16.5 to 105.5 ~g/ml for the cell lines tested. In general, cell lines that responded significantly at the lower dose levels also had lower EC 50's. Preliminary results indicate that the freezing procedure does not significantly alter the response of cultured cells to B C N U . This may have significance in several situations. For example, testing could be performed at a later date, in the case of a patient who either did not receive chemotherapy following surgery but who is recommended for chemotherapy later in the course of therapy, or who has a recurrent tumor which was not treated initially. As new chemotherapeutic agents are put into clinical use, frozen-cell suspensions could be thawed and tested with these agents as well. Although more testing along these lines must be done, the preliminary results are encouraging. We have used the term "cytotoxic index" to describe the fraction of the total cell number that does not remain in the test well after application of B C N U . By using the word "cytotoxic," we are not necessarily implying that actual cell death has occurred. The word is meant to indicate that toxic changes have occurred, resulting in a reduction in cell number in the treated wells. These changes may include some alterations to the cells that render them incapable of adhering to the plastic surface, resulting in a cell population that does not function in the usual manner of glial tumor cell cultures. These changes may also include actual cell death. Although further studies are necessary to determine more precisely what toxic effects B C N U has on cells in our assay, preliminary ultrastructural evaluation of treated cells suggests changes in mitochondrial density and number and increased lipofuscin granules comparable to those noted by Maxwell and Kruger 18 in radiated astrocytes. This study has d e m o n s t r a t e d that a microtiter assay can be used to test the response of patients' cultured glial cells to B C N U , and that specific cell lines do respond differently to B C N U . These differences have been detected after as few as two successful passages in vitro, representing an average time period of 6 to 8 weeks following surgery. J. Neurosurg. / Volume 48 / April, 1978
Preliminary testing suggests that freezing and thawing procedures do not affect the response of cultured cells, making testing at a later date possible, although more testing of thawed lines will be necessary to prove this. As many variables must be taken into account when evaluating an individual patient's clinical response to c h e m o t h e r a p y , the relationship between patients' clinical and in vitro responses with a relatively small sample size and at this early point in the study might be misleading, when based on the in vitro response to only one treatment parameter. Further in vitro testing and comparison of each individual patient's in vitro response with his clinical response in a larger group of patients, matched for location of lesion, clinical status before treatment, age, extent of surgery, and type and amount of radiation will be necessary before definitive conclusions can be drawn regarding a correlation. If a positive correlation can be established by comparing large groups of well matched, responding and non-responding patients, a basis exists for developing a program of more rationally planned nitrosourea therapy and, in addition, for selecting that agent most efficacious for each individual brain-tumor patient. Acknowledgments
The authors are indebted to Dr. William H. Sweet, Chief of Neurosurgical Service, for support and encouragement, to Ms. Kathleen Lowry for technical assistance, and to Drs. Eugene A. Quindlen and Philip C. Merker for their advice in the preparation of the manuscript. References
1. DeVita VT, Denham C, Davidson JD, et al: The physiological disposition of the carcinostatic 1,3-bis(2-chloroethyl)- l-nitrosourea (BCNU) in man and animals. Clin Pharmacol Ther 8:566-577, 1967 2. Dickson JA, Suzangar M: In vitro sensitivity testing of human tumour slices to chemotherapeutic agents - - its place in cancer therapy, in Dendy PP (ed): Human Tumours in Short Term Culture. New York: Academic Press, 1976, pp 107-138 3. Drewinko B, Novak JK, Barranco SC: The response of human lymphoma cells in vitro to bleomycin and 1,3-bis(2-chloroethyl)- 1nitrosourea. Cancer Res 32:1206-1208, 1972 4. Eagle H, Foley GE: The cytotoxic action of carcinolytic agents in tissue culture. Am J Med 21:739-749, 1956 585
