MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY NGUYEN KHAC TAN RESEARCH ON THE EXPRESSION AND RELATIONSHIP OF IMMUNE MARKERS OF CANCER STEM CELLS, HER2 IN GASTRIC ADENOCARCINOMA Speciality: Internal Medicine Code: 9720107 ABSTRACT OF MEDICAL PHD THESIS THAI NGUYEN - 2022 The thesis was done in: Thai Nguyen University of Medicine and Pharmacy Supervisor: 1. Luu Thi Binh 2. Phan Quoc Hoan Reviewer 1:………………. The thesis is defended in front of the Thesis Judging Committee at Thai Nguyen University, meeting at: Thai Nguyen University of Medicine and Pharmacy Day Month Year The thesis can be found at: 1.
Thai Nguyen University Library 2. Thai Nguyen University of Medicine and Pharmacy Library 1 INTRODUCTION Stomach cancer is one of the most common cancers and the fourth leading cause of death in the world. Gastric adenocarcinoma has a very poor prognosis, especially in patients with metastatic gastric adenocarcinoma. Evaluation of HER2 expression is an important criterion for choosing chemotherapy for patients.
However, the rate of HER2 positivity in gastric adenocarcinoma patients is quite low. Therefore, further understanding of other gastric adenocarcinoma markers besides HER2 is needed to improve the effectiveness of chemotherapy in patients with gastric adenocarcinoma. Cancer Stem Cells (CSCs) are resistant to chemotherapy and cause metastasis of cancer to other organs. Research on the expression of HER2 and gastric cancer stem cells such as CD44, ALDH opens up a great prospect in the treatment of this type of cancer as well as in the prognosis of treatment outcome and metastatic status of this cancer.
The expression of both HER2, CD44 and ALDH in gastric adenocarcinoma patients in Vietnam has not yet been published elaborately. In addition, immunohistochemical testing is currently still very expensive and not widely available to patients. Therefore, this thesis is conducted with the following objectives: 1. Evaluation of the expression of cancer stem cell immune markers (CD44, ALDH) and HER2 in gastric adenocarcinoma.
Analysis of the relationship between CD44, ALDH, HER2 immune phenotypes with clinical, endoscopic and histopathological characteristics in gastric adenocarcinoma. 2 NEW CONTRIBUTION OF THE THESIS - This is the first research on the simultaneous expression of HER2 and gastric cancer stem cells such as CD44, ALDH, conducted in Vietnam. - Research results have shown that the expression rate of a single marker is 29. Two markers simultaneously expressed the highest rate with 47.
The rate of expression of HER2, CD44 and ALDH concurrently was 10. - The expression of HER2 is different according to weight loss symptoms, Lauren's histopathological characteristics and WHO’s histopathological characteristics. Simultaneous expression of HER2, CD44 and ALDH differed according to symptoms of blood vomitting/black stools, Lauren's histopathological characteristics, and WHO’s histopathological characteristics. THESIS STRUCTURE The thesis consists of 122 pages (excluding references and appendices), 4 chapters (Introduction 2 pages, overview 39 pages, object and research methods 22 pages, research results 27 pages, discussion 30 pages, conclusion 1 pages, proposals 1 page), results has 42 tables, 4 charts, 19 figures, 139 references (9 Vietnamese, 130 English), 3 appendices.
ALDH Aldehyde Dehydrogenase 2. CSC Cancer Stem Cell 3. HER2 Human Epidermal growth factor Receptor 2 3 CHAPTER I OVERVIEW 1. The role of the HER2 in gastric cancer 1.
Role of HER2 in gastric cancer prognosis The rate of HER2 overexpression in gastric adenocarcinoma patients is variable, in the ToGA trial of 3,665 patients, the HER2- positive rate accounted for 810/3665 patients (22%). In a meta- analysis of 49 studies with 11,337 patients, the prevalence was 18%. Approximately 9–38% of all gastric cancer patients show overexpression of human epidermal growth factor receptor-2 (HER2), which may play a prognostic role in a more severe disease and poor outcome, although this issue remains unclear with conflicting results to date in different studies. Clinical application of HER2 markers in gastric cancer The ToGA study evaluated the combination of trastuzumab with a standard fluoropyrimidine plus cisplatin chemotherapy in HER2- positive advanced gastric cancer.
