PIK3CA mutations in patients with advanced cancers treated with PI3K/AKT/mTOR axis inhibitors

Preclinical data suggest that PIK3CA mutations predict response to PI3K/AKT/mTOR inhibitors. Concomitant KRAS or BRAF mutations may mediate resistance. Therefore, tumors from patients referred to the phase I program for targeted therapy starting in October 2008 were analyzed for PIK3CA mutations using PCR-based DNA sequencing of exons 9 and 20. Consecutive patients with diverse tumor types and PIK3CA mutation were treated whenever possible with agents targeting the PI3K/AKT/mTOR pathway. Overall, PIK3CA mutations were detected in 25 of 217 patients (11.5%; exon 9, n = 11; exon 20, n = 14). In tumor types with more than 10 patients tested, PIK3CA mutations were most frequent in endometrial (3 of 14, 21%), ovarian (5 of 30, 17%), colorectal (9 of 54, 17%), breast (2 of 14, 14%), cervical (2 of 15, 13%), and squamous cell cancer of the head and neck (1 of 11, 9%). Of the 25 patients with PIK3CA mutations, 17 (68%) were treated on a protocol that included a PI3K/AKT/mTOR pathway inhibitor, and 6 (35%) achieved a partial response. In contrast, only 15 of 241 patients (6%) without documented PIK3CA mutations treated on the same protocols responded (P = 0.001). Of the 17 patients with PIK3CA mutations, 6 (35%) had simultaneous KRAS or BRAF mutations (colorectal, n = 4; ovarian, n = 2). Colorectal cancer patients with PIK3CA and KRAS mutations did not respond to therapy, whereas both ovarian cancer patients with PIK3CA and KRAS or BRAF mutations did. In conclusion, PIK3CA mutations were detected in 11.5% of patients with diverse solid tumors. The response rate was significantly higher for patients with PIK3CA mutations treated with PI3K/AKT/mTOR pathway inhibitors than for those without documented mutations.     

结直肠癌患者血清外泌体与组织中KRAS，BRAF，NRAS 和PIK3CA 基因突变检测及临床意义的比较

目的 检测结直肠癌(colorectal cancer, CRC) 患者血清外泌体KRAS,BRAF,NRAS 和PIK3CA 相关基因位点的突变,分析其与癌组织基因突变的一致性及其可能的影响因素。方法 2019 年2 月～ 2021 年1 月中国人民解放军联勤保障部队第九二〇医院100 例结直肠癌患者作为研究对象。提取患者血清外泌体,并用蛋白免疫印迹（Westernblot,WB）法检测外泌体标志物CD63 和TSG101 的蛋白表达;聚合酶链式反应（polymerase chain reaction,PCR）检测血清外泌体及手术切除的癌组织中KRAS,BRAF,NRAS 和PIK3CA 基因突变情况,Kappa 一致性检验分析血清外泌体突变与组织突变的一致性,Logistic 单因素回归分析影响一致性的因素。结果 癌症基因组图谱（the cancer genomeatlas,TCGA）数据显示,KRAS,BRAF,NRAS 和PIK3CA 的结直肠癌突变率分别为35% ～ 96%,5% ～ 15%,5% ～ 30%和18% ～ 36%,且KRAS 和BRAF 的突变与结直肠癌患者的低存活率有关。血清外泌体中KRAS,BRAF,NRAS 和PIK3CA 的突变率分别为94.00%,11.00%,17.00% 和35.00%;结直肠癌组织KRAS,BRAF,NRAS 和PIK3CA 的突变率分别为34.00%,5.00%,6.00% 和16.00%;血清外泌体中KRAS,BRAF,NRAS 和PIK3CA 的突变率均高于组织,差异有统计学意义（χ2=101.027 ～ 256.250,均P ＜ 0.05）。血清外泌体与组织中KRAS 突变的一致率为40.00%（Kappa值=0.064,P>0.05）,BRAF 突变的一致率为99.00%（Kappa 值=0.599,P<0.05）,NRAS 突变的一致率为89.00%（Kappa 值=0.475,P<0.05）,KRAS 突变的一致率为81.00%（Kappa 值=0.523,P<0.05）。ECOG 评分、转移、临床分期是影响外泌体和组织KRAS,BRAF,NRAS 和PIK3CA 的突变检测一致性的因素。结论 结直肠癌患者血清外泌体KRAS,BRAF,NRAS 和PIK3CA 的突变率高于组织,二者BRAF,NRAS 和PIK3CA 的一致性中等,为外泌体的基因检测指导临床靶向治疗提供参考。     

