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Abstract

OBJECTIVE:

This study aims to investigate the significance of combined detection of HER2 gene amplification and chemosensitivity in gastric cancer.

METHODS:

Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and fluorescence reverse-transcription polymerase chain reaction (RT-PCR) were used to analyze the expression of HER2 protein, HER2 gene amplification and the mRNA expression of ERCC1, TUBB3 and TYMS genes in 135 cases of gastric carcinoma.

RESULTS:

The expression rate of HER2 protein was 39.3% (53/135). Among these positive cases, patients with HER2 protein (3+) accounted for 9.6% (13/135), patients with HER2 protein (2+) accounted for 13.3% (18/135), and patients with HER2 protein (1+) accounted for 16.3% (22/135). The amplification rate of the HER2 gene was 35.8% (19/53). In the detection of the mRNA expression of ERCC1, TUBB3 and TYMS, 45 patients had low and moderate single gene expression, 50 patients had low and moderate double gene expression, 22 patients had low and moderate mRNA expression for ERCC1, TUBB3 and TYMS, and 18 patients had no low and moderate expression. Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received chemotherapy and trastuzumab. Follow-up results revealed that HER2 gene status was positively correlated with the therapeutic effect of the combined treatment in patients with low mRNA expression of ERCC1, TUBB3 and TYMS. Among these patients, five patients with extensive HER2 (3+), HER2 cluster-specific amplification, and low mRNA expression of ERCC1, TUBB3 and TYMS had a total survival of up to 19.1 months.

CONCLUSIONS:

The detection of HER2 in gastric cancer is highly heterogeneity, and the combined detection of HER2 protein expression, HER2 gene amplification and chemosensitivity can provide important reference markers for the benefit of antitumor drugs.

Keywords

Stomach neoplasms HER2 gene HER2 protein FISH IHC fluorescence RT-PCR

1. Introduction

“Precision medicine” has become a hot topic, which advocates treating the same disease with different treatments. Hence, a patient needs different treatment plans at different stages. With the development of molecular pathology, each patient can be accurately typed and treated [1, 2]. In recent years, focus has been directed at signal transduction, growth factors and their receptors, several novel drugs have been developed, and important results have been achieved. One of these protein products is human epidermal growth factor receptor 2 (HER2). Its high expression can activate the signal transduction system of cells, and induce cell transformation and proliferation [3, 4]. HER2 is a widely used therapeutic target in breast cancer [5, 6, 7]. Accumulating evidence [1, 2, 3, 4, 5] suggests that HER2 is an important marker of gastric cancer, and a high HER2 expression is most beneficial to tumor patients [8, 9]. Compared with breast cancer, gastric cancer exhibits unique immunostaining characteristics, and there is high tumor heterogeneity in HER2 positive patients. Hence, standardizing the determination of the expression of HER2 in gastric cancer is a guarantee of correct medication. In addition, chemotherapy is the main treatment option for patients with middle and late stage gastric cancer. In clinical practice, chemotherapeutic agents are blindly selected. As a result, some patients fail to benefit from chemotherapy. Instead, their quality of life is affected by many adverse reactions, and the best time of treatment is missed. The common chemotherapeutic agents for gastric cancer include fluorouracil, platinum-based drugs, and paclitaxel. In the present study, immunohistochemistry (IHC) was used to detect the expression of HER2 protein in gastric cancer, fluorescence in situ hybridization (FISH) was used to detect the status of the HER2 gene, and fluorescence reverse-transcription polymerase chain reaction (RT-PCR) was used to detect the expression of fluorouracil, platinum-based drugs and paclitaxel, in order to evaluate the diagnostic criteria of FISH, IHC and fluorescence RT-PCR for gastric cancer, provide a quantitative reference for the benefit of antitumor drugs, and guide the individualized selection of drugs.

