MD Nadia Peparini
email: nadiapeparini@yahoo.it

PhD Piero Chirletti

To the Editor

We read with great interest the review by Pepels and colleagues on the safety of withholding axillary lymph node dissection (ALND) to patients with clinically node-negative breast cancer [1]. According to the survival and local control results from the NSABP B-32 randomised fase 3 trial [2] the Authors concluded that in patients with T1 or small T2 sentinel node (SN)-negative breast cancer omission of ALND can be regarded as safe. They pointed out that there is a shift toward omitting completion ALND in SN-positive patients but estimated that in SN-positive series the overall axillary recurrence rates were 2,8 times higher than for the SNnegative studies, and axillary recurrence rates after 5 and 8 years might be as high as 13% and 18% respectively. They highlighted that in many patients with a positive SN withholding ALND may be not safe and that the role of systemic therapy have to be elucidated to establish a potential role for avoiding ALND in selected SN-positive cases.

Most of published studies on SN associated with ALND reported not insignificant prevalence of falsenegative results, but the clinical consequences of these false-negative SN were fewer than expected. The discrepancy between prevalence of false-negative SN and lower prevalence of nodal recurrence in patients without definitive axillary treatment, the discrepancy between prevalence of axillary failure in clinically node-negative patients who do not undergo ALND and prevalence of positive nodes in clinically node-negative patients undergoing ALND have been partly attributed to the systemic therapies which are administered to the most SNnegative patients [3][4][5]. On the one hand in contemporary practice of breast cancer treatment on the one hand there is a shift toward using systemic therapies as a substitute for potentially incomplete surgery (i.e. SN biopsy alone in SN-positive cases) more than as a complement to complete surgery. On the other hand, ratio of involved to total number of LNs (LNR) was found to be a better prognostic factor than the number of positive lymph nodes (LNs) for staging node-positive breast cancer in series of patients treated in era of systemic therapy [6][7][8]. An association between survival and extension of nodal dissection evaluated by the total number of the removed LNs, the number of removed uninvolved LNs, LNR with improvement in outcome in cases of more extended lymphadenectomy has been reported for melanoma and for colon, lung, gastric, pancreatic, esophageal and breast cancers. LNR can minimize the stage migration produced by extended nodal dissection; using LNR rather than the absolute number of positive nodes reduces inter-institutional differences in outcome that may exist because of variations in the number of nodes excised. However the best cut-off points of LN ratio required for a staging classification of each malignant tumor is controversial. Better prognosis has been reported with higher number of dissected normal nodes in histologically node-positive and node-negative gastrointestinal tumors and several studies on breast cancer showed survival advantages after removal of many axillary nodes even if all regional nodes were pathologically negative [4,9]. Although the Will Rogers effect (i.e., stage migration) was the most evident confounding factor, the prognostic improvement was attributed mostly to removal of nodes with micrometastases that were undetectable by routine histologic examination but which had prognostic impact. The concept of better prognosis by increase of the total number of dissected nodes and mostly by lowering of LNR (which minimizes the "stage migration" effect) supports the thesis of the therapeutic value of extended nodal dissection. However whether or not ALND confers a survival advantage in breast cancer with micrometastatic or macrometastatic SNs in era of systemic therapy is questioned. Wasif and coll. showed that current ASCO guidelines recommending routine ALND following identification of micrometastasis in sentinel nodes (SNs) are being followed in only 60 % of cases and that the use of SN biopsy alone for axillary staging in cases of micrometastatic SNs may underestimate a 20% of patients with additional involved nonsentinel nodes revealed by ALND [10]. The prognostic value of micrometastasis and isolated tumor cells (ITCs) in SNs in terms of associated additional positive nodes and distant recurrences is controversial. In the preliminary report from the EORTC 10981-22023 AMAROS Trial the further nodal involvement in patients with macrometastasis, micrometastasis and ITCs in SNs was 41%, 18% and 18% respectively [START_REF] Pugliese | The clinical impact and outcomes of immunohistochemistry-only metastasis in breast cancer[END_REF], according to the results of other studies underlined in the review by Pepels and colleagues. Maaskant-Braat and colleagues in their report on this Journal found no statistically significant difference in survival between patients with micrometastasis or ITCs in SNs compared to those patients with tumor-free SNs [START_REF] Leidenius | Influence of isolated tumor cells in sentinel nodes on outcome in small, node-negative (pT1N0M0) breast cancer[END_REF]. However in the study by de Boer and coll. ITCs and micrometastasis in regional LNs resulted be associated with a reduced 5-year rate of disease-free survival among women with earlystage breast cancer who did not receive adjuvant therapy, without significant differences in outcome between ITCs and micrometastasis [START_REF] Hansen | Impact of micrometastases in the sentinel node of patients with invasive breast cancer[END_REF]. Truong and coll. showed that similar to patients with macroscopic positive nodes, mortality hazards in patients with micrometastatic nodal disease increased with increased number of positive nodes and LNR [8].

Although the prognostic impact of micrometastasis and ITCs is under debate, it is known that the occurrence of locoregional failures confers poor prognosis [START_REF] Maaskant-Braat | Sentinel node micrometastases in breast cancer do not affect prognosis: a population-based study[END_REF].

At present SN biopsy can be considered to be an effective procedure in the assessment of regional nodal status in clinically N0 cancer patients, and LNR considered to be the most effective prognostic factor that assesses the extent of therapeutic nodal dissection in node-positive tumors [START_REF] Andersson | Breast cancer survival in relation to the metastatic tumor burden in axillary lymph nodes[END_REF].In breast cancer the concept of LNR cannot be consistent with the concept of SN biopsy if completion ALND is considered unnecessary in SN-positive patients, and if staging by SN biopsy is considered to be the only necessary surgical treatment for these patients.

Moreover performing SN biopsy alone in pN1 mi SN patients cannot be consistent with the prognostic value of LNR in pN1 mi : if the therapeutic or prognostic value of LNR in microscopically SN-positive tumors is accepted, completion ALND will have to be done in all SN-positive tumors (apart from micrometastatic or macrometastatic SNs). In fact the risk of additional non-SN involvement associated to micrometastatic SN involvement is not negligible: SN biopsy improves pathological nodal assessment detecting otherwise misdiagnosed nodal micrometastases by adopting multistep sectioning, immunohistochemistry or RT-PCR but it should be considered that if these same techniques are applied to non-sentinel nodes, the false-negative rate of SNs will increase.

In conclusion avoiding completion ALND in patients with micrometastatic or macrometastatic SNs is not consistent with the concept of LNR as a prognostic factor in these patients. The potential role of systemic therapy for avoiding ALND in selected SN-positive patients need further to be elucidated but it should be considered that the prognostic value of LNR in identifying higher-risk node-positive cases warranting more aggressive systemic therapy may be estimated only by ALND. To the editor We like to thank dr. Peparini for her interesting letter-to-the-editor regarding our systematic review on the safety of avoiding routine use of axillary lymph node dissection (ALND) in early breast cancer patients [1,2]. She agrees on our conclusion that omitting ALND in sentinel node (SN) negative patients can be considered a safe strategy. Further, she also fully agrees with our second message, that is that in SN positive patients still many issues need to be solved before omission of ALND can be recommended to all these patients. Dr. Peparini argues that there are two additional reasons why ALND cannot be omitted in SN positive patients, besides low number of patients followed up and absence of well-defined selection criteria to be used. The first additional argument concerns the lack of information on the lymph node ratio (LNR) if no completion ALND is performed. The second argument concerns possible changes in systemic treatment choices depending on ALND, and more specifically treatment choices related to the presence of isolated tumor cells and micrometastases. Although we share the same vision on the preferred strategy in SN positive patients, we are not convinced that the aforementioned arguments really further support our conclusion.

The LNR is the ratio of positive to excised number of nodes. Many studies have reported that in breast cancer the LNR is a stronger prognosticator than the absolute number of positive nodes [3][4][5][6]. With more negative nodes being removed, the LNR reduces and prognosis improves. In addition, several studies have shown that also in node-negative patients clinical outcome is associated with number of (negative) nodes excised [7,8]. The argument in favour of an extensive ALND is that if more nodes are excised also microscopic disease is resected, preventing outgrowth and dissemination of disease. As such, ALND might offer therapeutic benefit. On the other hand, there may be stage migration when more nodes are removed, for example in patients with two resected SNs that are both positive, that could actually have a larger number of positive nodes if only more nodes would have been investigated. So, the meaning of "two positive" nodes is different for patients with more versus those with less nodes excised. This latter concept relates the improved outcome of extensive ALND to the more accurate staging process and not necessarily to a therapeutic effect of ALND. This latter hypothesis may, however, not be relevant for patients who have undergone an SN procedure. The SN is by definition already a more accurate staging procedure than conventional ALND, and therefore, the number of negative nodes removed may be less relevant. A recent study that compared survival between pN0 breast cancer patients who had undergone more or less extensive ALND and pN0 patients who had an SN procedure confirmed this. Although extensive ALND was associated with better survival than limited ALND, SN only patients also had an excellent survival [7].

