Predicting response of human solid tumors to chemotherapy

Cancer ‘Treatment Reviews

(1983)

10,203-219

Predicting response chemotherapy S. Tanneberger

of human

solid tumors

to

and E. Nissen

Central Institute for Cancer Research, Academy of Sciences, Berlin-Buch, Republic

German Democratic

In common with other groups, we have for more than 15 years emphasized that the biological individuality of human tumors at the clinical and cellular levels (92,95-97,99) demands individualized, ‘biology-adapted’ cancer treatment. The concept of individualized cancer treatment is now well established. It requires the pre-therapeutic characterization of the individual tumor biology and biochemistry (including cell kinetics, hormone receptors etc), and of the individual tumor-host-drug relationships (including biological markers, immuneprofile and pharmacokinetics). Work on the concept of individualized cancer treatment began in the early sixties in many institutes and hospitals, but this approach has had at best only a very limited effect on the general pattern of cancer treatment and little influence on clinical cancer chemotherapy. Even now, cancer chemotherapy is still the more or less empirical inauguration of new drug combination regimens and often means surgical adjuvant chemotherapy without sufficient scientific basis. In complete contrast to the patient-tailored approach to cancer chemotherapy, many chemotherapists have turned to aggressive, polychemotherapy. Unwanted side effects and enormously high costs are the price for this approach. Under some circumstances, however, aggressive multidrug treatment has a logical scientific basis, though the combination of several inactive drugs will certainly not overcome individual primary drug resistance. Why has individualized cancer chemotherapy not replaced the far less scientific, sometimes even unethical empirical approach to cancer chemotherapy? The reason is that methods for individualized cancer chemotherapy have been inadequate. Although some progress has been made during the last decade towards pretherapeutic characterization of human tumors (particularly hormone receptor assessment and the measurement of cell kinetics) many of the fundamental problems remain unresolved. In the present paper the current status of in vitro prediction of response of human solid tumors to chemotherapy will be reviewed. This ofcourse is only one aspect ofindividualized cancer chemotherapy but an important one. Efforts to predict drug sensitivity of human 0305-7372/83/040203

+ 17 $03.00/O

0 203

1983 Academic

Press Inc.

(London)

Limited

204

S. TANNEBERGER

AND

E. NISSEN

tumors by the use ofin vitro investigation of biopsy material date back to 1955 when Wright ( 117) and Cobb (12) studied the effect of triethylenemelamine (TEM) against human tumors in plasma clot cultures (11, 116, 117). Later, using other methods Limburg, Ambrose and others (2,46) continued the pioneering work in this field. The long history of predictive tests has repeatedly been reviewed (14, 24, 48, 55, 62, 93, 100) and our own studies on human tumor drug sensitivity prediction began in 1964. A summary is given in Table 1 in which the most important predictive tests are listed and classified by the principles of the technique used. The history of predictive tests is characterized by optimistic beginnings of investigators using relatively inadequate methodologies namely, monolayer or plasma clot cultures with

Table

1.

Prediction

of human

tumor

drug

sensitivity.

Conclusion:

Clinical

+

= valuable,

-

= no value

value

Correlation Approach Long-term tissue cultures + drug

Method Cell cultures/ morphology

Cell cultures/ transmembrane potential Cell cultures/ cell counting Cell cultures/ autoradiography Lymphocyte cultures/trypan blue dye exclusion Organ cultures/ DNA or RNA synthesis Organ cultures/ DNA synthesis Organ cultures/ histochemistry In vitrosoft-agar human tumor stem cell assay

Soft-agar culture assay/ scintillation counter

Patients 102 188 85 88 41 96 33 39 201 2.5

9/o

Conclusion

(+I (+I 65 63 46 83 81

(1, + + + + + +

Reference Lickiss et al. (1974) Cobb et al. (1964) Limburg (1973) Tanneberger el al. (1967) Krafft et al. (1981) Terentieva et al. (1976) Izsak et al. (1971) Marzotko et al. (1976) Walliser et al. (1981)

etal.

