An attempt to develop a radioactive drug

AN

ATTEMPT

TO

DEVELOP

A RADIOACTIVE

DRUG*

Professor J. S. M I T C H E L L , C.B.E., M.D., Ph.D., F.R.S., F.R.C.P., F.F.R.

The Radiotherapeutic Centre, Addenbrooke's Hospital, Trumpington Street, Cambridge IN 1915, Dr. Robert Knox published his important text-book entitled ' R a d i o g r a p h y , X-ray Therapeutics and Radium Therapy'. Fifty years later, it is of great interest to read in this book on page 263: 'The endeavour must be to procure a substance, radioactive or not, which, when introduced into the general circulation, will influence morbid processes in the tissues'. This is the earliest reference that I have been able to find to the idea of a radioactive drug, excluding of course the misguided efforts of that time to use inorganic preparations of radium, mesothorium and other natural radioactive elements internally in treatment. The possibility of practical development of radioactive drugs for use in the treatment of patients with cancer could only be envisaged many years later when artificial radio-active isotopes became widely available. Probably the most important stimulus to this development was the success of radio-iodine therapy in some cases of carcinoma of the thyroid, following the pioneer work of Keston et al. (1942). In the present context, the term radioactive drug is used to mean an organic compound which is concentrated or absorbed to some extent selectively by the malignant cells of tumours and carries incorporated atoms of radio-active isotopes in sufficiently high specific activity to produce therapeutic irradiation in situ, i.e., in the tumour cells without significant irradiation of normal cells. The development of a radioactive drug appears to depend upon the finding of substantial and exploitable biochemical differences between malignant and normal cells. Since about 1952, our team at Cambridge has been working on the scientific and clinical aspects of this problem. We came to the conclusion that tritium--radioactive hydrogen, aH or T--is the most suitable radioactive isotope for incorporation into a carrier molecule for this purpose. It emits only soft-particles, which have .a maximum range of about 6~ in unit density tissue, so that the biological effects are strictly localized and hazards in handling are reduced to a minimum. The radioactive half-life is 12-4 years but almost invariably the values of the biological half-life are very much less, usually a matter of days. Since B

about 1953, tritium 11as become easily available in pure form. Tritium gas now costs £10 for the first Curie, and then £2 10s. per Curie. Most of our investigations have concerned tritiated derivatives of the soluble compound 2-methyl-I, 4-naphthaquinol bis (disodium phosphate) (Fig. 1). This compound has been in use in medicine since 1941 as the synthetic vitamin K-substitute, Synkavit (Roche Products Ltd.) and had been investigated by us as a radiosensitizer since 1946 (see Mitchell and Simon-Reuss, 1947; Mitchell, 1960; Marrian, Marshall and Mitchell, 1961; Mitchell, Brinkley and Haybittle, 1965). We have studied a series of tritiated derivatives of this compound in the treatment of 102 patients with advanced recurrent and inoperable neoplastic diseases between 4.2.1959 and 1.3.1965. The first tritiated preparations used, described as T1, were made by the Wilzbach exchange method and often turned out to be rather toxic as a result of contamination by varying amounts of tritiated water. Then my colleague, Dr. D. H. Marrian, devised a method of tritiation which made it possible to obtain reliable preparations of the compound containing approximately one atom of tritium per molecule with the tritium firmly bound. The radioactive drugs used (see Fig. 2) have been prepared as a result of a collaborative effort of the Department of Radiotherapeutics, University of Cambridge, The Radiochemical Centre, Amersham, and Roche Products Ltd., Welwyn Garden City (Andrews, Bultitude, Evans, Gronow, Lambert and Marrian, 1962); the preparations are referred to as T R A 72, TRA 119 and T R K 219, .ONa O=P-oN a O

~

CH 3

.ONa O=P'oN a 2 - methyl -1.4- naphthaquinoL bis(disodiurn phosphate). Synkavit. FIG. 1 * The R o b e r t K n o x Memorial Lecture, 1965. 305

306

CLINICAL O p.ONa =, " O N a O

_OI / O N a 0 - P'ONa

T

TRA72 Maxima[ specific Activity Dale oF introduclien

29.1 Curies per mM (14"64 mg p e r Curie I

.ONa O=P.ONa O

O p.ONa : l "ONa O

~]ONa O =r.ON a

O.ON" 0 =P ' O N a

TRA119 87-3 Curies per mM (4"88 mg per Curie)

21sl September 1960 7th March 1962 THE RADIOACTIVE DRUGS

TRK219 58'2 Curies per mM [7-32mg per Curie) 24 th Seplember 1964

~IO. 2

OH

I

HO3S@SO3H I - lode - 8-naphthol-3, 6-disulphonic acid. ( K. Kottmann, 1935) FIG. 3

respectively, following the catalogue of The Radiochemical Centre. T R A 72 is 2-methyl-6tritio-1, 4-naphthaquinol bis (disodium phosphate) containing 1 atom of tritium per molecule; TRA 119 is 2-methyl-5, 6, 7-tritritio-1, 4-naphthaquinol bis (disodium phosphate) and theoretically contains 3 atoms of tritium per molecule. It now appears that depite the lower specific activity, the most reliable compound for investigation as a radioactive drug is T R K 219, 2-methyl-6, 7-ditritio-l, 4-naphthaquinol bis (disodium phosphate) incorporating 2 tritium atoms per molecule with a specific activity of 58-2 Curies per millimol and containing 1 Curie in 7.3~ mg. of compound. The Radiochemical Centre has been able to provide sufficient T R K 219 to meet our clinical needs, though not without some difficulty mainly on account of the problems of decomposition due to auto-irradiation in concentrated aqueous solution and of variation in the specific activity of different batches. Investigation of these problems continues. The solutions are sterilized by filtration into sterile rubber capped bottles and chlorocresol added as a bacteriostatic to give a concentration of 0.2%. The solutions are frozen immediately and have been kept under nitrogen at --78 + C. surrounded by solid CO2 in a Dewar vessel in a deep freeze at ambient temperature about --20 ° C. There is recent evidence that storage of T R K 219 in solution at the temperature of liquid nitrogen (--196 ° C.) reduces the rate of decomposition by a factor of at least 5 (Evans, 1965). Other Compounds and Related Studies.--The beginnings of this type of work may be of interest

RADIOLOGY

here. One of the first compounds with radioactive labelling developed for clinical localization of abscesses and tumours was disodium 1-amino-8. naphthol-3,6-disulphonate, b r o m i n a t e d and labelled with a2Br (Kroll, Strauss and Necheles, 1941). The choice of this compound was based on the work of Kottmann (1935), who showed that certain naphthalene derivatives concentrated selectively in experimental tumours: in addition, the compound, 1-iodo-8-naphthol-3, 6-disulphonie acid, was found to be a radiosensitizer; see Fig. 3. (This work has come to my notice only recently.) The most important compounds which have been studied as radioactive drugs are listed in Table 1. The localization of tetracyclin and tritiated tetracyclin has been examined again recently by Machado et al. (1964) who conclude that 'the actual site of affinity is the necrotic or prenecrotic area of the tumor'. The possibility of serious limitations to the development of radioactive drugs and perhaps to cancer chemotherapy generally, is raised by (1) the probable existence of a non-proliferating fraction of the tumour cells in some tumours (Mendelsohn, 1960, 1962a and b, 1963, 1964) and probably by (2) the inaccessibility to the drugs of parts of tumours containing viable cells as a result of defective blood supply even after continued intraarterial infusion (see Clarkson, e t a and Karnofsky, 1962; Goldacre and Syl6vn, 1962). On the basis of evidence suggesting that atebrin concentrated in some animal and human tumours (Lewis and Goland, 1948; Cramer, Brilmayer and Pabst, 1952; Brilmayer, Kohler, Mack and Stordeur, 1955, but also see Weissberg, 1959), my colleagues, Dr. F. Wild and Dr. J. M. Young, prepared atebrin tritiated in the side chain. Unfortunately, there was no selective concentration of the tritium in the Walker carcinoma 256 in rats, Ehrlich ascites tumour cells in mice or HeLa cells in tissue culture (Young, 1963; Young, Wild and Simon-Reuss, 1965). The tritiated side chain of the atebrin was removed in rive, probably as a result of reactions with thiol groups. Further, in the case of tritiated thiodiglycollic acid, the biological experiments were not encouraging (Young, 1963). A brief reference must be made to the development of lalI-carrying antibodies to fibrin. Recently, interesting results have been reported by Bale and Spar (1964) on 136 patients with known and usually advanced malignant disease to whom tracer doses of laaI-antibody were administered intravenously, followed by oral epsilon aminocaproic acid to depress fibrinolysis.

AN ATTEMPT

TO DEVELOP

A RADIOACTIVE

DRUG

307

TABLE 1 OTHER COMPOUNDSINVESTIGATEDAS RADIOACTIVEDRUGS Compound

References Rall et aL (1957); Titus, Loo & Rail (1958)

Tetracyclin and derivatives: An iodinated derivative incorporating 1~1I 7-1aq-iodo-6-deoxy-tetracyclin 7-tritio-tetracyclin Porphyrins : Derivatives of haematoporphyrin

D u n n et aL (1960). Hlavka & Buyske (1960). Machado et al. (1964).

Bis (2-iodoethyl-l~ll) deuteroporphyrin Rose Bengal, incorporating 131I Alizarin (Alizarin Red S),

Altman & Salomon (1960). Ariel & Pack (1960).

incorporating 131[ Dopa, tritiated Thymidine, tritiated

Atebrin (mepacrine, quinacrine), tritiated in the side chain Thiodiglycollic acid, tritiated Antifibrin-Antibody (rat), incorporating lali Antifibrin-Antibody (human), incorporating lzlI

Schwarz, Absolon & Vermund (1955); Lipson, Baldes & Olserl (1961).

