The age variation of HER2 immunohistochemistry positive rate in biopsy specimens of gastric cancer

Journal Pre-proof The age variation of HER2 immunohistochemistry positive rate in biopsy specimens of gastric cancer Yanfeng Xi (Resources) (Investigation) (Data curation) (Visualization) (Writing - original draft), Chen Xu (Resources) (Investigation) (Data curation) (Visualization) (Software) (Writing original draft), Yiqiang Liu (Resources) (Investigation) (Data curation), Xiaochu Yan (Resources) (Investigation) (Data curation), Chuansheng Huang (Resources) (Investigation) (Data curation), Yueping Liu (Resources) (Investigation) (Data curation), Jinhong Mei (Resources) (Investigation) (Data curation), Zhe Wang (Resources) (Investigation) (Data curation), Bin Liu (Resources) (Investigation) (Data curation), Xiaoming Li (Resources) (Investigation) (Data curation), Wencai Li (Resources) (Investigation) (Data curation), Jianyun Lan (Resources) (Investigation) (Data curation), Peng Gao (Resources) (Investigation) (Data curation), Jifeng Wu (Resources) (Investigation) (Data curation), Jianming Zheng (Resources) (Investigation) (Data curation), Yingyong Hou (Methodology) (Supervision) (Writing - review and editing)

PII:

S0344-0338(19)32313-1

DOI:

https://doi.org/10.1016/j.prp.2020.152882

Reference:

PRP 152882

To appear in:

Pathology - Research and Practice

Received Date:

19 October 2019

Revised Date:

23 January 2020

Accepted Date:

12 February 2020

Please cite this article as: Xi Y, Xu C, Liu Y, Yan X, Huang C, Liu Y, Mei J, Wang Z, Liu B, Li X, Li W, Lan J, Gao P, Wu J, Zheng J, Hou Y, The age variation of HER2 immunohistochemistry

positive rate in biopsy specimens of gastric cancer, Pathology - Research and Practice (2020), doi: https://doi.org/10.1016/j.prp.2020.152882

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

The age variation of HER2 immunohistochemistry positive rate in biopsy specimens of gastric cancer Yanfeng Xi1#, Chen Xu2#, Yiqiang Liu3, Xiaochu Yan4, Chuansheng Huang5, Yueping Liu6, Jinhong Mei7, Zhe Wang8, Bin Liu9, Xiaoming Li10, Wencai Li11, Jianyun Lan12,

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Peng Gao13, Jifeng Wu14, Jianming Zheng15, Yingyong Hou2*

Department of Pathology, Shanxi Cancer Hospital, Taiyuan, China.

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Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai,

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China.

Department of Pathology, Beijing Cancer Hospital, Beijing, China.

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Department of Pathology, Southwest Hospital, Chongqing, China.

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Department of Pathology, Jiangxi Cancer Hospital, Nanchang, China.

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Department of Pathology, Fourth Hospital of Hebei Medical University,

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Shijiazhuang, China. 7

Department of Pathology, The First Affiliated Hospital of Nanchang

University, Nanchang, China.

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Department of Pathology, Xijing Hospital, The Fourth Military Medical

University, Xi'an, China. 9

Department of Pathology, Lanzhou General Hospital of People's Liberation

Army, Lanzhou, China. Department of Pathology, Lanzhou University Second Hospital, Lanzhou,

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China.

Department of Pathology, The First Affiliated Hospital of Zhengzhou

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Department of Pathology, Yancheng City No.1 People's Hospital, Yancheng,

China.

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University, Zhengzhou, China.

Department of Pathology, Qilu Hospital of Shandong University, Jinan, China.

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Department of Pathology, The First Affiliated Hospital of Anhui Medical

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University, Hefei, China 15

Department of Pathology, Changhai Hospital, Shanghai, China.

# Yangfeng Xi and Chen Xu contributed equally to this work.

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*Correspondence: Dr. Yingyong Hou Department of Pathology, Zhongshan hospital, Fudan University, Shanghai, 20032, China Tel: +86-21-64041990, Fax: +86-21-64038038

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E-mail: [email protected]

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Abstract

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Aims: The aim of this study was to explore HER2 status and characteristics in biopsy specimens of gastric cancer (GC) in Chinese population.

