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Original article

PTEN deletion is rare but often homogeneous in gastric cancer

Sormeh Mina,1,2 Benjamin A Bohn,1,2 Ronald Simon,1 Antje Krohn,1 Matthias Reeh,2 Dirk Arnold,3 Carsten Bokemeyer,4 Guido Sauter,1 Jakob R Izbicki,2 Andreas Marx,1 Phillip R Stahl1
1

Institute of Pathology, University Medical Center HamburgeEppendorf, Hamburg, Germany 2 General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center HamburgeEppendorf, Hamburg, Germany 3 Hubertus Wald Tumor Center/University Cancer Center Hamburg (UCCH), University Medical Center HamburgeEppendorf, Hamburg, Germany 4 Department of Internal Medicine II and Clinic (Oncology Center), University Medical Center HamburgeEppendorf, Hamburg, Germany Correspondence to Benjamin A Bohn, General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center HamburgeEppendorf, Martinistrasse 52, 20246 Hamburg, Germany; b.bohn@uke.de SM and BAB have contributed equally to this work. Accepted 16 April 2012

ABSTRACT Background and aim Gastric carcinoma is the second most frequent cause of cancer-related death worldwide. As PTEN is a potential modier of tumour response to trastuzumab, a recently approved therapy in metastatic HER2 positive gastric cancer, the existence of PTEN deletions in primary gastric cancer was investigated. Methods 230 primary gastric cancers were analysed in a tissue microarray format by dual labelling uorescence in situ hybridisation for PTEN deletion. HER2 analysis was also performed. To study PTEN deletion heterogeneity, all available large tissue sections from primary cancer and corresponding metastases were analysed in seven patients. Results Eight of 180 interpretable primary gastric cancer spots showed PTEN deletions (4.4%), including seven hemizygous and one homozygous deletion. PTEN deletion was correlated with nodal (8 of 122 cases (6.6%); p0.041) and distant metastases (4 of 19 (21.1%); p<0.001). Large section validation showed a homogeneous distribution of PTEN deletion. HER2 positivity was seen in one PTEN deleted case. Conclusion Genomic PTEN deletion is a rare event in gastric adenocarcinoma but correlates with metastatic disease. The homogeneous distribution pattern indicates that this alteration occurs early in tumour development.

INTRODUCTION
Gastric adenocarcinoma is the second most frequent cause of cancer death worldwide, despite an ongoing decline of incidence and mortality.1 Adequate surgery with and without neoadjuvant and adjuvant radiotherapy and/or chemotherapy are the potentially curative therapeutic options.2 More recently, the spectrum of therapeutic options was complemented by trastuzumab and potentially also other anti-HER2 (human epidermal growth factor receptor 2) therapies.3 The ToGAtrial demonstrated an extended median overall survival in the trastuzumab plus chemotherapy group compared with the chemotherapy alone group in 594 patients with advanced or gastrooesophageal junction cancer. The adverse events were similar in the two groups and so the authors considered trastuzumab as a new standard option for advanced HER2 positive gastric cancer.4 The tumour suppressor gene PTEN (phosphatase and tensin homologue) is located on chromosome 10q23.3 and acts as a plasma membrane lipid phophatase.5 Its primary target for dephosphorylation is the second messenger phosphatidylinositol. J Clin Pathol 2012;65:693698. doi:10.1136/jclinpath-2011-200525

3,4,5-triphosphate, the product of phosphatidylinositol-3-kinase. Thereby PTEN negatively regulates the phosphatidylinositol-3-kinase/ATPdependant tyrosine kinase (Akt) pathway.6 ATPdependent tyrosine kinase activation suppresses apoptosis, induced by a number of different stimuli, and regulates cellular functions including cell proliferation, cell growth and metabolism, migration and resistance to hypoxia.7 Alterations of PTEN by inactivating mutations and/or chromosomal deletions have been described in many different tumour types including gastric cancer. Furthermore, reduced PTEN function has been associated with aggressive tumour phenotype and unfavourable disease course in various cancer types.8 Moreover, PTEN aberration has been associated with failure of anti-HER2 therapy in breast cancer.9 PTEN alterations may thus also become clinically relevant in gastric cancer. Most studies evaluating PTEN alterations are not very recent, and there are no studies using uorescence in situ hybridisation (FISH)dthe gold standard for deletion analysis. Given the potential relevance of PTEN alterations as a predictive tool for trastuzumab therapy, we designed this study to evaluate the frequency of chromosomal deletions in primary gastric cancers, and also to determine whether or not such alterations may be represented homogeneously or heterogeneously in affected cancers.

