Open Access

DNA copy number profiles of gastric cancer precursor lesions

  • Tineke E Buffart1,
  • Beatriz Carvalho1, 2Email author,
  • Thomas Mons1,
  • Rui M Reis3,
  • Cátia Moutinho2,
  • Paula Silva2,
  • Nicole CT van Grieken1,
  • Michael Vieth4,
  • Manfred Stolte4,
  • Cornelis JH van de Velde5,
  • Evelin Schrock6,
  • Anja Matthaei6,
  • Bauke Ylstra1,
  • Fátima Carneiro2, 7 and
  • Gerrit A Meijer1
BMC Genomics20078:345

DOI: 10.1186/1471-2164-8-345

Received: 25 April 2007

Accepted: 01 October 2007

Published: 01 October 2007

Abstract

Background

Chromosomal instability (CIN) is the most prevalent type of genomic instability in gastric tumours, but its role in malignant transformation of the gastric mucosa is still obscure. In the present study, we set out to study whether two morphologically distinct categories of gastric cancer precursor lesions, i.e. intestinal-type and pyloric gland adenomas, would carry different patterns of DNA copy number changes, possibly reflecting distinct genetic pathways of gastric carcinogenesis in these two adenoma types.

Results

Using a 5K BAC array CGH platform, we showed that the most common aberrations shared by the 11 intestinal-type and 10 pyloric gland adenomas were gains of chromosomes 9 (29%), 11q (29%) and 20 (33%), and losses of chromosomes 13q (48%), 6(48%), 5(43%) and 10 (33%). The most frequent aberrations in intestinal-type gastric adenoma were gains on 11q, 9q and 8, and losses on chromosomes 5q, 6, 10 and 13, whereas in pyloric gland gastric adenomas these were gains on chromosome 20 and losses on 5q and 6. However, no significant differences were observed between the two adenoma types.

Conclusion

The results suggest that gains on chromosomes 8, 9q, 11q and 20, and losses on chromosomes 5q, 6, 10 and 13, likely represent early events in gastric carcinogenesis. The phenotypical entities, intestinal-type and pyloric gland adenomas, however, do not differ significantly (P = 0.8) at the level of DNA copy number changes.

Background

Gastric cancer is the second most frequent malignancy worldwide and the prognosis of this malignancy remains very poor [1]. Gastric cancer incidence and mortality rates differ between different countries within the European Union [2]. In the Netherlands it ranks fifth as a cause of cancer death, with approximately 2,200 new cases each year [3]. Surgery with curative intent is the treatment of choice in advanced cases of gastric cancer, whereas local endoscopic mucosectomy can be curative in early gastric cancer. Detection and removal of gastric neoplasias in an early or even premalignant state will contribute to reduce death due to gastric cancer. To achieve this goal, better tests for early detection of gastric cancer are needed, and an improved understanding of the biology of gastric cancer progression is crucial in this respect.

According to the Correa model, pathogenesis of intestinal-type gastric adenocarcinoma follows a pathway of chronic active gastritis due to Helicobacter pylori infection, leading to mucosal atrophy, intestinal metaplasia followed by intraepithelial neoplasia and finally invasive adenocarcinoma [4]. Genetic characterization of tissue samples in intraepithelial neoplasia stage would substantially contribute to our understanding of the molecular pathogenesis of gastric cancer. However, these lesions are only rarely detected, possibly due to rapid progression through this stage towards cancer, and are usually present only in parts of biopsy specimens, hampering genomic analysis of these lesions. Analysis of alternative precursor lesions could therefore, at least partly, be a substitute. Development of gastric cancer through an adenoma stage, although less common, is such alternative route. These adenomas are occasionally detected during gastroscopy and present as large lesions that histologically show intra-epithelial neoplasia, which makes them suitable for genomic analysis. Gastric adenomas have a direct malignant potential and account for approximately 20% of all epithelial polyps [5, 6]. Gastric adenomas can have a classic tubular, tubulovillous, or villous morphology with a predominantly intestinal-type epithelium, but can also appear as pyloric gland adenomas [6]. Pyloric gland adenomas arise from deep mucoid glands in the stomach and are strongly positive for mucin 6 [7, 8]. A substantial number of gastric adenomas already show progression to adenocarcinoma. On first diagnosis around 30–40% of all pyloric gland adenomas already show a focus of carcinoma [9, 10]. For intestinal-type adenomas this number is lower and varies from 28,5% for villous adenomas and 29.4% for tubulovillous type adenomas to only 5.4% in the tubular adenomas [11]. Both adenocarcinomas, ex intestinal-type adenomas and ex pyloric gland adenomas, show glandular structures, in contrast to diffuse type gastric cancer.

A key feature in the pathogenesis of most gastric cancers, as in many other solid cancers, is chromosomal instability, resulting in gains and losses of parts or even whole chromosomes [12]. These chromosomal changes can be analyzed by comparative genomic hybridization (CGH). Several previous studies have detected genetic alterations in gastric adenomas using this technique, being gains on chromosome 7q, 8q, 13q, 20q, and losses on chromosome 4p, 5q, 9p 17p and 18q [1316]. Although uncommon and only observed in adenomas with high grade intraepithelial neoplasia, high level amplifications have been detected on chromosomes 7q, 8p, 13q, 17q and 20q [1316]. In gastric adenocarcinomas, consistently described chromosomal aberrations are gains on chromosome 3q, 7p, 7q, 8q, 13q, 17q and 20q and losses on chromosome 4q, 5q, 6q, 9p, 17p and 18q. High level amplifications have been repeatedly detected on 7q, 8p, 8q, 17q, 19q and 20q [14, 1723]. Yet, chromosomal aberrations, or DNA copy number changes, are not uniform in gastric cancer [24]. Subgroups with different patterns of DNA copy number alterations can be recognized, which have been shown to be associated with clinical outcome as well [25].

