论文部分内容阅读
Abstract Objective To analyze the difference of the positive rate of HER2 expression between gastroscopic and surgical specimens of gastric carcinoma and investigate the feasibility of HER2 detection in gastroscopic specimens to substitute surgical specimens.Methods Using immunohistochemical technique to detect HER2 expression in gastroscopic and surgical specimens of 200 cases with gastric carcinoma.Results In the test of 200 cases of gastric cancer tissues， 171 cases in the gastrosopic specimens showed HER2 protein expression （0/1+）， 6 cases showed （2+）， and 23 cases （3+）； in the surgical specimens 161 cases expressed （0/1+）， 18 cases （2+）， and 21 cases （3+）. The difference of the positive HER2 protein expression between gastroscopic and surgical specimens did not reach statistical significance （P<0.5）. The consistency of the both is ordinary （kappa=0.613， P<0.05）.Conclusion The positive rates of HER2 expression in gastroscopic and surgical specimens of gastric carcinoma are basically the same. For the patients with inoperable advanced gastric carcinoma， gastroscopic specimens may substitute for surgical specimens for HER2 examination. Thus， it may also provide reliable theoretical basis for targeted therapy.
Key words Gastric carcinoma； Gastroscopy； Immunohistochemistry； HER2
中图分类号：R737.9 文献标识码：A
Introduction
Gastric carcinoma is the fourth-common malignant tumor in the world [1]. Chemotherapy is the standard treatment for gastric carcinoma [1]， but the patients with advanced gastric carcinoma cannot adopt better chemotherapeutic strategy because HER2 in surgical specimens of gastric carcinoma cannot be examined， and oncologists cannot obtain accurate treatment evidence. In this study， we try to find a new testing method to substitute the test in the surgical specimens to provide reliable treatment evidence for clinic， so more inoperable patients with advanced gastric carcinoma may get proper treatment. This study focused on the HER2 expression status in gastroscopic tissue specimens to improve the living conditions of inoperable patients with advanced gastric carcinoma.
1 Materials and Methods
1.1 Materials
200 cases of gastric carcinoma pathologically diagnosed by gastroscopy and surgically removed in the Affiliated Hospital of Qingdao University in 2010-2013 were collected. Among them， there were 139 cases of males and 61 cases of females. They were 38-83 years of age， the median age was 60. In the Lauren’s types ， there were 55 cased of intestinal type， 115 cases of diffuse type and 30 cases of mixed type. About the location of the tumors， there are 26 cases in esophagogastric junction， 68 cases in gastric body， and 106 cases in antrum. All the cases had not been treated with preoperative radiotherapy or chemotherapy. All the gastroscopic specimens were fixed immediately after sampling. All the surgical specimens were fixed with 10 % neutral buffered formalin within half an hour to one hour after surgical removal. 1.2 Methods
ConfirmTM anti-HER2 （4B5） monoclonal antibody and ultraViewTM general DAB detection kit were used in immunohistochemistry. All the tissue specimens of gastric carcinoma were fixed with 10 % neutral buffered formalin， embedded with paraffin， and sliced with 3 m thickness. HER2 protein was examined on the Roche BenchMark XT and GT full-automatic immunohistochemical machine. The operation steps were all executed in strict accordance with the Roche diagnosis optimization program. After routine mounting， the sections were observed with light microscope. The known positive and negative cases of gastric carcinoma were used as external controls， and the normal mucous epithelium in the same section as internal control.
1.3 Analysis of HER2 expression
Hercep Test was adopted for IHC detection and analysis [1]. HER2 expression was scored according to the revised guidance for IHC investigation in gastric carcinoma. Positive control， negative control and blank control were set up. The gastric cancer tissue that had been confirmed to be HER2 3+ in IHC was adopted as positive control. The negative control was the epithelium of gastric mucosa with normal morphology adjacent to carcinoma in the tested sections. Phosphate buffer solution （PBS） was substituted for the primary antibody as blank control. In the guideline， IHC 3+ was HER2 positive， while IHC + and IHC 2+ were HER2 negative. HER2-positive staining appeared brown granules and was located in the cell membrane.
In the surgical specimens， no response or membranous staining in less than 10 % of tumor cells was 0 or negative. Weak and incomplete membranous staining in more than 10 % of tumor cells was 1+ or negative. Weak to moderate complete membranous staining in more than 10 % of tumor cells was 2+ or uncertain. Strong and complete membranous staining in more than 10 % of tumor cells was 3+ or positive.
