Brirbh Journalof L'mlogj~ (1989), 64, 25-29 1989 British Journal of Urology

Testing the Chemosensitivity of Renal Carcinoma in vitro

TERESALAI, M. A. FERR0,A.V. KAISARY, P. J. B. SMITH and M. 0. S Y M E S

Departments of Surgery and Urology. University of Bristol, Royal Infirmary, Bristol

Summary-Tumour cells from 23 renal carcinomas were exposed in vitro to increasing concentrations of medroxyprogesterone acetate (MPA) or one of several cytotoxic agents. Thereafter the uptake of 75Selenomethionine (75SeM) was compared to that of tumour cells not exposed to the drug.

Using drug concentrations similar to those seen following in vivo administration in patients, MPA produced 2 50% inhibition of 75SeM uptake in 5 of 23 cases. The comparable proportion for doxorubicin was 4 of 5 and for mitozantrone 7 of 12. Methotrexate and Vinblastine were much less effective. In all cases where 2 50% inhibition was obtained with doxorubicin and mitozantrone the carcinoma had spread beyond the kidney. Thus drug sensitivity may be associated with a high mitotic rate.

Renal carcinomas, with an incidence of 4/100,000, account for 3% of all malignant neoplasms (Kantor, 1977). Some 25% of these patients have metastatic disease at the time of diagnosis, with a death rate of 74% at 1 year and 96% at 3 years (Pate1 and Lavengood, 1978). The survival rate of patients with localised disease following radical nephrec- tomy has been reported as 62% at 5 years (Nurmi, 1984) and 56% at 10 years (Skinner et al., 1971).

Apart from surgery, no form of therapy is of proven value in the management of this neoplasm. A variety of hormonal and chemotherapeutic agents has been administered, alone or in combination, but the remission rates have been low (Harris, 1983). Thus a test to screen individual drugs for their effectiveness against particular tumours would be of value. Fleischmann et al. (1983) found that the ability of different chemotherapeutic drugs to inhibit the formation of neoplastic cell colonies when applied to renal carcinomas was so variable as to be unsuitable in the assessment of drug sensitivity. Day et al. (1981) found identical responsiveness in 2 renal carcinomas to drugs as measured in vitro by inhibition of 3H thymidine

Accepted for publication 27 October 1988

uptake and in cico by inhibition of tumour growth in athymic mice. Bazeed et al. (1988) found only occasional sensitivity of carcinomas to drugs as measured by inhibition of 3H thymidine and 3H uridine uptake in citro. Ferro et al. (1988) reported the use of a 7sSelenomethionine uptake assay (Liebold and Bridge, 1979) to investigate the in vitro cytotoxic effect of stilboestrol on prostate carcinoma cells. We describe the use of this assay to measure the inhibitory action of several hormonal and chemotherapeutic agents on protein synthesis by renal carcinoma cells.

Patients and Methods

General plan of the e-rperiments A total of 23 patients was studied. In each case a suspension of tumour cells was prepared from the renal carcinoma. The cells were then exposed to a hormonal or chemotherapeutic agent for 24 h, washed to remove the drug and cultured with 15Se (as Selenomethionine) in methionine-free medium for a further 48 hours. The radioactivity incorpo- rated in the cell pellet was an indicator of protein synthesis and was compared with that of cells not exposed to a drug.

25

26 BRITISH JOURNAL OF UROLOGY

Preparation of tumour cell suspensions This was as described by Ferro et al. (1988) except that incubation with enzymes, to disaggregate the solid neoplasm, was for 1 h. When the tumour cell band, separated following centrifugation on the Nycodenz column, was examined using a haemo- cytometer, only carcinoma cells were seen and in most cases their viability exceeded 90%.

E-uposure to drugs In the present experiments 1 from each of the major classes of cytotoxic agents was investigated. The drugs used were :

(1) Medroxyprogesterone acetate (Depo-Prov- era) - a hormonal agent.

(ii) Doxorubicin hychochloride (Adriamycin) - an antibiotic.

(iii) Vinblastine - a vinca alkaloid. (iv) Methotrexate - an antimetabolite.