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Methods and results: A total of 27787 biopsy specimens of GC from 103 hospitals were obtained. Immunohistochemistry (IHC) staining of HER2 was

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performed. Overall HER2 IHC positive rate was 11.2%. HER2 positive rate elevated with the increase of age in total patients and both genders. The rates were 7.1%, 8.1%, 9.0%, 10.9%, 11.8%, 12.6%, and 12.1% when patient age was ≤30, 31-40, 41-50, 51-60, 61-70, 71-80, and >80, respectively (P<0.001). In male,

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the rates were 6.5%, 8.4%, 9.6%, 11.5%, 12.4%, 13.3%, and 12.1% (P<0.001). In female, the rates were 7.4%, 7.9%, 8.0%, 9.0%, 9.6%, 10.6%, and 11.9% (P=0.128). The changes in male was more dramatic than in female (P<0.001). Furthermore, the proportion of the intestinal type GCs increased with age in total

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patients and both genders (P<0.001), and in male the changes were more dramatic (P<0.001). While the proportion of the diffuse type showed the

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opposite tendency to that of the intestinal type (P<0.001). HER2 IHC positive rate

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showed a positive correlation with the proportion of the intestinal type (r=0.986,

(r=0.984, P<0.001).

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P<0.001), and a negative correlation with the proportion of the diffuse type

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Conclusions: The HER2 IHC positive rate showed age variation in biopsy specimens of GC. In male the variation was more dramatic than in female. The

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variation of HER2 positive rate can be attributed to the age variation of the Lauren subtypes. Key words: gastric cancer, HER2, immunohistochemistry

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Introduction Gastric cancer (GC) is the fifth most common malignancy worldwide[7, 25]. In 2012, there were 952,000 new cases and 723,000 deaths[25]. In Chinese

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population, it is the second and the third most common cancer among male and female respectively, and the second leading cause of cancer death for both

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genders[5].

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As a pivotal predictive biomarker in GC, Human epidermal growth factor

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receptor 2 (HER2) is currently the only biomarker to select eligible patients for the molecular targeted therapy[31]. Since the ToGA trial, GC specific HER2

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testing protocols and guidelines have been standardized these years[2, 3, 22]. Both endoscopic biopsy and resected specimens are suitable for HER2

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analysis[3]. And for inoperable patients, biopsy is the only approach to obtain tumor tissues. Many studies have shown that HER2 status of biopsy specimens are highly consistent with that of resected specimens[14, 20, 26]. Several approaches including immunohistochemistry (IHC), fluorescence in

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situ hybridization (FISH), and silver in situ hybridization (SISH) have been used in HER2 assessment[8, 23]. Among them, IHC is an robust and effective method to reflect HER2 status suitable for most kinds of specimens[6, 23], and should be performed first in the assessment[3].

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Based on these methods, expression characteristics of HER2 have been studied extensively in GC. The studies showed that the HER2 positive rate varied in

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different regions and races[16]. China has the largest number of GC patients;

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therefore, it is important to explore HER2 status and characteristics in Chinese

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population. Up to now, there were only several related studies in Chinese population with relatively small patient number[9, 13, 27]. Studies with large

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sample number are needed to reflect the actual status of HER2 in China. Therefore, the current large-scale multi-center study was launched to explore the

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HER2 status and its characteristics in Chinese population.

Materials and Methods Study design

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The multicenter retrospective observational study was launched by the Chinese Anti-cancer Association (CACA). The study was conducted in 103 hospitals (local laboratories) across China. The research protocol was approved by the ethics board of each hospital. These hospitals were divided into 3 regions,

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namely the East region (44 hospitals), the north region (28 hospitals), and the South region (31 hospitals). One of the hospitals in each region was chosen as the

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central laboratory. In this study, biopsy specimens of GC and clinical data were

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obtained from February 2013 to June 2014. Based on the study design, cases

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diagnosed as adenocarcinoma were included, and those with the following conditions were excluded: special subtypes (adenosquamous carcinoma,

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squamous carcinoma, hepatoid adenocarcinoma, and carcinoma with lymphoid stroma, and neuroendocrine tumors), and having received neoadjuvant therapy

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before biopsy. Formalin-fixed paraffin-embedded (FFPE) samples were assessed for HER2 status using IHC staining. All the HER2 IHC sections were reviewed by two independent observers, and cases with discrepant HER2 results were further verified by a discussion panel including 3 observers. Ten percent of the cases

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were randomly selected in each hospital and were reviewed in the central laboratory of each region. Data of HER2 from the local laboratories with a concordance rate between the local and central laboratories ≥ 90% was adopted. Key clinical parameters including gender, age, Lauren classification[4],

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differentiation, histological subtypes[4], tumor location[15], number of tumor

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containing biopsy pieces (tumor pieces) [1, 27] were collected.

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Immunohistochemistry

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In all the laboratories, HER-2/neu (4B5) rabbit monoclonal antibody (Ventana Medical Systems, Inc., Tucson, AZ) was used, and the IHC staining was performed

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with iView DAB Detection Kits (Ventana, Tucson, AZ) on BenchMark XT automated platform (Ventana Medical Systems, Inc., Tucson, AZ). For each test,

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positive and negative controls were routinely used. HER2 results were evaluated based on the established scoring system for biopsy specimens of GC[12, 17, 22]. Briefly, no reactivity or no membranous reactivity in any tumor cell was recorded as 0; tumor cell cluster (≥5 cells) with a faint/barely perceptible membranous

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reactivity irrespective of percentage of tumor cells stained was recorded as 1+; tumor cell cluster (≥5 cells) with a weak to moderate complete, basolateral, or lateral membranous reactivity irrespective of percentage of tumor cells stained was recorded as 2+; tumor cell cluster (≥5 cells) with a strong complete,

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basolateral, or lateral membranous reactivity irrespective of percentage of tumor

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cells stained was recorded as 3+.

