ON THE CORRELATION BETWEEN MALIGNANCY AND THE RATE O F GROWTH O F TAR WARTS IN MICE

J. C. MOTTRAM, M.B. (LDN.)

(Prom the Radium Institute, London, and Mount Vernon Hospital)

Since the discovery that both benign and malignant warts follow the prolonged painting with tar of the skin of mice, much investigation has been carried out using this method; nevertheless, as far as I am aware, a detailed study of the natural history of individual warts has not been made, attention so fa r being directed to individual mice, rather than in- dividual warts.

While carrying ouf such an investigation on individual warts, a close correlation between malignancy and high rate of growth was ob- served, and vice versa. The present paper is concerned with this part of their natural history and allied considerations and is divided into four sections, as follows : A. Experimental Conditions ; B. Results Ob- tained ; C. Discussion ; D. Summary.

A. EXPERIMENTAL CONDITIONS

A mixed straiu of mice was used which had been bred in the labora- tory for a number of years; 60 of these mice were painted with a tar of high wart-producing potency for one hundred days on the nape of the neck, along a line 2 em. long; 54 mice survived the hundred days; one of them never showed any warts, though observed for two hundred and eighty-seven days ; upon the remaining 53 animals there grew two hundred and three warts. These warts formed the investigated ma- terial. The necks of the mice were examined and the warts measured to the nearest quarter of a millimeter three times a week. The major and minor dimensions and height of the warts were recorded, and from time to time drawings were made of the warts in elevation. Certain characters of the warts were also recorded: whether smooth or rough, scaly, ulcerated, horny, encrusted, exhibiting lateral or deep extension, etc. The records of their superficial dimensions, multiplied together, were plotted against time, so that the progress of the growth of the warts could always be seen, and closely followed.

From time to time warts were removed by operation, and histologic examination was done then or on the death of the animal. Very often, when removed by operation, half the wart was reserved f o r histologic study and the other half was used for autografts. No particular rule was adopted as regards time of operation except that an endeavour was made to operate at all stages of the life cycle. Generally warts were removed early from mice exhibiting many warts, in order to avoid co- aIition of two or more warts. For the investigation of the late stages,

801

802 J. C. MOTTUM

warts were generally obtained from mice having only a few lesions. A description of the technique of autografting is given on p. 820. If nodules appeared at the site of inoculation, these were removed by op- eration for histological observation. These nodules were not per- mitted to grow to such a large size that there was danger of recurrence after operation, for fear of having to kill the mouse in which probably warts or other sets of autografts were being observed.

The observations of many kinds thus obtained were brought to- gether in large tables, too extensive to publish, and from these tables, after the death of the last mouse, and from the charts representing the

CHART 1. TIME OF APPEARANCE OF WARTS mER TARE IN^ Sixteen warta, for instance, 5 benign, 3 malignant, a d 8 which disappeared, appeared

between aeventy-five and one hundred days after tarring was begun, and one benign wart appeared between four hundred and four hundred and twenty-five days after tarring.

growth of each wart, and the histological study of the warts and auto- grafts, the following results were obtained. In order to prevent one observation weighing upon others, each record was tabulated sep- arately: for instance, the histological slides were examined and tab- ulated all together (four days were required) without knowledge of the growth rate of the warts, the results of autografting, or any other observation ; and so all observations were tabulated.

B. EXPERIMENTAL RESULTS 1. Tiwe of Appearawe of Warts: The number of days from the be-

ginning of tarring to the first appearance of warts, has been recorded in the case of 186 warts, of which 90 were benign, 50 malignant, and 46 disappeared. This information is given in Chart 1,

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 803

Tarring was continued for only one hundred days. As the chart shows, a few warts appear between seventy-five and one hundred days after the beginning of tarring. Soon after one hundred days a great increase in warts occurs ; subsequently the warts appear in gradually decreasing numbers, very few appearing beyond three hundred days. The chart shows that the times of appearance of benign warts and of warts which disappeared (also presumably benign) are not appreciably different. Malignant warts, on the other hand, seem to appear some- what later than the benign, though the number of observations requires to be amplified to make sure of this difference.

t a l i I I M Y OF \s APPZARANCE.

CHART 2. TIME OF APPEARANCE OF WARTS A ~ E R T ~ R I N G The cxpressions “below

10”” and “above loo” refer t o a method of measuring growth rate described on p. 806. The chart shows, for instance, 28 slow-growing and 20 fast-growing warts appearing betweeu one hundred and one hundred and fifty days after tarring was begun.

Here the warts are divided into slow-growing and fast-growing.

It might be thought that warts which grow fast would appear sooner than slow-growing warts; this, however, is not the case, as shown in Chart 2, where it is seen that the slow-growing warts appear quite as early as the fast, indeed, on the whole, rather earlier. This is not an unexpected finding, since, as will be shown later, fast-growing warts are malignant, and already we have seen that malignant warts tend to appear later than benign.

2. Size of Warts when First Seean: Since the mice were examined three times a week, the size of the warts at their first appearance was approximately recorded. This information is given in Chart 3. This

804 J. C. MOTTRAM

chart shows that the majority of warts, when first seen, have a super- ficial area of from 1 to 3 sq. mm. This is remarkable when one con- siders that it would be easy to see warts or localised raised areas of skin, of a very much smaller size, say 0.25 sq. mm. It cannot be con- cluded, however, that the comparatively large size of the warts at first appearance is due to the warty change having taken place over a corresponding large area of the epidermis: the explanation may well be that local growth of the epidermis at first compresses the surround- ing tissues ; but that after a time a sudden dimpling out takes place, and thus a wart of about 2 sq. cm. suddenly appears.

3. Charncter of Warts When First Seen: The majority of warts, when first seen, are sessile. Within a few days many of these sessile warts become pedunculated, and, since two or three days may elapse

CHART 3. CLABSIFICATION OF 201 WART6 ACCORDING TO THEIR SIZE WHEN FIRST SEEN

Twenty warts, for instance, when first seen, were less than 1 sq. mm. in area, 79 warts were between 1 and 2 sq. mm., 54 between 2 and 3 sq. mm., ete.

between the first appearance of a wart and its observation, it follows that Borne warts when first seen were pedunculated. Of 199 warts, 157 were sessile, and 42 were pedunculated when first seen. The vast majority, 136, had a smooth surface. In 14 cases the surface was rough or scaly.

