EGFR and KRAS mutations in metastatic lung adenocarcinomas

In primary lung adenocarcinoma, EGFR and KRAS mutations are found in approximately 10% to 20% and 20% to 30%, respectively. Few studies have investigated these mutations in metastases. Patients with EGFR mutations have a 70% to 80% response rate to tyrosine-kinase inhibitors therapy and a longer progression-free survival rate in contrast to patients with KRAS mutations that are associated with virtually no response tyrosine-kinase inhibitors. In this study, we have investigated EGFR and KRAS mutations in metastatic lung adenocarcinoma. Using Johns Hopkins Hospital archives, 1966 lung adenocarcinomas were found from January 2007 to May 2010. A total of 60 metastatic adenocarcinomas (28 cytologic and 32 surgical cases) with EGFR and KRAS studies were identified. In addition, 18 cases of primary and matched metastases were also included. Exons 18 to 21 of EGFR and exon 2 of KRAS (codons 12 and 13) were sequenced. In our study, EGFR and KRAS mutations were found in 21.7% (13 of 60 cases) and 28.3% (17 of 60 cases), respectively, and occurred more often with advanced stage of primary tumors. KRAS mutations were associated with poor prognosis and occurred exclusively in smokers in comparison with EGFR mutation. Of 9 pairs, mutations were concordant in 77.8%; 1 pair displayed acquisition of KRAS mutation, whereas 1 pair showed loss of EGFR mutation in the corresponding metastasis. Our findings suggest that EGFR and KRAS status should be tested in metastasis regardless of known mutations of the primary tumor. Additional studies are needed to further investigate the mechanisms of discordances in metastatic tumors.     

EGFR, ERBB2, and KRAS mutations in Korean non-small cell lung cancer patients

The epidermal growth factor receptor (EGFR), and its family members play an important role in the development and progression of lung cancers. It has been reported that somatic mutations in the tyrosine kinase domain of the EGFR or ERBB2 genes occur in a subset of patients with lung cancer. We searched for mutations of the EGFR, ERBB2, and KRAS genes in surgically resected non-small cell lung cancers (NSCLCs) to determine the prevalence of these mutations in Korean lung cancer patients. In addition, we examined the relationship between the mutations and clinicopathologic features of lung cancers. Mutations of the EGFR, ERBB2, and KRAS genes were determined by polymerase chain reaction-based direct sequencing in 115 surgically resected non-small cell lung cancers. EGFR mutations were present in 20 patients (17.4%). The EGFR mutations were found only in adenocarcinomas (20 of 55 adenocarcinomas, 36.4%). The ERBB2 mutation was found in 1 adenocarcinoma of the 115 NSCLCs (0.9% overall; 1.8% of the 55 adenocarcinomas). KRAS mutations were found in 6 (5.2%) of the 115 NSCLCs (2 of 60 squamous cell carcinomas, or 3.3%, and 4 of 55 adenocarcinomas, or 7.3%). EGFR mutations in adenocarcinomas were more frequent in women (P = 0.02) and in never-smokers (P = 0.004). EGFR mutations in adenocarcinomas were not associated with pathologic stage in never-smokers, but were more frequent in pathologic stage II-IV than in stage I in ever-smokers (P = 0.01). Of the 55 adenocarcinomas, 25 (45.5%) had mutations of one or another of the three genes; EGFR mutations were never found in adenocarcinomas together with ERBB2 or KRAS mutations. These findings suggest that the EGFR mutation is frequent in Korean lung cancer patients, and that the ERBB2 mutation is rare. Further studies are needed to investigate the role of EGFR mutations in the carcinogenesis of adenocarcinoma among smokers.     

Detection of human papillomavirus from liquid-based cytology specimens by in-house PCR: a pilot study

The Papanicolaou smear remains the most common method for the detection of precancerous changes in cervical cytology. However, the introduction of a liquid-based cytology (LBC) technique expands the possibility of cervical intraepithelial neoplasia (CIN) diagnosis, and permits detection of precancerous changes and human papillomavirus (HPV) simultaneously. In the pilot study reported here, using an in-house polymerase chain reaction (PCR) method, high-grade HPV was detected in 32% of a cohort of 38 patients. This conventional PCR method could be developed for use on a real-time PCR platform or in a microtitre-well format and subsequently automated.     

