基于高通量靶向测序的常见实体肿瘤基因突变图谱研究

目的采用含有50个基因组合的高通量靶向测序技术检测多种实体肿瘤的基因突变情况,分析实体肿瘤的基因突变情况,了解实体肿瘤的基因突变图谱,为临床诊疗提供理论依据。方法收集367例多种类型实体肿瘤样本,采用BES 4000平台检测50个基因状态,分析各种肿瘤中的基因突变情况。结果 367例样本中,64.03%的样本至少存在1种基因的突变;共检测到28种基因突变,突变率从高到低的前9个基因分别为EGFR(32.15%)、TP53(15.53%)、K-ras(8.17%)、c-kit(5.72%)、APC(3.00%)、PTEN(3.00%)、B-raf(2.72%)、ERBB2(2.72%)和PIKC3CA(2.72%)。在非小细胞肺癌中突变率最高的基因为EGFR(40.10%),该基因突变在本研究其他恶性实体肿瘤中未被发现。在非小细胞肺癌(NSCLC)、结直肠癌、胃癌和卵巢癌中均检测到TP53的突变,突变率分别为13.61%、34.38%、33.30%和50.00%。在结直肠癌中突变率最高的基因为K-ras(42.86%),胃肠间质瘤中突变率最高的基因为c-kit(53.13%)。在被检测到突变的样本中,28.97%(68/235)的样本存在基因的共突变,TP53的共突变率最高为16.60%(39/235)。统计学分析结果显示,是否发生基因突变与患者性别和年龄无关,但TP53基因突变在≥58岁患者明显高于58岁患者(P0.001)。在非小细胞肺癌中,女性(P0.01)、肺腺癌(P0.001)患者EGFR突变率较高;ERBB2在58岁患者中突变率较高(P=0.001)。结论不同肿瘤中存在不同基因突变图谱,TP53基因突变发生多种实体肿瘤中并且与年龄相关,高通量靶向测序技术可同时检测多种基因的突变情况,为临床治疗决策提供全面指导。     

应用高通量测序技术检测甲状腺乳头状癌相关基因变异

摘要：目的?探讨高通量测序技术(next generation sequencing，NGS)在甲状腺乳头状癌(papillary thyroid cancer，PTC)相关的15个靶基因变异检测中的应用价值。方法?收集2019年1月至2020年6月就诊的188例PTC患者手术切除的癌组织石蜡包埋标本，采用NGS技术检测其中15个目标基因；利用GATK 4.0.2.0、VarScan.v2.3.9和 Factera 1.4.4软件分析基因变异的特点。结果?共计80.32%(151/188)的标本检出突变。其中74.47%(140/188)的标本检出1个点突变或基因融合突变，5.32%(10/188)的标本同时检出2个点突变，0.53%(1/188)的标本同时检出3个点突变。151例突变标本中共检出点突变及基因融合突变163个，其中BRAF基因检出率最高。在所有双突变的标本中，2个不同突变等位基因突变丰度基本一致。在3个基因同时发生突变的标本中，TERT和NRAS基因等位基因突变丰度基本一致，而TP53基因等位基因突变丰度显著低于其他2个基因。PTC患者基因突变与性别(χ2=0.585，P>0.05)、年龄(χ2=0.575，P>0.05)及临床分期(χ2=0.711，P>0.05)均无关。结论?NGS技术检测PTC相关基因突变位点具有经济、效率高和可定量的优势，可为患者的诊断、预后和个体化治疗提供依据。     

基于高通量测序技术的急性髓系白血病患者的基因突变谱和预后分析

目的:探讨急性髓系白血病(AML)患者的基因突变谱及其预后意义.方法:回顾性分析2014年3月至2018年4月于本院采用高通量测序技术(HTS)行基因突变检测的93例初治AML患者的临床资料,描述突变基因的分布情况并对中危组(IR-AML)患者进行预后分析.结果:93例AML患者中有88.17％至少发生1个基因突变,多...     

