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Chromosome studies on direct and cultured preparations from malignant tissues
Pathology
ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: http://www.tandfonline.com/loi/ipat20
Chromosome Studies on Direct and Cultured Preparations from Malignant Tissues Sandra Von Der Borch, Margaret A. Stanley & James A. Kirkland To cite this article: Sandra Von Der Borch, Margaret A. Stanley & James A. Kirkland (1969) Chromosome Studies on Direct and Cultured Preparations from Malignant Tissues, Pathology, 1:3, 243-250 To link to this article: http://dx.doi.org/10.3109/00313026909071302
Published online: 06 Jul 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ipat20 Download by: [FU Berlin]
Date: 25 January 2017, At: 18:07
Pathology (1969), 1, pp. 243-50
CHROMOSOME STUDIES ON DIRECT A N D CULTURED PREPARATIONS F R O M MALIGNANT TISSUES SANDRAVON DERBORCH,MARGARET A. STANLEY and A. KIRKLAND
JAMES
Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, Woodville, South Australia
Summary Cytogenetic data has been obtained from 1 6 primary cultures of nonmalignant cervical, uterine, and ovarian tissues. Normal diploid cells constituted the major population in these cultures, with a small tetraploid population. Forty-eight cultures were set up from biopsies derived from 26 malignant gynaecological turnours. The chromosome constitution of these tumours was also analysed using a direct squash technique. Of the in vitro cultures, 10 cases produced adequate data for comparison with the results from squash preparations. In vitro cultures were characterized by the presence of normal diploid populations in contrast t o the marked aneuploidy shown i n the squash preparations. I n only 1 case was evidence obtained suggesting that malignant cells similar t o those present in the in viva population were present i n culture.
Chromosomal studies of cervical malignant disease have been made with either tissue culture techniques or fresh squash preparations (Richart & Wilbanks, 1966; Wakonig-Vaartaja & Kirkland, 1965; Kirkland et al., 1967; Kirkland 81 Stanley, 1967; Spriggs, Boddington & Clarke, 1962), but there appear to be no reported studies using both techniques in the same case. Chromosome counts and karyotypes from 14 patients with carcinoma-in-situ of the uterine cervix have been reported using a tissue culture technique (Richart & Wilbanks, 1966) in which cultures were examined from the second to the seventh sub-culture. In each case a modal chromosome number of 46 was found, with predominantly normal karyotypes. In cervical malignant disease, however, a direct squash technique has revealed a wide range of chromosome numbers, with predominantly abnormal or pseudo-diploid karyotypes (Wakonig-Vaartaja 81 Kirkland, 1965; Kirkland et al., 1967; Kirkland & Stanley, 1967). In view of these differing results, we have used both techniques in the study of the same malignant neoplasms. Little work has been done on the chromosome patterns of normal cervical tissue in primary in vitro culture. Variations in the chromosome pattern of normal endometrium have been reported by Hughes & Csermely (1965) and in view of this, we have also compared the results of normal and neoplastic tissue cultures using the same techniques.
