Gastric cancers of Western European and African patients show different patterns of genomic instability

  • Tineke E Buffart1,

    Affiliated with

    • Melanie Louw2,

      Affiliated with

      • Nicole CT van Grieken1,

        Affiliated with

        • Marianne Tijssen1,

          Affiliated with

          • Beatriz Carvalho1,

            Affiliated with

            • Bauke Ylstra1,

              Affiliated with

              • Heike Grabsch3,

                Affiliated with

                • Chris JJ Mulder4,

                  Affiliated with

                  • Cornelis JH van de Velde5,

                    Affiliated with

                    • Schalk W van der Merwe6 and

                      Affiliated with

                      • Gerrit A Meijer1Email author

                        Affiliated with

                        BMC Medical Genomics20114:7

                        DOI: 10.1186/1755-8794-4-7

                        Received: 7 October 2010

                        Accepted: 13 January 2011

                        Published: 13 January 2011

                        Abstract

                        Background

                        Infection with H. pylori is important in the etiology of gastric cancer. Gastric cancer is infrequent in Africa, despite high frequencies of H. pylori infection, referred to as the African enigma. Variation in environmental and host factors influencing gastric cancer risk between different populations have been reported but little is known about the biological differences between gastric cancers from different geographic locations. We aim to study genomic instability patterns of gastric cancers obtained from patients from United Kingdom (UK) and South Africa (SA), in an attempt to support the African enigma hypothesis at the biological level.

                        Methods

                        DNA was isolated from 67 gastric adenocarcinomas, 33 UK patients, 9 Caucasian SA patients and 25 native SA patients. Microsatellite instability and chromosomal instability were analyzed by PCR and microarray comparative genomic hybridization, respectively. Data was analyzed by supervised univariate and multivariate analyses as well as unsupervised hierarchical cluster analysis.

                        Results

                        Tumors from Caucasian and native SA patients showed significantly more microsatellite instable tumors (p < 0.05). For the microsatellite stable tumors, geographical origin of the patients correlated with cluster membership, derived from unsupervised hierarchical cluster analysis (p = 0.001). Several chromosomal alterations showed significantly different frequencies in tumors from UK patients and native SA patients, but not between UK and Caucasian SA patients and between native and Caucasian SA patients.

                        Conclusions

                        Gastric cancers from SA and UK patients show differences in genetic instability patterns, indicating possible different biological mechanisms in patients from different geographical origin. This is of future clinical relevance for stratification of gastric cancer therapy.

                        Background

                        Gastric cancer is the second most common cause of cancer death worldwide, but incidence and mortality rates show large variations across different countries. Japan and China show the highest incidence rates of gastric cancer of 80-115 cancers/100,000 population and 32-59 cancers/100,000 population respectively, while in other Asian counties, such as India, Bangladesh, and Thailand, the incidence rates are much lower (10.6, 1.3, and 7.1 per 100,000 populations, respectively). Also within Europe, incidence and mortality rates differ between countries. Portugal has the highest incidence rates (33.2/100,000) whereas other countries in Western Europe show incidence rates of 19.4 per 100,000 populations. In the Netherlands it ranks fifth as a cause of cancer death with incidence rates of 14.6/100,000. In Africa, gastric cancer is infrequent, with incidence rates varying between 6.9/100,000 in Northern Africa, 12.9/100,000 in Eastern Africa, 11.9/100,000 in Southern Africa and 7.0/100,000 in Western Africa (Table 1) [13].
                        Table 1

                        Incidence rates of gastric cancers per 100,000 populations.

                         

                        Incidence rates

                         

                        Incidence rates

                        Japan

                        80-115

                        Netherlands

                        14.6

                        China

                        32-59

                        Western Europe

                        19.4

                        India

                        10.6

                        Northern Africa

                        6.9

                        Bangladesh

                        1.3

                        Eastern Africa

                        12.9

                        Thailand

                        7.1

                        Southern Africa

                        11.9

                        Portugal

                        33.2

                        Western Africa

                        7.0

                        According to the Correa model, intestinal type gastric cancers arise through a sequence of events, starting with chronic active gastritis due to infection with Helicobacter pylori (H. pylori). This chronic inflammatory process may lead to atrophy, intestinal metaplasia followed by dysplasia and eventually may lead to invasive adenocarcinoma [4].

                        The mechanism by which H. pylori contributes to gastric carcinogenesis is still largely unknown. However, we do know that gastric cancer is the result of accumulation of (epi)genetic changes. In gastric cancer, at least two types of genetic instability play a role. Microsatellite instability (MSI) occurs in cancers associated with Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC), and in 10-15% of sporadic gastric cancers due to hMLH1 promoter hypermethylation [5, 6]. However, the majority of gastric cancers show chromosomal instability, resulting in DNA copy number aberrations that can be analyzed in detail by high resolution array comparative genomic hybridization (array CGH). In a previous study using chromosome based comparative genomic hybridization (CGH), we were unable to demonstrate that there are specific chromosomal alterations which are associated with H. pylori infection [7].

                        Infection with H. pylori is important in the etiology of gastric cancer, consequently high incidences of gastric cancer are observed in areas with high prevalence of H. pylori infection, like Asia. However, despite high frequencies of H. pylori infection in Africa, gastric cancer is infrequent in Africa, a phenomenon often referred to as the 'African enigma' [8, 9]. We hypothesize that geographical differences in environmental factors, including infection with H. pylori, and host factors are reflected by different biological characteristics of the tumors from those areas. Therefore, we compared MSI status and DNA copy number profiles in gastric cancer patients from United Kingdom (UK) and South Africa (SA).

                        Methods

                        Material

                        A total of 67 gastric adenocarcinomas were included in this study. Of these, 33 gastric adenocarcinomas were obtained from Leeds (Leeds, General Infirmary, UK) and 34 gastric adenocarcinomas were obtained from Pretoria (Prinshof Campus, Pretoria, South Africa), of which 25 were obtained from native South African patients (native SA) and 9 from Caucasian South African patients (Caucasian SA), respectively. All tumors were randomly selected after testing for proper DNA quality as previously described [10]. All gastric adenocarcinomas were staged according to the TNM classification (5th edition) for the grading and to the Laurén's classification for morphology [11]. The study was approved by the Institutional Review Board and was in accordance with local medical ethical regulations.

                        DNA isolation procedure

                        DNA was isolated from formalin-fixed and paraffin embedded gastric cancer material as described previously,[12, 13] using the QIAamp microkit (Qiagen, Hilden, Germany). DNA concentrations were measured using a Nanodrop ND-1000 spectrophotometer (Isogen, IJsselstein, The Netherlands) and DNA quality was assessed by isothermal amplification [10]. Genomic DNA isolated from peripheral blood obtained from eighteen healthy females or males was pooled to use as normal reference.

                        Microsatellite instability (MSI) analysis

                        MSI analysis was performed using the MSI Analysis System (MSI Multiplex System Version 1.1, Promega) consisting of five nearly monomorphic mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24, MONO-27) according to the manufacturer's instructions. PCR products were separated by capillary electrophoresis using an ABI 3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA), and analyzed using GeneScan 3100 (Applied Biosystems, Foster City, CA, USA). An internal lane size standard was added to the PCR samples for accurate sizing of alleles and to adjust for run-to run variations. When all markers were stable, the tumor was interpreted as microsatellite stable (MSS). The tumor was interpreted as MSI-low (MSI-L) if one marker was instable and MSI-high (MSI-H) if two or more markers showed instability. MSI-L tumors were included in the MSS category in further analysis. Due to polymorphisms[14] in the South African population, native South African tumors were classified as MSI when three or more markers were instable.