P. L. Kornblith and P. E. Szypko 5. Fewer D, Wilson CB, Boldrey EB, et al: The chemotherapy of brain tumors. Clinical experience with carmustine (BCNU) and vincristine. J A M A 222:549-551, 1972 6. Hirschberg E, Gellhorn A, Reiner L, et al: In vivo and in vitro effects of quinacrine and related acridines on human and mouse brain tumors. Proc Am Assoc Cancer Res 2:117-118, 1956 (Abstract) 7. Holmes H L , Little JM: Tissue-culture microtest for predicting response of human cancer to chemotherapy. Lancet 2:985-987, 1974 8. Kessel D, Hall TC: Retention of 6mercaptopurine derivatives by intact cells as an index of drug response in human and murine leukemias. Cancer Res 29:2116-2119, 1969 9. Kornblith PL, Prieto A Jr, Pollen DA: Alterations in glial and mesenchymal tumor cell membrane resistance with heteroimmune and autologous sera. Ann NY Acad Sci 159 (Art. 2):585-590, 1969 10. Levin VA, Weinkam R J, Hoffman W, et al: Pharmacokinetics of BCNU in humans. Proc Am Assoc Cancer Res 18:76, 1977 (Abstract 302) 11. Lightbody J, Pfeiffer SE, Kornblith PL, et al: Biochemically differentiated clonal human glial cells in tissue culture. J Ncurobiol 1:411-417, 1970 12. Martuza RL, Proffitt MR, Moore MB, et al: E v a p o r a t i o n as a cause of positional differences in cell plating and growth in microtiter plates. Transplantation 21:271-273, 1976 13. Maxwell DS, Kruger L: The fine structure of astrocytes in the cerebral cortex and their response to focal injury produced by heavy ionizing particles. J Cell Biol 25 (No. 2 Part 2):141-157, 1965 14. Mealey J Jr, Chen TT, Shupe R: Response of cultured human glioblastomas to radiation and B C N U chemotherapy. J Neurosurg 41:339349, 1974 15. Schabel F M Jr, Johnston TP, McCaleb GS, et al: Experimental evaluation of potential anticancer agents. VIII. Effects of certain nitrosoureas on intracerebral L1210 leukemia. Cancer Res 23:725-733, 1963 16. Skipper HE, Schabel F M Jr, Trader MW, et al: Experimental evaluation of potential anticancer agents. VI. Anatomical distribution of
586
17. 18. 19.
20.
21.
22.
23.
24.
25.
leukemic cells and failure of chemotherapy. Cancer Res 21:1154-1164, 1961 Trachtenberg MC, Kornblith PL, H~tuptli J: Biophysical properties of cultured human glial cells. Brain Res 38:279-298, 1972 Walker MD: Nitrosoureas in central nervous system tumors. Cancer Chemother Rep Part 3 4(3):21-26, 1973 Walker MD, Gehan EA: Clinical studies in malignant gliomas and their treatment with the nitrosoureas. Cancer Treat Rep 60: 713-716, 1976 Walker MD, Hurwitz BS: BCNU (1,3-bis(2chloroethyl)-l-nitrosourea; NSC-409962) in the treatment of malignant brain t u m o r - - A preliminary report. Cancer Chemother Rep Part 1 54(4):263-271, 1970 Walsh JM, Cassady JR, Frei E III, et al: Recent advances in the treatment of primary brain tumors. A seminar. Arch Surg 110:696-702, 1975 Wheeler KT, Tel N, Williams ME, et al: Factors influencing the survival of rat brain tumor cells after in vitro treatment with 1,3-Bis(2chloroethyl)-l-nitrosourea. Cancer Res 35: 1464-1469, 1975 Wilson CB, Boldrey EB, Enot K J: 1,3-bis(2chloroethyl)-l-nitrosourea (NSC-409962) in the t r e a t m e n t of brain tumors. Cancer Chemother Rep 54:273-281, 1970 Wilson CB, Kornblith PL: Monograph: Experimental brain tumors and experimental chemotherapy, in: Tower DB (ed): The Nervous System, Vol. 2: The Clinical Neurosciences. New York: Raven Press, 1975, pp 175-181 Wright JC, Cobb JP, Gumport SL, et al: Investigation of the relation between clinical and tissue culture response to chemotherapeutic agents on human cancer. N Engl J Med 257:1207-1211, 1957
This investigation was supported by National Cancer Institute Grant CA-07368. Additional support was received from the family and friends of Albert H. Bartlett. Address reprint requests to: Paul L. Kornblith, M.D., Neurosurgical Service, Massachusetts General Hospital, 32 Fruit Street, Warren 460, Boston, Massachusetts 02114.
J. Neurosurg. / Volume 48 / April, 1978
Variations in response of human brain tumors to B C N U in vitro PAUL L. KORNBLITH, M . D . , AND PAULA E. SZYPKO, B.S.