In gastroscopic specimens， no staining in any tumor cells was 0 or negative. Weak or barely visible membranous staining of tumor cell cluster was 1+ or negative regardless of the percentage of pigmenting cells in the whole tissue. Weak to moderate staining of basolateral membrane or lateral membrane or complete membrane in at least five clustered tumor cells was 2+ or uncertain. Strong staining of basolateral membrane or lateral membrane or complete membrane in at least five clustered tumor cells was 3+ or positive.
1.4 Statistical treatment
Corrected 2 test was used for the ratio test. P<0.05 was considered significant. Kappa test was used for consistency check. Kappa>0.75 was considered well consistency between the both， 0.75>kappa>0.4 was considered ordinary consistency between the both， and kappa<0.4 was considered poor consistency between the both. 2 Results
In gastroscopic tissue specimens of 200 patients with gastric carcinoma tested by IHC， the positive expression of HER2 protein was mainly manifested as membranous staining or with granular cytoplasmic staining. HER2 protein was expressed neither in benign lesions nor in pericarcinomatous tissues of stomach. Its expression was heterogeneous. The positive rate in the surgical specimens of gastric carcinoma was 10.70 %， and the positive rate in gastroscopic specimens was 11.76 %. Different scores in surgical specimens and gastroscopic specimens were shown in table 1 and 2.
The coincidence rate of HER2 expression in surgical specimens and gastroscopic specimens of gastric carcinoma was 80.95 %. According to the statistical test， there was no statistical significance in the difference between surgical specimens and gastroscopic specimens， that is， there was consistency between the both. Kappa was 0.613， so the consistency between the both was ordinary.
3 Discuss
A lot of researches show that HER2 gene does not express or express with low level in normal cells. When HER2 gene is affected by certain carcinogenic factors， in some malignant cells， such as in gastric carcinoma or breast cancer， its structure or express gets out of control， and then it got tumor-transforming activity [2]. It mechanisms of action include inhibition of apoptosis， promotion of cell proliferation， invasiveness increase of tumor cells， acceleration of angiogenesis and lymphangiogenesis of tumors. The results from an international， randomized controlled， multi-center study on clinical phase Ⅲ （ToGA trial） revealed that the positive rate of HER2 in patients with gastric carcinoma was 22 %. The high expression of HER2 in gastric carcinoma provides a locus for targeted therapy of gastric carcinoma [3]. The expression or over-expression of HER2 gene in gastric carcinoma was a canceration marker of epithelial cells in gastric mucosa， and may act as a reference index of diagnosis and prognosis of gastric carcinoma.
HER2 expression level is an important biological marker to determine if a patient is suited to using the drug Trastuzumab [2]. Numerous scholars in the world discussed comprehensively the clinical significance reflected by HER2 expression and over-expression in surgical specimens of gastric carcinoma. The research scholars analyzed experimentally and statistically the relations between all the clinical and pathological features and HER2 expression level in surgical specimens of patients with gastric carcinoma， and draw a conclusion that there is a correlation between HER2 expression and tissue differentiation， clinical stages， infiltration depth or lymph node metastasis， and there is no close correlation between HER2 expression and gender or nationality. This experiment focused on the comparative study of HER2 expression in gastroscopic specimens and in the corresponding surgical specimens of patients with gastric carcinoma. This experiment studied the cases with surgical specimens and gastroscopic specimens of gastric carcinoma by statistical analysis. The difference was not statistically significant. It was computed statistically that the consistency between the both showed ordinary. Less material may influence the end result in sectioning and staining， so sampling should be in the most typical part of the tumor. Because the staining of cell membrane may be influenced by objective factors， at this point extensively sampling should be paid more attention to.
In some cases HER2 expression in surgical specimens was negative or uncertain， while the expression in gastroscopic tissue was positive. Many fixation-related objective factors， like that the surgical specimens were not fixed in time after leaving the body， or the fixation time or the concentration of fixative was substandard， lead to the difference in consistency of HER2 expression in surgical specimens and in gastroscopic tissues. Normative specimen fixation is the premise and guarantee of HER2 detection quality， which can keep good tissue appearance and prevent the intracellular protein and nucleic acid from degradation. In the guidance for HER2 investigations of gastric carcinoma [1]， it is mentioned that all the specimens should be processed initially by marking， incision and fixation in 20-30 min after leaving the body， freshly prepared 4% buffered neutral formalin should be used as fixative， and the volume of fixative should be 10 times that of the tissue. In practice， we can ensure that gastroscopic tissue specimens are fixed relatively in time， but sometimes the surgical specimens can not be fixed in time. Moreover， the fixative used in our hospital is 10 % neutral formalin. Thus， these factors influenced the membrane staining of tumor cells in the late surgical specimens.