In the present study, we set out to study whether two morphologically distinct categories of gastric cancer precursor lesions, i.e. intestinal-type and pyloric gland adenomas, would carry different patterns of DNA copy number changes, possibly reflecting distinct genetic pathways of gastric carcinogenesis in the two adenoma types.

Results

DNA copy number changes were observed in 10 out of 11 intestinal-type adenomas and 9 out of 10 pyloric gland adenomas. The mean number of chromosomal events, defined as gains and losses, per tumour was 6.0 (range 0–18), including 2.9 (range 0–14) gains and 3.0 (range 0–7) losses. In intestinal-type adenomas, the mean number of chromosomal events per tumour was 6.5 (range 0–18) of which 3.4 (range 0–14) gains and 3.1 (range 0–7) losses, and in the pyloric gland adenomas the mean numbers were 5.4 (range 0–9), 2.4 (range 0–7) and 3.0 (range 0–7) respectively.

In the intestinal-type gastric adenomas, the most common aberrations observed were gains on chromosomes 8, 9q and 11q, and losses on chromosomes 5q, 6, 10 and 13. In four adenomas (36.4%), gain of chromosome 11q23.3 was observed with a common region of overlap of 2.6 Mb. Gain of chromosome 9q was observed in four adenomas (36.4%) with a 12.6 Mb common region of overlap located on chromosome 9q33.1-q34.13. Gain of chromosome 8 was observed in three adenomas (31%), two of which adenomas showed gain of whole chromosome 8, and the third adenoma showed a gain of chromosome 8p-q22.3 with an additional 28.7 Mb gain on chromosome 8q24.11-qter. In addition, gains were observed on chromosomes 1, 3, 6p, 7, 11p, 12p, 13q, 16, 17, 19, 20 and 22q. No amplifications were seen in the intestinal-type adenomas.

Deletions on chromosome 13 were observed in seven intestinal-type adenomas (64%). Of these, five showed a 11.9 Mb deletion of chromosome 13q21.2-21.33 with an additional 7.7 Mb deletion on chromosome 13q31.1-31.3. The other two adenomas showed a 16.6 Mb deletion of 13q14.3-31. A deletion on chromosome 6 was observed in six adenomas (55%), with an overlapping region of 68.9 Mb located on 6cen-q22.1. A deletion of chromosome 5q was observed in four adenomas (36%) with a common region of overlap located on chromosome 5q22.1-q23.2. In addition, a deletion of whole chromosome 10 was observed in four adenomas (36%). Other losses observed in intestinal-type adenomas were located on chromosomes 8q, 9p, 10, 12q, 20q and 21. An overview of all DNA copy number aberrations of the intestinal-type adenomas is shown in Table 1.
Table 1

Overview of the DNA copy number changes in 11 intestinal-type adenomas

 

Chromosomal aberrations

 

Flanking clones

  

Tumour ID

Gains

Losses

Segment size (Mb)

Start

End

1

1p-p36.11

 

26.68

RP11-465B22

RP1-159A19

  

5q13.2-q23.2

55.26

RP11-115I6

CTB-1054G2

 

6p21.33-p21.1

 

13.78

RP11-346K8

RP11-227E22

  

6p21.1-q16.1

52.05

RP11-89I17

RP3-393D12

 

9q33.1-34.2

 

17.32

RP11-27I1

RP11-417A4

 

11q23.3

 

4.80

RP11-4N9

RP11-730K11

  

13q21.1-q31.3

39.63

RP11-200F15

RP11-62D23

2

1p-1p33

 

46.90

RP11-465B22

RP11-330M19

 

6p21.33-p21.1

 

14.12

RP11-346K8

RP11-121G20

  

6p21.1-q16.2

54.91

RP11-554O14

RP11-79G15

 

8p-q22.3

 

105.67

GS1-77L23

RP11-200A13

 

8q24.11-qter

 

28.65

RP11-278L8

RP5-1056B24

 

9q33.1-q34.2

 

13.63

RP11-85O21

RP11-417A4

 

11p11.2-q13.5

 

31.69

RP11-58K22

RP11-30J7

 

11q23.3

 

2.62

RP11-4N9

RP11-62A14

 

12q13.11-q14.1

 

10.57

RP11-493L12

RP11-571M6

  

13q21.1-q21.33

18.24

RP11-200F15

RP11-335N6

  

13q31.1-q31.3

12.49

RP11-533P8

RP11-62D23

 

16p13.3-q21

 

57.26

RP11-243K18

RP11-405F3

  

16q21-q22.1

5.97

RP11-105C20

RP11-298C15

 

16q22.1-q24.3

 

22.46

RP11-63M22

CTC-240G10

 

17

 

81.24

GS1-68F18

RP11-567O16

 

19

 

61.01

CTB-1031C16

GS1-1129C9

 

20q11.21-q11.23

 

5.09

RP3-324O17

RP5-977B1

 

20q13.12-qter

 

19.60

RP1-138B7

CTB81F12

3

-

-

   

4

6p21.1

 

3.32

RP11-79J5

RP11-121G20

  

6p12.3-q22.1

76.38

RP11-79G12

RP11-59D10

 

7

 

156.89

RP11-510K8

CTB-3K23

  

8q22.3-q23.3

9.69

RP11-142M8

RP11-261F23

 

9q33.1-q34.13

 

12.58

RP11-55P21

RP11-83N9

 

11q23.3

 

3.04

RP11-4N9

RP11-8K10

  

13q21.2-q21.33

17.05

RP11-240M20

RP11-77P3

  

13q31.1-q31.3

11.68

RP11-400M8

RP11-100A3

 

16q23.2-q24.3

 

8.92

RP11-303E16

RP4-597G12

 

20p-q13.2

 

53.40

CTB-106I1

RP5-1162C3

 

20q13.31-qter

 

8.06

RP5-1167H4

CTB-81F12

 

22q

 

33.72

XX-P8708

CTB-99K24

5

 