Schorr, Aviad & Laufer (1959). Hempel & Deimel (1963). Verly (1960); Lisco et ol. (1961); Kisieleski, Baserga & Lisco (1961) ; Mendelsohn (1960, 1962a and b, 1963, 1964); Clarksorl, Ota & Karnofsky (1962). Young (1963); Young, Wild & Simon-Reuss (1965. Young (1963). Bale, Spar & Goodland (1960). Spar et al. (1964): Bale & Spar (1964).

Further, mention must be made of the internal use of radioactive colloids and suspensions of radioactive particles and microspheres in radiotherapy (Miiller, 1962; Belcher et al., 1964; Blanchard et al., 1965). It is suggested that a practical advantage of the intra-arterial administration of a soluble tritiated drug such as T R K 219 over that of a suspension of large radioactive particles is likely to be the sparing of the normal tissues (Horwitz et al., 1959). Earlier Stages of Our Investigations.--We came to investigate this development as a result of the clinical observation of focal sensations and sometimes pain in the region of the tumour, in addition to various non-specific sensations, in some patients within a few minutes after intravenous injection of Synkavit as a radiosensitizer. It may be mentioned here that in a few patients, we have seen these focal reactions after intravenous injections of amounts of the radioactive drugs, containing about 80-90 mg. of chemical, thus confirming as expected that, in this respect, the body cannot distinguish tritium from ordinary hydrogen in the Synkavit molecule. A number of studies of the distribution of Synkavit in tumours and some other tissues were made, using the methods of fluorescence with Wood's light (Mitchell 1948, 1949, 1953, 1955) and of radioactive labelling with 14C, SZBr and 181I

(Maxwell 1954, 1955; Marrian and Maxwell 1956a and b). The radioactive halogen substituents proved labile under physiological conditions, as also had the a2p label studied by Neukomm et al. (1953). Our investigations with the compound, which chemically is Synkavit, labelled with 14C in the methyl group, confirmed the uptake and retention of this compound in some human tumours (Marrian and Maxwell, 1956a; Horwitz et al. 1959). When tritium became available, Dr. Marrian prepared the compound labelled at tracer levels with tritium in position 3 of the nucleus (Martian, 1957). This was the turning point in our investigations. Since then we have devoted our limited resources to the development and investigation of preparations of the parent compound, Synkavit, containing tritium in very high specific activity. Information of great value from the point of view of the clinical applications in man was obtained in studies of the treatment of spontaneous malignant tumours and leukaemias in cats and dogs (Silver, Cater, Marrian and Marshall, 1962), We started the use of the radioactive drugs, with the tritiated preparations T 1, in the treatment of patients with advanced recurrent and inoperable neoplastic diseases 'after all conventional treatment l:as failed' on 4.2.1959. At that time, we envisaged unit doses of up to 3 Curies, repeated if necessary for 3-4 times. We carried out many investigations

308

CLINICAL RADIOLOGY

to obtain information, especially measurements of the specific activity of tritium in biopsy and autopsy specimens. It became clear that when the tritium was firmly bound in the molecule, there was no clinical problem with toxic effects on the bone marrow. Then we slowly and cautiously increased the doses used. Throughout the whole of this investigation, the radioactive drugs have been administered by some form of intra-arterial injection whenever possible, otherwise by intravenous injection. The procedures for the intra-arterial injections and the methods used have been described elsewhere. (Horwitz, Gregg, Marrian, Marshall and Mitchell, 1959; Gregg, 1958, 1960, 1965; Horwitz, 1960a and b; Marrian, Marshall and Mitchell, 1961, p. 287; Marrian, Marshall, Mitchell and Simon-Reuss, 1965). In general preliminary arteriograms are carried out; Tolazoline (Priscol) has been used as a vasodilator in most cases (except the cerebral gliomata) before the intra-arterial injection of the radioactive drug. For both the intravenous and intra-arterial routes of administration, it is considered important

to inject the radioactive drug as rapidly as reasonably possible in order to minimise reaction with SH-compounds in the blood, particularly serum albumin, and to make certain that as high a proportion as possible reaches the tumour cells. A number of accounts of various aspects of the earlier laboratory and clinical investigations of the radioactive drugs have been publist~ed (Horwitz, Gregg, Marrian, Marshall and Mitchell, 1959; Mitchell, 1960, 1963a and b; Marrian, Marshall and Mitchell, 1961; Marrian, Marshall, Mitchell and Simon-Reuss 1961; Simon-Reuss, 1961; Mitchell, King, Marrian and Chipperfield, 1963; Marrian, Marshall, Mitchell and Simon-Reuss, 1965). It is of interest that T R A 72 produced mutations in the cruciferous plant, Arabidopsis thaliana, similar to those produced by filtered 220 kV x-rays (McKelvie, 1962, 1963).

Biological Testing of the Radioactive D r u g s . Until recently, when it n.o longer seemed necessary for the preparations of T R K 219, each batch of the radioactive drug has been tested biologically before clinical use in two ways: (I) to exclude acute

A Fro. 4 B Differential 'staining' of tumour cells by means of TRK 219. Smears of Ehrlich ascites tumour of the mouse (4A)and mouse femoralmarrow (4B)weremadeon the sa'aaeslide, air dried and treated together. 'Staining'with solution of TRK 219 containing 10inc. per ml. for 30 minutes at 37° C_The smears were then washed with normal saline, fixed with absolute ethyl alcohol for 2 minutes and air dried. Autoradiographswith G5 liquid emulsion, 16 hour exposure ( x 1,333).

AN A T T E M P T

TO D E V E L O P A R A D I O A C T I V E

toxicity--which hag never been observed--after intravenous injection in rabbits, which have been kept for the rest of their lifespan for studies of possible late radiation effects, especially on the bone marrow and the kidneys, and (2) on tissue cultures, mainly of H e L a cells, monkey kidney cells and embryonic mouse fibroblasts, to check sterility and to assess the uptake of the tritium into the tumour cells under standard conditions by means of autoradiograpb.y (Simon-Reuss, 1961). It would have been impossible to reach the present stage of this development without the work of Mrs. Simon-Reuss with tissue cultures to check the purity and the selective uptake into the tumour cells and lack of uptake into the normal cells of the various preparations of the radioactive drugs. With regard to storage, many batches of T R A 119 showed no substantial decrease in the labelling of the tumour cells, with only a little increase in background, after two weeks, though some batches deteriorated more quickly. In August 1964, I found that it was possible to 'stain' air-dried smears o f cells, including tumour and m a r r o w cells, with the radioactive drugs and demonstrate selective concentration in some tumour cells in vitro by means of autoradiography (see Fig. 4). Dr. Marrian has developed this method for routine testing of preparations of the radioactive drugs.

309

DRUG

CLINICAL INVESTIGATIONS The selection of patients for the investigation of the therapeutic possibilities of the radioactive drugs presents m a n y problems. In both the preliminary study and the subsequent follow-up trial which are described here, the use of the radioactive drugs has been restricted to selected, otherwise untreatable, cases, for which it was thought the drugs might be helpful and for which, in general, there were positive histological findings and objective clinical assessment of the results was practicable. In almost all these cases, the radioactive drugs were used only when conventional methods of surgery, radiotherapy and chemotherapy had failed. In these trials, 102 patients with advanced and recurrent inoperable malignant disease were treated by means of the radioactive drugs, T 1, T R A 72, T R A 119, and T R K 219 between 4 February 1959 and 1 March 1965 (see Table 2). In all these cases, the diagnosis was confirmed histologically and a total amount of 1 Curie or more was administered. Of these 102 patien.ts, the following were the numbers treated with the different preparations : - (a) T 1, 19 patients, between 42.1959 and 1.12.1959. (b) T R A 72, 19 patients, between 21.9.1960 and 5.5.1961.

TABLE 2 SUMMARY OF ALL PATIENTS TREATED ONE-HUNDRED-AND TWO PATIENTS WITH ADVANCED AND RECURRENT INOPERABLE MALIGNANT DISEASE WERE TREATED BY MEANS OF

THE RADIO-ACTIVEDRUGS, T 1, TRA 72, TRA 119 and T R K 219 BETWEEN4/2/1959 AND 1/3/1965. Number of Cases Intra-arterial routes

Carcinoma of gastro-intestinal tract (stomach, caecum, colon and rectum)--adenocarcinoma Cerebral glioma, in conjunction with surgery Carcinoma of breast Malignant melanoma of skin Carcinoma of skin, including pinna of ear Carcinoma of ovary Carcinoma of uterine cervix Carcinoma of vagina Carcinoma of bronchus--inoperable Primary tumours of bone Multiple myeloma Carcinoma of gall bladder with hepatic metases--adenocarcinoma Miscellaneous neoplasms 'Radiosensitive diseases' :-Seminoma of testis Hodgkin's disease Chronic lymphatic leukaemia Neuroblastoma

10 10

(o) (o?)

4 (2) 2 (0) 3 (0) 4 (1) 1 (1) 4 (0) 3 (2) 1 (o) 5

(o)

1

(1)

1

(o)

49 (7)

Intra-venous route

Total

12 (2)

22 (2)

8 (1)

10 (0?) s (1) 10 (3)

6 (1) 1 (0)

2 (1) 2 (0)

3 (0) 5 (1) 6 (1) 1 (1)

3 (o) 2 (o) 2 (o)

7 (0)

1 (l)

I (1) 14 (0)

1 (1)

2 (2) 3 (1) 1 (1)

9 (o) 3 (1) 1 (1)

5 (2) 3 (o)

1 (0) 53 (9)

102 (16)

The numbers in brackets give the number of cases showing reduction in the size of the tumour or parts of the tumour.