高通量测序在非小细胞肺癌基因突变研究中的应用

目的对非小细胞肺癌(NSCLC)精准化诊疗相关的28种肿瘤驱动基因进行高通量测序,分析多基因突变与疾病临床特征的关系。方法收集重庆大学附属肿瘤医院2017年1月至2018年10月396例NSCLC患者标本,对EGFR、ALK、ROS1、KRAS、NRAS、HRAS、PIK3CA、TP53、PTEN、BRAF、HER2、RET、MET等28种基因进行高通量测序分析。结果EGFR基因突变占基因突变总检出例数的35.35%,且基因突变频率最高的前5个基因占总突变的63.89%,表现出明显的肿瘤主基因突变聚集现象。EGFR基因亚型同样呈明显的主次分布规律,其中最常见的突变亚型是19del、L858R,分别占45.00%、41.43%。EGFR基因的19del、L858R和20ins 3个亚型多为单突变,而T790M、G719X、S768I、L861Q等亚型则常表现为共突变。T790M伴随L858R突变常常是原发性突变,T790M与19del共突变则常常是获得性突变。EGFR突变标本发生其他多基因突变的检出率显著低于无EGFR突变标本,可能是因为EGFR基因突变抑制了其他基因发生突变。结论高通量测序可高效检测NSCLC患者与靶向治疗相关驱动基因的突变情况,可为研究多基因突变的内在关系及突变负荷提供有效工具,为临床医生制订NSCLC患者的精准诊疗方案提供有力支撑。     

BRCA1/2基因突变乳腺癌患者的心理体验

目的 了解BRCA12基因突变乳腺癌患者的身心需求,探讨她们的心理体验。 方法 采用半结构式访谈,访谈基因突变乳腺癌患者13名,采用Colaizzi现象学研究法分析资料。 结果 BRCA12基因突变乳腺癌患者的心理体验可归纳为4个主题：①为罹患乳腺癌找到理由;②负性情绪;③行为改变;④信息缺失和对专业知识的渴求。 结论 了解基因突变乳腺癌患者的心理体验可以协助医护人员在基因遗传方面并提供完整和持续的信息和建议,舒缓患者的情绪,提高患者的生活质量。     

外显子捕获-高通量测序技术用于法洛四联症胎儿遗传学检测

目的用外显子捕获-高通量测序(exon-capture high-throughput sequencing,EC-HTS)技术寻找法洛四联症(tetralogy of fallot,TOF)胎儿可能存在的遗传学致病性证据,并探讨其在产前分子遗传学诊断中的价值。方法选择经超声心动图发现且G显带和微阵列比较基因组杂交(array-based comparative genomic hybridization,a CGH)分析结果均正常的9例TOF胎儿作为研究对象,以63个人类已知的先天性心脏病致病基因作为检测靶点制作捕获芯片,用EC-HTS技术进行检测,数据经过Calling、数据库比对、软件分析得到最终病理性突变位点;并用Sanger测序对其进行验证。结果在9例胎儿中共检测到单核苷酸多态性(SNP)位点732个,结果经过生物信息学分析得到致病性突变位点3个:CITED2 c.574_579 del AGCGGC(p.S192_G193del纯合突变,CHD7c.2550_2554 del GAGAA(p.K850Nfs*6)杂合突变,NOTCH2 c.4918TC(p.F1640L)杂合突变。Sanger测序结果验证了这3个突变位点的真实存在,父母溯源检测发现这3个点突变均为新发突变。结论用靶向性EC-HTS技术发现了3例TOF胎儿存在病理性基因突变,可能为其致病的遗传学病因。     

基于高通量测序技术的105例AML和MDS患者驱动基因突变谱差异性分析

目的 获得西南地区急性髓系白血病(AML)和骨髓增生异常综合征(MDS)患者的基因突变谱特征,分析其差异性,探讨AML和MDS的差异性指标。方法 回顾性分析2019年9月至2022年12月该院收治的71例AML患者和34例MDS患者的临床资料,将AML和MDS患者分别按年龄进行分组,年龄≥60岁纳入老年组,年龄<60岁纳入中青年组。患者住院期间均采用高通量测序技术进行基因突变谱检测,采用流式细胞术检测分化抗原。通过分析比较基因突变谱的特征,并结合细胞表面分化抗原,获得西南地区AML和MDS患者基因突变谱的差异性。结果 AML和MDS患者老年组与中青年组的中位年龄比较,差异有统计学意义(P<0.05)。AML患者老年组男性比例较高,为76.2%,明显高于中青年组(P<0.05)。AML患者中DNMT3A的突变频率与年龄有关(P<0.05)。CEBPA、FLT3、RUNX1基因突变频率在AML中分别为23.9%、32.4%、9.9%,在MDS中分别2.9%、11.8%、26.5%,差异均有统计学意义(P<0.05)。结论 髓系白血病患者的基因突变频率与患者的年...     

