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Metabolic activity in normal and abnormal vulvar epithelia
Metabolic activity in normal and abnormal vulvar epithelia An assessment
J.
by the use of tritiated
D.
WOODRUFF,
H.
I.
BORKOWF,
M.D.
G.
B.
HOLZMAN,
M.D.
E.
A.
ARNOLD,
JLJERGEN Baltimore,
M.D.*
KNAACK,
M.D.**
Maryland
Dermatologists have described lichen sclerosus et atrophicus as an entity occurring on many areas of the body beginning as bluish-white papules which eventually coalesce to form whitish plaques.3 This lesion microscopically simulates exactly the terminal stage of chronic atrophic vulvitis as described by Taussig. It is also similar in every respect to the findings characteristic of “kraurosis”-that diffuse primary atrophy of the vulvar skin eventuating in stenosis of the outlet. Although there may be, and undoubtedly is, more than one clinical course which may eventuate in this variety of atrophic lesion, the major concern is the same, namely: Is there a real potentiality of malignant change occurring in such a setting? One of the particularly complicating features in the study of many of these lesions has been the use of the term atrophicus which should semantically refer to wasting or degeneration and is generally caused by changes in nutrition and metabolism of the involved cells. This concept must have been predicated on the microscopic findings of a thin flattened epithelium and an acellular collagenized dermis. While it must be admitted that without serious investigation of the cellular detail, the epithelium commonly noted in lichen sclerosus et atrophicus ap-
From the Defiartments of Gynecology, Obstetrics, and Pathology, the Johns Hopkins University School of Medicine. Sujported in part by National Institute of Health Research Grant No. C5319 and the Nellie McCarty Research Grant. Presented at the Seventy-fifth dnnual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Sept. 10-12, 1964. address:
Research Fellow, of Pathology, 25, D. C.
Armed Forces Institute Washington **Present Montreal,
acid precursors
M.D.
C 0 N T R 0 v E R s Y as to the pathogenesis, clinical course, and particularly the malignant potential of many of the hyperkeratotic lesions has been a continuing issue in the study of vulvar disease. Special difficulties have arisen in an attempt to evahrate the so-called atrophic lesion. Bonneyl recognized 4 stages in the life history of the disease which he labeled “leukoplakic vulvitis,” the terminal one being an “atrophy or quiescence” during which the possibility of malignant alteration was essentially nonexistent. Taussig’ agreed in general with this thesis, however, he preferred the term chronic atrophic vulvitis and did not accept the quiescent stage, but rather believed that the malignant potentiality continued unabated.
*Present
nucleic
address: Quebec,
McGill Canada.
University, 809
810
Woodruff
et
al.
pears inactive because of its thinness and decreased cellularity, certain studies do not substantiate this opinion. Clark and co-workers,’ in their investigation of the lesions of the vulva hy the use of radioactixx~ phosphcwlls (P:,, i . recognized that the uptake
1
slle
e
Fig. 1. Hernatoxylin and eosin section of typical lichen srlerosus et atrophicus. Note the disturbed and indistinct basal layer with loss of polarity and tendency to early or rapid maturation (not wastin,? and deqxuzration 1.
Fig. 2. Autoradiogram of carcinoma in situ of the vulva showing diffuse nuclear Iabelin,g throughout all Iayers of the epithelium (incubation for 30 minutes).
of the radioactive material was just as great in the so-called lichen sclerosus as it was in the premalignant conditions, and much greater than in the normal epithelium. Recently? published data from a study in OUI laboratory which attempted to evaluate the tnetabolic activity by means of acridine orange fluoresccncc,5 revealed increased evidence of DNA in cells scattered in a disorderly fashion through the epithelium. Furthermore, although the epithelium is at first glance atrophic, a more detailed study reveals an ill-defined and disorderly basal layer with evidence of mature type cells in the deep epitheliurn (Fig. 1 ) . These findings su,qgest disturbed and over-zealous maturation. While it must bc admitted that metabolic activity cannot be directly correlated hvith anaplastic potential, nevertheless, such studies suggest that these tissues are not atrophic, but they actually rnetabolize more actively than the normal. Materials
and
methods
Study project. In addition to the acridine orange studies noted above, evaluation of the metabolic activity in the normal and pathologic vulvar epithelium was approached by the use of tagged precursors for nucleic acid synthesis. As noted above, Clark and co-workers’ have made similar investigations in viva by the use of radioactive phosphorus (P,,,) Phosphorus was actually the first nucleic acid label to be used.” As it is a component of both DNA and RNA, it was suitable for the study of the metabolism of both types of nucleic acids. Its great advantage is that very small amounts of the isotope can be detected. There are, however, several disadvantages of P,,. Recause of its high ener,y Beta particles, it gives poor autoradiographic resolution. Also since it enters both DNA and RNA, to study DNA metabolism the RNA must be removed. Furthermore, phospholipids, phosphoproteins, and other organic phosphates will also incorporate P,,, and, as a result, radioautographs rnay be misleading. Finally the short half-life (14 days) of P,? necessitates constant corrections to stock solutions
Volume Number
91 6
due to radioactive decay, and this, not infrequently, results in an inaccurate dosage of Pz2.c’ Since thymidine was first tritiated by Hughes in 1955, in the Brookhaven Laboratories,’ it has been used as a radioactive tag in a variety of in vivo and in vitro studies.8* o Although damage occurs with doses as low as 1 PC per gram, the low energy of the tritium Beta rays insures high resolution nuclear radioautographs, and accurate assessment of nuclear labeling can be obtained if sections are not thicker than 3 p.l”, I1 Tritiated thymidine reaches cells rapidly, is taken up selectively by cells synthesizing DNA, and remains incorporated in the DNA molecule for the lifetime of the nucleus and its progeny. Most of the labeled thymidine not taken up by the cells is eliminated in the first hour after injection. Furthermore, most of the tritiuml* that goes into stable compounds appears in DNA which was synthesized at the time of labeling and stays there as long as the DNA molecule remains intact. The questions that were entertained in this study were as follows: 1. What differences, if any, can be detected between the metabolic activity in the vulvar epithelia of the various disease states and that noted in the normal epithelium? 2. If present, can these differences be detected by the use of isotopic tracer methods? 3. Does metabolic activity correspond to anaplastic activity? The frequency of uptake in cell populations seemingly should follow the progression from a low in atrophy to a high in cancer with normal, hypertrophy, and carcinoma in situ occupying intermediate points. There is doubt, however, that such an ordered frequency exists. There are paradoxes in which normal tissues exhibit a higher degree of mitotic activity than their malignant counterparts. The suggested possible explanations for these paradoxes are as follows:+ A. Death of malignant progeny, due to genetic complements incompatible with survival. B. Increased DNA synthetic rate marked by longer absolute division time due to ploidy
Metabolic
activity
Fig. 3. Autoradiogram
in vulvar
epithelia
811
of normal vulvar epithelium
with sparsely scattered labeled nuclei in basal parabasal layer (incubation time, 1 hour).