310

CLINICAL

RADIOLOGY

(c) T R A 119, 41 patients, between 7.3.1962 and 12.12.1963, and (d) T R K 219, 23 patients, between 24.9.1964 and 1.3.1965. It appears reasonable to pool the results for the various stages of the development of the radioactive drug. The diagnoses and routes of administration of the radioactive drugs are summarized in Table 2. These 102 advanced and recurrent inoperable cases may be classified in three main groups as follows : - (i) Late cases, with advanced or generalized malignant disease including radio-resistant and refractory tumours, (ii) Locally advanced but otherwise untreatable cases, and (iii) Advanced cases of 'radiosensitive diseases' with malignant tumours and allied conditions, which are usually radiosensitive. In Table 2, the numbers in brackets give the numbers of cases showing reduction in size of the tumour or parts of the tumour. This criterion of response was accepted as objective, simple and apparently reliable, even though modest. It includes complete disappearance of secondary nodules and has never been accepted as positive if there was any doubt. In this investigation as with any new method in this field, it was necessary to start with a preliminary clinical study to see whether the agent was worth further examination. This appeared to be the case so that a follow-up trial was continued to estimate the effectiveness of the radioactive drugs and to obtain information about optimal dosage and time factors and about the clinical indications for their use. This problem can be considered quantitatively;

it must be noted that during the course of the trial the preparations of the radioactive drugs were improving, so that the conclusions may tend to be over-cautious. I f the drug were effective in 20 of patients or more, there would be a 95 % chance of one or more 'responses' in 14 consecutive cases (Gehan, 1961). In the particular example, there were 5 responses--on the basis of reduction in size of the tumour or part of the t u m o u r - - i n the first 14 cases. It can be shown that an additional 86 cases must be investigated in the follow-up trim in order to estimate a therapeutic effectiveness of 20% with a standard error of + 5%. This was done and, in fact, it was found that among the first 102 cases, 16 showed a positive response. Accordingly, it can be concluded that the drugs were effective, according to the modest criteria used, in 16 + 4 % of the cases in the whole group studied. It became clear that among these cases, those receiving the highest doses tended to show th~ most striking responses. The effects of dose are demonstrated in Table 3. The proportion of cases showing a positive response is 10/23, i.e., 43-5 %, with the high doses as specified, but only 6/79, i.e., 7.6 % with the low doses. This difference is highly significant statistically (P <0'001). Accordingly, it can be concluded that the radioactive drugs have some therapeutic effect and that this investigation must be continued with higher doses of the agent--which is now a practical possibility-and for particular types of tumour. The results for the 23 patients with advanced and recurrent inoperable malignant disease treated with high doses are summarized in Table 4. First, it is to be noted that in this small group, there is no

TABLE 3 EFFECTS OF DOSE PATIENTS WITH ADVANCED AND RECURRENT INOPERABLE MALIGNANT DISEASE TREATED BY MEANS OF THE RADIOACTIVE DRUGS, T 1,

T R A 72, T R A 119 AND T R K 219, BETWEEN 4/2/1959 AND 1/3/1965

Total a m o u n t o f radioactive drug administered

N u m b e r s of cases showing reduction in size of the t u m o u r or of parts of the t u m o u r and the corresp o n d i n g numbers in each relevant group lntra-arterial routes

L o w doses : - 1 ~ 9 . 5 Curies by intra-arterial routes 1--19-5 Curies by intra-venous route H i g h doses : - 10 Curies and m o r e b y intra-arterial routes 20 Curies and more by intravenous route All doses of 1 Curie and m o r e

lntra-venous route

Both routes 6/79(a)

3/36 3/43 10/23(b) 4/13

6/10 7/49

The difference between (a) and (b) is highly significant statistically. (For 1 d.f., ~c ~ = 14-74 a n d P < 0 - 0 0 1 ) .

9/53

16/102

AN

ATTEMPT

TO DEVELOP

A RADIOACTIVE

311

DRUG

TABLE 4 SUMMARY OF PATIENTS TREATED WITH HIGH DOSES TwENTY-THREE OF THE 102 PATIENTS WITH ADVANCED AND RECURRENT INOPERABLE MALIGNANT DISEASE WERE TREATFD BY MFANS OF THE RADIO-ACTIVEDRUGS, T 1, T R A 72, T R A 119 AND T R K 219 WITH DOSES OF l 0 CURIES OR MORE BY INTRA-ARTERIAL ROUTES AND 20 CURIES OR MORE BY THE INTRA-VENOUS ROUTE N u m b e r of Cases Intra-arterial routes

Diagnosis Carcinoma of colon Carcinoma of rectum Cerebral glioma, in coniunction with surgery Carcinoma of breast Malignant m e l a n o m a of skin Carcinoma o f skin, including p i n n a of ear Carcinoma of ovary Carcinoma of uterine cervix Carcinoma of v a g i n a C h o n d r o s a r c o m a of femur Carcinoma of gall bladder with hepatic m e t a s t a s e s - - a d e n o c a r c i n o m a C a r c i n o m a o f head of pancreas Secondary c a r c i n o m a in f e m u r - - u n k n o w n p r i m a r y Seminoma of testis Hodgkin's disease

3 (o)

Intra-venous route

2 (1)

1 (07) 1 (0) 2 (2) 2 (0)

2 (0)

1 (1) I (1) 1 (0)

1 (1) 1 (1) 1 (1) 1 (0)

1 (0) 1 (1) 1 (l) 13 (4)

10 (6)

Total

2 (1) 3 (0) 1 (0?) I (0)

3 (3) 2 (0) 1 (1)

3 (0) 1 1 1 1 1 1 1

(1) (1) (1) (0) (0) (1) (1)

23 (10)

The numbers in brackets give the n u m b e r of cases showing reduction in the size of the tulnour or parts of the t u m o u r

significant difference between the proportion of responses after intra-arterial and after intravenous administration. It may be relevant to mention that in all the intra-arterial cases but one (the case of advanced recurrent carcinoma of the vagina), showing a response, the radioactive drug was administered either in divided doses at intervals over periods of 2 to 3 days with the catheter in situ, or by 2 or 3 separate catheterizations. On the basis of the evidence summarized in Table 4, the types of advanced disease showing a response are as follows: carcinoma of colon, malignant melanoma of skin, carcinoma of ovary, carcinoma of vagina, chondrosarcoma of femur, carcinoma of gall bladder and the radiosensitive conditions, seminoma of testis and Hodgkin's disease. Turning to other criteria of response--which are usually less objective and much more difficult to measure--, in addition to improvement of the general condition and Performance Status (Karnofsky et al. 1951) in some patients, there has been useful relief of pain in at least 15 of the 102 patients. Relief of pain, as indicated by a great reduction of the analgesics required, in some instances discontinuance of the need for morphia, was observed in the following advanced cases: one case each of carcinoma of the stomach, colon, rectum, breast, ovary and gall bladder, malignant melanoma 2 cases, bone sarcoma 2, multiple

myeloma 2, seminoma 2 and chronic lymphatic leukaemia 1. In a case of advanced Hodgkin's disease, intractable generalized pruritus was relieved completely for about 3 weeks from 4 hours after a single intravenous injection of 3 Curies of T R A 119. Nevertheless, in attempting to assess any new drug, it seems wise not to attach too much importance to the relief of pain, despite its value to the individual patient. It is interesting to see the improvement in the general condition of a patient when the pain has been relieved so that morphia and similar drugs can be stopped. It must be emphasized that from the immediate practical point of view, the various responses observed are, in general, only of modest and usually temporary palliative value. However, they appear to be sufficiently encouraging to suggest that it is profitable to pursue this investigation with higher doses of the radioactive drugs and with repeated injections, both intra-arterial and intravenous, making every effort to find the optimal dose and time factors and to develop new methods for increasing the selective uptake of the radioactive drug into tumour cells. The types of disease mentioned above must be studied intensively. In addition, the evidence for selective concentration in tumour cells provided by autoradiography and the histological evidence for damage to turnout cells suggests that it is justifiable to make further attempts to treat locally advanced cases of

312

CLINICAL RADIOLOGY

s q u a m o u s carcinoma of the head and neck and skin and inoperable cerebral tumours by means of repeated intra-arterial injections with high doses. However, the most striking effects obtained so far have been with certain types of adenocareinoma, malignant melanoma of the skin and the radiosensitive conditions especially seminoma of the testis and Hodgkin's disease. In the whole group o f 102 patients with advanced and recurrent inoperable malignant disease, followup observations for 3 years and longer are available for 41 patients. O f these, 4 survived m o r e than 1 year, 3 more than 2 years and one 3 years. This result is not unexpected. It m a y be of interest to give a brief account o f the one patient still alive after three years. Mrs. E.A., aged 52 Disease and previous treatment--Metastases of malignant

melanoma of the skin involving the right leg below the knee; multiple recurrent nodules. Previous surgical excisions; mole removed 5.8.53; reexcision of primary site and block dissecqon of inguinal nodes (not involed) 10.9.53; excision of recurrent nodules 18.4.59 and in September 1960. Treatment--Three intra-arterial injections of TRA 119: 1.5 Curies on 19_4.62, 4-92 Curies on 1.6.62 and 9.8 Curies on 4.7.62. Total amount: 16.2 Curies. (The dose entering the general Circulation was considerably less.) Subsequent cour~e~There was slow retrogression of many of the recurrent nodules, especially after the last injection but a few of the nodules appeared to continue to grow. Accordingly, several of these were treated locally with small field x-ray therapy on 17.9.62, 26.9.62 and 15.5.63. These nodules certainly responded to the radiation treatment but other nodules, which were not treated with x-rays, also increased further in size and then decreased spontaneously and finally disappeared. Unfortunately, in the areas treated with x-rays, necrosis of the tissues appeared and it was necessary to carry out a series of operations with skin grafting. In one of these operations, when removing necrotic bone, the right tibia was fractured. In the necrotic area on the medial side of the ankle, there was recurrent melanoma in the site of the scar of one of the biopsies, which had subsequently become infected, broken down and then slowly healed. This area was excised with immediate skin grafting on 18.6_64. Since then, her general condition has improved; she has put on 9 lbs. in weight and re-established herself, using a walking caliper. On 9.6.65 she was very well. There is no evidence of damage to the bone marrow or the kidneys. Experience suggests that caution is always necessary in considering favourable results in the treatment of malignant melanoma. 'The eccentricities o f its clinical course' were emphasized by Ewing (1934, p. 919) and occasionally, a m e l a n o m a which has already metastasized undergoes spontaneous retrogression (Willis, 1948, p. 908). However, a definite objective response was observed in all 3 cases of m e l a n o m a treated with high doses of the radioactive drugs and in 2 further