HSVnbS
CHROMOSOMES I N SQUASH A N D CULTURE
245
TABLE 1 Chromosome counts from non-malignant tissues in primary culture
Case number
Biopsy
-S119 s120.1 s120.2 s122 S125 S129 S131.1 S131.2 S136.1 S136.2 S138.1 S138.2 S139 S140 S142 S143
Days in culture
-
I
<35
Normal cervix >>
>Y
2,
>Y
39
>¶
I>
>>
2,
Chromosome number 46
_ _ _ 35-56 57-78
1
~ 79-103
I1 counts Total 10
-
1
2
13 20 5 2 8 3 6 44 3
,,
Y
JJ
>Y
>Y
>J
YY
9,
J,
,I
>Y
,Y
Y>
11
>Y
J,
12
Non-malignant curettings Non-malignant curettings Fibroma of ovary
4
TOTAL l4
,
5 57 204
MATERIALS AND METHODS Punch and cone biopsies of the cervix uteri, uterine curettings and other specimens for pathological examination were obtained fresh from the operating theatre. All specimens were diagnosed histologically. Tissues for squash and culture were selected from adjacent sites on the specimen; from cone biopsies, this selection was made with a dissecting microscope, as described by Wakonig-Vaartaja & Kirkland (1965). Direct squash preparations were made according to Kirkland et al. (1967). Fragments of the tissue were then placed on a slide and squashed with a cover glass to spread the chromosomes in dividing cells for examination with a phase-contrast microscope, Tissues for culture were washed in Dulbecco phosphate solution to which was added Kanamycin (104 ,ug./ml.) and Fungizone (500 ,ug./ml.), and then minced finely with scissors before treatment in 0.25% trypsin (pH 7.4) for 15 min. at 37". The fragments of tissue were placed in small culture chambers similar to those used by Richart (1964), but the present cultures were grown directly on 7 x 5 cm. microscope slides. Perforated cellophane was placed over the minced tissue to hold the fragments in place and the medium (20% foetal calf serum in medium 199) was added to the chamber. Cultures were held at 37" in desiccators gassed with 5% CO, in air and the medium changed three times weekly until there was adequate outgrowth from the fragments (6-23 days). The cultures were then treated with 0.0005~0 colcemid for 6 hr. at 37" before the medium was replaced by a hypotonic solution (1 :5, foetal calfserum:water) for 35 min. at 37". Fixation for 45 min. in 3:1:6, ch1oroform:acetic acid:absolute ethanol was followed by air drying of the slides for 10 min. The preparations were stained with Giemsa. In both squash and cultured preparations, unbroken metaphases were counted and karyotypes prepared according to the Denver classification, either by visual or photographic analysis.
Borderline carcinomain-situ Borderline carcinomain-situ Carcinoma-in-situ Carcinoma-in-situ Invasive cervical carcinoma Embryonic teratoma
Cone
Cone
Cone
Cone
Punch : cervix
At hysterectomy
At hysterectomy
H483
H445
H474
H584
H590
H564
H521
TOTALS
Severe dysplasia
Cone: anterior lip
H565
Carcinoma of the ovary
Severe dysplasia
Cone: posterior lip
H565
~
Dysplasia
-
Lesion
Cone
-
Biopsy
H520
..
Case number
21 65 -
5
Squash Culture
TOTAL
5
21 4
86
26
24I
79 162
-
26
-
2 12 11
1
9
-
3
1
1
4
___
-
4 39 16 11 7 16 1 6 14 2 1 9 13 5 23 27
35-56
-_....
-
5 1 2 4
46
87
80 7
Chromosome number
Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture
rechnique
156
35
121
4
-
-
6 12 14
2
-
-
10 41 1 7 1 41
._
4 6 2 5
79-103
33
-
33
TABLE 2 Comparison of chromosome counts from fresh squash preparations with those from primary tissue culture
608
339 269
17 50 31 20 8 30 64 8 31 6 60 18 24 7 45 53 25 52 34 25
?-
Q
(D 0
CHROMOSOMES I N SQUASH A N D CULTURE
247
RESULTS Normal tissue T o establish normal values for non-malignant tissues, 22 cultures were set up with tissues from 13 normal cervices, two non-malignant uterine curettings and one fibroma of the ovary. Three specimens produced no outgrowth, and in one other culture no divisions were found. The results were pooled into ranges of chromosome numbers which included both accurate and estimated counts (Table 1). Most of the counts obtained from these primary cultures were in the diploid range (35-56), and, although relatively few accurate counts of 46 were obtained, all had apparently normal karyotypes. Of the total counts, 6.9% were in the tetraploid range (79-103), a value slightly exceeding that of Tjio & Puck (1958), who reported 0-3.4% polyploidy as normal for cultured diploid tissue.