2. Materials and methods

2.1 Materials

A total of 135 cases of radical resection of the stomach and esophagogastric junction were performed in the Pathology Department of the 152nd Central Hospital of Pingdingshan and the 159th Central Hospital of Zhumadian from January 2013 to October 2015, and samples were collected. The histological diagnostic criteria were based on the literature “Stomach Tumor Pathology” and the 2010 WHO Classification of Tumors of the Digestive System. The age of these patients ranged within 25–74 years, with an average age of 62.4 years old. Within 30 minutes after resection from the body, the samples were fixed with freshly prepared 10% neutral buffered formalin (NBF) for 8–48 hours. The fixation fluid-to-tissue volume ratio was 10:1. From the tumor tissues, 4–6 pieces was obtained (the proximal and distal margins were routinely cut, but the tumors, the adjacent gastric mucosa and other tissue blocks were not included), which included the deepest invasion point and the closest serosal layer. The samples were detected by hematoxylin and eosin (H&E), IHC, FISH and fluorescence RT-PCR, respectively.

2.1.1 Reagents

HER2 immunohistochemical staining, HER2 HercepTestTM kit and pharmDxTM kit approved by FDA were purchased from Dako Cytomation (Denmark).

2.1.2 Reagent and probe

Paraffin Pretreatment Kit II, which mainly contains the pretreatment solution and protease solution; PathVysionTM HER2 probe kit; Human ERCC1, TUBB3 and TYMS gene expression relative quantification kit (PCR, fluorescent probe method); ERCC1, TUBB3 and TYMS PCR reaction solution, primer/ probe mixture, positive controls, negative controls, internal PCR reaction solution, internal primer/probe mixture, internal positive control, and internal negative control. The same components between different batches could not be interchanged.

2.2 Methods

2.2.1 Immunohistochemical staining

Interpretation of the positive results of staining: when the cell membrane of tumor cells in all 4–6 slices of one case was unstained, the result was 0; when the membrane of tumor cells was slightly or faintly stained, the result was 1+; when the basal membrane, lateral membrane, or whole membrane of tumor cells were weakly or moderately stained, the result was 2+; when the basal membrane, lateral membrane, or the whole membrane of tumor cells had strongly positive staining, the result was 3+.

Interpretation of the positive area of staining: when the cell membrane of tumor cells in all 4–6 slices in one case was unstained, the result was negative; when the stained area was $\geqslant$ 80% the result was extensive type; when the stained area was within 79–21%, the result was partial type; when the stained area was within $\leqslant$ 20%, the result was focal type.

2.2.2 FISH test

Reagents, probes and ISH procedures: The pretreatment procedures and process steps for the paraffin-embedded gastric cancer tissue sections were carried out according to a related literature [9] and the instructions of the kits.

Determination of FISH results

FISH results were determined mainly through the HER2 positive regions on the sections detected by IHC. First, the positive region of cancer cells was identified on H&E stained sections. Then, the same field of view of H&E staining was found on the FISH slice under a 10 $\times$ objective, and the whole slice was observed under a 40 $\times$ objective. If more than 75% of these cancer cells have hybridization signals, the result was defined as satisfactory. Next, the objective was replaced with a 100 $\times$ objective, and at least 30 isolated, non-overlapping cancer cells with complete margins were counted. HER2 gene status evaluation criteria: Thirty cancer cells were randomly selected, and the ratio of the copy number of HER2 gene to the copy number of chromosome 17 in the nucleus was calculated. When the ratio was $>$ 2.2, the result was positive, suggesting that the HER2 gene was amplified in the sample. When the ratio was $<$ 1.8, the result was negative, suggesting no HER2 gene amplification was found in the sample. If the copy number of the HER2 gene was within 1.8–2.2, 30 other tumor cells were selected from other tumor tissue regions, and the counting was performed again.

2.2.3 Fluorescence RT-PCR

Paraffin sections should contain sufficient proportions of tumor cells, and have no protein or DNA contamination. Samples were processed using a commercially available RNA extraction kit, and the operation was carried out strictly in accordance with kit instructions. For ERCC1, TUBB3 and TYMS positive controls and internal positive controls, calibrated RNA controls did not need to be extracted. Instead, these were melted before use, subjected to vortex-induced oscillation for 10 seconds, centrifuged at 2,000 rpm for 15 seconds, and the number of reactions (the number of the first batch of samples to be inspected $+$ the number of controls; the number of the second batch of samples to be inspected $+$ the number of controls) was determined. Then, 6 $\mu$ l of the volume was taken from the negative control, and the sample was processed. The RNA control and positive control were calibrated and placed into an ERCC1 PCR reaction tube and internal control PCR reaction tube, respectively; and the cover of the tube was tightly fixed. Next, the sample was centrifuged at 2,000 rpm for 15 seconds, allowing all liquid attached to the tube wall to be thrown to the bottom of the tube, avoiding bubbles. The sample was centrifuged again at 2,000 rpm for 15 seconds, and PCR amplification was immediately performed.