                        Array CGH

                        Array CGH was performed as described before [12, 15]. Briefly, 600 ng tumor and normal reference DNAs were labeled by random priming (Bioprime DNA Labeling System, Invitrogen, Breda, The Netherlands) and hybridized onto a BAC array containing approximately 6000 clones, consisting of the Sanger BAC clone set with an average resolution along the whole genome of 1.0 Mb, the OncoBac set, containing approximately 600 clones corresponding to 200 cancer-related genes, and selected clones of interest obtained from the Children's Hospital Oakland Research Institute (CHORI) to fill gaps larger than 1 Mb on chromosome 6 and to have full coverage contigs of regions on chromosome 8, 13 and 20. All clones were printed in triplicate on Nexterion slides (Schott Nexterion, Jena, Germany). Subsequent analysis was performed according to the clone position from the UCSC May 2004 freeze of the Human Genome Golden Path http://​genome.​ucsc.​edu.

                        Image acquisition and data analysis

                        Images of the arrays were acquired by scanning (Agilent DNA Microarray scanner, Agilent Technologies, Palo Alto, USA) and Bluefuse software version 3.4 (BlueGnome, Cambridge, UK) was used for automatic feature extraction. Spots were excluded when the quality flag was below 1 or the confidence value was below 0.1. Log2 tumor to normal ratio was calculated for each clone and median block normalization was used to normalize the data. Quality of array CGH profiles was measured by calculating a median absolute deviation value of chromosome 2 (MAD2) [10]. Array CGH profiles with MAD2 values >0.18 were excluded from further analysis. For determining copy number gains and losses, the R package CGH call was used [16]. Output of the CGH call analysis was used for CGH region analysis to compress the data, using a threshold for average error rate of 0.001 [17]. Hierarchical cluster analysis was performed using the WECCA program, with the parameter total linkage [18].

                        Array data can be accessed using the Gene Expression Omnibus (GEO) http://​www.​ncbi.​nlm.​nih.​gov/​geo/​, under accession number GSE22789.

                        Statistical analysis

                        Significance of differences for categorical variables between different categories was tested using a chi-square test. One-way ANOVA with Bonferroni correction was used to calculate significant differences for continuous variables between Caucasian SA, native SA, and UK patients (SPSS 12.0.1 for Windows, SPSS Inc, Chicago, IL, USA). P values less than 0.05 were considered to be significant.

                        Supervised analysis was performed using the non-parametric Mann-Whitney two-sample test (CGH test [19]). Alterations in patterns between different tumor groups were compared using a binomial differential proportion test. The test procedure included a permutation-based false discovery rate correction for multiple testing [20]. Two-sided p values less than 0.05 and false discovery rates below 0.15 were considered to be significant.

                        Results

                        Clinicopathological data

                        The mean age of the UK gastric cancer patients was 73.3 years (range 51-96), mean age of the Caucasian SA patients was 68.0 years (range 56-84) and the mean age of the native SA patients was 56.5 years (range 29-79). One-way ANOVA with Bonferroni correction yielded a significant difference between the mean age of the patients between native and Caucasian SA patients (p = 0.03) and between native SA and UK patients (p < 0.001), but not between Caucasian SA and UK patients (n.s).

                        There was no significant difference between patients of different geographical location and gender, tumor stage (T-category) and lymph node stage (N-category). UK gastric cancers showed significantly more diffuse type morphology compared to South African gastric cancers (p = 0.002). Overview of patient and tumor characteristics is given in Table 2.
                        Table 2

                        Tumor and patient characteristics of the 67 tumors used for MSI and array CGH analysis.