Fig. 4. Autoradiogram
and
of lichen sclerosus (x49) showing the typical picture with hyperkeratosis, thinned epithelium, loss of rete pegs. and collagcnization of the dermis. Labeled nuclei can be recognized throughout the basal and parabasal layers (incubation time, 1 hour).
Fig. labeled
5.
Same nuclei.
as
Fig. ,.
4
(x100)
demonstrating
812
Woodruff
et
al.
Fig. 6. Autoradiogram of lichen srlerosus showing labeling of basal and parabasal nuclei at both edges but absence of labeling in center even though the epithelium is thinner and perhaps more easily penetrated.
Fig. ‘7. Diagram showing the divisions of the generative cycle including those romprising generation time, interphase, synthesis. and mitosis. i-2. Postsynthetic rest or premitotic gap; T,, presynthetic period or presynthetic Rap ; S. synthesis time ; M, mitotic time.
increase. C. Faulty division processes in malignant cells whereby giant cells or multinucleate cells represent the progeny (i.e., one daughter cell instead of two). Procedure. Fresh tissue was cut with a sharp knife, free-hand, at approximately 2 to 3 mm. These fragments of fresh tissue were then placed in Erlenmeyer flasks which contained a basic amount of solution MXg9, a mixture of approximately 50 ingredients commonly used in tissue culture. To this solution has been added 40 units of penicillin and 50 mg. of streptomycin per cubic centimeter of fluid. Tritiated thymidine, 1 PC per cubic centimeter, was added to one series of flasks, and tritiated uracil, 1 PC per cubic centimeter, was added to another. The total solution and its ingredients
h.
March 15, 196.~ J. Obrt. & Gynrc
amounted to 5 C.C. per flask. These were then incubated at 37v2’ C. on a rack-shaker with a gas phase consisting of 95 per cent oxygen and 5 per cent carbon dioxide at 5 L. per minute. Incubation was carried out for periods of time varying from 15 minutes to 72 hours. The reaction was then stopped by the addition of 10 per cent formalin solution. The tissues were washed, processed, and embedded in paraffin. The sections were cut at 5 \C from one surface and then reembedded in an effort to obtain an equal number of sections from opposite faces. The sections were then deparaffinized and placed in slide racks. The water bath was then equilibrated to 100’ C. All dipping and developing procedures were carried out in the darkroom; Kodak NTIS2 emulsion was melted and placed in the staining dish container for dipping. The entire rack was immersed in the emulsion, reversed, and dipped again to assure that ail slide areas were adequately covered. The slides were then placed in a fan box, without a heater, and allowed to dry for 30 to 45 minutes. The dry racks were then placed in a canister with a small bag of CaCI? and locked. The canisters were gassed with N?, placed in a refrigerator at 4’ C., and kept in this atmosphere for approximately 21 to 28 days. After this period of time, the developing process was carried out. All reagents were brought to 68’ C. in the sink. The racks were left in Dektol, the developing agent, for 2 minutes. They were then washed in water and placed in a 25 per cent solution of hypo for 3 minutes, rewashed. and stained. Results
The metabolic activity in the epithelia of a variety of disease states has been studied. Several examples of grossly normal vulvar skin,
either
front
the
tissue
adjacent
to
a
specific lesion or from patients who were surgically treated for other conditions, were [ised as controls. To obtain similar areas for evaluation, the labeled nuclei in the first section from each
Volume Numtm
91 6
Metabolic
Fig. 8. Autoradiogram of section taken approximately 1.5 cm. from carcinoma in situ. The percentage of labeled nuclei and the penetration of these nuclei through 50 per cent of the thickness of the epithelium evidences increased metabolic activity although the tissue was grossly normal (incubation time, 3 hours).