靶向捕获高通量测序技术在检测早期乳腺癌胚系突变中的应用价值

目的 应用靶向捕获高通量测序技术探究早期乳腺癌患者血浆胚系突变情况与临床病理特征的相关性.方法 选取99例早期乳腺癌女性患者,采用靶向捕获高通量测序技术对其血浆标本进行21个乳腺癌易感基因测序,对检测到的突变位点进行筛选和分类,并分析其与临床病理特征的相关性.结果 有48例患者检测出18个基因的55个突变位点,其中包含...     

1例男性乳腺癌患者的临床特征及基因突变分析

目的对1例异时性男性乳腺癌和结肠癌患者及其家系成员进行基因突变分析,以明确病因,便于指导临床风险管理决策。方法采用全外显子测序技术对先证者外周血样本进行基因突变分析,结合表型资料,确定候选基因的可能致病位点。应用Sanger测序技术对先证者及其家系成员候选突变位点进行共分离验证。结果在先证者BRCA2基因第11号外显子中发现c.64026406delTAACT (p.Asn2134fs)杂合突变,该突变在乳腺癌信息中心(BIC)、ClinVar数据库中已有报道,为乳腺癌致病性突变。Sanger测序证实其儿子也为该突变的携带者,而其患结肠癌的母亲未检测到该突变。结论 BRCA2基因c.64026406delTAACT突变是该先证者乳腺癌的致病突变位点,而先证者及其母亲所患结肠癌可能为散发。     

宁夏地区回族女性散发性乳腺癌易感基因BRCA1/BRCA2突变的研究

摘要：目的：研究宁夏地区回族女性散发性乳腺癌中BRCA1/BRCA2基因 (breast cancer susceptibility gene 1/2)的突变位点及携带情况。 方法：收集60例回族居民乳腺癌石蜡包埋组织标本及15例乳腺小叶增生或纤维腺瘤标本。PCR和DNA直接测序法检测BRCA1基因第2、11和20号外显子和BRCA2基因第11号部分外显子突变情况。 结果：60例乳腺癌BRCA1基因有10例突变,突变率为16.7%,突变位点均位于BRCA1基因。15例对照均未检出突变且淋巴结转移与未转移组间BRCA1基因突变率差异（30.8%与5.9%）有统计学意义（P＜0.05）。 结论：BRCA1基因突变可能与宁夏回族女性乳腺癌发生相关。     

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement–negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.The detection of recurrent chromosomal rearrangements by cytogenetics was one of the earliest clinical molecular oncology assays and continues to play a major role in cancer diagnosis and prognosis.^1,2 Although translocations in the clinical laboratory are generally detected by cytogenetics, fluorescence in situ hybridization (FISH), or RT-PCR, studies have demonstrated that they may also be detected by next-generation sequencing (NGS) of DNA or RNA.^3–5 DNA-level translocations can be detected in particular areas of interest by first performing hybrid capture enrichment to target one or both partner genes in a translocation, followed by NGS.^4,6 NGS-based translocation detection has several advantages over conventional clinical laboratory methods, such as the ability to precisely define the breakpoint region, detect cryptic rearrangements and unknown partner genes, and run in parallel with gene mutation detection.Chromosomal rearrangements are detected in the clinical laboratory by routine cytogenetics, FISH, or RT-PCR; however, these methods have limitations. Cytogenetic studies, including chromosome analysis and metaphase FISH, require actively dividing cells, which can be especially difficult to obtain from solid tumors. In addition, chromosome analysis is of limited resolution, particularly in oncology specimens, and is therefore insensitive to cryptic and complex rearrangements.^5,7,8 Some rearrangements can be assayed via RNA-based RT-PCR methods, but this approach is less useful for translocations with a large number of partner genes or those with potentially diverse breakpoints.^9,10 FISH is among the most commonly used laboratory methods for the detection of chromosomal rearrangements and offers high sensitivity and the ability to test routine interphase, formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, FISH relies on highly trained individuals to score rearrangements by fluorescent microcopy and is an inherently low-resolution method that may be confounded by complex, multiway rearrangements and may require numerous probes to fully elucidate translocation partners for promiscuous genes, such as KMT2A.^5,10 Finally, FISH results are generally difficult to validate by orthogonal methods, outside less sensitive cytogenetic assays.Two of the most commonly tested translocations in the clinical laboratory are for rearrangements of the anaplastic lymphoma kinase gene, ALK, in non–small cell lung cancer and of the mixed-lineage leukemia gene, KMT2A (formerly known as MLL), in acute leukemia. The EML4-ALK fusion results from an inversion event on chromosome 2p that generally causes an in-frame fusion of EML4 exons 1 to 13 to ALK exons 20 to 29, producing an aberrant fusion gene with constitutive kinase activity, sensitive to crizotinib.^11–14 The occurrence of ALK fusions and other common lung cancer gene mutations in KRAS and EGFR are generally considered to be mutually exclusive, arguing that these tumors represent a distinct subset of lung cancers.¹⁵ Although not pharmacologically targetable, KMT2A rearrangements are of diagnostic and prognostic significance in acute leukemias, including both acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL).^16,17 KMT2A rearrangements can be readily detected by FISH using break-apart probes; however, elucidation of the translocation partner gene may be difficult because >100 have been identified.^10,18NGS has had a tremendous effect on cancer discovery and is now becoming routine in the clinical molecular oncology laboratory.^3,19–21 NGS allows for the cost-effective, simultaneous evaluation of numerous sequence variants as part of focused clinical oncology panels or whole exomes. We and other groups have previously found that a range of DNA variants, including translocations, insertions or deletions, and copy number variants, can be detected from targeted NGS data and that it is possible to identify DNA-level breakpoints with single-nucleotide precision.^4,22,23 However, to be useful in the clinical setting, a thorough evaluation of the sensitivity and specificity of structural variation (SV) detection by NGS compared with standard methods is required. Given that numerous potential translocations can be evaluated by NGS simultaneously as part of a larger NGS cancer panel, for little to no additional cost, such methods could provide a significant savings for laboratories that perform multiple single-gene tests and multiple FISH assays on oncology specimens.We present a comprehensive evaluation of targeted translocation detection by NGS in the clinical laboratory by comparing four publicly available translocation detection tools (including the laboratory derived ClusterFAST) on targeted NGS data from 13 cases with ALK or KMT2A rearrangements (six lung carcinomas and one anaplastic large cell carcinoma with ALK rearrangements; six leukemias with KMT2A rearrangements) and 77 cancers negative for ALK and KMT2A rearrangements by FISH. We found that translocations can be reliably detected at the DNA level by targeted NGS panels and that such methods offer sensitivity and specificity similar to that of routine FISH with the advantage of single-nucleotide breakpoint resolution. Further, we examine approaches to designing capture probes for targeted NGS evaluation, evaluate the minimal coverage levels necessary to detect translocations, and explore methods to reduce false-positive translocation reports.     

