|Ahead of print publication
Is there any relationship between Helicobacter pylori infection and human epidermal growth factor receptor 2 expression in gastric cancer?
Efnan Algin1, Meltem Baykara2, Guldal Yilmaz3, Bulent Cetin4, Ozgur Ekinci3, Aytug Uner5, Ahmet Ozet5
1 Department of Medical Oncology, Ankara City Hospital, Sakarya, Turkey
2 Department of Medical Oncology, Sakarya Education and Research Hospital, Sakarya, Turkey
3 Department of Pathology, Gazi University Faculty of Medicine, Isparta, Turkey
4 Department of Medical Oncology, Suleyman Demirel University, Faculty of Medicine, Isparta, Turkey
5 Department of Internal Medicine, Division of Medical Oncology, Gazi University Faculty of Medicine, Ankara, Turkey
Department of Medical Oncology, Ankara City Hospital, Ankara 06490
Source of Support: None, Conflict of Interest: None
Purpose: Helicobacter pylori(HP) is a significant causative agent of gastric cancer (GC). However, the underlying mechanisms involved in its pathogenesis and association with oncoproteins are unclear. The aim of the present study was to evaluate the relationship between HP infection and human epidermal growth factor receptor 2 (HER2) expression in GC patients.
Materials and Methods: Surgery (173) or endoscopic biopsy (35) specimen of 208 patients diagnosed with GC was evaluated for the presence of HER2 and HP. HER2 expression was assessed by fluorescence in situ hybridization (FISH) method, whereas HP status was evaluated histologically. Giemsa stain was used to identify HP status, in case HP could not be recognized in routine H and E-stained sections despite careful examination
Results: The median age was 63 years (27–91), and most patients were male (male/female: 149/59). Of all the 208 patients, HP was positive in 87 (41.8%) and negative in 121 (58.2%) patients. FISH positivity for HER2 was observed in 41 (19.7%) patients, whereas FISH negativity was observed in 167 (80.3%) patients. According to the Chi-square test, patient distribution was 21 in HER2-positive HP-negative group, 20 in HER2-positive HP-positive group, 100 in HER2-negative HP-negative group, and 67 in HER2-negative HP-positive group. No correlation was found between HP and HER2 status (P = 0.314). HP positivity had significant effect on median overall survival (27.4 vs. 12.9 months, P = 0.046).
Conclusions: Our results suggest that there is no relationship between HP infection and HER2 status in patients with GC.
Keywords: Fluorescence in situ hybridization, gastric cancer, Giemsa, Helicobacter pylori, human epidermal growth factor receptor 2
|How to cite this URL:|
Algin E, Baykara M, Yilmaz G, Cetin B, Ekinci O, Uner A, Ozet A. Is there any relationship between Helicobacter pylori infection and human epidermal growth factor receptor 2 expression in gastric cancer?. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 21]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269750
| > Introduction|| |
Gastric cancer (GC) is one of the most common cancers. Despite the addition of targeted novel agents to cytotoxic chemotherapy, GC remains the second leading cause of cancer-related mortality. Among these, human epidermal growth factor receptor 2 (HER2) is the most popular target of investigation. HER2 is a transmembrane tyrosine kinase receptor and a member of the human epidermal growth factor receptor (EGFR) family. HER2 has an important role in the regulation of cell proliferation and differentiation and other activities associated with cell survival., The rate of HER2 overexpression in GC varies widely between 2% and 45%.,,,,
Helicobacter pylori (HP) is a Gram-negative bacterium that colonizes the gastric mucosa. In 1994, it has been accepted as a carcinogen by the World Health Organization in the development of GC. It has been demonstrated that HP induces the transcription of multiple host genes. However, little is known about the underlying molecular mechanisms of HP-related tumor development and its association with oncoproteins. Although it has been reported that EGF and EGFR levels increased by HP infection and decreased after eradication, there are conflicting results about the relationship between HP infection and HER2 expression., The limited number of studies suggested that there was no correlation between HP and HER2 in GC patients, whereas Shim et al. reported that HP may induce HER2 overexpression.,,,,
As is known, the ToGA trial reported improved overall survival (OS) with the addition of trastuzumab, a monoclonal antibody targeting the HER2 protein, to standard chemotherapy in patients with advanced-stage HER2-positive GC. Based on the results of this study, testing HER2 status is important to establish treatment strategy in these patients. Defining a biomarker for HER2 amplification and overexpression will be quite exciting. In this context, the present study aimed to investigate the relationship between HP infection and HER2 expression in patients diagnosed with GC. In addition, the association of HP infection with the clinical and pathological parameters and its impact on prognosis were evaluated.