12q24.31-qter

11.75

RP11-322N7

RP11-1K22

6

3

 

193.37

RP11-299N3

RP11-279P10

  

6cen-q24.1

88.49

RP11-91E17

RP11-86O4

 

7

 

156.09

RP11-510K8

RP11-518I12

 

8

 

144.26

RP11-91J19

RP5-1118A7

  

13q21.1-q21.33

11.86

RP11-640E11

RP11-452P23

  

13q31.1-q31.3

9.62

RP11-400M8

RP11-306O1

  

20q13.2-q13.31

1.41

RP11-212M6

RP4-586J11

7

 

5q21.1-qter

80.52

CTC-1564E20

RP11-281O15

  

10

132.19

RP11-29A19

RP11-45A17

 

13q21.33-31.1

 

8.76

RP11-209P2

RP11-470M1

8

 

5q22.1-q23.2

13.28

RP11-276O18

RP11-14L4

  

6p12.3-q22.1

74.37

RP11-89l17

RP11-149M1

  

9p21.1-pter

31.18

RP11-147I11

RP11-12K1

  

10

133.18

RP11-10D13

RP11-45A17

  

13q14.3-q31.3

39.71

RP11-211J11

RP11-306O1

 

17

 

77.65

GS1-68F18

RP11-398J5

 

19

 

63.31

CTC-546C11

CTD-3138B18

 

20

 

60.87

RP4-686C3

RP4-591C20

 

22q

 

31.25

XX-bac32

CTA-722E9

9

 

5q14.3-q23.2

33.06

RP11-302L17

RP11-14L4

  

6p22.2-q22.3

8.44

RP11-91n3

RP11-88h24

  

6p12.1-q24.1

88.89

RP11-7h16

RP11-368P1

 

8

 

145.95

GS1-77L23

CTC-489D14

 

9q33.1-qter

 

13.60

RP11-91G7

GS1-135I17

  

10

133.18

RP11-10D13

RP11-45A17

 

11q23.3

 

3.16

RP11-4N9

RP11-215D10

  

13q14.3-qter

58.59

RP11-240M20

RP11-480K16

  

20q13.2-q13.31

1.96

RP11-55E1

RP5-832E24

  

21cen-q21.3

17.39

RP11-193B6

RP11-41N19

10

 

8q22.3-q23.3

12.93

RP11-142M8

RP11-143P23

  

10

134.52

RP11-10D13

RP11-122K13

  

13q21.1-q21.33

18.03

RP11-322F18

RP11-335N6

  

13q31.1-q31.3

8.99

RP11-533P8

RP11-505P2

11

-

-

   

The most frequent aberration observed in pyloric gland adenomas were gains on chromosome 20 and losses on chromosomes 5q and 6. Gains on chromosome 20 were seen in four adenomas (40%). Three adenomas showed a 9.8 Mb gain of chromosome 20q13.12-q13.33, and gain of whole chromosome 20 was observed in the other adenoma. In addition, gains were seen on chromosomes 1, 3q, 5q, 7, 9q, 11q, 12q, 13q, 15q, 17 and 22q. One pyloric gland adenoma showed amplifications, located on 12q13.2-q21.1 and 20q13.3-q13.33.

Five pyloric gland adenomas (50%) showed loss of chromosome 5q, two of which had lost a whole chromosome arm, while two adenomas showed a 22.4 Mb deletion of 5q11.2-q13.3 and one adenoma a 40.3 Mb deletion of 5q21.1-q31.2. Loss of chromosome 6 was observed in four pyloric gland adenomas (40%), three of which showed a complete loss of 6q and one adenoma showed a 51.2 Mb deletion of 6p21.1-q16.3. Other chromosomal losses were observed on chromosomes 1p, 2q, 4, 9p, 10, 12q 13q, 14q, 16, 18q, 20q, and 21. An overview of DNA copy number aberrations of the pyloric gland adenomas is shown in Table 2.
Table 2

Overview of the DNA copy number changes in 10 pyloric gland adenomas

 

Chromosomal aberrations

 

Flanking clones

  

Tumour ID

Gains

Losses

Segment size (Mb)

Start

End

12

1q21.3-q23.3

 

9.95

RP11-98D18

RP11-5K23

 

1q42.13-q43

 

14.07

RP11-375H24

RP11-80B9

 

3q

 

111.59

RP11-312H1

RP11-23M2

 

5q35.1-q35.3

 

9.11

RP11-20O22

RP11-451H23

  

6q

115.76

RP11-524H19

RP5-1086L22

 

7

 

156.09

RP11-510K8

RP11-518I12

 

17

 

77.48

RP11-4F24

RP11-313F15

 

20

 

63.47

CTB-106I1

CTB-81F12

13

-

-

   

14

 

4

191.13

CTC-963K6

RP11-45F23

  

5q

128.59

CTD-2276O24

RP11-281O15

  

14q

83.81

RP11-98N22

RP11-73M18

  

16

89.71

RP11-344L6

RP4-597G12

 

20q13.2-q13.33

 

10.84

RP4-724E16

CTB-81F12

15

9q33.2-q34.3

 

16.81

RP11-57K1

RP11-83N9

 

11q23.2-q24.3

 

16.04

RP11-635F12

RP11-567M21

 

12q14.3-q15

 

2.58

RP11-30I11

RP11-444B24

 

20q13.31-q13.33

 

6.86

RP5-1153D9

RP5-963E22

 

22q

 

32.53

XX-p8708

CTA-722E9

16

9q33.3-qter

 

13.57

RP11-85C21

GS1-135I17

  

10p12.1-qter

110.28

RP11-379L21

RP11-45A17

 

11q23.1-q24.3

 

17.72

RP11-107P10

RP11-567M21

  

13q31.1-q32.1

10.84

RP11-661D17

RP11-40H10

  

20q13.2-q13.31

1.96

RP11-55E1

RP4-586J11

17

 

