two cases of renal carcinoma showing cytoplasmic inclusions

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616. 61-006. 6-091 .825 TWO CASES OF RENAL CARCINOMA SHOWING CYTOPLASMIC INCLUSIONS DAVID M. MCCLURE and C. F. Ross From the Pathology Departments of the University and Western Infcrmry, Glasgow, and Ashford Hospital, Middlesex (PLATE CXXV) CARCINOMA of the renal parenchyma is a fairly frequent disease in the human subject, comprising about 2.5 per cent. of all malignant tumours occurring between the ages of thirty and seventy. Two examples have recently been encountered in widely separated parts of the country, in which the malignant cells contained cytoplasmic inclusions bearing a certain resemblance to inclusion bodies of virus origin. Since no report of a similar finding in this common disease has been found in the literature, it appears worth while to place on record a detailed account of these neoplasms. CASE REPORTS Case I Clinical historg Mrs J. L., aged 41, was admitted to the Glasgow Western Inf%-mary suffering from mitral stenosis. Fourteen days after admission she complained of constant gnawing pain in the left loin. A firm irregular mass was palpable in the left hypochondrium, mobile and tender, and moving with respiration. The urine showed numerous red cells, but no casts or excess of leucocytes. A provisional diagnosis of renal tumour was made. Retrograde pyelography showed a filling defect of the left renal pelvis and at the pelvi-ureteral junction, suggestive of a neoplasm. At operation a tumour was found to occupy most of the lower half of the kidney, which was adherent to the peritoneum in this situation. The renal pelvis was slightly distended by a soft mass of growth and, in its upper third, the ureter was adherent to the psoas muscle in which a nodule about the size of an almond was felt. The kidney was removed with 75 mm. of the ureter, and the nodule in the psoas was excised. At no time before or after operation was there any evidence of urinary infection, and the patient made an uneventful recovery. Description of specimen The kidney weighed 260 g. In the lower pole was a firm nodular mass, occupying the entire thickness of the organ. On dissection, t,he tumour was seen to be roughly spherical and 55 mm. in diameter. J. PATE. BACT.-VOL. LXIII 719

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Page 1: Two cases of renal carcinoma showing cytoplasmic inclusions

616. 61-006. 6-091 . 8 2 5

TWO CASES O F RENAL CARCINOMA SHOWING CYTOPLASMIC INCLUSIONS

DAVID M. MCCLURE and C. F. Ross From the Pathology Departments of the University and Western

In fcrmry , Glasgow, and Ashford Hospital, Middlesex

(PLATE CXXV)

CARCINOMA of the renal parenchyma is a fairly frequent disease in the human subject, comprising about 2 .5 per cent. of all malignant tumours occurring between the ages of thirty and seventy. Two examples have recently been encountered in widely separated parts of the country, in which the malignant cells contained cytoplasmic inclusions bearing a certain resemblance to inclusion bodies of virus origin. Since no report of a similar finding in this common disease has been found in the literature, it appears worth while to place on record a detailed account of these neoplasms.

CASE REPORTS Case I

Clinical historg

Mrs J. L., aged 41, was admitted to the Glasgow Western Inf%-mary suffering from mitral stenosis. Fourteen days after admission she complained of constant gnawing pain in the left loin. A firm irregular mass was palpable in the left hypochondrium, mobile and tender, and moving with respiration. The urine showed numerous red cells, but no casts or excess of leucocytes. A provisional diagnosis of renal tumour was made. Retrograde pyelography showed a filling defect of the left renal pelvis and a t the pelvi-ureteral junction, suggestive of a neoplasm.

At operation a tumour was found to occupy most of the lower half of the kidney, which was adherent to the peritoneum in this situation. The renal pelvis was slightly distended by a soft mass of growth and, in its upper third, the ureter was adherent to the psoas muscle in which a nodule about the size of an almond was felt. The kidney was removed with 75 mm. of the ureter, and the nodule in the psoas was excised. At no time before or after operation was there any evidence of urinary infection, and the patient made an uneventful recovery.