Neurosurgical Service, Massachusetts General Hospital, Boston, Massachusetts
v" A microtiter assay was used to study the sensitivity of 24 cultured human astrocytomas to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). The tumor cell lines tested originated from patients who were being treated with postoperative chemotherapy within the past 24 months. At a dose level comparable to the maximum in vivo therapeutic dose level, 18 of the 24 cell lines tested showed a significant response to the drug. If in vitro response can eventually be correlated with clinical response, this microtiter assay could help to form a basis for planning more specific chemotherapeutic treatment. KEY WORDS tissue culture
A
9 BCNU
N in vitro determination that could predict the response of a specific patient's malignant brain tumor cells to anti-cancer agents would be a useful tool in the rational planning of therapy for patients with such tumors. The nitrosourea deriva tives are considered to possess pharmacophysiological characteristics favorable for the treatment of malignant brain tumors, x8,21 and in carefully studied controlled evaluations show m o d e r a t e but significant value in the treatment of malignant brain tumors. L9 Since approximately 45% of the brain-tumor patients t r e a t e d with these drugs respond clinically) ,ls,2~ it would be advantageous to determine which patients would benefit most from nitrosourea therapy, especially in view of the toxic side effects of these drugs, 18 and to identify those patients who should be offered alternative or additional drugs. In the present study, an in vitro microtiter assay has been used to measure the in vitro response of patient's cultured glial cells to 1,3bis(2-chloroethyt)-l-nitrosourea (BCNU). 580
brain tumor
9 chemotherapy
9
The series of cell lines tested were found to exhibit a range of responses to the drug. For this r e a s o n we have sought to s t u d y systematically the in vitro responses of individual patients' tumors to B C N U to identify those tumors which are responsive to the agent. This is the fi~'st step in establishing a system to correlate in vitro chemotherapeutic responses with the in vivo clinical response. Materials and Methods
Tissue obtained during surgery was minced into 1 cu m m pieces, and planted in plastic flasks,* using sterile technique. Cell cultures were grown as monolayers in F-10 nutrient medium containing 12% fetal calf s e r u m . t *Falcon No. 3024 flasks manufactured by Falcon Plastics, Division of Becton, Dickinson and Co., 1950 Williams Drive, Oxnard, California. tF-10 nutrient medium available from GIBCO (Grand Island Biological Company), Grand Island, New York. J. Neurosurg. / Volume 48 / April, 1978
H u m a n b r a i n t u m o r response to B C N U in vitro When a flask of cells reached confluency, subculturing was performed as follows: the cells were washed twice with 5 ml Hanks' balanced salt solution (Ca ++ and Mg++-free), and then treated with 1.5 mi of trypsin-EDTA solution (0.05% trypsin, 0.02% E D T A (ethylenediaminetetraacetate)) to remove the cells from the surface of the culture flask. Cells were characterized by morphological, ultrastructural, biophysical, and biochemical techniques. 9'H'~7'24 We have had success in minimizing non-astrocytic cell growth by incubating glial cultures at 35 ~ C, so cultures were maintained at that temperature. A total of 24 lines were tested for sensitivity to BCNU.:~ Fifteen of the cell lines tested were derived from tissue diagnosed as either glioblastoma multiforme or astrocytoma Grade III to IV; the remaining nine lines were derived from other intracranial tumor types, including mixed glioma, lowgrade a s t r o c y t o m a , and meningioma. Twenty-one of the lines tested were from patients who were being treated postoperatively with B C N U a n d / o r C C N U (1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea) within the past 24 months; chemotherapy had been planned for the other three patients, but was decided against following surgery. When a cell line had been successfully subcultured at least twice, it was considered ready for testing. To perform the microtiter assay, the cells in a flask of the selected test cell line were washed with Hanks' balanced salt solution (Ca ++ and Mg++-free) and treated with trypsin-EDTA solution (0.05% trypsin~ 0.02% EDTA) to remove the cells from the surface of the culture flask. A cell count was done on the resulting cell suspension. The suspension was diluted with F-10 medium containing 12% fetal calf serum supplemented with 100 units/ml penicillin and 100 u g / m l streptomycin to yield approximately 100 cells/well. A six-barrel Terasaki syringew was used to deliver 0.01 ml aliquots of the cell suspension into each of 60 wells of several Falcon
Microtest I plates. II Plates were incubated at 37 ~ C in a humidified atmosphere containing 5% CO2 for 20 to 30 minutes. The plates were then examined microscopically to insure that cells had begun to adhere to the surface of the well. A B C N U stock solution was prepared as for clinical use, first diluting the drug in absolute ethanol and then in sterile distilled water. Fresh stock solution was prepared for each experiment just before use. Ten dilutions were then prepared serially from the stock solution, using F-10 medium which had been warmed to 37 ~ C as a diluent. The dilution concentrations ranged from 11 to 330 # g / m l BCNU. The B C N U dilutions were added to the plates using a H a m i l t o n m i c r o t i t e r syringe with repeating dispenser* which delivers 0.01 ml of test solution to each well. Since 0.0! ml of nutrient medium was already present in each of the wells, the ultimate dose levels to which the cells were exposed ranged from 5.5 to 165 # g / m l B C N U . One row of six replicate wells was used for each test dilution; each plate also included three separate rows of six wells each containing F-10 medium as a control. To avoid possible effects at the edges of the plates, TM the wells in Rows 1 and 10 were not used, and the test solutions were added to the plates using a diagonal scheme. After a l-hour incubation period at 37 ~ C, the plates were blotted with a sterile gauze sponge to remove the fluid above the cells including the drug dilutions. The plates were washed twice with Hanks' balanced salt solution, then flooded with medium. As there is no standard means for determining the in vitro concentration that would be equivalent to the concentration achieved in vivo, we chose the method recommended by Dickson and Suzangar, ~ which takes into account the level of the agent to which the tumor cells would initially be exposed. In our study, the in vitro concentration of B C N U was correlated with the patient dosage of 100 mg/sq m on the basis of a median body sur-
:I:BCNU: NSC-409962, 1,3-bis(2-chloroethyl)-lnitrosourea, distributed by Division of Cancer Treatment, N.C.I., N.I.H., Bethesda, Maryland, manufactured for N.C.I. by Ben Venue Laboratories, Inc., Bedford, Ohio. w syringe manufactured by Hamilton Co., Reno, Nevada.