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Statistics

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χ2 test was used for univariate analysis; cross-tabulations with qualitative variables were analyzed with the Pearson χ2 test. Covariance analysis was used

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to compare the age variation between two groups. Spearmen analysis was used to evaluate the correlation between two factors.

Multivariate general linear

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model was used to analyze possible parameters that may affect HER2 positive rate. P value <0.05 was defined as statistically significant. No adjustments were made. All analyses were performed using the statistical package SPSS version 22.0 (SPSS, Inc, an IBM Company, Chicago, IL).

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Results Patient characteristics The distribution of the 103 hospitals was shown in Figure.1. A total of 27787

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biopsy specimens were obtained. Characteristics of the patients were shown in Table 1. There were 20347 males and 7440 females with a male to female ratio of

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2.73:1. The patient age ranged from 15 to 99 years old, with a median of 62 years

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old and a mean of 61.6 years old. The number of patients between 61 and 70

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years old was the largest (8941, 32.2%). Information of tumor location, histological subtypes, and Lauren subtypes was obtained in 14578, 9594 and

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14664 patients, respectively (Table 1).

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HER2 status

HER2 IHC staining was performed in all the 27787 patients. HER2 status was

shown in Table 1 and Figure.2A. The IHC positive rate of HER2 in male (11.9%) was higher than that in female (9.2%) (P<0.001) (Figure.2B). With regard to

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Lauren classification, the positive rate of intestinal type tumors was the highest (17.4%), followed by the mixed type tumors (10.0%), and the diffuse type demonstrated the lowest HER2 IHC positive rate (4.3%) (P<0.001) (Figure.2B). As to tumor location, HER2 IHC positive rate of tumors located in the EGJ was the

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highest (15.7%), followed by the tumors located in the upper third of the stomach (U) (13.3%), the middle third of the stomach (M) (10.5%), and the

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lower third of the stomach (L) (9.4%) (P<0.001) (Figure.2B). We further

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compared the HER2 IHC positive rate based on the number of tumor containing

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biopsy pieces. The rate of the cases with ≥4 tumor pieces (12.3%) was higher than that of those with <4 tumor pieces (10.3%) (P<0.001) (Fig.2B). With regard

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to region, the HER2 IHC positive rate of the east region (11.7%) was slightly higher than that of the north region (10.8%) and the south region (10.8%)

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(P=0.047) (Figure2. B).

The age variation of HER2 IHC positive rate The IHC positive rate of HER2 increased with age in total patients and both

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genders (P<0.001)(Figure.3A, Table 2).

The increase of male in the positive rate

was more obvious than that of female (P<0.001) (Figure.3A). In the age subgroup of 51-60, 61-70, and 71-80, the HER2 IHC positive rate of male was significantly higher than that of female (P=0.003, P<0.001, and P=0.01, respectively).

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We next evaluated the changes of the HER2 IHC positive rate with age based on the number of tumor containing pieces. When the number was <4, the

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positive rates of the age subgroups were 6.2%, 7.3%, 8.1%, 9.7%, 11.1%, 11.7%

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and 11.7% (P<0.001) (Figure.3B). When the number was ≥4, the rates were 8.1%,

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9.2%, 10.2%, 12.5%, 12.6%, 13.9%, and 12.6% respectively (P=0.005) (Figure.3B). In both sets, HER2 IHC positive rate showed similar elevation with

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the increase of age.

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The age variation of Lauren subtypes To find out the possible reason leading to the elevation of the HER2 IHC

positive rate with the increase of age, we assessed the changes of Lauren subtypes with age (Figure.4, Table 3). In total patients and both genders, the

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proportion of intestinal type GCs increased with age increasing (P<0.001) (Figure.4A). Although both sexes showed the age dependent changes in the proportion, the elevation in the male was more obvious than that in female (P<0.001)(Figure.4A). In age subgroups of 31-40, 41-50, 51-60, 61-70, and 71-80,

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the proportion of intestinal type was much higher in male than in female (P=0.01, P<0.001, P<0.001, and P=0.005, respectively).

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Conversely, the proportion of the diffuse type GCs decreased with the increase

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of age in total patients and both genders (P<0.001) (Figure.4B, Table.3). The

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decrease of the proportion in male was more obvious than in female (P<0.001). In age subgroups of 41-50, 51-60, 61-70, and 71-80, the proportion of the diffuse

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type was much lower in male than in female (P=0.01, P<0.001, P<0.001, and P=0.001).

