LYMPHOBLASTOMA IN MICE FOLLOWING ADMINISTRATIONOF CARCINOGENIC TAR 1

AUSTIN M. BRUES AND BEULA B. MARBLE

(From the Laboratories of the Collis P. Huntington Memorial Hospital of Harvard University)

In the course of an experiment designed to investigate the effect of certaindietary factors on the development of tar cancer, a strikingly high percentageof lymphoblastic tumors appeared in one of the strains of mice used. Thepresent report is an analysis of this material and of further experiments car­ried out on the same strain.

The relationship between leukemoid states and carcinogenic chemicalagents is an interesting one. A sufficient number of reports has appeared inthe literature to make it certain that a relationship of some sort exists. Yetthe very occasional nature of such reports makes it clear that this relation isnot the direct and sequential one which exists between carcinogenic agents andepithelioma or sarcoma, or between certain substances and hepatoma (2, 3).

Leukemia has been observed following the administration of benzol.Lignac (4) noted lymphoid hyperplasia in the spleen following injections ofbenzol in oil, and subsequently (S) observed eight leukemias in S4 mice simi­larly injected; 3 of these were lymphatic in type. Hess (6) obtained similarresults. Forkner (7) cites a few reports of human leukemia following in­dustrial exposure to benzol. Leukosis in the fowl has been seen, usually con­currently with sarcoma, after tar injections (8) . Bernard (9) reportederythroleukemia following injection of tar into the bone marrow of rats, andBarnes and Furth (10) have described an atypical leukemia produced by theinjection of benzpyrene in lard into the mouse spleen, the type cell here ap­peared to be the megakaryocyte. Injections of indol into mice (11) and ofhydroxylamine hydrochloride into rabbits (12) have been followed by leu­kemia; in the former instance most of the cases were myeloid in type and werepreceded by a leukopenic phase, as in the case of benzol treatment.

A clear-cut report of a leukemic state following the administration of thewater-soluble endo-succinate of dibenzanthracene was given by Burrows andCook (13), and it was subsequently shown by Parsons (14) that leukemoidblood pictures occurred in animals into which sarcomas induced by thisagent were grafted. It was further noted that animals which developed tu­mors as a result of injection of this compound showed an increase in cells ofthe myeloid series in the blood at the time of the development of the sarcomas.The leukemoid states noted were all characterized by lesions of the myeloidtype.

Perry and Ginzton (1 S), in a large series of mice painted with dibenzan­thracene, in some cases combined with theelin, found a case of lymphoblastomaand several cases of generalized. lymphadenitis in which a diagnosis of leu­

1 This investigation was materially aided through a grant from the Proctor Fund of HarvardUniversity. A preliminary report of this work has appeared (1).

45

46 AUSTIN M. BRUES AND BEULA B. MARBLE

kemia was not proved. Lawrence and Gardner (16) obtained a transplantablelymphoid leukemia in a strain A mouse which received prolonged injectionsof hydroxyestrin benzoate; the tissue used for transplantation was taken fromthe ovaries.

Hueper (17) has emphasized the occurrence of heterotopic foci of lymphoidand myeloid tissue in mice with spontaneous mammary tumors, frequentlysufficiently extensive to justify the diagnosis of leukemia. Irradiation of thesemice by small doses of x-rays resulted in a great increase in the incidence ofleukemia, especially in the myeloid forms. Many other observers have notedleukemia following the use of x-rays, and it has been thought that this destruc­tive agent may lead to exaggerated proliferation, as has been suggested in thecase of leukemias following benzol treatment (5).

FIG. 1. BLOOD SMEAR OF BAGG ALBINO MOUSE SHOWING LYMPHOCYTOSIS. WRIGHT'S STAIN­

HEMATOXYLIN. X 420

Morton and Mider (18) have recently published a preliminary account ofan experiment which demonstrates the production of lymphomatosis in 10 of48 dilute brown mice painted with methylcholanthrene in benzene over sev­eral areas. Their pathologic description of the lesions shows great similarityto those of the present report.

