Genomic profiling of rectal adenoma and carcinoma by array-based comparative genomic hybridization

  • Zhi-Zhou Shi1,

    Affiliated with

    • Yue-Ming Zhang2,

      Affiliated with

      • Li Shang1,

        Affiliated with

        • Jia-Jie Hao1,

          Affiliated with

          • Tong-Tong Zhang1,

            Affiliated with

            • Bo-Shi Wang1,

              Affiliated with

              • Jian-Wei Liang3,

                Affiliated with

                • Xi Chen1,

                  Affiliated with

                  • Ying Zhang1,

                    Affiliated with

                    • Gui-Qi Wang2,

                      Affiliated with

                      • Ming-Rong Wang1Email author and

                        Affiliated with

                        • Yu Zhang1Email author

                          Affiliated with

                          BMC Medical Genomics20125:52

                          DOI: 10.1186/1755-8794-5-52

                          Received: 7 December 2011

                          Accepted: 18 October 2012

                          Published: 16 November 2012

                          Abstract

                          Background

                          Rectal cancer is one of the most common cancers in the world. Early detection and early therapy are important for the control of death caused by rectal cancer. The present study aims to investigate the genomic alterations in rectal adenoma and carcinoma.

                          Methods

                          We detected the genomic changes of 8 rectal adenomas and 8 carcinomas using array CGH. Then 14 genes were selected for analyzing the expression between rectal tumor and paracancerous normal tissues as well as from adenoma to carcinoma by real-time PCR. The expression of GPNMB and DIS3 were further investigated in rectal adenoma and carcinoma tissues by immunohistochemistry.

                          Results

                          We indentified ten gains and 22 losses in rectal adenoma, and found 25 gains and 14 losses in carcinoma. Gains of 7p21.3-p15.3, 7q22.3-q32.1, 13q13.1-q14.11, 13q21.1-q32.1, 13q32.2-q34, 20p11.21 and 20q11.23-q12 and losses of 17p13.1-p11.2, 18p11.32-p11.21 and 18q11.1-q11.2 were shared by both rectal adenoma and carcinoma. Gains of 1q, 6p21.33-p21.31 and losses of 10p14-p11.21, 14q12-q21.1, 14q22.1-q24.3, 14q31.3-q32.1, 14q32.2-q32.32, 15q15.1-q21.1, 15q22.31 and 15q25.1-q25.2 were only detected in carcinoma but not in adenoma. Copy number and mRNA expression of EFNA1 increased from rectal adenoma to carcinoma. C13orf27 and PMEPA1 with increased copy number in both adenoma and carcinoma were over expressed in rectal cancer tissues. Protein and mRNA expression of GPNMB was significantly higher in cancer tissues than rectal adenoma tissues.

                          Conclusion

                          Our data may help to identify the driving genes involved in the adenoma-carcinoma progression.

                          Background

                          Rectal cancer is the 5th leading cause of cancer-related death and its incidence is increasing at a rate of 4.2% per year in China [1]. Early detection and early therapy are important for the control of death caused by rectal cancer.

                          The majority of epithelial cancers arise through a stepwise progression from normal cells, through dysplasia, into malignant cells that have invasive and metastatic potential. The classic example of this process is the colorectal adenoma to carcinoma progression [2, 3]. Genomic aberrations are found frequently in cancers and are believed to contribute to initiation and progression of cancer by deletion-induced down-expression of tumor suppressor genes or amplification and activation of oncogenes. In colorectal cancer the most frequent chromosomal aberrations were gains at 7p, 7q, 8q, 13q, and 20q and losses of 1p, 4p, 4q, 5q, 8p, 14q, 15q, 17p and 18q [49]. In particular, 8q, 13q and 20q gains and 8p, 15q and 18q losses are linked with colorectal adenoma to carcinoma progression. However, most of published reports are focused on colon cancer. Little information is available concerning the genomic aberrations of rectal carcinoma, especially DNA copy number changes in the progression from adenoma to tumor.

                          In the present study, we investigated the genomic aberrations of rectal adenoma and carcinoma by oligonucleotide-based array CGH, and identified common and different alterated chromosome regions between rectal adenoma and carcinoma. Then the expression of 15 genes at selected chromosome regions above was analyzed by real-time PCR or immunohistochemistry.