IIFalcon No. 3034 Microtest I plates manufactured by Falcon Plastics, Division of Becton, Dickinson and Co., 1950 Williams Drive, Oxnard, California. *Hamilton microtiter syringe manufactured by Hamilton Co., Reno, Nevada.
J. Neurosurg. / Volume 48 / April, 1978
581
P. L. Kornblith and P. E. Szypko 1.0 r 0.8
~
/ X~lt
CL
.,,,,.,.,B
~ 0.6 ~ 0.4 ~ 0.2 0 0
|
50
A
100
BCNU )tg/ml
I
150
FIG. I. BCNU concentration versus cytotoxic index for two representative cell lines: Case 1 (crosses) and Case 22 (circles).
face area of 1.65 sq m and a blood volume of 5 liters. Since such a patient would be treated with 165 mg B C N U , the following calculation can be made: 165 mg BCNU 5000 ml plasma • 10s pg/ml = 33 pg BCNU/ml plasma. Therefore, 33 p g / m l B C N U represents the maximum concentration attained in the blood, assuming that the total dose was uniformly distributed in the plasma only. As the concentration of B C N U in the plasma has been found to be about equal to that in tumor tissue, 1,2~ the in vitro dose level of 33 #g B C N U / m l medium was determined to be the closest estimate of the maximum dose level to which t u m o r tissue would be exposed in vivo. After approximately 20 hours of incubation, plates were rinsed three times with Hanks' balanced salt solution, fixed with methanol for 10 minutes, and stained with Giemsa. The intact cells remaining attached to the bottom of each well were counted by projecting the light microscopic image of the well onto a semi-opaque screen. The screen was m a r k e d at the position of each intact cell and the marks were automatically tabulated. All cell counting was done by observers who were unaware of the test solutions used on the plates. The cytotoxic index (CI) of the test dilutions was calculated by the following formula: CI = 1 -
582
average number of cells in test wells average number of cells in control wells"
For each dilution tested, the mean and the variance for the cell counts in each set of wells containing the test dilution and in the wells containing F-10 medium as a control were calculated; from these values, a t value was calculated which was used to determine the statistical significance, at the 99% level of confidence, of any observed differences in cell counts between test dilutions and the control. After several passages, many of o u r cell lines are frozen in nutrient medium with 10% glycerol and stored at - 8 0 ~ C. Frozen-cell suspensions may be thawed for testing at a later date. Such a procedure may be advantageous in the case of patients with recurrent tumors. To test whether this procedure would affect the in vitro response to B C N U , a Grade III astrocytoma cell line from Case 4 was tested approximately 14 weeks after surgical resection of the tumor. The line was then frozen in suspension, thawed nearly 1 year later, and retested for sensitivity to BCNU. The CI's for the individual dosages both before and after freezing were calculated and compared. Results The results of the microtiter tests are summarized in Table 1. The series of cell lines tested exhibited a range of responses. Treatment of cells with the highest dosage of BCNU, 165 p g / m l , resulted in cell numbers which for all lines were significantly different from controls (p < 0.01). For all cell lines, the number of cells remaining in the test wells was les~ than or equal to 30% of the numbers of cells in control wells. At the lowest B C N U dose level tested, 5.5 p g / m l , the drug had no significant effect on any of the cell lines tested. Cell numbers for this dose level were not significantly different from controls, even when the p value was raised from 0.01 to 0.05. At the intermediate dose levels, however, variations in response could be observed. These differences are especially apparent at the dose level calculated to approximate the maximum therapeutic level, 33 pg/ml, as well as at dose levels of 22 p g / m l and 16.5/~g/ml (Fig. 1). When a dosage of 33 # g / m l B C N U was administered to the cells, the cytotoxic index ranged from 0.80 for the cell line for Case l, derived from a Grade II astrocytoma, to 0.04 for the cell line for Case 24, derived from a Grade III to IV astrocytoma. Eighteen of the cell lines tested (75%) with 33 J. Neurosurg. / Volume 48 / April. 1978
H u m a n brain t u m o r r e s p o n s e to B C N U in vitro TABLE 1
Summary of in vitro cytotoxieity data Case No. 1 2 3 4t 4 5
Pathological Diagnosis
NO.