The second issue concerns the question of non-SN involvement, including macrometastatic disease, in case of SN micrometastases and if such additional information would change adjuvant systemic therapy choices. The chance of further lymph node metastases beyond the SN is 12.3% in case of SN isolated tumour cells (64% macrometastases) and 20% in case of micrometastases [9]. Previously, we have shown in the Dutch MIRROR study that isolated tumor cells and micrometastases as final nodal status are significantly associated with increased risk of disease-events at a hazard ratio of about 1.5 as compared with patients having node-negative disease [10]. Some other SN studies could [START_REF] Pugliese | The clinical impact and outcomes of immunohistochemistry-only metastasis in breast cancer[END_REF][START_REF] Leidenius | Influence of isolated tumor cells in sentinel nodes on outcome in small, node-negative (pT1N0M0) breast cancer[END_REF] but some could not confirm this association between micrometastatic disease and clinical outcome [START_REF] Hansen | Impact of micrometastases in the sentinel node of patients with invasive breast cancer[END_REF][START_REF] Maaskant-Braat | Sentinel node micrometastases in breast cancer do not affect prognosis: a population-based study[END_REF][START_REF] Andersson | Breast cancer survival in relation to the metastatic tumor burden in axillary lymph nodes[END_REF]. However, it is noted that the MIRROR study is by far the largest SN study on prognostic relevance of isolated tumor cells and micrometatases, with outcome being assessed in patients who had not received systemic therapy and SN classification on full central pathology revision, and by such offering the most robust data on this issue. Moreover, a comparable hazard ratio of around 1.45 was seen in a meta-analysis on studies from the pre-SN era [START_REF] De Boer | Breast cancer prognosis and occult lymph node metastases, isolated tumor cells, and micrometastases[END_REF]. Therefore, we recommend to consider the use of adjuvant systemic therapy in these patients, and think that systemic treatment choices should not depend on the number of positive nodes that have been detected.

To conclude, we agree with Peparini that for most patients it is yet too soon to routinely implement omission of ALND in SN positive patients. Although many questions still need to be addressed, we are not convinced that LNR is an important factor when the SN procedure is being used. phone +31 43 3877025; fax +31 43 3876281 email: vcg.tjan.heijnen@mumc.nl

  micrometastases in breast cancer. Arch Surg 145:161-166 11. Straver ME, Meijnen P, van Tienhoven G et al (2010) Sentinel node identification rate and nodal involvement in the EORTC 10981-22023 AMAROS Trial. Ann Surg Oncol 17:1854-1861 12.Maaskant-Braat AJ, van de Poll-Franse LV, Voogd AC et al (2010) Sentinel node micrometastases in breast cancer do not affect prognosis: a population-based study. Breast Cancer Res Treat doi:10.1007s10549-010-1086-6 13.de Boer M, van Deurzen C, van Dijck JAAM et al (2009) Micrometastases or isolated tumor cells and the outcome of breast cancer. N Engl J Med 361;653-663 14. Anderson SJ, Wapnir I, Dignam JJ et al (2009) Prognosis after ipsilateral breast tumor recurrence and locoregional recurrences in patients treated by breast-conserving therapy in five national surgical adjuvant breast and bowel project protocols of node-negative breast cancer. J Clin Oncol 27:2466-2473 15. Xing Y, Badgwell BD, Ross MI et al (2009) Lymph node ratio predicts disease-specific survival in melanoma patients. Cancer 115:2505-2513


B M Kronenwett

Müller

I Wirtz

Baumann

Grasshoff St

Ulmer Hu

Von Minckwitz

Dr Med Carsten Denkert
email: carsten.denkert@charite.de

Comparison of different approaches for assessment of HER2 expression on protein and mRNA level -prediction of chemotherapy response in the neoadjuvant GeparTrio trial (NCT00544765)

Keywords: HER2, kinetic RT-PCR, breast cancer, neoadjuvant therapy, pathological complete response

Comparison of different approaches for assessment of HER2 expression on protein and mRNA level: prediction of chemotherapy response in the neoadjuvant

GeparTrio trial (NCT00544765)

Introduction

Human epidermal growth factor receptor 2 (HER2) testing is the prototype of a predictive biomarker for therapeutic decisions in breast cancer. There are several studies that have compared different HER2 testing methods to find out the most accurate and precise technique [START_REF] Yaziji | Accuracy and precision in her2/neu testing in breast cancer: Are we there yet?[END_REF] , [START_REF] Press | Evaluation of her-2/neu gene amplification and overexpression: Comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens[END_REF] , [START_REF] Lebeau | Her-2/neu analysis in archival tissue samples of human breast cancer: Comparison of immunohistochemistry and fluorescence in situ hybridization[END_REF] , [START_REF] Thomson | Her-2/neu in breast cancer: Interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization[END_REF]. In these studies, a high correlation between HER2 protein level evaluated by immunohistochemistry and gene amplification assessed by fluorescence or silverenhanced in-situ-hybridization (FISH/SISH) was demonstrated. Therefore the current state-of-the-art approach is immunohistochemistry combined with additional testing of equivocal cases by in-situhybridization techniques.

However, several reports have pointed out considerable difficulties in the assessment of HER2 by immunohistochemistry, with discordance rates of up to 20% [5]. This is mainly related to the fact that the immunohistochemical assessment is only semiquantitative.

It has been suggested that HER2 expression is a continuous parameter that could be measured on mRNA level by RT-PCR in formalin-fixed, paraffin-embedded (FFPE) tissue [START_REF] Paik | Her2 status and benefit from adjuvant trastuzumab in breast cancer[END_REF]. On the basis of this observation, we have developed a quantitative method for determination of HER2 mRNA expression in FFPE tissue [START_REF] Koutras | Evaluation of the prognostic and predictive value of her family mrna expression in high-risk early breast cancer: A hellenic cooperative oncology group (hecog) study[END_REF][START_REF] Bohmann | Rna extraction from archival formalin-fixed paraffin-embedded tissue: A comparison of manual, semiautomated, and fully automated purification methods[END_REF]. The aim of this study was to compare different methods for HER2 testing and to identify the predictive value of HER2 expression for a pathological complete response to neoadjuvant anthracycline/taxane-based chemotherapy. To address this issue, we evaluated a cohort of 278 pretherapeutic FFPE core biopsies of patients from the GeparTrio neoadjuvant trial. The patients had not been treated with any anti-HER2 therapy regardless of individual HER2 status, since this was not standard treatment for HER2-positive tumors at the time the study was conducted. HER2 status was investigated by kinetic RT-PCR on 278 available RNA samples isolated from FFPE tissue to determine mRNA expression. Further, the samples were analyzed for HER2 expression by local pathological institutes of the participating centers as well as by central evaluation using immunohistochemistry and in-situ-hybridization.

Patients and methods

Clinical study background

The GeparTrio study enrolled women with operable as well as locally advanced breast carcinomas and was organized by the German Breast Group between 2002 and 2005 [START_REF] Von Minckwitz | Intensified neoadjuvant chemotherapy in early-responding breast cancer: Phase iii randomized gepartrio study[END_REF][START_REF] Von Minckwitz | Neoadjuvant vinorelbine-capecitabine versus docetaxel-doxorubicincyclophosphamide in early nonresponsive breast cancer: Phase iii randomized gepartrio trial[END_REF][START_REF] Huober | Effect of neoadjuvant anthracycline-taxane-based chemotherapy in different biological breast cancer phenotypes: Overall results from the gepartrio study[END_REF]. After two cycles with TAC (docetaxel 75 mg/m 2 , doxorubicin 50 mg/m 2 , cyclophosphamide 500 mg/m 2 ), patients were randomized depending on their clinical response. Patients with an early response were randomized to proceed with either 4 or 6 additional cycles of TAC, whereas non-responding patients were randomized either to continue with 4 cycles of TAC or 4 cycles of NX (vinorelbine 25 mg/m 2 plus capecitabine 1000 mg/m 2 ).

Breast cancer diagnosis was made by core needle biopsy and the pathological examination was performed by local pathologists of the participating sites. The histological type was defined according to the WHO classification. Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status were determined locally at each participating center by immunohistochemistry on tissue sections of the true/core-cutbiopsies obtained at the time of primary diagnosis before treatment. In case of moderate Her2 overexpression (Score 2+) by immunohistochemistry, confirmatory FISH testing was required. All pathology reports were centrally reviewed at the German Breast Group headquarters, Neu-Isenburg, Germany. This study includes essential elements of the REMARK criteria [START_REF] Hayes | New guidelines for reporting of tumor marker studies in breast cancer research and treatment: Remark[END_REF][START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies (remark)[END_REF].

