肿瘤原位液中肿瘤来源DNA实时监测脑胶质母细胞瘤术后替莫唑胺诱导超突变的临床应用价值

目的:探讨脑肿瘤原位液（tumor in situ fluid,TISF）来源的肿瘤DNA（tumor-derived DNA）测序实时监测脑胶质母细胞瘤（GBM）术后替莫唑胺（temozolomide,TMZ）诱导超突变的临床应用价值。方法:收集2019年3月至2021年6月于我院神经外科接受诊治的21例GBM患者的肿瘤组织标本及术后TMZ辅助治疗期间的TISF标本和CSF标本,共50个样本（4个CSF、19个组织标本、27个TISF）,从TISF和CSF中提取无细胞DNA（cell-free DNA,cfDNA）进行肿瘤来源DNA靶向测序,表征脑胶质母细胞瘤术后基因组景观,并与肿瘤组织标本测序结果对比,鉴定出在术后TMZ辅助化疗期间基因组发生超突变的GBM患者。结果:21例GBM患者平均年龄（53.2±13.5）岁,18例为IDH野生型,3例为IDH突变型,初始MGMT甲基化均阳性。共检测到67个不同基因的785个突变,每个个体平均突变数为15.7,最常见的改变基因为TP53（35%）、NF1（33%）、TSC2（29%）、PTEN（27%）、PTCH1（23%）,最常见的突变类型是错义突变（65%）,其次是多重突变（13%）和移码突变（12%）。与初始肿瘤组织标本测序结果对比,共有2例患者经TISF 肿瘤来源DNA测序检测出在用药期间发生基因组超突变,并证实为TMZ诱导产生的超突变。结论:通过TISF 肿瘤来源DNA在体动态监测,可实时在体表征脑胶质母细胞瘤术后肿瘤基因景观及监测替莫唑胺诱导的超突变,通过基因组分析,早期发现肿瘤耐药,提示临床调整后续治疗方案。     

DNA错配修复基因的遗传变异/突变与肿瘤治疗反应

DNA错配修复（mismatch repair,MMR）是一个复杂的生物学过程,在维持基因组完整性方面起着重要作用。微卫星不稳定性（microsatellite instability,MSI）由MMR蛋白功能缺陷导致。MMR基因的遗传变异或突变对肿瘤的发生、发展以及预后起到关键作用,其相关研究已取得显著成果。目前国内外多个指南建议对晚期实体瘤患者检测MMR/MSI,其结果可以预测肿瘤患者的预后,并可预测肿瘤辅助化疗及免疫治疗疗效,对治疗方案的选择具有指导意义。     

高通量基因测序技术检测外周血循环肿瘤DNA基因突变在非小细胞肺癌中的应用

目的:探究高通量基因测序技术检测非小细胞肺癌外周血循环肿瘤DNA基因突变的应用价值。方法:临床纳入2017年1月至2018年9月在我院就诊的40例晚期非小细胞肺癌患者作为研究对象,所有患者入院后均经肺组织活检或气管镜检查确诊为晚期非小细胞肺癌。对患者进行病理组织石蜡切片DNA（tDNA）检测,并采集患者肘静脉血使用高通量基因测序技术检测患者外周血循环肿瘤ctDNA基因情况。对比分析tDNA与ctDNA检测对患者DNA基因突变的准确性,探讨非小细胞肺癌患者进行高通量基因测序技术检测外周血循环肿瘤DNA基因突变的应用价值。结果:40例非小细胞肺癌的外周血循环肿瘤DNA基因突变检测与组织石蜡切片比较,两种方法检测率差异无统计学意义（P>0.05）。在高通量基因测序技术检查外周血循环肿瘤DNA中,21外显子测序结果:61号替代突变2573G→T,62/63/68号替代突变L858R（2573T→G）。19外显子测序结果:50号样品突变为del E746→A750+2235G→A,60号样品突变为del E746→A750,70号样品突变为del L747→T751,80号样品突变为del L747→S752＋2257C→T。结论:非小细胞肺癌外周血循环肿瘤DNA基因突变进行高通量基因测序技术对具体的基因突变或缺失具有较高准确性,可实时监测肿瘤DNA基因突变情况,且具有无创性、可重复应用等优点。     