36

92

+

Holmes

48 53 40 11

100 77 79 100

+ + + +

Murphy etal. (1975) Zittoun etal. (1975) Livingstone ct al. (1980) Durkin et al. (1979)

450 55 5 108 74 152 10 18 66 5 119 40 21 14 23

45

100 100 100 62/99 87.5

(1974)

(+I + c=i (+) (+) (+) +

(+) + + + (1) + -c

Mitchel et al. (1972) Wheeler et al. ( 1974) Shrivastav et al. (1980) Tschao et al. (1962) Tanneberger etal. (1977) Nissen et al. (1978) Peek et at. (1981) Hecker et al. (1976)

Salmon etal. (1978) v. Hoff (1979) Tveit et al. (1980, 1981) Tveit et al. (1982) Alberts et al. (1980) Schlag et al. ( 1982) Ludwig etal. (1981) Tanigawa et al. (1982)

PREDICTING

Table

RESPONSE

TO

CHEMOTHERAPY

205

I.-continued Clinical

Approach Short-term tissue incubation +drug

Method Cell suspension/ morphology Biopsy/ autoradiography Ccl1 suspension/ autoradiography Cell suspension/ DNA or RNA synthesis

Biopsy/ DNA synthesis Cell suspension/ enzyme assay

Determination of druginactivating or target enzymes

Cell kinetic data

In viuo/in Intro test

Patients

73 55

15

93

50 23 24 24 125 23 114 69 144 47 21

92 50 100 40

22 89 60

Bone marrow+ cell suspension/ DNA synthesis SH groups

11

DNA dependent RNA polymerase (Bleomycin inactivating enzymes) TMP-Synthetase FdUMP d-UMP Clumps/biopsy/ labeling index Cell suspension/ cytofluometry Diffusion chamber/ cell suspension Xenograftsl colony assay

23

subrenal capsule assay/ nude mice Xenografts/ nude mice

Correlation o/0

48 148 25

27

Conclusion

+ + .-

23

100 80

83

47

313 (5) 5 15

44

100 100 100

el al. (1976)

Kondo (1971) Di Paolo (1971) Knock et al. (1974) Tisman (1973)

27 70

82

Dendy et al. (1973) Wright et al. (1973) Wolberg (1971)

Andrysek (1973) Hirschmann (1973) Possinger et al. (1976) Mattern et al. (1976) Bastert et al. (1975) Volm (1975) Volm ( 1980) Schlag (1979) Sanfilippo el al. (1981, 1982) Daidone et al. ( 198 1) Kaufmann et at. (1971)

90 85

45

Reference

Thirlwell

+

Kulik

+

Miiller

(1977)

+

Moran

et al. (1979)

Kirmiss

et al (1976)

34

2 In viva test

value

(1977)

+

Smets et al. (1976)

+

Heckmann

++ (:I +

(1967)

Bateman (1980) Tveit (1980) Siegel ( 1980) Bogden ( 1982)

Bastert

(1977)

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morphological evaluation of drug effects. Despite subsequent progress in tissue culture technique and biochemistry (organ culture, metabolic monitoring of cultures) the problem is unresolved. As shown in Table 1 most authors have evaluated their assays enthusiastically; the fact that no assay has achieved general clinical applications is evidence enough that such evaluations have generally been over optimistic. The in vitro soft-agar human tumor stem cell assaystrongly advocated over the last few years by some (19,2 1,29,35, 76, 77-79,91) has also received a more sober and realistic evaluation by others (8,49, 70, 72, 83, 90). Our own experiences concerning the use of in vitro methods to predict tumor response to antineoplastic chemotherapy were similar. In 1964 we started very optimistically with a cell culture technique, evaluating drug effects by morphological criteria. Having cultivated several hundred tumor specimens, we recognized that the preparation of single cells, their characterization in vitro and finally their cultivation as a monolayer is possible only in a limited number of cases (Fig. 1). Moreover, the effects of antimetabolites cannot be definitively detected by morphological methods (95). In the light of this experience we developed a second type of assay using the organ culture technique and evaluating the drug effects by DNA-synthesis measurement (96). Undoubtedly this organ culture assay was a real advance in comparison to the former technique of in vitro drug prediction. As can be seen in Figure 1 for example, the rate of in vitro maintenance for breast cancer specimen rose to 88.7% in organ culture compared with