Expression level criteria: $<$ 37.5%, low expression; 37.5–62.5%, moderate expression; $>$ 62.5%, high expression.

Sensitive chemotherapies: DDP (C) for low and moderate expression of ERCC1, docetaxel (D) for low and moderate expression of TUBB3, and 5-FU (F) for low and moderate expression of TYMS.

2.3 Statistics analysis

Data were statistically analyzed using statistical software SPSS 13.0. HER2 gene status, HER2 gene amplification, and the mRNA expression levels of ERCC1, TUBB3 and TYMS were compared between the two groups using t-test and chi-square test. P $<$ 0.05 was considered statistically significant.

Table 1
Relationship between the expression of HER2 gene protein and histological type in 135 cases of gastric carcinoma

Type	Case	IHC
		Area/strength	–	1+	2+	3+	Masculine (%)
Papillary adenocarcinoma	25	$\geqslant$ 80%		1	1	2	4 (16.0)
		79–21%		1	2	2	5 (20.0)
		$\leqslant$ 20%		0	1	1	2 (8.0)
		Negative	14				14 (56.0)
Tubular adenocarcinoma	37	$\geqslant$ 80%		2	2	1	5 (13.5)
		79–21%		3	1	2	6 (16.2)
		$\leqslant$ 20%		2	2	1	5 (13.5)
		Negative	21				21 (56.8)
Mucinous adenocarcinoma	23	$\geqslant$ 80%		0	1	0	1 (4.3)
		79–21%		1	1	1	3 (13.0)
		$\leqslant$ 20%		1	1	0	2 (8.7)
		Negative	17				17 (73.9)
Low adhesion carcinoma	11	$\geqslant$ 80%		0	1	0	1 (9.1)
		79–21%		0	1	1	2 (18.1)
		$\leqslant$ 20%		1	0	0	1 (9.1)
		Negative	7				7 (63.6)
Mixed adenocarcinoma	39	$\geqslant$ 80%		4	0	0	4 (10.3)
		79–21%		3	2	1	6 (15.4)
		$\leqslant$ 20%		3	2	1	6 (15.4)
		Negative	23				23 (59.0)
Total	135		82 (60.7)	22 (16.3)	18 (13.3)	13 (9.6)	53 (39.3)

Figure 1.

The positive expression of HER2 protein in gastric adenocarcinoma, En Vision ( $\times$ 40). A: (3+) extensive type; B: (3+) partial type; C: (3+) focus type.

3. Results

3.1 Histomorphology of gastric cancer

Among the 135 patients with gastric cancer, 25 patients had papillary adenocarcinoma (18.5%), 37 patients had tubular adenoma (27.4%), 23 patients had mucinous adenocarcinoma (17%), 11 patients had low adhesion adenocarcinoma (8.1%), and 39 patients had mixed adenocarcinoma (28.9%). Mixed adenocarcinoma refers to the mix of different histological structures of papillary adenocarcinoma, tubular adenocarcinoma, mucinous adenocarcinoma, and low adhesion adenocarcinoma. In the present study, all patients were at stage pT2-4.

3.2 Expression rate and expression characteristics of HER2 protein in gastric cancer

The relationship of the expression rate of HER2 protein and its histological types in gastric cancer is shown in Table 1. The positive expression of HER2 protein was located in the cell membrane, and the expression rate was 39.3% (53/135). Among these patients, three patients were HER2 (3+) extensive type (Fig. 1), seven patients were (3+) partial type, and three patients were (3+) focus type. These patients accounted for 9.6% (13/135). Furthermore, five patients were HER2 protein (2+) extensive type, seven patients were (2+) partial type, and six patients were (2+) focus type. These patients accounted for 13.3% (18/135). Moreover, seven patients were HER2 positive (1+) extensive type, eight patients were (1+) partial type, and seven patients were (1+) focus type. These patients accounted for 16.3% (22/135).