MATERIALS AND METHODS Patients

Tissues from a total of 382 consecutive patients who had undergone either endoscopic treatment at the Interdisciplinary Endoscopy Department and Clinic of the University Medical Center HamburgeEppendorf or surgical therapy at the Department of General, Visceral and Thoracic Surgery between 1994 and 2006 were taken for tissue microarray (TMA) construction. Written informed consent for the use of resected samples was obtained from all patients and approval was obtained from the Ethics Committee of the Chamber of Physicians in Hamburg, Germany. Age, sex, tumour localisation, tumour size, lymphatic invasion, pTNM stage,10 grading, and tumour type according to Lauren were evaluated by a review of medical charts and pathological records. Glass slides were reviewed for determining the histological type. Clinical follow-up data were available from 143 patients. The median follow-up was 24.3626.8 months (range 1e145 months).
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TMA construction and large section validation
Tissue samples were xed in buffered 4% formalin, parafn embedded, and used for TMA construction as previously described.11 H&E stained histological sections from the gastrectomy specimens were used to mark representative tumour areas. A 0.6-mm tissue core was punched out from the tumour area of each specimen using a homemade semi-automated tissue arrayer and transferred in a TMA recipient block. The TMA contained 770 tissue samples, including 230 primary gastric tumours, 79 matched lymph node metastases, four distant metastases (ovary, skin, liver), 140 dysplasias (10 lowgrade, 77 high-grade, 53 intramucosal cancers), and 317 normal gastric tissue samples from patients with gastric cancer as well as from healthy patients. To study a potential heterogeneous distribution pattern of PTEN deletion, all available large tissue sections (4 mm) from primary cancer and corresponding metastases were analysed in seven patients.

Fluorescence in situ hybridisation

SpectrumGreen-labelled PTEN probes were manufactured by using two bacterial articial chromosome clones (RP11-38065 and RP11-81303). Bacterial articial chromosome DNA was Table 1 PTEN deletion and HER2 expression status in gastric adenocarcinoma
Clinicopathological features Gender Female Male Age <62 $62e<73 $73 Tumour pT1 pT2 pT3 pT4 Nodes pN0 pN+ Met. pM0 pM1 UICCz I+II III+IV WHO G1+G2 G3+G4 Lauren Intestinal Diffuse Mixed Localisation Cardia Fundus/corpus Antrum/pylorus Gastric stump NS Int. metaplasia () (+) Gastritis () (+) (HP neg) (+) (HP pos) PTEN FISH analysis n* 57 123 54 62 64 25 98 42 15 58 122 161 19 93 87 48 132 92 67 15 48 61 39 4 28 148 30 120 55 3 PTEN del 3 (5.3)y 5 (4.1) 4 (7.4)y 1 (1.6) 3 (4.7) 0 7 (7.1)y 1 (2.4) 0 0 8 (6.6)y 4 (2.5)y 4 (21.1) 3 (3.2)y 5 (5.7) 4 (8.3) 4 (3.0)y 7 (7.6)y 1 (1.5) 0 6 (12.5)y 1 (1.6) 1 (2.6) 0 0 8 (5.4)y 0 6 (5.0)y 2 (3.6) 0 p Value HER2 IHC analysis n* 75 141 69 74 73 29 113 52 22 64 152 192 24 103 113 65 151 114 80 15 53 82 43 5 33 174 40 144 66 4 HER2 pos 7 (9.3) 12 (8.5) 2 (2.9) 10 (13.5) 7 (9.6) 2 8 7 2 (6.9) (7.1) (13.5) (9.1) p Value