                          Methods

                          Patients and samples

                          Biopsy tissues from 22 rectal adenoma patients and 36 rectal carcinoma patients were collected by the Department of Endoscopy, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China. Biopsy samples were obtained by colonoscopy and stored at −80°C. Definitive pathological result from a biopsy was obtained at a later clinical course. An experienced pathologist confirmed that normal cell content of all the samples was less than 40% by HE staining. All the samples used in this study were residual specimens after diagnosis sampling. And all patients signed separate informed consent forms for sampling and research. The clinicopathological characteristics of the patients in array CGH assay are summarized in Table 1.
                          Table 1

                          Clinical Characteristics of 16 Patients Studied by Array CGH

                          Case No.

                          Sex

                          Age

                          Type

                          Location

                          1

                          F

                          52

                          Adenoma

                          Rectum

                          2

                          F

                          49

                          Adenoma

                          Rectum

                          3

                          M

                          75

                          Adenoma

                          Rectum

                          4

                          M

                          47

                          Adenoma

                          Rectum

                          5

                          M

                          57

                          Adenoma

                          Rectum

                          6

                          F

                          61

                          Adenoma

                          Rectum

                          7

                          M

                          69

                          Adenoma

                          Rectum

                          8

                          F

                          75

                          Adenoma

                          Rectum

                          9

                          M

                          69

                          Carcinoma

                          Rectum

                          10

                          M

                          61

                          Carcinoma

                          Rectum

                          11

                          F

                          70

                          Carcinoma

                          Rectum

                          12

                          F

                          73

                          Carcinoma

                          Rectum

                          13

                          M

                          42

                          Carcinoma

                          Rectum

                          14

                          M

                          32

                          Carcinoma

                          Rectum

                          15

                          F

                          31

                          Carcinoma

                          Rectum

                          16

                          M

                          66

                          Carcinoma

                          Rectum

                          Genomic DNA extraction and array-based CGH

                          Genomic DNA was isolated from tumor tissues using the Qiagen DNeasy Blood & Tissue Kit as described by the manufacturer (Qiagen, Hilden, Germany).

                          Array CGH experiments were performed using standard Agilent protocols (Agilent Technologies, Santa Clara, CA). Commercial human genomic DNA (PROMEGA, Warrington, UK) was used as reference. For each CGH hybridization, 500 ng of reference genomic DNA and the same amount of tumor DNA were digested with Alu I and RSA I restriction enzyme (PROMEGA, Warrington, UK). The digested reference DNA fragments were labeled with cyanine-3 dUTP and the tumor DNA with cyanine-5 dUTP (Agilent Technologies, Santa Clara, CA). After clean-up, reference and tumor DNA probes were mixed and hybridized onto Agilent 44K human genome CGH microarray (Agilent) for 40 h. Washing, scanning and data extraction procedures were performed following standard protocols.

                          Array CGH data set is available at Gene Expression Omnibus (GEO) http://​www.​ncbi.​nlm.​nih.​gov/​geo/​[10], accession number GSE34472.

                          Microarray data analysis

                          Microarray data were analyzed using Agilent Genomic Workbench (Agilent Technologies, Santa Clara, CA) and MD-SeeGH (http://​www.​flintbox.​ca). The Aberration Detection Method 2 algorithm with threshold at 6 (Agilent Genomic Workbench) was applied to identify common genomic aberrations. Mean Log2ratio of all probes in a chromosome region between 0.125 and 0.5 was classified as genomic gain, > 0.5 as high-level DNA amplification, < −0.125 as hemizygous loss, and < −0.5 as homozygous deletion. Minimal regions of gains or losses in our study defined as the smallest overlapping aberrant chromosomal regions identified by Agilent Genomic Workbench. Frequency plot comparison method (MD-SeeGH) was used to compare frequency of DNA copy number changes between rectal adenoma and carcinoma.

                          Total RNA extraction and real-time PCR

                          Total RNA was isolated from tissues using the RNeasy Mini Kit as described by the manufacturer (Qiagen, Hilden, Germany).