cases treated with lower doses, there was definite relief o f pain. Evidence has been obtained which confirms the low toxicity o f T R A 72, T R A 119 and T R K 219 in patients with normal bone m a r r o w undamaged by widespread metastases or previous chemotherapy. There were 10 cases in which the effects of single intravenous doses of 10.2 to 18.5 Curies per 70 kg. b o d y weight could be studied; only one o f these, a case of carcinoma of the head of the pancreas with extensive metastases in bones, where the dose was 13.6 Curies per 70 kg. b o d y weight, showed evidence o f a serious fall in the level of the blood platelets, with petechial haemorrhages. In none of these cases was there any evidence of renal damage. The highest total amount of radioactive drug given so far has been 68.3 Curies of T R K 219, corresponding to approx. 72 Curies per 70 kg. b o d y weight, and administered intravenously in 13 injections in 13 weeks 5 days. The patient, Mr. L.M., was a m a n o f 36 with extensive metastases f r o m an inoperable adenocareinoma o f the colon, referred 3 months after a palliative local resection and end-to-end anastomosis of the pelvic colon. There were recurrences in the surgical scar in the anterior abdominal wall, large masses on the posterior abdominal wall and pelvic floor, fixed left supraclavicular nodes and probably early pulmonary metastases. Following the injections of T R K 219, there was a gradual reduction in size o f the palpable secondary masses in the anterior wall (which were confirmed as adenocarcinoma histologically--see Fig. 5a), especially of the smaller masses, first noted as definite after about 2 weeks and then a slower decrease in size of the palpable masses in the abdomen. There is histological evidence of t u m o u r retrogression (see Fig. 5b and c). The largest masses responded least; also the left supraclavicular nodes changed very little. The patient improved in general condition and felt m u c h better for about 6 weeks, t h o u g h he slowly lost weight, but then he slowly deteriorated and developed severe pain in the right thigh apparently due to involvement of nerves by growth. A n intra-thecal phenol injection was unsuccessful in relieving this pain. He died 28 days after the last injection of T R K 219, despite transfusions following haemorrhages f r o m the gastrointestinal tract with severe anaemia and thrombopenia. However, f r o m the examinations of the sternal marrow, there appeared to be relatively little damage; the autopsy showed hypoplasia o f the bone marrow. There was no clinical evidence of renal damage. However, histologically at autopsy there was some evidence of interstitial fibrosis (see

AN A T T E M P T TO D E V E L O P A R A D I O A C T I V E - D R U G

A

313

B

C FIG. 5 D (A) Histology of recurrent mass in anterior abdominal wall, secondary to inoperable carcinoma of colon. Biopsy specimen before treatment (× 42). (B) and (c) Histological evidence of tumour retrogression after intravenous TRK 219. Autopsy specimen of comparable recurrent mass (× 110). (o) Histology of autopsy specimen of the kidney in the same case (× 110).

Fig. 5d). It is to be noted that the blood vessels showed little damage. These changes are m u c h less pronounced than the histological changes in the kidney described by Platt (1947) as an effect of therapeutic doses o f radiophosphorus, 3~p. I wish to thank Dr. G. A. Gresham for the following histological report on the autopsy specimens of the kidneys in this case: 'Histological examination of the right kidney shows a slight increase of interstitial collagen in the medulla. I cannot be sure that the glomeruli are abnormal; there may be a slight increase of interstitial material in the glomeruli but this cannot be readily assessed in necropsy material. There is a moderate increase of interstitial tissue in the cortex and medulla of the left kidney and some tubules are dilated. However, there is one area of secondary neoplasm in the kidney so that the cause of the interstitial fibrosis is difficult to assess'. ESTIMATION OF DOSE With these radioactive drugs as with all previous therapeutic applications o f radio-active isotopes

administered internally, the dosage has been largely empirical. Nevertheless, it is essential to try to correlate the a m o u n t of the radioactive drug administered with estimates of the dose of radiation received by malignant and normal cells, particularly the bone m a r r o w and kidneys, and on the basis of this evidence to try to find the optimal dose and time factors for the individual patient. The problem is difficult and complicated, primarily because o f the inhomogeneous distribution of the tritium, not only macroscopically but also a m o n g different cells and within the cells. The evidence collected is of 3 main types : - (i) measurements of the specific activity of the tritium of biopsy and autopsy specimens, usually expressed in microCuries or milliCuries per g. o f fresh tissue. (ii) estimates of the Biological Half-Life (B.H.L.) o f the tritium in the tumour and various normal tissues, based on the measurements of specific activity at different times; in addition, excretion

314

CLINICAL

RADIOLOGY TABLE 5

DOSIMETRY OF TRITIUM T r i t i u m (~H o r T) h a s a r a d i o a c t i v e half-life o f 12-43 + 0.04 years a n d emits soft f3-rays o f m a x i m u m energy 18-7 + 0.1 keV a n d c o r r e s p o n d i n g m a x i m u m r a n g e in u n i t density tissue a p p r o x . 6~. T h e m e a n energy o f the ~-particles is 5.73 _+ 0-03 keV, a n d the c o r r e s p o n d i n g r a n g e in unit density tissue a p p r o x . 1v F o r u n i f o r m l y distributed r a d i o - a c t i v e m a t e r i a l i milliCurie o f T r i t i u m per g. o f tissue delivers a d o s e o f 293 r a d in 24 h o u r s A s s u m i n g u n i f o r m d i s t r i b u t i o n t h r o u g h o u t the b o d y , o n e C u r i e o f Tritium administ e r e d to a 70 kg. p a t i e n t delivers a dose o f 18-12 r a d f o r a Biological Half-Life o f 3 days, 36.2 r a d f o r a B . H . L . o f 6 d a y s a n d 72-5 r a d f o r a B . H . L . o f 12 days.

studies give information for the body as a whole, and (iii) autoradiographic investigations, from which information about the microscopic and intracellular distribution of the tritium can be deduced. Extensive measurements of the specific activity of the tritium in biopsy and autopsy specimens have been made by Dr. Barbara Chipperfield and provide evidence for some degree of selective concentration of the radioactive drugs in certain human malignant turnouts in relation to the bone marrow after both intravenous and intra-arterial injection. The specific activity of the specimens of tumour, which of course contain stroma, is almost invariably higher than that of specimens of bone marrow, often by a factor of 3 and sometimes by a factor as high as 7. The methods used and many of the results have been described and discussed in some detail. For routine assay of tritium, the tritiated water obtained by combustion of specimens of tissue of weight 10 to 40 rag. has been counted in a Nuclear Enterprises NE 8301 liquid scintillation counter using either N E 213 or SP 2 (Nash and Thompson Ltd., Chessington, Surrey) scintillator (Horwitz, Gregg, Martian, Marshall and Mitchell, 1959; Chipperfield, 1961; Marrian, Marshall and Mitchell, 1961; Mitchell, King, Marrian and Chipperfield, 1963 ; Martian, Marshall, Mitchell and Simon-Reuss, 1965). Here it is only necessary to summarize the position. The general approach is to start with the assumption of homogeneity of distribution of the tritium and exponential decrease of specific activity with time, then introduce selective concentration in the tumour and different tissues with a separate Biological Half-Life (B.H.L.) for each of these and after that try to study, as far as possible, the effects of the observed inhomogeneity of distribution of the tritium, including the distribution among the tumour cells and within the cells, and more complex

processes of biological elimination of the tritium corresponding to the evidence available. Assuming a uniform distribution, the basic calculations for the dosimetry of tritium are given in Table 5. Further details are given by Mitchell (1963a) and Mitchell, King, Marrian and Chipperfield (1963). Radiation dose calculations for cells containing intra-nuclear tritium are available (Goodheart, 1961; Ktinkel, 1962; Bleecken, 1964; Stewart, 1964; see also Koch, 1965). The usual estimate of the relative biological effectiveness (R.B.E.) of the tritium f3-particles in relation to G°Co 7-radiation is 1 '7; however, this value cart only be regarded as provisional and may not be strictly relevant to the present application. On the basis of certain assumptions concerning time factors (Mitchell, 1960, p. 234) the approximate value of the biologically equivalent single acute dose of S°Co 7-radiation for one Curie of tritium administered to a 70 kg. patient, with uniform distribution, would be 14r for B.H.L. 3 days, 24r for B.H.L. 6 days, 35r for B.H.L. 9 days, and 46r for B.H.L. 12 days. The rate of excretion of the tritium of the radioactive drugs from the body as a whole varies very considerably in different patients; see Figure 6, for which I wish to thank Dr. Barbara Chipperfield. Other data suggest that the excretion of tritium cannot be represented by a single exponential function. For the 14 injections studied in Figure 6, 9 showed 50 ~ or more excretion in the first 3 days. Attention must be paid now to the gross inhomogeneity of distribution of the radioactive drugs within the human body. It is of interest to note that Dr. G. di Vita (1964, personal communication) has shown that in mice for a single intra-peritoneal injection, the LD 50/30 days was about 20 milliCuries per g of body weight for T R A 72 but only 1-I milliCuries per g for tritiated water; recently for T R K 219 he found that the LD 0/30 days for mice was approx. 10 milliCuries per g. Brues,

AN A T T E M P T TO D E V E L O P A R A D I O A C T I V E Patients no,

65

Dose curies

Injection route

82

82

84

88

90

90 90

92

94

95

97

9.6 2-9 4-95 12

%7 8.3

5

9.6

17.7 23.7 8

12

9.05

I-V. I-V. I-V.

