A radioautographic analysis of cellular proliferation in dysplasia and carcinoma in situ of the uterine cervix

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proliferation in dysplasia and carcinoma in situ of the uterine cervix RALPH :M. RICHART, M.D.* New York, New York

scribes the methods used and the results obtained.

T R I T I u M is extremely effective as a label for high resolution radioautography and, when combined with thymidine, selectively labels nuclei which are synthesizing deoxyribonucleic acid (DNA), preparatory to mitosis. It is essentially stable in the nucleus after incorporation, and produces relatively little disturbance in the mitotic cycle, at least

Materials and methods

On the basis of cytologic and colpomicroscopic findings a group of patients was selected who had epithelial abnormalities ranging from mild dysplasia to carcinoma in situ; a second group of patients was selected with no known cervical disease. After a cytologic diagnosis was made and the degree of severity of the neoplastic process was assessed by colpomicroscopy, areas were selected for biopsy on the basis of their toluidine blue staining reaction. 1 These selected areas were sampled with a Kevorkian punch and the biopsies immediately placed in Puck's tissue culture medium and taken to the laboratory. In the laboratory, under sterile conditions, each biopsy was bisected longitudinally with a scalpel, one half saved for histologic analysis, and the other half placed in 2 mi. of medium containing 1 p.c of tritiated thymidine per milliliter. The biopsies were placed at 37° C. for one hour, after which they were washed, fixed in neutral buffered formalin, and prepared for sectioning. When the sections were made, care was taken to include the first cuts from the block. The slides were prepared for

over a short period of time.

As part of a program investigating dysplasia (atypical hyperplasia) and carcinoma in situ of the uterine cervix, an attempt was made to quantitate the proliferative activity of normal cervical epithelium, dysplasia, and carcinoma in situ by computing a labeling index for each condition. This paper de-

From the Departments of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeons, Coluntbia University and the Obstetrical and Gynecological Service (The Sloane Hospital) of the Presbyterian Hospital, New York City. Work Performed in the Department of Pathology of the Medical College of Virginia undt·r United States Public Health Service Grants C-9800, GM-KJ-13975, and RG-8165. *Assistant Professor of Pathology, College of Physicians and Surgeons, Columbia University and Director of the Pathology and Cytology Laboiatories of the Obstetrical and Gynecolof.(ical Division (The Sloane Hospital) of the Presbyterian Hospital.

radioautography by the use of fluid emulsion,

as described by Joftes,2 and the coated slides exposed for 30 days at 4° C. All sections

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Fig. 1. Radioautograph of moderate dysplasia ( +;-). Note predominance of labeled cells in lower layers although a few labeled cells can he seen in the upper third. (Hematoxylin. x240.)

were stained through the emulsion with Harris' hematoxylin. When the radioautographs were analyzed only those sections which were cut in a plane perpendicular to the plane of the epithelium were used. These well-oriented sections were then scanned and an area was chosen for counting which was felt to contain the highest proportion of labeled cells (including all layers of the epithelium) in the section. When the areas for counting had been selected, a labeling index (labeled nuclei/total number of nuclei) was calculated for the total thickness of the epithelium in that area. The epithelium was further subdivided into three regions; the single layer of cells adjacent to the basement membrane was termed

Table I. Six point severity scale used m assigning histologic diagnoses

-----

1+ 2+ 3+ 4+ 5+ 6+

Normal Minimal dysplasia Mild dysplasia Moderate dysplasia Severe dysplasia Carcinoma in situ

the basal layer and the remaining thickp.ess of epithelium was visually di~id~d into halves, the lower portion being designated the parabasal layer and the upper pOrtion the intermediate layer. A labeled cell . was arbitrarily chosen as being repreSented by a nucleus overlaid by at least three grains. After the slides had been evaluated for the number of labeled nuclei they were ~;:x~ amined histologically and, without reference to the radioautographic data, a diagnosis was assigned to each of the areas that had been counted, on the basis of a severity scale ranging from normal to carcinoma in situ (Table I ) . In general the criteria used in assigning the diagnoses were those described by McKay and associates. 3 • 4 Representative radioautographs are seen in Figs. 1 and 2~ Results