                          The PCR reactions were performed in a total volume of 20 μl, including 10 μl of 2 X SYBR ® Green PCR Master Mix (Applied Biosystems, Warrington, UK), 2 μl of cDNA (5 ng/μl), 1 μl of primer mix (10 μM each). The PCR amplification and detection were carried out in a 7300 Real Time PCR System (Applied Biosystems) for 45 cycles, each with 15 s at 95 °C, 1 min at 60 °C, and initial denaturation with 10 min at 95 °C. The relative gene expression was calculated using the comparative CT Method [11]. The copy number of the target gene normalized to an endogenous reference (GAPDH), and relative to calibrator was given by the formula 2 − ΔΔCt. ΔCT was calculated by subtracting the average GAPDH CT from the average CT of the gene of interest. The ratio defines the level of relative expression of the target gene to that of GAPDH.

                          Immunohistochemical staining

                          Formalin-fixed, paraffin-embedded specimens of rectal adenoma and carcinoma were detected in immunohistochemistry assay. Tissues of each case were repeated for three times. The slides were deparaffinized, rehydrated, immersed in 3% hydrogen peroxide solution for 10 min, heated in citrate buffer (pH 6) for 25 min at 95°C, and cooled for 60 min at room temperature. The slides were blocked by 10% normal goat serum for 30 min at 37°C and then incubated with rabbit polyclonal antibody against DIS3 (PTGLab), rabbit polyclonal antibody against GPNMB (PTGLab) overnight at 4°C. After being washed with PBS, the slides were incubated with biotinylated secondary antibody (diluted 1:100) for 30 min at 37°C, followed by streptavidin-peroxidase (1:100 dilution) incubation for 30 min at 37°C. Immunolabeling was visualized with a mixture of 3,3'-diaminobenzidine solution. Counterstaining was carried out with hematoxylin.

                          Expression level was determined on the basis of staining intensity and percentage of immunoreactive cells. Negative expression (score = 0) was no or faint staining, or moderate to strong staining in <25% of cells. Weak expression (score = 1) was a moderate or strong staining in 25% to 50% of cells. And strong expression (score = 2) was > 50% of the cells with strong staining. Weak expression and strong expression defined as positive staining.

                          Statistical analysis

                          Statistical analyses were conducted using the Student’s t-test and performed with the statistical software SPSS 15.0. The differences were judged as statistically significant when the corresponding two-sided P value were <.05.

                          Results

                          Recurrent copy number alterations in rectal adenoma and carcinoma detected by array CGH

                          Seven out of eight adenomas and all of carcinomas had genomic aberrations. More alterations were observed in patients of rectal cancer than adenoma, and the numbers of changes were 39.13±20.48 and 14.3±6.164, respectively (Additional file 1: Figure S1). Array CGH results showed that the most frequent copy number alterations in rectal adenoma were gains of 7p21.3-p15.3 and 20p12.3-p11.21 and losses of 5q13.2, 7q11.23, 11q13.1-q14.1, 17q25.1 and 19p13.3-p13.11 (Figure 1A, Tables 2 and 3). And the most common genetic aberrations in rectal carcinoma were gains of 7p21.3-p15.3, 7p15.3-p14.1, 7p14.1-p13, 7p13-p11.2, 13q13.1-q14.11, 13q21.1-q32.1, 13q32.1-q34, 20p11.21, 20q11.23-q12 and 20q13.2-q13.33 and losses of 17p13.1-p11.2, 18p11.32-p11.21 and 18q11.1-q11.2 (Figure 1B, Tables 2 and 3).
                          http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-5-52/MediaObjects/12920_2011_330_Fig1_HTML.jpg
                          Figure 1

                          Genome-wide frequency plot of rectal adenoma (A) and adenocarcinoma (B) in array CGH assay. Line on the right of 0%-axis: gain; Line on the left of 0%-axis: loss.

                          Table 2

                          Genomic Gains in Rectal Adenoma and Adenocarcinoma

                          Chromosome Region

                          Rectal adenoma

                          Rectal adenocarcinoma

                           

                          Start

                          End

                          No. of probes

                          No. of cases

                          Start

                          End

                          No. of probes

                          No. of cases

                          1q21.3

                              

                          150819451

                          150852905

                          3

                          3

                          1q25.3-q31.3

                              

                          183720174

                          197184608

                          157

                          3

                          1q32.1-q41

                              

                          204180950

                          214439909

                          173

                          3

                          5p13.3-p12

                              

                          33503866

                          45681293

                          165

                          3

                          6p21.33-p21.31

                              

                          30737615

                          33655570

                          151

                          4

                          6q16.3-q27

                          100547312

                          168205989

                          848

                          2

                              