0.492

0.839

0.317

0.078

0.266

0.581

0.041

6 (9.4) 13 (8.6) 16 (8.3) 3 (12.5) 6 (5.8) 13 (11.5) 13 (20.0) 6 (4.0) 14 (12.3) 1 (1.3) 2 (13.3) 8 4 4 0 3 (15.1) (4.9) (9.3) (9.1)

0.846

<0.001

0.497

0.412

0.141

0.127

<0.001

0.223

0.016

0.036

0.321

0.193

19 (10.9) 0 17 (11.8) 1 (1.5) 1 (25.0)

0.029

0.858

0.027

Figure 1 Representative uorescence in-situ hybridisation images of gastric adenocarcinomas showing (A) normal PTEN status, (B) hemizygous PTEN gene deletion, and (C) homozygous PTEN gene deletion (arrows); green signals represent PTEN and orange signals represent CEP10.
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*Numbers of patients do not always add up to 180 (PTEN) or 216 (HER2) because of missing information; percentage of PTEN deleted or HER2-positive cases in parentheses. yIncluding one homogeneous PTEN deletion. zAccording to TNM-UICC, 6th edition. FISH, uorescence in-situ hybridisation; HP, Helicobacter pylori; IHC, immunohistochemistry; NS, not specied.

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Figure 2 (A) Association of PTEN status with median overall survival; (B) association of pT stage with median overall survival.

extracted and labelled with SpectrumGreen-dUTP (VysisAbbott, Chicago, Illinois, USA) using a commercial nick-translation kit (Vysis-Abbott). These homemade PTEN probes were used together with a SpectrumOrange-labelled centromere 10 (CEP10) reference probe (Vysis-Abbott). For proteolytic slide pretreatment, a commercial kit was used (parafn pretreatment reagent kit, Vysis-Abbott). Before hybridisation, sections were deparafnised, air dried and dehydrated in 70%, 85% and 100% ethanol, and then denatured for 15 min at 808C in 70% formamide-23 SSC solution. Following overnight hybridisation at 378C in a humidied chamber, slides were washed, counterstained with 0.2 mmol/l 49 ,6-diamidino-2-phenylindole, and mounted in antifade solution (Vector Labs, Vectashield, California, USA). Each spot was evaluated and the predominant signal numbers were recorded for each FISH probe. Homozygous deletion of PTEN was dened as unequivocal complete absence of PTEN signals in all tumour cell nuclei of the tissue spot, but presence of centromere signals in tumour cells and presence of PTEN and CEP10 signals in adjacent normal cells. Hemizygous deletion of PTEN was dened as presence of fewer PTEN signals than CEP10 probe signals (ratio PTEN:CEP10 <1) in $60% tumour cell nuclei. Tissue spots with lack of PTEN signals in all (tumour and normal cells) or lack of any normal cells as an internal control for successful hybridisation of the PTEN probe were excluded from analysis.

50 min. After peroxidase blocking the sections were incubated with the prediluted primary antibody. Immunostaining was scored following a standardised four-step scoring system.12 Tumours graded as 2+ and 3+ were evaluated as positive.

Statistical analysis
Continuous data are given as median with range or mean with SD. Cross-table analysis (c2 test) was used to study the relationship between FISH and IHC results and clinicopathological features. Categorical variables are shown as numbers and percentage. Overall survival was estimated by the KaplaneMeier method and evaluated by log-rank testing. Signicance statements refer to p values of 2-tailed tests that are <0.05. Statistical calculation was performed with the PASW V.18.0 (SPSS).

RESULTS Technical aspects

A total of 520 of 770 (67.5%) tissue spots were interpretable for PTEN copy number estimation by FISH. Samples were from 180 primary cancers, 54 lymph nodes, two distant metastases (ovary, liver), 82 dysplasias (6 low-grade, 45 high-grade and 31 intramucosal carcinomas) and 202 normal gastric tissues. The remaining 250 (32.5%) spots were non-informative due to lack of tissue, absence of unequivocal dysplasia or cancer tissue, or insufcient hybridisation.