Table
I. Nuclear
labeling
in certain
vulvar
activity
in vulvar
epithelia
813
Fig. 9. Autoradiogram of normal epithelium showing penetration of labeled nuclei through approximately 50 per cent of epithelial depth (incubation time, 30 hours). Compare with Fig. 8 which shows similar changes in normal-appearing epithelium adjacent carcinoma in situ (incubation, 3 hours).
diseases -
Labeled
nuclei
Epithelium
Low
1 High
Normal Lichen sclerosus Perianaplastic “normal” Perianaplastic “atypical” Carcinoma in situ
0.7 5.8 3.8
6.4 10.2 7.0
total
(‘%a)
( Average 2.5 8.5 5.2
in
the
sections
incubated
10 days 3 days 4.8 days
Low 2.5 8.9 5.6
1 High 7.6 20.9 16.7
17.0
1.5 days
23.5
17.3
1.5 days
22.9
side of the tissue slide were compared. Only the cells at the ends of the cut tissue were counted since penetration of the radioactive material was not routinely uniform in the central portions. This was probably due to the surface keratin and underlying dermis which isolated the epithelium and resulted in this uneven labeling. Adjacent material was sectioned in the routine fashion and was stained with hematoxylin-eosin for usual histologic evaluation. The study sections were, however, found to be quite satisfactory for morphologic evaluation. Percentage labeled cells. The labeled nuclei
Generation time average
Labeled nuclei parabasal
with
tritiated
thymidine were counted and compared in the study of each lesion. Since there were striking differences in the thickness of the
basal (70)
and
1 Averace
Generation time average
3.6 16.4 11.9
7 days 1.5 days 2.1 days 25.5
hours
26 hours
epithelium, similar counts were performed in only the basal and parabasal layers in an attempt to make the results more comparable. The percentages of labeled nuclei in both the entire population and in the basal and parabasal zones are noted in Table I. It is readily seen that, as might be expected, there was a marked increased labeling in the malignant epithelia (Fig. 2) as compared with the normal (Fig. 3). Of special interest are the “unexpected” counts in lichen scleroSW In spite of the so-called atrophy, the per cent labeled nuclei was approximately 3 to 6 times as great as the normal (Figs. 4 and 5), and almost as great as that noted in some anaplastic epithelia. The question was raised as to whether or not the apparent “increased” nuclear labeling noted in the
814
Woodruff
et al
Fig. 10. Autoradiogram ately adjacent carcinoma ing striking nuclear
of tissue taken immediin situ and demonstrat-
labeling
(incubation
time,
3 hours).
lichen sclerosus might really he due to easier penetration of radioactive tags as the result of the reduction in the cellular components. One study section aided in providing an answer to this query. In Fig. 6 it will be noted that the thinnest epithelium, in the center of the section, reveals no nuclear labeling, while on both sides there is a demonstrable uptake in the lower cell layers. It would appear that the unlabeled zone represents the true “atrophy” and is metabolically inactive. Although it must again be stated that increased metabolic activity cannot be correlated directly with the degree nevertheless the findings in of anaplasia, these cases suggest that the conditions labeled atrophic, in commonly used terminology, are in reality not inactive or degenerative. Of interest also has been the study of the “normal appearing” skin adjacent to malignant lesions of the vulva. Although this tissue has been grossly innocent, study has indicated an increased labeling of the nuclei in many instances. This finding needs furthel confirmation, but it may offer a solution to the common problem of local recurrence of vulvar cancer and multicentric foci of anaplastic change, namely that the remaining grossly normal skin edges have in reality already been affected by the malignant stimulus. Although the variations in the per cent
labeled nuclei suggested definite differences in DNA metabolism of the epithelia studied, attempts were also made to determine the generation and turnover times in each instance. These values have been more readily obtained by in viva studies, since more control of the experimental tissues is available, nevertheless certain known formulas and fixed times make possible relatively accurate determinations for in vitro studies. The generation time has been defined as the time necessary for the basal cells to complete one mitotic cycle. This includes the times for synthesis and mitoses as well as the pre- and postsynthetic gaps shown in Fig. 7. It has been determined in vivo by serially sacrificing animals after a single intravenous or intraperitoneal injection of the radioactive tag. This is an impractical approach to studies such as ours due to the danger of diffuse cellular radiation damageI and the esorbitant cost of the material for introduction into a human, even if a subject were available. Furthermore, it has, to date, been impossible to specifically outline mitosis in thr vulvar epithelium either in the study material or in the routine laboratory preparations except in malignant conditions. Formulas, howevf,r, have been established to determine, with some degree of accuracy, the kgeneration time in various tissues. Using the formula.
as suggested by ,Johnson,14 and the standard synthesis time of 6 hours’j. Ifi as determined for the HeLa cells in vitro and for normal mouse intestine, the generation times for the tissues compared in this study are r+ corded in Table I with per cent labeled nuclei. As would be expected, these times corresponded gchnerally to the percentage of nuclear labeling. The turnover time is designated as that time necessary for complete replacement of the epithelium, that is the shortest elapsed time
from
the
initial
uptake
of
the
radio-
active tag in the basal layer to the appearance of labeled nuclei in the most superficial
Volume Numb
91 6
Metabolic
activity
in vulvar
epithelia
815
Fig. 11. Autoradiogram of tissue adjacent to invasive cancer showing labeling throughout all layers, however the associated infection must play a role here (incubation time, 1 hour).