携带同源重组修复相关基因突变的消化道肿瘤的临床特点

目的:探讨消化道肿瘤中同源重组修复相关基因(homologous recombination repair related gene，HRR)突变的发生情况及临床意义。方法:共92例消化道肿瘤患者，79例患者进行了血液标本HRR检测，53例患者进行了组织标本HRR检测，40例患者同时行血液和组织的HRR基因检测，收集患者基因检测结果及临床相关资料。结果:在79例患者血液标本检测中发现10例（12.6%）有临床意义HRR突变，在53例患者组织标本检测中发现9例（17.0%）有临床意义HRR突变。40例同时行血液和组织的HRR基因检测患者中常见的有临床意义HRR突变为CDK12突变4例（10.0%）、ATM突变3例（7.5%）、BRCA1突变2例（5.0%）。13例有临床意义HRR突变患者中常见共存突变为TP53突变10例（76.9%）、APC突变5例（38.5%）、PIK3CA突变4例（30.8%）。40例患者中13例患者血液和/或组织中有临床意义HRR突变，27例患者血液和组织中均无任何临床意义HRR突变且两组相比，有临床意义HRR突变组肿瘤突变负荷（tumor mutational burden，TMB）为6.17(2.24～11.52)，而未携带HRR突变组TMB为0.4(0～3.75)，差异有统计学意义（P＜0.05）。40例患者组织检测中7例HRR有临床意义的突变，33例无HRR突变，血液检测中10例HRR有临床意义的突变，30例无HRR突变，一致性检验的Kappa值为0.333（P=0.031）。结论:携带有临床意义HRR突变的消化道肿瘤患者TMB更高，血液和组织检测HRR突变有较好的一致性。     

k-ras 基因突变与结直肠癌的关系

 目的　探讨k-ras 基因在结直肠癌患者中的突变状况,为k-ras基因突变检测提供理论依据。方法　提取基因组DNA,采用PCR扩增和双向直接测序法,检测56例结直肠癌k-ras基因的突变状况。结果　56例结直肠癌中k-ras基因的突变率为46.63 %（26／56）,其中20例（20／26,76.92 %）为密码子12的突变,6例（6／26,23.08 %）为密码子13的突变,未发现同时存在两个位点突变的样本。突变类型以G>A为主,密码子12的突变以GGT>GAT（G12D）为主。密码子13的突变以GGC>GAC为主。统计学分析发现,k-ras基因突变与患者的性别密切相关而与其他临床病理特征如：肿瘤发生部位、有无淋巴结转移等无关。结论　在结直肠癌患者中,k-ras基因突变主要发生于密码子12;突变类型以G>A为主;k-ras基因的突变可能与患者的性别相关。     

地西他滨对造血系统肿瘤细胞热休克蛋白22表达的影响

 目的　探讨地西他滨（Aza-C）对造血系统肿瘤细胞热休克蛋白22（HSP22）表达的影响及可能的机制。方法　半定量反转录聚合酶链反应（PCR）法检测HSP22在造血系统肿瘤细胞系以及造血系统肿瘤患者骨髓单个核细胞中的表达水平；去甲基化药物Aza-C（2 μmol／L）诱导上述细胞HSP22的表达；甲基化特异PCR法检测造血系统肿瘤细胞系、造血系统肿瘤患者和健康供者的骨髓单个核细胞中HSP22基因启动子甲基化状态。结果　在检测的13个造血系统肿瘤细胞系、20例不同类型的造血系统肿瘤患者以及10名健康供者骨髓单个核细胞中均未发现HSP22基因的表达；Aza-C能诱导造血系统肿瘤细胞系及造血系统肿瘤患者骨髓单个核细胞中的HSP22表达；Aza-C处理过的造血系统肿瘤细胞系中HSP22基因启动子处于部分去甲基化状态，健康供者及造血系统肿瘤患者骨髓单个核细胞HSP22基因启动子处于高甲基化状态。结论　Aza-C通过使HSP22基因启动子去甲基化诱导造血系统肿瘤细胞HSP22的表达。     