                        ID

                        gender

                        age

                        Tumor type

                        T

                        N

                        origin

                        MSI status

                        Cluster number

                        Cluster order

                        % events

                        % gains

                        % losses

                        1

                        F

                        62

                        intestinal

                        T2

                        N1

                        Cauc SA

                        MSS

                        5

                        34

                        3.2

                        3.2

                        0

                        2

                        M

                        73

                        intestinal

                        T2

                        N2

                        Cauc SA

                        MSS

                        5

                        28

                        16.9

                        13.4

                        3.4

                        3

                        F

                        59

                        intestinal

                        T2

                        N1

                        Cauc SA

                        MSS

                        5

                        26

                        26.5

                        15.1

                        11.4

                        4

                        F

                        74

                        intestinal

                        T2

                        N1

                        Cauc SA

                        MSS

                        5

                        27

                        15.7

                        9.3

                        6.5

                        5

                        M

                        56

                        intestinal

                        T3

                        N1

                        Cauc SA

                        MSI

                        -

                        -

                        15.6

                        10.3

                        5.3

                        6

                        M

                        57

                        intestinal

                        T2

                        N1

                        Cauc SA

                        MSS

                        6

                        48

                        3.4

                        2.4

                        1.0

                        7

                        F

                        84

                        intestinal

                        T1

                        N0

                        Cauc SA

                        MSS

                        6

                        43

                        10.9

                        7.9

                        3.1

                        8

                        F

                        79

                        intestinal

                        T3

                        N0

                        Cauc SA

                        MSI

                        -

                        -

                        17.6

                        16.8

                        0.8

                        9

                        M

                        68

                        intestinal

                        T3

                        N2

                        Cauc SA

                        MSS

                        5

                        25

                        32.1

                        24.3

                        7.7

                        10

                        M

                        65

                        intestinal

                        T2

                        N0

                        native SA

                        MSI

                        -

                        -

                        1.7

                        1.7

                        0

                        11

                        M

                        57

                        intestinal

                        T1

                        N0

                        native SA

                        MSI

                        -

                        -

                        0

                        0

                        0

                        12

                        F

                        29

                        intestinal

                        T4

                        N0

                        native SA

                        MSS

                        6

                        37

                        28.3

                        26.2

                        2.1

                        13

                        F

                        59

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        6

                        42

                        10.6

                        5.3

                        5.3

                        14

                        M

                        66

                        intestinal

                        T2

                        N1

                        native SA

                        MSS

                        5

                        31

                        6.4

                        6.4

                        0

                        15

                        M

                        46

                        intestinal

                        T3

                        N2

                        native SA

                        MSS

                        5

                        35

                        3.2

                        3.2

                        0

                        16

                        F

                        -

                        diffuse

                        T4

                        N2

                        native SA

                        MSS

                        6

                        44

                        13.2

                        12.4

                        0.7

                        17

                        M

                        51

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        5

                        32

                        6.1

                        6.1

                        0

                        18

                        F

                        49

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        4

                        20

                        29.1

                        18.2

                        10.9

                        19

                        M

                        56

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        4

                        19

                        26.0

                        10.3

                        15.7

                        20

                        M

                        48

                        intestinal

                        T3

                        N2

                        native SA

                        MSS

                        5

                        36

                        5.4

                        5.4

                        0

                        21

                        M

                        65

                        mixed

                        T3

                        N1

                        native SA

                        MSI

                        -

                        -

                        18.2

                        16.3

                        1.9

                        22

                        M

                        60

                        intestinal

                        T2

                        -

                        native SA

                        MSS

                        6

                        41

                        14.0

                        11.3

                        2.7

                        23

                        F

                        63

                        intestinal

                        -

                        -

                        native SA

                        MSS

                        6

                        38

                        17.1

                        10.7

                        6.4

                        24

                        F

                        54

                        papillary

                        T2

                        N0

                        native SA

                        MSS

                        3

                        14

                        44.8

                        20.6

                        24.2

                        25

                        M

                        67

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        -

                        -

                        -

                        -

                        -

                        26

                        M

                        31

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        5

                        24

                        41.1

                        28.4

                        12.7

                        27

                        M

                        43

                        intestinal

                        T3

                        N1

                        native SA

                        MSI

                        -

                        -

                        9.9

                        9.9

                        0

                        28

                        F

                        71

                        intestinal

                        T3

                        -

                        native SA

                        MSS

                        5

                        33

                        3.7

                        2.7

                        1.0

                        29

                        F

                        77

                        intestinal

                        T3

                        N1

                        native SA

                        MSS

                        5

                        30

                        12.7

                        7.1

                        5.5

                        30

                        M

                        57

                        intestinal

                        T2

                        N0

                        native SA

                        MSS

                        -

                        -

                        -

                        -

                        -

                        31

                        M

                        79

                        intestinal

                        T3

                        N2

                        native SA

                        MSI

                        -

                        -

                        2.5

                        2.5

                        0

                        32

                        M

                        57

                        intestinal

                        T4

                        N0

                        native SA

                        MSI

                        -

                        -

                        17.8

                        16.8

                        1.0

                        33

                        M

                        56

                        mixed

                        T3

                        N1

                        native SA

                        MSS

                        4

                        17

                        42.2

                        21.3

                        20.9

                        34

                        F

                        49

                        mixed

                        T3

                        N3

                        native SA

                        MSS

                        5

                        29

                        22.3

                        19.2

                        3.2

                        35

                        F

                        82

                        diffuse

                        T1

                        N0

                        UK

                        MSS

                        6

                        46

                        11.2

                        10.3

                        1.0

                        36

                        M

                        81

                        diffuse

                        T3

                        N2

                        UK

                        MSS

                        4

                        22

                        15.7

                        7.2

                        8.5

                        37

                        M

                        71

                        diffuse

                        T2

                        N1

                        UK

                        MSS

                        7

                        53

                        18.4

                        14.7

                        3.7

                        38

                        M

                        73

                        intestinal

                        T2

                        N0

                        UK

                        MSS

                        2

                        11

                        26.0

                        17.2

                        8.8

                        39

                        F

                        65

                        diffuse

                        T2

                        N3

                        UK

                        MSS

                        6

                        45

                        15.0

                        12.0

                        3.1

                        40

                        F

                        58

                        diffuse

                        T3

                        N3

                        UK

                        MSS

                        1

                        6

                        25.7

                        13.7

                        12.0

                        41

                        F

                        51

                        diffuse

                        T3

                        N3

                        UK

                        MSS

                        1

                        7

                        22.4

                        16.5

                        6.0

                        42

                        M

                        91

                        intestinal

                        T1

                        N0

                        UK

                        MSS

                        7

                        56

                        49.2

                        27.6

                        21.6

                        43

                        M

                        71

                        diffuse

                        T3

                        N2

                        UK

                        MSS

                        7

                        49

                        40.6

                        19.8

                        20.8

                        44

                        M

                        73

                        intestinal

                        T2

                        N1

                        UK

                        MSS

                        1

                        5

                        21.0

                        8.2

                        12.9

                        45

                        M

                        64

                        diffuse

                        T2

                        N0

                        UK

                        MSS

                        7

                        50

                        14.9

                        10.3

                        4.6

                        46

                        F

                        71

                        intestinal

                        T1

                        N0

                        UK

                        MSS

                        1

                        4

                        40.8

                        13.4

                        27.4

                        47

                        F

                        60

                        intestinal

                        T4

                        N1

                        UK

                        MSS

                        3

                        16

                        30.1

                        15.6

                        14.5

                        48

                        F

                        96

                        diffuse

                        T3

                        N0

                        UK

                        MSS

                        2

                        12

                        41.0

                        25.0

                        16.0

                        49

                        M

                        91

                        mixed

                        T3

                        N0

                        UK

                        MSS

                        6

                        40

                        16.0

                        11.4

                        4.6

                        50

                        M

                        81

                        diffuse

                        T2

                        N0

                        UK

                        MSS

                        2

                        10

                        37.7

                        17.2

                        20.4

                        51

                        F

                        83

                        intestinal

                        T2

                        N0

                        UK

                        MSS

                        7

                        51

                        14.5

                        10.5

                        4.0

                        52

                        F

                        82

                        intestinal

                        T3

                        N1

                        UK

                        MSS

                        6

                        47

                        2.2

                        2.2

                        0

                        53

                        F

                        77

                        intestinal

                        T3

                        N1

                        UK

                        MSS

                        6

                        39

                        19.5

                        11.1

                        8.5

                        54

                        M

                        74

                        mixed

                        T2

                        N1

                        UK

                        MSS

                        2

                        8

                        44.8

                        22.4

                        22.3

                        55

                        F

                        59

                        diffuse

                        T3

                        N1

                        UK

                        MSS

                        3

                        15

                        26.4

                        18.1

                        8.2

                        56

                        M

                        77

                        mixed

                        T3

                        N2

                        UK

                        MSS

                        1

                        3

                        24.1

                        13.7

                        10.3

                        57

                        M

                        75

                        intestinal

                        T2

                        N0

                        UK

                        MSS

                        2

                        9

                        36.9

                        18.2

                        18.7

                        58

                        M

                        64

                        diffuse

                        T3

                        N3

                        UK

                        MSS

                        2

                        13

                        35.3

                        20.2

                        15.1

                        59

                        M

                        71

                        intestinal

                        T3

                        N1

                        UK

                        MSS

                        1

                        2

                        31.0

                        11.7

                        19.3

                        60

                        F

                        74

                        diffuse

                        T3

                        N2

                        UK

                        MSS

                        4

                        23

                        23.2

                        14.7

                        8.4

                        61

                        M

                        81

                        intestinal

                        T2

                        N0

                        UK

                        MSI

                        -

                        -

                        10.7

                        8.7

                        2.0

                        62

                        F

                        74

                        mixed

                        T3

                        N2

                        UK

                        MSS

                        4

                        18

                        30.4

                        17.9

                        12.5

                        63

                        M

                        67

                        mixed

                        T3

                        N1

                        UK

                        MSS

                        7

                        52

                        12.3

                        6.4

                        5.9

                        64

                        M

                        73

                        intestinal

                        T3

                        N1

                        UK

                        MSS

                        4

                        21

                        15.2

                        7.3

                        7.9

                        65

                        F

                        66

                        mixed

                        T3

                        N2

                        UK

                        MSS

                        7

                        55

                        34.3

                        18.7

                        15.6

                        66

                        M

                        82

                        intestinal

                        T1

                        N1

                        UK

                        MSS

                        7

                        54

                        46.7

                        27.4

                        19.3

                        67

                        F

                        62

                        mixed

                        T3

                        N0

                        UK

                        MSS

                        1

                        1

                        44.0

                        20.7

                        23.3

                        Percentages of events, gains and losses are given for all tumors of sufficient array CGH quality. Cluster number and order are listed for the 56 cases included in the cluster analysis.

                        F: female, M: male, T: T-stage, N: N-stage MSS: microsatellite stable, MSI: microsatellite instable, Cauc SA: Caucasian South African patients, native SA: native South African patients, UK: patients from United Kingdom, -: unknown.

                        Microsatellite instability (MSI) analysis

                        Two out of nine (22%) Caucasian SA gastric cancers, six out of 25 (24%) native SA gastric cancers, and one out of 33 (3%) UK gastric cancers showed MSI. All other gastric cancers were MSS (Table 2). Pearson chi-square yielded a significant difference between the three different tumor groups and MSI status (p < 0.05).

                        Hierarchical cluster analysis

                        We analyzed DNA from all gastric cancers by genome-wide array CGH analysis to unravel DNA copy number changes in tumors from different geographical location. MSI positive gastric cancers and gastric cancers with array CGH profiles with a MAD2 value above 0.18 were excluded for cluster analysis leaving 56 tumors (from 32 UK, 17 native SA and 7 Caucasian SA patients) for further analysis.