                          7p21.3-p15.3

                          11041844

                          23202043

                          119

                          4

                          7671318

                          23172047

                          142

                          5

                          7p15.3-p14.1

                              

                          23821348

                          39813908

                          231

                          5

                          7p14.1-p13

                              

                          40099046

                          44497196

                          64

                          5

                          7p13-p11.2

                              

                          44890654

                          55242365

                          111

                          5

                          7q21.11-q21.12

                          81196827

                          86205180

                          42

                          2

                              

                          7q21.12-q21.3

                              

                          87207024

                          97321855

                          144

                          4

                          7q22.3-q32.1

                          106191096

                          127234809

                          245

                          2

                          105253205

                          127519635

                          260

                          4

                          8q12.1

                              

                          59565778

                          61340797

                          21

                          3

                          8q24.21-q24.22

                              

                          128816904

                          133653633

                          42

                          3

                          9p24.1-p21.1

                              

                          7058096

                          31463899

                          251

                          4

                          11p15.5

                              

                          192958

                          2278596

                          76

                          4

                          11q13.2

                              

                          66917525

                          67689856

                          30

                          4

                          12p13.31-p11.21

                          9053548

                          30700931

                          337

                          2

                              

                          12q12-q13.11

                          37052371

                          47174877

                          139

                          2

                              

                          12q13.13

                              

                          50568352

                          51486634

                          34

                          4

                          12q14.1-q22

                          57350276

                          91428773

                          354

                          2

                              

                          13q13.1-q14.11

                          13q21.1-q32.1

                          13q32.3-q34

                          21038984

                          109780488

                          909

                          2

                          32490193

                          39679219

                          79

                          7

                               

                          52774228

                          94079000

                          275

                          7

                               

                          100091512

                          114022929

                          148

                          7

                          19p13.2-p13.11

                              

                          9800520

                          19631574

                          473

                          3

                          19q13.13-q13.33

                              

                          43396893

                          55615310

                          550

                          3

                          20p11.21

                          7296794

                          23132344

                          189

                          3

                          22510206

                          23380542

                          15

                          8

                          20q11.23-q12

                          29592072

                          42681834

                          275

                          2

                          35467169

                          41087006

                          78

                          7

                          20q13.2-q13.33

                              

                          52017030

                          62323759

                          215

                          7

                          Note: The number of rectal adenoma and adenocarcinoma in Array CGH study are both 8 cases.

                          Table 3

                          Genomic Losses in Rectal Adenoma and Adenocarcinoma

                          Chromosome Region

                          Rectal adenoma

                          Rectal adenocarcinoma

                           

                          Start

                          End

                          No. of probes

                          No. of cases

                          Start

                          End

                          No. of probes

                          No. of cases

                          1p36.23-p36.22

                              

                          7804415

                          11633739

                          82

                          3

                          1p36.22-p36.13

                              

                          12600054

                          16167534

                          41

                          3

                          1p36.12-p35.3

                              

                          21802142

                          29525663

                          226

                          3

                          1q21.2-q21.3

                          148163183

                          149505863

                          60

                          2

                              

                          1q21.3-q23.1

                          151880217

                          155031244

                          154

                          2

                              

                          4q12

                          55913547

                          57653302

                          38

                          2

                              

                          5p15.33-p12

                          260981

                          45865412

                          433

                          2

                              

                          5q13.2

                          68434643

                          68900029

                          18

                          3

                              

                          7p22.2-p22.1

                          4298590

                          6547570

                          42

                          2

                              

                          7q11.23

                          72003839

                          75977276

                          77

                          3

                              

                          7q22.1

                          99538250

                          101895994

                          79

                          2

                              

                          8q22.2-q24.3

                          100781187

                          143914353

                          448

                          2

                              

                          8q24.3

                          143914353

                          146250824

                          75

                          2

                              

                          9q34.11

                          130111425

                          132321365

                          64

                          2

                              

                          10p14-p11.21

                              

                          11825924

                          35645512

                          315

                          3

                          11p15.2-p11.12

                          14750051

                          50638829

                          468

                          2

                              

                          11q13.1-q14.1

                          63802950

                          80046693

                          442

                          4

                              

                          12q24.23-q24.33

                          116956235

                          132193660

                          257

                          2

                              

                          14q12-q21.1

                              

                          30209271

                          38927323

                          130

                          3

                          14q22.1-q24.3

                              