二代测序在家族遗传性高危胃肠肿瘤筛查中的应用

  目的  探索二代测序技术对家族遗传性高危胃肠肿瘤患者进行遗传筛查的意义及高危因素在筛选患者中的价值。  方法  选取2016年3月至2016年4月收治于北京大学肿瘤医院的322例结直肠癌及胃癌患者,筛选出25例遗传性胃肠肿瘤高危患者,运用二代测序技术对患者的外周血白细胞DNA进行42个遗传性肿瘤综合征相关基因的胚系检测。  结果  24%（6/25）患者检测出遗传性肿瘤相关基因的病理性胚系突变,其中50%（3/6）患者肿瘤组织的免疫组织化学检测表现为错配修复蛋白表达缺失,83%（5/6）患者发病年龄≤50岁且具有恶性肿瘤家族史。发生胚系突变的6例遗传性肿瘤相关基因分别为MYH基因错义突变1例,APC基因缺失突变1例和遗传性非息肉病性结直肠癌（hereditary nonpolyposis colorectal cancer,HNPCC）相关基因的突变4例（包括MLH1、MLH3、TGFBR2的错义突变和MSH6的无义突变各1例）,且提供了MLH3的胚系致病突变的家系验证。  结论  通过二代测序技术对本研究入组的25例患者进行家族遗传性肿瘤综合征的筛查,检测出遗传性肿瘤相关基因的胚系致病突变6例,提示运用二代测序技术对家族遗传性高危消化道肿瘤患者进行遗传筛查具有提高检测阳性率的临床应用价值。      

CSF-ctDNA与TIF-ctDNA检测在脑胶质瘤中的临床应用研究

  目的  探讨脑脊液（cerebrospinal fluid，CSF）来源的循环肿瘤DNA（circulating tumor DNA，ctDNA）与肿瘤间质液（tumor interstitial fluid，TIF）ctDNA检测在脑胶质瘤中的诊断、复发监测、肿瘤标志物及靶向药物筛选方面的应用价值。  方法  收集2019年1月至12月河南省人民医院12例脑胶质瘤患者CSF标本、TIF标本及血液标本。CSF标本、TIF标本进行二代基因测序（next generation sequencing，NGS）检测ctDNA突变，血液标本进行白细胞胚系突变检测，二者对比筛选出肿瘤突变基因。与临床资料结合，对胶质瘤患者CSF-ctDNA与TIF-ctDNA检测结果进行对比及生物信息学分析。  结果  检测到57种基因变异类型共209个，标本总平均突变数为8.7个，CSF-ctDNA平均突变数为4.3个，TIF-ctDNA平均突变数为13个。9例CSF-ctDNA检测阳性率为（75.0%）、11例TIF-ctDNA检测阳性率（91.6%）。同1例患者ctDNA浓度TIF标本高于CSF标本，TIF标本ctDNA平均浓度（3.38 ng/μL）高于CSF标本中ctDNA平均浓度（0.58 ng/μL）。ctDNA检测发现肿瘤基因突变，较影像学发现复发时间平均提前2.9个月。突变基因PIK3CA、PTEN、KRAS、EGFR、TP53、NF1、BRCA1、BRCA2、TSC1、TSC2、IDH1、CDKN2A匹配到靶向潜在获益药物，突变基因PIK3CA、KRAS、EGFR匹配到潜在耐药药物。  结论  CSF-ctDNA与TIF-ctDNA能够比影像学更早发现肿瘤复发，是一种可行的能够在肿瘤的发生和复发的过程反复检测、筛选胶质瘤标志物的方法，对胶质瘤患者基因诊断及靶向治疗有一定临床指导作用。相对于CSF-ctDNA，TIF-ctDNA检测获得样本ctDNA浓度更高，检测患者基因平均突变数更多、检测阳性率与检测效力更高，能够提供更多、全面的肿瘤基因组信息，适合临床用于获取胶质瘤的基因表达信息。      