In view of the effectiveness of Adriamycin the less toxic, but structurally related agent mitozantrone (Novantrone), was also studied in later patients. Carcinoma cells in aliquots of lo5 were either exposed for 24 h to a single concentration of a particular agent, or to increasing concentrations.

Incorporation ~f '~Selenomethionine i ' 5 S e M ) This was as described by Ferro et al. (1988). From each 1 ml aliquot of carcinoma cells, incorporating '%eM following exposure to a given concentration of a particular drug, three 0.2ml samples were obtained and the radioactivity incorporated in the cells was determined. In a separate experiment (Lai, unpublished data), repeat determinations of protein synthesis inhibition following drug expo- sure were made using a carcinoma cell line HT29. This yielded consistent data. Using mitozantrone, tumour inhibition was directly related to drug concentration.

Statistics

For a given carcinoma all of the values for ' 3 e M incorporation were subjected to a one-way analysis of variance. The significance of any reduction in counts following exposure to a given concentration of a particular drug was determined in comparison to that for cells not exposed to any drug.

Results

The details of the 23 patients are shown in Table 1 . Although 17 patients had tumour spread beyond

the kidney, only 4 received drug therapy, 3 with Depo-Provera and 1 with methotrexate. Of the 17 patients, 9 survived for more than 2 years, a further 3 for more than 1 year; 10 of these 17 patients remain well.

The percentage inhibition of protein synthesis and its significance following exposure to drugs are shown for Depo-Provera (Table 2) , Adriamycin (Table 3), Novantrone (Table 4), vinblastine (Table 5 ) and methotrexate (Table 6). The peak plasma concentration and the terminal half-life following standard in cico administration were : Adriamycin 0.24-0.60 pg/ml and 28.1-43.6 h, Vinblastine 0.44- 1.37 pg/ml and 17.4-21.8 h, Methotrexate 2.75 pg/ml and 7.6 h (Alberts and Chen, 1980), Novan- trone 1.2-1.5 kg/ml and 38.0 h (Hulhoven et al., 1984).

At a concentration of 1 pg/ml Depo-Provera, carcinoma cells from 5 of 23 patients showed 250% inhibition of protein synthesis. The com- parable proportion for Adriamycin was 4 of 5 and for Novantrone 7 of 12 patients. The data for vinblastine and methotrexate are less relevant in that the concentrations of drugs used in vitro were greatly in excess of those seen following in cioo administration. Despite this, at 10 pg/ml vinblas- tine, carcinoma cells from only 3 of 12 patients showed > 50% inhibition of protein synthesis. At 400 kg/ml methotrexate, the comparable propor- tion was 5 of 12 patients. There was no relationship between responsiveness in vitro to a particular drug and the histological pattern of the neoplasm except in the cases of Novantrone (vide infra).

Discussion

Using a mouse mammary carcinoma, in z:itro inhibition of "SeM uptake was related to the in vitro anti-tumour acztion of the drugs tested (Lai et al., 1989). The unpredictable course of renal carcinoma (Holland, 1973) makes it difficult to evaluate the outcome of any therapy. The relatively long survival of patients with tumour spread beyond the kidney in the present series is a reflection of this. It also questions the necessity of therapy other than nephrectomy. However, if a clear indication of tumour responsiveness could be obtained, admin- istration of the appropriate drug might be indicated in patients with metastases.

In the present study, with the exception of patients 1, 5 and 18, no correlation between in uitro and in cico results of treatment was possible as no drug treatment was given. However, in these 3 cases there was good agreement, Harris (1983)

TESTING THE CHEMOSENSITIVITY OF RENAL CARCINOMA I N VITRO 27

Table 1 Details of Patients

Turnour Size Degree of No. Age Se.r (ern) spread (stage) Histology Chemotheraphy Results

1

2

3 4

5

6 7

8 9

10 1 1 12

13

14 15 16

17

18

19

20

21 22

23

63

68

74 60

57

67 70

52 69 68 70 70

70

67 51 63

77

40

65

51

54 54

50

M

M

M F

M

F M

F M F M M

M

M M M

F

M

M

M

M F

M

6 x 5 ~ 2 IV

IV

4 x 5 ~ 2 1 7 x 8 ~ 6 I1

Diam 10 IV

Pleomorphic adenocarcinoma. Some areas of clear cells. Numerous cysts Clear cell ca. with some oxyphil cells Granular and clear ca. cells Tubular arrangement of ca. cells with intervining areas of spindle cells Solid clear cell ca.