结直肠癌组织中KRAS、NRAS、BRAF、PIK3CA基因突变与临床病理特征及MMR蛋白、p53蛋白表达的相关性研究

目的 分析结直肠癌组织中KRAS、NRAS、BRAF、PIK3CA基因突变情况,探讨其与临床病理参数及MMR蛋白、p53蛋白表达的关系.方法 回顾性分析140例结直肠癌组织的临床病理特征,应用扩增阻碍突变系统(ARMS)进行实时荧光定量PCR检测140例结直肠癌组织KRAS、NRAS、BRAF、PIK3CA基因突变情况...     

HRM法检测结直肠癌组织KRAS基因密码子12和13点突变

目的 建立HRM法检测大肠癌患者肿瘤组织KRAS(v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)基因突变的方法 .方法 采用HRM法对含不同比例KRAS基因突变型质粒的系列混合样本进行检测,以评价其灵敏度.应用HRM法检测60份大肠癌患者新鲜肿瘤组织KRAS基因密码子12和13的突变状况,并与直接测序法的结果 进行比较分析.结果 HRM法只需在PCR结束后直接运行高分辨熔解,即可获得检测结果 .HRM法可检出系列混合样本中突变型质粒比例为10%的突变,其检测灵敏度达10%.HRM法从60份大肠癌患者组织标本中,检出17份KRAS基因密码子12或13突变(28.3%);直接测序法检出15份(25.0%)突变,2份未检出KRAS基因突变.HRM法检测的敏感度为100%(15/15),特异度为96%(43/45).结论 HRM法在筛选大肠癌标本的KRAS基因突变类型时,具有操作简便、快速、灵敏,单管避免污染等优点,完全符合临床个体化治疗的要求,值得推广.     

Multiplexed assays for detection of mutations in PIK3CA

BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.     

Coexistence of PIK3CA and other oncogene mutations in lung adenocarcinoma-rationale for comprehensive mutation profiling

Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110α subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.     

Molecular profiling of patients with colorectal cancer and matched targeted therapy in phase I clinical trials

Clinical experience increasingly suggests that molecular prescreening and biomarker enrichment strategies in phase I trials with targeted therapies will improve the outcomes of patients with cancer. In keeping with the exigencies of a personalized oncology program, tumors from patients with advanced chemorefractory colorectal cancer were analyzed for specific aberrations (KRAS/BRAF/PIK3CA mutations, PTEN and pMET expression). Patients were subsequently offered phase I trials with matched targeted agents (MTA) directed at the identified anomalies. During 2010 and 2011, tumor molecular analysis was conducted in 254 patients: KRAS mutations (80 of 254, 31.5%), BRAF mutations (24 of 196, 12.2%), PIK3CA mutations (15 of 114, 13.2%), KRAS and PIK3CA mutations (9 of 114, 7.9%), low PTEN expression (97 of 183, 53.0%), and high pMET expression (38 of 64, 59.4%). In total, 68 patients received 82 different MTAs: phosphoinositide 3-kinase (PI3K) pathway inhibitor (if PIK3CA mutation, n = 10; or low PTEN, n = 32), PI3K pathway inhibitor plus MEK inhibitor (if KRAS mutation, n = 10; or BRAF mutation, n = 1), second-generation anti-EGF receptor monoclonal antibodies (if wild-type KRAS, n = 11), anti-hepatocyte growth factor monoclonal antibody (if high pMET, n = 10), mTOR inhibitor plus anti-insulin-like growth factor-1 receptor monoclonal antibody (if low PTEN, n = 5), and BRAF inhibitor (if BRAF mutation, n = 3). Median time-to-treatment failure on MTA was 7.9 versus 16.3 weeks for their prior systemic antitumor therapy (P < 0.001). Partial response was seen in 1 patient [1.2%, PI3K inhibitor with PIK3CA mutation] and stable disease >16 weeks in 10 cases (12.2%). These results suggest that matching chemorefractory patients with colorectal cancer with targeted agents in phase I trials based on the current molecular profile does not confer a significant clinical benefit. Mol Cancer Ther; 11(9); 2062-71. ?2012 AACR.     