Tumour tissue Fresh squash preparations and 48 cultures were set up from 26 different malignant neoplasms;
2 cultures became infected, 12 produced no outgrowth and 2 other cultures produced less than 5 divisions. Few tumours produced sufficient data for consideration and only a limited number of accurate chromosome counts were obtained; the results are illustrated in Fig. 1. In other cases, considerable numbers of cells yielded counts accurate to within f2 chromosomes. These counts were pooled into ranges of chromosome numbers in which the accurate counts were included (Table 2). In all cases there was a definite tendency for in vitro cultures to exhibit populations which were predominantly diploid with a small tetraploid content. This was in direct contrast to the squash preparations in which aneuploidy predominated. Karyotypes of diploid cells from both squash and cultured preparations were prepared wherever possible. Ten metaphases from five cases were prepared from cultured material; all exhibited normal diploid karyotypes. Only two karyotypes of diploid cells in squash preparations were prepared in this series, but both exhibited pseudodiploid (abnormal diploid) karyotypes, a finding in accordance with the observations of Kirkland et al. (1967) that diploid cells in pre-invasive and invasive carcinoma are predominantly pseudodiploid. In Case H590 the distribution of chromosome counts between squash and cultured preparations was not dissimilar; both exhibited a predominantly diploid population with a smaller triploid population. Two ring chromosomes were found in squash preparations of this case and similar ring chromosomes were apparent in cultured material (Figs 2 & 3). In several other cases, H445, H521, H564, H584, marker chromosomes were evident in squash preparations, but similar chromosomes were not detected in cultured material.
DISCUSSION The most striking characteristic of the in vitro cultures was their tendency to revert to a normal diploid chromosome pattern. This feature could be due either to sampling of material for in vitro culture or to selective growth of normal diploid cells in culture. All the cultures derived from cervical neoplasms were epithelial in type; growth of stromal tissue, which would have produced fibroblasts with normal diploid karyotypes, was not observed. I n two cases, H564 and H521, the biopsies did not permit microscopic selection of the tissue. Selection of nonmalignant tissue for culture could therefore account for the presence of the normal diploid population which was observed in these cultures. Fibroblasts occurred in both cultures.
248
VON DER B O R C H
et al.
FIG.2a & b Case H590. Invasive carcinoma of the cervix; in vivo preparation, 40 chromosomes a. Metaphase from a squash preparation. A small ring chromosome is present b. Metaphase from a squash preparation. A second and larger ring chromosome is present in this metaphase
CHROMOSOMES I N SQUASH A N D CULTURE
249
FIG.3 Same as in Fig. 2 but after in oitro culture. Metaphase showing small and large rings similar to the ring chromosomes seen in squash preparations
All material from the pre-invasive cervical lesions was selected under the dissecting microscope and the biopsies for squash preparations and culture were selected from the same area. Due to this careful selection of material it is probable that, in these cases, selective growth led to the increase in the diploid population. All the available karyotypic data suggests that normal female diploid cells constitute the bulk of this population. This, plus the absence of marker chromosomes and the fact that no increase in abnormal metaphases occurred in these cultures, reinforces the assumption that selective growth and not initial sampling errors is responsible for the reversion to a diploid mode in culture. In only one case, H590,an invasive carcinoma of the cervix, was evidence obtained to suggest that malignant cells were present in culture. In this case the chromosome patterns were similar in both squash and cultured preparations and identical markers occurred in both types of preparation. There are few reports which describe the successful in vizro culture of pre-invasive cervical lesions and major difficulties have been encountered in the present study in obtaining actively dividing monolayer cultures from which well-spread metaphases could be analysed. Furthermore, in this report like that of Richart & Wilbanks (1966), only normal diploid cells appeared in culture, despite the fact that the in vivo dividing population was aneuploid or pseudodiploid. It seems possible that a specific in vivo micro-environment exists, essential for the growth of intra-epithelial neoplasms and which apparently cannot be reproduced in vitro. In view of these observations it seems evident that a more accurate analysis of the chromosome constitution
250
V O N DER B O R C H e t
at.
of the in vivo dividing population of pre-invasive cervical lesions is obtained by direct analysis of squash or air-dried preparations from carefully selected sites, than by in Vitro cultures. ACKNOWLEDGEMENTS We wish to thank Professor L. W. Cox and the members of the Department of Obstetrics and Gynaecology for the collection of material, and Miss K. Ormond, Mrs M. L. Tregloan and Miss S. Sims for considerable technical assistance. This investigation was supported by the Anti-Cancer Foundation, The University of Adelaide. Requests for reprints should be addressed to Dr J. A. Kirkland.