非小细胞肺癌患者肿瘤组织中EGFR和KARS基因突变的分子病理检测分析

目的：探讨非小细胞肺癌患者肿瘤组织中EGFR和KRAS基因各亚型突变情况。方法：应用直接测序方法检测非小细胞肺癌石蜡组织中1273例EGFR基因和1062例KRAS基因突变情况。结果：非小细胞肺癌肿瘤组织中EGFR基因总突变率为36.68%(467/1273),外显子18、19、20和21的突变率分别为1.02%(13/1273)、18.93%(241/1273)、2.59%(33/1273)和15.95%(203/1273);EGFR基因各外显子之间双重突变共17例(1.34%),其中18外显子与20外显子双重突变3例(0.24%),19外显子与20外显子双重突变7例(0.55%),19外显子与21外显子双重突变4例(0.31%)和20外显子与21外显子双重突变3例(0.24%);EGFR基因各外显子内双重突变共2例(2.18%),均为21外显子双重突变。KRAS基因总突变率为3.01%(32/1062),外显子2的密码子5、12、13和25的突变率分别为0.09%(1/1062)、2.64%(28/1062)、0.18%(2/1062)和0.09%(1/1062),外显子3密码子61的突变率为0.09%(1/1062)。结论：非小细胞肺癌患者中EGFR基因存在较高的突变率,尤其为19和21外显子突变,其基因突变亚型分类能指导EGFR-TKI的肿瘤靶向治疗,KRAS基因突变率虽低但不容忽视,其基因突变预示着EGFR-TKI原发耐药。     

KRAS gene mutations in lung cancer: Particulars established and issues unresolved

Lung cancer, like other cancers, is considered to develop through the accumulation of genetic alterations. Mutation of the KRAS gene is one of the most important events in carcinogenesis of the lung. The KRAS gene, belonging to the RAS gene family, encodes a membrane‐bound 21‐kd guanosine triphosphate (GTP)‐binding protein. Single point mutations in this protein result in continuous activation to transmit excessive signals, promoting a variety of biological events. In lung cancers, the mutations concentrate at codon 12 and mostly affect adenocarcinomas (ADCs). They also affect atypical adenomatous hyperplasia, the precursor of ADCs. Therefore, mutation of the KRAS gene is suggested to confer a growth advantage to airway epithelial cells enabling them to expand clonally early in the development of ADCs. The mutation is also a reliable marker of an unfavorable response to certain molecular‐targeting therapies. Furthermore, patients with ADCs affected by mutations have been reported to exhibit a significantly higher risk of postoperative disease recurrence. Thus, the significance of KRAS gene mutations has been investigated extensively. However, not all the details emerged. In this review, particulars that have been established are introduced, and important issues remaining to be resolved are discussed, with special reference to carcinogenesis of the lung.     

液基细胞学与经支气管针吸活检在肺癌诊断中应用

目的探讨液基细胞学(liquid-based cytology,LBC)与经支气管针吸活检(transbronchial needle aspiration,TBNA)在肺癌诊断中的应用价值。方法 TBNA取材共833例,其中674例常规制备传统涂片(conventional smears,CS)后制备LBC涂片,余159例仅制作CS,比较两种制片方法在TBNA细胞病理学诊断中的差异和优、缺点,并将细胞病理学诊断与组织病理学相比较。结果 CS标本不满意率为16.4%,高于LBC涂片的标本不满意率(10.2%)。TBNA标本中LBC诊断肺癌的敏感性和准确性分别为93.5%和95.3%,CS敏感性和准确性分别为93.8%、95.3%,统计学显示两种方法的敏感性及准确性均无差异。18.5%(5/27)可疑癌病例应用LBC可得以确诊。两种制片方法得出阳性病例的分型准确率无差异。鳞癌、腺癌和小细胞癌与组织病理诊断的符合率分别为93.5%、98.3%和100%。结论在TBNA标本中应用LBC诊断肺癌的敏感性和准确性高、分型准确,与组织病理诊断符合率高,且与CS合用有互补作用。此外,LBC标本不满意率低,细胞结构清楚、背景干净、涂片数量少、阅片时间明显缩短。     