Description of specimen

The kidney weighed 260 g. In the lower pole was a firm nodular mass, occupying the entire thickness of the organ. On dissection, t,he tumour was seen to be roughly spherical and 55 mm. in diameter.

J. PATE. BACT.-VOL. LXIII 719

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720 D. M . McCLURE AND C. F . ROSS

It was sharply demarcated by a capsule of compressed and fibrosed renal tissue. The cut surface showed golden-yellow areas of central necrosis. A few fibrous trabecuh traversed the growth. The tumour extended into the renal pelvis, and there were white papillomatous outgrowths from its lining epithelium. The lumen of the ureter was patent, and no extension along the renal vein was seen.

There was no cyst formation or mucoid change.

General histological features

Blocks were taken from the central and peripheral parts of the tumour, from the adjacent tumour-free renal tissue, from the pelvis and ureter and from the nodule in the psoas muscle. The tissues were fixed in corrosive formol and 10 per cent. formol saline. Paraffin and frozen sections were prepared.

Both the architecture of the growth and the degree of epithelial differentiation varied in different parts. The predominant structure was papilliform, and this was most highly developed in the peripheral parts, where the tumour was divided into lobules by fibrous septa derived from the capsule of compressed and fibrosed renal tissue. Elsewhere, the cells were arranged in tubular form. Extensive areas of necrosis with heavy polymorph infiltration were present in the central parts of the growth. Immediately adjacent to the necrotic areas there was an increase in the collagen of the stroma and infiltration with polymorphs. Thin-walled blood vessels were present at the periphery, but the main bulk of the tumour showed low vascularity.

The degree of differentiation in both the papilliform and tubular areas may conveniently be divided into three grades :-(1) areas with well defined epithelium, in which the cells show clearly defined cell membranes and are generally in single layers, but with fairly frequent reduplications. The intervening stroma is very delicate, and of low vascularity, so that the epithelial layers of each papilliform process are separated by little more than the basement membrane. (2) Areas with poorly differentiated epithelium showing great irregularity and heaped into multiple layers in which cell membranes are not distinguishable, but in which the basic papilliform or tubular structure is retained. The stroma in these areas is more abundant, and frequently contains bulky aggregations of very large foamy cells with small dense nuclei. (3) Frankly anaplastic areas in which the features of papilliform or tubular structure are lost, and which show marked nuclear aberrations and mitotic figures. The latter features are readily found only in such areas of maximum anaplasia.

Sections of the pelvi-ureteral junction and of the ureter showed lymphatic permeation of all the layers by malignant cells acconipanied by considerable polymorph infiltration.

The malignant cells in the nodule from the psoas muscle showed

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CYTOPLASMIC INCLUSIONS I N T U M O U R CELLS 721

no suggestion of papilliform or tubular structure, and had a much more abundant fibrous stroma, freely infiltrated with polymorphs.

The cytoplasmic inclusions

In the primary tumour, the cytoplasm of the epithelial cells unaffected by necrosis contained highly refractile eosinophilic bodies. Almost every cell had one or more of these inclusions, producing a very striking microscopic picture. The individual bodies varied considerably in shape and size, ranging from 2-15 p in their greatest diameter, with an average size rather less than that of the cell nuclei. Only the smallest of the inclusions were spherical. All the larger bodies showed various degrees of distortion-elongation, compression and, whenever closely applied to the nucleus, indentation. These appearances were taken to indicate a relatively low density and low fluid tension. Almost without exception, the cells containing cyto- plasmic inclusions had a large eosinophilic nucleolus.

The distribution of the bodies varied in different parts of the growth. In the areas with well differentiated epithelium, single cytoplasmic bodies of uniform size were localised a t the base of the cell between the nucleus and the basement membrane. This arrangement was best seen in the papilliform areas with a single layer of epithelium (fig. 1). The size of the inclusions in these areas was generally a little less than that of the nuclei. In the areas with poorly differentiated epithelium in multiple layers, there was a corresponding multiplicity of inclusions. There was also a greater variation in size, and a greater number of smaller forms, frequently with several bodies in a single cell (fig. 2 ) . In the most anaplastic areas with large cells and large aberrant nucleoli, the inclusions were proportionately larger and more distorted. Numbers of smaller forms were again often present in association with the larger bodies within a single cell. Where the tubules were small and closely packed, with low cubical type of epithelium, the basal location of the bodies was much less in evidence, and they were found more often lateral to the nucleus, and frequently occupied the whole depth of the cell.