Table 2
The positive intensity and area distribution of HER2 protein

	Generalized	Partial	Focal	Total
	type	type
Mono signal type	11	14	8	33 (62.3)
Multi signal type	4	10	6	20 (37.7)
Total	15 (28.3)	24 (45.3)	14 (26.4)	53 (100.0)

$\Delta$ There is only one type of the generalized type, partial type, focal type in each patient’s 4–6 slices; $\blacktriangle$ Patients with two or more of extensive type, partial type, and focal type in which some of the 4–6 sections of each patient were negative, while some were positive (3+), positive (2+) and positive (1+) for HER2.

Table 3

Comparison of HER2 protein positive and HER2 gene amplification results

HER2 strength	HER2 protein expression		Amplification status and number of HER2 gene
(case)	Range	Case	Cluster	Large granular	Point	High multi-body	Number of amplified cases (%)
3+ (13)	Wide range	3	2	1	1	1	12 (92.3)
	Partial type	7	2	1	1	1
	Focal type	3	0	0	0	0
2+ (18)	Wide range	5	1	1	1	1	6 (33.3)
	Partial type	7	1	1	0	1
	Focal type	6	0	0	0	0
1+ (22)	Wide range	7	0	1	0	0	1 (4.5)
	Partial type	8	1	0	0	0
	Focal type	7	0	0	0	0
Total		53	7 (13.2)	5 (9.4)	3 (5.6)	4 (7.5)	19 (35.8)

Table 4

Relationship between HER2 gene amplification and expression levels of ERCC1, TUBB3 and TYMS genes mRNA

	Case	HER2 Gene status
		Cluster	Large granular	Point	High multi-body
Low expression in single gene	45	2	2	1	1
Double gene, low expression	50	3	2	1	2
The three genes were middle and low expression	22	1	0	1	1
There were no middle and low expression of three genes	18	1	1	0	0
Total	135	7	5	3	4

Figure 2.

Detection of the HER2 gene in gastric cancer using the FISH method. The red area is the probe signal and the green area is chromosome 17 ( $\times$ 1,000). A: Cluster-specific amplification; B: Large particle amplification; C: Spotty amplification.

The significant feature of HER2 protein expression in gastric cancer is heterogeneity. If there was only either an extensive, partial, or focal type in 4–6 sections of one patient, it was defined as single-signal type; and single-signal type patients accounted for 62.3% (33/53). If there was two or more than two of HER2 (3+), HER2 (2+) and HER2 (1+) and the negative types in 4–6 sections of one patient, it was defined as multi-signal type; and multi-signal type patients accounted for 37.7% (20/53, Table 2).

Table 5

Comparison of HER2 gene amplification with chemotherapy alone and trastuzumab in combination with chemotherapy in 11 patients

Grouping	HER2 Gene status 53 cases	The three genes were middle	Average values of PFS and OS
		and low expression	meso-PFS (month)	meso-OS (month)
Joint programme	12	12	9.7	17.2
DCFf scheme	39	8	5.3	12.0

Figure 3.

The mRNA expression of ERCC1, TUBB3 and TYMS. A: Low expression of a single-gene. The low expression of the ERCC1 gene, and the high expression of the TUBB3 and TYMS genes are shown. B: Low expression of a double-gene. The low expression of the ERCC1 and TYMS genes, and the high expression of the TUBB3 gene are shown. C: Low expression of a three-gene. The low expression of the ERCC1, TYMS and TUBB3 genes are shown.

3.3 High copy number of chromosome 17 and HER2 gene amplification and their characteristics

The amplification rate of the HER2 gene was 35.8% (19/53, Table 3). Patients with HER2 protein (3+) accounted for 92.3% (12/13), patients with HER2 protein (2+) accounted for 33.3% (6/18), and patients with HER2 protein (1+) accounted for 4.5% (1/22). Morphological types of HER2 gene amplification in 19 patients: patients with HER2 gene cluster-specific amplifications accounted for 14.3% (7/19, Fig. 2), patients with large granular amplifications accounted for 10.2% (5/19), patients with spotty amplifications accounted for 6.1% (3/19), and patients with high multi-bodies accounted for 8.2% (4/19).