Table II lists the calculated labeling indices and their averages in per cent Jor each . of the 6 points on the severity se<1le. Similar indices were calculated in each ca8e for .~h of the 3 layers described above. In o.tcler to examine the relationship between the ·label-

Cellular proliferation in dysplasia and cancer

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Fig. 2. Radioautograph of carcinoma in situ ( 6+). Note presence of labeled cells throughout the full thickness of the epithelium including several at the surface. (Hematoxylin. x240.)

ing index and the severity, a regression analysis was performed with the logarithm of the labeling index as the dependent variable and the severity scale as the independent variable. This analysis revealed a strong linear relationship between the 2 parameters with no significant evidence of curvature. The slope of the line (Fig. 3) is given by B 0.1952 ± 0.0218 and the fitted line Y = 0.195X + 0.367. The correlation around the fitted line is given as R 2 = 94 per cent, that is, 94 per cent of the variability in the mitotic index is explained, on the average, by the severity index scale. A similar analysis was made for each of the 3 layers described above. The results are expressed graphically in Fig. 4. It will be noted that the slope of the line representing the basal layer is less steep than that of the parabasal layer and that the line representing the intermediate layer is the most steep. Figs. 3 and 4 are superimposed to form Fig. 5 and it will be seen from this figure that the increase in the slope of the labeling index, calculated for the total thickness of the epithelium, is largely

=

accounted for by the steepness of the slope of the line representing the intermediate layer. In Fig. 6 the per cent by which each of the three layers contributes to the total number of labeled nuclei is plotted against the

Table II. Labeling indices calculated* for the area of greatest proliferative activity in each specimen under the 6 categories of severity, in per cent

Average *IL =

]+

2+

3+

4+

5+

6+

2.7 4.5 4. 7 5.4 5.7

2.0 4.1 6.0 7.8 7.9

3.8 7.0 11.0 13.8 16.0 22.6 34.2

8.3 12.0 14.8 16.4 16.6 23 .0 35.3 :16.0

39.7 41.0 44.3 48 .0 49 .0 57 .0

4.6

5.6

3.7 5.3 7.0 7.5 8.3 8.5 10.0 12.0 16.0 17.0 18.0 10.3

15.2

20.3

46.5

Labeled cells Total cells

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2

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3 SEVERITY

4

5

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SCALE

Fig. 3. Graph of the relationship of the log of the labeling index and severity.

severity scale. There is relatively little change in the basal layer. The parabasal layer contributes less arid less, relatively, as car· cinoma in situ is approached and the intertnediate layer contributes more and more. At 6+ on the severity scale the parabasal and intermediate layers contribute essentially equal percentages and their labeling indices are approximately equal.

is least in carcinoma in situ. When carcinoma in situ is reached the calculated generation time is 11.3 hours, i.e., the full thickness of neoplastic epithelium is replaced approximately twice a day, or approximately 8 cells are produced each hour for each 100 cells that are proliferating. Because of the means by which the areas for examination were chosen all the figures represent the maximum rate of reproduction. If areas of carcinoma in situ do have a generation time of 12 hours this figure is quite remarkable. It is even more remarkable when the rate of reproduction of this neoplasm is compared to others that have been reported in the literature reached by similar methods and calculations. Johnson and co-workers6 • 7 have made estimates of the generation time of a number of human neoplasms and have reported the following figures, each representing a single case: glioblastoma multiforme, greater than one month; ovarian cystadenoma, 0.5 month: breast carcinoma, 3 months; leiomyoma of the spermatic cord, 5.5 months. In a separate publication Johnson and Bond" reported a breast fibroadenoma with a generation time

I

Comment

An estimate of the generation time can be made through the formula, given by Johnson and Bond, 5 which relates the generation time to the labeling index on the assumption that the period of DNA synthesis is equal to 6 hours. While the DNA synthesis period is not known for either the normal or neoplastic cervical epithelium, the application of this formula provides an interesting set of figures. Calculations utilizing the data presented in this paper indicate that the normal cervical epithelium is replaced every 5. 7 days. Since the relationship between the labeling index and the generation time is an inverse one, it can be seen from Fig. 2 that the generation time in cases of mild dysplasia is essentially the same as that for normal epithelium, and that it gradually decreases as the severity increases and

8

6 4

2

I

8

6 4

2

2

3 SEVERITY

4

5

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SCALE

Fig. 4. Graph of the relationship between the labeling index and the severity sc~ for each <:>f the three cellular layers chosen as described in the text.