                        Hierarchical cluster analysis yielded seven clusters which were significantly correlated with gastric cancers of different geographical origin (p < 0.001) (Figure 1). Clusters 1, 2 and 7 obtained only gastric cancers from UK patients. Clusters 3 and 4 comprised of gastric cancers from UK and native SA patients. Cluster 5 contained only gastric cancers from SA patients, and cluster 6 contained a mixture of tumors of all three groups (Table 2).
                        http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-4-7/MediaObjects/12920_2010_204_Fig1_HTML.jpg
                        Figure 1

                        Cluster analysis of 56 gastric adenocarcinomas of which 32, 17 and 7 were obtained from UK, native SA and Caucasian SA patients, respectively. Hierarchical cluster analysis yielded 7 clusters significantly correlated with geographical origin of the tumors (p < 0.001). Columns represent the different tumors and rows represent the different chromosomal regions, with chromosome 1 at the bottom and chromosome 22 at the top of the heatmap. DNA copy number gains and losses are indicated in green and red, respectively. The yellow and blue bar next to the cluster represents the chromosome separation.

                        UK patients showed significantly more gastric adenocarcinomas of the diffuse type according to the Laurén classification[11] compared to SA patients (p = 0.002). We therefore repeated the cluster analysis including only intestinal type gastric carcinomas. Cluster membership of the remaining 12 tumors from UK patients and 12 and 7 tumors from native SA and Caucasian SA patients, respectively, was again significantly correlated to geographical origin of the patient (p < 0.001). Moreover, when analyzing only UK gastric cancers, hierarchical cluster analysis did not separate intestinal and diffuse type gastric cancers, nor were any significant differences observed between these two morphological tumor types with supervised analysis using CGH test.

                        Cluster membership was independent of gender, tumor stage, lymph node stage and of age of the patients (categorized as < 50 years of age versus ≥ 50 years of age).

                        DNA copy number changes

                        We first compared the number of events, which was defined as percentage of clones showing a gain or loss. Gastric cancers from UK patients showed a higher number of events (27% (range 2-49%)) compared to cancers from Caucasian SA (16% (range 3-32%)) and native SA patients (16% (range 0-45%)) (p = 0.005). Cancers from UK, Causasian SA and native SA patients showed 15% (range 2-28%), 11% (range 2-24%) and 11% (range 0-28%) of gained clones respectively, and 12% (range 0-27%), 4% (range 0-11%) and 5% (0-24%) clones showing a loss, respectively. A significant difference in the percentage of clones showing a loss was observed between UK patients and Caucasian SA patients (p = 0.002) and between UK patients and native SA patients (p = 0.02).

                        Also, when looking only at microsatellite stable gastric cancers UK patients showed a higher number of events (27% (range 2-49%)) compared to microsatellite stable cancers from Caucasian SA and native SA patients (16% (range 3-32%) and 19% (range 3-45%), respectively; p = 0.04). Microsatellite stable cancers from UK, Caucasian SA and native SA patients showed 15% (range 2-28%), 12% (range 2-24%) and 13% (range 3-28%) of gained clones, respectively. There was again a significant difference in percentages of clones showing a loss between cancers from UK and Caucasian SA patients (12% (range 0-27%) and 4% (range 0-11%), respectively; p = 0.04) and between cancers from UK and native SA patients (12% (range 0-27%) and 7% (range 0-24%) respectively; p = 0.04).

                        An overview of frequently altered (>30%) chromosomal regions with gains and losses per tumor group is given in Tables 3, 4 and 5. Most frequently altered (>30%) chromosomal regions observed in the UK tumors were gains on chromosomes 1p, 1q, 5p, 6p, 7p, 7q, 8q, 9q, 10p, 10q, 11p, 11q, 13q, 14q, 16p, 16q, 17p, 17q, 19p, 19q, 20p, 20q, 21q and 22q and losses on chromosomes 1p, 1q, 3p, 4p, 4q, 5q, 9p, 12q, 13q, 14q, 15q, 17q, 18q and 21q (Table 3). Most frequent DNA copy number aberrations in the native SA patients were gains on the chromosomal regions 7p, 7q, 8q, 9q, 17q, 19p, 19q, 20p and 20q, and losses on 3p and 4q (Table 4). Most frequently altered chromosomal regions in Caucasian SA patients were gains on 3q, 5p, 7p, 7q, 8p, 8q, 9q, 11q, 16p, 17q, 19p, 19q, 20p and 20q and losses on 3p, 4q and 9p (Table 5). A summary of frequencies of gains and losses of all gastric cancers per tumor group is presented in Figures 2 (UK), 3 (native SA) and 4 (Caucasian SA).
                        Table 3

                        Detailed overview of frequent DNA copy number aberrations (>30%) of tumors from UK patients.

                        chromosomal aberrations

                        flanking clones

                        position (bp)

                         

                        segment size

                        gains

                        losses

                        start

                        end

                        start

                        end

                        (Mb)

                        1p36.33-p36.21

                         

                        RP11-206L10

                        RP4-636F13

                        672780

                        12417597

                        11.74

                         

                        1p31.1

                        RP5-944F13

                        RP11-246O4

                        69815162

                        83112098

                        13.30

                         

                        1p21.3-p13.3

                        RP11-146P11

                        RP5-1077K16

                        95695632

                        107389118

                        11.69

                        1q21.2-q23.1

                         

                        RP4-790G17

                        RP11-214H6

                        146971278

                        153444622

                        6.47

                         

                        1q31.1-q31.3

                        RP11-134C1

                        RP11-75C23

                        184717073

                        194242465

                        9.53

                        1q32.1-q32.2

                         

                        RP11-150l7

                        RP11-564A8

                        197877387

                        203602276

                        5.72

                         

                        3p26.3

                        RP11-385A18

                        RP11-129K1

                        46140

                        2377366

                        2.33

                         

                        3p25.1-p24.1

                        RP11-255O19

                        RP11-99M10

                        15780361

                        30799547

                        15.02

                         

                        3p14.2

                        RP11-170K19

                        RP11-114P15

                        59701329

                        62639806

                        2.94

                         

                        3p12.3-p11.2

                        RP11-103P13

                        RP11-91M15

                        75146113

                        96627928

                        21.48

                         

                        4p16.1-q35.2

                        RP11-61G19

                        CTC-963K6

                        10275012

                        191158370

                        180.88

                        5p15.33

                         

                        RP11-811I15

                        CTD-2265D9

                        70262

                        2671745

                        2.60

                         

                        5q11.1-q23.3

                        RP11-269M20

                        RP11-114H7

                        49913067

                        130460728

                        80.55

                        6p21.32-p21.1

                         

                        RP11-79I1

                        RP11-121G20

                        33123932

                        44385866

                        11.26

                        7p22.3-p22.1

                         

                        RP11-713A20

                        RP11-161C7

                        106471

                        6396697

                        6.29

                        7p11.2

                         

                        RP11-449G3

                        RP11-34J24

                        54413814

                        55403627

                        0.99

                        7q22.1

                         

                        RP11-10D8

                        RP11-163M5

                        98067793

                        101528379

                        3.46

                        7q36.1

                         

                        RP11-89P11

                        RP11-43l19

                        147485335

                        151131938

                        3.65

                        7q36.3

                         

                        RP11-58F7

                        RP11-120H14

                        157072238

                        158524109

                        1.45

                        8q24.12-q24.3

                         

                        RP11-22A24

                        RP5-1109M23

                        120711365

                        146238749

                        25.53

                         

                        9p24.3-p21.1

                        RP11-48M17

                        RP11-141J7

                        2136329

                        32469400

                        30.33

                        9q33.3-q34.3

                         

                        RP11-205K6

                        RP11-424E7

                        126296075

                        138363252

                        12.07

                        10p15.3

                         

                        RP11-631M21

                        RP11-74N14

                        50000

                        1789100

                        1.74

                        10p15.2

                         

