Gastric cancer is the 4th most common cancer and the 2nd most common cause of death worldwide, and the mortality rate of gastric cancer in China is higher than the world average for both men and women. 2010 China Health Statistical Yearbook shows that the mortality rate of gastric cancer was the 3rd highest among malignant tumors in China in 2005. With the clarification of molecular mechanisms of tumor growth and proliferation and the rapid development of molecular targeted drug research, it is increasingly important to find molecular markers that can predict tumor treatment effects and patient prognosis.
Based on the results of an international multicenter, randomized, controlled, phase III clinical study, ToGA, which showed that chemotherapy combined with trastuzumab significantly prolonged the survival of patients with progressive gastric cancer, the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) approved the use of trastuzumab in combination with chemotherapy for the treatment of human epidermal growth factor receptor 2 (HER2) positive stomach and gastric cancer in 2010. HER2)-positive gastric and gastroesophageal junction cancer (hereafter referred to as gastric cancer) patients, and the “Gastric Cancer Treatment Specification (2011 Edition)” issued by China’s Health and Family Planning Commission (hereafter referred to as the Health and Family Planning Commission) stipulates that patients with HER2-positive advanced gastric cancer may be considered for the combination of the molecularly targeted therapeutic drug trastuzumab on top of chemotherapy. Thus, pathological test reports of tissue specimens should also provide HER2 test results, and the accuracy of the test is a prerequisite for patient screening and efficacy prediction of HER2-targeted therapy for gastric cancer.
1. Comparison of HER2 detection methods in gastric cancer and breast cancer
The specimen production process, staining method and detection reagents for HER2 detection in gastric cancer are the same as those for breast cancer, and the judgment method is also roughly the same as the standard used for breast cancer, but the following points should be noted.
First, HER2 expression in breast cancer is interpreted according to the criteria specified by the American Society of Clinical Oncology/American College of Pathologists (ASCO/CAP) guidelines, while gastric cancer is judged according to the criteria for breast cancer interpretation (FDA standards) before the guidelines, using a 10% cut-off value.
Second, the HER2 expression patterns of gastric cancer and breast cancer are different, as the overexpressed Her-2 protein in breast cancer is localized in the intact cell membrane, whereas in gastric cancer, it is mainly expressed in the lateral or basolateral membranes (i.e., U-type) of cancer cells, and may not be expressed in the luminal mask.
Third, because tumor heterogeneity is more common in gastric cancer than in breast cancer, the cut-off value of the percentage of positive cells has been abolished in the interpretation criteria of endoscopic biopsy specimens of gastric cancer, except that a 10% cut-off value is used for surgical specimens, whereas the criteria for breast cancer puncture biopsy specimens are the same as those for surgical specimens.
Fourthly, gastric cancer advocates using immunohistochemical staining (IHC) method for detection first, while breast cancer does not emphasize which IHC or fluorescence in situ hybridization method (FISH or two-color silver-enhanced in situ hybridization, DSISH) is first, therefore, it is necessary to establish HER2 detection process and interpretation criteria that meet the characteristics of gastric cancer.
2.HER2 detection process
As the first test for screening the indications of HER2 molecular targeted therapy, it is recommended to use IHC method first, because the heterogeneity of gastric cancer itself and the heterogeneity of HER2 expression within the tumor are very strong, and IHC is easier to grasp the overall tumor situation than FISH (or DSISH). When the judgment result is 2+, FISH (or DSISH) must be retested. Cases judged to be 3+ or 2+/FISH (or DSISH) positive [HER2/chromosome 17 copy number (CEP17) ≥ 2.0] will be treated with trastuzumab
3. Preparation and selection of HER2 test specimens
Surgical resection and biopsy specimens of primary tumor foci or metastases can be used for testing specimens. When taking biopsy specimens endoscopically, granulation and necrotic tissues such as ulcerated bottom should be avoided, and slightly more tissues than usual (6~8 pieces if possible) should be clamped at different parts of the tumor respectively, and observed with magnification endoscopy when possible to determine papillary carcinoma or high grade differentiated tubular adenocarcinoma in order to take biopsies in a targeted manner.