结直肠癌患者血清外泌体代替肿瘤组织检测K-Ras基因突变的研究*

目的 探讨结直肠癌患者血清外泌体代替肿瘤组织进行DNA中K-Ras基因12密码子突变检测的价值。方法 收集2014年5月至2015年9月于我院病理组织学诊断为局部晚期或转移性结直肠癌患者的外周血标本90例,使用ExoQuick试剂提取血清外泌体,盐析法提取外泌体及血细胞DNA,采用PCR方法扩增K-Ras基因12密码子并通过高通量测序法检测突变,比较血清外泌体DNA与血细胞DNA突变的特异性,比较外泌体与组织学检测的K-Ras基因12密码子突变情况。结果 90例结直肠癌患者中检测出外泌体K-Ras基因12密码子突变43例,突变率为47.8％。全组共检出4种突变类型,以G12D突变率为最高。与组织学检测比较,灵敏度为90.6％,一致性为81.1％（Kappa值=0.62,P＜0.05）。结论 血清外泌体DNA用于检测肿瘤相关突变,与肿瘤组织相比一致性较高,可作为液体活检的来源指导肿瘤的个体化治疗。     

ATM与肿瘤的关系研究进展

共济失调毛细血管扩张征突变基因(ataxia-telangiectasia mutated gene,ATM gene)属于一种DNA修复基因,定位于染色体llq22-q23.ATM基因的胚系突变或基因多态性导致个体对多种肿瘤易感,包括淋巴样肿瘤和实体瘤.ATM基因编码产物——ATM蛋白属于一种自动磷酸化蛋白激酶,在DNA损伤修复、细胞周期阻滞和细胞凋亡过程中发挥着关键的调控作用.体外实验发现,许多肿瘤的发生和治疗过程涉及了ATM蛋白表达或活性的改变,提示ATM基因或许能成为肿瘤治疗过程中一个新的潜在作用靶点.     

Identification of fibroblast growth factor receptor 3 mutations in urine sediment DNA samples complements cytology in bladder tumor detection

BACKGROUND: Mutations in fibroblast growth factor 3 receptor (FGFR3) are frequent events in low-grade bladder tumors. To assess the potential utility of the detection of FGFR3 mutations in a screening modality, the authors analyzed urine sediment DNA samples from 192 patients in a retrospective study. METHODS: Urine sediment DNA samples from 192 patients were prepared. Seventy-two patients had undergone transurethral resection (TURBT group) of mainly Ta lesions and 120 patients had undergone cystectomy (cystectomy group). The majority of patients in the cystectomy group had more advanced tumors compared with patients in the TURBT group. DNA preparations were screened for FGFR3 mutations in exons 7, 10, and 15 using single-strand conformation polymorphism (SSCP) and DNA sequencing. RESULTS: Using SSCP, 67% of patients in the TURBT group and 28% in the cystectomy group displayed FGFR3 mutations. Comparative analysis of cytology results and FGFR3 mutational analysis were performed in 122 cases. Within the TURBT group, FGFR3 mutation analysis outperformed cytology. FGFR3 mutation analysis identified change in 68% of urine sediment DNA samples whereas cytology recorded the presence of tumor cells in 32% of the DNA samples. In the cystectomy group, cytology outperformed FGFR3 mutation analysis. Cytology recorded tumor detection in 90% of patients, while SSCP identified mutational change in 24%. CONCLUSIONS: Combining FGFR3 mutation results with cytology in both groups correctly identified tumor presence in 105 of 122 (86%) of patients. The greater sensitivity of FGFR3 mutation detection over cytology in identifying the presence of low-grade, superficial bladder tumors represents a potential new tool to complement standard cytology in screening patients for bladder tumors and recurrent disease.     

