【作者】 侯静；

【导师】 周桥；

【作者基本信息】 四川大学 ， 临床病理学（专业学位）， 2021， 硕士

【摘要】 背景与目的:TCEB1(transcription elongation factor B)基因突变型肾细胞癌是新发现的肾脏肿瘤,在2016版世界卫生组织(WHO)肾脏肿瘤分类中尚未纳入。该类肾细胞癌虽然与经典的透明细胞肾细胞癌在形态学上有重叠,但仍具有较独特的形态学特点、免疫表型和分子遗传学改变,可能是独立的肾细胞癌亚型。本研究的目的是(1)分析TCEB1基因突变型肾细胞癌的临床病理特点;(2)分析TCEB1基因突变型肾细胞癌的基因改变/分子遗传学特征,探讨其分子基础。材料和方法:(1)复习四川大学华西医院2014年1月至2020年12月确诊的肾细胞癌共3406例,筛选出免疫组化CAIX(+)、CK7(+)的病例263例。由两位泌尿病理亚专业医师评估形态学特点,筛选有胞质透明的肿瘤细胞构成的上皮成分并有纤维/平滑肌间质成分的病例共147例,进行TCEB1基因突变检测分析,确诊TCEB1基因突变型肾细胞癌30例。另纳入来自院外会诊并经基因突变检测确诊为TCEB1基因突变型肾细胞癌病例1例,以及经NGS检测发现的TCEB1基因突变型肾细胞癌2例。本研究共获得经分子检测确诊的TCEB1基因突变型肾细胞癌33例。对33例TCEB1基因突变型肾细胞癌进行FISH检测验证是否存在染色体3p缺失;总结这些病例的临床表现,评估组织形态学特点,进行CAIX、AMACR、CK7、CD10、PAX8、SDHB、FH、TFE3等免疫组织化学染色检查。获取并分析本组病例的随访结果。(2)选送22例经Sanger测序证实的TCEB1基因突变型肾细胞癌和6例Sanger测序阴性病例6例。镜下评估肿瘤占比,选择最佳的石蜡组织提取DNA进行二代测序。使用实体肿瘤539基因Panel进行测序,生物信息学技术分析突变类型、拷贝数变异等,计算肿瘤突变负荷(TMB)。运用DAVID在线工具对突变基因进行通路富集分析,探讨TCEB1基因突变型肾细胞癌发生发展的可能分子通路。结果:本研究共获得经分子检测确诊的TCEB1基因突变型肾细胞癌33例,临床病理资料及分子遗传学结果总结如下:(1)临床特点:患者年龄25.0～69.0岁(平均50.3),40～60岁居多。男性患者明显多于女性(M:F=32:1),33例病患中仅有一名女性患者。患者多无明显临床症状,常偶然发现肾脏占位(71.8%),少数患者(28.2%)出现腰腹部疼痛、尿频尿急等症状。肿瘤常累及单侧肾脏,未发现患者有任何遗传性综合征表现。TNM分期均为Ⅰ期,其中T1a期患者31例(94.0%),T1b期患者2例(6.0%)。33例患者均接受手术治疗,包括根治性肾切除术7例(21.2%),保留肾单位手术26例(78.8%)。(2)病理特征:肿瘤最大径1.0～5.0cm,平均直径2.5cm。切面呈实性(18例,54.5%)或伴囊性变(15例,45.5%)。镜下可见所有肿瘤周围有厚纤维被膜包裹,与周围正常肾脏组织分界清楚。肿瘤细胞形成的组织结构多样,可形成腺泡状、分支管状、乳头状、实性片状等结构。29例(87.9%)病例中可见到大小不等的富含甲状腺胶质样物的腺腔样结构,腺腔中可见到胞质透明的细胞构成的小乳头,构成乳头结构的细胞可见分泌现象。肿瘤细胞大多胞膜清楚,胞质透明或嗜酸性,含丰富的胞质,个别病例胞质稀少。肿瘤细胞胞核圆形或卵圆形,多位于基底部,5例(15.1%)肿瘤的局灶区域可见肿瘤细胞胞核远离基底部,形成类似于透明细胞乳头状肾细胞癌的经典组织结构。肿瘤细胞核级较低,按WHO/ISUP核分级标准:4例为1级,29例为2级。所有肿瘤均未见坏死和淋巴管、血管侵犯,核分裂少见。所有肿瘤均可见数量不等纤维/平滑肌间质成分,分割肿瘤。富于淋巴细胞和间质水肿等区域亦可在部分肿瘤中见到。(3)免疫表型:肿瘤细胞CAIX弥漫强阳性,胞膜呈“盒状”阳性模式(完整膜阳性)。所有病例均不同程度表达CK7,11例(33.3%)呈弥漫强阳性,13例(39.4%)部分肿瘤细胞中等强度阳性,9例(27.3%)表现为局灶弱阳性。CD10在多数病例中阳性,表达强度不等,15例(48.3%)肿瘤细胞呈弥漫强阳性,14例(45.2%)肿瘤细胞呈中等强度阳性或弱阳性,仅2例(6.5%)阴性。19例(79.2%)肿瘤细胞部分弱阳性表达AMACR;6例(20.8%)AMACR阴性。所有肿瘤细胞PAX8均为阳性。SDHB、FH均未见缺失。TFE3阴性。(4)分子遗传学特点:33例TCEB1基因突变型肾细胞癌突变密码子多样,其中79密码子为突变热点,共30例(28例Y79C,2例Y79S),另检测出95密码子突变(I95N)、92密码子(E92K)和112密码子(C112fs)各1例。所有病例均未见染色体3p缺失。NGS检测结果显示24例肿瘤共251个体细胞突变,涉及128个基因,错义突变最常见(169个),其余突变方式有同义突变、移码突变等。除TCEB1基因之外,TSC基因为其中突变率最高的基因,发生于13例(54.2%)病例中,其中6例为TSC1基因突变,6例为TSC2基因突变,1例为TSC1与TSC2基因共同突变。6例TSC1基因突变病例中2例为同义突变,余4例均为剪切变异伴错义突变:3例为322密码子突变(M322T);另1例有2个密码子突变:84密码子突变(A84T)和322密码子突变(M322T)。6例TSC2基因突变病例中3例为同义突变,余3例均为错义突变:1例为196密码子突变(A196T);1例为286密码子突变(M286V);1例为1036密码子突变(S1036T)。同时伴TSC1和TSC2基因的病例表现为:TSC1基因剪切变异伴322密码子(M322T)错义突变;TSC2基因1744密码子错义突变(L1744F)。拷贝数分析发现4例(16.7%)出现基因拷贝数增加,其中2例(8.3%)出现FGF3基因增加,1例出现ERCC1基因增加,1例为FGF3基因和FGF4基因增加。TMB分析发现23例TMB值有效,TMB范围为0.849～7.752 mut/Mb,中位值为3.917 mut/Mb,平均值3.763 mut/Mb,有8例(34.8%)患者显示TMB大于5。TMB值的高低与年龄(P=0.324)、有无TSC基因突变(P=0.166)、79号密码子突变类型(P=0.268)均无明显相关性。。(5)预后分析:本组病例随访时间4.0～82.0月,中位随访时间19.0月,平均随访时间26.7月。随访截止时,所有患者均存活,未见肿瘤复发和转移。结论:1.TCEB1基因突变型肾细胞癌是较为罕见的肾脏肿瘤,占本研究统计我院2014～2020年3406例肾细胞癌的0.94%,临床上常为偶然发现的肾脏占位,少数可有腰腹部疼痛、尿频尿急等症状。好发于40岁～60岁,男性明显多于女性。TCEB1基因突变型肾细胞癌预后较好,生物学行为多为惰性。2.TCEB1基因突变型肾细胞癌病理特征包括:大体边界较清,有纤维性被膜;镜下由胞质透明的肿瘤细胞形成腺泡状、分支管状、乳头状等多种结构,常可见甲状腺滤泡样胶质,伴纤维/平滑肌间质成分。组织学分级常为WHO/ISUP 1-2级。3.免疫组化示TCEB1基因突变型肾细胞癌呈CAIX弥漫完整膜阳性,CK7和CD10不同程度阳性,AMACR阴性或弱阳性。4.TCEB1基因突变型肾细胞癌常为TCEB1基因Y79C突变,少数为E92K、I95N或C112fs突变。不伴有染色体3p缺失。本组高通量基因测序研究发现54.2%病例伴有TSC1/TSC2基因突变;4.2%伴随FGF3基因拷贝数增加。这些新发现的突变可能参与部分TCEB1基因突变型肾细胞癌的发生,并可能有潜在治疗意义。更多还原