Immunohistochemistry
Immunohistochemical (IHC) staining was performed with the HercepTest (Dako, Glostrup, Denmark) according to the manufacturer s protocol. Sections were deparafnised and antigen retrieval was performed in a water bath at 95e998C for Table 2 Analysis of PTEN heterogeneity
Case 1 2 3 Primary tumour slides, n (informative) 4 (2) 5 (5) 4 (4) Metastasis, n (informative) NA 2 (2) (LN) 8 (8) (LN) FISH interpretation average (PTEN/CEP10) 1e2/2e6 1e3/3e8 1e3/2e7 Result All homogeneous All homogeneous Homogeneous deletion in primary tumour and 5/8 metastasis slides; in three metastasis slides polyploid PTEN/CEP10 with co-located cluster-like arrangement All homogeneous Homogeneous Homogeneous Homogeneous

PTEN deletion in gastric cancer by FISH

Representative images of PTEN FISH ndings are shown in gure 1. PTEN gene deletions were found in 8 of 180 (4.4%) evaluable primary gastric adenocarcinomas, including seven

4 5 6 7

2 (2) NA 1 (1) NA

1 (1) (LIV) 1 (1) (LIV) NA 1 (1) (LN)

0e2/2e7 1e3/2e8 1e2/3e6 1e2/4e8

NA, no tumour or metastasis blocks available; LN, lymph node metastasis; LIV, liver metastasis.

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Original article
tumours with a hemizygous deletion and one tumour with a homozygous deletion. Additionally, one lymph node metastasis and one high-grade dysplasia showed a hemizygous PTEN deletion. Clinicopathological associations are given in table 1. We were able to show a signicant correlation between PTEN deletion and lymph node metastases (p0.041) as well as distant metastases (p<0.001). Another signicant correlation was seen for PTEN deletion and tumour localisation (p0.036). Furthermore, we found a tendency towards higher rates of PTEN deletion in intestinal type cancers. Seven of eight deletions were found in intestinal type cancer, yet without statistical signicance. We were unable to show a relation between PTEN status and clinical outcome (gure 2).

HER2 expression in gastric cancer

HER2 positivity was seen in 19 of 216 (8.8%) analysable primary gastric cancers. We were able to show a signicant correlation between HER2 positivity and WHO grading (p<0.001) as well as Laurens classication (p0.016). Another signicant correlation was seen for HER2 positivity and intestinal metaplasia (p0.029) and gastritis (p0.027). One HER2 positive case showed a PTEN deletion.

Figure 3 Representative uorescence in-situ hybridisation image of non-deleted lymph node metastasis showing cluster-like arrangement of the PTEN gene; orange signals represent PTEN and green signals represent CEP10. by cell analysis which is not disturbed by an admixture of inammatory cells. That direct PTEN alterations are rather rare in gastric cancer is also supported from earlier studies nding PTEN mutations by direct sequencing in 2e10% of cases (summarised in table 415 17 20e25). A markedly higher mutation rate (18.8%, 28.3%) was only suggested in two studies using the single-strand conformation polymorphism technique and subsequent DNA sequencing of positive cases.20 23 A validation of positive cases by a second independent PCR was not reported in these studies, however. One study reported a promoter hypermethylation in 39% of examined cases with a correlation to advanced tumour stage.25 High rates of PTEN alterations have been suggested by IHC studies describing PTEN expression loss in 18e77% of cases (summarised in table 521 25e33). Despite considerable variability in antibody selection, scoring criteria and frequency of PTEN loss, multiple IHC studies proposed a link between reduced PTEN expression and unfavourable tumour phenotype and disease progression.26 27 31e33 It can be speculated that posttranscriptional modications of PTEN protein expression have a stronger impact on the cellular protein levels than copy number variations and are thus better distinguishable by IHC. Several mechanisms of post-transcriptional PTEN downregulation have been suggested in the literature. For example, human protein NEDD4-1 (neural precursor cell expressed, developmentally down-regulated-4-1) was identied as a negative regulator for PTEN stability by catalysing PTEN polyubiquitination.34 Tumour transforming growth factor-b1 (TGF-b1) and the necrosis factor-a/nuclear factor-kB (NF-kB)inducing kinase/NF-kB pathway were also shown to suppress PTEN transcription.35 36 It is noteworthy that a negative prognostic impact of PTEN alterations has been described for various other tumour types. Table 3 PTEN studies in gastric adenocarcinoma using loss of heterozygosity analysis
Association with Author Oki et al Oki et al14 Li et al15 Byun et al16 Chang et al17
13