3.4 ERCC1, TUBB3 and TYMS gene mRNA expression and its relationship with HER2 gene amplification

For the mRNA expression of ERCC1, TUBB3 and TYMS, 45 patients had low and moderate single-gene expression (Fig. 3), 50 patients had low and moderate double-gene expression, 22 patients had low and moderate expression of all ERCC1, TUBB3 and TYMS mRNAs, and 18 patients had no low and moderate expression. The relationship between HER2 gene amplification and the mRNA expression of ERCC1, TUBB3 and TYMS genes is shown in Table 4.

3.5 Treatment based on the combined detection of HER2 gene amplification and ERCC1, TUBB3 and TYMS gene mRNA expression levels versus simple chemotherapy regimen

The treatment regimen based on the detection of HER2 gene amplification combined with ERCC1, TUBB3 and TYMS gene mRNA expression levels was called the combination regimen, while the treatment regimen that used chemotherapeutic drugs alone was called simple chemotherapy (chemotherapy regimens C, D and F). Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received chemotherapy and trastuzumab (Table 5).

4. Discussion

In recent years, trastuzumab (trade name Herceptin), a monoclonal antibody targeting HER2 protein, has been verified to improve the survival rate of patients with primary and metastatic HER2 positive breast cancer [10]. In order to select the most suitable patient to receive this treatment, and avoid unnecessary medication for some patients, in 2007, the American Society of Clinical Oncology (ASCO)/American College of Pathologists (CAP) published an updated version of detection guidelines [11] based on the 2007 edition of the ASCO/CAP HER2 detection guideline [12]. This puts forward new requirements for the technical route, interpretation standard of results and quality control of HER2 detection in breast cancer, and is a reference with significance of evidence-based medicine, marking a major step towards the standardization of HER2 detection. For gastric cancer, related studies were subsequently carried out. A recent global multi-centre phase III clinical trial (ToGA) that involved 24 countries including China revealed the efficacy of trastuzumab for patients with HER2 positive advanced gastric cancer and gastroesophageal junction cancer [13, 14, 15]. Subsequently, the United States Food and Drug Administration (FDA) approved the drug for combination chemotherapy for the treatment of patients with HER2 positive metastatic gastric cancer and gastroesophageal junction cancer [11, 12]. At present, the IHC method is generally used to detect the HER2 protein expression status at home and abroad. However, in gastric cancer, the overexpression rates of HER2 protein in different studies were inconsistent, and most of these concentrated within 6.8–34.0% [16, 17, 18]. The detection of HER2 in gastric cancer is different from that in breast cancer, and these detection results are influenced by many factors, including the preoperative processing and fixation of samples, reagents and methods of detection, laboratory quality control, and the interpretation of staining results [19, 20]. In the present study, 135 patients with gastric cancer underwent combined detection using IHC and FISH techniques. The expression rate of HER2 protein was 39.3% (53/135). Among these positive cases, patients with HER2 protein (3+) accounted for 9.6% (13/135), patients with HER2 protein (2+) accounted for 13.3% (18/135), and patients with HER2 protein (1+) accounted for 16.3% (22/135). The amplification rate of the HER2 gene was 35.8% (19/53). Morphological types of HER2 gene amplification in 19 patients: patients with HER2 gene cluster-specific amplification accounted for 14.3% (7/19), patients with large granular amplification accounted for 10.2% (5/19), patients with spotty amplification accounted for 6.1% (3/19) and patients with high multi-body accounted for 8.2% (4/19). These results were consistent with those reported in related literatures [21, 22, 23, 24, 25]. The higher the expression level of HER2, the more likely the patient would benefit from the treatment of anti-tumor drugs targeting HER2. However, a literature stated that the interpretation standard for the expression of HER2 protein in gastric cancer is that patients with 3+ in $>$ 10% of tumor cells are considered to benefit from the treatment. However, it was revealed through clinical follow-ups that patients with 3+ in 5% of HER2 protein expression also achieved effective results. In addition, when the expression area and intensity of HER2 protein was $>$ 80%, the clinical therapeutic effects were more significant than that of 10%. As a result, we propose that the detection results of HER2 protein expression be classified into extensive, partial and focus types; that is, the detection results of HER2 gene amplification was classified into cluster-specific, large particles, spots and high multi-bodies types. This can provide a better quantized reference indicator for the benefit of the targeted therapy of antitumor drugs. If the 4–6 sections of one patient had only either the extensive, partial or focal type, it was defined as a single-signal type. If the 4–6 sections of one patient simultaneously had more than two of the extensive, partial and focal types, it was defined as multi-signal type. The purpose of this classification, which is based on single-signal and multi-signal types, is mainly to distinguish heterogeneous tumors. However, more signals for the benefit of treatment by targeting anticancer drugs are needed through more follow-ups on more patients. This would make it possible to perform effective individualized treatment by referring to an integral examination report.