EXPERIMENTAL METHOD

The mice in our series, in which lesions occurred, were of the Bagg albinostrain, inbred originally by Dr. H. ]. Bagg of the Memorial Hospital, NewYork, in 1912, and inbred from August 1929 by Mrs. Claire Masters in thislaboratory. The black mice which serve as controls were of the C57 strain,obtained from Dr. C. C. Little in August 1929, and inbred by Mrs. Mastersuntil the time of these experiments.

LYMPHOBLASTOMA IN MICE 47

At the beginning of tarring, all animals were between ten and twenty weeks of age, with a mean weight of 2 1 grams. They received a diet of special breads of constant composition, which were given ad libitum, supplemented twice a week by cod liver oil and lettuce. They were divided into four dietary groups, the first serving as control. In a second group, the diet contained, in addition to the bread, 12.6 mg. per cent of lead acetate. In the third and fourth groups, sodium aluminum sulphate was added: in one case, 1.83 per cent by weight was added; in the other, 0.375 per cent. The diets were instituted four weeks before tarring was begun, and continued throughout the life of the animals. No significant differences were found in the incidence of lymphoma or of skin tumors on the various diets.

The tar used was prepared according to the directions of Murphy.( 19). I t

FIG. 2. SPLEEN OF TAR-PAINTED MOUSE IN SERIES 4: DETAIL FROM A LARGE AREA OF LYMPHO- BLASTIC PROLWERATION, WITH MANY MITOSES. HEMATOXYLIN-EOSIN. X 420

was prepared separately in each of three experiments, and it appears clear that the carcinogenicity of the three samples was widely variant. Applications were made to the nape of the neck three times a week for four months in the case of the most active tar, and for six months in the other experiments, after which the mice were observed until death.. In all possible cases they were carefully autopsied.

The mice had been inbred in this laboratory by brother-to-sister matings for at least six generations. Unfortunately, complete pedigrees for all the ani- mals used are not available, but one line derived from the Bagg strain, desig- nated below as line 7 2 , has been tabulated separately. This line originated in a single brother-to-sister mating eight months before the beginning of the first experiment, following six generations of inbreeding in this laboratory, and comprises a large number of the mice used in each of the four experi- mental groups.

48 AUSTIN M. BRUES AND BEULA B. MARBLE

PATHOLOGICAL OBSERVATIONS

During life, affected animals could be recognized by general lymphadenop- athy and palpable enlarged spleens. In many cases the liver could be seen extending far down towards the pelvis. Blood counts on a few of these mice showed a leukocytosis of 15,000 to 80,000, with a high proportion of mature and immature cells of the lymphoid series (Fig. 1). As death approached, the mice frequently became dyspneic because of mediastinal obstruction. At autopsy, in addition to enlargement of lymph nodes, spleen, and liver, the thymus was usually found to be enlarged.

Microscopic examination of the lymph nodes and spleen showed more or

FIG. 3 . LIVER OF TAR-PAINTED MOUSE IN SERIES 3, SHOWING LYMPHOBLASTIC INFILTRATION. HEMATOXYLIN-EOSIN. X 420

less wide infiltration of these organs with small and large lymphocytes and lymphoblasts, in which a high proportion of mitoses appeared (Fig. Z), to- gether with loss of structure and invasion of the capsule. Similar abnormal cells appeared around the portal vessels in the liver and invaded this organ to a greater or less extent (Fig. 3 ) ; the lungs were occasionally involved; and in one tarred mouse a small focus of such cells was found microscopically in an epithelioma. The liver was infiltrated with great constancy and presumptive diagnosis was most readily made by examination of that organ.

Several borderline cases were seen in which the spleen contained minute foci of lymphocytic proliferation by cells somewhat larger than normal, with numerous mitoses (Fig. 4) , occasionally with an associated perivascular lymphocytic infiltration of the liver. These cases have been classed as ‘‘ early or questionable ” lymphoblastoma.

A number of sections of liver and spleen showed amyloidosis, and small

LYMPHOBLASTOMA IN MICE 49

focal necroses in the liver were occasionally noted. These lesions were found throughout all of the groups, and were equally common in the C57 mice, which did not show lymphoblastoma.