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Unlike the intestinal type and the diffuse type, the proportion of the mixed type did not show significant changes with age in total patients (P=0.792) and both genders (P=0.518 in male and P=0.659 in female) (Figure.4C).

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The associations between the HER2 IHC positive rate and Lauren subtypes We further analyzed the correlation between the HER2 IHC positive rate and the Lauren subtypes of different age groups. The proportion of the intestinal type GCs was positively correlated with the HER2 IHC positive rate (r=0.986, P<0.001)

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(Figure. 5A). and the proportion of the diffuse type GCs was negatively related with the HER2 IHC positive rate (r=0.984, P<0.001) (Figure. 5B).

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To rule out other factors that may affect the HER2 IHC positive rate, we next

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compared the rate based on the gender and age in each Lauren subtype including

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the intestinal type, the diffuse type and the mixed type. The results showed that in each Lauren subtype; the IHC positive rate did not exhibit statistical

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differences between genders or among the age subgroups (Table 4).

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Multivariate analysis of the relationships between HER2 positive rate and clinicopathological parameters To further analyze the relationships between HER2 positive rate and clinicopathological parameters including age, location and Lauren classification,

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multivariate analysis was also used (Table.5). The results indicated that age was not directly associated with the HER2 positivity (P=0.731), to the contrary, Lauren classification and tumor location demonstrated statistical association with HER2 positive rate (P<0.001 and P=0.001, respectively), indicating that age

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was not the direct cause of the age disparity of HER2 positive rate.

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Discussion

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To our knowledge, this study has the largest sample number among the

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studies related to HER2 in GC in the English literatures. Biopsy specimens were used in the study because, on one hand, they had been proved to be able to

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reflect actual HER2 status of GC [19, 20, 26], on the other hand, they were available in both the operable and inoperable patients, therefore, unlike resected

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specimens, they can represent HER2 status of late-stage GCs with inoperable lesions. Besides, biopsy specimens, due to their small size and instant fixation after biopsy, may minimize the discrepancies of HER2 caused by the inconsistent pre-test procedures in different hospitals [3].

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In the study, the IHC positive rate of HER2 was 11.2% in total patients. The data is close to the available previous studies from China [13, 21, 24, 27, 28]. Consistent with other studies, the HER2 IHC positive rate in the current one was higher in male patients, intestinal type GCs, and GCs located in the

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gastroesophageal junction (GEJ) and U [2, 10, 13, 21]. Since tumor containing piece number has been shown to be a crucial factor to affect HER2 analysis in

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biopsy specimens of GC and 4 was indicated to be an ideal piece number for the

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assessment [1, 27], we further compared HER2 IHC positive rate based on the

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piece number. And it turned out that GCs with ≥4 pieces demonstrated a higher HER2 positive rate than those with <4 pieces. The incidence of GC shows regional

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discrepancies, with most of the high-risk areas located in rural areas in the middle-western part of China [30], and we only identified slight regional

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discrepancies of HER2 positivity in this study. A crucial finding of the study is that HER2 IHC positive rate showed age

variation. The positive rate elevated with the increase of age. Both genders exhibited similar increasing tendency with age, and in male, the variation in

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HER2 positive rate was more dramatic than that in female. A recent multi-country study by Kim W-H et al. also found that HER2 positive rate was related to age and the rate was lower in the young age subgroup[18]. Unlike the study of Kim W-H et al. which quartered the patients according to age, the large

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patient number in this study made it possible to subdivide the patients at 10-year interval.

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To explore the cause leading to the age variation of HER2 positivity, we further

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analyzed distribution of the Lauren subtypes based on age. We found that the

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intestinal type and the diffuse type other than the mixed type showed age variation. The proportion of the intestinal type GCs increased with age, while the

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proportion of the diffuse type GCs showed the opposite tendency. The age variation of the proportion of intestinal type tumors was more obvious in male,

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while the variation of the diffuse type was more obvious in female. Furthermore, the HER2 IHC positive rate was positively correlated with the proportion of intestinal type and negatively correlated with the proportion of the diffuse type. We further analyzed the HER2 IHC positive rate in each Lauren subtype based on

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age and gender, which indicated that the HER2 positive rate did not show age or gender discrepancy within each of 3 Lauren subtypes.

These findings indicated

that the changes of the Lauren subtypes were responsible for the age variation of the HER2 IHC positive rate.

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In this study we revealed that the age variation of the HER2 IHC positive rate was caused by the age variation of the Lauren subtypes in GC.

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discrepancies in the age variation of the Lauren subtypes between the genders

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lead to the gender differences in variation trend of the HER2 IHC positive rate.