Cellular proliferation in dysplasia and cancer 929

Volume 116 Number 7

of 9 to 12 days and a breast carcinoma with a generation time of 31 to 45 days. Although none of the spontaneous neoplasms that have been reported have a generation time approaching that of carcinoma in situ, a transplantable mouse sarcoma with a generation time of 18 hours has been reported. 5 Several normal tissues, which depend on rapid replacement for their maintenance, have generation times within the same range as carcinoma in situ. These include the crypt epithelium of the mouse ileum with reports of generation time varying from 12 to 19 hours and the bone marrow elements with a generation time of from 20 to 30 hours. The in vitro methods utilized in these experiments have yielded data which are reproducible in widely spaced sections within a single block in areas of similar morphology. The methods described were worked out prior to the publication by Johnson and Bond" whose method is essentially identical to this one. Those authors compared the labeling index in a transplantable mouse tumor, in vivo and in vitro, and found that the results were in good agreement. In the data presented in this paper the

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Fig. 6. Graph illustrating the percentge by which each of the cellular layers contributes to the total proliferating pool in each of the six categories of severity.

labeling index has been shown to increase logarithmically with the severity of neoplasia as shown in Fig. 3. This relationship is linear with no evidence of curvature and, hence, with no sharp breaking point, or line of demarcation. The data derived from this single parameter, labeling index, suggest that the development of carcinoma in situ from normal cervical epithelium is a progressive and continuous process which may be thought of as a continuum. The observation that the labeling index of normal epithelium and that of mild dysplasia are essentially equivalent suggests that, at this stage, the process is not far advanced and may be spontaneously reversible. Similarly, certain biopsies in each of the severity categories 2+ to 5+ possess labeling indices within or near the range for normal epithelium, suggesting that certain cases which morphologically are felt to represent advanced stages in the continuum may be indeterminate biologically. Six plus on the severity scale, or carcinoma in situ, contains

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no points which even approach the labeling index in normal epithelium, is a relatively homogeneous group, and does not overlap with any other groups. These observations lend verification to the hypothesis that carcinoma in situ is a reproducibly recognizable lesion, at a microscopic level, and represents a distinct pathological entity. It is questionable whether the categories beneath 6+ represent distinct homogenous entities either as a group or within the groups chosen in this study. Summary

The stages in the development of carcinoma in situ in the human uterine cervix

REFERENCES 1. Richart, Ralph M.: AM. J. 0BsT. & GYNEC.

(In press.)

2. Joftes, L. D.: Lab. Invest. 8: 131, 1959. 3. McKay, D. G., Terjanian, B., Poschyachinda, D., Younge, P. A., and Hertig, A. T.: Obst. & Gynec. 13: 2, 1959. 4. Takeuchi, A., and McKay, D. G.: Obst. & Gynec. 15: 134, 1960. 5. Johnson, H. A., and Bond, V. P.: Cancer 14: 639, 1961.

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are accompanied by altered labeling indin·> that increase logarithmically with severin. There is a gradual replacement of the layers of epithelium, normally composed of cells in a mitotic end stage, by cells that retain the ability to produce daughter cells. Not only is there an increase in the number of cells capable of division, but there is an increase in the rate at which these cells divide. In carcinoma in situ the population appears to be essentially homogenous in terms of the labeling index. The excellent technica 1 assistance of M r~. Ethel Lovings was extremdy valuable during the course of this study.

6. Johnson, H. A., Haymaker, W. E., Rubini, J. R., Fliedner, T. M., Bond, V. P., Cronkite, E. P., and Hughes, W. L.: Cancer 13: 636,

1960. 7. Johnson, H. A., Rubini, S. R., Cronkite, E. P., and Bond, V. P.: Lab. Invest. 9: 460, 1960.

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