No organised internal structure was detected in the cytoplasmic bodies. With some staining methods the bodies were not homogeneous, but they never suggested the regular structural pattern of a group of elementary bodies.

On approaching the necrotic areas, a sharp transition was observed from cells containing inclusions and prominent nucleoli to cells showing neither of these features. In some instances nucleoli persisted in the zone beyond where the inclusions disappeared, but in general the absence of both was parallel to a striking degree. The majority of these cells were still apparently viable and formed a zone of varying width surrounding the necrotic areas. In these zones, the collagen of the stroma was more abundant. Occasionally, the transition

J. PATH. BACT.-VOL. LXIII 2 2 2

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722 D. 171. MCCLURE A N D C. P . ROSS

was so abrupt as to be a clearly defined line, but this may represent merely the point of contact of two lobules of turnour tissue having separate vascular supplies, one of which was failing. In the same way, there were occasional narrow prolongations of inclusion-bearing cells between two areas of necrosis.

In general, the greatest concentration of inclusions was found at the periphery of the tumour. At the extreme periphery, occasional areas were found where the entire cytoplasm of the cells was occupied by eosinophilic refractile material. In a very few places similar material was found extracellularly . The inclusions were wholly confined to the tumour and none was found in the renal substance proper.

Neither inclusions nor nucleoli were present in the tumour cells in the renal pelvis and ureter or in the nodule from the psoas muscle. In these sites the malignant cells were morphologically similar to those lacking nucleoli and inclusions in the primary tumour, namely, the cells adjacent to necrotic foci. The secondaries also showed marked polymorph infiltration.

Case 2

Clinical history Mr A. J. M., aged 46 years, was referred to Ashford Hospital in December

1949, with a history of severe cough of five months’ duration, accompanied by yellow non-offensive sputum, shortness of breath and fever, but no haemoptysis. He had been investigated as a possible case of pulmonary tuberculosis but X-ray examination of his chest suggested silicosis, though there had been no occupational risk. His sputum was consistently devoid of tubercle bacilli and the Mantoux reaction was negative in all dilutions.

In view of the condition of the patient, treatment was merely symptomatic, and he was discharged to attend the outpatient department. He was admitted to an associated hospital three weeks after his discharge, with a diagnosis of bronchopneumonia and died three weeks later.

Post-mortem Jindings

The right kidney contained a large tumour, 8 ~ 8 x 6 cm. in the upper pole, unattached to surrounding structures. On cutting, it showed much central fibrosis and areas of cyst formation. Its colour was yellowish-white, with some brownish areas of necrosis and hzemorrhage. The tumour was covered by the renal capsule, and was demarcated from the kidney by compressed renal substance. There was no involvement of the renal pelvis or vein. All the abdominal lymph nodes were neoplastic and continuous with those in the thorax. The rest of the urogenital tract was normal.

Apart from an old healed duodenal ulcer, the alimentary tract was normal. The spleen was “ toxic ”. The pancreas and adrenals were normal. The liver contained a small metastasis in the right lobe and one in the left. The gall-bladder and bile-ducts were healthy. The left kidney was congested but otherwise normal.

There was a little free fluid in the peritoneal cavity.

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J. PATH. BACT.-VOL. LXIII

CYTOPLASMIC INCLUSIONS IN TUMOUB CELLS

PLATE cxxv

CASE l.-Well-differentiated papillifom growth with predominantly subnuclear localisation of the cytoplasmic inclusions. Phloxin- tartrazine. x 350.

CASE 2.-Pleomorphic tumour growth with corresponding variation in the cytoplamic inclusions. Phloxin-tartrazine. x 350.