EGFR mutations in non-small-cell lung cancer among smokers and non-smokers: a meta-analysis

Mounting evidence has suggested somatic mutations in the EGFR gene are associated with better responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Some, but not all, studies have reported that the mutations were more frequently observed in patients without a smoking history. To comprehensively address this issue, we performed a meta-analysis to evaluate the association between cigarette-smoking history and mutation of the EGFR gene in NSCLC. Twenty-six studies, involving 3,688 patients with NSCLC were included in the analysis. The pooled analysis shows that the incidence of EGFR mutations in NSCLC differs according to cigarette-smoking history. The odds ratio (OR) for the EGFR mutation in non-smokers relative to smokers was 4.829 (95% confidence interval [CI]: 3.598-6.482; P < 0.001). These data may assist clinicians in assessing the likelihood of EGFR mutations in patients with NSCLC when mutational analysis is not feasible.     

液基细胞学检查系统在肺癌患者痰液检查中的应用价值

痰液脱落细胞学检查足早期诊断肺癌的重要方法之一,然而该方法的假阴性率较高,有报道达20%～60%,液基细胞学检测(LCT)技术在我国应用于妇科肿瘤的筛查和诊断取得公认的效果.     

非小细胞肺癌EGFR突变检测技术的研究进展

针对表皮生长因子受体(epidermal growth factor receptor,EGFR)突变基因的检测是肺癌靶向治疗的重要基石.目前临床中主要针对肺癌组织、脱落细胞以及液态活检标本开展检测工作,此外尿液标本近期也展现出良好的检测效率.针对上述标本,临床中开展多种检测技术,如以特定靶点为目的的技术,包括数字PCR、楔形探针扩增阻滞突变系统等;以筛查为目的的检测技术,包括二代测序(next generation sequencing,NGS)、Sanger测序等,这些技术在特定的标本类型和检测目的中具有独特的优势.一些新兴的技术,如拉曼光谱,也在临床检测中展现出良好的应用前景.     

xTAG液相芯片技术用于肺癌 EGFR基因突变检测的临床适用性

目的：分析肺癌EGFR基因第18~21号外显子突变,以Sanger测序法为参照,探讨xTAG液相芯片用于临床样本检测的适用性。方法随机选择1139例Ⅰ~Ⅳ期肺癌组织标本,提取DNA,分别采用xTAG液相芯片法和Sanger测序法检测EG-FR基因第18~21号外显子突变情况,评价xTAG液相芯片法检测的敏感性和特异性。结果液相芯片法：共1134例患者获得基因突变检测结果,Sanger测序法：共1105例患者获得基因突变检测结果,检测成功率分别为99．56%和97．01%。以San-ger测序法为参照,xTAG液相芯片法检测的敏感性和特异性分别为99．59%和94．54%,两种方法均检出少数样本存在双外显子突变。两种方法检测出的突变型别完全吻合。结论采用xTAG液相芯片法可有效检测肺癌EGFR基因突变,实现突变分型,且相对测序法更方便、高效,适合临床推广应用。     

Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients

OBJECTIVE:

Epidermal growth factor receptor is involved in the pathogenesis of non-small cell lung cancer and has recently emerged as an important target for molecular therapeutics. The KRAS oncogene also plays an important role in the development of lung cancer. The aim of this study was to evaluate the frequency of epidermal growth factor receptor and KRAS mutations in a population of Brazilian patients with non-small cell lung cancer.

METHODS:

A total of 207 specimens from Brazilian patients with non-small cell lung cancer were analyzed for activating epidermal growth factor receptor and KRAS somatic mutations, and their associations with clinicopathological characteristics (including age, gender, ethnicity, smoking habits, and histological subtype) were examined.