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Besides bringing new insights to the understanding of HER2, these findings reconfirmed that the Lauren classification is a crucial clinicopathological factor

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for HER2 assessment in GC. Lauren classification has been shown to be associated with not only the IHC positive rate, but also the prognosis[11, 21],

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even the efficacy to target treatment[29]. Further studies are needed to explore the mechanism underlying the age variation of Lauren subtypes in GC, which will provide new insights to understand the carcinogenesis and progression of GC. Our study has several limitations. First, its retrospective nature. Second, only

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biopsy specimens were analyzed in the study. Due to the limited biopsy tissue and HER2 heterogeneity, false negative results may be got. In the current research, we found that the IHC positive rate with ≥4 tumor containing biopsy pieces was higher than that with <4 pieces, but the age variation was similar in

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both sets. Third, only IHC was used for HER2 assessment in this study. In clinical practice, in situ hybridization is needed for HER2 equivocal cases.

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In conclusion, for the first time we demonstrate the age variation of the HER2

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positive rate in GC. The variation in male is more dramatic than that in female.

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Lauren subtypes.

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The age variation of the HER2 IHC positive rate is due to the age variation of

Abbreviations

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GC: gastric cancer, IHC: immunohistochemistry, GEJ: gastroesophageal junction, FISH: fluorescence in situ hybridization, SISH: silver in situ hybridization

Acknowledgement

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We thank the Committee of Oncopathology of Chinese Anti Cancer Association for the organization of the research. We thank Guimei Qu (Department of Pathology, Yuhuangding Hospital of Yantai, Yantai, China), Qi Sun (Department of Pathology, Nanjing Drum Tower Hospital,

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Nanjing, China), Jingshu Geng (Department of Pathology, Harbin Medical University Cancer Hospital, Harbin, China), Yejun Qin (Department of Pathology,

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Shandong Provincial Hospital, Jinan, China), Jianping Liu (Department of

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Pathology, West China Hospital, Sichuan University, Chengdu, China), Daiqiang Li

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(Department of Pathology, The Second Xiangya Hospital of Central South University, Changsha, China), Guoping Wang (Department of Pathology, Tongji

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Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China), Xiu Nie (Department of Pathology, Union Hospital,

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Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China), Yong Zhao (Department of Pathology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China), Fan Zhang (Department of Pathology, The First Affiliated Hospital of Wannan Medical College, Wuhu, China),

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Yujun Li (Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, China), Xiaojun Zhou (Department of Pathology, Nanjing General Hospital of Nanjing Military Command, Nanjing, China), Jianghua Zhou (Department of Pathology, Xiangya Hospital Central South University, Changsha,

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China), Enshan Han (Department of Pathology, General Hospital of Ningxia Medical University, Yinchuan, China), Wenyong Sun (Department of Pathology,

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Zhejiang Cancer Hospital, Hangzhou, China), Yinghong Yang (Department of

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Pathology, Fujian Medical University Union Hospital, Fuzhou, China), Yinghao Yu

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(Department of Pathology, Fuzhou General Hospital of Nanjing Military Command, Fuzhou, China), Qingkai Yu (Department of Pathology, Henan Cancer

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Hospital, Zhengzhou, China), Changqing Lu (Department of Pathology, The First People's Hospital of Changzhou, Changzhou, China), Jianguo Zhang (Department

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of Pathology, Affiliated Hospital of Nantong University, Nantong, China), Rongge Xing (Department of Pathology, Cangzhou Central Hospital, Cangzhou, China), Xiaowei Qi (Department of Pathology, Affiliated Hospital, Jiangnan University, Wuxi, China), Qiang Liu (Department of Pathology, Renji Hospital, School of

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Medicine, Shanghai Jiaotong University, Shanghai, China), Guoping Ren (Department of Pathology, The First Affiliated Hospital of Zhejiang University, Hangzhou, China), Lingfei Kong (Department of Pathology, Henan Provincial People's Hospital, Zhengzhou, China), Xiaoyan Chen (Department of Pathology,

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Fujian Provincial Hospital, Fuzhou, China), Hongyu Yu (Department of Pathology, Changzheng Hospital, Shanghai, China), Meifu Gan (Department of Pathology,

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Taizhou Hospital of Zhejiang Province, Taizhou, China), Hua Chen (Department of

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Pathology, Qingdao Municipal Hospital, Qingdao, China), Guorong Chen

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(Department of Pathology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China), Zhihong Zhang (Department of Pathology, Jiangsu

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Province Hospital, Nanjing, China), Mei Jin (Department of Pathology, Sir Run Run Shaw Hospital School of Medicine, Zhejiang University, Hangzhou, China),

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Hongwei Guan (Department of Pathology, First Affiliated Hospital of Dalian Medical University, Dalian, China), Yufei Jiao (Department of Pathology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China), Gang Chen (Department of Pathology, Fujian Provincial Cancer Hospital, Fuzhou, China),