Fig, 12. Autoradiogram of tissue taken from invasive cancer showing the absence of label in the pearls but diffuse activity in the disturbed more immature cells (incubation time, 30 minutes).
layer. Again, as with the generation time, this period has been determined in vivo by serially sacrificing animals. In vitro attempts to assess the turnover time in this study were made by incubation of the fresh tissue for varying periods of time from 15 minutes to 72 hours, and variations were noted in the penetration of the epithelium by labeled nuclei. For example, in 30 minutes there were such nuclei in the most superficial layer in cases of carcinoma in situ (Fig. 3). Such a finding may, however, represent the simultaneous metabolic activity of the immature cells throughout all layers in this anaplastic epithelium rather than an orderly progression of the basal cell to the surface by the normal processes of maturation. Furthermore, it was recognized that the tissues lose their cellular detail in approximately 30 hours and are probably reproductively inactive some time prior to this period. Better tissue culture methods may answer this problem of loss of viability. In spite of the difficulties in interpretation of results, particularly in the anaplasia, differences in penetration were noted between the normal vulvar epithelium, lichen sclerosus, and the normal-appearing tissue adjacent to the anaplasia. These results closely approximate those tabulated for per cent of nuclear labeling, but they are not as dramatic in degree. Particularly significant
however was the depth of penetration in the tissues adjacent to malignancy. Fig. 8 represents histologically normal tissue about 1 cm. from carcinoma in situ, but it reveals an increased percentage of labeled nuclei over that noted in other normals, as well as penetration of these nuclei through at least 50 per cent of the thickness of the epithelium in 3 hours. A comparable section from normal epithelium shows slightly less labeling and less penetration after 30 hours incubation (Fig. 9). Fig. 10 shows more striking changes and is immediately adjacent to the anaplastic epithelium in which cellular atypism can be readily recognized. Fig. 11 was taken in the vicinity of invasive cancer and shows nuclear labeling throughout all layers, In this section there is also striking infection, an added factor which is known to affect the metabolism of the cell. These findings, with proper evaluation, may offer a means of recognizing abnormal nuclear activity before the morphologic changes are visible if the results are confirmed in future studies. It was recognized that metabolic activity as determined by the percentage of labeled nuclei in total population was greater in the examples of carcinoma in situ than in cases of invasive disease. The very mature cells forming the keratin pearls demonstrated no labeling. The active and distorted cells
816
Woodruff
et al.
of the prekeratin and deeper zones, however, revealed striking activity (Fig. 12). Possible explanations for this “apparent” incongruity have been suggested earlier in this report. Summary
1. Metabolic activity of the vulvar epithelium, in the normal and in certain diseased states, was evaluated by incubation of the fresh tissue with tritiated precursors of ribonucleic acid (uracil) and deoxyribonucleic acid (thymidine) . 2. It was recognized that, in general, metabolic activity seemed to be directly correlated to the anaplastic activity. Invasive cancer was the lone exception.
its atrophic .cl. Lichen sclerosus, belying was metabolically active, as appearance recognized by the percentage of labeled cells which approach that noted in carcinoma in situ and invasive cancer. 4. Increased activity of normal-appearing skin adjacent to anaplastic lesions suggests a reason for the frequency of local recurrences, and may offer a means of evaluating the activity of the tissues at the limits of surgical excision. The authors would like to thank Mrs. Leah M. Carter and Mr. Richard Lewis, Jr., for their technical assistance and Mr. Raymond Lund for the excellent photography.
REFERENCES
Bonney, V.: Lancet 1: 1465, 1908. Taussig, F. J.: Diseases of the vulva, New York, 1923, D. Appleton and Company. 3. Hunt, Elizabeth: Disease Affecting the Vulva, St. Louis, 1954, The C. V. Mosby Company. 4. Clark, D. G. G., Zumoff, B., Brunschwig, A., and Hellman, L.: Cancer 13: 775, 1960. 5. Friedrichs, E. G., Julian, C. G., and Woodruff, J. D.: AM. J. OBST. & GYNEC. 90:
10.
1281, 1964. Lajtha, L. G., and Oliver, R.: Lab. Invest. 8: 214, 1959. Hughes, W. L., Bond, V. P., Brecher, G., Cronkhite, E. D., Quastler, H., and Sherman, F. G.: Proc. Nat. Acad. SC. 44: 476, 1958. Peckham, B., and Kiekhofer, Wm.: AM. J. OBST. & GYNEC. 83: 1021, 1962. Richart. R. M.: AM. J. OBST. & GYNEC. 86: 925, 1963.
14.
1. ?h.
6. 7.
8. 9.
11.
12. 13.
15. 16.
Lesher, S.: Fry, R. J. M., and Kohn, H. I.: Lab. Invest. 10: 291, 1959. Cronkhite, E. P., Bond, V. P., Fliedner, T. M., and Rubin, J. R.: Lab. Invest. 8: 263, 1959. LeBlond, C. P., Messier, B., and Kopriwa, B.: Lab. Invest. 8: 296, 1959. Oliver, R., and Lajtha, 1~. G.: Nature 186: 91, 1960. Johnson, H. A., and Bond, V. P.: Cancer 14: 639, 1961. Quastler, H., and Sherman, F. G.: Exper. Cell Res. 17: 420, 1959. Painter, R. B., and Drew, R. M.: Lab. Invest. 8: 278, 1959.
107 Cotswold Road Baltimore Ii?, Maryland
Discussion ARTHUR B. HUNT, Rochester, Minnesota. Although lichen sclerosus et atrophicus is admittedly a good example of atrophic change of the vulvar epithelium, it does have features that strongly suggest that it is a dermatologic entity of unknown etiology. These features are the peculiar “keyhole” distribution over the vulva and around the anus, the delling of the skin found at or near the periphery of the lesion, its relatively common appearance in the skin of other portions of the body, and its infrequent although not entirely rare occurrence in youthful patients. These features are wanting in kraurosis vulvac and simple senile atrophy as described
by
Adair. It would be interesting if Dr. Woodcould extend his studies in simple senih atrophy to include more than just one example of it-his Fig. 6. In this type of true atrophy he found cellular uptake to he the lowest in the entire study, as I interpret the photomicrographs. As a clinician I am neither knowledgeable nor experienced in the use of tritiated nucleic acid precursors, hut I feel, as do my colleagues who are experienced in this field, that one can only detrrmine that the percentage of cells that were labeled was considered to he higher than normal in the three diseases of the vulva. Any assumption that increased uptake of DNA prr-
ruff
J.
by the use of tritiated
D.
