基于生物信息学分析的自噬相关基因构建肺鳞状细胞癌预后模型及验证

目的基于生物信息学分析的自噬相关基因构建肺鳞状细胞癌(SqCLC)预后模型并验证。方法从癌症基因组图谱(TCGA)数据库下载268例SqCLC患者的表达谱数据和临床信息, 从基因型组织表达(GTEx)数据库下载336名健康人正常肺组织的数据集;从人类自噬数据库(HADb)及分子特征数据库(MSigDB)6.2中的GO_AUTOPHAGY基因组中获得自噬相关基因组。应用R 4.0.3软件分析TCGA数据库中SqCLC组织与GTEx数据库中正常肺组织间差异表达基因。使用R 4.0.3软件筛选TCGA数据库SqCLC组织和正常肺组织间差异表达的自噬相关基因(简称:差异表达自噬基因)。应用Cox比例风险模型分析TCGA数据库SqCLC患者差异表达自噬基因与预后的关系, 构建预后模型。依据预后模型风险评分中位数将TCGA数据库SqCLC患者分为高风险组和低风险组, 采用Kaplan-Meier法比较两组总生存;绘制应用预后模型预测的TCGA数据库268例患者3、5、10年总生存率的时间相关受试者工作特征(ROC)曲线。采用Cox回归分析TCGA数据库SqCLC患者总生存的独立...     

基于炎症反应相关基因构建结肠癌预后模型及验证

目的应用生物信息学方法筛选结肠癌预后相关的炎症反应相关差异表达基因, 构建并验证结肠癌预后模型。方法从癌症基因组图谱(TCGA)数据库中检索472例结肠癌患者及41名健康人正常结肠组织的RNA测序和临床数据。从国际癌症基因组联盟(ICGC)数据库中检索结肠癌预后相关基因表达及临床数据。检索时间均为建库至2022年11月。通过基因集富集分析(GSEA)数据库获取200个与炎症反应相关的基因, 将其与TCGA数据库中获得的结肠癌和正常结肠组织的RNA测序基因数据集进行对比, 获得炎症反应相关的差异表达基因。采用Cox比例风险模型分析评估TCGA数据库中与预后相关的差异表达基因, 炎症反应相关的差异表达基因与预后相关的差异表达基因取交集, 获得预后相关的炎症反应相关差异表达基因。通过LASSO Cox回归构建结肠癌预后模型。计算风险评分, 按风险评分的中位值将TCGA数据库结肠癌患者分为低风险(<中位值)和高风险(≥中位值)两组。对两组患者进行主成分分析(PCA), 采用Kaplan-Meier法进行生存分析。基于R软件timeROC程序包分析风险评分预测TCGA数据库结肠癌患者总生存...     

血液肿瘤患者IDH1/IDH2基因突变发生率及其血液病分布特点研究

目的研究血液肿瘤患者IDH1/IDH2基因突变发生率和其血液病的分布特点。方法选取300例不同病情的血液患者骨髓的样品,其中245例是血液肿瘤患者,55例是非血液肿瘤患者,对比检查IDH2和IDH1基因是否在血液肿瘤患者中有突变行为。结果实验共发现了7个错义的突变基因,其中2个为IDH2基因突变,4个为IDH1基因突变;还有2个IDH1同义突变。4个IDH1的突变类型是:199M、R132C、R131D及R132H,前3个突变患者都出现在3例急性髓系的白血病患者中,最后1例出现在急性淋巴细胞的白血病患者中。2个IDH2的错义突变类型是:P167R和R172K,这2例也都出现在急性髓系的白血病患者中。在发生IDH1错义基因突变的4例患者中,其体内白细胞的计数明显升高,且患者年龄均大于60岁。在发生IDH2基因突变的2例患者中,其体内白细胞的计数明显降低,且患者的年龄均小于60岁。结论 96例的急性白血病患者的检测中,共发现了9例突变患者,其IDH的突变率是9.3%,且7例发生于急性髓系的白血病患者中,说明急性髓系的白血病的发病和IDH的突变有紧密的联系。     