I.A. I.V. I.V. I-V. i.V. I.A- I.A. I-V. I-V. I-V.

I-V.

86

5

92

315

DRUG

KEY WITrilium 100. 908070Injected dose 6o5040302oIo-

~/~/A I t

~

;/,/ ;

in urine

[] Tritium in body water • Tritium in ta gee s

u//l

7/

B.C. 17 8 , 6 a . FIG. 6 Excretion of tritium by patients in the first three days after iniections of TRA 119. (Data obtained by Dr. Barbara Chipperfield).

Stroud and|Rietz (1952) found that the LD 50/30 days of tritiated water for CF No. 1 female mice is about 1 inc. per g of body weight when given in a :single injection; Verly (1960) concluded that the LD 50/30 days for man would be of the order of 10 Curies for 70 kg. body weight. Our clinical experience with the first preparations of the radioactive drug, T 1, which contained tritiated water as an impurity, provides evidence that no serious deleterious effects followed a single intravenous dose of 3 Curies of tritiated water, .even in these rather ill patients. It can be assumed that the B.H.L. of tritiated water is 9-12 days. According to the calculations given above for the uniform distribution of tritium, the biological effects of 3 Curies of tritiated water in a 70 kg. patient would be equivalent to a single acute dose of a°Co y-radiation of 105-138r, which, is likely to be tolerated. Assuming for T R K 219 values of the main B.H.L. for the body as a whole varying between 1 and 6 days, and considering these values in relation to the experiments with mice, it seems reasonable to expect that in man T R K 219 is less toxic than tritiated water by a factor of 10. Accordingly, no serious deleterious effects are likely to follow a single dose as high as 30 Curies in a 70 kg. patient though particular attention must be paid to the possibility of radiation effects on the kidney. The highest single intravenous dose given so far was 18.5 Curies per 70 kg body weight; this produced no evidence of renal damage during the subsequent 2 months before the patient died. It must be pointed out that not more than about 20 Curies of

T R K 219 per 70 kg of body weight is likely to be administered as a single intravenous injection because this corresponds to 146 mg of chemical Synkavit, which for many patients is near the upper limit of practical tolerance. The approximate macroscopic average values which have been found for the main Biological Half-Life are as follows :-- for different malignant tumours, 3-18 days, with a value of about 13 days in a number of cases; for bone marrow about 4 days, probably with a weak longer lived component perhaps of half-value about 20 days; and for the kidneys, small intestine, voluntary muscle, heart muscle, liver spleen and brain about 5 days, with great variability between different cases and frequently lower values for the normal tissues. Except for cases where the turnout shows a rapid turnover, repeated injections appear to be indicated. For B.H.L. of the tumour of 13 days, and corresponding mean life of 18.8 days, it would appear to be reasonable to give injections at intervals of 2-3 weeks. After 14 days, the level of radioactivity of the tumour would have fallen to about 47 % of the original maximum value, the level for the bone marrow to about 9 ~o and the level for the kidneys and other organs mentioned to about 14%. It is clear that a knowledge of the values of the B.H.L. of the tumour and normal tissues is necessary for planning optimal time factors in individual patients. The level of dosage must, of course, be taken into consideration. So far, information of this type has been incomplete; it is often very difficult to obtain. The levels of specific activity of biopsy specimens of tumour vary greatly. For most of the specimens

316

CL1NICAL

RADIOLOGY

of tumour taken at 30 minutes after administration of the radioactive drugs, the specific activity had values between 25 and 400 microCuries per g of wet tissue. In a case, Mrs. A.M., of recurrent melanoma with multiple metastases involving the left leg, the specific activity of the biopsy specimen 30 minutes after intra-arterial injection of 7.4 Curies of T R K 219 was 2-65 milliCuries per g. The autoradiographs in this case (Figures 7a & b) show clear evidence of selective concentration in the tumour with little radioactivity in the stroma. In areas of tumour such as those shown in these autoradiographs, the initial dose rate delivered by the tritium is obviously greater than 776 rad per 24 hours. In this case many of the small metastic lesions disappeared completely but the large metastases only showed temporary retrogression. The arteriograms (see Figure 8, for which I wish to thank Dr. Duncan Gregg) show apparently quite good vascularity of a small metastasis but areas of deficient vascularity in a large metastasis. It seems likely that deficiency of the blood supply is one of

A

the factors contributing to the lack of response of large metastases. Detailed considerations of the evidence provided by autoradiography (Mitchell, King, Marrian and and Chipperfield, 1963) suggest that, until much more precise information is available, the best approximation to make is to assume that the relevant values of the specific activity correspond to the values averaged over the tumour cells, on the basis of measurements of the proportion of the volume of the specimen occupied by tumour cells. Studies of the distribution of autoradiographia grain counts per cell give some evidence concerning the distribution of cell doses. There appear to be viable tumour cells with much less th.an the average uptake of tritium; however, such low uptake may be offset to a certain extent by the persistence of the tritium in the tumour cells irradiated at the lower dose rates. This problem of low dosage in some of the tumour cells is important and may prove to be one of the factors limiting the possibility of cure by means of radioactive drugs.

FIG. 7

B

A u t o r a d i o g r a p h s o f b i o p s y s p e c i m e n o f m e t a s t a t i c t u m o u r 30 m i n u t e s after i n t r a - a r t e r i a l iniection o f 7.4 Curies o f T R K 219 in a case o f r e c u r r e n t m e l a n o m a with multiple m e t a s t a s e s ( × 1,333). FIG. 7 (A) s h o w s a h i g h degree o f selective c o n c e n t r a t i o n in the t u m o u r a n d little in the s t r o m a . FIG. 7 (B) s h o w s a n a r e a o f t u m o u r , with c o n c e n t r a t i o n o f t h e t r i t i u m o f the T R K 219 in the t u m o u r cells. ( F o c u s s i n g o n the silver grains).

AN A'ITEMPT

TO D E V E L O P

A RADIOACTIVE

DRUG

317

Fio. 8 Arteriogram by Dr. Duncan Gregg in the case of recurrent melanoma, for which autoradiographs are shown in Figure 7. The small metastasis on the left shows apparently quite good vascularity but the large metastasis to the right of the centre of the soft tissues above the bones shows areas of deficient vascularity.

It can be concluded that therapeutic doses of radiation have been delivered in a few of the cases treated by means of the radioactive drugs. In one case, a man of 66 in not very good general condition with extensive recurrent squamous carcinoma of the left pinna involving a large area on the side of the head and neck, following intra-arterial injection of 10.8 Curies of T R A 119, the specific activity was 8.99 ( + 2.7 ~ ) milliCuries per g for a biopsy from the anterior part of the lesion after 30 minutes and 1 '82 ( + 3'8 ~ ) milliCuries per g for a biopsy from the posterior part of the lesion after 3½ hours. The patient unfortunately died with severe bronchopneumonia after 14 days. At autopsy, the histological examination of the edge of the ulcer showed severe degenerative changes in the tumour cells consistent with irradiation at a therapeutic level. The specific activity of the autopsy specimen was not inconsistent with a value of the B.H.L. of 4.4 days. Accordingly, on the basis of a value of about 6.3 days for the mean life of the tritium in the posterior part of the tumour, it was estimated that the total dose of radiation delivered to this area was equivalent to about 5700r of 6°Co-radiation. In general, the estimated mean doses of radiation delivered by the radioactive drugs are consistent with the clinical findings, in particular the palliative effects observed in certain cases of recurrent adenocarcinoma of the gastro-intestinal tract and in selected cases of the highly radiosensitive conditions, such as testicular seminoma and Hodgkin's disease, when all other methods of treatment lz.ave failed.

AUTORAD1OGRAPHY A N D R E L A T E D PROBLEMS Some of the most interesting evidence in this investigation has been obtained by means of autoradiography. We have already published autoradiographs of biopsy specimens from 8 patients with the following types of recurrent and metastatic tumours: carcinoma of stomach 1, carcinoma of caecum 1, carcinoma of rectum 2, squamous carcinoma of pinna 1, squamous carcinoma of vagina 1, carcinoma of breast 1, and the case of malignant melanoma, E.A., described earlier in this lecture (Mitchell, King, Marrian and Chipperfield, 1963 ; Mitchell, 1964; Mitchell, 1965). These and the autoradiographs which we have seen (Figure 7) show selective concentration of the tritium of the radioactive drug, T R A 119, in the tumour cells in relation to the normal cells of the stroma and also the bone marrow. The absence of serious toxic effects on the bone marrow, in cases with relatively normal haematopoietic marrow, after single intravenous doses of T R A 119 and T R K 219, of 10-2 to 18.5 Curies per 70 kg body weight, is consistent with the autoradiographic studies showing the absence of uptake of the tritium in the vast majority of the cells in the bone marrow, although a few rare, as yet unidentified cells, have been observed which show considerable uptake (Mitchell, 1964). The question, as yet unsolved, is, of course, whether any of these unidentified cells are tumour cells. Another point to be mentioned is that there appears to be little or no uptake in small lymphocytes.