非小细胞肺癌患者PIK3CA基因与其他致癌基因共突变

  目的  研究中国非小细胞肺癌患者PIK3CA基因与其他致癌基因共突变现象及特点。  方法  2009年9月至2012年4月全国25家医院病理证实为非小细胞肺癌并进行基因突变检测的患者纳入本研究。收集和分析9个基因位点数据, 包括PIK3CA E9、PIK3CA E20、KRAS E2、KRAS E3、BRAF, 以及EGFR E18、E19、E20、E21。  结果  在纳入研究的5125例患者中, 有161例(3.14%)存在多个突变, 其中77例存在PIK3CA突变, 包括50例E9突变和27例E20突变。与PIK3CA共存的其他致癌基因突变位点包括KRAS E2(11例)、KRAS E3(1例)、BRAF(2例)、EGFR E18(4例)、EGFR E20(5例)、EGFR E21(28例)和EGFR E19缺失突变(37例)。在存在PIK3CA共突变的病例中, E9与E20相比, 更容易产生共突变现象; 与EGFR E20相比, 更易与EGFR E21的L858R型突变共存。在PIK3CA E20突变中, H1047R型突变较H1047L型更为常见。与PIK3CA共突变的KRAS突变经常出现在E2的G12位点。BRAF V600E突变也存在与PIK3CA共突变的倾向。  结论  PIK3CA是中国非小细胞肺癌常见的共突变致癌基因, 其E9与E20突变相互排斥, 但均可与其他致癌基因同时存在。其在肺癌发生发展中的作用和对患者预后的意义有待进一步研究。     

PIK3CA基因突变状态在乳腺癌不同临床病理特征中的研究

目的检测乳腺癌组织磷脂酰肌醇-3-激酶催化亚单位α(PIK3CA)基因突变状态,并分析突变与临床病理特征的关系。方法选取176例原发性乳腺癌患者石蜡组织标本,提取组织DNA,对外显子9及20进行探针扩增阻滞突变系统聚合酶链反应(ARMS-PCR)扩增,检测PIK3CA基因的突变状态,另取20例乳腺腺病组织标本作为阴性对照。结果 176例乳腺癌组织中发现突变45例,突变率25.6%,其中以20号外显子H1047R突变率为主,突变率26.7%(12/45);PIK3CA基因突变在ER阳性和阴性、PR阳性和阴性患者中差异有统计学意义(P0.05),但在HER2阳性和阴性患者中差异无统计学意义(P0.05);PIK3CA基因突变与乳腺癌患者肿瘤大小、年龄、淋巴结状态之间无明显相关性,但与组织学分级有相关性。结论 PIK3CA基因突变与乳腺癌激素受体表达和肿瘤进展相关,PIK3CA基因突变检测对指导临床制订个体化治疗有重要意义。     

Mutation analysis of a 10-gene panel for colorectal cancer in Huizhou,Guangdong Province of China