For recurrent gastric cancer where de novo cancer tissue is difficult to obtain, wax blocks of primary foci from previous surgeries or biopsies can be tested, and unstained sections kept for more than 6 weeks should not be used; wax blocks of cancerous tissues, especially those of highly- and moderately-differentiated adenocarcinomas, should be screened by certified pathologists through previous HE staining results and sectioned simultaneously with peri-cancerous tissues to be used.
The optimal tissue thickness for both IHC and FISH (or DSISH) is 3~4 μm, and the positive IHC stained portion should be selected for testing as much as possible; due to different tissue fixation conditions, not all specimens may be suitable for FISH (or DSISH) testing.
Surgically excised specimens should be cut and fixed within 20-30 minutes after isolation, and biopsy tissues should be fixed immediately. 10% neutral formalin should be used as the fixative solution, and the volume should be 10 times the volume of the tissue, and it is not advisable to use microwave to fix the tissue quickly.
The fixation time for surgical excision specimens should be 6~48 hours, while biopsy specimens should be fixed according to the size of the sample (fixative penetration potency is 1mm/hr); attention should be paid to adequate fixation and thorough dehydration, paraffin melting point ≤ 60°C, avoiding prolonged placement in melted paraffin.
4.Interpretation of HER2 test results
IHC interpretation criteria for positive results are based on cell membrane U-shape staining and staining intensity. 10% cut-off value is chosen for surgical specimens (stained tumor cells >10% are considered positive); endoscopic biopsy specimens are judged without setting a cut-off value for cell positivity, and HER2 positivity can be judged by confirming more than 5 clusters of positive cancer cells.
The evaluation of staining intensity is often subjective, and Rüschoff et al. suggested that the intensity of cell membrane staining should be evaluated at different microscopic magnifications. The membrane staining is clearly visible at low magnification (×2.5 or ×5) and is 3+; at medium magnification (×10 or ×20) it is more obvious and is 2+; at high magnification (×40) it is faintly visible and is 1+; cells with nuclear staining are not evaluated or FISH (or DSISH) is recommended.
FISH or DSISH interpretation criteria should be verified by FISH or DSISH for critical cases or “difficult” cases, because DSISH has high compliance rate, low cost, long-term preservation of specimens, observation of morphology in bright field, and automated staining compared with FISH method, which is currently adopted by many testing institutions at home and abroad. In 2011, IHC combined with FISH or DSISH is recommended in the “Guidelines for HER2 detection in gastric cancer” of Chinese Medical Association.
The interpretation method and criteria are to count the ratio of HER2 gene copy number to CEP17 in at least 20 tumor cell nuclei.
The results were judged by the following criteria.
(i) The ratio of total HER2 signal to total CEP17 signal ≥ 2.2 is HER2 gene amplification.
(ii) The ratio of the two signals <1.8, as no gene amplification.
(iii) The ratio between 1.8 and 2.2 should be counted in 20 more nuclei or recounted by a different analyst.
If 40 cancer cells are counted, a ratio >2.0 is interpreted as positive, and a ratio <2.0 is considered negative. The average value of the two signals should be indicated in the test report. If the CEP17 signal of each tumor cell is ≥3, it is defined as chromosome 17 polyploidy, which should also be indicated.
5. Quality control of HER2 assay
Here, we recommend that gastric cancer HER2 test results be analyzed by trained and certified pathologists, that all testing laboratories be managed using certified processes, and that they actively participate in external quality control to validate testing capabilities in addition to internal quality control programs. External quality control for gastric cancer HER2 testing is performed by the UK External Quality Assessment Center, the Nordic National Immunohistochemistry Quality Control, and the Pathology Quality Control Evaluation Center of the Chinese Health and Family Planning Commission.
With the continuous progress of molecular biology research on gastric cancer, targeted therapy has become the focus of individualized treatment for gastric cancer, and standardized diagnosis can help gastric cancer patients to diagnose the disease early and carry out individualized treatment; the National Comprehensive Cancer Network (NCCN) Chinese version of the “Clinical Practice Guidelines for Gastric Cancer 2011, 1st Edition” has clearly stipulated that for gastric cancer patients to be treated with trastuzumab, they need to IHC and/or FISH (or other in situ hybridization methods) are required to detect HER2 expression status, and standardization and standardization of HER2 testing in gastric cancer can help improve the efficiency of individualized treatment for gastric cancer.