References HUGHES,E. C. & C~WMELY, T. V. (1965): Chromosome constitution of human endomerrium. Amer. 3. Obstet. Gynec. 93, 777790.
KIRKLAND, J. A. & STANLEY,M. A. (1967): The cytogenetics of carcinoma of the cervix. Aust. N.Z.3. Obstet. Gynaec. 7 , 189-193. KIRKLAND, J. A., STANLEY, M. A. & CELLIER, K. M. (1967): A comparative study of histologic and chromosomal abnormalities in cervical neoplasia. Cancer (Philad.).20,19341952.
RICHART,R. M. (1964): Growth of ‘pure’ cervical epithelium in vitro. Amer. 3. Obsret. G ~ w c 88,710-714. .
RICHART, R. M. & WILBANKS, G. D. (1966): T h e chromosomes of human intraepithelial neoplasia: report of 14 cases of cervical intraepithelial neoplasia and review. Cancer Res. 26, 60-74. SPRIGGS, A. I., BODDINGTON, M. M. & CLARKE, C. M. (1962): Chromosomes of human cancer cells. Brit. med. 3. 2, 1431-1435. TJIO, J. H. & PUCK,T. T. (1958): Genetics of somatic mammalian cells. 11. Chromosomal constitution of cells in tissue culture. 3. exp. Med. 108, 259-268. WAKONIG-VAARTAJA, R. & KIRKLAND, J. A. (1965) : A correlated chromosomal and histopathologic study of pre-invasive lesions of the cervix. Cancer (Philad.). 18, 1101-1112.
ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: http://www.tandfonline.com/loi/ipat20
Chromosome Studies on Direct and Cultured Preparations from Malignant Tissues Sandra Von Der Borch, Margaret A. Stanley & James A. Kirkland To cite this article: Sandra Von Der Borch, Margaret A. Stanley & James A. Kirkland (1969) Chromosome Studies on Direct and Cultured Preparations from Malignant Tissues, Pathology, 1:3, 243-250 To link to this article: http://dx.doi.org/10.3109/00313026909071302
Published online: 06 Jul 2009.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ipat20 Download by: [FU Berlin]
Date: 25 January 2017, At: 18:07
Pathology (1969), 1, pp. 243-50
CHROMOSOME STUDIES ON DIRECT A N D CULTURED PREPARATIONS F R O M MALIGNANT TISSUES SANDRAVON DERBORCH,MARGARET A. STANLEY and A. KIRKLAND
JAMES
Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, Woodville, South Australia
Summary Cytogenetic data has been obtained from 1 6 primary cultures of nonmalignant cervical, uterine, and ovarian tissues. Normal diploid cells constituted the major population in these cultures, with a small tetraploid population. Forty-eight cultures were set up from biopsies derived from 26 malignant gynaecological turnours. The chromosome constitution of these tumours was also analysed using a direct squash technique. Of the in vitro cultures, 10 cases produced adequate data for comparison with the results from squash preparations. In vitro cultures were characterized by the presence of normal diploid populations in contrast t o the marked aneuploidy shown i n the squash preparations. I n only 1 case was evidence obtained suggesting that malignant cells similar t o those present in the in viva population were present i n culture.