Ulceration commonly occurs in these tar warts and may begin very early, leading to an open sore or to an area covered by a crust of dried serum or blood. Thirty-eight warts were ulcerated when first seen; 27 of these had crusts and 11 were open sores. It will be seen later that ulceration is a reliable sign of malignancy, so that the very early ap- pearance of ulceration suggests that some warts are malignant from the start, as is, of course, well known. Warts have been examined histologically when first seen, and found to be malignant; also, they have been autografted and given rise to epitheliomas.

Many warts grow into horns, and in 5 cases the warts were horny

CORRELATION BETWEEN MALIGNANCY AND RATE O F GROWTH 805

when first seen. A few warts develop into cauliflower-like structures, and in 4 instances were of this nature when first seen.

This was observed in 'two cases, in both of which the cyst broke at the surface and grew into a wart, Fig. 1 is an example of a subepithelial cyst.

4. Subsequent Characters of Warts: In many cases, warts which start as sessile subsequently become pednnculated and later still may assume a cauliflower form. When epidermal cells throw off large num- bers of daughter cells outwardly to form keratin, then the wart grows into a horn, which itself conceals the contours of the living cells beneath, making it difficult to measure the warts for the purpose of obtaining growth rates (see also p. 809). More commonly ulceration supervenes, and the wart becomes a sore or an area covered by a crust of dried

Warts rarely begin as subepithelial cysts.

Flu. 1. SUBEPITHELIAL CYST FOUND ON REMOVING A SMALL ULCERATED WART, NOT BHOWN IN THE PHOTOQRAPII AS I T WAS A FEW FIELDS TO THE LEFT

Compare with subsoquent figures, as, for example, Fig. 10.

serum or blood. Thus the regular structure of the wart is lost, and also, as will be seen later, the regularity of its growth rate. Lastly, growth of the epidermal cells may extend deeply or laterally, or both, beneath the surrounding skin ; this lateral and deep extension is, how- ever, impossible to distinguish during life from inflammatory reactions which occur especially in ulcerated warts. Lateral and deep extensions diagnosed during life are not, therefore, the completely reliable signs of malignancy which some observers have assumed. Of 18 cases in which such extension was observed, in no less than 5 were the warts found to be benign and the reaction inflammatory,

The association of malignancy with ulceration is dealt with later. The other characters are summarised in Table I. This table shows that, whereas about half of the warts which remained sessile were

806 J. C. MOTTRAM

malignant, only two pedunculated warts were malignant. In one of the two malignant pedunculated warts (XXXIII.ll) autografting &cl not confirm the histological finding, since epithelial cysts instead of epitheliomas grew at the sites of inoculation. I n the other case- (XXI.2) the \;;art was described as “ slightIy pedunculated,” and in

CHAFLT 4. LENGTH ON’ LIFE OP WARTS WHIOH DISAPPEAIIED Twelve warts lived for twenty to forty days, 9 from forty to ahty days, eto.

this case two epitheliomas resulted from autografting. The number of warts which disappeared is high in the pedunculated, and low in the sessile group, indicating again that pedunculated warts are for the most part benign, since warts which disappear are probably not malignant. Horny warts, like pedunculated warts, are not prone to be malignant.

TABLE I

Malignant Benign Disappeared

Warts which became pedunculated . . . . . . . 2 (5%) 20 (49%) 19 ( 4 6 T Warts which remained sessile . . . . . . . . . . . 43 (47% ) 36 (39%) 13 (14%) Warts which became horny . . . . . . . . . . . . . 2 (7%) 19 (68%) 7 (25%)

5. Life of Warts: The length of life of warts cannot be stated, since all malignant and most benign warts survive the mouse. Only in the case of warts which disappear can the life-period be given. This varied from four to one hundred and ninety days for 42 warts ; the age distri- bution is given in Chart 4.

6. Growth Rate of Warts: The height of the warts was found to vary very widely, according as to whether or not they grew much or little horny material or became crusted or ulcerated. Since portions of horn or crust frequently dropped off, height was not employed in estimating growth, and reliance was placed on the superficial area obtained by multiplying together major and minor diameters. Diagrams were made by plotting these areas against time. Samples of slow, medium, and fast-growing warts are given in Chart 5. In order to obtain a single figure to express rates of growth, straight lines were ruled fol- lowing as near as possible the irregularities of growth of the warts, and the angles between these lines and the base lines were ascertained.

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 807

These angles were used to express growth rates. All warts growing at an angle of less than 3" were classified together. If desired the angles can be converted into an increase of sq. mm. in ten days by multiplying the tangent by ten.

It is clear that growth probably does not take place in this

:i ia 1 ' -

linear

CHART 5. SERIES OF WARTS FROM THE VERY FAST-GROWINQ XLI. 11, HAVINQ A RATE OF 7 3 O TO A VERY SLOW-GROWINQ WART, XX. 2

Note: I n all the charts dealing with the growth of warts, there is given, to the left, a vertical scale for areas in sq. mm.; upon the base line of each wart, periods of twenty-five days are shown. Upon the base line are drawings of the warts i n elevation to the lateral scale but in inm. instead of 89. mm. Notes along the base line describe the warts : CR. = crusted, ULC. = ulcerated, 8 = lateral exten- sion. The warts are named after the mouse in Roman numerals, and accordiilg to the order of their appearance, in Arabic numerals: XX. 2, for example, means the second wart appearing in mouse No. 20. "Wart = B" indicates benign, and " Wart = E" an epithelionia. Whether or not the wart was autografted is also indi- cated, and the histological result of each inoculation i e shown: E indicates a n epithelioma, EC an epithelial cyst, N no growth, and I an infective nodule. Lastly a straight line is drawn, approxiniately following the general trend of the growth, and the angle made between this and the base line is given in degrees.