ObjectiveThis study aimed to investigate the type and frequency of mutations in 10 genes in 85 colorectal cancer (CRC) patients in Huizhou and the guiding significance of targeted drug use.MethodsThe 10-gene panel next-generation sequencing (NGS) was used to assess genetic variants in 85 CRC patients from the Huizhou area combined with clinical information for a comprehensive analysis.ResultsUpon initial mutation testing, 68% (58/85) were positive. The mutation frequencies of these genes, including KRAS, PIK3CA, NRAS, ERBB2, BRAF, EGFR, and PDGFRA, were 51%, 20%, 5%, 4%, 4%, 1%, and 1%, respectively. Overall, 29 mutation types were detected from seven genes. More mutations were detected in more advanced cancers. There were three samples with multiple mutations of a single gene, including KRAS (n = 2) and ERBB2 (n = 2), 12 samples with multiple mutations of double genes, including KRAS/PIK3CA (n = 10), BRAF/PIK3CA (n = 1), and NRAS/PIK3CA (n = 1), and one sample with multiple mutations of three genes, including ERBB2/KRAS/PIK3CA (n = 1). Theoretically, 27 patients could receive targeted treatment. During the actual treatment, 10 patients received bevacizumab, cetuximab, or fruquintinib with no progression ranging from 12 to 24 months.ConclusionGene mutations detected by a 10-gene panel were useful for targeting therapy of CRC in Huizhou.     

Improved marker combination for detection of de novo genetic variation and aberrant DNA in colorectal neoplasia

BACKGROUND: The genetic heterogeneity of sporadic colorectal cancer (CRC) makes the choice of genetic markers and sequence variation-detection technologies critical to the performance of screening assays. We have previously described the effectiveness of a CRC assay composed of 22 known variants in KRAS, APC, TP53, and BAT-26 (V1). We introduce a new marker formulation (V2) that includes detection of de novo variation in APC, PIK3CA, and CTNNB1, hypermethylated sequences within SMARCA3 and VIM, and a single-base variation within BRAF. We compared the abilities of the V1 and V2 markers to detect aberrant DNA in colorectal neoplasias. METHODS: V1 and V2 marker formulations were used to analyze 144 colorectal tissue samples comprising 50 precancerous adenomas, 94 carcinomas, and 11 nonpathologic tissues. V1 analysis consisted of single-base extension analysis of the 22 V1 variants. V2 analysis consisted of DNA scanning of the APC mutation cluster region, PIK3CA exons 9 and 20, CTNNB1 exon 3, analysis for the BRAF Val600Glu substitution, and methylation-specific PCR analysis of VIM and SMARCA3. RESULTS: The V2 marker formulation had significantly higher sensitivity than the V1 markers for carcinomas (93.6% and 72.3%, respectively; P = 0.0002) and adenomas (92.0% and 62.0%, respectively; P = 0.0006). None of the nonpathologic samples were positive for any marker. CONCLUSIONS: We demonstrate improved sensitivity of a new marker formulation (V2) to detect aberrant DNA in CRC and precancerous adenoma tumor tissues.     

结肠癌PIK3CA基因G1876A突变的意义

目的寻找结肠癌中磷脂酰肌醇激酶-3(PIK3CA)基因的突变位点,结合结肠癌计算机断层扫描(CT)分期资料分析其意义。方法收集结肠癌组织78例和癌旁组织30例,通过聚合酶链反应(PCR)扩增PIK3CA第12和20外显子,单核苷酸多态性(SSCP)进行筛查,再用测序法确证,并对照经CT检查Dukes临床病理分期资料进行分析。结果结肠癌样本SSCP和DNA测序在PIK3CA第20外显子未发现突变,第12外显子有16例(20.51%)样本发生G1876A突变,突变样本均位于Dukes分期的A期和B期;而30例癌旁组织样本PIK3CA第12和20外显子均未发现突变。结论 PIK3CA基因G1876A突变可能在结肠癌发生中起着重要的作用,第12外显子基因分析对早期结肠癌患者的诊断有重要意义。     

An activating Pik3ca mutation coupled with Pten loss is sufficient to initiate ovarian tumorigenesis in mice