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CYTOPLASiMIC INCLUSIONS IN TUMOUR CELLS 723

There were some small neoplastic lymph nodes in the deep cervical chain, otherwise no abnormalities in the neck. Thoraz. Bilateral pleural effusion of about one litre was present with fibrinous adhesions over the lower lobe of the left lung. The parietal pleura showed scattered firm nodules from 5-10 mm. diameter, very numerous over the diaphragm; some were pedunculated. The lungs were almost completely solid, showing generalised multiple white discrete tumour nodules and intervening bronchopneumonia. The nodules were mostly rounded or lobulated and their size varied from 2-15 mm. diameter. Peribronchial spread was prominent. All the mediastinal lymph nodes were neoplastic, but were discrete. Nodules were scattered over the outer surface of the pericardial sac. The epicardium and the heart showed no abnormality.

General histological features A carcinoma which for the most part was growing in masses with

no attempt at differentiation was present in the right kidney. In some parts, tubule formation was seen and in others the structure approached that of the clear-celled variant. The bulk of the growth was made up of very large cells with abundant finely granular eosinophilic cytoplasm having large nuclei with prominent deeply eosinophilic nucleoli, often multiple. The size and shape of the tumour cells were very variable, and bizarre forms and tumour giant cells were common. In some places, necrosis and hzmorrhage were present. Mitoses were uncommon. Lymphatics and occasional venules con- tained growth.

All the lymph nodes examined (mediastinal, para-bronchial, celiac, para-aortic and mesenteric) were almost completely replaced by growth having similar characters t o those of the renal tumour. Large masses of growth replaced lung tissue which in some parts was infected. Dilated perivascular and peribronchial lymphatics were full of growth and, very rarely, small blood vessels contained carcinoma cells.

The cytoplasmic inclusions In the primary tumour, where the cells were not necrotic, in the

lung metastases and generally distributed throughout the growth in the lymph nodes wcre refractile deeply eosinophilic bodies. These were not) present in the turnour-free parts of the right kidney nor in any other organ examined. Nearly every malignant cell contained one or more of these inclusions, the number bearing an indirect relation to the size. Even where single cells in remote lymphatics were seen, the cytoplasm contained the bodies. In size, they varied from only just visible granules to large masses up to 2 5 x 8 p. The smallest bodies were round, but the larger ones oval, bean-shaped, horseshoe-shaped, dumb-bell-shaped or in irregular masses. In other characters the bodies were generally similar to those in case 1.

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724 D. M . MaCLURE AND C. F . ROSS

STAINING REACTIONS OF THE CYTOPLASMIC BODIES

The bodies were investigated by standard histological and histo- chemical techniques, including methods for the specific demonstration of various intracellular granules.

The bodies gave a negative reaction with the periodic acid-Schiff technique, thus eliminating glycogen, simple and complex muco- polysaccharides, mucoproteins, glycoproteins and glycolipids. Feulgen’s reaction was negative. Tests for iron and fat were also negative and under polarised light the bodies were isotropic.

With the general histological stains, the cytoplasmic bodies were uniformly eosinophilic. With Mallory’s phosphotungstic acid haema- toxylin and with iron haematoxylins they were blue-grey and blue-black respectively. Kiihne’s hyaline stain gave only a very faint blue, and Gram’s stain was not well retained. With van Gieson’s stain the bodies stained a bright yellow.

Of the various stains, carbacid fuchsin (Lendrum, 1945) and Laidlaw’s acid fuchsin-orange G were the most specific, the fuchsin staining being wholly confined to the bodies. With carbacid fuchsin the staining was patchy and inconstant, and uniform results were not readily obtained. The bodies did not appear homogeneous and gave an impression, not definitely confirmed by any other method, of a coagulum confined within a limiting pellicle. Phloxin-tartrazine (Lendrum, 1947) gave excellent definition, the bodies being homo- geneous and strongly phloxinophilic, retaining the red stain rather longer than the majority of the red cells. In the subsequent investiga- tions, this stain was adopted as a standard.