Following the discovery of lesions in tarred albinos, the entire strain from which the tarred animals had been taken was examined for clinical lympho- blastoma; and three mice out of about two hundred showed obvious lesions. Other sporadic cases were found later and followed for several months. Dur- ing the four subsequent years approximately a thousand of these mice have been followed, and about two out of each hundred have developed these le- sions. In all non-tarred animals observed, the disease followed a slowly pro- gressive, almost benign, course. Death in these animals rarely occurred within

FIG. 4. SPLEEN OF TAR-PAINTED MOUSE IN SERIES 4, SHOWING SOME PROLIFERATION OF LYMPHOID CELLS, BUT NOT CLASSED AS LYMPHOBLASTOMA. HEMATOXYLIN-EOSIN. X 420

six months of the time when lymphadenopathy was first noted, in contrast to the rapid course (one to two months) in tarred animals.

ANALYSIS OF DATA

The incidence of lesions in albino mice has been tabulated separately for four series: those receiving no tar application; those painted with a batch of tar which was virtually ineffective in producing malignant disease; and those painted with two separately prepared batches of tar which appeared to be significantly different in carcinogenicity as judged by the rapidity of appear- ance of skin tumors. The data on these groups are shown in Table I.

In order to give an index of the relative carcinogenicity of the three tars used, we have noted in each group the duration from the beginning of tarring to the appearance of the first papilloma, and the mean duration before ap- pearance of skin tumors in those animals which eventually developed malig- nancy. The cancer morbidity rate was higher by groups as the onset became

50 AUSTIN M. BRUES AND BEULA B. MARBLE

Bagg

40 50.0%

135.1 84

~

earlier, but because of the difficulty of making fair statistical corrections in the case of animals dying or lost during the experiment, no attempt has been made to express this in tabular form. The “ ineffective tar ’’ series (Group 3 ) gave rise to only two papillomata, at 441 and 445 days after tarring was begun.

TABLE I: Frequency of Lymfihoblastoma and Carcinogenic Potency of Tars

INo Tar1 Tar I Tar I Tar

C57

24 0

140.6 119

---

Series Number of mice Frank lymphoblastoma “Early or questionable” lymphoma Mean duration (days) from tarring to appearance of epithe-

lioma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Duration (days) till appearance of first papilloma

2 59

1.7% 10.270

3 60

1.7% 8.3%

- 44 1

4 30 20.0% 10.0%

202.4 179

1 40 50.00/1, 12.0%

135.1 84

The data in the table show clearly that as the tar used was more actively carcinogenic, the proportion of frank lymphoblastomas appearing became higher. It is noteworthy that the diagnosis OF “early or questionable ” lymphoma was made in about 10 per cent of all groups, whether tarred or not.

The results of tarring black (C57) mice were in definite contrast to those just recorded. In our laboratory, spontaneous lymphadenopathy in these mice has been extremely rare. In these experiments, C57 mice were treated with the two highly carcinogenic tars. As will be seen in Table 11, the activity of these two tars in producing cancer was about the same in each strain, yet lymphoblastoma did not appear in any of the black mice. In each group of black mice, one case of ‘‘ early questionable ” lymphoma, without criteria of malignancy, was found.

TABLE I1 : Effects of Carcinogenic Tars on Two Strains of Mice - 1 Tar Group 4 1 Tar Group 1

1 I I I

Number of mice Frank lymphoblastoma Mean duration to appearance of epithelioma (days) Duration to appearance of first papilloma (days)

w g

30 20.0%

202.4 179

c51

31 0

200.0 166

Despite the fact that the series of mice are not large, statistical examination shows that the increased incidence of lesions in groups 1 and 4 is highly sig- nificant, owing to the very low incidence in untreated mice of the strain used. Assuming, on the basis of 1000 untreated mice, a normal incidence of 2 per cent, the probability of finding 6 affected mice in 30 chosen at random from this population (as in Group 4) has been calculated according to the Poisson distribution (20), and is 3.6 X lo-‘. The likelihood of drawing 20 affected mice in 40 from this population, correspondingly (as in Group 1 ) , is extremely low. Calculation of the standard deviation, although perhaps a less appropri-

LYMPHOBLASTOMA IN MICE 51

ate mode of treatment of such data, yields probabilities of the same low order of magnitude.

