【作者】 冯莉娟；

【导师】 张桂英；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 研究背景原发性胃肠淋巴瘤(primary gastrointestinal lymphoma,PGIL)是最常见的结外淋巴瘤,临床表现无特异性,病理分类复杂,早期诊断比较困难,极易误诊和漏诊。基因诊断已逐渐成为淋巴瘤的重要诊断手段,基因异常在结性淋巴瘤的发生、发展、预后等方面均有重要意义。PGIL基因异常包括基因突变、缺失和染色体畸变等,它们在PGIL的发生、发展、预后及鉴别诊断等方面起着怎样的重要作用,目前尚无系统报道。荧光原位杂交(Fluorescence in situ hvbridization,FISH)技术为多种基因与染色体的检测提供了新的手段。FISH是一门新兴的分子细胞遗传学技术,其基本原理是用已知的标记单链核酸为探针,按照碱基互补的原则检测特定的DNA序列。FISH技术操作相对简单,重复性好,具有快速、准确、敏感等优点,适于临床医学检测。该方法尤其对回顾性分析病理切片中靶细胞分子检测有优势。用FISH技术检测实体瘤蜡块切片(如胃肠癌、乳腺癌等)和血液细胞涂片的方法均已稳定成熟,重复性好,但二者在操作方法上有较大区别。FISH技术已在淋巴瘤的发病机制、诊断、鉴别诊断及临床预后等诸多发面的研究中发挥作用。但目前FISH检测的淋巴瘤标本多局限于外周血、骨髓涂片及淋巴结穿刺涂片等,而结外淋巴瘤石蜡标本尚少涉及。结外淋巴瘤石蜡标本检测的目的细胞是组织蜡块中的血细胞,此区域淋巴细胞的消化比滴片细胞难但又易于其它组织细胞,具体操作方法不同于胃肠癌等实体瘤组织蜡块切片或血液细胞涂片的方法。因此建立一套用FISH技术检测PGIL等结外淋巴瘤的多种基因及染色体变异的稳定的、重复性好的实验方法十分必要。IGH基因重排检测对B-NHL的克隆性和细胞源性确定有重要价值,有研究认为IGH重排检测可以用于慢性胃炎及胃MALT淋巴瘤的鉴别诊断。但IGH重排检测在其他病理类型的胃淋巴瘤及原发性肠淋巴瘤中的临床意义尚无系统分析报告。IGH重排在PGIL的早期诊断、预后判断中起怎样的重要作用有待进一步研究。p53基因是一种重要的抑癌基因,有研究发现结性NHL中p53突变阳性的病例与病理分类、临床分期无关,但也有学者发现p53突变与MALT型淋巴瘤向恶性程度高的转变有关。PGIL中p53基因的突变与恶性程度、预后的关系报道较少。探讨p53基因在PGIL中的生物学行为极为重要。ATM(毛细血管扩张性共济失调症突变)基因是一种肿瘤抑制基因,ATM基因编码的蛋白参与了一系列细胞周期的调控、细胞凋亡及DNA修复活动,AT患者淋巴样恶性肿瘤的发生率高,ATM对PGIL的发生、发展、预后判断方面的作用值得深入研究。13q14缺失是多种淋巴细胞增殖性疾病常见的染色体异常,其突变或缺失,可导致细胞增殖、失去负调控而致肿瘤发生。D13S25是13q14.3上唯一编号为25的DNA片段,13q14缺失已知是B细胞慢性淋巴细胞白血病(B-CLL)最常见的染色体异常,国内外已有广泛报道,但鲜见结外淋巴瘤中13q14缺失的研究报告。PGIL是否存在13q14缺失?13q14缺失与PGIL预后的关系如何?需进一步探讨。本研究采用FISH检测PGIL患者胃肠组织石蜡切片中IGH重排、p53、ATM以及13q14等多基因及染色体异常情况,探讨PGIL中多基因及染色体异常与诊断及预后的关系,研究结果将为PGIL个体化的诊断与治疗提供实验依据。回顾性分析PGIL的临床表现、总结临床误诊原因,通过电话随访比较不同治疗方案的生存期,了解当前PGIL诊治现状,客观地解析各种治疗方案的优劣。第一章FISH技术检测胃肠淋巴瘤基因与染色体变异方法的建立目的:建立检测PGIL基因及染色体变异的荧光原位杂交技术(FISH)标准方法。方法:采用增强组织通透性、适当消化、控制变性时间、采用慢洗等方法摸索FISH检测胃肠组织淋巴瘤标本的方法。结果:改良的FISH方法获得了PGIL瘤细胞的P53基因探针(绿色荧光信号)、D13S25基因探针(红色荧光信号)、IGH基因探针(红/绿或黄色荧光信号)、ATM基因探针(红色荧光信号)清晰和稳定的图像。结论:建立了一套检测PGIL的基因及染色体变异的FISH检测技术,为临床研究结外淋巴瘤的基因及染色体变异提供了可供选择的新方法。第二章原发性胃肠淋巴瘤多基因与染色体检测及意义目的:探讨多基因与染色体变异对PGIL的早期诊断、预后判断、及治疗中的作用。方法:采用改良的FISH技术检测72例PGIL患者胃肠组织石蜡切片及30例淋巴结反应性增生石蜡切片中IGH重排、p53、ATM以及13q14等多基因与染色体变化,分析其与诊断及预后的关系。