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Baocun Sun (Department of Pathology, Tianjin Cancer Hospital, Tianjin, China), Chao Pan (Department of Pathology, Zhongshan Hospital Xiamen University, Xiamen, China), Lingchuan Guo (Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, China), Hualiang Xiao (Department of

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Pathology, Daping Hospital, Research Institute of Surgery Third Military Medical University, Chongqing, China), Xiaolong Jin (Department of Pathology, Ruijin

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Hospital Shanghai Jiao Tong University School of Medicine, Shanghai, China),

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Jianrong Wang (Department of Pathology, Jiangsu Province Hospital of TCM,

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Nanjing, China), Jiannong Wu (Department of Pathology, Affiliated Hospital of Jiangsu University, Zhenjiang, China), Xuenong Li (Department of Pathology,

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Nanfang Hospital, Southern Medical University, Guangzhou, China), Sheng Zhang (Department of Pathology, The First Affiliated Hospital of Fujian Medical

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University, Fuzhou, China), Rong Ying (Department of Pathology, Gansu Provincial Cancer Hospital, Lanzhou, China), Yonghong Shi (Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical University, Huhehaote, China), Dianbin Mu (Department of Pathology, Shandong Cancer

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Hospital and Institute, Jinan, China), Wenbin Huang (Department of Pathology, Nanjing First Hospital, Nanjing, China), Haiping Zhang (Department of Pathology, The First Affiliated Hospital of Xaimen University, Xiamen, China), Jinhui Shen (Department of Pathology, Shantou Central Hospital, Shantou, China), Aijing Sun

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(Department of Pathology, Shaoxing People's Hospital, Shaoxing, China), Lifeng Wang (Department of Pathology, Xinhua Hospital, Shanghai Jiaotong University,

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Shanghai, China), Xiaobing Li (Department of Pathology, The 307th Hospital of

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Chinese People's Liberation Army, Beijing, China), Lirong Chen (Department of

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Pathology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China), Feng Tang (Department of Pathology, Huashan

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Hospital, Fudan University, Shanghai, China), Gang Xu (Department of Pathology, Sichuan Provincial People's Hospital, Chengdu, China), Guoqing Pan(Department

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of Pathology, First Affiliated Hospital of Kunming Medical University, Kunming, China), Zhenzhu Sun (Department of Pathology, People's Hospital of Xinjiang Uygur Autonomous Region, Urumichi, China), Jingping Yun (Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, China), Guoping

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Zhong (Department of Pathology, Yinzhou Hospital Affiliated to Medical School of Ningbo University, Yinzhou, China), Zongkai Zou (Department of Pathology, Zhangzhou Municipal Hospital of Fujian Province, Zhangzhou, China), Ying Wang (Department of Pathology, The Second People's Hospital of Sichuan, Chengdu,

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China), Jiping Da (Department of Pathology, China-Japan Friendship Hospital, Beijing, China), Jing Yuan (Department of Pathology, The General Hospital of

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People’s Liberation Army(301 Hospital), Beijing, China), Guifang Yang

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(Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan,

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China), Xianghong Yang (Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, China), Jinliang Ping (Department of Pathology,

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Huzhou Central Hospital, Huzhou, China), Zhuo Wang (Department of Pathology, Shaanxi Provincial People's Hospital, Xi'an, China), Xiaofeng Li (Department of

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Pathology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China), Hongmin Li (Department of Pathology, Tangshan People's Hospital, Tangshan, China), Qiong Ye (Department of Pathology, Wenzhou Central Hospital, Wenzhou, China), Xueshan Qiu (Department of Pathology, The First Hospital of China

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Medical University, Shenyang, China), Mulan Jin (Department of Pathology, Beijing Chao-yang Hospital, Beijing, China), Quancai Cui (Department of Pathology, Peking Union Medical College Hospital, Beijing, China), Wei Yi (Department of Pathology, Guizhou Provincial People's Hospital, Guiyang, China),

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Haiyan Zhang (Department of Pathology, The 2nd Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China), Bing Liao

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(Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University,

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Guangzhou, China), Xiaolan Xiao (Department of Pathology, Hainan General

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Hospital, Haikou, China), Dawei Ma (Department of Pathology, Jiangsu Cancer Hospital, Nanjing, China), Yuan Luo (Department of Pathology, Affiliated Cancer

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Hospital of Guangxi Medical University, Nanning, China), Hao Chen (Department of Pathology, The First People's Hospital of Lianyungang, Lianyungang, China),

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Hongwen Gao (Department of Pathology, The Second Hospital of Jilin University, Changchun, China), Yulan Li (Department of Pathology, Affiliated Hospital of Hebei University, Baoding, China), Wanxin Wu (Department of Pathology, The First Hospital of Jiaxing, Jiaxing, China), Song He (Department of Pathology,

26

Nantong Tumor Hospital, Nantong, China), Zhihong Liu (Department of Pathology, Hunan Cancer Hospital, Changsha, China), Yunjie Zeng (Department of Pathology, Sun Yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China), and Lifen Wang (Department of Pathology, The Second Hospital of Dalian

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Medical University, Dalian, China) for providing the primary data from their hospitals.