318

CLINICAL

In most of the tumour cells showing uptake, the tritium of the radioactive drug is present in the nucleoli and in the cytoplasm, probably with concentration immediately outside and perhaps within the substance of the nuclear membrane. There is almost certainly a higher concentration in proliferating tumour cells than in the cells in non-proliferating parts of the tumour and in most cases there appears to be negligible uptake in degenerating tumour ceils. There is no obvious immediate relationship between uptake and the synthesis and presence of DNA. However, there appears to be some degree of parallelism between the distribution of the radioactive drug and that of total RNA. This is of interest in connection with the inhibition by Synkavit of part of the synthesis of RNA found by Dr. D. H. Marrian (1959). It is important to know whether ribosomal-RNA or perhaps messengerRNA is involved. However, the concentration of the tritium of the radioactive drugs produces localized irradiation of the nucleolus and nucleolusassociated chromatin and such localized irradiation is of great interest in view of the radiosensitivity of the nucleolus found by Dr. J. Seed (1960) in his microbeam irradiation studies. Recently, I have obtained evidence about the distribution of the tritium of T R K 219 in human tumour cells by means of electron microscopic autoradiography. The technique developed with Mr. E. A. King is to fix the ½ mm. fragments of frozen dehydrated biopsy material in 1 ~ OsO~ in water-free acetone for 30 minutes, to minimise diffusion artefact, and then embed in araldite. The sections were cut with a glass knife using a PorterBlum microtome set at 50 m~, then mounted on Formvar carbon coated copper specimen grids. The sections were stained with aqueous lead citrate solution for 30 minutes and then after washing and drying were thinly coated with Ilford L4 fluid emulsion. The autoradiographic technique followed the methods described by Moses (1964). The preparations were examined in a Superscope (Japanese Electron Optics Laboratory Co. Ltd.) Type JEM-SS electron microscope. Figure 9 shows an electron microscopic autoradiograph of a small part of the biopsy specimen in another case of recurrent melanoma 30 minutes after intravenous injection of 8"7 Curies of T R K 219. The E.M. autoradiographs in Fig. 9 were prepared with an exposure time of 4 days and subsequent development with Microdol X for 6 minutes. The primary magnification was X 2,000, with photographic enlargement to X 7,000. In the

RADIOLOGY

FIG. 9 Electron microscopic autoradiograph of biopsy specimen of metastatic malignant melanoma 30 minutes after intravenous injection of 8-7 Curies of TRK 219 (× 7,000). See text.

centre of Fig. 9 is the nucleus of a malignant melanocyte with dense silver grains over the nucJeolus. There are a number of silver grains at the edge of the nucleus, some of them clearly outside and in contact with the outer layer of the nuclear membrane. There are probably also grains at the edge of the cytoplasm of this cell. Much further work is required in this field, including biochemical investigations of nuclei and nucleoli. Returning to light microscopic autoradiographs, it may be of interest to discuss some new results for the initial biopsy and sternal marrow specimens from the case, L.M., with extensive metastases from an inoperable carcinoma of the colon. The techniques used have been described (Mitchell, King, Marrian and Chipperfield, 1963). After freeze-drying of the biopsy specimens and embedding in paraffin, 5 ~ sections were cut; with these sections and with the bone marrow smears, autoradiographs were prepared using Ilford G5 fluid emulsion. Figs. 10a and b show autoradiographs of the biopsy specimen of a relatively small metastasis in the anterior abdominal wall 30 minutes after intravenous injection of 3'7 Curies of T R K 219; the exposure time for these autoradiographs was 4 days. Figs. 10c and d shows autoradiographs of the sternal marrow smears taken at 24 hours after this dose of 3'7 Curies of T R K 219; the exposure time here was 6 days. In all these photographs, the focussing is on the silver grains so the cells are usually out of focus. It is obvious that there is a high concentration of the tritium of the radioactive drug in the tumour cells, with very little in the stroma. The normal bone marrow cells, including megakaryocytes, show very little uptake; however, there were a very few large

AN

ATTEMPT

TO DEVELOP

A RADIOACTIVE

DRUG

319

FIG. 10 A u t o r a d i o g r a p h s of b i o p s y specimens from the case, for which histology is shown in Figure 5 ( × 1,000). FIGS, 10A and n - - A u t o r a d i o g r a p h s of biopsy specimen of metastasis of a d e n o c a r c i n o m a of the colon 30 minutes after intravenous iniection of 3-7 Curies of T R K 219 (Exposure time of autor a d i o g r a p h 4 days). Fins. 10c and D: A u t o r a d i o g r a p h s of sternal m a r r o w smears in this case at 24 hours after the intravenous injection of 3'7 Curies of T R K 219. FIG. 10g. A further a u t o r a d i o g r a p h of the sternal m a r r o w smear in this case showing an unidentified cell with considerable u p t a k e of tritium. (Focussing on the silver grains).

D

E

unidentified ceils such as tbat in Fig. 10e which show considerable uptake of tritium. The low-power pb.otomicrograph (Fig. 5a) of the histology of the biopsy specimen of the metastasis in the anterior abdominal wall shows abundant stroma. Quantitative measurements including grain counts have been made. It was found that the mean specific activity of the tumour cells was 6-31 times the mean specific activity of the fixed whole specimen. The measured specific activity of the fresh biopsy specimen was 36-5 (+ 3 ~ ) microCuries per g so that the mean specific

activity of the tumour cells is about 230 microCuries per g corresponding to a dose of about 67 rads per 24 hours. It is to be noted that some of the tumour cells contained much less tritium than the average value; in 10 ~ of the cells, the grain counts were about 6-8 ~ of the average value. It was clear tbat much higher doses were necessary. After the total amount of 68-3 Curies given in 13 injections in 13 weeks 5 days in this case, it is reasonable to expect therapeutic effects especially in relatively small metastases. This is in fact the case. The histological appearances of the autopsy specimen

320

CLINICAL RADIOLOGY

of a metastasis of adenocarcinoma in the anterior abdominal wall are shown in Fig. 5b and c. There are areas where the carcinoma has completely or almost completely disappeared leaving largely fibrous stroma, and other areas where there are degenerative changes in the carcinoma although its general structure persists. In order to try to increase the uptake of the radioactive drug into the tumour cells, experiments on mice with the Ehrlich ascites tumour cells are in progress in which with the necessary controls, T R K 219 is injected intraperitoneally after the mice have been kept in oxygen at 3 atmospheres absolute pressure (29-30 p.s.i, gauge pressure) for 30 minutes. The autoradiographs appear to show considerable increase in the uptake of the radioactive drug under these conditions. However, further experimental work is necessary before reaching a decision concerning the clinical application of hyperbaric oxygen for this purpose. SOME B I O C H E M I C A L E V I D E N C E Evidence is accumulating to show that there are biochemical differences between some malignant cells and some normal cells in relation to the actions of Synkavit and the parent quinone, menadione, 2-methyl-I, 4-naphthaquinone which is almost certainly the effective form of the molecule within the cells (see Mitchell and Marrian, 1965). These differences are probably related to the deficiency of the quinone coenzymes, the ubiquinones (see Fig. 11) found in some malignant turnouts (Osterberg and Wattenberg, 1961; Wattenberg, 1961; Sugimura, Okabe and Baba, 1962). In this respect there appear to be significant differences between experimental hepatomas and the regenerating liver after partial hepatectomy in the rat. In our group in Cambridge, Dr. Barbara Chipperfield has found a consistent reduction of the ratio of the content of ubiquinones to that of D N A in a number of human malignant tumours, in relation o

o CH~O

CH~

H~-CH=C--CH, ],n

c~o

O 2 - m f t h y I - l, 4 - n o p h t haquinonE (Menad~on¢)

O Ca¢nzymz Qio LIT0iqu]nonz [so)

0

0

o Vitamin

KI (a-Phylloquinan¢)

o ViLamin

K a (1.)

Fie. 11 Menadione, Ubiquinone, and Vitamins K1, K2.

to the accompanying normal tissues. The possibility may be envisaged that in this respect menadione acts as a pseudo-ubiquinone rather than as a vitamin K analogue. Recently, Dr. D. H. Marrian has shown that in vitro incubation of HeLa cells in solutions of T R K 219 at 37 ° C. leads to a rapid breakdown of the T R K 219, easily observed after 3 minutes, while cultured monkey kidney cells and also embryonic mouse fibroblasts showed no detectable change even after similar incubation for 96 minutes. With the H e L a cells, no T R K 219 was left in solution after 36 minutes. The rapid breakdown of T R K 219 in solution by H e L a cells involves dephosphorylation. Paper chromatography showed two new peaks of radioactivity corresponding to the products, menadione and a menadiol monophosphate which is probably menadiol 1-phosphate. Further investigations are in progressl The probable importance of SH-groups in the biological actions of Synkavit has been recognized for many years (Friedmann, Marrian and SimonReuss, 1948a and b). Recently, Gronow (1963, 1965) has shown that in two strains of mouse ascites tumour in rive, Synkavit and the radioactive drugs T R A 72 and T R A 119 produce a decrease in the acid-soluble - - S H content of the cells after 30 minutes. The tritiated compounds gave a considerably greater decrease than Synkavit alone or a combination of Synkavit and tritiated water. No corresponding increase in soluble disulphide levels was found. H E A L T H A N D SAFETY P R E C A U T I O N S It has proved to be possible to organise the safe handling of the therapeutic amounts of the radioactive drugs and the treatment and care of the patients within the framework of the Recommendations of I.C.R.P. (1959). We administer the radioactive drugs almost invariably in an operating theatre and usually in the theatre of the Radiotherapeutic Centre, Addenbrooke's Hospital, which has an Isotope Cleansing R o o m immediately adjacent. Mr. J. L. Haybittle has drawn up the following: NOTES ON PRECAUTIONS TO BE TAKEN IN TREATMENTS WITH RADIOACTIVE DRUGS In Theatre

1. All persons must wear long-sleeved gowns, paper masks and plastic or rubber gloves. In addition, a thin polythene apron and polythene sleeve covers must be worn; (2)All working surfaces, inside of waste-bins and the operating

AN ATTEMPT TO DEVELOP A RADIOACTIVE DRUG table top must be covered with polythene; (3) Only specially marked linen and syringes must be used; (4) After the procedure is completed, used syringes and linen (excluding gowns) must be placed in plastic bags and transferred to Isotope Cleansing Room. All disposable items must be placed in a plastic bag and burned immediately in the incinerator, unless there is reason to suspect that the total activity of these items is more than 100 milficuries. (This is because of the limit in our authorisation of not burning more than 100 mc./day); (5) Linen sent to the isotope Cleansing Room is washed there initially before being sent to the Hospital Laundry. The syringes are similarly treated before being sent to the Central Sterile Supply Department for sterilising. On Wards 6. Patients must be kept in a sideward; (7) Only specially marked linen and crockery must be used; (8) All used linen must be placed in a plastic bag and sent to the Isotope Cleansing Room in the Radiotherapeutic Centre for initial laundering; (9) Urine must be collected for 3 days after the injection and sent to the Radiotherapeutic Centre for measurement and disposal; (10) The ward should be kept well ventilated; 11) Nursing staff should spend as little time in the ward as possible, but no particular precautions are necessary for visitors. Measurements of the concentration of tritium in air under representative conditions have shown that there is no serious hazard, but have emphasized the importance of good ventilation in the operating theatre and in the side-ward. For these measurements, we have used the Radioactive Gas Monitor Type 1762A (Isotope Developments Ltd.). In future, it will probably be wise to introduce routine monitoring of the urine of certain personnel for tritium at intervals. Concerning the disposal of tritium in liquid wastes into the drains, it may be mentioned that the daily effluent of the part of the hospital concerned is about 1 "4 × 105 litres (30,000 gallons) and that authorization has been given for disposal of up to 80 Curies of tritium per 4 weeks into the drains.

