Nuclear morphology and mitotic activity in the human endometrium observed in squash preparations

Nuclear morphology and mitotic activity in the human endometrium observed in squash preparations JERRY J. CARTER, PH.D. New York, New York

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E A R L Y detection of cancerous and precancerous lesions of the female reproductive tract has markedly increased with the screening of vaginal smears and exfoliative cytology. Great improvements have been made in screening projects with the smear technique following its use by Papanicolaou, 39-4 5 Papanicolaou and associates,4 7 - 49 Meigs and colleagues, 35 • 53 Gates and Warren, 26 and others. Cells from the vaginal and cervical regions have been studied extensively by smear methods. 1 •38 Although cells from the endometrium have been investigated, they have not been studied as thoroughly. Descriptions of normal cells from the endometrium and for the female genital tract generally as· described in sections are comprehensive. 5-7 • 19 • 49 Extensive accounts of endometrial cancer which are of great value in understanding the cytopathology of this organ have been described by Berg and Durfee9 and Boschann. 11 - 13 It is not always possible to detect total abnormal changes in cells which arise in

the endometrium and descend into the vagina. This is particularly true because of degeneration, red blood corpuscles, autolysis, and dilation of cells by the vaginal pool. 31 • 40 • 5° Cells which suggest but do not conform to the total picture of malignancy might result in confusing cytologic patterns. 30 It has been suggested 29 that before the true picture of the cell study of endometrial cancer can be brought into focus, further investigations should be made to differentiate the variability of normal cells from the variability of cancer cells. The aceto-orcein squash technique seems to be beneficial. Barigozzi and Dellepiane 3 employed the squash method as a tool in gynecologic cytology and explained that the procedure serves as a significant adjunct to the Papanicolaou smear method. They and their students2· 3• 20-25 found that the technique revealed nuclear details which permitted a high percentage of diagnostic accuracy. The technique was also used in studies of hyperplastic and neoplastic endometrial tissue by Barzilai and Oppenheim, 8 cyclic changes in endometrial nuclei by Glass, Barzilai and Oppenheim, 27 and was reported on favorably as a routine procedure in large public health screening programs by Carter, Barzilai, and Oppenheim. 14 The A and B nuclei, characteristic of cancer cells as described by Casperson and Santesson 17 and Casperson/ 5 • 16 and the mitotically active and inactive cells described by Koller 33 were found in these investigations.

From the Division of Cancer Control and Research, New York City Department of Health. Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Biology at Fordham University. Aided by a fellowship from the National Medical Fellowships Inc., through funds appropriated by The National Foundation.

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It would be useful to further investigate features which will eventually delineate differences in various cell types for diagnostic disposition. Cytologists and cytotechnicians might then be able to process more squashes and screen them routinely on the basis of few nuclear characteristics. The present study is concerned with variations in nuclear details of interphase nuclei in normal, atypical and neoplastic endometrial tissue as seen in squash preparations,

Fig. 1. Normal surface epithelial cell nuclei. Sex chromatin is distinct, seventh day of a normal menstrual cycle.

Fig. 2. The glandular epithelial nuclei on the left shows uniform distribution of chromatin. Sex chromatin is pronounced, ninth day of a normal 28 day cycle.

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and with the mitotic activity accompanying these changes in normal tissue.

