Set Up FISH Methods To Detect Genes And Chromosomes Mutation In Primary Gastrointestinal Lymphoma And Investigate The Clinical Significance Of It

BackgroundPrimary gastrointestinal lymphoma (PGIL) is the most common extra nodal lymphoma. PGIL has no specific clinical manifestations, and has a wide range of complex pathological types and classifications. It is difficult for early diagnosis and easy to misdiagnose. Gene analysis has gradually become an important diagnostic method for lymphoma, and gene abnormality has important significance in the occurrence, development and prognostic evaluations of in-nodal lymphoma. There are no system reports that how gene abnormalities such as gene mutation, deletion and chromosomal aberrations play an important role in the PGIL happen, development, prognosis and differential diagnosis.Fluorescence in situ hybridization (FISH) has provided new means for detection of genes and chromosomes. FISH is a new molecular cytogenetic techniques, the basic principle is to use known marked single-stranded nucleic acid probe to detect specific DNA sequence in according to the principle of complementary base. FISH technique is suitable for clinical testing, because of the characteristics of simple, good repeatability and the results are rapid, accurate and sensitive. In particular, the method is good for retrospective analysis. Methods of solid tumors paraffin section (such as gastrointestinal cancer, breast cancer, etc) and blood smears detected by FISH have been stable and good repeatability. However, there is greater difference in operation of the two methods. FISH technology has played an important role in the study of pathogenesis, diagnosis, differential diagnosis and clinical prognosis of lymphoma. At present, FISH detected specimens restricted to the peripheral blood samples, bone marrow smears and lymph node puncture smears, etc, paraffin specimens of extra nodal lymphoma are still less involved in. The detected purpose of extra nodal lymphoma paraffin specimens is blood cells in wax block, the operation method is difference with the method of gastrointestinal cancer paraffin section or blood cell smear. Therefore it is necessary to set up stability, good reproducibility FISH techniques to detect gene and chromosome mutation in extra nodal lymphoma such as PGIL.Immunoglobulin heavy chain gene (IGH) rearrangement has great value in determining the cloning and cytogenesis of B-NHL. Studies suggest that IGH rearrangement detection can be used for differential diagnosis of chronic gastritis and gastric MALT lymphoma. There is no systematic analytical report on the clinical significance of IGH rearrangement in other pathological types of gastric lymphoma or primary intestinal lymphoma. What role is IGH rearrangement in PGIL play in early diagnosis and prognosis is remains to be further studied. P53 gene is one of the important tumor suppressor gene. Study found p53 mutation has no relationship with the pathological classification and clinical stage in in-nodal NHL. However, scholars have also found p53 mutations relation with MALT lymphoma transform to a high degree of the malignant. There are few reports on the relationship between p53 mutation and the malignancy and prognosis of PGIL, hence it is important to investigate its biological influence on PGIL. Ataxia- telangiectasia mutated (ATM) gene is a tumor suppressor gene whose encoding proteins are involved in cell-cycle regulation, apoptosis and DNA repair. Studies have found lymphoid malignancy has high incidence in AT patients. It is also of importance to study its effect on the treatment and prognosis of PGIL. 13q14 deletion is a common chromosomal abnormality in many types of lymphoproliferative diseases. 13q14 mutation or deletion can lead to a dysfunction of negative regulation on cell proliferation and eventually result in tumor. D13S25 is the DNA fragment in the 13q14.3 only named 25. 13q14 deletion is a known chromosomal abnormality commonly seen in B-cell chronic lymphocytic leukemia (B-CLL), nevertheless, reports of 13q14 deletion in extra nodal lymphoma is rarely seen. The relationship between 13q14 deletion and PGIL and its prognosis needs further investigation.In this program, we analysis the clinical manifestations, clinical misdiagnosis of PGIL, and comparison different survival in different treatment cases retrospectively, and FISH was employed to detect IGH rearrangement, p53, ATM, 13q14 genes and chromosomal abnormalities in the gastrointestinal paraffin sections to investigate the relationship between polygenic and chromosomal abnormalities in PGIL and its diagnosis and prognosis. The results will provide experimental basis for individual diagnosis and treatment.The first chapter: Set up FISH methods to detect genes and chromosomes mutation in primary gastrointestinal lymphomaObjective: To establish a standardized method with fluorescence in situ hybridization (FISH) technology to detect the chromosomal and genic variations in extra nodal lymphoma such as PGIL. Method: Methods such as enhancing tissue permeability, proper digestion, control degeneration time and slow washing technique were used to optimize FISH detection rate in this type of specimens. Results: Utilizing the established FISH method, we got a clear picture of PGIL tumor cells with the P53 gene probe (green fluorescent signal), the D13S25 probe (red fluorescent signal), the IGH gene probe (red / green or yellow fluorescence signal), and the ATM gene probe(red fluorescent signal) Conclusion: we have established a set of FISH detection technology to detect the chromosomal and genic variations in extra-nodal lymphoma such as PGIL. Provide new methods to clinical research.The second chapter: significance of multiple genes and chromosomes detection in primary gastrointestinal lymphomaObjective: To discuss the