RESULTS:

We identified 63 cases (30.4%) with epidermal growth factor receptor mutations and 30 cases (14.6%) with KRAS mutations. The most frequent epidermal growth factor receptor mutation we detected was a deletion in exon 19 (60.3%, 38 patients), followed by an L858R amino acid substitution in exon 21 (27%, 17 patients). The most common types of KRAS mutations were found in codon 12. There were no significant differences in epidermal growth factor receptor or KRAS mutations by gender or primary versus metastatic lung cancer. There was a higher prevalence of KRAS mutations in the non-Asian patients. Epidermal growth factor receptor mutations were more prevalent in adenocarcinomas than in non-adenocarcinoma histological types. Being a non-smoker was significantly associated with the prevalence of epidermal growth factor receptor mutations, but the prevalence of KRAS mutations was significantly associated with smoking.

CONCLUSIONS:

This study is the first to examine the prevalence of epidermal growth factor receptor and KRAS mutations in a Brazilian population sample with non-small cell lung cancer.     

Cytomorphologic features of advanced lung adenocarcinomas tested for EGFR and KRAS mutations: A retrospective review of 50 cases

Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin‐embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real‐time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

The application of molecular diagnostic studies interrogating EGFR and KRAS mutations to stained cytologic smears of lung carcinoma

EGFR and KRAS mutation analyses are of increasing importance for guiding the treatment of non-small cell lung carcinomas. Insufficient cellularity of cell blocks can represent an impediment to the performance of these tests. We investigated the usefulness of cytologic direct smears as an alternative specimen source for mutation testing. Tumor cell-enriched areas from freshly prepared and archived rapid Romanowsky-stained direct smears in 33 cases of lung carcinoma were microdissected for DNA isolation and evaluated for EGFR and KRAS mutations. EGFR mutations were detected in 3 adenocarcinomas; 2 tumors had the L858R substitution and 1 an exon 19 deletion. KRAS mutations affecting codon 12, 13, or 61 were detected in 11 cases (8 adenocarcinomas and 3 non-small cell carcinomas). EGFR and KRAS mutations were mutually exclusive. Hence, archived and freshly prepared direct smears represent a robust and valuable specimen source for molecular studies, especially when cell blocks exhibit insufficient cellularity.     

非小细胞肺癌患者ALK,EGFR及KRAS基因的检测及临床病理特征

目的:研究汉族非小细胞肺癌患者中间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)、表皮生长因子受体(epidermal growth factor receptor,EGFR)及Kirsten鼠肉瘤基因(Kirstenrat sarcoma,KRAS)突变的阳性率及其与临床病理特征的关系.方法:采用免疫组织化学(immunohistochemistry,IHC)的方法检测ALK融合基因异常表达,采用PCR检测EGFR基因和KRAS基因突变,采用x2检验及Fisher精确概率法进行数据分析.结果:共2 267例进行了ALK融合基因检测,其中1 655例同时进行了EGFR突变检测,951例同时进行了KRAS检测.ALK融合基因、EGFR基因及KRAS基因突变阳性率分别为7.28％(165/2 267)、48.58％(804/1 655)、11.40％(108/947).ALK基因突变多见于年轻、腺癌患者;EGFR基因突变多见于女性、腺癌患者;KRAS基因突变多见于老年、男性、腺癌患者.1 655例同时进行了ALK与EGFR检测的病例中,共6例存在双基因突变(0.36％);947例同时进行了ALK与KRAS检测,共4例存在双基因突变(0.42％);943例同时进行了EGFR与KRAS突变检测,未发现双突变病例.结论:非小细胞肺癌患者ALK,EGFR和KRAS基因的突变与患者的年龄、性别、组织学类型均存在相应的联系,个别病例可以出现ALK融合基因与EGFR或KRAS突变共存.     