WOODRUFF,
H.
I.
BORKOWF,
M.D.
G.
B.
HOLZMAN,
M.D.
E.
A.
ARNOLD,
JLJERGEN Baltimore,
M.D.*
KNAACK,
M.D.**
Maryland
Dermatologists have described lichen sclerosus et atrophicus as an entity occurring on many areas of the body beginning as bluish-white papules which eventually coalesce to form whitish plaques.3 This lesion microscopically simulates exactly the terminal stage of chronic atrophic vulvitis as described by Taussig. It is also similar in every respect to the findings characteristic of “kraurosis”-that diffuse primary atrophy of the vulvar skin eventuating in stenosis of the outlet. Although there may be, and undoubtedly is, more than one clinical course which may eventuate in this variety of atrophic lesion, the major concern is the same, namely: Is there a real potentiality of malignant change occurring in such a setting? One of the particularly complicating features in the study of many of these lesions has been the use of the term atrophicus which should semantically refer to wasting or degeneration and is generally caused by changes in nutrition and metabolism of the involved cells. This concept must have been predicated on the microscopic findings of a thin flattened epithelium and an acellular collagenized dermis. While it must be admitted that without serious investigation of the cellular detail, the epithelium commonly noted in lichen sclerosus et atrophicus ap-
From the Defiartments of Gynecology, Obstetrics, and Pathology, the Johns Hopkins University School of Medicine. Sujported in part by National Institute of Health Research Grant No. C5319 and the Nellie McCarty Research Grant. Presented at the Seventy-fifth dnnual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Sept. 10-12, 1964. address:
Research Fellow, of Pathology, 25, D. C.
Armed Forces Institute Washington **Present Montreal,
acid precursors
M.D.
C 0 N T R 0 v E R s Y as to the pathogenesis, clinical course, and particularly the malignant potential of many of the hyperkeratotic lesions has been a continuing issue in the study of vulvar disease. Special difficulties have arisen in an attempt to evahrate the so-called atrophic lesion. Bonneyl recognized 4 stages in the life history of the disease which he labeled “leukoplakic vulvitis,” the terminal one being an “atrophy or quiescence” during which the possibility of malignant alteration was essentially nonexistent. Taussig’ agreed in general with this thesis, however, he preferred the term chronic atrophic vulvitis and did not accept the quiescent stage, but rather believed that the malignant potentiality continued unabated.
*Present
nucleic
address: Quebec,
McGill Canada.
University, 809
810
Woodruff
et
al.
pears inactive because of its thinness and decreased cellularity, certain studies do not substantiate this opinion. Clark and co-workers,’ in their investigation of the lesions of the vulva hy the use of radioactixx~ phosphcwlls (P:,, i . recognized that the uptake
1
slle
e
Fig. 1. Hernatoxylin and eosin section of typical lichen srlerosus et atrophicus. Note the disturbed and indistinct basal layer with loss of polarity and tendency to early or rapid maturation (not wastin,? and deqxuzration 1.
Fig. 2. Autoradiogram of carcinoma in situ of the vulva showing diffuse nuclear Iabelin,g throughout all Iayers of the epithelium (incubation for 30 minutes).
of the radioactive material was just as great in the so-called lichen sclerosus as it was in the premalignant conditions, and much greater than in the normal epithelium. Recently? published data from a study in OUI laboratory which attempted to evaluate the tnetabolic activity by means of acridine orange fluoresccncc,5 revealed increased evidence of DNA in cells scattered in a disorderly fashion through the epithelium. Furthermore, although the epithelium is at first glance atrophic, a more detailed study reveals an ill-defined and disorderly basal layer with evidence of mature type cells in the deep epitheliurn (Fig. 1 ) . These findings su,qgest disturbed and over-zealous maturation. While it must bc admitted that metabolic activity cannot be directly correlated hvith anaplastic potential, nevertheless, such studies suggest that these tissues are not atrophic, but they actually rnetabolize more actively than the normal. Materials
and
methods
Study project. In addition to the acridine orange studies noted above, evaluation of the metabolic activity in the normal and pathologic vulvar epithelium was approached by the use of tagged precursors for nucleic acid synthesis. As noted above, Clark and co-workers’ have made similar investigations in viva by the use of radioactive phosphorus (P,,,) Phosphorus was actually the first nucleic acid label to be used.” As it is a component of both DNA and RNA, it was suitable for the study of the metabolism of both types of nucleic acids. Its great advantage is that very small amounts of the isotope can be detected. There are, however, several disadvantages of P,,. Recause of its high ener,y Beta particles, it gives poor autoradiographic resolution. Also since it enters both DNA and RNA, to study DNA metabolism the RNA must be removed. Furthermore, phospholipids, phosphoproteins, and other organic phosphates will also incorporate P,,, and, as a result, radioautographs rnay be misleading. Finally the short half-life (14 days) of P,? necessitates constant corrections to stock solutions
Volume Number
91 6
due to radioactive decay, and this, not infrequently, results in an inaccurate dosage of Pz2.c’ Since thymidine was first tritiated by Hughes in 1955, in the Brookhaven Laboratories,’ it has been used as a radioactive tag in a variety of in vivo and in vitro studies.8* o Although damage occurs with doses as low as 1 PC per gram, the low energy of the tritium Beta rays insures high resolution nuclear radioautographs, and accurate assessment of nuclear labeling can be obtained if sections are not thicker than 3 p.l”, I1 Tritiated thymidine reaches cells rapidly, is taken up selectively by cells synthesizing DNA, and remains incorporated in the DNA molecule for the lifetime of the nucleus and its progeny. Most of the labeled thymidine not taken up by the cells is eliminated in the first hour after injection. Furthermore, most of the tritiuml* that goes into stable compounds appears in DNA which was synthesized at the time of labeling and stays there as long as the DNA molecule remains intact. The questions that were entertained in this study were as follows: 1. What differences, if any, can be detected between the metabolic activity in the vulvar epithelia of the various disease states and that noted in the normal epithelium? 2. If present, can these differences be detected by the use of isotopic tracer methods? 3. Does metabolic activity correspond to anaplastic activity? The frequency of uptake in cell populations seemingly should follow the progression from a low in atrophy to a high in cancer with normal, hypertrophy, and carcinoma in situ occupying intermediate points. There is doubt, however, that such an ordered frequency exists. There are paradoxes in which normal tissues exhibit a higher degree of mitotic activity than their malignant counterparts. The suggested possible explanations for these paradoxes are as follows:+ A. Death of malignant progeny, due to genetic complements incompatible with survival. B. Increased DNA synthetic rate marked by longer absolute division time due to ploidy
Metabolic
activity
Fig. 3. Autoradiogram
in vulvar
epithelia
811
of normal vulvar epithelium
with sparsely scattered labeled nuclei in basal parabasal layer (incubation time, 1 hour).