                        RP11-195B3

                         

                        3293007

                        3338470

                        0.05

                        10q22.1

                         

                        RP11-91A1

                        RP11-28E3

                        72033907

                        73573433

                        1.54

                        11p15.5-p15.4

                         

                        CTC-908H22

                        RP11-304P12

                        178227

                        3140168

                        2.96

                        11q12.2-q13.5

                         

                        RP11-286N22

                        RP11-30J7

                        60851860

                        76232373

                        15.38

                         

                        12q21.2-q22

                        RP1-97G4

                        RP11-2K12

                        76228586

                        91346299

                        15.12

                        13q11-q12.11

                         

                        RP11-94A1

                        RP11-61K9

                        18360157

                        19386914

                        1.03

                         

                        13q21.2-q21.33

                        RP11-310K10

                        RP11-451E2A

                        60721181

                        71574154

                        10.85

                        13q32.3

                         

                        RP11-19J14

                        RP11-113F15

                        97851594

                        99328275

                        1.48

                        13q33.3-q34

                         

                        RP11-61I17

                        RP11-569D9

                        108847725

                        114103243

                        5.26

                         

                        14q12

                        RP11-330O19

                        RP11-109D12

                        25538832

                        26345513

                        0.81

                         

                        14q21.1-q21.3

                        RP11-88D14

                        RP11-94K16

                        36949212

                        48298851

                        11.35

                         

                        14q31.1-q31.3

                        RP11-46l17

                        RP11-88N20

                        78630860

                        86772926

                        8.14

                        14q32.31-q32.33

                         

                        RP11-367F11

                        RP11-815P21

                        101467534

                        105159201

                        3.69

                         

                        15q14

                        RP11-294M6

                        RP11-79A5

                        33841939

                        35953471

                        2.11

                        16p13.3

                         

                        CTD-2148K8

                        RP11-89M4

                        87754

                        4697230

                        4.61

                        16p13.2-p13.11

                         

                        RP11-475D10

                        RP11-489O1

                        8598165

                        15572359

                        6.97

                        16p12.1-p11.2

                         

                        RP11-142A12

                        RP11-18H23

                        26595069

                        31443695

                        4.85

                        16q21-q22.1

                         

                        RP11-52B24

                        RP11-394B2

                        63708677

                        69365102

                        5.66

                        16q23.3-q24.3

                         

                        RP11-483P21

                        RP11-566K11

                        82361609

                        88613383

                        6.25

                        17p13.3-p13.1

                         

                        RP11-411G7

                        RP11-89A15

                        427024

                        8365794

                        7.94

                        17p11.2

                         

                        RP11-524F11

                        RP1-162E17

                        17343389

                        19251691

                        1.91

                        17q11.2-q21.31

                         

                        RP11-138P22

                        RP11-374N3

                        23133763

                        41096064

                        17.96

                        17q21.32-q21.33

                         

                        RP11-234J24

                        RP11-506D12

                        42655422

                        46333070

                        3.68

                         

                        17q22-q23.2

                        RP11-143M4

                        RP11-139B3

                        47607556

                        51363278

                        3.76

                        17q24.3-q25.3

                         

                        RP11-65C22

                        RP11-258N23

                        68165339

                        78308832

                        10.14

                         

                        18q11.2-q23

                        RP11-5G23

                        RP11-396D4

                        21431314

                        71337306

                        49.91

                        19p13.3-q13.43

                         

                        RP11-110A24

                        GS1-1129C9

                        134914

                        63771717

                        63.64

                        20p13-q13.33

                         

                        RP11-530N10

                        CTB-81F12

                        9943

                        62393015

                        62.38

                         

                        21q11.2-q22.11

                        RP11-72P4

                        RP11-41N19

                        13857799

                        30673984

                        16.82

                        22q11.1-q11.21

                         

                        RP11-81H21

                        RP11-586I18

                        14754982

                        18976359

                        4.22

                        22q12.3-q13.33

                         

                        RP11-90I17

                        CTB-99K24

                        35686144

                        49397088

                        13.71

                        Table 4

                        Detailed overview of frequent DNA copy number aberrations (>30%) of tumors from native SA patients.

                        chromosomal aberrations

                        flanking clones

                        position (bp)

                         

                        segment size

                        gains

                        losses

                        start

                        end

                        start

                        end

                        (Mb)

                         

                        3p14.2

                        RP11-734E15

                        RP11-137N22

                        59105371

                        61252524

                        2.15

                         

                        4q35.2

                        RP11-354H17

                        CTC-963K6

                        190095484

                        191158370

                        1.06

                        7p22.3-p11.2

                         

                        RP11-713A20

                        RP11-80l24

                        106471

                        55784518

                        55.68

                        7q22.1

                         

                        RP11-10D8

                        RP11-163M5

                        98067793

                        101528379

                        3.46

                        8q24.3

                         

                        RP11-472K18

                        RP5-1109M23

                        144481535

                        146238749

                        1.76

                        9q33.3-q34.3

                         

                        RP11-91G7

                        RP11-424E7

                        124316484

                        138363252

                        14.05

                        17q12-q21.31

                         

                        RP11-893G17

                        RP11-392O1

                        31506328

                        39091575

                        7.59

                        17q21.32-q21.33

                         

                        RP1-62O9

                        RP11-506D12

                        44647598

                        46333070

                        1.69

                        17q23.2-q25.3

                         

                        RP11-579A4

                        RP11-258N23

                        54149948

                        78451750

                        24.30

                        19p13.3

                         

                        RP11-110A24

                        CTC-1482H14

                        134914

                        5154803

                        5.02

                        19p13.2-p13.11

                         

                        RP11-197O4

                        RP11-88I12

                        10248852

                        19023254

                        8.77

                        19q12-q13.34

                         

                        CTC-1459F4

                        GS1-1129C9

                        32889410

                        63771717

                        30.88

                        20p13-q13.33

                         

                        RP11-530N10

                        CTB-81F12

                        9943

                        62393015

                        62.38

                        Table 5

                        Detailed overview of frequent DNA copy number aberrations (>30%) of tumors from Caucasian SA patients.

                        chromosomal aberrations

                        flanking clones

                        position (bp)

                         

                        segment size

                        gains

                        losses

                        start

                        end

                        start

                        end

                        (Mb)

                         

                        3p14.2

                        RP11-48E21

                        RP11-641C17

                        60380670

                        60705094

                        0.32

                        3q26.2-q26.31

                         

                        RP11-669J9

                        RP11-44A1

                        172392313

                        173855790

                        1.46

                         

                        4q32.1-q35.2

                        RP11-192D11

                        CTC-963K6

                        159886665

                        191158370

                        31.27

                        5p13.1-p12

                         

                        RP11-17J3

                        RP11-55O15

                        40113135

                        44396362

                        4.28

                        7p22.3-p21.3

                         

                        RP11-713A20

                        RP11-505D17

                        106471

                        7932634

                        7.83

                        7q22.1

                         

                        RP4-550A13

                        RP11-333G13

                        98512376

                        101153193

                        2.64

                        8p23.1

                         

                        RP11-241P12

                        RP11-589N15

                        9788949

                        11803111

                        2.01

                        8q22.1-q22.3

                         

                        RP11-664H21

                        RP11-132E3

                        98618965

                        105402542

                        6.78

                        8q24.21

                         

                        RP11-28I2

                        RP11-1142f3

                        127563658

                        129620230

                        2.06

                         

                        9p24.1-p23

                        RP11-165O14

                        RP11-91E3

                        5873408

                        9689968

                        3.82

                        9q33.3-q34.3

                         

                        RP11-62A6

                        RP11-424E7

                        124479347

                        138363252

                        13.88

                        11q13.3-q13.5

                         