Table 1. HER2 expression in surgical specimens and gastroscopic specimens of gastric carcinoma
Table 2. The consistency of HER2 between surgical specimens and gastroscopic specimens of gastric carcinoma
In the whole experimental results， the positive rate of HER2 expression in gastroscopic tissue of gastric carcinoma was higher than that in surgical specimens. Uncertain （2+） cases in gastroscopic specimens were less than that in surgical specimens， which is due to the difference of HER2 interpretation standard between gastroscopic tissue and surgical specimens. HER2-positive interpretation standard in gastroscopic specimens cancels the critical value of the cell-staining percentage. Staining of at least five tumor cells in a cluster can be interpreted to be positive. Moreover some surgical specimens were not fixed in time， which had some impact on the IHC detection at later stage. Therefore 3+ cases in gastroscopic tissues are more than that in surgical specimens， while 2+ cases are less than that in surgical specimens. Outlooks
The modern medical system advocates personalized medicine that varies from person to person [2]. Trastuzumab combination chemotherapy used in patients with gastric carcinoma whose HER2 gene expressions are 3+ or 2+ and whose gene amplification is ideal， shows survival advantage， and at the same time， does not increase the toxicity of chemotherapeutics. So to the patients with advanced gastric carcinoma and positive HER2， trastuzumab combination chemotherapy more and more trends to standardization. It is possible to use gastroscopic tissue specimens to substitute for surgical specimens， which provides an accurate， simple and quick method for HER2 test in gastric carcinoma， and provides important basis of selecting molecular targeted therapy and extending survival time for patients with advanced gastric carcinoma， especially the inoperable patients， to ultimately realize the real personalized medicine of gastric carcinoma.
References
[1] Hofmann M，Stoss O，Shi D，et al. Assessment of a HER2 scoring system for gastric cancer： results from a validation study [J].Histopathology 2008.52：797-805.
[2] Schechter AL， Hung MC， Vaidyanathan L. The neu gene： an erbBhomologous gene distinct from and unlinked to the gene encoding the EGF receptor[J].Science，1985.229（4717）：976-8.
[3] Bang YJ， Van culsem E， Feyereislova A et al.Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER-2-postivie advanced gastric or gastro- oesophageal junction cancer（ToGA）：a phase 3 open-label randomised controlled trial lancet，2011.376 （9742）：687-597.
Key words Gastric carcinoma； Gastroscopy； Immunohistochemistry； HER2
中图分类号：R737.9 文献标识码：A
Introduction
Gastric carcinoma is the fourth-common malignant tumor in the world [1]. Chemotherapy is the standard treatment for gastric carcinoma [1]， but the patients with advanced gastric carcinoma cannot adopt better chemotherapeutic strategy because HER2 in surgical specimens of gastric carcinoma cannot be examined， and oncologists cannot obtain accurate treatment evidence. In this study， we try to find a new testing method to substitute the test in the surgical specimens to provide reliable treatment evidence for clinic， so more inoperable patients with advanced gastric carcinoma may get proper treatment. This study focused on the HER2 expression status in gastroscopic tissue specimens to improve the living conditions of inoperable patients with advanced gastric carcinoma.
1 Materials and Methods
1.1 Materials
200 cases of gastric carcinoma pathologically diagnosed by gastroscopy and surgically removed in the Affiliated Hospital of Qingdao University in 2010-2013 were collected. Among them， there were 139 cases of males and 61 cases of females. They were 38-83 years of age， the median age was 60. In the Lauren’s types ， there were 55 cased of intestinal type， 115 cases of diffuse type and 30 cases of mixed type. About the location of the tumors， there are 26 cases in esophagogastric junction， 68 cases in gastric body， and 106 cases in antrum. All the cases had not been treated with preoperative radiotherapy or chemotherapy. All the gastroscopic specimens were fixed immediately after sampling. All the surgical specimens were fixed with 10 % neutral buffered formalin within half an hour to one hour after surgical removal. 1.2 Methods
ConfirmTM anti-HER2 （4B5） monoclonal antibody and ultraViewTM general DAB detection kit were used in immunohistochemistry. All the tissue specimens of gastric carcinoma were fixed with 10 % neutral buffered formalin， embedded with paraffin， and sliced with 3 m thickness. HER2 protein was examined on the Roche BenchMark XT and GT full-automatic immunohistochemical machine. The operation steps were all executed in strict accordance with the Roche diagnosis optimization program. After routine mounting， the sections were observed with light microscope. The known positive and negative cases of gastric carcinoma were used as external controls， and the normal mucous epithelium in the same section as internal control.