| > Materials and Methods|| |
Between years 2004 and 2013, 208 patients with GC who were treated at the Department of Medical Oncology, Gazi University School of Medicine, were retrospectively reviewed. Samples of all patients were obtained from surgical or endoscopic biopsy materials. Two experienced pathologists assessed the presence of HER2 expression using fluorescence in situ hybridization (FISH) method on adequate tissue samples from the patients, whereas HP status was evaluated histologically. The impact of HP infection on clinical and histopathological characteristics and survival was evaluated.
Helicobacter pylori assessment method
Routine formalin-fixed, paraffin-embedded 173 surgical and 35 endoscopic biopsy specimens from 208 GC patients were reviewed for HP assessment. In the first place, standard H and E-stained gastric tissue sections were evaluated in line with the literature. Of all the 208 patients, 72 were positive for HP, whereas the remaining 136 patients were negative. The histological sections of these 136 HP-negative patients were stained with Giemsa to re-identify HP status. Among them, 15 patients were positive for HP with Giemsa stain. As a result of Giemsa stain, 87 patients were positive for HP and the remaining 121 were negative [Figure 1]a, [Figure 1]b, [Figure 1]c.
|Figure 1: Poorly differentiated gastric adenocarcinoma with signet ring features (H and E, ×200) (a) Helicobacter pylori gastritis on the neighboring surface epithelium (H and E, ×400) (b) A group of Helicobacter pylori within the surface mucin of antral mucosa (Giemsa stain, ×2000) (c)|
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Fluorescence in situ hybridization method
Six-μm-thickness sections were obtained from formalin-fixed, paraffin-embedded tumor samples and mounted on SuperFrost™ +/+ slides. After deparaffinizing, protease digestion procedures were performed following the ABBOTT/Vysis PathVysion HER-2 DNA Probe Kit protocol (PathVysion CE Package Insert, Revision I, 2002), with additional monitoring of the progress of proteinase digestion by propidium iodide staining. After the hybridization of probe mixes at 37°C between 14 and 18 h, the slides were washed in × 2 saline-sodium citrate (SSC)/0.3% NP-40 at 72°C for 30 min, air-dried, and counterstained with diamidino-2-phenylindole (DAPI). A minimum of twenty nuclei of invasive tumor cells were scored by epifluorescence microscopes equipped with × 100 oil immersion objective and DAPI/Spectrum Green/Orange single and triple band-pass filters (AHF). The HER2 signals and the chromosome 17 signals were counted in at least twenty cells. Samples with a HER2/CEP 17 rate ≥ 2 were considered HER2 positive.
All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (version 21.0, SPSS Inc., Chicago, IL, USA). The Chi-square test was used to assess the correlation between HP infection and other parameters. Survival rates were calculated by using Kaplan–Meier survival analysis. OS was identified as the interval between the date of diagnosis and the date of death due to any reason or last visit. The impact of HP infection on survival was evaluated by log-rank test. P <0.05 was considered statistically significant for statistical analysis.
| > Results|| |
The median age of the 208 patients included in the study was 63 years (27–91), and most patients were male (male/female: 149/59). Histopathologic diagnosis was adenocarcinoma in 117 (56.2%) patients, signet ring cell adenocarcinoma in 51 (24.6%), and mixed adenocarcinoma-signet ring cell adenocarcinoma in 40 patients (19.2%). Of all the 208 patients, HP was positive in 87 (41.8%) and negative in 121 (58.2%) patients according to the results of histopathologic examination. FISH positivity for HER2 was observed in 41 (19.7%) patients, whereas FISH negativity was observed in 167 (80.3%) patients.
Pearson's Chi-square test was performed to identify the association between HP and HER2 status. In total, 21 of the 121 (17.4%) HP-negative patients were HER 2 positive and 100 (82.6%) patients were HER2 negative, whereas 20 of the 87 (23%) HP-positive patients were HER2 positive and 67 (77%) were HER2 negative. There was no correlation between HP and HER2 status (P = 0.314). In addition, there was no relationship between HP status and age, gender, histopathologic diagnosis, tumor location, tumor-node-metastasis stage, Eastern Cooperative Oncology Group performance status, grade, lymphovascular invasion, perineural invasion, and Lauren classification. The general patient characteristics and the relationship between these features and HP status are summarized in [Table 1].