1p34.3-pter

35.59

RP1-37J18

RP11-204L3

 

1p33-qter

 

203.62

RP4-739H11

RP11-551G24

  

2q31.1-qter

66.00

RP11-205B19

RP11-556H17

  

5q21.1-q31.2

40.27

CTD-2068C11

RP11-515C16

 

5q31.3-qter

 

39.06

CTD-2323H12

RP11-451H23

  

6q

113.61

RP11-89D6

CTB-57H24

  

10

134.52

RP11-10D13

RP11-122K13

  

13q31.1-qter

36.14

RP11-388E20

RP11-245B11

  

20q13.2-qter

11.24

RP11-15M15

RP5-1022E24

18

 

5q11.2-q21.2

51.24

CTC-1329H14

RP1-66P19

  

6p12.1-q16.3

51.24

RP11-7H16

RP11-438N24

  

9pter-q13

66.82

GS1-41L13

RP11-265B8

  

10

133.04

RP11-10D13

RP11-45A17

  

13q21.1-q21.33

18.39

RP11-240M20

RP11-335N6

  

13q31.1-q31.3

12.45

RP11-551D9

RP11-100A3

  

21cen-q21.3

17.39

RP11-193B6

RP11-41N19

19

 

1p32.3-p21.1

50.40

RP11-117D22

RP5-1108M17

  

5q11.2-q13.3

24.64

RP4-592P18

CTD-2200O3

 

13q12.11-q14.3

 

31.58

RP11-187L3

RP11-327P2

 

15q12-q26.3

 

77.21

RP11-131I21

CTB-154P1

  

18q21.1-q23

31.31

RP11-46D1

RP11-154H12

 

22q13.2-qter

 

10.02

CTA-229A8

CTA-799F10

20

 

9p-q13

66.57

GS1-41L13

RP11-274B18

 

12q13.2-q21.1 (amplification)

 

19.50

RP11-548L8

RP11-255I14

  

12q21.2-qter

55.56

RP11-25J3

RP11-1K22

  

18q21.31-q23

23.28

RP11-383D22

CTC-964M9

 

20q13.13-q13.33 (amplification)

 

14.62

RP5-1041C10

RP5-1022E24

21

5p

 

43.15

CTD-2265D9

RP11-28I9

  

5q

130.26

RP11-269M20

RP11-451H23

 

6p

 

62.57

CTB-62I11

RP11-506N21

  

6q

106.73

RP11-767J14

RP5-1086L22

The most common aberrations shared by both intestinal-type and pyloric gland adenomas were gain of chromosome 9q (29%), 11q (29%), and 20q (33%) and loss of chromosome 5 (43%), 6 (48%), 10 (33%) and 13q (48%). By comparing intestinal-type and pyloric gland adenomas, CGH Multiarray revealed eight clones to be significantly different, six of which were located at chromosome 6q14-q21 (p = 0.02 to 0.05) and two clones on chromosome 9p22-p23 (p = 0.02 and 0.04, respectively) (Figure 1). No genes located in the regions covered by these clones have been known to be involved in cancer related biological processes. Yet, CGH Multiarray Region, after correction for multiplicity, yielded a false discovery rate (FDR) of 1 for all these regions, indicating no significant differences between the two different types of adenomas at the chromosomal level. Unsupervised hierarchical cluster analysis yielded 2 clusters. No significant associations were found here (p = 0.8).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-345/MediaObjects/12864_2007_Article_1058_Fig1_HTML.jpg
Figure 1

Comparison of DNA copy number alterations in intestinal and pyloric gland type gastric adenomas. A p-value (Y-axis) was calculated for every clone, based on a Wilcoxon test with ties, and plotted in chromosomal order from chromosome 1 to 22 (X-axis). Eight clones reached the level of significance (p < 0.05), but failed to maintain a significantly low false discovery rate after correction for multiple comparison.

Discussion

Given the heterogeneous phenotype of gastric cancer, the present study primarily aimed to compare copy number changes between intestinal-type adenomas and pyloric gland adenomas, in order to find leads towards genetic pathways involved in the pathogenesis of gastric cancer. Adenoma-to-carcinoma progression is observed in 30–40% of the pyloric gland adenomas and in approximately 5–30% of the intestinal-type adenomas (varying from about 5% in tubular adenomas to almost 30% for tubulovillous and villous adenomas) [911], indicating the direct malignant potential of these two adenoma types and making gastric adenomas a suitable model for detecting early events in gastric carcinogenesis.

Pyloric gland adenomas constitute a recently recognized entity [8, 26]. To the best of our knowledge, this type of adenomas has never been analyzed by array CGH before. The mean number of events in this type of adenoma was 5.4 (0–9), with 2.4 (0–7) gains and 3 (0–7) losses. This is comparable with the mean number of aberrations in intestinal-type adenomas (6.5 (0–18), 3.4 (0–14) and 3.1 (0–7) respectively). In pyloric gland adenomas, frequent events were gain on chromosome 20 and losses on chromosomes 5q and 6, while intestinal-type adenomas mainly showed gain on chromosomes 8, 9q, and 11q, and losses on chromosomes 5q, 6, 10 and 13. In the present study, gain of chromosome 7 was less common than found previously [16]. Although these frequently altered regions differ between the two types of adenomas, hierarchical cluster analyses did not separate the groups. In addition, CGH Multiarray Region did not reveal any significant differences after correction for multiple comparisons. This lack of statistically significant differences could be due to the limited sample size combined with the fact that in general, adenomas show little chromosomal aberrations. On the other hand, it could simply be that these morphologically different entities do not differ in terms of chromosomal gains and losses. Finding no significant differences at the chromosomal level does not preclude other genetic and biological differences such as mutation or promoter methylation status of specific genes.

Aberrations already detected in adenomas may be early events in the stepwise process of accumulating changes which may cause progression of adenoma to carcinoma. As expected, the mean number of chromosomal events was lower in adenomas compared to the carcinomas [13, 14, 27]. Moreover, high level amplifications are uncommon in adenomas, while carcinomas frequently show high level amplifications [13, 16].

