616 . 455-006 . 465-033 . 2 : 612 . 621 . I (Mus)
ADRENAL CORTICAL CARCINOMA WITH METASTASES IN AN OVARIECTOMISED STRONG A MOUSE
J. FLAKS *
From the Department of Experimental Pathology and Cancer Research, University of Leeds
(PLATES LXVI and LXVII)
Spontaneous adrenal cortical carcinomata are very rare in mice. Slye et al. (1921) reported only 4 tumours of adrenal origin in 33,000 mice. A spontaneous adrenal cortical tumour approximately 4 mm. in diameter has also been reported in a strain C female mouse aged 24 months by Dalton et al. (1943-44). Although the primary tumour did not give rise to metastases, it was successfully trans- planted in the same strain of mice. Recently, spontaneous malignant cortical tumours have been found in 2 males of the Minnesota line of the NH strain at 16 and 24 months respectively by Kirschbaum et al. (1946). These tumours were about one-third the size of the kidney and did not produce metastases. Females of the same strain a year or more old developed adrenal adenomata, the glands attaining 2-4 times the normal size.
Tumours of the adrenal cortex in ovariectomised mice of the NH strain have been observed by Gardner (1941). Of 15 mice ovariectomised at 43-65 days, 13 showed tumours of the adrenals without metastases between approximately 600 and 700 days of age. These tumours were mainly of microscopic size, but in some mice they were almost two-thirds the size of the kidney. That œstrogen was present in the ovariectomised mice was shown by the condition of the uterus and mammary glands.
Woolley et al. (1943) observed a high percentage of adrenal cortical tumours in ovariectomised or castrated mice of the JAX ce strain. In a series of papers Woolley and Little (1945a-d, 1946) gave a detailed account of the changes preceding and accompanying the formation of adrenal cortical carcinomata. Development of the accessory sex organs in females and males following the appearance of adrenal tumours showed that œestrogenic and androgenic hormones may be produced in the adrenals or in the developing tumours.
A single macroscopic lung metastasis was observed in a tumour-bearing mouse at 12 months by Woolley and Little (1945a), and Fekete and Little (1945) found only 8 microscopic metastases in the lungs of 43 ovariectomised ce mice between 12 and 22 months of age. No grossly visible metastases were found in other mice bearing adrenal cortical tumours in these series.
The ætiology of adrenal cortical tumours is no doubt related to an endocrine imbalance. It seems possible that a pituitary hormone of adrenocorticotropic type may operate following the removal of the gonads, causing at first hyper- plasia, and finally adrenal cortical tumours. There is, however, no experimental proof for such an action of the pituitary hormone, as no increase of adreno- corticotropic hormone has so far been found in animals deprived of their gonads. It seemed, therefore, of considerable interest to inject an adrenocorticotropic
* Bertram Parkinson Research Fellow. Dr Flaks died, after a brief illness, on 16th May 1949, soon after this paper had been completed.
ADRENAL CARCINOMA IN A MOUSE
pituitary extract into normal and spayed mice of various strains in order to ascertain if this hormone exhibits any influence on the formation of adrenal cortical tumours.
Amongst a group of spayed mice of different strains which received what was thought to be adequate doses of adrenocorticotropic hormone * and developed adrenal cortical tumours, one Strong A female showed a highly malignant metastasising tumour at 537 days. This mouse was ovariectomised at 3 days, weaned at 3 weeks and treated over a period of 8} months.
At autopsy, a tumour approximately 20x 12 × 12 mm. was found in the left flank. The left kidney, which was embedded in the growth, contained numerous metastatic nodules and was displaced downwards. It could be easily enucleated from the main mass of the tumour, which covered its ventral surface like a sheet. Anterior to this a pyramid-like nodule 6 x 3 x 2 mm. was attached on a broad base to the tumour. This nodule was identified histologically as the left adrenal, from which the growth had evidently arisen.
The right kidney also contained numerous metastatic nodules. The right adrenal was found approximately in its normal position. It was somewhat enlarged, but not invaded macroscopically.