PTEN status in primary tumour, corresponding metastases and associated dysplasia

Two deleted cases showed interpretable FISH results in corresponding metastases and associated dysplasia. One case showed an identical PTEN status in the primary tumour, associated dysplasia and corresponding metastasis. In the other case a discordant PTEN result occurred. The primary tumour showed a PTEN deletion, while a normal PTEN status was recorded in the corresponding lymph node metastasis. This case (no. 3) was included in our large section heterogeneity analysis.

Analysis of PTEN heterogeneity

To assess potential heterogeneity of the PTEN status in gastric cancers, all available tumour blocks were analysed by FISH on large tissue sections in seven cases (table 2). This analysis showed a homogeneous distribution of PTEN deletions in six of these seven cases. Case 3 showed a homogeneous distribution in all sections (4/4) of the primary tumour and in 5/8 evaluable nodal metastases. The three remaining nodal metastases showed an aberrant nding with increased copy number of both PTEN and CEP10 in all tumour cells. PTEN and also CEP10 signals were often co-located in a cluster-like arrangement in these tumour cells (gure 3).

DISCUSSION
The results of our study show that PTEN deletions occur rarely in gastric cancer. Only 8 of 180 evaluable primary gastric cancers (4.4%) showed a PTEN deletion by FISH. This rate of deletion is markedly lower than described in earlier studies using loss of heterozygosity (LOH) analysis for assessing 10q23 deletions. LOH studies had reported 10q23 deletions in 16e47% of gastric cancers (summarised in table 313e17). It is possible that a high rate of polysomic cases (unequal numbers of paternal and maternal chromosomes mimic LOH) might have contributed to the higher number of deletions found by LOH. Substantial aneuploidy has been described in gastric cancer in earlier studies by cytometry. For example, Furuya et al described aneuploidy in 98 of 186 analysed gastric cancers18 and Osterheld et al have seen aneuploidy in 15 of 16 analysed gastric cancers.19 It is unlikely that FISH would underestimate the true rates of deletion substantially as FISH enables a cell
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n 76 76 60 30 15

Events (%) 17.1 16.2 25 46.7 0

pT NS NI NI NS NS

pN NS NI NI NI NS

UICC stage NS NI NI <0.001 NS

Grading WHO NS NI NI <0.001 NS

Lauren NS NI NI NS NS

Survival 0.0093 NI NI NI NS

NI, not investigated; NS, not signicant.

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Table 4
Author Wen et al
20

Analysis of PTEN alterations in gastric adenocarcinoma using different approaches

Association with N 144 Events (%) 18.6 Methods SSCP-PCR; DNA- sequencing SSCP-PCR; DNA- sequencing SSCP-PCR; DNA- sequencing SSCP-PCR; DNA- sequencing SSCP-PCR; DNA- sequencing Findings in detail 15 missense mutations in exons 1, 2, 4 and 9; 9 nonsense mutations in exons 1, 7 and 9 2 1-bp deletions in exon 4 1 stop mutation in exon 7; 2 point mutations in exons 5 and 7; 1 single base A mutation in exon 5 1 silent point mutation in exon 8 One base change in exons 5 and in exon 8 One 4-bp deletion in exon 8 All only in advanced cases 8 missense mutations in exons 3, 4, 5 and, 6; 5 silent mutations in exons 2 and 4; 2 nonsense mutations in exon 3; 1 12-bp deletion in exon 4 1 5-bp deletion in intron 7; No mutation in coding regions; No promoter methylation No point mutations or base changes; In 33% (5/15) smaller size transcripts 73% (19/26) in examined cases with negative PTEN expression (IHC staining) pT NI pN NI UICC stage <0.005 Grading WHO <0.05 Lauren NI