Mutations in the gene encoding the p110α subunit of PI3K (PIK3CA) that result in enhanced PI3K activity are frequently observed in human cancers. To better understand the role of mutant PIK3CA in the initiation or progression of tumorigenesis, we generated mice in which a PIK3CA mutation commonly detected in human cancers (the H1047R mutation) could be conditionally knocked into the endogenous Pik3ca locus. Activation of this mutation in the mouse ovary revealed that alone, Pik3caH1047R induced premalignant hyperplasia of the ovarian surface epithelium but no tumors. Concomitantly, we analyzed several human ovarian cancers and found PIK3CA mutations coexistent with KRAS and/or PTEN mutations, raising the possibility that a secondary defect in a co-regulator of PI3K activity may be required for mutant PIK3CA to promote transformation. Consistent with this notion, we found that Pik3caH1047R mutation plus Pten deletion in the mouse ovary led to the development of ovarian serous adenocarcinomas and granulosa cell tumors. Both mutational events were required for early, robust Akt activation. Pharmacological inhibition of PI3K/mTOR in these mice delayed tumor growth and prolonged survival. These results demonstrate that the Pik3caH1047R mutation with loss of Pten is enough to promote ovarian cell transformation and that we have developed a model system for studying possible therapies.     

High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles

The missense mutations at codons 12 and 13 of KRAS gene have been confirmed as a predictor of nonresponse to EGFR-targeted therapy with monoclonal antibodies cetuximab and panitumumab in patients with metastatic colorectal carcinoma (mCRC). Because of the intra-tumor heterogeneity at genetic levels, it is important to develop sensitive and selective assays to detect above KRAS mutation of rare mutated cells in the presence of large excess of wild-type cells. In the present study, wild-type blocking PCR (WTB-PCR) was developed to detect the aforementioned KRAS mutations, in which a chimera composed of locked nucleic acid (LNA) and DNA was used to inhibit with high sensitivity the amplification of wild-type KRAS alleles whereas it allowed the highly selective amplification of mutated KRAS alleles. Using mutated KRAS from HCT-116 as spiking DNA, the results showed that WTB-PCR could detect mutated alleles in a ratio of 1:10,000 (i.e., 0.01%) wild-type alleles and at a single copy level. For its further applications to detect aforementioned KRAS mutations in 20 cases of mCRC patients, the results showed that the detected mutation percentage of WTB-PCR (60%, 12/20) was higher than that of traditional PCR (45%, 9/20). Moreover, two patients respectively having synonymous mutated codons 13 (i.e., c.39C > T) and missense mutated codons 14 (i.e., c.40G > A) could be also only detected by WTB-PCR. In conclusion, the current WTB-PCR was a rapid, simple, and low-cost method to detect a trace amount of mutated KRAS gene.     

Multiplex mutation screening by mass spectrometry evaluation of 820 cases from a personalized cancer medicine registry

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.     

No duplicate KRAS mutation is identified on the same allele in gastric or colorectal cancer cells with multiple KRAS mutations

Codon 12 and 13 mutations in 170 colorectal cancer (CRC) and 66 gastric cancer (GC) specimens were analysed by an 'enriched' polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All identified mutations were verified by direct sequencing of the second PCR products. Among the 170 CRC specimens, mutations were identified in 47 (28%) and 13 (7.6%) cases in codons 12 and 13, respectively. In the 66 GC specimens examined, however, mutations in codons 12 and 13 were only detected in two (3.0%) and one (1.5%) cases, respectively. Mutations in both codon 12 and 13 were found in 3/170 (1.8%) CRCs and 1/66 (1.5%) GCs. Duplicate mutations were never identified in the same allele, which was confirmed by direct sequencing of the second amplified products. The majority of colorectal and gastric cancer cells with KRAS mutations are homogeneous because they have the same KRAS mutation. A few colorectal or gastric cancers, however, showed heterogeneity, as verified by the fact that single mutations were identified in the same allele.     

A platform for rapid detection of multiple oncogenic mutations with relevance to targeted therapy in non-small-cell lung cancer

The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.     