of Cytotoxic Index for Dosages of BCNU (t~g/ml) Pas- 165.0 66.0 41.5 33.0w 22.0 16.5 13.0 11.0 8.5 sages
astrocytoma Gr. II 5 astroeytoma Gr. III-IV 4 recurrent meningioma 5 astrocytoma Gr. Ill 6 astrocytoma Gr. III 11 mixed astrocytoma & oligodendroglioma Gr III-IV 7 6 glioblastoma 10 7 astrocytoma Gr. II 4 8 astrocytoma Gr. Ill-IV 3 9 astrocytoma Gr. III 2 10~ glioblastoma 152 11 astrocytoma Gr. III-IV 4 12 glioblastoma 5 13 astrocytoma Gr. III-IV 3 14:~ glioblastoma 5 15 meningioma metastatic to lung 6 16 astrocytoma Gr. II 5 17 astrocytoma Gr. Ill-IV 3 18:1: astrocytoma Gr. I 3 19 astrocytoma Gr. III-IV 7 20 mixed glioma 4 21 glioblastoma 4 22 astrocytoma Gr. III-IV 3 23 glioblastoma 40 24 astrocytoma Gr. Ill-IV 13
.99* 1.00" .98* 1.00" --
.96* .95* .87* 1.00" --
.94* .92* .82* .75* --
.80* .77* .69* .67* .73*
.59" .60* .66" .42* .57*
.38* .44* .49* .42* .37*
.32* .40* .37 .35* --
.31" .24 .30 .30*
.20 .22 .31 .18
.97* .94* 1.00" .99* .97* 1.00" 1.00" .82* .98* 1.00"
.98* .93* .99* .81" .89* .98* 1.00" .61" .99* .93*
.91" .75* .74* .68* .73* .77* -.69* .82* .74*
.76* .62* .58* .52* .51" .58* .52* .29* .42* .40*
.36* .39* .21 .29 .24 .15 .48* .18" .08* .16"
.21" .34* .32* .31" .27* .03 .24* .20* .03 .00
.13" .21" .10 .13 .26* .06 -.24* .05 .01
.15" .13" .14 .18 .05 .03 .42* .16 .06 .11
.07 .17" .04 .08 .17 .01 .46* .10 .01 .15
.98* .98* 1.00" .70* .88* .82* 1.00" .91" .99* .89*
.42* .63* .61" .47* ,41" .42* .52* .27 .44* .24*
.43* .51" .38* .28* .29* .20 .18" .20 .10 .11
.31" .26* .24* .15 .24* ,16 .23* .11 .22 .12 .24 .16 .09 .06 .05 --.03 .07 .05 .04 .04
.13 .17 .19 .14 .15 .16 .22 .18 .22* .10 .06 --.04 .09 .10 .14 .05 .09 .06 .07 .08
5.5
EC 50[I (~g/ml)
.10 .13 .12 .02 .02
19.5 18.5 16.5 25.5 --
.01 .04 --.02 .03 .23 .01 --.01 --.03 -.04
26.0 27.5 28.5 32.0 33.0 31.0 28.0 54.5 34.5 35.5
.22* .18" .02 .14 .01 .03 .05 .14 .13 .06 .11 .03 .18 .10 .08 .27 .07 .12 .06 .02 --.01 .05 - . 0 3 --.02 .15 .09 .04 .05 .00 --.02
78.5 41.5 54.5 67.0 85.0 86.0 64.5 101.5 75.0 105.5
*Significantly different from control (p < 0.01). tFrozen and thawed. ~;Not treated clinically. w therapeutic level. IJConcentration of BCNU effective in reducing cell number to 50%.