At present, chemotherapy for gastric cancer is usually based on combination regimens, and the common drugs of fluorouracil, platinum-based drugs and paclitaxel are commonly used. The combination of three drugs is a relatively intensive chemotherapy regimen, and the chemotherapy of a single-drug or a combination of two drugs is a non-intensive regimen. The combination of three drugs has significant advantages in survival, but the toxicity and side effects are greater. Since gastric cancer has a poorer prognosis in all solid tumors, heterogeneity is high, drug sensitivity is poor, and it has a higher requirement of individualized treatment. In clinical studies and clinical practice, attention must be given to these patients and tumor characteristics, in order to reasonably and individually select drugs. This is the only possible means that would really benefit patients and prolong their survival time. This would allow people focus more attention to the high heterogeneity of gastric cancer. Realizing an individualized treatment of cytotoxic drugs and targeted drugs in progressive gastric cancer has become a hotspot in this field [26, 27, 28]. In the present study, the combination of IHC, FISH and fluorescence RT-PCR was used to detect the targeted and chemotherapeutic agents for gastric cancer. Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received regimen of cetuximab combined with oxaliplatin, leucovorin or fluorouracil; and these patients were followed up for 27 months. Follow-up results revealed that the HER2 gene status was positively correlated with the therapeutic effect of the combined treatment in patients with low mRNA expression of ERCC1, TUBB3 and TYMS. Among these patients, five patients with extensive HER2 (3+), HER2 cluster-specific amplification, and low mRNA expression of ERCC1, TUBB3 and TYMS had a total survival time of up to 19.1 months. Furthermore, the maintenance treatment of targeted drugs was used, and the progression free survival (PFS) was 9.7 months. In addition, both objective response rate (ORR) and disease control rate (DCR) significantly improved. The detection of HER2 in gastric cancer is highly heterogenous, and the combined detection of HER2 protein expression, HER2 gene amplification and the chemosensitivity marker can provide important reference markers for the benefit of antitumor drugs. The combined detection of targeted and chemotherapeutic drugs for gastric cancer is worth to be popularized in the selection of individualized regimens.

In summary, the standardization of the detection criteria of HER2 and drug sensitivity markers in gastric cancer is a guarantee of correct medication. The detection of HER2 in gastric cancer is highly heterogeneity, and the specification and refinement of HER2 detection is helpful to guide the medication. The classification of extensive partial, and focal types, as well as the range and intensity of HER2 detection, can guide and help to evaluate the benefit of targeted drugs. In the targeted therapy of gastric cancer, it is necessary to simultaneously detect the chemosensitivity markers. It is an inevitable trend to select chemotherapy regimens for the individualized treatment of gastric cancer by detecting these chemosensitivity markers.

Footnotes

Acknowledgments

Key scientific and technological projects in Henan Province funded plan, No. 132102310008.

References

Wang

Y.K.

Gao

C.F.

Yun

Chen

Zhang

X.W.

X.X.

Meng

N.L.

and Zhao

W.Z.

, Assessment of ERBB2 and EGFR gene amplification and protein expression in gastric carcinoma byimmunohistochemistry and fluorescence in situhybridization, Mol Cytogenet (2011), 14.

Zhang

Yang

X.W.

L.H.

Sun

Wang

and Yu

X.T.

, Epidermal growth factor receptor expression and gene copy number analysis in gastric carcinoma samples from Chinese patients, Oncol Lett 11(1) (2016), 173–181.

Wang

Y.K.

Zhong

Yun

C.Y.

Jiang

X.X.

Chu

G.H.

Wang

S.N.

Yan

and Shi

L.F.

, human epidermal growth factor receptor 2 expression inmixed gastric carcinoma, World Journal of Gastroenterology 21(15) (2015), 4680–4687.

Wang

S.N.

Y.Y.

Z.S.