TO-

60:: 2 5 e p"

50-

40-

30-

7 $5

20IO-

/d c

6’

Stomach

Figure 1. Percentage

ofgrowing

cultures biopsies);

ofdifferent 0, organ

tumor culture

types cultivated in vitro. technique (589 biopsies).

a,

cell culture

technique

(196

PREDICTING

RESPONSE

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CHEMOTHERAPY

207

only 32.8% in monolayer cell culture. Furthermore, the detection of cell viability with the aid of DNA-synthesis measurement is much more precise than with morphological methods. Still unresolved with this assay is the problem of whether the material investigated in organ culture can be assumed to be representative of the tumor Zn vivo. Due to the heterogeneity of human tumors this is an important limitation of the assay. The results of some clinical trials we performed using the organ culture predictive assay were however in good accord with our theoretical expectations. In 1972 two randomized clinical trials (UICC trials Nos. 72-020 and 72-053) evaluating individualized drug treatment using the organ culture assay were initiated in our institute. In the first of these trials 50 patients with ovarian cancer (40 palliative operations; 10 not operated) were treated with triazequone (Trenimon) (a drug which was ofinterest at the time) and 37 (31 after palliative operations; six not operated) were treated with a drug selected on the basis of the predictive assay. The survival rate of the patients treated individually had a tendency to be higher, but the difference was not statistically significantly different (60). For lung cancer no dilIerence in outcome was found between non-individualized (31 patients) and individualized (33 patients) surgical adjuvant chemotherapy treatment arms. A very promising result was obtained incorporating principles of individualized treatment in a surgical adjuvant chemotherapy protocol for the treatment of breast cancer stage III (UICC trial nos. 77-051) (66). F rom January 1974 to February 1981, 200 patients entered this study. For the first 110 patients the control/treatment ratio was 1 to 1, thereafter 1 to 2. One hundred and ninety-three patients are now evaluable. According to the protocol all patients (control and treatment arm) are investigated in vitro for drug sensitivity and estrogen receptor content of the tumor. The control group is treated by radical mastectomy only, the treatment group receives the predicted hormone chemoor chemotherapy in case of therapy in the case of sensitivity and ER+, and no hormone drug resistance and ER-. In case ofinsufficient in vitro data for technical reasons, patients in the treatment arm receive surgical adjuvant treatment with cyclophosphamide alone. As shown in Figure 2 a statistically significant longer relapse free survival has been achieved if drug sensitivity, ER f patients are treated with the predicted hormone-chemotherapy (72 patients) in comparison to the relapse free survival ofdrug sensitive, ER f control patients who had no adjuvant treatment (35 patients). This difference was not observed (Fig. 3) comparing the total control (85 patients) with the total treatment arm (105 patients). The relevance of this trial to the problem of correlation between in vitro drug prediction and clinical response is limited due to the combination of hormonetherapy and chemotherapy. Present indications also suggest that premenopausal control patients should perhaps have CMF adjuvant chemotherapy instead of no chemotherapy. Nevertheless this trial, which addressed the general problem of individualized chemotherapy, confirmed our present standpoint regarding the role of drug prediction in antineoplastic therapy. From these 20 years’ experience of in vitro drug predictive tests, the following observations seem to be justified: There are some biologically and pharmacologically acceptable approaches of some predictive value for antineoplastic drug efficacy in experimental systems and in man, but these approaches are complicated, time-consuming and need special laboratory facilities. In contrast to some over-optimistic evaluations in the past, there is available at present no assay for the prediction of antineoplastic drug efficacy which could be recommended for use in clinical practice. This criticism applies also to the ‘tumor stem cell assays’ which have received wide publicity during the last few years. Accordingly, human tumor drug sensitivity prediction and individualized cancer chemotherapy remains a major problem for applied cancer research. Surgical adjuvant chemotherapy is currently

208

S. TANNEBERGER

1

0 ?