Chromosomal studies of cervical malignant disease have been made with either tissue culture techniques or fresh squash preparations (Richart & Wilbanks, 1966; Wakonig-Vaartaja & Kirkland, 1965; Kirkland et al., 1967; Kirkland 81 Stanley, 1967; Spriggs, Boddington & Clarke, 1962), but there appear to be no reported studies using both techniques in the same case. Chromosome counts and karyotypes from 14 patients with carcinoma-in-situ of the uterine cervix have been reported using a tissue culture technique (Richart & Wilbanks, 1966) in which cultures were examined from the second to the seventh sub-culture. In each case a modal chromosome number of 46 was found, with predominantly normal karyotypes. In cervical malignant disease, however, a direct squash technique has revealed a wide range of chromosome numbers, with predominantly abnormal or pseudo-diploid karyotypes (Wakonig-Vaartaja 81 Kirkland, 1965; Kirkland et al., 1967; Kirkland & Stanley, 1967). In view of these differing results, we have used both techniques in the study of the same malignant neoplasms. Little work has been done on the chromosome patterns of normal cervical tissue in primary in vitro culture. Variations in the chromosome pattern of normal endometrium have been reported by Hughes & Csermely (1965) and in view of this, we have also compared the results of normal and neoplastic tissue cultures using the same techniques.
HSVnbS
CHROMOSOMES I N SQUASH A N D CULTURE
245
TABLE 1 Chromosome counts from non-malignant tissues in primary culture
Case number
Biopsy
-S119 s120.1 s120.2 s122 S125 S129 S131.1 S131.2 S136.1 S136.2 S138.1 S138.2 S139 S140 S142 S143
Days in culture
-
I
<35
Normal cervix >>
>Y
2,
>Y
39
>¶
I>
>>
2,
Chromosome number 46
_ _ _ 35-56 57-78
1
~ 79-103
I1 counts Total 10
-
1
2
13 20 5 2 8 3 6 44 3
,,
Y
JJ
>Y
>Y
>J
YY
9,
J,
,I
>Y
,Y
Y>
11
>Y
J,
12
Non-malignant curettings Non-malignant curettings Fibroma of ovary
4
TOTAL l4
,
5 57 204
MATERIALS AND METHODS Punch and cone biopsies of the cervix uteri, uterine curettings and other specimens for pathological examination were obtained fresh from the operating theatre. All specimens were diagnosed histologically. Tissues for squash and culture were selected from adjacent sites on the specimen; from cone biopsies, this selection was made with a dissecting microscope, as described by Wakonig-Vaartaja & Kirkland (1965). Direct squash preparations were made according to Kirkland et al. (1967). Fragments of the tissue were then placed on a slide and squashed with a cover glass to spread the chromosomes in dividing cells for examination with a phase-contrast microscope, Tissues for culture were washed in Dulbecco phosphate solution to which was added Kanamycin (104 ,ug./ml.) and Fungizone (500 ,ug./ml.), and then minced finely with scissors before treatment in 0.25% trypsin (pH 7.4) for 15 min. at 37". The fragments of tissue were placed in small culture chambers similar to those used by Richart (1964), but the present cultures were grown directly on 7 x 5 cm. microscope slides. Perforated cellophane was placed over the minced tissue to hold the fragments in place and the medium (20% foetal calf serum in medium 199) was added to the chamber. Cultures were held at 37" in desiccators gassed with 5% CO, in air and the medium changed three times weekly until there was adequate outgrowth from the fragments (6-23 days). The cultures were then treated with 0.0005~0 colcemid for 6 hr. at 37" before the medium was replaced by a hypotonic solution (1 :5, foetal calfserum:water) for 35 min. at 37". Fixation for 45 min. in 3:1:6, ch1oroform:acetic acid:absolute ethanol was followed by air drying of the slides for 10 min. The preparations were stained with Giemsa. In both squash and cultured preparations, unbroken metaphases were counted and karyotypes prepared according to the Denver classification, either by visual or photographic analysis.
Borderline carcinomain-situ Borderline carcinomain-situ Carcinoma-in-situ Carcinoma-in-situ Invasive cervical carcinoma Embryonic teratoma
Cone
Cone
Cone
Cone
Punch : cervix
At hysterectomy
At hysterectomy
H483
H445
H474
H584
H590
H564
H521
TOTALS
Severe dysplasia
Cone: anterior lip
H565
Carcinoma of the ovary
Severe dysplasia
Cone: posterior lip
H565
~
Dysplasia
-
Lesion
Cone
-
Biopsy
H520
..