Guo et al21 Lima et al22 Li et al

15

53 48 60 60

9.4 2.1 5 28.3

<0.05* NI NI NI

<0.05* NI NI NI

<0.005* NI NI NI

NI NI NI NI

NI NI NI NI

Wang et al23

Sato et al24 Chang et al17 Kang et al25

58 15 66

1.7 0 39.4

SSCP-PCR; methylation-specic PCR; DNA- sequencing SSCP-PCR; DNA- sequencing Methylation-specic PCR;

NI NI <0.01*

NI NI <0.001*

NI NI <0.001*

NI NI NS*

NI NI NS*

*Referring to IHC-staining results. NI, not investigated; NS, not signicant.

Studies using FISH for PTEN deletion detection demonstrated the presence of PTEN genomic losses and its impact on development of advanced disease, for example in prostate cancer,37 gliomas38 and endometrial cancer.39 Perhaps due to a shortage of sufciently large clinical studies, it is not entirely clear whether PTEN alterations have clinical signicance in gastric cancer. Although only eight deleted cancers were found, our data seem to suggest a tendency towards more deletions in intestinal type and advanced stage gastric cancer. Similar observations were described in several IHC studies.21 25 28 31 32 A critical clinical signicance of PTEN changes in gastric cancer may arise from its interactions with the HER2 pathway, however. Since studies have demonstrated a signicant survival benet of trastuzumab treated HER2 positive gastric cancers,4 there is substantial interest in biomarkers that will predict response to trastuzumab in individual patients. PTEN inactivation is considered a potential mechanism of trastuzumab resistance in breast cancer.9 40 In our subset, one of eight tumours with a PTEN deletion was HER2 positive. This patient might be affected in case of PTEN deletion representing a resistance mechanism to trastuzumab therapy. Though PTEN deletion is rare in gastric cancer it seems to be of clinical and biological relevance. Our ndings of a higher Table 5
Author Cho et al Darwish et al27 Lee et al28 Kang et al25 Bai et al29 Deng et al30 Zheng et al31 Im et al32 Guo et al21 Zhou et al33
32

prevalence of lymphatic and haematogeneous metastases in PTEN deleted cases argue for a more aggressive behaviour of these tumours. The fact that PTEN deleted gastric cancer occurs more often in the cardia than in other parts of the stomach indicates a biological difference between cancers of the cardia and the distal stomach as, for example, suggested by Xue et al.41 However, in case of a predictive role of molecular features, it is important to determine whether a pivotal alteration is present in all or only a fraction of the tumour cells. Our nding of a homogeneous distribution of PTEN deletions in six of seven evaluated cases and also in corresponding dysplasia in one case makes it very likely that PTEN deletions occur early in gastric cancer. The highly homogeneous distribution makes it also unlikely that a signicant number of heterogeneous deletions were missed in this analysis. Therefore it validates the TMA technique as a useful tool in this assay. In another patient analysed for PTEN heterogeneity, a surprising nding of a cluster-like copy number increase was seen in three metastases, while the remaining cancer and metastases showed clear-cut PTEN deletion. It is possible that this unusual FISH nding reects a structural rearrangement, potentially including a duplication of genomic PTEN material.