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Author contribution

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Yanfeng Xi and Chen Xu analyzed the data and wrote the paper. Yiqiang Liu,

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Xiaochu Yan, Chuansheng Huang, Yulan Li, Jinhong Mei, Zhe Wang, Bin Liu, Xiaoming Li, Wencai Li, Jianyun Lan, Peng Gao, Jifeng Wu, and Jianming Zheng

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provided data and helped data analysis, Yingyong Hou designed the research.

Author statement

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Yanfeng Xi: Resources, Investigation, Data Curation, Visualization, Writing -

Original Draft

Chen Xu: Resources, Investigation, Data Curation, Visualization,

Softwar, Writing - Original Draft Yiqiang Liu: Resources, Investigation, Data

Curation Xiaochu Yan: Resources, Investigation, Data Curation Chuansheng Huang:

27

Resources, Investigation, Data Curation Yulan Li: Resources, Investigation, Data

Curation Jinhong Mei: Resources, Investigation, Data Curation

Resources, Investigation, Data Curation

Curation

Bin Liu: Resources, Investigation, Data

Xiaoming Li: Resources, Investigation, Data Curation

Jifeng Wu:

-p

Jianming Zheng: Resources, Investigation,

Yingyong Hou: Methodology, Supervision, Writing - Review &

re

Data Curation

Jianyun Lan: Resources, Investigation,

Peng Gao: Resources, Investigation, Data Curation

Resources, Investigation, Data Curation

Wencai Li:

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Resources, Investigation, Data Curation

Data Curation

Zhe Wang:

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Editing .

Conflict of interest statement

The authors declared that they had no conflict of interest to this work.

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Declaration of interests

The authors declare that they have no known competing financial interests or

personal relationships that could have appeared to influence the work reported in this paper.

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Figure legends

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Figure.1 The geographic distribution of the hospitals.

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Figure.2 A. Examples of HER2 IHC staining. Magnification ×400. B. Comparisons of HER2 IHC positive rate based on gender, Lauren classification, tumor location,

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tumor fragment number and region. *P<0.001, #P=0.047.

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Figure.3 The age variation of HER2 IHC positive rate. A. In total patients and both genders, the HER2 IHC positive rate increased with the increase of age (P<0.001). In male the age variation was more obvious than in female (P<0.001). B. In patients with <4 and ≥4 tumor containing biopsy pieces, the HER2 positive rate

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showed similar elevated tendency with the increase of age (P<0.001 and

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P=0.005).

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Figure.4 A. The age variation of the proportion of the intestinal type GCs. In total patients and both genders, the proportion increased with the increase of age

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(P<0.001). In male, the age variation of the proportion was more dramatic than in female (P<0.001). B. The age variation of the proportion of the diffuse type GCs. In total patients and both genders, the proportion decreased with the increase of age (P<0.001). In male the age variation of the proportion was more dramatic

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than in female (P<0.001). C. The age variation of the proportion of the mixed type GCs. The proportion did not show significant changes with age in total patients

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and both genders (P>0.05).

Figure.5 The relationships between HER2 IHC positive rate and Lauren subtypes

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in different age groups. Each plot represents an age group (from left to right: ≤30,

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41-50, 51-60, 61-70, 71-80, and >80). The horizontal axis indicates the

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proportion of intestinal type or diffuse type GCs in each age group. The vertical

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axis indicates the HER2 IHC positive rate of each age group. A. The HER2 IHC positive rate showed a positive correlation with the proportion of the intestinal type GCs (r=0.986, P<0.001). B. The HER2 IHC positive rate showed a negative

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correlation with the proportion of the diffuse type GCs (r=0.984, P<0.001).