Inclusion criteria for evaluation of HER2 expression

For this study, only patients of those arms of the GeparTrio trial that had received 6 cycles of TAC were included, to ensure a homogenous therapy. All samples were taken at the time of initial diagnosis and were collected prospectively at the tumor bank of the German Breast Group at the Institute of Pathology, Charité Hospital, Germany. The study was approved by the institutional review boards of the centers participating in the clinical trial as well as the Hospital Charité. All patients gave written informed consent on participating in the trials and biomaterial collection.

Inclusion criteria for this study were treatment with 6 cycles of TAC, available FFPE pretherapeutic core biopsies, and available RNA from the tissue biopsies containing more than 30% tumor cells. Of the total of 1025 patients that had received 6 cycles of TAC, mRNA samples from 278 patients were included. Data on clinical parameters were taken from the clinical trials database.

Pathological complete remission (pCR) was defined as a histopathological complete remission of all invasive tumor cells from both breast and axillary tissue removed at surgery (ypT0, ypTis, ypN0).

Assessment of HER2 status using different approaches

A total of three approaches were used for determination of HER2 status. First, HER2 expression was analyzed by local pathological institutes of the participating centers by immunohistochemistry and/or fluorescence in-situ-hybridization (FISH).

In addition, we performed a central assessment of HER2 expression by immunohistochemical staining in combination with silverenhanced in-situhybridization (SISH), according to the ASCO/CAP guidelines for HER2 testing in breast carcinomas and as described recently [14,[START_REF] Loibl | Identification of biology-based breast cancer types with distinct predictive and prognostic features: Role of steroid hormone and her2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy[END_REF]. For this evaluation, tissue microarrays were constructed from needle core biopsies as described recently [START_REF] Loibl | Identification of biology-based breast cancer types with distinct predictive and prognostic features: Role of steroid hormone and her2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy[END_REF].

For immunohistochemical determination of HER2 were incubated with the rabbit polyclonal antibody against human HER2 (HercepTest antibody, Dako, 1:500) using the Discovery XT autostainer (Ventana, Tuscon, AZ, USA). Stained slides were digitized by a slide scanner (MIRAX SCAN, Zeiss Jena, Germany). The central review was done by two independent pathologists using custom-made software for whole slide imaging of virtual slides (VMscope, Berlin, Germany). SISH analyses were carried out in cases with equivocal Her2 expression. This technique was performed on a Benchmark XT autostainer (Ventana) using the INFORM HER2 probe (Ventana) according to the manufacturer's instructions. Internal and external quality controls were performed to assure test accuracy.

RNA isolation and kinetic RT-PCR

As a third method, HER2 mRNA expression was measured by kinetic RT-PCR in the pretherapeutic core biopsies. RNA was isolated from formalin-fixed, paraffinembedded (FFPE) tumor tissue specimens employing a Siemens Healthcare Diagnostics proprietary experimental method based on magnetic beads (not commercially available) as previously described [START_REF] Bohmann | Rna extraction from archival formalin-fixed paraffin-embedded tissue: A comparison of manual, semiautomated, and fully automated purification methods[END_REF]. All tumor samples included in the study contained at least 30% tumor tissue, as evaluated by hematoxylin-eosin staining. RPL37A, a house keeping gene and HER2 mRNA expression were assessed by kinetic one-step RT-PCR on ABI7900 systems using primers and probes as described previously [START_REF] Bohmann | Rna extraction from archival formalin-fixed paraffin-embedded tissue: A comparison of manual, semiautomated, and fully automated purification methods[END_REF]. Forty amplification cycles were applied and the threshold (Ct) values of genes were determined by ABI PRISM software. Relative HER2 mRNA expression levels were calculated as ΔCt values (ΔCt=20-[Ct Her2 -Ct RPL37A ]). For mRNA-based classification of HER2 status, a ΔCt cutoff point of 18 was used. The cutoff value was pre-defined on the basis of the bi-modal distribution of HER2 expression in a previous project [START_REF] Müller | Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new fully automated method for rna isolation[END_REF].

Statistics

For the statistical calculations, the software package SPSSv17.0 was used.

Correlation analyses were performed by using binary logistic regression analysis, and as indicated by Fisher's exact test or chi-square test for trends. Correlations among the HER2 assessment methods were evaluated using the Spearman's correlation coefficient method and Mann-Whitney test. Agreement between the immunohistochemistry/ in-situ-hybridization methods and kRT-PCR uncorrected for chance was calculated as the number of samples which agree divided by the total number of samples. Agreement corrected for chance was determined by kappa statistics estimating Cohen's kappa coefficient. In general, κ > 0.91 is considered to represent almost perfect concordance. To identify parameters independently associated with pCR, a multivariate logistic regression was performed. In this analysis, all parameters that had been described as predictive in the primary clinical study cohort were included. Generally, p-values of <0.05 were considered significant.

Results

Characteristics of the study cohort

A cohort of 278 patients from the GeparTrio trial was selected (Table 1). Patient age ranged from 23.5 to 78.7 years (mean 50.8). The distribution of clinicopathological parameters and pCR rate was comparable to the full study population that received 6 cycles of TAC. We observed a significant association between all HER2 assessment methods using the Mann-Whitney test (p<0.0001) and Spearman's rho correlation. The correlation coefficient between HER2 mRNA and central HER2 expression was particularly high (0.867, p<0.0001). In contrast, the correlation between HER2 mRNA and local HER2 (0.449, p<0.001) as well as central and local HER2 (0.438, p<0.0001) was significant as well, but the correlation coefficients were much lower.

The concordance (Table 2) between central HER2 and HER2 mRNA status was 95.6% (219 out of 229 cases), while the results of local HER2 and HER2 mRNA testing were concordant in only 182 of 244 cases (74%). Local HER2 assessment found 52 additional positive cases that were negative by kRT-PCR. In these discrepant cases, the mean relative RNA expression was 16.6 indicating relatively high levels of HER2 mRNA. 4).

The predictive effect of central HER2 expression was confirmed in a multivariate binary logistic regression model using all clinicopathological parameters that were significant on univariate analysis (Table 5). However, the local HER2 status (OR 1.5 CI 0.67-3.35, p=0.32) as well as the HER2 mRNA status (OR 1.56 CI 0.66-3.68, p=0.311) was not significant in multivariate analysis. .

Discussion

Our study shows a significant correlation of HER2 levels by conventional immunohistochemistry and in-situ-hybridization methods as well as kinetic RT-PCR.

We found that central HER2 status and HER2 mRNA expression are predictors for a pathological complete response after neoadjuvant TAC treatment in a univariate binary logistic regression. Moreover, central HER2 status is an independent predictor for pCR in a multivariate binary logistic regression.

This study demonstrates that evaluation of HER2 mRNA levels is feasible in routine formalin-fixed, paraffin-embedded tissue from clinical multicenter studies, which is in line with other studies [START_REF] Müller | Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new fully automated method for rna isolation[END_REF][START_REF] Iverson | A single-tube quantitative assay for mrna levels of hormonal and growth factor receptors in breast cancer specimens[END_REF]. Since the interpretation of HER2 immunostaining and in-situ-hybridization may be influenced by laboratory and observer variability, the kRT-PCR could improve the assessment of HER2 status as an additional molecular test. This approach enables a relatively fast, reproducible quantification of HER2 expression and reduces inter-observer variability. Previous studies revealed an overall concordance from 82 to 93% between different HER2 assessment methods of protein, DNA, and mRNA levels [START_REF] Ginestier | Comparative multi-methodological measurement of erbb2 status in breast cancer[END_REF][START_REF] Vinatzer | Expression of her2 and the coamplified genes grb7 and mln64 in human breast cancer: Quantitative real-time reverse transcription-pcr as a diagnostic alternative to immunohistochemistry and fluorescence in situ hybridization[END_REF].