The aberrations found in both intestinal-type and pyloric gland adenomas, such as losses on chromosome 5q, are also frequently detected in gastric carcinomas [15, 19, 28]. Previous CGH results showed a significantly higher number of chromosome 5q losses in intestinal-type carcinoma compared to diffuse type carcinoma [29]. Chromosome 6, also lost in both types of adenomas, frequently is deleted in gastric carcinomas as determined by LOH studies [30, 31]. Moreover, chromosome 6q deletion has been reported to be involved in an early stage of gastric carcinogenesis, since chromosome 6q deletions are frequently detected in early gastric cancer and also in intestinal metaplasia [31, 32]. Losses of chromosomes 10 and 13 have been previously observed in adenomas at lower frequencies. In gastric carcinomas, both gains and losses of chromosome 10 and 13 have been observed by previous CGH studies [15, 19, 21, 33]. Chromosome 10 harbors the oncogene FGFR2 (10q26) and tumour suppressor genes PTEN/MMAC1 (10q23) and DMBT1 (10q25-q26), both involved in carcinogenesis, which could explain the observation of both gains and losses of chromosomes 10 in gastric carcinomas [3436]. Indeed chromosome 13 harbors tumour suppressor genes such as BRCA2 (13q12.3) and retinoblastoma gene (RB1) (13q14). In contrast, gain of chromosome 13q has been correlated to colorectal adenoma-to-carcinoma progression, and amplification of chromosome 13 has been observed in gastric adenomas with severe intraepithelial neoplasia [14, 37]. The precise role of chromosome 13 aberration in gastric cancer therefore remains to be resolved.

Most frequent copy number gains were observed on chromosomes 8, 9q, 11q and 20. Especially gains of chromosomes 8 and 20 are consistent with previous (array) CGH studies in both gastric adenomas and gastric carcinomas [1316, 19, 25], implicating this as early events in tumourigenesis. Although gain of chromosome 11q has not been described as a frequent event in adenomas, in carcinomas gain or amplification on chromosome 11q is common [1316]. In the present study gain of chromosome 11q was frequently observed in the adenomas, implying the malignant potential of these adenomas.

Conclusion

These data indicate that gains on chromosomes 8, 9q, 11q and 20 and losses on chromosomes 5q, 6, 10 and 13 are early events in gastric carcinogenesis. Despite the phenotypical differences, intestinal-type and pyloric gland adenoma do not differ significantly at the level of DNA copy number changes.

Methods

Material

Twenty-one paraffin-embedded gastric adenomas, 11 intestinal-type and 10 pyloric gland adenomas, were included in this study (Figure 2A and 2B). Tumour and patient data are given in Table 3. For each case, a tumour area consisting for at least 70% of tumour cells was demarcated on a 4 μm hematoxylin and eosin stained tissue section. Adjacent 10–15 serial tissue sections of 10 μm were stained with hematoxylin and the corresponding tumour area was microdissected using a surgical blade. A final 4μm "sandwich" section was made and stained with hemotoxylin and eosin, to compare with the first slide as a control. After deparaffinization, DNA was extracted by a column-based method (QIAamp DNA mini kit; Qiagen, Westburg, Leusden, NL) [38].
Table 3

Tumour and patient information

Tumour ID

Adenoma type

Grade of dysplasia

Gender

Age

Tumour ID

Adenoma type

Grade of dysplasie

Gender

Age

1

Intestinal

Moderate

Male

75

12

Pyloric gland

Moderate

Male

78

2

Intestinal

Moderate

Male

45

13

Pyloric gland

Mild

Male

50

3

Intestinal

Moderate

Male

80

14

Pyloric gland

Severe

Female

76

4

Intestinal

Moderate

Male

79

15

Pyloric gland

Moderate

Female

85

5

Intestinal

Moderate

Male

76

16

Pyloric gland

Moderate

Male

63

6

Intestinal

Moderate

Male

75

17

Pyloric gland

Mild

Female

86

7

Intestinal

Mild

Male

57

18

Pyloric gland

Moderate

Female

59

8

Intestinal

Moderate

Male

64

19

Pyloric gland

Moderate

Male

69

9

Intestinal

Mild

Male

63

20

Pyloric gland

Moderate

Female

78

10

Intestinal

Mild

Male

75

21

Pyloric gland

Moderate

Male

?

11

Intestinal

Moderate

Female

45

     
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-8-345/MediaObjects/12864_2007_Article_1058_Fig2_HTML.jpg
Figure 2

Haematoxilin and eosin staining (original magnification ×400) of intestinal-type (A) and pyloric gland (B) gastric adenomas. A. Intestinal-type adenoma of the stomach composed of irregularly arranged glands composed of intestinal-type epithelium with eosinophilic cytoplasm and enlarged nuclei. B. Pyloric gland adenoma of the stomach composed of densely back to back packed glands consisting of cells with pale cytoplasm and small round hyperchromatic nuclei.

Genomic DNA obtained from peripheral blood from ten normal individuals was pooled (either ten females or ten males, depending on the gender of the patient from which the adenoma was obtained) and used as control reference DNA.

