Original Paper Oncology 1992;49:358-362
3 Institut für Medizinische Strahlenbiologie und b Abteilung für Allgemeine Chirurgie der Chirurgischen Klinik und Poliklinik, Universitätsklinikum Essen; c Institut für Radiobiologie, Sanitätsakademie der Bundeswehr, München; d Chirurgische Klinik am Allgemeinen Krankenhaus Hamburg-Barmbeck, Hamburg, BRD
Flow Cytometric Analysis of Colorectal Mucosa from Patients with Crohn's Disease, Ulcerative Colitis and Cancer
K e y w o rd s
A b s tra c t
S-phase G 2-phase Proliferation Inflammation Age dependence
Samples of colorectal mucosa from patients with Crohn’s disease, ulcerative colitis and cancer were analyzed by means of flow cytometry. S- and Gr phase fractions were determined and mean values were calculated for different groups of patients. Almost identical results were obtained for inflamed and normal appearing mucosa from patients with Crohn's disease as well as inflamed mucosa from patients with ulcerative colitis. The mean S- and G2-phase frac tions in normal appearing mucosa from cancer patients, however, were signifi cantly higher. This seems to be due to the fact that patients with Crohn’s disease are between 15 and 45 years old, while cancer patients are mostly over 45. A detailed analysis of the S- and G,-phase fractions in different age groups re vealed a slight, but significant increase in colorectal proliferation between 25 and 75 years.
In trod u ction
posis [2, 9-11] and in patients with ulcerative colitis [12-16], both conditions being considered as precancerous. Typically, the mean labelling index was increased and a higher proportion of labelled cells appeared in the upper compartments of the intestinal crypts, which is not usually seen in healthy subjects. Very little is known about proliferative patterns in pa tients with Crohn’s disease. The ’H-thymidine labelling technique seems to have been used in only a few cases. Bleiberg et al. [6] observed changes as described above in 2
Enhanced proliferative activity of epithelial cells in the large bowel is generally assumed to be an indicator of a relatively high cancer risk [1-8]. A number of authors stu died 'H-thymidine incorporation and found characteristic changes not only in adenoma and carcinoma, but also in the normal appearing mucosa adjacent to such lesions [2-8]. More importantly, similar observations were made in symptom-free relatives of patients with familial poly
These investigations have been supported by ‘Deutsche Forschungsgemein schaft’ (SFB 102)
Dr. Friedo Zölzer Institut für Medizinische Strahlenbiologie Universitätsklinikum Essen Hufclandstrassc 55 D-W 4300 Essen (FRG)
0 1 9 9 2 S. Karger AG. Basel 0030-2414/92/ 0495-0358 $ 2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/14/2019 11:02:45 AM
Friedo Zölzer'' Christian Streffera Dirk van Benningena c Tamara Pelzera Eberhardt Gross M Friedrich- Wilhelm Eiglerh
M a te ria l and M e th o d s Twenty-four patients with Crohn’s disease were studied. In 6 cases two samples of inflamed mucosa were available, in another 10 cases one sample each of inflamed and normal appearing mucosa. We also analyzed material from 8 patients with ulcerative colitis and from 67 patients with different carcinoma of the large bowel. In the latter cases, only samples of normal appearing mucosa 5 cm away from the tum or were analyzed in the present study. Age. sex and sam pling site were recorded. Biopsies were taken during surgery. Histopathological diagnosis wascarried out in the Institute o f Pathology, Essen (Dir. Prof. L.-D. Leder). All samples of inflamed mucosa from patients with Crohn's disease showed edemas, fissures, and infiltration o f lymphocytes, plasma cells, macrophages as well as eosinophilic granulocytes. The inflammation extended into the submucosa and muscularis. No at tempt was made to stage the degree of inflammation. In 5 patients (seven samples) epithelial cell granulomas were observed, indicating a more severe level of disease. These samples did not show any par ticularity with respect to proliferation. In the cases of ulcerative co litis, varying degrees of inflammation were evident, but again no stag ing was attempted. Both more severe and less severe cases were seen to an equal extent. Biopsy material for the flow cytometric measurements was placed in physiological saline and stored at 4 C. As soon as possible (and no more than 24 h later), the material was cut into small pieces with scis
sors, minced through a 50 pm mesh, rinsed with physiological saline and fixed in 80% ethanol. For flow cytometric analysis, the cells were centrifuged, nuclei were isolated with pepsine (0,5% in 0.05 n HC1, pH 1.8, 10 min, room temperature), treated with RNAse (0.1% in 0.1 M Tris buffer, pH 7.4, 10 min, room temperature) and stained with ethidium iodide (2.5-10“5 M in Tris buffer). Measurements were performed with an impulse cytophotometer ICP 22 (Phywe, FRG). The DNA histograms were analyzed with a computer program developed by Dr. O. Ahrens (Labor für messtech nische Beratung und Entwicklung, Bargteheide. FRG). In principle, the background was approximated by an exponential curve and the cell cycle distribution fitted to thesum o f two Gaussian curves for the G r and G.-fractions and a broadened rectangle for the S-fraction. All values are given as the mean ± SD. Statistical significance was calculated using Student’s t test. Linear regression was compiled from individual data (instead o f means for certain age groups).
R e su lts
Our histograms had a reasonable quality with coeffi cients of variation between 2.9 and 6.7%. The percentage of S- and G2-phase cells was small in most cases, namely below 10% each, so that a certain scatter in the data is to be expected. Values for different samples from one and the same patient varied to a similar extent as values for sam ples from different patients. The mean percentage of S- and G,-phase cells was 5.4 + 1.8 and 8.1+ 2.6, respectively, for all cases of Crohn’s disease taken together (table 1). The sex of the patients did not seem to play a role, but dis crepancies were found between different age groups. While patients from 25 to 34 showed a relatively low percentage of S- and G 2-phase cells, those younger and older had slightly higher values. The differences of the latter groups to the one between 25 and 34 was significant (p < 0.05). Samples from inflamed mucosa had the same mean per centage of S- and G 2-phase cells as those from normal ap pearing mucosa of the same patients, and also results from patients with ulcerative colitis were identical (naturally, no material from affected mucosa was available in these cases). In the case of normal appearing mucosa from cancer patients, however, we found significantly higher values (p < 0.05), namely 6.4 + 2.5 and 9.3 + 2.8. Again, differ ences in sex and also in tumor location seemed to be ir relevant, but the mean percentage of S- and G2-phase cells was obviously related to age. Whereas the results for pa tients below 54 and above 75 did not differ from those for patients with Crohn’s disease, significantly higher values were found for the two groups in between (p < 0.05). Overall, there was a continuous increase (r = 0.359, p < 0.01) in the mean percentage of S- und G2-phase cells from the group between 25 and 34(4.7+ 1.5 and 6.2+ 2.1)
359
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patients, Terpsta et al. [7] did not find any significant in crease in labelling index or shift in the distribution of labelled cells in 6 patients. In view of the fact that Crohn’s disease as well as ulcerative colitis and familiar polyposis may be a precancerous condition, though probably less serious [17-20], the whole question needs further con sideration. Here we present data on proliferation in colorectal mucosa, as determined by means of flow cytometry. This method does not discriminate for epithelial cells, but even so should make possible the detection of abnormal growth kinetics [21,22]. We analyzed samples of inflamed mucosa from patients with Crohn’s disease and ulcerative colitis. For comparison, normal appearing mucosa was examined in cases of Crohn’s disease and various types of carcinoma. In this context, attention has to be paid to the question of age dependence. Patients with Crohn's disease are be tween 15 and 45 years old, while cancer patients are mostly over 45. There is evidence for an enhanced epithelial cell proliferation in normal elderly persons, similar to that ob served in patients with adenoma [23], Thus, the charac teristic changes described above may first of all be related to age and only under special circumstances, for example in thecase of ulcerative colitis or familial polyposis, appear earlier in life. We therefore studied proliferation in seven age groups from 15 to 85 years.
Fig. 1. Age dependence of (S + G :)-fraction. * = Crohn’s disease (samples from af fected and unaffected mucosa); + = cancer (samples from normal appearing mucosa).
n
S-fraction
G;-fraction
(S + G :)-fraction
30
5.4 ± 1.8
8.1 ± 2 .6
13.5 ± 3 .5
Unaffected mucosa Affected mucosa (Paired samples from the same patients)
10 10
5.6 ± 1.5 5.0 ± 1.9
7.2 ± 2 .3 7.4 ±3.1
12.7 ± 3 .0 12.4 ± 4 .7
Male Female (Paired samples from patients of the same age)
12 12
4.9 ± 1.7 5.2 ± 1.7
7.1 ± 2 .8 8.3 ± 2 .0
12.0 ± 4 .0 13.5 ± 3 .0
15-24 years 25-34 years 35-44 years (Including samples of unaffected mucosa)
15 15 10
5.8 ± 2 .0 4.7 ± 1.6 6 .0 + 1.3
8.8 ± 2 .3 6.2 ±2.1 7.8 ± 2.0
14.6 ± 3 .6 10.9 ± 2 .6 13.8 ± 2 .0
8
5.6 ± 1.9
6.8 ±2.1
12.4 ± 2 .4
67
6.4 ± 2 .5
9.3 ± 2 .8
15.7 ± 3 .5
Male Female (Paired samples from patients o f the same age)
22 22
6.4 ± 2 .5 6.6 ± 2 .6
9.0 ± 2 .8 9.6 ± 2 .2
15.4 ± 4 .3 16.2 ± 3 .2
Colon Rectum (Paired samples from patients of the same age)
16 16
6.0 ± 2.3 6.5 ± 3 .0
8.9 ± 2 .6 9.6 ± 3 .6
14.9 ± 3 .8 16.1 ± 6 .0
35-44 years 45-54 years 55-64 years 65-74 years 75-84 years
3 12 20 20 12
5.7 ± 0 .8 6.0 ± 2 .2 6.5 ± 2 .7 7.2 ± 2 .5 5.2 ± 2 .5
8.4 ± 3.7 9.0 ± 1.9 8.9 ± 3 .5 9.7 ± 2 .9 9.8 ± 2.0
14.1 ± 2 .8 15.0 + 2.9 15.4 ± 5 .3 16.9 ± 4 .2 15.0 ± 2 .8
Crolm "s disease All samples (including two samples each from 6 patients)
Ulcerative colitis All samples Cancer ( normal appearing mucosa ) All samples
360
Zölzer/Streffer/van Beuningen/Pelzer/ Gross/Eigler
Flow Cytometric Analysis of Colorectal Mucosa
Downloaded by: King's College London 137.73.144.138 - 1/14/2019 11:02:45 AM
Table 1. Mean percentages of S- and G 2-phase cells for different groups of patients
to the one between 65 and 74 (7.2 ±2.5 and 9.7 ± 2.9), while the patients below 24 and over 75 showed intermediate values (fig. 1,2).
D is c u ss io n
Our data are in general agreement with those of others, who found labelling indices between 5 and 20%. The mean percentage of S-phase cells determined by flow cytometry tend towards somewhat lower values, perhaps due to the fact that biopsy material contains a certain amount of non-epithelial cells [21], As mentioned above, this does not seem to preclude the detection of abnormal growth ki netics [22], It is therefore rather surprising that the mean percentage of S-phase cells was not particularly high in the case of ulcerative colitis, where increased labelling indices have been reported by others. Changes in proliferative ac tivity, however, seem to be related to the stage of the di sease, so that general conclusions cannot be based on just 8 patients. In the case of Crohn’s disease, it is clear from our results that inflammation alone has no influence on growth kinet ics, because samples of affected and unaffected mucosa were taken from the same patients. We had no opportunity to examine healthy subjects, so that the values reported here may still be elevated above normal levels. This seems unlikely, but needs further investigation. As far as the age dependence of proliferation is con cerned, our data support those of Roncucci et al. [23], who found an increased labelling index and an altered distri
Mean age of group, years
bution of labelled cells in normal elderly persons. These changes may constitute a precancerous condition and for no other reason be typically observed in the unaffected mucosa of cancer patients, as noticed in the Introduction. We therefore suspected that the comparatively lower pro liferation in the case of Crohn’s disease observed here was not primarily related to disease. Indeed our analysis led to similar results, where we compared people of about the same age.
361
Downloaded by: King's College London 137.73.144.138 - 1/14/2019 11:02:45 AM
Fig. 2. Age dependence of (S + G,)-fraction (mean values). G roups are as described in table 1 and symbols as in figure 1.
1 Desehner F.F.. Lewis CM. Lipkin M: In vitro study of human rectal epithelial cells. I. Atypi cal zone of ’H-thymidine incorporation in mucosa of multiple polyposis. J Clin Invest 1963;42:1922-1928. 2 Desehner EE. Lipkin M. Solomon C: Study of human rectal epithelial cells in vitro. II. 'IIthymidine incorporation into polyps and adja cent mucosa. J Natl Cancer Inst 1966:36: 849-857. 3 Lipkin M: Phase 1 and phase 2 proliferative le sions ofcolonic epithelial cells in diseases lead ing to colonic cancer. Cancer 1974;34:878-888. 4 Maskens AP, Desehner F.F.: Tritiatcd thy midine incorporation into epithelial cells of normal-appearingcolorcctal mucosa of cancer patients. J Natl Cancer Inst 1977:58:12211224. 5 Bleiberg H. Salhadin A. Galand P: Cell cycle parameters in human colon. Comparison be tween primary and recurrent adenocar cinomas. benign polyps and adjacent unaffect ed mucosa. Cancer 1977:39:1190-1194. 6 Bleiberg 11. Buyse M. Galand P: Cell kinetic in dicators of premalignant stages of colorectal cancer. Cancer 1985:56:124 129. 7 Terpsta OT. van Blankenstein M. Dees J. Eilers GAM: Abnormal pattern of cell pro liferation in the entire colonic mucosa of pa tients with colon adenoma or cancer. Gas troenterology 1987:92:704-708.
8 Strcfler C, van Bcuningen D. Gross E. Eigler FW. Pelzer T: Determination of DNA, mi cronuclei and vascular density in human rec tum carcinomas; in Chapman JD. Peters LJ, Withers H R(eds): Prediction of Tumor Treat ment Response. New York. Pcrgamon Press. 1989, pp 2 17-226. 9 Lipkin M: Biomarkers of increased suscep tibility to gastrointestinal cancer. Their de velopment and application to studies of cancer prevention. Gastroenterology 1987:92:1083— 1086. 10 Bleiberg H. Mainguet P, Galand P: Cell re newal in familial polyposis: Comparison be tween polyps and adjacent healthy mucosa. Gastroenterology 1972:63:240 245. 11 Desehner EE. Lipkin M: Proliferative patterns in colonic mucosa in familial polyposis. Cancer 1975;35:413-418. 12 Bleiberg H. Mainguet P. Galand P. Chretien J, Dopont-Maircssc N: Cell renewal in the hu man rectum. In vitro autoradiographic study on active ulcerative colitis. Gastroenterology 1970:58:851-855. 13 Eastwood G L. Trier J S: Epithelial cell renewal in cultured rectal biopsies in ulcerative colitis. Gastroenterology 1973;64:383-390. 14 Serafmi EP. Kirk AP. ChambersTJ: Rate and pattern of epithelial cell proliferation in ulcera tive colitis. Gut 1981;22:648-652. 15 Lehy T. Mignon M, Abitbol J L: Epithelial cell proliferation in the rectal stump of patients with ileorectal anastomosis for ulcerative co litis. Gut 1983:24:1048-1056.
16 Franklin WA. McDonaldGB.Stein HO,Gut ter KC, Jewell DP. Clarke LC, Mason DY: Immunohistologic demonstration of abnor mal colonic crypt cell kinetics in ulcerative co litis. Hum Pathol 1985:16:1129-1132. 17 Greenstein A J, Sachar D B, Smith H. Janowitz HD, Aufses AH: A comparison of cancer risk in Crohn's disease and ulcerative colitis. Can cer 1981;48:2742-2745. 18 Glotzer DJ: The risk of cancer in Crohn’s dis ease. Gastroenterology 1985:89:438-441. 19 Allen DC. Hughes DF, Calvert CH: Car cinoma in Crohn's disease of the colon. Dis Colon Rectum 1968;29:760-765. 20 Petras RE, Mir-Madjlessi SH, Farmer RG: Crohn's disease and intestinal cancer. A report of 11 cases with emphasis on associated epi thelial dysplasia. Gastroenterology I987;93: 1307-1314. 21 Cheng H, Bjerknes M. Amar J: Methods for the determination of epithelial cell kinetic parameters of human colonic epithelium iso lated from surgical and biopsy specimens. Gas troenterology 1984;86:78-85. 22 Borkje B. Hostmark J. Skagen DW. Schrumpf E. Maerum OD: Flow cytometry of biopsy spec:mens from ulcerative colitis, colorectal adenomas, and carcinomas. J Gastroenterol 1987;22:1231-1237. 23 Roncucci L, Ponz de Leon M. Scalmati A, Malagoli G. Pratissolli S. Perini M. Chahin N: The influence of age on colonic epithelial cell proliferation. Cancer 1988:62:2373-2377.
362
Zolzer/Streffer/van Beuningen/Pelzer Gross/Eigler
Flow Cytometric Analysis of Colorectal Mucosa
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R eferences
3 Institut für Medizinische Strahlenbiologie und b Abteilung für Allgemeine Chirurgie der Chirurgischen Klinik und Poliklinik, Universitätsklinikum Essen; c Institut für Radiobiologie, Sanitätsakademie der Bundeswehr, München; d Chirurgische Klinik am Allgemeinen Krankenhaus Hamburg-Barmbeck, Hamburg, BRD
Flow Cytometric Analysis of Colorectal Mucosa from Patients with Crohn's Disease, Ulcerative Colitis and Cancer
K e y w o rd s
A b s tra c t
S-phase G 2-phase Proliferation Inflammation Age dependence
Samples of colorectal mucosa from patients with Crohn’s disease, ulcerative colitis and cancer were analyzed by means of flow cytometry. S- and Gr phase fractions were determined and mean values were calculated for different groups of patients. Almost identical results were obtained for inflamed and normal appearing mucosa from patients with Crohn's disease as well as inflamed mucosa from patients with ulcerative colitis. The mean S- and G2-phase frac tions in normal appearing mucosa from cancer patients, however, were signifi cantly higher. This seems to be due to the fact that patients with Crohn’s disease are between 15 and 45 years old, while cancer patients are mostly over 45. A detailed analysis of the S- and G,-phase fractions in different age groups re vealed a slight, but significant increase in colorectal proliferation between 25 and 75 years.
In trod u ction
posis [2, 9-11] and in patients with ulcerative colitis [12-16], both conditions being considered as precancerous. Typically, the mean labelling index was increased and a higher proportion of labelled cells appeared in the upper compartments of the intestinal crypts, which is not usually seen in healthy subjects. Very little is known about proliferative patterns in pa tients with Crohn’s disease. The ’H-thymidine labelling technique seems to have been used in only a few cases. Bleiberg et al. [6] observed changes as described above in 2
Enhanced proliferative activity of epithelial cells in the large bowel is generally assumed to be an indicator of a relatively high cancer risk [1-8]. A number of authors stu died 'H-thymidine incorporation and found characteristic changes not only in adenoma and carcinoma, but also in the normal appearing mucosa adjacent to such lesions [2-8]. More importantly, similar observations were made in symptom-free relatives of patients with familial poly
These investigations have been supported by ‘Deutsche Forschungsgemein schaft’ (SFB 102)
Dr. Friedo Zölzer Institut für Medizinische Strahlenbiologie Universitätsklinikum Essen Hufclandstrassc 55 D-W 4300 Essen (FRG)
0 1 9 9 2 S. Karger AG. Basel 0030-2414/92/ 0495-0358 $ 2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/14/2019 11:02:45 AM
Friedo Zölzer'' Christian Streffera Dirk van Benningena c Tamara Pelzera Eberhardt Gross M Friedrich- Wilhelm Eiglerh
M a te ria l and M e th o d s Twenty-four patients with Crohn’s disease were studied. In 6 cases two samples of inflamed mucosa were available, in another 10 cases one sample each of inflamed and normal appearing mucosa. We also analyzed material from 8 patients with ulcerative colitis and from 67 patients with different carcinoma of the large bowel. In the latter cases, only samples of normal appearing mucosa 5 cm away from the tum or were analyzed in the present study. Age. sex and sam pling site were recorded. Biopsies were taken during surgery. Histopathological diagnosis wascarried out in the Institute o f Pathology, Essen (Dir. Prof. L.-D. Leder). All samples of inflamed mucosa from patients with Crohn's disease showed edemas, fissures, and infiltration o f lymphocytes, plasma cells, macrophages as well as eosinophilic granulocytes. The inflammation extended into the submucosa and muscularis. No at tempt was made to stage the degree of inflammation. In 5 patients (seven samples) epithelial cell granulomas were observed, indicating a more severe level of disease. These samples did not show any par ticularity with respect to proliferation. In the cases of ulcerative co litis, varying degrees of inflammation were evident, but again no stag ing was attempted. Both more severe and less severe cases were seen to an equal extent. Biopsy material for the flow cytometric measurements was placed in physiological saline and stored at 4 C. As soon as possible (and no more than 24 h later), the material was cut into small pieces with scis
sors, minced through a 50 pm mesh, rinsed with physiological saline and fixed in 80% ethanol. For flow cytometric analysis, the cells were centrifuged, nuclei were isolated with pepsine (0,5% in 0.05 n HC1, pH 1.8, 10 min, room temperature), treated with RNAse (0.1% in 0.1 M Tris buffer, pH 7.4, 10 min, room temperature) and stained with ethidium iodide (2.5-10“5 M in Tris buffer). Measurements were performed with an impulse cytophotometer ICP 22 (Phywe, FRG). The DNA histograms were analyzed with a computer program developed by Dr. O. Ahrens (Labor für messtech nische Beratung und Entwicklung, Bargteheide. FRG). In principle, the background was approximated by an exponential curve and the cell cycle distribution fitted to thesum o f two Gaussian curves for the G r and G.-fractions and a broadened rectangle for the S-fraction. All values are given as the mean ± SD. Statistical significance was calculated using Student’s t test. Linear regression was compiled from individual data (instead o f means for certain age groups).
R e su lts
Our histograms had a reasonable quality with coeffi cients of variation between 2.9 and 6.7%. The percentage of S- and G2-phase cells was small in most cases, namely below 10% each, so that a certain scatter in the data is to be expected. Values for different samples from one and the same patient varied to a similar extent as values for sam ples from different patients. The mean percentage of S- and G,-phase cells was 5.4 + 1.8 and 8.1+ 2.6, respectively, for all cases of Crohn’s disease taken together (table 1). The sex of the patients did not seem to play a role, but dis crepancies were found between different age groups. While patients from 25 to 34 showed a relatively low percentage of S- and G 2-phase cells, those younger and older had slightly higher values. The differences of the latter groups to the one between 25 and 34 was significant (p < 0.05). Samples from inflamed mucosa had the same mean per centage of S- and G 2-phase cells as those from normal ap pearing mucosa of the same patients, and also results from patients with ulcerative colitis were identical (naturally, no material from affected mucosa was available in these cases). In the case of normal appearing mucosa from cancer patients, however, we found significantly higher values (p < 0.05), namely 6.4 + 2.5 and 9.3 + 2.8. Again, differ ences in sex and also in tumor location seemed to be ir relevant, but the mean percentage of S- and G2-phase cells was obviously related to age. Whereas the results for pa tients below 54 and above 75 did not differ from those for patients with Crohn’s disease, significantly higher values were found for the two groups in between (p < 0.05). Overall, there was a continuous increase (r = 0.359, p < 0.01) in the mean percentage of S- und G2-phase cells from the group between 25 and 34(4.7+ 1.5 and 6.2+ 2.1)
359
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patients, Terpsta et al. [7] did not find any significant in crease in labelling index or shift in the distribution of labelled cells in 6 patients. In view of the fact that Crohn’s disease as well as ulcerative colitis and familiar polyposis may be a precancerous condition, though probably less serious [17-20], the whole question needs further con sideration. Here we present data on proliferation in colorectal mucosa, as determined by means of flow cytometry. This method does not discriminate for epithelial cells, but even so should make possible the detection of abnormal growth kinetics [21,22]. We analyzed samples of inflamed mucosa from patients with Crohn’s disease and ulcerative colitis. For comparison, normal appearing mucosa was examined in cases of Crohn’s disease and various types of carcinoma. In this context, attention has to be paid to the question of age dependence. Patients with Crohn's disease are be tween 15 and 45 years old, while cancer patients are mostly over 45. There is evidence for an enhanced epithelial cell proliferation in normal elderly persons, similar to that ob served in patients with adenoma [23], Thus, the charac teristic changes described above may first of all be related to age and only under special circumstances, for example in thecase of ulcerative colitis or familial polyposis, appear earlier in life. We therefore studied proliferation in seven age groups from 15 to 85 years.
Fig. 1. Age dependence of (S + G :)-fraction. * = Crohn’s disease (samples from af fected and unaffected mucosa); + = cancer (samples from normal appearing mucosa).
n
S-fraction
G;-fraction
(S + G :)-fraction
30
5.4 ± 1.8
8.1 ± 2 .6
13.5 ± 3 .5
Unaffected mucosa Affected mucosa (Paired samples from the same patients)
10 10
5.6 ± 1.5 5.0 ± 1.9
7.2 ± 2 .3 7.4 ±3.1
12.7 ± 3 .0 12.4 ± 4 .7
Male Female (Paired samples from patients of the same age)
12 12
4.9 ± 1.7 5.2 ± 1.7
7.1 ± 2 .8 8.3 ± 2 .0
12.0 ± 4 .0 13.5 ± 3 .0
15-24 years 25-34 years 35-44 years (Including samples of unaffected mucosa)
15 15 10
5.8 ± 2 .0 4.7 ± 1.6 6 .0 + 1.3
8.8 ± 2 .3 6.2 ±2.1 7.8 ± 2.0
14.6 ± 3 .6 10.9 ± 2 .6 13.8 ± 2 .0
8
5.6 ± 1.9
6.8 ±2.1
12.4 ± 2 .4
67
6.4 ± 2 .5
9.3 ± 2 .8
15.7 ± 3 .5
Male Female (Paired samples from patients o f the same age)
22 22
6.4 ± 2 .5 6.6 ± 2 .6
9.0 ± 2 .8 9.6 ± 2 .2
15.4 ± 4 .3 16.2 ± 3 .2
Colon Rectum (Paired samples from patients of the same age)
16 16
6.0 ± 2.3 6.5 ± 3 .0
8.9 ± 2 .6 9.6 ± 3 .6
14.9 ± 3 .8 16.1 ± 6 .0
35-44 years 45-54 years 55-64 years 65-74 years 75-84 years
3 12 20 20 12
5.7 ± 0 .8 6.0 ± 2 .2 6.5 ± 2 .7 7.2 ± 2 .5 5.2 ± 2 .5
8.4 ± 3.7 9.0 ± 1.9 8.9 ± 3 .5 9.7 ± 2 .9 9.8 ± 2.0
14.1 ± 2 .8 15.0 + 2.9 15.4 ± 5 .3 16.9 ± 4 .2 15.0 ± 2 .8
Crolm "s disease All samples (including two samples each from 6 patients)
Ulcerative colitis All samples Cancer ( normal appearing mucosa ) All samples
360
Zölzer/Streffer/van Beuningen/Pelzer/ Gross/Eigler
Flow Cytometric Analysis of Colorectal Mucosa
Downloaded by: King's College London 137.73.144.138 - 1/14/2019 11:02:45 AM
Table 1. Mean percentages of S- and G 2-phase cells for different groups of patients
to the one between 65 and 74 (7.2 ±2.5 and 9.7 ± 2.9), while the patients below 24 and over 75 showed intermediate values (fig. 1,2).
D is c u ss io n
Our data are in general agreement with those of others, who found labelling indices between 5 and 20%. The mean percentage of S-phase cells determined by flow cytometry tend towards somewhat lower values, perhaps due to the fact that biopsy material contains a certain amount of non-epithelial cells [21], As mentioned above, this does not seem to preclude the detection of abnormal growth ki netics [22], It is therefore rather surprising that the mean percentage of S-phase cells was not particularly high in the case of ulcerative colitis, where increased labelling indices have been reported by others. Changes in proliferative ac tivity, however, seem to be related to the stage of the di sease, so that general conclusions cannot be based on just 8 patients. In the case of Crohn’s disease, it is clear from our results that inflammation alone has no influence on growth kinet ics, because samples of affected and unaffected mucosa were taken from the same patients. We had no opportunity to examine healthy subjects, so that the values reported here may still be elevated above normal levels. This seems unlikely, but needs further investigation. As far as the age dependence of proliferation is con cerned, our data support those of Roncucci et al. [23], who found an increased labelling index and an altered distri
Mean age of group, years
bution of labelled cells in normal elderly persons. These changes may constitute a precancerous condition and for no other reason be typically observed in the unaffected mucosa of cancer patients, as noticed in the Introduction. We therefore suspected that the comparatively lower pro liferation in the case of Crohn’s disease observed here was not primarily related to disease. Indeed our analysis led to similar results, where we compared people of about the same age.
361
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Fig. 2. Age dependence of (S + G,)-fraction (mean values). G roups are as described in table 1 and symbols as in figure 1.
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Flow Cytometric Analysis of Colorectal Mucosa
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R eferences