                        RP11-554A11

                        RP11-98G24

                        68509550

                        77008323

                        8.50

                        16p11.2

                         

                        RP11-110P16

                        RP11-388M20

                        28675396

                        31163676

                        2.49

                        17q12-q21.1

                         

                        RP5-986F12

                        RP11-94L15

                        33099924

                        35227135

                        2.13

                        17q25.1-q25.3

                         

                        RP11-41E12

                        RP11-258N23

                        68729134

                        78451750

                        9.72

                        19p13.3-p13.11

                         

                        RP11-110A24

                        RP11-88I12

                        134914

                        19023254

                        18.89

                        19q13.11-q13.43

                         

                        CTC-1325L16

                        GS1-1129C9

                        37623641

                        63771717

                        26.15

                        20p13-q13.33

                         

                        RP11-48M7

                        CTB-81F12

                        3728265

                        62393015

                        58.66

                        http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-4-7/MediaObjects/12920_2010_204_Fig2_HTML.jpg
                        Figure 2

                        Frequencies of gains and losses throughout the genome in all gastric adenocarcinomas from UK patients. Clones are sorted by position per chromosome (1-22). Vertical lines indicate transition between chromosomes; dashed vertical lines indicate centromere position.

                        http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-4-7/MediaObjects/12920_2010_204_Fig3_HTML.jpg
                        Figure 3

                        Frequencies of gains and losses throughout the genome in all gastric adenocarcinomas from native SA patients. Clones are sorted by position per chromosome (1-22). Vertical lines indicate transition between chromosomes; dashed vertical lines indicate centromere position.

                        http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-4-7/MediaObjects/12920_2010_204_Fig4_HTML.jpg
                        Figure 4

                        Frequencies of gains and losses throughout the genome in all gastric adenocarcinomas from Caucasian SA patients. Clones are sorted by position per chromosome (1-22). Vertical lines indicate transition between chromosomes; dashed vertical lines indicate centromere position.

                        Supervised analysis

                        To identify biological differences between gastric cancers from different geographical origin, native SA tumors were compared with UK tumors using CGH test. Only MSS tumors were included in the supervised analysis. In total, 133 regions, located on different chromosomes, were significantly different (p < 0.05 and fdr≤0.15) between these two patient groups. An overview of the significant chromosomal regions, including the fdr rates, is given in Table 6. No significant differences were found between gastric cancers from UK and Caucasian SA patients or between gastric cancers from native and Caucasian SA patients.
                        Table 6

                        Detailed overview of the supervised analysis using CGH test.

                        cytoband

                        region start (bp)

                        region end (bp)

                        p-value

                        fdr

                        cytoband

                        region start (bp)

                        region end (bp)