Array CGH

Array CGH was performed essentially as described previously [39]. Briefly, 300 ng tumour and reference DNAs, sex-mismatch as experimental control, were labelled by random priming (Bioprime DNA Labelling System, Invitrogen, Breda, NL), each in a volume of 50μL. Non incorporated nucleotides were removed using ProbeQuant G-50 microcolumns (Amersham Biosciences). Cy3 labelled test genomic DNA and Cy5 labelled reference DNA were combined and co-precipitated with 100μg of human Cot-1 DNA (Invitrogen, Breda, NL) by adding 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% ethanol. The precipitate was collected by centrifugation at 14,000 rpm for 30 minutes at 4°C, and dissolved in 130 μl hybridization mixture containing 50% formamide, 2 × SCC and 4% SDS. The hybridization solution was heated for 10 minutes at 73°C to denature the DNA, followed by 60–120 minutes incubation at 37°C to allow the Cot-1 DNA to block repetitive sequences. The mixture was hybridized on an array containing approximately 5000 clones spotted in triplicate and spread along the whole genome with an average resolution of 1.0 Mb. The clones are comprised of the Sanger BAC clone set with an average resolution along the whole genome of 1.0 Mb [40], the OncoBac set [41], and selected clones of interest, obtained from the Children's Hospital Oakland Research Institute (CHORI). The selected clones comprise a collection of BAC clones on chromosome 6 filling the gaps larger than 1 Mb, and full-coverage contigs on specific regions on chromosomes 8, 13 and 20. Hybridization was performed in a in a hybridization station (Hybstation12 – Perkin Elmer Life Sciences, Zaventem, BE) and incubated for 38 h at 37°C. After hybridization, slides were washed in a solution containing 50% formamide, 2× SCC, pH 7 for 3 minutes at 45°C, followed by 1 minute wash steps at room temperature with PN buffer (PN: 0.1 M sodiumphosphate, 0.1% nonidet P40, pH 8), 0.2× SSC, 0.1× SCC and 0.01× SCC.

Image acquisition and data analysis

Images of the arrays were acquired by scanning (Agilent DNA Microarray scanner- Agilent technologies, Palo Alto, USA) and quantification of the signal and background intensities for each spot for the two channels Cy3 and Cy5 was performed by Imagene 5.6 software (Biodiscovery Ltd, Marina del Rey, CA, USA). Local background was subtracted from the signal median intensities and tumours to reference ratios were calculated. The ratios were normalized against the mode of the ratios of all autosomes. Clones with poor quality of one of the triplicates and hybridization with a standard deviation (SD) ≤ 0.22 and clones with > 50% missing values in all adenomas were excluded, leaving 4648 clones for further analysis. All subsequent analyses were done considering the clone position from the UCSC May2004 freeze of the Human Golden Path.

Array CGH smooth [42, 43], was used for automated detection of breakpoints to determine copy number gains and losses. Since variation in quality is observed in DNA obtained from formalin-fixed paraffin-embedded gastric tissues, different smoothing parameters were applied, depending on the quality of the hybridization. For array CGH profiles with a standard deviation smaller or equal to 0.15, between 0.15 and 0.20 or between 0.20 and 0.22, the applied smoothing parameters to determine gains and losses were 0.10, 0.15 and 0.20 respectively. Log2 tumour to reference ratio above 1 was regarded as amplification.

Statistical analysis

Unsupervised hierarchical cluster analysis was performed to analyze the distributions of the genomic profiles of all adenomas using TMEV software 3.0.3 [44]. Based on normalized smoothed log2 tumour to normal fluorescence intensity ratios, a hierarchical tree was constructed using the parameters complete linkage and euclidean distance. Pearson Chi-square test was used for analyzing correlations between cluster membership and adenoma type (SPSS 11.5.0 for windows, SPSS Inc, Chicago, IL, USA). P-values less than 0.05 were considered to be significant.

Supervised analysis was used for identifying chromosomal regions specific for the two adenoma types using CGH Multiarray and CGH Multiarray Region [45, 46]. Based on normalized smoothed log2 tumour to normal fluorescence intensity ratios, p-values were calculated for the significance of difference of values for each clone between pyloric gland and intestinal-type adenomas, using a Wilcoxon test with ties. To correct for multiple testing, a permutation-based false discovery rate (FDR) was calculated [47].

Declarations

Acknowledgements

We thank the Mapping Core and Map Finishing groups of the Wellcome Trust Sanger Institute for initial clone supply and verification. This work was financially supported by the Portuguese Foundation for Science and Technology (FCT), grant POCTI/CBO/41179/2001 and by Dutch Cancer Society grant-KWF 2004–3051.

Authors’ Affiliations

(1)
Department of Pathology, VU University Medical Center
(2)
Institute of Pathology and Molecular Immunology of University of Porto – IPATIMUP
(3)
Life and Health Sciences Research Institute (ICVS), Health Sciences School, University of Minho
(4)
Institute of Pathology, Klinikum Bayreuth
(5)
Dept. Surgery, Leiden University Medical Center
(6)
Institute of Clinical Genetics, University of Technology
(7)
Faculty of Medicine, University of Porto and Hospital