Analysis of PTEN expression in gastric adenocarcinoma using immunohistochemistry

n 265 290 310 248 91 118 249 94 53 70 Events (%) 21.5 20.6 20 25 29.7 23.7 27.7 18.1 34 77.1 PTEN-antibody clone; origin 6H2.1 mouse 6H2.1 mouse 6H2.1 mouse 6H2.1 mouse 2F4C9 mouse 17.A mouse 28H6 mouse 28H6 mouse Polyclonal rabbit Polyclonal rabbit Association with pT NI NI 0.25 <0.001 0.239 0.02 NI 0.001 0.025 <0.05 pN NI NI 0.215 <0.001 0.049 NS NI 0.04 0.01 NS UICC stage NI NI 0.253 <0.001 0.194 NI NI 0.01 0.005 NS Grading WHO NI NI 0.0034 NS 0.322 0.02 NI NI 0.025 NS Lauren NI NI 0.028 (int) NS 0.007 (dif.) NI <0.05 (dif.) NI NI NI Survival NI 0.0003 0.0014 NI 0.003 NI NI 0.0907 NI NI

NI, not investigated; NS, not signicant; dif, diffuse type; int, intestinal type.

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11. Simon R, Mirlacher M, Sauter G. Tissue microarrays. Methods Mol Med 2005;114:257e68. Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008;52:797e805. Oki E, Kakeji Y, Baba H, et al. Impact of loss of heterozygosity of encoding phosphate and tensin homolog on the prognosis of gastric cancer. J Gastroenterol Hepatol 2006;21:814e18. Oki E, Baba H, Tokunaga E, et al. Akt phosphorylation associates with LOH of PTEN and leads to chemoresistance for gastric cancer. Int J Cancer 2005;117:376e80. Li YL, Tian Z, Wu DY, et al. Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. World J Gastroenterol 2005;11:285e8. Byun DS, Cho K, Ryu BK, et al. Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplication in gastric carcinoma. Int J Cancer 2003;104:318e27. Chang JG, Chen YJ, Perng LI, et al. Mutation analysis of the PTEN/MMAC1 gene in cancers of the digestive tract. Eur J Cancer 1999;35:647e51. Furuya T, Uchiyama T, Murakami T, et al. Relationship between chromosomal instability and intratumoral regional DNA ploidy heterogeneity in primary gastric cancers. Clin Cancer Res 2000;6:2815e20. Osterheld MC, Caron L, Demierre M, et al. DNA-ploidy in advanced gastric carcinoma is less heterogeneous than in early gastric cancer. Cell Oncol 2004;26:21e9. Wen YG, Wang Q, Zhou CZ, et al. Mutation analysis of tumor suppressor gene PTEN in patients with gastric carcinomas and its impact on PI3K/AKT pathway. Oncol Rep 2010;24:89e95. Guo CY, Xu XF, Wu JY, et al. PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its signicance in human gastric cancer. World J Gastroenterol 2008;14:3804e11. jo JJ, Harada ML, et al. Molecular study of the tumour suppressor Lima EM, Arau gene PTEN in gastric adenocarcinoma in Brazil. Clin Exp Med 2005;5:129e32. Wang JY, Huang TJ, Chen FM, et al. Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in advanced gastric carcinomas. Virchows Arch 2003;442:437e43. Sato K, Tamura G, Tsuchiya T, et al. Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer. Virchows Arch 2002;440:160e5. Kang YH, Lee HS, Kim WH. Promoter methylation and silencing of PTEN in gastric carcinoma. Lab Invest 2002;82:285e91. Cho YJ, Kim JH, Yoon J, et al. Constitutive activation of glycogen synthase kinase3beta correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer. BMC Gastroenterol 2010;10:91. Darwish NS, Kim MA, Chang MS, et al. Prognostic signicance of CD24 expression in gastric carcinoma. Cancer Res Treat 2004;36:298e302. Lee HS, Lee HK, Kim HS, et al. Tumour suppressor gene expression correlates with gastric cancer prognosis. J Pathol 2003;200:39e46. Bai Z, Ye Y, Chen D, et al. Homeoprotein Cdx2 and nuclear PTEN expression proles are related to gastric cancer prognosis. APMIS 2007;115:1383e90. Deng H, Wu RL, Zhou HY, et al. Signicance of Survivin and PTEN expression in full lymph node-examined gastric cancer. World J Gastroenterol 2006;12:1013e17. Zheng H, Takahashi H, Murai Y, et al. Pathobiological characteristics of intestinal and diffuse-type gastric carcinoma in Japan: an immunostaining study on the tissue microarray. J Clin Pathol 2007;60:273e7. Im SA, Lee KE, Nam E, et al. Potential prognostic signicance of p185(HER2) overexpression with loss of PTEN expression in gastric carcinomas. Tumori 2005;91:513e21. Zhou YJ, Xiong YX, Wu XT, et al. Inactivation of PTEN is associated with increased angiogenesis and VEGF overexpression in gastric cancer. World J Gastroenterol 2004;10:3225e9. Wang X, Trotman LC, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 2007;128:129e39. Wang Q, Zhou Y, Wang X, et al. Regulation of PTEN expression in intestinal epithelial cells by c-Jun NH2-terminal kinase activation and nuclear factor-kappaB inhibition. Cancer Res 2007;67:7773e81. Yang Y, Zhou F, Fang Z, et al. Post-transcriptional and post-translational regulation of PTEN by transforming growth factor-beta1. J Cell Biochem 2009;106:1102e12. Sircar K, Yoshimoto M, Monzon FA, et al. PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer. J Pathol 2009;218:505e13. Korshunov A, Sycheva R, Gorelyshev S, et al. Clinical utility of uorescence in situ hybridization (FISH) in nonbrainstem glioblastomas of childhood. Mod Pathol 2005;18:1258e63. Dellas A, Jundt G, Sartorius G, et al. Combined PTEN and p27kip1 protein expression patterns are associated with obesity and prognosis in endometrial carcinomas. Clin Cancer Res 2009;15:2456e62. Nagata Y, Lan KH, Zhou X, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:117e27. Xue L, Zhang X, Li Y, et al. Differences of immunophenotypic markers and signaling molecules between adenocarcinomas of gastric cardia and distal stomach. Hum Pathol 2011;42:594e601. Berger MF, Lawrence MS, Demichelis F, et al. The genomic complexity of primary human prostate cancer. Nature 2011;470:214e20.