Pathological complete response (pCR) following neoadjuvant chemotherapy (NCT)

has been shown to be predictive of longer recurrence-free and overall survival [START_REF] Kaufmann | Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: An update[END_REF][START_REF] Rastogi | Preoperative chemotherapy: Updates of national surgical adjuvant breast and bowel project protocols b-18 and b-27[END_REF]. However, only 15 to 20% of the patients achieve a pathological complete response. In the last few years, several studies have shown a higher chemosensitivity in certain tumor subtypes. Tumors with high-grade, non-lobular invasive histology, and negative lymph node status correlate with a good response to NCT [START_REF] Kaufmann | Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: New perspectives 2006[END_REF], [START_REF] Tan | Predictors of complete pathological response after neoadjuvant systemic therapy for breast cancer[END_REF]. Additionally, several reports suggest a significance of the hormone receptor and HER2 status in assessment of chemotherapy response. Thus, negative hormone receptors are one of the strongest predictive markers [START_REF] Colleoni | Chemotherapy is more effective in patients with breast cancer not expressing steroid hormone receptors: A study of preoperative treatment[END_REF], and triplenegative breast carcinomas have higher pCR rates than non-triple-negative tumors [START_REF] Liedtke | Response to neoadjuvant therapy and long-term survival in patients with triplenegative breast cancer[END_REF] , [START_REF] Huober | Neoadjuvant chemotherapy in operable breast cancer with docetaxel, doxorubicin, and cyclophosphamide (tac) or tac followed by vinorelbine and capecitabine (nx): Final results and analysis of markers predicting response to treatment[END_REF].

Our findings are in line with other studies demonstrating a predictive value for HER2 expression in achieving a good response to neoadjuvant chemotherapy [START_REF] Rody | The erbb2+ cluster of the intrinsic gene set predicts tumor response of breast cancer patients receiving neoadjuvant chemotherapy with docetaxel, doxorubicin and cyclophosphamide within the gepartrio trial[END_REF][START_REF] Jones | Relationship between oestrogen receptor status and proliferation in predicting response and long-term outcome to neoadjuvant chemotherapy for breast cancer[END_REF]. Until now, the reports concerning the predictive value of HER2 were inconsistent due to heterogeneous study populations and different systemic treatment regimens.

Regarding the molecular mechanisms, it is not completely clear why HER2expressing breast carcinomas show a better response to NCT. Thus, it was suggested that HER2-positive tumors are more sensitive to anthracycline chemotherapy due to the frequent coamplification of HER2 and topoisomerase II [29].

Generally, basal-like and HER2+/HR-breast carcinomas have shown a higher sensitivity to neoadjuvant anthracycline-based therapy than the luminal subtype [START_REF] Tan | Predictors of complete pathological response after neoadjuvant systemic therapy for breast cancer[END_REF][START_REF] Rouzier | Breast cancer molecular subtypes respond differently to preoperative chemotherapy[END_REF][START_REF] Carey | The triple negative paradox: Primary tumor chemosensitivity of breast cancer subtypes[END_REF][START_REF] Guarneri | Prognostic value of pathologic complete response after primary chemotherapy in relation to hormone receptor status and other factors[END_REF]. However, the therapy response was higher in the so called luminal B (HER2+/HR+) than in the luminal A subtype (HER2-/HR+) [START_REF] Carey | The triple negative paradox: Primary tumor chemosensitivity of breast cancer subtypes[END_REF][START_REF] Guarneri | Prognostic value of pathologic complete response after primary chemotherapy in relation to hormone receptor status and other factors[END_REF]. In the study of Guarneri and colleagues, the pCR was higher in HER2+/HR+ than in HER2-/HR+ carcinomas (15.3% vs. 6%). This was also observed in a cohort of the GeparDuo trial on anthracycline/taxane-based neoadjuvant chemotherapy showing higher pCR rates in HER2+/HR+ than in HER2-/HR+ breast carcinomas [START_REF] Loibl | Identification of biology-based breast cancer types with distinct predictive and prognostic features: Role of steroid hormone and her2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy[END_REF] as well as in the whole analysis of GeparTrio [START_REF] Huober | Neoadjuvant chemotherapy in operable breast cancer with docetaxel, doxorubicin, and cyclophosphamide (tac) or tac followed by vinorelbine and capecitabine (nx): Final results and analysis of markers predicting response to treatment[END_REF].

HER2 overexpression is associated with a higher rate of pCR to sequential paclitaxel/FAC preoperative chemotherapy regardless of ER status in a cohort of 534 patients. Further, HER2 was a particularly strong predictor of pCR in ER-positive carcinomas (pCR rate 19% vs. 6%) [START_REF] Andre | Her2 expression and efficacy of preoperative paclitaxel/fac chemotherapy in breast cancer[END_REF]. The authors report also on an association of higher TOP2A mRNA, lower MAP-tau expression, and HER2 positivity in a subset of ER-positive breast carcinomas suggesting an increased sensitivity for neoadjuvant chemotherapy.

We are aware of the limitations our study: It is a retrospective analysis of pretherapeutic core biopsies in the neoadjuvant setting, which results in restrictions in the number of available samples as well as in comparably small amounts of tissue used for TMA construction resulting in false-negative results, and missing data from local histology reports. Further, only cases which were informative for kinetic RT-PCR were included in this investigation which may cause a potential selection bias.

However, we evaluated a study cohort of a large prospective clinical trial with similar well-documented clinical data and pre-defined cutoffs for HER2 mRNA. It is not completely clear why central HER2 status, but not HER2 mRNA status is an independent predictor in multivariate analysis, considering the fact that both factors are highly correlated and both are significant in univariate analysis. This result should be interpreted with caution and validated in additional cohorts.

Our study shows that the local HER2 status was not predictive for pCR in a univariate logistic regression analysis. The rate of HER2-positive breast carcinomas by local analysis (37.3%) was considerably higher as compared with the central HER2 status (17.9%) and HER2 mRNA (19.8%) expression. This is not unexpected because preanalytic and analytic process steps in HER2 testing as well as the inter-observer variability in evaluation of HER2 immunostaining may influence the testing accuracy.

Furthermore, at the time the GeparTrio trial was conducted, the experience with HER2 testing in pathology laboratories was much lower than today. As reviewed by Gown, HER2 studies performed at local and central laboratories showed significant levels of discordance [5]. However, the application of ASCO/CAP guidelines as well as proficiency tests have improved the accuracy of HER2 determination in the last few years [START_REF] Lebeau | Reliability of human epidermal growth factor receptor 2 immunohistochemistry in breast core needle biopsies[END_REF].

Conclusions

As the HER2 status plays a critical role in the treatment of breast cancer patients, a standardized HER2 assessment with implementation of the ASCO/CAP guidelines is essential for future clinical trials. Quality assurance standards in assessing hormone receptors and HER2 status are also needed in routine clinical practice. Furthermore, due to the false-positive rate in the determination of HER2 by immunohistochemistry and time-consuming ISH methods, reliable and accurate assessment approaches are essential. In addition to the established methods, the kinetic RT-PCR might be a diagnostic alternative in HER2 testing. Further studies are needed to approve the advantages of this quantitative approach.

Figure 1

 1 Figure 1 Relationship between HER2 assessment methods A: Distribution of HER2 expression assessed by local and central evaluation as well as by kinetic RT-PCR. B and C: Association of the different assessment methods presented in a scatter diagram.

Table 1

 1 Characteristics of the study cohort and results of the HER2 determination with three approaches

	Parameter	Study cohort No (%) GeparTrio 6xTAC No (%)
	cases	278	1025
	Pathological complete response
	pCR	56 (20.1)	(19.4)
	no pCR	222 (79.9)	(80.6)
	Tumor grade
	G1-2	165 (59.4)	(55.9)
	G3	81 (29.1)	(38.9)
	missing	32 (11.5)	(5.2)
	Tumor type
	invasive ductal/others	241 (86.7)	(86.2)
	invasive lobular	37 (13.3)	(13.8)
	ER/ PgR status
	ER-/ PgR-	63 (22.7)	(33.8)
	ER+ and/ or PgR+	181 (65.1)	(63.1)
	missing	34 (12.2)	(3.1)
	Palpable tumor size
	≤4 cm	122 (43.9)	(53.3)
	>4 cm	152 (54.7)	(45.3)
	unknown	4 (1.4)	(1.4)
	Nodal status
	cN0	133 (47.8)	(43.0)
	cN+	130 (46.8)	(52.9)
	missing	15 (5.4)	(4.1)
	Age
	≤40 yrs	49 (17.6)	(19.2)
	> 40 yrs	82.4 (82.4)	(80.8)
	central HER2 status
	negative	188 (67.6)	(58.7)
	positive	41 (14.7)	(25.8)

status by local and central IHC/ISH and quantitative RT- PCR

Results on the three different measurements of HER2 are summarized in Table

1

and Figure

1

. Each analysis method was performed blinded to the results of the other methods. Data on local HER2 analysis were available in 244 cases and revealed HER2 positivity in 37.3% (91 out of 244). However, in our central analysis, the rate of HER2-positive cases was considerably lower, with 17.9% (41 out of 229 interpretable samples). Assessment of HER2 mRNA by kinetic RT-PCR was carried out on 278 breast cancer samples. Normalized HER2 mRNA expression (ΔCT) ranged from 12.6 to 21.8, with a median of 16.3 ΔCT. We used a pre-defined cutoff value of a ΔCT of 18 that was based on independent studies and found a positive HER2 mRNA expression in 19.8% of tumors.