【Abstract】 Background and Objective:TCEB1-mutated renal cell carcinoma is a rare type of kidney tumor,which was not included in the 2016 World Health Organization(WHO)classification of renal tumor.The morphological characteristics,immunophenotype and molecular genetic changes of TCEB1-mutated renal cell carcinoma are different from classical clear cell renal cell carcinoma,which indicates it may be a novel entity of renal cell carcinoma.The objectives of this study are to analyze the clinicopathological features of TCEB1-mutated renal cell carcinoma and to analyze the genetic changes and explore the molecular basis of this type kidney tumor by next generation sequencing(NGS).Materials and Methods:(1)Pathological screening found 263 cases of i mmunohistochemically CAIX and CK7 positive cases of renal cell carcinomas(RCC)from a total of3406 RCCs diagnosed between 2014 and 2020 in West China Hospital of Sichuan University.Morphological features were evaluated by two genitourinary pathologists independently.147 tumors with both clear cell RCC cells and fibrous//leiomyomatous stromal components were further selected.DNA was extracted and Sanger sequencing was performed to detect whether there was TCEB1 gene mutation.31 cases were confirmed by Sanger sequencing,and additional 2 cases were discovered by NGS.FISH was performed to verify the status of chromosome 3p.The clinical features,morphologic characteristics and immunophenotype of these cases,as well as follow-up data,were analyzed.(2)24 cases of RCC with TCEB1 mutation were selected for NGS.The slides of twenty-four cases of TCEB1-mutated RCC were reviewed to select the best paraffin block to extract DNA for genetic analysis.A 539 gene panel was used for NGS.The sequencing data were analyzed by bioinformatics tools for mutation types and copy number variations,and to calculate the tumor mutation burden(TMB).The David tool was used to analyze the mutant geneenriched pathways to explore the possible molecular basis of the pathogenesis of TCEB1-mutated renal cell carcinoma.Results:A total of 33 TCEB1-mutated renal cell carcinomas were confirmed by molecular study.The clinicopathological characteristics and molecular genetic features were summarized as follows:(1)Clinical features: Patients were 25.0～69.0 years old(mean 50.3),and the majority of patients were middle-aged and elderly.Male patients outnumbered significantly female patients(M:F=32:1),and there was only one female patient among 33 patients.Most of the patients(71.8%)were found incidentally by physical examination with no obvious clinical symptoms,and a few patients(28.2%)showed lumbar and abdominal pain,urination disfunction and other symptoms.This tumor usually presents as unilateral lesion with no associated genetic syndromes.Most of the patients were TNM stage Ⅰ,of which 31 patients(94.0%)were diagnosed as T1 a and 2 patients(6.0%)were diagnosed as T1 b.All 33 patients received surgical treatment,including radical nephrectomy in 7 cases(21.2%)and partial nephrectomy surgery in 26 cases(78.8%).