Fig. 4. Autoradiogram
and
of lichen sclerosus (x49) showing the typical picture with hyperkeratosis, thinned epithelium, loss of rete pegs. and collagcnization of the dermis. Labeled nuclei can be recognized throughout the basal and parabasal layers (incubation time, 1 hour).
Fig. labeled
5.
Same nuclei.
as
Fig. ,.
4
(x100)
demonstrating
812
Woodruff
et
al.
Fig. 6. Autoradiogram of lichen srlerosus showing labeling of basal and parabasal nuclei at both edges but absence of labeling in center even though the epithelium is thinner and perhaps more easily penetrated.
Fig. ‘7. Diagram showing the divisions of the generative cycle including those romprising generation time, interphase, synthesis. and mitosis. i-2. Postsynthetic rest or premitotic gap; T,, presynthetic period or presynthetic Rap ; S. synthesis time ; M, mitotic time.
increase. C. Faulty division processes in malignant cells whereby giant cells or multinucleate cells represent the progeny (i.e., one daughter cell instead of two). Procedure. Fresh tissue was cut with a sharp knife, free-hand, at approximately 2 to 3 mm. These fragments of fresh tissue were then placed in Erlenmeyer flasks which contained a basic amount of solution MXg9, a mixture of approximately 50 ingredients commonly used in tissue culture. To this solution has been added 40 units of penicillin and 50 mg. of streptomycin per cubic centimeter of fluid. Tritiated thymidine, 1 PC per cubic centimeter, was added to one series of flasks, and tritiated uracil, 1 PC per cubic centimeter, was added to another. The total solution and its ingredients
h.
March 15, 196.~ J. Obrt. & Gynrc
amounted to 5 C.C. per flask. These were then incubated at 37v2’ C. on a rack-shaker with a gas phase consisting of 95 per cent oxygen and 5 per cent carbon dioxide at 5 L. per minute. Incubation was carried out for periods of time varying from 15 minutes to 72 hours. The reaction was then stopped by the addition of 10 per cent formalin solution. The tissues were washed, processed, and embedded in paraffin. The sections were cut at 5 \C from one surface and then reembedded in an effort to obtain an equal number of sections from opposite faces. The sections were then deparaffinized and placed in slide racks. The water bath was then equilibrated to 100’ C. All dipping and developing procedures were carried out in the darkroom; Kodak NTIS2 emulsion was melted and placed in the staining dish container for dipping. The entire rack was immersed in the emulsion, reversed, and dipped again to assure that ail slide areas were adequately covered. The slides were then placed in a fan box, without a heater, and allowed to dry for 30 to 45 minutes. The dry racks were then placed in a canister with a small bag of CaCI? and locked. The canisters were gassed with N?, placed in a refrigerator at 4’ C., and kept in this atmosphere for approximately 21 to 28 days. After this period of time, the developing process was carried out. All reagents were brought to 68’ C. in the sink. The racks were left in Dektol, the developing agent, for 2 minutes. They were then washed in water and placed in a 25 per cent solution of hypo for 3 minutes, rewashed. and stained. Results
The metabolic activity in the epithelia of a variety of disease states has been studied. Several examples of grossly normal vulvar skin,
either
front
the
tissue
adjacent
to
a
specific lesion or from patients who were surgically treated for other conditions, were [ised as controls. To obtain similar areas for evaluation, the labeled nuclei in the first section from each
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Metabolic
Fig. 8. Autoradiogram of section taken approximately 1.5 cm. from carcinoma in situ. The percentage of labeled nuclei and the penetration of these nuclei through 50 per cent of the thickness of the epithelium evidences increased metabolic activity although the tissue was grossly normal (incubation time, 3 hours).