’

6

I

I

I

12

18

24

AND

I

I

E. NISSEN

I

I

I

I

I

I

I

72

76

I

Time (months)

Figure 2. High risk breast cancer. CCT CICR Berlin Buch 1974-80. In vitro sensitive turnours, irrespective of age, menopausal and estrogen receptor state. Results of surgical adjuvant therapy, dfs, CT+HT12/80. Total: 107; treatment: 72 (I); control: 35 (0). ( ) = patients at risk x2 = 8.99; P < 0.005 (significant).

0

6

12

16

24

30

66

47 Time

46

54

60

66

72

76

I

(months)

Figure3. High risk breast cancer. CCT CICR Berlin Buch 1974-80. All cases irrespective ofage, menopausal state and results of ER-Assay and oncobiogram. Disease-free survival of patients with surgical adjuvant therapy according to predictive tests, resistant tumours included (12/80). Total: 193; treatment: 105 (I); control: 85 (0). ( ) = patients at risk x2 = 2.25; P > 0.1 (not significant).

PREDICTING

RESPONSE

TO

CHEMOTHERAPY

209

a field of increasing activity; the importance of drug sensitivity prediction is therefore that much the greater. There are some crucial obstacles in the way of an effective predictive test. Technical problems still exist in the in vitro methodology, but more fundamental problems result from the heterogeneity of human tumors in space and time (27, 31). Heterogeneity of human tumors refers to the balance between tumor and normal cells in each tumor and each part of a tumor. Many tumors are composed of mixed populations of tumor cells with different growth properties and different drug sensitivities. Taking into account tumor heterogeneity it seems clear that only those predictive techniques which ensure representative sampling of the entire tumor can be expected to produce useful answers. A very basic obstacle for the prediction of the tumor drug response is the great variability of human tumor drug sensitivity depending on the in vivo or in vitro biological environment of the tumor and of the stage oftumor cell development when testing occurs. In an experimental human cells in vitro nude mouse heterotransplantation system we have demonstrated that human tumor cells change such fundamental properties as growth rate and karyotype as well as the drug sensitivity pattern in a very dynamic way when growing under different conditions (6 1,65). The pharmacologic heterogeneity among different metastases, as well as between the metastasis and the primary tumor are probably related to this phenomenon. What then is the present status of the various tests presently available? The following summarizes the problems with regard to sampling and maintenance of human tumors in vitro. (1) Zsolated cells for short-term studies, monolayer cultures or soft-agar cloning frequently cannot be obtained in sufficient amounts, particularly in such clinically important tumor types as breast and stomach cancer (22). This still seems to be true, in spite of some recent positive results which may indicate promising new approaches (36). Furthermore, as emphasized by Dendy 1980 ( 15), the identification of isolated cells as neoplastic, essential for a successful assay, is still an unresolved problem. The risk of selection from mixed cell populations can never be overlooked. Selection of subpopulations able to grow in vitro may automatically introduce a false positive bias in the test procedure since tumor cells unable to grow under these conditions may also be insensitive to drugs (27). Thus it was to be expected that assays such as those described by us (92,95) or by Salmon (73377) would be much better at predicting drug insensitivity than drug sensitivity. Finally, the kinetics of isolated single cells are very different from those of the cells in tissues, a fact which can influence interpretation of drug sensitivity (22). (2) Tumor biopsies are used in short-term incubation techniques which avoid tissue disaggregation or digestion. Short-term incubation techniques have the disadvantage that the ‘dying tissue’ incubated may not be representative of a proliferating tumor. Another crucial point is the representativeness and nature of the biopsies themselves. Due to the heterogeneity of human tumors there is a high risk of biopsying connective tissue instead of neoplastic tissue. Knowledge of what is in the test tube is essential for a successful in vitro assay but this is difficult to acquire when working with biopsies. (3) In organ cultures on the other hand vital tumor tissue is under investigation (63). The tumor cells metabolize in the normal biological environment, the original biological architecture, cellular interrelationships and function are largely retained. There are only minimal deviations of the cell behaviour from the in uivo situation. To this extent organ