胶质瘤中异柠檬酸脱氢酶的检测及其临床意义

目的 分析异柠檬酸脱氢酶(IDH)在胶质瘤中的突变情况,探讨IDH基因突变对胶质瘤的诊断和预后意义.方法 通过Sanger DNA测序法检测胶质瘤中IDH基因突变情况,同时比较分析IDH突变在不同人群中的突变频率以及对预后的影响.结果 20例胶质瘤患者中发现IDH突变7例,均为IDH1 R132H(C.395G＞ A)突变,突变率为35.0％;存在IDH突变的胶质瘤患者无进展生存期和总生存期优于IDH野生型患者.结论 IDH突变对胶质瘤的辅助诊断和预后判断具有重要的临床意义.     

多发性骨髓瘤驱动基因的筛选

  目的  鉴定并比较不同测序方案中多发性骨髓瘤（multiple myeloma,MM）潜在的驱动基因,为探讨MM的发生机制提供研究基础。  方法  选择两套数据,一套数据为2016年5月至2020年11月苏州大学附属第二医院84例MM患者的基因组靶向突变测序数据,另一套数据从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库下载的205例MM全基因组测序数据。在R4.1.0环境下,利用驱动基因识别算法对MM的潜在驱动基因进行挖掘,分析并比较两套数据筛选出的驱动基因及具体突变信息。  结果  NRAS、KRAS、TP53、IDH1为两套数据共同识别出的驱动基因。两套数据中突变基因在NOTCH、RTK-RAS、Cell-Cycle、TP53、MYC及WNT通路均有富集。显著突变的NRAS、KRAS、TP53和IDH1在两套数据中发生突变的位点无显著性差异。另外,通过靶向突变测序方法,鉴定出MM新的驱动基因,CBL、BCOR和DNMT3A。  结论  通过本研究获得了MM可能的驱动基因,有助于从基因分子层面揭示MM发生发展机制。      

基于生物信息学筛选的结肠癌细胞焦亡相关基因的分析及预后模型的构建

目的探讨应用生物信息学筛选的结肠癌细胞焦亡相关基因特点, 对基于差异表达的细胞焦亡相关基因构建的结肠癌预后模型予以验证。方法从癌症基因组图谱(TCGA)数据库下载结肠癌患者RNA测序的基因数据和临床数据。通过检索文献确定52个与细胞焦亡相关基因, 将其与TCGA数据库中获得的结肠癌和正常结肠组织的RNA测序基因数据集进行对比, 获取差异表达的临床样本细胞焦亡基因, 应用STRING网站和R软件分析这些基因蛋白质互作网络。依据TCGA数据库临床样本中细胞焦亡基因的差异表达情况, 将TCGA数据库中结肠癌患者分为焦亡组和未焦亡组, 根据基因表达量, 以P<0.05筛选两组间差异表达明显的基因;基于这些差异表达基因, 采用LASSO Cox回归构建细胞焦亡相关的结肠癌预后模型。按照模型计算的风险评分的中位值将从TCGA数据库中收集的患者分为高风险(≥中位值)和低风险(<中位值)两组, 通过Kaplan-Meier生存函数分析两组总生存, 采用R软件的timeROC程序包分析应用风险评分预测TCGA数据库结肠癌患者生存不同时间的效能。采用多因素Cox回归分析临床病理因素及模型风险评...     