早期上皮性卵巢癌的临床与预后

[目的]分析早期上皮性卵巢癌的临床特点、预后及其影响因素,探讨早期患者的理想治疗方案.[方法]回顾性分析1993～1998年间湖南省肿瘤医院治疗的60例早期上皮性卵巢癌患者的临床资料.[结果]患者平均年龄41岁,≤35岁22例,占36.7%.多因素分析显示FIGO分期及组织病理分级是独立预后因素.FIGOⅠA-B期患者...     

非小细胞肺癌组织中NSE 的基因表达及与患者临床特征的相关性研究

目的　探讨非小细胞肺癌(non-small cell lung cancer, NSCLC) 患者肿瘤组织中神经元特异性烯醇化酶(neuron-specific enolase, NSE) 的基因表达及与患者临床特征的相关性。方法　选取2013 年1 月～ 2017 年12 月商洛市中心医院157 例NSCLC 患者为研究对象,采用电化学发光法检测患者血清中NSE 的水平,采用PCR 法比较NSCLC 组织和相应癌旁正常组织中NSE 的相对表达量,并且探究NSE 的表达水平与患者临床特征间( 年龄、性别、是否吸烟、是否饮酒、临床分期、是否原位癌、有无淋巴结和远端转移) 的相关性。结果　NSE 在NSCLC 组织中的相对表达量显著高于相应癌旁正常组织,其差异具有统计学意义 (P=0.0182)。NSE 表达水平与NSCLC 患者是否吸烟(χ2 ＝ 9.959,P=0.002)、是否饮酒(χ2 ＝ 4.893,P=0.032)、临床分期(χ2 ＝ 11.070,P=0.001)、是否原位癌(P<0.001)、有无淋巴结(P<0.001) 和远端转移显著相关(P=0.017)。结论　NSE 在NSCLC 组织中高表达,血清NSE 水平可作为NSCLC 诊断和预后的生物指标。     