Translational studies for target-based drugs

The biological background for the clinical and prognostic heterogeneity among tumors within the same histological subgroup is due to individual variations in the biology of tumors. The number of investigations looking at the application of novel technologies within the setting of clinical trials is increasing. The most promising way to improve cancer treatment is to build clinical research strategies on intricate biological evidence. New genomic technologies have been developed over recent years. These techniques are able to analyze thousands of genes and their expression profiles simultaneously. The purpose of this approach is to discover new cancer biomarkers, to improve diagnosis, predict clinical outcomes of disease and response to treatment, and to select new targets for novel agents with innovative mechanisms of action. Gene expression profiles are also used to assist in selecting biomarkers of pharmacodynamic effects of drugs in the clinical setting. Biomarker monitoring in surrogate tissues may allow researchers to assess "proof of principle" of new treatments. Clinical studies of biomarkers monitoring toxicity profiles have also been done. Such pharmacodynamic markers usually respond to treatment earlier than clinical response, and as such may be useful predictors of efficacy. Epidermal growth factor receptor (EGFR) mutation in lung cancer tissues is a strong predictive biomarker for EGFR-targeted protein tyrosine kinase inhibitors. Monitoring of EGFR mutation has been broadly performed in retrospective and prospective clinical studies. However, global standardization for the assay system is essential for such molecular correlative studies. A more sensitive assay for EGFR mutation is now under evaluation for small biopsy samples. Microdissection for tumor samples is also useful for the sensitive detection of EGFR mutation. Novel approaches for the detection of EGFR mutation in other clinical samples such as cytology, pleural effusion and circulating tumor cells are ongoing.     

Fibroblast Growth Factor Receptor 3 Mutations in Bladder Tumors Correlate with Low Frequency of Chromosome Alterations

The aim of this study was to analyze the distribution of FGFR3 mutations in bladder tumors of different grade and stage and determine the relation of mutations to chromosomal alterations detected by comparative genomic hybridization (CGH). One hundred bladder cancer samples served as templates for manual microdissection. DNA was isolated from dissected samples containing at least 80% tumor cells. Mutations in FGFR3 were analyzed by SNaPshot analysis. CGH was carried out according to standard protocols. FGFR3 mutations were detected in 45 of 92 samples (48.9%). Concerning T-category, the following mutation frequencies occurred: pTa, 69%; pT1, 38%; and pT2-3, 0%. The mutation frequency was significantly associated with tumor grade: G1, 72%; G2, 56%; and G3, 4%. In pTaG1 tumors, mutations were found in 74%. A significantly lower number of genetic alterations per tumor detected by CGH was associated with FGFR3 mutations (2 vs 8). This association was also seen in pTaG1 tumors: 2.5 (with mutation) vs 7.5 (without mutation). FGFR3 mutations characterize noninvasive low-risk tumors of low malignancy. The low malignant potential of these tumors is underlined by a low number of genetic alterations per tumor. Therefore, FGFR3 represents a valuable prognostic marker of tumors with low malignant potential and can be used as surrogate marker for the detection of genetically stable bladder tumors.     

Occurrence of H-ras codon 61 CAA to AAA mutation during mouse liver tumor progression

The initiating mutations of a tumor are present in each of the cancerous cells comprising the tumor. Identification and measurement of the subsequent mutations that occur during tumor progression, however, requires mutation detection in a smaller subset of the tumor cells. In this study, allele-specific competitive blocker PCR (ACB-PCR), a genotypic selection method with the sensitivity to detect a specific point mutation in the presence of a 10(5)-fold excess of wild-type DNA sequence, was used to measure H-ras codon 61 CAA to AAA mutation in mouse liver tumors that did not have this mutation as an initiating event. Twenty-one spontaneous or chemically induced mouse liver tumors, negative for the H-ras codon 61 CAA to AAA mutation by DNA sequencing or denaturing gradient gel electrophoresis, were analyzed for this mutation by ACB-PCR. The mutation was detected at some level in 71% of these tumors. The mutation was detected in adenomas and carcinomas more frequently (13 of 14 tumors) and at significantly higher mutant fractions than it was detected in histiocytic sarcomas (1 of 5 tumors). These data indicate that the same oncogenic point mutation that can be identified as a tumor-initiating event based on its clonal amplification in a tumor can also be present in only a small sub-population of tumor cells where the mutation must have been fixed at a later stage in tumor development. The occurrence of a mutation as a primary or secondary event probably reflects the stochastic nature of mutation and is likely to be affected by the mutation rate for each target site.     