基于单细胞RNA测序的膀胱癌预后风险模型的构建与验证

目的基于单细胞RNA测序(scRNA-seq)生物信息学分析的预后相关的差异表达基因构建膀胱癌预后风险模型并验证。方法从基因表达综合(GEO)数据库中下载膀胱癌scRNA-seq数据集GSE135337、GSE129845, 数据更新时间分别为2022年、2019年;下载常规转录组数据集GSE13507(数据更新时间为2020年)中165例膀胱癌样本的表达谱及其生存信息。从癌症基因组图谱(TCGA)数据库中下载414例膀胱癌样本和19例癌旁样本的表达谱数据及405例膀胱癌患者的临床信息。采用R 4.1.2软件对GEO数据库中的10例膀胱癌单细胞样本进行质量控制及降维聚类并对其进行细胞注释;采用CellChat分析GEO数据库中单细胞数据的细胞间通信。采用单因素Cox比例风险模型分析筛选与膀胱癌预后相关的差异表达基因, 并使用LASSO-Cox回归分析构建预后风险模型, 计算风险评分。根据中位风险评分, 以TCGA数据集中膀胱癌患者为训练集, 将患者分为低风险组和高风险组;采用GEO数据库GSE13507数据集为验证集进行验证, 通过Kaplan-Meier分析比较训练集、验证集低风险组...     

肿瘤坏死因子受体超家族成员21在肝细胞癌中的表达及临床意义

目的 基于生物信息学分析探讨肿瘤坏死因子受体超家族成员21(TNFRSF21)在肝细胞癌(HCC)中的表达和预后价值、潜在的生物学功能及对免疫微环境的影响.方法 从高通量基因表达(GEO)数据库获取HCC细胞株数据集筛选共交集基因.从癌症基因组图谱(TCGA)数据库获取HCC样本数据信息,分析TNFRSF21表达与总生...     

Hsa-miR-98-5p/DKK3信号轴对乳腺癌细胞生物学行为的影响

背景与目的:乳腺癌威胁着全世界女性的健康.虽然已发现大量微小RNA(microRNA,miRNA)在乳腺癌中异常表达,但仍需要构建一个完整的miRNA-信使RNA(messenger RNA,mRNA)网络.方法:利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库下载乳腺癌相关数据集,...     

胃癌组织的LCMT1-AS2水平及临床意义

目的 探讨胃癌组织中长链非编码RNA LCMT1反义RNA 2(LCMT1-AS2)的水平及临床意义.方法 基于UALCAN在线分析癌症基因组图谱(TCGA)数据库中375例胃癌组织和32例正常组织的LCMT1-AS2水平,通过NCBI提供的GEO2R平台检索GEO数据库GSE54129中111例胃癌组织和21例正常组...     

Immunohistochemical detection of IDH1 mutation, p53, and internexin as prognostic factors of glial tumors

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, are specific to arginine 132 (R132). Recently, we established monoclonal antibodies (mAbs) against IDH1 mutations: anti-IDH1-R132H and anti-IDH1-R132S. However, the importance of immunohistochemistry using the combination of those mAbs has not been elucidated. For this study, 164 cases of glioma were evaluated immunohistochemically for IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs (HMab-1 and SMab-1). IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary grade IV, grade III, and grade II gliomas. For each grade of glioma, prognostic factors for progression-free survival and overall survival were evaluated using clinical and pathological parameters in addition to IDH1 immunohistochemistry. IDH1 mutation, p53 overexpression, and internexin expression, as evaluated using immunohistochemistry with clinical parameters such as degree of surgical removal and preoperative Karnofsky Performance Status (KPS), might be of greater prognostic significance than histological grading alone in grade III as well as IDH1 mutation in grade IV gliomas.     

Comparative study of IDH1 mutations in gliomas by immunohistochemistry and DNA sequencing

Background

Mutations involving isocitrate dehydrogenase 1 (IDH 1) occur in a high proportion of diffuse gliomas, with implications on diagnosis and prognosis. About 90% involve exon 4 at codon 132, replacing amino acid arginine with histidine (R132H). Rarer ones include R132C, R132S, R132G, R132L, R132V, and R132P. Most authors have used DNA-based methods to assess IDH1 status. Preliminary studies comparing imunohistochemistry (IHC) with IDH1-R132H mutation-specific antibodies have shown concordance with DNA sequencing and no cross-reactivity with wild-type IDH1 or other mutant proteins. The present study compares results of IHC with DNA sequencing in diffuse gliomas.