Zhao

W.Z.

Yun

Zhu

W.L.

and Wang

Y.K.

, Study on the protein expression and amplification of HER2 gene in gastric cancer, Chinese-German J Clin Oncol (2014), 267–272.

Rieck

L.D.

Pottala

J.V.

Eliason

S.L.

Koch

M.R.

Egland

P.G.

and Egland

K.A.

, Status of HER-2 gene amplification in breast cancers from Native American women, Cancer Genet Cytogenet (2008), 54–56.

English

D.P.

Roque

D.M.

and Santin

A.D.

, HER2 expression beyond breast cancer: therapeutic implications for gynecologic malignancies, Mol Diagn Ther (2013), 85–99.

Kim

Lee

H.D.

Jeong

Lee

and Lee

K.A.

, Spectrum of EGFR gene copy number changes and KRAS gene mutation status in Korean triple negative breast cancer patients, PLoS One 8 (2013), e79014.

Berezowska

Novotny

Bauer

Feuchtinger

Slotta-Huspenina

Becker

Langer

and Walch

, Association between HSP90 and Her2 in gastric and gastroesophageal carcinomas, PLoS One (2013), e69098.

Matsuoka

and Yashiro

, Recent advances in the HER2 targeted therapy of gastric cancer, World J Clin Cases (2015), 42–51.

10.

Gordon

M.A.

Gundacker

H.M.

Benedetti

Macdonald

J.S.

Baranda

J.C.

Levin

W.J.

Blanke

C.D.

Elatre

Weng

Zhou

J.Y.

Lenz

H.J.

and Press

M.F.

, Assessment of HER2 gene amplification in adenocarcinomas of the stomach or gastroesophageal junction in the INT-0116/SWOG9008 clinical trial, Ann Oncol (2013), 1754–1761.

11.

Roa

de Toro

Schalper

de Aretxabala

Churi

and Javle

, Overexpression of the HER2/neu Gene: A New Therapeutic Possibility for Patients With Advanced Gallbladder Cancer, Gastrointest Cancer Res (2014), 42–48.

12.

Wolff

A.C.

Hammond

M.C.

Hicks

D.G.

Dowsett

McShane

L.M.

Allison

K.H.

Allred

D.C.

Bartlett

J.M.

Bilous

Fitzgibbons

Hanna

Jenkins

R.B.

Mangu

P.B.

Paik

Perez

E.A.

Press

M.F.

Spears

P.A.

Vance

G.H.

Viale

and Hayes

D.F.

, Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update, Arch Pathol Lab Med (2014), 241–256.

13.

Tang

D.Q.

Liu

Y.J.

Shen

D.P.

Fan

S.Q.

X.Y.

Gavine

P.R.

and Yin

X.L.

, Assessment and prognostic analysis of EGFR, HER2, and HER3 protein expression in surgically resected gastric adenocarcinomas, Onco Targets Ther (2015), 7–14.

14.

Werner

Battmann

Steinmetz

Jones

Lamb

Martinez

Altmannsberger

H.M.

and Al-Batran

S.E.

, The validation of a novel method combining both HER2 immunohistochemistry and HER2 dual-colour silver in situ hybridization on one slide for gastric carcinoma testing, J Transl Med (2014), 160.

15.

Dahle-Smith

Stevenson

Massie

Murray

G.I.

Dutton

S.J.

Roberts

Ferry

Osborne

Clark

Petty

R.D.

and Miedzybrodzka

, Epidermal Growth Factor (EGFR) copy number aberrations in esophageal and gastro-esophageal junctional carcinoma, Mol Cytogenet (2015), 78.

16.

Wang

Y.K.

Chen

Yun

Y.C.

Jiang

X.X.

Chu

G.H.

Wang

S.N.

Yan

and Shi

L.F.

, Human epidermal growth factor receptor 2 expression in mixed gastric carcinoma, World J Gastroenterol (2015), 4680–4687.

17.

Somlo

Chu

Frankel

Groshen

Doroshow

J.H.

Danenberg

and Danenberg

, Molecular profiling including epidermal growth factor receptor and p21 expression in high-risk breast cancer patients as indicators of outcome, Ann Oncol (2008), 1853–1859.

18.