Dots indicate the readings made three times a week.

The histological diagnosis is also given.

manner. Indeed, some rapidly growing warts show a growth curve upwardly concave ; examples are given in Chart 6.

There is a type of growth, sometimes difficult to distinguish from the upwardly concave type, in which a sudden instead of a gradual increase in growth rate takes place. Examples are shown in Chart 7.

CHART 6. EXAMPLES OF WARTS HAVING A GROWTH CURVE UPWARDLY CONUAVE

There is also to be seen the effect of ulceration in causing irregular growth and even ternporary shrinkage in size. For explanation of details, see Note on Chart 5.

CHART 7. WARTS HAVINQ AT FIRST A 8 W W QROWTH RATE WHICH LATER CHANGED SUDDENLY To A RAPID RATE

It is to be noted that all these warts were malignant aud all finally ahowed ulceration. Wart XLIII. 3 disappeared for a time. See Note on Chart 5.

CHART 8. WARTS WHICH DISAPPEARED In many cases disappearance was preceded by dropping off of most of the wart. See

Note on Chart 5.

808

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 809

It will be seen later that in the vast majority of cases, these warts are malignant.

So far, examples of progressive growth have been dealt with. It sometimes happens that growth is irregular and warts have been observed to grow smaller, and to disappear completely. Examples of warts which disappeared are given in Chart 8.

Irregular growth is also to be especially observed in warts which ulcerate, When, as usually happens, the center of the wart ulcerates, the sides fall in, and the superficial area of the wart, for a time, de- creases. This also happens, but to a less extent, when ulceration be- gins at the side of a wart. Examples are given in Chart 9.

CHART 9. WARTS WHICH ULCERATED AND AS A RESULT PRESENTED IRREQULAR GROWTH See also Chart 6 ; also Note 011 Chart 5.

Drying up or dry necrosis of a wart also rarely gives rise to irregu- larity in growth ; an example is given in Chart 9. Both this and ulcera- tion commonly cause the dropping off of a portion of a wart, or even the whole of it. On the ulcerated surface of warts, crusts of inspissated serum or blood collect and give rise to irregularities of measurement, since, from time to time, such crusts fall off. Care has been taken, as far as possible, t o measure the live wart beneath. The prodaction of much keratin to form horns also leads to irregularities in growth, since the growth of a horn produced by epithelial cells, rather than the growth of the cells, is likely to be measured.

On the whole these irregularities in growth are uncommon and occur late in the life history; indeed, the warts usually show growth which is remarkably regular and progressive. This is one of the outstanding features which this investigation has brought out, namely, that warts from their start have a characteristic growth rate which is maintained

810 J. C. MOTTRAM

over long periods of time, until ulceration or some other disturbing factor supervenes, and by means of which they can be readily classified.

7. CorreZatim of Growth Rccte With ~ ~ i g ~ ~ c ~ : It has been already mentioned that a histological diagnosis, independent of any knowledge of the life history, was made of every wart. The usual criteria of malignancy were used, and since the slides were all examined by a single observer, the varying opinion with regard to this controversial subject was ruled out. Furthermore, in many cases the warts were also tested by autografting, and it will be seen later that the two tests

BENIGN.

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Q ? ANGLE: OP S R O W i k /U =RdZ#. CHART 10. GROWTH RATES OF BENIQN AND MALIQNANT WABTB

Fourteen benign warts had a growth rate of between 10 and 20 degrees, and 14 malignant warts of between 20 and 30 degrees, etc. The results of autografting are alao shown: E= epithelioma; EC = epithelial cyst; N = negative on inoculation; I = inflammatory nodule. For instance, of the 4 benign warts having a growth rate of between 30 and 40 degrees, 2 were autografted and gave rise, one to an epithelioma and the other to an epithelial Cyst.

almost always led to the same diagnosis. It follows that the histological test was reliable in the vast majority of cases for distinguishing malignant from benign warts.

Histologically the warts were divided into 67 benign and 47 mnlig- nant. The growth rates of these warts are given in Chart 10. Here is to be seen a remarkable difference: whereas the majority of the benign warts, 43 out of 67, have a growth rate of under lo", most of the malignant warts, 40 out of 47, have a growth rate above 10". This is of such high importance that it is necessary to look closely into it. The chart also gives the results of autografting; on the whole, the

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 811

grafts confirm the histological diagnosis of the warts. Among the benign warts, oiily one gave rise to an epithelioma.

Let us deal first with the seven exceptional malignant warts having growth rates of less than 10". In two cases the grafts confirmed the diagnosis. Now there is much evidence, elsewhere and in this paper, that some warts start as benign and become malignant later, start at a low rate of growth and later become fast-growing (see examples in Chart 7) . It may well be, therefore, that these slow-growing malignant warts were removed just before they showed a change to a rapid growth

CHART 11. GROFTH OF SEVEN MALIQNANT WARTS OF Low GROWTH RATE See X o t e on (;*hart 5.

rate, and that, had they been left, they would subsequently have shown such a change. According to the diagrams (see Chart 11), in the case of XLI.5 and XXXIII. l l a rapid rate of growth seems to be just beginning shortly before the warts were removed, and this is true to a less extent of XXXIV.3. Further, in six of these warts ulceration occurred; now ulceration, as has been seen, greatly lowers the rate of growth of warts, so that this also may account for these exceptionally low rates. In wart XXXIII. l l there was no ulceration, but it shows an upward tendency of growth at the end. Further, since on auto- grafting it gave rise to an epithelial cyst and not to an epithelioma, it might be argued that the histological diagnosis was perhaps at fault; but since only half warts were used for autografting, and since there is evidence that when a benign wart becomes malignant not all of it changes, it follows that there is a good chance that while the half used for histologic study exhibited malignancy, the other half was benign and therefore gave rise to an epithelial cyst. This question will be dealt with at greater length subsequently. It is concluded, therefore, that these seven warts do not invalidate the rule that malignant warts grow fast.