1.3 Analysis of HER2 expression
Hercep Test was adopted for IHC detection and analysis [1]. HER2 expression was scored according to the revised guidance for IHC investigation in gastric carcinoma. Positive control， negative control and blank control were set up. The gastric cancer tissue that had been confirmed to be HER2 3+ in IHC was adopted as positive control. The negative control was the epithelium of gastric mucosa with normal morphology adjacent to carcinoma in the tested sections. Phosphate buffer solution （PBS） was substituted for the primary antibody as blank control. In the guideline， IHC 3+ was HER2 positive， while IHC + and IHC 2+ were HER2 negative. HER2-positive staining appeared brown granules and was located in the cell membrane.
In the surgical specimens， no response or membranous staining in less than 10 % of tumor cells was 0 or negative. Weak and incomplete membranous staining in more than 10 % of tumor cells was 1+ or negative. Weak to moderate complete membranous staining in more than 10 % of tumor cells was 2+ or uncertain. Strong and complete membranous staining in more than 10 % of tumor cells was 3+ or positive.
In gastroscopic specimens， no staining in any tumor cells was 0 or negative. Weak or barely visible membranous staining of tumor cell cluster was 1+ or negative regardless of the percentage of pigmenting cells in the whole tissue. Weak to moderate staining of basolateral membrane or lateral membrane or complete membrane in at least five clustered tumor cells was 2+ or uncertain. Strong staining of basolateral membrane or lateral membrane or complete membrane in at least five clustered tumor cells was 3+ or positive.
1.4 Statistical treatment
Corrected 2 test was used for the ratio test. P<0.05 was considered significant. Kappa test was used for consistency check. Kappa>0.75 was considered well consistency between the both， 0.75>kappa>0.4 was considered ordinary consistency between the both， and kappa<0.4 was considered poor consistency between the both. 2 Results
In gastroscopic tissue specimens of 200 patients with gastric carcinoma tested by IHC， the positive expression of HER2 protein was mainly manifested as membranous staining or with granular cytoplasmic staining. HER2 protein was expressed neither in benign lesions nor in pericarcinomatous tissues of stomach. Its expression was heterogeneous. The positive rate in the surgical specimens of gastric carcinoma was 10.70 %， and the positive rate in gastroscopic specimens was 11.76 %. Different scores in surgical specimens and gastroscopic specimens were shown in table 1 and 2.
The coincidence rate of HER2 expression in surgical specimens and gastroscopic specimens of gastric carcinoma was 80.95 %. According to the statistical test， there was no statistical significance in the difference between surgical specimens and gastroscopic specimens， that is， there was consistency between the both. Kappa was 0.613， so the consistency between the both was ordinary.
3 Discuss
A lot of researches show that HER2 gene does not express or express with low level in normal cells. When HER2 gene is affected by certain carcinogenic factors， in some malignant cells， such as in gastric carcinoma or breast cancer， its structure or express gets out of control， and then it got tumor-transforming activity [2]. It mechanisms of action include inhibition of apoptosis， promotion of cell proliferation， invasiveness increase of tumor cells， acceleration of angiogenesis and lymphangiogenesis of tumors. The results from an international， randomized controlled， multi-center study on clinical phase Ⅲ （ToGA trial） revealed that the positive rate of HER2 in patients with gastric carcinoma was 22 %. The high expression of HER2 in gastric carcinoma provides a locus for targeted therapy of gastric carcinoma [3]. The expression or over-expression of HER2 gene in gastric carcinoma was a canceration marker of epithelial cells in gastric mucosa， and may act as a reference index of diagnosis and prognosis of gastric carcinoma.