Inclusion bodies in the renal epithelium of the newborn have been described by various authors, and as material from a recent case (Cappell and McFarlane, 1947) was available, the staining reactions were compared with those of the present tumours. Material from the submaxillary virus disease of guinea-pigs was also employed, as this condition occasionally involves the kidneys, and the cytoplasmic globules of hyaline droplet change in renal epithelium were also studied. No tinctorial or morphological similarity was found between the inclusions in the newborn or in the guinea-pig and those of the present cases. Comparison of the staining reaction of the tumour inclusions with hyaline droplets in renal epithelium showed some tinctorial similarity, particularly with phloxin-tartrazine, but the larger hyaline droplets exhibited somewhat greater refractility and a more strongly phloxinophilic property. Fundamental differences were, however, observed. The hyaline droplets were P.A.S. positive, and also stained strongly with Kiihne’s hyaline stain and Weigert’s fibrin stain, in cpntrast to the tumour bodies.

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CYTOPLASMIC INCLUSIONS IN TUMOUR CELLS 7 2 5

CYTOPLASMIC BODIES IN OTHER RENAL TUMOURS

A specific search for cytoplasmic inclusions was made in a series of renal tumours, since their presence in small numbers in previous cases might have been overlooked in the routine haematoxylin and eosin preparations. Accordingly, sections from 70 cases of renal tumour were prepared, stained with phloxin-tartrazine and searched carefully for inclusions.

The series included 58 cases of renal carcinoma, 55 cases of renal adenoma, and 7 renal rests and miscellaneous cases. Fixation was, as before, by corrosive formol or formol saline.

In sixteen of the renal carcinomata, refractile spherical phloxinophil bodies were found in the malignant cells. In all cases, the bodies were scanty and appeared as multiple small spherical globules situated peripherally in large swollen cells, and lying between the nucleus and the free margin, or as large single spherical globules. The inclusions were never located at the base of the cells and were almost invariably spherical. In addition, they were rather more highly refractile, and even more phloxinophilic than those in the two tumours under review. Investigation of the staining reactions of the inclusions in this series shows that they were tinctorially identical with the hyaline droplets in renal epithelium. With carbacid fuchsin, the fuchsin staining was wholly conhed to the bodies, which presented a much more homo- geneous appearance than did those in cases 1 and 2. With phloxin- tartrazine the bodies were readily identifiable by virtue of their greater refractility, even when their size approximated to that of a red cell, and despite incomplete decolorisation of the red cells. The staining reactions of both large and small inclusions in the series were identical.

DISCUSSION Cellular inclusions are not infrequent in malignant turnours, and

in the past have given rise to speculation about the possible parasitic and virus aetiology of neoplasms. Various accounts of both cytoplasmic and intranuclear inclusion bodies in tumour cells have been given. The cytoplasmic bodies include the " bird's eye " inclusions of glandular carcinomata, which are characteristically located between the cell nucleus and the alveolar lumen, and contain a central basophilic portion surrounded by a protoplasmic rim or " halo '' (Ewing, 1940 ; Willis, 1948). Cytoplasmic inclusions have been most frequently reported in tumours of glandular origin, and as a rule are the result of disordered attempts at the formation of secretion. In the gliomata, the intranuclear inclusions are compatible with virus activity (Russell, 1932). In other cases, as in the muscle sarcomata, abnormal con- figurations of the nuclear chromatin may present a superficial resemblance to virus inclusion bodies or to protozoan agents. Prominent acidophil nucleoli are characteristic of rapidly growing cells, both simple and malignant.

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726 D. M . MCCLVRE AND C. P. ROSS

In the renal carcinoma of the leopard frog (Luck6, 1934a and b ; 1938a and b ) where the general architecture of the growth corresponds very closely to the papilliform areas of the tumour in case 1, the inclusions are again exclusively intranuclear , being of Cowdry’s type “ A ”.

In the present cases, the association of cytoplasmic inclusions with acidophil intranuclear bodies in a regular pattern suggested the activity of a virus. On detailed examination, however, the bodies differed in various fundamental respects from intranuclear and cyto- plasmic bodies of known virus origin.