roles of multiple genes and chromosomes variation in the early diagnosis, prognosis and therapeutic options of PGIL. Methods: With the refined FISH technology, the IGH rearrangement, p53, ATM, 13q14 gene and chromosomal variations were detected in the gastrointestinal paraffin sections of 72 cases of PGIL patients and the lymphatic paraffin sections of 30 patients with reactive lymphoid hyperplasia. Using t test and chi-square test, p <0.05 considered that there was a significant difference. Results:①IGH rearrangement accounted for 70.8% of B cell lymphoma, however, no IGH arrangement was observed in T cell lymphoma or reactive lymphoid hyperplasia(p<0.001);IGH arrangement was found in 59.5% MALT lymphoma and 91.3% non-MALT lymphoma (p= 0.007); IGH arrangement was found in 63.8% stageⅠ-Ⅱpatients and 88.9% stageⅢ-Ⅳpatients (p = 0.047); Average survival time of IGH rearrangement B-cell lymphoma is 16.85 months, shorter than non-IGH rearrangement cases whose average survival time is 39.81 months (p = 0.001).②p53 gene deletion was found in 28.6% MALT lymphoma and 53.3% non-MALT lymphoma (p = 0.034);p53 gene deletion was found in 30.2% stageⅠ-Ⅱpatients and 63.2% stageⅢ-Ⅳpatients (p = 0.011) ; Average survival time of p53 gene deletion patients is 10.21 months, shorter than p53 gene normal cases whose average survival time is 32.00 months (p< 0.001) .③ATM gene deletion was found in 23.8%MALT lymphoma and 46.7% non-MALT lymphoma (p = 0.043);ATM gene deletion was found in26.4% stageⅠ-Ⅱpatients and 52.6% stageⅢ-Ⅳpatients (p = 0.038); Average survival time of ATM gene deletion patients is 12.35 months, shorter than ATM gene normal cases whose average survival time is 31.42 months (p =0.001) .④13q14 deletion was found in 34.7% patients, significantly higher than that in patients with lymph node reactive hyperplasia (p 0.001). 13ql4 deletion was no correlation with tumor location, patient age, pathological type, clinical stage and average survival time.⑤The average survival time in MALT lymphoma of non-gene or single gene change is 39.25 months, while in poly-gene change is 9.11 months (p<0.001).⑥The average survival time in stageⅠ-Ⅱpatients of non-gene or single gene change is 40.33 months, while in poly-gene change patients is 20.61 months (p <0.001). Conclusions:①IGH rearrangement has a high incidence in PGIL derived from B cells.②There was a high incidence of IGH rearrangement or p53 and ATM gene deletion in non-MALT lymphoma and stageⅢ-Ⅳpatients. The average survival time is shorter in these patients.③In PGIL, the average survival time in patients with multiple genic and chromosomal abnormalities is shorter than non-gene or single gene change patients.The third chapter: Analysis Clinical features of 81 cases of primary gastrointestinal lymphomaObjective: To analyze the status quo of the diagnosis and treatments of PGIL, and to improve it. Method: 81 cases of PGIL patients were analyzed retrospectively in clinical manifestations, endoscopic features, pathological features, HP infection, treatment and prognosis. Using t test and ehi-square test, p <0.05 considered that there was a significant difference. Results:①The average age of patients with gastric lymphoma was 52.84±15.33 years old, Older than the average age of patients with intestinal lymphoma 42.09±15.28 years old ( t =3.087, p=0.003) .②Common symptoms included abdominal pain (76.5%); gastrointestinal bleeding (55.6%); anemia (54.3%); hypoproteinemia (40.7%); weight loss (33.3%); abdominal mass (25.9%); bowel obstruction (11.1%).③Frequent incidence locations were as follows: stomach, small intestine, ileocecal junction, and colon.④Endoscopic appearance were as follows: tumor type 67.7%, ulcer type 27.7%, diffuse type 4.6%.⑤Clinical diagnosis rate was 30.9%; clinical misdiagnosis rate was 69.1%.⑥Endoscopic biopsy confirmed rate was 73.8%; biopsy misdiagnosis rate was 26.2%.⑦B-cell lymphoma accounted for 91.4%, MALT lymphoma accounted for 61.7% of the patients.⑧HP detection rate was 39.5%, positive rate was 37.5%.⑨60 cases (74.1 %)have followed-up, 5-year survival rate was 42.9%, 3-year survival rate was 60.0%, and 1-year survival rate was 74.0%.⑩The average survival time of stageⅠ-Ⅱis 30.41 months, longer than 9.63 months in stageⅢ-Ⅳ( t =3.823, p <0.001).(?) The average survival time of MALT lymphoma is 24.24 months, and non-MALT lymphoma is 22.95 months ,there was no significant difference (t = 0.213, p = 0.832).(?) In stageⅠ-Ⅱ: the average survival time of surgery alone is 33.00 months, the average survival time of surgery plus chemotherapy is 31.95 months, the average survival time of chemotherapy plus HP eradication therapy is 33.25 months. In stageⅢ-Ⅳ: the average survival time of surgery alone is 4.00 months, the average survival time of surgery plus chemotherapy is 12.20 months, the average survival time of chemotherapy plus HP eradication therapy is 9.50months. There are no significant difference of these three treatments in stageⅠ-Ⅱpatients(p =0.897,0.986,0.916). Survival time of surgery alone patients in stageⅢ-Ⅳare shorter than the other two groups(p =0.006,0.009). And there are no significant difference of survival time in surgery plus chemotherapy and chemotherapy combined with HP eradication treatment groups (p = 0.558). Conclusion:①There is no specific clinical and endoscopic features in PGIL, so the misdiagnosis rate is high.②Most PGIL belong to B-NHL, the most common pathological type is MALT lymphoma.③There are no significant difference of survival time in surgery alone, surgery plus chemotherapy, chemotherapy combined with HP eradication treatment in stageⅠ-Ⅱpatients. Survival time of surgery alone patients in stageⅢ-Ⅳare shorter than the other two groups. And there are no significant difference of survival time in surgery plus chemotherapy and chemotherapy combined with HP eradication treatment groups.