CONCLUSIONS 1. There is quantitative evidence from the measurements of specific activity and autoradiographic studies that the tritiated derivatives of 2-methyl-I, 4-naphthaquinol bis (disodium phosphate) Synkavit, of high specific activity show some degree of selective concentration in the malignant cells of certain human tumours in relation to normal cells, especially those of the critical tissues. These prove to be bone marrow and kidney. 2. Under some conditions, therapeutic doses of the p-radiation of tritium have been administered by means of these radioactive drugs. Objective therapeutic effects have been demonstrated clinically and histologically. The practical clinical results obtained so far have proved to be in general relatively modest but sometimes useful. Small turnout masses and metastases respond much more satisfactorily than larger ones. 3. In the first 2 stages of the clinical trial, 102 patients with various types of advanced recurrent and inoperable malignant tumours and reticuloses

321

have been treated by means of the radioactive drugs when all other methods of treatment had failed. Reduction in size of the turnout or parts of the tumour was observed in 16 cases, corresponding to an effectiveness, by this relatively modest criterion, in 16 + 4 ~ of cases. High doses produced a response in a significantly greater proportion of cases than low doses. 4. Much more evidence is required concerning the optimal dose and time factors for the individual patient and the indications for the clinical use of the radioactive drugs. The final part of the trial has yet to be done. Meanwhile, it is necessary to reserve judgement. 5. Some biochemical differences between certain malignant cells and certain normal cells appear to be relevant. Of interest are the findings of rapid dephosphorylation-of the radioactive drugs by tumour cells and the apparently lower ratio of the content of ubiquinones to that of D N A in tumour cells. The concentration of the radioactive drugs in nucleoli especially of tumour cells may be important in relation to the radiosensitivity of nucleoli to localized irradiation and to inhibition of part of R N A synthesis. Direct experimental evidence has been obtained for reduction of the levels of soluble sulphydryl (--SH) compounds in certain turnout cells by Synkavit and to a greater degree by the tritiated derivatives. 6. The work as a whole is regarded as the beginning of an intensive study of the possibilities of radioactive drugs in the treatment of patients with neoplastic diseases. SUMMARY This lecture is an account of the work of a team in Cambridge since about 1953 to try to develop radioactive drugs for use in the treatment of patients with cancer. In addition, other work in this field is reviewed. A series of tritiated derivatives of 2-metbyl-1, 4-naphthaquinol bis (disodium phosphate), Synkavit, of high specific activity, have been prepared and investigated, first in animal experiments and by means of tissue culture techniques and then in clinical trials. There is evidence of selective concentration of the radioactive drugs in the malignant cells of some tumours in relation to normal cells especially of the bone marrow and kidneys. Special attention has been paid to autoradiographic and biochemical studies. In the first 2 stages of the clinical trials 102 patients with various types of advanced recurrent and inoperable malignant tumours and reticuloses

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CLINICAL RADIOLOGY

GOLDACRE, R. J. & SYLVAN, B. (1962). Brit. J. Cancer, 16, 306. GOODHEART, C. R. (1961). Radiat. Res., 15, 767. GREGG, D. McC. (1958). Postgrad. Med. J., 34, 149. GREGG, D. McC. (1960). Brit. J. RadiaL, 33, 531. GREGG, D. McC. (1965). in "The Treatment o f Cancer'. Ed. Acknowledgements. This investigation is the work of a Mitchell, J. S., Cambridge, University Press. p. 279. team. I wish to thank my colleagues, especially Dr. D. H. GRONOW, M. (1963). P h . D . Thesis, Univ. Cambridge. Marrian, Mrs. I. Sinaon-Reuss, Mr. E. A. King, Dr. Barbara GRONOW, M. (1965). Int. J. Rad. Biol. 9, 123. Chipperfield, Drs. D. Mc. Gregg, I. A. Silver, D. B. Cater, HEMPEL, K. & DEIMEL, M. (1963). Strahlentherapie, 121, 22. D. G. Bratherton, P. T. Chopping, Diana M. Brinkley and HLAVKA, J. J. & BUYSKE, D. A. (1960). Nature, Land., 186, Elizabeth M. Kingsley Pillers; further, Mr. B. McN. Truscott, 1064. Mr. Walpole Lewin and Drs. T. D. Hawkins, Aileen K. HORWITZ, H. (1960). Brit. J. Radial., 33, (a) 659, (b) 679. Adams, H. R. Youngman, and M. J. Greenberg, and Mr. HORWlTZ, H., GREGG, D. McC_, MARRIAN, D. /q., J. L. Haybittle, Mr. R. A. Pope, Miss J. Young, Sister A. M. MARSHALL, B. & MITCHELL, J. S. (1959). Acta radial. Mitchell and Sister E. J. Porter. Stockh., SuppL 188, 111. I wish to acknowledge with grateful thanks the most I.C.R.P. (1959). Publication 2. Recommendations of the helpful collaboration of the staff of the Radiochemical International Commission on Radiological Protection. Centre, Amersham, and of Roche Products, Welwyn Garden Report of Committee II on Permissible Dose for Internal City, and especially Drs. A. L. Harrison and K. J. Andrews. Radiation. New York, Pergamon Press. I wish to express my grateful thanks for financial assistance KARNOFSKY, D. A., BURCHENAL, J. H., ARM/STEAD, G. C., to the International Atomic Energy Agency, the British SOUTHAM, C. M., BERNSTEIN, J. L., CRAVER, L. F. & Empire Cancer Campaign, the Medical Research Council, RHOADS, C. P. (1951). Arch. intern. Med., 87, 477. the Beebe Fund (through the good offices of the Secretary KESTON, A. S., BALE, R. P., FRANTZ, V. K. • PALMER, W. W. General of the Faculties of the University of Cambridge), (1942). Science, 95, 362. the Wolfson Foundation, my own Research Fund and an KISIELESKI, W. E., BASERGA, R. & LISCO, H. (1961). Atomanonymous donor. praxis, 7, 81. KOCH, A. L. (1965). Radiat. Res., 24, 398. REFERENCES KOTTMANN, K. (1935). Schweiz. med. Wschr. 16, 533. KROLL, H. H., STRAUSS, S. F. & NECrlELES, H. (1941). J. ALTMAN, K. I. & SALOMON, K. (1960). Nature, Land. 187, Lab. Clin. Med., 27, 50. 1124. ANDREWS, K. J. M., BULTITUDE, F., EVANS, E. A., GRONOW, Ki_iNKEL, H. A. (1962). Strahlentherapie, 118, 46. LEWIS, M. R. & GOLAND, P. P. (1948). Amer. J. Med_ Sci. M., LAMBERT, R. W_ & MARmAN, D. H. (1962). J. Chem. 215, 282. Sac. 3440. LIPSON, R. L., BALDES, E_ J. & OLSEN, A. M. (1961)_ J. Nat. ARIEL, I. M. & PACK, G. T. (1960). Amer. J. RoentgenoL Cancer Inst., 26, 1. 83, 474. Llsco, H., NISHIMURA, E_ T., BASERGA, R. & KISlELSKI, BALE, W. F. & SEAR, I. L. (1964). --personal communication. W. E. (1961). Lab. Invest., 10, 435. BALE, W. F., SPAR, I. L. & GOODLAND, R. L. (1960). Cancer MACHADO, L., ZAIDMAN, I., GERSTEIN, J. F., LICHTENBERG, Res., 20, 1488. F. & GRAY, S. J. (1964). Cancer Res., 24. 1845. BELCHER, E. H., COHEN M., DUDLEY, R. A., PARKER, H. G., MARRIAN, D. H. (1957). J. Chem. Sac., 499. TSIEN, K. C. & WETTER, H. (1964). Third United Nations MARRIAN, D_ H. (1959). Brit. J. Cancer, 13, 461. Internat. Conf. on Peaceful Uses of Atomic Energy. MARR/AN, D. H., MARSHALL, B. & MITCHELL, J. S. (1961). 28/P/880. Chemotherapia, 3,225. BLANCHARD, R. J. W., GROTENHUIS, I_, LA FARE, J. W. & MARRIAN, D. H., MARSHALL, B., MITCHELL, J. S. & SIMONPERRY, J. F. (1965) Proc. Sac. Exp. Biol. Med., 118, 465. REUSS, I. (1961). In I.A.E.A_ Symposium on Tritium, BLEECKEN, S. (1964). Strahlentherapie, 125, 145. Vienna, Int. Atomic Energy Agency, p. 211. BRILMAYER, C., KOHLER, A., MACK, A. & STORDEUR, K. MARRIAN, D. H., MARSHALL, B., MITCHELL, J. S. & SIMON(1955) Zs. f. Krebsforsch. 60, 334. REUSS, I. (1965). In 'The Treatment o f Cancer', Ed. Mitchell, BRUES, A. M., STROUD, A. N. & RIETZ, L. (1952). Proc. Sac. J. S., Cambridge, University Press, p. 98. Exp. Biol. Med. 79, 174. MARRIAN, D. H. & MAXWELL, D. R. (1956). Brit. J. Cancer, CHrPVERFrELD (n6e MARSHALL), B. (1961). Ph.D. Thesis. 10, (a) 575, (b) 739. Univ. Cambridge. MAXWELL, D. R. (1954). In Second Radio-Isotope ConferCLARKSON, B. D., OTA, K. & KARNOESKY, D. A. (1962). ence. Vol. I, p. 200. Ed. Johnston, J. E., London, ButterProc. Amer. Ass. Cancer Res., 3, 311. worths Scientific Publications. CRAMER, H., BRILMAYER, C. & PABST, H. W. (1952). Schweiz. MAXWELL, D. R. (1955). Ph.D. Thesis. Univ. Cambridge. med. Wschr. 82, 76. MCKELVlE, A. D. (1959). Nature, Land_, 183, 1194. DI VITA, G. (1964). - - personal communication; letter dated MCKELVm, A. D. (1963). Radiation Botany, 3, 105. 12.5.1964. DUNN, A. L., ESKELSON, C. D., MCLEAY, J. F., OGBORN, MENDELSOHN, M. L. (1960). J. Nat Cancer Inst., 25, 485. MENDELSOHN, M. L. (1962a). Science, 135, 213. R. E. & WALSKE, B. R. (1960). Proc. Sac. Exp. Biol. Med., MENDELSOHN, M. L. (1962b). J. Nat. Cancer Inst., 28, 1015. 104, 12. MENDELSOHN, M. L. (1963). In Cell Proliferation and Tumor EVANS, E. A. (1965). Nature, Land. (in press). Growth. ed. Lamerton, L. F. & Fry, R. J. M. Oxford, EWING, J. (1934). Neoplastic Diseases. Philadelphia and Blackwe11 Scientific Publications, p. 190. London, W. B. Saunders & Co. MENDELSOHN, M. L. (1964). In Cellular Radiation Biology, FRIEDMANN, E., MARRIAN, D. H., & SIMON-REuss, I. (1948). Austin, Univ. Texas (in press). Brit. J. PharmacoL 3, (a) 263, (b) 335. MITCHELL, J. S. (1948). Brit. J. Cancer, 2, 351. GEHAN, E. A. (1961). J. Chron. Dis., 13, 346. h a v e b e e n t r e a t e d . D e f i n i t e o b j e c t i v e effects h a v e been observed. The clinical results have proved to b e i n g e n e r a l r e l a t i v e l y m o d e s t b u t s o m e t i m e s useful. T h e f i n a l p a r t o f t h e t r i a l h a s y e t t o b e d o n e .