Table 2

 2 Concordance analysis between the different HER2 testing methods

		mRNA vs. central	mRNA vs. local	central vs. local
	kappa (CI 95%)	0.862 (0.778-0.946) 0.401 (0.272-0.530)	0.339 (0.257-0.541)
	overall agreement	95.6%	74.6%	74.50%
	positive agreement	97.6%	42.9%	34.1%
	negative agreement	95.2%	93.5%	72.4%
	As shown in Table 3, there was no relationship between HER2 status and clinico-
	pathological features like tumor size, nodal stage, histological grade, and subtype or
	patient age.

Table 3

 3 Expression of HER2: relationship of different methods as well as correlation of mRNA HER2 status with outcome and clinical parameters

	Characteristic	n	HER2 mRNA	HER2 mRNA	p-value
			negative	positive
	cases	278	223 (80.2%)	55 (19.8%)
	Central HER2				<0.0005*
	negative	188	179 (95.2%)	9 (4.8%)
	positive	41	1 (2.4%)	40 (97.6%)
	Local HER2				<0.0005*
	negative	153	143 (93.5%)	10 (6.5%)
	positive	91	52 (57.1%)	39 (42.9%)
	Pathological response				0.005*
	pCR	56	37 (66.1%)	19 (33.9%)
	no pCR	222	186 (83.8%)	36 (16.2%)
	Tumor grade				ns
	G1-2	165	134 (81.25)	31 (18.8%)
	G3	81	62 (76.5%)	19 (23.5%)
	Tumor type				0.074*
	invasive ductal/other	241	189 (78.4%)	52 (21.6%)
	invasive lobular	37	34 (91.9%)	3 (8.1%)
	ER/PgR Status				ns
	ER+ and/or PgR+	181	148 (81.8%)	33 (18.2%)
	ER-/PR-	63	48 (76.2%)	15 (23.8%)
	Palpable tumor size				ns
	≤4 cm	122	98 (80.3%)	24 (19.7%)
	>4 cm	152	123 (80.9%)	29 (19.1%)
	Nodal status				ns
	cN0	133	107 (80.5%)	26 (19.5%)
	cN+	130	104 (80.0%)	26 (20.0%)
	Age				ns
	≤40 yrs	49	36 (16.1%)	13 (23.6%)
	>40 yrs	229	187 (83.9%)	42 (76.4%)

Table 4

 4 Univariate logistic regression analysis: factors predicting a pCR

	Characteristic	n	pCR (%)	Univariate analysis	p-value
				Odds ratio (95% CI)
	Central HER2	229			0.002
	negative	188	28 (14.9%)	1.00
	positive	41	15 (36.6%)	3.297 (1.555-6.991)
	HER2 mRNA	278			0.004
	negative	223	37 (16.6%)	1.00
	positive	55	19 (34.5%)	2.653 (1.374-5.125)
	Local HER2	244			0.678
	negative	153	27 (17.6%)	1.00
	positive	91	18 (19.8%)	1.151 (0.593-2.232)
	Hormone receptor status	244			<0.0001
	ER+ and/ or PgR+	181	23 (12.7%)	1.00
	ER-/PgR -	63	25 (39.7%)	4.519 (2.318-8.813)
	Age	278			0.006
	>40 yrs	229	39 (17.0%)	1.00
	≤40 yrs	49	17 (34.7%)	2.588 (1.309-5.117)
	Tumor type	278			0.138
	invasive lobular	37	4 (10.8%)	1.00
	invasive ductal /other	241	52 (21.6%)	2.270 (0.769-6.698)
	Tumor grade	246			0.003
	G1-2	165	21 (12.7%)	1.00
	G3	81	23 (28.4%)	2.719 (1.398-5.290)
	Palpable tumor size	274			0.024
	>4 cm	152	22 (14.5%)	1.00
	≤4 cm	122	31 (25.4%)	2.013 (1.095-3.699)
	Nodal status	263			0.833
	cN+	130	26 (20.0%)	1.00
	cN0	133	28 (21.1%)	1.067 (0.586-1.941)

Table 5

 5 Multivariate logistic regression analysis: factors predicting a pCR

	Characteristic	Odds Ratio (95% CI)	p-value
	Central HER2 (positive vs. negative)	3.04 (1.13-8.16)	0.027
	Hormone receptor status (ER-/PgR -vs. ER+ and/ or PgR+)	3.99 (1.58-10.06)	0.003
	Age (<40 vs. >40 yrs)	1.27 (0.44-3.60)	ns
	Tumor grade (G3 vs. G1-2)	2.86 (1.12-7.29)	0.028
	Palpable tumor size (< 4 cm vs. > 4 cm)	2.71 (1.09-6.72)	0.031

Acknowledgements

We would like to thank Mrs. Petra Wachs, Mrs. Gabriela Altmann and Mrs. Claudia Roth for her excellent technical assistance.

Abbreviations

Author contributions

AN, SL, SDE, MR, BMM, JS, RK, CT, RW, CD, GM conceived study design, data collection, data analysis and interpretation. IB, GH, GH, STG, HUU carried out data collection. All authors were involved in writing the paper and had final approval.

Competing interests

The authors declare that they have no competing interests. Ralf Kronenwett, Christian von Törne and Ralph Wirtz are employees of Siemens Healthcare Diagnostics, Cologne, Germany.


Mariam Abdullahi

Adamu • Melanie

Nicole Weck

Lei Gao

Hermann Brenner
email: h.brenner@dkfz-heidelberg.de

M A Adamu
email: m.adamu@dkfz-heidelberg.de

Á M N Weck

Á L Gao

Á H Brenner

Incidence of chronic atrophic gastritis: systematic review and meta-analysis of follow-up studies

Keywords: Chronic atrophic gastritis, Incidence, Follow-up studies Chronic atrophic gastritis cagA Cytotoxin associated gene A H. pylori Helicobacter pylori

Chronic atrophic gastritis (CAG) is an important precursor lesion of intestinal gastric cancer. As it is typically asymptomatic, epidemiological data on the incidence of CAG are sparse. We aimed to provide an overview of published data on CAG incidence (overall and according to risk factors) from follow-up studies. Articles with information on incidence of CAG published in English until 26th of July 2009 were identified through a systematic MEDLINE and EMBASE search. Data extracted include study characteristics and key findings regarding the incidence of CAG. A meta-analysis was performed on the association between Helicobacter pylori infection and CAG incidence. Overall, data on CAG incidence were available from 14 studies, in 7 studies incidence could be estimated according to H. pylori infection. Most studies were conducted in symptomatic or high risk populations and the maximum number of incident cases was 284. Incidence estimates ranged from 0 to 11% per year and were consistently below 1% in patients not infected with H. pylori. The highest incidence was observed in a special study conducted on ulcer patients treated by proximal gastric vagotomy. Rate ratios for the association between H. pylori infection and CAG incidence ranged from 2.4 to 7.6 with a summary estimate of 5.0 (95% confidence interval: 3.1-8.3). Incidence of CAG is very low in the absence of H. pylori infection. There is a need for more population-based studies to provide comparable estimates of incidence and the impact of risk factors in the development of CAG.

Introduction

Chronic atrophic gastritis (CAG) is an important precursor lesion in the development of intestinal type of gastric cancer [START_REF] Ohata | Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer[END_REF]. Even though there is a worldwide decline in the incidence of this type of cancer, its public health burden is still high, with gastric cancer ranking fourth in cancer incidence and being the second most common cause of cancer-related deaths worldwide [START_REF] Parkin | Global cancer statistics[END_REF].

The sequence of events that occur in the gastric mucosa before the manifestation of gastric cancer has been shown to take decades. Typically, the sequence is from chronic non-atrophic gastritis that progresses to CAG, which may eventually lead to intestinal metaplasia, dysplasia and finally adenocarcinoma [START_REF] Kuipers | Review article: exploring the link between Helicobacter pylori and gastric cancer[END_REF]. In order to understand the processes involved in the occurrence of this type of cancer and to establish risk factors for its development, it is necessary to understand its precursor lesions and the risk factors associated with their development.

The first follow-up studies on CAG have been conducted more than five decades ago. However, the majority of epidemiological studies on CAG and its risk factors have been cross-sectional [START_REF] Weck | Prevalence of chronic atrophic gastritis in different parts of the world[END_REF]. Infection with the gastric bacterium Helicobacter pylori was shown to be strongly associated with CAG in a recently published meta-analysis that relied on cross-sectional data [START_REF] Weck | Association of Helicobacter pylori infection with chronic atrophic gastritis: meta-analyses according to type of disease definition[END_REF]. However, to prove the causal role of H. pylori infection and other risk factors in the development of CAG, analysis of follow-up data is necessary.