HER2 expression level is an important biological marker to determine if a patient is suited to using the drug Trastuzumab [2]. Numerous scholars in the world discussed comprehensively the clinical significance reflected by HER2 expression and over-expression in surgical specimens of gastric carcinoma. The research scholars analyzed experimentally and statistically the relations between all the clinical and pathological features and HER2 expression level in surgical specimens of patients with gastric carcinoma， and draw a conclusion that there is a correlation between HER2 expression and tissue differentiation， clinical stages， infiltration depth or lymph node metastasis， and there is no close correlation between HER2 expression and gender or nationality. This experiment focused on the comparative study of HER2 expression in gastroscopic specimens and in the corresponding surgical specimens of patients with gastric carcinoma. This experiment studied the cases with surgical specimens and gastroscopic specimens of gastric carcinoma by statistical analysis. The difference was not statistically significant. It was computed statistically that the consistency between the both showed ordinary. Less material may influence the end result in sectioning and staining， so sampling should be in the most typical part of the tumor. Because the staining of cell membrane may be influenced by objective factors， at this point extensively sampling should be paid more attention to.
In some cases HER2 expression in surgical specimens was negative or uncertain， while the expression in gastroscopic tissue was positive. Many fixation-related objective factors， like that the surgical specimens were not fixed in time after leaving the body， or the fixation time or the concentration of fixative was substandard， lead to the difference in consistency of HER2 expression in surgical specimens and in gastroscopic tissues. Normative specimen fixation is the premise and guarantee of HER2 detection quality， which can keep good tissue appearance and prevent the intracellular protein and nucleic acid from degradation. In the guidance for HER2 investigations of gastric carcinoma [1]， it is mentioned that all the specimens should be processed initially by marking， incision and fixation in 20-30 min after leaving the body， freshly prepared 4% buffered neutral formalin should be used as fixative， and the volume of fixative should be 10 times that of the tissue. In practice， we can ensure that gastroscopic tissue specimens are fixed relatively in time， but sometimes the surgical specimens can not be fixed in time. Moreover， the fixative used in our hospital is 10 % neutral formalin. Thus， these factors influenced the membrane staining of tumor cells in the late surgical specimens.
Table 1. HER2 expression in surgical specimens and gastroscopic specimens of gastric carcinoma
Table 2. The consistency of HER2 between surgical specimens and gastroscopic specimens of gastric carcinoma
In the whole experimental results， the positive rate of HER2 expression in gastroscopic tissue of gastric carcinoma was higher than that in surgical specimens. Uncertain （2+） cases in gastroscopic specimens were less than that in surgical specimens， which is due to the difference of HER2 interpretation standard between gastroscopic tissue and surgical specimens. HER2-positive interpretation standard in gastroscopic specimens cancels the critical value of the cell-staining percentage. Staining of at least five tumor cells in a cluster can be interpreted to be positive. Moreover some surgical specimens were not fixed in time， which had some impact on the IHC detection at later stage. Therefore 3+ cases in gastroscopic tissues are more than that in surgical specimens， while 2+ cases are less than that in surgical specimens. Outlooks
The modern medical system advocates personalized medicine that varies from person to person [2]. Trastuzumab combination chemotherapy used in patients with gastric carcinoma whose HER2 gene expressions are 3+ or 2+ and whose gene amplification is ideal， shows survival advantage， and at the same time， does not increase the toxicity of chemotherapeutics. So to the patients with advanced gastric carcinoma and positive HER2， trastuzumab combination chemotherapy more and more trends to standardization. It is possible to use gastroscopic tissue specimens to substitute for surgical specimens， which provides an accurate， simple and quick method for HER2 test in gastric carcinoma， and provides important basis of selecting molecular targeted therapy and extending survival time for patients with advanced gastric carcinoma， especially the inoperable patients， to ultimately realize the real personalized medicine of gastric carcinoma.
References
[1] Hofmann M，Stoss O，Shi D，et al. Assessment of a HER2 scoring system for gastric cancer： results from a validation study [J].Histopathology 2008.52：797-805.
[2] Schechter AL， Hung MC， Vaidyanathan L. The neu gene： an erbBhomologous gene distinct from and unlinked to the gene encoding the EGF receptor[J].Science，1985.229（4717）：976-8.
[3] Bang YJ， Van culsem E， Feyereislova A et al.Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER-2-postivie advanced gastric or gastro- oesophageal junction cancer（ToGA）：a phase 3 open-label randomised controlled trial lancet，2011.376 （9742）：687-597.