u g / m l showed a significant ( p < 0 . 0 1 ) response to B C N U ( C I r a n g e 0.80 to 0.23); the r e m a i n i n g six lines showed no significant response. A t a dose level o f 22 t s g / m l B C N U , 11 lines r e s p o n d e d s i g n i f i c a n t l y , whereas 13 lines did not. A t a dose level o f 16.5 u g / m l , a level half that c o r r e s p o n d i n g to the m a x i m u m t h e r a p e u t i c level, 12 of the 24 lines responded significantly to the drug. A t d e c r e a s i n g dose levels, fewer cell lines were s h o w n to r e s p o n d significantly to B C N U t r e a t m e n t . T h e r e was n o t significant difference b e t w e e n the n u m b e r of cells in c o n t r o l wells a n d the n u m b e r of cells r e m a i n i n g in wells which had been treated with c o m p a r a b l e dilutions of ethanolic diluent. The c o n c e n t r a t i o n of B C N U which was effective in r e d u c i n g the
J. Neurosurg. / Volume 48 / April, 1978
cell n u m b e r 50% ( E C 50) was c a l c u l a t e d , a n d found to r a n g e from 16.5 u g / m l to 105.5 # g / m l ( T a b l e 1). T h e results for the cell line for C a s e 4, a G r a d e I I I a s t r o c y t o m a cell line which was tested both b e f o r e a n d after freezing, are included in T a b l e 1. A l t h o u g h only four d o s a g e s were tested b e f o r e freezing, the c y t o t o x i c indices before freezing c o r r e s p o n d closely to those o b t a i n e d after the freezing a n d t h a w i n g procedure. Discussion
The use o f in vitro d a t a to help g u i d e clinical t h e r a p y has been a n i m p o r t a n t goal o f c h e m o t h e r a p e u t i c research. Yet, a l t h o u g h 583
P. L. Kornblith and P. E. Szypko considerable in vitro testing of chemotherapeutic agents has been done using animal cell cultures, 8,15,aSa2 relatively little testing has been done using human cell cultures of solid tumors. The factors which have limited such studies have been 1) the difficulty of establishing lines from virtually all cultured tumors, 2) the difficulties in cellular characterization, 3) the slow rate of growth even in successful cases, thus making observations too late in the clinical course to be of any real value, and 4) the problems inherent in the extrapolation of in vitro data to the clinical situation. Previous studies of in vitro and in vivo effects of chemotherapeutic agents have yielded varying results. In early studies, Hirschberg, et al., e reported inhibition in tissue culture, but not in vivo, with the use of quinacrine and related acridines on human glioblastoma multiforme and mouse glioma 26. The discrepancy was believed to be due to quantitative differences in drug concentrations in the two situations. In our study, we have avoided this problem by including in the dilution series a concentration of BCNU that corresponds to the comparable therapeutic dose level. Eagle and Foley4 studied the effects of 13 compounds with known carcinolytic activity in either experimental animals or man and two carcinolytically inactive compounds on six non-glial human cell lines. All 13 carcinolytic compounds were found to be cytotoxic in vitro, while the two inactive compounds had no effect in vitro, even at the highest dosages administered. This study provided one of the first pieces of evidence that a correlation may exist between the activity of a chemotherapeutic agent in vivo and in vitro.
Wright, et al., 25 studied the response of non-glial human cell lines to the particular chemotherapeutic agent being administered clinically. In vitro and clinical response were graded somewhat subjectively, and a positive correlation between the responses was found in 26 out of 40 cases. Holmes and Littld have used an in vitro microcytotoxicity test similar to the technique used in our laboratory, to test various chemotherapeutic agents against non-glial human cell culture lines. Clinical response was evaluated by measuring the size of the tumor. When in vitro results were compared 584
with clinical response, a positive correlation was found in 11 out of 12 cases. Drewinko, et al., s have tested BCNU against human lymphoma cells in order to determine cell survival rates for the drug. For the cells treated with BCNU in this study, a broad-shouldered, threshold-type survival curve was obtained. Mealey, et al., 1~ have used an in vitro microtest to study the effects of BCNU and radiation, both separately and in combination, on five cultured human glioblastoma cell lines. These studies demonstrate the additive effects of BCNU and radiation, and suggest synergistic effects between the two modalities. O f the factors stated previously which have limited the study of in vitro and in vivo correlations, the first three problems now appear to be solved. We have had success in establishing monolayer cultures in over 95% of the glial tumor specimens we receive. These cultures can be accurately characterized as to their cellular origin by a panel of morphological, ultrastructural, biophysical, and biochemical parameters. 