References

  1. Parkin DM, Pisani P, Ferlay J: Global cancer statistics. CA Cancer J Clin. 1999, 49: 33-64, 1.PubMedView Article
  2. Black RJ, Bray F, Ferlay J, Parkin DM: Cancer incidence and mortality in the European Union: cancer registry data and estimates of national incidence for 1990. Eur J Cancer. 1997, 33: 1075-1107. 10.1016/S0959-8049(96)00492-3.PubMedView Article
  3. Visser O, Coebergh J, Schouten L, Dijck J: Incidence of Cancer in The Netherlands 1997. Utrecht. 2001
  4. Correa P, Haenszel W, Cuello C, Tannenbaum S, Archer M: A model for gastric cancer epidemiology. Lancet. 1975, 2: 58-60. 10.1016/S0140-6736(75)90498-5.PubMedView Article
  5. Oberhuber G, Stolte M: Gastric polyps: an update of their pathology and biological significance. Virchows Arch. 2000, 437: 581-590. 10.1007/s004280000330.PubMedView Article
  6. Stolte M, Sticht T, Eidt S, Ebert D, Finkenzeller G: Frequency, location, and age and sex distribution of various types of gastric polyp. Endoscopy. 1994, 26: 659-665.PubMedView Article
  7. Borchard F, Ghanei A, Koldovski U, Hengels KJ, Buckmann FW: Gastrale Differenzierung in Adenomen der Magenschleim. Immunohistochemische und elektronmikroskopische Untersuchen. Verh Dtsch Ges Pathol. 1990, 74: 528-524.
  8. Hamilton SR, Aaltonen LS: WHO Classification. Tumors of the gastrointestinal tract. Pathology and genetics. IARC Press, Lyon. 2000, --20.
  9. Stolte M: Clinical consequences of the endoscopic diagnosis of gastric polyps. Endoscopy. 1995, 27: 32-37.PubMedView Article
  10. Vieth M, Kushima R, Borchard F, Stolte M: Pyloric gland adenoma: a clinico-pathological analysis of 90 cases. Virchows Arch. 2003, 442: 317-321.PubMed
  11. Schmitz JM, Stolte M: Gastric polyps as precancerous lesions. Gastrointest Endosc Clin N Am. 1997, 7: 29-46.PubMed
  12. Lengauer C, Kinzler KW, Vogelstein B: Genetic instabilities in human cancers. Nature. 1998, 396: 643-649. 10.1038/25292.PubMedView Article
  13. Kim YH, Kim NG, Lim JG, Park C, Kim H: Chromosomal alterations in paired gastric adenomas and carcinomas. Am J Pathol. 2001, 158: 655-662.PubMed CentralPubMedView Article
  14. Kokkola A, Monni O, Puolakkainen P, Nordling S, Haapiainen R, Kivilaakso E, Knuutila S: Presence of high-level DNA copy number gains in gastric carcinoma and severely dysplastic adenomas but not in moderately dysplastic adenomas. Cancer Genet Cytogenet. 1998, 107: 32-36. 10.1016/S0165-4608(98)00092-2.PubMedView Article
  15. van Dekken H, Alers JC, Riegman PH, Rosenberg C, Tilanus HW, Vissers K: Molecular cytogenetic evaluation of gastric cardia adenocarcinoma and precursor lesions. Am J Pathol. 2001, 158: 1961-1967.PubMed CentralPubMedView Article
  16. Weiss MM, Kuipers EJ, Postma C, Snijders AM, Stolte M, Vieth M, Pinkel D, Meuwissen SG, Albertson D, Meijer GA: Genome wide array comparative genomic hybridisation analysis of premalignant lesions of the stomach. Mol Pathol. 2003, 56: 293-298. 10.1136/mp.56.5.293.PubMed CentralPubMedView Article
  17. Kimura Y, Noguchi T, Kawahara K, Kashima K, Daa T, Yokoyama S: Genetic alterations in 102 primary gastric cancers by comparative genomic hybridization: gain of 20q and loss of 18q are associated with tumor progression. Mod Pathol. 2004, 17: 1328-1337. 10.1038/modpathol.3800180.PubMedView Article
  18. Koo SH, Kwon KC, Shin SY, Jeon YM, Park JW, Kim SH, Noh SM: Genetic alterations of gastric cancer: comparative genomic hybridization and fluorescence In situ hybridization studies. Cancer Genet Cytogenet. 2000, 117: 97-103. 10.1016/S0165-4608(99)00152-1.PubMedView Article
  19. Sakakura C, Mori T, Sakabe T, Ariyama Y, Shinomiya T, Date K, Hagiwara A, Yamaguchi T, Takahashi T, Nakamura Y, Abe T, Inazawa J: Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer. 1999, 24: 299-305. 10.1002/(SICI)1098-2264(199904)24:4<299::AID-GCC2>3.0.CO;2-U.PubMedView Article
  20. Takada H, Imoto I, Tsuda H, Sonoda I, Ichikura T, Mochizuki H, Okanoue T, Inazawa J: Screening of DNA copy-number aberrations in gastric cancer cell lines by array-based comparative genomic hybridization. Cancer Sci. 2005, 96: 100-110. 10.1111/j.1349-7006.2005.00016.x.PubMedView Article
  21. van Grieken NC, Weiss MM, Meijer GA, Hermsen MA, Scholte GH, Lindeman J, Craanen ME, Bloemena E, Meuwissen SG, Baak JP, Kuipers EJ: Helicobacter pylori-related and -non-related gastric cancers do not differ with respect to chromosomal aberrations. J Pathol. 2000, 192: 301-306. 10.1002/1096-9896(2000)9999:9999<::AID-PATH697>3.0.CO;2-F.PubMedView Article
  22. Weiss MM, Kuipers EJ, Hermsen MA, van Grieken NC, Offerhaus J, Baak JP, Meuwissen SG, Meijer GA: Barrett's adenocarcinomas resemble adenocarcinomas of the gastric cardia in terms of chromosomal copy number changes, but relate to squamous cell carcinomas of the distal oesophagus with respect to the presence of high-level amplifications. J Pathol. 2003, 199: 157-165. 10.1002/path.1260.PubMedView Article
  23. Wu CW, Chen GD, Fann CS, Lee AF, Chi CW, Liu JM, Weier U, Chen JY: Clinical implications of chromosomal abnormalities in gastric adenocarcinomas. Genes Chromosomes Cancer. 2002, 35: 219-231. 10.1002/gcc.10106.PubMedView Article