The large eosinophilic nucleoli in the renal tumours bear a close resemblance to the earliest stages of development of the intranuclear inclusions of guinea-pig submaxillary virus disease, but the fully developed virus bodies differ radically from the nucleoli, which show no significant difference from the nucleoli of other renal carcinomata without cytoplasmic inclusions, and from the nucleoli in other carcino- mata and similar rapidly growing cells.

The tumours do not arise from glandular epithelium. Although the secretory function of renal tubule cells for certain metabolic products, dyes, etc., is now generally accepted, this activity is not associated with the formation of visible intracellular secretion products. Materials absorbed from a tubular lumen are most abundantly stored between the luminal margin of the tubule cells and the nucleus, but only rarely between the nucleus and the basement membrane. In case 1 the position of the material in the cells is in direct contrast to that of products of secretory activity. Also the subnuclear localisation of the bodies is opposed to the possibility that they have been formed by absorption from the fluid contents of the renal tubules. Moreover, in case 2, the bodies were found in metastases in widely different structures such as lymph nodes and lung. However, the possibility of absorption of some substance fiom the blood stream remains. It has been observed that the greatest concentration of these bodies is in the peripheral parts of the tumours, and that they appear to undergo lysis with the onset of necrosis. This indicates that the viability of the cells is necessary for their preservation and the presence of the bodies cannot be attributed to degenerative change.

Eosinophilic cytoplasmic inclusions occur in a number of human infections, but these differ fundamentally from the bodies in the present cases, being demonstrably composed of aggregations of elementary bodies.

It is concluded that the bodies in the tuniours do not correspond to known virus inclusions. There is no evidence that the material in the cells is a secretion, and degenerative processes are clearly not involved in the production of the inclusions. It seems certain that the bodies do not arise from absorption of extraneous matter. The remaining possibilities are that the cytoplasmic bodies may represent either material absorbed from the blood, which the cells are unable to

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CYTOPLASMIC INCLUSIOATS I N TUMOUR CELLS 727

metabolise, or that the bodies are accumulations of some by-product of the abnormal metabolism of the tumour cells. The greater con- centration and greater size of the bodies in the peripheral and most actively growing parts of the tumour in case 1 and in the metastases in case 2 may be taken as consistent with either hypothesis.

A notable feature in case 1 is the absence of inclusion bodies in the secondary growths. In these, all the cells showed features similar to the cells in the primary growth that were in the early stages of nutritional impairment, but it is not clear whether there was any actual impairment of vitality in the secondaries. The presence of a heavy polymorph infiltration in the absence of any demonstrable pyogenic infection, however, may indicate some such disturbance.

SUMMARY

Two cases of renal carcinoma are described in which the majority of the viable celh contain one or more cytoplasmic inclusions. The morphology and staining reactions of these bodies were compared with those of known virus origin, and with those of hyaline droplets in renal epithelium, In each instance fundamental differences were demonstrated. A series of renal carcinomata was investigated for corresponding structures with negative results.

Our thanks are due to Prof. D. F. Cappell for the use of material from the caae of renal inclusion disease, and for his advice and criticism, to Mr W. A. Galbraith and Dr A. Barham Carter for access to the respective case histories, and to Mr H. C . Gray, A.R.P.S., of The Victoria Infirmary, Glasgow, for the Ektachrome photomicrographs.

REFERENCES

CAPPELL. D. F., AND MCFARLANE, 1947. This Journal, lix, 385. MARJORY N.

EWING, J. . . . . . .

LENDRUM, A. C. . . . .

LUCK&, B. . . . . . . . . . . ,,

. . . . . . ,, 1 ,

9 ,

. . . . . .

. . . . . . RUSSELL, DOROTHY S. . . . WILLIS, R. A. . . . . .

1940. Neoplastic diseases, Phihdelphia and London, 4th ed., pp. 32 and 116.

1945. This J o u r m l , lvii, 267. 1947. This Journal, lix, 399. 1934a. Amer. J . Cancer, xx, 352. 1934b. Ibid., xxii, 326. 1938~ . Ibid., xxxiv, 15. 19386. J. Exp. Med., Ixviii, 457. 1932. This Journal, xxxv, 625. 1948. Pathology of tumours, London,

pp. 150, 195 and 819.