The results of the study suggest that trastuzumab in combination with chemotherapy could be considered a new standard option for patients with HER2-positive advanced gastroesophageal cancer. Based on the role of HER2 in the development and progression of many cancers in general and gastric adenocarcinoma in particular, especially through studies showing that HER2 expression is often associated with poor prognosis, they have applied drugs targeting these molecular targets to treat gastric adenocarcinoma. Anti-HER2 therapy is a promising treatment strategy for patients with gastric adenocarcinoma. The role of CD44 in cancer 1.
The role of CD44 in tumorigenesis CD44 activates and regulates several cell signaling systems, so it plays an important role in mediating the tumorigenic properties of cells leading to tumorigenesis, metastasis, and drug resistance. The function of CD44 in mesenchymal epithelial translocation has been investigated in several types of cancer. Increasing the expression of CD44 reduces the expression of E-cadherin, increasing N-cadherin, α-actin, vimentin, fibronectin inhibits the formation of the E- cadherin-β-catenin membrane complex, which increases the invasive process and cell migration. CD44 and drug resistance of cancer cells Clinical evidence suggests a positive correlation between CD44 expression and metastatic status.
In vitro, decreased expression of CD44 in breast cancer cell lines resulted in a significant reduction in tumor angiogenesis. Cell migration and invasion were prevented by knockout of CD44. Overexpression of CD44 increases tumorigenesis and cell turnover. In vivo, tumor formation in mice was inhibited when injected with CD44 knockout cells.
The number of osteoclasts decreased in metastatic bone with CD44 removed. These studies indicate that cancer cells acquire a tendency toward drug resistance when accompanied by forms of CD44 overexpression. Role of ALDH in gastric cancer 1. ALDH is overexpressed in some cancers Increased ALDH1 enzyme activity has been identified in several types of cancer such as multiple myeloma, acute leukemia, and some solid organ cancers.
Some stem cell markers including ALDH1, 5 CD166, LGR5 have very low concentrations in normal human gastric mucosa, but these markers are very high in gastric adenocarcinoma. ALDH1 expression was significantly correlated with invasion, lymph node metastasis, stage of disease, progression- free survival, and overall survival. However, the prognostic role of ALDH1 isozymes has not been determined. The study results of Chen J.
et al also showed that ALDH1A1 is overexpressed in gastric cancer patients and may play an important role in tumor invasion, metastasis and prognosis. ALDH in chemotherapy resistance ALDH is an enzyme involved in detoxification, protecting tissues from the toxic effects of aldehydes. ALDH1A1 and ALDH3A1 may protect cells against the toxic effects of drugs. The first observations several decades ago in hematopoietic and leukemic stem cells with ALDH were highly resistant to cyclophosphamide and the alkyls, ALDH1A1 and ALDH3A1 modified cyclophosphamide to the active form and 4-hydroperoxy cyclophosphamide into an inactive form for excretion.
Therefore, ALDH can cause drug resistance and resistance to radiation therapy. In recent years, growth factor receptors, tyrosine kinase inhibitors (EGFR-TKIs) have been used as a potential therapy in inhibiting the growth of cancer cells. 6 CHAPTER II SUBJECTS AND METHODS 2. Studying subjects The study subjects were 107 patients with confirmed diagnosis of gastric adenocarcinoma who underwent resection at K Hospital (Tan Trieu and Quan Su campuses), from July 2016 to July 2020.
Selection criteria for study patients - The patient was diagnosed with gastric adenocarcinoma based on histopathological results by Hematoxylin-Eosin (HE) staining according to ESMO 2016 criteria. - Postoperative primary tumor specimens were processed according to standard procedures and stained with immunohistochemistry to identify HER2 markers and cancer stem cell immune markers. - The patient consented and voluntarily participated in the study. Exclusive criteria - Cases of gastric cancer metastasized from other organs.