Zhang

Yang

Sun

Wang

B.O.

and Yu

, Epidermal growth factor receptor expression and gene copy number analysis in gastric carcinoma samples from Chinese patients, Oncol Lett (2016), 173–181.

19.

Tang

Liu

C.Y.

Shen

Fan

Gavine

P.R.

and Yin

, Assessment and prognostic analysis of EGFR, HER2, and HER3 protein expression in surgically resected gastric adenocarcinomas, Onco Targets Ther (2015), 7–14.

20.

Stahl

Seeschaaf

Lebok

Kutup

Bockhorn

Izbicki

J.R.

Bokemeyer

Simon

Sauter

and Marx

A.H.

, Heterogeneity of amplification of HER2, EGFR, CCND1 and MYC in gastric cancer, BMC Gastroenterol (2015), 7.

21.

Shen

Liu

Bao

Jia

Liang

and Shan

, Assessment and prognostic analysis of EGFR mutations or/andHER2 overexpression in Uygur’s Non-small Cell Lung Cancer, Int J Clin Exp Med (2015), 22300–22309.

22.

Liu

Zhu

Peng

Zheng

Mao

and Zheng

, Comparison of droplet digital PCR and IHC combined FISH indetection of breast carcinoma HER2 amplification, Zhonghua Bing Li Xue Za Zhi (2016), 117–119.

23.

Zhu

Guo

Zhang

Wang

and Xu

, Biomarker triplet NAMPT/VEGF/HER2 as a de novo detection panel for the diagnosis and prognosis of human breast cancer, Oncol Rep (2016), 454–462.

24.

Yin

Wang

Y.L.

Wang

Y.F.

Yang

Zhang

Tang

Xie

and Ma

, Correlation between clinical pathology of luminal b breast cancer and determination of estrogen receptor, progesterone receptor and HER2 expression combined with nuclear morphology, J Biol Regul Homeost Agents (2015), 579-587.

25.

Janiszewska

Liu

Almendro

Kuang

Paweletz

Sakr

R.A.

Weigelt

Hanker

A.B.

Chandarlapaty

King

T.A.

Reis-Filho

J.S.

Arteaga

C.L.

Park

S.Y.

Michor

and Polyak

, In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer, Nat Genet (2015), 1212–1219.

26.

Luo

Cui

Shen

Zhao

Wang

Chen

Liu

and Ri-Li

, Joint detection of ERCC1, TUBB3, and TYMS guidance selection of docetaxel, 5-fluorouracil and cisplatin (DDP) individual chemotherapy in advanced gastric cancer patients, Eur J Med Res (2014), 50.

27.

Liu

Wang

L.E.

Tan

Ajani

J.A.

and Wei

Q.Y.

, ERCC1 and ERCC2 variants predict survival in gastric cancer patients, PLoS One (2013), e71994.

28.

Baek

S.K.

Kim

S.Y.

Lee

J.J.

Kim

Y.W.

Yoon

H.J.

and Cho

K.S.

, Increased ERCC expression correlates with improved outcome of patients treated with cisplatin as an adjuvant therapy for curatively resected gastric cancer, Cancer Res Treat (2006), 19–24.

Methods and significance of the combined detection of HER2 gene amplification and chemosensitivity in gastric cancer

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Materials

2.1.1 Reagents

2.1.2 Reagent and probe

2.2 Methods

2.2.1 Immunohistochemical staining

2.2.2 FISH test

Determination of FISH results

2.2.3 Fluorescence RT-PCR

2.3 Statistics analysis

Table 1 Relationship between the expression of HER2 gene protein and histological type in 135 cases of gastric carcinoma

3.1 Histomorphology of gastric cancer

3.2 Expression rate and expression characteristics of HER2 protein in gastric cancer

Table 2 The positive intensity and area distribution of HER2 protein

3.4 ERCC1, TUBB3 and TYMS gene mRNA expression and its relationship with HER2 gene amplification

3.5 Treatment based on the combined detection of HER2 gene amplification and ERCC1, TUBB3 and TYMS gene mRNA expression levels versus simple chemotherapy regimen

4. Discussion

Footnotes

Acknowledgments

References

Table 1
Relationship between the expression of HER2 gene protein and histological type in 135 cases of gastric carcinoma

Table 2
The positive intensity and area distribution of HER2 protein