                        p-value

                        fdr

                        1p36.33

                        672780

                        1359795

                        0.04

                        0.15

                        11p14.2-p14.1

                        27033269

                        27371257

                        0.05

                        0.15

                        1p36.32-p36.31

                        3386389

                        6294064

                        0.03

                        0.12

                        11q13.3

                        68509550

                        69323966

                        0.04

                        0.14

                        1p36.21-p36.13

                        12798944

                        15683816

                        0.03

                        0.12

                        11q13.3-q13.4

                        69314721

                        70472869

                        0.04

                        0.14

                        1p31.2

                        67178936

                        69303906

                        0.04

                        0.14

                        11q22.1-q22.2

                        98930611

                        101405228

                        0.03

                        0.12

                        1p31.1

                        69815162

                        76679895

                        0.01

                        0.06

                        11q22.2-q22.3

                        102010610

                        102424014

                        0.03

                        0.12

                        1p31.1

                        77428804

                        77820126

                        <0.01

                        0.04

                        12q21.2

                        76570565

                        78724263

                        0.04

                        0.14

                        1p21.2-p21.1

                        101684496

                        104502748

                        0.03

                        0.12

                        13q21.31-q21.33

                        61335626

                        69275204

                        0.04

                        0.14

                        1q31.1-q31.2

                        184717073

                        188976520

                        0.01

                        0.09

                        14q21.1

                        39694531

                        42171623

                        0.02

                        0.10

                        1q31.2-q31.3

                        189822405

                        193082884

                        0.02

                        0.10

                        14q21.2

                        42965408

                        44043547

                        0.01

                        0.09

                        1q31.3

                        193336091

                        194242465

                        0.01

                        0.08

                        14q21.2-q21.3

                        45258184

                        48298851

                        0.03

                        0.12

                        1q31.3

                        195068870

                        195629725

                        0.03

                        0.12

                        15q14

                        33841939

                        35953471

                        0.04

                        0.14

                        3p26.3

                        46140

                        2377366

                        <0.01

                        0.03

                        15q22.2

                        57373165

                        61214280

                        0.02

                        0.09

                        3p24.3

                        17181327

                        18148477

                        <0.01

                        0.03

                        15q23

                        65816865

                        68768615

                        0.02

                        0.09

                        3p24.3

                        19033520

                        21742232

                        <0.01

                        0.03

                        15q23-q24.2

                        69188639

                        73645336

                        0.05

                        0.15

                        3p24.3-p24.1

                        22747912

                        27531283

                        0.02

                        0.10

                        15q24.2-q25.2

                        74334873

                        81024206

                        0.03

                        0.12

                        3p21.31

                        46545403

                        47371983

                        0.05

                        0.15

                        16p13.2-p13.12

                        8777494

                        12522798

                        0.05

                        0.15

                        3p21.31

                        47384745

                        50114898

                        0.04

                        0.14

                        16p11.2

                        28675396

                        31163676

                        <0.01

                        0.04

                        3p21.31-p21.2

                        50533656

                        51418837

                        0.01

                        0.09

                        16q24.1

                        82993991

                        83622386

                        0.05

                        0.15

                        3p21.31-p21.2

                        51390596

                        52007218

                        0.02

                        0.09

                        16q24.1

                        84123856

                        84922042

                        0.02

                        0.09

                        3p21.1

                        52658450

                        53621497

                        0.01

                        0.09

                        16q24.2-q24.3

                        86986672

                        87452904

                        0.03

                        0.12

                        3p12.3

                        76450939

                        79097142

                        0.02

                        0.09

                        16q24.3

                        87848795

                        88465228

                        0.02

                        0.09

                        3p12.3-p12.2

                        79197544

                        82066233

                        <0.01

                        0.04

                        16q24.3

                        88398231

                        88613383

                        0.01

                        0.07

                        3p12.2-p12.1

                        82657794

                        84801874

                        <0.01

                        0.03

                        17p13.3

                        427024

                        1071560

                        <0.01

                        0.03

                        3p12.1-p11.2

                        85234579

                        87730259

                        <0.01

                        0.03

                        17p13.3-p13.2

                        2026966

                        4169913

                        0.04

                        0.14

                        3p11.1

                        88915283

                        89771786

                        <0.01

                        0.04

                        17p13.2

                        4810523

                        5166678

                        0.01

                        0.09

                        3q11.2

                        95569618

                        96250439

                        0.01

                        0.05

                        17p13.1

                        6780962

                        7477414

                        <0.01

                        0.03

                        3q25.1-q25.33

                        151508469

                        161536133

                        0.03

                        0.12

                        17p13.1

                        7436435

                        7682367

                        <0.01

                        0.03

                        3q25.33-q26.1

                        162044657

                        164516640

                        0.03

                        0.12

                        17p11.2

                        17343389

                        19251691

                        0.04

                        0.14

                        3q26.1

                        165289729

                        167697600

                        0.05

                        0.15

                        17q11.2

                        23133763

                        28423820

                        0.02

                        0.09

                        3q26.31-q26.32

                        174848631

                        180449332

                        0.05

                        0.15

                        17q11.2-q12

                        28664889

                        29147086

                        0.02

                        0.09

                        4p15.32-p14

                        17799729

                        36260048

                        0.02

                        0.11

                        17q25.1

                        69709369

                        71238958

                        0.01

                        0.09

                        4p14

                        36998587

                        37497326

                        0.01

                        0.09

                        17q25.1-q25.3

                        71406319

                        77588058

                        0.03

                        0.12

                        4p14

                        37851044

                        38103870

                        0.02

                        0.11

                        18q11.2

                        21431314

                        21774952

                        0.01

                        0.05

                        4p14-p12

                        38087293

                        46781512

                        0.01

                        0.09

                        18q11.2

                        22104619

                        22931012

                        <0.01

                        0.04

                        4q12

                        58332155

                        59148507

                        0.04

                        0.14

                        18q12.1

                        23970882

                        24526449

                        0.02

                        0.10

                        4q12-q13.1

                        59679465

                        62653308

                        0.02

                        0.10

                        18q12.1

                        25034674

                        26878315

                        0.01

                        0.08

                        4q13.3

                        74322497

                        76429795

                        0.03

                        0.12

                        18q12.1

                        27447009

                        28415095

                        0.01

                        0.09

                        4q13.3-q21.21

                        76495364

                        79353372

                        0.02

                        0.09

                        18q12.1

                        29409483

                        30219417

                        0.01

                        0.09

                        4q21.21

                        79222718

                        80683287

                        0.03

                        0.12

                        18q12.1-q12.2

                        30773824

                        31588529

                        0.01

                        0.06

                        4q33-q34.3

                        172094999

                        178721484

                        0.02

                        0.10

                        18q22.1

                        60340433

                        61310295

                        0.03

                        0.12

                        4q34.3

                        178969859

                        179599683

                        0.01

                        0.09

                        18q22.1

                        61623805

                        62645202

                        0.03

                        0.12

                        4q34.3

                        180819690

                        183096645

                        0.02

                        0.10

                        18q22.1-q22.3

                        63092764

                        68041625

                        0.03

                        0.12

                        4q35.1

                        184503994

                        186178296

                        0.03

                        0.12

                        19p13.3

                        134914

                        913289

                        0.01

                        0.08

                        5q11.2

                        50971745

                        52055659

                        0.04

                        0.14

                        19p13.3

                        902641

                        5009969

                        <0.01

                        0.04

                        5q11.2

                        52909242

                        56437163

                        0.03

                        0.12

                        19p13.3

                        5663923

                        6519297

                        <0.01

                        0.01

                        5q11.2-q12.1

                        56921490

                        58947885

                        0.02

                        0.09

                        19p13.3-p13.2

                        6523443

                        9826740

                        <0.01

                        <0.01

                        5q14.3

                        82802677

                        86118543

                        0.05

                        0.15

                        19p13.2-p13.12

                        10248852

                        15116365

                        <0.01

                        0.03

                        5q23.2

                        122463056

                        123527915

                        0.05

                        0.15

                        19p13.12-p13.11

                        15415833

                        17777501

                        <0.01

                        0.01

                        7p22.1-p21.3

                        6983150

                        7932634

                        <0.01

                        0.02

                        19p13.11

                        18202507

                        19023254

                        <0.01

                        0.03

                        7p21.3-p21.2

                        9256088

                        14085902

                        <0.01

                        <0.01

                        19p12

                        19877150

                        21504328

                        0.01

                        0.05

                        7p21.2-p21.1

                        14342152

                        19957111

                        <0.01

                        0.01

                        19p12

                        22133662

                        22949959

                        0.01

                        0.05

                        7q22.1

                        98651132

                        100929260

                        <0.01

                        0.03

                        19q12

                        33315121

                        33507712

                        0.02

                        0.10

                        8q24.21

                        130562467

                        131126185

                        0.05

                        0.15

                        19q12

                        34159370

                        34664148

                        0.02

                        0.11

                        8q24.22

                        131641447

                        133561490

                        0.01

                        0.09

                        19q12

                        34960906

                        35766560

                        0.02

                        0.11

                        8q24.22

                        134537164

                        136154996

                        0.02

                        0.11

                        19q12-q13.11

                        36958851

                        37518517

                        0.05

                        0.15

                        8q24.22-q24.23

                        136472354

                        138543270

                        0.04

                        0.14

                        19q13.12-q13.13

                        40883472

                        43004503

                        0.03

                        0.13

                        8q24.3

                        141395868

                        142790557

                        0.01

                        0.05

                        19q13.2-q13.32

                        46498596

                        50725496

                        0.03

                        0.12

                        8q24.3

                        144790054

                        145357620

                        0.01

                        0.09

                        19q13.32-q13.33

                        52665374

                        54718281

                        0.03

                        0.12

                        8q24.3

                        145585590

                        145953950

                        0.02

                        0.11

                        19q13.33-q13.43

                        55461670

                        63771717

                        <0.01

                        0.01

                        8q24.3

                        145893230

                        146238749

                        0.04

                        0.14

                        21q11.2-q21.1

                        13857799

                        18774434

                        <0.01

                        0.03

                        9p24.1-p23

                        8398601

                        9689968

                        0.04

                        0.14

                        21q21.1

                        20982315

                        21411332

                        <0.01

                        0.03

                        9p23

                        9684353

                        10554235

                        0.04

                        0.14

                        21q21.1-q21.2

                        22151920

                        22943367

                        <0.01

                        0.03

                        10q22.1

                        70824040

                        71674097

                        0.03

                        0.12

                        21q21.2

                        23491399

                        25568510

                        <0.01

                        0.01

                        10q22.1

                        72033907

                        73573433

                        0.02

                        0.09

                        21q21.3

                        26174732

                        26923374

                        0.02

                        0.09

                        10q26.3

                        135110821

                        135301208

                        0.03

                        0.12

                        21q21.3-q22.11

                        28596969

                        30855176

                        0.03

                        0.12

                        11p15.5

                        178227

                        626401

                        0.01

                        0.05

                        22q13.33

                        48473404

                        49397088

                        0.04

                        0.15

                        11p15.5

                        1299306

                        1785278

                        <0.01

                        0.03

                             

                        In total, 133 regions were significantly different (p < 0.05 and fdr≤0.15) between UK and native SA tumors. The chromosomal regions, including the start and end positions, and the fdr rates are listed.

                        Discussion

                        One of the main risk factors contributing to gastric cancer is infection with H. pylori, which causes a chronic active gastritis [4, 21]. In South Africa, gastric cancer is infrequent, while the prevalence of H. pylori infection is very high. Although differences in genotypes of H. pylori exist in different geographic areas, this African enigma can not only be explained by differences in virulent strains of H. pylori [2224]. High prevalence of vacA s1b strain is observed in South Africa as well as in Brazil and Portugal, countries with high incidences of gastric cancer,[2527] and frequencies of CagA antibodies were similar between patients with gastric neoplasia compared to healthy controls [28]. The prevalence of the different virulent strains in the present study is unknown. Since H. pylori is thought to play a major role in the initiation phase of gastric cancer development and most often already disappeared at time of gastric cancer diagnosis, it is impossible to accurately retrieve this information.

                        Besides the virulence of the infecting H. pylori strain, other factors influence gastric cancer risk, including environmental factors such as diet and socioeconomic status, and host factors, such as polymorphisms, which are involved in the inflammatory response to the infection [29, 30]. Knowing that the prevalence of H. pylori infection and incidences of gastric cancer are different in South Africa and Western Europe, we aimed to study if this would reflect in different patterns of gastric carcinogenesis.