采用t检验及卡方检验,p<0.05认为差异有显著性。结果:①B细胞淋巴瘤中IGH基因重排占70.8%,而T细胞淋巴瘤及淋巴结反应性增生中均未见IGH基因重排(p<0.001);MALT淋巴瘤IGH基因重排率59.5%,非MALT淋巴瘤91.3%(p=0.007);Ⅰ-Ⅱ期IGH基因重排63.8%,Ⅲ-Ⅳ期88.9%(p=0.047);IGH重排的B细胞淋巴瘤平均生存期16.85月,明显短于无IGH重排的平均生存期39.81月(p=0.001)。②MALT淋巴瘤p53基因缺失28.6%,非MALT淋巴瘤53.3%(p=0.034);Ⅰ-Ⅱ期p53基因缺失30.2%,Ⅲ-Ⅳ期63.2%(p=0.011);p53基因缺失者平均生存期10.21月明显短于p53基因正常者32.00月(t=3.930,p<0.001)。③MALT淋巴瘤ATM基因缺失23.8%,非MALT淋巴瘤46.7%(p=0.043);Ⅰ-Ⅱ期ATM基因缺失26.4%,Ⅲ-Ⅳ期52.6%(p=0.038);ATM基因缺失平均生存期12.35月,明显短于ATM基因正常者31.42月(t=3.378,p=0.001)。④13q14缺失在PGIL发生率为34.7%,明显高于淋巴结反应性增生(p<0.001)。但与肿瘤发生部位、患者年龄、病理类型、临床分期以及平均生存期无相关。⑤MALT淋巴瘤中,无基因或单基因改变者平均生存期39.25月,明显长于多基因改变的平均生存期9.11月(p<0.001)。⑥Ⅰ期-Ⅱ期患者中,无基因或单基因改变者平均生存期40.33月,明显长于多基因改变的平均生存期20.61月(p=0.007)。结论:①IGH基因重排在B细胞来源的PGIL中发生率高。②IGH基因重排、p53、ATM基因缺失率在非MALT淋巴瘤及Ⅲ-Ⅳ期患者中发生率较高。IGH基因重排或p53、ATM基因缺失的PGIL患者平均生存期短。③同时存在多个基因与染色体异常的PGIL患者的平均生存期短于无基因或单基因改变者。第三章81例原发性胃肠淋巴瘤的临床分析目的:分析PGIL的临床资料、诊断与治疗现状,以期提高其诊治水平。方法:对81例PGIL的临床表现、内镜特征、病理特点、HP感染、治疗方案、随访进行回顾性分析。采用t检验及卡方检验,P<0.05认为差异有显著性。结果:①胃淋巴瘤平均年龄52.84±15.33岁,大于肠淋巴瘤平均年龄42.09±15.28岁(t=3.087,p=0.003)。②常见症状:腹痛76.5%;消化道出血55.6%;贫血54.3%;低蛋白血症40.7%;体重明显下降33.3%;腹部包块25.9%;肠梗阻11.1%。③发病部位依次为:胃、小肠、回盲部、结肠。④内镜下表现为肿块型67.7%,溃疡型27.7%,弥漫型4.6%。⑤门诊诊断率30.9%,误诊率69.1%。⑥内镜下活检病理确诊率73.8%,活检病理误诊漏诊率26.2%。⑦B细胞来源91.4%,MALT淋巴瘤61.7%。⑧HP检测率39.5%,阳性率37.5%。⑨随访60例,随访率74.1%。5年生存率42.9%,3年生存率60.0%,1年生存率74.0%。⑩随访Ⅰ-Ⅱ期41例平均生存时间30.41月,长于Ⅲ-Ⅳ期19例平均生存时间9.63月(t=3.823,p<0.001)。(?)随访41例MALT淋巴瘤平均生存时间24.24月,与19例非MALT淋巴瘤平均生存时间22.95月无明显差异(t=0.213,p=0.832)。(?)Ⅰ-Ⅱ期中:单纯手术组平均生存时间33.00月,手术加化疗组平均生存时间31.95月,化疗联合根除HP组平均生存时间33.25月。Ⅲ-Ⅳ期中:单纯手术组平均生存时间4.00月,手术加化疗组平均生存时间12.20月,化疗联合根除HP组平均生存时间9.50月。三种治疗方法的生存期在Ⅰ-Ⅱ期患者无差异(p值分别为0.897、0.986、0.916),Ⅲ-Ⅳ期患者中单纯手术组生存时间短于其他两组(p值分别为0.006、0.009),而手术加化疗与化疗联合HP根除治疗组无差异(p=0.558)。结论:①PGIL的临床及镜下表现缺乏特异性,临床误诊率高;②PGIL多属于B-NHL,最常见的病理类型为MALT淋巴瘤;③单纯手术、手术加化疗、化疗联合HP根除治疗三种方法的生存期在Ⅰ-Ⅱ期患者无差异,Ⅲ-Ⅳ期患者中单纯手术组生存时间短于其他两组,而手术加化疗与化疗联合HP根除治疗组无差异。更多还原

【Abstract】 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.更多还原