|Table 1: General patient characteristics and comparisons of these features according to helicobacter pylori status|
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The median OS of the entire population was 14.8 months (0.07–82.5). The median OS was 12.9 months (95% confidence interval [CI]: 7.7–18.0) in the HP-negative group and 27.4 months (95% CI: 16.4–38.4) in the HP-positive group, and the difference was statistically significant (P = 0.046). Survival curves for HP-positive and HP-negative patients are shown in [Figure 2].
|Figure 2: Kaplan–Meier curve for overall survival according to Helicobacter pylori status|
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| > Discussion|| |
HP has been determined as an etiologic factor of GC development. In the study by Forman, HP antibody was defined in 69% of GC cases, whereas Parsonnet et al. and Nomura et al. reported the concomitance rate of HP and GC to be 84% and 94%, respectively. It is shown that HP increases mucosal cellular proliferation. Nucleolar organizer regions and proliferated cellular nuclear antigen are defined as cellular proliferation markers in the HP-infected mucosa. After HP eradication, these proliferation markers as well as p53 immunoreactivity and EGFR levels return to normal values.,,
Although it is well known that HP infection is a significant causative agent of GC, the underlying mechanisms involved in its pathogenesis remain unclear. The spectrum of genetic alterations in neoplasms may serve clues to the molecular carcinogenesis of HP. For this purpose, the relationship between HP infection and several oncoproteins has been investigated for a long time. Coyle et al. reported that HP plays a major role in EGFR expression, and HP eradication reduces EGF and EGFR levels in gastric mucosal cells. This expression results in HER2 oncogene activities which can lead to GC development.
There are only a limited number of studies investigating the relationship between HP infection and HER2 status. In these studies, no correlation was found between these parameters.,,, In contrast to the literature, Shim et al. found that HER2 overexpression was closely related to HP cytotoxin-associated gene A (CagA) positivity, a bacterial virulence factor, and they concluded that CagA-positive HP may induce HER2 overexpression by increasing HER2 DNA and mRNA copy number. In our study, the largest series in this field to date, we could not find any correlation between HP infection and HER2 status, which is consistent with the majority of literatures. The reason for these conflicting results may be based on HER2 testing methods. Because of the heterogeneous tumor cell distribution in GC, unlike breast cancer, immunohistochemical staining for HER2 expression status can result in false positivities or negativities., Another technique to evaluate HER2 status is FISH method. Different from other studies, Shim et al. detected HER2 overexpression using DNA copy number testing, which is much easier and cheaper than that of FISH. To the best of our knowledge, the present study is the first report investigating the relationship between these parameters using FISH method for HER2 assessment.
The CagA protein of HP is a bacterial virulence factor, which is delivered into gastric epithelial cells via bacterial type IV secretion system. CagA-positive HP strains are known to be more virulent. Shim et al. revealed a positive correlation between HER2 amplification and CagA-positive HP, whereas other studies, including our study, could not find any relationship. The reason for these different results may be related to CagA positivity and CagA may be a biomarker for HER2 amplification. Further studies on large sample sizes are necessary to clarify the relationship of HER2 expression to CagA-positive and CagA-negative HP in GC.
Debate on the clinicopathologic features of HP-induced GC continues. It has been reported that HP plays an important role in the development of intestinal type and distal forms of GC. However, recent studies suggested that there was no difference in HP seroprevalence between the majority of intestinal and diffuse types as well as proximal and distal forms of GC patients. Similar with these recent studies, we did not find any relationship between HP status and these parameters.,
HP-negative patients with GC were reported to have poor prognosis, and it was recommended that these patients should be closely followed up after surgery.,, The reason for this difference is not clear. Meimarakis et al. hypothesized that tumor-specific immune responses might be downregulated in HP-negative patients, whereas Leung et al. reported that microsatellite instability is strongly related to HP status. Consistent with these studies, we found that HP-negative GC patients had significantly longer OS than HP-positive patients (median OS, 27.4 months vs. 12.9 months, P = 0.046). Finally, it is known that the eradication of HP infection is an effective strategy for the prevention of GC. The most important limitation of our study was the uncertainty in the past history of HP-related gastric diseases and eradication treatment, due to retrospective design.
| > Conclusions|| |
We found that there was no correlation between HER2 expression and HP. However, debate on this topic seems to continue. We consider that the relationship between HER2 status and virulence factors of HP should be clarified and for this reason, comprehensive prospective trials with higher number of patients are required.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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