9,xx,xT,~x,24 Since cell lines are usually subcultured 4 weeks after planting, various types of tests are possible as early as 6 to 8 weeks following surgery. In this series of experiments, 18 of the 24 cell lines studied by an in vitro microcytotoxicity test were found to have a significant response to the maximum therapeutic dose level, 33 ~g/ml, of the chemotherapeutic agent BCNU. This represented 75% of the overall test population. At half the maximum therapeutic dose level, 16.5 ug/ml, 50% of the test population responded significantly, a percentage closer to the 45% of patients reported to respond clinically in the past? ,18a~ Levin, et al., ~~ recently reported BCNU levels of-~ 1 # g / m l plasma in patients during the period 2 to 24 hours after drug treatment. In our test, however, dose levels of 5.5 #g/ml had no significant effect on the cells. As the in vivo dose level was measured by Levin, et aL, 2 hours after treatment, this level is probably lower than the maximum level achieved immediately after administration of the drug, since B C N U is known to have an in vivo half life of approximately 15 minutes? The possibility also exists, as has been suggested by Drewinko, et al., 3 that the nutrient medium exerts a partial inactivating effect when low concentrations of B C N U are tested. Our calculation of the maximum in J. Neurosurg. / Volume 48 / April, 1978
H u m a n b r a i n t u m o r r e s p o n s e to B C N U in vitro vivo B C N U level, however, is probably slightly high, as it does not take into account the fact that the drug may become diffuse throughout the tissue upon injection. The EC 50 ranged from 16.5 to 105.5 ~g/ml for the cell lines tested. In general, cell lines that responded significantly at the lower dose levels also had lower EC 50's. Preliminary results indicate that the freezing procedure does not significantly alter the response of cultured cells to B C N U . This may have significance in several situations. For example, testing could be performed at a later date, in the case of a patient who either did not receive chemotherapy following surgery but who is recommended for chemotherapy later in the course of therapy, or who has a recurrent tumor which was not treated initially. As new chemotherapeutic agents are put into clinical use, frozen-cell suspensions could be thawed and tested with these agents as well. Although more testing along these lines must be done, the preliminary results are encouraging. We have used the term "cytotoxic index" to describe the fraction of the total cell number that does not remain in the test well after application of B C N U . By using the word "cytotoxic," we are not necessarily implying that actual cell death has occurred. The word is meant to indicate that toxic changes have occurred, resulting in a reduction in cell number in the treated wells. These changes may include some alterations to the cells that render them incapable of adhering to the plastic surface, resulting in a cell population that does not function in the usual manner of glial tumor cell cultures. These changes may also include actual cell death. Although further studies are necessary to determine more precisely what toxic effects B C N U has on cells in our assay, preliminary ultrastructural evaluation of treated cells suggests changes in mitochondrial density and number and increased lipofuscin granules comparable to those noted by Maxwell and Kruger 18 in radiated astrocytes. This study has d e m o n s t r a t e d that a microtiter assay can be used to test the response of patients' cultured glial cells to B C N U , and that specific cell lines do respond differently to B C N U . These differences have been detected after as few as two successful passages in vitro, representing an average time period of 6 to 8 weeks following surgery. J. Neurosurg. / Volume 48 / April, 1978
Preliminary testing suggests that freezing and thawing procedures do not affect the response of cultured cells, making testing at a later date possible, although more testing of thawed lines will be necessary to prove this. As many variables must be taken into account when evaluating an individual patient's clinical response to c h e m o t h e r a p y , the relationship between patients' clinical and in vitro responses with a relatively small sample size and at this early point in the study might be misleading, when based on the in vitro response to only one treatment parameter. Further in vitro testing and comparison of each individual patient's in vitro response with his clinical response in a larger group of patients, matched for location of lesion, clinical status before treatment, age, extent of surgery, and type and amount of radiation will be necessary before definitive conclusions can be drawn regarding a correlation. If a positive correlation can be established by comparing large groups of well matched, responding and non-responding patients, a basis exists for developing a program of more rationally planned nitrosourea therapy and, in addition, for selecting that agent most efficacious for each individual brain-tumor patient. Acknowledgments