Case number
21 65 -
5
Squash Culture
TOTAL
5
21 4
86
26
24I
79 162
-
26
-
2 12 11
1
9
-
3
1
1
4
___
-
4 39 16 11 7 16 1 6 14 2 1 9 13 5 23 27
35-56
-_....
-
5 1 2 4
46
87
80 7
Chromosome number
Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture Squash Culture
rechnique
156
35
121
4
-
-
6 12 14
2
-
-
10 41 1 7 1 41
._
4 6 2 5
79-103
33
-
33
TABLE 2 Comparison of chromosome counts from fresh squash preparations with those from primary tissue culture
608
339 269
17 50 31 20 8 30 64 8 31 6 60 18 24 7 45 53 25 52 34 25
?-
Q
(D 0
CHROMOSOMES I N SQUASH A N D CULTURE
247
RESULTS Normal tissue T o establish normal values for non-malignant tissues, 22 cultures were set up with tissues from 13 normal cervices, two non-malignant uterine curettings and one fibroma of the ovary. Three specimens produced no outgrowth, and in one other culture no divisions were found. The results were pooled into ranges of chromosome numbers which included both accurate and estimated counts (Table 1). Most of the counts obtained from these primary cultures were in the diploid range (35-56), and, although relatively few accurate counts of 46 were obtained, all had apparently normal karyotypes. Of the total counts, 6.9% were in the tetraploid range (79-103), a value slightly exceeding that of Tjio & Puck (1958), who reported 0-3.4% polyploidy as normal for cultured diploid tissue.
Tumour tissue Fresh squash preparations and 48 cultures were set up from 26 different malignant neoplasms;
2 cultures became infected, 12 produced no outgrowth and 2 other cultures produced less than 5 divisions. Few tumours produced sufficient data for consideration and only a limited number of accurate chromosome counts were obtained; the results are illustrated in Fig. 1. In other cases, considerable numbers of cells yielded counts accurate to within f2 chromosomes. These counts were pooled into ranges of chromosome numbers in which the accurate counts were included (Table 2). In all cases there was a definite tendency for in vitro cultures to exhibit populations which were predominantly diploid with a small tetraploid content. This was in direct contrast to the squash preparations in which aneuploidy predominated. Karyotypes of diploid cells from both squash and cultured preparations were prepared wherever possible. Ten metaphases from five cases were prepared from cultured material; all exhibited normal diploid karyotypes. Only two karyotypes of diploid cells in squash preparations were prepared in this series, but both exhibited pseudodiploid (abnormal diploid) karyotypes, a finding in accordance with the observations of Kirkland et al. (1967) that diploid cells in pre-invasive and invasive carcinoma are predominantly pseudodiploid. In Case H590 the distribution of chromosome counts between squash and cultured preparations was not dissimilar; both exhibited a predominantly diploid population with a smaller triploid population. Two ring chromosomes were found in squash preparations of this case and similar ring chromosomes were apparent in cultured material (Figs 2 & 3). In several other cases, H445, H521, H564, H584, marker chromosomes were evident in squash preparations, but similar chromosomes were not detected in cultured material.
DISCUSSION The most striking characteristic of the in vitro cultures was their tendency to revert to a normal diploid chromosome pattern. This feature could be due either to sampling of material for in vitro culture or to selective growth of normal diploid cells in culture. All the cultures derived from cervical neoplasms were epithelial in type; growth of stromal tissue, which would have produced fibroblasts with normal diploid karyotypes, was not observed. I n two cases, H564 and H521, the biopsies did not permit microscopic selection of the tissue. Selection of nonmalignant tissue for culture could therefore account for the presence of the normal diploid population which was observed in these cultures. Fibroblasts occurred in both cultures.
248
VON DER B O R C H
et al.