Material and methods Two hundred and seventy-five cases w·ere studied. Nuclei from the tissue cells were classified as normaL atypical and/or suspicious for malignancy after screening. In 220 cases the tissue was classified as normal. One hundred and ninety-nine of these specimens were from actively menstruating patients and 21 were from menopausal patients. There were 39 cases which exhibited atypical and/or suspicious nonmalignant tissue cell nuclei. Sixteen cases involved tissues showing adenocarcinoma. Specimens were obtained through the Departments of Gynecology and Pathology at Long Island College Hospital and Harlem Hospital. Papanicolaou smears and histologic sections studied in this investigation were examined and controlled by members of the Department of Pathology at the Long Island College Hospital. Vaginal fluid was obtained from the endocervix and from the posterior fornix of the patients, smeared on clean glass slides, and immediately fixed in pqual parts of 95 per cent alcohol and ether. After the smears were obtained, endometrial microbiopsy specimens or aspirations were secured. Approximately half of the microbiopsy specimens were used for histologic sections and half for squash preparations. The portion used for sections was fixed m Zenker's fluid. Sections were cut at .f or 5 fL and stained in hematoxylin and eosin. Some cases were selected to determine the frequency of mitotic division. The total number of cells in division were counted in the surface epithelium, glandular epithelium, and connective tissue. Material used for squashes was stained in aceto-orceinhematoxylin made by refluxing 1 Grn. of natural orcein and 1 Gm. of hematoxylin in a 45 per cent solution of acetic acid for 6 hours. Specimens from each case were dropped into a vial containing 5 c.c. of the dye for approximately 1 hour. A small piece of tissue, approximately 0.5 to 1.0 sq. mm.,

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was placed on a clean glass slide and a drop of dye added. A clean cover glass was placed over the tissue which was teased out by gently tapping the cover glass with a dissecting needle. Aspirated cells were treated in the same manner after sedimentation. Squashing was performed by applying even pressure on the cover glass with the thumb. The nuclei were squashed as nearly as possible into one focal plane to reveal sharply outlined details. Kodak 35 mm. Panatomic-X film was used to obtain negatives of freshly prepared nuclei. The photographs were printed at a magnification of 2,500 diameters.

Observations and results The distinction between normal, atypical, suspicious, and malignant nuclei was made on the basis of ( 1 ) the number and arrangement of chromocenters and (2) the appearance of the nuclear background. From 1 to 4 small chromocenters were seen in normal nuclei. Nuclei exhibiting atypical features generally contained larger chromatin granules and 3 or more pronounced larger chromocenters. The nuclei suspicious for malignancy contained lobated, polarized chromatin, hyperchromatic chromocenters, and heterogeneous interiors. Malignant nuclei contained the greatest number of chromocenters, and presented the most incongruous nuclear chromatin backgrounds. Nuclei of cells from patients exhibiting normal endometrial tissue. The nuclear chromatin patterns appeared to vary with the influence of hormones on the menstrual cycle. During the menstrual and early postmenstrual periods, degenerative changes in chromatin were evident. Proliferative stage. In the early proliferative phase, surface and glandular epithelial nuclei were similar in appearance. The former were generally elliptical in shape. The latter appeared small, elliptical, spherical, and oval. The nuclei contained chromatin granules of medium size with 1 large and 2 or 3 smaller chromocenters; the glandular nuclei generally contained less (Figs. 1 and 2). Binucleate cells were some-

Nuclear morphology and mitotic activity 399

Fig. 3. Stromal nuclei from the compacta showing slightly enlarged chromatin granules and 3 or 4 distinct chromocenters, seventh day of a normal cycle.

Fig. 4. Surface epithelial nuclei showing small chromatin granules, fourteenth day of a normal cycle.

times present but rarely found. The sex chromatin mass described by Barr and coworkers4• 28 in animal and human tissue cells was seen throughout the cycle. It appeared closely appressed to the nuclear membrane and was present in 8 to 36 per cent of the nuclei (Figs. 1 and 2). Only stroma nuclei from the compacta exhibited cyclic changes. In the early proliferative phase, they appeared small with densely stained chromatin granules and 1 to 3 pronounced chromocenters (Fig. 3).

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During the late proliferative phase, surface epithelial nuclei were larger than in the early proliferative stage (Fig. 4). Chromatin granules were smaller at this stage, especially in the larger nuclei. Glandular epithelial nuclei stained less intensely during the late proliferative phase, contained small chromatin granules, and had regular membranes (Fig. 5). It was during this phase of the cycle that nuclei showed the greatest distention and variation in size. Large, oval nuclei with small chromatin granules were greater in number than

Fig. 5. Normal glandular epithelial nuclei. Note the absence of large distinct chromocenters, fourteenth day of a normal menstrual cycle.

Fig. 6. A large glandular epithelial nucleus showing a slightly thickened membrane. Note the size of this nucleus, seventeenth day of a normal menstrual cycle.

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at any other phase of the cycle. These nuclei contained practically no deeply stained heterochromatic regions. In some cases, the nuclear membranes were slightly thickened (Fig. 6). In instances of inflammation, nuclei showed maximum elongation at this stage. Chromatin granules were usually of equal size and evenly distributed within the nuclei (Fig. 7). Glandular nuclei contained only 1 or 2 pronounced chromocenters throughout the proliferative stage. During the late proliferative phase, stromal nuclei contained 1 or 2 chromocenters and had slightly thickened membranes. Secretory stage. In the secretory stage, nuclei from the surface epithelial cells did not appear very different from those seen in the late proliferative stage. Glandular epithelial nuclei of the early secretory phase contained unevenly distributed coarse chromatin, and 1 to 4 chromocenters which were slightly larger than during the proliferative stage. Nuclear details were usually more pronounced during this phase of the cycle (Fig. 8). Stromal nuclei from the compacta exhibited coarse chromatin granules and 3 or 4 chromocenters of even size during the secretory stage. Other stromal nuclei did not show cyclic variations but exhibited great variety in size and shape. Large nuclei which seemed to be extruding portions of chromatin were often encountered. Many contained chromatin polarized within the smaller regions of their large oval or leaf-shaped membranes (Fig. 9). Plasma cell nuclei (cartwheel or wagonwheel nuclei) were also present. Their chromatin appeared as dense hyperchromatic aggregations in close proximity to the nuclear membrane with 1 or 2 chromocenters in the interior (Fig. 10). Other nuclei such as histiocytes with enlarged chromocenters were often observed in the same microscopic field. Nuclei of cells from menopausal patients. Cyclic hormonal influence was not evident in patients exhibiting nuclei in this group. Early menopausal and postmenopausal cells

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contained nuclei which generally possessed the same characteristics. They differed from nuclei previously described, but were similar enough to be classified as menopausal nuclei. Chromatin granules of these nuclei were usually larger than those of cells from patients having active cyclic changes. The menopausal nuclei stained more intensely. Their membranes did not always exhibit elliptical, oval, or round contours. Small rounded nuclei with intensely stained granules resembled those from the compacta of early proliferating nuclei (Fig. 11). Many of the postmenopausal gland cell nuclei were round at one end, flattened on the other, and contained medium-sized chromatin granules which stained darkly, while others contained slightly larger granules (Fig. 12). Nuclei of cells exhibiting features atypical or suspicious for malignancy. Nuclei of this group did not show cyclic changes. Chromatin granules were larger and less regularly distributed than in normal nuclei. Bizarre-shaped, elongated, and irregular membranes were generally characteristic. The number of chromocenters increased to as many as 6 or 8 and occasionally more. They were usually more distinct than those seen in normal nuclei and many showed a tendency to aggregate in localized areas of the nucleus. Nuclear membranes were more irregular in nuclei of the atypical and suspicious cells than in those of normal cells. Some atypical and suspicious nuclei exhibited features not very different from normal, while others showed features of nuclei bordering on malignancy. Nuclei of binucleate and multinucleate cells, having small or medium-sized chromatin granules and slightly ragged interiors, were occasionally encountered. Several small atypical nuclei were lobated and had as many as 6 or 7 large chromocenters (Fig. 13). Suspicious nuclei with characteristics similar to the A type appeared in this group. Some contained as many as 8 chromocenters and exhibited heterogeneous backgrounds (Fig. 14) . Cancer cell nuclei. Nuclei present in malignant cells were similar to those described as atypical or suspicious. The anomalous

Nuclear morphology and mitotic activity 401

characteristics of the malignant cell nuclei were more extensive, however. Chromocenters showed fusion to a greater extent in cancer cell nuclei than in atypical or suspicious nuclei. In some nuclei from the cancerous group, as many as 20 chromocenters were seen. The distinction between atypical, suspicious, and cancerous nuclei could be made on the basis of number and size of chromocenters and the appearance of the nuclear background. The nuclear chromatin

Fig. 7. Glandular epithelial nuclei with slightly enlarged chromatin granules. Nuclei of this type were often seen in instances of inflammation, fifteenth day of a normal menstrual cycle.

Fig. 8. Glandular epithelial nuclei with an increased number of chromocenters distinctly outlined. The chromatin granules are slightly enlarged. The nuclear backgrounds are heterogeneous but not ragged, twenty-first day of a normal menstrual cycle.

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was less homogeneous in cancer nuclei than in atypical or suspicious nuclei. Many nuclei in the cancerous group possessed nuclear membranes with irregular outlines. Some, however, possessed fairly regular membranes. Nuclear chromatin of A type nuclei was heterogeneously distributed and the membranes generally regularly outlined (Fig. 15). The granules were often aggregated into 2 very large chromocenters surrounded by smaller chromatin granules giving the nuclei

Fig. 9. Small chromatin granules are distributed throughout the larger portion of this stromal cell nucleus; homogeneously dispersed granules are being extruded in the smaller portion.

Fig. 10. A plasma cell nucleus showing peripheral location of chromatin and a cartwheel or wagonwheel appearance.

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B type characteristics (Fig. 16). Intermediates between A and B types had heterogeneous backgrounds and irregular membranes with portions projected. Large multilobated nuclei with many chromocenters as well as grotesque elliptically shaped nuclei containing several large and small chromocenters were encountered. Mitotic activity. The rate of mitotic division could not be determined for all strata of the mucosa in all cases. Some tissue fragments were frayed and were not suitable for accurate counts. The frequency of mitotic divisions differed in each case studied. Glandular epithelial cells, as determined in sections, showed the highest number of mitotic divisions. Surface epithelial and stromal cells divided less frequently. In one case in which the patient had irregular bleeding, only 3 dividing cells were found in the entire tissue. The clinical diagnosis was a possible carcinoma. The lesion was cervical, however. Vaginal smears and squash preparations revealed endometrial nuclei with only slight atypical changes while cells from the cervical region showed nuclei of atypical and suspicious character. When the uterus was curetted, the Pathology Department reported that edipermization was present. No endometrial pathologic findings were reported. Other cases exhibited the same characteristics. The cervical aspects are reported here only to show these peculiarities. The absence of mitotic figures seemed to correspond to the histologic changes found within the tissues. The frequency of dividing endometrial cells and a case showing epidermization are given in Table I. All divisions appeared normal as in Fig. 17. On rare occasions, an anaphase division showing lagging chromosomes was observed. The number of metaphases found in each case was divided by the number of prophases to calculate the prophase index. Usually, a minimum of 100 dividing cells were counted. Ten cases from each group (normal, atypical, and/or suspicious, and cancer) were selected as shown in Table II. The values for cases containing normal cells ranged from 0.50 to 1.08 with a mean of 0.78. The

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Table I. Frequency of mitotic divisions in the proliferating endometrium in sections and in squashed preparations

No. of Dividing cells

Mitoses in squashes Total No. of Dividing cells

40 in 40,000 cells 0.1%

103 in 30,126 cells 0.34%

Mitoses in histologic sections Day of cycle

Case No.

Surface epithelium

I

Glandular epithelium

I

Stroma

No counts

I Total

Seventh

88

No counts

40 in 40,000 cells 0.1%

Ninth

93

No counts

41 in 28,980 cells 0.14%

13 in 17,320 cells 0.075%

54 in 46,300 cells 0.11%

118 in 69,428 cells 0.16%

Tenth

73

8 in 6,636 cells 0.12%

5 in 3,760 cells 0.132%

1 in 8,000 cells 0.012%

14 in 18,396 cells 0.07%

100 in 43,316 cells 0.23%

Eleventh

102

10 in 16,843 cells 0.059%

82 in 40,455 cells 0.2%

6 in 19,961 cells 0.03%

98 in 77,259 cells 0.11%

100 in 59,008 cells 0.17%

Fourteenth

126

3 in 10,262 cells 0.029%

35 in 10,390 cells 0.33%

5 in 10,094 cells 0.049%

43 in 30,746 cells 0.13%

61 in 23,353 cells 0.26%

1 in total count

2 in total count

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Epidermization; date-irregular

31

Table II. Frequency of prophase and metaphase divisions observed in squashes* Prophase

76 71 66 70 67 65 40 38 87 56

Index

38 39 41 44 50 56 35 36 85 61 Mean= 0.78

0.50 0.55 0.62 0.62 0.74 0.86 0.87 0.94 0.97 1.08

87 61 50 55 50 45 50 50 47 37

85 60 51 60 59 54 63 66 63 64 Mean= 1.20

0.97 0.98 1.02 1.09 1.18 1.20 1.26 1.32 1.34 1.72

64 40 60 37 40 30 46 31 23 34

82 61 100 67 82 69 111 83 79 145 Mean= 2.39

1.28 1.52 1.66 1.81 2.05 2.30 2.41 3.21 3.43 4.26

*The prophase index (metaphase/prophase) is given for 10 cases in each group.

values for cases contammg atypical and/or suspicious cells ranged from 0.97 to 1. 72 with a mean of 1.20. In cases with cancer cells, the values ranged from 1.28 to 4.26 with a mean of 2.39. Comment

Since screening procedures are primarily concerned with detecting cancer or lesions suspected of being cancerous, it is essential that normal and borderline cells be studied

and understood. The smear technique, while fast and accurate, is not completely suitable for such detailed study of nuclei. Supplementary methods which aid in screening projects are, therefore, advantageous. Techniques such as the Feulgen reaction, 34 ultraviolet cytochemical techniques/ 5 - 17 the cytoanalyzer,36• 37 • 55 fluorescent and interphase microscopy/ 0 • 18 • 32 • 52 phase microscopy, 56 • &T tissue culture methods, 46 and acetic squashing are now used in gynecologic cytology to

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Fig. 11. Early menopausal nuclei with mediumsized, darkly stained chromatin granules.

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Fig. 13. This small atypical m.cleus shows as many as 5 lobes and 6 large chromocenters. Note the thickened irregular membrane; from a patient with postmenopausal bleeding.

" Fig. 14. The enlarged atypical nucleus on the left exhibits a heterogeneous chromatin background, an irregular membrane and several chromocenters; from a patient with a history of an irregular discharge and a fibroid uterus.

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complement the smear method. Histology is the ultimate tool but it is not conducive to rapid screening of large numbers of patients, and the problem of obtaining sufficient material for sections is frequently encountered. 29 The squash technique reveals nuclear details which make screening easier, requires few cells, and can be used to complement smears or microbiopsies. Many of the technical obstacles which obscure cytologic patterns in smears are eliminated in squashes, thus facilitating establishment of simple criteria distinguishing normal and malignant cell nuclei. Koller 33 reported the maximum number or chromocenters in cancer nuclei as 36. He explained that smaller chromocenters might fuse forming larger ones. It appears that fusion does occur. Large chromocenters were seen which give evidence for this assumption. According to Koller, 33 the relative frequencies of mitotic stages might be taken as an indication of their relative duration. Timonen" 4 used the relative frequencies of metaphase and prophase divisions to calculate the prophase index and reported that this number differed in normal and cancerous endometrial tissues. Shortening of the prophase was reported as characteristic of dividing cancer cells. Studies on mitoses in the cervical epithelium during experimental inflammation and carcinogenesis in mice were conducted by Scarpelli and von Haam. 51 They stated that the increase in the prophase index values during the development of cervical carcinoma suggested a shortening of the prophase and lengthening of metaphase and malignancy. In the present investigation, the prophase index was lower in normal than in atypical or cancer cases. In normal cases, it was lower than that reported by Scarpelli and von Haam. However, the values reported by them for normal cases and those reported here are all less than unity. There was also an increase in values for cases containing atypical cells. Timonen suggested that the prophase in-

Nuclear morphology and mitotic activity 405

Fig. 15. A type nuclei with several distinct chromocenters, adenocarcinoma.

Fig. 16. B type nuclei showing condensation of chromatin, adenocarcinoma.

Fig. 17. Normal metaphase division, twelfth day of the menstrual cycle.

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dex was of theoretical importance. It appears that there is evidence for this assumption. With the progressive increase in prophase index values greater than unity, there might be some correlation between the prophase index and the presence of atypical and malignant nuclei. In cases where epidermization was present, however, few mitoses were encountered and atypical nuclei were observed. Further investigation on this subject appears necessary before any definite conclusions can be made. Summary

1. The morphology of nuclei from 275 cases of human uterine mucosa was investigated by use of the vaginal smear method, the aceta-orcein-hematoxylin squash technique and by histologic sections. The cytologic interpretations were based on the use of the squashed preparations. 2. Two types of chromatin patterns were seen in the nuclei based on the arrangement of the granules. In one type the granules appeared homogeneous with only a few small chromocenters and regular nuclear backgrounds were consistent. In the other type, the granules were larger and the small chromocenters gave the nucleus a heterogeneous background. Stromal nuclei of normal endometrial cells varied in shape an.d the chromatin generally was not homogeneously distributed. 3. During the menstrual cycle, nuclei of normal epithelial and stromal cells from the compacta of the endometrium underwent cyclic changes. In the early proliferative stage the chromatin appeared granular with 2 or 3 small chromocenters. During the late proliferative stage, distended nuclei with small chromatin granules were predominant. During the secretory stage, the nuclei showed small chromocenters. 4. Nuclei from cells of menopausal women

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contained chromatin granules largrr than those observed in nuclei of cells from women of childbearing age. The chromatin generally stained more intensely in nuclei from cells of menopausal women. 5. The regularly distributed chromatin pattern seen in normal nuclei disappeared in atypical nuclei and progressive anomalous changes were seen which were present in both atypical and malignant nuclei. The nuclei were often lobated and appeared to be hyperchromatic. 6. The patterns observed in nuclei classified as atypical were seen in malignant nuclei to a greater extent. The chromatin of malignant nuclei showed proliferative and regressive changes which corresponded to the A and B type nuclei described by Casperson and Santesson. Many nuclei contained 20 or more large chromocenters. 7. Mitotic activity was greater in glandular epithelial cells than in surface epithelial cells and stromal cells. The latter showed the least amount of mitotic divisions. In normal endometrial nuclei mitotic figures were normal. 8. In the normal endometrium, the prophase index values ranged from 0.50 to 1.08 with a mean of 0. 78. In tissue containing atypical cells the values ranged from 1.28 to 4.26 with a mean of 2.39. With the progressive increase of prophase index values above unity, there might be some correlation between the increase of the prophase index and the presence of atypical and malignant nuclei. This dissertation was done under the direction of Dr. Charles A. Berger in cooperation with Dr. Abraham Oppenheim, Director, Division of Cancer Control and Research, New York City Department of Health, the late Dr. Morris Glass, and Dr. Thomas G. Morrione at the Long Island College Hospital.

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