In order to rule out the possibility that lymphoblastoma might have arisen as a mutation in a single line of mice, and been selected in the experiments in which high incidence of this disease appeared, line 7 2 , for which the complete pedigree is available, has been followed through the course of the experiments. Fourteen of these mice were used in Experiment 1 , and 8 developed lymphoma. Several months later Experiments 2 and 3 were done, which resulted in a low incidence of tumors. Twenty-nine of the mice in this line were chosen for each experiment, all of which remained free from lymphoblastoma. Again after a long interval, 1 2 were used in Experiment 4, and 3 developed the dis- ease. These proportions of affected mice of line 7 2 in the several experiments are very close to the percentages of albino mice as a whole, which showed lesions.

No antagonism was obvious between the development of cancer and of lymphoblastoma. In Group 1 , of the 2 0 animals dying with lymphoblastoma, only 4 had failed to show skin tumors; these 4 died from lymphoma within 140 days of tarring, before many of the other mice had developed papillomata.

Two attempts have been made to transplant lymphoma, appearing in this strain spontaneously, into other mice; 30 recipients were used on each occa- sion, divided between the C 5 7 strain, the A (albino) strain, and the original strain. In each instance the donor showed characteristic gross and micro- scopic lesions. A much enlarged, heavily involved spleen was ground with an equal part of saline and injected subcutaneously. The suspension thus made was loaded with intact immature cells, many of which were in mitosis, but no lesions have appeared after nine and three months, respectively.

DISCUSSION

These results, in conjunction with those of Morton and Mider ( 1 8 ) , make it appear that under appropriate conditions the application of carcinogens may give rise to lymphoblastoma or lymphatic leukemia. Application of carcino- genic agents to the skin of an inbred strain of mice appears to be the common denominator.

It does not seem likely that these lesions are an accentuation of changes commonly seen after such treatment. A number of investigators have studied constitutional changes resulting from the use of tar or of known carcinogens. In general, either slight lymphocytosis has been noted in the blood picture ( 2 1 ) , or else a leukopenia or lymphopenia ( 2 2 , 2 3 ) . Picard (24) reported a decrease in size of the spleen, while Kinosita ( 2 5 ) and Lewis ( 2 6 ) have found myeloid hyperplasia following the administration of a variety of agents. Twort ( 2 7 ) likewise observed a splenic enlargement after the use of various tars and oils, although histologic findings were not recorded.

Some attention should be given to the nature of the spontaneous lymphoma appearing in the strain of mice used. The Bagg albino strain has been noted as one particularly free from lymphomatosis ( 2 8 ) . The very benign nature of the disease as it appears spontaneously, and the fact that it is not transmissible to mice of the same strain, seem to mark it off from the usual lymphomatosis

52 AUSTIN M. BRUES AND BEULA B. MARBLE

of mice as a different entity. The tar lymphoblastoma seen in this series of mice appears, therefore, to have occurred in a somewhat atypical substrate. Whether the abnormality of the substrate depends on genetics or environment cannot be proved; the former seems more likely, since no other mice in this laboratory have evidenced any similar condition. I t likewise would seem more probable that it arose as a mutation in this stock, rather than that it has been undiscovered throughout the many years during which the Bagg albino strain has been commonly used for research.

An observation by MacDowell (29) may throw some light on this ques- tion. After a long series of investigations relative to the genetics of mouse leukemia, he concludes that extrinsic factors must also be of importance in determining the incidence of the disease under given hereditary conditions. It may well be that carcinogenic agents, and perhaps estrogens, can play such a rde. It would be of interest to know whether these agents would have any such influence on a genetically controlled strain of animals having a moderate incidence of lymphomatosis.

SUMMARY

A strain of mice derived from Bagg albino stock shows occasional lympho- matosis with a subleukemic blood picture, which runs a chronic course and appears not to be transplantable into normal mice of the same or other strains. The incidence of this condition is normally 2 per cent.

Following cutaneous application of carcinogenic tar, the incidence of lymphoblastoma and lymphatic leukemia in these mice became as high as 50 per cent, and the disease followed a much more rapid course. The inci- dence of lymphoblastoma was closely correlated with the carcinogenic potency of three different tars used. A series of C57 mice treated by the same means failed to show any such lesions.

It is concluded that a carcinogenic agent may act as an extrinsic factor leading to the development of lymphoblastoma, in the presence of a latent predisposition.

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