Detection of APC and k-ras mutations in the serum of patients with colorectal cancer

Detection of tumor DNA in peripheral blood of patients with colorectal cancer (CRC) may allow early diagnosis of tumor disease and be of prognostic value. However, a reliable tumor marker detectable in the serum of patients with this disease is missing. Because k-ras and APC mutations occur frequently and at an early stage in CRCs, these mutations might also be detected in the serum of CRC patients and serve as tumor markers. Hence, tumor tissues of CRC patients were examined for the presence of mutations in the k-ras and APC genes. If a mutation was detected in the tumor, the serum of the patient was screened subsequently for the presence of this mutation. K-ray mutations were detected in 22 of 30 colorectal tumor tissues, but only in six patients was the mutation identified in their serum samples. Mutations of the APC gene were identified in 25 of 65 tumors: 20 of these 25 patients showed the respective mutation in their serum. Given their higher detection rate, APC mutations could be a more informative serum marker than k-ras in CRC patients.     

Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal stromal tumors under treatment with imatinib mesylate.

Gastrointestinal stromal tumors (GIST) are characterized by a strong KIT receptor activation most often resulting from KIT mutations. In a smaller subgroup of tumors without KIT mutations, analogous activating mutations are found in the platelet-derived growth factor receptor alpha (PDGFRalpha) gene. Both PDGFRalpha and KIT receptors are targets of the tyrosine kinase inhibitor imatinib (Glivec) which has improved the treatment of advanced GISTs significantly. However, a subgroup of tumors show a secondary progress under therapy with imatinib after initial response. One possible mechanism of secondary resistance is the development of newly acquired KIT mutations. In the present study, we evaluated the frequency of such secondary KIT mutations in a series of GIST patients in which tumor tissue was resected under treatment. We examined one to seven different tumor areas in 32 cases (total of 104 samples) and found up to four newly acquired KIT mutations in 14 patients (43.8%). These were always located in exons encoding the first or second tyrosine kinase domain (exon 13, 14, or 17). Mutations were found only in a subset of samples analyzed from each case whereas others retained the wild-type sequence in the same region. There was never more than one new mutation in the same sample. Consistent with a secondary clonal evolution, the primary mutation was always detectable in all samples from each tumor. According to our results, the identification of newly acquired KIT mutations in addition to the primary mutation is dependent on the number of tissue samples analyzed and has high implications for further therapeutic strategies.     

A simple and fast method for the simultaneous detection of nine fibroblast growth factor receptor 3 mutations in bladder cancer and voided urine.

PURPOSE: Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases. These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detection of recurrences in urine is an important clinical issue. In this paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations. EXPERIMENTAL DESIGN: The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide. The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of a mutation. RESULTS: The assay was found to be more sensitive than single-strand conformation polymorphism analysis. Mutations could still be detected with an input of only 1 ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%. CONCLUSIONS: We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.     

Prognostic value of codon 918 (ATG-->ACG) RET proto-oncogene mutations in sporadic medullary thyroid carcinoma