- Stomach cancer that has been treated with chemotherapy or radiation. Reseach methodology - Cross-sectional descriptive research method. The main criteria and classifications in the research - Tumor location in the stomach on gastroendoscopy. - Tumor morphology according to Borrmann: + Borrmann type I (Polyp form), Borrmann type II (Fungal form), Borrmann type III (Ulcerative form), Borrmann type IV (Infiltrative form).
7 - Tumor size on gastroendoscopy: + Group I: Tumor size < 2 cm. + Group II: Tumor size from 2 to < 5 cm. + Group III: Tumor size ≥ 5 cm. - Histopathological classification according to Lauren: + Intestinal type, diffuse type.
- Histopathological classification according to the WHO 2010 classification system: + Papillary adenocarcinoma. + Signet-ring cell adenocarcinoma. - Histopathological classification according to the degree of differentiation according to WHO: + Poor differentiation. - Diagnosis of TNM stage: According to the 7th AJCC system.
- Assess the expression level of marker HER2 in cancer samples and control samples according to levels: 0, 1+, 2+, 3+. The expression level of HER2 is 0 and 1+ evaluates to negative, the expression level of HER2 is 2+ and 3+ evaluates to positive. - Evaluate the expression level of marker CD44 in cancer samples and control samples according to levels: negative, positive. - Evaluate the expression level of marker ALDH in cancer samples and control samples according to levels: negative, positive.
The method of data collection 2. Clinical examination and subclinical indications - Patients who come to the hospital for examination will be asked about disease, clinical examination, indicated for hematology, biochemistry, coagulation, and immunological tests. - The patient is assigned to have a gastroscopy, when a stomach tumor is found, a biopsy will be performed during the endoscopy. Gastroendoscopy with biopsy - Put the scope through the mouth, throat into the esophagus, stomach, down the duodenum, inflate and observe.
It may be necessary to pump water to clear the mucus in the areas to be observed, and to drain the esophagus and stomach. - If lesions are detected, pump clean, then observe carefully with NBI mode and near focus to evaluate, biopsy for pathology. - Specimens are fixed into tubes containing Formol 20% neutral buffer. Tumor removal surgery and how to handle specimens - When the pathology results are gastric cancer.
The patient was consulted and transferred to the Department of Abdominal Surgery 1,2, K hospital to perform gastric tumor removal surgery. After surgery, the tumor was preserved and a sample was taken for histopathology at K Hospital. Methods of histopathological analysis - Place of implementation: Inserm U1053 laboratory. - Technique: Histopathological analysis by conventional HE staining method.
9 - Read results on HE-stained slide: The HE-stained slide is examined and photographed under a normal light microscope, at 10 - 40x magnification. Immunohistochemistry and staining methods - Place of implementation: Inserm U1053 Laboratory (Helicobacter pylori and gastric cancer research unit), National Institute of Health and Medical Research, Bordeaux, France. The testing process was supported by Prof. * Steps of immunohistochemical staining: - Cut the tissue mass cast in paraffin into slices with a thickness of 3-4 μm.
- Place slices on slides already filled with water and slides are always placed on a 37°C thermostatic plate. - Transfer the slides to a 56°C incubator overnight. - Antigen was expressed by incubation in a water bath at 96°C in citrate buffer pH 6. - Cool the slide for 20 min, wash with a spray nozzle of distilled water 2 times × 5 min.
- Reduce endogenous peroxidase with 3% H2O2 solution × 5 minutes. - Wash the slide with Tris-Buffer-Saline (TBS) solution pH 7. - Deactivation of nonspecific proteins with bovine serum albumin (BSA Bovin SerumAlbumine × 5 min. 10 - Wash the slide with TBS solution 2 times × 5 minutes, always keep the slide in the moist tray.
Cover with 1st antibody against antigen for 60 min. - Wash the slide with TBS solution 2 times × 5 minutes with a gentle spray. - Coat the second antibody with Biotin for 30 minutes. - Wash the slide with TBS solution 2 times × 5 minutes.
- Cover with Avidin-Biotin complex for 30 minutes. - Wash the slide with TBS solution 2 times × 5 minutes. - Cover with Diamino Benzidine (DAB) solution for 10 minutes. - Rinse the slide under running water for 5 minutes.
- Hematoxyline staining for 5 seconds.