                        The concept of the African enigma has been challenged since it has been suggested that the enigma could be explained due to lack of infrastructure and access to hospitals and care in African countries resulting in incomplete reporting of gastric cancer. However the incidence of gastric cancer would have been so dramatically underestimated that it has been stated that under-reporting by itself could not explain the lower frequencies of gastric cancer in African countries [31]. Also, when using the proportional frequency of gastric cancer compared to other cancer types in Africa, gastric cancer incidence remains very low [8]. Another criticism on the African enigma has been the high prevalence of HIV infection. A relatively large part of the African population would die of HIV before the age in which gastric cancer becomes more frequent. However, the low gastric cancer incidence in Africa was described before the HIV epidemic.

                        South African patients showed significantly more microsatellite instable gastric cancers compared to Western European patients. Also at the level of chromosomal instability clear differences were found, reflected by a significant correlation between cluster membership and geographical tumor origin, i.e. UK, native SA and Caucasian SA. Microsatellite instable gastric cancers are described to have fewer chromosomal aberrations compared to microsatellite stable gastric cancers [32, 33]. To rule out that tumors from South African patients cluster together by hierarchical cluster analysis due to the fact that these tumors show higher frequencies of microsatellite instability, only microsatellite stable gastric cancers were included in the hierarchical cluster analysis.

                        Not much has been reported about microsatellite status in gastric cancers from African patients. One study reported infrequent microsatellite instable gastric cancers in South African patients[34] which is in contrast with our findings which show a higher frequency of microsatellite instability in gastric cancers from SA patients compared to UK patients. Based on the present data, MSI does play an important role in gastric carcinogenesis in South Africa.

                        Several chromosomal aberrations are common in the three different tumor groups analyzed, including gains of chromosomes 7, 8q, 9q, 17q, 19 and 20 and losses of 3p and 4q, while other chromosomal changes are specific for each tumor group. In addition, gastric cancers from UK patients showed a significantly higher number of clones showing a loss compared to gastric cancers form South African patients. These results indicate different patterns of chromosomal instabilities in gastric cancers correlating to geographical origin of the patient.

                        The chromosomal aberrations of the UK tumors are comparable to other array CGH studies analyzing Western European tumors [12, 3537]. To the best of our knowledge, this is the first array CGH study on gastric cancers from South African patients. Since several chromosomal regions are significantly different between gastric cancers from different geographical origin, and each region comprises multiple genes, further studies are needed to pinpoint candidate genes contributing to the differences in genomic profiles.

                        The higher frequency of diffuse gastric cancers from UK patients compared to the SA patients in the present study could be considered as a confounding factor. Contradicting results have been published either describing different or similar patterns of DNA copy number aberrations between intestinal and diffuse type gastric cancers [7, 33, 3739]. In the context of the present study we believe that the differences in DNA copy number aberrations between UK and SA gastric cancers are independent of the histological tumor type. When repeating the cluster analysis with intestinal type carcinomas only, cluster membership again was significantly correlated with geographical origin of the tumors. Also supervised data analysis, i.e. testing copy number status of all genomic loci, did not reveal any significant differences in DNA copy number changes between intestinal and diffuse gastric cancers from UK patients. Furthermore, hierarchical cluster analysis including UK gastric cancers only did not separate intestinal and diffuse type gastric cancers. We therefore do not believe our findings to be influenced by distribution of histological types in this series. Question remains why diffuse type gastric cancers were more frequently observed in gastric cancers from UK patients compared to SA patients. Besides being a confounding factor, we can hypothesize that mutation of E-cadherin (CDH1), or other mechanisms disrupting the CDH1 gene function such as epigenetic mechanisms or miRNAs, playing an important role in diffuse type gastric cancer, might play a minor role in SA gastric cancer patients due to different pathways of carcinogenesis, as shown in the present study by differences in patterns of DNA copy number aberrations. Also, the prevalence of H. pylori infection is very high in South Africa, and H. pylori infection mainly plays a role in intestinal type gastric cancers. This could also explain the higher number of intestinal type gastric cancers in SA patients.

                        Further, with respect to copy number changes in relation to histological types, chromosomal gains of 8q and 17q and losses of 3p have been described to be associated with intestinal type gastric cancers [33]. On the other hand, gains of 8q and 17q have been reported to be altered predominantly in diffuse type gastric cancers [38]. In the present study gains on chromosomes 8q and 17q and losses on 3p were common to both intestinal and diffuse type gastric cancers. In addition, these aberrations also were common in tumors from both UK and SA patients. Gains on chromosomes 13q and 19q have been found more frequently in diffuse type gastric cancers [33, 36, 38]. Again, in the present study, gains of these chromosomes were observed equally in intestinal and diffuse type gastric cancers. Gain of 19q was frequently observed in tumors from both geographical origins. Although gain of 13q was observed less frequently in tumors from SA patients compared to tumors from UK patients, still around 20-25% of the tumors of native SA patients show a gain of chromosome 13q, making it unlikely that tumor type has influenced cluster membership.

                        A limitation of the present study is the fact that native SA gastric cancer patients were significantly younger compared to Caucasian SA and UK gastric cancer patients. We previously showed that gastric cancers of young and elderly patients have different patterns of chromosomal aberrations [12]. We cannot rule out that also in these series, age might contribute to differences in DNA copy number profiles, however cluster analysis showed that gastric cancers from native SA patients were more similar to cancers from Caucasian SA patients, who have similar age as UK patients, indicating that cluster membership is independent of age in this respect. Overall, most differences were observed between UK and native SA tumors.

                        We realize that the present study is based on a heterogeneous group of gastric cancer patients, with different genetic background and different environmental factors, including H. pylori, diet and socioeconomic status, influencing gastric cancer risk. Statements on genotype influencing gastric cancer are very difficult to make since the degree of heterogeneity within each different patient group, i.e. UK, native SA and Caucasian SA, is unknown.

                        The patterns of genomic alterations in gastric cancers from UK and SA patients could gain clinical relevance in the future. In addition to surgery, gastric cancer treatment increasingly includes (neo)adjuvant chemotherapy and/or radiotherapy, however still without patients being stratified based on biological characteristics of their tumors. Clinical trials are underway in which also the value of genetic markers for predicting response to therapy are studied. In the end, stratification for therapy may include genomic alterations observed in tumors of patients from different geographical origin.

                        Conclusions

                        We showed that gastric cancers of UK and SA patients are different in their patterns of genomic instability. Gastric cancers from SA patients show higher frequencies of microsatellite instability and different patterns of chromosomal aberrations compared to gastric cancers from UK patients. These results may suggest different molecular pathways of gastric carcinogenesis, consistent with the African enigma hypothesis. Further studies are needed to explore the link between H. pylori and other environmental factors, as well as host factors, such as polymorphisms influencing gastric cancer susceptibility, in relation to the patterns of genomic instability in gastric cancers from these different geographic areas.

                        Declarations

                        Acknowledgements

                        We thank the Mapping Core and Map Finishing groups of the Wellcome Trust Sanger Institute for initial clone supply and verification. We thank Peter van der Vlies and Klaas Kok for providing the BAC arrays. This work was financially supported by the Dutch Cancer Society grant-KWF 2004-3051 and the Pathological Society Pilot Study Grant - August 2007.

                        Authors’ Affiliations

                        (1)
                        Dept. of Pathology, VU University Medical Center
                        (2)
                        Dept. of Anatomical Pathology, University of Pretoria
                        (3)
                        Pathology and Tumour Biology, Leeds Institute of Molecular Medicine, University of Leeds
                        (4)
                        Dept. of Gastroenterology, VU University Medical Center
                        (5)
                        Dept. of Surgery, Leiden University Medical Center
                        (6)
                        Dept of Internal Medicine and Gastroenterology, University of Pretoria

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                        40. Pre-publication history

                          1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1755-8794/​4/​7/​prepub

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                        © Buffart et al; licensee BioMed Central Ltd. 2011

                        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.