(2)Pathological features: The maximum diameter of the tumors was1.0～5.0cm(mean: 2.5cm).The tumor was solid(18 cases,54.5%)or solidcystic(15 cases,45.5%).Microscopically,all the tumors were wellcircumscribed by thick fibrous capsule.Tumor cells with clear cytoplasm often form acinar,branching tubular,papillary,solid sheet structures.In 29 cases(87.9%),macro-and micro-follicular architectures,composed of vacuole-rich cells can be observed.These follicles were filled with colloid-like material,reminiscent of thyroid follicles.Some crowding cells resulted in the formation of small papillae,which projected into the follicular lumen with different sizes of glandular lumen.The membranes of the tumor cells were prominent.Cytoplasm of tumor cells were abundant and clear.However,hazy and eosinophilic cytoplasm can be seen in some areas of some cases.The tumor cell nuclei were round or oval.Most of the tumor nuclei were located at the base of the tumor cells.However,nucleus located far away from the basement can be observed in 5 cases(15.1%),forming a structure similar to clear cell papillary renal cell carcinoma(ccp RCC).The tumor nuclear grade was low with 29(87.9%)cases of WHO/ISUP grade 2,and 4 cases(12.1%)WHO/ISUP grade 1.No necrosis,lymphatic or vascular invasion was observed.Mitotic figures were rare.Tumor cell nests were separated by various amounts of fibrous/leiomyomatous stroma.Areas rich in lymphocytes and edematous interstitial can also be seen in some tumors.(3)Immunophenotype: All tumors were diffused and strongly positive for CAIX,in a "box-like" positive pattern.CK7 was expressed in all cases,however,only 11 cases(33.3%)showed diffusely strong positivity,13 cases(39.4%)showed moderately positivity in some tumor cells,and 9 cases(27.3%)showed weakly positive pattern in focal areas.CD10 was positive in most cases,but the expression intensity was variable.The tumor cells were diffusely and strongly positive in 15 cases(48.3%),moderately or weakly positive in 14cases(45.2%),and negative in only 2 cases(6.5%).Nineteen cases(79.2%)were weakly positive for AMACR in some cells and 5 cases(20.8%)were negative for AMACR.PAX8 was positive in all tumors.SDHB and FH were all positive(no loss of expression).No expression of TFE3 was observed.