Dealing now with the benign warts, can it be said that they are slow- growing? There were ten exceptional warts growing at over 200. If malignancy begins locally in a benign wart, then a diagnosis based

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CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 813

on sections only through the middle of the wart, as has been used here, is not reliable in excluding malignancy. In the case of one of these exceptional warts, XLV.1, the half used for grafting was evidently malignant since epitheliomas occurred at the site of inoculation, though the other half appeared to be histologically benign. In order, there- fore, to make the diagnosis more certain, serial sections of all these nine warts were made (excluding XLV.1). The growth rates are shown in Chart 12. The following statements can now be made in respect to these warts :

Wart I V . l (Chart 12), growth rate 3l0, grew into a large horn 8.5 mm. high and having a pedunculated base. Measurements, therefore, were not of the growing cells but of the keratin produced. Serial sections showed no malignancy, but a thick mass of keratin growing from a thin layer of buried epithelial cells. The wart was not in- oculated.

Wart VI.8 (Chart 12) , growth rate 20", grew into a long, thin horn with a wide base. Serial sections showed no malignancy and confirmed the horny structure. This wart was not autografted.

Wart XIX.2 (Chart 12) , growth rate 24", remained sessile and became ulcerated, strongly suggesting a malignant wart. Serial sections showed deflnite malignancy over an area not seen in the original section.

War t XXXVIII.4 (Chart 6), growth rate 39", was also ulcerated, and presented lateral extension under the skin (8.9. in chart), strongly suggesting a malignant wart. Serial sections showed no malignancy. Autografting gave rise to an epithelial cyst, but this was not a simple cyst lined by a thin layer of epithelial cells, but had thickened walls which, when this question is later dealt with, will be seen to be semi-malignant, inter- mediate between a benign cyst and definite epithelioma.

Wart XXXIX.2 (Chart 12), growth rate 28", was an ulcerated wart suggesting malignancy. The wart was not auto- grafted.

Wart XLIII.6 (Chart 12) , growth rate 25", grew into a horn which, on about the fiftieth day, dropped off and then suddenly decreased in size, showing that one had meas- ured the growth of the horn rather than the growth of living cells. Serial sections showed no malignancy. It was not autografted.

Wart XLI . l l (Chart 5), growth rate 73", grew into a very large horn, 1 6 mm. high. Serial sections showed no malignancy, and three autografts were all negative.

Wart XLIV.9 (Chart 12), growth rate 30°, was not a horny wart but one which ulcerated and bled, and was therefore likely to be malignant. Serial sections show un- doubted malignancy. I n the original section there was a large epithelial cyst under the wart which interfered with a proper view of its base. The wart was not autografted.

The growth was generally very irregular and unlike that of a malignant wart. Serial sections showed no malignancy. It was not autografted.

To summarise, 3 of the 9 warts, on serial section, showed local malignancy; five were horny warts in which growth of keratin was measured and not growth of epithelial cells; and in the last case an epithelial cyst of doubtful malignancy resulted from inoculation.

Much space has been devoted to the consideration of exceptions, but not wastefully, since they have helped to prove the rule that malignancy is closely associated with a fast growth rate, and vice versa. I n further proof of this, may be considered the growth rate of warts which disap- peared: 38 such warts were observed, and of these, 33 had very low rates, under 3" ; the other 5 had growth rates of 5", 6", 8", 25" and 27".

The top dropped off, leaving a pedunculated horn.

Serial sections showed undoubted malignancy.

Wart LI.3 (Chart 12) , growth rate 26", grew into a large antler-like horn.

814 J. C. MOTTRAM

Diagrams of the two exceptional marts growing at 25" and 27", warts JWII.2 and XXXVI.l, are given in Chart 8. The first was a peduncu- lated wart which dropped off about the thirty-fifth day, leaving an ulcer that healed. Even if the mass of the wart had been malignant, it seems quite possible that all the malignant cells could have fallen off when necrosis of the wart occurred. In the other exception, the wart was likewise pedunculated, likewise dropped off, and likewise left a tiny ulcer that healed in a few days. The consideration of these warts which disappeared and were therefore benign, thus confirms the rule that benign warts are slow-growing. Nevertheless, there is no sharp and definable demarcation of warts into slow-growing benign and fast- growing malignant. There are in this series (see Chart 10) 15 benign warts and 11 malignant warts growing between 10" and 20". Four of the benign warts were tested by autografts (see Chart 10) and gave

T A B L ~ I1

Original New rate in Day of rate in Histology Result of

Mouse Wart degrees alteration degrees of wart autograft

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1s

I I XVI XVIII xx XXIV xxv XXXI XXXIII XXXVIII XLII XLIII XLIV LI LIII

3 4 5 1 1 3 1 1 11 1 2 3 4 3 3

5 18 3 4

> 3 > 3 > 3 > 3 18

> 3 7

> 3 14

> 3 > 3

65 40 40 32 77 17 16 25 10 94 54 06 31 123 74

57 63 33 74 19 57 57 17 63 28 42 24 04 26 20

E E E E E E E E E E E E E B E

E E E E

not inoculated not inoculated not inoculated

E E N E E E

not inoculated not inoculated

(E = epithelioma. B = benign. N = negative.)

rise to four epithelial cysts and one negative result; and nine of the malignant warts to seven epitheliomas and two negatives. Evidently, therefore, the histological diagnosis of the warts was reliable. There is, therefore, without doubt a gradation between these two kipds of warts. Further evidence of this will be found subaequently.

Continuing the consideration of growth rate, it has been shown that warts sometimes show a sudden increase of growth rate. Examples have been illustrated in Chart 7. Fifteen of these warts were observed, and information with regard to them is given in Table 11. It is seen that, with a single exception, LI.3, all the warts were malignant and in 9 cases this was confirmed by autografts. The exceptional wart, LI.3, has been described previously (see Chart 12). It was a horny wart, and unusual in many respects. It is evident from this series that a fast growth rate occurring in a wart previously slow-growing is asso- ciat ed with malignancy,

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 815

The consideration of ulceration has been raised on several occa- sions, and already it has been shown that, ulceration causes irregu- larities in growth rate. The association of ulceration with growth rate and with benign and malignant conditions will now be dealt with.