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Table.1 Patient characteristics. Clinicopathologic parameters

n (%)

Gender Total

27787 (100)

Male

20347 (73.2)

Female

7440 (26.8) 27787 (100)

20-30

354 (1.3)

31-40

996 (3.6)

41-50

3567 (12.8)

51-60

7257 (26.1)

61-70

8941 (32.2)

71-80

5447 (19.6)

>80

1225 (4.4)

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Total

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Region Total

27787 (100)

South

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Location

12397 (53.9) 8803 (38.5)

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East North

6677 (29.2)

Total

14578 (100)

GEJ

1878 (12.9)

U M

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L

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Age

2704 (18.5) 3420 (23.5) 5573 (38.2)

UML

271 (1.9)

Others

732 (5)

Histological subtypes Total

9594 (100)

Papillary

158 (1.6)

Tubular

5203 (54.2)

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Mucinous

422 (4.4)

Poorly cohesive

2739 (28.5)

Mixed

1072 (11.2)

Grade Total

27787 (100)

Well

1856 (6.9)

Moderate

9791 (36.4)

Poorly

15222 (56.7) 14664 (100)

Intestinal

7114 (48.5)

Diffuse

5158 (35.2)

Mixed

2198 (15.0)

Indeterminate

194 (1.3)

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Total

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Lauren subtypes

Total

27787 (100)

0/1+

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HER2

18524 (66.7)

2+

6155 (22.2) 3108 (11.2)

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Abbreviations:

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3+

GEJ: gastroesophageal junction, U: upper third, M: middle third, L: lower third, papillary

adenocarcinoma,

Tubular:

tubular

adenocarcinoma,

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Papillary:

Mucinous: mucinous adenocarcinoma, Poorly cohesive: poorly cohesive adenocarcinoma, Mixed: mixed adenocarcinoma, Intestinal: intestinal type, Diffuse: diffuse type, Mixed: mixed type, Indeterminate: indeterminate type.

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Table. 2 The age variation of the HER2 IHC positive rate. Total, n (%)

Age

Male, n (%)

Fem

non-3+

P value

3+

non-3+

P value

3+

≤30

25 (7.1)

329 (92.9)

P<0.001

9 (6.5)

129 (93.5)

P<0.001

16 (

31-40

81 (8.1)

915 (91.9)

39 (8.4)

426 (91.6)

42 (

41-50

322 (9.0)

3245 (91.0)

218 (9.6)

2050 (90.4)

104

51-60

791 (10.9)

6466 (89.1)

628 (11.5)

4825 (88.5)

163

61-70

1052 (11.8)

7889 (88.2)

860 (12.4)

6071 (87.6)

192

71-80

689 (12.6)

4758 (87.4)

553 (13.3)

3607 (86.7)

136

>80

148 (12.1)

1077 (87.9)

113 (12.1)

819 (87.9)

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3+

Table. 3 The age variation of the Lauren subtypes. Total, n (%) Age

Male, n (%) Diffuse

Mixed

Diffuse

Mixed

In

≤30

29 (15.6)

125 (67.2)

27 (14.5)

15 (21.4)

46 (65.7)

6 (8.6)

1

31-40

124 (22.8)

340 (62.6)

71 (13.1)

72 (27.8)

151 (58.3)

31 (12.0)

5

41-50

699 (36.8)

897 (47.3)

533 (44.1)

481 (39.8)

177 (14.6)

1

51-60

1831 (47.3) 1397 (36.1) 579 (15.0)

1480 (50.9) 952 (32.8)

421 (14.5)

3

61-70

2457 (52.9) 1411 (30.4) 723 (15.5)

2010 (55.6) 1003 (27.7) 553 (15.2)

4

71-80

1602 (56.0) 800 (28.0)

425 (14.9)

1254 (57.5) 575 (26.4)

328 (15.0)

3

>80

372 (56.3)

97 (14.7)

287 (56.8)

70 (13.9)

8

re 276 (14.5)

lP 188 (28.4)

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Intestinal

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Intestinal

F

144 (28.5)

Table.4 Assessment of HER2 IHC positive rate in each Lauren subgroup based on gender and age.

HER2 positive rate

P value

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(Intestinal type)

Gender

HER2 positive rate P value (Diffuse type)

P=0.33

HER2 positive rate P value (Mixed type)

P=0.357

P=0.871

Male

17.6%

4.5%

9.9%

Female

16.5%

4.0%

10.1%

Age

P=0.136

P=0.949

P=0.541

≤30

13.8%

5.6%

14.8%

31-40

17.7%

4.1%

12.7%

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41-50

14.2%

4.7%

11.2%

51-60

16.8%

3.9%

9.8%

61-70

17.8%

4.6%

8.4%

71-80

19.2%

4.1%

10.4%

>80

16.7%

4.3%

13.4%

Table.5 Multivariate analysis of clinicopathological parameters HER2 positive rate

P<0.001

Intestinal

17.4%

Diffuse

4.3%

Mixed

10.0%

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Lauren subtypes

P=0.001

15.7%

U

13.3%

M

10.5%

L

9.4%

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EGJ

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41-50

P=0.731

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Age

31-40

-p

Location

≤30

P value

7.1% 8.0% 9.0%

51-60

10.9%

61-70

11.8%

71-80

12.6%

>80

12.1%

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Abbreviations:

GEJ: gastroesophageal junction, U: upper third, M: middle third, L: lower third, Intestinal: intestinal type, Diffuse: diffuse type, Mixed: mixed type.

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