210 cultures problems remain, Each unique

S. TANNEBERGERAND E. NISSEN have remarkable advantages in comparison to the other in vitro approaches. The of representativeness and the nature of the biopsied and cultured material however, an issue for conventional organ culture techniques. of the main approaches to assess drug-induced damage and death in vitro also has and important problems:

(1) Morphological methods are the simplest means to detect cell damage and cell death. Although measurement of the multiplication rate of a cell population without doubt provides definitive information on the viability of any cell population, the measurement of the multiplication rate of a given cell population is difficult and time-consuming to standardize. For these reasons alternative, technically easier methods have been sought. Morphological criteria of cell damage and death such as shrinking, granulation etc. can be used to assess the effects of some antineoplastic drugs but, unfortunately some compounds, e.g. antimetabolites, cause late and not early changes of cells and cell properties. Some groups have attempted to render morphologic assessment of cell kill less subjective by counting the proportion of cells remaining in culture than can exclude vital dyes such as trypan blue. Evaluation of these techniques leads to the conclusion that response to vital dyes is not a reliable indicator of drug-induced cytotoxicity. In vitro predictive tests dependent on interpretation of morphological changes in tumor cells therefore appear unpromising, even when efforts are made to make such interpretations semiquantitative (46). (2) Biochemical techniques for the measurement of cell damage and death are widely used. In vitro tests that measure inhibition of cellular metabolism, dehydrogenase activity or oxygen consumption, for example, were already proposed in the pioneer period ofdrug prediction. Despite the apparent logic behind this approach it appears that the metabolic parameters used were too unspecific to be used as indicators of the death of tumor cells. Furthermore the technical difficulties of measuring these parameters prevent the routine use of such assays. Radioactive precursor incorporation has also been used to measure cell damage and cell death. The measurement of DNA-, RNA- or protein synthesis after 1 to 2 days of drug influence on metabolizing cells no doubt adequately reflects cellular defects. Unfortunately, however, radioactive precursor incorporation is often used in so-called short-term incubation assays and this practice needs critical evaluation. Since not all antineoplastic compounds act directly against DNA-, RNAand protein synthesis, precursor incorporation inhibition has inherent limitations (84). Moreover, a realistic picture of ultimate drug effects cannot be expected to be apparent within a few hours (see also 48). In the presence of drugs such as 5-Fluorouracil in fact, an apparent increase of 3Hthymidine uptake is to be expected due to the increased use of metabolic salvage pathways. Only in cases where the mechanism of drug action is known, is short-term radioactive precursor incorporation justified. Furthermore the precursor to be incorporated has to be chosen according to the known mechanism ofdrug action. Another limitation ofshort-term precursor incorporation assays results from the failure of DNA precursors to be incorporated in cells with a long cycle time (7,69). After explanation to an in vitro system the cell cycle time is commonly prolonged. This phenomenon increases the risk of failure of a short-term precursor incorporation assay. (3) Measurement of the cloning ability of tumor cells in soft-agar is, theoretically, an interesting approach. Cloning increases the chance that from a mixed single cell population only