Materials and methods

Fifty diffuse gliomas with frozen tissue samples for DNA sequencing and adequate tissue in paraffin blocks for IHC using IDH1-R132H specific antibody were assessed for IDH1 mutations.

Results

Concordance of findings between IHC and DNA sequencing was noted in 88% (44/50) cases. All 6 cases with discrepancy were immunopositive with DIA-H09 antibody. While in 3 of these 6 cases, DNA sequencing failed to reveal any mutations, R132L (arginine replaced by leucine) mutation was found in the rest 3 cases. Interestingly, of the immunopositive cases, 46.6% (14/30) showed immunostaining in only a fraction of tumor cells.

Conclusions

IHC is an easy and quick method of detecting IDH1-R132H mutations, but there may be some discrepancies between IHC and DNA sequencing. Although there were no false-negative cases, cross-reactivity with IDH1-R132L was seen in 3, a finding not reported thus far. Because of more universal availability of IHC over genetic testing, cross-reactivity and staining heterogeneity may have bearing over its use in detecting IDH1-R132H mutation in gliomas.     

Comparison of immunohistochemistry, DNA sequencing and allele-specific PCR for the detection of IDH1 mutations in gliomas

Previous studies have identified mutations of the isocitrate dehydrogenase 1 (IDH1) gene in more than 70% of World Health Organization (WHO) grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (c.395G>A, p.R132H). IDH1 mutated tumors have a better prognosis than IDH1 non-mutated tumors. The aim of our study was to evaluate and compare the methods of mIDH1 R132H immunohistochemistry, allele-specific PCR and DNA sequencing for determination of IDH1 status. We performed a retrospective study of 91 patients with WHO grade II (n=43) and III (n=48) oligodendrogliomas. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced using standard conditions. Allele-specific amplification was performed using two forward primers with variations in their 3' nucleotides such that each was specific for the wild-type or the mutated variant, and one reverse primer. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H. DNA was extracted from FFPE sections following macrodissection. IDH1 mutations were found in 55/90 patients (61.1%) by direct sequencing. R132H mutations were found in 47/55 patients (85.4%). The results of the allele-specific PCR positively correlated with those from DNA sequencing. Other mutations (p.R132C, p.R132S and pR132G) were found by DNA sequencing in 3, 3 and 2 tumors, respectively (8/55 patients, 14.6%). mIDH1 R132H immunostaining was found in the 47 patients presenting the R132H mutation (sensitivity 47/47, 100% for this mutation). None of the tumors presenting a wild-type IDH1 gene were stained (specificity 35/35, 100%). Our results demonstrate that immunohistochemistry using the mIDH1 R132H antibody and allele-specific amplification are highly sensitive techniques to detect the most frequent mutation of the IDH1 gene.     

Analysis of IDH1 and IDH2 mutations in Japanese glioma patients

A recent study reported on mutations in the active site of the isocitrate dehydrogenase 1 ( IDH1 ) gene in several types of gliomas. All mutations detected resulted in an amino acid exchange at position 132. We analyzed the genomic region spanning wild-type R132 of IDH1 by direct sequencing in 125 glial tumors. A total of 39 IDH1 mutations were observed. Mutations of the IDH2 gene, homologous to IDH1 , were often detected in gliomas without IDH1 mutations. In the present study, R172 mutation of the IDH2 gene was detected in one anaplastic astrocytoma. IDH1 or IDH2 mutations were frequently in oligodendrogliomas (67%), anaplastic astrocytomas (62%), anaplastic oligoastrocytomas (75%), anaplastic oligodendrogliomas (50%), secondary glioblastomas (67%), gangliogliomas (38%), and anaplastic gangliogliomas (60%). Primary glioblastomas were characterized by a low frequency of mutations (5%) at amino acid position 132 of IDH1 . Mutations of the IDH1 or IDH2 genes were significantly associated with improved outcome in patients with anaplastic astrocytomas. Our data suggest that IDH1 or IDH2 mutation plays a role in early tumor progression of several types of glioma and might arise from a common glial precursor. The infrequency of IDH1 mutation in primary glioblastomas revealed that these subtypes are genetically distinct entities from other glial tumors. ( Cancer Sci  2009; 100: 1996–1998)     

Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors

Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors.     