The authors are indebted to Dr. William H. Sweet, Chief of Neurosurgical Service, for support and encouragement, to Ms. Kathleen Lowry for technical assistance, and to Drs. Eugene A. Quindlen and Philip C. Merker for their advice in the preparation of the manuscript. References
1. DeVita VT, Denham C, Davidson JD, et al: The physiological disposition of the carcinostatic 1,3-bis(2-chloroethyl)- l-nitrosourea (BCNU) in man and animals. Clin Pharmacol Ther 8:566-577, 1967 2. Dickson JA, Suzangar M: In vitro sensitivity testing of human tumour slices to chemotherapeutic agents - - its place in cancer therapy, in Dendy PP (ed): Human Tumours in Short Term Culture. New York: Academic Press, 1976, pp 107-138 3. Drewinko B, Novak JK, Barranco SC: The response of human lymphoma cells in vitro to bleomycin and 1,3-bis(2-chloroethyl)- 1nitrosourea. Cancer Res 32:1206-1208, 1972 4. Eagle H, Foley GE: The cytotoxic action of carcinolytic agents in tissue culture. Am J Med 21:739-749, 1956 585
P. L. Kornblith and P. E. Szypko 5. Fewer D, Wilson CB, Boldrey EB, et al: The chemotherapy of brain tumors. Clinical experience with carmustine (BCNU) and vincristine. J A M A 222:549-551, 1972 6. Hirschberg E, Gellhorn A, Reiner L, et al: In vivo and in vitro effects of quinacrine and related acridines on human and mouse brain tumors. Proc Am Assoc Cancer Res 2:117-118, 1956 (Abstract) 7. Holmes H L , Little JM: Tissue-culture microtest for predicting response of human cancer to chemotherapy. Lancet 2:985-987, 1974 8. Kessel D, Hall TC: Retention of 6mercaptopurine derivatives by intact cells as an index of drug response in human and murine leukemias. Cancer Res 29:2116-2119, 1969 9. Kornblith PL, Prieto A Jr, Pollen DA: Alterations in glial and mesenchymal tumor cell membrane resistance with heteroimmune and autologous sera. Ann NY Acad Sci 159 (Art. 2):585-590, 1969 10. Levin VA, Weinkam R J, Hoffman W, et al: Pharmacokinetics of BCNU in humans. Proc Am Assoc Cancer Res 18:76, 1977 (Abstract 302) 11. Lightbody J, Pfeiffer SE, Kornblith PL, et al: Biochemically differentiated clonal human glial cells in tissue culture. J Ncurobiol 1:411-417, 1970 12. Martuza RL, Proffitt MR, Moore MB, et al: E v a p o r a t i o n as a cause of positional differences in cell plating and growth in microtiter plates. Transplantation 21:271-273, 1976 13. Maxwell DS, Kruger L: The fine structure of astrocytes in the cerebral cortex and their response to focal injury produced by heavy ionizing particles. J Cell Biol 25 (No. 2 Part 2):141-157, 1965 14. Mealey J Jr, Chen TT, Shupe R: Response of cultured human glioblastomas to radiation and B C N U chemotherapy. J Neurosurg 41:339349, 1974 15. Schabel F M Jr, Johnston TP, McCaleb GS, et al: Experimental evaluation of potential anticancer agents. VIII. Effects of certain nitrosoureas on intracerebral L1210 leukemia. Cancer Res 23:725-733, 1963 16. Skipper HE, Schabel F M Jr, Trader MW, et al: Experimental evaluation of potential anticancer agents. VI. Anatomical distribution of
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leukemic cells and failure of chemotherapy. Cancer Res 21:1154-1164, 1961 Trachtenberg MC, Kornblith PL, H~tuptli J: Biophysical properties of cultured human glial cells. Brain Res 38:279-298, 1972 Walker MD: Nitrosoureas in central nervous system tumors. Cancer Chemother Rep Part 3 4(3):21-26, 1973 Walker MD, Gehan EA: Clinical studies in malignant gliomas and their treatment with the nitrosoureas. Cancer Treat Rep 60: 713-716, 1976 Walker MD, Hurwitz BS: BCNU (1,3-bis(2chloroethyl)-l-nitrosourea; NSC-409962) in the treatment of malignant brain t u m o r - - A preliminary report. Cancer Chemother Rep Part 1 54(4):263-271, 1970 Walsh JM, Cassady JR, Frei E III, et al: Recent advances in the treatment of primary brain tumors. A seminar. Arch Surg 110:696-702, 1975 Wheeler KT, Tel N, Williams ME, et al: Factors influencing the survival of rat brain tumor cells after in vitro treatment with 1,3-Bis(2chloroethyl)-l-nitrosourea. Cancer Res 35: 1464-1469, 1975 Wilson CB, Boldrey EB, Enot K J: 1,3-bis(2chloroethyl)-l-nitrosourea (NSC-409962) in the t r e a t m e n t of brain tumors. Cancer Chemother Rep 54:273-281, 1970 Wilson CB, Kornblith PL: Monograph: Experimental brain tumors and experimental chemotherapy, in: Tower DB (ed): The Nervous System, Vol. 2: The Clinical Neurosciences. New York: Raven Press, 1975, pp 175-181 Wright JC, Cobb JP, Gumport SL, et al: Investigation of the relation between clinical and tissue culture response to chemotherapeutic agents on human cancer. N Engl J Med 257:1207-1211, 1957
This investigation was supported by National Cancer Institute Grant CA-07368. Additional support was received from the family and friends of Albert H. Bartlett. Address reprint requests to: Paul L. Kornblith, M.D., Neurosurgical Service, Massachusetts General Hospital, 32 Fruit Street, Warren 460, Boston, Massachusetts 02114.
J. Neurosurg. / Volume 48 / April, 1978