The much enlarged liver contained numerous metastases. In addition, many small nodules were found in the pelvis, some of them adhering to the thread-like uterine horns. The growth had also extended into the left pleural cavity through the diaphragm, forming a nodule measuring 4x3 x 1.5 mm. in the upper part of the left pleural cavity. There were no metastases in the lungs or elsewhere and no general involvement of the lymphatic system. The spleen and thymus were tumour-free.
Histology
The structure of the tumour is seen to best advantage in the liver metastases fixed in Susa.
1. The liver contains numerous nodules disseminated throughout its parenchyma. The tumour cells (fig. 1) are mainly polyhedral and often without distinct cell borders. A follicle-like arrangement can be seen, but the cells are usually arranged in rows or cords. The protoplasm is slightly basophilic and the nuclei show variability in size and shape. Each contains one distinct or several less distinct nucleoli. Mitoses are abundant. Laidlaw’s stain reveals a fine reticulum encircling groups of cells or even single cells.
2. The left adrenal (fig. 2) is recognised by the presence of a few degenerate foamy cells of the zona glomerulosa and zona fasciculata lying beneath its capsule and forming a rim to a nodule composed of almost completely degenerated tumour cells and containing a few scattered calcified foci. Deeper areas show cells with fairly well preserved nuclei and typical arrangement. At the base the nodule is connected with the main tumour surrounding the left kidney.
3. The right adrenal (figs. 3 and 4) is free from malignant growth. There is, however, an extensive replacement of the cortex by the “type A ” cells of Woolley and Little (1945a), which are regarded by them as non-neoplastic. These irregular cells, with round or fusiform nuclei and scanty protoplasm, are found scattered over most of the cortical area. In some regions they were subcapsular in position, with finger-like processes spreading from the periphery into the zona glomerulosa (fig. 3). At one pole of the adrenal they occupy the whole area of the cortex as far as the medulla, ending abruptly in the boundary zone.
* Armour’s ” Adrenotrophic Factor ” and an adrenocorticotropic preparation of sheep and ox pituitaries prepared in this department in collaboration with Dr L. H. Stickland according to the method of Li et al. (1943).
4. Tumour tissue which had invaded the kidneys and the outer muscle layer of the stomach shows the same histological structure as the liver secondaries.
DISCUSSION
Some difficulty was experienced in tracing the origin of this tumour, which was so widely disseminated throughout the abdomen. In strong support of an adrenal cortical origin was the fact that the left adrenal was almost com- pletely changed into a tumour nodule showing advanced necrosis. This suggested that it was most probably the first organ to be involved in the malignant process. It would seem that the initial cortical tumour broke through the capsule at the caudal pole of the adrenal, giving rise to the main mass of growth outside the adrenal and remaining attached to it at the time of death. The right adrenal, although not neoplastic, was heavily invaded by Woolley and Little’s ” type A ” cells, indicating that a generally favourable state for the development of adrenal tumours existed within the body.
The structure and arrangement of the left adrenal tumour leave no doubt as to its cortical origin. When compared with a localised adrenal cortical tumour (4 x 2 mm.) found in the right adrenal of a gonadectomised and hormone- treated stock female mouse at the age of 514 days (figs. 5 and 6), the present tumour differs in that it is more anaplastic, shows more numerous mitotic figures, and has less well developed connective tissue septa.
The time of appearance of the tumour is well within the range of appearance of cortical tumours in gonadectomised mice of the JAX ce strain. It is, therefore, not possible to conclude that the injected hormone accelerated the development of this or other tumours which arose in our gonadectomised mice. But what seems to be unusual is the expansion and spreading capacity of the adrenal carcinoma here described. It is also interesting to note that malignant tumours may develop after gonadectomy, not only in the JAX ce and NH strains but also in Strong A and CBA strains and in mice of mixed stock, at advanced ages.