The aim of this systematic review is to provide an overview of the incidence of CAG, both overall and according to potential risk factors reported by follow-up epidemiological studies.

Materials and methods

Articles providing information on the incidence of CAG were identified through a systematic search in the MED-LINE and EMBASE databases by using various combinations of the terms 'atrophic', 'atrophy', 'chronic', 'gastritis', 'pepsinogen', 'incidence', 'cohort', 'follow-up', 'long term', 'prospective' and 'retrospective'. Additionally, references of retrieved articles were screened for further studies (cross-referencing).

All original research articles from human studies published in English until the 26th of July 2009 were included. Due to the few studies that could be identified, restrictions in terms of selection criteria of participants (e.g., hospital based studies or studies which included patients with a particular disease or treatment) were not taken. However, only studies with more than 15 study participants without CAG at baseline and with a mean or median of at least 2 years of follow-up were included. Furthermore, studies in which follow-up was made by indication only (rather than by design) were not included.

The following data were extracted from the articles: first author, year of publication, characteristics of the study population [country, population type, sample size, age (range and mean or median) of study participants], dates of baseline and of follow-up examinations, mean years of follow-up, H. pylori infection at baseline, method of diagnosis of CAG (both at baseline and at follow-up), absolute number of incident cases and incidence of CAG (in percent per year), overall and according to pertinent risk factors. If incidence rates of CAG per year were not given in the articles, they were calculated from case numbers and mean years of follow-up time. In order to enhance comparability of the studies only data from the corpus or fundus mucosa (where given separately) were used to calculate incidence rates in the studies with histological assessment of CAG even when data on multiple biopsy sites for histological assessment of CAG was provided. Extraction of data from the articles was performed independently by two investigators (MAA and LG), and minor discrepancies were resolved in consensus.

A meta-analysis on the association between H. pylori infection and incidence of CAG was conducted with the Comprehensive Meta-Analysis software (V2.0; Biostat, Englewood, NJ, USA) by using a random effects model. The meta-analysis was performed on all studies where incidence of CAG could be estimated according to H. pylori infection at baseline. A forest plot showing rate ratios for the incidence among H. pylori positive compared to H. pylori negative study participants was created. Heterogeneity was analysed by the Q-test [START_REF] Cochran | The combination of estimates from different experiments[END_REF] and by the I 2 -test [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]. Publication bias was analysed by Egger's weighted regression model [START_REF] Egger | Bias in metaanalysis detected by a simple, graphical test[END_REF] and by Begg's rank correlation method [START_REF] Begg | Operating characteristics of a rank correlation test for publication bias[END_REF].

Results

Figure 1 presents a flowchart of the literature search; numbers of articles and reasons for exclusion are given. The literature search initially resulted in 3,334 articles which were screened by title and abstract. Sixty-four articles were identified to potentially provide data on the incidence of CAG. After reading the full text of the 64 articles a number of them had to be excluded, these are given in an additional file as supplementary material. 8 articles were excluded because they did not present any data on CAG or its incidence. 15 articles had to be excluded because they only presented prevalence data at baseline and follow-up examinations or at either time period. In another 14 articles, distribution or changes in 3334 articles screened by title and abstract 64 articles potentially providing data on the incidence of CAG (full text read) Exclusions (n) -Prevalence rather than incidence (15) -Use of mean scores or grades of gastritis to estimate CAG [START_REF] Kuipers | Atrophic gastritis and Helicobacter pylori infection in patients with esophagitis treated with omeprazole or fundoplication[END_REF] -Absence of data on CAG or incidence of CAG [START_REF] Egger | Bias in metaanalysis detected by a simple, graphical test[END_REF] -Less than 15 study participants (8) -Other reasons (4) 15 articles (from 14 studies) included in the review Fig. 1 Flow chart representing the literature search. Abbreviations: CAG chronic atrophic gastritis severity of carditis or gastritis scores, or gastritis grades rather than incidence of CAG was reported. 8 articles were excluded because they had less than 15 study participants at baseline without CAG. Another 4 articles were excluded for the following reasons: all patients had either chronic gastritis or CAG at baseline, incidence of CAG was based on biopsies and not on study participants, incidence of CAG and metaplasia or either of them were grouped together and in one study, no mean follow-up time could be estimated. Due to these exclusions, only 14 studies (from 15 articles) with information on the incidence of CAG could be included in this review [START_REF] Jonsson | Histologic changes in the gastroduodenal mucosa after long-term medical treatment with cimetidine or parietal cell vagotomy in patients with juxtapyloric ulcer disease[END_REF][START_REF] Correa | Gastric precancerous process in a high risk population: cohort follow-up[END_REF][START_REF] Kuipers | Helicobacter pylori and atrophic gastritis: importance of the cagA status[END_REF][START_REF] Kuipers | Long-term sequelae of Helicobacter pylori gastritis[END_REF][START_REF] Kuipers | Atrophic gastritis and Helicobacter pylori infection in patients with esophagitis treated with omeprazole or fundoplication[END_REF][START_REF] Valle | Long-term course and consequences of Helicobacter pylori gastritis. Results of a 32-year follow-up study[END_REF][START_REF] Maaroos | Follow-up of Helicobacter pylori positive gastritis and argyrophil cells pattern during the natural course of gastric ulcer[END_REF][START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF][START_REF] Carter | Does acid suppression by antacids and H2 receptor antagonists increase the incidence of atrophic gastritis in patients with or without H. pylori gastritis?[END_REF][START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF][START_REF] Klinkenberg-Knol | Long-term omeprazole treatment in resistant gastroesophageal reflux disease: efficacy, safety, and influence on gastric mucosa[END_REF][START_REF] Lamberts | Effects of very long (up to 10 years) proton pump blockade on human gastric mucosa[END_REF][START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF][START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF][START_REF] Vorobjova | Immune response to Helicobacter pylori and its association with the dynamics of chronic gastritis in the antrum and corpus[END_REF]. According to the above described criteria, all of these studies included more than 15 study participants who were diagnosed to be free of CAG at baseline, either by histology ('normal' or 'superficial gastritis') or by serum pepsinogen (PG) levels.

A summary and characterization of the 14 studies included in this review is given in Table 1. The earliest study was started in 1952 in Finland [START_REF] Valle | Long-term course and consequences of Helicobacter pylori gastritis. Results of a 32-year follow-up study[END_REF]. The most recent one in 1990-1993 in Slovenia; [START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF] all studies were published between 1988 and 2008. 2 of the studies were conducted in multiple centres in different countries [START_REF] Klinkenberg-Knol | Long-term omeprazole treatment in resistant gastroesophageal reflux disease: efficacy, safety, and influence on gastric mucosa[END_REF][START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF]. Studies were carried out in Sweden, [START_REF] Jonsson | Histologic changes in the gastroduodenal mucosa after long-term medical treatment with cimetidine or parietal cell vagotomy in patients with juxtapyloric ulcer disease[END_REF] Columbia, [START_REF] Correa | Gastric precancerous process in a high risk population: cohort follow-up[END_REF] the Netherlands, [START_REF] Kuipers | Helicobacter pylori and atrophic gastritis: importance of the cagA status[END_REF][START_REF] Kuipers | Long-term sequelae of Helicobacter pylori gastritis[END_REF] the Netherlands and Sweden, [START_REF] Kuipers | Atrophic gastritis and Helicobacter pylori infection in patients with esophagitis treated with omeprazole or fundoplication[END_REF] Finland, [START_REF] Valle | Long-term course and consequences of Helicobacter pylori gastritis. Results of a 32-year follow-up study[END_REF] Estonia, [START_REF] Maaroos | Follow-up of Helicobacter pylori positive gastritis and argyrophil cells pattern during the natural course of gastric ulcer[END_REF][START_REF] Vorobjova | Immune response to Helicobacter pylori and its association with the dynamics of chronic gastritis in the antrum and corpus[END_REF] Japan, [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF][START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF] USA, [START_REF] Carter | Does acid suppression by antacids and H2 receptor antagonists increase the incidence of atrophic gastritis in patients with or without H. pylori gastritis?[END_REF] Slovenia, [START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF] Australia, Canada, Germany, and the Netherlands, [START_REF] Klinkenberg-Knol | Long-term omeprazole treatment in resistant gastroesophageal reflux disease: efficacy, safety, and influence on gastric mucosa[END_REF] Germany [START_REF] Lamberts | Effects of very long (up to 10 years) proton pump blockade on human gastric mucosa[END_REF] and Denmark, Finland, Norway and Sweden [START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF]. Most of the studies were not population-based and consisted of symptomatic patients. CAG was diagnosed by histology in twelve studies and by serum pepsinogen levels in two studies [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF][START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF]. Mean follow-up time of the study participants in the different studies ranged from 2 to 32 years. Sample sizes ranged from 17 to a maximum of 780 subjects. However, only 6 of the studies had more than 100 participants and only one study included more than 500 study participants. Numbers of incident cases ranged from 0 to 284. The age of the study participants at baseline ranged from 15 to at least 83 or older. Where reported, mean age at baseline ranged from 35 to 64 years.