Conventional and liquid-based cervicovaginal cytology: a comparison study with clinical and histologic follow-up

Increased rates of squamous intraepithelial lesion (SIL) diagnosis with liquid-based cervicovaginal cytology (CVC) methods are well documented. This retrospective study compares the ability of the ThinPrep Pap Test (TP) and the conventional Pap smear (CP) to detect biopsy-proven SIL and to exclude nonneoplastic disease. All CVC reports from January 1999 through December 2000 from seven community Family Medicine clinics affiliated with the University of Nebraska were reviewed. For women with at least one CVC diagnosis of epithelial cell abnormality (ECA), follow-up histology, cytology, and clinical data were obtained. Statistical analysis was performed using the chi-square method. SIL was diagnosed in 166 of 3,286 patients by TP (5.1%) and in 169 of 4,872 patients by CP (3.5%) (P < 0.001); 32 of the TP diagnoses (1.0%) and 34 of the CP diagnoses (0.7%) were high-grade SIL (HSIL). Atypical squamous or glandular cells of undetermined significance (ASCUS/AGUS) was the most severe abnormality diagnosed by TP in 218 patients (6.6%) and by CP in 279 patients (5.7%). Follow-up histology data on CVC SIL diagnoses showed evidence of cervical intraepithelial neoplasia in 94 patients screened by TP (2.9%) and in 79 patients screened by CP (1.6%) (P < 0.001); the biopsy diagnoses were CIN 2 or CIN 3 in 34 patients in the TP group (1.0%) and in 28 patients in the CP group (0.6%) (P < 0.025). Follow-up of patients in whom the first ECA CVC diagnosis was ASCUS or AGUS disclosed a positive predictive value for CIN of 22.8% for TP ASCUS/AGUS diagnoses and 11.9% for CP ASCUS/AGUS diagnoses (P < 0.005). In this population, TP was significantly better than CP in detecting biopsy- proven disease and in screening out benign abnormalities.     

Adenosquamous carcinoma of the lung: a microdissection study of KRAS and EGFR mutational and amplification status in a western patient population

Molecular testing of pulmonary adenocarcinomas for EGFR and KRAS mutations is becoming more common as tyrosine kinase inhibitor therapy is used for EGFR-mutated adenocarcinomas. Adenosquamous carcinomas represent a hybrid carcinoma, and there is no literature addressing the frequency of EGFR and KRAS mutations in this subset of lung carcinomas in Western populations. For this study, 23 adenosquamous carcinomas were microdissected with the glandular and squamous components analyzed for EGFR and KRAS mutations and EGFR amplification. In 3 cases (13%), there were EGFR mutations, with 2 having the identical mutation in the glandular and squamous elements. In 3 cases (13%), there were KRAS mutations in both histologic elements. Great heterogeneity existed in the rates of EGFR amplification in the 2 histologic components. Amplification was most common in both glandular and squamous components (11/23 [48%]). EGFR mutations occur in adenosquamous carcinoma in the same percentages as in conventional adenocarcinoma in the Western population. KRAS mutations are less common.     

液基细胞技术在术前检测乳腺癌ER和PR中的应用

雌激素受体(ER)和孕激素受体(PR)是乳腺癌重要的预后因素和预测激素治疗反应的指标.术前检测乳腺癌ER和PR的方法主要是用针吸细胞学标本进行免疫细胞化学染色,由于制片质量不稳定,常导致检测结果不理想.液基细胞制片技术是近几年发展起来的新技术,本实验把液基细胞制片技术引入到乳腺癌ER和PR的术前检测中,尝试对传统检测方法的进一步完善.     

Incision of AP sites in lung cancer patients: a pilot study

DNA base excision repair (BER) removes frequent DNA lesions of either endogenous or exogenous origin. Some indications point to BER defects in lung cancer patients. We have investigated the ability of ten lung cancer patients to repair natural AP sites, the most frequent genetic lesion, using an in vitro assay in which peripheral blood lymphocytes (PBL) extracts incise randomly depurinated plasmid DNA. The median value of repair activity in patients was lower than that of matched controls but the difference did not reach significance. Unlike other BER enzymatic steps, marked defects in incision of AP sites may not be associated with lung carcinogenesis.     

Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing

Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18–21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2–1.5 %, as opposed to 10–20 % detection limit of Sanger sequencing. High reproducibility (0–2.1 % variant frequency) and very low background level (0.4–0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9–10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.