表皮生长因子受体-酪氨酸激酶抑制剂治疗后耐药的肺腺癌患者二次组织活检与外周血基因特征一致性分析

目的对表皮生长因子受体(epidermal growth factor receptor,EGFR)-酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)治疗后耐药的肺腺癌患者行二次组织活检并采集外周血标本进行基因检测,分析二次组织活检与外周血标本基因特征的一致性。方法EGFR-TKIs治疗后耐药的原发性肺腺癌患者37例,均行二次组织活检并采集外周血标本,应用二代测序技术检测肺癌常见的10个基因突变情况,分析二次组织活检与外周血标本基因检测结果的一致性。结果二次组织活检EGFR基因T790M突变18例(48.6%),均合并初始EGFR基因敏感位点突变,未合并其他基因突变;另19例二次组织活检EGFR基因T790M突变阴性患者均合并初始EGFR基因敏感位点突变,其中4例合并TP53基因突变,1例合并磷脂酰肌醇3-激酶催化亚基α(phosphatidylinositol 3-kinase catalytic subunitα,PIK3CA)基因突变,1例合并Kirsten大鼠肉瘤病毒癌基因同源物(Kirsten rat sarcoma viral oncogene homolog,KRAS)基因突变,1例合并间变性大细胞激酶(anaplastic lymphoma kinase,ALK)基因突变。外周血EGFR基因T790M突变12例(32.4%),其中7例合并初始EGFR基因敏感位点突变,未合并其他基因突变;另25例外周血EGFR基因T790M突变阴性患者中,18例合并初始EGFR基因敏感位点突变,9例合并TP53基因突变,2例合并PIK3CA基因突变,1例合并KRAS基因突变,1例合并ALK基因突变。外周血标本检出的EGFR基因T790M突变12例均在组织活检标本中检出,以组织活检标本基因检测结果为参照,外周血标本EGFR基因T790M突变一致性为66.7%(12/18);组织活检标本EGFR基因T790M突变阳性率(48.6%)与外周血标本(32.4%)比较差异无统计学意义(P>0.05)。除EGFR基因突变外,其他9种基因突变于外周血标本中共检出13例,其中7例在组织活检标本中检出,基因突变一致性为53.8%(7/13)。二次组织活检标本TP53基因突变阳性率(10.8%)与外周血标本(24.3%)比较差异无统计学意义(P>0.05),PIK3CA基因突变阳性率(2.7%)与外周血标本(5.4%)比较差异无统计学意义(P>0.05)。结论外周血标本EGFR基因T790M突变阳性率相对较低,可作为组织活检的重要补充;外周血标本的基因检测结果一定程度上可反映患者整体的基因突变状态。     

KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary colorectal cancers diagnostic and therapeutic implications

Targeted therapy of advanced colorectal carcinoma (CRC) necessitates KRAS genotyping. Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases. We studied 21 patients with 43 synchronous and 2 metachronous adenocarcinomas of the colorectum (n = 20) and stomach (n = 1). Five patients had liver metastases and one had a distant lymph node metastasis. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed by Sanger sequencing and pyrosequencing. Fifty-seven different neoplastic lesions were genotyped, showing 18 (31.6%) KRAS, 2 (3.5%) NRAS, and 7 (12.3%) BRAF mutations, distributed among 10 (47.6%), 1 (4.8%), and 5 (23.8%) of the patients. An identical genotype of all synchronous primary CRCs was found only in 7 (35%) of the patients; the remainder had dissimilar genotypes in various combinations. Interestingly, a single patient had an unknown KRAS genotype (c.37_39dupGGC). Six patients with 13 primary carcinomas had distant metastases. In three of these patients, the metastasis shared the genotype only with one of the primary tumors, because the other primary tumors had another genotype. Synchronous and metachronous primary CRCs of the same patient have variable KRAS, NRAS, and BRAF genotypes. When metastases occur in these patients, the genotype has diagnostic and therapeutic implications and should be determined from the simultaneous or metachronous distant metastases.     

Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF

Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.