12 studies examined H. pylori infection at baseline by one or more of the following methods: bacterial culture, [START_REF] Kuipers | Helicobacter pylori and atrophic gastritis: importance of the cagA status[END_REF][START_REF] Kuipers | Long-term sequelae of Helicobacter pylori gastritis[END_REF][START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF] histology, [12-16, 18-22, 24] PCR (polymerase chain reaction), [START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF] rapid urease test, [START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF] and serology [12-14, 17, 19, 24].

Incidence rates of CAG ranged from 0 to 10.9% per year. The lowest incidence (0%) was observed in two study populations, one made up of a very special cohort of 71 patients with reflux esophagitis, who were treated by fundoplication (an endoscopic procedure of wrapping the fundus around the esophagus for treatment of reflux esophagitis) and followed for a mean period of 5 years [START_REF] Kuipers | Atrophic gastritis and Helicobacter pylori infection in patients with esophagitis treated with omeprazole or fundoplication[END_REF]. The other study population consisted of 45 patients who had undergone successful H. pylori eradication therapy before the start of the study [START_REF] Tepes | Two-to four-year histological follow-up of gastric mucosa after Helicobacter pylori eradication[END_REF]. However, there was also a lack of any incident case in subpopulations of 140 H. pylori negative patients who had undergone either fundoplication or who were treated by omeprazole for 7 years and were followed for 7 years [START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF]. The highest incidence was estimated in a cohort of patients who had undergone proximal gastric vagotomy for treatment of juxtapyloric ulcer [START_REF] Jonsson | Histologic changes in the gastroduodenal mucosa after long-term medical treatment with cimetidine or parietal cell vagotomy in patients with juxtapyloric ulcer disease[END_REF].

The two studies from Japan which diagnosed CAG by serum pepsinogen levels, a study from Estonia [START_REF] Vorobjova | Immune response to Helicobacter pylori and its association with the dynamics of chronic gastritis in the antrum and corpus[END_REF] and one from Colombia [START_REF] Correa | Gastric precancerous process in a high risk population: cohort follow-up[END_REF] were the only studies with an approximate population-based study sample. The incidence observed in the study from Estonia was 1.9% per year and that in the Columbian study 7.1%. The two Japanese studies used slightly different definitions of CAG. The incidence reported in the first of these studies using a less restrictive CAG definition (PGI \ 70ng/mL & PGI/ PGII \ 3) was rather high (2.7% per year) [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF] compared to the studies that assessed CAG by histology. The incidence in the second serology-based study was 1.3% per year [START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF].

In the study from Columbia [START_REF] Correa | Gastric precancerous process in a high risk population: cohort follow-up[END_REF], incidence of CAG was given according to age groups with a higher incidence in the older group (B40 years of age 7.0% per year and C41 years of age 8.5% per year). There was however, no such trend in another two studies in which incidence of CAG could be estimated by the given age groups [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF][START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF]. In two serological studies from Japan incidence was further estimated by gender, with males (3.2% per year) having higher CAG incidence compared to females (2.4% per year) in one of the studies [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF], whereas no such difference was seen in the other one (annual incidence in males and females: 1.3 and 1.4%, respectively) [START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF]. The study of Ozasa et al. [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF] from Japan was the only study in which incidence of CAG could be estimated by dietary and lifestyle factors. Daily consumption of green-yellow vegetables was associated with an increased incidence of CAG in both men and women. Overall, daily alcohol consumption was also associated with an increased incidence of CAG, however, results were not consistent across genders. Men who smoked daily had a higher incidence of CAG compared to those who did not smoke daily; the reverse was the case in the female study participants. Crude incidence of CAG according to these risk factors (daily smoking, daily alcohol consumption and daily consumption of green yellow-vegetables) is given as footnotes in Table 1.

In 7 studies, incidence rates could be estimated according to H. pylori infection at baseline. In all of these studies, incidence rates were substantially higher in H. pylori positive subjects (up to 8.1% per year) compared to the H. pylori negatives (up to 1.8% per year). Of the studies that assessed H. pylori infection, only one provided additional data on antibodies against cytotoxin associated gene A (cagA), an established virulence factor of H. pylori.

Among the H. pylori positive population in this study, incidence was substantially higher in subjects who showed antibodies against cagA (4.3% per year) compared to those who were cagA-negative (2.5% per year) [START_REF] Kuipers | Helicobacter pylori and atrophic gastritis: importance of the cagA status[END_REF].

Estimates of the impact of H. pylori infection on the incidence of CAG are shown in Fig. 2. The forest plot visualizes results of a meta-analysis conducted among all those studies which provided information on H. pylori infection of the study participants at baseline. The test for heterogeneity resulted in I 2 = 18.4 (Q-test: P [ 0.2) and there was no indication of publication bias according to both Egger's weighted regression model and Begg's rank correlation method (P [ 0.1). Rate ratios comparing the incidence of CAG in H. pylori positive subjects to the incidence in H. pylori negative subjects ranged from 2.4 to 7.6 (with an outlier at 95.5). In a sensitivity analysis excluding the study with a rate ratio of 95.5 [START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF], the summary estimate was only slightly lower [4.5, 95% confidence interval (CI): 2.9-7.0]. We furthermore, performed a meta-analysis including only study populations made up of patients with gastric complains or diseases in which CAG was diagnosed exclusively by gastroscopy. In this analysis, heterogeneity was further reduced [I 2 = 9.5 (Qtest: P [ 0.4)] and the summary estimate [6.1 95% CI (3.4-10.9)] was similar to the one obtained when all the studies were included.

Discussion

Overall, fourteen studies with data on the incidence of CAG could be identified. Among these, incidence rates varied widely, ranging from 0 to 10.9% per year. Substantially higher incidence rates were consistently observed in H. pylori positive participants compared to H. pylori negatives. The meta-analysis on the association between H. pylori infection and incidence of CAG resulted in a summary estimate of 5.0 (95% confidence interval: 3.1-8.3). Patients who had undergone proximal gastric vagotomy, as well as patients who have reflux esophagitis and who were on long-term omeprazole treatment showed a remarkable high incidence of CAG.

The much higher incidence rates of CAG that were observed in H. pylori positive individuals compared to H. pylori negative individuals and the according rate ratios observed in the meta-analysis are consistent with findings from a number of cross-sectional studies which disclosed a strong association between H. pylori infection and CAG [START_REF] Weck | Association of Helicobacter pylori infection with chronic atrophic gastritis: meta-analyses according to type of disease definition[END_REF]. Similarly, the H. pylori specific virulence factor cagA has consistently been shown to be associated with an increased risk of CAG in cross-sectional studies. In this review, only one of the studies took cagA seropositivity into account and within the group of H. pylori positive individuals, those who did not show antibodies against cagA showed a much lower incidence than those who were cagA-positive [START_REF] Kuipers | Helicobacter pylori and atrophic gastritis: importance of the cagA status[END_REF]. Even though these results have to be interpreted with caution due to the small sample size of the study, they support the importance and impact of cagA in the pathogenesis of H. pylori induced CAG.

The highest incidence of CAG was observed in a cohort of patients treated by vagotomy for juxtapyloric ulcer [START_REF] Jonsson | Histologic changes in the gastroduodenal mucosa after long-term medical treatment with cimetidine or parietal cell vagotomy in patients with juxtapyloric ulcer disease[END_REF]. High incidence rates of CAG were furthermore, observed in cohorts of H. pylori positive patients treated with omeprazole for reflux esophagitis [START_REF] Kuipers | Atrophic gastritis and Helicobacter pylori infection in patients with esophagitis treated with omeprazole or fundoplication[END_REF][START_REF] Klinkenberg-Knol | Long-term omeprazole treatment in resistant gastroesophageal reflux disease: efficacy, safety, and influence on gastric mucosa[END_REF][START_REF] Lundell | Changes of gastric mucosal architecture during long-term omeprazole therapy: results of a randomized clinical trial[END_REF]. This finding might be explained by the joint impact of long-term H. pylori infection and omeprazole therapy on lowering of the gastric acidity which makes the gastric mucosa more prone to atrophy. Furthermore, even though, the overall incidence of CAG in this group of patients was remarkably high, CAG incidence was lower and comparable to that of other study populations without such treatment in the H. pylori negative study participants, leading us to presuppose that infection with the bacterium plays a major role for the development of CAG in this group of patients.