(4)Molecular characteristics: Various mutated codon were detected in TCEB1-mutated renal cell carcinomas.Codon 79 was the hot spot detected in30 cases(28 Y79 C,2 Y79S).In addition,there was one with codon 95 mutation(I95N).And two cases detected novel mutation sites,one with codon 92(E92K)and one with codon 112(C112f S).No deletion of chromosome 3p was observed in all 33 cases.The results of NGS showed that there were 251 somatic mutations in 24 tumors,involving 128 genes.Missense mutations were the most common.In addition to TCEB1,the mutation of TSC was the most common,which occurred in 13 cases(54.2%),including 6 TSC1 mutations,6 TSC2 mutations,and 1case with TSC1 and TSC2 co-mutation.Among the 6 TSC1 mutations,2 cases were synonymous mutations,and the remaining 4 cases were missense mutations accompanied with splicing region variation: 3 cases were codon 322mutations(M322T).Another case had two mutations: codon 84 mutation(A84T)and codon 322 mutation(M322T).Among the 6 TSC2 mutations,3were synonymous mutations,and the remaining 3 were missense mutations:one case harbored codon 196 mutation(A196T).One case harbored codon286 mutation(M286V).One case harbored codon 1036 mutation(S1036T).The case harbored TSC1 and TSC2 co-mutation were characterized by TSC2L1744 F missense mutation and TSC2 M322 T missense mutation accompanied by splicing region variation.Copy number analysis revealed gene copy number gain could be seen in4 cases(16.7%),including FGF3 gain in 2 cases(8.3%),ERCC1 gain in 1 case,and FGF3 and FGF4 gain in 1 case.TMB analysis showed TMB range of 0.849 to 7.752 m ut/MB(median:3.917 mut/MB,mean: 3.763 mut/MB).Eight patients(34.8%)showed TMB greater than 5.There was no significant correlation between TMB and patient age(P=0.324),TSC mutation status(P=0.166),and TCEB1 mutation type(P=0.268).KEGG analysis showed that the mutant genes were mainly enriched in the PI3K-Akt signaling pathway and the RAS signaling pathway.(5)Prognostic analysis: The follow-up of 33 patients were available with a follow-up time of 4.0～82.0 months(median: 19.0 months,mean: 26.7months).All patients were alive without tumor recurrence or metastasis.Conclusion:(1)TCEB1-mutated renal cell carcinoma is a rare renal tumor(accounting for 0.94% of all RCCs in our series).Most of the patients have no clinical manifestations.Some patients may present lumbar and abdominal pain,urination abnormality.There was significant male predominance,and patient age mainly between 40 and 60.The tumors exhibited indolent behavior.(2)The pathological features of TCEB1-mutated renal cell carcinoma include:well-circumscription with fibrous capsule;tumor cells with clear cytoplasm forming various structures such as alveolar,branching tubular,and papillary structures;thyroid follicle-like colloid;variable amount of fibrous/leiomyomatous stroma.The nuclear grade is WHO/ISUP 1-2.(3)TCEB1-mutated renal cell carcinoma were strongly and diffusely positive for CAIX.The tumor cells were weakly/ moderately and focally positive for CK7CD10 was variably positive in most tumors.(4)TCEB1-mutated renal cell carcinoma most commonly harbored TCEB1Y79 C mutation.E92 K,I95N and C112 fs are rare mutation.Th ere was no loss of chromosome 3p.Our study found that 54.2% cases harbored TSC1/TSC2 mutations,and 4.2% could be found copy number gain of FGF3.更多还原