  24. Buffart T, Carvalho B, Hopmans E, Brehm V, Kranenbarg EK, Schaaij-Visser T, Eijk P, van Grieken N, Ylstra B, van de Velde CJ, Meijer G: Gastric cancers in young and elderly patients show different genomic profiles. J Pathol. 2007, 211: 45-51. 10.1002/path.2085.PubMedView Article
  25. Weiss MM, Kuipers EJ, Postma C, Snijders AM, Pinkel D, Meuwissen SG, Albertson D, Meijer GA: Genomic alterations in primary gastric adenocarcinomas correlate with clinicopathological characteristics and survival. Cell Oncol. 2004, 26: 307-317.PubMed
  26. Kushima R, Ruthlein HJ, Stolte M, Bamba M, Hattori T, Borchard F: 'Pyloric gland-type adenoma' arising in heterotopic gastric mucosa of the duodenum, with dysplastic progression of the gastric type. Virchows Arch. 1999, 435: 452-457. 10.1007/s004280050425.PubMedView Article
  27. Weiss MM, Kuipers EJ, Postma C, Snijders AM, Siccama I, Pinkel D, Westerga J, Meuwissen SG, Albertson DG, Meijer GA: Genomic profiling of gastric cancer predicts lymph node status and survival. Oncogene. 2003, 22: 1872-1879. 10.1038/sj.onc.1206350.PubMedView Article
  28. Oga A, Kong G, Ishii Y, Izumi H, Park CY, Sasaki K: Preferential loss of 5q14-21 in intestinal-type gastric cancer with DNA aneuploidy. Cytometry. 2001, 46: 57-62. 10.1002/1097-0320(20010215)46:1<57::AID-CYTO1038>3.0.CO;2-5.PubMedView Article
  29. Wu MS, Chang MC, Huang SP, Tseng CC, Sheu JC, Lin YW, Shun CT, Lin MT, Lin JT: Correlation of histologic subtypes and replication error phenotype with comparative genomic hybridization in gastric cancer. Genes Chromosomes Cancer. 2001, 30: 80-86. 10.1002/1098-2264(2000)9999:9999<::AID-GCC1062>3.0.CO;2-R.PubMedView Article
  30. Carvalho B, Seruca R, Carneiro F, Buys CH, Kok K: Substantial reduction of the gastric carcinoma critical region at 6q16.3-q23.1. Genes Chromosomes Cancer. 1999, 26: 29-34. 10.1002/(SICI)1098-2264(199909)26:1<29::AID-GCC4>3.0.CO;2-D.PubMedView Article
  31. Queimado L, Seruca R, Costa-Pereira A, Castedo S: Identification of two distinct regions of deletion at 6q in gastric carcinoma. Genes Chromosomes Cancer. 1995, 14: 28-34. 10.1002/gcc.2870140106.PubMedView Article
  32. Li BC, Chan WY, Li CY, Chow C, Ng EK, Chung SC: Allelic loss of chromosome 6q in gastric carcinoma. Diagn Mol Pathol. 2003, 12: 193-200. 10.1097/00019606-200312000-00003.PubMedView Article
  33. Peng DF, Sugihara H, Mukaisho K, Tsubosa Y, Hattori T: Alterations of chromosomal copy number during progression of diffuse-type gastric carcinomas: metaphase- and array-based comparative genomic hybridization analyses of multiple samples from individual tumours. J Pathol. 2003, 201: 439-450. 10.1002/path.1459.PubMedView Article
  34. Katoh M, Katoh M: FGFR2 and WDR11 are neighboring oncogene and tumor suppressor gene on human chromosome 10q26. Int J Oncol. 2003, 22: 1155-1159.PubMed
  35. Mori M, Shiraishi T, Tanaka S, Yamagata M, Mafune K, Tanaka Y, Ueo H, Barnard GF, Sugimachi K: Lack of DMBT1 expression in oesophageal, gastric and colon cancers. Br J Cancer. 1999, 79: 211-213.PubMed CentralPubMedView Article
  36. Wang JY, Huang TJ, Chen FM, Hsieh MC, Lin SR, Hou MF, Hsieh JS: Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in advanced gastric carcinomas. Virchows Arch. 2003, 442: 437-443.PubMed
  37. Hermsen M, Postma C, Baak J, Weiss M, Rapallo A, Sciutto A, Roemen G, Arends JW, Williams R, Giaretti W, De Goeij A, Meijer G: Colorectal adenoma to carcinoma progression follows multiple pathways of chromosomal instability. Gastroenterology. 2002, 123: 1109-1119. 10.1053/gast.2002.36051.PubMedView Article
  38. Weiss MM, Hermsen MA, Meijer GA, van Grieken NC, Baak JP, Kuipers EJ, van Diest PJ: Comparative genomic hybridisation. Mol Pathol. 1999, 52: 243-251.PubMed CentralPubMedView Article
  39. Snijders AM, Nowak N, Segraves R, Blackwood S, Brown N, Conroy J, Hamilton G, Hindle AK, Huey B, Kimura K, Law S, Myambo K, Palmer J, Ylstra B, Yue JP, Gray JW, Jain AN, Pinkel D, Albertson DG: Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet. 2001, 29: 263-264. 10.1038/ng754.PubMedView Article
  40. ensembl . 2007, [http://​www.​ensembl.​org/​Homo_​sapiens/​cytoview]
  41. informa. 2007, [http://​informa.​bio.​caltech.​edu/​Bac_​onc.​html]
  42. Jong K, Marchiori E, van der Vaart AV, Ylstra B, Weiss MM, Meijer G: Chromosomal breakpoint detection in human cancer. In LNCN. 2003, 2611: 54-65.
  43. Jong K, Marchiori E, Meijer G, Vaart AV, Ylstra B: Breakpoint identification and smoothing of array comparative genomic hybridization data. Bioinformatics. 2004, 20: 3636-3637. 10.1093/bioinformatics/bth355.PubMedView Article
  44. tigr . 2007, [http://​www.​tigr.​org/​software]
  45. CGH Multiarray. 2007, [http://​www.​few.​vu.​nl/​~mavdwiel/​]
  46. van de Wiel MA, Smeets SJ, Brakenhoff RH, Ylstra B: CGHMultiArray: exact P-values for multi-array comparative genomic hybridization data. Bioinformatics. 2005, 21: 3193-3194. 10.1093/bioinformatics/bti489.PubMedView Article
  47. Manduchi E, Grant GR, McKenzie SE, Overton GC, Surrey S, Stoeckert CJ: Generation of patterns from gene expression data by assigning confidence to differentially expressed genes. Bioinformatics. 2000, 16: 685-698. 10.1093/bioinformatics/16.8.685.PubMedView Article

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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