We have determined the frequency of 918 RET proto-oncogene mutations (ATG-->ACG) in primary MTC tumors and metastases and correlated the presence or absence of this mutation with the clinical outcome of patients suffering from sporadic medullary thyroid carcinoma (MTC). A total of 197 samples, consisting of both primary tumors and lymph node metastases from 34 patients with sporadic MTC, were collected for PCR analysis of the RET 918 mutation. In 75 of the samples (38%), codon 918 (ATG-->ACG) mutations could be detected. The mutations showed a heterogeneous distribution: 21/34 patients (62%) had mutations in at least 1 tumor sample, and in 13 patients (38%) the mutation was present in all examined samples. Patients were considered 918mt when at least 1 tumor sample showed the RET 918 mutation. These 918mt and 918 wild-type (918wt) patients did not differ significantly concerning sex, age at diagnosis, TNM stage at diagnosis, number of examined tumor samples or follow-up time. However, 918mt patients showed more aggressive development of distant metastases during follow-up (p = 0.032, Fisher's exact test) with decreased metastases-free survival (p < 0.005, log-rank test). Furthermore, 918mt patients had a significantly lower survival rate than 918wt patients (p = 0.048, log-rank test). These data show that the RET codon 918 mutation has a prognostic impact on patients with sporadic MTC which may influence follow-up treatment.     

Targeted Next-Generation Sequencing of Circulating Tumor DNA Mutations among Metastatic Breast Cancer Patients

Liquid biopsy through the detection of circulating tumor DNA (ctDNA) has potential advantages in cancer monitoring and prediction. However, most previous studies in this area were performed with a few hotspot genes, single time point detection, or insufficient sequencing depth. In this study, we performed targeted next-generation sequencing (NGS) with a customized panel in metastatic breast cancer (MBC) patients. Fifty-four plasma samples were taken before chemotherapy and after the third course of treatment for detection and analysis. Paired lymphocytes were also included to eliminate clonal hematopoiesis (CH)-related alternatives. A total of 1182 nonsynonymous mutations in 419 genes were identified. More ctDNA mutations were detected in patients with tumors > 3 cm (p = 0.035) and HER2(−) patients (p = 0.029). For a single gene, the distribution of ctDNA mutations was also correlated with clinical characteristics. Multivariate regression analysis revealed that HER2 status was significantly associated with mutation burden (OR 0.02, 95% CI 0–0.62, p = 0.025). The profiles of ctDNA mutations exhibited marked discrepancies between two time points, and baseline ctDNA was more sensitive and specific than that after chemotherapy. Finally, elevated ctDNA mutation level was positively correlated with poor survival (p < 0.001). Mutations in ctDNA could serve as a potential biomarker for the evaluation, prediction, and clinical management guidance of MBC patients with chemotherapy.     

Detecting colorectal cancer in stool with the use of multiple genetic targets

BACKGROUND: Colorectal cancer cells are shed into the stool, providing a potential means for the early detection of the disease using noninvasive approaches. Our goal was to develop reliable, specific molecular genetic tests for the detection of colorectal cancer in stool samples. METHODS: Stool DNA was isolated from paired stools and primary tumor samples from 51 colorectal cancer patients. Three genetic targets-TP53, BAT26, and K-RAS-were used to detect tumor-associated mutations in the stool prior to or without regard to the molecular analyses of the paired tumors. TP53 gene mutations were detected with a mismatch-ligation assay that detects nine common p53 gene mutations. Deletions within the BAT26 locus were detected by a modified solid-phase minisequencing method. Mutations in codons 12 and 13 of K-RAS were detected with a digital polymerase chain reaction-based method. RESULTS: TP53 gene mutations were detected in the tumor DNA of 30 patients, all of whom had the identical TP53 mutation in their stools. Tumors from three patients contained a noninherited deletion at the BAT26 locus, and the same alterations were identified in these patients' stool specimens. Nineteen of 50 tumors tested had a K-RAS mutation; identical mutations were detected in the paired stool DNA samples from eight patients. In no case was a mutation found in stool that was not also present in the primary tumor. Thus, the three genetic markers together detected 36 (71%) of 51 patients (95% confidence interval [CI] = 56% to 83%) with colorectal cancer and 36 (92%) of 39 patients (95% CI = 79% to 98%) whose tumors had an alteration. CONCLUSION: We were able to detect the majority of colorectal cancers by analyzing stool DNA for just three genetic markers. Additional work is needed to determine the specificity of these genetic tests for detecting colorectal neoplasia in asymptomatic patients and to more precisely estimate the prevalence of the mutations and sensitivity of the assay.