                          48874529

                          77750644

                          544

                          3

                          14q31.3-q32.1

                              

                          87763614

                          93260389

                          110

                          3

                          14q32.2-q32.32

                              

                          99254905

                          102592287

                          70

                          3

                          15q15.1-q21.1

                              

                          38653893

                          42843706

                          119

                          3

                          15q22.31

                              

                          61519869

                          64628895

                          74

                          3

                          15q25.1-q25.2

                              

                          76206143

                          79967204

                          77

                          3

                          17p13.1-p11.2

                          84287

                          21386319

                          606

                          2

                          8327645

                          20974722

                          266

                          4

                          17q25.1

                          70528777

                          71603516

                          61

                          3

                              

                          18p11.32-p11.21

                          170229

                          13875315

                          173

                          2

                          2580000

                          13752309

                          137

                          5

                          18q11.1-q11.2

                          16904187

                          76018409

                          684

                          2

                          16976046

                          20313378

                          51

                          4

                          19p13.3-p13.11

                          1432408

                          19699544

                          795

                          4

                              

                          19q13.11-q13.43

                          37554715

                          63672832

                          1114

                          2

                              

                          20q13.33

                          60039825

                          62320720

                          85

                          2

                              

                          22q13.1

                          37689058

                          37715431

                          3

                          2

                              

                          Note: The number of rectal adenoma and adenocarcinoma in Array CGH study are both 8 cases.

                          Common and distinct genomic events in rectal adenoma and carcinoma

                          By comparing the genomic aberrations of rectal adenoma and carcinoma, we found that gains of 7p21.3-p15.3, 7q22.3-q32.1, 13q13.1-q14.11, 13q21.1-q32.1, 13q32.3-q34, 20p11.21 and 20q11.23-q12 and losses of 17p13.1-p11.2, 18p11.32-p11.21, and 18q11.1-q11.2 were shared by rectal adenoma and carcinoma. However, gains of 1q, 6p21.33-p21.31 and losses of 10p14-p11.21, 14q12-q21.1, 14q22.1-q24.3, 14q31.3-q32.1, 14q32.2-q32.32, 15q15.1-q21.1, 15q22.31 and 15q25.1-q25.2 were detected in carcinoma but not in adenoma (Figure 2, Tables 2 and 3).
                          http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-5-52/MediaObjects/12920_2011_330_Fig2_HTML.jpg
                          Figure 2

                          Frequency plot comparison of rectal adenoma and carcinoma. Red: carcinoma; green: adenoma; yellow: shared by both. The presentation is per array probe; gains and losses are represented by the colors on the right and left, respectively. Vertical blue line represents 100% of the samples. Brown and blue arrows highlight the changed chromosomal areas that were common or distinct between rectal adenoma and carcinoma, respectively.

                          Candidate target genes of interesting gains and losses

                          Further, we selected 14 genes of 1q, 6p, 7p, 13q, 18q and 20q to analyze the mRNA expression by real-time PCR (Table 4). Array CGH found that copy number increase of GPNMB (7p15.2), OXGR1 (13q32.1), C13orf27 (13q32.2-q34), PMEPA1 (20q13.31), PHACTR3 (20q13.32) and decrease of SMAD4 (18q21.2), BCL2 (18q21.33) occurred in both rectal adenoma and carcinoma. Our real-time PCR results showed that C13orf27 and PMEPA1 were overexpressed in rectal cancer tissues comparing with paracancerous normal tissues. BCL2 and SMAD4 were underexpressed in tumor tissue (Figure 3A). And the expression level of C13orf27 and GPNMB was significantly higher in cancer tissues than rectal adenoma tissues (Figure 3B).
                          Table 4

                          Primers of genes in Real-time PCR assay

                          Gene

                          Forward primer

                          Backward primer

                          Size (bp)