Table
I. Nuclear
labeling
in certain
vulvar
activity
in vulvar
epithelia
813
Fig. 9. Autoradiogram of normal epithelium showing penetration of labeled nuclei through approximately 50 per cent of epithelial depth (incubation time, 30 hours). Compare with Fig. 8 which shows similar changes in normal-appearing epithelium adjacent carcinoma in situ (incubation, 3 hours).
diseases -
Labeled
nuclei
Epithelium
Low
1 High
Normal Lichen sclerosus Perianaplastic “normal” Perianaplastic “atypical” Carcinoma in situ
0.7 5.8 3.8
6.4 10.2 7.0
total
(‘%a)
( Average 2.5 8.5 5.2
in
the
sections
incubated
10 days 3 days 4.8 days
Low 2.5 8.9 5.6
1 High 7.6 20.9 16.7
17.0
1.5 days
23.5
17.3
1.5 days
22.9
side of the tissue slide were compared. Only the cells at the ends of the cut tissue were counted since penetration of the radioactive material was not routinely uniform in the central portions. This was probably due to the surface keratin and underlying dermis which isolated the epithelium and resulted in this uneven labeling. Adjacent material was sectioned in the routine fashion and was stained with hematoxylin-eosin for usual histologic evaluation. The study sections were, however, found to be quite satisfactory for morphologic evaluation. Percentage labeled cells. The labeled nuclei
Generation time average
Labeled nuclei parabasal
with
tritiated
thymidine were counted and compared in the study of each lesion. Since there were striking differences in the thickness of the
basal (70)
and
1 Averace
Generation time average
3.6 16.4 11.9
7 days 1.5 days 2.1 days 25.5
hours
26 hours
epithelium, similar counts were performed in only the basal and parabasal layers in an attempt to make the results more comparable. The percentages of labeled nuclei in both the entire population and in the basal and parabasal zones are noted in Table I. It is readily seen that, as might be expected, there was a marked increased labeling in the malignant epithelia (Fig. 2) as compared with the normal (Fig. 3). Of special interest are the “unexpected” counts in lichen scleroSW In spite of the so-called atrophy, the per cent labeled nuclei was approximately 3 to 6 times as great as the normal (Figs. 4 and 5), and almost as great as that noted in some anaplastic epithelia. The question was raised as to whether or not the apparent “increased” nuclear labeling noted in the
814
Woodruff
et al
Fig. 10. Autoradiogram ately adjacent carcinoma ing striking nuclear
of tissue taken immediin situ and demonstrat-
labeling
(incubation
time,
3 hours).
lichen sclerosus might really he due to easier penetration of radioactive tags as the result of the reduction in the cellular components. One study section aided in providing an answer to this query. In Fig. 6 it will be noted that the thinnest epithelium, in the center of the section, reveals no nuclear labeling, while on both sides there is a demonstrable uptake in the lower cell layers. It would appear that the unlabeled zone represents the true “atrophy” and is metabolically inactive. Although it must again be stated that increased metabolic activity cannot be correlated directly with the degree nevertheless the findings in of anaplasia, these cases suggest that the conditions labeled atrophic, in commonly used terminology, are in reality not inactive or degenerative. Of interest also has been the study of the “normal appearing” skin adjacent to malignant lesions of the vulva. Although this tissue has been grossly innocent, study has indicated an increased labeling of the nuclei in many instances. This finding needs furthel confirmation, but it may offer a solution to the common problem of local recurrence of vulvar cancer and multicentric foci of anaplastic change, namely that the remaining grossly normal skin edges have in reality already been affected by the malignant stimulus. Although the variations in the per cent
labeled nuclei suggested definite differences in DNA metabolism of the epithelia studied, attempts were also made to determine the generation and turnover times in each instance. These values have been more readily obtained by in viva studies, since more control of the experimental tissues is available, nevertheless certain known formulas and fixed times make possible relatively accurate determinations for in vitro studies. The generation time has been defined as the time necessary for the basal cells to complete one mitotic cycle. This includes the times for synthesis and mitoses as well as the pre- and postsynthetic gaps shown in Fig. 7. It has been determined in vivo by serially sacrificing animals after a single intravenous or intraperitoneal injection of the radioactive tag. This is an impractical approach to studies such as ours due to the danger of diffuse cellular radiation damageI and the esorbitant cost of the material for introduction into a human, even if a subject were available. Furthermore, it has, to date, been impossible to specifically outline mitosis in thr vulvar epithelium either in the study material or in the routine laboratory preparations except in malignant conditions. Formulas, howevf,r, have been established to determine, with some degree of accuracy, the kgeneration time in various tissues. Using the formula.
as suggested by ,Johnson,14 and the standard synthesis time of 6 hours’j. Ifi as determined for the HeLa cells in vitro and for normal mouse intestine, the generation times for the tissues compared in this study are r+ corded in Table I with per cent labeled nuclei. As would be expected, these times corresponded gchnerally to the percentage of nuclear labeling. The turnover time is designated as that time necessary for complete replacement of the epithelium, that is the shortest elapsed time
from
the
initial
uptake
of
the
radio-
active tag in the basal layer to the appearance of labeled nuclei in the most superficial
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815
Fig. 11. Autoradiogram of tissue adjacent to invasive cancer showing labeling throughout all layers, however the associated infection must play a role here (incubation time, 1 hour).
Fig, 12. Autoradiogram of tissue taken from invasive cancer showing the absence of label in the pearls but diffuse activity in the disturbed more immature cells (incubation time, 30 minutes).