5 x 4 x 2.5 1 I 1 x 1 0 x 6 111

3 0 x 1 2 x 15 111 Cyst diam 11.5 111 7 x 7 ~ 8 111 8 x 6 ~ 4 I11 6 x 6 ~ 6 111

7 X 7 X 5 1 v

Diam 5.5 I 1 4 X 1 2 X 7 I1 Diam 6 1 v

1 5 x 1 1 ~ 8 IV

1 9 X 1 2 X 9 IV

12X12X11 I V

1 8 x 9 ~ 8 IV

1 0 x 9 ~ 4 111 Diam 10 11

1 2 X 1 0 X 9 I1

Clear cell ca. Polycystic variant of renal ca. Tubulo-papillary ca. Clear cell ca. in wall of cyst Clear cell ca. Clear cell ca.

Moderately diff. adenocarcinoma

Clear cell cd. Clear cell ca. Few mitoses Poorly diff ca. Mainly clear cells. Some epithelial cells Moderatel) diff. ca. Clear cells Adenocarcinoma. Varying cell types and patterns. Focal sarcomatous areas Clear cell ca.

Mixed clear and granular cell ca. Clear cell C A .

Clear cell cd. Well differentiated Clear cell cd.

M P A t

MTX:

Nil Nil

MPA

Nil Nil

Nil Nil Nil Nil Nil

Nil

Nil Nil Nil (local DXR) Nil

MPA

Nil

DXR

Nil Nil

Nil

Relief of pain. Died 2 years 1 month. Progressive disease

A & W* 2 years 9 months A & W 3 years

Relief of pain. Died 8 months. Progressive disease A & W 3 years Died 2 days. Myocardial infarct A & W 3 years A & W 3 years 4 months A & W 2 years A & W I year 9 months A & W I year 1 I months with ca. oesophagus Died 4 days. Bronchopneumonia. Acute tubular necrosis A & W 2 years 2 months A & W 2 years 1 month 1 1 months. Unfit to attend clinic Died 3 days. Gastric perforation Died I month. Progressive disease

Died 5 months. Progressive disease A & W 1 year 10 months

A & W 3 months Awaiting first follow-up

First follow-up 1 month

All patients underwent a radical nephrectomy. * A & W =Alive and well. t MPA = Medroxyprogesterone acetate. 3 MTX = Methotrexate.

presented data for the number of objective re- response rate of 24% among 135 patients treated sponses in patients with renal carcinoma treated with vinblastine. This compares with 25% of renal withprogesterone. The figureof 10% in 300patients, carcinomas showing inhibition of protein synthesis compared with 22% for 2 50% inhibition of protein when exposed in citro to vinblastine (vide supra). synthesis recorded in the present study is of great Oliver et al. (personal communication) treated 12 interest. Heim et al. (1981) reported an objective patients with bulky rapidly progressive renal

BRITISH JOURNAL OF UROLOGY

Table 2 zitro Exposure to Depo-Provera

Putlei7l Conc ? I 0 w

Percentage Inhibition of -'SeM Uptake b) 111

I

7

5 6 7 X 9

10

I I 12 13 14 I 5 16 17 18 I9 20 21

23

, -

4

13 --

0.1 1.0 5.0 66.0s 16 0' x . n t 54.03

50.03 5.0

86.0s Nil

Nil Nil

culture 8 days Nil 77.06 Nil 20.0* Nil 15.0* 8.0 6.0 Nil

Nil Nil 22.0: 24.0:

2.3 Nil 20.8s 42.86

30.6 41.6

Nil Nil

0.8 7.4

culture 19 days 4.9 25.5

10.0

14.0 26.0: Nil Nil 81.03 69.06

7 .7 31 .1 26.0 Nil

+ * < n o s t < 0 0 1 :<I3005 4 < n no1

Cells were exposed to hormone for 24 h a n d -'SeM for 48 h

Table 3 I itro Exposure to Adriam>cint

Percentage Inhibition of - 3 e M Uptake bq rn

I 0 5 0 10.0

I I 89.0* 12 9.0 65.0* I3 9I).O* 95.O* 95.0* 15 7 0 . 0 * 55.0" ?9.0* 16 71.0* 93.0' 97.0* 17 57.0* 96.0' 98.0'

* P= <O.OOl t Cells were exposed t o drug tor 24 h and -'SeM for 48 h

carcinoma using Novantrone. They obtained 1 partial remission which lasted for 13 months. Further studies on the use of Novantrone in the treatment of metastatic renal carcinoma may therefore be of value.

Our results indicated that Adriamycin and the related drug Novantrone were more effective than

Table 4 ritro Exposure to Novantrone

Percentage Inhibition of '%eM Uptake by in

Patient no. Conc pg!ml

0 1 1.0 5.0 10.0

12 1.0 Nil mt 13 80.0: 97.0: 97.01 13 15.0' 5.0 17.o* 15 14.0 48.0: 52.01 16 77.0: 94.0: 98.01 17 81.0: 97.0: 97.01 18 61.2; 90.3: 88.lf 19 74.4: 93.7: 92.7: 20 13.2 55.4: 48.61 21 52.7 57.5* 90.3t I_ 71 13.2 2.9 13.1 23 Nil 20.8 39.6*

*P= <0.05. t P = <0.005. :P= <0.001.

Table 5 ritro Exposure to Vinblastine

Percentage Inhibition of 'SeM Uptake by in

Patient no Conc pg ml

1 .o 5.0 10.0

Nil

Nil 69.0t Nil

1 .o Ni l 74.01. N i l

Culture 8 days Nil

16.n*

a n t

56.0t 53.0t s3.w 11.0 2.0

other agents in inhibiting renal carcinoma cells in zitro. All patients whose tumours showed in ritro sensitivity to Adriamycin had extension of their disease beyond the kidney. The same was true for Novantrone and, by contrast, 3 of 4 patients whose carcinoma cells were not sensitive had tumours confined to the kidney. The spread of renal carcinoma cells beyond the kidney may be associ- ated with a higher mitotic rate and thus with an increased sensitivity to Adriamycin or Novantrone. Support for this hypothesis comes from the obser- vation that respectively 3 of 4 and 6 of 7 sensitive tumours showed a mixed cell pattern and/or moderate or poor differentiation.

TESTING THE CHEMOSENSITIVITY OF RENAL CARCINOMA I N VJTRO 29

Table 6 uitro Exposure to Methot rexa te

Percentage Inhibi t ion of 7SSeM U p t a k e by in

Patientno. 100 200 Concpgg/ml 400

1 Nil 2 59.0' 3 Nil 4 55.0* 7 52.0* 8 Nil 9 83.0*

10 culture 8 days 12.0 I I 5.0* 12 Nil Nil Nil 14 2.0 Nil Nil 16 Nil Nil 17 2.5 55.0* 57.0*

* P = <O.OOI.

Acknowledgments We thank Miss Doris Heinemann, FIMLS, for her assistance and Lederle Laboratories for their generous gift of mitozantrone.

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Patel, N. P. and Lavengood, R. W. (1978). Renal cell carcinoma: natural history and results of treatment. J . Urol., 119, 722- 726.

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The Authors Teresa Lai, MSc, MIBiol, Research Associate, Department of

M . A. Ferro, ChM, FRCS, Consultant Urologist, Royal

A. V. Kaisary, ChM, FRCS, Consultant Urologist, Royal Free

P. J. B. Smith, ChM, FRCS, Consultant Urologist, Bristol Royal

M. 0. Symes, MD, Senior Lecturer and Consultant in Imrnu-

Surgery, University of Bristol.

Infirmary, Huddersfield.

Hospital, London.

Infirmary.

nology, Department of Surgery, University of Bristol.

Requests for reprints to: M. 0. Symes, Department of Surgery, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW.