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Significance of lymphocytic sister chromatid exchange frequencies in ovarian cancer patients
ELSEVIER
Significance of Lymphocytic Sister Chromatid Exchange Frequencies in Ovarian Cancer Patients Pawan Kumar Dhar, Sulochna Devi, T. Ramesh Rao, Usha Kumari, Anita Joseph, M. R. Kumar, Satish Nayak, Y. Shreemati, S. M. Bhat, and K. R. Bhat
ABSTRACT: Very f e w studies report the analysis of sister chromatid exchanges in ovarian cancer patients. We tested the null hypothesis that SCE frequency increases with the advancing stages of ovarian cancer and Jbllows a Poisson distribution. As controls we examined age- and sex-matched healthy volunteers who had no such past history. An increased average SCE frequency was observed in ovarian cancer patients (6.34 ± 0.09) vis-&-vis controls (4.47 ± 0.12). Further, the data also suggested a stagewise increase in the SCE frequency. INTRODUCTION Sister chromatid exchanges are reciprocal exchanges of DNA segments between sister chromatids at identical loci [1] during S-phase of the cell cycle. The exact mechanism underlying SCE formation is still unclear, although its significance is well understood [2]. It has been suggested that female subjects are more influenced by biologic rhythms because of specific hormonal cycles and are sensitive to genetic damage during the menstrual cycle [3, 4]. Although many external agents are known to cause SCEs, in their absence SCEs are formed, which leads one to the conclusion that they are an integral part of DNA replication [5]. Due to the paucity of literature on SCEs in ovarian cancer patients, there is no information about their diagnostic importance in these patients. However, a higher spontaneous SCE rate in ovarian cancer patients has been reported [6].
Patients in their menstrual cycle were also omitted for analysis. The control group included 12 sex- and agematched healthy volunteers. Tumors were classified into various stages based on standard guidelines [7].
Cell and Culture Conditions
MATERIALS AND METHODS
Blood culture, harvesting, and slide preparation were done according to the established protocol [8], with some modifications. Heparinized whole blood (0.4 mL) was cultured in 10 mL of RPMI 1640 medium (without serum) supplemented with phytohemagglutinin and antibiotics. Bromodeoxyuridine was added at 0 hours at a final concentration of 10 ~g/mL. Cultures were incubated at 37°C in 5% CO2 in complete darkness, 68 hours, after which Colcemid (0.4 ~g/mL) was added to the cultures and incubation continued for another 2 hours. The cultures were harvested at 70 hours and slides prepared by the air-dry method. Chromosomes were stained by the FPG technique [9].
Subjects and Classification
Analysis and Statistical Methods
All 38 cancer patients selected in the present study were between 39 and 63 years of age (Table 1). Most of them under treatment were excluded from the research group.
Exchanges between the two interstitial chromatids were considered as two SCEs and terminal exchanges as one [10]. Analysis of the data was performed using Descriptive Statistics and one-way ANOVA method of SPSS-PC (SPSS Inc., Chicago, IL).
From the Departments of Anatomy (Human Genetics Section) (P.K.D., T.R.R.,A.J.,M.R.K., S.N., S.M.B.,K.R.B.),Physiology (S. D., U. K.), and Medical Education (Y S.), Kasturba Medical College, Manipal, Karnataka, India. Address reprint requests to: Pawan Kumar Dhar, Ph.D., Assistant Professor and Head, Human Genetics Section, Department of Anatomy, Kasturba Medical College, Manipal, Karnataka 576 119, India. Received November 14, 1994; accepted March 20, 1995. Cancer Genet Cytogenet 8 9 : 1 0 5 - 1 0 8 (1996) © Elsevier Science Inc., 1996 655 Avenue of the Americas, N e w York, NY 10010
RESULTS Suitable data were available from a total of 38 cancer patients and 12 normal controls. In aggregate, there were 10,025 SCEs in 1665 metaphases. The mean, SCE range, variance, and H value of all cancer cases (stage-wise) and controls are shown in Table 1. An average value of 6.34 ±
0165-4608/96/$15.00 SSDI 0165-4608(95)00071-V
106
Table 1.
P.K. Dhar et al.
Mean, range, variance, a n d H-values of SCE in ovarian cancer patients and control
SL. N o .
Patient Code
Cell Nos.
Total SCE
M e a n _+ S.E.
SCE Range
Stage I 01. 02.
HGS-05 HGS-06
30 40
152 216
5.07 ± 0.48 5 . 4 0 Jr 0 . 4 0
1-10 1-11
6.82 6.55
1.34 1.21
HGS-14 HGS-20 HGS-12 HGS-25 HGS-26 HGS-22
36 50 43 26 33 24
194 277 211 146 195 130
5.39 5.54 4.91 5.62 5.91 5.42
± 0.67 _+ 0 . 5 4 ± 0.43 ± 0.45 ± 0.43 ± 0.50
1-14 1-16 1-10 1-9 2-11 1-11
16.30 14.70 7.84 5.20 6.08 6.08
3.03 2.80 1.60 0.92 1.03 1.12
HGS-34 HGS-37 HGS-11 HGS-40 HGS-23 HGS-42 HGS-10 HGS-45
44 43 47 22 20 20 29 36
241 258 278 124 110 143 150 241
5.48 6.00 5.91 5.64 5.50 7.15 5.17 6.69
± 0.46 ± 0.57 ± 0.39 _+ 0 . 5 8 ± 0.59 ± 0.81 ± 0.44 ± 0.41
2-13 1-15 2-12 2-10 1-10 3-13 2-10 2-10
9.18 14.14 7.21 7.29 6.89 13.18 5.57 6.10
1.67 2.35 1.30 1.21 1.25 1.84 1.07 0.91
HGS-38 ° HGS-02 HGS-03 HGS-51 HGS-21 HGS-30 HGS-13 HGS-04 HGS-49 HGS-48
64 24 27 25 39 45 21 22 50 65
409 135 201 185 271 267 134 156 325 421
6.39 5.63 7.44 7.40 6.95 5.93 6.38 7.09 6.50 6.48
_+0.33 ± 0.60 ± 0.65 ± 0.51 ± 0.53 ± 0.57 ± 0.68 ± 0.64 -+ 0 . 5 3 ± 0.57
1-10 1-10 1-12 3-13 1-13 1-17 1-14 2-12 1-14 1-14
6.94 8.59 11.48 6.41 11.15 14.56 9.64 8.94 14.25 20.97
1.08 1.52 1.54 0.86 1.60 2.45 1.51 1.26 2.19 3.24
HGS-01 HGS-28 HGS-43 a HGS-07 HGS-44 HGS-24 HGS-27 HGS-08 a HGS-41 HGS-15 HGS-35 HGS-19
35 63 60 38 32 38 25 40 35 32 30 24
288 435 366 324 262 235 202 229 252 193 185 194
8.23 6.90 6.10 8.53 8.19 6.18 8.08 5.73 7.20 6.03 6.17 8.08
± ± ± ± ± ± ± ± ± ± ± ±
0.64 0.52 0.48 0.63 0.45 0.62 0.67 0.66 0.55 0.68 0.51 0.74
1-13 1-13 1-12 2-13 3-11 1-13 3-13 1-12 1-12 1-12 1-12 1-13
14.29 16.92 13.65 14.90 6.60 14.74 11.07 17.43 10.51 14.67 7.79 13.29
1.73 2.36 2.23 1.74 0.80 2.38 1.37 3.04 1.46 2.43 1.26 1.64
NOR-01 NOR-02 NOR-03 NOR-04 NOR-05 NOR-06 NOR-07 NOR-08 NOR-09 NOR-IO NOR-11 NOR-12
19 31 22 20 22 31 18 20 27 19 38 21
84 113 105 93 102 154 73 84 139 74 174 95
4.42 3.65 4.77 4.65 4.64 4.97 4.06 4.20 5.15 3.89 4.58 4.52
± ± ± ± ± ± ± ± ± ± ± ±
0.67 0.50 0.51 0.56 0.48 0.27 0.42 0.30 0.36 0.34 0.37 0.30
2-11 1-9 1-10 1-10 1-12 3-8 1-7 1-6 1-9 1-7 2-12 1-6
8.59 7.70 5.61 6.43 5.00 2.29 3.23 1.85 3.59 2.21 5.33 1.86
1.94 2.11 1.17 1.36 1.07 0.46 0.79 0.44 0.69 0.56 1.16 0.41
03. 04. 05. 06. 07. 08. S t a g e II 01. 02. 03. 04. 05. 06. 07. 08. S t a g e III 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. S t a g e IV 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. Control (age & sex matched) 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12.
~Indicates those patients w h o were u n d e r treatment at the time of c h r o m o s o m a l analysis.
Variance
H Value
SCE F r e q u e n c y in Ovarian Cancer
Table 2
107
A n overall statistical c o m p a r i s o n of cancer patients a n d control Stages of cancer Control
Control Stage I Stage II Stage III Stage IV
I
II
III
IV
* * * *
*Denotes groups with statistically significant (p < 0.05) difference in SCE frequency
0.09 was observed in 38 cancer cases regarding the control of 4.47 -+ 0.12 observed in 12 subjects. Figure 1 shows a change in the m e a n frequency _ SEM of SCEs from controis to cancer patients (stage-wise). The average SCE rate per cell in ovarian cancer cases of stage I, II, III, and IV was 5.39 -+ 0.17, 5.91 -+ 0.18, 6.55 -+ 0.17, and 7.00 - 0.17, respectively. One-way analysis of variance followed b y Duncan's m u l t i p l e range test s h o w e d the data to be statistically highly significant b e t w e e n control and cancer patients (sum of squares = 1342.42, F = 32.36, p < 0.0001). The group-wise c o m p a r i s o n (Table 2) s h o w e d a significance level of < 0.05 b e t w e e n stages II, III, a n d IV vis-a-vis control. However, no statistical significance was observed b e t w e e n stages I and II. DISCUSSION
There has not been m u c h u n a n i m i t y in the rates of SCEs expressed as n o r m a l ranges, varying from 3 to 11 SCEs/
7
...................................................................................................
6.55
6
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......................................
4.47
4
3
-
....
I
5.39
5
5,9!.
-
i
. . . . . . . . . . . . . . .
.....
_
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
i .........
We thank the management of K. M. C., whose generous financial support made this work possible. We also gratefully acknowledge the expert technical assistance by Ms. Shoba.
2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
l
. . . . . . . . . . . . . . . . . . . . . . .
I
control
I
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cell in different laboratories [11]. Using n o n s e r u m - s u p p l e m e n t e d w h o l e b l o o d cultures the m e a n SCE value has been found to be 4.47 in our laboratory. SCE frequencies have also been observed to vary with the tissue culture m e d i u m a n d serum concentrations u s e d [12]. In our study, w e a d h e r e d to a strict timing schedule for drawing b l o o d from patients followed b y its culture in a n o n s e r u m - s u p p l e m e n t e d m e d i u m , to cut d o w n both the r h y t h m i c variations and also those due to a d d i t i o n of ser u m to the media. Further, data from a few female subjects in their menstrual cycle (both patients and controls) were not c o n s i d e r e d for statistical analysis [4]. Insufficient data were available for c o m p a r i s o n b e t w e e n these two groups. Only those patients in stages I a n d II w h o d i d not receive any treatment prior to c h r o m o s o m a l analysis were considered. Nevertheless, it was not entirely possible to select the untreated patients from later stages a n d a few of t h e m d i d receive some c h e m o t h e r a p e u t i c treatment for associated symptoms, but not for malignancy. The present study confirmed our h y p o t h e s i s that there is an a p p a r e n t increase in the frequency of SCEs as the stage of the cancer increases vis-a-vis control. However, the data d i d not follow Poisson distribution, confirming earlier observations [13]. A statistically significant increase in the average SCE frequency was observed between stages II, III, a n d IV. To the best of our knowledge, this is the first detailed stagewise description of the SCEs in ovarian cancer patients. The variance in patients was quite high, indicating a large distribution of SCE data. The reason for the nonsignificant relationship b e t w e e n stages I a n d II is not clearly discernible. It m a y be due to the similarity of s y m p t o m s and, thus, possible overlapping of cancer stages. Perhaps analysis of more data will clarify the picture further. The present s t u d y tries to find a diagnostic use of SCE in cancer patients. On excluding chemotherap e u t i c a l l y treated Stage III and IV patients (Table 1) from the present study, a progressive stagewise increase in the SCE frequency is still observed, although the m e a n frequency in both groups is slightly higher (Stage I I I = 6.58 -+ 0.19, Stage IV = 7.30 -+ 0.19). However, it is not k n o w n w h e t h e r the ovarian cancer patients on anticancer drugs w o u l d s h o w a similar or different alteration in SCE frequency. On the basis of the a c c u m u l a t e d data, it is clear that patients showing even m i l d s y m p t o m s b u t high SCE values s h o u l d be m o n i t o r e d closely for possible malignancy. SCE analysis m a y help in p r o p e r diagnosis of cancer stages in some cases.
REFERENCES
I
I
I
stage II
stage III
stage IV
Figure 1 Mean SCE frequency + / - S.E.M. in controls and ovarian cancer patients of different stages.
1. Latt SA (1977): The detection and induction of sister chromatid exchange (INC-UCLA Symposium on Molecular and Cellular Biology). Mol Hum Cytogenet Vli:315-334. 2. Shiraishi Y (1990): Nature and role of high sister chromatid exchanges in Bloom syndrome cells. Some cytogenetic and immunological aspects. Cancer Genet Cytogenet 50:175-187.
108
3. D'Souza, Thomas IM, Das BC (1988): Variation in spontaneous chromosomal damage as a function of biologic rhythms in women. Hum C,enet 79:83-85. 4. Lerner NJ, Fejgin M, Ben-Nun I, Legum C, Amiel A (1993): The correlation between the frequency of sister chromatid exchange and human reproductive hormones. Mutation Res 300:247-252. 5. Tucker ]D, Christansen ML, Strout CL, Carrano AV (1986): Determination of the baseline sister chromatid exchange frequency in humans and mouse peripheral lymphocytes using monoclonal antibodies and very low dose of bromodeoxyuridine. Cytogenet Cell Genet 43:38-42. 6. Adhvaryu SG, Rawal UM, Patel IV (1988): Enhancement of lymphocytic SCE frequencies in patients with ovarian cancer. Neoplasma 35:103-108. 7. Fox H, Buckley CH (1992): The Ovary. In: Mc Gee JO'D, PI Issacson, NA Wright, eds. Oxford Textbook of Pathology. Oxford University Press, pp. 1614-1632.
E K. Dhar et al.
8. Moorhead PS, Nowell PC, Mellman WJ, Batiips DM, Hungerford DA (1960): Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613-616. 9. Perry E Wolff S (1974): New Giemsa method for the differential staining of sister chromatids. Nature 251:156-158. 10. Vereantern E Maulapas E, Viietink R, Cassiman JJ (1986): Non-uniform distribution of sister chromatid exchanges in human lymphocytes. Chromosoma 93:197-202. 11. Husain SA, Bamezai R (1988): Sister chromatid exchange (SCE) rate in normal and abnormal sexual development in males and females. Mutation Res 206:261-270. 12. Morgan WF, Crossen PE [1981): Factors influencing SCE rate in cultured human lymphocytes. Mutat Res 81:395-402. 13. Moore DH, Carrano AV (1984): Statistical analysis of high SCE frequency cells in human lymphocytes, In: Sister Chromatid Exchanges. 25 Years of Experimental Research. Plenum, New York, pp. 469-480.
Significance of Lymphocytic Sister Chromatid Exchange Frequencies in Ovarian Cancer Patients Pawan Kumar Dhar, Sulochna Devi, T. Ramesh Rao, Usha Kumari, Anita Joseph, M. R. Kumar, Satish Nayak, Y. Shreemati, S. M. Bhat, and K. R. Bhat
ABSTRACT: Very f e w studies report the analysis of sister chromatid exchanges in ovarian cancer patients. We tested the null hypothesis that SCE frequency increases with the advancing stages of ovarian cancer and Jbllows a Poisson distribution. As controls we examined age- and sex-matched healthy volunteers who had no such past history. An increased average SCE frequency was observed in ovarian cancer patients (6.34 ± 0.09) vis-&-vis controls (4.47 ± 0.12). Further, the data also suggested a stagewise increase in the SCE frequency. INTRODUCTION Sister chromatid exchanges are reciprocal exchanges of DNA segments between sister chromatids at identical loci [1] during S-phase of the cell cycle. The exact mechanism underlying SCE formation is still unclear, although its significance is well understood [2]. It has been suggested that female subjects are more influenced by biologic rhythms because of specific hormonal cycles and are sensitive to genetic damage during the menstrual cycle [3, 4]. Although many external agents are known to cause SCEs, in their absence SCEs are formed, which leads one to the conclusion that they are an integral part of DNA replication [5]. Due to the paucity of literature on SCEs in ovarian cancer patients, there is no information about their diagnostic importance in these patients. However, a higher spontaneous SCE rate in ovarian cancer patients has been reported [6].
Patients in their menstrual cycle were also omitted for analysis. The control group included 12 sex- and agematched healthy volunteers. Tumors were classified into various stages based on standard guidelines [7].
Cell and Culture Conditions
MATERIALS AND METHODS
Blood culture, harvesting, and slide preparation were done according to the established protocol [8], with some modifications. Heparinized whole blood (0.4 mL) was cultured in 10 mL of RPMI 1640 medium (without serum) supplemented with phytohemagglutinin and antibiotics. Bromodeoxyuridine was added at 0 hours at a final concentration of 10 ~g/mL. Cultures were incubated at 37°C in 5% CO2 in complete darkness, 68 hours, after which Colcemid (0.4 ~g/mL) was added to the cultures and incubation continued for another 2 hours. The cultures were harvested at 70 hours and slides prepared by the air-dry method. Chromosomes were stained by the FPG technique [9].
Subjects and Classification
Analysis and Statistical Methods
All 38 cancer patients selected in the present study were between 39 and 63 years of age (Table 1). Most of them under treatment were excluded from the research group.
Exchanges between the two interstitial chromatids were considered as two SCEs and terminal exchanges as one [10]. Analysis of the data was performed using Descriptive Statistics and one-way ANOVA method of SPSS-PC (SPSS Inc., Chicago, IL).
From the Departments of Anatomy (Human Genetics Section) (P.K.D., T.R.R.,A.J.,M.R.K., S.N., S.M.B.,K.R.B.),Physiology (S. D., U. K.), and Medical Education (Y S.), Kasturba Medical College, Manipal, Karnataka, India. Address reprint requests to: Pawan Kumar Dhar, Ph.D., Assistant Professor and Head, Human Genetics Section, Department of Anatomy, Kasturba Medical College, Manipal, Karnataka 576 119, India. Received November 14, 1994; accepted March 20, 1995. Cancer Genet Cytogenet 8 9 : 1 0 5 - 1 0 8 (1996) © Elsevier Science Inc., 1996 655 Avenue of the Americas, N e w York, NY 10010
RESULTS Suitable data were available from a total of 38 cancer patients and 12 normal controls. In aggregate, there were 10,025 SCEs in 1665 metaphases. The mean, SCE range, variance, and H value of all cancer cases (stage-wise) and controls are shown in Table 1. An average value of 6.34 ±
0165-4608/96/$15.00 SSDI 0165-4608(95)00071-V
106
Table 1.
P.K. Dhar et al.
Mean, range, variance, a n d H-values of SCE in ovarian cancer patients and control
SL. N o .
Patient Code
Cell Nos.
Total SCE
M e a n _+ S.E.
SCE Range
Stage I 01. 02.
HGS-05 HGS-06
30 40
152 216
5.07 ± 0.48 5 . 4 0 Jr 0 . 4 0
1-10 1-11
6.82 6.55
1.34 1.21
HGS-14 HGS-20 HGS-12 HGS-25 HGS-26 HGS-22
36 50 43 26 33 24
194 277 211 146 195 130
5.39 5.54 4.91 5.62 5.91 5.42
± 0.67 _+ 0 . 5 4 ± 0.43 ± 0.45 ± 0.43 ± 0.50
1-14 1-16 1-10 1-9 2-11 1-11
16.30 14.70 7.84 5.20 6.08 6.08
3.03 2.80 1.60 0.92 1.03 1.12
HGS-34 HGS-37 HGS-11 HGS-40 HGS-23 HGS-42 HGS-10 HGS-45
44 43 47 22 20 20 29 36
241 258 278 124 110 143 150 241
5.48 6.00 5.91 5.64 5.50 7.15 5.17 6.69
± 0.46 ± 0.57 ± 0.39 _+ 0 . 5 8 ± 0.59 ± 0.81 ± 0.44 ± 0.41
2-13 1-15 2-12 2-10 1-10 3-13 2-10 2-10
9.18 14.14 7.21 7.29 6.89 13.18 5.57 6.10
1.67 2.35 1.30 1.21 1.25 1.84 1.07 0.91
HGS-38 ° HGS-02 HGS-03 HGS-51 HGS-21 HGS-30 HGS-13 HGS-04 HGS-49 HGS-48
64 24 27 25 39 45 21 22 50 65
409 135 201 185 271 267 134 156 325 421
6.39 5.63 7.44 7.40 6.95 5.93 6.38 7.09 6.50 6.48
_+0.33 ± 0.60 ± 0.65 ± 0.51 ± 0.53 ± 0.57 ± 0.68 ± 0.64 -+ 0 . 5 3 ± 0.57
1-10 1-10 1-12 3-13 1-13 1-17 1-14 2-12 1-14 1-14
6.94 8.59 11.48 6.41 11.15 14.56 9.64 8.94 14.25 20.97
1.08 1.52 1.54 0.86 1.60 2.45 1.51 1.26 2.19 3.24
HGS-01 HGS-28 HGS-43 a HGS-07 HGS-44 HGS-24 HGS-27 HGS-08 a HGS-41 HGS-15 HGS-35 HGS-19
35 63 60 38 32 38 25 40 35 32 30 24
288 435 366 324 262 235 202 229 252 193 185 194
8.23 6.90 6.10 8.53 8.19 6.18 8.08 5.73 7.20 6.03 6.17 8.08
± ± ± ± ± ± ± ± ± ± ± ±
0.64 0.52 0.48 0.63 0.45 0.62 0.67 0.66 0.55 0.68 0.51 0.74
1-13 1-13 1-12 2-13 3-11 1-13 3-13 1-12 1-12 1-12 1-12 1-13
14.29 16.92 13.65 14.90 6.60 14.74 11.07 17.43 10.51 14.67 7.79 13.29
1.73 2.36 2.23 1.74 0.80 2.38 1.37 3.04 1.46 2.43 1.26 1.64
NOR-01 NOR-02 NOR-03 NOR-04 NOR-05 NOR-06 NOR-07 NOR-08 NOR-09 NOR-IO NOR-11 NOR-12
19 31 22 20 22 31 18 20 27 19 38 21
84 113 105 93 102 154 73 84 139 74 174 95
4.42 3.65 4.77 4.65 4.64 4.97 4.06 4.20 5.15 3.89 4.58 4.52
± ± ± ± ± ± ± ± ± ± ± ±
0.67 0.50 0.51 0.56 0.48 0.27 0.42 0.30 0.36 0.34 0.37 0.30
2-11 1-9 1-10 1-10 1-12 3-8 1-7 1-6 1-9 1-7 2-12 1-6
8.59 7.70 5.61 6.43 5.00 2.29 3.23 1.85 3.59 2.21 5.33 1.86
1.94 2.11 1.17 1.36 1.07 0.46 0.79 0.44 0.69 0.56 1.16 0.41
03. 04. 05. 06. 07. 08. S t a g e II 01. 02. 03. 04. 05. 06. 07. 08. S t a g e III 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. S t a g e IV 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. Control (age & sex matched) 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12.
~Indicates those patients w h o were u n d e r treatment at the time of c h r o m o s o m a l analysis.
Variance
H Value
SCE F r e q u e n c y in Ovarian Cancer
Table 2
107
A n overall statistical c o m p a r i s o n of cancer patients a n d control Stages of cancer Control
Control Stage I Stage II Stage III Stage IV
I
II
III
IV
* * * *
*Denotes groups with statistically significant (p < 0.05) difference in SCE frequency
0.09 was observed in 38 cancer cases regarding the control of 4.47 -+ 0.12 observed in 12 subjects. Figure 1 shows a change in the m e a n frequency _ SEM of SCEs from controis to cancer patients (stage-wise). The average SCE rate per cell in ovarian cancer cases of stage I, II, III, and IV was 5.39 -+ 0.17, 5.91 -+ 0.18, 6.55 -+ 0.17, and 7.00 - 0.17, respectively. One-way analysis of variance followed b y Duncan's m u l t i p l e range test s h o w e d the data to be statistically highly significant b e t w e e n control and cancer patients (sum of squares = 1342.42, F = 32.36, p < 0.0001). The group-wise c o m p a r i s o n (Table 2) s h o w e d a significance level of < 0.05 b e t w e e n stages II, III, a n d IV vis-a-vis control. However, no statistical significance was observed b e t w e e n stages I and II. DISCUSSION
There has not been m u c h u n a n i m i t y in the rates of SCEs expressed as n o r m a l ranges, varying from 3 to 11 SCEs/
7
...................................................................................................
6.55
6
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......................................
4.47
4
3
-
....
I
5.39
5
5,9!.
-
i
. . . . . . . . . . . . . . .
.....
_
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
i .........
We thank the management of K. M. C., whose generous financial support made this work possible. We also gratefully acknowledge the expert technical assistance by Ms. Shoba.
2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
l
. . . . . . . . . . . . . . . . . . . . . . .
I
control
I
stage
cell in different laboratories [11]. Using n o n s e r u m - s u p p l e m e n t e d w h o l e b l o o d cultures the m e a n SCE value has been found to be 4.47 in our laboratory. SCE frequencies have also been observed to vary with the tissue culture m e d i u m a n d serum concentrations u s e d [12]. In our study, w e a d h e r e d to a strict timing schedule for drawing b l o o d from patients followed b y its culture in a n o n s e r u m - s u p p l e m e n t e d m e d i u m , to cut d o w n both the r h y t h m i c variations and also those due to a d d i t i o n of ser u m to the media. Further, data from a few female subjects in their menstrual cycle (both patients and controls) were not c o n s i d e r e d for statistical analysis [4]. Insufficient data were available for c o m p a r i s o n b e t w e e n these two groups. Only those patients in stages I a n d II w h o d i d not receive any treatment prior to c h r o m o s o m a l analysis were considered. Nevertheless, it was not entirely possible to select the untreated patients from later stages a n d a few of t h e m d i d receive some c h e m o t h e r a p e u t i c treatment for associated symptoms, but not for malignancy. The present study confirmed our h y p o t h e s i s that there is an a p p a r e n t increase in the frequency of SCEs as the stage of the cancer increases vis-a-vis control. However, the data d i d not follow Poisson distribution, confirming earlier observations [13]. A statistically significant increase in the average SCE frequency was observed between stages II, III, a n d IV. To the best of our knowledge, this is the first detailed stagewise description of the SCEs in ovarian cancer patients. The variance in patients was quite high, indicating a large distribution of SCE data. The reason for the nonsignificant relationship b e t w e e n stages I a n d II is not clearly discernible. It m a y be due to the similarity of s y m p t o m s and, thus, possible overlapping of cancer stages. Perhaps analysis of more data will clarify the picture further. The present s t u d y tries to find a diagnostic use of SCE in cancer patients. On excluding chemotherap e u t i c a l l y treated Stage III and IV patients (Table 1) from the present study, a progressive stagewise increase in the SCE frequency is still observed, although the m e a n frequency in both groups is slightly higher (Stage I I I = 6.58 -+ 0.19, Stage IV = 7.30 -+ 0.19). However, it is not k n o w n w h e t h e r the ovarian cancer patients on anticancer drugs w o u l d s h o w a similar or different alteration in SCE frequency. On the basis of the a c c u m u l a t e d data, it is clear that patients showing even m i l d s y m p t o m s b u t high SCE values s h o u l d be m o n i t o r e d closely for possible malignancy. SCE analysis m a y help in p r o p e r diagnosis of cancer stages in some cases.
REFERENCES
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Figure 1 Mean SCE frequency + / - S.E.M. in controls and ovarian cancer patients of different stages.
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