On reference to Chart 13, dealing with growth rate, it is seen that the distribution of growth rate among ulcerated warts is quite different than among t.hose not ulcerated : there is an enormous preponderance of slow-growing warts among the non-ulcerated.

CHART 13. SHOWING THE ASSOCIATION OF ULCERATION WITH GROWTH RATE Eighty-three out of 116 non-ulcerated warts had a growth rate between 0" and lo",

whereas 9 out of 43 ulcerated warts grew within the same rate limits.

The information with regard to the awociatioii of ulceration with The observation of bleeding is also malignancy is given in Table 111.

TABLE I11

Ulcerated Not ulcerated Bleeding Not bleeding Total

Benign 13 67 9 71 80 Malignant 33 19 11 41 58

dealt with here, since bleeding indicates severe ulceration. It is seen that malignant warts present ulceration very much more often than do benign ones. We therefore have ulceration closely associated with rapid growth on the one hand, and with malignancy on the other, with however much overlapping of these characters.

The lateral extension of warts heneath surrounding normal epi- dermis, and the deep extension of warts, are often to be observed during life. Generally histologic examination of these warts shows them to be malignant, but by 110 means always, since the lateral or deep

816 J. C. MOTTBAM

extension is often found to be an inflammatory reaction of polymorphs around a benign wart. As signs of malignancy, therefore, these fea- tures are not reliable.

FIG. 2. AUTOORAFT REMOVED ON THE 2 6 ~ ~ DAY, SHOWING A SEMI-MALIGNANT CYST HAVING AT ONE END FINGERS OF EPITHELIAL CELLS EXTENDING INTO THE REACTION TISSUE

Inset S h O \ w the region covered by the photograph.

8. Consideratimi of Autogrufts: It will be convenient here to con- sider in detail the diagnosis of malignancy in respect to these tar warts. Tt has already been seen that, to exclude malignancy, serial sections are required. In several cases, warts diagnosed benign on single sections were shown to he malignant when serial sections were studied, In many warts found to be malignant, some parts were dis- tinctly different in structure and decidedly benign. There is no need to illustrate such findings, which are well recognized. It may there- fore be concluded that malignancy often begins locally in a benign wart.

It may sometimes happen that a new malignant wart starts so close to an old established benign wart that its separate existence is over- looked, and a wrong conclusion drawn that the benign wart has become malignant. There are, however, good reasons why this must be a very uncommon occurrence. In any single mouse, only one or two malignant warts usually occur. In this series of mice the maximum number was five, and in only five cases did more than two malignant warts appear. The chance, therefore, of a malignant wart appearing so close to a benign wart as to be unrecognized as a separate lesion must be extremely small.

On the other hand, for beiiigii warts to become malignant is a rela- tively common occurrence. Nevertheless, there is still the possibility that when such an event takes place, it is normal epithelial cells in the immediate vicinity of the benign wart which undergo malignant change,

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 817

and not the epithelial cells of the benign wart. This cannot be excluded, and when examining serial sections of benign warts, looking for a local malignant change, one certainly does find, sometimes, such a change

FIG. 3. AUTOGRAFT REMOVED ON THE 44TH DAY Note the generally thiu epithelial lining, and outside a thick reaetiou tissue invnded by

islands of epithelial cells.

FIG. 4. AUToGRAFT REMOVED ON THE 53D DAY Note that the epithelial w:ills of tlic cyst are thick aiid villous, and that fingers of epi-

thelial cells extend into the rrnvtion tissue.

occurring at the edge of the b e n i p wart, so that it is impossible to say whether it began in the wart or 111 the neighbouring normal epidermis.

In spite of this, it follows that if malignancy is to be tested by autografting, then, to exclude malignancy, the whole wart must be

818 J. C. MOTTRAM

inoculated. It must be divided into a number of pieces of convenient size and every piece must be autografted. In this investigation only three or four pieces taken from half the wart were inoculated, yet in spite of this, in a few cases, some of the pieces from the same wart gave rise to epitheliomas and some to non-malignant cysts. For instance, four pieces of wart IX.l, were inoculated; one piece gave rise to an epithelioma, one to an epithelial cyst, and two were negative. Further examples of such occurrences will be given later. In the meanwhile it may be said that four different results follow autografting: (1) no growth, (2) a local inflammatory nodule, (3) an epithelioma, (4) an epithelial cyst filled with keratin or cell debris and lined by a thin layer of epithelial cells having a surrounding connective-tissue capsule,

PIQ. 6. AUTOORAFT REMOVED ON THE 36Ta DAY, SHOWING A MALIGNAET CYST LINED BY VEBY THICK WALLS OF EPITHELIAL CELLS EXTENDING DEEPLY INTO THE SUEROUNDINQ RE- ACTION TISSUE

These epithelial cysts are evidence of non-malignancy and exactly resemble the cysts which follow the inoculation of embryo skin. Un- fortunately for the use of autografting as a test for malignancy, the demarcation between epithelial cysts and epitheliomas is not to be defined. The wall of the cyst may be thickened (Fig. 2) and groups of epithelial cells may extend into the surrounding connective tissue. Indeed, just as with the warts themselves, we are confronted with many semi-malignant (for want of a better word) epithelial cysts. Alto- gether eight examples of such semi-malignant cysts were encountered. Photographs of seven of these (Figs. 2-8) and of three innocent cysts (Figs. 9-11) are given. Here again we have evidence not of two dis- continuous series of warts, benign and malignant, but of a single gratlu- ated series.

FIQ. 6. AUTOGRAFT RENOVED ON THE 21ST DAY, SHOWINQ A SEMI-MALIQNANT CYST WITH

THELIAL CELLS PREsENT I N THE REACTION TISSUE, WHICH IS EXTENBIVE AND CONTAINS ABUNDANT LYMPHOCYTES

A WALL OF EPITHELIAL CELLS GREATLY THICKENED LOCALLY, A N D WITH ISLANDS OF EPI-

Inset gives the rc.gioii covered by the photograph.