AN A T T E M P T TO DEVELOP A R A D I O A C T I V E D R U G

MITCHELL, J. S. (1949). Twenty-seventh Annual Report, British Empire Cancer Campaign, p. 214 (see p. 217). MITCHELL,J. S. (1953). Acta radiol. Stockh. Suppl. 116, 431. MITCHELL, J- S. (1955). In Radiobiology Symposium, Liege, London, Butterworths Scientific Publications, p. 170. MITCHELL, J- S. (1960). Studies in Radiotherapeutics. Oxford Blackwell Scientific Publications, and Cambridge, Massachusetts, Harvard Univ. Press. MITCHELL, J. S. (1963a). In Strahlenschutz in Forschung und Praxis. Ed. Melching, H. J., Beck, H. R., Ladner, H.-A. & Scherer, E. Vol. 2, p. 139. Freiburg i. Br., Rombach. MITCHELL, J- S. (1963b). Proc. Roy. Soc. Med., 56, 651. MITCHELL, J- S. (1964). Anglo-German Med. Rev. 2, 524. MITCHELL, J. S. (1965a). In Progress in Biochemical Pharmacology. Vol. I. Basle, Karger (in press). MITCHELL, J. S. (1965b). Strahlentherapie 127, 497. MITCHELL, J. S., BRINKLEY, D. & HAYBITTLE, J. L. (1965). Acta radiol. Stockh. (in press). MITCHELL, J. S., KING, E. A,, MARRIAN, D. H. & CHIPPERFIELD, B. (1963). Acta radiol. Stockh. (New series), 1, 321. MITCHELL, J. S. & MARRIAN, D. H. (1965). In Biochemistry of Quinones. Ed. Morton, R. A. London, Academic Press, Chapter 15, p. 503. MITCHELL, J- S. & SIMON-REUSS, I. (1947). Nature, Lond., 160, 98. MosEs, M. J. (1964). J. Histochem. & Cytochem., 12, 115. Mf3LLER, J. H. (1962). Review Series No. 27. Vienna, Int. Atomic Energy Agency. NEUKOMM, S,, P~GUmON, L., LERCH, P. & RICHARD, M. (1953). Arch. int. Pharmacodyn., 93, 373.

BOOK Roentgenography and Roentgenology of the Middle Ear and Mastoid Process. By LEWIS E. ETTER, B.S., M.D., F,A.C.R. 149 pp. 134 illustrations. Springfield: Thomas. Price (not stated). PROFESSORETTER has written this short book in collaboration with two x-ray technicians, Lawrence C. Cross and Merle J. Stuart. The material is presented in atlas form. The author states that, in the United States, the standard of radiography of the temporal bone, including the middle ear and mastoid process, is not as high as it might be. He contends that this is due to lack of a clearly defined method of examination and to the belief that specialised equipment is necessary to produce satisfactory results. He sets out to demonstrate that a routine and acceptable radiographic examination of the temporal bone can be obtained by Using only apparatus normally available in any x-ray department, with the addition of a protractor or "Angligner" to measure tube and patient angles accurately and a two inch diameter cylinder for collimating the x-ray beam and improving the detail. The radiographs are taken with kilovoltages up to 110° without a Potter-Bucky diaphragm. The author considers that the routine examination of the temporal bone consists of six pairs of radiographs taken in the Schuller (lateral oblique), Owen's Stenvers', Mayer's, ChamberlainTowne's and basal (submentovertical) projections. The radiographic positioning of the patient and tube for each of these projections is illustrated by photographs with the "Angligner" in position and the method of obtaining each radiograph is described in the text. Unfortunately, the descriptions are brief and the terminology difficult for those on this side of the Atlantic to understand. In addition, the

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OSTERBERG, K. A. & WATTENBERG, L. W. (1961). Proc. Soc. Exp. Biol. Med., 108, 300. PLATT, W. R. (1947). Arch. Path., 43, 1. RALL, D. P., Loo, T. L., LAN~, M., & KELLY, M. (1957). J. Nat. Cancer Inst., 19, 97. SCHORR, S., AWAD, I. & LAUFER, A. (1959). Radiology, 73, 410. SCHWARZ, S., ABSOLON, K. & VERMUND, H. (1955). Univ. Minnesota Medical Bulletin, 27. SEED, J. (1960). Proc. Roy. Soc. B., 152, 387. SILVER,I. A., CATER,D. B., MARRZaN, D. H., & MARSHALL, B. (1962). Acta radioL Stockh., 58, 381. SIMON-REuss, I. (1961). Acta radiol. Stockh., 56, 49. SPAR, L L., BALE, W. F., GOODLAND, R. L., CASARETT, G. W. • MICHAELSON, S. M. (1960). Cancer Res., 20, 1501. STEWART,F. S- (1964). Int. J. Rad. Biol., 8, 545. SUGIMURA, T., OKABE, K. & BABA, T. (1962). Gann, 53, 171. TITUS, E. D., Loo, Z. L. & RALL, O. P. (1958). Antibiot. Ann., p. 949. VERLY, W. G. (1960). Review Series. No. 2, Vienna, Int. Atomic Energy Agency. WATTENBERG, L. W. (1961). In Ciba Foundation Symposium on Quinones in Electron Transport. Ed. Wolstenholme, G. E. W. & O'Connor, C_ M. London, J. & A. Churchill Ltd. p. 367. WEISSBERG, M. (1959). Zs. f Krebsforsch. 62, 668. WILLIS, R. A. (1948). Pathology o f Turnouts. London, Butterworth & Co. YOUNG, J. M. (1963). Ph.D. Thesis. Univ. Cambridge. YOUNG, J. M_, WILD, F. & SIMON-REusS, I. (1965). Brit. J. Cancer 19, 370.

REVIEW angles at which the patient is placed are described differently in the text and in the corresponding photographs in some instances (Fig. 7, 31, 38). No reasons are advanced for advocating these particular projections and no attempt is made to assess their value in demonstrating the structures in the temporal bone, Radiographs for each projection are reproduced, with the superimposed names of the visible structures obscuring the detail, and corresponding four-fold enlargements are used to show the structures more clearly. More than half of the pathological temporal bones pictured are affected with cbolesteatomata and the differential diagnosis is not discussed. The last section of the book consists of a description of Professor Etter's direct radiographic enlargement technique using a 0.3 mrn. focal spot tube. The film is placed at a distance from the patient either in a box with an adjustable shelf for examinations in the recumbent positions or with the variable-angle positioner designed by Lawrence Cross for examinations in the upright position. The author explains that three-fold enlargement gives the most consistent results with minimum penumbra and maximum preservation of detail but illustrates the book with fourfold enlargements. He considers that the enlargement technique is a definite teaching aid. It is also claimed that features not apparent on the conventional radiographs are occasionally shown on the enlargements but it is emphasised that they should be used only to supplement and not to replace conventional radiographs. A useful bibliography is given. In the reviewer's opinion this book will not raise the standard of radiography of the temporal bone in this country, nor does it advance the interpretation of temporal bone lesions, F. G. M. Ross