Interlaboratory comparison of IDH mutation detection

Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.     

Prevalence of IDH1/2 Mutations in Different Subtypes of Glioma in the North-East Population of Morocco

Background: Genetic alterations in gliomas have increasing importance for classification purposes. Thus, we are especially interested in studying IDH mutations which may feature potential roles in diagnosis, prognosis and response to treatment. Our aim was to investigate IDH mutations in diffuse glioma patients diagnosed in university hospital centre of Fez in Morocco. Materials and Methods: IDH1 codon 132 and IDH2 codon 172 were direct-sequenced in 117 diffuse glioma samples diagnosed and treated in University Hospital Hassan II between 2010 and 2014. Results: The R132H IDH1 mutation was identified in 43/117 tumor samples and R172K IDH2 mutation was detected in only one anaplastic oligodendroglioma. IDH mutations were observed in 63.2% of astrocytomas, 73.3% of diffuse oligodendrogliomas and 12.90% of glioblastomas. Conclusions: Our results confirmed other studies published earlier for other populations with some small discrepancies.     

Frequent IDH1/2 mutations in intracranial chondrosarcoma: a possible diagnostic clue for its differentiation from chordoma

Mutations in the genes encoding isocitrate dehydrogenase (IDH) 1/2 have been detected in a significant proportion of diffuse gliomas and in a small fraction of acute myeloid leukemia (AML) cases. Recently, in an examination of various types of mesenchymal tumor, IDH1/2 mutations were only found in cartilaginous tumors including central conventional and periosteal enchondromas/chondrosarcomas. The frequency of IDH1/2 mutations was 56%, and the IDH1 R132C mutation, which is not common in diffuse gliomas or AML, accounted for 40% of these mutations. In this study, we investigated the IDH1/2 mutation status of intracranial chondrosarcomas and chordomas, which are morphologically similar and affect similar regions of the cranial cavity. Of the 13 chondrosarcomas analyzed, six (46.1%) displayed IDH1/2 mutations (the predominant type was IDH1 R132C). Also, an IDH2 mutation (R172S) was observed in one case. Conversely, none of the ten chordomas analyzed displayed any IDH1 or IDH2 mutations. Our data suggest that the IDH1/2 mutation status could be valuable for distinguishing intracranial chondrosarcomas from chordomas.     

Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone

Giant cell tumors of bone (GCTB) are benign and locally destructive tumors that include osteoclast‐type multinuclear giant cells. No available treatment is definitively effective in curing GCTB, especially in surgically unresectable cases. Isocitrate dehydrogenase (IDH) mutations have been reported not only in gliomas and acute myeloid leukemias, but also in cartilaginous tumors and osteosarcomas. However, IDH mutations in GCTB have not been investigated. The IDH mutations are remarkably specific to arginine 132 (R132) in IDH1 and arginine 172 (R172) or arginine 140 (R140) in IDH2; IDH1/2 mutations are known to convert α‐ketoglutarate to oncometabolite R(‐)‐2‐hydroxyglutarate. We recently reported that the most frequent IDH mutation in osteosarcomas is IDH2‐R172S, which was detected by MsMab‐1, a multispecific anti‐IDH1/2 mAb. Herein, we newly report the IDH mutations in GCTB, which were stained by MsMab‐1 in immunohistochemistry. DNA direct sequencing and subcloning identified IDH mutations of GCTB as IDH2‐R172S (16 of 20; 80%). This is the first report to describe IDH mutations in GCTB, and MsMab‐1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation‐bearing GCTB.