FIQ. 7. AUTOGRAFT R E M O V ~ ON THE 57TH DAY, SHOWINQ A CYST FILLED WITH CELL DBBKIS AND HAVINQ WALLS OF XPITHELIAL CELLS ABOUT 20 CELLS THICK, AND A TRICKER CELL Mnss AT THE LOWER END

Inset shows the size of the eyst, and the regiou covered by the photograph.

The reaction tissue consists of a definite fibrous connective-tissue capsule.

819

820 J. C. MOTTRAM

The value of autografts as a test for malignancy is further reduced by the possibility of infection. Infection at the site of inoculation, if common, would greatly lower the value of negative results. This can

FIO. 8. AUTOQUFT REMOVED ON THE 47mi DAY This consisted of four small rysts (see inset), lined by a layer of epithelial cells about 12

cells thick.

FIO. 9. AIJTOQBAFT REMOVED ON THE 21ST DAY Note the very thin lining of epithelial cells, except at one place where the lining is about

a dozen cells thick.

be largely avoided, however, by adopting the following technique. The wart and surrounding skin are swabbed with ether; crusts are then loosened and removed; sterile water is applied, and the wart is next removed and placed, inner surface downwards, on a glass plate. All surrounding skin is then cut off, using two tenotomy knives like scissors.

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 821

The wart is next cut in half with the knives (AB Fig. 12). The two halves, C and D, are now turned cut surface downwards and the deep parts of the wart separated by cuts EF and GH approximately 1 mm,

FIG. 10. AUTOGRAFT REMOVED ON THE 21ST DAY Note the very thin lining of epithelial celle and the connective-tissue capsule; at one place

the epithelial cells are about six deep.

E’IQ. 11. AUTOGRAFT REMOVED ON THE 37TH DAY, 8HOWINQ AN EPITHELIAL LINING ABOUT BIX TO TEN CELLS THICK, BURROUNDED BY AN ABUNDANT REACTION TISSUE

thick; the superficial parts I and J are discarded, the deep parts K and L are laid base downwards, and cross-cut with the knives with cuts XX, YY approximately 1 mm. apart. Thus there are obtained for

822 J. C. MOTTEAM

autografting, pieces of the deep layers of the wart of about one cubic millimeter in volume. If this technique is adopted, infection at the site of inoculation is rarely seen; this gives considerable value to negative results and justifies their further consideration. I n order to exclude malignancy, all the pieces thus obtained should be inoculated. In these experiments, as has been stated, only half the wart was used, and no matter how large the wart, only four pieces were inoculated,

When the skin taken from early embryos of rats is inoculated into near relatives, using a very inbred strain of rats, epithelial cysts grow at the sites of inoculation (see Chart 14). Figs. 13, 14, and 15 illus-

X

A

FIQ. 12. TECHNIQUE 03’ CUTTINQ AUTOQRAFTS (SEE TEXT)

trate these cysts. If late embryos are used, only a few small epithelial cysts develop; if adult skin is used, cysts are not found. Thus we see that the ability to grow into cysts depends upon the power of the epi- thelial cells to reproduce: rapidly growing cells from early embryos produce cysts; less rapidly growing cells from late embryos only small cysts ; and slowly growing cells from adult skins give negative results. If this applies to warts, then a negative finding indicates a very low growth capacity, and an epithelial cyst a higher growth capacity, com- parable with that of the skin of embryos. Next in this series come the semi-malignant cysts, already described, next the differentiated epi- theliomas, and finally the undifferentiated, rapidly growing, non-kera- tinising spindle- or round-celled epitheliomas. Once again, therefore, these warts can be arranged in a continuous series according to their behaviour on autografting; and further, jTst as we find some warts histologically exhibiting in some parts malignancy and in others inno- cence, so do we find some inoculations giving rise to epitheliomas and others from the 8ame wart giving rise to negative results or to epi- thelial cysts. These findings, which are given in Table IV, demon- strate that warts sometimes show malignancy at one place, and capacity to grow only a cyst at another (A in the table) ; also that often not even a cyst results (C in the table). Therefore, malignant warts are not always malignant throughout, as is, of course, to be expected if benign warts become malignant locally. Not only this, but it seems also that benign warts have not the same growth capacity throughout, since, on inoculation, some pieces will give rise to epithelial cysts while others

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 823

from the same wart produce negative results (B in the table). If the occurrence of semi-malignant cysts had also been taken into account, still more evidence of the complexity of these warts would have been shown.

It is to be noted that infection, I in the table, has spoiled the results in a few cases.

This completes the descriptions of the life history of the warts as regards features having a bearing on growth-rate. They also show that the intensive study of warts along these lines is likely to give much information about both malignant and benign warts.

o o o o o o o o o m 0 O O *0000000000 0

b s 2. 1 a- 1 o~ooooooooooo 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 CEART 14. GROWTH OF EPITHELIAL CYSTS IN FOUR RATS FOLLOWINQ INOCULATION OF SKIN

OF EARLY EMBRYOS Measurements wcre made weekly for thirteen weeks, the last being made in the twentieth

week.

C. DISCUSSION

It has been concluded that the painting of mice with tar gives rise to a series of warts varying from benign at one end to malignant a t the other, with semi-malignant warts in between. The consideration of autografts also shows that we are not dealing with the overlapping of two kinds of warts, malignant and benign, since the presence of semi-malignant epithelial cysts proves the presence of intermediate forms; a number of other considerations favour this conclusion. It is evident, therefore, that from the point of view of these warts (and there is no reason to think that new growths and turnours in general are essentially different) there are in the cancer problem two sets of oc- currences to bc accounted for. In the first place we must account for the occurrence of turnours, in this case of warts, forming a single series from benign to malignant; in the second place, within this series we require to know why some tumours are malignant, some semi-malignant, and some benign. I propose to consider these two subsidiary problems separately.

824 J. C. MOTTRAM

Mouse Wnrt Inoeulationa

IX XVI

A . Combinationa of Bpithelioms and Epithelial Cyets 1 4

E. EC. N. N. E. E. E. EC.

XXXIII 11 E. EC. N. XLI 4 E. EC. EC. EC. XLIV 4 E. E. EC. XLIV 6 E. EC. N. LIII 2 E. E. EC.