FIG.2a & b Case H590. Invasive carcinoma of the cervix; in vivo preparation, 40 chromosomes a. Metaphase from a squash preparation. A small ring chromosome is present b. Metaphase from a squash preparation. A second and larger ring chromosome is present in this metaphase
CHROMOSOMES I N SQUASH A N D CULTURE
249
FIG.3 Same as in Fig. 2 but after in oitro culture. Metaphase showing small and large rings similar to the ring chromosomes seen in squash preparations
All material from the pre-invasive cervical lesions was selected under the dissecting microscope and the biopsies for squash preparations and culture were selected from the same area. Due to this careful selection of material it is probable that, in these cases, selective growth led to the increase in the diploid population. All the available karyotypic data suggests that normal female diploid cells constitute the bulk of this population. This, plus the absence of marker chromosomes and the fact that no increase in abnormal metaphases occurred in these cultures, reinforces the assumption that selective growth and not initial sampling errors is responsible for the reversion to a diploid mode in culture. In only one case, H590,an invasive carcinoma of the cervix, was evidence obtained to suggest that malignant cells were present in culture. In this case the chromosome patterns were similar in both squash and cultured preparations and identical markers occurred in both types of preparation. There are few reports which describe the successful in vizro culture of pre-invasive cervical lesions and major difficulties have been encountered in the present study in obtaining actively dividing monolayer cultures from which well-spread metaphases could be analysed. Furthermore, in this report like that of Richart & Wilbanks (1966), only normal diploid cells appeared in culture, despite the fact that the in vivo dividing population was aneuploid or pseudodiploid. It seems possible that a specific in vivo micro-environment exists, essential for the growth of intra-epithelial neoplasms and which apparently cannot be reproduced in vitro. In view of these observations it seems evident that a more accurate analysis of the chromosome constitution
250
V O N DER B O R C H e t
at.
of the in vivo dividing population of pre-invasive cervical lesions is obtained by direct analysis of squash or air-dried preparations from carefully selected sites, than by in Vitro cultures. ACKNOWLEDGEMENTS We wish to thank Professor L. W. Cox and the members of the Department of Obstetrics and Gynaecology for the collection of material, and Miss K. Ormond, Mrs M. L. Tregloan and Miss S. Sims for considerable technical assistance. This investigation was supported by the Anti-Cancer Foundation, The University of Adelaide. Requests for reprints should be addressed to Dr J. A. Kirkland.
References HUGHES,E. C. & C~WMELY, T. V. (1965): Chromosome constitution of human endomerrium. Amer. 3. Obstet. Gynec. 93, 777790.
KIRKLAND, J. A. & STANLEY,M. A. (1967): The cytogenetics of carcinoma of the cervix. Aust. N.Z.3. Obstet. Gynaec. 7 , 189-193. KIRKLAND, J. A., STANLEY, M. A. & CELLIER, K. M. (1967): A comparative study of histologic and chromosomal abnormalities in cervical neoplasia. Cancer (Philad.).20,19341952.
RICHART,R. M. (1964): Growth of ‘pure’ cervical epithelium in vitro. Amer. 3. Obsret. G ~ w c 88,710-714. .
RICHART, R. M. & WILBANKS, G. D. (1966): T h e chromosomes of human intraepithelial neoplasia: report of 14 cases of cervical intraepithelial neoplasia and review. Cancer Res. 26, 60-74. SPRIGGS, A. I., BODDINGTON, M. M. & CLARKE, C. M. (1962): Chromosomes of human cancer cells. Brit. med. 3. 2, 1431-1435. TJIO, J. H. & PUCK,T. T. (1958): Genetics of somatic mammalian cells. 11. Chromosomal constitution of cells in tissue culture. 3. exp. Med. 108, 259-268. WAKONIG-VAARTAJA, R. & KIRKLAND, J. A. (1965) : A correlated chromosomal and histopathologic study of pre-invasive lesions of the cervix. Cancer (Philad.). 18, 1101-1112.