                          GAPDH

                          GGTCGTATTGGGCGCCTGGTC

                          TGACGGTGCCATGGAATTTGCCA

                          148

                          KIFC1

                          TCTCTGGGTGGTAGTGCTAAGA

                          TAAGTCACTTCCTGTTGGCCTG

                          148

                          SOX4

                          GACCGGGACCTGGATTTTAACT

                          TGAAAACCAGGTTGGAGATGCT

                          133

                          PBX2

                          AAGTTCCAAGAGGAGGCAAACA

                          TCCTGAGAGATTGAAAGAGCCG

                          132

                          ESRRG

                          GCTATCCTGCAGCTGGTAAAGA

                          GCTATCCTGCAGCTGGTAAAGA

                          133

                          KDM5B

                          CCCTCAGACACATCCTATTCCG

                          CAGTCCACCTCATCTCCTTCTG

                          101

                          PTGS2

                          TGTATCCTGCCCTTCTGGTAGA

                          AAGGAGAATGGTGCTCCAACTT

                          85

                          EFNA1

                          GTGGCAAAATCACTCACAGTCC

                          CTATGTAGAACCCGCACCTCTG

                          91

                          BCL2

                          AGGATTGTGGCCTTCTTTGAGT

                          CGGTTCAGGTACTCAGTCATCC

                          113

                          SMAD4

                          TGTTGATGACCTTCGTCGCTTA

                          ATGCTCTGTCTTGGGTAATCCG

                          81

                          PHACTR3

                          TATGACAGGAGGGCAGACAAAC

                          GCTTGCTTGATGCATGTACCTC

                          118

                          C13orf27

                          TCAGGCTCAGCAGATGAAATGT

                          TCCAGTGGATTTTATGGGGAGC

                          85

                          PMEPA

                          CTGAGCCACTACAAGCTGTCTG

                          CTTCTGAGGACAGGGCATCTTC

                          85

                          OXGR1

                          ATCTTGAGGGTCATTCGGATCG

                          TGTCGCTGACCACCACATATAG

                          148

                          GPNMB

                          GTCACTGTGATCTCCCTCTTGG

                          TTTGCACGGTTGAGAAAGACAC

                          116

                          http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-5-52/MediaObjects/12920_2011_330_Fig3_HTML.jpg
                          Figure 3

                          Expression of genes which were located on the common aberrant chromosomal regions in rectal adenoma and carcinoma. N: paracancerous normal tissues; T: rectal cancer tissues.

                          Copy number increase of EFNA1 (1q22), PTGS2 (1q31.1), KDM5B (1q32.1), ESRRG (1q41), KIFC1 (6p21.32), PBX2 (6p21.32) and SOX4 (6p22.3) were only detected in rectal cancer in array CGH. Among them, EFNA1 had increased expression in carcinoma compared with adenoma, and KIFC1 had an upward trend but not significant in statistical analysis (Figure 4A). Of these genes KIFC1 and SOX4 were also significantly overexpressed in rectal tumor tissues than paracancerous tissues (Figure 4B).
                          http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-5-52/MediaObjects/12920_2011_330_Fig4_HTML.jpg
                          Figure 4

                          Expression of genes which were located on the distinct aberrant chromosomal regions in rectal adenoma and carcinoma. N: paracancerous normal tissues; T: rectal cancer tissues.

                          We also analyzed the protein expression of GPNMB (7p15.2) and DIS3 (13q22.1) by immunohistochemistry. Of all six detected rectal adenoma tissues, GPNMB and DIS3 had no expression. In twenty rectal cancer tissues, GPNMB and DIS3 were positively stained in six and five cases, respectively (Figure 5).
                          http://static-content.springer.com/image/art%3A10.1186%2F1755-8794-5-52/MediaObjects/12920_2011_330_Fig5_HTML.jpg
                          Figure 5

                          Expression of GPNMB and DIS3 by immunohistochemistry assay.

                          Discussion

                          In the past decades, a number of genomic changes were found in colorectal adenoma and carcinoma, but the target genes are limited and molecular mechanism of adenoma to carcinoma progression is still unknown.

                          Previous studies found that 8q, 13q and 20q gains and 8p, 15q and 18q losses are linked with colorectal adenoma to carcinoma progression [49]. Our study narrowed down the gain regions to 13q13.1-q14.11, 13q21.1-q32.1, 13q32.2-q34 and 20q11.23-q12 and the loss regions to 18q11.2. Furthermore, gains of 7p21.3-p15.3 and 7q22.3-q32.1 and losses of 17p13.1-p11.2, 18p11.32-p11.21 were also found in both rectal adenoma and carcinoma.