肺癌患者血浆miR-3151 表达水平与临床病理特征的相关性研究

目的　探讨肺癌患者血浆中miR-3151 的表达及其与患者临床病理特征的关系。方法　收集十堰市太和医院2018 年10 月～ 2019 年10 月确诊的120 例肺癌患者（肺癌组）及同期120 例体检健康者（对照组）的血浆样本,实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）技术检测两组血浆miR-3151 的表达水平,分析血浆miR-3151 的表达与肺癌患者临床病理特征的关系。受试者工作特征曲线（receiver operating characteristic curve,ROC）用以评估血浆miR-3151 单独及联合细胞角蛋白19 片段（cytokeratin-19 fragment,CYFRA21-1）、神经元特异性烯醇化酶（neuronspecificenolase,NSE）和癌胚抗原（carcinoembryonic antigen,CEA）对肺癌的诊断价值。结果　肺癌组血浆miR-3151（nmol/L）显著高于对照组[86.356（19.893,305.155）vs 8.871（4.620,14.037）],差异有统计学意义（Z=-10.039,P ＜ 0.001）。血浆miR-3151 的表达上调与肺癌患者淋巴结转移（Z=-2.336,P=0.019）和临床分期（Z=-2.628,P=0.009）相关。血浆miR-3151,CYFRA21-1,NSE 及CEA 单独用于诊断肺癌的曲线下面积（area under the cure,AUC）分别为0.875（95%CI:0.830 ～ 0.919）,0.704（95%CI:0.639 ～ 0.770）,0.769（95%CI:0.707 ～ 0.830）和0.632（95%CI:0.562 ～ 0.702）,当miR-3151 联合NSE 时,诊断肺癌的AUC 增加至0.909（95%CI:0.872 ～ 0.946）,较单独诊断时有明显提高。结论　miR-3151 在肺癌患者血浆中表达上调,与肿瘤进展相关,有成为肺癌诊断标志物的潜能。     

鼻咽癌患者血清同型半胱氨酸水平与临床病理特征的相关性分析

目的 研究鼻咽癌患者血清同型半胱氨酸(HCY)水平与临床病理特征的相关性,以及HCY在鼻咽癌疗效监测及预后判断中的作用。方法 选取郴州市第一人民医院南院2017年3月～2018年3月初次经病理确诊未经治疗的鼻咽癌患者作为观察组(n=72),体检中心就诊的健康体检人群作为对照组(n=81)。采集鼻咽癌患者治疗前、健康体检者当日空腹静脉血,离心分离血清,采用循环酶法检测血清HCY。比较两组人群的血清HCY水平,分析血清HCY水平与鼻咽癌患者临床病理特征的相关性。结果 鼻咽癌患者血清HCY为14.44±4.24 μmol/L,健康体检人群血清HCY为10.96±2.06 μmol/L。鼻咽癌患者、健康体检人群血清HCY异常率分别为93.05%(67/72)和58.02%(47/81)。鼻咽癌患者血清HCY水平、HCY异常率明显高于健康体检人群,差异有统计学意义(t=-6.331,P<0.05; t=24.629,P<0.05)。血清HCY诊断鼻咽癌的敏感度为93.05%(67/72),特异度为41.98%(34/81)。血清HCY水平与鼻咽癌患者年龄、性别、肿瘤的分化程度以及临床分期相关(t=2.011,-2.673,-2.303,-2.409,均P<0.05); 与鼻咽癌浸润深度、淋巴结转移及远处转移无关(t=0.042,0.537,0.238,均P>0.05)。结论 鼻咽癌患者血清HCY水平明显升高,且与年龄、性别、肿瘤分化程度以及临床分期相关。血清HCY有望成为鼻咽癌疗效监测及预后判断的肿瘤标志物。     

卵巢癌患者的症状群与其生活质量的相关性

目的调查卵巢癌患者的症状群,并探讨症状群与其生活质量的相关性。方法便利抽样法选择2012年5-10月在山东省济南市某三级甲等医院妇科病房接受治疗的卵巢癌患者130例,采用安德森症状评估表(M.D.Anderson symptom inventory,MDASI)和卵巢癌治疗功能评估量表(the functional assessment of cancer therapy scale-ovarian,FACT-O)对其进行调查。结果卵巢癌患者有较高内部一致性的4个症状群分别为情感性症状、躯体性症状、胃肠道症状及治疗不良反应,其Cronbachα系数分别为0.838、0.632、0.715和0.613。患者生活质量评分均值为(96.78±17.13)分,与4个症状群均呈负相关(均P0.01)。其中情感性症状群、躯体性症状群及胃肠道症状群被纳入回归方程。结论卵巢癌患者存在情感性症状、躯体性症状、治疗不良反应及胃肠道症状四个主要症状群,其中情感性症状群、躯体性症状群、胃肠道症状群是生活质量的重要影响因素。因此,医护人员可以进行有针对性的治疗及心理疏导,以提高患者的生活质量。