layer. Again, as with the generation time, this period has been determined in vivo by serially sacrificing animals. In vitro attempts to assess the turnover time in this study were made by incubation of the fresh tissue for varying periods of time from 15 minutes to 72 hours, and variations were noted in the penetration of the epithelium by labeled nuclei. For example, in 30 minutes there were such nuclei in the most superficial layer in cases of carcinoma in situ (Fig. 3). Such a finding may, however, represent the simultaneous metabolic activity of the immature cells throughout all layers in this anaplastic epithelium rather than an orderly progression of the basal cell to the surface by the normal processes of maturation. Furthermore, it was recognized that the tissues lose their cellular detail in approximately 30 hours and are probably reproductively inactive some time prior to this period. Better tissue culture methods may answer this problem of loss of viability. In spite of the difficulties in interpretation of results, particularly in the anaplasia, differences in penetration were noted between the normal vulvar epithelium, lichen sclerosus, and the normal-appearing tissue adjacent to the anaplasia. These results closely approximate those tabulated for per cent of nuclear labeling, but they are not as dramatic in degree. Particularly significant
however was the depth of penetration in the tissues adjacent to malignancy. Fig. 8 represents histologically normal tissue about 1 cm. from carcinoma in situ, but it reveals an increased percentage of labeled nuclei over that noted in other normals, as well as penetration of these nuclei through at least 50 per cent of the thickness of the epithelium in 3 hours. A comparable section from normal epithelium shows slightly less labeling and less penetration after 30 hours incubation (Fig. 9). Fig. 10 shows more striking changes and is immediately adjacent to the anaplastic epithelium in which cellular atypism can be readily recognized. Fig. 11 was taken in the vicinity of invasive cancer and shows nuclear labeling throughout all layers, In this section there is also striking infection, an added factor which is known to affect the metabolism of the cell. These findings, with proper evaluation, may offer a means of recognizing abnormal nuclear activity before the morphologic changes are visible if the results are confirmed in future studies. It was recognized that metabolic activity as determined by the percentage of labeled nuclei in total population was greater in the examples of carcinoma in situ than in cases of invasive disease. The very mature cells forming the keratin pearls demonstrated no labeling. The active and distorted cells
816
Woodruff
et al.
of the prekeratin and deeper zones, however, revealed striking activity (Fig. 12). Possible explanations for this “apparent” incongruity have been suggested earlier in this report. Summary
1. Metabolic activity of the vulvar epithelium, in the normal and in certain diseased states, was evaluated by incubation of the fresh tissue with tritiated precursors of ribonucleic acid (uracil) and deoxyribonucleic acid (thymidine) . 2. It was recognized that, in general, metabolic activity seemed to be directly correlated to the anaplastic activity. Invasive cancer was the lone exception.
its atrophic .cl. Lichen sclerosus, belying was metabolically active, as appearance recognized by the percentage of labeled cells which approach that noted in carcinoma in situ and invasive cancer. 4. Increased activity of normal-appearing skin adjacent to anaplastic lesions suggests a reason for the frequency of local recurrences, and may offer a means of evaluating the activity of the tissues at the limits of surgical excision. The authors would like to thank Mrs. Leah M. Carter and Mr. Richard Lewis, Jr., for their technical assistance and Mr. Raymond Lund for the excellent photography.
REFERENCES
Bonney, V.: Lancet 1: 1465, 1908. Taussig, F. J.: Diseases of the vulva, New York, 1923, D. Appleton and Company. 3. Hunt, Elizabeth: Disease Affecting the Vulva, St. Louis, 1954, The C. V. Mosby Company. 4. Clark, D. G. G., Zumoff, B., Brunschwig, A., and Hellman, L.: Cancer 13: 775, 1960. 5. Friedrichs, E. G., Julian, C. G., and Woodruff, J. D.: AM. J. OBST. & GYNEC. 90:
10.
1281, 1964. Lajtha, L. G., and Oliver, R.: Lab. Invest. 8: 214, 1959. Hughes, W. L., Bond, V. P., Brecher, G., Cronkhite, E. D., Quastler, H., and Sherman, F. G.: Proc. Nat. Acad. SC. 44: 476, 1958. Peckham, B., and Kiekhofer, Wm.: AM. J. OBST. & GYNEC. 83: 1021, 1962. Richart. R. M.: AM. J. OBST. & GYNEC. 86: 925, 1963.
14.
1. ?h.
6. 7.
8. 9.
11.
12. 13.
15. 16.
Lesher, S.: Fry, R. J. M., and Kohn, H. I.: Lab. Invest. 10: 291, 1959. Cronkhite, E. P., Bond, V. P., Fliedner, T. M., and Rubin, J. R.: Lab. Invest. 8: 263, 1959. LeBlond, C. P., Messier, B., and Kopriwa, B.: Lab. Invest. 8: 296, 1959. Oliver, R., and Lajtha, 1~. G.: Nature 186: 91, 1960. Johnson, H. A., and Bond, V. P.: Cancer 14: 639, 1961. Quastler, H., and Sherman, F. G.: Exper. Cell Res. 17: 420, 1959. Painter, R. B., and Drew, R. M.: Lab. Invest. 8: 278, 1959.
107 Cotswold Road Baltimore Ii?, Maryland
Discussion ARTHUR B. HUNT, Rochester, Minnesota. Although lichen sclerosus et atrophicus is admittedly a good example of atrophic change of the vulvar epithelium, it does have features that strongly suggest that it is a dermatologic entity of unknown etiology. These features are the peculiar “keyhole” distribution over the vulva and around the anus, the delling of the skin found at or near the periphery of the lesion, its relatively common appearance in the skin of other portions of the body, and its infrequent although not entirely rare occurrence in youthful patients. These features are wanting in kraurosis vulvac and simple senile atrophy as described
by
Adair. It would be interesting if Dr. Woodcould extend his studies in simple senih atrophy to include more than just one example of it-his Fig. 6. In this type of true atrophy he found cellular uptake to he the lowest in the entire study, as I interpret the photomicrographs. As a clinician I am neither knowledgeable nor experienced in the use of tritiated nucleic acid precursors, hut I feel, as do my colleagues who are experienced in this field, that one can only detrrmine that the percentage of cells that were labeled was considered to he higher than normal in the three diseases of the vulva. Any assumption that increased uptake of DNA prr-
ruff