PREDICTING

RESPONSE

TO

CHEMOTHERAPY

211

neoplastic cells have to be considered since neoplastic cells have a selectively higher potential for cloning in soft-agar. Moreover, cloning is a very precise test for cell viability (23, 24, 38, 68). Nevertheless there are increasing doubts as to whether the optimism of some groups working with the so-called human tumor stem cell assays is justified. There are theoretical and practical doubts concerning this approach. First of all, the hypothesis that clonogenic cells are representative human tumor stem cells needs further support. Neither is there at present evidence enough to show that the cells isolated from human tumors which clone are representative of the entire in vivo stem cell population. For this reason terms like colony forming assay (48) or clonogenic tumor cell assay (24) for this approach seem to be more realistic than the term ‘human tumor stem cell assay’ often used by Salmon (73, 75). For all populations used for cloning the same considerations apply as for populations of isolated cells (see above). The well known problems (failure of disaggregation, cell identification, selection of tumor cell subpopulations etc.) are likely also to play a role in the clonogenic tumor cell assay. Furthermore, limitations of the cloning ability of tumor cells in have also been described (8, 22, 49, 5 1, 70). Steel (89) p ointed out a special problem using cloning ability as an approach to assess drug-induced cell damage. The cell suspensions are made by conventional methods and plating efficiencies are generally below 0. l%, leaving open the possibility that the colonies that form arise predominantly from cell clumps. In view of the possible existence of clumps in the plated cell suspensions and the low (30 cells) criterion often used to score positive growth, it could be that the resistant components reflect a cell cloning artifact rather than true cellular resistance. In general this means that the clonogenic tumor cell assay is restricted with respect to the prediction ‘sensitive’ cells by tumor heterogeneity (27, 83) and, with respect to the prediction ‘resistant’ cells by cell cloning artifacts which possibly exist (69). Furthermore, a number of problems of cell isolation and characterization are unsolved. Finally the long duration of a clonogenic stem cell assay ( 10 days on the average) limits clinical application. Despite much publicity to the contrary doubts remain that the cloning ability approach is very promising and the technique demands more critical evaluation. Soft-agar cell colony assays may however eventually be useful tools for preclinical drug screening and drug development (36, 71, 77). (4) A more general problem playing a role in all approaches to in vitro drug sensitivity assessment is the dependency ofdrug effects on a number of conditions of the in vitro systems. Among these are the influence of medium composition, medium pH and serum quality (64). (5) Finally, it should be interaction with neoplastic vitro, if a realistic assay is to this direction (34, 86, 108)

emphasized that drugs requiring in vivo activation before cells such as cyclophosphamide must be artificially activated in be performed. There are a number of promising approaches in including the application of post-injection serum of patients

(47). Summarizing this critical review of the main obstacles to the development of an effective in vitro sensitivity prediction assay it is possible to formulate (10) a series of desirable characteristics which the ‘ideal’ assay ought to have. (a) Sampling and maintenance of human tumors for drug prediction should guarantee that only well-characterized, representative neoplastic cells which behave biologically and biochemically as they do in vivo are investigated. Particularly important is that cells

212

(b) (c) (d) (e) (f) (g)

S. TANNEBERGER.

AND

E. NISSEN

should metabolize and proliferate as in vivo, thus having comparable cell kinetic parameters. The main properties of the tumor cells should be stable in the in vitro system. The antineoplastic drug effects should be assayed by methods which objectively evaluate the viability of the neoplastic cells in vitro. The antineoplastic drugs should be tested in concentrations comparable to those achieved in vivo and should be in the active in vivo form and concentration. Not more than one week should be needed to obtain the results from the assay for the clinic. Drug resistance should be predictive with at least 90% probability, and drug sensitivity with 75% probability. The assay should be cheap enough to be used for the majority of patients in all hospitals.