B. Combinations of Epithelial Cysts and Negative Results I 2 EC. N. I 5 EC. N. XIV 1 EC. I.* XIV 2 EC. N. XIX 1 EC. N. N. N. XXII 1 EC. EC. N. N. xxx 3 EC. N. XXXIII XXXVIII XXXIX XXXIX XL XLIII XLVIII

C. I XIV XV XVI XVIII XXXIII XXXVI XLI XLII XLVII L L

11A EC. N. N. 4 EC. EC. I.*

EC. EC. N. N. EC. N. N, EC. EC. N. EU. EC. N. EC. N. N.

Combinations of Epitheliomaa and Negative Reeults 4 E. I." 1 E. I.* 3 E. E. N. 5 E. E. N. 1 E. I.*

10 E. E. N. 2 E. N. 9 E. N. I.* 2 E. N. N. 3 " E. N. N. 2 E. E. N. 4 E. E. N.

* I. = Infection.

( A ) To account for the occurrerice of a single series of tumours var!yin,g from benign to malignunt : Our observations do not throw any new light upon this problem. They do, however, stress once again that the cancer problem is unlikely to be solved by the consideration alone of malignant tumours, since there is very much evidence that these are only part of a series of tumours varying continuously from benign through semi-malignant to malignant. Yet, in spite of this, we And many propagandists, for instance, of the virus hypothesis, giving long dissertations on the cancer problem without a single reference to benign turnours. Those who favour the mutation hypothesis do not, of course, fail in this respect, since that hypothesis sets out to account for all new growths.

The evidence that both benign and malignant warts vary locally and

FIU. 13, CY6T RENOVED 279 DAYS AFTER PHE INOCULATION INTO A RAT OF AN EMULSION OF TEE SKIN OF RAT EMBRYOS

Tho cyst is filled with keratin and hairs, lined by a t h h layer of epithelial cells, and bounded by a fibrous connective-tissue capsule. Inset shows the region photographed.

FIG. 14. CYST REMOVED 241 DAYB AFTER THE INOCULATION INN A RAT OF AN EMULSION OF

The cyst is filled with cell dbbris, lined by a very thin layer of epithelial cells, and bounded THE SKIN OF EARLY RAT EMBRYOS

by a slight fibrous c*omiertivn-tissue rapsule. Inset ShOWS the region photographed.

825

826 J. G. MOTTRAM

that inoculations from the same wart often give rise to epitheliomas, to semi-malignant cysts, to benign cysts, and to negative results must likewise be taken into account. It appears that the tar produces a whole series of cells of varying growth capacity, and that the most actively growing finally obtain the upper hand.

( B ) To account for the occurrence o f both bemnign and maligmamt warts with& a single series of warts: It does not matter how warts are produced, be it by virus or mutation, it will still be necessary to con- sider why one cell is cancerous, invades its host and kills it, while an- other cell remains localised and gives rise only to a localised tumour. The present investigation brings out a fact which may be of much

FIQ. 16. CYST REMOVED 64 DAYS AFTER THE INOCULATION INVO A RAT OF AN Ea6umroN OF

The wall of the cyst is shown where it has a considerable thickness of epithelium, of hair follicles, and o f reaction tissue invaded locally by lymphocytes (arrows). Inset uhows the region photographed.

THE SKIN OF RAT EMBRYOS

importance in this connection: it has been seen that high growth rate is characteristic of malignant warts, and low growth rate of benign warts. The question, therefore, arises: could high growth rate be in itself the sole cause of malignancy? If this be so, then it is necessary to take into account the host as well as the cancer cell, since malignancy is an observed result of the interplay between cell and host, and to look closely into the reactions occurring around autografts.

When epithelial cells are inoculated under the skin, a reaction tissue is always found consisting of monocytes, fibroblasts, and capillaries, which later develops into fibrous tissue. When cells from a slow- growing benign wart or from embryo skin are inoculated, they become surrounded by this reaction tissue, and by the time the epithelial cells have slowly grown into an epithelial cyst, the reaction tissue has become

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 827

a fibrous tissue capsule separating the cyst from the surrounding tis- sues. If the inoculated cells grow very slowly, the fibrous capsule may be present before they have had time to form even a very small cyst, and then only a group of degenerated epithelial cells embedded in fibrous tissue (Figs. 16 and 17) is found, in which case the inoculation is said to be negative.

When, however, cells from a fast-growing wart are inoculated, exactly the same reaction tissue is found, but before this has had time to become fibrosed the epithelial cells have greatly increased in number, and have been forced, fo r want of space, into the reaction tissue. Fol- lowing this, fresh reaction tissue is formed, into which again the epi-

FIG. 16. AUTOORAFT REMOVED ON THE 41ST DAY, SHOWINO A FEW FLATTENED AND DEGENER- ATED EPITHELIAL CELLS EMBEDDED IN A RINQ OF FIBROUS CONNECTIVE TISSUE

thelial cells find their way before fibrosis has had time to develop. The epithelial cancer cells do not directly invade and destroy tissues, as, for instance, the layer of muscle in the skin, or liver tissue, but indirectly by means of the reaction tissue which their presence calls forth and which ever precedes them.

Lastly, in the semi-malignant epithelial cysts we have a nice balance between epithelial cell and host; it is touch and go whether a fibrous capsule will be established in time to check the cells, or whether they will grow a little too rapidly for this to be accomplished. Thus we find a controlled spread of cells into the reaction tissue.

This may appear to be no more than a speculation but it is also a short description of what is seen histologically when small pieces of warts are inoculated. It postulates that growth rate decides whether or not a wart is malignant or benign.

The reaction of the host towards inoculated cells also takes place in the dermis beneath the warts. There also is to be seen invasion of

828 J. C. MOTTRAM

the tissue with lymphocytes and other monocytes, with fibroblasts, and with new capillaries. In the case of benign warts, this develops into fibrous tissue, whereas in the malignant warts the epithelial cells grow- ing rapidly are budded off into this new tissue before fibrosis can supervene. I n many cancers it is only at the growing edge that this balance between host and cell is seen; within the mass of the tumour fibrous tissue has had time to develop and we find strings or islands of cancer cells embedded in well-formed fibrous tissue. In those rare cases where cancer is spontaneously cured, it appears that fibrosis, at first a loser, finally wins the race.