Even though H. pylori can survive the acidic gastric environment, due to its ability to break down urea to carbon dioxide and ammonia by urease activity, it has been shown to have an optimal growth at neutral pH [START_REF] Sidebotham | How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in vitro studies[END_REF][START_REF] Burne | Bacterial ureases in infectious diseases[END_REF]. In H. pylori positive patients, treatment with omeprazole or proximal gastric vagotomy reduces the gastric acidity and might, enhance bacterial growth. Bacterial overgrowth might be a key factor in the development of atrophy in such patients and therefore, maybe this higher incidence of CAG seen in this group of patients. Therefore, eradication of H. pylori infection, which has been shown to be able to reverse gastritis without exacerbation of reflux disease, maybe particularly important in this group of patients [START_REF] Kuipers | Cure of Helicobacter pylori infection in patients with reflux oesophagitis treated with long term omeprazole reverses gastritis without exacerbation of reflux disease: results of a randomised controlled trial[END_REF].

A number of limitations of this systematic review have to be kept in mind. Only studies published in English as full articles were included. Possibly, additional results have been presented at conferences and published as meeting proceedings. However, very few follow-up studies on the incidence of CAG have been conducted so far and it seems rather unlikely that further relevant studies exclusively reported in abstract form exist from which appropriate details on incidence could be derived.

Several other aspects have to also be considered when interpreting the study results. The determination of CAG requires specific attention. Even though, almost all of the studies included in this review assessed CAG by histological assessment of biopsy specimen of the gastric mucosa, which is considered the gold standard for clinical diagnoses of CAG, it should be noted that incidence of CAG was estimated solely on the corpus or fundus mucosa and did not take into account the antral mucosa (where data were separately given for each). It has been suggested, that the antral mucosa might be more prone to regressive CAG changes and that atrophy of the body mucosa might be associated with healing of the antrum [START_REF] Ihamaki | The sequelae and course of chronic gastritis during a 30-to 34-year bioptic followup study[END_REF]. On the other hand, atrophy has been reported to start in the antrum and to spread proximally (thus leading to an increased incidence in the antrum compared to the corpus) [START_REF] El-Zimaity | Patterns of gastric atrophy in intestinal type gastric carcinoma[END_REF]. In the two Japanese studies, different cut points of serum PG levels were used to define CAG [START_REF] Ozasa | Helicobacter pylori infection and atrophic gastritis: a nested case-control study in a rural town in Japan[END_REF][START_REF] Xie | Serum pepsinogen levels in the Japanese population: prospective study of 9 years of follow-up[END_REF]. This difference might explain the variation of the reported incidence by a factor of more than 2 (lower incidence in the study with the more stringent definition of disease), despite the similarity in setting, age range and recruitment years of the study populations. These findings underline the importance of the choice of the cut-off level [START_REF] Weck | Epidemiology of chronic atrophic gastritis: population-based study among 9444 older adults from Germany[END_REF][START_REF] Brenner | Epidemiologic findings on serologically defined chronic atrophic gastritis strongly depend on the choice of the cutoff-value[END_REF].

The main limitations regarding existing evidence are related to the heterogeneity of the existing studies. For example, the use of different diagnostic methods for assessment of CAG hampers direct comparisons between the studies. Similarly, H. pylori infection was assessed by different methods. As sensitivity and specificity of the different tests used to determine infection vary, misclassification of infection status might occur, leading to underestimation of the impact of H. pylori on the development of CAG. Most of the studies that could be included in the review showed rather small sample sizes. Additionally, only one of the study populations was sampled from the general population and most participants were lost to follow-up. Some studies had a rather short follow-up time and information on incidence according to potential risk factors other than H. pylori was lacking almost entirely. Despite these limitations, there is consistent evidence that incidence of CAG is very low in the absence of H. pylori infection. These findings are consistent with a major role of H. pylori infection suggested previously by cross-sectional studies. Proximal gastric vagotomy and long-term treatment with omeprazole of H. pylori positive patients with reflux esophagitis might further increase the risk to develop CAG, requiring eradication of the infection. However, this review also clearly shows the lack of largescale and population-based studies on the incidence of CAG. Ideally, studies could be designed in such a way that they allow for assessment of international variation as well as temporal trends. Using non-invasive tests (such as serum PG concentrations) [START_REF] Iijima | Serum biomarker tests are useful in delineating between patients with gastric atrophy and normal, healthy stomach[END_REF] for the diagnosis of CAG might greatly enhance possibilities to conduct such studies in population-based (asymptomatic) study samples. In order to further clarify the role of age, gender, socio-demographic factors, diet and lifestyle factors, such as smoking on the incidence of CAG future follow-up studies should address these, as well as the role of any other potential risk factors.

Table 1

 1 Summary of follow-up studies with data on the incidence of chronic atrophic gastritis

Table 1 continued

 1

	Comments/further subgroup analyses						Blind suction endoscopy at baseline,	visual endoscopy at follow-ups							Incidence of CAG per year	Sex [% (95% CI)]	male 3.2 (2.1-4.8)	female 2.4 (1.8-3.3)	Age group [% (95% CI)]	35-54 2.1 (1.2-3.4)	55-74 3.0 (2.2-4.0)	75 ? 2.5 (0.8-6.0)	Patients on anti-acids, H 2 -blockers,	proton pump inhibitors				Patients were successfully treated for	H. pylori infection before	recruition into the study, incidence	was estimated only for patients	followed-up for up to 2 years due	to loss to follow-up
	Incidence of CAG per year	[% (95% CI)]	Overall and by infection with	Helicobacter pylori		Total 0.8 (0.5-1.2)	Hp-0.4 (0.1-1.2)	Hp? 0.9 (0.5-1.3)		Total 4.5 (2.3-7.8)	Hp-(-)	Hp? (-)		Total 2.7 (2.1-3.4)	Hp-1.1 (0.5-2.0)	Hp? 3.6 (2.7-4.6)							Total 4.2 (2.4-6.9)	Hp-1.8 (0.5-4.8)	Hp? 8.1 (4.3-14.1)			Total (-)	Hp-0.0 (0.0-3.3)	Hp? (-)
	Determination	of CAG					Histology				Histology				Serology	PGI B 70 ng/	mL	&, PGI/PGII	B 3					Histology					Histology
	Years of follow-up (mean)	Dates of assessments	(baseline, follow-up)				32 (32)	1952, 1983			10-19 (14.5)	1972-1974,	(-)		5 (5)	1987, 1992									7-19 (12.3)	1970, 1992				2 (2)	1990-1993,	(-)
	Sample size a (m/f)	Population type	Age range at baseline	(mean or median)	Incident cases	89 (-/-)	Out-patients	16-55, 35	22	17 (-/-)	Gastric ulcer patients	28-76; (-)	11	464 (143/321)	Annual health examination	35-75?, (-)	62					27 (-/-)	Patients with gastric	diseases, private practice	(38-84), 64	14	45 (-/-)	Patients with duodenal	ulcer (severe or	complicated),	Hospital-based	19-78, 45	0
	Country						Finland				Estonia				Japan										USA					Slovenia
	First author	Year of	publication		Reference		Valle	1996	[15]		Maaroos	1998	[16]		Ozasa b	1999	[17]							Carter c	1999	[18]			Tepes	1999	[19]

b Additional results from the study of Ozasa et al. [17]; incidence of CAG per year according to: Daily smoking [% (95% CI)]: no: c Incidence of CAG in H. pylori positive and negative patients is estimated according to infection at baseline and d Might include in the Dutch sample of the study population the Dutch study population of reference 14

Study RR 95% CI H. pylori +* H. pylori -*

  Meta-analysis on the association between H. pylori and incidence of chronic atrophic gastritis (CAG). * Incident CAG-cases per personyears among the group of H. pylori positive and H. pylori negative study participants, respectively. Abbreviation: CI confidence interval, RR rate ratio

	Fig. 2				Rate ratio and 95% CI
	Valle 1996	2.4 0.6 -10.1 20/2304	2/544
	Kuipers 1995	7.6 1.8-33.2 16/506	2/483
	Kuipers 1996	6.8 1.6-29.5 18/500	2/380
	Carter 1999	4.6 1.3 -16.4 11/136	3/170
	Ozasa 1999	3.3 1.6-6.6	53/1490	9/830
	Klinkenberg-Knol 2000 6.7 2.7-16.3	24/507	6/845
	Lundell 2006	95.5 5.5-1671.3 7/154	0/980
	Summary estimate	5.0 3.1-8.3
				0.01	0.1	1	10	100

Acknowledgements This work was supported by a grant from the Baden-Wu ¨rttemberg State Ministry of Research, Science and Arts. The work of Gao Lei was supported by a scholarship from the German Academic Exchange Service.