Based on these ideal characteristics a new predictive test has been developed in our institute (Fig. 4) (63). The new test was based on our positive experience with organ cultures, and aimed to overcome the very critical problem ofinsufficient characterization of the tumor samples using the conventional organ culture technique. Organ cultures seem to us to be the most promising approach for sampling and maintenance of human tumors for in Ambrose (2, 104) have underlined that vitro drug prediction. Foley (20) and particularly organ cultures meet the essential requirement of an in vitro predictive test much better than other approaches. As mentioned above, organ cultures preserve the cellular interrelationships and, in contrast to single cell culture, there is no risk of selecting a small and unrepresentative proportion of the stem or total cell population. The organ culture method has since been successfully reproduced by some groups (17, 53). There are some alternative means to overcome the problem of insufficient characterization of tumor samples investigated in organ culture. Theoretically autoradiographic assessment of the drug effects appear to be such an alternative, but this approach is not practical particularly because of the long exposure time. We sought therefore a way to combine morphological control of the cultures and quantitative biochemical assessment of the drug effects. Based on earlier work (94) using tissue slices instead of human tumor Bio sy r Slicer + 48 h organ culture

Slice 1

control

48 h organ

culture

by microscopy

1 1 2 h with 3H-Thymidin 4 + measurement 3H-Thymidin /pg

SLices incubated 4 DNA extract Control

# before cultivation

Figure 4.

Slice organ

culture

and

Control

+ after cultivation

assay for human

tumor

drug

sensitivity

DNA

1 Drug influenced prediction.

PREDICTING

Table

2.

RESPONSE

DNA synthesis of human breast cancer and after (48 h) organ culture of biopsies

Tumor Patient

Histology

Before

TO

CHEMOTHERAPY

213

(dpm 3H-Tbymidine/pg and slices

DNA)

biopsies

culture

measured

Tumor

After

culture

Before

before

slices

culture

After

culture

64.6 100.9

75.6 82.8

2335.8 2006.4

3142.3 3941.6

St. E.

126.1 94.7

63.6 89.3

1949.5 2271.8

4197.6 3838.5

F.R.

291.3 304.1

191.6 206.7

3377.8 3885.0

2175.5 373 1.9

Sch. D.

220. I 169.5

402.1 206.6

2697.6 3689.5

2266.2 2548.7

P.A.

166.4 134.1

119.5 115.0

2308.3 4383.3

3990.2 4909.8

L.I.

291.4 244.3

472.9 308.5

2338.2 1459.0

1162.8 1439.9

Sch. E.

350.8 468.5

541.6 813.4

2191.2 4524.4

3039.7 4413.5

E.S.

119.3 205.3

151.8 182.8

10,624.2 7860.2

2420.8 3882.5

331.3 234.5

895.5 836.2

1889.4 1758.0

15,743.7 17,701.7

1091.7 1624.4

292.6 193.1

29,773.3 28,087.5

6445.6 3245.1

F.I.

Simple, undifferentiated

sp. s.

Medullary

U.1.

Table

3.

Tumor drug response in vitro measured by conventional assay (COCA) or slice, organ culture assay (SOCA). Histotogy: non-diff., medull., simple breast cancer

dpm/pg ~~___

Concentration Ml 1

Drug

DNA-synthesis DNA (“/

74.7 (78.3%) 112.4 (119.4%)

200.0 20.0

56.2 (59.7%) 555.6 (590.1%)

DBD

10.0 1.0

175.9 (186.8%) 208.6 (221.6%)

MTX

6.0 0.6

52.0 (55.2%) 391.8 (416.1%)

60.0

69.6 (73.9%) 218.3 (231.90/)

FU

L-PAM Control

after culture

Control

before culture

VLB, L-PAM,

vinblastine; melphalan.

5-fluorouracil;

DBD,

SOCA 321.0 544.0

(32.8%) (55.6%)

438.3 (44.8%) 1166.1 (119.20/) 161.4 389.3

(26.5%) (39.8%)

646.1 (66.0%) 1824.7 (186.5%) 170.0 318.9

(17.4%) (32.6%)

(91.7%)

978.3

(164.2%)

102.7 (100.0%)

595.9

(100.0%)

94.2

FU,

control)

COCA

2.0 0.2

VLB

organ culture Patient: H.A.

daunomycin;

MTX,

methotrexate;

214

S. TANNEBERGER

AND

E. NISSEN

Tumour

~~

Host

intoxication .