If it be true that the host always reacts similarly and that the growth rate of the epithelial cell alone decides whether or not a wart remains

FIQ. 17. AUTOQRART REbfOVED ON THE 30TH DAY The inoculation w a ~ classifled “negative,” but on section the knot of flbrous tissue WM

found to contain small islands of degencrated epithelial cells (arrowe in the photograph) em- bedded in reaction tissue largely flbrosed.

local or invades the host, then, while malignancy remains a clinical entity, pathologically; it loses its identity, and for the research worker the phenomenon of cancer becomes of much less importance than the study of tumours in general.

This thesis that malignancy and non-malignancy are determined by growth rate could be debated at length; but it may be a greater gain to allow pathologists to turn this over in their minds, unprejudiced. There is, however, one aspect which may perhaps be referred to with profit. I t has been suggested that the reaction tissue of an individual mouse is always the same towards all tumour cells, whether malign or benign. It does not follow that the reaction tissue will be the same if we compare one mowe with another. One mouse may be able to keep benign a wart of moderate growth rate, which in another mouse would

CORRELATION BETWEEN MALIGNANCY AND RATE OF GROWTH 829

exhibit malignancy. It may be that the varying hereditary incidence of cancer in different strains of mice and races of men, resides in the reaction tissue, in the resistance to invasion, and not in a varying disposition of the cells to become cancerous and invade.

Lastly, it may be asked why reference has not been made to our knowledge of tumours in man, and especially to the common knowledge that with few exceptions malignant tumours are characterized by rapid, and benign tumours by slow growth. Is it because this information is so easily come by, and so undisputed that its importance is over- looked? Do cells grow fast because they are malignant, or are they malignant because they grow fast 1 Which is the horse and which the cart 1

D. SUMMARY (1) A few warts appear within 100 days after the beginning of

tarring, the majority between 100 and 150 days, very few after 300 days. The time of appearance of benign warts and of warts which disappear is not different ; but malignant warts appear somewhat later. Fast-growing warts do not appear earlier than slow growing, as might be expected, but later, like malignant warts.

(2) Very few warts when first seen have superficial dimensions of less than 1 sq. mm. ; the vast majority are from 1 to 3 sq. mm. in size.

(3) Warts when first seen are for the most part sessile, a few are pedunculated. A small proportion of warts are ulcerated when first seen, and begin as sores; many of these are then malignant, Occa- sionally warts have other characters when first seen, such as being horny, but usually they have a smooth surface.

(4) Many warts a t first sessile later become pedunculated. Fur- ther, many changes subsequently take place : they may grow into horns, or become ulcerated, or extend deeply or laterally. Analysis of these characters showed that pedunculated warts are almost always benign, as are also horny warts; whereas warts which remain sessile are largely malignant. The lateral and deep extension of warts observed during life is not a completely reliable sign of malignancy, since infec- tive conditions lead to exactly similar signs on palpation.

(5) The length of life of warts which disappeared varied from four to one hundred and ninety days.

(6) The height of warts was found to be very variable, since horns and criists frequently dropped off ; superficial dimensions were there- fore relied upon for estimating rate of growth.

Warts varied much in growth rate: some presented no appreciable increase in size during months of observation, while others doubled their size within a few days. As a rule, growth was linear or with a slight upward concavity. Sometimes a sudden increase in growth rate was observed, and in all these cases the wart was then found to be malignant. In the early stages of the wart’s life, growth was progres- sive, but later such changes as ulceration interfered with growth and

$30 3. C. MOTTRAM

gave rise to irregularities in growth rate; indeed, ulceration often led temporarily to decrease in size. Apart from such irregularities, and especially in their early life, warts present a regular and characteristic growth rate.

(7) The vast majority of benign warts have a low growth rate, while malignant warts have a high rate. The exceptional warts, fast- growing benign and slow-growing malignant, have been considered in detail; and it is concluded that these exceptions do not invalidate this rule. In support of this, it was found that warts which disappear are slow-growing, and presumably these are benign warts.

In spite of this general rule, it is shown that the two sets of warts, fast-growing malignant and slow-growing benign, cannot be separated, but grade one into the other, semi-malignant warts of moderate growth rate forming the connecting link, and the whole forming a single series of warts. Ulceration was observed. among fast-growing warts ; slow- growing warts only very exceptionally become ulcerated. Further there is a close association between ulceration and malignancy.

(8) The study of autografts showed that many warts are only malignant locally, since both epitheliomas and simple epithelial cysts resulted from the inoculation of different fragments of the same wart; also that benign warts likewise vary from fragment to fragment. Auto- grafts also give rise to semi-malignant epithelial cysts and thus show that warts form a continuous series from malignant, through semi- malignant, to benign. The value of autografts as a test for malignancy is discussed and the conclusion reached that the whole wart must be divided in fragments and all the fragments inoculated in order to exclude malignancy. It was shown that the inoculation of embryo skin, in the case of rats, gives rise to a simple epithelial cyst similar in all respects to the cysts following the autografting of benign warts.

Under Discussion, the cancer problem is reviewed and the conclusion drawn that it embraces two subsidiary problems: the necessity to ac- count (1) for the occurrence of a single series of tumours varying from benign to malignant, and (2) the occurrence of what determines inno- cence and what malignancy, within this series.

As regards the first problem, the present findings do no more than stress once more the fact that in the cancer problem benign must be considered side by side with malignant tumours.

As regards the second problem, the theory is put forward that the growth rate of the tumour cell alone determines innocence or malig- nancy, and that the individual host responds to the presence of tumour cells, whether malignant or benign, always in the same way.

Nom: This research wm carried out under a grant made by the Trustees of the late T. C. Fitton. I am greatly indebted to Mr. ct. Burgb, the senior laboratory assistant of these laboratories, for his skilful help in this research.