                          Our study also showed that some genomic aberrations were present in rectal tumor but not in adenoma. They are gains of 1q and 6p21.33 and losses of 10p14-p11.21, 14q12-q21.1, 14q22.1-q24.3, 14q31.3-q32.1, 14q32.2-q32.32, 15q15.1-q21.1, 15q22.31 and 15q25.1-q25.2. These aberrations occurred at the later stages of rectal carcinogenesis, and may contribute the progression from adenoma to carcinoma.

                          Identifying the candidate targets underlying the genomic aberrations was important for understanding the mechanism of carcinogenesis. Carvalho et al. found that the overexpressions of C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCRL5 in carcinomas compared with adenomas were correlated with 20q gain [4]. Habermann et al. showed that copy number changes of 7q, 8p, 8q, 13q, 18p, 18q, 20p and 20q deregulated the average expression levels of the genes on these chromosome arms [12]. However, most of samples detected in these reports were colon cancer which had some different genomic aberrations compared with rectal cancer [13], expression-dysregulated genes in the carcinogenesis of rectum were still limited. By literature analyses, we selected 14 genes to compare their expression between in tumor and paracancerous tissues or between in rectal adenoma and carcinoma tissues. Of them, copy number and mRNA expression of EFNA1 increased from rectal adenoma to carcinoma, and C13orf27 and PMEPA1 with gains in both adenoma and carcinoma were overexpressed in rectal cancer tissues. These results revealed that copy number increase maybe the reason of expression up-regulation. Interestingly, both mRNA and protein expression of GPNMB was higher in cancer tissues than rectal adenoma tissues.

                          GPNMB is a type I transmembrane protein and overexpressed in several malignant human tissues relative to the corresponding normal tissues. Ectopic overexpression of GPNMB/osteoactivin can promote the metastasis and invasion of glioma, breast and hepatocellular carcinoma [1417]. EFNA1 was overexpressed in hepatocellular carcinoma and can inhibit growth of malignant mesothelioma by phosphorylating EPHA2 [18, 19]. C13orf27 was overexpressed in rectal tumor in our study, but the function of C13orf27 was unknown. PMEPA1 was also identified in our study, which is mapped to 20q13.3 is a TGF-beta inducible gene and encodes a NEDD4 E3 ubiguitin ligase binding protein. PMEPA1 is over-expressed in prostate, breast, renal cell, stomach and rectal carcinomas [2022]. But little is known about the function of PMEPA1, Further study should be conducted to investigate the roles of the above genes in human colorectal cancer.

                          Loss of 18q is a common event in colorectal cancer, and 18q deletion and loss of SMAD4 expression are associated with liver metastasis. In colorectal cancer, patients with reduced SMAD4 expression frequently presented an unfavorable survival because of liver metastasis [2326]. High expression level of SMAD4 reflected significantly longer overall and disease-free survival time than low expression level [27]. Bixiang et al. found that transgenic expression of SMAD4 can significantly reduce the oncogenic potential of MC38 and SW620 cells [28]. Our study confirmed the decreased expression of SMAD4 in rectal cancer.

                          In summary, we identified EFNA1 (1q), C13orf27 (13q), PMEPA1 (20q), GPNMB (7q) as candidate driving genes of genomic aberrations in rectal cancer. Further study was needed to reveal the mechanisms by which these genes may be involved in the carcinogenesis of the rectum.

                          Conclusions

                          Our data provide detailed information on genomic aberrations present in rectal adenoma or carcinoma, especially both in two groups or only in rectal cancer. Real-time PCR and immunohistochemistry assay selected EFNA1, C13orf27, PMEPA1 and GPNMB as candidate amplification targets. Our results may help to identify the driving genes involved in the adenoma-carcinoma progression.

                          Declarations

                          Acknowledgements

                          The authors would like to thank Kai-Tai Zhang, Department of Etiology and Carcinogenesis of Peking Union Medical College as the help of array CGH experiment.

                          Funding

                          Supported by: This work was supported by Special Public Health Fund of China (200902002-5) and Chinese Hi-Tech R&D Program Grant (2011AA022706).

                          Authors’ Affiliations

                          (1)
                          State Key Laboratory of Molecular Oncology, Cancer Institute /Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences
                          (2)
                          Department of Endoscopy, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences
                          (3)
                          Department of Abdominal Surgery, Cancer Institute/Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences

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                          29. Pre-publication history

                            1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1755-8794/​5/​52/​prepub

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                          © Shi et al.; licensee BioMed Central Ltd. 2012

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