Drug Metabolism

of drug

excretion, Figure 5. Tumor-drug-host

Host

resorption

interaction

in cancer

chemotherapy.

biopsies for organ culture, a new technique has been developed. It is based on Krumdieck’s (42) method of cutting uniform slices of human tumors which can be characterized morphologically and then cultivated in organ culture. The technique guarantees, in contrast to the conventional organ culture technique, a strict morphological control of all cultures. Furthermore, as shown in Table 2 the metabolic activity of the tissue slices maintained in vitro is much higher than biopsies similarly maintained due to an improved cell-medium interaction and improved oxygenation. Cell viability was assessed with 3Hthymidine inrorporation measurement after 48 h drug exposure and this technique was then incorporated into the assay. As shown in Table 3 antineoplastic drug effects and the individual tumor sensitivity are readily recognized with the 3H-thymidine uptake measurement technique applied to drug exposed tumor slices cultivated in organ culture. The results are more precise than in the conventional oral culture assay. Our first experience with the new assay suggest that this approach is a realistic route to individualized cancer chemotherapy based on accurate predictive tests. Our knowledge of the previous history of such tests however tempers our optimism. Here, as everywhere in drug prediction (and science in general) what Hamburger (24) emphasized in an excellent review of predictive cancer chemotherapy holds true, namely ‘That a system must be utilized by many investigators before it can be effectively used as a clinical tool’. Furthermore, it must not be forgotten that drug sensitivity prediction is only a part of individualized antineoplastic drug treatment (Fig. 5). References 1. Alberts, assay--in

D. S., Chen, r&o results

H. S. G., Soehnlen, B., Salmon, S. E., Surwit, and clinical correlation. Laacct ii: 34&342.

2. Ambrose, E. J., Andrew, 2: 499-504. 3. Andreysek, 0. (1975)

R. D. & Easty, Sensibilitatstestungen

D. M. (1974) van

Drug

E. A. & Young,

assay on organ

Zytostatika

an

Jododeoxyuridin. In (Wiist. G., ed.) Aktuelle Problemeder Them&e maligner pp. 8+85. 4. Bastert, G., Schmidt-Matthiesen, H., Gerner, R., Michel, R. T., Nord, Testung der Sensibilitat van Mamma-Karzinomen gegen Zytostatika. 2035 2043.

cultures

menschlichen

L. (1980)

Agar

of biopsies. Tumoren

‘Twnoren. Stuttgart:

Georg

cloning

Br. Med. mit

3.

“sJ-

Thieme,

D. & Leppien, G. (1975) In vitro Dtsch. med. Wochmrchriit 100:

PREDICTING

5. Bastert,

RESPONSE

G., Schmidt-Matthiesen,

(1977)

Human

mammary

H., Michel,

cancer

in nu-nu

TO

CHEMOTHERAPY

R. T., Fortmeyer,

mice.

A model

215

H. P. Sturm,

for testing

in research

R., Nord,

D. & Gerner,

and clinic.

Klin.

R.

Wochenschr.

55: 83-84. 6. Bateman, A. E., Selby, P. J., Stell, G. M. & Towse, G. D. W. (1980) human melanoma. Br. J. Cancer 41: 189198. 7. Beth-Hausen, T. N., Sara+, F., Sutherland, D. J. & Ling, V. (1977) of tumor cells. 3. N&l. Cancer Inst. 59: 21-27. I., Bradley, T. R., Campbell, J. J., Day, A. J., M c D onald,

sensitivity 8. Bertoncello,

In vitro sensitivity Rapid

test on xenografted

assays for evaluating

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