There was no evidence of an œestrogenic stimulation as judged by the condition of the uterus, but it was rather surprising that one week before death a pure œstrous smear occurred, and became di-œstrous on the following day.
SUMMARY
A case of a highly malignant metastasising carcinoma of the adrenal cortex in a gonadectomised Strong A female mouse treated with adrenocorticotropic hormone is described. Adrenal cortical tumours can easily be induced by gonadectomy in the JAX ce and NH strains of mice, but, so far as we are aware, no cortical tumour has been described in the Strong A strain, and in no strain one of such malignancy as to involve the liver, kidneys, outer muscle layer of the stomach and pleural cavity.
The appearance of such a tumour in a spayed mouse treated with adreno- corticotropic hormone may however be coincidental.
REFERENCES
DALTON, A. J., EDWARDS, J. E., 1943-44. J. Nat. Cancer Inst., iv, 329. AND ANDERVONT, H. B.
FEKETE, ELIZABETH, AND LITTLE, 1945. Cancer Res., v, 220. C. C.
GARDNER, W. U. 1941. Ibid., i, 632.
KIRSCHBAUM, A., FRANTZ, MARTH- 1946. Ibid., vi, 707. ELLA, AND WILLIAMS, W. L.
ADRENAL CARCINOMA IN A MOUSE
LI, C. H., EVANS, H. M., AND 1943. J. Biol. Chem., cxlix, 413. SIMPSON, MIRIAM E.
SLYE, MAUD, HOLMES, HARRIET 1921. J. Cancer Res., vi, 305. F., AND WELLS, H. G.
WOOLLEY, G. W., FEKETE, ELIZA- BETH, AND LITTLE, C. C.
WOOLLEY, G. W., AND LITTLE, 1945a. Cancer Res., v, 193. C. C.
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1945b. Ibid., v, 203.
”,
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”, 1945c. Ibid., v, 211.
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”, 1945d. Ibid., v, 506.
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1946. Ibid., vi, 712.
578 .65 : 576 . 852 . 211
THE STAINING OF TUBERCLE BACILLI WITH SUDAN BLACK B
H. L. SHEEHAN and F. WHITWELL Department of Pathology, University of Liverpool (PLATE LXVIII)
In recent years a number of workers have used ” broad ” techniques of bacteriological staining with sudan black B, designed to demonstrate non- specifically all the types of fatty material which occur in a great variety of bacteria (Hartman, 1940; Burdon, Stokes and Kimbrough, 1942; Burdon, 1946). The present note deals with a different application of sudan black- its use as a specific and non-fading stain for the demonstration of tubercle bacilli. This is based on the fact that these are the only organisms which, after staining, have been found to be quite resistant to decolourisation with acetone.
Method
1. The sputum or other material is smeared not too thickly on a slide and is fixed, while still wet, with Carnoy’s fluid for half a minute. Heat fixation after drying the smear is a satisfactory but tedious method. Most other fixatives of wet smears are also quite good if not allowed to act for too long. The sudano- phil lipids can be removed by treatment with chloroform or carbon tetrachloride in about half-an-hour, with xylol in about 3 hours and with alcohol in about 8 hours, but they are unaffected by treatment with acetone for over 24 hours. Antiformin concentration methods do not interfere with the staining. Paraffin sections of tissues are always negative, as would be expected.
2. The fixative is drained off, and the slide is half covered with a saturated solution of sudan black B in 70 per cent. alcohol. The stain is lighted and allowed to burn out, and the slide is then swilled with water. The stain is made by adding 3 parts of water to 7 parts of a thoroughly saturated solution of sudan black B in absolute alcohol and filtering the mixture. The diluted stain deteriorates slowly in the course of a few weeks. The stain is efficient over a pH range of about 5 to 9, and with various strengths of alcohol between 60 and 80 per cent. Other solvents such as dilutions of acetone, chloroform, dioxane or ethylene glycol are less satisfactory. Prolonged exposure of films to heated sudan solutions tends to stain other organisms, which may then be partially acetone-fast.