Take-home messages
< PTEN deletions occur rarely in gastric cancer. Only eight of

12. 13. 14. 15. 16. 17. 18.

180 evaluable primary gastric cancers (4.4%) showed a PTEN deletion by uorescence in-situ hybridisation. < The higher prevalence of lymphatic and haematogeneous metastases in PTEN deleted gastric cancers suggests a more aggressive behaviour of these tumours. < PTEN deletions may represent a critical event occurring early in a small subset of gastric cancers.

Recent studies using next generation sequencing methods have demonstrated recurrent genomic disruption of the PTEN gene in prostate cancer.42

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

CONCLUSION
In summary, a thorough examination of 180 gastric cancers using FISH analysis showed PTEN gene deletion in 4.4% of carcinomas and a tendency towards tumour progression in such cases. Homogeneous deletion in six of seven cancers evaluated on large sections may suggest that PTEN deletions represent a critical event occurring early in a small subset of gastric cancers.
Acknowledgements The authors thank Werner Otto Stiftung, Hamburg for their nancial support. We thank Sascha Eghtessadi and Sylvia Schno ger for their support with the FISH technique. Contributors All authors have read and approved the nal version of the text. SM and BAB performed the analysis, analysed the data and prepared the manuscript. BAB is a guarantor. RS, AK, MR, DA, CB, JRI and AM devised the study and revised the manuscript. GS and PRS designed the study, interpreted the data and revised the manuscript critically. GS and PRS are guarantors. Funding Financial support was provided by Werner Otto Stiftung, Hamburg. Competing interests None. Ethics approval Ethics approval was provided by the Ethics Committee of the Chamber of Physicians in Hamburg, Germany. Provenance and peer review Not commissioned; externally peer reviewed.

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J Clin Pathol 2012;65:693698. doi:10.1136/jclinpath-2011-200525

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PTEN deletion is rare but often homogeneous in gastric cancer

Sormeh Mina, Benjamin A Bohn, Ronald Simon, et al. J Clin Pathol 2012 65: 693-698 originally published online May 25, 2012

doi: 10.1136/jclinpath-2011-200525

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